ASTM E951-94(2005)

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.

General Information

• Status: Historical
• Publication Date: 31-Oct-2005
• ICS: 71.100.99 - Other products of the chemical industry
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents

Relations

• Replaces: ASTM E951-94(2000) - Standard Test Methods for Laboratory Testing of Non-Commercial Mosquito Repellent Formulations On the Skin
• Effective Date: 01-Nov-2005
• Replaced By: ASTM E951-94(2006) - Standard Test Methods for Laboratory Testing of Non-Commercial Mosquito Repellent Formulations On the Skin (Withdrawn 2015)
• Effective Date: 01-Apr-2006

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:E951–94(Reapproved 2005)
Standard Test Methods for
Laboratory Testing of Non-Commercial Mosquito Repellent
Formulations On the Skin
This standard is issued under the fixed designation E951; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
1.1 These test methods apply to repellent compounds and
bility of regulatory limitations prior to use.
formulations that can be appropriately diluted with ethanol,
acetone, or a similar inert carrier for test purposes. The test
2. Referenced Document
methods described are not suitable for testing powders, sticks
2.1 ASTM Standards:
or other solid formulations, or for testing thixotropic or other
E939 Test Method of FieldTestingTopicalApplications of
fluids whose physical properties would be modified by dilu-
Compounds as Repellents for Medically Important and
tion.
Pest Arthropods (Including Insects, Ticks, and Mites): I
1.2 These test methods are designed and intended for use as
Mosquitoes
aresearchstandardtodevelopdataontheefficacyofrepellents
2.2 Other Documents:
applied to the skin of humans against laboratory-reared or
Directions for Abstractors and Section Editors of Chemical
field-collected mosquitoes. The use of these test methods will
Abstracts
provide for the development of a data base whereby all
Consolidated List of Approved Common Names of Insecti-
investigators generate comparable data. Modifications of the
cides and Other Pesticides
equipment or procedures, or both, may be needed for tests
Common Names of Insects and Related Organisms
against other kinds of biting arthropods.
1.3 The test methods are intended for use in testing mate-
3. Apparatus
rials that are in an advanced stage of development, for which
3.1 Test Cage—The following design and materials have
human-use trials can be fully justified on scientific and ethical
been found suitable for construction of the mosquito cage (see
grounds. The test methods are not designed for the testing of
Fig. 1):
commercial formulations where registration or advertising
3.1.1 The cage is rectangular in shape, length, width, and
claims data are required.
height is approximately 7.2 by 2 by 1.6 in. (18 by 5 by 4 cm).
1.3.1 A repellent should not be considered for testing on
The top of the cage (5 by 18 cm) is made of metal or plastic
humans before its efficacy has been demonstrated in in vitro,
mosquito screening, and the sides, ends, and floor are made of
animal, or other nonhuman test systems.
⁄8 in. (3.2 mm) clear acrylic plastic.
1.3.2 Arepellent should not be applied to the skin before its
3.1.2 Five 1 ⁄8 in. (29 mm) circular openings are drilled in
safetyhasbeenestablishedinappropriatetoxicologicaltestson
line on 1 ⁄8 in. (35 mm) centers in the floor of the cage.
animals or other test organisms.
3.1.3 The two sides and one of the ends of the cage are
1.3.3 No repellent should be tested on humans without the
groovedandslottedtoreceiveaflexiblerectangularslidemade
written consent of the test subjects and prior approval of
of 0.012 in. (0.3 mm) cellulose acetate sheeting. The slide
competent authority, as designated in the applicable laws and
shouldmovefreelyoverthefloorofthecagetoopenandclose
regulations governing experimentation on humans.
the five openings.
1.4 The values stated in inch-pound units are to be regarded
3.1.4 One end of the cage is fitted with a No. 3 stopper in a
as the standard. The values given in parentheses are for
⁄2 in. (13 mm) hole for insertion of the test mosquitoes.
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
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
These test methods are under the jurisdiction of ASTM Committee E35 on the ASTM website.
Pesticides and Alternative Control Agents and are the direct responsibility of Available from the American Chemical Society, 1155 16th St., N.W., Wash-
Subcommittee 35.12 on Insect Control Agents. ington, D.C. 20036.
Current edition approved Nov. 1, 2005. Published November 2005. Originally Available from the Entomological Society of America, College Park, MD
approved in 1983. Last previous edition approved in 2000 as E951–94 (2000). 20740.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E951–94 (2005)
FIG. 1 Test Cage
NOTE 1—The age-range specified for the test mosquitoes is arbitrary
3.2 Harness—Two belts are used to secure the test cage to
and is intended only as an aid in standardization of the test among users.
the forearm during the test. These should be approximately 1
Users have reason to adopt a different age-range for testing should report
in. (2.5 cm) wide by 12 in. (30 cm) long. If an elastic material
the age-range actually used (12.3).
is used for the belts, snap or friction type fasteners should be
7.2 Where adequate facilities are available, rearing and
provided for joining the ends. If an inelastic material is used,
testing conditions should be controlled and standardized at
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,andwind.Wherepossible,allcomparativetestsshouldbe
4. Reagents and Materials made under similar conditions.
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 Nothinginthissectionshouldbeconstruedtomeanthat
