36054 
Federal Register / Vol. 49, No. 179 / Thursday, September 13. 1984 / Notices 
Defense (DOD) and the Arms Control and 
Disarmament Agency (ACDA) asking for an 
Arms Control Impact Statement (ACIS) for 
the Shiga-like toxin experiment and all other 
recombinant DNA related research presently 
being conducted by the DOD and agencies 
and institutions related to the DOD. 
Mr. Rifkin contended that: 
. . . experiments like the proposed Shiga 
experiment being considered today are 
technologies ‘with potential military 
application or weapons systems applications' 
and therefore are subject to section 36(a)(3) 
of the Arms Control and Disarmament Act. It 
is my understanding that the DOD and the 
Arms Control and Disarmament Agency are 
in non-compliance with this act in regard to 
the Shiga experiment and other biological 
experiments involving recombinant DNA 
technology. Therefore, it would be improper 
for the RAC to consider this proposed 
experiment to clone a Shiga toxin until such 
time as a weapons impact statement covering 
this experiment and or the category of 
experiments it is part of, is forthcoming from 
the Arms Control and Disarmament Agency. 
He further stated that: 
... 1 urge this committee to place a 
moratorium on all further authorizations of 
DOD related toxin experiments until such 
time as this committee engages in a full 
dialogue with all other interested agencies in 
the Executive and Congressional branches 
relative to the potential uses of this 
technology for biological warfare purposes. 
Dr. Jay P. Sanford, President, USUHS, 
in a letter dated May 15, 1984, wrote 
that: 
At the RAC meeting on 6 February 1964, 
Mr. Jeremy Rifkin, President, Foundation on 
Economic Trends, submitted a press release, 
'Arms Control Impact Statement requested 
for DoD Toxin Experiment.’ The statute from 
which he quoted is as follows: 22 U.S.C. 2576, 
'(a) In order to assist the Director in the 
performance of his duties with respect to 
arms control and disarmament policy and 
negotiations, any Government agency 
preparing any legislative or budgetary 
proposal for — ' ‘(3) any other programs 
involving technology with potential military 
application or weapon systems which such 
Government agency or the Director believes 
may have a significant impact on arms 
control and disarmament policy or 
negotiations shall, on a continuing basis, 
provide the Director with full and timely 
access to detailed information, in accordance 
with the procedures established pursuant to 
section 2575 of this title, with respect to the 
nature, scope and purpose of such proposal.' 
The Department of Defense does not 
believe that the O'Brien-Holmes proposal 
(program) has (would have) a significant 
impact on arms control and disarmament 
policy or negotiations. 
As a technical point, the current O'Brien- 
Holmes request to RAC was neither a 
legislative nor budgetary proposal. Their 
original budgetary proposal was reviewed 
and approved by the National Institute of 
Allergy and Infectious Diseases (AI 20148). 
A fact sheet concerning the Shiga-like 
toxin proposal was distributed by Ms. 
Patricia Campbell, Public Affairs 
Officer, USUHS, at the June 1 , 1984, 
RAC meeting. The fact sheet offered the 
following information: 
Randall Holmes, Ph.D., M.D., Chairman and 
Professor, and Alison O'Brien, Ph.D., 
Associate Professor, Department of 
Microbiology, Uniformed Services University 
of the Health Sciences (USUHS), are studying 
a substance (toxin) produced by a bacterium 
(Escherichia coli). This bacterium is one of 
many bacteria that can cause diarrhea in 
humans and animals. Diarrheal diseases are 
the number one killer of children in third 
world countries. 
The purpose of the O’Brien/Holmes 
research is to determine whether the toxin 
(called Shiga-like toxin) has a role in diarrhea 
due to E. coli. Such information may help in 
the development of vaccines against such 
diarrheas. 
I-A-3. Excerpts From the Minutes of the 
May 11, 1984, Meeting of the RAC 
Working Group on Toxins. 
The NIH convened a meeting of the 
RAC Working Group on Toxins on May 
11, 1984, to review available scientific 
data with regard to Drs. O'Brien's and 
Holmes' April 4, 1984, request. The 
following excerpts from the minutes of 
that meeting indicate the basis for the 
recommendations made by the working 
group to the RAC at the June 1 , 1984, 
RAC meeting. 
