A copy of the above memo is included in exhibit #72. Dr. Rao and Dr. Youkilis are no longer employed by Searle.
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When an animal spilled an excessive amount of food, this was noted on the observation records by means of an asterisk in the “appetite/thirst” column. The asterisk was also used to denote food spillage on the Teletype sheets for body/feeder weight data. The amount of food spillage was not quantitatively determined by the technicians assigned to observe, feed and weigh the animals, but we were told that they made an effort to return spilled food to the food cups whenever possible. WE were also told that food consumption data for those erats marked with an asterisk on the body/feeder weight sheets was not used in Searle’s statistical analysis of the data.
The “palpation record” in the “Tissue Masses and Deaths” volume shows that tissue masses were sometimes excised from the animals. The record indicates that a tissue mass measuring 1.5×1.0cm was excised from animal B31HF on 2-10-72. The record also shows that a “skin incision over mass” was performed on animals C22LM and G25LM on Feb. 10, 1972.
DOSAGE, BODY WEIGHT AND FOOD CONSUMPTION
DKP levels for the feeding study were multiples of 100, 200 and 400 times the estimated human dose. The levels in g DKP/kg body weight/day were 0,0.75, 1.5 and 3.0 for the control, low, medium and high treatment groups, respectively. The doses were mixed in the diet as described in Calculating Diet Concentration and Blending of Treatment Mixtures.
Individual body weights were recorded weekly for the first four weeks, once every two weeks for the next eight weeks and once every four weeks thereafter. The amount of food consumed was measured every week. An automated weighing system was employed consisting of an Intec balance and a Teletype machine. The Teletype produces a typewritten sheet and a machine-readable punched paper tape. All the typewritten sheets for the study were available. Xerox copies of these sheets were taken to the Division of Mathematics and technical Operations Staff of the form and calculated by a computer program designed by Dennis Wilson, Division of Mathematics.
In designing the computer program it was necessary to make certain assumptions on the handling of the data. One assumption concerned missing data, e.g. the empty feed cups weights were missing for the “D” housing group at the 12th week. Dr. George Clay, Group Leader, CNS Pharmacology, Searle and scientific co-ordinator for the FDA team, was unable to determine whether these animals were omitted from the food consumption calculations for that week, or whether the data for these animals from…
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…the 11th and 13th weeks were averaged and the average substituted for the missing data. Employees of Searle’s Math-Stat Department who had worked on the program for this experiment are no longer with the company. Dr. Clay calculated a few of the figures from the 11th and 13th weeks and stated that it appeared that the data had been averaged. For the FDA recalculation it was chosen to omit the animals with the missing weights from the calculations. In several instances (For example, C group males, mid and high levels for the 13th week; A group males, high level for the 99th week) the dietary concentration shown on the weight sheets did not agree with the concentration listed for that level in the other housing groups. Dr. Clay assured us that all the animals of the same sex in a given experimental group received the same dose for the same week on the experiment. He also assured us that the Searle computer program did not pick up the doses from the weight sheets. In the FDA program, the dietary concentrations were taken from the diet calculation sheets (Exhibit #34). Certain animals on the raw data sheets were marked with an asterisk. Dr. Clay explained that the asterisk indicated spillage and such animals were omitted from the food consumption calculations. This practice was followed in the FD computer program. In calculating the food consumption (g food eaten/day/ kg body weight) and the dosage (mg test compound/day/kg body weight), the body weight used was the weight at the end of the period under consideration, i.e. the current weight.
In addition to the calculations which were included in the Searle submission, the FDA program included calculation of the actual amount of food ingested, i.e., the total amount of diet ingested minus the test compound, and of the food efficiency (g weight gained/100 g actual food eaten). The food efficiency was calculated in order to determine whether the volume of DKP in the diet (which exceeded 7% of the diet for the high dose males at intervals during the study) was contributing to the body weight depression seen with DKP> This explanation of the body weight depression was discussed by Dr. John H. Rust, a Searle consultant, in a memo dated April 5, l976 to Dr. R. McConnell; in a memo to the file dated September 30, l974 by Dr. McConnell; and in a memo to Dr. K. S. Rao dated August 29, l974 by Dr. G. L. Schoenhard. (Exhibits #36-38).
The average body weights and weight gain (% change/week) from the FDA analysis of the Searle raw data are presented in Table 1 (Exhibit #39) which corresponds to Table 3 of the Searle submission.
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Weights which differ from the Searle submission by one (1) g or more and weight gains by 0.1 percentage point, or more are underlined. Fifteen differences were noted as follows:
Average Body Weight Discrepancies
Dose Searle
Days Sex Level Submission Calculated
280 M M 591.7 589.2
364 F L 353.2 345.1
420 M M 613.2 614.4
700 M C 595.4 579.3
728 M C 594.4 597.2
728 F H 343.1 341.2
784 F C 453.4 459.9
Percent Weight Gain Discrepancies
Dose Searle
Days Sex Level Submission Calculated
14 M C 35.11 35.23
21 M C 22.80 22.69
280 M M 0.33 0.21
364 F L -0.16 0.04
392 F L 1.13 0.85
728 F H 0.32 -0.18
756 F H -0.14 0.08
805 F C -0.04 -0.39
The food intake (in g/day and in g/kg/day) and dosage (in mg/kg/day) from the FDA analysis are presented in Table 2. This table corresponds to Table 4 of the Searle submission. there are numerous discrepancies (in excess of 80) of one (1) gram or greater in the food intake expressed in grams/day. Many of the discrepancies are probably the result of an error in the Searle computer program (see Exhibit #76). Through this error there was a failure to adjust the food intake for the precise number of days between weighings for the individual housing groups. This programming error had been pointed out to Searle by the Task Force but no amendment to the Searle submission was made. There are more than forty discrepancies of 5 or more grams when the food intake is expressed in g/kg/day. The Searle programming error would contribute to discrepancies in this expression of the food intake. The use of the current body weight in the FD analysis may also be a contributing factor. Most of the dosage…
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…calculations from the FDA program differ from the Searle submission by 10 or more mg. The two factors of the Searle programming error and the use of the current body weight in the FDA analysis would contribute to discrepancies between the FDA analysis and the Searle submission. Despite the discrepancies the FDA analysis shows dosage levels corresponding to the intended levels of 0.75, 1.5 and 3.0 g/kg/day. The test compound would have to be homogeneously mixed into the basal diet in order for these calculated dosage levels to be actually consumed. All discrepancies between the Searle submission and the FDA analysis shown in Tables 1 and 2 are underlined.
Table 3 presents the food efficiency (g gained/100 g actual food consumed) calculated in the FDA analysis. There is no corresponding table in the Searle submission. Tables 1, 2 and 3 and the computer printout of the FDA analysis are Exhibits # 39-42. Statistical analysis of the body weight and food consumption data was made and is shown as exhibit #73.
ORGAN WEIGHTS
Organ Weights were entered on the gross pathology sheets at the time of autopsy. We compared all of the individual organ weights on appendix table 5 in the submission to FDA (Vol 1, pp. 222-226) with the original data on the gross pathology sheets. A total of eleven (11) errors were noted in transcribing the raw data from the pathology sheets, to the tables in the submission to FDA.
The errors are tabulated below:
Wt. Shown In Wt. Recorded on
Animal No. Organ Submission Original Pathology Sheet
A12CM Kidneys 3.75 G 3.45 G
L28LM Ven. Prostrate 747 mg. 474.7 mg.
C0lMM Kidneys 9.40 G 9.219 G.
C02HM Kidneys 1.46 G 4.259 G
E14HM Kidneys 11.74 G 4.746 G
J12HM Pituitary 3.0 mg. 3.3 mg.
J30HM Ven. Prostrate 444 mg. 444.8 mg.
F17CF Ovaries 36.7 mg. 233.5 & 36.7 mg.
H30CF Liver 9.4 G 9.493 G
B20HF Uterus 1115 mg. 1155 mg.
