II. Containment

Effective biological safety programs .have been operative in a variety
of laboratories for many years. Considerable information therefore already
exists for the design of physical containment facilities and the selection of
laboratory procedures applicable to organisms carrying recombinant DNAs (4-17),
The existing programs rely upon mechanisms that, for convenience, can be
divided into two categories: (i) a set of standard practices that are
generally used in microbiological laboratories, and (ii) special procedures,
equipment, and laboratory installations that provide physical barriers
which are applied in varying degrees according to the estimated biohazard.

Experiments on recombinant DNAs by their very nature lend themselves
to a third containment nechanism--nanely the application of highly |
Specific biological barriers. In fact, natural barriers do exist which
either limit the infectivity of a vector or vehicle (plasmid, bacterio-
phage or virus) to specific hosts, or its dissemination and survival in
the environment. The vectors that provide the means for replication of
the recombinant DNAs and/or the host cells in which they replicate can be
genetically designed to decrease by many orders of magnitude the probability
of dissemination of recombinant DNAs outside the laboratory.

As these three means of containment are complementary, different levels
of containment appropriate for experiments with different recombinants can
be established by applying different combinations of the physical and
biological barriers to a constant use of the standard practices. We con-
sider these categories of containment separately here in order that such
combinations can be conveniently expressed in the guidelines for research

on the different kinds of recombinant DNAs (Section III).
A.. Standard practices and training - The first principle of. containment
is a strict adherence to good microbiological practices (4-13). Consequently,
all personnel directly or indirectly involved in experiments on recombinant
DNAs must receive adequate instruction. This should include at least train-
ing in aseptic techniques and instruction in the biology of the organisms
used in the experiments so that the potential biohazards can be understood
and appreciated.

Any research group working with agents with a known or potential bio-
hazard should have an emergency plan which describes the procedures to be
followed if an accident contaminates personnel or environment. The principal
investigator must ensure that everyone in the laboratory is familiar with
both the potential hazards of the work and the emergency plan. If a research
group is working with a known pathogen for which an effective vaccine is
_ available, all workers should be immunized. Serological monitoring, where
appropriate, should be provided.

B. Physical containment levels - A variety of combinations (levels)

 

of special practices, equipment, and laboratory installations that provide
additional physical barriers can be formed. For example, 31 combinations

are listed in "Laboratory Safety at the Center for Disease Control" (4);

four levels are associated with the "Classification of Etiologic Agents

on the Basis of Hazard" (5), four levels were recommended in the “Summary
Statement of the Asilomar Conference on Recombinant DNA Molecules" (3); and
the National Cancer Institute uses three levels for research on oncogenic
viruses (6). We emphasize that tnese are an aid to, and not a substitute for,

good technique. Personnel must be competent in the effective use of all
equipment needed for the required containment level as described below. We
define only four levels of physical containment here, both because the accuracy
with which one can presently assess the biohazards that may result from recom-
binant DNAs does not warrant a more detailed classification, and because addi-
tional flexibility can be obtained by combination of the physical with the
biological barriers. Though different in detail, these four levels (P1 < P2 <
P3 < P4) approximate those given for human etiologic agents by the Center for
Disease Control (i.e., classes 1 through 4; ref. 5), in the Asilomar summary
statement (i.e., minimal, low, moderate, and high; ref. 3), and by the National
Cancer Institute for oncogenic viruses (i.e., low, moderate, and high; ref. 6),
as is indicated by the P-number or adjective in the following headings. It
should be emphasized that the descriptions and assignments of physical con-
tainment' detailed below are based on existing approaches to containment of
hazardous organisms.

We anticipate, and indeed already know of, procedures (14) which enhance
physical containment capability in novel ways. For example, miniaturization
of screening, handling, and analytical procedures provides substantial con-
tainment of a given host-vector system. Thus, such procedures should reduce
the need for the standard types of physical containment, and such innovations
will be considered by the Recombinant DNA Molecule Program Advisory Committee.

The special practices, equipment and facility installations indicated for
each level of physical containment are required for the safety of laboratory
workers, other persons, and for the protection of the environment. Optional
items have been excluded; only those items deemed absolutely necessary for

Safety are presented. Thus, the listed requirements present basic safety
criteria for each level of physical containment. Other microbiological prac-
tices and laboratory techniques which promote safety are to be encouraged.
Additional information giving further guidance on physical containment is
provided in a supplement to the guidelines (Appendix D).

P1 Level (Minimal) - A laboratory suitable for experiments involving
recombinant DNA molecules requiring physical containment at the Pl level
is a laboratory that possesses no special engineering design features. It
is a laboratory commonly used for microorganisms of no or minimal biohazard
under ordinary conditions of handling. Work in this laboratory is generally
conducted on open bench tops. Special containment equipment is neither
required nor generally available in this laboratory. The laboratory is not
separated from the general traffic patterns of the building. Public access
is permitted.

