21 
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 reccm re ndat ions to RAC on Dr. O'Brien's April 4, 1984, proposal. 
Dr. Gottesrran 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 bo accept the working 
group recarme ndat ions . 
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 Eh_ coli into coli K-12 frcrn 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: 
(1) The data generated through experiments with 140 human volunteers 
fed Shiga toxin-producing Shigella lacking invasive characteris- 
tics. No disease symptoms were observed in 139 individuals; in 
one individual, the strain reverte! to an invasive form and the 
volunteer developed shigellosis. Since Ej_ coli K-12 neither 
adheres nor is invasive, no disease should be caused by coli 
K-12 containing the Shiga toxin gene. 
(2) The evidence generated by Branham, Dack, and Riggs vhich 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 produce! in the huiran gut. This amount is 
roughly equivalent to approximately 14,000 lethal doses for humans if the 
toxin were to be administered parenteral ly. 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 Mae 
request. The working group recommends that host-vector systers expressing 
the Shiga toxin gene may be removed from P3 to P2 containment corxiitions 
under the following condi tions: 
(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 ih_ coli 0157H7, grown aid measured under optino) condi- 
tions; and 
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