NOTICES 
60115 
cific instructions concerning the type 
of data to be submitted to NIH for 
proposed EK2 systems involving either 
plasmids or bacteriophage • in E. coli 
K-12 are available from ORDA. 
II-D-2-b-<3). HV3 Systems. Putative 
HV3 systems must, as the first step in 
certification, be certified as HV2 sys- 
tems. Systems which meet the criteria 
given above under II-D-l-(c>-l, II-D- 
l-(c)-2, II-D-l-(c)-3 will then be rec- 
ommended for HV3 testing. Tests to 
evaluate various HV2 host-vector sys- 
tems for HV3 certification will be per- 
formed by contractors selected by 
NIH. These contractors will repeat 
tests performed by individuals propos- 
ing the HV2 system and, in addition, 
will conduct more extensive tests on 
conditions likely to be encountered in 
nature. The genotypic and phenotypic 
traits of HV2 systems will be evaluat- 
ed. Tests on survival and transmissibil- 
ity in and on animals, including pri- 
mates, will be performed, as well as 
tests on survival in certain specified 
natural environments. 
II-D-3. Distribution of Certified 
Host- Vectors. Certified HV2 and HV3 
host-vector systems (plus appropriate 
control strains) must be obtained from 
the NIH or its designees, one of whom 
will be the investigator who developed 
the system. NIH shall announce the 
availability of the system by publica- 
tion of notices in appropriate Journals. 
Plasmid vectors will be provided in a 
suitable host strain, and phage vectors 
will be distributed as small-volume ly- 
sates. If NIH propagates any of the 
host strains or phage, a sample will be 
sent to the investigator who developed 
the system or to an appropriate con- 
tractor. prior to distribution, for verifi- 
cation that the material is free from 
contamination and unchanged in 
phenotypic properties. 
In distributing the certified HV2 and 
HV3 host-vector systems, NIH or its 
designee will (i) send out a complete 
description of the system: (il) enumer- 
ate and describe the tests to be per- 
formed by the user in order to verify 
important phenotyic traits; (iii) 
remind the user that any modification 
of the system necessitates independ- 
ent approval of the system by the 
NIH: and (iv) remind the user of re- 
sponsibility for notifying ORDA of 
any discrepancies with the reported 
properties or any problems in the safe 
use of the system. 
NIH may also distribute certified 
HV1 host- vector systems. 
III. Containment Guidelines for 
Covered Experiments 
Part III discusses experiments cov- 
ered by the Guidelines. The reader 
must first consult Part I. where list- 
ings are given of prohibited and 
exempt experiments. 
Containment guidelines for permissi- 
ble experiments are given in Part III. 
Changes in these levels for specific ex- 
periments (or the assignment of levels 
to experiments not explicitly consid- 
ered here) may not be instituted with- 
out the express approval of the Direc- 
tor. NIH. (See Sections IV-E-l-b-(l)- 
(a) , IV-E-l-b-(l)-(b), IV-E-l-b-(2)-(b), 
IV-E-l-b-(2)-(c). and IV-E-l-b-(3)- 
(b) .) 
III-A. Classification of Experiments 
Using the E. coli K-12 Host-Vector Sys- 
tems. Most recombinant DNA experi- 
ments currently being done employ E. 
coli K-12 host-vector systems. These 
are the systems for which we have the 
most experience and knowledge (i) re- 
garding the effectiveness of biological 
containment provided by existing 
hosts and vectors and (ii) necessary for 
the construction of more effective bio- 
logical barriers. We therefore consider 
DNA recombinants in E. coli K-12 
before proceeding to other host-vector 
systems. The levels of biological con- 
tainment for E. coli K-12 systems are 
designated EK1, EK2. and EK3 in as- 
cending order. 
It has been necessary, throughout 
this section, to use words and such 
terms are marked with footnote refer- 
ence numbers. These footnotes (Part 
V) define more fully what the terms 
denote. 
In the following classification of con- 
tainment criteria for different kinds of 
recombinant DNAs, the stated levels 
of physical and biological containment 
are minimal for the experiments desig- 
nated. The use of higher levels of bio- 
logical containment (EK3>EK2> 
EK1) is encouraged if they are availa- 
ble and equally appropriate for the 
purposes of the experiment. 
ni-A-1. Shotgun Experiments. 
