The toxicity of Cry1F and Cry1Ac proteins has been previously evaluated on several species of invertebrates including: lady beetle (Hippodamia convergens), green lacewing (Chrysoperla rufilabris), honey bee (Apis mellifera), collembola (Folsomia candida), parasitic wasp (Nasonia vitripennis) and earthworm (Eisenia fetida). Two new dietary toxicity studies were submitted in support for event DAS-81419-2 soybean, which includes Northern Bobwhite (Colinus viginianus) and Rainbow Trout, (Oncorhynchus Mykiss). The individual results for the cited and newly submitted nontarget organism studies in support for Cry1F and Cry1Ac are summarized in Table 1. The studies are described in more detail below, and full reviews of each study can be found in the individual Data Evaluation Records.

To support data bridging from the previously conducted environmental risk assessment for Cry1F and Cry1Ac insecticidal proteins co-expressed in WideStrike PIP cotton line, an environmental risk assessment was conducted using a "weight-of-evidence" approach based on the following lines of evidence to support the registration of event DAS-81419-2 soybean:

     1) Confirmation of biological equivalency of Cry1F and Cry1Ac proteins expressed in event DAS-81419-2 soybean plant and previously characterized microbial-derived Cry1F and Cry1Ac protein test substances;
     2) No significant increase in exposure to NTOs based on calculated margins of exposure for protein concentrations in event DAS-81419-2 plant tissue matrices relative to the dose concentrations used in the NTO laboratory toxicity tests conducted on Cry1Ac and Cry1F; and
     3) No adverse effects on nontarget arthropods on a population and community level observed from a field monitoring study and other supporting supplemental information (e.g. meta-analysis, target feeding studies, and biological specificity to the target pest).

 Exposure estimates were compared with endpoints reported from Tier I toxicity studies previously conducted on various representative nontarget test species to determine a Margin of Exposure (MOE), the ratio of the endpoint value to the EEC. The MOEs for representative NTOs were recalculated based on high-end exposure estimates (HEEEs) (US EPA, 2009) derived from Cry1Ac and Cry1F protein expression levels quantified from various plant tissues of event DAS-81419-2 soybean in a field study. A MOE of 50% mortality at 10X the estimated environmental exposure is regarded as sufficient to demonstrate negligible risk and was also the threshold for consideration of uncertainties in the risk assessment (US EPA 1998; US EPA 2007). If subsequent expression characterization in the commercial event leads to a safety margin of <10X, then the consequences are considered in terms of uncertainties in the ecological risk assessment below (US EPA 2007).

Table B.1. Summary of Environmental Effects Studies conducted for PIP Event DAS-81419-2 soybean (EPA Reg. No. 68467-EN) and Scientific Rationales in support for bridging Cry1F and Cry1Ac data from the Agency database on WideStrike(R) PIP cotton (EPA Reg. No. 68467-3)

                                OCSPP Guideline
                                  Study Type
                     SUMMARY OF RESULTS AND CLASSIFICATION
                                   MRID No.
              WideStrike(R) PIP cotton data (EPA Reg. No. 68467-3)
885.4150
Wild Mammal Testing, Tier I
Not required; Mammalian wildlife exposure to Cry1F and Cry1Ac proteins is considered likely; however, the results from two acute oral toxicity studies indicated no adverse effects on mice from microbial-derived Cry1F and Cry1Ac proteins administered via oral gavage at the maximum hazard dose. The NOAEL exceeded 600 mg/kg body weight for Cry1F (MRID No. 45542312) and 700 mg/kg bw for Cry1Ac (MRID No. 45542313). These data are sufficiently representative of toxicity that would be expected in wild mammals, and based on the results of this study, risk to wild mammals resulting from exposure to Cry1F and Cry1Ac proteins is not expected.

Classification:  ACCEPTABLE for data bridging
                                   45542312
                                      and
                                   45542313
850.1010
Aquatic Invertebrate Acute Toxicity Test,
Daphnia magna
In a 48-hour static test with freshwater daphnids (Daphnia magna) there were no observed adverse effects with Cry1F and Cry1Ac in combination at respective concentrations of 510 and 2,500 μg/L. No immobility or other adverse effects were seen during the study. The 24-hour and 48hour EC50s for daphnia exposed to the Cry1F + Cry1Ac mixture were >510 μg Cry1F/L and >2500 μg Cry1Ac/L (represents a worst-case exposure of one kg of transgenic cotton pollen per liter of pond water). This rate of fortification represents 298X and >23,000X the anticipated EEC for Cry1F and Cry1Ac protein in surface water. Therefore, no hazard to aquatic invertebrates is expected from incidental exposure to WideStrike cotton pollen.
Classification:   ACCEPTABLE for WideStrike Cotton; SUPPLEMENTAL for DAS-81419-2 soybean.
                                   45808412
885.4280
Estuarine and Marine Animal testing, Tier I
Not required. Exposure in marine and/or estuarine environments is not expected to be significant.
                                      N/A
885.4300
Nontarget Plant Studies, Tier I
Not required. Bt and its proteins are not known plant pathogens or toxins, and adverse effects in plants are not anticipated. Exposure to nontarget plants is expected to be minimal.
                                      N/A
885.4380
Honey Bee Larva Testing, Tier I
(Apis mellifera)
At 1.98 μg Cry1F + 11.94 μg Cry1Ac per mL sugar water no effect on survival of larvae to adult emergence was seen. The  LC50 is >4X pollen expression. Therefore no hazard to honey bee larvae and adult bee emergence is anticipated.

Classification:  ACCEPTABLE for data bridging
                                   45542316
885.4340
Nontarget Insect Testing, Tier I
(Nasonia vitripennis)
At 5.2 μg Cry1F + 46.8 μg Cry1Ac per mL sugar water at 10 days, no effect of limit dose with LC50 > 13X pollen expression was seen on parasitic hymenoptera larva. Minimal exposure and no hazard to parasitic Hymenoptera from Cry1F and Cry1Ac proteins are expected. Testing of a species more common to cotton fields is recommended.

Classification:  ACCEPTABLE for data bridging
                                   45808411
885.4340
Nontarget Insect Testing, Tier I
(Chryosperla camea)
A dietary toxicity study on green lacewing (Chrysoperla rufilabris) larvae was conducted by presenting the green lacewings with a moth egg (Sitotroga sp.) diet incorporated with Bt Cry1F and Cry1Ac proteins. The dietary LC50 was  >48.2 ug/g diet for Cry1Ac and >5.2 ug/g diet for Cry1F, which represented at least at 14X the concentration found in pollen. No effect was  noted at the Cry1F and Cry1Ac protein levels expressed in pollen that would be encountered by green lacewings in the field. Because of questionable ingestion of the test material another species (e.g, minute pirate bug), which is more likely to be exposed, should be tested.

Classification:  ACCEPTABLE for data bridging
                                   45808410
885.4340
Adult Lady Beetle Testing, Tier I
(Hippodamia convergens)
At 300 μg Cry1F + 22.5 μg Cry1Ac per mL sugar water, no effect of limit dose with LC50> 780X Cry1F pollen expression and > 8X Cry1Ac pollen expression on adult lady beetles. Based on these results, no hazard to H. convergens is expected when feeding on WideStrike(R) cotton pollen in the field.

Classification:  ACCEPTABLE for data bridging
                                   45542315
885.4340
Collembola Chronic Dietary Toxicity Study, Tier I
(Folsomia candida)
The combination of 709 μg Cry1F + 22.6 μg Cry1Ac per g diet and cotton leaf tissue showed no effect on adult survival and reproduction at up to 10X the anticipated field level of expression. Therefore, no hazard to decomposers represented by collembola is expected from exposure to WideStrike cotton in the field.

Classification:  ACCEPTABLE for data bridging
                                   45808409
OECD Guideline 207
Earthworm Toxicity Study, (Eisenia foetida)
A 14-day study on earthworms exposed to soils treated with microbial-produced Cry1Ac and Cry1F, individually and in combination, was performed. The 14-day LC50s were >247 mg a.i./kg for Cry1F; >107 mg a.i./kg for Cry1Ac, and > 247 mg a.i./kg Cry1F + >107 mg a.i./kg Cry1Ac in the test with the two proteins combined. There were no overt signs of toxicity to earthworms exposed to soils containing nominal concentrations of Cry1F and Cry1Ac at 50X the expected worst case EEC [this represents concentrations which are 792X and 5479X higher than the expected EEC for incorporation of defoliated cotton plants into the top 15 cm of soil].

Classification:  SUPPLEMENTAL
                                   45580701
885.5200
Expression in a terrestrial Environment Tier II
The soil half-life of the plant expressing Cry1F and Cry1Ac was estimated as 1.3 days in a laboratory study with a representative soil from a cotton growing region. The Cry proteins were not detectable after 14 days. These results verify that the Cry1F and Cry1Ac proteins degrade rapidly in cotton soil.

Classification:  ACCEPTABLE for WideStrike Cotton; SUPPLEMENTAL for DAS-81419-2 Soybean.
                                   45556801
               DAS-81419-2 Soybean data (EPA Reg. No. 68467-EN)
885.4050
Avian Dietary Toxicity Study, Tier I
(Colinus viginianus)
In a 14-day acute dietary toxicity study, Northern bobwhites (Colinus virginianus)  were exposed to avian diet fortified with a 7.9:1.0  mixture of Cry1F and Cry1Ac proteins  with a test substance concentration of 128 mg /kg body weight (24.52 mg a.i./kg body weight based on percent a.i.) by oral gavage. Results showed no apparent effects on mortality, sublethal observations, body weight, and food consumption at this treatment level. The 14 day acute oral LD50 for the test substance was > 128 mg /kg bw (24.52 mg a.i./kg body weight based on percent a.i.) and the 14 day NOEL for the test substance was > 128 mg /kg bw  (24.52 mg a.i./kg body weight based on percent a.i.). Monogastric animals like birds cannot safely consume raw soybean seed since it contains endogenous antinutrients (Friedman et al., 1991). The estimated feed consumption per bird was 33 grams/day. Therefore, in relation to the daily diet, Cry1F was dosed at 0.660 mg AI/gm (660 ppm) and Cry1Ac was dosed at 0.083 mg AI/gm (83 ppm). The HEEE concentration of Cry1F in raw DAS-81419-2 soybean seed is 14.34 ng/mg dw (ppm) and 1.08 ng/mg dw (ppm) for Cry1Ac (MRID No. 48828413). The MOEs was calculated for Cry1F and Cry1Ac in the quail study relative to raw soybean seed consumed as 100% of the diet was 46X (660 ppm/14.34 ppm) and 77X (83 ppm/1.08 ppm), respectively, which provides a large safety margin even if bird could safely ingest raw soybean as their sole source of food.
Thus, no adverse effects on avian wildlife are expected from incidental field exposure to DAS-81419-2 soybean expressing Bt Cry1F and Cry1Ac insecticidal proteins.
Classification:  ACCEPTABLE
                                   48828410
885.4200
Freshwater Fish Testing, Tier I
 (Onchorynchus mykiss)

An acute oral dietary toxicity study on rainbow trout (Onchorynchus mykiss) was conducted to assess the effects of a basal fish diet fortified with a mixture of microbial-derived Cry1F and Cry1Ac proteins for eight days. No fish mortality and no sublethal effects were observed in any of the control or test treatment organisms during this study.  Results show the 8-day LD50 value with rainbow trout was > 100 mg a.i./kg diet wet weight (>200 mg a.i./kg diet dry weight).  The NOEC was reported as > 100 mg a.i./kg diet weight (>200 mg a.i./kg diet dry weight) based on the lack of mortalities, sublethal effects, and apparent lack of effects on standard length and body weight. In addition, exposure to freshwater fish in aquatic environments is also considered to be limited . In view of the lack of toxicity and minimal aquatic exposure, no adverse effects to freshwater fish as a result of the cultivation of DAS-81419-2 soybeans are expected.

Classification:  ACCEPTABLE
                                   48828411
Non-guideline
Nontarget Arthropod Census Field Study
A field study was conducted to monitor nontarget arthropods in fields of DAS-81419-2 soybean in comparison to non-transgenic fields for any statistically significant differences in the types of taxa collected and their abundance for determining any adverse effects on a community level to nontarget arthropods. DAS-81419-2 soybean, a near-isogenic non-transgenic control (Maverick) and four non-transgenic reference lines (DSR 3590; IL 3505; Porter 75148; Williams 82) were monitored in replicated field trials at two locations in the USA, Richland, Iowa and York, Nebraska.  Each trial site included the six soybean lines arranged in a randomized complete block design with four replicate blocks.  Three sampling methods were used to monitor arthropod abundance:  1) pitfall trapping, 2) sticky card trapping and 3) vertical beat sheet sampling. A total of 74 taxonomic groupings were detected and assessed across field sites. To determine biological relevance of any statistical differences, results were interpreted in the context of the magnitude of differences, observations in reference plots, and trend consistency across sampling methods, sampling periods, and locations. Analysis of data found no statistically significant differences of biological relevance in the nontarget arthropod populations present in fields planted with DAS-81419-2 soybean and the non-transgenic control soybean lines.

