OCT 17 2003 MEMORANDUM SUBJECT: Assessment of the product characterization of the Cry1Ab / Cry3Bb1 combined trait, insect control proteins as expressed in maize, a Bacillus thuringiensis  based plant incorporated protectant for control of the corn borer and rootworm complexes, and consideration of a data waiver request for further acute oral toxicity evaluation.

TO: Mike Mendelsohn, Product Manager Microbial Pesticides Branch Biopesticides and Pollution Prevention Division FROM: Chris A. Wozniak, Ph. D., Biologist Microbial Pesticides Branch Biopesticides and Pollution Prevention Division THROUGH: John L. Kough, Ph. D., Senior Scientist Microbial Pesticides Branch Biopesticides and Pollution Prevention Division ACTION REQUESTED To review the product characterization considerations associated with the approval of a Section 3 registration for the insect control proteins Cry3Bb1 and Cry1Ab from Bacillus thuringiensis as expressed in maize as a dual trait product.

BACKGROUND Monsanto corporation has submitted a registration request for the Cry1Ab / Cry3Bb1   endotoxins as a combined trait product in maize as a means of controlling the European corn borer ( Ostrinia nubilalis) and other lepidopteran pests ( i. e., Diatraea grandosiella, D. crambidoides,, Helicoverpa zea, Spodoptera frugiperda, Papaipema nebris) and the corn rootworm complex, Diabrotica spp. These products are based on the insecticidal protein of B. thuringiensis strain HD 1 / HD 73 for Cry1Ab protein and strain EG11098, a modified Cry3Bb1 2 protein, which represents the active ingredient of this plant incorporated protectant. The combined trait product will be known as YieldGard ® Plus Corn and was developed through traditional breeding of maize plants containing cry1Ab ( MON 810) and cry3Bb1 ( MON 863). Maize plants also express neomycin phosphotransferase ( nptII) which was used for tolerance to the antibiotic kanamycin during in vitro culture and selection.

DATA REVIEW RECORD Active Ingredient: Bacillus thuringiensis Cry1Ab / Cry3Bb1 protein as expressed in maize Product Name: Event MON 810 x MON 863, Cry1Ab / Cry3Bb1 proteins as expressed in maize ID No.: 000524 LUL Submission No.: S627345 Chemical No.: 006484 Bacillus thuringiensis Cry3Bb1 protein and genetic material for its production. DP Barcode: D287601 Sponsor: Monsanto Corporation, 700 Chesterfield Parkway, St. Louis, MO 63198 MRID/ Study Titles: 457917 01  Confirmation of the molecular identity of YieldGard ® and corn rootworm protected combines trait corn hybrid MON 810 x MON 863 by Southern blot analysis. 457917 02  Cry3Bb1, Cry1Ab and NPTII protein levels in the dual trait maize hybrid MON 863 x MON 810 produced in Argentina field trials conducted during the 1999 2000 growing season. Data Waiver Request: Acute toxicity and irritation data requirements for the end use product, YieldGard ® Plus Corn seed containing Cry1Ab and Cry3Bb1 proteins from Bacillus thuringiensis.

SUMMARIES OF REVIEW MRID# 457917 01  Confirmation of the molecular identity of YieldGard ® and corn rootworm protected combines trait corn hybrid MON 810 x MON 863 by Southern blot analysis. The corn hybrid MON 810 x MON 863 containing the two transformation events, MON 810 ( cry1Ab) and MON 863 ( cry3Bb1) was examined for the presence of these two genes encoding   endotoxins in the resulting hybrid. Probes for the cry1Ab and cry3Bb1 genes were obtained from previous studies and corresponded to the first 900 bp ( cry1Ab) or the entire length of the gene ( cry3Bb1). The radiolabelled ( 32P) probe for cry1Ab hybridized to restricted DNA samples on nylon membranes and resulted in a lack of any signal detection for the samples from MON 846 ( non transgenic) and MON 863 samples, but did detect the presence of cry1Ab in the MON 810 and MON 810 x MON 863 hybrid plant samples. When DNA samples were probed 3 with the cry3Bb1 sequence, hybridization confirmed the presence of this gene in MON 863 plants and the MON 810 x MON 863 hybrid, but again failed to detect the presence of this gene in the negative control MON 846. Plasmid DNA from plasmids containing either gene separately did react positively with the appropriate probes when the restricted plasmid DNA was coelectrophoresed with MON 846 DNA and hybridized with the respective probe. From these results it is evident that the hybrid MON 810 x MON 863 contains the cry1Ab and cry3Bb1 genes. Additionally, the restriction patterns noted on the Southern blot provided suggest that there have been no major alterations or rearrangements in the conventional cross of these two events ( hybrids) for these two gene inserts.

457917 02  Cry3Bb1, Cry1Ab and NPTII protein levels in the dual trait maize hybrid MON 863 x MON 810 produced in Argentina field trials conducted during the 1999 2000 growing season. The hybrid maize resulting from the MON 863 x MON 810 cross represents the test substance for this study. This dual trait or stacked trait hybrid ( also known as MT 02) results from the contribution of the cry3Bb1 and nptII genes from MON 863 and the cry1Ab gene from MON 810. The Cry3Bb1 protein from MON 863 is also known as Cry3Bb1.11098, but will be referred to in this review as simply Cry3Bb1. Reference protein Cry3Bb1standard for the ELISA was produced by fermentation of Bacillus thuringiensis encoding Cry3Bb1.11231 and stored as a lyophilized powder. Cry1Ab protein, produced in E. coli, was trypsinized and established as the reference standard. The coding sequence for nptII was cloned and transferred to E. coli for fermentation. Four field sites within the province of Buenos Aires ( Argentina) were used to culture plants for sample generation: Fontezuela 1, Fontezuela 2, Salto and Rojas. Young leaves, pollen, mature root, over season root, grain and forage samples were taken for ELISA analysis. For each protein, a validated double antibody sandwich ELISA has been developed. Antibodies to these proteins were raised in rabbits and Protein A selected. The trypsinized core of Cry1Ab was used rather than the full length protein for antibody generation. For Cry3Bb1 and NPTII protein levels, a tissue specific correction factor was used to optimize the accuracy and reduce bias in the estimation of protein content of samples based upon extraction efficiency and recovery. Samples for forage, grain and root analysis of Cry3Bb1 protein content were stored longer than their intended period of known stability in some instances. An increase in apparent Cry3Bb1 protein levels resulted from the estimated 10 to 20 % instability evidenced in these samples stored beyond their intended dates of analysis. The net result is that the Cry3Bb1 levels reported may be overestimated by as much as 20 %. Data reported from forage samples were not adjusted as presented for this instability factor. Root tissues were estimated to exhibit a 20 to 30 % instability which resulted in a decrease in apparent Cry3Bb1 levels in roots. Hence, the Cry3Bb1 levels reported may be underestimated by up to 30 %. Data from grain samples were estimated to be overestimated by up to 20 % due to instability in stored samples. The reported root and grain data were not adjusted, as reported, for this instability factor. Data regarding the stability or instability of extracts stored beyond their intended expiration dates were not presented. The ranges of protein levels observed for Cry3Bb1, Cry1Ab and NPTII were similar across all four sites for the MON 863 x MON 810 hybrid and the MON 863 or MON 810 hybrids as appropriate for the 4 transgene in question. Averages for the Cry3Bb1 and Cry1Ab proteins were, however, somewhat higher in the MON 863 x MON 810 hybrid versus the single trait hybrids. NPTII protein levels and ranges in the MON 863 x MON 810 hybrid and the MON 863 hybrid were similar. The highest levels of expression of Cry3Bb1 or Cry1Ab were found to occur in pollen for Cry3Bb1 ( 79.6 µ g/ g FWT) and in leaf ( 17.9 µ g/ g FWT) for Cry1Ab.

