Appendix I to TOX/79/47

ASPARTAME — BIOLOGICAL STUDIES

I. BIOCImMICAL STUDIES

i. PHARMACOLOGY (Studies on aspartame and its diketopiperazine(D1®)

There were no effects in rats on appetite at 200 mg/kg, gastric secretion at225 mg/kg, bovine pepsin inhibition in vitro at 143 pg/tel, panoreatio lipaseinhibition at 1.25 mg/nd abd gastric ulcer formation in rats at approximately200 mg/kg. Cardiovascular studies in anaesthetised dogs showed a trans’.entincrease in blood pressure upon iv administration of 5 mg/kg aspartame (but notwith DKP at this level). In unanaesthetised dogs, no effects were noted Sn bloodpressure or heart rate after oral administration of up to 200 mg/kg of aspartameor DKP. Furthermore, neither compound possessed in vitro blood anticoagulantactivity. No effects were noted in rats on pressor responses to angiotensinafter iv administration of 10 leg/kg of material and in the isolated rabbit heart,the sweetener was unable to affect aconitine—induced ventricular arrhythmia. Ina range of studies on the CNS in mice, no effects were noted at the 200 mg/kglevel (single dose, oral administration) in antidepressant, anticonvulsant andanticholinergic activities, hexobarbita]. hypnosis and motor inca—ordination, and atthe 100 mg/kg level (highest dose aiimln4ctered) on analgesic activity; in ratsno behavioural effects were noted after a single dose of 200 mg/kg. Otherphanuacological studies showed that in rats, both compounds at oral doses of100 mg/kg were inactive as diuretic agents and failed to affect blood glucoselevels; after nine days oral administration of up to 200 mg/kg to hypercholesterolemic

, bodyweight gain and serum cholesterol were not affectd, neither compoundpossessed antiacetyl-choline or antihistamine activity in vitro and studies on theperipheral nervous system in rats and mice at up to 2g/~ had no adverse effect.(Searle Project Appendix 7).

A wide variety of studies were undertaken to assess the potential side effects of bothaspartame and DiG’ on the endocrine system and on hormonally dependent target tissues.Aspartame failed to display any oestrogenic, progestational, androgenic, wy~trophicor glucocorticord activity after oral a&n4nittration and adverse effects were not notedon the pituitary ovarian axis, in addition, the sweetener did not antagonise normalphysiologic responses to an oestrogen, progestogen and androgen. Lç’artame also didnot significantly affect rat or hamster fertility and was devoid of any anti—inflainnatoor isamuno—suppressive activity. DKP elicited similar results under identicaltest conditions to aspartame with the exception of minimal anti—inflammatory activitywhen orally administered at 65 mg/day to rats • (Searle Project Appendix 11).

ii. METABOLISM (Studies an aspartame and DiG’)

Studies on the pharmacokinetics and metabolism of aspartame were carried out in rats,mice, dogs, rabbits, rhesus monkeys and man. The methyl group is bydrolysed in ratsand nnnkeys by esterases in the intestine, absorbed and then metabolised in theone-carbon pool of the body. The hydrolysis rate in the monkey was found to be slowerthan that of the rat. Administration of aspartame, radiolabeiled in the aspartic acidja,iety, indicated that the compound was metabolised in a similar manner to that offree L—aspartic acid — lkC, with 60—70% of l4C being excreted in expired air and theremainder incorporated into body protein. Similar studies in experimental animalsand man, with the phenylalanine (phe) moiety labelled, indicated that the compoundwas metabolised in a likewise manner to that of free L—phe—lkC; however the rate ofabsorption of natural phe was found, in subsequent animal studies, to be more rapidthan that of radiolabelled aspartame. The difference was thought to be due to theneed for prior cleavage of the dipeptide bond in the gut before absorption of theresultant amino acids could occur. Comparative studies in man on the metabolismof aspartame—phe-14C and phe-14C were not performed; however the metabolisth andpharmacokinetics of aspartwne—phe-14C in man and monkey were found to be similar

4I

in all aspects. Studies in rabbits indicated that the rates of absorption anddigestion of aspartame were slower than in the other animal species investigated.

The metabolites of aspartame—phe—l4C found in the plasma were the same (bothquantitatively and qualitatively) in all animal species studied. 86—9’+% ofthe radioactivity was associated with complex phenylalanixae containing substancesand 2—3% exhibited similar mobilities on TLC to those of phenylalanineand tyrosine. The plasma half—life in dogs after oral administration of aspartamephe—14C was approximately 12 days.

