60090 
NOTICES 
the total DNA In the preparation. In 
addition, at least two biochemical or 
physical procedures are required for 
verification of purity. 
Footnote 3 gives guidance to the 
IBCs on many factors to be considered 
before deciding that DNA recombin- 
ants are "free of harmful genes." Fur- 
ther specificity seems unwarranted. I 
believe that to broaden the definition 
to Include the concept of "harmful" 
when a gene is expressed out of the 
normal physiological context would be 
contrary to the Intent of Footnote 3. 
In considering this issue. I have noted 
that in the proposed revised Guide- 
lines. different terms are used: l.e.. In 
Section III-A-3. "and the absence of 
harmful genes established”; In Section 
III-A-3-a, “are free of harmful genes"; 
in section III-A-3-b. "and there Is suf- 
ficient evidence that It is free of harm- 
ful genes." These have all been 
changed (also In Footnote 3 and Sec- 
tion I-D-8) to "and the absence of 
harmful sequences established." I 
have also decided that the 99 percent 
criterion should be retained in the pre- 
sent revision of the guidelines: but this 
point will be reconsidered by the RAC 
as a possible Item for future revision. 
Section Ill-B Experiments with Other 
Prokaryotic Host-Vectors 
8everal correspondents addressed 
the use of hosts other than E. coli K 
13. They pointed out that many ex- 
periments with such hosts are "inad- 
vertently prohibited." and suggested 
wording to allow experiments In hosts 
other than E. coli K 12 which do not 
meet the criteria for HV1. 
In the proposed revised Guidelines, 
experiments with prokaryotic hosts 
other than E. colt K 12 fall Into the 
following classes: (i) Self -cloning, 
exempted under the exemption I-E-3: 
(11) return of DNA segments to non- 
HV1 host of origin. Section III-B-2; 
and (ill) use of HV1 systems. Section 
III-B-1. I agree that there are many 
safe experiments which fall into none 
of the above three classes but which 
should be allowed under specified con- 
tainment levels. The proposed revised 
Guidelines, at the beginning of Sec- 
tion III. stated. "* * *(or the assign- 
ment of levels to experiments not ex- 
plicitly considered here) may be ex- 
pressly approved by the Director. NID. 
on the recommendation of the Recom- 
binant DNA Advisory Committee 
(RAC)." This language has been re- 
tained in a slightly modified form in 
the final Guidelines. In addition, simi- 
lar language is now repeated in a new 
Section III-B-3 and at the end of Sec- 
tion III-C-5. 
A specific example of this type of 
problem was provided by a commenta- 
tor who cited experiments he would 
like to perform involving recombinant 
DNA from Bacillus popiUiae, a patho- 
gen for the Japanese beetle, and Bacil- 
lus thuringiensis, a pathogen of pest 
caterpillar larvae. He asked for clarifi- 
cation of what containment levels 
would apply under the proposed re- 
vised Guidelines. 
The experiment could be considered 
under several different provisions. If 
data are submittted on natural ex- 
change of DNA between B. popilliae 
and B. thuringiensis, these organisms 
could be listed in a future version of 
Appendix A as falling under exemp- 
tion I-E-4. Or under the new para- 
graph III-B-3 which has been added 
to the Guidelines, containment levels 
could be set for these experiments. 
Section IH-C. Experiments with Eu- 
karyotic Host-Vectors 
A general issue raised by one re- 
spondent concerns the stipulation that 
some experiments will be assigned con- 
tainment levels on a case-by-case basis. 
The commentator Is concerned that 
this approach Ignores the need for 
mlnimumm standards which can serve 
as a guide for research workers. 
I believe that the case-by-case analy- 
sis prescribed for many experiments 
Involving the employment of viral 
DNA as a vector reflects the caution 
exercised over the use of such DNA. 
