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    ASPARTAME

    Explanation

         Aspartame was first evaluated by JECFA in 1975 (WHO, 1975).
    At that time a special problem was posed by the presence of the
    conversion product, 5-benzyl-3,6-dioxo-2-piperazine (diketopiperazine,
    DKP) and no ADI for man was allocated. It was again considered by
    JECFA in 1976 and its consideration was deferred in view of the
    incompleteness of the information available (WHO, 1976). In 1977
    JECFA had evidence that the problem with diketopiperazine was of no
    significance and concluded that the safety of aspartame had been
    adequately demonstrated; the Committee was prepared to establish an
    ADI for man, but because of the assertion that the data base from
    which the conclusions were drawn required validation the Committee
    deferred its decision pending an assurance that the toxicological data
    were valid (WHO, 1978). In 1979 JECFA was presented with evidence of
    validation of the toxicological data and accepted the validation;
    however, the Committee did not have sufficient time to reassess the
    data on aspartame which were evaluated by the previous meeting (WHO,
    1980).

         The present monograph contains summaries of data examined by
    JECFA in 1975, 1976, 1977 and 1980.

    BIOLOGICAL DATA

    BIOCHEMICAL STUDIES

         Aspartame (10 mg) was incubated with pepsin (0.4 mg) in a KCl-HCl
    buffer pH 1.0 at 37C for 15 min. Pepsin showed neither esterase nor
    peptidase activity when aspartame was the substrate. Aspartame was not
    hydrolysed when incubated with dog gastric juice. Methyl 14C-labelled
    aspartame was administered to rats whose stomach had been ligated
    at the pylorus. Examination of the stomach contents 4 h after
    administration of the test compound, showed no hydrolyses of the
    methyl group and essentially complete recovery of the administered
    dose. Incubation of Methyl 14C-labelled aspartame with fresh rat
    plasma resulted in demethylation of the aspartame (Anonymous, 1972a).

    Rat

         Male rats (300 g) were dosed orally with 0.5 ml of an aqueous
    solution of phenylalanine 14C-aspartame (10 mg/ml). Blood samples
    were taken 1, 2 and 4 h after administration. Afterwards the animals
    were sacrificed and GI tract removed and divided into stomach, small
    intestine and colon. About 50% of the administered dose was absorbed
    within 4 h, and 28-30% remained in the GI tract, mainly in the colon.
    Chromatographic separation of the plasma radioactivity showed that
    most of the radioactivity was probably associated with proteins,
    peptides and amino acid conjugates. In another study rats were
    administered a single dose of phenylalanine 14C-aspartame, and urine

    samples collected for up to 17 days post-dosing. Less than 5% of the
    administered radiolabel was excreted in the urine during this period,
    with most being excreted within the first two days post-dosing
    (Anonymous, 1972a).

         In another study the metabolism of methyl 14C-aspartame,
    phenylalanine 14C-aspartame, and aspartyl 14C-aspartame was studied
    and compared with that of methanol 14C, phenylalanine 14C and
    aspartic acid 14C. In these studies male albino rats (Charles River
    strain), ca 250-300 g, were dosed orally with a single dose of the
    test compound, at a level equivalent to 20-30 mg/kg. The CO2 in the
    expired air was collected for a period 8 h post-dosing. Urine and
    faeces were collected separately. Rats used for plasma studies weighed
    300-400 g; indwelling carotid catheters had been inserted 2 days
    before dosing. The methyl group of aspartame was metabolized in a
    manner similar to that of methanol, with major portion of the
    radiolabel appearing in the expired CO2, (53-73%)/(48-64%),
    with traces in the urine (2-7.5%)/(1.4-2.4%), and in the faeces
    (0.5-1%)/(0.02-0.09%) for methanol/aspartame respectively. The plasma
    concentration of the radiolabel was similar for methanol and the
    aspartame. Comparison of the metabolism of phenylalanine and aspartame
    showed that the amount of label converted to CO2, and excreted into
    the urine from the amino acid was greater than that of the aspartame
    (13-24%)/(7-15%) for CO2, and (2.65-4.62%)/(0.7-4.3%) for urine, for
    phenylalanine/aspartame respectively. Only trace amounts of radiolabel
    were present in the faeces in one study. However, in a subsequent
    study, with phenylalanine-14C-aspartame the level of radiolabel
    recovered in the faeces ranged from 3-48%. Forty-seven to fifty-three
    per cent. radiolabel from the phenyl-alanine 14C and 2.0-17% from the
    aspartame was incorporated into the carcass. Total recovery of 14C in
    these studies ranged from 14 to 85%. A direct comparison of aspartic
    acid 14C, aspartyl 14C-aspartame is not possible because of different
    times of collection of CO2. However, 68% of the radiolabel from the
    aspartame was excreted as CO2 in a 48 h period (Anonymous, 1972a).

         Groups of six adult male rats (S-D derived Charles River
    CD-strain) were dosed by gavage with a 3:1 mixture of aspartame/DKP at
    a dose level of 27 mg/kg bw for five days. On day 4, one group of rats
    was cannulated to permit the removal of blood for plasma studies. On
    day 6, following an overnight fast, the rats were dosed with the same
    dose of 14C-aspartame/DKP mixture previously used. Blood samples were
    taken from the cannulated rats at 0.5, 1, 2, 3, 4, 6 and 24 h post-
    dosing. The other group of rats was used for collection of expired
    14CO2 (7 h) and measurement of 14C excreted in urine and faeces (24,
    48, 72 and 96 h post-dosing). Five rats respired maximum amounts of
    14CO2 in 1 h, the sixth peaked at 30 min. The cumulative 14CO2
    expired ranged from 12 to 23% of the administered dose. Two to four
    per cent. of the total administered 14C dose was excreted in the
    urine, with maximum excretion within the first 24 h. Total faecal
    excretion of 14C ranged from 1.6 to 3.1% of the total dose. Peak
    blood levels of 14C were attained in 3 to 4 h and showed little

    change during the course of the study. The maximum level was estimated
    as 0.7% of the administered dose. No aspartame or DKP was detected in
    the plasma, at 0.5 h post-dosing. At subsequent time intervals through
    24 h the major 14C components in hydrolysed serum protein were
    present as phenylalanine or its metabolite tyrosine (Anonymous,
    1972b).

    Mice

         Six young adult male HAM/ICR-derived (Charles River CD-1 strain)
    outbred albino mice, were given by gavage aspartame at a dose level of
    20 mg/kg bw daily for six days. The mice were fasted overnight and
    then administered a single oral dose of 14C-labelled aspartame. CO2
    from expired air was collected up to 7 h post-dosing, and urine and
    faeces collected at 24 h intervals up to 96 h post-dosing. At
    termination of the study the animals were sacrificed and autopsied.
    Eleven to twenty-six per cent. of the administered dose of radiolabel
    was expired as CO2, during the 7 h period, with 4.5-8.18% being
    excreted within the first 30 min. Only small amounts of 14C were
    excreted in the urine (less than 1-3.7%), with peak excretion during
    the first 7 or 24 h. The cumulative faecal excretion was 4.4-7.49% of
    the administered dose. For the study of plasma levels a group of 14
    male mice were dosed with aspartame and then 14C-labelled aspartame
    as described in the previous study. Two mice were sacrificed 0.5, 1,
    2, 3, 4, 6 and 24 h post-dosing, and plasma samples prepared. Levels
    of 14C in the plasma showed little change during the 0.5 and 24 h
    period, but were slightly higher at the 3 h period. Chromatographic
    separation of the 14C in the serum showed that the 14C was
    incorporated into slowly migrating plasma components. Acid hyrolysis
    of the plasma gave rise to peaks containing tyrosine and phenylalanine
    (Anonymous, 1972b).

         Male Charles River rats were treated for two weeks with a
    standard diet or with diets containing 0.85% L-phenylalanine or 1.5%
    aspartame. At the end of the test period rats from each group were
    sacrificed at 08.00 (day 1), 12.00, 16.00, 20.00, 24.00, 04.00 (day 2)
    and 08.00 h. Hepatic phenylalanine hydroxylase activity was decreased
    during the 24 h period, and there was no change in the circadian
    rhythm of the hydroxylase. Plasma phenylalanine and tyrosine levels
    were directly related to the hepatic phenylalanine hydroxylase
    concentration (Anonymous, 1974a).

    Rabbit

         Eight young adult female rabbits (New Zealand White) were given
    by gavage 20 mg/kg/day aspartame for five days, and then after an
    overnight fast a single dose of 14C-labelled aspartame. Four of the
    rabbits were used to measure plasma 14C levels and metabolites,
    samples of blood being taken from the ear vein at 0.5, 1, 2, 3, 4, 6
    and 24 h post-dosing. The remaining four rabbits were used to measure
    14CO2, urinary 14C and faecal 14C. Cumulative 14CO2 production
    during 7 h post-dosing period ranged from 2.9 to 10.4% of the

    administered dose, with large variations between individual animals.
    Cumulative urinary 14C during the 96 h post-dosing period ranged from
    3.5 to 6.0% with maximum excretion occurring in the first 7-24 h
    period. The cumulative faecal excretion ranged from 2.6 to 5.9% of the
    dose. Plasma concentration of 14C was low, reaching a maximum 3 h
    post-dosing and then remaining relatively constant throughout the
    study. Chromatographic separation of the 14C in the plasma indicated
    a single component in the unhydrolysed plasma, and the presence of
    14C phenylalanine and tyrosine in the acid hydrolysed plasma
    (Anonymous, 1972b).

         Male New Zealand White Leunberg strain rabbits, which had been
    administered orally 20 mg/kg aspartame/day for five days were dosed
    with a single dose of 14C-phenylalanine aspartame (20 mg/kg). Blood
    samples were taken at 0.5 and 3 h post-dosing. Chromatographic
    separation of the 14C-labelled products in the plasma shows that the
    radiolabel was rapidly incorporated into protein, and by three hours
    all the 14C was associated with this fraction. The 14C in the protein
    was associated with phenylalanine and tyrosine (Anonymous, 1974a).

         Mature New Zealand female rabbits on day 6 of pregnancy were
    given a diet containing 6% aspartame. On days 6 and 9 of gestation
    blood samples were taken. On day 16 of gestation animals were
    anaesthetized and four foetuses (two from each uterine horn) removed,
    then the mothers and remaining foetuses sacrificed. On day 20 the
    remaining animals were autopsied in the same manner. All samples were
    analysed for phenylalanine and tyrosine. Since it was not possible to
    collect blood from the 16-day-old foetuses, homogenates of the whole
    foetus were prepared. Maternal plasma phenylalanine and tyrosine were
    significantly elevated in the early treatment period reaching peak
    levels at day 9 of gestation (three days after commencement of
    treatment). The subsequent values returned to normal. The ratio of
    foetal:maternal plasma phenylalanine was 1.58 in control rabbits and
    1.40 in treated animals. For plasma tyrosine the ratio was 2.29 in
    controls and 2.06 in the treated animals. The aromatic amino acid
    level of the amniotic fluid was consistently higher in treated animals
    than in controls (Anonymous, 1974a).

