unexpected non-target effects other than subtle shifts in the arthropod
community caused by the effective control of the target pests (Romeis et
al., 2006).  Slight reductions in some invertebrate predator populations
are an inevitable result of all pest management practices, which result
in reductions in the abundance of the pests as prey.

Overall, the Agency is in agreement with the conclusions of these
studies and collectively, these results provide extensive data to
support that Bt crops have not caused long term environmental effects on
a population level to organisms not targeted by Bt proteins. Based on
these considerations, regulatory testing of the specialist predators and
parasitoids of target pests may eventually be considered unnecessary.

B.  Environmental Exposure Assessment

The EPA risk assessment is centered only on adverse effects at the field
exposure rates (1X EEC), and not on adverse effects at greater
concentrations. Although it is recommended that non-target testing be
conducted at a test dose 10 X the EEC whenever possible, the test dose
margin can be less than 10X where uncertainty in the system is low or
where high concentrations of test material are not possible to achieve
due to test organism feeding habits.  High dose testing also may not be
necessary where many species are tested or tests are very sensitive,
although the concentration used must exceed 1X EEC.  It is important to
note that Tier I screen testing is not “safety factor testing”.  In
a traditional “10X safety factor” test any adverse effect noted is a
“level of concern”, whereas in the EPA environmental risk assessment
scenario any adverse effect is viewed as a concern only at 1X the field
exposure.

For the purposes of the non-target organism (NTO) studies submitted in
support of Event COT102 and Event COT67B, the test material dose levels
were based on the estimated concentration of Vip3Aa and full-length
Cry1Ab protein expressed in the tissue(s) that NTO would most likely be
exposed to in the environment (see Matten, 2007; Edelstein, 2008 for
protein expression levels).   The Agency has determined that the NTOs
most likely to be exposed to the Vip3Aa and FLCry1Ab protein in
transgenic cotton fields were beneficial insects feeding on cotton
pollen. Consequently, test material dose levels were based on the
maximum level of measured protein expression in pollen (3.47 ug/g dwt
for Vip3Aa and 12.1 ug/g dwt for Cry1Ab).  The principal route of Vip3Aa
and full-length Cry1Ab protein exposure for soil-dwelling organisms
(such as collembola, earthworms, and/or rove beetles) is assumed to be
from decomposing plant tissue and plant exudates in soil.  Consequently,
the dose levels of the test material were based on the maximum level of
estimated protein expression in the soil environment.

C.   Non-Target Wildlife Hazard Assessments for Event COT102 and Event
COT67B

Two separate SAP reports (October 2000 and August 2002) recommended that
non-target testing of Bt Cry proteins should focus on invertebrate
species exposed to the crop being registered.  Following SAP
recommendations, the EPA determined that non-target organisms with the
greatest exposure potential to Cry protein in transgenic corn fields are
beneficial insects, which feed on corn pollen and nectar, and soil
invertebrates, particularly Lepidoptera species. The Agency recommended
using this same approach for testing the effects of Vip protein in Event
COT102 and Cry protein in Event COT67B on beneficial insects in
transgenic cotton fields. Therefore, toxicity testing using the maximum
hazard dose on representative beneficial organisms from several taxa was
performed in support of both Section 3 FIFRA cotton registrations. The
toxicity of the Vip3Aa and Cry1Ab have been evaluated on several species
of invertebrates including the lady beetle, minute pirate bug,
collembola, daphnia, honey bee, rove beetle, and/or earthworm.
Reproductive and developmental observations were also examined in the
lady beetle, rove beetle, minute pirate bug, and honeybee studies.

As previously noted, Vip3Aa protein in Event COT102 and Cry1Ab protein
in Event COT67B are very host specific, conferring toxic effects on
cotton bollworm, tobacco budworm, fall armyworm, beet armyworm, and
cabbage looper.   SEQ CHAPTER \h \r 1 Despite the October 2000 and
August 2002 SAP’s recommendations against testing of non-target
species not related to susceptible target pests, EPA has completed a
risk assessment on a range of non-target wildlife to comply with the
Agency’s published non-target data requirements.  In the absence of
PIP-specific risk assessment guidance, EPA requires applicants for PIP
registrations to meet the 40 CFR Part 158 data requirements for
microbial toxins. These requirements include birds, mammals, plants, and
aquatic species.  In addition, earthworm, springtail, and/or rove beetle
studies were voluntarily submitted to the Agency to ascertain the
potential effects of Vip3Aa and FLCry1Ab proteins on beneficial
decomposer species.

The October 2000 SAP recommended that while actual plant material is the
preferred test material, bacterial-derived protein is also a valid test
substance, particularly in scenarios where test animals do not normally
consume cotton plant tissue and where large amounts of Cry protein (Cry
protein concentrations that exceed levels present in plant tissue) are
needed for maximum hazard dose testing. For Event COT102, an insect
feeding study, which compared the relative potency of plant-derived
Vip3Aa protein in both Event COT102 cotton and Event Pacha corn to the
microbial-derived proteins, indicated that plant-derived protein was
similar in toxicity to the microbial-derived protein (MRID No. 458358-12
and Edelstein, 2008). Similarly, for Event COT67B, an insect feeding
study, which compared the relative potency of plant-derived FLCry1Ab
protein in COT67B cotton to the microbial-derived protein, indicated
that plant-derived protein was similar in toxicity to the
microbial-derived protein (MRID No. 470176-08 and Edelstein, 2008).
Therefore, these data indicate that the microbial-derived proteins for
each event are substantially equivalent to the plant-derived proteins
expressed in cotton plants based on the similar insecticidal activity
for studying any potential toxicity on NTOs for the purposes of the
environmental risk assessment.

The Agency has also determined that toxicity studies using corn-derived
plant material rather than cotton-derived plant material is acceptable
because cotton contains gossypol and other possible plant toxicants that
may adversely affect non-target organisms. Furthermore, the non-target
species in the cotton agroecosystem are comparable to those in corn;
Specifically for Vip3Aa protein toxicity tests, Event COT102 cotton
expresses the same vip3A(a) gene as is expressed in Event Pacha corn,
and the expression level of pollen of Event Pacha corn is much higher
than that of Event COT102 cotton.

In support of the COT102 registration, test substances used in the
submitted studies included bacterial-produced purified Vip3Aa19 and
Vip3Aa1 protein, in addition to Vip3Aa19 as expressed in COT102 cotton
pollen and Event Pacha maize grain, pollen, and leaves. Likewise, in
support of the COT67B registration, test substances used in the
submitted studies included bacterial-produced purified full-length
Cry1Ab and truncated Cry1Ab protein, in addition to Cry1Ab protein as
expressed in Event Bt11 maize grain, pollen, and leaves. The individual
results for each study on ecological effects for Vip3Aa and Cry1Ab are
summarized in Tables 9 and 10, respectively.  The results are also
presented in a more descriptive format in subsequent sections of the
risk assessment document. Full reviews of each study for each event can
be found in the individual Data Evaluation Reports (DERs/MRID#s).

Table 9.  Summary of environmental effects studies and waiver
justifications for COT102 submitted to comply with data requirements
published in 40 CFR § 158.2150(d).

Data Requirement 	OPPTS

Guideline	Test Substance	Results Summary and Classification	MRID No.

Avian dietary testing,

broiler chicken, Gallus domesticus

	885.4050	Vip3Aa19 maize grain

 (Event Pacha)  	A 49-day dietary study showed no adverse affects to
broiler chickens when fed a 50% diet composed of Event Pacha maize grain
(containing VIP3A).  Therefore, the NOEC was 0.588 µg VIP3A/g corn feed
and the LC50 was > 0.588 µg VIP3A/g feed corn grain.

Classification:  Acceptable	470176-23

Avian injection testing	885.4100

	N/A	Acceptable waiver rationale	N/A

Avian oral testing, bobwhite quail,

Colinus virginianus	850.2100	Microbial Vip3Aa1 (VIP3A-0198)	A 14-day
study showed no adverse effects to bobwhite quail from VIP3A-0198, after
a single oral dose via gavage.  The NOEL was 400 mg VIP3A/kg and the
LD50 was > 400 mg VIP3A/kg bird body weight.

Classification:  Acceptable	457665-08

Wild mammal testing	885.4150

	N/A	Acceptable bridging rationale to acute oral  toxicity test on mice
(MRID No. 457665-05).	N/A

Freshwater fish testing,

channel catfish, Ictalurus punctatus	885.4200

	Vip3Aa19 maize grain (FFPACHA-0100)	A 30-day study showed no adverse
effects on juvenile catfish after exposure to Vip3Aa protein from Event
Pacha corn grain.  Therefore, the NOEC was 7.10 µg Vip3Aa19/g fish feed
and the LC50 was > 7.10 µg Vip3Aa19/g

Classification:  Acceptable	470176-24

Freshwater aquatic invertebrate testing,

water flea, Daphnia  magna 	885.4240

 	Vip3Aa19 maize pollen

(PHOPACHA-0199)	In a 48-hour static renewal limit bioassay, VIP3A maize
pollen (containing 83.8 µg VIP3A protein/g) had no adverse effects on
the survival of Daphnia magna, when suspended in 120 mg pollen/L.  The
LC50 was > 83.8 µg VIP3A protein/g.

Classification:  Unacceptable. The 885 Series Guidelines call for a 21
day study. The submitted 48 hour acute study is inadequate.	457921-01

Estuarine and marine animal testing 	885.4280

	N/A	Acceptable waiver rationale	N/A

Non-target plant testing	885.4300

	N/A	Acceptable waiver rationale	N/A

Non-target insect testing, minute pirate/insidious flower  bug , Orius
insidiosus	885.4340

	Microbial Vip3Aa19

(VIP3A-0104)	Orius insidiosus nymphs fed a meat-based diet containing
microbial-derived 7.25 mg Vip3Aa19 protein/ g diet showed no adverse
effects after 21 days.  The NOEC was 7.25 mg Vip3Aa19 protein/ g and the
LC50  was > 7.25 mg Vip3Aa19 protein/ g.

Classification:  Acceptable	468648-14

Non-target insect testing, pink-spotted lady beetle, Coleomegilla
maculata 	885.4340

	Vip3Aa19 maize pollen (PHOPACHA-0100)	Coleomegilla maculata adults were
fed a diet containing 5.0% VIP3A maize pollen (containing 144.8 µg
VIP3A protein/g pollen) for 21 days with no adverse effects observed.
The NOEC was 7.24 µg VIP3A protein/g pollen and the LC50  was > 7.24
µg/g VIP3A/g pollen.

Classification:  Acceptable	457665-09

Non-target insect testing, seven-spotted ladybird beetle, Coccinella
septempunctata 	885.4340

	Microbial Vip3Aa19 (VIP3A-0204)	C. septempunctata adults fed a 50%
sucrose diet containing 7250 µg Vip3Aa19 protein/g diet for showed no
adverse effects after 15 days.  The NOEC was 7250 µg Vip3Aa19 protein/g
diet and the LC50  was > 7250 µg/g Vip3Aa19 protein/g diet.

Classification:  Acceptable	468848-02

Non-target insect testing, green lacewing,  Chyrsoperla carnea	885.4340
Microbial Vip3Aa19 (VIP3A-0104)	Chyrsoperla carnea larvae fed a
meat-based diet containing 7250 µg Vip3Aa19 protein/g diet showed no
adverse effects.  The NOEC of 7250 µg Vip3Aa19 protein/g diet and the
LC50 was > 7250 µg Vip3Aa19 protein/g diet at day 14, when the control
mortality reached 20%.  There were no statistically significant
differences between the VIP3A-0104 group and the negative control group.

Classification:  Acceptable	468848-15

Non-target insect testing, collembolan, Folsomia candida	885.4340

	Vip3Aa19 maize leaves

(LLPACHA-0100)	Collembola were fed a diet containing 50% yeast and 50%
leaf tissue for 28 days.  No statistically significant effects on
survival or reproduction were found among the test and negative control
groups.  The NOEC was 43.2 µg Vip3Aa19 protein/g diet and the LC50 was
 > 43.2 µg Vip3Aa19 protein/g diet.

Classification:  Acceptable	458358-10

Honeybee testing, Honeybee larvae,

Apis mellifera	885.4380

	Vip3Aa19 maize pollen

(PHOPACHA-0199)	Three-to-five day old honeybee larvae were administered
a single dose of ca.2 mg of pollen moistened with a drop of 30% sucrose
solution (containing the equivalent of 168 µg of Vip3Aa) in their
individual brood cells.  After 19 days, there were no significant
differences between the treatment and control groups in survival to
capping, survival to emergence of adults, and the behavior and
morphology of the emerged adults.  The NOEL was 83.8 µg Vip3Aa19
protein/g diet and the LD50  was  > 83.8 µg Vip3Aa19 protein/g diet.

Classification:  Acceptable	458358-09

Earthworm toxicity,

Eisenia foetida	OECD Guideline 207, 850.6200	Vip3Aa19 maize leaves

(LPPACHA-0199)	Adult earthworms were exposed to artificial soil
containing 3.60 µg VIP3A protein/g soil for 14 days.  No mortality or
differences in body weights were observed in the test group.  The NOEC
was 3.60 µg VIP3A protein/g soil and the LC50  > 3.60 µg VIP3A
protein/g soil.

Classification:  Acceptable	457921-02

Soil fate and degradation	885.5200	Vip3Aa19 maize leaves

(LPPACHA-0199)	Results of this degradation study indicate that the DT50
of 16 mg/g concentration of the Vip3Aa19 test material protein do not
persist in various types of soil from 6 days to 12.6 days via measuring
the loss of bioactivity in black cutworm.

