ASTM E2406-04
(Test Method)Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations
Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations
SIGNIFICANCE AND USE
The procedure in this test method is used to evaluate the effectiveness of a test reagent (antimicrobial agent/active ingredient) or formulation to reduce or completely kill bacterial populations on contaminated fabrics and in wash water following a single wash under simulated low wash volume conditions. (See Table 1.)
TABLE 1 Typical Use Patterns Usage PatternFabric:
Wash Water
RatioWash Water
VolumeSpindle Wire
Requirement Deep Fill Top Load Washers≥ 1:5-1575-225 mLRetain High Efficiency/Horizontal Front
Load Washers 1:5 75 mLOmit
SCOPE
1.1 This test method is designed to evaluate sanitizing/disinfectant laundry detergents/additives for use in high efficiency (HE) automatic clothes washing operations that typically utilize very low wash water volumes. This test method is designed to provide testing with representative vegetative bacteria but can also be designed to accommodate the testing of fungi and viruses.
Note 1—Standard test method Test Method E 2274 is the recommended method to evaluate sanitizing/disinfectant laundry detergent/additives for use in traditional high wash water volume automatic clothes washing operations.
1.2 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.
1.4 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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Standards Content (Sample)
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Designation:E2406–04
Standard Test Method for
Evaluation of Laundry Sanitizers and Disinfectants for Use
in High Efficiency Washing Operations
This standard is issued under the fixed designation E2406; 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 Test Method 70-1997 Water Repellency; Tumble Jar Dy-
namic Absorption Test
1.1 This test method is designed to evaluate sanitizing/
2.3 AOAC Standard:
disinfectant laundry detergents/additives for use in high effi-
Official Methods ofAnalysis ofAOAC International Chap-
ciency (HE) automatic clothes washing operations that typi-
ter 6: Disinfectants, 17th ed., 2000
cally utilize very low wash water volumes.This test method is
2.4 EPA Standard:
designed to provide testing with representative vegetative
DIS/TSS13 LaundryAdditives—DisinfectionandSanitiza-
bacteriabutcanalsobedesignedtoaccommodatethetestingof
tion, U.S. Environmental Protection Agency, Office of
fungi and viruses.
Pesticide Programs, April 1980
NOTE 1—StandardtestmethodTestMethodE2274istherecommended
2.5 Federal Standard:
method to evaluate sanitizing/disinfectant laundry detergent/additives for
40 CFR, Part 160 Good Laboratory Practice Standards
use in traditional high wash water volume automatic clothes washing
2.6 Canadian Standard:
operations.
T-1-215 Canadian Pest Management Regulatory Standards
1.2 Knowledge of microbiological techniques is required 7
Trade Memorandum Oct, 1980.
for these procedures.
1.3 In this method metric units are used for all applications,
3. Terminology
except for distance in which case inches are used.
3.1 Definitions:
1.4 This standard does not purport to address all of the
3.1.1 active antimicrobial ingredient—a substance added to
safety concerns, if any, associated with its use. It is the
a formulation intended specifically for the inhibition or inac-
responsibility of the user of this standard to establish appro-
tivation of microorganisms.
priate safety and health practices and determine the applica-
3.1.2 antimicrobial agent(s)—an active ingredient designed
bility of regulatory limitations prior to use.
to suppress the growth or action of microorganisms.
3.1.3 carrier count control—procedure used to determine
2. Referenced Documents
the initial number of microorganisms on a fabric carrier
2.1 ASTM Standards:
following the inoculation and drying procedure.
D1193 Specification for Reagent Water
3.1.4 diluent—sterile deionized water, sterile distilled water
E1054 Test Methods for Evaluation of Inactivators of An-
or sterile synthetic AOAC hard water that may be used to
timicrobial Agents
prepare the active test formulation, vehicle control or product
E2274 Test Method for Evaluation of Laundry Sanitizers
control for use in the test procedure.
and Disinfectants
3.1.5 diluted product solution—test formulation, vehicle
2.2 AATCC Standard:
control, or product control diluted to use concentration.
