The policy clarifies that the Pasteur strain of Bacillus anthracis is included in these requirements.

The policy includes the recently added HHS Tier 1 select agent Bacillus cereus biovar anthracis.  B. cereus biovar anthracis is included in this policy because of its similarity to B. anthracis in terms of virulence, and because it possesses plasmids similar to pXO1 and pXO2 which are associated with virulence.  [Note: this provision takes effect on October 14, 2016, as this is the effective date that B. cereus biovar anthracis will be considered a select agent.]

The policy updates the definition of “safety margin”.  A safety margin is the treatment amount designed into an inactivation procedure beyond that required to reach the limit of assay detection intended to reduce the probability of inactivation failure.

The policy removes the requirement for generating statistically defensible inactivation criteria until further guidance can be provided.

The policy clarifies the requirements of the neutralization process for chemically inactivated material

• Allows for the addition of spores from other B. anthracis strains (e.g. Sterne, Pasteur, Ames) to determine if the chemical (or antimicrobial activity) interferes with the viability test.
• Generation of a neutralization curve using 100% of the sample during initial validation is required only if the residual chemical or antimicrobial activity interferes with the viability test.

The policy clarifies the validation process for chemically inactivated material and cellular extracts to include procedures for large volume cultures.

• For large volume cultures of chemically inactivated material, after initial validation testing 100% of the sample, entities can use a 0.22 µm (or smaller pore size) filter to test 10% of the inactivated material and culture the filter to test for viability.
• For large volume cultures used to create cellular extracts, entities can use a 0.22 µm (or smaller pore size) filter to test 10% of the extracted material, and then culture the filter to test for any viable cells present after the extraction and filtration processes.

The policy clarifies that to qualify for an exclusion an entity must maintain a permanent record of the validation data for the inactivation process being used.

No. Samples that were inactivated and excluded from the requirements of the select agent regulations based on the conditions of the previous policy (dated November 30, 2015, as revised June 29, 2016) remain excluded. This policy applies to future inactivation of materials effective September 30, 2016.  For B. cereus biovar anthracis the effective date is October 14, 2016.

No. As in the previous version of this policy, an entity must perform in-house validation. While published methods can be used to set up an inactivation protocol, the protocol must be validated in-house.

It depends. Entities may not use old data unless the validation conditions are exactly those that are described in the new policy. Further, controls must be used when validating the protocol. Validation data must be recorded and maintained on-site.

Chemical treatments or antimicrobial activity in inactivated samples may interfere with the viability test by prohibiting growth of select agents potentially present in the sample. Neutralization inactivates residual chemicals or antimicrobial activity. Therefore, viability tests should be performed once chemical or antimicrobial treatments have been subjected to a neutralizing substance or have been shown not to interfere with the viability test.

The policy excludes extracts (e.g., nucleic acid extracts, antigens, or lysates) from regulated strains of B. anthracis orB. cereus biovar anthracis, or material containing regulated strains of B. anthracis orB. cereus biovar anthracis (e.g., serum or culture) that have:

• Been filtered through a 0.22 µm or smaller pore size filter
• Passed a subsequent viability check on at least 10% of the sample or production lot and includes required documentation.

No. Any inactivated B. anthracis materials created on or before June 2, 2015 would not be subject to the policy or considered a select agent, and are excluded from the requirements of the regulations found at 42 CFR part 73 and 9 CFR part 121.

This time was selected based on the date that the Federal Select Agent Program (FSAP) issued a request for a moratorium to registered entities that produce and ship on or after that date inactivated B. anthracis to other laboratories.

No. Any inactivated B. cereus biovar anthracis materials created before October 14, 2016 would not be subject to the policy or considered a select agent, and are excluded from the requirements of the regulations found at 42 CFR part 73.  This is the date that B. cereus biovar anthracis will be considered an HHS select agent.

If testing of B. anthracis or B. cereus biovar anthracis that has been subjected to an inactivation process and was determined nonviable and therefore treated as a non-select agent, results in the growth of B. anthracis, the entity must report the inactivation failure within 24 hours of detection to FSAP.

1. Submit APHIS/CDC Form 3 (Report of Theft, Loss, or Release of Select Agents and Toxins).
2. Question 14 can be left blank.
3. Use Question 25 to describe the inactivation failure.
4. If the entity:
• Decides to destroy the materials, in addition to the completed APHIS/CDC Form 3, provide the following to either AgSAS or DSAT:
• Photographs of item after autoclaving showing the turning of an autoclave indicator and of the autoclave strip that shows the successful completion of the run.
• A brief letter stating the date and time of the destruction and listing the names of witnesses.
• Decides to transfer the agent to another entity that is registered with DSAT or AgSAS, submit a completed APHIS/CDC Form 2 (Request to Transfer Select Agents and Toxins). If you need assistance to identify a registered recipient institution, please contact APHIS/AgSAS at 301-851-3300 option 3 (voice only) or CDC/DSAT at 404-718-2000.
• Is registered and chooses to retain the agent in a registered space, include in the APHIS/CDC Form 3 the registered space that the materials were transferred to. The entity’s registration should be amended to include the change in inventory.