Classification:  SUPPLEMENTAL
                                   48828412
Non-guideline
 Environmental Risk Assessment Summary on Nontarget Arthropods
An environmental risk assessment was separately prepared by the registrant to summarize the potential for adverse effects of cultivating PIP event DAS-81419-2 soybean on nontarget arthropods associated with soybean agroecosystems with a special emphasis on beneficial taxa and threatened or endangered species. Dow requested data bridging to the existing ecotoxicological studies on the effects of Bt Cry1F and Cry1Ac proteins expressed in WideStrike(R) PIP cotton on representative nontarget insect species, including beneficials, pollinators, detritivores, and incidentals. Exposure estimates for Cry1F and Cry1Ac protein concentrations in PIP event DAS-81419-2 soybean were compared with endpoints reported from toxicity studies conducted for WideStrike cotton on select test species to determine the MOE, the ratio of the endpoint value to the EEC. Most MOEs exceeded 10X the estimated environmental concentrations.  None of the estimated exposure values exceeded NOECs for any of the tested species, indicating that the most sensitive individual in a population is not likely to be adversely affected.
Other supplemental information (i.e. meta-analyses, biological specificity to the pest species, targeted toxicity feeding studies), the biology of the crop and lack of exposure (due to feeding habits/food preferences) support the conclusion of no adverse effects to NTO species from PIP event DAS-81419-2 soybean. Other supporting data includes environmental fate data of the Cry1Ac and Cry1F proteins, results from a field survey showing no effects on the populations of nontarget arthropods associated with PIP event DAS-81419-2, and supplemental information from the FIFRA Endangered Species Task Force databases for identifying threatened and endangered species that may be exposed to PIP event DAS-81419-2 soybean.

Classification:  SUPPLEMENTAL A soil fate study is needed to demonstrate rapid protein degradation of Cry1F and Cry1Ac in a variety of soil types that are common in soybean cultivation as confirmatory data.
                                   48828413
Non-guideline
Supporting document
A response to EPA Comments on Cry1F(synpro) ICP and Cry1Ac(synpro) insecticidal crystal protein was submitted for the
acute oral toxicity study with the northern bobwhite (MRID No. 488284-10).  Protein identity and analytical assays confirmed the equivalence of and their respective certificates of analysis were submitted to demonstrate the equivalence of Dow's test material #101811 to plant-produced protein and provide a justification of the test dosage.

Classification:  SUPPLEMENTAL to MRID No. 48828410
                                   49044807
Non-guideline
Supporting document
A response to EPA Comments on Cry1F(synpro) and Cry1Ac(synpro) insecticidal crystal proteins was submitted to demonstrate  the equivalence of Dow's test material #101811 to the plant-produced protein used in the 8-day dietary study with the rainbow trout (Oncorhynchus mykiss) WALBAUM (MRID No. 488284-11).

Classification:  SUPPLEMENTAL to MRID No. 48828411
                                   49044808
Non-guideline
Supporting document
A response to EPA Comments on Cry1F(synpro) ICP and Cry1Ac(synpro) insecticidal crystal protein was submitted to address several  data gaps in the registrant's environmental risk summary (MRID No. 48828413)  for PIP event DAS-81419-2 soybean. Additional data was needed to complete the risk assessment on the environmental fate of Cry1F and Cry1Ac proteins expressed in PIP event DAS-81419-2 soybean.  Supporting literature and data waiver rationales were presented for evaluating soil fate, gene flow and the potential for weediness.

Classification:  SUPPLEMENTAL to MRID No. 48828413
                                   49044809

Additional support for a complete environmental risk assessment is gained from an overview of the assessment conducted to support the WideStrike(R) cotton EPA Sec. 3 registration (EPA Reg. No. 68467-3). A brief summary of the EPA's environmental risk assessment is provided in the following section:

      C. WideStrike cotton (lepidopteran active) Environmental Risk Assessment Summary (EPA Reg. No. 68467-3)

Potential adverse effects to nontarget organisms by Cry1F and Cry1Ac proteins have been reviewed in Bt Cry1F/Cry1Ac WideStrike cotton BRAD (US EPA 2005). The following is a summary of the environmental risk assessment for Cry1F and Cry1Ac. EPA performed risk assessments on plants, wild mammals, birds, fish, aquatic invertebrates, earthworms, terrestrial nontarget insects (including honey bee, parasitic wasps, green lacewings, ladybird beetle, springtails [Collembola toxicity/reproduction], and monarch butterflies), as well as field evaluations of the effects of Cry1F and Cry1Ac exposure on nontarget insects in cotton fields, soil degradation/persistence studies, and an endangered species impact assessment, particularly for Lepidoptera. In addition, gene flow and weediness assessments via pollen and Cry protein DNA uptake by plants were also performed. Cry1F and Cry1Ac protein in soil has been shown to degrade rapidly to very low levels. EPA concluded that there is sufficient information to believe that there is no risk from the uses of Cry1F and Cry1Ac cotton to nontarget wildlife, aquatic, and soil organisms. These studies were individually reviewed by the Agency and reported in Data Evaluation Reports (DERs). For detailed information of each study, refer to the DER according to its respective assigned MRID number in the associated environmental effects data summary tables for WideStrike cotton (reviewed in US EPA 2005).

At present, the Agency is aware of no identified significant adverse effects of Cry1F and Cry1Ac proteins on the abundance of nontarget organisms in any population in the aquatic or terrestrial field environment. Field testing and field census data submitted to the Agency show minimal to undetectable changes in the beneficial insect abundance or diversity. To date the available field test data show that compared to crops treated with conventional chemical pesticides, the transgenic crops have no detrimental effect on the abundance of nontarget invertebrate populations. In addition, no direct or indirect effects on Federally listed endangered and threatened species or effects on their critical habitat are expected. The EPA has reviewed the potential for gene capture and expression of Cry1F and Cry1Ac proteins by wild or weedy relatives of cotton in the United States, its possessions or territories and has found that there is no significant risk in the United States, its possessions or territories (US EPA 2005).

In conclusion, the risk assessment found no unreasonable adverse effects on the environment from cultivation of WideStrike cotton expressing Cry1F and Cry1Ac proteins for use as a PIP.

      D. Protein Equivalence

The October 2000 SAP recommended that while actual plant material is the preferred test material, microbial-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 are needed for maximum hazard dose testing. In support of event DAS-81419-2 soybean registration, several of the nontarget toxicological studies used microbial-produced, purified Cry1F and Cry1Ac protein test substances that were originally developed for the Widestrike PIP cotton registration; in addition to plant tissue (lypholized leaf tissue) from event DAS-81419-2 soybean.

In support of bridging data from previously reviewed NTO toxicological studies conducted for WideStrike cotton (US EPA 2005), the biochemical properties the microbial-derived Cry1F and Cry1Ac protein test substances were compared with plant-produced Cry1F and Cry1Ac proteins expressed in event DAS-81419-2 soybean to determine that the proteins from the two host sources were biologically equivalent. The proteins were characterized and equivalence was evaluated based on a panel of analytical tests and assays. The results of these evaluations provide a detailed characterization of the Cry1F and Cry1Ac proteins isolated from event DAS-81419-2 soybean and confirmed their equivalence to the microbial-derived Cry1F and Cry1Ac proteins (MRID Nos. 48828403 and 49044803; reviewed in US EPA 2013). Therefore, the results of the NTO toxicity studies that utilized the microbial-derived Cry1F and Cry1Ac test protein substances can be bridged to support the environmental risk assessment of event DAS-81419-2 soybean. Furthermore, development of additional nontarget organism toxicity testing can be waived for PIP event DAS-81419-2 soybean and the results and conclusions from the environmental risk assessment conducted for the WideStrike cotton PIP line can be bridged to support event DAS-81419-2 soybean (US EPA 2005).

      	E. Nontarget Organism Effects Cry1F and Cry1Ac insecticidal proteins expressed in Event DAS-81419-2 Soybean (EPA Reg. No. 68467-EN)

      1. Avian Wildlife

The primary routes of exposure of birds to Cry1F and Cry1Ac insecticidal proteins expressed in PIP event DAS-81419-2 soybean plants expected to occur through consumption of event DAS-81419-2 plant material (e.g., leaf foliage and pods) and invertebrates that feed on event DAS-81419-2 soybean plant material. Exposure via consumption of soybean seeds may be limited, however, by compounds that interfere with digestion and nutrient uptake, such as trypsin inhibitors. Other routes of exposure (e.g., inhalation) are not expected to be significant, since Cry1F and Cry1Ac proteins are contained primarily within the plant.

In a 14-day acute dietary toxicity study, Northern bobwhites (Colinus virginianus) were exposed to avian diet fortified with a 7.9:1.0 mixture of Cry1F and Cry1Ac proteins by oral gavage (MRID No. 48828410). Results showed no apparent effects on mortality, sublethal observations, body weight, and food consumption at this treatment level. The 14 day acute oral LD50 for the test substance was > 128 mg/kg bw (24.52 mg a.i./kg body weight based on percent a.i.) and the 14 day NOEL for the test substance was > 128 mg /kg bw test substance (24.52 mg a.i./kg body weight based on percent a.i.).

BPPD estimates the exposure to birds in the field for soybeans is minimal. Monogastric animals like birds cannot safely consume raw soybean seed since it contains endogenous antinutrients (Friedman et al., 1991). The estimated feed consumption per bird was 33 grams/day. Therefore, in relation to the daily diet, Cry1F was dosed at 0.660 mg a.i./gm (660 ppm) and Cry1Ac was dosed at 0.083 mg a.i./gm (83 ppm). The HEEE concentration of Cry1F in raw DAS-81419-2 soybean seed is 14.34 ng/mg dw (ppm) and 1.08 ng/mg dw (ppm) for Cry1Ac (MRID No. 48828413). The MOE was calculated for Cry1F and Cry1Ac in the quail study relative to raw soybean seed consumed as 100% of the diet was 46X (660 ppm/14.34 ppm) and 77X (83 ppm/1.08 ppm), respectively, which provides a large safety margin even if birds could safely ingest raw soybean as their sole source of food.

Furthermore, the Cry1F and Cry1Ac proteins have been registered as PIPs in several crops with no identified concerns for birds, and there is a history of use with no reported incidents involving birds in the field, including use in a crop that is typically ingested by birds (corn). Therefore, based on this history, current toxicity data, and the BPPD's assumptions of the lack of avian exposure, no adverse effects to birds are anticipated as a result of the registration of DAS-81419-2 Soybean.

    	2. Wild Mammals

Wild mammals could be exposed to Cry1F and Cry1Ac proteins produced by event DAS-81419-2 soybean plants by consumption of plant material and invertebrates that feed on event DAS-81419-2 soybean plant material. As with birds, these are expected to be the primary sources for exposure.

Data are available with which to determine the risk of Cry1F and Cry1Ac proteins from DAS-81419-2 soybeans to wild mammals. The results from the acute oral toxicity studies showed no significant toxicity to mice from acute oral testing with the Cry1F and Cry1Ac proteins, with a NOAEL that exceeds 600 mg/kg body weight for Cry1F (MRID No. 45542312) and 700 mg/kg for Cry1Ac (MRID No. 45542313). Based on the estimated expression level in leaf (V5) tissue (63.3 ng/mg dw) converted to dose (0.00153 mg/kg bw), adverse effects would not be expected at concentrations up to a MOE of >3.9 x 10[5]X for Cry1F. Based on the estimated expression level in leaf (V5) tissue (28.3 ng/mg dw) converted to dose (0.000683 mg/kg bw), adverse effects would not be expected at concentrations up to a MOE of >1.0 x 10[6]X for Cry1Ac. Thus, the MOEs greatly exceed the required 10X EEC showing no adverse effects when tested at the maximum hazard dose. These data are sufficiently representative of toxicity that would be expected in wild mammals, and based on the results of this study risk to
wild mammals resulting from exposure to Cry1F and Cry1Ac proteins expressed in DAS-81419-2  Soybean is not expected.

         	3. Freshwater Animals

The registrant submitted an acute oral dietary toxicity study on rainbow trout (Onchorynchus mykiss) to assess the effects of consuming a basal fish diet fortified with a mixture of microbial-derived Cry1F and Cry1Ac proteins for eight days (MRID No. 48828411). No fish mortality and no sublethal effects were observed in any of the control or test treatment organisms during this study. Results show the 8-day LD50 value with rainbow trout was > 100 mg a.i./kg diet wet weight (>200 mg a.i./kg diet dry weight). The NOEC was reported as > 100 mg a.i./kg diet weight (>200 mg a.i./kg diet dry weight) based on the lack of mortalities, sublethal effects, and apparent lack of effects on standard length and body weight. In addition, exposure to freshwater fish in aquatic environments is also considered to be minimal.

For freshwater invertebrates, a 48-hour static toxicity study on daphnids (Daphnia magna)  showed no adverse effects after exposure to  WideStrike(R) cotton pollen expressing Cry1F and Cry1Ac in combination at respective concentrations of 510 and 2,500 μg/L (MRID No. 45808412). Based on a worst-case exposure of one kg of transgenic cotton pollen per liter of pond water, the rate of fortification at these concentration are 298X and >23,000X the anticipated EEC for Cry1F and Cry1Ac protein in surface water. However, this study was determined of minimal value for evaluating the potential effects to nontartget freshwater invertebrates because  the length of the study was not conducted at the Agency's recommendation of at least 7-14 days, since the mode of action for Bt would likely take effect after several days of exposure. In addition, EPA determined that exposure to Cry proteins in the freshwater environment were expected to be low, based on the assumption that exposure in aquatic environments primarily resulted from pollen deposition (see US EPA 2010a)  and that post-harvest crop residue is the most likely route of exposure to aquatic organisms (Carstens et al. 2011). However, in light of published studies showing reduced growth in caddis flies exposed to anti-lepidopteran Cry1A protein (Rosi-Marshall et al. 2007), concerns regarding the potential for exposure to shredder invertebrate species via consumption of Bt crop plant litter in aquatic environments. EPA determined exposure to shredder invertebrate species from Bt crops is likely to be low, since the concentrations of Cry proteins in post-harvest crop tissues are limited temporally and spatially (Swan et al. 2009; Jensen et al. 2010; Wolt and Peterson 2010; Carstens et al. 2011). Nevertheless, EPA has required aquatic invertebrate testing lasting 7-10 days with Daphnia spp. to reduce uncertainty in past PIP registrations of Bt crops. A study with shredder species is even more appropriate since it would directly test the invertebrate species expected to be affected by Bt plant litter that may enter streams.

