Bollgard II Cotton Safety Summary

Transcription

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Safety Assessment of Bollgard II Cotton Event 15985
Executive Summary
Bollgard II cotton event 15985 was developed by Monsanto Company to produce the
Cry2Ab2 insect control protein, which provides effective season-long control of key
lepidopteran insect pests. This product was produced by re-transformation of Bollgard®
cotton event 531, which produces the Cry1Ac insect-control protein and the NPTII
selectable marker protein. Therefore, Bollgard II cotton produces two proteins for
effective control of the major lepidopteran insect pests of cotton, including the cotton
bollworm, tobacco budworm, pink bollworm, and armyworm. Bollgard II cotton also
produces the β-D-glucuronidase (GUS) marker protein. In addition, Bollgard II cotton
provides a more effective insect resistance management program compared to single gene
products.
Bollgard cotton has been grown globally on more than 32 million acres since commercial
introduction in 1996 (James, 2002). The primary benefits that have resulted from the use
of Bollgard cotton are reduced insecticide use, improved control of target insect pests,
improved yield, reduced production costs, and improved profitability for cotton growers
(Edge et al., 2001; Carpenter and Gianessi, 2001; Betz et al., 2000; Economic Research
Service/USDA, 2000; Falck-Zepeda et al., 1998; Falck-Zepeda et al., 2000; FernandezCornjeo and McBride, 2000; Klotz-Ingram et al., 1999; Traxler and Falck-Zepeda, 1999;
Xia et al., 1999). With the addition of a second insect protection protein, Bollgard II cotton
provides increased control of cotton bollworm, as well as certain secondary insect pests of
cotton, including armyworm (U.S. EPA, 2002). Furthermore, along with the other
components of Monsanto’s insect resistance management program, combining the
Cry2Ab2 and Cry1Ac proteins in a single product provides an additional tool to delay the
development of insect resistance to Cry proteins in cotton.
The Cry2Ab2 protein produced in Bollgard II cotton event 15985 is derived from the
naturally occurring soil bacterium Bacillus thuringiensis (B.t.). Microbial formulations of
Bacillus thuringiensis, which include the Cry2A class of proteins, have been registered in
numerous countries worldwide and have been safely used for control of lepidopteran insect
pests for more than 40 years (Lüthy et al., 1982; Baum et al., 1999; IPCS, 1999; Betz et al.,
2000). B.t. microbial formulations have been shown to be specific to the target insect pests
and do not have deleterious effects to non-target organisms such as beneficial insects, birds,
fish, and mammals, including humans (U.S. EPA, 1988; U.S. EPA, 1998). Therefore, there is
a history of safe dietary and occupational exposure to Cry proteins derived from B.t.,
including those of the Cry2A class.
The GUS protein present in Bollgard II cotton was used as a marker to facilitate the
selection of Cry2Ab2-producing plants. The GUS protein served no other purpose and has
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Bollgard and Bollgard II are registered trademarks of Monsanto Technology LLC.
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no known insect control properties. The history of safe use of the GUS protein is
extensive. Human exposure to the GUS protein is commonplace through intestinal
epithelial cells and intestinal microflora, bacterial exposure, and numerous foods containing
the GUS protein with no known harmful effects (Gilissen et al., 1998).
The Cry2Ab2 and GUS proteins in Bollgard II cotton event 15985 are present at very low
levels in cottonseed and are expected to be absent or inactivated in highly processed cotton
food and feed products. The safety of the introduced proteins has been assessed through
the confirmation of a history of safe food and feed use of the proteins or highly similar
proteins, the determination of no significant allergenic potential of the introduced proteins,
and the determination of no significant toxic potential of the introduced proteins.
Furthermore, there will be no significant consumption of these proteins in foods derived
from Bollgard II cotton due to the extensive processing and refinement of cottonseed oil
and cotton-derived food products.
An assessment of the nutritional and compositional equivalence of Bollgard II cotton to
conventional cotton varieties was performed on 48 components of cottonseed, oil, and
meal. These analyses included protein, fat, moisture, calories, minerals, amino acids,
cyclopropenoid fatty acid, and gossypol levels. Results of these extensive compositional
analyses demonstrated that the levels of the important nutritional and anti-nutritional
components in Bollgard II cotton event 15985 are comparable to the parental variety and
are within established ranges for commercial cotton varieties. It is concluded that Bollgard
II cotton event 15985 is not materially different in composition, safety, or any relevant
parameter from cotton now grown, marketed, and consumed.
The following summary provides information on the methods used to develop Bollgard II
cotton event 15985 and a summary of the food, feed, and environmental safety studies
performed. On the basis of these evaluations, Bollgard II cotton and its processed fractions
were found to be substantially equivalent to conventionally bred cotton, taking into
consideration the natural variation observed among cotton varieties, with the exception of
the production of the Cry1Ac, Cry2Ab2, NPTII, and GUS proteins. Previous studies
established the food, feed, and environmental safety of the Cry1Ac and NPTII proteins
produced in Bollgard cotton, and more recent studies have confirmed that the Cry2Ab2 and
GUS proteins produced in Bollgard II cotton are also safe for human and animal
consumption and to the environment.
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Introduction
Cotton is the leading plant fiber crop produced in the world and the most important in the
United States. Cotton production in the United States is located primarily in the tier of 15
southern states stretching from California to North Carolina, with approximately 13 million
acres grown annually (James, 2002). Lepidopteran insects are the main insect pest problem
in cotton. The primary lepidopteran pests infesting cotton are cotton bollworm, tobacco
budworm, and pink bollworm. The average percent yield loss due to bollworm and
budworm infestation between 1985 and 1995 was 3.7 % (Gianessi and Carpenter, 1999).
During the growing season other insects (e.g., cotton boll weevil, lygus bugs, fleahoppers,
spider mites, thrips, and aphids) are also present.
Bollgard® cotton, which produces the Cry1Ac insect control protein, has been adopted
broadly by growers since its commercial introduction in 1996, as it provides effective
protection from feeding damage by lepidopteran insect pests such as tobacco budworm,
pink bollworm, and cotton bollworm (Carpenter and Gianessi, 2001). Bollgard cotton
growers typically apply significantly less insecticide to control these pests, realize higher
yields, and achieve greater profitability compared to growers using conventional cotton
varieties (Fernandez-Cornejo and McBride, 2000). Bollgard cotton has been grown on
more than 32 million acres globally since it was introduced in the United States in 1996
(James, 2002). The food, feed, and environmental safety of Bollgard cotton has been
reviewed (Hamilton et al., 2002; Monsanto, 2002).
The introduction of Bollgard II cotton, producing both the Cry1Ac and Cry2Ab2 proteins,
is expected to expand the range of benefits to both growers and the environment. Bollgard
II cotton provides equivalent or increased control of the major insect pests of cotton
(tobacco budworm, pink bollworm, and cotton bollworm) compared to Bollgard cotton,
with additional control of secondary lepidopteran insect pests such as beet and fall
armyworm. Combining the Cry2Ab protein with the Cry1Ac protein in Bollgard cotton
will also provide an additional tool to delay the development of resistance since these two
protein classes have different modes of action (Crickmore et al., 1998). In general, if the
second insecticidal protein is sufficiently different in its mechanism of action from the first,
and is itself highly efficacious against the target pest species, then insects would need to
develop two distinct modes of resistance to survive both proteins, which is highly unlikely.
Therefore, Bollgard II cotton, containing both the Cry1Ac and Cry2Ab proteins, provides
added protection against the risk of resistance developing in the primary target insect
species and is expected to extend the effectiveness of this technology for the grower and
prolong the overall benefits already documented for Bollgard cotton.
In conclusion, lepidopteran insect pests -- cotton bollworm, tobacco budworm, and pink
bollworm -- are the main insect pest problem in cotton production. Bollgard cotton,
producing the insecticidal protein Cry1Ac, has been widely adopted by growers because of
its efficacy against these pests and demonstrated environmental and economic benefits.
The introduction of Bollgard II cotton, producing both the Cry1Ac and Cry2Ab2 proteins,
will expand the range of benefits. Furthermore, Bollgard II cotton, in combination with
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other components of the insect resistance management program, is expected to significantly
delay the development of insect resistance.
The following sections describe the molecular characterization of the inserted DNA, the
levels of the Cry2Ab2 and GUS proteins, the safety assessment of the Cry2Ab2 and GUS
proteins, the compositional analyses of cottonseed, cottonseed oil and cottonseed meal
alone and for Bollgard II cotton compared to other cotton varieties and the environmental
risk assessment of Bollgard II cotton.
