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Federal Register / Vol. 46, No. 126 / Wednesday, July 1, 1981 / Notices 
from insects, snakes etc.).” an Ad hoc 
Working Group on Toxins composed of 
Drs. Werner Maas and Alan Bernheimer 
of New York University, Dr. John Collier 
of Yale University, Dr. Michael Gill of 
Tufts University, Dr. Susan Gottesman 
of NIH, Dr. Myron Levine of the 
University of Maryland, and Dr. James 
Mason of the Utah State Department of 
Health, was convened to recommend 
containment conditions for recombinant 
DNA experiments with genes coding for 
toxins. The group conversed by 
telephone on November 4 and 
November 21, 1980, was convened at the 
National Institutes of Health on January 
7, 1981, and participated in telephone 
conference calls on February 23 and 
March 2, 1981. The following proposal, 
developed by the ad hoc group, 
appeared in the Federal Register of 
March 20, 1981 (46 FR 17996): 
"A. Section I-D-2 of Section I-D, 
Prohibitions, would be amended to read as 
follows: 
“l-D-2. Deliberate formation of 
recombinant DNAs containing genes for the 
biosynthesis of toxins lethal for vertebrates 
at an LDu of less than 100 nanograms per 
kilogram body weight (e.g., the botulinum 
toxins, tetanus toxin, Shigella dysenteriae 
neurotoxin). Guidelines for the cloning of 
DNAs containing genes coding for the 
biosynthesis of toxins which are lethal to 
vertebrates at 100 nanograms to 100 
micrograms per kilogram body weight are 
specified in Appendix G. 
B. A new Appendix G. would be added to 
the Guidelines as follows: 
Appendix G — Containment Conditions for 
Cloning of Cenes Coding for the Biosynthesis 
of Toxins for Vertebrates 
1. General Information 
“Appendix G specifies the containment to 
be used for the cloning of genes coding for 
the biosynthesis of toxins for vertebrates. 
Gloning 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, Shigella 
dysenteriae neurotoxin) is prohibited. No 
specific restrictions 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. 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 shall 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-{3Hi)). 
2. 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., diphtheria toxin, 
abrin, Clostridium perfringens epsilon toxin) 
may proceed under P2 + EK2 or P3 + EKl 
containment conditions. 
(b) Cloning of genps for the biosynthesis of 
toxins for vertebrates with an LDso in the 
range of 1 microgram to 100 micrograms per 
kilogram body weight may proceed under 
Section Ill-O (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, 
whose effects are confined to the stimulation 
of intestinal secretion and whose effects can 
be entirely reversed by administration of 
electrolyte solutions, shall be subject to 
Section III-O. These are cholera toxin, the 
heat labile toxins of E. coli, Klebsiella, and 
other related proteins that may be identitied 
by neutralization with an antiserum 
monospecific for cholera toxin, and the heat 
stable toxins of E. coli and of Yersinia 
enterocolitica. 
3. Containment Conditions for Cloning of 
Toxins 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 will be 
evaluated by ORDA, which will consult with 
the ad hoc working group on toxins. (See 
Section IV-E-l-b-(3)-(j)). 
C. Section V, Footnote 2A would be 
modified to delete the words: ‘toxins potent 
for vertebrates (Section I-D-2).' 
D. A new Section IV-E-l-b^3)-(i) would 
be added as follows: ‘IV-E-l-b-(3)-(i). 
Adding new entries to the list of toxins for 
vertebrates. (See Appendix G.)’ 
E. A new Section IV-E-l-b-(3)-(j) would 
be added as follows: ‘IV-E-l-b(3)-(j). 
Approving the cloning of toxins genes in host- 
vector systems other than E. coli K-12. (See 
Appendix G.)’" 
During the thirty day comment period, 
no comments were received. However, 
two letters vyere received after the thirty 
day comment period. One expressed 
concern about experiments involving 
genes which control bacterial toxins 
recommended “against relaxation of the 
Guidelines.” The other urged the 
proposed changes not be made “until 
adequate risk assessment studies have 
been performed.” 
Several members of the ad hoc 
Working Group on Toxins, who were 
members of the RAG, presented the 
proposal to the RAC at the April 23-24, 
1981, meeting. Working group members 
characterized the proposal as 
conservative. 
Noting the RAC recommendation at 
this meeting concerning containment 
conditions for E. coli K-12 and 
Saccharomyces cerevisiae host-vector 
systems currently covered under Section 
III-O of the Guidelines, ad hoc Working 
Group members recommended that 
language in the proposed Appendix G be 
amended. They said the original intent 
of the ad hoc group with regard to 
cloning genes for toxins with an LDso in 
the range of 1 to 100 micrograms, as well 
as for cloning enterotoxin genes, was to 
specify Pi -(-EKl containment. Thy felt it 
would be inappropriate to “exempt” 
from the Guidelines the cloning of toxin 
genes at this time. In response, the RAC 
recommended that Section 2-(b) and 2- 
(c) in proposed Appendix G be amended 
by substituting "Pi -|- EKl” for “Section 
III-O.” 
RAC also discussed the 
appropriateness of the introductory 
language “. . . whose effects are 
confined to the stimulation of intestinal 
secretion and whose effects can be 
entirely reversed by administration of 
electrolyte solutions” in Section 2-(c) of 
proposed Appendix G, and 
recommended its deletion. | 
RAC also discussed the intention of I 
the working group that Appendix G | 
specifications override other I 
specifications of the Guidelines (e.g., 1 
exemptions or return to host of origin i 
experiments). RAC recommended this j 
conservative approach at this time, and I 
specified wording to be added at the end j 
of Section I-D-2 indicating this. 
Finally, RAC indicated a desire that • 
the NIH be kept abreast of experiments I 
dealing with the cloning of toxin genes, | 
and amended the proposed language to ' ■ 
require prior registration with ORDA of j j 
experiments involving the cloning of i j 
toxin genes. i , 
After incorporating these changes, the i 
RAC by a vote of 18 in favor, 0 opposed, " 
with 1 abstention, recommended the j “ 
following amended language: ^ 
A. Section I-D-2 of Section I-D, ' j 
Prohibitions, would be amended to read 1» 
as follows: j « 
l-D-2. Deliberate formation of recombinant 
DNAs containing genes for the biosynthesis ^ 
-of toxins lethal for vertebrates at an LDso of u 
less than 100 nanograms per kilogram body n; 
weight (e.g.. the botulinum toxins, tetanus ti 
toxin. Shigella dysenteriae neurotoxin). | ^ 
Guidelines for the cloning of DNAs 
containing genes coding for the biosynthesis 
of toxins which are lethal to vertebrates at | 
100 nanograms to 100 micrograms per ^ 
kilogram body weight are specified in 
Appendix G. which overrides other parts of , 
the Guidelines (e.g., exemptions, return to ij, 
host of origin, etc.]. 
B. A new Appendix G, would read as ( 
follows: ' Ho 
[ 144 ] 
