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The "Bressler Report"
Note: This is the text of an FDA report on Searle
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
SUMMARY OF FINDINGS
Authentication of this study was performed primarily by com-
paring available raw data with the submission to FDA. This
was a problem, at times, due to the lack of some data and
difficulty in locating other material. The majority of
material relating to Aspartame was already under FDA seal
at Searle. However, during this investigation we discovered
various documents and notebooks that were not.
In some cases original data could be recorded in several areas,
making it difficult, and sometimes impossible to determine
which was actually the original. This was a particular problem
in dealing with dates of deaths, as some conflicted on the
"source" documents. Many of the responsible individuals invol-
ved with the study, including stability testing of DKP, are no
longer employed by Searle. Dr. K.S. Rao, Study Monitor,
the only individual who could have possible answered some quest-
tions, had left Searle. He was contacted, but permission for
an interview was refused by his attorney. Due to the absence
of various individuals it was not always possible to a accurately
determine methods used in some analyses and operations carried
out in conducting this study. In a number of areas, including
chemistry, statistics, diet preparation and feeding, it was
necessary to use assumptions, or information supplied by current
employees who were not involved with the study.
At the beginning of this investigation , Mr. James R. Phelps,
Vice-President and General Counsel for G.D. Searle & Co., advised
us that an attorney and scientific coordinator would have to be
present at all times to protect their interest in the data.
This did not present any insurmountable problems, but on several
occasions an attorney would question our request for data,
stating that it was not relevant for authentication. At no time
did we make any statement to the effect that our goal was to authen-
ticate the study. Two memos were discovered dealing with reaction
of animals to the diet. This was a significant factor in the
study. Permission to copy them was initially refused, but
finally granted after Searle was contacted by FDA General Coun-
sel. We were not allowed to make xerox copies of any documents
for about two and one-half weeks, due to Searle's concern over
confidentiality. This was eventually reconciled between Searle
and FDA General Counsel.
(1)
The major discrepancies concerning Study PD 988S73, SC-19192:
115 Week Oral Tumorigenicity Study in the Rat, are as follows:
A. Design and Conduct of Study
1) Control and treated animals were randomly distributed
on the same rack. (See diagram of housing group
attached as exhibit 7.)
2) No ear clips or other methods of uniquely identifying
each animal were used. Identification consisted of two
types of cards attached to the front of each cage.
3) Compound inventory cards were deficient in that only one of
18 such cards stated the purpose (study 988S73) for with-
drawing the compound from inventory. Three of the cards
did not include the date withdrawn, amount withdrawn, or
signature of requestor. Therefore it was impossible to
Reconcile the amount withdrawn and the amount used.
(See exhibit #28.)
4) Food jars were not individually identified, yet all the
filled jars for a given housing group (control, low, mid,
and high dose) were placed on a mobile cart, which was
wheeled to the housing rack. The position of the jar
(in rows) on the cart was the only means of identifying
the proper dose level. The arrangement of the food cups
on the cart is shown in exhibit #8.
5) A total of 79 "observations for drug effects" records were
not signed or initialed.
6) Observation records indicated that animal A23LM was alive at
week 88, dead from week 92 through week 104, alive at week
108, and dead at week 112.
7) Records indicated that at the scheduled 104 week bleeding,
animal E2CM was substituted for animal A11CM. Records also indica-
ted that animal A11CM was alive on this date and therefore
should have been bled as scheduled.
8) Records indicated that penicillin was administered to four
rats beginning on May 16, 1973, and continuing daily through
May 28, 1973. This third occurrence of infections disease
and penicillin administration was not reported in the sub-
mission to FDA.
(2)
9) 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.
10) Ophthalmoscopic examination records were present for animals
H26MF and J29CM, yet the findings were not reported in the
submission to FDA. Two other discrepancies of this type
were noted.
11) Records indicate that a tissue mass measuring 1.5 x 1.0 cm
was excised from animal B3HF on 2/12/72, and that a "skin
incision over mass" was performed on animals C22LM and G25LM
on Feb. 10, 1972.
