SECTION IV 

Vertebrate Animal Biosafety Level Criteria

	If experimental animals are used, institutional management must provide facilities and staff and establish practices which reasonably assure appropriate levels of environmental quality, safety, and care.  Laboratory animal facilities in many ways are extensions of the 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. It is well to remember, however, that the animal room is not the laboratory, and can present some unique problems.  In the laboratory, hazardous conditions are caused by personnel or the equipment that is being used.  In the animal room the activities of the animals themselves can introduce new hazards.  Animals may produce aerosols, and they may also infect and traumatize animal handlers by biting and scratching. 

	These recommendations presuppose that laboratory animal facilities, operational practices, and quality of animal care meet applicable standards and regulations and that appropriate species have been selected for animal experiments (e.g., Guide for the Care and Use of Laboratory Animals, HEW Publication No. (NIH) 86-23, Rev. 1985, and Laboratory Animal Welfare Regulations - 9 CFR, Subchapter A, Parts 1, 2 and 3).

	Ideally, facilities for laboratory animals used for 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 that provide patient care.  Animal facilities should be designed and constructed to facilitate cleaning and housekeeping.  Traffic flow that will minimize the risk of cross contamination should be considered in the plans.  A "clean/dirty hall" layout is useful in achieving this.  Floor drains should be installed in animal facilities only on the basis of clearly defined needs.  If floor drains are installed, the drain trap should always contain water or a suitable disinfectant.

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

	Facility standards and practices for invertebrate vectors and hosts are not specifically addressed in standards written for commonly used laboratory animals.  "Laboratory Safety for Arboviruses and Certain other Viruses of Vertebrates,"178 prepared by the Subcommittee on Arbovirus Laboratory Safety 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

	A.	Standard Practices

	1.	Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

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

	3.	Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in animal rooms.  Persons who wear contact lenses in animal rooms should also wear goggles or a face shield.  

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

	5.	Work surfaces are decontaminated after use or after any spill of viable materials.
	6.	Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

	7.	All wastes from the animal room are appropriately decontaminated, preferably by autoclaving, before disposal.  Infected animal carcasses are incinerated after being transported from the animal room in leakproof, covered containers.  

	8.	An insect and rodent control program is in effect.

	B.	Special Practices

	1.	The laboratory or animal facility director limits access to the animal room to personnel who have been advised of the potential hazard and who need to enter the room for program or service purposes when work is in progress.  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 room. 

	2.	The laboratory or animal facility director establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet any specific requirements (e.g., immunization) may enter the animal room.

	3.	Bedding materials from animal cages are removed in such a manner as to minimize the creation of aerosols, and are disposed of in compliance with applicable institutional or local requirements.  

	4.	Cages are washed manually or in a cage washer. Temperature of final rinse water in a mechanical washer should be 180oF.

	5.	The wearing of laboratory coats, gowns, or uniforms in the animal facility is recommended.  It is further recommended that laboratory coats worn in the animal facility not be worn in other areas.

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

	C.	Safety Equipment (Primary Barriers)

Special containment equipment is not required for animals infected with agents assigned to Biosafety Level 1.

	D.	Animal Facilities (Secondary Barriers)

	1.	The animal facility is designed and constructed to facilitate cleaning and housekeeping.

	2.	A handwashing sink is available in the animal facility.

	3.	If the animal facility has windows that open, they are fitted with fly screens.

	4.	Exhaust air is discharged to the outside without being recirculated to other rooms, and it is recommended, but not required, that the direction of airflow in the animal facility is inward.

Animal Biosafety Level 2

	A.	Standard Practices

	1.	Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

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

	3.	Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in animal rooms.  Persons who wear contact lenses in animal rooms should also wear goggles or a face shield.  

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

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

	6.	Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

	7.	All wastes from the animal room are appropriately decontaminated, preferably by autoclaving, before disposal.  Infected animal carcasses are incinerated after being transported from the animal room in leakproof, covered containers.  

	8.	An insect and rodent control program is in effect.

	B.	Special Practices

	1.	The laboratory or animal facility director limits access to the animal room to personnel who have been advised of the potential hazard and who need to enter the room for program or service purposes when work is in progress.  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 room. 

	2.	The laboratory or animal facility director establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet any specific requirements (e.g., immunization) may enter the animal room.

	3.	When the infectious agent(s) in use in the animal room requires special entry provisions (e.g., the need for immunizations and respirators) a hazard warning sign, incorporating the universal biohazard symbol, is posted on the access door to the animal room.  The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the animal facility supervisor or other responsible person(s), and indicates the special requirement(s) for entering the animal room.

