APPENDIX
P. PHYSICAL
AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT DNA RESEARCH INVOLVING PLANTS
Appendix
P-I. General
Plant Biosafety Levels
Appendix
P-II. Physical
Containment Levels
Appendix
P-II-A. Biosafety
Level 1 - Plants (BL1-P)
Appendix
P-II-A-1. Standard
Practices (BL1-P)
Appendix
P-II-A-1-a. Greenhouse
Access (BL1-P)
Appendix
P-II-A-1-b. Records
(BL1-P)
Appendix
P-II-A-1-c. Decontamination
and Inactivation (BL1-P)
Appendix
P-II-A-1-d. Control
of Undesired Species and Motile Macroorganisms (BL1-P)
Appendix
P-II-A-1-e. Concurrent
Experiments Conducted in the Greenhouse (BL1-P)
Appendix
P-II-A-2. Facilities
(BL1-P)
Appendix
P-II-A-2-a. Definitions
(BL1-P)
Appendix
P-II-A-2-b. Greenhouse
Design (BL1-P)
Appendix
P-II-B. Biosafety
Level 2 - Plants (BL2-P)
Appendix
P-II-B-1. Standard
Practices (BL2-P)
Appendix
P-II-B-1-a. Greenhouse
Access (BL2-P)
Appendix
P-II-B-1-b. Records
(BL2-P)
Appendix
P-II-B-1-c. Decontamination
and Inactivation (BL2-P)
Appendix
P-II-B-1-d. Control
of Undesired Species and Motile Macroorganisms (BL2-P)
Appendix
P-II-B-1-e. Concurrent
Experiments Conducted in the Greenhouse (BL2-P)
Appendix
P-II-B-1-f. Signs
(BL2-P)
Appendix
P-II-B-1-g. Transfer
of Materials (BL2-P)
Appendix
P-II-B-1-h. Greenhouse
Practices Manual (BL2-P)
Appendix
P-II-B-2. Facilities
(BL2-P)
Appendix
P-II-B-2-a. Definitions
(BL2-P)
Appendix
P-II-B-2-b. Greenhouse
Design (BL2-P)
Appendix
P-II-B-2-c. Autoclaves
(BL2-P)
Appendix
P-II-B-2-d. Supply
and Exhaust Air Ventilation Systems (BL2-P)
Appendix
P-II-B-2-e. Other
(BL2-P)
Appendix
P-II-C. Biosafety
Level 3 - Plants (BL3-P)
Appendix
P-II-C-1. Standard
Practices (BL3-P)
Appendix
P-II-C-1-a. Greenhouse
Access (BL3-P)
Appendix
P-II-C-1-b. Records
(BL3-P)
Appendix
P-II-C-1-c. Decontamination
and Inactivation (BL3-P)
Appendix
P-II-C-1-d. Control
of Undesired Species and Motile Macroorganisms (BL3-P)
Appendix
P-II-C-1-e. Concurrent
Experiments Conducted in the Greenhouse (BL3-P)
Appendix
P-II-C-1-f. Signs
(BL3-P)
Appendix
P-II-C-1-g. Transfer
of Materials (BL3-P)
Appendix
P-II-C-1-h. Greenhouse
Practices Manual (BL3-P)
Appendix
P-II-C-1-i. Protective
Clothing (BL3-P)
Appendix
P-II-C-1-j. Other
(BL3-P)
Appendix
P-II-C-2. Facilities
(BL3-P)
Appendix
P-II-C-2-a. Definitions
(BL3-P)
Appendix
P-II-C-2-b. Greenhouse
Design (BL3-P)
Appendix
P-II-C-2-c. Autoclaves
(BL3-P)
Appendix
P-II-C-2-d. Supply
and Exhaust Air Ventilation Systems (BL3-P)
Appendix
P-II-C-2-e. Other
(BL3-P)
Appendix
P-II-D. Biosafety
Level 4 - Plants (BL4-P)
Appendix
P-II-D-1. Standard
Practices (BL4-P)
Appendix
P-II-D-1-a. Greenhouse
Access (BL4-P)
Appendix
P-II-D-1-b. Records
(BL4-P)
Appendix
P-II-D-1-c. Decontamination
and Inactivation (BL4-P)
Appendix
P-II-D-1-d. Control
of Undesired Species and Motile Macroorganisms (BL4-P)
Appendix
P-II-D-1-e. Concurrent
Experiments Conducted in the Greenhouse (BL4-P)
Appendix
P-II-D-1-f. Signs
(BL4-P)
Appendix
P-II-D-1-g. Transfer
of Materials (BL4-P)
Appendix
P-II-D-1-h. Greenhouse
Practices Manual (BL4-P)
Appendix
P-II-D-1-i. Protective
Clothing (BL4-P)
Appendix
P-II-D-2. Facilities
(BL4-P)
Appendix
P-II-D-2-a. Greenhouse
Design (BL4-P)
Appendix
P-II-D-2-b. Autoclaves
(BL4-P)
Appendix
P-II-D-2-c. Supply
and Exhaust Air Ventilation Systems (BL4-P)
Appendix
P-II-D-2-d. Other
(BL4-P)
Appendix
P-III. Biological
Containment Practices
Appendix
P-III-A. Biological
Containment Practices (Plants)
Appendix
P-III-B. Biological
Containment Practices (Microorganisms)
Appendix
P-III-C. Biological
Containment Practices (Macroorganisms)
Appendix P specifies
physical and biological containment conditions and practices suitable to the
greenhouse conduct of experiments involving recombinant DNA-containing plants,
plant-associated microorganisms, and small animals. All provisions of the NIH Guidelines apply to plant
research activities with the following modifications:
Appendix P shall
supersede Appendix G (Physical
Containment) when the research plants are of a size, number, or have growth
requirements that preclude the use of containment conditions described in
Appendix G. The plants covered in
Appendix P include but are not limited to mosses, liverworts, macroscopic
algae, and vascular plants including terrestrial crops, forest, and ornamental
species.
