CHAPTER 9

BIOLOGICAL SAFETY

19.0 APPENDICES

 

19.1  Appendix A - Risk Group 5 (RG5) - Animal Viral Etiological Agents in Common Use

19.2  Appendix B - Pathogens of Veterinary Significance

19.3  Appendix C - Footnotes & References

19.4  Appendix D - Laboratory Biosafety Level Criteria

19.5  Appendix E - Vertegrate Animal Biosafety Level Criteria

19.6  Appendix F - Biosafety in Microbiological and Biomedical Laboratories (MBML)-5th Editioon Table of Contents

19.7  Appendix G - UTMB Bloodborne Pathogen Exposure Control Policy


19.1       Appendix A - Rish Group 5 (RG5) - Animal Viral Etiological Agents in Common Use

The following list of animal etiologic agents is appended to the list of human etiologic agents.  None of these agents is associated with disease in healthy adult humans; they are commonly used in laboratory experimental work.

 

A containment level appropriate for RG1 human agents is recommended for their use.  For agents that are infectious to human cell, e.g., amphotropic and xenotropic strains of murine leukemia virus, a containment level appropriate for RG2 human agents is recommended.

 

            Baculoviruses

            Herpesviruses

                        Herpesvirus ateles

                        Herpesvirus saimiri

                        Marek’s disease virus

                        Murine cytomegalovirus

            Papovaviruses

                        Bovine papilloma virus

                        Polyoma virus

                        Shope papilloma virus

                        Simian virus 40 (SV40)

            Retroviruses

                        Avian leukosis virus

                        Avian sarcoma virus

                        Bovine leukemia virus

                        Feline leukemia virus

                        Feline sarcoma virus

                        Gibbon leukemia virus

                        Mason-Pfizer monkey virus

                        Mouse mammary tumor virus

                        Murine leukemia virus

                        Murine sarcoma  virus

                        Rat leukemia virus

 

19.2       Appendix B - Pathogens of Veterinary Significance

Nonindigenous pathogens of domestic livestock and poultry may require special laboratory design, operation, and containment features not generally addressed in this document.  The importation, possession, or use of the following agents is prohibited or restricted by law or by U.S. Department of Agriculture regulations or administrative policies:

African horse sickness virus

Louping ill virus

African swine fever virus

Lumpy skin disease virus

Akabane virus

Malignant catarrhal fever virus (exotic strains or alcelaphine herpesvirus type 1)

Avian influenza virus (highly pathogenic)

Menangle virus

Bacillus anthracis

Mycobacterium bovis

Besnoitia besnoiti

Mycoplasma agalactiae

Bluetongue virus (exotic)

Mycoplasma mycoides subsp. mycoides, (small colony type)

Borna disease virus

Mycoplasma capricolum

Bovine infectious petechial fever agent

Nairobi sheep disease virus (Ganjam virus)

Bovine spongiform encephalopathy prion

Newcastle disease virus (velogenic strains)

Brucella abortus

Nipah virus

Brucella melitensis

Peste des petits ruminants virus (plague of small ruminants)

Brucella suis

Rift Valley fever virus

Burkholderia mallei/Pseudomonas mallei (Glanders)

Rinderpest virus

Burkholderia pseudomallei

Sheep pox virus

Camelpox virus

Spring Viremia of Carp virus

Classical swine fever virus

Swine vesicular disease virus

Coccidioides immitis

Teschen disease virus

Cochliomyia hominivorax (Screwworm)

Theileria annulata

Coxiella burnetti (Q fever)

Theileria lawrencei

Ephemeral fever virus

Theileria bovis

Ehrlichia (Cowdria) ruminantium (heartwater)

Theileria hirci

Eastern equine encephalitis virus

Trypanosoma brucei

Foot and mouth disease virus

Trypanosoma congolense

Francisella tularensis

Trypanosoma equiperdum (dourine)

Goat pox

Trypanosoma evansi

Hemorrhagic disease of rabbits virus

Trypanosoma vivax

Hendra virus

Venezuelan equine encephalomyelitis virus

Histoplasma (Zymonema) farciminosum

Vesicular exanthema virus

Infectious salmon anemia virus

Vesicular stomatitis virus (exotic)

Japanese encephalitis virus

Wesselsbron disease virus

 

19.3       Appendix C - Footnotes & References

a)      The original reference for this classification was the publication Classification of Etiologic Agents on the Basis of Hazard, 4th edition, July 1974, U.S. Department of Health, Education and Welfare, Public Health Service, Center for Disease Control, Office of Biosafety, Atlanta, Georgia 30333.  For the purpose of these Guidelines, this list has been revised by the NIH. 

b)      A USDA permit, required for import and interstate transport of pathogens, may be obtained from:

U. S. Department of Agriculture

Animal and Plant Health Inspection Service

Veterinary Services

import-export Products Staff

Room 756,  Federal Building

6505 Blecrest Road

Hyattsville, MD 20782.

Telephone:  (301) 436-7830 or (301) 436-8499

FAX:          (301) 436-8226 

c)      All activities, including storage of variola and whitepox, are restricted to the single national facility (World Health Organization-WHO Collaborating Center for Smallpox Research, Center for Disease Control, in Atlanta). 

d)      National Cancer Institute Safety Standards for Research Involving Oncogenic Viruses (October, 1974).  U.S. Department of Health, Education and Welfare Publication No. (NIH) 75-790. 

e)      U.S. Department of Agriculture, Animal and Plant Health Inspection Service. 

f)       Biosafety in Microbiological and Biomedical Laboratories, Centers for Disease Control and National Institutes of Health, 4th edition, May 1999.

 

19.4       Appendix D - Laboratory Biosafety Level Criteria

The essential elements of the four biosafety levels for activities involving infectious microorganisms and laboratory animals are summarized in Tables 1 of this section and Section IV.  The levels are designated in ascending order, by degree of protection provided to personnel, the environment, and the community.     

Biosafety Level 1 (BSL-1)  

Biosafety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is neither required nor generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science. 

The following standard and special practices, safety equipment and facilities apply to agents assigned to Biosafety Level 1:  

Standard Microbiological Practices  

·         Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress.

·         Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.

·         Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.

·         Mouth pipetting is prohibited; mechanical pipetting devices are used.

·         Policies for the safe handling of sharps are instituted.

·         All procedures are performed carefully to minimize the creation of splashes or aerosols.

·         Work surfaces are decontaminated at least once a day and after any spill of viable material.

·         All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated outside of the immediate laboratory are packaged in accordance with applicable local, state, and federal regulations before removal from the facility.

·         A biohazard sign can be posted at the entrance to the laboratory whenever infectious agents are present. The sign may include the name of the agent(s) in use and the name and phone number of the investigator.

·         An insect and rodent control program is in effect (see Appendix G of BMBL, 5th Ed).  

 

Special Practices 

None  

 

Safety Equipment (Primary Barriers)  

·         Special containment devices or equipment such as biological safety cabinets are generally not required for manipulations of agents assigned to Biosafety Level 1.

·         It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.

·         Gloves should be worn if the skin on the hands is broken or if a rash is present. Alternatives to powdered latex gloves should be available.

·         Protective eyewear should be worn for conduct of procedures in which splashes of microorganisms or other hazardous materials is anticipated. 

 

Laboratory Facilities (Secondary Barriers)

 

·         Laboratories should have doors for access control.

·         Each laboratory contains a sink for handwashing.

·         The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are prohibited.

·         Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surface and equipment.

·         Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning.

·         If the laboratory has windows that open to the exterior, they are fitted with fly screens.  

 

Biosafety Level 2 (BSL-2) 

Biosafety Level 2 is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential   hazard to personnel and the environment. It differs from BSL-1 in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; (2) access to the laboratory is limited when work is being conducted; (3) extreme precautions are taken with contaminated sharp items; and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment. 

The following standard and special practices, safety equipment, and facilities apply to agents assigned to Biosafety Level 2:

 

Standard Microbiological Practices 

·         Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.

·         Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.

·         Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.

·         Mouth pipetting is prohibited; mechanical pipetting devices are used.

·         Policies for the safe handling of sharps are instituted.

·         All procedures are performed carefully to minimize the creation of splashes or aerosols.

·         Work surfaces are decontaminated on completion of work or at the end of the day and after any spill or splash of viable material with disinfectants that are effective against the agents of concern.

·         All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated off-site from the facility are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility.

·         An insect and rodent control program is in effect (see Appendix G of BMBL, 5th Ed.). 

 

Special Practices 

·         Access to the laboratory is limited or restricted by the laboratory director when work with infectious agents is in progress. In general, persons who are at increased risk of acquiring infection, or for whom infection may have serious consequences, are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosuppressed may be at increased risk of acquiring infections. The laboratory director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory or animal room.

·         The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential hazards and meet specific entry requirements (e.g., immunization) may enter the laboratory.

·         A biohazard sign must be posted on the entrance to the laboratory when etiologic agents are in use.  Appropriate information to be posted includes the agent(s) in use, the biosafety level, the required immunizations, the investigator's name and telephone number, any personal protective equipment that must be worn in the laboratory, and any procedures required for exiting the laboratory.

·         Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).

·         When appropriate, considering the agent(s) handled, baseline serum samples for laboratory and other at-risk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the facility.

·         Biosafety procedures are incorporated into standard operating procedures or in a biosafety manual adopted or prepared specifically for the laboratory by the laboratory director. Personnel are advised of special hazards and are required to read and follow instructions on practices and procedures.

·         The laboratory director ensures that laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates or additional training as necessary for procedural or policy changes.

·         A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. 

Ø      Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plastic ware should be substituted for glassware whenever possible.

Ø      Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.

Ø      Syringes, which re-sheathe the needle, needle-less systems, and other safety devices are used when appropriate.

Ø      Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal, according to any local, state, or federal regulations. 

·         Cultures, tissues, specimens of body fluids, or potentially infectious wastes are placed in a container with a cover that prevents leakage during collection, handling, processing, storage, transport, or shipping.

·         Laboratory equipment and work surfaces should be decontaminated with an effective disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials.  Contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility.

·         Spills and accidents that result in overt exposures to infectious materials are immediately reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

·         Animals not involved in the work being performed are not permitted in the lab. 

 

Safety Equipment (Primary Barriers) 

·         Properly maintained biological safety cabinets, preferably Class II, or other appropriate personal protective equipment or physical containment devices are used whenever: 

Ø      Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or embryonated eggs.

Ø      High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a biological safety cabinet. 

