U. S. ENVIRONMENTAL PROTECTION AGENCY

WHITE PAPER

CONCERNING DIETARY EXPOSURE TO CRY9C PROTEIN PRODUCED BY STARLINK® CORN
AND THE POTENTIAL RISKS ASSOCIATED WITH SUCH EXPOSURE

Executive Summary

	StarLink® refers to a variety of yellow corn genetically engineered to
express the protein Cry9C.  Cry9C is toxic to various insect pests of
corn and acts as a pesticide, therefore its sale and or distribution is
subject to regulation by the U.S. Environmental Protection Agency (EPA
or Agency) under the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) and the Federal Food, Drug, and Cosmetic Act (FFDCA).  Under
FIFRA and FFDCA, a company seeking to sell or distribute a pesticide
must submit data demonstrating that it will not cause unreasonable
adverse effects on the environment and that any residues in food will be
safe, i.e., that there is a reasonable certainty that no harm will
result from aggregate exposure to the pesticide chemical residue,
including all anticipated dietary exposures and all other exposures for
which there is reliable information. 

	Aventis Agroscience, Inc. (Aventis) submitted data on the safety of
StarLink® and applied for approvals under FIFRA and FFDCA.  EPA
concluded that the available data did not provide enough information to
support a conclusion that Cry9C was not a potential human allergen, but
that all other information indicated that it would not pose any other
types of risks to human health or the environment.  Accordingly, in 1998
EPA registered StarLink® for commercial use, provided that all grain
derived from StarLink® corn was directed to domestic animal feed or to
industrial uses (e.g., biofuels). The intent of requiring all StarLink®
to be segregated as either domestic animal feed or for industrial use
was to preclude any occurrence of the potentially allergenic Cry9C in
human food.  The registration contained several specific requirements
designed to ensure that no StarLink® grain entered the human food
supply.  Following registration, relatively small quantities of
StarLink® were planted in the United States: 9,018 acres in 1998,
247,694 acres in 1999, and 350,000 acres in 2000, with the largest
planting representing less than half a percent of the total acreage
planted to corn in the United States.

	In September 2000, residues from StarLink® were detected in taco
shells, indicating that it had entered the human food supply.  In
response to these detections, Aventis requested cancellation of the
StarLink® registration.  In addition, working with U.S. Department of
Agriculture (USDA), Food and Drug Administration (FDA), EPA, and the
food industry, Aventis undertook a program to remove all StarLink® from
the food supply.  Among other measures, FDA issued guidance “for
sampling and testing yellow corn and dry-milled yellow corn shipments
intended for human food use for Cry9C protein residues” that indicated
that “manufacturers who detect Cry9C-containing corn in any lot should
divert the lot to animal feed or industrial use”.

	At the same time, Aventis requested that EPA reconsider its position
that the available data did not provide enough information to support a
conclusion that Cry9C was not a potential human allergen.  Aventis
provided additional data and analysis to support its position that the
allergenic risks of Cry9C were very small.  Most of the arguments
advanced by Aventis involved the assertion that exposure to Cry9C was so
low, especially after the full implementation of the containment and
removal program, that there would be no threat to public health.  EPA
convened a meeting of its FIFRA Scientific Advisory Panel (SAP or Panel)
on November 28, 2000, to consider a series of questions concerning the
potential of Cry9C to cause a human allergic response. 

 Subsequent to the November 2000 SAP meeting, both Aventis and EPA
developed additional information and analyses.  The Agency convened
another meeting of the SAP in July 2001 to review the new information
and analyses.  Among the materials evaluated by the 2001 Panel was a
White Paper developed by EPA that described the corn wet milling process
and documented that it removes virtually all of the protein present in
corn grain from the various processed food forms produced by wet milling
for human consumption - primarily corn syrup, corn oil, alcohol, and
corn starch.  The SAP commented favorably on this White Paper in which
EPA stated that “it is reasonable to conclude that there is virtually
no Cry9C protein in wet milled products and that there is no likely
health concern for the public associated with the consumption of any
food fraction produced by wet milling of corn as long as reasonable
steps are taken to ensure that StarLink® corn is not diverted into wet
milling.”

	Following the cancellation of the StarLink® registration, Aventis
established a separate corporate entity, StarLink Logistics Inc. (SLLI),
as the successor to Aventis’ interest in StarLink® products.  SLLI
oversees the StarLink® Enhanced Stewardship Program, through which SLLI
and the U.S. corn millers have continued the efforts to contain and
remove Cry9C from the human food supply.  SLLI also maintains a
monitoring database containing the test results from more than 4 million
tests from over 4 billion bushels of corn collected by dry milling
facilities and other corn handling operations.  These tests were carried
out according to guidance developed by FDA and USDA’s Grain
Inspection, Packers, and Stockyards Administration (GIPSA), and the
federal government considers the data reliable.  

In 2005, SLLI commissioned Exponent, Inc., to prepare a new exposure
assessment of the levels of Cry9C present in the U.S. food supply for
submission to EPA.  SLLI provided supplemental information in 2006 that
updates the 2005 exposure assessment and that quantitatively
characterizes the impact of the monitoring and diversion program on
exposure to Cry9C.   The USDA’s Agricultural Research Service (ARS)
provided the analytical data on Cry9C concentrations in corn grain used
in Exponent’s exposure analysis.  In addition, the ARS provided
results from testing corn seeds from the 1970s and 1980s (that is,
before Cry9C was ever bioengineered into corn) for the possible presence
of naturally occurring Cry9C or other proteins that give a positive
reaction in the Cry9C test.  GIPSA conducted additional testing to
verify the results of the ARS laboratory.

A careful review of this information shows that the cancellation of the
StarLink® registration and the program to contain and remove StarLink®
from the corn supply have produced a dramatic decrease in the level of
Cry9C estimated to be in the human food supply – from an upper bound
estimate of 57 µg/person/day in 2000 to an estimate of high end
exposure of 0.007 µg/person/day in 2006. 

	

	Conclusion.  Based on the following lines of evidence, EPA has
concluded that, as of 2006, the potential exposure of the U.S.
population to Cry9C in the U.S. food supply is extremely low. 
Specifically, EPA finds that:

Levels of exposure to Cry9C estimated in 2006 are dramatically lower
than 2000 exposure estimates (~ 8,000 lower at the 99.9th percentile and
over 200,000 fold at the 95th percentile).

A comparison of the estimated exposures to Cry9C from all dietary
sources indicates that today they are comparable to or lower than the
upper bound estimates of exposure that could result from consumption of
corn-based food produced in 2001 by the wet-milling process. For these
estimates EPA concluded, based in part on the advice of its SAP, that
“there is no likely health concern for the public associated with the
consumption of any food fraction produced by wet milling of corn.” 
During the time frame between 2001 and 2007, there have been no reports
reliably linking allergic reactions to exposure to Cry9C, suggesting
that Cry9C at these levels is not causing allergic responses. 

A probabilistic estimation methodology assessment shows that diversion
from the human food supply of corn grain testing positive for Cry9C
would have an extremely small effect on reducing the already very low
levels of Cry9C in the food supply.  That is, very little difference in
exposure is observed between: (1) the estimated 2006 exposures that
occur when grain that tests positive for the presence of Cry9C is
removed from the food supply, and (2) the exposures that could have
occurred if such grain had not been tested and diverted but had been
allowed to remain in the human food supply. 

	While each of these lines of evidence has limitations, which are
discussed in detail in the White Paper, taken together they strongly
support a determination that testing corn grain for Cry9C at dry mills
and masa operations is unnecessary since current estimates of potential
exposure are such that there is no likely health concern for the public
associated with the consumption of corn-based food products, including
food products from the dry milled process or masa operations. 
Therefore, EPA recommends that: 

FDA withdraw its guidance for dry milling facilities and masa operations
that recommends sampling and testing yellow corn and dry-milled yellow
corn shipments intended for human food use for Cry9C protein residues. 



I.  Purpose 

	StarLink® is a variety of corn genetically engineered to express the
protein, Cry9C.  Because Cry9C was intended to be toxic to various
insect pests of corn, Cry9C and the genetic material necessary to
produce it are considered a “pesticide” and are regulated by the
U.S. Environmental Protection Agency (EPA or Agency) under the Federal
Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Federal
Food, Drug, and Cosmetic Act (FFDCA).  Cry9C from StarLink® is slow to
degrade in solutions that mimic human digestive fluids, a characteristic
that raised concerns about the potential of this protein to elicit
allergic responses in humans.  Therefore, when EPA approved the sale and
distribution of StarLink® in 1998, EPA imposed a series of conditions
designed to ensure that all StarLink® grain was directed to channels of
trade where it would be used only as domestic animal feed or for
industrial purposes.  Nonetheless, in 2000 Cry9C was detected in human
food.  This discovery led to the cancellation of the StarLink®
registration and a concerted effort to remove all grain testing positive
for Cry9C from the human food supply.  

	This paper (1) assesses the effect of those efforts on the levels of
exposure by comparing the levels of exposure to Cry9C that are occurring
today to the levels of exposure estimated to occur in 2000; (2)
discusses the significance of these levels of exposure for public
health; (3) reviews new information about analytical measurements of
Cry9C; (4) responds to public comment on its analysis; and (5) makes
public policy recommendations in light of its analysis.

This paper has eight sections.  The brief description of the purpose of
the paper in this Section is followed by a description in Section II of
the legal authority under which EPA regulates pesticides.  Section III
summarizes the state of the science used to assess the potential
allergenic risks to humans posed by the introduction of the StarLink®
protein, Cry9C, into the diet.  Section IV of the paper presents a full
description of the regulatory history of StarLink® corn and
particularly emphasizes the various scientific analyses and reviews that
were performed to evaluate the potential for risk to public health posed
by residues of Cry9C in the food supply.  This historical review
concludes with the SAP meeting in 2001, the most recent public and
external peer review of issues relating to the potential allergenic risk
of Cry9C.  Section V discusses the new information and analyses
developed since 2001, primarily by StarLink Logistics, Inc., (SLLI) and
its contractor, Exponent, Inc.  Section VI of the White Paper explains
how the information and analyses led to EPA’s conclusions on the
levels of Cry9C currently in the human food supply.  Section VII reviews
and responds to public comment.  Section VIII offers EPA’s summary
recommendation.  



II.  Legal Framework

	The Agency regulates pesticides primarily under two statutes:  FIFRA
and FFDCA.  These laws address the sale, distribution, and use of
pesticides (FIFRA) and the safety of pesticide residues in food (FFDCA).

A.  FIFRA  

StarLink® is the trade name for a variety of corn that has been
genetically engineered to produce a protein, Cry9C, that is toxic to
certain insect pests of corn.  Because the Cry9C protein was intended to
prevent, destroy, repel, or mitigate a pest, EPA considered the Cry9C
protein and the genetic material necessary for its production, to be a
pesticide under the Federal Insecticide, Fungicide and Rodenticide Act
(FIFRA).  7 U.S.C. §§ 136-136y.  (When plants have been genetically
engineered to express a pesticidal protein, the Agency refers to the
pesticide as a “plant-incorporated protectant” or “PIP.”) 
Section 3(a) of FIFRA requires that before a pesticide may be sold or
distributed in commerce, it must be registered, or licensed.  7 U.S.C.
§ 136a(a).  Pursuant to Section 3(c)(5) of FIFRA, the Agency may
register a pesticide under FIFRA only if EPA concludes, among other
things, that use of the pesticide will not cause “unreasonable adverse
effects on the environment.”  7 U.S.C. § 136a(c)(5)(C).  FIFRA
defines “unreasonable adverse effects on the environment as “. . .
any unreasonable risk to man or the environment, taking into account the
economic, social, and environmental costs and benefits of the use of any
pesticide, or a human dietary risk from residues that result from a use
of a pesticide in or on any food inconsistent with the standard in
section 408 of the Federal Food, Drug, and Cosmetic Act. . . .”  7
U.S.C. § 136(bb).  Thus, a pesticide must meet two tests in order to be
registered:  the benefit of using the pesticide must outweigh its risk,
and any residues in food or feed resulting from use of the pesticide
must meet the safety standard in FFDCA Section 408.

	B.  FFDCA

The Agency regulates the safety of any residual amounts of a pesticide
or substances resulting from the metabolism or other degradation of a
pesticide (collectively referred to as a “pesticide chemical
residue”) that occur in or on food under the Food, Drug and Cosmetic
Act (FFDCA).  21 U.S.C. § 301 et seq.  The FFDCA makes it unlawful to
sell food – a term that includes food for both humans and animals –
if it is “adulterated.”  Food is adulterated if, among other things,
it contains a pesticide chemical residue for which there is no
“tolerance” or “exemption from the requirement of a tolerance”
or if the food contains a pesticide chemical residue in excess of the
level specified in the tolerance applicable to that type of food. 
Section 408 of the FFDCA provides that EPA “may establish or leave in
effect a tolerance for a pesticide chemical residue in or on a food only
if the Administrator determines that the tolerance is safe.”  21 U.S.C
§ 346a(b)(2)(A)(i).  Pursuant to Section 408, “safe” is defined  to
mean that “there is a reasonable certainty that no harm will result
from aggregate exposure to the pesticide chemical residue, including all
anticipated dietary exposures and all other exposures for which there is
reliable information.”  21 U.S.C. § 346a(b)(2)(A)(ii) or
346a(c)(2)(A)(ii).  The FFDCA also allows EPA to establish an exemption
from the requirement of a tolerance for a pesticide chemical residue if
EPA determines that such residue would be safe.  21 U.S.C. §
346a(c)(2)(A)(i). When EPA establishes an exemption for the requirement
of a tolerance for a pesticide chemical residue, a food may contain any
amount of that residue.  The Agency generally establishes tolerances and
exemptions for specific pesticide chemical residues and specific types
of food.  