Specifically for soybean, the Agency has determined that the exposure of aquatic systems to Cry1F and Cry1Ac proteins produced in PIP event DAS-81419-2 soybean are negligible. Movement of Cry proteins from off-field pollen or post-harvest crop residues to aquatic habitats is not expected given that soybeans are primarily self-pollinated and cultivated soybean plants release pollen prior to flower opening (Abud et al. 2007; Caviness 1966). The Agency previously concluded that plant litter from both Bt corn and Bt cotton crops are not deposited in amounts high enough to result in adverse effects (US EPA 2001b). Since soybean produces less above-ground residue under cultivation (Green and Blackmer 1995), this conclusion is also made for the cultivation of DAS-81419-2 Soybean.

Based on the acute oral toxicity study on rainbow trout, adverse effects to freshwater fish are not anticipated as a result of the registration of Cry1F and Cry1Ac as expressed in event DAS-81419-2 soybean. Risk posed to aquatic invertebrates (including the aquatic Lepidoptera) through DAS-81419-2 cultivation was concluded to be much reduced due to a low likelihood of exposure (Carstens et al. 2011) and no verified instances of sensitive species. In view of the lack of toxicity and minimal aquatic exposure, the rainbow trout study and the data waiver rationale  for freshwater invertebrates are considered to be adequate to support the conclusion that deployment of event DAS-81419-2 for use as a PIP in soybean poses little risk to aquatic organisms.

         4. Estuarine and Marine Animals

BPPD typically waives the requirement of studies with estuarine/marine animals for Bt Cry proteins because of an expected lack of exposure in these environments. Therefore, data were not required for Cry1F and Cry1Ac for the registration of DAS-81419-2 Soybean, and based on expected lack of exposure; adverse effects to these species are not anticipated.

      5. Terrestrial and Aquatic Plant Species

BPPD typically waives nontarget plant testing for Bt Cry proteins, since the active ingredients are insect toxins (Bt δ-endotoxin) that have never shown any toxicity to plants. Therefore, BPPD has concluded that adverse effects of Cry1F and Cry1Ac insecticidal proteins expressed in DAS-81419-2 Soybean to terrestrial and aquatic plants are not anticipated.

The active ingredients Cry1Ac and Cry1F are part of a larger group of insect toxins produced a naturally-occurring Bacillus thuringiensis (Bt -endotoxins) that have not shown toxicity to plants. Effects to nontarget plants in the terrestrial and aquatic environments are not expected, and BPPD concludes that adverse effects to terrestrial and aquatic plants as a result of the registration and use of Cry1F and Cry1Ac insecticidal proteins expressed in event DAS-81419-2 Soybean are not anticipated.

      6. Invertebrate Species

BPPD assumes that nontarget insects receive exposure primarily through consumption of DAS-81419-2 Soybean leaf foliage, pods, and occasionally other soybean plant tissues as well as indirect exposure through consumption of insects that feed on soybean plant tissue. The principal route of exposure to soil-dwelling invertebrates, such as collembola and earthworms, is assumed to be consumption of decomposing plant tissue, and also possibly plant exudates, in soil during feeding.

The registrant requested data bridging to the existing ecotoxicological studies on the effects of Bt Cry1F and Cry1Ac proteins expressed in WideStrike(R) PIP cotton on representative nontarget species, including nontarget beneficial insects, pollinators, detritivores, and incidentals (MRID No. 48828413). As previously discussed, the Cry1F and Cry1Ac proteins produced in DAS-81419-2  soybeans were determined biologically equivalent with the microbial-derived Cry1F and Cry1Ac protein test substances used for producing toxicological data to support  WideStrike(R)  cotton (MRID No. 48828403 and 49044803; reviewed in US EPA 2013 ). The high-end exposure estimates (HEEEs) were derived from protein expression levels of Cry1F and Cry1Ac via Enzyme-linked immunosorbent assay (ELISA) analyses on various soybean plant tissue parts of PIP event DAS-81419-2. Exposure estimates for Cry1F and Cry1Ac protein concentrations in PIP event DAS-81419-2 soybean were compared with endpoints reported from toxicity studies previously conducted for WideStrike(R) cotton on various representative nontarget test species to determine a Margin of Exposure (MOE). The results of the MOE calculations are presented in Tables B.2. and B.3.

A MOE of 50% mortality at 10X the estimated environmental exposure is regarded as sufficient to demonstrate negligible risk and was also the threshold for consideration of uncertainties in the risk assessment (US EPA 1998; US EPA 2007). If subsequent expression characterization in the commercial event leads to a safety margin of <10X, then the consequences are considered in terms of uncertainties in the ecological risk assessment below (US EPA 2007). The ratio of the endpoint value to the EEC and are presented in Tables B.2. and B.3. for Cry1F and Cry1Ac, respectively. The published EPA level of concern is 50% mortality at 5x MEEC (US EPA 1998). It should also be noted that the dose margin can be less than 10x where uncertainty in the test system is low. 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 MEEC (US EPA 1998; US EPA 2007).  The registrant note that the concentrations tested were much higher than those encountered under field conditions. Since most MOEs exceeded 10X the estimated environmental concentrations (Tables B.2. and B.3.), the results of the MOE calculations indicates the most sensitive individual in a population is not likely to be adversely affected.

                   a. 	Beneficial Arthropods and Pollinators

Data from a dietary toxicity study on lady bird beetle previously conducted for WideStrike cotton showed that adverse effects did not occur to the test insects at >300 μg/mL diet for Cry1F and >22.5 μg/mL diet for Cry1Ac assuming a maximum expected environmental concentration of 10.6 ng/mg fresh weight for Cry1F and 4.76 ng/mg fresh weight for Cry1Ac (see MRID No. 45542315). Dow did not analyze protein expression levels for pollen or soybean floral nectar, as Cry1 protein expression in this secretion in four cotton lines is negligible (Wolt 2002; MRID No. 45808420). Therefore, the MOE calculation for lady beetle relies on the highest level of protein expression found in the field expression study (V5 leaf). Based on the estimated expression level in leaf (V5) tissue, adverse effects would not be expected at concentrations up to 25.6X for Cry1F and 4.3X Cry1Ac.

The registrant requested data bridging to the dietary toxicity study on green lacewing (Chrysoperla rufilabris) larvae, which was conducted by presenting the green lacewings with a moth egg (Sitotroga sp.) diet incorporated with Bt Cry1F and Cry1Ac proteins (MRID No. 45808410). Results showed that adverse effects did not occur to the test insects at >48.2 ug/g diet for Cry1Ac and >5.2 ug/g diet for Cry1F. However, this study was considered supplemental. A previous EPA FIFRA Scientific Advisory Panel (SAP) determined that it is inappropriate to test the activity of insecticidal proteins by incorporating the Bt protein into a moth egg diet, since Bt protein may bind to the surface of the moth eggs, resulting in limited exposure to lacewings that feed with piercing sucking mouthparts (US EPA-SAP 2002).  Furthermore, green lacewings do not consume much pollen in the field (US EPA-SAP 2002) and are not exposed to Bt proteins via consumption of aphids (Head et al. 2001). In the Agency's environmental risk assessment of WideStrike cotton, this study was classified as supplemental and the Agency recommended other generalist predators (e.g. minute pirate bug) as a representative surrogate test organism for Tier I testing the effects of Cry1F and Cry1Ac proteins (US EPA 2005). Thus, the green lacewing study on the effects of Cry1Ac and Cry1F proteins expressed in DAS-81419-2 soybean can be bridged, but only offer supplemental information towards the environmental risk assessment of DAS-81419-2 soybean.  Other information on the effects of Cry1Ac and Cry1F exist  on green lacewing larvae concludes Cry1Ac and Cry1F toxins do not have a detrimental effect on the species when they are ingested either directly or through prey (Rodrigo-Simón 2006; Tian et al. 2013).

A dietary toxicity study that tested the effects of Cry1F and Cry1Ac expressed in DAS-81419-2 soybean in honey bee (Apis mellifera) was also cited for data bridging. Results showed no effect on survival of larvae to adult emergence at >1.98 μg/mL sugar water diet for Cry1F and >11.9 μg/mL sugar water diet for Cry1Ac (MRID No. 45542316). Pollen expression data for DAS-81419-2 soybean was not available, however, the MOE previously calculated for WideStrike(R) cotton  showed the LC50 was >4X the levels of pollen expressed. These data can be inferred to represent the MOE for event DAS-81419-2 soybean, since the same genetic construct that was used to transform WideStrike(R) cotton was used for event DAS-81419-2 soybean and the constitutive expression in pollen should be similar.   In addition, other information from a meta-analysis (Duan et al. 2008) of several laboratory acute oral toxicity studies on honeybee is available, which consistently demonstrated no biological activity in Hymenoptera order of insects from Cry proteins that target lepidopteran pests. Six Cry1F and Cry1Ac studies were included in the meta-analysis, suggesting that regardless of expression level in event DAS-81419-2 soybean pollen the MOE will be acceptable.

An acute toxicity study on parasitic wasp species (Nasonia vitripennis) was cited for data bridging. Results showed adverse effects did not occur to the test insects at >5.2 μg/g diet for Cry1F, respectively, and >46.8 μg/g diet for Cry1Ac, which represented at least at 14X the concentration found in WideStrike(R) cotton pollen (see MRID No. 45808411).  Similar to the green lacewing and a honey bee study, the MOE was not determined for the parasitic wasp species due to the lack of pollen and nectar expression data, and some uncertainty remains. . However, the HEEE of Cry1F and Cry1Ac from V5 leaf sample (as opposed to pollen) relative to the dose concentrations used on honeybees and nontarget arthropods (likely to be exposed via pollen ingestion) in toxicity studies is considered highly conservative and the margins of exposure (see Tables B.2. and B.3.) are well above dose levels of Cry1Ac and Cry1F protein toxicity testing showing no adverse effects.

The registrant noted that the concentrations tested were much higher than those encountered under field conditions. Since most MOEs exceeded 10X the estimated environmental concentrations (Tables B.2. and B.3.), the results of the MOE calculations indicates the most sensitive individual in a population is not likely to be adversely affected.

Further, in regards to exposure of Cry1F and Cry1Ac proteins expressed in event DAS-81419-2 soybean pollen, pollinators and other beneficial arthropods are anticipated to be less exposed based upon the cleistogamous nature of its flowers (in most cultivars). Soybean is predominantly self-pollinated and its pollen is essentially contained in the flower, since anthers usually dehisce and release pollen before flowers open (Caviness 1966; OECD 2000). Typical outcrossing rates are less than 1% (CPC 2004), so this suggests that contact of honeybees and other beneficial and predatory invertebrates to soybean pollen is minimal. So although the MOE to honeybees, green lacewings, and lady beetle were calculated from the HEEEs from Cry1F and Cry1Ac proteins in leaf tissue (V5) stage instead of pollen, the likelihood of actual field exposure to many nontarget invertebrates via pollen consumption or indirectly through consumption of pollen-feeding invertebrates is expected to be minimal. Since no pollen is expected to travel outside the field borders, off-field exposure to many nontarget invertebrates is further reduced.

In any case, no adverse effects were found on previously conducted toxicity testing of Cry1F and Cry1Ac proteins on representative nontarget beneficial arthropods and pollinators species (e.g. honeybees, lady beetles, green lacewings) based on the majority of the MOEs exceeded 10X the estimated environmental concentrations (Tables B.2. and B.3.). The results of the MOE calculations indicate the most sensitive individual in a population is not likely to be adversely affected. Risk to honeybees and the nontarget arthropods is further reduced due to minimal potential for exposure via pollen. Further, published meta-analysis of laboratory and field studies showed no significant differences were noted on populations of nontarget invertebrates on a community level for Bt crops expressing Cry1F and/or Cry1Ac vs. non-transgenic crops (Marvier, et al 2007; Duan et al. 2008). Lastly, an independent field census study showed no differences in the population of nontarget arthropods (MRID No. 48828412) in field sites containing event DAS-81419-2 soybean vs. non-transgenic conventional soybean lines. Among the taxa showing no significant population impacts were two that were also tested in the laboratory:  Coccinellidae (lady beetles) and Parasitica (parasitoid wasps). In view of the lack of toxicity and limited exposure via pollen consumption, no adverse effects are anticipated on nontarget beneficial arthropods and pollinators from the cultivation of PIP event DAS-81419-2 Soybean.

                  b. 	Soil Dwelling Invertebrates

An earthworm study conducted for WideStrike(R) cotton was also cited for data bridging. Earthworms (Eisenia foetida) were exposed to artificial soils treated with 247 mg/kg and 107 mg/kg of microbial-produced Cry1Ac and Cry1F proteins, respectively, for 14 days (MRID No. 45580701). The study showed no effects on mortality or burrowing time resulting from exposure to Cry1F and Cry1Ac present in artificial soil at approximately 716X for Cry1F and 1176X for Cry1Ac at the maximum EEC. The maximum EEC of the soil was 0.345 mg/kg for Cry1F and 0.091 mg/kg for Cry1Ac based on soybean plant residue in the top 15cm of soil.

