Date: September 30, 2016

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 June 29, 2016.

The Federal Select Agent Program (FSAP) is comprised of the Centers for Disease Control and Prevention (CDC), Division of Select Agents and Toxins (DSAT) and the Animal and Plant Health Inspection Service (APHIS) Agriculture Select Agent Services (AgSAS). FSAP regulates the possession, use, and transfer of biological agents listed in 7 C.F.R. Part 331, 9 C.F.R. Part 121, and 42 C.F.R. Part 73 (select agents and toxins). FSAP administers the select agents and toxins regulations in close coordination with the Federal Bureau of Investigation’s Criminal Justice Information Services.

Bacillus anthracis exists in both a vegetative cell and spore form. Spores are hardy and have been involved in multiple episodes of inactivation failures, raising questions about the adequacy of treatment methods (e.g. chemical, irradiation) used in some inactivation protocols. Currently used treatments may not inactivate Bacillus anthracis spores completely, and standard viability testing procedures may not allow for the detection of sub-lethally injured spores (Ref. 1).

On November 30, 2015, FSAP issued the policy statement “Inactivated Bacillus anthracis,” stating:

On June 29, 2016, FSAP revised and reissued the policy to improve clarity and provide additional exclusion criteria.

On September 30, 2016, FSAP revised and reissued the policy to improve clarity and:

1. Clarify that the policy is applicable to Bacillus anthracis Pasteur strain.
2. Update the definition of safety margin.
3. Remove the requirement for statistically defensible inactivation criteria until guidance can be provided.
4. Clarify the neutralization step for chemically inactivated material (2c).
5. Clarify the sample volume sections to include procedures for large volume cultures.
6. Clarify that to qualify for an exclusion an entity must maintain a permanent record of the validation data for the inactivation process being used.
7. Include the recently added HHS Tier 1 select agent B. cereus biovar anthracis (effective October 14, 2016).  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. 

The categories for exclusion of inactivated material (heat-treated or chemically-inactivated) from the select agent regulations requires initially validating inactivation protocols and viability testing by one of two methods:

1. Using 100% of the inactivated material, or
2. Filtering 100% of the inactivated material, and then culturing the filter.

The second option addresses concerns with the feasibility of culturing 100% of the inactivated material for large-volume cultures. There is a small probability that an inactivation procedure, even with a validated method, will not achieve complete nonviability of Bacillus anthracis or Bacillus cereus biovar anthracis material. This is due to variability in staff performance of inactivation procedures and limits of detection of the viability testing procedures (related to the assay and the sampling). An entity must determine an appropriate safety margin derived from the kill curve to account for this variation. 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. A kill curve is the result 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.

Authority:

The U.S. Department of Health and Human Services (HHS) select agents and toxins regulations are found at 42 CFR Part 73. Pursuant to subtitle A of title II of the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 (the 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 CFR §§ 73.3, 73.4). Section 3(d)(2) and section 4(d)(2) of the HHS select agents and toxins regulations provide that nonviable select agents are excluded from the HHS select agents and toxins regulations. The Act directs the promulgation of 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 (Ref. 2).

The U.S. Department of Agriculture select agents and toxins regulations are found at 7 CFR Part 331 and 9 CFR 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. Section 3(d)(2) of Part 331 and sections 3(d)(2) and 4(d)(2) of Part 121 of the USDA select agents and toxins regulations provide that nonviable select agents are excluded from the USDA select agents and toxins regulations. The Agricultural Bioterrorism 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 (Ref. 3).

Bacillus anthracis, the bacteria that causes anthrax disease, and B. cereus biovar anthracis, the bacteria that causes anthrax-like disease,are on the list of overlap select agents and toxins as Tier 1 select agents (See 42 CFR 73.4 and 9 CFR 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” (Ref. 4). This policy also applies to Bacillus anthracis Pasteur strain which is not a Tier 1 agent.