K11HF Adrenals 799.1 mg. 797.1 mg.
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Copies of the applicable pages of the submission, appendix table 5, with errors indicated, are attached along with copies of the gross pathology sheets documenting the errors. (See exhibit #83)
DISEASES
The submission to FDA (Vol. 1, P. 10) reported that an unidentified infectious disease spread among the animals between 12 and 14 weeks of treatment, and that a second unidentified infectious disease occurred in high incidence between 48 and 52 weeks of treatment. In both cases, the control and treated rats were reportedly affected with equal frequency and severity. The same page of the submission also stated that over a period of two weeks, a total of 17 animals (8 control, 3 low dose, 4 medium dose, and 2 high dose) died. A memorandum dated October 13, 1972, and that more animals were morbid. Dr. Rao reported that this primary antemortem symptom observed was inappetance and labored respiration. Postmortem examination of dead animals revealed primary lesions in the lungs, and lungs exhibited patchy pneumonia, according to Dr. Rao. The memo indicates that Dr. Rao intended to administer 10,000 units of penicillin G, intramuscularly, to all the animals 2 to 3 times per day beginning 10/30/72. A copy of Dr. Rao’s memo is attached to the protocol (See Exhibit #77, Section 1).
The submission to FDA (Vol. 1, P. 10) stated that, “to prevent further loss of animals, all morbid rats were injected IN with 20,000 units of potassium penicillin G daily for 4-8 days.”
A review of the injection records (attached to Vol. A of Exhibit #75) showed that some animals were treated between approximately 51 and 60 weeks, and in one instance, a high dose animal, kB3HF, received at least 10 injections. In addition, some animals received 30,000 units per day (10,000 units 3 times per day) rather than the 20,000 units reported in the submission.
The records also indicated that penicillin was administered to four rats beginning on May 16, l973, and continued daily through May 28, l973. This third occurrence of infectious disease and penicillin administration was not reported in the submission to FDA.
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SURVIVAL
An attempt was made to construct a Survival Table using data from the “Tissue Masses and Deaths” book.
We were unable to determine the exact method used in constructing the table in the FDA submission. There was some survival data in the “Tissue Masses and Deaths” book (Exhibit 65), but this only extended through week 109 and consisted solely of running totals. According to Tony Martinez deaths purportedly were initially recorded in any one of the following documents:
(1) Body/Feeder Weight Sheets
(2) Autopsy/Pathology Sheets
(3) Observation Sheets
(4) Palpable Mass Sheet
He said that animals found dead at feeding/observation intervals were usually recorded on the observation, or Body/Feeder Weight Sheets. At other times, the death was recorded on a “scrap” of paper and then later transcribed to one of the documents. The term “scrap of paper” was used by Searle personnel both during the Task Force and current investigations. No notebooks containing observations or deaths ever surfaced during either investigation. Animals killed “in extremis” were recorded on Autopsy sheets. The least likely source for original death recording would be the Body/Feeder Weight Sheets.
Dates of death sometimes differed on the various records, making it impossible to determine which one was correct. A survival table was finally constructed for weeks 40-115, using the Body/Feeder weight teletype (hard copy) sheets and dates on which animals no longer appeared as a base (Exhibit 68). In this manner, the number of days on study was calculated for each animal (Exhibit 66). Using starting dates for each group, a calendar was made to encompass the entire duration of this study (Exhibit 67). Toward the end of the study, some feedings/observations were made at intervals such as 109 3/7, 110 6/7 and 111 6/7 weeks, so some differences are anticipated between this table and the one in the FDA submission. However, the final number of animals in each dosage group and sex do coincide. The table constructed for this report was on a weekly basis; that in the submission covering only weeks 40, 46, 52, 60, 68, 76, 84, 88, 92, 96, 100, 104, 108 and 115.
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A Life Table Analysis was performed from the Survival Table by Dennis Wilson, Department of Mathematics, Bureau of Foods (Exhibit #73). The female control population differed from the high level population p < 0.05. The male control population differed from both the medium and high dose levels (p. 0.05 in both cases). In all cases, the differences are due to the higher mortality level of controls.
CLINICAL LABORATORY ANALYSIS
A. Clinical laboratory procedures.
Hematologic, clinical, chemical and urinalysis examinations are described on pages 5-7 of Volume I of the submission. The same rats were employed for all clinical laboratory examinations throughout the study. In cases where one of these rats died during the study, another rat chosen from a corresponding group was substituted.
The following hematology parameters were measured at treatment days 42,92,189,364,547 and 734: hematocrit, hemoglobin, total RBC, total WBC, differential WBC, and prothrombin time.
The following clinical chemistry (serum) measurements were made: pyruvic transaminase (days 42,92,189,364,547,736), glutamic oxaloacetic transaminase (days 41,92,189,364,547 and 734), alkaline phosphatase (days 42,92,189,364,547.734), total bilirubin (days 42,,92,189,364,547,734) blood (serum) urea nitrogen (days 42,92,189,364), total cholesterol (days 42,92,189,364,734), L-phenylalanine (days 42,92,189,364,547,734) sodium (day 734), potassium (day 734), calcium (day 734), protein electrophoresis (day 734).
The following urinalysis (2 hour collection) measurements were made at days 42,92,190,364,547, & 734: specific gravity, pH, occult blood, protein, bilirubin, microscopic on sediment, and phenylketones; glucose and ketones were determined at days 42,92,190,364, & 734; urobilinogen was measured at day 42,190,364 and 547.
We noted that some of the data sheets for urinalysis had erroneously labeled the phenylketones test values as “phenylalanine” (see exhibit #84).
Some cholesterol and BUN determinations were carried out which were not described in the submission to FDA. They were as follows:
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1) Serum cholesterol determinations were done at days 796 & 798 (terminal bleeding), but not included in the submission to FDA.
The protocol indicated that clinical chemistry determinations, including serum cholesterol, were to have been performed at termination. 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. (Copies of these data were obtained and are attached as exhibit #77, Section V.
2. BUN determinations were done at day 546 but not reported in the submission to FDA (see exhibit #77 Section V).
3) Serum cholesterols were also done on day 546 and not reported in the submission (see exhibit #77). These determinations were only done for females, and only for a few animals, reportedly due to insufficient quantity of sample.
4) BUN’s were also done on day 735 and not reported in the submission. This data was not complete for all animals at day 735.
5) Additional animals (other than those designated) were bled at the regularly scheduled times and determinations were made. These determinations were not reported and we could not determine why the animals were bled. (See Exhibit #77)
B. A list of persons involved with lab analysis along with their responsibilities and duties is as follows:
1) Judith R. Hehmal? – Nov l971 to present, Supv., Clinical Chemistry, section of Bioanalytical laboratory.
2) Judith A. Beauchamp – Supervisor, Hematology Laboratory, April 1971 to present.
3) Denise Prikins? – Supervisor Hematology Laboratory until April l971 (no longer employed by Searle).
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Bart Tangonan
Tony Martinez
David Kie
Robert Spaet
The above four persons in the toxicology department were involved with assembling data for clinical chemistry and hematology determinations for April 1973 to Feb. l974.
Joyce Schulmann – performed urinalysis and hematology determinations from April l973 to Feb. l974.
Philip Muellner – Technician in Path-Tox Dept. July l970 till end of study.
Janet Praal – Technician, prepared individual work sheets for urinalysis. No longer employed by Searle.
C. The following employees were interviewed regarding clinical lab procedures, and methods for recording clinical lab. data.
1) Bart Tangonan on 6/1/77 regarding the recording of data.
2) Judith Beauchamp, on 6/2/77 regarding hematology and urinalysis.
3) Judith Schmal, on 6/2/77, 6/7/77, and 7/29/77 regarding clinical chemistry.
4) Tony Martinez, on 6/3/77 regarding urine and blood collection, and recording of data.
