ASTM E951-94
(Test Method)Standard Test Methods for Laboratory Testing of Non-Commercial Mosquito Repellent Formulations On the Skin
Standard Test Methods for Laboratory Testing of Non-Commercial Mosquito Repellent Formulations On the Skin
SCOPE
1.1 These test methods apply to repellent compounds and formulations that can be appropriately diluted with ethanol, acetone, or a similar inert carrier for test purposes. The test methods described are not suitable for testing powders, sticks or other solid formulations, or for testing thixotropic or other fluids whose physical properties would be modified by dilution.
1.2 These test methods are designed and intended for use as a research standard to develop data on the efficacy of repellents applied to the skin of humans against laboratory-reared or field-collected mosquitoes. The use of these test methods will provide for the development of a data base whereby all investigators generate comparable data. Modifications of the equipment or procedures, or both, may be needed for tests against other kinds of biting arthropods.
1.3 The test methods are intended for use in testing materials that are in an advanced stage of development, for which human-use trials can be fully justified on scientific and ethical grounds. The test methods are not designed for the testing of commercial formulations where registration or advertising claims data are required.
1.3.1 A repellent should not be considered for testing on humans before its efficacy has been demonstrated in in vitro, animal, or other nonhuman test systems.
1.3.2 A repellent should not be applied to the skin before its safety has been established in appropriate toxicological tests on animals or other test organisms.
1.3.3 No repellent should be tested on humans without the written consent of the test subjects and prior approval of competent authority, as designated in the applicable laws and regulations governing experimentation on humans.
1.4 The values stated in inch-pound units are to be regarded as the standard. The values given in parentheses are for information only.
1.5 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 951 – 94
Standard Test Methods for
Laboratory Testing of Non-Commercial Mosquito Repellent
Formulations On the Skin
This standard is issued under the fixed designation E 951; 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 priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
1.1 These test methods apply to repellent compounds and
formulations that can be appropriately diluted with ethanol,
2. Referenced Document
acetone, or a similar inert carrier for test purposes. The test
2.1 ASTM Standards:
methods described are not suitable for testing powders, sticks
E 939 Test Method of Field Testing Topical Applications of
or other solid formulations, or for testing thixotropic or other
Compounds as Repellents for Medically Important and
fluids whose physical properties would be modified by dilu-
Pest Arthropods (Including Insects, Ticks, and Mites): I
tion.
Mosquitoes
1.2 These test methods are designed and intended for use as
2.2 Other Documents:
a research standard to develop data on the efficacy of repellents
Directions for Abstractors and Section Editors of Chemical
applied to the skin of humans against laboratory-reared or
Abstracts
field-collected mosquitoes. The use of these test methods will
Consolidated List of Approved Common Names of Insecti-
provide for the development of a data base whereby all
cides and Other Pesticides
investigators generate comparable data. Modifications of the
Common Names of Insects and Related Organisms
equipment or procedures, or both, may be needed for tests
against other kinds of biting arthropods.
3. Apparatus
1.3 The test methods are intended for use in testing mate-
3.1 Test Cage—The following design and materials have
rials that are in an advanced stage of development, for which
been found suitable for construction of the mosquito cage (see
human-use trials can be fully justified on scientific and ethical
Fig. 1):
grounds. The test methods are not designed for the testing of
3.1.1 The cage is rectangular in shape, length, width, and
commercial formulations where registration or advertising
height is approximately 7.2 by 2 by 1.6 in. (18 by 5 by 4 cm).
claims data are required.
The top of the cage (5 by 18 cm) is made of metal or plastic
1.3.1 A repellent should not be considered for testing on
mosquito screening, and the sides, ends, and floor are made of
humans before its efficacy has been demonstrated in in vitro,
⁄8 in. (3.2 mm) clear acrylic plastic.
animal, or other nonhuman test systems.
3.1.2 Five 1 ⁄8 in. (29 mm) circular openings are drilled in
1.3.2 A repellent should not be applied to the skin before its
line on 1 ⁄8 in. (35 mm) centers in the floor of the cage.
safety has been established in appropriate toxicological tests on
3.1.3 The two sides and one of the ends of the cage are
animals or other test organisms.
grooved and slotted to receive a flexible rectangular slide made
1.3.3 No repellent should be tested on humans without the
of 0.012 in. (0.3 mm) cellulose acetate sheeting. The slide
written consent of the test subjects and prior approval of
should move freely over the floor of the cage to open and close
competent authority, as designated in the applicable laws and
the five openings.
regulations governing experimentation on humans.