Plant-associated
microorganisms include viroids, virusoids, viruses, bacteria, fungi,
protozoans, certain small algae, and microorganisms that have a benign or
beneficial association with plants, such as certain Rhizobium species
and microorganisms known to cause plant diseases. The appendix applies to microorganisms which are being modified
with the objective of fostering an association with plants.
Plant-associated
small animals include those arthropods that:
(i) are in obligate association with plants, (ii) are plant pests, (iii)
are plant pollinators, or (iv) transmit plant disease agents, as well as other
small animals such as nematodes for which tests of biological properties
necessitate the use of plants.
Microorganisms associated with such small animals (e.g., pathogens or
symbionts) are included.
The Institutional
Biosafety Committee shall include at least one individual with expertise in
plant, plant pathogen, or plant pest containment principles when experiments
utilizing Appendix P require prior approval by the Institutional Biosafety
Committee.
Appendix P-I-A. The principal purpose of plant containment
is to avoid the unintentional transmission of a recombinant DNA-containing plant
genome, including nuclear or organelle hereditary material or release of
recombinant DNA-derived organisms associated with plants.
Appendix P-I-B. The containment principles are based on the
recognition that the organisms that are used pose no health threat to humans or
higher animals (unless deliberately modified for that purpose), and that the
containment conditions minimize the possibility of an unanticipated deleterious
effect on organisms and ecosystems outside of the experimental facility, e.g.,
the inadvertent spread of a serious pathogen from a greenhouse to a local
agricultural crop or the unintentional introduction and establishment of an
organism in a new ecosystem.
Appendix P-I-C. Four biosafety levels, referred to as
Biosafety Level (BL) 1 - Plants (P), BL2-P, BL3-P, and BL4-P, are established
in Appendix P-II, Physical
Containment Levels. The selection
of containment levels required for research involving recombinant DNA molecules
in plants or associated with plants is specified in Appendix P-III, Biological
Containment Practices. These
biosafety levels are described in Appendix P-II, Physical Containment Levels. This appendix describes greenhouse practices
and special greenhouse facilities for physical containment.
Appendix P-I-D. BL1-P through BL4-P are designed to provide
differential levels of biosafety for plants in the absence or presence of other
experimental organisms that contain recombinant DNA. These biosafety levels, in conjunction with biological
containment conditions described in Appendix
P-III, Biological Containment Practices, provide flexible approaches
to ensure the safe conduct of research.
Appendix P-I-E. For experiments in which plants are grown at
the BL1 through BL4 laboratory settings, containment practices shall be
followed as described in Appendix G,
Physical Containment. These
containment practices include the use of plant tissue culture rooms, growth
chambers within laboratory facilities, or experiments performed on open
benches. Additional biological
containment practices should be added by the Greenhouse Director or
Institutional Biosafety Committee as necessary (see Appendix P-III, Biological
Containment Practices), if botanical reproductive structures are produced
that have the potential of being released.
Appendix
P-II-A-1-a-(1). Access to the
greenhouse shall be limited or restricted, at the discretion of the Greenhouse
Director, when experiments are in progress.
Appendix
P-II-A-1-a-(2). Prior to entering
the greenhouse, personnel shall be required to read and follow instructions on
BL1-P greenhouse practices and procedures.
