APPENDIX
Q. PHYSICAL
AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT DNA RESEARCH INVOLVING ANIMALS
Appendix
Q-I. General
Considerations
Appendix
Q-I-A. Containment
Levels
Appendix
Q-I-B. Disposal
of Animals (BL1-N through BL4-N)
Appendix
Q-II. Physical
and Biological Containment Levels
Appendix
Q-II-A. Biosafety
Level 1 - Animals (BL1-N)
Appendix
Q-II-A-1. Standard
Practices (BL1-N)
Appendix
Q-II-A-1-a. Animal
Facility Access (BL1-N)
Appendix
Q-II-A-1-b. Other
(BL1-N)
Appendix
Q-II-A-2. Animal
Facilities (BL1-N)
Appendix
Q-II-B-1. Standard
Practices (BL2-N)
Appendix
Q-II-B-1-a. Animal
Facility Access (BL2-N)
Appendix
Q-II-B-1-b. Decontamination
and Inactivation (BL2-N)
Appendix
Q-II-B-1-c. Signs
(BL2-N)
Appendix
Q-II-B-1-d. Protective
Clothing (BL2-N)
Appendix
Q-II-B-1-e. Records
(BL2-N)
Appendix
Q-II-B-1-f. Transfer
of Materials (BL2-N)
Appendix
Q-II-B-1-g. Other
(BL2-N)
Appendix
Q-II-B-2. Animal
Facilities (BL2-N)
Appendix
Q-II-C-1. Standard
Practices (BL3-N)
Appendix
Q-II-C-1-a. Animal
Facility Access (BL3-N)
Appendix
Q-II-C-1-b. Decontamination
and Inactivation (BL3-N)
Appendix
Q-II-C-1-c. Signs
(BL3-N)
Appendix
Q-II-C-1-d. Protective
Clothing (BL3-N)
Appendix
Q-II-C-1-e. Records
(BL3-N)
Appendix
Q-II-C-1-f. Transfer
of Materials (BL3-N)
Appendix
Q-II-C-1-g. Other
(BL3-N)
Appendix
Q-II-C-2. Animal
Facilities (BL3-N)
Appendix
Q-II-D-1. Standard
Practices (BL4-N)
Appendix
Q-II-D-1-a. Animal
Facility Access (BL4-N)
Appendix
Q-II-D-1-b. Decontamination
and Inactivation (BL4-N)
Appendix
Q-II-D-1-c. Signs
(BL4-N)
Appendix
Q-II-D-1-d. Protective
Clothing (BL4-N)
Appendix
Q-II-D-1-e. Records
(BL4-N)
Appendix
Q-II-D-1-f. Transfer
of Materials (BL4-N)
Appendix
Q-II-D-1-g. Other
(BL4-N)
Appendix
Q-II-D-2. Animal
Facilities (BL4-N)
Appendix
Q-III. Footnotes
and References for Appendix Q
Appendix Q specifies
containment and confinement practices for research involving whole animals,
both those in which the animal's genome has been altered by stable introduction
of recombinant DNA, or DNA derived therefrom, into the germ-line (transgenic
animals) and experiments involving viable recombinant DNA-modified
microorganisms tested on whole animals.
The appendix applies to animal research activities with the following
modifications:
Appendix Q shall
supersede Appendix G (Physical
Containment) when research animals are of a size or have growth
requirements that preclude the use of containment for laboratory animals. Some animals may require other types of
containment (see Appendix Q-III-D, Footnotes
and References for Appendix Q). The
animals covered in Appendix Q are those species normally categorized as animals
including but not limited to cattle, swine, sheep, goats, horses, and poultry.
The Institutional
Biosafety Committee shall include at least one scientist with expertise in
animal containment principles when experiments utilizing Appendix Q require
Institutional Biosafety Committee prior approval.
The institution shall
establish and maintain a health surveillance program for personnel engaged in
animal research involving viable recombinant DNA-containing microorganisms that
require Biosafety Level (BL) 3 or greater containment in the laboratory.
The containment
levels required for research involving recombinant DNA associated with or in
animals is based on classification of experiments in Section
III, Experiments Covered by the NIH Guidelines. For the purpose of animal research, four
levels of containment are established.
These are referred to as BL1-Animals (N), BL2-N, BL3-N, and BL4-N and
are described in the following appendices of Appendix Q. The descriptions include: (i) standard practices for physical and
biological containment, and (ii) animal facilities.
Appendix Q-I-B-1. When an animal covered by Appendix Q
containing recombinant DNA or a recombinant DNA-derived organism is euthanized or
dies, the carcass shall be disposed of to avoid its use as food for human
beings or animals unless food use is specifically authorized by an appropriate
Federal agency.
Appendix Q-I-B-2. A permanent record shall be maintained of
the experimental use and disposal of each animal or group of animals.
Appendix
Q-II-A-1-a-(1). The containment
area shall be locked.
Appendix
Q-II-A-1-a-(2). Access to the
containment area shall be limited or restricted when experimental animals are
being held.
Appendix
Q-II-A-1-a-(3). The containment
area shall be patrolled or monitored at frequent intervals.
Appendix
Q-II-A-1-b-(1). All genetically
engineered neonates shall be permanently marked within 72 hours after birth, if
their size permits. If their size does
not permit marking, their containers should be marked. In addition, transgenic animals should
contain distinct and biochemically assayable DNA sequences that allow
identification of transgenic animals from among non-transgenic animals.