3.1.4 One end of the cage is fitted with a No. 3 stopper in a
1.4 The values stated in inch-pound units are to be regarded
⁄2 in. (13 mm) hole for insertion of the test mosquitoes.
as the standard. The values given in parentheses are for
3.2 Harness—Two belts are used to secure the test cage to
information only.
the forearm during the test. These should be approximately 1
1.5 This standard does not purport to address all of the
in. (2.5 cm) wide by 12 in. (30 cm) long. If an elastic material
safety concerns, if any, associated with its use. It is the
is used for the belts, snap or friction type fasteners should be
responsibility of the user of this standard to establish appro-
provided for joining the ends. If an inelastic material is used,
1 2
These test methods are under the jurisdiction of ASTM Committee E-35 on Annual Book of ASTM Standards, Vol 11.05.
Pesticides and are the direct responsibility of Subcommittee 35.12 on Insect Control Available from the American Chemical Society, 1155 16th St., N.W., Wash-
Agents. ington, D.C. 20036.
Current edition approved June 15, 1994. Published August 1994. Originally Available from the Entomological Society of America, College Park, MD
published as E 951 – 83. Last previous edition E 951 – 89. 20740.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 951
FIG. 1 Test Cage
slides or buckles should be provided. levels appropriate for the species under test. In areas or
3.3 Template—A template made of ⁄8 in. (3.2 mm) acrylic localities where suitable laboratory facilities are not available,
plastic to match exactly the floor of the test cage is used as a rearing and testing should be carried out, if possible, in a
guide to outline the five circular treatment areas on the building free of extremes of temperature, humidity, illumina-
forearm. tion, and wind. Where possible, all comparative tests should be
made under similar conditions.
4. Reagents and Materials
7.3 In the case of the four-hour ED50 test (14.1-19.1), the
4.1 The diluent used in the test will ordinarily be ethanol.
forearm should not be washed, rubbed, scratched, or otherwise
However, for some repellents (for example, water-based or
treated in such a way as to nullify the repellent treatments
lanolin-based formulations) dilution in ethanol may be inap-
during the four-hour test period. However, the test participants
propriate. In such cases, the appropriate diluent will be used
should be normally active during the test period to ensure that
instead.
the factors of perspiration and abrasion are incorporated in the
test.
5. Sampling
7.4 Nothing in this section should be construed to mean that
5.1 Take a bulk sample and a laboratory sample as directed
special test conditions may not be adopted for tests having a
in any applicable material specifications. In the absence of such
special purpose. For example, the wear resistance of the test
specifications, take a laboratory sample believed to be repre-
repellent can be measured by abrading the treated forearm in a
sentative of the lot to be tested. In the case of a suspension,
controlled manner before the test mosquitoes are applied to
emulsion or similar formulation, thoroughly mix the material
bioassay. In such special-purpose tests, the applicable parts of
to be sampled before the sample is taken.
the standard should be followed as closely as possible.
7.5 Since test conditions may vary to some extent under this
6. Test Specimen and Sample
test method, it is essential that full information on all variables
6.1 Take test specimens by pipet as required (see 10.4 and
relating to the test repellent, test mosquitoes, procedures, and
16.1.1). Take a new test specimen for each trial and replicate
test conditions be made part of the final report (12.1-12.3).
needed in the test. The number of trials and replicates needed
depends on the variability of the results obtained and the TEST METHOD E50 (A)
degree of precision required. See 13.1 and 13.2.
8. Summary of Test Method
6.2 In the case of a suspension, emulsion, or similar
8.1 Five circular test areas are outlined on the flexor region
formulation, the sample must be thoroughly mixed before the
of the forearm and treated with the diluent (as the control) and
test specimen is taken.
four serial dilutions of the test repellent (the repellent treat-
7. Conditioning
ments). A cage having matching cutouts in its floor and
7.1 The mosquitoes used in the test should be conspecific containing ten mosquitoes is then applied to the forearm, and
females maintained on 10 % sucrose solution prior to testing. the numbers of mosquitoes feeding on the control and the
Laboratory-reared mosquitoes should be mated nullipars in the repellent treatments is recorded. In subsequent trials, the range
age-range of 5 to 15 days. Field-collected mosquitoes should of dosages applied to the forearm is adjusted to bracket the
be held in the laboratory for 48 h or longer prior to testing to median effective dosage (ED50) of the test repellent. The test
is replicated at that range of dosages until a valid estimate of
allow for transport mortality and accommodation to laboratory
conditions. During the period the mosquitoes should be pro- the ED50 can be obtained. The data are analyzed by established
vided with an appropriate substratum for oviposition. methods of biological assay.
NOTE 1—The age-range specified for the test mosquitoes is arbitrary
9. Significance and Use
and is intended only as an aid in standardization of the test among users.
9.1 The ED50 test provides an estimate of the amount of
Users have reason to adopt a different age-range for testing should report
the age-range actually used (12.3).
7.2 Where adequate facilities are available, rearing and
Gerberg, E. J., Manual for Mosquito Rearing and Experimental Techniques,
testing conditions should be controlled and standardized at Amer. Mosquito Control Assoc., Fresno, CA, 1970, 109 pp.