Classification:  Acceptable	470176-30

Table 10.  Summary of environmental effects studies and waiver
justifications for COT67B submitted to comply with data requirements
published in 40 CFR § 158.2150 (d).

Data Requirement 	OPPTS

Guideline	Test Substance	Results Summary and Classification	MRID No.

Avian dietary testing,

broiler chicken, Gallus domesticus 	885.4050	Bt11 maize grain	A 42-day
dietary study showed no deleterious effects on broiler chicken survival
or carcass yield when fed a 50% diet composed of Bt11 maize grain
(containing Cry1Ab).

Classification:   Acceptable	4565251-01

Avian injection testing	885.4100

	N/A	Acceptable waiver rationale	N/A

Avian oral testing, bobwhite quail,

Colinus virginianus	850.2100	Bt176 Maize leaf protein

(LP176-0194)	A 14-day study on bobwhite quail showed no adverse effects
after a single oral dose of Bt176 grain, containing Cry1Ab.  The NOEL
was 140 mg Cry1Ab/kg bodyweight and the LD50 was  > 140 mg Cry1Ab/kg
bodyweight.

Classification:   Acceptable	433236-09

Wild mammal testing	885.4150

	N/A	Acceptable bridging rationale to acute oral toxicity test on mice
(MRID No. 47017614)	N/A

Freshwater fish testing,

channel catfish, Ictalurus punctatus 	885.4200

	Microbial FLCry1Ab

(FLCRY1AB-0103) 	A 30-day study showed no adverse effects to juvenile
channel catfish.  The NOAEC was 7.10 µg  FLCry1Ab/g fish feed and  the
LC50  was > 7.10 µg  FLCry1Ab/g fish feed.

Classification:   Acceptable 	470176-25

Freshwater aquatic invertebrate, water flea neonate, Daphnia magna
885.4240

 	Bt176 maize pollen (PHO176-0194)	In a 48-hour static renewal limit
bioassay, Event 176 maize pollen containing 12.36 µg Cry1Ab protein/g
had no adverse effects on the survival of Daphnia  magna, when suspended
in 150 mg pollen/L.  The LC50  was >  12.36 µg Cry1Ab protein/L.

Classification:   Unacceptable. The 885 Series Guidelines call for a 21
day study. The submitted 48 hour acute study is inadequate	433236-10

Estuarine and marine animal  testing	885.4280

	N/A	Acceptable waiver rationale	N/A

Non-target plant testing 	885.4300

	N/A	Acceptable waiver rationale	N/A

Non-target insect testing, predatory bug, Orius laevigatus	885.4340

	Microbial FLCry1Ab

(FLCRY1AB-0103) and

Microbial Vip3Aa19

(VIP3A-0204)	Orius laevigatus had no adverse effects after fed
meat-based artificial diets, containing either 1.0039 mg  FLCry1Ab/g
diet or 1.0039 mg  FLCry1Ab + 0.1950 mg Vip3Aa19/g diet  for 14 days.
Only the results from the first study were valid.  The NOEC for O.
laevigatus was 1003.9 µg FLCry1Ab/g diet and 1003.9 µg FLCry1Ab +
195.0 µg Vip3Aa19/g diet for Event COT67 and Event COT102 x COT67B
cotton leaves, respectively.  Furthermore, the LC50 was greater than
1003.9 µg FLCry1Ab/g diet and 1003.9 µg FLCry1Ab + 195.0 µg
Vip3Aa19/g diet for COT67B and COT102 x COT67B cotton, respectively.

Classification:  Supplemental- see discussion below (Section III.2.e.ii)
	470176-28

Non-target insect testing, pink-spotted lady beetle, Coleomegilla
maculata 	885.4340

	Microbial FLCry1Ab

(FLCRY1AB-0103) and

Microbial Vip3Aa19

(VIP3A-0204)	Coleomegilla maculata larvae were fed prepared diets
containing bee pollen, Ephestia eggs, and either FLCRY1AB-0103
(containing 1000 µg FLCry1Ab protein/g diet) or FLCRY1AB-0103 and
VIP3A-0204 (containing 1000 µg FLCry1Ab and 250 µg Vip3Aa protein/g
diet) for 21 days.  No adverse effects were observed on larval, pupal,
or adult survival from either test material diet.  The NOAEC for
FLCry1Ab was 1000 µg FLCry1Ab/g diet and the LC50 was greater than 1000
µg FLCry1Ab/g diet. The NOAC for FLCry1Ab + Vip3Aa19 proteins tested in
combination was 1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet and
the LC50 was greater than 1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g
diet.

Classification:  Acceptable	470176-26

Non-target insect testing, rove beetle, Aleochara bilineata 	885.4340

	Microbial FLCry1Ab

(FLCRY1AB-0103)	A. bilineata adults were fed a meat diet containing
1298.7 g FLCry1Ab protein/g diet for 35 days with a LC50  >  1298.7 g
FLCry1Ab /g.  Reproductive effects were also assessed by counting the
number of second-generation adult beetles emerging from parasitized
pupae of the onion fly (Delia antique).  There were no differences noted
between the treatment and negative control groups.

Classification:  Acceptable	470176-27

Non-target insect testing, collembolan, Folsomia candida	885.4340

	Lyophilized Bt11 maize leaf

(LLBt11-0100)	Collembola were fed a diet containing 50% yeast and 50%
Bt11 leaf tissue for 28 days.  No statistically significant effects on
survival or reproduction were found among the test and negative control
groups.  The NOEC for the survival and reproduction of F. candida was
17.1 µg Cry1Ab protein/g diet and the LC50  was  > 17.1 µg Cry1Ab
protein/g diet.

Classification:  Acceptable	458358-10

Honeybee  testing,

Apis mellifera larvae, adults, and whole hive conditions 	885.4380

	Microbial FLCry1Ab

(FLCRY1AB-0103)	Honeybees were exposed via oral ingestion to
microbial-derived FLCry1Ab test material in a sucrose solution, using
in-hive commercial bee feeders.  The treatments consisted of: a sucrose
solution containing 107.82 mg/L FLCRY1AB-0103 test material/g sucrose
solution (representing 92.4 µg FLCry1Ab/mL and 10X EEC in FlCry1Ab in
Event COT67B  pollen), a negative control of 50% w/v sucrose solution,
or a positive control of 6.35 g/L diflubenzuron insect growth regulator
in sucrose solution. The test consisted of a single application of one
liter of the appropriate solution per hive and the hives were observed
for 24 days for percent successful brood development to adults and
colony conditions.  There was no significant difference in mortality
between the test and negative control groups for cells with eggs and
young or old larvae. There was also no significant difference in pre-
and post-test hive conditions between the test and negative control
treatments. Results for the positive control treatment were
significantly different from the other treatments.  Adult bees were not
affected by any of the treatments. Despite some experimental
shortcomings, there is enough certainty to indicate exposure of the
FLCry1Ab to adult worker honeybees and larvae, via direct and incidental
oral ingestion.  Furthermore, the results of the study may be considered
as weight-of-evidence for determining effects on honeybees for the
purposes of the environmental risk assessment. Therefore, the NOEL was
92.4 µg FLCry1Ab/mL and the LD50 was greater than 92.4 µg FLCry1Ab/mL.

Classification:  Acceptable-for the purposes of the environmental risk
assessment	470176-29

Soil fate and degradation	885.5200	Microbial FLCry1Ab

(FLCRY1AB-0103)	The degradation of FLCry1Ab protein (incorporated at a
rate equivalent to 80 µg FLCry1Ab/g dry wt soil) in a sandy loam soil
was assessed by measuring the loss of bioactivity to European corn
borer. The estimated DT50 and DT90 values were 17 and 52 days,
respectively, indicating that FLCry1Ab protein in plant residues
incorporated into sandy loam soil is not likely to persist or accumulate
in soil.

Classification:  Acceptable	470176-31

3-year Soil Degradation 	885.5200	Soil from Bt11 corn cultivated fields
Soil samples were collected from five fields, representing four
different soil types, in five different states, in which Bt corn
expressing Cry1Ab had been grown for three consecutive years.  Results
showed that European corn borer (ECB) larvae exhibited no toxic response
to a diet mixture, containing 15% Bt corn soil. Overall, results support
use of corn expressing the Cry1Ab protein does not result in the
accumulation and persistence of this protein in soil.

Classification:  Acceptable	460224-01

1.   Non-target Wildlife Study Summaries for COT102 expressing Vip3Aa

   a.   Avian species

Published data and studies on file at EPA show that consumption of Bt
plants have no measurable deleterious effects on avian species. However,
to comply with published data requirements, the following studies were
submitted to EPA in support of Vip3Aa protein as expressed in Event
COT102 product registration. The broiler chicken study was published in
a peer-reviewed journal and not subject to GLP standards, while the
Northern Bobwhite quail study was GLP compliant.  When considered
together, these studies meet EPA data requirements for avian species
risk assessment.

Broiler Chicken (MRID No. 470176-23)

For the first 49 days of life, commercial broiler chickens (Gallus
domesticus) were fed a prepared diet based on 50% corn grain from
transgenic Event Pacha containing VIP3A protein, grain from an isoline
non-transgenic corn, or grain from one of two locally grown reference
corns. There were no treatment-related differences for mortality, body
weight, feed conversion ratio, carcass yield, or clinical chemistry
parameters. The diet containing VIP3A had no deleterious effects on
broiler performance or carcass yield.  A separate study determined the
concentration of the transgenic Event Pacha grain as 0.588 µg
Vip3Aa19/g feed for this study (MRID No. 470176-20).  Therefore, the
NOEC was 0.588 µg VIP3A/g feed and the 49-day LC50  for broilers is
greater than 0.588 µg VIP3A/g feed.

Conclusions/Recommendations:  No adverse effects were observed on Gallus
domesticus after a 49-day chronic dietary study after exposure to a 50%
diet containing Event Pacha corn grain, expressing VIP3A. The NOEC was
0.588 µg VIP3A /g feed and the LC50 for broilers is greater than 0.588
µg VIP3A /g feed. Based on the information presented, this study is
acceptable.

Northern Bobwhite Quail (MRID No. 457665-08)

Five male and five female (Colinus virginianus) quails were administered
a single oral dose of 2000 mg VIP3A-0198 /kg, via gelatin capsules. The
VIP3A-0198 test substance (microbial-derived protein) represented 400 mg
VIP3A /kg body weight.  No mortalities occurred during the study period.
 There were no clinical signs of toxicity in any birds during the study.
 There were no statistically significant changes in body weights after
dosing.  Additionally, gross pathological examinations of all birds at
study termination revealed no abnormalities.  The results indicate that
the NOEL was 400 VIP3A mg/kg and the 14-day LD50 was > 400 VIP3A mg/kg
body weight for northern bobwhite for 14 days.

Conclusions/Recommendations:  No adverse effects or mortalities were
found after a 14-day acute oral study after exposure to the test
substance (VIP3A-0198, microbial-derived containing Vip3Aa1). The NOEL
was 400 VIP3A mg/kg and the 14-day LD50 was > 400 VIP3A mg/kg body
weight for northern bobwhite for 14 days. Based on the information
presented, this study is acceptable.

   b. Wild mammalian species

Mammalian wildlife exposure to Vip3Aa protein is considered likely;
however, mammalian toxicology information gathered to date on Bt Cry and
Vip proteins does not show a hazard to wild mammals. In addition, acute
oral toxicity studies submitted to EPA in support of the COT102
registration indicated that no significant toxicity was seen when
rodents were exposed to microbial-derived Vip3Aa19 (VIP3A-0100) protein
at the maximum hazard dose level (MRID No. 457665-05). Therefore, no
hazard from COT102 cotton expressing Vip3Aa protein to mammalian
wildlife is anticipated and data on wild mammal testing is not required
for this registration.

   c. Aquatic species

There is no reported toxicity to aquatic organisms from exposure to
anti-coleopeteran Cry proteins in Bt plants.  However, a published
laboratory study with lepidopteran-active Cry proteins has revealed that
the leaf shredding (caddis fly) trichopteran, Lepidostoma liba, had 50%
lower growth rate when fed Bt corn litter (Rosi-Marshall, et al. 2007).
Two previous field study reports by the same authors did not find
adverse effects on head stream invertebrates.  The Agency’s position
on this matter is that until Tier III and Tier IV field studies are
performed, there is not enough information to assert that sufficient
corn plant litter enters streams to cause unreasonable adverse effects
on stream invertebrate populations or communities (See Section B.I.
above - Tiered Testing Hazard and Risk Assessment Process). Two years
ago the Iowa State University and the University of Maryland received
Research grants to study the effects of Bt corn cultivation on streams
and to develop methods for aquatic hazard assessment. The results of
these studies are pending. When the study reports are reviewed the
Agency will respond with action commensurate with the outcome of the
studies. Therefore, the Agency’s current position is that there is no
evidence to conclude that there is sufficient aquatic exposure to Cry
proteins in corn plant litter to result in adverse effects on stream
invertebrate populations or communities.  In regards to Bt cotton plant
litter expressing lepidopteran-active Vip proteins, the Agency maintains
the same position at this time.

Farmed fish may be exposed to Bt protein in fish feed. However, Bt
protein activity is generally destroyed during typical fish food
manufacturing processes due to protein degradation from with the high
temperatures. Consequently, exposure of farmed fish to active Bt
proteins is not expected.  Overall, aquatic animal exposure to Bt crops
is extremely small.

