USDA/APHIS Environmental Assessment

In response to the Planet Biotechnology permit application

05-354-03r for an environmental release to produce antibodies in
genetically engineered

*N. tabacum* X *N. glauca* hybrid plants

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

Biotechnology Regulatory Services

**TABLE OF CONTENTS**

[I. SUMMARY 3](#i.-summary)

[II. INTRODUCTION 3](#ii.-introduction)

[A. Purpose of the Environmental Release
3](#a.-purpose-of-the-environmental-release)

[B. USDA-APHIS Regulatory Authority
4](#b.-usda-aphis-regulatory-authority)

[III. NEED FOR THE PROPOSED ACTION
4](#iii.-need-for-the-proposed-action)

[IV. ALTERNATIVES TO THE PROPOSED ACTION
5](#iv.-alternatives-to-the-proposed-action)

[A. No Action 5](#a.-no-action)

[B. Issue the Permit with Supplemental Permit Conditions
5](#b.-issue-the-permit-with-supplemental-permit-conditions)

[V. AFFECTED ENVIRONMENT 6](#v.-affected-environment)

[A. Description of the Regulated Article
6](#a.-description-of-the-regulated-article)

[B. Biology of *Nicotiana* and of *N. tabacum* X *N. glauca* Hybrids
7](#b.-biology-of-nicotiana-and-of-n.-tabacum-x-n.-glauca-hybrids)

[C. Description of the Field Release
8](#c.-description-of-the-field-release)

[VI. POTENTIAL ENVIRONMENTAL IMPACTS
9](#vi.-potential-environmental-impacts)

[A. Potential for Persistence of the Engineered Plants in the
Environment
9](#a.-potential-for-persistence-of-the-engineered-plants-in-the-environment)

[1. Potential for Human Error 9](#potential-for-human-error)

[2. Potential for Seed Production 10](#potential-for-seed-production)

[3. Potential for Outcrossing to Cultivated Tobacco
10](#potential-for-outcrossing-to-cultivated-tobacco)

[4. Potential for Outcrossing to Tobacco Grown for Seed Production
11](#potential-for-outcrossing-to-tobacco-grown-for-seed-production)

[5. Potential for Outcrossing to Naturalized or Ornamental Species of
Nicotiana
11](#potential-for-outcrossing-to-naturalized-or-ornamental-species-of-nicotiana)

[B. Impacts from the Presence of *npt*II, *nos, add*3, *trf*A and ColEI
and RK2 Origin of Replication.
12](#b.-impacts-from-the-presence-of-nptii-nos-add3-trfa-and-colei-and-rk2-origin-of-replication.)

[C. Impact on animals 12](#c.-impact-on-animals)

[D. Alteration in susceptibility to disease or insects
13](#d.-alteration-in-susceptibility-to-disease-or-insects)

[E. Weediness 13](#e.-weediness)

[F. Impact on Existing Agricultural Practices
13](#f.-impact-on-existing-agricultural-practices)

[G. Horizontal Gene Transfer to Other Organisms
13](#g.-horizontal-gene-transfer-to-other-organisms)

[H. Impacts on Human Health 14](#h.-impacts-on-human-health)

[I. Impacts on Threatened and Endangered Species
14](#i.-impacts-on-threatened-and-endangered-species)

[J. Cumulative Environmental Effects
15](#j.-cumulative-environmental-effects)

[K. Special Considerations: Other Environmental Statutes and
Considerations
15](#k.-special-considerations-other-environmental-statutes-and-considerations)

[VII. CITED REFERENCES 16](#vii.-cited-references)

[VIII. PREPARERS AND REVIEWERS 18](#viii.-preparers-and-reviewers)

[IX. AGENCY CONTACT 18](#ix.-agency-contact)

[APPENDIX A: ANTIBODIES 19](#appendix-a-antibodies)

[APPENDIX B: STANDARD PERMIT CONDITIONS FROM 7 CFR § 340.4
25](#appendix-b-standard-permit-conditions-from-7-cfr-340.4)

[APPENDIX C: SUPPLEMENTAL PERMIT CONDITIONS
27](#appendix-c-supplemental-permit-conditions)

[APPENDIX D: APPLICANT SUPPLIED TES WORKSHEET
34](#appendix-d-applicant-supplied-tes-worksheet)

#   {#section .unnumbered}

# I. SUMMARY

The Animal and Plant Health Inspection Service (APHIS) of the United
States Department of Agriculture, has prepared an environmental
assessment (EA) in response to a request for a permit (APHIS Number
05-354-03r) submitted by Planet Biotechnology for environmental release
of a genetically engineered *Nicotiana* interspecies hybrid*.* Planet
Biotechnology crossed a genetically engineered *Nicotiana tabacum* that
produces an antimicrobial antibody which binds to a bacterium
(*Streptococcus mutans*) associated with tooth decay in humans, with *N.
glauca* (*N. tabacum* X *N. glauca*) to generate an interspecific
*Nicotiana* hybrid line, 06PBCarHG1.

Prior to submission of this permit, Planet Biotechnology obtained APHIS
permits for small-scale field tests of 06PBCarHG1. In permit application
05-354-03r Planet Biotechnology seeks approval to grow 06PBCarHG1 at a
larger-scale of production. The mitigation measures imposed to prevent
outcrossing for previous field tests included removal of flower buds.
More recent data on the fertility of these hybrids have indicated that
06PBCarHG1 is sterile under certain circumstances (see Section V.
AFFECTED ENVIRONMENT, part B. Biology of *Nicotiana* and of *N. tabacum*
X *N. glauca* Hybrids, for a discussion of sterility factors).
Therefore, during the proposed field trial, Planet Biotechnology has
proposed that the *N. tabacum* X *N. glauca* hybrid 06PBCarHG1 plants be
allowed to flower openly. This EA discusses the contribution of the
sterility of these hybrids to the overall confinement of the regulated
article. Additional physical confinement procedures and processes will
also contribute to the confinement of the regulated article to the field
test site. This EA examines whether there are potential adverse
environmental impacts associated with the proposed action.

APHIS has reviewed the information submitted by Planet Biotechnology and
is considering whether to deny the permit (Alternative A), or issue the
permit with the requirements proposed in the application and the
supplemental permit conditions (Alternative B). This EA and the comments
received from the public will serve to inform this decisionmaking
process to allow or not to allow this environmental release.

The preferred alternative that APHIS selected is "Alternative B," *Issue
the Permit with Supplemental Permit Conditions*. This decision is based
upon the conclusion that the mitigation measures described in the
environmental assessment are adequate to prevent significant
environmental impacts.

# II. INTRODUCTION

## 

## A. Purpose of the Environmental Release

On December 21, 2005, APHIS received a permit application (05-354-03r)
from Planet Biotechnology of Hayward, CA. The application requests
permission to release a genetically engineered *N. tabacum* X *N.
glauca* hybrid line, 06PBCarHG1, into the environment in a confined
field trial. The purpose of the environmental release is to grow the
genetically engineered *Nicotiana* hybrid line, 06PBCarHG1, which
produces an antibody, designated by Planet Biotechnology as CaroRx™. The
environmental release, in Daviess County, Kentucky, will begin in June
2007 and will conclude in the fall of 2007. Following harvest of the
*Nicotiana* hybrid leaves, the permittee will extract and purify the
CaroRx™ antibody. Application of CaroRx™ to human teeth is intended to
prevent tooth decay (Ma 1998). In the United States, CaroRx™ is an
Investigational New Drug (BB-IND \# 7526) and in the European Union, it
is a registered Medical Device.

## B. USDA-APHIS Regulatory Authority

The authorities for regulation of genetically engineered plants is the
Plant Protection Act, 7 U.S.C. 7701-7772, and USDA-APHIS regulations
under Title 7 of the Code of Federal Regulations, Part 340 (7 CFR part
340), "Introduction of Organisms and Products Altered or Produced
Through Genetic Engineering Which are Plant Pests or Which There is
Reason to Believe are Plant Pests." A genetically engineered organism is
considered a regulated article if the donor organism, recipient
organism, vector, or vector agent used in engineering the organism
belongs to one of the taxonomic groups listed in the regulation and is
also a plant pest, or if there is a reason to believe it is a plant
pest. The permit application submitted to APHIS by Planet Biotechnology
requests approval for environmental release of transgenic *Nicotiana*
hybrid line, 06PBCarHG1, which contains regulatory genes from the
cauliflower mosaic virus (CaMV). Because Number range CHAPTERCaMV is
listed as a plant pest under 7 CFR 340.2, the organism is deemed a
regulated article.

This EA was prepared in accordance with: (1) The National Environmental
Policy Act of 1969 (NEPA), as amended (42 U.S.C. 4321 *et seq*.); (2)
regulations of the Council on Environmental Quality for implementing the
procedural provisions of NEPA (40 CFR 1500-1508); and (3) APHIS' NEPA
Implementing Procedures (7 CFR part 372).

Generally, the issuance of permits for confined field tests of regulated
articles is categorically excluded from the requirements for an EA under
APHIS' NEPA implementing procedures (7 CFR 372.5(c)(3)(ii)). In certain
cases, when APHIS determines that a confined field release of a
genetically engineered organism has the potential to significantly
affect the quality of the human environment as those terms are defined
in 40 CFR 1508.27 and 1508.14, an EA or environmental impact statement
is prepared pursuant to 7 CFR 372.5(d). Accordingly, APHIS prepared this
EA because the permit applicant intends to grow *N. tabacum* X *N.
glauca* hybrid plants, which are genetically engineered to produce
antibodies, at a large scale for a plant made pharmaceutical. The
applicant designed the production practices for the proposed
environmental release that raises new issues. The permittee proposes use
of no mitigation measures to remove the flower buds because *N. tabacum*
X *N. glauca* hybrids exhibit male sterility. Consequently, APHIS has
prepared this EA to determine whether there are adverse environmental
effects associated with the proposed release of *N. tabacum X N. glauca*
hybrid line, 06PBCarHG1, genetically engineered to express an antibody
as well as the marker gene, neomycin phosphotransferase (NPTII), and
nopaline synthase (NOS).

