Guidance for the Inactivation of Select Agents and Toxins and Rendering Samples Free of Select Agents and Toxins

The federal regulations for the possession, use, and transfer of select agents and toxins (i.e., 9 CFR Part 121, 42 CFR Part 73 and 7 CFR Part 331) state that non-viable select agents or nonfunctional toxins are to be excluded from these regulations. The specific regulations excluding non-viable select agents and nonfunctional select toxins are found in 9 CFR §121.3(d)(2), 42 CFR §73.3(d)(2) , and 7 CFR §331.3(d)(2) for agency specific agents; and 9 CFR §121.4(d)(2) and 42 CFR §73.4(d)(2) for overlap agents. Since implementation of the regulations governing select agents and toxins, known as the Select Agent Regulations (SAR), there have been several notable events that have occurred at entities related to select agents and toxins that were presumed to be non-viable. These events required special attention of the Federal Select Agent Program (FSAP). For example, the shipments of presumed non-viable select agent material that subsequently resulted in human and laboratory animal infections; the commingling of presumed inactivated select agent inventory with non-select agent inventory and having insufficient laboratory safeguards; and importing and using biological samples for diagnostic testing from high risk countries in lower containment laboratories with insufficient laboratory safeguards. As a result, the FSAP finds it necessary to provide guidance addressing when to categorize select agents and toxins as non-viable or nonfunctional. This guidance does not apply to products that have been licensed or approved by a federal regulatory agency (i.e., The Federal Food, Drug, and Cosmetic Act (21 U.S.C. § 301 et seq.), Section 351 of the Public Health Service Act pertaining to biological products (42 U.S.C. § 262), or The Virus-Serum-Toxin Act (21 U.S.C. §§ 151-159)) as they would be exempt under other provisions of the SAR as provided for in Sections 5 and 6.

"Non-viable" and "nonfunctional" are similar terms that refer to the permanent loss of biological activity. For a select agent, non-viable means that a select agent is no longer capable of growing, replicating, infecting, or causing disease; for a toxin, nonfunctional means a toxin is no longer capable of exerting its toxic effect. In general, the major categories utilized to inactivate agents, rendering them non-viable or nonfunctional fall into one of two broad categories: physical (e.g., heat or ultraviolet light) or chemical. Different processes (e.g., heat, radiation or chemicals) work by different mechanisms. For example, some of these processes denature surface proteins to prevent cellular attachment and others damage nucleic acid to prevent replication. Thus, for a select agent or toxin to be properly characterized as non-viable or nonfunctional after treatment, there must be zero risk of acquiring infection, disease, or toxicity after exposure.

By contrast, with respect to the SAR, the attenuation of a select agent or toxin has a different meaning. Attenuations are usually specific manipulations targeted at the genomic level to alter gene expression rendering a select agent less virulent or avirulent, or a toxin less toxic or non-toxic; however, the agent or toxin usually remains viable or functional to some extent (e.g., the knockout of an essential virulence gene). The SAR establish a process by which an attenuated strain of a select agent or toxin that does not present a severe threat to public health and safety, animal or plant health, or animal or plant products may be excluded from the select agents and toxins list (See 9 CFR §121.3(e), 9 CFR §121.4(e), 42 CFR §73.3(e), 42 CFR §73.4(e) and 7 CFR §331.3(e)). To apply for an exclusion, an applicant must submit a letter to the FSAP that provides information which establishes its eligibility for exclusion. Information should include at a minimum: strain; how the strain was derived; how it was determined that strain is attenuated; and all citation or pertinent data to support the request. After reviewing the request for exclusion, the FSAP will provide a written decision granting or denying the request. Approval of exclusion is effective on the date of the written notification to the applicant. A list of current exclusions may be found at http://www.selectagents.gov/ under the "Select Agents and Toxin" tab. Until such time that exclusions are considered and granted, the agent in question is subject to the requirements of the SAR. If an excluded strain is subjected to any manipulation that restores or enhances its virulence or toxicity, the resulting select agent or toxin will be subject to the SAR. It is also important to remember that inactive and attenuated select agents may still be subject to APHIS permitting requirements for interstate transport or importation as required under 9 CFR Part 122.

As a matter of biosafety and containment, select agents and toxins should not be treated as non-viable or nonfunctional until subjected to a scientifically supportable method (i.e., a validated method) that will cause permanent loss of biological activity. As a matter of Federal law a select agent or toxin must not be treated as non-viable or nonfunctional until it has been subjected to such a scientifically supportable method because the burden remains on the individual or entity possessing the select agent or toxin to avoid possessing, using, or transferring a select agent or toxin for which they are not registered. The method used to cause permanent loss of biological activity must be reliable and based on the agent's or toxin's sensitivity to the inactivant. An acceptable method may consist of traditional methods that have been generally recognized in the scientific community and published in the scientific literature or a method developed in-house, but that method should be validated as applied. The method may stand as a separate laboratory protocol or standard operating procedure or be incorporated into the laboratory biosafety manual. While there is no requirement to notify the FSAP prior to rendering a select agent or toxin non-viable or nonfunctional, an individual or entity should incorporate a practice of maintaining records on file in support of materials no longer subject to the SAR.

Scenarios

Example 1
A laboratory in the U.S. has received 50 cloacal and tracheal swab samples collected from wild waterfowl in Vietnam under a valid Veterinary Services import permit. The laboratory wants to know if it would be acceptable to perform a real-time reverse transcriptase PCR assay on these samples in its BSL-2 laboratory to detect avian influenza A viral RNA.

Highly pathogenic avian influenza virus is endemic in Vietnam. The origin of the samples presents potential risk that the samples are not free of virus of concern, and it should not be assumed that the method used to extract viral nucleic acids will render the samples non-infectious, unless the method used is a validated one. Strict import controls are necessary to avoid accidental introduction of the virus. Thus, the diagnostic work with the samples collected from a high risk area should be conducted in a BSL-3 laboratory with enhancements. Additionally, if highly pathogenic avian influenza virus is confirmed in any of the samples, then Section 1 of the APHIS/CDC Form 4 must be completed and sent to the FSAP within 7 calendar days of identification of the select agent as a result of diagnosis. Use of a validated protocol that extracts nucleic acid and inactivates the virus would permit the work in a BSL-2 laboratory.

Example 2
A registered entity would like to revise its registration application to remove virulent Newcastle disease virus (vNDV) as an agent held in its possession in storage. Approximately 100 Cryo-Micro Tubes were steam autoclaved at 120 degrees Celsius at 15 psi for 90 minutes according to the protocol present in its biosafety manual. The entity has submitted an amendment to revise its registration as well as documentation for the destruction of the Cryo-Micro Tubes, and final disposal as medical waste.

This would be appropriate because steam autoclaving is an acceptable method to inactivate vNDV, the autoclave cycle configured for the size of the load, and the performance of the autoclave has been checked on a regular basis. Submission of the revised amendment would be sufficient notification for the FSAP.

Example 3
A registered ABSL-3 laboratory would like to distribute nucleic acid extracted from cell cultures infected with Rift Valley fever virus to several US laboratories to assist them in the development of in-vitro assays. Prior to shipping any samples, the laboratory has conducted safety studies in Vero-E6 cells for cytopathic effects. No cytopathic effects have been observed and the entity would like to know if it is appropriate to ship the samples to unregistered laboratories.

The use of Vero-E6 cells is a valid method for safety testing. It would be appropriate to distribute the samples to the unregistered laboratories, provided appropriate safeguards are practiced to remove the samples from ABSL-3 containment. Also, the recipients of the material would be required to have a valid Veterinary Services transport permit.