    Dog

         Groups each of four female beagle dogs (5.4-7.9 kg) were
    pre-treated for five days by oral intubation with 0.068 mmol/kg
    aspartame or L-phenylalanine. On day 6, a dose of 0.068 mmol 14C-
    phenylalanine aspartame was administered to four dogs via the
    saphenous vein, and 0.068 mmol 14C-phenylalanine to the other four
    dogs by gavage. 14CO2 elimination was measured for the period 7 h
    post-dosing and urinary and faecal 14C excretion for the 96 h post-
    dosing period. Plasma 14C levels were determined at 15 min intervals
    during the first hour post-dosing, then hourly up to 3 h post-dosing,
    and then daily for 15 days post-dosing. Phenylalanine was oxidized
    more rapidly to CO2 than aspartame, the maximum level of elimination
    of 14CO2 occurring within 60 min in the case of phenylalanine and

    90 min in the case of aspartame. Urinary and faecal excretion of 14C
    was low and similar for phenylalanine and aspartame: for urine
    5.32  0.43/2.63  0.3 and for faeces 5.38  9.2/6.17  0.77, for
    phenylalanine/aspartame respectively. Most of the 14C in the urine
    and faeces from the phenylalanine treated animals was excreted in
    the first 48 h, whereas in the case of aspartame the low level of
    excretion occurred during the 96 h test period. Plasma 14C levels
    were similar for phenylalanine and aspartame treated animals, with
    peak values occurring at 5 h post-treatment. The plasma 14C half-time
    for phenylalanine was 10.8 days, and for aspartame 12.1 days.
    Fractionation of plasma into low and high molecular weight 14C-
    containing compounds showed that the low molecular weight component
    was being converted to a high molecular weight component.
    Chromatographic studies of the low molecular weight 14C plasma
    component from aspartame treated animals showed that during the first
    hour post-dosing the radiolabel was associated mainly with
    phenylalanine and its metabolite tyrosine (Anonymous, 1972c).

    Monkey

         Four young female rhesus monkeys (2-3 kg) were administered
    orally either 14C-methanol, 14C-L-phenylalanine, methyl 14C-
    aspartame or phenylalanine 14C-aspartame at a dose level equivalent
    to 0.068 mmol. 14CO2 elimination and 14C excretion in urine and
    faeces was measured for a 8 h post-dosing period in monkeys treated
    with the 14C-methyl-labelled compounds, and 24 h for the 14C-
    phenylalanine compound. Plasma 14C determinations were made up to
    72 h post-dosing. The cumulative 14CO2 excretion expressed as a
    percentage of total administered dose was 72-88% and 60-75% for
    methanol and aspartame (methyl 14C label) treated animals and
    9.00-21.70% and 13.17-22.60% for the phenylalanine and aspartame
    (phenylalanine 14C label). Cumulative 14C excretion in the urine was
    3.17% and 1.57% for methanol and aspartame (methyl 14C label) treated
    animals. For phenylalanine and aspartame (phenylalanine 14C label)
    treated animals the respective cumulative 14C excretion in the urine
    was 1.3-4.6%, and 1.5-3.7%, and for faeces 0.6-8.8% and 0.5-3.3%.
    Plasma 14C levels following administration of methanol or aspartame
    (14C-methyl label) was low. The loss of 14C was slow. In the case of
    phenylalanine and aspartame (14C-phenylalanine) treated animals, 14C
    plasma levels reached a maximum at 5 h and showed a slow loss during
    the next 75 h of the test (Anonymous, 1972a).

         In another study four female rhesus monkeys (5-7 kg bw) were
    dosed via the saphenous vein, with a single dose of either
    14C-L-aspartic acid or 14C-aspartyl aspartame at a dose level
    equivalent to 0.068 mmol/kg. 14CO2 excretion was measured for 12 h
    and urinary and faecal 14C excretion for 72 h post-dosing and plasma
    14C for 24 h post-dosing. Peak 14CO2 excretion occurred within 1 h
    post-dosing. The cumulative mean 14CO2 excretion during the 12 h
    period was 77% and 67% of the dose of the aspartame and aspartic acid,
    respectively. Faecal excretion accounted for less than 2% of the total

    dose for both compounds. Urinary excretion was low, 2.1 and 3.8% of
    the doses of aspartame and aspartic acid respectively. There was
    considerable individual variation in the plasma 14C levels. Maximum
    levels were observed 1 h post-dosing, followed by a biphasic loss of
    14C. Chromatographic separation of 14C from the plasma of aspartame
    or aspartic acid treated animals (5 h post-dosing) showed similar
    radioactive peaks. Analysis of acid hydrolysates of serum showed that
    the major 14C component was aspartic acid (Anonymous, 1972b).

         Groups each of three female rhesus monkeys between 4.2 and 6.4 kg
    were fed daily for 10 days, 0, 15 or 60 mg/kg aspartame in peanut
    butter. On day 11, a catheter was passed into the saphenous vein to
    permit withdrawal of blood samples, as well as the administration of
    L-phenylalanine-u-C14 (0.068 mmol/kg). After administration of the
    C14-phenylalanine blood samples were collected via the catheter for
    up to 7 h post-dosing and then by venous puncture for up to 10 days
    post-dosing. CO2 was collected for 7 h post-dosing. Rates of
    excretion of 14CO2 were nearly identical for animals pre-dosed with
    0 or 60 mg/kg aspartame for 10 days. Slightly less 14CO2 was expired
    from animals receiving 15 mg/kg aspartame. Plasma 14C levels
    declined during the first 30 min then peaked at 3 h. The plasma
    14C-phenylalanine disappearance in the three groups was similar. The
    appearance and disappearance of free plasma 14C-tyrosine was similar
    in all groups. The plasma 14C levels in all groups showed a similar
    pattern over the 10 day study. However, the levels of 14C in plasma
    of the group not previously exposed to aspartame was slightly lower
    than that of the test groups. Three hours post-dosing more than 90% of
    the 14C in the plasma of all groups was incorporated into protein
    (Anonymous, 1972b).

         Infant Macaque monkeys (species Macaca mulatta, M.
    fascicularis and M. arctoides) were fasted 3-4 h, lightly
    tranquillized and dosed via stomach tube with 2 g/kg bw of aspartame
    (APM) or 2 g/kg aspartame plus 1 g/kg monosodium glutamate (MSG).
    Control infant monkeys received either no fluid or water. Blood
    samples were obtained from the umbilical vein (four-day-old or younger
    monkeys) or saphenous vein (older infants) at 0, 20, 40, 60, 90, 120,
    180 and 240 min post-dosing and analysed for amino acid content. At
    the termination of the last measurement the monkeys were sacrificed
    and the hypothalami examined by light and electron microscopy. Peak
    plasma levels of aspartate in the 14 infant monkeys dosed with APM or
    APM plus MSG occurred at 60 min (23  33 mol/dl versus 0.69  0.43
    mol/dl for controls). Ninety minutes after aspartame loading, peak
    plasma phenylalanine levels reached 95  59 mol/dl versus 5.93  2.81
    mol/dl control values. The monkeys metabolized the amino acids
    somewhat more rapidly than the human. No signs of pyknotic nuclei,
    neuronal degeneration or dendritic swelling were noted in the
    hypothalami of treated infant monkeys (Reynolds et al., 1979a).

         A developmental study was performed on newborn Macaca arctoides
    reared for nine months on infant formula diets containing 1, 2 or
    3 g/kg bw of APM. Control monkeys received only infant formula or
    formula plus 1.65 g/kg bw of phenylalanine per day. There were four
    monkeys in each group and formula and water were available ad lib.
    All animals were monitored for formula and water intake, weight and
    growth. Blood samples were taken from the saphenous veins of all
    monkeys at 2, 4, 6, 8 and 9 months on study after a 4 h fast. The
    samples were analysed for serum electrolytes, osmolality, CBC and
    glucose as well as plasma amino acid content. Urine was analysed
    periodically for pH, occult blood, protein, glucose, ketones,
    bilirubin and phenylketones. Once a month the following developmental
    milestones were assessed: extent of teething; ability to vocalize;
    alertness; tractability; and general behaviour. Socialization
    with laboratory technicians was allowed to occur freely.
    Electroencephalograms were performed at the time of first dosing and
    at four and nine months on study. After removal of the monkeys from
    the experimental diets, eight animals were continued on regular
    formula for an additional month and given EEGs. Some infants were
    tested at quarterly intervals after cessation of the experimental
    diet.

         While no significant differences were noted in any groups with
    respect to formula and water intake over the course of the study, when
    a milk to water intake ratio was calculated the 1 g/kg APM group had a
    ratio significantly different (p <.05) from the other groups at five
    months on study. The 3 g/kg APM and 1.65 g/kg phenylalanine groups had
    the highest ratio of water to formula consumption. All treatment and
    control groups had similar growth rates. Blood indices, urinalyses and
    EEGs were within normal limits for all monkeys during and after the
    study. No significant differences in plasma aspartate or glutamate
    levels were seen between animal groups. Fasting plasma phenylalanine
    levels are given below:

    Plasma phenylalanine levels

                                                                        

    Treatment and dose    mol/dl  s.d.           Reference
                                                                        

    1 g/kg bw APM         8.88  5.15

    2 g/kg bw APM         28.7  48

    3 g/kg bw APM         66.2  83.3         Reynolds et al., 1979b

    1.65 g/kg bw PHE      54.4  65

    None                  5.49  1.49
                                                                        

         No behavioural deficits in treated versus control, untreated
    monkeys were found.

    TOXICOLOGICAL STUDIES

    Special studies

    Reproduction/teratological studies

    Chicken embryo

         Four hundred selected fertile eggs (White Leghorn) were randomly
    distributed into eight groups of 50 eggs each. Three control groups
    were assigned; one such group was untreated, one received only the
    thrust of the hypodermic needle, and one received a 0.05 ml injection
    of distilled water. The remaining five groups of 50 eggs each received
    one of the following agents: aspartame, 0.25 mg/egg; aspartame,
    0.5 mg/egg; calcium cyclamate, 0.5 mg/egg; calcium cyclamate,
    2.5 mg/egg; or sucrose, 0.5 mg/egg. All compounds were dissolved in
    sufficient distilled water so the total dose was contained in 0.05 ml.

         A vertical injection technique directly through the air cell
    enabled the injection of 0.05 ml of the prescribed solution directly
    into the yolk sac. The shell puncture was sealed and all eggs were
    candled daily beginning on day 6 post-injection. On day 19, all eggs
    with live embryos were transferred to hatching trays and returned to
    the incubator. On day 21, all hatched chicks were examined closely for
    gross signs of malformations. Additionally, all dead embryos observed
    during the candling procedure were examined grossly for signs of
    abnormality. On day 23, all surviving chicks were sacrificed and a
    complete gross necropsy was performed.

         Mortality rates were similar in the untreated control and the
    needle-thrust control groups (40%), in the vehicle control and the
    sucrose groups (60%) and in both aspartame treated groups and both
    calcium cyclamate treated groups (80%). No morphological abnormalities
    were observed in the embryos or hatched chicks of both aspartame
    treated groups, the sucrose group, or the intact or needle-thrust
    control groups. One malformed embryo was found in each of the vehicle
    control and cyclamate treated groups; due to the frequency (single
    incident) and the nature of the abnormalities little biological
    significance was attached, however, to these findings. No
    abnormalities were observed in any of the cyclamate treated hatched
    chicks (Anonymous, 1970a).