Data from a toxicity study on Collembolas (Folsomia candida) previously conducted for WideStrike cotton showed that adverse effects did not occur to the test insects at >702 mg/kg soil for Cry1F and >22.6 mg/kg soil for Cry1Ac (MRID No. 45808409). The MOE for Cry1Ac was calculated as >2035X and  >248X for Cry1F, assuming a maximum EEC of  0.345 mg/kg for Cry1F and 0.091 mg/kg for Cry1Ac based on soybean plant residue in the top 15cm of soil. A lack of adverse effects following exposure to Cry1Ac protein has also been shown for collembolans (Folsomia candida) (Sims and Martin, 1997).

Based on the studies submitted for DAS-81419-2, as well as other information for Cry1F and Cry1Ac, risk to nontarget soil invertebrates resulting from the cultivation of PIP event DAS-81419-2 Soybean is not expected.

Table B.2. Margins of Exposure calculated from Cry1F Protein Concentrations in PIP Event DAS-81419-2  soybean plant tissues relative to Dose Concentrations used in the Nontarget Arthropod Ecotoxicological Studies previously conducted on WideStrike(R) PIP cotton plant- and microbial- derived Cry1F protein [a]

                                   MRID No.
                                  Study Type
                                    Species
                                 Tissue sample
                                    HEEE[b]
                                 Tox. Endpoint
                                      MOE
                                    Comment
                                   45542316
                        Honey Bee Larva Testing, Tier I
                                  (885.4380)
                                   Honeybee
                               (Apis mellifera)
                                    Pollen
                                      ND
                           >1.98 ug/mL diet NOEC
                                      ND
No effects found for honey bee larvae and adults in published meta-analysis with Cry1F and/or Cry1Ac expressing Bt crops (Duan et al. 2008)
                                   45808410
                 Nontarget Insect Testing, Tier I  (885.4340)
                                Green lacewing
                              (Chryosperla camea)
                                Nectar, pollen
                                      ND
                      >5.2 ug/g diet acute oral LC50
                                      ND
No effects found in Cry1Ac and Cry1F protein lab study (Rodrigo-Simón et al. 2006) and tritrophic feeding study on green lacewings exposed to a diet of Cry1F or Cry1Ac resistant prey  (Tian et al. 2013)
                                   45542315
                  Nontarget Insect Testing, Tier I (885.4340)
                                  Lady beetle
                            (Hippodamia convergens)
                             Pollen (used V5 Leaf)
                                 10.6 ng/mg fw
                      >300 ug/mL diet acute oral LC50
                                   >25.6
Pollen expression data is not available; Highest expression in leaf converted to fw for MOE calculation.
                                   45808411
                 Nontarget Insect Testing, Tier I  (885.4340)
                                Parasitic wasp
                             (Nasonia vitripennis)
                                    Nectar
                                      ND
                      >5.2 ug/mL diet acute oral LC50
                                      ND
No feeding in narrow soybean flowers
                                   45808409
                  Nontarget Insect Testing, Tier I (885.4340)

                                  Collembola
                              (Folsomia candida)
                            R3 Forage soil residue
                               0.345 mg/kg soil
                            >702 mg/kg soil NOEC
                                   >2035
Plant residue in top 15 cm soil
                                   45580701
                           Earthworm Toxicity Study
                                   Earthworm
                               (Eisenia foetida)
                            R3 Forage soil residue
                               0.345 mg/kg soil
                              247 mg/kg soil NOEC
                                      716
Plant residue in top 15 cm soil
[a] Microbe-derived Cry1F protein used in laboratory studies was demonstrated to be equivalent with the Cry1F protein expressed in DAS-81419-2
[b] HEEE = mean expression level + (t0.1, upper tail, n-1 x std. dev.) / n[1/2]. HEEE values based on Cry1F expression level determined for DAS-81419-2 plant tissue relevant to the potential exposure route for the organism of interest.
ND = Not Determined (data not available).
Table B.2 reproduced from Table 3, page 26 from MRID No. 48828413

Table B. 3. Margins of Exposure calculated from Cry1Ac protein concentrations in DAS-81419-2 Soybean plant tissues relative to dose concentrations used in the Nontarget Arthropod Ecotoxicological Studies previously conducted on WideStrike(R) PIP cotton plant- and microbial- derived Cry1Ac protein [a]

                                   MRID No.
                      Study Type and OCSPP Guideline No.
                                    Species
                                 Tissue sample
                                    HEEE[b]
                                 Tox. Endpoint
                                      MOE
                                    Comment
                                   45580701
                           Earthworm Toxicity Study
                                   Earthworm
                               (Eisenia foetida)
                            R3 Forage soil residue
                               0.091 mg/kg soil
                              107 mg/kg soil NOEC
                                     1176
Plant residue in top 15 cm soil
                                   45808409
                  Nontarget Insect Testing, Tier I (885.4340)

                                  Collembola
                              (Folsomia candida)
                            R3 Forage soil residue
                               0.091 mg/kg soil
                           >22.6 mg/kg soil NOEC
                                    >248
Plant residue in top 15 cm soil
                                   45542316
                       Honey Bee Larva Testing, Tier I
                                  (885.4380)
                                   Honeybee
                               (Apis mellifera)
                                    Pollen
                                      ND
                           >11.9ug/mL diet NOEC
                                      ND
No effects found for honey bee larvae and adults in published meta-analysis with Cry1F and/or Cry1Ac expressing Bt crops (Duan et al. 2008)
                                   45808410
                 Nontarget Insect Testing, Tier I  (885.4340)
                                Green lacewing
                              (Chryosperla camea)
                                Nectar, pollen
                                      ND
                      >46.8 ug/g diet acute oral LC50
                                      ND
No effects found in Cry protein lab study (Rodrigo-Simón et al. 2006) and tritrophic feeding studies on green lacewings exposed to a diet of Cry1F or Cry1Ac resistant prey  (Tian et al. 2013)
                                   45542315
                  Nontarget Insect Testing, Tier I (885.4340)
                                  Lady beetle
                            (Hippodamia convergens)
                             Pollen (used V5 Leaf)
                                 4.76 ng/mg fw
                     >22.5 ug/mL diet acute oral LC50
                                    >4.3
Highest expression in leaf converted to fw. No effect found in Cry1Ac feeding study in Coleomegilla maculata (Li et al. 2011)
                                   45808411
                 Nontarget Insect Testing, Tier I  (885.4340)
                                Parasitic wasp
                             (Nasonia vitripennis)
                                    Nectar
                                      ND
                     >46.8 ug/mL diet acute oral LC50
                                      ND
No feeding in narrow soybean flowers
[a] Microbe-derived Cry1Ac protein used in laboratory studies have been demonstrated to be equivalent with the Cry1Ac protein expressed in DAS-81419-2
[b] HEEE = mean expression level + (t0.1, upper tail, n-1 x std. dev.) / n[1/2]. HEEE values based on Cry1Ac expression level determined for DAS-81419-2 plant tissue relevant to the potential exposure route for the organism of interest.
ND = Not Determined (data not available).  Table B.3 reproduced from Table 3, page 26 from MRID No. 488284-13

	7. Supplemental Information

Where a lack of expression data prevented calculation of high-end exposure values, other supplemental information (i.e. meta-analyses, biological specificity to the pest species, targeted toxicity studies, or food preferences) was used as a supporting line of evidence to support the conclusion of no adverse effects on NTOs from Cry1F and Cry1Ac proteins expressed in DAS-81419-2 soybean. To address any uncertainties in the risk assessment focused on taxonomic groups that were most likely to be exposed to Cry1F and Cry1Ac proteins expressed in PIP event DAS-81419-2 soybean in field settings, the potential for adverse effects of DAS-81419-2 on nontarget arthropod populations was assessed in a US field study (MRID No. 48828412). A nontarget arthropod field study with PIP event DAS-81419-2 soybean, a near-isogenic non-transgenic control (Maverick) and four non-transgenic reference lines (DSR 3590; IL 3505; Porter 75148; Williams 82) was conducted in 2011 at two field sites in the U.S. located in Richland, Iowa and York, Nebraska. Each trial site included the six soybean lines arranged in a randomized complete block design with four replicate blocks.

Arthropod abundance was sampled during the vegetative and reproductive soybean stages and monitored using three sampling methods: pitfall trapping, sticky card trapping, and vertical beat sheet sampling. Arthropods collected during the study included representatives from multiple orders and diverse ecological roles, including phytophagous, predatory, parasitic, and saprophagous modes of feeding. The analyses encompassed several taxonomic groups, including: Oribatida (soil mites), Araneae (foraging and web spiders), Collembola (springtails), Hemiptera (e.g. Orius spp.), Coleoptera (e.g. ground beetles, lady beetles), Neuroptera (e.g. Chrysopa spp.), Lepidoptera, Diptera, and Hymenoptera (e.g. parasitoid wasps). Consistent across locations and sampling methods, results showed arthropod populations associated with DAS-81419-2 soybean were similar to those of non-transgenic soybean. Therefore, the lack of differences in between populations of nontarget arthropod taxa and their abundance from the field monitoring suggests no adverse effects on nontarget arthropod communities from deployment of event DAS-81419-2 soybean cultivation.

      F. Soil Fate

Since proteins are not known to persist in the environment due to the ubiquitous nature of proteases in microbes, the registrant submitted a rationale for demonstrating rapid degradation of Cry1F and Cry1Ac proteins in various crop soils. Data from a previously submitted laboratory study showed that Cry1F and Cry1Ac expressed in cotton plants degrades quickly and does not accumulate in soil for WideStrike(R) PIP cotton (MRID No. 45556801). The soil half-life of the plant expressing Cry1F and Cry1Ac was estimated as 1.3 days and the Cry proteins were not detectable after 14 days. These results verify that the Cry1F and Cry1Ac proteins degrade rapidly in soils sampled from cotton growing regions.

The registrant also provided a data waiver rationale to support rapid degradation of Bt Cry1F and Cry1Ac proteins in soil based on the history of safe use in sprayable Bt formulations. Cry1Ac is present in both WideStrike(R) cotton event DAS-21023-5 (also described as 3006-210-23) and Bollgard(R) cotton (event MON 531, as well as a discontinued corn event: DBT418Cry1Ac corn) (Mendelsohn et al. 2003; Sanahuja et al. 2011). Cry1F is also present in WideStrike(R) cotton event DAS-24236-5 (also described as 281-24-236), as well as, in Herculex(R) and SmartStax(R)  corn (event TC1507) (Mendelsohn et al. 2003; Sanahuja et al. 2011). Other supporting information included previously conducted field studies showing no accumulation (i.e. persistence) of the proteins as a result of continuous planting of other Bt crops containing these proteins (Head et al. 2002; Herman et al. 2002; Shan et al. 2008). As a result, the Agency has determined that no additional long-term field studies are required for PIP event DAS-81419-2 soybeans because currently registered PIPs have not been shown to persist in soil.

Although the registrant submitted a sufficient rationale of rapid degradation of Cry1F and Cry1Ac proteins in other crop soil systems, a soil degradation study has not been evaluated in soybean for this specific PIP event. A previous EPA FIFRA SAP noted that in soil degradation and field persistence studies, toxin degradation rates may vary with the crop, the toxin produced by the transformation event, the microbial community in the recipient soil microcosm, the statistical characterization of toxin persistence, and/or the initial dose of toxin in the experiment (US EPA 2001a). These differences in Cry toxin concentration may be due to the fact that different genetic constructs were used and/or inserted at different locations in the plant genome (Yu et al. 2012). Plant genotype and parental backgrounds have also been recognized as important factors influencing protein expression (Kranthi et al. 2005; Adamczyk and Sumerford 2001; and Torres et al. 2006). Other important factors that affect degradation of Cry toxins include environmental conditions such as temperature, light, drought, and soil properties (e.g. pH, texture, clay content) (Adamczyk et al. 2004; Chen et al. 2005; Jiang et al. 2006; Rochester 2006; Dong et al. 2008; Hallikeri et al. 2009; Addison and Rogers 2010; Chen et al. 2011; Ranjithkumar et al. 2011).   To reduce this uncertainty, a soil fate study to determine the degradation rate (DT50) of Cry1F and Cry1Ac proteins sampled from representative soils of soybean growing regions is to be conducted as confirmatory data.

         G. 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, the soil degradation studies discussed in the WideStrike cotton BRAD show Cry1F and Cry1Ac proteins degrade quickly in soil (US EPA 2005).

With regard to the impact of genetically engineered crops on soil, EPA has previously noted (US EPA 2010a) that agricultural practices themselves cause large changes in soil and soil microbial composition. Furthermore, factors such as 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. If reports of adverse effects became available, the Agency will take appropriate action to mitigate potential risks.

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

The EPA has evaluated the potential for horizontal gene transfer (HGT) from Bt crops to soil organisms and has considered possible risk implications if such a transfer were to occur. Genes that have been engineered into Bt crops are mostly found, or have their origin, in soil-inhabiting bacteria. Soil is also the habitat of other toxin-producing bacteria, and transfer of these genes and/or toxins to other microorganisms or plants has not been detected. There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to microbes (Conner et al. 2003). Furthermore, several published experiments, that were conducted to assess the likelihood of HGT, have been unable to detect gene transfer under typical environmental conditions. Horizontal gene transfer to soil organisms has only been detected with very promiscuous microbes under laboratory conditions designed to favor transfer. As a result of these findings and the fact that the Bt toxins engineered into event DAS-81419-2 are derived from soil-inhabiting bacteria, the EPA has concluded that the risk of HGT of transgenes found in event DAS-81419-2 soybean expressing Cry1F and Cry1Ac proteins are low.

