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The "Bressler Report" Note: This is the text of an FDA report on Searle (Part 2 of 5 ... Pages 11-30) EIR 4/25/77 to 8/4/77 Searle Laboratories JSA/DME/JT/LF Div. G.D. Searle & Co. 4901 Searle Parkway Skokie, Illinois 60076 (page 11) Significant interviews are attached as Exhibits, as referenced. Individuals interviewed were as follows: 1. Donna Helms - Administrative Assistant to Dr. McConnell on 5-18-77, 6-30-77 and 7/1/77 (Exh. #46). 2. Judith Beauchamp - Hematology Lab Supervisor on 6-2-77 (Exh. #47). 3. Barbara Bickford (Nee Ross) - Technician, Department of Analytical Research on 6-1-77 and 6-2-77 (Exh. #48). 4. Clifford J. Suel - Supervisor, Department of Analytical Research and Development on 6-2-77 (Exh. #49). 5. Bartolome R. Tangonan - Research Technician, Pathology Toxicology Department on 6-1-77 (Exh. #50). 6. Tony Martinez - Research Assistant and Toxicology Lab Supervisor on 5-19-77, 6-3-77, 7-7-77, 7-20-77 and 8-2-77 (Exh. #51). 7. Ted Reichert - Supervisory Systems Analyst on 5-24-77 (Exh. #52). 8. Phil Polli - Systems Analyst on 5-24-77 (Exh. #53). 9. Judith Schmal - Clinical Chemistry Section Supervisor on 6-2-77 and 6-7-77 (Exh. #54). 10. Jane Drury - Analytical Chemist, Bioanalytical Dept. 6-7-77. 11. Alan Mitchell - Teratologist on 7-20-77 (Exh. #56). 12. Raymond G. Schroeder - Former Searle Teratologist on 7-18-77 (Exh. #57). 13. Dr. Rudolph Stejskal - Pathologist on 6-23-77. 14. Patricia Erdenberger - Research Assistant and Histopathology Lab Supervisor on various dates (Exh. #58). Dr. Robert McConnell, Pathology-Toxicology Advisor at the time of this study, was not directly involved with daily procedures. He is no longer employed at Searle. An attempt was made to interview Dr. K. S. Rao, Monitor of Study P. T. #988S73 on 7-25-77. We were referred to Dr. Rao's attorney, who refused permission for an interview (see Jerome Bressler's memo dated 7-27-77, Exh. #33). (11) PURPOSE OF STUDY PT 988S73 (E-77/78) SC-19192: 115 Week Oral Tumorigenicity Study in the Rat According to the submission to FDA, this study was intended to evaluate the safety and tumorigenic potential of SC-19192, diketopiperazine (5-benzyl-3, 6-dioxo-2-piperazine-acetic acid), which is a conversion product of aspartame, and to induce and define such adverse effects as might occur only at prodigious multiples of the estimated daily human intake. The commercial grade of aspartame (SC-18862) may contain up to 2 percent of the conversion product (DKP), according to Searle's specifications. DATES Study e-77/78 (PT #988S73) was initiated on November 8, l971. The study was to be terminated at 104 weeks, but was extended to 115 weeks. The reason for extending the study was stated as follows in protocol amendment #3 dated September 6, l973: "it was decided to extend or continue the study until the mortality of either sex reduced the control group to 20 animals per sex, provided the survival in the treated groups is not less than 10 animals/sex/treated group prior to that period. This approach is consistent with current FDA desires." A copy of the study protocol is attached as exhibit #11. Initiation of treatment was staggered over a two week period as follows: HOUSING DATE PLACED SCHEDULED DAYS ON GROUP ON STUDY SACRIFICE STUDY A - Male 11/8/71 1/21/74 805 B - Female 11/9/71 1/22/74 805 C - Male 11/9/71 1/22/74 805 D - Female 11/10/71 1/23/74 805 E - Male 11/11/71 1/24/74 805 F - Female 11/12/71 1/25/74 805 G - Male 11/15/71 1/28/74 805 H - Female 11/16/71 1/29/74 805 J - Male 11/17/71 1/30/74 805 K - Female 11/17/71 1/30/74 805 L - Male 11/18/71 1/31/74 805 M - Female 11/19/71 2/1/74 805 (12) PROTOCOL AND AMENDMENTS A copy of the protocol for this study was obtained and is attached to this report (See Exhibit #11). The protocol includes 4 amendments which are dated Aug 20, l973, (amendments #1 and 2), Sept. 6, l973 and Jan 9, l974. Amendment #1 dated Aug 20, l973 specified 4 additional clinical chemistry laboratory measurements: 1.) serum insulin, 2.)serum ornithine carbamyl transferase, 3.) serum protein electrophoresis, 4.) serum total protein. Two of the above assays (serum insulin, and serum ornithine carbamyl transferase) were apparently not done, because no data for these two parameters was submitted to FDA, and we could find no raw data or other evidence that they were done. Amendment #2 dated Aug 20, l973, specified 8 coronal sections of brain to be examined microscopically, and also described the procedure for sectioning the urinary bladder. Four transverse sections