ASTM E951-94(2006)
(Test Method)Standard Test Methods for Laboratory Testing of Non-Commercial Mosquito Repellent Formulations On the Skin (Withdrawn 2015)
Standard Test Methods for Laboratory Testing of Non-Commercial Mosquito Repellent Formulations On the Skin (Withdrawn 2015)
SIGNIFICANCE AND USE
The ED50 test provides an estimate of the amount of repellent that must be applied to the skin to produce a given level of effectiveness against the mosquito test population. The levels of effectiveness that are usually of interest are the 50 % level (for comparative purposes) and the 95 % level (for practical purposes). The ED50 test method is used to determine the effectiveness of a repellent against different kinds of mosquitoes or to compare the effectiveness of different repellents against any particular kind of mosquito. It may also be used to establish the dosages needed to provide protection under special conditions of climate, weather, activity, etc.
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.
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.
WITHDRAWN RATIONALE
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.
Formerly under the jurisdiction of Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents, these test methods were withdrawn in January 2015 in accordance with section 10.6.3 of the Regulations Governing ASTM Technical Committees, which requires that standards shall be updated by the end of the eighth year since the last approval date.
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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 (Reapproved2006)
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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.5 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 These test methods apply to repellent compounds and
responsibility of the user of this standard to establish appro-
formulations that can be appropriately diluted with ethanol,
priate safety and health practices and determine the applica-
acetone, or a similar inert carrier for test purposes. The test
bility of regulatory limitations prior to use.
methods described are not suitable for testing powders, sticks
or other solid formulations, or for testing thixotropic or other
2. Referenced Documents
fluids whose physical properties would be modified by dilu-
2.1 ASTM Standards:
tion.
E939Test Method of Field Testing Topical Applications of
1.2 These test methods are designed and intended for use as
Compounds as Repellents for Medically Important and
aresearchstandardtodevelopdataontheefficacyofrepellents
Pest Arthropods (Including Insects, Ticks, and Mites):I
applied to the skin of humans against laboratory-reared or
Mosquitoes
field-collected mosquitoes. The use of these test methods will
2.2 Other Documents:
provide for the development of a data base whereby all
Directions for Abstractors and Section Editors of Chemical
investigators generate comparable data. Modifications of the 3
Abstracts
equipment or procedures, or both, may be needed for tests
Consolidated List of Approved Common Names of Insecti-
against other kinds of biting arthropods. 4
cides and Other Pesticides
1.3 The test methods are intended for use in testing mate- Common Names of Insects and Related Organisms
rials that are in an advanced stage of development, for which
3. Apparatus
human-use trials can be fully justified on scientific and ethical
grounds. The test methods are not designed for the testing of
3.1 Test Cage—The following design and materials have
commercial formulations where registration or advertising
been found suitable for construction of the mosquito cage (see
claims data are required. Fig. 1):
1.3.1 A repellent should not be considered for testing on
3.1.1 The cage is rectangular in shape, length, width, and
humans before its efficacy has been demonstrated in in vitro, height is approximately 7.2 by 2 by 1.6 in. (18 by 5 by 4 cm).
animal, or other nonhuman test systems.
The top of the cage (5 by 18 cm) is made of metal or plastic
1.3.2 Arepellent should not be applied to the skin before its mosquito screening, and the sides, ends, and floor are made of
safetyhasbeenestablishedinappropriatetoxicologicaltestson
⁄8 in. (3.2 mm) clear acrylic plastic.
animals or other test organisms. 3.1.2 Five 1 ⁄8 in. (29 mm) circular openings are drilled in
1.3.3 No repellent should be tested on humans without the
line on 1 ⁄8 in. (35 mm) centers in the floor of the cage.
written consent of the test subjects and prior approval of 3.1.3 The two sides and one of the ends of the cage are
competent authority, as designated in the applicable laws and
groovedandslottedtoreceiveaflexiblerectangularslidemade
regulations governing experimentation on humans. of 0.012 in. (0.3 mm) cellulose acetate sheeting. The slide
shouldmovefreelyoverthefloorofthecagetoopenandclose
1.4 The values stated in inch-pound units are to be regarded
the five openings.
as the standard. The values given in parentheses are for
information only.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
These test methods are under the jurisdiction of ASTM Committee E35 on Standards volume information, refer to the standard’s Document Summary page on
Pesticides, Antimicrobials, and Alternative Control Agents and are the direct the ASTM website.
responsibility of Subcommittee E35.12 on Insect Control Agents. Available from the American Chemical Society, 1155 16th St., N.W.,
Current edition approved April 1, 2006. Published April 2006. Originally Washington, D.C. 20036.
approved in 1983. Last previous edition approved in 2005 as E951–94 (2005). Available from the Entomological Society ofAmerica, 10001 Derekwood Ln.,
DOI: 10.1520/E0951-94R06. Ste. 100, Lanham, MD 20706–4876.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E951 − 94 (2006)
FIG. 1 Test Cage
3.1.4 One end of the cage is fitted with a No. 3 stopper in a allow for transport mortality and accommodation to laboratory
⁄2 in. (13 mm) hole for insertion of the test mosquitoes. conditions. During the period the mosquitoes should be pro-
vided with an appropriate substratum for oviposition.
