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‘j I,, ,,ll_r ., .i ..“. .I . I ?‘.C’ ~^The Agricultural Group of Monsanto Company Study #: 92-01-36-22New Products Division I MSL #: 13299Regulatory SciendesPIt ikdFbStudy Number:Experiment Number:Title:Facility:Study Director:Contributors:Study Start Date:92-01-36-2292-427-728Assessment of the In vitro Digestive Fate ofBacillus thuringiensis var. kurstaki HD-73 ProteinMonsanto CompanyAgricultural Group - New Products Division700 Chesterfield Parkway NorthChesterfield, Missouri 63198Joel E. ReamSteven R. SimsJohn N. LeachMay 25,1993ExperimentalTerminationDate:June, 1993RecordsRetention:All study specific raw data, protocols, final reportsand facility records will be retained at Monsanto - St.LouisSignaturesof Approval:r;,Ic 1 *LStudy @ onsorPooor;;ot-0043
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22M@L #: 13299PAGE +. I ‘-.’TABLE OF CONTENTS. .. -Confidexitiality Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Statement of Compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 -Quality Assurance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Signatures of Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.*........... 5Abbreviations .*...*...............................*............... 8SummarY . ..*...................................................... 9Lntroduction . . . . . . . . . . . . . . . . . . . . . . ..*.......*...................... 11Materials .......................................................... 11Test Substance ................. ..*...................~ ....... 11Control Substance ............................................ 11Reference Substance .......................................... 11Test <strong>System</strong> .................................................. 11Test <strong>System</strong> Justification ...................................... 12Reagents ..................................................... 12Methods ........................................................... 12Assessment of Gastric and Intestinal Fluid Activity .............. 12Addition of Test Substance ..................................... 12Incubation .................................................... 13Part I. Assessment by Western Blot Analysis .................... 13Part II. Assessment by Insect Bioassay ......................... 13ControlofBias ................................................ 14Data Analysis ................................................ 14Results ............................................................ 14Part I. Assessment by Western Blot Analysis .................... 14GastricFluid ............................................ 14Intes~~Fluid .......................................... 15Part II. Assessment by Insect Bioassay ......................... 16Investigation of Potential Assay Interference .............. 16GastricFluid ....... ...................................... 16Intestinal Fluid .......................................... 16Discussion ........................................................ 17---
A___..;The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299Conclusions ....................................................... 18References ........................................................ 19Tables1 Effect of Simulated Gastric and Intestinal Fluids onTobacco Budworm Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 Dissipation of B.t.k. ND-73 Protein Bioactivity in Simulated Gastric Fluid . . . . 223 Dissipation of B.t.K. HD-73 Protein Bioactivity in Simulated Intestinal Fluid . . 23Figures1 Western Blot Analysis of B.t.k. HD-73 Protein Degradationin Simulated Gastric Fluid.. . . . . . . . . . . . . . . . . . . . . . . . . . , , . . . . . . . . . . . , 242 Western Blot Analysis of B.&k. HD-73 Protein Degradation ’in Simulated Intestinal Fluid . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . , . . . 25List of appendices1. Soplist...................................................... 262. Protocol .,,......._...........................,................ 28
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory Sciences .Study #: 92-01-36-22MSL #: 13299ABBREVIATIONS-CE. coliFig.hMwminmln&PAGEbSSGFSIFSOPTCA5, . . .var.TBWELISATCACentigradeEscherichiu coliFigurehoursIllO1Ei.Tmilligramminutemilliliternanogrampolyacrylamide gel electrophoresissecondsodium dodecyl sulfatesimulated’gastric fluidsimulated intestinal fluidStandard Operating Proceduretrichloroacetic acidmicrogrammicroliterBacillus thuringiensis VS. kurstakivariety, synonymous with subspeciestobacco budwormenzyme-linked immunosorbent assaytrichloroacetic acid----c_
‘ .i _+ J,‘,mwf I*u ,ksm:The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatorySciencesINTRODUCTIONStudy #: 92-01-36-22MSL #: 13299smL,I; i ,The purpose of this study was to assess the degradation of the Bacillusthuringiensis var. kurstaki HD-73 [Cry IA(c)] (Hofte and WhiteJy, 1989)protein (abbreviated as B.t.k. m-73) and its biological activity using in vitromammalian digestion models. Degradation of the B. t. k. HD-73 protein wasassessed using western blot analysis. Degradation of the biological activity ofthe B.t.k, HD-73 protein was assessed using an insect bioassay. The B. t.k.HD-73 protein, expressed in cotton plants, confers resistance to Lepidopteraninsect damage. The B.t.k. HD-73 degrades in the presence of trypsin to form atrypsin-resistant core protein (called tryptic core in this study) and thistryptic core is the insecticidally active component of the B.t.k. HD-73 protein(Bielot et al., 1989).MATERIALSrt. IFc ie:Ptt ’Test substance. The test substance for this study was the B. t. k. HD-73protein that was purified corn Escherichia coli (Lot Number 5025385) to 67%purity by ELISA (<strong>Volume</strong> 5) and shown to be equivalent to the