ASTM E2406-16

Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations

SIGNIFICANCE AND USE
5.1 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. The water to fabric ratio in common front loading machines is dynamic and varies by region and machine used. The proper water to fabric ratio and temperature for the worse-case scenario for product use should be determined and documented prior to testing.
SCOPE
1.1 This test method is designed to evaluate sanitizing/disinfectant laundry detergents/additives for use in front loading 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.  
1.2 This test method is intended to compliment Test Method E2274 and is to be used in the cases where this test method is determined to be the worse case scenario for product usage.
Note 1: Test Method E2274 is the recommended method to evaluate sanitizing/disinfectant laundry detergent/additives for use in traditional high wash water volume automatic clothes washing operations.  
1.3 Knowledge of microbiological techniques is required for these procedures.  
1.4 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLP) are required and to follow them where appropriate (see section 40 CFR, 160 or as revised.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
Note 2: In this test method, metric units are used for all applications, except for distance, in which case inches are used.  
1.6 Appropriate modifications to the test method may be required when the testing organisms are not specified herein.  
1.7 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.

General Information

• Status: Published
• Publication Date: 14-Apr-2016
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Replaces: ASTM E2406-09 - Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations
• Effective Date: 15-Apr-2016
• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM E2274-16 - Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants
• Effective Date: 15-Apr-2016
• Refers: ASTM E177-14 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2014
• Refers: ASTM E177-13 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2013
• Refers: ASTM E691-13 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-May-2013
• Refers: ASTM E691-11 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2011
• Refers: ASTM E177-10 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-Oct-2010
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010
• Refers: ASTM E2274-09 - Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants
• Effective Date: 01-Nov-2009
• Refers: ASTM E691-08 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Oct-2008
• Refers: ASTM E177-08 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-Oct-2008
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008
• Refers: ASTM E177-06b - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 15-Nov-2006

Overview

ASTM E2406-16 is the Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations. Developed by ASTM International, this standard provides a comprehensive procedure for evaluating the performance of antimicrobial agents, laundry sanitizers, and disinfectant additives in front-loading, high-efficiency (HE) washing machines that use low water volumes. By simulating real-world HE laundry conditions, the method assesses the ability of a product to reduce or eliminate bacterial populations on contaminated fabrics and in wash water after a single wash cycle.

Compliance with ASTM E2406-16 ensures accurate and repeatable results for manufacturers, testing labs, and regulatory agencies, aiding in the approval and proper labeling of laundry hygiene products intended for efficient washing systems.

Key Topics

• Antimicrobial Efficacy: The standard measures both the reduction and inactivation of microorganisms-primarily vegetative bacteria, but the methodology can be extended to include fungi and viruses.
• Simulated HE Conditions: Testing replicates the dynamic and variable water-to-fabric ratios found in front-loading, high-efficiency washing machines, ensuring methods are applicable to real-world use.
• Control Procedures: The test design includes various controls, such as numbers control, neutralizer efficacy, and carrier count control, to validate results and ensure accuracy.
• Required Expertise: Microbiological techniques are integral to the procedure, underlining the need for skilled personnel and compliance with Good Laboratory Practices (GLP) when appropriate.
• Reporting and Compliance: Results are reported as percent reduction in viable organisms, offering a standard metric for efficacy claims.

Applications

The ASTM E2406-16 test method is critical for:

• Product Development: Enables manufacturers of laundry sanitizers and disinfectant additives to test and validate the efficacy of new formulations in high-efficiency machines. This supports claims of antimicrobial action on product labels.
• Regulatory Approval: Provides data necessary for regulatory submissions to agencies such as the US Environmental Protection Agency (EPA), especially when demonstrating worst-case scenario results in low water volume conditions.
• Quality Assurance: Ensures consistent product performance by offering a repeatable and reproducible methodology for testing different production batches.
• Comparative Testing: Supports benchmarking against other products or formulations to select the most effective technology for HE washers.
• Lab and Industrial Use: Ideal for both independent laboratories offering contract testing and in-house R&D labs within detergent and cleaning product companies.

The method's adaptability to different organisms and flexible water-to-fabric ratios make it suitable for evaluating a wide range of laundry additive types, from household laundry sanitizers to industrial disinfectants.