Under certain conditions, small amounts of aspartame can be transformed bydemethylation and cyclization to DiG’. Small amounts of this product were found infoods containing aspartame and hence its metabolism in rats, rabbits, monkeysand man was studied. D1CP—phe—lUC was found to be poorly absorbed from the gutand the l’+C then rapidly excreted in the twine. DiG’ is absorbed by the bodyunchanged and radiotracer techniques revealed that its major metabolite wasphenylacetylglutamine, (a metabolite associated with ph.enylalanine metabolism);however phenylacetylglutamjne was not formed when OK? was injected iv intomonkeys and it was thought therefore that the metaholite arose from bacterialdegradation of OK? in the gut. Further studies with germ-free rats showed thatonly a small amount of the metabolite was formed in the urine. A third metaboliteof DiG’ was identified, in the faeces of monkeys (after oral administration), asphenylacetic acid. Both phenylacetylglutamjne and phenylacetic acid were identifiedin man in the urine and faeces respectively as the major metabolities of DX?.

In vivo and in vitro studies were performed to investigate the potential of DIG’Ca cyclic amide) to react with dietary nitrite and form nitrosamines. DX? failedto react with nitrite under conditions in which piperidine Ca secondary amine)reacted with nitrite to form N—nitrosopiperidine.

Neither aspartalne nor DX? stimulated or inl4bited hepatic enzyme activities in modelin vivo and in vitro studies, but liver phenylalanine hydroxylase was inhibitedby both compounds tn a dose—related manner. This effect was also observed whenanimals were fed diets which were supplemented with equimolar amounts of phenylalanine;however a similar supplementation in vivo in the monkey did not affect plasmaphenylalanine or tyr’osine levels • Further studies showed that the inhibition wasdue to absolute changes in activity rather than a shift in the known circadianrhythm of the enzyme.

The effects of dietary aspartame on maternal and fetal phenylalani.ne were studiedin rabbits. The feeding of diets containing 6% aspartame to pregnant rabbits resultedin a maximal increase in maternal plasma and tyz’osine levels on day 9 of gestationfoliowed by a return to normal values by day 20, Fetal/maternal plasma amino—acidratios were not significantly different in control and treated groups; however, asthe increase in tyrosine levels in maternal plasma exceeded those of phenylalaninethe phenylalanine/tyrosine ratio fell; this Indicated that most probably, thephenylalanine hydroxylase activity was unaffected by treatment. In an in vitrostudy with maternal phenylala~ine hydr’oxylase, the findings in the above rabbitstudy were verified.

It was thus concluded that the metabolism of aspartame in man was similar to that ofphenylal~ine and aspartic acid. (Searle Project Appendi.c.es 8,9, 10, bA).

Appendix I to Ta/79/47

II ACtJI’E TOXICITY

Species Sex Route LD~ (mg/kg) Reference (Appendix)

Rat—Charles River llale ig > sooo Searle (12)

Rat—Charles River Male iv > 100 Sear’je No. 1179 3714 (12k)

Rat—Sprague~Daw1ey Male ip > 2033 Searle (12)

Mouse—Sprague—Dawley Male ig > 5000 Searle (12)

Mouse—Sprague—Dawley Male ip ) 1000 Searle (12)

Rabbit—New Zealand Male ig > 5000 Searle (12)White Luenberg

Dog—Beagle Male iv ) 100 Searle No. 1178 374 (122)

C”

Appendix I to TQX/79/47

III SUB—ACUTE TOXICITY S7t.WIES

U) Species Mouse

Strain FIA(ICR)

Group Size 5

Route Dietary administration

Dose/Dietary Levels 0, 3, 5, 13 gAg/day

Duration of Treatment ~4 weeks

RESULTS

Appearance and Behaviour Monitored daily; no adverse effects

Growth Measured on days 0, 3, 7, 14, 21 and 28 oftreatment No adverse effects.

Food and Water Consumption Measured as above for growth; no adverse effects

Haematology Not performed

Serum Analyses Not performed

Urine Studies Not performed

Organ Weights Not measured

Post—mortem observations 3 males + 2 females (controls) t all animalsof high dose group. Gross observations only.Mucosa of stomach, duodenum and jejunum oftreated animals were heavily coated withclear viscous fluid.

Other Observations None

No—untoward—effect level 13 g/kg

Reference : Searle Project No. 815 239 (Appendix 13)

ii) Species Rat

Strain Charles River CD

Group Size sC÷ sE

Route Dietary administration

Dose/Dietary Levels 0, 2, 4, lOg/kg/day

Duration of Treatment 4 weeks

RESULTS

Appearance and Behaviour : Monitored daily: no adverse effects

Growth : Measured on days 0, 3, 7, 14, 21 and 28of treatment; no adverse effects.

