A damning report used by the FDA as povital proof of safety

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[Paranormal_Research] A damning report used by the FDA as " povital

proof of safety "

 

 

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SC-18862

 

52 WEEK ORAL TOXICITY STUDY IN THE

INFANT MONKEY

 

K. S. RAO a, R. G. Mc Connell a, and H. A. Waisman b

 

a) Department of Biological Research (Pathology-Toxicology)

Searle Laboratories, Chicago, Illinois

 

b) Pediatrics Department, University of Wisconsin Medical

Center, Madison, Wisconsin (deceased)

 

October 10, 1972 Pathology—Toxicology

Project No. 856ot70

 

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TABLE OF CONTENTS PAGE NO.

INTRODUCTION 3

METHODS 3

Material evaluated 3

Animals, housing and diet 4

Compound administration 4

Experimental design 5

Physical examinations and observations 5

Clinical laboratory procedures 6

Hematology 6

Clinical chemistry 6

Urinalysis 7

RESULTS 7

ANTEMORTEM OBSERVATIONS 7

Compound consumption 7

Growth and food consumption 8

Observations, physical and behavioral signs 9

Clinical laboratory findings 9

Hematology 9

Clinical chemistry 9

Serum phenylalanine and tyrosine 10

Urinalysis 10

POSTMORTEM OBSERVATIONS 10

SUMMARY AND CONCLUSIONS 10

REFERENCES 12

 

 

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SC-18862: 52 WEEK ORAL TOXICITY STUDY IN THE INFANT MONKEY

K. S. Rao, R. C. McConnell and H. A. Waisman

Department of Biological Research (Pathology-Toxicology)

Searle Laboratories

and

University of Wisconsin Medical Center

Madison, Wisconsin

 

INTRODUCTION

 

In this toxicity study SC-l8862, a nutritive artificial sweetening

agent, was administered orally in the milk formula to infant Rhesus

monkeys for 52 consecutive weeks. SC-18862 is a dipeptide and is split

to its constituent moieties by peptidases in the digestive tract.

 

This study was designed to determine the adverse effects, If any, of SC-

18862 ingestion on the neonatal Rhesus monkey, and also whether all such

effects were identical in nature and magnitude to those produced by an

equimolar molar quantity of L-phenylalanine.1

 

A research project involving repeated daily oral administration of any

agent to a sizable population of baby monkeys, commencing at birth and

continuing uninterrupted throughout the 1st year of life, is a major

undertaking fraught with hazard, even for the partially initiated.

 

Thus, this study was performed at the Primate Research Center, Madison,

Wisconsin under the direction of the late Dr. Harry A. Waisman, Prof. of

Pediatrics and Director, Joseph P. Kennedy Memorial Laboratories. His

established expertise in research involving phenylalanine and the

neonatal Rhesus monkey was invaluable, and his unfortunate demise

necessitated revision of the initial objectives of this study. This

report does provide valuable physical examination and clinical

laboratory data enabling comparison of SC-l8862 with known effects of

Lphenylalanine.

 

METHODS

 

Material evaluated

 

SC-l8862 is a fine white powder with the chemical name L-aspartyl,

Lphenylalanine methyl ester. Three lots (74O2C, 75060B, 74060) were used

throughout this study. These lots contained from 0.2 to 1% of SC-l9l92

(Diketopiperazine; DKP), a conversion product of SC-18862.

 

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Animals, housing and diet

 

Infant Rhesus monkeys (Macaca mulatta) from full-term, normal

pregnancies were separated from their mothers within 6 hours after birth

and transferred to individual heated cages.

 

During the first 24 hours of life, the infants were fed a 10% glucose

solution at four-hour intervals; during the second day, this diet was

supplemented with equal volumes of a commercial milk preparation

(Similac, Ross Laboratories, Columbus, Ohio; Control diet, CD).