Molecular Characterization of Bollgard II Cotton
Bollgard II cotton event 15985 was generated by re-transformation of cotton meristems of
Bollgard cotton event 531, variety DP50B. A particle acceleration plant transformation
procedure was used to insert the cry2Ab2 insect control coding sequence and the uidA
marker coding sequence into the Bollgard cotton genome. The purified plasmid vector,
PV-GHBK11, is a 8.7Kb high copy number based plasmid containing well-characterized
DNA elements for selection and replication of the plasmid in bacteria (Figure 1). The
purified, linear DNA was inserted into the Bollgard cotton genome. The linear plasmid
fragment only contains two plant gene expression cassettes, each using separate controlling
DNA elements essential for production in the cotton plant cells and does not contain the
nptII selectable marker gene or origin of replication. The first cassette contains a copy of
the cry2Ab2 gene encoding the B.t. insecticidal protein Cry2Ab2 and the second cassette
contains the uidA gene encoding the β-D-glucuronidase (GUS) marker protein to facilitate
selection of Cry2Ab2-producing plants. The GUS protein serves no other purpose and has
no known insect control properties.
The cry2Ab2 and uidA genes are both under the regulation of the enhanced cauliflower
mosaic virus 35S promoter (e35S) (Odell et al., 1985) and the 3’ untranslated region of the
nopaline synthase gene (NOS 3’) from Agrobacterium tumifaciens, which provides the
signal for mRNA polyadenylation. The e35S promoter driving the cry2Ab2 gene is also
fused to the 5’ untranslated leader sequence from the petunia heat shock protein 70
(HSP70) and the chloroplast transit peptide from the Arabidopsis thaliana 5-enolpyruvyl
shikimate-3-phosphate synthase gene (CPT2), which is used to direct the protein to the
chloroplasts.
Integration of DNA into cotton germlings was detected by histochemical staining for GUS
protein activity in vascular tissue. Non-transformed tissue was removed and growth of
meristems containing the introduced DNA was promoted. The resulting seed from these
plants was screened for the production of Cry2Ab2 protein.
The molecular characterization of Bollgard II cotton demonstrated that there is one DNA
cry2Ab2 insert. The single DNA insert in Bollgard II cotton event 15985 contains one
copy of the cry2Ab and uidA gene cassettes from the linear DNA PV-GHBK11 used for
transformation containing:
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•
•
the complete cry2Ab coding region and cassette, although the restriction site
following the NOS 3’ polyadenylation sequence in the cassette is not present;
and
the complete uidA coding region and cassette, except that 260 bp of the 5’ end
of the enhanced CaMV 35S promoter is not present; however, the truncated
promoter is functional as demonstrated by production of the GUS protein.
Sequencing of the DNA inserted into Bollgard II cotton confirmed the molecular details
above. PCR and DNA sequencing verified the 5’ and 3’ ends of the insert and confirmed
that the DNA flanking the insert was native to cotton. Production of the full-length
Cry2Ab2 and GUS proteins was confirmed by western blot analysis.
Inheritance analysis of the cry2Ab2 insert conforms to the expected Mendelian segregation
pattern for a single genetic locus. The stability of the insert was demonstrated by Southern
blot over four generations of selfing and two generations of backcrossing. In addition,
progeny of Bollgard II cotton event 15985 have been field tested at multiple sites in the
U.S. since 1998. No instability of the DNA cry2Ab2 insert has been detected during
extensive field-testing and commercial seed production of Bollgard II cotton based on the
following results:
•
•
•
•
analyses of seed obtained from multi-site trials over four years showed similar levels of
the Cry2Ab2 and GUS proteins;
the production of the Cry2Ab2 protein has been confirmed by immuno-detection and/or
efficacy data under various environmental conditions and in numerous Bollgard II
cotton varieties;
the insecticidal efficacy has been maintained during the development of this product in
the U.S. and other world areas where this product will be commercialized; and
the production of the Cry2Ab2 protein has been maintained after transfer of the
cry2Ab2 gene into different varieties of cotton.
These data confirm that the Bollgard II cotton insert is stably integrated in the cotton
genome.
Cry2Ab2 and GUS Protein Levels in Bollgard II Cotton Plants
Enzyme-linked immunosorbent assays (ELISA) (Harlow and Lane, 1988) were developed
and optimized to estimate the Cry2Ab2 and GUS protein levels in cottonseed and cotton
leaf matrices. Cry2Ab2 and GUS proteins were detected in various plant tissues of
Bollgard II cotton plants during the 1998 growing season across eight locations
representative of major cotton production regions (Table 1). The Cry2Ab2 and GUS
proteins were detected in Bollgard II cotton plants but, as expected, neither protein was
detected in the parental control, Bollgard cotton, or in the non-transgenic control. The
Cry2Ab2 protein levels estimated in Bollgard II cotton leaf and seed were 23.9 and 43.2
µg/g fresh weight, respectively. The mean protein levels for GUS were 106 and 58.8 µg
fresh weight in leaf and cottonseed, respectively. These protein levels are low in
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comparison to total protein levels. Levels of the Cry2Ab2 protein were also measured in
whole plants collected at the end of the season, and in pollen. In field tests from the 1998
season, mature Bollgard II cotton plants contained an estimated 8.8 µg Cry2Ab2 protein/g
fresh weight. The Cry2Ab2 protein was not detected in pollen collected from Bollgard II
cotton plants above the limit of detection of the assay (0.25 µg/g fresh weight).
Safety Assessment of Cry2Ab2 and GUS Proteins in Bollgard II Cotton
Safety assessments of the Cry2Ab2 and GUS proteins produced in Bollgard II cotton event
15985 included protein characterization, demonstration of the lack of similarity to known
allergens and toxins, the long history of safe consumption of similar proteins, in vitro
digestibility, and the lack of acute oral toxicity in mice.
Cry2Ab2 is a protein derived from Bacillus thuringiensis and has also been designated
Cry2Ab2, CryIIB, CryB2 or CryIIAb (Liang and Dean, 1994; Widner and Whiteley, 1990;
Crickmore et al., 1998). In the current nomenclature scheme, Cry protein names are
assigned according to amino acid similarity to establish holotype proteins as defined by
Crickmore et al. (1998). In this nomenclature, Cry proteins with similar amino acid
sequences are grouped together. Cry proteins with the same Arabic numeral, e.g., Cry2,
share at least 45% amino acid sequence identity. Those with the Arabic numeral and upper
case letter, e.g., Cry2A, share at least 75% sequence identity. Finally, Cry proteins with
the same Arabic numeral, upper case letter and lower case letter, e.g., Cry2Ab, share
greater than 95% sequence identity.
Bacillus thuringiensis (B.t.) is a gram-positive bacterium commonly present in soil and has
been used commercially in the U.S. since 1958 in microbially derived products with
insecticidal activity (U.S. EPA, 1988). Bacillus thuringiensis subsp. kurstaki, present in
commercial microbial pest control products such as DiPel® and Crymax®, contain both the
cry2Aa and cry2Ab2 genes. Although the Cry2Aa protein is produced in these commercial
products, the cry2A2b gene is a pseudo gene, meaning that although the coding sequence is
present, Cry2Ab protein is not produced due to an inefficient promoter (Dankocsik et al.,
1990). Therefore, the Cry2Ab2 protein is not naturally produced in soil bacteria or sprayable
microbial formulations (Widner and Whiteley, 1990; Crickmore et al., 1994). Both the
cry2Aa and cry2Ab2 genes are located on the same 100 MDa plasmid (Donovan, et al., 1988;
1989) and the sequence of the cry2Ab2 gene has been fully characterized (Widner and
Whiteley, 1990). The Cry2Ab2 protein is derived from Bacillus thuringiensis, and is 88%
amino acid sequence identical to the Cry2Aa protein produced by the B. thuringiensis
kurstaki bacterium. This bacterial strain controls insect pests by the production of crystalline
insecticidal proteins known as delta-toxins.
Mode of Action and Specificity of the Cry2Ab2 Protein
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DiPel is a registered trademark of Abbott Laboratories.
Crymax is a registered trademark of Ecogen, Inc.
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The Cry proteins exhibit a complex, multi-component mode of action. Insecticidal activity
of the Cry proteins requires that the protein be ingested by the target insect pest. In the
insect gut, the protein is solubilized due to the high pH of the insect gut and is
proteolytically cleaved to the active core of the protein, which is resistant to further
degradation by the insect gut proteases (Lilley et al., 1980; English and Slatin, 1992). The
core protein binds to specific receptors on the mid-gut epithelium cells of susceptible
insects, inserts into the membrane, and forms ion-specific pores (English and Slatin, 1992).