B. t.k. HD-73protein expressed in insect resistant cotton plants (<strong>Volume</strong> 4). Concentratedstock solutions of B. t.k. HID-73 protein were prepared from solid materialdissolved in buffer (10 mM sodium carbonate-/bicarbonate, pH 10.5).Control substance, The control substance for this study was the buffer usedto prepare the B. t.k. HD-73 protein solutions (10 mM sodium carbonate/bicarbonate, pH 10.5, prepared by dilution from a 1 M stock solution).Reference substance. Incubated samples were referenced to unincubated(time = 0) samples. Analytical standards of the B.t.k. HD-73 protein and thetryptic core of the B.t.k. HD-73 protein were used in the western blot analysisfor reference to the digestion samples. Two types of standards were usedbecause the B. t.k. HID-73 protein was known to degrade to the biologicallyactivetryptic core upon exposure to trypsin (Bietlot et aZ., 19891, a componentof the simulated intestinal fluid used in this study.Test system. The test systems for this study were simulated gastric fluid andsimulated intestinal fluid prepared as described in SOP Number GEN-PRO-055-00. Simulated digestion solutions were used in this in vitro study tosimulate digestion, simplify analysis of protein degradation and increasereproducibility relative to in uiuo systems. These digestion fluids were usedwithin 24 hours of preparation, stored at approximately 4OC and assayed forprotease activity before use. The test system incubation tubes were clearly00110f0043
‘l%e Agriculturai Group of Monsanto CompanyNew Products DivisionRegulatory SciekesStudy #: 92-01-36-22MSL #: 13299-marked using a permanent marking pen with unique alphanumericreflecting the treatments.codesTest system justifkation. In vitro studies with simulated digestion solution3are widely used as models of animal digestion. Simulated digestion solutionshave been used to investigate the digestibility of plant proteins (Nielson, 1988,Marquez et al., J.981>, animal proteins (Zikakis et al., 1977), and food additives(Tilch and Elias, 1984); to assess protein quality (Akeson and Stahmann,1964); to study digestion in pigs and poultry (Fuller, 1991); to measure tabletdissolution rates to assess bioavailability for pharmaceuticals (Alam et al.,1980); and to investigate the controlled-release properties of experimentalpharmaceuticals (Doherty et al., 1991). The method of preparation of thesimulated digestion solutions to be used in this study is described in the UnitedStates Pharmacopeia (USP, 1989), a frequently cited reference for in vitrodigestion studies.Reagents. Pepsin (porcine, Product Number P-7000), pancreatin (porcine,Product Number P-1500), and hemoglobin (bovine, Product Number H-2625)were obtained from Sigma Chemical Company (St. Louis, MO). Resorufinlabeledcasein (Product Number 1080733) was obtained from BoehringerMannheim Corporation (<strong>Indian</strong>apolis, IN).METHODSAssessment of gastric and intestinal fluid activity. Pepsin activity wabmeasured in simulated gastric fluid (SGF) according to SOP Number GEN-PRO-057-00 by measuring spectrophotometrically the increase insupernatant absorbance at 280 nm following TCA precipitation of SGFincubations with hemoglobin. Protease activity in simulated intestinal fluid(SIF) was measured according to SOP Number GEN-PRO-058-00 bymeasuring spectrophotometrically the increase in supernatant absorbance at574 nm following TCA precipitation of SIF incubations with resorufin-labelledcasein.Addition of Test Substance. Aliquots of purified B.t.k. HD-73 protein wasadded to temperature-equilibrated (approximately 37OC) digestion solutions bypipetting from concentrated B. t. k. HD-73 protein stock solutions. This met&&.of administration of the test substance was selected to provide reproducibledosing of small amounts of protein. One ml volumes of digestion fluids wereincubated in 15 ml plastic test tubes with the following exceptions: 1) a 0.1 rtilincubation volume in a 1.5 ml plastic test tube were used for the t = 0 sampfek;of l3.t.k. HD-73 protein in SGF and SIF in Part I; and 2) a 0.1 ml incubationvolume in a 1.5 ml plastic test tube was used for the whole incubation samplssfor both SGF and SIF in Part I. These treatments used a reduced volume to-----
,, i #< ‘7 : .I;. ,. y;,F%;r;. jt”1. *:p7iBc’if-k ”y,i, ,F!eP-i ;I: ;The Agricultural Group of Monsanto Company Study #: 92-01-36-22New Products Division MSL #: 13299Regulatory Scienceskeep final quenched sample volumes compatible with equipment used foraliquots sampled from the l-ml digestion incubations. The digestion solutionswere thoroughly mixed immediately after the addition of the B. t.k. HD-73protein.Incubation. All digestion solutions were incubated at approximately 37°C in ashaker-incubating water bath (Model G76, New Brunswick Scientific Co,Edison, NJ.). Incubation solutions were agitated continuously throughout theincubation period with the following exceptions: agitation was interruptedbriefly to facilitate sampling; and no agitation occurred between samplingintervals that were less than 2 minutes. In the latter case, incubationsamples were agitated manually just prior to sampling.Part I. Assessment by western blot analysis. In this part, the B.t.k. ED-73protein was added to digestion fluids to a final concentration of approximately 2l.