# III. NEED FOR THE PROPOSED ACTION

Under APHIS regulations (7 CFR 340.4(e)), the receipt of a permit
application to introduce a genetically engineered organism requires a
response from the Administrator:

> *"Administrative action on applications*. After receipt and review by
> APHIS of the application and the data submitted pursuant to paragraph
> (a) of this section, including any additional information requested by
> APHIS, a permit shall be granted or denied."

# 

# IV. ALTERNATIVES TO THE PROPOSED ACTION

## A. No Action

For the purposes of this EA, the "no action" alternative would be denial
of permit application 05-354-03r. This would be the preferred
alternative if, after review of the processes and procedures to prevent
the dissemination and establishment of plant pests as described in the
permit, APHIS concluded that the permit conditions would not be adequate
to prevent significant environmental impacts.

## B. Issue the Permit with Supplemental Permit Conditions

If APHIS made the decision to issue this permit with supplemental permit
conditions, the permittee would be allowed to proceed with the
environmental release in Daviess County, Kentucky, (see Section V.
AFFECTED ENVIRONMENT, part C. Description of the Field Release), but
would have to adhere to supplemental permit conditions specified by
APHIS in addition to the standard permit conditions and those proposed
in the application. APHIS would base the supplemental permit conditions
on its scientific analysis of the permit application, input from the
state of Kentucky, and public comment on this EA (see Appendix C:
Supplemental Permit Conditions). If warranted, APHIS would require
mitigating measures, stipulated in the final supplemental permit
conditions, to prevent dissemination of the organism outside the field
production area.

Currently, APHIS proposes to include the following measures to promote a
confined field release and to ensure no significant harm to the
environment:

1.  The permittee must document activities related to the field test and
    movement of the regulated article and make them available for APHIS
    inspections.

2.  The permittee will report unintended releases according to
    timeframes and procedures provided in the supplemental permit
    conditions.

3.  The permittee will provide reports to APHIS according to the
    guidance in the supplemental permit conditions.

Under APHIS regulations, compliance with all mitigating measures is
required:

> "*Permit conditions.* A person who is issued a permit and his/her
> employees or agents shall comply with the following conditions
> (Standard Permit Conditions, Appendix B), and any supplemental permit
> conditions (Supplemental Permit Conditions, Appendix C) which shall be
> listed on the permit, as deemed by the Deputy Administrator to be
> necessary to prevent the dissemination and establishment of plant
> pests" 7 CFR 340.4(f)

This would be the preferred alternative if, after review of the
processes and procedures to prevent the dissemination and establishment
of plant pests as described in the permit and in the supplemental permit
conditions, APHIS concluded that the permit conditions would adequately
prevent any significant environmental impacts.

# V. AFFECTED ENVIRONMENT

## A. Description of the Regulated Article

The interspecific *Nicotiana* hybrid, 06PBCarHG1, is genetically
engineered to produce the antibody CaroRx™ for use as a treatment to
prevent tooth decay. CaroRx™ specifically binds to the bacterium, *S.
mutans,* which has been identified as the major organism causing tooth
decay (Loesche 1975). When the attachment of *S. mutans* to the tooth
surface is blocked by an antibody, such as CaroRx™, which binds to the
*S. mutans* SAI/II surface adhesion protein, subsequent infection and
resulting tooth decay may be prevented (Ma 1998).

An antibody is a protein that binds specifically to a particular
substance, known as an antigen. While each antibody is unique in its
ability to bind to its corresponding antigen, antibodies, in general,
have the same overall structure. Typically, antibodies exist as one or
more copies of a Y-shaped unit composed of four polypeptide chains
called immunoglobulins (Ig). Each Y-shaped Ig contains two identical
copies of a heavy chain, and two identical copies of a light chain (see
Appendix A, Fig.1A). An antibody can also be fragmented via enzymatic
digestion into the Fab, Fab2, and Fc fragments (see Appendix A, Fig.
1B). Secretory antibodies comprise the most abundant class of antibodies
produced in humans (Ma 2005). They exist in the dimeric form with two
Y-shaped Ig molecules bound to a J chain and to a secretory component
(see Appendix A, Fig. 2). The J chain serves to dimerize the two Ig
molecules and the secretory component serves to protect the
immunoglobulin from proteases (Ma 1998). The Ig molecules in CaroRx^TM^
were derived from Guy's 13-immunoglobulin G (IgG) monoclonal antibody
that specifically binds to *S. mutans* (Ma 1994). The CaroRx™ antibody
differs from Guy's 13 antibody as the former is engineered to contain an
Fc portion of the heavy chain from an immunoglobulin A (IgA). This
chimeric IgA/IgG antibody binds to a J chain and further assembly with a
secretory component (Ma 1995, Ma 1998). The secretory antibody CaroRx™
prevents bacterial colonization to protect humans against oral *S.
mutans* infection (Ma 1998).

Planet Biotechnology generated the transformed *Nicotiana* interspecies
hybrid line, 06PBCarHG1, by first cloning and expressing the CaroRx^TM^
gene in *N. tabacum* line H8-105 and then crossing H8-105 with *N.
glauca.* The *Nicotiana* hybrid line, 06PBCarHG1, is genetically
engineered with the constituent parts of the secretory CaroRx^TM^
antibody (light chain from mouse, heavy chain from mouse, J chain from
mouse, and secretory component from rabbit), all driven by the
cauliflower mosaic virus (CaMV) promoter. These constituent parts were
cloned and expressed in tobacco by independent transformation events.
The events were combined into a single line by classical breeding
methods (Ma 1995). Hybrid line 06PBCarHG1 contains two additional
protein products that are expressed under the control of a plant
recognized NOS promoter (one of very few bacterial promoters known to be
expressed in plants). These proteins are NPTII (from *E. coli*), an
enzyme that confers resistance to kanamycin, which is used as a
selectable marker, and NOS (from *A. tumefaciens*), an enzyme that forms
nopaline from the amino acid arginine and alpha-ketoglutaric acid. NOS,
although present in the plasmid vector used in the production
06PBCarHG1, was not utilized as a selectable marker in the construction
of 06PBCarHG1. Line 06PBCarHG1 also contains *trf*A (from *E. coli*)
that encodes for a DNA-binding protein important for plasmid DNA
replication and *add*3 (from *E. coli*) that encodes for resistance to
the antibiotic streptomycin/spectinomycin. However, because *trf*A and
*add*3 are driven by bacterial promoters that are not recognized by
plants, these genes are not expressed in 06PBCarHG1. Additional
non-coding sequences (sequences contained in the transformed plant, but
not converted into protein products) present in 06PBCarHG1 are ColEI and
RK2 origin of replication sequences (from *E. coli*) and the *nos*
terminator from *A. tumefaciens.*

In the transformed line 06PBCarHG1, the expression level of CaroRx^TM^
can be as high as 35.0 mg per kilogram of fresh weight in leaves (mg/kg
FW), up to 8.0 mg/kg FW in stems, and up to 4.1 mg/kg FW roots, but it
is not detectable in pollen (data submitted by the applicant with the
05-354-03r permit).

## B. Biology of *Nicotiana* and of *N. tabacum* X *N. glauca* Hybrids 

The genus, *Nicotiana*, is composed of some 76 naturally occurring
species (Chase 2003). Many *Nicotiana* species are native to South
America with the remainder distributed throughout Central America,
western North America, Australia, and various islands of the South
Pacific. *N. tabacum* L. (tobacco) probably originated in Argentina by
hybridization of *N. sylvestris* Speg. & Comes, and *N. tomentosiformis*
Goods, where the progenitors are native. Most *Nicotiana* species are
not cultivated, with the exception of *N. tabacum,* and to a lesser
extent, *N. rustica* (Chaplan 1979). *N. glauca* Graham, also a native
of Argentina, is not naturalized in Kentucky (USDA, NRCS 2006), although
it does naturalize in warm temperate regions throughout the world.

The subject of this EA is a *N. tabacum* X *N. glauca* hybrid line
06PBCarHG1 that resulted from crossing *N. tabacum* cultivar Petite
Havana line H8-105 (female parent) to a wild type *N. glauca* (male
parent). The Kentucky Tobacco Research and Development Center (KTRDC)
made all crosses at the University of Kentucky. The strategy of
production of *N. tabacum* X *N. glauca* hybrid plants having various
phenotypic characteristics, which are optimal for plant made
pharmaceutical applications, is property of the University of Kentucky,
and the subject of a U.S. patent application (20,060,236,433) by KTRDC.

