36052 
Federal Register / Vol. 49, No. 179 / Thursday, September 13, 1984 / Notices 
DEPARTMENT OF HEALTH AND 
HUMAN SERVICES 
National Institutes of Health 
Recombinant DNA Research; Actions 
Under Guidelines 
agency: National Institutes of Health, 
PHS, DHHS. 
action: Notice of actions under NIH 
guidelines for research involving 
recombinant DNA Molecules. 
summary: This notice sets forth actions 
taken by the Director, National 
Institutes of Health (NIH), under the 
June 1983 NIH Guidelines for Research 
Involving Recombinant DNA.Molecules 
(48 FR 24556). 
EFFECTIVE date: September 13, 1984. 
FOR FURTHER INFORMATION CONTACT: 
Additional information can be obtained 
from Dr. William J. Gartland, Office of 
Recombinant DNA Activities (ORDA). 
National Institutes of Health, Bethesda, 
Maryland 20205, (301) 469-6051. 
SUPPLEMENTARY INFORMATION: Two 
major actions under the NIH Guidelines 
for Research Involving Recombinant 
DNA Molecules are being promulgated 
today. These proposed actions were 
published for comment in the Federal 
Register of April 24, 1984 (49 FR 17674), 
and reviewed and recommended for 
approval by the Recombinant DNA 
Advisory Committee (RAC) at its 
meeting on June 1, 1984. In accordance 
with section IV-C-l-b of the NIH 
Guidelines, these actions have been 
found to comply with the Guidelines and 
to present no significant risk to health or 
the environment 
Part I of this announcement provides 
background information on the actions. 
Part II provides a summary of the 
actions of the Director, NIH. 
Action on other items revieweo and 
recommended for approval at the June 1, 
1984, RAC meeting will be announced at 
a later date. 
I. Decision on Actions Under Guidelines 
1-A. Proposal To Clone Shiga-Like 
Toxin Gene From E. coli 
Dr. Alison O’Brien of the Uniformed 
Services University of the Health 
Sciences (USUHS) in collaboration with 
Dr. Randall Holmes (USUHS) proposed 
to clone in Escherichia coli K-12 the 
structural gene of the Shiga-like toxin of 
E. coli. Shiga-like toxin has activity 
similar to the activity of Shigella 
dysenteriae toxin. 
The description of the review resulting 
in this decision is organized as follows: 
l-A-l. History of the Proposal 
I-A-2. Comments on the Proposal in 
Response to the April 24. 1984, Federal 
Register Notice 
I-A-2. Excerpts From the Minutes of the May 
11, 1984, Meeting of the RAC Working 
Croup on Toxins 
l-A-4. Excerpts of Drafts Minutes of the June 
1, 1984, RAC Discussion of the Proposal 
I-A-5. Decision 
I-A-l. History of the Proposal 
In a first submission in September 
1982, the investigators proposed to clone 
the Shiga-like toxin gene in S', coli EKl 
host-vector systems using plasmid, 
cosmid, or lambda cloning vectors. Irv 
support of their proposals, Drs. O’Brien 
and Holmes offered the following 
arguments: 
a. 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. 
b. Human volunteers fed large 
numbers of Shigella dysenteriae 1 
organisms that produced Shiga toxin btft 
could not colonize the bowel did not 
become ill. Therefore, any accidental 
ingestion of the organism to be 
manufactured, a loxin-producing E. coli 
K-12 strain that cannot colonize the 
human intestinal tract, would pose little 
hazard to man. 
c. Purification of Shiga toxin in 
several laboratories and E. coli Shiga- 
like toxin in the investigator’s laboratory 
has not.identified any excessive risk 
from the aerosolization of toxin that 
probably occurs during the process of 
toxin preparation. In orrf laboratory, 
toxin was isolated from 500 liters of 
culture with only Pi physical 
containment. 
d. 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, or 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. 
e. 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. 
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. 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 when the Shiga toxin gene is to 
be cloned in E. coli K-12. 
The RAC discussed the request 
submitted by Drs. O'Brien and Holmes 
at the October 25, 1982, meeting. 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 safety the exeriments proposed 
by Drs. O'Brien and Holmes and 
appropriate language was added to the 
Guidelines at Appendix F-IV-H, 
„In December 1983, Drs. O’Brien and 
Holmes requested reconsideration of 
containment levels in view of 
information which had recently become 
available. They requested approval at 
the P2 level of physical containment for 
the following reasons: 
a. Epidemiology studies have been 
performed on over 150 E. coli strains 
isolated from human and animals 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. 
b. Production of low levels of Shiga- 
like toxin was observed in 2 of 15 
normal human gut flora E. coli strains 
from asymptomatic infants. 
c. Strains of Vibrio cholerae and 
Vibrio parahaemolyticus were tested 
and shown to produce the Shiga-like 
toxin. Thus, the gene(s) for Shiga-like 
toxin are present in naturally occurring 
isolates of the family Vibrionaceae and 
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 virulence in humans 
challenged by the oral route. 
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