ASTM E1115-02
(Test Method)Standard Test Method for Evaluation of Surgical Hand Scrub Formulations
Standard Test Method for Evaluation of Surgical Hand Scrub Formulations
SIGNIFICANCE AND USE
The procedure in this test method should be used to evaluate the activity of the test formulation in reducing the bacterial population of the hands immediately after a single use and to determine persistent activity (inhibition of growth) after 6 h. Optionally, measurements of persistent activity after a 3 h period and measurements of cumulative activity may be made after repetitive uses over a five day period.
SCOPE
1.1 This test method is designed to measure the reduction of microbial flora on the skin. It is intended for determining both immediate and persistent microbial reductions, after single or repetitive treatments, or both. It may also be used to measure cumulative antimicrobial activity after repetitive treatments.
1.2 A knowledge of microbiological techniques is required for these procedures.
1.3 In this method, metric units are used for all applications, except for distance, in which case inches are used and metric units follow in parentheses.
1.4 Performance of this procedure requires the knowledge of regulations pertaining to the protection of human subjects.
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:E1115–02
Standard Test Method for
Evaluation of Surgical Hand Scrub Formulations
This standard is issued under the fixed designation E1115; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope macopeial Convention, Inc., Rockville, MD. Chapter 61
“Microbial Limits Test”
1.1 Thistestmethodisdesignedtomeasurethereductionof
microbial flora on the skin. It is intended for determining both
3. Terminology
immediate and persistent microbial reductions, after single or
3.1 Definitions:
repetitive treatments, or both. It may also be used to measure
3.1.1 active ingredient—a substance added to a formulation
cumulative antimicrobial activity after repetitive treatments.
specifically for the inhibition or inactivation of microorgan-
1.2 A knowledge of microbiological techniques is required
isms.
for these procedures.
3.1.2 cleansing wash—a non-antimicrobial wash intended
1.3 Inthismethod,metricunitsareusedforallapplications,
to remove gross soil or residues from the hands of the subjects
except for distance, in which case inches are used and metric
prior to collecting baseline samples.
units follow in parentheses.
3.1.3 cleansing wash formulation —a liquid castile soap or
1.4 Performance of this procedure requires the knowledge
2 other liquid soap with neutral pH which does not contain an
of regulations pertaining to the protection of human subjects.
antimicrobial.
1.5 This standard does not purport to address all of the
3.1.4 cumulative effect—a progressive decrease in the num-
safety concerns, if any, associated with its use. It is the
ber of microorganisms recovered following repeated applica-
responsibility of the user of this standard to establish appro-
tions.
priate safety and health practices and determine the applica-
3.1.5 internal reference formulation—a formulation with
bility of regulatory limitations prior to use.
demonstrated performance characteristics within the labora-
2. Referenced Documents tory.
3.1.6 neutralization—a process that results in quenching or
2.1 ASTM Standards:
inactivation of the antimicrobial activity of a formulation. This
D1193 Specification for Reagent Water
maybeachievedthroughdilutionoftheformulationorthrough
E1054 Test Methods for Evaluation of Inactivators of An-
the use of chemical agents called neutralizers.
timicrobial Agents
3.1.7 persistence—prolonged or extended antimicrobial ac-
2.2 Other Documents:
4 tivity that prevents or inhibits the proliferation or survival of
21 CFR Parts 50 and 56
microorganisms after treatment.
AATCC Test Method 147 1993AntibacterialAssessment of
5 3.1.8 sampling fluid—a buffered solution that aids in recov-
Textile Materials: Parallel Streak Method
ery of microorganisms from the skin and neutralization of the
Horowitz, W. (Ed.), 2000, Official Methods of Analysis of
active ingredient in test and internal reference formulations.
AOAC International 17th Ed., Ch 17, p. 4, Sec. 17.2.01
3.1.9 test formulation—a formulation containing an active
(m). Assoc. of Off. Anal. Chemist, Washington, D.C.
ingredient(s).
United States Pharmacopeia, 25, 2001, United States Phar-
4. Summary of Test Method
This test method is under the jurisdiction of ASTM Committee E35 on
4.1 This test method is conducted on subjects selected from
Pesticides and is the direct responsibility of Subcommittee E35.15 onAntimicrobial
a group of volunteers who have refrained from using any
Agents.
antimicrobials for at least two weeks prior to initiation of the
Current edition approved February 10, 2002. Published May 2002. Originally
published as E1115 – 86. Discontinued June 2000 and reinstated with revision as
test. Subjects are selected from this group on the basis of high
E1115–02. DOI: 10.1520/E1115-02.
initial bacterial count,$ 1 3 10 CFU/per hand as determined
21 CFR Ch. 1, Parts 50 and 56.
3 by baseline measurements of the bacteria on their hands using
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
the recovery techniques in this method.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
United States Code of Federal Regulations.