@rnl. This dose was selected based on the detection range of the western blotanalysis. The B.t.k. HD-73 protein was incubated for five time intervals(including t = 0) up to 20 minutes in SGF and up to 21 hours in SIF. For one-mlincubation volumes, 50 fl aliquots of the incubation solution were removed andthe digestion terminated. For SGF incubations, termination was byneutralization with 0.2 M sodium carbonate (15 fl sodium carbonate per 50 ~1digestion solution.) For SIF incubations, termination was by 1:l dilution withprotein-denaturing 2X SDS-PAGE sample buffer (Laemmli, 1970) followed byheating to about 100°C for approximately 5 minutes. Neutralized SGFincubation samples were diluted 1:l with 2X SDS-PAGE sample buffer andheated to approximately 100°C for approximately 5 minutes. For 0.1-mlincubation solutions, the entire incubation solution was terminated, dilutedwith 2X SDS-PAGE sample buffer and heated as described for aliquots fromone-ml incubations. All samples were stored at approximately -80°C untilanalyzed for B.&k. FfD-73 content by western blot analysis. Western blottingwas completed according to SOP Number BtC-PRO-002-02 (<strong>Volume</strong> 3,Appendix 3)Part II. Assessment by insect bioassay. In this part, the amount ofdegradation of B.t.k. HD-73 protein bioactivity over a fixed time interval indigestion fluids was assessed using the tobacco budworm (Heliothis uirescem)(TBW). The B.t.k. MD-73 protein was added to digestion fluids to finalconcentrations of 0.75,7.5 and 75 l.@ml. These concentrations were selectedbased on the sensitivity of the TBW assay. These samples were incubated fora single time interval -- 5 min for SGF and 20.7 hr for SIF -- approximatelycorresponding to one time interval used in Part I. The one-ml incubationvolumes were terminated by neutralization with sodium carbonate for SGFand by freezing on dry ice for SIF. The B.t.k. ND-73 protein content of thesesamples was also assessed by western blot analysis to allow a directcomparison with the TBW bioassay results. 50 ~1 samples were taken from
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299each incubation mixture in Part II and the B,t.k. HD-73 protein concentrationestimated by western blot analysis as described for Part I. Terminateddigestion samples to be evaluated for TBW activity were stored atapproximately -8OOC. On the day of the TBW assay, performed according toSOP Number.BUG-PRO-022-02, samples were thawed, diluted to 6 ml withdistilled water, brought to approximately 30 ml with TBW and poured into traywells. Individual TBW larvae were added to each well (24 larvae per sample),incubated 7 days at approximately 28OC and scored for mortality.Control ofBias, Three replicate digestion incubations were prepared for eachtreatment. SGF and SIF samples without B.t.iz. HD-73 protein added wereprepared to assess whether these digestion fluids interfere with the analyticalmethods used in this study. Buffer plus B.t.k. HD-73 protein (no SGF or SIP)treatments were prepared to allow an estimate of the recovery of the B.t.k.HD-73 protein and its bioactivity from the digestion solutions. In Part I, asample was prepared in which the entire digestion solution was quenched anddiluted with SDS-PAGE sample buffer to allow an assessment if the 50 flaliquots sampled from the 1 ml digestion solutions are representative of theentire digestion solution.Data analysis. For Part I, BLK. HD-73 protein concentration for each assaysample was estimated by visual comparison of the sample band intensity tothe band intensity of two concentrations, of the B.&k. HD-73 protein andtryptic fragment standards. The mean 23.t.k. HD-73 protein concentrationwas calculated from the three replicates of each treatment. The degradation ofthe B.t.k. HD-73 protein was reported as a percentage of B.t.k. ED-73 proteindegraded at each time interval relative to the estimated B.t.k. HD-73 proteinconcentration in the unincubated treatment.h-For Part II, the B.t.k. HD-73 bioactivity of each assay sample was reported asTBW mortality, calculated as the percentage of TBW larvae that died afier theTBW larvae incubation period relative the initial number of larvae added to theinsect diet. The mean and standard error of TBW mortality was calculated forthe three replicates from each treatment.RESULTS. . .Part I. Assessment of &m&xon bv western blot a&Gastric fluid. B. t, k. HD-73 protein degraded rapidly in SGF (Figure 1). NoB.t.k. HD-73 protein was detected afker 30 seconds incubation, the earliesttime point tested (lane 8). The western blot procedure used can typically beused to quantitate B.t.k. HD-73 protein levels down to 2 ng/well and trypticcore levels down to 0.5 ng/ml. The tryptic core of B.&k. HD-73 protein was
cE”,I JEli I)rpt.,,. ‘.,.-‘,S *,cy i
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299There is no human oral exposure to the B. t. k. HD-73 protein due to itsexpression in insect resistant cotton plants. The results of this study supportthe safety of the B.t.k. HD-73 protein to mammals by establishing thatsubstantial degradation of B.&k. HD-73 protein will occur in the stomachbefore it reaches the intestine. Furthermore, mammals do not have l3.t.k.protein receptors in the intestine (Hofmann, ef al., 1988; Sac&i et aZ., 1986).Finally, this combination of rapid degradation of the B.t.k. HD-73 protein andthe lack of receptors in mammals are consistent with the acute gavage study(<strong>Volume</strong> 15) which established the safety of the B.t.k. HD-73 protein aftermammalian consumption.-CQNCLUSIONSThe results of this study establish that the B.t.k. HD-73 protein and itsassociated insecticidal activity will readily degrade upon exposure to gastricfluid in the mammalian digestive tract. The stability of the insecticidallyactive tryptic core of the B. t.k. HD-73 protein for 20.8 hours in intestinal fluidwas expected; the tryptic fragment of this and other Bacillus insecticidalproteins have been shown to be relatively resistant to digestion by trypsin, akey protease in intestinal fluid (Guyton, 1981).--
