59386 
Federal Register / Vol. 46, No. 233 / Friday, December 4, 1981 / Notices 
organisms, the altered individual would 
not necessarily sit on the inventor's 
shelf, like some new and terribly 
explosive chemical, but might be able to 
establish itself in the environment and. 
' because of its ability to replicate, 
magnify any harmful effect. Three 
< assumptions are therefore involved in 
the decision to call recombinant DNA a 
unique danger; each one of these 
{ assumptions can be separately 
discussed and evaluated. These 
assumptions are: 
j (1) That a unique organism, never 
I found in nature, might be constructed by 
!i recombinant DNA techniques; 
I (2) That such a unique organism might 
I be able to establish itself in the 
! environment outside the laboratory; 
i (.1) That such an organism, established 
I in the environment and possessing 
j unique properties bestowed upon it by 
recombinant DNA techniques, might be 
harmful, either to man. animals, or 
‘ plants. 
If one could prove that any one of 
I these three assumptions is totally false, 
the dire expectations of the recombinant 
DNA technique could be discarded and 
no special precautions would need to be 
I taken. In the initial assessment, 
however, these assumptions seemed 
. reasonable, in that the technique 
appeared to allow any organism to be 
mofidied in any way. 
Over the last seven years, some 
changes in perception and in knowledge 
of recombinant DNA hazards have 
occurred: 
(1) The possibilities and limits of 
assumption 1 are somewhat better 
understood in (a) the limits to which 
recombinant DNA can bestow new 
characteristics on organisms, and (b) the 
ways by which organisms normally 
I exchange genetic information in 
I anticipation of the recombinant DNA 
I technique, thereby negating the 
I ’uniqueness" component of the 
I assumption. 
(2) A great deal of discussion in the 
I last few years has centered on 
. assumption 2; the whole concept of 
! biological containment is based upon 
! circumventing the organisms' 
establishment in the environment by 
t modifying the properties of the 
I organism. In the case of E. coli K-12, 
I specific tests to asses the likelihood of 
I establishment, with or without 
i recombinant DNA. have been carried 
j out (see appendix), and have suggested 
I that such establishment is unlikely. In 
j the more general sense, can any change 
. made in the laboratory either 
I intentionally or unintentionally endow 
inn organism with a competitive 
I advantage in a very competitive outside 
; environment? Seven years experience 
l 
with recombinant DNA has suggested 
that recombinant DNA is frequently not 
stably maintained in the host organism 
in the absence of selective pressures. 
(3) Assumption 3. that such a unique 
established organism will cause harm, 
remains primarily one of discussion, 
since experiments where one might 
explicitly predict harm have been for the 
most part forbidden or discouraged. 
Some explicit experiments to assess 
risks have been performed: I.e., 
examination of whether E. coli can act 
as a vector to introduce viral DNA into 
animal cells (Israel et al., 1979, a, b; 
Chan et al., 1979), and whether E. coli 
manufacturing active hormones can 
affect the physiology of animals 
(Stebbing et al.. 1980). Much of the other 
discussion involves unknowns, although 
understanding of the basis for 
pathogenicity of some organisms can 
help in predicting the probable effects of 
introducing new genetic information. 
We will try to analyze (1) the kinds of 
information available to support or 
disprove each of these assumptions, (2) 
how these assumptions have been dealt 
with in past Guidelines, and (3) how 
they might be dealt with in the future. 
■The early Guidelines presumed that, 
since the possibility of harm could not 
be properly evaluated, all recombinants 
were potentially harmful; the Guidelines 
controlled the establishment of 
recombinant DNA containing organisms 
in the environment by indicating levels 
of physical and biological containment. 
More recently, the Guidelines have 
evolved to include the concept that only 
unique organisms should be of concern. 
This concept is the basis for the long list 
of exemptions in the 1978 Guideline 
revision. Other relaxations in the 
Guidelines concerning E. coii K-12 host- 
vector systems have resulted from 
further examination of the second 
assumption, as evidence accumulated 
on the inability of E. coii K-12 to 
establish itself in the environment. 
Acciderftai vs. intentionai Cioning of 
a Harmfui Segment. One other aspect of 
the change from early to more recent 
attitudes can be traced to the qualitative 
difference between accidental 
constructs of unknown recombinants 
and the intentional cloning of a specific 
gene. The early Guidelines in their 
concern for inadvertant combinations of 
potentially harmful genes, required high 
containment for experiments in which 
random pieces of DNA from one 
organism are inserted into a second 
organism (shotgun experiment). 
Advances in our understanding of how 
expression of mammalian genes 
(eukaryotes) differs from expression of 
genes in lower forms of life such as 
bacterid (prokaryotes), has led to a 
[275] 
perception that the accidental altering of 
an organism is very unlikely to produce 
a harmful result. Thus, some lessening of 
containment for such "shotgun” 
experiments has seemed justified. The 
other concern, and probably the basis 
for the remaining concerns, centers on 
the deliberate construction of a 
recombinant organism expressing a 
foreign product. The rapidly developing 
techniques of recombinant DNA 
technology can be utilized to insure that 
the recombinant DNA in fact efficiently 
expresses a product in its new host. The 
organism can, in addition, be engineered 
to excrete the product into the outside 
environment. In such a case, questions 
about possible harm can be posed 
without considering assumptions on the 
likelihood of expression or the 
possibility of one DNA fragment out of 
many establishing itself. 
1. Uniqueness of Organisms Created 
by Recombinant DNA Techniques. 
A. Limits of the techniques. 
The original assumption that DNA 
from essentially any source could be 
introduced into essentially any organism 
has been supported by research 
advances of the last six years. Methods 
have been developed for introducing 
foreign DNA stably into a variety of 
bacteria, yeasts, plants and animal cells. 
In some organisms, the nature of the 
vector or the growth pressures on the 
organism limit the amount of new 
information which can be introduced as 
recombinant DNA. For example, many 
viral vectors have size limits on the 
amount of introduced DNA; only a 
specific amount can be packaged into 
viral the coat (Blattner et al., 1977). 
Some bacterial plasmids become harder 
to maintain stably in cells as their size 
increases. In practice, many 
recombinant DNA experiments are more 
productive when relatively small, 
defined pieces of the foreign DNA of 
interest are used. In considering the 
general limits for the purposes of risk 
considerations, it seems fair to say that 
there is no limit to either the source of 
DNA (donor) or of the host (recipient) 
but that size of the donor DNA may be 
limited, especially where viral vectors 
are used. 
b. Naturai exchange mechanisms. 
The earliest discussions of the 
recombinant DNA technique considered 
the well known ability of E. coii to 
exchange DNA with other organisms as 
a possible hazard of using the 
recombinant DNA technique in that 
host-vector system. Such exchange 
might disseminate recombinant DNA 
from an E. coii K-12 host to other less 
well characterized and less disabled 
hosts; therefore, use of plasmids that 
:r 
