ASTM E2361-04(2007)
(Guide)Standard Guide for Testing Leave-On Products Using In-Situ Methods
Standard Guide for Testing Leave-On Products Using In-Situ Methods
SCOPE
1.1 This guide covers test methods and sampling procedure options for leave-on products for consumer and hospital personnel. Leave-on products, such as alcohol hand rubs and lotions containing antimicrobial ingredients, are increasingly marketed and used by consumers and health care personnel. These products are distinguished from conventional washing and scrubbing preparations in that they do not rely on the rinsing, physical removal, and antimicrobial action in determining their effectiveness. Although agitation and friction may serve to release organisms from the skin and folds and crevices, organisms are then killed in situ and are not rinsed from the skin surface before sampling. Appropriate test methods for the hands have been published, while other sampling methods will be needed for testing body areas other than the hands.
1.2 Methods of recovery after application of the contaminating organisms to a part of the body other than by the agitation/rubbing of the hands against a glass petri plate also need examination. Consideration should be given to contact plating, controlled swabbing with a template, and cup scrubbing (detergent/agitation used) since the target organisms for recovery are likely to be on the superficial layers of skin.
1.3 The values stated in SI units are to be regarded as the standard.
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 requirements prior to use.
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Designation: E2361 − 04(Reapproved 2007)
Standard Guide for
Testing Leave-On Products Using In-Situ Methods
This standard is issued under the fixed designation E2361; 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 Health Care Personnel Handwash Formulations
E1327 Test Method for Evaluation of Antimicrobial Hand-
1.1 This guide covers test methods and sampling procedure
wash Formulations by Utilizing Fingernail Regions
options for leave-on products for consumer and hospital
2.2 European Standard:
personnel. Leave-on products, such as alcohol hand rubs and
EN1500 Chemical Disinfectants and Antiseptics-Hygienic
lotions containing antimicrobial ingredients, are increasingly
Handrub-Test Method Requirements (phase 2/step 2)
marketed and used by consumers and health care personnel.
approved by CEN (Comité Européen de Normalisation)
These products are distinguished from conventional washing
and scrubbing preparations in that they do not rely on the
3. Summary of Guide
rinsing, physical removal, and antimicrobial action in deter-
mining their effectiveness.Although agitation and friction may
3.1 In this guide, choices of recovery techniques after the
servetoreleaseorganismsfromtheskinandfoldsandcrevices, use of antimicrobial products will be considered. By the nature
organisms are then killed in situ and are not rinsed from the
of the distribution of the skin flora, these sampling techniques
skin surface before sampling.Appropriate test methods for the estimate the flora remaining after antimicrobial use; some of it
hands have been published, while other sampling methods will
is superficial and some hidden. An appropriate sampling
be needed for testing body areas other than the hands. method can be selected depending on product use and the
importance of superficial (transient) and hidden or deep
1.2 Methods of recovery after application of the contami-
(mostly resident) flora.
nating organisms to a part of the body other than by the
agitation/rubbing of the hands against a glass petri plate also
3.2 This guide was written because ASTM Subcommittee
need examination. Consideration should be given to contact E35.15 worked on its own test method for leave-on products
plating, controlled swabbing with a template, and cup scrub-
used without water, but found that the EN1500 protocol
bing (detergent/agitation used) since the target organisms for encompassed the test method that had been developed.
recovery are likely to be on the superficial layers of skin.
3.3 This CEN type of test methodology is widely used in
1.3 The values stated in SI units are to be regarded as the
European and Scandinavian countries but has not been widely
standard. used in the United States, although the use of alcohol/alcohol
gel hand rubs has expanded greatly here in the last few years.
1.4 This standard does not purport to address all of the
The underlying question is whether a test method designed for
safety concerns, if any, associated with its use. It is the
a leave-on product like alcohol or the conventional hand
responsibility of the user of this standard to establish appro-
washingfollowedbysamplinginagloveorplasticbagismore
priate safety and health practices and determine the applica-
appropriate. There have been criticisms of test methods, such
bility of regulatory requirements prior to use.
as EN1500, which was based on Rotter’s methods (1), but
2. Referenced Documents published data confirm that the test is highly reliable in
showing consistent reduction levels with low variation from
2.1 ASTM Standards:
subject to subject. Leave-on products that are not rinsed or
E1174 Test Method for Evaluation of the Effectiveness of
washed off in use are primarily represented by alcohol-based
hand rubs. However, other leave-on formulations have been
1 introduced and, undoubtedly, their number will increase in the
This guide is under the jurisdiction ofASTM Committee E35 on Pesticides and
Alternative ControlAgents and is the direct responsibility of Subcommittee E35.15
future. Often test methods designed for washing/rinsing pro-
on Antimicrobial Agents.
cedures have been used for these products. When different
Current edition approved Nov. 1, 2007. Published November 2007. Originally
approved in 2004. Last previous edition approved in 2004 as E2361 – 04. DOI:
10.1520/E2361-04R07.
