USDA-APHIS

Environmental Assessment

In response to permit application (06-363-103r), received from
SemBioSys, Inc. for a field-test to produce human proinsulin (line
4438-5A) in genetically engineered safflower (*Carthamus tinctorius*)
seeds

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

Biotechnology Regulatory Services

[]{#__RefHeading___Toc161479866 .anchor}TABLE OF CONTENTS

**TABLE OF CONTENTS [2](#__RefHeading___Toc161479866)**

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)

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
[13](#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
[14](#potential-impact-on-non-target-organisms-including-beneficial-organisms-and-threatened-or-endangered-species)

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

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

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

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

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

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

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

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

Appendix I. Biology of Safflower [22](#appendix-i.-biology-of-safflower)

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

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

Appendix IV. Human Proinsulin [27](#appendix-iv.-human-proinsulin)

Appendix V. Threatened and Endangered Species Analysis
[28](#__RefHeading___Toc161479893)

Appendix VI. Standard Permit Conditions for APHIS Form 2000 (7 CFR
340.4) [33](#__RefHeading___Toc161479894)

Appendix VII. Supplemental Permit Conditions for APHIS Form 2000 (7 CFR
340.4)
[35](#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 a request for a permit (APHIS Number
06-363-103r) 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-human proinsulin
protein exclusively within its seed. This transformed safflower is
currently a regulated article under USDA regulations in 7 CFR part 340,
and as such, any field tests of transformed safflower have to be
conducted under a permit issued by APHIS.

# **INTRODUCTION**

The World Health Organization (WHO) estimates that more than 180 million
people worldwide have diabetes. This number is likely to more than
double by 2030. The increase in diabetes worldwide translates into an
estimated 8,000 to 11,000 pounds of insulin needed for the year 2005 and
is projected to increase to 35,000 pounds by 2010
(<http://www.who.int/mediacentre/factsheets/fs312/en/index.html>). This
demand is expected to grow due to earlier diagnosis, increased incidence
based on demographic trends, and consumption and behavioral habits.
Insulin use is also expected to increase from new alternative delivery
methods, including inhaled insulin devices that require between five and
ten times the amount of insulin as injection methods. Existing
commercial insulin production methods typically rely on yeast
(*Saccharomyces cerevisiae*) or bacteria (*E. coli*) genetically
engineered to produce synthetic human insulin. These organisms are grown
in large bioreactors, and the insulin is then extracted and purified for
final formulation.

SemBioSys believes that safflower-produced insulin will help meet the
growing market demands while reducing equipment and manufacturing costs
compared to current processes. The transgenic safflower seeds produced
in the proposed planting will be ground and used in the development of
techniques to extract insulin and is not for commercial production. A
technical review of human proinsulin can be found in Appendix IV.

## USDA Regulatory Authority

APHIS regulates genetically engineered safflower under the authority of
the Plant Protection Act, 7 U.S.C. 7701 *et seq.*, and USDA-APHIS
regulations in 7 CFR part 340, "Introduction of Organisms and Products
Altered or Produced Through Genetic Engineering Which are Plant Pests or
Which There is Reason to Believe are Plant Pests." A genetically
engineered organism is considered a regulated article if the donor
organism, recipient organism, vector or vector agent used in engineering
the organism belongs to one of the taxonomic groups listed in the
regulation and is also a plant pest, or if there is a reason to believe
it is a plant pest. In this submission, safflower has been genetically
engineered using disarmed *Agrobacterium tumefaciens*. *Number range
CHAPTERA. tumefaciens* is a plant pest listed under 7 CFR part 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. This planting is not for commercial
distribution, but for development of proinsulin purification techniques.

The Federal Food, Drug and Cosmetics Act (U.S.C. Title 21, Chapter 9),
addresses the FDA regulations concerning pharmaceuticals and new drugs
(as amended on December 31, 2004). Under these regulations, FDA allows
an exemption for investigational new drugs provided required information
and protocols have been submitted. SemBioSys has begun the application
process needed to apply for an Investigational New Drug (IND) with the
FDA in order to commence clinical trials for future dates.

