Policy Statement

Date: August 14, 2017

Subject: Revised FSAP Policy Statement: Inactivated Bacillus anthracis and Bacillus cereus Biovar anthracis

This policy statement replaces the FSAP Policy Statement: “Inactivated Bacillus anthracis,” dated
April 19, 2017.


Authority:

The U.S. Department of Health and Human Services (HHS) select agents and toxins regulations are found at 42 C.F.R. Part 73. Pursuant to subtitle A of title II of the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 (the Bioterrorism Response Act), the HHS Secretary has established a list of biological agents and toxins which have the potential to pose a severe threat to the public health and safety (See 42 U.S.C. § 262a). This list is found in section 3 (HHS select agents and toxins) and section 4 (Overlap select agents and toxins) of the HHS select agents and toxins regulations (See 42 C.F.R. §§ 73.3, 73.4).

  • Sections 3(d)(2) and 4(d)(2) of Part 73 of the HHS select agents and toxins regulations provide that non-viable select agents are excluded from the HHS select agents and toxins regulations.  
  • Sections 3(d)(4) and 4(d)(4) of Part 73 of the HHS select agents and toxins regulations exclude a select agent or regulated nucleic acids that can produce infectious forms of any select agent virus that has been subjected to a validated inactivation procedure that is confirmed through a viability testing protocol. 
  • Sections 3(d)(5) and 4(d)(5) of Part 73 of the HHS select agents and toxins regulations exclude material containing a select agent that is subjected to a procedure that removes all viable select agent cells, spores, or virus particles if the material is subjected to a viability testing protocol to ensure that the removal method has rendered the material free of all viable select agent. 

The Bioterrorism Response Act also directs the HHS Secretary to promulgate regulations to establish and enforce safety and security procedures for the possession and use of select agents and toxins, including measures to ensure proper training and appropriate skills to handle such select agents and toxins.  See generally, 42 C.F.R. Part 73. 

The U.S. Department of Agriculture select agents and toxins regulations are found at 7 C.F.R. Part 331 and 9 C.F.R. Part 121. Pursuant to subtitle B of title II of the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 (the Agricultural Bioterrorism Protection Act), the USDA Secretary has established a list of biological agents and toxins which have the potential to pose a severe threat to animal and plant health, or to animal or plant products (See 7 USC § 8041). This list is found in section 3 (PPQ select agents and toxins) of Part 331 and in section 3 (VS select agents and toxins) and section 4 (Overlap select agents and toxins) of Part 121 of the USDA select agents and toxins regulations.

  • Sections  3(d)(2), 4(d)(2) of Part 121 of the USDA select agents and toxins regulations provide that non-viable select agents are excluded from the USDA select agents and toxins regulations.  
  • Sections 3(d)(4) and 4(d)(4) of Part 121 of the USDA select agent regulations exclude a select agent or regulated nucleic acids that can produce infectious forms of any select agent virus that has been subjected to a validated inactivation procedure that is confirmed through a viability testing protocol. 
  • Sections 3(d)(5) and 4(d)(5) of the USDA select agent and toxin regulations exclude material containing a select agent that is subjected to a procedure that removes all viable select agent cells, spores, or virus particles if the material is subjected to a viability testing protocol to ensure that the removal method has rendered the material free of all viable select agent.

The Agricultural Bioterrorism Protection Act also directs the USDA Secretary to promulgate regulations to establish and enforce safety and security procedures for the possession and use of select agents and toxins, including measures to ensure proper training and appropriate skills to handle such select agents and toxins.  See generally, 7 C.F.R. Part 331 and 9 C.F.R. Part 121.

Bacillus anthracis, the bacteria that causes anthrax disease, and B. cereus Biovar anthracis, the bacteria shown to cause anthrax-like disease in animals,are on the list of select agents and toxins as Tier 1 select agents (See 42 C.F.R. 73.4 and 9 C.F.R. 121.4). Tier 1 select agents and toxins are those that have the “greatest risk of deliberate misuse with significant potential for mass casualties or devastating effect to the economy, critical infrastructure, or public confidence.” B. anthracis Pasteur strain is also on the list of select agents and toxins but is not a Tier 1 agent.


Top of page

 

Policy Statement:

Because some inactivation protocols have failed to inactivate B. anthracis spores completely, as evidenced by episodes of inactivation failures that have occurred in the recent past, unless  material containing B. anthracis, including B. anthracis Pasteur strain, or B. cereus Biovar anthracis  meets one of the exclusion requirements listed in this policy or is otherwise waived  by the APHIS Administrator or HHS Secretary (see 42 CFR §§ 73.3(6), 73.4(6); and 9 CFR §§ 121.3(6), 121.4(6)), it is the policy of FSAP that:

  • all vegetative cell and spore preparations that were subject to an inactivation procedure on or after June 2, 2015 for B. anthracis strains, including B. anthracis Pasteur strain, and
  • on or after October 14, 2016 for B. cereus Biovar anthracisare considered a select agent.

