NOTICES 
33115 
xl776) at the June 23 meeting, and 
will do so for all future submissions. 
These criteria are clearly more strin- 
gent than previous ones, and this 
means that EK2 host-vector systems 
approved now and in the future will be 
even safer than those approved previ- 
ously. 
KV3 Level Requirements for HV3 
systems have been made more strin- 
gent that those in the 1976 guidelines. 
The additional requirements mean 
that only some HV2 systems are eligi- 
ble for consideration as HV3 systems. 
This should significantly increase the 
containment of HV3 systems and 
therefore increase the safety of ex- 
perimentation. In addition, attempts 
to develop HV3 systems that meet 
these criteria should simultaneously 
upgrade the HV2 systems in use. since 
it is to the experimenter’s advantage 
to use the HV2 systems that have the 
greatest likelihood of meeting HV3 cri- 
teria. 
Certification of HV Systems. A new 
section has been added to clarify re- 
sponsibility for certification of all 
host-vector systems, the types of data 
to be submitted, and the mechanisms 
for distributing strains once certified. 
The section delineates procedures 
used by the RAC for the past year and 
thus represents no change from prac- 
tices under the 1976 guidelines. 
Under the PRG-NTH, HV1 systems 
other than E. coli K-12 and HV2 and 
HV3 systems are considered by an 
expert working group and then by the 
RAC. which makes appropriate recom- 
mendations to the Director, NTH. 
Modifications of HV2 and HV3 sys- 
tems must be independently certified 
by NTH. Data to be submitted are de- 
tailed. All HY2 and HV3 systems are 
to be obtained from NTH or its desig- 
nee, and recipients are to report to 
NTH any discrepancies from the ex- 
pected properties. If the strains are 
propagated by NTH, a sample will be 
tested for relevant properties prior to 
distribution. The requirements assure 
adequate controls in the certification 
and distribution of host-vector systems 
and provide sufficient protection 
against potential hazards to the envi- 
ronment. 
Containment Properties of Hosts 
and Vectors. In regard to containment 
properties of individual org anisms 
used in recombinant DNA research, 
recent experimental evidence supports 
the view that biological containment 
works well This is particularly borne 
out by results from experiments spe- 
cifically designed to test the survivabi- 
lity and colonizing ability of E. coli K- 
12 and EK2 host-vector systems and 
the transmissibility of plasmids and 
phage vectors. 
At the time of the release of the cur- 
rent guidelines in 1976, EK2 systems 
were defined but none existed. An ex- 
tramural contract program was initiat- 
ed to develop safer host-vector systems 
and to verify their genotypic constitu- 
tion and phenotypic traits. The pro- 
gram is administered by the National 
Institute of Allergy and Infectious Dis- 
eases. The ability of these systems to 
survive in laboratory and natural envi- 
ronments was determined. As a result 
of this contract program and of work 
by other investigators, a series of EK2 
host-vector systems was developed. 
The RAC subjected these to great 
scrutiny and finally recommended 
them for certification. A list of certi- 
fied EK2 host-vector systems appears 
in appendix H of the October 1977 
EIS. 
III. Containment Guidelines for 
Covered Experiments 
Analysis of Current Guidelines 
E. Coli K-12 Host-Vector Systems 
The several levels of physical and 
biological containment are defined in 
part H, and specific recommendations 
are given for experiments using the E. 
coli K-12 host- vector systems. Each 
type of experiment is assigned both a 
physical containment level (that is. a 
P level) and a biological containment 
level (an EK level). The particular 
combination of the two reflects the se- 
verity of the estimated potential 
hazard. 1 
The guidelines are organized, for the 
E. Coli systems, according to the 
source and nature of the foreign DNA. 
A sample of DNA containing essential- 
ly all the genetic information of an or- 
ganism can be isolated and fragment- 
ed. If the experiment involves such a 
mixture of DNA fragments, it is re- 
ferred to as a "shotgun” and will call 
for a certain level of containment. Ex- 
periments involving such mixtures of 
DNA fragments are assumed to be of 
higher potential hazard than those 
done with a single, purified fragment, 
because of the greater likelihood of 
dangerous and unknown genes being 
introduced into a recipient cell Puri- 
fied fragments containing mainly 
genes whose properties are known and 
are not harmful offer less potential 
hazard than a shotgun experiment. 
In some instances, the foreign DNA 
will be derived from extra chromoso- 
mal genetic elements. Such elements 
include the DNA of animal viruses, 
plant viruses, other eukaryote organ- 
elles such as mitochondria and chloro- 
p lasts, as well as prokaryote plasmids 
or bacteriophages of the same type 
used as vectors. Each of these cases is 
treated separately in the guidelines. 
The prokaryote sources are treated 
differently, depending on whether the 
•In the PRG-NTH the EK systems sue In- 
cluded in the broader HV (host-vector) cate- 
gories created to accommodate potential 
systems ranging beyond E. Coli K-12. 
"foreign” DNA is from an organism 
that does or does not exchange genetic 
information with E. Coli in nature. 
The physical and biological contain- 
ment is listed for various possible DNA 
sources: both must be used, as they 
complement each other. For example. 
DNA from primates requires the most 
stringent containment, since the esti- 
mated potential hazard, either from 
genes that might function in humans 
with untoward effects or from patho- 
genic viral DNA's residing in primate 
tissue, is judged to be most serious. 
The experiments now require either 
P3 + EK3 or P4 + EK2; and it should 
be noted that only the latter combina- 
tion is feasible at present, and then 
only at the limited number of P4 fa- 
cilities. 
In two instances— primates and cold- 
blooded vertebrates — containment re- 
quirements are lower if the DNA is iso- 
lated from embryonic tissue, or germ- 
line material, since such material is 
less likely to be contaminated by 
pathogenic viruses than is adult tissue. 
Thus, if the foreign DNA is from cold- 
blooded vertebrates, P2 and EK2 are 
required, but P2 and EK1 can be used 
if the DNA is from embroyonic or 
germ-line tissues. If the cold-blooded 
vertebrate is known to produce a 
potent toxin, P3 and EK2 must be 
used. In some instances — lower eukar- 
yotes, for example— the guidelines re- 
quire more or less stringent condi- 
tions, depending on whether or not 
the source of foreign DNA is known to 
be pathogenic or toxigenic, or might 
be infected with a pathogen, or is 
known to make a harmful product. 
The guidelines for shotgun experi- 
ments, when the source of the DNA is 
a prokaryotic organism, may be sum- 
marized. First those prokaryotes that 
are known to exchange genetic infor- 
mation with E. Coli in nature are con- 
sidered. The containment require- 
ments are low for this group, since it is 
unlikely that the experiments will 
create new genetic combinations. Re- 
quirements vary with the pathogeni- 
city of the source of foreign DNA. 
When the source is a prokaryote that 
does not naturally exchange genetic 
material with E. Coli, the containment 
recommendations are high, for there 
is a greater potential for new genetic 
combinations to be formed and ex- 
pressed. Further, it is assumed that 
the more similar the DNA’s of donor 
and host, the greater the probability 
of expression of foreign DNA or of 
derepression of host genes. 
Characterized clones obtained from 
shotgun experiments may not be as 
potentially hazardous as the original 
mixture of cells. Cloning of the recipi- 
ent host cell containing the DNA frag- 
ment of interest will be one of the 
normal aims of any recombinant DNA 
experiment. The guidelines state that 
FEDERAL REGISTER, VOL 43, NO. 146— FRIDAY, JULY 28, 1978 
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