The cells swell due to an influx of ions and water, leading to cell lysis and ultimately the
death of the insect (Höfte and Whitely, 1989). The digestive tract tissues of non-target
insects, mammals, birds, and fish do not contain receptors that bind the Cry proteins
(Noteborn, 1994; Sacchi et al., 1986; Van Mellaert et al., 1988). Therefore, the Cry
proteins cannot disrupt digestion in on-target species. Cry proteins are considered nontoxic to species other than lepidopteran and dipteran insects because there is a strong
correlation between toxicity and specific binding of Cry proteins (Siegel et al., 2001; Betz
et al., 2000; Hofmann et al., 1988).
Characterization and History of Safe Consumption of Cry2Ab2 and GUS Proteins
There is a history of safe use of Cry proteins in microbial B.t.-based products (U.S. EPA,
1998; IPCS, 1999). EPA and WHO have concluded that the potential dietary exposure to
Cry proteins from use of microbial sprays on food crops does not raise any concerns: “The
use patterns for B. thuringiensis may result in dietary exposure with possible residues of
the bacterial spores on raw agricultural commodities. However, in the absence of any
toxicological concerns, risk from the consumption of treated commodities is not expected
for both the general population and infants and children” (U.S. EPA, 1998) and “B.t. has
not been reported to cause adverse effects on human health when present in drinking-water
or food.” (IPCS, 1999).
The amino acid sequence of the Cry2Ab2 protein produced in Bollgard II cotton has been
predicted based on nucleotide sequence of the coding sequence. The Cry2Aa protein
exhibits a high degree of amino acid similarity (97%) with the 88% amino acid identical
Cry2Ab2 protein produced in Bollgard II cotton. Thus, safety studies conducted with
microbial B.t. products containing Cry2A proteins are relevant to the safety assessment of
the Cry2Ab2 protein present in Bollgard II cotton. The Cry2A protein as a component of
B.t. microbial products has been shown to have no deleterious effects on fish, avian
species, mammals, and other non-target organisms (U.S. EPA, 1998; Betz et al., 2000).
The lack of acute toxicity of the Cry proteins to non-target species is attributed to the
highly specific mode of action and rapid digestibility.
The GUS protein produced in Bollgard II cotton has an extensive history of safe use.
Exposure of humans to the GUS protein is common, because GUS is present in intestinal
epithelial cells, intestinal microflora bacteria, and numerous foods, and no harmful effects
have been reported (Gilissen et al, 1998). GUS activity has been detected in over 50 plant
species (Hu et al., 1990). These species include a number of human food sources,
including potato, apple, almond, rye, rhubarb, and sugar beet (Schulz and Weissenbock,
1987; Hodal et al., 1992; Wozniak and Owens, 1994). GUS is also present in beef and in a
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number of invertebrate species, including nematodes, mollusks, snails, and insects
(Gilissen et al., 1998). Even when ingested in raw foods such as shellfish or apples, GUS
is not known to cause harmful effects (Gilissen et al., 1998). Likewise, the metabolites of
E. coli-derived GUS are non-toxic (Gilissen et al., 1998). The E. coli-derived GUS
enzyme produced by Bollgard II cotton is 99.8% homologous and functionally equivalent
to the GUS enzyme from E. coli naturally present in the human gut.
Digestion of Cry2Ab2 and GUS Proteins in Simulated Gastric and Intestinal Fluids
In vitro, simulated mammalian gastric and intestinal digestive mixtures were used to assess
the susceptibility of the Cry2Ab2 and GUS proteins to proteolytic digestion. Rapidly
digested proteins represent a minimal risk of conferring novel toxicity or allergy,
comparable to other safe dietary proteins (Astwood et al., 1996; Astwood and Fuchs,
2000). The rate of degradation of the Cry2Ab2 and GUS proteins was evaluated
separately in simulated gastric (pepsin, pH 1.2) and intestinal (pancreatin, pH 7.5) fluids.
The method of preparation of the simulated digestion solutions used is described in the
United States Pharmacopoeia (1995).
The degradation of the Cry2Ab2 protein was assessed by SDS-PAGE, western blot
analysis and insect bioassay. SDS-PAGE analysis of simulated gastric fluid (SGF)
demonstrated that greater than 98% of the Cry2Ab2 protein was digested within 15
seconds and that no fragments >2kDa of the parent protein were resolved. The acid
conditions of the stomach denature the native conformation of the Cry2Ab2 protein,
facilitating its rapid digestion. Western blot analysis of simulated intestinal fluid (SIF),
showed that within one minute the Cry2Ab2 protein was degraded to a relatively stable
protein fragment (≈50kDa) that was bioactive for at least 24 hours. This result was
expected because protease-resistant core proteins of B.t. insecticidal proteins are known to
be resistant to further trypsin digestion (Lilley et al., 1980). In vivo, the Cry2Ab2 protein
would be exposed to gastric conditions prior to entering the intestinal lumen. The low pH
and pepsin in the stomach would be expected to either fully digest the protein or cause it to
become susceptible to intestinal digestion.
The degradation of the GUS protein was assessed by western blot analysis and enzymatic
activity assays. Within 15 seconds of exposure to SGF, there was no detectable GUS
protein in either assay. After two hours in SIF, 91% of the original GUS activity was lost
in the enzyme assay, with only a faint band detected in the western blot analysis. Based on
these results, it was concluded that any GUS protein ingested by humans would be readily
degraded in the digestive tract (Fuchs and Astwood, 1996).
Human exposure to either the Cry2Ab2 or the GUS protein from cotton-derived products
would not be expected because cotton processing removes or denatures both the Cry2Ab2
and GUS proteins (refer to section below on Assessment of Human Exposure to Cry2Ab2
and GUS Proteins from Bollgard II Cotton).
Assessment of Acute Oral Toxicity of Cry2Ab2 and GUS Proteins in Mice
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Few proteins are toxic when ingested. Those that are toxic, typically act acutely (Sjoblad
et al., 1992). Results of mammalian acute oral toxicity studies of Cry2Ab2 and GUS
proteins support their specificity and lack of acute toxicity. There was no evidence of
toxicity even at extremely high dose levels. There were no treatment-related adverse
effects in mice administered Cry2Ab2 protein by oral gavage at doses up to 1450 mg/kg of
body weight. Similarly, the GUS protein caused no deleterious effects when administered
by oral gavage at doses up to 69 mg/kg. Results from these studies demonstrated that the
Cry2Ab2 and GUS proteins are not acutely toxic to mammals. This result was expected
because both the Cry2Ab2 and GUS proteins are readily digested in gastric and intestinal
fluids in vitro and both proteins are from families of proteins with a history of safe
consumption.
Assessment of Sequence Similarity of Cry2Ab2 and GUS Proteins to Known Protein Toxins
Another aspect used for the assessment of potential toxic effects of proteins introduced into
plants is to compare the amino acid sequence of the protein to sequences of known toxic
proteins. Homologous proteins derived from a common ancestor have similar amino acid
sequences, are structurally similar and share common function. Therefore, it is undesirable to
introduce DNA that encodes a protein that is homologous to any toxin. Homology is
determined by comparing the degree of amino acid similarity between proteins using
published criteria (Doolittle et al., 1990). The Cry2Ab2 protein does not show meaningful
amino acid sequence similarity when compared to known protein toxins present in the PIR,
EMBL, SwissProt, and GenBank protein databases, with the exception of other Cry proteins.
The GUS protein does not show any meaningful amino acid sequence similarity when
compared to known protein toxins present in these protein databases.
Assessment of Potential Allergenicity of Cry2Ab2 and GUS Proteins
Although there are no single predictive bioassays available to assess the allergenic
potential of proteins in humans (U.S. FDA, 1992), the physicochemical and human
exposure profile of the protein provides a basis for assessing potential allergenicity by
comparing it to known protein allergens. Thus, important considerations contributing to
the allergenicity of proteins ingested orally include exposure and an assessment of the
factors that contribute to exposure, such as stability to digestion, prevalence in the food,
and consumption patterns (amount) of the specific food (Metcalfe et al., 1996; Kimber et
al., 1999).
A key parameter contributing to the systemic allergenicity of certain food proteins appears
to be stability to gastrointestinal digestion, especially stability to acid proteases like pepsin
found in the stomach (Astwood et al., 1996; Astwood and Fuchs, 1996; Fuchs and
Astwood, 1996; FAO, 1995; Kimber et al., 1999). Important protein allergens tend to be
stable to peptic digestion and the acidic conditions of the stomach if they are to reach the
intestinal mucosa where an immune response can be initiated. As noted above, the in vitro
assessment of the Cry2Ab2 and GUS proteins digestibility indicates that these proteins are
readily digested.
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Another significant factor contributing to the allergenicity of certain food proteins is their
high concentration in foods (Taylor et al., 1987; Taylor, 1992; Fuchs and Astwood, 1996).
