Federal Register / Vol. 49, No. 81 / Wednesday, April 25, 1984 / Notices 
17845 
"containment" be retained in the title of 
Section III. He went on to write, "The 
reference to the need for environmental 
impact statements and/or 
environmental assessment and a 
requirement of such by N1H is entirely 
out of place in these guidelines * * * 
We are opposed to the suggested 
change." 
The RAC reviewed the proposals at 
the February 6, 1984, meeting. RAC 
members agreed that it is not 
appropriate for the RAC to recommend 
incorporation of the added paragraphs 
proposed by Messrs. Rifkin and Rogers 
into the Guidelines. They agreed, 
however, that the proposal to amend the 
title of Part III of the Guidelines was 
appropriate. The RAC recommended by 
a vote of sixteen in favor, none opposed, 
and no abstentions, acceptance of the 
first part of the proposal, ue., to change 
the title of Part III from "Containment 
Guidelines for Covered Experiments" to 
"Guidelines for Covered Experiments," 
but rejection of the proposed additions 
to Section 1U-A of the Guidelines. 
I accept this recommendation of the 
RAC and the tide of Section III will be 
changed to read, “Guidelines for 
Covered Experiments." 
B. Request for Permission to Lower 
Containment Conditions for the Cloning 
of the Gene for Shiga-Like Toxin From 
E. coli 
In September 1982, Dr. Alison O’Brien 
of the Uniformed Services University of 
the Health Sciences (USUHS) requested 
permission in collaboration with Dr. 
Randall Holmes (USUHS) to clone in 
Escherichia coli K— 12 the structural 
gene of the Shiga-like toxin from 
clinically isolated strains of E coli. The 
E. coli Shiga-like toxin has activity 
similar to the activity of Shigella 
dysenteriae toxin. The investigators 
proposed to clone the Shiga-Kke toxin 
gene in E coli EKl host-vector systems 
using plasmid, cosmid, or lambda 
cloning vectors. In support of their 
proposal, Drs. O’Brien and Holmes 
offered the following arguments: 
1. Clinical isolates of E. coli have 
already been demonstrated to elaborate 
large amounts of toxin indistinguishable 
from that produced by Shigella 
dysenteriae 1 (Shiga). Therefore, the 
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 
colionize the bowel did not become ill. 
Therefore, any accidental ingestion of 
the organism to be manufactured, a 
toxin-producing E. coh K-12 strain that 
cannot colonize the human intestinal 
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 aerosolization of toxin that 
probably occurs during the process of 
toxin preparation. In one laboratory, 
toxin was iolated 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 neurotoxin. 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 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. 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 
Shigella and Escherichia are 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 argued, 
therefore, 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. At the 
meeting, 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 insufficient 
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. 
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 then 
requested in a letter dated December 8, 
1983, reconsideration of containment 
levels in view of information which has 
recently become available. They 
requested approval to conduct the 
experiments 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 introduction 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 Bora 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 Entcrobacteriaceae. In 
volunteer studies, some of the strains of 
V. cholerae that produce Shiga-like 
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