Document ID: EPA-HQ-OPP-2007-0306-0045
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2008-10-29T04:00Z

OFFICE OF

PREVENTION, PESTICIDES, AND

TOXIC SUBSTANCES

MEMORANDUM					Date: February 11, 2008

DP Barcode: 337040

							

SUBJECT:  EPA Review of Syngenta Seed’s VipCot™ Cotton Insect
Resistance Management Plan for Section 3 Full Commercial Registration
[EPA Reg. No. 67979-O, MRID 470176-34] 

TO:	Alan Reynolds, Regulatory Action Leader

	Microbial Pesticide Branch

	Biopesticides and Pollution Prevention Division (7511P)

FROM: 	Jeannette Martinez, M.S., Biologist

	Microbial Pesticide Branch

	Biopesticides and Pollution Prevention Division (7511P)

ACTION REQUESTED:

Cot™ cotton.

CONCLUSIONS AND RECOMMENDATIONS

The BPPD/IRM team concludes the following based on the review of
‘dose’ studies including some information from the efficacy
submission (MRID 470176-33):

COT67B expresses a ‘high-dose’ against PBW 

VipCot™ expresses a ‘high-dose’ against PBW 

COT67B expresses a ‘near high-dose’ against TBW and CBW 

VipCot™ expresses a ‘near high-dose’ but may express a
‘high-dose’ against TBW and CBW. While results of any two
verification methods together did not meet the exact definition of a
high-dose toxin, the overall dose results together appear to indicate
that VipCot™ may still fit into the existing paradigm. However, field
efficacy data submitted for 2005 and 2006 indicate that under field
conditions, VipCot™ cotton may not always express a ‘high-dose’
against TBW and CBW.

COT102 does not express a ‘high-dose’ nor a ‘near high-dose’
against any of the three target pests

t structural information is not available for the Vip3A protein,
available information gives no indication of a similar domain
organization or α-helical bundle region within the polypeptide sequence
as exists for the Cry proteins.

3. 	Syngenta commissioned Dr. Michael Caprio to 1) evaluate the risk of
resistance evolving in TBW and CBW to VipCot™ cotton and 2) explore
the impact of VipCot™ on other single gene Bt cotton events such as
for Cry1Ac. 

BPPD does not request that further modeling be done and concludes that
the provided simulation results are sufficient to support the refuge
strategies requested for VipCot™ cotton. The refuge options proposed
by Syngenta match the presently in use refuge strategies for cotton (see
section VII). While BPPD has reservations about the ‘high-dose’
assumptions for some toxins against TBW and CBW (see dose section IV for
specifics), it appears that VipCot™ may sometimes fall into the
existing paradigm for what constitutes an ‘effective’ high-dose
pyramided product. However, since the conclusion of delayed resistance
in TBW to Cry1A toxins hinges greatly on the assumption that Vip3A
mortality is high, the evolution of resistance in TBW following
introduction of VipCot™ can be expected to occur in less time than
predicted by the model. Similarly for CBW, the evolution of resistance
to Cry1A toxins may not be delayed by the introduction of VipCot™
based on Vip3A mortality.

he approach has proven successful, and pests are susceptible and
population variance is small. In addition, follow-up testing of larval
survivors needs to be conducted for all toxins where field population
survivorship (≥2 instar) is significantly different from lab/reference
colony’s survivorship. 

Specifically for CBW (but not only), BPPD has the following
recommendations: if a good amount of effort has been put into developing
a discriminating or diagnostic concentration for CRW and FLCry1Ab and
there is evidence that the diagnostic concentration cannot be achieved
due to i.e. high-variability in response to the toxin, then a comparison
in baseline susceptibility (i.e. LC50s) may be a feasible approach to
monitoring. Estimated LC50s may serve well as a baseline monitoring tool
for shifts in susceptibility to Bt toxins; however, the LC50 approach is
not useful in discriminating resistant from susceptible individuals.
Therefore, this approach must then be linked with follow-up testing of
populations with elevated LC50s relative to previously established
baseline susceptibility. Furthermore, BPPD recommends that Syngenta
consider head capsule width assay and DNA markers in lieu of mortality
based diagnostic concentrations.

5. 	BPPD concludes that Syngenta has included the major requirements
needed by a compliance program and outlined by Agency in the first
paragraph of section X and the 2001 Bt crop reassessment document.
Syngenta’s proposed CAP resembles CAPs for other introduced Bt PIPs
and appears to meet the Agency’s requirements at this time. 

 to VipCot™ cotton.

7.	BPPD requests for future reports that Syngenta state all assumptions
and procedures clearly followed by a thorough description of the full
range of study results in order to avoid confusion and guessing by the
BPPD IRM team. In addition, if there are unusual events taking place at
any time during an experiment that are reflected in the end result, such
as for example high control mortality, BPPD requests that Syngenta
explain what they think the underlying cause may have been.



BACKGROUND 

™ cotton is a pyramided transgenic cotton trait that expresses
full-length Cry1Ab insecticidal protein (COT67B event) and Vip3Aa19
(COT102 event). The Vip3A is different from Cry proteins as it is
produced during vegetative growth of the bacteria, does not form
parasporal crystal proteins, and is secreted (but not processed upon
secretion) from the cell as a soluble protein. Its physical
manifestations of intoxication, however, resemble those of Cry proteins
(gut paralysis and lysis of midgut epithelial cells) (Schnepf et al.
1998). Activated Vip3A does not bind to the same receptors as Cry1Ab
(APN and cadherin-like receptor); the two Bt proteins do not seem to
share binding sites. Lee et al. (2003) have investigated the mode of
action of the Vip3A protein and determined that it involves a number of
steps much like the mode of actions for the δ-endotoxins. Following
ingestion by the lepidopteran target pest, the Vip3A protein becomes
soluble in the gut and is then processed into four dominant bands
(retaining activity). The authors propose that this processing is
required for the bioactivity of the toxin (activation step). Interaction
with the midgut epithelium is the next likely step in the mode of action
of Vip3A. However, Vip3A does not bind to APN and cadherin-like
glycoprotein receptors as does Cry1Ab (as already stated by Schnepf et
al. (1998)) and supported by the researchers’ competition study. Upon
binding to midgut epithelial receptors, data supports the existence of a
pore-forming step that creates ion channels which are structurally and
functionally distinct from those of Cry1Ab. Direct structural
information is missing for Vip3A; however, preliminary data do not
support the notion that the two proteins share similar domain
organization or an α-helical bundle region.