5) Jane Drury, on 6/7/77 regarding electrophoresis.
Accounts of these interviews are attached as exhibits #47-54.
D. Other Documents and Procedures Used to Authenticate Clinical Laboratory Data values in Submission were as follows:
1) One loose leaf volume entitled “SC-19192: 104 Week Oral Toxicity Study In The Rat. PT – 988573 Protocols, Organ Weights, Dosage, Hematology, Urinalysis, Blood Chemistry, Protein Electrophoresis.” The volume was subdivided into sections according to the above parameters. The individual…
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…pages (See Exhibit #77, Section IX for example) are composed of forms containing the appropriate measurements and units printed on the left side of the page onto which data on “sticky back sheets” corresponding to each of these measurements were pasted in columns representing the various time periods. These pages, in addition to other information, were headed by the identifying number of the rat for which the measurements were made. The information on the sticky back sheets (see Exhibit #77, Sec. IX) was copied (handwritten) from laboratory notebooks, sheets, Auto-analyzer Charts, teletype sheets (on line data generated by analytical instruments) or computer printouts (containing raw and calculated data resulting from on line or off line input data from instruments) by individuals of General Toxicology Section (see interview with Bart Tangonan). Many of the pages were initialed “BRT” (apparently by Bart R. Tangonan). Most of the final values transcribed into the sticky back sheets resulted apparently from calculations made directly by the analytical instruments or by external computer using the appropriate stored equations and data for the reference standards.
(2) Since the values appearing in the volume referred to in the above section were copied from other sources, an attempt was made to verify these values by examining the information in these sources. No attempt was made to recover the teletype sheets, or computer printouts which we were told were no longer available or could not be recovered (see interview with Judith R. Schmal, Exhibit #54). All laboratory notebooks that might contain the original data were requested. Notebooks dated prior to the dates of the DKP study were excluded. The appropriate laboratory notebooks were then identified by BA numbers which were listed on the top sections of the sticky back sheets included in the volume referred to above. Examination of these few laboratory notebooks revealed only a very small amount of data would could be used for additional verification of the values in the submission. It was necessary to obtain the consultation of Judith Schmal to clarify the system used to relate the values in these books with the corresponding rat and period of time of bleeding. The following notebooks, as designated by information on the front covers, were examined:
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1) Lab. notebook #N-26375 (hematology), 25 June 71 to 1/21/72.
2) Lab notebook #127133 (phenylalanine), 10/8/71 to 4/21/72.
3) *Lab notebook #113239 (cholesterol), dated 5/1/72.
4) *Lab notebook #17, BA #0007118926 (SGOT), 12/27/71 to 2/25/72.
5) Lab notebook #126472 (phenylalanine), 4/21/72 to 6/8/72.
6) Blue Book #1591, identified “JF VON – 70″ (hematology).
7) Columnar book #21, identified “JF VON 27″ (Differential cell counts).
8) Spiral notebook identified “JABEA-” (coagulation/prothrombin) dated 7/23/71.
9) *Spiral notebook #16, (SGOT), 8/27/71 to 12/16/71.
*Those books (3,4, & 9 above) marked with an asterisk provided us with no useable data, because a formula or standard curve (no longer available) was necessary to convert the data.
Copies of the applicable pages from all of the above notebooks were obtained, and are included in exhibit #77.
The following data were cross checked against available data from original entries (in addition to being checked against transcribed data on “sticky back sheets” in bound volume):
1. Hematology – Erythrocytes: Treatment days 42,91,364 & 546 Males and Females.
2. Hematology – Leucocytes, WBC: Treatment Days 42, 91, 364, & 546 Males and Females.
3. Hematology – Leucocytes, Differential: Treatment Days 42,91,189,364, & 546, Males and Females.
4. Hematology – Coagulogram, Prothrombin Time: Treatment Days 42 & 91, Males and Females.
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5. Phenylalanine. Treatment Days 42,189 Males and Females, Day 91 Males.
E. Discrepancies were found between the clinical laboratory methods described on pages 5-7 of submission Volume 1 (referenced on page 120) and those actually carried out. These discrepancies were documented by the interviews described in Section C and in a document (Exhibit #77, Section II) voluntarily submitted by Jutidy Schmal, June 7, 1977 in response to requests for clarification of the clinical chemistry procedures as they were actually conducted in regard to analytical methology instrumentation, and processing and recording of data.
1) Glutamic Pyruvic Transaminase.
Reference: Russell, C.D. and Cotlove, E. (1971)/ Clin. Chem. 17; 1114
The reference describes a coupled reaction U.V. assay for serum glutamic oxaloacetic transaminase in which malicdehydrogenase is used.
As described by Judith R. Schmal (June 7,1977) glutamicpyruvic transaminase was assayed by a method adopted from Sigma Kit Technical bulletin #410 – U. V. using Lactic acid dehydrogenase.
2) Glutamic Oxaloacetic Transaminase
Reference: Same as in (1) above.
As described by Judith R. Schmal (June 7, 1977), from November 1971 to March 15, 1972 a manual colorimetric method (Fermco Kit) was used (employing dinitrophenylhydrazine). From March 15, 1972 the method used was adapted from Sigma Kit, Technical bulletin #410 – U. V. using Lactic acid dehydrogenase.
3) Blood (serum) urea nitrogen.
Reference: Marsh, (Marsh in Submission) W.H., Fingerhut, B. and Miller, H. (1965). Clin Chem 11, 624.
The referenced method calls for reaction of urea with diacetyl monoxime in the presence of thiosemicarbazide and ferricions in a relatively weak acid solution.
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As described by Judith R. Schmal (June 7, 1977) the method used from November 1971 to February 1, 1974 was adapted from Fermco Kit Bulletins #20 and 20-1. Urea is hydrolyzed to ammonia and carbonic acid in the presence of urease. Ammonia is detected by the Berthelot reaction to produce indophenol. From February 1, 1974 the “direct serum methond” modified from the method of Marsh et al was used.
4) Phenylalanine
Reference: Hill, J. B., Summer, G. K. Pencer, M. W. Resz N.O. (1965) Chin Chem 11, 541
From Nov. 1971 to about Septembe 1972 there is no documentation in file as to method used. From about September 1972 the method used was a flurometric determination in the presence of ninhydrin and l-leucyl-l-alanine as adapted from McCaman and Rubins. (This is a manual method modified for automation by Hill et al – reverenced above) (Judith R. Schmal, June 7, 1977)
5) Calcium: Reference Pybus J., (Pylrus in submission), Feldman, F. J., and Browers (Borrers in Submission) Jr., G. N. (1970) Clin. Chem. 16 (11 in submission), 998.
The referenced method involves the measurement of total calcium in serum by atomic absorption spectrophotometry. As described by Judith R. Schmal (June 7, 1977) from November, 1961 to February 1974 the procedure used was a colorimetric procedure using Corinth dye as adapted from Kingsley and Robnet. From May 21, 1973 the method used was atomic absorption spectrophotometry, as adapted from Pybus et all (reference above)
6) Total Cholesterol
Reference: Levine J. S., Morgenstern, S., and Vlastelica, D. (1968). Automation Anal Chem. pp 25-28, Technicon Symposia 1968.
We were unable to check the above reference because of difficulty up to now in obtaining a copy of the publication, but as shown below two different procedures were employed to measure total serum cholesterol at different times during the study.
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(Judith R. Schmal, June 7, 1977). From November 1971 to July 2, 1973 the method involved reacting an isopropanol extract of serum with ferric chloride (modified from Block, Tirret and Levine). From July 2, 1973 the method used was a direct serum method using a modified Lieberman – Burchard reaction.
7) Glucose
Reference: Frings, C.S., Ratliff, C.R. and Dunn, R.T. (1969) Advances in Automated Analysis 1, 73
This reference was not checked (because of difficulty up to now in obtaining a copy of the publication) but as shown below two different procedures were employed to measure serum glucose at different times during the study (Judith R. Schmal, June 7, 1977).
