Document ID: EPA-HQ-OPP-2007-0306-0042
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2008-10-29T04:00Z

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY			WASHINGTON, D.C. 20460

                        OFFICE OF PREVENTION,                           
                                                                        
      

                        		   PESTICIDES AND TOXIC  

          		  		 SUBSTANCES  

MAY 15 2008

MEMORANDUM

SUBJECT:	Environmental Risk Assessment for Bacillus thuringiensis (Bt)
Vip3Aa insect control protein as expressed in Event COT102 cotton, Bt
full-length Cry1Ab insect control protein as expressed in Event COT67B,
and its associated breeding stack, COT102 x COT67B cotton [EPA Reg. No.
67979-O], MRID No: 470176 -20 through -32,  submitted by Syngenta Seeds,
Inc.- Field Corps- NAFTA

FROM:	Annabel Waggoner, Environmental Protection Specialist  [signed]

			Microbial Pesticides Branch

			Biopesticides and Pollution Prevention Division, 7511P

THROUGH:        	Zigfridas Vaituzis, Ph.D. Senior Scientist		        
[signed]	

	Microbial Pesticides Branch

		Biopesticides and Pollution Prevention Division, 7511P

TO:			Alan Reynolds, M.S. Regulatory Action Leader 

			Microbial Pesticides Branch

			Biopesticides and Pollution Prevention Division, 7511P

™ cotton hybrid.

CONCLUSION:

Syngenta is applying for Sec. 3 registration for transgenic cotton
[Gossypium hirsutum] plant lines: Event COT102 (which express Bt Vip3Aa
protein), Event COT67B (which expresses Bt FLCry1Ab protein), and COT102
x COT67B or VipCOT™, its associated breeding stack. At present, the
Agency has not identified any significant adverse effects of the Vip3Aa
and FLCry1Ab proteins on the abundance of non-target organisms (NTOs) in
any field population.  It is unlikely that direct or indirect harmful
effects to NTOs, including federally-listed threatened or endangered
species, would result from exposure to the insecticidal proteins Vip3Aa
in Event COT102 cotton, FLCry1Ab in Event COT67B cotton, and VipCot™
cotton hybrid, as a result of the proposed Sec. 3 registration. The
Agency anticipates that for full commercial cultivation, no hazard will
result to the environment.

BACKGROUND			

Vip3A is a novel class of recently discovered insecticidal proteins that
occur naturally in Bacillus thuringiensis (Bt), a gram-positive soil
bacterium (Estruch, et al. 1996).  The vegetative insecticidal proteins
are produced during vegetative bacterial growth and are secreted as
soluble proteins into the extracellular environment.  Syngenta Seeds,
Inc. has developed Event COT102, a cotton line that expresses Bt  insect
control protein, known as Vip3Aa.  In addition, Syngenta Seeds, Inc. has
also developed Event COT67B, a cotton line that expresses a modified Bt
insect control Cry1Ab protein, expressing an additional 26 amino acids
(hereafter, referred to as FLCry1Ab).

These proteins are intended to control several lepidopteran pests of
cotton including:  Helicoverpa zea (cotton bollworm), Heliothis
virescens (tobacco budworm), Spodoptera frugiperda (fall armyworm),
Spodoptera exigua (beet armyworm), and Trichoplusia ni (cabbage looper).

lso known as VipCot™, EPA Reg. No. 67979-O] cotton (which combines
Vip3Aa and FLCry1Ab proteins), crossed via traditional breeding.  An 
experimental use permit (EUP) was granted by the Agency to conduct field
tests on Event COT102, Event COT67B, and its associated breeding stack
COT102 x COT67B (Matten, 2007).  

