• The potential biohazard associat- 
ed with the DNA of the cell or micro- 
organism that serves as the DNA 
source (e.g.. genes for toxin produc- 
tion), 
• The potential biohazard associat- 
ed with the vector that serves to trans- 
mit the source DNA to a recipient host 
cell, 
• The ability of the vector to sur- 
vive in natural environments or habi- 
tats, 
• The kinds and number of organ- 
isms that are susceptible to infection 
by the vector or recipient, 
• The potential biohazard of the re- 
cipient host cell that serves to repli- 
cate the recombinant DNA molecules, 
• The ability of the host ceil to sur- 
vive in natural environments or habi- 
tats, 
• The ability of the host cell to 
transmit the recombinant DNA mole- 
cule to other cells capable of surviving 
in natural environments or habitats, 
• The potential of the host cell to 
obtain the source DNA by natural 
means, and 
• The evolutionary relatedness of 
the DNA source to humans. The po- 
tential dangers are considered to in- 
crease as the organism providing the 
source DNA approaches humans phy- 
logenetically. For example, source 
DNA from primate cells is considered 
to be more potentially dangerous than 
that from prokaryotes. 
In an effort to present more clearly 
the changes in containment levels pro- 
posed by the PRG-RAC, a table was 
prepared for use at the December 1977 
DAC meeting' which compared the 
containment levels in the original 
(current) Guidelines and the PRG- 
RAC. This table has now been expand- 
ed with a third column to show the 
containment levels of the PRG-NIH 
(Appendix A). The remainder of this 
section summarizes a number of the 
proposed changes, comparing the cur- 
rent Guidelines with the PRG-RAC. 
(Not all the changes are discussed 
here; certain items in which the FRG- 
NIH differs significantly from the 
PRG-RAC are discussed below under 
"Alternatives: Public Commentators.” 
The numbers in parentheses indicate 
the line numbers on the table to 
which the proposed revision applies. 
• Several categories of experiments 
(primarily those involving prokaryotes 
that are exchangers of genetic infor- 
mation with E. coli in nature) would 
no longer be subject to the Guidelines 
because of the changes in the defini- 
tion. (See lines 20, 21. 27, 46, and 47.) 
• Shotgun experiments involving 
birds and mammals other than pri- 
mates were the subject of lowering of 
containment from P3+EK2 to 
P2+EK2. This action reflects the in- 
creased confidence of the RAC in the 
NOTICES 
EK2 host-vector systems. (See lines 4 
and 5.) 
• Another category which the RAC 
decided was in need of revision was 
that pertaining to the cloning of DNA 
from organisms producing a toxic 
product. This was clarified in the 
PRG-RAC by setting containment 
levels according to whether polypep- 
tide toxins are produced. Polypeptide 
toxins are specified, since they might 
be encoded by a single gene or cluster 
of genes, whereas toxins of other 
chemical structure would not. (See 
lines 8, 9, 10, 11, 12, 16. 17, and 19.) 
•For several categories of experi- 
ments, it is proposed that the investi- 
gator have the option of working at 
P2 + EK1 or P1+EK2 rather than the 
P2 + EK1 levels previously specified. 
This again reflects confidence in the 
EK2 systems. (See lines 7, 14, and 15.) 
•The lowering of containment for 
experiments with rigorously charac- 
terized clones free of harmful genes 
has been revised to provide more flexi- 
bility. Under the PRG-RAC, Institu- 
tional Biosafety Committees (IBC’s) 
would be able to lower containment by 
a single level. The IBC should consider 
the purity, extent of characterization, 
and harmlessness of the clone before 
allowing such lowering. Reduction of 
containment by more than one level 
would require approval by NIH. Under 
the 1976 Guidelines, NIH had the 
option of lowering containment down 
to certain specified levels or not lower- 
ing it at all. The PRG-RAC would 
allow NIH to consider all available 
data for the clone and to lower con- 
tainment accordingly. 