B. Stability and Homogeneity of DKP in Diet Mixtures
1) There were no batch records to show the quantities of DKP
and basal diet weighted, type of mixer used, mixing time,
dates, or names of individuals performing the weighing and
blending operations.
2) There was no evidence that any tests had been done to deter-
mine the blending characteristics of the mixer, or to vali-
date the mixing time.
3) No homogeneity tests were performed on any batches of diet
used in the study, and to stability study assay reports
(A7738 and A7739) indicated that samples were not homogene-
ous. (See exhibit #29.)
4) A stability study was conducted with DKP
in 1972. However, the 115 week rat stud employed
Basal Diet from week 2 to its conclusion, and to stability
studies had been conducted with Basal Diet.
5) Methods of assay for DKP in the diet were deficient in that:
The titration method was discontinued after 1 week of the
stability study. Some of the TLC photographs showed no DKP
reference standards and photographs also showed that there
was something in the basal diet itself producing a spot
on the TLC plate which had an Rf value corresponding to
DKP. Only one solvent system was used for development of
the TLC Plates. Some of the chromatograms showed poor separa-
tion.
(3)
6) No reserve samples of any of the lots of DKP used in this study
were retained by Searle.
7) Three different sets of specifications for DKP were found, and
Searle could not determine with any degree of certainty which
of the three were applicable to the 7 lots of DKP used in the
study.
8) The analytical records for DKP lots IR through 5R refer to
reference standard IR #3701. None of the three sets of DKP
specifications lists reference #3701. No data was made avail-
able as to dates, methods of preparation and authentication
of DKP reference standards.
9) Analytical records A-9129 for DKP lot 5R showed an assay of
1000%. Examination of laboratory notebooks showed
that eleven (11) samples had been analyzed from this lot, and
the analytical record only reflected an average of the last
three of theses. The other assays (not reported) ranged from
87.93% to 114.83%.
C. Dosage, Body Weight and Food Consumption
1) Examination of the raw data sheets revealed the following
discrepancies:
a. Empty feed cup weights were missing for the D hous-
ing group at the 12th week, in the raw data sheets. (See
exhibit #75.)
b. In several instances, the dietary concentration shown
on the weight sheets did not agree with the concentra-
tion listed for the same level in the other housing
groups. (For example; C group Males, mid & high levels
for week 13,; A group Males, high levels for week 99.)
2) Comparison of the Searle submission and the independent FDA
analysis of the raw body weight and food consumption data
revealed the following discrepancies:
a. We found a total of 15 differences of 1 gram or more in
the average body weight and of 0.1 percentage points
or more in weight gain. (See table 1.)
(4)
b. We found approximately 82 discrepancies of one gram or
or more in the food intake when expressed in grams/day.
(See table 2.)
c. We found approximately 40 errors of 5 or more grams in
food intake when expressed in grams/kg./day. (See
table 2.)
d. Most of our dosage calculations differed from Searle's
dosage calculations by 10 or more mg., when the dosage
is expressed as mg/kg/day. (See table 2.)
D. Gross and Microscopic Pathology
1) 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.
2) 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.
3) Records indicated that animal F6HF, a high dose female, was
found dead at 787 days of treatment and the gross pathology
sheet reported a tissue mass measuring 5.0 X 4.5 X 2.5 cm.
The submission to FDA reported no tissue mass and the
animal was excluded from the study due to marked autolysis.
4) Records for approximately 30 animals showed substantial
differences between gross observations on pathology sheets,
when compared with the gross observations on pathology sheets
submitted to FDA. A detailed description of 10 of these is
included in the report. Copies of all the gross pathology
sheets, and the pathology summaries submitted to FDA are
attached as exhibits.