No. If a product is cleared, approved, licensed, or registered under any of the following laws, it is exempt from this policy:

• The Federal Food, Drug, and Cosmetic Act (21 U.S.C. 301 et seq.), or
• Section 351 of the Public Health Service Act pertaining to biological products (42 U.S.C. 262), or
• The Act commonly known as the Virus-Serum-Toxin Act (21 U.S.C. 151-159), or
• The Federal Insecticide, Fungicide, and Rodenticide Act (7 U.S.C. 136 et seq.).

Yes. This policy includes only B. anthracis strains (including the Pasteur strain) and the B. cereus biovar anthracis strain subject to the select agent regulations. The policy does not apply to the Sterne strain.

Yes. Below is a list of material and conditions that must be met for exclusion of inactivated B. anthracis or B. cereus biovar anthracis:

• Any inactivated B. anthracis materials created on or before June 2, 2015, or any inactivated materials of B. cereus biovar anthracis created before October 14, 2016.
• Extracts from regulated strains of B. anthracis, B. cereus biovar anthracis, or material containing regulated strains of B. anthracis or B. cereus biovar anthracis (where viable agent is removed) that have been filtered and viability tested as outlined in the policy.
• Chemically-treated vegetative and spore preparations that meet all of the conditions in the policy.
• Chemically-treated whole tissue specimens (such as formalin fixed tissue) that meet all of the conditions in the policy.
• Heat treated vegetative cell and spore preparations for future use that meet all of the conditions in the policy.
• Materials for which a waiver of the policy has been granted by AgSAS or DSAT.

Yes. FSAP is requiring the following documents regarding viability testing of materials be maintained on-site, and they may be reviewed during an inspection.

• Process or procedures used for inactivation or removal,
• Results of the final viability testing including the date of those results,
• Name of the individual who performed the viability testing, and
• Names of recipients of these materials. 

Yes, entities must maintain a permanent record of the initial validation data for as long as the procedure is used to inactivate material.

Submit a letter to FSAP at lrsat@cdc.gov or AgSAS@aphis.usda.gov describing what material is to be waived, the inactivation protocol and viability test used, validation data, and any other supporting information/references.

This policy is effective immediately for B. anthracis, and effective on October 14, 2016 for B. cereus biovar anthracis. However, entities may continue to follow the more stringent requirements for extracts in the previous policy, if desired.

• Viability testing with 10% of every subsequent sample must be conducted for:
• Chemically-treated vegetative and spore preparations.
• Extracts (e.g., nucleic acid extracts, antigens, or lysates) from regulated strains of B. anthracis or B. cereus biovar anthracis.
• Material containing regulated strains of B. anthracis or B. cereus biovar anthracis (e.g., serum or culture) where viable agent is removed.
• For tissue samples, subsequent viability testing does not need to be conducted once initial validation is complete.
• For heat treated samples, subsequent viability testing can be performed with an appropriate Bacillus species spore-based indicator under conditions that accurately represent the types of material that are treated and results in no growth of Bacillus species.

A neutralization curve is performed by using different amounts of the neutralization substance to determine how much is needed to completely neutralize the chemical or antimicrobial activity. An inactivation kill curve represents the results of a dose-response experiment where a select agent is subjected to increasing levels of the inactivating method in order to determine the minimum conditions required to render it nonviable.

Yes. However, an entity must demonstrate that dialysis or dilution will neutralize residual chemical activity. This must be accomplished with a neutralization curve to determine the appropriate conditions for dialysis or dilution and the positive control that is stipulated in the policy.

Yes. Preparations of B. anthracis that were irradiated prior to June 2, 2015 are not subject to the policy and can be treated as non-select agents so long as the entity validated the material as non-viable. Entities that irradiate B. anthracis samples after June 2, 2015 must treat those samples as select agents. There is no exclusion for B. anthracis samples irradiated after June 2, 2015 unless they receive a waiver of the policy. Entities that irradiate B. cereus biovar anthracis samples after October 14, 2016 must treat those samples as select agents. There is no exclusion for B. cereus biovar anthracis samples irradiated after October 14, 2016 unless they receive a waiver of the policy

No. Entities that inactivate B. anthracis or B. cereus biovar anthracis must follow the procedures outlined in the policy. Each entity must maintain records of the kill curve, viability tests, and neutralization curve, and have the records available for inspectors upon request.

Entities should select an appropriate Bacillus species spore based indicator. For example, Geobacillus stearothermophilus, formerly known as Bacillus stearothermophilus, can be used to validate subsequent inactivated samples.

If an entity chooses to inactivate a higher concentration of cells, then the method needs to be revalidated with a new kill curve. If the entity chooses to inactivate a lower concentration of cells, then the entity must validate by culturing 100% of a treated sample or the filter through which 100% of the sample was run.

No, a formal death certificate indicating the material is inactivated is not a required record. The required records are listed in FAQ #13.