ASTM E1882-00
(Test Method)Standard Test Method for Evaluation of Antimicrobial Formulations by the Agar Patch Technique
Standard Test Method for Evaluation of Antimicrobial Formulations by the Agar Patch Technique
SCOPE
1.1 This test method determines the antibacterial activity and persistence of test formulations, as measured by the inhibition of a test organism on an agar surface exposed to test sites on human skin treated with the formulations.
1.2 A knowledge of microbiological techniques is required for these procedures.
1.3 It is the responsibility of the investigator to determine if Good Laboratory Practice (GLP) and Good Clinical Practice (GCP) are required and to adhere to these practices, as appropriate.
1.4 In this test method, metric units are used for all applications except linear measure. In that case, inches are used and metric units follow in parentheses.
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. Performance of this procedure requires the knowledge of regulations pertaining to the protection of human subjects (see 21 CFR, Parts 50 and 56).
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Designation:E1882–00
Standard Test Method for
Evaluation of Antimicrobial Formulations by the Agar Patch
Technique
This standard is issued under the fixed designation E 1882; 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 3. Terminology
3.1 active formulation—a formulation containing active
1.1 This test method determines the antibacterial activity
and persistence of test formulations, as measured by the ingredient(s).
inhibition of a test organism on an agar surface exposed to test 3.2 active ingredient—a substance added to a formulation
sites on human skin treated with the formulations. specifically for the inhibition or inactivation of microorgan-
1.2 A knowledge of microbiological techniques is required isms.
for these procedures. 3.3 active plate—inoculated plate that has been attached to
1.3 It is the responsibility of the investigator to determine if a skin site treated with an active formulation.
Good Laboratory Practice (GLP) and Good Clinical Practice 3.4 antibacterial activity—bacteriostatic and/or bacterio-
(GCP) are required and to adhere to these practices, as cidal activity.
appropriate. 3.5 control formulation—a formulation that does not con-
1.4 In this test method, metric units are used for all tain an active ingredient.
applicationsexceptlinearmeasure.Inthatcase,inchesareused 3.6 control plate—inoculated plate that has been attached to
and metric units follow in parentheses. anuntreatedskinsite,oronetreatedwithacontrolformulation.
1.5 This standard does not purport to address all of the 3.7 inhibition—prevention of bacterial population growth,
safety concerns, if any, associated with its use. It is the either through lethality or through prevention of bacterial
responsibility of the user of this standard to establish appro- reproduction.
priate safety and health practices and determine the applica- 3.8 inoculum determination plate—an inoculated plate that
bility of regulatory limitations prior to use.Performanceofthis has not been attached to any skin test site.
procedure requires the knowledge of regulations pertaining to 3.9 persistence—effectiveness of a test formulation in in-
the protection of human subjects (see 21 CFR, Parts 50 and hibiting bacteria, defined in terms of time elapsed between
56). application of test formulation and application of test plates.
3.10 resident microorganisms—microorganisms that nor-
2. Referenced Documents
mally live and multiply on skin, forming a permanent popula-
2.1 Federal Standard: Protection of Human Subjects, 21 tion.
CFR Ch. I, Parts 50 and 56 .
3.11 transient microorganisms—organisms that contami-
2.2 Yackovich, F., C.A. Wagner, and J.E. Heinze. 1989. nate, but do not normally colonize skin permanently.
Validity of the agar patch test with an antibacterial liquid soap
3.12 volar aspect of the forearms—the inside of the forearm
and comparison with the finger imprint method. J. Soc. Cos. on the same side as the palm of the hand.
Chem. 40:263-271.
4. Summary of Test Method
2.3 U.S. Pharmacopeia XXIV, NF 19. 2000. United States
Pharmacopeial Convention Inc., Rockville, MD. Chapter 61, 4.1 This test method is conducted on subjects selected from
entitled “Microbial Limits Test.” a group of volunteers who have refrained from using topical
2.4 Horowitz, W. (Ed.). 1980. Offıcial Methods of Analysis antimicrobials for at least one week and have minimal hair on
th
of the AOAC,13 Ed. Sec. 46.013(m), p. 825. Assoc. of the test site. The test site should normally have a low number
4 2
Official Analytical Chemists, Washington, D.C. 1018 pp. of resident microorganisms (approximately 10 CFU/cm or
fewer) and be easily sampled.
4.2 The surfaces of agar contact plates are inoculated with
This test method is under the jurisdiction of ASTM Committee E-35 on
the selected organism and placed in contact with skin sites that
Pesticides and is the direct responsibility of Subcommittee E35.15 onAntimicrobial
have been treated with active or control formulations, or left
Agents.
Current edition approved May 10, 2000. Published August 2000. Originally
published as E 1882 – 97. Last previous edition E 1882 – 97.