Freshwater fish - Channel Catfish (MRID No. 470176-24)

The objective of this study was to determine the potential for adverse
effects of Vip3Aa protein to freshwater fish, using the channel catfish,
Ictalurus punctatus, as a representative test species, in a 30-day
feeding study.  The study compared survival and growth of juvenile
channel catfish fed commercial fish feed formulated with transgenic
maize grain with test substance FFPACHA-0100 (containing 7.1 µg
Vip3Aa19 protein/g diet) or with non-transgenic maize grain for 30 days.
Both feeds contained approximately 50% maize grain by weight.  The diet
was formulated using a “cold pelleting” process to minimize exposure
to temperatures that might degrade VIP3A protein. The formulation,
nutrient composition, characterization, homogeneity, and stability of
the fish feed test substance was also analyzed.  After 30 days, there
was no test material-related mortality. Fish fed either the VIP3A maize
grain or the control maize grain gained equal amounts of weight, and no
abnormal behavior was observed in either group. The activity and
stability of VIP3A in grain and fish feed was confirmed via fall
armyworm insect bioassay and analyzed by ELISA to confirm the presence
and amount of the test material.  There were no adverse effects on
growth or behavior of juvenile catfish exposed for 30 days.  Therefore,
the NOEC was 7.1 µg Vip3Aa19/g diet and the 30-day LC50 was greater
than 7.1 µg Vip3Aa19/g diet fish feed made from Event Pacha maize
grain.

Conclusions/Recommendations: No observed adverse effects were noted in
Ictalurus punctatu after exposure to Vip3Aa via commercial feed
formulated from Event Pacha grain.  The NOEC was 7.1 µg Vip3Aa19/g diet
and the LC50 was greater than 7.1 µg Vip3Aa19/g diet.   Based on the
information presented, this study is acceptable.

Freshwater aquatic invertebrates (MRID No.  457921-01)

The objective of this study was to determine the potential for acute
effects to the aquatic organism, Daphnia magna, during a static renewal
exposure to VIP3A via the Pacha maize pollen.  The test was conducted as
a limit test using test substance PHOPACHA-0199, containing 83.8 µg
VIP3A protein/g pollen. Daphnids were exposed to a single nominal test
concentration of 120 mg pollen/L for 48 hours with renewal of the test
solution at approximately 24 hours.  Two control groups were included: a
group in water exposed to pollen (120 mg/L) from non-transgenic,
near-isogenic maize, and an assay control group exposed to water only.
Each treatment was replicated three times and each replicate contained
10 neonate daphnids.  Observations of mortality, immobility and other
sub-lethal effects were made during the test. At test termination, there
was 100% survival in each group with no sign of immobilization or any
other adverse effects. Therefore, the NOEC was 120 mg VIP3A pollen/L and
the LC50 was greater than 120 mg VIP3A pollen/L.

Conclusions/Recommendations: Results of the 48-hour limit test showed
the LC50 was greater than 120 mg PHOPACHA-0199/L, representing 10.1 µg
VIP3A /L. Based on the information presented, this study is
unacceptable.  The 48 hour test duration is not sufficient to show
mortality for Bt toxins. The mode of action of the toxin would take more
than 48 hours for target insect pests to succumb to Cry proteins,
therefore, mortality or reproductive effects to aquatic invertebrates
(e.g., daphnids) are not expected to show within 48 hours. Because Vip
proteins are also derived from Bt and susceptible species display
similar symptoms upon ingestion, a 7-14 day Daphnia study (OPPTS
Guideline 885.4240 Series) must be performed. This study can be
submitted as a condition of registration.  Alternatively, a dietary
study of the effects on an aquatic invertebrate, representing the
functional group of a leaf shredder in headwater streams, can be
performed and submitted in lieu of the 7-14 day Daphnia study.

	iii. Estuarine and marine animals - Waiver granted

Estuarine and marine animal studies were not required for this product,
because of the low probability that estuarine or marine systems will be
exposed to Vip3Aa protein produced in event COT102 cotton plant tissues
and pollen.

   d. Terrestrial and aquatic plant species - Waiver granted

Plant toxicity studies were not required for this product because the
active ingredient is an insect toxin (Bt (-endotoxin) that has never
shown any toxicity to plants.

   e. Invertebrate species

The Vip3Aa protein is meant to target species within the order
Lepidoptera (moths and butterflies). Bt toxins are known to have a
limited host range, however, to address any unforeseen change in
activity spectrum as a result of laboratory protein synthesis and to
fulfill the published registration data requirements EPA requires that
test species used for non-target insect evaluations should include
several species that are not related to the target pests. Earthworm
studies are also recommended.

Ladybird beetle

MRID No. 457665-09

The purpose of this study was to determine the potential dietary effects
of the Vip3Aa protein on the mortality and development of the ladybird
beetle, Coleomegilla maculata.  The protocol for the non-target lady
beetle study was based on OPPTS Guideline 885.4340. Eight- to nine-day
old ladybird beetles were exposed to Vip3Aa via Pacha maize pollen test
substance (PHOPACHA-0100), incorporated into an artificial diet at 5%
weight by weight (w/w).  The negative control diet comprised 5% w/w
pollen from non-transgenic, near-isogenic maize, and a positive control
diet contained 50 µg thiobendacarb/g diet.  The treatment and control
groups each comprised three replicates of 25 beetles, which received
fresh diet daily.  After 21 days, there were no statistically
significant differences in survival, development, and growth between the
treatment and negative control groups (P(0.05), while there was 100%
mortality in the positive control group.   Therefore, the NOEC was 7.24
µg Vip3Aa19/g diet and the LC50 was greater than 7.24 µg Vip3Aa19/g
diet.

Conclusions/Recommendations: The results indicate that Vip3Aa protein
had no adverse effect on the survival, development, and growth of the
ladybird beetles.  The NOEC was 7.24 µg Vip3Aa19/g of diet and the LC50
was greater than 7.24 µg Vip3Aa19/g of diet.  This study was previously
reviewed and found acceptable (Rose and Vaituzis, 2003).

MRID No. 468848-02

The objective of this study was to determine the potential dietary
effects of Vip3Aa protein on the mortality and development of the
five-spotted ladybird beetle, Coccinella septempunctata. The test
substance, VIP3A-0204, was produced by recombinant E. coli fermentation
system and contained 7.25 mg Vip3Aa19/g before addition to a 50% sucrose
diet.  The negative control diet comprised of sucrose only, and a
positive control diet contained 0.3333 mg dimethoate/g diet.  Treatment
and control groups, each comprising of 40 beetles, were fed fresh diet
daily and the endpoints evaluated were survival and development through
15 days.  At study end, mortality in the Vip3Aa-treated group was not
statistically significantly different from that of the untreated
controls (0% vs. 5%, respectively). Positive control mortality was 100%.
The NOEC was 7.25 mg Vip3Aa19 protein/g diet and the LC50 was > 7.25 mg
Vip3Aa19 protein/g diet.

Conclusions/Recommendations: No adverse effects were seen in C.
septempunctata after exposure to Vip3Aa protein in a sucrose diet.  The
NOEC was 7.25 mg Vip3Aa19 protein/g diet and LC50 was > 7.25 mg Vip3Aa19
protein/g diet.  This study was previously reviewed and found acceptable
(Milofsky and Vaituzis, 2007).

Minute pirate bug  (MRID No. 468848-14)

The purpose of this study was to determine the potential dietary effects
of Vip3Aa protein on mortality and development of Orius insidiosus, the
minute pirate bug or insidious flower bug.

The test substance was VIP3A-0104, a 63.1 % pure preparation of
microbial-derived Vip3Aa19.  The test substance was dissolved in buffer
and incorporated at a rate of 11.49 mg/g diet (7.25 mg Vip3Aa19/g of
artificial diet -- approximately 310X the highest mean concentration of
Vip3Aa in COT102) and was continuously supplied to predatory bug (Orius
insidiosus) nymphs for 21 days. Control nymphs were fed untreated diet,
and positive control nymphs were fed diet treated with 10 µg
teflubenzuron/g of diet.  At study end, mortality in the Vip3Aa treated
nymphs was not significantly different from that of the untreated
controls (15% vs. 13%, respectively).  Positive control mortality was
100%.  The NOEC was 7.25 mg Vip3Aa19 protein/g diet and the LC50 value
was determined to be greater than 7.25 mg Vip3Aa19 protein/g diet.

Conclusions/Recommendations:  No adverse effects were seen in Orius
insidiosus after exposure to Vip3Aa protein in an artificial diet. The
NOEC was 7.25 mg Vip3Aa19 protein/g diet and the LC50 value was
determined to be greater than 7.25 mg Vip3Aa19 protein/g diet.  This
study was previously reviewed and found acceptable (Milofsky and
Vaituzis, 2007).

	iii. Green Lacewing (MRID No. 468848-15)

The purpose of this study was to determine the potential dietary effects
of Vip3Aa protein on mortality and development of Chrysoperla carnea
larvae, the green lacewing.  The test substance, VIP3A-0104, consisted
of 7.25 mg aVip3Aa19/g of artificial diet was continuously supplied to
green lacewing (Chrysoperla carnea) larvae for 21 days. Control larvae
were fed untreated diet, and positive control larvae were fed diet
treated with 10 µg teflubenzuron/g diet. At study end, mortality in the
Vip3Aa-treated larvae was not statistically significantly different from
that of the untreated controls (37.5% vs. 35.0%, respectively). Positive
control mortality was 100%. Although the control mortality exceeded the
25% criterion for the test to be considered valid, mortality did not
differ significantly between the test and control groups. Furthermore,
the control mortality was <25% through day 21, which was judged to be a
sufficient exposure period to observe acute and developmental effects on
lacewing larvae. Therefore, the NOEC was 7.25 mg Vip3Aa19 protein/g diet
and the LC50 value was greater than 7.25 mg Vip3Aa19 protein/g diet.

Conclusions/Recommendations:  No adverse effects were seen in
Chrysoperla carnea after exposure to Vip3Aa protein mixed in an
artificial diet. The NOEC was 7.25 mg Vip3Aa19 protein/g diet and the
LC50 value was determined to be greater than 7.25 mg Vip3Aa19 protein/g
diet.  This study was previously reviewed and found acceptable (Milofsky
and Vaituzis, 2007).

iv.   Collembola (MRID No. 458358-10)

The purpose of this study was to determine the potential dietary effects
of Vip3Aa protein on mortality and reproduction on Folsomia candida
(springtail; Collembola). The test substances included: LLPACHA-0100,
containing 43.4 µg Vip3Aa19 protein/g leaf tissue diet from Event
Pacha, distilled water as a negative control and thiodicarb as a
positive control.  There were 4 replicates of 10 juvenile collembola per
replicate per treatment and fresh diet was provided daily.  Vip3Aa
protein had no detectable impact on the survival or reproduction of the
collembola after 28 days of continuous exposure.  The NOEC of
lyophilized Vip3Aa protein from Event Pacha corn leaves was 50% of the
diet which was the highest concentration tested.  Therefore, the NOEC
was 43.4 µg Vip3Aa19 protein/g diet and the LC50 was greater than 43.4
µg Vip3Aa19 protein/g diet.

Conclusions/Recommendations: No adverse effects were seen on Folsomia
candida after exposure to Vip3Aa protein in Event Pacha maize leaf
tissue.  The NOEC was 43.4 µg Vip3Aa19 protein/g diet and the LC50 was
greater than 43.4 µg Vip3Aa19 protein/g diet. This study was previously
reviewed and found acceptable (Rose and Vaituzis, 2003).

v.     Honeybee (MRID No. 458358-09)

The objective of this study was to evaluate potential dietary effects of
transgenic Vip3Aa pollen from Event Pacha corn on honeybee larvae (Apis
mellifera) survival and adult emergence in a single dose study.  The
test substance (PHOPACHA-0199) contained 2 mg of pollen moistened with
30% sucrose solution and was estimated to contain 83.8 µg Vip3Aa19/g
pollen.  The study included three controls:  one group of larvae were
fed 2 mg inbred maize pollen (PIPACHA-0299C) moistened with 30% sucrose
solution, one group received 2 mg inbred maize pollen (PIPACHA-0299C)
moistened with 30% sucrose solution  and mixed with potassium arsenate
at 1000 ppm (positive control), and the third group received no
treatment at all.  Eighty, three- to five-day old larvae (four
replicates of 20) were allowed to consume the pollen and then returned
to their source hives for capping of the brood cells by nurse bees.  The
hives were maintained under natural environmental conditions.   After 19
days, mean survival to capping and mean survival to adult emergence were
76.3% in the Vip3Aa corn pollen group and 77.5% in the control corn
pollen group.  Mean survival to capping and mean survival to adult
emergence were 87.5% for the negative control group.  There were no
statistically significant differences among these three study groups.
Mean survival to capping and mean survival to adult emergence were 20%
in the positive control group, which was statistically significantly
lower than in the other two study groups.  No behavioral or
morphological abnormalities were noted among the emerged adult bees, and
no differences in mean emergence times were observed.  Therefore, no
adverse effects from Vip3Aa pollen in Event Pacha corn were seen on
honeybee larvae and adult emergency.  The NOEC was 83.8 µg Vip3Aa19/g
pollen and the LC50 was greater than 83.8 µg Vip3Aa19/g pollen.

