ASTM E2011-99

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Designation:E2011–99
Standard Test Method for
Evaluation of Handwashing Formulations for Virus-
Eliminating Activity Using the Entire Hand
This standard is issued under the fixed designation E 2011; 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.
INTRODUCTION
Physical removal, inactivation in situ, elimination and a combination of these actions to reduce the
spread of viral infections with effective handwashing has become an achievable goal. Artificial
contaminationofhandswithselectedtestvirusesprovidesausablemodel.Thisguideiscloselyrelated
to another ASTM test of handwash agents which restricts contamination and sampling to a smaller
area of the hand, Test Method E 1836, Determining the Virus Eliminating Effectiveness of Liquid
Hygienic HandwashAgents Using the Fingerpads ofAdult Volunteers. This test method tests a larger
area of the hands than the fingerpad method; however, reported results are comparable. (1)
1. Scope in Virucidal Efficacy Evaluations
E 1836 Test Method for Determining the Virus-Eliminating
1.1 This test method is designed to evaluate antimicrobial
Effectiveness of Liquid Hygienic HandwashAgents Using
agents in formulations for utility and effectiveness for virus-
3 the Fingerpads of Adult Volunteers
eliminating activity using human subjects.
2.2 AOAC Standard:
1.2 This standard may involve hazardous materials, opera-
AOAC 960.9
tionsandequipment.Thisstandarddoesnotpurporttoaddress
all of the safety concerns, if any, associated with its use. It is
3. Summary of Test Method
the responsibility of the user of this standard to establish
3.1 This test method is conducted on subjects selected from
appropriate safety and health practices and determine the
a group of adult volunteers who have provided a written
applicability of regulatory limitations prior to use. The user
informedconsentandwhosehandshavebeendeterminedtobe
should consult a reference for laboratory safety recommenda-
free from any apparent damage. All subjects should have
tions. (2, 3)
refrained from the use of any antimicrobials for at least one
2. Referenced Documents weekpriortoinitiationofthetestandbesuppliedwithselected
products free from antimicrobials for use during this week. At
2.1 ASTM Standards:
least 12 to 15 subjects are selected from this group for the test.
2.1.1 Use the most current editions of the standards refer-
The number of required subjects may vary with the virus
enced herein.
tested.
E 1052 Test Method for Efficacy of Virucidal Agents
3.2 Apreparedsuspensionoftheselectedtestvirusisgrown
Against Viruses in Suspension
and diluted or concentrated to produce a titer with a minimum
E 1053 Test Method for Efficacy of Virucidal Agents In-
of10 infectiveunits/mL.Thecontaminatingvirusisappliedto
tended for Inanimate Environmental Surfaces
the hands and the hands are washed with the test formulation
E 1482 Test Method for Neutralization of Virucidal Agents
according to the manufacturer’s directions or with a set test
regimen.
3.3 The virus titer recovered after washing with the test
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides and is the direct responsibility of Subcommittee E35.15 onAntimicrobial
product is compared to a control titer of virus. For the control,
Agents.
a titer of virus is applied to the hands and recovered from
Current edition approved March 10, 1999. Published September 1999.
2 subjects washing with standard hard water (200 ppm as
The boldface numbers in parentheses refer to a list of references at the end of
this test method. calcium carbonate) or vehicle, or both, instead of the formu-
A knowledge of virological techniques is required for this test method.
lation.
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
Standards volume information, refer to the standard’s Document Summary page on Available from Association of Organic and Analytical Chemists International,
the ASTM website. Gaithersburg, MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2011–99
3.4 The virus is exposed to the virucide for the length of 5.6 Freezers—A freezer at –20 6 2°C is required for the
time that is representative of actual use conditions of the storage of fetal bovine serum and other additives for cell
product,forexample,from10to20sforahandsoap.Thevirus culture media. A second freezer at –70°C or lower is required
to be recovered after exposure to the test germicide is assayed to store viruses.
in a cell culture system appropriate to the test virus. The virus 5.7 Refrigerator—Arefrigerator at 4 6 2°C is necessary for
titerofthestock,testsamplesandcontrolsisdeterminedbythe storage of prepared cell culture media and reagents.
a suitable infectivity assay. Cytotoxicity of the host cell culture 5.8 Timer—Any stop watch that can be read in minutes and
system caused by the test germicide at the tested concentration seconds is acceptable.
is also determined. The virus-germicide mixture is assayed in 5.9 Magnetic Stirrer and Magnets—Magnetic stirrer and
numerous units of the host system at a dilution just beyond the magnets must be large enough to hold a 5-L beaker or
cytotoxicity range of the germicide. At least three replicate Erlenmeyer flask for preparing cell culture media or other
determinations are performed on controls (untreated) and test solutions.
samples (treated) to confirm virus elimination by a sample of 5.10 Handwashing Sink—Asink of sufficient size to permit
the test germicide. Results are recorded as the median value of subjects to wash hands without touching hands to sink surface
log - reduction in virus infectivity. is required.