Alternatives: Public Commentators 
During the development of the origi- 
nal Guidelines in early 1976, Part III 
w T as the section most commented 
upon. There was also much comment 
on this section in the PRG-RAC. 
Many of the issues raised, however, 
did not address the specific proposals 
to alter the containment levels but 
more general topics, such as the need 
for a rationale for each of the 
changes. 
Rationale 
A number of commentators asked 
that the rationale for the classifica- 
tion of permissible experiments be elu- 
cidated. Concern was expressed that: 
• It was difficult for a layman to un- 
derstand the entire section on permis- 
sible experiments because the ratio- 
nale is not detailed in either the cur- 
rent Guidelines or the PRG-RAC; 
• The whole categorization is de- 
pendent upon investigatorial confi- 
dence rather than documented fact; 
and 
• The quantification of containment 
levels, the means whereby they were 
33117 
decided, and the rationale for raising 
and lowering them is not clear. 
In general, the classification is some- 
what arbitrary. It depends in large 
part on the scientific judgment of the 
RAC rather than on demonstrable 
risk, because there is in fact no actual 
scientific evidence that there is a 
hazard in any recombinant DNA ex- 
periment. The rationale for classifying 
recombinant DNA experiments at sev- 
eral containment levels was explained 
in the "Decision of the Director. Na- 
tional Institutes of Health, To Release 
Guidelines for Research on Re- com- 
binant DNA Molecules,” which was 
published along with the current 
Guidelines in the Federal Register on 
July 7, 1976 (page 27908), as follows: 
The guidelines assign different levels of 
containment for experiments in which DNA 
from different sources is to be introduced 
Into an E. coli K-12 host-vector system. The 
variation is based on both facts and assump- 
tions. There are some prokaryotes (bacteria) 
that constantly exchange DNA with E. coli. 
Here it is assumed that experimental condi- 
tions beyond those obtained in careful, rou- 
tined microbiology laboratories are super- 
fluous. because any exchange experiments 
have undoubtedly been performed already 
in nature. 
In every instance of artificial re- combina- 
tion, consideration must be given to the pos- 
sibility that foreign DNA may be translated 
into protein (expressed), and also to the pos- 
sibility that normally repressed genes of the 
host may be expressed and thus change, un- 
desirably. the characteristics of the cell. It 
is assumed that the more similar the DNAs 
of donor and host, the greater the probabil- 
ity of expression of foreign DNA, or of pos- 
sible derepression of host genes. In those 
cases where the donor exchanges DNA with 
E. coli in nature, it is unlikely that recom- 
bination experiments will create new genet- 
ic combinations. When prokaryote donors 
not known to exchange DNA with E. coli in 
nature are used, however, there is a greater 
potential for new genetic combinations to be 
formed and be expressed. Therefore it is re- 
quired that experiments involving prokaryo- 
tic DNA from a donor that is not known to 
exchange DNA with E. coli in nature be car- 
ried out at a higher level of containment. 
Recombination using prokaryotic DNA from 
an organism known to be highly pathogenic 
is prohibited. 
There are only limited data available con- 
cerning the expression of DNA from higher 
forms of life (eukaryotes) in E. coli (or any 
other prokaryote). Therefore, the contain- 
ment prescriptions for experiments insert- 
ing eukaryotic DNA into prokaryotes are 
based on risks having quite uncertain prob- 
abilities. 
On the assumption that a prokaryotic 
host might translate eukaryotic DNA, it is 
further presumed that the product of that 
foreign gene would be most harmful to man 
if it were an enzyme, hormone, or other pro- 
tein that was similar (homologous) to pro- 
teins already produced by or active in man. 
An example is a bacterium that could pro- 
duce insulin. Such a "rogue'’ bacterium 
could be of benefit if contained, a nuisance 
or possibly dangerbus if capable of surviving 
in nature. This is one reason that the 
higher the phylogenetic order of the eukar- 
FEDERAL REGISTER, VOL 43, NO. 144— FRIDAY, JULY 28, 1978 
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