Food and Water Consumption : Measured as for growth; food intake decreasedsignificantly after weeks 2 and 3 ofadministration in females receiving lOg/kg.

Haematology : Not performed

Serum Analyses : Net performed

Urine Studies : Not performed

Organ Weights : Not measured

Post-mortem observations 3 males ÷ 2 females of controls + all animalsof Sigh dose group. Gross observations only.Nucosa of stomach, duodenum and jejunum oftreated animals were heavily coated with a clearviscous fluid.

Other Observations : None

No—untoward—effect level : lOg/kg

Reference : Searle Project No 8l~69 (Appendix 14)

I.

Appendix I to TOX/79/47 •IV. SHORT-TERN TOXICITY STUDIES

i. Species Rat

Strain Charles River CD

Weight range Hales 353-425g; females 223—306g

Group Size icd’+io ~

Route Dietary administration

Dose/Dietary Levels 0, 5, 125 mg/kg/day

Duration of Treatment a weeks

RESULTS

Appearance and Behaviour Monitored weekly; no adverse effects

Growth Monitored weekly; no adverse effects

Food and Water Consumption Monitored weekly; no adverse effects

Haematolo~r Exythrocy-te and total and differentialwhite cell counts, microhaematocrjtand haemoglobin levels arid coagulationand prothrombln tine after I and 2months. No adverse effects.

Serum Analyses No adverse effects on BUN, glucose,SGPT, SAP, biirubin, protein content,Na, K, Ca, Cl, C02; SCOT levelsreduced in a dose-related manner inboth sexes.

Urine Studies No adverse effects in appearance, pHspecific gravity, protein, glucose,ketones, bilirubin and microscopicexamination of sediment.

Organ Weights Only change observed was statisticallysignificant increase in relative liverweight in wales gives 125 mg/kg.

Histopatholou Chronic murine pneumonia was observedIn several animals/group. No adverseeffects attributable to treatment.

Other observations Ophthalmoscopy on ail rats prior toand at end of treatment; no adverseeffects.

Ro-imtoward-effect level 125 mg/kg.

Reference Searle Project Mo PT 719H68 (Appendix 15).

ii. Species Dog

Strain Beagle

Bodyweight range 7.2 — 13.2 kg

Group Size 2 1Route Oral (gelatin capsule)

Dose/Dietary Levels o, 5, 125 mg/kg/day

Duration of Treatment 8 weeks

RESULTS

Appearance and Behaviour Monitored daily; no adverse effects

Growth Monitored weekly; no adverse effects

Food and Water Consumption Monitored weekly; no adverse effects

Haematology No adverse effects attributable totreatment in erythr’ocy-te and totaland differential white cell coimts,haematocrit, haemoglobin, coagulationand protbrombin time.

Serum Analy~s No adverse effects on sugar, BUN, ES?±‘etentiou, SOFT, SOOT, SAP, Na, K, Cl,Ca, Protein, C02, bilirubin.

Urine Studies No adverse effects on pH, specificgravity, sugar, acetone, protein,bilirubin, occult blood, or microscopic examination of the sediment.

Organ Weights There was a dose—related increase inrelative spleen weight in females only;no other changes were observed.

Histopatholo~ No adverse effects noted in a widerange of tissues and organs examined.

Other Observations Ophthalmoscopy on ail dogs prior toand at the end of treatment; noadverse effects.

No-untoward—effect level 125 mg/kg

Reference Searle Project No P—T 7201468

iii. Species Rat

Strain Charles River Cl) Cweanijng rats)

Group Size 5

Route Dietary administration

Dose/Dietary Levels 0 (controls), 5% L-phenylalanjne,9% aspartame

Duration of Treatment 9 weeks

RESULTS I

Appearance and Behaviour Monitored twice weekly for’ first 4weeks, then at weekly interva].s; noadverse effects

Growth Monitored as above; bodyweights werereduced in both treatment groups, andto a greater extent in males

Food and Water Consumption Monitored as above; food consumptionwas reduced in a manner similar tothat of rate of bodyweight gain

Uaematology Monitored for PCV, haemoglobin,ery-throcyte and total and differentialwhite cell counts, prothombin timeafter 9 weeks; no adverse effects

Serum Analysas After 9 weeks no adven~ effects on BUN,uric acid, SAP, bilirubin or sugar;

decrease in GPT in both male treatmentgroups and decrease in Ca and Cl inmales given aspartame

