ASTM E2897-22

Standard Guide for Evaluation of the Effectiveness of Hand Hygiene Topical Antimicrobial Products using <emph type="bdit">ex vivo</emph> Porcine Skin

SIGNIFICANCE AND USE
5.1 The guide may be used to demonstrate the effectiveness of topical antimicrobial products using pigskin as a surrogate for human skin and the cup scrub technique for sampling.  
5.2 The techniques described can be used to simulate Test Method E1174 and will use the pigskin substrate to overcome limitations posed by exposure of human subjects to potentially pathogenic microorganisms, while offering the benefit of applicability to a wide variety of hand-washing conditions that cannot be simulated in test tubes.  
5.3 Use of the pigskin surrogate offers less expensive and higher throughput screening.
SCOPE
1.1 This guide is designed to demonstrate the effectiveness of hand hygiene topical antimicrobial products using pigskin as a surrogate model.  
1.2 Knowledge of microbiological techniques is required for these procedures.  
1.3 This standard guide can be used to evaluate topical antimicrobial handwash or handrub formulations.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 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.

General Information

• Status: Published
• Publication Date: 31-Mar-2022
• ICS: 07.100.99 - Other standards related to microbiology
• 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

• Refers: ASTM E1874-09 - Standard Test Method for Recovery of Microorganisms from Skin using the Cup Scrub Technique
• Effective Date: 01-Oct-2009
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008
• Refers: ASTM E1174-06 - Standard Test Method for Evaluation of the Effectiveness of Health Care Personnel Handwash Formulations
• Effective Date: 01-Mar-2006
• Refers: ASTM E1054-02 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 10-May-2002
• Refers: ASTM E1174-00 - Standard Test Method for Evaluation of the Effectiveness of Health Care Personnel or Consumer Handwash Formulations
• Effective Date: 10-Aug-2000
• Refers: ASTM E1874-97 - Standard Test Method for Evaluation of Antibacterial Washes by Cup Scrub Technique (Withdrawn 2006)
• Effective Date: 01-Jan-1997

Overview

ASTM E2897-22 is a standard guide developed by ASTM International for evaluating the effectiveness of hand hygiene topical antimicrobial products using ex vivo porcine (pig) skin. This guide provides a reliable, practical, and safe alternative to human skin testing, leveraging pigskin as a surrogate model to assess antimicrobial efficacy. The standard details procedures using the cup scrub technique for sampling and is especially valuable for evaluating both handwash and handrub formulations. This approach addresses ethical and safety concerns by avoiding the exposure of human subjects to potentially pathogenic microorganisms and offers cost-effective, higher-throughput screening options for manufacturers and quality control laboratories.

Key Topics

• Pigskin as a Human Skin Surrogate: Pigskin closely mimics the structural properties of human skin, making it an effective substrate for testing without the risks associated with human research subjects.
• Cup Scrub Technique: A standardized sampling method that allows for quantitative recovery of microorganisms from skin surfaces, ensuring accurate measurement of antimicrobial performance.
• Antimicrobial Efficacy Evaluation: The primary focus is to determine the reduction of test organisms on pigskin after treatment with various hand hygiene products.
• Safety and Regulatory Considerations: Emphasizes the importance of using proper microbiological techniques and establishing safety, health, and environmental practices before testing.
• Applicability Across Formulations: Suitable for both leave-on and rinse-off (wash-off) antiseptic hand products, expanding the scope of test conditions and simulation of real-world scenarios.

Applications

The ASTM E2897-22 standard is widely used in the following contexts:

• Product Development and Screening: Allows manufacturers to efficiently screen and compare the antimicrobial efficacy of hand hygiene formulations early in development, reducing both cost and resource requirements.
• Regulatory Submissions: Provides scientifically validated data to support the effectiveness claims required for regulatory approval of hand hygiene products.
• Quality Assurance: Used by quality control laboratories to verify batch-to-batch consistency and ongoing product performance.
• Research and Innovation: Facilitates the study of novel antimicrobial agents, alternative hand hygiene formulations, and the impact of various washing conditions within a controlled and reproducible framework.
• Healthcare and Public Health: Ensures that products intended for healthcare personnel and the general public meet efficacy standards against a range of microorganisms.