5) Dr. Charles H. Frith, D.V.M., Ph.D., Directory, Pathology
Services, NCTR, examined slides for a total of 150 animals,
or about 42 percent of the animals on study. He noted
the following discrepancies:
a. The reporting of a mass (by Searle) as missing which was
actually present (animal M1LF.)
(5)
b. The finding of a polyp of the uterus which was not
diagnosed by Searle (animal K9MF). The finding of
this additional uterine polyp by Dr. Frith increases
the incidence in the midi dose to 5 of 34. (15 percent.)
c. The finding of ovarian neoplasms in animals H19CF, H19C,
and H7HF, and the finding of diffuse hyperplasia in animal
D29CF, which were not diagnosed by Searle.
d. The finding of additional inconsistencies in 21 animals.
6) No microscopic worksheets or other "raw data" relating to
microscopic pathology could be found for this study.
7) A mammary tumor found in animal F27CF was described as a
papillary cystadenoma on the pathology summary sheet, (page
105, Vol. II of the submission) and as an adenocarcinoma on
summary table 12 (p. 95, Vol. I of the submission).
8) 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.
E. Organ Weights
1) 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,
pgs. 222-226) with the original data on the gross pathology
sheets. A total of eleven (11) errors were noted in transcrib-
ing the raw data from the pathology sheets to the tables in the
submission to FDA.
F. Survival
1. We were unable to determine the exact method used by Searle in
constructing the survival table in the submission to FDA.
We constructed a survival table using the body/feeder weight
Teletype sheets. A Life Table Analysis was constructed from
our survival table by Dennis Wilson, FDA Department of Mathe-
matics. The female control population differed from the high
level population (p 0.05) and the mail control population
differed from the mid and high level population (p 0.05). In
all cases the differences are due to higher mortality in
the controls.
(6)
G. Clinical Laboratory Procedures
1. Laboratory records of one sort or another for all assays report-
ted in the submission were obtained. In some cases data sheets
were noted with results of assays carried out at treatment days
not indicated in the protocol or protocol amendment. For example,
serum cholesterol determinations were done at days 796 and 798
(terminal bleeding) but not included in the submission to FDA.
Because the submission to FDA (Vol. 1 p. 286) reported a signi-
ficant decrease in serum cholesterol that was more perceptible
towards the end of the study, and may have been related to
compound administration, the omitted data is of some importance.
2. No data was seen for two assays (serum insulin and serum ornit-
hine carbamyl transferase) which were called for in an amend-
mend to the protocol.
3. Original data was not always available for authentication of
results or examination of procedures for conversion of raw
data into the calculated values submitted to FDA.
4. Data pages for clinical chemistry and urinalysis were initialled
by a technician who transcribed data but apparently was not
directly involved in the assays indicated. He stated in an
interview that Dr. Rao told him to initial the data sheets.
5. The methodology as referenced in the submission to FDA is incom-
plete and not always an accurate reflection of the methodology
actually used in the study. The fact that more than one method
was sometimes used for a particular assay during different times
of the study was not indicated in the submission to FDA.
6. 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.
7. A total of 49 disparities were noted between statistical
computations reported by Searle in the submission and
those calculated by FDA. The disparities are constituted
by the values for 6 means, 23 standard errors, and 20 signi-
ficant differences (as measured by T tests).
8. Some of the data sheets for urinalysis had erroneously
labeled the phenylketones test values as "phenylalanine".
(7)
PURPOSE OF INVESTIGATION
Assignment memo dated may 16, 1977 from Donald Healton, Acting
Director of Regional Operations, confirmed an earlier oral
assignment to Chicago District for a directed inspection of cer-
tain non-clinical studies submitted to FDA in support of a food
additive petition for the sweetener aspartame.
The investigation began on 4/25/77, and encompassed the authen-
tication of all data, both raw and summary, relating to the
studies jointly chosen for review by Bureau of Foods and EDRO.
Two studies actually done at G.D. Searle were selected for initial
coverage, and a decision to expand the investigation to a third
study was made at a later date.