In regard to the first request in the 
April 4, 1984, letter from Drs. O’Brien 
and Holmes, the following discussion 
ensured: 
Dr. Levine said he had participated in 
formulating Appendix F [to the Guidelines). 
In his opinion, cloning of the Shiga toxin gene 
was paced under Section F-l which requires 
NIH review and approval because the 
working group at that time did not possess 
sufficient data to evaluate pertinent 
questions. Dr. Levine thought pertinent dpta 
were now available. He cited the data 
generated through feeding experiments with 
140 human volunteers. These volunteers were 
fed Shiga toxin-producing Shigella which 
lacked invasive characteristics. No disease 
symptoms were observed in 139 individuals: 
in one individual the strain reverted to an 
invasive form and the volunteer developed 
shigellosis. He also cited the evidence 
generated by Branham, Dack, and Riggs 
which shows that large amounts of Shiga 
toxin instilled directly into monkey intestinal 
pouches has no effect. Dr. Levine said the 
containment specified for cloning Shiga toxin 
in E. coli K-12 should be lowered on the 
basis of these data. 
Dr. Gottesman calculated that in a worst 
case scenario, 10 * engineered E. coli with the 
Shiga toxin gene on a high expression, high 
copy number plasmid might produce one 
milligram of toxin in the human gut. Dr. Gill 
calculated that this amount is roughly 
equivalent to approximately 14,000 lethal 
doses for humans when the toxin is 
administered parenterally. He said that 
100. 000 times more tetanus toxin is required 
enterally to kill animals than is required 
parenterally. Dr. Gottesman noted that 
Branham. Dack. and Riggs had administered 
20.000 lethal doses to monkey intestinal 
pouches with no observed effect. 
Dr. Levine moved that the working group 
recommend to RAC that cloning of the Shiga 
toxin gene in E coli be permitted at P3 
containment. Dr. Gill seconded the motion. 
By a vote of five in favor, none opposed, and 
no abstentions, the working group accepted 
the motion. 
In regard to the second item in the 
request in the April 4, 1984, letter from 
Drs. O'Brien and Holmes, the following 
discussion ensued: 
Dr. Levine suggested that under the 
conditions specified by Dr. O'Brien, P2 
containment would be acceptable. He felt 
highly toxigenic isolates should be handled 
under conditions equivalent to P2 in the 
clinical laboratory. 
Dr. Gill said some novel considerations 
arise when a gene is cloned in a new genetic 
background. These include: (1) Has the 
potential for genetic transfer of the gene to 
other organisms been increased; and (2) how 
does the cloning affect the amount of gene 
product expressed? He felt these issues 
should be considered in evaluating the 
second item in the proposal. 
Dr. Gill said he would like Dr. Levine’s 
motion to require the investigators to select 
organisms with lower levels of toxin 
expression than 10’ 50% cytotoxic doses/mg 
protein in cell lysates and 10*50% cytotoxic 
doses/ml in culture supernatants. 
Dr. Levine pointed out that use of poorly 
mobilizable plasmid vectors such as pBR322 
add an additional measure of safety; 
language specifying use of this type of 
plasmid vector might meet Dr. Gill’s 
concerns. 
After some discussion as to 
appropriate language the following 
motion was developed: 
The Working Group recommends to RAC 
that E. coli host-vector systems expressing 
the Shiga toxin gene may be removed from P3 
to P2 containment conditions under the 
following conditions: 
(1) that the amount of toxin produced -by 
the modified host-vector systems be no 
greater than that produced by the positive 
control strain 933 E. co/; 0157H7. grown and 
measured under optimal conditions: and 
(2) the cloning vehicle is to be an EKl 
vector preferrably belonging to the class of 
poorly mobilizable plasmids such as pBR322, 
pBR328, and pBR325. 
By a vote of five in favor, none opposed, 
and no abstentions, the working group 
accepted this motion. 
In regard to the third item in the 
request of the April 4, 1984, letter, the 
following discussion ensued: 
Dr. Gill suggested the working group 
approve the third item of the request with the 
added clarification that under pl + ERl 
conditions the modified organism will not 
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