All procedures shall be performed in accordance with accepted greenhouse
practices that are appropriate to the experimental organism.
Appendix
P-II-A-1-b-(1). A record shall be kept
of experiments currently in progress in the greenhouse facility.
Appendix
P-II-A-1-c-(1). Experimental
organisms shall be rendered biologically inactive by appropriate methods before
disposal outside of the greenhouse facility.
Appendix
P-II-A-1-d-(1). A program shall be
implemented to control undesired species (e.g., weed, rodent, or arthropod
pests and pathogens), by methods appropriate to the organisms and in accordance
with applicable state and Federal laws.
Appendix
P-II-A-1-d-(2). Arthropods and
other motile macroorganisms shall be housed in appropriate cages. If macroorganisms (e.g., flying arthropods
or nematodes) are released within the greenhouse, precautions shall be taken to
minimize escape from the greenhouse facility.
Appendix
P-II-A-1-e-(1). Experiments involving
other organisms that require a containment level lower than BL1-P may be
conducted in the greenhouse concurrently with experiments that require BL1-P
containment, provided that all work is conducted in accordance with BL1-P
greenhouse practices.
Appendix
P-II-A-2-a-(1). The term
"greenhouse" refers to a structure with walls, a roof, and a floor
designed and used principally for growing plants in a controlled and protected
environment. The walls and roof are
usually constructed of transparent or translucent material to allow passage of
sunlight for plant growth.
Appendix
P-II-A-2-a-(2). The term
"greenhouse facility" includes the actual greenhouse rooms or
compartments for growing plants, including all immediately contiguous hallways
and head-house areas, and is considered part of the confinement area.
Appendix
P-II-A-2-b-(1). The greenhouse floor
may be composed of gravel or other porous material. At a minimum, impervious (e.g., concrete) walkways are
recommended.
Appendix
P-II-A-2-b-(2). Windows and other
openings in the walls and roof of the greenhouse facility may be open for
ventilation as needed for proper operation and do not require any special
barrier to contain or exclude pollen, microorganisms, or small flying animals
(e.g., arthropods and birds); however, screens are recommended.
Appendix
P-II-B-1-a-(1). Access to the
greenhouse shall be limited or restricted, at the discretion of the Greenhouse
Director, to individuals directly involved with the experiments when they are
in progress.
Appendix
P-II-B-1-a-(2). Personnel shall be
required to read and follow instructions on BL2-P practices and
procedures. All procedures shall be conducted
in accordance with accepted greenhouse practices that are appropriate to the
experimental organisms.
Appendix
P-II-B-1-b-(1). A record shall be
kept of experimental plants, microorganisms, or small animals that are brought
into or removed from the greenhouse facility.
Appendix
P-II-B-1-b-(2). A record shall be
kept of experiments currently in progress in the greenhouse facility.
Appendix
P-II-B-1-b-(3). The Principal
Investigator shall report any greenhouse accident involving the inadvertent
release or spill of microorganisms to the Greenhouse Director, Institutional
Biosafety Committee, NIH/OBA and other appropriate authorities immediately (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).
Documentation of any such accident shall be prepared and maintained.
Appendix
P-II-B-1-c-(1). Experimental
organisms shall be rendered biologically inactive by appropriate methods before
disposal outside of the greenhouse facility.
Appendix P-II-B-1-c-(2). Decontamination of run-off water is not
necessarily required. If part of the
greenhouse is composed of gravel or similar material, appropriate treatments
should be made periodically to eliminate, or render inactive, any organisms
potentially entrapped by the gravel.
Appendix
P-II-B-1-d-(1). A program shall be
implemented to control undesired species (e.g., weed, rodent, or arthropod
pests and pathogens) by methods appropriate to the organisms and in accordance
with applicable state and Federal laws.
Appendix
P-II-B-1-d-(2). Arthropods and
other motile macroorganisms shall be housed in appropriate cages. If macroorganisms (e.g., flying arthropods
or nematodes) are released within the greenhouse, precautions shall be taken to
minimize escape from the greenhouse facility.
Appendix
P-II-B-1-e-(1). Experiments
involving other organisms that require a containment level lower than BL2-P may
be conducted in the greenhouse concurrently with experiments that require BL2-P
containment provided that all work is conducted in accordance with BL2-P
greenhouse practices.
Appendix
P-II-B-1-f-(1). A sign shall be
posted indicating that a restricted experiment is in progress. The sign shall indicate the following: (i) the name of the responsible individual,
(ii) the plants in use, and (iii) any special requirements for using the
area.
Appendix
P-II-B-1-f-(2). If organisms are
used that have a recognized potential for causing serious detrimental impacts
on managed or natural ecosystems, their presence shall be indicated on a sign
posted on the greenhouse access doors.