Urine Studies No adverse effects on specific gravity,pH, occult blood, protein, glucose,ketones or microscopic examination ofthe sediment

Organ Weights No adverse effects were noted inseveral organs with the exception of aslight increase in absolute and relativethyroid weights in both male treatmentgroups

Histopatho1o~r No treatment-related changes were~served

Other Observations None

No—untoward—effect level 9% aspartame C a 6.7 g/kg/day)

Reference Searle Project No 847570 (Appendix 17)

e

iv. Species ; Rat

Strain Long—Evans — 21 days old

Group Size : 32C” + 32

Route : Dietary administration

Dose/Dietary Levels : 0 (control), 2.5 ox’ 5.0 g/kg/dayL’-phenylalanine or 4.5 or 9.0 g/kg/dayaspartame

Duration of Treatment 13 weeks

RESULTS

Appearance and Behaviour Observed daily for survival. AU otherobservations made when bodyweights wererecorded. During days 66-86 of treatmentthe rats were subjected to 3 types ofbehavioural study:

a. t 12$ rats selected fordetermination of general activitylevels.

b. 20d’+ 20 rats selected forevaluation of learning of conditionedavoidance response in a two-wayshuttle box.

c. + 8 rats selected forevaluation of non-discriminatedavoidance response ~idnian).

No adverse effects noted in genera].appearance or behaviour. Asparta,ne—treated rats tended to have a slightlyhigher general activity than controls,but the difference was not significant.No adverse effects were noted inconditioned responses in groups given2.5 g/kg/day L-phenylalanine or 11.5g/kg/day aspartame, but at the higherlevels, significant decreases inavoidance were observed. No adverseeffects were noted in the Sidman testat any treatment level.

Growth : Bodyweights recorded twice weekly fox’2 weeks, weekly for 5 weeks and fort-nightly thereafter. Full data notreported; on day 66 dose-relatedreductions were noted in all treatmentgroups.

Food and Water Consumption Measured at same time as bodyweights.Results not reported.

Survival Several animals died during the courseof the study1 but mortality was neitherdose nor treatment—related.

Serum Analyses Not performed

Naematology Not performed

Urine Studies Not performed

Organ Weights Not performed

Histopathology Not performed

Other Obervatjons None

No-untoward-effect level 2 .5 g/kg

Reference Searle Project No (Appendix 23)

I,

Species and Strain : Rat - Charles River CD. (The animalswere selected from the F2 litters ofthe 2—generation reproduc$ion study.

Age at Start of Study 1 day

GroupSize 20(f’t20?

Route Maternal milk: in addition, the mothersdiets, which contained the treatmentagent,were accessible to the litters.

Dose/Dietary Levels o, 2, 4 g/kg/day (mothers diet)

Duration of Treatment 21 days

RESULTS

Appearance and Behaviour Observed regularly; no treatment-related effects

Growth Bodyweights recorded on all animalskilled by design. No treatment—related effects

Haematology : Performed on 5 & + 5 ? prior to deathon days 5, 15 and. 21; haematocrit,haemoglobin, erythrocyte and total anddifferential white cell counts. Onlyeffect noted was in total leucocytecount which was significantly decreasedin males on day 15 and females on day21 from both treatment groups.

Serum Analyses : Performed on 5 t ~ ? prior to deathon days 15 and 21; fasting blood sugar,BUN, total protein, bilirubin, GPT, AP.No adverse effects noted.

Urine Studies Not performed

Organ Weights : Not performed

Gross Observations s (P + ~ killed after periods of 24houz 5, 15 and 21 days and subjectedto autopsy together with any rats founddead or killed in extremis. No adverseeffects noted.

Histopathology : Heart, liver, lungs bladder and kidneyexamined. All organs unremarkableexcept the kidney where nuclear hypertrophy and nuclear vesiculation werenoted in males and females (given4 g/kg aspartame) on days 15 and 21.The severity was greater on day 15.These changes were also noted in onecontrol male on day 15.

No—untoward-effect level 2 g/kg -

Reference Searle Project No P-P 8931171 (Appendix 22)

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vi. Species and Strain : Rhesus monkey. Macaca mulatta.

Group Size 1 2-3 animals

Route : In milk, from age of 1-9 days

Dose levels i, ~ or 4—6 g/kg/day — Ro conventionalcontrols

Duration of Treatment 29—SDweeks for low dose group and 52weeks for medium and high dose groups.