Related Standards

• ASTM E2274: Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in Conventional Washing Operations (higher water volumes); recommended for top-loading machines.
• ASTM E1054: Test Methods for Evaluation of Inactivators of Antimicrobial Agents, relevant for validating neutralization steps.
• AATCC 70: Water Repellency; Tumble Jar Dynamic Absorption Test, referenced for equipment in the test procedure.
• 40 CFR Part 160: Good Laboratory Practice Standards, relevant for ensuring compliance in laboratory settings.
• EPA DIS/TSS 13: Guidelines for efficacy data submissions for laundry additives.

Practical Value

By following ASTM E2406-16, stakeholders in the laundry and cleaning product industry can reliably demonstrate antimicrobial efficacy, support regulatory compliance, and ensure consumer safety for products used in resource-efficient HE washing machines. The standard’s rigorous approach underlines its significance for manufacturers targeting high-efficiency appliance markets and laboratories seeking validated methods for antimicrobial performance testing.

Keywords: ASTM E2406-16, high efficiency washing, laundry sanitizer test, laundry disinfectant evaluation, antimicrobial efficacy, low wash water volume, washing machine standards, product validation.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E2406 − 16
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 2. Referenced Documents
2.1 ASTM Standards:
1.1 This test method is designed to evaluate sanitizing/
D1193Specification for Reagent Water
disinfectant laundry detergents/additives for use in front load-
E177Practice for Use of the Terms Precision and Bias in
ing high efficiency (HE) automatic clothes washing operations
ASTM Test Methods
that typically utilize very low wash water volumes. This test
E691Practice for Conducting an Interlaboratory Study to
method is designed to provide testing with representative
Determine the Precision of a Test Method
vegetative bacteria but can also be designed to accommodate
E1054Test Methods for Evaluation of Inactivators of Anti-
the testing of fungi and viruses.
microbial Agents
1.2 ThistestmethodisintendedtocomplimentTestMethod
E2274Test Method for Evaluation of Laundry Sanitizers
E2274 and is to be used in the cases where this test method is
and Disinfectants
determined to be the worse case scenario for product usage.
E2756Terminology Relating toAntimicrobial andAntiviral
Agents
NOTE 1—Test Method E2274 is the recommended method to evaluate
sanitizing/disinfectant laundry detergent/additives for use in traditional
2.2 Other Standards:
high wash water volume automatic clothes washing operations.
AATCC70WaterRepellency;TumbleJarDynamicAbsorp-
1.3 Knowledge of microbiological techniques is required tion Test
OSCPP 810.2400: Disinfectants and Sanitizers for Use on
for these procedures.
Fabrics and Textiles – Efficacy Data Recommendations
1.4 It is the responsibility of the investigator to determine
40 CFR, Part 160Good Laboratory Practice Standards
whether Good Laboratory Practices (GLP) are required and to
follow them where appropriate (see section 40 CFR, 160 or as
3. Terminology
revised.
3.1 For definitions of terms used in this test method refer to
1.5 The values stated in SI units are to be regarded as
Terminology E2756.
standard. No other units of measurement are included in this
3.2 Definitions of Terms Specific to This Standard:
standard.
3.2.1 active antimicrobial ingredient—a substance added to
NOTE 2—In this test method, metric units are used for all applications,
a formulation intended specifically for the inhibition or inac-
except for distance, in which case inches are used.
tivation of microorganisms.
1.6 Appropriate modifications to the test method may be
3.2.2 antimicrobial agent(s)—an active ingredient designed
required when the testing organisms are not specified herein.
to suppress the growth or action of microorganisms.
1.7 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 appro-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
priate safety and health practices and determine the applica-
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
bility of regulatory limitations prior to use.
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
Available from American Association of Textile Chemists and Colorists
(AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http://
This test method is under the jurisdiction of ASTM Committee E35 on www.aatcc.org.
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct Available from United States Environmental Protection Agency (EPA), Ariel