from each urinary bladder were to be examined microscopically. Amendment #3 dated Sept. 6, l973 extended the study until it reached a point where mortality reduced the control group to 20 animals per sex, provided survival of treated groups was not less than 10 per sex per group. (This represented a survival of approximately 30%). Amendment #4 dated Jan 9, l974 added serum cholesterol to the clinical chemistry measurements to be made at terminal sacrifice, and terminated the study after 114 weeks of treatment. Terminal sacrifice was to begin on 1-24-74 and continue through 2-1-74. Our examination of the original data showed that serum cholesterol determinations were done at day 796 and 798 (terminal bleeding) as specified in the above amendment, but the data was not included in the submission to FDA. The submission to FDA (Vol. 1 p. 286) reported a significant decrease in serum cholesterol that was more perceptible towards the end of the study, and may have been related to compound administration. Therefore, the omitted data may have been important. Serum cholesterol determinations were also done at day 546 (78 weeks) and not reported in the submission to FDA. (13) The protocol for Clinical Chemistry procedures specified that BUN determinations were to be done at 78 weeks (546 days). The submission to FDA contained no BUN data for day 546, but our review of the raw data indicated that BUN's had been done at day 546. Some BUN's were also done at day 735 (105 weeks) and not reported in the submission to FDA, but this data was not complete for all animals. Attached to the protocol is a memo dated Oct. 31, l972 which describes an acute infection spreading in the rat colony, and the administration of penicillin to combat the infection, and a memo dated May 8, l973 listing scheduled dates to be added to Body and Feeder Weights of housing groups A & B. The final Histology Lab Protocol, dated 1-21-74, specifies 24 organs to be embedded for control and high dose animals, and 19 organs to be embedded for low and mid dose groups. The organs which were to be embedded for the control and high dose groups but to be omitted in the low and mid dose groups include: lymph node, nerve, bone, eye, and salivary glands. Pathology sheets (blank forms) to be used at terminal sacrifice were reproduced (xeroxed) with check marks, time (death to tissue fix), fixative, study, and project number already entered. Twenty-seven (27) organs were checked off, to be embedded. However, as stated above, the control and high dose animals were to have 24 organs embedded, according to the protocol, and the mid and low dose 19. Therefore, all pathology sheets for animals killed by design have incorrectly identified the specific organs and tissues to be embedded. In addition to the above error, in many cases the actual number of tissues embedded was less than the 24 (control and high dose) or 19 (low and mid dose) specified in the final Histology Lab Protocol dated 1-21-74. Specific figures for numbers of tissues embedded at terminal sacrifice are as follows: NUMBER SPECI- NO. OF ANIMALS ACTUAL ACTUAL FIED IN PRO- NOT IN ACCORD RANGE AVERAGE TOCOL WITH PROTOCOL CONTROLS 10-24 20 24 129 of 144 LOW DOSE 12-23 19 19 19 of 72 MID DOSE 4-24 18 19 28 of 72 HIGH DOSE 9-25 22 24 51 of 72 (14) PERSONNEL AND RESPONSIBILITY The names of Dr. K.S. Rao, Dr. R. Stejskal, and Dr. R.G. McConnell appear on the final study report, indicating that they are the authors of this report, and were responsible for the study. Following are the principal person involved with study E-77/78 and their specific areas of responsibility: 1.) Dr. Robert G. McConnell - Director, Pathology-Toxicology Laboratory 1970 through 1974. Dr. McConnell functioned as the Path- Tox advisor on study E-77/78. He is no longer employed by Searle. 2.) Dr. Suryanarayana K. Rao - Manager, General Toxicology Laboratory, June 1971 until he left Searle in May of 1977. Dr. Rao was the Path-Tox monitor for study E-77/78. In 1971 Dr. Rao monitored 30 studies, in 1972 forty-seven (47) studies, in 1973 twenty-nine (29) studies and in 1974twenty-five (25) studies. 