Each such experiment Is thus prohib- 
ited until the RAC has had a chance 
to weigh the scientific evidence and 
propose whether the experiment 
should proceed and. if so. to assign ap- 
propriate physclal and biological con- 
tainment levels. 
I have accepted several other recom- 
mendations for changes in this sec- 
tion. Two commentators offered new 
language for the section dealing with 
requirements for the employment of 
defective adenoviruses as cloning vec- 
tors. They noted that new mutants of 
Ad5 or 2 In which the entire trans- 
forming region has been deleted have 
recently been Isolated. These mutants 
can only be propagated in adenovirus- 
transformed cells. The commentators 
suggested that the language in Section 
III-C-l-c-< lMa) be generalized to 
state: "Human adenoviruses 2 and 5. 
rendered unconditionally defective by 
deletion of at least two essential genes, 
(pith appropriate helper, can be used 
under P3 conditions to propagate DNA 
sequences from * * I believe this 1s 
Justified, and the Section has been 
modified by changing the word 
"capsid" to "essential.” 
A correspondent stated that Section 
III-C erroneously equates nonproduc- 
tive infections with non permissive 
cells. He pointed out that in certain 
situations nonproductive infections 
may result from Infection of permis- 
sive cells. I agree. Accordingly, the 
phrase "to transform nonperm issive 
cells In culture" has been eliminated 
at a number of places In Section III-C. 
and more appropriate language has 
been substituted. 
The same correspondent pointed out 
that in Section III-C the word 
"Intact” is not appropriate because as 
soon as a foreign sequence is intro- 
duced. the viral DNA is no longer 
Intact. I agree. The wording in the rel- 
evant sections has been changed from 
"intact" to "whole.” 
Section III-C-2. Invertebrate Host- 
Vector Systems in Which Insect 
Viruses Are Used to Propagate 
Other DNA Segments. Section Ill- 
C-2. Plant Viral Host-Vector Sys- 
tems. Section III-C-4, Plant Host- 
Vector Systems Other than Viruses 
I have considered the comments for 
these three sections in a single group. 
A witness questions "the necessity 
for EPA registration of an entomo- 
pathogenlc organism in lieu of simply 
meeting the criteria of EPA for a tem- 
porary exemption from a requirement 
of tolerance in the environment." 
In the proposed revised Guidelines 
published In the Federal Register on 
September 27. 1977, "baculovtruses 
which have been registered by the En- 
vironmental Protection Agency" are 
specifically discussed in the section 
dealing with Invertebrate host-vector 
systems in which Insect viruses are 
used to propagate other DNA seg- 
ments. The writer is apparently ad- 
dressing this section and requesting 
that the EPA registration not be re- 
quired but merely "the criteria of EPA 
for a temporary exemption from a re- 
quirement of tolerance in the environ- 
ment.” The analogous section (III-C- 
2) In the proposed revised Guidelines 
published in the Federal Register on 
July 28. 1978. does not in fact refer to 
EPA registration as a specific crite- 
rion. It Indicates that experiments in 
which Insect viruses are used to propa- 
gate other DNA segments will be eval- 
uated on a case-by-case basis by the 
Recombinant Advisory Committee. In- 
formation required for a Judgment in- 
cludes host range restrictions, and in- 
fectlvity. persistence, and integration 
of the viral DNA. Data submitted by 
the requesting investigator on wheth- 
er EPA has registered a given insect 
virus or whether it meets the EPA cri- 
teria for a temporary exemption from 
a requirement of tolerance in the envi- 
ronment will be considered by the 
RAC. 
The same witness also questioned 
the validity of the statement in Sec- 
tion III-C-3 that "the plants should be 
grown under PI conditions— that is. in 
either a limited access greenhouse or 
plant growth cabinet which is insect- 
proof” and suggested substitution of 
the term "insect- restrictive" rather 
than "insect-proof." since the' latter 
term Implies higher containment. I 
FEDERAL REGISTER. VOL 43. NO. 247— FRJOAV, DECEMBER 22. 1971 
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