1、Designation: E2406 16Standard Test Method forEvaluation of Laundry Sanitizers and Disinfectants for Usein High Efficiency Washing Operations1This standard is issued under the fixed designation E2406; the number immediately following the designation indicates the year oforiginal adoption or, in the c
2、ase of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is designed to evaluate sanitizing/disinfectant laundry detergents/addit
3、ives for use in front load-ing high efficiency (HE) automatic clothes washing operationsthat typically utilize very low wash water volumes. This testmethod is designed to provide testing with representativevegetative bacteria but can also be designed to accommodatethe testing of fungi and viruses.1.
4、2 This test method is intended to compliment Test MethodE2274 and is to be used in the cases where this test method isdetermined to be the worse case scenario for product usage.NOTE 1Test Method E2274 is the recommended method to evaluatesanitizing/disinfectant laundry detergent/additives for use in
5、 traditionalhigh wash water volume automatic clothes washing operations.1.3 Knowledge of microbiological techniques is requiredfor these procedures.1.4 It is the responsibility of the investigator to determinewhether Good Laboratory Practices (GLP) are required and tofollow them where appropriate (s
6、ee section 40 CFR, 160 or asrevised.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.NOTE 2In this test method, metric units are used for all applications,except for distance, in which case inches are used.1.6 Appropriate mod
7、ifications to the test method may berequired when the testing organisms are not specified herein.1.7 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health
8、practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine
9、 the Precision of a Test MethodE1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE2274 Test Method for Evaluation of Laundry Sanitizersand DisinfectantsE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 Other Standards:AATCC 70 Water Repellency; Tumble Jar Dyn
10、amicAbsorp-tion Test3OSCPP 810.2400 : Disinfectants and Sanitizers for Use onFabrics and Textiles Efficacy Data Recommendations440 CFR, Part 160 Good Laboratory Practice Standards53. Terminology3.1 For definitions of terms used in this test method refer toTerminology E2756.3.2 Definitions of Terms S
11、pecific to This Standard:3.2.1 active antimicrobial ingredienta substance added toa formulation intended specifically for the inhibition or inac-tivation of microorganisms.3.2.2 antimicrobial agent(s)an active ingredient designedto suppress the growth or action of microorganisms.1This test method is
12、 under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 15, 2016. Published May 2016. Originallyapproved in 2004. Last previous edition app
13、roved in 2009 as E2406 09. DOI:10.1520/E2406-16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available fr
14、om American Association of Textile Chemists and Colorists(AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http:/www.aatcc.org.4Available from United States Environmental Protection Agency (EPA), ArielRios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http:/www.epa.gov.5Available
15、 from U.S. Government Publishing Office Bookstore 710 NorthCapitol Street N.W. Washington, DC, http:/www.gpo.gov/about/bookstore.htmCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.2.3 carrier count controlprocedure used to determine
16、the initial number of microorganisms on a fabric carrierfollowing the inoculation and drying procedure.3.2.4 diluentsterile deionized water, sterile distilled wateror sterile synthetic AOAC hard water that may be used toprepare the active test formulation, vehicle control or productcontrol for use i
17、n the test procedure.3.2.5 diluted product solutiontest formulation, vehiclecontrol, or product control diluted to use concentration.3.2.6 numbers controlin assessing sanitizer levelperformance, procedure used to determine the number ofmicroorganisms remaining on the fabric carriers and in thewash w
18、ater following the test procedure in the presence of thediluent. This may also be performed using diluent or phosphatebuffer dilution water with surfactant.3.2.7 product controla formulation with or without anactive ingredient(s) used for comparison to the test formula-tion.3.2.8 test formulationa f
