Guidance on the Inactivation or Removal of Select Agents and Toxins for Future Use: Inactivation Failure Reporting Requirements

Reporting requirements regarding inactivation failures require the Responsible Official (RO) to:

The intent of this requirement is to create an entity environment where inactivation or select agent removal failures are investigated to determine the reason for the failure as opposed to merely re-subjecting the material to the inactivation or select agent removal method. Human error is a significant contributor when inactivation or select agent removal methods fail. These situations demand careful attention by the entity to ensure training and reevaluation of the inactivation procedure in order to minimize the likelihood that the situation would reoccur in the future. The regulations only require reporting of inactivation or select agent removal failures to FSAP when the RO cannot establish the reason of the failure of the validated inactivation or select agent removal method. It is recommended that inactivation failures be investigated immediately to prevent the use of a failed procedure. Removal of samples that fail inactivation out of registered space would be a violation of the regulations.

Record keeping requirements

For select agents or material containing select agents or regulated nucleic acids, the registered individual or entity must keep the following records for 3 years:

• A written description of the:
  • Validated inactivation procedure or viable select agent removal method used, including validation data.
  • Viability testing protocol used.
  • Investigation conducted by the entity RO involving an inactivation or viable select agent removal failure and the corrective actions taken.
• The name of each individual performing the validated inactivation or viable select agent removal method.
• The date(s) the validated inactivation or viable select agent removal method was completed.
• The location where the validated inactivation or viable select agent removal method was performed.
• A certificate, signed by the Principal Investigator, that includes the date of inactivation or viable select agent removal, the validated inactivation or viable select agent removal method used, and the name of the Principal Investigator. A copy of the certificate must accompany any transfer of inactivated or select agent removed material.

The information specified in section 17 (a)(8)(vii) must be retained by the entity that performs the inactivation or removal of viable select agent and must accompany the inactivated sample if it is transferred to another entity. The certificate needs to be a written certification of the information but doesn’t require any specific format. The required information can be recorded outside of containment. Further, the PI does not have to be present during the performance of the inactivation or select agent removal procedure. A copy of the certificate must accompany any transfer of the inactivated samples to another entity.  However, FSAP recommends a certificate of inactivation accompany all transfers of inactivated select agents (includes intra-entity transfers). The PI who signs the certificate is the one individual who is designated by the entity to direct a project or program and who is responsible to the entity for the scientific and technical direction of that project or program (including all inactivation procedures or removal procedures associated with that project). In the absence of that PI, an individual designated by that PI and approved by the entity’s Responsible Official can sign the certificate but only during the duration of the PI’s absence. Each absence of a PI requires a new delegation.

In order for an individual to be the PI’s designee to sign the certificate, a person must:

• Be listed on the entity’s registration
• Have the knowledge and expertise to provide scientific and technical direction regarding the validated inactivation procedure or the procedure for removal of viable select agent to which the certificate refers. If this requirement causes unintended consequences please contact FSAP.

The signature denotes that the PI or designee that is responsible for the specific agent, has reviewed the inactivation procedure used and the validation or verification data. The certificate should be signed as close to the date of inactivation as possible. There is no regulatory requirement for the receiver to keep the certificate but it is strongly recommended that this information is kept so long as the inactivated sample exists. However, the sender or the PI that performed the inactivation procedure must keep the certificate for 3 years. A PI can review and sign documents electronically.

For electronic signature, the method used should:

• Identify and authenticate a person using at least two factors of authentication, including something the person knows (i.e., email password) and something the person has (e.g., a mobile phone with SMS text message access);
• Provide a means to preserve the integrity of the signed record that is (a) portable, (b) independently verifiable, (c) tamper-evident, (d) granular, and (d) verifiable in the long-term; and
• Meet the requirements of Government Paperwork Elimination Act, the Electronic Signatures in Global and National Commerce Act (ESIGN, 15 U.S.C. ch. 96), and the Uniform Electronic Transactions Act.

In addition, the electronic form of signature must be executed or adopted by a person with the intent to sign the electronic record, (e.g., to indicate a person’s approval of the information contained in the electronic record). Not only must this intent be captured at the time of each signature, but it must also be captured for each individual signature and be provided as granular evidence within the electronic signature system’s audit trail.  Each signed document must be backed by an audit trail that captures intent to sign for each individual signature and provides granular, consistent, timestamped evidence as to every step in the entire signature process. The electronic form of signature must be attached to or associated with the electronic record being signed, and each signature should be produced according to established standards. The signature should be independently verifiable, without relying on the electronic signature service or website to be validated (Reference https://www.signix.com/blog/bid/109223/7-Rules-for-E-Signature-Legality-and-Compliance-Part-3external icon).

Once non-viability or non-infectivity, have been demonstrated, and the record requirements are met, the material is no longer subject to the regulations. While it is not a regulatory requirement, it is recommended that entities maintain records that identify the recipients of inactivated materials.

