TSCA Environmental Release Application (TERA) for Pseudomonas putida (P. Putida)

On October 11, 2000, the Office of Pollution Prevention and Toxics approved the TSCA Environmental Release Application (TERA) under the biotechnology regulations promulgated under the Toxic Substances Control Act (TSCA). The TERA, submitted by Oak Ridge National Laboratory, involves field trials of a modified strain of Pseudomonas putida (P. Putida). The TERA was given the tracking designations of R-00-0002. The identical strain was the subject of TERA R-98-0005. The microorganism will be tested on three 0.25 acre areas of Edwards Air Force Base to determine whether it will produce light in the presence of trinitrotoluene (TNT) and whether the light produced is a practical means for the detection of TNT in soil. The eventual application of such technology would be in detection of land mines and unexploded ordnance in desert environments. The field trials were set to begin the week of October 22, 2000.

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 the land mines and UXO is to detect the presence of TNT in the soil that has leaked from the devices. If the field tests are successful, the microorganism which is the subject of this TERA 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 strain is expected to have no effect on pathogenicity, it is believed that the strain presents 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 small site with limited access;
• This strain is not expected to survive long outside of the laboratory when in the presence of sunlight (ultraviolet); 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 the intergeneric microorganism will not present an unreasonable risk of injury to health or the environment. In the TERA approval letter, EPA advised the submitter that this antibiotic is among the least desirable antibiotic resistance markers. The Agency has previously granted TERA R-98-0005 for the same use. The letter of approval advised the submitter to insert the genetic material into the recipient microorganism in a more stable manner. The submitter was also advised to avoid using antibiotic resistance markers that are associated with clinically significant antibiotics.

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.