The purpose of this study was to determine the presence of any vector backbone sequences in Event GHB119  cotton after insertion of the intended cry2Ae gene cassette via the pTEM12 transformation vector. Isolated genomic DNA samples from leaves of cotton Event GHB119  and from the non-transgenic negative control were subjected to Southern blot analysis, after digestion with restriction enzymes EcoRV and NotI, using seven overlapping vector backbone probes that encompassed the entire backbone of the pTEM12 transformation vector used to produce this transformation event. The vector backbone probes were then stripped from the membranes so that they could be re-hybridized with the T-DNA probe, as a positive control. Genomic DNA from the Event GHB119 cotton showed no hybridization with any of the vector backbone probes, while the positive control showed hybridization as expected. The results confirmed the absence of vector backbone sequences after insertion of the intended cry2Ae gene cassette via the pTEM12 transformation vector to produce Event GHB119 cotton.

CLASSIFICATION:  ACCEPTABLE
                                   48480002

                   [This study supersedes MRID No. 47641901]
Characterization of Cry2Ae and PAT/bar proteins expressed in Gossypium hirsutum transformation Event GHB119
The purpose of this study was to determine the biochemical and functional equivalence of plant-expressed Cry2Ae protein produced in Event GHB119cotton plants with bacterial-expressed Cry2Ae proteins (Batch Nos. VMLV1041-1 and NB210806P25 produced from recombinant Bacillus thuringiensis  and  Escherichia coli over-expression system, respectively) for their use as suitable test substance surrogates in toxicological testing. Equivalence studies were also conducted for plant- expressed PAT protein in Event GHB119 plants with bacterial-expressed PAT protein (Batch Nos. NB010905P44P2) for use as a test substance surrogate. Six analytical tests were used to assess the biochemical and functional  equivalence, which included: 1) estimation of molecular weight by observing protein mobility in SDS-PAGE, 2) verification of immunoreactivity using Western blot analysis,  3) analysis of glycosylation using the reaction with glycostain, 4) mass spectroscopic peptide mapping using HPLC/Electrospray Mass Spectrometry (LC/MS) of peptides, 5) analysis of N-terminal sequencing using Edman degradation, and , 3) analysis of biological activity using a sensitive insect bioactivity feeding assay to determine the  insecticidal activity for the Cry2Ae proteins and  enzymatic activity for the PAT/bar protein,

The Cry2Ae protein purified from GHB119 cotton plants and the bacterial-produced batches were found to have a similar molecular weight after electrophoresis, similar immunoreactive bands based on Western Blot analyses and non-glycosylated. Identity of the plant-produced Cry2Ae protein and the bacterial-produced protein batches was confirmed by peptide mapping using electrospray mass spectrometry. After CNBr treatment and tryptic digest, 66% of the plant Cry2Ae protein, 73% of bacterial-produced batch VMLV1041-1 and 75% of bacterial-produced batch NB210806P25 were identified. The N-terminal sequence of the Cry2Ae protein batches was confirmed by Edman degradation. N-terminal sequencing also confirmed that the transit peptide of the plant form was cleaved at the expected residue. Biological activity was assessed by an insect bioassay using Heliothis virescens first instar larvae and the obtained LC50 for the plant-produced Cry2Ae protein was 296 ng/cm[2], while the bacterial-produced Cry2Ae proteins from Batch Nos. VMLV1041-1 and NB210806P25 were 311 ng/cm[2] and 346 ng/cm[2], respectively, demonstrating that the plant- and bacterial-produced proteins have comparable biological activity.

Thus, both the bacterial-expressed Cry2Ae (Batch No. VMLV1041-1 and NB210806P25) and PAT (Batch No. NB010905P44P2) proteins are suitable test substance surrogates for use in toxicological testing for establishing the safety of the plant-produced Cry2Ae and PAT proteins as expressed in Event GHB119 cotton.

CLASSIFICATION:  ACCEPTABLE
                                   48480006

            [This study supersedes MRID No. 47641905 and 47641906]

B. II.  	HUMAN HEALTH ASSESSMENT  -   Cry2Ae PIP

Section 408(c)(2)(A)(i) of the Federal Food, Drug, and Cosmetic Act (FFDCA) allows EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if EPA determines that the exemption is "safe." Section 408(c)(2)(A)(ii) of the FFDCA defines "safe" to mean that "there is a reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other exposures for which there is reliable information." This includes exposure through drinking water and in residential settings, but does not include occupational exposure. Pursuant to section 408(c)(2)(B), in establishing or maintaining in effect an exemption from the requirement of a tolerance, EPA must take into account the factors set forth in section 408(b)(2)(C), which require EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance and to "ensure that there is a reasonable certainty that no harm will result to infants and children from aggregate exposure to the pesticide chemical residue... ."

Additionally, section 408(b)(2)(D) of the FFDCA requires that the Agency consider "available information concerning the cumulative effects of a particular pesticide's residues" and "other substances that have a common mechanism of toxicity." EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide residues. First, EPA determines the toxicity of pesticides. Second, EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings.

a.  Toxicological Profile

BCS submitted a pesticide tolerance petition (PP 9F7514) under the FFDCA, requesting 40 CFR Part 174 be amended  by establishing an exemption from the requirement of a tolerance for residues of Bacillus thuringiensis (Bt) Cry2Ae insect control protein and the genetic material necessary for its production in or on all food commodities. The toxicological profile of the protein was previously described in the Federal Register of September 10, 2008 (73FR 52591: FRL-8380-1) establishing a temporary tolerance exemption for Cry2Ae protein in or on cotton food/feed commodities (40 CFR §174.530). The data reviewed to support the petition for establishing a permanent tolerance exemption are based upon the biochemical and functional characteristics of Bt Cry2Ae protein and its expression analyses in cotton. No data were submitted for use of this protein as a PIP in other crops nor do any currently approved PIP Events express Cry2Ae protein. Since the submitted data are specific to Cry2Ae protein expressed in cotton, the exemption from the requirement of a tolerance of Cry2Ae protein residues may only be supported in or on cotton food/feed commodities.

Consistent with section 408(b)(2)(D) of the FFDCA, EPA has reviewed the available scientific data and other relevant information in support of this action and considered its validity, completeness and reliability, and the relationship of this information to human risk. EPA has also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.

1) Mammalian Toxicity and Allergenicity Assessment  -  Cry2Ae PIP

BCS submitted acute oral toxicity data to demonstrate the lack of mammalian toxicity at high levels of exposure to the pure Cry2Ae protein (MRID No. 47076902). The acute oral toxicity of Cry2Ae protein was assessed by orally dosing (via gavage) to five female mice with 2,000 milligrams/kilograms bodyweight of purified bacterial-produced Cry2Ae protein test substance (Batch No. NB210806P25,  >93% purity). All treated animals gained weight and had no test material-related clinical signs and no test material-related findings at necropsy. The acute oral LD50 of the Cry2Ae protein is greater than 2000 mg/kg body weight. The Cry2Ae protein does not appear to cause any significant adverse effects at an exposure level of up to 2000 mg/kg bodyweight and supports the finding that the Cry2Ae protein would be non-toxic to mammals.

These data demonstrate the safety of the product at a level of 2000 mg/kg b.w. that is well above maximum possible ppm exposure levels that are reasonably anticipated in the crop (see Table B.1. above). Basing this conclusion on acute oral toxicity data without requiring further toxicity testing and residue data is similar to the Agency position regarding toxicity testing and the requirement of residue data for the microbial Bacillus thuringiensis products from which this plant-incorporated protectant was derived (see 40 CFR §§ 158.2130(d)(1)(i) and 158.2140(d)(7)). For microbial products, further toxicity testing and residue data are triggered by significant adverse acute effects in studies such as the acute oral toxicity study, to verify the observed adverse effects and clarify the source of these effects (Tiers II & III). Since there were no adverse effects in the Tier I acute oral study, further Tiers II and III toxicity testing and residue data were not required.

2) Allergenicity Assessment

When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad et al. 1992). Since no acute effects were shown to be caused by Cry2Ae protein, even at relatively high dose levels, the Cry2Ae protein is not considered toxic. Further, amino acid sequence comparisons showed no similarities that would raise a safety concern between the Cry2Ae protein and known toxins in protein databases.

Since Cry2Ae is a protein, allergenic sensitivities were considered. Currently, no definitive tests exist for determining the allergenic potential of novel proteins. Therefore, EPA uses a weight-of- evidence approach where the following factors are considered: source of the trait; amino acid sequence similarity with known allergens; prevalence in food; and biochemical properties of the protein, including in vitro digestibility in simulated gastric fluid (SGF) and glycosylation (as recommended by Codex Alimentarius Commission, 2003). Current scientific knowledge suggests that common food allergens tend to be resistant to degradation by acid and proteases; may be glycosylated; and present at high concentrations in the food.

Summaries of the allergenicity assessment for Cry2Ae protein include:

      1. Source of the trait. Bacillus thuringiensis, the microorganism from which Cry2Ae protein is derived, is not considered to be a source of allergenic proteins (MRID No. 47125101).
2. Amino acid sequence. A comparison of the amino acid sequence of Cry2Ae with known allergens showed no overall sequence similarity using the criterion of 35% identity over an 80 amino acid segment or identity at the level of eight contiguous amino acid residues (MRIDs No. 47641908 and 47641909).
      3. Prevalence in food. Protein expression level analyses of Cry2Ae protein confirm its presence at relatively low levels. Dietary exposure is extremely limited and expected to be correspondingly low. Expression in Event GHB119 ranged from 0.99 to 1.55 ug/g or ppm for cottonseed. Thus, the expression has been shown to be in the parts per million range (MRID No. 47641903).
      4. Digestibility. The Cry2Ae protein was rapidly digested within 30 seconds in simulated mammalian gastric fluid containing pepsin at a pH of 1.2 (MRID No. 47125102).
5. Glycosylation. The Cry2Ae protein expressed in cotton was shown not to be glycosylated. (MRIDs No. 48471901 and 48480006).

Considering all of the available information, EPA concluded that the potential for Cry2Ae protein to be a food allergen is minimal.

b. Aggregate Exposures

Pursuant to FFDCA section 408(b)(2)(D)(vi), EPA considers available information concerning aggregate exposures from the pesticide residue in food and all other non-occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses).

The Agency considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other related substances. These considerations include dietary exposure under the tolerance exemption and all other tolerances or exemptions in effect for the plant-incorporated protectants chemical residue, and exposure from non-occupational sources. Exposure via the skin or inhalation is not likely since the plant- incorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to negligible. The amino acid homology assessment included similarity to known aeroallergens. It has been demonstrated that there is no evidence of occupationally related respiratory symptoms, based on a health survey on migrant workers after exposure to Bt pesticides (Bernstein et al. 1999). Exposure via residential or lawn use to infants and children is also not expected because the use sites for the Cry2Ae protein are all agricultural for control of specific insect pest species. Although cotton is not a directly consumed food commodity, humans may be potentially exposed to extremely low levels in the diet, from ingestion of processed cotton products (e.g. cottonseed flour and oil) and, potentially, drinking water.

The oral toxicity study conducted at a dose of 2,000 mg/kg b.w. showed no adverse effects. Cottonseed may be processed to edible food commodities, cottonseed flour and cottonseed oil. In cottonseed from the parental strain and TwinLink(TM), Cry2Ae protein is expressed in the ppm levels (see Allergenicity Assessment above, and Tables B.1 and B.4 in this Appendix). These levels are several orders of magnitude lower than the amounts of Cry2Ae protein shown to have no toxicity. Even if low levels of dietary exposure should occur, no harm is expected, based upon the lack of mammalian toxicity and the rapid digestibility demonstrated for the Cry2Ae protein.

c. Cumulative Effects from Substances with a Common Mechanism of Toxicity

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance, the Agency consider "available information" concerning the cumulative effects of a particular pesticide's residues and "other substances that have a common mechanism of toxicity."