Data Waiver Request: Acute toxicity and irritation data requirements for the end use product, YieldGard ® Plus Corn seed containing Cry1Ab and Cry3Bb1 proteins from Bacillus thuringiensis  The registrant requests a waiver for the following studies: Acute Oral Toxicity ( Guideline Reference No. 152 30); Acute Dermal Toxicity ( Guideline Reference No. 152 31); Acute Inhalation Toxicity ( Guideline Reference No. 152 32); Primary Dermal Irritation ( Guideline Reference No. 152 34); Primary Eye Irritation ( Guideline Reference No. 152 35); Hypersensitivity ( Guideline Reference No. 152 36). The combination of a lack of human exposure to these proteins as expressed in maize and a safe history for   endotoxins derived from Bacillus thuringiensis are the main reasons that waivers were requested. All of the above studies which are required for registration of a biological pesticide may be waived due to lack of human exposure to the insecticidal proteins, or in the case of the acute oral toxicity test, previous evaluation of these proteins. Since the primary route of exposure for these proteins, Cry1Ab and Cry3Bb1, is through consumption of the maize grain or forage expressing these proteins, the acute oral toxicity tests previously reviewed for these proteins will be sufficient to conclude that there is no acute oral toxicity to mammals associated with consumption of these proteins. These proteins are known to act individually to effect a typical midgut pathology in susceptible insects which is characteristic of previously studied   endotoxins of Bacillus thuringiensis. No synergistic action or interaction of these proteins is known or expected to occur. 5 DATA EVALUATION REPORT Reviewed by: Chris A. Wozniak, Ph. D., Biologist, OPP / BPPD Secondary Reviewer: John L. Kough, Ph. D., Biologist, OPP / BPPD MRID No.: 457917 01 Active Ingredients: Bacillus thuringiensis Cry1Ab / Cry3Bb1 protein as expressed in maize Product Name: Event MON 810 x MON 863, Cry1Ab / Cry3Bb1as expressed in maize Confidentiality: No claim of confidentiality is made under FIFRA 10( d)( 1)( A), ( B) or ( C). ID No.: 000524 LEI Submission No.: S600950 Chemical No.: 006484 Bacillus thuringiensis Cry3Bb protein and genetic material for its production. DP Barcode: D276610 Sponsor: Monsanto Corporation, 700 Chesterfield Parkway, St. Louis, MO 63198. Authors: I. G. Borovkov, T. A. Cavato, R. P. Lirette Testing Facility: Monsanto Corporation, Product Characterization Center, Biotechnology Regulatory Services, 700 Chesterfield Parkway North, St. Louis, MO 63198 Study Titles: Confirmation of the molecular identity of YieldGard ® and corn rootworm protected combines trait corn hybrid MON 810 x MON 863 by Southern blot analysis. Study Date: October 19, 2001 Study No.: 01 01 39 39; MSL 17466 Conclusion: The corn hybrid MON 810 x MON 863 containing the two transformation events, MON 810 ( cry1Ab) and MON 863 ( cry3Bb1) was examined for the presence of these two genes encoding   endotoxins in the resulting hybrid. Probes for the cry1Ab and cry3Bb1 genes were obtained from previous studies and corresponded to the first 900 bp ( cry1Ab) or the entire 1.96 kbp length of the gene ( cry3Bb1). The radiolabelled ( 32P) probe for cry1Ab hybridized to DNA samples on nylon membranes and resulted in a lack of any signal detection for the samples from MON 846 ( non transgenic) and MON 863 samples, but did detect the presence of cry1Ab in the MON 810 and MON 810 x MON 863 hybrid plant samples. When DNA samples were probed with the cry3Bb1 sequence, hybridization confirmed the presence of this gene in MON 863 plants and the MON 810 x MON 863 hybrid, but again failed to detect the presence of this gene in the negative control MON 846. Plasmid DNA from plasmids containing either gene separately did react positively with the appropriate probes when the restricted plasmid DNA was co electrophoresed with MON 846 DNA and hybridized with the respective probe. From these results it is evident that the hybrid MON 810 x MON 863 contains the cry1Ab and cry3Bb1 genes. Additionally, the restriction patterns noted on the Southern blot provided 6 suggest that there have been no major alterations or rearrangements in the conventional cross of these two events ( lines) for these two gene inserts. Classification: Acceptable.

Good Laboratory Practices: This submission was conducted in compliance with the Certification of Good Laboratory Practices as outlined in 40 CFR 160.

Purpose: To describe and confirm the presence of the combined trait genotype ( i. e., cry1Ab and cry3Bb1) in the MON 810 x MON 863 cross, known as YieldGard ® Plus.

METHODS: Test substance  The corn hybrid MON 810 x MON 863 ( lot# TII 0108 11672 S) containing the two transformation events, MON 810 ( cry1Ab) and MON 863 ( cry3Bb1). Grain was from Production Plan # 99 04 39 01 and was stored at  80

C after grinding.

Control substances  Both single transformation event maize lines, MON 810 ( lot# TIO 0108 11670 S) and MON 863 ( lot# TIO 0108 11669 S), as well as a non transformed control hybrid MON 846 ( lot# TNT 0108 11671 S) were included as controls for comparison. Grain from these, as produced under Production Plan # 99 04 39 01, were ground and stored at  80

C until needed.

Reference substances  Plasmid PV ZMBK07 was included as a positive control for Southern blotting as it contains the cry1Ab gene as present in MON 810. Similarly, plasmid PV ZMIR13 was used in Southern blotting as a positive control for cry3Bb1, as found in event MON 863. Molecular weight markers from Roche Diagnostics ( Indianapolis, IN) were used to estimate DNA size following hybridization.

All control and test substances were verified by event specific PCR for MON 810 and MON 863 prior to use. Data supporting this evidence were not provided in this submission.

DNA extraction  Approximately 6 g of ground grain sample was added to a conical tube. CTAB extraction buffer ( 1.5 % CTAB, 75 mM Tris pH 8.0, 100 mM EDTA pH 8.0, 1.05 M NaCl, 0.75 % PVP [ MW 40000]; 16 mL) was added to each tube and these were allowed to incubate at 65

C for approximately 30 minutes. Chloroform: Isoamyl alcohol ( 24: 1) was added to the sample after allowing 5 minutes for sample cooling. Following mixing and centrifugation ( 5 min., 16,139 x g), the upper phase ( aqueous) was transferred to a new conical tube and combined with 1.6 mL of CTAB buffer. An equal volume of Chloroform: Isoamyl alcohol ( 24: 1) was added to the separated phase and centrifugation repeated. The upper phase was removed to a new tube and DNA precipitated with CTAB precipitation buffer. DNA was pelleted at 10,329 x g and 7 resuspended in 2 mL of high salt TE ( 10 mM Tris, pH 8.0; 10 mM EDTA, pH 8.0, 1 M NaCl) and incubated for 2 h at 37

C. The suspended DNA sample was then centrifuged ( 23,240 x g) and the supernatant transferred to new tube. Sodium acetate ( 3 M) and ethanol were added to the supernatant to reprecipitate the DNA. Following inversion the DNA was spooled out and transferred to two ethanol washes, then centrifuged to pellet and vacuum dried. The pellet was dissolved in TE and stored at 4

C .

DNA digestion  Approximately 10 µ g of genomic DNA was digested overnight at 37

C with one or more restriction enzyme( s). Following digestion, the DNA was precipitated with sodium acetate and ethanol, then suspended in water.

DNA probes  Probes for the cry1Ab and cry3Bb1 genes were obtained from previous studies and corresponded to the first 900 bp ( cry1Ab) or the entire length ( 1.96 kbp) of the gene ( cry3Bb1). Each probe template was labeled with 32P dCTP ( 6000 Ci/ mmol) using a random priming kit ( Gibco BRL).

Southern blot analysis  DNA samples from ground grain were electrophoresed on an 0.8 % agarose gel and then stained with ethidium bromide. The samples were depurinated with a 0.125 N HCl solution for 10 minutes, then denatured with a 0.5 M NaOH, 1.5 M NaCl solution for 30 minutes. The gel was then neutralized with 0.5 M Tris HCl, pH 7.0, 1.5 M NaCl for 30 minutes and eventually equilibrated in 20 X SSC transfer buffer. DNA samples were then transferred to Hybond N membrane in 20 x SSC for 20 hours. Transferred DNA fragments were crosslinked to the membrane with an ultraviolet light treatment.