Natural hybridization has frequently occurred in the evolution of the
genus *Nicotiana* (Goodspeed 1954). More recently, to create improved
varieties of *N. tabacum* (commonly cultivated tobacco), breeders have
taken advantage of the interspecific transfer of genes from wild
relatives of *N. tabacum* for traits such as disease and insect
resistance. These interspecific *Nicotiana* hybrids generally have
reduced fertility (therefore some level of sterility) such as reduced
pollen viability and/or reduced seed production and seed viability
(Al-Almad 2006, Burk 1979, Nikova 1997). When there is a large
difference in the numbers of chromosomes between the two parents, there
is likely to be very little chromosome pairing at meiosis resulting in
progeny that are sterile. For *N. tabacum*, which has twice the number
of chromosomes as *N. glauca,* sterile progeny are expected*.*
Interspecific breeding allowed for production of male sterile lines that
lack viable pollen. Such lines are useful in breeding *Nicotiana,*
species, such as *N. tabacum,* which produce flowers that are selfing
(are fertilized by their own pollen) (Burk 1979). Previously, *N.
tabacum* crossed with N. *bigelovii*, *N. debneyi*, *N. glutinosa, N.
megalosiphon*, *N. plumbaginifolia*, *N. rustica*, *N. suaveolens*, and
*N. undulata* also resulted in sterile *Nicotiana* hybrids (Chaplin
1964). In the present case, KTRDC produced sterile hybrids as a
confinement measure for field testing genetically engineered *Nicotiana*
hybrids (Mundell 2005).

As discussed in the section VI. POTENTIAL ENVIRONMENTAL IMPACTS, the
only potential plants to which outcrossing could occur, that are likely
present in the area surrounding the release site, are cultivated tobacco
(*N. tabacum*). The sterility of 06PBCarHG1 and *N. tabacum* X *N.
glauca* hybrids was analyzed in crossing tests in the greenhouse, where
manual hand pollination was evaluated, and in the field, where natural
bird and insect pollination was evaluated (Mundell 2005, data submitted
with permit 05-354-03r). Under field conditions, 06PBCarHG1 is highly
likely to be both female and male sterile.

## C. Description of the Field Release

The affected environment will be limited to the release site, as the
applicant proposes a confined environmental release in the permit. The
applicant shall perform mitigation measures designed to confine the
regulated material to the release site and apart from nonregulated
material.

The applicant proposed the following measures in the submitted permit to
confine the release to the field test site:

1.  The applicant shall use dedicated facilities (locked or secured
    buildings, bins, or areas, posted as "Restricted To Authorized
    Personnel Only") for storage of equipment and regulated articles for
    the duration of the field test.

2.  The applicant shall tag all field plots and record and communicate
    Global Positioning System (GPS) coordinates to APHIS in order to
    precisely locate the field (helps to locate the field to monitor for
    volunteers).

3.  The applicant shall border the field plots shall on all four sides
    with 50 feet of perimeter fallow zone (not in production) to allow
    farm machinery to move around the site and will prevent physical
    mixing of the regulated plants with surrounding plants that may be
    used for food or feed.

4.  The applicant shall maintain an isolation distance of at least
    ½-mile between the regulated plots and non-regulated tobacco. The
    applicant will monitor this area throughout the field test period to
    ensure the minimum ½-mile distance to non-regulated tobacco.

5.  Applicants will maintain at least a 1-mile distance between the
    field plots and any seed production. The applicant will monitor this
    area throughout the field test period to ensure the minimum 1-mile
    separation from seed production.

6.  Applicants shall germinate seeds in a greenhouse and transplant
    plantlets out into the field.

7.  APHIS will inspect the plants during the growing season for traits
    such as weediness, resistance/susceptibility to insects or disease,
    or unusual differences in plant growth or morphology.

8.  The applicant shall clean all field equipment or vehicles entering
    the field that are used for harvest, transport, and pest/weed
    control both prior to use and after use, according to the
    APHIS-approved Standard Operating Procedures (SOPs).

9.  The applicant will monitor all surrounding land within a ½-mile
    radius during the period of time when the regulated article is
    producing pollen to ensure that the area has no reproductively
    compatible plants.

10. The applicant shall disk into the soil the remaining plant material
    within 2 weeks following harvest and antibody extraction.

11. Harvesting equipment must be dedicated for use in the permitted test
    site(s).

12. The applicant shall monitor the field plot and 50-foot border area
    for volunteers for 12 months. Applicants will uproot any volunteers,
    destroy them, and incorporate them into the soil.

13. Applicants shall provide instruction to personnel who handle the
    regulated material so that they are aware of handling procedures.
    The applicant shall document this training and make it available to
    APHIS inspectors. This training shall encompass conditions
    stipulated in the permit, the APHIS permit conditions, the
    APHIS-supplemental permit conditions, and the pertinent Federal
    regulations.

14. The applicant shall document activities related to the field test
    and movement of the regulated article.

In addition, APHIS performs the following activities to reduce the
possibility of human error:

1)  Inspection of records that cover multiple aspects of the field
    trial.

2)  Multiple inspections of field trials timed to occur at critical
    steps in the production process.

# VI. POTENTIAL ENVIRONMENTAL IMPACTS

The proposed action is to conduct a confined environmental release with
a regulated article, a genetically engineered organism. The permit
application describes the procedures that the applicant has submitted to
APHIS to confine the regulated organism to the release site according to
the requirements under 7 CFR part 340. APHIS evaluated the proposed
action based on the biology of the *N. tabacum* X *N. glauca* hybrid
plants, any potential hazards associated with the transgenes, and the
likelihood that the transgenes could persist and potentially harm the
environment.

## A. Potential for Persistence of the Engineered Plants in the Environment

### Potential for Human Error

APHIS considered the likelihood that humans could inadvertently move the
regulated article from the environmental release site. In a recent
workshop hosted by APHIS dealing with gene confinement issues in
genetically engineered crops (USDA-APHIS 2004) one of the more likely
mechanisms contributing to the breakdown of confinement and movement of
seed was identified as human error, and the most reliable means of
preventing this is to maintain and enforce stringent SOPs. APHIS
requests and reviews SOPs for equipment and processes related to the
movement, planting, monitoring, and harvest of genetically engineered
plants. APHIS verifies implementation of these procedures during
multiple inspections. In addition, APHIS requires that the personnel who
handle the regulated material receive training according to
APHIS-approved training processes to further ensure that the SOPs are
carried out during the course of the environmental release. Therefore,
APHIS believes that measures are in place to ensure that unauthorized
movements resulting from human error are very unlikely.

### Potential for Seed Production

APHIS evaluated the potential for the *N. tabacum* X *N. glauca* hybrid
line, 06PBCarHG1, plants or their progeny to survive in the environment
at the conclusion of the environmental release. *Nicotiana* hybrid
production starts in the greenhouse, where seeds are germinated under
controlled conditions. *Nicotiana* hybrid seedlings are then
transplanted into the field and grown to maturity. Researchers at the
University of Kentucky performed several years of field trials with
non-genetically engineered *N. tabacum* X *N. glauca* hybrids, where the
plants flowered. The fields used for these trials were surveyed for
seedpods. No viable seeds were observed after these trails ended
(Richard Mundell, KTRDC, University of Kentucky, personal communication
to Orlando Chambers KTRDC, University of Kentucky, December 7, 2006).
Additional greenhouse testing and field experiments indicated *N.
tabacum* X *N. glauca* hybrids and 06PBCarHG1, when crossed with pollen
from *N. tabacum* did not produce viable seeds (Mundell 2005, data
submitted with permit 05-354-03r). APHIS therefore determines that the
genetically engineered hybrid line 06PBCarHG1 is not likely to persist
because viable seeds are not likely to be produced. Even if the hybrid
*Nicotiana* produces seeds, they would likely not survive the winter and
establish, because neither parental species of the hybrid overwinters in
the state of Kentucky (USDA-NRCS 2006). The applicant will monitor the
environmental release site for volunteers the following year. APHIS
determines that viable seed production is not likely and even if seed
production occurs, the resulting plants are not likely to persist in the
environment.

### Potential for Outcrossing to Cultivated Tobacco

APHIS further evaluated the potential for genes from 06PBCarGH1 to
persist in the environment due to the possibility that the transgenic
pollen would outcross to surrounding tobacco plants. In nature,
pollination of *Nicotiana* occurs via honeybees (*Apis mellifera* L.),
sweat flies (*Didea fasciata* Macq.), bumblebees (*Bombus* species),
hawkmoths (*Theretra tersa* L.), hummingbirds (*Archilochus colubris*
L), and in some cases by bats (Hodges 1952, McMurtrey 1960, Nattero
2003, Poehlman 1959). Greenhouse pollination tests are typically much
more efficient than crosses in nature, especially when pollen of
doubtful viability is involved, because the hand crosses involve larger
quantities of pollen than would be transferred by an animal pollinator
and are from a single source. When grown in the greenhouse or out in the
field, *N. tabacum* X *N. glauca* hybrids were not able to fertilize *N.
tabacum* plants placed in proximity when pollinated by insects or birds
nor when pollinated by hand (Mundell 2005). Furthermore, less than 1
percent of the pollen germinated indicating that the hybrid pollen is
predominantly non-viable. Similarly*,* the genetically engineered hybrid
06PBCarHG1 line was analyzed in greenhouse studies by hand pollinating
06PBCarHG1 pollen onto *N. tabacum* plants (data submitted with permit
05-354-03r). Most attempts to pollinate *N. tabacum* using 06PBCarHG1 as
the male parent also failed. In 1 experiment, out of 32 attempted
crosses, a single seedpod produced viable seeds, (05-354-04r field test
report). When these seeds were germinated and resulting plants were
tested for CaroRx, no CaroRx was detected. The greenhouse and field test
results suggest that outcrossing will not occur. Even if the 06PBCarHG1
was capable of outcrossing to tobacco plants, in nature, animal
pollinators will frequent different flowers and will bring a mixture of
pollen to the flower including pollen of much higher viability than that
produced by 06PBCarHG1 and with a much higher probably of successfully
fertilizing flowers.

