II-B-4-c. Special Laboratory Design. 
II-B-4-c-( 1 ). The laboratory shall be 
located in a restricted-access facility 
which is either a separate building or 
a clearly demarcated and isolated zone 
within a building. Clothing-change 
areas and shower rooms shall be pro- 
vided for personnel entry and egress. 
These rooms shall be arranged so that 
personnel leave through the shower 
area to the change room. A double- 
door ventilated vestibule or ultraviolet 
air lock shall be provided for passage 
of materials, supplies, and equipment 
which are not brought into the P4 fa- 
cility through the change room area. 
II-B-4-c-<2). Walls, floors, and ceil- 
ings of the P4 facility are constructed 
to form an internal shell which readily 
allows vapor-phase decontamination 
and is animal- and insect-proof. All 
penetrations through these structures 
and surfaces are sealed. (The integrity 
of the walls, floors, ceilings, and pene- 
tration seals should ensure adequate 
containment of a vapor-phase deconta- 
minant under static pressure condi- 
tions. This requirement does not imply 
that these surfaces must be airtight.) 
II-B-4-c-<3). A foot-, elbow-, or auto- 
matically-operated handwashing facili- 
ty shall be provided near the door 
within each laboratory in which ex- 
periments involving recombinant DNA 
are conducted in openface biological 
safety cabinets. 
II-B-4-c-(4). Central vacuum sys- 
tems are permitted. The system, if 
provided, shall not serve areas outside 
the P4 facility. The vacuum system 
shall include in-line HEPA filters near 
each use point or service cock. The fil- 
ters shall be Installed so as to permit 
in-place decontamination and replace- 
ment. Water supply and liquid and 
gaseous servies provided to the P4 fa- 
cility shall be protected by devices 
that prevent backflow. 
II-B-4-c-<5). Drinking water foun- 
tains shall not be installed in labora- 
tory or animal rooms of the P4 facili- 
ty. Foot-operated water fountains are 
permitted in the corridors of the P4 
facility. The water service provided to 
such fountains shall be protected from 
the water services to the laboratory 
areas of the P4 facility. 
II-B-4-c-<6). Laboratory doors shall 
be self-closing. 
II-B-4-c-(7). A double-door auto- 
clave shall be provided for sterilization 
of material passing out of the P4 fa- 
cility. The autoclave doors shall be in- 
terlocked so that both doors will not 
be open at the same time. 
II-B-4-c-<8). A pass-through dunk 
tank or fumigation chamber shall be 
provided for removal from the P4 fa- 
cility of material and equipment that 
cannot be heat-sterilized. 
II-B-4-c-<9). All liquid effluents 
from the P4 facility shall be collected 
and decontaminated before disposal. 
FEDERAL 
NOTICES 
Liquid effluents from biological safety 
cabinets and laboratory sinks shall be 
sterilized by heat. Liquid effluents 
from the shower and hand washing 
facilities may be inactivated by chemi- 
cal treatment. HEPA filters shall be 
installed in all vents from effluent 
drains. 
II-B-4-c-(10). An individual supply 
and exhaust-air ventilation system 
shall be provided. The system shall 
maintain pressure differentials and di- 
rectional air flow as required to ensure 
inflow from areas outside the facility 
toward areas of highest potential risk 
within the facility. The system shall 
be designed to prevent the reversal of 
air flow. The system shall sound an 
alarm in the event of system malfunc- 
tion. 
II-B-4-c-(ll). Air within individual 
laboratories of the P4 facility may be 
recirculated if HEPA filtered. 
II-B-4-c-<12). The exhaust air from 
the P4 facility shall be HEPA filtered 
and discharged to the outdoors so that 
it is dispersed clear of occupied build- 
ings and air intakes. The filter cham- 
bers shall be designed to allow in situ 
decontamination before removal and 
to facilitate certification testing after 
replacement. 