These experiments involve the produc- 
tion of recombinant DNAs between 
the vector and portions of the speci- 
fied cellular source, preferably a par- 
tially purified fraction. Care should be 
taken either to preclude or eliminate 
contaminating microorganisms before 
isolating the DNA. 
III-A-l-a. Eukaryotic DNA Recom- 
binants. 
III-A-l-a-(I). Primates. P2 physical 
containment + an EK2 host- vector. 
Any lowering of containment below 
these levels (i.e., for purified DNA or 
characterized clones) cannot be made 
solely by an institutional biosafety 
committee but requires NIH approval. 
(See Section IV-E-l-b-(3)-<e).) 
III-A-l-a-<2). Other Mammals. P2 
physical containment + an EK2 host- 
vector. 
III-A-l-aO<3). Birds. P2 physical 
containment + an EK2 host-vector, or 
P3 + EK1. 
III-A-l-a-(4). Cold-Blooded Verte- 
brates. P2 physical containment + an 
EK1 host-vector or P1 + EK2. If the 
eukaryote is known to produce a 
potent polypeptide toxin, [34] the con- 
tainment shall be increased to 
P3 + EK2. 
III-A-l-a-(5). Other Cold-Blooded 
Animals and Lower Eukaryotes. This 
large class of eukaryotes is divided 
into two groups: 
III-A-l-a-(5)-(a). Species that are 
known to produce a potent polypep- 
tide toxin [34] that acts in vertebrates, 
or are known pathogens listed in Class 
2, [1] or are known to carry such path- 
ogens must use P3 physical 
containment + an EK2 host-vector. 
When the potent toxin is not a poly- 
peptide and is likely not to be the 
product of closely linked eukaryote 
genes, containment may be reduced to 
P3 + EK1 or P2 + EK2. Species that 
produce potent toxins that affect in- 
vertebrates or plants but not verte- 
brates require P2 + EK2 or P3 + EK1. 
Any species that has a demonstrated 
capacity for carrying particular patho- 
genic microorganisms is included in 
this group, unless the organisms used 
as the source of DNA have been showm 
not to contain those agents, in which 
case they may be placed in the follow- 
ing group. [2A] 
III-A-l-a-(5)-<b). The remainder of 
the species in this class including 
plant pathogenic or symbiotic fungi 
that do not produce potent toxins: P2 
+ EK1 or PI + EK2. However, any 
Insect in this group must be either (i) 
grown under laboratory conditions for 
at least 10 generations prior to its use 
as a source of DNA, or (ii) if caught in 
the wild, must be shown to be free of 
disease-causing microorganisms or 
must belong to a species that does not 
carry microorganisms causing disease 
in vertebrates or plants. [2A] If these 
conditions cannot be met, experiments 
must be done under P3 + EK1 or P2 + 
EK2 containment. 
III-A-l-a-(6). Plants. P2 physical 
containment + an EK1 host-vector, or 
PI + EK2. If the plant source makes a 
potent polypeptide toxin, [34] the con- 
tainment must be raised to P3 physi- 
cal containment + an EK2 host- 
vector. When the potent toxin is not a 
polypeptide and is likely not to be the 
product of closely linked plant genes, 
containment may be reduced to P3 + 
EK1 or P2 + EK2. [2A] 
III-A-l-b. Prokaryotic DNA Recom- 
binants. 
III-A-l-b-(l). Prokaryotes That Ex- 
change Genetic Information [35] with 
E. Colt Those prokaryotes that ex- 
change genetic information with E. 
coli by knowm physiological processes 
will be exempted from these Guide- 
lines if they appear on the "list of, ex- 
changers” set forth in Appendix A (see 
Section I-E-4). 
For those not on the list, the con- 
tainment levels are PI physical con- 
tainment + an EK1 host- vector. In 
FEDERAL REGISTER, VOL 43, NO. 247— FRIDAY, DECEMBER 22, 1978 
[37] 