Appendix
Q-II-A-1-b-(2) A double barrier
shall be provided to separate male and female animals unless reproductive
studies are part of the experiment or other measures are taken to avoid
reproductive transmission. Reproductive
incapacitation may be used.
Appendix
Q-II-A-1-b-(3). The containment
area shall be in accordance with state and Federal laws and animal care
requirements.
Appendix
Q-II-A-2-a. Animals shall be
confined to securely fenced areas or be in enclosed structures (animal rooms)
to minimize the possibility of theft or unintentional release.
Appendix
Q-II-B-1-a-(1). The containment
area shall be locked.
Appendix
Q-II-B-1-a-(2). The containment
area shall be patrolled or monitored at frequent intervals.
Appendix
Q-II-B-1-a-(3). The containment
building shall be controlled and have a locking access.
Appendix
Q-II-B-1-a-(4). The Animal Facility
Director shall establish policies and procedures whereby only persons who have
been advised of the potential hazard and who meet any specific entry
requirements (e.g., vaccination) may enter the laboratory or animal rooms.
Appendix
Q-II-B-1-a-(5). Animals of the same
or different species, which are not involved in the work being performed, shall
not be permitted in the animal area.
Appendix
Q-II-B-1-b-(1). Contaminated
materials that are decontaminated at a site away from the laboratory shall be
placed in a closed durable leak-proof container prior to removal from the
laboratory.
Appendix
Q-II-B-1-b-(2). Needles and
syringes shall be promptly placed in a puncture-resistant container and
decontaminated, preferably by autoclaving, before discard or reuse.
Appendix
Q-II-B-1-c-(1). When the animal
research requires special provisions for entry (e.g., vaccination), a warning
sign incorporating the universal biosafety symbol shall be posted on all access
doors to the animal work area. The sign
shall indicate: (i) the agent, (ii) the
animal species, (iii) the name and telephone number of the Animal Facility
Director or other responsible individual, and (iv) any special requirements for
entering the laboratory.
Appendix
Q-II-B-1-d-(1). Laboratory coats,
gowns, smocks, or uniforms shall be worn while in the animal area or attached
laboratory. Before entering
non-laboratory areas (e.g., cafeteria, library, administrative offices),
protective clothing shall be removed and kept in the work entrance area.
Appendix
Q-II-B-1-d-(2). Special care shall
be taken to avoid skin contamination with microorganisms containing recombinant
DNA. Impervious and/or protective
gloves shall be worn when handling experimental animals and when skin contact
with an infectious agent is unavoidable.
Appendix
Q-II-B-1-e-(1). Any incident
involving spills and accidents that result in environmental release or
exposures of animals or laboratory workers to organisms containing recombinant
DNA molecules shall be reported immediately to the Animal Facility Director,
Institutional Biosafety Committee, NIH/OBA, and other appropriate authorities
(if applicable). Reports to the NIH/OBA
shall be sent to the Office of Biotechnology Activities, National Institutes of
Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail),
301-496-9838, 301-496-9839 (fax).
Medical evaluation, surveillance, and treatment shall be provided as
appropriate and written records maintained.
If necessary, the area shall be appropriately decontaminated.
Appendix
Q-II-B-1-e-(2). When appropriate
and giving consideration to the agent handled, baseline serum samples shall be
collected and stored for animal care and other at-risk personnel. Additional serum specimens may be collected
periodically depending on the agent handled and the function of the animal
facility.
Appendix
Q-II-B-1-f-(1). Biological
materials removed from the animal containment area in a viable or intact state
shall be transferred to a non-breakable sealed primary container and then
enclosed in a non-breakable sealed secondary container. All containers, primary and secondary, shall
be disinfected before removal from the animal facility. Advance approval for transfer of material
shall be obtained from the Animal Facility Director. Packages containing viable agents may only be opened in a
facility having an equivalent or higher level of physical containment unless
the agent is biologically inactivated or incapable of reproduction.
Appendix
Q-II-B-1-g-(1). All genetically
engineered neonates shall be permanently marked within 72 hours after birth, if
their size permits. If their size does
not permit marking, their containers should be marked. In addition, transgenic animals should
contain distinct and biochemically assayable DNA sequences that allow
identification of transgenic animals from among non-transgenic animals.
Appendix
Q-II-B-1-g-(2). Needles and
syringes shall be used only for parenteral injection and aspiration of fluids
from laboratory animals and diaphragm bottles.
Only needle-locking syringes or disposable syringe-needle units (i.e.,
needle is integral to the syringe) shall be used for the injection or
aspiration of fluids containing organisms that contain recombinant DNA. Extreme caution shall be used when handling
needles and syringes to avoid autoinoculation and the generation of aerosols
during use and disposal. Following use,
needles shall not be bent, sheared, replaced in the needle sheath or guard, or
removed from the syringe. Needles and syringes
shall be promptly placed in a puncture-resistant container and decontaminated,
preferably by autoclaving, before discard or reuse.
Appendix
Q-II-B-1-g-(3). Appropriate steps
should be taken to prevent horizontal transmission or exposure of laboratory
personnel. If the agent used as a
vector is known to be transmitted by a particular route (e.g., arthropods),
special attention should be given to preventing spread by that route. In the absence of specific knowledge of a
particular route of transmission, all potential means of horizontal
transmission (e.g., arthropods, contaminated bedding, or animal waste, etc.)
should be prevented.
Appendix
Q-II-B-1-g-(4). Eating, drinking,
smoking, and applying cosmetics shall not be permitted in the work area.
Appendix
Q-II-B-1-g-(5). Individuals who handle
materials and animals containing recombinant DNA molecules shall be required to
wash their hands before exiting the containment area.