From November 1971 to October 16, 1972 the method was a glucose oxidase determination (modified Gertrud Acrow) using protein free filtrates. From October 16, 1972 the method was a direct serum O-Toluidine reaction as modified from Frings, Ratlif and Dunn.
In the case of four of the above parameters (glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, blood urea nitrogen and calcium) different methodology was used during part of the study then was indicated in the submission. For one parameter (phenylalanine), there was no documentation as to the method used for one period of the study and for two other parameters (total cholesterol and glucose), two different methods were used for each of the parameters while only one was referenced in the submission.
Alkaline phosphatase was measured generally as referenced in the submission (McComb, R.B. and Borrers, G.N. (1972). Clin. CHem., 18, 97 in that the method involved measuring the production of p-nitrophenol from p-nitrophenylphosphate However starting July, 1973 there was a “re-optimization of reagent concentrations” (Judith R. Small, June 7, 1977).
The above changes in procedure could conceivably result in differences in the apparent absolute values for the concentration of the substances measured. Changes in the method of conversion of raw data to calculated values as was don in the determination of sodium and potassium by atomic absorption spectrophotometry during different periods of the study, (Judth R.Schmal, June 7, 1977) could also possibly produce differences in final values.
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In an interview with Judith Schmal on June 2, 1977, she did state in response to a question that two levels of “Serum Controls” were used in each run to check the method and instruments and that the data was not reported if the values were more than two standard deviations greater than that for the expected values.
NO evidence was obtained that any attempts were made to determine whether or not DKP cold interfere with any of the clinical laboratory tests conducted. For that matter no information was made available to us as to whether DKP itself or related compounds did appear in the blood or the urine of rats fed diet containing this compound.
Neither, as a result of interviews held or reference to available laboratory notebooks were we able to obtain information helpful in explaining the unusually low values for BUN for the control males at treatment days 189 and 364 and for all the treated male groups at treatment day 364. No raw laboratory data in reference to this could be found and may have been recorded on discarded teletype sheets referred to previously. In reference to the low BUN values, Page 29 of the submission contains the following statement: “BUN” values for the control males at treatment day 189 were unusually low and may possibly be related to a technical artefact; as a result, the group mean values for all treated males at this interval were significantly higher but, in fact, these values were in the normal range. BUN values both in control and al treated male groups at treatment day 364 were unusually low; this again reflects a possible technical artefact.”
F. A total of 21 disparities between individual clinical laboratory analysis values appearing in the submission Volume I and those values appearing in data sheets and/ or laboratory notebooks were found (Table 4). Of these, 17 were in hematology, one in clinical chemistry, and three in urinalysis. As a result of the discussion with Robert Bost, it was apparent that some of the hematology discrepancies may have resulted for Searle personnel mistaking recorded instrument readings for calculated values. In two cases no value or crossed out values appeared in the laboratory notebooks while values were found entered onto the appropriate places in the data sheets. For animal number A01HM and treatment day 546 four discrepancies (hematocrit, hemoglobin, RBC and WBC) were noted.
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G. Discrepancies Found In Statistical Analysis:
The mean and standard errors for the three dose levels and the controls for the various measurements using the values in the submission Volume I or values noted in the data sheets (where there values differed from those found in the submission) were calculated by the Division of Mathematics, FDA. Also supplied were the results of the T-TEsts comparing the controls to the treated groups. See memo to Leonard Friedman from Dennis Wilson, dated July 20, 1977 with attached Tables 1 and 2 (Exhibit #87).
A total of 49 disparities were found, which were comprised of 6 means, 23 standard errors and 20 significant differences. As stated in the memo, in all cases where there is a disparity, it appears to be due to differences in the data.
Calculations were also carried out for cholesterol data found in the data sheets but not reported in the submission. As shown in Table 5 the mean values for the median and high level treated females and the high level treated males were significantly lower than the mean values for the respective controls. To illustrate the possible significance of these changes and disparities between the values calculated by Searle and FDA for cholesterol data at the other time periods of treatment, table 5 was constructed. Very few disparities are seen between the calculated values obtained by FDA and those in the submission but a fairly consistent trend is seen for treatment related lowering of serum cholesterol, particularly at the two highest dose levels and for the female rats.
Because additional disparities were recently noted in individual hematology values after these statistical computations by FDA were completed (due to the discovery of additional laboratory notebooks), and addendum to this report regarding the statistical disparities reported here will be forthcoming.
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TABLE 5
TOTAL SERUM CHOLESTEROL (MG/DL)
TREATMENT 42 92 189 364 734 798
GROUP
-
-
-
-
-
-
THIS ENTIRE PAGE OF DATA GUTTED BEFORE RELEASE OF THIS REPORT UNDER THE FREEDOM OF INFORATION ACT!
WHAT DID THE FDA DECIDE TO KEEP FROM THE PUBLIC IT “PROTECTS”?
-
-
-
-
-
-
-
-
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GROSS PATHOLOGY
The pathologists responsible for the microscopic examination (Rudolph Stejskal and Joseph smith) did not perform the necropsies. Necropsies were performed by Tony Martinez, David Kie and Robert Spaet, with the two pathologists avilable for consultation.
The submission to FDA (Vol 1, p. 7) reported that “Rats found dead during the study were autopsied immediately whenever possible. In cases where the ncropsy could not be performed promptly, the thoracic and abdominal cavities of dead rats were opened and the entire animal was immersed in neutral buffered formalin fixative for subsequent gross examination and dissection”.
Our examination of gross pathology records showed that 98 of the 196 animals that died during the study were fixed in toto and autopsied at some later date, in some cases more than one year later.
A total of 20 animals were excluded from the study due to excessive autolysis. Of these, 17 had been fixed in toto and autopsied at a later date. Following are the twenty animals excluded from the study:
Animal No Date Found Dead Date Autopsied
C21CM 7/3/73 1/11/74
G16CM 9/21/73 1/11/74
G18CM 8/11/73 10/4/73
G26CM 4/2/73 1/11/74
J2CM 5/21/73 1/11/74
J5CM 10/30/72 11/8/72
L10CM 3/29/73 1/11/74
L15/CM 9/9/73 1/11/74
L21CM 4/13/73 1/11/74
L11LM 5/6/73 1/9/74
A14MM 5/21/73 1/9/74
G28MM 1/5/74 1/7/74
J25MM 5/24/73 5/24/73
A3HN 6/17/73 1/9/74
C15HM 1/7/74 1/7/74
(56)
Animal No Date Found Dead Date Autopsied
G13HM 7/25/73 **1/9/74
H24CF 4/29/73 1/11/74
D4HF 7/11/73 7/11/73
D16HF *4/2/73 1/8/74
F6HF 1/5/74 1/7/74
*Although the date found dead was listed as 4/12/73 on the gross pathology sheet, the “Tissue Masses & Deaths” book listed this date as 4/1/73.
**Although the date found dead was listed as 1/9/74 on the gross pathology sheet, the “Tissue Masses & Deaths” book listed this date as 7/25/73.
The gross pathology sheet for one of the above animals, F6HF, described a tissue mass measuring 5.0 X 4.5X2.5 cm. This tissue mass was first observed on 8/24/73 according to the pathology sheet (Exhibit #79), the observation records (Exhibit #70), and the palpation record in the “Tissue Masses and Deaths” book (Exhibit #65). The submission to FDA (Exhibit #8) reported no tissue mass and the animal was excluded from the study due to marked autolysis.
In addition to the above twenty animals that were excluded from the study, many other animals exhibited marked autolysis. For example, D27LF, M25CF, and H12CF are all described grossly in the submission to FDA as follows; “all organs examined grossly were markedly autolyzed”.
Records for approximately 30 animals showed substantial differences between gross observations on pathology sheets, when compared with the individual pathology summaries submitted to FDA. Following is a detailed comparison of ten of these. (Copies of all the gross pathology sheets, and the pathology summaries submitted to FDA are attached as Exhibits #78, #79, and #86).