III.  Scientific Considerations Regarding Allergenicity for the Initial
StarLink® Decision  

While plant-incorporated protectants generally promise a potentially
more benign means of pest control than conventional chemical pesticide
alternatives, as part of the risk assessment conducted under FIFRA and
FFDCA, EPA must assess whether any protein portion of the PIP could pose
an allergenic risk to humans.  Beginning with the first field tests of
crops genetically engineered to express new traits, the three federal
agencies involved in agricultural biotechnology oversight, the United
States Department of Agriculture (USDA), the Food and Drug
Administration (FDA) and EPA, have sought the most current scientific
input on allergenicity through workshops and other public meetings. A
1994 conference in Annapolis, MD on Scientific Issues Related to
Potential Allergenicity in Transgenic Food Crops led to the development
of an understanding of how sensitization develops into food allergy and
the development of a list of features characterizing food allergenic
proteins.  More recently, the U. S. has participated in the development
in the Codex Alimentarius of guidelines for assessing the food safety of
foods derived from biotechnology, including the assessment of potential
allergenicity of newly expressed proteins in recombinant-DNA plants. 

	A food allergy is a reaction of the immune system to an otherwise
harmless food or food component.  Reactions can have a highly variable
clinical presentation from as little as mild itching or rash to
anaphylactic shock.  All allergens are proteins or chemical sensitizers
that interact with proteins.  In the development of food allergy, a
sensitization phase is required where exposure to a substance primes the
immune system to react.  If sensitization occurs, subsequent exposures
to the same substance at sufficient concentrations can then elicit an
allergic response.  Only a limited number of foods are known to cause
the majority of food allergies, and many of the major food proteins
responsible for these reactions have been described.  

There are no definitive methods to assess potential allergenicity of
proteins originating from sources not known to produce food allergy. 
There are no toxicology tests in the harmonized guidelines used for
pesticide registration to evaluate food hypersensitivity reactions. In
addition, there is no established animal testing system to evaluate
proteins for potential food allergenicity in humans.  However, there are
some recognized procedures that can be used to evaluate a new protein to
assess its allergenic potential.  These procedures include evaluating
the similarity of the new protein to known food allergens with respect
to amino acid sequence and examining its biochemical characteristics
such as molecular size, resistance to enzymatic and acid degradation,
and heat stability.  These procedures together form the basis for
evaluating whether a given protein is likely to be, or become, an
allergen.  Even known food allergens do not always share all these
characteristics, however, and no one feature is predictive.  Therefore,
allergenicity assessments, including EPA’s, consider a suite of
characteristics and use a weight of the evidence approach for the safety
determination.

	Until the application for registering Cry9C in StarLink® from AgrEvo
(subsequently merged into Aventis CropScience), all the registered
plant-incorporated protectants reviewed by EPA were proteins that had
appeared in microbial pesticides, had some previous dietary exposure,
and had no similarities or shared biochemical features to known food
allergens.  While there was no amino acid sequence similarity between
Cry9C and known allergens, the company had submitted studies that
indicated Cry9C had stability to gastric digestion and heat denaturation
and an animal study that suggested the possibility of Cry9C
allergenicity.  In order to proceed with the registration process, EPA
issued an exemption from the requirement of a tolerance for feed uses of
StarLink®, incorporated into the registration strict terms and
conditions requiring that Aventis take specific measures to ensure that
StarLink® corn would be segregated from the human food supply, and
prepared for a meeting of its FIFRA Scientific Advisory Panel (SAP or
Panel) to address questions about digestive stability and human
allergenicity.  (The SAP is an advisory committee, chartered under the
Federal Advisory Committee Act, composed of independent, external
experts in the science of assessing the risks of pesticides.)

IV.  History of the Scientific Evaluation and Regulation of StarLink®

A.  Registration & Tolerance Exemption for StarLink® 

In 1998, EPA received an application to register StarLink® corn under
FIFRA and a petition to establish an exemption from the requirement of a
tolerance for Cry9C and the genetic material necessary to produce it
under the FFDCA from AgrEvo. In 1998, following a thorough scientific
review of the safety of StarLink®, EPA concluded that it was unable to
determine whether Cry9C would elicit an allergic response in humans. 
The Agency did determine, however, that aside from this unresolved human
allergenicity issue, StarLink® would pose no other risks to public
health or the environment, including a finding that there was no risk to
humans from eating food from animals that had consumed StarLink® corn.

Because StarLink® appeared to offer benefits in controlling important
corn pests and the risks of using StarLink® were limited, the Agency
concluded that StarLink®’s benefits outweighed the risks of its use
– if its use was conditioned to preclude the occurrence of Cry9C in
the human food supply.  Therefore, in 1998 EPA issued a registration for
StarLink® to AgrEvo.  EPA limited the registration by requiring that
all grain and other products derived from StarLink® corn be used only
in domestic animal feed and for industrial purposes. EPA did not approve
the use of StarLink® corn in foods destined for human consumption
because of unanswered questions about the potential allergenicity of
Cry9C.  As part of its approval of the registration of StarLink®, the
Agency imposed specific terms and conditions on AgrEvo (and its
successor, Aventis) that were intended to ensure that the registrant and
growers would take appropriate steps to preclude the presence of Cry9C
in the human food supply.

Consistent with its regulatory decisions on StarLink® under FIFRA, in
1998 the Agency established an exemption from the requirement of a
tolerance for the residues of Cry9C, and the genetic material necessary
to produce it, in domestic animal feed (40 CFR 180.1192).  Because the
exemption did not apply to food products intended for human consumption,
the presence of Cry9C from StarLink® in human food products would make
such food adulterated.

Aventis began to market StarLink® commercially in 1998, and farmers
bought and planted small but increasing amounts of StarLink® corn in
1998, 1999, and 2000.  The following table provides information on the
amount of StarLink® planted in these years:

Table 1:  Corn Acreage Planted to StarLink® by Year

Year	Acres of StarLink®	Percent of U.S. Corn

1998	9,018	0.01

1999	247,694	0.32

2000	350,420	0.43



The degree to which StarLink® corn penetrated the commercial seed
market varied by locality.  The highest penetration recorded in any year
in any state was 1.5% of the corn planted.

B.  February 2000 SAP Meeting

Because of Aventis’ continuing interest in obtaining approval for use
of StarLink® grain in the production of human food and the novel
scientific issues raised concerning the assessment of potential
allergenicity, EPA called a meeting of the FIFRA SAP on February 29,
2000, regarding Cry9C.  The February 29, 2000, SAP report stated that,
with the data available, the SAP could not determine whether or not
Cry9C is an allergenic protein.  Specifically, “[t]he Panel agreed
that based on the available data, there is no evidence to indicate that
Cry9C is or is not a potential food allergen.” Later the Panel’s
report stated “The question that must be addressed is what additional
data should be collected to assess if Cry9C or any other protein [in]
plant-pesticides is a potential food allergen. The level of the allergen
in the environment would be useful for its potential as an inhalant or
ingestant allergen. Exposure levels are important[.]” 

C.  2000 Cancellation of StarLink® and Initiation of Efforts to Remove
StarLink® from the Grain and Seed Supply

In September 2000, cry9c DNA was detected in a finished food product for
human consumption – taco shells. Subsequently, the cry9c DNA and Cry9C
were found both in corn grain and in other corn products in the human
food supply. These detections demonstrated that, despite the EPA
restrictions, some quantities of StarLink® had directly entered the
human food chain.  On September 26, 2000, Aventis discontinued sale of
StarLink® for the 2001 growing season and three days later announced an
agreement with EPA, FDA, and USDA to locate and contain StarLink® and
direct it to approved uses under USDA oversight. The program
implementing this agreement is known as the StarLink® Enhanced
Stewardship (SES) Program. On October 12, 2000, Aventis requested that
the registration for their StarLink® corn product be voluntarily
cancelled. Upon EPA’s acceptance of the cancellation request in 2000,
it became unlawful to sell or distribute StarLink® seed for planting. 
In addition, USDA, EPA, and FDA worked with the National Corn Growers
Association, the American Seed Trade Association, and individual seed
companies to institute a testing program to ensure that corn seed for
planting in 2001 and future years would be free of Cry9C.  

D.  November 2000 SAP Meeting

On October 25, 2000, Aventis submitted to EPA an amendment to its
pending petition for Cry9C in human food.  This amended petition,
submitted under the FFDCA, requested a time-limited (four years)
tolerance to cover residues of Cry9C that could be present in human food
made from StarLink® corn planted in 1998, 1999, and 2000. Aventis
submitted a variety of additional information with its petition to
support its contention that Cry9C posed no allergenic risk to public
health. 

EPA convened an SAP meeting on November 28, 2000, to consider a series
of questions concerning the potential of Cry9C to be a human allergen,
whether there was an amount of the protein in corn sufficient to cause
sensitization, and to what levels of Cry9C the public might be exposed
if this time-limited tolerance were to be approved. Among other things,
EPA prepared a series of “bounding estimates” of the amount of Cry9C
exposure possible for different population groups.  EPA’s estimate
indicated that for the general U.S. population, 99.9% of the population
would have an exposure lower than 57 µg/person/day.  It should be noted
that EPA’s assessment indicated that, because of different food
consumption patterns, members of some subgroups appeared to receive
higher exposure than the general U.S. population, but EPA lacked data
adequate to develop reliable estimates of exposure above the 99th
percentile of exposure for these subgroups.  More information, including
the EPA documents submitted for SAP review, other background
information, and the SAP final reports can be found on the following web
site: http://www.epa.gov/oscpmont/sap/meetings/2000/index.htm#112800.

E.  Report from the November 2000 SAP Meeting

The November 28, 2000, SAP concluded that “there is a medium
likelihood that the Cry9C protein is a potential allergen based on the
biochemical properties of Cry9C protein itself . . .  .”   (Emphasis
in original.)  The November 2000 Panel report cited the February 29,
2000 Panel report and found that Cry9C had multiple characteristics
associated with known allergens.  While noting that these
characteristics were not considered “definitive evidence,” the Panel
concluded that, in light of these factors “[t]aken together, . . .
Cry9C protein has a medium probability of being a potential allergen.”
 

At the same time the Panel also concluded that the extent of StarLink®
in the human food supply at that time had a “low probability to
sensitize some individuals to Cry9C protein.”  (Emphasis added.)  This
conclusion rested on three factors: the expression level of Cry9C in
corn, the amount of StarLink® corn expected to have entered the human
food supply, and the fact that StarLink ®grain was expected to be
commingled with grain that did not contain Cry9C. The Panel further
stated “lowering the levels of Cry9C in the food supply makes
sensitization less probable.”  

The Panel’s November 2000 report also concluded that “the
methodology used by the Agency to estimate exposure is defendable.” 
The SAP also noted, however, that the “Agency’s analysis results in
an upper bound estimate that is considerably high and could justifiably
be reduced if [EPA factored several considerations cited by the SAP into
its estimate.]”  The Panel noted that a more refined estimate of
exposure would take into account:  (1) a more realistic approach to
assessing the extent and impacts of blending StarLink® grain with
non-StarLink grain; (2) data from actual monitoring programs to detect
StarLink® in grain intended for the human food supply; and (3) the
impacts of processing on the likelihood that Cry9C would be present and
detected in different, corn-based, processed food products.  The Panel
indicated that the levels of Cry9C present in such processed foods would
vary considerably, depending especially on the method of processing and
whether the product was from white or yellow corn. (The cry9c DNA was
only engineered into certain yellow corn varieties.)  The SAP report
observed that items produced by “wet milling” of corn grain – such
as corn syrup, corn oil, and corn starch – contain virtually no
protein and therefore would contain virtually no Cry9C.

In conclusion, the SAP responded to EPA’s question about the overall
probability of the risk of significant allergic reactions to Cry9C as
follows:  

“The Panel assessed the currently submitted data and concurred, while
not conclusive, that the likely levels of Cry9C protein in the U.S. diet
of [sic] provide sufficient evidence of a low probability of
allergenicity in the exposed population.  

“This conclusion was based on taking [into] consideration several
factors:

1.  The moderate allergenicity prediction of the protein.

2.  The low levels of protein expression in corn products.

3.  The low levels of estimated exposure in the diet.

4.  The accepted conservative approach used by the Agency to estimate
exposure levels.

5.  The epidemiological data from workers and consumer surveys are not
conclusive for allergenicity of Cry9C.

6. The clinical responses reported to date have been inconclusive in
establishing allergic reactions to Cry9C.”

Finally, the Panel observed that “[i]f corn-derived food products
contain a highly potent allergen then allergic reactions should appear
within a few years.”

F.  USDA & FDA Guidance Documents on Testing Corn Grain for Cry9C

In response to concern from the milling and food processing industries,
during the months following the SAP meeting both the USDA and the FDA
developed guidance documents addressing how private entities could
reduce the chance that StarLink® grain would be used in the production
of human food.

First, in December 2000 the USDA Grain Inspection, Packers, and
Stockyards Administration (GIPSA) issued guidance containing a protocol
for sampling and testing corn grain to detect the possible presence of
Cry9C.  This guidance reported that USDA GIPSA had validated two
different test kit systems for detecting the presence of Cry9C in corn
grain – the TraitCheck Bt9 Lateral Flow Strip (LFS) Test from
Strategic Diagnostics, Inc., and the Cry9C QuickStixTM Test Kit from
EnviroLogix, Inc.  The GIPSA guidance also recommended using the test
kits as part of a specific sampling and testing strategy that would
detect the presence of StarLink® grain 99% of the time in a sample that
contained more than 0.19% StarLink® grain. This is equivalent to a
limit of detection (LOD) for Cry9C in the range of 20 parts per billion
(ppb).

Also in December 2000, FDA issued draft guidance to corn dry-milling and
masa operations recommending that they perform testing to detect the
possible presence of Cry9C in yellow corn and, in certain circumstances,
milled yellow corn (i.e., corn meal or corn flour) intended for human
food use.  The guidance advised corn dry-milling and masa operations to
analyze representative samples from every incoming yellow corn shipment.
 FDA also recommended that corn dry milling and masa operations screen
stored inventories of corn or milled corn (meal or flours), if incoming
corn shipments or milled corn produced from those shipments had not been
screened on arrival.  The FDA guidance advised private entities
conducting such testing to follow the analytical procedures described in
the USDA GIPSA guidance  NOTEREF _Ref177364391 \h  \* MERGEFORMAT  8 . 
In the event that a sample tested positive for the presence of Cry9C,
the FDA guidance indicated that the lot from which the sample was taken
should be diverted to animal feed or industrial use (e.g., the
production of ethanol).  

