**Attachment**

**Finding of No Significant Impact**

**Response to Comments**

**APHIS 06-250-01r**

On July 18, 2007, APHIS published a notice in the *Federal Register* (72
FR 39378--39379, Docket No. APHIS--2007--0018) announcing the
availability of an Environmental Assessment for public comment prepared
in response to permit application 06--250--01r for a controlled release
of *Populus* species and hybrids. During the 30-day comment period,
which ended on August 17, 2007, APHIS received 5 comments. Comments
opposed to APHIS granting the permit were submitted by two individuals
and a public interest group. Comments that supported APHIS granting the
permit were submitted by the permit applicant and a limited liability
company. The pertinent issues that were raised during the comment period
and APHIS' responses to those issues follow:

1.  Two commenters submitted comments reiterating the findings of the EA
    and also outlined the benefits of using forest trees for cellulose
    feedstock for production of ethanol in the development of a
    renewables-based energy and materials economy. The commenters also
    listed the benefits of the study which included, among others, that
    the study has the potential to provide substantial benefits to the
    nation by providing information enabling improved yields in
    plantations, reduce the risk of spread of transgenic trees, and
    reducing many of the biological and legal issues that have plagued
    GE crops.

> **Response:** While the results of the field test might aid in meeting
> the demands of the nation's biofuel industry and aid in addressing
> research questions, these comments do not deal with questions
> addressing plant pest risk and are irrelevant to APHIS's
> decision-making process. APHIS does not judge the merits of a field
> test or the necessity of the research being conducted. APHIS evaluates
> the environmental impacts of a field test, regardless of the merits of
> the field test.

2.  One commenter expressed a concern about the presence of barnase in
    the trees since it is a toxin aimed at inducing sterility. The
    commenter was concerned that barnase will be present in the leaves,
    stems, and roots of trees and this will adversely affect not only
    the transgenic plant, but also the fauna and flora of the forest
    ecosystem. The commenter indicates that "The toxicity of barnase to
    mammals is well known" and gives citations from the journal Science
    and Society in support of these claims.

> **Response:** As pointed out in the EA, these genes have been
> engineered to be expressed during flower development. In this case
> barnase (a ribonuclease) is expressed primarily in the cells of
> developing flowers. Barnase is expressed in a small number of cells in
> developing flowers which last only a short time in the environment.
> There may be a very low level expression in vegetative tissues since
> some of the promoters that drive the genes are not exclusively
> expressed in floral tissues. However the levels expressed in
> vegetative tissues are significantly lower than that expressed in
> developing flower parts. Barstar, a specific inhibitor of barnase, is
> also being expressed in the vegetative tissues to counteract the low
> level of barnase activity that might inhibit vegetative growth (1).
>
> Barnase is not toxic when ingested. Ribonucleases, such as barnase,
> are naturally expressed in all plant tissues and therefore are already
> part of human and animal diets. (see FDA consultations BNF No. 000031
> <http://www.cfsan.fda.gov/~rdb/bnfm031.html>, BNF No. 000032
> <http://www.cfsan.fda.gov/~rdb/bnfm032.html> BNF No. 000057
> <http://www.cfsan.fda.gov/~rdb/bnfm057.html> and BNF No. 000066
> <http://www.cfsan.fda.gov/~rdb/bnfM066.html>.). Barnase and barstar
> have also been in products previously deregulated by APHIS and which
> have gone through a full FDA food and feed safety consultation, for
> example corn (Petitions 95-228-01p and 98-349-01p), *Cichorium
> intybus* (Petition 97-148-01p) and Rapeseed (Petitions 98-278-01r and
> 01-206-01p).
>
> APHIS disputes the claim that the toxicity of barnase to mammals is
> well known. The articles the commenter cites are not from a credible
> science source but from a journal of Marxist thought and analysis. As
> stated above, no adverse effects to wildlife or humans are expected
> from expressing the barnase in the developing flowers. Because the
> hazard of the protein is extremely low and has been consumed by
> animals and humans with no adverse effects, APHIS reasonably concludes
> that there should not be a significant adverse effect on wildlife from
> the expression of barnase and barstar in this GE field release.

3.  One commenter was concerned about the use of the diphtheria toxin
    A-chain to induce sterile flowers and the lack of any published
    studies on the safety in animals from eating transgenic plants
    modified with the diphtheria A-chain.