Available from Superintendent of Documents, U.S. Govenment Printing Office,
Washington, D.C. 20402. Yackovich, F., Wagner C.A., Heinze J.E. 1989.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1882
untreated.After contact with the treated skin sites, these plates 7.12 Test Formulations—Directions for application of ac-
are incubated and the colonies enumerated. Inhibitory activity tive and control formulations should be followed, if available.
oftheactiveformulationismeasuredbycomparingdifferences If directions are not available, the directions provided in this
in microbial colony counts between plates that were in contact test method may be applied.
with sites treated with an active formulation and plates that
8. Test and Control Skin Sites
were in contact with untreated sites, or sites treated with a
control formulation. Results are expressed as percent inhibi- The volar aspect of the forearm is commonly used as the
location of the skin sites, but other areas such as the back or
tion.
forehead may be used for test sites. Application of test and
5. Significance and Use
control formulations (or no treatment) will be assigned by a
predetermined randomization so that either forearm (or either
5.1 This procedure can be used to evaluate formulations
side, right or left, of other anatomical areas) may receive active
containingingredientsintendedtoinhibitgrowthofbacteriaon
or control formulations (or none).
intact skin and measures the difference, post-product-exposure,
between numbers of bacterial colonies on active plates and
9. Subjects
numbers on control plates, expressed as percent inhibition.
9.1 Number of Subjects—The number of subjects used in
5.2 This procedure may also be used to test for persistence
the test depends on the statistical significance required for the
ofactivity,asafunctionoftimeelapsedbetweenapplicationof
expected results, the sampling variability encountered in the
formulation and application of plates.
study, and the relative efficacy of the active formulation being
5.3 Because no procedure for neutralization of the antimi-
evaluated.
crobial action of active ingredients can be included in the test,
9.1.1 Recruit a sufficient number of healthy adult subjects
the agar patch method is limited to the extent that results
who have no clinical evidence of dermatoses, open wounds, or
expressed as percent inhibition do not differentiate between
other skin disorders that may affect the integrity of the test.
bacteriostatic and bacteriocidal effects and, hence, must not be
9.2 Instructthesubjectstoavoidcontactwithantimicrobials
portrayed as “reductions.”
for at least the week prior to testing and, other than the active
formulation, for the duration of the test. This restriction
6. Apparatus
includes spray antiperspirants and deodorants, shampoos, lo-
6.1 Colony Counter—Any of several types may be used. A
tions, dishwashing detergents, and soaps containing antimicro-
magnifying device, such as a dissecting microscope, may be
bial compounds, and materials such as acids, bases, and
used for manual enumeration of colonies.
solvents. Bathing in biocide-treated pools, hot tubs, or spas
6.2 Incubator—Any incubator capable of maintaining a
should be avoided. Subjects may be provided with a kit of
suitable temperature 6 2°C may be used.
non-antimicrobial personal hygiene products for exclusive use
6.3 Sterilizer—Any steam sterilizer capable of producing
during the test period and rubber gloves to be worn when
the conditions of sterility.
contact with antimicrobial agents cannot be avoided.
6.4 Timer (Stop Watch)—One that can be read for hours,
minutes, and seconds.
10. Procedure
10.1 Preparation of Agar Patch Contact Plates:
7. Reagents and Materials
10.1.1 Asepticallyplacethree35mmby10mmPetridishes
7.1 Bacteriological Pipettes, 10.0 and 2.2 or 1.1 mL capac-
with lids removed into a sterile 100 mm by 20 mm Petri dish.
ity.
NOTE 1—Three 35 mm by 10 mm plates are needed per active or
7.2 Pipetter, with disposable tips capable of delivering 10
control formulation per organism per subject. Three additional plates are
µL.
needed per organism for an inoculum determination count.
7.3 Plating Medium, soybean-casein digest agar, or equiva-
lent. 10.1.2 Using a sterile pipette, aseptically dispense approxi-
7.4 Dilution Fluid, Butterfield’s phosphate buffer,or mately 11.5 mL of soybean-casein digest agar (or other
equivalent. appropriate solid medium) into each of the small Petri dishes.
7.5 Isopropanol or Ethanol, 60 to 75% (v/v) The dishes are filled to form a convex meniscus elevated above
7.6 Sterile Disposable Culture Dishes, 1.4 3 0.4 in (35 mm the rim of the plate.
by 10 mm) and 4.0 3 0.8 in (100 mm by 20 mm). 10.1.3 Allow the agar to solidify and the surface to dry
7.7 Sterile Test Tubes. before inoculation.
7.8 Surgical Adhesive Tape, or equivalent.
10.2 Preparation of Test Organisms:
7.9 Disposable Examining Gloves. 10.2.1 The test organisms selected should be representative
7.10 Inoculating Loop or Glass Spreader.
ofthemicrobialfloraoftheskinoroftransientmicroorganisms
7.11 Appropriate Bacterial Cultures. that may contaminate human skin under certain conditions. A
partial list of organisms that have been used or recommended
for use in this test includes Staphylococcus epidermidisATCC
Presterilized/disp
...
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