Appendix
Q-II-B-1-g-(6). A double barrier
shall be provided to separate male and female animals unless reproductive
studies are part of the experiment or other measures are taken to avoid
reproductive transmission. Reproductive
incapacitation may be used.
Appendix
Q-II-B-1-g-(7). The containment
area shall be in accordance with state and Federal laws and animal care
requirements.
Appendix
Q-II-B-1-g-(8). A biosafety manual
shall be prepared or adopted. Personnel
shall be advised of special hazards and required to read and follow
instructions on practices and procedures.
Appendix
Q-II-B-2-a. Animals shall be
contained within an enclosed structure (animal room or equivalent) to minimize
the possibility of theft or unintentional release and to avoid arthropod
access. The special provision to avoid
the entry or escape of arthropods from the animal areas may be waived if the
agent in use is not known to be transmitted by arthropods.
Appendix
Q-II-B-2-b. Surfaces shall be
impervious to water and resistant to acids, alkalis, organic solvents, and
moderate heat.
Appendix
Q-II-B-2-c. The animal containment
area shall be designed so that it can be easily cleaned.
Appendix
Q-II-B-2-d. Windows that open shall
be fitted with fly screens.
Appendix
Q-II-B-2-e. An autoclave shall be
available for decontamination of laboratory wastes.
Appendix
Q-II-B-2-f. If arthropods are used
in the experiment or the agent under study can be transmitted by an arthropod,
interior work areas shall be appropriately screened (52 mesh). All perimeter joints and openings shall be
sealed and additional arthropod control mechanisms used to minimize arthropod
entry and propagation, including appropriate screening of access doors or the
equivalent.
Appendix
Q-II-C-1-a-(1). The containment
area shall be locked.
Appendix
Q-II-C-1-a-(2). The containment
area shall be patrolled or monitored at frequent intervals.
Appendix
Q-II-C-1-a-(3). The containment
building shall be controlled and have a locking access.
Appendix
Q-II-C-1-a-(4). The Animal Facility
Director shall establish policies and procedures whereby only persons who have
been advised of the potential hazard and who meet any specific entry
requirements (e.g., vaccination) shall enter the laboratory or animal rooms.
Appendix
Q-II-C-1-a-(5). Animal room doors,
gates, or other closures shall be kept closed when experiments are in progress.
Appendix
Q-II-C-1-b-(1). The work surfaces
of containment equipment shall be decontaminated when work with organisms
containing recombinant DNA molecules is finished. Where feasible, plastic-backed paper toweling shall be used on
nonporous work surfaces to facilitate clean-up.
Appendix
Q-II-C-1-b-(2). All animals shall
be euthanized at the end of their experimental usefulness and the carcasses
decontaminated before disposal in an approved manner.
Appendix
Q-II-C-1-b-(3). Needles and
syringes shall be promptly placed in a puncture-resistant container and
decontaminated, preferably by autoclaving, before discard or reuse.
Appendix
Q-II-C-1-b-(4). Special safety
testing, decontamination procedures, and Institutional Biosafety Committee
approval shall be required to transfer agents or tissue/organ specimens from a
BL3-N animal facility to a facility with a lower containment classification.
Appendix
Q-II-C-1-b-(5). Liquid effluent
from containment equipment, sinks, biological safety cabinets, animal rooms,
primary barriers, floor drains, and sterilizers shall be decontaminated by heat
treatment before being released into the sanitary system. The procedure used for heat decontamination
of liquid wastes shall be monitored with a recording thermometer. The effectiveness of the heat
decontamination process system shall be revalidated every 30 days with an
indicator organism.
Appendix
Q-II-C-1-c-(1). When the animal
research requires special provisions for entry (e.g., vaccination), a warning
sign incorporating the universal biosafety symbol shall be posted on all access
doors to the animal work area. The sign
shall indicate: (i) the agent, (ii) the
animal species, (iii) the name and telephone number of the Animal Facility
Director or other responsible individual, and (iv) any special requirements for
entering the laboratory.
Appendix
Q-II-C-1-d-(1). Full protective
clothing that protects the individual (e.g., scrub suits, coveralls, uniforms)
shall be worn in the animal area. Clothing
shall not be worn outside the animal containment area and shall be
decontaminated before laundering or disposal.
Personnel shall be required to shower before exiting the BL3-N area and
wearing of personal clothing.
Appendix
Q-II-C-1-d-(2). Special care shall
be taken to avoid skin contamination with microorganisms containing recombinant
DNA. Impervious and/or protective
gloves shall be worn when handling experimental animals and when skin contact
with an infectious agent is unavoidable.
Appendix
Q-II-C-1-d-(3). Appropriate
respiratory protection shall be worn in rooms containing experimental animals.
Appendix
Q-II-C-1-e-(1). Documents regarding
experimental animal use and disposal shall be maintained in a permanent record
book.
Appendix
Q-II-C-1-e-(2). Any incident
involving spills and accidents that result in environmental release or exposure
of animals or laboratory workers to organisms containing recombinant DNA shall
be reported immediately to the Biological Safety Office, Animal Facility
Director, Institutional Biosafety Committee, NIH/OBA, and other appropriate
authorities (if applicable). Reports to
the NIH/OBA shall be sent to the Office of Biotechnology Activities, National
Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda,
MD 20892-7985 (20817 for non-USPS
mail), 301-496-9838, 301-496-9839 (fax).
Medical evaluation, surveillance, and treatment shall be provided as
appropriate and written records maintained.
If necessary, the area shall be appropriately decontaminated.