# **PURPOSE AND NEED**

The proposed action is for APHIS, Biotechnology Regulatory Services
(BRS), to issue a permit for field-testing safflower S-317 in Lincoln
County, WA. The safflower is genetically engineered to express, within
its seeds, human proinsulin fused to an *Arabidopsis* oleosin. The
purpose of this introduction is to obtain a source of seed containing
proinsulin to be used to develop an insulin purification process. The
permit application was received by APHIS-BRS on December 18, 2006. It
was submitted by SemBioSys Genetics, Inc., West Sacramento, CA. The
application number is 06-363-103r.

APHIS has prepared this EA before making a determination on whether to
allow the planting of transformed safflower under APHIS regulations at
the request of SemBioSys. 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 U.S.C. 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

For the purposes of this Environmental Assessment, the No Action
alternative would be the denial of permit application 06-363-103r. APHIS
might choose this alternative if there were evidence to demonstrate a
plant pest risk from the cultivation of safflower engineered to express
human proinsulin within its seeds.

Under the No Action Alternative, the transgenic safflower plants will
not be planted.

## Issue the Permit: Allow planting with supplemental conditions

In this alternative, issuing this permit would allow the proposed
research trial to proceed at a field site in Lincoln County, WA (see
Appendix III for additional details). In this alternative, input from
the State of Washington and public comment from this environmental
assessment would be required. If APHIS chooses this alternative,
additional mitigating measures specified within the Supplemental Permit
Conditions (Appendix VII) would be required to prevent the persistence
of the organism outside the field production area.

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 Standard 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 site will be geographically isolated from any commercial
    safflower production fields or SemBioSys experimental safflower
    fields, ensuring no cross pollination between the GE safflower and
    food or feed safflower crops.** The location of the field site has
    been claimed CBI. Crops surrounding the field site will be barley
    and wheat with the closest commercial safflower production field
    expected to be at least 15 miles away. APHIS Supplemental Permit
    Conditions (Appendix VII) would require an isolation 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 safflower seed. The 15 mile distance between the proposed
    SemBioSys field test site and any commercial safflower fields would
    exceed the requirements specified by AOSCO and APHIS.

b.  **The field test site will be geographically isolated from any
    sexually compatible wild safflower species to ensure no cross
    pollination between the GE safflower and 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*) is only sexually compatible with the federally
    listed noxious weed known as wild safflower or jeweled distaff
    thistle (*C.* *oxyacanthus*). *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 field will be located because of the Noxious Weed
    Surveillance Program set up by APHIS Plant Protection and Quarantine
    (PPQ).

c.  **There are no apiaries within a ten mile radius of the field test
    site ensuring no pollen flow of safflower by honey bees.** 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 Lincoln
    County. No apiaries are found within a 10 mile radius of the test
    plot site. Wild bees and pollinating insects are not expected to be
    present due to insecticides used on adjacent wheat and barley fields
    to prevent thrips, wireworms, cutworms and other insect pests. Based
    on the expected absence of beehives in the vicinity of the field
    trial and the use of insecticides to control insect pests in the
    area, insect pollination is expected to be less than 10%.
    (<http://www.ipmcenters.org/CropProfiles/docs/wabarley.html>,
    <http://www.ipmcenters.org/cropprofiles/docs/IDwheat.html>).

d.  **The test plot will be surrounded by a 50 ft fallow (bare ground)
    border to facilitate the detection of any growth of transgenic
    safflower plants or volunteers that may occur as dedicated equipment
    is moved throughout 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:

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 outcrossing
    of the GE safflower with any food or feed safflower crop.

