NOTICES 
27907 
was urged by some that the prohibition 
be broadened to include experiments that 
result in resistance to any antibiotic, ir- 
respective of its use in medicine or agri- 
culture. Consideration of such a sugges- 
tion must take into account that anti- 
biotic resistance occurs naturally among 
bacteria, and that resistance is a valu- 
able marker in the study of microbial 
genetics in general, and recombinants 
in particular. 
In view of these concerns, however, the 
Recombinant Advisory Committee was 
asked to reconsider carefully the prohi- 
bition and related sections concerning 
antibiotic resistance. The committee 
noted that the prohibition relating to 
drug resistance was intended to ban 
those experiments that could compro- 
mise drug use in controlling disease 
agents in veterinary as well as human 
medicine and this is now clearly stated. 
In the draft guidelines there were two 
statements concerning resistance to 
drugs which related to experiments with 
E. coli. The statements appeared to allow 
experiments that would extend the range 
of resistance of this bacterium to thera- 
peutically useful drugs and disinfectants, 
and thus seemed to be in conflict with 
the general prohibition on such research. 
There are numerous reports in the scien- 
tific literature indicating that E. coli can 
acquire resistance to all antibiotics 
known to act against it. Since E. coli ac- 
quires resistance naturally, the prohibi- 
tion directed against increasing resist- 
ance does not apply. The ambiguous 
statements have been deleted from the 
present guidelines. On the other hand, 
new language has been inserted in the 
section dealing with other prokaryote 
species to set containment levels for per- 
mitted experiments. 1 
2. The Recombinant Advisory Com- 
mittee was also asked to clarify whether 
the prohibition of use of DNA derived 
from pathogenic organisms (those classi- 
fied as 3, 4, and 5 by the Center for 
Disease Control, USPHS) also included 
the DNA from any host infected with 
these organisms. The committee ex- 
plained that this prohibition did extend 
to experiments with cells known to be so 
infected. To avoid misunderstanding, the 
prohibition as now worded includes such 
cells. In addition, the prohibitions have 
been extended to include moderate-risk 
oncogenic viruses, as defined by the Na- 
tional Cancer Institute, and cells known 
to be infected with them. 
3. Two other issues relating to the sec- 
tion on prohibited experiments were 
raised by Roy Curtiss III, Ph.D., Profes- 
sor, Department of Microbiology, Uni- 
versity of Alabama School of Medicine, 
Birmingham, who is a member of the Re- 
combinant Advisory Committee. Dr. 
Curtiss noted that for the class of expe- 
riments prohibited on the basis of pro- 
duction of highly toxic substances, only 
1 Specifically, experiments that would ex- 
tend resistance to therapeutically useful 
drugs must use P3 physical containment 
plus a host-vector comparable to EK1, or 
P2 containment plus a host-vector compa- 
rable to EK2. 
substances from micro-organisms were 
cited as examples. He suggested that 
other examples be included, such as ven- 
oms from insects and snakes. The com- 
mittee approved the suggestion and I 
concur. 
In the proposed guidelines, release of 
organisms containing recombinant E^NA 
molecules into the environment was pro- 
hibited unless a series of controlled tests 
had been done to leave no reasonable 
doubt of safety. Dr. Curtiss felt that the 
guidelines should provide greater spec- 
ificity for testing and should include 
some form of review prior to release of 
the organism. I have decided that the 
guidelines should, for the present, pro- 
hibit any deliberate release of organisms 
containing recombinant DNA into the 
environment. With the present limited 
state of knowledge, it seems highly un- 
likely that there will be in the near 
future, any recombinant organism that is 
universally accepted as being beneficial 
to introduce into the environment. When 
the scientific evidence becomes available 
that the potential benefits of recom- 
binant organisms, particularly for agri- 
culture, are about to be realized, then 
the guidelines can be altered to meet the 
needs for release. It is most important 
that the potential environmental impact 
of the release be considered. 
