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.

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 or B. cereus biovar anthracis, or material containing regulated strains of B. anthracis or B. 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 (including Pasteur strain) materials created on or before June 2, 2015 or inactivated B. cereus Biovar anthracis created on or after 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 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 (including Pasteur strain) to other laboratories and the date B. cereus Biovar anthracis was added as a select agent.

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.).

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 (including Pasteur strain) or B. cereus biovar anthracis:

• Any inactivated B. anthracis (including Pasteur strain) 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 (including Pasteur strain), 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 DASAT or DRSC.

Yes.  FSAP is requiring the following documents regarding inactivation of B. anthracis (including Pasteur strain) and B. cereus biovar anthracis be maintained on-site, and they may be reviewed during an inspection:

• The record of the validation data for the inactivation process being used must be available for review by the Federal Select Agent Program, even if the record is older than 3 years.
• Documentation maintained for any validated select agent inactivation procedure or select agent removal method should include:
  • A written description of the method used, including validation data;
  • The date(s) the validated inactivation or viable select agent removal method was completed and the location where the procedures were conducted;
  • The name of each individual performing the select agent inactivation or viable select agent removal method; and
  • Results of the final viability testing including the date.
• A certificate of inactivation reviewed and signed by the Principal Investigator, or designee, that includes the date of inactivation or viable select agent removal, the validated inactivation or viable select agent removal method used, and the names of individuals who conducted the inactivation and/or viability testing. A copy of the certificate must accompany any transfer of inactivated or select agent removed material.
• Names and location of recipients of inactivated select agent material or material from which select agents were removed.
• A written description of the investigation conducted by the entity Responsible Official involving a select agent inactivation or viable select agent removal failure and the corrective actions taken.

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 DASAT@usda.gov describing what material is to be waived, the inactivation protocol and viability test used, validation data, and any other supporting information/references.

• 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.