responsibility of Subcommittee E35.15 on Antimicrobial Agents. Rios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http://
Current edition approved April 15, 2016. Published May 2016. Originally www.epa.gov.
approved in 2004. Last previous edition approved in 2009 as E2406–09. DOI: Available from U.S. Government Publishing Office Bookstore 710 North
10.1520/E2406-16. Capitol Street N.W. Washington, DC, http://www.gpo.gov/about/bookstore.htm
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2406 − 16
3.2.3 carrier count control—procedure used to determine
the initial number of microorganisms on a fabric carrier
following the inoculation and drying procedure.
3.2.4 diluent—sterile deionized water, sterile distilled water
or sterile synthetic AOAC hard water that may be used to
prepare the active test formulation, vehicle control or product
control for use in the test procedure.
3.2.5 diluted product solution—test formulation, vehicle
control, or product control diluted to use concentration.
3.2.6 numbers control—in assessing sanitizer level
performance, procedure used to determine the number of
microorganisms remaining on the fabric carriers and in the
wash water following the test procedure in the presence of the
diluent.Thismayalsobeperformedusingdiluentorphosphate
FIG. 1 Stainless Steel Spindle Schematic
(Top View and Side View)
buffer dilution water with surfactant.
3.2.7 product control—a formulation with or without an
6.4 Timer (Stop-clock)—Any calibrated device that can be
active ingredient(s) used for comparison to the test formula-
read for minutes and seconds.
tion.
6.5 Exposure Chamber—Container with closure that can
3.2.8 test formulation—a formulation containing an antimi-
withstandsterilization.Dimensionandvolumecapacityshould
crobial agent(s).
be consistent for use in Test Method E2274.
3.2.9 vehicle control—the test formulation without the ac-
NOTE 4—Standard lids may form a vacuum seal when steam sterilized.
tive ingredient(s) used for comparison to the test formulation. To avoid, prior to sterilization place a piece of paper between lid and jar.
3.2.10 wash water—the liquid contained in the exposure 6.6 Stainless Steel Spindles—Spindles are fabricated from a
chamber previously exposed to either uninoculated fabric or single continuous piece of stainless steel wire ( ⁄16 in. diameter
fabric inoculated with the challenge microorganism. and bent to contain 3 horizontal extensions, 2 in. long
connected by 2 vertical sections approximately 2 in. long).
4. Summary of Test Method
They are shaped so that vertical sections form 150° angle
where the free ends of the 2 outer horizontal extensions are
4.1 Under simulated laundry conditions, sets of inoculated
sharpenedtoapoint.Thiswillbeusedasscaffoldingforinitial
fabricswatchesareplacedintolowvolumesofdilutedproduct
wrapping of fabric ballast. See Fig. 1.
solution and agitated. After a specified contact time, the wash
water and the test fabric are individually cultured either
6.7 Agitator—Tumbling device intended to rotate Exposure
quantitatively (sanitizer efficacy) or qualitatively (disinfectant
Chamberthrough360°verticalorbitof4to8in.diameterat45
efficacy).
to 60 rpm or a comparable tumbling devices such as Launder-
NOTE 3—See appropriate regulatory guidance document for the mini-
ometer or Tumble Jar described in AATCC 70.
mum number of replicates required to make a specific claim.
6.8 Micropipettor (and Pipet Tips), suitable to deliver 0.01
5. Significance and Use to 0.03 mL volume.
6.9 Forceps, large and small, sterile.
5.1 Theprocedureinthistestmethodisusedtoevaluatethe
effectiveness of a test reagent (antimicrobial agent/active
6.10 Safety Pins, sterile.
ingredient)orformulationtoreduceorcompletelykillbacterial
6.11 Stapler and Staples.
populationsoncontaminatedfabricsandinwashwaterfollow-
ing a single wash under simulated low wash volume condi- 6.12 Balance, with a platform to accommodate 15 6 0.1 g
tions. The water to fabric ratio in common front loading of test fabric.
machines is dynamic and varies by region and machine used.
6.13 Sterile Glass Beads, Average size 3 to 4 mm.
The proper water to fabric ratio and temperature for the
6.14 Filter Sterilization System for Media and Reagents—A
worse-case scenario for product use should be determined and
membrane or cartridge filtration system (0.22 µm pore diam-
documented prior to testing.
eter). Required for sterilizing heat-sensitive solutions.
6. Apparatus
6.15 Membrane Filtration System for Capture of the Test