3.) Dr. Rudolf Stejskal - Senior Research Investigator, Pathologist. Dr. Stejskal was responsible for the micro- scopic findings and accuracy of these findings in the study report of E-77/78. Because Dr. Stejskal joined Searle in July, 1973, he had no input into the pathology protocol. Also, he did not examine all of the slides for this study, but was assisted in that task by Dr. Joseph H. Smith M. D.. 4.) Dr. Joseph H. Smith, M.D. - Group Leader and Senior Patholo- gist at Michael Reese Hospital, Chicago, IL., before joining Searle in June of 1973. Dr. Smith examined some of the slides for study E-77/78, and supervised the necropsy labora- tory. 5.) Tony Martinez - Toxicology Laboratory Supervisor, 1970 through 1973. Mr. Martinez participated in twelve (12) studies in 1971, Seventeen (17) studies in 1972, and thirteen (13) studies in 1973. Mr. Martinez supervised the technicians who worked on the study. He also performed some necropsies. (15) 6.) David K.T. Kie, B.S., Research Assistant in Pathology Laboratory. He performed some of the necropsies on E-77/78. 7.) Robert Spaet - Research Assistant. He also performed necropsies. 8.) Bartolome R. Tangonan - Research Technician II - He was involved with preparation of diet mixtures, daily observa- tions, weighing and feeding animals, etc. 9.) Donna K. Helms - Manager, Safety Evaluation, Project Sche- duling, Reporting, and Data Storage, Path-Tox Dept. and Administrative Assistant to Dr. McConnell. 10.) Patrica Erdenberger - Research Assistant, and Histology lab Supervisor. 11.) Dr. Eugene Joseph Youkilis - Senior Research Investigator. He performed the opthalmoscopic examinations in study E-77/78. 12.) Judy A Henderson - August 1972 to present, Research Tech- nician III, Histopathology Dept. She was involved with tissue processing on study E-77/78. 13.) Judith R. Schmal - Nov. 1971 to present, Supervisor, Clini- cal Chemistry Section of Bioanalytical Laboratory. 14.) Judith A. Beauchamp - Employed Aug, 1970 to present; Supervisor Hematology laboratory since April 1973. 15.) Barbara (Ross) Bickford - Research Technician, Quality Control Department. She performed analyses of DKP diet mixtures for study E-77/78. 16.) Clifford J. Seul - Supervisor, Method Development, Stability Evaluation Laboratory. He was Barbara Bickford's supervisor at the time the DKP stability study was performed. 17.) Jack Drogt - 1967 to present, Senior Research Assistant, Chemical Development. Mr. Drogt manufactured the 7 lots of DKP used in the study E-77/78. 18.) Dr. John E. Dutt - Math-Stat. Dept. 19.) John Mellman, Math-Stat Dept. (16) 20.) Fred Hunter, Technician. Since the Task Force investigation in 1975, there has been a major internal reorganization. The current organization of Worldwide Pharmaceutical Research & Development is attached as Exhibit #3. The only change has been the resignation of Dr. Robert A Moe, Executive Vice-President. Mr. George V. O'Bleness, Corporate Vice-President, is temporarily filling this position. Organizational charts for Preclinical Research and Development of Products safety Assessment are also attached as Exhibits 4 & 5. There have been no changes in these areas to date. Worldwide Pharmaceutical Research and Development is respon- sible for research and development of Aspartame and is a part of the Pharmaceutical/Consumer Product group. The group President is O.B. Parrish, who reports to James A Buzard, Executive Vice-President for Operations, G.D. Searle & Co. The current corporate structure of G.D. Searle & Co. has been discussed under History of Business. P.T. No. 988S73, 115 Week Oral Tumorigenicity Study in the Rat was conducted between November 10\971 and February 1974. The final FDA submission was dated September 1974. Following is a yearly breakdown of key personnel during this study: 1971 Robert Moe - Director, Biological Research Department. Robert McConnell - Director, Pathology - Toxicology Section K.S. Rao (June, 1971) - Manager, Toxicology Section. Tony Martinez - Toxicology Laboratory Supervisor. 1972 Robert Moe - Director Biological Research Department (January through April) F. Saunders - Director, Biological Research Department (May through December). Robert McConnell - Director, Pathology - Toxicology Section K.S. Rao - Manager, Toxicology Section. Tony Martinez - Toxicology Laboratory Supervisor. (17) 1973 (January to June) Francis Saunders - Director, Biological Research Department. Robert McConnell - Director, Pathology-Toxicology Section. K.S. Rao - Manager, General Toxicology Laboratory. Tony Martinez - Toxicology Laboratory Supervisor. 1972 (July to December) Paul Klimstra - Director, Pre-clinical Research & Development Department. Robert McConnell - Director, Pathology-Toxicology Section. K.S. Rao - Manager, General Toxicology Laboratory. Tony Martinez - Toxicology Laboratory Supervisor. 