Cry2Ae protein is not considered to be toxic, and does not share a common mechanism of toxicity with any other substance. In addition, Cry2Ae protein does not appear to produce a toxic metabolite produced by other substances. EPA concludes that there are no cumulative effects associated with Cry2Ae protein that need be considered. For information regarding EPA's efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see EPA's website at http://www.epa.gov/pesticides/cumulative

d. Determination of Safety for U.S. Population, Infants and Children

The data submitted and cited regarding potential health effects for the Cry2Ae protein include the characterization of the expressed Cry2Ae protein in cotton, as well as the acute oral toxicity, heat stability, and in vitro digestibility of the protein. The results of these studies were used to evaluate human risk, and the validity, completeness, and reliability of the available data from the studies were also considered.
As discussed more fully in Appendix B.II.a., above, the acute oral toxicity data submitted supports the prediction that the Cry2Ae protein would be nontoxic to humans. Moreover, Cry2Ae protein has no sequence similarity to any known toxin. Because of this lack of demonstrated mammalian toxicity, no protein residue chemistry data for Cry2Ae were required for a human health effects assessment. Even so, expression level analysis showed Cry2Ae protein is present at relatively low levels. Dietary exposure is expected to be correspondingly low.
Since Cry2Ae is a protein, its allergenic potential was considered as part of the toxicity assessment. Data considered as part of the allergenicity assessment include that the Cry2Ae protein:
   a) came from Bacillus thuringiensis, which is not a known allergenic source;
   b) showed no sequence similarity to known allergens;
   c) was readily degraded by pepsin; and
   d) was not glycosylated when expressed in the plant.
On this basis, EPA concluded there is a reasonable certainty that Cry2Ae protein will not be a food allergen. Considered together, the lack of mammalian toxicity at high levels of exposure to the Cry2Ae protein and the minimal potential for that protein to be a food allergen demonstrate the safety of the product at levels well above possible maximum exposure levels anticipated in the crop.

Finally, and specifically with regards to infants and children, FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pesticide chemical residues, and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity. In addition, FFDCA section 408(b)(2)(C) provides that EPA shall apply an additional tenfold margin of safety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the data base unless EPA determines that a different margin of safety will be safe for infants and children.

Based on all of the available information described in this BRAD,, the Agency concludes that there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of the Cry2Ae protein and the genetic material necessary for its production in cotton. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency also concluded that there are no threshold effects of concern and, as a result, that an additional margin of safety for infants and children is unnecessary in this instance.

e. Other Considerations

1) Endocrine Disruptors

The pesticidal active ingredient is a protein that breaks down readily in dietary exposure and is derived from a source not known to exert an influence on the endocrine system. Therefore, the Agency is not requiring information on the endocrine effects of the plant-incorporated protectant at this time.

2) Analytical Method

BCS provided a validation study for EnviroLogix's QuantiPlate(TM) Kit for Cry2A (Cat. No. AP005), which uses enzyme-linked immunosorbent assay (ELISA) analyses for the detection of Cry2Ae protein residues in Event GHB119 cotton. Results showed the test kit is able to detect Cry2Ae protein residues in cotton with sufficient accuracy, precision, and sensitivity. However, a validation study conducted by an independent 3[rd] party laboratory is still needed to evaluate the performance of the ELISA test kit as the designated residue analytical method for the detection of Cry2Ae protein expressed in Event GHB119 cotton.

3) International Residue Limits

In making its tolerance decisions, EPA seeks to harmonize U.S. tolerances with international standards whenever possible, consistent with U.S. food safety standards and agricultural practices. In this context, EPA considers the international maximum residue limits (MRLs) established by the Codex Alimentarius Commission (Codex), as required by FFDCA section 408(b)(4). The Codex Alimentarius is a joint U.N. Food and Agriculture Organization/World Health Organization food standards program, and it is recognized as an international food safety standards-setting organization in trade agreements to which the United States is a party. EPA may establish a tolerance that is different from a Codex MRL; however, FFDCA section 408(b)(4) requires that EPA explain the reasons for departing from the Codex level. No Codex maximum residue level exists for the plant-incorporated protectant Bacillus thuringiensis Cry2Ae protein.

f. Overall Safety Conclusion

There is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, including infants and children, to the residues of Cry2Ae protein and the genetic material necessary for its production. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency has arrived at this conclusion because, as discussed above, no toxicity to mammals has been observed, nor any indication of allergenicity potential for the plant-incorporated protectant.
The data submitted and reviewed for Cry2Ae protein support the petition for an exemption from the requirement of tolerance for Bacillus thuringiensis Cry2Ae protein when used as plant -  incorporated protectant in or on cotton food/feed commodities.
G. Supporting Data

The human health studies submitted to support the safety of Cry2Ae protein are summarized in Table B.3 below. For detailed information of each study, please refer to the individually reviewed studies in the Data Evaluation Reports (DERs) according to its respective assigned MRID number).

Table B.3. Human Health Data for Event GHB119 cotton expressing Bt Cry2Ae protein
                          (EPA File Symbol 264-RNOL)

                                  Study Title
                 B.3. Summary  -  Human health  -  Cry2Ae PIP
                                    MRID No
Description of the Amino Acid Sequence of the Cry2Ae Protein
The 631 amino acid sequence of the Cry2Ae protein is provided in this submission. No information is provided on how the sequence was determined, though it was presumably deduced from the inserted sequence of the gene, and no information is provided on whether the sequence was verified by mass spectrometry or N-terminal amino acid sequencing. SDS-PAGE and western blot analyses of the plant-produced protein are provided in MRID 46708907, which indicate that the protein has the expected molecular weight.
Classification: SUPPLEMENTAL; Preliminary study used to support EUP
                                   46708902
Cry2Ae (GEM2) Protein: Overall Amino Acid Sequence Homology Search with Known Toxins and Allergens[4]
The amino acid sequence of the Cry2Ae protein was compared to sequences contained in publicly available databases (SwissProt, trEMBL, GeneSeq-Prot, PIR, PDB, DAD, and GenPept.) using the BLASTP 2.2.2 algorithm and the BLOSUM62 scoring matrix. No similarities between the Cry2Ae protein and known allergens or toxins, other than Cry toxins, were found using the criterion of 35% identity over an 80 amino acid segment.

Classification: SUPPLEMENTAL; Sufficient to support EUP and tolerance exemption; Data gaps addressed for Sec. 3 registration in MRID No. 47641910
                                   46708903
Cry2Ae (GEM2) Protein: Epitope Homology and N-Glycosylation Searches[4]
The amino acid sequence of the Cry2Ae protein was compared to known protein sequences in the Allergen database to identify any similarities between short segments of amino acids. Searches were also performed to identify any possible N-glycosylation motifs. No significant similarities between Cry2Ae protein and known allergens based on a 100% identity over an 8 amino acid segment were found. Eleven potential N-glycosylation sites were found in the Cry2Ae protein sequence.

Classification: ACCEPTABLE
                                   46708904
Cry2Ae (GEM2) Protein in vitro Digestibility Study in Simulated Gastric Fluid[4]
The digestibility of the trypsin resistant core of microbially produced Cry2Ae protein was investigated in simulated gastric fluid containing pepsin. Horseradish peroxidase and ovalbumin were included in the study as controls. The trypsinized Cry2Ae protein was degraded within two minutes of incubation in simulated gastric fluid at pH 1.2. Horseradish peroxidase and ovalbumin were degraded rapidly and slowly, respectively, as expected. However, because the protein that is produced in cotton is the full-length Cry2Ae protein, digestibility of the full-length protein is more relevant for the risk assessment.
Classification: SUPPLEMENTAL- Data gaps were addressed for EUP and Sec. 3 registration in MRID No. 47125102
                                   46708905

Cry2Ae (GEM2) Protein - Acute Toxicity by Intravenous Injection in the Mouse[4]
The acute toxicity of the trypsin resistant core of microbially produced Cry2Ae was investigated by intravenous injection in mice. The protein (>95% purity) was suspended in PBS buffer (phosphate buffered saline) and administered as a single 1 or 5 mg/kg body weight intravenous dose. Melittin protein was used as a positive control, and aprotinin protein and buffer alone were used as negative controls. Mice were observed for clinical signs daily for the duration of the study (15 days), and gross necropsy was performed at the end of the study. Four out of five mice treated with melittin at a dose of 5 mg/kg died on day 1. No other deaths occurred during the study. No treatment-related adverse effects were observed in the mice administered Cry2Ae protein at either dose.
Classification: SUPPLEMENTAL; Data gaps were addressed for EUP in MRID No. 47076902  and no additional data were required
                                   46708906
Analysis to Determine if the Cry2Ae (GEM2) Protein from Cotton Leaves is Glycosylated[4]
Cry2Ae protein, purified from Bacillus thuringiensis and from cotton leaves from Events GHB119, GHB714, and GHB866, was analyzed for glycosylation using a commercially available glycosylation test kit. The results indicate that the Cry2Ae protein is not glycosylated.
Classification: ACCEPTABLE
                                   48471901
                   [This study supersedes MRID No. 46708907]
Cry2Ae (GEM2) Protein: Acute Toxicity by Oral Gavage in Mice[4]
The acute oral toxicity of Cry2Ae protein was assessed by administering groups of five female mice Bacillus thuringiensis-produced Cry2Ae protein by oral gavage at a dose of 2000 mg/kg body weight. There were no mortalities, and no treatment-related adverse effects observed. Therefore, the acute oral LD50 of the Cry2Ae protein is greater than 2000 mg/kg body weight.
Classification: ACCEPTABLE
                                   47076902
Cry2Ae (GEM2) Protein: In Vitro Digestibility Study in Simulated Gastric Fluid[4]
The susceptibility of the Cry2Ae protein to proteolytic degradation in simulated gastric fluid containing pepsin (SGF) was investigated using Bacillus thuringiensis-produced full-length Cry2Ae protein. The band for the Cry2Ae protein was no longer visible on the Coomassie blue stained gel after incubation in SGF for 0.5 minutes. In addition, smaller degradation fragments were not observed. Horseradish peroxidase and ovalbumin were degraded rapidly and slowly, respectively, as expected. This study indicates that the Cry2Ae protein is rapidly digested in SGF at pH 1.2.
Classification: ACCEPTABLE
                                   47125102
    [This study supersedes MRID No. 46708905 and confirms MRID No. 47125102]
Cry2Ae Protein: Heat stability study
An in vitro heat stability study was conducted on bacterial-expressed Cry2Ae protein at temperatures of 60, 75 and  90°C for incubation periods of 0, 10, 30 and 60 minutes and examined by SDS-PAGE and by Western blot analysis. Results of the SDS-PAGE showed a ~59.3 kDa band for Cry2Ae protein and no significant deterioration after incu -  - ba - tion at 60ºC at all time points, but partial degradation at 75ºC for 10 through 60 minutes. After 30 minutes at 90ºC, the band was visible, but markedly decreased and by 60 minutes was no longer present. Results of the Western blot analyses showed a ~59.3 kDa immunoreactive band for Cry2Ae protein and no signifi - cant deterioration after incu - bation at 60ºC for 10, 30, or 60 minutes. At incubation of 75ºC, the protein was partially stable for 10 minutes and further decreased in stain intensity through 60 minutes. At incubation of 90ºC, the band was visible at 10 minutes, but no longer present after 30 through 60 minutes. Results showed consistent patterns of decreasing structural stability of the Cry2Ae protein in between both analyses tested. This study indicates that the Cry2Ae protein is heat labile at 90°C at 60 minutes.
Classification: ACCEPTABLE
                                   47641911
Toxicology (Human Health Assessment) Summary of Analysis of Open Reading Frame Homology Searches
The purpose of this study was to evaluate newly created ORFs that span the 5' or 3' junction of the transgenic insert (T-DNA) in cotton Event GHB119 and determine if any newly created chimeric ORFs could give rise to amino acid sequences with toxic or allergenic properties. The ORF search was performed by means of the GetORF search program from the EMBOSS (European Molecular Biology Open Software Suite). BLASTP or FindPatterns algorithms were used to search publically available databases (Uniprot-Swissprot, Uniprot-TrEMB, DAD, PDB, and GenPept). Three ORFs were found, one spanning the 5' junction and two spanning the 3' junction. No significant similarities were identified between any of the putative amino acid sequences generated by these ORFs and sequences found in databases, including known allergens and toxins.
Classification: ACCEPTABLE
                                   47641906
Cry2Ae Protein: In vitro digestibility study in simulated intestinal fluid
An in vitro digestibility study was conducted on bacterial-expressed Cry2Ae protein in simu - lated human intestinal fluid (SIF) containing pancreatin (pH 7.5) to assess the susceptibility of Cry2Ae protein to proteolytic degradation. The Cry2Ae protein was degraded into several stable fragments in the presence of pancreatin, at pH 7.5 during the 60 minute digestion.
Classification: ACCEPTABLE
                                   47641907