Furthermore, the transgenic hybrid 06PBCarGH1 site is isolated from
other tobacco plants by at least ½-mile. This distance is twice the
AOSCA standard to produce tobacco seed of 0.01 percent varietal purity.
The AOSCA standard assumes pollen of full viability, and 06PBCarHG1 has
been shown to have pollen of only about 1% viability. Another factor
that will reduce the likelihood of outcrossing is that the common
practice for the production of commercial tobacco in Kentucky involves
topping (flower removal) and sucker control methods (Dr. Robert Pearce,
Extension Tobacco Specialist, University of Kentucky, Communicated to
APHIS on 11/03/06). These production methods will reduce or eliminate
the availability of receptive cultivated tobacco flowers for
cross-hybridization.

APHIS believes that the field studies have shown that hybrid 06PBCarGH1
plants are sterile when the hybrid 06PBCarGH1 is grown and pollinated
under natural conditions. However, because some of the greenhouse
crossing experiments indicate some low level of fertility, the proposed
field release will include trap plants to obtain additional field data
on the sterility of the 06PBCarGH1 under field conditions. Planet
Biotechnology will place male sterile/female fertile tobacco plants 8-15
feet from the edge of each field release site. The male sterile tobacco
plants will be monitored for seed formation to assess the outcrossing
potential from the hybrid plants. In this way, the expectation that
transgenic hybrid 06PBCarGH1 plants will not outcross with surrounding
cultivated tobacco will be directly tested.

### Potential for Outcrossing to Tobacco Grown for Seed Production

Another concern that APHIS must consider is the proximity of tobacco
seed production in the area of the environmental release. Such tobacco
plants will not be topped and the seed will be saved for planting. The
closest known tobacco seed production area is at least 10 miles from
Daviess County, Kentucky (Dr. Robert Pearce, Extension Tobacco
Specialist, University of Kentucky, 11/03/06). This distance is 20 times
greater than the distance specified by the AOSCA standard for 0.01
percent varietal purity (AOSCA 2004). APHIS concludes there is a
negligible likelihood that the transgenic hybrid 06PBCarGH1 plants will
outcross to tobacco grown for seed production purposes.

### Potential for Outcrossing to Naturalized or Ornamental Species of *Nicotiana* 

APHIS evaluated the potential for the genetically engineered hybrid line
06PBCarHG1 to outcross to other *Nicotiana* species, other than that of
cultivated tobacco. No wild and weedy *Nicotiana* plants that could
potentially cross with 06PBCarHG1 reside in Kentucky (Kartesz 2005,
USDA-NRCS 2006). Ornamental *Nicotiana* species are not frequently grown
in Kentucky (Robert G. Anderson**,** Extension Professor**,** Department
of Horticulture, University of Kentucky**,** Lexington, KY, personal
communication). Even if ornamental *Nicotiana* species were grown near
this field plot, seed production between interspecific hybrids is
unlikely for reasons already discussed above (Al-Ahmad 2006, Nikova
1997). Even if seeds were produced, APHIS does not foresee that these
seeds would be collected and saved for planting the following year.

APHIS concludes that gene introgression from 06PBCarHG1 into cultivated
tobacco and ornamental *Nicotiana* species and persistence of the
regulated article in the environment is extremely unlikely to occur as a
result of the proposed environmental release.

## B. Impacts from the Presence of *npt*II, *nos, add*3, *trf*A and ColEI and RK2 Origin of Replication.

The selectable marker gene for kanamycin resistance (*nptII*) is
expressed in 06PBCarHG1. Because NPTII is not toxic, shares no homology
with proteins known to be toxic or allergenic (U.S. FDA 1998), and is
present in many plant lines previously deregulated by USDA, the
expression of NPTII in *Nicotiana* hybrid plants is not expected to have
deleterious effects or significant effects on nontarget organisms,
including beneficial organisms and either listed or proposed-for-listing
threatened and endangered species (TES). The NOS protein, which produces
nopaline, has no known sequence homology to known toxins or allergens
(Canadian Food Inspection Agency, 1998), and many plant species, such as
soybeans and cotton, naturally produce nopaline (Christou 1986).
Similarly, the expression of NOS in *Nicotiana* hybrid plants is not
expected to have deleterious effects or significant effects on nontarget
organisms, including beneficial organisms and TES.

Other DNA sequences are present, but are not expected to be expressed in
06PBCarHG1. Because the genes *trf*A (encodes for a DNA-binding protein
that initiates replication from the RK2 origin of replication) and
*add*3 (encodes for the resistance to streptomycin/spectinomycin) are
under the control of bacterial promoters, they are unlikely to be
expressed in the nuclear plant genome. Similarly ColEI and RK2, origin
of replication sequences (from *E. coli*), and the *nos* terminator
(from *A. tumefaciens*) are not expressed in 06PBCarHG1. Therefore,
APHIS has determined the presence of the NPTII and NOS proteins and the
non-expressed DNA will have no significant environmental impacts.

## C. Impact on animals

Animals, other than the occasional skunk foraging on insects, do not
generally consume *Nicotiana* hybrids planted in an agricultural setting
(Dr. Orlando Chambers, University of Kentucky, personal communication).
In the rare situation that an animal might forage on a 06PBCarHG1 plant,
antibodies as a class are non-toxic. Antibodies are ubiquitous in
nature, so insects and animals that consume eggs and milk are routinely
exposed to antibodies (see Appendix A: Antibodies). Any antibody in
plant debris that was produced via genetic engineering would have the
same fate as antibodies in any decaying animal tissue or animal
by-product, and would be quickly degraded (see Appendix A: Antibodies,
for more detailed information on the degradation of antibodies in the
environment). The genetically engineered CaroRx™ has been found to be
non-toxic and non-allergenic. During clinical trials where CaroRx™ was
administered to animals and human volunteers, no adverse effects were
detected suggesting that plant preparations containing the purified
antibodies did not induce an allergic response when given orally (Ma
1998, Ma 2005, Weintraub 2004). Therefore, APHIS believes that the
genetically engineered *Nicotiana* hybrids present no increased risk to
animals compared to the non-GE *Nicotiana* hybrid.

## D. Alteration in susceptibility to disease or insects

The presence of CaroRx™ in *Nicotiana* hybrids is not expected to alter
the susceptibility of *Nicotiana* hybrid plants to diseases or to
insects, as the antibody binds specifically to the *S. mutans* SAI/II
surface adhesion protein in the bacterium *S. mutans*. *S. mutans* is an
organism that causes tooth decay but has no reported effects on plants
and due to its specificity will not bind to plant pathogens or insect
pests. In addition, previous field tests conducted with *N. tabacum* X
*N. glauca* hybrids in 2005 (05-053-01r, 05-354-04r, and in 2006
(06-037-0r) did not reveal increased susceptibility to disease or
insects. Therefore, APHIS believes that this environmental release will
not increase *Nicotiana* hybrid disease or susceptibility of *Nicotiana*
hybrids to insects.

## E. Weediness

APHIS evaluated the potential for the genetically engineered *N.
tabacum* X *N. glauca* hybrid line 06PBCarHG1 to be weedy. Previous
field tests over a number of years have indicated that the *N. tabacum*
X *N. glauca* hybrid 06PBCarHG1 does not produce viable seeds. Because
*Nicotiana* plants propagate by the production of seeds and little to no
seeds will be produced, the field release of hybrid line 06PBCarHG1 is
not expected to result in plants that could potentially be weedy. As
neither parental species of the hybrid overwinters in the state of
Kentucky, even if plants produced viable seeds, the resulting plants are
not likely to persist in the environment. The addition of the transgenes
is not likely to render the *N. tabacum* and *N. glauca* hybrid line,
weedier. None of the gene products is likely to increase the fitness,
alter reproductive capacity, or affect other traits associated with
weediness.

## F. Impact on Existing Agricultural Practices 

The transgenic *Nicotiana* hybrid plants engineered to produce
antibodies will be cultivated using standard cultivation practices
generally used for tobacco production in Kentucky. Additional measures
will be taken to ensure that the regulated article is confined to the
site of the environmental release. These measures are described in part
V, section C, Affected Environment Description of the Field Release, and
in Appendix C, Supplemental Permit Conditions, of this EA. Planet
Biotechnology will monitor the fields throughout the growing season for
deleterious effects on plants, non-target organisms, or the environment,
and during the following year for volunteer plants. The use of these
plants in the proposed environmental release should not affect current
agricultural practices with tobacco.

## G. Horizontal Gene Transfer to Other Organisms 

Following harvest of the *Nicotiana* hybrid plants, some plant material
will remain at the environmental release site and will be subject to
natural degradation by soil-inhabiting microbes. APHIS has assessed the
likelihood of whether DNA transfer could occur to soil-inhabiting
microbes through a process known as horizontal transfer. Horizontal gene
transfer of DNA from the *Nicotiana* hybrid plants to bacteria and
expression in bacteria is unlikely to occur. First, many genomes have
been sequenced from bacteria that are closely associated with plants,
including *Agrobacterium* and *Rhizobium* (Kaneko 2000, Wood 2001,
Kaneko 2002). There is no evidence that these organisms contain genes
derived from plants. Second, in cases where review of sequence data
implied that horizontal gene transfer occurred, these events are
inferred to occur on an evolutionary time scale, on the order of
millions of years (Koonin 2001, Brown 2003). Third, FDA has evaluated
horizontal gene transfer from the use of antibiotic resistance marker
genes in genetically engineered plants and concluded that the likelihood
of transfer of antibiotic resistance genes from plant genomes to
microorganisms, in the gastrointestinal tract of humans or animals or in
the environment, is remote (<http://vm.cfsan.fda.gov/~dms/opa-armg.html>
), and APHIS concurs with this finding. Therefore, APHIS concludes that
horizontal gene transfer is unlikely to occur.