Event COT102 cotton specifically expresses Vip3Aa19, a variant of the
naturally occurring Vip3Aa1 protein isolated from Bacillus thuringiensis
strain AB88, differing from the Vip3Aa1 protein by one amino acid.  The
same protein variant present in Event COT102 cotton is also expressed as
Vip3Aa19 in Syngenta’s experimental Event Pacha corn. The Agency
previously determined that “all proteins designated as Vip3Aa are more
than 95% identical,” and “there is sufficient information to support
the safety of all Vip3Aa proteins, provided that they do not have any
significant sequence similarity with known allergens” (Edelstein,
2008). Therefore, in addition to the data reviewed in this report, all
the previously submitted data developed for Vip3Aa protein can be cited
in support of the registration of Event COT102.

Although Vip3Aa protein shares no homology with FLCry1Ab or other known
Cry proteins, extensive testing by Syngenta has established that Vip3Aa
has demonstrated a similar toxicity against larvae of certain
lepidopteran species, including key pests of cotton.  While the modes of
action differ between the two proteins,  the general symptoms displayed
by sensitive lepidopteran larvae following ingestion of Vip proteins
resembles that caused by Cry proteins (i.e., cessation of feeding, loss
of gut peristalsis, overall paralysis of the insect, and death) (Yu, et
al, 1997).  Since the effects of Vip and Cry proteins are considered
similar, the studies submitted on non-target organisms for Event COT102
were conducted and evaluated according to the same environmental risk
assessment criteria of previously reviewed PIP products containing Cry
protein.

The modified Cry1Ab protein (or FLCry1Ab protein) expressed in COT67B
cotton and native Cry1Ab protein are both derived from Bacillus
thuringiensis subsp. kurstaki strain HD-1 (B.t.k.).  FLCry1Ab differs
from the naturally occurring Cry1Ab protein in that FLCry1Ab was
modified to contain 26 additional consecutive amino acids (described as
the ‘Geiser motif”) in the C-terminal portion (Geiser et al., 1986).
The ‘Geiser motif’ is also expressed in another registered PIP
cotton product containing Cry1Ac.  FLCry1Ab protein in Event COT67B is
also similar to the truncated protein variants of Cry1Ab as expressed in
transgenic maize.  The Agency previously determined that Syngenta’s
Event Bt11 corn produces a truncated Cry1Ab protein that has the same
insecticidal active region of amino acids as FLCry1Ab produced in COT67B
cotton (Matten, 2007). In addition, there are numerous laboratory
studies, field studies, and scientific literature on the mode of action
of Cry1Ab-expressing maize and Cry1Ac-expressing cotton (US EPA, 2001b;
Naranjo et al., 2005; Romeis et al., 2006; Cattaneo et al., 2006; and
Torres and Ruberson, 2007).  These data provide a large
weight-of-evidence that these proteins demonstrate very similar
insecticidal activity against several lepidopteran cotton pests at
concentrations found in transgenic plants. Furthermore, the Agency also
determined that field efficacy data submitted with the registration
application (MRID No. 470176-33) and reports provided with the Public
Interest Document (MRID No. 470176-35) demonstrate a similar
insecticidal spectrum of the truncated and full-length Cry1Ab proteins
(Martinez, 2008).  Therefore, the effects of truncated Cry1Ab proteins
are considered predictive of the effects of FLCry1Ab protein as
expressed in COT67B cotton to non-target organisms for the purposes of
the environmental risk assessment.  

The Agency has conducted an environmental risk assessment of the Vip3Aa
and FLCry1Ab proteins producing COT102 and COT67B cotton lines,
respectively.  The general topics covered include gene flow to related
wild plants, potential of weediness, effects on wildlife, and fate of
Vip3Aa and Cry1Ab proteins in the environment.  The assessment is based
on data submitted to the Agency during the development of the cotton
lines, additional data submitted for registration, Federal Insecticide
Fungicide and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP)
recommendations, consultations with scientific experts, and public
comments on Plant Incorporated Protectant (PIP) regulation.