COT102 cotton expresses the vegetative insecticidal protein (VIP3A),
which was isolated from Bacillus thuringiensis strain AB88. The cotton
line Coker 312 (Gossypium hirsutum L. cv Coker 312) was transformed via
Agrobacterium transformation procedures with synthetic vip3A(a) gene
encoding VIP3A protein and the selectable marker gene aph4 encoding the
enzyme APH4. The transformation event that produced the transgenic
cotton line, designated COT102, was transformed with plasmid pCOT1.
COT102 is intended to protect cotton from feeding by the primary
lepidopteran pests: tobacco budworm (Heliothis virescens, TBW), cotton
bollworm (Helicoverpa zea, CBW), and pink bollworm (Pectinophora
gossypiella, PBW). Based on cotton insect loss data from 1991-2000, the
primary target pests, TBW, CBW, and PBW, account for more than 77% of
the yield loss and 84% of the insecticide use due to lepidopteran
infestation in cotton.

In 2004, BPPD reviewed Syngenta Seed’s IRM plan for VipCot™ and
determined that the registrant did not provide sufficient data to
formulate an IRM strategy. Specifically, The BPPD IRM team concluded
that data or published literature was needed to address the pest biology
of each target pest; additional data was required to make high-dose
claim for COT102 using two of the five techniques described by 1998
FIFRA Science Advisory Panel; baseline susceptibility and diagnostic
concentrations needed to be established for all target pests; estimates
of initial resistant allele frequency as well as models of evolution of
resistance for Vip3A19 should be provided; additional cross-resistance
data was required for target pests using Vip3A19, Cry1Ac, and Cry2Ab2;
and specific monitoring plans, remedial action strategies, grower
education program, compliance assurance program and research activities
for COT102 needed to be provided.

PEST BIOLOGY AND ECOLOGY

A Summary of the biology and ecology for major Bt cotton target pest,
such as TBW, CBW, and PBW, can be viewed in IRM section of the
Agency’s 2001 Bt crop reassessment document at   HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm .

DOSE

The determination of dose, or the amount of toxin expressed by the
transgenic crop relative to the susceptibility of the target pests, is a
critical component of IRM.  Models have shown that a high-dose of toxin,
coupled with a non-transgenic refuge to provide a supply of susceptible
insects, is the most effective strategy for delaying resistance in Bt
crops.  The high-dose/refuge strategy assumes that resistance to Bt is
recessive and is conferred by a single locus with two alleles resulting
in three genotypes: susceptible homozygotes (SS), heterozygotes (RS),
and resistant homozygotes (RR).  The high-dose/refuge strategy also
assumes that there will be a low initial resistance allele frequency and
extensive random mating between resistant and susceptible adults.  In
practice, a high-dose PIP should express sufficient quantities of toxin
to kill all susceptible insects (SS) as well as heterozygous insects
with one resistance allele (RS).  Lower dose PIPs might allow for
survival of insects with at least one susceptibility allele (SS or RS),
although effective IRM may still be possible with a suitable refuge
strategy.  To be able to demonstrate high-dose, it is recommended that
registrants generate data by at least two of the five laboratory and
field approaches as outlined by the SAP (1998) and described by the
Agency in the 1998 Bt Plant-Pesticides and Resistance Management
document (US EPA, 1998) and 2001 Biopesticide Registration Action
document (US EPA, 2001). For procedures of high-dose determination, see
Appendix A at the end of BPPD’s review.

It must be noted that both the high-dose definition and verification
techniques were developed in 1998 when all of the registered Bt crops
were single toxin products targeted against lepidopteran pests.  In
recent years, PIPs in Bt cotton have been approved that contain two
genes targeted at the same insect pest.  These “pyramided” products
can be beneficial for IRM since target pests must overcome two toxins to
develop field resistance to the PIP.  The benefits are greatest for two
toxins with unrelated modes of action (i.e. binding to different Bt
receptor sites in the midgut) that are expressed at high-doses in the
plant (Roush 1994).  

For pyramided products, the dose of each toxin should be evaluated
separately.  This can be easily accomplished if the pyramided product is
created through conventional breeding -- in this case, the dose of the
single toxin products has already been established and the combined dose
in the pyramided PIP can be determined with comparative efficacy
studies.  However, for pyramids created by non-conventional breeding
(e.g. recombinant DNA techniques), defining the dose can be more
complicated since single toxin lines may not be available (or
commercialized) for comparisons. The dual toxins can also be evaluated
collectively to determine an “effective” high-dose.  In some
examples, each toxin by itself may not supply a high-dose, but in
combination a sufficient control (>95% of heterozygotes) is provided and
can be considered high-dose.

The native vip3Aa1 gene was cloned from Bt strain AB88 originally
isolated from milk. The Vip3A protein expressed in COT102 plants is
produced by a synthetic vip3Aa19 gene that encodes a polypeptide of 789
amino acids. This protein is identical to the native Bt Vip3Aa1 protein
aside from a single conservative amino acid change that has no apparent
effect on Vip3A protein function.

The full-length Cry1Ab protein encoded in COT67B is similar to that
produced in Bt subsp. kurstaki strain HD-1 and only differs from its
native FLCry1Ab protein in that it contains 26 additional consecutive
amino acids in the C-terminal portion (Geiser motif).

02, and VipCot™ cotton plant material. Three sets of experiments were
conducted: 1) bioassays with the single proteins expressed in
lyophilized plant material and both proteins expressed in lyophilized
plant material and combined as VipCot™ to determine target pest
susceptibility (TBW, CBW, PBW), 2) field tests on VipCot™ and COT67B
plants and control plants (artificial infestation) during the 2006
growing season, and 3) tolerance assays with single proteins expressed
in fresh plant material and both proteins expressed in fresh plant
material and combined as VipCot™ to determine susceptibility of
neonates as compared to fourth instar larvae (N.C. State University lab
colony). A description of procedures for high-dose determination is
provided in Appendix A at the end of this review. 

Verification Methods:

of COT102, COT67B, VipCot™, and Coker 312 cotton. Mortality data for
H. zea, H. virescens, and P. gossypiella were collected. 

At 25X dilution, percent mortality on COT102 for TBW ranges from 66.7-
95.0%, and for CBW mean corrected mortality reported (one lab only) is
72.4%. For PBW, mean corrected mortality reported (by one lab only) is
16.7%. These data seem to indicate that COT102 alone has minimal
efficacy against PBW and an efficacy against TBW ranging from
intermediate to high. COT102 efficacy against CBW does not appear to be
very high judging from these test results. Based on the range of
susceptibility observed in CBW for other Bt PIPs, it can be expected
that the range of susceptibility for COT102 in this target pest is
rather wide as well and that 72% mortality likely represents an upper
value in this potential range. 

At 25X dilution, percent mortality on COT67B for TBW and PBW is reported
to be 100% and for CBW ranges from 98.3- 100%. The dilution bioassay for
VipCot™ was conducted at SBI only; their results suggest that at 25X
dilution, mortality on VipCot™ for TBW, CBW, and PBW is 100%. Based on
this verification method alone, VipCot™ provides an ‘effective’
high-dose against all three target pests, while COT102 provides no
high-dose to all three target pests and COT67B expresses a high-dose
against TBW and PBW (but not CBW). 