3.2 Harness—Two belts are used to secure the test cage to
the forearm during the test. These should be approximately 1
NOTE 1—The age-range specified for the test mosquitoes is arbitrary
and is intended only as an aid in standardization of the test among users.
in. (2.5 cm) wide by 12 in. (30 cm) long. If an elastic material
Users have reason to adopt a different age-range for testing should report
is used for the belts, snap or friction type fasteners should be
the age-range actually used (12.3).
provided for joining the ends. If an inelastic material is used,
7.2 Where adequate facilities are available, rearing and
slides or buckles should be provided.
testing conditions should be controlled and standardized at
3.3 Template—A template made of ⁄8 in. (3.2 mm) acrylic
levels appropriate for the species under test. In areas or
plastic to match exactly the floor of the test cage is used as a
localities where suitable laboratory facilities are not available,
guide to outline the five circular treatment areas on the
rearing and testing should be carried out, if possible, in a
forearm.
building free of extremes of temperature, humidity, illumina-
tion,andwind.Wherepossible,allcomparativetestsshouldbe
4. Reagents and Materials
made under similar conditions.
4.1 The diluent used in the test will ordinarily be ethanol.
7.3 In the case of the four-hour ED50 test (14.1-19.1), the
However, for some repellents (for example, water-based or
forearm should not be washed, rubbed, scratched, or otherwise
lanolin-based formulations) dilution in ethanol may be inap-
treated in such a way as to nullify the repellent treatments
propriate. In such cases, the appropriate diluent will be used
during the four-hour test period. However, the test participants
instead.
should be normally active during the test period to ensure that
5. Sampling the factors of perspiration and abrasion are incorporated in the
test.
5.1 Take a bulk sample and a laboratory sample as directed
inanyapplicablematerialspecifications.Intheabsenceofsuch 7.4 Nothinginthissectionshouldbeconstruedtomeanthat
specifications, take a laboratory sample believed to be repre- special test conditions may not be adopted for tests having a
sentative of the lot to be tested. In the case of a suspension, special purpose. For example, the wear resistance of the test
emulsion or similar formulation, thoroughly mix the material repellent can be measured by abrading the treated forearm in a
controlled manner before the test mosquitoes are applied to
to be sampled before the sample is taken.
bioassay. In such special-purpose tests, the applicable parts of
6. Test Specimen and Sample
the standard should be followed as closely as possible.
6.1 Take test specimens by pipet as required (see 10.4 and
7.5 Sincetestconditionsmayvarytosomeextentunderthis
16.1.1). Take a new test specimen for each trial and replicate
test method, it is essential that full information on all variables
needed in the test. The number of trials and replicates needed
relating to the test repellent, test mosquitoes, procedures, and
depends on the variability of the results obtained and the
test conditions be made part of the final report (12.1-12.3).
degree of precision required. See 13.1 and 13.2.
TEST METHOD ED50 (A)
6.2 In the case of a suspension, emulsion, or similar
formulation, the sample must be thoroughly mixed before the
8. Summary of Test Method
test specimen is taken.
8.1 Five circular test areas are outlined on the flexor region
of the forearm and treated with the diluent (as the control) and
7. Conditioning
four serial dilutions of the test repellent (the repellent treat-
7.1 The mosquitoes used in the test should be conspecific
ments). A cage having matching cutouts in its floor and
females maintained on 10% sucrose solution prior to testing.
Laboratory-reared mosquitoes should be mated nullipars in the
age-range of 5 to 15 days. Field-collected mosquitoes should
Gerberg, E. J., Manual for Mosquito Rearing and Experimental Techniques,
be held in the laboratory for 48 h or longer prior to testing to Amer. Mosquito Control Assoc., Fresno, CA, 1970, 109 pp.
E951 − 94 (2006)
containing ten mosquitoes is then applied to the forearm, and 10.9 Assign the control and the four repellent treatments
the numbers of mosquitoes feeding on the control and the (tubes 1 to 4) to the five test areas (A to E) at random.
repellent treatments is recorded. In subsequent trials, the range
NOTE6—Anewrandomizationisrequiredforeachtrialandreplicateof
of dosages applied to the forearm is adjusted to bracket the
thetest.Randomizationcanbeaccomplishedbycomputerorwithadieor
median effective dosage (ED50) of the test repellent. The test
tableofrandomnumbers.Itisconvenienttomakeenoughrandomizations
at one time for use in a number of trials.
is replicated at that range of dosages until a valid estimate of
theED50canbeobtained.Thedataareanalyzedbyestablished
10.10 Apply 0.025 mL of diluent with a micropipet to the
methods of biological assay.
test area designated as the control. Spread evenly with the tip
of a glass rod.