      I. Gene Flow and Weediness Potential

The movement of transgenes from the host plant into weeds has been a significant concern for EPA due to the possibility of novel exposures to the pesticidal substance. This concern has been considered for each of the Bt plant-incorporated protectants currently registered, and was extensively reexamined in 2010 (see US EPA 2010a), and EPA believes that these concerns have been satisfactorily addressed.
The FIFRA EPA Scientific Advisory Panel meeting held on October 18-20, 2000 discussed the matter of gene flow and offered some issues for consideration in this matter. The panel agreed that the potential for gene transfer between Gossypium hirsutum (cotton) and any receptive plants within the U.S., its possessions and territories, was of limited probability and nearly risks free (US EPA 2000). Since soybean is also predominantly a self-pollinating species, the dispersal of pollen is limited because the anthers mature in the bud and directly pollinate the stigma of the same flower (OECD 2000). Thus, the risk of potential gene transfer between soybean and any receptive plants is further negligible. Lastly, there are no known wild or weedy relatives exist in the U.S. with which Glycine max (L.) Merr. can form viable hybrids in nature, and soybean is not weedy in character or invasive (US EPA 2007). Therefore, BPPD determined that there is no significant risk of gene capture and expression of any Bt endotoxin by wild or weedy relatives of soybean in the U.S., its possessions or territories.

      J. Endangered Species Considerations

Because of the selectivity of Cry1F and Cry1Ac insecticidal crystal proteins for lepidopteran species and lack of evidence of effects on other nontarget species, the Agency has investigated concerns for Federally listed threatened and endangered insect species in the order Lepidoptera. Because soybean pollen is not expected to move beyond the planted soybean field and its immediate margins, as discussed above, any exposure to lepidopterans would be expected to occur within those areas. Exposure could occur via direct consumption of PIP event DAS-81419-2 soybean plants or consumption of DAS-81419-2 pollen that falls on non-soybean plants within the soybean field and its immediate margins. However, oral exposure to significant amounts of Cry1F and Cry1Ac via pollen consumption is not likely. Little pollen is expected to be released from soybean flowers, since soybean plants are predominantly self-pollinated and anthers usually dehisce and release pollen before flowers open. Airborne pollen concentrations have been measured at very low levels within soybean fields (mean of 0.18 grains/cm[2]/day) (Yoshimura et al. 2006). Based on this analysis, BPPD concludes that exposure resulting from pollen falling on potential non-soybean food plants in the field and immediate margins is not sufficient to cause effects in listed lepidopterans. Therefore, any significant exposure would have to occur through consumption of the DAS-81419-2 soybean plants on the field or via tritrophic effects, in which predatory beneficial arthropod species consume soybean pest species feeding on PIP event DAS-81419-2 soybean plant tissues.

A search of EPA's LOCATES database indicates that three species of listed lepidopterans are present in U.S. counties in which soybeans are grown. These are the Karner blue butterfly (Lycaeides melissa samuelis), St. Francis's Satyr Butterfly (Neonympha mitchellii fransisci), and Mitchell's Satyr Butterfly (Neonympha mitchellii mitchellii). The potential effects of Bt PIPs in corn on the Karner blue butterfly was extensively analyzed in BPPD's Bt Crops Reassessment (USEPA 2001b, 2004b and 2010a and b) and in the endangered species assessment section in the BRAD for event MON87701 soybean expressing Cry1Ac protein (see US EPA 2010c). As previously discussed, soybean pollen is not expected to be deposited on plants in or around soybean fields in amounts sufficient to cause effects in sensitive lepidopterans. Ecology and life history information demonstrated that habitat requirements for larvae and adults of two of the identified species (Mitchell's Satyr butterfly and Saint Francis' Satyr butterfly) do not overlap with commercial soybean acreage, and are therefore not expected to be impacted by PIP event DAS-81419-2 cultivation (Bartel and Sexton 2009; Barton and Bach 2005; US FWS 1997). There is a possible overlap between the geographic range for Karner blue butterfly and soybean use sites. EPA has previously reviewed information on the proximity of Karner blue habitats, and none are known to exist immediately adjacent to agricultural fields ("adjacent to" was defined as 0-3 m for corn fields, which is reasonably applied to soybean fields) (US EPA 2001b). Karner blue larvae feed only on lupines (Lupinus spp.) and soybean is neither identified as a larval food source, nor is soybean open-pollinated. Thus, pollen from event DAS-81419-2 soybean is highly unlikely to be deposited on lupine leaves (NatureServe 2013). Furthermore, soybean is not identified as a nectaring source for Karner blue adults. Therefore, the risk posed to the Karner blue butterfly was considered to be negligible.

Based on the above analysis, the EPA determines that there will be no direct effect to listed lepidopteran species as a result of the cultivation of DAS-81419-2 soybeans as proposed. Obligate relationships between insectivorous listed species with lepidopterans that are expected to be found in soybean fields, especially pest species that feed on DAS-81419-2 plants, are not currently known. Since the Cry1F and Cry1Ac in DAS-81419-2 soybeans targets only lepidopteran insects, loss of the pest insects as a result of DAS-81419-2 are expected to be offset by the presence of other insects that could act as food sources for listed species, including beneficial insects that are known not to be affected by Cry1F and Cry1Ac. Effects on species other than insects have also been determined to be very unlikely because of the specificity of Cry1F and Cry1Ac.

Lastly, since the Agency previously determined Cry1F and Cry1Ac proteins are not expected to have adverse effects on mammals, birds, plants, freshwater and estuarine/marine fish and invertebrates, nontarget insects and other invertebrate species at the EEC, a "No Effect" determination is made for direct and indirect effects to Federally listed threatened and endangered species and their designated Critical Habitats. In addition, EPA does not expect that any threatened or endangered plant species will be affected by outcrossing to wild relatives or by competition with such entities. Hybrid soybean does not exist in the wild in the United States, nor do any wild plants that can interbreed with soybean in the United States.

      K.   Data Needed to Confirm DAS-81419-2 Soybean Environmental Fate Assessment

Although the registrant submitted a sufficient rationale of rapid degradation of Cry1F and Cry1Ac proteins in other  crop soil systems, the degradation rate (DT50) of Cry1F and Cry1Ac proteins sampled from representative soils of soybean growing regions has not been evaluated and must be conducted as confirmatory data. The soil degradation study is to be submitted to the EPA within 18 months of the issuance date of the DAS-81419-2 Soybean Notice of Pesticide Registration.

		II. Conclusion

The biological equivalency of Cry1F and Cry1Ac proteins expressed in event DAS-81419-2 soybean plant was confirmed with the previously characterized microbial-derived Cry1F and Cry1Ac protein test substances. No significant increase in exposure to NTOs was demonstrated based on calculated margins of exposure for protein concentrations in event DAS-81419-2 plant tissue matrices relative to the dose concentrations used in the NTO laboratory toxicity tests conducted on Cry1Ac and Cry1F. Lastly, a field monitoring study showing no differences on nontarget arthropods on a population and community level and other supporting supplemental information (e.g. meta-analysis, target feeding studies, and biological specificity to the target pest) provided additional certainty to support the "weight-of-evidence" that there is negligible risk to NTOs representing diverse taxonomic groups and exposure scenarios from the cultivation of DAS-81419-2 soybean.

 Therefore, the submitted data supports the applicant's request and the findings from the environmental risk assessment conducted for WideStrike(R) cotton can be bridged to support a breeding/seed increase registration for PIP event DAS-81419-2 (US EPA 2005). Based on extrapolating from previous studies on Bt proteins, which tend to be very species specific in their activity, the Agency concludes that the cultivation of event DAS-81419-2 soybean will not result in adverse effects to nontarget organisms, are not likely to persist in the soil, and pose no risk of gene flow and development of weediness in wild relatives, and additionally will have no effect, direct or indirect, on Federally listed threatened and endangered species and  their designated Critical Habitats.

APPENDIX C: Insect Resistance Management

Cry1Ac and Cry1F proteins as DAS-81419-2 Soybean

Discussions of EPA's risk assessments for Insect Resistance Management (IRM) are presented in this Appendix. Immediately below is the risk assessment completed by EPA's A. Reynolds on November 7, 2013.

I. Insect Resistance Management  Review

Below is the BPPD review of Dow's Insect Resistance Management (IRM) plan and supporting data for the application to register DAS-81419-2 Soybean PIP for seed production and research purposes, as well as supporting documentation concerning natural refuge for Bt cotton (U.S. EPA memorandum 11/7/13; EPA File Symbol 68467-EN, MRID 49024110 replaced MRID 48828415).

CONCLUSION AND REQUIREMENTS

1) Because DAS-81419-2 soybean is intended for breeding/seed increase, Dow did not propose a species-specific IRM plan for the product.  Rather, the company proposed to mitigate resistance risk by acreage limitations on a county (1,000, 10,000, or 25,000 acres) and national (250,000 acres) scale. BPPD agrees that given the low acreage projected by Dow, it is unlikely that there will be a significant risk of resistance to the main soybean lepidopteran pests in the United States. These insects include soybean looper, velvetbean caterpillar, beet armyworm, cotton bollworm, green cloverworm, tobacco budworm, and cutworms (Agrotis spp.). This conclusion is further supported by the biology of the target insects, which are highly polyphagous (feeding on a number of wild hosts and cultivated crops) and (for some species) have limited overwintering in non-tropical areas.

2) Should a full commercial registration or expanded acreage be sought in the future, a complete, species-specific IRM assessment for DAS-81419-2 will be required. Data needed to support such a review should include additional information on pest biology, dose, simulation modeling, cross resistance, resistance monitoring, and potential impacts on the natural refuge strategy for Bt cotton.

3) The use of DAS-81419-2 on a limited basis for seed increase purposes should not significantly impact the natural refuge strategy in place for Bt cotton PIPs. Soybean is one of the non-cotton crops that have been considered as part of natural refuge for tobacco budworm and cotton bollworm. While it can be expected that plantings of DAS-81419-2 soybean will remove some of the currently-available natural refuge for Bt cotton, this potential reduction will likely be small relative to the total amount of natural refuge present in southern cotton regions. Dow has proposed to further mitigate the impact on natural refuge by limiting DAS-81419-2 to 10,000 acres in counties with > 25,000 total acres of soybean and 1,000 acres in counties with < 25,000 total acres of soybean.

4) Dow must institute a stewardship program for DAS-81419-2 including resistance monitoring and remedial action (in the event of resistance). A resistance monitoring plan limited to investigations of unexpected pest damage is appropriate for a capped acreage registration.  BPPD notes that monitoring of two potential target insects, tobacco budworm and cotton bollworm, is already being conducted as part of the WideStrike Bt cotton monitoring program. Dow must also compile annual sales and acreage data for each state and provide a report to EPA if requested. Since DAS-81419-2 will be deployed without a structured refuge requirement, a compliance assurance plan and grower education program should not be necessary.

IRM Rationale and Proposed Plan for DAS-81419-2 Bt Soybean (MRID No. 49024110)

Dow's proposed DAS-81419-2 soybean is intended for breeding, seed production, and research in the United States. Therefore, acreage of the product grown in the United States will be limited to a total of 250,000 per year. Individual counties would also be annually limited to either 20,000 (in non-cotton growing regions), 10,000 (in cotton growing counties with at least 25,000 acres of soybean), or 1,000 acres (in cotton-growing counties with < 25,000 soybean acres).

Dow's IRM rationale for DAS-81419-2 is based on the limited commercialization of the product in the United States. The company reasons that selection pressure should be limited by the acreage caps and notes that EPA has not mandated refuges for similarly-capped Bt corn breeding registrations. Further, the product provides two modes of action (Cry1F and Cry1Ac) for control of lepidopteran pests.

Soybean is one of the lepidopteran crop hosts that have been considered as natural refuge for Bt cotton. Dow's acreage proposal for DAS-81419-2 could therefore reduce the amount of soybean available for cotton refuge in areas where both crops are planted. To estimate the potential effects of DAS-81419-2 on natural refuge, Dow calculated the refuge contribution of soybean relative to the amount of cotton as such:  [(acres of soybean  -  proposed DAS-81419-2 acres)/(acres of soybean + cotton) * 100]. DAS acreage was set at either 10,000 or 1,000 per county as described above. Acreage data for county cotton and soybean were obtained from USDA-NASS (http://quickstats.nass.usda.gov/) for the 2010 season. Calculations were conducted for all counties with plantings of both soybean and cotton in the states of Alabama, Arkansas, Georgia, Kansas, Louisiana, Mississippi, Missouri, North Carolina, Oklahoma, South Carolina, Tennessee, and Georgia.

A total of 183 counties were surveyed by Dow (data contained in Tables 1-7 in MRID No. 49024110). Of these, 84 had at least 25,000 acres of soybean planted during the 2010 season. In these counties up to 10,000 acres of DAS-81419-2 has been proposed. Even with this potential reduction in refuge, the amount of available soybean refuge averaged 62% among the 84 counties. Dow noted that this level of refuge is well above the 20% structured refuge that was required for Bt cotton prior to approval of natural refuge. In addition, commercial soybean fields are rarely treated for cotton bollworm which should further enhance production of susceptible insects.

The remaining 99 counties analyzed by Dow, with less than 25,000 total soybean acres, would have a limit of 1,000 DAS-81419-2 acres. Some of these counties grow little soybean (< 2000 acres) such that the relative contribution to natural refuge from the crop is likely low, regardless of the adoption of DAS-81419-2. Other counties grow larger amounts of soybean; however, in these counties full adoption of DAS-81419-2 (1,000 acres) would still have a small impact on natural refuge.

