USDA-APHIS

Draft Environmental Assessment

In response to permit application (06-250-02r)

received from SemBioSys, Inc. for a field-test to produce carp growth
hormone in genetically engineered safflower (*Carthamus tinctorius*)
seeds

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

Biotechnology Regulatory Services

[]{#__RefHeading___Toc156125251 .anchor}TABLE OF CONTENTS

**TABLE OF CONTENTS [2](#__RefHeading___Toc156125251)**

I. SUMMARY [3](#summary)

II\. INTRODUCTION [3](#introduction)

A. USDA Regulatory Authority [3](#usda-regulatory-authority)

B. Food and Drug Administration (FDA) Regulatory Authority
[4](#food-and-drug-administration-fda-regulatory-authority)

III\. PURPOSE AND NEED [4](#purpose-and-need)

IV\. ALTERNATIVES [4](#alternatives)

A. No Action: Do not allow planting to increase seed production
[5](#no-action-do-not-allow-planting-to-increase-seed-production)

B. Issue the Permit: Allow planting with supplemental conditions
[5](#issue-the-permit-allow-planting-with-supplemental-conditions)

C. Preferred Alternative [7](#preferred-alternative)

V. AFFECTED ENVIRONMENT [7](#affected-environment)

VI\. POTENTIAL ENVIRONMENTAL IMPACTS
[10](#potential-environmental-impacts)

1\. Potential impacts from gene introgression from GE safflower into its
sexually compatible relatives
[10](#potential-impacts-from-gene-introgression-from-ge-safflower-into-its-sexually-compatible-relatives)

2\. Potential impacts based on the relative weediness of GE safflower
[12](#potential-impacts-based-on-the-relative-weediness-of-ge-safflower)

3\. Potential impact on non-target organisms, including beneficial
organisms and threatened or endangered species
[12](#potential-impact-on-non-target-organisms-including-beneficial-organisms-and-threatened-or-endangered-species)

4\. Potential impacts on biodiversity
[13](#potential-impacts-on-biodiversity)

5\. Potential impacts on agricultural practices
[13](#potential-impacts-on-agricultural-practices)

6\. Potential impacts on organic farming
[14](#potential-impacts-on-organic-farming)

7\. Potential impacts on raw or processed agricultural commodities
[14](#potential-impacts-on-raw-or-processed-agricultural-commodities)

VII\. CONSIDERATION OF EXECUTIVE ORDERS, STANDARDS AND TREATIES RELATING
TO ENVIRONMENTAL IMPACTS
[15](#consideration-of-executive-orders-standards-and-treaties-relating-to-environmental-impacts)

VIII\. PREPARERS AND REVIEWERS [16](#preparers-and-reviewers)

IX\. AGENCY CONTACT [16](#agency-contact)

X. APPENDICES: Summary of permit data and information considered in
completing environmental assessment
[20](#appendices-summary-of-permit-data-and-information-considered-in-completing-environmental-assessment)

Appendix I. Biology of Safflower [20](#appendix-i.-biology-of-safflower)

Appendix II. Description of the Regulated Safflower Plant
[21](#appendix-ii.-description-of-the-regulated-safflower-plant)

Appendix III. Field Test Release and Agricultural Practices
[23](#appendix-iii.-field-test-release-and-agricultural-practices)

Appendix IV. Carp Growth Hormone [24](#appendix-iv.-carp-growth-hormone)

Appendix V. Threatened and Endangered Species Analysis
[25](#__RefHeading___Toc156125278)

Appendix VI. Standard Permit Conditions for APHIS Form 2000 (7 CFR
340.4) [30](#__RefHeading___Toc156125279)

Appendix VII. Supplemental Permit Conditions for APHIS Form 2000 (7 CFR
340.4)
[32](#appendix-vii.-supplemental-permit-conditions-for-aphis-form-2000-7-cfr-340.4)

#  {#section .unnumbered}

# **SUMMARY**

The Animal and Plant Health Inspection Service of the United States
Department of Agriculture (USDA-APHIS), has prepared an Environmental
Assessment (EA) in response to request for a permit (APHIS Number
06-250-02r) submitted by SemBioSys Genetics, Inc. (SemBioSys) for a
release of genetically engineered (transformed) safflower *(Carthamus
tinctorius)*. The genetically engineered (GE) safflower *(Carthamus
tinctorius)* was developed to express an oleosin-carp growth hormone
(somatotropin from *Cyprinus carpio*) exclusively within its seed to be
ground and incorporated into experimental aquaculture feed. This
transformed safflower is currently a regulated article under USDA
regulations at 7 CFR Part 340, and as such, field tests of transformed
safflower have been conducted under a permit issued by APHIS (Permit
#05-320-01r). SemBioSys has submitted a permit application to plant GE
safflower to increase seed production.

# **INTRODUCTION**

Demand for seafood has increased dramatically in recent years (Worm et
al., 2006). The aquaculture industry seeks to raise seafood in managed
environments often at higher densities than are supported in the wild.
High density production methods may lead to detrimental health
consequences including more rapid spread of disease and decreased growth
rates. The aquaculture industry has been trying to find ways to improve
their managed environments without the detrimental health consequences
of over-crowding. One current practice that promotes health in farm
raised seafood is to include carp growth somatotropin in aquaculture
feed. SemBioSys has genetically engineered the safflower cultivar S-317
(moderate oleic acid oil producer) to produce carp growth somatotropin
as a fusion protein to oleosin, a naturally occurring seed storage
protein. The fusion protein is expressed exclusively within its seed,
which will then be ground and incorporated into aquaculture feed. The
ground seed supplement in aquaculture feed has been shown to increase
experimental farmed fish growth rates and health as well as increasing
the immuno-resistance of farmed shrimp to common shrimp diseases such as
white-spot virus (unpublished SemBioSys, Inc. internal data). Currently,
there are no U.S. Food and Drug Administration (FDA) approved hormones
for growth promotion of aquatic animals. The transgenic safflower seed
meal is to be used in experimental fish feeding studies by SemBioSys and
is not for commercial production. A technical review of carp growth
hormone can be found in Appendix IV.

## USDA Regulatory Authority

The authorities for regulation of genetically engineered safflower is
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, safflower has been genetically
engineered using the recombinant DNA technique using the vector,
disarmed *Agrobacterium tumefaciens*. *Number range CHAPTERA.
tumefaciens* is a plant pest listed under 7 CFR § 340. Thus, the
genetically engineered organism in this submission is deemed a regulated
article.

## Food and Drug Administration (FDA) Regulatory Authority

The FDA policy statement concerning regulation of products derived from
new plant varieties, including those genetically engineered, was
published in the Federal Register on May 29, 1992, and appears at 57 FR
22984-23005. Under this policy, FDA uses what is termed a consultation
process to ensure that human food and animal feed safety issues or other
regulatory issues (e.g., labeling) are resolved prior to commercial
distribution of bioengineered food. Since this planting is not for
commercial distribution, SemBioSys has not submitted a food and feed
safety and nutritional assessment summary to FDA for the transformed
safflower.

# **PURPOSE AND NEED**

The proposed action is for APHIS, Biotechnology Regulatory Services
(BRS), to issue a permit for field-testing safflower S-317 in Douglas,
Grant and Lincoln Counties, WA. The safflower is genetically engineered
to express, within its seeds, the carp growth hormone fused to an
*Arabidopsis* oleosin. The purpose of this introduction is to obtain a
seed increase of material for future use as a supplement in aquaculture
meal. The permit application was received by APHIS, BRS on September 5,
2006. It was submitted by SemBioSys Genetics, Inc., W. Sacramento, CA.
The application number is 06-250-02r.

APHIS has prepared this EA before making a determination on whether to
allow the planting of transformed safflower under APHIS regulations. The
developer of this transformed safflower, SemBioSys, submitted a permit
application to USDA-APHIS requesting that APHIS make a determination
whether to allow planting for seed increase under 7 CFR Part 340. Under
regulations in 7 CFR Part 340, APHIS is required to give a determination
on the permit application before release into the environment.

This EA was prepared in compliance with the National Environmental
Policy Act (NEPA) of 1969 as amended, (42 USC 4321 *et seq.*) and the
pursuant implementing regulations (40 CFR 1500-1508; 7 CFR Part 1b; 7
CFR Part 372).

# **ALTERNATIVES**

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).

## No Action: Do not allow planting to increase seed production

For the purposes of this Environmental Assessment, the No Action
alternative would be the denial of permit application 06-250-02r. APHIS
might choose this alternative if there were evidence to demonstrate a
plant pest risk from the cultivation of safflower engineered to express
carp growth hormone within its seeds.