A2CM
Submission to FDA:
Lung – Focal adhesion
Adrenal – Moderately enlarged
(57)
All other organs examined grossly were unremarkable.
Original Pathology Sheet:
Pituitary – Missing
Lung – Left, mid-portion adheres to the medial area of the rib cage by a “fibrous” type of tissue. (Submitted together with relevant portion of the rib cage).
Right, post-caval lobe has undergone consolidation. Contains grayish-yellowish nodules measuring 2 x 2 mm. (Entire lung submitted in toto)
Lymph NOdes, Pancreatic – Slightly enlarged
Adrenal – Left, moderately enlarged. Right and left, covered with tiny yellow spots measuring 1.0 x 1.0 mm.
Lymph Nodes, Mesenteric – moderately enlarged. Mass – previously described on 8/20/73 has since then regressed.
Prostate – Marked atrophy, all lobes
Seminal Vesicles – Marked atrophy, bilaterally
All other organs examined were grossly normal and unremarkable.
M15CF
Submission:
Mammary gland – subcutaneous mass located in mid-thoracic
region measuring 7 x 6 x 2.5 cm.
Urinary bladder – papillary growth in the lumen.
All other organs examined grossly were unremarkable.
Original:
Mass #1 – Previously described in the left inguinal region
on 2/9/73 has since then regressed.
(58)
Masses #2 and # – Located in the mid-axillary-cervical regions
are all on mass now measuring 7.0 x 6.0 x 2.5 cm
and may be described as irregular in shape, multi-
nodular, smooth-surfaced, non-glistening,
No Spinal Cord yellowish-purpulish in color, non-adherent to the
VL underlying muscle and containing a whitish-yellowish
firm tissue within. (Submitted in toto together with
remainder of tissue).
Heart – Left Ventricle – dilitation and walls thin.
Spleen – Slightly enlarged
Liver – Prominent lobular architecture.
Adrenal – Left, slightly enlarged. Right, unremarkable.
Ovary – Right, small cyst measuring 4.0 x 4.0 mm and
distended with a clear yellow fluid.
All other organs examined were grossly normal and unremarkable.
G10LM
Submission:
Testis – Marked atrophy, unilaterally.
Kidney – Moderate enlargement, mottled appearance, bilaterally.
Small and large intestine exhibited moderate autolysis, no sections
submitted.
All other organs examined were grossly normal and unremarkable.
Original:
Mass which was initially palpated on 2/9/72 (86 days Rx)
in the left inguinal area was actually the left testis which
ascended and went thru weakened left inguinal ring into the
subcutaneous area.
Testis – Left (ascended) appears atrophied (submitted in toto).
Kidney – MOderate, diffuse and uniform enlargement, mottled,
bilaterally (submitted in toto).
Small and large intestines are moderately autolyzed (no sections
submitted).
Thyroid – Moderately enlarged, bilaterally. A 2 mm in dia., dis-
crete, sl raised, moderately firm yellowish-grey lesion
is located in the posterior tip, bilaterally. (Thyroid
submitted in toto wrapped in a lens paper).
All other organs examined were grossly normal and unremarkable.
(59)
L11LM
Submission:
Kidney – Mottled appearance
Testes – Marked atrophy, bilaterally
Prostate – Marked atrophy
All other organs examined grossly exhibited marked autolysis.
Original:
Adrenal – Pale yellow, bilaterally
Kidney – Pale yellow, bilaterally, rough-surfaced, bilaterally,
moderately autolyzed, bilaterally, tiny spaces in the
cortex region measuring about 1 mm in diameter,
bilaterally.
Testes – Marked atrophy, bilaterally, marked autolysis,
bilaterally.
Prostate – Marked atrophy, all lobes
Seminal Vesicles – Marked atrophy, bilaterally
Spleen – Marked autolysis
Pancreas – Marked autolysis
Stomach – Marked autolysis. A glandular portion – numerous, tiny,
pitted ulcerations measuring 1 -4 mm in diameter.
Lymph Nodes, Mesenteric – Marked autolysis
Heart – Wall of left ventricle thin
Brain – Marked autolysis
Pituitary – Marked autolysis
Liver – Marked autolysis
All other organs examined were grossly normal and unremarkable.
M17LF
Submission:
Pituitary – Marked enlargement.
Adrenal – Markedly enlarged and hyperemic, bilaterally.
Mammary Gland – Mass 1, located subcutaneously in left axillary
region, measuring 3 X 3 X 2.5 cm; mass 2, located
subcutaneously adjacent to mass 1, measuring 3 X 2
X 1 cm; mass 3, located subcutaneously in the right
axillary region, measuring 2.5 X 2 X 1 cm; mass 4,
(60)
located subcutaneously in the left inguinal region,
measuring 3 X 1 X 1 cm; mass 5, located subcutane-
ously in the right inguinal region, measuring 2 X
1.5 X 1 cm.
All other organs examined grossly were unremarkable.
Original
Pit – appears markedly hyperemic
Adrenal – Exhibits numerous minute greyish spots on the serosal
surface bilaterally. It appears markedly enlarged.
Mass (1) – A 3 X 3 X 2.5 cm. spheroidal, multinodular, yellowish
white, slightly firm mass located subcutaneously in
the left axillary area. Mass non-adherent to the
surrounding muscles or tissue (submitted in toto).
Mass (2) – A 2.5 X 2 X 1 cm spheroidal, smooth, yellowish white
firm mass located subcutaneously and adjacent to the
above described mass (submitted in toto) mass non-
adherent to the surrounding muscles or tissues.
Mass (3) – A 2.3 X 2 X 1 cm. irregularly shaped, multinodular,
yellowish white, firm mass located subcutaneously on
the rt. axillary area. Mass non-adherent to the
surrounding muscles or tissues (submitted in toto).
Mass (4) – A 3 X 1 X 1 cm. elongated, multinodular, yellowish
white, firm mass located subcutaneously on the left
inguinal area. Mass non-adherent to the surrounding
muscles or tissues (submitted in toto).
Mass (5) – A 2 X 1.5 X 1 cm. flat, multinodular, yellowish white, firm mass located subcutaneously on the rt. inguinal area. Mass non-adherent to the surrounding muscles or tissues (submitted in toto).
All other organs examined were grossly normal and unremarkable.
C1MM
Submission
Kidney Marked enlargement with yellowish discoloration.
Testis Marked atrophy, bilaterally.
(61)
Tissue mass located subcutaneously in the right inguinal area measuring 2.5 X 1 cm.
All other organs examined grossly were unremarkable.
Original:
Mass – Previously described on 12/9/72 and located subcutaneously in the right inguinal area now measures 2.5 X 2.0 X 1.0 cm and may be described as smooth-surfaced, purplish-yellowish in color, non-glistening, firm, multi-nodular, non-adherent to the underlying muscles and containing a firm yellowish-whitish tissue. (Submitted in toto together with a portion of the skin and underlying muscle with remainder of tissue).
Heart – Left ventricle has undergone a moderate amount of dilitation. Wall, left ventricle is thin.
Liver – Prominent lobular architecture.
Lung – Right, post-caval lobe-consolidation.
Kidney – Markedly enlarged, yellow and rough-surfaced, bilaterally. Dilitation of the pelvis.
Adrenal – Covered with tiny yellow spots measuring 1 mm in diameter, bilaterally.
Testes – Marked atrophy, bilaterally.
All other organs examined were grossly normal and unremarkable.
Tiss. Trimming – Nodules discovered immediately posterior (2.0 cm) to the pyloric portion of the stomach within the adipose tissue. Nodules may be described as firm, yellowish brownish in color. Non-glistening measuring 1.2 X 1.0 mm to 4.0 X 4.0 mm.
E27MM
Submission:
Lung – Moderate diffuse hyperemia.
