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Federal Register / Vol. 47, No. 102 / Wednesday, May 26, 1982 / Notices 
App«f>dix I— Biological Contaimnenl 
Appendix 1-L 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 call) in which 
the vector is propagated in the laboratory 
will be considered together. Any combination 
of vector and host which is to provide 
biological contaiiunent 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 Wost- V'ector. system) for 
prokaryotes will be establish: specific criteria 
will depend on the organisms to be used. 
Appendix l-l-A. HVl. A host-vector 
system which provides a moderate level of 
containment Specific systems: 
Appendix l-t-A-l. EKl. The host is always 
£ coh K-12 or a derivative thereof, and the 
vectors Include noncon|ugative plasmids (e.g., 
pSClOl. CollEl. or derivatives thereof |1-7|) 
and variants of bacteriophage, such as 
lambda |S-15]. The £ coH K-12 host shall not 
contain conjugation-proficient plasmids, 
whether autonomous or integrated, or 
generalised transducing phages. 
Appendix W-A-2. Other HVl. Hosts and 
vectors shall be, at a minimum, comparable 
In containment to £ coU K-12 with a non 
conjugatlve plasmid or bacteriophage vector. 
The data to be consklared and a mechanism 
for approval of such HVl systems are 
described below (Appendix l-U). 
Appendix l-l-B. HVZ These are host- 
vector systems shown to provide a high level 
of biological containment as demonstrated by 
data from suitable testa performed in the 
laboratory. Escape of the recombinant DNA 
either vie survial of the organisms or via 
transmission of recombinant DNA to other 
organisms should be less than l/lOhinder 
specified conditions. Specific systems: 
Appendix l-l-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 nonpermisaive laboratory 
conditions designed to represent the natural 
environment either by survival of the original 
host or as a consequences of transmission of 
the cloned DNA fragment. 
Appendix M-B-2. For EK2 host-vector 
systems in which the vector is a phage, no 
more than one in 10* phage partides should 
be able to perpetuate a doned DNA fragment 
under the specified nonpermlssivs laboratory 
conditions designed to represent the natural 
environment either (i) as a prophage (in the 
iruerted or plasmid form) in the laboratory 
host used for phage propagation or (ii) by 
surviving in natural environments and 
transferring a doned DNA fragment to other 
hosts (or their resident prophages). 
Appendix HI. Certification of Host-Vector 
Systems. 
Appendix Hl-A. Responsibility. HVl 
systems other than £ coh K-12. and HV2 
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- 
C-l-b-(l)-(e).) This will first involve review 
of the data on construction, properties, and 
testing of the proposed host-vector system by 
a Working Croup 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 regiJar 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 nunimal or no 
consequence to the properties relevant to 
containment may be certified by the Director. 
NIK without review by the RAC (See 
Section rV-C-l-b-(3)-(c).) 
When new host-vector systems are 
certified, notice of the certification will be 
sent by the Office of Recombinant DNA 
Activities (ORDA) to the applicant and to all 
Institutional Biosafety Committees (IBCs) and 
will be published in the Recombinant DNA 
Technical Bulletin. Copies of a list of all 
currently certified host-vector systems may 
be obtained from ORDA at any time. 
The Director. NIH. may at any time rescind 
the certlficatloo of any host-vector system. 
(See Section IV-C-l-M3Hd) ) If 
certification of a host-vector system is 
rescinded. NIH will instruct investigators to 
transfer cloned DNA into a different system, 
or use the clones at a higher physical 
containment level unless NIH determines that 
the already constructed clones Incorporate 
adequate biological containment 
Certification of a given system docs not 
extend to modifications of either the host or 
vector component of that system. Such 
modified systems must be independently 
certified by the Director. NIH. If 
modifications are minor, it may only be 
necessary for the investigator to submit data 
showing that the modirications have either 
improved or not Impaired the major 
phenotypic traits on which the containment 
of the system depends. Substantial 
modifications of a certified system require 
the submission of complete testing data. 
Appendix l-U-B. Data To Be Submitted for 
Certification. 
Appendix HI-B-1. HVl Systems Other 
than £ coli K-12. The following types of data 
shall be submitted, modlfled as appropriate 
for the particular system under consideration, 
(i) A description of the organism and vector, 
the strain's natural habitat and growth 
requirements; its physiological properties, 
particularly those related to its reproduction 
and survival and the mechanisms by which it 
exchanges genetic information: the range of 
organisms with which this organism normally 
exchange genetic information and what sort 
of Information is exchanged; and any 
relevant information on its pathogenicity or 
toxicity, (ii) A description of the Ustory of the 
particular strains and vectors to be used. 
including data on any mutations which 
render this organism less able to survive or 
transmit genetic information, (iii) A general 
description of the range of experiments 
contemplated, with emphasis on the need for 
developing such an HVl system. 
Appendix I-II-B-2. HV2 Systems. 
Investigators planning to request HV2 
certiHcation for host-vector systems can 
obtain instructions from ORDA concerning 
data to be submitted [14-15]. 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 containment of 
the system, (iii) Data on the survival of the 
host-vector system under nonpermisaive 
laboratory conditions designed to represent 
the relevant natural environment, (iv) Data 
on transmissibility of the vector and/or a 
cloned DNA fragment under both permissive 
and nonpermissive conditions, (v) Data on all 
other properties of the system which affect 
containment and utility, including 
information on yields of phage or plasmid 
molecules, ease of DNA isolation, and ease of 
transfection or transformation, (vi) In some 
cases, the investigator may be asked to 
submit data on survival and vector 
transmissibility from experiments in which 
the host-vector is fed to laboratory animals 
and human subjects. Such in vivo data may 
be required to confirm the validity of 
predicting in vivo survival on the basis of in 
vitro experiments. 
Data must be submitted In writing to 
ORDA Ten to twelve weeks are normally 
required for review and circulation of the 
data prior to the meeting at which such data 
can be considered by the RAC. Investigators 
are encouraged to publish their data on the 
construction, properties, and testing of 
proposed HV2 systems prior to consideration 
of the system by the RAC and its 
subcommittee. More specific instructions 
concerning the type of data to be submitted 
to NTH for proposed EK2 systems Involving 
either plasmids or bacteriophage in £ coli K- 
12 are available from ORDA. 
Appendix HII. Footnotes and References 
of Appendix I. 
1. Hershtield. V„ H. W., Boyer, C 
Yanofsky. M. A. Lovett and D. R. Hellnskl 
(1974). Plasmid Col El as a Molecular Vehicle 
for Cloning and Amplification of DNA. Proc. 
Nat. Acad. Sci. USA 71. 3455-3459. 
2. Wensink. P. C.. D. ). Finnegan, ). E. 
Donelson. and D. S. Hogness (1974). A System 
for Mapping DNA Sequences in the 
Chromosomes of Drosophila Melanogaster 
Cell 3. 315-335. 
3. Tanaka, T., and B. Weisbium (1975). 
Construction of a Col id n El-R Factor 
Composite Plasmid in Vitro: Means for 
Amplification of Deoxyribonucleic Acid. ]. 
BacterioL 121, 354-362. 
4. Armstrong. K. A.. V. Hershfield, and D. 
R. Helinski (1977). Gene Cloning and 
Containment Properties of Plasmid Col El 
and Its Derivatives, Science 196, 172-174. 
5. Bolivar. F., R. L Rodriquez, M. C. 
Betlach, and H. W. Boyer (1977). Construction 
and Characterization of New Cloning 
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