after 35 days was 17.5% in the controls and 20% in the eCry3.1Ab groups,
and did not differ significantly. No difference in the number of F1
progeny produced was observed between these groups.  Mortality was only
18.8% in the positive control group; however, no offspring were produced
in this group, which was expected. The study indicates that at the level
tested, eCry3.1Ab does not produce adverse effects. The LC50 for this
species is >400 µg eCry3.1Ab/g diet. MRID 48442536.

An additional study was conducted with the predatory bug, Orius
laevigatus. Nymphs were provided an artificial meat-based diet
containing a nominal concentration of 400 µg eCry3.1Ab/g diet. An
untreated control diet and a toxic reference diet containing
teflubenzuron were also included in the test. All nymphs developed to
adults or died by test day 12. Pre-imaginal mortality was 0% and 5% in
the control and test groups, respectively, which was not significantly
different. Mortality was 100% in the toxic reference group. Based on the
results, eCry3.1Ab had no adverse effects on O. laevigatus when provided
at a rate of 400 µg/g diet. The LC50 for this species is >400 µg
eCry3.1Ab/g diet. MRID 48442537.

A semi-field study with honey bee (Apis mellifera) was also conducted to
determine the effects of eCry3.1Ab on adults and on brood development.
Adults were fed a sucrose solution containing 50 µg eCry3.1Ab/g for 5
days. A sucrose solution-only assay control and a sucrose solution
containing fenoxycarb as a positive control were also included.
Mortality was observed for eight days prior to feeding. The test diets
were provided to the bees daily within the hive for five days and then
observations of mortality and brood development were made up to 22 days
after initial feeding. Mortality was monitored with a dead bee trap
placed on the hive, and flight intensity (the number of worker bees
entering and leaving the hive) was also observed.  Brood development was
monitored after the initial feeding day and assessed using a brood
development index based on expected and observed development stage at
the time of observation (brood index). An additional development index
(compensation index) was calculated to account for eggs or larvae that
were placed in cells as a result of a previous mortality. Mortality and
development indices did not differ significantly between the negative
control group and the group exposed to eCry3.1Ab. Significant mortality
and brood losses were observed in the positive control compared to the
negative control. Based on the results adverse effects on honey bee
adults and brood development are not expected when hives are exposed to
eCry3.1Ab at 50 µg eCry3.1Ab under these conditions. MRID 44842534.

The effects of eCry3.1Ab on honey bees was also observed in another
semi-field study in which bees were similarly exposed to 50 µg
eCry3.1Ab/g in a sucrose solution for five days. A negative control and
a positive control containing diflubenzuron were also included. The
endpoints evaluated were: a) the pre-imaginal mortality of pre-marked
broods; b) the number of dead adult bees, larvae and pupae expelled from
the hive; and c) an assessment of whether the treatments affected the
relative proportions of brood and food cells within the hives over the
duration of the test (24 days). For cells that contained eggs at the
time of initial feeding, mortality was 25.5% in the (negative) control,
compared with 38.2% (17.0% corrected mortality) in the eCry3.1Ab
treatment group and 99.8% in the toxic reference treatment group. For
cells that contained larvae, mortality was mortality was 16.8% in the
control group, compared with 2.3% (0.0% corrected mortality) in the
eCry3.1Ab treatment group and 100% in the toxic reference treatment
group. Mortality between the negative control and eCry3.1Ab treated
groups was not significant. The numbers of dead bees and brood deposited
in the dead bee traps during the treatment and post-treatment periods
were 22.4, 12.0 and 25 .3 per hive for the control, eCry3.1Ab and toxic
reference treatments, respectively. The study authors noted that wasps
were taking cadavers out of the bee traps, which may have affected the
results. However, the study reliably indicates that overall hive
condition and brood development are not affected by eCry3.1Ab at the
level tested under the exposure conditions. MRID 48442538.

Toxicity to a non-arthropod invertebrate was also examined in a study to
determine the toxicity of eCry3.1Ab to the earthworm (Eisenia fetida).
Artificial soil was treated at a rate of 50 µg eCry3.1Ab/g of soil dry
weight, which was intended to represent approximately 10X the average
expression rate of eCry3.1Ab in roots of Event 5307 plants. Control
artificial soil was treated with purified water only. Ten adult
earthworms were placed on the soil surface in each jar, and the jars
were maintained under controlled conditions for 14 days. At test end,
none of the earthworms in either group had died, and all showed normal
condition and behavior. The change in mean earthworm weight was -9.1% in
the control group, and -7.2% in the test material group, and the
difference between these was not statistically significant. Worms likely
lost weight because no food was included in the treatment. The
no-observed-effect concentration in this study was 50 µg eCry3.1Ab/g
soil dry weight. The presence and activity of eCry3.1Ab in the
artificial soil used in the test were confirmed in a separate study.
MRID 48442530.

b.  Soil Fate Studies for eCry3.1Ab

Soil organisms may be exposed to eCry3.1Ab through contact with corn
roots (by direct feeding), plant root exudates, incorporation of
above-ground plant tissues into soil following harvest, or by
soil-deposited pollen. Some evidence suggests that soils that are high
in clays and humic acids are more likely to bind Cry protein.  However,
neutral pH soils tend to have high microbial activity and microbes
contribute to Cry protein degradation.  In addition, a study on the
release of Cry proteins in the root exudates of Bt cotton has shown that
no Cry proteins were detected immunologically or by larvicidal assay in
any soil or hydroponic solution in which Bt cotton had been grown
(Saxena and Stotzky, 2001).  The weight of evidence indicates that Cry
proteins do not accumulate in soil to arthropod-toxic levels. Because
Cry proteins are toxins derived from soil-inhabiting bacteria, Bacillus
thuringiensis, the degradation of eCry3.1Ab from Event 5307 corn is
expected to be similar to that of these proteins that are produced
naturally from soil bacteria.  Nonetheless, BPPD required the following
soil fate evaluations to support this registration.

A bioassay system (first instar larvae of the Colorado potato beetle,
CPB) was used to investigate the loss of insecticidal activity of
eCry3.1Ab protein in soil over 120 days under laboratory conditions.
The test soil, characterized as a loam, was collected from the top six
inches of soil over an area of approximately four acres in Newtown, IN.
The site had been used for growing corn over the three previous years,
and was currently being used for soybeans.  Soil samples were dosed with
a concentration of approximately 3.125 mg eCry3.1Ab/mL. Controls were
dosed with 1 mL of the buffer. Additional incubation bottles, containing
dry weight equivalents of soil, were designated for biomass
measurements. All samples were mixed well, and maintained in an
incubation chamber under aerobic conditions at a temperature of
15-30°C. Each incubation bottle was connected to hydration bottle and
airflow system.  The bottles were checked every one to two weeks, and
moisture levels were adjusted with purified water to maintain proper
hydration levels. Treated soils were incubated for 0, 1, 3, 7, 14, 29,
60, 90, and 120 days (NOTE: the 90-day treated soils were dosed
incorrectly, and results from the sample were not used). Untreated
controls were incubated for 0, 60 and 120 days. Results from the 60 and
120 day control incubations were also not available. Following
incubation, the soil samples were incorporated into a commercially
available CPB diet at a concentration of 10% (w/v). A total of 150 μL
of soil/diet mixture was fed to each of 24 larvae. The containers with
larvae were sealed and in the dark at ambient temperature. Mortality
readings were taken daily starting at 72 hours and continuing through
120 hours. Corrected mortality was 72%, 63%, 48%, and 40% on Days 0, 1,
3, and 7, and then were -4%, 3%, 1% and 0% for Days 14, 29, 60, and 120.
The data are not sufficient to calculate a half-life. However, based on
the data provided, the half-life of the eCry3.1Ab in the soil tested
appears to be between 7 and 14 days.  This is comparable to half lives
observed with other Cry proteins (e.g., see MRID 48442526). The study
design could be improved to provide a more robust estimate of soil half
life, and a range of soil types should have been tested, including those
with high clay and humic acid content, due to their known binding
affinity for proteins. The data do show that eCry3.1Ab is not likely to
persist in loam soils. However, additional studies would be useful in
evaluating insecticidal protein degradation, accumulation, and
persistence in a variety of soil types and also for refining the
estimate of the DT50 for eCry3.1Ab in soil.

Based on FIFRA Scientific Advisory Panel recommendations and public
comments, the Agency has required three year soil fate studies for the
currently registered Cry protein producing crops grown in a variety of
soils and environmental conditions, as a condition of registration. , A
comprehensive review of all available scientific data on ecological
effects of commercially grown GM crops over ten years was published in
2007 (Sanvido, et al. 2007).  The review concluded “none of the
laboratory or field studies suggest accumulation of Bt-toxins in soil
over several years of cultivation” and “experience from commercial
cultivation indicates that Bt-toxin will not persist for long periods
under natural conditions.”  The Agency agrees with these conclusions.
Collectively, the long-term field studies for Bt crops also confirm the
previous SAP conclusion that “bioaccumulation is not expected to occur
with transgenic proteins because biodegredation mechanisms for proteins
are ubiquitous” (US EPA, 2000).  More importantly, the numerous
laboratory studies that demonstrated rapid protein degradation in soil
of Bt proteins produced in Bt crops (when performed under realistic
environmental conditions) can be considered predictive that Bt protein
in soil is not likely to persist or accumulate in soil after continuous
cultivation.

In light of these published findings and the relatively rapid
degradation of eCry3.1Ab protein in soil as demonstrated in the insect
bioassay described above, there is no indication that the eCry3.1Ab
protein expressed in Event 5307 corn is likely to persist in soil after
continuous cultivation. Therefore, no additional long-term field studies
are required for these PIP products.

	c.  Supplemental Information

Syngenta submitted a summary of nontarget organism testing with
eCry3.1Ab (MRID 44842526), which summarized the information above, and
included an assessment of ecological risk and some discussion of
environmental fate.  These summaries were determined to be supplemental,
and relevant information was utilized in this risk assessment.

3.  Environmental Risk Assessment for eCry3.1Ab

		a.  Effects to Nontarget Wildlife, Invertebrates, and Plants

   		1.   Avian Wildlife

Birds are known to forage in corn fields, and may be exposed to
eCry3.1Ab. An estimate of dietary exposure for birds is provided in MRID
44842526, which is based on estimates of daily dietary dose based on
consumption of Event 5307 grain and mean expression levels (fresh
weight) in this plant tissue using a method developed by Crocker et al.
(2002). The worst case exposure, based on 100% consumption of grain was
estimated to be 1.94 mg eCry3.1Ab/kg bodyweight for birds with body
weights ranging from 22g (e.g., a sparrow) to 953g (e.g., a pheasant).
Exposure through other foods is not well characterized, however, and to
account for uncertainties related to their possible consumption, a more
conservative estimate of exposure may be determined using estimates of
protein expression in tissues that express the highest amounts (e.g.,
leaves). Based on the method and mean fresh weight expression levels
used in MRID 44842526, assuming 100% consumption of a diet containing
material with an eCry3.1Ab concentration equivalent to the mean fresh
weight expression level in leaves (51.74 µg/g), a worst-case exposure
would range from 5.69 to 18.11 mg eCry3.1Ab/kg bodyweight for birds with
the same body weights.

An avian oral toxicity study was submitted, wherein the LD50 for
eCry3.1Ab was determined to be >900 mg eCry3.1Ab/kg bodyweight. This
exposure level is much higher than the maximum exposure estimates
discussed above, and minimizes any uncertainties related to exposure in
the field. Additional data provided in the study with broiler chickens
shows that longer-term dietary exposure at low levels is unlikely to
affect the parameters examined in that study.  Based on these data,
cultivation of Event 5307 corn expressing eCry3.1Ab is not anticipated
to cause adverse effects in birds.

    		2. Wild mammals

Wild mammals also forage in corn fields and may be exposed to eCry3.1Ab.
Syngenta provided an estimated maximum dose of 1.82 mg eCry3.1Ab/kg
bodyweight for mammals, based on the same methods as described above for
birds. Using these methods and the mean fresh weight expression in
leaves as given above, a maximum exposure for mammals would be 17.07 mg
eCry3.1Ab/kg bodyweight.

Toxicity testing with laboratory mice showed no adverse effects with
exposure to eCry3.1Ab at a dose of 2000 mg/kg bodyweight.  This amount
is much higher than concentrations that wild mammals are expected to
encounter in the field; therefore, any uncertainties related to exposure
are minimized by testing at this level. Based on this information,
adverse effects to wild mammals are not expected as a result of the
registration and use of eCry3.1Ab as expressed in Event 5307 corn.

		3. Freshwater animals

BPPD previously determined that exposure to Cry proteins in the
freshwater environment were expected to be low, based on the assumption
that exposure in aquatic environments primarily resulted from pollen
deposition. In light of published studies showing reduced growth in
caddis flies exposed to anti-lepidopteran Cry1A protein (Rosi-Marshall
et al. 2007), concerns were raised regarding the potential for exposure
to shredder invertebrate species via consumption of corn plant litter in
aquatic environments. EPA determined (see USEPA 2010a) that post-harvest
crop residue is the most likely route of exposure to aquatic organisms
(Carstens et al. 2011); however, their exposure to biotech crops is
likely to be low, since the concentrations of Cry proteins in
post-harvest crop tissues  are  limited temporally and spatially (Swan
et al. 2009, Jensen et al. 2010, Wolt and Peterson 2010, Carstens et al.
2011). Nonetheless, because of concerns that have been raised over this
issue, EPA has required aquatic invertebrate testing.

Studies lasting 7-10 days with Daphnia spp. have typically been
required. Studies with shredder species would test directly the
invertebrate species expected to be affected by Bt corn plant litter
that may enter streams. A study with a shredder species (Gammarus
fasicatus) was submitted that showed that this species of isopod is not
affected when exposed to Event 5307 corn leaves. The leaves were
collected during a growth stage in which leaves express higher amounts
of the eCry3.1Ab protein compared to other growth stages. Event 5307
corn leaves also express greater amounts of eCry3.1Ab compared to other
tissues (based on MRID 48442509). Therefore, the isopods in this study
were exposed to eCry3.1Ab at or near a maximum EEC. Additionally the
study with channel catfish described above found no effects of eCry3.1Ab
in this species when fed Event 5307 corn grain at 41% in the diet. Based
on the results of this study, adverse effects to freshwater fish and
invertebrates are not anticipated as a result of the registration and of
eCry3.1Ab as expressed in Event 5307 corn.

		4. Estuarine and marine animals

As discussed above, significant amounts of eCry3.1Ab use of Event 5307
corn are not expected to reach marine/estuarine areas.  Therefore, BPPD
concludes that adverse effects to fish and invertebrates in these
environments as a result of the registration and cultivation of corn
containing eCry3.1Ab are not expected because exposure is expected to be
minimal.