2)  Unlike the artificial diet bioassay, the second method to verify
high-dose was conducted on VipCot™ and COT67B plants in the field (US
EPA method #4) using artificial infestations of laboratory strains of
TBW and CBW (Kurtz (2006), Appendix 2). The purpose of this approach was
to determine whether the dose of toxins expressed in VipCot™ and
COT67B was at or greater than the LD99 for the key target pests.
Specifically, TBW survival was measured on both COT67B and VipCot™,
while CBW survival was measured on VipCot™ cotton only.

In 2005, two field trials were conducted for COT67B of which one was
held in Mississippi and the other in Florida. Unreplicated blocks of
>1,000 plants were planted with one control block of Coker 312 plants at
each site. Infestation with TBW was simulated by spraying eggs onto
cotton plants (greater infestation was conducted on Bt variety due to
low survival expectancy). Survival on control plants was estimated by
collecting leaves containing TBW eggs from Coker 312 plants (50% and 75%
of field inspected in MS and FL, respectively) and counting successful
larval hatching. Survival of TBW larvae on COT67B plants was estimated
by visually assessing Bt- plants for larval presence in each field in MS
and FL, respectively. When survivors were found, the plants were marked
and reexamined for further larval survival after four to seven days.
Syngenta states in their report that in MS and FL one and two surviving
TBW larvae, respectively, were found after seven days of artificial
infestation (7DAI) and zero survivors after 14 days (14DAI). The second
set of observations (14 DAI) may not be as reliable as the initial
assessment of survivors because it appears that bolls were not
‘caged’ to prevent larval loss or escape. Thus, BPPD will base its
review and conclusion on the first set of observations (7DAI). In
addition, it is unclear to BPPD from reading the report what fraction of
COT67 fields were surveyed in each location. Syngenta states that only a
fraction of the control plots were searched for survivors due to
limiting resources but fails to clarify in their report whether the same
restrictions apply for surveys conducted in treatment plots.

VipCot™ provides an ‘effective’ high-dose for TBW or CBW under
this method. 

3)  The third verification method for high-dose was conducted as a
tolerance bioassay with purified toxins as well as leaf disks to
determine if a later larval instar of the targeted pest could be found
with an LD50 that was approximately 25-fold higher than that of the
neonate larvae (US EPA method #5) on VipCot™, COT 102, and COT67B
plants for CBW and on COT102 for TBW (Kurtz (2006), Appendix 3). The SBI
lab conducted the purified toxin (with Vip3A and FLCry1Ab) and leaf disk
study (on COT102, COT67B, and VipCot™) for CBW; JH lab conducted the
purified toxin (with Vip3A) and leaf disk study (on COT102) for TBW.

Syngenta Biotechnology, Inc. provided leaf disk results on COT67B,
COT102, and VipCot™ cotton for CBW that cannot be interpreted for
following reasons: while each experiment had two replicates, two of the
three tests conducted for each Bt variety had excessive control
mortalities which ranged from 28% to 81%. This indicates that some
conditions were suboptimal during the course of the experiment. BPPD
speculates that the additional mortality is due to the laboratory colony
being adapted to their artificial diet and that insects are no longer
fit enough to survive on this natural host, i.e. cotton. While there is
evidence that the two toxins alone and the combined events cause
mortality in CBW, BPPD finds the results inconclusive and cannot
determine if each toxin alone or as a pyramided event express a
‘high-dose’ against CBW.

Syngenta reports that the tolerance assay conducted with the purified
toxin Vip3A and tested on CBW did not allow estimating LC50 for later
instars because the estimates were well in excess of the highest
concentration tested. Syngenta concludes that later instars are at least
25X more tolerant to Vip3A than neonate larvae. BPPD recognizes that
there are several issues with the tolerance assay data reported: 1)
Mortality data for neonates are highly variable from bioassay to
bioassay with LC50 estimates ranging from 504.6ng/cm2 to 2669ng/cm2. 2)
Mortality data for neonates within any test are not steadily increasing
with increasing toxin concentrations but show a trend to oscillate.
Mortality data for later instar larvae, of course, follow the same trend
as described for neonates under 1 and 2. 3) Where an LC50 could be
estimated for both, neonates and later instars, the difference between
estimates is only 8 fold as opposed to the 25 fold requirement. 4) While
Syngenta reports that no LC50 could be estimated for later instars in 4
of the 5 tests because the estimate was well in excess of the highest
concentration tested, the report lacks a further description of the
data. At a minimum, the data show that two of the four tests had actual
mortality data (older larvae) near the 50% level at some of the measured
concentrations where the estimated LC50s were predicted to be in excess
of the concentrations tested (Table 1 and 4, Appendix 3) and that the
same measured mortalities were not linearly increasing. In addition, CBW
susceptibility, especially for older instars but also for neonates,
appears to be highly variable from bioassay to bioassay as visible in
Tables 1 through 5 for Vip3A. Considering the weight of evidence and
uncertainties from above, BPPD cannot conclude that Vip3A express a
high-dose toxin against CBW.

Two tolerance bioassays were conducted with FLCry1Ab on CBW (Tables 6
and 7, Appendix 3). In these tests, mortality data for later instars and
neonates were more consistently increasing with increasing
concentrations (when compared to the Vip3A neonate and later instar
mortality data). Later instar LC50 estimates were greater than neonate
LC50 estimate (>25-fold); BPPD agrees that these data support that
FLCry1Ab expresses a high-dose toxin against CBW under method #5. 

Syngenta reports that the tolerance bioassays conducted with Vip3A on
TBW neonate and 2nd instar larvae show a 36 fold difference in
susceptibility/ LC50 estimates. BPPD would like to add that there were
two estimated LC50s for older larvae of which one showed a 21-fold and
the other 36-fold difference from neonate susceptibility. The 21-fold
difference does not support the high-dose claim. In addition, BPPD is
concerned about the quality of data provided for this bioassay. There
are data gaps at some concentrations for neonates as well as second
instar larvae tested, and the fluctuations in mortality (not steadily
increasing with increasing toxin concentration) do not seem indicative
of a high-dose toxin. Considering the weight of evidence and
uncertainties mentioned above, BPPD cannot conclude that Vip3A is a
high-dose but at the most a ‘near high-dose’ toxin for TBW under
method #5.

With respect to the leaf disk bioassay conducted (COT102) with TBW, BPPD
recognizes similar deficiencies for the JH as for SBI laboratory.
Control mortality for neonates or second instar larvae is above 10%
(ranging from 14%-27%) in two of the three experiments. Again, this
indicates that conditions for the experiments were suboptimal, and
therefore, the 100% mortality reported for neonates and later instar
larvae is not likely to reflect actual toxin mortality alone; mortality
results are confounded by an external factor. BPPD speculates, as
mentioned above, that the additional mortality is due to the laboratory
colony being no longer being fit enough to survive on this natural host,
i.e. cotton. BPPD concludes that these data are inconclusive because of
the confounding factor in the observed mortality.