Appendix
P-II-B-1-f-(3). If there is a risk
to human health, a sign shall be posted incorporating the universal biosafety
symbol.
Appendix
P-II-B-1-g-(1). Materials
containing experimental microorganisms, which are brought into or removed from
the greenhouse facility in a viable or intact state, shall be transferred in a
closed non-breakable container.
Appendix
P-II-B-1-h-(1). A greenhouse
practices manual shall be prepared or adopted.
This manual shall: (i) advise
personnel of the potential consequences if such practices are not followed, and
(ii) outline contingency plans to be implemented in the event of the
unintentional release of organisms.
Appendix
P-II-B-2-a-(1). The term
"greenhouse" refers to a structure with walls, a roof, and a floor designed
and used principally for growing plants in a controlled and protected
environment. The walls and roof are
usually constructed of transparent or translucent material to allow passage of
sunlight for plant growth.
Appendix
P-II-B-2-a-(2). The term
"greenhouse facility" includes the actual greenhouse rooms or
compartments for growing plants, including all immediately contiguous hallways
and head-house areas and is considered part of the confinement area.
Appendix
P-II-B-2-b-(1). A greenhouse floor
composed of an impervious material.
Concrete is recommended, but gravel or other porous material under
benches is acceptable unless propagules of experimental organisms are readily
disseminated through soil. Soil beds
are acceptable unless propagules of experimental organisms are readily
disseminated through soil.
Appendix
P-II-B-2-b-(2). Windows and other
openings in the walls and roof of the greenhouse facility may be open for
ventilation as needed for proper operation and do not require any special
barrier to exclude pollen or microorganisms; however, screens are required to
exclude small flying animals (e.g., arthropods and birds).
Appendix
P-II-B-2-c-(1). An autoclave shall
be available for the treatment of contaminated greenhouse materials.
Appendix
P-II-B-2-d-(1). If intake fans are
used, measures shall be taken to minimize the ingress of arthropods. Louvers or fans shall be constructed such
that they can only be opened when the fan is in operation.
Appendix
P-II-B-2-e-(1). BL2-P greenhouse
containment requirements may be satisfied by using a growth chamber or growth
room within a building provided that the external physical structure limits
access and escape of microorganisms and macroorganisms in a manner that
satisfies the intent of the foregoing clauses.
Appendix
P-II-C-1-a-(1). Authorized entry
into the greenhouse shall be restricted to individuals who are required for program
or support purposes. The Greenhouse
Director shall be responsible for assessing each circumstance and determining
those individuals who are authorized to enter the greenhouse facility.
Appendix
P-II-C-1-a-(2). Prior to entering the
greenhouse, personnel shall be required to read and follow instructions on
BL3-P practices and procedures. All
procedures shall be conducted in accordance with accepted greenhouse practices
that are appropriate to the experimental organisms.
Appendix
P-II-C-1-b-(1). A record shall be
kept of experimental plants, microorganisms, or small animals that are brought
into or removed from the greenhouse facility.
Appendix
P-II-C-1-b-(2). A record shall be
kept of experiments currently in progress in the greenhouse facility.
Appendix
P-II-C-1-b-(3). The Principal
Investigator shall report any greenhouse accident involving the inadvertent
release or spill of microorganisms to the Biological Safety Officer, Greenhouse
Director, Institutional Biosafety Committee, NIH/OBA, and other appropriate
authorities immediately (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). Documentation of any such accident shall be prepared and
maintained.
Appendix P-II-C-1-c-(1). All experimental materials shall be
sterilized in an autoclave or rendered biologically inactive by appropriate
methods before disposal, except those that are to remain in a viable or intact
state for experimental purposes; including water that comes in contact with
experimental microorganisms or with material exposed to such microorganisms,
and contaminated equipment and supplies.
Appendix
P-II-C-1-d-(1). A program shall be
implemented to control undesired species (e.g., weed, rodent, or arthropod
pests and pathogens) by methods appropriate to the organisms and in accordance
with applicable state and Federal laws.
Appendix
P-II-C-1-d-(2). Arthropods and
other motile macroorganisms shall be housed in appropriate cages. When appropriate to the organism,
experiments shall be conducted within cages designed to contain the motile
organisms.
Appendix
P-II-C-1-e-(1). Experiments
involving organisms that require a containment level lower than BL3-P may be
conducted in the greenhouse concurrently with experiments that require BL3-P
containment provided that all work is conducted in accordance with BL3-P
greenhouse practices.
