ASTM E2720-10

Standard Test Method for Evaluation of Effectiveness of Decontamination Procedures for Air-Permeable Materials when Challenged with Biological Aerosols Containing Human Pathogenic Viruses

SIGNIFICANCE AND USE
The efficacy of disinfection technologies can be evaluated on finished products, as well as on developmental items.
This test method defines procedures for validation of the aerosol generator, preparation of the test specimen, application of the challenge virus, enumeration of viable viruses, assessing data quality, and calculation of decontamination efficacy.
This test method provides defined procedures for creating droplet nuclei that approximate those produced by human respiratory secretions with particular emphasis on particle size distribution and aerosolization media.
Safety concerns associated with aerosolizing microbial agents are not addressed as part of this test method. Individual users should consult with their local safety authority, and a detailed biological aerosol safety plan and risk assessment should be conducted prior to using this method. Users are encouraged to consult the manual Biosafety in Microbiological and Biomedical Laboratories published by the U.S. Centers for Disease Control and Prevention (CDC).  
This test method differs from Test Methods E1052 and E2197 in the presentation of the virus to surface. The aforementioned test methods use liquid inoculum to contaminate carrier surfaces, whereas this test method presents the virus in the absence of water as droplet nuclei.
This test method differs from Test Method  because (1) smaller particles are being formed, (2) the droplets will be dried, thus forming droplet nuclei, prior to application to air-permeable materials, and (3) unique equipment is required to create the droplet nuclei.
SCOPE
1.1 This test method is designed to evaluate decontamination methods (physical, chemical, self-decontaminating materials) when used on air-permeable materials contaminated with virus-containing droplet nuclei.
1.2 This test method defines the conditions for simulating respiratory droplet nuclei produced by humans.
1.3 The method is specific to influenza viruses but could be adapted for work with other types of respiratory viruses or surrogates (Appendix X6).
1.4 This test method is suitable only for air-permeable materials.
1.5 This test method does not address the performance of decontaminants against microbes expelled via blood splatter, vomit, or fecal contamination.
1.6 This test method should be performed only by those trained in bioaerosols, microbiology, or virology, or combinations thereof.
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 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: Historical
• Publication Date: 30-Sep-2010
• ICS: 11.080.99 - Other standards related to sterilization and disinfection
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Referred By: ASTM E2721-16 - Standard Practice for Evaluation of Effectiveness of Decontamination Procedures for Surfaces When Challenged with Droplets Containing Human Pathogenic Viruses (Withdrawn 2024)
• Effective Date: 01-Oct-2010
• Referred By: ASTM E3179-18 - Standard Test Method for Determining Antimicrobial Efficacy of Ultraviolet Germicidal Irradiation against Influenza Virus on Fabric Carriers with Simulated Soil
• Effective Date: 01-Oct-2010

Standards Content (Sample)

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: E2720 − 10
StandardTest Method for
Evaluation of Effectiveness of Decontamination Procedures
for Air-Permeable Materials when Challenged with Biological
Aerosols Containing Human Pathogenic Viruses
This standard is issued under the fixed designation E2720; 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.
INTRODUCTION
Many communicable diseases are often spread through the aerosol route of exposure. The droplet
nuclei formed in these aerosols may infect susceptible individuals directly or contaminate environ-
mental surfaces and render them fomites for further spread of disease. The characteristics of the
droplet nuclei (particle size and composition) will influence the viability of microorganisms when
exposed to environmental stresses but may also shield them from physical and chemical decontami-
nants. The wide variations in the types and levels of such protective/shielding ingredients can have
impact on the effectiveness of surface decontaminants. This test method is designed to simulate the
deposition of droplet nuclei from human respiratory secretions onto and into air-permeable
membranes. It is primarily focused on influenza viruses but other respiratory viruses or surrogate
viruses could be used. Protocols for assessing the microbicidal activity of disinfectants are also
described.
1. Scope 1.7 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
1.1 This test method is designed to evaluate decontamina-
standard.
tion methods (physical, chemical, self-decontaminating mate-
rials)whenusedonair-permeablematerialscontaminatedwith 1.8 This standard does not purport to address all of the
virus-containing droplet nuclei.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
1.2 This test method defines the conditions for simulating
priate safety and health practices and determine the applica-
respiratory droplet nuclei produced by humans.
bility of regulatory limitations prior to use.