”k !@, df?The Agriculkral Group of Monsanto CompanyNew Products DivisionRegulatory SciencesREFERENCESi.kl’I*- IFt h dStudy #: 92-01-36-22MSL #: 13299Akeson, W. R. and Stahmann, M. A. 1964. “A pepsin pancreatin digest indexof protein quality evaluation,” J. Nutrition 83: 257-261.Alam, A. S., Hager-man, L. M., and Imondi, A. R. 1980. “Bioavailability ofsulphiride tablet and capsule in dogs,” Arch. Int. Pharmodyn. Ther. 247:180-189.Bietlot, H., Carey, P. R., Choma, C., Kaplan, H., Lessard, T., and Pozsgay, 2.1989. “Facile preparation and characterization of the toxin from Bacillusthuringiensis var. kurstaki,” Biochem. J. 260: 87-91.Chroma, C. T. and Kaplan, H. 1990. “title,” Biochemistry 29: 1071-1077.Doherty, A. M., Kaltenbronn, J. S., Hudspeth, J. P., Repine, J. T., Roark, W. H.,Sircar, I., Tinney, F. J., Connolly, C. J., Hodges, J. C., Taylor, M. D.,Batley, B. L., Ryan, M. J., Essenburg, A. D., Rapundalo, S. T., Weishaar,R. E., Humblet, C. and Lunney, E. A. 1991. “New inhibitors of human reninthat contain novel replacements at the P2 site,” J. Med. Chem. 34:1258-1271.Guyton, A. C. 1981. “Digestion and absorption in the gastrointestinalIn Textbook of Medicinal Physiology. W. B. Saunders Co., Philadelphia.816-826.tract,”Hofmann, C., Vanderbruggen, H. V., HoRe, H., Van Rie, J., Jansens, S., andVan Mellaert, H. 1988. “Specificity of B. thuringiensis delta-endotoxins iscorrelated with the presence of high afsnity binding sites in the brush bordermembrane of target insect midguts,” Proc. Natl. Acad. Sci. USA 85: 7844-7848.Hofte, H. and H. R. Whitely. 1989. “Insecticidal crystal proteins of Bacillusthuringiensis,” Microbiological Reviews 53: 242-255.Laemmli, U. K. 1970. “Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4,” Nature 227: 680-685.Marquez, U. M. L. and Lajolo, F. M. 1981. “Composition and digestibility of,albumins, globulins, and glutelins from Phuseolus vulgaris,” J. Agric. FoodChem. 29: 1068-1074.Nielson, S. S. 1988. “Degradation of bean proteins by endogenous andexogenous proteases - A review,” Cereal Chemistry 65: 435-442.00190~0043
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299-Sac&i, V. F., Parenti, P., Hanozet, G. M., Giordana, B., Luthy, P. AdWolfersberger, M. G. 1986. “BaciUus thuringiensis toxin inhibits K+gradient-dependentamino acid transport across the brush bordermembrane of Pieris brassicae midgut cells,” FEBS Lett. 204: 213-218.Tilch, C. and Elias, P. S. 1984. “Investigation of the mutagenicity ofethylphenylglycidate,” Mutation <strong>Research</strong> 138: 1-8.Zikakis, J. P., Rzucidlo, S. J., and Biasotto, N. 0. 1977. Persistence of bovinemilk xanthine oxidase activity after gastric digestion in viuo and in uitro,” J.Dairy Science 60: 533-541.Fuller, M. ed. 1991.1h vitro digestion for pigs and poultry. C.A.B. International,Walling-ford, Oxon, U.K., ,--1989. The United States Pharmacopeia, Vol. XXII, NF XVII. United StatesPharmacopeial Convention, Inc., Rockville, MD. pp. 1788-1789.--
The Agricult&al Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299Table 1. Effect,of +.mqlated gastric and intestinal fluids on tobaccobudworm mortality.SGF and SIF samples were incubated at approximately 37°C and quenched asdescribed under “Methods”. Incubated and non-incubated SGF and SIF wereincorporated into the diet of TBW at 1 ml digestion fluid/ 30 ml insect diet.Data represents the mean and standard error ( > of TBW larval mortality fromthree replicate samples each evaluated with 24 larvae.TreatmentTBW % mortalityWater controlBuffer controlt=Omin 5.6t=Omin 1.4t 1.4)(1.4)SGFt=Omin 5.6t=5min 0.0a.41(0.0)SIFt=Omin 1.4t = 20.7 h 1.4(1.4)(1.4)
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299Table 2. Dissipation of J3.t.k. HD-‘73 protein bioactivity in simulatedgastric fluid.Three levels of purified B.t.k. HD-73 protein were incubated in SGF for 5 min atapproximately 37”C, quenched by neutralization with sodium carbonate andincorporated into TBW diet. Data represent the mean and standard error ( ) ofthree replicated digestion samples, each assayed for mortality with 24 larvae.---B. t. k. HD-73(clg/ml)DigestionfluidIncubation(mildtimeTBW % mortality--0.75Buffer control01.4 0.4)SGF08.3 (‘4.2)u50.0 (0.0)-7.5Buffer control033.3 (4.2)SGF038.9 (7.4)“50.0 (0.0)-75Buffer control083.3 6.4SGF“0579.2 (6.4)6.9 (5.0)--00zYlf0043-
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299Table 3. Dissipation of B.&k. HD-73 protein bioactivity in simulat@dintestinal fiuid,Three levels of purified B. t. k. HD-73 protein were incubated in SE’ for 20.7 hrat approximately 37”C, quenched by freezing on dry ice and incorporated intoTBW diet. Data represent the mean and standard error t > of three replicateddigestion samples, each assayed for TBW mortality with 24 larvae.B. t. k. HD-73(P.&a-DigestionfluidIncubation time(W TBW % mortality0.75 Buffer control01.4(1.4)SIF66020.711.18.3(6.1)(4.8)7.5 BufEer controlSIF“0020.733.3 (4.2)36.1 (7.4)59.7 (6.1)75 Buffer controlSIF“00.20.783.3 (6.4)98.6 (1.4)100.0 (0.0)
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy W: 92-01-36-22MSL #: 13299Figure 1, ,_ Western blot analysis of B,t.k. HD-73 protein degradation insimulated gastric fluid.-31 2 3 4 5 6 7 8 9 101112-3---‘-Western blot shown is one of three replicate digestions. B. t.k. HD-73 proteinwas added to SGF to a final concentration of approximately 2 ~gknl, incubatedat approximately 37”C, quenched by neutralization with 0.2 M sodiumcarbonate and evaluated by western blot analysis. Lanes 1 and 2 are B.t.k.HD-73 protein standard added at 5 and 10 ngkne, respectively. Lanes 3 and 4are tryptic core of B.&k. HD-73 protein standard added at 5 and 10 ngkne,respectively. Lane 5 is SGF plus buffer at t = 0 min. Lane 6 is buffer plusB.t.k. HD-73 protein at t = 0 min. Lanes 7 through 11 are B.t.k. HD-73 proteinafter 0,0.5,2,7 and 20 min incubation in SGF, respectively. Lane 12 is SGFplus l3.t.k. HD-73 after 20 min incubation where the entire incubation samplewas quenched and prepared for western blot analysis. The gel shown is gelnumber G3 from 6/l/93.-.