Appendix
P-II-C-1-f-(1). A sign shall be
posted indicating that a restricted experiment is in progress. The sign shall indicate the following: (i) the name of the responsible individual,
(ii) the plants in use, and (iii) any special requirements for using the
area.
Appendix
P-II-C-1-f-(2). If organisms are
used that have a recognized potential for causing serious detrimental impacts
on managed or natural ecosystems, their presence should be indicated on a sign
posted on the greenhouse access doors.
Appendix
P-II-C-1-f-(3). If there is a risk
to human health, a sign shall be posted incorporating the universal biosafety
symbol.
Appendix
P-II-C-1-g-(1). Experimental
materials that are brought into or removed from the greenhouse facility in a
viable or intact state shall be transferred to a non-breakable sealed secondary
container. At the time of transfer, if
the same plant species, host, or vector are present within the effective
dissemination distance of propagules of the experimental organism, the surface
of the secondary container shall be decontaminated. Decontamination may be accomplished by passage through a chemical
disinfectant or fumigation chamber or by an alternative procedure that has
demonstrated effective inactivation of the experimental organism.
Appendix
P-II-C-1-h-(1). A greenhouse
practices manual shall be prepared or adopted.
This manual shall: (i) advise
personnel of the potential consequences if such practices are not followed, and
(ii) outline contingency plans to be implemented in the event of the
unintentional release of organisms with recognized potential for serious
detrimental impact.
Appendix
P-II-C-1-i-(1). Disposable clothing
(e.g., solid front or wrap-around gowns, scrub suits, or other appropriate
clothing) shall be worn in the greenhouse if deemed necessary by the Greenhouse
Director because of potential dissemination of the experimental
microorganisms.
Appendix
P-II-C-1-i-(2). Protective clothing
shall be removed before exiting the greenhouse and decontaminated prior to
laundering or disposal.
Appendix
P-II-C-1-j-(1). Personnel are
required to thoroughly wash their hands upon exiting the greenhouse.
Appendix
P-II-C-1-j-(2). All procedures shall
be performed carefully to minimize the creation of aerosols and excessive
splashing of potting material/soil during watering, transplanting, and all
experimental manipulations.
Appendix
P-II-C-2-a-(1). The term
"greenhouse" refers to a structure with walls, roof, and floor
designed and used principally for growing plants in a controlled and protected
environment. The walls and roof are
usually constructed of transparent or translucent material to allow passage of
sunlight for plant growth.
Appendix
P-II-C-2-a-(2). The term
"greenhouse facility" includes the actual greenhouse rooms or
compartments for growing plants, including all immediately contiguous hallways
and head-house areas, and is considered part of the confinement area. The need to maintain negative pressure
should be considered when constructing or renovating the greenhouse.
Appendix
P-II-C-2-b-(1). The greenhouse
floor shall be composed of concrete or other impervious material with provision
for collection and decontamination of liquid run-off.
Appendix
P-II-C-2-b-(2). Windows shall be
closed and sealed. All glazing shall be
resistant to breakage (e.g., double-pane tempered glass or equivalent).
Appendix
P-II-C-2-b-(3). The greenhouse
shall be a closed self-contained structure with a continuous covering that is
separated from areas that are open to unrestricted traffic flow. The minimum requirement for greenhouse entry
shall be passage through two sets of self-closing locking doors.
Appendix
P-II-C-2-b-(4). The greenhouse
facility shall be surrounded by a security fence or protected by equivalent
security measures.
Appendix
P-II-C-2-b-(5). Internal walls,
ceilings, and floors shall be resistant to penetration by liquids and chemicals
to facilitate cleaning and decontamination of the area. All penetrations into these structures and
surfaces (e.g., plumbing and utilities) shall be sealed.
Appendix
P-II-C-2-b-(6). Bench tops and
other work surfaces should have seamless surfaces that are impervious to water
and resistant to acids, alkalis, organic solvents, and moderate heat.
Appendix
P-II-C-2-b-(7). The greenhouse
contains a foot, elbow, or automatically operated sink, which is located near
the exit door for hand washing.
Appendix
P-II-C-2-c-(1). An autoclave shall
be available for decontaminating materials within the greenhouse facility. A double-door autoclave is recommended (not
required) for the decontamination of materials passing out of the greenhouse
facility.
Appendix
P-II-C-2-d-(1). An individual
supply and exhaust air ventilation system shall be provided. The system maintains pressure differentials
and directional airflow, as required, to assure inward (or zero) airflow from
areas outside of the greenhouse.