Cry2Ae protein with transit peptide epitope homology and N-glycosylation searches

No significant amino acid sequence similarities between eight linear contiguous amino acid blocks of the Cry2Ae protein with known aller - gens in the publically available AllergenOnline database (release 8.0, 1313 sequences) were found. Results suggest the Cry2Ae protein should not cross-react with known allergenic epitopes. Eleven potential N-glycosylation sites were identified.
Classification: ACCEPTABLE
                                   47641908
                   [This study supersedes MRID No. 47076904]
Cry2Ae protein with transit peptide overall amino acid sequence homology search with known toxins and allergens
A. Bioinformatic searches were conducted to evaluate any significant amino acid sequence similarities between the Cry2Ae protein and any known toxins in public available databases (Uniprot_SwissProt, Uniprot_TrEMBL, PDB, DAD, and GenPept.) using the FASTA algorithm and the BLOSUM62 scoring matrix; and any known allergens in the AllergenOnline database (release 8.0) using the FASTA algorithm and  BLOSUM50 scoring matrix. Comparisons were conducted on the complete amino acid sequence of the test protein with matches of >35% identity over an 80 amino acid sequence segment; and then sequentially compared using sub-divided sequences of the test protein into 80 amino acid segments with all known allergens present in the database. No similarities between the Cry2Ae protein and known allergens or toxins, other than Cry toxins, were found.
Classification: ACCEPTABLE
                                   47641909
                   [This study supersedes MRID No. 47076903]
Update on Cry2Ae protein: In vitro digestibility study in human simulated gastric fluid, overall amino acid sequence homology search with known toxins and allergens, and epitope homology and N-glycosylation searches
Bioinformatic studies on Cry2Ae protein were repeated in 2008 and showed no amino acid sequence similarities between Cry2Ae protein and known toxins and allergens (MRID No. 47641909); and no amino acid sequence similarities with known allergen epitopes and N-glyco - sylation sites (MRID No. 47641908), thus confirming original results from previously conducted studies done in 2005 (MRIDs No. 46708903 and MRID 46708904, respectively). An in vitro digestibility study of Cry2Ae protein in human simulated gastric fluid containing pepsin was repeated in 2007 (MRID No. 47125102) and showed rapid degradation of Cry2Ae protein to pepsin, thus confirming original results from the previously conducted in vitro digestibility SGF study in 2005 (MRID No. 46708905).
Classification: SUPPLEMENTAL- This study is a supplemental  to MRIDs 46708905, 46708903 and 46708904
                                   47641910
[This study is a supplemental report to MRIDs 46708905, 46708903 and 46708904]

B.III. Product characterization and human health effects for TwinLink(TM) (T304-40 x GHB119) Combination PIP Cotton Product (OECD Unique Identifier: BCS-GH4-7 x BCS-GH5-8) Expressing Cry1Ab, PAT, and Cry2Ae Proteins

BCS submitted data in support of FIFRA Sec. 3 registration of TwinLink(TM) cotton (EPA File Symbol 264-RNOA) for use as an alternative insect resistant and herbicide tolerant combination PIP cotton product. TwinLink(TM) cotton was developed by conventional breeding of the parental Events T304-40 x GHB119 to express Cry1Ab, Cry2Ae and PAT proteins. Event T304-40 cotton encodes the cry1Ab gene derived from Bt subsp. berliner (described in Höfte et al. 1986) to express Cry1Ab insecticidal protein, which also confers resistance to lepidopteran pest species. The safety of Cry1Ab protein, as expressed in the cotton Event T304-40, was previously reviewed by the Agency and the data were found acceptable to support registration of Event T304-40 cotton (US EPA 2006, 2008, and 2011a and c). A tolerance exemption exists for Cry1Ab protein residues when used as plant-incorporated protectant in all food commodities (40 CFR § 174.511) and all final data submitted for its registration were found acceptable (US EPA 2011c). Considerable safety data have been established for both Cry2Ae protein expressed in GHB119 and Cry1Ab protein expressed in T304-40 cotton. The applicant submitted the following equivalency studies in order to bridge from these existing acceptable toxicological data for the parental Events for registration of TwinLink(TM) Cotton.

a. Product Characterization

TwinLink(TM) cotton (T304-40 x GHB119 combination PIP product) contains cry1Ab, cry2Ae, and bar transgenes. Events T304-40 and GHB119 cotton were individually developed by Agrobacterium-mediated transformation to insert T-DNA containing the cry1Ab and cry2Ae gene cassettes via plasmid vectors pTDL008 and pTEM12, respectively. The resulting cotton lines were then crossed by conventional breeding to create TwinLink(TM) (GHB119 x T304-40) cotton.

Characterization of DNA isolated from TwinLink(TM) (T304-40 x GHB119) cotton using restriction enzyme digests and Southern blot analyses were used to confirm the genetic stability and integrity of the T304-40 and GHB119 transgenic inserts in TwinLink(TM) cotton. The analyses showed the presence of the expected hybridization fragments in all tested transgenic DNA samples when hybridized with each of the three probes. In addition, results showed the cry1Ab, cry2Ae, and bar DNA inserts are stably integrated in TwinLink(TM) cotton, based on Southern blot analyses of combination PIP product plants obtained after five generations from the original TwinLink(TM) cross. Data from the comparative Southern blot analyses confirmed the integrity and genetic stability of the transgenic cry1Ab, cry2Ae, and bar inserts from the individual parental events as preserved during conventional breeding to produce TwinLink(TM) cotton.

b. Analytical Detection Methods

BCS provided validation studies for commercial enzyme-linked immunosorbent assay (ELISA) test kits for the analytical detection of Cry2Ae and Cry1Ab protein residues in cottonseed and cotton leaf tissue. The studies showed the test kits are able to detect Cry2Ae and Cry1Ab protein in Events GHB119 and T304-40 cotton, respectively, with sufficient accuracy, precision, and sensitivity. Verification of the specific ELISA test kit method to be used as the proposed residue analytical method for the detection of Cry1Ab protein in Event T304-40 cotton is needed. In addition, individual validation studies conducted by an independent third party laboratory are still needed to evaluate the performance of each ELISA test kit as the designated residue analytical method for the detection of Cry2Ae and Cry1Ab proteins expressed in cotton Events GHB119 and T304-40, respectively.

c. Protein Expression

Protein expression levels were provided for Cry2Ae, PAT, and Cry1Ab in different plant tissues from T304-40 x GHB119 cotton tested at key developmental growth stages at 4 field sites. The average protein expression levels are shown below in Table 4. The Cry2Ae and Cry1Ab protein levels in TwinLink(TM) cotton are comparable to those observed in plant tissues from its parental Events GHB119 and T304-40 cotton. The PAT protein expression levels in TwinLink(TM) cotton were higher than those expressed in its parental events. These results were expected since TwinLink(TM) cotton inherited two copies of the bar gene, which were individually inserted in its parental events.

Table B.4. Mean Expression Levels of Cry2Ae, PAT and Cry1Ab Proteins from TwinLink(TM) cotton

                                  Tissue Type
                                    Cry2Ae
                    (ug/g dry weight + standard deviation)
                                      PAT
                    (ug/g dry weight + standard deviation)
                                    Cry1Ab
                    (ug/g dry weight + standard deviation)
                                Leaves (T1-T3)*
                            18 +- 6.4  -  70  +- 35
                           250 +-120  -  1200 +- 650
                           3.0 +- 2.1  -  8.8 +- 3.8
                                 Squares (T3)
                                   12 +- 4.7
                                  370 +- 160
                                  8.2 +- 3.7
                                  Bolls (T3)
                                   4.7 +-1.7
                                  240 +- 100
                                  6.5 +- 2.9
                                Cottonseed (T4)
                                  6.7 +- 3.3
                                  280 +- 130
                                   13 +- 6.5
                                Roots (T1-T3)*
                           3.9 +- 1.9  -  11 +- 5.3
                           170 +- 80  -  290 +- 130
                           2.5 +- 1.4  -  3.8 +- 1.9
      *Ranges reflect means tested at different growth stages

d. Conclusion

The DNA characterization study and protein expression analyses field study considered with comparable biological activity in between the parental cotton Events GHB119 and T304-40 and no evidence of either a synergistic or antagonistic interaction between Cry2Ae and Cry1Ab proteins in tobacco budworm (TBW, Heliothis virescens) (see Dean, B. et al. 2010; MRID No. 48480009) reviewed in the ecological risk assessment memo for this product (US EPA 2011b) demonstrate that the Cry2Ae and Cry1Ab proteins expressed in the parental cotton Events GHB119 and T304-40 are biochemically and functionally equivalent to the Cry2Ae and Cry1Ab proteins expressed in TwinLink(TM) cotton. Thus, the existing toxicological data of parental Events GHB119 and T304-40 cotton can be bridged to support the safety of TwinLink(TM) Cotton.

e. Supporting Data

The product characterization studies submitted to support the biochemical equivalence of Cry2Ae and Cry1Ab proteins expressed in TwinLink(TM) cotton to Cry2Ae and Cry1Ab expressed in its constituent parental Events GHB119 and T304-40, respectively, are summarized in Table 5 below. For detailed information of each study, please refer to the individually reviewed studies in the Data Evaluation Reports (DERs) according to its respective assigned MRID number).

   Table B.5. Product Characterization Data for Combination PIP TwinLink(TM)
                          (EPA File Symbol 264-RNOA)

                                  Study Title
             B.5. Summary - TwinLink(TM) - Product Characterization
                                   MRID No.
TwinLink(TM)  Product Characterization - Molecular Characterization
Southern blot analyses were used to characterize the genomic DNA to confirm the integrity of the GHB119 and T304-40 inserts in the combination PIP product T304-40 x GHB119. Samples from T304-40 x GHB119 cotton showed band patterns obtained using each probe were repeatable as those observed for the individual events, indicating that no DNA rearrangements occurred during the production of TwinLink(TM) developed by conventional breeding of the parental Events T304-40 x GHB119.

However, no Southern blot analyses were conducted using the pat/bar as a probe on the parental events and TwinLink(TM) Cotton. In addition, details were missing on the test materials, methods used to conduct the study. There were also no plasmid maps or schematic depictions  included to show the location of the cleavage sites for each restriction enzyme, the probe positions, and the predicted sizes of each DNA fragments after digestion. Lastly, the lanes for the negative control and the positive control appear to be mislabeled, since the negative and positive control results were opposite to those expected in both gel figures.

CLASSIFICATION:  SUPPLEMENTAL- Data gaps were addressed in MRID No. 47640007
                                  47640001
Protein Expression Analysis for T304-40, GHB119 and TwinLink(TM) Transgenic Cotton[5]
This study was conducted to characterize and compare the levels of protein expression for Cry1Ab, Cry2Ae, and PAT expressed in Events T304-40, GHB119,  and the combination PIP TwinLink(TM) cotton, developed by conventional breeding of the parental Events T304-40 x GHB119. The trial contained 50 plants for each cotton line grown under typical greenhouse conditions including the application of insecticides and fertilizer. Samples of leaves, squares and grain were collected from five randomly selected cotton plants from each genotype. The Cry1Ab, Cry2Ae and PAT proteins were extracted from the plant tissue matrices to measure the amount of protein expressed in the cotton lines, using commercially available enzyme-linked immunosorbent assay (ELISA) test kits. There was considerable variation in the levels of protein expression reported for each PIP protein expressed in the parental events compared to the levels of protein expression in TwinLink(TM) Cotton; as well as  significant differences reported within the ranges of protein expression levels for certain tissue types, although not statistically analyzed.

However, the significant differences may be attributed to the limited number of samples, replicates, and trials since this study was conducted as a single trial in a greenhouse. Therefore, the dataset used to characterize the protein expression levels may not be representative of the protein levels expressed in the field. In addition, the study does not account for variability in the levels of protein expressed under field conditions, where different environmental factors may also have an effect.