G.  EPA White Paper on Wet Milling of Corn

Field corn that is made into processed foods for human consumption first
undergoes milling. There are two primary types of milling: “dry
milling,” which primarily produces corn flour and corn meal, and
“wet milling,” which primarily produces high fructose corn syrup,
corn oil, corn starch, some animal feed products, and ethanol.  As the
November 2000 SAP noted, depending on the food fraction, the wet milling
process removes all or nearly all of the protein in corn.  In contrast,
dry milling processes do not remove protein from products intended for
human consumption.  (Not surprisingly, all of the food products in which
Cry9C had been detected contained dry-milled processing fractions. No
samples of food containing only wet milled food fractions have tested
positive using the analytical method recommended by FDA and USDA GIPSA.)


In March 2001, EPA issued a White Paper in response to the November 2000
SAP recommendation to examine the impact of wet milling on the levels of
Cry9C potentially present in human food made from StarLink® grain.  The
Agency’s White Paper described the wet milling process and documented
that it removes virtually all of the protein present in corn grain from
the various processed food forms produced for human consumption,
primarily corn syrup, corn oil, alcohol, and corn starch.  Data showed
that corn protein will not be present in corn syrup, corn oil, or
alcohol, and that corn starch only contains approximately 0.01% protein.
 Moreover, Cry9C comprised a very small part of the overall protein
content of StarLink® corn grain (approximately 0.13%), and StarLink®
represented only a small proportion of the total corn planted in 1998,
1999 or 2000, the only years StarLink® was commercially grown (see
Table 1).  Therefore, the White Paper concluded that there would be, at
most, extremely low amounts of Cry9C in food products made from wet
milling of StarLink® corn.  As StarLink® was removed from the corn
supply, even these low levels would fall until there would be
essentially no Cry9C in the processed food for human consumption derived
from wet milling.  

The 2001 EPA White Paper contained upper bound estimates of the levels
of daily exposure to Cry9C that different population groups (the U.S.
population, infants, children aged 1-6 years, and children aged 7-12
years) might experience in 2000 as the result of consuming corn starch. 
Based on this analysis, the highest exposure value estimated was for the
general U.S. population in 2000 with 99.5% of the population estimated
to have an exposure lower than 0.019 µg/day.  

Based on the foregoing analysis, EPA concluded that “it is reasonable
to conclude that there is virtually no Cry9C protein in wet milled
products and that there is no likely health concern for the public
associated with the consumption of any food fraction produced by wet
milling of corn as long as reasonable steps are taken to ensure that
StarLink® corn is not diverted into wet milling.”  

H.  July 2001 SAP Meeting

The Agency convened another meeting of the SAP on July 17-18, 2001 to
review new information and analyses developed subsequent to the November
2000 SAP meeting.  The new information included:  

data on the impact of processing and cooking on the levels of Cry9C in
various types of finished foods made from corn; 

a description of the Aventis – USDA program to purchase StarLink®
corn and to direct such corn to permissible uses; 

a description of the efforts of corn handlers, millers, and food
processors to ensure that corn grain was being tested for the possible
presence of Cry9C and that quantities testing positive are redirected
away from the human food chain; 

data on the presence of antibodies in individuals who reported adverse
effects following alleged exposure to StarLink®; and 

reports from the medical community on individuals who claimed to have
experienced adverse effects following alleged exposure to StarLink®.

Additional analyses included:  

The 2001 EPA White Paper on the effects of the wet milling process on
human exposure to Cry9C; and

Aventis’ revised assessment of exposure to Cry9C in light of the steps
taken to prevent further amounts of StarLink® from entering the food
supply.

	The Aventis revised assessment estimated the 99th percentile of
exposure to StarLink® derived Cry9C in the diet was 0.39 µg/person/day
for the general U.S. population  NOTEREF _Ref177365391 \h  \*
MERGEFORMAT  11 .  Consistent with the SAP’s advice in its November
2000 report, this revised estimate reflected several improvements:
actual measurements of Cry9C in finished foods, other data measuring the
effects of processing, inclusion of corn starch as a potential source of
protein, and the assumption that all grain containing more than 0.125%
StarLink® was being successfully diverted from the human food supply. 
Aventis characterized its assessment as being conservative, i.e., likely
to overestimate potential exposure.

I.  July 2001 SAP Meeting Report

The SAP reviewed the materials described above and addressed a series of
questions from the Agency that asked for scientific comment on the 2001
EPA White Paper, the Aventis exposure assessment, EPA projections about
levels of StarLink® in the corn supply in future years, and the
implications of this and other new information on the potential
allergenic risks Cry9C might pose in the human food supply.

The SAP commented favorably on EPA’s White Paper describing the levels
of Cry9C exposure that could result from consumption of food fractions
derived from the wet milling of corn. Their report stated that “[t]he
Panel concluded EPA used a reasonable approach in determining human
dietary exposure from corn starch produced from Cry9C protein containing
corn via the wet milling process.”  The Panel noted that if grain
handlers and millers conduct the recommended screening of lots in the
corn supply, they should rarely, if ever, process corn containing more
than 0.125% StarLink®  NOTEREF _Ref177365717 \h  12 .  If incoming
grain is assumed to contain 0.125% StarLink® instead of EPA’s
assumption of 1.5% StarLink® corn, the estimated exposure to Cry9C
protein from consumption of corn starch for the 99.5th percentile of the
general U.S. population would be 0.0013 µg/person/day, i.e.,
approximately 10 fold lower than EPA’s initial estimate of 0.019
µg/person/day. 

The SAP also commented on the Aventis exposure assessment, which they
described as “similar to that used by EPA in calculating exposures to
Cry9C protein from corn starch consumption, except for the multiple
sources of exposure that substantially complicated the analysis and that
carried some important implications for the final result.”  The Panel
questioned the accuracy of the values used by Aventis in the assessment
for Cry9C in finished foods.  Because Aventis used data from processing
studies, as measured by an Enzyme-Linked ImmunoSorbent Assay (ELISA)
methodology developed by EnviroLogix, the SAP had concerns that the
method might underestimate the amount of Cry9C actually present in the
processed food fractions.  As a result the Panel concluded
“[e]stimated upper bound exposure generated by use of Cry9C protein
content of corn intermediates rather than the available finished food
data could be several fold greater than the 0.37 µg/day presented in
the revised Aventis dietary exposure assessment.”  Offsetting this
consideration, the Panel noted that the assumption that grain contained
0.125% StarLink® was “highly conservative,” in view of the
documented effectiveness of the StarLink® containment program. 
Finally, the Panel noted that there is considerable variability in the
amount of corn-derived ingredients used in recipes for the same food,
e.g., corn meal used to make corn bread.  The Panel suggested such
variability could change estimated exposure by as much as 50%.

In response to an EPA question about the levels of Cry9C likely to be
detectable in future years, the SAP stated, “The impact of 1998 and
1999 production on Cry9C protein levels in current U.S. grain stocks is
small and rapidly diminishing. With continued testing under the GIPSA
protocol, redirection of grain testing positive for Cry9C protein,
producer control of volunteer corn occurrences, and removal of seed
testing positive for Cry9C protein, EPA estimates that Cry9C protein
will essentially be gone from corn grain in 2 to 3 years and from
finished food products made from such corn in 4 to 5 years.  The Panel
concurs, although trace amounts of cry9c DNA may be detectable far
beyond these time frames.”  (Citation omitted.)  In general the Panel
complimented the past and ongoing efforts of the government and the
private sector to contain StarLink® and recommended that they continue.

The Panel reviewed information from FDA and the Centers for Disease
Control (CDC) that followed up on incident reports made by individuals
who had experienced physical symptoms that they thought might have been
caused by eating foods containing Cry9C.  The Panel agreed with the
government scientists’ assessment that the information was
inconclusive, i.e., that it failed to establish any link between
exposures to Cry9C and allergic responses.  The Panel recommended that
the federal government continue to monitor incident reports and
investigate any for which such an association appeared possible.

Finally, the Panel addressed the implications of the new information for
understanding potential allergenic risks to the U.S. population.  The
SAP agreed there was no reason to change the earlier conclusion that
“there is a medium likelihood that the Cry9C protein is a potential
allergen based on the biochemical properties of Cry9C protein itself –
not its levels in food.”  The Panel also affirmed the earlier
conclusion regarding the probability of an allergenic risk:  “the
likely levels of Cry9C protein in the U.S. diet provide sufficient
evidence of a low probability of allergenicity in the exposed
population.”  While the SAP was aware that new estimates suggested
exposure to Cry9C could be lower than previously predicted, the Panel
raised several questions about the basis of the new estimates. 
Specifically, the Panel pointed out possible limitations in the
analytical methodology used to measure Cry9C levels in finished food
products and the fact that there was no exposure estimate for infants
who consume a high corn content diet.  

As part of its comments on the potential allergenic risks of Cry9C in
the diet, the Panel discussed the ability to estimate a threshold level,
below which an allergic response was not expected to occur.  The SAP
summarized its thinking as follows:

“. . . the Panel could not determine a threshold level of Cry9C
protein where there would be a reasonable scientific certainty that
exposure would not be harmful to public health.  No reliable data are
available on threshold levels of isolated food proteins for inducing
allergic response in highly sensitive individuals.  Thus, the Panel
concluded that based on reasonable scientific certainty, there is no
identifiable maximum level of Cry9C protein that can be suggested that
would not provoke an allergic response and thus would not be harmful to
the public.”

J.  EPA’s July 27, 2001 Note to Correspondents

Concurrently with the release of the July 2001 SAP report, EPA issued a
statement in a “Note to Correspondents” in which it characterized
several of the report’s key conclusions as follows:  

“The Panel also stated that, since there is inadequate information to
establish a reasonable scientific certainty that exposure would not be
harmful to public heath, they could not recommend establishing a
specific tolerance level for StarLink.”  Accordingly, EPA announced
that “establishing a tolerance for StarLink in human food is not
currently supported.” 

“[T]he Panel endorsed EPA’s conclusion that the process of
wet-milling corn removes virtually all of the StarLink protein (called
Cry9C) from products made for human food.  This supports the Agency’s
determination that there is no public health risk from eating products
manufactured from StarLink corn through the wet-milling process,
provided that corn utilized in the wet-milling process does not contain
significant levels of StarLink.” 

“The SAP agreed with EPA estimates that the Cry9C protein in domestic
corn supply will decline rapidly after the 2001 crop is harvested and
with each subsequent production year.”

V.  Information Generated Since 2001 on StarLink®

Since the 2001 SAP Meeting, EPA has received several studies and
analyses addressing Cry9C.  These studies and analyses include: results
from the SLLI Quality Check Program (QCP), assessments of dietary
exposure to Cry9C by Exponent, Inc., (a contractor of SLLI) in 2005 and
2006, a report from USDA’s Agricultural Research Service (ARS) Market
Quality and Handling Research laboratory in Raleigh, NC on testing corn
grain samples pre-dating StarLink®, a 2007 report from a GIPSA
Technical Services Division laboratory on retesting of selected samples,
and a “tumbled sample” experiment performed in 2007 by the GIPSA
laboratory.

A.  Ongoing Results of the SLLI Quality Check Program (QCP)

In October 2000 Aventis initiated the StarLink Enhanced Stewardship
Program to purchase corn that was determined to contain StarLink®
grain.  Once purchased, Aventis arranged for the grain to go to domestic
animal feed or industrial uses  NOTEREF _Ref177452138 \h  \* MERGEFORMAT
 13 .  Since 2000 and continuing to the present, Aventis and its
successor entity, SLLI, have provided LFS testing kits to dry milling
facilities and other grain handling operations so that they could, as
recommended by FDA, conduct testing on shipments of corn to detect the
possible presence of Cry9C.  Over 400 facilities have participated and
have conducted over 4 million tests on over 4 billion bushels of grain. 


The results represent the most comprehensive database on the levels of
any substance in the food supply.  The database shows detection of Cry9C
in a shipment of corn grain becoming an increasingly rare event as
measured by the LFS test methodology: in the testing period from
September 2004 to August 2005, 99.96% of all grain sampled tested
negative, and in the testing period from October 2005 to September 2006,
99.99% of all grain sampled tested negative.  In contrast, in April and
August 2001 only ~ 90.5% of the grain tested was negative.  From October
2005 through September 2006, positives represented approximately 0.01%
of the approximately 70,000 tests using the LFS test conducted each
month  NOTEREF _Ref175642037 \h  \* MERGEFORMAT  16 .  This means that
the percentage of samples testing positive for Cry9C has declined 950
fold since 2001.  The results of testing collected by SLLI from January
2000 through September 2005 are depicted graphically in Figure 1 (from
Exponent 2005 report).  The data obtained since September 2005 show
further declines in detection frequency. 

In addition, individual seed companies producing seed corn for planting
in years after the cancellation of StarLink® have reported to EPA that
they have conducted testing of their seed lines to ensure they were free
of Cry9C.  While the Agency does not have access to the results of the
seed companies’ tests, it seems apparent from the overall low
frequency of detection of Cry9C in grain in recent years that new corn
seed planted after 2000 contains at most minute amounts, and, possibly,
no StarLink® derived Cry9C.  

B.  Exponent’s 2005 and 2006 Assessments of Dietary Exposure to Cry9C 

In November 2005, Exponent, a contractor for SLLI, prepared an analysis 
NOTEREF _Ref175642241 \h  \* MERGEFORMAT  15  estimating the levels of
exposure to Cry9C in the U.S. food supply.  The Exponent 2005 assessment
estimated consumer exposure by utilizing the results of monitoring of
Cry9C residues in the US corn supply by government and industry using
the LFS test on samples collected “in market” as corn is stored,
shipped, and/or milled; additional confirmation and quantification
studies performed by the USDA ARS using the ELISA plate test; and food
consumption surveys for the U.S. population.  EPA, USDA, and FDA have
reviewed the Exponent analysis and conclude that it provides a reliable
basis for characterizing the distribution of potential exposure to Cry9C
for the general U.S. population and significant subgroups during 2005.  

