77388 
Federal Register / Vol. 45, No. 227 / Friday, November 21, 1980 / Notices 
Table I .— Combinations of Containment Safeguards 
Classification of experiment 
According to guidelines 
Alternate combinations of physical and biological containment 
Physical containment 
Biological 
containment 
Physical 
containment 
Biological ' 
containment 
Laboratory 
design 
specified for— 
Laboratory 
practices 
specified for— 
Containment 
equipment 
specified for— 
P3 
HV2 
P3 
P3 
P4 
HV1 
P3 
HV1 
P3 
P3 
P3 
HV1 
P3 
HV1 
P3 
P3 
P2 
MV2 
1 See Section ll-D for deception of biological containment 
II-B-3-c. Special Laboratory Design. 
II— B— 3— c— (1). The laboratory shall be 
separated by a controlled access area 
from areas that are open to unrestricted 
traffic flow. A controlled access area is 
an anteroom, a change room, an air lock 
or any other double-door arrangement 
that separates the laboratory from areas 
open to unrestricted traffic flow. 
II— B— 3— c— (2). The surfaces of walls, 
floors, and ceilings shall be readily 
cleanable. Penetrations through these 
surfaces shall be sealed or capable of 
being sealed to facilitate space 
decontamination. 
Il-B-3-c-(3). A foot-, elbow-, or 
automatically-operated hand-washing 
facility shall be provided near each 
primary laboratory exit area. 
II— B— 3— c— (4J. Windows in the 
laboratory shall be sealed. 
II-B-3-c-(5). An autoclave for 
sterilization of wastes and contaminated 
materials shall be available in the same 
building (and preferably within the 
controlled laboratory area) in which 
organisms containing recombinant DNA 
molecules are used. 
II-B-3-c-(6). The laboratory shall 
have a ventilation system that is 
capable of controlling air movement. 
The movement of air shall be from areas 
of lower contamination potential to 
areas of higher contamination potential 
(i.e., from the controlled access area to 
the laboratory area). If the ventilation 
system provides positive pressure 
supply air, the system shall operate in a 
manner that prevents the reversal of the 
direction of air movement or shall be 
equipped with an alarm that would be 
actuated in the event that reversal in the 
direction of air movement were to occur. 
The exhaust air from the laboratory area 
shall not be recirculated to other areas 
of the building unless the exhaust air is 
filtered by HEPA filters or equivalent. 
The exhaust air from the laboratory area 
can be discharged to the outdoors 
without filtration or other means for 
effectively reducing an accidental 
aerosol burden provided that it can be 
dispersed clear of occupied buildings 
and air intakes. 
II-B-3-c-(7). The treated exhaust-air 
from Class I and Class II biological 
safety cabinets [20]may be discharged 
either to the laboratory or to the 
outdoors. The treated exhaust-air from a 
Class III cabinet shall be discharged 
directly to the outdoors. If the treated 
exhaust-air from these cabinets is to be 
discharged to the outdoors through a 
building exhaust air system, it shall be 
connected to this system so as to avoid 
any interference with the air balance of 
the cabinet and the building ventilation 
system. 
II-B-4. P4 Level. 
II-B-4-a. Laboratory Practices. 
II— B — 4— a— (1). Laboratory doors shall 
be kept closed while experiments are in 
progress. 
II-B-4-a-(2). Work surfaces shall be 
decontaminated following the 
completion of the experimental activity 
and immediately following spills of 
organisms containing recombinant DNA 
molecules. 
II-B-4-a-(3). All laboratory wastes 
shall be steam-sterilized (autoclaved) 
before disposal. Other contaminated 
materials such as glassware, animal 
cages, laboratory equipment, and 
radioactive wastes shall be 
decontaminated by a method 
demonstrated to be effective before 
washing, reuse, or disposal. 
II-B-4-a-(4). Mechanical pipetting 
devices shall be used; pipetting by 
mouth is prohibited. 
II-B-4-a-(5). Eating, drinking, 
smoking, and storage of food are not 
permitted in the P4 facility. 
II-B-4~a-(6). Persons shall wash their 
hands after handling organisms 
containing recombinant DNA molecules 
and when they leave the laboratory. 
II-B-4-a-(7). Care shall be exercised 
to minimize the creation of aerosols. For 
example, manipulations such as 
inserting a hot inoculating loop or 
needle into a culture, flaming an 
inoculation loop or needle so that it 
splatters, and forceful ejection of fluids 
from pipettes or syringes shall be 
avoided. 
II-B-4-a-(8). Biological materials to 
be removed from the P4 facility in a 
viable or intact state shall be 
transferred to a nonbreakable sealed 
container, which is then removed from 
the P4 facility through a pass-through 
disinfectant dunk tank or fumigation 
chamber. 
II-B-4-a-(9). No materials, except for 
biological materials that are to remain in 
a viable or intact state, shall be removed 
from the P4 facility unless they have 
been steam-sterilized (autoclaved) or 
decontaminated by a means 
demonstrated to be effective as they 
pass out of the P4 facility. All wastes 
and other materials as well as 
equipment not damaged by high 
temperature or steam shall be. steam 
sterilized in the double-door autoclave 
of the P4 facility. Other materials which 
may be damaged by temperature or 
steam shall be removed from the P4 
facility through a pass-through 
fumigation chamber. 
Il-B-4-a-(10). Materials within the 
Class III cabinets shall be removed from 
the cabinet system only after being 
steam-sterilized in an attached double- 
door autoclave or after being contained 
in a nonbreakable sealed container, 
which is then passed through a 
disinfectant dunk tank or a fumigation 
chamber. 
II-B-4-a-(ll). Only persons whose 
entry into the P4 facility is required to 
meet program or support needs shall be 
authorized to enter. Before entering, 
such persons shall be advised of the 
nature of the research being conducted 
and shall be instructed as to the 
appropriate safeguards to ensure their 
safety. They shall comply with 
instructions and all other required 
procedures. 
II-B-4-a-(12). Persons under 18 years 
of age shall not enter the P4 facility. 
II— B— 4— a— (13). Personnel shall enter 
into and exit from the P4 facility only 
through the clothing change and shower 
rooms. Personnel shall shower at each 
egress from the P4 facility. Air locks 
shail not be used for personnel entry or 
exit except for emergencies. 
II-B-4-a-(14). Street clothing shall be 
removed in the outer side of the 
clothing-change area and kept there. 
-Complete laboratory clothing, including 
undergarments, head cover, shoes, and 
either pants and shirts or jumpsuits, 
shall be used by all persons who enter 
the P4 facility. Upon exit, personnel 
shall store this clothing in lockers 
provided for this purpose or discard it 
into collection hampers before entering 
the shower area. 
II-B-4-a-(15). the universal biohazard 
sign is required on the P4 facility access 
doors and on all interior doors to 
individual laboratory rooms where 
experiments are conducted. The sign 
shall also be posted on freezers, 
refrigerators, or other units used to store 
organisms containing recombinant DNA 
molecules. 
II-B-4-a-(16). An insect and rodent 
control program shall be instituted. 
II-B-4-a-(17). Animals and plants not 
related to the experiment shall not be 
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