77390 
Federal Register / Vol. 45, No. 227 / Friday, November 21, 1980 / Notices 
air system, it shall be connected to this 
system so as to avoid any interference 
with the air balance of the cabinets of 
the facility exhaust air system. 
II-B-4-c-(14). As noted in Section II- 
B-4-b-(l), the P4 facility may contain 
specially designed areas in which all 
personnel are required to wear one- 
piece positive-pressure isolation suits. 
Such areas shall be airtight. The 
exhaust-air from the suit area shall be 
Altered by two sets of HEPA filters 
installed in series, and a duplicate 
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 emergency lighting 
system, communication systems, and 
power source shall be provided. A 
double-door autoclave shall be provided 
for sterilization of all waste materials 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 system shall be 
provided with alarms and emergency 
backup air. Entry to this area is through 
an airlock fitted with airtight doors. A 
chemical shower area shall be provided 
to decontaminate the surfaces of tbe suit 
before removal. 
II-C. Shipment. Recombinant DNA 
molecules contained in an organism or 
virus shall be shipped only as an 
etiologic agent under requirements of 
the U.S. Public Health Service, and the 
U.S. Department of Transportation 
(§ 72.25, Part 72. Title 42, and 
§5 173.386-.388, Part 173, Title 49. U.S. 
Code of federal Regulations (CFR)) as 
specified below; 
II-C-1. Recombinant DNA molecules 
contained in an organism or viTus 
requiring PI, P2, or P3 physical 
containment, when offered for 
transportation or transported, are 
subject to all requirements of 
§* 72.25(c) (1 (5), Part 72, Title 42 CFR, 
and § § 173.388-.388, Part 173, Title 49 
CFR. 
II-C-2. Recombinant DNA molecules 
contained in an organism or virus 
requiring P4 physical containment, when 
offered for transportation or 
^transported, are subject to the 
requirements listed above under II-C-1 
and are also subject to § 72.25(c)(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 
Recombinant DNA Research." 
Il-D. Biological Containment. 
Il-D-1. Levels of Biological 
Containment. In consideration of 
biological containment, the vector 
(plasmid, organelle, 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 outside the 
laboratory and (ii) transmission of the 
vector from the propagation host to 
other nonlaboratory hosts. 
The following levels of biological 
containment (HV, or Host- Victor, 
systems) for prokaryotes will be 
established; specific' criteria will depend 
on the organisms to be used. Eukaryotic 
host-vector systems are considered in 
Part III. 
II-D-l-a. HVl. A host-vector system 
which provides a moderate level of 
containment. Specific systems: 
II-D-l-a-(l). EKl. The host is always 
E. coli K-12 or a derivative thereof, and 
the vectors include nonconjugative 
plasmids (e.g., pSClOl, ColEl, or 
derivatives thereof [21-27]) and variants 
of bacteriophage, such as lambda [28- 
33). The E. coli K-12 hosts shall not 
contain conjugation-proficient plasmids, 
whether autonomous or integrated, or 
generalized transducing phages, except 
as specified in Section III— 0. 
fl-D-l-a-(2). Other Prokaryotes. 
Hosts and vectors shall be, at a 
minimum, comparable in containment to 
E. coli K-12 with a non conjugative 
plasmid or bacteriophage vector. The 
data to be considered and a mechanism 
for approval of such HVl systems are 
described below (Section II — D-2). 
II-D-l-b. HV2. These are host-vector 
systems shown to provide a high level of 
biological containment as demonstrated 
by data from suitable tests performed in 
the laboratory. Escape of the 
recombinant DNA either via survival of 
the organisms or via transmission of 
recombinant DNA to other organisms 
should be less than 1/10* under 
specified conditions. Specific systems: 
II— D— 1— b— (1 ) . For EK2 host-vector 
systems in which the vector is a 
plasmid, 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 environment, either by survival 
of the original host or as a consequence 
of transmission of the cloned DNA 
fragment 
II-D-l-b-(2). For EK2 host-vector 
systems in which the vector is a phage, 
no more than one in 10 8 phage particles 
should be able to perpetuate a cloned 
DNA fragment under the specified 
nonpermissive laboratory conditions 
designed to represent the natural 
environment either (i) as a prophage (in 
the inserted or plasmid form) in the 
laboratory host used for phage 
propagation or (ii) by surviving in 
natural environments and transferring a 
cloned DNA fragment to other hosts (or 
their resident prophages). 
n-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 dependent 
on its propagation host or is highly 
defective in mobilizability. Reversion 
to host-independence must be less than 
l/l0 8 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 expression of such 
markers is dependent on the 
propagating host or on unique 
laboratory-controlled conditions or is 
blocked by the inserted DNA. 
II-D-l-c-{4). The specified 
containment shown by laboratory tests 
has been independently confirmed by 
specified tests in animals, including 
primates, and in other relevant 
environments. 
II-D-l-c-{5). The relevant genotypic 
and phenotypic traits have been 
independently confirmed. 
II-D-2. Certification of Host-Vector 
Systems. 
II-D-2-a. Responsibility. HVl 
systems other than E. coli K-12, and 
HV2 and HV3 host-vector systems, may 
not be designated as such until they 
have been certified by the director, NIH. 
Application for certification of a host- 
vector system is made by written 
application to the Office of Recombinant 
DNA Activities, National Institutes of 
Health, Bethesda, Maryland 20205. 
Host-vector systems that are proposed 
for certification will be reviewed by the 
National Institutes of Health (NIH) 
Recombinant DNA Advisory Committee 
(RAC). (See Section IV-E-l-b-(l)-(c).) 
This will first involve review of the data 
on construction, properties, and testing 
of the proposed host-vector system by a 
Working Group composed of one or 
more members of the RAC and other 
persons chosen because of their 
expertise in evaluating such data. The 
Committee will then evaluate the report 
of the Working Group and any other 
available information at a regular 
meeting. The Director, NIH, is 
responsible for certification after 
receiving the advice of the RAC. Minor 
modifications of existing certified host- 
vector systems, where the modifications 
are of minimal or no consequence to the 
properties relevant to containment may 
be certified by the Director, NIH, 
[9] 