The U.S. Department of Agriculture's Animal and Plant Health Inspection
Service, Biotechnology Regulatory Services has previously allowed the
SemBioSys Genetics, Inc. to plant transgenic safflower plants containing
the carp growth hormone-oleosin fusion gene under permit 05-320-01r for
a total of 20 acres which is set to expire in April 2007. Under the No
Action Alternative, if submitted permit 06-250-02r is denied, the
transgenic safflower plants will not be planted. Any safflower plants
currently planted under permit 05-320-01r will be removed from the field
or devitalized before the expiration of that permit, as required by
APHIS regulations in 7 CFR 340.3(c).

## Issue the Permit: Allow planting with supplemental conditions

In this alternative, issuing this permit would allow the following
research to proceed at field sites in Douglas, Grant and Lincoln
Counties, WA (see Appendix III for additional details). In this
alternative, Supplemental Permit Conditions (Appendix VII), input from
the State of Washington and public comment from this environmental
assessment would be required. If APHIS chooses this alternative,
additional mitigating measures would be required to prevent spread of
the organism outside the field production area based on environmental
risk of escape of the engineered organism.

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 VI), and any supplemental
> conditions (Supplemental Permit Conditions, Appendix VII) 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)

SemBioSys has submitted Standing Operating Procedures (SOP) for this
permit \[CBI, reviewed by APHIS\] detailing protocols for planting,
harvesting and storing genetically-engineered material to ensure a
confined field release. These SOPs follow the requirements set up within
the Supplemental Permit Conditions (Appendix VII) and are specific to
SemBioSys facilities, equipment and employees. The following mitigation
measures also have been incorporated into the permit's experimental
procedures:

a.  The field sites will be geographically isolated from any commercial
    safflower production fields, ensuring no out-crossing of the GE
    safflower to food or feed safflower crops. The locations of the
    field sites are considered CBI. Crops surrounding the field sites
    will be barley and wheat with the closest commercial safflower
    production field at least ten miles away; however, APHIS permit
    conditions only require a distance of two miles from any commercial
    safflower fields. The Association of Official Seed Certifying
    Agencies (AOSCA) mandates a distance of 1320 ft from contaminating
    sources for a 99.9% purity of foundation certified seed. The ten
    mile distance between the SemBioSys field test sites and any
    commercial safflower fields far exceeds these requirements.

b.  The field test sites will be geographically isolated from any
    sexually compatible wild safflower species to ensure no out-crossing
    of the GE safflower with any wild safflower species. There exists
    about 25 species of wild safflower divided into four sections based
    on chromosome number (Ashri and Knowles, 1960). Of all the species
    of wild safflower, cultivated safflower (*C. tinctorius* with an
    n=12 chromosome number) is only sexually compatible with the
    federally listed noxious weed known as wild safflower or jeweled
    distaff thistle (*C.* *oxyacanthus* with an n=12 chromosome number).
    *C.* *oxyacanthus* is normally distributed in Afghanistan,
    Azerbaijan, India, Iran, Iraq, Kyrgyzstan, Pakistan, Tajikistan,
    Turkmenistan ([www.plants.usda.gov](http://www.plants.usda.gov/)).
    According to the Federal Noxious Weed website
    ([www.invasive.org](http://www.invasive.org/)), Monterey County, CA
    (southern-central coast) is the only known area where this noxious
    weed has been found. There are no known wild safflower relatives in
    Washington State. There is little chance of wild safflower (jeweled
    distaff thistle) establishing itself in the agriculture area where
    the SemBioSys fields will be located.

c.  There are no apiaries within a ten mile radius of the field test
    sites ensuring no pollen flow of safflower by bees and preventing
    out-crossing of GE safflower in the unlikely event that any
    safflower (wild or cultivated) is present in the 2 mile isolation
    zone around the field sites. The S-317 safflower cultivar is 85-90%
    self-pollinating with insects and bees being responsible for the
    remaining 10-15%. Of insects and bees, honey bees are the primary
    pollinator for safflower (Eckert, 1962). SemBioSys has provided a
    list of registered apiaries in Douglas, Lincoln and Grant Counties.
    No apiaries are found within a 10 mile radius of the test plot
    sites. Wild bees and pollinating insects are not expected to be a
    problem due to insecticides used on adjacent wheat and barley fields
    to prevent thrips, wireworms, cutworms and other insect pests
    (<http://www.ipmcenters.org/CropProfiles/docs/wabarley.html>,
    <http://www.ipmcenters.org/cropprofiles/docs/IDwheat.html>).

d.  Each test plot will be surrounded by a 50 ft fallow (bare ground)
    border to detect any potential vegetative reproduction by the
    transgenic safflower plants or volunteers that may occur as
    dedicated equipment is moved through out the field site.

If APHIS chooses alternative B, APHIS would require the following safety
measures in the Supplemental Permit Conditions (Appendix VII) to promote
a confined field release and to ensure no significant harm to the
environment:

a.  In addition to any removed transgenic plant material, any
    non-transgenic plant material removed from the test field plot will
    be treated as a regulated article. This includes any viable stems,
    whole plants, seedlings, and seeds.

b.  APHIS requires that the field site be at least 2 miles from any
    commercial safflower production field sites to ensure no
    out-crossing of the GE safflower with any food or feed safflower
    crop.

## Preferred Alternative

APHIS has chosen Alternative B as the preferred alternative. This is
based on APHIS' scientific analysis of the permit application.

# **AFFECTED ENVIRONMENT**

Safflower is a minor crop of North America and is grown mainly for its
seed, which is used as edible oil, meal and birdseed. India produces
about half of the world's safflower each year (430,000 tons) compared to
89,000 tons in the combined United States Great Plains area and
southwestern prairies of Canada. In the United States, this annual
oilseed crop is adapted primarily to the cereal grain areas of North and
South Dakota as well as Montana. In North Dakota, acreage has been
concentrated in the western part of the state. It also grows well in the
southwestern United States, most notably Arizona and New Mexico (USDA
APHIS, 2004).

Traditionally, safflower was grown for its flowers to be used in
medicines as well as coloring and flavoring in foods until cheaper
aniline dyes came on the market. Safflower oil is used by both food
producers and by industry. There are two types of safflower oil with
corresponding types of safflower varieties: those high in
monounsaturated fatty acid (oleic) and those high in polyunsaturated
fatty acid (linoleic). Currently the predominant oil market is for those
varieties that produce seed high in oleic acid and very low in saturated
fatty acids. High oleic safflower oil is lower in saturates and higher
in monounsaturates than olive oil. High oleic safflower is used as a
heat stable cooking oil to fry such food items as french fries, chips
and other snack items and is also used in cosmetics, food coatings, and
infant food formulations. High linoleic safflower oil is also used in
human nutrition, but in recent years market demand has drastically
shifted from the traditional high linoleic oils to high oleic oil. High
linoleic oil is valued as a drying agent in paints and varnishes because
of its non-yellowing characteristic. The meal, which is about 24 percent
protein and high in fiber, is used as a protein supplement for livestock
and poultry feed. The S-317 variety that was transformed by SemBioSys to
contain the carp growth hormone-oleosin fusion gene is a moderate oleic
oil producer (\~39% oleic oil production).

Out-crossing between safflower crops has been reported to be anywhere
from 0 to 100% (Claassen, 1950; Knowles, 1980) with an average between
15 and 20% (based on dominant flower-color markers). The S-317 safflower
cultivar is 85-90% self-pollinating with bees being primarily
responsible for the remaining 10-15 of out-crossing.

Wild relatives of cultivated safflower, *Carthamus creticus* and *C.
oxyacantha,* have been reported to occur sporadically in several U.S.
states (Kartesz, 2004) and are listed as noxious weeds. *C. creticus* is
not sexually compatible with cultivated safflower due to its chromosome
number (2n=20 or 4n=44 compared to that of cultivated safflower with a
chromosome number of n=12). Although, *C. oxyacantha* has been reported
in California (specifically in Monterey County), overall this sexually
compatible species is rare and none have been detected in Washington.
(Kartesz, 2004); (Kiel and Turner, 1993). Noxious weeds are carefully
monitored, quarantined, and subject to eradication efforts, thereby
minimizing the possibility these species will establish. Because they
are listed as noxious weeds, there are detailed records of their
distribution.

Up to ten field sites (50-100 ac each site) will be located on private
property in Douglas, Grant and Lincoln Counties, WA. These counties
contain a mix of agricultural sagebrush-steppe ecosystems with an
average rainfall between 7-10 inches per year
([www.worldclimate.com](http://www.worldclimate.com/)). The adjacent
agricultural lands are expected to grow barley and wheat. There are no
bodies of water (lakes, streams or rivers) within a 3 mile radius of any
of the proposed field test sites. Since the carp growth hormone is only
expressed in the safflower seeds and not the pollen, any safflower
pollen that could be blown far enough away during a rogue wind event
will not affect any aquaculture in the area. Wind is also not known to
be a significant pollen dispersal agent as the pollen is large, having a
mean diameter of 53-56 μm (Berglund et al., 1998).