A.	Environmental Risk Assessment for COT102 and COT67B (lepidopteran
active)

I.	Tiered Testing and Risk Assessment Process 

To minimize data requirements and avoid unnecessary tests, risk
assessments are structured such that risk is determined first from
estimates of hazard under “worst-case” exposure conditions.  A lack
of adverse effects under these conditions would provide enough
confidence that there is no risk and no further data would be needed. 
Hence, such screening tests conducted early in an investigation tend to
be broad in scope but relatively simple in design, and can be used to
demonstrate acceptable risk under most conceivable conditions.  When
screening studies suggest potentially unacceptable risk additional
studies are designed to assess risk under more realistic field exposure
conditions.  These later tests are more complex than earlier screening
studies. Use of this “tiered” testing framework saves valuable time
and resources by organizing the studies in a cohesive and coherent
manner and eliminating unnecessary lines of investigation.  Lower tier,
high dose screening studies also allow tighter control over experimental
variables and exposure conditions, resulting in a greater ability to
produce statistically reliable results at relatively low cost.  

Tiered tests are designed to first represent unrealistic worst case
scenarios and ONLY progress to real world field scenarios if the earlier
tiered tests fail to indicate adequate certainty of acceptable risk. 
Screening (Tier I) non-target organism hazard tests are conducted at
exposure concentrations several times higher than the highest
concentrations expected to occur under realistic field exposure
scenarios.  This has allowed an endpoint of 50% mortality to be used as
a trigger for additional higher-tier testing.  Less than 50% mortality
under these conditions of extreme exposure suggest that population
effects are likely to be negligible given realistic field exposure
scenarios. 

The EPA uses a tiered (Tiers I-IV) testing system to assess the toxicity
of a PIP to representative non-target organisms that could be exposed to
the toxin in the field environment. Tier I high dose studies reflect a
screening approach to testing designed to maximize any toxic effects of
the test substance on the test (non-target) organism.  The screening
tests evaluate single species in a laboratory setting with mortality as
the end point.  Tiers II – IV generally encompass definitive hazard
level determinations, longer term greenhouse or field testing, and are
implemented when unacceptable effects are seen at the Tier I screening
level.

Testing methods which utilize the tiered approach were last published by
the EPA as Harmonized OPPTS Testing Guidelines, Series 850 and 885 (EPA
712-C-96-280, February 1996). These guidelines, as defined in 40 CFR
152.20, apply to microbes and microbial toxins when used as pesticides,
including those that are naturally occurring, and those that are
strain-improved, either by natural selection or by deliberate genetic
manipulation.  Therefore, PIPs containing microbial toxins are also
covered by these testing guidelines. 

The Tier I screening maximum hazard dose (MHD) approach to environmental
hazard assessment is based on some factor (whenever possible >10) times
the maximum amount of active ingredient expected to be available to
terrestrial and aquatic non-target organisms in the environment (EEC).
Tier I tests serve to identify potential hazards and are conducted in
the laboratory at high dose levels which increase the statistical power
to test the hypotheses.  Elevated test doses, therefore, add certainty
to the assessment, and such tests can be well standardized. The
Guidelines call for initial screening testing of a single group or
several groups of test animals at the maximum hazard dose level. The
Guidelines call for testing of one treatment group of at least 30
animals or three groups of 10 test animals at the screening test
concentration. The Guidelines further state that the duration of all
Tier I tests should be approximately 30 days. Some test species, notably
non-target insects, may be difficult to culture and the suggested test
duration has been adjusted accordingly. Control and treated insects
should be observed for at least 30 days, or in cases where an insect
species cannot be cultured for 30 days, until negative control mortality
rises above 20 percent. 