The control: of biohazards at the Pl level is provided by standard micro-
biological practices of which the following are examples: (i) Laboratory
doors should be kept closed while experiments are in progress. (ii) Work
surfaces should be decontaminated daily and following spills of recombinant
DNA materials. (iii) Liquid wastes containing recombinant DNA materials should
be decontaminated before disposal. (iv) Solid wastes contaminated with recom-
binant DNA materials should be decontaminated or packaged in a durable
leak-proof container before removal from the laboratory. (v) Although
pipetting by mouth is permitted, it is preferable that mechanical pipetting .
devices be used. When pipetting by mouth, cotton-plugged pipettes shall
be employed. (vi) Eating, drinking, smoking, and storage of food in the

working area should be discouraged. (vii) Facilities to wash hands should
be available. (viii) An insect and rodent control program should be pro-
vided. (ix) The use of laboratory gowns, coats, or uniforms is discretionary
with the laboratory supervisor.

P2 Level (Low) - A laboratory suitable for experiments involving re-
combinant DNA molecules requiring physical containment at the P2 level is
similar in construction and design to the Pl laboratory. The P2. laboratory
must have access to an autoclave within the bufiding; it may have a Bio-
logical Safety Cabinet. Work which does not produce a considerable aerosol
is conducted on the open bench. Although this laboratory is not separated
from the general traffic patterns of the building, access to the laboratory
is limited when experiments requiring P2 level physical containment are being
conducted. Experiments of lesser biohazard potential can be carried out
concurrently in carefully demarcated areas of the same laboratory.

The P2 laboratory is commonly used for experiments involving micro-
Organisms of low biohazard such as. those which have been classified by the
Center for Disease Control] as Class 2 agents (5).

The following practices shall apply to all experiments requiring P2
level physical containment: (i) Laboratory doors shall be kept closed while
experiments are in progress. (ii) Only persons who have been advised of the
potential biohazard shall enter the laboratory. (443) Children under 12
years of age shall not enter the laboratory. (iv) Work surfaces shal] be
decontaminated daily and immediately following spills of recombinant DNA
materials. (v) Liquid wastes of recombinant DNA materials shall be decon-
taminated before disposal. (vi) Solid wastes contaminated with recombinant

DNA materials shall be decontaminated or packaged in a durable leak-proof
container before removal from the laboratory. Packaged materials shall be
disposed of by incineration or sterilized before disposal by other methods .
Contaminated materials that are to be processed and reused (i.e., glassware)
shall be decontaminated before removal from the laboratory. (vii) Pipetting
by mouth is prohibited; mechanical pipetting devices shall be used. (viii)
Eating, drinking, smoking, and storage of food are not permitted in the working
area. (ix) Facilities to wash hands shall be available within the laboratory.
Persons handling recombinant DNA materials should be encouraged to wash their
hands frequently and when they leave the laboratory. (x) An insect and rodent
control program shal] be provided. (xi) The use of laboratory gowns, coats,

or uniforms is required. Such clothing shall not be worn to the lunch room or
outside the building. (xii) Animals not related to the experiment shall not

be permitted in the laboratory. (xiii) Biological Safety Cabinets’ and/or
other physical containment equipment shall be used to minimize the hazard of
aerosolization of recombinant DNA materials from operations or devices that
produce a considerable aerosol (e.g., blender, lyophilizer, sonicator, shaking
machine, etc.). (xiv) Use of the hypodermic needle and syringe shall be avoided
when alternate methods are available.

P3 Level (Moderate) - A laboratory suitable for experiments involving
recombinant DNA molecules requiring physical containment at the P3 level has
special engineering design features and physical containment equipment. The
laboratory is separated from areas which are open to the general public. Sep-
aration is generally achieved by controlled access corridors, air locks,
locker rooms or other double-doored facilities which are not available for use

by the general public. Access to the laboratory is controlled. Biological
10

Safety Cabinets” are available within the controlled laboratory area. An
autoclave shall be available within the building and preferably within the
controlled laboratory area. The surfaces of walls, floors, bench tops,
and ceilings are easily cleanable to facilitate housekeeping and space
decontamination. |

Directional air flow is provided within the controlled laboratory
area. The ventilation system is balanced to provide for an inflow of supply
air from the access corridor into the laboratory. The general exhaust air
from the laboratory is discharged outdoors and so dispersed to the atmos-
phere as to prevent reentry into the building. No recirculation of the
exhaust air shall be permitted without appropriate treatment.

No work jn open vessels involving hosts or vectors containing recom-
binant DNA molecules requiring P3 physical containment is conducted on the
Open bench. All such procedures are confined to Biological Safety Cabinets.’

