Federal Register / Vol. 46, No. 233 / Friday, December 4, 1981 / Notices 
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Appendix D [Deleted] 
Appendix E [Deleted] 
Appendix F. — Certified Host-Vector 
Systems 
A listing of host-vector systems 
previously classified as HVl or HV2 
follows. 
HVl — The following plasmids are 
accepted as the vector components of 
certified B. subtilis HVl systems; 
pUBllO, pCl94, pSl94, pSA2100, pE194, 
pTl27, pUBll2, pC221, pC223, and 
pABl24. B. subtilis strains RUB 331 and 
BGSC 1S53 have been certified as the 
host component of HVl systems based 
on these plasmids. 
HVl — The following specified strains 
of Neurospora crassa which have been 
modified to prevent aerial dispersion: 
(1) ini (inositolless) strains 37102, 
37401, 46316, 64001, and 89601. 
(2) csp-1 strain UCLA and csp-2 
strains FS 590, UCLAlOl (these are 
conidial separation mutants). 
(3) eas strain UCLAlOl (an “easily 
wettable” mutant). 
HVl — The following Streptomyces 
species: Streptomyces coelicolor, S. 
lividans, S. parvulus, and S. griseus. The 
following are accepted as vector 
components of certified Streptomyces 
HVl systems: Streptomyces plasmids 
SCP2, SLP1.2, pIJlOl, actinophage phi 
C31, and their derivatives. 
HV2 — The asporogenic mutant 
derivative of Bacillus subtilis, ASB 298, 
with the following plasmids as the 
vector component: pUBllO, pCl94, 
pSl94, pSA2100, pEl94, pTl27, pUB112, 
pC221, pC223, and pABl24. 
HV2 — The following sterile strains of 
Saccharomyces cerevisiae, all of which 
have the ste-VC9 mutation, SHYl, 
SHY2, SHY3, and SHY4. The following 
plasmids are certified for use: YIpl, 
YEp2, YEp4, YIp5, YEp6, YRp7, YEp20, 
YEp21, YEp24, YIp25, YIp26, YIp27, 
YIp28, YIp29, YIp30, YIp31, YIp32 and 
YIp33. 
EK2 Plasmid Systems. The E. coli K- 
12 strain chi-1776. The following 
plasmids are certified for use: pSClOl, 
pMB9, pBR313, pBR322, pDH24, pBR327, 
pGLlOl, pHBl. The following E. coli/S. 
cerevisiae hybrid plasmids are certified 
as EK2 vectors when used in E. coli chi- 
1776 or in the sterile yeast strains, 
SHYl, SHY2, SHY3 and SHY4: YIpl, 
YEp2, YEp4, YIp5, YEp6, YRp7, YEp20, 
YEp21, YEp24, YIp25, YIp26, YIp27, 
YIp28, YIp29, YIp30, YIp31, YIp32, YIp33. 
EK2 Bacteriophage Systems. The 
following are certified EK2 systems 
based on bacteriophage lambda: 
Vector Host 
AgttV'£'S. AB' DPSOsupF 
AglWfiS. AB* DPSOsupF 
AgtZ)v/r. AB’ 