	4.	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).

	5.	When appropriate, considering the agents handled, baseline serum samples from animal care and other at-risk personnel are collected and stored. Additional serum samples may be collected periodically depending on the agents handled or the function of the facility.  The decision to establish a serologic surveillance program must take into account the availability of methods for the assessment of antibody to the agent(s) of concern.  The program should provide for the testing of serum samples at each collection interval and the communication of results to the participants.

	6.	A 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. 

	7.	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.

	8.	A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.  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.  Plasticware should be substituted for glassware whenever possible.

	a.  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.

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

c.  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, according to any local, state, or federal regulations.

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

	10.	Cages are appropriately decontaminated, preferably by autoclaving, before they are cleaned and washed.  Equipment and work surfaces should be decontaminated with an appropriate 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.

	11.	Spills and accidents which 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.

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

	C.	Safety Equipment (Primary Barriers)

	1.	Biological safety cabinets, other physical containment devices, and/or personal protective equipment (e.g., respirators, face shields) are used whenever procedures with a high potential for creating aerosols are conducted.139  These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, intranasal inoculation of animals, and manipulations of high concentrations or large volumes of infectious materials.

	2.	Appropriate face/eye and respiratory protection is worn by all personnel entering animal rooms housing nonhuman primates.

	3.	Laboratory coats, gowns, or uniforms are worn while in the animal room.  This protective clothing is removed before leaving the animal facility.

	4.	Special care is taken to avoid skin contamination with infectious materials; gloves are worn when handling infected animals and when skin contact with infectious materials is unavoidable. 

	D.	Animal Facilities (Secondary Barriers)

	1.	The animal facility is designed and constructed to facilitate cleaning and housekeeping.

	2.	A handwashing sink is available in the room where infected animals are housed.

	3.	If the animal facility has windows that open, they are fitted with fly screens.

	4.	If floor drains are provided, the drain traps are always filled with water or a suitable disinfectant.

	5.	Exhaust air is discharged to the outside without being recirculated to other rooms, and it is recommended, but not required, that the direction of airflow in the animal facility is inward.
	6.	An autoclave which can be used for decontaminating infectious laboratory waste is available in the building with the animal facility.

Animal Biosafety Level 3

	A.	Standard Practices

	1.	Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

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

	3.	Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in animal rooms.  Persons who wear contact lenses in animal rooms should also wear goggles or a face shield.  

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

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

	6.	Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

	7.	All wastes from the animal room are appropriately decontaminated, preferably by autoclaving, before disposal.  Infected animal carcasses are incinerated after being transported from the animal room in leakproof, covered containers.

	8.	An insect and rodent control program is in effect.

	B.	Special Practices

	1.	The laboratory director or other responsible person restricts access to the animal room to personnel who have been advised of the potential hazard and who need to enter the room for program or service purposes when  infected animals are present.  Persons who are at increased risk of acquiring infection, or for whom infection might be unusually hazardous, are not allowed in the animal room.  Persons at increased risk may include children, pregnant women, and persons who are immunodeficient or immunosuppressed.  The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the facility. 

	2.	The laboratory director or other responsible person establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet any specific requirements (e.g., for immunization) may enter the animal room.

	3.	When the infectious agent(s) in use in the animal room requires special entry provisions (e.g., the need for immunizations and respirators) a hazard warning sign, incorporating the universal biohazard symbol, is posted on the access door to the animal room.  The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the animal facility supervisor or other responsible person(s), and indicates the special requirement(s) for entering the animal room.

	4.	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).

	5.	Baseline serum samples from all personnel working in the facility and other at-risk personnel should be collected and stored.  Additional serum samples may be collected periodically and stored.  The serum surveillance program must take into account the availability of methods for the assessment of antibody to the agent(s) of concern.  The program should provide for the testing of serum samples at each collection interval and the communication of results to the participants.

	6.	A 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. 

	7.	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.

	8.	A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.  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, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles.  Plasticware should be substituted for glassware whenever possible.

	a.  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, preferably containing a suitable disinfectant, for transport to a processing area for decontamination, preferably by autoclaving.

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

	c.  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, according to any local, state, or federal regulations.

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

	10.	Cages are autoclaved or thoroughly decontaminated before bedding is removed or before they are cleaned and washed.  Equipment and work surfaces should be decontaminated with an appropriate 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.