Pre hybridization treatment of the membrane was carried out for 3 hours in 500 mM disodium phosphate pentahydrate, 7 % sodium dodecyl sulfate, and 10 mg/ mL tRNA from E. coli. Hybridization was performed in fresh pre hybridization solution with radiolabelled probe for 17 to 20 hours at 65

C. Membranes were subsequently washed in 0.1 % SDS, 0.1 % SSC for two 15 min periods and two 20 min periods at 65

C. Autoradiography was performed using a Kodak Biomax intensifying screen and Kodak Biomax MS 2 film.

RESULTS: MON 810 and MON 863 event specific fingerprints  Restriction enzymes NcoI and EcoRI were used to digest test and control DNA samples. DNA from MON 846, the non transgenic control, was mixed with plasmid PV ZMBK07 and digested similarly. This plasmid is cleaved at three sites by the two restriction enzymes and one of the fragments produced ( 3.5 kbp) would be detected by the 900 bp probe for cry1Ab coding region. The radiolabelled ( 32P) 900 bp probe was hybridized to DNA samples on a nylon membrane and resulted in a lack of any signal detection for the samples from MON 846 or MON 863 plants. This was expected since neither of these genotypes carry the cry1Ab gene. The primary signal detected was from a 3.5 kbp band present in the lane containing the restricted PV ZMB07 DNA, which does contain the cry1Ab gene. Less intense signal was detected in bands hybridizing with the probe at 9.0, 6.5 and 6.0 kbp. It is 8 suggested that these results are a reflection of star activity by the restriction enzymes ( i. e., nonspecific cutting by enzymes due to chemical or physical conditions present at the time of digestion) or an incomplete digestion by the enzymes. Some non specific hybridization was reported as well around 4.4 kbp in between lanes 1 and 2 of the Southern blot. The shape of the signal as it appears following autoradiography indicates that it is not due to direct hybridization with an electrophoresed band or fragment of DNA. The most likely explanation for this aberrant signal is a non specific binding of the probe to the membrane where incomplete blocking of the surface occurred.

DNA samples from MON 810 and MON 810 x MON 863 plants produced the predicted 3.1 kbp fragment as a hybridization signal. The size of this fragment for the cry1Ab coding region differs from that expected from the digestion of the plasmid PV ZMB07 due to the loss of an EcoRI site on the integrated gene or construct. This was previously noted for MON 810 according to internal Monsanto documents. As a result, the EcoRI enzyme cleaves the DNA adjacent to the insert in the host flanking chromosomal DNA.

EcoRV was used to test for the presence of the MON 863 event in test and control samples. DNA from MON 846 was mixed with plasmid DNA PV ZMIR13 and restricted with EcoRV. This enzyme cleaves PV ZMIR13 once producing a 7.3 kb fragment which will hybridize with the probe for the coding region of MON 863 gene cry3Bb1. When the nylon membrane was probed using a 1.96 kbp radiolabelled ( 32P) probe comprising the cry3Bb1 coding region, hybridization signal was detected in a band at 9.6 kbp in lanes containing DNA from MON 863 and MON 810 x MON 863 plants. This fragment size differs from that predicted by EcoRV digestion of the plasmid PV ZMBIR13 ( 7.3 kbp) as the second EcoRV site which results in fragment production from digestion of the genomic DNA must come from the flanking DNA near the inserted cry3Bb1 gene ( i. e., the plasmid contains only a single EcoRV site). DNA samples from MON 810 or the nontransgenic MON 846 did not produce any detectable hybridization signal when probed similarly. This was expected from previous studies conducted in house by Monsanto and is predictable from the plasmid restriction map provided with this submission.

From these results it is evident that the hybrid MON 810 x MON 863 contains the cry1Ab and cry3Bb1 genes. Additionally, the restriction patterns noted on the Southern blot provided suggest that there have been no major alterations or rearrangements in the conventional cross of these two events ( hybrids) for these two gene inserts.

DATA EVALUATION REPORT 9 Reviewed by: Chris A. Wozniak, Ph. D., Biologist, OPP / BPPD Secondary Reviewer: John L. Kough, Ph. D., Biologist, OPP / BPPD MRID No.: 457917 02 Active Ingredients: Bacillus thuringiensis Cry1Ab / Cry3Bb1 protein as expressed in maize Product Name: Event MON863 x MON 810, Cry3Bb1 / Cry1Ab as expressed in maize Confidentiality: No claim of confidentiality is made under FIFRA 10( d)( 1)( A), ( B) or ( C). ID No.: 000524 LEI Submission No.: S600950 Chemical No.: 006484 Bacillus thuringiensis Cry3Bb protein and genetic material for its production. DP Barcode: D276610 Sponsor: Monsanto Corporation, 700 Chesterfield Parkway, St. Louis, MO 63198. Authors: Y. A. Dudin, B P Tonnu, R. P. Lirette Testing Facility: Monsanto Corporation, Product Characterization Center, Biotechnology Regulatory Services, 700 Chesterfield Parkway North, St. Louis, MO 63198 Study Titles: Cry3Bb1, Cry1Ab and NPTII protein levels in the dual trait maize hybrid MON 863 x MON 810 produced in Argentina field trials conducted during the 1999 2000 growing season. Study Date: December 12, 2001 Study No.: 00 01 39 44; MSL 17266 Conclusion: ELISA values for field grown maize samples ( grain, forage, root, leaf and pollen) were examined with a double antibody sandwich technique and Cry3Bb1 and NPTII protein values adjusted for method bias to optimize the accuracy based upon extraction efficiency, and for variance in storage stability of extracts in some instances. The ranges of protein levels observed for Cry3Bb1, Cry1Ab and NPTII were similar across all four field sites for the MON 863 x MON 810 hybrid and the MON 863 or MON 810 hybrids as appropriate for the transgene in question. Averages for the Cry3Bb1 and Cry1Ab proteins were, however, somewhat higher in the MON 863 x MON 810 hybrid versus the single trait hybrids. NPTII protein levels and ranges in the MON 863 x MON 810 hybrid and the MON 863 hybrid were similar. The highest average levels of expression of Cry3Bb1 or Cry1Ab were found to occur in pollen for Cry3Bb1 ( 79.6 µ g/ g FWT) and in leaf ( 17.9 µ g/ g FWT) for Cry1Ab. Classification: Acceptable.

Good Laboratory Practices: This submission was conducted in compliance with the Certification of Good Laboratory Practices as outlined in 40 CFR 160. 10 Purpose: To describe the levels of Cry3Bb1, Cry1Ab and NPTII proteins in various tissues collected from the insect protected MON 863 x MON 810 maize hybrid. The plants tested resulted from a multi site field trial in Argentina during the 1999 2000 growing season.

METHODS: Test substance  The hybrid maize resulting from the MON 863 x MON 810 cross represents the test substance for this study. This dual trait or stacked trait hybrid ( also known as MT 02) results from the contribution of the cry3Bb1 and nptII genes from MON 863 and the cry1Ab gene from MON 810. The Cry3Bb1 protein from MON 863 is also known as Cry3Bb1.11098, but will be referred to in this review as simply Cry3Bb1.

Control substances  MON 863, MON 810 and MON 846 represent the hybrids used as controls for this analysis. The last hybrid has a genetic background similar to that of the other hybrids, but does not contain the cry3Bb1, cry1Ab or nptII genes. Event specific PCR was conducted to ascertain the presence of the transgenes in MON 863, MON 810 and to confirm the absence of transgenes in MON 846. Additionally, other events which were planted in the field at the time of hybrid production were screened for in these hybrids to ensure their absence in the test and control substances ( hybrids).

Reference substances  Purified Cry3Bb1, Cry1Ab and NPTII were used as protein standards in the ELISA assays.

Cry3Bb1 protein standard  Variant Cry3Bb1.11231 ( lot# 6312812) protein was used as a standard in the Cry3Bb1 ELISA. This variant differs from the wild type Cry3Bb1 by four amino acids from the wild type form. This variant was used as a standard in the ELISA assays. The variant of the Cry3Bb1 protein used in the MON 863 event differs from the Cry3Bb1.11231 form by 3 amino acids and from the wild type by seven amino acids. The variant in MON 863 ( Cry3Bb1.11098) and the Cry3Bb1.11231 proteins share > 99% homology. The polyclonal antibodies used in the ELISA were produced against the wild type form of Cry3Bb1.

