17 
strain that cannot colonize the human intestinal tract, would pose 
little hazard to man. 
(3) Purification of Shiga toxin in several laboratories and Eb_ coli 
Shiga-like toxin in the investigators' laboratory has not identi- 
fied any excessive risk frcm the aerosolization of toxin that 
probably occurs during the process of toxin preparation. In one 
laboratory, toxin was isolated frcm 500 liters of culture with 
only PI physical containment . 
(4) Shiga toxin is a potent cytotoxin for a subline of HeLa cells (a 
human cervical carcinoma tissue culture cell line), but the toxin 
has no effect on many other human, monkey, and rodent tissue culture 
cells. Therefore, the toxin is quite cell-type specific; and this 
limited spectrum of activity suggests that it would be non-toxic 
for most cells in the human body. 
(5) Contrary to the old literature, Shiga toxin is not a neurotoxin. 
By 1955, it was established that the paralysis observed in rabbits 
and mice (but not monkeys, guinea pigs, hamsters, or rats) vhen 
toxin is given intravenously is a reflection of the effect of 
toxin on the endothelium of small blood vessels, not a direct 
effect on nerve cells. 
This first submission was summarized in the Federal Register of 
September 22, 1982 (47 FR 41924). 
One comment on a related issue was received during the comment period. 
Dr. K. N. Timrnis of the Universite de Geneva suggested that the NIH 
Guidelines for Research Involving Recombinant DNA Molecules as they 
relate to the cloning of the Shiga toxin gene be revised. Dr. Tirrmis 
argued that Shigella and Escherichia are closely related, and that the 
NIH recognizes the high degree of relatedness by including these tvo 
genera in Sublist A, Appendix A, of the NIH Guidelines. Dr. Timrnis argued, 
therefore, that no NIH review should be required (as new specified by 
Section III-A and Appendix F) vhen the Shiga toxin gene is to be cloned in 
E. coli K-12. 
The RAC discussed the request submitted by Dr. O'Brien at the October 25, 
1982, meeting. The carmittee, by a vote of twelve in favor, none opposed, 
and one abstention, recommended that the initial experiments be performed 
under P4 4- EK1 containment conditions. The NIH accepted the RAC's recom- 
mendation that P4 + EK1 containment is adequate to contain safely the 
experiments proposed by Drs. O'Brien and Holmes and appropriate language 
was added to Appendix F of the NIH Guidelines. 
In December 1983, Drs. O'Brien and Holmes requested reconsideration of 
containment levels in view of information which had recently became 
available. They requested approval at the P2 level of physical containment 
for the following reasons: 
[470] 