Related Standards

Several ASTM standards complement and inform practices described in ASTM E2897-22:

• ASTM E1054 – Practices for Evaluation of Inactivators of Antimicrobial Agents: Outlines procedures for validating agents used to neutralize residual antimicrobials during efficacy testing.
• ASTM E1174 – Test Method for Evaluation of the Effectiveness of Healthcare Personnel Handwash Formulations: Serves as a reference for simulating handwashing conditions and performance metrics.
• ASTM E1874 – Test Method for Recovery of Microorganisms From Skin using the Cup Scrub Technique: Provides detailed guidance on the method used for sampling microorganisms from skin substrates.

Practical Value

Adopting ASTM E2897-22 enables:

• Safer Testing: Avoids the need for potentially hazardous tests on human volunteers.
• Cost Efficiency: Offers a less expensive method for large-scale or high-throughput product screening.
• Versatility: Applicable to a wide range of product types and test organisms, simulating clinical and consumer hand hygiene scenarios.
• Quality and Consistency: Ensures antimicrobial hand hygiene products are effective, contributing to infection prevention in healthcare and community settings.

Keywords: hand hygiene, antimicrobial efficacy, pigskin model, cup scrub technique, handwash formulations, handrub testing, ASTM standards, surrogate model, quality assurance in antimicrobials