## Preferred Alternative

APHIS has chosen Alternative B as the preferred alternative based on
APHIS' scientific analysis of the permit application. This decision was
based on the following points (discussed in depth in the following
sections of this EA)

1.  The experimental test site is geographically isolated.

2.  There are no known wild relatives of safflower in WA.

3.  Safflower is not weedy and will not establish itself readily without
    human intervention.

4.  Dedicated equipment will be used during the field test.

5.  There are no commercial seed grinding processing plants in Lincoln
    County and therefore, no commercially produced safflower seed oil
    products in the area. This ensures the experimental crop will not
    accidentally be mixed with feed or food sources of safflower.

6.  The proinsulin hormone is only found in seeds and not in pollen or
    elsewhere in plant parts.

7.  There are no registered apiaries in a 10 mile radius to add to
    pollen flow if safflower is unexpectedly within a two mile radius
    from the test site.

8.  APHIS requires all transgenic material to be tracked and labeled
    after it leaves the test site for further processing, ensuring a
    chain of custody and prevention of accidental mixing into the food
    or feed supply chain.

9.  Proinsulin lacks biological activity when seeds are ingested.

# **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. Safflower 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 human proinsulin-oleosin fusion gene is a
moderate oleic oil producer (\~39% oleic oil production).

Outcrossing between safflower plants 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% cross pollination.

Wild relatives of cultivated safflower, *Carthamus creticus* and *C.
oxyacanthus,* 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. oxyacanthus* has been reported
in California (specifically in Monterey County), 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.

The field site (\<1 acre) will be located on private property in Lincoln
County, WA. This county contains 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 will grow barley and wheat. There are no bodies of
water (lakes, streams or rivers) within a 5 mile radius of the proposed
field test site. Since the human proinsulin 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).

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 heads (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 Lincoln County in WA (The closest the
Pacific Flyway comes to Lincoln County 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 nearby
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 site, 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 Calgary, Canada following SemBioSys' standard
operating procedure \[CBI, reviewed by APHIS\] and as described in BRS
Variance #06-027 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.

A more detailed description of the biology of safflower, including
genetics and out-crossing is available in Appendix I, page 22.

# **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*., 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 2 miles and up to 15 miles from any
    commercially produced safflower fields.

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 human proinsulin
    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).

Human proinsulin is not expressed in pollen or any of the vegetative
material except seeds (see Figure 1). Because it is not expressed in the
pollen, no cumulative effects of human proinsulin are expected if APHIS
chooses to allow planting and the pollen falls outside the field test
site.

![](media/image1.jpeg){width="5.999305555555556in" height="2.89375in"}

Figure 1. The proinsulin fusion protein accumulates in seed tissue only.
4438-5A refers to the proinsulin fusion safflower line and WT is the
non-transgenic safflower.

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

Movement of whole seed by animals is one way by which 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
site 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. 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 be ground up and digested and
are not expected to pass through the avian digestive tract while
remaining viable.

Any foraging animals that ingest the transgenic safflower seeds will
effectively digest the seed and proinsulin protein. Proinsulin is the
inactive form of insulin (See Appendix IV for a more detailed review on
proinsulin) and, like insulin, has no biological activity when ingested
(Crane et al., 1968). Like most peptides and proteins that animals eat
as food sources, the seed and proinsulin will be broken into amino acids
or small peptides within the stomach before they can be absorbed in the
intestine. This breakdown of protein begins in the stomach where
hydrochloric acid (HCl) denatures the protein and facilitates the action
of pepsin, the major gastric enzyme that splits the peptide bonds. Other
proteolytic enzymes (enzymes that break down protein bonds) involved in
the gastric process are trypsin, chymotrypsin, carboxypeptidase and
elastase (Zeitlin et al., 1999). In simulated digestive studies, active
insulin is completely digested in 60 minutes whereas the proinsulin seed
fusion protein is completely digested in 15 minutes (SemBioSys, permit
application data). In the unlikely event of an exclusive diet of
transgenic proinsulin safflower seeds, no effects of human proinsulin
are expected on animals due to the digestion process.