Reference protein standard for the ELISA was produced by fermentation of Bacillus thuringiensis encoding Cry3Bb1.11231 and stored as a lyophilized powder. This variant and the one found in MON 863 were previously shown to be physicochemically and functionally equivalent. This lyophilized powder was prepared in 50 mM sodium bicarbonate with 0.05 % ( v/ v) Tween 20, pH 9.6 and assigned lot # 6307780. Storage was at  80 C. Amino acid composition analysis was used to estimate the concentration of this protein to be 0.291 mg/ mL and purity was found to be 93.2 % by SDS PAGE and densitometry. Adjusting for the purity of the preparation, the final concentration of the Cry3Bb1 protein in the assay was 0.271 mg/ mL.

Cry1Ab protein standard  Cry1Ab protein ( lot# 6700409), produced in E. coli, was trypsinized and established as the reference standard. Purity was determined as 94 % by SDS PAGE and densitometry. Total protein concentration was determined to be 3.41 mg/ mL by amino acid 11 composition analysis, but following adjustment for purity, the final concentration is 3.21 mg/ mL. Aliquots of this protein were stored at  80

C in 50 mM carbonate bicarbonate ( pH 10.25) buffer and 50 mM sodium chloride.

NPTII protein standard  The coding sequence for nptII ( neomycin phosphotransferase) was cloned and transferred to E. coli for fermentation. Lyophilized protein powder ( lot# 4821020) was solubilized in 50 mM carbonate bicarbonate buffer and assigned lot # 6340505 and stored at  80

C. Amino acid composition analysis was used to estimate the protein concentration at 1.15 mg/ mL and the purity was determined as 99.5 % based upon SDS PAGE and subsequent densitometry. No adjustment for purity was performed.

Generation of plant samples for analysis  Four field sites within the province of Buenos Aires ( Argentina) were used to culture plants for sample generation: Fontezuela 1, Fontezuela 2, Salto and Rojas. Agronomic practices and field conditions were typical for maize production at the four sites. A randomized complete block design was used with two row plots and four replications per site All samples collected were maintained on dry ice within 30 minutes of collection. Grain, however, was handled at ambient temperatures.

Young leaf  Young leaves ( YL) were pooled from 15 plants at the V 4 growth stage ( approximately 18 days post planting) for each of the four replications.

Forage  Two entire plants, including both aerial and root portions, were harvested from each of the four replicates for forage analysis. Plants were taken at the early dent stage, approximately 90 days post planting. Plant material was cut into 10 to 15 cm segments and stored frozen on dry ice.

Mature root and over season root  The root mass from two plants collected at the same time as the forage samples to represent the mature root samples. Only one sample of over season roots was collected at 46 days post planting for Cry3Bb1 analysis.

Pollen  Pollen samples were collected at all sites approximately 60 days post planting which represents 50 % pollen shed. Test and control samples were prepared by collecting 20 to 25 g of pollen from 10 to 15 plants for each of the four replicated plots at each of the four sites.

Grain  Self pollinated primary ears were collected from all four sites ( four replicates / site) and air dried to moisture below 15 %. Grain was collected at maturity ( approximately 117 days post planting).

Sample storage, processing and shipping  Storage and processing of plant samples occurred at both Fontezuela, Argentina and St. Louis, MO, USA. Tissue samples were handled on dry ice and stored frozen, however, grain samples were handled at ambient temperatures. Samples shipped to St. Louis were shipped on dry ice and stored at  80

C upon receipt. All tissues, except pollen, were processed prior to extraction. Samples processed in Argentina were stored at  80

C and 12 then shipped on dry ice to St. Louis for further analysis.

ELISA analytical methods  Extraction of protein from maize tissues  Control samples were extracted prior to test samples to minimize contamination. Forage tissues were extracted with triborate buffer containing 0.2 % ( w/ v) ascorbic acid ( TBA) at a 1: 50 ( g/ mL) ratio. Grain was extracted at a 1: 100 ratio ( g/ mL) in phosphate buffered saline with Tween 20 ( PBST). Pollen samples are extracted at a 1: 40 ratio using TBA for Cry1Ab and PBST with 0.1 % BSA ( bovine serum albumin) for Cry3BB1 analysis. Leaf samples analyzed for NPTII and Cry3Bb1 proteins were processed in PBST with 0.1 % BSA at a 1: 100 ( g/ mL) ratio and in PBST at a 1: 50 ratio for CryAb1. Mature root and over season root were extracted in PBST with 0.1 % ( w/ v) BSA at a 1: 100 ( g/ mL) ratio for Cry3Bb1 using a Polytron ® homogenizer ( Brinkmann Instruments). Pollen was extracted with a Wheaton 2000 homogenizer for the smaller sample sizes associated with this tissue. Insoluble materials were removed by use of a Serum Filter System ( Fisher Scientific) or centrifugation. Clarified extracts were stored at  80

C.

Cry3Bb1, Cry1Ab and NPTII protein ELISA  For each protein, a validated double antibody sandwich ELISA has been developed. The validation data is presented in the appendix 2 of this volume. For Cry3Bb1 protein the following LODs are: 0.087 µ g/ g FWT for leaf, 0.22 µ g/ g FWT for forage, 0.096 µ g/ g FWT for grain and 0.76 µ g/ g FWT for roots. The LOD for pollen was estimated from non transgenic field samples to be 0.7 µ g/ g. For Cry1Ab protein the following LODs were established: 0.55 µ g/ g FWT for leaf, 0.26 µ g/ g FWT for forage, 0.13 µ g/ g FWT for grain, and 0.08 µ g/ g FWT for pollen. The LODs for NPTII protein were established at: 0.093 µ g/ g FWT for leaf, 0.075 µ g/ g FWT for forage, and 0.076 µ g/ g FWT for grain.

Reagents for Cry3Bb1 ELISA  A rabbit anti Cry3Bb1 antibody was purified by Protein A selection and used as the capture antibody. Two lots of coating antibody were using during the testing: # 6199830A and # 6199830B, at concentrations of 3.95 mg/ mL and 3.21 mg/ mL, respectively. The rabbit polyclonal antibody was also conjugated to horseradish peroxidase ( HRP) to serve as the detection antibody. The final concentration of this antibody was 1.1 mg/ mL and it was stored in a buffer containing 0.02 M potassium phosphate, 0.15 M sodium chloride, and 0.01 % thimerosal.

Reagents for Cry1Ab ELISA  A rabbit anti Cry1Ab antibody was purified by Protein A selection and used as the capture antibody. One lot of coating antibody was using during the testing: # 6178568A at a concentration of 7.7 mg/ mL. The rabbit polyclonal antibody was also conjugated to alkaline phosphatase ( AP) to serve as the detection antibody. The final concentration of this antibody was 4.5 mg/ mL and it was stored in a buffer containing 50 mM Tris HCl, pH 8.0, 1 mM magnesium chloride, 1 % BSA and 0.1 % sodium azide.

Reagents for the NPTII ELISA  A rabbit anti NPTII antibody was purified by Protein A selection and used as the capture antibody. Two lots of coating antibody were using during the 13 testing: # 6549463A at concentration of 4.7 mg/ mL and 3.21 mg/ mL, respectively. The rabbit polyclonal antibody was also conjugated to horseradish peroxidase to serve as the detection antibody. The final concentration of this antibody was 3.0 mg/ mL and it was stored in a buffer containing 0.02 M potassium phosphate, 0.15 M sodium chloride, and 0.01 % thimerosal.