Organism(s)—Sterile 47 mm diameter membrane Polyether-
6.1 Colony Counter—Anyofseveraltypesmaybeused,for
sulfone (PES) filters (0.45 µm pore diameter) and holders for
example, Quebec.
such filters.
6.2 Incubator—Any incubator that can maintain the opti-
mum temperature 62°C for growth of the challenge microor- 7. Reagents and Materials
ganism(s).
7.1 Petri Dishes, sterile 100 × 15 mm glass and plastic.
6.3 Sterilizer—Any suitable steam sterilizer producing the Required for performing standard plate counts and used in
conditions of sterility, is accetable. preparation of contaminated fabric carriers.
E2406 − 16
7.2 Bacteriological Pipets, sterile, various sizes. 7.9.5 To obtain a 500 µL inoculum of the challenge
microorganism, add to 340 µL of the microbial suspension 25
7.3 Test Fabric, approximately 80 by 80 threads/in.
µL, 100 µL and 35 µL of BSA, mucin and tryptone stock
bleached, desized, plain-weave cotton print cloth and without
solutions, respectively.
bluing or optical brighteners.
NOTE 6—The quality of the above materials may vary among manu-
NOTE 5—Other test fabrics/blends may be used at the discretion of the
facturersorproductlots.Therefore,preliminaryscreeningofsuchitemsis
investigator.
recommended to ensure compatibility with the test microorganism(s).
7.4 Dilution Fluid, AOAC Phosphate buffer dilution water NOTE 7—The investigator should confirm the appropriate organic soil
usage with the appropriate regulatory agency prior to initiating testing.
orothersuitablediluentcontainingappropriateneutralizersfor
serial dilution of test samples.
8. Test Microorganisms (810,2400)
7.5 Water for Dilution of Formulations Under Test:
8.1 Klebsiella pneumoniae, ATCC 4352.
7.5.1 Water, sterile, deionized or distilled, equivalent to or
8.2 Staphylococcus aureus, ATCC 6538.
better than Type 3, see Specification D1193.
7.5.2 AOAC Synthetic Hard Water .
8.3 Pseudomonas aeruginosa, ATCC 15442.
7.5.3 Allwaterusedforpreparationoftestsolutionsshallbe
8.4 Other microorganisms, as applicable. 15442, or other
sterile.
microorganisms, as applicable.
7.6 Purity of Reagents—Reagent grade chemicals shall be
9. Preparation of Test Microorganisms
used in all tests.
7.6.1 Sodium carbonate.
9.1 Subculture microorganism(s) on Nutrient Agar A
7.6.2 Alkaline nonionic wetting agent with HLB
through at least one daily transfer, incubating at 35 6 2°C.
(hydrophilic-lipophilic balance) value of approximately 13.
9.2 On the day prior to testing, wash the slant and transfer
Prepare solution containing 0.5% nonoxynol-10 class of
the cells into French square bottles containing 20 mLNutrient
ethoxylated alkyl phenols, for example Tergitol NP-10 or
Agar B. Incubate 18 to 24 h at 35 6 2°C, agar side down.
Triton X-100 and 0.5% Na CO .
2 3
9.3 Remove growth from the French square bottles using
7.7 Neutralizing Subculture Media—Aneutralizingmedium
three-mL dilution fluid and five sterile glass beads to suspend
capable of supporting the growth of the test organism (for
growth. The cultures will be standardized to yield approxi-
disinfection testing) following exposure to the test material in
mately 10 colony forming units (CFU) per mL of S. aureus
accordance with Test Methods E1054. Alternatively, the neu-
and 10 CFU/mL of K. pneumoniae and P. aeruginosa.
tralizing broths may be of sufficient volume to reduce the
NOTE 8—The initial inoculum concentration for these and other
concentration of the antimicrobials to below active levels. See
challenge microorganisms may vary and should be determined from
step 12.8.
carrier and wash water numbers control recovery (see Section 12).
9.4 A soil load may be added to each inoculum (see 7.9).
7.8 Culture Media:
7.8.1 Nutrient Agar A .
10. Fabric and Spindle Preparation
7.8.2 Nutrient Agar B .
7.8.3 Media suitable for identification of microorganism(s)
10.1 Scour test fabric by boiling approximately 300 g of
used in the study. materialfor1hin3Lofdistilledordeionizedwatercontaining
7.8.4 Soybean casein digest medium or other suitable 1.5-g sodium carbonate and 1.5-g nonionic wetting agent.
media,withorwithoutspecificneutralizers,forrecoveryofthe Rinse fabric, first in boiling water and then in cold water, until
challenge microorganism(s). all visual traces of wetting agent are removed (that is, foam-
ing). Remove as much water as possible from fabric.
7.9 Organic Soil Load—When an organic soil load is to be
10.2 Air dry for at least 24 h at ambient room temperature
incorporated in the suspension of the challenge