The Exponent analysis presents a distribution of potential exposures
based on a probabilistic estimation methodology that takes into account
the fact that any individual’s exposure depends on two independent
factors:  (1) how much Cry9C (if any) is present in food derived from
yellow corn, and (2) how much food derived from yellow corn the person
consumes.  The following paragraphs provide a brief description of
Exponent’s exposure analysis.

As the first step in estimating levels of Cry9C in food, Exponent
created a representative distribution of Cry9C in the corn grain supply
based on:

(1) The QCP database, a compilation of the results of extensive
screening for the presence of Cry9C in the human grain supply, provided
some three hundred samples testing positive for Cry9C in the field from
March 2004 to September 2005: The USDA ARS laboratory in Raleigh, NC
reanalyzed samples in order to confirm and quantify the amount of Cry9C
using both the LFS test (LOD ~ 20 ppb) and the more sensitive ELISA
plate test (detection threshold = 0.0655 optical density units,
corresponding to about 0.1 ppb); and 

(2) The Federal Grain Inspection Service (FGIS) of USDA GIPSA collected
more than a thousand one hundred corn samples from mid-April through
mid-August 2005 from a variety of locations throughout the corn
industry: The USDA ARS laboratory in Raleigh, NC analyzed these samples
using both the LFS test (LOD ~ 20 ppb) and the more sensitive ELISA
plate test (detection threshold = 0.0655 optical density units,
corresponding to about 0.1 ppb).

Exponent then divided the resulting data into six categories
(“distributions” in the Exponent report) as follows:

Table 2:  Description of “Distributions” for Exponent’s Exposure
Assessment

Distribution	Field Test Using Lateral Flow Strip	Laboratory Test Using
Lateral Flow Strip	Laboratory Test Using ELISA Plate Test

1	n.a.*	–	–

2	n.a.*	–	+

3	+	–	–

4	+	–	+

5	+	+	–

6	+	+	+

*Not available but assumed to be negative for purposes of Exponent’s
analysis.

Corn in distribution 1 is assumed to test negative in the field for
Cry9C using the LFS test, would test negative in the laboratory when
retested for Cry9C using the LFS test and the more sensitive ELISA plate
test.  

Corn in distribution 2 is assumed to test negative in the field for
Cry9C using the LFS test, would test negative with the LFS test in the
laboratory but positive for Cry9C when retested in the laboratory using
the ELISA plate test.

Corn in distribution 3 would test positive in the field for Cry9C using
the LFS test, negative for Cry9C when retested in the laboratory using
the LFS test and negative for Cry9C when retested in the laboratory
using the ELISA plate test.

Corn in distribution 4 would test positive in the field for Cry9C using
the LFS test method, negative when retested in the laboratory using the
LFS test but positive for Cry9C when retested in the laboratory using
the ELISA plate test.

Corn in distribution 5 would test positive in the field for Cry9C using
the LFS test, positive for Cry9C when retested in the laboratory using
the LFS test, but negative for Cry9C when retested in the laboratory
using the ELISA plate test.

Corn in distribution 6 would test positive in the field for Cry9C using
the LFS test, positive when rested in the laboratory using the LFS test,
and positive for Cry9C when retested in the laboratory using the ELISA
plate test.

The likelihood grain would produce a positive in a LFS test under field
conditions was based on the sampling results from the QCP for the time
period, September 2004 through August 2005.  In order to confirm
positive results, samples testing positive in the QCP were sent for
laboratory analysis at the USDA ARS laboratory in Raleigh, NC.  These
samples underwent both a new round of the LFS test under laboratory
controlled conditions, as well as an ELISA plate test.  These testings
allowed for the identification of any false positives in the field
testing and quantification of the amount of Cry9C present in positive
samples, since the LFS test method only provides a qualitative answer
regarding the presence of Cry9C at or above the LOD of ~ 20 ppb. The
results of these tests were used, as shown in Figure 2, to characterize
the levels of Cry9C in “distributions” 3 through 6.



Figure 2 (from 2005 Exponent Report):  Derivation of Exponent’s
Distributions 3-6*

   Distribution 3	Distribution 4	Distribution 5	Distribution 6

*QCP = Quality Check Program; LOD = Limit of Detection; ND = non-detect;
U indicates that the Exponent assessment assumed Cry9C residues were
uniformly distributed between the indicated upper and lower limits.

The second source of samples was the USDA GIPSA Federal Grain Inspection
Service (FGIS).  FGIS randomly collected, in the period from mid-April
to mid-August 2005, over 1100 samples of corn grain from throughout the
U.S.  These samples were shipped to the USDA ARS laboratory at Raleigh,
NC.  ARS analyzed them in the laboratory using the LFS test and the more
sensitive ELISA plate test to characterize the levels of Cry9C in grain.
 Samples were collected from locations and at a time of the year that
would enhance finding Cry9C if it were present in the market.  All
samples tested negative by the LFS test.  For these ELISA plate tests
conducted by ARS, the LOD for Cry9C was ~ 0.11 ppb.  The amounts
detected ranged from the LOD up to 2.73 ppb.  These results were used,
as shown in Figure 3, to characterize the levels of Cry9C in
“distributions” 1 and 2. 

Figure 3 (from 2005 Exponent Report): Derivation of Exponent’s
Distributions 1-2* 

	Distribution 1		Distribution 2

*FGIS = USDA Federal Grain Inspection Service; LOD = Limit of Detection;
ND = non-detect.

The estimated proportion of the corn grain supply for each distribution
is shown in Table 3. For each of the distributions, Exponent assigned a
residue value or range of values to reflect the amount of Cry9C likely
to be present in grain derived from that distribution.



Table 3:  Proportion of Corn Supply Assigned by Exponent to
Distributions and Residue Values of Cry9C in 2005 Exponent Assessment

Distribution 	% Occurrence in Corn Supply 	Residue value  

1 	88.569% [= 99.96% x 88.6%]	All values = 0 ppb

2 	11.391% [= 99.96% x 11.4%]	Values = empirically observed values in
2005 testing  Mean = 0.26 ppb, Min = 0.10 ppb, Max = 2.73 ppb

3 	0.008% [= 0.04% x 75% x 26.1%]	Values = a uniform distribution from 0
ppb to the LOD for the ELISA plate test, where LOD ranges from 0 to
0.156 ppb

4 	0.022% [= 0.04% x 75% x 73.9%]	Values = empirically observed values
in 2005 testing  Mean = 7.6 ppb, Min = 0.14 ppb, Max = 18.3 ppb

5 	0.000% [= 0.04% x 25% x 0%]	No samples fell into this distribution
(see Figure 2); all samples testing positive in the field and laboratory
LFS tests also tested positive in the ELISA test and were assigned to
distribution 6.

6 	0.010% [= 0.04% x 25% x 100%]	Values = empirically observed values in
2004-05 monitoring database (Mean = 53.8 ppb, Min = 5.3 ppb, Max = 96.4
ppb), supplemented by an exponential “tail” (Because of the limited
number of data points in the database, Exponent supplemented the
empirical values to address the possibility that the food supply
potentially contained some grain with higher levels of Cry9C than the
maximum (96 ppb) seen.)



	

Exponent then estimated the levels of Cry9C in food products commonly
consumed by humans (e.g., hush puppies, corn bread, polenta, tortillas,
taco shells, corn meal and flour) by adjusting the values obtained above
with data showing how the level of Cry9C potentially present in the raw
corn grain would be affected by the processing used to produce the food.
 These processes include the “masa” processing and the blending,
grinding, intense cooking, and extruding processes used to produce
corn-based cereals.  

To estimate consumption of corn-derived food, Exponent used a USDA
database on food consumption in the United States, the Continuing Survey
of Food Intakes by Individuals (CSFII), and a second database developed
jointly by EPA and USDA, the Food Consumption Intake Database, that
contains recipes for calculating the amounts and forms of constituent
agricultural commodities (corn oil, corn meal, sugar, etc.) that are
present in various types of finished foods (e.g., cornbread), which
people report in the CSFII having eaten.  Exponent’s approach to
estimation of food consumption of corn-derived food follows standard EPA
estimation methodology.

	Exponent used the data described above to develop a probabilistic
exposure assessment for dietary exposure to Cry9C.  A probabilistic
exposure assessment is designed to capture the variability in exposure
that can result from the fact that each person’s daily intake of Cry9C
will depend on multiple factors: which foods the person eats, how much
of the specific foods he or she eats, and how much (if any) Cry9C is
present in the different foods. Because there are a huge number of
possible combinations of foods and residues, the variability is assessed
using a computer program that randomly combines different diets with
different residue levels.  The computer program performs tens of
thousands of estimates combining the randomly selected values to
generate a large distribution of potential exposure values.  Because
multiple individual exposures comprise the distribution, it is possible
to estimate the specific level of exposure received by a particular
percentile of the population.  The results of Exponent’s assessment
are shown in Table 4.  

Table 4:  Exponent’s 2005 Dietary Intake Estimates of Cry9C Protein
(µg/person/day)

Population Group	Mean	95th Percentile	99.5th Percentile	99.9th
Percentile

U.S. Population	0.00007	0.00005	0.00303	0.00821

U.S. Children, 1 – 6 	0.00005	0.00004	0.00210	0.00577

U.S. Children, 7 – 12 	0.00006	0.00007	0.00246	0.00677

Hispanic Population 	0.00006	0.00006	0.00233	0.00642



	Exponent’s assessment uses a number of assumptions that can affect
the resulting exposure estimates.  To illustrate the effect different
assumptions might have on the exposure estimate, Exponent performed four
analyses, substituting one alternative assumption in each while holding
the other assumptions constant:   

Assumption substitution 1. As noted in Table 3, Exponent assigned a
uniform or empirical distribution for each of the “distributions”
except for distribution 6 for which they used both empirical data and an
exponential tail. Exponent conducted a sensitivity analysis making an
alternative assumption, i.e., that all of the observed residue values in
distributions 2, 4, and 6 were part of a single, lognormal distribution.
 Rather than assuming the residue values were as described in Table 3,
Exponent used modeling techniques to generate the best estimate of a
single, lognormal distribution for Cry9C residues. Fitting the data to a
lognormal distribution would likely overestimate the amount of Cry9C in
the food supply. Using this assumption and keeping other assumptions
constant, Exponent estimated exposures as seen in Table 5.

Table 5:  Exponent’s 2005 Dietary Intake Estimates of Cry9C
(µg/person/day), Assuming Residues are Lognormally Distributed

Population Group	Mean	95th Percentile	99.5th Percentile	99.9th
Percentile

U.S. Population	0.000178	0.000269	0.0070821	0.023896



The values in Table 5 are 2.5 fold (mean); 5.4 fold (95th percentile);
2.3 fold (99.5th percentile); and 2.9 fold (99.9th percentile) higher
than the estimates reported in Table 4 for the corresponding
percentiles.  A comparison of the exposures estimated for the other
population subgroups shows a similar range of differences. 

Assumption substitution 2.  In the original assessment, it was assumed
that there were no detectable residues of Cry9C in cereals due to the
extensive blending and mixing involved in their preparation.  Exponent
performed a sensitivity analysis assuming that some residual level of
Cry9C could be found in processed cereals.  For this analysis, Exponent
assigned a Cry9C level of 0.036 ppb to all cereals.  This level was
determined as the weighted average of the Cry9C levels from the six
distributions in the 2005 report.  All other assumptions were kept
constant.  Exponent exposure estimates under this scenario are presented
in Table 6.

Table 6.  Exponent’s 2005 Dietary Intake Estimates of Cry9C
(µg/person/day), Assuming Some Residual Level of Cry9C Would be Found
in Cereals  NOTEREF _Ref175539029 \h  23 

Population Group	Mean	95th Percentile	99.5th Percentile	99.9th
Percentile

U.S. Population	0.000205	0.000999	0.003386	0.008526



Assumption substitution 3.  Extensive mixing of corn occurs as it moves
from the farm to elevators to mills and finally to food processors.
Through storage, tempering, multiple grinding/sifting operations,
transfer to storage bins, further processing into retail products, there
are at least 7 to 8 distinct points of dilution during the entire voyage
from field to end-user. An estimate of the commingling /dilution factor
for grain at one specific dilution point, e.g., in an elevator, is on
the order of 3 to 5 times, while dilution at the mill is probably much
greater.  In the Exponent estimate leading to Table 4, only a single
occasion of commingling, with 5 fold dilution, was incorporated into the
assessment.  The process of commingling and the potential for “hot
spots” was addressed by dividing the distribution of Cry9C levels into
10 strata representing the 10 deciles of the distribution before
adjustment for dilution and commingling.  This approach is a worst case
estimate in that it forms composite samples within strata that have
similar levels, and thus is not likely to form composite samples from
corn with low Cry9C levels with corn with high Cry9C levels.  Exponent
performed a sensitivity analysis assuming no commingling; such an
assumption is likely to result in an overestimate of exposure.  Keeping
all other assumptions constant, the exposure estimates resulting from
this analysis are presented in Table 7. 

Table 7.  Exponent’s 2005 Dietary Intake Estimates of Cry9C
(µg/person/day), Assuming No Commingling of Grain  NOTEREF
_Ref175539029 \h  23 

Population Group	Mean	95th Percentile	99.5th Percentile	99.9th
Percentile

U.S. Population	0.000069	0.00003	0.002677	0.008257



Assumption substitution 4.  In the original assessment, corn testing
negative with the LFS test in the field and subsequently testing
negative with the ELISA plate test was assigned a 0 ppb Cry9C level
(distribution 1).  Exponent performed a sensitivity analysis in which
distribution 1 was assigned a uniform distribution ranging from 0 ppb to
0.156 ppb, the highest LOD seen with the ELISA plate test. All other
assumptions were kept constant.  The exposure estimates resulting from
this analysis are presented in Table 8.

