NOTICES 
60119 
II- A-2-a-(2McM2Ma). PI physical 
containment + an EK1 host-vector 
shall be used for DNA recombinants 
produced with purified subgenomic 
cDNA segments.[381 
III- A-2-a-<2Wc>-<2>-<6). P2 physical 
containment + an EK1 host and a 
vector certified for use in an EK2 
system, or P3 + EK1, shall be used for 
DNA recombinants produced with (i) 
cDNA copies of the whole genome, or 
(ii) purified cDNA copies of viral 
mRNA.[37] 
III-A-2-a-(2Md). Double-Stranded 
Segmented RNA Viruses. PI physical 
containment + an EK1 host-vector 
shall be used for DNA recombinants 
produced with (i) mixtures of subgeno- 
mic cDNA segments, (ii) a specific sub- 
genomic cDNA segment, or (iii) puri- 
fied cDNA copies of viral mRNA.[37] 
III-A-2-a-(2Me). RNA Plant Viruses 
and Plant Viroids. PI physical con- 
tainment + an EK1 host-vector shall 
be used for DNA recombinants pro- 
duced with (i) cDNA copies of the 
whole viral genome, (ii) subgenomic 
cDNA segments, or (ill) purified cDNA 
copies of viral mRNA.[37] 
III-A-2-a-(3). Intracellular Viral 
DNA. Physical and biological contain- 
ment specified for shotgun experi- 
ments with eukaryotic cellular DNA 
[see Section III-A-< l)-(a)l shall be 
used for DNA recombinants produced 
with integrated viral DNA or viral gen- 
omes present in Infected cells. 
III-A-2-b. Eukaryotic Organelle 
DNAs. P2 physical containment + an 
EK1 host-vector, or PI + EK2, for mi- 
tochondrial or chloroplast DNA from 
eukaryotes when the organelle DNA 
has been obtained from Isolated organ- 
elles. Otherwise, the conditions given 
for shotgun experiments apply. 
III-A-2-c. Prokaryotic Plasmid and 
Phage DNAs. The containment levels 
required for shotgun experiments with 
DNA from prokaryotes apply to their 
plasmids or phages. 
III-A-3. Lowering of Containment 
Levels for characterized or Purified 
DNA Preparations and Clones. Many 
of the risks which might conceivably 
arise from some types of recombinant 
DNA experiments, particularly shot- 
gun experiments, would result from 
the inadvertent cloning of a harmful 
sequence. Therefore, in case where the 
risk of inadvertently cloning the 
"wrong’' DNA is reduced by prior en- 
richment for the desired piece, or in 
which a clone made from a random as- 
sortment of DNAs has been purified 
and the absence of harmful sequences 
established, the containment condi- 
tions for further work may be reduced. 
The following section outlines the 
mechanisms for such reductions. 
III-A-3-a. Purified DNA Other than 
Plasmids. Bacteriophages, and Other 
Virtues. The formation of DNA recom- 
binants from cellular DNAs that have 
been purified [411 and in which the ab- 
sence of harmful sequences had been 
established [3] can be carried out 
under lower containment conditions 
than used for the corresponding shot- 
gun experiment. [42] The containment 
may be decreased one step in physical 
containment (P4— »P3; P3--P2; P2— PI) 
while maintianing the biological con- 
tainment specified for the shotgun ex- 
periment, or one step in biological con- 
tainment (EK3 - EK2; EK2 — EK1) 
while maintaining the specified physi- 
cal containment. The institutional bio- 
safety committee (IBC) must review’ 
such a reduction and the approval of 
the IBC must be secured before such a 
reduction may be put into effect. (See 
Section IV-D-3-b.) The IBC must 
notify the NIH Office of Recombinant 
DNA activities (ORDA) in writing of 
all such approvals within 30 days after 
they take place. IBC approval is suffi- 
cient for such a reduction except for 
(i) primate DNA. which also requires 
prior NIH approval (see Section III-A- 
I-a-(l)), or (ii) any lowering of con- 
tainment under Section III-A-3-a to 
levels below PI + EK1, which also re- 
quires prior NIH approval. (See Sec- 
tions IV-D-l-c, IV-E-l-b-(3)-(e). and 
IV-E-l-b-(3Mf).) 