## H. Impacts on Human Health

The *Nicotiana* hybrid plants in this environmental release will be used
for the processing and extraction of antibodies, but will not be used
directly for other purposes. After extraction of CaroRx^TM^, the
remaining *Nicotiana* hybrid plant material will be incorporated into
the soil for natural decomposition. CaroRx^TM^ has been the subject of
clinical trials where its safety was demonstrated. No adverse effects to
humans were reported when CaroRx^TM^ antibodies were applied orally (Ma
1990). Because there may be some flowers produced, and honeybees are
known to pollinate *Nicotiana* species, APHIS considered the possibility
that the gene product would be present in honey. APHIS concludes that
the CaroRx^TM^ will not be detectable in honey because CaroRx^TM^ is not
expressed in pollen or nectar at levels that can be measured with a
sensitive antibody assay (data submitted with the 05-354-03r permit by
Planet Biotechnology). No potential impact on people living in the area
of the environmental release, or any other human population, can be
identified. Therefore, APHIS concludes that there should not be any
significant effects on the human environment as a result of this
proposed field trial.

## I. Impacts on Threatened and Endangered Species

APHIS evaluated the potential for impacts on TES proposed and listed
with the U.S. Fish and Wildlife Service (FWS) using the U.S. Fish and
Wildlife database <http://www.fws.gov/endangered/wildlife.html> and
NatureServe database: <http://www.natureserve.org/explorer/>. APHIS
analyzed the published data and studies supplied by the applicant and
supports the applicant's conclusion that the confined release of
06PBCarHG1 would not harm any Federally listed (or proposed) TES (see
Appendix D: The Threatened and Endangered Species worksheet that was
prepared by the permit applicant). The analyses found that none of the
listed (or proposed) TES are associated with *Nicotiana* hybrid fields
in Kentucky. Even if any of the listed (or proposed) TES frequented the
environmental releases, none of the species would likely be exposed to
the engineered products because they do not consume tobacco plants and
the engineered products are not detectable in pollen. Even if the listed
species were exposed to the engineered products (antibody, NOS, NPTII),
the risk would be negligible because there is no reported toxicity of
the products and, therefore, the *Nicotiana* hybrids would not be any
more hazardous to these organisms than unmodified tobacco.

APHIS has reached a determination that the proposed environmental
release should have no effect on federally listed TES or species
proposed for listing, and no effect on designated critical habitat or
habitat proposed for designation in the action area. Consequently,
consultation under Section 7 of the Endangered Species Act with FWS is
not required for the action described in the preferred alternative of
this EA.

## J. Cumulative Environmental Effects

The vast majority of tobacco grown in the U.S. for leaf and seed
purposes is non-transgenic. A small portion of the tobacco currently
grown is a deregulated transformed line (Vector 21-41) that was
engineered to produce reduced levels of nicotine. Relative to the total
tobacco acreage (110,734 acres in Kentucky and 428,631 total acres in
the U.S., in 2001, according to the USDA National Agricultural
Statistics Service) the proposed 100 acres will represent a very small
percentage of the acreage that will be grown in Kentucky in 2007. This
genetically engineered line differs from non-transformed tobacco, by its
ability to produce an antibody. Compared to field testing and/or large
scale plantings that may raise issues for loss of effectiveness of an
engineered herbicide because of the preponderance of use, or for the
build up of resistance in target organisms due to plant incorporated
protectants, this field release of an antibody does not raise similar
issues. Any antibody in plant debris that was produced via genetic
engineering would have the same fate as antibodies in any decaying
animal tissue or by-product, and would be quickly degraded and
incorporated into the nitrogen cycle (see Appendix A: Antibodies, for
more detailed information on the degradation of antibodies in the
environment). However, if any unidentified cumulative effects should be
found in the future, APHIS would take the appropriate action to curtail
potential significant environmental effects. There are no other past,
present, or reasonably foreseeable actions, which could, in aggregation
with the environmental release of 06PBCarHG1, cause any significant
cumulative impacts on the environment.

## K. Special Considerations: Other Environmental Statutes and Considerations 

Executive Order (EO) 12898, \"Federal Actions to Address Environmental
Justice in Minority Populations and Low-Income Populations,\" requires
Federal agencies to conduct their programs, policies, and activities
that substantially affect human health or the environment in a manner so
as not to exclude persons and populations from participation in or
benefiting from such programs. It also enforces existing statutes to
prevent minority and low-income communities from being subjected to
disproportionately high and significant human health or environmental
effects. Each alternative was analyzed in its ability to affect minority
and low-income populations. None of the alternatives were found to pose
disproportionately high or significant human health or environmental
effects to any specific minority or low-income group.

EO 13045, "Protection of Children from Environmental Health Risks and
Safety Risks," acknowledges that children may suffer disproportionately
from environmental health and safety risks because of their
developmental stage, greater metabolic activity levels, and behavior
patterns, as compared to adults. The EO (to the extent permitted by law
and consistent with APHIS's mission) requires each Federal agency to
identify, assess, and address environmental health risks and safety
risks that may disproportionately affect children. None of the
alternatives are expected to have disproportionately high or significant
human health or environmental effects to children.

EO 13112, "Invasive Species," states that Federal agencies take action
to prevent the introduction of invasive species and provide for their
control and to minimize the economic, ecological, and human health
impacts that invasive species cause. The engineered *Nicotiana* hybrid
is not expected to produce viable seeds, and is also not expected to
survive Kentucky winters, so it is not thought that it will persist in
the environment and become invasive. Based on the data submitted by the
applicant and reviewed by APHIS, the engineered plant is not
significantly different in any fitness characteristics from its parent
in ways that might increase its invasive potential.

# VII. CITED REFERENCES

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hybrids between transgenically mitigated *Nicotiana tabacum* and
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**AOSCA** (2004) \"Yellow Books\" 2004 Operational Procedures, Crop
Standards and Service Programs. AOSCA, Meridian, Idaho.

**Brown JR (**2003) Ancient horizontal gene transfer. Genetics
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**Burk G, Chaplan JF** (1979) Hybridization. pp. 23-27. In: *Nicotiana*:
Procedures for Experimental Use. Durbin RD (ed.) USDA Technical Bulletin
Number 1586.

**Canadian Food Inspection Agency** (1998) Decision Document 98-24:
Determination of the Safety of the Crop Development Centre\'s \'CDC
Triffid\', a Flax (*Linum usitatissimum* L.) Variety Tolerant to Soil
Residues of Triasulfuron and Metsulfuron-methyl
<http://www.inspection.gc.ca/english/plaveg/bio/dd/dd9824e.shtml>.

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**Goodspeed T H** 1954. The Genus *Nicotiana*, Chronica Botanica
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Complete genome structure of the nitrogen-fixing symbiotic bacterium
*Mesorhizobium loti*. DNA Research 7:331-338.

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Watanabe A, Idesawa K, Iriguchi M, Kawashima K, Kohara M, Matsumoto M,
Shimpo S, Tsuruoka H, Wada T, Yamada M, Tabata S.** (2002) Complete
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**Kartesz JT (**2005) Synthesis of the North American Flora, 2nd Ed.,
Version 1.0.2190.25485, ms. (12/30/2005), CD-ROM. Biota of North America
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monoclonal antibodies with IgG1 and IgA heavy chain domains in
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C, Mostov K, Lehner T (**1995) Generation and assembly of secretory
antibodies in plants. Science 268:716-719.

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progenitors share long-tongued hawkmoths as pollen vectors. Plant Syst.
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(http://plants.usda.gov). Data compiled from various sources by Mark W.
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**Wood DW, Setubal JC, Kaul R, Monks DE, Kitajima JP, Okura VK, Zhou Y,
Chen L.** *et al.* (2001) The genome of the natural genetic engineer
*Agrobacterium tumefaciens* C58. Science 294:2317-2323.

# VIII. PREPARERS AND REVIEWERS

**Biotechnology Regulatory Services**

Rebecca Bech, Deputy Administrator

**BRS, Environmental Risk Analysis Program**

Patricia Beetham, Ph.D., Biotechnologist (Preparer of Antibody Appendix)

Neil Hoffman, Ph.D., Director (Reviewer)

Margaret Jones, Ph.D., Senior Biotechnologist (Preparer of EA)

Michael Watson, Ph.D. Supervisory Biotechnologist (Reviewer)

# IX. AGENCY CONTACT

Cynthia Eck, Document Control Officer

USDA, APHIS, BRS

4700 River Road, Unit 147

Riverdale, MD 20737-1237

Phone: (301) 734-0667

Fax: (301) 734-8669

cynthia.a.eck@aphis.usda.gov

# APPENDIX A: ANTIBODIES

**Introduction**

The following section is an in depth description of the structure and
function of antibodies as well as their uses in research and disease
treatment.

**The Immune System**

Healthy humans are born with an innate (or natural) immunity, a type of
immunity that occurs naturally as a result of a person\'s genetic
constitution or physiology and does not arise from a previous infection
or vaccination. Innate immunity also includes the external barriers of
the body, like the skin and mucous membranes (like those that line the
nose, throat, and gastrointestinal tract). There is also a second kind
of protection called adaptive (or active) immunity. Adaptive immunity
evolves as a person or animal is exposed to diseases or immunized
against diseases through vaccination and generally produces long-term
immunity. Passive immunity is the transfer of antibodies from another
individual, as through injection or placental transfer to a fetus; it
essentially is \"borrowed\" from another source and it lasts for a short
time.