5.1 Take a bulk sample and a laboratory sample as directed
special test conditions may not be adopted for tests having a
inanyapplicablematerialspecifications.Intheabsenceofsuch
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 Sincetestconditionsmayvarytosomeextentunderthis
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.
6.2 In the case of a suspension, emulsion, or similar
8. Summary of Test Method
formulation, the sample must be thoroughly mixed before the
8.1 Five circular test areas are outlined on the flexor region
test specimen is taken.
of the forearm and treated with the diluent (as the control) and
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
allow for transport mortality and accommodation to laboratory
conditions. During the period the mosquitoes should be pro-
Gerberg, E. J., Manual for Mosquito Rearing and Experimental Techniques,
vided with an appropriate substratum for oviposition. Amer. Mosquito Control Assoc., Fresno, CA, 1970, 109 pp.
E951–94 (2005)
at one time for use in a number of trials.
median effective dosage (ED50) of the test repellent. The test
is replicated at that range of dosages until a valid estimate of
10.10 Apply 0.025 mL of diluent with a micropipet to the
theED50canbeobtained.Thedataareanalyzedbyestablished
test area designated as the control. Spread evenly with the tip
methods of biological assay.
of a glass rod.
10.11 Apply 0.025 mL of the material in Tube 4 to the test
9. Significance and Use
area designated for that treatment. Spread evenly.
9.1 The ED50 test provides an estimate of the amount of
10.12 Apply 0.025 mL of the material in Tube 3 to the test
repellent that must be applied to the skin to produce a given
area designated for that treatment. Spread evenly.
levelofeffectivenessagainstthemosquitotestpopulation.The
10.13 Apply 0.025 mL of the material in Tube 2 to the test
levels of effectiveness that are usually of interest are the 50%
area designated for that treatment. Spread evenly.
level (for comparative purposes) and the 95% level (for
10.14 Apply 0.025 mL of the material in Tube 1 to the test
practicalpurposes).TheED50testmethodisusedtodetermine
area designated for that treatment. Spread evenly.
the effectiveness of a repellent against different kinds of
10.15 After 4 min, fit the five cutouts in the floor of the test
mosquitoes or to compare the effectiveness of different repel-
cagetothefivetestareasoutlinedonthetestsubject’sforearm,
lents against any particular kind of mosquito. It may also be
and secure the test cage to the forearm with the two belts
used to establish the dosages needed to provide protection
provided.
under special conditions of climate, weather, activity, etc.
10.16 After one additional minute, withdraw the slide from
the test cage to expose the test areas on the forearm to the
10. Procedure
mosquitoes.
10.1 Load the test cage with 10 to 20 female mosquitoes
10.17 At 1.5 min after the slide is withdrawn, record the
from the population to be tested.
numbers of mosquitoes feeding on each of the five test areas.
10.18 In subsequent trials, adjust the range of dosages to
NOTE 2—This number of mosquitoes is intended as a limitation on the
brackettheED50ofthetestrepellentbysuccessivelydoubling
numberofbitesreceivedbythetestsubject.Intestsagainstspecieshaving
characteristically low laboratory feeding rates, larger numbers of mosqui- or halving the concentration of repellent made up in Tube 1
toes should be used.
(10.4).
10.2 Using the plastic template as a guide, outline five
NOTE 7—The general equivalents calculated in accord once with Note
circular test areas on the flexor region of the test subject’s
4 are as follows: (1)Aconcentration of 1.0% exactly repellent in Tube 1
forearm with a fine-tipped felt pen. Label the test areas “A” provides a dosage of 0.03898 mg/cm on the forearm. (2)Aconcentration
of 25.65% repellent inTube 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.
10.3 Set up four test tubes in a test tube rack and label them
However, modification of the equipment or procedures described herein
“1” through “4”, from left to right.
may necessitate recalculation of the conversion factors.
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.
acceptablypreciseestimateoftheED50ofthetestrepellent.If
NOTE 3—The diluent used will ordinarily be ethanol, but in some cases
a low dilution of repellent is being applied, it may tend to
it may be appropriate to use a different material. See 4.1.
spread beyond the boundary of the test area. This can often be
NOTE 4—This strength (0.41%) is calculated to provide a dosage of
prevented by applying undiluted repellents at the desired rate
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
calculation assumes that the specific gravity of the test repellent is equal
that can be applied to a given area of skin will ultimately be
to one. If the actual specific gravity of the test repellent is known, a
limitedbyrunoff.Thedoseatwhichrunoffoccursisafunction
correction to the nominal dosage of 0.016 mg/cm can be calculated.
of the viscosity of the repellent. The number of trials and
10.5 Put 0.5 mL of diluent into each of tubes, 2, 3, and 4. replicates needed depends on the variability of the results
10.6 Transfer 0.5 mLof material from tube 1 to tube 2. Mix
obtained and the degree of precision required. See 13.1 and
with the vortical mixer. 13.2.
10.7 Transfer 0.5 mLof material from tube 2 to tube 3. Mix
NOTE 8—It is best to limit the test participants to not more than one or
with the vortical mixer.
two trials per day on each forearm. If the same subject is used in
10.8 Transfer 0.5 mLof material from tube 3 to tube 4. Mix
subsequent trials to bracket the median effective dose, care must be taken
with the vortical mixer. to cleanse comp
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