Cry3Bb1 and NPTII ELISA  Polyclonal antibody preparations for these two proteins were diluted in carbonate bicarbonate buffer at a final concentration of 5.0 mg/ mL. Microtiter plate wells were coated overnight at 4

C with 100 µ L of the diluted antibody solutions ( separately). Plates were washed in PBST three times prior to assay. Antibody binding was carried out at 37

C for 60 to 70 minutes using 50 µ L of the antibody preparation and 50 µ L of the secondary conjugated antibody simultaneously. Plates were then washed again with PBST three times. Tetramethylbenzidine ( TMB) was used as the development agent for these reactions at room temperature for 10 to 15 minutes. Reactions were terminated with 6 M phosphoric acid. Absorbance readings were taken at 450 nm and 650 nm as reference wavelengths and a correlation between color development and degree of secondary antibody binding was established using standard calibration curves. A seven point standard curve, ranging from 1 to 64 ng/ mL, for each ELISA was used to estimate the concentration of protein in the ELISA by interpolation.

Cry1Ab ELISA  Antibody to Cry1Ab was raised in rabbits using the tryptic core of the protein as an antigen. The antibody was diluted in plate coating buffer and add to wells at 2.0 µ g/ mL in 250 µ L / well and plates were incubated overnight at 4

C. Prior to assay plates were washed two times with PBST. Tissue extracts were digested with trypsin prior to assay ( 175 µ g/ mL) and the reaction terminated by the addition of 50 mM phenylmethylsulfonyl fluoride to a final concentration of 1.21 mM. Trypsinized sample extracts were incubated ( 200 µ L) overnight at 4

C with 50 µ L of rabbit anti Cry1Ab antibody conjugated to AP. Plates were washed three times with PBST. Wells were subsequently developed using p nitrophenyl phosphate ( pNPP) in ethanolamine buffer, pH 9.7 for 20 to 25 minutes at room temperature. Absorbance readings were taken at 450 nm and 650 nm as reference wavelengths and a correlation between color development and degree of secondary antibody binding was established using standard calibration curves. A seven point standard curve, ranging from 1 to 64 ng/ mL, for each ELISA was used to estimate the concentration of protein in the ELISA by interpolation. The standard curve ranged from 0.4 to 16 ng/ mL.

To estimate the final concentration of Cry1Ab, it has historically required a multiplication by a factor of 2 to account for the difference in reaction of the trypsinized core of the protein versus the full length moiety. No data was presented to corroborate this statement.

Control of bias  A tissue specific correction factor was used to optimize the accuracy and reduce bias in the estimation of protein content of samples. The following equation describes the calculations: 14 Bias Correction Factor = (% extraction efficiency) x (% spike and recovery) ( 100) ( 100)

In order to maintain consistency with previously reported data, the Cry1Ab data were not adjusted for method bias. For Cry3Bb1 and NPTII an estimation of protein content in a sample was obtained by using the observed µ g/ g FWT divided by the tissue specific method bias correction factor as follows:

Observed µ g/ g FWT = Corrected µ g/ g FWT Bias Correction Factor Data reduction and statistical analysis  ELISA plates were measured for absorbance on a microplate reader. HRP readings were taken at 450 nm, whereas the AP absorbance readings were taken at 405 nm. A reference wavelength of 650 nm was used in both cases; this value was then subtracted from the sample reading at 405 nm or 450 nm as appropriate. Data reduction analysis was performed using SOFTmax PRO version 2.4.1 ( Molecular Devices, Sunnyvale, CA). For all three ELISA methods, a four parameter logistic curve was used to fit the data for absorbance readings of standard curve measurements and sample data. Interpolating from the standard curve, the amount of protein in the tissue extracts was converted to µ g/ g FWT using dilution factors, tissue to buffer ratios, and the bias correction factors described above.

RESULTS: Test and control substance characterization  Event specific PCR was used to characterize grain samples harvested from all sites. PCR was used to identify MON 863 and MON 810 events in samples as well as other events present in the fields but not part of this submission. Grain identity was confirmed in all sites with one exception where in a sample from the MON 863 plants and one from the MON 863 x MON 810 hybrid were excluded due to the presence of a contaminating event. For the control hybrid, MON 846, 13 of 16 grain samples were determined to be ` event free'. In the three control samples excluded, the Cry3Bb1 and NPTII protein levels were below their respective LODs, they were still excluded from analysis.

Test and control substance stability  Some of the Cry3Bb1 samples ( tissues and extracts) were stored beyond their recommended date of storage. From previous work on the stability of Cry3Bb1 at  80

C, it was possible to determine the method bias using experimental time points which covered the time frame considered. This was also true for some NPTII sample extracts. All tissues and extracts were stored at  80

C to prevent any further degradation throughout the study.

Cry3Bb1 protein stability  Samples for forage, grain and root analysis of Cry3Bb1 protein content were stored longer than their intended period of known stability in some instances. An analysis of forage tissue stability using time points ( i. e., storage periods) similar to those present in 15 this analysis. An increase in apparent Cry3Bb1 protein levels resulted from the estimated 10 to 20 % instability evidenced in these samples stored beyond their intended dates of analysis. The net result is that the Cry3Bb1 levels reported may be overestimated by as much as 20 %. Data reported from forage samples were not adjusted as presented for this instability factor. Root tissues were estimated to exhibit a 20 to 30 % instability which resulted in a decrease in apparent Cry3Bb1 levels in roots. Hence, the Cry3Bb1 levels reported may be underestimated by up to 30 %. Data from grain samples were estimated to be overestimated by up to 20 % due to instability in stored samples. The reported root and grain data were not adjusted, as reported, for this instability factor.

NPTII protein extract stability  Grain extracts analyzed for NPTII levels were initially analyzed during the stability period and then again following expiration of that period. The first data set was accepted with one deviation from the accepted protocol ( SOP) in that blank readings were slightly above the SOP specification. The positive and negative control samples were found to be within their acceptable ranges as defined by the SOP. This deviation was not considered to have compromised the analysis. When this analysis was repeated, all of the ELISA criteria were met for the SOP, however, the extracts were then outside the stability period. This second data set confirmed the initial data set in that the levels of NPTII were determined to be below the quantifiable NPTII protein level. The first data set was then used as a valid assessment of the NPTII protein levels observed in grain.

Two forage extracts were analyzed beyond their known stability periods to determine that the NPTII levels were below the LOD. A second analysis reached the same conclusion; NPTII levels reported for the MON 863 samples were determined to be valid estimates of this protein's levels in forage.

Protein levels in maize tissues  Average values are reported for the levels of Cry3Bb1, Cry1Ab and NPTII as tabulated below. These are reported as µ g/ g FWT with a total number of samples analyzed being 16, except where noted. Average values and standard deviations reported omit the samples found to be below the LOD, however, these samples were included in the ranges reported.

For the dual trait samples analyzed for Cry1Ab in grain, 15 samples were analyzed since one was discarded due to contamination. Grain analyzed for Cry3Bb1 in MON 863 consisted of 14 samples as one was omitted for contamination and one was found to be below the LOD. For the MON 863 x MON 810 hybrid, 15 samples were analyzed as one was discarded for contamination. Root and forage data for the MON 863 hybrid consisted of 13 plant samples as three samples were below the LOD. For both the MON 863 and MON 863 x MON 810 hybrids, the NPTII data set included 15 extracts as one sample was excluded in each case due contamination.

Pollen analyses for Cry3Bb1 protein levels in MON 863 contained 16 samples and levels were similar across all four field sites.

MON 846 ( non transgenic) samples were not included in the Cry1Ab ELISA as required by the 16 SOP. A negative control ( QC) sample was included in every ELISA to detect any problems with the assay. This negative QC sample was within the guidelines established in the SOP.

Cry3Bb1 protein levels  Levels in the MON 863 x MON 810 hybrid presented below are overestimates of Cry3Bb1 protein levels since some of the samples were beyond their documented stability period.

Tissue Type and Collection Time ( Days post planting*) Average Cry3Bb1 protein levels ( µ g/ g FWT)

MON 863 x MON 810 Average Cry3Bb1 protein levels ( µ g/ g FWT)

MON 863 Young leaf ( 18) 46.7 ( 35.5  53.2) 30.0 ( 21.3  47.2)

Forage ( 90) 23.6 ( 6.7  39.7) 12.8 (< 0.22  28.8)

Grain ( 117) 61.1 ( 38.5  83.1) 43.7 (< 0.096  84.1)

Pollen ( 60) 79.6 ( 65.1  96.5) 60.4 ( 29.7  90.7)

Mature root ( 90) 19.7 ( 6.0  41.7) 16.2 (< 0.76  49.8)

Over season root ( 46) 22.0 ( N/ A) 20.0 ( N/ A)

* Days are approximate.

