Page 1 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 Page 2 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 Page 3 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. Page 4 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. Page 5 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. Page 6 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 Page 7 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 Page 8 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 Page 9 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 Page 10 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. Page 13 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@aspartame.ca for converting the often difficult to read hard copy into a useful file format. 4-12-2002