Biosafety/Biocontainment Plan Guidance: Provision Requirements

Hazardous Characteristics of Select Agents and Toxins

It is important that the biosafety/biocontainment plan contain the hazardous characteristics of each agent or toxin listed on the entity’s registration and the biosafety/biocontainment risk associated with laboratory procedures related to the select agent or toxin.

To assist with identifying the hazardous characteristics of each agent or toxin and the biosafety risk associated with laboratory procedures related to the select agent or toxin, the BMBL is an excellent reference and includes agent summary statements that describe the hazards, recommended precautions, additional risks, and levels of containment appropriate for handling select agents and toxins in the laboratory. The BMBL also states that HEPA filtration of exhaust air should be required when working with all BSL-4 select agents, and other agents to include: Classical swine fever virus, Reconstructed 1918 influenza virus, Rift Valley fever virus, Venezuelan equine encephalitis virus, and Highly pathogenic avian influenza virus.

The NIH Guidelines provide further guidance on risk assessment, physical containment, and biological containment provisions relating to genetic elements, recombinant nucleic acids and recombinant select agents and toxins.

In addition, DASAT has developed Guidelines for Avian Influenza Viruses to assist individuals and entities with developing policies and implementing procedures for working safely with these viruses.

In considering hazardous characteristics of each agent or toxin, the entity should discuss the hazards of agent cross-contamination in laboratories performing work with multiple select agents and agent strains to prevent the accidental transfer of agents. Areas to consider are:

• Characteristics  of agent or toxin (e.g., virus, bacteria; [e.g., spore or non-spore forming], mode of transmission, etc.), and work being performed
• Work practices to prevent dissemination outside primary containment and exposure
• Engineering Controls to prevent dissemination
• Decontamination of laboratory work surfaces, equipment, and select agent and toxin waste to prevent dissemination

Safeguards for Protecting Against Exposure to Select Agents and Toxins

Section 12(a)(2) of the select agent regulations state that the biosafety/biocontainment plan must include Safeguards in place with associated work practices to protect entity personnel, the public, and the environment from exposure to the select agent or toxin including, but not limited to:

• Engineering controls such as containment equipment; including, but not limited to:
  • Biological safety cabinets
  • Animal caging systems
  • Centrifuge safety containers
• Administrative Controls such as vaccinations and the creation of biosafety plans and procedures
• Work Practices such as procedures that describes safe and proper work practices
• Personal protective equipment (PPE)

Please note that the entity should focus and think about the agents and procedures specific to their facility to ensure that they are using the appropriate equipment and practices for the conditions of their laboratory.

Engineering controls

The basic concept behind engineering controls is that, to the extent feasible, the work environment and the biosafety/biocontainment risk associated with the laboratory procedures should be designed to eliminate hazards or reduce exposure to hazards. Engineering controls should be based on the following principles:

• If feasible, design the facility, equipment, or process to remove the hazard.
• If removal is not feasible, enclose the hazard to prevent exposure during normal operations.
• Where complete enclosure is not feasible, establish barriers or local ventilation to reduce exposure to the hazard during normal operations.

The basic types of engineering controls are:

• Process control
• Enclosure and/or isolation of source
• Ventilation

Some examples of engineering controls may include:

• Building ventilation/exhaust or HVAC (heating, ventilation and air conditioning) must provide safe, comfortable, breathable environments for all employees and the public, and to minimize exposures to hazardous air contaminants. At BSL-3 and BSL-4, exhaust laboratory air must be directly exhausted to the outside since it is considered potentially contaminated. The exhausted room air can be high-efficiency particulate air (HEPA)-filtered to prevent the hazards from being released to the outside environment.  The HVAC exhaust system must be sized to handle both the room exhaust and the exhaust requirements of all containment devices that may be present. Adequate supply air must be provided to ensure proper function of the exhaust system.
• Biological safety cabinet (BSC) is an enclosed, ventilated laboratory workspace for safely working with materials contaminated with BSAT.  The BMBL is an excellent reference to assist with identifying and selecting a BSC.
• Effluent Decontamination System (EDS) is defined as a system that sterilizes biohazardous liquid waste generated from biocontainment laboratories or other facilities prior to discharge.
• Pathological incinerators, alkaline hydrolysis digesters, or other approved means, must be provided for the safe disposal of the large carcasses of infected animals. Redundancy and the use of multiple technologies need to be considered and evaluated.
• Anaerobic digesters use a biochemical process in which organic matter is decomposed by bacteria in the absence of oxygen. Digesters must be airtight (no oxygen) for anaerobic digestion to occur.

Containment equipment

The containment equipment should focus on:

• Biological safety cabinets Evers et. al. (2013) Laboratory Decontamination of HHS-listed and HHS/USDA Overlap Select Agents and Toxins.  Applied Biosafety. 18: 2, pp. 59-72.
• Animal/arthropod caging systems
• Plant growth chambers
• Centrifuge safety containers

Secondary Containment

Secondary containment is the protection of the environment external to the laboratory from exposure to infectious materials and is provided by a combination of facility design and operational practices. Secondary containment may include separation of the laboratory work area from public access, availability of decontamination equipment (e.g., autoclave), separate clean and dirty corridors, double entry ways, air locks, hand washing facilities, etc.