Table 8.  Exponent’s 2005 Dietary Intake Estimates of Cry9C
(µg/person/day), Assuming a Uniform Distribution in Distribution 1 of
Cry9C Ranging from 0 ppb to 0.156 ppb  NOTEREF _Ref175539029 \h  23 

Population Group	Mean	95th Percentile	99.5th Percentile	99.9th
Percentile

U.S. Population	0.000207	0.00106	0.004543	0.009298



As can be seen from Tables 5 through 8, all these analyses produced
exposure estimates at or below the upper bound exposure estimate from
wet-milled corn products alone (i.e., less than 0.01959 µg/person/day).

 In EPA’s view, most of the other assumptions in Exponent’s
assessment tend either to overstate somewhat or to have no effect on the
estimate of potential exposure.  The assumptions and the manner in which
they tend to bias Exponent’s exposure assessment are discussed in
Table 9.



Table 9:  Effect of Key Assumptions on Exponent’s 2005 Exposure
Assessment

Exponent Assumption	Effect of Assumption

Different assumptions regarding the choice of residue values for each
distribution (see Table 3).	Compared to an assumption that all residues
are part of a single, log normal distribution, understates potential
exposure by < 3 fold at the highest percentiles.

All grain testing positive for Cry9C  remains in the human food supply.
If anything, slightly overstates potential exposure. 

The results of the ELISA assay at low levels reflect levels of Cry9C
produced by StarLink®.	If some residues in corn grain detected in the
2005 USDA FGIS survey (distribution 2) are not attributable to Cry9C
from StarLink®, the Exponent estimates would overstate the potential
exposure to Cry9C.

Commingling of grain occurs at a single point and results in a mixing of
each shipment with 5 other shipments of comparable size.	An assumption
of no commingling yields similar exposure assessments.  Assuming greater
rates of commingling would likely yield somewhat lower estimates of
exposure at higher percentiles.

Heat processing (e.g., cooking) does not reduce levels of Cry9C. 
Overstates exposure to the extent heat processing destroys Cry9C.

Processing of breakfast cereal destroys all Cry9C.	Assuming breakfast
cereals retain low levels of Cry9C yields similar exposure estimate.  

Samples assigned to distribution 1 contain 0 ppb Cry9C. 	If the
distribution of residues in distribution 1 samples was assumed to be
uniform and to range between 0 and the LOD of the ELISA plate test, the
mean would be three fold higher and 99.9th percentile 10% higher (0.008
vs. 0.009 µg/person).



	In December 2006  NOTEREF _Ref175642037 \h  \* MERGEFORMAT  16 ,
Exponent updated its estimates of exposure to Cry9C in the diet, relying
on data collected from October 2005 through September 2006 from the
Quality Check Program.  These data showed the frequency of positive test
results using the LFS test on samples of grain in the corn transport and
processing system declined from 0.04% to 0.01%.  Based on these data,
Exponent adjusted the percentages in Table 3 in the various
distributions to reflect the less frequent detection of Cry9C in field
LFS tests, and using the same assumptions and methodology as it employed
in the 2005 assessment, estimated dietary exposure.  The results of the
updated assessment appear in Table 10.  A comparison of Table 10 with
Table 4 shows a decrease in dietary exposure in all percentiles of all
population groups.



Table 10:  Exponent’s 2006 Dietary Intake Estimates of Cry9C
(µg/person/day)

Population Group	Mean	95th Percentile	99.5th Percentile	99.9th
Percentile

U.S. Population	0.00006	0.00005	0.00288	0.00747

U.S. Children, 1 – 6 	0.00004	0.00004	0.00199	0.00535

U.S. Children, 7 – 12 	0.00005	0.00007	0.00234	0.00597

Hispanic Population 	0.00004	0.00006	0.00217	0.00589



	C.  Exponent’s 2006 Analysis of the Influence on Human Dietary
Exposure of Continued Monitoring and Diversion of Lots Testing Positive
for Cry9C

	At EPA’s suggestion, in 2006 Exponent performed additional analysis
to assess the effect on exposure of the ongoing program to test corn
grain for the presence of Cry9C in order to divert all lots testing
positive to domestic animal feed or industrial uses.  Exponent executed
this assessment by evaluating the effect on the exposure estimates of
omitting from the analysis all samples testing positive in the field. 
Table 11 incorporates the estimates resulting from this analysis with
the 2006 exposure estimates of Table 10.  Because Table 10 estimates the
amount of Cry9C that may be present in the corn transport and processing
system prior to diversion  NOTEREF _Ref175548428 \h  \* MERGEFORMAT  19
, this comparison essentially offers an estimate of the effect of
diversion on human dietary exposure.  The comparison shows that
estimated exposures should not be substantially increased if testing and
diversion were to be terminated.  For example, for the 99.9th percentile
of the U.S. population, Exponent’s analysis estimates exposure could
increase by 0.000185 µg/person/day if testing and diversion ceased.



Table 11:  Comparison of Exponent’s 2006 Dietary Intake Estimates of
Cry9C Assuming Lots Testing Positive are Diverted with Exponent’s 2006
Dietary Intake Estimates of Cry9C Assuming Lots Testing Positive Remain
in the Food Supply (µg/person/day)

Population Group	Mean	95th Percentile	99.5th Percentile	99.9th
Percentile

U.S. Population (Without diversion)	0.00006	0.00005	0.00288	0.00747

U.S. Population   (With diversion)	0.000054	0.000048	0.002863	0.007285

U.S. Children, 1 – 6 (Without diversion)	0.00004	0.00004	0.00199
0.00535

U.S. Children, 1 – 6 (With diversion)	0.000037	0.000034	0.001936
0.005135

U.S. Children, 7 – 12 (Without diversion)	0.00005	0.00007	0.00234
0.00597

U.S. Children, 7 – 12 (With diversion)	0.000047	0.000066	0.002296
0.005685

Hispanic Population (Without diversion)	0.00004	0.00006	0.00217	0.00589

Hispanic Population (With diversion)	0.000042	0.000057	0.002167	0.005955



D.  Results of Monitoring for StarLink® Since August 2005

The data from SLLI’s ongoing Quality Check Program confirm that levels
of Cry9C in corn grain continue to diminish.  As noted above, the
percentage of grain testing positive using the analytical method
recommended by FDA and GIPSA (three 800 kernel samples tested using the
LFS test) in the 12 month period from October 2005 through September
2006 is only 0.01%, compared to 0.04% positive grain for the period
September 2004 through August 2005.  In addition, GIPSA continues to
fulfill requests to analyze corn grain for the presence of StarLink®
using the analytical method recommended by FDA and GIPSA and reports the
results of its testing data monthly.  GIPSA has not had a positive test
since April 2005.  Like the QCP program, these results indicate that
detection of Cry9C in samples is increasingly rare.

E.  Results of Testing Corn Grain Samples Pre-Dating StarLink®

	In addition to the results discussed above, SLLI supplied EPA with the
results of testing performed by the USDA’s ARS Laboratory in Raleigh,
NC.  ARS analyzed several samples of corn seed for the possible presence
of Cry9C using the ELISA plate test, EnviroLogixTM  QuantiPlateTM Kit
for Cry9C High Sensitivity Protocol.  ARS tested 31 breeding lines of
corn developed in the 1970s and early 1980s, well before research and
development began on StarLink® or any other varieties of genetically
engineered corn, and stored from that time by a corn breeder.  For each
of the 31 lines, five seeds were taken and tested by ARS (total of 155
kernels).  Two separate aliquots were analyzed from each single kernel
sample. Of the 155 kernels, 135 single kernels registered an optical
density above the background level, but only 16 single kernels (~ 10%)
produced results greater than the LOD of ~ 0.52 ppb in at least one of
the aliquots. Of these 16, there was only one sample in which both
aliquots produced results greater than the limit of detection (1.07 ppb
and 1.19 ppb). When the results of the two aliquots were averaged for
each of the 16 seed samples, the results suggested Cry9C levels up to
5.22 ppb, with 14 of the seeds containing less than 2 ppb.  However, in
most cases, the two aliquots gave highly variable (and, therefore,
suspect) results.  For example, one seed produced 10.11 ppb in one
aliquot and 0.33 ppb in another. Other examples of variable aliquot
pairs include: 2.99 and 0.14 ppb; 5.03 and 0.16 ppb; 1.45 and 0.02 ppb.

	Neither ARS nor EPA can definitively explain these results.  Since the
corn seeds were grown before StarLink® was first created, it seems
impossible that an ELISA plate test is measuring Cry9C produced by
StarLink®.  EPA has considered three other possible explanations.
First, there is some suggestion that variation among reagents and
materials is affecting the results. It is also possible that at this
level of sensitivity, the ELISA plate test is detecting the presence in
corn samples of other substances that react with Cry9C antibodies in the
ELISA plate test, yielding apparently positive results.  Third, these
old samples of corn could contain trace background levels of naturally
occurring Cry9C because some strains of the natural bacterium Bacillus
thuringiensis, which are natural components of plant surfaces, have the
gene for Cry9C and can express this protein in certain circumstances.
Neither the ELISA plate test nor the LFS test is able to distinguish
between StarLink® Cry9C protein and Cry9C protein from other sources. 
Any of these possibilities could result in a “false positive”
reading for Cry9C in corn seed grown before StarLink® was created.

	In an attempt to better understand these results, EPA asked GIPSA to
retest seeds from the pre-StarLink seed lots that generated positive
ELISA plate test results in the ARS laboratory.  Using the same
Envirologix high sensitivity ELISA protocol with an empirically
determined LOD lower than that of the ARS laboratory, GIPSA was unable
to verify the earlier findings of the ARS laboratory on the seeds.  None
of the seeds GIPSA tested were positive for Cry9C above the estimated
LOD.  

At this point, it is uncertain why GIPSA was unable to verify the ARS
testing data for pre-StarLink corn seeds.  Possible explanations include
the following: 

Since both ARS and GIPSA conducted tests on individual seeds, ARS may
have tested seeds that could give a positive ELISA reading while GIPSA
tested seeds that did not contain material that could give a positive
reading (sample variability).  

The ARS laboratory may have been using faulty reagents (e.g., poor
quality ELISA plates) or had a source of sample contamination (e.g.,
StarLink® derived Cry9C inadvertently introduced into the test sample),
or observed a reaction between other protein(s) present in the sample
(not StarLink® Cry9C) with the antibodies of the ELISA plate test
(cross-reaction). 

F.  Retesting Grain Samples Used to Estimate the Level of Cry9C in
Exponent’s Distribution 2

	Some of the data on Cry9C residues generated by the ARS laboratory with
ELISA plate testing and used by Exponent in its 2005 exposure analysis
(i.e., to characterize distribution 2) might be interpreted as showing
that low levels of Cry9C may have been missed on test and retest with
the LFS assay, thus raising the question of whether as much as 11.4% of
the corn grain supply could contain anywhere from 0.10 ppb to 2.73 ppb
Cry9C.  Because of the variability observed at the LOD for the ELISA
plate test, EPA asked the GIPSA laboratory to retest these samples in
order to better understand the limitations of the ELISA plate test
analytical method and the frequency with which Cry9C occurs in the grain
supply. 

	Using the ELISA plate assay, the GIPSA laboratory retested 158 survey
samples  NOTEREF _Ref175632762 \h  \* MERGEFORMAT  26  originally tested
by the ARS laboratory in Raleigh, NC and used to characterize
distribution 2.  All the samples were assessed by the GIPSA laboratory
to be negative for the presence of the Cry9C protein, since none of the
values obtained were above the LOD for the ELISA plate method. As noted
above, the LOD for the ELISA plate test in the GIPSA laboratory was
lower, and therefore able to detect for lower levels of Cry9C, than the
LOD for this same test in the ARS laboratory. 

G.  Tumbled Sample Experiment: Can Cry9C be detected in non-StarLink®
corn after it has been tumbled overnight with StarLink® corn?

To determine whether the handling (mixing, moving, pouring, etc, of the
grain supply system, e.g., in the grain elevator) of non-StarLink® corn
admixed with small amounts of StarLink®  corn could produce dust or
fragments that could be responsible for data indicating the presence of
Cry9C (i.e., distribution 2), GIPSA conducted a “tumbled sample”
experiment  NOTEREF _Ref175632762 \h  \* MERGEFORMAT  26 .  This
experiment examined whether tumbling non-Cry9C kernels spiked with
either 1% or 10 % StarLink® kernels could generate a Cry9C positive
test with the ELISA plate test once the individual StarLink® kernels
were removed.  GIPSA was able to show that while the 10% mixture did
generate positive ELISA plate test results, the 1% mixture did not 
NOTEREF _Ref175632762 \h  \* MERGEFORMAT  26 .  Given that under current
commercial conditions where positive tests for StarLink® represent less
than 0.01% of all tests and those that do test positive indicate very
low levels, admixtures of StarLink® and non-StarLink® kernels are
highly unlikely to occur at levels as high as 1% StarLink®.  These
results demonstrate that residual intact StarLink® kernels are unlikely
to be the source of dust or fragments that might result in detectable
Cry9C below 20 ppb on the ELISA plate test. This increases the
probability that one of the other reasons discussed in Unit V.H. of this
White Paper may explain the variability of results observed with the
ELISA plate test at or near the LOD. 

 

H.  Interpretation of Test Results at or near LOD

Variability in test results at or near the LOD for Cry9C for both the
LFS test and ELISA plate test appears to be a consistent feature of the
testing that formed the basis of the Exponent 2005 study  NOTEREF
_Ref175642241 \h  \* MERGEFORMAT  15  and the tests on single corn
kernels  NOTEREF _Ref175644524 \h  \* MERGEFORMAT  25 ,  NOTEREF
_Ref175632762 \h  \* MERGEFORMAT  26  grown prior to the development of
StarLink®.  This variability is expressed in both qualitative (positive
or negative for the presence of Cry9C) and quantitative (how much Cry9C
is present) terms.  Qualitative differences can be observed for example:

In the Exponent 2005 study, of the 1132 USDA FGIS samples that tested
negative for Cry9C using the LFS test, 129 (11.4%) retested positive in
the USDA ARS laboratory using the more sensitive ELISA plate test, with
a mean of 0.26 ppb.  (See Figure 3 of this White Paper). Of these 129
samples, 73 samples were positive in the two aliquots tested for each
sample, while 56 of the 129 samples tested positive in one aliquot and
negative in the second aliquot.  One hundred fifty-eight of the 1132
samples were retested by the USDA GIPSA laboratory using the ELISA plate
test. All of the retested samples gave negative results. 