Appendix
Q-II-C-1-e-(3). When appropriate
and giving consideration to the agent handled, baseline serum samples shall be
collected and stored for animal care and other at-risk personnel. Additional serum specimens may be collected
periodically depending on the agent handled or the function of the facility.
Appendix
Q-II-C-1-f-(1). Biological
materials removed from the animal containment laboratory in a viable or intact
state shall be transferred to a non-breakable sealed primary container and then
enclosed in a non-breakable sealed secondary container. All containers, primary and secondary, shall
be disinfected before removal from the animal facility. Advance approval for transfer of material
shall be obtained from the Animal Facility Director. Packages containing viable agents may be opened only in a
facility having an equivalent or higher level of physical containment unless
the agent is biologically inactivated or incapable of reproduction.
Appendix
Q-II-C-1-f-(2). Special safety
testing, decontamination procedures, and Institutional Biosafety Committee
approval shall be required to transfer agents or tissue/organ specimens from a
BL3-N animal facility to a facility with a lower containment classification.
Appendix
Q-II-C-1-g-(1). All genetically
engineered neonates shall be permanently marked within 72 hours after birth, if
their size permits. If their size does
not permit marking, their containers should be marked. In addition, transgenic animals should
contain distinct and biochemically assayable DNA sequences that allow
identification of transgenic animals from among non-transgenic animals.
Appendix
Q-II-C-1-g-(2). Appropriate steps
should be taken to prevent horizontal transmission or exposure of laboratory
personnel. If the agent used as the
vector is known to be transmitted by a particular route (e.g., arthropods),
special attention should be given to preventing spread by that route. In the absence of specific knowledge of a
particular route of transmission, all potential means of horizontal
transmission (e.g., arthropods, contaminated bedding, or animal waste) should
be prevented.
Appendix
Q-II-C-1-g-(3). Eating, drinking,
smoking, and applying cosmetics shall not be permitted in the work area.
Appendix
Q-II-C-1-g-(4). Individuals who
handle materials and animals containing recombinant DNA molecules shall be
required to wash their hands before exiting the containment area.
Appendix
Q-II-C-1-g-(5). Experiments
involving other organisms that require containment levels lower than BL3-N may
be conducted in the same area concurrently with experiments requiring BL3-N
containment provided that they are conducted in accordance with BL3-N
practices.
Appendix
Q-II-C-1-g-(6). Animal holding
areas shall be cleaned at least once a day and decontaminated immediately
following any spill of viable materials.
Appendix
Q-II-C-1-g-(7). All procedures
shall be performed carefully to minimize the creation of aerosols.
Appendix
Q-II-C-1-g-(8). A double barrier
shall be provided to separate male and female animals unless reproductive
studies are part of the experiment or other measures are taken to avoid
reproductive transmission. Reproductive
incapacitation may be used.
Appendix
Q-II-C-1-g-(9). The containment
area shall be in accordance with state and Federal laws and animal care
requirements.
Appendix
Q-II-C-1-g-(10). All animals shall
be euthanized at the end of their experimental usefulness and the carcasses
decontaminated before disposal in an approved manner.
Appendix
Q-II-C-1-g-(11). Personnel shall be
required to shower before exiting the BL3-N area and wearing personal clothing.
Appendix
Q-II-C-1-g-(12). Animals of the
same or different species, which are not involved in the work being performed,
shall not be permitted in the animal area.
Appendix
Q-II-C-1-g-(13). Needles and
syringes shall be used only for parenteral injection and aspiration of fluids
from laboratory animals and diaphragm bottles.
Only needle-locking syringes or disposable syringe-needle units (i.e.,
needle is integral to the syringe) shall be used for the injection or
aspiration of fluids containing organisms that contain recombinant DNA. Extreme caution shall be used when handling
needles and syringes to avoid autoinoculation and the generation of aerosols
during use and disposal. Following use,
needles shall not be bent, sheared, replaced in the needle sheath or guard or
removed from the syringe. The needles
and syringes shall be promptly placed in a puncture-resistant container and
decontaminated, preferably by autoclaving, before discard or reuse.
Appendix
Q-II-C-1-g-(14). A biosafety manual
shall be prepared or adopted. Personnel
shall be advised of special hazards and required to read and follow
instructions on practices and procedures.
Appendix
Q-II-C-2-a. Animals shall be
contained within an enclosed structure (animal room or equivalent) to minimize
the possibility of theft or unintentional release and avoid arthropod
access. The special provision to avoid
the entry or escape of arthropods from the animal areas may be waived if the
agent in use is not known to be transmitted by arthropods.
Appendix
Q-II-C-2-b. The interior walls,
floors, and ceilings shall be impervious to water and resistant to acids,
alkalis, organic solvents, and moderate heat, to facilitate cleaning. Penetrations in these structures and
surfaces (e.g., plumbing and utilities) shall be sealed.
Appendix
Q-II-C-2-c. Windows in the animal
facility shall be closed, sealed, and breakage resistant (e.g., double-pane
tempered glass or equivalent). The need
to maintain negative pressure should be considered when constructing or
renovating the animal facility.
Appendix
Q-II-C-2-d. An autoclave,
incinerator, or other effective means to decontaminate animals and waste shall
be available, preferably within the containment area. If feasible, a double-door autoclave is preferred and should be
positioned to allow removal of material from the containment area.
Appendix
Q-II-C-2-e. If arthropods are used
in the experiment or the agent under study can be transmitted by an arthropod,
the interior work area shall be appropriately screened (52 mesh). All perimeter joints and openings shall be
sealed, and additional arthropod control mechanisms used to minimize arthropod
entry and propagation, including appropriate screening, or the equivalent of
access doors.
