ASTM E2197-17 Standard Quantitative Disk Carrier Test Method for Determining Bactericidal,Virucidal,Fungicidal

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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:E2197-17

INTERNATIONAL

Standard Quantitative Disk Carrier Test Method for

Determining Bactericidal,Virucidal,Fungicidal,

Mycobactericidal,and Sporicidal Activities of Chemicals¹

This standard is issued under the fixed designation E2197;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(ε) indicates an editorial change since the last revision or reapproval.

INTRODUCTION

The quantitative test method described here uses disks of stainless steel(1 cm in diameter)as

carriers.It employs the same basic set of materials and procedures to assess the ability of liquid

chemicals to inactivate vegetative bacteria,viruses,fungi,mycobacteria,and bacterial spores(1-7).²

Performance standards for test substances,the level of water hardness,the type and level of a soil load,

the test organism(s),and other test conditions may vary depending on the target regulatory agency.

This basic test can also be adapted for use with other carrier materials of similar dimensions.

The development of this test method was made possible with financial support from the

Antimicrobials Division of the U.S.Environmental Protection Agency.

1. Scope

1.1 This test method is designed to evaluate the ability of

test substances to inactivate vegetative bacteria,viruses,fungi,

mycobacteria,and bacterial spores(1-7)on disk carriers of

brushed stainless steel that represent hard,nonporous environ-

mental surfaces and medical devices.It is also designed to have

survivors that can be compared to the mean of no less than

three control carriers to determine if the performance standard

has been met.For proper statistical evaluation of the results,

the number of viable organisms in the test inoculum should be

sufficiently high to take into account both the performance

standard and the experimental variations in the results.

1.2 The test protocol does not include any wiping or rubbing

action.It is,therefore,not designed for testing wipes.

1.3 This test method should be performed by persons with

training in microbiology in facilities designed and equipped for

work with infectious agents at the appropriate biosafety level

(8).

1.4 It is the responsibility of the investigator to determine

whether Good Laboratory Practice Regulations(GLPs)are

1This 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 Dec.1,2017.Published December 2017.Originally

approved in 2002.Last previous edition approved in 2011 as E2197-11.DOI:

10.1520/E2197-17.

2 The boldface numbers in parenthesis refer to the list of references at the end of

this standard.

required and to follow them where appropriate (40 CFR,Part

160 for EPA submissions and 21 CFR,Part 58 for FDA

submissions).

1.5 In this test method,SI units are used for all applications,

except for distance in which case inches are used and metric

units follow.

1.6 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-

priate safety,health,and environmental practices and deter-

mine the applicability of regulatory limitations prior to use.

1.7 This international standard was developed in accor-

dance with internationally recognizedprinciples on standard-

ization established in the Decision on Principles for the

Development of International Standards,Guides and Recom-

mendations issued by the World Trade Organization Technical

Barriers to Trade(TBT)Committee.

2.Referenced Documents

2.1 ASTM Standards:3

A967/A967M Specification for Chemical Passivation Treat-

ments for Stainless Steel Parts

D1129 Terminology Relating to Water

D1193 Specification for Reagent Water

3For referenced ASTM standards,visit the ASTM website,www.astm.org,or

contact ASTM Customer Service at service@astm.org.For Annual Book ofASTM

Standards volume information,refer to the standard's Document Summary page on

the ASTM website.

E2756 Terminology Relating to Antimicrobial and Antiviral

Agents

2.2 CFR Standard:⁴

21 CFR,Part 58 Laboratory Practice for Nonclinical Labo-

ratory Studies

40 CFR,Part 160 Good Laboratory Practice Standards

2.3 CEN Standard:5

EN 10088-21J/2J Stainless steels -Part 2:Technical deliv-

ery conditions for sheet/plate and strip of corrosion

resisting steels for general purposes

3.Terminology

3.1 Definitions—For definitions of general terms used in this

test method,refer to Terminology E2756.

3.2 Definitions of Terms Specific to This Standard:

3.2.1 carrier,n—an inanimate surface or object inoculated

with the test organism.

3.2.2 eluate,n—an eluent,which contains the recovered

organism(s).

3.2.3 eluent, n—any solution that is harmless to the test

organism(s)and that is added to a carrier to recover the

organism(s)in or on it.

3.2.4 neutralization, n—a process to quench the antimicro-

bial activity of a test substance.This process may be achieved

by dilution of the organism/test substance mixture and/or by

adding to it one or more chemical neutralizers.