   		5. Terrestrial and aquatic plant species

The active ingredient, eCry3.1Ab is part of a larger group of insect
toxins produced a naturally-occuring Bacillus thuringiensis (Bt
(-endotoxins) that have not shown toxicity to plants.  Effects to
nontarget plants in the terrestrial and aquatic environments are not
expected, and BPPD concludes that adverse effects to terrestrial and
aquatic plants as a result of the registration and use of eCry3.1Ab in
Event 5307 corn are not anticipated.

   		6. Invertebrate species

Nontarget insects are expected to receive exposure primarily through
consumption of Bt cotton pollen and/or nectar, though they may also
consume pest insects that feed on cotton plant tissue or rarely other
corn plant tissues themselves.  The principal exposure route of
soil-dwelling invertebrates, such as collembola, earthworms, and rove
beetles, to eCry3.1Ab is assumed to be from decomposing plant tissue and
plant exudates in soil. Studies submitted to EPA that examined the
effects of eCry3.1Ab in lady beetle, rove beetle, predatory bug, and
earthworm showed no adverse effects at concentrations well above those
that would be encountered by nontarget insects in the environment, even
if directly consuming tissues with the highest levels of eCry3.1Ab
protein expression. No mortality was observed in carabid beetles fed
eCry3.1Ab at 400 µg eCry3.1Ab/g diet (~8X EEC); however, adult body
weight was significantly reduced at this exposure level. Effects in this
study are not necessarily unexpected, since eCry3.1Ab is a
coleopteran-active protein. This sublethal effect could affect
individual reproductive success and possibly population size for
sensitive species, and additional testing to establish a sublethal
endpoint (e.g., EC50 or NOAEC) would reduce any uncertainty related to
its observation in this study. Since other studies with nontarget
coleopteran species showed no adverse effects with exposure to
eCry3.1Ab, and because exposure is expected to be much lower in the
field, BPPD does not anticipate commercial cultivation of Event 5307
corn plants to cause significant effects to populations of nontarget
insects and other invertebrates in the field, including coleopteran
species.

Two studies examining the effects of eCry3.1Ab on adult honey bees,
honey bee brood development, and overall hive health showed no adverse
effects to honey bees at exposure levels to eCry3.1Ab above those
expected to be encountered in the environment.  Therefore risk to honey
bees as a result of the proposed registrations is also expected to be
minimal.

It should be noted that effects to invertebrates that are recognized as
corn pests (i.e., insects that feed detrimentally on corn plants) that
are not targeted by the eCry3.1Ab protein (but are controlled by other
means) are not included in BPPD’s assessment of risk to nontarget
insects.

		b.   Effects on Soil Microorganisms

Numerous published studies indicate that exposure to Cry proteins
produced in Bt PIP crop plants does not adversely affect soil
microorganisms (Sanvido et al., 2007). Although a minimal transient
increase and shift in microbial populations may result from the presence
of transgenic plant tissue in soil, no adverse effects have been
attributed to the Cry proteins.  In addition, the soil degradation study
discussed above shows that the eCry3.1Ab degrades quickly in soil.

With regard to the impact of genetically engineered crops on soil, EPA
has previously noted (USEPA 2010a) that agricultural practices
themselves cause large changes in soil and soil microbial composition.
Furthermore, factors such variations in seasons and weather, plant
growth stage, and plant varieties, independent of being genetically
engineered, are also responsible for significant shifts in soil
microbial communities. Most studies with genetically engineered crops to
date have shown minor or no effects on soil microbes beyond the
variation caused by the factors listed above.

		c.  Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms

The EPA has evaluated the potential for horizontal gene transfer (HGT)
from Bt crops to soil organisms and has considered possible risk
implications if such a transfer were to occur. Genes that have been
engineered into Bt crops are mostly found, or have their origin, in
soil-inhabiting bacteria. Soil is also the habitat of other
toxin-producing bacteria, and transfer of these genes and/or toxins to
other microorganisms or plants has not been detected. Furthermore,
several published experiments, that were conducted to assess the
likelihood of HGT, have been unable to detect gene transfer under
typical environmental conditions.  Horizontal gene transfer to soil
organisms has only been detected with very promiscuous microbes under
laboratory conditions designed to favor transfer.

As a result of these findings and the fact that the Bt toxin engineered
into Event 5307 is derived from soil-inhabiting bacteria, the EPA has
concluded that the risk of HGT of transgenes found in Event 5307 corn
expressing eCry3.1Ab is low.

		d.  Gene Flow and Weediness Potential

The movement of transgenes from the host plant into weeds has been a
significant concern for EPA due to the possibility of novel exposures to
the pesticidal substance. This concern has been considered for each of
the Bt plant-incorporated protectants currently registered, and was
extensively reexamined in 2010 (see USEPA 2010a), and EPA believes that
these concerns have been satisfactorily addressed. The Agency has
determined that there is no significant risk of gene capture and
expression of any Bt endotoxin by wild or weedy relatives of corn in the
U.S., its possessions or territories. In addition, the USDA/APHIS has
made this same determination under its statutory authority under the
Plant Pest Act.

The FIFRA EPA Scientific Advisory Panel meeting held on October 18-20,
2000, further discussed the matter of gene flow and offered some issues
for consideration in this matter. The panel agreed that the potential
for gene transfer between corn (maize) and any receptive plants within
the U.S., its possessions and territories was of limited probability and
nearly risk free.

Based on these extensive analyses, the potential for gene flow and
development of weediness is expected to be low for Event 5307 corn
expressing eCry3.1Ab protein.

		e.  Conclusions

BPPD concludes that significant adverse effects on birds, mammals,
nontarget insects, honey bees, freshwater and marine/estuarine fish and
invertebrates, and terrestrial and aquatic plants are not expected as a
result of the use of Event 5307 corn expressing eCry3.1Ab. EPA has also
determined that there is no significant risk of gene capture and
expression of eCry3.1Ab protein by wild or weedy relatives of corn
within the U.S. or its territories. Available data do not indicate that
Cry proteins have any measurable adverse effect on microbial populations
in the soil, nor has horizontal transfer of genes from transgenic plants
to soil bacteria been demonstrated.

f.  Impacts on Endangered Species

The primary route of exposure to eCry3.1Ab protein in corn is through
ingestion of corn tissue. Since eCry3.1Ab protein is not expected to
have adverse effects on mammals, birds, plants, freshwater and
estuarine/marine fish and invertebrates, nontarget insects (see
discussion below) and other invertebrate species at the Estimated
Environmental Concentration (EEC), a “No Effect” determination is
made for direct and indirect effects to federally listed threatened and
endangered (“listed”) species. In addition, EPA does not expect that
any threatened or endangered plant species will be affected by
outcrossing to wild relatives or by competition with such entities.
Hybrid corn does not exist in the wild, nor are there wild plants that
can interbreed with corn in the United States.

Because of the selectivity of eCry3.1Ab protein for some coleopteran
species, there exists some concern for listed species in the order
Coleoptera. The potential risk to listed threatened or endangered
coleopteran species has been previously addressed for Bt Cry proteins in
corn (USEPA 2009, 2010b, 2010c). At present, 18 coleopteran species are
listed as threatened or endangered, and 16 of these occur in counties in
which corn is grown. Eight of these species are subterranean, and
exposure to Cry proteins expressed in plant tissues is not expected in
these environments. The remaining species existing above ground, in
terrestrial or aquatic habitats, are habitat specialists that are
largely excluded from agricultural areas due to their specific habitat
requirements. While habitats of some of these species may occur nearby
to agricultural areas where Event 5307 corn may be cultivated, exposure
via consumption of Event 5307 corn tissue is not anticipated due to
their specific food requirements that would exclude consumption of corn
plant material (i.e., they are predators/scavengers or consume specific
food plants). BPPD also has previously determined that exposure to Cry
proteins in pollen outside of areas where Bt corn is cultivated is
limited (USEPA 2010a).  Furthermore, the protein expression study for
Event 5307 corn (MRID 48442509), which is pending review, shows that
very little eCry3.1Ab is expressed in Event 5307 pollen, so exposure to
eCry3.1Ab resulting from pollen that lands on food items within or
outside of areas where Event 5307 corn is grown is expected to be very
low.

As discussed in previous assessments of coleopteran-active Cry proteins,
the American burying beetle (Nicrophorus americanus) is the only species
that may potentially utilize areas where corn is grown. Exposure to this
species has been discussed previously, and BPPD determined that this
species is not expected to be exposed because it largely feeds on
carrion (USEPA 2010c).

Based on the available information, exposure of listed coleopteran
species to eCry3.1Ab protein produced by Event 5307 corn is not
expected. Therefore, a “No Effect” determination is made for direct
effects to these species. Since effects on other insects or other animal
or plant taxa are not anticipated, as discussed above, a “No Effect”
determination is also made for indirect effects to listed coleopterans
and for effects to their critical habitat.

D.   Environmental Risk Assessment for eCry3.1Ab, Cry1Ab, mCry3A, and
Cry1F Expressed

       Bt11 x MIR604 x TC1507 x 5307 Corn Hybrid

1.  Background

Syngenta has applied for a FIFRA Section 3 Registration for a combined
trait corn product Bt11 x MIR604 x TC1507 x 5307 (EPA File Symbol
67979-EU) that confers lepidopteran and coleopteran insect resistance
and herbicide tolerance (with the Event GA21 trait). The combined trait
corn Bt11 x MIR604 x TC1507 x 5307 product was developed by crosses (via
traditional breeding methods) with each of the following individual PIP
events: Bt11 corn (expressing Cry1Ab), MIR604 (expressing mCry3A),
TC1507 (expressing Cry1F) and 5307 (expressing eCry3.1Ab). This
registration is supported by acceptable data approved previously for the
individual parental lines, and data for the eCry3.1Ab protein expressed
in Event 5307.,.  A synergism study involving the Cry1Ab, Vip3Aa20, and
Cry1F proteins was previously submitted and reviewed by EPA (MRID No.
48235204; reviewed in USEPA 2011) . To support the current applications
for these stacks, a synergism study was submitted to study potential
interactions in between mCry3A and eCry3.1Ab proteins. Another study was
also submitted that tested the interaction of the combination of Cry1Ab,
mCry3A, Vip3Aa20, Cry1F and eCry3.1Ab proteins in susceptible
lepidopteran and coleopteran species. These studies provide support for
bridging to data for the individual PIP events that are already
contained in the Agency’s database.

2.   Environmental Assessment

The hazard of each of the individual events in Bt11 x MIR604 x TC1507 x
5307 has been addressed for previous registrations and for Event 5307 as
discussed above. EPA has previously determined that the database for
ecological risk assessment is complete for the Cry1Ab, Cry1F, and mCry3A
proteins expressed in this hybrid, and also for eCry3.1Ab based on the
assessment above. Synergism is a unique concern with the environmental
risk assessment of stacked PIPs is the potential for synergistic effects
that may result from interaction between the Cry proteins contained in
the hybrid plants. Additionally, stacked hybrids are assessed for
protein expression comparability with their individual parent lines to
ensure that exposure is not significantly higher in the hybrid.
Therefore, this assessment contains an evaluation of the potential for
synergism in the Bt11 x MIR604 x TC1507 x 5307 corn hybrid and a
discussion of protein expression comparability.

 Event Bt11 (lepidopteran active) Environmental Risk Assessment Summary

Potential adverse effects to non-target organisms by the Cry1Ab protein
expressed by event Bt11 have been previously reviewed and was recently
updated (USEPA 2010a).  The following is a summary of the Bt11
environmental risk assessment.

The Cry1Ab protein expressed in Bt11 corn is intended to control
lepidopteran pests. The hazard of the Cry1Ab protein was evaluated for
the ladybird beetle, green lacewing, parasitic hymenoptera, collembolan,
Daphnia spp., honey bee, earthworm, Monarch butterfly, and birds,
mammals, fish, and non-target plants. Soil degradation/persistence
studies were also evaluated.  Additionally, gene flow and weediness
assessments via pollen and Cry protein DNA uptake by plants and soil
microorganisms previously have been performed and are discussed
extensively in the update to the Cry1Ab BRAD (USEPA 2010a).  This
document also discusses field surveys of effects on nontarget insects
and effects on nontarget lepidopterans that were conducted for several
Bt Cry protein registrations in corn. Based on this information, EPA
concluded that risk to non-target wildlife, aquatic, and soil organisms
is not expected from Cry1Ab expressed in Event Bt11 corn.

At present, the Agency is aware of no identified significant adverse
effects of Cry1Ab on the abundance of non-target organisms in any
population in the aquatic or terrestrial field environment. In addition,
no direct or indirect effects on Federally listed endangered and
threatened species or effects on their critical habitat are expected.
Further, the EPA has determined that there is no significant risk of
gene capture and expression of Cry1Ab protein by wild or weedy relatives
of corn in the U.S., its possessions, or territories, available data do
not indicate that Cry proteins have any measurable adverse effect on
microbial populations in the soil, nor has horizontal transfer of genes
from transgenic plants to soil bacteria been demonstrated.

In conclusion, the risk assessment found no risk to the environment from
cultivation of Event Bt11 corn expressing Cry1Ab.

Event TC1507 (lepidopteran active) Environmental Risk Assessment Summary

Potential adverse effects to non-target organisms by Cry1F protein were
reviewed and updated in the Cry1F BRAD, in 2010 (USEPA 2010a).  The
following is a summary of the Cry1F environmental risk assessment. EPA
performed risk assessments on plants, wild mammals, birds, fish, aquatic
invertebrates, earthworms, terrestrial non-target insects (including
honey bee, parasitic wasps, green lacewings, ladybird beetle,
springtails [Collembola toxicity/reproduction], and monarch
butterflies), as well as field evaluations of the effects of Cry1F
exposure on non-target insects in corn fields, soil
degradation/persistence studies, and an endangered species impact
assessment, particularly for Lepidoptera.  In addition, gene flow and
weediness assessments via pollen and Cry protein DNA uptake by plants
were also performed.  Cry1F protein in soil has been shown to degrade
rapidly to very low levels.  EPA concluded that there is sufficient
information to believe that there is no risk from the uses of Cry1F corn
to non-target wildlife, aquatic, and soil organisms.

At present, the Agency is aware of no identified significant adverse
effects of Cry1F protein on the abundance of non-target organisms in any
population in the aquatic or terrestrial field environment. Field
testing and field census data submitted to the Agency show minimal to
undetectable changes in the beneficial insect abundance or diversity.
To date the available field test data show that compared to crops
treated with conventional chemical pesticides, the transgenic crops have
no detrimental effect on the abundance of non-target invertebrate
populations. In addition, no direct or indirect effects on Federally
listed endangered and threatened species or effects on their critical
habitat are expected. The EPA has reviewed the potential for gene
capture and expression of Cry1F protein by wild or weedy relatives of
corn in the United States, its possessions or territories and has found
that there is no significant risk in the United States, its possessions
or territories (USEPA 2010a).

In conclusion, the risk assessment found no risk to the environment from
cultivation of Event TC1507 corn expressing Cry1F protein.