Of the QCP samples used in the 2005 Exponent report (see Figure 2 of
this White Paper) that tested positive in the field using the LFS test
(341), retesting a portion of them (56) resulted in 75% (42 of the 56)
producing a negative result for the LFS test. When a subset (23) of the
42 that produced a negative LFS test result were assayed using the more
sensitive ELISA plate test, 6 produced a negative result and 17 a
positive result with a mean of 7.6 ppb. 

  

In the ELISA plate test analysis of single corn seeds grown prior to the
development of StarLink®  NOTEREF _Ref175644524 \h  25 , the USDA ARS
laboratory obtained 16 positive results for Cry9C.  Fifteen of these
samples tested positive in one aliquot taken from a single sample but
negative in the other, while one tested positive in both aliquots.  The
average reading for all 16 samples was 1.36 ppb.  Ten of the single seed
samples were retested by the USDA GIPSA laboratory using the ELISA plate
test and all retested negative for Cry9C  NOTEREF _Ref175632762 \h  26 .


Examining the quantitative results in these studies also reveals
variability.  Quantitative differences can be observed, for example,
between the two aliquots that are routinely taken of a single sample for
the ELISA plate test:

In the ELISA plate testing of the USDA FGIS samples for the Exponent
2005 study  NOTEREF _Ref175648461 \h  30 , the two aliquots for each
sample testing positive could vary by as much as 2.73 ppb (average
difference 1.51 ppb, LOD ~ 0.125 ppb). 

In the ARS laboratory ELISA plate testing of the samples for the single
seed analysis  NOTEREF _Ref175644524 \h  25 , the two aliquots for each
sample could vary by as much as 9.78 ppb, with an average difference of
2.29 ppb (LOD ~ 0.4 ppb).

These observations indicate a degree of both qualitative and
quantitative variability that would dictate caution in interpreting
readings near the LOD for Cry9C of the LFS test and the LOD of the ELISA
plate test .

In addition, the Agency also considered whether reproducible positives
at very low levels between the LOD for the ELISA plate test and the LOD
for the LFS test are actually detecting Cry9C from StarLink®, or
whether the elevated optical density readings can be attributed to other
sources, i.e., Cry9C from naturally occurring Bacillus thuringiensis, a
cross-reacting protein (e.g., Cry1F), or some other source of elevated
optical density readings (e.g., laboratory contamination or faulty
reagents).  To address these issues, the Agency considered the following
two questions:

What is the likelihood that positive readings in the range between the
LOD of the LFS test (~ 20 ppb) and the ELISA plate test (~ 0.115 ppb)
could be due to StarLink® derived Cry9C?

What are other potential sources of positive readings in the range
between the LOD of the LFS test (~ 20 ppb) and the LOD of the ELISA
plate test (~ 0.115 ppb)?

1) What is the likelihood that positive readings in the range between
the LOD of the LFS test (~ 20 ppb) and the ELISA plate test (~ 0.115
ppb) could be due to StarLink® derived Cry9C?

There are three possible sources of StarLink® derived Cry9C in current
grain stocks that could result in readings in the range between the LOD
for the LFS test and the LOD of the ELISA plate test.  The first is from
actual StarLink® kernels from corn planted before 2001 still lingering
in grain storage or transport systems.  The second source would be from
grain dust or other types of contamination containing fragments of
StarLink® corn.  The third possibility would be if the StarLink® cry9c
gene introgressed into other corn lines.  Available information casts
doubt on each of these possible explanations. 

If kernels of StarLink® corn still lingered in the grain transport or
storage systems, and a single kernel was present in an 800 kernel sample
tested by the ELISA plate test, the level detected should be
approximately 20 ppb, given Cry9C residues in a StarLink® kernel
average approximately 13,000 ppb.  The levels detected in the ARS and
GIPSA laboratories are well below 20 ppb, suggesting that the positive
readings are not due to a whole kernel of StarLink® corn.

The results of GIPSA’s 2007 “tumbled sample” experiment described
in section V. G. of this White Paper addressed the question of whether
dust or other residues could contribute to data indicating detection of
Cry9C at a mean of 0.26 ppb in 11.4% of samples that first tested
negative in a LFS test, as reported in the 2005 Exponent report  NOTEREF
_Ref175642241 \h  \* MERGEFORMAT  15 .  GIPSA conducted its “tumbled
sample” experiment to simulate conditions found in the corn grain
handling system by tumbling non-StarLink® kernels spiked with either 1%
or 10% StarLink® kernels overnight, removing all the StarLink® kernels
and then testing with the ELISA plate test for the presence of Cry9C. 
GIPSA was able to show that while the 10% mixture did generate positive
ELISA plate test results, the 1% mixture did not. This outcome indicates
that in order for dust or residue from StarLink® kernels that are
residual in the grain system to be the source of the positive Cry9C
readings below ~ 20 ppb, the amount of StarLink® in the grain supply
would have to be above 1%.  The 2005 and 2006 Exponent reports and the
GIPSA follow-up testing suggest it is highly unlikely on both a
qualitative and quantitative basis that StarLink® comprises 1% of the
grain supply.  No StarLink® corn has been harvested since the 2000
growing season and extensive efforts have been used since then to remove
StarLink® from the human corn grain supply.  Even if such dust were
originally present, as the years pass, it becomes increasingly unlikely
that such possible sources of contamination can occur in quantities
sufficient to be detected by the ELISA plate test. Rather, such residues
would decrease over time as they are carried out of the system by
non-StarLink® corn moving through the grain storage and transport
system.  

	With regard to the possibility of the cry9c gene introgressing into
other corn lines, any Quality Assurance/Quality Control procedures
conducted by seed companies should further diminish the likelihood of
cry9c gene introgression being a significant source of Cry9C in corn
grain. Normally, seed companies conduct their own internal seed testing
to meet labeling and other regulatory requirements and market needs. In
addition to standardized testing protocols involving germination and
varietal purity, tests may also be conducted for the presence or absence
of a particular trait or genotype. These tests may include protein
detection (e.g., LFS, ELISA plate) or DNA (e.g., polymerase chain
reaction or PCR) tests.

2) What are other potential sources of positive readings in the range
between the LOD of the LFS test (~ 20 ppb) and the LOD of the ELISA
plate test (~ 0.115 ppb)?

 

In addition to considering whether the positive readings in the ELISA
plate test below ~ 20 ppb in corn grain can be attributed to StarLink®
kernels, fragments, or dust, EPA also evaluated other possible
explanations.  Specifically, EPA looked at whether the results could be
attributed to:

Contamination or variability of test reagents,

Presence of other proteins that can cross-react with Cry9C antibodies in
the ELISA test, and

Naturally occurring Bacillus thuringiensis expressing the cry9c gene.

Potential contamination or variability of test reagents.  Interpreting
the results of any test, including the ELISA plate test for field corn
samples tested for Cry9C derived from StarLink®, at or near the
estimated LOD can be challenging and the interpretations involve
significant uncertainty.  Some of the challenge and uncertainty may be
attributed to the fact that small variations in reagent production and
protocol execution can substantially affect the results observed with
very sensitive assay systems at their LOD.  It is extremely difficult,
if not impossible, to remove such variability from any assay system.
Within the range where analytical variability is observed for a
particular testing system, e.g., at or near the LOD of the LFS test and
the LOD of the ELISA plate test for Cry9C, a positive result does not
definitively demonstrate that the substance that the assay is testing
for is present.  

Presence of other proteins that can cross-react with Cry9C antibodies in
the ELISA test.  EnviroLogix’ literature for the QuantiPlateTM Kit for
Cry9C test kit indicates there can be some cross-reactivity with at
least four other Cry proteins (Cry1Ab, Cry1C, Cry2A, Cry1F).  While
three of these are detectable only at tens of thousands of ppb, Cry1F is
detectable at levels of 66 ppb and legally present in corn commercially
available today. EPA has established a tolerance exemption for Cry 1F,
and therefore the presence of Cry1F in corn would not cause the corn to
be adulterated.  It is unclear from the detection kit product insert if
any other proteins potentially present in corn may cross-react and
potentially interfere with the test for Cry9C.  Further, from analytical
method validation work at the EPA Environmental Science Center at Fort
Meade, MD, there are suggestions that it may be difficult to obtain a
corn sample completely free of Cry proteins.  This would suggest that
cross-reactive proteins could be common in the grain supply.  However,
without additional testing to establish the identity of the proteins
interacting with the ELISA plate test antibodies targeted at Cry9C, it
would not be possible to determine whether the protein interacting with
the Cry9C-targeted antibodies was indeed Cry9C.

Naturally occurring Bacillus thuringiensis expressing the cry9c gene. 
Cry9C is a protein that is produced by a naturally occurring bacterium,
Bacillus thuringiensis.  It has been demonstrated that naturally
occurring B. thuringiensis variants (i.e., not from commercially
available insecticidal preparations) can be isolated from the surfaces
of living plants,.  Indeed, some scientists have argued that the
observed population densities of B. thuringiensis on plant surfaces
suggest that individuals of this species should be considered part of
the natural microbial flora on the plant surface  NOTEREF _Ref174515206
\h  \* MERGEFORMAT  40 .  In addition, one of the richest sources of B.
thuringiensis variants is grain storage and processing mills, and a
higher percentage of B. thuringiensis isolates from North American maize
dust are toxic to lepidopterans than to other insects,,.  The original
natural source of the isolate that provided the cry9c gene was grain
dust in the Philippines.  Thus, it is possible that pre-StarLink® seeds
may have tested positive at these very low levels for Cry9C because of
the natural presence on the kernels of B. thuringiensis producing Cry9C
or a protein(s) cross-reactive with the Cry9C antibodies used in the
ELISA plate test.  Naturally occurring Cry9C from sources such as
Bacillus thuringiensis bacteria would not be subject to FIFRA and the
tolerance requirements of FFDCA section 408.  Therefore, it is important
in assessing the reliability of the testing method to consider whether
it could be detecting lawful, naturally occurring substances, instead of
an unlawful pesticide chemical residue.

In sum, it would appear that caution should be exercised when
interpreting ELISA plate test data when optical density readings suggest
very low levels of Cry9C.  Consideration should be given to the LOD of
the testing method and known analytical variability for each testing
method at or near the LOD.  Consideration should also be given to
whether there may be other proteins in the sample that may cross-react
with the antibodies in the ELISA test for Cry9C to give a positive
reading.  Given the above analyses, when positive results are obtained
for Cry9C at or near the LOD for a particular test method, confirmatory
retesting would be prudent. 

VI.  Conclusions

	The foregoing review leads EPA to conclude that the potential for
exposure of the U.S. population, and significant subgroups in the
population, to Cry9C in the U.S. food supply is currently so low that
continued testing by grain handlers and millers of yellow corn grain for
the presence of Cry9C provides no additional human health protection. 
This conclusion rests on careful consideration of the foregoing history
of the scientific and regulatory review of StarLink® and the
information accumulated since the last SAP meeting in 2001, particularly
the new data and analyses concerning the levels of Cry9C currently in
the food supply.  Although uncertainties remain, four considerations
indicate that risks are (and will remain) low and therefore the
discontinuation of testing for StarLink® by grain handlers and millers
is justified.

A.  Dramatically Lower Exposure Levels

The company responsible for StarLink® (Aventis, later it successor
entity SLLI) and EPA (as reviewed by the SAP) have all offered, at
various time points, estimates of the amount of Cry9C from StarLink® to
which the U.S. population may be exposed as a consequence of consuming
food derived from corn grain.  A comparison of these estimates – the
estimates generated by EPA in 2000 and the estimates generated by
Exponent (on behalf of SLLI) in 2005 and 2006 – provides very useful
insights into the changes in the level of Cry9C remaining in human food
as a result of coordinated efforts to contain and remove StarLink® from
the human food supply.

A comparison of these estimates indicates that there has been a very
large decline in exposure to Cry9C since 2000.  Some of the differences
between EPA’s 2000 estimate and Exponent’s 2005 and 2006 estimates
may be attributable to differences in the ways in which the estimates
were derived, but, by far, the most significant part of the difference
results from the success of government and industry containment efforts
in removing StarLink® from the human food supply.  For example, in the
most highly exposed group, the 99.9th percentile of exposure estimated
by Exponent in 2006 (0.00747 µg/person/day) is about 8,000 times lower
than the 99.9th percentile of exposure estimated by EPA in 2000 (57
µg/person/day).  Even when Exponent in its 2005 study modified its
assumptions in ways that would overestimate the levels of Cry9C in the
corn grain supply (see Tables 5 through 8), the estimates showed levels
that are thousands of time lower than the exposure estimated by EPA in
2000.  In addition, it should be noted that Exponent’s 2005 analysis,
and by extension its 2006 estimate, are based upon residue values that
fall for the most part at or near the LOD for ELISA plate testing.  As
discussed in Unit V.H. of this White Paper, the variability observed
with these sensitive assay systems at or near the LOD and the fact that
the positive ARS samples in distribution 2 when reassayed by the GIPSA
laboratory tested negative, suggest that Exponent’s 2005 and 2006
analyses could have resulted in estimates of far more Cry9C than was
actually present in the corn grain supply in 2005 and 2006.