4) Method #4 was conducted to verify high-dose in the field on COT67B
plants (and COT69D plants) under artificial infestation of laboratory
strains of PBW (Kurtz (2006), Appendix 4). The purpose of this approach
was to determine whether the dose of toxins expressed in COT67B was at
or greater than the LD99 for this key target pest.

The experiment was conducted at the University of California, Desert
Research and Extension Center. The experiment was set up as a randomized
complete block design with four replicates, COT67B plants as treatment,
and Coker 312 plants as controls. One-hundred bolls per plot were
artificially infested (twice) with PBW eggs supplied by USDA/ARS in
Phoenix, AZ. Eight days after infestation, 75 bolls from each plot got
harvested and evaluated for damage such as warts, mines, dead larvae,
and exit holes (indicating survival of larva). Surviving larvae and exit
hole data collected on Coker 312 plants served as a baseline to assess
infestation levels and PBW populations. 

No live larva larger than 1st instar was found on COT67B cotton bolls.
One exit hole in one boll was found out of 1,120 bolls of COT67B towards
the later time of the season and is possibly attributed to lower
expression levels of the toxin in aged plants. Mortality on COT67B
plants is estimated at 99.9%. Mortality in Coker 312 is significantly
lower and estimated at 40.3% with first instar larvae included. These
data support the conclusion that COT67B expresses a high-dose against
PBW.

BPPD’s Conclusions on High-Dose:

To be able to demonstrate high-dose, registrants are required to provide
data generated by at least two of the five laboratory and field
approaches as outlined by the SAP (1998) and described by the Agency in
the 1998 Bt Plant-Pesticides and Resistance Management document (US EPA,
1998). The BPPD/IRM team concludes the following based on the review of
‘dose’ data including information from the efficacy submission (MRID
470176-33):

COT67B expresses a high-dose against PBW based on data from verification
method1 and 4

VipCot™ expresses a high-dose against PBW based on data from
verification method 1 (verified only once by experiment, but conclusion
is reached based on COT67B determination). 

COT67B expresses a near high-dose against TBW and CBW based on data
provided by verification method 1 and 5

VipCot™ expresses a ‘near high-dose’ but may express a
‘high-dose’ against TBW. While results of any two methods together
did not meet the exact definition of a high-dose toxin/pyramided event,
the overall picture indicates that VipCot™ may still fit into the
existing paradigm. However, efficacy data submitted for 2005 and 2006
indicate that under field conditions, VipCot™ cotton may not always
express a high-dose against this target pest.

 VipCot™ expresses a ‘near high-dose’ but may express a
‘high-dose’ against CBW. However, efficacy data submitted for 2005
and 2006 indicate that under field conditions, VipCot™ cotton may not
always express a high-dose against this target pest.

COT102 does not express a high-dose against any of the three target
pests



Table   SEQ Table \* ARABIC  1  BPPD’s High-Dose Determination for
TBW, CBW, and PBW based on Experiments Conducted by Registrant

Species	Method 1	Method 4	Method 5

	COT102	COT67B	VipCot	COT67B	VipCot	COT102	COT67B	VipCot

TBW	No high-dose	High-dose	High-dose	***	***	Near high-dose, or ***1
Near high dose	---

CBW	No high-dose	Near high dose	High-dose	---	***	No high-dose, or ***1
High-dose1	***1

PBW	No high-dose	High dose	High-dose	High dose	---	---	---	---

Shaded fields indicate high-dose determinations by BPPD for single
toxins or stacked Bt product 

*** indicates that results were inconclusive

--- indicates Bt variety not tested under a particular method

1 either the tolerance assay or leaf disk bioassay results or both were
inconclusive

CROSS-RESISTANCE POTENTIAL

Analyses of resistance to Bt Cry proteins indicate that cross-resistance
occurs most often with proteins that are similar in structure
(Tabashnik, 1994; Gould et al., 1995). While direct structural
information of the Vip3A protein expressed in VipCot™ is missing (Lee
et al. 2003), this novel Bt protein does not share any sequence homology
with the known Bt Cry protein genes, and the predicted secondary
structure give no indication of a similar domain organization or
α-helical bundle region within the polypeptide sequence of Vip3A as
exists for the Cry proteins. Protein folding blasts reveal that Vip3A
may be a pore forming protein that has a structure of β-barrels
(Syngenta unpublished data). In order to further investigate the
potential for cross-resistance of Vip3A to Cry proteins, Syngenta
examined the mode of action of Vip3A at selected steps critical to the
mode of action of Bt Cry proteins: proteolytic activation, receptor
binding, and pore forming.

The first piece of analysis relates to the proteolytic activation in
both Bt toxins and shows that Vip3A and Cry1A proteins are
proteolytically activated upon solubilization in the midgut.
Syngenta’s experiments further demonstrate that both Vip3A and two Cry
proteins (Cry1Ac and Cry2Ab2) can be processed by either trypsin or gut
juice extracts. However, in Vip3A proteolysis occurs in susceptible as
well as non-susceptible insects and alone does not appear to be a key
factor in insect toxicity and specificity. Based on this information and
published literature stating that high levels of resistance have not
been found to correlate with the toxin activation step, Syngenta
speculates that the theoretical risk of cross-resistance is very small
at this particular step of the mode of action. 

Second, Syngenta investigated whether Vip3A and Cry proteins (Cry1Ab,
Cry1Ac, and Cry2Ab) shared the same receptor sites in Lepidoptera
species (M. sexta, H. virescens, and H. zea) by conducting receptor
binding studies with Amino Peptidase N and cadherin-like glycoproteins
(identified as putative Cry1A protein receptors) as well as others
identified to be Cry1Ac and Cry2Ab2 binding sites. Those studies show
that the protease activated form Vip3A does not bind to APN, the
ectodomaine of the cadherin-like protein, or other putative Cry1A toxin
binding proteins. In yet another study with H. zea and H. virescens, the
non-specific binding of Cry2Ab was not inhibited by the addition of
unlabeled Vip3A indicating that Vip3A does not bind to the Cry2Ab
binding sites. Syngenta further demonstrated that activated Vip3A bound
to two proteins of ca. 80 and 110 kDa and not to APN and cadherin-like
proteins. These binding studies demonstrate that there is little risk of
cross-resistance between Vip3A and Cry1Ab, Cry1Ac, and Cry2Ab2.

gives no indication of a similar domain organization or α-helical
bundle region within the polypeptide sequence as exists for the Cry
proteins. 