Thereafter, the infants were fed CD ad libitum at four hour intervals

until they were placed on the experimental liquid formula. During the

training period, the infant was gently wrapped in a cloth diaper and

held while fed from a toy nursing bottle and nipple. Four feedings per

day was the preferred number for this experiment. Later, between days 12

and 30, the animals were weaned and fed from a small cup: on or after

day 31 they were fed from a large cup.

 

Compound administration.

 

Similac formula was supplemented with SC-18862 on a “phenylalanine

equivalent” basis: 1.83 g L-aspartyl, L-phenylalanine methyl ester

contains 1.0 g Lphenylalanine.

 

The SC-18862 concentration was incrementally increased, based on

acceptance by the infant.

 

___________

Age Code Aspartyl Phenylalanine = L-Phenylalanine

Day 3- Day 9 1/8th .0029 g/cc .0016 g/cc

10- 19 1/4 .0057 g/cc .0031 g/cc

20- 29 3/8 .0086 g/cc .0046 g/cc

30- 119 1/2 .0114 g/cc .0063 g/cc

120- 179 5/8 .0143 g/cc .007S g/cc

180 229 3/4 .0171 g/cc .0094 g/cc

230 269 7/8 .02 g/cc .011 g/cc

270 365 1 .022 g/cc .012 g/cc

 

Milk intake was carefully recorded for each feeding, so that the amount

of SC-18862 consumed per day per kg of body weight could be calculated,

allowance being made for spillage. When the animals were 3 months old, a

quarter of an apple and a quarter of an orange were placed in the cage

once a day. The infant monkeys were fed SC-18862 with the milk formula.

Water was available ad libitum.

 

Animal quarters were air-conditioned with thermostats set to maintain a

room temperature of 720F; artificial fluorescent lighting was provided

on a 14 hour daily photoperiod.

 

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Experimental design.

 

Seven newborn Rhesus monkeys, five males (M34, M38, M64, M79, P53) and

two females (N14, P60), were randomly divided into three groups.

 

Intended Multiple of Start Treatment Dosage Estimated Daily Animal Date

of Supplement Group g/kg/day Human Intake* No. Sex Birth Age (Days)

 

Low 1 33 P53 M 08-28-70 6

P60 F 09-06-70 3

Medium 3 100 M64 M 03-19-70 3

M79 M 04-05-70 3

Nl4 F 04-26-70 2

High 4-6 133-200 M34 M 01-05-70 9

M38 M 01-13-70 1

* Based on 30 mg/kg oral intake daily to a 27 kg child.

 

The treatment was arbitrarily terminated by the late Dr. Waisman’s staff

as indicated below.

 

Total

 

Treatment Animal Treatment Treatment Days on

Group No. Initiated Terminated Treatment

Low P53 09-03-70 03-31-71 210

P60 09-09-70 03-31-71 204

Medium M64 03-23-70 03-18-71 363

M79 04-08-70 04- 4-71 362

N14 04-28-70 04-25-71 363

High M34 01-14-70 01-05-71 357

M38 01-14-70 10-20-70 279

Physical examinations and observations.

 

Animals were observed daily at the time of dosing and intermittently

between dosing periods for survival arid behavioral changes. Body

weights were recorded each day in the morning. Head circumference and

body length (crown to heel length) were recorded at 4 week intervals. An

evaluation of general motor and behavioral activity, locomotion,

external appearance of teeth, nose, eyes, ears, perineum, hair coat and

digital palpation for tissue masses was conducted immediately prior to

the initiation of compound administration, and subsequently concurrent

with each body weigh: measurement. Unusual signs, including indications

of systemic pharmacologic or toxicologic effects, were routinely

recorded at this time and whenever warranted.

 

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Clinical laboratory procedures.

 

Hematologic and clinical chemical examinations which were performed on

blood specimens of all animals, were collected via the saphenous vein at

3, 6, 9 and 12 months of compound administration.

 

Hematology.

 

The following hematologic parameters were measured:

 

Parameter Method

 

Hematocrit (micro) Micro method 2

Hemoglobin Cyanrnethemoglobin 3

Total RBC count Coulter Counter 4

Total W3C count Coulter Counter 4

Diff. WBC count Smear 5

Clinical chemistry.