·         Face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays of infectious or other hazardous materials to the face when the microorganisms must be manipulated outside the BSC.

·         Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn while in the laboratory. This protective clothing is removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). All protective clothing is either disposed of in the laboratory or laundered by the institution; personnel should never take it home.

·         Gloves are worn when hands may contact potentially infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate. Gloves are disposed of when overtly contaminated, and removed when work with infectious materials is completed or when the integrity of the glove is compromised. Disposable gloves are not washed, reused, or used for touching "clean" surfaces (keyboards, telephones, etc.), and they should not be worn outside the lab. Alternatives to powdered latex gloves should be available. Hands are washed following removal of gloves. 

 

Laboratory Facilities (Secondary Barriers) 

·         Provide lockable doors for facilities that house restricted agents (as defined in 42 CFR 72.6).

·         Consider locating new laboratories away from public areas.

·         Each laboratory contains a sink for handwashing.

·         The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are inappropriate.

·         Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.

·         Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning.  Chairs and other furniture used in laboratory work should be covered with a non-fabric material that can be easily decontaminated.

·         Install biological safety cabinets in such a manner that fluctuations of the room supply and exhaust air do not cause the biological safety cabinets to operate outside their parameters for containment. Locate biological safety cabinets away from doors, from windows that can be opened, from heavily traveled laboratory areas, and from other potentially disruptive equipment so as to maintain the biological safety cabinets' air flow parameters for containment.

·         An eyewash station is readily available.

·         Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.

·         There are no specific ventilation requirements.  However, planning of new facilities should consider mechanical ventilation systems that provide an inward flow of air without recirculation to spaces outside of the laboratory. If the laboratory has windows that open to the exterior, they are fitted with fly screens.  

 

 Biosafety Level 3 (BSL-3)  

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents, which may cause serious or potentially lethal disease as a result of exposure, by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. 

Personnel wearing appropriate personal protective clothing and equipment conduct within biological safety cabinets or other physical containment devices, or all procedures involving the manipulation of infectious materials. The laboratory has special engineering and design features. 

It is recognized, however, that some existing facilities may not have all the facility features recommended for Biosafety Level 3 (i.e., double-door access zone and sealed penetrations). In this circumstance, an acceptable level of safety for the conduct of routine procedures, (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, susceptibility testing, etc.), may be achieved in a Biosafety Level 2 facility, providing 1) the exhaust air from the laboratory room is discharged to the outdoors, 2) the ventilation to the laboratory is balanced to provide directional airflow into the room, 3) access to the laboratory is restricted when work is in progress, and 4) the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the laboratory director. 

The following standard and special safety practices, equipment and facilities apply to agents assigned to Biosafety Level 3: 

 

Standard Microbiological Practices 

·         Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.

·         Persons wash their hands after handling infectious materials, after removing gloves, and when they leave the laboratory.

·         Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the laboratory. Persons who wear contact lenses in laboratories should also wear goggles or a face shield.  Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.

·         Mouth pipetting is prohibited; mechanical pipetting devices are used.

·         Policies for the safe handling of sharps are instituted.

·         All procedures are performed carefully to minimize the creation of aerosols.

·         Work surfaces are decontaminated at least once a day and after any spill of viable material.

·         All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are placed in a durable, leakproof container and closed for transport from the laboratory. Infectious waste from BSL-3 laboratories should be decontaminated before removal for off-site disposal.

·         An insect and rodent control program is in effect (see Appendix G of BMBL, 5th Ed.).  

 

Special Practices  

·         Laboratory doors are kept closed when experiments are in progress.

·         The laboratory director controls access to the laboratory and restricts access to persons whose presence is required for program or support purposes.  Persons who are at increased risk of acquiring infection or for whom infection may have serious consequences are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosurppressed may be at risk of acquiring infections. The director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. No minors should be allowed in the laboratory.

·         The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential biohazard, who meet any specific entry requirements (e.g., immunization), and who comply with all entry and exit procedures, enter the laboratory or animal rooms.

·         When infectious materials or infected animals are present in the laboratory or containment module, a hazard warning sign, incorporating the universal biohazard symbol, is posted on all laboratory and animal room access doors. The hazard warning sign identifies the agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates any special requirements for entering the laboratory, such as the need for immunizations, respirators, or other personal protective measures.

·         Laboratory personnel receive the appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing), and periodic testing as recommended for the agent being handled.

·         Baseline serum samples are collected as appropriate and stored for all laboratory and other at-risk personnel. Additional serum specimens may be periodically collected, depending on the agents handled or the function of the laboratory.

·         A biosafety manual specific to the laboratory is prepared or adopted by the laboratory director and biosafety precautions are incorporated into standard operating procedures. Personnel are advised of special hazards and are required to read and follow instructions on practices and procedures.

·         Laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates or additional training as necessary for procedural changes.

·         The laboratory director is responsible for ensuring that, before working with organisms at Biosafety Level 3, all personnel demonstrate proficiency in standard microbiological practices and techniques, and in the practices and operations specific to the laboratory facility. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in safe microbiological practices and techniques.

·         A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. 

Ø      Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plastic ware should be substituted for glassware whenever possible.

Ø      Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.

Ø      Syringes, which re-sheathe the needle, needle-less systems, and other safe devices are used when appropriate.

Ø      Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, and disposed of according to any local, state, or federal regulations.  

·         All open manipulations involving infectious materials are conducted in biological safety cabinets or other physical containment devices within the containment module. No work in open vessels is conducted on the open bench. Clean-up is facilitated by using plastic-backed paper toweling on non-perforated work surfaces within biological safety cabinets.

·         Laboratory equipment and work surfaces should be decontaminated routinely with an effective disinfectant, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials. 

Ø      Spills of infectious materials are decontaminated, contained and cleaned up by appropriate professional staff, or others properly trained and equipped to work with concentrated infectious material. Spill procedures are developed and posted.

Ø      Contaminated equipment must be decontaminated before removal from the facility for repair or maintenance or packaging for transport, in accordance with applicable local, state, or federal regulations. 

·         Cultures, tissues, specimens of body fluids, or wastes are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.

·         All potentially contaminated waste materials (e.g., gloves, lab coats, etc.) from laboratories are decontaminated before disposal or reuse.

·         Spills and accidents that result in overt or potential exposures to infectious materials are immediately reported to the laboratory director.  Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained.

·         Animals and plants not related to the work being conducted are not permitted in the laboratory. 

 

Safety Equipment (Primary Barriers)  

·         Protective laboratory clothing such as solid-front or wrap-around gowns, scrub suits, or coveralls are worn by workers when in the laboratory. Protective clothing is not worn outside the laboratory. Reusable clothing is decontaminated before being laundered. Clothing is changed when overtly contaminated.

·         Gloves must be worn when handling infectious materials, infected animals, and contaminated equipment.

·         Frequent changing of gloves accompanied by hand washing is recommended. Disposable gloves are not reused.

·         All manipulations of infectious materials, necropsy of infected animals, harvesting of tissues or fluids from infected animals or embryonated eggs, etc., are conducted in a Class II or Class III biological safety cabinet (see Appendix A of BMBL, 5th Ed.).

·         When a procedure or process cannot be conducted within a biological safety cabinet, then appropriate combinations of personal protective equipment (e.g., respirators, face shields) and physical containment devices (e.g., centrifuge safety cups or sealed rotors) are used.

·         Respiratory and face protection are used when in rooms containing infected animals. 

 

Laboratory Facilities (Secondary Barriers)  

·         The laboratory is separated from areas that are open to unrestricted traffic flow within the building, and access to the laboratory is restricted. Passage through a series of two self-closing doors is the basic requirement for entry into the laboratory from access corridors. Doors are lockable (see Appendix F of BMBL, 5th Ed.). A clothes change room may be included in the passageway.

·         Each laboratory room contains a sink for handwashing. The sink is hands-free or automatically operated and is located near the room exit door.

·         The interior surfaces of walls, floors, and ceilings of areas where BSL-3 agents are handled are constructed for easy cleaning and decontamination. Seams, if present, must be sealed. Walls, ceilings, and floors should be smooth, impermeable to liquids and resistant to the chemicals and disinfectants normally used in the laboratory. Floors should be monolithic and slip-resistant. Consideration should be given to the use of covered floor coverings. Penetrations in floors, walls, and ceiling surfaces are sealed. Openings such as around ducts and the spaces between doors and frames are capable of being sealed to facilitate decontamination.

·         Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and those chemicals used to decontaminate the work surfaces and equipment.

·         Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning.  Chairs and other furniture used in laboratory work should be covered with a non-fabric material that can be easily decontaminated.

·         All windows in the laboratory are closed and sealed.

·         A method for decontaminating all laboratory wastes is available in the facility and utilized, preferably within the laboratory (i.e., autoclave, chemical disinfection, incineration, or other approved decontamination method). Consideration should be given to means of decontaminating equipment. If waste is transported out of the laboratory, it should be properly sealed and not transported in public corridors.

·         Biological safety cabinets are required and are located away from doors, from room supply louvers, and from heavily-traveled laboratory areas. 

·         A ducted exhaust air ventilation system is provided.  This system creates directional airflow, which draws air into the laboratory from "clean" areas and toward "contaminated" areas. The exhaust air is not recirculated to any other area of the building.  Filtration and other treatments of the exhaust air are not required, but may be considered based on site requirements, and specific agent manipulations and use conditions. The outside exhaust must be dispersed away from occupied areas and air intakes, or the exhaust must be HEPA-filtered. Laboratory personnel must verify that the direction of the airflow (into the laboratory) is proper. It is recommended that a visual monitoring device that indicates and confirms directional inward airflow be provided at the laboratory entry.  Consideration should be given to installing an HVAC control system to prevent sustained positive pressurization of the laboratory. Audible alarms should be considered to notify personnel of HVAC system failure.

·         HEPA-filtered exhaust air from a Class II biological safety cabinet can be recirculated into the laboratory if the cabinet is tested and certified at least annually. When exhaust air from Class II safety cabinets is to be discharged to the outside through the building exhaust air system, the cabinets must be connected in a manner that avoids any interference with the air balance of the cabinets or the building exhaust system (e.g., an air gap between the cabinet exhaust and the exhaust duct). When Class III biological safety cabinets are used they should be directly connected to the exhaust system. If the Class III cabinets are connected to the supply system, it is done in a manner that prevents positive pressurization of the cabinets (see Appendix A of BMBL, 5th Ed.).