IV. PERMISSIBLE EXPERIMENTS: E. COLIK-12 
HOST-VECTOR SYSTEMS 
The continued use of E. coli as a host 
has drawn considerable comment, in 
eluding some suggestions that its use be 
prohibited presently or within a specified 
time limit. It should be stressed that the 
use of E. coli as detailed in the guidelines 
is limited to E. coli K-12, a strain that 
has been carried in the laboratory for 
decades and does not involve the use of 
any strain of E. coli that is freshly iso- 
lated from a natural source. E. coli K-12 
does not usually colonize the normal 
bowel, even when given in large doses, 
and exhibits little if any multiplication 
while passing through the alimentary 
canal. For years it has been the subject 
of more intense investigation than any 
other single organism, and knowledge of 
its genetic makeup and recombinant be- 
havior exceeds greatly that pertaining to 
any other organism. I believe that be-^ 
cause of this experience, E. coli K-12 will 
provide a host-vector system that is safer 
than other candidate microorganisms. 
NIH recognizes the importance of sup- 
porting the development of alternative 
host-vector systems (such as B. subtilis, 
which has no ecological niche in man) 
and will encourage such development. It 
should be noted, however, that for each 
new host-vector system, the same ques- 
tions of risk from altered properties at- 
tendant upon the presence of recom- 
binant genes will apply as apply to E. coli. 
NIH does not believe it wise to set a 
time limit on replacement of E. coli sys- 
tems by other organisms. 
There were specific suggestions con- 
cerning the three levels of biological con- 
tainment prescribed for use of E. coli 
K-12 host-vectors. Some commentators 
requested a more detailed explanation of 
the adequacy of protection for labora- 
tory personnel with the first level of con- 
tainment (EK1) .* Sections of the guide- 
lines dealing with physical containment 
and roles and responsibilities now specify 
the need for safety practices and acci- 
dent plans. 
For the second level of containment 
(EK2), it is required that a cloned DNA 
fragment be contained in a host-vector 
system that has no greater than a 10 ® 
probability of survival in a nonpermis- 
sive or natural environment. It was sug- 
gested that the selection of this level of 
biological containment and the appro- 
priate tests for verification be more fully 
explained in the guidelines. The commit- 
tee, in responding to a request for fur- 
ther examination of this point, reviewed 
at considerable length the testing for an 
EK2 system and recommended certain 
modifications. We have accepted the 
committee’s new language that better ex- 
plains testing of 'survival of a genetic 
marker carried on the vector, preferably 
on an inserted NDA fragment. 
Possible tests to determine the level of 
biological containment afforded by these 
altered host-vector systems are outlined 
in this section. Because this is such a new 
area of scientific research and develop- 
ment, however, it is inappropriate to 
standardize such testing at the present 
time. Standards will gradually be set as 
more experience with EK2 host-vector " 
systems is acquired. The committee, for 
example, during its April 1976 meetings 
gave its first approval to an EK2 host- 
vector system. What is necessary is that 
new and more effective tests be devised 
by investigators, and this effort is very 
likely to occur under the present guide- 
lines. For example, one task recognized 
by the committee is to clarify how sur- 
vival of the organism and the cloned 
DNA should be. defined in terms of tem- 
perature, medium, and other variables. 
It is also very important to note here 
that the stringent requirements set by 
the committee for EK2 biological con- 
tainment jeopardize considerably the 
capacity of such crippled organisms to 
survive and replicate even under permis- 
sive laboratory conditions. More experi- 
ence will be required to determine 
whether EK2 containment will permit 
'some lines of important research to be 
followed. 
Several commentators suggested that 
methods and procedures to confirm an 
3 The EKl system presently consists of a 
battery of different vectors and of E. coli 
K-12 mutants, all of which afford a consid- 
erable degree of biological containment. The 
diversity of vectors and of host mutants in 
this battery has permitted a wide range of 
important scientific questions to be attacked. 
For example, the availability of different vec- 
tors with cleavage sites for different restric- 
tion endonucleases have increased the kind 
of DNA segments that can be cloned. By con- 
trast, the first EK2 host-vector systems are 
only now being considered by the Recom- 
binant Advisory Committee. While NIH is 
supporting the development of more EK2 
host-vector systems, it is not expected that a. 
battery equivalent to that available for the 
EKl system will be certified by the Recom- 
binant Advisory Committee in the near 
future. 
FEDERAL REGISTER, VOL. 41, NO. 131 — WEDNESDAY, JULY 7, 1976 
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