Appendix
Q-II-C-2-f. Access doors to the
containment area shall be self-closing.
Appendix
Q-II-C-2-g. The animal area shall
be separated from all other areas.
Passage through two sets of doors shall be the basic requirement for
entry into the animal area from access corridors or other contiguous
areas. The animal containment area
shall be physically separated from access corridors and other laboratories or
areas by a double-door clothes change room, equipped with integral showers and
airlock.
Appendix
Q-II-C-2-h. Liquid effluent from
containment equipment, sinks, biological safety cabinets, animal rooms, primary
barriers, floor drains, and sterilizers shall be decontaminated by heat
treatment before being released into the sanitary system. The procedure used for heat decontamination
of liquid wastes shall be monitored with a recording thermometer. The effectiveness of the heat
decontamination process system shall be revalidated every 30 days with an indicator
organism.
Appendix
Q-II-C-2-i. An exhaust air
ventilation system shall be provided.
This system shall create directional airflow that draws air into the
animal room through the entry area. The
building exhaust, or the exhaust from primary containment units, may be used
for this purpose if the exhaust air is discharged to the outside and shall be
dispersed away from occupied areas and air intakes. Personnel shall verify that the direction of the airflow (into
the animal room) is proper.
Appendix
Q-II-C-2-j. If the agent is
transmitted by aerosol, then the exhaust air shall pass through a high
efficiency particulate air/HEPA filter.
Appendix
Q-II-C-2-k. Vacuum lines shall be
protected with high efficiency particulate air/HEPA filters and liquid
disinfectant traps.
Appendix
Q-II-C-2-l. In lieu of open housing
in the special animal room, animals held in a BL3-N area may be housed in
partial-containment caging systems (e.g., Horsfall units or gnotobiotic
systems, or other special containment primary barriers). Prudent judgment must be exercised to
implement this ventilation system (e.g., animal species) and its discharge
location.
Appendix
Q-II-C-2-m. Each animal area shall
contain a foot, elbow, or automatically operated sink for hand washing. The sink shall be located near the exit
door.
Appendix
Q-II-C-2-n. Restraining devices for
animals may be required to avoid damage to the integrity of the animal
containment facility.
Appendix
Q-II-D-1-a-(1). Individuals under 16
years of age shall not be permitted to enter the animal area.
Appendix
Q-II-D-1-a-(2). The containment
area shall be locked.
Appendix
Q-II-D-1-a-(3). The containment
area shall be patrolled or monitored at frequent intervals.
Appendix
Q-II-D-1-a-(4). The containment
building shall be controlled and have a locking access.
Appendix
Q-II-D-1-a-(5). The Animal Facility
Director shall establish policies and procedures whereby only persons who have
been advised of the potential hazard and who meet any specific entry
requirements (e.g., vaccination) may enter the laboratory or animal room.
Appendix
Q-II-D-1-a-(6). Individuals shall
enter and exit the animal facility only through the clothing change and shower
rooms.
Appendix
Q-II-D-1-a-(7). Personnel shall use
the airlocks to enter or exit the laboratory only in an emergency.
Appendix
Q-II-D-1-a-(8). Animal room doors,
gates, and other closures shall be kept closed when experiments are in
progress.
Appendix
Q-II-D-1-b-(1). All contaminated
liquid or solid wastes shall be decontaminated before disposal.
Appendix
Q-II-D-1-b-(2). The work surfaces
and containment equipment shall be decontaminated when work with organisms
containing recombinant DNA molecules is finished. Where feasible, plastic-backed paper toweling shall be used on
nonporous work surfaces to facilitate clean-up.
Appendix
Q-II-D-1-b-(3). All wastes from animal
rooms and laboratories shall be appropriately decontaminated before disposal in
an approved manner.
Appendix
Q-II-D-1-b-(4). No materials,
except for biological materials that are to remain in a viable or intact state,
shall be removed from the maximum containment laboratory unless they have been
autoclaved or decontaminated. Equipment
or material that might be damaged by high temperatures or steam shall be
decontaminated by gaseous or vapor methods in an airlock or chamber designed
for this purpose.
Appendix
Q-II-D-1-b-(5). When ventilated
suits are required, the animal personnel shower entrance/exit area shall be
equipped with a chemical disinfectant shower to decontaminate the surface of
the suit before exiting the area. A
neutralization or water dilution device shall be integral with the chemical
disinfectant discharge piping before entering the heat sterilization
system. Entry to this area shall be
through an airlock fitted with airtight doors.
Appendix
Q-II-D-1-b-(6). Needles and syringes
shall be promptly placed in a puncture-resistant container and decontaminated,
preferably by autoclaving, before discard or reuse.
Appendix
Q-II-D-1-b-(7). Supplies and
materials needed in the animal facility shall be brought in by way of the
double-door autoclave, fumigation chamber, or airlock that shall be
appropriately decontaminated between each use.
Appendix
Q-II-D-1-b-(8). An autoclave,
incinerator, or other effective means to decontaminate animals and wastes shall
be available, preferably within the containment area. If feasible, a double-door autoclave is preferred and should be
positioned to allow removal of material from the containment area.
Appendix
Q-II-D-1-b-(9). Liquid effluent
from containment equipment, sinks, biological safety cabinets, animal rooms,
primary barriers, floor drains, and sterilizers shall be decontaminated by heat
treatment before being released into the sanitary system. Liquid wastes from shower rooms and toilets
shall be decontaminated with chemical disinfectants or heat by methods
demonstrated to be effective. The
procedure used for heat decontamination of liquid wastes shall be monitored
with a recording thermometer. The
effectiveness of the heat decontamination process system shall be revalidated
every 30 days with an indicator organism.