Most allergens are present as major protein components in the specific food, representing
from 2-3% to as high as 80% of total protein (Fuchs and Astwood, 1996). In contrast, the
Cry2Ab2 and GUS proteins are present at relatively low levels in Bollgard II cotton plants.
The Cry2Ab2 and GUS proteins represent approximately <0.004% and 0.007%,
respectively, of the total protein in seed.
It is also important to establish that the protein does not represent a previously described
allergen and does not share potentially cross-reactive amino acid sequence segments or
structure with a known allergen. An efficient way to assess whether the added protein is an
allergen or is likely to contain cross-reactive structures is to compare the amino acid
sequence with that of all known allergens. A database of protein sequences associated with
allergy and coeliac disease has been assembled from publicly available genetic databases
(GenBank, EMBL, PIR, and SwissProt). The amino acid sequences of the Cry2Ab2 and
GUS proteins were compared to these sequences. Neither the Cry2Ab2 protein nor the
GUS protein shares any meaningful amino acid sequence similarity with the known
allergens (Astwood et al., 1996).
In summary, these data and analyses support the conclusion that Cry2Ab2 and GUS
proteins do not pose a significant allergenic risk, are not derived from allergenic sources,
do not possess immunologically-relevant sequence similarity with known allergens, and do
not possess the characteristics of known protein allergens. In addition, as discussed in the
next section, these proteins are not detectable in cotton products used for human food.
Characteristics of known allergenic proteins
Characteristic
Allergens1
Stable to digestion
Stable to processing
Similarity to known allergens
Prevalent protein in food
1
yes
yes
yes
yes
Cry2Ab2
no
no
no
no
GUS
no
no
no
no
As described in Taylor (1992) and Taylor et al. (1987)
Assessment of Human Exposure to Cry2Ab2 and GUS Proteins in Bollgard II Cotton
Cottonseed oil and processed cotton linters are the major cotton products used for human
food (National Cottonseed Products Association, 1989). Analysis of refined cottonseed oil
and processed cotton linters derived from both the parental Coker control line and Bollgard
cotton event 531 confirmed that there is no detectable protein in cottonseed oil (detection
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limit = 1.3 ppm total protein). This is consistent with other reports that conclude that there
is an absence of protein in cottonseed oil (Cottonseed Oil, 1993). Analysis of processed
linters also confirmed there is no detectable protein (Sims et al., 1996). Likewise, there is
no reason to expect that the Cry2Ab2 or the GUS proteins would be present in cottonseed
oil or linters of Bollgard II cotton. Therefore, significant human consumption of the
Cry2Ab2 and GUS proteins present in Bollgard II cotton varieties is extremely unlikely.
Furthermore, direct food challenge of individuals allergic to proteins contained in the meal
derived from oilseed crops (e.g., soybean, peanut, and sunflower) with the oil from these
respective crops has established that refined oil does not elicit an allergenic response (Bush
et al., 1985; Halsey et al., 1986; Taylor et al., 1981). This lack of response is consistent
with the lack of detectable protein in the oil (Tattrie and Yaguchi, 1973). This information
supports the conclusion that there is insignificant human exposure to the Cry2Ab2 and
GUS proteins in Bollgard II cotton, and that Bollgard II cottonseed oil poses no significant
allergenic concerns.
Compositional Analysis and Nutritional Assessment of Bollgard II Cotton
The design of a food and feed safety assessment program for a genetically engineered crop
requires detailed understanding of the uses of the crop and crop products in animal and
human nutrition. Cotton is the leading plant fiber crop produced in the world and is grown
primarily for its fiber. Cottonseed is processed to produce animal feed ingredients.
Cottonseed meal is primarily used as cattle feed, with smaller proportions of meal fractions
used in feed for poultry, sheep, catfish, and swine. Cottonseed serves as an excellent
source of fiber and protein in animal feed, particularly due to its high lysine content. Oil is
the main food ingredient derived from cottonseed and is used for frying oil and in salad
dressings.
Compositional Analysis
To assess whether the composition of Bollgard II cotton is comparable to conventional
cotton present in the marketplace, with the exception of the introduced trait, compositional
analyses were performed on the cottonseed from Bollgard II cotton event 15985, the
DP50B parental variety, the DP50 non-transgenic control variety, and commercial cotton
varieties produced in 1998 from eight locations within six states in the U.S. (Texas,
Arizona, Mississippi, South Carolina, Louisiana, and Alabama). Forty-eight different
compositional components were evaluated. These analyses included:
• Proximate analysis: protein, fat, ash, water, carbohydrate, calories (Table 2);
• Amino acid composition: levels of individual amino acids (Table 3);
• Fatty acid profile: total lipid content and percentage of individual fatty acids in raw
seed (Table 4) and refined cottonseed oil (Table 5);
• Minerals: calcium, copper, iron, magnesium, manganese, phosphorus, potassium,
sodium, and zinc (Table 6);
• Anti-nutrients: levels of gossypol, cyclopropenoid fatty acids, and aflatoxins in seed
(Table 7); levels of gossypol in refined oil and cottonseed meal (Table 8); and
cyclopropenoid fatty acids in refined oil (Table 9).
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Statistical analyses were conducted on the data using a mixed model analysis of variance
for a combination of all sites for 1998. The results of these analyses, summarized in Tables
3 to 9, demonstrate that seed from Bollgard II cotton is compositionally equivalent to seed
from the DP50B parent variety and other commercial cotton varieties. In the 50
comparisons, there were six instances where the mean values of Bollgard II cotton were
statistically significantly different from the parental line (Table 10). In all of these
instances, the means were within the range of levels found for commercial cotton.
Furthermore, the statistical differences were not observed consistently at all locations and
as a result were not considered to be biologically relevant.
Fatty acid profiles were evaluated in cottonseed for Bollgard II cotton and there were no
statistically significant differences in palmitic, palmitoleic, oleic, linolenic, and gamma
linoleic, arachidic, behenic, or lignoceric acids compared to DP50B (Table 4). Small but
statistically significant differences were observed for myristic, stearic, and linoleic acids
between Bollgard II cotton and the DP50B control. All significantly different mean values
for Bollgard II cotton were within the non-transgenic and commercial cotton reference
ranges (Table 10), as well as within the ranges published in the literature (Berberich et al.,
1996). Therefore, these differences were not considered biologically relevant.
Mineral levels were measured in cottonseed (Table 6). There were no statistically
significant differences in mineral levels obtained for Bollgard II cotton compared to the
DP50B control and the means were within the non-transgenic and commercial reference
ranges.
Levels of anti-nutrients contained in cottonseed, such as gossypol, were comparable for
Bollgard II cotton and the parental cotton line. The primary aflatoxins (B1, B2, G1, G2)
were undetected in the Bollgard II, DP50B control, and the reference cotton lines at an
LOD of 0.1 ppb (Table 7). Statistically significant differences were observed for the mean
values of the cyclopropenoid fatty acids, malvalic, diydrosterculic, and sterculic acids, in
comparisons of values for Bollgard II cotton to the parental control (Table 10). All the
significantly different comparisons of mean values were within the ranges for the parental
and commercial reference ranges, as well as literature ranges. Additionally, only one of the
four replicated field locations showed the statistically significant differences in the mean
comparisons of Bollgard II cotton to the control. Therefore, these differences were not
considered biologically relevant.
The major cottonseed processed products, refined oil and meal, were also shown to be
equivalent to those products produced from the control cotton line. The refined oil was
evaluated for fatty acid profile, free gossypol content, and cyclopropenoid fatty acid levels.
The fatty acid profile of the refined oil was typical of commercial cottonseed oil (Table 5).
Free gossypol was not detectable and cyclopropenoid fatty acid levels were similar to
levels previously reported in the literature for both cottonseed meal and oil (Table 8 and 9).
The full fat flour and toasted meal were analyzed for total gossypol levels. When
cottonseed is flaked and heated during processing to oil and meal, the cotton lysigenous
glands are ruptured and gossypol is released. Some of the gossypol binds to seed
August 2003
12
components, primarily to proteins through the free amino groups of lysine. The binding of
gossypol during processing is important because the free form of gossypol is considered
toxic, whereas the bound form is unavailable and essentially inactive (Martin, 1990;
Berardi and Goldblatt, 1980). As expected, there was no detectable free gossypol in
toasted meal (Table 8). Therefore, insertion of the cry2Ab2 and uidA genes in the cotton
genome did not alter the processing characteristics of the cottonseed.
In summary, the results of numerous analytical measurements of composition demonstrate
that Bollgard II cottonseed is compositionally equivalent to the parental variety and
conventional cotton varieties. Processing is unlikely to alter the compositional components
of cotton and, therefore, products derived from cottonseed will also be compositionally
equivalent to and as safe as current cotton-derived products.
