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TSCA Environmental Release Application (TERA) for Alcaligenes xylosoxidans subspecies denitrificans strain AL6.1, R05-01

On May 26, 2005, 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 the University of California, Riverside, involves field trials of a modified strain of Alcaligenes xylosoxidans subspecies denitrificans (Axd) strain AL6.1. The strain was given the tracking designation of R05-0001 (previously reviewed under the tracking designations R03-0001 and R04-0003). The microorganism will be tested at the same three test sites as TERA R04-0003 in California to determine its biology and behavior in vineyard ecosystems as part of a research program to prevent or cure Pierce's disease in grapes. Pierce’s disease is an infection caused by the bacterium Xylella fastidiosa (X. fastidiosa) and is transmitted by leafhopper insects, the glassy-winged sharpshooter (GWSS). The TERA microorganism is considered a pesticide intermediate. The field trials were set for May 2005 through the growing season until pruning in the fall of 2005, with continued field data analysis through the winter to the target end date of March 2006.

PROPOSED USE AND FIELD STUDY

The bacterium will be used for small-scale research and development field trails to continue studying its biology and behavior in vineyard ecosystems at the three sites used in the previous submission, R04-0003. The X. fastidiosa pathogen is spread throughout grape growing regions by the glassy-winged sharpshooter (GWSS), a large leafhopper insect. Since the parental strain of Axd AL6.1 was originally isolated from the GWSS, the researchers ultimately plan to use the GWSS as a delivery vector of Axd carrying a pesticidal gene antagonist to X. fastidiosa to the xylem of grapevines where the pathogenic bacterium resides.

This field study differs from the two previous years (R03-0001 and R04-0003) for two reasons. First, the researchers are requesting that the experimental grapevines be left intact after harvest (fall 2005) so that sampling can continue throughout the winter and into the spring (March 2006). Secondly, they are requesting that the previous termination procedures not be required for the current submission since they have concluded that strain AL6.1 does not survive for long, less than two week, in the xylem of grapevines.

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

Based on available data and the two previous submissions (R03-0001 and R04-0003), there is low concern for potential toxicity of the recipient microorganism and the introduced genetic material. Although there are some uncertainties associated with the genetic construct, the risk to humans or the environment is low due to the low exposure conditions of the contained field tests. Additionally, the recipient species, Axd, is not a frank pathogen, although it has been associated with some opportunistic infections. No data is available regarding the toxicity associated with exposure to the DsRed fluorescent protein. However, the DsRed fluorescent protein may present some cytotoxicity when expressed in certain cells.

EPA has determined that a potential for widespread dissemination of the TERA bacterium exists if the field study were conducted according to the protocol described in the TERA. Specifically, at the end of the field study, if the containment procedures were no longer employed the small-scale field study could then be considered a large-scale field release. Available information does not support a conclusion that the TERA bacterium will have died off by the following year; therefore, the remaining population may be transported off site by the insect vector GWSS to the large numbers and variety of crops that the GWSS feeds on.

CONCLUSION

EPA recommends that the TERA submission R05-0001 be granted with two conditions. First, the pot in which the sweet orange tree is planted should be covered as part of the containment protocol. To satisfy this requirement, a separate fabric enclosing the pot should be connected to the above ground fabric enclosure. To reduce deterioration of the fabric due to contact with moist soil, the enclosed pot can be placed into an empty pot. Secondly, termination procedures following the conclusion of the field study should include three processes: fumigation of all the "cages" to kill the GWSS; incineration or autoclaving of all experimental plants inoculated with the TERA bacterium; and removal or autoclaving of the soil surrounding the root system of all experimental plants.

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 202-566-0282.


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