Cry1Ab protein levels  Levels in the MON 863 x MON 810 hybrid presented below are overestimates of Cry1Ab protein levels since some of the samples were beyond their documented stability period.

Tissue Type and Collection Time ( Days post planting*) Average CryAb1 protein levels ( µ g/ g FWT)

MON 863 x MON 810 Average CryAb1 protein levels ( µ g/ g FWT)

MON 810 Young leaf ( 18) 17.9 ( 14.1  27.5) 13.0 ( 9.8  15.4)

Forage ( 90) 7.9 ( 3.9  11.9) 5.6 ( 3.0  8.2)

Grain ( 117) 0.84 ( 0.63  1.2) 0.46 ( 0.24  0.77)

Pollen ( 60) < 0.08 (< 0.08  0.18) < 0.08 (< 0.08)

* Days are approximate. 17 NPTII protein levels  Similar levels of NPTII protein were found in MON 863 and MON 863 x MON 810.

Tissue Type and Collection Time ( Days post planting*) Average NPTII protein levels ( µ g/ g FWT)

MON 863 x MON 810 Average NPTII protein levels ( µ g/ g FWT)

MON 810 Young leaf ( 18) 1.6 ( 0.53  2.3) 1.06 ( 0.58  1.6)

Forage ( 90) 0.19 ( 0.13  0.27) 0.17 (< 0.075  0.33)

Grain ( 117) < 0.076 (< 0.076) < 0.076 (< 0.1)

* Days are approximate.

Conclusions  The ranges of protein levels observed for Cry3Bb1, Cry1Ab and NPTII were similar across all four sites for the MON 863 x MON 810 hybrid and the MON 863 or MON 810 hybrids as appropriate for the transgene in question. Averages for the Cry3Bb1 and Cry1Ab proteins were, however, somewhat higher in the MON 863 x MON 810 hybrid versus the single trait hybrids. NPTII protein levels and ranges in the MON 863 x MON 810 hybrid and the MON 863 hybrid were similar. For Cry3Bb1 and NPTII protein levels, a tissue specific correction factor was used to optimize the accuracy and reduce bias in the estimation of protein content of samples based upon extraction efficiency and recovery. The highest average levels of expression of Cry3Bb1 or Cry1Ab were found to occur in pollen for Cry3Bb1 ( 79.6 µ g/ g FWT) and in leaf ( 17.9 µ g/ g FWT) for Cry1Ab.

DATA EVALUATION REPORT 18 Reviewed by: Chris A. Wozniak, Ph. D., Biologist, OPP / BPPD Secondary Reviewer: John L. Kough, Ph. D., Biologist, OPP / BPPD MRID No.: Administrative Materials Active Ingredients: Bacillus thuringiensis Cry1Ab / Cry3Bb1 protein as expressed in maize Product Name: Event MON863 x MON 810, Cry3Bb1 / Cry1Ab as expressed in maize Confidentiality: No claim of confidentiality is made under FIFRA 10( d)( 1)( A), ( B) or ( C). ID No.: 000524 LEI Submission No.: S600950 Chemical No.: 006484 Bacillus thuringiensis Cry3Bb protein and genetic material for its production. DP Barcode: D276610 Sponsor: Monsanto Corporation, 700 Chesterfield Parkway, St. Louis, MO 63198. Authors: B. G. Hammond, W. F. Heydens, D. P. Ward Testing Facility: Monsanto Company, 600 13th Street N. W., Suite 660, Washington, D. C. 20005 Study Titles: Data Waiver Request: Acute toxicity and irritation data requirements for the end use product, YieldGard ® Plus Corn seed containing Cry1Ab and Cry3Bb1 proteins from Bacillus thuringiensis. Study Date: August 16, 2002 Study No.: MSL 18126 Conclusion: The registrant requests a waiver for the following studies: Acute Oral Toxicity ( Guideline Reference No. 152 30); Acute Dermal Toxicity ( Guideline Reference No. 152 31); Acute Inhalation Toxicity ( Guideline Reference No. 152 32); Primary Dermal Irritation ( Guideline Reference No. 152 34); Primary Eye Irritation ( Guideline Reference No. 152 35); Hypersensitivity ( Guideline Reference No. 152 36). All of the above studies which are required for registration of a biological pesticide may be waived due to lack of exposure or previous evaluation of toxicity in the case of oral consumption of these proteins. Since the primary route of exposure for these proteins, Cry1Ab and Cry3Bb1, is through consumption of the maize expressing these proteins, the acute oral toxicity tests previously reviewed for these proteins will be sufficient to conclude that there is no acute toxicity associated with consumption of these proteins. These proteins are known to act individually to effect a typical midgut pathology in susceptible insects which is characteristic of previously studied   endotoxins of Bacillus thuringiensis. No synergistic action or interaction of these proteins is known or expected to occur. Classification: Acceptable.

Good Laboratory Practices: This submission was not conducted in compliance with the 19 Certification of Good Laboratory Practices as outlined in 40 CFR 160 as it falls outside the scope of the Good Laboratory Practice Standards. The submission represents a request for waiver for certain toxicity studies.

Purpose: The registrant requests a waiver for the following studies: Acute Oral Toxicity ( Guideline Reference No. 152 30); Acute Dermal Toxicity ( Guideline Reference No. 152 31); Acute Inhalation Toxicity ( Guideline Reference No. 152 32); Primary Dermal Irritation ( Guideline Reference No. 152 34); Primary Eye Irritation ( Guideline Reference No. 152 35); Hypersensitivity ( Guideline Reference No. 152 36).

Data Requirement: Acute Oral Toxicity ( Guideline Reference No. 152 30)  In previous testing of the Cry1Ab and Cry3Bb1 proteins in acute oral toxicity studies using rodents as test animals, no adverse effects were noted in either case. The maximum dose evaluated was 4000 mg/ kg body weight and 3200 mg/ kg body weight for the Cry1Ab and Cry3Bb1 proteins, respectively. Neither of these proteins indicated any potential for adverse effects at the doses tested, which far exceed those found in a plant incorporated protectant. The probability of a synergistic reaction between the two proteins as it relates to mammalian toxicity seems very low. Since the primary route of exposure for these proteins, Cry1Ab and Cry3Bb1, is through consumption of the maize expressing these proteins, the acute oral toxicity tests previously reviewed for these proteins will be sufficient to conclude that there is no acute toxicity associated with consumption of these proteins. These proteins are known to act individually to effect a typical midgut pathology in susceptible insects which is characteristic of previously studied   endotoxins of Bacillus thuringiensis. No synergistic action or interaction of these proteins is known or expected to occur. From studies on other   endotoxins from Bacillus thuringiensis we do know that these insecticidal proteins specifically target unique receptors to effect a mid gut pathology typical of the insecticidal proteins from this bacterium and that competitive binding or cross resistance from these two classes of crystal proteins does not occur.

Acute Dermal Toxicity ( Guideline Reference No. 152 31)  The proteins Cry1Ab and Cry3Bb1 are intended to be produced within the plant incorporated protectant YieldGard ® Plus maize. As such, the potential for exposure of the skin to the active ingredient proteins is negligible. Additionally, these proteins are not known to harbor any properties which would suggest that they represent dermal irritants or toxicants.

Acute Inhalation Toxicity ( Guideline Reference No. 152 32)  The proteins Cry1Ab and Cry3Bb1 are intended to be produced within the plant incorporated protectant YieldGard ® Plus maize. As such, the potential for inhalation exposure to the active ingredient proteins is negligible. Additionally, these proteins are not known to harbor any properties which would suggest that they represent pulmonary irritants or toxicants.

Primary Dermal Irritation ( Guideline Reference No. 152 34)  The proteins Cry1Ab and Cry3Bb1 are intended to be produced within the plant incorporated protectant YieldGard ® Plus 20 maize. As such, the potential for exposure of the skin to the active ingredient proteins is negligible. Additionally, these proteins are not known to harbor any properties which would suggest that they represent dermal irritants or toxicants.