BPPD agrees with Syngenta that the potential risk for cross-resistance
between Cry1Ab (and other Cry1A proteins as well as Cry2Ab2) and Vip3A
appears low considering that: 1) Vip3A does not bind to APN and
cadherin-like proteins and thus, the two types of Bt toxins do not share
binding sites; and 2) Vip3A pore channels formed in the midgut of
insects are structurally and functionally distinct from Cry1Ab (and
maybe other Cry proteins). 

For general information regarding cross-resistance, Appendix B can be
consulted at the end of BPPD’s review.

MODELING 

EPA has used predictive models to compare IRM strategies for Bt crops.
Because models cannot be validated without actual field resistance,
models have limitations and the information gained from the use of
models is only a part of the weight of evidence used by EPA in assessing
the risks of resistance development. It was the consensus of the 2000
SAP Subpanel that models were an important tool in determining
appropriate Bt crop IRM strategies. They agreed that models were “the
only scientifically rigorous way to integrate all of the biological
information available, and that without these models, the Agency would
have little scientific basis for choosing among alternative resistance
management options.” They also recommended that models must have an
agreed upon time frame for resistance protection. For example,
conventional growers may desire a maximum planning horizon of five
years, while organic growers may desire an indefinite planning horizon.
The Subpanel recommended that model design should be peer reviewed and
parameters validated. Models should also include such factors as level
of Bt crop adoption, level of compliance, economics, fitness costs of
resistance, alternate hosts, spatial components, stochasticity, and pest
population dynamics. 

 to VipCot™ cotton. In the next few paragraphs, BPPD summarizes the
most important features and assumptions of the model and simulation
results for CBW and TBW. Later, BPPD comments on the input parameter
assumptions and applicability of the simulation results. 

≤100% of the Bt cotton planted was assumed to be VipCot™. The rate
at which resistance evolved was estimated by determining the amount of
time required until the average resistance allele frequency across all
fields exceeded 0.5. The second modeling approach explored the impact of
VipCot™ on other single gene Bt cotton events such as for Cry1Ac.
These second simulations assumed complete cross-resistance between Cry1A
and VipCot™ (making Cry1Ac and Cry1Ab functionally the same because of
reported cross-resistance) and no cross-resistance between Cry1A and
Vip3A.

Simulation results for H. zea indicate that there were few cases of
resistance (0.3%) for Cry1A toxin over 1000 simulations when 80% of all
cotton acres were assumed to be VipCot™ (with 10% of total corn
acreage planted to Cry1A hybrid); no resistance to Vip3A evolved in any
of the simulations. When 50% of the total corn acreage was planted to a
pyramided Vip3A x Cry1A hybrid, resistance to either protein did not
evolve after 400 model runs. In the first five years of the simulations,
the rate of increase for the Cry1A allele was lower than the rate of
increase for the Vip3A resistance allele; however, the rate measure was
strongly affected by the assumption of the initial resistance allele
frequency. The overall simulation results suggest that the introduction
of VipCot™ is not likely to select for resistance to Cry1A(b/c) and
Vip3A within 20-25 years.

 evolved in the mosaic (VipCot™ and Cry1A) within the same time frame.
Under no circumstances did evolution to Vip3A occur, which means,
product failure (resistance to both toxins) did not occur. 

Simulation results for H. virescens indicate that there are few cases of
resistance evolving to the Vip3A toxin and that most values (number of
occurrences) clump around the frequency of 10-3. There is a 0.2% chance
of resistance evolving to Vip3A and Cry1Ab (product failure) within a 25
year time. The most likely outcome for the resistance allele was either
‘no change’ or ‘slight decline’ in frequency. Equilibrium for
Vip3A resistance allele at around 0.0032 except for when resistance
evolved for Cry1A allele leading to considerable variation in the final
resistance allele frequency for Vip3A. This suggests that the
equilibrium value for the Vip3A allele is dependent on interactions
between two loci which generate an effect similar to overdominance.
Whether this is an error in the model or an effect to multi-locus
overdominance still needs to be further investigated. Dr. Caprio
concludes that for the moment it appears that high-dose in combination
with fitness cost may lead to unusual results.

™ on a Cry1A gene expressed in other Bt-cotton products (mosaic ratio
1:1 vs. Cry1A only) and indicate that there may be rapid evolution of
resistance to a Cry1A cotton product in absence of VipCot™ (despite
high-dose against H. virescens). Like in the case of H. zea, the
introduction of VipCot™ is expected to decrease the risk of resistance
in TBW to the Cry1A toxin in a single trait cotton product. In the
simulations, there was a 1% chance of resistance evolving within 20
years and 4% chance that resistance would evolve in 25 years to
VipCot™. 

For H. zea and H. virescens, comparison of VipCot™ simulations alone
versus the mosaic simulation results indicate that the presence of a
Cry1A single gene cotton product may seriously reduce the effectiveness
of resistance management strategies for dual gene products.

Syngenta provided Dr. Caprio with the following critical information
based on interpretations of laboratory and field results: Vip3A
mortality in H. zea and H. virescens was assumed to have a maximum of
0.975 (near high-dose assumption) and minimum of 0.875 with the most
likely mortality being at 0.92. Similarly, COT67B mortality in H. zea
and H. virescens was assumed to have a maximum of 0.999 (high-dose
assumption for both species) and a minimum of 0.95 (for H. zea). For H.
zea, the most likely mortality was chosen to be 0.999, the high-dose.
For H. virescens the assumption was that COT67B provides only a
high-dose. BPPD has reservations about some of these assumptions that
went into the model. 1) The high-dose assumption for COT67B in CBW
cannot be backed with the data submitted. For high-dose verification
method #1, the reported CBW mortality ranged from 98 -100%. For method
#5, high-dose expression of COT67B was demonstrated. Therefore, BPPD
feels more comfortable assuming the following: at most, COT67B expresses
a ‘near high-dose’ concentration against CBW but likely expresses
less than a ‘near high-dose’. It is unlikely based on the data
provided by Syngenta (and what is known about variation in
susceptibility of CBW towards other toxins) that the most likely CBW
mortality due to COT67B will be 99.9%. In addition, the ‘near
high-dose’ assumption for Vip3A against CBW is not supported by the
data. 2) The COT67B high-dose assumption for TBW has been verified by
method #1 only. The second verification method (#5) produced a near
high-dose only for COT67B. Method #4 still has some outstanding
questions associated with the data that were submitted to Syngenta, and
BPPD is still awaiting a response. The most likely mortality value
chosen for COT67B of 0.999 is questionable as well, and BPPD feels that
until Syngenta is able to provide answers to BPPD’s outstanding
questions, the more conservative value of 0.95, together with a maximum
and minimum mortality value of 0.999 and 0.90 respectively, are more
appropriate mortality input values. BPPD presently assumes that COT67B
expresses a ‘near high-dose’ for TBW. Much like for CBW, BPPD
concludes that the ‘near high-dose’ assumption for Vip3A against TBW
is not supported by the data. 