Nutritional Assessment and Toxicological Assessment of Cottonseed
In addition to the compositional studies, the nutritional wholesomeness of seed from
Bollgard II cotton was demonstrated by feeding rats, channel catfish, and dairy cows diets
which contained cottonseed from both the Bollgard cotton and control cotton cultivars. At
completion of the rat feeding study, there were no significant differences in weight gain or
feed intake between rats consuming Bollgard II cotton and the control cotton diet.
Similarly, there were no significant differences in survival, weight gain, feed conversion
ratio, or fillet composition between channel catfish fed a diet containing Bollgard cotton
compared to catfish fed the control cottonseed diet. Results of a cow study also showed
that cottonseed of Bollgard cotton is as wholesome and nutritious as control cottonseed for
cows based on similar feed intakes, general health and milk production and composition
(Castillo et al., 2001). Results of these studies confirm the food and feed safety and
nutritional equivalence of diets from Bollgard II cotton event 15985 to diets from
conventional cotton varieties.
Horizontal Gene Transfer and the Assessment of Marker Genes
Horizontal gene transfer is defined as the transfer of DNA from one species to another.
With respect to crop plants that are developed through biotechnology, a number of
assessments have been performed to evaluate the possibility that antibiotic resistance
marker genes used to facilitate the selection of the transformed plants might be transferred
to bacteria either in the field or in animals that have consumed the crop. The reason for the
assessment is that some species of bacteria found in soil, in the rumen or in the intestine
can receive DNA from other organisms through three mechanisms of transfer (Morrison,
1996; Davison, 1999). However, transformation is the only relevant mechanism to the
possible transfer of DNA from plants to bacteria and subsequent expression of the encoded
protein product. The other two mechanisms, conjugation (exchange of plasmid DNA
between compatible bacteria) and transduction (viral transfer of DNA into bacteria) are
specific to restricted forms of transfer and are not relevant to the potential transfer of DNA
from plants (Thomson, 2000).
August 2003
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Because Bollgard II cotton is a re-transformation of Bollgard cotton, the assessment of
possible horizontal gene transfer of the antibiotic resistance genes between Bollgard II cotton
and other organisms producing antibiotic resistance marker genes and microorganisms has
previously been discussed (Monsanto, 2002). In general, bacterial species differ markedly in
their ability to accept DNA from the environment, and the frequency of transformation,
even under ideal circumstances, is very low. The DNA that was transferred into cotton to
produce Bollgard II cotton was incorporated into the genomic DNA of the plant and
represents a small fraction of the cotton genome.
For Bollgard II cotton, the origin of replication for plasmid maintenance at high copy number
in E. coli, ori322, was contained on the plasmid PV-GHBK11 used for transformation, but was
not transferred into the cotton plant genome. Therefore, the antibiotic resistance genes in
Bollgard II cotton cannot be mobilized by excision of the marker gene to create a functional
plasmid. The DNA would have to be integrated into the recipient’s genome or plasmid in
order to replicate and be passed on through reproduction. Studies have addressed this potential
for the horizontal transfer of antibiotic selectable marker genes and concluded the probability
of this event occurring is virtually zero (Prins and Zadoks, 1994; Schlüter et al., 1995; Nielsen
et al., 1998; Beever and Kempe, 2000; Jelenić, 2003).
Environmental Assessment
Cotton
Cotton is of the genus Gossypium, of the tribe Gossypieae, and of the family Malvaceae.
Worldwide, four species of cotton are of agronomic importance: the two diploid Asiatic
species, G. arboreum and G. herbaceum, and the two-allotetraploid New World species, G.
barbadense and G. hirsutum. Although the diploid species remain important in restricted
areas of India, Asia, and Africa, the two New World species account for approximately
98% of world cotton fiber production. Wild species of Gossypium typically occur in arid
parts of the tropics and sub-tropics. Wild populations of G. hirsutum are relatively rare and
tend to be widely dispersed. All grow on beach strands or on small islands.
Cotton is normally considered a self-pollinating crop but can be cross-pollinated by certain
insects. However, outcrossing of the cry2Ab2 gene from Bollgard II cotton to other
Gossypium species or to other Malvaceous genera is extremely unlikely for the following
reasons (Percival et al., 1999):
• cultivated cotton is an allotetraploid and is incompatible with cultivated or wild diploid
cotton species; therefore, it cannot cross and produce fertile offspring.
• although outcrossing to wild or feral allotetraploid Gossypium species can occur, cotton
production generally does not occur in the same geographical locations as the wild
relatives. For example, outcrossing to G. tomentosum in Hawaii is possible, but cotton is
not grown commercially in Hawaii.
• there are no known plant species other than those of the genus Gossypium that are
sexually compatible with cultivated cotton.
August 2003
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If the cry2Ab2 gene were transferred to a wild population of a tetraploid cotton species, and
if this was considered undesirable, the size of the plants, their perennial growth habit, their
restricted habitat, and their low natural fecundity would make them easy to control.
Crossing of insect protection genes to other cultivated cotton genotypes is possible should
the plants be in close proximity; however, studies have shown that this occurs at a very low
frequency and is not considered to be a concern as it is unlikely to cause any adverse
impact to the environment (Green and Jones, 1953; Mehetre, 1992).
Assessment of Agronomic Performance
Bollgard II cotton has been grown and observed at multiple locations for weediness, plant
growth characteristics, susceptibility to insects, and disease infection. Based on results of
the field monitoring programs, there were no significant differences in agronomic
characteristics between Bollgard II cotton and the parental DB50 variety. Bollgard II
cotton does not pose any different plant pest risk to other plants and the environment than
non-transformed cotton varieties. Bollgard II cotton meets all morphological, yield, and
quality characteristics of cotton varieties produced in the United States.
Cotton is not considered to have weedy characteristics as an annual plant grown in the
United States. It does not possess any of the attributes commonly associated with weeds
such as seed dormancy, long soil persistence, germination under diverse environmental
conditions, rapid vegetative growth, a short life cycle, high seed output, high seed
dispersal, or long distance dispersal of seeds. Multiple genes typically control these
characteristics of weeds.
Wild populations of cotton are rare, widely dispersed and confined to beach strands or to
small islands (Lee, 1984). Cotton appears to be somewhat opportunistic towards disturbed
land and is not especially effective in invading established ecosystems.
There is little probability that Bollgard II cotton or any Gossypium species crossing with
Bollgard II cotton could become a weed. All wild and feral relatives of cotton are tropical,
woody, perennial shrubs (Percival et al., 1999), other than a few herbaceous perennials in
northwest Australia. With the exception of G. thurberi and G. sturtianum in Australia,
these cannot naturally exist even in the milder temperate regions. In most instances the
distribution of these species is determined by soil and climatic conditions. As perennials,
the plants do not tend to produce seed each year. In fact, they tend to drop fruit in response
to stress. It is unlikely that production of the Cry2Ab2 protein would impact survival
either way.
Bollgard II cotton does not have any different weediness characteristics than other
conventional cotton varieties. Bollgard II cotton does not exhibit different agronomic or
morphological traits compared to controls, which would confer a competitive advantage
over other species in the ecosystem in which it is grown. Based on these mechanistic
arguments and field experience, there is no indication that insertion of the cry2Ab2 gene
into the cotton genome would have any effect on the weediness traits of the cotton plant.
August 2003
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Assessment of Effect to Non-Target Organisms
There is extensive information about microbial preparations of Bacillus thuringiensis
subsp. kurstaki (B.t.k) containing Cry proteins that demonstrate that these proteins are nontoxic to non-target organisms (U.S. EPA, 1988; Betz et al., 2000). The literature has
established that the Cry proteins are extremely selective for lepidopteran insects, bind
specifically to receptors on the mid-gut of lepidopteran insects, and have no deleterious
effect on beneficial/non-target insects (Hofmann et al., 1988; English and Slatin, 1992;
Betz et al., 2000; Siegel et al., 2001).
To confirm and expand on results obtained for the microbial products, the potential impact
of the Cry2Ab protein on non-target organisms was assessed on several representative
organisms. The non-target organism species included larval and adult honey bee (Apis
mellifera L.), a beneficial insect pollinator; green lacewing larvae (Chrysopa carnea), a
beneficial predaceous insect commonly found on cotton and other cultivated crops;
parasitic Hymenoptera (Nasonia vitripennis), a beneficial parasitic wasp of the housefly;
the ladybird beetle (Hippodamia convergens), a beneficial predacious insect which feeds
on aphids and other plant bugs commonly found on stems and foliage of weeds and
cultivated plants; Collembola (Folsomia candida) and earthworm (Eisemia fetida) nontarget soil organisms; and northern bobwhite quail (Table 11).