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: E2897 − 22
Standard Guide for
Evaluation of the Effectiveness of Hand Hygiene Topical
Antimicrobial Products using ex vivo Porcine Skin
This standard is issued under the fixed designation E2897; 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
Use of ex vivo animal skin models such as pigskin has widely been used as surrogate for human
skin. Pigskin model is a safe, inexpensive, accurate, and reliable platform of testing antiseptic
2,3,4
efficacy. The test guide described here utilizes sterilized pigskin to evaluate the effectiveness of
hand hygiene topical antimicrobial products. The pigskin substrate is used to overcome limitations
posed by exposure of human subjects to potentially pathogenic microorganisms, while offering the
benefit of applicability to a wide variety of hand-washing conditions that cannot be simulated in test
tubes.The microbial reduction is the difference in log value obtained from artificially contaminated
pigskins after use of test formulation to the log value obtained from contaminated pigskins not
exposed to the test formulation.
1. Scope 1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 This guide is designed to demonstrate the effectiveness
ization established in the Decision on Principles for the
ofhandhygienetopicalantimicrobialproductsusingpigskinas
Development of International Standards, Guides and Recom-
a surrogate model.
mendations issued by the World Trade Organization Technical
1.2 Knowledge of microbiological techniques is required
Barriers to Trade (TBT) Committee.
for these procedures.
2. Referenced Documents
1.3 This standard guide can be used to evaluate topical
2.1 ASTM Standards:
antimicrobial handwash or handrub formulations.
E1054PracticesforEvaluationofInactivatorsofAntimicro-
1.4 The values stated in SI units are to be regarded as
bial Agents
standard. No other units of measurement are included in this
E1174Test Method for Evaluation of the Effectiveness of
standard.
Healthcare Personnel Handwash Formulations
1.5 This standard does not purport to address all of the
E1874Test Method for Recovery of Microorganisms From
safety concerns, if any, associated with its use. It is the
Skin using the Cup Scrub Technique
responsibility of the user of this standard to establish appro-
3. Terminology
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
3.1 Definitions:
3.1.1 antimicrobial ingredient, n—a substance added to a
formulation specifically for the inhibition or inactivation of
This guide is under the jurisdiction of ASTM Committee E35 on Pesticides,
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
microorganisms.
Subcommittee E35.15 on Antimicrobial Agents.
3.1.2 neutralization, n—the process for inactivating or
Current edition approved April 1, 2022. Published May 2022. Originally
approvedin2012.Lastpreviouseditionapprovedin2017asE2897–12(2017).DOI:
quenchingtheactivityofamicrobicide,oftenachievedthrough
10.1520/E2897-22.
physical(forexample,filtrationordilution)orchemicalmeans.
Woolwine, J. D., and Gerberding, J. L., “Effect ofTesting Method onApparent
Activities of Antiviral Disinfectants and Antiseptics,” Antimicrobial Agents and 3.1.3 resident microorganisms, n—microorganismsthatsur-
Chemotherapy, Vol 39, 1999, pp. 921–923.
vive and multiply on the skin, forming a stable population.
Bush, L. W., Benson, L. M., and White, J. H., “Pigskin as a Test Substrate for
EvaluatingTopicalAntimicrobialActivity,” J. Clinical Microbiology,Vol 24, 1986,
pp. 343–348. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
McDonnel, G., Haines, K., Klein, D., Rippon, M., Walmsley, R., and Pretzer, contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
D., “Clinical Correlation of a SkinAntisepsis Model,” J. Microbiological Methods, Standards volume information, refer to the standard’s Document Summary page on
Vol 35, 1999, pp. 31–35. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2897 − 22
3.1.4 transient microorganisms, n—microorganisms that 6.7 Water Temperature Regulator and Temperature
contaminate the skin but do not form a stable population. Monitor—To set and maintain the water temperature at 40°C
6 2°C.
3.1.5 test organism, n—an applied inoculum of an organism
