60088 
NOTICES 
DNA fragment under the specified 
nonpermissive laboratory conditions 
designed to represent the natural envi- 
ronment. either by survival of the 
original host or as a consequence of 
the transmission of the cloned DNA 
fragments." 
A concern expressed by the commen- 
tators is that plasmids containing re- 
combinant DNA might be transferred 
to other, wild-type organisms in the 
gut. Some appropriate risk-assessment 
studies concerning this possibility are 
being carried out. However, the Fal- 
mouth report and tests of EK2 host- 
vector systems present the following 
data to indicate that the probability of 
such transfer of the plasmids is ex- 
tremely low: 
1. H.W. Smith, looking specifically 
for transfer of a conjugative plasmid 
from E. coli K-12 to normal gut flora, 
could find no evidence that transfer 
occurred (Falmouth report, pages 655- 
660). For biological containment to be 
breached, transfer of a conjugative 
plasmid to E. coli K-12 would have to 
occur to be followed by transfer of the 
poorly mobillzable recombinant plas- 
mid from E. coli K-12 to other organ- 
isms. (Conjugative plasmids them- 
selves are not allowed to be used as 
vectors. ) 
2. Gene transfer in vivo greatly in- 
creases with colonization by both 
donor and recipient organisms, and 
colonization by E. coli K-12 in general 
and xl776 in particular is very rare. 
3. Mobilization of the plasmids used 
in recombinant DNA experiments is 
extremely low. even under optimal in 
vitro conditions. 
4. Transfer of the recombinant-con- 
taining vector would require a trtpar- 
ental mating in rtvo. which even 
under the most favorable of in vitro 
conditions is not a high-frequency 
event. Such transfer has not been ob- 
served in vivo. 
5. One commentator suggests that 
transduction might be an Important 
mode of gene transfer. "Generalized 
transduction" by phage lambda does 
not occur. Generalized transduction 
by phage PI Is a low-frequency event 
(less that 1/10* Infected organisms are 
transduced for a particular marker) 
under the very best of in uifno condi- 
tions. 
Additional information on plasmid 
transfer may be found In the Environ- 
mental Impact Assessment (Federal 
Register. July 28. 1978. p. 33123) and 
the previous October 1977 Environ- 
mental Impact Statement, both of 
which consider the possibility of trans- 
fer of foreign DNA from E. coli K-12 
as well as the ability of E. coli K-12 to 
survive and spread in nature. As noted 
in both these documents, the maxi- 
mum probability for transmission of 
nonconjugatlve plasmid vectors from 
E. coli K-12 was estimated at "less 
that 1 to 1016 K-12s surviving per day 
in the intestine of warm-blooded ani- 
mals. The probability is even lower in 
sewers, sewage treatment plants, and 
waterways." The EIA further states 
that E. coli K-12 survives poorly and 
is outcompeted by wild-type enteric 
bacteria. 
The concern expressed by the com- 
mentators about the possibility of au- 
toimmune disease will only prove true 
if a series of events occurs. The very 
low probability of the establishment 
of E. coli K-12 containing recombin- 
ant DNA in the Intestinal flora is dis- 
cussed above. In addition, the inserted 
eukaryotic gene must be transcribed, 
translated, and transported to a place 
where it can Induce an Immune re- 
sponse. These steps are highly unlike- 
ly to occur. 
RISK ASSESSMENT 
N1H Is supporting a number of risk- 
assessment activities. The Rowe- 
Martin polyoma experiments are dis- 
cussed elsewhere in this document. In 
addition, intramural NIH scientists are 
collaborating with scientists from 
other Institutions testing the virulence 
in mice of E. coli K-12 containing 
"shotgun clones" of recombinant DNA 
derived from other species. 