    Rat

         Low and high dose groups of 14 male and 30 female rats (Charles
    River Caesarean derived) received 2 or 4 g/kg/day of aspartame
    administered in the diet throughout the pre-mating, gestation and
    lactation period, and intragastrically during the mating period. A
    concurrent control group of 14 males, 48 females received basal diet

    or vehicle, as appropriate. For mating purposes, 12 males from each
    group were randomly subdivided into 36 units and mated with three
    females. This mating design (CC, LL, HH; CL, CH, LC, HC) enables
    comparison within each mating unit as well as between treatment levels
    and facilitates precise identification of any affected parent. Sires
    were sacrificed after completion of the mating period. Fifty per cent.
    of the dams from each dose level were sacrificed on day 13 of
    gestation; ovaries, uterus and uterine contents were examined. The
    remaining dams proceeded through natural delivery and lactation. The
    progeny were thoroughly examined at birth and periodically thereafter
    for evidence of maldevelopment. Ophthalmoscopic examinations were
    performed on all weaned pups. Pups were sacrificed at weaning (21 days
    old) or shortly thereafter. Compound ingestion closely approximated
    the indicated dosages, except during the latter half of lactation,
    when it increased to 2.7 and 5.9 g/kg/day. Aspartame had no effect on
    parental survival rate, mating performance, fertility, or paternal
    body weight gain. Paternal food consumption was significantly
    depressed during approximately one-half of the pre-mating period.
    Maternal food consumption was unremarkable during the pre-mating and
    lactation periods, but showed a variable, statistically significant
    reduction at the low dose level only during much of gestation.
    Maternal body weight was unremarkable during the pre-mating, gestation
    and lactation periods, but exhibited a variable, statistically
    significant reduction at the high dose level only during
    mid-gestation. Hysterotomy, litter examination, and the neonatal
    examination data were all unremarkable. Evaluation of the reproductive
    performance of the neonates (F2 generation study) was also normal
    (Schroeder et al., 1972).

         A two-generation reproduction study was performed in the rat to
    evaluate general reproductive performance in P1 and P2 generations
    continuously ingesting aspartame. Dosage levels of approximately 2 and
    4 g/kg/day were employed throughout the study, with a concurrent
    control group of equal size receiving the basal diet only. Rats were
    randomly distributed into three groups of 12 males and 24 females
    each; this constituted the first parental generation. Each group
    received the appropriate diet for nine weeks prior to mating, then
    throughout the mating phase and the interval prior to sacrifice. Each
    litter produced was arbitrarily reduced to a maximum of 10 pups within
    24 h of birth. Thirty males and 60 females from this F1 group were
    utilized as the P2 generation for continuation of the study.
    Following a nine week pre-mating treatment period the P2 animals were
    mated to produce F2A litters. All F2A litters were reduced to a
    maximum of eight pups within 24 h of birth. Five F2A litters per group
    were utilized for a separate neonatal clinical pathology study (Entry
    E-9); the remaining 15 litters per group were utilized for producing
    F2A weaning data. All pups were sacrificed at 21 days of age and
    gross necropsies performed on approximately 35% of the pups from 10
    litters per group. Full sets of wet tissues were preserved. All
    remaining weanlings and all P2 generation males and females were
    sacrificed and discarded. Mean food consumption body weight, survival,

    physical appearance and behaviour of the parental generations were
    comparable between control and both treated groups. Indices of
    fertility, gestation, live birth, litter size at weaning, as well as
    appearance, behaviour, physical examination data and gross necropsy
    data from weanling animals, were also comparable between control and
    both treated groups. Growth among the low dose level pups compared
    well to the controls during both reproduction phases. The only
    evidence of a treatment-related effect was a statistically significant
    reduction in the body weight of weanling rats of both generations
    (F1A and F2A) in the high dose group (Anonymous, 1971).

         Female rats were housed in groups of three plus a male of proven
    fertility. Twenty-four mated females were assigned to each of three
    groups, receiving 0, 2 or 4 g/kg/day of aspartame in the diet from
    gestation days 6 through 15. On gestation day 20 each female was
    sacrificed and the ovaries, uterus and uterine contents examined.
    Foetuses were examined externally and preserved intact for subsequent
    examination for soft tissue abnormalities or for skeletal anomalies.
    Forty-seven litters (589 term foetuses) from treated females were
    examined. Maternal survival, body weight, and necropsy findings were
    comparable between control and treated groups. Food consumption was
    unremarkable at the low dose level, but significantly decreased in the
    high dose group throughout the treatment period (gestation days 6-15).
    The mean number of resorption sites, foetuses per pregnant female, and
    viable foetuses was similar in both treatment groups. Likewise, foetal
    sex distribution, body weight and length, and crown-rump distance were
    unremarkable at all dose levels. In short, no evidence of treatment-
    related anatomical alterations was observed.  Thus, continuous dietary
    administration of aspartame to the primigravid rat, employing dosages
    up to 4 g/kg/day during the sixth to fifteenth day of gestation,
    exerts neither embryotoxic nor teratogenic effects in the developing
    foetus, nor does it affect the maternal rat adversely (Schroeder &
    McConnell, 1970).

         Three groups of 24 pregnant rats each received 0, 2.5 or
    4.4 g/kg/day of aspartame administered in the diet from gestation
    days 14 through 21 (parturition), and 0, 3.6 or 6.8 g/kg/day from
    postpartum days 1 to 21 (weaning). Twenty-one litters (246 pups) from
    the control group level, 20 litters (236 pups) from the low dose group
    level, and 22 litters (289 pups) from the high dose group level were
    available and received physical examinations at birth. Maternal food
    consumption, behaviour, morbidity, and mortality were comparable
    between control and both treated groups. Maternal body weights were
    also comparable between groups during gestation, but body weight gain
    during lactation was significantly depressed in the high dose group.
    Duration of gestation, litter size and live birth indices and physical
    examination data at birth were unremarkable at both treatment levels.
    Pup body weights at birth were significantly reduced at the high dose
    level; at weaning, body weights of males were unremarkable but females
    weighed significantly less at both dose levels. Weanling pups survival
    was significantly depressed at the high dose level. Weanling physical

    examination data revealed that the low dose pups were unremarkable but
    the 3% (5/164) of the high dose pups exhibited incompletely opened
    eyelids and 1% (2/164; a single litter) exhibited grossly observable
    lens opacities (Schroeder et al., 1973e).

         In another study aspartame and DKP in a 3:1 ratio was fed to
    rats. Females were housed in pairs with a male of proven fertility.
    Thirty mated females were assigned to each of four groups, receiving
    0, 1, 2 or 3 g/kg/day of the 3:1 mixture in the diet from the sixth
    through the fourteenth day of gestation. On gestation day 19 each
    female was sacrificed and the ovaries, uterus and uterine contents
    examined. Foetuses were examined externally and preserved intact for
    subsequent examination; approximately 33% of each litter was examined
    for soft-tissue abnormalities and the remaining 67% for skeletal
    anomalies. Eighty-two litters (1026 term foetuses) from treated
    females were examined. Maternal survival, body weight and food
    consumption were comparable between control and treated groups. The
    mean number of implantation sites, resorption sites, number of
    foetuses per pregnant female, live foetuses, or dead foetuses were
    similar in all treated groups. Foetal sex distribution, body weight
    and crown-rump distance exhibited no biologically significant
    alterations at any dosage level, nor was there any treatment-related
    anatomical alterations (Schroeder et al., 1972).

    Rabbit

         A total of six studies have been performed on the New Zealand
    White rabbit; five studies involved the administration of aspartame
    only while the sixth study involved administration of a 3:1 (w/w)
    mixture of aspartame:DKP. In all studies pooled sperm specimens were
    employed in the artificial insemination procedure.

         Food consumption was recorded daily and animals weighed
    periodically. All animals were sacrificed at term, partial necropsies
    performed and foetuses examined externally. Approximately one-half of
    the foetuses from each litter were processed for soft tissue
    examination. Viscera were removed and examined from the remaining
    animals and the carcasses processed for skeletal examination.

         Three Segment II rabbit studies involving intragastric
    administration were carried out. In each study an aqueous suspension
    of the test material was administered in equal quantities twice daily
    at 3-5 h intervals, employing a vehicle of 1% aqueous Tween 80 (v/v).
    Treatment was carried out on days 6-18 of gestation. Survival rates
    were frequently suboptimal, resulting both from technical difficulties
    encountered in intubating the rabbit and from intercurrent pulmonary
    infections. In the initial study a mixture of aspartame and DKP (3:1
    ratio) was administered at daily dosage levels of 1, 2 or 3 g/kg/day
    of the mixture. The concurrent control group was fed ad lib. In the
    other studies aspartame was administered at a single dose level of 2
    g/kg/day and the control group was pair-fed with those treated females
    consuming the lowest amount of food. There was a non-dose related
    decrease in the maternal survival rate of treated animals in one

    study. It is unlikely this was treatment related, but rather reflects
    intercurrent disease and technical difficulties with the compound
    administration procedure.

         The cumulative mean conception rate was slightly lower in the
    treated animals (70%) than in the concurrent control groups (85%).
    Much of this disparity can be traced to one study, which exhibited a
    reduced ovulation rate especially notable in the treated group.
    Cumulative mean rates of survival to term, abortion and premature
    delivery were similar between control and treated groups. The mean
    number of non-viable foetuses per litter was reduced in the treated
    groups. Other parameters evaluated were essentially comparable between
    control and treated animals.

         The foetal examination data reveal that the incidence of major
    foetal malformations was lower in the aspartame treated rabbits than
    in the controls, but was similar to the incidence in a historical
    control group. The incidence of anomalies in the concurrent control
    group of one study was unusually high, however, and this is reflected
    in the increased incidence observed in the compiled concurrent control
    data. The total number of litters and foetuses examined from aspartame
    treated animals (63 and 486, respectively) closely approximates the
    number in the historical control group (79 and 573, respectively) and
    is notably larger than the concurrent control group (34 and 263,
    respectively). Roughly similar fractions of each litter were examined
    by the two techniques employed (soft tissue examination by the
    Wilson's procedure, and skeletal examination of the maxillary bones,
    vomer irregularities, and cleft palate); one foetus exhibited
    underdeveloped frontal skull bones, a partial cleft palate, and
    eyelids fully opened at delivery; one foetus showed absence of the
    maxillary and mandibular bones, two missing cervical vertebrae and two
    split thoracic vertebral centra; and six foetuses exhibited
    microstomia and cleft palate and/or syn- or oligo-dactyly. Thus
    aspartame or aspartame/DKP mixture was neither embryotoxic nor
    teratogenic to the albino rabbit foetus when administered
    intragastrically at the doses studied during the mid-portion of
    gestation. It also lacked notable adverse effects on the maternal
    animal. The remaining three teratology studies on the rabbit involved
    administration of specially pelleted rabbit diet containing 3.28% or
    6.08% aspartame. These pellets were prepared commercially (Teklad,
    Inc., Monmouth, Ill.). Chemical and microbiological analyses of the
    specially pelleted diets were performed at periodic intervals
    throughout the study. The rabbits received the test diet from days
    6-18 of gestation. The ingested mean dosage of aspartame was 1.1 and
    1.9 g/kg/day.

         All rabbits were sacrificed at term and the uterine contents
    examined. Parameters evaluated in this study included maternal
    survival rates, conception rates, body weights and hysterotomy
    findings; litter size and viability; foetal size, sex distribution and
    morphological development.

         Maternal survival, conception, premature delivery rate and body
    weight data during gestation were comparable between the control and
    treated groups.

         Mean food consumption during the treatment period was similar for
    the two treatment groups.

         In utero litter size and resorption data were comparable
    between the control and both treated groups. Similarly, foetal sex
    distribution, body weight and crown-rump distance data were comparable
    between these same groups.

         External, soft tissue and skeletal examinations of the 181
    control, 151 low and 146 high dose foetuses recovered at term
    sacrifice were generally unremarkable. Major malformations were
    observed, in one foetus each from the control, low and high dose
    groups. Minor malformations were observed in an additional four high
    dose foetuses from two litters. A minor foetal malformation designated
    as separated eyelids was observed in three of five foetuses from one
    high dose litter. This malformation was not observed in the concurrent
    control group nor has it been observed historically in this
    laboratory. Its significance is not clear. No major compound-related
    embryotoxic or teratogenic effects were observed (Anonymous, 1973a and
    b, 1974b; Schroeder & McConnell, 1973; Schroeder et al., 1973f and g).

