NOTICES 
49601 
(T) Data on all other properties of the sys- 
tem which affect containment and utility, 
including Information on yields ol phage or 
plasmid molecules, ease of DNA isolation, 
and ease of transfection or transformation 
(▼i) In some cases, the Investigator may 
be asked to submit data on survival and vec- 
tor tranamiaelMUty from experiments in 
which the host- vector is fed to laboratory 
animals (eg. rodents). Such In vivo data 
may be required to confirm the validity of 
predicting in vivo survival on the basis of 
In vitro experiments. 
Data must be submitted In writing to the 
NTH Office of Recombinant DNA Activities 
Ten to twelve weeks are normally required 
dor review and circulation of the data prior 
to the meeting at which such data can be 
considered by the Recombinant DNA Mole- 
cule Program Advisory Committee Investi- 
gators are encouraged to publish their data 
on the construction, properties, and testing 
of proposed HV3 systems prior to considera- 
tion of the system by the NIH Recombinant 
DNA Moleculy Program Advisory Committee 
and Its subcommittee 
(•) HVJ ryttrsu Putative HVJ systems 
will, as the first step In certification, be cer- 
tified as HV2 systems Systems which meet 
criteria (1), (1). and (S) for HV3 (See page 
11-34) will then be recommended for HV3 
testing Tests to evaluate various HV2 host- 
vector systems for HVJ certification will be 
performed by contractors selected by NIH 
These contractors will repeat tests performed 
by Individuals proposing the HV2 system and. 
in addition will conduct more extensive tests 
on conditions likely to be encountered in 
nature The genotypic end phenotypic traits 
of HVJ systems will be evaluated, testa on 
survival and tranemlaslblHty In and on ani- 
mals. Including primates, will be performed, 
ee well es tests on survival In eartaln speci- 
fied natural environments 
3. Distribution of certiAed Host-rectors 
Certified HVJ and HVJ host-vector systems 
(plus appropriate control strains) must be 
obtained from the NTH or Its designees, one 
of whom wQl be the tnveetlgator who devel- 
oped the system. NTH shall announce the 
availability of the system by publication of 
notices in appropriate Journals 
Plasmid vectors will be provided In e suit- 
able bast strain, and phage vectors will 
be distributed ee small-volume lyvetee 
If NTH propagate* any of the host strains 
or phage, a sample will be cent to the In- 
vestigator who developed the system or to an 
appropriate contractor, prior to distribution, 
for verification that the material la free from 
contamination and unchanged in pheno- 
typic properties. 
In distributing the certified HVJ and HV3 
host-vector systems. NIH or Its designee will 
( 1 ) send out a complete description of the 
system; ( 11 ) enumerate and describe the 
testa to be performed by the user In order 
to verify Important phenotypic traits: (ill) 
remind the user that any modification of the 
system necessitates independent approval of 
the system by the NTH on recommendation 
of the Recombinant DNA Molecule Program 
Advisory Committee: and (Iv) remind the 
user of responsibility for notifying the NTH 
Office of Recombinant DNA Activities of 
any discrepancies with the reported proper- 
ties or any problems in the safe use of the 
system. 
HI. bmnovTU. Ovmemvxs 
A a a general rule, the level of containment 
required for experiments with novel recom- 
binant DNA shall not be lees than that 
required for handling the most hazardous 
component, t.e.. vector host, or Inserted DNA. 
Handling the purified DNA will require only 
minimal precautions. 
The above rule by Itself effectively pre- 
cludes certain experiments — namely, those In 
which one of the components is In Class o 
of the "Classification of Etlologlc Agents 
on the Basis of Hazard " (See Appendix B). 
as these are excluded from the United States 
by law and U 6 DA administrative policy 
There are additional experiments which are 
not to be performed at this time. These are 
considered prior to presentation of the con- 
tainment guidelines for permissible experi- 
ments. 
A. Experiments that are not to be per- 
formed We recognize that certain of the 
recombinants placed In this category might 
pose nc biohazard and oould be adequately 
contained at this time Nonetheless. It Is 
prudent to defer experiments on these recom- 
binant DNAs until there la more informa- 
tion to assess accurately the danger or lack 
thereof, and to allow the construction of 
more effective biological barriers In this re- 
spect. these Guidelines probably are more 
stringent than those that ultimately will be 
necessary. 
The following experiments are not to be 
Initiated at the present time: (1) Cloning of 
recombinant DNAs derived from the patho- 
genic organisms in Classes 3. 4. and 5 of the 
"Classification of Etlologlc Agents on the 
Baals of Hazard" (See Appendix B). or onco- 
genic viruses classified by NCI as moderate 
risk (See Appendix B). or cells known to be 
Infected with such agents, regardless of the 
host -rector system used (U) Deliberate for- 
mation of recombinant DNAs containing 
genes for the biosynthesis of potent toxins 
(eg. botullnum or diphtheria toxins, 
venoms from Insects, snakes, etc ). (Ill) De- 
liberate creation by the use of recombinant 
DNA of a plant pathogen with Increased 
virulence and host range beyond that which 
occurs by natural genetic enebange (tv) De- 
liberate release Into the environment of any 
organism containing novel recombinant 
DNA. (v) Transfer of e drug resistance trait 
to ml cr oorg an is m s that are not known to ac- 
quire It naturally If such acquisition could 
compromise the use of a drug to control 
dis ea se agents In human or veterinary medi- 
cine or agriculture < vl) Large-scale experi- 
ments (eg. more than 10 liters of culture) 
with organisms containing novel recombi- 
nant DNAs unless the recombinant DNAs 
are rigorously characterised and are shown 
to be free of harmful genes j We differentiate 
between small- and large-scale experiments 
with organisms containing recombinant 
DNAs because the probability of escape from 
containment barriers normally Increases with 
Increasing scale. 