1.3 The method is specific to influenza viruses but could be
adapted for work with other types of respiratory viruses or
2. Referenced Documents
surrogates (Appendix X6).
2.1 ASTM Standards:
1.4 This test method is suitable only for air-permeable
E1052Test Method to Assess the Activity of Microbicides
materials.
against Viruses in Suspension
1.5 This test method does not address the performance of
E2197Quantitative Disk Carrier Test Method for Determin-
decontaminants against microbes expelled via blood splatter,
ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
vomit, or fecal contamination.
and Sporicidal Activities of Chemicals
E2721Test Method for Evaluation of Effectiveness of De-
1.6 This test method should be performed only by those
contamination Procedures for Surfaces When Challenged
trained in bioaerosols, microbiology, or virology, or combina-
with Droplets Containing Human Pathogenic Viruses
tions thereof.
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct For referenced ASTM standards, visit the ASTM website, www.astm.org, or
responsibility of Subcommittee E35.15 on Antimicrobial Agents. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Current edition approved Oct. 1, 2010. Published February 2011. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
E2720–10. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2720 − 10
2.2 IEST Standards: 4.1.1 Using an aerosol device capable of meeting the data
IEST-RP-CC003.3 Garment System Considerations for quality objectives set for in this test method, influenza virus or
Clean Rooms and Other Controlled Environments surrogates are aerosolized to form droplet nuclei that are
subsequently applied to air-permeable materials.
2.3 Department of Defense Standards:
CA06PRO411Method for EvaluatingAir Purification Tech-
4.1.2 The virus-contaminated carriers are subjected to dis-
nologies for Collective Protections Using Viable Micro-
infection protocols and incubated for the specified time and
bial Aerosols
conditions. Control samples are incubated under identical
2.4 EPA Standards:
conditions but are not exposed to the disinfection protocols.
EPA600⁄4-84⁄013(N16) USEPA Manual of Methods for
5 NOTE 1—Carriers with incorporated microbicides do not receive any
Virology
additional disinfection treatment.An untreated control is needed to assess
2.5 WHO Standards:
antimicrobial efficacy.
WHO Manual on Animal Influenza Diagnosis and Surveil-
6 4.1.3 Virus particles are eluted from the test and control
lance
carriers and viability is assessed by tissue culture 50%
infectious dose assay (log TCID ).
3. Terminology 10 50
3.1 Definitions:
NOTE 2—Nonviable quantification techniques for viral enumeration
such as polymerase chain reaction (PCR) or hemagglutination cannot be
3.1.1 aerosol, n—a suspension of solid or liquid particles in
used.
a gas medium.
4.1.4 The virucidal activity of the decontamination proce-
3.1.2 air-permeable material, n—no standard definition is
dure is determined from the log difference in viability between
available; for the purpose of this test method, air-permeable
treated and control carriers.
materialisdescribedasanymembranethathasapressuredrop
≤ twice that of high efficiency particulate air (HEPA) media in
5. Significance and Use
the same test environment.
3.1.3 biological aerosol, n—aerosol comprising particles of
5.1 The efficacy of disinfection technologies can be evalu-
biological origin or activity which may affect living things
ated on finished products, as well as on developmental items.
through infectivity, allergencity, toxicity, or pharmacological
5.2 Thistestmethoddefinesproceduresforvalidationofthe
and other processes.
aerosol generator, preparation of the test specimen, application
3.1.4 influenza, n—an infectious disease of birds and mam-
ofthechallengevirus,enumerationofviableviruses,assessing
mals caused by RNAviruses of the family Orthomyxoviridae.
data quality, and calculation of decontamination efficacy.
3.1.5 protective factor, n—soluble or insoluble material
5.3 This test method provides defined procedures for creat-
co-deposited with microorganisms that directly protects the
ing droplet nuclei that approximate those produced by human
microorganism from environmental stresses or decontami-
respiratory secretions with particular emphasis on particle size
nants.
distribution and aerosolization media.
