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Federal Register / Vol. 46, No. 126 / Wednesday, July 1, 1981 / Notices 
(IBC) a registration document that 
contains a description of (a) the 
sourcefs) of DNA, (b) the nature of the 
inserted DNA sequences, (c) the hosts 
and vectors to be used, (d) whether a 
deliberate attempt will be made to 
obtain expression of a foreign gene in 
the cloning vehicle and if so, what 
protein, and (e) the containment 
conditions specified by these 
Guidelines. 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: IBC review prior to initiation 
of the experiment is not required for 
experiments described in Section III-O. 
Prior IBC review is required for all other 
experiments described in the 
subsections of Part III, including III-O-l, 
UI-0-2, etc. 
Changes from the levels specified in 
Part III 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- 1 - 
b-(2)-(b), IV-E-l-b-(2)-(c), and IV-E-1- 
M3Wb).) 
In the classiHcation 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. 
When the reader finds that the 
containment level given for the same 
experiment is different in two different 
sections within Part III, he may choose 
whichever of the two levels he wishes to 
use for the experiment. 
III-O. Classification of Experiments 
Using Certain Host-Vector Systems. 
Experiments listed in Appendix H may 
be performed at Pi physical 
containment. For these experiments IBC 
review prior to initiation of the 
experiment is not required. 
IIl-O-l. Experiments Involving Class 
3 organisms. Experiments involving 
recombinant DNA from Class 3 
Organisms [1) or from cells known to be 
infected with these agents may be 
conducted at P3 containment in E. coli 
K-12 EKl hosts (see Appendix C). 
Containment levels for all other 
experiments with Class 3 organisms'or 
with recombinant DNA which increases 
the virulence and host range of a plant 
pathogen beyond that which occurs by 
natural genetic exchange will be 
determined by NIH. (See Section IV-E- 
l_b-2-(e)). 
III-0-2. Experiments Involving 
Prokaryotes Nonpathogenic for Man, 
Animals or Plants, and/or Lower 
Eukaryotes Nonpathogenic for Man, 
Animals or Plants. Recombinant DNA 
experiments evolving prokaryotes 
nonpathogenic for man, animals or 
plants, and/or lower eukaryotes 
nonpathogenic for man, animals or 
plants, and only DNA from such 
sources, can be conducted under P3 
containment [2A). Lower levels of 
physical containment may be assigned 
by ORDA on a case-by-case basis for 
specific donor-recipient combinations 
(see Section IV-E-l-b-(3)-(h)). 
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 
Recombinant DNA Activities, National 
Institutes of Health, Bethesda, Maryland 
20205. 
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. 
• III-A-l-a-(l). Primates. P2 physical 
containment -|- an HV2 host-vector or 
P3 -f- HVl. 
III-A-l-a-(2). Other Mammals. P2 
physical containment -|- an HV2 host- 
vector or P3 -I- HVl. 
III-A-l-a-(3). Birds. P2 physical 
containment + an HV2 host-vector, or 
P3 -t- HVl. 
III-A-l-a-(4). Cold-Blooded 
Vertebrates. P2 physical containment 
an HVl host-vector or Pi -t- HV2. If the 
eukaryote is known to produce a potent 
polypeptide toxin, [34] the containment 
shall be increased to P3 -I- HV2. 
III-A-l-a-(5). Other Cold-Blooded 
Animals and Lower Eukaryotes. This 
large class of eukaryotes is divided into 
two groups; 
III-A-l-a-(5)-(a). Species that are 
known to produce a potent polypeptide 
toxin [34] that acts in vertebrates, or are 
known pathogens listed in Class 2,(1] or 
are known to carry such pathogens must 
use P3 physical containment -|- an HV2 
host-vector. When the potent toxin is 
not a polyJ)eptide and is likely not to be 
the product of closely linked eukaryote 
genes, containment may be reduced to 
P3-I-HV1 or P2-(-HV2. Species that 
produce potent toxins that affect 
invertebrates or plants but not 
vertebrates require P2-(-HV2 or 
P3-I-HV1. 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-l-a-(5)-(b). The remainder of 
the species in this class including plant 
pathogenic or symbiotic fungi that do 
not produce potent toxins; P2-)-HVl or 
Pl-t-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 
vertebrates or plants. [2A] If these 
conditions cannot be met, experiments 
must be done under P3-I-HV1 or 
P2-f Hv2 containment. 
III-A-l-a-(6). Plants. P2 physical 
containment -|- an HVl host-vector, or 
P1-I-HV2. If the plant source makes a 
potent polypeptide toxin, [34] the 
containment must be raised to P3 
physical containment -t- 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-f HVl or P2-I-HV2. [2A] 
III-A-l-b. Prokaryotic DNA 
Recombinants. P2-I-HV1 or PH-HV2 for 
experiments with phages, plasmids and 
DNA from nonpathogenic prokaryotes 
which do not produce polypeptide 
toxins. [34] P3-f-HV2 for experiments 
with phages, plasmids and DNA from 
Class 2 agents. [1] 
III-A-2-a. Viruses of Eukaryotes 
(summary given in Table III; see also 
exception given at asterisk at end of 
Appendix D). 
IIl-A-2-a-(l). DNA Viruses. 
III-A-2-a-(l)-(a). Nontransforming 
viruses. 
III-A-2-a-(l)-(a)-(l). Adeno- 
Associated Viruses, Minute Virus of 
Mice, Mouse Adenovirus (Strain FLJ, 
and Plant Viruses. (48) Pi physical 
containment -|- and HVl host-vector 
shall be used for DNA recombinants 
produced with (i) the whole viral 
genome, (ii) subgenomic DNA segments, 
or (iii) purified cDNA copies of viral 
mRNA. [37] 
III-A-2-a-(l)-(a)-(2). Hepatitis B. 
IIl-A-2-a-(l)-(a)-(2)-(o). PI physical 
containment -t- an HVl host-vector shall 
[158] 