Enclosure and Isolation

An enclosure keeps a selected hazard “physically” separated from workers. Enclosed equipment, for example, is tightly sealed and it is typically only opened for moving samples/cultures or for cleaning and maintenance. Examples include closed animal caging, “glove boxes” or Class III biosafety cabinets. Care must be taken when the enclosure is opened for maintenance as exposure could occur if adequate precautions are not taken. The enclosure itself must be well maintained to prevent leaks.

Isolation places the hazardous process “geographically” away from the majority of the workers. Common isolation techniques are to create a contaminant-free area either around the equipment or around the employee workstations.

Administrative Controls

Administrative controls are those that modify workers’ work schedules and tasks in ways that minimize
their exposure to workplace hazards.  Examples include vaccinations and developing plans and procedures to reduce the risk to the worker.  The plan should explain the following:

• Process controls should be appropriate for the activities performed and the select agent or toxin in use. Biosafety/biocontainment levels are dependent on the risks of the work being performed.
  • For example, the BMBL recommends BSL-3 practices, containment equipment and facilities for all manipulations of suspect cultures of Francisella tularensis. In contrast, BSL-2 practices, containment equipment, and facilities are recommended for diagnostic activities involving infectious cultures of Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, where there is no propagation of the agent or risk of aerosol or droplet formation only. All other activities with these agents are to be conducted at BSL-3.
• Describe detailed safety measures to ensure that primary and secondary containment are maintained during especially hazardous procedures (e.g., intentional production of select agent infectious aerosols or select toxin aerosols).

Work Practices

Work practices should involve procedures to reduce the risk of exposure (e.g., hand washing, spill procedures, using foot decontamination methods, maintaining the concept of clean and dirty spaces, etc.). Monitoring should be done before and after any change is implemented to make sure a change results in lower exposures. The plan should also describe the biosafety and containment procedures employed for experimentally exposed or infected animals or plants, if applicable:

• When animals or plants are to be infected with or exposed to select agents, describe the administration route(s) employed and the equipment used.
• Describe in detail appropriate containment of all organic material (select agent-infected carcasses, tissues, plant biomass) until final destruction (e.g., autoclave, incineration, etc.).
• Describe or reference procedures to monitor animals or plants for accidental infection.
• Describe procedures to ensure containment of animals accidentally exposed to or infected with select agents. Considerations for developing these procedures include but are not limited to, situations where an airflow reversal has occurred from a room harboring experimentally infected animals to an adjacent room housing native animals; or movement of personnel, equipment, or laboratory waste from a select agent area to a non-select agent area has resulted in accidental exposure.
• When animals infected with select agents are either loosely housed or housed in open caging, there is an increased potential of room-level select agent contamination. Unless it can be demonstrated that the animal model does not shed the agent, the increased hazard of not using containment caging must be mitigated by procedural or facility enhancements.

Personal protective equipment (PPE)

In determining the PPE and other safety equipment needed, consider the hazardous characteristics of each agent or toxin listed on the entity’s registration and the risk associated with laboratory procedures related to the select agent or toxin. The PPE and other safety equipment should focus on:

• Breathing or respiratory protection
• Eye and face protection
• Head protection
• Hearing protection
• Hand/arm protection (gloves, sleeves)
• Foot protection
• Full body protection

When considering laboratory clothing, the entity needs to determine what PPE should be worn to prevent hazards from leaving the laboratory (i.e., how clothing can be a fomite to carry BSAT out of laboratories and how the clothing should be cleaned, disinfected, or disposed, should street clothes being worn or wearing scrubs or full body PPE, should individuals shower out, include general principles for separation of clean and dirty boundaries).  Employees should be educated that PPE must not be worn outside the containment laboratory except when transporting samples between laboratories within containment. It must not be worn (or stored) in break rooms, office areas, toilets, or outside the building.  Employees must be properly instructed on how to don (put on) required PPE before entering an area with a potential hazard that requires the use of the PPE. Procedures for employees to remove (doff) required PPE before leaving the area of potential exposure should be structured to prevent transfer of infectious material outside laboratory room, as well as protect workers from exposure to infectious agents during exit procedures.

Biological Safety – Personal Protective Equipment (PPE) Requirements

Disinfection, Decontamination or Destruction of Select Agent and Toxin [1]

See the Inactivation guidance for more information on the inactivation of and rendering samples free of select agents and select toxins for future use.  In addition, reference Appendix I: Principles of decontamination, sterilization, and disinfection provides additional information regarding decontamination and disinfection.