No adverse effects were observed at the maximum expected environmental concentrations
to which these non-target organisms would be exposed. In all studies conducted, a NOEC
(no observed effect concentration) was established and found to exceed predicted
maximum environmental concentrations. In most studies, the NOEC exceeded the
maximum predicted environmental concentration by 10- to over 100-fold, demonstrating a
wide margin of safety for these organisms.
In summary, Cry proteins exhibit a high degree of specificity and therefore do not pose a
significant hazard to non-target animals such as mammals, birds, fish, water fleas,
earthworms, and beneficial insects. Although several endangered lepidopteran and
dipteran species may potentially be susceptible to Cry proteins, no exposure is predicted
because of their feeding habit or because the habitats of these endangered species in cottongrowing areas do not overlap with cotton fields.
Fate of Cry2Ab2 Protein in Soil
The results of a soil degradation study demonstrate that the Cry2Ab2 protein dissipates
rapidly in the soil environment. Analysis of soils from Mississippi, Arizona and Alabama
treated with purified Cry2Ab2 protein by insect bioassay established a DT50 range of 1.13.5 days; the DT90 range was 1.9-5.3 days. These results support the conclusion that the
Cry2Ab2 protein derived from Bollgard II cotton degrades rapidly in soil. In addition, the
short DT50 and DT90 values obtained in soils dosed with a solution of pure Cry2Ab2 protein
suggest that any Cry2Ab2 protein that would reach soil as the pure protein (e.g., by root
exudation, or otherwise not combined with tissue) would be degraded in less than 6 days.
August 2003
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The rapid degradation of the Cry2Ab2 protein in soil ensures exposure risk for soil
dwelling organisms will be minimal.
Assessment of Genetic Stability
The cry2Ab2 gene conferring insect protection in Bollgard II cotton event 15985 was
demonstrated as stably integrated into the chromosome. This conclusion is based on
molecular analyses, data on phenotypic expression, and inheritance patterns. The results of
these studies are summarized as follows:
•
•
•
•
•
•
molecular analyses of plants from the R3 to R5 generations establish that the introduced
genes are maintained in the same chromosomal location;
analyses of seed obtained from multi-site trials using R4 and R5 generations showed no
marked change in production of Cry2Ab2 protein;
the level of insect protection has been maintained for at least six generations and during
testing in the US over the last six years under diverse environmental conditions and in
many cotton lines with different genetic backgrounds;
production of Cry2Ab2 protein has been confirmed under different environmental
conditions and in many cotton lines with different genetic backgrounds;
Mendelian inheritance of the Cry2Ab2 protein production is observed after selfpollination or backcrossing with other cotton varieties; and
seed quality (germination, vigor) of Bollgard II cotton is maintained after transfer of the
cry2Ab2 gene into cotton from different genetic backgrounds.
In summary, it is concluded that the inserted genes in Bollgard II cotton event 15985 are
stably integrated and the line is phenotypically and genetically stable over several
generations, and in various environments.
Insect Resistance Management
Effective insect resistance management (IRM) programs for B.t. crops are a vital part of
responsible product stewardship and should be instituted based on the best available
knowledge, employing what is known about the trait, the mode of action, the targeted
insects and the environment in which the product is introduced, while being properly
respectful of uncertainties so as to make B.t. technologies available to growers as an
additional pest management tool. Such programs must strike a balance between available
knowledge and practicality, with grower acceptance and implementation of the plan as
critical components. Monsanto supports the development and implementation of an
effective and practical IRM plan for all B.t. crops in all markets where these products are
introduced. Each plan includes the following elements:
•
•
Baseline susceptibility determination for the target pests and surveillance for changes in
susceptibility;
An adequate supply of susceptible insects to mate with any resistant insects (achieved
through appropriate practical programs such as structured refuge, natural or cultivated
alternate hosts, grower practices, etc.);
August 2003
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•
•
Mitigation plans; and
Grower awareness, education of IRM concepts, and some means of assessing grower
behavior when particular IRM practices are required of them.
These plans vary according to geography, pest and overlapping crops, and are reviewed on
a regular basis with updated information available from interested stakeholders.
Combining the Cry2Ab2 protein with the Cry1Ac protein already in the marketplace
further strengthens the IRM strategy for Bollgard II cotton. The second insecticidal protein
in Bollgard II cotton is sufficiently different in its mechanism of action from the Cry1Ac
and is highly efficacious against the target pest species. Insects would need to develop two
distinct modes of resistance to survive both proteins, which is highly unlikely. Therefore,
if a target insect should develop resistance to one of the proteins, the other protein will
provide control of that resistant insect.
Environmental Assessment Conclusions
In summary, comparisons of Bollgard II cotton event 15985 plants were made to
conventional cotton plants with regard to disease and pest characteristics, yield,
morphology, weediness, impact on non-target organisms, and other characteristics. Based
on results of these extensive studies, it was concluded that the trait for protection from
lepidopteran insect pests is stably inherited and that Bollgard II cotton event 15985 does
not pose any increased plant pest risks or environmental risks compared to conventional
cotton varieties. Furthermore, the combination of the Cry2Ab2 and the Cry1Ac proteins
provide an enhanced IRM strategy to delay the development of resistance in lepidopteran
insects.
Summary
Bollgard II cotton, which has two modes of action for improved lepidopteran control and
increased spectrum of activity over Bollgard cotton, is expected to provide significant
benefits to cotton production including the reduction in pesticide use, improved control of
target insect pests, improved yield, reduced production costs, and improved profitability for
cotton growers. Detailed food, feed, and environmental safety assessments confirm the safety
of this product. The analyses included: 1) detailed molecular characterization of the
introduced DNA; 2) safety assessments of the produced Cry2Ab2 and GUS proteins; 3)
compositional analysis of cottonseed, oil, and meal; and 4) environmental impact assessment
of the cotton plants. These studies demonstrate that the Cry2Ab2 protein is safe to non-target
organisms, including humans, animals, and beneficial insects. Additionally, Bollgard II cotton
plants and cottonseed were shown to be as safe and nutritious as conventional cotton varieties.
Information and data contained within this document have been provided to regulatory
authorities for review. Regulatory review continues as we update regulatory files and
make submissions to additional countries globally.
August 2003
18
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23
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August 2003
25
Figure 1. Plasmid Map of PV-GHBK11.
KpnI 159
PstI 182
BglII 8394
PstI 8185
SphI 7833
NcoI 7798
BglII 821
NcoI 827
P-kan
P-e35S
kan
uidA
ori-pUC
PV-GHBK11
8718 bp
KpnI 6258
EcoRI 6206
PstI 6204
BamHI 6188
XbaI 6176
EcoRI 5892
NcoI 5886
NOS 3'
NOS 3'
cry2Ab2
PetHSP70-leader
P-e35S
AEPSPS/CTP2
EcoRI 5207
SphI 3959
NcoI 3964
PstI 4574
EcoRI 2688
BamHI 2951
PstI 3013
Used in
transformation
The KpnI segment of PV-GHBK11 plasmid used to generate insect-protected cotton event
15985.
August 2003
26
Table 1. Levels of Cry2Ab2 and GUS protein in leaf and seed samples collected
in the 1998 growing season. Mean protein levels (µg / g fwt)1 ± standard
deviation2 and range3.
Leaf
Seed
23.96
43.2
6.3
5.7
4
Cry2Ab2
Bollgard II
Mean
Standard deviation
Range
10.1-33.3
31.8-50.7
ND
ND
ND
ND
106
58.8
32
13.0
51.7-176
ND
37.2-82.3
ND
ND
ND
7
DP50B
DP50
GUS 5
Bollgard II
Mean
Standard
deviation
Range
DP50B
DP50
NA = Not Analyzed
ND = Not Detected
1: Protein levels are reported as microgram of protein per gram fresh weight of tissue and have been
corrected for overall assay bias.
2: The mean and standard deviation were calculated from the analyses of plant samples, one from
each of eight field sites.
3: Minimum and maximum values from the analyses of samples across sites.
4: The Limit of Detection for the Cry2Ab2 assay is 2.65 µg/g in leaf tissue and 2.31 µg/g in seed
tissue. The Limit of Quantification for the Cry2Ab2 assay is 1.24 µg/g in whole plant tissue and
0.25 µg/g in pollen tissue.
5: The Limit of Detection for the GUS assay is 0.91 µg/g in leaf tissue and 4.42 µg/g in
seed tissue
6: The mean level of Cry2Ab2 protein production in leaf samples peaked at 55 days after planting and
subsequently declined over the growing season to a mean of 16.7 µg/g fwt at 108 days after
planting
7: DP50B is a Delta and Pine Land Company commercial cotton variety, containing the Bollgard
cotton event 531 cry1Ac and nptII genes and was the recipient cotton tissue for the Bollgard II
cotton transformation
August 2003
27
Table 2. Summary of Proximate Analyses of Bollgard II Event 15985 Cottonseed Samples from the 1998 U.S. Field
Trials.
