ASTM E939-94
(Test Method)Standard Test Method of Field Testing Topical Applications of Compounds as Repellents for Medically Important and Pest Arthropods (Including Insects, Ticks, and Mites):I Mosquitoes
Standard Test Method of Field Testing Topical Applications of Compounds as Repellents for Medically Important and Pest Arthropods (Including Insects, Ticks, and Mites):I Mosquitoes
SCOPE
1.1 This test method is used to evaluate the repellency of promising compounds that have undergone primary laboratory studies and have been approved for skin application for secondary testing.
1.2 This test method is designed for the study of mosquito repellents, but with some modifications this test method can be used to determine the repellency of candidate compounds for other flying insects that attack humans.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: E 939 – 94
Standard Test Method of
Field Testing Topical Applications of Compounds as
Repellents for Medically Important and Pest Arthropods
(Including Insects, Ticks, and Mites):I Mosquitoes
This standard is issued under the fixed designation E 939; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 4.3 The simplicity of the test offers flexibility under a
relatively wide range of circumstances and geographical loca-
1.1 This test method is used to evaluate the repellency of
tions. By following this test method, international testing with
promising compounds that have undergone primary laboratory
a variety of vector mosquito populations is no more difficult to
studies and have been approved for skin application for
accomplish than tests with various domestic species.
secondary testing.
4.4 A number of people test topical applications of a
1.2 This test method is designed for the study of mosquito
repellent for the following reasons:
repellents, but with some modifications this test method can be
4.4.1 To determine how long the repellent is effective;
used to determine the repellency of candidate compounds for
4.4.2 To establish the effective dosage range;
other flying insects that attack humans.
4.4.3 To establish the range of effectiveness on several
1.3 This standard does not purport to address all of the
mosquito genera and species in a number of geographical
safety concerns, if any, associated with its use. It is the
areas; and
responsibility of the user of this standard to establish appro-
4.4.4 To identify the material in terms of odor, staining
priate safety and health practices and determine the applica-
capability, plasticizing effect, and oiliness or greasiness.
bility of regulatory limitations prior to use.
4.5 No repellent should be tested on humans without the
2. Terminology written consent of the test volunteers (hereafter referred to as
test subjects) and prior approval of competent authority, as
2.1 Description of Term Specific to This Standard:
designated in applicable laws and regulations governing ex-
2.1.1 complete protection time (CPT)—the time from appli-
perimentation on humans.
cation of the repellent to the time of the first confirmed bite (a
second bite by the same species within 30 min of the first). This
5. Apparatus
permits any number of unconfirmed bites during the CPT.
5.1 Insect collection vials.
3. Summary of Test Method 5.2 Aspirator.
5.3 Stereoscope (optional).
3.1 A measured amount of the candidate material is applied
5.4 Standard References for Mosquito Identification, for
to the forearm or sometimes the lower leg. These areas are then
determining species present in the field (optional).
protected from rubbing and are continuously exposed to
5.5 Temperature and Humidity Reading Equipment—
mosquitoes in the field to determine the length of time the
Ideally, a continuous recording device such as a hygrothermo-
treatment provides either complete protection or a high level of
graph should be used to record conditions during tests. If such
protection.
equipment is not available, readings should be made immedi-
4. Significance and Use
ately before, midway during, and immediately after the tests
are made, using a sling psychrometer.
4.1 This test method is an important part of the final phase
5.6 Air Speed Indicator and Light Meter, optional but
of study in the development of mosquito repellents for personal
preferred if equipment is available.
use.
5.7 Watch.
4.2 This test method is primarily designed to simulate a
5.8 Headnets.
situation in which a person treated with a repellent is exposed
5.9 Cotton Gloves.
to natural populations of attacking mosquitoes.
5.10 Battery-Operated Head Lamps, with red filters for tests
with nocturnally active species.
This test method is under the jurisdiction of ASTM Committee E-35 on
Pesticides and is the direct responsibility of Subcommittee E35.12 on Insect Control 5.11 Notebook, Test Sheets, and Pencils, for recording
Agents.
species, test data, date, and locality of the test. A sample work
Current edition approved June 15, 1994. Published August 1994. Originally
sheet is attached with recorded results (see Annex A1.).
e1
published as E 939 – 83. Last previous edition E 939 – 83 (1988) .
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 939
5.12 Water Supply, Mild Soap, and Paper Towels, for skin between the ankle and knee.
washing treated skin.
8.5 Determine the surface area of the limbs of each test
5.13 Clothing, should be appropriate to the season and
subject so that treatment rates of candidate repellents and the
geographical area.
standard are uniformly applied. Adjust the application rates for
differences in arm or leg size of different subjects.