    Mutagenicity studies

    Microbial systems

         Aspartame at doses of 10-5000 g/plate was tested in the Ames
    Salmonella/microsome mutagenicity screening assay in strains
    TA-1535, TA-1537, TA-1538, TA-98 and TA-100, with and without S-9
    activation. The assay was repeated once using DMSO as the solvent and
    negative control both times. No toxicity to the organisms was seen
    even at the highest dose tested, but the compound precipitated on the
    agar plates at 10 000 g. No aspartame induced increase in HIS- to
    HIS+ reversions was seen either in the presence or absence of the
    activating system. The positive control mutagens (sodium azide,
    9-aminoacridine, 2-nitrofluorine and 2-anthramine) did induce
    significant numbers of reversions in the tester strains (Simmon &
    Shan, 1978).

    Dominant lethal

         Aspartame was administered orally as a 10% suspension at 2 g/kg
    to 15 male rats (Charles River CD strain) in two divided dosages two
    hours apart on a single day. The vehicle was 1% Tween 80 in distilled
    water. The aspartame used contained 0.2% diketopiperazine (DKP). The
    negative control was Tween 80 as a 1% solution (v/v) and was given to
    15 males by the same route and dosage regimen as the aspartame. The
    positive control methylmethane sulfonate (MMS) was given to five males
    i.p., as a 0.7% suspension (w/v) in corn oil. The dosage was 25 mg/kg.

    Two females per week were mated to each male for eight weeks and
    autopsied 14 days after positive indications of mating. Fertility was
    reduced in the fourth week in males treated with aspartame. The
    fertility in all other time periods was normal. Females mated in the
    third and eighth week to aspartame treated males had a reduced number
    of corpora lutea, and significantly greater number of corpora lutea,
    respectively. The number of corpora lutea for all other time periods
    was normal. The number of implantations per female was unaffected by
    aspartame as well as by the positive control MMS.

         The positive control (MMS) produced a statistically significantly
    greater number of foetal deaths in weeks 2, 3 and 4. MMS also produced
    a statistically significant increase in the number of foetal
    deaths/pregnant female in week 6. Females mated to aspartame treated
    males in week 3 showed a statistically significant decrease in the
    number of live embryos, but the decrease is a result of fewer
    ovulations and as such is not considered important. Females mated to
    males in weeks 2, 3 and 4 showed a statistically significant decrease
    in the number of live embryos which indicates that the positive
    control was eliciting strong mutagenic response. This study was
    repeated with rats using MMS (positive control) at a dose level
    equivalent to 50 mg/kg. The aspartame contained 0.75% DKP. The
    fertility was significantly reduced in the fourth week in males
    treated with the positive control. All other time periods showed
    normal fertility. The number of corpora lutea was significantly
    reduced in weeks 2, 3 and 4 in females mated to positive control-
    treated males. There was no significant fluctuations in this parameter
    at any other time period. There was a significant decrease in the
    number of implantations in the fourth week in females mated to
    aspartame treated males. This parameter was significantly reduced in
    weeks 2, 3, 4 and 5 in females mated to positive control-treated
    males. All other time periods were unaffected. Females mated to
    positive control-treated males showed a significant increase in this
    parameter in weeks 1, 2, 3 and 5. All other time periods were
    unaffected. Females mated to aspartame treated males in week 4 showed
    a significant decrease in the number of viable foetal swellings. This
    effect appears spurious. Females mated to positive control treated
    males in weeks 1, 2, 3, 4 and 5 showed significant decreases in this
    parameter (Schroeder et al., 1973a).

    In vivo cytogenetic studies

         Holtzman strain rats were divided into eight groups of 10 rats
    each and received either no treatment, positive control triethylene-
    melamine, 0.5 mg/kg i.p./day if only positive control, days 1-5
    cyclohexylamine 10 mg/kg/day or 50 mg/kg/day or SC-18862 days 1-5,
    0.4 g/kg/day, 0.8 g/kg/day, 1.2 g/kg/day or 1.6 g/kg/day by gastric
    intubation for five days. Chromosomal aberrations in bone marrow and
    spermatogonial cells were evaluated. The positive control,
    triethylene-melamine produced a statistically significant increase
    in the number of cells with chromosomal aberrations. Neither
    cyclohexylamine nor aspartame had any significant cytogenetic effects
    (Anonymous, 1970b, 1972k).

         In another study aspartame (10% w/v) suspended in 10% Tween 80
    was administered intragastrically in three equally divided dosages
    every three hours to Purina Caesarian derived strain male albino rats.
    Dosages were 0.5, 1.0, 2.0 and 4.0 g/kg/day for five days. The
    positive control was triethylenemelamine 0.5 mg/kg. It was
    administered i.p., as a suspension in 1% Tween 80 in distilled water,
    on the final day of treatment for the other groups. The vehicle
    control received 40 ml/kg/day. Chromosomal aberrations in bone marrow
    were evaluated. All groups except the positive control lost weight
    during the five day treatment. The negative controls consumed less
    food than any other group. The positive control produced a
    "statistically significant" increase in cells with aberrations. No
    significant increases were observed utilizing aspartame (Anonymous,
    1970a).

    Host mediated assay

         Aspartame was administered orally to male rats (Purina Caesarean
    derived) approximately 12 weeks old, at four dose levels: 0.25, 0.50,
    1.0 and 2.0 g/kg/day in three equally divided doses for five days;
    the vehicle control was given at a level of 40 ml/kg/day. DMNA was
    administered i.p. at a level of 100.00 mg/kg on day 5 only.
    S. typhimurium G-46 was injected i.p. on day 5, 30 min after
    administration of the test compounds. The rats were sacrificed 3 h
    after S. typhimurium injection; their peritoneal cavities were
    aseptically exposed and washed with 2.0 ml sterile saline. As much
    fluid as possible was removed from the cavity. Peritoneal washings
    were diluted and plated in accordance with generally accepted
    procedures. A total of 6  108 CFU of S. typhimurium G-46 was
    injected into each rat. Recoveries from the peritoneal cavity were
    extremely low - approximately 1/10-1/1000 of the original inoculum -
    indicating that the bacteria were being rapidly killed. A number of
    the recoveries, 0.08, 0.10, and 0.12, and 0.16  107 CFU/ml of
    exudate translate to counts of 5, 7 and 10 colonies/three plates at
    the 105 dilution, respectively. Mutant cell values of 1.6, 3.3 and
    5.0 CFU/ml of exudate translate to counts of 1, 2 and 3 colonies/three
    plates, respectively. As a consequence of the low cell recoveries,
    only seven of 10 rats were usable from the 0.25 and 0.50 g/kg/day
    groups and 5 of 10 from the 2.0 g/kg/day group.

         The mean mutation frequencies (MF) were:

         Negative control:    4.39    4.47   10-7
         Positive control:   35.97   22.18   10-7
         0.5 g/kg/day:        2.48    1.78   10-7
         1.0 g/kg/day:        6.40    3.67   10-7
         2.0 g/kg/day:        2.93    4.82   10-7
         4.0 g/kg/day:       10.96    6.48   10-7

    (Anonymous, 1972e and l).

    Carcinogenicity studies (see also long-term studies)

         Groups each of 200 female 60-90-day-old Swiss albino mice were
    used for a urinary bladder tumorigenicity study by the intravescical
    pellet implant technique. Pellets of 20-22 mg of purified cholesterol
    (80%) and aspartame (20%, 4.0-4.4 mg) were prepared and surgically
    placed into the urinary bladder. The negative control group was
    exposed to pellets of cholesterol and the positive control group to
    pellets of cholesterol and the 8-methyl ether of zanthurenic acid. The
    study was for 56 weeks. Parameters measured included morbidity,
    mortality, motor and behavioural activity, growth, general external
    features and digital palpation of protruding tissue masses. All
    animals dying during the experiment, or at the termination of the
    study were subjected to necropsy and histopathological inspection of
    the bladder. No bladder neoplasia were observed in animals dying or
    killed prior to 175 days of the study. The following incidence of
    bladder neoplasia was recorded in mice surviving 175 days or more:
    negative control 17/155, aspartame 13/123 and positive control 40/111
    (Bryan, 1974a).

    Neurological effect studies

         Mice A/JAX-ICR hybrids between six and 10 days of age of both
    sexes, were administered by gastric intubation, aspartame as a 10%
    aqueous solution at dose levels equivalent to 0.25, 0.5, 1.0, 1.5 and
    2 mg/g. Three hours post-dosing the mice were killed and the brains
    prepared for microscopy. At 2 mg/g and 1.5 mg/g lesions were observed
    in midline structures, namely the hypothalamic arcuate nucleus, the
    subfornical organ and the area of the postrema. Less damage was
    observed at the 1.0 mg/g level, and at 0.5 and 0.25 mg/g no neuronal
    lesions were observed (Lemkey-Johnson et al., 1977).

    Special studies on DKP (5-benzyl-3,6-dioxo-2-piperazine acetic acid)

    BIOCHEMICAL STUDIES

    Metabolism

         All studies were carried out with 14C-labelled DKP prepared from
    14C phenylalanine aspartame.

    Rat

         14C DKP was incubated with rat plasma. There was no significant
    effect. Male rats (300 g) were dosed orally with 0.5 ml of an aqueous
    solution of 14C DKP (10 mg/animal). Blood samples were taken at 2,
    3 and 4 h post-dosing. Four hours post-dosing the animals were
    sacrificed and the GI tract removed and divided into stomach, small
    intestine and colon. The major part of the administered dose of 14C
    was present in the colon (40-50%), and stomach (8-18%). Less than 5%
    being present in the small intestine. Only trace amounts of 14C were

    detected in the plasma (less than 0.1% of the administered dose).
    Separation of the plasma 14C by chromatographic techniques indicated
    the presence of many components (Anonymous, 1972a).

         In another study fasted rats were administered a single dose of
    14C DKP (10 mg/animal), and tissue distribution of 14C determined 2,
    4, and 6 h post-dosing. The tissues studied were lung, spleen, kidney,
    skeletal muscle, brain, heart, plasma, stomach, small intestine and
    colon. The contents of the stomach, small intestine and colon were
    also sampled. Only trace amounts of 14C were present in the tissues;
    lungs and spleen contained little radiolabel. The colon, plasma, liver
    and small intestine contained small but significant amounts of 14C.
    Analysis of the stomach, colon and small intestine for 14C compounds,
    indicated significant amounts of aspartyl-phenylalanine in the stomach
    contents, significant amounts of unchanged DKP, aspartyl-phenylalanine
    and tyrosine were found in the contents of the colon, and
    phenylalanine methyl ester was present in the intestinal contents.
    Plasma radiolabel at 2 h or more presumably contained 14C
    phenylalanine-containing protein. Liver 14C consisted of
    phenylalanine and a phenylalanine-containing protein (Anonymous,
    1972a).

         In another study male rats (Charles River strain) were dosed
    orally with 14C DKP at a level equivalent to 10 mg/animal. Urine was
    collected for 18 days, and plasma samples for four days, post-dosing.
    About 25% of the dose was excreted in the first 24 h, with smaller
    amounts during the rest of the test period (cumulate excretion was
    less than 30% of the administered dose). Plasma 14C levels were low,
    but there was a possible biphasic response (Anonymous, 1972a).