III-A-3-b. Characterized Clones of 
DNA Recombinants. When a cloned 
DNA recombinant has been rigorously 
characterized and the absence of 
harmful sequences has been estab- 
lished (3), experiments involving this 
recombinant DNA may be carried out 
under lower containment conditions, 
as described below. 
III-A-3-b-(l). Institutional biosafety 
committees (IBCs) may give approval 
for a single-step reduction in physical 
or biological containment on receipt of 
evidence of characterization of a clone 
derived from a shotgun experiment 
and Its probable freedom from harm- 
ful genes. (See Section IV-D-3-b.) The 
IBC must notify ORDA in writing of 
all such approvals within 30 days after 
they take place. IBC approval is suffi- 
cient for such a reduction except for 
(i) primate DNA. which requires prior 
NIH approval (see Section III-A-l-a- 
(1)). or (ii) any lowering of contain- 
ment under Section III-A-3-b to levels 
below PI + EK1. which also requires 
prior NIH Approval. (See Sections IV- 
D-l-c, IV-E-l-b-3-(e) and IV-E-l-b- 
(3 )— ( f ).> 
III-A-3-b-<2). Reduction of contain- 
ment levels by more than one step, or 
cases involving primate DNA, or cases 
involving lowering of containment 
under Section III-A-3-b to levels 
below PI + EK1, will require prior ap- 
proval by NIH. (See Sections IV-E-1- 
b-(3)-(e), -(f) and -(g).) 
III-B. Experiments with Other Pro- 
karyotic Host-Vectors. 
III-B- 1. HV1 Systems. Host-vector 
systems which have been approved as 
HV1 systems may be used under P2 
containment conditions for shotgun 
experiments with phages, plasmids, 
and DNA from nonpathogenic prokar- 
yotes which do not produce polypep- 
tide toxins.[341 
Other classes of recombinant DNA 
experiments with these HV1 systems 
will require prior approval and classifi- 
cation by NIH. Experiments with 
DNAs from eukaryotes (and their plas- 
mids or viruses) will generally follow 
the criteria for the corresponding ex- 
periments with E. coli K-12 host-vec- 
tors if the major habitats of the given 
host-vector overlap those of E. coli. 
The habitats of other host-vector sys- 
tems should also be considered in rela- 
tion to containment. 
II- B-2. Return of DNA Segments to 
Non-HVl Host of Origin. Those pro- 
karyotes tht exchange genetic infor- 
mation with E. coli by known physio- 
logical processes will be exempt from 
these Guidelines if they appear on the 
"list of exchangers” set forth in Ap- 
pendix A (see Section I-E-4). For a 
prokaryote which can exchange genet- 
ic information [351 with E. coli under 
laboratory conditions but which is not 
on the list (Host A), the following type 
of experiment may be carried out 
under P-1 conditions without Host A 
having been approved as an HV1 host: 
DNA from Host A may be inserted 
into a vector and propagated in E. coli 
K-12 under P-1 conditions. Subse- 
quently, this recombinant DNA may 
be returned to Host A by mobilization, 
transformation, or transduction and 
may then be propagated in Host A in 
any desired vector under PI condi- 
tions. 
For a prokaryote which does not ex- 
change genetic information with E. 
coli (Host B). the following type of ex- 
periment may be carried out without 
Host B having been approved as an 
HV1 host: DNA from Host B may be 
inserted into a vector from a certified 
EK2 host-vector system and propagat- 
ed in E. coli K-12 under the appropri- 
ate containment conditions [see Sec- 
tion III-A-l-b-(2)l. Subsequently, this 
recombinant DNA may be returned to 
Host B and propagated in Host B 
under PI conditions. [431 
III- B-3. Non-HVl Systems. Contain- 
ment levels for other classes of experi- 
ments involving non-HVl systems may 
be approved by the Director, NIH. 
(See Sections IV-E-l-b-O)-(b), IV-E- 
l-b-(2)-(c) and IV-E-l-b-(3)-(bU 
III-C. Experiments with Eukaryotic 
Host-Vectors. 
III-C-1. Vertebrate Host-Vector Sys- 
tems. 144] (Summary given in Table 
IV). 
III-C-1-a. Polyoma Virus. 
IH-C-l-a-(l). Productive Virus-Cell 
Interactions. 
III-C-l-a-(l)-(a). Defective or whole 
polyoma virus genomes, with appropri- 
FEDERAl REGISTER. VOL 43. NO. 247— FRIDAY, DECEMBER 22, 1978 
[ 41 ] 