Liquid wastes from the shower shall be chemically decontaminated using
an Environmental Protection Agency-approved germicide. The efficacy of the chemical treatment
process shall be validated with an indicator organism. Chemical disinfectants shall be neutralized
or diluted before release into general effluent waste systems.
Appendix
Q-II-D-1-c-(1). When the animal
research requires special provisions for entry (e.g., vaccination), a warning
sign incorporating the universal biosafety symbol shall be posted on all access
doors to the animal work area. The sign
shall indicate: (i) the agent, (ii) the
animal species, (iii) the name and telephone number of the Animal Facility
Director, or other responsible individual, and (iv) any special requirements
for entering the laboratory.
Appendix
Q-II-D-1-d-(1). Individuals shall
enter and exit the animal facility only through the clothing change and shower
rooms. Street clothing shall be removed
and kept in the outer clothing change room.
Complete laboratory clothing (may be disposable), including
undergarments, pants, shirts, jump suits, and shoes shall be provided for all
personnel entering the animal facility.
When exiting the BL4-N area and before proceeding into the shower area,
personnel shall remove their laboratory clothing in the inner change room. All laboratory clothing shall be autoclaved
before laundering. Personnel shall
shower each time they exit the animal facility.
Appendix
Q-II-D-1-d-(2). A ventilated
head-hood or a one-piece positive pressure suit, which is ventilated by a
life-support system, shall be worn by all personnel entering rooms that contain
experimental animals when appropriate.
When ventilated suits are required, the animal personnel shower
entrance/exit area shall be equipped with a chemical disinfectant shower to
decontaminate the surface of the suit before exiting the area. A neutralization or water dilution device
shall be integral with the chemical disinfectant discharge piping before
entering the heat sterilization system.
Entry to this area shall be through an airlock fitted with airtight
doors.
Appendix
Q-II-D-1-d-(3). Appropriate
respiratory protection shall be worn in rooms containing experimental animals.
Appendix
Q-II-D-1-e-(1). Documents regarding
experimental animal use and disposal shall be maintained in a permanent record
book.
Appendix Q-II-D-1-e-(2). A system shall be established for: (i) reporting laboratory accidents and
exposures that are a result of overt exposures to organisms containing
recombinant DNA, (ii) employee absenteeism, and (iii) medical surveillance of
potential laboratory-associated illnesses.
Permanent records shall be prepared and maintained. Any incident involving spills and accidents
that results in environmental release or exposures of animals or laboratory
workers to organisms containing recombinant DNA molecules shall be reported
immediately to the Biological Safety Officer, Animal Facility Director,
Institutional Biosafety Committee, NIH/OBA, and other appropriate authorities
(if applicable). Reports to the NIH/OBA
shall be sent to the Office of Biotechnology Activities, National Institutes of
Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail),
301-496-9838, 301-496-9839 (fax). Medical
evaluation, surveillance, and treatment shall be provided as appropriate and
written records maintained. If
necessary, the area shall be appropriately decontaminated.
Appendix
Q-II-D-1-e-(3). When appropriate
and giving consideration to the agents handled, baseline serum samples shall be
collected and stored for animal care and other at-risk personnel. Additional serum specimens may be collected
periodically depending on the agents handled or the function of the facility.
Appendix
Q-II-D-1-e-(4). A permanent record
book indicating the date and time of each entry and exit shall be signed by all
personnel.
Appendix
Q-II-D-1-f-(1). No materials,
except for biological materials that are to remain in a viable or intact state,
shall be removed from the maximum containment laboratory unless they have been
autoclaved or decontaminated. Equipment
or material that might be damaged by high temperatures or steam shall be
decontaminated by gaseous or vapor methods in an airlock or chamber designed
for this purpose.
Appendix Q-II-D-1-f-(2). Biological materials removed from the animal
maximum containment laboratory in a viable or intact state shall be transferred
to a non-breakable sealed primary container and then enclosed in a
non-breakable sealed secondary container that shall be removed from the animal
facility through a disinfectant dunk tank, fumigation chamber, or an airlock
designed for this purpose. Advance
approval for transfer of material shall be obtained from the Animal Facility Director. Such packages containing viable agents can
only be opened in another BL4-N animal facility if the agent is biologically
inactivated or incapable of reproduction.
Special safety testing, decontamination procedures, and Institutional
Biosafety Committee approval shall be required to transfer agents or
tissue/organ specimens from a BL4-N animal facility to one with a lower
containment classification.
Appendix
Q-II-D-1-f-(3). Supplies and
materials needed in the animal facility shall be brought in by way of the
double-door autoclave, fumigation chamber, or airlock that shall be
appropriately decontaminated between each use.
After securing the outer doors, personnel within the animal facility
retrieve the materials by opening the interior doors of the autoclave,
fumigation chamber, or airlock. These
doors shall be secured after materials are brought into the animal facility.
Appendix
Q-II-D-1-g-(1). All genetically
engineered neonates shall be permanently marked within 72 hours after birth, if
their size permits. If their size does
not permit marking, their containers should be marked. In addition, transgenic animals should
contain distinct and biochemically assayable DNA sequences that allow
identification of transgenic animals from among non-transgenic animals.
Appendix
Q-II-D-1-g-(2). Eating, drinking,
smoking, and applying cosmetics shall not be permitted in the work area.