NOTE 1—There are differences of opinion as to whether the clothing
8.6 Expose the treated arms or legs continuously to natural
worn should be uniform in color and type, however, data available do not
reject or confirm this contention.
populations of the mosquito species being tested.
8.7 The duration of effectiveness of the repellent is indi-
5.14 List of Chemical Names, identifying the compounds or
cated by the CPT.
mixtures, or both, to be tested.
8.8 Employ a balanced incomplete block (BIB) experimen-
NOTE 2—In the event of a medical emergency, the chemical list along
tal design (round robin) when three to five chemicals are to be
with pertinent toxicological data may be required.
tested. With this design, test each repellent in the series on
6. Reagents and Materials opposite arms of a given number of subjects.
6.1 All test solutions are formulated on a weight to volume
NOTE 5—Ideally, the number of subjects should equal the number of
basis (usually with 95% ethanol).
candidate compounds, excluding the standard. Thus, if four compounds
6.2 Test chemicals in 25% ethanol solutions. are to be tested, including the standard, three test subjects would be
required. To illustrate, the pairings would be: AB, AC, AD, BC, BD, and
6.3 Test standard deet (N,N-diethyl-meta-toluamide) in a
CD. Subject 1 would test AB and CD; Subject 2, AC and BD, and Subject
25% ethanol solution.
3, AD and BC.
6.4 For final development studies, formulations of lotion,
liquids, creams, solids, or pressurized aerosols containing
8.9 Because of their variability in attractiveness, assign
ingredients for field evaluation are included.
repellents to test subjects in a randomized and balanced fashion
so that each subject does not wear the same combination of
7. Sampling
materials more than once in a single BIB test series. For
7.1 Each candidate repellent is paired with each other
example, if compounds A, B, C, D, and E are tested with a
repellent or a standard on the arms of a subject and exposed
standard F, then Subject 1 will test AB, CD, and EF; Subject 2
simultaneously to the same insect population (See Annex A1).
will test AC, BE, and DF; Subject 3 will test AD, BF, and CE;
7.2 A round-robin or paired test is used in the experiments,
Subject 4 will test AE, BC, and CF; and Subject 5 will test AF,
usually based on the number of experimental materials being
BC, and DE.
evaluated.
NOTE 6—If four or five compounds, excluding the standard, are tested
7.3 Treatments are exposed to the mosquito population for
in a BIB series, one replication of the BIB is sufficient for a statistical
as long as the repellents are effective, and the biting activity
analysis. If three compounds are to be tested in this fashion, two
continues.
replications of the round robin will be necessary.
8. Procedure
8.10 A direct comparison of the candidate versus the stan-
8.1 Determine the identity of species of mosquito in the test
dard repellent deet is used when fewer than three chemicals are
area prior to the test. Determine the time to begin and end tests
to be tested. For these tests, four or more replications of tests
each day by the activity of the species to be tested. Diurnal
with each chemical on at least three different subjects are
nocturnal or other patterns of mosquito behavior will govern
necessary.
the scheduling of tests.
8.11 Express the results obtained by paired tests or BIB
8.2 For field tests, make the initial studies with a 25%
comparisons at CPT in minutes or hours.
ethanol solution of the candidate repellents (250 mg AI/mL). A
8.12 During the experiment, subjects shall avoid contact
compound or formulation may be retested at reduced or
with the treated skin surfaces. Touching, rubbing, or abrasive
increased concentrations, or at full strength, if warranted.
action on the treated skin can affect the results. Avoid undue
NOTE 3—If the complete protection times (CPTs) of repellents are too sweating or wetting of the treated skin except in special tests
long for the available testing period or if the CPTs are too short for
designed to study the durability of repellents under adverse
evaluation, adjust the concentrations accordingly. The termination of an
conditions. Loss due to evaporation and absorption is, of
evaluation before a confirmed bite occurs is termed a “plus out” and
course, unavoidable but should be the only known reason for
should be avoided.
loss in these tests, if the above precautions are taken.
8.3 Spread 1 mL of the repellent formulation or repellent
8.13 Test each species with its biting behavior in mind. For
solution evenly over the forearm of the subject and compare
example, if a test subject remains immobile, some diurnally
directly with another repellent of the same concentration on the
active mosquito species become less aggressive and biting
other arm (see Note 4). The behavior of some species neces-
pressure on the repellent treated skin will be reduced.
sitates the use of the legs instead of the arms as treatment sites.
8.14 Intermittent walking, standing, and squatting, as well
This can be determined by observation before the tests begin.
as raising and lowering the arms periodically, are very effective
NOTE 4—The concentration of a compound being tested is not as
in attracting many of the daytime biting species. Test supervi-
important as the assurance that
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