Evolution of resistance to Cry1A toxin in CBW is predicted to occur well
beyond the life-time expectancy for any Bt-product. As expressed
earlier, BPPD has reservations about the mortality parameters and does
not agree that the Cry1Ab toxin is expressed at or near high-dose in
CBW. It is unclear how much or how little such a change in mortality
affects the outcome of evolution of resistance in CBW.

Evolution of resistance to Cry1A and Vip3A toxin in TBW (assuming
high-dose and near high-dose, respectively) is predicted to occur well
beyond the life-time expectancy for any Bt-product. BPPD has outstanding
questions about the COT67B dose data submitted by Syngenta and has not
been able to reach the same conclusion as the registrant. If COT67B was
expressed at near high-dose instead of the high-dose assumption in the
model, it is unclear how much or how little such a change in mortality
affects the evolution of resistance in TBW.

™ hinges greatly on the assumption that mortality caused by Vip3A
(COT102) is high (ranging between 0.875 – 0.975 with most likely
mortality being 0.92). BPPD notes that this particular assumption of
high mortality does not appear to be supported by the submitted dose and
efficacy studies. For CBW, the reported mortality on COT102 with
verification method #1 was 72.4%. Data from other verification methods
could not be relied on due to questions or sub-standard conditions
during the tests. For TBW, the reported mortality on COT102 with
verification method #1 ranged from 66.7 – 95.0% and with method #5 was
near high-dose. There are indications that COT102 is not very
efficacious against TBW under some conditions. Therefore, to what degree
the introduction of VipCot™ can delay resistance to Cry1A toxins in
TBW and CBW is uncertain.

™ can be expected to occur in less time than predicted by the model.
Similarly for CBW, the evolution of resistance to Cry1A toxins may not
be delayed by the introduction of VipCot™ when Vip3A mortality is low.

REFUGE STRATEGY

The size, placement, and management of the refuge are critical to the
success of the high-dose/structured refuge strategy to mitigate insect
resistance to Bt proteins produced in cotton (as well as corn and
potatoes). The 1998 SAP Subpanel defined structured refuges to
“include all suitable non-Bt host plants for a targeted pest that are
planted and managed by people. These refuges could be planted to offer
refuges at the same time when the Bt crops are available to the pests or
at times when the Bt crops are not available.” The 1998 Subpanel
suggested that a production of 500 susceptible adults in the refuge for
every adult in the transgenic crop area (assuming a resistance allele
frequency of 5 x 10-2) would be a suitable goal. The placement and size
of the structured refuge employed should be based on the current
understanding of the pest biology data and the technology. The 2000 SAP
Subpanel echoed the 1998 SAP’s recommendations that the refuge should
produce 500:1 susceptible to resistant insects and that regional IRM
working groups would be helpful in developing policies. (US EPA, 2001)

Under the established refuge strategy for cotton, growers can choose
from three structured refuge options, which are thoroughly described in
the Agency’s 2001 Bt crop reassessment document and briefly listed
here:

Option 1:   95:5 external structured, unsprayed refuge; 150 ft wide,
within ½ mile of edge of field

Option 2:   80:20 external sprayed refuge; within 1 linear mile,
preferably ½ mile, of edge of field	

Option 3:   95:5 embedded refuge; contiguous or within 1 mile2 of field
and 150 ft wide

According to their IRM plan (MRID 470176-34), Syngenta requests
identical refuge requirements as for currently registered cotton PIPs.
In addition, Syngenta requests that VipCot™ be considered for the
community refuge plan that allows multiple growers to contribute to the
overall required refuge acres by planting 20% external, sprayed or 5%
external, unsprayed refuge. 

BPPD notes that the simulations run by Dr. Caprio addressed refuge
option 2 only, the 20% external sprayed refuge (Appendix 5). BPPD would
like to expand on this apparent deficiency and clarify that the 20%
refuge option may actually be considered the least conservative approach
of all three options, and thus, the modeling assumptions could
potentially represent a worst case scenario for IRM because non-Bt
cotton refugia are often sprayed with multiple applications of
insecticides during a growing season. Shelton et al. (2000) indicate
that great care should be used to ensure that refuges sprayed with
highly efficacious insecticides produce adequate numbers of susceptible
alleles; thus, the 20% external sprayed refuge option for non-Bt cotton,
if over sprayed, may not produce a great amount of susceptible adults
that could potentially mate with resistant survivors from the Bt-field.
Gould & Tabashnik (1998) in their evaluation of Bt cotton IRM options
commented that a 20% external refuge that can be extensively treated
with insecticidal sprays may result in almost no refuge because all of
the susceptible target larvae would be killed. Therefore, the 5%
external unsprayed refuge as well as the embedded refuge can be expected
to generate the greatest number of susceptible insects that are able to
potentially mate with resistant survivors from adjacent Bt-fields in
comparison with the worst case scenario for the 20% external sprayed
cotton refuge. BPPD requests that in future reports Syngenta be clear as
to why certain assumptions were not included in the modeling efforts.

, the requested refuge options 1-3 and community refuge plan are
acceptable when VipCot™ cotton is planted.

RESISTANCE MONITORING PROGRAM

The need for proactive resistance detection and monitoring is critical
to the survival of Bt technology. The Agency mandates that registrants
monitor for insect resistance (measurement of resistance-conferring
alleles) to the Bt toxins as an important early warning sign to
developing resistance in the field and whether IRM strategies are
working. Grower participation (e.g., reports of unexpected damage) is
also important for monitoring. Resistance monitoring is also important
because it provides validation of biological parameters used in models.
However, resistance detection/monitoring is a difficult and imprecise
task. It requires both high sensitivity and accuracy. Good resistance
monitoring should have well-established baseline susceptibility data
prior to introduction of Bt crops. The chances of finding a resistant
larva in a Bt crop depend on the level of pest pressure, the frequency
of resistant individuals, the location and number of samples that are
collected, and the sensitivity of the detection technique. Therefore, as
the frequency of resistant individuals or the number of collected
samples increases, the likelihood of locating a resistant individual
increases (Roush & Miller 1986). If the phenotypic frequency of
resistance is one in 1,000, then more than 3,000 individuals must be
sampled to have a 95% probability of one resistant individual (Roush &
Miller 1986). Current sampling strategies have a target of 100 to 200
individuals per location. Previous experience with conventional
insecticides has shown than once resistant phenotypes are detected at a
frequency >10%, control or crop failures are common (Roush & Miller
1986). Because of sampling limitations and monitoring technique
sensitivity, resistance could develop to Bt toxins prior to it being
easily detected in the field. (  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm )

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R assemble baseline susceptibility data for TBW and CBW. Collection of
insects to be used in bioassays to fulfill the Agency’s annual
monitoring requirement will focus on cotton growing regions where
VipCot™ cotton sales are expected to be the highest. 