         In another study 300 g male rats were subjected to bile duct
    cannulation and were given 10 mg of 14C DKP intragastrically. Bile
    was collected for 48 h. About 1% of the administered 14C was excreted
    in the bile in this period (Anonymous 1972a).

         In another study germ-free rats (Charles River, CD strain) were
    removed from their germ-free environment and administered a single
    oral dose of 20 mg 14C DKP. Urine and faeces were collected for 48 h
    post-dosing. The metabolic profile (urinary) was quite different from
    that of neomycin or control animals. 89% of the 14C in the urinary
    extracts was unchanged SAIB. However, after keeping the rats for 30
    days in a regular animal room environment, administration of 14C DKP
    gave rise to metabolic urinary products similar to those of control
    animals (Anonymous, 1974a).

         DKP was incubated under aerobic or anaerobic conditions with
    faecal suspensions prepared from the faeces of male Charles River
    rats. No bacterial degradation occurred (Anonymous, 1974a).

         Male Charles River rats that had previously been dosed with
    neomycin sulfate, 25 mg/day for five days, received by oral intubation
    20 mg/kg 14C DKP. Urine and faeces were collected for 48 h. Unchanged
    DKP and two metabolites were identified in urine, hippuric acid and
    benzoic acid. Quantitatively and qualitatively, the amount of these
    compounds present in urine was similar to that observed in rats that
    had not been treated with neomycin (Anonymous, 1974a).

    Rabbit

         Eight young adult female New Zealand white rabbits were dosed by
    gavage, daily, with DKP (17 mg/kg bw) then following an overnight fast
    with a dose of 14C DKP. Four rabbits were used to measure 14C levels
    and plasma metabolites, blood samples being taken at 0.5, 1, 2, 3, 4,
    6 and 24 h post-dosing. The other four rabbits were used to measure
    14CO2 and urinary and faecal excretion of 14C. CO2 was collected up
    to 7 h post-dosing, and urine and faeces up to 96 h post-dosing.
    At the termination of the study all animals were sacrificed and
    autopsied. 14CO2 expiration was extremely low. The total cumulative
    14CO2 collected was 0.2-0.65% of the administered 14C. Plasma 14C
    levels were extremely low, 0.1-0.2% of the administered 14C dose. No
    characterization of the 14C content of the plasma was possible. 5-33%
    of the 14C was excreted in the urine in 96 h, with a maximum
    occurring in the 24-48 h period. Chromatographic separation of
    extracts of the urine indicated the presence of a single unidentified
    peak. The rabbits excreted about 19% of the 14C in the faeces in
    96 h, with the maximum levels occurring in the first 24 h post-dosing.
    Methanol extracts of the faeces showed a steady decrease of the major
    14C peak with time and appearance of other peaks. 14C DKP was
    identified in the faeces but the other metabolites have not been
    characterized (Anonymous, 1972b).

    Monkey

         Four female rhesus monkeys (4-6 kg) were given by intubation
    0.068 mmol 14C DKP. CO2 was collected for 12 h post-dosing. Plasma
    14C and excretion of 14C in the urine and faeces, was determined
    during the 120 h post-dosing period. About 1% of the administered dose
    was excreted as CO2. 48% of the administered 14C was excreted in
    the urine, with 30% being excreted in the first 24 h. 36% of the
    administered DKP was excreted in the faeces, the majority being
    excreted in the first 48 h. The 14C level in plasma was low, the peak
    level at 12 h was 1.61% dose/litre plasma. The 14C exhibited a
    biphasic response. One peak occurred at 2 h, another at 12 post-
    dosing. 26% of the extractable urinary radioactivity was identified as
    unchanged DKP and 58% as the major metabolite phenylacetylglutamine.
    Methanol extracts of the 14C-labelled material in the faeces
    contained 13% phenylalanine and 62% unchanged DKP. The other 14C
    materials were not identified (Anonymous, 1972a). In a later study
    phenylacetic acid was identified as a metabolite of DKP in the rhesus
    monkey (Anonymous, 1974a).

         In another study two female rhesus monkeys were infused via the
    saphenous vein with 14C DKP at a dose level equivalent to 0.068
    mmol/kg. 14C plasma levels were determined up to 12 h post-dosing.
    Urine was collected at one hour intervals, for up to 8 h post-dosing,
    by removing urine collected in bladder and then rinsing with 10 ml
    saline. Urinary collection was continued in a normal fasting for a
    further 64 h. Faeces were collected for 14C assay for a 72 h period.
    14C peaked in the plasma in less than a minute, and then showed a
    very rapid decrease. There were 3 phases of elimination of 14C from
    the plasma, namely, at t1/2 of 0.042 h, 0.40 h and 9 h. Approximately
    100% of the administered 14C was recovered in the urine with most of
    the radiolabel being excreted in the first 3 h. Chromatographic
    examination of the 14C in the serum and urine indicated that most of
    the 14C (approximately 97%) was present in unchanged DKP (Anonymous,
    1972a).

    Man

         Three male subjects (age 25-55 years) were fasted overnight prior
    to administration by mouth 14C DKP (97 mg in 120 ml water). Blood and
    urine samples were taken prior to dosing and then at various intervals
    up to 72 h post-dosing,. Maximum 14C levels were observed in the
    plasma 1 h post-dosing, with a second peak being observed 12 h post-
    dosing. It was estimated the plasma level of 14C DKP at 1 h was
    70 g/l. The plasma 14C disappearance half life of the first phase
    was about 2 h and the second phase 30 h. 14C was present in the urine
    within 4 h post-dosing. At the end of three days no additional 14C
    was excreted in the urine. A mean of 48.3% of the administered 14C
    was excreted during this period. Analysis of the 14C compounds
    present in the pooled 3-day samples of urine showed that about 12% of
    the extractable 14C was in the form of unchanged DKP, 29% was in the
    form of phenylacetylglutamine. In addition there were two other
    unidentified 14C compounds (Anonymous, 1972a). In a later study
    phenylacetylglutamine was identified as major urinary metabolite of
    DKP in man (Anonymous 1974a).

    Acute toxicity

                                                                        

                                LD50                Reference
    Animal        Route      (mg/kg bw)
                                                                        

    Rat           Oral         >5000           Andress et al., 1973a
                  i.p.         >1562           Andress et al., 1973a

    Mouse         Oral         >5000           Andress et al., 1973a

    Rabbit        Oral         >5000           Andress et al., 1973a
                                                                        

    Short-term studies

    Mouse

         Groups of 10 male mice received 0 and 1000 mg/kg/day of DKP
    intragastrically for two weeks. All animals survived. No significant
    differences were noted between control and test animals in terms of
    body weight, food consumption, physical appearance and behaviour.
    Haematology showed a decrease in total wbc due apparently to a marked
    decrease in polymorphonuclears and blood chemistry showed a
    significant decrease in glucose and non-significant decrease in BUN
    and bilirubin. Aside from a significantly increased weight of the
    seminal vesicles no other effects were noted upon organ weights nor
    was there any increase noted grossly or microscopically in the
    incidence of severity of lesions seen in treated as compared to
    control animals (Rao et al., 1971a).

    Rat

         Groups of 5 male and 5 female rats received 0 and 1000 mg/kg/day
    of DKP intragastrically for 2 weeks. No deaths or adverse physical or
    behavioural effects were noted. No effects were seen in the urinalysis
    findings. Transient depression of weight gain and food consumption was
    seen in treated females at one week. Haematology showed only a small
    percentage increase in polymorphonuclear leucocytes and decrease in
    lymphoocytes. A non-statistically significant lowering of serum BUN,
    SGPT, bilirubin and small but significant lowering of K+ was seen in
    the treated groups. Decreased male heart and increased prostate
    weights were noted at autopsy. No treated related evidence of
    microscopic tissue lesions were noted (Rao et al., 1971b).

         Groups of 5 male and 5 female rats received dietary levels of 0,
    1000, 2000, 4000 and 6000 mg/kg/day of DKP for 5 weeks. Each dietary
    group was run in duplicate. No mortality occurred. No dose-related
    variations in body weights of food consumption were observed. Terminal
    body weights and food consumption were decreased at the highest dose
    level, this effect being statistically significant for the females. No
    adverse physical or behavioural effects were noted in the treated
    animals nor were compound related eye lesions apparent in the
    ophthalmoscopic examination made at termination. Haematology and
    plasma chemistry did not indicate any consistent treatment related
    variations. No clear dose-related effects were noted in terms of
    organ weights aside from decreased male, heart weight. This was
    statistically significant only at the high dose level but was dosage
    related except for the 1000 mg/kg/day level. Other gross and
    microscopic pathology observations were not indicative of compound
    related effects (Rao et al., 1972a).

    Long-term studies

    Rat

         Groups of 6 male and 6 female rats received dietary levels of
    750, 1500 and 3000 mg/kg/day of DKP for 115 weeks. A group of 12 male
    and 12 female rats served as controls. Each group was replicated 6
    times. No effects were reported as being seen in terms of physical
    appearance and behaviour, nor was there evidence that the compound
    produced any effects in terms of survival. A consistent pattern of
    dosage-related decreased weight gain was seen in both sexes. These
    were reported to be statistically significant as follows:

                                                                        

                              Weeks of decreased body weight gain
      Dosage                                                            
    (mg/kg/day)                Males                 Females
                                                                        

      3000                     2-100              16-termination
      1500                     24-64              27-84
       750                     24-68              Not significant
                                                                        

         A fairly consistent, statistically significant pattern of
    increased food consumption was noted for high level males. Increased
    food consumption for the high level females was seen only during the
    second experimental year. For the lower feeding levels food intakes
    which differed significantly from control values were sporadic. No
    evidence of effect was seen in terms of haematology. Clinical
    chemistry findings were similarly without evidence of compound effect
    aside from an apparent statistically significant decrease in serum
    cholesterol seen to persist in the high level groups. Urinalysis
    findings were not remarkable aside from a significant drop in pH seen
    persistently in the high level females and sporadically in other
    groups. Ophthalmological findings also failed to disclose any evidence
    of compound related changes, on autopsy, findings with respect to
    organ weights were not remarkable. Gross and microscopic pathology as
    reported did not indicate the presence of tumorigenic or non-
    tumorigenic changes which would be attributable to the administration
    of DKP except for a dosage related increase in uterine polyps whose
    numbers were significantly increased over controls for both the
    intermediate and high dosage level groups. This observation will
    require further clarification (Rao et al., 1974).

    Mouse

         Groups of 36 male and 36 female mice received dietary levels of
    250, 500 and 1000 mg/kg/day of DKP for 110 weeks. A group of 72 male
    and 72 female rats served as controls. No evidence of compound induced
    effect was reported as being evident in terms of appearance and

    behaviour as well as weight gain and food consumption or eye lesions.
    Blood counts were not remarkable. Clinical chemistry results were not
    suggestive of compound related effect. At sacrifice thyroid weight and
    ratio of thyroid weight to body weight for the intermediate and high
    dose level females were significantly elevated over control values.
    Gross and microscopic pathology reported as not suggestive of
    tumorigenic or non-tumorigenic changes which might be attributable to
    the feeding of DKP (Anonymous, 1974c).

