Two TSCA Environmental Release Applications (TERAs) for Pseudomonas putida (P. Putida)

On May 30, 2001, the Office of Pollution Prevention and Toxics approved the TSCA Experimental Release Applications (TERAs) under the biotechnology regulations promulgated under the Toxic Substances Control Act (TSCA). The TERAs, submitted by Oak Ridge National Laboratory, involve field trials of modified strains of Pseudomonas putida (P. Putida). The strains were given the tracking designations of R-01-0003 and R-01-0004. The microorganisms will be tested at the Ravenna Army Ammunition Plant in Ohio to determine whether they can detect traces of trinitrotoluene (TNT) in soil. The eventual application of such technology would be in detection of land mines and unexploded ordnance. The field trials were set to begin the week of June 4, 2001.

Land Mines and Unexploded Ordnance (UXO)

It has been estimated that there are 70 million land mines presently active in the world, with more added every day. In addition, many areas are endangered by unexploded ordance (UXO) which include bombs, grenades, shells, raw explosives and other related devices which were fired, but did not detonate. Such devices can remain active for many years after firing. One approach to locate land mines and UXO is by detecting the presence of TNT in the soil which has leaked from the devices. If the field tests are successful, the microorganisms which are the subjects of these TERAs will provide an inexpensive means to detect TNT in the soil.

Regulatory Background

The EPA requirements concerning microbial products subject to TSCA (15 U.S.C. Section 2601, et seq.) are set forth in "Microbial Products of Biotechnology; Final Regulation under the Toxic Substances Control Act" (62 FR 17910 (April 11, 1997)) and codified at 40 C.F.R. Part 725. Microorganisms resulting from the deliberate combination of genetic material originally isolated from organisms of different taxonomic genera (intergeneric microorganisms) constitute "new" microorganisms subject to TSCA Section 5 notification requirements. Persons who manufacture, import, or process intergeneric microorganisms for commercial purposes subject to EPA jurisdiction under TSCA, are required to submit a Microbial Commercial Activity Notice (MCAN). Persons conducting commercial research and development activities may submit a TERA, instead of an MCAN, before initiation of such testing. EPA conducts a review of these submissions to determine whether the intergeneric microorganisms present an unreasonable risk to health or the environment. The Agency can impose regulatory controls under section 5 of TSCA.

Summary of the Risk Assessment

P. putida is a common, wide-spread, soil bacterium that is not pathogenic to plants or animals. Since the genetic modifications involved in creating the TERA strains are expected to have no effect on pathogenicity (i.e., on the ability of the microorganisms to infect and to cause disease), it is believed that these strains present a low ecological hazard for small-scale field testing.

Because the antibiotic has wide-spread clinical use, EPA's review identified a concern for the antibiotic resistance marker genes to transfer to other microorganisms in the environment which may be human, animal or plant pathogens. EPA believes, however, that the probability for this event is extremely low in the field trial because:

• The field trials will occur on a limited access site for a brief period of time;
• This strain is not expected to survive long outside of the laboratory when in the presence of sunlight (ultraviolet);
• The genetic transfer rates of the bacterial construct in the environment are extremely low; and
• The naturally occurring parent bacteria, P. putida, are not human inhabiting organisms.

Conclusion

EPA has determined that the proposed small scale field trials of these intergeneric microorganisms will not present an unreasonable risk of injury to health or the environment. In the TERA approval letter, EPA advised the submitter that the antibiotics are among the least desirable antibiotic resistance markers. In addition, the submitter is informed that the issue of gene transfer must be addressed in greater detail if the microorganisms are ever proposed for large scale testing or submitted as MCANs.

For a copy of the original nonconfidential TERAs and the nonconfidential approval letter, please contact the TSCA Non-Confidential Information Center (NCIC) by phone at (202) 566-0280, or by fax at (202) 566-9744.