 

The following (plasma chemistry) parameters were measured for all

groups:

 

Parameter Method

 

Blood (plasma) urea nitrogen Urograph method 6

Uric acid Brown 7

Glutamic oxalacetic transaminase Reitman & Frankel 8

Alkaline phosphatase Klein et al.9

Bilirubin Malloy & Evelyn 10, 11

Glucose Nelson & Somogyi 12, l3

Calcium Barr 14

Inorganic phosphate Fiske & Subbahow 15

Cholesterol Abell et al. 16

Total protein TS Meter 17

Phenylalanine Undenfriend & Cooper 18

Tyrosine La Du & Michael 19

 

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Serum phenylalanine and tyrosine were monitored twice a week for the

first 13 weeks; weekly for the next 17 weeks and once every two weeks

thereafter.

 

Urinalysis.

 

Spontaneously voided urine specimens from individually housed monkeys

were collected at 3, 6, 9 and 12 months of treatment. The following

parameters were measured.

 

Parameter Method

 

Specific gravity Total solids meter

pH Labstix (Ames)

Occult blood Labstix (Ames)

Protein Labstix (Ames)

Glucose Labstix (Ames)

Ketones Labstix (Ames)

Bilirubin Labstix (Ames)

Phenylketones Phenistix (Ames)

 

RESULTS

 

ANTEMORTEM OBSERVATIONS

 

The availability of acceptable historical and contemporary data on

untreated control monkeys from the Waisman group reduced the necessity

of a concurrent control group. The extremely limited availability of

newborn Rhesus, as well as limitations in adequately skilled laboratory

personnel, likewise contributed to our decision to eliminate the

requirement of a concurrent control group in this study.

 

For comparative purposes the normal range of values from 14 historical

control monkeys is superimposed on Figures 1-9.

 

Compound consumption.

 

The treatment of monkeys with SC-l8862 was initiated on the basis of

availability of newborn monkeys as indicated on page 3. The sudden

demise of Dr.

 

Waisman necessitated termination of the study. At that point in time,

the medium and high dose monkeys had completed 52 weeks of treatment,

and the low dose monkeys had completed 29-30 weeks of treatment.

 

Mean values for SC-18862 ingestion by the low and medium dose group

animals over the treatment period (Table 1) were within 5% of the

proposed doses of 1.0 and 3.0 g/kg. The intended dosage of SC-l8862 for

the high dose group was

 

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4 to 6 g/kg; because of an unanticipated decrease in the intake of milk

formula, presumably due to the intense sweetness of SC-18862, the

realized mean intake of SC-l8862 over the entire study was 3.6 g/kg

(range 1.21 to 5.33 g/kg). Hence, the SC-l8862 intake of high dose group

animals was not notably different from the medium dose group animals.

Irrespective of the actual intake of SC-l8862 levels, the results of

this study are presented as data for the low dose group (0.97 g/kg

intake), medium dose group (3.01 g/kg intake), and high dose group (3.62

g/kg intake), according to the original placement of animals within each

group.

 

As pointed out in the methods section, the SC-18862 lots employed in

this study contained 0.2 to 1% SC-19192, a conversion product of SC-

1S362.

 

The actual group mean daily ingestion of SC-19l92 (Table 2) was computed

from the actual intake of SC-18862 and from analytical data (Quality

Control Department, Searle Laboratories) indicating the SC-19l92 content

of each individual lot of SC-l8862 employed in this study. The group

mean intake of SC-19192 over the entire study was 4.84, 15.07 and 18.12

mg/kg/day for the low, medium and high dose groups, respectively.

 

Growth and food consumption.

 

Absolute body weight and weight gain (g/kg/day) of individual monkeys in

each group are presented in Figures 1, 2 and 3. Body weight gain per ml

milk formula consumed and actual intake of liquid diet over the 52 week

treatment period are depicted in Figures 4, 5 and 6.

 

The body weight in kilograms was within normal limits for P60, M64 and

M34.