Event MIR604 (coleopteran active) Environmental Risk Assessment Summary

A risk assessment was performed for modified Cry3A (mCry3A) protein
expressed in Event MIR604 corn when it was originally registered, and
the BRAD for this PIP was also updated in 2010 (USEPA 2010b).  EPA
examined data on birds (Northern bobwhite, broiler chicken), mammals
(from laboratory studies submitted for toxicology review), freshwater
fish (rainbow trout), lady beetle, carabid beetle, rove beetle,
insidious flower bug, honey bee, and earthworm. Data were not required
for estuarine/marine fish and invertebrates due to expected lack of
exposure.  Similarly, data were not required for freshwater
invertebrates because mCry3A was not detectable in MIR604 corn pollen,
and exposure was expected to be minimal in these environments. A soil
degradation study was also submitted.  Based on these data, EPA
performed an assessment of the risk of mCry3A to nontarget organisms. In
addition, gene flow and weediness assessments were performed when MIR604
was initially registered, which was extensively updated in 2010. Based
on the data submitted, EPA concluded that adverse effects to nontarget
organisms, including listed species, are not expected as a result of the
cultivation of Event MIR604 corn plants expressing mCry3A protein. Based
on recommendations from a Science Advisory Panel meeting that was
convened to comment on EPA’s risk assessment and proposed findings for
the mCry3A registration, EPA required a full-scale field study to
examine long-range effects of cultivation of corn expressing mCry3A on
invertebrate communities as a condition of registration (USEPA 2010b).
This study has been submitted and a review is pending.

At present, the Agency is aware of no identified significant adverse
effects of mCry3A protein on the abundance of non-target organisms in
any population in the aquatic or terrestrial field environment. In
addition, no direct or indirect effects on Federally listed endangered
and threatened species or effects on their critical habitat are
expected. The EPA has reviewed the potential for gene capture and
expression of mCry3A protein by wild or weedy relatives of corn in the
United States, its possessions or territories and has found that there
is no significant risk in the United States, its possessions or
territories (USEPA 2010b).

In conclusion, the risk assessment found no risk to the environment from
cultivation of Event MIR604 corn expressing mCry3A protein.

Event 5307 (coleopteran active) Environmental Risk Assessment Summary

The full risk assessment for eCry3.1Ab is provided above; a summary of
this assessment is presented here.  The eCry3.1Ab protein expressed in
Event 5307 corn is a new active ingredient that has not been registered.
Application for its registration has been made concurrently with that
for this Bt11 x MIR604 x TC1507 x 5307 corn hybrid. Syngenta has
submitted studies with Northern bobwhite, broiler chicken, mammals (from
laboratory studies), channel catfish, a Gammarus species (an aquatic
shredder invertebrate), ladybird beetle, rove beetle, carabid beetle,
predatory bug, honey bee, and earthworm to assess the effects to
nontarget organisms. A soil persistence study was also submitted. Based
on the data provided, BPPD found that no adverse effects to nontarget
birds, mammals, fish, aquatic invertebrates, nontarget insects and honey
bees, and nontarget plants were assessed are expected as a result of the
cultivation of Event 5307 corn plants expressing eCry3.1Ab.
Additionally, no direct or indirect effects on Federally listed
endangered and threatened species or effects on their critical habitat
are expected. The potential for gene capture and expression of Cry
proteins has been extensively examined by EPA (USEPA 2010a). This
analysis is relevant to the eCry3.1Ab protein as well, and it was
determined that the risk of gene flow and development of weediness is
low.

In conclusion, the risk assessment found no risk to the environment from
cultivation of Event 5307 corn expressing eCry3.1Ab protein.

Synergism Studies

The purpose of synergism studies is to characterize the potential for
interaction between the Bt Cry proteins contained in combined trait corn
event Bt11 x MIR604 x TC1507 x 5307. To bridge the ecological effects
and environmental fate data of the individual parental events to the
combined PIP products, the combined trait PIP product must be
demonstrated as biochemically and functionally equivalent to their
respective parental PIP events.  The functional equivalence is
established by demonstrating that the effects of the pesticidal mixture
of the combined PIP product on a susceptible pest species are comparable
to the effects of each protein tested individually. Interactions between
the test materials can be assessed by comparing the larval mortality
observed for the mixed proteins with the predicted responses based on
the bioassay of each protein individually, via diet-incorporation
sensitive insect bioassays. If there is no greater mortality than
expected over the range of concentrations in a sensitive pest species,
it is likely that there will be no synergism of the mixture against
non-target organisms and the effect of a mixture of proteins on
non-target organisms can be predicted from the effects of the individual
proteins alone.

The hypothesis of no synergism of the insecticidal proteins when
combined in Bt11 x MIR604 x TC1507 x 5307 was tested in three stages: 1)
a test for synergism among the lepidopteran-active proteins (Cry1Ab and
Cry1F), 2) a test for synergism among the coleopteran-active proteins
(mCry3A and eCry3.1Ab), and 3) a test for synergism between the mixture
Cry1Ab + Cry1F and the mixture mCry3A + eCry3.1Ab.

Bioassays for Interaction Between Cry1Ab and Cry1F

Tests for synergism among Cry1Ab and Cry1F were previously conducted in
two bioassays (MRID 48235204): one using European corn borer (Ostrinia
nubilalis, ECB), which is sensitive to Cry1Ab and Cry1F, and one using
fall armyworm (Spodoptera frugiperda, FAW), which is sensitive to Cry1F
only. Both bioassays also included an additional component, Vip3Aa20,
which is present in Event MIR162 corn, and is active against FAW but not
ECB. Vip3Aa20 was included because interactions among Cry1Ab, Vip3Aa20,
and Cry1F were also being investigated as part of the development of a
different stacked hybrid corn. This study was previously reviewed and
found to be acceptable by EPA (USEPA 2011).

In each bioassay, freshly-hatched larvae were exposed to the proteins
incorporated into commercial insect diet. The treatments consisted of
various individual concentrations of microbially-produced Cry1Ab,
Vip3Aa20, and Cry1F, and of mixtures of microbially-produced Cry1Ab +
Vip3Aa20 + Cry1F. The protein concentrations used were chosen to give a
range of predicted responses to the mixtures, based on preliminary tests
and historical data. The presence of synergism or antagonism among the
proteins was assessed by comparing the observed mortality of larvae on
diets containing the protein mixtures with the predicted mortality based
on bioassays using each protein individually. The predicted responses
were calculated based on an assumption of independent action, using an
extension of the Colby method (Colby, 1967) as described in the study
report.

Based on the results for ECB and FAW, there was no tendency for an
excess or deficit in observed mortality in any test at any time,
indicating that there is no synergism or antagonism among Cry1Ab,
Vip3Aa20, and Cry1F.

Bioassays for Interaction Between mCry3A and eCry3.1Ab

Tests for interactions between coleopteran-active mCry3A and eCry3.1Ab
insecticidal proteins were conducted with similar bioassays using the
Colorado potato beetle (Leptinosara decemlineata, CPB) (MRID 48442909).
Microbially-produced mCry3A and eCry3.1Ab protein test substances were
used in a series of concentrations chosen to give a range of predicted
responses to the combined proteins based on preliminary tests and
historical data. The presence of synergism or antagonism among the
proteins was assessed by comparing the observed mortality of larvae on
diets containing the protein mixtures with the predicted mortality based
on bioassays using each protein individually. As in the bioassays
previously performed to assess the interaction of Cry1Ab, Vip3Aa20, and
Cry1F (MRID 48235204, discussed above), the predicted responses were
calculated based on an assumption of independent action using an
extension of the Colby method (Colby 1967). This study was classified as
acceptable.

The observed mortality results were lower than expected in the combined
protein mixture bioassays in comparison to the predicted mortality
calculations, which presumed an additive effect due to the independent
action of mCry3A and eCry3.1Ab proteins. However, the results of the
combined protein mixtures were comparable to the observed mortality for
the proteins when tested individually against CPB, which corroborates
the hypothesis synergism is not expected between mCry3A and eCry3.1Ab
insecticidal proteins when expressed in combination. The results also
support the functional equivalence of the mCry3A and eCry3.1Ab
insecticidal proteins expressed in combined PIP Bt11 x MIR604 x MIR162 x
TC1507 x 5307 corn product with its respective parental corn events
MIR604 and 5307 corn. Therefore, this combination of proteins is not
likely to cause unexpected increased effects in nontarget organisms.

Bioassays for Interaction of Cry1Ab, Cry1F, mCry3A, and eCry3.1Ab

Diet incorporation feeding assays were conducted to determine whether a
mixture of lepidopteran-active proteins (Cry1Ab and Cry1F, which also
included Vip3Aa20) and a mixture of coleopteran-active proteins (mCry3A
and eCry3.1Ab) interact when used in combination (MRID 48442908). A
study had been previously conducted to determine the dose-response
relationships of the lepidopteran-active and coleopteran-active proteins
using ECB and CPB, and establish appropriate dosage levels for the
interaction study (MRID 48202317). Using these dosage levels, an
interaction study was performed in which ECB and CPB were exposed to a
mixture containing all of the lepidopteran- and coleopteran-active
proteins. The effect of the coleopteran-active mixture on the
insecticidal activity of the lepidopteran-active mixture was determined
using ECB assays. Two doses of the lepidopteran-active mixture (ECB 1
[lower dose] and ECB 2 [higher dose]) were used alone and in combination
with the higher dose of the coleopteran-active mixture (CPB 2).
Mortality results for the lepidopteran-active mixture alone were
compared to those for the combined lepidopteran-active and
coleopteran-active mixtures. The effect of the lepidopteran-active
mixture on the insecticidal activity of the coleopteran-active mixture
was determined using CPB assays. Two doses of the coleopteran-active
mixture (CPB 1 [lower dose] and CPB 2 [higher dose]) were used alone and
in combination with the higher dose of the lepidopteran-active mixture
(ECB 2). Mortality results for the coleopteran-active mixture alone were
compared to those for the combined coleopteran-active and
lepidopteran-active mixtures. For each interaction bioassay, F-tests
were used to assess the statistical significance of the effects of dose,
inactive ingredient, and dose x inactive ingredient interaction at the
5% probability level.

In the interaction studies with ECB, the ECB 2 dose produced higher
mortality than the ECB 1 dose, as expected. There were no statistically
significant differences in ECB mortality from addition of the
coleopteran-active (ECB-inactive) ingredient (CPB 2) and no
statistically significant dose x ECB-inactive ingredient interactions.
Similarly, the CPB 2 dose produced higher mortality than the CPB 1 dose
in CPB, as expected. There were no statistically significant differences
in CPB mortality from addition of the lepidopteran-active (CPB-inactive)
ingredient (ECB 2) and no statistically significant dose x CPB-inactive
ingredient interactions.

The study reviewer noted that there was some increase in the activity in
the CPB assays that was related to the high ECB dose, but it is not
statistically significant. Based on the results of the study, the
combination of these insecticidal proteins does not indicate potential
for synergistic activity in the two sensitive insect species tested. The
results also support the functional equivalence of the combined PIP
Bt11 x MIR604 x TC1507 x 5307 corn product with its respective parental
corn events Bt11, MIR604, TC1507 and 5307 corn, respectively. Therefore,
the combination of these proteins is unlikely to produce unexpected
effects in nontarget organisms as a result of the cultivation of Bt11 x
MIR604 x TC1507 x 5307 corn plants.

Protein Expression in Bt11 x MIR604 x TC1507 x 5307 Corn Plants

The expression of Cry1Ab, mCry3A, Cry1F, and eCry3.1Ab individually in
single-trait Bt11, MIR604, TC1507, and 5307 plants, respectively, was
compared with the expression of each of these proteins in the combined
trait Bt11 x MIR604 x TC1507 x 5307 corn plants (MRID 48443001).  This
study is also discussed in the environmental risk assessment submitted
by Syngenta for Bt11 x MIR604 x TC1507 x 5307 corn (MRID 48443004). The
data show that several proteins are expressed at significantly higher
amounts in some tissues of the stacked hybrid corn plants compared to
the individual trait plants. In the hybrid, Cry1Ab is expressed at
levels that are significantly higher in R1 leaves and in R1 whole
plants, mCry3A expression is significantly higher in pollen (also see
discussion below), Cry1F expression is significantly higher in V5
leaves, and eCry3.1Ab is significantly higher in V5 roots.  EPA
communicated concerns for these increased levels of expression in Bt11 x
MIR604 x TC1507 x 5307 corn plants in a letter to Syngenta dated March
15, 2012, and asked Syngenta to provide further information to justify
equivalence of protein expression. Syngenta provided a response (MRID
48802701), which discussed the relevance of these increased levels of
expression to margins of exposure for nontarget organisms and overall
exposure during the life of the plant.

In the letter, EPA also raised concern over the presence of mCry3A
protein in pollen of the Bt11 x MIR604 x TC1507 x 5307 stack. In the
original risk assessment for this protein, mCry3A was not detectable in
MIR604 corn pollen, and tests for effects of mCry3A in aquatic
invertebrates were not required due to the expected lack of exposure.
MRID 48802701 also contains Syngenta’s response for these concerns,
which is based on a discussion of new detection methods that enhanced
the sensitivity of detection assays below the previous level of
quantitation. Syngenta additionally discussed the exposure of aquatic
invertebrates, and concluded that the previous findings are still
relevant to the risk assessment for Bt11 x MIR604 x TC1507 x 5307 corn.

The comparative protein expression study for Bt11 x MIR604 x TC1507 x
5307 corn and Syngenta’s response to the Agency’s concerns is
currently under review, and conclusions of its biochemical equivalence
to its respective parental events are to be provided in the product
characterization memo for Bt11 x MIR604 x TC1507 x 5307 corn (USEPA
2012a). Provided that the expression study and supplemental information
are found to be acceptable, a conclusion can be made that significant
increased exposure of nontarget organisms to the Cry1Ab, mCry3A, Cry1F,
and eCry3.1Ab proteins is not expected as a result of cultivation of
Bt11 x MIR604 x TC1507 x 5307 corn plants.

Risk Assessment Conclusions for Cry1Ab, mCry3A, Cry1F, and eCry3.1Ab as
Expressed in Bt11 x MIR604 x TC1507 x 5307 Corn

EPA has concluded that the individual proteins expressed in Bt11 x
MIR604 x TC1507 x 5307 corn will have no adverse effect on nontarget
organisms, do not persist in the soil, and pose no risk of gene flow and
development of weediness in wild relatives. Synergism studies submitted
to EPA also show that increased toxicity is not expected from the
combination of these proteins. While protein expression data are
currently under review, a significant increase in nontarget exposure is
not anticipated. Therefore, a conclusion is made that cultivation of
Bt11 x MIR604 x TC1507 x 5307 corn will not result in adverse effects to
nontarget organisms or the environment, and additionally will have no
effect, direct or indirect, on Federally listed threatened and
endangered species or their designated Critical Habitats. This
conclusion is dependent on a finding that the expression study for Bt11
x MIR604 x TC1507 x 5307 corn and its associated supplemental
information are found to be acceptable and to support the conclusion of
no significant increase in exposure.

