Federal Register / Vol. 46. No. 126 / Wednesday, July 1. 1981 / Notices 
34457 
L Appendix C— ConUiomenI Conditioas for 
I ciociing of G«ne« Coding'tor the Bioaynihetia 
of Toxin* for Vertebrate* 
I. General Information 
Appendix C *pecine* the containment to 
be u*ed for the cloning of gene* coding for 
I the bio*ynihe*i* of toxin* for vertebrate*. 
Cloning of gene* coding for toxin* for 
vertebrate* that have an LOm of le** than 100 
I nanogram* per kilogram body weight (e g., 
I the botulinum ,‘oxin*. tetanu* toxin. Shigella 
dytenteriae neuroloxin) i* prohibited. No 
apecific realriction* ahall apply to the cloning 
of gene* if the protein apeciRed by the gene 
ha* an LDw of 100 microgram* or more per 
I kilogram of body weight. Experiment* 
involving gene* coding for toxiru with an 
LDw of 100 microgram* or lea* per kilogram 
body weight ahall be regialered with OROA 
prior to initiating the experiment*. A liat of 
toxin* claaaified a* to LDw i* available from 
OROA. Teating procedure* for determining 
toxicity of toxin* not on the liat are available 
from OROA. The reault* of auch teat* ahall be 
forwarded to OROA, which will conault with 
the ad hoc Working Croup on toxin* prior to 
incluaion of the toxin on the liat. (See Section 
IV-8-MH3Hl)l 
Z Containment Condition* for Cloning of 
Toxin Genes in £ coli K-JZ 
(a) Doning of gene* coding for toxin* for 
vertebrate* that have an LOm in the range of 
100 nanogram* to 1000 nanogram* per 
kilogram body weight (e.g.. diphtheria toxin, 
adbrin. Clostridium perfnngens epailon 
toxin) may proceed under P2 ■¥■ EJC2 or 
P3 > EKI containment condition*. 
(b) Cloning of gene* for the bioayntheai* of 
toxin* (or vertebrate* with an LOm in the 
range of I microgram to 100 microgram* per 
kilogram body weight may proceed under 
PI -f EKI containment condition* (e.q.. 
Staphylococcus aureus alpha toxin. 
Staphylococcus aureus beta toxin, ridn. 
Pseudomonas Aeruginosa exotoxin A. 
Bordatella pertussis toxin, the lethal factor of 
Bacillus anthracis. the Pasteurella pestis 
munne toxin*, the oxygen-labile hemoly*in* 
auch a* atreptolyain O. and certain 
neurotoxiiu present in anake venom* and 
other venuma). 
(c) Some enterotoxlns are aubatantially 
more toxic when adminiatered enterally than 
parenterally. The following enterotoxin* ahall 
be aubjecl to Pi 4- EKl containment 
condition*: cholera toxin, the heat labile 
toxin* of £ cali. Klebsiella, and other related 
protein* that may be identified by 
neutralization with an antiaerum 
monoapeciflc for cholera toxin, and the heat 
stable toxin* of £ coH and of Yersinia 
enterocolitica. 
3. Containment Conditions for Cloning of 
Toxins Genes in Organisms Other Than £ 
coh K-12 
Request* involving the cloning of gene* 
coding for toxin* for vertebrate* in host- 
vector system* other than £ coh K-12 will be 
evaluated by OROA. which will conault with 
the ad hoc working group on toxin*. (See 
Section |V-E-l-b-(3Hi)) 
C. Section V. Footnote 2A would be 
modified to delete the words; 
'toxins potent for vertebrates' (Section 1-D- 
2 ). 
D. A new Section IV-E-l-b-{3)-{i) 
would be added as follows; 
IV-E-l-b-(3HI)- Adding new entries to the 
list of toxins for vertebrates. (See Appendix 
G.) 
E. A new Section lV-E-l-l>-{3}-(j) 
would be added as follows; 
IV-E-l-b-(3)-(j). Approving the cloning of 
toxin genes in host-vector systems other than 
£ coh K-12. (See Appendix G.) 
I accept these recommendations with 
additional modifications as stated 
below. 
