Federal Register / Vol. 44, No. 232 / Friday, November 30, 1979 / Notices 
69237 
(d) Become established in an environment 
permitting its growth and multiplication, 
(e) Contact other living organisms in a 
significant manner, including contact by a 
sufficient number of organisms to ensure 
survival and growth and to cause infection. 
Note that the environment in (d) may be a 
living organism itself. 
In those cases where the detrimental effect 
would result from the formation of a harmful 
protein, the organism containing the 
recombinant DNA would have to — 
(f) Contain a gene for a potentially harmful 
protein. 
(g) Be able to express the foreign gene (that 
is. synthesize the corresponding protein) and 
(h) Synthesize the protein in sufficient 
quantity to be deleterious to the infected 
organism. 
The overall probability of a detrimental 
effect resulting from the formation of a 
harmful protein is then equal to the product 
obtained by multiplying together the 
probability of each event, (a) through (h). 
In those cases where the foreign DNA itself 
might be the cause of undesirable effects, 
another set of events must be considered. 
Where the foreign DNA is presumed to 
increase the pathogenicity of the initial host 
cell or virus, the inserted DNA must — 
(i) Impart a selective advantage for growth 
to the carrier of the recombinant DNA as 
compared with the original cell or virus. 
(j) Alter the metabolism of the carrier so 
that it becomes disease-producing. 
The overall probability of a detrimental 
effect by this mechanism is then equal to the 
product obtained by multiplying together the 
possibilities of event^ (a) through (e) times (i) 
through (j). 
In the case where the foreign DNA is 
presumed to cause undesirable effects by 
virtue of its transfer out of the original 
recipient and reinsertion into cells of another 
species, the DNA must — 
(k) Leave the original recipient without 
being destroyed. 
reversion of 
^ disabling properties ^ 
-> escape 
4 persistence 
\ 
transfer of plasmid 
to wild type strain 
(l) Survive transfer to another cell, 
(m) Become associated with the other cell 
in a stable manner, either as an independent 
element or by natural recombination. 
The overall probability of an undesirable 
effect arising by means of the secondary 
transfer mechanism is equal to the product 
obtained by multiplying together the 
probabilities of events (a) through (e) and (k) 
through (m). 
A more recent framework for 
assessing the risks of recombinant DNA 
experiments was prepared by Dr. Sidney 
Brenner for the United Kingdom Genetic 
Manipulation Advisory Group. This 
document, which is part of the 
"Background Documents on E. coli K- 
12/Pl Recommendation” available from 
ORDA, was discussed by the RAC at 
their September, 1979, meeting. The 
Brenner document uses "generation 
trees." He gives examples such as: 
outcome 
acquisition of 
a plasmid from - 
a natural donor 
transfer by 
mobilization 
to wild type 
strain 
outcome 
or 
escape > persistence 
As Dr. Brenner points out in a letter of 
July 26, "It is important to recognize that 
if any of the factors which are used can 
be assigned zero, then everything must 
be zero for that particular event. Thus if 
something does not express, that is 
enough: if it expresses and if the product 
has no biological target, that too is 
enough: and if it expresses and has a 
biological target and cannot gain access 
to it, that also is of no consequence." 
Both frameworks (i.e„ that of the EIS 
or of Brenner) lead to the conclusion 
that if any of the multiple steps leading 
to the final outcome has a zero 
probability, then the probability of the 
final outcome is zero. If no step has zero 
probability but if each sequential step 
leading to the final outcome has a low 
probability, then the final outcome has 
an exceedingly low probability. 
What is the Likelihood of E. coli K-12 
Escape From A Pi Laboratory? 
For an organism containing 
reco 'binant DNA to cause a public 
health hazard, it must first escape from 
the laboratory. 
The NIH Guidelines specify in Section 
II— A that: 
The first principle of containment is a strict 
adherence to good microbiological practices. 
Consequently, all personnel directly or 
indirectly involved in experiments on 
recombinant DNAs must receive adequate 
instruction. This shall as a minimum include 
instructions in aseptic techniques and in the 
biology of the organisms used in the 
experiments, so that the potential biohazards 
can be understood and appreciated. 
The definition of Pi physical 
containment in addition requires the 
following: 
"II— B— 1— a— (1). Laboratory doors shall be 
kept closed while experiments are in 
progress. 
II— B— 1— a— (2). Work surfaces shall be 
decontaminated daily, and immediately 
following spills of organisms containing 
recombinant DNA molecules. 
II— B— 1— a— (3). All biological wastes shall be 
decontaminated before disposal. Other 
contaminated materials, such as glassware, 
[ 223 ] 
animal cages, and laboratory equipment 
shall be decontaminated before washing, 
reuse, or disposal. 
II— B— 1— a— (4). Mechanical pipetting devices 
shall be used: pipetting by mouth is 
prohibited. 
II— B— 1— a— (5). Eating, drinking, smoking, and 
storage of foods are not permitted in the 
laboratory area in which recombinant DNA 
materials are handled. 
II— B— 1— a— (6). Persons shall wash their 
hands after handling organisms containing 
recombinant DNA molecules and when they 
leave the laboratory. 
II— B— 1— a— (7). Care shall be taken in the 
conduct of all procedures to minimize the 
creation of aerosols. 
II— B— 1— a— (8). Contaminated materials that 
are to be decontaminated at a site away from 
the laboratory shall be placed in a durable 
leak-proof container, which is closed before 
removal from the laboratory. 
II— B— 1— a— (9). An insect and rodent control 
program shall be instituted. 
II— B— 1— a— (10). The use of laboratory gowns 
coats, or uniforms is discretionary with the 
laboratory supervisor. 
II— B— 1— a— (11). Use of the hypodermic 
needle and syringe shall be avoided when 
alternative methods are available. 