·         Continuous flow centrifuges or other equipment that may produce aerosols are contained in devices that exhaust air through HEPA filters before discharge into the laboratory. These HEPA systems are tested at least annually. Alternatively, the exhaust from such equipment may be vented to the outside if it is dispersed away from occupied areas and air intakes.

·         Vacuum lines are protected with liquid disinfectant traps and HEPA filters, or their equivalent. Filters must be replaced as needed. An alternative is to use portable vacuum pumps (also properly protected with traps and filters).

·         An eyewash station is readily available inside the laboratory.

·         Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.

·         The Biosafety Level 3 facility design and operational procedures must be documented. The facility must be tested for verification that the design and operational parameters have been met prior to operation.  Facilities should be re-verified, at least annually, against these procedures as modified by operational experience.

·         Additional environmental protection (e.g., personnel showers, HEPA filtration of exhaust air, containment of other piped services and the provision of effluent decontamination) should be considered if recommended by the agent summary statement, as determined by risk assessment, the site conditions, or other applicable federal, state, or local regulations.  

 

Biosafety Level 4 (BSL-4)  

Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. Agents with a close or identical antigenic relationship to Biosafety Level 4 agents are handled at this level until sufficient data are obtained either to confirm continued work at this level, or to work with them at a lower level. Members of the laboratory staff have specific and thorough training in handling extremely hazardous infectious agents and they understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics. They are supervised by competent scientists who are trained and experienced in working with these agents. Access to the laboratory is strictly controlled by the laboratory director. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. A specific facility operations manual is prepared or adopted.  

Within work areas of the facility, all activities are confined to Class III biological safety cabinets, or Class II biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. The Biosafety Level 4 laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment.  

The following standard and special safety practices equipment, and facilities apply to agents assigned to Biosafety Level 4:  

 

Standard Microbiological Practices  

·         Access to the laboratory is limited by the laboratory director when experiments are in progress.

·         Policies for safe handling of sharps are instituted.

·         All procedures are performed carefully to minimize the creation of aerosols.

·         Work surfaces are decontaminated at least once a day and after any spill of viable material.

·         All waste is decontaminated before disposal by an approved method such as autoclaving.

·         An insect and rodent control program is in effect (see Appendix G of BMBL, 5th Ed.).  

 

Special Practices  

·         Only persons whose presence in the facility or individual laboratory rooms is required for program or support purposes are authorized to enter. Persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. Therefore, persons who may be at increased risk of acquiring infection or for whom infection may be unusually hazardous, such as children or pregnant women, are not allowed in the laboratory or animal rooms.  The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. Access to the facility is limited by means of secure, locked doors; accessibility is managed by the laboratory director, biohazard control officer, or other person responsible for the physical security of the facility. Before entering, persons are advised of the potential biohazards and instructed as to appropriate safeguards for ensuring their safety. Authorized persons comply with the instructions and all other applicable entry and exit procedures. A logbook, signed by all personnel, indicates the date and time of each entry and exit.  Practical and effective protocols for emergency situations are established.

·         When infectious materials or infected animals are present in the laboratory or animal rooms, hazard warning signs, incorporating the universal biohazard  symbol, are posted on all access doors. The sign identifies the agent, lists the name of the laboratory director or other responsible person(s), and indicates any special requirements for entering the area (e.g., the need for immunizations or respirators).

·         The laboratory director is responsible for ensuring that, before working with organisms at Biosafety Level 4, all personnel demonstrate a high proficiency in standard microbiological practices and techniques, and in the special practices and operations specific to the laboratory facility. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in these unique safe microbiological practices and techniques.

·         Laboratory personnel receive available immunizations for the agents handed or potentially present in the laboratory.

·         Baseline serum samples for all laboratory and other at-risk personnel are collected and stored. Additional serum specimens may be periodically collected, depending on the agents handled or the function of the laboratory.  The decision to establish a serologic surveillance program takes into account the availability of methods for the assessment of antibody to the agent(s) of concern. The program provides for the testing of serum samples at each collection interval and the communication of results to the participants.

·         A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and follow instructions on practices and procedures.

·         Laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures.  Personnel receive annual updates or additional training as necessary for procedural changes.

·         Personnel enter and leave the laboratory only through the clothing change and shower rooms. They take a decontaminating shower each time they leave the laboratory. Personnel use the airlocks to enter or leave the laboratory only in an emergency. 

·         Personal clothing is removed in the outer clothing change room and kept there. Complete laboratory clothing, including undergarments, pants and shirts or jumpsuits, shoes, and gloves, is provided and used by all personnel entering the laboratory.

·         When leaving the laboratory and before proceeding into the shower area, personnel remove their laboratory clothing in the inner change room. Soiled clothing is autoclaved before laundering.

·         Supplies and materials needed in the facility are brought in by way of the double-doored autoclave, fumigation chamber, or airlock, which is appropriately decontaminated between each use.

·         After securing the outer doors, personnel within the facility retrieve the materials by opening the interior doors of the autoclave, fumigation chamber, or airlock. These doors are secured after materials are brought into the facility.

·         A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. 

Ø      Needles and syringes or other sharp instruments are restricted in the laboratory for use only when there is no alternative, such as for parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plastic ware should be substituted for glassware whenever possible.

Ø      Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.

Ø      Syringes that re-sheath the needle, needle less systems, and other safety devices are used when appropriate.

Ø      Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass must be decontaminated before disposal, according to any local, state, or federal regulations.  

·         Biological materials to be removed from the Class III cabinet or from the Biosafety Level 4 laboratory in a viable or intact state are transferred to a nonbreakable, sealed primary container and then enclosed in a nonbreakable, sealed secondary container. This is removed from the facility through a disinfectant dunk tank, fumigation chamber, or an airlock designed for this purpose.

·         No materials, except biological materials that are to remain in a viable or intact state, are removed from the Biosafety Level 4 laboratory unless they have been autoclaved or decontaminated before they leave the laboratory. Equipment or material that might be damaged by high temperatures or steam may be decontaminated by gaseous or vapor methods in an airlock or chamber designed for this purpose.

·         Laboratory equipment is decontaminated routinely after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials. Equipment is decontaminated before it is sent for repair or maintenance.

·         Spills of infectious materials are contained and cleaned up by appropriate professional staff or others properly trained and equipped to work with concentrated infectious material. A spill procedure is developed and posted within the laboratory.

·         A system is established for reporting laboratory accidents and exposures and employee absenteeism, and for the medical surveillance of potential laboratory-associated illnesses. Written records are prepared and maintained. An essential adjunct to such a reporting-surveillance system is the availability of a facility for the quarantine, isolation, and medical care of personnel with potential or known laboratory-associated illnesses.

·         Materials not related to the experiment being conducted (e.g., plants, animals, and clothing) are not permitted in the facility.  

 

Safety Equipment (Primary Barriers) 

All procedures within the facility are conducted in the Class III biological safety cabinet or in Class II biological safety cabinets used in conjunction with one-piece positive pressure personnel suits ventilated by a life support system.  

 

Laboratory Facility (Secondary Barriers) 

There are two models for Biosafety Level 4 laboratories: (A) the Cabinet Laboratory where all handling of the agent is performed in a Class III Biological Safety Cabinet, and (B) the Suit Laboratory where personnel wear a protective suit. Biosafety Level-4 laboratories may be based on either model or a combination of both models in the same facility. If a combination is used, each type must meet all the requirements identified for that type.  

(A)   Cabinet Laboratory (See Appendix A of BMBL, 5th Ed.)  

·         The Biosafety Level 4 facility consists of either a separate building or a clearly demarcated and isolated zone within a building. The rooms in the facility are arranged to ensure passage through a minimum of two doors prior to entering the room(s) containing the Class III biological safety cabinet (cabinet room). Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the cabinet room. A double-door autoclave, dunk tank, fumigation chamber, or ventilated anteroom for decontamination is provided at the containment barrier for passage of those materials, supplies, or equipment that are not brought into the cabinet room through the change room.

·         Daily inspections of all containment parameters (e.g., directional airflow) and life support systems are completed before laboratory work is initiated to ensure that the laboratory is operating according to its operating parameters.

·         Walls, floors, and ceilings of the cabinet room and inner change room are constructed to form a sealed internal shell, which facilitates fumigation and is resistant to entry and exit of animals and insects.  Floors are integrally sealed and coved. The internal surfaces of this shell are resistant to liquids and chemicals to facilitate cleaning and decontamination of the area. All penetrations in these structures and surfaces are sealed. Openings around doors into the cabinet room and inner change room are minimized and are capable of being sealed to facilitate decontamination.  Any drains in the cabinet room floor are connected directly to the liquid waste decontamination system.  Sewer vents and other service lines contain HEPA filters and protection against vermin.

·         Bench tops have seamless or sealed surfaces which are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.

·         Laboratory furniture is of simple open construction, capable of supporting anticipated loading and uses.  Spaces between benches, cabinets, and equipment are accessible for cleaning and decontamination.  Chairs and other furniture used in laboratory work should be covered with a non-fabric material that can be easily decontaminated.

·         A hands-free or automatically operated handwashing sink is provided near the door of the cabinet room(s) and the outer and inner change rooms.

·         If there is a central vacuum system, it does not serve areas outside the cabinet room. In-line HEPA filters are placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement. Other liquid and gas services to the cabinet room are protected by devices that prevent backflow.

·         If water fountains are provided, they are automatically or foot-operated and are located in the facility corridors outside the laboratory. The water service to the fountain is isolated from the distribution system supplying water to the laboratory areas and is equipped with a backflow preventer.

·         Access doors to the laboratory are self-closing and lockable.

·         Any windows are breakage-resistant and sealed.

·         Double-door autoclaves are provided for decontaminating materials passing out of both the Class III biological safety cabinet(s) and the cabinet room(s). Autoclaves that open outside of the containment barrier must be sealed to the wall of the containment barrier. The autoclave doors are automatically controlled so that the outside door can only be opened after the autoclave "sterilization" cycle has been completed.

·         Pass-through dunk tanks, fumigation chambers, or equivalent decontamination methods are provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from both the Class III biological safety cabinet(s) and the cabinet room(s).

·         Liquid effluents from the dirty-side inner change room (including toilets) and cabinet room sinks, floor drains (if used), autoclave chambers, and other sources within the cabinet room are decontaminated by a proven method, preferably heat treatment, before being discharged to the sanitary sewer. Effluents from showers and clean-side toilets may be discharged to the sanitary sewer without treatment. The process used for decontamination of liquid wastes must be validated physically and biologically.