5 6
Technical Manual of the American Association of Textile Chemists and Johnson’sBabyWashHead–to–ToeyJohnson’sandJohnson’sInc.,Skillman,
Colorists, P.O. Box 12215, Research Triangle Park, NC 27709. NJ 08558–9418.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1115 – 02
4.2 Theselectedsubjectsperformasimulatedsurgicalscrub 7. Reagents and Materials
under the supervision of an individual competent in aseptic
7.1 Petri Dishes—100 by 15 mm. Required for performing
technique. One hand of each subject is sampled immediately 10
Standard Plate Count.
after the scrub (within 1 min), and the other hand, 6 h after
7.2 Bacteriological Pipets—10.0 and 2.2 or 1.1-mL capac-
scrubbing. Only one hand of a subject is sampled at a specified 11
ity.
time. Optionally, another sampling time, 3 h for example, can
7.3 Water-Dilution Bottles—Any sterilizable container hav-
beaddedbetweentheimmediateand6hsamplingtimes.Ifthis
inga150to200-mLcapacityandtightclosuresmaybeused.
is desired, the panel size must be increased by 50 % to obtain
7.4 Cleansing Wash Formulation—A formulation without
the same number of data points at each designated sampling
an active ingredient.
interval. Also, a sampling time randomization must be gener-
7.5 Gloves for Sampling—Loose-fitting, unlined, powder-
ated such that one-third of the hands are sampled at each
free latex gloves which do not demonstrate antimicrobial
sampling interval with only one hand of a subject being
activity, or equivalent glove. An equivalent is a glove com-
sampled at a sampling time interval.
posed of any material that is unlined, does not leak and does
4.3 If demonstration of cumulative activity is desired,
notdemonstrateantimicrobialactivity.Azoneofinhibitiontest
eleven additional scrubs are performed over a 5-day period,
such asAATCC Test Method 147 may be used to evaluate the
one additional time on Day 1, three times on Days 2, 3, and 4
antibacterial activity.
and once on Day 5. The hands are sampled again after the last
7.6 Test Formulation—Directions for use of active test
scheduled scrub.
formulation should be utilized if available. If not available, use
directions provided in this test method (see 11.3).
NOTE 1—The researcher should be cautioned that components of
7.7 Water—Sterile deionized water or equivalent (Specifi-
chemical neutralizer systems such as lecithin and polysorbate 80 may
, ,
7 8 9
interfere in the determination of cumulative effect on the skin.
cation D1193, Type III).
7.8 Sampling Fluid —Dissolve 0.4 g KH PO , 10.1 g
2 4
5. Significance and Use
Na HPO and 1.0 g isooctylphenoxypolyethoxyethanol in 1
2 4
5.1 The procedure in this test method should be used to
L of water. Adjust to obtain a final pH 7.8 6 0.1. Dispense to
evaluate the activity of the test formulation in reducing the
achieve a final volume of 75 6 1 mL into water dilution
bacterialpopulationofthehandsimmediatelyafterasingleuse
bottles, or other suitable containers, and sterilize. Optionally,
and to determine persistent activity (inhibition of growth) after
the sampling fluid may be sterilized before dispensing into
6 h. Optionally, measurements of persistent activity after a 3 h
sterilecontainers.Theabilityofthesamplingfluidtoneutralize
period and measurements of cumulative activity may be made
or quench the antimicrobial activity of the test formulation
after repetitive uses over a five day period.
must be validated (see Practices E1054). The validation test
should be conducted in vivo in accordance with how the
6. Apparatus
surgical hand scrub study is conducted. If the sampling fluid
6.1 Colony Counter—Anyofseveraltypesmaybeused,for
does not quench the antimicrobial activity of the test formula-
example, Quebec Colony Counter. tion and the internal reference formulation, an antimicrobial
6.2 Incubator—Any incubator that can maintain a tempera- inactivator should be included if required.
ture of 30 6 2°C may be used. 7.9 Dilution Fluid—Butterfield’s buffered phosphate dilu-
6.3 Sterilizer—Any suitable steam sterilizer that can pro- ent adjusted to pH 7.2 6 0.1 (or other suitable diluent) and
duce the conditions of sterility is acceptable. containing an antimicrobial inactivator if required.
6.4 Timer (stop-clock)—That can be read for minutes and 7.10 Soybean-Casein Digest Agar, or equivalent —
seconds. Containing an antimicrobial inactivator if required.
6.5 Hand Washing Sink—A sink of sufficient size to permit
NOTE 2—Inadequate neutralization may result in false interpretation of
subjects to wash without touching hands to sink surface or
the test data. The use of excess chemical neutralizers may exert a toxic
other subjects.
effect on the recovery of bacterial cells. The goal, therefore, is to stop
6.5.1 Water Faucet(s)—To be located above the sink at a antimicrobial activity as early as possible in the sampling/plating process.
If it can be demonstrated that antimicrobial activity is quenched or
height that permits the hands to be held higher than the elbows
during the washing procedure. (It is desirable for the height of
the faucet(s) to be adjustable.)
6.6 Tap Water Temperature Regulator and Temperature
Pre-sterilized/disposable plastic petri dishes are available from most local
Monitor—To monitor and regulate water temperature to 40 6
laboratory supply houses.