Primary Eye Irritation ( Guideline Reference No. 152 35)  The proteins Cry1Ab and Cry3Bb1 are intended to be produced within the plant incorporated protectant YieldGard ® Plus maize. As such, the potential for exposure of the eyes to the active ingredient proteins is negligible. Additionally, these proteins are not known to harbor any properties which would suggest that they represent ocular irritants or toxicants.

Hypersensitivity ( Guideline Reference No. 152 36)  The proteins Cry1Ab and Cry3Bb1 are intended to be produced within the plant incorporated protectant YieldGard ® Plus maize. As such, the potential for exposure of the skin to the active ingredient proteins is negligible. Additionally, these proteins are not known to harbor any properties which would suggest that they represent dermal irritants, toxicants or sensitizers.


[Federal Register: May 11, 2006 (Volume 71, Number 91)]
[Notices]
[Page 27497]
[DOCID:fr11my06-76]

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DEPARTMENT OF ENERGY

Federal Energy Regulatory Commission

[Project No. 2213]

Public Utility District No. 1 of Cowlitz County, WA; Notice of
Authorization for Continued Project Operation

May 5, 2006.
    On April 23, 2004, Public Utility District No. 1 of Cowlitz County,
licensee for the Swift No. 2 Hydroelectric Project, filed an
application for a new or subsequent license pursuant to the Federal
Power Act (FPA) and the Commission's regulations thereunder. The Swift
No. 2 Project is located on the North Fork Lewis River in Cowlitz and
Skamania County, Washington.
    The license for Project No. 2213 was issued for a period ending
April 30, 2006. Section 15(a)(1) of the FPA, 16 U.S.C. 808(a)(1),
requires the Commission, at the expiration of a license term, to issue
from year-to-year an annual license to the then licensee under the
terms and conditions of the prior license until a new license is
issued, or the project is otherwise disposed of as provided in section
15 or any other applicable Section of the FPA. If the project's prior
license waived the applicability of section 15 of the FPA, then, based
on section 9(b) of the Administrative Procedure Act, 5 U.S.C. 558(c),
and as set forth at 18 CFR 16.21(a), if the licensee of such project
has filed an application for a subsequent license, the licensee may
continue to operate the project in accordance with the terms and
conditions of the license after the minor or minor part license
expires, until the Commission acts on its application. If the licensee
of such a project has not filed an application for a subsequent
license, then it may be required, pursuant to 18 CFR 16.21(b), to
continue project operations until the Commission issues someone else a
license for the project or otherwise orders disposition of the project.
    If the project is subject to Section 15 of the FPA, notice is
hereby given that an annual license for Project No. 2213 is issued to
PacifiCorp for a period effective May 1, 2006 through April 30, 2007,
or until the issuance of a new license for the project or other
disposition under the FPA, whichever comes first. If issuance of a new
license (or other disposition) does not take place on or before April
30, 2007, notice is hereby given that, pursuant to 18 CFR 16.18(c), an
annual license under section 15(a)(1) of the FPA is renewed
automatically without further order or notice by the Commission, unless
the Commission orders otherwise. If the project is not subject to
section 15 of the FPA, notice is hereby given that Public Utility
District No. 1 of Cowlitz County, is authorized to continue operation
of the Swift No. 2 Project until such time as the Commission acts on
its application for a subsequent license.

Magalie R. Salas,
Secretary.
 [FR Doc. E6-7198 Filed 5-10-06; 8:45 am]

BILLING CODE 6717-01-P


[Federal Register: January 8, 2008 (Volume 73, Number 5)]
[Notices]
[Page 1392-1394]
[DOCID:fr08ja08-100]

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SECURITIES AND EXCHANGE COMMISSION

[Release No. 34-57069; File No. SR-NYSEArca-2007-126]

Self-Regulatory Organizations; NYSE Arca, Inc.; Notice of Filing
and Immediate Effectiveness of a Proposed Rule Change Amending Certain
Transaction Fees and Credits

December 31, 2007.
    Pursuant to Section 19(b)(1) of the Securities Exchange Act of 1934

(``Act'') \1\ and Rule 19b-4 thereunder,\2\ notice is hereby given that
on December 11, 2007, NYSE Arca, Inc. (``NYSE Arca'' or ``Exchange'')
filed with the Securities and Exchange Commission (``Commission'') the
proposed rule change as described in Items I, II and III below, which
Items have been prepared substantially by the Exchange. NYSE Arca has
designated this proposal as one establishing or changing a member due,
fee, or other charge imposed by the Exchange under Section
19(b)(3)(A)(ii) of the Act \3\ and Rule 19b-4(f)(2) thereunder,\4\
which renders the proposal effective upon filing with the Commission.
The Commission is publishing this notice to solicit comments on the
proposed rule change from interested persons.
---------------------------------------------------------------------------

    \1\ 15 U.S.C. 78s(b)(1).
    \2\ 17 CFR 240.19b-4.
    \3\ 15 U.S.C. 78s(b)(3)(A)(ii).
    \4\ 17 CFR 240.19b-4(f)(2).
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I. Self-Regulatory Organization's Statement of the Terms of Substance
of the Proposed Rule Change

    The Exchange, through its wholly-owned subsidiary NYSE Arca
Equities, proposes to amend the section of its Schedule of Fees and
Charges for Exchange Services (the ``Fee Schedule'') that applies to
orders submitted by ETP Holders.\5\ While changes to the Fee Schedule
pursuant to this proposal will be effective upon filing, the changes
will become operative on January 2, 2008. The text of the proposed rule
change is available at http://www.nyse.com, the principal offices of

the Exchange, and the Commission's Public Reference Room.
---------------------------------------------------------------------------

    \5\ See NYSE Arca Equities Rule 1.1(n).
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II. Self-Regulatory Organization's Statement of the Purpose of, and
Statutory Basis for, the Proposed Rule Change

    In its filing with the Commission, the Exchange included statements
concerning the purpose of and basis for the proposed rule change and
discussed any comments it received on the proposed rule change. The
text of these statements may be examined at the places specified in
Item IV below. NYSE Arca has prepared summaries, set forth in Sections
A, B, and C below, of the most significant aspects of such statements.

A. Self-Regulatory Organization's Statement of the Purpose of, and
Statutory Basis for, the Proposed Rule Change

1. Purpose
    As part of its continuing efforts to enhance participation on the
Exchange, NYSE Arca Equities proposes to amend certain sections of its
Fee Schedule that apply to orders submitted for securities listed on
The NASDAQ Stock Market, LLC (``Nasdaq'') or Tape B listed securities.
Primarily, these changes will increase the rebate (or credit) earned by
ETP Holders for providing significant liquidity in Nasdaq-listed
securities.
    Currently, the credit for round lot orders of Nasdaq-listed
securities that provide liquidity is $0.002 per share. Pursuant to this
proposal, for Nasdaq-listed securities, the Exchange will increase the
$0.002 per share credit to $0.0024 per share if certain volume
thresholds are met. Specifically, if an ETP Holder (i) transacts an
average daily share volume per month greater than 30 million shares
(including transactions that take liquidity, provide liquidity, or
route to away market centers) and also (ii) provides liquidity an
average daily share volume per month greater than 15 million, then the
ETP Holder will earn a credit of $.0024 for its orders that provide
liquidity.
    Additionally, the Exchange proposes to reduce the fee for round lot
orders of Tape B listed securities submitted to the Exchange by ETP
Holders that are subsequently routed away from the Exchange and
executed by another market center or participant, from $0.004 per share
to $0.0035 per share.
    While changes to the Fee Schedule pursuant to this proposal will be
effective upon filing, the changes will become operative on January 2,
2008.
2. Statutory Basis
    The Exchange believes the proposed rule change is consistent with
Section 6(b) of the Act \6\ in general, and furthers the objectives of
Section 6(b)(4) of the Act \7\ in particular, in that it is intended to
provide for the equitable allocation of reasonable dues, fees, and
other charges among its members and other persons using its facilities.
---------------------------------------------------------------------------

    \6\ 15 U.S.C. 78f(b).
    \7\ 15 U.S.C. 78f(b)(4).
---------------------------------------------------------------------------

B. Self-Regulatory Organization's Statement on Burden on Competition

    The Exchange does not believe that the proposed rule change will
impose any burden on competition that is not necessary or appropriate
in furtherance of the purposes of the Act.