microorganism(s), defibrinated heat-inactivated animal serum ensuring that the material is completely dry.
may be used or a mixture of the following stock solutions in
10.3 Cutscoureddryfabricintostrips2in.(5cm)wideand
phosphatebufferdilutionwater(pH7.2)maybeused(see7.4).
weighing 15 6 0.1 g each. For cotton fabrics, pierce one end
7.9.1 Add 0.5 g of tryptone to 10 mL phosphate buffer.
ofthe15-gtestfabricstripandsecureontotheouterhorizontal
7.9.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mL
extensionofastainlesssteelspindle.Windthestriparoundthe
of phosphate buffer.
three horizontal extensions with sufficient tension to obtain 12
7.9.3 Add 0.04 g of bovine mucin to 10 mL of phosphate
but not 13 laps while using the entire 15 6 0.1 g of fabric.
buffer.
Staples,apin,orautoclavablefabrictagmaybeusedtosecure
7.9.4 Prepare the solutions separately and sterilize by pas-
the fabric strip end.Apply additional staples to the 6th and 7th
sagethrougha0.22µmporediametermembranefilter,aliquote
folds along one horizontal side of the fabric bundle to create
and store at either 4 6 2°C or −20 6 2°C for no longer than 3
“pockets” that will secure individual fabric swatches during
months.
tumbling. Fabric wrapped spindles may be sterilized in indi-
vidual exposure chambers. Alternatively, fabric wrapped
spindles may be sterilized separately from exposure chambers.
Ensure fabric on spindles and exposure chambers are dry prior
Official Methods ofAnalysis of theAOAC International (AOAC) Washington,
DC, Chapter 6: Disinfectants. to testing.
E2406 − 16
NOTE 9—Fabric may be purchased in pre-cut strips and then scoured.
12.8 Addconcentratedneutralizingbrothtowashwaterand
mixwell.Alternatively,0.5mlwashwatermaybeaddedto9.5
10.4 Fabriccarriersofapproximately1by1.5in.willbecut
mL of neutralizer –10 mL of neutralizer.
from the remaining scoured fabric. A nontoxic permanent
NOTE 13—The specific neutralizer and concentrations should be deter-
markermaybeusedtoplaceamarkontheedgeofeachcarrier.
minedpriortotesting.Foradditiontowashwater,concentrationshouldbe
Alternatively, attach a pin to the short side of each carrier.
increased in order to reduce the amount of neutralizer added to wash
Place fabric carriers in glass petri dishes and sterilize. Ensure
water. Otherwise, the volume of neutralizer needed may overflow the
dryness of fabric prior to testing. exposure chamber.
12.9 Addition of fabric carrier to neutralizing broth and
10.5 For each exposure chamber, prepare at least 3 fabric
concentrated neutralizing broth to wash water completes the
carriers and 1 fabric wrapped spindle for each active test
exposure time.
formulation/product and control/numbers control.
12.10 All tubes containing fabric carriers will be mixed
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2406 − 09 E2406 − 16
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. 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
1.1 This test method is designed to evaluate sanitizing/disinfectant laundry detergents/additives for use in front loading 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—Test Method E2274 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 This test method is intended to compliment Test Method E2274 and is to be used in the cases where this test method is
determined to be the worse case scenario for product usage.
NOTE 1—Test Method E2274 is the recommended method to evaluate sanitizing/disinfectant laundry detergent/additives for use in traditional high wash
water volume automatic clothes washing operations.
1.3 Knowledge of microbiological techniques is required for these procedures.
1.4 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLP) are required and to follow
them where appropriate (see section 40 CFR, 160 or as revised.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
NOTE 2—In this test method, metric units are used for all applications, except for distance, in which case inches are used.
1.6 Appropriate modifications to the test method may be required when the testing organisms are not specified herein.
1.7 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.
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents
E2274 Test Method for Evaluation of Laundry Sanitizers and Disinfectants
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Nov. 1, 2009April 15, 2016. Published December 2009May 2016. Originally approved in 2004. Last previous edition approved in 20042009 as
E2406 – 04.E2406 – 09. DOI: 10.1520/E2406-09.10.1520/E2406-16.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2406 − 16