Component
Bollgard II
cotton event
15985
DP50
(non-transgenic
control)
Nontransgenic
reference
range 1
Commercial
reference
range 2
26.06
(21.93-28.15)
25.96
(21.76-27.79)
21.76-27.79
21.76-28.15
20.52
(17.54-27.42)
20.37
(16.04-23.48)
19.74
(15.44-23.64)
15.44-23.64
15.44-23.83
4.36
(3.93-4.81)
4.38
(4.06-4.67)
4.34
(3.76-4.85)
3.76-4.85
3.76-4.85
16.83
(14.93-17.95)
17.17
(15.42-19.69)
17.19
(15.38-19.31)
15.38-19.31
15.38-20.89
Carbohydrate,
%
49.09
(42.97-52.69)
49.23
(46.85-51.93)
49.94
(45.64-52.44)
45.64-53.62
45.64-53.62
Calories/100g
DW
485.33
(468.50-520.01)
Moisture, %
5.99
(4.34-7.59)
Protein, %
Fat, %
Ash, %
Fiber, crude %
26.13
(21.45-28.82)
DP50B
(parent)
484.45
481.57
(463.09-498.71) (457.77-499.84) 457.77-499.84
6.05
(4.22-7.28)
6.03
(3.97-7.26)
3.97-7.49
457.77-500.49
3.97-8.47
Underlined values are statistically significant relative to the DP50B control (p≤ 0.05). Values represent samples taken from eight U.S. field sites in
1998.
1
Range includes data from four commercially available cotton varieties.
2
Range includes data from ten commercially available transgenic and non-transgenic cotton varieties.
August 2003
28
Table 3. Summary of Amino Acid Analyses of Bollgard II Event 15985 Cottonseed Samples from the 1998 U.S.
Field Trials.
DP50
Non-transgenic Commercial
(non-transgenic
reference
reference
1
range2
control)
range
9.95
(9.78-10.45)
9.75-10.45
9.75-10.45
Amino Acid
(% total AA)
15985
DP50B
(parent)
aspartic acid 3
10.02
(9.74-10.49)
9.98
(9.76-10.39)
threonine
3.56
(3.37-3.77)
3.56
(3.40-3.90)
3.55
(3.38-3.73)
3.38-3.73
3.38-3.90
4.77
(4.23-5.04)
4.77
(4.21-5.20)
4.78
(4.16-5.08)
4.16-5.08
4.16-5.20
20.82
(20.09-21.27)
20.95
(20.09-21.68)
20.93
(20.24-21.25)
20.24-21.25
20.09-21.68
4.17
(4.03-4.46)
4.14
(4.00-4.50)
4.12
(3.93-4.38)
3.93-4.38
3.93-4.50
4.61
(4.51-4.72)
4.62
(4.51-4.88)
4.60
(4.54-4.68)
4.54-4.68
4.50-4.88
4.32
(4.20-4.48)
4.31
(4.18-4.60)
4.27
(4.15-4.41)
4.15-4.41
4.15-4.60
1.79
(1.68-2.03)
1.85
(1.46-2.12)
1.87
(1.67-1.99)
1.67-1.99
1.46-2.12
serine
glutamic acid 3
proline
glycine
alanine
cystine
1998.
1
2
3
Asparagine and glutamine are converted to aspartic acid and glutamic acid during the hydrolytic portion of the method.
August 2003
29
Table 3 (continued). Summary of Amino Acid Analyses of Bollgard II Event 15985 Cottonseed Samples from
the1998 U.S. Field Trials.
Amino Acid
(% total AA)
valine
methionine
isoleucine
leucine
tyrosine
phenylalanine
lysine
histidine
Nontransgenic
reference
range1
Commercial
reference
range2
4.72-5.22
4.72-5.34
1.75
(1.49-1.98)
1.49-1.98
1.46-2.03
3.56
(3.45-3.78)
3.53
(3.38-3.71)
3.38-3.71
3.38-3.78
6.58
(6.45-6.86)
6.56
(6.44-6.94)
6.52
(6.43-6.65)
6.42-6.65
6.38-6.94
2.85
(2.73-2.91)
2.85
(2.66-3.05)
2.83
(2.72-2.96)
2.72-2.96
2.66-3.05
5.68
(5.54-5.79)
5.70
(5.58-5.84)
5.66
(5.51-5.75)
5.51-5.75
5.51-5.84
5.10
(4.81-5.46)
5.08
(4.84-5.50)
5.11
(4.90-5.55)
4.88-5.55
4.83-5.55
3.07
(3.00-3.13)
3.09
(3.01-3.23)
3.09
(3.06-3.12)
3.06-3.12
3.01-3.23
15985
DP50B
(parent)
4.97
(4.77-5.34)
4.94
(4.72-5.34)
DP50
(nontransgenic
control)
4.89
(4.72-5.22)
1.71
(1.55-1.97)
1.75
(1.46-2.03)
3.58
(3.47-3.79)
1998.
1
Range includes data from four commercially available non-transgenic cotton varieties.
2
August 2003
30
Table 3 (continued). Summary of Amino Acid Analyses of Bollgard II Event 15985 Cottonseed Samples from the 1998
U.S. Field Trials.
Amino Acid
Bollgard II
(% total AA) cotton event
15985
arginine
tryptophan
DP50B
(parent)
DP50
(non-transgenic
control)
Nontransgenic
reference
range1
Commercial
reference
range2
11.37
(10.69-11.95)
11.24
(6.88-11.96)
11.49
(10.98-11.80)
10.98-12.10
6.88-12.17
1.02
(0.95-1.23)
1.03
(0.93-1.20)
1.03
(0.94-1.22)
0.94-1.22
0.93-1.26
1998.
1
2
August 2003
31
Table 4. Summary of Fatty Acid Analyses of Bollgard II Event 15985 Cottonseed Samples from the 1998 U.S.
Field Trials.
Nontransgenic
reference
range1
Commercial
reference
range2
0.77-2.40
0.64-2.40
25.81
(24.30-28.10)
24.30-28.10
23.40-28.10
0.58
(0.43-0.68)
0.63
(0.43-0.98)
0.43-0.98
0.43-0.98
2.63
(2.41-3.10)
2.38
(2.24-2.60)
2.30
(2.06-2.71)
2.06-3.11
2.06-3.11
15.58
(13.60-18.10)
15.59
(13.30-18.10)
15.40
(12.90-17.40)
12.90-20.10
12.90-20.10
linoleic
(18:2)
52.52
(47.70-55.50)
53.10
(49.00-55.80)
53.31
(49.50-57.10)
46.00-57.10
46.00-57.10
linolenic and
gamma
linoleic
(18:3)
0.13
(0.050-0.29)
0.14
(0.05-0.55)
0.11
(0.05-0.31)
0.05-0.31
0.05-0.55
Fatty Acid
(% total
fatty acids)
Bollgard II
cotton event
15985
DP50B
(parent)
myristic
(14:0)
1.26
(0.88-2.94)
0.92
(0.74-1.91)
DP50
(nontransgenic
control)
1.02
(0.77-2.15)
palmitic
(16:0)
25.80
(24.50-27.90)
25.92
(24.90-27.60)
palmitoleic
(16:1)
0.56
(0.33-0.65)
stearic (18:0)
oleic (18:1)
1998.
1
2
August 2003
32
Table 4 (continued). Summary of Fatty Acid Analyses of Bollgard II Event 15985 Cottonseed Samples from the
1998 U.S. Field Trials.
Fatty Acid
(% total
fatty acids)
Nontransgenic
reference
range1
Commercial
reference
range2
0.27
(0.24-0.34)
0.24-0.34
0.24-0.36
0.15
(0.11-0.23)
0.14
(0.12-0.24)
0.12-0.24
0.11-0.24
0.12
(0.05-0.26)
0.14
(0.05-0.29)
0.05-0.29
0.05-0.29
Bollgard II
cotton event
15985
DP50B
(parent)
DP50
(non-transgenic
control)
arachidic
(20:0)
0.30
(0.25-0.43)
0.29
(0.25-0.36)
behenic
(22.0)
0.14
(0.12-0.21)
lignoceric
(24:0)
0.14
(0.05-0.26)
1998.
1
2
August 2003
33
Table 5. Summary of Fatty Acid Analyses of Bollgard II Event 15985 Cottonseed Oil Samples from the 1998
U.S. Field Trials1.