1974 Paul Klimstra - Director, Pre-clinical Research & Development Department. Robert McConnell - Director, Pathology-Toxicology Section. K.S. Rao - Manager, General Toxicology Laboratory. D. Semler - Toxicology Laboratory Supervisor. A more complete listing of personnel in the Department of Science, from 1971-1975 is attached as Exhibit No. 64. This includes the Pathology - Toxicology Department and other ancillary areas. Curriculum vitae for individuals performing significant func- tions in the study are attached as Exhibit 12. MANUFACTURE AND TESTING OF SC-19192 Seven batches of SC-19192 (diketopiperazine) were used in this study. All batches were manufactured in-house by Searle Chemist jack Drogt. The lot numbers, analytical numbers, and quantities are as follows. (18) Lot Number Analytical Number Quantity (After Milling) 1R 6906 2R 7274 3R 7273 4R 7291 This data removed in released report 5R (JDR-5-18A) 9129 6R (JDR-5-30A) 9805 7R (JDR-5-30B) 9829 Bach records covering the manufacture of lots 1R through 5R were reviewed. Batch records for lot 6R and 7R could not be located by Searle personnel. analytical reports for all seven batches were reviewed. Copies of the batch records and analytical records were obtained and are attached to this report, along with copies of pages from Jack Drogt's laboratory notebook, and other labora- tory notebooks relating to the analysis of lots 1R through 7R of DKP. (See Exhibits 13-23.) We obtained copies of three different specification sheets for DKP. (See Exhibits 16-18.) We could not determine with certainty which of the three specifications sheets was in effect at the time that the 7 lots of DKP used in this study were assayed, because only one of the three specification sheets was dated. This resulted in ambiguities for two of the parameters measured: melting point and identity (IR Spectrum). Specification memorandum dated Dec. 4, 1969 listed a melting range of 252-256 degrees C. Another specification sheet (not dated) entitled "Tentative Specification For SC-19192", listed a melting range of 241-246 degrees C. A third specification sheet entitled "Specification for SC-19192, Specification No. C 40606C" (not dated) listed a melting range "at about 243 degrees C.". For identity (IR Spectrum) the first sheet (dated 12/4/69) speci- fied that "The reference standard shall be considered to be TJT-12-32 until something better comes along". The second and third sheets specify that the DKP "Conforms to IR #2358". No data was made available as to dates, method of preparation and authentication of DKP references standards used. Searle attorney Roger Thies was contacted about this point Aug. 1, 1977 and said he would attempt to obtain information regarding this point but later registered doubt as to whether anything would be found. We asked Searle personnel to tell us which of the specification sheets was valid for the DKP used in study E-77/78. We were (19) told that the third sheet, identified with "No. C4060C", could not have been used since the number corresponded to a date in June, 1974. It is not clear as to the exact date that the first sheet (dated 12/4/69) was superceded by the second one, identi- fied "tentative specifications for SC-19192" because the second sheet was not dated or numbered. However, Searle attorney Roger Thies told us that their "best guess" was that the sheet marked "tentative specifications for SC-19192" was the one used. Accordingly, we have used the specifications from the sheet marked "tentative specifications" for the following chart, which com- pares the specifications with the actual results of analysis. DKP LOTS Specifications 1R 2R 3R 4R 5R 6R 7R - - - - - - - Note... This entire table was expunged from the delivered document, by parties unknown. - - - - - - - (20) Specifications 1R 2R 3R 4R 5R 6R 7R - - - - - - - Note... This entire table was expunged from the delivered document, by parties unknown. - - - - - - - The only discrepancy apparent in the above chart is in the criteria for identity. The specification lists reference standard 1R #2358, while the analytical record for lots 1R through 5R refer to Reference #3701. Examination of the laboratory notebooks referenced on the analytical records revealed other possible discrepancies. For example, the analytical record A-9129 for DKP Lot 5R showed an assay (titration) of 100.0 percent. The analytical record referenced two different lab notebooks assigned to two different analysts. Examination (21) of lab notebook AR-68 assigned to Sandra Ann Carey revealed that she had analyzed 3 samples of lot 5R on 11/9/72. Results of the analysis showed that sample one had an assay (by titration) of 89.70 percent, sample two, 87.93 percent and sample three was discarded. Apparently