A number of contractors of the Na- 
tional Institute of Allergy and Infec- 
tious Diseases are testing the biologi- 
cal containment capabilities of various 
derivatives of E. coli K-12. Some are 
testing the survival and capacity of 
plasmid and phage vectors to be trans- 
mitted to secondary bacterial hosts in 
the gastrointestinal tract of mice and 
man. Others are assessing these pa- 
rameters in model sewage treatment 
systems and in situations simulating 
accidential spills and other types of ac- 
cidental release of the organisms from 
experimental procedures. 
In addition, investigators proposing 
systems to be certified by NIH as HV1 
or HV2 must perform certain specified 
tests on these systems relevant to 
their survival and transmission proper- 
ties. It is also anticipated in the event 
that investigators request exceptions 
to the prohibitions for specified 
clones. NIH will request substantial 
risk assessment experiments to be per- 
formed to evaluate claims of safety. 
SPECIFIC CONCERNS 
In addition to their general remarks 
about the experimental section of the 
Guidelines, many commentators raised 
questions about the containment 
levels set for specific experiments. 
Others suggested clarifying language 
for certain sections. I have taken all of 
these recommendations under consid- 
eration. In some Instances I have con- 
curred and the Guidelines reflect the 
change. In others I have decided not 
to act or have deferred actions pend- 
ing futher analysis and discussion by 
the Recombinant Advisory Commit- 
tee. In all cases I have attempted to 
respond and to explain the decision. 
Section HI-A-1: Shotgun Experiments 
Comments from respondents on this 
section of the Guidelines reflected dia- 
metrically opposed points of view. 
Some commentators questioned the 
rationale for lowering of containment 
levels for shotgun experiments. 
Other correspondents took a differ- 
ent view and requested that NIH fur- 
ther reduce the containment level for 
shotgun experiments. One advanced 
the argument that "these pieces of 
DNA in E. coli cannot be more danger- 
ous than their original source.” 
I have decided to retain those levels 
of physical and biological containment 
described in the proposed revised 
Guidelines. I believe the specified con- 
tainment levels represent a prudent, 
albeit most conservative, response to 
the hypothetical hazards. Rationale 
for them is presented in the July 28. 
1978. Decision document and Environ- 
mental Impact Assessment. 
Risk analysis by NIH is continuing. 
One important area of analysis in- 
volves the appropriateness of higher 
containment levels for shotgun experi- 
ments. with the tremendous dilution 
of potentially harmful genes, in con- 
trast to purified clones that have been 
"engineered" for efficient transcrip- 
tion and translation of DNA Inserts. 
Section lII-A-l-a. Eukaryotic DNA Re- 
combinants Including Primates. 
Other Mammals, and Birds 
Twelve commentators, the largest 
number of comment on a single issue, 
wrote to express their views on the 
limitation of P2 + EK2 under the pro- 
posed revised Guidelines for the doing 
in E. coli K-12 of shotgun DNA from 
primates, other mammals, and birds. 
All requested that the option of 
P2-t-EK2 or P3 + EK1 be offered. 
One group argues that to restrict 
cloning of these classes of DNA to P2 
+ EK2 is - inconsistent, since many 
viral genomes could be cldhed at 
either P2 + EK2 or P3 + EK1. Other 
respondents pointed out that use of P3 
+ EK1 conditions would provide ade- 
quate containment and would permit 
the inclusion of lysogenic lambda sys- 
tems. which cannot be employed in an 
EK2 system. One stated that propa- 
gating DNA fragments in EK1 host- 
vector systems permits a 5-10 times 
greater yield of DNA than in an EK2 
system. “Thus the advantage of EK2 
containment should be weighed 
against the necessity of handling 
much larger volumes of cells.” 
I referred this matter to the RAC at 
their October 30-31, 1978, meeting. 
The RAC advised that the phrase “or 
P3 + EK1” be added to Section III-A- 
l-a-(3) (i.e.. for DNA from birds) and 
FEDERAL REGISTER. VOC 43. NO. 247— fRIOAY, DECEMBER 22. 1V7R 
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