The proposed field test sites will not be artificially irrigated and
will rely on natural precipitation for water. Safflower grows best with
low atmospheric humidity and in deep soils with good internal drainage.
It also has good drought and heat tolerance and does not survive in
standing water, especially in warm weather (air temperature above
68^o^F), where it will die in less than two hours (Berglund et al.,
1998). Most of the diseases that safflower is susceptible to occur when
soil moisture and humidity conditions exceed the optimal. Crop rotation,
careful irrigation practices, and planting treated and disease free seed
are important methods for controlling losses from disease.

Large or small animals are unlikely to browse the safflower field sites
during plant growth due to the sharp spines that S-317 safflower
cultivar produces. Animals would most likely only browse for loose seeds
after harvest. Smaller animals such as rodents and rabbits normally
would only scavenge for dropped seeds after harvest, as the seed head is
tough and difficult to access during maturation compared to the cereal
grains that are to be planted in adjacent fields. The transformed
safflower seed is of the striped variety, which is associated with an
unpalatable color and odor. More palatable cereal grains in nearby
fields would more likely be the target of any seed scavenging animals.
Another built-in deterrent will be the proposed 50 ft fallow (bare
ground) zone around the field test site. Many small seed-scavenging
mammals are unwilling to be exposed to predators over such distances in
order to reach a food source when there are more accessible food sources
nearby such as wheat and barley. Birds rarely feed on standing mature
safflower fields due to the tough, tightly held seed head (Berglund et
al., 1998). Birds could scavenge loose seeds after harvest, but do not
prefer the striped seed safflower variety (white hulled variety is
readily eaten by birds and is sold as birdseed) and would most likely be
more attracted to the nearby cereal grain fields. SemBioSys contract
farmers have not reported any problems with birds during the maturation
stage of the safflower plants. The closest migratory pathway for birds
is the Pacific Flyway. The territory of this flyway comprises the
western Arctic, including Alaska and the Aleutian Islands and the Rocky
Mountain and Pacific coast regions of Canada, the United States and
Mexico, south to where it becomes blended with other flyways in Central
and South America. (<http://www.birdnature.com/flyways.html>). This
flyway does not pass through Grant, Douglas or Lincoln Counties in WA
(The closest the Pacific Flyway comes to the listed counties is
approximate 125 mi in either easterly or westerly directions.

Safflower has relatively few insect pests that cause economic damage and
the use of insecticides against safflower pests generally is not
required. Insect damage to safflower can occur at crop establishment,
during seedling and stem growth, and during the bud to flower stage. The
most susceptible period likely is the bud to flower stage. Thrips and
lygus bugs potentially are the most damaging pests to the plant, but do
not injure the seed head other than superficially. The head becomes too
tough for more than localized injury and seed loss is rare
(<http://agric.ucdavis.edu/crops/oilseed/saff11insect.htm>). In
practice, wireworms and cutworms, which affect stand establishment
(germination and seedling development), and lygus bugs, which migrate
from safflower to cotton, are the only insects commonly controlled with
pesticides in safflower. Wireworms can reduce stands but can be
controlled with Lindane either as a planter box treatment or as a
combination with seed treatment fungicides. In cotton growing regions,
however, safflower may be sprayed to control lygus bugs as the crop
begins to mature, to prevent the migration of those insects to near-by
cotton fields. This control is for the sake of the cotton, rather than
the safflower. Since no cotton is expected to be planted nearby the
safflower field test sites, it is unlikely that insecticides will be
used at all.

The most serious pests of safflower are weeds. Early weeds may compete
with safflower for moisture, sunlight, and nutrients, lowering
production and increasing cultivation costs. Heavy infestations of weeds
later in the season may interfere with mechanical harvesting. Since
safflower often matures before many common weed species, green weed
matter taken in by the harvester impairs quality and must be cleaned at
the grower\'s expense.

After harvest, seeds will be shipped to a storage and processing plant
owned by SemBioSys in WA following SemBioSys' standard operating
procedure \[CBI, reviewed by APHIS\] to prevent dissemination of
genetically engineered plants. All grinding and processing equipment
will be dedicated for use only with GE safflower and will not process
food or feed from non-transformed safflower.

# **POTENTIAL ENVIRONMENTAL IMPACTS**

### Potential impacts from gene introgression from GE safflower into its sexually compatible relatives

Safflower genes may escape from the test plots in two ways. The first
pathway of escape is by pollen transfer. The second is by movement of
propagative material, *i.e*., the whole seeds or by vegetative growth.

#### Pollen Movement {#pollen-movement .unnumbered}

Pollen gene flow is expected to be limited in safflower for the
following reasons:

a)  The field site will be at least 10 miles from any commercially
    produced safflower fields, far exceeding the 2 mile requirement set
    up by APHIS.

b)  Cultivated safflower is not sexually compatible with plant species
    outside of the *Carthamus* genus. The only wild relative it could
    hybridize with is listed as a noxious weed
    (<http://www.invasive.org/browse/subject.cfm?sub=4553>) and is not
    found in Washington State.

c)  The S-317 safflower cultivar is 85-90% self-pollinating with insects
    and honey bees being responsible for the remaining 10-15%. Of
    insects and bees, honey bees are the primary pollinator for
    safflower (Eckert, 1962). The average foraging radius of honey bees
    from the colony is only a few hundred meters in agricultural areas,
    and they typically do not move beyond 1.6 km (1 mi) (Winston, 1987).
    However, foragers may fly up to 10 km (6.25 mi and cover a 100 km^2^
    (38 sq mi) area around the hive (Seeley, 1995), and there is
    evidence of honey bees flying several kilometers (2-3 mi) between
    apiaries and to safflower fields (Gary et al., 1977). No apiaries
    are found within a 10 mile radius of the proposed test plot sites. A
    caged study was done comparing honey bees to convergent lady beetles
    (*Hippodamia convergens*), lygus (*Lygus hesperus*), and flower
    beetle (*Notoxus calcaratus*) as pollinators of safflower. This
    study showed that honey bees, followed by paper wasps (*Polistes
    exclamans*; a wasp of the Atlantic seaboard states) were the primary
    pollinators of safflower while the other insects demonstrated little
    or no pollinator activity (Levin et al., 1967). Any insect
    pollinators will not be affected by expression of carp growth
    hormone because it is only expressed in the seed of safflower.

d)  Wind is not known to be a significant pollen dispersal agent, most
    likely due to the pollen's large size (mean diameter of 53-56 μm).

Carp growth hormone is not expressed in pollen or any of the vegetative
material except seeds. Because it is not expressed in the pollen, no
cumulative effects of carp growth hormone are expected if APHIS chooses
to allow planting and the pollen falls outside the field test site,
by-passing the redundant mitigation measures in place.

#### Seed Movement {#seed-movement .unnumbered}

Movement of whole seed by animals is the major way safflower seed is
disseminated. For example, rodents may carry seed to new areas where it
could become established; however, eaten seed is not expected to survive
the digestive processes (USDA APHIS, 2004). Safflower seeds generally
lack dormancy and are light sensitive; allowing for monitoring of any
volunteers in the short distances that small animals could carry viable
seed away from the field site. As mentioned above, the proposed field
test sites will have a 50 ft fallow (bare ground) zone around the field
test site. Many small seed-scavenging mammals are unwilling to be
exposed to predators over such distances in order to reach a food source
when there are more accessible food sources nearby such as wheat and
barley. The S-317 cultivar is a spiny variety (comparable to many other
spiny thistles) that discourages large animals to browse the fields
after flowering which would carry the seeds away from the field site.
The S-317 safflower also has a striped hull, and is not as palatable
(due to odor and color) to birds as the white hulled variety. The nearby
fields of wheat and barley would more likely be targeted by scavenging
by birds than the maturing safflower field. Due to the large seed size,
any seeds eaten by birds will most likely be ground up and digested and
are not expected to pass through the avian digestive tract while
remaining viable. No cumulative effects of carp growth hormone are
expected if any small animals ingest GE safflower seed.

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 reinforce stringent standard
operating procedures. In this study, the applicant will follow detailed
SOPs \[CBI, reviewed by APHIS\] to prevent accidental dispersal of the
seeds or plants into the environment.

The seeds are the only place where the gene of interest (carp growth
hormone) is expressed. Due to the reasons listed above, APHIS believes
this field test will remain contained. Since there are no bodies of
water within a three mile radius of any of the proposed field test
sites, it is unlikely that aquatic animals will be exposed to any carp
growth hormone should GE safflower seeds be transported outside the
field test site despite strict confinement measures. Should APHIS choose
Alternative B and allow planting, APHIS is confident that the current
permit and supplementary permit conditions will confine the crop and
alleviate the risk of gene flow through seed movement and mitigate any
cumulative effects of the expressed gene within the seeds themselves.