not satisfied with her results she repeated the assay on the same day (11/9/72) and obtain 93.23 percent (the average of 3 samples), still well below the specification of 99.0 percent. The other lab notebook referenced was AR-57, assigned to E. Aranda. This notebook showed that analyst Aranda performed an assay (titration) of lot 5R on 12/1/72 the results of which were 114.83 percent for 3 samples. Apparently not satisfied with the results, he repeated the assay on 12/6/72 and obtained 100.4, 99.9 and 99.8 percent for an average of 100.0 percent. This result (100.0 percent) was the only one reported on the analytical record A-9129. The analytical record (A-7291) for DKP lot R shows a result of "less than 20 PPM" for the heavy metals test. Two laboratory notebooks are referenced: VSH-I, pages 260-263, and AR-23, page 269. Examination of both of these books revealed no mention of a heavy metals test. The analytical record (A-9805) for DKP lot 6R (JDR-5-30A) also showed a result of "less than 20 PPM" for heavy metals test. Examination of the referenced laboratory notebook (AR-77, page 83-86) revealed no evidence of a test for heavy metals. The analytical record (A-9829) for DKP lot 7R (JDR-5-30B) again showed "less than 20 PPM" heavy metals. Examination of the re- ferenced lab notebook (AR-93) again showed no evidence of a heavy metals test. The above discrepancies were the only ones noted with respect to lots 1R through 7R of DKP. All other criteria for identity and purity of DKP as shown in the reports of analysis, conforms to Searle specification sheet marked "tentative specifications for SC-19192". It should be noted however that none of the seven lots of DKP met the specifications on the sheet dated 12/4/69, with respect to melting range. STABILITY AND HOMOGENEITY OF DIET MIXTURES A stability study was initiated in January 1972, 2 months after the rat study (E-77/78) had begun. The objective of the study (22) was to evaluate the stability of SC-19192 (DKP) when mixed with Rockland mouse/rat diet and held at room temperature (73 degrees F.). Two concentrations of diet mixture were tested: 3.0% and 6.0% DKP. A preliminary analysis was performed on 1-31-72 to test the analytical method (T.L.C.), and recovery of DKP. Assays were performed at one-week intervals on 2-16-72, 2-23-72, 3-1-72, 3-8-72, 3-15-72, 3-23-72, and 3-29-72. Copies of all analytical reports were obtained and are attached to this report, along with a copy of the protocol. (see exhibits #24-27). The titration method of DKP analysis was used initially, along with the TLC method. The titration method was discontinued after the 1-week analysis on 2.-23-72. Thin layer chromoto- graphy was used thereafter. It should be noted that the titra- tion method was the only reliable quantification method for DKP analysis. Page #54 of the laboratory notebook #51 (See Exhibit #26) indicated (from the photograph) that there was something in the basal diet itself producing a spot on the TLC plate which had an Rf. value corresponding to DKP. This would make quantification of DKP by this method difficult. Some of the photographs of the TLC plates approached to labor- atory notebook #51 showed no DKP reference standards. The analysis described on pages #69-72 did use a DKP standard but those on pages #88-89, #106-107, #144-145, and #284- 285 showed no reference standard. (See Exhibit #26) Only one solvent system was used for development of TLC plates throughout the study, even though it was apparent that some material in the basal diet was producing a spot on the TLC plate with an Rf. value corresponding to DKP. with the above method of analysis, only materials reacting with the potassium iodine starch reagent would be detected. Another solvent system was available for TLC analysis of DKP (See Exhibit #19) but apparently was not used in the stability study. It should also be noted that some of the chromatograms showed poor separation (day 28 on pages #144-145, and day 35 on pages #156-157 of notebook #51). See Exhibit #26) In general, the data described in the reports of analysis corresponded well with the laboratory notebooks, although the poor chromatograms were not mentioned in the reports of analysis. (23) The level of impurities as indicated by TLC was low; the major impurity, an unknown substance, represented about 2% of the DKP. The remaining impurities were also low, as apparent from the density of the TLC spots compared with the DKP spots, but were not quantified. A glossary of terms for aspartame and its diketopiperazine is attached as exhibit #9 and copies of specifications for DKP are attached as exhibits #16-18. No homogeneity tests were performed on any batches of diet mix used in E-77-78, and evidence exists tat homogeneity was a problem with the DKP diet mixtures. Two of the stability study assay reports, analytical numbers A7728 and A7739 both dated 2-16-72, contained the statement: "These samples were not homogeneous. They had to be reground before they could be sampled". The assay reports were signed by Barbara Bickford, a Searle analyst. We examined the laboratory notebook #51 assigned to Barbara Bickford and noted that a B & W polaroid photograph of the non- homogeneous sample in question was attached to page #58 of the notebook. The photograph clearly shows discrete lighter colored particles of diverse size and shape distributed nonuniformly throughout the mixture. These lighter colored particles appear to be distinct from the fairly find granular nature of the chow itself. A copy of this photograph was made and is attached to the report as exhibit #29. When questioned about the size of the white square sheet of paper in the photograph (on which the diet mixture was placed) Ms. Bickford and C. Seul both stated that it was 6"by6", when we interviewed them on 6-2-77. When the photograph was enlarged until the sample paper was 6"by6" (actual size) we measured the large particles (which were identified as DKP by Ms. Bickford) and found them to be 4 to 6mm in size. When we interviewed Ms. Bickford on 6-1 and 6-2-77, she stated that she had nothing to do with the preparation of the diet mixtures. She said that the samples had probably been received from the toxicology lab and stored at room temperature. Her procedure was to weigh out a predetermined amount of the sample, and if not a uniform powder she would re-grind it with a mortar and pestle, and would make a note of this in her lab notebook. We asked Ms. Bickford if she ever reported this lack of homo- geneity to Dr.Rao, and she replied that she did not. (24) We could not determine whether the samples assayed in the stability study were from diet mixtures actually fed to the animals, in spite of the fact that we were told so by some employees. On 6-2-77, we interviewed Analyst Barbara Bickford and Clifford Seul, who was Mrs. Bickford's supervisor at the time that the stability samples were analyzed (Feb. 16, 1972). Clifford Seul told us that the samples analyzed on 2-16-72 and described on page #58 of laboratory notebook #51, were obtained from the admixture being fed the rats on study, and not a special mix- ture prepared for the stability study. On 6-1-77 we interviewed Bart Tangonan, whose duties included observing, weighing, and feeding the animals, and mixing the diet for study E-77/78. Mr. Tangonan did not remember if there were any written instructions for mixing the diets but thought that it was mixed for a specified length of time. He said that if the diet appeared to need more mixing, it was mixed longer. He could not remember anything about the samples obtained for the stability study. On 6-3-77 we interviewed Tony Martinez who was a supervisor in the Toxicology Laboratory in 1972. He told us that although the analytical report indicated that the sample was submitted by Dr. Rao, actually anyone in the toxicology laboratory could have submitted the sample. According to Mr. Martinez, the normal procedure in such cases was to collect a sample just after mixing compound and diet and then repeat this in four weeks. He could not specifically recall what was done with regard to the stability study in question, and could not remember whether the samples had been taken from the diets being fed the animals on study P.T. 98873 (E-77/78). He did not remember any problems with mixing, bud did say that a longer mixing time was required at higher compound concentrations. A point to be considered, however, is that although the analytical report states that the material analyzed was pre- pared to contain 3.0 and 6.0% DKP, none of the diets reported to be fed contained these exact amounts of DKP according to the records of food concentration calculations, which were used to prepare the diets for study #E-77/78. (see chart attached to Exhibit #30.) In addition, the stability study protocol (Exhibit #24) specified that the test batches would be 1 kg. in size. If the protocol was followed, the small (1 kg.) test batches would not have been sufficient in size to feed a single dose group of the animals on study. (See Protocol, Exhibit #24). (25) Additional evidence of homogeneity problems was