Specific experiments In these categories 
may be excepted from these rules: Provided. 
That these experiments are expressly ap- 
proved by the Director. NIH. on recommen- 
dation of the Recombinant DNA Molecule 
Program Advisory Committee. In making 
such exceptions, weight will be given both to 
scientific and societal benefits and to poten- 
tial risks 
B Containment Gutdeiinej Tor Permis- 
sible Experiments — 1 Classification Of Ex- 
periments Using The E. co U K-12 Host-Vec- 
tor Systems — Most recombinant DNA ex- 
periments currently being done employ E. 
colt K-12 host-vector systems These are also 
the systems for which we have the most ex- 
perience and knowledge ( 1 ) regarding the 
effectiveness at biological containment pro- 
vided by existing basis and vectors, and (II) 
necessary for the construction of more effec- 
tive biological barriers 
We therefore consider DNA recombinants 
In E. coll K-12 before proceeding to other 
host-vector systems 
It has been necessary, throughout this sec- 
tion. to use words end phrases such as "•puri- 
fied" or "rigorously characterised " In the 
teat such terms are marked with footnote 
reference numbers These footnotes (Section 
FIDftAl HGISTft. VOL 41, NO. 1I7_ TUESDAY. SEPTEMBER 
V) define more fully what Is Intended by the 
use of these terms. 
In the following classification of contain- 
ment criteria for different kinds of novel 
recombinant DNAs. the stated levels of phys- 
ical and biological containment are mini- 
mum levels The use of higher levels of bio- 
logical containment i EK3'--EK2' EK1 ) Is en- 
couraged If they are available and are equally 
appropriate for the purposes of the experi- 
ment. 
a Shotgun experiments These experi- 
ments Involve' the production of novel re- 
combinant DNAs between the vector and 
portions of the total DNA or (preferably) 
any partially purified fraction thereof from 
the specified cellular source Care should be 
taken either to preclude or eliminate con- 
taminating microorganisms before isolation 
of the DNA. 
(1) Eukaryotic DSA Recombinants. — (a) 
Pnmate* P3 physical containment -an 
EK2 host-vector for DNA from uninfected 
(le. not productively Infected) cells: other- 
wise. f*4 — EKl or P3 - EK3 must be used 
(b! Other mammals P2 physical con- 
tainment -an EK2 host-vector 
(c) Birds P3 physical containment - an 
EK2 host-vector 
(d) Cold-Blooded Vertebrates P3 physi- 
cal containment -an EK2 host-vector or 
P3 - EK 1 except for uncontaminated em- 
bryonic or germllne DNA from laboratory- 
reared animals which requires P2 - EK 1 or 
PI + EKJ If the eukaryote Is known to pro- 
duce a potent polypeptide toxin . 1 the con- 
tainment shall be Increased to P3-EK2 
(ei Other Cold-Blooded Animals And 
Veneer Eukaryotes This large class of 
eukaryotes is divided Into two groups 
1 Species that are known to produce a 
potent polypeptide toxin 1 for vertebrates, or 
are known pathogens listed In Class 2 (See 
Appendix Bi. or are known to carry such 
pathogens must use P3 physical contain- 
ment - an EK2 host-vector When the potent 
toxin Is not polypeptide and Is likely not to 
be the product of closely linked eukaryote 
gene*, containment may be reduced to P3+- 
EKl or P2 - EK2 Species that produce potent 
toxins that affect Invertebrates or plants: 
P2-EK3 or P 3 -EK 1 
Any species that has a demonstrated capac- 
ity for carrying particular pathogenic micro- 
organisms Is Included In this group unless 
It has been shown that those organisms used 
as the source of DNA do not contain these 
agents. In this case they may be placed In 
the following group 
l. The remainder of the species In this 
class P24-EK1 or PI — EK2 However, any 
insect In this group must be either ( 1 ) grown 
under laboratory conditions for at least 10 
generations prior to Its use as a source of 
DNA. or (11) If caught In the wild, must be 
shown to be free of disease-causing micro- 
organisms or must belong to a species that 
does not carry microorganisms causing dis- 
ease in vertebrates or plants. If these con- 
ditions cannot be met. experiments must be 
done under P3+EK1 or P2-EK2 contain- 
ment. 
(f) Plants. P2 physical containment 4 . an 
EK 1 host- vector or P1 + EK2 If the plant 
source carries a known pathogenic micro- 
organism or makes a potent polypeptide tox- 
in . 1 the containment must be raised to P3 
physical containment - 1 - an EK2 host-vector. 
When the potent toxin is not polypeptide and 
is likely not to be the product of closely 
linked eukaryote genes, containment may be 
reduced to P3-EK1 or P2-EK2 
(g) Cloning of tiro! genomes from eukary- 
otic cell DNA Efforts to Isolate and amplify 
any part or all of an endogenous Retrovirus 
genome, or nucleotide sequences identified by 
their homology with a known Retrovirus ge- 
Dome. shall be carried out according to the 
17, 1977 
[ 169 ] 
