49600 
NOTICES 
•(10) An Individual supply and exhaust 
air ventilation system shall be provided for 
the Pi facility. The system shall maintain 
pressure differentials and directional air flow 
as required to assure Inflow from areas out- 
side of the P4 facility toward areas of high- 
est potential risk within the Pi facility. The 
system shall be designed to prevent the re- 
versal of air flow. The system shall sound an 
alarm In the event of system malfunction. 
•(11) Recirculation of air within Individ- 
ual laboratories of the Pi facility Is permis- 
sible provided that this air Is filtered by a 
HE PA filter. 
•(12) The exhaust air from the facility 
6hall be filtered by HEPA filters and dis- 
charged to the outdoors so that It Is dis- 
persed clear of occupied buildings and air 
Intakes. The filter chambers shall be de- 
signed to allow In situ decontamination be- 
fore removal and to facilitate certification 
testing after replacement. 
(13 ■ The treated exhaust air from Class I 
and Class II biological safety cabinets : may 
be discharged directly to the laboratory room 
environment or to the outdoors. The treated 
exhaust air from Class HI cabinets shall be 
discharged to the outdoors. If the treated ex- 
haust air from these cabinets Is to be dis- 
charged to the outdoors through the Pi fa- 
cility exhaust air system, It shall be con- 
nected to this system so as to avoid any In- 
terference with the air balance of the cabi- 
nets or the facility exhaust air system. 
• (14) A specially designed suit area may 
be provided In the facility. Personnel who 
enter this area shall wear a one-piece posi- 
tive pressure suit that Is ventilated by a life 
support system. The life support system shall 
be provided with alarms and emergency back- 
up tank air. Entry to this area Is through 
an air-lock fitted with airtight doors. A 
chemical shower area shall be provided to 
decontaminate the surfaces of the suit before 
removal. The exhaust air from the suit area 
shall be filtered by two sets of HEPA filters 
Installed in series. A duplicate filtration 
unit and exhaust fan shall be provided. An 
emergency power source shall be provided. 
The air pressure within the suit area shall be 
less than that In any adjacent area. Emer- 
gency lighting and communication systems 
shall be provided. 
The Internal shell of the suit area shall be 
airtight. A double door autoclave shall be 
provided for sterilization of all waste mate- 
rials to be removed from the suit area. 
C. Shipment. Novel 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 
fl 72.25 of Part 72, Title 42. Code of Federal 
Regulations!. Department of Transportation 
(5 173.387 fb) of Part 173, Title 49, Code of 
Federal Regulations) . and the Civil Aero- 
nautics Board (C_A3. No. 82. Official Air 
Transport Restricted Articles Tariff No. 6-D) 
for shipment of etlologlc agents (Appendix 
A). 
The packaging and shipment of organisms 
and viruses containing recombinant DNA 
molecules shall be In compliance with all 
requirements specified In subparagraphs (1)- 
(5) of paragraph (c), “Transportation: etlo- 
loglc agents subject to additional require- 
ments". of 5 72.25 of Part 72. Title 42. Code 
of Federal Regulations. Subparagraph 16) 
of paragraph (c) of 5 72.25 of Part 72, Title 
42, Code of Federal Regulations shall apply 
to the shipment of all viable host and vector 
organisms which require P4 physical con- 
tainment. 
Additional Information on packaging and 
shipment Is given in ADpendlx A. 
D. Biological containment — 1. Levels of 
biolooical containment. In considering bio- 
logical containment, the vector fplasmld or 
virus) for the recombinant DNA and the 
host (bacterial, plant or animal cell) In 
which the vector Is propagated In the labo- 
ratory will be considered together. Any vector 
and host which are to provide biological con- 
tainment must be constructed so that the 
following types of “escape" are minimized: 
Survival of the vector In Its host outside 
of the laboratory. Transmission of the vector 
from the propagation host to other non- 
laboratory hosts. 
The following levels of biological contain- 
ment (Host-Vector Systems) for prokaryotes 
will be established; specific criteria will de- 
pend on the organism to be used. Eukaryotic 
host-vector systems are considered In Sec- 
tion m. 
a. HV1. A host-vector system which pro- 
vides a moderate level of containment. The 
host should have a low potential for survival 
In Its natural environment, and the vector 
should have a low potential for transmission 
of its recombinant DNA. 
Specific systems — (1) EK1. The host Is 
always E. coli K-12 or a derivative thereof, 
and the vectors Include nonconjugatlve 
plasmld3 (e.g., pSClOl, ColEl, or derivatives 
thereof (15-21)) and variants of bacterio- 
phage, such as X (22-27). The E. coli K-12 
hosts should not contain conjugation-pro- 
ficient plasmids, whether autonomous or 
Integrated, or generalized transducing 
phages. 