that has characteristics which allow it to be readily identified.
6.8 Vortex Mixer—Any suitable vortex mixer capable of
The test organism is used to simulate a transient topical mixing sample and diluent.
microbial contaminant. It may also be referred to as a marker
6.9 Spectrophotometer—An instrument that can measure
organism, bacterial simulant, or bacterial contaminant.
optical density at a wavelength of 620 nm.
3.1.6 test substance, n—a leave-on or wash-off product or
formulation which incorporates antimicrobial ingredient(s).
7. Reagents and Materials
7.1 Sterile Bacteriological Pipettes—10mL capacity; 1mL
4. Summary of Guide
capacity and 0.1mL capacity.
4.1 This guide describes procedures for testing using non-
7.2 Inoculating Loops or Sterile Swabs.
living pigskin substrates that have been shaved to remove the
7.3 Gamma Sterilized Pigskins—Pig hides can be obtained
hair and sterilized using gamma radiation prior to use in
from local source, defatted, washed with water and sterilized
testing. The specific procedures allow use of a test microor-
by gamma-irradiation (or other acceptable method) and stored
ganismoftheinvestigator’schoice.Activityofatestsubstance
in a freezer (–20°C) prior to use.
is measured by comparing the number of test microorganisms
recovered from artificially contaminated pigskins after use of 7.4 Industrial Grade Adhesive—Anyofseveraltypescanbe
anantimicrobialformulationtothenumberoftestmicroorgan-
used; for example, epoxy or other suitable glue.
isms recovered from contaminated pigskins not exposed to the
7.5 Suitable Carrier—Forholdingpigskininplaceallowing
test formulation. The antimicrobial activity of the test sub-
mechanical manipulation (washing, rubbing, and so forth); for
stance can be measured following a single wash or multiple
example, phenolic caps/closures.
washes, or both, in a single day. Sampling is performed using
7.6 Scalpel—Or any other appropriate cutting tool.
the cup scrub technique and a fluid shown to neutralize
effectively the antimicrobial activity of the test formulation,
7.7 Tissues or Paper Towels—Any sterile tissue or paper
and to be non-toxic to the test microorganism. towel that can be used to dry the pigskins.
7.8 Sterile Container—Any sterile or sterilizable container
5. Significance and Use
havingthecapacitytoculturethevolumeofinoculumrequired
5.1 The guide may be used to demonstrate the effectiveness for testing.
of topical antimicrobial products using pigskin as a surrogate
7.9 Scrub Cups—Sterileplastic/polypropyleneorothersuit-
for human skin and the cup scrub technique for sampling.
able cylinders, height approximately 2.5 cm, inside diameter
approximately 4.2 cm. Useful sizes range from approximately
5.2 The techniques described can be used to simulate Test
1.5cm to 4.0 cm.
Method E1174 and will use the pigskin substrate to overcome
limitations posed by exposure of human subjects to potentially
7.10 Sterile Polytetrafluoroethylene (PTFE) Scraper—Can
pathogenic microorganisms, while offering the benefit of
be fashioned in the laboratory or purchased.
applicability to a wide variety of hand-washing conditions that
7.11 Sterile Culture Tubes, or equivalent.
cannot be simulated in test tubes.
7.12 Appropriate Bacterial Cultures.
5.3 Use of the pigskin surrogate offers less expensive and
7.13 Test Formulation/Substance—Manufacturer directions
higher throughput screening.
for use of the test substance should be utilized and, prior to
testing,theappropriatevolumetobeappliedtothesurfacearea
6. Apparatus
of the pigskin substrate should be calculated as a ratio of the
6.1 Colony Counter—Anyofseveraltypesmaybeused,for
estimated volume that would exist on the hands of a human
example, Quebec Colony Counter or similar devices.
subject. If directions are not available, the investigator must
6.2 Incubator—Any incubator capable of maintaining de- determine an appropriately realistic volume.
sired temperature range.
7.14 Sampling and Dilution Fluid—Sterile Butterfield’s
6.3 Sterilizer—Any suitable steam sterilizer capable of pro- phosphate buffered diluent, containing an antimicrobial inac-
tivator specific for the test formulation.
ducing the conditions of steri
...