#### Vegetative Growth {#vegetative-growth .unnumbered}

Safflower is not known to spread vegetatively and propagates only
through seed germination. The inclusion of a 50 ft fallow zone (bare
ground) around the experimental field sites is primarily to be used as
an equipment staging and turn-around area. Any seeds dropped during
harvest would germinate in this zone and be easily seen and destroyed.
The fallow zone also serves as a deterrent for small animals looking to
forage seed from the safflower field sites. Few small animals will cross
such a distance and be exposed to predation when there are more easily
accessible cereal fields nearby (wheat and barley). No cumulative
effects of carp growth hormone are expected as volunteers in this area
will be monitored and properly destroyed before seed set.

#### Horizontal Gene Transfer {#horizontal-gene-transfer .unnumbered}

Transfer and expression of DNA from the plant to bacteria is unlikely to
occur. Gebhard and Smalla (Gebhard and Smalla, 1999) and Schlüter *et
al.* (Schlüter et al., 1995) have studied transgenic DNA movement to
bacteria and although theoretically possible, it occurs at extremely low
rates (approximately 1 in 10^-14^). Many genomes (or parts thereof) have
been sequenced from bacteria that are closely associated with plants
including *Agrobacterium* and *Rhizobium* (Kaneko et al., 2000). There
is no evidence that these organisms contain genes derived from plants.
Koonin *et al.* (Koonin et al., 2001) and Brown (Brown, 2003) presented
reviews based on sequencing data that revealed horizontal gene transfer
occurs occasionally on an evolutionary time scale of millions of years.
Even in the unlikely event transfer were to occur, the gene would be
poorly expressed at best because transgene promoters and coding
sequences are optimized for plant expression, not prokaryotic bacterial
expression.

If APHIS chooses the no action alternative (Alternative A), APHIS would
not permit the environmental release of this GE safflower. If APHIS
chooses Alternative B, no gene introgression is expected due to
safflower biology and geographical location of the field test sites. No
cumulative effects of carp growth hormone would be expected with either
alternative.

### Potential impacts based on the relative weediness of GE safflower

During early stages of growth, cultivated safflower is slow growing and
a poor competitor with fast growing weeds. If not controlled, weeds can
grow taller than safflower and compete for light, nutrients and moisture
and ultimately can cause complete crop losses if left uncontrolled
(Dajue and Mündel, 1996). The lack of seed dormancy also decreases the
weediness potential of cultivated safflower and volunteers after harvest
are not common. The transgenic safflower plants are no weedier than
traditional cultivated safflower; which has limited weediness potential
due to its biology (also see Appendix I for the detailed description
safflower biology).

Because cultivated safflower is not described as a weedy species and
none of its sexually compatible weedy species are present in Washington
State, there would be no weedy impact from allowing the field test for
seed increase to occur (Alternative B). If APHIS chooses the no action
alternative (Alternative A) there would also be no weed impact from this
variety.

### Potential impact on non-target organisms, including beneficial organisms and threatened or endangered species

APHIS has reached a determination that the proposed environmental
release will have no effect on federally listed threatened or endangered
species 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 the United States Fish and Wildlife Service is not required for the
action described in the preferred alternative of this EA. Appendix V
includes the BRS analysis of threatened and endangered species in the
area of the field release.

If APHIS chooses the no action alternative (Alternative A) there would
be no impact on non-target organisms or Federally-listed threatened or
endangered species.

Safflower has minimal insect pests and none that feed directly on the
seed; therefore, APHIS has determined there will be no impact of carp
growth hormone on insects should Alternative B be chosen. APHIS has
determined there will be no impact of carp growth hormone on birds or
scavenging animals that could possibly ingest seed (refer to Seed
Movement section). Based on the lack of toxicity of the proteins that
will be produced, APHIS concludes that if it chooses Alternative B,
there will be no significant effect on any native floral or faunal
species for Douglas, Grant and Lincoln Counties.

### Potential impacts on biodiversity

Analysis of available information indicates that SemBioSys' GE safflower
containing the carp growth hormone-oleosin fusion gene exhibits no
traits that would cause increased weediness in the proposed planting
area; nor should it lead to increased weediness of other cultivated
safflower or other sexually compatible relatives. Furthermore, it is
unlikely to harm non-target organisms common to the agricultural
ecosystem or threatened or endangered species recognized by the U.S.
Fish and Wildlife Service. There has been no intentional genetic change
in these plants to affect their susceptibility to disease or insect
damage. Neither the selectable marker gene \[CBI, reviewed by APHIS\],
nor the oleosin-carp growth hormone fusion gene is expected to change
any plant pest characteristics. There is no reason to believe that
weediness or plant pest characteristics are different between the
genetically engineered and non-engineered plants. The selectable marker
gene \[CBI, reviewed by APHIS\] is not expected to alter the
susceptibility of the transgenic safflower 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. SemBioSys, Inc. is required to report any
such unanticipated effects to APHIS under the terms of the permit. See 7
CFR § 340.4(f)(10)(ii).

If APHIS chooses either of the alternatives, there would also be no
impact on biodiversity.

### Potential impacts on agricultural practices

No impact on existing agricultural practices is expected if APHIS allows
planting (Alternative B). SemBioSys will employ agricultural practices
consistent with growing healthy safflower plants. Weeds will be
controlled using herbicide applications such as glyphosate, both pre-,
during, and post-harvest.

No environmental impacts on nearby crops are expected if APHIS chooses
to allow planting (Alternative B). No safflower seed production plots
are adjacent to the field test area. Barley and wheat are the only crops
that will be grown in the adjacent agricultural land. The closest
commercial safflower seed production field is expected to be greater
than 10 miles away from the closest test plot site and can be no closer
than 2 miles from the test plot.

### Potential impacts on organic farming

The National Organic Program (NOP) administered by USDA's Agricultural
Marketing Service (AMS) requires organic production operations to have
distinct, defined boundaries and buffer zones to prevent unintended
contact with prohibited substances from adjoining land that is not under
organic management. Organic production operations must also develop and
maintain an organic production system plan approved by their accredited
certifying agent. This plan enables the production operation to achieve
and document compliance with the National Organic Standards, including
the prohibition on the use of excluded methods. Excluded methods include
a variety of methods used to genetically modify organisms or influence
their growth and development by means that are not possible under
natural conditions or processes.

Organic certification involves oversight by an accredited certifying
agent of the materials and practices used to produce or handle an
organic agricultural product. This oversight includes an annual review
of the certified operation's organic system plan and on-site inspections
of the certified operation and its records. Although the National
Organic Standards prohibit the use of excluded methods, they do not
require testing of inputs or products for the presence of excluded
methods.

The presence of a detectable residue of a product of excluded methods
alone does not necessarily constitute a violation of the National
Organic Standards. The unintentional presence of the products of
excluded methods will not affect the status of an organic product or
operation when the operation has not used excluded methods and has taken
reasonable steps to avoid contact with the products of excluded methods
as detailed in their approved organic system plan. Organic certification
of a production or handling operation is a process claim, not a product
claim.

It is not likely that organic farmers or other farmers will be
significantly impacted by the expected planting of this product since
this proposed planting is greater than two miles away from any
cultivated safflower fields.

This particular product should not present new and different issues
regarding the use of pesticides and other organic cultivation practices.
APHIS has considered that gene transfer to cultivated *Carthamus*
species in the U.S. would be limited due to its presence as a minor
North American crop and the geographical distance between the proposed
field test sites and any other conventional safflower production fields.

If APHIS chooses either of the proposed alternatives, there would be no
impact on organic farmers and the current cultivation practices are
unlikely to change.

### Potential impacts on raw or processed agricultural commodities

The genetically engineered safflower is not being planted for commercial
sale or use and therefore, it will not have any opportunity to come into
contact with any commercialized raw or processed commodities.
Nevertheless, the field test sites will be confined by strict
requirements and conditions pursuant the permit to prevent its presence
in such commodities.

There would be no impacts on raw or processed agricultural commodities
if APHIS chooses the No Action option regarding the genetically
engineered safflower (Alternative A). There would also be no impacts
from allowing the GE safflower to be planted (Alternative B), should
APHIS choose to allow planting with supplemental conditions.

# **CONSIDERATION OF EXECUTIVE ORDERS, STANDARDS AND TREATIES RELATING TO ENVIRONMENTAL IMPACTS** 

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 adverse human health or environmental
effects.

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 the agency's mission) requires each Federal agency
to identify, assess, and address environmental health risks and safety
risks that may disproportionately affect children. Each alternative was
analyzed with respect to EO 12898 and 13045. None of the alternatives
are expected to have a disproportionate adverse effect on minorities,
low-income populations, or 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. Safflower is a minor crop in the
United States. Based on historical experience with safflower and the
data submitted by the applicant and reviewed by APHIS, the engineered
plant is sufficiently similar in fitness characteristics to other
safflower varieties currently grown and it is not expected to have an
increased invasive potential.

