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Federal Register / Vol. 45, No. 20 / Tuesday, January 29, 1980 / Notices 
reported properties or any problems in 
the safe use of the system. 
NIH may also distribute certified HVl 
host-vector systems. 
III. Containment Guidelines for Covered 
Experiments 
Part III discusses experiments covered 
by the Guidelines. The reader must first 
consult Part I, where listings are given of 
prohibited and exempt experiments. 
Containment guidelines for 
permissible experiments are given in 
Part III. Changes in these levels for 
specific experiments (or the assignment 
of levels to experiments not explicitly 
considered here) may not be instituted 
without the express approval of the 
Director, NIH. (See Sections IV-E-l-b- 
(l)-(a), IV-E-l-b-(l)-(b), IV-E-l-b-(2}- 
(b). IV-E-l-b-(2)-(c), and IV-E-l-b-(3)- 
(b)-) 
In the following classification of 
containment criteria for different kinds 
of recombinant DNAs, the stated levels 
of physical and biological containment 
are minimal for the experiments 
designated. The use of higher levels of 
biological containment 
(HV3>HV2>HVl) is encouraged if they 
are available and equally appropriate 
for the purposes of the experiment. 
Ill— O. Classification of Experiments 
Using the E. coli K-12 Host- Vector 
Systems. Most recombinant DNA 
experiments currently being done 
employ E. coli K-12 host-vector systems. 
These are the systems for which we 
have the most experience and 
knowledge. 
Some experiments using E. coli K-12 
host-vector systems are prohibited (see 
Section I-D). 
Some experiments using E. coli K-12 
host-vector systems are exempt from the 
Guidelines (see Section I-E). 
Other experiments using E. coli K-12 
shall use Pi physical containment and, 
except as specified in the last paragraph 
of this section, an EKl host-vector 
system (i.e. (a) the host shall not contain 
conjugation-proficient plasmids or 
generalized transducing phages, and (b) 
lambda or lambdoid bacteriophages or 
non-conjugative plasmids shall be used 
as vectors). For these experiments no 
Memorandum of Understanding and 
Agreement (MUA) as described in 
Section IV-D-l-c need be submitted, 
nor is any registration with NIH 
necessary. However, for these 
experiments, prior to their initiation, 
investigators must submit to their 
Institutional Biosafety Committee (IBC) 
a registration document that contains a 
description of (a) the source(s) of DNA, 
(b) the nature of the inserted DNA 
sequences, and (c) the hosts and vectors 
to be used. This registration document 
must be dated and signed by the 
investigator and filed only with the local 
IBC. The IBC shall review all such 
proposals but such review is not 
required prior to initiation of 
experiments. An exception, however, 
which does require prior review and 
approval by the IBC is any experiment 
in which there is a deliberate attempt to 
have the E. coli K-12 efficiently express 
any gene coding for a eukaryotic 
protein. 
Experiments involving the insertion 
into E. coli K-12 of DNA from 
prokaryotes that exchange genetic 
information with E. coli by known 
physiological processes will be 
exempted from these Guidelines if they 
appear on the “list of exchangers" set 
forth in Appendix A (see Section I-E-4). 
For those not on the Appendix A list 
but which exchange genetic information 
[35] with E. coli, experiments may be 
performed with any E. coli K-12 vector 
(e.g. conjugative plasmid). When a non- 
conjugative vector is used, the E. coli K- 
12 host may contain conjugation- 
proficient plasmids, either autonomous 
or integrated, or generalized transducing 
phages. 
III-A. Classification of Experiments 
Using Certain HVl and HV2 Host- 
Vector Systems. Certain HVl and HV2 
host-vector systems are assigned 
containment levels as specified in the 
subsections of this Section III-A. Those 
so classified as of publication of these 
revised Guidelines are listed in 
Appendix D. An updated list may be 
obtained from the Office of 
Recumbinant DNA Activities, National 
Institutes of Health, Bethesda, Maryland 
20205. 
It has been necessary, throughout this 
section, to use words and terms marked 
with footnote reference numbers. The 
footnotes (Part V) define more fully 
what the terms denote. 
III-A-1. Shotgun Experiments. These 
experiments involve the production of 
recombinant DNAs between the vector 
and portions of the specified cellular 
source, preferably a partially purified 
fraction. Care should be taken either to 
preclude or eliminate contaminating 
microorganisms before isolating the 
DNA. 
III-A-l-a. Eukaryotic DNA 
Recombinants. 
Ill— A— 1— a— (1). Primates. P2 physical 
containment + an HV2 host-vector or 
P3 + HV1. 
Ill— A— 1— a— (2). Other Mammals. P2 
physical containment + an HV2 host- 
vector or P3 + HVl. 
III-A-l-a-(3). Birds. P2 physical 
containment -)- an HV2 host-vector, or 
P3 + HVl. 
Ill— A— 1 — a— ( 4 ) . Cold-Blooded 
Vertebrates. P2 physical containment + 
an HVl host-vector or Pi + HV2. If the 
eukaryote is known to produce a potent 
polypeptide toxin, [34) the containment 
shall be increased to P3 + HV2. 
Ill— A— 1— a— (5). Other Cold-Blooded 
Animals and Lower Eukaryotes. This 
large class of eukaryotes is divided into 
two groups: 
1 1 1— A— 1— a— ( 5 )— { a ) . Species that are 
known to produce a potent polypeptide 
toxin(.M) that acts in vertebrates, or are 
known pathogens listed in Class 2,(7) or 
are known to carry such pathogens must 
use P3 physical containment + an HV2 
host-vector. When the potent toxin is 
not a polypeptide and is likely not to be 
the product of closely linked eukaryote 
genes, containment may be reduced to 
P3 + HVl or P2 + HV2. Species that 
produce potent toxins that affect 
invertebrates or plants but not 
vertebrates require P2 + HV2 or R3 + 
HVl. Any species that has a 
demonstrated capacity for carrying 
particular pathogenic microorganisms is 
included in this group, unless the 
organisms used as the source of DNA 
have been shown not to contain those 
agents, in which case they may be 
placed in the following group.(2A) 
III— A— 1— a— (5)— (b). The remainder of 
the species in this class including plant 
pathogenic or symbiotic lungi that do 
not produce potent toxins: P2 + HVl or 
PI + HV2. However, any insect in this 
group must be either (i) grown under 
laboratory conditions for at least 10 
generations prior to its use as a source 
of DNA, or (ii) if caught in the wild, must 
be shown to be free of disease-causing 
microorganisms or must belong to a 
species that does not carry 
microorganisms causing disease in 
veterbrates or plants.(2A) If these 
conditions cannot be met, experiments 
must be done under P3 + HVl or P2 + 
HV2 containment 
III-A-l-a-(6). Plants. P2 physical 
containment -|- an HVl host-vector, or 
PI + HV2. If the plant source makes a 
potent polypeptide toxin, [34] the 
containment must be raised to P3 
physical containment + an HV2 host- 
vector. When the potent toxin is not a 
polypeptide and is likely not to be the 
product of closely linked plant genes, 
containment may be reduced to P3 + 
HVl or P2 + HV2 \2A) 
III-A-l-b. Prokaryotic DNA 
Recombinants. P2 + HVl or PI + HV2 
for experiments with phages, plasmids 
and DNA from nonpathogenic 
prokaryotes which do not produce 
polypeptide toxins(3-7). P3 -t- HV2 for 
experiments with phages, plasmids and 
DNA from Class 2 agents(7). 
[ 24 ] 