Policy Statement:

Some inactivation protocols have failed to inactivate Bacillus anthracis spores completely, as evidenced by episodes of inactivation failures that have occurred recently. Unless waived by the APHIS Administrator or HHS Secretary, it is the policy of FSAP that all vegetative cell and spore preparations of Bacillus anthracis strains, including B. anthracis Pasteur and B. cereus biovar anthracis, that were subject to an inactivation procedure on or after June 2, 2015 are considered a select agent. The storage, transfer, or work with this material must comply with regulations found at 42 CFR Part 73 and 9 CFR Part 121 until a more effective protocol for inactivation of B. anthracis and confirmation of nonviability can be established and validated. This time period was selected based on the date FSAP issued a moratorium to entities that produce and ship inactivated B. anthracis to other laboratories. The following must be reported within 24 hours of discovery to FSAP:

1. Possession of such material by an entity not registered to possess the regulated strain of B. anthracis or B. cereus biovar anthracis, or
2. Location of such material in a non-registered room in a registered entity.

The APHIS/CDC Form 3 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 room.

The following is not subject to this policy:

1. 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
2. Specimens presented for diagnosis or verification (See section 5(d) of the select agents and toxins regulations).

Criteria:

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 section 3(d)(2) and section 4(d)(2) 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 and spore preparations:
1. Use of an inactivation method initially validated with a kill curve to determine an appropriate contact time to inactivate 100% of the organisms within the sample. If the final method chosen to achieve zero growth was not specifically tested during the development of the kill curve, the method should be further validated by culturing 100% of a treated sample or the filter through which 100% of the sample was run.
2. Use of a validated inactivation contact time that is derived from the kill curve and includes a safety margin for subsequent inactivation of samples.
3. Determination that residual chemical or antimicrobial activity does not interfere with the 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.
1. If the residual chemical or antimicrobial activity interferes with the viability test, then use neutralization methods initially validated with neutralization curves by using 100% of the sample.
4. For subsequent chemical inactivation of regulated vegetative and spore preparations, use of validated viability testing 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 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 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 37ºC and then at least 100 μl of the broth culture is spread plated on an agar plate medium that supports the growth of B. anthracis or Bacillus cereus biovar anthracis.
• The agar plate is incubated at 37ºC for at least 7 days, and no B. anthracis or Bacillus 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 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 validated with a kill curve to determine an appropriate autoclave time to inactivate 100% of the organisms within the sample. If the final method chosen to achieve zero growth was not specifically tested during the development of the kill curve, the method should be further validated by culturing 100% of the heat treated sample or the filter through which 100% of the heat treated sample was run.
2. Use of a validated autoclave time that is derived from the kill curve and includes a safety margin for subsequent inactivation of samples.
3. For subsequent inactivation of samples, use of validated viability testing 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.

Material containing B. anthracis or Bacillus cereus biovar anthracis (vegetative cell or spore preparations) that are described in the following criteria meet the exclusion found in section 3(d)(2) and section 4(d)(2) of the select agents and toxins regulations:

1. 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 validated viability testing and 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 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 10% of the inactivated material and culture the 0.22 µm filter. Note: Perform the viability test on the filtered material that contained 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 37ºC, and then at least 100 μl of the broth culture is spread plated on an agar plate medium that supports the growth of B. anthracis or B. cereus biovar anthracis.
• The agar plate is incubated at 37ºC for at least 48 hours and no B. anthracis or B. cereus biovar anthracis colonies are observed at the end of the incubation period.

In order for an entity to qualify for an exclusion it must maintain a permanent record of the validation data for the inactivation process being used.     

For all of the material described above a record is created to document any inactivation use to include:

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

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.

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.

Policy Statement:

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. Public Health Security and Bioterrorism Preparedness and Response Act of 2002, 42 U.S.C. § 262a.
   
3. Agricultural Bioterrorism Protection Act of 2002, 7 U.S.C. § 8401c.
4. Executive Order 13546, “Optimizing the Security of Biological Select Agents and Toxins in the United States,” July, 2010.

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.

Samuel S. Edwin, PhD
Director, Division of Select Agents and Toxins
Department of Health and Human Services
Centers for Disease Control and Prevention

Freeda E. Isaac, DVM
Director, Agriculture Select Agent Services
United States Department of Agriculture
Animal and Plant Health Inspection Service