, ,,- .iThe Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299Figure 2. Western blot analysis of B.&k. HD-73 protein degradation in.. simulated intestinal fluidiT?f 1L /Western blot shown is one of three replicate digestions. B.&k. HD-73 proteinwas added to SIF to a final concentration of approximately 2 ~gknl, incubatedat approximately 37”C, quenched by heating to approximately 100°C in 2XSDS-PAGE sample buffer and evaluated by western blot analysis. Lanes 1and 2 are B.&k. I-ID-73 protein standard added at 5 and 10 ngkne,respectively. Lanes 3 and 4 are tryptic core of B.t.k. HD-73 protein standardadded at 5 and 10 ngkne, respectively. Lane 5 is SIF plus buffer at t = 0 min.Lane 6 is buffer plus B.t.k. IID- protein at t = 0 min. Lanes 7 through 11 areB.t. k. HD-73 protein after 0,0.5,1.9,3.8 and 20.8 hr incubation in SIF,respectively. Lane 12 is SIF plus B. t.k. IID- after 20.8 hr incubation wherethe entire incubation sample was quenched and prepared for western blotanalysis. The gel shown is gel number 13 from 6/l/93.F
-The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92.01-36~22MSL #: 13299-APPENDIX 1SOP List-
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299.GEN-PRO-055-00 Preparation of simulated gastric and intestinal fluidGEN-PRO-057-00 Assay for pepsin activity in simulated gastric fluidGEN-PRO-058-00 Assay for protease activity in simulated intestinalfluidBtC-PRO-002-02 Procedure for western blotting: Alkalinephosphatase developmentBUG-PRO-022-02 Diet incorporation for measurement of insecticidalactivity of purified Bacillus thuringknsk endotoxinproteins and endotoxin proteins expressed in cottonseed002t70F0043
The Agricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy #: 92-01-36-22MSL #: 13299-APPENDIX 2ProtocolStudy Number 92-01-36-22Experiment Number 92-427-728---
The Agricultural Group of Monsanto Company Study I: 92-01-36-22New Pro&&a Division Experiment #: 92-427-728Regulatory Science9 Page 1 of 13Study #: 92-0136-22Experiment #: 92427-728study TitleAssessment of the in vitro digestive fate of Bacillusthuringiensis var. kurstaki HD-73 proteinSpOIlS0.E The Agricultural Group of Monsanto CompanyNew Products Division700 Chesterfield Parkway NorthSt. Lams, MO 63198The Agricultural Group of Monsanto CompanyNew Products Division700 Chesterfield Parkway NorthSt. Louis, MO 63198Study DirectorJoel E. Ream<strong>Research</strong> SpecialistThe Agricultural Group of Monsanto Company700 Chesterfield Parkway North - GG4KSt. Louis, MO 63198(314) 537-6678Date:R. L. Fuchs, Ph.D.Associate Fellow, Regulatory ScienceGG4G, (314) 537-6438The Agricultural Group of Monsanto Company
The Aghcultural Group of Monsanto Company Study I: 92-01.36-22New Products Division Experiment U: 92-427-728Regulatory Sciences Page 2 of 13--JJ. E. Ream<strong>Research</strong> Specialist, Regulatory ScienceGGdK, (314) 537-6678The Agricultural Group of Monsanto Company--Sr. Quality Assurance AuditorMonsanto CompanyDate:.GLP/QC CoordinatorThe Agricultural Group of Monsantostatistical support:k MA Date: (1;/2
The Agricultural Group of Monsanto C?mpany Study W: 92-01.36-22New Products Division Experiment 0: 92-427-726Regulatory Sciences Psge 3 of x3The purpose of this study is to assess the degradation of the Bacillusthuringiensis var. kurstaki HD-73 [Cry IA(c)11 protein (abbreviated as“B.t.k. HD-73”) and its biological activity using in uitro mammaliandigestion models. Degradation of the B.t.k. HD-73 protein will be assessedusing western blot analysis. Degradation of the biological activity of theB.t.k. HD-73 protein will be assessed using an insect bioassay. The B.t.k.HD-73 protein, expressed in cotton plants, confers resistance toLepidopteran insect damage..2. J%mosedEhemnentaIStartDat~ . May, 1993 ’. . .3. e$al Termmatmn Date, l June,19934.0 Tesk Conid & Refesq .al Test SubstanceThe test substance for this study will be the B.t.k. HD-73 protein thathas been purified from E. coli (Lot Number 5025385). Concentratedstock solutions of B,t.k. HD-73 protein will be prepared from solidmaterial dissolved in buffer (10 mM sodium carbonate/bicarbonate,pH 10.5). A description of the preparation of the stock solutions of thetest substance will be documented and included with the study.43 Ckmtrol SubstanceThe control substance for this study will be the buffer used to preparethe B.t.k. HD-73 protein solutions (10 mM sodium carbonate/bicarbonate, pH 10.5, prepared by dilution from a 1 M stock solution).43 Merence SubstanceIncubated substances will be referenced to unincubated (time = 01samples. Analytical standards of the full-length B. t.k. HD-73 proteinand the tryptic fragment of the full-length B.t.k. HD-73 protein will beused in the western blot analysis for reference to the digestion
The Agricuiturd Group of Monsanto Camp-y Study I: 92-01.36-22New Products Division Experiment Y: 92-427-72sReNatory Sciences Page 4 of 13--samples. Two standards will be used because the full-length B.t.k.HD-73 protein is known to degrade to a biologically-active trypticfragment upon exposure to trypsin, a component of the simulatedintestinal fluid used in this study.5. Test Svstem:The test systems for this study will be simulated gastric fluid and simulatedintestinal fluid prepared as described in The United States Pharmacopeiaz.Simulated digestion solutions will be used in this in vitro study to simulatedigestion, simplify analysis of protein degradation and increase”reproducibility relative to in uivo systems. These digestion fluids will beused within 24 hours of preparation, stored at approximately 4°C andassayed for protease activity according to applicable SOPS prior to use. Thetest system incubation tubes will be clearly marked using a permanentmarking pen with unique alphanumeric codes reflecting the treatments,--6. Te e~TZ vitro studies with simulated digestion solutions are widely used asmodels of animal digestion. Simulated digestion solutions have been usedto investigate the digestibility of plant protein&, animal proteins5 and foodadditive&; to assess protein quality’;; to study digestion in pigs and poultry8;to measure tablet dissolution rates to assess bioavailability forpharmaceuticals9; and to investigate the controlled-release properties ofexperimental pharmaceuticalslo. The method of preparation of thesimulated digestion solutions to be used in this study is described in theUnited States Pharmacopeia, a frequently cited reference for in vitrodigestion studies.