Appendix
P-II-C-2-d-(2). The exhaust air
from the greenhouse facility shall be filtered through high efficiency
particulate air-HEPA filters and discharged to the outside. The filter chambers shall be designed to
allow in situ decontamination before filters are removed and to
facilitate certification testing after they are replaced. Air filters shall be 80-85% average
efficiency by the American Society of Heating, Refrigerating, and Air
Conditioning Engineers (ASHRAE) Standard 52-68 test method using atmosphere dust. Air supply fans shall be equipped with a
back-flow damper that closes when the air supply fan is off. Alternatively, a HEPA filter may be used on
the air supply system instead of the filters and damper. The supply and exhaust airflow shall be
interlocked to assure inward (or zero) airflow at all times.
Appendix
P-II-C-2-e-(1). BL3-P greenhouse
containment requirements may be satisfied using a growth chamber or growth room
within a building provided that the location, access, airflow patterns, and
provisions for decontamination of experimental materials and supplies meet the
intent of the foregoing clauses.
Appendix
P-II-C-2-e-(2). Vacuum lines shall
be protected with high efficiency particulate air/HEPA or equivalent filters
and liquid disinfectant traps.
Appendix
P-II-D-1-a-(1). Authorized entry
into the greenhouse shall be restricted to individuals who are required for
program or support purposes. The
Greenhouse Director shall be responsible for assessing each circumstance and
determining those individuals who are authorized to enter the greenhouse
facility or work in the greenhouse during experiments.
Appendix
P-II-D-1-a-(2). Access shall be
managed by the Greenhouse Director, Biological Safety Officer, or other
individual responsible for physical security of the greenhouse facility; and
access limited by means of secure, locked doors.
Appendix P-II-D-1-a-(3). Prior to entering, individuals shall be
advised of the potential environmental hazards and instructed on appropriate
safeguards for ensuring environmental safety.
Individuals authorized to enter the greenhouse facility shall comply
with the instructions and all other applicable entry/exit procedures.
Appendix
P-II-D-1-a-(4). Personnel shall
enter and exit the greenhouse facility only through the clothing change and shower
rooms and shall shower each time they exit the greenhouse facility. Personnel shall use the airlocks to enter or
exit the laboratory only in an emergency.
In the event of an emergency, every reasonable effort should be made to
prevent the possible transport of viable propagules from containment.
Appendix
P-II-D-1-a-(5). Prior to entering
the greenhouse, personnel shall be required to read and follow instructions on
BL4-P practices and procedures.
Appendix P-II-D-1-b-(1). A record shall be kept of all experimental
materials brought into or removed from the greenhouse.
Appendix
P-II-D-1-b-(2). A record shall be
kept of experiments currently in progress in the greenhouse facility.
Appendix
P-II-D-1-b-(3). A record shall be
kept of all personnel entering and exiting the greenhouse facility, including
the date and time of each entry.
Appendix
P-II-D-1-b-(4). The Principal
Investigator shall report any greenhouse accident involving the inadvertent
release or spill of microorganisms to the Biological Safety Officer, Greenhouse
Director, Institutional Biosafety Committee, NIH/OBA, and other appropriate
authorities immediately (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). Documentation of any such accident shall be prepared and
maintained.
Appendix
P-II-D-1-c-(1). All materials,
except for those that are to remain in a viable or intact state for
experimental purposes, shall be autoclaved prior to removal from the maximum
containment greenhouse. Equipment or
material that could be damaged by high temperatures or steam shall be
decontaminated by alternative methods (e.g., gas or vapor sterilization) in an
airlock or chamber designed for this purpose.
Appendix
P-II-D-1-c-(2). Water that comes in
contact with experimental microorganisms or with material exposed to such
microorganisms (e.g., run-off from watering plants) shall be collected and
decontaminated before disposal.
Appendix
P-II-D-1-c-(3). Standard
microbiological procedures shall be followed for decontamination of equipment
and materials. Spray or liquid waste or
rinse water from containers used to apply the experimental microorganisms shall
be decontaminated before disposal.
Appendix
P-II-D-1-d-(1). A chemical control
program shall be implemented to eliminate undesired pests and pathogens in
accordance with applicable state and Federal laws.
Appendix
P-II-D-1-d-(2). Arthropods and
other motile macroorganisms used in conjunction with experiments requiring
BL4-P level physical containment shall be housed in appropriate cages. When appropriate to the organism,
experiments shall be conducted within cages designed to contain the motile
organisms.
Appendix
P-II-D-1-e-(1). Experiments
involving organisms that require a containment level lower than BL4-P may be conducted
in the greenhouse concurrently with experiments that require BL4-P containment
provided that all work is conducted in accordance with BL4-P greenhouse
practices. When the experimental
microorganisms in use require a containment level lower than BL4-P, greenhouse
practices reflect the level of containment required by the highest containment
level microorganisms being tested.