CLASSIFICATION:  SUPPLEMENTAL- Data gaps were addressed in MRID No. 47640008
                                 47640003
Analyses of Raw Agricultural Commodity (Fuzzy Seed) of TwinLink(TM) Cotton for PAT/bar, Cry2Ae and Cry1Ab and its Non-transgenic Counterpart for PAT/bar, Cry2Ae and Cry1Ab Proteins
The purpose of this study was to quantify the expression of Cry1Ab, Cry2Ae and PAT proteins in the fuzzy seeds of transgenic TwinLink(TM) cotton and the non-transgenic Coker 315 cotton line using enzyme-linked immunoabsorbent assay (ELISA). Fuzzy seed were separated into lint coats and kernels and these fractions were analyzed separately for Cry1Ab, Cry2Ae and PAT proteins and for total extractable protein. The respective values for the kernel and lint coat fractions were combined on a weight basis to calculate the respective values for the fuzzy seed that was received from the field. The limit of detection (LOD) and limits of quantitation (LOQ), the extract dilution factors, extraction efficiencies, standard curves (ng/mL), and the calculations for the protein extractions from each tissue samples were reported to establish the validity (sufficient sensitivity and extraction efficiencies) of the ELISA method used in this study.

The average Cry1Ab protein content of transgenic fuzzy seed from all test sites, without and with herbicide spraying, on a dry weight basis, was 1.32 +- 0.517 ug/g and 1.57 +- 0.317ug/g, respectively. As a percent of total crude protein, Cry1Ab protein comprised an average of 0.000577 +- 0.000204% and 0.000707 +- 0.000155% of the total crude protein in unsprayed and sprayed fuzzy seed, respectively.

The average Cry2Ae protein content of transgenic fuzzy seed from all test sites, without and with herbicide spraying, on a dry weight basis, was 9.35 +- 2.93ug/g and 10.7 +- 4.17 ug/g, respectively. As a percent of total crude protein, Cry2Ae protein comprised an average of 0.00420 +- 0.00129 % and 0.00485 +- 0.00204 % of the total crude protein in unsprayed and sprayed fuzzy seed, respectively.
The average PAT protein content of transgenic fuzzy seed from all test sites, without and with herbicide spraying, on a dry weight basis, was 162 +- 32.5 ug/g and 163 +- 27.4 ug/g, respectively. As a percent of total crude protein, PAT protein comprised an average of 0.0726 +- 0.0121% and 0.0737 +- 0.0151% of the total crude protein in unsprayed and sprayed fuzzy seed, respectively.

CLASSIFICATION:  ACCEPTABLE
                                   47640004
Structural stability analysis of Gossypium hirsutum combined Event TwinLink(TM) x GlyTol (GHB614)
The purpose of this study was to confirm the integrity and genetic stability of the intended transgenic inserts contained in combination PIP TwinLink(TM) cotton plants developed by conventional breeding of the parental cotton Events T304-40 (expressing Cry1Ab and PAT proteins) and GHB119 (expressing Cry2Ae and PAT proteins). TwinLink(TM) cotton contains the cry1Ab and bar (cry1Ab-bar) genes on one DNA insert from T304-40 cotton and the cry2Ae and bar (cry2Ae-bar) genes on a second DNA insert from GHB119 cotton. Genomic DNA was prepared from 24 individual combination PIP product cotton plants and was subjected to Southern blot analysis using the cry1Ab-bar TDNA probe, the cry1Ab gene probe, and the cry2Ae-bar TDNA probe. The analyses showed the presence of the expected hybridization fragments in all tested transgenic DNA samples when tested with each of the three probes. In addition, results showed the cry1Ab, cry2Ae, and bar DNA inserts are stably integrated in TwinLink(TM) cotton, based on Southern blot analyses of combination PIP product plants obtained after five generations from the original TwinLink(TM) cross. Therefore, data from the comparative Southern blot analyses confirmed the integrity and genetic stability of the transgenic cry1Ab, cry2Ae, and bar inserts from the individual parental events as preserved during conventional breeding to produce TwinLink(TM) Cotton.

CLASSIFICATION:  ACCEPTABLE
                                   48480007
                   [This study supersedes MRID No. 47640001]
Protein expression analysis for GlyTol x TwinLink(TM) (GHB614 x T304-40 x GHB119) cotton grown in the field, USA, 2010
The purpose of this study was to use enzyme-linked immunosorbent assay (ELISA) methods to determine the expression of CrylAb, Cry2Ae, and PAT/bar proteins in the combination PIP product: GlyTol x TwinLink(TM) (GHB614 x T304-40 x GHB119). Cotton Event T304-40 expresses the Cry1Ab and PAT/bar proteins, Event GHB119 expresses the Cry2Ae and PAT/bar proteins, and Event GHB614[1] expresses the 2mEPSPS (5-enolpyruvyl-shikimate-3-phosphate synthase) protein. Combination PIP product cotton tissue samples from leaves, roots, squares, bolls, and fuzzy seeds were collected from three different field trial sites at one or more developmental stages (T1 though T4). A total of 36 samples of each tissue type (matrix) were assayed for protein concentrations. Expression of all proteins (CrylAb, Cry2Ae and PAT/bar) was observed in all matrices (leaves (T1, T2, and T3), roots (T1, T2, and T3), squares (T3), bolls (T3), and fuzzy seed (T4)).

Detailed information was provided on the limit of detection (LOD) and limits of quantitation (LOQ), the extract dilution factors, extraction efficiencies, standard curves (ng/mL), and the calculations for the protein extractions from each tissue samples for the purposes of validating the ELISA methods used in this study. Overall, the levels of protein expression for Cry2Ae, Cry1Ab, and PAT proteins in the combination PIP maize product were comparable to the protein expression levels in its respective parental cotton Events GHB119 and T304-40. These results can also be used to establish the biochemical equivalence of the Cry2Ae, Cry1Ab and PAT proteins expressed in combination PIP relative to the PIP parental Events GHB119 and T304-40, when these parental lines are combined via traditional breeding methods.

CLASSIFICATION:  ACCEPTABLE
                                   48480008
                   [This study supersedes MRID No. 47871604]
REFERENCES  -  Cry2Ae and TwinLink(TM) Cotton PIPs
     Bernstein, I.L., Bernstein, J.A., Miller, M., Tierzieva, S., Bernstein, D.I., Lummus, Z., Selgrade, M.K., Doerfler DL, Seligy VL. (1999) Immune responses in farm workers after exposure to Bacillus thuringiensis pesticides. Environ Health Perspect. 107(7):575-82

CAC (2003) Alinorm 03/34: Joint FAO/WHO Food Standard Programme. Codex Alimentarius Commission, Twenty-Fifth Session, 30 July 2003. Rome, Italy. Appendix III: Guideline for Conduct of Food Safety Assessments of Foods Derived from Recombinant-DNA Plants; Appendix IV: Annex on Assessment of Possible Allergenicity. CAC, 47 - 60
Dean, B., Jansen., S., Becker, A. (2010) Insect bioassay of plant material expressing cotton Events T304-40 GHB119 and TwinLink(TM) (T304-40 x GHB119). Bayer CropScience Internal Report -Insect Bioassay from Monheim. MRID No. 48480009
Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J.,
      Vanderbruggen H., van Montagu M., Zabeau M.,Vaeck M. (1986) Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur. J. Biochem. 161:273-280
Sjoblad, Roy D. et al. (1992) Toxicological Considerations for Protein Components of Biological Pesticide Products" Regulatory Toxicology and Pharmacology 15: 3-9
Thompson C., Van Montagu M. and Leemans J. (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO Journal. 6(9): 2513-2518
U.S. EPA. 2006. "The product characterization and human health data submitted in support of the EUP."  Memorandum from R. Edelstein, through J. Kough, to S. Matten; dated January 27, 2006. US Environmental Protection Agency. Washington DC
U.S. EPA. 2008. Review of Product Characterization and Human health Data for an EUP Application for Bt. Cry2Ae Insecticidal Protein and Cry2Ae x Cry1Ab as Expressed in Cotton. Memorandum from R. Edelstein, through J. Kough, to S. Bacchus; dated February 12, 2008. US EPA. Washington DC
U.S. EPA. 2009. Residue Chemistry Test Guidelines: OPPTS 860.1340 Residue Analytical Method (EPA 712 - C - 96 - 174); Document ID: EPA-HQ-OPPT-2009-0155-0007; Docket ID: EPA-HQ-OPPT-2009-0155
U.S. EPA. 2011a. Review of Product Characterization Data in support of Sec. 3 Registration of Plant-Incorporated Protectant (PIP) cotton Events: GHB119 cotton expressing Bacillus thuringiensis Cry2Ae protein (EPA File Symbol 264-RNOU), T304-40 cotton expressing Bt CrylAb (EPA File Symbol 264-RNOL); and TwinLink(TM) cotton (EPA File Symbol 264-RNOA), its associated combination PIP product developed by conventional breeding of cotton Events GHB119 and T304-40."  Memorandum from A. Waggoner, through J. Kough, to S. Bacchus and D. Greenway; dated February 4, 2011. US EPA. Washington DC
U.S. EPA. 2011b. Environmental Risk Assessment for Bacillus thuringiensis (Bt) Cry1Ab and Bt Cry2Ae insect control proteins as expressed in Event T304-40 cotton and GHB119 cotton, respectively, and in the combined Event, T304-40 x GHB119 (TwinLink(TM)) cotton (EPA File Symbols 264-RNOU, -RNOL, -RNOA), submitted by Bayer CropScience LP. Memorandum from S. Borges to S. Bacchus; dated October 13, 2011. US EPA. Washington DC
US EPA. 2011c. Review of Product Characterization and Human Health Data in support of Sec. 3 Registration of Plant-Incorporated Protectant (PIP) Event T304-40 cotton expressing Bacillus thuringiensis Cry1Ab insecticidal protein (EPA File Symbol 264-RNOU). Memorandum from A. Waggoner, through J. Kough, to D. Greenway; dated November 30, 2011. US EPA. Washington DC
U.S. FDA. 1992. Statement of Policy: Foods Derived From New Plant Varieties. Federal Register: Vol. 57 No. 104 p. 22984. Dated: of May 29, 1992. Department Of Health And Human Services- Food and Drug Administration (Docket No. 92N-0139) http://www.cfsan.fda.gov/~lrd/biotechm.html#reg
Van Rie J. 2004. Comparative analysis of the Cry1Ab protein amino acid sequence. Project Number: None; Unpublished study prepared by Bayer CropScience LP. MRID No. 46474406.
APPENDIX C: Ecological and environmental effects  -  Bacillus thuringiensis Cry1Ab, Cry2Ae and TwinLink(TM) Cotton plant incorporated protectant

 BCS applied for a FIFRA Section 3 registration for two plant incorporated protectants (PIPs). These include:
   * Cry1Ab protein and the genetic material necessary for its production in Event T304-40 transgenic cotton [Gossypium hirsutum] plant line;
   * Cry2Ae protein and the genetic material necessary for its production in transgenic Event GHB119 cotton plant line; and
   * the associated pyramid in Event T304-40 x GHB119 (referred to hereafter as TwinLink(TM)) Cotton, which produces both proteins.
 BPPD does not anticipate any adverse effects of the Cry1Ab and Cry2Ae proteins on nontarget organisms or on their abundance in any field population. BPPD additionally concludes that the proposed Section 3 registrations of Cry1Ab and Cry2Ae proteins as expressed in Event T304-40 x Event GHB119, or TwinLink(TM) Cotton will not result in direct or indirect effects to nontarget organisms, including federally-listed threatened or endangered species.

 This assessment references data that were reviewed and found acceptable to demonstrate functional equivalence between the bacteria-derived Cry1Ab and Cry2Ae proteins used in toxicity testing and plant-expressed Cry1Ab and Cry2Ae proteins. The ecological risk assessment for the combined Events expressed in TwinLink(TM) cotton is also dependent on equivalent expression levels of Cry1Ab and Cry2Ae protein in TwinLink(TM) cotton plants compared to the T304-40 and GHB119 parent lines [see Product Characterization - Appendix A (Cry1Ab) and B (Cry2Ae)].

BACKGROUND

BCS developed Event T304-40 and Event GHB119 transgenic cotton [Gossypium hirsutum] plant lines, which each express a Cry protein insect control gene. Event T304-40 expresses a Cry1Ab gene, which is derived from the soil bacterium, Bacillus thuringiensis (Bt) subsp. berliner, and Event GHB119 expresses a cry2Ae gene derived from Bt subsp. dakota. Both genes have been modified for their expression in plants. Cry1Ab and Cry2Ae proteins are intended to control several lepidopteran pests of cotton including cotton bollworm (Helicoverpa zea), pink bollworm (Pectinophora gossypiella), tobacco budworm (Heliothis virenscens) larvae, beet armyworm (Spodoptera exigua), and fall armyworm (Spodoptera frugiperda). Events T304-40 and GHB119 also express the PAT gene, which confers herbicide tolerance.