E.   Environmental Risk Assessment for Cry1Ab, Vip3Aa20, mCry3A, Cry1F,
and eCry3.1Ab

       Expressed Bt11 x MIR162 x MIR604 x TC1507 x 5307 Corn Hybrid

1.  Background

Syngenta has applied for a FIFRA Section 3 Registration for a combined
trait corn product Bt11 x MIR162 x MIR604 x TC1507 x 5307 (EPA File
Symbol 67979-EG) that confers lepidopteran and coleopteran insect
resistance and herbicide tolerance (with the Event GA21 trait). The
combined trait corn Bt11 x MIR162 x MIR604 x TC1507 x 5307 product was
developed by crosses (via traditional breeding methods) with each of the
individual events. The data to support this registration are based on
data submitted and approved previously for the individual parental
lines, as well as the data for the eCry3.1Ab protein expressed in Event
5307, as discussed above. Synergism studies involving these proteins
were also submitted. These studies provide support for bridging to data
for the individual PIP events that are already contained in the
Agency’s database.

2.   Environmental Assessment

The hazard of each of the individual events in Bt11 x MIR162 x MIR604 x
TC1507 x 5307 has been addressed for previous registrations. Event 5307
is a new active ingredient, and the risk assessment performed for this
active ingredient also applies to this stack. EPA has previously
determined that the database for ecological risk assessment is complete
for the Cry1Ab, Vip3Aa20, Cry1F, and mCry3A proteins expressed in this
hybrid, and also for eCry3.1Ab based on the assessment above. Summaries
of the risk assessments for the individual Cry1Ab, mCry3A, Cry1F, and
eCry3.1Ab are provided above in the risk assessment for the Bt11 x
MIR604 x TC1507 x 5307 combined trait corn product. A summary of the
environmental assessment of Vip3Aa20 is provided below. This assessment
also addresses the potential for synergism in the Bt11 x MIR162 x MIR604
x TC1507 x 5307 corn hybrid, as well as protein expression comparability
between the combined trait hybrid and corn plants expressing the
individual traits only.

 Event MIR162 (lepidopteran active) Environmental Risk Assessment
Summary

Potential adverse effects to non-target organisms by the Vip3Aa20
protein expressed by event  MIR162 have been previously reviewed and was
recently updated (USEPA 2009).  The following is a summary of the MIR162
environmental risk assessment.

The Vip3Aa20 protein expressed in MIR162 corn is a Bt-derived vegetative
insecticidal protein intended to control lepidopteran pests; however, it
is not active against all primary lepidopteran corn pests. This protein
is known to act individually to affect a typical midgut pathology in
susceptible insects like previously studied Bt delta-endotoxins (Lee, et
al. 2003). Although the general symptoms displayed by sensitive
lepidopteran larvae following ingestion of Vip proteins resembles that
caused by Cry1Ab proteins (i.e., cessation of feeding, loss of gut
peristalsis, overall paralysis of the insect, and death) (Yu, et al.
1997; and Lee et al. 2008), the Vip3Aa protein variants share no
homology with Cry1Ab and other known Cry proteins and the modes of
action differ between the Vip and Cry proteins. Therefore, while event
MIR162 is active against a wide spectrum of lepidopteran pests, it is
intended for use in creating combinations with other PIPs instead of
commercial distribution in single-trait seed. The potential hazard of
Vip3Aa20 protein was evaluated for Northern bobwhite, broiler chicken,
mammals (based on acute oral toxicological laboratory studies on mice),
channel catfish, Daphnia magna, insidious flower bug, lady beetle, green
lacewing, rove beetle, collembolan, honey bee, and earthworm. In support
of the Event MIR162 registration, test substances used in the submitted
studies included bacterial-produced purified Vip3Aa19 and Vip3Aa1
protein, plant-expressed Vip3Aa20 protein as expressed in Event Pacha
maize leaves, in addition to plant-expressed Vip3Aa20 protein as
expressed in Event MIR162 maize grain, pollen, and leaves. Since
biochemical and functional equivalence was shown among Vip3Aa1, Vip3Aa19
and Vip3Aa20 proteins, some of the studies conducted with Vip3Aa1 and
Vip3Aa19  were bridged to the assessment for Vip3Aa20. A soil
degradation study was also submitted that allowed EPA to conclude that
Vip3Aa20 is not likely to persist in soil. Additionally, gene flow and
weediness assessments were performed, and the potential for these events
has also been discussed extensively for Cry proteins in corn in updated
Bt corn BRADs (e.g., see USEPA 2010).  Based on this information, EPA
concluded that risk to non-target wildlife, aquatic, and soil organisms
is not expected from Vip3Aa20 expressed in Event MIR162 corn.

At present, the Agency is aware of no identified significant adverse
effects of Vip3Aa20 on the abundance of non-target organisms in any
population in the aquatic or terrestrial field environment. In addition,
no direct or indirect effects on Federally listed endangered and
threatened species or effects on their critical habitat are expected.
Further, the EPA has determined that there is no significant risk of
gene capture and expression of Vip3Aa20 protein by wild or weedy
relatives of corn in the U.S., its possessions, or territories,
available data do not indicate that Cry proteins have any measurable
adverse effect on microbial populations in the soil, nor has horizontal
transfer of genes from transgenic plants to soil bacteria been
demonstrated.

In conclusion, the risk assessment found no risk to the environment from
cultivation of Event MIR162 corn expressing Vip3Aa20.

Synergism Studies

The synergism studies discussed in the risk assessment of the Cry
proteins expressed in the Bt11 x MIR604 x TC1507 x 5307 hybrid above
(MRIDs 48442908 and 48442909) are also applicable to the environmental
risk assessment of Bt11 x MIR162 x MIR604 x TC1507 x 5307 hybrid corn
because the Vip3Aa20 protein was also included in these studies. These
studies showed no increase in toxicity to sensitive insect species as a
result of the combination of the Cry1Ab, Vip3Aa20, mCry3A, Cry1F, and
eCry3.1Ab proteins. The results also support the functional equivalence
of the combined PIP  Bt11 x MIR604 x MIR162 x TC1507 x 5307 corn product
with its respective parental corn events Bt11, MIR604, MIR162, TC1507
and 5307 corn, respectively. Therefore, increased toxicity is not
expected in nontarget organisms as a result of the combination of these
proteins in Bt11 x MIR162 x MIR604 x TC1507 x 5307 hybrid corn.

Protein Expression in Bt11 x MIR162 x MIR604 x TC1507 x 5307 Corn Plants

The expression of Cry1Ab, Vip3Aa20, mCry3A, Cry1F, and eCry3.1Ab
individually in single-trait Bt11, MIR162, MIR604, TC1507, and 5307 corn
plants, respectively, was compared with the expression of each of these
proteins in the combined trait Bt11 x MIR604 x TC1507 x 5307 corn plants
(MRID 48442901).  This study is also discussed in the environmental risk
assessment submitted by Syngenta for Bt11 x MIR162 x MIR604 x TC1507 x
5307 corn (MRID 48442904). The data show that Cry1Ab is expressed at
levels that are significantly higher in R1 leaves and in R1 whole
plants. EPA communicated concerns for these increased levels of
expression in Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn plants in a
letter to Syngenta dated March 15, 2012, and asked Syngenta to provide
further information to justify equivalence of protein expression between
the single and combined trait plants. Syngenta provided a response (MRID
48802801), which discussed the relevance of these increased levels of
expression to margins of exposure for nontarget organisms and overall
exposure during the life of the plant.

In the letter, EPA also raised concern over the presence of mCry3A
protein in pollen of the Bt11 x MIR162 x MIR604 x TC1507 x 5307 stack.
As discussed above, in the original risk assessment for this protein,
mCry3A was not detectable in MIR604 corn pollen, and tests for effects
of mCry3A in aquatic invertebrates were not required due to the expected
lack of exposure. MRID 48802801 also contains Syngenta’s response for
these concerns, which are identical to their justification and
conclusion discussed above for Bt11 x MIR604 x TC1507 x 5307 corn.

The comparative protein expression study for Bt11 x MIR162 x MIR604 x
TC1507 x 5307 corn and Syngenta’s response to the Agency’s concerns
is currently under review, and conclusions of its biochemical
equivalence to its respective parental events are to be provided in the
product characterization memo for Bt11 x MIR162 x MIR604 x TC1507 x 5307
corn (USEPA 2012b). Provided that the expression study and supplemental
information are found to be acceptable, a conclusion can be made that
significant increased exposure of nontarget organisms to the Cry1Ab,
Vip3Aa20, mCry3A, Cry1F, and eCry3.1Ab proteins is not expected as a
result of cultivation of Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn
plants.

Risk Assessment Conclusions for Cry1Ab, Vip3Aa20, mCry3A, Cry1F, and

       eCry3.1Ab as Expressed in Bt11 x MIR162 x MIR604 x TC1507 x 5307
Corn

EPA has concluded that the individual proteins expressed in Bt11 x
MIR162 x MIR604 x TC1507 x 5307 corn will have no adverse effect on
nontarget organisms, do not persist in the soil, and pose no risk of
gene flow and development of weediness in wild relatives. Synergism
studies submitted to EPA also show that increased toxicity is not
expected from the combination of these proteins. While data are
currently under review to show that these proteins are not expressed in
Bt11 x MIR162 x MIR604 x TC1507 x 5307 hybrid corn at levels that
significantly increase nontarget exposure above levels of concern, a
significant increase in nontarget exposure is not anticipated.
Therefore, a conclusion is made that cultivation of Bt11 x MIR604 x
TC1507 x 5307 corn will not result in adverse effects to nontarget
organisms or the environment, and additionally will have No Effect,
direct or indirect, on Federally listed threatened and endangered
species or their designated Critical Habitats. This conclusion is
dependent on a finding that the expression study for Bt11 x MIR162 x
MIR604 x TC1507 x 5307 corn and its associated supplemental information
are found to be acceptable and to support the conclusion of no
significant increase in exposure.

IV.  References

Carstens, K.L., Anderson, J.A., Bachman, P., DeShrijver, A., Dively, G.
Federici, B. Hamer, M., Gielkens, M., Jensen, P., Lamp, W., Raushen, S.,
Ridley, G., Romeis, J., Waggoner, A. 2011. Genetically modified crops
and aquatic ecosystems: considerations for environmental risk assessment
and non-target organism testing. Transgenic Res., published online 26
November 2011.

Colby, S.R. 1967. Calculating synergistic and antagonistic responses of
herbicide combinations. Weeds 15:20-22.10.

Crocker, D., Hart, A., Gurney, J., McCoy, C. 2002. Project PN0908:
Methods for estimating daily food intake of wild birds and mammals.
Central Science Laboratory. Unpublished report to the (UK) Department
for Environment, Food and Rural Affairs. Included as an appendix to US
EPA MRID 46155615.

Jensen, P.D., Dively, G.P., Swan, C.M., Lamp, W.O. 2010. Exposure and
non-target effects of transgenic Bt corn debris in streams. Environ.
Entomol. 39(2):707-714.

Lee, M.K., Walters, F.S. , Hart, H., Palekar, N., Chen, J-S. 2003. The
mode of action of the

	Bacillus thuringiensis vegetative insecticidal protein Vip3A differs
from that of Cry1Ab δ-endotoxin.  App. Environ. Micro.  69(8):
4648-4657.

Marvier, M., McCreedy, C., Regetz, J. & Kareiva, P.  2007.  A
meta-analysis of effects of Bt

cotton and maize on nontarget invertebrates. Science 316: 1475–1477.

National Academy of Science (NAS).  2000.  Environmental Effects of
Transgenic Plants: The Scope and Adequacy of Regulation is available
from the National Academy Press, 2101 Constitution Avenue, N.W., Lockbox
285, Washington, DC 20055;<PHONE_NUMBER> or<PHONE_NUMBER> (in the
Washington metropolitan area);  HYPERLINK "http://www.nap.edu"
http://www.nap.edu .

Romeis, J., Meissle, M., Bigler, F.  2006.  Transgenic Crops expressing
Bacillus

thuringiensis toxins and biological control. Nature Biotechnology 24:
63-71.

Rosi-Marshall E. J., Tank, J. L., Royer, T. V., Whiles, M. R.,
Evans-White, M., Chambers, C., Griffiths, N. A., Pokelsek, J., Stephen,
M.L. 2007. Toxins in transgenic crop byproducts may affect headwater
stream ecosystems. PNAS:  104(41): 16204–16208.

Sanvido, O., Romeis, J., Bigler, F. (2007). Ecological Impacts of
Genetically Modified Crops: Ten Years of Field Research and Commercial
Cultivation. Adv Biochem Engin/Biotechnol 107: 235–278.

Saxena, D. and Stotzky, G. (2001) Bacillus thuringiensis (Bt) toxin
released from root exudates and biomass of Bt corn has no apparent
effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil.
Soil Biol. Biochem. 33: 1225–1230.

Swan, C.M., Jensen, P.D. , Dively, G.P., Lamp, W.O.. 2009. Processing of
transgenic crop residues in stream ecosystems. J. App. Ecol.
46:1304-1313.

United States Environmental Protection Agency (USEPA) (1998).
“Guidelines for Ecological Risk Assessment.”  EPA 630/R-95-002F.
Washington, DC, USA. [Federal Register, May 14, 1998. 63(93):
26846-26924.]

USEPA.  2000. SAP report No 99-06. Sets of scientific issues being
considered by the Environmental Protection Agency regarding: Section I -
Characterization and Nontarget Organism Data Requirements for Protein
Plant Pesticides. Dated February 4, 2000. Available at the EPA website:
http://www.epa.gov/scipoly/sap/1999/index.htm#december

USEPA. 2001. SAP Report No. 2000-07. Sets of scientific issues being
considered by the Environmental Protection Agency regarding: Bt
plant-pesticides risk and benefit assessments. Dated March 12, 2001. Web
site:  HYPERLINK
"http://www.epa.gov/scipoly/sap/2000/october/octoberfinal.pdf"
http://www.epa.gov/scipoly/sap/2000/october/octoberfinal.pdf

USEPA.  2002. SAP Report No. 2002-05. A set of scientific issues being
considered by the Environmental Protection Agency regarding: Corn
rootworm plant-incorporated protectant nontarget insect and insect
resistance management issues. Dated November 6, 2002.   HYPERLINK
"http://www.epa.gov/scipoly/sap/2002/august/august2002final.pdf"
http://www.epa.gov/scipoly/sap/2002/august/august2002final.pdf

USEPA.  2004. SAP Report No.2004-05.  Product characterization, human
health risk,

ecological risk, and insect resistance management for Bt cotton
products. Dated August 19, 2004.   HYPERLINK
"http://www.epa.gov/scipoly/sap/meetings/2004/june/final1a.pdf"
http://www.epa.gov/scipoly/sap/meetings/2004/june/final1a.pdf

USEPA, 2009. Biopesticides Registration Action Document – Bacillus
thuringiensis Vip3Aa20 Insecticidal Protein and the Genetic Material
Necessary for Its Production (via Elements of Vector pNOV1300) in Event
MIR162 Maize (OECD Unique Identifier: SYN-IR162-4),  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_00
6599.pdf"
http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006
599.pdf .