Conclusions/Recommendations: No adverse effects from Vip3Aa pollen in
Event Pacha corn were seen on the survival of Apis mellifera honeybee
larvae and adult emergence.  The NOEC was 83.8 µg Vip3Aa19/g pollen and
the LC50 was greater than 83.8 µg Vip3Aa19/g pollen. This study was
previously reviewed and found acceptable (Rose and Vaituzis, 2003).

vi.    Earthworm (MRID No. 457921-02)

The objective of this study was to evaluate the potential effects of
Vip3Aa from Event Pacha administered to earthworms (Eisenia fetida) via
an artificial soil substrate during a 14-day exposure period.  The
testing was conducted based on OPPTS Series 850.6200 Earthworm
Sub-chronic Toxicity Test and OECD Guideline 207. In the test,
earthworms were exposed to a single concentration of VIP3A protein
derived from Event Pacha maize leaf (test substance LPPACHA-0199) and
incorporated into an artificial soil substrate at 3.60 µg VIP3A/g soil.
 There were no mortalities in the assay control group, buffer control
group, or VIP3A protein group.  Analysis of the test soil showed that
VIP3A was present in the soil and was biologically active against
Agrotis ipsilon (black cutworm).  Therefore, no adverse effects on
earthworms were observed after exposure to VIP3A protein via Event Pacha
maize leaf tissue.  The NOEC was 3.60 µg VIP3A protein/kg dry soil and
the14-day LC50 for earthworms was determined to be greater than 3.60 µg
VIP3A protein/kg dry soil.

Conclusions/Recommendations: No adverse effects from Vip3Aa maize leaf
tissue in Event Pacha were seen on the survival of Eisenia fetida, via
an artificial soil substrate after 14 days. The NOEC was 3.60 µg VIP3A
protein/kg dry soil and the14-day LC50 for earthworms was determined to
be greater than 3.60 µg VIP3A protein/kg dry soil.  Based on the
information presented, this study is acceptable.

2.   Non-target Wildlife Study Summaries for COT67B expressing FLCry1Ab

   a.   Avian species

Published data and studies on file at EPA show that consumption of Bt
plants have no measurable deleterious effects on avian species. However,
to comply with published data requirements, the following studies were
submitted to EPA in support of Event COT67B registration. The broiler
chicken study was published in a peer-reviewed journal and not subject
to GLP standards, while the Northern Bobwhite quail study was GLP
compliant.  When considered together, these studies meet EPA data
requirements for avian species.

Broiler Chicken (MRID No. 456521-01)

For the first 42 days of life, commercial broiler chickens (Gallus
domesticus) were fed a prepared diet based on 50% grain from either
transgenic Bt11 corn containing Cry1Ab protein, transgenic Bt11 corn
sprayed with Liberty herbicide, grain from an isoline, non-transgenic
corn, or grain from a locally grown reference corn.  There were no
treatment-related differences for mortality, body weight, feed
conversion ratio, carcass yield, or clinical chemistry parameters. The
corn diet containing the test substance had no deleterious effects on
broiler performance or carcass yield in this study.  It should also be
noted that the concentration of Cry1Ab in Bt11 grain used to formulate
the diet was 0.8 µg/g seed, however, the concentration of Cry1Ab in the
feed was not determined.  In a similar broiler chicken study, the
concentration of Cry1Ab in Bt176 corn was less than 0.005 µg/g grain.
Therefore, while an official NOEC was not determined, exposure to Cry1Ab
was very likely during the experiment since it is expected that Cry1Ab
in Bt11 grain would behave similarly to Cry1Ab in Bt176 grain during
preparation of broiler diets.

Conclusions/Recommendations:  No adverse effects were found in the
42-day dietary study with Gallus domesticus when fed transgenic Bt11
grain, containing Cry1Ab. This study was previously reviewed and found
acceptable (Hunter and Vaituzis, 2007).

Northern Bobwhite Quail (MRID No. 433236-09)

Five male and five female juvenile bobwhite quails (Colinus virginianus)
were administered a single oral dose of 140 mg of Cry1Ab protein/kg body
weight, via oral gavage.  The test substance was LP176-0194 (Bt176 maize
leaf protein).  No mortalities occurred during the study period.  There
were no clinical signs of toxicity in any birds during the study.  There
were no statistically significant changes in body weights at any
weighing interval (3, 7 or 14 days) after dosing.  Additionally, gross
pathological examinations of all birds at study termination revealed no
abnormalities.  The results indicate that the NOEL was 140 mg of Cry1Ab
protein/kg body weight and the 14-day LD50 was greater than 140 Cry1Ab
mg/kg body weight for bobwhite quail.

Conclusions/Recommendations:  No adverse effects were found in the
14-day dietary study with Colinus virginianus when administered a single
oral dose of Vip3A.  The NOEL was 140 mg of Cry1Ab protein/kg body
weight and the 14-day LD50 was greater than 140 Cry1Ab mg/kg body weight
for bobwhite quail.  This study was reassessed in the 2001 Bt PIPs
Reassessment BRAD (US EPA, 2001b).

   b. Wild mammalian species

Mammalian wildlife exposure to Cry1Ab protein is considered likely;
however, mammalian toxicology information gathered to date on Bt Cry
proteins does not show a hazard to wild mammals. In addition, acute oral
toxicity studies submitted to EPA in support of the COT67B registration
indicated that no significant toxicity was seen when rodents were
exposed to microbial-derived full-length Cry1Ab (FLCRY1AB-0103) protein
at the maximum hazard dose level (MRID No. 470176-14). Therefore, no
hazard to mammalian wildlife is anticipated and data on wild mammal
testing is not required for this registration.

   c. Aquatic species

There is no reported toxicity to aquatic organisms from exposure to
anti-coleopeteran Cry proteins in Bt plants.  However, a published
laboratory study with lepidopteran-active Cry proteins has revealed that
the leaf shredding (caddis fly) trichopteran, Lepidostoma liba, had 50%
lower growth rate when fed Bt corn litter (Rosi-Marshall, et al. 2007).
Two previous field study reports by the same authors did not find
adverse effects on headwater stream invertebrates.  The Agency’s
position on this matter is that until Tier III and Tier IV field studies
are performed, there is not enough information to assert that sufficient
corn plant litter enters streams to cause unreasonable adverse effects
on stream invertebrate populations or communities (See Section B.I.
above - Tiered Testing Hazard and Risk Assessment Process). Two years
ago the Iowa State University and the University of Maryland received
Research grants to study the effects of Bt corn cultivation on streams
and to develop methods for aquatic hazard assessment. The results of
these studies are pending. When the study reports are reviewed, the
Agency will respond with action commensurate with the outcome of the
studies. Therefore, the Agency’s current position is that there is no
evidence to conclude that there is sufficient aquatic exposure to Cry
proteins in corn plant litter to result in adverse effects on stream
invertebrate populations or communities.  In regards to
lepidopteran-active Bt cotton plant litter, the Agency maintains the
same position at this time.

Farmed fish may be exposed to Bt protein in fish feed. However, Bt
protein activity is generally destroyed during typical fish food
manufacturing processes due to protein degradation in high temperatures
associated and consequently, exposure of farmed fish to active Bt
proteins is not expected. Overall, aquatic animal exposure to Bt crops
is negligible.

i.     Freshwater fish- Channel Catfish (MRID No. 470176-25)

The objective of this study was to determine the potential for adverse
effects of full-length Cry1Ab to freshwater fish, using the channel
catfish, Ictalurus punctatus, as a representative test species in a 28
day feeding study. The study compared survival and growth of juvenile
channel catfish fed commercial catfish diet containing a purified
preparation of FLCRY1AB-0103 (a microbial-derived test substance,
representing 15.4 µg FLCry1Ab protein/g diet) or standard untreated
diet for 28 days. The diet was formulated using a “cold pelleting”
process to minimize exposure to temperatures that might degrade FLCry1Ab
protein. After 28 days, no mortalities or abnormalities were seen in
fish either fed the test material or control diet. The activity and
stability of FLCry1Ab fish feed was confirmed via European corn borer
insect bioassay and analyzed by ELISA to confirm the presence and amount
of the test material in a separate study.  Overall, there were no
adverse effects and no mortality observed for juvenile catfish fed with
the commercial catfish diet containing FLCry1Ab after 28 days.  The
NOAEC was 15.4 µg FLCry1Ab/g fish food diet and the LD50 was greater
than 15.4 µg FLCry1Ab/g diet for juvenile channel catfish.

Conclusions/Recommendations: No observed adverse effects were noted in
Ictalurus punctatus.  Therefore, the NOAEC was 15.4 µg FLCry1Ab/g fish
food diet and the LD50 was greater than 15.4 µg FLCry1Ab/g diet for
juvenile channel catfish.  Based on the information presented, this
study is acceptable.

ii.      Freshwater aquatic invertebrates (MRID No.  433236-10)

The objective of this study was to determine the potential for acute
effects to the aquatic organism, Daphnia magna, during a static renewal
exposure to Cry1Ab via the Bt176 maize pollen test substance
(PHO176-0194- containing 12.36 µg Cry1Ab/g).  The test was conducted as
a limit test using one test concentration, representing 1.85 µg
Cry1Ab/L. Daphnids were exposed to a single nominal test concentration
of 150 mg pollen/L for 48 hours with renewal of the test solution at
approximately 24 hours.  Two control groups were included: a group in
water exposed to pollen (150 mg/L) from non-transgenic, near-isogenic
maize, and an assay control group exposed to water only.  Each treatment
was replicated three times and each replicate contained 10 neonate
daphnids.  Observations of any mortality, immobility and other
sub-lethal effects were recorded.  At test termination there was 100%
survival in each group with no sign of immobilization or other effects.
The NOEC was 150 mg PHO176-0194/L and the LC50 was greater than 150 mg
PHO176-0194/L, representing 1.85 µg Cry1Ab/L.

Conclusions/Recommendations:  After 48 hours, the results of the limit
test showed no adverse effects to Daphinia. The NOEC was 150 mg
PHO176-0194/L and the LC50 was greater than 150 mg PHO176-0194/L,
representing 1.85 µg Cry1Ab/L. However, based on the information
presented, this study is unacceptable.  The 48 hour test duration is not
sufficient to show mortality for Bt toxins. The mode of action of the
toxin would take more than 48 hrs. for target insect pests to succumb to
Cry proteins, therefore, mortality or reproductive effects to aquatic
invertebrates e.g., daphnids, are not expected to show within 48 hours.
Therefore, a 7-14 day Daphnia study (OPPTS Guideline 885.4240 Series)
needs to be performed. This study can be submitted as a condition of
registration.  Alternatively, a dietary study of the effects Cry1Ab on
an aquatic invertebrate, representing the functional group of a leaf
shredder in headwater streams, may be performed and submitted in lieu of
the 7-14 day Daphnia study.

	iii.        Estuarine and marine animals-Waiver granted

Estuarine and marine animal studies were not required for this product,
because of the low probability that estuarine or marine systems will be
exposed to Cry1Ab protein produced in event COT67B cotton plant tissues
and pollen.

   d. Terrestrial and aquatic plant species-Waiver granted

Plant toxicity studies were not required for this product because the
active ingredient is an insect toxin (Bt endotoxin) that has never shown
any toxicity to plants.

   e. Invertebrate species

The Cry1Ab protein is meant to target species within the order
Lepidoptera (moths and butterflies). Bt toxins are known to have a
limited host range; however, to address any unforeseen change in
activity spectrum as a result of laboratory protein synthesis and to
fulfill the published registration data requirements, EPA requires that
test species used for non-target insect evaluations should include
several species that are not related to the target pests.  Earthworm
studies are also recommended.

Ladybird beetle (MRID No. 470176-26)

The purpose of this study was to determine the potential dietary effects
of FLCry1Ab protein test alone and FLCry1Ab and Vip3Aa19 tested in
combination on the survival and development of the pink-spotted ladybird
beetle (Coleomegilla maculata). The protocol for the non-target lady
beetle study was based on OPPTS Guideline 885.4340. Five-day old, second
instar ladybird beetles were exposed to a diet of bee pollen, Esphestia
(moth) eggs, and either FLCRY1AB-0103 test material (at 1000 µg
FLCry1Ab/g diet) or FLCRY1AB-0103 + VIP3A-0204 test materials (at 1000
µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet).  The negative control
diet was the pollen and moth egg diet only, and a positive control diet
contained 250 µg potassium arsentate/g diet.  The treatment and control
groups each comprised of 40 beetles, which received fresh diet every
other day.  After 21 days, there were no statistically significant
differences in larval, pupal, and adult survival between the treatment
and negative control groups (P(0.05), while there was 100% mortality in
the positive control group.   Therefore, the NOAEC for FLCry1Ab was 1000
µg FLCry1Ab/g diet and the LC50 was greater than 1000 µg FLCry1Ab/g
diet. The NOAC for FLCry1Ab + Vip3Aa19 proteins tested in combination
was 1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet and the LC50 was
greater than 1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet.

Conclusions/Recommendations: The results indicate that the FLCry1Ab
protein tested alone or in combination with Vip3Aa19 had no adverse
effect on the survival, development, and growth of the ladybird beetles.
 In conclusion, the NOAEC for FLCry1Ab was 1000 µg FLCry1Ab/g diet and
the LC50 was greater than 1000 µg FLCry1Ab/g diet. The NOAC for
FLCry1Ab + Vip3Aa19 proteins tested in combination was 1000 µg
FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet and the LC50 was greater than
1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet.  Based on the
information presented, this study is acceptable.

Minute pirate bug  (MRID No. 470176-28)

The purpose of this study was to determine the potential dietary effects
of FLCry1Ab protein as expressed in Event COT67B and FLCry1Ab and
Vip3Aa19 proteins tested in combination, as expressed in Event COT102 X
COT67B, on mortality and development of Orius laevigatus, a predatory
bug which is closely-related and ecologically very similar to O.
insidiosus.