Experimental use permits (EUP) were previously granted by EPA to conduct field tests on Event T304-40, Event GHB119, and T304-40 x GHB119 cotton (EPA Reg. Nos. 264-EUP-140 and 264-EUP-143). BCS is currently requesting a FIFRA Sec. 3 registration for Bt insect control protein Cry1Ab as expressed in Event T304-40 cotton, Cry2Ae as expressed in Event GHB119 cotton, and the associated breeding stack, TwinLink(TM) cotton, produced by a traditional breeding cross. This memorandum contains the Agency's environmental risk assessment of the Cry1Ab and Cry2Ae proteins produced in T304-40 and GHB119 cotton lines, respectively, as well as in TwinLink(TM) Cotton.
I.	Environmental Risk Assessment for Cry1Ab as Expressed in T304-40 Cotton and Cry2Ae as Expressed in GHB119 Cotton PIPs

The paragraphs below describe the process and rationale developed by BPPD for evaluating hazard of PIPs to nontarget organisms. This process is described in several of BPPD's documents and is presented again here as background information.

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

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

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

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

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

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

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

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

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

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

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

Since the commercialization of Bt crops, the number of field studies published in scientific literature in combination with the post-registration field studies submitted to the Agency has accumulated to a level where empirical conclusions can be made. As a result, the issue of long range effects of cultivation of these Cry proteins on the invertebrate community structure in Bt crop fields has since been adequately addressed. Specifically, a meta-analysis of the data collected from 42 field studies indicated that non-target invertebrates are generally more abundant in Bt cotton and Bt maize fields than in non-transgenic fields managed with insecticides (Marvier et al., 2007). In addition, a comprehensive review of short and long term field studies on the effects of invertebrate populations in Bt corn and cotton fields indicated that no unreasonable adverse effects are taking place as a result of wide scale Bt crop cultivation (Sanvido et al. 2007). Another review of field tests published to date concluded that the large-scale studies in commercial Bt cotton have not revealed any unexpected non-target effects other than subtle shifts in the arthropod community caused by the effective control of the target pests (Romeis et al., 2006). Slight reductions in some invertebrate predator populations are an inevitable result of all pest management practices, which result in reductions in the abundance of the pests as prey.

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

      A. Environmental Exposure Assessment

Two separate SAP reports (October 2000 and August 2002) recommended that non-target testing of Bt Cry proteins should focus on invertebrate species exposed to the crop in which the protein(s) will be expressed. Following SAP recommendations, the EPA determined that non-target organisms with the greatest exposure potential to Cry protein in transgenic cotton fields are beneficial insects that feed on cotton pollen and nectar, particularly lepidopteran insects, and soil invertebrates. While EPA's risk assessments of Bt cotton have focused primarily on these taxa, BPPD recognizes that exposure to other nontarget organisms can occur and has required testing on representative species.

The EPA risk assessment is centered on adverse effects at the field exposure rates, which are typically based on protein expression levels within the plant for PIPs. Although it is recommended that non-target testing be conducted at a test dose 10X the EEC whenever possible, the test dose margin can be less than 10X where uncertainty in the system is low or where high concentrations of test material are not possible to achieve due to test organism feeding habits. BPPD may also allow for testing at lower doses in cases where many species are tested or tests are very sensitive, although the concentration used must exceed the EEC. For the purposes of the non-target organism studies submitted in support of Event T304-40 and Event GHB119, the test material dose levels were based on an estimated concentration of Cry1Ab or Cry2Ae protein expressed in the tissue(s) with the highest concentration based on protein expression studies (MRIDs 47634803, 47641903).
      B. Ecological Effects Data for Event T304-40 and Event GHB119

In the absence of PIP-specific risk assessment guidance, EPA requires applicants for PIP registrations to meet the 40 CFR Part 158 data requirements for microbial toxins. These requirements include testing on birds, mammals, nontarget insects, honey bee, plants, and aquatic species, and information has been submitted to address these requirements. Limit dose testing on representative organisms from several taxa was performed in support of the T304-40 and GHB119 Section 3 FIFRA registrations. As stated above, BPPD's risk assessments focus heavily on beneficial nontarget invertebrates, since they are most closely related to organisms susceptible to the insecticidal action of Bt toxins. The Cry1Ab and Cry2Ae proteins are meant to target species within the order Lepidoptera (moths and butterflies). Bt toxins are known typically to have a limited host range. However, to address any unforeseen change in activity spectrum as a result of laboratory protein synthesis and to fulfill the published registration data requirements, EPA requires that test species used for non-target insect evaluations should include several invertebrate species that are not related to the target pests. Earthworm studies are also recommended. The toxicity of the Cry1Ab and Cry2Ae proteins has been evaluated on several species of invertebrates including the lady beetle, green lacewing, collembola, Daphnia, honey bee, and earthworm. Reproductive and/or developmental observations were also examined in the lady beetle, Daphnia, collembola, and honeybee studies.

Since exposure may also occur to other nontarget organisms, EPA has received data to comply with the Agency's published non-target data requirements on other nontarget organisms. Tests to determine soil degradation of Cry1Ab and Cry2Ae proteins have also been submitted. The individual results for nontarget organism and soil degradation testing for Cry1Ab and Cry2Ae proteins are summarized in Tables 1 and 2, respectively. The studies are described in more detail below, and full reviews of each study for each event can be found in the individual Data Evaluation Records.

 The October 2000 SAP recommended that while actual plant material is the preferred test material, bacteria-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 the T304-40 and GHB119 registrations, test substances used in the submitted studies included bacteria-produced purified Cry1Ab and Cry2Ae protein, respectively. Comparative analyses have been performed to verify the equivalence of the bacterially-produced and purified Cry1Ab and Cry2Ae and the PIP Cry1Ab and Cry2Ae proteins produced in T304-40 or GHB119, respectively, and TwinLink(TM) cotton plants. Product characterization data (see Appendices A and B) demonstrate functional equivalence between the bacteria-derived Cry1Ab and Cry2Ae proteins used in toxicity testing and plant-expressed Cry1Ab and Cry2Ae proteins. The ecological risk assessment for the combined events expressed in TwinLink(TM) cotton is also dependent on equivalent expression levels of Cry1Ab and Cry2Ae protein in TwinLink(TM) cotton plants compared to the T304-40 and GHB119 parent lines. This bioequivalence has been demonstrated [see Product Characterization - Appendices A (Cry1Ab) and B (Cry2Ae and TwinLink(TM) Cotton)].

Table C.1. Summary of environmental effects studies and waiver justifications for Cry1Ab as expressed in T304-40 cotton submitted to comply with data requirements published in 40 CFR § 158.2150(d).

                               Data Requirement
                                     OPPTS
                                   Guideline
                                Test Substance
   C.1. Cry1Ab PIP  -  Ecological Effects - Results Summary and Classification
                                   MRID No.
Avian dietary testing,
broiler chicken, Gallus domesticus

885.4050
TwinLink(TM) (T304-40 x GHB119) cotton seed meal
A 42-day dietary study showed no adverse affects to broiler chickens when fed a diet composed of 10% TwinLink(TM) cottonseed meal containing both Cry1Ab x Cry2Ae proteins. However, the presence of the proteins in the test material was not confirmed.
Classification:  Unacceptable
 47640011
Avian inhalation testing
885.4100

N/A
Acceptable waiver rationale
47871403
Avian oral testing, Northern bobwhite
Colinus virginianus
850.2100
N/A
Data waiver rationale was submitted to justify that testing is not necessary on the basis of lack of exposure and information on the toxicity of registered Cry proteins to birds.
Classification:  Acceptable
47903201
Wild mammal testing
885.4150

N/A
Acceptable bridging rationale to acute oral  toxicity test on laboratory mice
47871403
Freshwater fish testing,
885.4200

N/A
Acceptable waiver rationale
47871403
Freshwater aquatic invertebrate testing,
water flea, Daphnia  magna
885.4240

Microbially-produced Cry1Ab
No adverse effects on survival or reproduction of Daphnia magna exposed to 48 ug Cry1Ab protein/L in a 10-day static renewal test. The 48-hr and 10-day EC50 values were determined to be > 48 ug Cry1Ab protein/L; the NOAEC is >= 48 ug Cry1Ab protein/L.
Classification:  Acceptable
 47634819
Estuarine and marine animal testing
885.4280

N/A
Acceptable waiver rationale
 47871403
Non-target plant testing
885.4300

N/A
Acceptable waiver rationale
 47871403
Non-target insect testing, pink-spotted lady beetle, Coleomegilla maculata
885.4340

Microbially-produced Cry1Ab protein
No adverse effects on survival, time to pupation, and mean adult weight for Coleomegilla maculata larvae exposed to 19 ug Cry1Ab protein/g diet over 21 days. The LC50 was >19 ug Cry1Ab protein/g diet and the NOAEC is >= 19 ug Cry1Ab protein/g diet.
Classification:  Acceptable
 47634815
Non-target insect testing, green lacewing,  Chyrsoperla rufilabris
885.4340
Microbially-produced Cry1Ab protein
No adverse effects on survival or pupation were observed in Chrysoperla rufilabris exposed to 20 ug Cry1Ab protein/g diet (nominal) in a 10-day laboratory bioassay. The nominal LC50 is >20 ug Cry1Ab protein/g diet and the NOAEC is >=20 ug Cry1Ab protein/g diet.
Classification:  Acceptable
 47692701
Non-target insect testing, collembola, Folsomia candida
885.4340

Microbially-produced Cry1Ab protein
No adverse effects on survival or reproduction of collembola (Folsomia candida) exposed to 20 ug of Cry1Ab protein /g diet in a 28-day laboratory bioassay. The nominal LC50 was determined to be > 20 ug Cry1Ab protein/g diet and the nominal NOAEC is >=20 ug.
Classification:  Acceptable
 47651106
Honeybee testing, Honeybee larvae,
Apis mellifera
885.4380

Microbially-produced Cry1Ab protein
No adverse effects on survival were observed in honey bee larvae treated in natal cells with 20 ug/L solution of Cry1Ab protein. The LC50 was determined to be > 20 ug Cry1Ab protein/mL.
Classification:  Acceptable
 47651105
Earthworm toxicity,
Eisenia foetida
850.6200
N/A
Acceptable Waiver Rationale
 47634818
Soil fate and degradation
885.5200
Microbially-produced Cry1Ab protein
Based on the measured bioactivity in tobacco budworm (Heliothis virescens), the average DT50 for Cry1Ab protein in three different soils was 3.6 days.
Classification:  Acceptable
 47634820

Table C.2. Summary of environmental effects studies and waiver justifications for GHB119 submitted to comply with data requirements published in 40 CFR § 158.2150 (d).
                               Data Requirement
                                     OPPTS
                                   Guideline
                                Test Substance
                   C.2. Cry2Ae PIP - Ecological Effects  -
                      Results Summary and Classification
                                   MRID No.
Avian dietary testing,
broiler chicken, Gallus domesticus

885.4050
TwinLink(TM) (T304-40 x GHB119) cotton seed meal
A 42-day dietary study showed no adverse affects to broiler chickens when fed a diet composed of 10% TwinLink(TM) cottonseed meal containing both Cry1Ab and Cry2Ae proteins. However, the presence of the proteins in the test material was not confirmed.
Classification:  Unacceptable
 47640011
 (same as above)
Avian inhalation testing
885.4100

N/A
Acceptable waiver rationale
47871503
Avian oral testing, Northern bobwhite
Colinus virginianus
850.2100
N/A

GHB119 cotton seed meal (raw)
Data waiver rationale was submitted to justify that testing is not necessary on the basis of lack of exposure.
Classification:  Supplemental
A 5-day feeding study with Northern bobwhite showed no adverse effects at 10% raw GHB119 cotton seed meal in the diet.
Classification: Acceptable
47903301

48177501

Wild mammal testing
885.4150

N/A
Acceptable bridging rationale to acute oral  toxicity test on laboratory mice
47871503
Freshwater fish testing
885.4200

N/A
Acceptable waiver rationale
47871503
Freshwater aquatic invertebrate, water flea neonate, Daphnia magna
885.4240

Microbially-produced Cry2Ae protein
Daphnia magna were exposed to a nominal concentration of 330 ug Cry2Ae protein/L both in water and in food for 10 days. No adverse effects on immobilization/survival or reproduction were observed. The nominal 48-hr and 10-day EC50 values were >330 ug a.i./L and the nominal NOAEC was >= 330 ug a.i./L.
Classification:   Acceptable
 47641919
Estuarine and marine animal  testing
885.4280