5.10.1 Water faucet(s) are to be located above the sink at a
3.4.1 This test method is designed to be performed by a
height that permits the hands to be held higher than the elbow
trained microbiologist or virologist who is responsible for
duringthewashingprocedures.Faucetswithelectronicsensors
choosing the appropriate host system for the test virus, and
or those that are wrist-, elbow-, knee-, or foot-operated are
applying the techniques necessary for propagation and main-
preferred to avoid recontamination of the washed hands.
tenance for host and test virus. For a reference text, see Ref.
5.10.2 Bland, proven non-antimicrobial soap, preferably
(4).
liquid, is needed.
5.10.3 Tap water temperature regulator and temperature
4. Significance and Use
monitortomonitorandregulatewatertemperatureat40 62°C
4.1 This test method should be used to evaluate the virus-
is needed.
eliminatingeffectivenessoftheseformulationsafterhandwash-
5.11 Liquid Nitrogen Storage for Cells—An appropriate
ing. Effective formulations can be further evaluated in a
liquid nitrogen container and liquid nitrogen for cryopreserva-
clinical trial on human subjects. Published data have shown(1)
tion of the stocks of cell lines are needed.
that results of in vitro tests do not accurately reflect what
5.12 Inverted Microscope—An inverted microscope with
occurs when this class of products is used in the health care
10X eye pieces and 5X, 10X and 40X objectives.
facility. This test method involves the incorporation of whole
5.13 Serological Pipettes—Sterile reusable or single-use
hand exposure and friction from washing, reflecting actual use
pipettes of 10.0, 5.0 and 1.0 mL capacity are needed.
conditions in human subjects. It is meant to confirm the results
5.14 Cell Culture Flasks —Plastic cell culture flasks of 25
of testing with Test Method E 1836. This method gives precise
2 2
cm or 75 cm capacity for culturing cells and for preparing
reductions on a limited area of the finger, the fingerpads.
virus pools are needed.
4.2 This test method is not meant for use with surgical hand
NOTE 1—Each flask for growing cell monolayers can be reused by
scrubs or preoperative skin preparations.
reseeding with new cell cultures 10 or more times before being discarded.
5.15 Plastic and Glass Vials, Medication (Medicant)—
5. Equipment and Apparatus
Sterile screw-capped vials will be required or post test sam-
5.1 Laminar Flow Cabinet—A Class II biological safety
pling of hands.
cabinet is required for virus work. The procedures for the
5.16 Miscellaneous Labware—Required are: automatic pi-
proper maintenance and use of such cabinets are given in Ref.
pettes, pipette tips, plastic vials for storing cell and virus
(5).
stocks, dilution tubes, cluster plates or flasks for virus titration.
5.2 Incubator—An incubator at 36 6 1°C is needed for
5.17 Sterile Glass beads—Beads that are 3.5 mm in diam-
growinghostcellsandforincubatingvirus-infectedcultures.If
eter are needed.
an open system is used for cell culture, a CO incubator will be
required. Work with rhinoviruses will require an incubator at
6. Materials and Reagents
33 6 1°C.
6.1 Cell Culture Media and Supplements—Culture media
5.3 Positive Displacement Pipette—A pipette and pipette
and the types and ratios of supplements will vary depending on
tips that can accurately dispense 10 to 20 µL volumes is
the cell line. Eagle’s minimal essential medium (EMEN) with
required.
5to10 %fetalbovineserum(virus-andmycoplasma-tested)is
5.4 Sterilizer—Any steam sterilizer suitable for processing
used for growing a wide variety of cells (see Note 2).
cell culture media and reagents is acceptable. The steam
Antibiotics may be required in the medium to suppress
supplied to the sterilizer must be free from additives toxic to
bacterial growth.
cell cultures.
5.5 Filter Sterilization System—A membrane or cartridge
filtration system (0.22 µm pore diameter) is required for
Plasticcellculturewareandotherrelatedsuppliesmaybepurchasedfrommost
sterilization of heat-sensitive media and solutions. laboratory supply houses.