 

One high dose monkey, M38, and two medium dose monkeys, N14 and M79,

showed slightly lower body weight, but there seemed to be a leveling off

in the weight as the animals approached one year on the diet.

 

Low dose monkey P53 exhibited evidence of physical deficiencies,

apparently congenital in origin, shortly after birth. The animal was

examined by selected consultants, and its suitability for inclusion in

the study was questioned. A precise account of their findings is not

available. The animal was continued on study irrespectively, however,

since the supply of baby Rhesus was very limited.

 

Subsequent poor growth of this animal (Fig. 1) was due to inappetance

and may reflect the initial difficulties. Relative weight gain

(g/kg/day) of all treated animals except monkey P53 was comparable to

historical controls.

 

Rate of growth expressed per unit of diet intake (Figs. 4, 5, 6) was

within normal limits despite the falling off of absolute body weight

(Figs. 1, 2, 3). This indicates that the dipeptide was utilized

efficiently and did not effect the efficiency of food conversion.

 

There was a marked decrease in total intake of milk formula in all the

treated animals (Figs. 4, 5, 6). This could be attributed to the intense

sweetness (200 x sucrose) of the dipeptide.

 

Individual daily body weight and milk formula intake of each

experimental monkey may be found in Figures 1, 2 and 3 Body length of

all treated animals is essentially within the historical control

 

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range; head circumference is likewise within historical control range

for 1/2 low level, 1/3 medium level and 2/2 high level monkeys, but is

below control level in the remaining animals (Figs. 7, 8, 9). The

decrease in head circumference during treatment in low dose monkey P53

(Fig. 7) could be attributed to a proportional decrease in the relative

weight gain (g/kg/day) of this monkey. Underdevelopment of this monkey

is presumably related to the physical deficiencies observed at birth.

 

An apparent decrease in the head circumference observed during treatment

in two medium dose monkeys, M79 and N14 (Fig. 8), is attributed to a

relatively lower head circumference at birth.

 

Observations, physical and behavioral signs.

 

All animals in the medium and high dosage groups exhibited seizure

activity. Seizures were observed for the first time following 218 days

of treatment. Thereafter, sporadic convulsions occurred inconsistently

at various times during the treatment period. Seizures occurred most

frequently during physical handling of the animal for body weight

measurements. The convulsions were of grand mal type similar to those

induced by feeding L-phenylalanine to infant monkeys.

 

All animals in the medium and high dosage groups contracted a Shigella

infection at various times during the treatment period. In an effort to

treat the Shigella infection, these anima1s received appropriate

antibiotic and intravenous fluid therapy.

 

One monkey, M38, of the high dose group, died after 300 days of

treatment.

 

The cause of death was not determined.. All other animals survived the

treatment period.

 

General posture and locomotion, pelage, body orifices and excretions

were otherwise unremarkable.

 

Clinical laboratory findings.

 

Hematology.

 

Individual values of hematology parameters evaluated are presented in

Tables 3 and 4. The Primate Research Center, Madison, Wisconsin,

supplied mean hematologic values of 16 historical control monkeys of the

same age group as the experimental animals; these values are presented

in Table 5. In general, hematologic values for individual treated

animals were unremarkable; no biologically significant deviation from

control ranges was observed. Statistical analysis was not performed due

to the lack of individual values for the historical controls.

 

Clinical chemistry.

 

Individual values of clinical chemistry parameters evaluated are

presented in Table 6. The Primate Research Center, Madison, Wisconsin,

supplied clinical chemistry values of 5 historical control monkeys of

the same age group as the experimental animals; these values are

presented in Table 7. Clinical chemistry values from SC-18862 fed

animals, in general, were comparable with the historical

 

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control values. No obvious compound related changes were evident.

 

Serum phenylalanine and tyrosine.

 

The serum phenylalanine and tyrosine values from SC-18862 fed animals

were monitored at frequent intervals and are depicted in Figures 10, 11

and 12.