The following practices shall apply to all experiments requiring P3
level physical containment: (i) The universal biohazard sign is required
on all laboratory access doors. Only persons whose entry into the laboratory
is required on the basis of program or Support needs shall be authorized ‘to
enter. Such persons shall be advised of the potential biohazards before entry
and they shall comply with posted entry and exit procedures. Children under
12 years of age shall not enter the laboratory. (ii) Laboratory doors shall
be kept closed while experiments are in progress. (iii) Biological safety.
Cabinets’ and other physical containment equipment shall be used for all pro-

cedures that produce aerosols of recombinant DNA materials (e.g., pipetting,
11

plating, flaming, transfer operations, grinding, blending, drying, soni-
cating, shaking, etc.). (iv) The work surfaces of Biological Safety

Cabinets and other equipment shall be decontaminated following the com-
pletion of the experimental activity contained within them. (v) Liquid

wastes containing recombinant DNA materials shall be decontaminated

before disposal. Solid wastes contaminated with recombinant DNA materials
shall be decontaminated or packaged in a durable leak-proof container before
removal from the laboratory. Packaged material shall be sterilized before
disposal. Contaminated materials that are to be processed and reused (i.e.,
glassware) shall be sterilized in the controlled laboratory area or placed in a
durable leak-proof container before removal from the controlled laboratory area.
This container shall be sterilized before the materials are processed. (vii)
Pipetting by.mouth is prohibited; mechanical pipetting devices shall be used.
(viii) Eating, drinking, smoking, and storage of food are not permitted in

the laboratory. (ix) Facilities to wash hands shall be available within the
laboratory. Persons shall wash hands after experiments involving recombinant
DNA materials and before leaving the laboratory. (x) An insect and rodent
control program shall be provided. . (xi) Laboratory clothing that protects
street clothing (i.e., long sleeve solid-front or wrap-around gowns, no-button
or slipover jackets, etc.) shall be worn in the laboratory. FRONT-BUTTON
LABORATORY COATS ARE UNSUITABLE. Gloves shall be worn when handling recombi -
nant DNA materials. Provision for laboratory shoes is recommended. Laboratory
clothing shall not be worn outside the laboratory and shall be decontaminated
before it is sent to the laundry. (xii) Raincoats, overcoats, topcoats, coats,

hats, caps, and such street outerwear shall not be kept in the laboratory.
12

(xiii) Animals and plants not .related to the experiment shall not be permitted

jn the laboratory. (xiv) Vacuum lines shall be protected by filters and liquid
traps. (xv) Use of the hypodermic needle and syringe shall be avoided when
alternate methods are available. (xvi) If experiments of lesser biohazard
potential are to be conducted in the same laboratory concurrently with ex-
periments requiring P3 level physical containment they shall be conducted only

in accordance with all P3 level requirements. (xvii) Experiments requiring P3
level physical containment can be conducted in laboratories where the directional
air' flow and general exhaust air conditions described above cannot be achieved,
provided that this work is conducted in accordance with all other requirements
listed and is contained in a Biological Safety Cabinet? with attached glove ports —
and gloves. All materials before removal from the Biological Safety Cabinet! shall
be sterilized or transferred to a non=breakable, sealed container, which is then
removed from the cabinet through a chemical decontamination tank, autoclave,
ultraviolet air lock, or after the entire cabinet has been decontaminated.

P4 Level (High) - Experiments involving recombinant DNA molecules requiring
physical containment at the P4 level shall be confined to work areas in a facility
of the type designed to contain microorganisms that are extremely hazardous to
man or may cause serious epidemic disease. The facility is either a separate
building or it is a controlled area, within a building, which js completely
isolated from all other areas of the building. Access to the facility is under
strict control. A specific facility operations manual is available. Class IIT
Biological Safety Cabinets: are available within work areas of the facility.

A P4 facility has engineering features which are designed to prevent the

escape of microorganisms to the environment (14, 15, 16, 17). These features
13

include: (i) monolithic walls, floors, and ceilings in which all penetra-
tions such as for air ducts, electrical conduits, and utility pipes are

sealed to assure the physical isolation of the work area and to facilitate
housekeeping and space decontamination; (ii) air locks through which supplies
and materials can be brought safely into the facility; (441) contiguous cloth-
ing change and shower rooms through which personnel enter into and exit from
the facility; (iv) double-door autoclaves to sterilize and safely remove
wastes and other materials from the facility; (v) a biowaste treatment system
to sterilize liquid effluents if facility drains are installed; (vi) a separate
ventilation system which maintains negative air pressures and directional air
flow within the facility; and (vii) a treatment system to decontaminate ex-
haust air before it is dispersed to the atmosphere. A central vacuum utility
system is not encouraged; if one is installed, each branch line leading to a
laboratory shall be protected by a high efficiency particulate air filter.