2 3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Available from British Standards Institute (BSI), 389 Chiswick High Rd.,
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM London W4 4AL, U.K.
Standards volume information, refer to the standard’s Document Summary page on The boldface numbers in parentheses refer to the list of references at the end of
the ASTM website. this standard.
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E2361 − 04 (2007)
more specific methods are required for testing, questions of is then added for sampling to increase the release of viable
methodology become clearer, and the selection of a new or organisms to be recovered. In contrast, in testing for hand rubs
different sampling method is necessary. or leave-on products, glove sampling would seem appropriate
only if sampling were performed after each contamination and
3.4 When a typical hand-washing product is used, the hands
product application. Since changes have been made in Test
are wet; scrubbing and manipulation are pursued, often vigor-
Method E1327 to sample only after the first and last applica-
ously; and rinsing follows. Agitation here is to remove organ-
tions, the applicability of this test method for products rubbed
isms and particulate and oily soil physically. Any residue of
into the skin and used repeatedly without water may not be
active ingredient remaining on the skin is a small fraction of
applicable for these leave-on products.
the amount applied and assumed to be attached to the stratum
corneum. The residual may also be absorbed over time. 3.9 EN1500 is an adaptation of a test developed by Rotter
Ultimately, the reduction in microbial count is a combination known as the Vienna Model (6).
of kill from the antimicrobial and the physical removal by
3.10 There are many publications describing and evaluating
agitation and rinsing.
fingertip-sampling methods. One of the major criticisms of the
methods is the procedure used for sampling. The tips of the
3.5 In contrast, leave-on products, such as alcohol products
intended to be applied and not rinsed off, present a different fingers and thumb are sampled by rubbing against the bottom
of a glass petri dish to release contaminating bacteria from
situation.There are two distinct techniques when sampling: (1)
sampling by washing target organisms off with detergent, these areas before and after treatment. The sampled areas are
only portions of the areas treated. However, published results
assuming that most of removal is transient flora, and (2)
sampling in situ, for example, the cup scrub, swab, contact have shown consistent, statistically valid data. With the
EN1500 test procedure, sampling is performed after a single
plate, or velvet block/pad that sample bacteria by impression
and contact or by using fluid to remove samples so that the use of the product (divided into two portions for application).
volume of the sample is restricted to a very small size. These
4. Significance and Use
different sampling methods disturb the deep or hidden flora to
differing degrees. There has been an overwhelming concentra- 4.1 The United States has concentrated attention and testing
tion of the cup-scrub sampling method as various test methods
efforts on surgical scrubbing far more than on hand care in
have been developed. The combination of detergent and patient-to-patient routines. Great Britain, the originators of
agitation attempts to remove as much remaining flora as
infection control nursing, have always had their focus on
possible.The best effort, however, only removes about 15 % of infection transmission. In the United States, published articles
the full thickness flora (2). When other contact sampling or
have documented the short exposure time for health care
tape stripping are used, the distribution of bacterial colonies on
personnel who do wash their hands between patients. The
the skin are mirrored as they occur; whereas, if detergent/
average is less than 10 s.The ideal product for the reduction of
scrubbing techniques are used, the microcolonies are dispersed
transient flora is one that rapidly kills or removes or both the
yielding higher counts. Washing/scrubbing methods stir up the
microbial load acquired during health care activities. The
cells and bacteria from the deeper skin layers and release more
emphasis on rapidity is essential simply because health care
of the hidden flora (described by Reybrouck (3)). This is also
personnel will not take the necessary time when using conven-
true of the cup scrub method that uses detergent/surfactants to
tional hand-washing products. The use of products not in-
detach bacteria from the skin. Contact methods sample the
tended for use with water has increased dramatically and their
flora that can easily be transferred and that is conceded to be
use is common in European countries largely because of
the most important in disease transmission. Williams (4) has
convenience and effectiveness. A second characteristic is the
stated that, “although the distinction between residents and
level of antimicrobial action. The use of a rapid and potent
transients must certainly be a real one, it is not to allocate the
active product to reduce work-acquired microbial flora is ideal.
various bacterial species to one or other class with regularity.”