E 951
repellent that must be applied to the skin to produce a given 10.13 Apply 0.025 mL of the material in Tube 2 to the test
level of effectiveness against the mosquito test population. The area designated for that treatment. Spread evenly.
levels of effectiveness that are usually of interest are the 50 % 10.14 Apply 0.025 mL of the material in Tube 1 to the test
level (for comparative purposes) and the 95 % level (for area designated for that treatment. Spread evenly.
practical purposes). The ED50 test method is used to determine 10.15 After 4 min, fit the five cutouts in the floor of the test
the effectiveness of a repellent against different kinds of cage to the five test areas outlined on the test subject’s forearm,
mosquitoes or to compare the effectiveness of different repel- and secure the test cage to the forearm with the two belts
lents against any particular kind of mosquito. It may also be provided.
used to establish the dosages needed to provide protection 10.16 After one additional minute, withdraw the slide from
under special conditions of climate, weather, activity, etc. the test cage to expose the test areas on the forearm to the
mosquitoes.
10. Procedure
10.17 At 1.5 min after the slide is withdrawn, record the
10.1 Load the test cage with 10 to 20 female mosquitoes
numbers of mosquitoes feeding on each of the five test areas.
from the population to be tested.
10.18 In subsequent trials, adjust the range of dosages to
bracket the ED50 of the test repellent by successively doubling
NOTE 2—This number of mosquitoes is intended as a limitation on the
number of bites received by the test subject. In tests against species having
or halving the concentration of repellent made up in Tube 1
characteristically low laboratory feeding rates, larger numbers of mosqui-
(10.4).
toes should be used.
NOTE 7—The general equivalents calculated in accord once with Note
10.2 Using the plastic template as a guide, outline five
4 are as follows: (1) A concentration of 1.0 % exactly repellent in Tube 1
circular test areas on the flexor region of the test subject’s
provides a dosage of 0.03898 mg/cm on the forearm. (2) A concentration
forearm with a fine-tipped felt pen. Label the test areas “A”
of 25.65 % repellent in Tube 1 provides a dosage of 1.000 mg/cm on the
through “E”, beginning with the test area nearest the elbow.
forearm. These figures may be used as conversion factors where needed.
However, modification of the equipment or procedures described herein
10.3 Set up four test tubes in a test tube rack and label them
may necessitate recalculation of the conversion factors.
“1” through “4”, from left to right.
10.4 Make up approximately 1 mL of 0.41 % of the test 10.19 When the appropriate range of dosages has been
repellent in Tube 1 (Notes 3 and 4). Mix the preparation with determined (10.18), replicate the test as necessary to obtain an
a vortical mixer. acceptably precise estimate of the ED50 of the test repellent. If
a low dilution of repellent is being applied, it may tend to
NOTE 3—The diluent used will ordinarily be ethanol, but in some cases
spread beyond the boundary of the test area. This can often be
it may be appropriate to use a different material. See 4.1.
prevented by applying undiluted repellents at the desired rate
NOTE 4—This strength (0.41 %) is calculated to provide a dosage of
0.016 mg of repellent per cm of skin surface when 0.025 mL of the with an adjustable pipet. However, the amount of a repellent
solution is spread over a 1 ⁄8 in.-(29 mm) diameter circular test area. The
that can be applied to a given area of skin will ultimately be
calculation assumes that the specific gravity of the test repellent is equal
limited by runoff. The dose at which runoff occurs is a function
to one. If the actual specific gravity of the test repellent is known, a
of the viscosity of the repellent. The number of trials and
correction to the nominal dosage of 0.016 mg/cm can be calculated.
replicates needed depends on the variability of the results
10.5 Put 0.5 mL of diluent into each of tubes, 2, 3, and 4.
obtained and the degree of precision required. See 13.1 and
10.6 Transfer 0.5 mL of material from tube 1 to tube 2. Mix
13.2.
with the vortical mixer.
NOTE 8—It is best to limit the test participants to not more than one or
10.7 Transfer 0.5 mL of material from tube 2 to tube 3. Mix
two trials per day on each forearm. If the same subject is used in
with the vortical mixer.
subsequent trials to bracket the median effective dose, care must be taken
10.8 Transfer 0.5 mL of material from tube 3 to tube 4. Mix
to cleanse completely the skin where the repellent had previously been
with the vortical mixer.
applied. Otherwise, residues from the initial repellent application might
compound the effects and results. Since there is reason to believe that the
NOTE 5—Steps described in 10.5-10.8 are a serial dilution procedure.
test repellent performs unequally on different test subjects, the replicates
The solutions contained in Tubes 1 to 4 will provide repellent dosages of
2 of the test should be divided equally among two or more subjects.
0.016, 0.008, 0.004, and 0.002
...
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