(2) Other prokaryotes. Hosts and vectors 
should be, at a ‘minimum, comparable In 
containment to E. coli K-12 with a non- 
conjugatlve plasmid 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 ). 
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. 
Speciflic systems — (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 non-permlsslve labora- 
tory conditions designed to represent the 
natural environment, either by survival of 
the original host as a consequence of trans- 
mission of the cloned DNA fragment. 
(2) For FK 2 host- vector systems In which 
the vector Is a phage, no more than one In 
10* phage particles should be able to per- 
petuate a cloned DNA fragment under the 
specified non-permlsslve laboratory condi- 
tions designed to represent the natural 
environment either (a) as a prophage or 
plasmid In the laboratory host used for phage 
propagation or (b) by surviving In natural 
environments and transferring a cloned DNA 
fragment to other hosts (or their resident 
prophages) . 
c. HV3. These are host-vector systems In 
which: 
(1) All HV2 criteria are met. 
(2) The vector Is dependent on Its propa- 
gation host or Is highly defective In mo- 
billzablllty. Reversion to host-independence 
must be less than 1/10* per vector genome 
per generation. 
(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 unique laboratory 
controlled conditions or Is blocked by the 
Inserted DNA. 
(4) The specified containment shown by 
laboratory tests has been Independently con- 
firmed by specified tests In animals, Includ- 
ing primates, and In other relevant environ- 
ments. 
(5) The relevant genotypic and pheno- 
typic traits of such HV3 systems have been 
Independently confirmed. 
This general description can be applied 
directly to the specific case of an EK3 system. 
2. Certification of Host-Vector Systems — 
a. Responsibility. Recommendation to the 
Director of NTH for certification of HV1 sys- 
tems other than E. coli K-12, and HV2 and 
HV3 host-vector systems, is the responsibil- 
ity of the NIH Recombinant DNA Molecule 
Program Advisory Committee. Data on con- 
struction, properties, and testing of pro- 
posed host-vector systems will be analyzed 
and reviewed by a subcommittee composed 
of one or more members of the NIH Recom- 
binant DNA Molecule Program Advisory 
Committee and other individuals chosen be- 
cause of their expertise In evaluating such 
data. Such subcommittees shall provide a 
written report to the NIH Recombinant DNA 
Molecule Program Advisory Committee. The 
Committee will evaluate this report and 
any other available Information at a regu- 
lar meeting. 
The NIH Recombinant DNA Molecule Pro- 
gram Advisory Committee can recommend 
that a certification be rescinded at any time, 
should new data or new considerations In- 
validate the previous decision. In such cases. 
Investigators may be asked to transfer their 
cloned DNA Into a different approved system. 
Certification of a given system does not 
extend to modifications of either the host 
or vector component of that system. Such 
modified systems must be Independently ap- 
proved. If modifications are minor. It may 
only be necessary for the Investigator to sub- 
mit data showing that the modifications have 
either Improved or not Impaired the major 
phenotypic traits on which the containment 
of the system depends. More substantial 
modifications of a certified system may 
necessitate submission of complete testing 
data. 
b. Data to be submitted for certification — 
(1) HV1 systems other than E. Coli K-12. 
The following types of data should be sub- 
mitted, modified as appropriate for the par- 
ticular system under consideration: 
(1) A description of the organism and vec- 
tor, the strain’s natural habitat and growth 
requirements; the range of organisms with 
which this organism normally exchanges 
genetic Information and what sort of ln- 
fornqation Is exchanged; any relevant In- 
formation on Its pathogenicity or toxicity. 
(II) A description of the history of the 
particular strains and vectors to be used; In- 
formation on any mutations which render 
this organism less able to survive or trans- 
mit genetic Information. 
(III) A general description of the range of 
experiments contemplated, with emphasis on 
the need for developing such an HV1 system. 
(2) HV2 systems. Investigators planning to 
request HV2 certification for host-vector sys- 
tems can obtain Instructions from the NIH 
Office of Recombinant DNA Activities con- 
cerning data to be submitted. In general, the 
following types of data are required: 
(I) Description of construction steps, with 
Indication of source, properties, and manner 
of Introduction of genetic traits. 
(II) Quantitative data on the stability of 
genetic traits that contribute to the contain- 
ment of the system. 
(III) Data on the survival of the host- 
vector system under non-permlsslve In vitro 
conditions designed to represent the natural 
environment. 
(lv) Data on transmlsslblllty of the vector 
and/or a cloned DNA fragment under both 
permissive and non-permlsslve conditions. 
FEDERAL REGISTER, VOL 42, NO. 187— TUESDAY. SEPTEMBER 27 t 1977 
[ 168 ] 