Following are the titles of the three studies selected for review:
1.) E-5 (P.T. #851S70), Evaluation of the Embryotoxic and Teatogenic
Potential in the Rat, conducted with SC-18862 (aspar-
tame).
2.) E-89 (PT #1218S75), An evaluation of the Embryotosic and Tetato-
genic Potential in the Mouse, conducted with SC-18862 (aspar-
tame).
3.) E-77/78 (PT #988S73), 115 Week Oral Tumorigenicity Study in the
Rat, conducted with SC-19192 (diketopiperazine).
This report is concerned only with study E-77/78. The report of
E-5 and E-89 was submitted separately.
HISTORY OF BUSINESS
G. D. Searle & Co. provides a wide range of health care products
and services on a worldwide basis. Its business is divided
among three principal areas: pharmaceuticals, medical instru-
ments and optical products, and hospital and laboratory products.
The firm's corporate offices are located in Skokie, Illinois,
with various branches and facilities throughout the world.
Effective June 1, 1977, Donald H. Rumsfeld assumed duties as
President and Chief Executive Officer. Mr. Daniel C. Searle,
formerly Chief Executive Officer is now Chairman of the Board,
while William L. Searle and Wesley M. Dixon, former Chairman
and President respectively, are now Vice-Chairmen.
(8)
Effective March 1, 1977, the firm underwent a major realignment,
shifting to a managerial system based on product lines. This
resulted in the establishment of four main product-line groups,
which are: Pharmaceutical/Consumer Products, Diagnostics,
Hospital Supplies and Optical Products. Each group is headed
by a President who will report to Searle's Executive Vice-Presi-
dent for Operations, Dr. James A. Buzard. A copy of the G. D.
Searle & Co. annual report for 1976 which is attached as Exhibit
#1 further expands on the firm's operations and lists Corpor-
ate Officers.
Mr. O. B. Parrish is President of the Pharmaceutical/Consumer
Products Group and also a Corporate Vice-President. An organi-
zational chart for this group is attached as Exhibit #2. Mr.
Guy Labrosse is now Group Executive Vice-President for U. S.
Commercial Pharmaceutical Operations. In the U. S., this is known
as Searle Laboratories. The facility at 4901 Searle Parkway,
Skokie, Illinois is a part of the U. S. Operations, e. g. Searle
Laboratories, yet houses the majority of the Research and
Development Group.
Worldwide Pharmaceutical Research and Development is also a
part of the Pharmaceutical/Consumer Products Group, but not of
Searle Laboratories. The Research and Development OF aspartame
is a function of this group. Copies of organizational charts
for this group are attached as Exhibit #3. Dr. Robert A. Moe
recently resigned and his position is temporarily being filled
by George V. O'Bleness, Corporate Vice-President for Compliance
and Administration.
Commercial aspects of Aspartame are being handled by an "aspar-
tame Division", under the direction of Elwood H. Ensor, Corpor-
ate Vice-President. There is no longer a division entitled
"New Ventures".
PERSONS INTERVIEWED
Credentials were shown and a written Notice of Inspection was
issued to Dr. William M. Merino, Directory, Domestic Pharmaceu-
tical Products, Regulatory Affairs Department on April 25,
1977. The following Searle personnel were present at the
initial meeting on 4-25-1977.
(9)
Robert A. Moe, PhD. - Executive Vice-President
George Clay, PhD. - Group Leader, CNS Pharmacology
Robert Bost, PhD. - Director of Food Products,
Regulatory Affairs
Holly Ru Probst - Director, Corporation Information
Management Group.
Dave Britton - Director Corporation Information
Department
William Merino, PhD. - Director, Domestic Pharmaceutical
Products
Richard Viktora - Attorney
James Phelps - Vice-President, General Counsel
Elwood H. Ensor, PhD. - Vice-President
Paul Klimstra, PhD. - Vice-President Pre-Clinical
Research and Development
Roger Thies - Attorney
During the course of our investigation one or more of the
following Searle personnel were present in the Conference Room
which we used for our data review.