	11.	Spills and accidents which 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.

	12.	All wastes from the animal room are autoclaved before disposal.  All animal carcasses are incinerated. Dead animals are transported from the animal room to the incinerator in leakproof covered containers.

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

	C.	Safety Equipment (Primary Barriers)

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

	a.  Wrap-around or solid-front gowns or uniforms are worn by personnel entering the animal room.  Front-button laboratory coats are unsuitable.  Protective gowns should be appropriately contained until decontamination or disposal.

	b.  Personnel wear gloves when handling infected animals. Gloves are removed aseptically and autoclaved with other animal room wastes before disposal.

		c.  Appropriate face/eye and respiratory protection is worn by all personnel entering animal rooms housing nonhuman primates.

		d.  Boots, shoe covers, or other protective footwear, and disinfectant footbaths are available and used when indicated.

	2.	Physical containment devices and equipment appropriate for the animal species are used for all procedures and manipulations of infectious materials or infected animals.

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

	D.	Animal Facilities (Secondary Barriers)

	1.	The animal facility is designed and constructed to facilitate cleaning and housekeeping, and is separated from areas which are open to unrestricted personnel traffic within the building.  Passage through two sets of doors is the basic requirement for entry into the animal room from access corridors or other contiguous areas.  Physical separation of the animal room from access corridors or other activities may also be provided by a double-doored clothes change room (showers may be included), airlock, or other access facility which requires passage through two sets of doors before entering the animal room.

	2.	The interior surfaces of walls, floors, and ceilings are water resistant so that they may be easily cleaned.  Penetrations in these surfaces are sealed or capable of being sealed to facilitate fumigation or space decontamination.

	3.	A foot, elbow, or automatically operated handwashing sink is provided in each animal room near the exit door.

	4.	If vacuum service (i.e., central or local) is provided, each service connection should be fitted with liquid disinfectant traps and a HEPA filter. 

	5.	If floor drains are provided, they are protected with liquid traps that are always filled with water or disinfectant.

	6.	Windows in the animal room are non-operating and sealed.

	7.	Animal room doors are self-closing and are kept closed when infected animals are present.

	8.	An autoclave for decontaminating wastes is available, preferably within the animal facility.  Materials are transferred to the autoclave in a covered leakproof container whose outer surface has been decontaminated.

	9.	A non-recirculating ventilation system is provided.  The supply and exhaust components of the system are balanced to provide for directional flow of air into the animal room.  The exhaust air is discharged directly to the outside and clear of occupied areas and air intakes.  Exhaust air from the room can be discharged without filtration or other treatment.  Personnel must periodically validate that proper directional airflow is maintained.  

	10.	The HEPA filtered exhaust air from Class I or Class II biological safety cabinets or other primary containment devices is discharged directly to the outside or through the building exhaust system. Exhaust air from these primary containment devices may be recirculated within the animal room if the device is tested and certified at least every 12 months.  If the HEPA filtered exhaust air from Class I or Class II biological safety cabinets is discharged to the outside through the building exhaust system, it is connected to this system in a manner (e.g., thimble unit connection)134 that avoids any interference with the performance of either the cabinet or building exhaust system

Animal Biosafety Level 4

	A.  Standard Practices

	1.	Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

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

	3.	Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in animal rooms.  Persons who wear contact lenses in animal rooms should also wear goggles or a face shield.  

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

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

	6.	Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

	7.	All wastes from the animal room are appropriately decontaminated, preferably by autoclaving, before disposal.  Infected animal carcasses are incinerated after being transported from the animal room in leakproof, covered containers.  

	8.	Cages are autoclaved before bedding is removed and before they are cleaned and washed.  When feasible, disposable cages that do not require cleaning are recommended; however, these cages also autoclaved before disposal.  Equipment and work surfaces should be decontaminated with an appropriate 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.

	9.	An insect and rodent control program is in effect.

	B.	Special Practices

	1.	Only persons whose entry into the facility or individual animal room is required for program or support purposes are authorized to enter.  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.  Persons at increased risk may include children, pregnant women, and persons who are immunodeficient or immunosuppressed.  The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the facility.  Access to the facility is limited by secure, locked doors; accessibility is controlled by the animal facility supervisor, biohazards 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.  Personnel comply with the instructions and all other applicable entry and exit procedures. Practical and effective protocols for emergency situations are established.

	2.	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).

	3.	Baseline serum samples are collected and stored for all laboratory and other at-risk personnel. Additional serum specimens may be collected periodically, 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.