N/A
Acceptable waiver rationale
 47871503
Non-target plant testing
885.4300

N/A
Acceptable waiver rationale
 47871503
Non-target insect testing, pink-spotted lady beetle, Coleomegilla maculata
885.4340

Microbially-produced Cry2Ae protein
No statistically significant effects on mortality, body weight, mean number of days to pupation, and mean number of days to adulthood were observed in Coleomegilla maculata exposed to 64 ug Cry2Ae protein/g of diet for 21 days. The nominal LC50 is > 64 ug Cry2Ae protein/g diet and the NOAEC is >= 64 ug Cry2Ae protein/g diet.
Classification:  Acceptable
 47641915
Non-target insect testing, green lacewing, Chrysoperla rufilabris
885.4340

Microbially-produced Cry2Ae protein
No statistically significant differences in mortality or % pupation were observed in green lacewing exposed to a nominal concentration of 10 or 110 ug Cry2Ae protein/g of diet for 10 days. The nominal LC50 is >110 ug Cry2Ae protein/g diet and the nominal NOAEC is >= 110 ug Cry2Ae protein/g diet.
Classification:  Acceptable
 47692501
Non-target insect testing, collembolan, Folsomia candida
885.4340

Microbially-produced Cry2Ae protein
Limit test:  The number of offspring per adult exposed to Cry2Ae protein was significantly reduced in Collembola exposed to 66 ug Cry2Ae protein/g diet for 28 days.
Classification:  Supplemental
Definitive test:  No statistically significant differences in survival or reproduction among the test material and negative control groups were observed in Collembola exposed to 9.9, 20, 40, 80, or 160 ug of Cry2Ae protein/g diet. The nominal LC50 is >160 ug Cry2Ae protein/g diet and the nominal NOAEC is >=160 ug Cry2Ae protein/g diet.
Classification:  Acceptable
 47641917a
 47641917b
Earthworm toxicity,
Eisenia foetida
850.6200
Microbially-produced
Cry2Ae protein
No adverse effects were observed on burrowing time, mortality, and weight change for earthworms (Eisenia fetida) were exposed to a nominal concentration of 100 mg Cry2Ae protein/kg of artificial soil for 14 days. The nominal 7- and 14-day LC50 was determined to be >100 mg a.i./kg, and the NOAEC was >=100 mg a.i./kg.
Classification:  Acceptable
 47641918
Honeybee  testing,
Apis mellifera
885.4380

Microbially-produced Cry2Ae protein
Larval survival did not differ significantly between the Cry2Ae protein treated group and the controls at capping and at emergence for honey bee (Apis mellifera) larvae exposed to 50 ug Cry2Ae protein/mL. Therefore, the LC50 is >50 ug Cry2Ae protein/mL.
Classification:  Acceptable
 47641914
Soil fate and degradation
885.5200
Microbially-produced Cry2Ae protein
Based on the measured bioactivity in tobacco budworm (Heliothis virescens), the average DT50 for Cry2Ae protein in three different soils was 3.4 days.
Classification:  Acceptable
 47641920

	1. Non-target Organism Study Summaries for T304-40 expressing Cry1Ab Protein

   	a.  Avian Wildlife

	Avian Dietary Toxicity

In a 42-day study (MRID 47640011), newly-hatched Ross #308 broiler chickens were fed formulated diet containing 10% TwinLink(TM) cottonseed meal  Control groups were fed formulated diet containing either 10% near isogenic cottonseed meal or 10% non-transgenic commercial cottonseed meal. There were no statistically significant treatment-related effects on survival, body weight, body weight gain, feed consumption, feed conversion ratio, or weight of chilled carcass, leg, thigh, wing, or breast among any of the test groups. DNA analysis of the prepared diets confirmed the presence of transgenic genetic material in the TwinLink(TM) diet, however the presence of the Cry1Ab and Cry2Ae proteins were not confirmed. The study report stated that the cottonseed meal was toasted, and this heating may have degraded the proteins. Thus, there is no assurance that the test animals were exposed to Cry1Ab or Cry2Ae proteins in the diet throughout the study, and the study cannot be used for avian risk assessment. A request to bridge to data from this study to the assessment of Cry1Ab protein expressed in T304-40 cotton (MRID 47871404) was not reviewed because the study was determined to be unacceptable.

	Avian Oral Toxicity

Rationale was presented to justify waiving the avian oral toxicity data requirement for the Cry1Ab protein expressed in Event T304-40 cotton (MRID 47903201). The justification rested primarily on the expected minimal exposure of birds to Cry1Ab protein in T304-40 cotton as a result of their avoidance of cotton seed consumption, as well as the lack of observed effects of other Bt proteins that have been observed on birds (Mendelsohn et al. 2003).

		Avian Inhalation Toxicity

Avian inhalation toxicity testing is typically not required for Bt cotton event registrations, since exposure to Cry proteins via inhalation is not expected. The applicant submitted data waiver rationale (MRID 47871403) that is consistent with BPPD's position on inhalation exposure for birds in Bt cotton, and this rationale is acceptable. An avian inhalation test is not required.

    	b. Wild mammals

BCS submitted acceptable data waiver rationale (MRID 47871403) to justify that testing with wild mammals is not necessary. An acute oral toxicity study with mice (MRID 47651103) has been submitted to EPA in support of the T304-40 registration, and in this study no mortalities, clinical signs of treatment-related effects, or treatment-related effects on body weight were observed on mice dosed at 2000 mg/kg body weight. This study has been determined to be acceptable. There is no indication that laboratory species would not be representative of wild mammals in this case; therefore, this information is suitable for use in assessment of risk to wild mammals, and no additional testing with wild species is necessary.

   	c. Aquatic species

	Freshwater animals

Data waiver rationale (MRID 47871403) was submitted to fulfill the data requirements for freshwater fish for Cry1Ab protein in T304-40 cotton. Justification rested primarily on the lack of expected exposure of aquatic environments to Cry proteins produced in cotton, and this rationale was determined to be sufficient. Cotton pollen is widely known to be "sticky" (i.e., to form clumps) and too heavy to be dispersed by wind. As a result, little pollen is expected to move off field and enter aquatic systems. BPPD has also concluded that little Bt cotton plant matter is expected to move off field (e.g., see U.S. EPA 2008a). Therefore, exposure in aquatic systems is expected to be low, and testing of freshwater fish is determined to be unnecessary.

Because of concerns raised in a study with the leaf shredding (caddis fly) trichopteran, Lepidostoma liba (Rosi-Marshall, et al. 2007), EPA is  requiring applicants of Bt corn and cotton to submit 7-10 day freshwater invertebrate toxicity studies to reduce uncertainty regarding this issue. A 10-day study with Daphnia is available with which to determine the risk to freshwater invertebrates as a result of exposure to Cry1Ab protein in T304-40 cotton. Daphnia were exposed to a nominal concentration of 48 ug Cry1Ab protein/L, which is based on the mean expression level of protein present in one kilogram of pollen, dispersed in one liter of water (see MRID 47634803). At this concentration, with exposure in both water and in food treated with Cry1Ab protein, no mortalities and no significant differences in the number of offspring produced were observed. Therefore, the EC50 was determined to be > 48 ug Cry1Ab protein/L, and the NOAEC was determined to be >= 48 ug Cry1Ab protein/L.

	Estuarine and marine animals

BPPD typically waives toxicity studies with estuarine/marine fish and invertebrates based on an expected minimum exposure in these environments; however, the applicant submitted data waiver rationale (MRID 47871403) to fulfill data requirements for estuarine and marine animals. The waiver rationale presented was the same as that discussed above for freshwater fish and was determined to be sufficient based on BPPD's previous conclusions about exposure to Cry proteins in aquatic environments.

   	d. Terrestrial and aquatic plant species

BPPD typically waives nontarget plant testing for Bt Cry proteins, since the active ingredient is an insect toxin (Bt -endotoxin) that has never shown any toxicity to plants. The applicant submitted acceptable data waiver rationale (MRID 47871403) that was consistent with BPPD's position on nontarget plant risks to Bt -endotoxin.

   	e. Invertebrate species

In a study with the ladybird beetle (Coleomegilla maculata, MRID 47634815), larvae were exposed to a nominal concentration of 19 ug Cry1Ab protein/g of artificial diet for 21 days. This amount is meant to represent 10X the mean expression level of Cry1Ab protein in the cotton plants. Larval and adult mortality, time to pupation, time to adulthood, and adult weight did not differ significantly from the negative control group. Larval mortality was 7.5% and 12.5% in the treated and control groups, respectively; adult mortality was 0% and 2.9% in the treated and control groups, respectively. The nominal LC50 was determined to be >19 ug Cry1Ab protein/g of diet and the NOAEC was determined to be >= 19 ug Cry1Ab protein/g of diet.

A study with green lacewing (Chrysoperla rufilabris, MRID 47692701) larvae exposed 25 individuals to a nominal concentration of 20 ug Cry1Ab protein/g of artificial diet for 10 days. The amount to which the larvae were exposed throughout the test was meant to represent 10X the mean fresh weight expression level of Cry1Ab protein in T304-40 cotton plants. Mortality and pupation measured in the test group (94% and 76%, respectively) did not differ significantly from the negative control (96% and 76%, respectively). Based on the results, the LC50 for Cry1Ab protein in C. rufilabris is >20 ug a.i./g and the NOAEC was determined to be >= 20 ug Cry1Ab protein /g of diet.

Collembola (Folsomia candida) are soil dwelling insects that consume detritus. In a 28-day study (MRID 47651106), 40 collembola were exposed to 20 ug Cry1Ab protein/g diet and observed for mortality and number of offspring produced per adult. Mortality of the collembola was 25% for the groups exposed to Cry1Ab protein and 12.5% for the control groups. The number of offspring produced per adult was 46.9 and 43.9 for the Cry1Ab treated and control groups, respectively. Neither of these endpoints differed significantly between treated and control groups. Based on the results, the, the LC50 based on the nominal concentration is > 20 ug Cry1Ab protein/g diet and the NOAEC was established to be >= 20 ug Cry1Ab protein/g diet.

Data waiver rationale (MRID 47634818) was presented to justify that toxicity testing for Cry1Ab protein as expressed in T304-40 cotton is not necessary with the earthworm. The 14-day LC50 for earthworms exposed to Cry1Ab protein derived from E. coli was previously determined to be > 200 mg/kg of artificial soil (U.S. EPA 2010, Mendelsohn et al. 2003). Since there is no evidence of toxicity of Cry1Ab protein to earthworms, BPPD determined that additional testing with earthworms is not needed.

A study with honey bee (Apis mellifera) was also performed. In this study, 2-3 day old honey bee larvae were exposed to a 10 uL droplet of solution containing 20 ug/mL Cry1Ab protein, which was intended to represent >10X the mean fresh weight expression concentration of Cry1Ab protein in T304-40 cotton leaves, and > 400 times the exposure to the amount of Cry1Ab protein expressed in T304-40 pollen. Mortality at capping and at emergence was observed, and neither of these differed significantly from controls. Emergence was considered to be complete on Day 16. Of the larvae treated with Cry1Ab protein, 85.25% survived to capping, and all of these survived to emergence; 82.5% of the controls survived to capping and all of these also survived to emergence. Therefore, the LC50 was determined to be > 20 ug Cry1Ab protein/mL.

      2. Non-target Organism Study Summaries for GHB119 expressing Cry2Ae protein

   	a.  Avian Wildlife

	Avian Dietary Toxicity

The broiler chicken dietary study described in the summaries above for T304-40 (MRID 47640011) was also submitted for this data requirement for Cry2Ae protein expressed in GHB119 cotton. Since there is no assurance that the test animals were exposed to Cry1Ab or Cry2Ae protein in the diet throughout the study, it cannot be used for avian risk assessment for Cry2Ae protein. A request to bridge to data from this study to the assessment of Cry2Ae protein expressed in GHB119 cotton (MRID 47871504) was not reviewed because the study was determined to be unacceptable.

	Avian Oral Toxicity

Rationale was presented to justify waiving the avian oral toxicity data requirement for the Cry2Ae protein expressed in Event GHB119 cotton (MRID 47903301). The justification rested primarily on the expected minimal exposure of birds to Cry2Ae protein in GHB119 cotton as a result of their avoidance of cotton seed consumption, as well as the lack of observed effects of other Bt proteins that have been observed on birds. However, this rationale does not account for other routes of oral exposure in birds (e.g., consumption of insects).