The report contained two dietary studies studying the effects on O.
laevigatus, after exposure via meat-based artificial diets containing
either FLCry1Ab insecticidal protein alone or in combination with
Vip3Aa19 insecticidal protein.  Only the results of the second run of
the first sudy were considered valid and are presented in this summary.
After 14 days, O. laevigatus fed 1.0039 mg FLCry1Ab/g diet (7X the
maximum concentration in COT67B cotton leaves) had pre-imaginal
mortality of 17.95%. O. laevigatus fed the combined proteins of 1.0039
mg FLCry1Ab + 0.1950 mg Vip3Aa19/g diet (corresponding to 10X the
highest mean concentrations of FLCry1Ab and Vip3Aa19 found in COT67B and
COT02 cotton leaves) had pre-imaginal mortality of 39.47%, which was a
statistically significant increase in mortality. The control
pre-imaginal mortality was 12.82%, while the toxic reference standard
had 100% mortality, as expected. The NOEC for O. laevigatus was 1003.9
µg FLCry1Ab/g diet and 1003.9 µg FLCry1Ab + 195.0 µg Vip3Aa19/g diet
for Event COT67 at 7X EEC and Event COT102 x COT67B at 10X EEC cotton
leaves, respectively.  Furthermore, the LC50 was greater than 1003.9 µg
FLCry1Ab/g diet and 1003.9 µg FLCry1Ab + 195.0 µg Vip3Aa19/g diet for
COT67B and COT102 x COT67B cotton, respectively.

Conclusions/Recommendations:  The overall results of the two studies
were inconsistent due to the high control mortality, implicating the use
of Orius laevigatus is equivocal, as a representative indicator species
for studying the effects of Bt PIP proteins.  In the only valid study,
there was a statistically significant increase in mortality of O.
laevigatus exposed to FLCYR1AB-0103 + VIP3A-0204 at 10X EEC for COT102 x
COT67B cotton leaves, which may represent an interaction between
FLCry1Ab and Vip3Aa19. However, the EPA established Level of Concern
(LOC) is 50% mortality when tested at 5X EEC and a less than 50%
mortality effect at the MHD is indicative of a minimal risk for the
purposes of the environmental risk assessment (US EPA, 1998).
Therefore, no adverse effects on O. laevigatus are expected at
concentrations encountered in field crops.  The NOEC for O. laevigatus
was 1003.9 µg FLCry1Ab/g diet and 1003.9 µg FLCry1Ab + 195.0 µg
Vip3Aa19/g diet for Event COT67 at 7X EEC and Event COT102 x COT67B at
10X EEC cotton leaves, respectively.  Furthermore, the LC50 was greater
than 1003.9 µg FLCry1Ab/g diet and 1003.9 µg FLCry1Ab + 195.0 µg
Vip3Aa19/g diet for COT67B and COT102 x COT67B cotton, respectively.
Based on the information submitted, this study is supplemental for the
purposes of the environmental risk assessment.

In addition, a three-year field study conducted on Event Bt11 x Event
Pacha maize (expressing Cry1Ab and VIP3A proteins) showed no differences
on densities of  non-target arthropod communities, including Orius
insidiosus, when compared with an isogenic conventional corn control
(Dively et al. 2005). The results also showed that biodiversity and
community-level responses were not significantly affected by expression
of the stacked VIP3A and Cry1Ab proteins.

When the results of the second run of the first study on O. laevigatus
are considered in combination with the three-year field Dively, et al.
(2005) study, the weight-of-evidence indicates there are no adverse
effects on Orius species from FLCry1Ab protein as expressed in COT67B or
its associated stacked product, Event COT102 x COT67B cotton. The Agency
also notes that there are several published field studies on the effects
of Bt crops on insect predators showing no significant differences in
the density of beneficial insects, including  Orius insidiosus (Pilcher
et al., 1997a, 1997b, and 2005; and Al-Deeb et al., 2001).

iii. 	Rove Beetle (MRID No. 470176-27)

The purpose of this study was to determine any reproductive effects of
FLCry1Ab protein on Aleochara bilineata (rove beetle).  In a laboratory
bioassay, adult rove beetles (Aleochara bilineata) were exposed to a
prepared meat diet containing 1298.7 g FLCRY1AB-0103/g of diet for 35
days. The FLCry1Ab concentration fed to the beetles was approximately 10
times that which occurs in fresh leaf tissue of Event COT67B cotton
plants. A negative control diet and a reference control diet were also
included in the test. To assess reproduction of the beetles, onion fly
(Delia antique) pupae were provided to be parasitized by the beetles
during the test. Second-generation beetles emerging from the parasitized
pupae were counted until emergence stopped on test day 86. The results
of the reproductive success of the beetles showed no statistically
significant differences between the number of beetles that emerged from
the FLCry1Ab test treatment, when compared to the control. The IOBC
validity criteria were met (Grimm, et al., 2000) and the stability and
bioactivity of the test material in the prepared diet were also
confirmed. Therefore, no adverse effects were noted on the reproductive
effects of FLCry1Ab protein on A. bilineata.  Furthermore, the NOEC was
1000 µg FLCry1Ab/g diet for the reproduction of Aleochara bilineata and
the LC50 was greater than 1000 µg FLCry1Ab/g diet, when exposed orally
via a treated meat-based diet

Conclusions/Recommendations:  No adverse effects were noted on the
reproductive effects of FLCry1Ab protein on rove beetles.  Therefore,
the NOEC was 1000 µg FLCry1Ab/g diet for the reproduction of Aleochara
bilineata and the LC50 was greater than 1000 µg FLCry1Ab/g diet, when
exposed orally via a treated meat-based diet.  Based on the information
presented, this study is acceptable.

iv.        Collembola (MRID No. 458358-10)

The purpose of this study was to determine the potential dietary effects
of Cry1Ab protein on mortality and reproduction on Folsomia candida
(springtail; Collembola). The treatments included: 17.1 µg Cry1Ab/g
diet of equal parts LLBt11-0100 test substance (lyophilized leaf from
Bt11 maize) and yeast, a control diet containing equal parts yeast and
lyophilized leaves of non-transgenic, near-isogenic mazie, a diet to
control the effects of maize leaves consisting of yeast only, and a
positive control of yeast with 500 µg thiodicarb/g diet.  There were 4
replicates of 10 juvenile collembola per replicate per treatment and
fresh diet was provided daily.

After 28 days, mean survival was 83%, 78%, and 80% in the
LLBt11-0100-treated group, the non-transgenic maize leaf-treated group,
and the group fed yeast only, respectively.  The mean survival for the
positive control group was 3%, which was statistically significant from
the other treatment groups.  The mean number of juveniles was 446.5,
343.5, and 218.5 in the LLBt11-0100-treated group, the non-transgenic
maize leaf-treated group, and the group fed yeast only, respectively.
The positive control was significantly different from the other groups.
Therefore, Cry1Ab protein had no detectable impact on the survival or
reproduction of the collembola after 28 days of continuous exposure.
The NOEC for the survival and reproduction of F. candida of lyophilized
Bt11 corn leaves was 17.1 µg Cry1Ab protein/g diet and the LC50 was
greater than 17.1 µg Cry1Ab protein/g diet.

Conclusions/Recommendations: No adverse effects of Cry1Ab were observed
on Folsomia candida from Bt11 corn leaf tissue. The NOEC for the
survival and reproduction of F. candida of lyophilized Bt11 corn leaves
was 17.1 µg Cry1Ab protein/g diet and the LC50 was greater than 17.1
µg Cry1Ab protein/g diet.  This study was previously reviewed and found
acceptable (Vaituzis.and Rose, 2000).

v.          Honeybee (MRID No. 470176-29)

A semi-field whole-hive feeding study was conducted based on the
recommendations in EPPO Bulletin 22 (Oomen, et al., 1992), in accordance
with UK Good Laboratory Practice regulations of 1999 and OECD principles
[Revised 1997].

The objective of this study was to evaluate potential dietary effects of
transgenic microbial-derived full-length Cry1Ab on honeybee (Apis
mellifera) larvae survival, adult emergence, exposed adult worker bee
survival, and whole-hive conditions in a semi-field study. Honeybees
were exposed, via oral ingestion using in-hive commercial bee feeders.
The treatments consisted of: a sucrose solution containing 107.82 mg/L
FLCRY1AB-0103 test material/g sucrose solution (representing 92.4 µg
FLCry1Ab/mL and 10X EEC in FlCry1Ab in Event COT67B  pollen), a negative
control of 50% w/v sucrose solution, or a positive control of 6.35 g/L
diflubenzuron insect growth regulator in sucrose solution. The test
consisted of a single application of one liter of the appropriate
solution per hive and the hives were observed for 24 days for percent
successful brood development to adults and colony conditions.  There was
no significant difference in mortality between the test and negative
control groups for brood development.  There was also no significant
difference in pre- and post-test hive conditions between the test and
negative control treatments. Results for the positive control treatment
were significantly different from the other treatments for brood
development and hive condition (as indicated by the significantly
reduced mean percentage of comb covered by life stages). Adult bees were
not affected by any of the treatments. These results indicate direct and
incidental ingestion of FLCry1Ab proteins did not adversely affect brood
development, exposed worker bees, and the hive condition.  Therefore,
the NOEL was 92.4 µg FLCry1Ab/mL and the LD50 was greater than 92.4 µg
FLCry1Ab/mL.

Conclusions/Recommendations: No adverse effects were observed after a
single-dose application of FLCRY1AB-0103 test material mixed with a
sucrose solution were observed on Apis mellifera honeybee larvae, adult
emergence, exposed adult worker bee survival, and whole-hive conditions
after 24 days.  Despite some experimental shortcomings, there is enough
certainty to indicate exposure of the FLCry1Ab to adult worker honeybees
and larvae, via direct and incidental oral ingestion.  Therefore, the
NOEL was 92.4 µg FLCry1Ab/mL and the LD50 was greater than 92.4 µg
FLCry1Ab/mL.Therefore, the NOEL was 92.4 µg FLCry1Ab/mL and the LD50
was greater than 92.4 µg FLCry1Ab/mL.  Therefore, this study is rated
acceptable for the purposes of the environmental risk assessment.

In addition to this study, a recent meta-analysis of 25 studies that
independently assessed potential effects of Bt Cry proteins on honeybee
survival showed that Bt Cry proteins used in genetically modified crops
commercialized for control of lepidopteran and coleopteran pests do not
negatively affect the survival of either honeybee larvae or adults in
laboratory settings (Duan, et al., 2008). A semi-field study also showed
no adverse effects of Bt corn pollen containing high levels of Cry1Ab
protein on adult honeybee survival, foraging frequency, behavior or
brood development during the 7-day period of pollen shed and no adverse
effects on brood development after an additional 30 days following
pollen shed (Schur et al., 2000).

Therefore, the weight-of-evidence demonstrates that there are no adverse
effects of FLCry1Ab protein on honeybee brood development and adults in
either the laboratory or field setting. This conclusion was determined
by the two semi-field studies (showing no adverse effects of FLCry1Ab
and Bt Cry1Ab on brood development, adult survival, and whole hive
conditions) in combination with the meta-analysis of various laboratory
studies (demonstrating no adverse effects of Bt Cry proteins on honeybee
larvae and adults).

3. Soil Fate

Soil organisms may be exposed to Vip3Aa and FLCry1Ab protein through
contact with cotton plant roots (by direct feeding), cotton plant root
exudates, incorporation of above-ground plant tissues into soil
following harvest, or by soil-deposited pollen. Some evidence suggests
that soils which 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 Vip and Cry proteins are both toxins
derived from soil-inhabiting bacteria, Bacillus thuringiensis and found
in commercial microbial insecticides (De Maagd et al., 2003 and Graser
and Song, 2006), Vip protein degradation would also be similar to Cry
protein degradation.   Nonetheless, the Agency required the following
soil fate evaluations to support the Event COT102 and COT67B Bt cotton
registrations.

MRID No. 470176-30

The purpose of this study was to investigate the degradation of Vip3Aa
protein in various types of soils (clay, sandy clay loam, sandy loam,
silt loam, and artificial soils) by assessing the loss of bioactivity,
via insect bioassay.  The test substance LPPACHA-0199 (maize leaf
protein, containing ca. 0.36% Vip3Aa19) was incorporated at
concentrations of 16 or 4 Vip3Aa19 mg/g of soil and incubated under
controlled conditions for 29 days. During the incubation, soil samples
were collected weekly and used in black cutworm (BCW, Agrotis ipsilon)
bioassays to determine biological activity of the test substance against
the insect over time. The loss of bioactivity was measured by BCW
mortality, which was used to estimate the DT50 (time to dissipation of
50% of the initial bioactivity) of the 16 mg/g concentration of the test
material in each soil. The estimated DT50 values ranged from 6.0 days in
the silt loam to 12.6 days in one of the clays, indicating that Vip3Aa
protein in plant residues incorporated into soil is not likely to
persist or accumulate in soil.