2.2 AATCC Standard:Other Standards:
AATCC 70-199770 Water Repellency; Tumble Jar Dynamic Absorption Test
OSCPP 810.2400 : Disinfectants and Sanitizers for Use on Fabrics and Textiles – Efficacy Data Recommendations
40 CFR, Part 160 Good Laboratory Practice Standards
2.3 EPA Standard:
DIS/TSS 13 Laundry Additives—Disinfection and Sanitization, U.S. Environmental Protection Agency, Office of Pesticide
Programs, April 1980
3. Terminology
3.1 For definitions of terms used in this test method refer to Terminology E2756.
3.2 Definitions:Definitions of Terms Specific to This Standard:
3.2.1 active antimicrobial ingredient—a substance added to a formulation intended specifically for the inhibition or inactivation
of microorganisms.
3.2.2 antimicrobial agent(s)—an active ingredient designed to suppress the growth or action of microorganisms.
3.2.3 carrier count control—procedure used to determine the initial number of microorganisms on a fabric carrier following the
inoculation and drying procedure.
3.2.4 diluent—sterile deionized water, sterile distilled water or sterile synthetic AOAC hard water that may be used to prepare
the active test formulation, vehicle control or product control for use in the test procedure.
3.2.5 diluted product solution—test formulation, vehicle control, or product control diluted to use concentration.
3.1.6 neutralization—a process that results in quenching the antimicrobial activity of a test formulation. This may be achieved
by dilution of the test formulation(s) to reduce the concentration of the antimicrobials, or through the use of chemical agents, called
neutralizers, to suppress antibacterial activity.
3.2.6 numbers control—in assessing sanitizer level performance, procedure used to determine the number of microorganisms
remaining on the fabric carriers and in the wash water following the test procedure in the presence of the diluent. This may also
be performed using diluent or phosphate buffer dilution water with surfactant.
3.2.7 product control—a formulation with or without an active ingredient(s) used for comparison to the test formulation.
3.2.8 test formulation—a formulation containing an antimicrobial agent(s).
3.2.9 vehicle control—the test formulation without the active ingredient(s) used for comparison to the test formulation.
3.2.10 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 low volumes of diluted product
solution and agitated. After a specified contact time, the wash water and the test fabric are individually cultured either
quantitatively (sanitizer efficacy) or qualitatively (disinfectant efficacy).
NOTE 3—See appropriate regulatory guidance document for the minimum number of replicates required to make a specific claim.
5. Significance and Use
5.1 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. (SeeThe Table 1.)water to fabric ratio in common front loading
machines is dynamic and varies by region and machine used. The proper water to fabric ratio and temperature for the worse-case
scenario for product use should be determined and documented prior to testing.
6. Apparatus
6.1 Colony Counter—Any of several types may be used, for example, Quebec.
6.2 Incubator—Any incubator that can maintain the optimum temperature 62°C for growth of the challenge microorganism(s).
6.3 Sterilizer—Any suitable steam sterilizer producing the conditions of sterility.sterility, is accetable.
6.4 Timer (Stop-clock)—Any calibrated device that can be read for minutes and seconds.
Available from American Association of Textile Chemists and Colorists (AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http://www.aatcc.org.
Available from United States Environmental Protection Agency (EPA), Ariel Rios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http://www.epa.gov.
Available from U.S. Government Publishing Office Bookstore 710 North Capitol Street N.W. Washington, DC, http://www.gpo.gov/about/bookstore.htm
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FIG. 1 Stainless Steel Spindle Schematic
(Top View and Side View)
6.5 Exposure Chamber—Container with closure that can withstand sterilization. Dimension and volume capacity should be
consistent for use in Test Method E2274.
NOTE 4—Standard lids may form a vacuum seal when steam sterilized. To avoid, prior to sterilization place a piece of paper between lid and jar.
6.6 Stainless Steel Spindles—Spindles are fabricated from a single continuous piece of stainless steel wire ( ⁄16 in. diameter and
bent to contain 3 horizontal extensions, 2 in. long connected by 2 vertical sections approximately 2 in. long). They are shaped so