Another way that seeds could be dispersed outside the field test site is
by human error. 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. These
SOPs detail the chain of custody of the GE safflower material (labeling
and record keeping procedures) as well as procedures necessary to clean
dedicated equipment used at the field site. All site managers, farmers
and technicians have been given a copy of these SOPs and are trained in
their implementation. The SOPs, while CBI, are in keeping with the
requirements listed in the Supplemental Permit Conditions (pages 35-41).

The seeds are the only place where the gene of interest (human
proinsulin) 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 5 mile radius of any of the proposed field test
sites, it is unlikely that aquatic animals will be exposed to any human
proinsulin should GE safflower seeds be transported outside the field
test site despite strict confinement measures. Should APHIS choose
Alternative B and allow planting, APHIS has determined 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 human proinsulin 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, if at all, because transgene promoters and coding
sequences are optimized for plant expression, not prokaryotic bacterial
expression. A more detailed description of the transforming method and
the genes involved can be found in Appendix II, page 24.

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.

### 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 a detailed
description of safflower biology).

Because cultivated safflower is not described as a weedy species and
none of its sexually compatible weedy species are known to be 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

No commercially grown safflower for seed oil is grown in WA due to the
lack of processing (seed grinding) facilities. Since the proinsulin
hormone is only found in the seeds, the only way for the seeds to end up
in the food and feed supply would be for the transgenic seeds to be
inadvertently shipped to a seed processor. APHIS requires comprehensive
procedures and safeguards be implemented by SemBioSys to prevent
contamination, release, and dissemination of the regulated article
during any shipping activities. After harvest, seeds will be shipped to
a storage and processing plant owned by SemBioSys in Calgary, Canada
following SemBioSys' standard operating procedure \[CBI, reviewed by
APHIS\] and as described in BRS Variance #06-027. 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.

Dermal exposure and ingestion are not viable delivery routes of
proinsulin. The proinsulin protein is associated exclusively with the
endosperm of the seed and not found in the seed coat or other plant
parts (see Figure 1, page 11). Inhalation of endosperm seed dust from
grinding would be the only way exposure to proinsulin could occur.
SemBioSys has reported that each safflower seed contains approximately
66 μg of proinsulin-oleosin fusion protein. The literature has been
reported that proinsulin has approximately 10% biological activity of
the active hormone, insulin on *in vitro* cell culture (Kitabchi, 1970;
Yu and Kitabchi, 1973). The mean weight of 1000 safflower seeds is 32g
(about 1 oz) (Camas and Esendal, 2006) and would potentially contain 66
mg of proinsulin. That amount would translate to 6.6 mg of insulin-type
activity. It has been reported that one unit of pure insulin is 38.5 μg
(Volund, 1993). Therefore, one ounce of dust has the potential to
deliver 171 U of insulin activity. Inhaled delivery of insulin has been
method recently approved by the FDA
(http://www.fda.gov/bbs/topics/news/2006/NEW01304.html). One of the
potential problems is that only 10% of insulin delivered nasally or via
lungs is transported across the membranes, requiring large amounts to be
inhaled in order to be effective unless surfactants or adjuvants are
added (Moses et al., 1983; Hilsted et al., 1995; Hite et al., 2006).
Therefore the one ounce of seed dust inhaled will deliver 17.1 U of
insulin-type activity from the proinsulin hormone provided all the dust
is inhaled and retained. This potential route of exposure will require
SemBioSys to provide adequate respiratory protection to workers during
the processing of its transgenic safflower seeds. A more detailed
description of proinsulin can be found in Appendix IV, page 27.

Safflower has minimal insect pests and none that feed directly on the
seed; therefore, APHIS has determined there will be no impact of human
proinsulin on insects should Alternative B be chosen. APHIS has
determined there will be no impact of human proinsulin on birds or
scavenging animals that could possibly ingest seed (refer to Seed
Movement section). Due to the lack of ingested toxicity of the protein
that will be produced, APHIS concludes that if it chooses Alternative B,
there will be no significant effect on any native faunal species for
Lincoln County.