RESULTS

Appearance and Behaviour Observed daily: from day 218, animalsin medium and high dose groupsexhibited seizure activity in whichthe convulsions were described as grandmal and occurred inconsistently thoughusually when the animal was being handled,Animals from these 2 groups also becameinfected with Shigella during the studyfor which they were treated. One monkeyfrom the high dose group died on day 300but the cause of death was not determined.

Growth I Sody~ieights recorded daily; weightgain in all animals was comparablewith the exception of one monkey fromthe low dose group which gainedaDtroximately half that of the others- this animal showed evidence of“physical deficiencies” at birth.Head circumference and body lengthwere recorded at 4—weeklyintervals; again all values werecomparable with the exception of theone monkey from the low dose group.

Food Consumption Milk consumption was reduced in the highdose group, such that the average intakeof aspartame was 3.6g/kg/day. Foodconsumptions in the other groups werecomparable.

Haeniatology Monitored for haernatocrit, haemoglobin,exythrocyte and total and differentialwhite cefl counts after 3, 6, 9 and 12months of treatment; no adverse effects.

Serum Analyses Monitored after 3, 6, 9 and 12 monthsfor BUN, uric acid, GOT, A?, bilirubin,glucose, Ca, inorganic phosphate,cholesterol and protein; no adverseeffects. Phenylalanine and tyrosinelevels were measured twice weekly for13 weeks, weekly for 17 weeks and

3

Serum Analys ~ fortnightly thereafter and their(continued) results were compared with those

of a group of monkeys fed 2-25 glkgldayof L-phenylalànine. The amino acidlevels were very low in the group given1 g/kg aspartame; levels in the mediumand high dose groups were comparablewith those of the treated controls.

Urine Studies Specific gravity, pH occult blood,protein, glucose, ketones, bilirubinand phenylketone levels were measuredafter 3, 6, 9 and 12 months; no adverseeffects were noted with the exception ofa significant Increase in the excretionof phenylketones in the medium and highdose roups.

Post-Mortem Studies No post-mortem observations were madeas the animals were not killed at theend of the study.

Other Observations Monkeys of the medium and high doseaspartame groups were maintained on apowdered similac diet for 3 months aftercessation of treatment; no furtherconvulsions were observed.

No—intoward-effect level 1 g/kg

Reference Searle Project No 856ot70 (Appendix 19)

Appendix ito

V LONG-TflN TOXICITY (CARCINOGENICIrY) STUD~S

i. SPEC]~S AID STRAIN: Hamster - Golden Syrian (Weanling)

GROUP SIZE: 70&t 7O~. (controls), 356tr35? (treatment groups).

RQTJ’I’E AND DOSE Dietary administration providing 0, 1, 2, 4 or 12gAg/day. (High dose group given Eg/kg/day duringweeks 1-14, 8g/kgfday during weeks 15-18, 10g./kg/dayduring weeks 19-22 end l2gflcg/day from week 23 onwards.)

DURATION OFTREAThENT: 46 weeks.

RESULTS

SURVIVAL: Monitored daily. The whole colony became infected bya disease thought to be “wet tail” soon after the studycommenced; the study was terminated when 80% mortalityhad been reached in the control and low dose femalegroups after 46 weeks. (The study had originally beendesigned to last 104 weeks.)

Psr~AnnIcE ANDBEHAVIOUR: Observed at weekly intervals for 4 weeks, fortnightly inter

vals for a further 10 weeks and then every 4 weeks for therem~nder of the study; no adverse effects attributable totreatment.

GROWTH: Bodyweights measured at same time intervals as observationson appearance and behaviour; rate of bodyweight gain wassimilar in control and treated groups.

FOOD AND WATERCONSUIWTION: Measured at same time intervals as growth; values for all

treatment groups comparable with those of controls.

HA~4AT0LOG7• PCV, haen,oglobin, erythrocyte,total and differential whitecoil and platelet counts measured after 14, 26 and 45 weeksof treatment, reticulocyte counts after 14 and 26 weeksand prothrombin time and activated Pfl after 4~ weeks, all onapproximately I of the animals per group. No adverseeffects were noted.

SERUM ANALYSES: Monitored after weeks 26 and 4~ for EON, SAP, SGPT,bilirubin, glucose, sodium, potassium and calcium; noadverse effects.

URINE STUDIY~S: Samples collected at end of treatment period and analysedfor specific gravity, pH, occult blood, protein, glucose,ketones, bilirubin, phenylketones and microscopicexamination of sediment; no adverse effects.

ORGAN WEIGHTS: No adverse effects noted in absolute or relative weightsof the major organs.