MRID No. 470176-31

The purpose of this study was to investigate the degradation of FLCry1Ab
protein in a viable microbial agricultural soil typical of a
cotton-growing region by assessing the loss of bioactivity, via insect
bioassay.  The test substance FLCRY1AB-0103 (microbial-derived protein,
containing 103 µg FLCRYCRY1AB-0103/g soil) was applied to sandy loam
soil at a rate equivalent to 80 µg FLCry1Ab/g dry wt of soil, which
would be 160 times the estimated soil concentration that would result
from incorporation of pre-harvest stage COT67B cotton crop residue in
the field. The soil was incubated under controlled conditions for 0, 1,
3, 7, 14, 30, 62, 94, or 120 days after dosing, with samples collected
at each time point for use in the bioassays. The dosed soil samples were
incorporated into insect diet at a concentration of 10% (w/v) and
provided to first instar European corn borer (ECB, Ostrinia nubilalis)
larvae for approximately five days. Degradation of FLCry1Ab was assessed
by the loss of bioactivity, measured by ECB mortality. Mortality was
plotted against incubation time to estimate the DT50 and DT90 (time to
dissipation of 50% and 90% of the initial bioactivity, respectively) of
the test material in the soil. The estimated DT50 and DT90 values were
17 and 52 days, respectively, indicating that FLCry1Ab protein in plant
residues incorporated into sandy loam soil is not likely to persist or
accumulate in soil.

Conclusions/Recommendations:  These studies utilized field soil spiked
with purified insecticidal protein derived from either plant- or
microbial-derived protein. This approach is useful because dose
responses can be easily quantified. However, the degradation and
accumulation of Bt Cry proteins found within decaying plant tissue may
behave differently than proteins in artificially spiked soil. Because
Vip protein is derived from Bt and display similar insecticidal
activity, the behavior of Vip protein is expected to be similar to Cry
proteins as well. Thus, the presence of low levels of Bt Cry and Vip
proteins in the soil (at or below the level of detection) is anticipated
until all plant tissue is ‘mineralized’. However, the reviewed data
show that Cry and Vip proteins will be quickly degraded upon release
from decaying plant tissue. In addition, a study that evaluated Cry1Ab
protein accumulation in a field with three years of continuous Cry1Ab
field corn production showed that the protein had not accumulated in
soil to a level that would elicit a toxic response from ECB larvae, a
species that is highly susceptible to Cry1Ab protein (MRID No.
460224-01; Milofsky and Vaituzis, 2006).

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. The
results of these studies show that there is no detectable Cry protein
accumulation in agricultural soils during commercial planting of
currently registered Cry protein producing crops (Milofsky and Vaituzis,
2006).

More recently, a comprehensive review of all available scientific data
on ecological effects of commercially grown GM crops over the last ten
years was completed (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) are 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 rapid degradation of Vip3Aa
and FLCry1Ab proteins in soil as demonstrated in the insect bioassays,
there is no indication that the proteins expressed in Event COT102 and
Event COT67B are likely to persist or accumulate in soil after
continuous cultivation. Therefore, no additional long-term field studies
are required for these PIP products.

4.   Effects on Soil Microorganisms

Numerous published studies indicate that exposure to Cry protein
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 protein. In addition, comparisons of microbial
biomass in FLCry1Ab dosed and undosed soil prior to and during the study
showed that microbial activity was maintained throughout the test
period.   Vip protein had similar DT50 or degradation time to Cry
proteins and these proteins are both Bt toxins.

In addition, there are several ongoing U.S. Department of Agriculture
and EPA Office of Research and Development funded research projects
evaluating the effects of Cry protein crops on soil microbial flora. If
adverse effects are seen from this or any other research, the Agency
will take appropriate action to mitigate potential risks.

With regard to the impact of genetically engineered crops on soil, it is
important to note 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.

5.  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 in, or have their origin in,
soil-inhabiting bacteria. Soil is also the habitat of anthrax, tetanus
and botulinum toxin-producing bacteria. Transfer of these genes and/or
toxins to other microorganisms or plants has not been detected.
Furthermore, several experiments (published in scientific journals),
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, which suggest that HGT is at most an
artificial event, and the fact that the Bt toxins engineered into COT102
and COT67B were derived from soil-inhabiting bacteria, EPA has concluded
that there is no risk of HGT from Vip3Aa or Cry1Ab producing cotton.

6.  Gene Flow and Weediness Potential

Movement of transgenes from crop plants into weeds is a significant
concern, due to uncertainty regarding the effect that a new pest
resistance gene may have on plant populations in the wild. Under FIFRA,
the Agency has reviewed the potential for gene capture and expression of
Cry proteins in commercial Bt cotton by wild or weedy relatives of
cotton in the United States, its possessions or territories. Because Vip
proteins are Bt toxins and have similarities to Cry proteins in its
insecticidal activity on similar target species, the Agency maintains
the same approach in evaluation of gene flow and weediness potential.

There is a possibility for gene transfer in locations where wild or
feral cotton relatives exist.  Therefore, EPA requires stringent sales
and distribution restrictions on Bt cotton within these areas to
preclude outcrossing or hybridization from the crop to sexually
compatible relatives.  There are only four areas in the United States
and its territories wherein cultivated cotton has the opportunity to
outcross to wild or feral species, which are genetically compatible: (1)
southern Arizona, (2) Hawaiian islands, (3) southern Florida and 4)
Puerto Rico. G. thurberi (Arizona Wild Cotton) is present in the
elevated regions of Arizona and does not grow in areas of commercial
cotton production. G. thurberi is a diploid and produces sterile,
triploid progeny when crossed with the tetraploids G. hirsutum or G.
barbadense. In the very south of Florida, feral G. hirsutum exists in
apparently self-sustaining populations. Since these would readily cross
with cultivated cotton, sale of Bt-Cotton is restricted south of
Interstate 60. There is currently no commercial cotton production in the
southern part of Florida. Evidence from germplasm collections indicates
that feral G. barbadense and possibly G. hirsutum exist in the U.S.
Virgin Islands. There is presently no production of commercial cotton in
either of these places; hence, outcrossing is not an issue. For a
detailed review of the Agency’s assessment of the potential for gene
capture and expression of Bt endotoxins by wild or weedy relatives of
cotton in the U.S., its possessions or territories, see the EPA
Biopesticides Registration Action Document (BRAD) for the Bacillus
thuringiensis (Bt) Plant-Incorporated Protectants, dated October 15,
2001.

7.  Impacts on Endangered Species

The primary route of exposure to Vip3Aa and FLCry1Ab proteins in cotton
is through ingestion of cotton tissue or pollen.  There are no reports
of threatened or endangered species feeding on cotton plants; therefore,
such species would not be exposed to cotton tissue containing these
proteins. Since Vip3Aa and FLCry1Ab proteins have not been shown to have
toxic effects on mammals, birds, plants, aquatic species, insects and
other invertebrate species at the Estimated Environmental Concentration
(EEC), a "may affect" situation for endangered land and aquatic species
is not anticipated. As previously noted, there is a possibility for gene
transfer in locations where wild or feral cotton relatives exist.  As a
result, EPA requires stringent sales and distribution restrictions on Bt
cotton within these areas to preclude outcrossing or hybridization from
the crop to sexually compatible relatives.  Therefore, EPA does not
expect that any threatened or endangered species will be affected by
outcrossing to wild relatives or by competition with such entities.

There are extensive data that demonstrate the lack of hazard of Cry1Ab
to non-Lepidoptera and the environmental safety of Bt11 corn (US EPA,
2001b).  Because of the selectivity of Vip3Aa and FLCry1Ab proteins for
lepidopteran species, endangered species concerns are mainly restricted
to the order Lepidoptera. Examination of an overlay map showing the
county level distribution of endangered/threatened lepidopteran species
(currently listed by the U.S. Fish and Wildlife Service) relative to
cotton production counties in the United States clearly indicated that
any potential concern regarding range overlap with cotton production was
mainly restricted to the Kern primrose sphinx moth (Euproserpinus
euterpe). However, cotton is not a host plant for this species nor do
host-range considerations place habitat in or near cotton fields.

Likewise, other insect species in the orders Diptera, Hemiptera,
Coleoptera,  Donata, and Orthoptera that are listed as
endangered/threatened species are found in dune, meadow/prairie or open
forest habitats and are not closely associated with row crop production,
often times due to the specificity of the habitat of their host plants.
Furthermore, the reviewed toxicological data shows the relative
insensitivity of a range of insects in non-lepidopteran orders to the
Vip3Aa and FLCry1Ab proteins, indicating that COT102 and COT67B cotton
plants are not likely to have detrimental effects on non-lepidopteran
insects included on the endangered/threatened species list.

In light of the above considerations (based on no spatial and temporal
overlap), the Agency has determined that registered uses of Event COT102
and Event COT67B cotton plants will have No Effect (NE), direct or
indirect, on endangered and threatened species or their habitat as
listed by the United States Fish and Wildlife Service (USFWS) and the
National Marine Fisheries Services (NMFS), including mammals, birds or
terrestrial and aquatic plants and invertebrate species. Therefore, no
consultation with the USFWS is required under the Endangered Species
Act.

II. C. 2.   Environmental Risk Assessment for Event COT102 and Event
COT67B

The EPA uses a Maximum Hazard Dose Tiered system for biopesticide
non-target wildlife hazard assessment. When no adverse effects at the
maximum hazard screening dose are observed, the Agency concludes that
there are no unreasonable adverse effects from the use of the pesticide.

A.  Direct effects

At present, the Agency is aware of no identified significant adverse
effects of Vip3Aa and/or FLCry1Ab proteins on the abundance of
non-target beneficial organisms in any population in the field
environment, whether they are pest parasites, pest predators, or
pollinators. Further, the EPA believes that cultivation of Event COT102
and/or Event COT67B cotton may have fewer adverse impacts on non-target
organisms than use of chemical pesticides for cotton production, because
under normal circumstances, COT102 and COT67B cotton requires
substantially fewer applications of chemical pesticides, compared to
production of non-Bt cotton. Fewer chemical insecticide applications
generally result in increased populations of beneficial organisms that
control secondary pests, such as aphids and leafhoppers. In addition, no
adverse effect on Federally-listed endangered and threatened species is
expected from the proposed lepidopteran-resistant cotton registration
(see part 7 in the preceding section above). Furthermore, the EPA has
determined that there is no significant risk of gene capture and
expression of Vip3Aa and FLCry1Ab protein by wild or weedy relatives of
cotton in the U.S., its possessions, or territories (see part 6 in the
preceding section above).  Available data do not indicate that Cry or
Vip proteins have any measurable adverse effect on microbial populations
in the soil (see part 4 in the preceding section above), nor has
horizontal transfer of genes from transgenic plants to soil bacteria
been demonstrated (see part 5 in the preceding section above). In
conclusion, this risk assessment finds no hazard to the environment at
the present time from cultivation of Event COT102 and Event COT67B
cotton in support for the Sec. 3 registration.

B.  Indirect effects:

The purpose of using PIP plants is the same as for any other pest
management tactic, i.e., to reduce pest populations below economic
injury levels. As a result, the abundance of pest insects should be
significantly reduced and this will have corresponding implications for
those organisms that exploit these pests as prey and hosts. Thus, the
potential for these indirect ecological effects on biological control
organisms should not be regarded as a unique ecological risk associated
with the PIP crop. Some reductions, however, should be expected if the
pest management strategy is effective. Since PIP crops are often grown
in vicinity with conventional crops to prevent resistance build-up by
the target pest(s), specialist antagonists can persist in these
‘refuges’, in other crops and in non-crop habitats and retain the
potential for recolonization of the PIP crop area. Based on these
considerations, regulatory testing of the specialist predators and
parasitoids of target pests may eventually be considered unnecessary.

II. C. 3.  Supplemental Data Needed to Confirm COT102 and COT67B
Non-Target Hazard Assessment

The Agency has sufficient information to believe that there is no risk
from the proposed uses of Event COT102 and Event COT67B cotton to
non-target wildlife, aquatic, and soil organisms. In previous Section 3
registrations of PIPs, the Agency required registrants to conduct
post-registration long term invertebrate population/community studies
and Cry protein accumulation in soils studies. However, the issue of
long range effects of cultivation of these Cry proteins on the
invertebrate community structure in corn and cotton fields has since
been adequately addressed by the meta-analysis of field studies
performed during the last 10 years (Marvier, et al. 2007; Sanvido, et
al. 2007).  No unexpected adverse effects on invertebrate community
structure were reported. The Agency is in agreement with these
conclusions Likewise, no unexpected accumulation of Cry or Vip proteins
in agricultural soils was seen in published studies (Icoz and Stotzky,
2007; Sanvido, et al. 2007) and in numerous studies submitted directly
to the EPA for the currently registered Cry proteins (Milofsky, 2006;
Part 3 in the preceding section above).

However, in light of recently published laboratory studies showing
reduced growth in shredding caddis flies exposed to anti-lepidopteran
Cry1A protein corn litter (Rosi-Marshall, et al. 2007), additional
aquatic invertebrate data are required. The submitted Daphnia magna
study is unacceptable because it is an 850 Series OPPTS Guideline study.
The 48 hour duration of this study is not sufficient to detect
mortality.  It takes more than 48 hours for the target pests to succumb
to Bt (-endotoxins, such as Cry or Vip proteins, therefore 48 hours is
also not expected to show mortality or reproductive effects on Daphnia.
A 7-14 day Daphnia study as per the OPPTS Series 885.4240 guideline must
be performed (see Tables 11 and 12) for Event COT102 and Event COT67B.
Alternatively, a dietary study of the effects on an aquatic
invertebrate, representing the functional group of a leaf shredder in
headwater streams, may be performed and submitted in lieu of the 7-14
day Daphnia study. These studies can be submitted as a condition of
registration.