Key features of Syngenta’s monitoring plan include:

EPA receives monitoring plan for approval, revised monitoring plan
within three months of the date of product registration, and detailed
resistance monitoring

 by Jan 31 of the year after VipCot™ cotton is registered

Follow-up on grower, extension specialist, or consultant reports of
unexpected damage or control failure of the three main target pests

BPPD considers the monitoring plan adequate for this step of the
VipCot™ registration process. In order to facilitate future
communication between BPPD and the registrant, the IRM team makes the
following recommendations for monitoring procedures: Syngenta should use
the diagnostic concentration (LC99) for both toxins and target pests
where the approach has proven successful, and pests are susceptible and
population variance is small. In addition, follow-up testing of larval
survivors needs to be conducted for all toxins where field population
survivorship (≥2 instar) is significantly different from lab/reference
colony’s survivorship. 

Specifically for CBW (but not only), BPPD has the following
recommendations for Syngenta: if a good amount of effort has been put
into developing a discriminating or diagnostic concentration for CRW and
FLCry1Ab and there is evidence that the diagnostic concentration cannot
be achieved due to i.e. high-variability in response to the toxin, then
a comparison in baseline susceptibility (i.e. LC50s) may be a feasible
approach to monitoring. Estimated LC50s may serve well as a baseline
monitoring tool for shifts in susceptibility to Bt toxins; however, the
LC50 approach is not useful in discriminating resistant from susceptible
individuals. Therefore, this approach must then be linked with follow-up
testing of populations with elevated LC50s relative to previously
established baseline susceptibility. Furthermore, BPPD recommends that
Syngenta consider head capsule width assay and DNA markers in lieu of
mortality based diagnostic concentrations.

GROWER EDUCATION

Syngenta proposes to use the following methods to educate growers which
have already been established for other PIPs:

Signing of grower agreement with purchase of VipCot™

Grower agreement and/or stewardship documents referenced in the grower
agreement will set forth terms of current IRM program and contractually
bind grower to comply with IRM requirements

Annual affirmation system for VipCot™ cotton growers to ensure they
understand that they are contractually bound to comply to  requirements

Syngenta proposes to 1) submit within 90 days from product registration
a copy of the grower agreement/stewardship documents and written
description of a system assuring that growers will sign grower
agreement; 2) revise and expand as
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3) maintain records of all signed VipCot™ cotton grower agreements for
three years.

BPPD concludes that the proposed grower education plan meets the
Agency’s present requirement and appears acceptable.

GROWER COMPLIANCE PROGRAM

Grower compliance with refuge and IRM requirements is a critical element
for resistance management. Significant non-compliance with IRM among
growers may increase the risk of resistance for Bt crops. To minimize
the effects of non-compliance, it is necessary to develop a broad
compliance program as part of the IRM strategy. Such a program has to
include 1) an understanding of the effect of non-compliance on IRM; 2)
identification of compliance mechanisms to maximize adoption of IRM
requirements; 3) measurement of the level of compliance; and 4)
establishment of an enforcement structure to ensure compliance and
penalize non-compliance.

Syngenta commits to implementing a compliance assurance program designed
to 1) evaluate the extent to which growers of VipCot™ cotton are
complying with the IRM requirements and 2) take reasonable actions
necessary to assure that non-compliant growers become compliant with
those requirements and submit within 90 days of the date of registration
a written description of their compliance assurance program. Consistent
with the registration of other cotton Bt PIPs, there are several key
elements to the CAP that Syngenta will employ:

Establish and publish a phased compliance approach that outlines
instances of non-compliance to IRM terms and options of responding to
non-compliant growers

Annual survey conducted by third party will measure degree of compliance
by growers in different cotton growing regions and consider potential
impact of non-response

Survey will obtain grower feedback on usefulness of educational tools
and initiatives and provide understanding of any difficulties growers
encounter with IRM requirements 

Annual on-farm assessment followed by appropriate action consistent with
the ‘phased compliance approach’ for non-compliant growers

‘Tips and complaints’ line with follow-up investigations and
appropriate actions taken consistent with the ‘phased compliance
approach’ for non-compliant growers

Syngenta proposes to revise and expand, as necessary, its compliance
assurance program to take into account information collected through the
compliance survey and allow a review of the compliance records by EPA or
by a State pesticide regulatory agency.

BPPD concludes that Syngenta has included the major requirements needed
by a compliance program and outlined by Agency in the first paragraph of
section X and the 2001 Bt crop reassessment document. Syngenta’s
proposed CAP resembles CAPs for other introduced Bt PIPs and appears to
meet the Agency’s requirements at this time. 

REMEDIAL ACTION PLAN

Remedial action plans are a potential response measure should resistance
develop to Bt crops. 

Since resistance may develop in “localized” pest populations, it may
be possible to contain the resistance outbreak before it becomes
widespread. A specific remedial action plan should clearly indicate what
actions the registrant will take in cases of “suspected” resistance
(i.e., unexpected damage) and “confirmed” resistance. The remedial
action plan can also include appropriate adaptations for regional
variation and the inclusion of appropriate stakeholders. To fully
mitigate resistance, a critical element of any remedial action plan
should be that once pest resistance is confirmed, sales of all Bt cotton
hybrids that express a similar protein or a protein in which
cross-resistance potential has been demonstrated would be ceased in the
affected region. . (  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm )

Syngenta states that it if resistance to any of the three major target
pests is suspected, growers will be informed to use alternate pest
control measures such as pesticide treatment, crop rotation the
following year, or use of soil or seed insecticides the following year.

Syngenta states that the following steps in order of events will be
taken if resistance to any of the three major target pests is confirmed:

Notify the Agency within 30 days of resistance confirmation

Notify affected customers and extension agents about confirmed
resistance

Encourage affected customers and extension agents to employ alternative
lepidopteran control measures

Cease sale and distribution of VipCot™ cotton in affected area

Devise long-term resistance management action plan according to
characteristics of resistance event and local agronomic needs

VipCot™ cotton is preserved for a long time, Syngenta will need to
play a more aggressive and supportive role in case of confirmed
resistance to VipCot™ cotton.

REPORTING REQUIREMENTS

Syngenta commits to providing an initial report to EPA summarizing
activities carried out under the grower education program for the prior
year with annual updates thereafter of any substantive changes.

ar, beginning the year after VipCot™ cotton is registered; 2) an
annual report to the Agency after January 31 of each year summarizing
results of their CAP, activities carried out under the CAP for the prior
year, and plans for the current year; 3) annual sales summed by state;
4) number of VipCot™ cotton seeds shipped or sold and not returned,
and number of such units sold to persons who have signed grower
agreements.