C. Self-Regulatory Organization's Statement on Comments on the Proposed
Rule Change Received From Members, Participants or Others

    The Exchange has neither solicited nor received written comments on
the proposed rule change.

III. Date of Effectiveness of the Proposed Rule Change and Timing for
Commission Action

    The foregoing proposed rule change has been designated as a fee
change pursuant to Section 19(b)(3)(A)(ii) of the Act \8\ and Rule 19b-
4(f)(2) \9\ thereunder, because it establishes or changes a due, fee,
or other change imposed on members by the Exchange. Accordingly, the
proposal is effective upon filing with the Commission. At any time
within 60 days of the filing of the proposed rule change, the
Commission may summarily abrogate such rule change if it appears to the
Commission that such action is necessary or appropriate in the public
interest, for the protection of investors, or otherwise in furtherance
of the purposes of the Act.
---------------------------------------------------------------------------

    \8\ 15 U.S.C. 78s(b)(3)(A)(ii).
    \9\ 17 CFR 240.19b-4(f)(2).
---------------------------------------------------------------------------

IV. Solicitation of Comments

    Interested persons are invited to submit written data, views, and
arguments concerning the foregoing, including whether the proposed rule
change is consistent with the Act. Comments may be submitted by any of
the following methods:

Electronic Comments

     Use the Commission's Internet comment form (http://www.sec.gov/rules/sro.shtml.
); or     Send an e-mail to<EMAIL_ADDRESS>Please include

File Number SR-NYSEArca-2007-126 on the subject line.

Paper Comments

     Send paper comments in triplicate to Nancy M. Morris,
Secretary, Securities and Exchange Commission, Station Place, 100 F
Street, NE., Washington, DC 20549-1090.

All submissions should refer to File Number SR-NYSEArca-2007-126. This
file number should be included on the subject line if e-mail is used.
To help the Commission process and review your comments more
efficiently, please use only one method. The Commission will post all
comments on the Commission's Internet Web site (http://www.sec.gov/rules/sro.shtml
). Copies of the submission, all subsequent

amendments, all written statements with respect to the proposed rule
change that are filed with the Commission, and all written
communications relating to the proposed rule change between the
Commission and any person, other than those that may be withheld from
the public in accordance with the provisions of 5 U.S.C. 552, will be
available for inspection and copying in the Commission's Public
Reference Room, 100 F Street, NE., Washington, DC 20549, on official
business days between the hours of 10 a.m. and 3 p.m. Copies of such
filing also will be available for inspection and copying at the
principal office of the Exchange. All comments received will be posted
without change; the Commission does not edit personal identifying
information from submissions. You should submit only information that
you wish to make publicly available. All submissions should refer to
File Number SR-NYSEArca-2007-126 and should be submitted on or before
January 29, 2008.

    For the Commission, by the Division of Trading and Markets,
pursuant to delegated authority.\10\
Florence E. Harmon,
Deputy Secretary.
---------------------------------------------------------------------------

    \10\ 17 CFR 200.30-3(a)(12).
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 [FR Doc. E8-85 Filed 1-7-08; 8:45 am]

BILLING CODE 8011-01-P


                                           UNITED STATES OF AMERICA

                                  DEPARTMENT OF TRANSPORTATION

                                           OFFICE OF THE SECRETARY

                                                    WASHINGTON, DC

       Issued by the Department of Transportation on September 12, 2005

NOTICE OF ACTION TAKEN

DOCKET OST-2003-16101

This serves as notice to the public of the action described below, taken
by the Department official indicated (no additional confirming order
will be issued in this matter).

Application of  American Eagle Airlines, Inc. (American Eagle) filed
7/14/05 for:

XX  Renewal of exemption for indefinite duration under 49 U.S.C. 40109
to provide the following service:

Scheduled foreign air transportation of persons, property, and mail
between Dallas/Ft. Worth, Texas, and Monterrey, Mexico.  American Eagle
states that it currently provides year-round service in the market with
two-daily nonstop flights.

Applicant rep:  Carl B. Nelson, Jr.<PHONE_NUMBER>   DOT Analyst:  Linda
Lundell<PHONE_NUMBER>

D I S P O S I T I O N

XX  Granted in part (two-year duration)(subject to conditions, see
below)

XX  Dismissed request for longer-term authority (see remarks, below)

The above action granting exemption authority was effective when taken:
September 12, 2005,

through September 12, 2007.

The above action dismissing the request for longer-term authority was
effective when

taken: September 12, 2005.

Action taken by:   Paul L. Gretch, Director

                               Office of International Aviation

XX  The authority granted is consistent with the aviation agreement
between the United States and Mexico.

Except to the extent exempted or waived, this authority is subject to
the terms, conditions, and limitations indicated:

XX Holder’s certificates of public convenience and necessity

XX  Standard exemption conditions (attached)

________________________________________________________________________
_______

Conditions:  The U.S.-Mexico exemption authority granted is subject to
the dormancy notice requirements set forth in condition 7 of Appendix A
of Order 88-10-2.

Remarks:  In its application, American Eagle requested that its
Dallas/Ft. Worth-Monterrey exemption authority be renewed for an
indefinite period.  Consistent with our standard practice for this type
of application, we renewed American Eagle’s authority for two years
and dismissed its request for longer-term authority.

________________________________________________________________________
_______

On the basis of data officially noticeable under Rule 24(g) of the
Department's regulations, we found the applicant qualified to provide
the exemption services authorized.

Under authority assigned by the Department in its regulations, 14 CFR
Part 385, we found that (1) our action was consistent with Department
policy; (2) grant of the authority was consistent with the public
interest; and (3) grant of the authority would not constitute a major
regulatory action under the Energy Policy and Conservation Act of 1975.
To the extent not granted or dismissed, we denied all requests in the
referenced Docket.  We may amend, modify, or revoke the authority
granted in this Notice at any time without hearing at our discretion.

Persons entitled to petition the Department for review of the action set
forth in this Notice under the Department’s regulations, 14 CFR
§385.30, may file their petitions within seven (7) days after the date
of issuance of this Notice.  This action was effective when taken, and
the filing of a petition for review will not alter such effectiveness.

An electronic version of this document is available on the World Wide
Web at:

  HYPERLINK "http://dms.dot.gov//reports/reports_aviation.asp"
http://dms.dot.gov//reports/reports_aviation.asp

			APPENDIX

U.S. Carrier Exemption Conditions

In the conduct of the operations authorized, the U.S. carrier
applicant(s) shall:

(1)  Hold at all times effective operating authority from the government
of each country served;

(2)  Comply with applicable requirements concerning oversales contained
in 14 CFR 250 (for scheduled operations, if authorized);

(3)  Comply with the requirements for reporting data contained in 14 CFR
241;

(4)  Comply with requirements for minimum insurance coverage, and for
certifying that coverage to the Department, contained in 14 CFR 205;

(5)  Except as specifically exempted or otherwise provided for in a
Department Order, comply with the requirements of 14 CFR 203, concerning
waiver of Warsaw Convention liability limits and defenses;

(6)  Comply with all applicable requirements of the Federal Aviation
Administration and with all applicable U.S. Government requirements
concerning security, including, but not limited to, 49 CFR Part 1544.
To assure compliance with all applicable U.S. Government requirements
concerning security, the holder shall, before commencing any new service
(including charter flights) to or from a foreign airport, contact its
International Principal Security Inspector (IPSI) to advise the IPSI of
its plans and to find out whether the Transportation Security
Administration has determined that security is adequate to allow such
airport(s) to be served; and

(7)  Comply with such other reasonable terms, conditions, and
limitations required by the public interest as may be prescribed by the
Department of Transportation, with all applicable orders and regulations
of other U.S. agencies and courts, and with all applicable laws of the
United States.

The authority granted shall be effective only during the period when the
holder is in compliance with the conditions imposed above.

05/2004

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