A further issue concerning cloning of 
toxin genes was raised by the ad hoc 
Working Croup and discussed by RAC 
during the evaluation of a proposal from 
Dr. John Murphy of Harvard University, 
i.e., appropriate containment for cloning 
the diphtheria toxin gene in E. coli K-12 
(See below. Part VI of this document). 
The new Section I-D-2 and Appendix C 
of the Cuidelines uses a cutoff of LDm of 
100 nanograms per kilogram of body 
weight to separate those toxins the 
cloning of whose genes is prohibited 
under Section I-D-2 from those allowed 
to be cloned at P2 -f- EK2 or P3 EKt. 
Diphtheria toxin falls very close to 
this cut-off line. Pharmacological 
toxicity data for diphtheria toxin 
demonstrates that the LDm for the most 
sensitive animal tested, the guinea pig. 
Is 160 nanograms per kilogram body 
weight. The LDm in humans is estimated 
to be equal to or less than 100 
nanograms per kilogram body weight. 
This figure was extrapolated from an 
incident in Japan in which children were 
inadvertently injected with diphtheria 
toxin rather than diphtheria toxoid. 
After much discussion. RAC 
recommended that experiments by Dr. 
John Murphy involving the cloning of the 
gene for the biosynthesis of diphtheria 
toxin be conducted under P4 physical 
containment. The RAC realized that this 
recommendation results in an 
inconsistency between the 
recommendation in Appendix C 
concerning cloning of diphtheria toxin 
and the recommendation concerning Dr. 
Murphy's proposal. They noted that 
cloning the gene for diphtheria toxin 
could be moved to the prohibited 
category in general, and Dr. Murphy's 
proposal could be considered an 
exemption to the prohibition. Based 
upon the RAC recommendation and the 
data available concerning the 
pharmacological toxicity of diphtheria 
toxin. I am removing diphtheria toxin 
from category 2-{a) of Appendix G. 
“genes coding for toxins that have an 
LDm in the range of 100 nanograms to 
1000 nanograms per kilogram body 
weight," and placing it under section 1- 
D-2. 
Accordingly, revised Section l-D-2 
will read as follows; 
I-D-2. Deliberate formation of recombinant 
DNAs containing genes for the biosynthesis 
of toxins lethal for vertebrates at an LDm of 
less than 100 nanograms per kilogram body 
weight (e.g.. the botulinum toxins, tetanus 
toxin, diphtheria toxin. Shigella dysenteriae 
neurotoxin). Cuidelines 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 Cuidelines (e.g.. 
exemptions, return to host of origin, etc.). 
Section 2-{a) of Appendix G will read 
as follows; 
(s) Cloning of genes coding for toxins for 
vertebrates that have an LDm in the range of 
100 nanogram* to 1000 nanograms per 
kilogram body weight (e.g.. abrin, Clostridium 
perfringens epsilon toxin) may proceed under 
P2 + EK2 or P3 ->- EKl containment conditions. 
With these modirications, I accept the 
RAC recommendations concerning the 
cloning of genes for the biosynthesis of 
toxins for vertebrates. 
Experiments currently underway in E. 
coli K-12 host-vector systems involving 
genes coding for toxins with an LDm of 
100 micrograms or less per kilogram of 
body weight should be registered with 
ORDA within 90 days. 
It is recognized that the requirements 
of revised Section I-D-2 and new 
Appendix G may result in more 
stringent standards for the cloning of 
toxin genes than interpretations under 
the previous Guidelines. Appendix G 
allows the cloning of toxin genes in E. 
coli K-12 only, and overrides other parts 
of the Guidelines. Under interpretations 
of the previous Guidelines, for example, 
investigators may be conducting 
recombinant DNA experiments with 
toxins not listed in Appendix G. or 
conducting experiments at lower 
containment levels than now specified 
in Appendix G, or using host-vector 
systems other than E. coli. K-12 as in 
"self cloning" experiments. Investigators 
currently conducting experiments that 
do not meet the requirements of new 
Appendix G and that have not already 
been acted upon individually by NIH 
must contact ORDA for a case-by-case 
review within 90 days. However, 
investigators currently conducting such 
experiments may continue with their 
ongoing projects until these projects 
have been evaluated by NIH. 
(145J 