Syngenta will provide to the Agency an annual resistance monitoring
report (by August 31 of each year beginning with the year after
VipCot™ cotton is registered) conducted on populations collected the
following year.

At this point in the VipCot™ registration process, the Agency is
satisfied with Syngenta’s commitment to fulfill the reporting
requirements.

REFERENCES

Andow, D.A. and Hutchison, W.D., Corn resistance management, in Now or
Never:  Serious Plans to Save a Natural Pest Control, Mellon, M. and
Rissler, J., Eds., Union of Concerned Scientists, Washington, D.C.,
1998, chap. 2.

Ferré, J. and J. Van Rie.  2002.  Biochemistry and genetics of insect
resistance to Bacillus thuringiensis.  Annu. Rev. Entomol. 47:  501-533.

Gould, F., Anderson, A., Reynolds, A., Bumgarner, L., and Moar, W. 1995.
Selection and genetic analysis of a Heliothis virescens (Lepidoptera:
Noctuidae) strain with high levels of resistance to Bacillus
thuringiensis toxins. J. Econ. Entomol. 88:1545-1559.

2006. Insect resistance management considerations for VipCot™ Cotton.
Report submitted from Syngenta Biotechnology, Inc. MRID 470176-34

Kurtz, R.W., 2006. Determining the dose of VipCot™ cotton and its
component events, COT67B and COT102, using US EPA Method #1 for
Helicoverpa zea and Heliothis virescens. Appendix 1 in report submitted
from Syngenta Biotechnology, Inc. MRID 470176-34

ve insecticidal protein Vip3A differs from that of Cry1Ab δ-endotoxin.
Applied and Environmental Microbiology, Vol. 69 (8): 4648-4657

Piggott, C.R. and D.J. Ellar.  2007.  Role of receptors in Bacillus
thuringiensis crystal toxin activity.  Micro. Molec. Bio. Reviews.  71: 
255-281.

Roush, R.T. 1994. Managing pests and their resistance to Bacillus
thuringiensis: Can transgenic crops be better than sprays? Biocontrol
Science Technology, Vol. 4:501-516.

Roush, R. T., and G. L. Miller, 1986. Considerations for design of
insecticide resistance monitoring programs. Journal of Economic
Entomology, Vol. 79: 293-298.

 (October 18-20, 2000), 2001. Report: sets of scientific issues being
considered by the Environmental Protection Agency regarding: Bt
plant-pesticides risk and benefits assessments. Report dated, March 12,
2001 (Pp. 5-53).

Schnepf, E., Crickmore, N., Van Rie, D. Lereclus, D., Baum, J.,
Feitelson, J., Zeigler, D.R., Dean, D.H. 1998. Bacillus thuringiensis
and its pesticidal crystal proteins. Microbiology and Molecular Biology
Reviews, Vol. 62 (3): 775-806.

Shelton, A.M., J.D. Tang, R.T. Roush, T. D. Metz, and E.D. Earle, 2000.
Field tests on managing resistance to Bt-engineered plants. Nature
Biotechnology. 18: 339-342.

Tabashnik, B.E. 1994. Evolution of resistance to Bacillus thuringiensis.
Annu. Rev. Entomol. 39:47-79. 

Others:

BPPD, 2004. EPA Review of Syngenta Seed’s Vip3A Cotton Insect
Resistance Management Plan for Section 3 Full Commercial Registration.
S. Matten memorandum to L. Cole, January 05, 2004.

US EPA, 1998. FIFRA Scientific Advisory Panel Subpanel on Bacillus
thuringiensis (Bt) Plant-Pesticides and Resistance Management, February
9 and 10, 1998

US EPA, 2001. Biopesticides Registration Action Document – Bacillus
thuringiensis Plant Incorporated Protectants,   HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm 

Appendix A.   Procedure for High-Dose Determination

The 1998 SAP defined high-dose as a level of toxin 25 times greater than
is needed to kill all susceptible insects.  The SAP also outlined five
techniques to determine high dose:  1) Serial dilution bioassay with
artificial diet containing lyophilized tissues of Bt plants using
tissues from non-Bt plants as controls; 2) Bioassays using plant lines
with expression levels approximately 25-fold lower than the commercial
cultivar determined by quantitative ELISA or some more reliable
technique; 3) Survey large numbers of commercial plants in the field to
make sure that the cultivar is at the LD99.9 or higher to assure that
95% of heterozygotes would be killed (see Andow & Hutchison 1998);  4)
Similar to #3 above, but would use controlled infestation with a
laboratory strain of the pest that had an LD50 value similar to field
strains; and 5) Determine if a later larval instar of the targeted pest
could be found with an LD50 that was about 25-fold higher than that of
the neonate larvae.  If so, the later stage could be tested on the Bt
crop plants to determine if 95% or more of the later stage larvae were
killed.  

Appendix B. Cross-Resistance Models and Mechanisms

There are three models that have been proposed to explain the mode of
action of Cry1A toxin mode of action (see discussion in Piggott and
Ellar, 2007).  The most accepted Bravo model proposes that both the
cadherin and aminopeptidase (APN) receptors are required for full Cry1A
toxicity.  This model suggests that receptor binding is sequential: 1)
ingestion of the protein inclusions by a susceptible insect larva, 2)
solubilization of the protein in the insect midgut, 3) cleavage of the
protoxin by host proteases and release of the active toxin, 4) binding
of the active toxin to specific receptors on the midgut epithelieum, 5)
oligomerization of toxin subunits to form pore structures that insect
into the membrane, 6) passage of ions and water through the pores,
resulting in swelling, lysis, and the eventual death of the host. 
Differences in any of these steps will reduce the probability of
cross-resistance between any two Cry proteins. The more controversial
Zhang model suggests that receptor binding activates a Mg+-dependent
signaling cascade that promotes cell death.  The Jurat-Fuentes model
suggests that cytotoxicity is due to the combined effects of osmotic
lysis and cell signaling.  The later two models are, at present, more
speculative.  

Resistance associated with modification of the binding site receptor has
been the primary Bt resistance mechanism reported to date (reviewed in
Ferré & Van Rie 2002). Other Bt resistance mechanisms have been
reported that are based on alterations in the proteases that cleave the
protoxin processing it into a smaller active toxin (Candas et al. 2003)
and most recently, the discovery that esterases can bind and detoxify Bt
toxins (Gunning et al. 2005).  Only the binding reduction mechanism has
a demonstrated causal link between the biochemical modification and
resistance (Ferré and Van Rie 2002).  Ferré and Van Rie (2002)
indicate that in all cases of binding site modification, resistance is
due to a recessive or partially recessive mutation in a major autosomal
gene, and cross-resistance extends only to Cry proteins sharing binding
sites. Cry proteins that do not share high levels of sequence similarity
tend to have different binding sites and different modes of action.  

 The use of BPPD in this review refers to the BPPD IRM team.

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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460