For material that is disinfected, decontaminated, or destroyed as waste, section 12(a)(3) states that the biosafety/biocontainment plan must contain written procedures for each validated method used for disinfection, decontamination, or destruction, as appropriate, of all contaminated or presumptively contaminated material including, but not limited to:

• Cultures and other materials related to the propagation of select agents or toxins
• Items related to the analysis of select agents and/or toxins
• Personal protective equipment
• Animal caging systems and bedding (if applicable)
• Animal carcasses or extracted tissues and fluids (if applicable)
• Plant biomass (if applicable)
• Laboratory surfaces and equipment
• Surfaces of transport containers
• Effluent material

The plan should describe the following:

• Adherence to the concentration and contact time specified by the manufacturer of a disinfectant during laboratory surface decontamination procedures to be effective in decontaminating the select agent and toxin material.
• Ensure that procedures follow any equipment manufacturer guidance on the disinfectants compatible with their equipment.
• Define waste management procedures based on the types of waste generated (e.g., PPE, plates, liquids, eggs, animal caging, carcasses, sharps) and the containers most appropriate for the types of waste being produced.
• Describe in detail safety procedures for decontaminating reusable sharps.
• Describe the procedure for safe transport of waste to the decontamination site, including the location of the decontamination equipment in relation to the laboratory generating the waste. Transport procedures must take into account any safety requirements to protect personnel and the environment during transport.
• Specify the actual method(s) used to decontaminate select agent and toxin waste (e.g., autoclave, incinerators, renderers, tissue digester, chemical, etc.).
• Describe the means of verifying that decontamination equipment is operating correctly, and how often verification is performed (i.e., biological indicators [BIs], confirmation of cycle parameters).
  • For autoclave verification, BIs or parametric monitors should be placed in the center of the load in a manner expected to provide the maximum challenge for steam penetration. When BIs are used, they should be incubated for the length of time stated by the manufacturer and a positive control should be used. The temperature of the material to be autoclaved must be considered when verifying the autoclave parameters (e.g., frozen carcasses will require a longer sterilization time than non-frozen carcasses).
  • For chemical decontamination, the chemical used must be appropriate for the select agent or toxin (Manufacturers normally test surrogates and not select agents and toxins.  This would be acceptable), and the chemical concentration and contact time must be defined in the procedure. The procedure should also address whether chemicals used for decontamination must be freshly prepared or can be stored, and the shelf life if stored.
• Describe the method(s) used to decontaminate laboratory surfaces and equipment (e.g., chemical surface decontamination, or space fumigation using Vaporized Hydrogen Peroxide, paraformaldehyde, or chlorine dioxide). The method selected must be appropriate for the equipment and the select agents and toxins used in the laboratory. Procedures should indicate contact time required which may be variable depending on agent and equipment.
  • Fumigation used as a means to inactivate select toxins requires the use of a published method or method validation.
  • Fumigation procedures for select agent inactivation should include the use of biological indicators to verify adequate decontamination.
• Describe how entity personnel are notified of the status of decontamination of laboratory surfaces and equipment.
• Describe how entity personnel are notified of ongoing or completed decontamination activities for laboratory spaces.
• Describe when laboratory surfaces and equipment should be decontaminated.

Note: Although the regulations require written procedures for each validated method used for disinfection, decontamination or destruction, as appropriate, of all contaminated or presumptively contaminated materials, an entity does not have to validate the method in-house. A validated method is a method that has been shown to render materials safe to handle (i.e., safe in the context of being reasonably free from a risk of disease transmission). The validation of methods for disinfection, decontamination or destruction of select agent waste does not have to occur in-house since this material is not for future use. Further, validation does not have to be performed on select agents but can be performed on surrogates. However, entities must use the concentrations and conditions prescribed by manufacturers, the BMBL, or other government regulations, such as those promulgated by the Environmental Protection Agency (EPA). The inactivation provisions for future use material do not apply to disinfection, decontamination or destruction of select agent waste.

---

[1] Evers et. al. (2013) Laboratory Decontamination of HHS-listed and HHS/USDA Overlap Select Agents and Toxins.  Applied Biosafety. 18: 2, pp. 59-72.

---

Handling Select Agents and Toxins in Shared Spaces

Section 12(a)(4) of the regulations requires the entity to describe procedures for the handling of select agents and toxins in the same spaces with non-select agents and toxins in order to prevent unintentional contamination. For example:

• Laboratory work surfaces, equipment, and all select agent and toxin waste that must be decontaminated prior to transitioning to work with non-select agents or toxins.
• How personnel are made aware of the status of any particular room or laboratory at any given time.
• Spatial and/or temporal considerations when performing tissue culture studies.
• Any concurrent work with Reconstructed 1918 Influenza virus and highly pathogenic avian influenza virus.
• Sterilization of all samples at the end of the study/experiment/procedure.

Precautions should be taken to prevent cross-contamination of viral select agents in cell cultures. Some means of preventing accidental transfer of agents between cultures include:

• Working with only one select agent at a time.
• Decontaminating biosafety cabinet with a surface disinfectant between select agents and toxins.
• Allowing a certain amount of time to pass between decontamination and beginning work with another select agent.
• Changing gloves when changing from one select agent to another.
• Aliquoting growth medium and other reagents so that the same vessel is not used for more than one select agent.