3.2.5 soil load, n—a solution of one or more organic,or

inorganic substances,or both,added to the suspension of the

test organism to simulate the presence of body secretions,

excretions,or other extraneous substances.

3.2.6 test organism,n—an organism that has characteristics

that allow it to be readily identified.It also may be referred to

as a surrogate,a simulant,or a marker organism.

3.2.7 test substance,n—a formulation that incorporates

antimicrobial ingredients.

4.Summary of Test Method

4.1 Each disk(1 cm in diameter)receives 10 μL of the test

organism with a soil load.The inoculum is dried,and then the

disk is placed on the inside bottom surface of a sterile plastic

vial prior to contact with 50 μL of the use-dilution of test

substance.The contact time and temperature may vary as

required.Control carriers receive 50μL of a fluid harmless to

the test organism(s)and its host cells,if any,but are otherwise

treated in the same way as test carriers.

4.2 For tests against vegetative bacteria, fungi,

mycobacteria,and bacterial spores,the test substance is then

neutralized and the inoculum eluted.The eluate and subsequent

rinses ofthe carrier and its vial are membrane filtered.Culture

plates with the filters are incubated,colonies counted,and log10

reductions calculated.

⁴Available from U.S.Government Printing Office Superintendent of Documents,

732 N.Capitol St.,NW,Mail Stop:SDE,Washington,DC 20401,http://

Www.access.gpo.goV.

5Available from European Committee for Standardization(CEN),Avenue

Marnix 17,B-1000,Brussels,Belgium,http://www.cen.eu.

4.3 For tests with viruses,appropriate dilutions ofthe eluate

are inoculated into suitable cell cultures,the cultures are

examined for cytopathology/infectious foci,which are esti-

mated as the most probable number(MPN)or counted as foci

or plaques,and log10 are calculated.

5.Significance and Use

5.1 The design of this test eliminates any loss of viable

organisms through wash off,thus making it possible to produce

statistically valid data using many fewer test carriers than

needed for methods based on simple MPN estimates.

5.2 The stringency in the test is provided by the use of a soil

load,the microtopography of the brushed stainless steel carrier

surface,and the smaller ratio of test substance to surface area

typical for many disinfectant applications.Thus,th e test

substance being assessed is presented with a reasonable chal-

lenge while allowing for efficient recovery of the test organ-

isms from the inoculated carriers.The metal disks in the basic

test are also compatible with a wide variety of actives.

5.3 The design of the carriers makes it possible to place onto

each a precisely measured volume of the test organism(10μL)

as well as the control fluid or test substance (50 μL).

5.4 The inoculum is placed at the center of each disk

whereas the volumes of the test substance covers nearly the

entire disk surface,thus virtually eliminating the risk of any

organisms remaining unexposed.

5.5 In all tests,other than those against viruses,the addition

of 10 mL of an eluent/diluent gives a 1:200 dilution of the test

substance immediately at the end of the contact time.While

this step in itself may be sufficient to arrest the microbicidal

activity of most actives,the test protocol permits the addition

of a specific neutralizer to the eluent/diluent,if required.

Except for viruses,the membrane filtration step also allows

processing of the entire eluate from the test carriers and,

therefore,the capture and subsequent detection of even low

numbers of viable organisms that may be present.Subsequent

rinsing of the membrane filters with saline also reduces the risk

of carrying any inhibitory residues over to the recovery

medium.Validation of the process of neutralization of the test

substance is required by challenge with low numbers of the test

organism.

5.6 In tests against viruses,addition of 1 mL of buffer at the

end of the contact time achieves a 1:20 dilution of the test

substance while keeping the volume of the eluate reasonably

small to allow for the titration of most or all ofthe eluate in cell

cultures.Confirmation of neutralization of the test substance is

required by challenge of a residual disinfection load with low

numbers of infective units of the test virus.Since the virus

assay system is indirect,an additional step is required to

demonstrate that prior exposure of the appropriate cell line to

any residual disinfectant or disinfectant/neutralizer mixture

does not interfere with the detection of a low level of virus

challenge (See Appendix).

NoTE 1—In 5.5 and 5.6,volumes of 10mLand 1mLare recommended

instead of 9.95 mL and 950 μL,respectively,for ease of dispensing the

eluent.