that vertical sections form 150° angle where the free ends of the 2 outer horizontal extensions are sharpened to a point. This will
be used as scaffolding for initial wrapping of fabric ballast. See Fig. 1.
6.7 Agitator—Tumbling device intended to rotate Exposure Chamber through 360° vertical orbit of 4 to 8 in. diameter at 45 to
60 rpm or a comparable tumbling devices such as Launderometer or Tumble Jar described in AATCC 70-1997.70.
6.8 Micropipettor (and Pipet Tips), suitable to deliver 0.01 to 0.03 mL volume.
6.9 Forceps, large and small, sterile.
6.10 Safety Pins, sterile.
6.11 Stapler and Staples.
6.12 Balance, with a platform to accommodate 15 6 0.1 g of test fabric.
6.13 Sterile Glass Beads, Average size 3 to 4 mm.
6.14 Filter Sterilization System for Media and Reagents—A membrane or cartridge filtration system (0.22 μm pore diameter).
Required for sterilizing heat-sensitive solutions.
6.15 Membrane Filtration System for Capture of the Test Organism(s)—Sterile 47 mm diameter membrane Polyethersulfone
(PES) filters (0.45 μm pore diameter) and holders for such filters.
7. Reagents and Materials
7.1 Petri Dishes, sterile 100 by 15 mm. × 15 mm glass and plastic. Required for performing standard plate counts and used in
preparation of contaminated fabric carriers.
7.2 Bacteriological Pipets, sterile, various sizes.
7.3 Test Fabric, approximately 80 by 80 threads/in. bleached, desized, plain-weave cotton print cloth and without bluing or
optical brighteners.
NOTE 5—Other test fabrics/blends may be used at the discretion of the investigator.
7.4 Dilution Fluid, AOAC Phosphate buffer dilution water or other suitable diluent containing appropriate neutralizers for
serial dilution of test samples.
7.5 Water for Dilution of Formulations Under Test:
7.5.1 Water, sterile, deionized or distilled, equivalent to or better than Type 3, see Specification D1193.
7.5.2 AOAC Synthetic Hard Water.Water .
7.5.3 All water used for preparation of test solutions shall be sterile.
7.6 Purity of Reagents—Reagent grade chemicals shall be used in all tests.
Official Methods of Analysis of the AOAC International (AOAC) Washington, DC, Chapter 6: Disinfectants.
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7.6.1 Sodium carbonate.
7.6.2 Alkaline nonionic wetting agent with HLB (hydrophilic-lipophilic balance) value of approximately 13. Prepare solution
containing 0.5 % nonoxynol-10 class of ethoxylated alkyl phenols, for example Tergitol NP-10 or Triton X-100 and 0.5 % Na CO .
2 3
7.7 Neutralizing Broths—Subculture Media—Growth media appropriate for the challenge microorganism containing chemical
agents to suppress antibacterial activity. A neutralizing medium capable of supporting the growth of the test organism (for
disinfection testing) following exposure to the test material in accordance with Test Methods E1054. Alternatively, the neutralizing
broths may be of sufficient volume to reduce the concentration of the antimicrobials to below active levels. See step 11.812.8.
7.8 Challenge Microorganisms (DIS/TSS 13):
7.8.1 Klebsiella pneumoniae, ATCC 4352.
7.8.2 Staphylococcus aureus, ATCC 6538.
7.8.3 Pseudomonas aeruginosa, ATCC 15442.
7.8.4 Other microorganisms, as applicable.
7.8 Culture Media:
7.8.1 Nutrient Agar A.A .
7.8.2 Nutrient Agar B.B .
7.8.3 Media suitable for identification of microorganism(s) used in the study.
7.8.4 Soybean casein digest medium or other suitable media, with or without specific neutralizers, for recovery of the challenge
microorganism(s).
7.9 Organic Soil Load—When an organic soil load is to be incorporated in the suspension of the challenge microorganism(s),
defibrinated heat-inactivated animal serum may be used or a mixture of the following stock solutions in phosphate buffer dilution
water (pH 7.2) may be used (see 7.4).
7.9.1 Add 0.5 g of tryptone to 10 mL phosphate buffer.
7.9.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mL of phosphate buffer.
7.9.3 Add 0.04 g of bovine mucin to 10 mL of phosphate buffer.
7.9.4 Prepare the solutions separately and sterilize by passage through a 0.22 μm pore diameter membrane filter, aliquote and
store at either 4 6 2°C or −20 6 2°C for no longer than 3 months.
7.9.5 To obtain a 500 μL inoculum of the challenge microorganism, add to 340 μL of the microbial suspension 25 μL, 100 μL
and 35 μL of BSA, mucin and tryptone stock solutions, respectively.
NOTE 6—The quality of the above materials may vary among manufacturers or product lots. Therefore, preliminary screening of such items is