B.  Relation of Exposure to Allergenic Risk 

In understanding the significance of the foregoing comparisons, it is
useful to examine how exposure may affect allergenic risk, since
exposure to the allergen is an important consideration for both the
sensitization and elicitation phases of the food allergic response.  The
first stage in the development of an allergic response to some component
of food is a “sensitization” period where the immune system is
primed to recognize a substance in food.  The SAP said in 2000, shortly
after StarLink® was first detected in food products intended for human
consumption, that the amount of StarLink® in the human food supply has
“a low probability to sensitize some individuals to Cry9C protein.” 
This conclusion rested on the Panel’s judgment that overall daily
dietary exposure was relatively limited, perhaps, in all likelihood,
below EPA’s estimated upper bound of 57 µg/person/day.  The Panel
further commented that “[l]owering the levels of Cry9C in the food
supply makes sensitization less probable.”  In July 2001, the SAP
reaffirmed its November 2000 conclusion that there was a “low
probability to sensitize individuals to Cry9C protein.”

EPA believes that the exposures to Cry9C occurring in 2005 were (and
continue to be) dramatically lower than were occurring in 2000 or 2001. 
The Agency agrees with Exponent’s estimate developed for SLLI that
mean exposure to Cry9C in 2005 is likely to be in the range of 0.00007
µg/person/day and that the 99.9th percentile of the exposure
distribution is approximately 0.008 µg/person/day.  These values are
about four to six orders of magnitude below the corresponding levels of
daily exposure estimated five years earlier in 2000 when the SAP
concluded that the levels of Cry9C estimated to be in the food supply
were so low that that there “was a low probability of allergenicity in
the exposed population.” 

 

Nonetheless, the dose or cumulative exposure resulting in sensitization
to any dietary protein is not generally known.  At the same time,
evidence of sensitization to an allergen alone does not necessarily
correlate with clinical reactivity or responsiveness to an allergen, and
doses causing sensitization may therefore not be relevant to assessing
hazard from allergenic exposure.  In this regard, the elicitation or
reactive phase of the allergic response best represents the hazard from
allergenic exposure, since associated doses result in clinical
symptomatology and risk to human health. The recent experience with
human consumption of corn since 2000 and the most recent exposure
estimates for Cry9C in the corn supply provide solid evidence to
evaluate the risk for allergenic reactivity posed by the current maximum
exposures to Cry9C.

Although the dose of allergen necessary to elicit allergic responses
(i.e., reactivity thresholds), has not been established for any known
allergen, data describing minimal doses to elicit allergic responses, or
lowest observed adverse event levels (LOAELs), to major food allergens
(i.e. peanut, milk, egg, etc.) in sensitive individuals are available.
These data have shown the LOAELs for any allergen to induce at least
mild, subjective allergic reactions to be in the 10 to 100 (g/meal
range. Although these doses correspond to foods with multiple allergenic
proteins rather than a single protein allergen and safety considerations
based on these doses are subject to uncertainties due to challenge study
design and materials as well as inclusion of sensitive populations, the
reported LOAELS of major food allergens are at least 3 to 4 orders of
magnitude higher than the amounts of Cry9C Exponent estimated in 2005
the most highly exposed individuals would consume (99.9th percentile of
the U.S. population at 0.00821 µg/person/day). 

Despite the fact that the levels of Cry9C estimated to be currently in
the food supply are far below the lowest levels ever shown to provoke an
allergic response in people allergic to known food allergens such as
peanuts, milk and tree nuts  NOTEREF _Ref177554310 \h  \* MERGEFORMAT 
46 , EPA cannot provide a definitive threshold for exposure to Cry9C
below which it could be proven to pose no allergenic risk human health,
if it were indeed a food allergen. Generally speaking, the only way to
prove definitively that a substance is a human allergen is to identify
an individual who exhibits an allergic response following exposure to
the substance, and who has antibodies in his or her blood against the
putative allergen.  Such individuals are initially identified through
incident reports in which a person claims to have experienced an
allergic reaction in temporal association with exposure to the allergen.
 Given the limitations on incident reporting and the general absence of
systematic follow-up, incident reporting is most likely to identify
substances that are potent allergens affecting significant numbers of
people.  In 2000, the SAP observed that “[i]f corn-derived food
products contain a highly potent allergen then allergic reactions should
appear within a few years.”  (Emphasis added.)  In 2001, FDA and CDC
invested considerable effort to identify individuals who alleged they
had allergic reactions associated with exposure to Cry9C, and
investigate these claims.  The information collected by FDA and CDC
failed to identify any instance in which it appeared a person had
experienced an allergic reaction to Cry9C.  Since 2001, no other
incidents reliably connecting allergic reactions and exposure to Cry9C
have been reported to, much less documented by FDA or EPA.  Moreover,
although the exact prevalence of allergy to corn is not known, there is
no evidence that allergies or reported allergic events to corn in
general have increased in the years since StarLink® corn was introduced
into the food supply. 

Given that exposure has decreased by an estimated 4 to 6 orders of
magnitude since 2000, it follows that current levels are unlikely to
lead to sensitization of even small numbers of individuals. In EPA’s
view, the reduction of exposure that has occurred since 2001 means that
it is unlikely anyone will become allergic (i.e., sensitized) to Cry9C
as a result of the low level of exposure encountered in today’s food
supply.  Moreover, should there be any individuals who were sensitized
to StarLink® derived Cry9C during 2000 and 2001, these individuals are
now unlikely to be exposed to any more than very low levels of Cry9C, if
any at all. There is little or no reason to expect the very low levels
of Cry9C residues estimated to be in the food supply at this time to
cause serious adverse allergic reactions. 

In sum, estimated levels of Cry9C exposure are orders of magnitude lower
than the minimal eliciting doses reported for most major food allergens.
Consistent with the SAP’s reasoning, the absence of reports
associating Cry9C with allergic reactions, while certainly not
conclusive, provides supporting evidence that Cry9C at the levels
present in the food supply from 2001 to the present is not triggering
significant, if any, allergic responses. We therefore conclude that
there is little or no reason to expect the very low levels of Cry9C
residues estimated to be in the food supply at this time to cause
serious adverse allergic reactions.

C.  Current Exposures Lower than 2001 Wet-Milling Estimates

EPA’s estimate of potential exposure to Cry9C through consumption of
food products containing ingredients derived from wet-milled corn grain
and EPA’s comments about the levels of allergenic risk posed by such
exposure create a context that is useful in evaluating the potential
allergenic risk posed by the levels of Cry9C being detected in the human
food supply since 2005.  Because EPA judged the levels of Cry9C coming
from consumption of food ingredients from wet-milling in 2001 to pose no
risk to public health, it follows that as the potential for exposure to
Cry9C from all sources as estimated in 2005 and 2006 is comparable or
lower, such exposure also poses no risk to public health.  The
wet-milling process removes virtually all protein content from the
various processed fractions produced for human food consumption, and as
analyzed in the 2001 White Paper, since in StarLink® grain only a very
small percentage of total protein is Cry9C, the total daily exposure
that could result from consumption of the various wet milling fractions
would be very low.  EPA’s upper bound estimate for exposure to Cry9C
from wet-milled food ingredients in 2001 for the 99.5th percentile
consumer was 0.019 µg/person/day.  The Agency’s 2001 White Paper on
the effects of wet-milling concluded that “it is reasonable to
conclude that there is virtually no Cry9C protein in wet milled products
and that there is no likely health concern for the public associated
with the consumption of any food fraction produced by wet milling of
corn as long as reasonable steps are taken to ensure that StarLink® is
not diverted into wet milling.”  In July 2001, EPA reaffirmed this
conclusion saying, “there is no public health risk from eating
products manufactured from StarLink corn through the wet-milling
process, provided that corn utilized in the wet-milling process does not
contain significant levels of StarLink.” 

In Exponent’s 2005 report, the levels of exposure to Cry9C from all
sources in 2005 are estimated to be lower than the exposure to Cry9C
from wet-milled products alone in 2001.  Specifically, a comparison of
the levels occurring at the 99.5th percentiles of the two relevant
estimates shows about a six fold difference:  2005 Exponent estimate of
0.003 µg/person/day vs. 2001 EPA estimate of 0.019 µg/person/day. 
Even the highest values estimated in any of the sensitivity analyses
performed by Exponent in 2005 were lower than the levels estimated in
2001 in food derived from wet milling.  (See Tables 5 through 8).  It is
important to recognize that EPA and Exponent used different
methodologies to develop estimates of Cry9C exposure.  Thus, any
comparison will not produce a highly precise estimate of the relative
levels of exposure at these different time points.  Nonetheless, EPA
believes that the EPA 2001 and Exponent 2005 analyses are sufficiently
comparable in methodology to conclude that 2005 estimate of exposure to
Cry9C from all sources lies in the same range as (or falls below) the
levels of Cry9C estimated exposure from wet-milled products alone in
2001.  Further, while some portion of any apparent difference in
estimated exposures may result from different estimation methodologies,
EPA believes that, by far, the most significant factor accounting for
the different estimates is the virtually complete removal of StarLink®
grain from the food supply. 

In 2006, Exponent provided additional information updating its estimates
of exposure to Cry9C.  The 2006 information indicates that detections of
Cry9C from October 2005 through September 2006 occurred less frequently
than they did in 2005. Using this 2006 information, Exponent developed
estimates of potential exposure to Cry9C in the diet.  Not surprisingly,
these estimates indicate that exposures to Cry9C in 2006 (0.00288
µg/person/day for the 99.5th percentile of the U.S. population (See
Table 10)) are lower than in 2005.  As the 2001 White Paper judged the
upper bound of exposure for wet milled fractions not to pose a risk to
public health or to raise a likely health concern, the levels of Cry9C
estimated to be in the corn grain supply today should similarly be
regarded as posing no risk to public health.

D.  Terminating Diversion of Grain Testing Positive for Cry9C from the
Human Food Supply Negligibly Affects Exposure 

At EPA’s suggestion, in 2006 SLLI directed Exponent to perform some
additional analysis to provide a quantitative assessment of the impact
on exposure to Cry9C of ongoing monitoring and diversion efforts. 
Exponent performed a probabilistic exposure assessment estimating the
effect on human dietary exposure of the efforts to identify and divert
lots of corn testing positive on the LFS test to domestic animal feed or
industrial uses. 

When instituted in 2000, the monitoring program recommended by FDA
resulted in the identification and diversion of numerous shipments of
corn testing positive for Cry9C to domestic animal feed or industrial
uses.  From 2000 onward, millions of bushels of grain have been
redirected as a result of the StarLink® containment program.  These
actions reduced the levels of Cry9C in the human food supply and lowered
the likelihood that, if StarLink® derived Cry9C were a human allergen,
any individual would receive enough exposure to become sensitized, and
that, if any person did develop an allergy to StarLink® derived Cry9C,
the sensitized individual would not be exposed to sufficient levels of
the allergen to produce an allergic response.  

The analysis that Exponent performed in 2006 comparing estimates made
assuming that lots testing positive for Cry9C were diverted from the
human food supply to acceptable uses to estimates made assuming that
lots testing positive would remain in the food supply (Table 11), shows
that the continuation of monitoring all shipments of corn grain coming
into dry milling facilities and masa operations as recommended by FDA is
no longer significantly reducing exposure to Cry9C.  The comparison
shows that, because positive tests are so extraordinarily rare,
monitoring and diversion of grain from the human food supply has no
measurable effect (about 0.000017 µg/day difference) on the levels of
exposure received by the overwhelming majority (99.5th percentile) of
consumers.  The greatest difference in estimated exposure was an
additional 0.000185 µg/day, and that difference affected only 0.1% of
all consumers (i.e., 99.9th percentile).  Therefore, the Agency
concludes that continuation of the monitoring program likely is having
no effect on the potential for exposure and thus having no effect on the
potential for allergenic risk. 

VII.  Public Comments and EPA Response

A draft version of this document was made available for public comment
on October 17, 2007, with a docket identification number of
EPA-HQ-OPP-2007-0832.  The open comment period from October 17, 2007, to
December 3, 2007, was held to allow members of the general public to
submit letters and/or documents to the Agency on this White Paper.  The
Agency received comment from the North American Export Grain
Association, Inc., the Corn Refiners Association, the Biotechnology
Industry Organization (BIO), the National Grain and Feed Association,
the U. S. Grains Council, the North American Millers’ Association, the
Food Corn Industry Coalition, the National Corn Growers, and two
unsigned comments.

Comments from the North American Export Grain Association, Inc., the
Corn Refiners Association, the Biotechnology Industry Organization
(BIO), the National Grain and Feed Association, the U. S. Grains
Council, the North American Millers’ Association, the Food Corn
Industry Coalition, and the National Corn Growers, supported the
Agency’s analysis and recommendation.  For example, the North American
Export Grain Association, Inc., noted that the “industry has tested
more than three million corn samples since 2000, and has not found Cry9C
since one sample tested positive in October 2004.”  They also stated
that the “method used to detect Cry9C is capable of detecting the
protein in single parts per billion levels (1 part per billion is
equivalent to 1 second in 32 years).  The US Department of Agriculture
has tested more than 450,000 corn samples since 2000, and even with such
highly sensitive testing, has not found Cry9C since one sample tested
positive in April 2005.”  The U. S. Grains Council offered that since
“2000 when the last StarLink corn was last grown, the US has produced
and utilized 6 complete crop cycles, totaling more than 75 billion
bushels of corn.  Therefore, the entire corn storage system, both
on-farm and commercial, has been effectively emptied and refilled 5-6
times.  This massive quantity of grain has effectively ensured that any
relevant trace of StarLink corn no longer exists in the US corn
supply.”   These eight commenters all urged that the EPA
recommendation be implemented at the earliest possible time.

The two anonymous commenters on the other hand opposed EPA’s
recommendation.  One of these commenters stated that “it is vitally
important that we monitor our food supply for genetically modified
products due to their unknown health effects.”  The other stated that
it is “strange for the testing to cease when employees who were
subject to inhaling the inert dusts from the dry milling process at the
facilities such as Bunge Milling were never tested, monitored,
evaluated, or even asked if they had an onset of any new health
problems.”  The commenter went on to say that the “study is
short-sighted as employees [at the dry milling facilities] did not fall
within the group of subjects being studied, so the results are
incomplete at best”. 