2°C. Pre-sterilized/disposable bacteriological pipets are available from most local
laboratory supply houses.
Dilution bottles of 160-mL capacity having a screw-cap closure are available
from Corning Glass Co., Kimble Glass Co., or most local laboratory supply houses.
7 13
Bently, M.V., Kedor,E.R., Vianna, R.F., Collett, J.H., Influence of lecithin and Peterson, A. F., “The Microbiology of the Hands: Evaluating the Effects of
urea on the in vitro permeation of hydrocortisone acetate through skin from hairless Surgical Scrubs,” Developments in Industrial Microbiology, Vol 14, 1973, pp.
mouse. International Journal of Pharmaceutics. Vol. 146: 255–262. 125-130.
8 14
Kato, A., Ishibahi, Y., Effect of egg yolk lecithin on transdermal delivery of Horowitz, W. (Ed.) 2000, Offıcial Methods of Analysis of AOAC International
bunazosin. Journal of Pharmacy and Pharmacology. Vol 39: 399–400. 17th Ed., Ch 17, p. 4, Sec. 17.2.01 (m).Assoc. of Off.Anal. Chemist, Washington,
RejendranD.,Sivabalan,M.,Dhanaraj,S.A.,Ponnusankar,S.,Dube,R.,Suresh, D.C.
B., Trandermal delivery of prazoosin HCL with non–ionic surfactants. Indian United States Pharmacopeia, 23, 1995, United States Pharmacopeial Conven-
Journal of Pharmaceutical Sciences. Vol. 150–153. tion, Inc., Rockville, MD. Chapter 61 “Microbial Limits Test.”
E1115 – 02
inactivated in the sampling fluid then, to reduce the chance of possible
procedure on Days 3 and 7, Days 3 and 5, or Days 5 and 7 of
toxic effects, inactivators should not be added to the dilution fluid or
“Baseline Period” to obtain three estimates of baseline popu-
plating media.
lation. After obtaining the first and second estimates of the
7.11 Scrub Sponge and Nail Cleaner Stick—Such as E-Z
baseline populations, select subjects who exhibited at each
16 5
Scrub 160 or any equivalent may be used.
sampling time counts$1 3 10 per hand. The three estimates
of the baseline population obtained for each of the selected
8. Subjects
subjects are averaged to obtain the mean baseline counts.
8.1 Sample size calculations should be done to determine
9.2 A basic random sampling plan should be followed. The
thenumberofsubjectsnecessarytofindstatisticallysignificant
number of subjects and sampling times depend on the test
differences (reductions) from baseline. The number of subjects
formulation but must establish the onset and extent of the
required depends on the statistical confidence required for the
bacterialsuppressionandthedurationofsuppressionbelowthe
expected results, the variability encountered in the data collec-
baseline counts. Equal numbers of subjects should be assigned
tion (that is, variability in reductions from baseline), and the
per sampling time, test formulation and hand. A typical
expected efficacy of the test product (that is, its expected
balanced randomization plan for testing a block of six subjects
reduction from baseline). This number of subjects (n) can be
follows with sampling at 0 h, 3 h (optional), and 6 h.
estimated from the following equation:
Subject No. Post Scrub Sampling Time, h
0-h 3-h 6-h
~Z 1 Z !
a/2 b
n . S 5 1 left hand right hand
F G
D
2 left hand right hand
3 right hand left hand
where: 4 right hand left hand
5 left hand right hand
S = estimate of variance (of reduction from baseline
6 right hand left hand
based on in-house data pool),
Z = cumulative probability of the standard normal dis-
a/2 If only 0 h and 6 h post scrub samples are collected the 0 h
tribution = 1.96 for a = 0.05,
will be randomized to the right or left hand.
Z = power of the test = 0.842 for b = 0.80, and
b
9.2.1 The number of subjects per block may vary but must
D = expected efficacy (expected reduction from base-
be divisible by two and by the number of sampling times in
line).
order to assign equal number of left and right hands to each
8.2 Recruitasufficientnumberofhealthysubjectswhohave
sampling time.
no clinical evidence of dermatoses, open wounds, or other skin
9.3 No sooner than 24 h and no longer than 96 h after
disorders. Exclude any individual receiving antibiotic therapy
completion of the baseline determination, subjects perform
and any individual sensitive to natural rubber or latex or to a
scrub with the test formulation. The starting interval should be
component of the formulation(s) being tested.
same for all subjects participating in the study. According to
8.3 Instructthesubjectstoavoidcontactwithantimicrobials
the random sampling plan, determine the bacterial populations
(otherthanthetestformulationasdispensedforeachscrub)for
on the subjects’ hands at the assigned sampling times after
at least two weeks prior to obtaining the first baseline sampling
scrubbing. Determine bacterial population by sampling hands
and for the duration of the test. This restriction includes
and enumerating the bacteria in the sampling fluid as specified
antimicrobial-containing antiperspirants, deod
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