**USDA/APHIS**

**Draft Environmental Assessment**

In response to Permit applications 06-278-01r, 06-278-02r and 06-285-02r
received from Ventria Bioscience

To conduct field plantings of rice (*Oryza sativa*) genetically
engineered to express human lysozyme, lactoferrin and serum albumin

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

Biotechnology Regulatory Services

**[Table of Contents]{.underline}**

Page

**I. Summary** 3

**II. Introduction** 4

**A. Intended uses for the developed rice lines** 4

**B. APHIS Regulatory Authority** 4

**III. Purpose and Need** 5

A.  **Proposed Action** 5

B.  **Purpose of the Environmental Assessment** 5

C.  **Need for This Action** 6

**IV. Alternatives to the Proposed Action** 6

**A. No Action** 6

**B. Issue permits as received** 6

**C. Issue permits with supplemental conditions** 6

**V. Description of Field Plantings/ Affected Environment** 7

**VI. Potential Environmental Impacts** 10

**VII. Preparers and Reviewers** 11

**VIII. Agency Contact** 11

**Appendix 1: Description of Rice Biology** 13

**Appendix 2: Description of the Regulated Rice Plants** 13

**Appendix 3: Threatened and Endangered Species for Kansas** 15

# Appendix 4: Proposed Supplemental Permit Conditions 16

**I. Summary**

The U.S. Department of Agriculture's (USDA) Animal and Plant Health
Inspection Service, Biotechnology Regulatory Services (APHIS/BRS) has
prepared an environmental assessment (EA) in response to permit
applications (APHIS numbers 06-278-01r, 06-278-02r and 06-285-02r)
received from Ventria Bioscience, Sacramento, California, to grow
genetically engineered rice (*Oryza sativa* L.) in Geary County, Kansas.
These transgenic plants have been modified to express the human (*Homo
sapiens*) proteins lysozyme, lactoferrin or serum albumin. The plants
expressing lysozyme and lactoferrin have also been engineered with the
selectable marker gene *hpt*[^1] which encodes for the enzyme hygromycin
B phosphotransferase (Hpt). Hpt inactivates the antibiotic hygromycin.
Plants containing serum albumin also contain either the *hpt* marker
gene and/or the phosphinothricin acetyltransferase (*pat*) gene which
allows laboratory selection of transformed tissues using bialaphos
herbicide. None of the marker genes are expressed in mature plants due
to the nature of the gene promoters.

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

These plantings in Kansas should be harvested in the fall of 2007.
Similar plantings are planned in future years.

The bases of confinement for these field tests are:

-   The field test sites are located on private lands in Geary County,
    KS. There is no commercial rice grown in the State of Kansas.

-   In nature, chromosomal genetic material of rice can only be
    transferred to other sexually compatible plants by
    cross-pollination. Rice is primarily self pollinating and outcrosses
    at a very low frequency. Furthermore, the only sexually compatible
    plants in the U.S. are cultivated rice and red rice. Wild rice is a
    different genus and is not sexually compatible with cultivated rice.
    As there is no commercial rice grown in Kansas and red rice only
    grows where there is cultivated rice, the possibility of outcrossing
    is effectively zero.

-   To prevent intermingling of seeds with other crop plants, Ventria
    will use dedicated equipment, storage and processing facilities. The
    majority of the harvested seeds will be milled to flour and will not
    be shipped to any outside milling facilities.

-   None of the introduced genes provide the engineered rice plants with
    any selective advantage over non-engineered rice in the ability to
    be disseminated or to become established in the environment.

The proposed field plantings are a controlled release of the regulated
article into the environment. The protocols and field plot design as
well as the procedures for termination of the field tests have been
deemed sufficient to ensure that none of the engineered rice plants
persist in the environment beyond the termination of the plantings. The
proposed field tests do not present a significant impact on populations
of non-target animal species, including any threatened or endangered
species or critical habitat in Kansas. APHIS review and analyses of the
data packages presented by the applicant indicate that the proposed
field plantings do not present a risk of introduction and dissemination
of a plant pest and should not have a significant impact, either
individually or cumulatively, on the quality of the human environment.