Dow indicated a number of other factors add to the conservatism of the IRM rationale:
   * Seed production for soybean (the primary intended purpose for DAS-81419-2 in the U.S.) occurs in areas where the crop is favored  -  i.e., in counties with large amounts of soybean (>25,000 acres).
   * Counties with not as much soybean (<25,000 acres) are less likely to be used for seed production.  Nonetheless, a "regional" natural refuge may provide susceptible insects from other counties since H. zea is known to be highly mobile (Jackson et al. 2008).
   * Soybean for seed production (non-commercial production) is frequently treated with insecticides for pod-feeding insects. Therefore, seed production soybean is likely to be limited in value as natural refuge regardless of the adoption of DAS-81419-2.
   * In addition to soybean, H. zea is known to utilize numerous other crop and plant hosts including corn, peanuts, sorghum, and weed species. Accordingly, natural refuge for the insect is not limited solely to soybean acres and the county refuge estimates provided by Dow will be underestimates of the actual amounts.

BPPD Review

Dow's proposal to limit the overall acreage and distribution of DAS-81419-2 in the U.S. should be sufficient to mitigate potential resistance development for a seed increase registration. It should be noted, however, that BPPD does not have sufficient information in the submitted materials to completely assess the risk of resistance for a full commercial registration of DAS-81419-2.

Target Pests

Dow's submission did not indicate the primary insect pests targeted by DAS-81419-2 soybean.  BPPD presumes this is because the product is intended for commercialization in South America; U.S. plantings will be solely for seed production. Nonetheless, DAS-81419-2 can be expected to confer protection against susceptible soybean-feeding lepidoptera while grown for seed production in the U.S.

Soybeans can be affected by a number of lepidopteran insects, including (among others) soybean looper (Pseudoplusia includens), velvetbean caterpillar (Anticarsia gemmatalis), beet armyworm (Spodoptera exigua), cotton bollworm/corn earworm (Helicoverpa zea), green cloverworm (Plathypena scabra), and cutworms (Agrotis spp.). Though not common on soybean, tobacco budworm (Heliothis virescens) has also been known to infest the crop (Sheck and Gould 1993).  Other occasional lepidopteran pests include fall armyworm (Spodoptera frugiperda) and saltmarsh caterpillar (Estigmene acrea).

Soybean looper occurs throughout the U.S. where soybeans are grown, but overwinters only in tropical regions migrating annually from Mexico, Central America, and the Caribbean (Pedigo 1999).  The insect is multivoltine with two (northern regions) to seven (southern states) generations per season.  Soybean looper is known to feed on a wide range of vegetables, field crops (including corn and cotton), and weeds in addition to soybean (Capinera 2005).

Velvetbean caterpillar, similar to soybean looper, is a tropical species that migrates annually northward into the U.S.  It can range as far north as New England, but overwinters only in the southern end of the Florida peninsula (Pedigo 1999; Barbara 2008).  Velvetbean caterpillar is polyphagous and feeds on a number of field crops (soybean, alfalfa, cotton, peanut, other legumes) and weeds (Barbara 2008).

Green cloverworm also follows a southern overwintering/migration pattern from Gulf Coast areas.  The insect feeds on a variety of legumes, including soybean, and has up to four generations per season (http://ipm.ncsu.edu/AG271/soybeans/green_cloverworm.html).

Cotton bollworm and tobacco budworm are polyphagous Noctuid species that are primary target pests of Bt cotton registrations (see descriptions in BPPD 2001 and 2006). Fall armyworm and cutworms are secondary pests targeted by some Bt corn registrations (see BPPD 2010).

IRM Risk Considerations

No data were provided in the submission to evaluate the efficacy of DAS-81419-2 against any of the potential U.S. target pests. Published data for another Bt soybean cultivar (TIC107 with a cry1A gene) showed high activity against velvetbean caterpillar and soybean looper (McRae et al., 2005). Cry1Ac and Cry1F are also known to have toxicity to cotton bollworm and tobacco budworm (BPPD 2005), while fall armyworm is affected by Cry1F (BPPD 2010). However, without specific data for DAS-81419-2, it is not possible to evaluate dose considerations for the product or determine the resistance risk for targeted insect pests.

Rather than formulate an insect-specific IRM approach for DAS-81419-2, Dow has proposed acreage limitations to lower selection pressure for resistance development. A number of other factors should further reduce the risk of resistance such that a formalized refuge strategy is unnecessary. These considerations include biological aspects of soybean lepidopteran pests, the abundance of alternative hosts (natural refuge) for these insects, the pyramiding of two toxins in DAS-81419-2, and anticipated limited plantings of the product for seed increase purposes.

Most of the lepidopteran insects that infest soybean are polyphagous and feed on numerous other wild and cultivated hosts which should provide a source of natural refuge to reduce selection pressure for Cry1Ac resistance. Further, soybean looper, velvetbean caterpillar, and fall armyworm overwinter only in tropical areas (e.g., Florida in the U. S.); population migrate annually from the Caribbean, Mexico and Central America northward through the U.S. Other key pests, cotton bollworm and green cloverworm, overwinter in southern Gulf Coast states. Because of this, it is less likely that any resistant individuals that evolve during the growing season would be able to successfully overwinter and propagate the resistant trait to future generations.

As a pyramid, DAS-81419-2 expresses two toxins (Cry1Ac and Cry1F) for lepidopteran control.  Pyramided PIPs are thought to increase the respective durability of each toxin relative to mosaics of single toxin PIPs (Roush 1998, Caprio 1998, Zhao et al. 2003, others). BPPD notes that the potential for some cross resistance exists between Cry1Ac and Cry1F in cotton bollworm and tobacco budworm (Gould et al. 1995; BPPD 2005, 2010), which could limit the effectiveness of the pyramid for those insects. Nonetheless, pyramiding two toxins in DAS-81419-2 should further decrease the likelihood of resistance development.

Acreage limitations have been employed for seed/breeding registrations issued for Bt corn PIPs as a means to lower overall selection pressure for resistance. These restrictions have been implemented on county (up to 20,000 acres) and national level (250,000 acres total). Dow's proposed acreage for DAS-81419-2 generally follows this standard, with up to 25,000 acres per county (250,000 nationally) except in counties where soybean is currently planted (see discussion below).

Based on the proposed acreage-limited (breeding) registration and in consideration of the biological aspects of the soybean pests discussed above, BPPD concludes the risk of resistance to Cry1Ac and Cry1F in DAS-81419-2 should be low. Therefore, a refuge strategy is not recommended at this time for DAS-81419-2. However, should any request be made to expand the scope of DAS-81419-2 to a commercial registration (with large or unlimited acreage), BPPD recommends a formal IRM assessment to better evaluate the resistance risk and develop appropriate mitigation measures. Such a review should include appropriate data for dose expression, simulation modeling, and cross resistance.

Considerations for Bt Cotton Natural Refuge

In addition to the primary target pests of soybean, BPPD is also concerned about other insect pests that are targeted by Bt cotton but may also be exposed to Bt soybean.  These insects, tobacco budworm (TBW, Heliothis virescens) and cotton bollworm (CBW, Helicoverpa zea), are highly polyphagous and are known to exploit numerous crop and wild plant hosts. Cry1Ac and Cry1F are expressed in both DAS-81419-2 and WideStrike Bt cotton (Bollgard II also expresses Cry1Ac).

The established resistance management plan for Bt cotton involves the use of "natural refuge" in which non-PIP hosts (such as weeds and other non-cotton cultivated crops) provide sources of susceptible insects to dilute any potential resistance genes arising from transgenic cotton. One of the non-cotton crops included in calculations of natural refuge is soybean. Should a significant portion of the soybean crop be devoted to a Bt variety, the potential amount of natural refuge available to TBW and CBW could be reduced.

To evaluate potential impacts to cotton natural refuge, Dow conducted an analysis of counties in southern states growing soybean. The company tabulated soybean acreage in 2010 (USDA NASS data) and projected the impact DAS-81419-2 would have on the natural refuge contribution of soybean. In the surveyed counties with large soybean acreage (>25,000), full adoption of DAS-81419-2 would still leave a sizable (average 62%) contribution to natural refuge from non-Bt soybean. As could be expected, the potential reduction in natural refuge was greater in counties with less than 25,000, particularly those with less than 5,000 acres. Soybean contribution in 19 counties (of 99 total) would be reduced to less than 10% with the full addition of DAS-81419-2. In counties with less than 1,000 soybean acres (3 total), refuge contribution from soybean could potentially be eliminated. Generally, soybean refuge was lower in Georgia and Texas than the other surveyed states. This was largely because these states each have a number of counties with low overall soybean acreage but relatively high (> 20,000) cotton planting.

Dow's analysis was limited by considering only soybean as a contributor to cotton natural refuge. BPPD agrees with the company that numerous other crops (e.g., tobacco, peanut, corn, sorghum, alfalfa) and wild or weedy hosts that support TBW and CBW are present in cotton-growing regions. As such, Dow's assessment of the impact of DAS-81419-2 on natural refuge was likely highly conservative and underestimated the amount of refuge relative to cotton.

Nonetheless, it can be expected that plantings of DAS-81419-2 soybean will remove some of the currently-available natural refuge for Bt cotton. But this potential reduction will likely be small relative to the total amount of natural refuge available in southern cotton regions. As part of the natural refuge assessment in 2006-2007, BPPD reviewed extensive host utilization data developed by Monsanto (see BPPD 2006, 2007; Jackson et al. 2008). TBW and CBW were sampled for host preference by testing insect bodies for the presence of bound gossypol (TBW) or insect wings for the ratio of C3 to C4 plant isotopes (CBW). These data showed that TBW and CBW in cotton-growing areas fed much more frequently on non-cotton hosts than on cotton. In many of the sampled states, 50% or more of sampled TBW developed on crops other than cotton.  While soybean is a component of these non-cotton hosts, it is unlikely that it is the sole (or even a majority) source of natural refuge, given the relatively low county acreages (< 25,000 soybean acres in most counties). This is the case in Georgia, where low soybean acreage (relative to cotton) in Dow's analysis appeared to indicate a small natural refuge contribution from soybean (< 10% in many counties). However, Monsanto's host utilization data clearly showed a high proportion of natural refuge in the state, as very few sampled TBW (0 to 10%) developed on cotton (MRID No. 46717201, reviewed in BPPD 2006, 2007).

BPPD's assessment of the impact of DAS-81419-2 on Bt cotton natural refuge is contingent upon the acreage projections provided by Dow (as discussed in the previous section). A full commercial registration of DAS-81419-2 without acreage limitations would require a more detailed assessment potentially including revised natural refuge calculations and simulation modeling.

Stewardship Activities

BPPD's review indicated that other aspects of IRM including resistance monitoring and remedial action plan are needed for DAS-81419-2. A full resistance monitoring plan with pest sampling and detection bioassays should not be warranted for a seed increase registration; rather an approach based on investigations of unexpected pest damage reports should be appropriate. In addition, both TBW and CBW are monitored for Cry1Ac and Cry1F susceptibility as part of the WideStrike cotton monitoring program (see BPPD 2005). A remedial action plan, in the event of documented resistance, could mimic those in place for Bt corn or cotton or could rely on a "stop sale" approach if resistance develops.  It was also noted that Dow will need to compile DAS-81419-2 - specific annual sales and total acreage data for each state and provide a report to EPA if requested. Since DAS-81419-2 will be deployed without a structured refuge requirement, a compliance assurance plan and grower education program should not be necessary.

APPENDIX D: References

Product Characterization and Human Health

Lerouge, P. Cabanes-Macheteau, M., Rayon, C., Fichette-Lainé, A-C., Gomord, V., and Faye, L., (1998) N-Glycoprotein biosynthesis in plants: recent developments and future trends. Plant Molecular Biology 38: 31-48.
Sjoblad, Roy D., et al., (1992) Toxicological Considerations for Protein Components of Biological Pesticide Products. Regulatory Toxicology and Pharmacology 15:3-9.
U.S. Environmental Protection Agency (2005) BIOPESTICIDES REGISTRATION ACTION DOCUMENT (Bacillus thuringiensis Cry1F (synpro and Cry1Ac (synpro) Construct 281/3006 Insecticidal Crystal Proteins as expressed in cotton) (Chemical PC Codes 006512 and 006513, respectively) May 2005 Office of Pesticide Programs http://www.epa.gov/oppbppd1/biopesticides/ingredients_keep/tech_docs/brad_006512.pdf
US Environmental Protection Agency (2010a) BIOPESTICIDE REGISTRATION ACTION DOCUMENT, Bacillus thuringiensis Cry1Ac Protein and the Genetic Material (Vector PV-GMIR9) Necessary for Its Production in MON 87701 (OECD Unique Identifier: MON 877Ø1-2) Soybean [PC Code 006532]  http://www.epa.gov/pesticides/biopesticides/pips/bt-cry1ac-protien.pdf
US Environmental Protection Agency (2010b) BIOPESTICIDES REGISTRATION ACTION DOCUMENT Cry1Ab and Cry1F Bacillus thuringiensis (Bt) Corn Plant-Incorporated Protectants  http://www.epa.gov/pesticides/biopesticides/pips/cry1f-cry1ab-brad.pdf