The following practices shall apply to all experiments requiring P4 level
physical containment: (i) The universal biohazard sign is required on all
facility access doors and al] interior doors to individual Taboratory rooms
where experiments are conducted. Only persons whose entry into the facility
or individual laboratory rooms is required on the basis of program or support
needs shall be authorized to enter. Such persons shall be advised of the
potential biohazards and instructed as to the appropriate safeguards to ensure
their safety before entry. Such persons shall comply with the instructions
and all other posted entry and exit procedures. Under no condition shall
children under 15 years of age be allowed entry. (ii) Personnel shall enter

into and exit from the facility only through the clothing change.and shower
14

rooms. Personnel shall shower at each exit from the facility. The air

locks shall not be used for personnel entry or exit except for emergencies.
(iii) Street clothing shall be removed in the outer facility side of the
clothing change area and kept there. Complete laboratory clothing including
undergarments, pants and shirts or jumpsuits, shoes, head cover, and gloves
shall be provided and used by all persons who enter into the facility. Upon
exit, this clothing shall be stored in lockers provided for this purpose or
discarded into collection hampers before personnel enter into the shower area.
(iv) Supplies and materials to be taken into the facility shall be placed in
an entry air lock. After the outer door (opening to the corridor outside of
facility) has been secured, personnel occupying the facility shall retrieve
the supplies and materials by opening the interior air lock door. This door
shall be secured after supplies and materials are brought into the facility.
(v) Doors to laboratory rooms within the facility shall be kept closed while
experiments are in progress. (vi) Experimental procedures requiring P4 level
physical containment shall be confined to Class III Biological Safety Cabinets. ?
All materials, before removal from these cabinets, shall be sterilized or
transferred to a non-breakable sealed container, which is then removed from
the system through a chemical decontaminated tank, autoclave, or after the
entire system has been decontaminated. (vii) No materials shall be removed
from the facility unless they have been sterilized or decontaminated in a
manner to prevent the release of agents requiring P4 physical containment.

All wastes and other materials and equipment not damaged by high temperature
or steam shall be sterilized in the double-door autoclave. Biological materials

to be renioved from the facility shall be transferred to a non-breakable sealed
15

container which is then removed from the facility through a chemical de-
contamination tank or a chamber designed for gas sterilization. Other
materials which may be damaged by temperature or steam shall be sterilized
by gaseous or vapor methods in an air lock or chamber designed for this
purpose. (viii) Eating, drinking, smoking, and storage of food are not
permitted in the facility. Foot-operated water fountains located in the
facility corridors are permitted. Separate potable water piping shall be
provided for these water fountains. (ix) Facilities to wash hands shall

be available within the facility. Persons shall wash hands after experi-
ments. (x) An insect and rodent control program shall be provided. (xi)
Animals and plants not related to the experiment shall not be permitted in
the facility. (xii) If a central vacuum system is provided, each vacuum
outlet shall be protected by a filter and liquid trap in addition to the
branch line HEPA filter mentioned above. (xiii) Use of the hypodermic needle
and syringe shall be avoided when alternate methods are available. (xiv)

If experiments of lesser biohazard potential are to be conducted in the
facility concurrently with experiments requiring P4 level containment, they
shall be confined in Class I or Class II Biological Safety Cabinets’ or
isolated by other physical containment equipment. Work surfaces of Biologi-
cal Safety Cabinets” and other equipment shall be decontaminated following
the completion of the experimental activity contained within them. Mechani-
cal pipetting devices shall be used. All other practices listed above with
the exception of (vi) shall apply.

C. Shipment - To protect product, personnel, and the environment,

all recombinant DNA material will be shipped in containers that meet the
16

requirements issued by the U.S. Public Health Service (Section 72.25 of
Part 72, Title 42, Code of Federal Regulations), Department of Transporta-
tion (Section 173.387 (b) of Part 173, Title 49, Code of Federal Regulations)
and the Civil Aeronautics Board (C.A.B. No. 82, Official Air Transport
Restricted Articles Tariff No. 6-D) for shipment of etiologic agents.
Labeling requirements specified in these Federal regulations and tariffs
will apply to all viable recombinant DNA materials in which any portion of
the material is derived from an etiologic agent listed in paragraph (c) of
42 CFR 72.25. Additional information on packing and shipping is given in
a supplement to the guidelines (Appendix D, part X).

D. Biological containment levels - Biological barriers are specific
to each host-vector system. Hence the criteria for this mechanism of
containment cannot be generalized to the same extent as for physical con-
tainment. This is particularly true at the present time when our experience
with existing host-vector systems and our predictive knowledge about projected
Systems are sparse. The classification of experiments with recombinant DNAs
that is necessary for the construction of the experimental guidelines
(Section III) can be accomplished with least confusion if we use the host-
vector system as the primary element and the source of the inserted DNA as
the secondary element in the classification. It is therefore convenient to
specify the nature of the biological containment under host-vector headings

such as those given below for Escherichia coli K-12.