	4.	A 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. 

	5.	When the infectious agent(s) in use in the animal room requires special entry provisions (e.g., the need for immunizations and respirators) a hazard warning sign, incorporating the universal biohazard symbol, is posted on the access door to the animal room.  The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the animal facility supervisor or other responsible person(s), and indicates the special requirement(s) for entering the animal room.

	6.	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.

	7.	Hypodermic needles and syringes are used only for gavage, for parenteral injection, and aspiration of fluids from diaphragm bottles or well-restrained laboratory animals.

	a.  A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.  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, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles.  Plasticware is substituted for glassware whenever possible.

	b.  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 are not bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they are carefully placed in conveniently located puncture-resistant containers used for sharps disposal.  Non-disposable sharps are placed in a hard-walled container, preferably containing a suitable disinfectant, for transport to a processing area for decontamination, preferably by autoclaving.

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

	d.  Broken glassware is not handled directly by hand, but is 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.

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

	9.	Spills and accidents which 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.

	10.	Personnel enter and leave the facility only through the clothing change and shower rooms.  Personnel shower each time they leave the facility.  Head covers are provided to personnel who do not wash their hair during the exit shower.  Except in an emergency, personnel do not enter or leave the facility through the airlocks.

	11.	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, are provided and used by all personnel entering the facility.  When exiting, personnel remove laboratory clothing in the inner change room before entering the shower area.  Soiled clothing is autoclaved before laundering.

	12.	Supplies and materials are brought into the facility by way of a double-door autoclave, fumigation chamber, or airlock.  After securing the outer doors, personnel inside the facility retrieve the materials by opening the interior door of the autoclave, fumigation chamber, or airlock.  This inner door is secured after materials are brought into the facility.  The autoclave fumigation chamber or airlock is decontaminated before the outer door is opened.

	13.	A system is established for the reporting of animal facility accidents and exposures, 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.

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

	C.	Safety Equipment (Primary Barriers)

Laboratory animals, infected with agents assigned to Biosafety Level 4, are housed a Class III biological safety cabinet or in a partial containment caging system (such as open cages placed in ventilated enclosures, solid wall and bottom cages covered with filter bonnets, or other equivalent primary containment systems), in specially designed areas in which all personnel are required to wear one-piece positive pressure suits ventilated with a life support system.  

Animal work with viral agents that require Biosafety Level 4 secondary containment, and for which highly effective vaccines are available and used, may be conducted with partial containment cages and without the one-piece positive pressure personnel suit if: the facility has been decontaminated, no concurrent experiments are being done in the facility which require Biosafety Level 4 primary and secondary containment, and all other standard and special practices are followed.

	D.	Animal Facility (Secondary Barriers)

	1.	The animal rooms are located in a separate building or in a clearly demarcated and isolated zone within a building.  Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the facility.  A double-doored autoclave, fumigation chamber, or ventilated airlock is provided for passage of materials, supplies, or equipment which are not brought into the facility through the change room.

	2.	Walls, floors, and ceilings of the facility are constructed to form a sealed internal shell which facilitates decontamination and is animal and insect proof. The internal surfaces of this shell are resistant to liquids and chemicals, thus facilitating cleaning and decontamination of the area.  All penetrations in these structures and surfaces are sealed.

	3.	Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas on which dust can settle.

	4.	A foot, elbow, or automatically operated handwashing sink is provided in each animal room near the exit door.

	5.	If there is a central vacuum system, it does not serve areas outside of the facility.  The vacuum system has in-line HEPA filters 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 for the facility are protected by devices that prevent backflow.

	6.	External animal facility doors are self-closing and selflocking.

	7.	Any windows must be resistant to breakage and sealed.

	8.	A double-doored autoclave is provided for decontaminating materials that leave the facility. The autoclave door which opens to the area external to the facility is automatically controlled so that it can only be opened after the autoclave "sterilization" cycle is completed.

	9.	A pass-through dunk tank, fumigation chamber, or an equivalent decontamination method is provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the facility.

	10.	Liquid effluents from laboratory sinks, biological safety cabinets, floor drains (if used), and autoclave chambers are decontaminated by 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 by use of a constant recording temperature sensor in conjunction with an indicator microorganism having a defined heat susceptibility profile.  

	11.	A dedicated non-recirculating ventilation system is provided.  The supply and exhaust components of the system are balanced to assure 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 malfunction of the system.  The airflow in the supply and exhaust components is monitored and the components interlocked to assure inward (or zero) airflow is maintained.  