Ecological and Environmental Effects

Abud, S., de Souza, P.I.M.,  Vianna, G.R.,  Leonardecz, E., Moreira, C.T.,  Faleiro, F.G., Junior, J.N., Monteiro, P.M.F.O., Rech, E.L. and F.J.L. Aragao. (2007) Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil. Genetics and Molecular Research 6: 445-452.
Adamczyk, J.J. Jr, and D.V. Sumerford. (2001) Potential factors impacting season long expression of Cry1Ac in 13 commercial varieties of Bollgard cotton. J Insect Sci 1: 1 - 6.
Adamczyk, J.J., Jr, William, R. and J. Meredith. (2004) Genetic bases for viability of Cry1Ac expression among commercial transgenic Bacillus thuringensis (Bt) cotton cultivars in the United States. J Insect Sci 8: 17 - 23.
Addison, S.J. and D.J. Rogers. (2010) Potential impact of differential production of the Cry2Ab and Cry1Ac proteins in transgenic cotton in response to cold stress. J Econ Entomol 103: 1206 - 1215.
Bartel, R.A. and J.O. Sexton. (2009) Monitoring habitat dynamics for rare and endangered species using satellite images and niche-based models. Ecography 32: 888-896.
Barton, B.J. and C.E. Bach. (2005) Habitat use by the federally endangered Mitchell's satyr butterfly (Neonympha mitchellii mitchellii) in a Michigan prairie fen. American Midland Naturalist 153:41-51.
Carstens, K.L., Anderson, J.A., Bachman, P., DeShrijver, A., Dively, G. Federici, B. Hamer, M., Gielkens, M., Jensen, P., Lamp, W., Raushen, S., Ridley, G., Romeis, J., and A. Waggoner. (2011) Genetically modified crops and aquatic ecosystems: considerations for environmental risk assessment and non-target organism testing. Transgenic Res. 21(4): 813-42.
Caviness, C.E. (1966) Estimates of natural cross-pollination in Jackson soybeans in Arkansas. Crop Sci. 6: 211.
Chen, D.H., Ye, G.Y., Yang, C.Q., Chen, Y. and Y.K. Wu. (2005) The effect of high temperature on the insecticidal properties of Bt cotton. Environ Exp Bot 53: 333 - 342.
Chen, F.J., Wu, G., Ge, F. and M.N. Parajulee. (2011) Relationships between exogenous-toxin quantity and increased biomass of transgenic Bt crops under elevated carbon dioxide. Ecotoxicol Environ Saf.  74: 1074 - 1080.
Conner, A.J., Glare, T.R., and J.P. Nap. (2003) The release of genetically modified crops into the environment. Part II. Overview of ecological risk assessment. The Plant Journal 33: 19-46.
Crop Protection Compendium (2004) Glycine max (soybean). CD-ROM edition, Cab International 2004, Nosworthy way, Wallingford, U.K.
Dong, H.Z., Tang, W., Li, W.J., Li, Z.H., Niu, Y.H. and D.M. Zhang. (2008) Yield, leaf senescence, and Cry1Ac expression in response to removal of early fruiting branches in transgenic Bt cotton. Agric Sci Chin 7: 692 - 702.
Duan, J.J., Marvier, M., Huesing, J., Dively, G., and Z.Y. Huang. (2008) A Meta-Analysis of Effects of Bt Crops on Honey Bees (Hymenoptera: Apidae). PLoS ONE 3(1): e1415.
Friedman, M., Brandon, D.L., Bates, A.H., and T. Hymowitz. (1991) Comparison of a commercial soybean cultivar and an isoline lacking the Kunitz trypsin inhibitor: Composition, nutritional value and effects of heating. Journal of Agricultural and Food Chemistry 39: 327-335.
Green, C.J. and A.M. Blackmer. (1995) Residue decomposition effects on nitrogen availability to corn following corn or soybean. Soil Science Society of America Journal 59: 1065-1070.
Hallikeri, S.S., Halemani, H.L., Katageri, I.S., Patil, B.C., Patil, V.C. and Y.B. Palled. (2009) Influence of sowing time and moisture regimes on Cry protein concentration and related parameters of Bt-cotton. Karnataka J Agric Sci 22: 995 - 1000.
Head, G., Surber, J.B., Watson, J.A., Martin, J.W., and J.J. Duan. (2002). No detection of Cry1Ac protein in soil after multiple years of transgenic Bt cotton (Bollgard) use. Environmental Entomology 31: 30-36.
Richard L. Hellmich, R.L.,  Siegfried, B.D., Sears, M.K., Stanley-Horn, D.E., Daniels, M.J., Mattila, H.R., Spencer, T., Bidne, K.G. and L.C. Lewis (2001) Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen. Proc Natl Acad Sc 98(21) 11925-11930
Herman, R.A., Scherer, P.N., Young, D.L., Mihaliak, C.A., Meade, T., Woodsworth, A.T., Stockhoff, B.A., and K.E. Narva. (2002) Binary insecticidal crystal protein from Bacillus thuringiensis, strain PS149B1: Effects of individual protein components and mixtures in laboratory bioassays. Journal of Economic Entomology 95: 635-639.
Icoz, I. and G. Stotzky. (2008) Fate and effects of insect-resistant Bt crops in soil ecosystems. Soil Biology and Biochemistry 40: 559-586.
Jensen, P.D., Dively, G.P., Swan, C.M., and W.O. Lamp. (2010) Exposure and non-target effects of transgenic Bt corn debris in streams. Environ. Entomol. 39(2): 707-714.
Jiang, L.J., Duan, L.S., Tian, X.L., Wang, B.M., Zhang, H.F., Zhang, M.C. et al. (2006) NaCl salinity stress decreased Bacillus thuringiensis (Bt) protein content of transgenic Bt cotton (Gossypium hirsutum L.) seedlings. Environ Exp Bot 55: 315 - 320.
Kranthi, K.R., Naidu, S., Dhawad, C.S., Tatwawadi, A., Mate, K. Patil E, et al. (2005) Temporal and intra-plant variability of Cry1Ac expression in Bt cotton and its influence on the survival of the cotton bollworm, Helicoverpa armigera (Hübner) (Noctuidae: Lepidoptera). Curr Sci 89: 291 - 298.
Marvier, M., McCreedy, C., Regetz, J. and P. Kareiva.  (2007)  A meta-analysis of effects of Bt corn and cotton on nontarget invertebrates. Science 316: 1475 - 1477.
McClintock, J.T., Schaffer, C.R., and R.D. Sjoblad. (1995) A comparative review of the mammalian toxicity of Bacillus thuringiensis-based pesticides. Pesticide Science 45: 95-105.
Mendelsohn, M., Kough, J., Vaituzis, Z., and K. Matthews. (2003) Are Bt crops safe? Nature Biotechnology 21: 1003-1009.
National Academy of Science (2000)  Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation is available from the National Academy Press, 2101 Constitution Avenue, N.W., Lockbox 285, Washington, DC 20055;<PHONE_NUMBER> or<PHONE_NUMBER> (in the Washington metropolitan area); http://www.nap.edu.
NatureServe ( 2013) NatureServe Explorer: An online encyclopedia of life [web application]. Version 7.1. NatureServe, Arlington, Virginia. Available http://www.natureserve.org/explorer  [Accessed: November 6, 2013].
OECD (2000). Consensus document on the biology of Glycine max (L.) Merr. (Soybean). Organisation for Economic Co-operation and Development. Series on Harmonization of Regulatory Oversight in Biotechnology No. 15. ENV/JM/MONO(2000)9 http://www.oecd.org/dataoecd/16/56/46815668.pdf.
Ranjithkumar, L., Patil, B.V. and N.G. Vijaykumar (2011) Impact of irrigation and fertilizer levels on Cry1Ac protein content in Bt cotton. Res J Agric Sci 2: 33 - 35.
Raybould, A. (2006) Problem formulation and hypothesis testing for environmental risk assessments of genetically modified crops. Environmental Biosafety Research 5: 119-125.
Rochester, I.J. (2006) Effect of genotype, edaphic, environmental conditions and agronomic practices on Cry1A protein expression in transgenic cotton. J Cotton Sci 10: 252 - 262.
Rodrigo-Simón, A., de Maagd, R.A., Avilla, C., Bakker, P.L., Molthoff, J., González- Zamora, J.E., and J. Ferré, (2006) Lack of Detrimental Effects of Bacillus thuringiensis Cry Toxins on the Insect Predator Chrysoperla carnea: a Toxicological, Histopathological, and Biochemical Analysis. Environ Microbiol 72: 1595-1603.
Rosi-Marshall E. J., Tank, J. L., Royer, T. V., Whiles, M. R., Evans-White, M.,  Chambers, C., Griffiths, N. A., Pokelsek, J. and M.L. Stephen. (2007) Toxins in transgenic crop byproducts in Bacillus thuringiensis Cry1Ac may affect headwater stream ecosystems. Proc Natl Acad Sc 104: 16204 - 16208.
Romeis, J., M. Meissle, and F. Bigler (2006) Transgenic Crops expressing Bacillus thuringiensis toxins and biological control. Nature Biotechnology 24:  63-71.
Sanvido,O., Romeis, J., and F. Bigler. (2007) Ecological Impacts of Genetically Modified Crops: Ten Years of Field Research and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107: 235 - 278.
Shan, G., Embrey, S.K., Herman, R.A., and R. McCormick. (2008) Cry1F protein not detected in soil after three years of transgenic Bt corn (1507 corn) use. Environmental Entomology 37: 255-262.
Sims, S.R. and J.W. Martin. (1997) Effect of the Bacillus thuringiensis insecticidal proteins Cry1A(b), Cry1A(c), cryiia, and cryiiia on folsomia candida and xenylla grisea (insecta: Collembola). Pedobiologia 41: 412-416.
Swan, C.M., Jensen, P.D., Dively, G.P., and W.O. Lamp, W.O. (2009) Processing of transgenic crop residues in stream ecosystems. J. App. Ecol. 46: 1304-1313.
Tian, J.C., Wang, X.P., Long, L.P., Romeis, J., Naranjo, S.E. et al. (2013) Bt Crops Producing Cry1Ac, Cry2Ab and Cry1F Do Not Harm the Green Lacewing, Chrysoperla rufilabris. PLoS ONE 8(3): e60125.
Torres, J.B., Ruberson, J.R. and M.J. Adang. (2006) Expression of Bacillus thuringiensis Cry1Ac protein in cotton plants, acquisition by pests and predators: a tritrophic analysis. Agric For Entomol 8: 191 - 202.
United States Environmental Protection Agency (US EPA) (1998). "Guidelines for Ecological Risk Assessment."  EPA 630/R-95-002F. Washington, DC, USA. [Federal Register, May 14, 1998. 63(93): 26846-26924.]
US EPA (2000) Scientific Advisory Panel (SAP) Report No. 99-06. Sets of scientific issues being considered by the Environmental Protection Agency regarding: Section I - Characterization and Nontarget Organism Data Requirements for Protein Plant Pesticides. Dated February 4, 2000. Available at the EPA website: http://www.epa.gov/scipoly/sap/1999/index.htm#december
US EPA (2001a) SAP Report No. 2000-07. Sets of scientific issues being considered by the Environmental Protection Agency regarding: Bt plant-pesticides risk and benefit assessments. Dated March 12, 2001.
US EPA (2001b) Biopesticides Registration Action Document for Bacillus thuringiensis (Bt) Plant-Incorporated Protectants, dated October 21, 2001. US Environmental Protection Agency, Washington, D.C.
US EPA (2002) SAP Report No. 2002-05. A set of scientific issues being considered by the Environmental Protection Agency regarding: Corn rootworm plant-incorporated protectant nontarget insect and insect resistance management issues. Dated November 6, 2002.
US EPA (2004a) SAP Report No.2004-05.  Product characterization, human health risk, ecological risk, and insect resistance management for Bt cotton products. Dated August 19, 2004.
US EPA (2004b) Biopesticides Registration Action Document for  event TC1507 (Trade name: Herculex[TM] I) and DAS-06275-8 Maize expressing Bacillus thuringiensis Cry1F . (Chemical PC Codes 006481 and 006491, respectively); US Environmental Protection Agency, Washington, D.C.
US EPA (2005) Biopesticides Registration Action Document for WideStrike cotton expressing Bacillus thuringiensis Cry1F (synpro) and Cry1Ac (synpro) Construct 281/3006 Insecticidal Crystal Proteins as expressed in cotton (Trade name: WideStrike cotton; OECD Unique ID No: DAS-21Ø23-5 x DAS-24236-5 (Chemical PC Codes 006512 and 006513, respectively); US Environmental Protection Agency, Washington, D.C.
US EPA (2007) White Paper on Tier-Based Testing for the Effects of Proteinaceous Insecticidal Plant-Incorporated Protectants on Non-Target Arthropods for Regulatory Risk Assessments. April 6, 2007. US Environmental Protection Agency, Washington, D.C.
US EPA (2010a) Biopesticides Registration Action Document for  Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Genetic Material Necessary for Their Production (PHP17662 T-DNA) in Event DAS-59122-7 Maize (OECD Unique Identifier: DAS-59122-7). US Environmental Protection Agency, Washington, D.C.
US EPA (2010b) Biopesticides Registration Action Document  -  Modified Cry3A Protein and the Genetic Material Necessary for its Production (Via Elements of PZM26) in Event MIR604 Maize SYN-IR604-8. US Environmental Protection Agency, Washington, D.C.
US EPA (2010c) Biopesticides Registration Action Document  -  Bacillus thuringiensis Cry1Ac Protein and the Genetic Material Necessary for its Production in Event MON8771  (OECD Unique Identifier: MON 877Ø1-2) Soybean. US Environmental Protection Agency, Washington, D.C.
US EPA (2013) Review of Product Characterization and Human Health data in support for Sec. 3 Registration of PIP event DAS-81419-2 soybean expressing Cry1F and Cry1Ac insecticidal proteins; U.S. EPA Memorandum from C. Wozniak, through J. Kough, to D. Greenway. US Environmental Protection Agency, Washington, D.C.
US FWS (1997) Recovery Plan for Mitchell's Satyr Butterfly (Neonympha mitchellii mitchellii French), Ft. Snelling, MN. United States Fish and Wildlife Service. US Fish and Wildlife Services (2009) Mitchell's Satyr Butterfly Spotlight Species Action Plan. Ft. Snelling, MN. http://ecos.fws.gov/docs/action_plans/doc3038.pdf
Wolt, J.D. and R.K.D. Peterson (2010) Prospective formulation of environmental risk assessments: Probabilistic screening for Cry1A(b) maize risk to aquatic insects. Ecotoxicol. Environ. Safety 73: 1182-1188.
Yoshimura, Y., K. Matsuo, and K. Yasuda. (2006) Gene flow from GM glyphosate-tolerant to conventional soybeans under field conditions in Japan. Environmental Biosafety Research 5:169-173.
Yu, H., Li, Y., Xiangju, L., Romeis, J. and K. Wu. (2013) Expression of Cry1Ac in transgenic Bt soybean lines and their efficiency in controlling lepidopteran pests. Pest Manag Sci doi: 10.1002/ps.3508.