Richard Viktora - Attorney
Roger Thies - Attorney
George Clay, PhD. - Group Leader, CNS Pharmacology
Robert Bost, PhD. - Director of Food Products
Regulatory affairs
Don Cook, PhD. - Associate Director, Department of
Bio Research
Dick Aspinol, PhD. - I. I. D. Group Leader
Bill Jenkins, PhD. - Director, Product Affairs
Fred McIlreath, PhD. - Director, Regulatory Affairs
Paul Landefeld, - Attorney
Most of the time one attorney (R. Viktora or R. Thies) and
one scientist were present. During our initial meeting with
Searle personnel, James Phelps stated that a Searle monitor
must be with us at all times during our data review in order
to "protect the data".
During the course of our investigation, various individuals
were interviewed in an attempt to obtain all available raw
data and reconstruct the manner in which the study was con-
ducted, as accurately as possible. Since many employees
involved in the study or support areas are no longer employed
at Searle, others were interviewed who had general knowledge
(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.
(Page 31)
ANIMALS UNDER TEST
Three hundred and sixty weanling albino rats, CD strain, 180 of each sex,
were used. The rats were 21 days old when received from the
Copies of shipping labels were obtained, and
are attached as Exhibit #10.
The rats were housed individually in wire cages an were given a one-week
acclimation period before being placed on treatment at the age of four
weeks.
Rockland Rat/Mouse Diet (complete), , was fed for the first
62 weeks, and /Rat Chow was used from week 63 until the study was
terminated at 114 weeks.
The animals were housed in air conditioned rooms maintained at 72 degrees
F, with artificial fluorescent lighting at 12 hours per day exposure.
The rats were divided into 12 housing groups, (6 groups per sex), 30 rats
in each housing group. Initiation of treatment was staggered over a 2
week period, beginning 11/8/71.
Each housing group was composed of dosage groups as follows:
Multiples of No. of Rats Per
Treatment Dosage Estimated Daily Housing Group Total
Group gm/kg/day Human Dosage Male Female Rats
Control
Low
This entire table was blotted out prior to delivery
Medium
High
RANDOMIZATION OF ANIMALS
Computer-generated randomization tables were used for assigning the dose
and housing groups (copies of these tables were obtained and are attached
as Exhibit #6). Each housing group consisted of 30 animals (12 controls,
6 low, 6 medium, and 6 high dose). Each animal was assigned a letter to
(31)
designate the housing group (A through M), a cage number (1 through 30),
a letter to indicate dose group (C,L,M & H), and a letter to indicate sex
(M or F). For example, animal A30CM would be a control male, in housing
group A, occupying cage number 30 (Exhibit #69).
Each rack (30 animals) contained a random distribution of control and
treated animals. An example of a typical housing group is shown in the
diagram attached as Exhibit #7.
The specific problems of feeding animals housed in the above manner were
discussed in the report generated by the task force investigation of
Aspartame in l975/l976. We will reiterate them here:
Housing experimental animals in this manner (controls, low, medium, & high
dose animals randomly distributed on the same rack) would greatly increase
the chance of administering the wrong diet to the animals. The chance of
error was compounded by the method used to feed the animals which was as
follows: At the specified intervals, the animals were weighed, and the
empty food jars were removed, weighed and new food jars placed in the
cages. The new (filled) food jars were placed on a mobile cart in rows
corresponding to dose group (See Exhibit #8). The cart was wheeled to the
Intec Unit and placed up against it with the rows of high dose jars
farthest away from the operator. The operator started from the upper left
corner of the housing rack, (See Exhibit #7), removed the mylar card from
the cage and inserted it into the Intec Unit. This printed out the
animal's identification number. A color coded card for dose level, also
bearing the animal number, remained on the cage. The technician then
opened the cage, removed the animal, placed it on the scale pan, pushed
the button to register the weight and returned the animal to its cage. He
then removed the empty food jar, placed it on the scale and pushed the
button to record the empty feeder weight. The empty jar was placed on
another mobile cart provided for that purpose. The new (filled) jar was
selected from the appropriate row according to dose level (Exhibit #8),
placed on the scale, weight recorded, and the jaw placed in the cage. The
Card was then removed from the Unit and replaced on the cage.
