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Federal Register / Vol. 48, No. 6 / Monday, January 10, 1983 / Notices 
DEPARTMENT OF HEALTH AND 
HUMAN SERVICES 
National Institutes of Health 
Recombinant DNA Research; Actions 
Under Guidelines 
AGENCY: National Institutes of Health, 
PHS, HHS. 
action: Notice of Actions under NIH 
Guidelines for Research Involving 
Recombinant DNA Molecules. 
summary: This notice sets forth actions 
taken by the Director, National Institute 
of Allergy and Infectious Diseases, by 
authority of the Director, NIH, under the 
August 1982 NIH Guidelines for 
Research Involving Recombinant DNA 
Molecules (47 FR 38048). 
EFFECTIVE date: January 10, 1983. 
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) 496-6051. 
SUPPLEMENTARY information: Several 
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 September 22, 1982 (47 FR 
41924), and reviewed by the NIH 
Recombinant DNA Advisory Committee 
(RAC) at its meeting on October 25, 
1982. In accordance with Section IV-C- 
1-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 and an additional 
announcement of the Director, NLAID. 
I. Decisions on Actions Under 
Guidelines 
A. Revision of Appendix F 
NIH staff proposed to revise 
Appendix F, Section F-l, second 
sentence, to clarify that the subject 
experiments are not in fact "prohibited," 
but rather fall under Section III— A of the 
Guidelines, which requires that the 
experiments receive RAC review and 
NIH and IBC approval before initiation. 
The current language is inaccurate. 
The relevant sentence in Appendix F 
currently reads as follows: 
Cloning of genes coding for molecules toxic 
for vertebrates that have an LD*, of less than 
100 nanograms per kilogram body weight 
[e.g., microbial toxins such as the botulinum 
toxins, tetanus toxin, diphtheria toxin. 
Shigella dysenteriue neurotoxin) is 
prohibited. 
NIH staff proposed that this sentence 
be amended to read as follows: 
The cloning of genes coding for molecules 
toxic for vertebrates that have an I.D, 0 of less 
than 100 nanograms per kilogram bodyweight 
(e.g., microbial toxins Such as the botulinum 
toxins, tetanus toxin, diphtheria toxin, 
Shigella dysenteriae neurotoxin| is covered 
under Section III— A— 1 of the Guidelines und 
requires RAC review and NIH arid IDC 
approval before initiation. 
The proposal was published in the 
September 22, 1982, Federal Register (47 
FR 41924). During the comment period, 
no comments were received. 
At its October 25, 1982, meeting, the 
RAC, by a vote of one in favor, thirteen 
opposed, and no abstentions, rejected a 
proposal to add additional wording to 
this section to indicate that such 
experiments would not ordinarily be 
allowed. The RAC then agreed by a vote 
of fourteen in favor, none opposed, and 
no abstentions, to recommend the 
change as published in the Federal 
Register on September 22, 1982, to make 
Appendix F consistent with the main 
text of the Guidelines. 
I accept this recommendation to 
revise Appendix F. 
B. Request for Permission To Clone a 
Shiga-like Toxin Structural Gene from 
E. coli 
In a letter dated September 29, 1902, 
Dr. Alison O’Brien of the Uniformed 
Services University of the Health 
Sciences requested permission, in 
collaboration with Dr. Randall Holmes, 
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 neurotoxin. The 
investigators proposed to clone the 
Shiga-like toxin gene in E. coli EKl host- 
vector systems using plasmid, cosmid, or 
lambda cloning vectors under PI 
conditions. 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 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 intestinal tract, should pose little 
hazard to man. 
3. Purification of Shiga toxin in 
several laboratories and E. coli Shign- 
hke 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 isolated from 500 liters of 
culture with only PI physical 
containment. 
4. Shiga toxin is a potent cytotoxin for 
a sublir.e 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, 
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 close 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 undoubtedly already resulted 
in cloning of the toxin gene. One RAC 
member pointed out that in Shigella the 
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