·         A dedicated non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to ensure directional airflow from the area of least hazard to the area(s) of greatest potential hazard. The differential pressure/directional airflow between adjacent areas is monitored and alarmed to indicate any system malfunction. An appropriate visual pressure monitoring device that indicates and confirms the pressure differential of the cabinet room is provided and located at the entry to the clean change room. The airflow in the supply and exhaust components is monitored and the HVAC control system is designed to prevent sustained positive pressurization of the laboratory. The Class III cabinet should be directly connected to the exhaust system. If the Class III cabinet is connected to the supply system, it is done in a manner that prevents positive pressurization of the cabinet.

·         The supply air to and exhaust air from the cabinet room, inner change room, and anteroom pass through HEPA filter(s). The air is discharged away from occupied spaces and air intakes. The HEPA filter(s) are located as near as practicable to the source in order to minimize the length of potentially contaminated ductwork. All HEPA filters need to be tested and certified annually. The HEPA filter housings are designed to allow for in situ decontamination of the filter prior to removal, or removal of the filter in a sealed, gas-tight primary container for subsequent decontamination and/or destruction by incineration. The design of the HEPA filter housing should facilitate validation of the filter installation. The use of pre-certified HEPA filters can be an advantage. The service life of the exhaust HEPA filters can be extended through adequate prefiltration of the supply air.

·         The Biosafety Level 4 facility design and operational procedures must be documented. The facility must be tested for verification that the design and operational parameters have been met prior to operation.  Facilities should be re-verified annually against these procedures as modified by operational experience.

·         Appropriate communication systems are provided between the laboratory and the outside (e.g., voice, fax, computer).

 

(B)   Suit Laboratory  

·         The Biosafety Level 4 facility consists of either a separate building or a clearly demarcated and isolated zone within a building. The rooms in the facility are arranged to ensure passage through the changing and decontamination areas prior to entering the room(s) where work is done with BSL-4 agents (suit area). Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the suit area. A specially designed suit area is maintained in the facility to provide personnel protection equivalent to that provided by Class III biological safety cabinets. Personnel who enter this area wear a one-piece positive pressure suit that is ventilated by a life-support system protected by HEPA filtration. The life support system includes redundant breathing air compressors, alarms and emergency backup breathing air tanks. Entry to this area is through an airlock fitted with airtight doors. A chemical shower is provided to decontaminate the surface of the suit before the worker leaves the area. An automatically starting emergency power source is provided at a minimum for the exhaust system, life support systems, alarms, lighting, entry and exit controls, and BSCs.  The air pressure within the suit is positive to the surrounding laboratory. The air pressure within the suit area is lower than that of any adjacent area. Emergency lighting and communication systems are provided. All penetrations into the internal shell of the suit area, chemical shower, and airlocks, are sealed.

·         A daily inspection of all containment parameters (e.g., directional airflow, chemical showers) and life support systems is completed before laboratory work is initiated to ensure that the laboratory is operating according to its operating parameters.

·         A double-doored autoclave is provided at the containment barrier for decontaminating waste materials to be removed from the suit area. The autoclave door, which opens to the area external to the suit area, is sealed to the outer wall of the suit area and is automatically controlled so that the outside door can be opened only after the autoclave "sterilization" cycle. A dunk tank, fumigation chamber, or ventilated airlock for decontamination is provided for passage of materials, supplies, or equipment that are not brought into the suit area through the change room. These devices can be also used for the safe removal of materials, supplies, or equipment from the laboratory that cannot be decontaminated in the autoclave.

·         Walls, floors, and ceilings of the suit area are constructed to form a sealed internal shell, which facilitates fumigation and is animal and insect prohibitive (see Appendix G of BMBL, 5th Ed.). The internal surfaces of this shell are resistant to liquids and chemicals, facilitating cleaning and decontamination of the area. All penetrations in these structures and surfaces are sealed. Any drains in the floor of the suit area contain traps filled with a chemical disinfectant of demonstrated efficacy against the target agent, and they are connected directly to the liquid waste decontamination system. Sewer vents and other service lines contain HEPA filters.

·         Internal facility appurtenances in the suit area, such as light fixtures, air ducts, and utility pipes, are arranged to minimize the horizontal surface area.

·         Bench tops have seamless surfaces which are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.

·         Laboratory furniture is of simple open construction capable of supporting anticipated loading and uses.  Non-porous materials are preferable. Spaces between benches, cabinets, and equipment are accessible for cleaning and decontamination. Chairs and other furniture used in laboratory work should be covered with a non-fabric material that can be easily decontaminated.

·         A hands-free or automatically operated handwashing sink is provided in the suit area(s); handwashing sinks in the outer and inner change rooms should be considered based on the risk assessment.

·         If there is a central vacuum system, it does not serve areas outside the suit area. In-line HEPA filters are placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement. Other liquid and gas services to the suit area are protected by devices that prevent backflow.

·         Access doors to the laboratory are self-closing and lockable. Inner and outer doors to the chemical shower and inner and outer doors to airlocks are interlocked to prevent both doors from being opened simultaneously.

·         Any windows are breakage-resistant and are sealed.

·         Liquid effluents from sinks, floor drains (if used), autoclave chambers and other sources within the containment barrier are decontaminated by a proven method, preferably heat treatment, before being discharged to the sanitary sewer. Effluents from showers and toilets may be discharged to the sanitary sewer without treatment. The process used for decontamination of liquid wastes must be validated physically and biologically.

·         A dedicated non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to ensure directional airflow from the area of least hazard to the area(s) of greatest potential hazard. Redundant supply fans are recommended. Redundant exhaust fans are required. The differential pressure/directional airflow between adjacent areas is monitored and alarmed to indicate malfunction of the system. An appropriate visual pressure monitoring device that indicates and confirms the pressure differential of the suit area must be provided and located at the entry to the clean change room. The airflow in the supply and exhaust components is monitored and an HVAC control system is installed to prevent positive pressurization of the laboratory.

·         The supply air to the suit area, decontamination shower, and decontamination airlock is protected by passage through a HEPA filter. The general room exhaust air from the suit area, decontamination shower and decontamination airlock is treated by a passage through two HEPA filters in series prior to discharge to the outside. The air is discharged away from occupied spaces and air intakes. The HEPA filters are located as near as practicable to the source in order to minimize the length of potentially contaminated ductwork. All HEPA filters need to be tested and certified annually. The HEPA filter housings are designed to allow for in situ decontamination of the filter prior to removal.  Alternatively, the filter can be removed in a sealed, gas-tight primary container for subsequent decontamination and/or destruction by incineration. The design of the HEPA filter housing should facilitate validation of the filter installation. The use of pre-certified HEPA filters can be an advantage. The service life of the exhaust HEPA filters can be extended through adequate prefiltration of the supply air.

·         The positioning of the supply and exhaust points should be such that dead air space in the suit room is minimized.

·         The treated exhaust air from Class II biological safety cabinets, located in a facility where workers wear a positive pressure suit, may be discharged into the room environment or to the outside through the facility air exhaust system. If the treated exhaust is discharged to the outside through the facility exhaust system, it is connected to this system in a manner that avoids any interference with the air balance of the cabinets or the facility exhaust system.

·         The Biosafety Level 4 facility design and operational procedures must be documented. The facility must be tested for verification that the design and operational parameters have been met prior to operation.  Facilities should be re-verified annually against these procedures as modified by operational experience.

·         Appropriate communication systems should be provided between the laboratory and the outside.

 

 TABLE 1.       SUMMARY OF RECOMMENDED BIOSAFETY LEVELS FOR INFECTIOUS AGENTS

Agents

Practices

Safety Equipment (Primary Barriers)

Facilities(Secondary Barriers)

Not known to consistently cause disease in healthy adults

Standard Microbiological

 

Practices

None required

Open bench top sink required

Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure

BSL-1 practice plus:

 

Limited access

 

Biohazard warning signs

 

"Sharps" precautions

 

Biosafety manual defining any needed waste decontamination or medical surveillance policies

Primary barriers = Class I or II BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials.

 

 PPEs: laboratory coats; gloves; face protection as needed

BSL-1 plus:

 

Autoclave available

Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

BSL-2 practice plus: 

 

Controlled access

 

Decontamination of all waste

 

Decontamination of lab clothing before laundering

 

Baseline serum

Primary barriers = Class I or II BCSs or other physical containment devices used for all open manipulations of agents.

 

PPEs: protective lab clothing; gloves; respiratory protection as needed

BSL-2 plus:

 

Physical separation from access corridors

 

Self-closing, double-door access

 

Exhausted air not recirculated

 

Negative airflow into laboratory

Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission

BSL-3 practices plus:

 

Clothing change before entering

 

Shower on exit

 

All material decontaminated on exit from facility

Primary barriers = All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure personnel suit

BSL-3 plus:

 

Separate building or isolated zone

 

Dedicated supply and exhaust, vacuum, and decon systems

 

Other requirements outlined in the text

 

 

19.5       Appendix E - Vertebrate Animal Biosafety Level Criteria

If experimental animals are used, institutional management must provide facilities, staff, and established practices that reasonably ensure appropriate levels of environmental quality, safety, and care. Laboratory animal facilities are simply a special type of laboratory. As a general principle, the biosafety level (facilities, practices, and operational requirements) recommended for working with infectious agents in vivo and in vitro are comparable.  

However, it is best to remember that the animal room can present some unique problems. In the microbiological laboratory, hazardous conditions are caused by personnel or by the equipment being used.  In the animal room, the activities of the animals themselves can present new hazards. Animals may generate aerosols, they may bite and scratch, and they may be infected with a zoonotic disease. 

These recommendations presuppose that laboratory animal facilities, operational practices, and quality of animal care meet applicable standards and regulations (e.g., “Guide for the Care and Use of Laboratory Animals” and Laboratory Animal Welfare Regulations) and that appropriate species have been selected for animal experiments. In addition, the organization should have an occupational health and safety plan. The recent publication of the Institute of Medicine, “Occupational Health and Safety in the Care of Research Animals” is most helpful in this regard.

Ideally, facilities for laboratory animals used in studies of infectious or noninfectious disease should be physically separate from other activities such as animal production and quarantine, clinical laboratories, and especially from facilities providing patient care. Traffic flow that will minimize the risk of cross contamination should be considered in the plans. A "clean/dirty hall" layout may be useful to minimize this risk.  