**II. Introduction**

**A. Intended uses for the developed rice lines**

Ventria Bioscience (formerly Applied Phytologics) has been developing
genetically engineered plants producing pharmaceutical and novel
proteins since 1996. They intend to continue this work in 2007, growing
several different rice lines containing three distinct novel proteins
(described later in this Environmental Assessment), which will be
extracted from rice seed.

Ventria intends to extract recombinant human lysozyme and lactoferrin
from rice flour to be used as supplements in yogurts, meal replacement
and performance beverages, bars (for example granola bars) and in
nutritional supplement drinks. They also intend to use lysozyme and
lactoferrin in the preparation of medical foods such as oral rehydration
solutions. Ventria will also extract recombinant human serum albumin
(HSA) protein to be used primarily for cell culture use.

**B. Regulatory Authority**

The authorities for regulation of genetically engineered rice are the
Plant Protection Act of 2000, 7 U.S.C. 7701-7772, and USDA, APHIS
regulations under 7 CFR § 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. In this submission, rice lines have been genetically
engineered using techniques of microprojectile bombardment or disarmed
*Agrobacterium*-mediated transformation using genes from at least one
plant pest. The introduced DNA contains human, bacterial and synthetic
gene sequences that have been engineered for optimal protein expression
in rice. Regulatory and selectable antibiotic or herbicide marker gene
sequences from *E.coli*, *Agrobacterium tumefaciens*, rice, and/or
*Streptomyces hygroscopicus* are also incorporated into these rice
lines.

Generally, issuance of a permit for field trials of regulated articles
is categorically excluded from requirements for an environmental
assessment (EA) under APHIS NEPA implementing procedures (7 C.F.R. §
372.5(c)(3)(i)). However, when APHIS determines that a confined field
release of genetically engineered organisms has the potential to
significantly affect the quality of the human environment, as those
terms are defined in 40 C.F.R. §§ 1508.27 and 1509.14, an environmental
assessment or environmental impact statement will be prepared, pursuant
to 7 C.F.R. § 372.5(d). This EA was prepared because the applicant
intends to have plantings of these engineered plants in Geary County,
Kansas for the next several years. The potential for cumulative impacts
of these plantings in the same area raises new issues that this EA
addresses. Future plantings are anticipated to increase in size and will
be required to meet all the performance and mitigation measures
described in the permit applications and all permit conditions.

**III. Purpose and Need**

**A. Proposed Action**

The proposed action is for APHIS, Biotechnology Regulatory Services
(BRS), to issue permits for confined field plantings of several lines of
rice that have been genetically engineered to express proteins for human
lysozyme, lactoferrin or serum albumin. Lysozyme and lactoferrin lines
contain a variety of regulatory gene sequences (i.e., promoters,
terminators and enhancers) and selectable marker genes for hygromycin
phosphotransferase (*hpt*). Serum albumin lines also contain similar
regulatory gene sequences and may contain selectable marker genes for
*hpt* and/or phosphinothricin acetyltransferase (*pat*).

The plantings will occur in at least 2 locations and individual rice
lines will be physically isolated from each other to maintain product
purity. This environmental assessment (EA) will evaluate the regulated
articles in three permit applications: 06-278-01r, 06-278-02r and
06-285-02r.

**B. Purpose**

The purpose of this EA is to assess potential environmental effects
resulting from confined field plantings of the engineered rice lines in
Geary County, Kansas. Permit applications were submitted by Ventria
Bioscience to USDA/APHIS/BRS on October 5 and October 12, 2006.

Identical or nearly identical rice lines have been the subject of
previous Environmental Assessments by APHIS and portions of those
assessments, when appropriate, will be incorporated in this EA or
appendices by reference.

**C. Need for This Action**

Under APHIS regulations, 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. 7 CFR 340.4(e)

**IV. Alternatives to the Proposed Action**

APHIS has considered the following three alternatives in response to the
applicant\'s request for a permit:

**Alternative 1:**

**No action/deny the permit applications**. Under this alternative,
field releases of the engineered rice plants would not be authorized.