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: E2897 − 12 (Reapproved 2017) E2897 − 22
Standard Guide for
Evaluation of the Effectiveness of Hand Hygiene Topical
Antimicrobial Products using ex vivo Porcine Skin
This standard is issued under the fixed designation E2897; 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.
INTRODUCTION
Use of ex vivo animal skin models such as pigskin has widely been used as surrogate for human
skin. Pigskin model is a safe, inexpensive, accurate, and reliable platform of testing antiseptic
2,3,4
efficacy. The test guide described here utilizes sterilized pigskin to evaluate the effectiveness of
hand hygiene topical antimicrobial products. The pigskin substrate is used to overcome limitations
posed by exposure of human subjects to potentially pathogenic microorganisms, while offering the
benefit of applicability to a wide variety of hand-washing conditions that cannot be simulated in test
tubes. The microbial reduction is the difference in log value obtained from artificially contaminated
pigskins after use of test formulation to the log value obtained from contaminated pigskins not
exposed to the test formulation.
1. Scope
1.1 This guide is designed to demonstrate the effectiveness of hand hygiene topical antimicrobial products using pigskin as a
surrogate model.
1.2 Knowledge of microbiological techniques is required for these procedures.
1.3 This standard guide can be used to evaluate topical antimicrobial handwash or handrub formulations.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.6 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.
This guide 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 April 1, 2017April 1, 2022. Published April 2017May 2022. Originally approved in 2012. Last previous edition approved in 20122017 as
E2897E2897–12(2017).–12. DOI: 10.1520/E2897-12R17.10.1520/E2897-22.
Woolwine, J. D., and Gerberding, J. L., “Effect of Testing Method on Apparent Activities of Antiviral Disinfectants and Antiseptics,” Antimicrobial Agents and
Chemotherapy, Vol 39, 1999, pp. 921–923.
Bush, L. W., Benson, L. M., and White, J. H., “Pigskin as a Test Substrate for Evaluating Topical Antimicrobial Activity,” J. Clinical Microbiology, Vol 24, 1986, pp.
343–348.
McDonnel, G., Haines, K., Klein, D., Rippon, M., Walmsley, R., and Pretzer, D., “Clinical Correlation of a Skin Antisepsis Model,” J. Microbiological Methods, Vol
35, 1999, pp. 31–35.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2897 − 22
2. Referenced Documents
2.1 ASTM Standards:
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
E1174 Test Method for Evaluation of the Effectiveness of Healthcare Personnel Handwash Formulations
E1874 Test Method for Recovery of Microorganisms From Skin using the Cup Scrub Technique
3. Terminology
3.1 Definitions:
3.1.1 antimicrobial ingredient, n—a substance added to a formulation specifically for the inhibition or inactivation of
microorganisms.
3.1.2 neutralization, n—the process for inactivating or quenching the activity of a microbicide, often achieved through physical
(for example, filtration or dilution) or chemical means.
3.1.3 resident microorganisms, n—microorganisms that survive and multiply on the skin, forming a stable population.
3.1.4 transient microorganisms, n—microorganisms that contaminate the skin but do not form a stable population.
3.1.5 test organism, n—an applied inoculum of an organism that has characteristics which allow it to be readily identified. The
test organism is used to simulate a transient topical microbial contaminant. It may also be referred to as a marker organism,
bacterial simulant, or bacterial contaminant.
3.1.6 test substance, n—a leave-on or wash-off product or formulation which incorporates antimicrobial ingredient(s).
4. Summary of Guide
4.1 This guide describes procedures for testing using non-living pigskin substrates that have been shaved to remove the hair and
sterilized using gamma radiation prior to use in testing. The specific procedures allow use of a test microorganism of the
investigator’s choice. Activity of a test substance is measured by comparing the number of test microorganisms recovered from
artificially contaminated pigskins after use of an antimicrobial formulation to the number of test microorganisms recovered from
contaminated pigskins not exposed to the test formulation. The antimicrobial activity of the test substance can be measured
following a single wash or multiple washes, or both, in a single day. Sampling is performed using the cup scrub technique and a
fluid shown to neutralize effectively the antimicrobial activity of the test formulation, and to be non-toxic to the test microorganism.
5. Significance and Use
5.1 The guide may be used to demonstrate the effectiveness of topical antimicrobial products using pigskin as a surrogate for
human skin and the cup scrub technique for sampling.
5.2 The techniques described can be used to simulate Test Method E1174 and will use the pigskin substrate to overcome
limitations posed by exposure of human subjects to potentially pathogenic microorganisms, while offering the benefit of
applicability to a wide variety of hand-washing conditions that cannot be simulated in test tubes.
5.3 Use of the pigskin surrogate offers less expensive and higher throughput screening.
6. Apparatus
6.1 Colony Counter—Any of several types may be used, for example, Quebec Colony Counter or similar devices.
6.2 Incubator—Any incubator capable of maintaining desired temperature range.
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.
E2897 − 22
6.3 Sterilizer—Any suitable steam sterilizer capable of producing the conditions of sterilization.
6.4 Timer (Stop-Clock)—Type that can be read for minutes and seconds.
6.5 Sink—A sink of sufficient size to permit the washing of pigskins without touching the sink surface.
6.6 Water Faucet(s)—To be located above the sink at height which permits the rubbing of the pigskins during the washing
procedure. Faucet should maintain a constant flow rate.
6.7 Water Temperature Regulator and Temperature Monitor—To set and maintain the water temperature at 4040 °C 6 2°C.2 °C.
6.8 Vortex Mixer—Any suitable vortex mixer capable of mixing sample and diluent.
6.9 Spectrophotometer—An instrument that can measure optical density at a wavelength of 620 nm.
7. Reagents and Materials
7.1 Sterile Bacteriological Pipettes—10-mL10 mL capacity; 1-mL1 mL capacity and 0.1-mL0.1 mL capacity.
7.2 Inoculating Loops or Sterile Swabs.
7.3 Gamma Sterilized Pigskins—Pig hides can be obtained from local source, defatted, washed with water and sterilized by
gamma-irradiation (or other acceptable method) and stored in a freezer (–20°C)(–20 °C) prior to use.
7.4 Industrial Grade Adhesive—Any of several types can be used; for example, epoxy or other suitable glue.
7.5 Suitable Carrier—For holding pigskin in place allowing mechanical manipulation (washing, rubbing, and so forth); for
example, phenolic caps/closures.
7.6 Scalpel—Or any other appropriate cutting tool.
7.7 Tissues or Paper Towels—Any sterile tissue or paper towel that can be used to dry the pigskins.
7.8 Sterile Container—Any sterile or sterilizable container having the capacity to culture the volume of inoculum required for
testing.
7.9 Scrub Cups—Sterile plastic/polypropylene or other suitable cylinders, height approximately 2.5 cm, inside diameter
approximately 4.2 cm. Useful sizes range from approximately 1.51.5 cm to 4.0 cm.
7.10 Sterile Polytetrafluoroethylene (PTFE) Scraper—Can be fashioned in the laboratory or purchased.
7.11 Sterile Culture Tubes, or equivalent.
7.12 Appropriate Bacterial Cultures.
7.13 Test Formulation/Substance—Manufacturer directions for use of the test substance should be utilized and, prior to testing,
the appropriate volume
...