Executive Order 12114, "Environmental Effects Abroad of Major Federal
Actions" requires Federal officials to take into consideration any
potential environmental effects outside the U.S., its territories and
possessions that result from actions being taken. APHIS has given this
due consideration and does not expect a significant environmental impact
outside the United States should APHIS choose any of the listed
alternatives to permit# 06-250-02r. Any international traffic of
genetically engineered safflower subsequent to a determination of
regulated status for GE safflower would be fully subject to national
phytosanitary requirements and be in accordance with phytosanitary
standards developed under the International Plant Protection Convention
(IPPC).

APHIS continues to work toward harmonization of biosafety and
biotechnology consensus documents, guidelines and regulations, including
within the North American Plant Protection Organization (NAPPO), which
includes Mexico, Canada, and the United States and in the Organization
for Economic Cooperation and Development. NAPPO has completed three
modules of a standard for the *Importation and Release into the
Environment of Transgenic Plants in NAPPO Member Countries* (see
http://www.nappo.org/Standards/Std-e.html). APHIS also participates in
the North American Biotechnology Initiative (NABI), a forum for
information exchange and cooperation on agricultural biotechnology
issues for the U.S., Mexico and Canada. In addition, bilateral
discussions on biotechnology regulatory issues are held regularly with
other countries including: Argentina, Brazil, Japan, China, and Korea.
Many countries, e.g. Argentina, Australia, Canada, China, Japan, Korea,
Philippines, South Africa, Switzerland, the United Kingdom

# **PREPARERS AND REVIEWERS** 

**Biotechnology Regulatory Services**

Cindy Smith, Deputy Administrator

Rebecca Bech, Associate Deputy Administrator for Emerging and
International Programs

**Permits and Risk Assessment Staff**

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

**BRS, Policy and Coordination Division**

John Turner, Ph.D., Director

Patricia Beetham, Ph.D. Biotechnologist (EA Preparer)

Rebecca Stankiewicz Gable, PhD. Biotechnologist (reviewer)

# **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

Email: cynthia.a.eck@aphis.usda.gov

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Bechtold, N., Ellis, J. and Pelletier, G. (1993) *In planta
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Berglund, D.R., Riveland, N. and Bergman, J. (1998) Safflower Production
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Brown, J.R. (2003) Ancient horizontal gene transfer. Genetics 4,
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Carapetian, J. (1994) Effects of safflower sterility genes on the
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325-384.

Claassen, C.E. (1950) Natural and controlled crossing in safflower,
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Dajue, L. and Mündel, H.-H. (1996) Safflower. *Carthamus tinctorius* L.
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Ekin, Z. (2005) Resurence of safflower (*Carthamus tinctorius* L.)
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Fine, M., Sakal, E., Vashdi, D., Daniel, V., Levanon, A., Lipshitz, O.
and Gertler, A. (1993) Recombinant carp (*Cyprinus carpio*) growth
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89, 54-61.

Garcia-Jacas, N., Garnatje, T., Susanna, A. and Vilatersana, R. (2002)
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Gary, N.E., Witherell, P.C., Lorenzen, K. and Marston, J.M. (1977) The
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Gebhard, F. and Smalla, K. (1999) Monitoring field releases of
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Hertz, Y., Tchelet, A., Madar, Z. and Gertler, A. (1991) Absorption of
bioactive human growth hormone after oral administration in the common
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Comparative Physiology 161-159-63.

Kaneko, T., Nakamura, Y., Sato, S., Asamizu, E., Kato, T., Sasamoto, S.,
Watanabe, A., Idesawa, K., Ishikawa, A., Kawashima, K., Kimura, T.,
Kishida, Y., Kiyokawa, C., Kohara, M., Matsumoto, M., Matsuno, A.,
Mochizuki, Y., Nakayama, S., Nakazaki, N., Shimpo, S., Sugimoto, M.,
Takeuchi, C., Yamada, M. and Tabata, S. (2000) Complete Genome Structure
of the Nitrogen-fixing Symbiotic Bacterium Mesorhizobium loti. DNA
Research 7, 331-338.

Kartesz, J.T. (2004) A synonymized checklist and atlas with biological
attributes for the vascular flora of the United States, Canada, and
Greenland, 2nd Edition. In: Synthesis of the North American Flora.
University of North Carolina, Chapel Hill and University of California,
Berkley.

Kiel, D.J. and Turner, C.E. (1993) *Carthamus*, distaff thistle. In:
J.C. Hickman (Ed) The Jepson Manual: Higher Plants of California.
University of California Press, Berkley, p. 220-227.

Knowles, P.F. (1980) Safflower. In: W.R. Fehr and H.H. Hadley (Eds),
Hybridization of Crop Plants. American Society of Agronomy and Crop
Science Society of America, Madison, WI, p. 535-548.

Koonin, E.V., Makarova, K.S. and Aravind, L. (2001) Horizontal gene
transfer in prokaryotes: Quantification and classification. Annual
Review of Microbiology 55, 709-742.

Langridge, D.F. and Goodman, R.D. (1980) A study on pollination of
safflower (*Carthamus tinctorius*) cv. Gila. Australian Journal of
Experimental Agriculture and Animal Husbandry 20, 105-107.

Levin, M.D., Butler, G.D.J. and Rubis, D.D. (1967) Pollination of
safflower by insects other than honey bees. Journal of Economic
Entomology 60, 1481-1482.

Mahmoud, S.S., Wang, S., Moloney, M.M. and Habibi, H.R. (1998)
Production of a biologically active novel goldfish growth hormone in
*Escherichia coli*. Comparative Biochemistry and Physiology 120,
657-663.

McLean, E. and Donaldson, E.M. (1990) Absorption of Bioactive Proteins
by the Gastrointestinal Tract of Fish: A Review. Journal of Aquatic
Animal Health 2, 1-11.

McPherson, M.A., Good, A.G., Topinka, A.K.C. and Hall, L.M. (2004)
Theoretical hybridization potential of transgenic safflower (*Carthamus
tinctorius* L.) with weedy relatives in the New World. Canadian Journal
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Celis, S., Gómez-Requeni, P., Saera-Vila, A., Astola, A. and Valdivia,
M.M. (2002) Overview of fish growth hormone family. New insights in
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Physiology and Biochemistry 27, 243-258.

Peter, R.E. and Marchant, T.A. (1995) The endocrinology of growth in
carp and related species. Aquaculture 129, 299-321.

PEW (2003) Future Fish: Issues in Science and Regulation of Transgenic
Fish. Pew Initiative on Food and Biotechnology, Washington, DC.

Schlüter, K., Fütterer, J. and Potrykus, I. (1995) Horizontal gene
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chrysanthemi) occurs if at all at an extremely low frequency.
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Honey Bee Colonies. Harvard University Press, Cambridge.

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Worm, B., Barbier, E.B., Beaumont, N., Duffy, J.E., Folke, C., Halpern,
B.S., Jackson, J.B.C., Lotze, H.K., Micheli, F., Palumbi, S.R., Sala,
E., Selkoe, K.A., Stachowicz, J.J. and Watson, R. (2006) Impacts of
biodiversity loss on ocean ecosystem services. Science 314, 787-790.

# **APPENDICES: Summary of permit data and information considered in completing environmental assessment**

## Appendix I. Biology of Safflower  {#appendix-i.-biology-of-safflower .unnumbered}

In this section of the environmental assessment, the biology of
safflower and plants related to safflower are considered along with
potential routes of gene escape. Because the mechanism by which genes
are moved from one flowering plant to another is through
cross-pollination of sexually compatible plants, the plants with which
safflower can cross-pollinate are also described. Below is an analysis
of the biology of safflower. This review focuses solely on safflower in
the United States.

#### Systematics of Safflower {#systematics-of-safflower .unnumbered}

Safflower, *Carthamus tinctorius* L., is a member of the family
Compositae (Asteraceae) tribe Cardueae, and subtribe Centaureinae
(Garcia-Jacas et al., 2002). Safflower is a highly branched herbaceous
annual thistle, usually with sharp spines on the leaves. Plants are
30-150 cm tall with globular flower heads (capitula) and, commonly,
brilliant yellow, orange or red flowers. Each branch usually has one to
five flower heads, and the flower head typically has 15 to 20 (but up to
180) florets, each of which can produce a dry fruit (achene) with a
single large seed (Dajue and Mündel, 1996). The taproot can penetrate to
8 to 10 ft if subsoil temperature and moisture permit. As a result,
safflower is more tolerant to drought than small grains (Berglund et
al., 1998). The florets are self-pollinating, but seedset can be
increased by bees or other insects.

