01-20191. Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH Guidelines

AGENCY:

National Institutes of Health (NIH), PHS, DHHS.

ACTION:

Notice of proposed actions under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines).

SUMMARY:

The NIH is proposing to amend Appendix B-I of the NIH Guidelines to establish criteria for designating strains of E. coli as risk group 1 agents.

DATES:

The public is encouraged to submit written comments on the proposed change. Comments may be submitted to the NIH Office of Biotechnology Activities (OBA) in paper or electronic form. Comments received on or before September 12, 2001 will be considered by NIH. All comments received in response to this notice will be available for public inspection in the NIH OBA office, 6705 Rockledge Drive, Suite 750, Bethesda, MD 20892-7985, 301-496-9838, weekdays between the hours of 8:30 a.m. and 5 p.m.

FOR FURTHER INFORMATION CONTACT:

If you have questions, or want additional information about these proposed changes, please contact OBA by e-mail at [e-mail], or telephone at 301-496-9838. Comments can be submitted to the same e-mail address, by fax to 301-496-9839, or mail to the Office of Biotechnology Activities address set forth above.

SUPPLEMENTARY INFORMATION:

The University of Florida has asked OBA to set the risk group level for strain B of the common bacterium E. coli, which is non-virulent. Strain B is widely used in industry for large-scale work (greater than 10 liters of culture) due to increased stability of cloned sequences in this strain versus E. coli K-12. Currently, the only non-virulent strain of E. coli designated as a risk group 1 agent (agents not associated with disease in healthy adult humans) in the NIH Guidelines is strain K-12. Potentially pathogenic strains of E. coli are designated as risk group 2 agents in the NIH Guidelines.

At the March 2001 RAC meeting, a recommendation was made to define the criteria for designating strains of E. coli as risk group 1 agents. The establishment of general criteria is preferable to narrowly addressing a single strain. The suggested criteria were: “(1) they [the E. coli strain] carry deletions in metabolic genes to engender the requirement for specialized laboratory media; and (2) they do not pose a threat of disease: they do not carry any active virulence markers nor do they make any toxins (nor do they carry the genes for these toxins).”

Following the March 2001 meeting, the University of Florida Institutional Biosafety Committee responded that the investigator, Dr. Luli (Adjunct Professor in Microbiology and Cell Science at the University of Florida and also Research Director for BC International Corp.), who made the initial request had reservations regarding the requirement for deletions in metabolic genes. Dr. Luli stated that the use of specialized laboratory media would pose a problem for large-scale, industrial work. Dr. Luli suggested that instead of an absolute requirement for specialized laboratory media, that the “* * * scope of the [first] requirement be broadened to simply demonstrate “crippled” or adversely affected metabolism.” The rationale for this modification is that the strains of E. coli B that Dr. Luli proposes to use have reduced growth rates compared to wild type E. coli even in complete, rich laboratory media.

The proposed criteria for designating an E. coli strain as a risk group 1 agent were revisited at the June 2001 RAC meeting. Ad hoc consultant, Dr. James Kaper, University of Maryland School of Medicine, also participated in the RAC review and discussion. During the June meeting discussion, it was pointed out that a growth requirement is not a current criteria in the NIH Guidelines for designation of E. coli K-12 as a risk group 1 agent. Accordingly, the criteria for designating strains of E. coli as risk group 1 agents were revised as follows: “(1) they [the E. coli strain] do not possess a complete lipopolysaccharide (i.e., they lack the O antigen and have a “rough” colony morphology); and (2) they do not carry any active virulence factors—such as—toxin, or colonization factors nor do they carry genes for these factors.” A “rough” colony morphology is indicative of the absence of a complete coat that aids in survival in the intestine and environment.

The proposed use of general criteria for designating strains of E. coli as risk group 1 agents is not intended to eliminate the need for case-by-case consideration of the potential effects of a biological agent on those who may be exposed to it (Section II-A-2 of the NIH Guidelines) and any general criteria will be subject to reevaluation and change in light of evidence that a strain meeting those criteria is associated with disease in healthy adult humans.

Proposed Amendments to the NIH Guidelines

For the reasons stated above, it is proposed to amend Appendix B-I, Risk Group (RG1) Agents, to state:

Appendix B-I. Risk Group (RG1) Agents

RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis (see Appendix C-IV-A, Bacillus subtilis or Bacillus licheniformis Host-Vector Systems, Exceptions); adeno-associated virus (AAV) types 1 through 4; and recombinant AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus. A strain of Escherichia coli (see Appendix C-II-A, Escherichia coli K-12 Host Vector Systems, Exceptions) is an RG1 agent if it (1) does not possess a complete lipopolysaccharide (i.e., lacks the O antigen and has a “rough” colony morphology); and (2) does not carry any active virulence factor (e.g., toxins) or colonization factors and does not carry any genes encoding these factors.

Those agents not listed in Risk Groups (RGs) 2, 3 and 4 are not automatically or implicitly classified in RG1; a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed.

Dated: August 3, 2001.

Ruth L. Kirschstein,

Acting Director, National Institutes of Health.