ASTM E2274-03

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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:E2274–03
Standard Test Method for
Evaluation of Laundry Sanitizers and Disinfectants
This standard is issued under the fixed designation E2274; 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 Federal Standard 40 CFR Part 160, Good Laboratory Prac-
tice Standards
1.1 This test method is designed to evaluate sanitizing/
Canadian Pest Management Regulatory Standards Trade
disinfectant laundry detergents/additives for use in top-loading
Memorandum T-1-215
automatic clothes washing operations. This test method is
designed predominantly to provide testing with representative
3. Terminology
vegetative bacteria but can also be designed to accommodate
3.1 Definitions:
the testing of fungi and viruses.
3.1.1 active antimicrobial ingredient—a substance added to
NOTE 1—This test method does not evaluate sanitizing/disinfectant
a formulation intended specifically for the inhibition or inac-
laundry detergent/additives for use in front-loading, low water volume
tivation of microorganisms.
automatic clothes washing operations.
3.1.2 antimicrobial agent(s)—an active ingredient designed
1.2 Knowledge of microbiological techniques is required
to suppress the growth or action of microorganisms.
for these procedures.
3.1.3 carrier count control—procedure used to determine
1.3 In this method metric units are used for all applications,
the initial number of microorganisms on a fabric carrier
except for distance in which case inches are used.
following the inoculation and drying procedure.
1.4 This standard does not purport to address all of the
3.1.4 diluent—sterile deionized water, sterile distilled water
safety concerns, if any, associated with its use. It is the
or sterile synthetic AOAC hard water that may be used to
responsibility of the user of this standard to establish appro-
prepare the active test formulation, vehicle control or product
priate safety and health practices and determine the applica-
control for use in the test procedure.
bility of regulatory limitations prior to use.
3.1.5 diluted product solution—test formulation, vehicle
control, or product control diluted to use concentration.
2. Referenced Documents
3.1.6 neutralization—aprocessthatresultsinquenchingthe
2.1 ASTM Standards:
antimicrobial activity of a test formulation. This may be
D1193 Specification for Reagent Water
achieved by dilution of the test formulation(s) to reduce the
E1054 Test Methods for Evaluation of Inactivators of An-
concentration of the antimicrobials, or through the use of
timicrobial Agents
chemical agents, called neutralizers, to suppress antibacterial
2.2 Other Documents:
activity.
TheAmericanAssociationofTextileChemistsAndColorists
3.1.7 numbers control—in assessing sanitizer level perfor-
(AATCC) Test Method 70-1997 Water Repellency;
mance, procedure used to determine the number of microor-
Tumble Jar Dynamic Absorption Test
ganismsremainingonthefabriccarriersandinthewashwater
Official Methods of Analysis of AOAC Internation-
followingthetestprocedureinthepresenceofthediluent.This
al AOAC, Washington, D.C., Chapter 6: Disinfectants,
may also be performed using diluent or phosphate buffer
17th ed., 2000.
dilution water with surfactant.
DIS/TSS13 LaundryAdditives—DisinfectionandSanitiza-
3.1.8 product control—a formulation with or without an
tion, U.S. Environmental Protection Agency, Office of
active ingredient(s) used for comparison to the test formula-
Pesticide Programs, April 1980
tion.
3.1.9 test formulation—a formulation containing an antimi-
crobial agent(s).
This test method is under the jurisdiction of ASTM Committee E35 on
3.1.10 vehicle control—the test formulation without the
Pesticides and is the direct responsibility of Subcommittee E35.15 onAntibacterial
active ingredient(s) used for comparison to the test formula-
Agents.
Current edition approved Dec. 1, 2003. Published January 2004. DOI: 10.1520/ tion.
E2274-03.
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
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E2274–03
3.1.11 wash water—the liquid contained in the exposure
chamber previously exposed to either uninoculated fabric or
fabric inoculated with the challenge microorganism.
4. Summary of Test Method
4.1 Under simulated laundry conditions, sets of inoculated
fabric swatches are placed into diluted product solution and
agitated.After a specified contact time, the wash water and the
test fabric are individually cultured either quantitatively (sani-
tizer efficacy) or qualitatively (disinfectant efficacy).
NOTE 2—See appropriate regulatory guidance document for the mini-
mum number of replicates required to meet a specific claim.
5. Significance and Use
5.1 Theprocedureinthistestmethodisusedtoevaluatethe
activityofatestreagent(antimicrobialagent/activeingredient)
orformulationinthereductionorcompletekillofthebacterial
population in fabric and wash water following a single wash.
FIG. 1 Stainless Steel Spindel Schematic (top view and side
view).
6. Apparatus
6.1 Colony Counter, any of several types may be used, for
6.15 Membrane Filtration System for Capture of the Test
example, Quebec.
