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EK2 containment . Increasing numbers after the designation "EK" 
specify increasing levels of biological containment for E. coli systems. 
The next level is called ''EK2. " Its host-vector combinations must 
be demonstrated by suitable laboratory tests to provide a high level 
of biological containment. Such combinations are obtained by classical 
genetic modification (not recombinant DNA) of E. coli K-12 host cells, 
the relevant plasmids and bacteriophages, or both. More specifically, 
the Guidelines state that in order to qualify as EK2 the modified system 
composed of derivatives of E. coli K-12 and a particular vector should 
not permit survival of a genetic marker carried on the vector in other 
than specially designed laboratory environments at a frequency greater 
-8 
than 10 (1 in 100, 000, 000). Various examples of the types of necessary 
modifications are suggested in the Guidelines. 
EK3 containment; other biological information . One additional level 
of contained E. coli host-vector systems is defined in the Guidelines 
and is called EK~3"! EK3 systems are EK2 systems for which the specified 
containment properties have been demonstrated not only by microbiological 
and genetic analysis but by appropriate tests in animals, including humans 
or primates, and other relevant environments. 
EK2 and EK3 host-vector systems must be certified by the Director 
of NIH after evaluation and recommendation by the Recombinant Advisory 
Committee. Detailed data on the relevant properties of the system 
must be submitted for consideration by the Committee. Detailed state- 
ments of the criteria that must be met for EK2 certification are included 
in Appendix H (I and II). Appendix H-III contains a list of already 
certified EK2 host-vector systems. 
0 
As yet no EK3 systems have been submitted for certification. A 
Workshop on Design of Tests for EK3 Host-Phage -Vector Systems was 
held at NIH on July 19, 1977. A forthcoming report will be available 
from the NIH Office of Recombinant DNA Activities. 
In the case of a vector derived from an animal virus, the virus 
itself must be a low-risk agent (as classified by CDC or the National 
Cancer Institute), and a strain of the virus that is defective in infection 
must serve as the source of the vector DNA. 
D. Combination of Physical and Biological Containment in Practice 
Permissible experiments are assigned required levels of both physical 
and biological containment. The greatest level of safety can be obtained 
by the complementary use of both. It is precisely because neither form 
of containment alone is infallible that the complementary use of the two, 
for every experiment, significantly decreases the likelihood that 
potentially hazardous organisms will escape and cause significant harm. 