> **Response:** As explained in the EA, there should be very limited
> exposure of animals to the A-chain component of *DTA.* That is because
> the A-chain is primarily expressed in the cells of developing flowers.
> During flower development there would be very few cells expressing the
> protein and for only a short period of time. There could be a very low
> level of expression in vegetative tissues based on the promoter being
> used to drive the gene. The promoter has been shown to impart
> vegetative expression, but at a level approximately 100-fold below
> that in floral tissues (2).
>
> The diphtheria toxin A-chain is not expected to present a hazard to
> animals consuming it because the toxin activity is dependent on two
> components, an A‑chain and a B-chain. The B-chain component allows
> movement of the holotoxin into cells while the A-chain component
> disrupts protein synthesis in eukaryotic organisms by inhibiting
> translocase, the enzyme involved in the elongation phase of protein
> synthesis. As the GE trees lack the B-chain, the inhibition of protein
> synthesis is restricted to a few cells in the developing flowers. In
> studies conducted by the applicant, no adverse effect on vegetative
> growth was observed indicating that the expression of the DTA protein
> was too low to be active in vegetative cells (2). The *DTA* is
> disarmed from entry into other cells and is expected to only cause
> rapid death of the cells in which it is expressed. For animals
> consuming the tissues that contain the A-chain component, A-toxin is
> not expected to be absorbed into the cells of the GI tract in the
> absence of the B-chain. Therefore there should be no toxic effect to
> animals consuming the flowers of plant parts. Together with the low
> exposure, APHIS reasonably concludes that there should not be a
> significant adverse effect on wildlife from the expression of
> diphtheria toxin A in this GE field release.

4.  One commenter was concerned about MADS-box genes being present in
    the transgenic trees and indicated that "MADS-box transgenes should
    not be presumed safe, as they are related to the extensively studied
    animal homeotic genes that regulate development and may well be
    active in animals."

> **Response:** APHIS does not agree that the introduction of an
> additional MADS-box gene into poplar trees should be presumed unsafe
> because they are related to animal homeotic genes and may be active in
> animals. First, there is no scientific reason to believe that plant
> MADS proteins could have a significant impact on animals. APHIS is
> unaware of and the commenter has not provided any evidence that plant
> MADS box proteins have any activity on animals. Second, animals are
> unlikely to get the plant MADS box genes through horizontal gene
> transfer from plants to animals as this process does not occur except
> perhaps on an evolutionary time scale. Third, animals are unlikely to
> be exposed to active plant MADS box proteins as these will be digested
> upon consumption and are unlikely to remain active in the GI tract of
> animals. Finally, plants are estimated to have about 100 MADS box
> genes (3) so animals are naturally and continuously exposed to plant
> MADS box genes and proteins. The addition of one more plant derived
> MADS box gene into Poplar trees through genetic engineering should
> have no incremental impact on activity in animals even in the unlikely
> case that such proteins may be active in animals.

5.  One comment points out the use of RNA interference (RNAi) gene
    therapy and its potential for adverse health effects based on
    studies with mice. RNAi involves the use of a small interfering
    double stranded RNA of approximately 21-15 nucleotides that is
    complimentary to a known messenger RNA that is used to block its
    expression. In this case Populus RNAi genes have been used in an
    attempt to induce sterile flowers.

> **Response:** The safety of nucleic acids is widely accepted. Both RNA
> and DNA are part of all food products that we consume. Gene therapy is
> a technique for correcting defective genes responsible for disease
> development
> (http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml).
> It is still in the early experimental stages and has not proven
> successful in clinical trials. Researchers may use one of several
> approaches for correcting faulty genes including RNAi. In the current
> case, RNAi is being used to inactivate a gene involved in flower
> development and thereby induce sterile flowers in a plant and not to
> correct a defective gene in a patient (or mammalian model organism)
> for gene therapy. The potential adverse health effects of gene therapy
> are not relevant to this field test as the situations are so entirely
> different that a comparison is without meaning.

6.  One commenter was concerned about the fact that the Poplars were
    engineered with genes involved with light response and gibberellin
    metabolism genes and that the transgenes in these releases have not
    been studied regarding any potential untoward effects.

> **Response:** The gibberellin gene family and phytochrome receptor
> genes have been studied in many other plant species and include some
> of the most extensively studied genes in plant science due to their
> effects on growth and development. Natural and induced mutations in
> gibberellin biosynthesis have been isolated and have been exploited in
> conventional plant breeding to produce dwarf varieties in many crops
> including fruit trees. Similarly, gibberellins are routinely applied
> as growth regulators to many food and feed crops and ornamentals to
> increase the yield and quality of crops. As with any field trial, the
> responsible party is required to report any unusual effects to APHIS
> should they occur, and the field trial is confined to prevent the
> establishment of the regulated material outside the test site. Thus
> while there is no reason to believe that the light response and
> gibberellin genes will have any untoward effects, the experiment is
> conducted in a way that even if they did, the material will be
> confined to the test site.