**Alternative 2:**

**Issue the permits for growing under the conditions proposed by the
applicant.** Under this alternative, field release of the engineered
rice plants would be authorized at the specified locations with no
additional conditions outside of those the applicant provided in the
request. Standard permit conditions under 7 CFR § 340.4 would be
required. Standard management practices, including use of some
pesticidal and herbicidal sprays, will be included as part of the
planting design.

**Alternative 3:**

**Issue the permits with additional conditions for carrying out the
field plantings**. Under this alternative, field release of the
engineered plants would be authorized. Supplemental permit conditions,
based on APHIS analysis, the State of Kansas, and public comment on this
environmental assessment, would be required. If warranted, based on
environmental risk of escape of the engineered organism, APHIS will
require mitigating measures to prevent spread of the organism outside
the field production area. Standard management practices, including use
of some pesticidal and herbicidal sprays, will be included as part of
the planting design.

APHIS field test permits typically include supplemental permit
conditions that may reflect input from the appropriate State regulatory
officials. The supplemental permit conditions also specify requirements
for additional post-planting and post-harvest volunteer monitoring
reports. These post-planting and monitoring reports assist APHIS in
evaluating specific field tests under permit and also provide guidance
for evaluating future proposed field tests. APHIS' proposed Supplemental
Permit Conditions are described in Appendix 4. Several of these
conditions are described:

-   The plantings shall be at least ¼ mile from any commercial rice
    plantings.

-   The applicant shall provide APHIS and State regulatory officials
    information on the location of the nearest commercial rice plants
    that are not part of the field test.

-   The applicant shall provide APHIS and State regulatory officials a
    detailed map of the proposed plantings. One month after planting,
    the applicant will submit a detailed map of the planted sites.

-   The applicant shall use screens on irrigation outlets to prevent
    movement of seeds/seedlings out of the field with water used to
    flood the field. They shall also employ flooding methods which
    create a closed system so that ungerminated seeds cannot leave the
    field site.

-   A zone of 50 feet shall be maintained surrounding the field test
    site. A non-food or non-feed cover crop may be planted in this zone
    to prevent erosion or this area may remain fallow.

-   In the subsequent growing season, the production sites and the 50
    foot fallow zone may not be planted with rice unless similar
    transgenic rice is repeated. If the same crop does not follow in
    subsequent years, the site shall be monitored for volunteer rice
    plants at least 3 times during the following growing season. Any
    volunteer rice plants shall be destroyed before flowering.

-   Ventria shall use equipment dedicated to these plantings as outlined
    in their SOPs. This equipment shall not be used for any other
    purposes during the course of the field test. After the field test
    is completed, all equipment shall be thoroughly cleaned and
    inspected to ensure that all genetically engineered seed and other
    plant material has been removed and destroyed.

-   Ventria shall scout for red rice during the entire growing season
    both within the growing plots and for ¼ mile from the production
    fields. Any red rice found shall be destroyed and shall not be
    allowed to flower. Ventria shall also inform APHIS if any red rice
    is found within the ¼ mile zone or in their production plots.

Ventria is also required to maintain at least 1320 feet isolation from
other rice. The Association of Official Seed Certifying Agencies (AOSCA)
standards for foundation certified rice seed require at least 10 feet
from other rice. In practice, there is no known commercial rice grown in
the state of Kansas.

**V. Description of the Field Plantings/ Affected Environment**

## Purpose

The purpose of this proposed introduction is multi-fold:

-   For continued testing of gene stability over several generations of
    seed production,

-   For further molecular and protein testing,

-   To develop seed stock for product development and breeding, and

-   For scale-up.

The regulated introduction is proposed for planting between March and
May 2007 and would grow in the field until harvest (estimated
September-November 2007). Ventria has proposed to plant acreages
comparable in size and scope to those grown in North Carolina in 2006
([http://www.aphis.usda.gov/brs/ph_permits.html]{.underline}. See permit
numbers 05-293-01r, 05-332-01r, and 05-332-02r). One noted change is the
addition of rice lines developed to produce human serum albumin.