9. Significance and Use
10.11 Apply 0.025 mL of the material in Tube 4 to the test
9.1 The ED50 test provides an estimate of the amount of
area designated for that treatment. Spread evenly.
repellent that must be applied to the skin to produce a given
10.12 Apply 0.025 mL of the material in Tube 3 to the test
levelofeffectivenessagainstthemosquitotestpopulation.The
area designated for that treatment. Spread evenly.
levels of effectiveness that are usually of interest are the 50%
level (for comparative purposes) and the 95% level (for
10.13 Apply 0.025 mL of the material in Tube 2 to the test
practicalpurposes).TheED50testmethodisusedtodetermine
area designated for that treatment. Spread evenly.
the effectiveness of a repellent against different kinds of
10.14 Apply 0.025 mL of the material in Tube 1 to the test
mosquitoes or to compare the effectiveness of different repel-
area designated for that treatment. Spread evenly.
lents against any particular kind of mosquito. It may also be
used to establish the dosages needed to provide protection
10.15 After 4 min, fit the five cutouts in the floor of the test
under special conditions of climate, weather, activity, etc. cagetothefivetestareasoutlinedonthetestsubject’sforearm,
and secure the test cage to the forearm with the two belts
10. Procedure
provided.
10.1 Load the test cage with 10 to 20 female mosquitoes
10.16 After one additional minute, withdraw the slide from
from the population to be tested.
the test cage to expose the test areas on the forearm to the
mosquitoes.
NOTE 2—This number of mosquitoes is intended as a limitation on the
numberofbitesreceivedbythetestsubject.Intestsagainstspecieshaving
10.17 At 1.5 min after the slide is withdrawn, record the
characteristically low laboratory feeding rates, larger numbers of mosqui-
numbers of mosquitoes feeding on each of the five test areas.
toes should be used.
10.18 In subsequent trials, adjust the range of dosages to
10.2 Using the plastic template as a guide, outline five
brackettheED50ofthetestrepellentbysuccessivelydoubling
circular test areas on the flexor region of the test subject’s
or halving the concentration of repellent made up in Tube 1
forearm with a fine-tipped felt pen. Label the test areas “A”
(10.4).
through “E”, beginning with the test area nearest the elbow.
NOTE 7—The general equivalents calculated in accord once with Note
10.3 Set up four test tubes in a test tube rack and label them
4 are as follows: (1)Aconcentration of 1.0% exactly repellent in Tube 1
“1” through “4”, from left to right.
provides a dosage of 0.03898 mg/cm on the forearm. (2)Aconcentration
10.4 Make up approximately 1 mL of 0.41% of the test of 25.65% repellent inTube 1 provides a dosage of 1.000 mg/cm on the
forearm. These figures may be used as conversion factors where needed.
repellent in Tube 1 (Notes 3 and 4). Mix the preparation with
However, modification of the equipment or procedures described herein
a vortical mixer.
may necessitate recalculation of the conversion factors.
NOTE 3—The diluent used will ordinarily be ethanol, but in some cases
10.19 When the appropriate range of dosages has been
it may be appropriate to use a different material. See 4.1.
determined (10.18), replicate the test as necessary to obtain an
NOTE 4—This strength (0.41%) is calculated to provide a dosage of
acceptablypreciseestimateoftheED50ofthetestrepellent.If
0.016 mg of repellent per cm of skin surface when 0.025 mL of the
solution is spread over a 1 ⁄8 in.-(29 mm) diameter circular test area. The
a low dilution of repellent is being applied, it may tend to
calculation assumes that the specific gravity of the test repellent is equal
spread beyond the boundary of the test area. This can often be
to one. If the actual specific gravity of the test repellent is known, a
prevented by applying undiluted repellents at the desired rate
correction to the nominal dosage of 0.016 mg/cm can be calculated.
with an adjustable pipet. However, the amount of a repellent
10.5 Put 0.5 mL of diluent into each of tubes, 2, 3, and 4.
that can be applied to a given area of skin will ultimately be
10.6 Transfer 0.5 mLof material from tube 1 to tube 2. Mix limitedbyrunoff.Thedoseatwhichrunoffoccursisafunction
with the vortical mixer. of the viscosity of the repellent. The number of trials and
replicates needed depends on the variability of the results
10.7 Transfer 0.5 mLof material from tube 2 to tube 3. Mix
obtained and the degree of pr
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