The recommendations detailed below describe four combinations of practices, safety equipment, and facilities for experiments with animals infected with agents that cause, or may cause, human infection. These four combinations, designated Animal Biosafety Levels (ABSL) 1-4, provide increasing levels of protection to personnel and to the environment, and are recommended as minimal standards for activities involving infected laboratory animals. The four ABSLs describe animal facilities and practices applicable to work with animals infected with agents assigned to Biosafety Levels 1-4, respectively.  

Investigators inexperienced in conducting these types of experiments should seek help in designing their experiments from individuals who are experienced in this special work.  

Facility standards and practices for invertebrate vectors and hosts are not specifically addressed in the standards for commonly used laboratory animals. “Laboratory Safety for Arboviruses and Certain Other Viruses of Vertebrates,” prepared by the Subcommittee on Arbovirus Laboratory Safety (SALS) of the American Committee on Arthropod-Borne Viruses, serves as a useful reference in the design and operation of facilities using arthropods.
 

Animal Biosafety Level 1 (ABSL-1)  

Animal Biosafety Level 1 (ABSL-1) is suitable for work involving well characterized agents that are not known to cause disease in healthy adult humans, and that are of minimal potential hazard to laboratory personnel and the environment.

 

Standard Practices 

·         The animal facility director establishes policies, procedures, and protocols for emergency situations. Each project is subject to pre-approval by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). Any special practices are approved at this time.

·         Only those persons required for program or support purposes are authorized to enter the facility. Before entering, persons are advised of the potential biohazards and are instructed on the appropriate safeguards.

·         An appropriate medical surveillance program is in place.

·         A safety manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and follow instructions on practices and procedures.

·         Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use should only be done in designated areas and are not permitted in animal or procedure rooms.

·         All procedures are carefully performed to minimize the creation of aerosols or splatters.

·         Work surfaces are decontaminated after use or after any spill of viable materials.

·         All wastes from the animal room (including animal tissues, carcasses, and contaminated bedding) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements.  Incineration is recommended.

·         Policies for the safe handling of sharps are instituted.

·         Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility.

·         A biohazard sign must be posted on the entrance to the animal room whenever infectious agents are present.  The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements for entering the animal room (e.g., the need for immunizations and respirators).

·         An insect and rodent control program is in effect (see Appendix G of BMBL, 5th Ed.).  

 

Special Practices 

None.  

 

Safety Equipment (Primary Barriers) 

·         The wearing of laboratory coats, gowns, and/or uniforms in the facility is recommended. Laboratory coats remain in the animal room. Gowns and uniforms are not worn outside the facility.

·         Persons having contact with non-human primates should assess their risk of mucous membrane exposure and wear appropriate eye and face protection (See Ref. 5 at the end of this section).  

 

Facilities (Secondary Barriers) 

·         The animal facility is separated from areas that are open to unrestricted personnel traffic within the building.

·         External facility doors are self-closing and self-locking. Doors to animal rooms open inward, are self-closing, and are kept closed when experimental animals are present. Cubicle room inner doors may open outward or be horizontal or vertical sliding.

·         The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are water resistant.

·         Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas.

·         Windows are not recommended. Any windows must be resistant to breakage. Where possible, windows should be sealed. If the animal facility has windows that open, they are fitted with fly screens.

·         If floor drains are provided, the traps are always filled with water and/or an appropriate disinfectant.

·         Ventilation should be provided in accordance with the “Guide for Care and Use of Laboratory Animals,” latest edition (See Reference #6 at the end of this section). No recirculation of exhaust air should occur. It is recommended that animal rooms maintain negative pressure compared to adjoining hallways.

·         The facility has a hand washing sink.

·         Cages are washed manually or in a cage washer. The mechanical cage washer should have a final rinse temperature of at least 180°F.

·         Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.  

 

Animal Biosafety Level 2 (ABSL-2) 

Animal Biosafety Level 2 involves practices for work with those agents associated with human disease. It addresses hazards from ingestion as well as from percutaneous and mucous membrane exposure. ABSL-2 builds upon the practices, procedures, containment equipment, and facility requirements of ABSL-1.  

 

Standard Practices  

·         Aside from the standard policies, procedures, and protocols for emergency situations established by the facility director, appropriate special policies and procedures should be developed as needed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biohazard Committee (IBC).

·         Access to the animal room is limited to the fewest number of individuals possible. Personnel who must enter the room for program or service purposes when work is in progress are advised of the potential hazard.

·         An appropriate medical surveillance program is in place. All personnel receive appropriate immunizations or tests for the agents handled or potentially present (e.g., hepatitis B vaccine, TB skin testing). When appropriate, a serum surveillance system should be implemented (See Reference # 7 at the end of this section).

·         A biosafety manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and follow instructions on practices and procedures.

·         Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use should only be done in designated areas and are not permitted in animal or procedure rooms.

·         All procedures are carefully performed to minimize the creation of aerosols or splatters.

·         Equipment and work surfaces in the room are routinely decontaminated with an effective disinfectant after work with the infectious agent, and especially after overt spills, splashes, or other contamination by infectious materials.

·         All infectious samples are collected, labeled, transported, and processed in a manner that contains and prevents transmission of the agent(s). All wastes from the animal room (including animal tissues, carcasses, contaminated bedding, unused feed, sharps, and other refuse) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements. The outer surface of the containers is disinfected prior to moving the material. Autoclaving of the contents prior to incineration is recommended.

·         Policies for the safe handling of sharps are instituted.

·         Needles and syringes or other sharp instruments are restricted for use in the animal facility only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles.

·         Syringes that re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

·         Plastic ware should be substituted for glassware whenever possible.

·         Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility.

·         A biohazard sign must be posted on the entrance to the animal room whenever infectious agents are present.  The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements (e.g., the need for immunizations and respirators) for entering the animal room.

·         An insect and rodent control program is in effect (see Appendix G of BMBL, 5th Ed.).  

Animal Biosafety Level 1 (ABSL-1) is suitable for work involving well characterized agents that are not known to cause disease in healthy adult humans, and that are of minimal potential hazard to laboratory personnel and the environment.

 

Special Practices

·         Animal care laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural or policy changes. Records of all training provided are maintained. In general, persons who may be at increased risk of acquiring infection, or for whom infection might be unusually hazardous, are not allowed in the animal facility unless special procedures can eliminate the extra risk.

·         Only animals used for the experiment(s) are allowed in the room.

·         All equipment must be appropriately decontaminated prior to removal from the room.

·         Spills and accidents, which result in overt exposures to infectious materials, must be immediately reported to the facility director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

Animal Biosafety Level 1 (ABSL-1) is suitable for work involving well characterized agents that are not known to cause disease in healthy adult humans, and that are of minimal potential hazard to laboratory personnel and the environment.

 

Safety Equipment (Primary Barriers)

·         Gowns, uniforms, or laboratory coats are worn while in the animal room. The laboratory coat is removed and left in the animal room. Gowns, uniforms, and laboratory coats are removed before leaving the animal facility.  Gloves are worn when handling infected animals and when skin contact with infectious materials is unavoidable.

·         Personal protective equipment is used based on risk assessment determinations (see Section V of BMBL, 5th Ed.). Appropriate face/eye and respiratory protection is worn by all personnel entering animal rooms that house nonhuman primates (See Reference #8 at the end of this section).

·         Biological safety cabinets, other physical containment devices, and/or personal protective equipment (e.g., respirators, face shields) are used whenever conducting procedures with a high potential for creating aerosols. These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, or intranasal inoculation of animals.

·         When needed, animals are housed in primary biosafety containment equipment appropriate for the animal species. Filter top cages are always handled in properly designed and operating animal biocontainment cabinets recommended for rodents.  

 

Animal Biosafety Level 1 (ABSL-1) is suitable for work involving well characterized agents that are not known to cause disease in healthy adult humans, and that are of minimal potential hazard to laboratory personnel and the environment.

 

Facilities (Secondary Barriers)

·         The animal facility is separated from areas that are open to unrestricted personnel traffic within the building.

·         Access to the facility is limited by secure locked doors. External doors are self-closing and self-locking.  Doors to animal rooms open inward, are self-closing, and are kept closed when experimental animals are present.  Cubicle room inner doors may open outward or be horizontal or vertical sliding.

·         The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are water resistant.

·         Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas.

·         Any windows must be resistant to breakage. Where possible, windows should be sealed. If the animal facility has windows that open, they are fitted with fly screens.

·         If floor drains are provided, the traps are always filled with an appropriate disinfectant.

·         Exhaust air is discharged to the outside without being recirculated to other rooms. Ventilation should be provided in accordance with criteria from “Guide for Care and Use of Laboratory Animals,” latest edition. The direction of airflow in the animal facility is inward; animal rooms should maintain negative pressure compared to adjoining hallways.

·         Cages are washed manually or in an appropriate cage washer. The mechanical cage washer should have a final rinse temperature of at least 180°F.

·         An autoclave is available in the animal facility to decontaminate infectious waste.

·         A hand washing sink is in the animal room where infected animals are housed, as well as elsewhere in the facility.

·         Illumination is adequate for all activities, avoiding reflections and glare that could impede vision. 

 

Animal Biosafety Level 3 (ABSL-3)  

Animal Biosafety Level 3 involves practices suitable for work with animals infected with indigenous or exotic agents that present the potential of aerosol transmission and of causing serious or potentially lethal disease. ABSL-3 builds upon the standard practices, procedures, containment equipment, and facility requirements of ABSL-2.  

 

Standard Practices  

·         Aside from the standard policies, procedures, and protocols for emergency situations established by the facility director, appropriate special policies and procedures should be developed as needed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC).

·         The laboratory or animal facility director limits access to the animal room to the fewest number of individuals possible. Personnel who must enter the room for program or service purposes when work is in progress are advised of the potential hazard.

·         An appropriate medical surveillance program is in place. All personnel receive appropriate immunizations or tests for the agents handled or potentially present (e.g., hepatitis B vaccine, TB skin testing). When appropriate, a serum surveillance system should be implemented (see Reference #9 at the end of this section). In general, persons who may be at increased risk of acquiring infection, or for whom infection might have serious consequences, are not allowed in the animal facility unless special procedures can eliminate the extra risk. Assessment should be made by the occupational health physician.

·         A biosafety manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and follow instructions on practices and procedures.

·         Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use should be done only in designated areas and are not permitted in animal or procedure rooms.

·         All procedures are carefully performed to minimize the creation of aerosols or splatters.

