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Federal Register / Vol. 49, No. 3 / Thursday, January 5, 1984 / Notices 
genes for Shiga-like toxin production are 
present in the E. coli gene pool found in 
nature. 
2. Human volunteers fed large 
numbers of Shigella dysenteriae 1 
organisms that produced Shiga toxin but 
could not colonize the bowel did not 
become ill. Therefore, any accidental 
ingestion of the organism to be 
manufactured, a toxin-producing E. coli 
K-12 strain that cannot colonize the 
human intestine tract, would pose little 
hazard to man. 
3. Purification of Shiga toxin in 
several laboratories and E. coli Shiga- 
like toxin in the investigators' laboratory 
has not identified any excessive risk 
from the aerosolizatioin of toxin that 
probably occurs during the process of 
toxin preparation. In one laboratory, 
toxin was isolated from 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 neurotizxin. By 1955, it 
was established that the paralysis 
observed in rabbits and mice (but not 
monkeys, guinea pigs, hamsters, or rats) 
when 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 tbe 
Federal Register of September 22, 1982 
(47 FR 41924). One comment on a related 
issue was received during the comment 
period. Dr. K. N. Timmis of the 
Universite de Geneve 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. Timmis argued that 
Sigella and Escherichia closely related, 
and that the NIH recognizes the high 
degree of relatedness 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 now specified by Section 
III— A and Appendix F) when 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. During that meeting, it 
was stated that taxonomically Shigella 
and Escherichia coli are so closely 
related that in the future they may be 
classified as the same organism. The 
toxin administered intravenously to 
rabbits and monkeys is very toxic; it is 
not very toxic to mice when 
administered intravenously. Many E. 
coli isolates, both pathogenic and 
nonpathogenic. express some toxin; 
therefore, shotgun cloning of E. coli into 
E. 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 what effect introducing that 
gene into a high copy number plasmid 
would have. Finally questions were 
raised concerning the relationship of 
invasiveness to pathogenicity and to 
toxin toxicity. Most of these questions 
could not be answered as inadequate 
data exist. However, there was general 
agreement that P4 containment would 
be adequate. After hearing the 
arguments, the committee by a vote of 
twelve in favor, none opposed, and one 
abstention, recommended that the initial 
experiments be performed under P4 + 
EKl containment conditions. A motion 
to approve the experiments at P3 + EKl 
failed by a vote of five in favor, seven 
opposed, and one abstention. A motion 
to approve the experiments using P3 
laboratory practices and containment 
equipment in a P4 facility failed to pass 
by a vote of five in favor, seven 
opposed, and one abstention. 
The NIH accepted the RAC 
recommendation that P4 + EKl 
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 Guidelines. The language stipulated 
that if the investigators wish to proceed 
with the experiments in the NIH P4 
facility a prior review would be 
conducted to advise NIH whether the 
proposal had sufficient scientific merit 
to justify the use of the NIH P4 facility. 
A subsequent review by the 
Bacteriology and Mycology Study 
Section of the NIH indicated that the 
proposal had sufficient scientific merit. 
Drs. O'Brien and Holmes have now 
requested reconsideration of 
containment levels in view of 
information which has recently become 
available. They are requesting approval 
at the P2 level of physical containment 
for the following reasons: 
1. Epidemiology studies have been 
performed on over 150 E. coli strains 
isolated from human and animal stools. 
These studies have shown that the 
majority (80%) of the strains made 
detectable levels of Shiga-like toxin. 
Moreover, four of four substrains of the 
well-characterized bacterium E. coli K- 
12 were shown to make low levels of the 
toxin. Thus, cloning of the Shiga-like 
toxin gene from clinical isolates of E. 
coli into laboratory strains of E. coli K- 
12 will not involve the introductions of a 
"foreign" toxin into the organism. 
2. Production of low levels of Shiga- 
like toxin was observed in 2 of 15 
normal human gut flora E. coli strains 
from asymptomatic infants. 
3. Strains of Vibrio cholerae and 
Vibrio parahaemolyticus were tested 
and shown to produce Shiga-like toxin. 
Thus, the gene(s) for Shiga-like toxin are 
present in naturally occurring isolates of 
the family Vibrionaceae and are not 
restricted to the Enterobacteriaceae. In 
volunteer studies, some of the strains of 
V. cholerae that produce Shiga-like 
toxin did not cause disease. Therefore, 
the ability to produce Shiga-like toxin is 
not equivalent with vihjlence in humans 
challenged by the oral route. 
4. Phages from two clinical isolates of 
E. coli have been shown to control high- 
level production of Shiga-like toxin in E. 
coli K-12 host strains by phage 
conversion. Thus, either the structural 
gene(s) for the Shiga-like toxin or 
regulatory genes that control high-level 
production of the toxin are present on 
wild-type phages from clinical isolates 
of E. coli. In this sense, "cloning" of 
genes that affect production of Shiga- 
like toxin onto phage genames has 
already occurred in nature. 
Based on the occurrence of the gene(s) 
for Shiga-like toxin in several different 
bacterial genera, the avirulence of some 
bacterial strains that produce Shiga-like 
toxin in human subjects challenged 
orully, and occurrence of converting 
phages in clinical isolates of E. coli that 
control high-level production of Shiga- 
like toxin, the investigators believe there 
is little justification to require the 
maximum possible level of physical 
containment for the proposed studies of 
cloned genes for Shiga-like toxin in 
laboratory strains of E. coli that lack the 
ability to colonize or invade the 
intestinal mucosa in man. 
In addition, the U.S. Cholera Panel of 
the National Institute of Allergy and 
Infectious Disease (NIAID) in a meeting 
on October 16, 1983, reviewed and 
critiqued the Microbiology and 
Infectious Diseases Program dealing 
with cholera and E. coli (ETEC) vaccine 
development. It was the consensus of 
the Panel that it be recommended that 
NIH reconsider the ban "on shiga toxin 
cloning experiments in containment 
facilities other than P4. This strict 
requirement will prevent most 
laboratories from deleting the Shiga 
gene from candidate V. cholera and 
ETEC vaccine strains. Shiga toxin is 
now found in many nonpathogenic E. 
coli, including the common vector host, 
E. coli K-12." 
[241] 
