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Federal Register / Vol. 49, No. 81 / Wednesday, April 25, 1984 / Notices 
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. 
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. ccli. In this sense, “cloning” of 
genes that affect production of Shiga- 
like toxin onto phage genomes 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 
orally, and the occurence of converting 
phages in clinical isolates of E. coli that 
control high-level production of Shiga- 
like toxin, the investigators reasoned 
that there is little justification for 
requiring the maximum possible level of 
physical containment for the proposed 
studies. 
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." 
The proposal was published in the 
January 5, 1984, Federal Register (49 FR 
696). A letter was received from Dr. 
Werner Arber, the chairman of the 
Swiss Commission for Experimental 
Genetics, which is in charge of questions 
related to research involving 
recombinant DNA molecules. Dr. Arbei 
wrote that a Swiss ad hoc committee of 
experts requested by the Commission 
for Experimental Genetics had reviewed 
proposed research involving cloning of 
the Shiga toxin gene in an E. coli host- 
vector system. Dr. Arber wrote this 
committee concluded that: 
Work with recombinant DNA could not be 
expected to present a more severe biohazard 
than work with the natural pathogens * * * 
recent investigations had shown that a 
number of bacterial strains related to 
Shigella, in particular E. coli strains, carried 
genes homologous to the gene for Shiga toxin 
* * although Shigellosis is a serious 
disease, It does not represent a serious 
danger for an epidemic. 
A later letter from Dr. Kenneth 
Timmis of the Universile de Geneve 
said: 
An ad hoc committee of medical 
microbiologists specifically constituted in 
Switzerland to evaluate the possible danger 
of cloning in E. coli K-12 the gene for Shiga 
toxin, concluded that the experiment 
represented no greater danger than did wor k 
on Shigella itself and, as a result, 
recommended P2/EK1 containment 
conditions* * *. A different committee of 
medical microbiologists set up for the same 
purpose in Western Germany arrived at 
precisely the same conclusion. 
The RAC reviewed the proposal of 
Drs. O’Brien and Holmes at its February 
6, 1984, meeting. During the discussion, 
the history of the development of 
information about this toxin was 
summarized. Also, the genesis of 
Appendix F of the NIH Guidelines for 
Research Involving Recombinant DNA 
Molecules which is entitled. 
Cointainment Conditions for Cloning of 
Genes Coding for the Biosynthesis of 
Molecules Toxic for Vertebrates, was 
described: in Appendix F, toxins and the 
conditions for the cloning of their genes 
are classified by the pharmacological 
potency of the toxin. 
It was stated that there is no evidence 
that Shiga-like toxin causes disease in 
the way that a few molecules of 
botulinum toxin or tetanus toxin can. 
Several RAC members noted that the 
proposed experiments essentially 
involve cloning of E. coli DNA into E. 
coli host-vector systems since many E. 
coli strains produce Shiga-like toxin. 
However, it was pointed out that 
Section III— A of the Guidelines 
"overrides" other Sections of the 
Guidelines; questions of gene regulation 
and copy number must also be taken 
into account when considering potential 
hazard. One member said it appears 
from the data that organisms elaborating 
ralatively low levels of the toxin (IX 10* 
CD&o per milligram of protein) are not 
associated with desease while 
organisms elaborating somewhat higher 
doses (1 X 10 s CD»o per milligram of 
protein) appear to be assoicated with 
hemorrhagic colitis. Again, questions 
were raised concerning the effect of 
introducing the gene into a high copy 
number plasmid and of the relationship 
between invasiveness and 
pathogenicity. 
Other questions posed by the 
committee were: (1) Would the Shiga- 
like toxin molecule be excreted by the 
host-vector system; (2) is the mechanism 
of toxin entry into target cells known: (3) 
does the phage mentioned in the 
submittal carry the gene for Shiga-like 
toxin or does it carry regulatory genes? 
By a vole of nine in favor, five 
opposed, and four abstentions, the RAC 
recommended that Dra. O'Brien and 
Holmes and coworkers be allowed to 
proceed with cloning the gene for Shiga- 
like toxin under P2 physical containment 
conditions in E. col : K-12, restricted to 
using F.K2 plasmid vectors, commencing 
first with the use of pBR325 and pBR322, 
and proceeding to other EK2 plasmid 
vectors only if those are unsatisfactory. 
By a vote of eight in favor, four 
opposed, and five abstentions, the RAC 
passed the same motion but with the 
names of the investigators deleted from 
the motion. 
It has been the practice of the NIH not 
to accept RAC recommendations that do 
not indicate a clear consensus. 
Accordingly, this recommendation is not 
accepted. The investigators already 
have approval to conduct these 
experiments at the P4 level of 
containment. If they wish approval at a 
lower containment level, they may bring 
this proposal or a modified proposal 
back for consideration at a future RAC 
meeting and may at that time wish to 
submit additional data. In the meantime, 
they may proceed under previous 
permission which appears in the 
Guidelines (48 FR 24569) under 
Appendix F-IV-H. 
C. Proposal To Add a New Section 111 - 
A-4 to the Guidelines and a Footnote to 
Section IlI-B—4-b 
At its April 11, 1983, meeting, the 
Recombinant DNA Advisory Committee 
(RAC) endorsed a proposal to form a 
working group to comment and report to 
RAC on the "Report on the Social and 
Ethical Issues of Genetic Engineering 
with Human Beings” issued by the 
President's Commission for the Study of 
Ethical Problems in Medicine and 
Biomedical and Behavioral Research. 
The President’s Commission began its 
study in September 1980 in response to a 
request of the President's Science 
Advisor. Concern had been expressed 
earlier that year by the nation's three 
major religious associations that no 
governmental body was "exercising 
adequate oversight or control, nor 
addressing the fundamental ethical 
questions in a major way." The 
Commission’s report, issued in 
November 1982, concluded that 
continuing oversight of the field is 
desirable and suggested that one 
possible oversight mechanism could be 
revising RAC’s responsibilities. 
The RAC Working Group for 
Development of Response to President’s 
Commission Report on Ethical and 
[ 407 ] 
