Federal Register / Vol. 49, No. 179 / Thursday, September 13, 1984 / Notices 
36055 
contain overlapping fragments which together 
would encompass the structural gene(s). This 
specification will eliminate the possibility 
that the structural gene might be regenerated 
through recombinational events. 
By a vote of five in favor, none opposed, 
and no abstentions, the working group 
approved the motion to offer this 
recommendation to RAC. 
In regard to the fourth item in the 
April 4, 1984, proposal: 
It was the consensus of the group that 
these experiments would not fall under 
Appendix F of the Guidelines, and no action 
need be taken. 
In regard to the fifth item in the April 
4, 1984, letter from Drs. O'Brien and 
Holmes: 
It was the consensus of the group that 
because no working group could predict all 
potential scenarios each specific 
nontoxinogpnic allele should be considered 
individually on a case-by-case basis. A 
system is in place within the N1H to perform 
this type of evaluation. 
I-A-4. Excerpts of Draft Minutes of the, 
June 1, 1984, RAC Discussion of the 
Proposal 
At its June 1, 1984, meeting, the RAC 
discussed the requests in the April 4, 
1984, letter from Drs. O’Brien and 
Holmes and the recommendations made 
by the RAC Working Group on Toxins 
at its May 11, 1984, meeting. The 
following draft minutes from the June 1, 
1984, RAC meeting summarize the 
pertinent points raised during the 
discussion: 
Dr. Gottesman introduced the proposal 
(tabs 1153, 1165, 1162, 1156/11. 1168. 1170) of 
Drs. Alison O'Brien and Randall Holmes of 
the Uniformed Services University of the 
Health Sciences (USUHS) to clone at P3 
containment the Shiga-like toxin gene of E. 
coli in E. coli K-12 host-vector systems. 
Shiga-like toxin has activity similar to the 
activity of Shigella dysenteriae toxin. 
Dr. Gottesman said that at the February 6. 
1964, RAC meeting, she had voted against the 
motion to lower containment from P4 to P2 
because she felt certain questions had not 
been fully addressed. Her preception of the 
sentiment of the committee at that meeting, 
however, was that RAC overwhelmingly 
favored the motion in spite of the split vote. 
She felt the split vote partially reflected a 
disagreement over whether the motion should 
provide an exclusive approval for Dr. 
O'Brien's group. 
Dr. Gottesman said subsequent to the 
February 6 RAC meeting, Dr. O'Brien had 
submitted a revised proposal on April 4 and 
that NIH had convened the RAC Working 
Group on Toxins on May 11, 1984, to review 
the new proposal in the light of available 
scientific data on Shiga toxin. Dr. Gottesman 
said a great deal of discussion occurred at the 
working group meeting. This discussion 
clarified the scientific issues and resulted in 
working group recommendations to RAC on 
Dr. O'Brien's April 4, 1984, proposal. 
Dr. Gottesman said these recommendations 
were unanimously approved by the working 
group and represent a consensus between 
individuals holding very different points of 
view. She strongly urged the RAC to accept 
the working group recommendations. 
Dr. Gottesman said the first request of Dr. 
O'Brien's April 4 proposal was to lower 
containment conditions for cloning the intact 
structural gene(s) for Shiga-like toxin of E. 
coli into E. coli K-12 from P4 + EK1 to P3 + 
EK1. Dr. Gottesman said this proposal was 
accepted by the Working Group on Toxins on 
the basis of two sets of data: 
a. The data generated through experiments 
with 140 human volunteers fed Shiga toxin- 
producing Shigella lacking 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. 
Since E. coli K-12 neither adheres nor is 
invasive, no disease should be caused by E. 
coli K-12 containing the Shiga toxin gene. 
b. 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. Gottesman said that in the worst case 
scenario, in which all the E. coli in the human 
intestine (estimated to be 10") were 
expressing the Shiga toxin gene on a high 
expression, high copy number plasmid, one 
milligram of toxin might be produced in the 
human gut This amount is roughly equivalent 
to approximately 14,000 lethal does for 
humans if the toxin were to be administered 
parenterally. However, Branham, Dack, and 
Riggs had administered 20,000 lethal doses 
enterally to monkey intestinal pouches with 
no observed effect 
In regard to the second item of Dr. 