A gtALO. A B 
Charon 3A 
Charon 4A 
Charon 16A 
Charon 2lA 
Charon 23A 
Charon 24A 
E. coli K-12 
DPSOsupF 
DPSO or DPSOsupF 
DPSO or DPSOsupF 
DPSO or DPSOsupF 
DPSOsupF 
DPSO or DPSOsupF 
DPSO or DPSOsupF 
Appendix G — Containment Conditions 
for Cloning of Genes Coding for the 
Biosynthesis of Toxins for Vertebrates 
1. General Information. 
Appendix G specifies the containment 
to be used for the deliberate cloning of 
genes coding for the biosynthesis of 
toxins for vertebrates. Cloning of genes 
coding for toxins for vertebrates that 
have an LDso of less than 100 nanograms 
per kilogram body weight (e.g., the 
botulinum toxins, tetanus toxin, 
diphtheria toxin. Shigella dysenteriae 
neurotoxin) should not be performed. No 
specific recommendations other than 
those given in Part III of the Guidelines 
shall apply to the cloning of genes if the 
protein specified by the gene has an 
LDso of 100 micrograms or more per 
kilogram of body weight. Experiments 
involving genes coding for toxins with 
an LDso of 100 micrograms or less per 
kilogram body weight should be 
registered with ORDA prior to initiating 
the experiments. A list of toxins 
classified as to LDso is available from 
ORDA. Testing procedures for 
determining toxicity of toxins not on the 
list are available from ORDA. The 
results of such tests should be 
forwarded to ORDA, which will consult 
with the ad hoc Working Group on 
toxins prior to inclusion of the toxin on 
the list. (See Section IV-E-l-b-(3)-(i).) 
2. Recommended Containment 
Conditions for Cloning of Toxin Genes 
in E. coli K-12. 
(a) Cloning of genes coding for toxins 
for vertebrates that have an LDso in the 
range of 100 nanograms to 1000 
nanograms per kilogram body weight 
(e.g., abrin, Clostridium perfringens 
epsilon toxin) should proceed under 
P2 -f EK2 or P3 + EKl containment 
conditions. 
(b) Cloning of genes for the 
biosynthesis of toxins for vertebrates 
with an LDso in the range of 1 microgram 
to 100 micrograms per kilogram body 
weight should proceed under Pi -t-EKl 
containment conditions (e.g.. 
Staphylococcus aureus alpha toxin. 
Staphylococcus aureus beta toxin, ricin. 
Pseudomonas aeruginosa exotoxin A, 
Bordatella pertussis toxin, the lethal 
factor of Bacillus anthracis, the 
Pasteurella pestis murine toxins, the 
oxygen-labile hemolysins such as 
streptolysin O, and certain neurotoxins 
present in snake venoms and other 
venoms). 
(c) Some enterotoxins are 
substantially more toxic when 
administered enterally than 
parenterally. The following enterotoxins 
should be subject to Pi -|- EKl 
containment conditions: cholera toxin, 
the heat labile toxins of E. coli, 
Klebsiella, and other related proteins 
that may be identified by neutralization 
with an antiserum monospecibc for 
cholera toxin, and the heat stable toxins 
of E. coli and of Yersinia enterocolitica. 
3. Containment Conditions for t 
Cloning of Toxin Genes in Organisms 
Other than E. coli K-12. Requests 
involving the cloning of genes coding for 
toxins for vertebrates in host-vector 
systems other than E. coli K-12 should 
be submitted to ORDA for evaluation. 
ORDA will consult with the ad hoc 
working group on toxins. (See Section 
IV-E-l-b-(3)-(j).) 
Appendix H [Deleted] 
Annex A. — Original Proposal of Drs. 
David Baltimore & Allan Campell; 
Proposal To Convert the NIH Guidelines 
Into a Non-regulatory Code of Standard 
Practice and To Reduce the 
Recommended containment Levels For 
Some Experiments 
Proposals 
(1) Section I-A of the NIH Guidelines 
will be replaced with the following: 
"I-A. Purpose. The purpose of these 
Guidelines is to specify standard practices 
for constructing and handling (i) recombinant 
DNA molecules and (ii) organisms and 
viruses containing recombinant DNA 
molecules. Adherence to these standards by 
all laboratories using recombinant DNA is 
recommended.” 
(2) Part I-C of the NIH Guidelines 
shall be eliminated. 
(3) Part III of the Guidelines will be 
replaced with the following: 
"Part III discuss experiments covered by 
the Guidelines. The reader must first consult 
Part I, where listings are given of prohibited 
and exempt experiments. 
“Where there are existing recommended 
physical containment levels applicable to 
non-recombinant DNA experiments with 
either the host or the vector (such as those 
specified by the GDC Guidelines), 
recombinant DNA experiments should be 
carried out at containment levels at least as 
high as those recommended for non- 
recombinant DNA experiments. Otherwise, 
all non-prohibited experiments may be 
carried out under conditions of PI physical 
containment. As a general practice, 
investigators should use the highest level of 
biological containment (HV3>HV2>HVl) 
which is available and appropriate for the 
purposes of the experiment. 
"Specific exceptions to the prohibitions 
may be approved by the Director NIH 
(section I-D). The Director will consider 
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