Failing the Tier I (10 X EEC) screening at the MHD dose does not
necessarily indicate the presence of an unacceptable risk in the field
but it triggers the need for additional testing. A less than 50%
mortality effect at the MHD is taken to indicate minimal risk.  However,
greater than 50% mortality does not necessarily indicate the existence
of unacceptable risk in the field, but it does trigger the need to
collect additional dose-response information and a refinement of the
exposure estimation before deciding if the risk is acceptable or
unacceptable. Where potential hazards are detected in Tier I testing
(i.e. mortality is greater than  50%), additional information at lower
test doses is required which can serve to confirm whether any effect
might still be detected at more realistic field [1X EEC] concentrations
and routes of exposure.  

When screening tests indicate a need for additional data, the OPPTS
Harmonized Guidelines call for testing at incrementally lower doses in
order to establish a definitive LD50 and to quantify the hazard.  In the
definitive testing, the number of doses and test organisms evaluated
must be sufficient to determine an LD50 value and, when necessary, the
Lowest Observed Effect Concentration (LOEC), No Observed Adverse Effect
Level (NOAEL) , or reproductive and behavioral effects such as feeding
inhibition,  weight loss, etc.  In the final analysis, a risk assessment
is made by comparing the LOAEC to the EEC; when the EEC is lower than
the LOAEC, a no risk conclusion is made. These tests offer greater
environmental realism, but they may have lower statistical power.
Appropriate statistical methods, and appropriate statistical power, must
be employed to evaluate the data from the definitive tests. Higher
levels of replication, the number of test species, and/or repetition are
needed to enhance statistical power in these circumstances. 

Data that shows less than 50 % mortality at the maximum hazard dosage
level – (i.e. LC50, ED50, or LD50 >10 X EEC) is sufficient to evaluate
adverse effects, making lower field exposure dose definitive testing
unnecessary.   It is also notable that the recommended >10X EEC maximum
hazard dose level is a highly conservative factor.  The published EPA
Level of Concern [LOC] is 50% mortality at 5X EEC  (US EPA, 1998).  

Validation:  The tiered hazard assessment approach was developed for the
EPA by the American Institute of Biological Sciences (AIBS) and
confirmed in 1996 as an acceptable method of environmental hazard
assessment by a FIFRA Scientific Advisory Panel (SAP) on microbial
pesticides and microbial toxins. The December 9, 1999 SAP agreed that
the Tiered approach was suitable for use with Plant-Incorporated
Protectants (PIPs); however, this panel recommended that, for PIPs with
insecticidal properties, additional testing of beneficial invertebrates
closely related to target species and/or likely to be present in GM crop
fields should be conducted. Testing of Bt Cry proteins on species not
closely related to the target insect pest was not recommended, although
it is still performed to fulfill the published EPA non-target species
data requirements.  In October 2000, another SAP also recommended that
field testing should be used to evaluate population-level effects on
non-target organisms. The August 2002 SAP, and some public comments,
generally agreed with this approach, with the additional recommendation
that indicator organisms should be selected on the basis of potential
for field exposure to the subject protein (US EPA, 2000, 2001a, 2002,
and 2004). 

Chronic studies: Since delayed adverse effects and/or accumulation of
toxins through the food chain are not expected to result from exposure
to proteins, protein toxins are not routinely tested for chronic effects
on non-target organisms.  However, the 30 day test duration requirement
does amount to subchronic testing when performed at field exposure test
doses. Proteins do not bioaccumulate. The biological nature of proteins
makes them readily susceptible to metabolic, microbial, and abiotic
degradation once they are ingested or excreted into the environment. 
Although there are reports that some proteins (Cry proteins) bind to
soil particles, it has also been shown that these proteins are degraded
rapidly by soil microbial flora upon elution from soil particles.  