-7.0 Exnerimental;7.1 Experimental SummaryThis study will be conducted in two parts:-Part I. Assessment of degradation of B. t.k, HD-73 proteinas dettcted by western blot analysisPart II. Assessment of degradation of B.t.k. HD-73 proteinbioactivity as detected by insect bioassay-
’ The Agriculturzd Group of Monsanto Company Study 8: 92-01-36-22New Products Division Experiment t: 92-427-72sRegulatory Sciences Page 6 of 13The B.t.k. HD-73 protein will be added to simulated gastric fluid(SGF) and simulated intestinal fluid (SIF), incubated atapproximately 37”C, the reaction terminated, and the B.t.B. HD-73content of the incubation solutions evaluated by western blot analysis(Part I) or insect bioactivity (Part II). Part I will consist of 17separate treatments with 3 replicate digestion incubations pertreatment (total samples = 51). Part II will consist of 20 separatetreatments with three replicate digestion incubations per treatment(total samples = 60). A list of all treatments, incubation labels andsample labels is attached (Attachment #l).72 Addition ofTest SubstanceAliquots of purified l3.t.k. HD-73 will be added to temperature -equilibrated (approximately 37°C) digestion solutions by pipetting‘from concentrated B.&h. HD-73 stock solutions. This method ofadministration of the test substance was selected to providereproducible dosing of small amounts of protein. One-ml volumes ofdigestive fluids will be incubated in l&ml plastic test tubes with thefollowing exceptions: 1) a &l-ml incubation volume in a l&mlplastic test tube will be used for the t = 0 samples of B.t.k. HD-73protein in SGF and SIF (incubation samples GP-0 and IP-0, Part I);and 2) a O-l-ml incubation volume in a 1.5-ml plastic test tube will beused for the whole incubation samples for both SGF and SIF(incubation samples GPW and IPW, Part I). These treatments use areduced volume to keep final quenched sample volumes compatiblewith equipment used for aliquots sampled from the l-ml digestionincubations. The digestion solutions will be thoroughly mixedimmediately after the addition of the l3.t.k. hD-73 protein.7.3 IncubationAll digestion solutions will be incubated at approximately 37°C in ashaker-incubating water bath. Incubation solutions will be agitatedcontinuously throughout the incubation period with the followingexceptions: agitation may be interrupted briefly to facilitatesampling; and no agitation will occur between sampling intervalsthat are less than 2 minutes. In the latter case, incubation sampleswill be agitated manually just prior to sampling.
The Agricultural Group of Monsanto Company Study t: 92-01-36.22New Products Division Experiment I: 92427.728Reguiatory Sciences Page 6 of 13_-7.4 Part I. Assessment by Western Blot AnalysisIn this part, the B.t.k. HD-73 protein will be added to digestion fluidsto a final concentration of approximately 2 &ml. This dose wasselected based on the detection range of the western blot analysis.The B. t. k. HD-73 protein will be incubated for five time intervals(including t = 0) with a total duration not to exceed 20 minutes in SGFand 24 hours in SIF. For one-ml incubation volumes, 50-~1.1 aliquotsof the incubation solution will be removed and the digestionterminated. For SGF, incubation termination will be byneutralization with 0.2 M sodium carbonate (15~1 sodium*carbonateper 50 pl digestion solution.) For SIF, incubation termination will beby 1:l dilution with protein-denaturing 2X SDS-PAGE samplebufferll followed by heating to about 100°C for approximately 5minutes. Neutralized SGF incubation samples will be diluted 1:lwith 2X SDS-PAGE sample buffer and heated to approximately 100°Cfor approximately 5 minutes. For O.l-ml incubation solutions, theentire incubation solution will be terminated, diluted with 2X SDS-PAGE sample buffer and heated as described for aliquots from onemlincubations. All samples will be stored at approximately -80°Cuntil analyzed for B.&k. HD-73 content by western blot analysis.Western blot analysis will be conducted according to SOP.7.5 Part II. Assessment by Insect Bioassay----->--In this part, the amount of degradation of B.&k. HD-73 proteinbioactivity over a fured time interval in digestion fluids will beassessed using the tobacco budworm Heliothis uirescens (TBW)assay. The B.t.k. HD-73 protein will be added to digestion fluids tofinal concentrations of 0.75, 7.5 and 75 pg/ml. These concentrationswere selected based on the sensitivity of the TBW assay. Thesesamples will be incubated for a single time interval (two time pointsincluding t = 0). The incubation time interval will be chosen based onthe results of Part I; it will be approximately the same as one of thetime intervals from Part I. This interval will be chosen based on thefollowing criteria: the time interval is one that indicates substantialdegradation of the B.t.k. HD-73 protein by western blot analysis, orthe time interval is the longest one used in Part I if no substantialdegradation is observed. The one-ml incubation volumes will beterminated by neutralization with sodium carbonate for SGF and byfreezing on dry ice for SIF. 50-pl samples will be taken from eachincubation mixture in Part II and the B.t.k. HD-73 proteinconcentration estimated by western blot analysis. Terminated