BCS submitted a feeding study in which Northern bobwhite were exposed to raw Event GHB119 cotton seed meal (containing Cry2Ae protein) at 10% of their daily diet for five days (MRID 48177501). No mortality or behavioral abnormalities were observed, and significant differences in body weight gain or food consumption were not detected. This study is sufficient to show that hazard to birds exposed to the dietary concentration tested (up to 0.241 ppm diet or 10% of GHB119 cottonseed) is not expected. This study does not sufficiently test hazard at higher dietary concentrations. EPA has previously determined that an exposure level of 10% raw cottonseed meal containing Cry protein is a sufficient exposure level for determining the effects of Cry proteins expressed in cotton in birds (e.g., U.S. EPA 2005a, U.S. EPA 2005b, U.S. EPA 2008). Based on these exposure assumptions, the study was determined to be acceptable; however, there is uncertainty about the potential effects to birds if field exposure occurs at higher concentrations. It is noted that no avian toxicity data submitted to the Agency to date has shown detrimental effects of Cry proteins on birds, including data on the Cry2Ab2 protein (U.S. EPA 2005a). Therefore, BPPD concludes that exposure to Cry2Ae protein is not likely to cause adverse effects in birds. However, a study in which birds are exposed to higher doses of Cry2Ae protein would reduce uncertainties related to actual field exposure.

		Avian Inhalation Toxicity

Avian inhalation toxicity testing is typically not required for Bt cotton event registrations, since exposure to Cry proteins via inhalation is not expected. The applicant submitted data waiver rationale (MRID 47871503) that is consistent with BPPD's position on inhalation exposure for birds in Bt cotton, and this rationale is acceptable. An avian inhalation test is not required.

    	b. Wild mammals

The applicant submitted acceptable data waiver rationale (MRID 47871503) to justify that testing with wild mammals is not necessary. An acute oral toxicity study with mice (MRID 47076902) has been submitted to EPA in support of the GHB119 registration, and in this study no mortalities or treatment-related signs of adverse effects were observed on mice dosed at 2000 mg/kg body weight. This study was determined to be acceptable. This information is suitable for use in assessment of risk to wild mammals, and no additional testing with wild species is necessary at this time.

		c. Aquatic animals

Data waiver rationale (MRID 47871503) was submitted to fulfill the data requirements for freshwater fish for Cry2Ae protein in GHB119 cotton. Justification rested primarily on the lack of expected exposure of aquatic environments to Cry proteins produced in cotton, and this rationale was determined to be sufficient. The same rationale as presented above for Cry1Ab protein was presented for Cry2Ae protein, and this is consistent with BPPD's previous conclusions that little aquatic exposure is expected from the use of cotton expressing Cry proteins. Since BPPD has concluded that exposure in aquatic systems is expected to be very low, testing of freshwater fish is unnecessary.

A 10-day study with Daphnia magna is available with which to determine the risk to freshwater invertebrates as a result of exposure to Cry1Ab protein in T304-40 cotton. Daphnia were exposed to a nominal concentration of 330 ug Cry2Ae protein/L contained in water alone and water and food. This exposure level is based on the fresh weight expression level of protein present in one kilogram of pollen (see MRID 47641933), dispersed in one liter of water. No mortalities and no significant differences in the number of offspring produced were observed. Therefore, the EC50 was determined to be > 330 ug Cry2Ae protein/L, and the NOAEC was determined to be >= 330 ug Cry2Ae protein/L.

		 Estuarine and marine animals

BPPD typically waives toxicity studies with estuarine/marine fish and invertebrates; however, the applicant submitted data waiver rationale (MRID 47871503) to fulfill data requirements for estuarine and marine animals. The waiver rationale presented was the same as that discussed above for exposure of freshwater fish to Cry1Ab protein and was determined to be sufficient based on BPPD's conclusions about exposure in aquatic environments.

		d. Terrestrial and aquatic plant species

BPPD typically waives nontarget plant testing for Bt Cry proteins, since the active ingredient is an insect toxin (Bt -endotoxin) that has never shown any toxicity to plants. The applicant submitted acceptable data waiver rationale (MRID 47871503) that was consistent with BPPD's position on nontarget plant risks to Bt -endotoxin.

   	e. Invertebrate species

A study with ladybird beetle (Coleomegilla maculata, MRID 47641915) larvae exposed 40 individuals to a nominal concentration of 64 ug Cry1Ab protein/g of artificial diet for 21 days. This amount was approximately 7X the fresh weight expression level of Cry2Ae protein in the leaves of GHB119 cotton plants (see MRID 47641903). Larval and adult mortality, time to pupation, time to adulthood, and adult body weight did not differ significantly from the negative control group. Larval mortality was 5.0% and 12.8% in the treated and corresponding control group, respectively; adult mortality was 0% and 3.3% in the treated and control groups, respectively. The nominal LC50 was determined to be >64 ug Cry1Ab protein/g diet and the NOAEC based on development and body weight is >= 64 ug Cry2Ae protein/g diet.

A study with green lacewing (Chrysoperla rufilabris, MRID 47692501) larvae exposed 25 individuals to a nominal concentration of 10 ug and 100 ug Cry2Ae protein/g of artificial diet for 10 days. These concentrations were meant to test effects at 1X and 10X the fresh weight expression level of Cry2Ae protein as measured in the leaves of GHB119 cotton plants (see MRID 47641903). Mortality and percent pupation were measured and compared to negative and buffer controls, and no significant differences were observed. Survival of the 10 ug and 100 ug Cry2Ae protein/g diet test groups and the buffer controls was 100%, 96%, and 100%, respectively. Pupation in these groups was 84%, 72%, and 88%, respectively. The results establish a nominal LC50 of >100 ug Cry2Ae protein/g diet and a nominal NOAEC of >= 100 ug Cry2Ae protein/g diet.

Collembola (Folsomia candida) are soil dwelling insects that consume detritus. In a 28-day single-dose study (MRID 47641917a), collembola were exposed to 66 ug Cry2Ae protein/g diet and observed for mortality and number of offspring produced per adult. Mortality of the collembola was 27.5% for the groups exposed to Cry2Ae protein and 22.5% for the control groups. The number of offspring produced per adult was 10.3 and 44.5 for the Cry2Ae protein treated and control groups, respectively, and the difference was statistically significant. Because of this outcome, a definitive test was performed on collembolan exposed to nominal concentrations of 9.9, 20, 40, 80, or 160 ug Cry2Ae protein/g diet (MRID 47641917b). Negative and positive controls were also included, and a bioassay on a sensitive species (Heliothis virescens) confirmed bioactivity. Mortality of the test groups from lowest to highest concentration was 2.5%, 0%, 0%, 13%, and 0%, and mortality in the negative controls was 7.5%. Mean number of offspring did not differ significantly among any of these groups. With this test, the nominal LC50 was determined to be >160 ug Cry2Ae protein/g diet and the NOAEC was determined to be >=160 ug Cry2Ae protein/g diet.

A 14-day earthworm toxicity study (MRID 47641918) was submitted, wherein earthworms were exposed to 100 mg Cry2Ae protein/gram of artificial soil. This concentration was intended to be approximately 10X the fresh weight concentration of Cry2Ae protein expressed in the leaves of GHB119 cotton plants (see MRID 47641903). The average burrowing time was equal for both the treated and control groups, and no mortalities were observed in either group. Mean weight decreased by 22% in the control group and by 20% in the test material group, which was expected since the artificial soil did not contain nutritive material. Based on the results the nominal LC50 was determined to be >100 mg Cry2Ae protein/kg, and the NOAEC was >=100 mg Cry2Ae protein/kg.

A study with honey bee (Apis mellifera) was also performed. In this study, 2-3 day old honey bee larvae were exposed to a 10 uL droplet of solution containing 50 ug/mL Cry1Ab protein, which was intended to represent >50X the exposure to the amount of Cry2Ae protein expressed in GHB119 cotton pollen (assuming the test solution is approximately the same weight, see MRID 47641913). Mortality at capping and at emergence were observed, and neither of these differed significantly from controls. Emergence was considered to be complete on Day 16. Of the larvae treated with Cry2Ae protein, 93.75% survived to capping, and all of these survived to emergence; 95% of the controls survived to capping and all of these also survived to emergence. Therefore, the LC50 is >50 ug Cry2Ae protein/mL.

      3. Soil Fate Studies for Cry1Ab and Cry2Ae PIPs

Soil organisms may be exposed to Cry1Ab and Cry2Ae protein through contact with cotton plant roots (by direct feeding), cotton plant root exudates, incorporation of above-ground plant tissues into soil following harvest, or by soil-deposited pollen. Some evidence suggests that soils which are high in clays and humic acids are more likely to bind Cry protein. However, neutral pH soils tend to have high microbial activity and microbes contribute to Cry protein degradation. In addition, a study on the release of Cry proteins in the root exudates of Bt cotton has shown that no Cry proteins were detected immunologically or by larvicidal assay in any soil or hydroponic solution in which Bt cotton had been grown (Saxena and Stotzky, 2001). The weight of evidence indicates that Cry proteins do not accumulate in soil to arthropod-toxic levels. Because Cry proteins are toxins derived from soil-inhabiting bacteria, Bacillus thuringiensis, and found in commercial microbial insecticides (De Maagd et al., 2003 and Graser and Song, 2006), the degradation of Cry1Ab and Cry2Ae from Event T304-40 and Event GHB119 cotton is expected to be similar to that of these proteins that are produced naturally from soil bacteria. Nonetheless, BPPD required the following soil fate evaluations to support the Event T304-40 and GHB119 Bt cotton registrations. These studies are summarized here, and detailed evaluations are provided in their associated DERs.

A laboratory aerobic soil degradation study was conducted to determine the DT50 (time for 50% of the initial concentration of bioactive material to dissipate) for Cry1Ab protein. Soil used in the study was collected from three locations throughout the region where transgenic cotton is grown, and each was of a different texture (Proctor, AR: clay loam; Senatobia, MS: silt loam; East Bernard, TX: sandy clay loam). Each soil was treated once with 15 ug/g bacterially-produced Cry1Ab protein, and then sampled periodically over 45 days. These samples were used in sensitive insect bioassays. The bioassay used tobacco budworm (Heliothis virescens) larvae exposed to the soil samples incorporated into lepidopteran diet. A concurrent six-day dose response study with H. virescens larvae exposed to 0.004 to 0.28 ug/g Cry1Ab protein in the soils was conducted to generate a dose-response curve to determine the degradation of Cry1Ab protein over time based on the bioactivity response. The DT50 was determined from the Cry1Ab protein concentration first-order exponential regression curve. The average DT50 for the three soils was 3.6 days. The soils were also analyzed for Cry1Ab protein content by enzyme linked immunosorbent assay (ELISA), which showed that Cry1Ab protein steadily degraded in soil during the study.

A similar study was conducted to determine the DT50 for Cry2Ae protein in soil. Soils were collected from the same locations as above for the study with Cry1Ab protein. The soils were treated once with 50 ug/g Cry2Ae protein and then sampled periodically over 45 days. A sensitive insect bioassay using the same species (Heliothis virescens) was conducted using identical methods as with Cry1Ab protein, except that the concurrent six-day dose response study exposed H. virescens larvae to 0.056 to 3.4 ug/g Cry2Ae protein to generate a standard curve for determination of Cry2Ae protein concentration based on bioactivity response. Based on the bioassays, the average DT50 was determined to be 3.4 days. The soils were also analyzed for Cry2Ae protein content by ELISA, which showed that Cry2Ae protein steadily degraded in soil during the study.

These studies utilized field soil spiked with purified insecticidal protein produced by bacteria. This approach is useful because dose responses can be easily quantified. However, the degradation and accumulation of Bt Cry proteins found within decaying plant tissue may behave differently than proteins in artificially spiked soil. Thus, the presence of low levels of Bt Cry proteins in the soil (at or below the level of detection) is anticipated until all plant tissue is degraded. However, the reviewed data show that Cry1Ab and Cry2Ae proteins will be quickly degraded upon release from decaying plant tissue.

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

In light of these published findings and the rapid degradation of Cry1Ab and Cry2Ae proteins in soil as demonstrated in the insect bioassays described above, there is no indication that the proteins expressed in Event T304-40 and Event GHB119 cotton are likely to persist in soil after continuous cultivation. Therefore, no additional long-term field studies are required for these PIP products.