7.  One commenter was concerned about the use of the 4CL1 gene from
    *Populus tremuloides* inserted to alter lignin levels. The commenter
    is concerned that low lignin trees are likely to be more susceptible
    to pests and to be prone to wind damage because they lack mechanical
    strength.

> **Response:** One of the purposes of these field studies is designed
> to answer the above question. To date the permittee has observed no
> changes in the incidence of pests, beneficial insects or pathogens in
> the existing field tests. However, the test will be used to gather
> data to answer the above question, and would be important information
> to gather to determine if indeed low lignin leads to a greater
> incidence of plant pests on these transgenic trees. Since there are
> very few trees, and the test is a confined field trial, there should
> be no impact on the environment should these few trees be found to be
> more susceptible to pests or wind. The trees in the field test will be
> monitored for any increased disease or pest susceptibility at least
> once a year and any unusual occurrence must be reported to APHIS.

8.  One commenter was concerned about the use of activation tagging.
    Activation tagging is insertional mutagenesis using insertion
    vectors that contain a strong transcription enhancer to up-regulate
    a gene near the insertion site. The insertions appear randomly in
    the genome, resulting in gain of function dominant mutations. The
    commenter does not believe that it is safe for field test releases
    because "it is likely to cause unintended insertional mutagenesis in
    a range of microorganisms and animals that interact with the
    transgenic plants."

> **Response:** APHIS disagrees with this comment. The commenter did not
> provide any refereed citations that would substantiate their
> assumption. The inserted DNA used for the activation tagging is stably
> integrated into the Poplar genome. It is no more likely to cause
> unintended insertional mutagenesis in another organism than any other
> DNA within the Poplar genome.

9.  One commenter is concerned about the ability of the applicant to
    monitor the field sites, indicates that mechanical pruning to
    prevent flowering seems risky in a large complex array of
    experimental trees, and believes that it will be inevitable that the
    transgenes will be dispersed.

> **Response:** APHIS disagrees with this comment. Pruning of poplar
> trees on a large scale is very easy and effective. The trees are all
> planted in clonal blocks and are easily maintained to prevent
> flowering. It is a common and well-established practice to maintain
> poplar clones at close spacing with severe pruning to prevent
> flowering and to maintain material for research purposes. In addition,
> APHIS will be inspecting the field trial to verify compliance to
> permit conditions.

10. One commenter is concerned about horizontal gene transfer and
    indicates that it is a distinct possibility because "the extensive
    root system of trees is a hotbed for horizontal gene transfer and
    recombination."

> **Response:** APHIS disagrees with this comment. The commenter
> provides no evidence to support the claim that the root system of
> trees is a hotbed for horizontal gene transfer and recombination.
> APHIS has reviewed the scientific literature on horizontal gene
> transfer and concluded that it is very unlikely to occur from trees to
> any other organisms (discussed in the EA on page 12).

11. One commenter indicated that the proposal made no attempt to present
    the genetic modifications of the many transgenic lines in a rational
    and coherent manner, with diagrams detailing the transgenic
    constructs in each line being tested along with an explanation of
    the function of each gene.

> **Response:** A list of all the genes and donors that are in the field
> test are listed in Table 1 of the EA. In addition, a description of
> each of the genes is covered in Appendix III of the EA.

12. One commenter indicated that there are so many separate lines being
    tested in one big 320 acre site that transgene escape form the site
    is bound to happen due to human error.

> **Response:** The commenter is mistaken in that the field tests are on
> three different sites totaling 30 acres and not 320. This permittee
> has an excellent compliance record, and has been inspected a number of
> times with no compliance issues. APHIS has confirmed that the
> applicant has the appropriate resources to maintain the test and
> monitor for volunteers. In addition, numerous redundant confinement
> measures are employed to minimize the likelihood of escape such as
> isolation distances, use of male sterility, removal of flowers,
> inhospitable environment (others)

References

1.  Wei, H., R. Meilan, et al. (2007). Field trial detects incomplete
    barstar attenuation of vegetative cytotoxicity in Populus trees
    containing a poplar LEAFY promoter:barnase sterility transgene.
    Molecular Breeding **19**: 69-85.

2.  Skinner, J. S., R. Meilan, et al. (2003). The *Populus PTD* promoter
    imparts floral-predominant expression and enables high levels of
    floral-organ ablation in *Populus*, *Nicotiana* and *Arabidopsis*.
    Molecular Breeding **12**: 119-132.

3.  Nam, J., J. Kim, et al. (2004). Type I MADS-box genes have
    experienced faster birth-and-death evolution than type II MADS-box
    genes in angiosperms. PNAS 101: 1910-1915.