The immune system (both active and innate) is the body\'s defense
against infectious organisms and other invaders. It is made up of a
network of cells, tissues, and organs that work together to protect the
body. The cells that are part of the adaptive defense system are white
blood cells, or leukocytes. One type of leukocyte is called a
lymphocyte, which allows the body to remember and recognize previous
invaders. There are two kinds of lymphocytes: B lymphocytes (B cells)
and T lymphocytes (T cells). Lymphocytes start out in the bone marrow
and either stay there and mature into B cells, or they leave for the
thymus gland, where they mature into T cells. One of the main jobs of B
cells is the production of antibodies. The part of the immune system
that involves antibodies secreted by B cells is called humoral immunity.
A substance introduced into the body that stimulates the production of
an antibody is called an antigen. Antigens include toxins, bacteria,
foreign blood cells, and the cells of transplanted organs. When an
organism detects an antigen, several types of cells work together to
recognize and respond to it. These cells trigger the B cells to produce
antibodies. Antibodies are specialized proteins that lock onto specific
antigens. Although antibodies can recognize an antigen and lock onto it,
they are not capable of destroying it without the help of T cells. T
cells are part of the system that destroys antigens that have been
tagged by antibodies or cells that have been infected or somehow
changed. Antibodies can also neutralize toxins (poisonous or damaging
substances) produced by different organisms.

**Antibodies**

An antibody is a protein that binds specifically to a particular
substance (an antigen). While each antibody is unique in its ability to
bind to its corresponding antigen, antibodies in general have the same
overall structure and are referred to collectively as immunoglobulins.
Antibodies exist as one or more copies of a Y-shaped unit, composed of
four polypeptide chains. Each Y contains two identical copies of a
"heavy" chain, and two identical copies of a "light" chain, named as
such by their relative molecular weights (Fig.1A). An antibody can also
be broken into two pieces (via enzymatic digestion) called the Fab,
Fab2, and Fc fragments. The Fab fragment is the portion of the
immunoglobulin where the relevant antigen binds and the Fc fragment is
the other section of the immunoglobulin (Fig 1B). Antibodies can be
divided into five classes: IgG (IgY in birds), IgM, IgA, IgD and IgE,
based on the number of Y units and the type of heavy chain. Antibodies
are produced by plasma cells (B cells) in response to infection or
immunization. By binding to an antigen (or pathogen), the antibody
either neutralizes the antigen or prepares it (or pathogen) for uptake
and destruction by phagocytes (Janeway 2001).

**Polyclonal versus Monoclonal Antibodies**

Antibodies produced by the immune system are, by definition, polyclonal;
meaning many different B cells produced the antibodies in response to
antigen stimulation. A monoclonal antibody is produced by cloning a
single B cell. Large amounts of monoclonal antibodies are produced by a
hybridoma; a cell line that is made by fusing a B cell with a myeloma
cell (Köhler 1975). Monoclonal antibodies can also be produced in
genetically transformed microbial cells. Recombinant antibodies have
been successfully made in microbial expression systems and mammalian
cell cultures for over two decades. The estimated cost to produce a
monoclonal antibody using hybridoma technology or microbial expression
systems is US \$5000/gram (Institute for Laboratory Animal Research and
Council 1999). The cost to produce recombinant proteins (such as
antibodies) is reduced 80-98 percent in plants as compared to
traditional microbial and mammalian cell systems (Institute for
Laboratory Animal Research and Council 1999, Giddings 2001), with much
of the costs focused on downstream purification systems.

**Importance of Monoclonal Antibody Production**

Some of the early applications of monoclonal antibodies were blood-group
typing, pregnancy testing, and identifying viruses, cancers, blood
clots, and heart disease. Today, along with multiple diagnostic test
uses, monoclonal antibodies are part of many cancer treatments, as well
as treatments for arthritis, a variety of viruses, diabetes, and
multiple sclerosis
(<http://users.path.ox.ac.uk/~scobbold/tig/new1/mabth.html>). The FDA
has approved many monoclonal antibodies for use in cancer therapy
([www.cancer.org](http://www.cancer.org/)):

  ----------------------------- ----------------------------- --------------
  **Trade Name (Generic Name)** **Cancer Treated**            **Approved**

  Rituxan (Rituximab)           Non-Hodgkin lymphoma          1997

  Herceptin (Trastuzumab)       Breast cancer                 1998

  Mylotarg (Gemtuzumab          Acute myelogenous leukemia    2000
  ozogamicin)                   (AML)                         

  Campath (Alemtuzumab)         Chronic lymphocytic leukemia  2001
                                (CLL)                         

  Zevalin (Ibritumomab          Non-Hodgkin lymphoma          2002
  tiuxetan)                                                   

  Bexxar (Tositumomab)          Non-Hodgkin lymphoma          2003

  Erbitux (Cetuximab)           Colorectal cancer             2004

  Avastin (Bevacizumab)         Colorectal cancer             2004
  ----------------------------- ----------------------------- --------------

Another important therapeutic use of antibodies is the post-exposure
rabies treatment to prevent disease outbreak in a bitten human
(considered category III exposure by WHO,
[http://www.who.int](http://www.who.int/)). The administration of
antibody to an unimmunized individual is called passive immunization.
One of the first monoclonal antibodies to be marketed for passive
immunization was palivizumab, which is used to prevent serious lower
respiratory tract infections caused by respiratory syncytial virus (RSV)
in infants. Passive immunization of *Campylobacter jejuni*, infected
chickens with oral monoclonal antibodies, was found to be successful as
both prophylaxis and therapy (Tsubokura 1997). With rising medical costs
and increasing use of monoclonal antibodies in the medical and
industrial field, researchers have found ways to produce monoclonal
antibodies at a reduced cost to the consumer by creating plants
containing the monoclonal antibody gene construct.

**Ubiquitous Presence of Antibodies in Nature**

Humans consume 50-100g of protein per day as part of their normal
dietary intake (USDA and HHS 2005). Foods that contain naturally present
polyclonal antibodies include eggs, meat, milk, and milk products. Egg
yolks contain approximately 100-150 mg of total IgY antibody per yolk,
while the egg whites contain trace amounts of IgM and IgA (Polson 1990).
Polyclonal antibodies are a normal component of animal blood (Tizard
2000); and therefore are present in all meat. Cow's milk (not colostrum,
whose antibody content is considerably higher) contains 50-100 mg
IgA/dl, 5-10 mg IgM/ dl, and 20-50 mg IgG/dl (Tizard 2000).

**Antibody Degradation in the Environment**

The growth of all organisms depends on the availability of nitrogen,
which is required in large amounts as an essential component of
proteins, nucleic acids, and other cellular constituents. Nitrogen is
often the limiting factor for growth and biomass production in all
environments where there is suitable climate and availability of water
to support life. Along with nitrogen fixation (the conversion of
atmospheric nitrogen to ammonium or nitrate ions), microbes degrade
organic material and debris in the soil, releasing nitrogen for reuse by
other organisms (<http://soil.gsfc.nasa.gov/NFTG/nitrocyc.htm>).
Microbes are known to degrade many different types of complex organic
and inorganic molecules such as TNT, Dioxins, and polychlorobiphenyls
(PCBs) (Tiedje 1993, Wittich 1998, Lewis 2004). Antibodies are
relatively simple molecules (see Figure 1) consisting of a chain of
amino acids that fold into a three-dimensional shape due to amino acid
interactions and disulfide bonds. Any antibody in plant debris that was
produced via genetic engineering would have the same fate as antibodies
in any decaying animal tissue or by-product and would be quickly
degraded and incorporated into the nitrogen cycle.

**Antibody Degradation in the Digestive Tract**

Without degradation into smaller peptides, proteins (such as antibodies)
are too large to pass intact through the intestinal wall. They need to
be broken down into amino acids or small peptides before they can be
absorbed. The breakdown of protein begins in the stomach where
hydrochloric acid (HCl) denatures the protein and facilitates the action
of pepsin, the major gastric enzyme that splits the peptide bonds. Other
proteolytic enzymes (enzymes that break down protein bonds) involved in
the gastric process are trypsin, chymotrypsin, carboxypeptidase, and
elastase. Most monoclonal antibody immunizations and therapies are given
intravenously and not orally due to the instability of the antibody
during digestion (Zeitlin 1999).

Orally administered antibodies break into F(ab)2, Fab, and Fc fragments
in the digestive system (Fig. 1B). It has been found that 19 percent of
these fragments retain their neutralizing activity in the ileum of
healthy adults (Roos 1995). Another study (Bogstedt 1997) analyzed the
amount of neutralizing antibody activity in the fecal material of
healthy adults orally administered antibodies and only found minute
amounts present (\<0.01 percent of the ingested antibodies), suggesting
the majority of the peptides are absorbed in the small intestine. When
the time of passage through the gastrointestinal tract is short (as with
patients who have diarrhea) antibodies can retain more activity in the
lower gastrointestinal tract and be more effective in a therapeutic
setting (Hammarström 1994).

![](media/image1.wmf){width="4.127083333333333in"
height="2.545138888888889in"}

**Figure 1. Basic Antibody Structure.** **A**. Typical Y structure with
two light chains (in black) and two heavy chains (in red) bound by
disulfide bonds (dotted lines); **B.** Location of the Fab and Fc
fragments on the antibody molecule. The two Fab antibody regions are
typically indicated as (Fab)~2~.

**Secretory Antibody Structure**

![](media/image2.wmf){width="2.1590277777777778in"
height="2.129861111111111in"}

**Figure 2. Secretory Antibody Structure.** Complex antibody where two Y
shaped antibodies are joined by a J Chain to a secretory component.

**References:**

**Bogstedt A, Hammarström L, and Robertson A.** (1997) Survival of
immunoglobulins from different species through the gastrointestinal
tract in healthy adult volunteers: implications for human therapy.
Antimicrobial Agents and Chemotherapy 41, 2320.