APHIS has reviewed and approved protocols that are proposed by Ventria
to prevent the escape and dissemination of these plants, submitted on
APHIS Form 2000. In addition, Standard Operating Procedures (SOPs)
submitted by Ventria identify more detailed instructions and provide
additional guidance.

**Field Plot Design, Breeding Procedures and Agricultural Practices**

**Plot Design and Location**

Ventria's plantings are proposed in Geary County, Kansas. There are no
known commercial rice fields in Geary County or neighboring counties.
The area has not had a history of rice farming; soybean, corn and winter
wheat have been grown in the area for many years. Ventria will monitor
for commercial rice production and scout for weedy red rice within ¼
mile of its plantings.

Rice is highly self-pollinating and is not generally pollinated by
insects. AOSCA-certified seed regulations for foundation rice seed
require a minimum isolation distance from other rice varieties of at
least ten feet when hand- or machine-planted. A 50 foot fallow zone and
a separation distance of ¼ mile from any other rice (one hundred thirty
two times the AOSCA standard) as proposed by the applicant should be
more than adequate to prevent unintended release of the transgenic rice
into adjacent fields. To prevent erosion, the fallow zone may be planted
with a low-growing crop that will not be used for food or feed. The rice
will be grown in flooded/water soaked fields.

**Agricultural Practices**

The rice will be allowed to self pollinate to produce seed. No breeding
operations are planned under this permit. Agricultural practices
consistent with growing healthy rice plants will be used. Weeds will be
controlled by herbicide applications. If necessary, pesticides such as
insecticides and/or fungicides will be used to control insect pests and
disease. Any pesticides used will be applied by appropriately trained
personnel. If necessary, EPA-registered pesticides will be used to
control insect pests. Fields will be monitored for noxious weeds and
other plant pests during the growing season. Three times during the
growing season the plants will be inspected for traits such as
weediness, resistance/susceptibility to insects or disease, or unusual
differences in plant growth or morphology. The plot will be inspected
weekly while personnel also conduct agricultural practices. Nearby
fields will likely have crops such as corn, soybean and winter wheat.

**Field Observation and Monitoring**

The applicant has thoroughly described field site monitoring and
management practices that should provide the necessary degree of
biological and physical confinement. Confinement practices and permit
conditions are described in section IV Alternative 3 listed above.
Additionally:

-   there are no known fields of commercial rice in Geary or surrounding
    counties in Kansas.

-   there are no known populations of red rice in Kansas.

**Alteration in susceptibility to disease or insects**

Since Ventria first began planting these rice lines, they have not noted
increased susceptibility to disease or insect pests. There has been no
intentional genetic change in these plants to affect their
susceptibility to disease or insect damage. Neither the selectable
marker genes, *hpt and pat*, nor the lysozyme, lactoferrin or *hsa*
genes are expected to alter the susceptibility of the transgenic rice
plants to disease or insect damage. Execution of the prescribed periodic
monitoring of the field plots will allow the detection of any unexpected
infestation by plant disease organisms or animal pests. Ventria is
required to report any such unanticipated effects to APHIS.

**Termination of the field test and final disposition of the test
plants**

Seed will be machine-harvested using a dedicated combine and dried and
cleaned in a designated/dedicated staging area in the same county using
a dedicated dryer and cleaner. The seeds will be stored in dedicated
storage bins until processed. During the process, the seed will be
dehusked using a dedicated dehusker and will be milled in a dedicated
staging area using a dedicated mill. Milled rice flour will be shipped
to designated locations for subsequent processing. Any devitalized waste
material from the milling operation will be returned to the field test
site and incorporated into the soil or otherwise properly disposed of by
an alternate, APHIS-approved method. All the operations up to milling
will be performed in an APHIS-inspected dedicated area using dedicated
equipment. Most material will be shipped only after milling. Some viable
seed may be stored in APHIS-inspected locations and/or shipped to other
locations for analysis or planting in subsequent seasons. All interstate
shipments will be done under APHIS permits.