USEPA, 2010a. Biopesticides Registration Action Document – Cry1Ab and
Cry1F Bacillus thuringiensis (Bt) Corn Plant-Incorporated Protectants,
HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/cry1f-cry1ab-brad.pdf"
http://www.epa.gov/oppbppd1/biopesticides/pips/cry1f-cry1ab-brad.pdf .

USEPA, 2010b. Biopesticides Registration Action Document – Modified
Cry3A Protein and the Genetic Material Necessary for its Production (Via
Elements of PZM26) in Event MIR604 Corn SYN-IR604-8,  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/mcry3a-brad.pdf"
http://www.epa.gov/oppbppd1/biopesticides/pips/mcry3a-brad.pdf .

USEPA, 2010c. Biopesticides Registration Action Document – Bacillus
thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Genetic Material
Necessary for Their Production (PHP17662 T-DNA) in Event DAS-59122-7
Corn (OECD Unique Identifier: DAS-59122-7),  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/cry3435ab1-brad.pdf"
http://www.epa.gov/oppbppd1/biopesticides/pips/cry3435ab1-brad.pdf .

USEPA, 2011. Review of Molecular Characterization data, Test Substance
Characterizations, Protein Expression Analyses and Synergism  Study for
Combination PIP Bt11 x MIR162 x TC1507 corn, Agrisure ™ 3100 [EPA Reg.
No. 67979-RL, OECD Unique ID. SYN-BTØ11-1 x SYN-IR162-4 x
DAS-Ø15Ø7-1] in support for Sec. 3 Registration, submitted by Syngenta
Seeds, Inc. – Field Crops- NAFTA. Memorandum from A. Waggoner, through
J. Kough, to J. Kausch. Dated March 25, 2011. US Environmental
Protection Agency, Washington, D.C.

USEPA, 2012a. Review of Molecular Characterization Data, Test Substance
Characterizations and Protein Expression Analyses for Combination PIP Bt
11 x MIR604, TC1507 x 5307 corn [EPA Reg. No. 67979-EU; OECD Unique ID.
SYN-BTØ11-1 x SYN-IR-604-5 x DAS-Ø15Ø7-1 x SYN-05037-1] in support
for FIFRA Section 3 Registration, submitted by Syngenta Seeds, Inc. –
Field Crops- NAFTA. Memorandum from J. Facey, through J. Kough, to M.
Mendelsohn. US Environmental Protection Agency, Washington, D.C.

USEPA, 2012b. Review of Molecular Characterization Data, Test Substance
Characterizations, Protein Expression Analyses and Synergism  Study for
Combination PIP Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn [EPA Reg.
No. 67979-EG, OECD Unique ID. SYN-BTØ11-1 x SYN-IR162-4 x SYN-IR-604-5
x DAS-Ø15Ø7-1 x SYN-05307-1] in support for Sec. 3 Registration,
submitted by Syngenta Seeds, Inc. – Field Crops- NAFTA. Memorandum
from A. Balen, through J. Kough, to M. Mendelsohn. US Environmental
Protection Agency, Washington, D.C.

Wolt, J.D. and Peterson, R.K.D. 2010. Prospective formulation of
environmental risk assessments: Probabilistic screening for Cry1A(b)
maize risk to aquatic insects. Ecotoxicol. Environ. Safety 73:
1182-1188.

Yu, C. Mullins, M.A., Warren, G.W., Koziel, M.G. and Estruch, J.J.
(1997).  The Bacillus

thuringiensis Vegetative Insecticidal Protein Vip3A Lyses Midgut
Epithelium Cells of Susceptible Insects. Applied and Environmental
Microbiology 63 (2): 532-53.

F. 	Insect Resistance Management (IRM)

Background

BPPD   SEQ CHAPTER \h \r 1 has reviewed the Insect Resistance Management
(IRM) plan and supporting data submitted by Syngenta for two proposed
commercial Bt corn products containing the new active ingredient
eCry3.1Ab (event 5307).  The two products (EPA Reg No. 67979-EU and
67979-EG) are “stacked pyramids” consisting of eCry3.1Ab and
previously-registered Bt corn toxins for control of lepidopteran stalk
borers and corn rootworm.  Separate IRM volumes were submitted for each
proposed product:  Bt11 x MIR604 x TC1507 x 5307 (MRID# 484430-08) and
Bt11 x MIR162 x MIR604 x TC1507 x 5307 (MRID# 484429-10).

Conclusions and Recommendations

1) Syngenta has submitted adequate data and modeling to demonstrate that
the 5307 pyramided products (Bt11 x MIR604 x TC1507 x 5307 and Bt11 x
MIR162 x MIR604 x TC1507 x 5307) will have high durability with a 5%
refuge in the Corn Belt (relative to single toxin products deployed with
20% refuge).  There should be strong durability for both of the target
pest complexes:  lepidopteran stalk/ear borers (European corn borer,
Southwestern corn borer, and corn earworm) and corn rootworm.

2) For southern regions in which cotton is also grown, Syngenta proposed
a 20% refuge for Bt11 x MIR162 x MIR604 x TC1507 x 5307 (67979-EG) but a
50% refuge for Bt11 x MIR604 x TC1507 x 5307 (67979-EU), presumably
because the latter product has one fewer lepidopteran-active toxin.
Larger refuge sizes in this region are driven by concerns with corn
earworm (a corn and cotton pest).   BPPD notes that a 20% refuge is
compatible with the requirements for other similar Bt corn pyramids
(including two Syngenta products).  Syngenta may be able to support a
20% refuge for the Bt11 x MIR604 x TC1507 x 5307 pyramid with a
relatively simple model (as was done to support the other registered
pyramids).

3) Syngenta did not propose a seed blend with initial product
registration for either of the two 5307 pyramids.  BPPD notes that
additional information and modeling may be necessary to support such a
refuge strategy.

4) Syngenta provided data to demonstrate that the risk of cross
resistance between eCry3.1Ab and mCry3A (Syngenta’s other registered
rootworm toxin) should be low.  The company did not, however, provide an
assessment of the cross resistance potential between eCry3.1Ab and other
rootworm toxins not registered by Syngenta (i.e., Cry3Bb1 and
Cry34/35Ab1).  BPPD recognizes that eCry3.1Ab (an engineered toxin)
should have significant structural differences with Cry3Bb1 and
Cry34/35Ab1, and therefore, a low likelihood of cross resistance.  But
no information was provided in the submission to verify this assumption.
 Accordingly, BPPD recommends that Syngenta provide additional data or
analysis to address the cross resistance potential of eCry3.1Ab with
Cry3Bb1 and Cry35/35Ab1.  As part of this effort, BPPD also recommends
that Syngenta work to develop a eCry3.1Ab-resistant colony (similar to
the work done for mCry3A -- see Meihls et al. 2011).

5) Existing programs for resistance monitoring (lepidopteran pests),
remedial action, and compliance assurance should be compatible with the
5307 pyramids.  Additional data and information, however, are
recommended for corn rootworm resistance monitoring (see #6 below).
BPPD recommends that Syngenta update and submit grower education
materials (e.g., technology use guides) to include the 5307 product line
and the appropriate refuge requirements.  In addition, Syngenta should
submit annual IRM reports for resistance monitoring, compliance, and
sales.

6) BPPD recommends that Syngenta submit the following additional data
for corn rootworm resistance monitoring with the eCry3.1Ab toxin:

Baseline susceptibility data;

A proposal for a plant-based diagnostic assay including an investigation
into the feasibility of using the “Sublethal Seedling Assay”
(Nowatzki et al. 2008);

A monitoring plan for northern corn rootworm (Diabrotica barberi);

Corn rootworm damage guidelines (for unexpected pest damage
investigations of eCry3.1Ab corn).

Background

Syngenta Seed, Inc. has proposed to register three Bt corn products
containing the new active ingredient event 5307, which expresses the
eCry3A.1Ab toxin.  Applications were submitted for registration of Event
5307 alone as a breeding intermediate and in two stacked/pyramided end
products (referred to as the “5307” pyramids in this review).  The
proposed products are as follows:

5307 (breeding intermediate); EPA Reg. No. 67979-EE

Bt11 x MIR604 x TC1507 x 5307; EPA Reg. No. 67979-EU

Bt11 x MIR162 x MIR604 x TC1507 x 5307; EPA Reg. No. 67979-EG

The eCry3A.1Ab toxin in event 5307 was engineered from “variable
regions” of mCry3A and Cry1Ab (Walters et al. 2010).  It is active
against the corn rootworm (CRW) complex, including western corn rootworm
(WCRW, Diabrotica virgifera virgifera) and northern corn rootworm (NCRW,
Diabrotica barberi).  MIR604 (mCry3A) is also targeted against CRW and
has been previously registered in a number of stacked and pyramided
products (see BRAD -- BPPD 2006).

Bt11 (Cry1Ab) and TC1507 (Cry1F) x TC1507 are effective against European
corn borer (ECB, Ostrinia nubilalis), the major lepidopteran target pest
in the Corn Belt, as well as corn earworm (CEW, Helicoverpa zea),
southwestern corn borer (SWCB, Diatraea grandiosella), and fall armyworm
(FAW, Spodoptera frugiperda).  MIR162 (Vip3Aa20) is also targeted
against the lepidopteran pests CEW, SWCB, and FAW but does not provide
efficacy against ECB.  All three of the lepidopteran toxins have been
previously registered and deployed in stacked/pyramided products (BRADs:
 BPPD 2001 and 2009a).   Syngenta has indicated that a number of other
secondary lepidopteran pests will be targeted including black cutworm
(Agrotis ipsilon), dingy cutworm (Feltia jaculifera), lesser corn stalk
borer (Elasmopalpus lignosellus), southern corn stalk borer (Diatrea
crambidoides), common stalk borer (Papaipema nebris), sugarcane borer
(Diatrea saccharalis), and western bean cutworm (WBCW, Richia
albicosta).

Because 5307 (EPA Reg. No. 67979-EE) is intended for breeding purposes
only (and not for commercial corn production), a specific IRM plan is
not required for the product (and it is not addressed further in this
review).  Although no refuge is typically planted with breeding corn,
such registrations have been limited in terms of overall acreage (at
county and national levels) to mitigate potential resistance.

Summary of Syngenta’s IRM Submissions for 5307 Pyramided Products

Two separate IRM plans were submitted for the stacked/pyramided
registrations containing the new active ingredient 5307, though much of
the same data was shared between the two applications.  Each of these
listed studies listed is reviewed in this memorandum and the assessment
of the shared data is applicable to both products.

MRID# 484429-10:	Insect Resistance Management Plan for Bt11 x MIR162 x
MIR604 x TC1507 x 5307 Maize

MRID# 484430-08:	Insect Resistance Management Plan for Bt11 x MIR604 x
5307 x TC1507 Maize

The major components of Syngenta’s IRM plan included dose
determinations, investigation of cross resistance, and simulation
modeling.  These sections and BPPD’s review of the overall IRM plan
are described below.

1.  Dose Considerations

Lepidopteran Toxins

Because all of the lepidopteran-active toxins in the two proposed
products have been previously registered, Syngenta did not conduct new
dose studies for ECB, SWCB, or CEW.  Rather, data (MRID# 480506-06)
developed for a separate pyramided product containing Cry1Ab, Cry1F, and
Vip3Aa20 were cited.  In conducting these studies, Syngenta employed the
methodology prescribed by the 1998 Science Advisory Panel (SAP 1998),
using techniques “1” and “4” for each of the insects.
Specifically, the two SAP procedures are as follows:

1)  Serial dilution bioassay with artificial diet containing lyophilized
tissues of Bt plants using tissues from non-Bt plants as controls;

4)  Survey large numbers of commercial plants (using a controlled
infestation with a susceptible laboratory strain) 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.

Syngenta reported the results of these tests in their IRM submission for
the registration of Bt11 x MIR162 x TC1507 corn (EPA Reg. No. 67979-15).
 Based on those data, Syngenta concluded that Bt11 x MIR162 x TC1507
corn has high dose expression for ECB and SWCB.  This conclusion was
supported by the data obtained from the Bt11 x MIR162 x TC1507 pyramid
and also from the single toxin components (Bt11 and TC1507) which showed
similar high dose activity.  MIR162 has no activity against ECB, but
showed high activity for SWCB in one test (technique “4”) but not
the other (technique “1”).  Against CEW, Bt11 x MIR162 x TC1507
expresses an “effective” high dose as a pyramid, though the three
single toxin events were shown to have less than high dose expression by
themselves.  Effective high dose indicates that the combined activity of
the toxin in the pyramid produces complete mortality (though the single
toxin constituents may allow some survival).

In the current submission, Syngenta concluded that these Cry1Ab and
Cry1F dose results should also be valid for Bt11 x MIR162 x MIR604 x
TC1507 x 5307 and Bt11 x MIR604 x 5307 x TC1507 since field expression
data show comparable toxin levels between the pyramided and constituent
single toxin products.  The lepidopteran dose data were fully described
in MRID# 480506-06 (reviewed in BPPD 2011).

BPPD Review - Lepidopteran Dose Considerations

Data for lepidopteran dose expression were previously reviewed for the
registration of Bt11 x MIR162 x TC1507 corn (see review in BPPD 2011).
An excerpt of the main conclusions in BPPD’s review is as follows:

BPPD generally agrees with Syngenta’s characterization of the dose
profile for Bt11 x MIR162 x TC1507 corn -- i.e., high dose activity
against the major target pests (with CEW considered an “effective”
high dose).  Dose testing was not reported for FAW in this submission
for Cry1Ab or Cry1F.  The results obtained for the other toxin
combinations in these studies, including the single trait (Bt11, MIR162,
TC1507) and dual toxin (Bt11 x MIR162, Bt11 x TC1507) constituent
products, support previously-determined dose conclusions for MIR162
(summarized in BPPD 2009a).

BPPD questions, however, whether the sample sizes tested in SAP method
#1 were adequate for a definitive “high dose” conclusion.  Syngenta
tested only 20 - 24 insects per treatment (with 100% mortality on Bt).
It is unclear if these small scale tests are sufficient to conclude that
each toxin will produce 99.99% mortality to the larger ECB and SWCB
populations.

