NOTICES 
33075 
sterilized by heat. Liquid effluents 
from the shower and hand washing fa- 
cilities may be inactivated by chemical 
treatment. HEPA filters shall be in- 
stalled in all effluent drain vent lines. 
II-B-4-c-(10). ‘An individual supply 
and exhaust -air ventilation system 
shall be provided for the P4 facility. 
The system shall maintain pressure 
differemials and directional airflow as 
required to assure inflow from areas 
outside the P4 facility toward areas of 
highest potential risk within the facili- 
ty. The system shall be designed to 
prevent the reversal of airflow. The 
system shall sound an alarm in the 
event of system malfunction. 
II-B-4-c-(ll). ‘Recirculation of air 
within individual laboratories of the 
P4 facility is permissible if this air is 
filtered by a HEPA filter. 
II-B-4-c-(12). ‘The exhaust air from 
the P4 facility shall be filtered and 
discharged to the outdoors so that it is 
dispersed clear of occupied buildings 
and air intakes. The filter chambers 
shall be designed to allow in situ de- 
contamination before removal and to 
facilitate certification testing after re- 
placement. 
II-B-4-c-(13). The treated exhaust 
air from Class I and Class II biological 
safety cabinets(20) 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-c-<14). *A specially designed 
suit area may be provided in the facili- 
ty. Personnel who enter this area shall 
wear a one-piece positive-pressure suit 
that is ventilated by a life-support 
system. The life-support system shall 
be pro tided with alarms and emergen- 
cy backup air. Entry to this area is 
through an air-lock fitted with air- 
tight doors. A chemical shower area 
shall be provided to decontaminate 
the surfaces of the suit before remov- 
al. The exhaust air from the suit area 
shall be filtered by two sets of HEPA 
filters installed in series, and a dupli- 
cate filtration unit and exhaust fan 
shall be provided. The air pressure 
within the suit area shall be less than 
that in any adjacent area. An emer- 
gency lighting system, communication 
systems, and power source shall be 
provided. 
The internal shell of the suit area 
shall be airtight. A doubiedoor auto- 
clave shall be provided for sterilization 
of all waste materials to be removed 
from the suit area. 
II-C. Shipment Recombinant DNA 
when contained in an organism or 
virus shall be shipped in compliance 
with the requirements issued by the 
U.S. Public Health Service (section 
72.25 of Part 72. Title 42. Code of Fed- 
eral Regulations). Department of 
Transportation (section 173.387(b) of 
Part 173. Title 49. Code of Federal 
Regulations), and the Civil Aeronau- 
tics Board (C.A.B. No. 82. Official Air 
Transport Restricted Articles Tariff 
No. 6-D) for shipment of etiologic 
agents. 
The packaging and shipment of or- 
ganism and viruses containing recom- 
binant DNA molecules shall be in com- 
pliance with all requirements specified 
in subparagraphs ( i )— (5 ) of paragraph 
(c). ••Transportation; etiologic agents 
subject to additional requirements," of 
section 72.25 of Part 72. Title 42, Code 
of Federal Regulations. Subparagraph 
(6) of paragraph (c) of section 72.25 of 
Part 72. Title 42. Code of Federal Reg- 
ulations shall apply to the shipment 
of all viable host and vector organisms 
which require F4 physical contain- 
ment. 
Additional information on packaging 
and shipment is given in ' Labor tory 
Safety Monograph— A Supplement to 
the NIH Guidelines for Recombinant 
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 are to pro- 
vide biological containment must be 
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. 
H-D-l-a. HV1. A host-vector system 
which provides a moderate level of 
containment. Specific systems: 
H-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, 
CoiEl. or derivatives thereof 121-27) 
and variants of bacteriophage, such as 
0 (.28-33). The E. coli K-12 hosts 
should not contain conjugation-profi- 
cient plasmids, whether autonomous 
or integrated, or generalized transduc- 
ing phages. 
II-D-l-a-(2). Other Prokaryotes. 
Hosts and vectors should be. at a mini- 
mum, comparable in containment to E. 
coli K-12 with a nonconjugative plas- 
mid or bacteriophage vector. The data 
to be considered and a mechanism for 
approval of such HV1 systems are de- 
scribed below (section II-D-2). 
II-D-l-b. HV2. These are host-vector 
systems showm to provide a high level 
of biological containment as demon- 
strated by data from suitable tests per- 
formed in the laboratory. Escape of 
the recombinant DNA either via sur- 
vival of the organisms or via transmis- 
sion of recombinant DNA to other or- 
ganisms should be less than Vio* under 
specified conditions. Specific systems: 
II-D-l-b-(l). For EK2 host-vector 
systems in which the vector is a plas- 
mid. no more than one in 10* host cells 
should be able to perpetuate a cloned 
DNA fragment under the specified 
nonpermissive laboratory conditions 
designed to represent the natural envi- 
ronment, either by survival of the 
original host or as a consequence of 
transmission of the cloned DNA frag- 
ment, 
II-D-l-b-(2). For EK2 host-vector 
systems in which the vector is a phage, 
no more than one in 10* phage parti- 
cles should be able to perpetuate a 
cloned DNA fragment under the speci- 
fied nonpermissive laboratory condi- 
tions designed to represent the natural 
environment either (i) as a prophage 
or plasmid in the laboratory host used 
for phage propagation or (ii) by surviv- 
ing in natural environments and trans- 
ferring a cloned DNA fragment to 
other hosts (or their resident pro- 
phages). 
II-D-l-c. HV3. These are host-vector 
systems in which: 
II-D-l-c-(l). All HV2 criteria are 
met. 
II-D-l-c-(2). The vector is depend- 
ent on its propagation host or is 
highly defective in mobilizability. Re- 
version to host-independence must be 
less than yio s per vector genome per 
generation. 
II-D-l-c-(3). No markers conferring 
resistance to antibiotics commonly 
used clinically or in agriculture are 
carried by the vector, unless expres- 
sion of such markers is dependent on 
the propagating host or on unique lab- 
oratory controlled conditions or is 
blocked by the inserted DNA. 
n-D-l-c-(4). The specified contain- 
ment shown by laboratory tests has 
been independently confirmed by 
specified tests in animals, including 
primates, and in other relevant envi- 
ronments. 
II-D-l-c-(5). The relevant genotypic 
and phenotypic traits have been inde- 
pendently confirmed. 
II-D-2. Certification of host-vector 
systems. 
II-D-2-a. Responsibility. HV1 sys- 
tems other than E. coli K-12, and HV2 
and HV3 host-vector systems may not 
FEDERAL REGISTER, VOL 43, MO. 146 — FRIDAY, Y 28. 1978 
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