The A&cultural Group of Monsanto Company Study 8: 92.01.36-22New Products Division Experiment #: 92-427-728Regulatory Sciences Page 7 of 13samples to be evaluated for TBW activity will be stored atapproximately -8OOC. On the day of the TBW assay, samples will bethawed, incorporated into artificial TBW diet and assayed for activityagainst TBW according to applicable SOPS.7.6 Control of BiasThree replicate digestion incubations will be prepared for eachtreatment.For Part I and Part II, the following treatments will be prepared toassess whether the SGF and/or SIF solutions are interfering with theanalytical methods used in this study:SGFSIF+ buffer (- B.t.k. HD-73 protein)+ buffer (- B.t.k. HD-73 protein)To estimate the recovery of added B.t.k. HD-73 from SGF and SIF inPart I and Part II, the following treatments will be compared:Buffer + B.&k. HD-73 protein (t = 0)SGF/SIF + B.t.k. HD-73 protein (t = 0)For Part I, to assess whether the 50-JLI aliquots sampled from thel-ml digestion solutions are representative of the entire digestionsolution, the following treatments will be compared:SGF/SIF + B.t.k. HD-73 protein, time interval #4 (50-~1aliquot of l-ml incubation sample)SGF/SIF + B.t.k. HD-73 protein, time interval #4 (O.l-mlincubation sample in which entire incubationsample is prepared for western blot analysis)7.7 Proposed Sta~dMethodsFor Part I, B.t.k. HD-73 protein concentration for each assay samplewill be estimated by visual comparison of the sample band intensity tothe band intensity of two concentrations of the 3.0~. HD-73 proteinstandard. The mean 13.t.k. HD-73 protein concentration will bereported from the three replicates of each treatment. Degradation ofB.t.k. HD-73 protein will be reported as a percentage of B.t.k. HD-73
--The Agricuhrd Group of Monsanto Company Study W: 92-01-36.22New Products Division Experiment I: 92-427-728Regulatory Sciences Page 6 cif 13-protein degraded at each time interval relative to the estimated B.t.k.HD-73 protein concentration in the unincubated treatment.For Part II, the B.&k. HD-73 bioactivity of each assay sample will bereported as TBW mortality, calculated as the percentage of TBWlarvae that died after the TBW larvae incubation period relative theinitial number of larvae added to the insect diet. The mean andstandard error of TBW mortality will be calculated for the threereplicates from each treatment. Degradation of B. t.k. HD-73 proteinbioactivity will be related to the percent change in TBW mortalitywithin the digestion incubation time interval tested.---8. RecordstobeMaintajnedRecords specific to this study that will be maintainedare:-Stock solution preparationDosing solution preparationDosing descriptionsIncubation interval dataWestern blot gel loading formsOriginal western blots and electronically-scanned copiesTBW assay sample data formsSample transfer formsStatistical analyses9. Studv Conduct StatementqThis experiment shall be conducted in accordance with the.protocol. Anychange, revision or deviation from this protocol will be documentedproperly and communicated to the Study Director immediately. (If theStudy Director is unavailable, deviations will be communicated to theGLP/QC Coordinator who will inform the Study Director as soon aspossible.) All specimens will be identified clearly with the Study andExperiment numbers and a unique identifier. All data generated duringthe conduct of this study will be recorded directly and promptly usingindelible ink, preferably black. Entries, at least once per page, will beidentified with the protocol number, dated on the day of entry and signed orinitialed by the person entering the information. Data generated byautomated data collection systems will include the protocol number, initialsor signature of the individual responsible for the direct data input and thedate. Any change in data entries will be made so as not to obscure the-------
The &ricultural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy 8: 92-01-36-22Experiment B: 92-427-728Page 9 of 13original entry, will indicate the reason for the change, will be dated and theresponsible individual will be identified.10. Kev Personnel / Co-investifrators:Steven R. Sims <strong>Research</strong> Specialist Monsanto CompanyJohn N. Leach <strong>Research</strong> Technician Today’s Temporary11. ConfidentialikNo raw data, worksheets, data or other information summaries, reports. orother information related to this study may be revealed or released to anythird party without prior notification and authorization of the AgriculturalGroup of Monsanto.12. C&P ComxAiance; LThis experiment will be conducted in compliance with the United StatesEPA FIFRA Good Laboratory Practice Regulations (40 CFR Part 1601, FDAGood Laboratory Practice Regulations (21CFR Part 58) and OECb GoodLaboratory Practice Standards and principles.13. References, .1. Hofte, H. & Whiteley, H. R. 1989. Microbiological Reviews 53: 242-255.2. The United States Pharmacopeia, Vol. XXII. 1990. United StatesPharmacopeial Convention, Inc. Rockville, MD, pp. 17881789.3. Nielson, S. S. 1988. Cereal Chem. 65: 435442.4. Marquez, U. M. L. & F. M. Lajolo. 1981. J. Agric. Food Chem. 29: 1068.1074.5. Zikakis, J. P., Rzucidlo, S, J. & Biasotto, N. 0. 1977. J. Dairy Science 60:533-541.6. Tilch, C. & Elias, P. S. 1984. Mutation <strong>Research</strong> 138: l-8.7. Akeson, W. R. & Stahmann, M. A. 1964. J. Nutrition 83: 257-261.8. Fuller, M. F., ed. 1991. In vitro digestion for pigs and poultry, C.A.B.9.International, U.K.Alam, A. S., Hagerman, L. lli. & Imondi, A. R. 1980. Arch. Int.Pharmodyn. Ther. 247: 180-189.10. Doherty, A. M., et al. 1991. J. Med. Chem. 34: 1258-1271.11. Laemmli, U. K. 1970. Nature 227: 680-685.