BPPD notes that no new dose data were developed specifically for the
either of the “5307” pyramided products.  Syngenta’s dose
conclusions for these products are largely supported by a reference to
field expression data showing comparable levels of protein in the
pyramided products to the single toxin events.  These data were
submitted as part of the “5307” applications, but were not
summarized in the IRM volume.  Field efficacy trials (another means to
verify dose performance in pyramided/stacked products) against the major
target pests were also not submitted or referenced in the report.
However, provided that the protein expression levels are comparable (as
described by Syngenta), BPPD agrees with the dose characterization of
the “5307” pyramided products as having a high dose for ECB and SWCB
and an “effective” high dose for CEW.

Rootworm Toxins (MRID# 484429-10, Appendix A)

Dose studies for the CRW-active toxins eCry3.1Ab and mCry3A were
conducted by Dr. Bruce Hibbard (USDA-ARS, Columbia, MO).  CRW larval
mortality to single toxin and pyramided events was assessed from
artificially-infested plots in Central Missouri over a three year
period.  Selection intensity (survival relative to the control) was
calculated to provide an indication of the effectiveness (dose) of each
toxin and the pyramid.

Within each test field (1-3 locations with 3-5 replicates were tested
per year) plots were established of eCry3A.1Ab (designated event SYN101
in the report), mCry3A (MIR604), SYN101 x MIR604, and a non-Bt isoline.
During the seasons prior to testing, soybeans were planted to reduce the
possibility of wild CRW infesting the plots.  Insecticides were not used
in the test fields (including seed treatments) to avoid confounding
factors, though the fields were otherwise managed with corn agronomic
practices typical of the region.  In the Bt test plots, each plant was
verified for Bt expression using gene strip tests.  Buffers
(vegetation-free areas) were established outside each test plot to
prevent CRW larval movement between plots.  Prior to testing, plots were
thinned to 16 plants per row (3.05 m row length).

WCRW eggs (obtained from wild populations) were artificially infested in
each plot when the corn plants reached the V2-V3 growth stage.  The
infestation rate was 1,380 eggs per 30.5 cm (row foot) for 2007-2008
tests, but was reduced to 875 eggs/foot in 2009 testing to reduce
potential density dependent mortality in the control plots (based on
work by Hibbard et al. 2010).  Test plots were fitted with screen tents
to collect emerging adult CRW approximately 5-6 weeks after infestation.
 After initial beetle emergence, all but four plants in each plot were
cut off at 1 m and stripped of leaves (presumably for easier beetle
collection).  Beetles were collected 2-3 times per week for the duration
of the study.

Survival of WCRW was assessed in terms of “selection intensity,” or
the percentage of beetles emerging from the Bt treatments relative to
the non-Bt control plots.   In addition to WCRW, any northern corn
rootworm (NCRW) and southern corn rootworm (SCRW) beetles (presumably
from wild populations) were also collected from the field tents.

The results from the trials showed that both the single toxin (SYN101
and MIR604) and pyramided (MIR604 x SYN101) treatments significantly
reduced WCRW and SCRW populations relative to control plots.  NCRW
beetle emergence was also reduced by the Bt treatments, though in most
of the treatments sites no beetles were collected in the non-Bt tents.
The mCry3A and eCry3.1Ab toxins appeared to have a greater impact on
WCRW emergence than SCRW.  In terms of selection intensity, single trait
SYN101 reduced beetle survival by 99.79%, while pyramided MIR604 x
SYN101 lowered emergence by 99.91% over the five test locations/years.
MIR604 alone resulted in an average selection intensity of 97.84%.
There appeared to be little difference in beetle counts or selection
intensity between males and females.  The study authors did not report
selection intensity for SCRW (though the average beetle counts were
provided), but noted selection intensities for NCRW of 97.13% (MIR604),
98.85% (SYN101) and 100% (MIR604 x SYN101).  Based on the results,
Syngenta concluded that the mCry3A x eCry3.1Ab pyramid expresses a
“highly effective dose” for WCRW and NCRW.  Data from the dose
investigations are summarized in Table 1 below.

To determine whether each toxin was acting independently, the
researchers conducted a statistical analysis of beetle emergence.  Had
the two toxins acted independently, 0.0982 beetles would have been
expected to emerge from the MIR604 x SYN101 plots (based on the survival
results from the single toxin plots).  In the experiments, 0.8 beetles
emerged (averaged across all five locations), a statistically higher
amount than the 0.0982 expected.

Table 7.  Mortality (Selection Intensity) of CRW to eCry3.1Ab and mCry3A
in Single Toxin and Pyramided Toxin Deployments (compiled from data
provided in Tables 3 and 4 of MRID# 484429-10, Appendix A)

Treatment	SCRW	NCRW 1	WCRW

	Beetle Ct.2 (avg.)	Sel. Int. (%)	Beetle Ct.2 (avg.)	Sel. Int. (%)
Beetle Ct.2 (avg.)	Sel. Int. (%)

MIR604	13.1 b	NR	0.2 b	97.13	19.0 b	97.84

SYN101	13.6 b	NR	0.1 b	98.85	1.9 c	99.79

MIR604 x SYN101	10.6 b	NR	0.0 b	100	1.9 c	99.91

Isoline	36.8 a	NR	7.9 a	0	879.0 a	0

1 NCRW beetles were collected at only two of the five test sites.

2 Total beetle counts for both sexes are reported. For each treatment,
mean values with separate letters are statistically different by
Fisher’s Least Square Difference test.

BPPD Review - Corn Rootworm Dose Considerations

Syngenta’s dose studies for CRW follow the methodology employed for
other rootworm-active PIP toxins including Cry34/35, mCry3A, and Cry3Bb1
(see methodology and data published in Storer et al. 2006; Hibbard et
al. 2010a, b; and data summarized in BPPD 2009b).  BPPD agrees with
Syngenta that the events containing eCry3.1Ab have high activity against
WCRW, although it is unlikely that the toxin is expressed at a “high
dose” level (as defined by the 1998 SAP).

None of the other registered CRW toxins (Cry34/35, mCry3A, and Cry3Bb1)
is known to express a true high dose for CRW, even in pyramided toxin
deployments.  Still, eCry3.1Ab greatly reduced WCRW survival (>99%) and
may be among the most efficacious toxins for rootworm registered to
date.  The study authors compared the eCry3.1Ab results with data from
other toxins and noted that selection intensity for Cry34/35 was 96.47%
(Storer et al. 2006) and 94.88% for mCry3A (Hibbard et al. 2010b).  Data
analyzed for a separate product (SmartStax) showed selection intensity
ranges of 96.17 - 99.96% for Cry3Bb1, 94.20 - 99.18% for Cry34/35, and
98.22 - 99.97% for Cry3Bb1 x Cry34/35 pyramid (reviewed in BPPD 2009b).

Fewer conclusions can be drawn for either NCRW or SCRW.  Although the
researchers reported high reductions in NCRW emergence (see Table 1),
the data were based on only two locations (no beetles were collected in
the other locations).  No artificial infestations were performed, so it
is assumed that the NCRW observed in the two locations were from wild
populations.  Even in the plots where NCRW were found, overall beetle
numbers were low (7.9 avg. beetles/non-Bt field).  Similarly, SCRW
collections presumably arose from feral populations, though beetles were
fairly numerous (36.8 avg. beetles/non-Bt field).  Although not
reported, the selection intensity appeared to be lower with SCRW than
with either WCRW or NCRW (e.g., 71.2% for the pyramid).  However,
because infestation was not controlled, any conclusions regarding the
efficacy of eCry3.1Ab against either NCRW or SCRW are limited.

2.  Cross Resistance

Lepidopteran Toxins

Syngenta’s conclusions regarding potential cross resistance between
Cry1F, Cry1Ab, and Vip3A were based upon data and rationales that were
previously submitted for Bt11 x MIR162 x TC1507 (EPA Reg. No. 67979-15),
a lepidopteran-active pyramid containing the same toxins as the proposed
products.  These cross resistance data were assessed in BPPD’s review
of the Bt11 x MIR162 x TC1507 submission (MRID# 480506-07 and -08;
reviewed in BPPD 2011).  Previously, the cross resistance potential for
Vip3A was assessed for the initial registration of MIR162 (see
discussion in BPPD 2009).

For ECB, Syngenta cited a number of published studies to support the
lack of cross resistance between Cry1Ab and Cry1F.  Vip3A (MIR162) is
not active against ECB and was therefore not considered in the cross
resistance assessment.  Syngenta noted the following points for ECB:

Resistance is most likely to result from a decrease in Bt (Cry) toxin
binding with epithelial cells in the insect midgut.  Other mechanisms
(e.g. metabolic adaptations) would likely be too weak to overcome the
high levels of mortality causes by PIP toxins in the field.

Midgut binding assays have shown that Cry1Ab and Cry1F do not strongly
compete for the same binding sites in ECB (Hua et al. 2001).

Studies with Cry1Ab and Cry1F-resistant ECB colonies did not show high
levels of cross resistance to the reciprocal toxin (Pereira et al. 2008;
Siqueira et al. 2004)

Fewer data are available for CEW and the three toxins.  Syngenta cited
work with Cry1F and Cry1Ac (developed for Dow AgroScience’s WideStrike
cotton product, see Storer 2002), based on the rationale that Cry1Ab and
Cry1Ac share binding sites and likely would have similar patterns of
cross resistance.  Work with resistant colonies has been limited since
stable field-relevant resistance has not been established.  However,
Syngenta noted that a Cry1Ab-resistant laboratory CEW strain was still
susceptible to Vip3A (Konasale et al. 2008).  [BPPD note:  After review,
BPPD believes Syngenta intended to reference Anilkumar et al. 2008, a
study in which Cry1Ac-resistant CEW were tested.]

Syngenta referenced previously-submitted resistant colony studies for
FAW (MRID# 480506-07; reviewed in BPPD 2011).  The company concluded
that these data showed incomplete cross resistance between Cry1F and
Cry1Ab, but no cross resistance with Cry1F and Vip3A.

Overall, Syngenta concluded that:  1) Based on midgut binding
characterizations and studies with resistant colonies, Cry1F likely has
“little or no potential cross resistance” with Vip3A in CEW or FAW;
2) Similarly, there is low potential for “field relevant” cross
resistance between Cry1F and Cry1Ab in ECB or FAW; 3) More limited
information on CEW (midgut binding studies with Cry1F and Cry1Ac)
suggests there is “potential for moderate levels of cross resistance
between Cry1F and Cry1Ab.....however, this risk is highly mitigated by
the presence of Vip3A.”

BPPD Review - Lepidopteran Cross Resistance Potential

BPPD generally agrees with the overall conclusion that there is low
potential for cross resistance between the toxins among the lepidopteran
pests, despite the cursory information provided by Syngenta in its
previous submission (MRID# 480506-07 and -08; see review in BPPD 2011).
No new data were provided with the two 5307 pyramid applications.

Cry1F and Cry1Ab may have some risk for cross resistance.  Both are part
of the Cry1 family of Bt toxins that share some structural similarities
and several studies have found cross resistance in various insects
between different toxins in the group (e.g., Denholf et al. 1993;
Granero et al. 1993; Gould et al. 1995, Bolin et al. 1999; others).
Nevertheless, the weight of evidence from midgut receptor binding
characterizations and resistant colony work suggests that among the
major corn pests (ECB and FAW), the potential for cross resistance
between Cry1F and CryAb1 should be low.  There are other factors that
may reduce the probability for cross resistance among the targeted
pests.  As discussed in BPPD (2011), recent research (Pereira et al.
2010) suggests that resistance to Cry1F may be the result of a unique
process different from identified resistance mechanisms in other Bt cry
toxins.  Also, an analysis of separate data by BPPD (2010a) concluded
that there is low cross resistance potential between Cry1Ab and Cry1F
for the major target pests of Bt corn.

Fewer data and/or information regarding CEW were available or cited by
Syngenta.  Previous binding work (Storer 2002) was conducted with Cry1F
and Cry1Ac (data developed by Dow AgroSciences); Syngenta believes that
Cry1Ac and Cry1Ab can be expected to have similar binding patterns.
BPPD cannot confirm this hypothesis with the information cited by
Syngenta but notes that a separate analysis (BPPD 2010b) concluded that
it is likely that Cry1Ab and Cry1F will have a low cross resistance
potential in CEW.

The presence of Vip3A in one of the proposed 5307 pyramids (Bt11 x
MIR162 x MIR604 x TC1507 x 5307) will likely help mitigate the potential
risk of Cry1F - Cry1Ab cross resistance for CEW and FAW.  For example, a
Cry1Ac-resistant CEW colony was tested and found to be cross resistant
to Cry1Ab, but not Vip3A (Anilkumar et al. 2008).  Similarly,
Cry1F-resistant FAW were even more susceptible to Vip3A than an
unselected colony (MRID# 480506-07; reviewed in BPPD 2011).  On the
other hand, ECB may have a greater concern for cross resistance in a
Bt11 x MIR162 x TC1507 lepidopteran pyramid because Vip3A is not active
against the insect.  Cry1Ab and Cry1F, therefore, must maintain separate
pathways of toxicity to realize the benefits of the pyramid.

Syngenta has not addressed cross resistance potential with SWCB in this
or previous submissions.  It is commonly assumed that SWCB behaves
similar to ECB and that conclusions made for ECB can be applied to this
insect as well.  However, the 2010 SAP noted key differences between the
insects, particularly in response to Bt toxins (SAP 2011).  To
illustrate, Vip3A has at least some activity against SWCB while having
no activity against ECB (BPPD 2009a).  Therefore, it is unclear to BPPD
whether the assumption that SWCB will also be at low risk for cross
resistance with Cry1Ab, Cry1F, and/or Vip3A will have validity for a
lepidopteran pyramid containing the toxins.

While the likelihood of cross resistance may be low in terms of modified
midgut receptors, it is possible that other mechanisms of cross
resistance (e.g., behavioral modifications or expression of
cry-proteases) could arise with pyramided Bt corn.  The 2010 SAP
concluded that the prediction of low cross resistance potential between
Cry1F and the Cry1A toxins for ECB and SWCB remains uncertain and
outlined a number of epistatic mechanisms that could lead to increased
survival on pyramided Bt corn (SAP 2011).   For these reasons, cross
resistance should be considered in predictive modeling to evaluate the
durability of a Cry1F/Cry1Ab/Vip3A pyramid (particularly for worst case
scenarios).

Corn Rootworm Toxins

Syngenta tested the cross resistance potential between eCry3.1Ab and
mCry3A by investigating midgut binding patterns in WCRW.  Potential
interactions of the toxins with WCRW binding sites were investigated in
qualitative and quantitative competitive binding assays.