After harvest, as soon as the weather allows, Ventria will burn and disk
the fields to encourage rapid decomposition of all plant and seed
materials that may remain. Off-season irrigation may also be used to
accelerate the germination of any remaining viable seed prior to winter.
Ventria plans to grow these rice varieties in the same locations in
subsequent years. However, if a change to a different crop is
anticipated, the field will be fallowed for one cropping season after
the harvest of the transgenic lines. During this 18 month period (the
period between harvest year 1 to planting year 3), the field will be
monitored at least three times in the following growing season and
managed to induce germination and/or decomposition of any viable seed.
Ventria employs a "pureland" procedure to control volunteers. This
includes flushing the field with water during the growing season to
induce germination of weed and rice seed, drying the field to kill
seedlings, and then disking to dry and kill seedlings. This process is
repeated three times.

**Security of the planting sites**

The planting sites are expected to provide adequate physical security.
The contract farmers own the fields to be planted with the transgenic
rice. The surrounding fields outside the food/feed crop fallow zone will
be planted to soybean, corn or winter wheat. The sites are not prone to
flooding. The closest body of water is the Smokey Hill River which is
located approximately 1 mile to the south of one proposed plot and
approximately 1 mile west of another proposed plot. In both locations,
the river is separated from fields by a steep embankment and these two
particular locations do not have a history of flooding. The Kansas River
is located about 3-4 miles from the proposed planting sites. If other
sites in Geary County are proposed prior to issuance of these permits
and subsequent planting, APHIS will assess them to determine whether
they may also be appropriate for these plantings.

**VI. Potential Environmental Impacts**

Other than location-specific references, descriptions of the potential
environmental impacts to Alternatives 1, 2 and 3 are incorporated here
by reference to Environmental Assessments (EA) completed for permits
05-117-01r (lactoferrin, Section VIII, pp 16-24), 05-117-02r (lysozyme,
Section VIII, pp 16-24) and 96-355-01r (human serum albumin, Section V,
pp 5-7). These documents can be found at the following links:
<http://www.aphis.usda.gov/brs/ph_permits.html> and
<http://www.isb.vt.edu/cfdocs/fieldtests3.cfm>. Issues addressed
included the potential for persistence in the environment, the potential
for gene transfer, potential impacts from use of the marker genes,
potential impact on native floral and faunal communities, potential
alteration in susceptibility to disease or insects, potential impacts on
existing agricultural practices, potential impact on adjacent row crops,
fate of transgenic DNA, potential impacts on human health, potential
cumulative environmental effects and special considerations regarding
other environmental statutes. A discussion of potential effects of the
plantings on Threatened and Endangered species (TES) and critical
habitat specific to Kansas follows.

## Effects of field plantings on Threatened and Endangered Species

The proposed field plantings are confined releases of the regulated
articles into the environment in Geary County, Kansas. The proposed
planting sites are within 10 miles of Ventria's storage and processing
facility in Junction City, KS. Some sites may be within ½ mile of
Dickinson County to the west. Lists of TES and proposed TES, as well as
an analysis of designated critical habitat and proposed designated
habitat, were obtained from the U.S. Fish and Wildlife Service (USFWS).
Upon review of the information obtained from USFWS, it was determined
that there are seventeen TES in the state and no critical habitat in the
proposed planting areas. These documents can be found at the following
links:

<http://ecos.fws.gov/tess_public/StateListing.do?state=KS&status=listed>
and

<http://crithab.fws.gov/>

The TES listed include the Indiana bat (*Myotis sodalis*), gray bat
(*Myotis* *grisescens*), American burying beetle (*Nicrophorus
americanus*), gray wolf (*Canis lupus*); 6 bird species: whooping crane
(*Grus americana*), Eskimo curlew (*Numenius borealis*), bald eagle
(*Haliaeetus leucocephalus*), Least tern (*Sterna antillarum)*,
black-capped Vireo (*Vireo atricapilla*), piping plover (*Charadrius
melodus*); 4 fish species: Neosho madtom (*Noturus placidus*), Pallid
sturgeon (*Scaphirhynchus albus*)*,* Arkansas River shiner (*Notropis
girardi*), Topeka shiner (*Notropis topeka*); and three endangered
plants: running buffalo clover (*Trifolium stolonifera*), Mead's
milkweed (*Asclepias meadii*) and the western prairie fringed orchid
(*Platanthera praeclara*) as existing or once existing in Kansas. Only 2
of these species have been recorded as identified in Geary County: the
Topeka shiner and the Least tern. Neither of these would likely be
exposed to or consume material from these plantings. The Topeka shiner
is a small minnow, occurring typically in quiet, clear, open pools of
river headwaters and creeks. It primarily consumes midge larvae and
aquatic invertebrates. The Least tern is considered a shore bird and
primarily consumes insects and fish. Several of the other species listed
are presumed absent from Kansas. Given the non-toxic nature of the rice
and proteins produced, other species, even if exposed, would not be
expected to be affected.

BRS has reviewed this data in accordance with a process mutually agreed
upon with the U.S. Fish and Wildlife Service (USFWS) to determine when a
consultation, as required under Section 7 of the Endangered Species Act,
is needed. APHIS has reached a determination that release under these
permits (06-278-01r, 06-278-02r and 06-285-02r) would have no effect on
designated critical habitat or listed threatened or endangered species
and consequently, a written concurrence or formal consultation with the
USFWS is not required for this EA.

**VII. Preparers and Reviewers**

John M. Cordts, M.S., M.B.A. (preparer)

Biotechnologist

USDA/APHIS/BRS

Riverdale, MD 20737

Michael Watson, Ph.D. (reviewer)

Branch Chief

USDA/APHIS/BRS

Riverdale, MD

Neil E. Hoffman, Ph.D. (reviewer)

Director

USDA/APHIS/BRS

Riverdale, MD

Richard Coker (reviewer)

Regulatory Analyst

USDA/ APHIS/BRS

Riverdale, MD

**VIII. Agency Contact**

Ms. 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 1: Description of Rice Biology**

A description of rice biology is incorporated here by reference to the
Environmental Assessment (EA) completed for permit 05-117-01r (Section
V, pp 7-9). This document is applicable to all three permit applications
that are the subject of this EA and can be found here:
<http://www.aphis.usda.gov/brs/ph_permits.html>. Topics covered in the
referenced Environmental Assessment discuss rice systematics, genetic
improvement, weediness, modes of gene escape, outcrossing, movement of
seed by animals, movement of seed by water and movement by human error.

**Appendix 2: Description of the Regulated Rice Plants**

Ventria has engineered these rice plants to produce human lysozyme,
lactoferrin or human serum albumin in the seeds. The engineered rice
(*Oryza sativa* ssp *japonica* var Taipei 309, M202 or M103) varieties
are medium grain, Japonica types. They are not grown widely in the U.S.
In each case, gene expression is targeted to the developing seed so the
different proteins are not produced in other parts of the plant.

Detailed descriptions of the lactoferrin and lysozyme producing rice
plants are incorporated here by reference to Environmental Assessments
(EA) completed for permits 05-117-01r (lactoferrin, Section VI, pp 9-13)
and 05-117-02r (lysozyme, Section VI, pp 9-13). These documents can be
found here: <http://www.aphis.usda.gov/brs/ph_permits.html>. Topics
covered in the referenced EAs discussed gene vectors, selectable
markers, the genes of interest, characterization of the engineered
plants, molecular characterization, protein characterization,
assessments of protein toxicity and allergenicity, thermal and gastric
stability and potential environmental exposure levels.

Ventria has made minor changes to its gene constructs and method of
transformation for its HSA-containing rice lines. The plants developed
for current use (permit application 06-278-02r) were generated using
*Agrobacterium*-mediated transformation and the use of two selectable
marker genes. The marker genes hygromycin phosphotransferase and
phosphinothricin acetyltransferase (*hpt* and *pat*) are included to
allow selection of transgenic tissues in the laboratory using the
antibiotic hygromycin and/or the herbicide bialaphos. Neither selectable
marker gene is expressed in mature rice tissues. In many years of
testing, these rice plants have not exhibited plant pest characteristics
when modified by the introduction of these genes and their associated
regulatory sequences. These sequences do not have any inherent plant
pest characteristics and are not known to enhance gene transfer from
plants to other organisms.