Eye – Opaque cornea, bilaterally.
All other organs examined grossly were unremarkable
Original:
Lungs – All lobes exhibit moderate diffuse and uniform hyperemia.
(62)
Kidney – Moderate autolysis.
Eye – The entire cornea is opaque, bilaterally.
Spleen – Moderately autolyzed.
Stomach – Numerous 1-2 mm. hemorrhagic ulcerations are located on the glandular mucosa. Entire small and large intestines are moderately autolyzed.
Brain & Pituitary – Moderately autolyzed.
All other organs examined were grossly normal and unremarkable.
A1HM
Submission:
All organs examined were grossly unremarkable.
Original:
Testes – Markedly atrophy, bilaterally
Lung, Rt – Middle lobe exhibits a 1 X 1 cm consolidation on the posterior portion.
Liver – All lobes appear olive green otherwise unremarkable.
All other organs examined were grossly normal and unremarkable.
L27HM
Submission:
Testes – Right, slightly enlarged; left, mild atrophy.
All other organs examined grossly were unremarkable.
Original:
Testes – lt./appears markedly atrophy rt./appears to be distended with yellowish white substance
Seminal V- Appears markedly atrophy bilaterally.
Intestinal – Large, markedly distended with “gas.”
All other organs examined were grossly normal and unremarkable.
P.M. Testes – Also, small black areas are noted within along with the yellowish areas. Black areas measuring 1.0 X 1.0 to 4.0 X 4.0 mm in diameter.
(63)
J30HM
Submission:
Lung – Moderate consolidation of all right lobes.
Testis – Moderate atrophy
All other organs examined grossly were unremarkable.
Original:
Pituitary – Markedly enlarged; slightly hyperemic.
Heart – Left Ventricle has undergone dilitation walls thin.
Lung – Right, anterior, medial and post-caval lobe have undergone consolidation.
Testes – Marked atrophy, bilaterally.
Seminal Vesicle – Marked atrophy, bilaterally.
All other organs examined were grossly normal and unremarkable.
Dr. Stejskal told us that the other pathologist (Dr. Joseph Smith) who made microscopic evaluations of the slides, came from a hospital background (human pathology) and therefore his descriptions and terminology were a little bit different than one would expect from a veterinary pathologist.
MICROSCOPIC PATHOLOGY
We have assisted in our review of the Microscopic Pathology of Study E-77/78 by Charles H. Frith, D.V.M., Ph.D, Director, Pathology Services, NCTR. Dr. Frith arrived on 6/22/77 and spent 3 days with the FDA team. He examined slides for a representative number of animals, the selection of which was made jointly by Dr. Frith and the other members of the FDA team. A Searle Pathologist was not present during Dr. Frith’s review of the slides. However, Dr. Frith did meet with Dr. Rudolf Stejskal, SEarle Pathologist, at the conclusion of this review and discussed some of his findings with him.
The first phase of Dr. Frith’s review consisted of the examination of the tissues of 25 of the surviving control females and 11 of the non-surviving control females for a total of 36 animals. All of the slides were examined for each animal and the results were compared to the microscopic reports provided by Searle Laboratories. The inconsistencies (findings that differed from those reported by Searle) are listed below:
(64)
In most cases the inconsistencies represent findings that were not diagnosed or reported by Searle. Copies of Searle’s microscopic pathology reports for each of the animals listed below are attached as exhibit #60.
Female Rat No. F13CF (Path. No. 95617)
Small Intestine – Diverticulum with mucosal necrosis and cellular inflammatory infiltrate.
Female Rat No. F15CF (Path No. 95618)
Pancreas – Focal hyperplasia
Female Rat No. F16CF (Path No. 95619)
Heart – Focal Fibrosis.
Kidney – Mild chronic nephritis.
Female Rat No. H10CF (Path 95624)
Ovary – Neoplasm – probably granulosa cell tumor.
Female Rat No. H19CF (Path. No. 95626)
Kidney – Focal calcification.
Ovary – Neoplasm – probably granulosa cell tumor.
Female Rat No. H30CF (Path. No. 95628)
Kidney – Focal calcification.
Female Rat – No. K25CF (Path No. 95630)
Kidney – Focal calcification.
Female Rat No. K29CF (Path No. 95631)
Heart – Focal fibrosis
Kidney – Focal calcification
Female Rat No. M4CF (Path No. 95632)
Liver – Focal hyperplasia
Female Rat No. M10CF (Path No. 95634)
Kidney – Focal calcification.
Pituitary – Adenoma
Ovary – Fibrosis and Pigmentation.
(65)
Female Rat No. M15CF (Path No. 95635)
Pituitary – Adenoma.
Ovary – Cyst.
Female Rat No. B30CF (Path No. 95801)
Kidney – Focal calcification.
Female Rat D29CF (Path No. 95803)
Urinary Bladder (1) Chronic diffuse inflammation. (2) Diffuse mild hyperplasia.
The second phase of the review consisted of the microscopic examination of all tissues from the high dose females – a total of 36 animals. The inconsistencies are listed below:
Female Rat No. B14HF (Path. No. 95657)
Eye was reported as not examined but eye was present and normal.
Female Rat No. F25HF (Path. No. 95823)
Urinary Bladder – Mild diffuse hyperplasia.
Female Rat No. H7HF (Path No. 95623)
Ovary – Neoplasm – probably granulosa cell tumor.
Female Rat No. H9HF (Path No. 95665)
Heart – Focal fibrosis.
Urinary Bladder – Mild focal hyperplasia.
Female Rat No. H15HF (Path No. 95665)
Lymph Node – The diagnosis of lymphoma, benign, was present on the Searle microscopic report. According to Dr. Frith, lymphoma is generally not considered to be benign and he would diagnose lympphosarcoma.
Female Rat NO. H18HF (Path No. 95667)
Pituitary – Adenoma.
Brain – Mild bilateral hydrocephalus.
Female Rat No. K18HF (Path No. 95824)
Pituitary – Adenoma
Female Rat No. K24HF (Path. No. 95671)
Mass noted grossly – nothing consistent with mass reported microscopically.
(66)
Female Rat No. – M2HF (Path. No. 95672)
Uterus – Chronic mild endometritis.
Female Rat No. M30HF (Path. No. 95343)
Kidney – Focal calcification. Uterus – Chronic mild endometritis.
Female Rat No. M30HF (Path. No. 95675)
Pancreas – Focal hyperplasia.
The third phase of this review consisted of microscopic verification of all masses reported grossly at necropsy from all female animals not examined in phases 1 and 2 and included a total of 73 animals. The inconsistencies are listed below:
Female Rat No. D10Lf (Path No. 92521)
Subcutaneous mass was diagnosed as an angiofibroma on Searle report. The lesion is more consistent with an angiosarcoma.
Female Rat No. K9MF (Path. No. 95707)
Uterus – Polyp.
Female Rat No. M1LF (Path. No. 95844)
Tissue mass seen grossly was reported as missing and not available for microscopic examination. The tissue was present and was a mammary fibroadenoma.
In summary, Dr. Frith reviewed:
1) All 36 high dose females (all slides) including 3 that had been excluded from the study due to autolysis.
2) 36 (one-half) of the control females (all slides) including 1 animal that had been excluded from the study due to autolysis.
3) Remaining 73 female animals with grossly observed masses. (sufficient slides were reviewed to substantiate the masses)
4) 5 additional animals selected by the investigators (A1HM, A9HM, A29HM, C2CM, C24HM).
(67)
The slides reviewed in the first two categories above constituted 20% of the total animals on the study. Dr. Frith reviewed these slides blindly and then compared his findings with the Searle microscopic reports. According to Dr. Frith, his findings were in agreement with those of SEarle, for the most part. In his opinion, some of the lesions that he reported as inconsistencies were small, and might be considered insignificant by some pathologists. Dr. Frith did feel, however, that the ovarian neoplasms (animals H10CF, H19CF, and H7HP, and chronic cystitis and diffuse hyperplasia (animal D29CF) should have been reported.