·         Equipment and work surfaces in the room are routinely decontaminated with an effective disinfectant after work with the infectious agent, and especially after overt spills, splashes, or other contamination by infectious materials.

·         All wastes from the animal room (including animal tissues, carcasses, contaminated bedding, unused feed, sharps, and other refuse animal tissues) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements. Incineration is recommended. The outer surface of the containers is disinfected prior to moving the material (see Special Practices, bullet #3 below).

·         Policies for the safe handling of sharps are instituted.  

Ø      Needles and syringes or other sharp instruments are restricted in the animal facility for use only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles.

Ø      Syringes that re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

Ø      Plastic ware should be substituted for glassware whenever possible.  

·         Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility.

·         A biohazard sign must be posted on the entrance to the animal room whenever infectious agents are present.  The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements for entering the animal room (e.g., the need for immunizations and respirators).

·         All infectious samples are collected, labeled, transported, and processed in a manner that contains and prevents transmission of the agent(s).

·         Laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. As necessary, personnel receive updates and/or additional training on procedural or policy changes. Records of all training provided are maintained.

·         An insect and rodent control program is in effect.  

Animal Biosafety Level 1 (ABSL-1) is suitable for work involving well characterized agents that are not known to cause disease in healthy adult humans, and that are of minimal potential hazard to laboratory personnel and the environment.

 

Special Practices

·         Cages are autoclaved or thoroughly decontaminated before bedding is removed and before they are cleaned and washed. Equipment must be decontaminated according to any local, state, or federal regulations before being packaged for transport or removal from the facility for repair or maintenance.

·         A spill procedure is developed and posted. Only personnel properly trained and equipped to work with infectious materials are to clean up spills. Spills and accidents that result in overt exposures to infectious materials must be immediately reported to the facility director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

·         All wastes from the animal room must be autoclaved prior to incineration or other appropriate terminal treatment.

·         Materials not related to the experiment (e.g., plants, animals) are not permitted in the animal room.

 

Safety Equipment (Primary Barriers)

·         Uniforms or scrub suits are worn by personnel entering the animal room. Wrap-around or solid-front gowns should be worn over this clothing. Front-button laboratory coats are unsuitable. The gown must be removed and left in the animal room. Before leaving the animal facility, scrub suits and uniforms are removed and appropriately contained and decontaminated prior to laundering or disposal.  

·         Personal protective equipment used is based on risk assessment determinations. 

Ø      Personal protective equipment is used for all activities involving manipulations of infectious material or infected animals.

Ø      Personnel wear gloves when handling infected animals.

Ø      Gloves are removed aseptically and autoclaved with other animal room wastes before disposal.

Ø      Appropriate face/eye and respiratory protection (e.g., respirators and face shields) is worn by all personnel entering animal rooms.

Ø      Boots, shoe covers, or other protective footwear, and disinfectant foot baths are available and used where indicated.  

·         The risk of infectious aerosols from infected animals or their bedding also can be reduced if animals are housed in containment caging systems, such as open cages placed in inward flow ventilated enclosures (e.g., laminar flow cabinets), solid wall and bottom cages covered with filter bonnets, or other equivalent primary containment systems.

·         Biological safety cabinets and other physical containment devices are used whenever conducting procedures with a potential for creating aerosols. These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, or intranasal inoculation of animals. At BSL-3, all work should be done in a primary barrier; otherwise respirators should be worn by personnel in the room.  

 

Facilities (Secondary Barriers)

·         The animal facility is separated from areas that are open to unrestricted personnel traffic within the building.

·         Access to the facility is limited by a self-closing and self-locking door. This exterior entry door may be controlled by a key lock, card key, or proximity reader.  Entry into the animal room is via a double-door entry, which includes a change room and shower(s). An additional double-door access (air-lock) or double-doored autoclave may be provided for movement of supplies and wastes into and out of the facility, respectively. Doors to animal rooms open inward and are self-closing. Doors to cubicles inside an animal room may open outward or slide horizontally or vertically.

·         The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are water resistant. Penetrations in floors, walls and ceiling surfaces are sealed and openings around ducts and the spaces between doors and frames are capable of being sealed to facilitate decontamination.

·         A hands-free or automatically operated hand washing sink is provided in each animal room near the exit door.  The sink trap is filled with an appropriate disinfectant after each use.

·         Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas.

·         Windows are not recommended.  Any windows must be resistant to breakage and must be sealed.

·         If floor drains are provided, they are always filled with an appropriate disinfectant.

·         Ventilation should be provided in accordance with criteria from the Guide for Care and Use of Laboratory Animals, latest edition. A ducted exhaust air ventilation system is provided. This system creates directional airflow, which draws air into the laboratory from “clean” areas and toward “contaminated” areas. The exhaust air is not recirculated to any other area of the building. Filtration and other treatments of the exhaust air may not be required, but should be considered based on site requirements, and specific agent manipulations and use conditions. The exhaust must be dispersed away from occupied areas and air intakes, or the exhaust must be HEPA-filtered. Personnel must verify that the direction of the airflow (into the animal areas) is proper. It is recommended that a visual monitoring device that indicates and confirms directional inward airflow be provided at the animal room entry.  Consideration should be given to installing an HVAC control system to prevent sustained positive pressurization of the animal spaces. Audible alarms should be considered to notify personnel of HVAC system failure.

·         HEPA-filtered exhaust air from a Class II biological safety cabinet can be recirculated into the animal room if the cabinet is tested and certified at least annually. When exhaust air from Class II safety cabinets is to be discharged to the outside through the building exhaust air system, the cabinets must be connected in a manner that avoids any interference with the air balance of the cabinets or the building exhaust system (e.g., an air gap between the cabinet exhaust and the exhaust duct). When Class III biological safety cabinets are used, they should be directly connected to the exhaust system. If the Class III cabinets are connected to the supply system, it is done in a manner that prevents positive pressurization of the cabinets (see Appendix A of BMBL, 5th Ed.).

·         Cages are washed in a cage washer. The mechanical cage washer has a final rinse temperature of at least 180°F.

·         An autoclave is available which is convenient to the animal rooms where the biohazard is contained. The autoclave is utilized to decontaminate infectious waste before moving it to other areas of the facility.

·         If vacuum service (i.e., central or local) is provided, each service connection should be fitted with liquid disinfectant traps and an in-line HEPA filter, placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement.

·         Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.

·         The completed Biosafety Level 3 facility design and operational procedures must be documented. The facility must be tested for verification that the design and operational parameters have been met prior to operation.  Facilities should be re-verified at least annually against these procedures as modified by operational experience.

·         Additional environmental protection (e.g., personnel showers, HEPA filtration of exhaust air, containment of other piped services, and the provision of effluent decontamination) should be considered if recommended by the agent summary statement, as determined by risk assessment of the site conditions, or other applicable federal, state, or local regulations.

   

Animal Biosafety Level 4 (ABSL-4)  

Animal Biosafety Level 4 involves practices suitable for addressing dangerous or exotic agents that pose high risk of life threatening disease, aerosol transmission, or related agents with unknown risk of transmission.  ABSL-4 builds upon the standard practices, procedures, containment equipment, and facility requirements of ABSL-3. Procedures must be developed locally to address specific operations of the Class III cabinet line or the suit laboratory.

Animal Biosafety Level 1 (ABSL-1) is suitable for work involving well characterized agents that are not known to cause disease in healthy adult humans, and that are of minimal potential hazard to laboratory personnel and the environment.

 

Standard Practices

·         Aside from the standard policies, procedures, and protocols for emergency situations established by the facility director, appropriate special policies and procedures should be developed as needed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC).

·         The laboratory or animal facility director limits access to the animal room to the fewest individuals possible. Personnel who must enter the room for program or service purposes when work is in progress are advised of the potential hazard.

·         A medical surveillance program must be instituted for all persons entering an ABSL-4 facility. This program must include appropriate immunizations, serum collection, and availability of post-exposure counseling and potential prophylaxis (Reference #10 at the end of this section). In general, persons who may be at increased risk of acquiring infection, or for whom infection might have serious consequences, are not allowed in the animal facility unless special procedures can eliminate the extra risk. Assessment should be made by the occupational health physician.

·         A site-specific biosafety manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and to follow instructions on practices and procedures.

·         Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use should be done only in designated areas and are not permitted in animal or procedure rooms.

·         All procedures are carefully performed to minimize the creation of aerosols or splatters.

·         Equipment and work surfaces in the room are routinely decontaminated with an appropriate disinfectant after work with the infectious agent, and especially after overt spills, splashes, or other contamination by infectious materials.

·         A spill procedure is developed and posted. Only personnel properly trained and equipped to work with infectious materials are to clean up spills. Spills and accidents that result in overt exposures to infectious materials must be immediately reported to the facility director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

·         All wastes (including animal tissues, carcasses, and contaminated bedding), other materials for disposal, and clothing to be laundered, are sterilized in a double-door autoclave located in the secondary barrier wall of the facility (see Special Practices, bullet #4 below). Disposable wastes are incinerated.

·         Policies for the safe handling of sharps are instituted.  

Ø      Needles and syringes or other sharp instruments are restricted in the animal facility for use only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles.

Ø      Syringes that re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

Ø      Plastic ware should be substituted for glassware whenever possible

·         A biohazard sign must be posted on the entrance to the animal room whenever infectious agents are present.  The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements for entering the animal room (e.g., the need for immunizations and respirators).

·         Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural or policy changes. Records are maintained on all training provided.

·         Cages are autoclaved or thoroughly decontaminated before bedding is removed and before they are cleaned and washed. Equipment and work surfaces are routinely decontaminated with an appropriate disinfectant after work with infectious materials, and especially after spills, splashes, or other contamination by infectious materials. Equipment must be decontaminated according to any local, state, or federal regulations before removal from the facility for repair or maintenance.

·         Personnel assigned to work with infected animals should work in pairs. Based on the risk assessment (see Section V of BMBL, 5th Ed.), use of squeeze cages, working only with anesthetized animals, or other appropriate procedures to reduce possible worker exposure must be instituted.

·         Materials not related to the experiment (e.g., plants, animals) are not permitted in the facility.

 

 

Special Practices

·         Additional measures are affected to control access (e.g., 24-hour guard and check in/out system). Personnel enter and leave the facility only through the clothing change and shower rooms. Personnel shower each time they leave the facility. Personnel should not enter or leave the facility through the air locks, except in an emergency.