	12.	The general room exhaust air from a facility in which the work is conducted in a Class III cabinet system is treated by a passage through a HEPA filter(s) prior to discharge to the outside.  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.  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 filtration of the supply air.

	13.	The treated exhaust air from Class II biological safety cabinets located in a facility in which workers wear a positive pressure suit may be discharged into the animal room environment or to the outside through the facility air exhaust system.  The biological safety cabinets are tested and certified at 9-month intervals.  The air exhausted from Class III biological safety cabinets is passaged through two HEPA filter systems (in series) prior to discharge to the outside.  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.  

	14.	A specially designed suit area may be provided in the facility.  Personnel who enter this area wear a one-piece positive pressure suit that is ventilated by a life support system.  The life support system is provided with 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.  The exhaust air from the area in which the suit is used is filtered by two sets of HEPA filters installed in series.  Duplicate filtration units and exhaust fans are provided.  An automatically starting emergency power source is provided.  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 inner shell of the suit area are sealed.  A doubledoored autoclave is provided for decontaminating waste materials to be removed from the suit area.
SECTION V

Recommended Biosafety Levels For Infectious Agents and Infected Animals

	Selection of an appropriate biosafety level for work with a particular agent or animal study depends upon a number of factors.  Some of the most important are: the virulence, pathogenicity, biological stability, route of spread, and communicability of the agent; the nature or function of the laboratory; the procedures and manipulations involving the agent; the endemicity of the agent; and the availability of effective vaccines or therapeutic measures.

	Agent summary statements in this section provide guidance for the selection of appropriate biosafety levels.  Specific information on laboratory hazards associated with a particular agent, and recommendations regarding practical safeguards that can significantly reduce the risk of laboratory-associated diseases, are included.  Agent summary statements are presented for agents which meet one or more of the following criteria: the agent is a proven hazard to laboratory personnel working with infectious materials (e.g., hepatitis B virus, M. tuberculosis); the potential for laboratory associated infections is high, even in the absence of previously documented laboratory-associated infections (e.g., exotic arboviruses); or, the consequences of infection are grave.

	Recommendations for the use of vaccines and toxoids are included in agent summary statements when such products are available, either as licensed or Investigational New Drug (IND) products.  When applicable, recommendations for the use of these products are based on current recommendations of the Public Health Service Advisory Committee on Immunization Practice, and are specifically targeted to at-risk laboratory personnel and others who must work in or enter laboratory areas.  These specific recommendations should in no way preclude the routine use of such products as diphtheria-tetanus toxoids, poliovirus vaccine, influenza vaccine and others, because of the potential risk of community exposures irrespective of any laboratory risks. Appropriate precautions should be taken in the administration of live attenuated virus vaccines in individuals with altered immunocompetence, or other medical condition (e.g., pregnancy), in which a viral infection could result in adverse consequences.

	Risk assessments and biosafety levels recommended in the agent summary statements presuppose a population of immunocompetent individuals.  Persons with altered immunocompetence may be at a increased risk when exposed to infectious agents.  Immunodeficiency may be hereditary, congenital, or induced by a number of neoplastic or infectious diseases, by therapy, or by radiation.  The risk of becoming infected or the consequence of infection may also be influenced by such factors as age, sex, race, pregnancy, surgery (e.g., splenectomy, gastrectomy), predisposing diseases (e.g., diabetes, lupus erythematosus) or altered physiological function.  These and other variables must be considered in applying the generic risk assessments of the agent summary statements to specific activities of selected individuals.

	The biosafety level assigned to an agent is based on the activities typically associated with the growth and manipulation of the quantities and concentrations of infectious agents required to accomplish identification or typing.  If activities with clinical materials pose a lesser risk to personnel than those activities associated with manipulation of cultures, a lower biosafety level is recommended.  On the other hand, if the activities involve large volumes and/or concentrated preparations ("production quantities"), or manipulations which are likely to produce aerosols or which are otherwise intrinsically hazardous, additional personnel precautions and increased levels of primary and secondary containment may be indicated.  

	"Production quantities" refers to large volumes or concentrations of infectious agents considerably in excess of those typically used for identification and typing activities. Propagation and concentration of infectious agents as occurs in large-scale fermentations, antigen and vaccine production, and a variety of other commercial and research activities, clearly deal with significant masses of infectious agents that are reasonably considered "production quantities".  However, in terms of potentially increased risk as a function of the mass of infectious agents, it is not possible to define "production quantities" in finite volumes or concentrations for any given agent.  Therefore, the laboratory director must make an assessment of the activities conducted and select practices, containment equipment, and facilities appropriate to the risk, irrespective of the volume or concentration of agent involved.