Appendix
Q-II-D-1-g-(3). Individuals who
handle materials and animals containing recombinant DNA molecules shall be
required to wash their hands before exiting the containment area.
Appendix
Q-II-D-1-g-(4). Experiments
involving other organisms that require containment levels lower than BL4-N may
be conducted in the same area concurrently with experiments requiring BL4-N containment
provided that they are conducted in accordance with BL4-N practices.
Appendix
Q-II-D-1-g-(5). Animal holding
areas shall be cleaned at least once a day and decontaminated immediately
following any spill of viable materials.
Appendix Q-II-D-1-g-(6). All procedures shall be performed carefully
to minimize the creation of aerosols.
Appendix
Q-II-D-1-g-(7). A double barrier
shall be provided to separate male and female animals. Animal isolation barriers shall be sturdy
and accessible for cleaning.
Reproductive incapacitation may be used.
Appendix
Q-II-D-1-g-(8). The containment
area shall be in accordance with state and Federal laws and animal care
requirements.
Appendix
Q-II-D-1-g-(9). The life support
system for the ventilated suit or head hood is equipped with alarms and
emergency back-up air tanks. The
exhaust air from the suit area shall be filtered by two sets of high efficiency
particulate air/HEPA filters installed in series or incinerated. A duplicate filtration unit, exhaust fan,
and an automatically starting emergency power source shall be provided. The air pressure within the suit shall be
greater than that of any adjacent area.
Emergency lighting and communication systems shall be provided. A double-door autoclave shall be provided
for decontamination of waste materials to be removed from the suit area.
Appendix
Q-II-D-1-g-(10). Needles and
syringes shall be used only for parenteral injection and aspiration of fluids
from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units
(i.e., needle is integral to the syringe) shall be used for the injection or
aspiration of fluids containing organisms that contain recombinant DNA. Extreme caution shall be used when handling
needles and syringes to avoid autoinoculation and the generation of aerosols
during use and disposal. Following use,
needles shall
not be bent, sheared,
replaced in the needle sheath or guard, or removed from the syringe. The needles and syringes shall be promptly
placed in a puncture-resistant container and decontaminated, preferably by
autoclaving, before discard or reuse.
Appendix
Q-II-D-1-g-(11). An essential
adjunct to the reporting-surveillance system is the availability of a facility
for quarantine, isolation, and medical care of personnel with potential or
known laboratory-associated illnesses.
Appendix
Q-II-D-1-g-(12). A biosafety manual
shall be prepared or adopted. Personnel
shall be advised of special hazards and required to read and follow
instructions on practices and procedures.
Appendix
Q-II-D-1-g-(13). Vacuum lines shall
be protected with high efficiency particulate air/HEPA filters and liquid
disinfectant traps.
Appendix Q-II-D-2-a. Animals shall be contained within an
enclosed structure (animal room or equivalent) to minimize the possibility of
theft or unintentional release and avoid arthropod access.
Appendix
Q-II-D-2-b. The interior walls,
floors, and ceilings shall be impervious to water and resistant to acids,
alkalis, organic solvents, and moderate heat, to facilitate cleaning. Penetrations in these structures and
surfaces (e.g., plumbing and utilities) shall be sealed.
Appendix
Q-II-D-2-c. Windows in the animal
facility shall be closed, sealed, and breakage resistant (e.g., double-pane
tempered glass or equivalent).
Appendix
Q-II-D-2-d. An autoclave,
incinerator, or other effective means to decontaminate animals and wastes shall
be available, preferably within the containment area. If feasible, a double-door autoclave is preferred and should be
positioned to allow removal of material from the containment area.
Appendix
Q-II-D-2-e. Access doors to the
containment area shall be self-closing.
Appendix
Q-II-D-2-f. All perimeter joints
and openings shall be sealed to form an arthropod-proof structure.
Appendix
Q-II-D-2-g. The BL4-N laboratory
provides a double barrier to prevent the release of recombinant DNA containing
microorganisms into the environment.
Design of the animal facility shall be such that if the barrier of the
inner facility is breached, the outer barrier will prevent release into the
environment. The animal area shall be
separated from all other areas. Passage
through two sets of doors shall be the basic requirement for entry into the
animal area from access corridors or other contiguous areas. Physical separation of the animal
containment area from access corridors or other laboratories or activities shall
be provided by a double-door clothes change room equipped with integral showers
and airlock.
Appendix
Q-II-D-2-h. A necropsy room shall
be provided within the BL4-N containment area.
Appendix
Q-II-D-2-i. Liquid effluent from
containment equipment, sinks, biological safety cabinets, animal rooms, primary
barriers, floor drains, and sterilizers shall be decontaminated by heat
treatment before being released into the sanitary system. Liquid wastes from shower rooms and toilets
shall be decontaminated with chemical disinfectants or heat by methods
demonstrated to be effective. The
procedure used for heat decontamination of liquid wastes shall be monitored
with a recording thermometer. The
effectiveness of the heat decontamination process system shall be revalidated
every 30 days with an indicator organism.
Liquid wastes from the shower shall be chemically decontaminated using
an Environmental Protection Agency-approved germicide. The efficacy of the chemical treatment
process shall be validated with an indicator organism. Chemical disinfectants shall be neutralized
or diluted before release into general effluent waste systems.
Appendix
Q-II-D-2-j. A ducted exhaust air
ventilation system shall be provided that creates directional airflow that
draws air into the laboratory through the entry area. The exhaust air, which is not recirculated to any other area of
the building, shall be discharged to the outside and dispersed away from the
occupied areas and air intakes.
