Level 3 (ABSL3)
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.
A. Standard Practices
B. Special 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
- 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.(9) 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
- 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 #3
- Policies for the safe handling of sharps are
- 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
- Syringes that re-sheathe the needle,
needle-less systems, and other safe devices should be used when
- 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
- 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
C. Safety Equipment (Primary
- 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
- Materials not related to the
experiment (e.g., plants, animals) are not permitted in the animal
D. Facilities (Secondary
- 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.
- Personal protective equipment is used for
all activities involving manipulations of infectious material or
- Personnel wear gloves when handling
infected animals. Gloves are removed aseptically and autoclaved
with other animal room wastes before disposal.
- Appropriate face/eye and respiratory
protection (e.g., respirators and face shields) is worn by all
personnel entering animal rooms.
- Boots, shoe covers, or other protective
footwear, and disinfectant foot baths are available and used where
- 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.
- The animal facility is separated
from areas that are open to unrestricted personnel traffic within the
- 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.
- 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,
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.
- Cages are washed in a cage
washer. The mechanical cage washer has a final rinse temperature
of at least 180F.
- 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.
Reproduced from "Biosafety in Microbiological and Biomedical
Laboratories, BMBL 4th Edition" with permission from the Center for
Disease Control (CDC).