**Giddings G** (2001) Transgenic plants as protein factories. Current
Opinion in Biotechnology 12, 450-454.

**Hammarström L, Gardulf A, Hammarström V, Janson A, Lindberg K and
Smith CI** (1994) Systemic and topical immunoglobulin treatment in
immunocompromised patients. Immunological Review 139: 43-70.

**Institute for Laboratory Animal Research and Council, N.R.** (1999)
Large-scale production of monoclonal antibodies. In: Monoclonal Antibody
Production. The National Academy Press, Washington, D.C., p. 25-32.

**Janeway CA, Travers P, Walport M, Shlomchik M** (2001) Immunobiology:
The Immune System in Health and Disease. Garland Publishing, New York,
N.Y.

**Köhler G, Milstein C** (1975) Continuous cultures of fused cells
secreting antibody of predefined specificity. Nature 256: 495-497.

**Lewis TA, Newcombe DA, Crawford RL** (2004) Bioremediation of soils
contaminated with explosives. Journal of Environmental Management
70:291.

**Polson A** (1990) Isolation of IgY from the yolk of eggs by a
chloroform polyethylene glycol procedure. Immunological Investigations
19:253-258.

**Roos N, Mahe S, Benamouzig R, Sick H, Rautureau J and Tome D** (1995)
15N-labeled immunoglobulins from bovine colostrum are partially
resistant to digestion in human intestine. Journal of Nutrition
125:1238-1244.

**Tiedje JM, Quensen JF, Chee-Sanford J, Schimel JP, Boyd SA** (1993)
Microbial reductive dechlorination of PCBs. Biodegradation 4:231-240.

**Tizard IR, Schubot RM** (2000) Veterinary Immunology: An Introduction.
W.B. Saunders Company, Philadelphia, P.A.

**Tsubokura, K., Berndtson, E., Bogstedt, A., Kaijser, B., Kim, M.,
Ozeki, M. and Hammarström, L.** (1997) Oral administration of antibodies
as prophylaxis and therapy in Campylobacter jejuni-infected chickens.
Clinical and Experimental Immunology 108, 451-455.

**USDA and HHS** (2005) Dietary Guidelines for Americans, 2005, 6th
Edition. US Government Printing Office, Washington, D.C.

**Wittich, R.M.** (1998) Degradation of dioxin-like compounds by
microorganisms. Applied Microbiology and Biotechnology 49, 489.

**Zeitlin, L., Cone, R.A. and Whaley, K.J.** (1999) Using monoclonal
antibodies to prevent mucosal transmission of epidemic infectious
diseases. Emerging Infectious Diseases 5, 54-64.

#  

# APPENDIX B: STANDARD PERMIT CONDITIONS FROM 7 CFR 340.4

\(f\) *Permit conditions*. A person who is issued a permit and his/her
employees or agents shall comply with the following conditions, and any
supplemental permit conditions which shall be listed on the permit, as
deemed by the Administrator to be necessary to prevent the dissemination
and establishment of plant pests:

1.  The regulated article shall be maintained and disposed of (when
    necessary) in a manner so as to prevent the dissemination and
    establishment of plant pests.

2.  All packing material, shipping containers, and any other material
    accompanying the regulated article shall be treated or disposed of
    in such a manner so as to prevent the dissemination and
    establishment of plant pests.

3.  The regulated article shall be kept separate from other organisms,
    except as specifically allowed in the permit;

4.  The regulated article shall be maintained only in areas and premises
    specified in the permit;

5.  An inspector shall be allowed access, during regular business hours,
    to the place where the regulated article is located and to any
    records relating to the introduction of a regulated article;

6.  The regulated article shall, when possible, be kept identified with
    a label showing the name of the regulated article;

7.  The regulated article shall be subject to the application of
    measures determined by the Administrator to be necessary to prevent
    the accidental or unauthorized release of the regulated article;

8.  The regulated article shall be subject to the application of
    remedial measures (including disposal) determined by the
    Administrator to be necessary to prevent the spread of plant pests;

9.  A person who has been issued a permit shall submit to APHIS a field
    test report within 6 months after the termination of the field test.
    A field test report shall include the APHIS reference number,
    methods of observation, resulting data, and analysis regarding all
    deleterious effects on plants, non-target organisms, or the
    environment;

10. APHIS shall be notified within the time periods and manner specified
    below, in the event of the following occurrences:

-   Orally notified immediately upon discovery and notify in writing
    within 24 hours in the event of any accidental or unauthorized
    release of the regulated article;

-   In writing as soon as possible but not later than within 5 working
    days if the regulated article or associated host organism is found
    to have characteristics substantially different from those listed in
    the application for a permit or suffers any unusual occurrence
    (excessive mortality or morbidity, or unanticipated effect on
    non-target organisms);

11. A permittee or his/her agent and any person who seeks to import a
    regulated article into the United States shall:

    -   Import or offer the regulated article for entry only at a port
        of entry which is designated by an asterisk in 7 CFR
        319.37-14(b);

    -   Notify APHIS promptly upon arrival of any regulated article at a
        port of entry, of its arrival by such means as a manifest,
        customs entry document, commercial invoice, waybill, a broker\'s
        document, or a notice form provided for such purpose; and

    -   Mark and identify the regulated article in accordance with 7 CFR
        340.5.

# APPENDIX C: SUPPLEMENTAL PERMIT CONDITIONS

I.  **Compliance with Regulations**

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1.  Any regulated article introduced not in compliance with the
    requirements of Title 7 of the Code of Federal Regulations, Part
    340, or any standard or supplemental permit conditions, shall be
    subject to the immediate application of such remedial measures or
    safeguards as an inspector determines necessary, to prevent the
    introduction of such plant pests. The responsible party may be
    subject to fines or penalties as authorized by the Plant Protection
    Act (7 U.S.C. 7701-7772).

2.  This Permit (APHIS form 2000) does not eliminate the permittee's
    legal responsibility to obtain all necessary Federal and State
    approvals, including: (A) for the use of any non-genetically
    engineered plant pest or pathogens as challenge inoculum; (B)
    plants, plant parts or seeds which are under existing Federal or
    State quarantine or restricted use; (C) experimental use of
    unregistered chemicals; and (D) food, feed, pharmacological,
    biologic, or industrial use of regulated articles or their products
    and co-mingled plant material. In the latter case, depending on the
    use, reviews by APHIS, the U.S. Food and Drug Administration, or the
    U.S. Environmental Protection Agency may be necessary.

3.  The procedures, processes, and safeguards used to prevent escape,
    dissemination, and persistence of the regulated article as described
    in the permit application, in APHIS-approved Standard Operating
    Procedures (SOPs) and, in the supplemental permit conditions must be
    strictly followed. The permittee must maintain records sufficient to
    verify compliance with these procedures, including information
    regarding who performed the activity. Persons performing such
    activities shall have received training as described in a training
    program submitted to and approved by APHIS. These records are
    subject to examination by APHIS. APHIS, BRS must be notified of any
    proposed changes to the protocol referenced in the permit
    application.

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II. **Reporting Unauthorized Releases and Unintended Effects**

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1.  According to the regulation in 7 CFR 340.4(f)(10)(i), APHIS shall be
    notified orally immediately upon discovery and notified in writing
    within 24 hours in the event of any accidental or unauthorized
    release of the regulated article.

-   For immediate oral notification, contact APHIS/BRS Compliance Staff
    at (301) 734-5690 and ask to speak to a Compliance and Inspection
    staff member.

-   In the event of an emergency and you are unable to reach the BRS
    Compliance Staff at the above number, you may call:

> The APHIS/BRS Regional Biotechnology Coordinator assigned to the
> state, where the field test occurs
>
> **[For Western Region]{.underline}**, contact Ralph Stoaks by phone at
> (970) 494-7573 or e-mail <Ralph.D.Stoaks@aphis.usda.gov>
>
> **[For Eastern Region]{.underline}**, contact Ashima SenGupta by phone
> at (919) 855-7622 or e-mail
> [Ashima.SenGupta]{.underline}[\@aphis.usda.gov](mailto:Roger.L.Holman@aphis.usda.gov)
>
> Or
>
> The APHIS/PPQ Regional Biotechnology Coordinator assigned to the state
> where the field test occurs
>
> **[For Western Region]{.underline}**, contact Stacy Scott by phone at
> 970-494-7577 or e-mail <Stacy.E.Scott@aphis.usda.gov>
>
> **[For Eastern Region]{.underline}**, contact Susan Dublinski by phone
> at (919) 855-7324 or e-mail
> [Susan.G.Dublinski]{.underline}[\@aphis.usda.gov](mailto:?????????@aphis.usda.gov)
>
> Or
>
> The APHIS State Plant Health Director for the state where the field
> test occurs. The list of APHIS State Plant Health Director is
> available at <http://ceris.purdue.edu/napis/names/sphdXstate.html>

  ---- ---------------- ---------- ---------- ------------------------------------
  KY   Mike Madryga,    \(502\)    \(502\)    <michael.b.madryga@aphis.usda.gov>
       Prospect         228-8224   228-6306   

  ---- ---------------- ---------- ---------- ------------------------------------

2.  According to 7 CFR 340.4(f)(10)(ii), APHIS shall be notified in
    writing as soon as possible but within 5 working days if the
    regulated article or associated host organism is found to have
    characteristics substantially different from those listed in the
    permit application or suffers any unusual occurrence (excessive
    mortality or morbidity, or unanticipated effect on non-target
    organisms).

