34466 
Federal Register / Vol. 46, No. 126 / Wednesday, July 1. 1981 / Notices 
physical containment. Experimental 
procedures involving a host-vector 
system that provides a one-step lower 
level of biological containment than that 
specified in Part III can be conducted in 
II-B-3-C. Special Laboratory Design. 
Il-B-3-c-(l). 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 facilities space 
decontamination. 
II-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-(4). 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 perferably 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 
areas 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 
the P3 laboratory using containment 
equipment specified for the P4 level of 
physical containment. Alternative 
combinations of containment safeguards 
are shown in Table I. 
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 dipersed 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-(l). 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 experiemental 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 dispoal. 
U-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. 
Il-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 stream shall be steam 
sterilzed 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. 
II-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-r4-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 P4 facility only 
through the clothing change and shower 
rooms. Personnel shall shower at each 
egress from the P4 facility. Air locks 
shall 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 areas and kept there. 
Table \.— Combinations of Containment Safeguards 
Oassificatiorr of experiment according to 
guidelines 
Physical 
containmefU 
Biological ' 
containment 
Alternate combinations of physical and biological containment 
Physical containment 
Laboratory design 
specified for 
Laboratory 
practices specified 
for 
Containment 
equipment 
specified for 
Biological 
containment 
P3 
.... HV3 
.... P3 
,. P3 
P3 
HV3. 
P3 
.... HV3 
... P3 
. P3 
P4 
HV2 
P3 
.... HV2 
... P3 
. P3 
P3 
HV2. 
P3 
.... HV2 
.... P3 
. P3 
P2 
HV3 
P3 
.... HV2 
.... P3 
. P3 
P4 
HV1 
P3 
.... HV1 
P3 
. P3 
P3 
HV1 
P3 
.... HV1 
... P3 
. P3 
P2 
HV2. 
t See Section II- 
-D for description of biological containment 
[154] 