-The Agricultural Gronp of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy I: 92-01.36-22Experiment Y: 92-427-726Page 10 of 13-Attac&r.nent I: Incubation and Assay Sample LabelsPart I. Assessmentby Western Blot Analysis-Incubationsamnle label*BPAssaylabel*BP-OsampleDescriptionBuffer + protein, t = 0 min--GBGB-0GB-4SGF + btier, t = 0 min ”SGF + buffer, time interval #4GP-0GPGP-0GP-1GP-2GP-3GP-4SGF + protein, t = 0 minSGF + protein, time interval #1SGF + protein, time interval #2SGF + protein, time interval #3SGF + protein, time interval #4-GPW GPW-4 SGF + protein, whole incubationsample control, time interval #4ISIB-0X13-4SIF + buffer, t = 0 hrSIF + buffer, time interval #4IP-0IPIP-0SIF + protein, t = 0 hrIP-1 SIF + protein, time interval #1IP-2 SIF + protein, time interval #2IP-3 SIF + protein, time interval #3rp-4 SIF + protein, time interval #4IPWIPW-4SIF + protein, whole incubationsample control, time interval #4-* An additional numeral (A, B or C) wiil be added to reflect replicatedigestion solutions.SGF = simulated gastric fluid; SIF = simulated intestinal fluid-+4-
The Agricultural Group of Monsanto Company Study 6: 92-01-36-22New Products Division Experiment #: 92-427-728Regulatory Sciences Page 11 of 13Attachment1 (continued)Part II. Assessment by Insect BioassayIncubationsample label*B-O::yI-OI-lB-LOG-L-OG-L- 1I-L-OI-L-1B-M-OG-M-OG-M-lI-M-OI-M-1B-H-OG-H-OG-H- 1I-M-OI-M- 1DescridionBuffer,SGF,t=Ot=oSGF, time intervai #lSIF, t=oSIF, time interval #lBuf’fer + 0.75 pg/ml B.t.k. HD-73,SGF +(1,SGF +u,SIF +u,SE +u,Btier + 7.5 pg/ml B.t.k. HD-73,SGF +64,SGF +(L,SIF +u9SrF +a9Buffer + 75 pg/ml B.t.k. HD-73,SGF cu,SGF cu,SIF +cc7SIF +uIt=ot=otime interval #1t=otime interval #lt=ot=otime interval #lt=otime interval #lt=ot=otime interval #lt=otime interval #l* An additional numeral (A, B or C) will be added to reflect replicatedigestion solutionsSGF = simulated gastric fluid; SIF = simulated intestinal fluid
The Agricuitural Group of Monsanto Company Study Cy: 92-01-36.22New Products Division Erperiment b: 92-427-728Regulatory Sciences Page 12 of 13Attachment 2: Abbmviationsa...“.,.._-2T.t.1B.t.k.E. coliZPSGFSIF“,1”mlSDSPAGETl3WGLPQCEPAFIFRAOECDTreatmentBPGIWiiHBacillus thuringiensis var. kurstaktEscherichia colidegrees CentigradeStandardsimulatedOperating Proceduregastric fluidsimulated intestinal fluidmicrogrammicrolitermillilitersodium dodecyl sulfatepoiyacryiamide gel electrophoresistobacco budwormGood Laboratory PracticeQuality ControlEnvironmental Protection AgencyFederal Insecticide, Fungicide, and Rodenticide ActOrganization for Economic Cooperation and Developmentcodes:bufferprotein f= B.t.k. HID-73 protein)gastric fluidintestinal fluidwhole incubation samplelow dosemedium dosehigh dose-------
The Agricuitural Group of Monsanto CompanyNew Products DivisionRegulatory SciencesStudy UI: 92-01-36-22Experiment II: 92427.728Page 13 of 13Attachment. 3: Study co-invebr acknowkdgemex~ of study pnotillrrrt :1c. 1Study: Assessment of the in vitro digestive fate of Bacillusthuringiensis var. kurstaki HD-73 proteinSteven R. SimsMonsanto Agricultural Group, New Products Division(314) 537-6549eIr*c !. ISignature: Date: war/q^ John N. LeachToday’s Temporary, contracted toMonsanto Agriculturai(314) 537 7460Group, New Products Divisionf
.MonsantoThaAgricul~GroupNew Products Division - Regdatory Scienoer,Study Number: 92-0136-22 Amendment #: 1Date change implementi 06/17/93Experhmt’s af!fected by this amendment:92-427-728-Page Nch. &or Se&ion/s: p.6, se& 7.4 ori@nally stat&For SIF, incubation termination wiIl be by 1~1 dilution with proteindenaturing 2XSDS-PAGE sample buffer followed by heating to about 100 C for approximately 5 &i&es.This section is amended as followIf necessary, au akemative quench procedure wilI be used for SIF incubations. For thisaltemative procedure, the SIF incubation sohxtion witl be tarmine&& by heating atapproximately 100 C for five minutee before the addition of 2X SDS-PAGE sampIe buffer andheating as described originally. This alternative quench wiIl be used if the results using theoriginal quench procedure described suggest it is not optimal, In this case, only the BP-O,IP-0 and E-1 treatments wiII be repeat& using the al&native quench procedure.F&ason for amendment:Initial results from Part I. suggest a low recovery of the fulI-length B.t.k.HD-73 proteid &omintestinal fluid as observed by comparison of the BP-O and IP-0 treatments. There appears tobe a rapid degradation of the full-length B.&k. HD-73 protein towards the tryptic fkagmentduring the initial assay termination steps.This change will impact the Stxxdy in the following waysThis change may provide data supporting a high recovery of the M-length B.t.k. MD-73protein. This would enhance this study. The single time point incubation treatment wilisllow a comparison of the results using the aitemative quench to those using the originalquench procedure.signatureof Approv&Study Director:x-7--.----Quality Assurance: % \c. &ULU-Not Applicable for this ProtocolNot Applicable for this Protocol Date:Not Applicable for this Protocol Date:Cc:
E”:,Monsanto‘IkeAgridturaIGroupNew E&Acts Division - Regulators &Iences-1AmendmentStudy Number 9241-36-22 Amendment #: 2Date change implemented: 06’17193,?-Experiment’s af&ctedbytbis ;aIkle&h.enk92427-728Page Nds. &.‘or Sectionk p.11, Attach #1, Part II ox+gbaUy stat&Incubation Sample Labels:B-O, . . . I-IB-LO, . . . I-L-1B-M-O, . . . I-M-lF*rItir.IB-H-O, G-H-O, G-H-1, I-M-O. I-M-1This section is amended as followaIncubation Sample Labels:B-O, . . . I-1B-LO, . . . I-L1B-M-O, . . . 1-M-lP:, YB-H-O, G-I-I-0, G-H-f, I-H-O, I-H-1Reason for amendmerl~The indicated sample labels in the protocol were inamect.Thfschangewillimpact~studyinchef&~wayls:No impact; comet labele were placed on appropriate samples.Signatureof ApprovakStudy Director: Date. b+-2 s-93.mF -\\. Isignaturw ofAcknowk?dgem~~Sponsor:n&;**Quality Assurance: a-5. h&kNot Applicable for this Protocol Date; -Not Applicable for this Protocol Date:Not Applicable for this Protocol Date:cc:30043OFUO43Ph 1