	4. Supplemental Information

BCS submitted summaries of nontarget organism testing with Cry1Ab and Cry2Ae proteins (MRIDs 47634813 and 47641913, respectively), which summarized the information above, and included an assessment of ecological risk and some discussion of environmental fate. These summaries were determined to be supplemental, but provided little additional information for use in the risk assessment.

      C. Environmental Risk Assessment for Event T304-40 and Event GHB119

		1. Effects to Nontarget Wildlife, Invertebrates, and Plants

   		a.  Avian Wildlife

Birds are known to forage in cotton fields. While birds are unlikely to consume cotton plant material, particularly cotton seeds, due to high concentrations of gossypol, they could be exposed to Cry1Ab and/or Cry2Ae proteins through consumption of invertebrates that feed on Bt cotton plant tissues. Exposure is expected to be low, and data and rationale were sufficient to conclude that Cry1Ab and Cry2Ae proteins are not expected to cause adverse effects in birds. There is some uncertainty related to the exposure level in the testing with Cry2Ae protein compared to field exposures, but EPA previously determined that the exposure level in the study was sufficient for avian testing. Testing at higher doses would reduce this uncertainty.

    		b. Wild mammals

Wild mammals may be exposed to Cry1Ab protein in Event T304-40 cotton or Cry2Ae protein in Event GHB119 cotton by consuming plant parts and/or invertebrates that feed on the cotton plants. Toxicity testing with laboratory mice showed no adverse of Cry1Ab or Cry2Ae proteins at doses of 2000 mg/kg bodyweight each. This amount is much higher than concentrations that wild mammals are expected to encounter in the field. Therefore, based on this information, adverse effects to wild mammals are not expected as a result of the use of Event T304-40 and Event GHB119 cotton.

		c. Freshwater animals

BPPD has concluded that exposure in the aquatic environment to the Cry proteins in Bt cotton is expected to be low. Cotton plants are not wind-pollinated, and cotton pollen is widely understood to be spiny and "sticky," leading to the formation of clumps of pollen that are not easily dispersed by wind. As a result, the majority of the cotton pollen that is produced is not expected to travel very far off-field. BPPD has previously determined that exposure in aquatic environments resulting from deposition of other cotton plant tissues is minimal (e.g., see U.S. EPA 2008a). Therefore, exposure of Cry1Ab or Cry2Ae protein from use of Event T304-40 or Event GHB119 cotton, respectively, to freshwater fish and invertebrates is expected to be minimal, and adverse effects to these species are not expected to occur.

		d. Estuarine and marine animals

Significant amounts of Cry1Ab and Cry2Ae protein resulting from Event T304-40 or GHB119 cotton use, respectively, are not expected to reach marine/estuarine areas. BPPD concludes that adverse effects to fish and invertebrates in these environments are not expected.

   		e. Terrestrial and aquatic plant species

BPPD typically waives nontarget plant testing for Bt Cry proteins, since the active ingredient is an insect toxin (Bt -endotoxin) that has never shown any toxicity to plants. BPPD has concluded that adverse effects to terrestrial and aquatic plants are not anticipated.

   		f. Invertebrate species

Nontarget insects are expected to receive exposure primarily through consumption of Bt cotton pollen and/or nectar, though they may also consume pest insects that feed on cotton plant tissue or rarely consume other cotton plant tissues themselves. The principal route of soil-dwelling invertebrates, such as collembola, earthworms, and rove beetles, is assumed to be from decomposing plant tissue and plant exudates in soil. Studies submitted to the Agency with Cry1Ab and Cry2Ae proteins in lady bird beetle, green lacewing, collembola, and earthworm showed no adverse effects at concentrations well above those that would be encountered by nontarget insects in the environment. Therefore, BPPD does not anticipate the occurrence of adverse effects to nontarget invertebrates as a result of the use of T304-40 or GHB119 cotton. Additionally, studies with honey bee larvae also showed no adverse effects to honey bees at exposure levels to Cry1Ab and Cry2Ae proteins above those expected to be encountered in the environment. Thus, risk to honey bees as a result of the proposed registrations is also expected to be minimal.

It should be noted that effects to invertebrates that are recognized as cotton pests (i.e., insects that feed detrimentally on cotton plants) that are not targeted by the Cry1Ab and Cry2Ae proteins (but are controlled by other means) are not included in BPPD's assessment of risk to nontarget insects.

		2. 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 above show that the Cry1Ab and Cry2Ae proteins degrade quickly in soil.

In addition, there are several ongoing U.S. Department of Agriculture and EPA Office of Research and Development funded research projects evaluating the effects of Cry protein crops on soil microbial flora. If adverse effects are seen from this or any other research, the Agency will take appropriate action to mitigate potential risks (U.S. EPA 2010).

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

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

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

As a result of these findings and the fact that the Bt toxins engineered into T304-40 and GHB119 were derived from soil-inhabiting bacteria, the EPA has concluded that the risk of HGT of transgenes found in Cry1Ab or Cry2Ae producing cotton is low.

		4. Gene Flow and Weediness Potential

Movement of transgenes from crop plants into weeds is a significant concern, due to uncertainty regarding the effect that a new pest resistance gene may have on plant populations in the wild. Under FIFRA, the Agency has reviewed the potential for gene capture and expression of Cry proteins in commercial Bt cotton by wild or weedy relatives of cotton in the United States, its possessions or territories.

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

		5. Conclusions

BPPD concludes that significant adverse effects on birds, mammals, nontarget insects, honey bees, freshwater and marine/estuarine fish and invertebrates, and terrestrial and aquatic plants are not expected as a result of the use of Event T304-40 and Event GHB119 in cotton. EPA has also determined that there is no significant risk of gene capture and expression of Cry1Ab or Cry2Ae protein by wild or weedy relatives of cotton within the U.S. or its territories as long as the current sales and distribution restrictions on Bt cotton are in place. Available data do not indicate that Cry proteins have any measurable adverse effect on microbial populations in the soil, nor has horizontal transfer of genes from transgenic plants to soil bacteria been demonstrated.

      6. Impacts on Endangered Species

Because of the selectivity of Cry1Ab and Cry2Ae proteins for lepidopteran species, endangered species concerns are also expressed for insect species in the order Lepidoptera. The Agency has previously determined that any potential concern regarding range overlap of endangered Lepidoptera with cotton production was mainly restricted to the Kern primrose sphinx moth (Euproserpinus euterpe). However, the Agency determined that cotton is not a host plant for this species nor do host-range considerations for this species or other endangered insects place habitat in or near cotton fields (US EPA 2008a).

As previously noted, there is a possibility for gene transfer in locations where wild or feral cotton relatives exist. As a result, EPA requires stringent sales and distribution restrictions on Bt cotton within these areas to preclude outcrossing or hybridization from the crop to sexually compatible relatives. Therefore, EPA does not expect that any threatened or endangered species will be affected by outcrossing to wild relatives or by competition with such entities.

Since Cry1Ab protein has not been shown to have toxic effects on mammals, birds, freshwater invertebrates, terrestrial nontarget invertebrates, and plants, and exposure is not anticipated in aquatic environments. Therefore, the Agency makes a "No Effect" determination for direct and indirect effects to listed species of these taxa for Cry1Ab protein expressed in Event T304-40 cotton.

Since Cry2Ae protein has not been shown to have toxic effects on birds, mammals, freshwater invertebrates, terrestrial nontarget invertebrates, and plants, and also because exposure is not anticipated in aquatic environments, the Agency makes a "No Effect" determination for direct and indirect effects to listed species of these taxa for Cry2Ae protein expressed in Event GHB119 cotton.

      D. Data Needed to Confirm T304-40 and GHB119 Non-Target Hazard Assessment

BPPD has sufficient information to believe that there is no significant risk from the proposed uses of Event T304-40 and Event GHB119 cotton to non-target avian and mammalian wildlife, terrestrial invertebrates, fish and aquatic invertebrates, and plants and soil organisms. As described above, uncertainty related to testing with Cry2Ae protein in birds would be reduced by additional testing at higher doses.

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II.  Environmental Risk Assessment for Cry1Ab and Cry2Ae proteins expressed in T304-40 X GHB119 (TwinLink(TM)) Cotton Hybrid
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      A. Background

BCS developed T304-40 x GHB119 (TwinLink(TM)) cotton by conventional breeding of transgenic Event T304-40 cotton and transgenic Event GHB119 cotton, which express the Cry1Ab and Cry2Ae insecticidal proteins, respectively, for control of certain lepidopteran pests. Event T304-40 expresses a cry1Ab gene, which is derived from the soil bacterium, Bacillus thuringiensis (Bt) subsp. berliner, and Event GHB119 expresses a cry2Ae gene derived from Bt subsp. dakota.

	B. Ecological effects data for T304-40 x GHB119 (TwinLink(TM)) cotton hybrid

Environmental effects data are available for studies conducted with Cry1Ab and Cry2Ae proteins. These can be used in an assessment of risk for TwinLink(TM) cotton, provided there is no evidence of a synergistic association between Cry1Ab and Cry2Ae protein and protein expression levels of Cry1Ab and Cry2Ae are equivalent between the T304-40 and GHB119 parent lines and the TwinLink(TM) hybrid. Data have been submitted with which to determine the potential for synergy and protein expression levels. Additional toxicity data have been submitted in which the combination of these proteins has been tested (TwinLink(TM) plant material was used as the test substance) with some nontarget species, including honeybee, collembola, and broiler chicken. Additionally, data waiver rationales were presented for freshwater fish, marine/estuarine fish and invertebrates, nontarget plants, avian oral and inhalation toxicity, and wild mammals. Data available on TwinLink(TM) cotton are summarized in Table C.3, and descriptions of these studies and waiver rationales are provided below. Additional review of these studies is provided in their associated DERs.

For most studies required, BCS is seeking to bridge from data generated on the proteins expressed individually in the parent lines to the combined event. Data showing a lack of synergism between the Cry1Ab and Cry2Ae proteins are necessary for bridging, as well as data showing equivalent expression of the proteins within the TwinLink(TM) plants and the individual parent lines. A synergism study was submitted and is discussed below. Studies showing equivalence were reviewed and found acceptable in the product characterization review for TwinLink(TM) (BRAD Appendices A and B). BPPD concluded that the data from the expression of the proteins in the parent lines can be bridged to those expressed in the combined event.

Table C.3. Summary of environmental effects studies and waiver justifications for T304-40 x GHB119 (TwinLink(TM)) submitted to comply with data requirements published in 40 CFR § 158.2150 (d).
                               Data Requirement
                                     OPPTS
                                   Guideline
                                Test Substance
  C.3. TwinLink(TM) - Ecological Effects - Results Summary and Classification
                                   MRID No.
Avian dietary testing,
broiler chicken, Gallus domesticus

885.4050
TwinLink(TM) (T304-40 x GHB119) cotton seed meal
A 42-day dietary study showed no adverse affects to broiler chickens when fed a diet composed of 10% TwinLink(TM) cottonseed meal containing both Cry1Ab and Cry2Ae protein. However, the presence of the proteins in the test material was not confirmed.
Classification:  Unacceptable
 47640011
Avian inhalation testing
885.4100

N/A
Acceptable waiver rationale
47871603
Avian oral testing, Northern bobwhite
Colinus virginianus
850.2100
N/A
Data waiver rationale was submitted to justify that testing is not necessary on the basis of lack of exposure.
Classification:  Supplemental (subsequent testing with Cry2Ae protein was performed)
47903101
Freshwater fish testing,
885.4200

N/A
Acceptable waiver rationale
47871603
Estuarine and marine animal testing
885.4280

N/A
Acceptable waiver rationale
 47871603
Non-target plant testing
885.4300

N/A
Acceptable waiver rationale
 47871603
Non-target insect testing, collembola, Folsomia candida
885.4340

Lyophilized TwinLink(TM) cotton leaves
Collembola (Folsomia candida) were fed diet containing nominal concentrations of 0, 5, 20, or 50% lyophilized TwinLink(TM) cotton leaf, or 50% lyophilized near isoline cotton leaf for 28 days. No statistically significant differences in survival or reproduction among the test material and control groups were observed, indicating that exposure to TwinLink(TM) plant tissue at the levels tested has no effect on survival or reproduction of Collembola.
Classification:  Acceptable
 47640010
Honeybee testing, Honeybee larvae,
Apis mellifera
885.4380

TwinLink(TM) pollen