(32)
This procedure was repeated for all cages proceeding from left to right.
It is important to note that none of the food jars were identified in any
manner, as to animal number or dose level. The position of the jar on the
cart was the only means of identifying the proper dose level.
This procedure was used when concentrations were changed. At other times,
the feeder jars were weighed; filled with the appropriate diet/dose,
weighed and replaced in the cage. If a feed container was almost empty,
or contained feces, it would be replaced with a new container.
IDENTIFICATION OF ANIMALS
No ear clips or other methods of uniquely identifying each animal were
used. Animals were individually housed (one animal per cage) and a color
coded card containing the cage number, compound number, project number
(Path-Tox No.), and dose level was attached to each cage. When an animal
died during the study, the color coded identification card was removed
from the cage and accompanied the animal to the necropsy laboratory.
Also attached to each cage was a Mylar Card which identified the an imals
for the Computer System.
At the time of death or sacrifice the animals were assigned a pathology
number which was used to identify the animal during tissue processing, and
on all records pertaining to pathology. The pathology number was a
five-digit sequential number assigned by the pathology department. An
example of a pathology number is 94.893. The number was etched on all
slides as a permanent identification.
All animals that died during the study were assigned an additional number
called a "Master Number". The master numbers denote the chronological
order in which the animals died during the study, by dose group. For
example, CM38 would be the 38th Control Male to die during the study.
Master numbers were noted only on the gross pathology sheets and not
recorded for all animals. Four animals that died during the study had no
master numbers. We were not able to locate any other records providing
the missing numbers. Also, two of the master numbers (CM28 and CM29) were
out of sequence.
(33)
A chart was made by the FDA team, which shows the animal number (cage #),
pathology number, master number, and the complete pathology history of
each animal. The chart is organized by dose group, and is attached as
Exhibit #35.
ANTE-MORTEM OBSERVATIONS
"Observations for Drug Effects" records are attached as exhibits #70 & 71.
These records were completed on a weekly basis for the first four weeks,
every two weeks through week 12, and every 4 weeks thereafter. each
record lists the animals in a specific housing group, and entries are made
for the following parameters: appearance and awareness, rales, eyes, motor
activity sensory loss, urine/feces, appetite/thirst, and tissue
masses/lesions. There is also a space for notes, and masses are routinely
described on the reverse side of the sheet. The top of the sheet has
blocks for entering the date of observations, number of weeks on
treatment, and signature or initials of the person making the
observations. It was noted that many of the observation records were not
signed or initiated. Following is a tabulation of the numbers of records
in which the person making the observations is not identified.
HOUSING
GROUP NO. OF RECORDS NOT SIGNED OR INITIALED
A 6 (wks 80, 92, 84, 88, 93, 94)
B 6 (wks 80, 84, 88, 92, 93, 96)
C 5 (wks 80, 84, 88, 92, 96)
D 7 (wks 79, 80, 84, 88, 92, 94, 96)
E 6 (wks 76, 80, 84, 88, 92, 96)
F 9 (wks 76, 77, 80, 81, 84, 88, 92, 93, 96)
G 5 (wks 80, 84, 88, 92, 96)
H 11 (wks 1, 68, 76, 80, 81, 83, 84, 88, 92,
95, 96)
J 6 (wks 76, 80, 84, 88, 92, 96)
K 8 (wks 71, 76, 80, 84, 88, 92, 93, 96)
L 5 (wks 76, 80, 84, 92, 96)
M 5 (wks 76, 80, 84, 92, 96)
TOTAL 79 records not signed or initialed
In addition to the lack of signatures, it was noted that many of the
records were not originals, but appeared to be xerox copies of the
originals. Surprisingly, some of the xerox copies had "original"
(34)
initials. It was obvious that the initials had been placed on these
sheets sometime after the sheets had been filled out, and after they had
been copied.