Table 11.  Supplemental non-target data requirements for COT102
expressing Vip3Aa

Testing Category	Type of Data

Aquatic invertebrate 	A 7-14 day Daphnia study as per the OPPTS 885.4240
guideline has to be submitted as a condition of registration.
Alternatively, a dietary study of the effects on an aquatic
invertebrate, representing the functional group of a leaf shredder in
headwater streams, can be performed and submitted in lieu of the 7-14
day Daphnia study.

Table 12.  Supplemental non-target data requirements for COT67B
expressing FLCry1Ab

Testing Category	Type of Data

Aquatic invertebrate 	A 7-14 day Daphnia study as per the OPPTS 885.4240
guideline has to be submitted as a condition of registration.
Alternatively, a dietary study of the effects on an aquatic
invertebrate, representing the functional group of a leaf shredder in
headwater streams, can be performed and submitted in lieu of the 7-14
day Daphnia study.

II. C. 4.   Event COT102 X COT67B (VipCot) Environmental Risk Assessment

SUMMARY

differs by one amino acid substitution. FLCry1Ab is a δ-endotoxin
identical to a protein produced by B. thuringiensis subsp. kurstaki HD-1
except for an additional 26 amino acids at the C-terminal region. The
VipCot stack expresses both the Vip3Aa and FLCry1Ab proteins.

It was previously established by the Agency that the relative potency of
plant-produced Vip3Aa and full-length Cry1Ab proteins is similar to
their corresponding microbial-produced proteins, indicated that
plant-produced protein was similar in toxicity to the microbial-produced
protein (Matten, 2007 and Edelstein, 2008).  Each event also had
comparable  protein expression levels to the COT102 x COT67B breeding
stack (MRID No. 470176-07 and Edelstein, 2008).

Although the general symptomatology of Vip3Aa displayed by sensitive
lepidopteran larvae following ingestion of Bt (-endotoxins resembles
that of Cry proteins (Yu et al., 1997), Vip3Aa contains significantly
different receptor binding properties than the Cry proteins (Lee et al.,
2003).  Therefore, since the proteins have different modes of action,
the predicted effect of the mixture was calculated using a model called
independent joint action (Raybould, 2007; Colby, 1967). The observed and
expected mortalities were compared over a range of concentrations. Since
there is no test to identify statistical significance, the predicted
dose response curves were compared with the expected dose response
curves.  If there is greater mortality than expected over the range of
concentrations in a sensitive pest species, the hypothesis of synergism
is falsified and subsequently it is likely that there will be no
synergism of the mixture against non-target organisms.

Syngenta submitted additional data on the potential synergistic
interaction between Vip3Aa and FLCry1Ab proteins and are summarized in
this report to support the hypothesis of no synergism between the two
proteins. If no synergism is indicated, then development of new
non-target species data are not required because the reviewed non-target
data and the environmental risk assessments for the single indicated PIP
lines are applicable to the COT102 x COT67B cotton line. The results of
ecological effects studies submitted in support of the Section 3
full-commercial registration of Event COT012 and Event COT67B were
previously summarized in Tables 1 and 2, respectively, and presented in
a more descriptive format in previous sections of this risk assessment
document.

Synergism Studies

The purpose of these studies was to characterize the potential for
interaction between the lepidopteran-active proteins Vip3Aa and
FLCry1Ab.  The Vip3Aa and FLCry1Ab proteins were tested alone and in
combination against tobacco budworm (TBW, Heliothis virescens) and
cotton bollworm (CBW, Helicoverpa zea), respectively, in diet
incorporation studies.

MRID No. 470176-21

Four laboratory feeding bioassays were conducted to assess any
synergistic or antagonistic interactions between Vip3Aa and full-length
Cry1Ab proteins in a key lepidopteran pest, tobacco budworm (Heliothis
virescens). Five dilution series of the test materials were prepared in
buffer for each test: one series each of Vip3Aa and FLCry1Ab alone, and
three series of the two proteins mixed together in different ratios (up
to 1600 µg/mL Vip3Aa and 100 µg/mL FLCry1Ab together). The treatments
were applied to non-transgenic cotton leaves which were fed to H.
virescens larvae. Interaction between the two test materials was
assessed by comparing the larval mortality observed for the mixed
proteins with the predicted responses based on the bioassay of each
protein individually. The predicted responses were calculated based on
the assumption of “independent action” (Raybould, 2007) and there
was no evidence of either a synergistic or an antagonistic interaction
between Vip3Aa and FLCry1Ab in H. virescens, indicating that the effect
of a mixture of Vip3Aa and FLCry1Ab on non-target Lepidoptera can be
predicted from the effects of the individual proteins alone.

MRID No. 470176-22

Three laboratory feeding bioassays were conducted to assess any
synergistic or antagonistic interactions between Vip3Aa and full-length
Cry1Ab proteins in a key lepidopteran pest, cotton bollworm (Helicoverpa
zea). Five dilution series of the test materials were prepared in buffer
for each test: one series each of Vip3Aa and FLCry1Ab alone, and three
series of the two proteins mixed in different ratios (up to 25,600
ng/cm2 Vip3Aa and 12,800 ng/cm2 FLCry1Ab together). The test materials
were added to standard lepidopteran diet and fed to H. zea larvae.
Interaction between the two test materials was assessed by comparing the
larval mortality observed for the mixed proteins with the predicted
responses based on the bioassay of each protein individually. Since
previous evidence indicates that Vip3Aa and FLCry1Ab act at different
binding sites, the predicted responses were calculated based on the
assumption of “independent action” (Raybould, 2007). The results
were compared and there was no evidence of either a synergistic or an
antagonistic interaction between Vip3Aa and FLCry1Ab in H. zea,
indicating that the effect of a mixture of Vip3Aa and FLCry1Ab on
non-target Lepidoptera can be predicted from the effects of the
individual proteins alone.

Conclusions/Recommendations: The results of the interaction studies of
the combined proteins (Vip3Aa and FLCry1Ab) indicate that there is no
change in the level of activity among susceptible insects.  Collectively
these data provide evidence that Vip3Aa and FLCry1Ab proteins do not
interact in an antagonistic or synergistic manner. These studies, along
with the single-species, NTO toxicity testing and the Vip3Aa and Cry1Ab
protein long-term field studies, reviewed for the parental Event COT102
and Event COT67B, indicate its associated breeding stack, COT102 x
COT67B cotton, will not result in any unexpected interaction related to
an antagonistic or synergistic action to target and non-target insects.
Therefore, it is extremely unlikely that the Vip3Aa and FLCry1Ab
proteins contained in a single plant will impart any hazard to
non-target organisms exposed to these hybrids in the environment. In
conclusion, the Agency has determined that the environmental risk
assessment of Event COT102 expressing Vip3Aa protein and Event COT67B
expressing FLCry1Ab protein indicate there will be no unreasonable
adverse effects to the environment, including federally-listed
threatened and endangered species, by VipCot (COT102 x COT67B) cotton
hybird, crossed via traditional breeding.

CONCLUSION

The environmental risk assessment indicates for the VipCot cotton
breeding stack (COT102 x COT67B), based on prior assessments conducted
on Vip3Aa and Cry1Ab proteins individually, that no unreasonable harm
will result to the environment or any federally-listed threatened or
endangered species from commercial cultivation of COT102 x COT67B
cotton. Furthermore, the Agency has determined that Events COT102,
COT67B, and VipCot cotton will have No Effect (NE) on endangered and/or
threatened species listed by the US Fish and Wildlife Service (USFWS)
and the National Marine Fisheries Services (NMFS), including mammals,
birds, terrestrial and aquatic plants, and invertebrate species.
Therefore, no consultation with the USFWS is required under the
Endangered Species Act.

The Agency believes that cultivation of VipCot cotton may result in
fewer adverse impacts to non-target organisms than result from the use
of chemical pesticides. Under normal circumstances, Bt cotton requires
substantially fewer applications of chemical pesticides. This should
result in fewer adverse impacts to non-target organisms because
application of nonspecific conventional chemical pesticides is known to
have an adverse effect on non-target beneficial organisms found living
in the complex environment of an agricultural field. Many of these
beneficial organisms are important integrated pest management controls
(IPM) for secondary pests such as aphids and leafhoppers. Therefore, the
overall result of cultivation of VipCot cotton, expressing Vip3Aa and
FLCry1Ab proteins, is that the number of chemical insecticide
applications for non-target pest control will be reduced for management
of multiple pest problems.

II. C. 5.  References

Al-Deeb, et al. (2001). No effect of Bacillus thuringiensis corn and
Bacillus thuringiensis on

the predator Orius insidiosus. Environmental Entomology, v.30,
p.624-629.

Colby, S.R. (1967). Calculating synergistic and antagonistic responses
of herbicide

combinations. Weeds 15: 20-22.

DeMaagd, et al. (2003) Structure, diversity and evolution of protein
toxins from spore-forming entomopathogenic bacteria.  Annual Review of
Genetics 37:  409-433.

Dively, G.P. (2005).  Impact of transgenic VIP3A x Cry1Ab
lepidopteran-resistant field corn on the nontarget arthropod community.
Environmental Entomology 34, 1267-1291.  MRID 46784601

Duan J.J., Marvier M., Huesing J., Dively G., Huang Z.Y. (2008). A
Meta-Analysis of Effects of Bt Crops on Honey Bees (Hymenoptera:
Apidae). PLoS ONE 3(1):

e1415.doi:10.1371/journal.pone.0001415

Edelstein, R. (2008). Review of Human Health and Product
Characterization Data for Registration for B. thuringiensis Vip3Aa19 and
Cry1Ab Proteins and the Genetic Material Necessary for their Production
in Event COT102 x COT67B. U.S. Environmental Protection Agency.
Washington, D.C. Memorandum dated February 7, 2008.

Estruch, J.J., G.W. Warren, M.A. Mullins, G.J. Nye, J.A. Craig, and M.G.
Koziel. (1996). Vip3A, a novel Bacillus thuringiensis vegetative
insecticidal protein with a wide spectrum of activities against
lepidopteran insects. Proc. Natl. Acad. Sci., vol. 93: pp. 5389-5394.

Geiser, M. Schweizer, S. & Grimm, C. (1986). The hypervariable region in
the genes coding or entomopathic crystal proteins of Bacillus
thuringiensis: nucleotide sequence of the kurhd1 gene of subsp. kurstaki
HD1. Gene 48: 109-118.

Graser, et al. (2006).  Analysis of Vip3A or Vip3A-like Proteins in Six
Difference

Commercial Microbial Bacillus thuringiensis Products. Syngenta Seeds
Biotechnology

Report # SSB-036-6. Unpublished. MRID No. 457665-01.

Grimm, et al. (2000) “A test for evaluating the chronic effects of
plant protection products on

the rove beetle Aleochara bilineata (Coleoptera: Staphylinidae) under
laboratory and extended laboratory conditions,” In:  Candolfi, et al.
(2000). Guidelines to evaluate side-effects of plant protection products
to non-target arthropods. IOBC, BART and EPPO Joint Initiative, pp.
1-13. ISBN 92-9067-129-7.

Hanley ,et al. (2003) Effects of dietary transgenic Bt corn pollen on
larvae of Apis mellifera

and Galleria mellonella. Journal of Apicultural Research 42(4): 77-81.

Holm, L, J.V. Pancho, J.P. Herberger, and D.L. Plucknett. (1979). In: A
geographical atlas of world weeds. (pp. 391). John Wiley and Sons, New
York.

Hunter, M. and Z. Vaituzis. (2007). Environmental Risk Assessment for
Bt11 x MIR604 Maize (Stacked Hybrid Maize Containing Cry1Ab Protein and
Modified Cry3A Protein (mCry3A)). Biopesticides and Pollution Prevention
Division. U.S. Environmental Protection Agency. Washington, D.C.
Memorandum dated January 22, 2007.

Icoz, I, and G. Stotzky (2007). Cry3Bb1 protein from Bacillus
thuringiensis in root exudates and biomass of transgenic corn does not
persist in soil. Transgenic Research, September 13, 2007.

Lee, M.K., F.S. Walters, H. Hart, N. Palekar and J-S Chen. (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.

Martinez, J. (2008). Review of Insect Resistance Management (IRM) for
Sec 3 registration for VipCot™ (COT102 x COT67B).  U.S. Environmental
Protection Agency. Washington, D.C. Memorandum dated February11, 2008.

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.

Matten, S. and J. Kough (2007). Review of Product Characterization and
Human Health Data for PIP Bacillus thuringiensis (Bt) Insect Control
Proteins Full-length Cry1Ab and Vip3Aa19 and the Genetic Material
Necessary for their Production in Event COT67B, Event COT102, and COT67B
X COT102 Cotton in Support of the EUP. Biopesticides and Pollution
Prevention Division. U.S. Environmental Protection Agency. Washington,
D.C. Memorandum dated April 4, 2007.

Milofsky, T. and Z. Vaituzis (2006). Review the soil fate study
submitted in support of ABSTC’s Cry1Ab corn registrations.
Biopesticides and Pollution Prevention Division. U.S. Environmental
Protection Agency. Washington, D.C. Memorandum dated March 29, 2006.

Milofsky, T. and Z. Vaituzis (2007a). Environmental effects risk
assessment for Syngenta’s MIR162 Bt corn EUP. Biopesticides and
Pollution Prevention Division. U.S. Environmental Protection Agency.
Washington, D.C. Memorandum dated January 3, 2007.

Milofsky, T. and Z. Vaituzis (2007b). Environmental Risk Assessment for
Syngenta’s COT102 x COT67B Bacillus thuringiensis Cotton Experimental
Use Permit. U.S. Environmental Protection Agency. Washington, D.C.
Memorandum dated March 21, 2007.