The qualitative binding studies were published in Walters et al. (2010).
 Brush border membrane vesicles preparations were made from first instar
WCRW larvae and exposed to activated (chymotrypsin-processed) mCry3A or
eCry3.1Ab.  Heterologous and homologous competition experiments were
performed for each of the toxins.  The resulting western blots showed
that both unlabeled mCry3A and eCry3.1Ab competed with their respective
labeled homologues.   On the other hand, in the heterologous
competitions unlabeled eCry3.1Ab did not inhibit labeled mCry3A binding,
while unlabeled mCry3A failed to diminish the binding of labeled
eCry3.1Ab.  Based on these competition studies, the study authors
concluded that mCry3A and eCry3.1Ab recognize different binding sites in
WCRW.  This novel activity is likely due to the exchange of Cry1Ab and
Cry3A variable regions resulted in the engineered toxin (eCry3.1Ab).

A second set of experiments was conducted to quantify the level of
competition among mCry3A and eCry3.1Ab for binding sites in WCRW.  The
studies were performed by Drs. Juan Jurat-Fuentes and Siva Rama Krishna
Jakka of the University of Tennessee (Appendix C of MRID# 484429-10).
Both toxins were chymotrypsin-activated and radiolabeled with I125 for
detection by photographic plates.  In initial testing, both toxins were
shown to specifically bind to the BBMV preparations, which suggested
limited binding sites in WCRW.  The level of specific binding was higher
for eCry3.1Ab (70-80%) than for mCry3A (50%).  Three separate binding
competition assays were conducted:  1) labeled mCry3A with unlabeled
eCry3.1Ab and mCry3A; 2) labeled eCry3.1Ab with unlabeled mCry3A and
eCry3.1Ab, and 3) labeled mCry3A with unlabeled mCry3A (non-activated)
and eCry3.1Ab (activated).

Results from the assays showed little or no competition between mCry3A
and eCry3.1Ab for BBMV binding sites.  In the first competition, labeled
mCry3A binding was reduced slightly (to ~ 80%) when exposed to the
higher concentrations of unlabeled eCry3.1Ab.  Homologous competition
with unlabeled mCry3A resulted in a significantly greater reduction in
labeled mCry3A binding (to ~ 40-60% at the two highest concentrations).
The authors suggested that the low level of competition observed could
be explained if there are multiple binding sites that recognize mCry3A
with one that is shared with eCry3.1Ab.  However, the shared binding
site would have low affinity for eCry3A.1Ab since competition was
observed at only at the peak concentrations tested.  No competition was
observed in the second assay:  eCry3.1Ab binding was not reduced by even
the highest concentrations of unlabeled mCry3A.  These results suggest
that the specific binding site(s) for eCry3.1Ab are not recognized by
mCry3A.  Competition experiments with non-activated protoxins showed
similar patterns as were observed in the first competition, indicating
that both chymotrypsin-activated and non-activated mCry3A and eCry3.1Ab
toxins recognize the same binding sites.

Considering the results of both studies, Syngenta concluded that
eCry3.1Ab and mCry3A maintain independent binding sites in WCRW and that
separate genetic mutations would be needed to develop resistance to both
toxins.  As such, the toxins are viable in a pyramided product for
resistance management of CRW.

BPPD Review - Corn Rootworm Cross Resistance Potential

Syngenta’s cross resistance analysis was based on the assumption that
Bt resistance will develop through an alteration of the toxin binding
receptors in the target, a mechanism that has been documented for a
number of pests (Ferre et al. 1991; Tabashnik 1994; Lee et al. 1995;
Ferre & Van Rie 2002).  Syngenta showed through an analysis of WCRW
midgut binding activity and competitive binding assays that eCry3.1Ab
and mCry3A largely recognize unique binding sites.  As such, cross
resistance between the two toxins is not expected according to the
traditional Bt resistance model of binding site modification.  This is
perhaps not surprising, considering that eCry3.1Ab is an engineered
toxin with different structural elements (transferred from Cry1Ab) than
mCry3A (Walters et al. 2010).

Syngenta did not consider alternate resistance mechanisms or cross
resistance scenarios in addition to toxin - receptor binding.  It has
been suggested that resistance in CRW to Bt may evolve differently than
lepidopteran pests on which the toxin-receptor mechanism of resistance
is based.  Resistance to Bt in CRW may involve multiple loci exerting
small changes in susceptibility as opposed to a single locus creating
high level resistance (as is assumed for lepidoptera) (SAP 2011).
Several selection experiments with CRW have suggested that resistance
may be incomplete (Meihls et al. 2008, 2011) or could occur at low
levels (Lefko et al. 2008).  The SAP also noted the potential for
epistatic processes in which a single genetic change (e.g., protease
expression or regulation of cadherin levels) could confer broader
resistance to Bt toxins.  BPPD notes, however, that specific cases of
epistasis (or broader mechanisms of cross resistance) have not yet been
documented in CRW or other Bt corn pests.  In addition, one study of a
Cry3Bb1-resistant colony showed no evidence of cross resistance to
Cry34/35Ab1 (Gassmann et al. 2011).

Syngenta’s assessment of cross resistance was limited to the novel
eCry3.1Ab and existing mCry3A proteins, both of which are proprietary to
the company.  No analysis of the other registered CRW toxins, Cry3Bb1
and Cry34/35Ab1, was included in the submission.  Customarily, the
registrant of a new PIP active ingredient will analyze cross resistance
for all existing toxins in the landscape and not just the toxin(s) in
the proposed product or pyramid (e.g., refer to the cross resistance
discussion for mCry3A in BPPD 2006).  BPPD recognizes that because
eCry3.1Ab is an engineered toxin, it is likely to have significant
structural differences with Cry3Bb1 and Cry34/35Ab1 and perhaps
different midgut binding patterns in CRW.  But no information was
provided in the submission to verify this assumption; therefore, BPPD
recommends that Syngenta provide additional data or analysis to address
the cross resistance potential of eCry3.1Ab with Cry3Bb1 and
Cry35/35Ab1.

In addition to midgut binding analysis (as submitted by Syngenta), tests
with resistant colonies can be useful to assess cross resistance,
provided such colonies are available.  Resistant colonies have been
selected with Cry3Bb1 (Meihls et al. 2008), Cry34/35Ab1 (Lefko et al.
2008), and mCry3A (Meihls et al. 2011), though it is unknown whether
such colonies could be accessed for future testing.  Should they be
available, BPPD recommends testing with the resistant colonies to
improve the cross resistance analysis for mCry3A.  BPPD is unaware of
any selection experiments that have been conducted for the relatively
new eCry3A.1Ab protein, though it is recommended that selection
experiments be initiated with this protein as well.

3. Simulation Modeling

Lepidoptera

Since the lepidopteran-active toxins (Cry1Ab, Cry1F, and Vip3Aa20) in
Syngenta’s proposed 5307 products have been previously registered, the
applicant cited existing modeling for CEW, ECB, and SWCB.  For CEW,
Syngenta cited simulations conducted for the initial registration of
Vip3Aa20 (MIR162).  These simulations, described in BPPD (2009a), were
used to support deployment of a 20% refuge in cotton-growing regions,
where the risk of resistance in CEW may be high. Syngenta did not
conduct additional simulations for CEW because the company is proposing
to maintain the existing 20% refuge standard for the new
Cry1Ab/Cry1F/Vip3Aa20 pyramid.

Modeling for ECB and SWCB was previously developed for the registration
of Syngenta’s Bt11 x MIR162 x 1507 pyramid (EPA Reg. No. 67979-15).
The model (detailed in MRID# 480506-06) was deterministic for two loci
(Cry1Ab and Cry1F) and was similar in design to the single locus
simulation created by Caprio (1998).  Vip3A was not included in the
simulations since it is not active against ECB.  Results from the
modeling showed that resistance did not develop (within 100 years) to
the Cry1Ab/Cry1F pyramid with either a 5% or 20% refuge.  Syngenta
asserted that the model included a high degree of conservatism including
assumptions that resistance carries no fitness costs, resistance is
complete, no natural refuge in the system, and that single trait
versions of Cry1Ab and Cry1F are present.  The company concluded that
the simulations support the use of Cry1Ab, Cry1F, and Vip3Aa20 in a
pyramid with a 5% refuge.

BPPD Review - Lepidopteran Modeling

Syngenta’s ECB/SWCB models were assessed for the registration of Bt11
x MIR162 x 1507 (see full review in BPPD 2011).  BPPD generally agreed
with Syngenta that, under the modeling conditions investigated, Bt11 x
TC1507 x MIR162 was highly durable relative to the corresponding single
trait PIPs (i.e., resistance did not evolve within the time frame of the
model).  Furthermore, BPPD agreed that some of the parameter choices
were conservative for the model, including assumptions of complete
resistance, no fitness costs for resistance, and no natural refuge.
Also, the exclusion of MIR162 (Vip3Aa20) from the modeling adds
conservatism for SWCB, which has susceptibility to the toxin.

Despite these conclusions, BPPD’s analysis was limited by the cursory
description provided for the model (Appendix F, MRID# 480506-06), which
lacked a number of critical details in the methodology description.
BPPD had additional questions regarding some of the parameter
assumptions and was unable to find descriptions of others.  The scope of
the modeling appeared to be limited, as no alternate scenarios (i.e.,
worst cases hypotheses) or sensitivity analysis were conducted (or
reported in the submission).  For these reasons, it was difficult for
BPPD to assess the relative durability of refuge options for Bt11 x
TC1507 x MIR162 based solely on the limited modeling submitted by
Syngenta.  BPPD recommended that Syngenta provide an improved modeling
analysis to address the concerns so that the expected durability of Bt11
x TC1507 x MIR162 could be confirmed (BPPD 2011).  A revised modeling
report was required as a term of registration, which was subsequently
submitted to EPA (MRID# 485509-02).  As of the date of this memorandum,
this submission has not yet been formally reviewed by BPPD.

Syngenta’s CEW model was submitted and reviewed for the registration
of MIR162 (Vip3Aa20).  The model was designed by Dr. Michael Caprio
(Mississippi State University) and included several scenarios in which
Vip3Aa20 x Cry1Ab corn (with either 20% or 50% refuge) was planted in a
landscape containing Bt cotton (Vip3Aa19 x Cry1Ab).  Simulations with
20% or 50% corn refuge did not evolve resistance during the 25-year
model timeframe.  BPPD’s assessment of the model concluded that it,
coupled with other evidence, provided sufficient support to warrant the
use of a 20% refuge for the MIR162 pyramid in southern cotton-growing
regions (BPPD 2009a).   Syngenta is proposing to employ the same 20%
refuge strategy in cotton regions with the 5307 pyramids (containing
Cry1Ab, Cry1F, and/or Vip3Aa20) and did not conduct any new CEW modeling
for these new products.  BPPD agrees that the modeling originally
submitted for MIR162 is adequate to support Syngenta’s proposed
lepidopteran refuge strategy for the 5307 applications.

Corn Rootworm

Syngenta partnered with Dr. Michael Caprio (Mississippi State
University) to conduct simulations for CRW.  Dr. Caprio (in
collaboration with U.S EPA’s Office of Research development) had
previously designed a stochastic model that was used for an analysis of
a “seed blend” IRM strategy (Caprio and Glaser 2010).  For
Syngenta’s simulations, Caprio modeled the time to resistance for a
MIR604 (mCry3A) x 5307 (eCry3.1Ab) pyramid with a 5% block or seed blend
refuge.  Single trait events (MIR 604 and 5307) were also modeled as a
mosaic with 20% block refuges.

Caprio’s model employed a landscape structure with four 36 ha fields,
with each of the fields including a 20 x 20 grid of patches (each 30m x
30m).  The grid allowed for structured refuges to be placed within the
Bt fields so that adult dispersal could be modeled.  Fields were
populated at the start of each year in the model run with the PIP crop
and refuges.  For the single toxin mosaic simulations, each of two
toxins was randomly assigned to two of the fields.  Refuges were located
at one side of the Bt field (randomly selected for each field).  Bt
fields had a 30% probability of not having a refuge each year of the
simulation (corresponding with a non-compliance rate of 30%).  No
partial non-compliance was incorporated and Caprio noted that there was
a small probability (0.81%) in any given year of there being no refuge
in any of the four fields (an unlikely scenario in the real world).  If
resistance to one of the two toxins developed, in subsequent years all
fields were planted exclusively with the second toxin.  Dual toxin
simulations for the pyramid (with 5% structured refuge or seed blend)
were modeled using the same landscape features, though no single toxin
PIPs were included (all four fields were planted with the same dual gene
PIP).

Adult dispersal was modeled in a routine that included both population
and individual parameters.  For each dispersal routine, a certain
proportion of the total population was determined to move at least one
patch (randomly drawn from binomial distribution).  Movement on an
individual basis included vector (direction) and distance, both of which
were randomly generated (dispersal distance was based on data developed
by Nowatski et al. 2003).  A second routine covered “aging” in which
individuals aged sequentially by one day in the simulations.  This
feature allowed for the modeling of a developmental delay for insects
exposed to PIP toxins, which was accomplished by holding back 12% of
such individuals from progressing through life stages each day
(resulting in an average overall delay of 7 days for the population).

Larval movement included a base movement rate (the same for both Bt and
non-Bt plants) and asymmetrical movement away from Bt plants.  Base
larval movement was sampled for each simulation from a distribution that
ranged from 1 to 10% with 5% being the most likely value.  Asymmetrical
movement incorporated the genotype of the insect and mortality on Bt,
such that susceptible genotypes (more likely to be killed on Bt) had
greater movement rates on Bt plants.  The frequency (ratio) of
asymmetrical movement was determined for each simulation from a
probability distribution (0.1 to 1.0, 0.5 most likely).

Many of the other parameters were populated using uncertainty
distributions, as was done for larval movement.  Values for toxin
mortality (used to derive genotype fitness) were provided by Syngenta,
while other biological parameters were carried over from previous
modeling (Caprio and Glaser 2010).  Information on the parameter
assumptions and model features is detailed in Table 2 below.

Table 8.  Parameter Values and Assumptions for Syngenta’s (Caprio)
Stochastic Model to Evaluate the Durability of 5307 x MIR604 (taken from
data provided in Table 1 and other information in Appendix D of MRID#
484429-10)

Parameter	Value or Assumption	Notes

	Min	Most Likely	Max

	Initial R-allele freq.	0.001	0.005	0.02	Same values for both toxins but
drawn independently for each locus

Mortality due to toxin 1	0.95	0.9808	0.9997	Toxin not specified; assumed
to be eCry3.1Ab

Mortality due to toxin 2	0.833	0.95	0.9906	Toxin not specified; assumed
to be mCry3A

Overall mortality	0.99165	0.99904	0.9997

	Base larval movement	0.01	0.05	0.1	Total movement for the lifetime of
the larvae

Asymmetrical movement ratio	0.1	0.5	1.0

	Insect aging	One day per simulated day; 12% on Bt plants have one day
developmental  delay

	Cross resistance	Not reported 	BPPD assumes to be zero

Non compliance (structured refuges)	30% (no partial non-compliance)

	Model duration	4440 days (31.7 years)

	Definition of resistance	R allele > 0.5	Model continued to run if
resistance developed to one toxin - all subsequent fields were assumed
to be planted to 2nd toxin

Several hundred simulations were conducted for each toxin deployment
scenario with each simulation run for the equivalent of 4440 days (31.7
years).  Resistance did not evolve to either toxin in the pyramid seed
blend scenario (510 total simulations).  Similarly, resistance did not
evolve to the pyramid in the structured refuge scenario (535
simulations) though in three simulations resistance evolved to the
second toxin.  Resistance to both toxins evolved only in simulations
with the single toxin mosaic scenario.  The average time to resistance
was 26.7 years for toxin 1, 28.7 years for toxin 2, and 29.9 years for
both toxins; however, resistance did not evolve to both toxins in 62.7%
of the simulations (311 total).  Caprio also presented a risk profile
for the single toxin modeling with levels of risk ranging from 0% to
50%.  For a 10% level of risk, the time to resistance would be ≥ 25.3
years for both toxins.  This means that 90% of the simulations evolved
resistance after 25.3 years, while in the other 10% of simulations
resistance developed more quickly (the researcher essentially assumes a
10% chance of being “wrong” about the estimated durability).  As the
level of risk drops, the resistance values also drop (more outlying
simulations are included); at the 0% level of risk the time to
resistance was 17.4 years for both toxins (i.e., none of the simulations
had resistance develop earlier than 17.4 years).  Results from the
modeling are summarized in Table 3.