APHIS has analyzed the direct and indirect effects of Alternative B on
federally listed threatened and endangered species and proposed species
and designated critical habitat and habitat proposed for designation as
required by Section 7 of the Endangered Species Act (ESA) of 1973 (see
Appendix V). In conclusion, APHIS has determined that the proposed
environmental release described in Alternative B will have no effect on
federally listed threatened and endangered species or species proposed
for listing, and will have no effect on designated critical habitat or
habitat proposed for designation. Likewise, if APHIS chooses the no
action alternative (Alternative A) there will be no impact on non-target
organisms, and no effect on federally listed threatened and endangered
species or species proposed for listing, and will have no effect on
designated critical habitat or habitat proposed for designation.
Consequently, consultation under Section 7 of the Endangered Species Act
with the U.S. Fish and Wildlife Service will not be required.

### Potential impacts on biodiversity

Analysis of available information indicates that SemBioSys' GE safflower
containing the human proinsulin-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 human proinsulin-oleosin
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. A description of SemBioSys' agricultural
practices can be found in Appendix III, page 26.

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 15 miles away from the closest test plot site and may 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, which is 8 times the distance required to
produce certified safflower seed of 99.9% purity.

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 status 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 their 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 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# 06-363-103r. 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, Australia, Canada, China, Japan,
Korea, Philippines, South Africa, Switzerland, and 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)

# **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

**REFERENCES CITED**

Ashri, A. and Knowles, P.F. (1960) Cytogenetics of safflower
(*Carthamus* L.) species and their hybrids. Agronomy Journal 52, 11-17.

Bechtold, N., Ellis, J. and Pelletier, G. (1993) *In planta
Agrobacterium* mediated gene transfer by infiltration of adult
*Arabidopsis thaliana* plants, Vol. 316. C R Acad Sci, Paris.

Berglund, D.R., Riveland, N. and Bergman, J. (1998) Safflower Production
A-870. In. North Dakota State University Agriculture and University
Extension, Fargo, ND.

Bolinder, J., Fernlund, P., Borg, H., Arnqvist, H.J., Bjork, E., Blohme,
G., Eriksson, J.W., Nystrom, L., Ostman, J. and Sundkvist, G. (2005)
Hyperproinsulinemia segregates young adult patients with newly diagnosed
autoimmune (type 1) and non-autoimmune (type 2) diabetes. Scand J Clin
Lab Invest 65, 585-94.

Brown, J.R. (2003) Ancient horizontal gene transfer. Genetics 4,
121-132.

Camas, N. and Esendal, E. (2006) Estimates of broad-sense heritability
for seed yield and yield components of safflower (*Carthamus tinctorius*
L.). Hereditas 143, 55-57.

Carapetian, J. (1994) Effects of safflower sterility genes on the
inflorescences and pollen grains. Australian Journal of Botany 42,
325-384.

Claassen, C.E. (1950) Natural and controlled crossing in safflower,
*Carthamus tinctorius* L. Agronomy Journal 42, 381-384.

Crane, C.W., Path, M.C. and Luntz, G.R. (1968) Absorption of insulin
from the human small intestine. Diabetes 17, 625-627.

Dajue, L. and Mündel, H.-H. (1996) Safflower. *Carthamus tinctorius* L.
Promoting the conservation and use of underutilized and neglected crops.
7, pp.1-83.

Eckert, J.E. (1962) The Relation of Honey Bees to Safflower. American
Bee Journal 102, 349-350.

Ekin, Z. (2005) Resurgence of safflower (*Carthamus tinctorius* L.)
utilization: A global view. Journal of Agronomy 4, 83-87.

Galloway, J.A., Hooper, S.A., Spradlin, C.T., Howey, D.C., Frank, B.H.,
R.R., B. and Anderson, J.H. (1992) Biosynthetic human proinsulin. Review
of chemistry, *in vitro* and *in vivo* receptor binding, animal and
human pharmacology studies, and clinical trial experience. Diabetes Care
15, 666-692.