Conclusion: The tiered approach to test guidelines ensures, to the
greatest extent possible, that the Agency requires the minimum amount of
data needed to make scientifically sound regulatory decisions. The EPA
believes that maximum hazard dose Tier I screening testing presents a
reasonable approach for evaluating hazards related to the use of
biological pesticides and for identifying negative results with a high
degree of confidence. The Agency expects that Tier 1 testing for
short-term hazard assessment will be sufficient for most studies
submitted in support of PIP registrations. However, if long range
adverse effects must be ascertained, then higher-tier longer-term field
testing will be required.    As noted above, the October 2000 SAP and
the National Academy of Sciences (NAS, 2000) recommended testing
non-target organisms directly in the field. This approach, with an
emphasis on testing invertebrates found in corn fields, was also
recommended by the August 2002 SAP and was supported by several public
comments. Based on these recommendations, the Agency has required field
studies on long term invertebrate population/community and Cry protein
accumulation in soils as a condition of registration due to the lack of
baseline data on the potential for long-term environmental effects from
the cultivation of PIP-producing plants.

Since the commercialization of Bt crops, the number of field studies
published in scientific literature in combination with the
post-registration field studies submitted to the Agency has accumulated
to a level where empirical conclusions can be made.  As a result, the
issue of long range effects of cultivation of these Cry proteins on the
invertebrate community structure in Bt crop fields has since been
adequately addressed.  Specifically, a meta-analysis of the data
collected from 42 field studies indicated that non-target invertebrates
are generally more abundant in Bt cotton and Bt maize fields than in
non-transgenic fields managed with insecticides (Marvier, et al., 2007).
 In addition, a comprehensive review of short and long term field
studies on the effects of invertebrate populations in Bt corn and cotton
fields indicated that no unreasonable adverse effects are taking place
as a result of wide scale Bt crop cultivation (Sanvido, et al. 2007). 
Another review of field tests published to date concluded that the
large-scale studies in commercial Bt cotton have not revealed any
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.   

    

	

II.	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.

III.   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 1 and 2, 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 1.  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 10.1 µg VIP3A protein/L) had no adverse effects on
the survival of Daphnia magna, when suspended in 120 mg pollen/L.  The
LC50 was > 10.1 µg VIP3A protein/L.

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 2.  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

 	Bt11 maize pollen (PHO176-0194)	In a 48-hour static renewal limit
bioassay, Cry1Ab maize pollen containing 10.1 µg Cry1Ab protein/L had
no adverse effects on the survival of Daphnia  magna, when suspended in
120 mg pollen/L.  The LC50  was >  10.1 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 COT102 x COT67B cotton
hybrid 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 hybrid,
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 three 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 risk assessment.

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 COT102 X COT67B
hybrid, 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 COT67B at 7X EEC and COT102 x COT67B cotton hybrid 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 as a representative test species 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 for
terrestrial organisms 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 COT102 x COT67B cotton 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 Bt11 x Event Pacha
maize hybrid (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, COT102 x COT67B cotton hybrid. 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, the EPA has
concluded that there is a low probability of risk from HGT of transgenes
found in 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.

B.	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.

1. 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 Section B.III.7 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 Section B.III.6 above).  Available
data do not indicate that Cry or Vip proteins have any measurable
adverse effect on microbial populations in the soil (see Section B.III.4
above), nor has horizontal transfer of genes from transgenic plants to
soil bacteria been demonstrated (see Section B.III.5 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.  

2.  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.  

C.	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;
Section B.III.3 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 3 and 4) 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 3.  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 4.  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. 

ot™) Cotton Hybrid 

SUMMARY 

Syngenta Seeds, Inc. developed COT102 x COT67B cotton (VipCot™) by
conventional breeding of transgenic event COT102 cotton and transgenic
event COT67B cotton, which express the Vip3Aa and FLCry1Ab insecticidal
proteins, respectively, for control of certain lepidopteran pests.
Vip3Aa is a protein variant of Vip3Aa1 (originally identified in
Bacillus thuringiensis strain AB88), from which it 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™ cotton
hybrid 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 hybrid
(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 hybrid. 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 hybrid, 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
hybrid, crossed via traditional breeding.

CONCLUSION

Cot™ 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. 