3.1.6 respiratory droplet nuclei, n—evaporatively
5.4 Safety concerns associated with aerosolizing microbial
condensed, pathogen-containing particles of respiratory secre-
tions expelled into the air by coughing, sneezing, or talking, agents are not addressed as part of this test method. Individual
users should consult with their local safety authority, and a
which can remain airborne for long periods of time.
detailed biological aerosol safety plan and risk assessment
3.1.7 self-sanitizing material, n—a substrate containing an
should be conducted prior to using this method. Users are
antimicrobial agent that collectively acts as a germicide.
encouragedtoconsultthemanual Biosafety in Microbiological
and Biomedical Laboratories published by the U.S. Centers
4. Summary of Test Method
for Disease Control and Prevention (CDC).
4.1 The test method describes the steps required to deposit
droplet nuclei onto air-permeable membranes and quantita-
5.5 This test method differs from Test Methods E1052 and
tively assess decontamination efficiency.
E2197 in the presentation of the virus to surface. The afore-
mentioned test methods use liquid inoculum to contaminate
carrier surfaces, whereas this test method presents the virus in
Available from Institute of Environmental Sciences and Technology (IEST),
the absence of water as droplet nuclei.
Arlington Place One, 2340 S.Arlington Heights Rd., Suite 100,Arlington Heights,
IL 60005-4516, http://www.iest.org.
5.6 This test method differs from Test Method E2721
Foarde, K., Heimbuch, B. K., Maxwell, A., VanOsdell, D., “Method for
because (1) smaller particles are being formed, (2) the droplets
Evaluating Air Purification Technologies for Collective Protection Using Viable
Microbial Aerosols,” Test Operating Procedure (TOP) Under the Army Test and
will be dried, thus forming droplet nuclei, prior to application
Evaluation Command (ATEC), Edgewood Chemical and Biological Center,
to air-permeable materials, and (3) unique equipment is re-
Edgewood, Md., 2010 in press.
5 quired to create the droplet nuclei.
Available from United States Environmental Protection Agency (EPA), Ariel
Rios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http://
www.epa.gov.
Webster, R., Cox, N., Stohr, K. WHO Manual on Animal Influenza Diagnosis
and Surveillance. World Health Organization, Department of Communicable Dis- CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories, 5th
ease Surveillance and Response. WHO/CDS/CDR/2002.5 Rev. 1. Edition, U.S. Department of Health and Human Services, Washington, D.C., 2009.
E2720 − 10
6. Apparatus formulation and recipe listing the protective factors and par-
ticle size must be reported.
6.1 Biological Aerosol Generators—The apparatus to load
microorganisms onto a substrate is composed of several 6.2 Other Equipment—The list is specific for influenza
commercially available components and can be readily con- virus. Other equipment may be needed if a different virus is
4,8,9
structed (see IEST-RP-CC003.3). The overall design of the used.
apparatus can take various forms and can be fashioned in 6.2.1 Autoclave, capable of maintaining 121 to 123°C and
differentdimensionswhilemeetingthevalidationrequirements [15 to 17 lbs per in. –gauge (psig)].
and data quality objectives listed below. Appendix X1 and 6.2.2 CO Incubator,capableofmaintaining35to37°Cand
Appendix X2 contain the description of a prototypical device 5 6 0.5% CO .
thatcanbeusedtoloaddropletnucleiontosurfaces.However, 6.2.3 Vortex Mixer.
itistheresponsibilityoftheuserofthisstandardtovalidatethe 6.2.4 Analytical Balance, capable of weighing 0.001 g.
performance of the device prior to use. 6.2.5 Refrigerator, capable of maintaining 2 to 8°C.
6.1.1 Validation requirements and baseline testing. 6.2.6 Stopwatch or Electronic Timer.
6.1.1.1 Environmental Conditions—Generator must be ca- 6.2.7 Pipettor, with a precision of 0.001 mL.
pable of delivering air with a relative humidity of 70 6 10%.
7. Reagents and Materials
6.1.1.2 Leak Test—The device must maintain a positive
pressure of ~ 50 cm of water for at least 10 min.
7.1 Reagents—The list is specific for influenza use. Other
6.1.1.3 Flow Rate Consistency—All ports containing speci-
reagents may be needed if a different virus is used.
menholdersmustmaintainaconstantflowwithacoefficientof
7.1.1 Influenzavirus (H1N1; A/PR/8/34)—cell culture
variation (CV) ≤ 10% over the duration of the sampling
adapted, ATCC VR-146.
period.
