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Federal Register / Vol. 46, No. Ill / Wednesday, June 10. 1961 / Notices 
hormones) by E. coll K-12 should they 
colonize higher organisms. 
In the charge to the Workshop it was 
noted that these potential risks were 
under consideration because there is 
still debate over the degree of possible 
risk, even though E. coli K-12 has 
apparently lost those characteristics 
that are required for colonization of the 
normal intestinal tract. 
The purpose was to decide whether 
the two specific possibilities are valid 
scientific hypotheses if such an event 
did occur; and if valid, to determine if 
sufficient experimental data already 
exist to make a final judgment 
concerning possible risk. If data were 
insufficient, the participants were asked 
to outline those types of studies 
necessary to develop the definitive 
information on these issues. 
The meeting brought together 92 
scientists from the fields of immunology, 
endocrinology, physiology, 
microbiology, infectious diseases and 
other appropriate disciplines. The 
Chairmen’s Summary and a full 
transcript of the Final Plenary Session 
were distributed to all participants and 
also to the RAC for their review at the 
June 1980 meeting. In addition to the 
report of the Director, NIAID, Drs. 
Setlow and Campbell, members of the 
RAC, prepared a separate report and the 
RAC Risk Assessment Subcommittee 
alos reported their analysis of the 
Workshop. 
NIAID plans in implementing the four 
prime recommendations were influenced 
by the RAC discussions. At this time we 
propose to respond to the 
recommendations as follows: 
(1) It was recommended that 
experiments of the following general 
type should be performed: “E. coli cells 
carrying recombinant DNA and making 
a mammalian protein are introduced 
into living animals under conditions 
where they cause genuine infection (e.g., 
an abscess^, with subsequent monitoring 
for breakdown of immune tolerance.” 
This subject stimulated the most 
discussion dining the fmal plenary 
session of the Workshop. Tlie sentiment 
was to acquire the data as a matter of 
scientific interest rather than to answer 
the risk assessment question. An RFP . 
was developed to solicit proposals from 
interested investigators. Immunologists 
from the NIAID staff designed the 
experimental protocol for the workscope 
and other specifications of the study. 
(2) Additional information was 
requested on the handling and 
absorption of polypeptides from the 
normal and pathologic colon, and the 
potential effects of synthetic peptides on 
the bowel itself. 
We have confirmed that this is an 
issue about which there are little direct 
data. The Workshop participants opined 
that although their own calculations 
revealed that only extremely small 
amounts of hormones would be 
produced under the most ideal 
conditions, data should still be sought 
for potential future needs. The issue of 
the potential effects of synthetic 
peptides on the bowel itself is different 
from that of hormones, and that 
discussion was centered on interferon 
and its direct effect on cells. 
Although this will be a difficult study 
to initiate because of the technical 
difficulties as well as the process for 
approval of research in human subjects, 
NIAID is considering solicitation of 
grant proposals through a Request for 
Applications (RFA). The study will have 
as its objective a determination of the 
fate of peptide hormones when 
deposited in the distal small intestine 
and large intestine of humans. These 
sites are relevant to the production of 
hormones by recombinant DNA 
technology because they represent the 
regions of colonization by E. coli. 
(3) It weis suggested that additional 
information is needed on the potential 
transfer of plasmids to anaerobic 
bacteria from £. coli. This 
recommendation differs ffom the 
findings of the ad hoc groiq) convened in 
August 1979 vdiich RAC reviewed at the 
march 1980 meetmg. That expert Group 
noted that by studying the epidemiology 
of plasmids the conjugative plasmids of 
E. coli are not found within the 
anaerobic flora of the gut. The uniform 
use of DODconjugative plasmids in rDNA 
research furdim reduces the likelihood 
of transfer 
It is likely that the requested data will 
be forthcoming from NIH (NIAIDJ 
supported stu^s on plasmids d^ugh 
the regular grant-supporter research 
programs and that new initiatives are 
not imeded. In fact, the NIAID Advisory 
Council identified a grant application 
(AI 15279) for selective payment that 
addressed some aspects of this issue; 
this action will initiate the acquisition of 
the data at an early date. There is no 
plan to develop a RFA at this time but 
NIAID staff will continue to identify all 
appropriate iimoming applications as 
potential sources of data. 
(4) It was recommended the NIH 
communicate with the Centers for 
Disease Control (CDC) about possible 
types of health surveillance for workers 
using recombinant DNA. 
A CDC representative was present at 
the Pasadena meeting and discussed 
some approaches to this issue. 
Furthermore, representatives of the 
National Institute for Occupational 
[ 138 ] 
Safety and Health and the Occupational 
Safety and Health Administration were 
also present and therefore, are cognizant 
of the Workshop recommendation. All 
of these agencies are members of The 
Industrial Practices Subcommittee of 
The Federal Interagency Advisory 
Committee on Recombinant DNA 
Research. 
e. NIH scientists have continued a 
further evaluation of the biological 
activity of polyoma virus DNA cloned in 
E. coli host-vector systems. It had been 
reported (Science 203: 883-892, 1979), 
when the Final Plan was initially 
published, that polyoma DNA was 
noninfectious when cloned in EK2 
plasmid and bacteriophage host-vector 
systems. 
As a further step in evaluating the 
biologic activity of the polyoma-plasmid 
and polyoma-lambda recombinant DNA 
host-vector systems, the scientists tested 
the ability of the EK2 systems containing 
polyoma DNA to induce tumors in 
suckling hamsters. 
These animals are highly sensitive to 
tumor induction by polyoma virus and in 
some cases even subgenomic fragments 
of viral DNA can induce tumors in them. 
The results (Science 205: 1140-T142, 
1979) indicated that inoculation of 
suckling hamsters with 2 x 10* live cells 
of E. coli K-12 strain Chi 1776 carrying 
the complete genome of polyoma virus 
in a recombinant plasmid, failed to 
induce tumors in any of 32 recipients. 
Also, lambda phage DNA and particles 
with a monomeric insert of polyoma 
DNA did not induce tumors. Purified j 
recombinant plasmid DNA, as well as [ 
phage particles and DNA containing a | 
head-to-tail dimer of polyoma DNA, |j 
showed a low degree of oncogenicity, i. 
comparable to that of polyoma DNA L 
prepared from mouse cells. These J 
findings support the previous J 
conclusions, based on infectivity assays [J 
in mi(x, that propagation of polyoma 
virus DNA as a component of fi 
recombinant DNA molecules in E. coli i 
K-12 reduces its biologic activity many |f|i 
orders of magnitude relative to the virus i| 
itself. fir 
Currently both the infectivity and ® 
tumorigenicity experiments are beii^ 
extended. In one, E. coli K-12 (EKl) 
containing recombinant plasmids ^ 
consisting of two copies of polyoma 
virus DNA and one copy of pBR322 N 
plasmid DNA are being incoculated into 1 
newborn hanaters and weanling mice, it p 
is also planned to test lambdaphage ' 
lysogens [E. coli K-12 containing one or j'i 
two copies of a lambda bacteriophage- 
polyoma virus DNA recombinant 
integrated into the bacterial ] 
chromosome) by inoculation into 
1 
I 
1 