 

For comparative purposes the range of serum phenylalanine and tyrosine

values from 4 historical positive control monkeys fed 2 to 2.5 g/kg/day

L-phenylalanine are superimposed in the Figures. In the low dose (1

g/kg/day SC-18862) animals there was no appreciable change in the serum

phenylalanine and tyrosine levels (Fig. 10). There was a significant

increase in serum phenylalanine and tyrosine values in the medium and

high dose monkeys (Figs. 11 and 12). These increased serum phenylalanine

and tyrosine values are comparable to positive control Lphenylalanine

fed animals. It is interesting to note that in the medium and high dose

groups very high levels of serum phenylalanine were achieved after 200

days of feeding SC-l8862 (Figs. 11 and 12). As mentioned earlier, the

convulsions in the medium and high dose animals were observed initially

at 218 and 219 days on the experiment. Hence, the convulsions In the

monkeys are correlated with and can be attributed to high serum

phenylalanine levels. In the low dose monkeys (1 g/kg/day) serum

phenylalanine levels were at a basal level (Fig.10) and no convulsions

had been observed when the study was terminated (30 weeks of treatment).

 

Following the termination of treatment, medium and high dose monkeys

were kept under observation for 3 months on powdered Similac. No further

convulsions were detected during this period.

 

Serum phenylalanine and tyrosine values of individual animals monitored

at various times in the study may be found in the Appendix.

 

Urinalysis.

 

The results of urinalyses performed on individual monkeys are presented

in Table 8. No meaningful variations were consistently present in the

parameters measured: pH, Sp. Gr., blood, protein, glucose, ketones,

bilirubin. There was a significant increase in the urinary excretion of

phenylketones in the medium and high dose group monkeys. This was

consistent with a concomitant increase in serum phenylalanine levels in

these monkeys.

 

P0STM0RTEM OBSERVATIONS

 

Animals were not available for sacrifice and necropsy at the termination

of compound administration, due to a shortage of personnel and

supervision following Dr. Waisman’s death. Likewise, necropsy data on

the one non-survivor, high dose monkey M38 that died after 300 days of

compound ingestion, was lost for similar reasons.

 

SUMMARY AND CONCLUSIONS

 

A 52 week oral toxicity study of SC-18562 was conducted, employing oral

administration of the compound to newborn Rhesus monkeys. SC-18862 was

mixed with Similac milk formula and fed four times daily. Mean daily

dosage levels of

 

Page 11

 

0.97, 3.01 and 3.62 g/kg were attained incrementally. These levels are

multiples of 32, 100 and 120 times the estimated maximal human daily

intake (30 mg/kg/day for 27 kg child). Physical examinations were

performed regularly. Body weight and milk formula intake were recorded

monthly. Hematology and clinical chemistry parameters were evaluated

every three months. Serum phenylalanine and tyrosine levels were

monitored at frequent intervals.

 

Survival was 100% in all treated groups except the high dose group; one

monkey, M38 in the high dose group, died after 300 days of treatment.

The cause of death is unknown. Animals in both the medium and high dose

groups experienced grand mal convulsions after about 220 days of

treatment. Similar convulsions may be induced in the monkey by feeding

L-phenylalanine alone in equimolar quantities1. Occurrence of seizures

coincided with the attainment of high serum phenylalanine levels. In the

low dose group (1 g/kg/day) there was no appreciable increase in serum

phenylalanine; thus, convulsions would not be expected irrespective of

the duration of treatment. Physical examination findings were otherwise

unremarkable.

 

Food intake and growth rate were mildly reduced by SC-18862 treatment.

 

The head circumference of one low dose monkey (P53) and two medium dose

monkeys (M79 and Nl4) was lower than the historical control range. This

was attributed to physical deficiencies evident at birth and subsequent

partial inanition in the former animal, and to unusually low head

circumference measurements at birth in the latter two. The head

circumference of all other monkeys was within normal range. The body

length of all treated monkeys was within the historical control range.