19、ormulation containing an antimi-crobial agent(s).3.2.9 vehicle controlthe test formulation without the ac-tive ingredient(s) used for comparison to the test formulation.3.2.10 wash waterthe liquid contained in the exposurechamber previously exposed to either uninoculated fabric orfabric inoculated w
20、ith the challenge microorganism.4. Summary of Test Method4.1 Under simulated laundry conditions, sets of inoculatedfabric swatches are placed into low volumes of diluted productsolution and agitated. After a specified contact time, the washwater and the test fabric are individually cultured eitherqu
21、antitatively (sanitizer efficacy) or qualitatively (disinfectantefficacy).NOTE 3See appropriate regulatory guidance document for the mini-mum number of replicates required to make a specific claim.5. Significance and Use5.1 The procedure in this test method is used to evaluate theeffectiveness of a
22、test reagent (antimicrobial agent/activeingredient) or formulation to reduce or completely kill bacterialpopulations on contaminated fabrics and in wash water follow-ing a single wash under simulated low wash volume condi-tions. The water to fabric ratio in common front loadingmachines is dynamic an
23、d varies by region and machine used.The proper water to fabric ratio and temperature for theworse-case scenario for product use should be determined anddocumented prior to testing.6. Apparatus6.1 Colony CounterAny of several types may be used, forexample, Quebec.6.2 IncubatorAny incubator that can m
24、aintain the opti-mum temperature 62C for growth of the challenge microor-ganism(s).6.3 SterilizerAny suitable steam sterilizer producing theconditions of sterility, is accetable.6.4 Timer (Stop-clock)Any calibrated device that can beread for minutes and seconds.6.5 Exposure ChamberContainer with clo
25、sure that canwithstand sterilization. Dimension and volume capacity shouldbe consistent for use in Test Method E2274.NOTE 4Standard 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 SpindlesSpindles are f
26、abricated from asingle continuous piece of stainless steel wire (116 in. diameterand bent to contain 3 horizontal extensions, 2 in. longconnected by 2 vertical sections approximately 2 in. long).They are shaped so that vertical sections form 150 anglewhere the free ends of the 2 outer horizontal ext
27、ensions aresharpened to a point. This will be used as scaffolding for initialwrapping of fabric ballast. See Fig. 1.6.7 AgitatorTumbling device intended to rotate ExposureChamber through 360 vertical orbit of 4 to 8 in. diameter at 45to 60 rpm or a comparable tumbling devices such as Launder-ometer
28、or Tumble Jar described in AATCC 70.6.8 Micropipettor (and Pipet Tips), suitable to deliver 0.01to 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 gof test fabric.6.13 Sterile Glass Beads, A
29、verage size 3 to 4 mm.6.14 Filter Sterilization System for Media and ReagentsAmembrane or cartridge filtration system (0.22 m pore diam-eter). Required for sterilizing heat-sensitive solutions.6.15 Membrane Filtration System for Capture of the TestOrganism(s)Sterile 47 mm diameter membrane Polyether
30、-sulfone (PES) filters (0.45 m pore diameter) and holders forsuch filters.7. Reagents and Materials7.1 Petri Dishes, sterile 100 15 mm glass and plastic.Required for performing standard plate counts and used inpreparation of contaminated fabric carriers.FIG. 1 Stainless Steel Spindle Schematic(Top V
31、iew and Side View)E2406 1627.2 Bacteriological Pipets, sterile, various sizes.7.3 Test Fabric, approximately 80 by 80 threads/in.bleached, desized, plain-weave cotton print cloth and withoutbluing or optical brighteners.NOTE 5Other test fabrics/blends may be used at the discretion of theinvestigator
32、.7.4 Dilution Fluid, AOAC Phosphate buffer dilution water6or other suitable diluent containing appropriate neutralizers forserial dilution of test samples.7.5 Water for Dilution of Formulations Under Test:7.5.1 Water, sterile, deionized or distilled, equivalent to orbetter than Type 3, see Specifica
33、tion D1193.7.5.2 AOAC Synthetic Hard Water6.7.5.3 All water used for preparation of test solutions shall besterile.7.6 Purity of ReagentsReagent grade chemicals shall beused in all tests.7.6.1 Sodium carbonate.7.6.2 Alkaline nonionic wetting agent with HLB(hydrophilic-lipophilic balance) value of ap
34、proximately 13.Prepare solution containing 0.5 % nonoxynol-10 class ofethoxylated alkyl phenols, for example Tergitol NP-10 orTriton X-100 and 0.5 % Na2CO3.7.7 Neutralizing Subculture MediaAneutralizing mediumcapable of supporting the growth of the test organism (fordisinfection testing) following e
