Appendix F-I. General Information
Appendix F-II. Cloning of Toxin Molecule Genes in Escherichia coli K-12
Appendix F-III. Cloning of Toxic Molecule Genes in Organisms Other Than Escherichia coli K-12
Appendix F-IV. Specific Approvals
Appendix F specifies the containment to be used for the
deliberate cloning of genes coding for the biosynthesis of molecules toxic for
vertebrates. The cloning of genes
coding for molecules toxic for vertebrates that have an LD50 of <
100 nanograms per kilograms body weight (e.g., microbial toxins such as the
botulinum toxins, tetanus toxin, diphtheria toxin, Shigella dysenteriae
neurotoxin) are covered under Section
III-B-1 (Experiments Involving the Cloning of Toxin Molecules with LD50
of Less than 100 Nanograms Per Kilogram Body Weight) and require
Institutional Biosafety Committee and NIH/OBA approval before initiation. No specific restrictions shall apply to the
cloning of genes if the protein specified by the gene has an LD50 ≥ 100 micrograms per kilograms
of body weight. Experiments involving
genes coding for toxin molecules with an LD50 of < 100 micrograms
per kilograms and > 100 nanograms per kilograms body weight require
Institutional Biosafety Committee approval and registration with NIH/OBA prior
to initiating the experiments. A list
of toxin molecules classified as to LD50 is available from
NIH/OBA. Testing procedures for
determining toxicity of toxin molecules not on the list are available from the
Office of Biotechnology Activities, National Institutes of Health, 6705
Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839
(fax). The results of such tests shall
be forwarded to NIH/OBA, which will consult with ad hoc experts, prior
to inclusion of the molecules on the list (see Section
IV-C-1-b-(2)-(c), Minor Actions).
Appendix F-II-A.
Cloning of genes coding for molecules toxic for vertebrates that have an
LD50 of >100 nanograms per kilograms and <1000 nanograms per
kilograms body weight (e.g., abrin, Clostridium perfringens epsilon
toxin) may proceed under Biosafety Level (BL) 2 + EK2 or BL3 + EK1 containment
conditions.
Appendix F-II-B.
Cloning of genes for the biosynthesis of molecules toxic for vertebrates
that have an LD50 of >1 microgram per kilogram and <100
microgram per kilogram body weight may proceed under BL1 + EK1 containment
conditions (e.g., Staphylococcus aureus alpha toxin, Staphylococcus
aureus beta toxin, ricin, Pseudomonas aeruginosa exotoxin A, Bordetella
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).
Appendix F-II-C.
Some enterotoxins are substantially more toxic when administered
enterally than parenterally. The
following enterotoxins shall be subject to BL1 + EK1 containment
conditions: cholera toxin, the heat
labile toxins of Escherichia coli, Klebsiella, and other
related proteins that may be identified by neutralization with an antiserum
monospecific for cholera toxin, and the heat stable toxins of Escherichia
coli and of Yersinia enterocolitica.
Requests involving the cloning of genes coding for toxin
molecules for vertebrates at an LD50 of <100 nanograms per
kilogram body weight in host-vector systems other than Escherichia coli
K-12 will be evaluated by NIH/OBA in consultation with ad hoc toxin
experts (see Sections III-B-1, Experiments Involving the Cloning of
Toxin Molecules with LD50 of Less than 100 Nanograms Per Kilogram
Body Weight, and IV-C-1-b-(2)-(c),
Minor Actions).
An updated list of experiments involving the deliberate formation
of recombinant DNA containing genes coding for toxins lethal for vertebrates at
an LD50 of <100 nanograms per kilogram body weight is available
from the Office of Biotechnology Activities, National Institutes of Health,
6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail),
301-496-9838, 301-496-9839 (fax).