#### Origin and Distribution of Safflower {#origin-and-distribution-of-safflower .unnumbered}

Safflower originated in southwest Asia (where the genus is native) and
moved to India and China. It is a minor crop of North America and is
grown mainly for its seed, which is used as edible oil, meal and
birdseed. India produces about half of the world's safflower each year
(430,000 tons) compared to 89,000 tons in the combined United States
Great Plains area and southwestern prairies of Canada (Ekin, 2005). The
U.S. safflower acreage varies widely (annual acreage ranges between
100,000 and 200,000 acres) with the majority of production in California
(USDA APHIS, 2004). The oilseed crop is also adapted to the cereal grain
areas of North and South Dakota as well as Montana.

#### Out-crossing {#out-crossing .unnumbered}

Out-crossing between safflower crops has been reported to be anywhere
from 0 to 100% (Claassen, 1950) with an average between 15 and 20%
(based on dominant flower-color markers) (Knowles, 1980). The S-317
safflower cultivar is 85-90% self-pollinating with bees being primarily
responsible for the remaining 10-15% of out-crossing.

Cultivated safflower originated in the Euphrates Basin and from this
center of origin expanded to Egypt, Ethiopia, southern Europe and the
Far East (McPherson et al., 2004). It can potentially hybridize with at
least six species of wild *Carthamus* (McPherson et al., 2004). Of the
four naturalized wild relatives in the New World, only *C. oxyacantha*
(or *C. oxyacanthus*) and *C. creticus* (*C. lanatus* subsp. *creticus*,
*C. baeticus*), have produced fertile F1 hybrids when crossed with *C.
tinctorius* (McPherson et al., 2004). *Carthamus creticus* and *C.
oxyacantha* have been reported to occur in several U.S. states (Kartesz,
2004) and are listed as noxious weeds, so they must be removed wherever
they are found (thus also minimizing the potential for out-crossing).

There are some areas in the United States and Canada where no cultivated
or wild *Carthamus* are currently found; hence cultivation in isolation
is possible. For example, no wild relatives of safflower occur in
Washington State, whereas *C. oxyacantha* has been reported in
California (Kartesz, 2004; Kiel and Turner, 1993). Overall, the sexually
compatible weedy wild *Carthamus* are quite rare and their presence can
be verified in each county because they are noxious weeds.

#### Dispersal/Pollination Mechanisms {#dispersalpollination-mechanisms .unnumbered}

Safflower is largely self-pollinated, and bees are occasionally a pollen
vector, whereas wind is not known to be a significant dispersal agent
(Knowles, 1980; Langridge and Goodman, 1980). Morphological
characteristics (e.g., pollen grain's moderately large size, with a mean
diameter of 53-56 μm) and the behavior of the floret in pollen
presentation (Knowles, 1980) help to explain the apparent lack of
significant wind dispersal (Carapetian, 1994). Most pollen movement has
been thought to occur within 2 m of the source (USDA APHIS, 2004).

Bees are the main pollinator moving pollen among flowers (florets) and
flower heads (Langridge and Goodman, 1980). Most honey bee colonies are
managed, as there are very few feral populations left in the United
States due to invertebrate pests (e.g., varroa mites and wax moths) and
disease (e.g., foul brood and chalk brood). Honey bees are the most
important contributors to long-distance pollen movement. The average
foraging radius of honey bees from the colony is only a few hundred
meters in agricultural areas, and they typically do not move beyond 1.6
km (1 mi) (Winston, 1987). However, foragers may fly up to 10 km (6.25
mi) and cover a 100 km^2^ (38 sq mi) area around the hive (Seeley,
1995), and there is evidence of honey bees flying several kilometers
(2-3 mi) between apiaries and to safflower fields (Gary et al., 1977).
Native bees tend to fly shorter distances than honey bees. Although
bumble bees typically forage close to their nests, they may travel 5 km
(3 mi) from the nest (USDA APHIS, 2004).

#### Pollen Competition and Viability {#pollen-competition-and-viability .unnumbered}

Issues related to pollen viability include how far pollen can travel and
how long it will stay viable on the pollinator. Bees tend to prefer
foraging on viable pollen. Pollen viability is influenced by
environmental factors such as relative humidity and temperature, and can
vary among cultivars. Safflower is typically grown in dry conditions,
where pollen is expected to desiccate rapidly. The viability of pollen
is variable between safflower varieties and probably is very short,
lasting less than 24 hours and perhaps into the following day (Knowles,
1980).

## Appendix II. Description of the Regulated Safflower Plant {#appendix-ii.-description-of-the-regulated-safflower-plant .unnumbered}

SemBioSys, Inc has engineered safflower to contain the carp growth
hormone gene fused with an oleosin gene from *Arabidopsis*. The
recipient organism, *Carthamus tinctorius*, cv. S-317, is a common
commercial cultivar and is grown mainly for its seed, which is used for
its oil in both food and industrial processing. The resulting transgenic
safflower seed will be used as a feed additive in aquaculture.

#### The Vectors {#the-vectors .unnumbered}

The genes were transferred into safflower plants via a vector system,
disarmed *Agrobacterium tumefaciens*. This process is a well
characterized transformation system which integrates the donor genes
into the chromosome of the recipient plant cell (Bechtold et al., 1993).
The donor DNA sequences are stably and irreversibly integrated into the
plant\'s chromosomal DNA, where they are maintained and inherited as any
other genes of the plant cell.

Parsley (*Petroselinum crispum* L.) and common bean (*Phaseolus
vulgaris*) are donors for non-coding DNA regulatory sequences that are
associated with the introduced genes to facilitate expression in plants.
Number range CHAPTERThe regulatory sequences from parsley are the
ubiquitin (*ubi*) promoter and terminator regions for the selectable
marker. The regulatory sequences from bean are the phaseolin promoter
and terminator regions for the gene of interest. None of the DNA
regulatory sequences can cause plant disease by themselves or in
conjunction with the genes that were introduced into the transgenic
safflower plant.

#### The Selectable Marker {#the-selectable-marker .unnumbered}

To facilitate the selection of transformed plants, the safflower plants
were engineered with the \[CBI, reviewed by APHIS\] gene flanked by the
ubiquitin promoter and terminator from parsley (*Petroselinum crispum*
L.).

The \[CBI, reviewed by APHIS\] gene is devoid of inherent plant pest
characteristics and is the most commonly used selective marker in plants
has been safely used in many previous field trials (CBI).

#### The Gene of Interest {#the-gene-of-interest .unnumbered}

Safflower plants were engineered so the carp growth hormone (carp
somatotropin from *Cyprinus carpio*) fused to an *Arabidopsis* oleosin
gene is exclusively expressed in the seeds. SemBioSys has determined
that the attachment of fish growth hormone and similar proteins to plant
oilbodies (e.g. *Arabidopsis* oleosin gene) allows proper folding and
stable accumulation of the protein within seeds while preventing
unwanted glycosylation. Expression of this gene is controlled by the
phaseolin promoter and terminator sequences from *Phaseolus vulgaris* L.
(common bean). The phaseolin promoter drives the tight, seed-specific
transcription of carp growth hormone. As expected, no detectable fusion
protein was found in leaf, root and floral (including pollen) material
when analyzed by western blots. See Appendix IV for a more in-depth
review of carp growth hormone.

## Appendix III. Field Test Release and Agricultural Practices {#appendix-iii.-field-test-release-and-agricultural-practices .unnumbered}

SemBioSys has provided APHIS with detailed SOPs \[CBI, reviewed by
APHIS\] for field release, movement and containment of GE plants. Below
is a summary of the permit information provided by SemBioSys.

#### Plot Design and Location {#plot-design-and-location .unnumbered}

Up to 10 field sites (50-100 ac each site) are located on private
property in Douglas, Grant and Lincoln Counties, WA. The experimental
plots will be bordered on all sides by a 50 ft fallow strip. The
adjacent agricultural lands will be planted with barley and wheat.

#### Agricultural Practices {#agricultural-practices .unnumbered}

Agricultural practices consistent with growing healthy safflower plants
will be used; weeds will be controlled by herbicide applications.

There are no serious insect threats to safflower in the United States.
If necessary, pesticides such as insecticides and/or fungicides will be
used to control insect pests and disease that would diminish the health
of the plant and subsequent seed yield. Any pesticides or herbicides
used will be applied by personnel trained in their use and application.
The field will be monitored for noxious weeds and other plant pests
during the growing season.

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.

#### Termination of the field test and final disposition of the test plants {#termination-of-the-field-test-and-final-disposition-of-the-test-plants .unnumbered}

The harvested seeds will be placed in dedicated storage bags on site and
immediately shipped for further processing in aquaculture feed.

After harvest, as soon as possible as the weather allows, SemBioSys,
Inc. will till the field and apply the herbicide glyphosate to kill all
plants remaining in the field. The field site will be monitored
quarterly for one year following the termination of the field test, and
any volunteer safflower plants will be destroyed by application of
glyphosate, removed and autoclaved.

