FDA report on Searle's submission for NutraSweet approval 1977 - Part 16

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As described by Judith R. Schmal (June 7, 1977) the method

used from November 1971 to February 1, 1974 was adapted

from Fermco Kit Bulletins #20 and 20-1. Urea is hydrolyzed

to ammonia and carbonic acid in the presence of urease.

Ammonia is detected by the Berthelot reaction to produce

indophenol. From February 1, 1974 the "direct serum methond"

modified from the method of Marsh et al was used.


4) Phenylalanine


Reference: Hill, J. B., Summer, G. K. Pencer, M. W. Resz

N.O. (1965) Chin Chem 11, 541


From Nov. 1971 to about Septembe 1972 there is no 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.




(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



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.




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



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.


Continue to Part 17


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