Annual review requirements

Inactivation and select agent removal methods must be monitored continuously to ensure adherence to validated methods. Oversight should include investigation of select agent inactivation or removal failures, ensuring that validated procedure are used, training for staff that inactivate the agents, and appropriate record keeping.

The RO is required to review, and revise as necessary, each of the entity’s validated inactivation procedures or viable select agent removal methods. The RO must conduct the review annually or after any change in Principal Investigator, change in the validated inactivation procedure or viable select agent removal method, or failure of the validated inactivation procedure or viable select agent removal method. The RO must document the review and ensure training occurs if there are any changes to the validated inactivation procedure, viable select agent removal method, or viability testing protocol. The annual review does not mean the procedures have to be revalidated. For example, the RO could review the inactivation procedures and determine that they are still being used and work as intended. In this situation, revalidation would not be necessary.

Waiver

Entities must inactivate select agents prior to treating them as non-select agents. However, there may be rare situations where there is a need to take material out of containment without following the exact requirements for viability testing. For example, consider a situation where an entity filters serum that contains a select agent but the recovered volume is so low that the requirement to viability test every sample would further deplete the sample volume and not leave enough for experiments. In these situations, an entity may submit a request to FSAP to apply an alternative select agent removal or inactivation procedure and/or an alternative means of validation. To apply for this determination, a registered individual or entity must submit a written request including:

• A justification regarding the alternative procedure including a description of what material is to be waived.
• The inactivation protocol and viability test to be used
• Validation data.
• Any other supporting information/references, such as scientific references.

FSAP will issue a written decision granting or denying the request.

Scenarios

Example 1:
A laboratory uses aqueous formaldehyde to inactivate Francisella tularensis, and wants to ship these inactivated cells to unregistered entities. They plan on changing the concentration of cells to be inactivated. Should the entity revalidate their inactivation procedure?

It depends. If the concentration of cells is decreased or the incubation time of inactivation is increased, then the inactivation procedure would not need to be revalidated. However, any time there has been a change to an inactivation procedure (e.g. equipment changes, a different strain used with known resistance to inactivation method, or a change in the volume, or increased concentration of cells, or decrease in incubation time) the protocol needs to be revalidated to ensure there are no viable cells present in the sample. Revalidation via a kill curve or bioburden reduction should be performed to determine the conditions needed to inactivate the material. If there are strain-to-strain variations in sensitivity to the inactivation procedure, validate the procedure on the more resistant strain. Also, use a viability testing procedure for subsequent inactivation to verify the effectiveness of the inactivation procedure. In addition, positive controls should be included to ensure that the chemical (formaldehyde) does not interfere with the viability test. If it does interfere, then perform a step to neutralize the formaldehyde.

Example 2:
A laboratory in the U.S. has received highly pathogenic avian influenza virus (HPAIV) samples from Vietnam under a valid APHIS/CDC Form 2 Transfer authorization. What can the laboratory do in order to safely perform a real-time reverse transcriptase PCR assay on these samples in its unregistered BSL-2 laboratory to further analyze the avian influenza A viral RNA?

Work with HPAIV must be conducted in a BSL-3 laboratory with enhancements. Nucleic acids that encode for HPAIV are not subject to the select agent regulations. You could use a validated protocol that extracts nucleic acids and renders the virus nonviable and confirm the protocol by viability testing. Subsequent work could then be conducted in an unregistered BSL-2 laboratory.

Example 3:
A registered entity is inactivating 500 milliliters (ml) of Yersinia pestis by gamma irradiation and performs viability tests that show no growth of Yersinia pestis. During initial development of this inactivation method, the entity validated non-viability using a starting volume of 150 ml of Yersinia pestis. Can the 500 ml preparation be worked with as non-select agent material outside of registered space?

No, the entity tested viability on material that did not represent the exact conditions (volume) set by the initial validation of inactivation. The entity would have to generate a kill curve or bioburden reduction to demonstrate that the protocol will inactivate a 500 ml sample.

Example 4:
A registered ABSL-3 laboratory extracts nucleic acid from cell cultures infected with Rift Valley fever virus (RVFV) using a commercially available kit. The entity would like to distribute the nucleic acids to several US laboratories to assist them in the development of in vitro assays. The laboratory would like to know what it should consider when validating that the kit inactivates the RVFV present in the sample.

The entity should perform viability tests to validate that the kit inactivates the virus and no select agent material remains. When validating the ability of the kit to inactivate RVFV in-house, the entity should consider viability test conditions such as the use of permissive cells to culture material, appropriate controls, and incubation time of the culture. Once the entity has validated that the kit inactivates RVFV, the nucleic acids, which are not subject to the select agent regulations, can be distributed to unregistered laboratories.