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Federal Register / Vol. 45, No. 182 / Wednesday, September 17, 1980 / Notices 
unanimously recommended that the 
N1AID not initiate new Btudies to pursue 
the investigations as written in Protocol 
I. This judgment was based on a review 
of data that existed at the time of the 
Falmouth Workshop, a consideration of 
some newer published data and the 
results of contracts that N1AID was 
supporting. As written by the Falmouth 
Workshop Participants, the experiments 
were to be based on £ coli K-12 and 
this was judged to not be a fruitful 
experimental model. It was a clear 
consensus of the Croup that, based on 
the available data, it can be predicted 
that only negative results will be 
obtained and that limited resources 
could be better expended in other 
pursuits. 
Protocol II was designed to study the 
transmission of plasmids from £. coli K- 
12, including Chi 1776, into the normal 
intestinal flora utilizing a germ-free 
mouse model. The Working Group 
unanimously supported the view that 
the NIAID should not initiate new 
studies for the Protocol as it was written 
but to rely on the contracts to supply 
some additional data. This judgment 
was based on the same reasoning and 
data base considered for Protocol I. 
During discussion of the issues cited 
above it was obvious that the Working 
Group felt that a more beneficial use of 
monies would be to support the training 
of workers in good microbiological 
laboratory practices and to support 
research aimed at gaining a better basic 
scientific understanding of bacterial 
colonization and plasmid mobilization. 
Toward this last goal the Working 
Group strongly recommended that the 
NIAID support studies that could obtain 
quantitative data, expand scientific 
knowledge in an important area and 
which may prove useful at some future 
date for risk assessment. The Group felt 
that such studies should be performed 
directly in humans and employ wild 
type E. coli (not K-12). Strain HS 
containing pBR325 was suggested as a 
good initial combination and the study 
should be developed to assay for both 
survival and transfer to the indigenous 
flora. [These studies are now in their 
early stages and being performed by an 
NIAID contractor at the University of 
Maryland Medical School.] 
The full transcript of this meeting and 
a verbal report was made to RAC at 
their March 1980 meeting and the 
recommendations were approved by 
that Committee. 
c. NIAID identified two relevant grant 
applications and the National Advisory 
Allergy and Infectious Diseases Council 
supported selective payment of these 
projects for inclusion into the risk 
assessment program. 
One grantee will focus on the 
mechanisms that control human and 
animal gut flora. Of the four stated 
proposed aims of the research plan three 
relate to issues of importance to the NIH 
Recombinant DNA Risk Assessment 
Program. They are: (1) characterize and 
extend the application of anaerobic 
continuous flow cultures, (2) analyze the 
efficiency of plasmid and bacteriophage 
transfer, and (3) determine whether 
human microflora can be maintained in 
gnotobiotic mice and in anaerobic 
continuous flow cultures. The issue of 
mobilization of vector plasmids to the 
indigenous flora has always been a 
concern when considering the use of E. 
coli K-12 based host-vector systems. 
Most recently concern has been 
expressed over the potential for 
exchange of plasmids between the 
Enterobacteriaceae and the anaerobic' 
flora, principally members of the genus 
Bacteroides. This project has the 
capacity for filling a significant void in 
experimental data. 
A second grantee will be exploring a 
related issue which focuses on the 
molecular mechanisms of £. coli 
colonization of the intestine, specifically 
on the relative importance of plasmid or 
chromosomal determinants of 
colonization. 
At this point in time the majority of 
experiments using recombinant DNA 
technology employ host-vector systems 
based on £. coli K-12 and its plasmids 
or bacteriophages. Prominent among the 
scenarios raised early in the debate over 
use of this technology was the possible 
colonization of the intestinal tract by 
host-vector systems followed by various 
consequences depending on the 
elaboration of a product which would 
cause harm to the individual by either 
direct or indirect mechanisms. There 
now are a considerable number of 
studies describing the survival of 
various types of £. coli in the intestinal 
tracts of man and mice and they 
demonstrate a tremendous disparity in 
the survivability and colonization 
potential of such strains. A complete 
understanding of those factors that 
control survival and colonization may 
permit the development of both safer 
and more useful £. coli hosts in the 
future as well as perhaps provide data 
suggesting adjustments in the physical 
containment requirements of the NIH 
Guidelines governing use of this 
technology. 
3. Experiments testing £. coli K-12 
host-vector systems carrying 
recombinant DNA for virulence have 
been done by NIH scientists. The 
studies were designed to determine the 
pathogenicity and stability of shotgun 
clones of Saccharomyces DNA when 
used with both plasmid and 
bacteriophage vectors. In this 
experimental model, which utilized 
mice, there was no evidence that the 
presence of segments of the entire yeast 
genome altered the inherently low 
pathogenicity of £. coli K-12 in any way. 
4. A Workshop was convened to 
consider two areas [within the NIH 
Program] recommended by the Risk 
Assessment Subcommittee of RAC. This 
Workshop was designed to define the 
scientific issues and assess the potential 
risks of: (a) Possible direct adverse 
effects of hormone-producing strains of 
*£. coli K-12, and (b) the possible 
occurrence of autoantibodies or 
autoreactive cells due to the production 
of eukaryotic polypeptides (including 
hormones) by £. coli 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 £. coli K-12 has 
apparently lost those known 
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 
exists 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 
was distributed to all participants and 
also the RAC for their review at the June 
1980 meeting. In addition to the report of 
the Director, NIAID. Drs. Setlow and 
Campbell prepared a separate report 
and the RAC Risk Assessment 
Subcommittee reported their analysis of 
the Workshop. 
NIAID Plans in implementing the four 
prime recommendations was influenced 
by the RAC discussions. At this time we 
propose to respond to the 
recommendations as follows: 
a. It was recommended that 
experiments of the folowing general type 
should be performed: “£. coli cells 
carrying recombinant NDA and making 
a mammalian protein are introduced 
into living animals under conditions 
where they cause genuine infection (e.g. 
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