Caprio (and Syngenta) concluded that the pyramid of mCry3A and eCry3.1Ab
with 5% refuge (seed blend or block refuge) is more durable (“less
risky”) than a mosaic of single toxin products with a 20% refuge.
Given that resistance did evolve to toxin 2 in three simulations of the
pyramid/structured refuge scenario, Caprio also concluded that the seed
blend (in which resistance never evolved) was somewhat more durable than
the structured refuge.  This was likely due to the non-compliance rate
(30%) that was assumed for structured refuges.

Table 9.  Results from Syngenta’s modeling (taken from data provided
in Table 2 and other information in Appendix D of MRID# 484429-10)

Scenario	Simulations	Mean Time to Resistance (years)

Toxin 1	Toxin 2	Both Toxins

Single toxin mosaic:  50% toxin 1, 50% toxin 2	311	28.67	26.73	29.92

Pyramid with 5% structured refuge	535	No resistance	*	No resistance

Pyramid with 5% seed blend refuge	510	No resistance evolved to either
toxin

* Resistance evolved in three simulations to toxin 2.  The time to
resistance was not reported for these simulations.

BPPD Review - Corn Rootworm Modeling

Syngenta’s modeling largely produced expected results, in that
pyramided deployment of toxins were relatively more durable than single
toxin PIP despite having lower percent refuges.  This output is
consistent with other models that have analyzed pyramids as a resistance
management approach for Bt crops (Roush 1998, Caprio 1998, Zhao et al.
2003, Gould et al. 2006, Onstad and Meinke 2010).

Caprio’s stochastic model differs from other models by using a
probabilistic approach for assigning parameter in which values are drawn
from a distribution (with a most likely value) for each simulation.
This allows the model to incorporate variability and uncertainty
associated with biological parameters (e.g., resistance allele
frequencies, dose mortality, and movement).  Many simulations are run
for each scenario from which a “risk profile” of estimated
durability can be drawn for different levels of uncertainty (selected by
the risk assessor).  As the criterion for certainty in the durability
estimate is raised, the time to resistance decreases.  BPPD has
previously reviewed and utilized Caprio’s model in evaluations of
other products (see BPPD 2010a) and has found it to be a rigorous risk
assessment tool.

While BPPD agrees with Syngenta regarding the overall conclusions, the
modeling was limited in scope and the handling of some parameters.
Three general scenarios were run that included landscapes of either
single toxin or pyramided toxin PIPs.  The single toxin simulations
included a mosaic of both traits (eCry3.1Ab and mCry3a) but both pyramid
scenarios (seed blend and block refuge) were run with no single trait
PIPs in the landscape.  This was a realistic choice in the case of
eCry3.1Ab since no products (single trait or pyramid) have been
previously approved with the toxin.  In the case of mCry3A, however,
single toxin PIPs expressing the trait have been registered since 2006
and would likely be grown along with 5307 pyramided products should they
be registered.  Single trait products can act as a “stepping stone”
to resistance for pyramids, particularly if non-compliance with single
trait refuge requirements is high.  It is uncertain how the durability
of the toxins would have been affected in a simulation including both
single traits and pyramids.  BPPD expects that resistance would have
evolved to the single trait mCry3A (as occurred in the single toxin
mosaic simulations), though it is unknown whether resistance would have
evolved to the pyramid within the modeled time frame.  One possible way
to address this issue would be to increase the resistance allele
frequency for mCry3A to account for previous selection to the single
toxin.  It is unclear, however, whether such an approach was considered
in Syngenta’s simulations.

The model duration (31.7 years) may have also limited the analysis
between the 5% pyramid block and 5% pyramid seed blend refuges.  For the
vast majority of simulations (including the single toxin mosaics),
resistance did not evolve during the model time period.  In only three
simulations (out of over 1,000 total) was resistance observed in the
pyramid toxin scenarios.  Because of this, it is difficult to make any
relative comparisons between the pyramid refuge options (i.e., blocks
vs. blends); in essence, the only conclusion that can be drawn is that
pyramided deployments are superior to a mosaic of single toxin PIPs.
The study author (Caprio) suggested that blended refuge may be superior
to block refuge for pyramids, though this was based on the three cases
of resistance to one of the toxins that arose during the block refuge
simulations.  BPPD suspects, however, that this result is not
statistically relevant given that only 0.56% of the simulations (3 of
535) showed resistance.  Expanding the time horizon of the model could
have helped to tease out differences between block refuge and seed
blends and perhaps further flesh out the role non-compliance plays on
block refuge durability.

Though not explicitly stated in the report, cross resistance was
presumably assumed to be negligible and not included in the modeling.
Data submitted by Syngenta (and reviewed earlier in this memorandum)
support the assumption of little cross resistance potential through the
traditional toxin-midgut binding site model.  BPPD cannot, however,
entirely eliminate the possibility of cross resistance through means
other than toxin-receptor interactions. The 2011 SAP identified
epistasis, in which a single genetic change (e.g., protease expression
or regulation of cadherin levels) exerts a broad effect, as a potential
mechanism that could result in resistance to multiple toxins.  Because
of this, Syngenta could have assumed a low level of cross resistance as
a worst case scenario in the modeling (it is unclear if the model
assumed epistasis as part of genotype survival).  This would likely have
reduced the durability of both traits in all scenarios, though the
relative impact of cross resistance/epistasis on durability between seed
blend and block refuges is unknown.

Other potential parameters and considerations, such as mating
(random/non-random), pre-ovipositional dispersal, oviposition, and
density-dependent effects, were not discussed in the report.  These
parameters can be important for relative evaluations of block and seed
blend refuges, though Syngenta’s application proposes only block
refuge for the 5307 pyramids.  BPPD assumes that these parameters were
addressed in the same manner as Caprio’s previous modeling (Caprio and
Glaser 2010), though this cannot be confirmed.

Despite the limitations in the model and lack of clarity in the report,
BPPD concludes that Syngenta’s modeling demonstrates high durability
of the mCry3A x eCry3.1Ab pyramid with a 5% block refuge.  A number of
other published and unpublished models have also shown that multi-toxin
PIPs with high efficacy and low cross resistance have superior
durability to single toxin PIPs.  On the other hand, additional
information may be need to fully assess the durability of a 5% seed
blend, including more complete descriptions of how parameters such as
genotypic fitness/movement, assumptions regarding heterozygote survival,
and oviposition were addressed in the model.   BPPD notes, however, that
Syngenta has not proposed a seed blend strategy for 5307 corn at the
time of their application.

4. Proposed IRM Plan

For Bt11 x MIR604 x TC1507 x 5307 (EPA Reg. No. 67979-EU) and Bt11 x
MIR162 x MIR604 x TC1507 x 5307 (67979-EG), Syngenta has proposed
similar IRM strategies.  The plans are based on reduced (5%) refuges for
pyramided Bt corn and established paradigms for stewardship (i.e.,
resistance monitoring, remedial action, and compliance).

Refuge

Separate refuge requirements were proposed for the Corn Belt (i.e.,
non-cotton growing regions) and cotton-growing regions.  Larger refuges
have been required in cotton regions because of CEW, which can have up
to two generations on corn before moving onto cotton during the same
growing season.  In the Corn Belt, resistance management is primarily
geared towards stalk borers (ECB and SWCB) and CRW.

	Corn Belt Refuge (non-cotton growing regions)

For both Bt11 x MIR604 x TC1507 x 5307 and Bt11 x MIR162 x MIR604 x
TC1507 x 5307, Syngenta proposes a 5% structured refuge.  The refuge can
be planted as either a “common refuge” for both ECB/SWCB and CRW or
as two separate (5%) refuges for each pest complex.

The common refuge must be planted adjacent to or within the Bt field.
In-field refuges must be at least four strips wide and if crop rotation
is used, it must be employed for both the refuge and Bt fields.
Insecticide treatments can be made in the refuge for corn rootworm
larvae with soil insecticides.  Non-rootworm late season pests can be
treated with foliar (non-Bt) insecticides in the refuge provided adult
CRW are not present and economic thresholds are reached.  If adult CRW
are present, both the refuge and Bt field must be treated in the same
manner.  Economic thresholds are to be determined by local or regional
professionals.  Syngenta also proposed that “pests on the [Bt] maize
acres can be treated as needed without having to treat the common
refuge.”

With the separate refuge option, discrete refuges (each 5%) must be
planted for lepidoptera and rootworm.  The lepidopteran refuge must be
planted within ½ mile of the Bt field and in-field strips must be at
least four rows wide.  Insecticides can be used in the refuge for 1)
corn rootworm larvae (soil insecticides), or 2) corn borers (non-Bt
foliar applications) provided economic thresholds are reached.  The corn
rootworm refuge has essentially the same restrictions as the common
refuge in terms of deployment (adjacent to or within the Bt field),
in-field strip width (at least four rows), and insecticide use for
rootworm and other pests.

Cotton-Growing Regions

Syngenta proposed different refuge requirements for each product in
cotton regions, though both strategies maintain the common and separate
refuge options.  For Bt11 x MIR162 x MIR604 x TC1507 x 5307, Syngenta
proposed a 20% lepidopteran refuge while for Bt11 x MIR604 x TC1507 x
5307, a 50% lepidopteran refuge would be required.

The common refuge (20% for Bt11 x MIR162 x MIR604 x TC1507 x 5307 and
50% for Bt11 x MIR604 x TC1507 x 5307) must be planted with the same
deployment and insecticide use restrictions as the common refuge for the
Corn Belt.  For the separate refuges, both products must have a 20% corn
rootworm refuge.  Each product must also have a lepidopteran refuge:
20% for Bt11 x MIR162 x MIR604 x TC1507 x 5307 and 50% for Bt11 x MIR604
x TC1507 x 5307.  The separate refuges must also be planted with the
same restrictions as the separate refuges for the Corn Belt region.

Syngenta concluded that their proposal for the 5% refuge is supported by
a number of factors, including submitted data reviewed in this
memorandum.  Specifically, Syngenta cited the following justification:

EPA has set a precedent of lower refuge for pyramided products for both
lepidoptera and corn rootworm (e.g., MON 89034, SmartStax);

Two of the toxins in the pyramids, Bt11 (Cry1Ab) and TC1507 (Cry1F), are
expressed at high dose levels for ECB and SWCB;

For CEW, the Bt11 x TC1507 x MIR162 pyramid offers three modes of action
and a functional high dose;

For CRW, MIR604 (mCry3A) x 5307 (eCry3.1Ab) offers two effective modes
of action;

Cross resistance potential for both lepidoptera and corn rootworm among
the toxin groups in the pyramids is low;

Simulation modeling demonstrates the durability of the Bt11 x MIR162 x
MIR604 x TC1507 x 5307 pyramid relative to single toxin deployments.

BPPD Review - Refuge

As discussed in the modeling section, BPPD concludes that there is
adequate scientific support to demonstrate that the 5307 pyramids will
have high durability with a 5% structured refuge (relative to single
trait PIPs with 20% refuge).  Syngenta has not proposed a seed blend for
any of the 5307 products; BPPD notes that additional information and
modeling may be necessary to support such a refuge strategy.

Syngenta’s proposed language for the refuge requirement includes the
following statement:  “Pests on the Bt11 x MIR162 x MIR604 x TC1507 x
5307 [or Bt11 x MIR604 x TC1507 x 5307] maize acres can be treated as
needed without having to treat the common refuge.”   This language has
not been specifically included in the registration terms and conditions
of similar Bt corn products, but the registrations do not prohibit the
practice.  Treatment of Bt acres should not increase the risk of
resistance since most pest insects present would presumably be resistant
to the toxin(s) (or otherwise have avoided lethal exposure to the
toxin).  Therefore, differential insecticide usage in which the PIP
field is treated but the refuge is not would not be expected to create a
selection gradient disfavoring susceptible insects (as would be the case
if only the refuge was treated).

For cotton-growing regions, Syngenta proposed a 50% lepidopteran refuge
for Bt11 x MIR604 x TC1507 x 5307, while requesting a 20% refuge for
Bt11 x MIR162 x MIR604 x TC1507 x 5307.  BPPD assumes the difference is
due to the fact that the latter product has three active toxins against
lepidoptera (CEW in particular) while the former has two.  CEW has been
the primary target pest of concern in cotton areas (given its annual
life cycle on corn and cotton) and larger refuges (50%) were required
for single toxin corn PIPs in the region.  However, EPA has approved a
number of two toxin pyramids with a 20% refuge in cotton regions,
including two Syngenta products (EPA Reg. Nos. 67979-12 and 67979-15)
and another registrant’s product containing Cry1Ab and Cry1F
(29964-7).  Syngenta may be able to support a 20% refuge for the Bt11 x
MIR604 x TC1507 x 5307 pyramid with a relatively simple model (as was
done to support the other registered pyramids).

Syngenta’s refuge proposal did not specifically define the
cotton-growing region that would require 20% (or 50%) refuges.  Based on
the paradigm established for previously-registered Bt corn PIPs, this
region should be defined as follows:  Alabama, Arkansas, Georgia,
Florida, Louisiana, Mississippi, North Carolina, South Carolina,
Oklahoma (only the counties of Beckham, Caddo, Comanche, Custer, Greer,
Harmon, Jackson, Kay, Kiowa, Tillman, Washita), Tennessee (only the
counties of Carroll, Chester, Crockett, Dyer, Fayette, Franklin, Gibson,
Hardeman, Hardin, Haywood, Lake, Lauderdale, Lincoln, Madison, Obion,
Rutherford, Shelby, and Tipton), Texas (except the counties of Carson,
Dallam, Hansford, Hartley, Hutchinson, Lipscomb, Moore, Ochiltree,