revealed when a former Searle employee, Raymond Schroeder, was interviewed buy the other FDA team on 6-22-77 concerning teratology studies E-5 and E-89. At that time Mr. Schroeder volunteered the information that homogeneity may have been a problem in the DKP diet mixtures, but not ion the aspartame diet mixtures. A follow-up phone call to Mr. Schroeder was made on 7-13-77, and at that time he stated that he observed the DKP diet mixtures being fed to the animals, and that in his opinion, the particles of DKP were large enough to allow the rats to discriminate between the DKP and basal diet. (See Thomas F. X. Collins memos (2) dated 7-14-77 (attached as Exhibit #31). An interview was arranged for July 18, 1977 between Mr. Schroeder and members of the FDA team investigating study E-77/78. The interview was conducted at [information blanked out to protect the individual], Mr. Schroeder's current place of employment. Also participating in the interview by means of a conference phone were Thomas F.X. Collins, and Leonard Friedman. Mr. Schroeder stated that he was not certain of the date, or even the year, when he observed the rats being fed DKP diets. He further stated that he could not be absolutely certain that the rats he observed were on study E-77/78. He was not certain about the dose levels of the diets he observed, and could not remember how many times he observed the DKP diets. He estimated that he observed the DKP diets "one or two times". When he was shown an actual-size enlargement of the DKP diet mixture (See Exhibit #29) he stated that to the best of his knowledge, the white particles that he observed were not as large as the largest particles in the photo, but may have been similar to the smaller white particles. He said that he may have mentioned the appearance of the DKP diets to Dr. Rao. Mr. Schroeder seemed reluctant to make any positive state- ments during this interview. Dr. Collins reminded Mr. Schroeder that he had previously volunteered the information that the DKP diets appeared to be non-homogeneous and that the rats could probably discriminate between the DKP particles and the basal diet. Mr. Schroeder replied that he had had some time to think over his previous statements and now wasn't sure about them. He told us that there must be people at Searle who know more about the DKP diets than he did. (see memo dated 7-19-77, attached as exhibit #32,which describes our interview with Mr. Schroeder). (26) When we arrived at [ address expunged ] on 7-18-77 at approximately 2:40 P.M., we were asked by the receptionist to sign a log book. While signing the log, we noted that a G.D. Searle employee (W. R. Pool) had signed in on 7-15-77. W. R. Pool works in the Toxicology Section (Safety Assessment Division) at Searle Laboratories. During our interview, we asked Mr. Schroeder if he had been contacted by anyone from Searle during the period from June 22, 1977-July 18, 1977. He replied that he had not. We again interviewed Tony Martinez on 7-19-77, and specific- ally asked him if he was aware of any homogeneity problems with the DKP diet mixtures fed the rats in study #988S73 (E-77/78). He replied that he was not aware of any problems. We asked whether any samples of DKP had been retained by Searle Laboratories. We were told that a small quantity of DKP remained in the compound file, but that it was a lot other than those used in study E-77/78. Upon request, we were then shown a jar containing 4.9 grams of DKP, lot #TJT-12-32. Its appearance was that of a fine white crystalline material with a tendency to adhere to the sides of the jar. Mr. Martinez said that this was the only lot of DKP remaining at Searle. We also interviewed Teratologist Alan Mitchell, on 7-19-77. We had previously noticed his name on one of the DKP com- pound inventory cards, and his name had also been mentioned by Raymond Schroeder, in connection with DKP. Mr. Mitchell stated that he had done two teratology studies with DKP, both with rats, and both in 1972. In one study the DKP was administered I.G. (as a suspension), and the other was a dietary feeding study. Mr. Mitchell told us that he didn't recall any problems with homogeneity in the dietary feeding study. He said he never remixed or reground any DKP diets. He admitted, however, that when he prepared the diet mixtures, he first sifted the DKP through a hand flour sifter. We attempted to interview a former Searle employee, Dr. Rao, after learning the the still lived in the Chicago area. Dr. Rao had been in charge of the DKP stability study and was the monitor for study