Federal Register / Vol. 49, No. 179 / Thursday, September 13, 1964 / Notices 
36059 
by experimentation with these organisms at 
low volumes and no reason that the 
production of large volumes will lead to any 
greater risk to personnel or the environment. 
. . . We believe that the proposed 
amendments are well conceived and 
recommend their acceptance by N1H for 
incorporation into the guidelines. We suggest, 
however, that the placement of the paragraph 
under modification #3 ... be reconsidered. 
Adding the proposed paragraph after the 
second paragraph would, in effect, place this 
paragraph dealing with large-scale 
fermentation experiments under the 
exemption experiments. Since large scale 
experiments are not exempted but rather are 
listed as exceptions to the exemptions, we 
recommend the proposed paragraph be 
added to the third paragraph under the 
"Exceptions" section of Appendix C— II. 
Appendix C— III. and Appendix C-IV. 
Dr. Judith A. Hautala of Cenex 
Corporation, Gaithersburg, Maryland 
wrote: 
The proposed modification of Appendix K- 
II— D to require that exhaust gases be treated 
so as to "minimize” rather than "prevent" the 
release of viable organisms is necessary in 
order to make the requirement realistic. Even 
HEPA filters, which are specifically 
recommended in Appendix K-II-D, are rated 
as removing only 99.97% of the particles 
larger than 0.2 microns. Thus, the term 
"prevent" is too restrictive to be 
operationally useful. . . . Part 3 of the 
proposed amendment, which explicitly grants 
to the Institutional Biosafety Committee (IBC) 
the right to exercise some latitude in the 
application'of Pl-LS containment 
requirements to organisms covered by 
Appendix C, is appropriate for several 
reasons. These organisms are exempt from 
the NIH Guidelines at the laboratory level 
because they have been-shown to present no 
significant risk to health or the environment. 
Therefore, it would seem reasonable to allow, 
the IBC to relax certain aspects of the Pl-LS 
requirements after careful review of the host- 
vector system, the large-scale process 
description, the equipment to be utilized, and 
the emergency spill procedures. For example, 
in order to accommodate process and 
equipment realities, It may in some instances 
be appropriate for the IBC to allow certain 
large-scale transfer or processing operations 
involving viable organisms to be carried out 
without absolute containment. Providing that 
worker exposure and environmental losses 
are minimized, this type of latitude should 
present no significant risk. 
Dr. J. Allan Waitz of Schering 
Corporation, Bloomfield, New Jersey, 
wrote: 
HEPA filters are only 99.97% efficient for 
particles of 0.3 u diameter, so to say that 
HEPA filtration ‘prevents’ release of 
recombinant organisms is incorrect. Use of 
the word 'minimize' would more accurately 
reflect the removal properties of a UEPA 
filter. If this is the intent of the proposed 
change, we support it. . . . 
The Pl-LS recommendations should be the 
minimum requirements for handling volumes 
of recombinant organisms. While we agree 
that the lBCs should have the flexibility 
necessary to carry out their responsibilities, 
we are opposed to the IBC having so much 
flexibility that the Guidelines could be 
ignored completely. Accordingly, we urge the 
RAC to reject this proposed change. This 
includes rejection of the change proposed in 
Item 1-2, since the current wording in 
Appendix C-U, C— III, and C-IV is adequate. 
The RAC discussed this proposal at 
the June 1, 1984, meeting. Drs. 
McKinney, McGarrity, and Wensink 
spoke in favor of the proposal. 
Alternative wording to that which 
appeared in the April 24 Federal 
Register was considered but rejected. By 
a vote of twenty in favor, none opposed, 
and no abstentions, the RAC adopted 
the recommendation of the Large-Scale 
Review Working Group to substitute the 
word “minimize" for the word “prevent" 
in the Appendix K-II-D. . 
By a vote of twenty-one in favor, none 
opposed, and no absentions, the RAC 
adopted the recommendation of the 
Large-Scale Review Working Group that 
the second paragraph of Appendices C- 
II, C— III. and C-IV be modified to read: 
For these exempt laboratory experiments 
Pi physical containment conditions are 
recommended. 