Brief descriptions of the proteins to be produced in the engineered rice
plants are included here.

Lysozyme is ubiquitous in the human body where it acts as a protective
barrier against environmental agents and, in doing so, helps prevent
infection. Lysozyme is a small enzyme that attacks the protective cell
walls of bacteria. It breaks the carbohydrate chains in bacterial cell
walls, destroys the structural integrity, and causes the bacteria to
burst under their own internal pressure. Lysozyme plays a role in
antibacterial disease defense, particularly against gram-positive
bacteria. Both antiviral (O\'Neil et al., 2001) and antifungal
(Samaranayake et al., 2001) activity has been reported. Lysozyme in
cattle plays a role in gastric digestion and in chicken egg whites
functions as an antibiotic. Lysozyme occurs in tears, nasal mucus, milk,
saliva, blood serum, many types of tissues and secretions of different
animals, including vertebrates and invertebrates, and in plant latex
(O\'Neil et al., 2001).

Lactoferrin is an iron-binding glycoprotein (\~80 kDa) consisting of
approximately 700 amino acids and is found in milk, tears, saliva and
other mammalian body secretions (Ekstrand, 1994). In human milk, it is a
major component of whey protein with 6-8 grams/liter in colostrum
(foremilk) and 2-4 grams/liter in "mature" milk (Harper, accessed 2006).
Bovine (cow) milk contains 5-20 times less by weight. Lactoferrin is
reported to have broad antimicrobial properties (against bacteria,
fungi, and viruses), an immune system regulatory function,
anti-inflammatory properties, antioxidant activity, toxin binding
properties and anti-cancer activity (Harper, accessed 2006). Lactoferrin
may exist in several forms: iron deficient (apo-form), iron sufficient
(holo-form), and an "activated" form (Naidu, 2002). The form it takes
depends primarily upon pH, the citrate/bicarbonate ion ratios, and the
iron content of the medium. Treatment of lactoferrin under specific
chemical conditions results in "activation" of lactoferrin. The iron
deficient (apo-) and activated forms, due primarily to their ability to
sequester iron, are most active in their antimicrobial properties
(Naidu, 2002).

Human serum albumin (HSA) is a soluble, monomeric protein which
comprises about one-half of the blood serum protein. The protein is
encoded by the *alb* gene and is produced in the liver. It functions
primarily as a carrier protein for steroids, fatty acids, and thyroid
hormones and plays a role in stabilizing extracellular fluid volume. It
is used in medical practice to replace blood volume in burn victims,
patients suffering acute traumatic shock, and those undergoing certain
types of surgery. It has no reported oral or dermal activities.

References

Ekstrand B, Dillon VM and Board RG (1994) Lactoperoxidase and
lactoferrin. Natural Antimicrobial Systems and Food Preservation.
Pp15-63.

Harper, accessed 12/2006.
<http://www-fst.ag.ohio-state.edu/People/HARPER/Functional->
foods/Milk%20Components/LACTOFERRIN.html.

Naidu, AS. 2002. Activated Lactoferrin - A New Approach to Meat Safety.
Food Technol. 56: (3) 40-46.

O\'Neil MJ, Smith A, Heckelman PE (2001) Lysozyme *In*: The Merck Index.
13th edition, Vol 13th edition. Merck & Co., Inc., Rahway, NJ. p. 1011.

Samaranayake YH, Samaranayake LP, Pow EH, Beena VT, Yeung KW (2001)
Antifungal effects of lysozyme and lactoferrin against genetically
similar sequential *Candida albicans* isolates from a human
immunodeficiency virus-infected southern Chinese cohort. J. Clin.
Microbiol. **39:** 3296-3302.