O'Brien's April 4 letter requesting lowering of 
certain characterized clones to PI + EK1 
conditions, Dr. Gottesman said the working 
group recommends modifications In the 
request. The working group recommends that 
host-vector systems expressing the Shiga 
toxin gene may be removed from P3 to P2 
containment conditions under the following 
conditions: 
a. 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. coli 0157H7, grown and 
measured under optimal conditions; and 
b. The cloning vehicle is to be an EKl 
vector preferably belonging to the class of 
poorly mobillzable plasmids such pBR322, 
pBR328, and pBR325. 
Dr. Landy asked if the working group 
recommendation specified that both the host- 
vector system and strain 933 E. coli 0157H7 
were to be grown under optimal conditions. 
Dr. Gottesman replied that both strains 
should be grown under optimal toxin 
producing conditions. 
Dr. Gottesman said the working group 
recommended approval of the third item of 
the Apirl 4 request with the clarification that 
the modified organism will not contain 
overlapping fragments which together would 
encompass the structural gene(s). This 
specification will eliminate the possibility 
that the structural gene might be regenerated 
through recombinational events. 
In regard to the fourth item in the April 4 , 
1984. proposal Dr. Gottesman said it was the 
consensus of the working group that these 
experiments would not fall under Appendix F 
of the Guidelines, and no action need be 
taken by the RAC. 
In regard to the fifth item in the April 4 , 
1984, letter from Drs. O'Brien and Holmes. Dr. 
Cottesman said it was the consensus of the 
group that no working group could predict all 
potential scenarios; thus, each specific 
nontoxinogenic allele should be considered 
individually on a case-by-case basis. A 
system is in place within the NIH to perform 
this type of evaluation, so no specific action 
need be taken by the RAC. 
Dr. King Holmes said the proposed 
research is extremely important and should 
be pursued. He had, however, several 
concerns which he felt should be addressed: 
(1) He noted that only four individual animals 
of one primate species had been tested by 
Branham, Dack, and Riggs. He asked whether 
primate species might differ in their response 
to the toxin. (2) He also questioned the 
calculations developed by the working group 
in a worst case scenario; he wondered 
whether this scenario would correspond to 
the in vivo situation. (3) He noted that data 
presented at an earlier RAC meeting by Dr. 
O’Brien suggested a toxin dose-effect; i.e., E. 
coli isolates from patients who have 
hemorrhagic colitis produced more toxin in 
vitro than did E. coli isolates from patients 
who did not have hemorrhagic colitis. (4) He 
questioned what would be the effect of 
feeding "non-healthy" individuals E. coli K- 
12 producing Shiga toxin. 
Dr. Holmes felt the apparent lack of 
toxicity for intestinal epithelial cells is not 
entirely reassuring in terms of toxicities for 
other epithelial cell types such as HeLa cells. 
He pointed out that the' toxin is presumed to 
be toxic for endothelial vascular cells. He 
asked what would be the effect on humans If 
toxin producing E. coli is inhaled? What if 
toxin producing E. coli colonizes the skin or 
urogenital tract? 
Dr. Holmes questioned the effect the toxin 
might have on comeal or conjunctival cells in 
neonates bom vaginally of women vaginally 
colonized by E. coli producing Shiga toxin. 
What might be the effect on the endocervix 
or endometrium of women vaginally 
colonized by E. coli producing the toxin? 
What would be the effect on the male whose 
prostate might be colonized? 
Dr Holmes questioned the language of the 
third recommendation which specifies that 
the modified host-sector system will not 
contain overlapping fragments which together 
would encompass the structural gene(s); he 
noted that E. coli K-12 host-vector systems 
may contain a chromosomal gene encoding 
Shiga toxin. 
Dr. Holmes said he was not persuaded that 
the proposed experiments require an Arms 
Control Impact Statement (ACIS) as argued 
by Mr. Rifkin in his May 15, 1984, letter. Dr. 
O’Brien's proposed experiments are NIF 
funded and will be performed by civilian 
investigators associated with the USUHS 
medical school. He said he was not 
persuaded that the affiliation of the 
[526] 