3.1.6 neutralization—aprocessthatresultsinquenchingthe
antimicrobial activity of a test formulation. This may be
Available from American Association of Textile Chemists and Colorists
This test method is under the jurisdiction of ASTM Committee E35 on (AATCC), One Davis Dr., P.O. Box 12215, Research Triangle Park, NC 27709-
Pesticides and is the direct responsibility of Subcommittee E35.15 onAntibacterial 2215.
Agents. Available from AOAC International, Washington, DC.
Current edition approved Oct. 1, 2004. Published November 2004. DOI: Available from United States Environmental Protection Association (EPA),
10.1520/E2406-04. Ariel Rios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460.
2 6
For referenced ASTM standards, visit the ASTM website, www.astm.org, or AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.
Standards volume information, refer to the standard’s Document Summary page on Available from Canadian Standards Association (CSA), 178 Rexdale Blvd.,
the ASTM website. Toronto, ON Canada M9W1R3.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2406–04
achieved by dilution of the test formulation(s) to reduce the 6.5 Exposure Chamber—Container with closure that can
concentration of the antimicrobials, or through the use of withstandsterilization.Dimensionandvolumecapacityshould
chemical agents, called neutralizers, to suppress antibacterial be consistent for use in Test Method E2274.
activity.
NOTE 3—Standard lids may form a vacuum seal when steam sterilized.
3.1.7 numbers control—in assessing sanitizer level perfor-
To avoid, prior to sterilization place a piece of paper between lid and jar.
mance, procedure used to determine the number of microor-
ganismsremainingonthefabriccarriersandinthewashwater 6.6 Stainless Steel Spindles—Spindlesarefabricatedfroma
followingthetestprocedureinthepresenceofthediluent.This single continuous piece of stainless steel wire ( ⁄16 in. diameter
may also be performed using diluent or phosphate buffer and bent to contain 3 horizontal extensions, 2 in. long
dilution water with surfactant. connected by 2 vertical sections approximately 2 in. long).
3.1.8 product control—a formulation with or without an They are shaped so that vertical sections form 150° angle
active ingredient(s) used for comparison to the test formula- where the free ends of the 2 outer horizontal extensions are
tion.
sharpenedtoapoint.Thiswillbeusedasscaffoldingforinitial
3.1.9 test formulation—a formulation containing an antimi- wrapping of fabric ballast.
crobial agent(s).
6.7 Agitator—Tumbling device intended to rotate Exposure
3.1.10 vehicle control—the test formulation without the
Chamberthrough360°verticalorbitof4to8in.diameterat45
active ingredient(s) used for comparison to the test formula-
to 60 rpm or a comparable tumbling devices such as Launder-
tion.
ometer or Tumble Jar described in AATCC 70-1997.
3.1.11 wash water—the liquid contained in the exposure
6.8 Micropipettor (and Pipet Tips), suitable to deliver 0.01
chamber previously exposed to either uninoculated fabric or
to 0.03 mL volume.
fabric inoculated with the challenge microorganism.
6.9 Forceps, large and small, sterile.
4. Summary of Test Method
6.10 Safety Pins, sterile.
4.1 Under simulated laundry conditions, sets of inoculated 6.11 Stapler and Staples.
fabricswatchesareplacedintolowvolumesofdilutedproduct
6.12 Balance, with a platform to accommodate 15 6 0.1 g
solution and agitated. After a specified contact time, the wash
of test fabric.
water and the test fabric are individually cultured either
6.13 Sterile Glass Beads,3to4mm.
quantitatively (sanitizer efficacy) or qualitatively (disinfectant
6.14 Filter Sterilization System for Media and Reagents—A
efficacy).
membrane or cartridge filtration system (0.22 µm pore diam-
NOTE 2—See appropriate regulatory guidance document for the mini-
eter). Required for sterilizing heat-sensitive solutions.
mum number of replicates required to make a specific claim.
6.15 Membrane Filtration System for Capture of the Test
Organism(s)—Sterile 47 mm diameter membrane filters (0.45
5. Significance and Use
µm pore diameter) and holders for such filters.