**Appendix 3: Threatened or Endangered Species for Kansas (17 species)**

(<http://ecos.fws.gov/tess_public/StateListingAndOccurrence.do?state=KS>)

[Animals]{.underline}

-   [Bat, gray (*Myotis
    grisescens*)](http://ecos.fws.gov/SpeciesProfile?spcode=A04J)

-   [Bat, Indiana (*Myotis
    sodalis*)](http://ecos.fws.gov/SpeciesProfile?spcode=A000)

-   [Beetle, American burying (*Nicrophorus
    americanus*)](http://ecos.fws.gov/SpeciesProfile?spcode=I028)

-   [Crane, whooping (*Grus
    americana*)](http://ecos.fws.gov/SpeciesProfile?spcode=B003)

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-   [Curlew, Eskimo (*Numenius borealis*)
    (shorebird)](http://ecos.fws.gov/SpeciesProfile?spcode=B01A)

-   [Eagle, bald (*Haliaeetus
    leucocephalus*)](http://ecos.fws.gov/SpeciesProfile?spcode=B008)

-   [Madtom, Neosho (*Noturus
    placidus*)(fish)](http://ecos.fws.gov/SpeciesProfile?spcode=E03S)

-   [Plover, piping (*Charadrius
    melodus*)(shorebird)](http://ecos.fws.gov/SpeciesProfile?spcode=B079)

-   [Shiner, Arkansas (*Notropis
    girardi*)(fish)](http://ecos.fws.gov/SpeciesProfile?spcode=E05X)

-   [Shiner, Topeka (*Notropis topeka*
    (=*tristis*))](http://ecos.fws.gov/SpeciesProfile?spcode=E07R)

-   [Sturgeon, pallid (Scaphirhynchus
    albus)](http://ecos.fws.gov/SpeciesProfile?spcode=E06X)

-   [Tern, least (*Sterna
    antillarum*)](http://ecos.fws.gov/SpeciesProfile?spcode=B07N)

-   [Vireo, black-capped (*Vireo
    atricapilla*)](http://ecos.fws.gov/SpeciesProfile?spcode=B07T)

-   [Wolf, gray (*Canis
    lupus*)](http://ecos.fws.gov/SpeciesProfile?spcode=A00D)

[Plants]{.underline}

-   [Clover, running buffalo (*Trifolium
    stoloniferum*)](http://ecos.fws.gov/SpeciesProfile?spcode=Q2RE)

-   [Milkweed, Mead\'s (*Asclepias
    meadii*)](http://ecos.fws.gov/SpeciesProfile?spcode=Q1T6)

-   [Orchid, western prairie fringed (*Platanthera
    praeclara*)](http://ecos.fws.gov/SpeciesProfile?spcode=Q2YD)

**Appendix 4: APHIS' Proposed Supplemental Permit Conditions**

I.  **Compliance with Regulations**

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1.  Any regulated article introduced not in compliance with the
    requirements of 7 Code of Federal Regulation 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 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 APHIS/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>
>
> 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>
>
> Or
>
> The APHIS State Plant Health Director for the state where the field
> tests occur.
>
> Mr. Russell McKinney
>
> Phone: 785-270-1381
>
> Fax: 785-235-1464
>
> Russell.A.McKinney@aphis.usda.gov

2.  According to the regulation in 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 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, planting and harvesting equipment must be dedicated
    for use in the permitted test site(s) from the time of planting
    through the end of harvest.

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 this planting and
    harvesting equipment can be used on sites planted to crops not
    included under this permit. Ventria 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 test 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. **Ventria 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 should be received before
    facilities are returned to general use.

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VII. **Post Harvest Monitoring**

> The field test site including the perimeter fallow zone must be
> monitored for the presence of volunteer rice plants for 1 year after
> termination of the field test. Viable plant material should not remain
> at the test site following termination.

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(s)**
>
> **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/BRS 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;

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 which will be used for food, feed, or seed
    production. A survey should be done within at least ¼ mile of the
    planting sites.

v.  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.

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XI. **Additional Data Requirements**

> No additional requirements.

[^1]: By convention, the gene is designated by small italic letters and
    the protein produced by that gene is designated by non-italicized
    letters, first letter capitalized.