On April 20, 2007, the Office of Pollution Prevention and Toxics approved the TSCA Experimental Release Application (TERA) under the biotechnology regulations promulgated under the Toxic Substances Control Act (TSCA). The TERA, submitted by Micro Systems Technologies (MST), Dayton, OH, and the Purdue University, West Lafayette, IN, involves field trials of a modified strain of Pseudomonas putida strain TV8. The strain was given the tracking designation of R07-0001. The objective of the application is to update approved TERA R04-0001 and -0002, and Premanufacture Notice (PMN) P-95-1601, and to obtain an extension form EPA for the demonstration and continued testing of biosensor systems that use genetically-engineered bioluminescent microorganisms. This microorganism will be used in an enclosed biosensor device for detection of trichloroethylene (TCE).

Proposed Use and Field Study

The parental microorganism Pseudomonas putida was chosen for its indigenous tod (toluene dioxygenase) operon, which is a multicomponent enzyme system responsible for the degradation of toluene in P. putida strains. This strain was modified by the introduction of a promoter - luxCDABE gene fusion that allows for bioluminescence in the presence of TCE, as well as BTEX compounds (benzene, toluene, ethylbenzene, and xylene). These lux genes are from the marine bacterium Vibrio fischeri.

A small quantity of the microorganism is to be encapsulated onto a membrane which is enclosed within a metal capsule sampling device that also contains electronic circuitry for the detection of the bioluminescence. Since the bacteria are first encapsulated in an alginate matrix, then enclosed within a 0.45 μm filter unit, and finally enclosed in metal capsule devices, there is no direct release of the microorganisms to the environment expected.

The proposed field tests, demonstrations for a number of government agencies (DOD, CDC, EPA, DOE, DOT) and contractors (System Research Development Corp. and Northrup Grumman, Boeing, Cartwright, etc.), are similar to field tests of a different intergeneric microorganism (HK44) in a previous MST submission (R02-0001), and to tests conducted, again with HK44 and another intergeneric strain 5RL, in another submission (R04-0001).

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

The proposed field tests using the intergeneric bacterium P. putida TVA8 pose low risk to human health and the environment. The parent P. putida F1 does not pose concerns for human health; it is not a frank human pathogen, and like many other microorganisms in the environment, is capable of causing infection only in immunocompromised individuals. P. putida is widespread in the environment, occurring predominately in soils, particularly rhizosphere soil, and in water. It is not a frank pathogen to animals or plants. The strain F1 has a long history of study for its metabolic capabilities in degradation of chlorinated compounds and other xenobiotics, especially BTEX compounds.

The genetic modifications made to the parental microorganism to create the bioluminescent reporter bacterium TVA8 do not pose great concern. The presence in the microorganism of the lux genes from Vibrio fishceri does not pose any risk. The inclusion of a kanamycin resistance gene as a selection marker is not necessarily desirable for microorganisms intended to release to the environment. Transfer of an antibiotic resistance gene to pathogenic microorganisms whose infections are treated with that particular antibiotic may compromise the antibiotic's therapeutic value, not only for humans, but also for animals and plants. However, in strain TVA8, the introduced genes are stably incorporated into the chromosome of the recipient microorganism which lessens the potential for horizontal gene transfer. Of course, concern over any potential gene transfer is alleviated by the fact that there is actually no release of TVA8 to the environment, and consequently, no exposure to other microorganisms to which genes might potentially be transferred.

The design of the biosensor capsule units prevents release of the microorganism into the environment by three levels of containment. First, the bacterium is encapsulated in a gel matrix on a membrane which is then sandwiched between 0.45 Φm membranes that prevent passage of the bacterial cells outside the inner chamber. The membrane unit with the bacterial cells, along with the electronic circuitry for light detection, is then enclosed in a stainless steel housing with a screw-on top portion. Only small holes in the metal housing allow for air transport through the device (to detect volatilized TCE).

Conclusion

EPA decided to grant the TERA submission R07-0001 on April 20, 2007. There is low risk of injury to human health and the environment with the proposed field tests for TCE detection using the submission microorganism TVA8, as both the potential hazards associated with the submission microorganism and potential exposures to the submission microorganism are low.

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.