#### Volunteer Monitoring {#volunteer-monitoring .unnumbered}

Volunteer monitoring by the researcher will be done for 1 year (1
growing season) after the end of the field test site. Safflower seeds
generally lack dormancy and can even germinate in the seed head if too
much rain occurs before harvest. Despite limited potential for seed
dormancy, the field site will be monitored quarterly for one year (one
growing season) following the termination of the field test and any
volunteer safflower plants will be destroyed by application of
glyphosate, removed and autoclaved.

## Appendix IV. Carp Growth Hormone {#appendix-iv.-carp-growth-hormone .unnumbered}

Growth hormone (GH) is a single chain peptide produced in the anterior
pituitary of all vertebrates. The primary function of GH is to stimulate
growth and development either directly or indirectly through the
production of insulin-like growth factors (IGFs). GH also influences
reproduction and food conversion efficiency as well as osmoregulation
and sea water adaptation in fish (Mahmoud et al., 1998).

Carp growth hormone (somatotropin protein; 21.5 kDa) is found naturally
within common carp as a product of the pituitary gland regulation of the
growth of these fish (Mahmoud et al., 1998). The growth hormones of
cyprinids (carp-like fish) belong to a family possessing similar primary
structure but are not closely related to those of other animals and
share only 36% identity with human and about 40% with bovine
somatotropins (Mahmoud et al., 1998).

While the safety of carp growth hormone to humans and animals other than
fish has never been investigated, the long history of fish ingestion by
numerous mammalian and avian species would indicate that it is not toxic
at its natural biological levels. A comparison was made of the safety of
the transgenic protein as it relates to recombinant bovine somatotropin,
which is very well characterized. As mentioned above, carp growth
hormone shares some homology with its bovine counterpart and performs
the same basic function within its host. The ingestion safety of bovine
somatotropin to mammals such as mice, cattle and humans has been
described and it is deemed a safe protein even at high levels of
exposure due to the breakdown of proteins in the mammalian and avian
digestive process (USFDA, 1993). Part of the difference between the oral
efficacy of these hormones on fish versus mammals and birds is that the
fish gut can absorb intact proteins into circulation before digestion
occurs (McLean and Donaldson, 1990). Another interesting feature of
somatotropins is that those belonging to higher organisms such as
mammals have a definite biological effect on lower animals such as fish
while the fish counterpart does not appear to affect mammals (Hertz et
al., 1991; Peter and Marchant, 1995; Pérez-Sánchez et al., 2002).

[]{#__RefHeading___Toc156125278 .anchor}**Appendix V. Threatened and
Endangered Species Analysis**

The proposed field sites are for the confined releases of the regulated
article into the environment in Grant, Douglas and Lincoln Counties, WA.
There are no listed critical habitats for any threatened and endangered
animal species in Grant, Douglas and Lincoln Counties, WA according to
the U.S. Fish and Wildlife database (<http://criticalhabitat.fws.gov/>)
and therefore the confined release of the regulated article is not
expected to affect any critical habitats.

On the basis of our review of permit 06-250-02r, we conclude that
controlled field testing of the genetically engineered safflower plants
described in this application would not present any risk of new plant
pest introduction, would have no significant impact on non-target
organisms and on the threatened or endangered species, and therefore
constitutes a confined field trial. Furthermore, if the field test is
performed with conditions outlined here and in the permit, the risk to
the environment would be exceedingly low.

APHIS evaluated plant pest impacts related to the transformation method
used in this permit and concluded that the DNA inserted into the plants
does not have any inherent plant pest characteristics and is not likely
to pose a plant pest risk for the following reasons:

1.  The safflower plants were transformed using a disarmed
    *Agrobacterium tumefaciens* protocol which does not cause plant
    disease.

2.  The selectable marker gene and all of the non-translated regulatory
    elements are well characterized.

3.  The introduced DNA will not lead to the expression of a toxin or
    other product that is known to affect the metabolism, growth,
    development, or reproduction of animals, plants, or microbes. A
    BLAST search using the amino acid sequence of the carp growth
    hormone-oleosin domains did not reveal any significant homology (\>
    50%) to the amino acid sequence of proteins other than oleosin and
    carp growth hormone proteins.

4.  Although part of the inserted oleosin gene shares a 70% sequence
    homology within its hydrophobic domain (central part) to a filbert
    (hazelnut) oleosin that has been implicated as a candidate allergen,
    this should not be of a concern since the hydrophobic domain is
    highly conserved among oleosins of many food species that are
    consumed by humans and animals. The three domains (C-terminal,
    central core, and the N-terminal) of the *Arabidopsis* oleosin
    protein share a significant overall homology to the oleosin protein
    of many food species such as corn, rice, canola, sesame seed and
    rye) with the highest sequence homology to canola oleosin.

5.  Literature searches of the NCBI's PubMed databases did not reveal
    any evidence for toxic or allergenic effects of the carp
    somatotropin growth hormone on human or animals. Fish somatotropin
    does not appear to have a biological effect on mammals (Fine et al.,
    1993).

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1.  

> APHIS evaluated potential plant pest impacts related to the quarantine
> and final disposal of transgenic plants and concluded that the field
> trial is a confined release and has no significant impact on the
> environment. The following containment measures should be sufficient
> to prevent any unplanned release of the transgenic plant material or
> transgenic seed or the persistence of the transgenic material or its
> progeny in the environment:

1.  Dedicated equipment will be used for planting and harvesting and
    will be labeled accordingly. This precaution ensures that the
    transgenic safflower plants are not inadvertently removed from the
    field and therefore eliminates dispersal and gene flow of the
    transgenic safflower plants.

2.  A perimeter fallow zone of 50 feet will be maintained around the
    transgenic test site to ensure that transgenic safflower are not
    inadvertently commingled with plants to be used for food or feed.

3.  In addition to the large degree of self-pollination of safflower
    plants, other mitigating measures are implemented to prevent gene
    flow through pollen dispersal to any compatible species or by seed
    dispersal. The field site will be isolated from sexually compatible
    wild safflower or any other commercial safflower seed production
    areas by at least 2 miles. Additionally, the applicant presented a
    procedure to report to APHIS any unauthorized or accidental release
    of the transgenic material. These measures would further ensure that
    the transgenes do not enter the commercial safflower seed supply.

4.  The test plots will be monitored weekly for weed, disease, and
    insect infestation.

5.  In addition to lack of seed dormancy of safflower where seed can
    germinate in the head if rain fall occurs at harvest time
    ([http://www.ipgri.cgiar.org/publications/pdf/498.pdf]{.underline}),
    the field will be monitored for safflower volunteers for one growing
    season after harvest. In the growing season following the harvest,
    the test area will be left fallow.

6.  It is unlikely for safflower (*Carthamus tinctorius*) to become a
    weed under most agricultural situations. Safflower is unable to
    persist in the environment without continuous human intervention and
    is not reported to be an agricultural weed. Wild relatives of
    safflower are not found in Washington and the wild safflower
    (*Carthamus oxyacanthus*), which is on the Federal Noxious Weed
    list, is not sexually compatible with *Carthamus tinctorius*. The
    gene function is known and the phenotype will not confer any traits
    associated with weediness to safflower.

7.  The proposed cultivation practices involved in growing these
    transgenic safflower plants are similar to growing practices for
    normal commercial safflower and as a result no "unusual" growing
    practices should be expected to increase weediness or volunteers.

APHIS evaluated the potential impacts on non-target organisms, including
threatened or endangered species (TES). An examination of the U.S. Fish
and Wildlife threatened and endangered species system (TESS)
[http://ecos.fws.gov/tess_public/StateListingAndOccurrence.do?state=WA]{.underline}
on December 2006 showed that 9 threatened or endangered plant species
and 28 animal species exist in Washington. Of the 28 listed, only four
animals potentially reside in Douglas, Grant and Lincoln Counties, WA:

-   Bald eagle (*Haliaeetus leucocephalus*)

-   Pygmy rabbit (*Brachylagus idahoenis*)

-   Columbian white-tailed deer (*Odocoileus virginianus leucurus*)

-   Gray wolf (*Canis lupus*)

The bald eagle (*Haliaeetus leucocephalus*) lives near large bodies of
open water such as lakes, marshes, seacoasts and rivers, where there are
plenty of fish to eat and tall trees for nesting and roosting. The
nearest sources of waters with potential roosting places for the bald
eagle are the Grand Coulee River, Columbia River and Banks Lake. All
three water sources are not near the field test sites, so it is unlikely
that this species would be impacted by the field tests.

The pygmy rabbit (*Brachylagus idahoenis*) inhabits areas throughout the
Great Basin and prefers dense, tall stands of big sagebrush that are
usually along intermittent streams or riparian areas in sagebrush
grasslands. Ninety nine percent of their diet in the winter consists of
sagebrush. The pygmy rabbit is not expected to be impacted by the field
test sites due to its preference for sagebrush and riparian habitats.

