6 
(3) Purification of Shiga toxin in several laboratories and coli 
Shiga-like toxin in the investigators' laboratory has not identified 
any excessive risk fran the aerosolization of toxin that probably 
occurs during the process of toxin preparation. In one laboratory, 
toxin was isolated fron 500 liters of culture with only PI physical 
containment. 
(4) Shiga toxin is a potent cytotoxin for a sub line 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. 
The request was summarized in the Federal Register of September 22, 1982 
(47 FR 41924). Cue caiment on a related issue was received during the 
carment period. Dr. K. N. Tiimds of the Universite de Geneve suggested that 
the National Institutes of Health (NIH) Guidelines for Research Involving 
Recombinant ENA. Molecules as they relate to the cloning of the Shiga toxin 
gene be revised. Dr. Timmis argued that Shigella and Escherichia are 
closely related, and that the NIH recognizes the high degree of r elatedness 
by including these two genera in Sublist A, Appendix A, of the Guidelines. 
Dr. Timmis, therefore, argued 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 Ih coli K-12. 
The RAC discussed the request submitted by Dr. O'Brien at the October 25, 
1982, meeting. During that meeting, it was stated that taxonanically 
Shigella and Escherichia coli are so closely related that in the future 
they may be classified as the same organism. Many Ej_ coli isolates, both 
pathogenic and nonpathogenic , express some toxin? therefore, shotgun 
dealing of Eh coli into Eh coli has undoubtly already resulted in cloning 
of the toxin gene. One RAC member pointed out that in Shigella the Shiga 
toxin gene is chromosomal, and he questioned the effect of introducing that 
gene into a high copy nuntoer plasmid. Finally, questions were raised 
concerning the relationship of invasiveness to pathogen ici ty and to toxin 
toxicity. However, there was general agreement that P4 containment would 
be adequate. After hearing the arguments, the carmittee by a vote of 
twelve in favor, none opposed, and one abstention, recommended that the 
initial experiments be performed under P4 + E3CL containment conditions. 
The NIH accepted the RAC's recommendation that P4 + E3CL containment is 
adequate to contain safely the experiments proposed by Drs. O'Brien and 
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