·         In a Class III cabinet operation, personal clothing is removed in the outer clothing change room and kept there. Complete laboratory clothing, including undergarments, pants and shirts or jump suits, shoes, and gloves, is provided and used by personnel entering the facility. When exiting, personnel remove laboratory clothing in the inner change room before entering the shower area. Soiled clothing is sterilized in an autoclave.

·         In an ABSL-4 suit operation, a complete clothing change is required. A personal shower is required following removal of the decontaminated suit. Soiled lab clothing is autoclaved before laundering.

·         Supplies and materials are introduced into the facility via a double-door autoclave or fumigation chamber. After the outer door is secure, personnel inside the facility open the inner door to retrieve the materials. The doors of the autoclave and fumigation chamber are interlocked in a manner that prevents opening of the outer door unless the autoclave has been operated through a “sterilization cycle” or the fumigation chamber has been decontaminated.

·         A system is established for the reporting of accidents, incidents, exposures, and employee absenteeism, and for the medical surveillance of potential laboratory-associated illnesses. An essential adjunct to such a reporting/surveillance system is the availability of a facility for the quarantine, isolation, and medical care of persons with potential or known laboratory-associated illnesses.

·         The serum samples collected are analyzed at intervals. The results are communicated to the participants.  

 

Safety Equipment (Primary Barriers)

·         Laboratory animals infected with Biosafety Level 4 agents must be housed within a Class III biological safety cabinet in a BSL-4 Cabinet Laboratory. In a BSL-4 Suit Laboratory, all personnel are required to wear one-piece positive pressure suits ventilated with a life support system. Infected animals should be housed in a partial containment system (such as open cages placed in ventilated enclosures, solid wall and bottom cages covered with filter bonnets and opened in laminar flow hoods, or other equivalent primary containment systems).

·         The use of disposable material that does not require cleaning, including animal caging, should be considered.  Disposable materials must be autoclaved on exit from the facility and then incinerated.

 

Facilities (Secondary Barriers)

BSL-4 animal areas may be included as an integral part of BSL-4 Cabinet Laboratories or Suit Laboratories as described in Section III of this document. The facility requirements described in the BSL-4 Laboratory section should be utilized in conjunction with the caging described in the equipment section above.

References

1.   Guide for the Care and Use of Laboratory Animals, National

Academy Press, Washington, DC, 1996

2.   U.S. Department of Agriculture. Laboratory Animal Welfare Regulations - 9 CFR, Subchapter A, Parts 1, 2, and 3.

3.   Occupational Health and Safety in the Care of Research Animals.

National Academy Press, Washington, D.C., 1996.

4.   Subcommittee on Arbovirus Laboratory Safety for Arboviruses and Certain Other Viruses of Vertebrates. 1980. Am J Trop Med Hyg 29(6):1359-1381.

5.   Centers for Disease Control and Prevention. 1998. Fatal Cercopithecine herpesvirus 1 (B Virus) Infection Following a Mucocutaneous Exposure and Interim Recommendations for Worker Protection. MMWR 47(49); 1073-6,1083

6.   Guide for the Care and Use of Laboratory Animals (1)

7.   Occupational Health and Safety in the Care of Research Animals (3)

8.   Centers for Disease Control and Prevention. 1998. (5)

9.   Occupational Health and Safety in the Care of Research Animals (3)

10.  Occupational Health and Safety in the Care of Research Animals. (3)

 

TABLE 1.       SUMMARY OF RECOMMENDED BIOSAFETY LEVELS FOR ACTIVITIES IN WHICH EXPERIMENTALLY OR NATURALLY INFECTED VERTEBRATE ANIMALS ARE USED

BSL

Agents

Practices

Safety Equipment (Primary Barriers)

Facilities (Secondary Barriers)

1

Not known to consistently cause disease in healthy human adults

Standard animal care and management practices, including appropriate medical surveillance programs

As required for normal care of each species

·         Standard animal facility;

·         No recirculation of exhaust air;

·         Directional air flow recommended;

·         Handwashing sink recommended

2

Associated with human disease. Hazard: percutaneous exposure, ingestion, mucous membrane exposure.

ABSL-1 practices plus:

·                      Limited access

·         Biohazard warning signs

·         Sharps precautions

·         Biosafety manual

·         Decontamination of all infectious wastes and of animal cages prior to washing

ABSL-1 equipment plus:

·         Primary barriers containment equipment appropriate for animal species

·         PPES: laboratory coats, gloves, face and respiratory protection as needed

ABSL-1 facility plus:

·         Autoclave available

·         Handwashing sink available in the animal room

·         Mechanical cage washer used

3

Indigenous or exotic agents with potential for aerosol transmission; disease may have serious health effects

ABSL-2 practices plus:

·         Controlled access

·         Decontamination of clothing before laundering

·         Cages decontaminated before bedding removed

·         Disinfectant foot bath as needed

ABSL-2 equipment plus:

·         Containment equipment for housing animals and cage dumping activities

·         Class I or II BSCs available for manipulative procedures (inoculation, necropsy) that may create infectious aerosols

·         PPEs: appropriate respiratory protection

·          

ABSL-2 facility plus:

·         Physical separation from access corridors

·         Self-closing, double-door access

·         Sealed penetrations

·         Sealed windows

·         Autoclave available in facility

4

Dangerous/exotic agents that pose high risk of life threatening disease; aerosol transmission, or related agents with unknown risk of transmission

ABSL-3 practices plus:

·         Entrance through change room where personal clothing is removed and laboratory clothing is put on; shower on exiting

·         All wastes are decontaminated before removal from the facility

ABSL-3 equipment plus:

·         Maximum containment equipment (i.e., Class III BSC or partial containment equipment in combination with full body, air-supplied positive-pressure personnel suit) used for all procedures and activities

ABSL-3 facility plus:

·         Separate building or isolated zone

·         Dedicated supply and exhaust, vacuum and decontamination systems

·         Other requirements outlined in the text

 

19.6       Appendix F - Biosafety in Microbiological and Biomedical Laboratories (BMBL) - 5th Edition Table of Contents

Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition 

NOTE: - Printed booklets, HTML and Spanish versions not yet available

http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

Visit this web address to link to the document.

U.S. Department of Health and Human Services
Centers for Disease Control and Prevention
and
National Institutes of Health
Fifth Edition, Feb 2007

US Government Printing Office
Washington: 2007

Complete 5th Edition of BMBL (All Sections) PDF
(PDF - 2.4 MB)

Table of Contents

Title Page and Credits

Biosafety in Microbiological and Biomedical Laboratories, 5th Edition

SECTION I

Introduction

SECTION II

Biological Risk Assessment

SECTION III

Principles of Biosafety

SECTION IV

Laboratory Biosafety Level Criteria

Table 1 (Sect.IV)

Summary of Recommended Biosafety Levels for Infectious Agents

SECTION V

Vertebrate Animal Biosafety Level Criteria, for Vivarium Research Facilities

Table 1 (Sect.V)

Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used.

SECTION VI

Principles of Laboratory Biosecurity

SECTION VII

Occupational Health and Immunoprophylaxis

SECTION VIII

Agent Summary Statements

SECTION VIII-A

Bacterial Agents

SECTION VIII-B

Fungal Agents

SECTION VIII-C

Parasitic Agents

SECTION VIII-D

Rickettsial Agents

SECTION VIII-E

Viral Agents

SECTION VIII-F

Arboviruses and Related Zoonotic Viruses

Table 3

Alphabetic Listing of 597 Arboviruses and Hemorrhagic Fever Viruses

SECTION VIII-G

Toxin Agents

SECTION VIII-H

Prion Diseases

APPENDIX A

Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets

APPENDIX B

Decontamination and Disinfection

APPENDIX C

Transportation of Infectious Substances

APPENDIX D

Agriculture Pathogen Biosafety

APPENDIX E

Arthropod Containment Guidelines

APPENDIX F

Select Agents and Toxins

APPENDIX G

Integrated Pest Management

APPENDIX H

Working with Human, NHP and Other Mammalian Cells and Tissues

APPENDIX I

Guidelines for Work with Toxins of Biological Origin

APPENDIX J

NIH Oversight of Research Involving Recombinant Biosafety Issues

APPENDIX K

Resources for Information

APPENDIX L

Acronyms

 

19.7       Appendix G - UTMB Bloodborne Pathogen Exposure Control Policy

University of Texas Medical Branch at Galveston                       Revised: 1/07/06

                                                                                                    4/10/08

 

Bloodborne Pathogen Exposure Control Policy: Standard (Universal) Precautions for Handling Human Products in the Laboratory

Purpose                        To outline a plan for the prevention of exposure to human products:  human blood, tissue, body fluids, primary cell lines, commercially purchased human cell lines and bloodborne pathogens designated at Biological Safety Level 2, 3 and 4 (BSL2/3/4). 

Audience                      Laboratorians, to include UTMB faculty, visiting faculty/scientists, students, graduate students and post-doctoral fellows that handle human products and bloodborne pathogens. 

Policy                       Standard/universal precautions is a program based on the assumption that all human products (tissue, blood, body fluids, primary cell lines, commercially purchased human cell lines at BSL 2) are potentially infectious.  Application of standard precautions will protect the laboratorian from bloodborne infectious agents such as Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus as well as other bloodborne pathogens.  Standard precautions require that barrier precautions be used when contact with these materials is anticipated. Standard precautions do not rely on the diagnosis of disease to be made before precautions are instituted.  It is assumed that all human products are potentially infected and that measures are to be taken to safely handle these materials.  Consistent use of personal protective equipment, safety devices and application of BSL2/3 guidelines will reduce the potential for exposure to bloodborne pathogens. 

Exposure                      The bloodborne pathogen exposure control plan is

Control                         based on Standard Precautions, OSHA regulation (29

Plan                              CFR Part 190.1030), the Texas Administrative Code Chapter 81, Health and Safety Code, Subchapter H. and the HHS/CDC Biosafety in Microbiological and Biomedical Laboratories, 4th Edition, 1999.  The plan will be reviewed periodically and revised as necessary. 

Institutional       Human products and bloodborne pathogens have

Biological                      been designated by the UTMB Institutional Biological

Safety                           Safety Committee to be handled at Biological Safety

Committee                    Level 2 or higher as appropriate.  This requires the principal investigator and/or laboratory director to submit a Notification of Use for Human Products or a Notification of Use for Biological Agents to the institutional biological safety committee for review and approval. A minimum of BSL2 microbiological practices, safety equipment and facilities shall be in place for use of human products and BSL2/3 for bloodborne pathogens as deemed appropriate by the UTMB Institutional Biological Safety Committee. 