5.7 The soil load in this test is a mixture of three types of

proteins (high molecular weight proteins,low molecular

weight peptides,and mucous material)designed to represent

body secretions,excretions,or other extraneous substances that

microbicidal chemicals may encounter under field conditions.

It is suitable for working with all types of test organisms

included here.The components of the soil load are readily

available and subject to much less variability than animal sera.

5.8 If distilled water or other diluent is not to be specified on

the product label,the diluent for the test substance is assumed

to be tap water.Since the quality of tap water varies consid-

erably both geographically and temporally,this test method

incorporates the use of water with a specified and documented

level of hardness to prepare use-dilutions oftest substance that

require dilution in water before use.While water with a

hardness of at least 300 ppm as CaCO₃is recommended

consult local regulations regarding use of hard water prior to

testing.

5.9 The Annex contains a list of those organisms that are

often used in assessing the microbicidal activities of disinfec-

tants for use on environmental surfaces or medical devices.

Culture conditions for each organism are also included in the

Annex.Depending on the label claim(s)desired and the

requirements of the target regulatory agency,one or more of

the organisms listed may be selected for the testing.If

organisms other than those listed are to be used (for example,

in the dairy or brewing industries),a clear justification must be

provided and details of the culture media and growth condi-

tions must be validated and clearly specified in test reports.

6.General Equipment and Labware

6.1 Air Displacement Pipettes,Eppendorf or equivalent,

100 to 1000 μL with disposable tips.

6.2 Analytical Balance, to weigh chemicals and to standard-

ize inoculum delivery volumes by pipettes.

6.3 Cell Culture Flasks and Other Plastic-warefor Viruses,

(see Note 2) plastic cell culture flasks of 25-and 75-cm²

capacity for culturing cells and for preparing virus pools;

12-well or 96-well plastic plates for titrating virus infectivity.

NoTE 2—Plastic culture ware may be purchased from most laboratory

supply houses.

6.4 Centrifuge,to allow for the sedimentation of the cells/

spores of the test organism(s)for concentration,or washing,or

both.

6.5 Colony Counter,for example,Quebec Colony Counter.

6.6 Desiccator, recommended size is 25 cm wide by 20 cm

deep,with an active desiccant for drying the inocula on the

carriers.

6.7 Dissecting Microscope,for the screening of the metal

disks for damage to surface topography.

6.8 Environmental Chamber or Incubator; to hold the car-

riers at the desired test temperature.

6.9 Filter Sterilization System for Media and Reagents,a

membrane or cartridge filtration system (0.22-μm pore diam-

eter)is required for sterilizing heat-sensitive solutions.

6.10 Forceps, straight or curved,(1)with smooth tips to

handle membrane filters,and (2)to pick up the metal disk

carriers for placement in plastic vials.

6.11 Freezers,a freezer at -20±2℃ is required for the

storage of media and additives.A second freezer at-70℃ or

lower is required to store the stocks of test organisms.

6.12 Glassware,1-L flasks with a side-arm and appropriate

tubing to capture the filtrates from 47-mm diameter membrane

filters;250-mL Erlenmeyer flasks for culture media.

6.13 Hemocytometer, for counting fungal conidia,and/or for

use in the preparation of suitable cell numbers for seeding

monolayers.

6.14 Hot Air Oven,an oven at 60℃ to dry clean and sterile

glassware.

6.15 Incubators,an ordinary incubator,an anaerobic

incubator,and a CO₂incubator to incubate cell cultures in a

5%CO₂atmosphere.If only one ordinary incubator is

available,its temperature will require adjustment depending on

the type of organism under test.

6.16 Inverted Microscope,an inverted microscope with 10×

eyepiece and 5×,10x,and 40×objectives to examine cell

cultures.

6.17 Laminar Flow Cabinet,a Class Ⅱ(Type A)biological

safety cabinet.The procedures for the proper maintenance and

use of such cabinets are given in Ref (8).

6.18 Liquid Nitrogen Storage for Cells, a proper liquid

nitrogen container and liquid nitrogen supply for cryopreser-

vation of the stocks of cell lines.

6.19 Magnetic Stir Plate and Stir Bars,large enough for a

5-L beaker or Erlenmeyer flask for preparing culture media or

ASTM E2197-17 Standard Quantitative Disk Carrier Test Method for Determining Bactericidal,Virucidal,Fungicidal

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