33112 
NOTICES 
logical containment and are defined as 
EK1. 
As with physical containment levels, 
increasing numbers specify increasing 
levels of biological containment for E. 
coli systems. For the next level, called 
EK2, host-vector combinations must 
be demonstrated to provide a high 
level of biological containment by suit- 
able laboratory tests. Such combina- 
tions are obtained by genetic modifica- 
tion of either E. coli K-12 host cells or 
the relevant plasmids and bacterio- 
phage or both. Various examples of 
the types of necessary modifications 
are suggested in the guidelines. 
One additional level of contained E. 
coli host-vector systems, defined in 
the guidelines, is called EK3. EK3 sys- 
tems are EK2 systems for which the 
specified containment properties have 
been demonstrated, not only by micro- 
biological and genetic analysis but by 
appropriate tests In animals, including 
humans or primates and other rele- 
vant environments. 
EK2 and EK3 host-vector systems 
must be certified as such by the Direc- 
tor. N1H. after evaluation and recom- 
mendation by the Recombinant Advi- 
sory Committee. 
Alternative *: RAC -Proposed Revisions 
Physical containment 
Two major changes wpre proposed in 
the physical containment section of 
the PRG-KAC. One deals with the or- 
ganization of the section: the other in- 
corporates Into the PRO-RAC the 
philosophy and guidance of the report 
of the NTH/European Molecular Biol- 
ogy Organization (EMBO) workshop 
on parameters of physical contain- 
ment. 
Physical containment requirements 
for each P-level have been organized 
under the topic headings "laboratory 
Practices.” "Containment Equip- 
ment.” and “Special Laboratory 
Design.” This was done to emphasize 
the importance of laboratory practices 
and containment equipment in achiev- 
ing the desired safety objective. 
Other proposed revisions contained 
in the physical containment section 
reflect a conscious effort to encourage 
international uniformity with respect 
to recombinant DNA guidelines. This 
has been achieved by revising the con- 
tainment descriptions so that they are 
consistent with the guidance provided 
In the NIH/EMBO report. In addition, 
some statements have been rewritten 
and others added in order to clarify 
the basic requirements for each level 
of containment. The most significant 
clarifications have been made in the 
areas on containment equipment and 
special facility design. The revisions, 
however, have not resulted In chang- 
ing the purpose or intent of the physi- 
cal containment descriptions in the 
1976 guidelines. 
One specific addition to the PRG- 
RAC that has origuiated from the 
NIH, EMRO report is the inclusion of 
design criteria for an area of the labo- 
ratory in which personnel wear posi- 
tive-pressure suits ventilated by life- 
support systems. This added approach 
provides a level of physical contain- 
ment equivalent to that afforded by 
glove-box cabinets at the P4 level. 
Other Important recommended 
changes include— 
• Certain good microbiological prac- 
tices are mandatory at the PI level Li 
the PRG-RAC (the 1976 guidelines en- 
courage but do not require such prac- 
tices): 
• At the P2 level, prohibitions 
against eating, drinking, smoking, and 
storage of foods have been extended 
from the work area to the entire labo- 
ratory; 
• The universal biohazard sign is 
now required at the P2 let el. and its 
use has been extended to equipment 
such as freezers and refrigerators in 
which organisms containing recom- 
binant DNA molecules are stored; 
• Access procedures have been spec- 
ified for controlled areas adjacent to 
P3 laboratories; 
• Installation of foot-, elbow-, or 
automatically-operated facilities for 
washing hands Is now required for all 
laboratcries in which P3-level work is 
done: 
• Specific guidance on containment 
equipment appropriate for laboratory 
animals has been added to the P3 and 
P4 sections; and 
• The labeling requirements for 
shipment of etioicgic agents now 
apply to all organisms containing re- 
combinant DNA moleciles. Thus, the 
Center for Disease Control. U.S. 
Public Health Service, must be noti- 
fied in the event of any accidental 
breakage during shipment. ALso, 
agents requiring P4 containment must 
be packaged according to strict Feder- 
al standards and shipped by registered 
mail or an equivalent system that pro- 
vides for sending notifications to the 
shipper upon delivery. 
The PRG-NIH adopt these sugges- 
tions in large part. Thus, they 
strengthen the safety standards and 
procedures for physical containment 
and move toward international agree- 
ment. 
Biological containment 
The PRG-RAC describe the catego- 
ries of hosts and vectors to be used in 
minimizing the spread of organisms 
containing recombinant DNA. The 
PRG-RAC differ from the 1976 guide- 
lines in that they were expanded to in- 
clude (1) further definitions of host- 
vector systems, (2) a more restrictive 
set of requirements for HV3 systems 
(see below), and (3) a new section de- 
scribing mechanisms for certification. 
Definitions of Host-Vector Systems. 
A new nomenclature— 1TV1. HV2, and 
HV3— was developed to incorporate a 
variety of hosts and vectors into the 
framework initially established for E. 
coli K-12. In particular, the PRG- 
RAC provide criteria for HV1 systems 
oiher than E. coli K-12. In the 1976 
Guidelines, cloning systems other E. 
cop FI-12 were to be considered only if 
superior to K-12 in containment prop- 
erties: but it is now recognized that 
many useful experiments can only be 
conducted using host-vector systems 
other than those based on E. coli K- 
12. and that such experiments should 
be permitted so long as the proposed 
system provides equivalent biological 
containment. The new HV1 criteria 
provide a structure for approval of sys- 
tems that meet these requirements.' 
Requirements for HV3 Systems. 
These have been made more stringent 
than the corresponding requirements 
for EK3 in the 1976 guidelines. The 
PRG-RAC require that the vector be 
dependent on its propagation host or 
be highly defective in mobilizability. 
Also, it may carry no markers confer- 
ring resistance to antibiotics used 
clinically or in agriculture. This provi- 
sion should preclude any inadvertent 
advantage for recomblnant-DNA-bcar- 
ing vectors that encounter antibiotics 
in the environment. 
Certification of Host-Vector Systems. 
A ntw section has been added detail- 
ing the responsibility for certification 
of HV1. HV2. and HV3 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 2 years and therefore represents 
no change from practices under the 
1976 guidelines. 
These recommendations, reflected in 
the proposed revisions, set improved 
procedures and standards to meet en- 
vironmental and occupational con- 
cerns. 
Physical and biological interaction 
Another specific addition to the 
guidelines that has originated from 
the NIH/EMBO report is particularly 
notable— the addition of tables I and 
II to the P3 and P4 sections, respec- 
tively. These tables show combinations 
of physical and biological safeguards 
that provide similar protection. This 
approach allows flexibility in selecting 
comtainment equipment for a particu- 
lar study without compromising 
safety. 
Alternatives: Public Commentators 
Commentators have expressed par- 
ticular concern over (1) the flexibility 
•Under the proposed revisions, new HVl's 
need not offer a distinct advantage over E. 
coli K-12 host-vectors, need not be capable 
of modification to HV2 and HV3. and need 
not be class 1 etiologic agents. 
FEDERAL REGISTER, VOL 43, NO. 144 — FRIDAY, JULY 28, 1978 
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