3.  Written notification should be sent by one of the following means:

> [By e-mail]{.underline}:
>
> <BRSCompliance@aphis.usda.gov>
>
> [By mail]{.underline}:
>
> Biotechnology Regulatory Services (BRS)
>
> Compliance and Inspection Branch
>
> USDA/APHIS
>
> 4700 River Rd. Unit 147
>
> Riverdale, MD 20737

III. **Perimeter Fallow Zone**

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1.  To ensure that transgenic plants are not inadvertently commingled
    with plants to be used for food or feed, a perimeter fallow zone of
    at least 50 ft. must be maintained around the transgenic test site
    in which no crops are grown to be harvested or used for food or
    feed.

2.  The permitted border rows of non-transgenic plants that are the same
    as, or sexually compatible with, the regulated article are
    considered part of the field test. The perimeter fallow zone shall
    start outside the border rows.

3.  The perimeter fallow zone shall be managed in a way that allows
    detection and destruction of volunteer plants that are the same as,
    or sexually compatible with, the transgenic plants.

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IV. **Dedicated Planting and Harvesting**

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1.  To ensure that the regulated article is not inadvertently removed
    from the site, harvesting equipment must be dedicated for use in the
    permitted test site(s) or used on non-regulated research tobacco
    that are not used for food or feed (BRS Variance 05-001) from the
    time of planting through the end of harvesting.

2.  After harvest, you will not be required to obtain APHIS
    authorization to use this equipment on APHIS -permitted sites (same
    sites or different sites) planted with same transgenic crop, with
    the target protein(s) authorized under this permit, in subsequent
    growing seasons under an extension of this permit or a different
    permit.

3.  Authorization is required from APHIS before harvesting equipment
    used during this field test can be used on sites planted to crops
    not included under this permit. The permittee must notify APHIS/BRS
    and the State Regulatory Official at least 21 calendar days in
    advance of cleaning this equipment for this purpose so that APHIS
    may schedule an inspection to ensure that the equipment has been
    cleaned appropriately.

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V.  **Cleaning of Equipment**

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1.  To minimize the risk of seed movement and commingling, equipment
    used for planting and harvesting, as well as other field equipment
    (e.g. tractors and tillage attachments, such as disks, plows,
    harrows, and subsoilers) used at any time from the time of planting
    through the post-harvest monitoring period must be cleaned in
    accordance with procedures submitted to and approved by APHIS before
    they are moved off of the environmental release site.

2.  Equipment used to transport seeds or harvested material must be
    cleaned prior to loading and after transportation to the authorized
    site in accordance with procedures submitted to and approved by
    APHIS.

3.  Seed cleaning and drying must be performed in accordance with the
    procedures submitted to and approved by APHIS to confine the plant
    material and minimize the risk of seed loss, spillage, or
    commingling.

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VI. **Use of Dedicated Storage Facilities**

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1.  Dedicated facilities (locked or secured buildings, bins, or areas,
    posted as restricted to authorized personnel only) must be used for
    storage of equipment and regulated articles for the duration of the
    field test.

2.  Before returning these facilities to general use, they must be
    cleaned in accordance with procedures submitted to and approved by
    APHIS. **The permittee must notify** APHIS/BRS and the State
    Regulatory Official at least 21 calendar days in advance to allow
    for APHIS to schedule an inspection to ensure that the facilities
    have been cleaned appropriately. APHIS authorization must be
    received before facilities are returned to general use.

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VII. **Post Harvest Monitoring**

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1.  The field test site including the perimeter fallow zone must be
    monitored for the presence of volunteer ***Nicotiana* hybrid**
    plants for **one year** after termination of the field test. Viable
    plant material should not remain at the test site following
    termination. Volunteers, if found, will be uprooted by hand and
    destroyed by dismemberment and incorporation into the soil.

2.  Fields must be checked for volunteers once every 2 weeks, over a
    period of 4 weeks, immediately post harvest. Then, before the Fall
    and Winter months arrive, (the first frost) the fields will be
    checked once every 6 weeks. During the Fall and Winter months the
    fields will be checked once every 8 weeks. During Spring and the
    following Summer the fields will be checked once every three weeks.

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VIII. **Post Harvest Land Use Restrictions**

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1.  Production of food and feed crops at the field test site and the
    perimeter fallow zone is restricted during the growing season that
    follows harvest or termination of the field test.

2.  Permission must be obtained from APHIS/BRS prior to planting any
    food or feed crop at the field test site and perimeter fallow zone
    during the post-harvest monitoring period. Requests for such
    permission are not encouraged and will not be granted in cases where
    there is a reasonable potential for plant material derived from, or
    originating from, the regulated articles to become mixed with the
    proposed food or feed crop during harvesting.

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IX. **Inspections**

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1.  APHIS Biotechnology Regulatory Services (BRS) and/or an APHIS/PPQ
    Regional Biotechnologist, APHIS/BRS Regional Biotechnology
    Coordinator or APHIS State Plant Health Director may conduct
    inspections of the test site, facilities, and/or records at any
    time.

2.  APHIS may invite the FDA or State Regulatory Officials to
    participate in these inspections.

3.  Inspections will likely correspond to the beginning of the field
    test, mid-season or during flowering, at and/or following harvest,
    and during the post-harvest monitoring period.

4.  Inspections will include examination of records that verify
    compliance with regulations and SOPs.

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X.  **Reports and Notices**

> **Send notices and all reports (CBI and CBI-deleted or non-CBI copies)
> to BRS by e-mail, mail, or fax.**
>
> **BRS E-mail:**
>
> <BRSCompliance@aphis.usda.gov>
>
> **BRS Mail:**
>
> Biotechnology Regulatory Services (BRS)
>
> Compliance and Inspection Branch
>
> USDA/APHIS
>
> 4700 River Rd. Unit 147
>
> Riverdale, MD 20737
>
> **BRS Fax:**
>
> Compliance and Inspection Branch
>
> \(301\) 734-8669
>
> **In addition, fax the CBI deleted or non CBI version of the
> pre-planting and pre-harvest (termination) notices to the State
> Regulatory Official:**

  ------------------------------- ---------------------------------------------------------
  **State Plant Regulatory        John Obrycki, State Entomologist
  Official**                      

  **Mailing Address**             Department of Entomology\
                                  S-225 Ag. Science Center North\
                                  University of Kentucky\
                                  Lexington, KY 40546-0091

  **Phone**                       859-257-5838

  **Fax**                         859-257-3807

  **Email**                       <john.obrycki@uky.edu>

  **Web Site**                    [http://www.KyStateEnt.org](http://www.KyStateEnt.org/)
  ------------------------------- ---------------------------------------------------------

> **Contact information for State Officials**
>
> [**http://www.nationalplantboard.org/member/index.html**](http://www.nationalplantboard.org/member/index.html)

1.  **Pre-Planting Notice**

> At least 7 calendar days before planting, submit a Pre-Planting notice
> that includes the following information for each field test site:

i.  Provide APHIS with the contact information for each field test site.

ii. Indicate if planting and harvesting equipment will be moved between
    authorized field test sites.

iii. A map that clearly identifies the site location to facilitate any
     inspections by USDA personnel.

iv. The planned number of acres for each gene construct.

v.  The planned planting date

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2.  **Planting Report**

> Within 28 calendar days after planting, submit a planting report that
> includes the following information for each field test site:

i.  A map of the site, with sufficient information to locate it, that
    includes: the state, county, address, GPS coordinates for each
    corner of the plot (inclusive of the border rows of any sexually
    compatible plants);

ii. The location and the approximate number and/or acres of transgenic
    plants which were actually planted at the test site for each of the
    target proteins;

iii. The total acreage of the test plot (exclude border rows, if any);

iv. The distance from the genetically engineered plants to the nearest
    plants of the same crop that will be used for food, feed, or seed
    production. A survey should be done within the distance specified in
    the permit.

v.  A list of the specific confinement option(s) selected at each site
    if your permit allows different confinement options (e.g. bagging
    flowers, border rows, or isolation distance.).

vi. The actual planting date.

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3.  **Pre-Harvest/ Termination Notice**

> At least 21 calendar days prior to the anticipated harvest or
> termination, submit a Notice indicating the planned date of harvest
> **or** termination and the contact information for each field test
> site. For multiple harvests, submit the notice prior to the initial
> harvest.

4.  **Field Test Report**

> Within 6 months after the end of the field test (final harvest or crop
> destruct), the permittee is required to submit a field test report.
> Field test reports shall include:

i.  APHIS reference number

ii. Methods of observation.

iii. Resulting data.

iv. Analysis of all deleterious effects on plants, non-target organisms,
    or the environment.

v.  A list of the lines planted at each site

vi. Disposition table

> The disposition table should contain the following information: site
> name (or GPS), crop, gene, harvest date, and disposition of harvested
> material.
>
> The disposition table is a formal record of how the regulated material
> was removed from the environment. An accounting of the harvested
> material should be provided with regards to what material is
> harvested, how much material is harvested per site,
>
> what is done to devitalize residual and harvested material at the
> site, where the harvested material is transported, stored and further
> processed up to the time it is taken to a contained facility.

**5**. **Monitoring Report**

> Within 3 months after the end of the monitoring period, submit a
> volunteer monitoring report. The report must include:

1.  

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i.  Dates when the field site and perimeter fallow zone were inspected
    for volunteers.

ii. Number of volunteers observed.

iii. Any actions taken to remove or destroy volunteers.

# APPENDIX D: APPLICANT SUPPLIED TES WORKSHEET

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height="8.519444444444444in"}

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height="8.540972222222223in"}

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height="7.978472222222222in"}

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height="3.5597222222222222in"}