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 chromotography was used thereafter.  It should be noted that the titration 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 laboratory 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.

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 homogeneity to Dr.Rao, and she replied that she did not.

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 mixture 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 prepared 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).

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 statements 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).

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 specifically 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 compound 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.