7.1.1.1 The WHO Manual on Animal Influenza Diagnosis
6.1.1.4 Loading uniformity across the diameter of the test
and Surveillance contains specific procedures for preparing
specimen is required to ensure the even distribution of the
influenza virus and titering samples. Appendix X3 also has
droplet nuclei over the surface of the carrier. A standard
specificinformationontitratingviableinfluenzaviruses.Other
deviation of 60.5 log TCID is desired.
viruses may be used, but conditions for propagation and
10 50
6.1.1.5 Sample-to-Sample Variation—The variability of vi-
enumeration are not provided in this method.
rus loading for multiple samples loaded for a single test must
7.1.2 Madin–Darby Canine Kidney (MDCK) Cell Line,
have a standard deviation of 60.5 log TCID .
10 50 ATCC CRL-34.
6.1.1.6 Droplet Nuclei Characteristics—The droplet nuclei
7.1.3 Artificial Saliva, see Table 1.
generated for this method will have a count median diameter
7.1.4 Minimal Essential Medium With Earle’s Balanced
(CMD) of ~0.8 µm. The virus will be aerosolized in a saliva
Salts (EMEM).
substitute (Table 1) that will add the appropriate “protective
7.1.5 Heat-Inactivated Fetal Bovine Serum (45 min at
56°C).
7.1.6 Penicillin/Streptomycin,10000unitspenicillinand10
TABLE 1 Artificial Saliva
mg streptomycin per mL.
Reagent Amount
7.1.7 L-Glutamine, 200 mM in 0.85% NaCl.
MgCl ·7HO0.04g
2 2
CaCl ·HO0.13g
7.1.8 Crystal Violet.
2 2
NaHCO 0.42 g
7.1.9 Glutaraldehyde.
0.2MKH PO 7.70 mL
2 4
7.1.10 TPCK–Trypsin.
0.2MK HPO 12.3 mL
2 4
NH Cl 0.11 g
4 7.1.11 Phosphate Buffered Saline (PBS).
KSCN 0.19 g
7.1.12 Bovine Serum Albumin.
(NH ) CO 0.12 g
2 2
7.1.13 Trypsin–EDTA Solution, 0.05% trypsin, 0.53 mM
NaCl 0.88 g
KCl 1.04 g
EDTA in Hanks balanced salts solution without sodium
Mucin 3.00 g
bicarbonate, calcium, and magnesium.
Distilled water 1000 mL
7.1.14 Distilled Water and Purified Water.
pH7
7.1.15 Ethanol, laboratory grade.
7.1.16 Bleach.
7.2 Materials—The list is specific for influenza use. Other
factors.” This test method would be suitable for simulating
reagents may be needed if a different virus is used.
other fluids of interest; however, if a different fluid is used, the
7.2.1 Tissue Culture Treated Flasks—T-25, T-75, T-175,
24-well plate.
Heimbuch B. K., Wallace, W. H., Kinney, K., Lumley, A. E., Wu, C-Y, Woo,
7.2.2 Pipettes, 1, 5, 10, and 25 mL.
M-H, Wander, J. D., “APandemic Influenza Preparedness Study: Use of Energetic
7.2.3 Test Tube Rack.
Methods to Decontaminate Filtering Facepiece Respirators Contaminated with
7.2.4 Micropipettes, capable of delivering 0.001 mL accu-
H1N1 Aerosols and Droplets,” American Journal of Infection Control, 2010, DOI
10.1016/j.ajic.2010.07.004.
rately and consistently.
Fisher E, Rengasamy S, Viscusi DJ, Vo E, Shaffer R., Development of a test
7.2.5 1.7-mL Sterile Microcentrifuge Tubes.
system to apply virus-containing particles to filtering facepiece respirators for the
7.2.6 15-mL Sterile Centrifuge Tubes.
evaluation of decontamination procedures, Appl Environ Microbiol, Vol 75, No. 6,
2009, pp. 1500–1507. 7.2.7 50-mL Sterile Centrifuge Tubes.
E2720 − 10
7.2.8 Air-Permeable Test Materials. 10.4 Decontamination—Remove samples from the aerosol
generatorandexposeasubset(atleastthree)tothedecontami-
nation
...