1
2
Fatty Acid
(% total fatty
acids)
Bollgard II
cotton event
15985
DP50B
(parent)
Commercial
reference
range2
0.980
DP50
(nontransgenic
control)
1.06
myristic (14:0)
1.32
pentadecanoic
(15:0)
palmitic (16:0)
<0.100
<0.100
<0.100
<0.100
23.9
25.2
25.3
22.7-26.3
palmitoleic (16:1)
0.832
0.735
0.78
0.735-0.954
heptadecanoic
(17:0)
stearic (18:0)
<0.100
<0.100
<0.100
<0.100
2.04
2.34
2.04
1.98-2.34
oleic (18:1)
15.1
15.7
14.7
14.7-17.8
linoleic (18:2)
55.6
53.7
54.9
51-54.9
linolenic and
gamma linoleic
(18:3)
arachidic (20:0)
0.171
0.152
0.145
0.120-0.152
0.176
0.244
0.178
0.178-0.244
behenic (22:0)
<0.100
0.103
<0.100
<0.100-0.103
0.923-1.45
Values represent samples pooled from eight U.S. field sites in 1998.
August 2003
34
Table 5 (continued). Summary of Fatty Acid Analyses of Bollgard II Event 15985 Cottonseed Oil Samples from
the 1998 U.S. Field Trials 1.
Fatty Acid
(% total fatty
acids)
lignoceric (24:0)
1
2
Bollgard II
cotton event
15985
<0.100
DP50B
(parent)
DP50
Commercial
(non-transgenic
reference
control)
range2
<0.100
<0.100
<0.100
August 2003
35
Table 6. Summary of Mineral Analyses of Bollgard II Event 15985 Cottonseed Samples from the 1998 U.S. Field
Trials1.
Nontransgenic
reference
range2
Commercial
reference
range3
0.12-0.33
0.12-0.33
7.48
(4.39-10.35)
4.39-10.35
4.39-10.35
51.13
(41.84-60.76)
54.13
(42.57-72.15)
42.57-72.15
41.84-72.15
0.41
(0.37-0.47)
0.41
(0.37-0.49)
0.41
(0.37-0.47)
0.37-0.47
0.37-0.49
manganese
(mg/kg DW)
14.11
(11.96-16.53)
14.10
(11.17-16.81)
14.11
(12.16-16.39)
12.16-18.31
11.17-18.31
phosphorus
(% DW)
0.70
(0.58-0.83)
0.71
(0.61-0.88)
0.73
(0.63-0.86)
0.63-0.86
0.61-0.88
potassium
(% DW)
1.16
(1.07-1.24)
1.15
(1.09-1.22)
1.15
(1.08-1.23)
1.08-1.24
1.08-1.25
Mineral
Bollgard II
cotton event
15985
DP50B
(parent)
calcium
(% DW)
0.15
(0.13-0.19)
0.15
(0.13-0.20)
DP50
(nontransgenic
control)
0.15
(0.12-0.20)
copper
(mg/kg DW)
7.18
(4.27-10.12)
7.24
(4.39-9.51)
iron
(mg/kg DW)
50.83
(43.92-57.56)
magnesium
(% DW)
1
Values represent samples taken from eight U.S. field sites in 1998.
3
2
August 2003
36
Table 6 (continued). Summary of Mineral Analyses of Bollgard II Event 15985 Cottonseed Samples from the
1998 U.S. Field Trials1.
Mineral
15985
DP50B
(parent)
sodium
(% DW)
0.14
(0.067-0.21)
0.15
(0.039-0.30)
DP50
(nontransgenic
control)
0.14
(0.04-0.25)
zinc
(mg/kg DW)
40.30
(27.70-52.50)
41.06
(27.39-51.20)
40.97
(31.66-48.62)
1
Nontransgenic
reference
range2
0.0054-0.25
0.0054-0.30
31.66-48.62
27.39-51.20
3
2
August 2003
37
Commercial
reference
range3
Table 7. Summary of Toxicant Analyses of Bollgard II Event 15985 Cottonseed Samples from the 1998 U.S. Field
Trials1.
Total gossypol
(% DW)
CPFA
malvalic (C-17)
(% total fatty acids)
CPFA
sterculic (C-18)
CPFA
dihydrosterculic (C-19)
Aflatoxin B1 (ppb)
Bollgard II
cotton event
15985
DP50B
(parent)
1.00
(0.79-1.29)
0.45
(0.26-0.71)
0.97
(0.78-1.24)
0.39
(0.22-0.51)
DP50
(nontransgenic
control)
0.96
(0.72-1.23)
0.39
(0.17-0.61)
Nontransgenic
reference
range2
Commercial
reference
range3
0.72-1.23
0.71-1.24
0.17-0.61
0.17-0.61
0.30
(0.21-0.58)
0.25
(0.16-0.44)
0.24
(0.13-0.43)
0.13-0.56
0.13-0.66
0.18
(0.12-0.22)
0.15
(0.11-0.17)
0.16
(0.12-0.19)
0.12-0.22
0.11-0.22
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
<1.00
Aflatoxin B2 (ppb)
Aflatoxin G1 (ppb)
Aflatoxin G2 (ppb)
1
2
3
August 2003
38
Table 8. Summary of Analyses of Bollgard II Event 15985 Cottonseed Meal and Oil Samples from the 1998
U.S. Field Trials1.
OIL
Free
gossypol
(%FW)
OIL
Total
gossypol
(%FW)
OIL
Vitamin E
(mg/100g)
MEAL
Free
gossypol
(%FW)
MEAL
Total
gossypol
(%FW)
1
2
August 2003
Bollgard II
cotton event
15985
<0.005
DP50B
(parent)
Commercial
reference range2
<0.005
DP50
(non-transgenic
control)
<0.005
<0.005
<0.005
<0.005
<0.005
59.8
45.1
53.4
45.1-58.5
0.037
0.042
0.041
0.025-0.068
0.986
1.05
1.04
0.933 - 1.43
Range includes data from five commercially available cotton varieties.
39
<0.005
Table 9. Summary of Cyclopropenoid Fatty Acid Analyses of Bollgard II Event 15985 Cottonseed Oil Samples
from the 1998 U.S. Field Trials1.
Cyclopropenoid
Fatty Acid
1
2
Bollgard II
cotton event
15985
DP50B
(parent)
Commercial
reference
range2
0.384
DP50
(nontransgenic
control)
0.377
malvalic (C-17)
0.378
sterculic (C-18)
0.205
0.227
0.217
0.216-0.289
dihydrosterculic
(C-19)
0.165
0.169
0.146
0.146-0.202
Range includes data from five commercially available cotton varieties.
August 2003
40
0.294-0.405
Table 10. Summary of Statistically Significant Differences in Composition for Bollgard II Event 15985
Cottonseed
Samples from the 1998 U.S. Field Trials.
p
Difference
Significant 15985 DP50B
Mean
Number of Commercial
Range 2
Value as Percent
Parameter Mean (Control) Difference Sites with
of Control
Mean
Significant
Differences1
myristic 1.26
0.92
0.33
2
0.64-2.40
0.004
36%
acid
1
2
stearic
acid
2.63
2.38
0.25
3
2.06-3.11
<0.001
11%
linoleic
acid
52.52
53.1
-0.58
1
46-57.10
0.038
1%
malvalic
acid
0.45
0.39
0.058
0
0.17-0.61
0.024
15%
sterculic
acid
0.30
0.25
0.054
0
0.13-0.66
0.034
22%
dihydrosterculic
acid
0.18
0.15
0.036
3
0.11-0.22
<0.001
24%
Data is from four replicated sites.
August 2003
41
Table 11. Summary of Cry2Ab Protein Studies on Non-Target Organisms
Test Organism
Test Substance
Bobwhite Quail
Cry2Ab corn
grain
Cry2Ab
cottonseed
Results1
Conclusions
No mortality or toxic effects in birds
consuming Cry2Ab corn grain or
Cry2Ab cottonseed at greater than
100,000 ppm
Cry2Ab corn grain or cottonseed
poses minimal risk.
Adult Honey
Bee
Cry2Ab protein
NOEC = 68 µg Cry2Ab/ml diet
No effects at maximum environmental
concentration.
Larval Honey
Bee
Cry2Ab
2
protein
NOEC = 170 µg Cry2Ab/ml , single
dose
concentration.
Ladybird Beetle
Cry2Ab protein
concentration.
Collembola
Cry2Ab protein
NOEC = 313 µg Cry2Ab/g diet
Minimal risk indicated.
Green
Lacewing
Larvae
Cry2Ab protein
NOEC = 1100 µg Cry2Ab/g diet
concentration.
Parasitic
Hymenoptera
(Wasp)
Cry2Ab protein
concentration.
Earthworm
Cry2Ab protein
NOEC = 330 mg Cry2Ab/kg dry
soil
concentration.
1
NOEC is the no observable effect concentration
August 2003
42

Bollgard II Cotton Safety Summary

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