Garcia-Jacas, N., Garnatje, T., Susanna, A. and Vilatersana, R. (2002)
Tribal and subtribal delimitation and phylogeny of the Cardueae
(Asteraceae): a combined nuclear and chloroplast DNA analysis. Molecular
Phylogentics and Evolution 22, 51-64.

Gary, N.E., Witherell, P.C., Lorenzen, K. and Marston, J.M. (1977) The
interfield distribution of honey bees foraging on carrots, onions and
safflower. Environmental Entomology 6, 637-640.

Gebhard, F. and Smalla, K. (1999) Monitoring field releases of
genetically modified sugar beets for persistence of transgenic plant DNA
and horizontal gene transfer. FEMS Microbiology Ecology 28, 261-272.

Glauber, H.S., Henry, R.R., Wallace, P., Frank, B.H., Galloway, J.A.,
Cohen, R.M. and Olefsky, J.M. (1987) The effects of biosynthetic human
proinsulin on carbohydrate metabolism in non-insulin-dependent diabetes
mellitus. New England Journal of Medicine 316, 443-449.

Hilsted, J., Madsbad, S., Hvidberg, A., Rasmussen, M.H., Krarup, T.,
Ispsen, H., Hansen, B., Pedersen, M., Djurup, R. and Oxenboll, B. (1995)
Intranasal insulin therapy: the clinical realities. Diabetologia 38,
680-684.

Hite, P.F., Barnes, A.M. and Johnston, P.E. (2006) Exhuberance over
Exubera. Clinical Diabetes 24, 110-114.

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.

Kitabchi, A.E. (1970) The biological and immunological properties of
pork and beef insulin, proinsulin, and connecting peptides. The Journal
of Clinical Investigation 49, 979-987.

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.

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
of Plant Science 84, 923-934.

Moses, A.C., Gordon, G.S., Carey, M.C. and Flier, J.S. (1983) Insulin
administered intranasally as an insulin-bile salt aerosol. Effectiveness
and reporducibility in normal and diabetic subjects. Diabetes 32,
1040-1047.

Rosak, C., Boehm, B.O., Althoff, P.H. and Schoffling, K. (1988)
Biosynthetic human proinsulin, a new therapeutic compound for diabetics?
A comparative study of biosynthetic human proinsulin with biosynthetic
human insulin. Horm Metab Res Suppl 18, 16-21.

Schlüter, K., Fütterer, J. and Potrykus, I. (1995) Horizontal gene
transfer from a transgenic potato line to a bacterial pathogen (*Erwinia
chrysanthemi*) occurs if at all at an extremely low frequency.
Biotechnology 13, 1094-1098.

Seeley, T.D. (1995) The Wisdom of the Hive: The Social Physiology of
Honey Bee Colonies. Harvard University Press, Cambridge.

Tillil, H., Frank, B.H., Pekar, A.H., Broelsch, C., Rubenstein, A.H. and
Polonsky, K.S. (1990) Hypoglycemic potency and metabolic clearance rate
of intravenously administered human proinsulin and metabolites.
Endocrinology 127, 2418-2422.

USDA APHIS. (2004) Workshop on the Confinement of Genetically Engineered
Crops during Field Testing, September 13-15, 2004. In, Washington, D.C.

USFWS. (2005) Draft Recovery Plan for *Silene spaldingii* (Spalding's
Catchfly). In. U.S. Fish and Wildlife Service, Denver, Colorado, p. 121.

Volund, A. (1993) Conversion of insulin units to SI units. The American
Journal of Clinical Nutrition 58, 714-715.

Winston, M.L. (1987) The Biology of the Honey Bee. Harvard University
Press, Cambridge.

You, S. and Chatenoud, L. (2006) Proinsulin: a unique autoantigen
triggering autoimmune diabetes. The Journal of Clinical Investigation
116, 3108-3110.

Yu, S.S. and Kitabchi, A.E. (1973) Biological activity of proinsulin and
related polypeptides in the fat tissue. The Journal of Biological
Chemistry 248, 3753-3761.