Some examples of discrepancies of this type are as follows:
1.) In housing group A, 26 of the 39 observation records were xerox
copies with original initials.
2.) In housing group B, 27 of the 43 observation records were xerox
copies with original initials.
3.) The record for week 76 of housing group A was a xerox copy but the
date, initials, an week are all original.
4.) For week 96 of housing group K, both an original and xerox copy of
the observation record are present. The xerox copy has original
initials and a "B" entered in the "tissue masses and lesions"
column. There is also an entry in the "notes" column for rat
#K25CF. The original record also has a "B" entered in the
"notes" column. All of the above entries had obviously been
made sometime after their original record had been completed and
the xerox copy made.
5.) A record dated 4-27-73 for housing group M does not have the date
entered. The observations were made for this animal on sheets
covering weeks 92, 96, 100 and 104, indicating that the animal
was dead. The record for week 108, however, shows that the
animal is alive, with motor activity, appetite, and thirst. The
record for week 112 again shows that the animal is dead.
In addition to the discrepancy noted above, there is also an obvious error
in the dating of these records; the observation sheet for week 92 is dated
June 13, l973, and the observation sheet for week 88 is dated July 16,
l973.
Ante-mortem observations were also made on other types of records. A
volume entitled "Tissue Masses and Deaths" (exhibit #65) has a record of
the date that each animal died during the study. The deaths are recorded
in two different ways in this volume. One record has a chronological list
of deaths, and another record has a list of deaths organized by housing
(35)
group. This volume also has a "palpation Record", which describes each
tissue mass, and lists the date that it was initially detected.
It was noted that many of the animals in the sequential record of deaths
were listed out of sequence. Following is a tabulation of the animals
that were out of sequence.
(* indicates animal is listed out of sequence)
ANIMAL NO. DATE FOUND DEAD NO. OF DAYS ON TREATMENT
E6HN 8-12-73 640
E24HM 8-16-73 640
G10LM *8-14-73 638
L11CM 5-6-73 535
C17CM *5-4-73 542
A14MM 5-21-73 560
A24HM 10-15-73 707
C14HM *10-27-73 718
E22CM 10-19-73 707
E29LM 10-25-73 714
A4CM 10-26-73 718
E26CM 11-15-73 735
C12LM *11-14-73 736
L20CM 11-19-73 732
AllCM 11-25-73 748
A18CM *11-18-73 741
J19MM 12-10-73 754
F25HF 6-10-73 576
D21CF *6-9-73 577
H6CF 6-17-73 579
M12CF *6-16-73 575
F18CF 7-22-73 615
H22LF *8-20-73 641
D25MF 7-25-73 623
G2LM 10-5-73 689
G7CM *6-14-73 567
(36)
The log of animal deaths in the "tissue masses and deaths" book was
considered the primary data. Animals were allegedly recorded in this book
as they died. When the above discrepancies were pointed out to Searle
personnel, we were told by Tony Martinez on 4-28-77 that this log was
compiled from the body and feeder weight data. When it was pointed out
that the exact date of death could not be determined from the body and
feeder weight data, or from the observation records, we were told that the
primary record of animal deaths was the log organized by Housing Group in
the "Tissue Masses and Deaths" book. We were told that the chronological
log of animal deaths was made by transferring the data from the log
organized by housing group, and therefore the animals being recorded out
of sequence was not significant.
We then pointed out that the chronological record of deaths also contained
the date fixed "in toto" and the date autopsied, neither of which were
found on the log organized by housing group. If the data was transferred
from one record to the other, we wondered where the "date fixed in toto"
and "date autopsied" came from. We posed this question to Searle
personnel and we were finally told on 4-29-77 that the "fixed in toto" and
"date autopsied" columns on the chronological record of deaths was
considered to be primary data, although it was prepared simultaneously
with the pathology sheets, which contained the same data.