	Occasions will arise when the laboratory director should select a biosafety level higher than that recommended.  For example, a higher biosafety level may be indicated by the unique nature of the proposed activity (e.g., the need for special containment for experimentally generated aerosols for inhalation studies) or by the proximity of the laboratory to areas of special concern (e.g., a diagnostic laboratory located near patient care areas).  Similarly, a recommended biosafety level may be adapted to compensate for the absence of certain recommended safeguards.  For example, in those situations where Biosafety Level 3 is recommended, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures involving the propagation of an agent for identification, typing and susceptibility testing) in laboratories where facility features satisfy Biosafety Level 2 recommendations, provided the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed.  

	One example involves work with the Human Immunodeficiency Viruses (HIVs).  Routine diagnostic work with clinical specimens can be done safely at Biosafety Level 2, using Biosafety Level 2 practices and procedures. Research work (including co-cultivation, virus replication studies, or manipulations involving concentrated virus) can be done in a BSL-2 facility, using BSL-3 practices and procedures.  Virus production activities, including virus concentrations, require a BSL-3 facility and use of BSL-3 practices and procedures (see Agent Summary Statement).

	The decision to adapt Biosafety Level 3 recommendations in this manner should be made only by the laboratory director.  This adaptation, however, is not suggested for agent production operations or activities where procedures are frequently changing.  The laboratory director should also give special consideration to selecting appropriate safeguards for materials that may contain a suspected agent.  For example, sera of human origin may contain hepatitis B virus, and therefore, all blood or blood-derived fluids should be handled under conditions which reasonably preclude cutaneous, mucous membrane or parenteral exposure of personnel.  Sputa submitted to the laboratory for tubercle bacilli assay should be handled under conditions which reasonably preclude the generation of aerosols during the manipulation of clinical materials or cultures. 

	The infectious agents which meet the previously stated criteria are listed by category of agent in Section VII.  To use these summaries, first locate the agent in the listing under the appropriate category of agent.  Second, utilize the practices, safety equipment, and type of facilities recommended in the agent summary statement as described in Section VII for working with clinical materials, cultures or infectious agents, or infected animals.

	The laboratory director is also responsible for appropriate risk assessment and for utilization of appropriate practices, containment equipment, and facilities for agents not included in the agent summary statements.

SECTION VI

Risk Assessment

	The assessment of risks associated with laboratory activities involving the use of infectious microorganisms is ultimately a subjective process.  The risks associated with the agent, as well as with the activity to be conducted, must be considered in the assessment.  The characteristics of infectious agents and the primary laboratory hazards of working with the agents are described generically for agents in Biosafety Levels 1-4 and specifically for individual agents or groups of agents in Section VII of this publication.

	Hepatitis B (HBv) is an appropriate model for illustrating the risk assessment process.  HBv is among the most ubiquitous of human pathogens and most prevalent of laboratory-associated infections.  The agent has been demonstrated in a variety of body secretions and excretions.  Blood, saliva, and semen have been shown to contain the virus.  Natural transmission is associated with parenteral inoculation or with contamination of broken skin or mucous membranes with infectious body fluids. There is no evidence of airborne or interpersonal spread through casual contact. Prophylactic measures include the use of a licensed vaccine in high-risk groups and the use of hepatitis B immune globulin following overt exposures.

	The primary risks of HBv infection for laboratory personnel are accidental parenteral inoculation, exposure of broken skin or the mucous membranes of eyes, nose, or mouth.   These risks are typical of those described for Biosafety Level 2 agents, and are addressed by using the recommended standard and special microbiological practices to minimize or eliminate these overt exposures.

	The routes of infection with hepatitis C virus (non A-non B) and human immunodeficiency virus are similar for laboratory personnel.  The prudent practices recommended for HBv are applicable to these two pathogens, as well as to the routine laboratory manipulation of clinical materials of domestic origin.

	The described risk assessment process is also applicable to laboratory operations other than those involving the use of primary agents of human disease. Microbiological studies of animal host-specific pathogens, soil, water, food, feeds, and other natural or manufactured materials, by comparison, pose substantially lower risks for the laboratory worker. Microbiologists and other scientists working with such materials may, nevertheless, find the practices, containment equipment, and facility recommendations described in this publication of value in developing operational standards to meet their own assessed needs.

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