Insect Resistance Management

Barbara, K.A., 2008.  University of Florida, Department of Entomology and Nematology informational web page for velvetbean caterpillar (Anticarsia gemmatalis):  http://entnemdept.ufl.edu/creatures/field/velvetbean.htm.   Publication Number: EENY-151.
BPPD, 2001.  Bt plant-incorporated protectants October 15, 2001 Biopesticides Registration Action Document.  Available at http://www.epa.gov/pesticides/biopesticides.
BPPD, 2005. Biopesticides Registration Action Document:  Bacillus thuringiensis var. aizawai Cry1F and the genetic material (from the insert of plasmid pgma281) necessary for its production in cotton and Bacillus thuringiensis var. kurstaki Cry1Ac and the genetic material (from the insert of plasmid pmyc3006) necessary for its production in cotton) (Chemical PC Codes 006512 and 006513, respectively).  September, 2005.  Available at http://www.epa.gov/opp00001/chem_search/reg_actions/registration/decision_PC-006512_1-Sep-05.pdf.
BPPD, 2006.  Review of data and support materials submitted by Monsanto to amend the insect resistance management requirements for Bollgard II Bt cotton.   A. Reynolds and S. Matten memorandum to L. Cole, May 16, 2006.
BPPD, 2007.   Scientific review of a proposal by Monsanto to replace structured refuge with natural refuge for Bollgard II Bt cotton (resubmitted amendment request to address the concerns of the FIFRA SAP).  A. Reynolds and S. Matten memorandum to M. Mendelsohn, May 3, 2007.
BPPD, 2010. Biopesticide Registration Action Document:  Cry1Ab and Cry1F Bacillus thuringiensis (Bt) Corn Plant-Incorporated Protectants.  Updated September, 2010.  Available at http://www.epa.gov/pesticides/biopesticides/pips/cry1f-cry1ab-brad.pdf.
Capinera, J.L., 2005.  University of Florida, Department of Entomology and Nematology informational web page for soybean looper (Pseudoplusia includens):  http://entnemdept.ufl.edu/creatures/veg/leaf/cabbage_looper.htm.  Publication Number: EENY-116.
Caprio, M., 1998. Evaluating resistance management strategies for multiple toxins in the presence of external refuges.  J. Econ. Entomol.  91: 1021-1031.
Gould, F., A. Anderson, A. Reynolds, L. Bumgarner, and W. Moar, 1995. Selection and genetic analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high levels of resistance to Bacillus thuringiensis toxins. J. Econ. Entomol. 88: 1545-59.
Jackson, R. E., J. R. Bradley, J. W. Van Duyn, B. R. Leonard, R. Luttrell, J. Ruberson, J. J. Adamczyk, J. Gore, D. Hardee, R. Voth, S. Sivasupramaniam, J. W. Mullins and G. Head, 2008. Regional assessment of Helicoverpa zea (Lepidoptera:  Noctuidae) populations on cotton and non-cotton crop hosts.  Entomol. Exp. Appl. 126 (2): 89-106.
MacRae T.C., M.E. Baur, D.J. Boethel, B.J. Fitzpatrick, A.G. Gao, J.C. Gamundi, L.A. Harrison, V.T. Kabuye, R.M. McPherson, J.A. Miklos, M.S. Paradise, A.S. Toedebusch, A. Viegas, 2005.  Laboratory and field evaluations of transgenic soybean exhibiting high-dose expression of a synthetic Bacillus thuringiensis cry1A gene for control of Lepidoptera.  J Econ Entomol.  98 (2):  577-87.
Pedigo, L.P., 1999.  Entomology and Pest Management (Third Edition).  Prentice Hall: New Jersey.  691 pp.
Roush, R.T., 1998.  Two toxin strategies for management of insecticidal transgenic crops:  will pyramiding succeed where pesticide mixtures have not?  Phil. Trans. R. Soc. Lond. 353:  1777-1786.
Sheck, A. L. and F. Gould, 1993. The genetic basis of host range in Heliothis virescens: larval survival and growth. Entomol. Exp. Appl. 69: 157 - 172.
Zhao, J., J. Cao, Y. Li, H. Collins, R. Roush, E. Earle, and A. Shelton, 2003. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution.  Nature Biotechnology.  21: 1493-1497.

Other

Ferré, J and J. Van Rie, 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis, J Annu Rev Entomol, 47:501-33.

Appendix E. Bibliography of data submitted for registration of Bacillus thuringiensis Cry1Ac and Cry1F Proteins expressed in Event DAS-81419-2 Soybean.
Table E. 1.	Data Submitted for Cry 1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean
                                   MRID No.
                                   Citation
                                 Receipt Date
49024100
Dow AgroSciences LLC (2012) Submission of Pesticide Use, Product Chemistry and Toxicity Data in Support of the Application for Registration of DAS-81419-2 Soybean. Transmittal of 10 Studies.
18-Dec-2012
49024101
Unknown (2012) Supplement to MRID 488284-01: Molecular Characterization of DAS/81419-2 Soybean. Unpublished study prepared by Dow AgroSciences. 5p.
18-Dec-2012
49024102
Embrey, S. (2012) Characterization for Soy Leaf Tissue Samples (TSN303074, TSN303075). Project Number: 120416. Unpublished study prepared by Dow AgroSciences LLC. 2p.
18-Dec-2012
49024103
Unknown (2012) Supplement to MRID 48828409: Similarity Assessment of Cry1Ac Protein to Known Toxins by Bioinformatics Analysis: (DAS-81419-2 Soybean). Project Number: 120416. Unpublished study prepared by Dow AgroSciences LLC. 1p.
18-Dec-2012
49024104
Unknown (2012) Supplement to MRID 48848216: Characterization of the Cry1Ac Protein Derived from Transgenic Soybean Event DAS-81419-2. Project Number: 110840. Unpublished study prepared by Dow AgroSciences LLC. 3p.
18-Dec-2012
49024105
Unknown (2012) Supplement to MRID 48828417: Characterization of the Cry1F Protein Derived from Transgenic Soybean Event DAS-81419-2. Unpublished study prepared by Dow AgroSciences LLC. 3p.
18-Dec-2012
49024106
Unknown (2002) Certificate of Analysis for Test/Reference/Control Substances (TSN103748): (DAS-81419-2 Soybean). Project Number: BIOT023159. Unpublished study prepared by Dow AgroSciences LLC. 7p.
18-Dec-2012
49024107
Unknown (2012) Supplement to MRID 488284-10: An Acute Oral Toxicity Study with the Northern Bobwhite: (DAS-81419-2 Soybean). Unpublished study prepared by Dow AgroSciences LLC. 1p.
18-Dec-2012
49024108
Unknown (2002) Certificate of Analysis for Test/Reference/Control Substances (TSN103748): (DAS-81419-2 Soybean). Project Number: BIOT023159. Unpublished study prepared by Dow AgroSciences LLC. 7p.
18-Dec-2012
49024109
Unknown (2012) Supplement to MRID 48828413: Potential Impact of Cry1F and Cry1Ac in DAS-81419-2 Soybean on Non-Target Organisms and Threatened and Endangered Species. Unpublished study prepared by Dow AgroSciences LLC. 2p.
18-Dec-2012
49024110
Storer, N. (2012) Insect Resistance Management Implications of Seed-Production and Research Registration for DAS-81419-2 Soybean - Revised. Project Number: IRM/2012/08. Unpublished study prepared by Dow AgroSciences LLC. 19p.
18-Dec-2012
49044800
Dow AgroSciences, LLC (2013) Submission of Product Chemistry, Efficacy, Toxicity and Exposure/Risk Data in Support of the Application for Registration of DAS-81419-2 Soybean. Transmittal of 9 Studies.
25-Jan-2013
49044801
Storer, N. (2012) Insect Resistance Management Implications of Seed-Production and Research Registration for DAS-81419-2 Soybean - Revised. Project Number: IRM/2012/08. Unpublished study prepared by Dow AgroSciences, LLC. 19p.
25-Jan-2013
49044802
Guttikonda, S. (2013) Response to EPA Comment on Molecular Characterization of DAS-81419-2 Soybean Study - Provide Information Concerning the Source of the Promoters and Terminators; Provide the Actual construction of the Cry Proteins; Related to MRID 488284-01. Project Number: IRSOY/2013/01. Unpublished study prepared by Dow AgroSciences, LLC. 8p.
25-Jan-2013
49044803
Han, L. (2013) Response to EPA Comment on Biological Equivalency of (DAS-81419-2) and Microbe-Produced Cry1Ac and Cry1F Proteins Study - Provide Explanation to Establish the Equivalence of Dow''s Cry1Ac and Cry1F Proteins to those Expressed in the Soybean; Identify the Leaf Powder Diluent Used in the Insect Bioassays: Related to MRID 488284-03. Project Number: IRSOY/2013/02. Unpublished study prepared by Dow AgroSciences, LLC. 8p.
25-Jan-2013
49044804
Han, L. (2013) Response to EPA Comment on Similarity Assessment of Cry1Ac Protein to Known Toxins Bioinformatics Analysis Study - Identify the Protein Toxin Source; Explain the Error on Page 11 of 16: Related to MRID 488284-09. Project Number: IRSOY/2013/03. Unpublished study prepared by Dow AgroSciences, LLC. 5p.
25-Jan-2013
49044805
Han, L. (2013) Response to EPA Comment on Characterization of the Cry1Ac Protein Derived from Transgenic Soybean Event DAS-81419-2 Study - Address Amino Acid Sequence Similarity between Dow''s Cry1Ac Microbial Protein and Soybean-Produced Cry1Ac; Provide a Replacement for the Illegible Figure on Page 55: Related to MRID 488284-16. Project Number: IRSOY/2013/04. Unpublished study prepared by Dow AgroSciences, LLC. 8p.
25-Jan-2013
49044806
Han, L. (2013) Response to EPA Comment on Characterization of the Cry1F Protein Derived from Transgenic Soybean Event DAS-81419-2 Study - Address Amino Acid Sequence Similarity between Dow''s Cry1F Microbial Protein and Soybean-Produced Cry1F: Related to MRID 488284-14. Project Number: IRSOY/2013/05. Unpublished study prepared by Dow AgroSciences, LLC. 7p.
25-Jan-2013
49044807
Herman, R. (2013) Response to EPA Comment on Cry1F(Synpro) ICP and Cry1Ac(Synpro) ICP: An Acute Oral Toxicity Study with the Northern Bobwhite - Provide Data Demonstrating the Equivalence of Dow''''s Test Material #101811 to Plant-produced Protein; Provide a Justification of the Test Dosage: Related to MRID 488284-10. Project Number: IRSOY/2013/06. Unpublished study prepared by Dow AgroSciences, LLC. 14p.
25-Jan-2013
49044808
Han, L. (2013) Response to EPA Comment on Cry1F(Synpro) and Cry1Ac(Synpro) Insecticidal Crystal Proteins: An 8-Day Dietary Study with the Rainbow Trout Oncorhynchus mykiss, WALBAUM - Provide Data Demonstrating the Equivalence of Dow''s Test Material #101811 to Plant-produced Protein: Related to MRID 488284-11. Project Number: IRSOY/2013/07. Unpublished study prepared by Dow AgroSciences, LLC. 13p.
25-Jan-2013
49044809
Han, L. (2013) Response to EPA Comment on Potential Impact of Cry1F and Cry1Ac in DAS-81419-2 Soybean on Non-target Organisms and Threatened and Endangered Species Study - Provide Gene Flow and Weediness Information to Address Environmental Fate Concerns; Address Soil Degradation: Related to MRID 488284-13. Project Number: IRSOY/2013/08. Unpublished study prepared by Dow AgroSciences, LLC. 6p.
25-Jan-2013
49081500
Dow AgroSciences, LLC (2013) Submission of Pesticide Use Data in Support of the Application for Registration of DAS-81419-2 Soybean. Transmittal of 1 Study.
14-Mar-2013
49081501
Han, L. (2013) Amended Response to the EPA Comment on Characterization of the Cry1F Protein Derived from Transgenic Soybean Event DAS-81419-2 Study - Address Amino Acid Sequence Similarity Between Dow's Cry1F Microbial Protein and Soybean-Produced Cry1F. Project Number: IRSOY/2013/05. Unpublished study prepared by Dow AgroSciences, LLC. 7p.
14-Mar-2013