Dr. Frith also considered two other discrepancies to be significant. They were:
1) The reporting of a mass (by Searle) as missing which was actually present (MlLF).
2) The finding of a polyp of the uterus which was not diagnosed by Searle (K9MF)
The second of the above two discrepancies assumes even more significance in view of the following:
The Histopathologic Summary table (table 11) in Volume I of the submission to FDA lists the following incidence of Uterine Polyps on page 87:
Incidence of Uterine Polyps
Controls Low Medium High
1 of 69 1 of 34 4 of 34 6 of 33
(1%) (3%) (12%) (18%)
The finding of one additional uterine polyp by Dr. Frith (in animal K9MF) increases the incidence in the mid dose to 5 of 34 (15%).
On page 82 of Volume I of the submission to FDA, is the statement: “other sporadic findings is included endometrial hyperplasia, polyp, cyst, congestion and squamous metaplasia.” The term “sporadic findings” was used to characterize the incidence of uterine polyps, in spite of the fact that Searle had done a statistical analysis of these findings.
(68)
When this study was reviewed by the Bureau of Foods in l975, the dose-related incidence of uterine polyps was noted. The appropriate slides were requested by FDA at that time and were reviewed by three groups of pathologists: 1.) The Division of Pathology, Bureau of Foods, 2.) Armed Forces Institute of Pathology, 3.) Massachusetts Institute of Technology. Copies of the reports submitted by the 3 groups and related correspondence were obtained and are attached as exhibits #43-45.
Dr. Rudolph Stejskal was responsible for the microscopic findings and accuracy on these findings in the submission to FDA. Only Dr. Stejskal’s name appears on the submission. However, a Dr. Joseph H. Smith, M.D. also read slides for this study and his initials appear on some of the microscopic examination sheets. Dr. Frith questioned some of the terminology used in describing tissues. Dr. Stejskal stated that Dr. Smith had come directly to SEarle from a hospital situation. Due to his human pathology background, his description of animal tissues was somewhat different than that used by veterinary pathologists.
Dr. Stejskal joined SEarle in July of l973, therefore, he had no input into the pathology protocol, since E-77/78 was initiated in November of l971.
No microscopic worksheets or other “raw data” relating to microscopic pathology could be found for study E-77/78. We were told by Searle personnel that the original microscopic findings were dictated by the pathologists (Stejskal & Smith) onto belts, and then typed onto sheets which were placed in a binder. The belts were then discarded and apparently the bound microscopic pathology sheets were either discarded or lost, after the study report was written. Therefore our verification of the microscopic findings submitted to FDA was limited to a complete inventory of the slides and tissue blocks and microscopic examination of a representative number of slides by Dr. Frith.
Our inventory of the slides and tissue blocks for each animal included a complete list on the tissues sectioned, the number of slides made from each tissue, and a complete count of the total number of slides and blocks for each animal. We also checked the identification numbers on every slide and tissue block. We examined a total of 7,872 slides and 7,360 tissue blocks. The average number of organs submitted for tissue processing was…
(69)
…20 per animal. No errors in slide identification were noted, although in many cases the number of organs submitted for sectioning was less than specified in the protocol. A detailed discussion of this can be found under the heading PROTOCOL.
In addition to the discrepancies noted by Dr. Frith, some other errors were noted in the submission to FDA. A mammary tumor found in rat F27CF was described as a papillary cystadenoma on the individual pathology sheet (page 105, Volume II of the submission to FDA) and as an adenocarcinoma on the summary table 12, page 96, Volume I of the submission to FDA.
Page 92, Volume I of the submission to FDA (a summary table) reports that animal J23CM was found dead after 754 days on study, while the individual pathology sheet for this animal (page 56, Volume II of the submission to FDA) reported that the animal was found dead after 620 days on study. The correct figure is 620 days, since J23CM was placed on the study on 11/17/72 and was found dead on 7/29 /73.
In several instances the histopathology technician made notes at the bottom of the gross pathology sheet to indicate that certain organs were not present in the bottle of fixative. (and were therefore not available for sectioning). Yet in three of these instances (animals A4CM, K23CF, and J3CM) a diagnosis appears in the submission to FDA.
CHARTS, DIAGRAMS AND TABLES
It was necessary to construct a number of charts, diagrams and tables to facilitate our review of the data. For example we constructed a chart, by housing group, showing the identification and complete pathology history of each of the 360 animals. We also rearranged this chart into dosage groups, a copy of which is attached as exhibit #35.
To compare survival data it was necessary to construct a survival table. This also involved devising a calendar to show days and weeks on study for each housing group, taking into account the starting dates for each group. This also included tables showing the numbers of days and weeks animals were on study and a table comparing the survival data from various sources.
We constructed a chart showing diet calculations (gm./kg) and total amounts of DKP used (gm./batch). This is attached as exhibit #30.
(70)
Three tables were constructed which summarize the FDA statistical analysis of body/feeder weight data. They are attached as exhibits #39-41.
All of the charts, diagrams and tables that we constructed are attached to the report as exhibits and are referred to in various sections of the report.
EXHIBITS
#1. G.D. Searle & Company Annual Report for l976.
#2. Organizational Chart of Pharmaceutical/Consumer Products Group.
#3. Organizational Chart of World Wide Pharmaceutical R&D Group.
#4. Organizational Chart of Preclinical R&D Group.
#5. Organizational Chart of Product Safety Assessment Group.
#6. Copies of Computer-Generated Randomization Tables used by Searle to assign the Dose & Housing Groups.
#7. Diagram showing Typical Housing Group of 30 animals, containing a random distribution of control and treated animals.
#8. Diagram showing arrangement of food cups on cart, used in feeding the animals.
#9. Copy of “Glossary of Terms for Aspartame and its Diketopiperazine” and “Analytical Data and Specifications of Food Grade Aspartame.”
#10. Copy of shipping labels for rats received from
#11. Copy of protocol with amendments for Study P.T. 988S73 (E-77/78).
#12. Copies of CV’s for principal persons involved in study E-77/78.
#13. Copies of Batch Records for the manufacture of DKP, lots 1R through 5R.
#14. Copies of pages from SEarle chemist Jack Drogt’s notebook, concerning the manufacture of DKP.
#15. Copies of Analytical Reports for DKP, lots 1R through 7R.
#16. Copy of Searle memorandum dated 12/4/69, concerning DKP Specifications.
#17. Copy of DKP Specification Sheet (not dated) entitled “Tenative Specifications for SC-19192″.
(71)
#18. Copy of DKP Specification Sheet entitled “Specifications for SC-19192, Specification #C40606C”.
#19. Copies of pages 75-84 & 285 of lab notebook #AR-39, concerning assay of DKP, lots 1R, 2R & 3R.
#20. Copies of pages 60-63 of lab notebook VSH-I, and page 269 of lab notebook book #AR-23, pertaining to analysis of DKP lot 4R.
#21. Copies of pages 250, 251 and 257 of lab notebook #AR-57, and pages 44-49 of lab notebook #AR-68, pertaining to analysis of DKP lot 5R.
#22. Copies of pages 83-86 of lab notebook #AR-77, concerning analysis of DKP lot 6R.
#23. Copy of page 31 of lab notebook #AR-93, concerning analysis of DKP lot 7R.
#24. Copy of protocol for DKP stability study, dated 1/13/72.
#25. Copies of pages 51-56 of laboratory notebook #AR-49, assigned to
C. Seul. These pages describe a preliminary TLC Test for recovery of DKP from the diet mixture.
#26. Copies of pages 53-59, 67-72, 88-89, 106-107, 144-145, 156-157, and 284-285 of laboratory notebook #AR-51, assigned to Barbara Bickford. These pages refer to the assay procedure and methods for the DKP Stability Study.