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

# **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 feet 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 seed set 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 for 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.

#### Outcrossing {#outcrossing .unnumbered}

Outcrossing 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 outcrossing.

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. oxyacanthus*
(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.
oxyacanthus* 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
outcrossing).

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. oxyacanthus* 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., moderately large pollen grains, 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 meters 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, but pollen viability 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 human proinsulin
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 to develop isolation techniques for human proinsulin
for future clinical trials.

#### 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 a modified human proinsulin gene was
fused to an *Arabidopsis* oleosin gene to be exclusively expressed in
the seeds. SemBioSys has determined that the attachment human proinsulin
and other similar experimental proteins to plant oilbodies (e.g.
*Arabidopsis* oleosin gene) allows proper folding and stable
accumulation of the protein within seeds while preventing unwanted
glycosylation.

The modified human proinsulin protein has two basic amino acids removed
for added stability in plants plus eleven C-terminal amino acids. The
added C-terminal amino acids act as a protein signal that ensures the
retention of the fusion protein in the endoplasmic reticulum of the
plant seed cell and is removed in downstream processing. SemBioSys has
provided data that demonstrates that proinsulin is only produced in the
developing seeds and nowhere else in the plant.

Expression of this gene is controlled by the phaseolin promoter and
terminator sequences from *Phaseolus vulgaris* L. (common bean). The
phaseolin promoter drives the exclusively seed-specific transcription of
human proinsulin. 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 human
proinsulin.

## 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}

A field site (\<1 acre) will be located on private property in Lincoln
County, WA. The experimental plot 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. EPA registered chemical
pesticides are likely to be used to control insect pests on this crop.
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 a non-food or feed seed
grinding facility owned by SemBioSys in Canada.

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. 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. Human Proinsulin {#appendix-iv.-human-proinsulin .unnumbered}

With the growing market demand for insulin, SemBioSys has developed a
genetically engineered safflower that produces an oleosin-human
proinsulin fusion protein exclusively within its seeds. SemBioSys
believes that safflower-produced insulin will help meet the growing
market demands while reducing equipment and manufacturing costs compared
to current processes.

Proinsulin is produced in the beta cells of the pancreas and is the
precursor molecule to the active forms of insulin and C-peptide.
Proinsulin itself only has 10% of the activity of insulin and works
slower to decrease blood sugar when injected subcutaneously (Rosak et
al., 1988). Because of its longer half-life, injected human proinsulin
was initially tested as a substitute for faster acting insulin in the
late 1980s and early 1990s (Glauber et al., 1987; Tillil et al., 1990;
Galloway et al., 1992). More recently, the role of naturally existing
proinsulin levels in the blood have been used to distinguish type 1 and
type 2 diabetes in patients with newly diagnosed high blood sugar
(Bolinder et al., 2005). It has also been suggested that high levels of
proinsulin found in the blood may become an autoantigen; a protein that
the body unfavorably reacts to causing an allergic response (e.g.
inflammation) (You and Chatenoud, 2006).

Proinsulin has no biological activity when ingested. Even the task of
developing an oral insulin delivery method has been unsuccessful in the
last several decades because both insulin and proinsulin are peptides
that are easily and quickly digested. Like most peptides and proteins
that animals eat as food sources, the proinsulin within the safflower
seed will be broken into amino acids or small peptides within the
stomach before they can be absorbed in the intestine. This breakdown of
protein begins in the stomach where hydrochloric acid (HCl) denatures
the protein and facilitates the action of pepsin, the major gastric
enzyme that splits the peptide bonds. Other proteolytic enzymes (enzymes
that break down protein bonds) involved in the gastric process are
trypsin, chymotrypsin, carboxypeptidase and elastase (Zeitlin et al.,
1999). In simulated digestive studies, active insulin is completely
digested in 60 minutes whereas the proinsulin seed fusion protein is
completely digested in 15 minutes (SemBioSys, permit application data).
In the unlikely event of an exclusive diet of transgenic proinsulin
safflower seeds, no effects of human proinsulin are expected on animals
due to the digestion process.