With regard to the comment on monitoring the food supply for unknown
health effects from genetically engineered products, the commenter
provided no information to indicate that such products in the food
supply pose any potential health effects that differ from those posed by
conventionally-derived food products, and EPA is aware of no such
information.  In any event, the White Paper is not addressing
hypothetical health effects of genetically engineered foods generally,
but only whether continued testing of corn grain for Cry9C at dry mills
and masa operations is warranted.  The commenter provided no information
addressing the issues posed in the White Paper.  In addition, the U. S.
also has mechanisms for individuals who believe they have been adversely
affected by exposure to a substance, including substances in food, to
report such effects to the Federal government.  EPA examined these
reports as part of this analysis; no adverse effects were determined to
be associated with StarLink® corn.

With regard to the comment on employees of Bunge Grain, Bunge North
America mills, along with other dry milling and masa operations, were
part of the network that tested lots of corn for the presence of Cry9C. 
Beginning in 2000, lots of corn that tested positive for Cry9C were then
diverted from milling operations to domestic animal feed or industrial
uses.  EPA estimates that maximum exposure in mills at the time the
testing/diversion program was instituted was low   NOTEREF _Ref185837453
\h  \* MERGEFORMAT  4 .  Even at these low levels of exposure, routine
grain handling procedures would have reduced worker exposure to corn
dust, and to any dust containing Cry9C.  The potential to be exposed to
Cry9C would have been dramatically reduced immediately in the mills
subsequent to institution of the testing/diversion program.  Efforts
from 2000 to the present to remove StarLink® from the grain supply
would have further reduced the potential for mill workers to be exposed
to Cry9C.  As described in this White Paper, the levels of Cry9C in the
grain supply were estimated in 2005 and 2006 to be thousands of times
lower than the estimates made in 2000 and 2001.  During the time frame
between 2001 and 2007, there have been no reports reliably linking
allergic responses to exposure to Cry9C.  The absence of such reports,
while certainly not conclusive, provides supporting evidence that the
extremely low levels of Cry9C estimated to be present in corn is not
triggering detectable, if any, allergic responses.

VIII.  Summary Recommendation

In light of the above analyses EPA recommends:

FDA should withdraw its guidance for dry milling facilities and masa
operations that recommends sampling and testing yellow corn and
dry-milled yellow corn shipments intended for human food use for Cry9C
protein residues. 

Codex Alimentarius Guidelines for the Conduct of Food Safety Assessment
of Foods Derived from Recombinant-DNA Plants (CAC/GL 45-2003) available
at: 
http://www.codexalimentarius.net/download/standards/10021/CXG_045e.pdf

 "Development of new methods for safety evaluation of transgenic food
crops"  MRID 447140-02. Relevant materials can also be found in the
February 29, 2000 SAP meeting at:   HYPERLINK
"http://www.epa.gov/scipoly/sap/meetings/2000/february/foodal.pdf" 
http://www.epa.gov/scipoly/sap/meetings/2000/february/foodal.pdf 

 EPA in the July 19, 2001 Federal Register (59 FR 60542) established a
tolerance exemption for all DNA and other genetic material that are part
of a plant-incorporated protectant.  See Title 40 of the Code of Federal
Regulations (CFR) at 174.475.  However, the AgrEvo petition addressed
both an exemption for the residues of Cry9C and the genetic material
necessary to produce it as the AgrEvo petition was submitted prior to
issuance of the July 19, 2001 Federal Register.

   HYPERLINK
"http://www.epa.gov/scipoly/sap/meetings/2000/february/foodal.pdf" 
http://www.epa.gov/scipoly/sap/meetings/2000/february/foodal.pdf 

 Aventis’ amended petition also included estimates of potential
exposure to Cry9C that were approximately 7 fold lower than EPA
estimates.  The difference between the EPA and Aventis estimates was due
primarily to differences in assumptions about the extent of commingling
of StarLink® grain with non-StarLink® grain during grain handling and
processing.

 EPA estimated that at several percentiles of exposure the subgroup
identified as the general Hispanic population would have exposure to
Cry9C higher than the general U. S. population.  For example, at the
99th percentile of exposure, the general Hispanic subgroup was estimated
to receive approximately 133% of the amount of exposure of U.S.
population – 33 µg/person/day vs. 25 µg/person/day.  

 EPA did not include corn syrup and corn oil in its dietary assessment
presented at the November 28, 2000 SAP meeting because EPA had judged
protein to be absent or virtually undetectable in these food products,
and therefore that these commodities would not contribute meaningfully
to the overall estimate of potential exposure to Cry9C.  These materials
are available at:  http://www.epa.gov/scipoly/sap/meetings/2000/#112800 

   HYPERLINK "http://archive.gipsa.usda.gov/rdd/cry9csampling.pdf" 
http://archive.gipsa.usda.gov/rdd/cry9csampling.pdf 

 The final guidance, issued in January 2001, can be found at:   
HYPERLINK "http://www.cfsan.fda.gov/~dms/starguid.html" 
http://www.cfsan.fda.gov/~dms/starguid.html 

 http://www.epa.gov/oppbppd1/biopesticides/pips/wetmill18.pdf

 http://www.epa.gov/scipoly/sap/meetings/2001/index.htm#july

 The GIPSA validated test systems are capable of detecting down to ~ 20
ppb Cry9C; this level of detection corresponds to approximately 0.125%
StarLink® grain, or 1 in 800 kernels. 

 Following the cancellation of the StarLink® registration, Aventis
established a separate corporate entity, StarLink Logistics Inc. (SLLI),
as the successor to Aventis’ interest in StarLink® products.  SLLI
oversees the StarLink® Enhanced Stewardship Program, including the
Quality Check Program (QCP), through which SLLI and the U.S. corn
millers have continued the efforts to contain and remove Cry9C from the
human food supply.  SLLI also maintains a monitoring database containing
the test results from more than 4 million tests from over 4 billion
bushels of corn collected by dry milling facilities and other corn
handling operations.  These tests were carried out according to guidance
developed by FDA and USDA GIPSA.

 The LFS test is an immunoassay test based on antibodies specifically
directed at the protein Cry9C.  It does not quantify the amount of
protein present, but rather indicates the presence of a substance that
reacts with the antibodies. The LFS test has a limit of detection (LOD)
of ~ 20ppb.  The LFS test is designed to be a quick immunoassay that can
be used in the field.

 Exponent November 16, 2005 Report. “Assessing exposure to Cry9C
protein in StarLink corn”.

 Exponent December 6, 2006 External Memorandum. “Revised StarLink corn
risk assessment and updated QCP data through October 2006”.

 Exponent November 16, 2005 report.  “Assessing exposure to Cry9C
protein in StarLink corn.”  Independent testing in 2001 by USDA
reported a high value of ~ 14% of grain testing positive (86% negative)
for Cry9C (as reported in Exponent 2005 report).

 None of the samples that tested negative with the LFS test in the field
tested positive in an LFS test performed in the laboratory. Thus, there
are only six distributions rather than eight.

 Two types of locations test for StarLink® corn within the QCP –
mills and elevators (including export terminals).  Elevator data are
classified into corn tested inbound to the elevator and corn tested
outbound from the elevator.  According to the 2005 Exponent report, in
the QCP 0.020% of yellow corn tested positive for Cry9C using the LFS
test.  Of the corn tested at elevators, 0.024% of corn tested positive
(0.019% inbound, 0.028% outbound). Of the corn tested at mills, 0%
tested positive.  Since the same corn may have been tested at multiple
locations, Exponent attempted to allow for this by depending on results
from a single site in its assessment, i.e., at elevators.  Results from
testing conducted on outbound corn samples at elevators were used as an
estimate of the prevalence of Cry9C in the U.S. corn supply.  As a
further conservative measure, all positive results from corn testing
positive inbound to elevators was added to the positive results of corn
testing positive outbound from elevators, but the denominator was not
changed.  This resulted in the estimate of percentage positive
increasing to 0.04%.  The mill results in the QCP were not included
since mill testing historically shows a lower prevalence of positive
results; most likely because the corn has previously been screened at
the elevator and lots testing positive diverted.

 The ELISA plate test, also used in assessing the amount of Cry9C in the
corn supply, is based on antibodies specifically directed against Cry9C
protein.  The ELISA plate test, however, can be used to quantify the
amount of Cry9C present in a sample.  It generally has lower limits of
detection than the LFS test.  Current laboratory results indicate that
the average LOD is approximately 0.115 ppb but can range from 0.076 to
0.156 ppb.

 It is worth noting that all of the quantified positive detections in
this USDA survey correspond to amounts of residue that represent
substantially less than would be contributed by a single kernel of corn
in the sample of 800 kernels.  Since the level of Cry9C in a kernel of
StarLink® corn is approximately 13,000 ppb, a single kernel of
StarLink® among 800 kernels of corn would correspond to ~ 20 ppb.  

 See Table 6 of the Exponent 2005 report which is based on the study
submitted by Aventis (MRID 453866-03) and reviewed by the SAP at the
July 17-18, 2001 meeting. “Estimate dietary intake of Cry9C protein
based on measurements of Cry9C in process foods made from 100%
StarLinkTM corn.”

 Estimates for U.S. children 1 to 6 years of age, U.S. children 7 to 12
years of age, and for Hispanic populations can be found in the 2005
Exponent report. 

 GIPSA June 26, 2007 StarLink Test Results.

 Whitaker/Slate November 14, 2006 Memorandum. “ELISA plate test
results for Cry9C in corn grown prior to 1990”.

 GIPSA June 20, 2007 Analysis and July 12, 2007 Addendum. “Analysis of
individual corn kernels using ELISA-based technology to detect Cry9C in
StarLink®”.

 GIPSA, however, did confirm a positive result for the one sample where
ARS was able to provide to GIPSA test material that ARS had extracted
and tested (described in the November 14, 2006 memorandum) and found
positive.[Personal communication  GIPSA].

 While these estimates are useful for Exponent’s exposure assessment,
they probably do not reflect the actual occurrence of Cry9C in the
current grain supply.  

 The GIPSA LOD was estimated to be 0.37 ppb from GIPSA June 20, 2007
Analysis and July 7, 2007 Addendum. “Analysis of individual corn
kernels using ELISA-based technology to detect Cry9C in StarLink”. 
The ARS LOD is estimated to be ~ 0.4 ppb from Whitaker/Slate November
14, 2006 Memorandum. “ELISA plate test results for Cry9C in corn grown
prior to 1990.” 

 Whitaker March 21, 2007 Memorandum. “ELISA plate results of Cry9C
protein in commercial corn.”

 GIPSA analyzed 158 survey samples originally assayed by USDA ARS. The
retested samples contained all of the samples that had tested positive
in distribution 2 in the ARS laboratory and some of the samples that
tested negative in the ARS laboratory. 

 These survey samples were analyzed blind by the GIPSA laboratory
without any prior knowledge of the USDA ARS results.

 Table 1 of the Exponent November 16, 2005 Report. “Assessing exposure
to Cry9C protein in StarLink® corn”

 Table 2 of the Exponent November 16, 2005 Report. “Assessing exposure
to Cry9C protein in StarLink® corn.”

 See Unit V.E. of this White Paper and the Whitaker/Slate November 14,
2006 Memorandum. “ELISA plate test results for Cry9C in corn grown
prior to 1990.”

 Although the data generated at or near the LOD should be interpreted
with caution, it is reasonable to use such information to estimate the
upper bounds of human dietary exposure to Cry9C.

 EnvirologixTM Catalog Number AP 008 QT “QuantiPlateTM Kit for
Cry9C”.

 In 2004, 2% of U.S. corn acres were planted to corn containing Cry1F.
(From Biotechnology –Derived Crops Planted in 2004 – Impacts on US
Agriculture. December 2005 S. Sankula, G. Marmon, E. Blumenthal,
National Center for Food and Agricultural Policy) at:
http://www.ncfap.org/whatwedo/pdf/2004biotechimpacts.pdf

 Additional information on Cry1F can be found at:
http://www.epa.gov/pesticides/biopesticides/pips/bt_brad.htm

http://www.epa.gov/fedrgstr/EPA-PEST/2001/June/Day-06/p13837.htm

 Smith, R.A. and G.A. Couche. 1991. The phylloplane as a source of
Bacillus thuringiensis variants. Applied and Environmental Microbiology
57:311-315.

 Frederiksen, K., H. Rosenquist, K. Jorgensen, and A Wilkes. 2006.
Occurrence of natural Bacillus thuringiensis contaminants and residues
of  Bacillus thurginiensis-based insecticides on fresh fruits and
vegetables. Applied and Environmental Microbiology 72:3435-3440.

 Bernhard, K., P. Jarrett, M. Meadows, J. Butt, D.J. Ellis, G.M.
Roberts, S. Pauli, P. Rodgers, and H.D. Burges. 1997. Natural isolates
of Bacillus thuringiensis: worldwide distribution, characterization, and
activity against insect pests. Journal of Invertebrate Pathology
70:59-68

 DeLucca, A.J., M.S. Palmgren, and A. Ciegier. 1982. Bacillus
thuringiensis in grain elevator dusts. Canadian Journal of Microbiology
28:452-456.

 Meadows, M.P., D.B Ellis, J. Butt, P. Jarrett, and H.D. Burges. 1992.
Distribution, Frequency, and Diversity of Bacillus thuringiensis in an
Animal Feed Mill. Applied and Environmental Microbiology 58:1344-1350.

 Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J.
Seurinck, K. Van Audenhove, J. Van Rie, A. Van Vliet, and M. Peferoen.
1996. A Bacillus thuringiensis insectidical crystal protein with a high
activity against members of the family Noctuidae. Applied and
Environmental Microbiology 62:80-86.

 Report “Approaches to establish thresholds for major food allergens
and for gluten in food” at http://www.cfsan.fda.gov/~dms/alrgn2.html

  E.g.,   HYPERLINK "http://www.epa.gov/pesticides/health/reporting.htm"
 http://www.epa.gov/pesticides/health/reporting.htm ; http://
www.fda.gov/medwatch/how.htm

  http://www.osha.gov/SLTC/grain handling/recognition.html;
http;//www.cdc.gov/niosh/pdfs/83-126d.pdf

March 28, 2008 

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