 

Hematology and clinical chemistry parameters were generally unremarkable

in treated animals, as compared with data from historical control

animals of the same age from the same laboratory. No biologically

significant alterations were observed except, as mentioned earlier,

there was a significant increase in serum phenylalanine and tyrosine

levels at the medium and high dose levels. Urinalysis parameters were

generally unremarkable, except for a significant excretion of

phenylketones in both medium and high dose groups after 6 months. This

increase coincided with the increase of serum phenylalanine levels.

Thus, the SC-18862 treated monkeys exhibited increased serum

phenylalanine levels, increased urinary phenylketone levels, and

episodes of grand mal seizures in relation to the phenylalanine moiety

of the compound administered. At the low dose level (1 g/kg/day), none

of the above alterations were observed through 30 weeks of treatment, at

which point the study terminated.

 

It is concluded that dietary administration of SC-18862 to infant

monkeys starting at birth and continuing for 30 consecutive weeks at

approximately 1 g/kg/day, caused no biologically meaningful alterations

in physical or behavioral findings or in clinical laboratory parameters.

At higher dosages a significant increase in serum phenylalanine and

tyrosine levels, an increase in urinary phenylketone excretion and

episodes of grand mal type seizure activity were observed at this point,

and continued through the 52 weeks of treatment. Both the nature and

magnitude of the changes observed were comparable to historical positive

control animals fed equivalent quantities of L-phenylalanine alone.

 

Page 12

 

REFERENCES

 

1. Waisman, H. A. and Harlow, B. F. (1965). Science 147, p. 685.

 

2. Bauer, J. D., Ackermann, P. G. and Toro, G. (1962). Bray’s Clinical

Laboratory Methods. The C. V. Mosby Company, Sc. Louis. p. 149.

 

3. Kolmer, J. A., Spaulding, E.H. and Robinson, H. W. (1951). Approved

Laboratory Technic. Appleton-Century-Crofts, Inc., New York. p. 52.

 

4. Instruction and Service Manual for the Model “B” Coulter Counter, 5th

edit., April, 1969.

 

5. Bauer, J. D., Ackermann, P.G. and Toro, C. (1962). Bray’s Clinical

Laboratory Methods. The C. V. Mosby Company, St. Louis. p. 143.

 

6. " Urograph”, Quantitative Urea Nitrogen Assay System, General

Diagnostics, May, 1963.

 

7. Brown, H. (1945). J. Biol. Chem. 158, p. 601.

 

8. Reitman, S. and Frankel, S. (1957). Am. J. Clin. Path. 28, p. 56.

 

9. Klein, B., Read, P. A. and Babson, A. L. (1960). Clin. Chem. 6, p.

269; 10. Malloy, H. T. and Evelyn, K. A. (1937). J. Biol. Chem. 119, p.

480.

 

11. Standard Methods of Clinical Chemistry, Vol. I (1953), p. 11.

 

12. Nelson, J. (1944). J. Biol. Chem., 153, p. 375.

 

13. Somgyi, M. (1945). J. Biol. Chem., 160, p. 62.

 

14. Kingsley, G. R. and Robnett, 0. (1961). Anal. Chem. 33, p. 552.

 

15. Fiske, C. H. and Subbarow, Y. (1925). J. Biol Chem. 66, p. 375.

 

16. Abell, L. L., Levy, B. B., Brodie, B. B., et al. (1952). J. Biol.

Chem. 195, p. 357.

 

17. Instruction and Service Manual for the Model 10400 Meter, 1964.

American Optical Corporation, Scientific Instrument Division, Buffalo,

New York.

 

18. Udenfriend, S. and Cooper, J. R. (1953). J. Biol. Chem. 203, p. 953.

 

19. LaDu, B. N. and Michael, P. J. (1960). J. Lab. Clin. Med. 55, p.

491.

 

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Those interested in tables and graphs will have to view the Adobe

version 5 PDF file. URL is: http://www.dorway.com/raoreport.pdf

 

Thanks to Mr. John T. Linnell admin for converting

the often difficult to read hard copy into a useful file format.

 

4-12-2002

 

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