[]{.mark}

[]{#__RefHeading___Toc161479893 .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 Lincoln County, WA. There are no listed
critical habitats for any threatened and endangered animal species in
Lincoln County, 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 06-363-103r and the analysis of the
threatened and endangered species found below, 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 negligible.

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 plants to
    exhibit 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. A
    BLAST search using the amino acid sequence of the human
    proinsulin-oleosin domains did not reveal any significant homology
    (\> 50%) to the amino acid sequence of proteins other than oleosin
    and human proinsulin 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.

```{=html}
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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).

16 U.S.C., Section 1531, Endangered Species Act (ESA) of 1973, as
amended, requires Federal agencies to utilize their authorities to
conserve listed species, and to ensure their actions do not jeopardize
the continued existence of a listed species. Section 7 (a) (2) of the
ESA states that each Federal agency shall, in consultation with and with
the assistance of the Secretary (Interior), insure that any action
authorized, funded, or carried out by such agency is not likely to
jeopardize the continued existence of a listed threatened or endangered
species, or result in the destruction or adverse modification of
designated critical habitat. Regulations are found in 50 CFR Part 402,
Interagency Cooperation Regulations.

APHIS has analyzed the effects of Alternative B on federally listed
threatened and endangered species and proposed species and designated
critical habitat and habitat proposed for designation.

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 20, 2006 showed that 9 threatened or endangered plant
species and 28 animal species exist in Washington. Of the 28 animal
species listed, only four animals potentially reside in Lincoln County,
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 \>25 miles from the field test site, so it is
unlikely that this species would be found in the area of the field
tests. Therefore, it is unlikely there would be any effect on this
threatened bird.

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 while grasses and forbs are eaten in mid- to late summer. The
pygmy rabbit is not expected to be impacted by the field test site due
to its preference for sagebrush and riparian habitats. Therefore, it is
unlikely there would be any effect on this endangered mammal.

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. Because the Columbia Whitetail
Deer would be unlikely to feed on S-317 safflower, it is unlikely there
would be any effect on this endangered mammal.

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. It is unlikely that the Gray Wolf would be
found on agricultural land under cultivation, or feed on the genetically
engineered plants if it were to enter the release site. Therefore, it is
unlikely there would be any effect on this endangered mammal.

Of the nine plants listed, only one threatened species potentially
resides in Lincoln 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

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. In addition, the land
is currently used for agriculture. Therefore, it is unlikely there would
be any effect on this threatened plant.

Based on the reasons listed below, APHIS is confident that these field
trials will not harm or have any direct or indirect 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 human
    proinsulin-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 human proinsulin
    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 proinsulin on human
    or animals. The publications that were brought up using the search
    terms allergy, allergic, allergenicity, and immunogenicity were
    decades old. These publications investigated the common allergic
    responses to bovine and porcine insulins that were administered to
    diabetics at that time.

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 #06-363-103r 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.

The proposed field sites for the confined releases of the regulated
article into the environment are in Lincoln County, WA. No federally
listed threatened or endangered species has designated critical habitat
within Lincoln County, WA. (U.S. Fish and Wildlife database
<http://criticalhabitat.fws.gov/>).

In conclusion, APHIS has determined that choosing Alternative B will
have no effect on federally listed threatened and endangered species or
species proposed for listing, and will have no effect on designated
critical habitat or habitat proposed for designation. Likewise, the
selection of Alternative A, the no-action alternative, will have no
effect on federally listed threatened and endangered species or species
proposed for listing, and will have no effect on designated critical
habitat or habitat proposed for designation. Consequently, consultation
under Section 7 of the Endangered Species Act with the U.S. Fish and
Wildlife Service will not be required.

[]{#__RefHeading___Toc161479894 .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.**     |
| e2.png |                                                             |
| ){widt | **USDA-APHIS-BRS Permit 06-363-103r**                       |
| 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 Title 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
    *et seq.*).

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.