FAQ: Inactivated Bacillus Anthracis

This policy has been revised and reissued to:

1. Clarify that the policy includes exclusion for certain inactivated material
2. Remove the kill curve and neutralization curve requirements
3. Clarify other language throughout (non-substantive changes)

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, September 30, 2016, or April 19, 2017) remain excluded. This policy applies to future inactivation of materials effective August 14, 2017.

No. 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.

If testing of B. anthracis (including Pasteur strain) 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 (including Pasteur strain),the entity must report the inactivation failure within 24 hours of detection to FSAP.

1. Submit APHIS/CDC Form 3 [PDF – 291 KB] (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 DASAT or DRSC:
     • 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 DRSC or DASAT, submit a completed APHIS/CDC Form 2 [PDF – 492 KB] (Request to Transfer Select Agents and Toxins). If you need assistance to identify a registered recipient institution, please contact APHIS/DASAT at 301-851-2070 or CDC/DRSC 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.).

• Viability testing with at least 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 (including Pasteur strain) or B. cereus biovar anthracis.
  • Material containing regulated strains of B. anthracis (including Pasteur strain)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.

Yes. However, an entity must demonstrate that dialysis or dilution will neutralize residual chemical activity by using the positive control that is stipulated in the policy.

Yes. Preparations of B. anthracis (including Pasteur strain) 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 (including Pasteur strain) samples after June 2, 2015 must treat those samples as select agents. There is no exclusion for B. anthracis (including Pasteur strain) 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 (including Pasteur strain) or B. cereus biovar anthracis must follow the procedures outlined in the policy. Each entity must maintain records of the validation data and viability tests, 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. If the entity chooses to inactivate a lower concentration of cells, then the entity does not need to revalidate

• For initial validation, the following culture conditions are required:

  1. Sample volume: The tested material consists of 100% of the sample of inactivated material directly inoculated into a broth medium (e.g., trypticase soy broth, nutrient broth) that supports B. anthracis or B. cereus Biovar anthracis growth so that the volume of the test sample is not more than 10% of the volume of the medium (i.e., 1/10 or greater dilution). For large volume cultures, use a 0.22 µm filter to filter 100% of the inactivated material and culture the 0.22 µm filter.
     • Note: Perform the viability test once chemical and/or antimicrobial treatments have been subjected to a validated neutralizing substance or have been shown not to interfere with the viability test.
  2. Culture conditions: The broth culture is incubated for at least 7 days at 35°±2ºC and then at least 100 microliters of the broth culture is spread on an agar plate medium that supports the growth of B. anthracis or B. cereus Biovar anthracis.

  The agar plate is incubated at 35°±2ºC for at least 7 days, and no B. anthracis or B. cereus Biovar anthracis colonies are observed at the end of the incubation period.