35、xposure to the test material inaccordance with Test Methods E1054. Alternatively, the neu-tralizing broths may be of sufficient volume to reduce theconcentration of the antimicrobials to below active levels. Seestep 12.8.7.8 Culture Media:7.8.1 Nutrient Agar A6.7.8.2 Nutrient Agar B6.7.8.3 Media sui
36、table for identification of microorganism(s)used in the study.7.8.4 Soybean casein digest medium or other suitablemedia, with or without specific neutralizers, for recovery of thechallenge microorganism(s).7.9 Organic Soil LoadWhen an organic soil load is to beincorporated in the suspension of the c
37、hallengemicroorganism(s), defibrinated heat-inactivated animal serummay be used or a mixture of the following stock solutions inphosphate 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 mLo
38、f phosphate buffer.7.9.3 Add 0.04 g of bovine mucin to 10 mL of phosphatebuffer.7.9.4 Prepare the solutions separately and sterilize by pas-sage through a 0.22 m pore diameter membrane filter, aliquoteand store at either 4 6 2C or 20 6 2C for no longer than 3months.7.9.5 To obtain a 500 L inoculum o
39、f the challengemicroorganism, add to 340 L of the microbial suspension 25L, 100 L and 35 L of BSA, mucin and tryptone stocksolutions, respectively.NOTE 6The quality of the above materials may vary among manu-facturers or product lots. Therefore, preliminary screening of such items isrecommended to e
40、nsure compatibility with the test microorganism(s).NOTE 7The investigator should confirm the appropriate organic soilusage 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
41、 Pseudomonas aeruginosa, ATCC 15442.8.4 Other microorganisms, as applicable. 15442, or othermicroorganisms, as applicable.9. Preparation of Test Microorganisms9.1 Subculture microorganism(s) on Nutrient Agar Athrough at least one daily transfer, incubating at 35 6 2C.9.2 On the day prior to testing,
42、 wash the slant and transferthe cells into French square bottles containing 20 mL NutrientAgar B. Incubate 18 to 24 h at 35 6 2C, agar side down.9.3 Remove growth from the French square bottles usingthree-mL dilution fluid and five sterile glass beads to suspendgrowth. The cultures will be standardi
43、zed to yield approxi-mately 108colony forming units (CFU) per mL of S. aureusand 109CFU/mL of K. pneumoniae and P. aeruginosa.NOTE 8The initial inoculum concentration for these and otherchallenge microorganisms may vary and should be determined fromcarrier and wash water numbers control recovery (se
44、e Section 12).9.4 A soil load may be added to each inoculum (see 7.9).10. Fabric and Spindle Preparation10.1 Scour test fabric by boiling approximately 300 g ofmaterial for1hin3Lofdistilled or deionized water containing1.5-g sodium carbonate and 1.5-g nonionic wetting agent.Rinse fabric, first in bo
45、iling water and then in cold water, untilall visual traces of wetting agent are removed (that is, foam-ing). Remove as much water as possible from fabric.10.2 Air dry for at least 24 h at ambient room temperatureensuring that the material is completely dry.10.3 Cut scoured dry fabric into strips 2 i
46、n. (5 cm) wide andweighing 15 6 0.1 g each. For cotton fabrics, pierce one endof the 15-g test fabric strip and secure onto the outer horizontalextension of a stainless steel spindle. Wind the strip around thethree horizontal extensions with sufficient tension to obtain 12but not 13 laps while using
47、 the entire 15 6 0.1 g of fabric.Staples, a pin, or autoclavable fabric tag may be used to securethe fabric strip end. Apply additional staples to the 6th and 7thfolds along one horizontal side of the fabric bundle to create“pockets” that will secure individual fabric swatches duringtumbling. Fabric
48、 wrapped spindles may be sterilized in indi-vidual exposure chambers. Alternatively, fabric wrappedspindles may be sterilized separately from exposure chambers.Ensure fabric on spindles and exposure chambers are dry priorto testing.6Official Methods of Analysis of the AOAC International (AOAC) Washi
49、ngton,DC, Chapter 6: Disinfectants.E2406 163NOTE 9Fabric may be purchased in pre-cut strips and then scoured.10.4 Fabric carriers of approximately 1 by 1.5 in. will be cutfrom the remaining scoured fabric. A nontoxic permanentmarker 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. Ensuredryness of fabric prior to testing.10.5 For each exposure chamber, prepare at least 3 fabriccarriers and 1 fabric wr