6.2 Incubator, any incubator that can maintain the optimum Organism(s), sterile 47 mm diameter membrane filters (0.45
µm pore diameter) and holders for such filters.
temperature, 62°C, for growth of the challenge microorgan-
ism(s).
7. Reagents and Materials
6.3 Sterilizer, any suitable steam sterilizer producing the
conditions of sterility.
7.1 Petri Dishes, sterile 100 by 15 mm. Required for
6.4 Timer (Stop-clock), any device that can be read for
performing standard plate counts and used in preparation of
minutes and seconds.
contaminated fabric carriers.
6.5 Exposure Chamber, container with closure that can
7.2 Bacteriological Pipets, sterile, various sizes.
withstandsterilization.Shouldbelargeenoughtoholdasingle
7.3 Test Fabric, approximately 80 by 80 threads/in.
stainless steel spindle yet allow diluted product solution to
bleached, desized, plain-weave cotton print cloth and without
completely contact the entire fabric spindle during the tum-
bluing or optical brighteners.
bling period.
NOTE 4—Other test fabrics/blends may be used at the discretion of the
NOTE 3—Standard lids may form a vacuum seal when steam sterilized.
investigator.
To avoid, prior to sterilization place a piece of paper between lid and jar.
7.4 Dilution Fluid,AOAC Phosphate buffer dilution water
6.6 Stainless Steel spindles, Spindles are fabricated from a
orothersuitablediluentcontainingappropriateneutralizersfor
singlecontinuouspieceofstainlesssteelwire,( ⁄16in.diameter
serial dilution of test samples.
and bent to contain 3 horizontal extensions, 2 in. long
7.5 Water for Dilution of Formulations under Test:
connected by 2 vertical sections approximately 2 in. long.)
7.5.1 Water, sterile, deionized or distilled, equivalent to or
Theyareshapedsothatverticalsectionsform150°angles,free
better than Type 3, see Specification D1193.
ends of 2 outer horizontal extensions are sharpened to a point.
7.5.2 AOAC Synthetic Hard Water.
Use as carrier for wrapping fabric ballast. See Fig. 1.
7.5.3 Allwaterusedforpreparationoftestsolutionsshallbe
6.7 Agitator, tumbling device to rotate Exposure Chamber
sterile.
through 360° vertical orbit of 4 to 8 in. diameter at 45 to 60
7.6 Purity of Reagents—reagent grade chemicals shall be
rpm or comparable tumbling devices such as, Launderometer
used in all tests.
or Tumble Jar described in AATCC 70-1997.
7.6.1 Sodium carbonate.
6.8 Micropipettor (and Pipet Tips), suitable to deliver 0.01
7.6.2 Alkaline nonionic wetting agent with HLB
to 0.03 mL volume.
(hydrophilic-lipophilic balance) value of approximately 13.
6.9 Forceps, large and small, sterile.
Prepare solution containing 0.5% nonoxynol-10 class of
6.10 Safety Pins, sterile.
ethoxylated alkyl phenols, for example Tergitol NP-10 or
6.11 Stapler and Staples.
Triton X-100 an 0.5% Na CO .
2 3
6.12 Balance, with a platform to accommodate 15 6 0.1 g
of test fabric.
6.13 Sterile Glass Beads,3to4mm.
Fabric#4008-1978)obtainedfromTestFabric,Inc.,P.O.Box26,WestPittson,
6.14 Filter Sterilization System for Media and Reagents,a
PA 18643 or supplier of comparable specifications.
membrane or cartridge filtration system (0.22 µm pore diam-
Offıcial Methods of Analysis of the AOAC International, AOAC, Washington,
eter). Required for sterilizing heat-sensitive solutions. DC, 16th 16th ed, Chapter 6: Disinfectants, 1995.
E2274–03
7.7 Neutralizing Broths—growth media appropriate for the but not 13 laps while using the entire 15 6 0.1 g of fabric.
challenge microorganism containing chemical agents to sup- Staples or a pin may be used to secure the fabric. Fabric
press antibacterial activity. Alternatively, the neutralizing wrapped spindles may be sterilized in individual Exposure
broths may be of sufficient volume to reduce the concentration Chambers. Alternatively, fabric wrapped spindles may be
of the antimicrobials to below active levels. See 11.8. sterilized separately from Exposure Chambers. Ensure dryness
7.8 Challenge Microorganisms, of fabric on spindles and Exposure Chambers prior to testing.
7.8.1 Klebsiella pneumoniae, ATCC 4352.
NOTE 7—Fabric may be purchased in pre-cut strips and then scoured.
7.8.2 Staphylococcus aureus, ATCC 6538.
8.4 Fabric carriers of approximately 1 by 1.5 in. will be cut
7.8.3 Pseudomonas aeruginosa, ATCC 15442.
from the remaining scoured fabric. Nontoxic permanent
7.8.4 Other microorganisms, as applicable.
markermaybeusedtoplaceamarkontheedgeofeachcarrier.