E2011–99
6.2 Organic Load: same system to be used for virus recovery following virus
6.2.1 Fetal bovine serum, at a final concentration of 5 % in challenge of the test germicide. The virus pool should contain
he virus inoculum (see Note 2), if required for the test. not less than 10 infective unit/mL.
6.2.2 Peptone, a pancreatic digest of casein as an alternative 8.2 Cell Culture Technique—1.0 mL of virus is allowed to
to serum, is made by dissolving 7.6 g of tryptone powder in 1L adsorb on the cell sheet of a 0.75 cm flask for about1hatan
physiological saline (0.85 % NaCl). Sterilize by autoclaving or appropriate incubation temperature after which 25 mL of
membrane filtration. This peptone solution should contain maintenance media is added. The flask is then re-incubated
approximately 2.0 g of total protein/L, which is approximately until about 75 % of the cell monolayer shows virus cytopathol-
equivalent to the protein content of a 5 % solution of fetal ogy. The flask is frozen and thawed three times in a dry
bovine serum. ice-alcohol bath and the disrupted cells centrifuged 20 min at
about 1000 xg. The supernatant is collected and divided into
NOTE 2—Fetal bovine serum is considered unsuitable for use as an
appropriate volumes for test and may be used fresh or stored at
organic load when working with rotaviruses because of its rotavirus-
–70°C.
inhibitory and trypsin-neutralizing activity.
8.3 Virus titer of the stock is the titer of the test virus after
6.3 Standard Hard Water—Water prepared according to
growth and dilution or concentration. A titer of 10 infective
AOAC 960.09 E and F (4) to a standard hardness of 200 ppm
units/1 mL is required for the test. The control virus titer is
as calcium carbonate is used for dilution of test products. This
determined on subjects using hard water (200 ppm as calcium
is the control solution to determine the baseline level of virus
carbonate) or EBSS (with or without peptone) or the vehicle of
elimination, and to rinse the fingerpads after exposure to the
the test product for handwashing of virus-contaminated hands.
test product.
The initial titer of the virus is the virus recovered by sampling
6.4 Test Products—Duplicate samples of the product shall
the inoculated hands after washing and recovery with EBSS
be tested.
solution.
6.5 Diluent for Virus Titration—Earle’s balanced saline
8.4 The initial or control titers, or both, can be determined
solution (EBSS) with a pH of 7.2 to 7.4.
using the virus application techniques and recovery samples
6.6 Eluent for Virus Recovery from Hands and Fingers—
after washing with buffer, hard water or the vehicle of the test
Use EBSS containing peptone.
product.
7. Test Viruses and Cell Cultures
9. Cytotoxicity Testing Antimicrobial
7.1 The selection of the following test viruses is based on
9.1 Prior to studying the effects of the test product on
their (a) relative safety to the volunteers as well as experiment-
viruses, determine its cytotoxic effect on the host system. Use
ers, (b) ability to grow to titers sufficiently high for testing, (c)
serial 2-fold dilutions and inoculate a minimum of 4 units/
property to produce cytopathic effects or plaques, or both, in
dilution.
cell cultures, (d) potential to spread through contaminated
9.2 A technique recommended for reduction of cytotoxic
hands, and (e) relative resistance to agents used in hygienic
effects is based on gel filtration (see Test Method E 1482 and
handwashing. Other strains or types of viruses may be substi-
(5)). The eluates from virus-contaminated hands are passed
tuted provided they meet the above criteria.
through separate columns of Sephadex LH-20 gel to remove
residues of the antimicrobial which may be cytotoxic. The
NOTE 3—There is insufficient information on whether the passage
history, culture conditions and strain differences of viruses can influence filtrates are centrifuged at 10 000 xg to remove bacteria before
the efficiency of their elimination by hygienic handwash agents. There-
virus infectivity assay.
fore, caution must be exercised when substituting viruses as this may lead
to variations in results from one laboratory to another.
10. Conditioning
NOTE 4—Poliovirus is scheduled for eradication.The goal is to achieve
10.1 Prior to testing the virus-eliminating activity of the test
a world free from polio.At that time, laboratory work with the poliovirus
product or agent, wash the subject’s hands with a mild, proven
will not be possible.
non-antimicrobial soap for 1 min under running water to
7.2 Human Rotavirus Wa (ATCC VR-201 8) of the cell line
remove transient microflora. Rinse thoroughly and dry
...