The storage, transfer, or work with such material must comply with regulations found at 42 CFR Part 73 and 9 CFR Part 121. The time period was selected based on the date FSAP issued a moratorium to entities on working with and shipping inactivated B. anthracis and the date B. cereus Biovar anthracis became a select agent.

The following must be reported within 24 hours of discovery to FSAP via lrsat@cdc.gov or AgSAS@aphis.usda.gov:

  • Possession of regulated strain of B. anthracis, including B. anthracis Pasteur strainor B. cereus Biovar anthracis by an entity not registered with FSAP to possess these agents, and/or
  • Location of such material in a non-registered laboratory and/or storage room in a registered entity.

The APHIS/CDC Form 3 (https://www.selectagents.gov/form3.html) must be used for reporting so that FSAP can ensure that such material is appropriately destroyed, transferred to a registered entity, or moved to a registered laboratory and/or storage room within a registered entity.

The following is not subject to this policy:

  • Inactivated material from strains of B. anthracis or B. cereus Biovar anthracis listed as excluded from the select agent regulations, found at Select Agent and Toxins Exclusions, and
  • Specimens presented to a clinical or diagnostic laboratory for diagnosis or verification (See section 5(b) of the select agents and toxins regulations).

Top of page


Exclusion Criteria:

All categories for exclusion of material subjected to an inactivation or removal procedure from the select agent regulations require initially validating select agent inactivation or select agent removal procedures (using one of the two options listed below) and viability testing using appropriate positive, negative, and process controls,

  • Using 100% of the inactivated material for viability testing, or
  • Filtering (pore size ≤ 0.22 micron) 100% of the inactivated material, and then culturing the filter (for larger volume cultures).

The second option addresses concerns with the feasibility of culturing 100% of the inactivated material for large-volume cultures. An entity must determine an appropriate safety margin for select agent inactivation. A safety margin is the treatment amount designed into an inactivation procedure beyond that required to reach complete inactivation (e.g. if inactivating 107cells/ml, develop inactivation assay parameters to inactivate 109 cells/ml) to reduce the probability of inactivation failure.

regulated strains of B. anthracis, including B. anthracis Pasteur, and B. cereus Biovar anthracis (vegetative cell or spore preparations) inactivated as described below meet the exclusion found in sections 3(d)(4), 3(d)(5), 4(d)(4), and 4(d)(5) of the select agents and toxins regulations:

  1. Preparations of regulated strains of B. anthracis, including B. anthracis Pasteur, that were subjected to an inactivation procedure prior to June 2, 2015, when FSAP issued through email notification the “Request for immediate moratorium on all work with, and shipments of, inactivated Bacillus
    anthracis
    .”
  2. Chemically-treated vegetative cells and spore preparations :
    1. Use of an inactivation method developed to determine an appropriate concentration and contact time for the chemical to inactivate 100% of the organisms within the sample.
    2. Use of a validated inactivation method that includes a safety margin for subsequent inactivation of samples.
    3. Determination that residual chemical or antimicrobial activity does not interfere with viability test by using this positive control: Split the chemically treated sample into two portions. To one, add ≥100 B. anthracis (e.g. Sterne, Pasteur, Ames) spores to determine if the chemical (or antimicrobial activity if present) interferes with the viability test. If the residual chemical or antimicrobial activity interferes with the viability test, then use neutralization methods initially validated by using 100% of the sample.
    4. For subsequent chemical inactivation of regulated vegetative cells and spore preparations, use of a viability testing protocol that meets or exceeds the following:
      1. Sample volume: The tested material consists of at least 10% of the sample or production lot of inactivated material directly inocolated 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 10% 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.
  3. Chemically-treated whole tissue specimens (such as formalin fixed tissue): Use of an inactivation method developed and/or validated initially (as described in 2a-c above) to the exact conditions to be used for subsequent inactivation.
  4. Heat treated (autoclave) vegetative cell and spore preparations for future use that meet all of the following:
    1. Use of an inactivation method initially developed to determine an appropriate autoclave time to inactivate 100% of the organisms within the sample.  
    2. Use of a validated autoclave temperature and time that includes a safety margin for subsequent inactivation of samples.
    3. For subsequent inactivation of samples, use of a viability testing protocol that includes the use of an appropriate Bacillus species spore based indicator under conditions that accurately represent the types of material that are treated, and shows no growth of Bacillus species.
  5. Extracts (e.g., nucleic acid extracts, antigens, lysates, etc.) 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, culture) where viable agent is removed, if the procedure:
    1. Includes filtration through a 0.22 μm or smaller pore size filter, and
    2. Shows no growth of B. anthracis or B. cereus Biovar anthracis after using a viability testing protocol that meets or exceeds the following:
      1. Sample volume: The tested material consists of ≥10% of the sample or production lot of inactivated material directly inocolated 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 10% of the inactivated material and culture the 0.22 µm filter. Note: Perform the viability test on the filtered material that contained inactivated regulated strains of B. anthracis or B. cereus Biovar anthracis once 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 48 hours at 35º±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 48 hours and no B. anthracis or B. cereus Biovar anthracis colonies are observed at the end of the incubation period.