5.1 Theprocedureinthistestmethodisusedtoevaluatethe
effectiveness of a test reagent (antimicrobial agent/active
7. Reagents and Materials
ingredient)orformulationtoreduceorcompletelykillbacterial
7.1 Petri Dishes, sterile 100 by 15 mm. Required for
populationsoncontaminatedfabricsandinwashwaterfollow-
ing a single wash under simulated low wash volume condi- performing standard plate counts and used in preparation of
contaminated fabric carriers.
tions. (See Table 1.)
7.2 Bacteriological Pipets, sterile, various sizes.
TABLE 1 Typical Use Patterns
Fabric:
Wash Water Spindle Wire
Usage Pattern Wash Water
Volume Requirement
Ratio
Deep Fill Top Load Washers $ 1:5-15 75-225 mL Retain
High Efficiency/Horizontal Front <1:5 <75mL Omit
Load Washers
6. Apparatus
6.1 Colony Counter—Anyofseveraltypesmaybeused,for
example, Quebec.
6.2 Incubator—Any incubator that can maintain the opti-
mum temperature 6 2°C for growth of the challenge microor-
ganism(s).
6.3 Sterilizer—Any suitable steam sterilizer producing the
conditions of sterility.
6.4 Timer (Stop-clock)—Any device that can be read for
FIG. 1 Stainless Steel Spindle Schematic
minutes and seconds. (Top View and Side View)
E2406–04
7.3 Test Fabric, approximately 80 by 80 threads/in. 7.10.4 Prepare the solutions separately and sterilize by
bleached, desized, plain-weave cotton print cloth and without passage through a 0.22 µm pore diameter membrane filter,
bluing or optical brighteners. aliquoteandstoreateither4 62°Cor−20 62°Cfornolonger
than 3 months.
NOTE 4—Other test fabrics/blends may be used at the discretion of the
7.10.5 To obtain a 500 µL inoculum of the challenge
investigator.
microorganism, add to 340 µL of the microbial suspension 25
7.4 Dilution Fluid, AOAC Phosphate buffer dilution water
µL, 100 µL and 35 µL of BSA, mucin and tryptone stock
orothersuitablediluentcontainingappropriateneutralizersfor
solutions, respectively.
serial dilution of test samples.
NOTE 5—The quality of the above materials may vary among manu-
7.5 Water for Dilution of Formulations Under Test:
facturersorproductlots.Therefore,preliminaryscreeningofsuchitemsis
7.5.1 Water, sterile, deionized or distilled, equivalent to or
recommended to ensure compatibility with the test microorganism(s).
better than Type 3, see Specification D1193.
NOTE 6—The investigator should confirm the appropriate organic soil
7.5.2 AOAC Synthetic Hard Water.
usage with the appropriate regulatory agency prior to initiating testing.
7.5.3 Allwaterusedforpreparationoftestsolutionsshallbe
8. Fabric and Spindle Preparation
sterile.
7.6 Purity of Reagents—Reagent grade chemicals shall be 8.1 Scour test fabric by boiling approximately 300 g of
materialfor1hin3Lofdistilledordeionizedwatercontaining
used in all tests.
1.5-g sodium carbonate and 1.5-g nonionic wetting agent.
7.6.1 Sodium carbonate.
Rinse fabric, first in boiling water and then in cold water, until
7.6.2 Alkaline nonionic wetting agent with HLB
all visual traces of wetting agent are removed (that is, foam-
(hydrophilic-lipophilic balance) value of approximately 13.
ing). Remove as much water as possible from fabric.
Prepare solution containing 0.5% nonoxynol-10 class of
8.2 Air dry for at least 24 h at ambient room temperature.
ethoxylated alkyl phenols, for example Tergitol NP-10 or
8.3 Cut scoured dry fabric into strips 2 in. (5 cm) wide and
Triton X-100 and 0.5% Na CO .