II-B-4-c-(13). The treated exhaust- 
air from Class I and Class II bioligical 
safety cabinets [201 may be discharged 
directly to the laboratory room envi- 
ronment or to the outdoors. The treat- 
ed exhaust-air from Class III cabinets 
shall be discharged to the outdoors. If 
the treated exhaust-air from these 
cabinets is to be discharged to the out- 
doors through the P4 facility exhaust 
air system, it shall be connected to 
this system so as to avoid any interfer- 
ence with the air balance of the cabi- 
nets or the facility exhaust air system. 
II-B-4-b-(14). As noted in Section 
II-B-4-c-d), the P4 facility may con- 
tain specially designed areas in which 
all personnel are required to wear one- 
piece positive-pressure isolation suits. 
Such areas shall be airtight. The ex- 
haust-air from the suit area shall be 
filtered by two sets of HEPA filters in- 
stalled in series, and a duplicate filtra- 
tion unit and exhaust fan shall be pro- 
vided. The air pressure within the suit 
area shall be less than that in any ad- 
jacent area. An emergency lighting 
system, communication systems, and 
power source shall be provided. A 
double-door autoclave shall be pro- 
vided for sterilization of all waste ma- 
terials to be removed from the suit 
area. 
Personnel who enter this area shall 
wear a one-piece positive-pressure suit 
that is ventilated by a life-support 
system. The life-support sysem shall 
be provided with alarms and emergen- 
cy backup air. Entry to this area is 
through an airlock fitted with airtight 
doors. A chemical shower area shall be 
REGISTER, VOL 43, NO. 247— FRIDAY, DECEMBER 
60113 
provided to decontaminate the sur- 
faces of the suit before removal. 
II-C. Shipment Recombinant DNA 
molecules contained in an organism or 
virus shall be shipped only as an etio- 
logic agent under requirements of the 
U.S. Public Health Service and the 
U.S. Department of Transportation 
(§72.25, Part 72, Title 42, and 
§§ 173. 386-. 388, Part 173, Title 49, U.S. 
Code of Federal Regulations) as speci- 
fied below: 
II-C-1. Recombinant DNA molecules 
contained in an organism or virus re- 
quiring PI, P2. or P3 physical contain- 
ment, when offered for transportation 
or transported, are subject to all re- 
quirements of § 72.25(c)(l )— (5). Part 
72. Title 42 CFR, and §§ 173.386-.388, 
Part 173, Title 49 CFR. 
II-C-2. Recombinant DNA molecules 
contained in an organism or virus re- 
quiring P4 physical containment, 
when offerd for transportation or 
transported, are subject to the require- 
ments listed above under II-C-1 and 
are also subject to §72.25(0(6), Part 
72. Title 42 CFR. 
II-C-3. Additional information on 
packaging and shipment is given in 
the “Laboratory Safety Monograph— 
A Supplement to the NIH Guidelines 
for Reombinant DNA Research.” 
II-D. Biological Containment 
II-D-1. Levels of Biological Contain- 
ment In consideration of biological 
containment, the vector (Plasmid, or- 
ganelle, or virus) for the recombinant 
DNA and the host (bacterial, plant, or 
animal cell) in which the vector is 
propagated in the laboratory will be 
considered together. Any combination 
of vector and host which is to provide 
biological containment must be chosen 
or constructed so that the following 
types of "escape” are minimized: (i) 
Survival of the vector in its host out- 
side the laboratory and (ii) transmis- 
sion of the vector from the propaga- 
tion host to other nonlaboratory 
hosts. 
The following levels of biological 
containment (HV, or Host- Vector, sys- 
tems) for prokaryotes will be estab- 
lished: specific criteria will depend on 
the organisms to be used. Eukaryotic 
host-vector systems are considered in 
Part III. 
II-D-l-a. HV1. A host-vector system 
which provides a moderate level of 
containment. Specific systems: 
II-D-l-a-(l). EK1. The host is 
always E. Coli K-12 or a derivative 
thereof, and the vectors include non- 
conjugative plasmids (e.g., pSClOl, 
ColEl, or derivatives thereof [21-27]) 
and variants of bacteriophage, such as 
[28-33]. The E. Coli K-12 hosts shall 
not contain conjugation-proficient 
plasmids, whether autonomous or in- 
tegrated, or generalized transducing 
phages. 
22, 1978 
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