In the same motion, RAC adopted the 
recommendation of the Large-Scale 
Review Working Group that a paragraph 
be added following the second 
paragraph of Appendix C— II, Appendix 
C— HI, and Appendix C-IV. The 
paragraph would read as follows: 
For large-scale fermentation experiments 
Pl-LS physical containment conditions are 
recommended. However, following review by 
the IBC of appropriate data for a particular 
host-vector system, some latitude in the 
application of Pl-LS requirements as outlined 
in Appendix K-II-A through K-II-F is 
permitted. 
By a vote of twenty in favor, none 
opposed, and no abstentions, the RAC 
recommended the NIH staff proposal to 
add the following language to Appendix 
K-I: 
(The Pl-LS level of physical containment is 
recommended for large-scale research or 
production of viable organisms for which PI 
is recommended at the laboratory scale such 
as those described in Appendix C.) 
I accept these recommendations and 
Appendix C— II, Experiments Involving 
E. coli K-12 Host- Vector Systems, 
Appendix C— III, Experiments Involving 
Saccharomyces cerevisiae Host-Vector 
Systems, Appendix C-IV, Experiments 
Involving Bacillus subtilis Host-Vector 
Systems, Appendix K-I, Selection of 
Physical Containment Levels, and 
Appendix K-II-D of Appendix K— II, Pl- 
LS Level, will be modified as 
recommended. 
II. Summary of Actions 
l I- A. Proposal to Clone Shiga-Like 
Toxin Gene From E. coli 
Appendix F-IV-H of the Guidelines is 
modified to read as follows: 
Appendix F-IV-H. The intact structural 
gene(s) of the Shiga-like toxin from E. coli 
may be cloned in E. coli K-12 under P3 + EKl 
containment conditions. 
E. coli host-vector systems expressing the 
Shiga-like toxin gene may be moved from P3 
to P2 containment conditions provided that 
(1) the amount of toxin produced by the 
modified host-vector systems is no greater 
than that produced by the positive control 
strain 933 £. coli 0157H7, grown and 
measured under optimal conditions; and (2) 
the cloning vehicle is to be an EKl vector 
preferably belonging to the class of poorly 
mobilizable plasmids such as pBR322, 
pBR328, and pBR325. 
“Nontoxinogenic fragments of the Shiga- 
like toxin structural gene(s) may be moved 
from P3 + EKl to P2 + EKl containment 
conditions or such nontoxic fragments may 
be directly cloned in E. coli K-12 under P2 + 
EKl conditions provided that the £. coli host- 
vector systems containing the fragments do 
not contain overlapping fragments which 
together would encompass the Shiga-like 
toxin structural gene(s). 
II-B. Proposed Amendment of 
Procedures for Scale-Up of Organisms 
Listed in Appendix C 
1. In Appendix K-II-D of Appendix K- 
II, Pl-LS Level, the word "minimize" is 
substituted for the word “prevent." 
Appendix K-II-D reads as follows: 
Appendix K-II-D. Exhaust gases removed 
from a closed system or other primary 
containment shall be treated by filters which 
have efficiencies equivalent to HEPA filters 
or by other equivalent procedures (e.g., 
incineration) to minimize the release of 
viable organisms containing recombinant 
DNA molecules to the environment. 
2. The second paragraph of Appendix 
C— II, Experiments Involving E. coli K-12 
Host- Vector Systems; Appendix C— III. 
Experiments Involving Saccharomyces 
cerevisiae Host-Vector Systems; and 
Appendix C-IV, Experiments Involving 
Bacillus subtilis Host-Vector System is 
modified to read as follows: 
For these exempt laboratory experiments, 
Pi physical containment conditions are 
recommended. 
3. A paragraph is added following the 
second paragraph of Appendix C— II. 
Appendix C— III. and Appendix C-IV. 
That paragraph reads as follows: 
For large-scale fermentation experiments 
Pl-LS physical containment conditions are 
recommended. However, following review by 
the IBC of appropriate data for a particular 
host-vector system, some latitude in the 
application of Pl-LS requirements as outlined 
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