    Reproduction studies

    Rat

         Low, medium and high dose groups of rats (Charles River CD
    strain) 14 males and 28 females received 0.45, 0.9 or 1.8 g/kg/day of
    DKP administered in the diet throughout the premating, gestation, and
    lactation periods, and intragastrically during the mating period. A
    concurrent control group of 14 males and 60 females received either
    basal diet or the diluent. For mating purposes, the rats were randomly
    subdivided into 48 mating units each containing one male and three
    females. This male-female cross-mating design is the same as that
    described in the aspartame studies. Sires are sacrificed after
    completion of the mating period. 50% of the dams from each group are
    sacrificed on gestation day 14; ovaries, uterus, and uterine contents
    are examined. The remaining dams proceed through natural delivery
    and lactation. The progeny are thoroughly examined at birth
    and periodically thereafter for evidence of maldevelopment.
    Ophthalmoscopic examinations are included. Pups are sacrificed at
    weaning (21 days old) or shortly thereafter. DKP had no effect on
    parental survival rate, food consumption, mating performance,
    fertility, or on paternal body weight gain. Maternal body weights were
    unremarkable at low and medium dose levels, but significantly
    depressed during mid-gestation (day 14) and lactation (days 14 and 21)
    in the high dose animals. Hysterotomy, litter examination, and
    neonatal data were all unremarkable, excepting a slight but
    significant decrease in mean viable litter size in the high dose group
    (Schroeder et al., 1973c).

         Four groups of 20 pregnant rats (Charles River CD strain) each
    received 0, 0.7, 1.3 or 2.5 g/kg/day of DKP administered in the diet
    from gestation day 14 through postpartum day 21 (weaning). Following
    physical examination at birth, each litter was arbitrarily reduced to
    a maximum of 8 pups. Maternal food consumption, body weight gain,
    behaviour, morbidity, and mortality were comparable between control
    and all treated groups. Likewise, duration of gestation, litter size
    and live birth indices, and weanling pup survival, body weight gain,
    and physical examination data (including ophthalmoscopic exams) were
    normal in all treatment groups. No treatment-related anatomical
    abnormalities were observed (Schroeder et al., 1973c).

    Teratological studies in the rat (Charles River CD strain)
    Diketopiperazine (DKP)

         Female rats were housed in groups of four with a male of proven
    fertility. Twenty-four mated females were assigned to each of four
    groups, receiving 0, 1, 2, or 4 g/kg/day of DKP in the diet from the
    sixth through the fifteenth day of gestation. On day 20 each female
    was sacrificed and the ovaries, uterus, and uterine contents examined.
    Foetuses were examined externally and preserved intact for subsequent
    examination for soft tissue abnormalities or for skeletal anomalies.
    Fifty-seven litters (711 term foetuses) from treated females were
    examined. Maternal survival, conception, body weight, and food
    consumption were comparable between control and treated groups. Mean
    number of resorption sites was unremarkable at the low and medium dose
    levels, and significantly decreased at the high dose level. The mean
    number of foetuses per pregnant female was likewise unremarkable at
    the low and medium dose levels, and significantly increased at the
    high dose level. Foetal sex distribution, body weight, and crown-rump
    distance were unremarkable at all dose levels. No evidence of
    treatment-related anatomical alterations was observed (Schroeder et
    al., 1973d).

    Teratological studies in the rabbit

         Four groups comprised of 21 artificially inseminated females of
    the New Zealand white strain received aqueous suspension of DKP by
    gastric intubation at dosages of 0.5, 1, or 2 g/kg/day from the sixth
    day of gestation through day 18. Controls received diluent only. Body
    weights were recorded periodically (7 intervals) and food consumption
    was measured daily. All animals were sacrificed at term (gestation day
    28 or 29). Partial necropsies were performed, and foetuses examined
    externally. Approximately one-half the foetuses from each litter were
    processed for soft tissue examination. Viscera from the remainder were
    removed and examined. The carcasses were processed for skeletal
    examination. Approximately 40-50 foetuses were processed from each
    group except the high dose from which only 4-5 foetuses were available
    for each type of examination. Survival rates were sub-optimal but
    comparable in the control, low, and medium dose groups, with 20-30%
    mortality per group. At the high dose level 90% of the rabbits died.
    Pulmonary aspiration of compound suspension and gastric perforation,
    resulting from technical difficulties encountered in intubating the
    animals, clearly contributed notably to the mortality rate in all
    groups. However, in the high dose group marked anorexia and weight
    loss occurred secondary to pyloric obstruction by a gastric concretion
    composed ostensibly of DKP intermixed with rabbit hair. Data on the
    mean number of implantation sites, resorption sites, viable and
    non-viable foetuses, and foetal length and weight were unremarkable
    for control, low, medium dose groups. There were insufficient data
    from the high dose group to permit a proper evaluation. External,
    visceral, and skeletal examinations of foetuses from 10-14 litters per
    group (except the high dose group) showed no treatment-related effects
    (Anonymous, 1972f).

    Mutagenicity studies

    Dominant lethal study

         DKP was administered intragastrically as a freshly prepared 5%
    suspension w/v in a 1% solution of Tween-80 (v/v) in distilled water
    to 15 male rats (Charles River CD strain). Two equally divided dosages
    were administered two hours apart on a single day. The negative
    control was Tween-80, as a 1% solution (v/v) and was administered to
    15 males by the same route and dosage regimen as the DKP. The positive
    control methylmethane sulfonate (mms) was given to 10 males i.p., as
    an 0.7% suspension (w/v) in corn oil. The dosage was 50 mg/ml. Two
    females per week were mated to each male for 8 weeks and autopsied 14
    days after positive indications of mating. Males treated with the
    positive control had statistically significant decreases in fertility
    in the fourth week. There was no evidence that DKP affected the
    fertility of male rats. There were significant decreases in the number
    of corpora lutea in females mated to positive control-treated animals,
    in the second, third and fourth weeks. Females mated to DKP treated
    males showed a decrease in the number of corpora lutea in the seventh
    week. The number of implantations in females mated to positive
    control-treated males was significantly decreased in the second,
    third, fourth and fifth week. Females mated to DKP treated males in
    the fifth week showed a significant decrease in the number of
    implantations. The results in all other time periods were normal. The
    number of foetal deaths was significantly increased in females mated
    to positive control-treated males in weeks 1, 2, 3 and 5. Females
    mated to DKP-treated males were unaffected. The number of viable
    foetal swellings was significantly decreased in females mated to
    positive control-treated males in weeks 1, 2, 3, 4 and 5. Females
    mated to DKP treated males in week 5 showed a significant reduction in
    the number of viable foetal swellings (Schroeder et al., 1973b).

    In vivo cytogenetics

         DKP was administered intragastrically to four groups of 10 male
    Purina Caesarian derived albino rats for five consecutive days. The
    dosages were 0.25, 0.5, 1.0 and 2.0 g/kg/day given in three equally
    divided daily dosages. The negative control received the Tween-80-
    water vehicle (1% Tween-80 in distilled water). The positive control
    received a single i.p. dose of triethylene melamine. Chromosomal
    aberrations in bone marrow were evaluated. The mean body weight was
    reduced in all groups, but more significantly at the very high dosage
    level (2.0 g/kg/day). Food consumption was also reduced in the groups
    receiving five days of treatment. The mean percentage of cells with
    aberrations was significantly higher in the positive control-treated
    groups. There were increases above control level in other DKP treated
    groups, but these apparently were not evaluated statistically
    (Anonymous, 1972g).

    Host mediated assay

         DKP was administered orally to male rats (Purina Caesarian
    derived) approximately 12 weeks old at 4 dose levels; 0.25, 0.50, 1.0
    and 2.0 g/kg/day in 3 equally divided doses for 5 days. The vehicle
    control was given at a level of 40 ml/kg/day. DMNA was administered
    i.p. at a level of 100.00 mg/kg on day 5 only. S. typhimurium G-46
    was injected i.p. on day 5, 30 min after administration of the test
    compounds.

         The rats were sacrificed 3 hours after S. typhimurium
    injection; their peritoneal cavities were aseptically exposed and
    washed with 2.0 ml sterile saline. As much fluid as possible was
    removed from the cavity. Peritoneal washings were diluted and plated
    in accordance with generally accepted procedures.

         A total of 16.0  108 CFU of S. typhimurium G-46 was injected
    into each rat. Recoveries from the peritoneal cavity were extremely
    low, approximately 1/3-1/100 of the original inoculum.

         The mean mutation frequencies (MF) were:

                                                                        

    Negative control:        1.26        0.64               10-7
    Positive control:       23.36       10.58               10-7
    0.25 g/kg/day            2.90        6.33               10-7
    0.50 g/kg/day            0.83        0.84               10-7
    1.0 g/kg/day             1.34        1.21               10-7
    2.0 g/kg/day             2.19        1.88               10-7

    (Anonymous, 1972e)
                                                                        

    Special studies

    Urinary bladder tumorigenicity study in the mouse by the intravesical
    pellet implant technique (DKP)

         Groups each of 200 female 60-90 day-old Swiss albino mice were
    used. Pellets of 20-22 mg of purified cholesterol (80%) and DKP (20%,
    4.0-4.4 mg) were prepared and surgically placed into the urinary
    bladder. The negative control group was exposed to pellets of
    cholesterol, and the positive control group to pellets of cholesterol
    and the 8-methyl ether of xanthurenic acid. The study was for 56
    weeks. Parameters measured included morbidity, mortality, motor and
    behavioural activity, growth, general external features and digital
    palpation of protruding tissue masses. All animals dying during the
    experiment, or at the termination of the study were subject to

    necropsy, and histopathological inspection of the bladder. No bladder
    neoplasma was observed in animals dying to 175 days of the study. The
    following incidence of bladder neoplasia was recorded in mice
    surviving 175 days or more: control 17/155 (10.6%), DKP 17/125 (13.6%)
    and positive control 40/111 (36.0%) (Bryan, 1974b).

    Special studies on the possible nitrosation of DKP

    Reaction of DKP and sodium nitrite in vitro

         1250 mg of piperidine and 25 mg of Na nitrite were dissolved in
    distilled water; pH was adjusted to 4 and volume to 10 ml. Duplicate
    flasks were capped and incubated at 37C with constant shaking. At
    0.5, 1, 2 and 4 hours, 2 ml samples were transferred to tubes
    containing 3 ml of ether with extraction for 15 min. The ether phase
    was used for GLC detection of N-nitroso-piperidine. In duplicate
    vials, 10 mg of DKP-14C-phenylalanine and 3 mg of Na nitrite were
    dissolved in 0.25 M phthalic acid buffer, pH 4. The volume was
    adjusted to 10 ml, the vials gassed with N2 and capped. Control vials
    were prepared containing DKP but no Na nitrite. Vials were incubated
    with shaking at 37C and at 0.5, 1, 2 and 4 hours, samples (were taken
    and analysed for the presence of nitroso compounds by use of multiple
    sampling procedures) and the use of two different thin chromatographic
    systems. Nitrosopiperidine formation occurred by reaction of
    piperidine with sodium nitrite (3-6 mg or 0.2 to 0.5% of the possible
    yield). DKP did not react with nitrite to form compounds that could be
    detected by the methods used (Anonymous, 1972a).

    A study of the possible reaction of DKP with aqueous nitrous acid

         A 0.1% solution of DKP, containing 0.13% (5 molar excess) Na
    nitrite was prepared in 0.1 N HCl. The mixture was allowed to stand at
    room temperature for 1.5 hours. Excess nitrous acid, formed by the
    interaction of HCl and Na nitrite was distilled off from the mixture
    at 30 under vacuum. Following distillation, a 25 ml aliquot of the
    solution was taken and to it 0.062 mg of N-ethyl, N-nitrosourethane
    was added. The UV spectrum of this solution was taken against a blank
    of 0.1% DKP in 0.1 N HCl. An aliquot of the solution was subjected to
    UV spectral analysis (against a blank of 0.1% DKP in 0.1 N HCl) prior
    to addition of N-ethyl, N-nitrosourethane. A 0.158% solution of
    NaNO2 in 0.1 N HCl was also distilled and the UV spectrum taken
    against 0.1 N HCl. At 245 nm, the DKP showed no increase in absorbance
    as compared to the NaNO2 solution alone, suggesting that DKP had not
    been nitrosated to form N-Nitroso-DKP (Anonymous, 1972h).