E-77/78. After reaching Dr. Rao by telephone on July 25, 1977 he stated that he would like to talk to his attorney before consenting to the interview. We then received a call from his attorney, Mr. John H. Bickley, Jr., who told us that the interview would be of no advantage to his client, and he therefore refused to allow it. A memo (27) of telephone conversation betwen J. Bressler and Mr. Bickley is attached as Exhibit #33. CALCULATING DIET CONCENTRATION & BLENDING OF TREATMENT MIXTURES There were no batch records to show the quantities of DKP and basal diet weighed, type of mixer used, mixing time, dates, or names of individuals performing the weighing and blending operations. We were told that mixing was performed in a Hobard mixer, and that mixing times were about 10 minutes. There was no evidence that any tests had been done to deter- mine theblending charactoeristics of the mixer, or to validate the 10 minute mix time. Fresh batches were mixed on a weekly, bi-weekly, or monthly basis, and batch size ranged from 6 kilograms to 28 kilograms during the study. The concentration of DKP in the basal diet was calculated by the Mat-Stat Department on a weeklly, bi-weekly, or monthly basis (based on the food consumtion for the previous time period), and submitted to the Path-Tox Department as a Food Concentration Prediction record. The concentration was ex- pressed as grams of DKP per kilogram of basal diet. The Path-Tox Department PErsonnel thenmultiplied the grams of compound indicated on the prediction record by the number of kilograms of diet mix needed to arrive at the proper quantities of DKP and basal diet to be blended. THe concen- trations were calculated to yield the proper dosage levels of 9.75, 1.5, and 3.0 grams of DKP per kilograms of body weight per day, for the low, medium, and high dose groups. (Copies of Diet Calculation Records are attached as Exhibit #34). At the end of each treatment period, the remaining treatment mistures were discaded and fresh batches were made. Now reserve sample of either the DKP or the DKP/diet mixtuures used in this study were retained according to Searle. DKP was withdrawin from stock by means of a compound inven- tory card, which was filled out by the person requesting the material. Tony Martinez was the person that usually requested DKP for use in study E-77/78. We examined eighteen (18) compound inventory cards which accounted for 177.0 kg of DKP withdrawon from stock. According to our calculations a total of 152.81 kg of DKP would have been necessary (28) achieve the diet concentrations and batch sizes that were reportedly used in the study. A total of 230.0 kg of DKP was manufacturered by Searle Chemist Jack Drogt. Following are tables showing the quantities of DKP manufactured, calculalted quantity required for the study, and quantities withdrawin from stock. QUANTITIES MANUFACTURED Lot #. Quantity (After Milling) 1R 2R 3R 4R Another FDA gutted table! 5R 6R 7R Total CALCULATED QUANTITIES REQUIRED FOR THE STUDY Dose Group Calculated Quantity Required Low Dose Males Mid Dose Males High Dose Males Low Dose Females Another FDA gutted table! Mid Dose Females High Dose Females (29) QUANTITIES WITHDRAWN FROM STOCK (FROM COMPOUND INVENTORY CARDS) Date Withdrawn From Stock Quantity Lot 10/29/71 kg 1R 1/4/72 kg 1R 2/28/72 kg 4R 3/11/72 kg 3R 3/29/72 kg 2R 9/11/72 kg 3R 10/10/72 kg 2R * kg 2R 12/1/72 kg 3R * kg 4R 12/27/72 kg 5R * kg 2R 1/25/73 kg 5R 3/22/73 kg 6R 4/18/73 kg 5R 7/10/73 )5 kg 6R 8/10/73 )5 kg 6R 9/7/73 kg 6R 11/2/73 kg 7R Another FDA gutted table! TOTAL kg * These three cards were not signed or dated. It should be noted that only two of the 18 compound inventory cards specified that the SKP withdrawn from stock was to be used in study E-77/78 (PT 988S73). Thirteen of the cards list "Toxicity" or "Toxicology" as the reason for withdrawal. Three of the cards have no entries at all, except for the word "empty". (Copies of the compound inventory cards are attached as Exhibit #28). The total quantity withdrawn from stock is kg in excess of the amount necessary to achieve the diet concentrations used in the study. (Based on the diet calculation records attached as Exhibit #34, which we used to construct the diet calculation summary table attached as Exhibit #30). It is not known whether any of the kg of DKP accounted for on the 18 compound inventory cards was withdrawn for use in studies other than E-77/78. We could find no other records to verify the amount of DKP withdrawn for, or used in this study. (30)
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