E.	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
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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. EPA, Washington, D.C. Memorandum dated
Feb. 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 

	for 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. EPA. 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 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. EPA.
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
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Washington, DC 20055; (800) 624-6242 or (202) 334-3313 (in the
Washington metropolitan area);   HYPERLINK "http://www.nap.edu" 
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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
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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.
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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.
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Romeis, J., Meissle, M. and Bigler, F. (2006).  Transgenic Crops
expressing Bacillus 

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

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Stephen. (2007). Toxins in transgenic crop byproducts may affect
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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.

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released from root exudates and biomass of Bt corn has no apparent
effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil.
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HYPERLINK
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health risk, 

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

Vaituzis, Z. and R.Rose. (2000). Reassessment of Bt crop effects on
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Yu, C. Mullins, M.A., Warren, G.W., Koziel, M.G. and Estruch, J.J.
(1997).  The Bacillus

 

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!and Environmental Microbiology 63 (2): 532-53.

 Prior to receiving the Crickmore designation of Vip3Aa19, the protein
produced in Events COT102 and Pacha were referred to as VIP3A, Vip3A or
Vip3Aa.

 Non-target invertebrate hazard tests often are conducted at exposure
concentrations several times higher than the maximum concentrations
expected to occur under realistic exposure scenarios.  This has
customarily allowed an endpoint of 50% mortality to be used as a trigger
for additional higher-tier testing.  Lower levels of mortality under
these conditions of extreme exposure suggest that population effects are
likely to be negligible given realistic exposure scenarios.  Thus, it
follows that the observed proportion of responding individuals can be
compared to a 50% effect to determine if the observed proportion is
significantly lower than 50%.  For example, using a binomial approach, a
sample size of 30 individuals is sufficient to allow a treatment effect
of 30% to be differentiated from a 50% effect with 95% confidence using
a one-sided Z test.  A one-sided test is appropriate because only
effects of less than 50% indicate that further experiments are not
needed to evaluate risk.  

 OPPTS Testing Guidelines, Series 850 and 885 website: 

  HYPERLINK "http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/"
 http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/
885Microbial_Pesticide_Test_Guidelines/Series

 The 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 or other factors. High dose
testing also may not be necessary where many species are tested or tests
are very sensitive, although the test concentration used must exceed 1X
EEC.

 It is notable that that the 10 X EEC MHD testing approach is not
equivalent to what is commonly known as “testing at a 10X SAFETY
FACTOR” where any adverse effect is considered significant. Tier I
screen testing is not ‘safety factor testing’.  In a “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.   

 The 1X EEC test dose is based on plant tissue content and is considered
a high worst case dose (sometimes referred to as HEEC). This 1X  EEC is
still much greater than any amount which any given non-target organism
may be ingesting in the field because most non-target organisms do not
ingest plant tissue.

 The established peer and EPA Science Board reviewed guidance on
screening test levels of concern is 50% mortality at 5X environmental
concentration. The appropriate endpoints in high dose limit/screening
testing are based on mortality of the treated, as compared to the
untreated (control) non-target organisms. A single group of 30 test
animals may be tested at the maximum hazard dose.

 This research was funded by Environmental Protection Agency grant
CR-832147-01.  The Bt crop non-target effects database can be found on
the National Center for Ecological Analysis and Synthesis (NCEAS).
Website. (  HYPERLINK "http://delphi.nceas.ucsb.edu/btcrops/" 
http://delphi.nceas.ucsb.edu/btcrops/ ).

 Model hypothesis:  if a certain amount of protein A alone kills x% of a
sample, and a certain amount of protein B kills y%, the predicted
percentage kill of a mixture of these amounts of protein is given by x +
y – (xy/100).  

 Bridging of data between the variants of Vip3Aa as well as the Cry1Ab
proteins was addressed in the Agency’ reviews of the VipCot™
Experimental Use Permit (see memoranda:  Matten, 2006; Milofsky and
Vaituzis, 2007b).   

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