4.2 Since the change from strictly in-vitro testing of topical
3.6 There has been a long-time focus on the cup-scrub antimicrobials for use on skin to simulated use testing in hand
technique only, and it would be beneficial to look at sampling
washing, prepping, site access testing, and sampling, emphasis
specific areas, such as Test Method E1327, which samples has always been on washing hands, agitating, rubbing, and
around the fingernail region using a toothbrush, or the use of
brushing with liquid on the skin site to estimate bacteria
direct contact plating when washing is not involved (5),asin removed after testing.
skin prepared for surgery. This guide is intended to assess the
4.3 The use of hard agitation has diminished with surgical
effectiveness of application of products rubbed into the skin or
scrubs without brushes or with only mild agitation and friction.
on the hands when these sites are not washed between uses.
4.4 There is a history of microbial dispersal (7) and increase
3.7 Superficially, the testing method is the same as with
insurfacebacteriafromdeeperlayersresultingfromshowering
products that are used to scrub and wash the hands or skin in
(8-10), washing, scrubbing, and agitation. In the normal
that the hands are contaminated with a recoverable transient
situation on the skin, there is a superficial, surface flora and a
organismandthetestproductapplied.Thesimilarityendshere.
deeper or hidden flora (3). The proportion of one to the other
3.8 If the hands are sampled after application of organisms has been addressed by Selwyn (2) and his judgment is that
and the test product in sequence, they are dried or gloved wet from 20 to 50 % of the flora is “deep.” The intent in skin
and are sampled after extensive rinsing. The stripping solution sampling has almost always been to scrub, agitate, and use
E2361 − 04 (2007)
surfactant to remove as many organisms as we can. In doing 5.2.2 This test method offers a procedure for reliably
this, we have completely ignored the two types of flora. samplingadifficultarea,forinstance,comparedtothefingertip
sampling of Rotter (6) and in EN1500. Furthermore, individual
4.5 Further, when the skin is treated with a cleansing agent
fingersorcombinationsoffingerscanbeusedtotestmorethan
or an antimicrobial that is subsequently rinsed away, the
one test product.
“deep” or “hidden” flora is pushed to the surface as the sebum
5.2.3 Any of the test methods for sampling the microflora of
replenishes the sebum from the sebaceous glands removed in
the skin recovers only a small fraction of that flora. For
washing. Many early investigators have looked at simpler
example, the most often used cup-scrub procedure recovers
sampling methods that we now recognize were sampling
approximately 15 %; template swabbing 3 to 20 %; and im-
primarily the superficial transient flora.
pression plating with contact plating, velvet block/pad, or tape
transfer, all under 0.5 %. Selwyn (2) developed these data by
5. Published Testing Methods for Leave-On Products
comparing a sampling method to the culture results of a full
thickness biopsy of the skin designated as 100 %. Another
5.1 Hygienic Hand Rub—Vienna Model:
technique developed and published by Leyden et al (13) may
5.1.1 When viable organisms are captured in the sampling
enhance the use of some contact methods. This test method
fluid after exposure to a test product, sampling like that used in
involves improved counting methodology using computer
theglovejuicetestusesamuchlargeramountoffluidfollowed
imaging of colonies on contact plates from fingerprint/
by microbiological analysis on a small sample. While in the
handprint techniques.
test method in EN1500 and Rotter’s procedure (6), the volume
5.2.4 At any rate, we are sampling only a fraction of the
in the plate after rubbing the fingertips on the plate’s bottom is
total skin flora and sampling the hands presents even a bigger
either cultured in toto or sampled and diluted.
dilemma. There are nail folds, cuticles, and fingernail spaces
5.1.2 With this in-situ procedure, only the fingertips are
that collect bacteria. Price (14) himself showed that bacteria
sampled in contrast to the whole hand in the glove juice are released in each of a sequential series of twelve basins, so
procedure. The agitation to the fingertips in the in-situ testing that a technique like the glove fluid sampling method is still
only releasing a fractional portion of the total flora. The
is more intense than 1 min of massage of the whole hand.
fingertips are the part of the hand most frequently in contact
5.1.3 This test method has been legally mandated as the
with people and hard surfaces. The important question be-
officialCENmethod for their member c
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