7.9 Culture Media:
Alternatively, attach a pin to the short side of each carrier.
7.9.1 Nutrient Agar A.
Place fabric carriers in glass Petri dishes and sterilize. Ensure
7.9.2 Nutrient Agar B.
dryness of fabric prior to testing.
7.9.3 Media suitable for identification of microorganism(s)
8.5 For each challenge microorganism, prepare at least 3
used in the study.
fabriccarriersand1fabricwrappedspindleforeachactivetest
7.9.4 Soybean casein digest medium or other suitable me-
formulation/product and control/numbers control.
dia, with or without specific neutralizers, for recovery of the
challenge microorganism(s).
9. Preparation of Challenge Microorganisms
7.10 Organic Soil Load—when an organic soil load is to be
9.1 Subculture microorganism(s) on Nutrient Agar A
incorporated in the suspension of the challenge microorgan-
through at least three daily transfers, incubating at 35 6 2°C.
ism(s),defibrinatedheat-inactivatedanimalserummaybeused
Ifonlyonedailytransferismissed,itisnotnecessarytorepeat
or a mixture of the following stock solutions in phosphate
the three consecutive transfers prior to use in testing.
buffer dilution water (pH 7.2) may be used (see 7.4):
9.2 Onthedaypriortotesting,transferthecellsintoFrench
7.10.1 Add 0.5 g of tryptone to 10 mL phosphate buffer.
square bottles containing 20 mLof solidified NutrientAgar B.
7.10.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mL
Incubate 18 to 24 h at 35 6 2°C, agar side down.
of phosphate buffer.
9.3 Remove growth from the French square bottles using
7.10.3 Add 0.04 g of bovine mucin to 10 mL of phosphate
three-mL dilution fluid and five sterile glass beads to suspend
buffer.
growth. The cultures will be standardized to yield approxi-
7.10.4 Prepare the solutions separately and sterilize by
mately 10 colony forming units (CFU) per mL of S. aureus
passage through a 0.22 µm pore diameter membrane filter,
and 10 CFU/mL of K. pneumoniae and P. aeruginosa.
apportioned and stored at either 4 6 2°C or -20 6 2°C for no
longer than 3 months.
NOTE 8—The initial inoculum concentration for different challenge
microorganisms may vary and should be determined from carrier and
7.10.5 To obtain a 500 µL inoculum of the challenge
wash water numbers control recovery (see Section 12).
microorganism, add to 340 µL of the microbial suspension 25
µL, 100 µL, and 35 µL of BSA, mucin and Tryptone stock
9.4 A soil load may be added to each inoculum (see 7.10).
solutions, respectively.
10. Preparation of Test Sample
NOTE 5—The quality of the above materials may vary among manu-
10.1 Prepare a sufficient volume of diluted active test
facturersorproductlots.Therefore,preliminaryscreeningofsuchitemsis
formulation and product control (at least 1 L) according to
recommended to ensure compatibility with the test microorganism(s).
NOTE 6—The investigator should confirm the appropriate organic soil manufacturerinstructions,usingdiluentpre-equilibratedtotest
usage with the appropriate regulatory agency prior to initiating testing.
temperature.
NOTE 9— Fabric to wash-water ratios based on usage patterns must be
8. Fabric and Spindle Preparation
considered in this step (see DIS/TSS 13 and Table 1.)
8.1 Scour test fabric by boiling approximately 300 g of
NOTE 10—When appropriate use AOAC hard water in preparation of
materialfor1hin3Lofdistilledordeionizedwatercontaining
test product (see 7.5.2).
1.5-g sodium carbonate and 1.5-g nonionic wetting agent.
10.2 Using diluent at test temperature, prepare test product
Rinse fabric, first in boiling water and then in cold water, until
dilution no more than 3 h prior to use and maintain solution at
all visual traces of wetting agent are removed (that is, foam-
test temperature. Some active ingredients may require prepa-
ing). Remove as much water as possible from fabric.
ration and usage in less than 3 h.
8.2 Air dry for at least 24 h at ambient room temperature.
8.3 Cut scoured dry fabric into strips 2 in. (5 cm) wide and
11. Procedure
weighing 15 6 0.1 g each. For cotton fabrics, pierce one end
11.1 Inoculate three sterile fabric carriers (in a single sterile
ofthe15-gtestfabricstripandsecureontotheouterhorizontal
Petri dish) with 0.01 to 0.03 mL of prepared inoculum per
extensionofastainlesssteelspindle.Windthestriparoundthe
three horizontal extensions with sufficient tension to obtain 12
TABLE 1 Typical Use Patterns
Usage Pattern Fabric: Wash-water Ratio
Home or coin-operated laundering 1:10
DIS/TSS 13. LaundryAdditives—Disinfection and Sanitization. U.S. Environ- Industrial laundering 1:5
mental Pr
...