Top of page

 

Investigation of inactivation or viable select agent removal failures

The Responsible Official must investigate to determine the reason for any failure of a validated inactivation procedure or any failure to remove viable select agent from material. If the Responsible Official is unable to determine the cause of the failure of a validated inactivation procedure or a viable select agent removal method; or receives a report of any inactivation failure after the movement of material to another location, the Responsible Official must report immediately by telephone or email the inactivation or viable agent removal method failure to CDC or APHIS.

Top of page



Records:

In order for an entity to qualify for an exclusion, the record of the validation data for the inactivation process being used must be available to be reviewed 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:

  1. A written description of the method used, including validation data;
  2. The date(s) the validated inactivation or viable select agent removal method was completed and the location where the procedures were conducted;
  3. The name of each individual performing the select agent inactivation or viable select agent removal method;
  4. 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.
  5. Names and location of recipients of inactivated select agent material or material from which select agents were removed.
  6. 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.

Top of page

 
Annual Review Requirements

The Responsible Official must review, and revise as necessary; each of the entity’s validated inactivation procedures or viable select agent removal methods. The review must be conducted annually or after any change in Principal Investigator responsible for inactivation, 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 review must be documented and training must be conducted if there are any changes to the validated inactivation procedure, viable select agent removal method, or viability testing protocol.

Top of page


Waiver:

To request a waiver to this policy, submit a letter to FSAP at lrsat@cdc.gov or AgSAS@aphis.usda.gov. Describe what material is to be waived, and provide the inactivation protocol and viability test used, validation data, and any other supporting information/references.

Top of page


References:
  1. Weller SA, Stokes MGM, and Lukaszewski RA. Observations on the inactivation efficacy of a MALDI-TOF MS chemical extraction method on Bacillus anthracis vegetative cells and spores. PLoS One 2015; 10(12): e0143870. doi: 10.1371/journal.pone.0143870.
  2. Brezillon, C, Hauslant, M, Dupke, S, Corre, JP, Lander, A, Franz, T, Monot, M, Couture-Tosi, E, Jouvion, G, Leendertz, FH, Grunow, R, Mock, ME, Klee, SR, and Goossens, L. (2015) Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus Biovar anthracis. PLOS Negl. Trop. Dis. 9(4):e0003455.

Top of page


Additional information on sterilization methods for waste containing B. anthracis can be found in the following references:
  1. Whitney E, Beatty M, Taylor T, Weyant R, Sobel J, Arduino M, and Ashford D. Inactivation of Bacillus anthracis spores. Emerg Infect Dis 2003; 9(6): 623-627.
  2. Rutala W, Stiegel M, and Sarubbi F. Decontamination of laboratory microbiological waste by steam sterilization. Appl Environ Microbiol1982; 43(6): 1311-6.
  3. Lauer J, Battles D, and Vesley D. Decontaminating infectious laboratory waste by autoclaving. Appl Environ Microbiol. 1982; 44(3): 690-4.
  4. Wood J, Lemieux P, Betancourt D, Kariher P, and Gatchalian N. Dry thermal resistance of Bacillus anthracis (Sterne) spores and spores of other Bacillus species: implications for biological agent destruction via waste incineration. J Appl Microbiol 2010; 109(1):99-106.
  5. United States Pharmacopoeial Chapter (1035). Biological Indicators for Sterilization. USP38-NF33, United States Pharmacopoeial Convention, Rockville, MD, 2015.
  6. Association for the Advancement of Medical Instrumentation/International Org. for Standardization. Sterilization of health care products - Biological indicators - Part 5: Biological indicators for low-temperature steam and formaldehyde sterilization processes. Association for the Advancement of Medical Instrumentation, Arlington, VA, 2006.

This policy statement will be provided to the Responsible Official via Select Agent (SA) Gram for each registered entity and to the Responsible Official for each newly registered entity during the registration process. A copy of this policy statement may also be found at http://www.selectagents.gov.

Any questions concerning this policy may be addressed by contacting the Federal Select Agent Program at lrsat@cdc.gov or AgSAS@aphis.usda.gov.

Top of page