#27. Copies of Analytical Reports for DKP Stability Study.
#28. Copies of DKP Compound Inventory Cards.
#29. Two photographs showing a non-homogeneous sample of DKP diet mixture.
#30. Chart showing diet calculations (gm.kg.) and total amounts of DKP used (gm./batch).
#31. Two memos dated 7/14/77 from Thomas F.X. Collins concerning interview with Ray Schroeder.
#32. Memo dated 7/19/77 from Thomas F. X. Collins describing the 7/18/77 interview with Ray Schroeder.
#33. Memo of Telephone Conversation between Jerome Bressler and Attorney John H. Bickley Jr., dated July 25, l977.
#34. Copies of records concerning calculation of diet concentrations, food concentrations prediction records, dates of bath mixing, and calculation of mean food intake values.
#35. Charts organized by dose group, showing the identification and pathology history of each of the 360 animals on study.
#36. Memo dated April 5, l976, from Dr. John H. Rust to Dr. R. McConnell.
#37. Searle memo dated September 30, l974, by Dr. McConnell.
(72)
#38. Memo dated August 29, l974 from Dr. G. L. Schoenhard to Dr. K.S. Rao.
#39. Table 1 – Summary of Average Body Weights and Weight gain (%change/week) from the FDA Statistical Analysis.
#40. Table 2 – Summary of food intake (g/day and g/kg./day and dosage (mg./kg./day) from the FDA STatistical Analysis
#41. Table 3 – Summary of Food Efficiency (g. gained/100g. actual food consumed) calculated in the FDA STatistical Analysis.
#42. Computer printout of FDA Statistical Analysis of food intake and body weight data.
#43. Pathology report from Division of Pathology, Bureau of Foods, concerning uterine polyps, along with correspondence, and memo from Janet Springer.
#44. Pathology report from Armed Forces Institute of Pathology, concerning uterine polyps.
#45. Pathology report from Massachusetts Institute of Technology, concerning uterine polyps.
#46. Written account of interviews with Dr. Jean Taylor on 6/2/77, 6/3/77, and 6/7/77.
#47. Written account of interview with Judy Beauchamp on 6/2/77.
#48. Written account of interview with Barbara Bickford on 6/1/77.
#49. Written account of interview with Clifford J. Seul on 6/2/77.
#50. Written account of interview with Bartolome R. Tangonan on 6/1/77.
#51. Written account of interviews with Tony Martinez on 5/19/77,6/3/77, 7/7/77, and 8/2/77.
#52. Written account of interview with Ted Reichert on 5/24/77.
#53. Written account of interview with Barbara Bickford and Clif Seul on 6/2/77.
#54. Written account of interview with Judith Schmal on 6/2/77 and 6/7/77.
#55. List of animals bled at 104 and 114 weeks.
#56. Written account of interview with Alan Mitchell on 7/20/77.
#57. Written account of interview with Raymond G. Schroeder on 7/18/77.
#58. Injection records showing administration of penicillin, dates of administration, rat numbers, and units injected.
#59. Methodology for “Phenistix” determination of phenylketones in urine.
(73)
#60. Dr. Frith’s report of examination of slides for DKP study (E-77/78).
#61. Key for animal identification card numbers used on the body/feeder weight teletype sheets.
#62. Chart correlating animal cage numbers with pathology numbers, arranged by dose group.
#63. Chronological list of pathology numbers and corresponding animal cage numbers.
#64. Organizational charts showing responsibility during the time that study E-77/78 was conducted.
#65. Volume entitled “tissue masses & deaths”. Chronological list of all animals that died, during study, and dates that masses were first observed.
#66. Charts of days on study for each animal.
#67. Calendar for duration of study showing starting dates, days and weeks for each group.
#68. Survival table.
#69. Charts of Housing/Dosage Groups.
#70. “Observations for Drug Effects” records for housing groups A through F.
#71. “Observations for Drug Effects” records for housing groups G through M.
#72. Ophthalmoscopic records and copies of pathology sheets that have ophthalmoscopic findings.
#73. Life table analysis and statistics on body weight and food consumption data by Dennis Wilson, Div. of Mathematics, Bureau of Foods.
#74. Evaluation of feeding study on DKP, a conversion product of Aspartame, by Janet Springer/Ann Ducca, FDA Division on Mathematics.
#75. Volume A – teletype sheets for body and feeder weight data, housing groups.
Volume B – ”
Volume C – ”
#76. Copy of Searle Computer Program.
#77. Volume of protocols, organ weights, dosage, hematology, urinalysis, blood chemistry, and protein electrophoresis.
#78. Complete gross pathology sheets, males.
#79. Complete gross pathology sheets, females.
#80. Key to slide tissue identification numbers and abbreviations.
#81. Key to stain abbreviations.
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#82. Copies of submission appendix tables relating to hematology, clinical chemistry, urinalysis, and electrophoresis, along with check marks showing errors, and attached copies of raw data sheets documenting the errors.
#83. Copies of submission appendix tables for organ weights, with errors indicated, and copies of pathology sheets documenting the errors.
#84. Data sheets showing the phenylketones test erroneously labeled “phenylalanine”.
Signed by:
John S. Arnold, Investigator
David M. Eerspamer, Investigator
Dr. Jean Taylor, Toxicologist
Dr. Leonard Friedman, Biochemist
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Addendum
Exhibit #85 – Copy of Volume 1 of the submission to FDA.
Exhibit #86 – Copy of Volume 2 of the submission to FDA, consisting of the individual pathology summaries, both gross and microscopic.
Exhibit #87 – Statistical analysis of Blood and Clinical Chemistry Data by Dennis Wilson, Division of Mathematics, HFF-110.
Exhibit #88 – Copies of pages from Histology accession book #770C, and Histology inventory sheets.
Exhibit #89 – Documents from Searle’s Math-Stat. Dept. concerning statistical analysis of study 988S73.
Signed
Jerome Bressler
Team Leader
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Wanna Unload?
Name: Jerome Bressler
SUPVRY CONSUMER SAF FDA
Chicago Office
Mail stop HFR-MW140
Phone: 312-353-5863
Fax: 312-353-0947
Internet e-mail Dr. Jerome Bressler JBRESSLE@ORA.FDA.GOV
Or, communicate with HIS boss:
Name: Michael A. Friedman
DEPUTY COMMISSIONER FDA
Building PKLN Room 1471 Mail stop HF-28
5600 Fishers Ln.
Rockville MD 20857
Phone 301-827-3310
Fax 301-443-3100
Internet E-mail Dr. Michael Friedman MFRIEDMAN@OC.FDA.GOV
Or, communicate with HIS boss:
DHHS Donna Shalala hhsmail@os.dhhs.gov (Boss over FDA)
Or, communicate with HER boss:
The President president@whitehouse.gov
1600 Pennsylvania Ave.
Washington, D.C. 20500
Phone: (202) 456-1414
Fax: (202) 456-2883
Or… how about somebody in congress?
Congress http://www.dorway.com/congress.html
Last… but it should NOT be least…
Department Of Justice (DoJ) Janet Reno (DoJ) web@usdoj.gov
A few words about this document
This document appears on the Internet courtesy of whomever excercized the Freedom of Information Act request (and paid the money to spring it), and the combined typing efforts of Betty Martini and myself. The reader will note, that for whatever reasons a number of tables were blanked out before releasing this document. To the best of my knowledge, errors, poor grammar and other errors were kept intact.
“Aspartame is a Pandora’s box of chameleon-like toxic chemicals, that causes cumulative physical and mental damage that hammer the unsuspecting victim into the ground… micro-dose by micro-dose!”
“Aspartam är en Pandoras ask med kameleont-liknande giftiga kemikalier, som orsakar kumulativa fysiska och mentala skador som slår det intet ont anande offret till marken… mikro-dos för mikro-dos!
Translation to Swedish courtesy of: Cyrilla & Tomas Högberg cyrilla@algonet.se
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