Summary of the ASTM E1053 Method
- The stock virus to be used is thawed and diluted to an appropriate concentration.
- If requested by the Study Sponsor, the test virus is loaded with organic soil to the appropriate level. This is typically done when the test product is intended to be marketed as a one-step disinfectant.
- The EPA requires an organic soil load of 5% for a one-step disinfectant claim.
- The test product is prepared to the use-dilution specified by the Study Sponsor, if applicable. An equal volume of buffered saline or cell culture media is prepared to serve as a virus recovery control.
- The prepared viral inoculum is spread over the entire surface of a glass Petri dish carrier and allowed to dry.
- At the conclusion of the dry time the test product is applied to the viral film.
- Typically 2.0 ml of a use-dilution test product will be added. If a spray product is used it will be applied as 4 sprays at a distance of 6 to 8 inches, or according to Sponsor directions as appropriate.
- Upon closure of the study contact time, the test and recovery suspensions are harvested by use of a cell scraper and neutralized by a method most suitable for the active ingredient(s) present in the test substance.
- Common methods of neutralization include dilution into chemical neutralizers, filtration through a physical gel matrix (e.g. Sephacryl gel columns), or a combination thereof.
- An aliquot of the use-dilution of the test substance is neutralized in an identical manner to the test suspension. This is used to generate the cytotoxicity control to determine the lowest dilution at which, if any, the test substance causes damage to the host cells.
- An aliquot is removed from the cytotoxicity control and inoculated with a low-concentration viral suspension. This is used to generate the neutralization control to confirm the efficacy of the selected neutralization method.
- The neutralized test, recovery, cytotoxicity control, and neutralization control suspensions are serially diluted in the appropriate media. Each dilution is plated in quadruplicate onto host cell monolayers. Media is added to each monolayer and the host cell-virus system is allowed to incubate for the appropriate time. Most assays incubate for a 7 to 10 day period.
- At the close of the incubation time, the assay is scored using standard cell culture methods.
- Each well in the tray is examined under a microscope for the presence of cytopathic effects (CPE) of infection, such as cell rounding, sloughing, and monolayer degradation. Cytotoxicity control wells are examined for damage caused by the test product. Confirmatory assays are used as necessary.
- The Spearman-Karber method, or another appropriate statistical method, is used to quantify the amount of infectious virus present in the assay.
Virucidal Efficacy Success Criteria
- At least 4-log10 infectious units per carrier must be recovered from the virus recovery control.
- The test product demonstrates complete inactivation of the test virus at all dilutions of the assay, or,
- if the neutralized test product causes cytotoxicity in the assay, a 3-log10 reduction past the level of cytotoxicity observed.
- Neutralization controls indicate that the selected neutralization method was successful - typically through demonstration of similar levels of cytopathic effects relative to a control.
Strengths of the ASTM E1053 Method
- Data generated will conform to U.S. EPA guidelines for efficacy data required for disinfectant claims.
- Dried viral films present a significant challenge, providing "worst case" scenario data.
- The test method may be modified to accomodate different types of test product application (spray, wipes, use-dilution) and different use conditions (soiled surfaces, varied temperatures and humidities).
Weaknesses of the ASTM E1053 Method
- Data generated for formulations against dried virus films on hard, nonporous surfaces may not translate to efficacy against viruses in suspension or other applications.
- Limits of detection can be impacted by cytotoxicity and recovery media volumes, requiring the use of a higher viral titer which places an additional level of challenge on the test products.
- Data generated for submission to the U.S. EPA may not be sufficient for regulatory agencies in other countries.
ASTM E1053 Virus Suspension Time-Kill Testing according to U.S. EPA and FDA criteria can be performed at Microchem Laboratory as a simple screen (non-GLP) and under more comprehensive GLP study conditions.