Appendix
P-II-D-1-f-(1). A sign shall be
posted indicating that a restricted experiment is in progress. The sign shall indicate the following: (i) the name of the responsible individual,
(ii) the plants in use, and (iii) any special requirements for using the area.
Appendix
P-II-D-1-f-(2). If organisms are
used that have a recognized potential for causing serious detrimental impacts
on managed or natural ecosystems, their presence shall be indicated by a sign
posted on the greenhouse access doors.
Appendix
P-II-D-1-f-(3). If there is a risk
to human health, a sign shall be posted incorporating the universal biosafety
symbol.
Appendix
P-II-D-1-g-(1). Experimental
materials that are brought into or removed from the greenhouse in a viable or
intact state shall be transferred to a non-breakable, sealed, primary container
then enclosed in a non-breakable, sealed secondary container. These containers shall be removed from the
greenhouse facility through a chemical disinfectant, fumigation chamber, or an
airlock designed for this purpose.
Appendix
P-II-D-1-g-(2). Supplies and
materials shall be brought into the greenhouse facility through a double-door
autoclave, fumigation chamber, or airlock that is appropriately decontaminated
between each use. After securing the
outer doors, personnel within the greenhouse facility shall retrieve the
materials by opening the interior door of the autoclave, fumigation chamber, or
airlock. These doors shall be secured
after the materials are brought into the greenhouse facility.
Appendix
P-II-D-1-h-(1). A greenhouse
practices manual shall be prepared or adopted.
This manual shall include contingency plans to be implemented in the
event of the unintentional release of experimental organisms.
Appendix
P-II-D-1-i-(1). Street clothing
shall be removed in the outer clothing change room. Complete laboratory clothing (may be disposable) including
undergarments, pants, and shirts, jump suits, shoes, and hats shall be provided
and worn by all personnel entering the greenhouse facility.
Appendix
P-II-D-1-i-(2). Personnel shall
remove laboratory clothing when exiting the greenhouse facility and before
entering the shower area. This clothing
shall be stored in a locker or hamper in the inner change room.
Appendix
P-II-D-1-i-(3). All laboratory
clothing shall be autoclaved before laundering.
Appendix P-II-D-2-a-(1). The maximum containment greenhouse facility
shall consist of a separate building or a clearly demarcated and isolated area
within a building. The need to maintain
negative pressure should be considered when constructing or renovating the greenhouse
facility.
Appendix
P-II-D-2-a-(2). Outer and inner
change rooms, separated by a shower, shall be provided for personnel entering
and exiting the greenhouse facility.
Appendix
P-II-D-2-a-(3). Windows shall be
closed and sealed. All glazing shall be
resistant to breakage (e.g., double-pane tempered glass or equivalent).
Appendix
P-II-D-2-a-(4). Access doors to the
greenhouse shall be self-closing and locking.
Appendix
P-II-D-2-a-(5). The greenhouse
facility shall be surrounded by a security fence or protected by equivalent
security measures.
Appendix
P-II-D-2-a-(6). The walls, floors,
and ceilings of the greenhouse shall be constructed to form a sealed internal
shell that facilitates fumigation and is animal and arthropod-proof. These internal surfaces shall be resistant
to penetration and degradation by liquids and chemicals to facilitate cleaning
and decontamination of the area. All
penetrations into these structures and surfaces (e.g., plumbing and utilities)
shall be sealed.
Appendix
P-II-D-2-a-(7). Bench tops and
other work surfaces shall have seamless surfaces impervious to water and
resistant to acids, alkalis, organic solvents, and moderate heat.
Appendix
P-II-D-2-a-(8). A double-door
autoclave, fumigation chamber, or ventilated airlock shall be provided for
passage of all materials, supplies, or equipment that are not brought into the
greenhouse facility through the change room.
Appendix
P-II-D-2-b-(1). A double-door
autoclave shall be provided for the decontamination of materials removed from
the greenhouse facility. The autoclave
door, which opens to the area external to the greenhouse facility, shall be
sealed to the outer wall and automatically controlled so that it can only be
opened upon completion of the sterilization cycle.
Appendix
P-II-D-2-c-(1). An individual
supply and exhaust air ventilation system shall be provided. The system shall maintain pressure
differentials and directional airflow as required to assure inward (or zero)
airflow from areas outside of the greenhouse.
Differential pressure transducers shall be used to sense pressure
levels. If a system malfunctions, the
transducers shall sound an alarm. A
backup source of power should be considered.