2 3
weighing 15 6 0.1 g each. For cotton fabrics, pierce one end
7.7 Neutralizing Broths—Growth media appropriate for the
ofthe15-gtestfabricstripandsecureontotheouterhorizontal
challenge microorganism containing chemical agents to sup-
extensionofastainlesssteelspindle.Windthestriparoundthe
press antibacterial activity. Alternatively, the neutralizing
three horizontal extensions with sufficient tension to obtain 12
broths may be of sufficient volume to reduce the concentration
but not 13 laps while using the entire 15 6 0.1 g of fabric.
of the antimicrobials to below active levels. See step 11.8.
9 Staples or a pin may be used to secure the fabric strip end.
7.8 Challenge Microorganisms:
Apply additional staples to the 6th and 7th folds along one
7.8.1 Klebsiella pneumoniae, ATCC 4352.
horizontalsideofthefabricbundletocreate“pockets”thatwill
7.8.2 Staphylococcus aureus, ATCC 6538.
secure individual fabric swatches during tumbling. Fabric
7.8.3 Pseudomonas aeruginosa, ATCC 15442.
wrapped spindles may be sterilized in individual Exposure
7.8.4 Other microorganisms, as applicable.
Chambers. Alternatively, fabric wrapped spindles may be
7.9 Culture Media:
sterilized separately from Exposure Chambers. Ensure dryness
7.9.1 Nutrient Agar A.
of fabric on spindles and Exposure Chambers prior to testing.
7.9.2 Nutrient Agar B.
NOTE 7—Fabric may be purchased in pre-cut strips and then scoured.
7.9.3 Media suitable for identification of microorganism(s)
8.4 Fabric carriers of approximately 1 by 1.5 in. will be cut
used in the study.
from the remaining scoured fabric. A nontoxic permanent
7.9.4 Soybean casein digest medium or other suitable me-
markermaybeusedtoplaceamarkontheedgeofeachcarrier.
dia, with or without specific neutralizers, for recovery of the
Alternatively, attach a pin to the short side of each carrier.
challenge microorganism(s).
Place fabric carriers in glass Petri dishes and sterilize. Ensure
7.10 Organic Soil Load—Whenanorganicsoilloadistobe
dryness of fabric prior to testing.
incorporated in the suspension of the challenge microorgan-
8.5 For each exposure chamber, prepare at least 3 fabric
ism(s),defibrinatedheat-inactivatedanimalserummaybeused
carriers and 1 fabric wrapped spindle for each active test
or a mixture of the following stock solutions in phosphate
formulation/product and control/numbers control.
buffer dilution water (pH 7.2) may be used (see 7.4).
7.10.1 Add 0.5 g of tryptone to 10 mL phosphate buffer.
9. Preparation of Challenge Microorganisms
7.10.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mL
9.1 Subculture microorganism(s) on Nutrient Agar A
of phosphate buffer.
through at least three daily transfers, incubating at 35 6 2°C.
7.10.3 Add 0.04 g of bovine mucin to 10 mL of phosphate
Ifonlyonedailytransferismissed,itisnotnecessarytorepeat
buffer.
the three consecutive transfers prior to use in testing.
9.2 Onthedaypriortotesting,transferthecellsintoFrench
square bottles containing 20 mL NutrientAgar B. Incubate 18
Fabric#4008-1978)obtainedfromTestFabric,Inc.,P.O.Box26,WestPittson,
to24hat35 6 2°C, agar side down.
PA 18643 or supplier of fabric with comparable specifications.
9.3 Remove growth from the French square bottles using
DIS/TSS 13. LaundryAdditives—Disinfection and Sanitization. U.S. Environ-
mental Protection Agency, Office of Pesticide Programs, April 1980. three-mL dilution fluid and five sterile glass beads to suspend
E2406–04
growth. The cultures will be standardized to yield approxi- 11.9 Addition of fabric carrier to neutralizing broth and
mately 10 colony forming units (CFU) per mL of S. aureus concentrated neutralizing broth to wash water completes the
and 10 CFU/mL of K. pneumoniae and P. aeruginosa. exposure time.
11.10 All
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