Another source of ante-mortem data was the ophthalmoscopic examination
record. Dr. Youkilis performed the eye examinations at periodic intervals
and recorded his findings on the ophthalmoscopic record sheets. These eye
exam sheets were eventually attached to the pathology records, and the
results of the eye exam was incorporated into the Clinical History of the
animal on the pathology sheet.
During our review of the pathology records we noted that there were no eye
exam sheets for 15 animals, all of which had been included in the
individual Ophthalmoscopic Findings in the submission to FDA (Vol. 1,
table 1, pages 122-133). All of these missing eye exam sheets were for
animals that had died during the study. On 6-30-77 we interviewed Donna
Helms, who told us that she would make an attempt to find the missing
records. We advised her that Dr. R. Stejskal had told us on 6-29-77 that
not all of the eye exam sheets were attached to the pathology records, and
that there may be another file of eye exam sheets somewhere.
(37)
On 7-1-77 Donna Helms reported that she had found the missing eye exam
sheets in the K-1 File Room in K-Building. After reviewing these records
we found that a few discrepancies still existed. They are as follows:
1.) It appears that animal J3CM on page 125, Vol. l of the
submission to FDA is in error. We could find no records
to substantiate the listed corneal scar and haziness for
this animals. Also, the observation records indicate that
J3CM died at week 78. It appears that the correct animal
on page 125 should be J2CM and not J3CM.
2.) We found eye exam sheets for H26MF and J29CM, yet the
findings were not reported in the submission to FDA.
3.) There seems to be a discrepancy between G16CM and G12CM.
The pathology sheets for both of these animals report the
identical ophthalmoscopic finding, yet there is no eye
exam sheet for G12CM, and only the finding for G16CM was
reported in the submission to FDA (Vol. 1, p. 125).
During our data review, we found an internal memo from Dr. Youkilis to
K.S. Rao, dated 4-28-74. The text of this memo is as follows: "
-
-
-
-
-
-
Note... This entire memo was expunged from the delivered
document, by parties unknown.
-
-
-
-
-
-
-
-
A copy of the above memo is included in exhibit #72. Dr. Rao and Dr.
Youkilis are no longer employed by Searle.
(38)
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 1.5x1.0cm 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
(39)
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.
(40)
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
(41)
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.
(42)
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.
(43)
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.
(44)
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 <>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:
(45)
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 termina-
tion. 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 administra-
tion. 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 insuffi-
cient 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).
(46)
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
(47)
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 (hand
written) 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:
(48)
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.
(49)
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 malic
dehydrogenase is used.
As described by Judith R. Schmal (June 7,1977) glutamic
pyruvic 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 Novem-
ber 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 ferric
(Page 51)
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 documenta-
tion in file as to method used. From about September 1972
the method used was a flurometric determination in the pre-
sence 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 cal-
cium 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 absorp-
tion 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 diffi-
culty 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.
(51)
(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 modi-
fied 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 dif-
ferences in the apparent absolute values for the concentration
of the substances measured. Changes in the method of conver-
sion 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.
(52)
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 lab-
oratory 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.
(53)
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 submis-
sion) were calculated by the Division of Mathematics, FDA.
Also supplied were the results of the T-TEsts comparing the con-
trols 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.
(54)
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"?
-
-
-
-
-
-
-
-
(55)
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. (Sub-
mitted 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 dili-
tation. 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 auto-
lysis.
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
(Page 69)
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 sec-
tioning was less than specified in the protocol. A detailed dis-
cussion 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, con-
cerning 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 Specifica-
tions.
#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 calcu-
lation 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, con-
cerning 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, concern-
ing 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.
(74)
#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
(75)
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
that Searle had done a statistical analysis of these findings.
ions in a relatively weak acid solution.
(30)
of such parameters as statistics and chemistry.
A few words about this document:
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