The supply and exhaust airflow shall be interlocked to assure inward (or
zero) airflow at all times. The
integrity of the greenhouse shall have an air leak rate (decay rate) not to
exceed 7 percent per minute (logarithm of pressure against time) over a
20-minute period at 2 inches of water gauge pressure. Nominally, this is 0.05 inches of water gauge pressure loss in 1
minute at 2 inches water gauge pressure.
Appendix
P-II-D-2-c-(2). Exhaust air from
the greenhouse facility shall be filtered through high efficiency particulate
air/HEPA filters and discharged to the outside and dispersed away from occupied
buildings and air intakes. Filter
chambers shall be designed to allow in situ decontamination before
filters are removed and to facilitate certification testing after they are
replaced. HEPA filters shall be
provided to treat air supplied to the greenhouse facility. HEPA filters shall be certified annually.
Appendix
P-II-D-2-d-(1). Sewer vents and
other ventilation lines contain high efficiency particulate air/HEPA
filters. HEPA filters shall be
certified annually.
Appendix
P-II-D-2-d-(2). A pass-through dunk
tank, fumigation chamber, or an equivalent method of decontamination shall be
provided to ensure decontamination of materials and equipment that cannot be
decontaminated in the autoclave.
Appendix
P-II-D-2-d-(3). Liquid effluent from
sinks, floors, and autoclave chambers shall be decontaminated by heat or
chemical treatment before being released from the maximum containment
greenhouse facility. Liquid wastes from
shower rooms and toilets may be decontaminated by heat or chemical treatment. Autoclave and chemical decontamination of
liquid wastes shall be evaluated by appropriate standard procedures for
autoclaved wastes. Decontamination
shall be evaluated mechanically and biologically using a recording thermometer
and an indicator microorganism with a defined heat susceptibility pattern. If liquid wastes are decontaminated with
chemical disinfectants, the chemicals used must have demonstrated efficacy
against the target or indicator microorganisms.
Appendix
P-II-D-2-d-(4). If there is a
central vacuum system, it shall not serve areas outside of the greenhouse
facility. In-line high efficiency
particulate air/HEPA filters shall be placed as near as practicable to each use
point or vacuum service cock. Other
liquid and gas services to the greenhouse facility shall be protected by
devices that prevent back-flow. HEPA
filters shall be certified annually.
Appropriate selection
of the following biological containment practices may be used to meet the
containment requirements for a given organism.
The present list is not exhaustive; there may be other ways of
preventing effective dissemination that could possibly lead to the
establishment of the organism or its genetic material in the environment
resulting in deleterious consequences to managed or natural ecosystems.
Appendix
P-III-A-1. Effective dissemination
of plants by pollen or seed can be prevented by one or more of the following
procedures: (i) cover the reproductive
structures to prevent pollen dissemination at flowering and seed dissemination
at maturity; (ii) remove reproductive structures by employing male sterile
strains, or harvest the plant material prior to the reproductive stage; (iii)
ensure that experimental plants flower at a time of year when cross-fertile
plants are not flowering within the normal pollen dispersal range of the
experimental plant; or (iv) ensure that cross-fertile plants are not growing within
the known pollen dispersal range of the experimental plant.
Appendix
P-III-B-1. Effective dissemination
of microorganisms beyond the confines of the greenhouse can be prevented by one
or more of the following procedures:
(i) confine all operations to injections of microorganisms or other
biological procedures (including genetic manipulation) that limit replication
or reproduction of viruses and microorganisms or sequences derived from
microorganisms, and confine these injections to internal plant parts or
adherent plant surfaces; (ii) ensure that organisms, which can serve as hosts
or promote the transmission of the virus or microorganism, are not present
within the farthest distance that the airborne virus or microorganism may be
expected to be effectively disseminated; (iii) conduct experiments at a time of
year when plants that can serve as hosts are either not growing or are not
susceptible to productive infection; (iv) use viruses and other microorganisms
or their genomes that have known arthropod or animal vectors, in the absence of
such vectors; (v) use microorganisms that have an obligate association with the
plant; or (vi) use microorganisms that are genetically disabled to minimize
survival outside of the research facility and whose natural mode of
transmission requires injury of the target organism, or assures that
inadvertent release is unlikely to initiate productive infection of organisms
outside of the experimental facility.
Appendix
P-III-C-1. Effective dissemination
of arthropods and other small animals can be prevented by using one or more of
the following procedures: (i) use non-flying,
flight-impaired, or sterile arthropods; (ii) use non-motile or sterile strains
of small animals; (iii) conduct experiments at a time of year that precludes
the survival of escaping organisms; (iv) use animals that have an obligate
association with a plant that is not present within the dispersal range of the
organism; or (v) prevent the escape of organisms present in run-off water by
chemical treatment or evaporation of run-off water.