    Reaction of DKP and Na Nitrite in vivo

         Non-fasted male Charles River rats (340-360 g) were anaesthetized
    with Na pentobarbital. The stomach was ligated at the cardiac and
    pyloric junctions. At the pyloric junction, an incision was made
    through which coarse food particles were manually expressed from the
    stomach and through which a polyethylene cannula was inserted. 25 mg
    of Na nitrite in 0.5 ml water followed by 5 mg 14C DKP in phthalic

    acid buffer were administered. The cannula was removed, the ligature
    tightened, and stomach returned to abdominal cavity. Controls received
    either piperidine, HCl or Na nitrite alone with saline. Two rats were
    used for each experiment. After 60 min, the animals were sacrificed
    with ether, gastric contents removed, and stomach rinsed with saline
    and the contents were assayed for the presence of n-nitroso compounds.
    In vivo nitrosation of piperidine was demonstrated and only the
    stomachs of rats given piperidine and nitrite. No reaction products of
    sodium nitrite and DKP were detected by the assay methods used
    (radiochromatography by several solvent systems) (Anonymous, 1972a).

    Special studies with aspartame and DPK

    Enzyme induction studies with aspartame and DPK

         Male albino rats (Charles River strain) of 80-100 g weights were
    pretreated with either saline (1 mg/kg), phenobarbital (60 mg/kg), or
    aspartame (3.5 g/kg) for 4 days. Saline and phenobarbital were
    administered once daily by i.p. injection. Aspartame was given twice
    daily as an aqueous suspension. On the day following the last
    pre-treatment day, the animals were challenged with either
    hexobarbital (100 mg/kg, i.p.) or zoxazolamine (60 mg/kg, i.p.). The
    duration of hexobarbitol sleeping time or zoxazolamine paralysis time
    was determined, using loss and return of the righting reflex as end
    points. In vitro studies were carried out on liver preparations
    derived from the livers of rat following the last pretreatment day, to
    measure the effect of aspartame administration on hepatic amino-N-
    demethylase, p-nitrosoamisole, O-demethylase, zoxazolamine hydroxylase
    and hexobarbital oxidase activity. Aspartame administration to the
    rats had no effect on the in vivo and in vitro systems studied
    (Anonymous, 1972a). Similar results were observed when DPK (2.0 g/kg)
    was used in the diet (Anonymous, 1972a).

    Effect of dietary aspartame and phenylalanine on hepatic
    phenylalanine hydrolase activity in the rat

         Groups each of 8 male Charles River strain rats (170 g) had free
    access to one of 4 diets and water. Diets used were: (1) powdered
    Rockland diet, (2) powdered diet plus 0.15% aspartame, (3) powdered
    diet plus 1.5% aspartame and powdered diets and 0.85% phenylalanine
    (equimolar with 1.5% aspartame). Animals were weighed at 2-day
    intervals and food consumption recorded every second day. Treatment
    period varied from 1 to 8 weeks. The animals were sacrificed and blood
    samples taken for phenylalanine analysis. A liver preparation
    consisting of the supernatant from homogenates centrifuged 45 min 
    16 000 g, was used for the enzyme assay. Feeding diets of 0.85%
    phenylalanine or 1.5% aspartame for one or more weeks resulted in
    decreases in phenylalanine hydroxylase activity and increases in
    plasma phenylalanine levels. The hydroxylase activities were more
    sensitive to the presence of the dietary aspartame and phenylalanine
    than were the plasma phenylalanine level (Anonymous, 1972i).

    Gastrointestinal system

    Appetite inhibition studies in rats

         Ten male adult Charles River rats were trained to eat during a
    two-hour period each day for 4 consecutive days. Tap water was allowed
    throughout the experiment. On day 5, 1 h prior to feeding, rats were
    dosed intragastrically with 200 mg/kg of aspartame, DKP (an aspartame
    breakdown product), or vehicle (30% propylene glycol) only. One group
    received no treatment. One hour later, all except the control group,
    which was fasted, were allowed food ad lib. for 2 h. The body weight
    was taken before compound administration and 24 h after feeding.
    Neither aspartame or DKP had any effect on food consumption or weight
    loss, indicating no effect on appetite (Anonymous, 1972j).

    Effects on gastric secretion in rats

         Groups each of 6 male Charles River strain rats (175-225 g) were
    fasted for 48 h. The animals were anaesthetized with ether and
    subjected to pyloric ligation. Aspartame, DKP, or distilled water
    (control) were administered in 1 ml of water intragastrically
    (250 mg/kg). Five hours later, the stomachs were removed, contents
    measured, and centrifuged. Acid concentration and proteolytic activity
    of the gastric juice was measured. Neither aspartame nor DKP had any
    marked effect on gastric juice volume, acidity or proteolytic activity
    (Anonymous, 1972j).

    Pepsin inhibition in vitro

         Bovine pepsin was incubated with bovine haemoglobin in the
    presence of aspartame or DKP at 143 g/ml. There was no inhibition of
    pepsin activity (Anonymous, 1972j).

    Pancreatic lipase inhibition in vitro

         Emulsified triglyceride (olive oil) containing 0.4 mg/ml of
    pancreatic lipase was incubated for 2 h in the presence or absence of
    aspartame or DKP at 1.25 mg/ml. There was no inhibition of lipase
    activity (Anonymous, 1972j).

    Effects on gastric ulceration in rats

         Male Charles River rats (200-250 g) were anaesthetized with ether
    and stomach ligated at the pyloric junction. The animals were given
    50 mg of aspartame or DKP or vehicle only. 17-1/2 hours later, the
    stomachs were removed and examined microscopically. The numbers of
    ulcers in the non-secretory portion of the stomach were counted
    according to size to obtain the Z score. Six rats were in each
    treatment group. A known anti-ulcer agent was used as a positive
    control. Neither aspartame nor DKP significantly increased or
    decreased the severity of gastric ulceration (Anonymous, 1972j).

    Cardiovascular system

    Effects on blood pressure in anaesthetized dogs following intravenous
    administration

         Two mongrel dogs were anaesthetized with sodium pentobarbital and
    the blood pressure monitored continuously from a femoral artery.
    Aspartame or DKP was dissolved in propylene glycol and injected at
    concentrations of 0.1, 1.0, and 5.0 mg/kg. Aspartame had no effect on
    blood pressure at 0.1 or 1.0 mg/kg. At 5 mg/kg, one dog (of 2) had a
    slightly lowered blood pressure temporarily. DKP had no effects at any
    concentration (Anonymous, 1972j).

    Effects on blood pressure and heart rates following oral
    administration of aspartame or DKP in unanaesthetized normotensive
    dogs

         Blood pressure was recorded via a surgically implanted aortic
    cannula. Following surgery, the animals were isolated from noise.
    Systolic, diastolic, and mean arterial pressures were recorded. Heart
    rate was derived from the blood pressure tracing. Blood pressure and
    heart rate were determined at 5 min intervals during a 30 min control
    period prior to compound administration. Aspartame, DKP, and placebo
    were administered in capsule form at levels of 100 and 200 mg/kg. Two
    dogs of each sex were in each treatment series. Measurements were made
    at 10, 20, 30, 40, 50, 60, 120, 180, 240 min and 24 h after treatment.
    Neither aspartame nor DKP showed any consistent effects on blood
    pressure or heart rates (Rozek, 1972).

    Inhibition of the pressor response to angiotensin in rats

         Adult male Charles River rats were anaesthetized by i.p.
    injection of sodium pentobarbital. Cardiovascular reflexes were
    blocked with atropine and pentolinium subcutaneous injections. A
    femoral vein was cannulated for injections, a femoral artery for
    blood pressure measurements. Five consecutive doses of 0.01 g of
    angiotensin were given intravenously at 3 min intervals. Three minutes
    after the last injection, Aspartame or DKP was injected at 10 mg/kg.
    Following 15 min, the angiotensin protocol was repeated. The mean
    pressor responses were compared before and after test compound
    administration. A known antihypertensive agent was used as a positive
    control. Neither aspartame nor DKP had any effect on the pressor
    response to angiotensin in rats (Anonymous, 1972j).

    Antiarrhythmic activity using the isolated rabbit heart

         The ability of aspartame and DKP at concentrations of 10, 20, and
    40 mg/l to affect aconitine-induced ventricular arrhythmia in the
    isolated rabbit heart was determined. No significant effects of
    aspartame or DKP were observed (Anonymous, 1972j).

    Effects on blood coagulation in vitro

         Saline solutions of aspartame or DKP were added to freshly drawn
    rabbit blood and the mixture incubated at 37C. The coagulation time
    was compared to controls (no treatment) and coagulation time in the
    presence of heparin. DKP or aspartame had no effects on coagulation
    time (Anonymous, 1972j).

    Central nervous system

    General observable effects in mice

         Groups of 4 mice each were injected i.p. or s.c. with aspartame
    or DKP at dose levels of 0, 5, 20, 40, 80, and 320 mg/kg. Observations
    and tests were carried out just prior to compound administration and
    an 0.5, 1, 2, 3, and 4 h treatment. The spontaneous elicited behaviour
    was rated for each mouse and tests for locomotor ataxia were carried
    out by observing the behaviour of the mouse for 30 sec when placed on
    a horizontal rod. Neither aspartame nor DKP caused excitation or
    depression at any of the doses used.  Very slight ataxia was observed
    for aspartame at i.p. doses of 20, 40, 80 and 320 mg/kg and
    subcutaneous doses of 40, 80 and 320 mg/kg. Similar observations were
    reported for DKP following i.p. doses of 5-320 mg/kg and subcutaneous
    doses of 40 and 320 mg/kg (Anonymous, 1972j).

    Antidepressant activity in mice

         Aspartame and DKP were checked for antidepressant activity by
    determining their abilities in antagonizing the drooping of the upper
    eyelid (ptosis) caused by the administration of RO-4-1284. Ten mice
    were used in each group. Mice received 0, 25, or 200 mg/kg of
    aspartame or DKP intragastrically. One hour later, RO-4-1284 was
    administered i.p. at 20 mg/kg. The abilities of the two compounds to
    antagonize the eyelid drooping caused by RO-4-1284 were rated.
    Positive controls with known antidepressants were run. Aspartame or
    DKP had essentially no antidepressant effects (Anonymous, 1972j).

    Effects on hexobarbital hypnosis in mice

         Groups of 16 male HAM/ICR mice (18-25 g) were administered saline
    (control), or DKP or aspartame at dosage levels of 250, 500, or
    1000 mg/kg intragastrically. Thirty minutes later, hexobarbital was
    administered i.p. at 100 mg/kg. Sleeptime was defined as the time from
    the loss of righting reflex until a 2-time spontaneous righting in a
    15 sec interval. Aspartame did not show any effect at the doses
    studied. DKP caused a significant increase in sleeping time at the
    highest dose level test (1000 mg/kg) (Anonymous, 1972j).

    Effect on motor coordination in mice

         To groups of male HAM/ICR mice (20-30 g), aspartame or DKP was
    administered intragastrically at 0, 50, 100 or 200 mg/kg. Diazepam
    (positive control) was administered at 10 mg/kg. Mice were scored
    after 2-1/2 hours on the ability to stay on a rotating rod (4.5 rpm)
    for 1 min or longer. Neither aspartame nor DKP produced motor
    incoordination. Diazepam was significantly active in this test
    (Anonymous, 1972j).