recommended to ensure compatibility with the test microorganism(s).
NOTE 7—The investigator should confirm the appropriate organic soil usage with the appropriate regulatory agency prior to initiating testing.
8. Test Microorganisms (810,2400)
8.1 Klebsiella pneumoniae, ATCC 4352.
8.2 Staphylococcus aureus, ATCC 6538.
8.3 Pseudomonas aeruginosa, ATCC 15442.
8.4 Other microorganisms, as applicable. 15442, or other microorganisms, as applicable.
9. Preparation of Test Microorganisms
9.1 Subculture microorganism(s) on Nutrient Agar A through at least one daily transfer, incubating at 35 6 2°C.
9.2 On the day prior to testing, wash the slant and transfer the cells into French square bottles containing 20 mL Nutrient Agar
B. Incubate 18 to 24 h at 35 6 2°C, agar side down.
9.3 Remove growth from the French square bottles using three-mL dilution fluid and five sterile glass beads to suspend growth.
8 9
The cultures will be standardized to yield approximately 10 colony forming units (CFU) per mL of S. aureus and 10 CFU/mL
of K. pneumoniae and P. aeruginosa.
NOTE 8—The initial inoculum concentration for these and other challenge microorganisms may vary and should be determined from carrier and wash
water numbers control recovery (see Section 12).
9.4 A soil load may be added to each inoculum (see 7.9).
10. Fabric and Spindle Preparation
10.1 Scour test fabric by boiling approximately 300 g of material for 1 h in 3 L of distilled or deionized water containing 1.5-g
sodium carbonate and 1.5-g nonionic wetting agent. Rinse fabric, first in boiling water and then in cold water, until all visual traces
of wetting agent are removed (that is, foaming). Remove as much water as possible from fabric.
10.2 Air dry for at least 24 h at ambient room temperature.temperature ensuring that the material is completely dry.
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10.3 Cut scoured dry fabric into strips 2 in. (5 cm) wide and weighing 15 6 0.1 g each. For cotton fabrics, pierce one end of
the 15-g test fabric strip and secure onto the outer horizontal extension of a stainless steel spindle. Wind the strip around the three
horizontal extensions with sufficient tension to obtain 12 but not 13 laps while using the entire 15 6 0.1 g of fabric. Staples or
a pin Staples, a pin, or autoclavable fabric tag may be used to secure the fabric strip end. Apply additional staples to the 6th and
7th folds along one horizontal side of the fabric bundle to create “pockets” that will secure individual fabric swatches during
tumbling. Fabric wrapped spindles may be sterilized in individual exposure chambers. Alternatively, fabric wrapped spindles may
be sterilized separately from exposure chambers. Ensure dryness of fabric on spindles and exposure chambers are dry prior to
testing.
NOTE 9—Fabric may be purchased in pre-cut strips and then scoured.
10.4 Fabric carriers of approximately 1 by 1.5 in. will be cut from the remaining scoured fabric. A nontoxic permanent marker
may be used to place a mark on the edge of each carrier. Alternatively, attach a pin to the short side of each carrier. Place fabric
carriers in glass petri dishes and sterilize. Ensure dryness of fabric prior to testing.
10.5 For each exposure chamber, prepare at least 3 fabric carriers and 1 fabric wrapped spindle for each active test
formulation/product and control/numbers control.
9. Preparation of Challenge Microorganisms
9.1 Subculture microorganism(s) on Nutrient Agar A through at least three daily transfers, incubating at 35 6 2°C. If only one
daily transfer is missed, it is not necessary to repeat the three consecutive transfers prior to use in testing.
9.2 On the day prior to testing, transfer the cells into French square bottles containing 20 mL Nutrient Agar B. Incubate 18 to
24 h at 35 6 2°C, agar side down.
9.3 Remove growth from the French square bottles using three-mL dilution fluid and five sterile glass beads to suspend growth.
8 9
The cultures will be standardized to yield approximately 10 colony forming units (CFU) per mL of S. aureus and 10 CFU/mL
of K. pneumoniae and P. aeruginosa.
NOTE 9—The initial inoculum concentration for different challenge microorganisms may vary and should be determined from carrier and wash water
numbers control recovery (see Section 12).
9.4 A soil load may be added to each inoculum (see 7.10).
11. Preparation of Test Sample
11.1 Prepare a sufficient volume of active test formulation and product control (at least 1 L) according to manufacturer
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