Standard Microbiological Practices 

·         A biohazard sign must be posted on the laboratory entrance.  The sign must include the biosafety level, name of the agent in use, special handling procedures, immunizations, principal investigator/ lab director name, phone number and emergency contact information.  The sign must be updated to reflect changes in lab staff. Contact EHS B&C at x 21781

·         Laboratory equipment (freezers, refrigerators, incubators etc.) where human products and bloodborne pathogens are stored, cultured or manipulated shall be labeled with the biohazard symbol.

·         Access to the laboratory is limited or restricted when experiments or work with cultures and specimens are in progress.

·         Hands shall be washed with soap and water after removing gloves,

after handling viable material and before leaving the laboratory.

·         Eating, drinking, handling contact lenses, applying cosmetics

and storing food for human use is not permitted.  Persons

wearing contact lenses in laboratories should also wear eye protection

or a face shield.

·         Mouth pipetting is prohibited, mechanical pipetting devices are used.

·         All procedures are performed carefully to minimize the creation of

splashes or aerosols.

·         Work surfaces are decontaminated at completion of work or at the end of the day and after any spill of viable material with disinfectants that are effective against the agents of concern.

·         Liquid human products and bloodborne pathogens (blood, bloody fluids, cell cultures) are to be decontaminated prior to drain disposal.  Solid disposable materials will be placed in a red bag lined biohazard box or autoclaved prior to disposal in the regular trash.

·         Disposable instruments/equipment saturated with blood/bloody fluids or contaminated with infectious agents shall be red bagged and boxed as biohazardous for incineration or autoclaved (clear or orange autoclave bags) prior to disposal in the regular trash.

·         Re-usable contaminated equipment shall be decontaminated by application of an approved disinfectant prior to final cleaning (contact Environmental Health and Safety ext. 2-1781 for information on disinfectants) or autoclaved.

·         Equipment that cannot be decontaminated or autoclaved prior to servicing shall have a notice displaying the biohazard symbol attached.  The notice shall identify the contaminated site.

·         Laboratorians receive appropriate immunizations or antibody titers for agents handled or potentially present in the laboratory.

·         The principal investigator/ lab director ensures that the laboratory personnel receive appropriate training on the potential hazards associated with the work involved and the exposure evaluation procedures prior to starting work.

Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative. Plasticware should be substituted for glassware whenever possible.

·         Sharps include the following items:  needles, syringes, capillary tubes, glass slide and cover slips, glass pasteur pipettes, scalpel and razor blades whether contaminated or not contaminated.

·         Sharps products, which are engineered to reduce or prevent injury, are strongly encouraged.  These products include but are not limited to safety needles and sharps with engineered sharps protection and needle less systems.

·         Used sharps must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.  They must be carefully placed in conveniently located puncture-resistant containers specifically designed for sharps disposal.

·         Non-disposable sharps must be placed in a hard-walled closable container for transport to a processing area for decontamination preferably by autoclaving.  Sharps containers are to be closed when they are 3/4 full.  They may be placed into the red bag lined biohazard box/container for disposal.

·         With two exceptions, needles shall never be removed from a syringe or vacutainer holder, and they shall be disposed of as a unit in a sharps container designed for sharps disposal.  One exception to this rule is that needles may be removed from vacutainer holders using the "keyhole" device on red plastic needle disposal containers.  The only other exception to this rule is removal of needles from syringes used to obtain arterial blood, or recapping dental needles used for local anesthesia or needles used for triturating IV solution.   In the instance that needles are required to be recapped the one-handed technique shall be used.  The cap is placed on a flat surface and then picked up with the needle and syringe.  The cap shall be snapped into place by gently pushing the cap against a flat vertical inanimate surface (not the other hand).

·         Venipuncture and insertion of steel needles or plastic catheters into any intra-vascular space shall be carried out with great care.

¨       Gloves shall be worn

¨       Vacutainers shall be used for Venipuncture as appropriate.

¨       In the extraordinary circumstance where blood cannot be obtained using a vacutainer, a needle and syringe shall be used.  However, in transferring blood from syringes to vacutainer, NEVER FORCIBLY INJECT blood into the tubes.  Forcible injection of blood through the rubber stopper of tubes without a vacuum may cause the top to pop off and spray blood on the operator.  Tubes without a vacuum shall be discarded and replaced by tubes with a vacuum.

 

Safety Equipment (primary barriers) 

Disposable or re-usable gloves shall be used for procedures involving all contact with human products and/or bloodborne pathogens.

·         Wearing two pair of gloves may be appropriate.

·         Disposable gloves shall be replaced when they become overtly contaminated or if they are torn, punctured, or when the integrity of the barrier is compromised.

·         Disposable gloves shall not be washed or disinfected for reuse or used for touching "clean" surfaces.

·         Gloves are never to be worn outside the laboratory.

·         Re-usable gloves (heavy duty latex or nitrile gloves) may be decontaminated with an appropriate disinfectant and reused but shall be discarded if they are peeling, cracked or discolored, or if they have punctures, tears, or other evidence of deterioration.

·         Discovery of an allergic reaction to glove material shall be reported to the principal investigator/lab director or lab supervisor immediately.  Alternative glove material shall be identified and appropriate gloves then provided to the employee.

Lab coats or cover gowns designated for lab use are worn while in the laboratory.

·         Laboratory coats and gowns will be removed prior to leaving the laboratory.

·         Disposable coats or gowns shall be discarded as regular waste unless significantly contaminated with infectious material in which case they shall be placed in a red bag as biohazardous waste or in a clear or orange autoclave bag for autoclaving. 

·         Re-usable coats or gowns shall be placed in an impervious laundry bag and sent to the UTMB laundry or for BSL3 facilities autoclaved first and then sent to the laundry.

·         Gloves shall be worn when handling contaminated instruments or equipment.

Face protection (goggles & mask, face shield or other splatter guard) is used for anticipated splashes or sprays of infectious materials to the face when the microorganisms must be manipulated outside the biological safety cabinet.

Spills and accidents that result in overt exposures to infectious materials are immediately reported and spill procedures are implemented.  Refer to the UTMB Safety Manual, Chapter 9 Biological Safety for spill procedure information.

Properly maintained biological safety cabinets, Class II type A/B3 or B2 cabinets, or other appropriate personal protective equipment or physical containment devices are used whenever:

·         procedures are conducted that have potential for producing infectious droplet aerosols or splashes. This includes but is not limited to centrifuging, pipetting, grinding, sonic disruption, vortexing, vigorous mixing, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals.

·         high concentration or large volumes of infectious agents are used.  Such materials may be centrifuged in the open laboratory only if sealed rotor heads or centrifuge safety cups are used and if these rotors or safety cups are opened only in a biological safety cabinet.

·         face protection as described above or a shield is to be used whenever manipulating material outside the biological safety cabinet.

 

Human Products and Bloodborne Pathogen Exposure 

Exposed laboratorians shall report to the Employee Health Center or the Student Health Services during normal working hours and the Trauma Center/emergency room after hours or on weekends as soon as possible.  Exposure of a laboratorian is defined as follows:

·         Puncture of skin or laceration by a sharp object contaminated with blood, blood tinged fluids, potentially infectious body fluids, or bloodborne pathogens.

·         Contamination of mucous membranes (eyes, nose, mouth) with blood, blood tinged fluids, potentially infectious body fluids, or bloodborne pathogens.

·         Contamination of non-intact skin (cuts, scratches, abrasions, dermatitis, etc.) by blood, blood tinged fluids, potentially infectious body fluids, or bloodborne pathogens.

·         Contamination of intact skin by blood, blood tinged fluids, potentially infectious body fluids, or bloodborne pathogens that is prolonged or involves an extensive area.

·         Should a needle stick or injury with another sharp instrument occur, the wound should be cleansed immediately with povidone-iodine, chlorohexidine, or 70% isopropyl alcohol and washed off.

·         All injuries and exposures shall be reported to Employee Health Center or to Student Health Service and appropriate paper work (Bloodborne Pathogen Post Exposure Packet) completed.

 

Biohazardous Waste Disposal

Biohazardous waste includes:

·         Microbiological waste

·         Pathological waste

·         Human blood and blood products

·         Bulk blood (100 mls or more)

·         Sharps in sharps containers

·         Containers used for disposal of biohazardous waste shall be closable, leak resistant and lined with a red biohazard bag.  These containers shall be labeled with a biohazard symbol or a red container may be substituted for a labeled container.

·         Sharps containers shall be specifically designed for sharps disposal.

·         Disposable biohazard boxes will be lined with a red plastic bag and

additional absorbent material added if necessary to contain liquids.

·         Reusable biohazardous waste containers shall be washed out with

soap and water and disinfected with an appropriate disinfectant. 

·         Gloves shall be worn for washing out containers.

State of Texas, CDC and OSHA Documents

Copies of regulations and guidelines are available in Environmental Health and Safety, Biological & Chemical Safety. 

Transportation of Human Products and Bloodborne Pathogens/Infectious Substances.

·         Transport on campus:

Human products and bloodborne pathogens will be contained for transport on campus by placing the material in a leakproof non-breakable primary container labeled with a biohazard symbol and then placed into a biohazard labeled secondary transport container.

·         Transport off campus:

Any person placing human products or bloodborne pathogens/infectious substances into transport shall be trained and certified to do so.  Training shall meet the requirements set forth by either the U.S. Department of Transportation (DOT) or the International Air Transport Association (IATA). Contact Environmental Health and Safety, Biological & Chemical Safety at x 21781 for information.

Compliance Monitoring

·         It is the responsibility of all laboratorians to comply with the Exposure Control Plan.

·         Non-compliance by a UTMB Employee shall be subject to appropriate disciplinary measure as outlined by the Human Resources Department

·         Environmental Health and Safety, Biological & Chemical Safety shall randomly monitor compliance.

·         Non-compliance incident reports shall be sent to the appropriate principal investigator/laboratory director.

·         The UTMB Institutional Biological Safety Committee shall review non-compliance reports.

 

References:

HHS/CDC, Biosafety in Microbiological and Biomedical Laboratories, 4th Edition, May 1999.

Texas Administrative Code, Chapter 81, subchapter H Health and Safety Code.  Title 25 Heath Services, Chapter 96 Bloodborne Pathogen Control.

Federal Register, December 6, 1991, Part II: department of Labor, Occupational Safety and Health Administration:  29 CFR 1910.1030 Occupational Exposure to Bloodborne Pathogens; Final Rule.