Personnel shall verify that the direction of the airflow (into the
animal room) is proper.
Appendix
Q-II-D-2-k. Exhaust air from BL4-N
containment area shall be double high efficiency particulate air/HEPA filtered
or treated by passing through a certified HEPA filter and an air incinerator
before release to the atmosphere. Double
HEPA filters shall be required for the supply air system in a BL4-N containment
area.
Appendix
Q-II-D-2-l. All high efficiency
particulate air/HEPA filters' frames and housings shall be certified to have no
detectable smoke [dioctylphthalate] leaks when the exit face (direction of
flow) of the filter is scanned above 0.01 percent when measured by a linear or
logarithmic photometer. The instrument
must demonstrate a threshold sensitivity of at least 1x10-3
micrograms per liter for 0.3 micrometer diameter dioctylphthalate particles and
a challenge concentration of 80-120 micrograms per liter. The air sampling rate should be at least 1
cfm (28.3 liters per minute).
Appendix
Q-II-D-2-m. If an air incinerator
is used in lieu of the second high efficiency particulate air/HEPA filter, it
shall be biologically challenged to prove all viable test agents are
sterilized. The biological challenge
must be minimally 1x108 organisms per cubic foot of airflow through
the incinerator. It is universally
accepted if bacterial spores are used to challenge and verify that the
equipment is capable of killing spores, then assurance is provided that all
other known agents are inactivated by the parameters established to operate the
equipment. Test spores meeting this criterion
are Bacillus subtilis var. niger or Bacillus stearothermophilis. The operating temperature of the incinerator
shall be continuously monitored and recorded during use.
Appendix
Q-II-D-2-n. All equipment and floor
drains shall be equipped with deep traps (minimally 5 inches). Floor drains shall be fitted with isolation
plugs or fitted with automatic water fill devices.
Appendix
Q-II-D-2-o. Each animal area shall
contain a foot, elbow, or automatically operated sink for hand washing. The sink shall be located near the exit
door.
Appendix
Q-II-D-2-p. Restraining devices for
animals may be required to avoid damage to the integrity of the containment
animal facility.
Appendix
Q-II-D-2-q. The supply water
distribution system shall be fitted with a back-flow preventer or break tank.
Appendix
Q-II-D-2-r. All utilities, liquid
and gas services, shall be protected with devices that avoid back-flow.
Appendix
Q-II-D-2-s. Sewer and other
atmospheric ventilation lines shall be equipped minimally with a single high
efficiency particulate/HEPA filter.
Condensate drains from these type housings shall be appropriately
connected to a contaminated or sanitary drain system. The drain position in the housing dictates the appropriate system
to be used.
Appendix Q-III-A. If recombinant DNA is derived from a Class 2
organism requiring BL2 containment, personnel shall be required to have
specific training in handling pathogenic agents and directed by knowledgeable
scientists.
Appendix Q-III-B. Personnel who handle pathogenic and
potentially lethal agents shall be required to have specific training and be
supervised by knowledgeable scientists who are experienced in working with
these agents. BL3-N containment also
minimizes escape of recombinant DNA-containing organisms from exhaust air or
waste material from the containment area.
Appendix Q-III-C. Risk Group 4 and restricted microorganisms
(see Appendix B, Classification
of Human Etiologic Agents on the Basis of Hazard, and Sections
V-G and V-L, Footnotes and References of Sections I through IV) pose
a high level of individual risk for acquiring life-threatening diseases to
personnel and/or animals. To import
animal or plant pathogens, special approval must be obtained from U.S. Department of Agriculture, Animal and
Plant Health Inspection Service (APHIS), Veterinary Services, National
Center for Import-Export, Products Program, 4700 River Road, Unit 40,
Riverdale, MD 20737. Phone: (301) 734-8499; Fax: (301) 734-8226.
Laboratory staff
shall be required to have specific and thorough training in handling extremely
hazardous infectious agents, primary and secondary containment, standard and
special practices, and laboratory design characteristics. The laboratory staff shall be supervised by
knowledgeable scientists who are trained and experienced in working with these
agents and in the special containment facilities.
Within work areas of
the animal facility, all activities shall be confined to the specially equipped
animal rooms or support areas. The
maximum animal containment area and support areas shall have special
engineering and design features to prevent the dissemination of microorganisms
into the environment via exhaust air or waste disposal.
Appendix Q-III-D. Other research with non-laboratory animals,
which may not appropriately be conducted under conditions described in Appendix Q, may be conducted safely by
applying practices routinely used for controlled culture of these biota. In aquatic systems, for example, BL1
equivalent conditions could be met by utilizing growth tanks that provide
adequate physical means to avoid the escape of the aquatic species, its
gametes, and introduced exogenous genetic material. A mechanism shall be provided to ensure that neither the
organisms nor their gametes can escape into the supply or discharge system of
the rearing container (e.g., tank, aquarium, etc.) Acceptable barriers include appropriate filtration, irradiation,
heat treatment, chemical treatment, etc.
Moreover, the top of the rearing container shall be covered to avoid
escape of the organism and its gametes.
In the event of tank rupture, leakage, or overflow, the construction of
the room containing these tanks should prevent the organisms and gametes from
entering the building's drains before the organism and its gametes have been
inactivated.
Other types of
non-laboratory animals (e.g., nematodes, arthropods, and certain forms of
smaller animals) may be accommodated by using the appropriate BL1 through BL4
or BL1-P through BL4-P containment practices and procedures as specified in
Appendices G and P.