The Columbian white-tailed deer (*Odocoileus virginianus leucurus*)
occurs in farmlands, brush areas, woods, suburbs and gardens. They feed
on green plants, acorns, beechnuts, and other nuts and corn in the fall
and in winter they feed on woody vegetation, including the twigs and
buds of viburnum, birch, maple, and many conifers. The S-317 cultivar is
a spiny variety (comparable to many other spiny thistles) that
discourages large animals to browse the fields after flowering. S-317
safflower also has a striped hull, and is not as palatable (due to odor
and color) as the white hulled variety. Columbian White-tailed Deer are
not expected to be impacted by the field test sites.

The Gray Wolf (*Canis lupus*) The Northern Rocky Mountain gray wolf is
currently endangered in Colorado, Idaho, Michigan, Montana, North
Dakota, South Dakota, Washington and Wisconsin and threatened in
Minnesota. Wolves are carnivorous but may also feed on earthworms,
berries or grasshoppers. Since the Gray Wolf habitat does not include
maintained agricultural land, the field test site is not expected to
impact this endangered species.

Of the nine plants listed, only one threatened species potentially
resides in Lincoln County and another threatened species in Douglas
County
([http://ecos.fws.gov/](http://ecos.fws.gov/species_profile/servlet/gov.doi.species_profile.servlets.SpeciesProfile?spcode=Q1P9)):

-   Spalding's Catchfly (*Silene spaldingii*) -- Lincoln County

-   Ladies' Tresses (*Spiranthes diluvialis*) -- Douglas County

Spalding's catchfly is endemic and is restricted to remnants of the
prairie grasslands of eastern Washington, northeastern Oregon, northern
Idaho, and western Montana (barely extending into British Columbia,
Canada). This species is restricted to Palouse Prairies, sometimes
extending into areas where the grasslands are intermingled with
ponderosa pine (*Pinus ponderosa*) woodlands. Sites are often near lower
treeline, or near scattered ponderosa pine trees. A significant amount
of habitat has been lost to conversion to agriculture, restricting most
remaining occurrences to small, isolated fragments of native vegetation,
where they are vulnerable to degradation. Most remaining populations are
small and threatened by weed invasion (including yellow starthistle in
places), herbicide treatment (particularly because many populations are
small and located near farmlands and roads), and livestock grazing.
Activities such as road construction and maintenance, gravel mining,
off-road vehicles, and urban developments are additional threats.
Recovery of this species requires habitat improvement through site
monitoring and restricting public access to areas where they occur. The
FWS recovery plan (USFWS, 2005) indicates no occurrences of Spalding's
catchfly have been noted in the areas proposed by SemBioSys, Inc \[CBI,
reviewed by APHIS\] within Lincoln County and thus, the field trial
sites, all agricultural areas, are not expected to impact this
threatened species.

Ladies' Tresses (*Spiranthes diluvialis*) is a threatened species listed
as potentially occurring in Douglas County, WA; however, Natureserve
distribution and the U.S. FWS recovery plan only indicate potential
growth sites in Chelan and Okanogan Counties
([www.natureserver.org](http://www.natureserver.org/); (USFWS, 1995).
Its preferred habitat is moist to very wet meadows along streams or in
abandoned stream meanders that still retain ample ground water. It is
also found near springs, seeps, and lakeshores. Because of the preferred
habitat locations, Ladies' Tresses are not expected to be impacted by
the field trial sites which are all areas previously used for
agricultural production.

Based on the reasons listed below, APHIS is confident that these field
trials will not harm or have any significant adverse effects on
threatened or endangered species either by direct or indirect exposure.

1.  The introduced DNA will not lead to the expression of a toxin or
    other product that is known to affect the metabolism, growth,
    development, or reproduction of animals, plants, or microbes.
    Sequence alignments and homology searches of the carp growth
    hormone-oleosin protein using the BLAST search of non-redundant
    GeneBank coding sequence translations plus RefSeq, SwissProt and PDB
    showed more than 50% similarity to oleosin and carp growth hormone
    proteins. It did not show significant similarity to proteins that
    are known toxins or allergens. Therefore, the protein expressed in
    the transgenic safflower plants should have no known or foreseeable
    toxic or allergenic effects to humans or animals.

2.  Literature searches of the NCBI's PubMed databases did not reveal
    any evidence for toxic or allergenic effects of the carp growth
    hormone on human or animals. The long history of fish ingestion by
    numerous mammalian and avian species also confirms that carp growth
    hormone protein is not toxic at its natural biological levels.

3.  The expression of the transgene is driven by a seed specific
    promoter and the protein does not accumulate in any safflower
    tissues other than the seed. Because safflower has tightly closed
    seed heads, predation by birds is minimized.

4.  Only trained employees will perform activities related to this
    permit including planting and harvesting of the transgenic
    safflower. All activities will be conducted according to the
    procedures described in the field guide that the applicant submitted
    for APHIS' approval. This will also minimize any accidental release
    or possible animal exposure.

5.  Several field trials have been performed with transgenic safflower
    plants under APHIS authority, and APHIS is familiar with safflower
    biology and methods to manage confined safflower field trials.

6.  Safflower does not outcross with any of the plant species that are
    on the Federal list of threatened or endangered species.

This field release does not involve new species or organisms or novel
genes that raise new issues. Many field trials have been performed with
transgenic safflower plants under APHIS authority. APHIS is familiar
with the biology of safflower and methods to manage confined safflower
trials.

For the above reasons, APHIS has determined that (1) pursuant to 7
C.F.R. §372, the field trials proposed under permit #06-250-02r will not
significantly affect the physical environment and (2) there are no
applicable, extraordinary, or other reasonably foreseeable circumstances
under which significant environmental effects could occur given the
protective and ameliorative measures specified above. Therefore, this
field test is deemed confined within the meaning of 7 C.F.R. §372.5.

[]{#__RefHeading___Toc156125279 .anchor}**Appendix VI. Standard Permit
Conditions for APHIS Form 2000 (7 CFR 340.4)**

Permit conditions: A person who is issued a permit and his/her employees
or agents shall comply with the following conditions, and any
supplemental conditions 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:

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 packaging 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, and the date of
    importation.

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 follower occurrences:

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

    ii. 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:

    i.  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);

    ii. Notify APHIS promptly upon arrive of any regulated article at a
        port of entry, or 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

    iii. Mark and identify the regulated article in accordance with 7
         CFR 340.7.

Rev. 3/2003

## Appendix VII. Supplemental Permit Conditions for APHIS Form 2000 (7 CFR 340.4) {#appendix-vii.-supplemental-permit-conditions-for-aphis-form-2000-7-cfr-340.4 .unnumbered}

+--------+-------------------------------------------------------------+
| ![     | **SUPPLEMENTAL PERMIT CONDITIONS**                          |
| ](medi |                                                             |
| a/imag | **For Release of Safflower, *Carthamus tinctorius* L.**     |
| e1.png |                                                             |
| ){widt | **USDA-APHIS-BRS Permit 06-250-02r**                        |
| h="1.0 |                                                             |
| 034722 |                                                             |
| 222222 |                                                             |
| 223in" |                                                             |
| height |                                                             |
| ="0.78 |                                                             |
| 472222 |                                                             |
| 222222 |                                                             |
| 22in"} |                                                             |
+--------+-------------------------------------------------------------+

Compliance with Regulations

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 Standing 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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I.  **Reporting Unauthorized Releases and Unintended Effects**

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>
>
> **[For Washington State:]{.underline}**
>
> Barbara Chambers, Seattle
>
> Phone: (206) 592-9057
>
> Fax: (206) 592-9043
>
> Email: <barbara.a.chambers@aphis.usda.gov>

1.  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).

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

3.  

> [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

Perimeter Fallow Zone

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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II. **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 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 this planting and
    harvesting equipment 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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III. **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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IV. **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 should be
    received before facilities are returned to general use.

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V.  **Post Harvest Monitoring**

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

VI. **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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VII. **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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VIII. **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>
>
> and please cc the review biotechnologist:
> <Patricia.K.Beetham@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):**
>
> Brad White, Ph.D., Program Manager
>
> Plant Services Division\
> Washington State Dept. of Agriculture\
> P. O. Box 42560\
> Olympia, WA 98504-2560
>
> Phone: 360-902-2071
>
> Fax: 360-902-2094
>
> Email: <bwhite@agr.wa.gov>

A.  **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 numbers of acres for each gene construct.

v.  The planned planting date

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B.  **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 which will be used for food, feed, or seed
    production. A survey should be done within the distance specified in
    the permit. APHIS requires a distance of two (2) miles from any
    commercially planted safflower fields.

v.  The actual planting date.

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C.  **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.

D.  **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.
>
> **E**. **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.