Naranjo, S.E., Head, G. and Dively, G.P. (2005). Field Studies assessing
arthropod nontarget effects in Bt transgenic crops:  Introduction.
Environmental Entomology 34:  1178-1180.

National Academy of Science. (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 .

Oomen, et al. (1992). Method for honeybee brood feeding tests with
insect growth-

regulating insecticides. OEPP/EPPO Bulletin, 22:  613-616.

Pilcher et al. (1997a). Preimaginal development, survival, and field
abundance of insect predators on transgenic Bacillus thuringiensis corn.
Environmental Entomology 26(2): 446-454.

Pilcher, C. D., M. E. Rice, J. J. Obrycki & L. C. Lewis. (1997b). Field
and laboratory evaluations of transgenic Bacillus thuringiensis corn on
secondary Lepidopteran pests (Lepidoptera: Noctuidae). J. Econ. Entomol.
90: 669-678.

Pilcher et al. (2005) Impact of transgenic Bacillus thuringiensis corn
and crop phenology on five nontarget arthropods. Environmental
Entomology 34(5): 1302-1316.

Raybould, A. (2007) Environmental Risk Assessment of Genetically
Modified Crops:

General Principles and Risks to Non-target Organisms BioAssay 2:8, pg.
1-15

Rose R. and Z. Vaituzis. (2003). Review of non-target terrestrial
arthropod studies (lady

beetle, honey bee, Collembola & green lacewing) submitted by Syngenta
Seeds, Inc. to EPA for the registration of Bacillus thuringiensis VIP3A
protein expressed in cotton. U.S. Environmental Protection Agency.
Washington, D.C. Memorandum dated November 18, 2003.

Rose et al. (ed.) (2007). White Paper on Tier-Based Testing for the
Effects of Proteinaceous Insecticidal Plant-Incorporated Protectants on
Non-Target Arthropods for Regulatory Risk Assessments. U.S.
Environmental Protection Agency. Washington, D.C.

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

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

Rosi-Marshall E. J., J. L. Tank, T. V. Royer, M. R. Whiles, M.
Evans-White, C. Chambers, N. A. Griffiths, J. Pokelsek, and M. L.
Stephen. (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.

Schnepf, E., Crickmore, N., van Rie, J., Lereclus, D., Baum, J.,
Feitelson, J., Zeigler, D.R. and

Dean, D.H. (1998).  Bacillus thuringiensis and its pesticidal proteins.
Microbiology and Molecular Biology Reviews 62:  775-806.

Schur A., Tornier I., and Neumann C. (2000). Bt-Mais und non Bt-Mais:
vergleichende Untersuchungen an Honigbienen (Tunnelzeltversuch). 47th
annual meeting bee research, April 3-5, 2000. Blaubeuren bei Ulm,
Germany. Poster.

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

US EPA (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

US EPA (2001a). 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: http://www.epa.gov/scipoly/sap/2000/october/octoberfinal.pdf

US EPA (2001b). Biopesticides Registration Action Document for the
Bacillus thuringiensis (Bt) Plant-Incorporated Protectants, Dated
October 15, 2001.

US EPA (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. Web site:
HYPERLINK
"http://www.epa.gov/scipoly/sap/2002/august/august2002final.pdf"
http://www.epa.gov/scipoly/sap/2002/august/august2002final.pdf

US EPA (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 Web site:
http://www.epa.gov/scipoly/sap/meetings/2004/june/final1a.pdf

Vaituzis, Z. and R.Rose. (2000). Reassessment of Bt crop effects on
non-target wildlife. Biopesticides and Pollution Prevention Division.
U.S. Environmental Protection Agency. Washington, D.C.

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.

II. D. Insect Resistance Management

1.  Background

etermined that it involves a number of steps much like the mode of
actions for the δ-endotoxins. Following ingestion by the lepidopteran
target pest, the Vip3A protein becomes soluble in the gut and is then
processed into four dominant bands (retaining activity).  The authors
propose that this processing is required for the bioactivity of the
toxin (activation step).  Interaction with the midgut epithelium is the
next likely step in the mode of action of Vip3A.  However, Vip3A does
not bind to APN and cadherin-like glycoprotein receptors as does Cry1Ab
(as already stated by Schnepf et al. (1998)) and supported by the
researchers’ competition study.  Upon binding to midgut epithelial
receptors, data supports the existence of a pore-forming step that
creates ion channels which are structurally and functionally distinct
from those of Cry1Ab.  Direct structural information is missing for
Vip3A; however, preliminary data do not support the notion that the two
proteins share similar domain organization or an α-helical bundle
region.

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

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

A revised IRM plan (MRID 470176-34) was submitted by Syngenta to support
registration of VipCot in 2006 and has been reviewed by BPPD (see
Martinez 2008 a, b).  An additional e-mail correspondence clarifying
aspects of the dose evaluation was submitted by Syngenta (Reed 2008).
The details of resistance management plan as reviewed by BPPD are
described below.

2. Pest Biology

A clear understanding of pest biology and ecology is essential to the
development of a sound IRM plan.  The target pests of VipCot (TBW, CBW,
and PBW) have been well studied since Bt cotton was first introduced and
is available in the scientific literature.  A summary of the biology and
ecology for these insects can be found in the Agency’s 2001 Bt crop
reassessment document (EPA 2001).

3. Dose

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

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

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

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

To evaluate dose, Syngenta conducted a number of laboratory and field
studies with diet bioassays and COT67B, COT102, and VipCot cotton plant
material. Three sets of experiments were conducted: 1) bioassays with
the single proteins expressed in lyophilized plant material and both
proteins expressed in lyophilized plant material and combined as VipCot
to determine target pest susceptibility (TBW, CBW, PBW), 2) field tests
on VipCot and COT67B plants and control plants (artificial infestation)
during the 2005 and 2006 growing seasons, and 3) tolerance assays with
single proteins expressed in fresh plant material and both proteins
expressed in fresh plant material and combined as VipCot to determine
susceptibility of neonates as compared to fourth instar larvae (N.C.
State University lab colony).

Verification Methods:

1)  Bioassays (Syngenta’s submission, MRID 470176-34, Appendix 1)
using US EPA method #1 (serial dilution assays) were conducted by two
laboratories (Jealott’s Hill International Research Center and
Syngenta Biotechnology, Inc) using lyophilized tissue from terminal
leaves and bolls of COT102, COT67B, VipCot, and Coker 312 cotton.
Mortality data for H. zea, H. virescens, and P. gossypiella were
collected.

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

At 25X dilution, percent mortality on COT67B for TBW and PBW is reported
to be 100% and for CBW ranges from 98.3- 100%. The dilution bioassay for
VipCot was conducted at SBI only; their results suggest that at 25X
dilution, mortality on VipCot for TBW, CBW, and PBW is 100%. Based on
this verification method alone, VipCot provides an ‘effective’
high-dose against all three target pests, while COT102 provides no
high-dose to all three target pests and COT67B expresses a high-dose
against TBW and PBW and is nearly high dose for CBW.

2)  Unlike the artificial diet bioassay, the second method to verify
high-dose was conducted on VipCot and COT67B plants in the field (US EPA
method #4) using artificial infestations of laboratory strains of TBW
and CBW (MRID 470176-34, Appendix 2; additional clarification provided
in Reed 2008).  The purpose of this approach was to determine whether
the dose of toxins expressed in VipCot and COT67B was at or greater than
the LD99 for the key target pests. Specifically, TBW survival was
measured on both COT67B and VipCot, while CBW survival was measured on
VipCot cotton only.

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

In 2006, six VipCot cotton field trials were held at three locations in
the US (1,600 -2,000 plants each trial) and, like for COT67B, did not
get replicated. Experimental design, artificial infestation for TBW and
CBW, egg hatching, and larval survival estimation methods resembled
those of 2005.  The number of survivors of TBW and CBW (7DAI) were 32
and 21, respectively, at the Texas site and 14 and 5, respectively, at
the Louisiana site.  For Mississippi, no CBW and TBW survivors on VipCot
plants were reported.  In Tables 2 and 3 of Appendix 2 (MRID 470176-34),
BPPD notes that there is large difference in number of insects observed
in the control plots 7DAI (i.e. 363 TBW observed, TX) and 14DAI (i.e. 17
TBW observed, TX).  Based on such a decrease of survivors in the control
plots, BPPD concludes that other outside effects add to the natural
mortality when laboratory reared insects are exposed to cotton plants
(i.e. fed on artificial diets for many generations and no longer fit to
survive on natural host).  Thus, the low number of TBW survivors
reported from Bt-plants 7DAI is very likely confounded by this
additional ‘non-Bt exposure’ mortality. Similar results were
reported for CBW, and therefore, the same conclusion is extended to this
pest. Based on the discrepancy observed in survivorship on control
plants, BPPD concludes that the 2006 data alone are inconclusive.
However, given the results observed in 2005 for TBW, it can be concluded
that COT67B likely provides a high-dose for TBW under this method.

3)  The third verification method for high-dose was conducted as a
tolerance bioassay with purified toxins as well as leaf disks to
determine if a later larval instar of the targeted pest could be found
with an LD50 that was approximately 25-fold higher than that of the
neonate larvae (US EPA method #5) on VipCot, COT 102, and COT67B plants
for CBW and on COT102 for TBW (MRID 470176-34, Appendix 3). The SBI lab
conducted the purified toxin (with Vip3A and FLCry1Ab) and leaf disk
study (on COT102, COT67B, and VipCot) for CBW; JH lab conducted the
purified toxin (with Vip3A) and leaf disk study (on COT102) for TBW.

Leaf Disk Bioassays for CBW

While Syngenta reports 100% mortality for both neonates and older CBW
larvae for leave disk bioassays (method #5), Syngenta does not mention
that control mortality for neonates ranged from 0% to 81% for neonates.
While neonates may have only served as a reference, it is inappropriate
to list these results to support the conclusion that VipCot, as well as
its individual events, is highly effective against CBW. However, control
mortality for later instar larvae was much lower and ranged from 0% to
28% (only one control had very high mortality results). The results from
leaf disk bioassays show that later instar larvae of CBW are
experiencing 100% mortality when exposed to VipCot, COT102, and COT67B.

Leaf Disk Bioassays for TBW

Mortality for TBW later instar larvae was 100% when tested on COT102.
Control mortality results for later instar larvae were 14%, 15%, and
27%; treatment mortality for later instars was 100%. The results from
leave disk bioassays show that older larvae of TBW are experiencing 100%
mortality when exposed to COT102.

Tolerance Assays for CBW

Syngenta reports that the tolerance assay conducted with the purified
toxin Vip3A and tested on CBW did not allow estimating LC50 for later
instars because the estimates were well in excess of the highest
concentration tested. Syngenta concludes that later instars are at least
25X more tolerant to Vip3A than neonate larvae. BPPD recognizes that
there are several issues with the tolerance assay data reported: 1)
Mortality data for neonates are highly variable from bioassay to
bioassay, and LC50 estimates for CBW neonates range from 504.6ng/cm2 to
2669ng/cm2. 2) Mortality data for neonates within any test are not
steadily increasing with increasing toxin concentrations but show a
fluctuating trend and data gaps at some concentrations tested. Mortality
data for later instar larvae follow the same trend as described for
neonates under 1 and 2. 3) Where an LC50 could be estimated for both CBW
neonates and later instars, the difference between estimates is only 8
fold as opposed to the desirable 25-fold factor. The variability
reported in estimated LC50s for neonates and mortality data for neonates
and older instars are most likely due to CBW’s wide range in response
to Bt toxins. Overall, the tolerance assay data support that it is
difficult to achieve mortality in neonates and older instars of CBW when
exposed to Vip3A.

Two tolerance bioassays were conducted with FLCry1Ab on CBW (Tables 6
and 7, Appendix 3). In these tests, mortality data for later instars and
neonates were more consistently increasing with increasing
concentrations. Later instar LC50 estimates were greater than neonate
LC50 estimates by more than 25-fold; BPPD concludes that older CBW
larvae are >25-fold more tolerant than neonates when exposed to
FLCry1Ab.

Leaf Disk Assays for TBW

For the leaf disk assays, Syngenta reported 100% mortality for older TBW
larvae. Although control mortality for older larvae was somewhat higher
than desirable (ranging from 15%-27%), BPPD concludes that COT102
appears to have very good activity against TBW.

Tolerance Assays for TBW

Syngenta reports that the tolerance bioassays conducted with Vip3A on
TBW neonate and 2nd instar larvae show a 36-fold difference in
susceptibility/ LC50 estimates. BPPD would like to add that there were
two estimated LC50s for older larvae of which one showed a 21-fold and
the other 36-fold difference from neonate susceptibility. Overall, BPPD
concludes that older TBW larvae are approximately ≥25-fold more
tolerant than neonates when exposed to Vip3A alone.

4) Method #4 was conducted to verify high-dose in the field on COT67B
plants (and COT69D plants) under artificial infestation of laboratory
strains of PBW (MRID 470176-34, Appendix 4). The purpose of this
approach was to determine whether the dose of toxins expressed in COT67B
was at or greater than the LD99 for this key target pest.

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

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

Overall, BPPD’s conclusions for the VipCot dose trials are as follows:

COT67B expresses a probable high-dose against TBW (methods 1 and 4) and
CBW (methods 1 and 5).

VipCot expresses a probable high-dose against TBW (using method 1;
method 4 was considered inconclusive) and CBW (methods 1 and 5).

COT102 does not appear to express a high-dose against any of three