    Anticonvulsant activity in mice

         Groups of male HAM/ICR mice (20-30 g) were administered aspartame
    or DKP intragastrically at 0, 50, 100, or 200 mg/kg. 2-1/2 hours
    later, the animals were exposed to a current of 50 milliamperes
    delivered by corneal electrodes. Anticonvulsant effects are judged by
    protections from the hindlimb extension component of the seizure.
    Diphenylhydantoin was used as a positive control. Neither aspartame
    nor DKP had any anticonvulsant effects at any dosage level. Groups of
    the same strain of mice were given aspartame or DKP intragastrically
    at dosage levels of 0, 100, or 200 mg/kg. Trimethadione and diazepam
    were used for positive controls. 1-1/2 hours later, 35 mg/kg of
    metrazol was administered i.p. Anticonvulsant activity was judged by
    abolition of seizures. Neither aspartame nor DKP had any effect
    (Anonymous, 1972j).

    Analgesic activity in mice

         Groups of 10 male HAM/ICR mice (18-25 g) were administered
    aspartame or DKP intragastrically at levels of 0, 50, and 100 mg/kg.
    The reaction time of each mouse to lick a foot or jump was measured
    at 60, 40, and 20 min before, and 30, 60, 90, and 120 min after
    administration of the test compound. The mice were scored on foot
    licking or jumping when placed on a hot plate at 55C, as compared
    with controls (Anonymous, 1972j).

         Adult male HAM/ICR mice (18-25 g) were administered aspartame or
    DKP at levels of 0, 50, or 100 mg/kg intragastrically. A pressure-
    standardized artery clip was placed one inch from the base of the
    tail. Response to the clip was measured as compared to controls.
    Neither aspartame nor DKP showed any analgesic effects (Anonymous,
    1972j).

    Central anticholinergic activity in mice

         Groups of 10 adult male HAM/ICR mice were given i.p. or oral
    doses of aspartame or DKP at levels of 0, 20, or 200 mg/kg. 20 min
    later, the mice received 20 mg/kg i.p. doses of tremorine. The mice
    were placed on a rotating rod 10 min after tremorine treatment and
    scored on the ability to remain on the rod for 2 min as compared to
    controls. Neither compound showed any effect (Anonymous, 1972j).

    Effects on behaviour in rats

         Naive male Fischer rats (90 days of age) were treated
    intragastrically with saline or 50, 100 or 200 mg/kg of aspartame,
    DKP or L-phenylalanine; 30 min later, rats were placed in a
    two-compartment shuttle box equipped with electrified grid floor and
    insulated from noise. A 5 sec conditioned stimulus (a tone and a
    light) preceded a 0.2 milliampere footshock delivered via the grid.
    The shock was terminated in 30 sec if the rat did not respond. If the
    rat moved to the other chamber, the shock was avoided and the response
    scored as an avoidance response. A movement to the other chamber
    during the shock was scored as an escape response. If no response
    was made, it was recorded as a failure response. 15 sec intervals were
    given before another conditioned stimulus was given. If a shuttle
    response was made during this rest interval, the shock and conditioned
    stimulus were applied until the rat returned to the other side. Each
    rat received 100 trials. 12 rats per dose were tested. Neither
    aspartame nor L-phenylalanine had any significant effect at any dose
    tested. DKP had a significant effect on the number of foot avoidances
    at 50 and 200 mg/kg dose. However this effect may not be significant
    since it was not dose related nor was it accompanied by a significant
    increase in intertrial interval responses (Potts, 1973).

    Miscellaneous pharmacological activity

    Diuretic activity in rats

         Six groups of 4 male Sprague-Dawley rats (188-228 g) were
    administered saline or aspartame or DKP at a level of 100 mg/kg. The
    rats had been maintained on a normal commercial diet, with water
    ad lib. Food was withdrawn 18 h before, and water withdrawn during
    the 5 h test period. Urine was collected for 5 h, and voiding induced
    by bladder palpation. The volume of urine, and Na and K contents of
    urine were measured. Neither aspartame or DKP showed any diuretic
    activity (Anonymous, 1972j).

    Effects on blood glucose in rats

         Adult male Charles River rats (180-230 g) were fasted for 24 h.
    Blood samples were obtained via the tail veins and blood analysed
    for glucose by a published procedure. DKP or aspartame was
    administered intragastrically at 100 mg/kg. Serum samples were then
    obtained at 2 and 4 h post-treatment, and analysed for glucose
    content. Neither aspartame nor DKP had any effect on the blood glucose
    level (Anonymous, 1972j).

    Effects on body weight gain and blood cholesterol in
    hypercholesterolaemic rats

         Adult male Charles River rats (200-250 g) were made hyper-
    cholesterolaemic by receiving 0.02% of propylthiouracil in their
    drinking water. Groups of 8 rats each were treated intragastrically
    daily for 9 days with 0, 5, 10, 30 or 200 mg/kg of aspartame or DKP in

    30% propylene glycol. On the tenth day the rats were anaesthetized
    with ether, and blood samples withdrawn from the abdominal aorta.
    Serum samples were analysed for cholesterol by a published procedure.
    Body weights were taken on the first and tenth day of the test period.
    Neither aspartame nor DKP had any significant effect on body weight
    gain or blood serum cholesterol (Anonymous, 1972j).

    Anti-acetylcholine activity in vitro

         Segments of rabbit ileum were cleansed of adipose tissue and
    suspended in Tyrode's solution. Muscle movement was recorded on a
    physiograph. Maximum muscle contraction was recorded as that produced
    by adding acetylcholine at a final concentration of 5 g/ml in the
    bath. Aspartame, DKP, or atropine sulfate was then added until the
    contraction had been reduced by 50% of the maximum value. Each
    concentration of test compound was tested over a 7 min interval with
    at least 8 min intervals between tests. Concentrations of aspartame
    and DKP one thousand times those used for atropine sulfate failed to
    reduce the contraction by 50% indicating that anticholinergic
    activity, if present at all, is less than 0.001% that of atropine
    (Anonymous, 1972j).

    Antihistamine activity in vitro

         Segments of guinea-pig ileum were suspended in Tyrode's solution
    and muscle movement recorded on a physiograph. Maximal muscle
    contraction was induced by histamine diphosphate at a concentration of
    50 g/ml of bath solution. Test compounds, including the known
    antihistamine agent diphenhydramine-HBr, were introduced to determine
    what concentration induced a 50% relaxation of maximal contraction.
    Each concentration of test compound was tested for 7 min with 8 min
    periods between tests during which the bath solution was changed. Both
    aspartame and DKP at a PD concentration of 4.6 failed to relax the
    tissue by 50% indicating that both compounds are essentially devoid of
    antihistamine activity (Anonymous, 1972j).

    Autonomic ganglionic blockade effects in cats

         Aspartame and DKP were tested for their effects on the superior
    cervical ganglia of cats. Each cat was anaesthetized with Na
    pentobarbital and (1) the right superior sympathetic nerve was
    exteriorized and sectioned caudal to the superior cervical ganglion;
    (2) the left common carotid artery was cannulated to monitor blood
    pressure; (3) a femoral vein was cannulated for test compound
    injections. The rostial stump of the cervical sympathetic nerve was
    stimulated electrically and the sustained contraction of the
    ipsilateral nictitating membrane was recorded on a physiograph.
    Aspartame and DKP were given at a single intravenous dose of 6.4 mg/kg
    in saline. Tetraethylammonium bromide was used as a positive control.
    Blocking agents decrease the nictitating membrane contraction. Neither
    aspartame nor DKP were effective as autonomic ganglionic blocking
    agents (Anonymous, 1972j).

    Effect of short-term dietary administration of DKP and aspartame on
    serum levels of glucose, insulin, triglycerides, free fatty acids,
    and cholesterol in rats

         Male and female Charles River rats (150-200 g) were placed in
    metabolism cages and fed a commercial diet containing 0, 0.2% or 2% of
    aspartame or DKP. Ten rats of each sex were used at the 2 treatment
    levels for both compounds; controls received commercial diet only. A
    paired-feeding technique was used in which a rat of the same sex and
    similar weight was given one more gram of food than that eaten by its
    pair-fed rat. Body weight, food and water consumption were measured
    daily. After 7 days, the rats were sacrificed by decapitation and the
    serum obtained after clotting by centrifugation. The serum was
    analysed for insulin (by radioimmunoassay, with rat insulin as a
    standard), for fatty acids, triglycerides and cholesterol by standard
    methods. Serum glucose was determined with the Beckman Glucose
    Analyser. For treated rats the mean daily consumption of aspartame
    was, for females, low dose/high dose 0.152/1.48 g/kg, and for males,
    low dose/high dose 0.2/2.01 g/kg. Ingestion of these levels of
    aspartame for one week did not significantly affect the parameters
    measured. For DKP the daily dose ingested was females, low/high,
    0.153/1.57 g/kg and for males, low/high, 0.197/1.88. Consumption of
    the levels of DKP for one week had no significant effect on the
    parameters studied (Saunders, 1972).

    Endocrinological studies

    Hormonal properties

    Estrogenic activity

         Twenty-one day-old female mice (8-10 per group), maintained
    on an estrogen free diet, were orally administered aspartame or DKP
    (dissolved in corn oil) at a daily total dose of 1.35 mg for 3 days.
    Controls received corn oil. A positive control group received estrone
    subcutaneously at doses of 0.1 or 0.3 g. On the day after the last
    injection, the mice were sacrificed and the uteri weighed. Aspartame
    and DKP did not stimulate uterine weights in contrast to the marked
    stimulation at both dose levels of estrone (Nutting, 1972).

    Estrogen antagonism

         The test was similar to that used to study estrogen activity
    except that all groups were treated simultaneously with a total dose
    of 0.3 g, estrone. A positive effect consists of a limitation of
    uterine growth induced by estrone. Aspartame and DKP were given orally
    in total doses of 450 and 1350 g (10-20 mice/dose). Untreated
    controls (38 mice) received corn oil. Progesterone in total doses of
    50, 100 and 200 g, s.c., was used as a positive control for estrogen
    antagonistic activity. Neither aspartame nor DKP significantly

    antagonized uterine growth induced by estrone. In contrast,
    progesterone, injected s.c., significantly decreased uterine weight
    at all doses (Nutting, 1972).

    Progesterone-like activity

         Immature female rabbits were primed for 6 days with daily s.c.
    doses of estradiol-17-Beta. After priming, aspartame or DKP in corn
    oil or corn oil alone was administered bucally for 5 days. Test
    compounds were given at 300 mg/day. The positive control,
    progesterone, was injected s.c. at doses of 0.02, 0.05 or 0.1 mg/day.
    On the day after the last injection, the rabbits were sacrificed and
    a segment of uterus was examined histologically and rated as to
    the degree of arborization of the endometrial glands (glandular
    proliferation is a progestational effect). Increased uterine carbonic
    anhydrase activity has been correlated with glandular proliferation.
    Accordingly, a uterine segment was also analysed for carbonic
    anhydrase activity. Neither aspartame nor DKP caused an increase in
    the degree of glandular arborization or the concentration of carbonic
    anhydrase in the uterus. In comparison progesterone at a dose of
    0.05 mg/day or greater significantly increased both measures (Nutting,
    1972).

    Progesterone antagonism

         The test was similar to that for progesterone-like activity
    except that all groups received a daily dose of 0.1 mg progesterone in
    addition to the test compounds. Aspartame (4 rabbits) and DKP (4
    rabbits) were given at a dose of 300 mg/day. Controls (15 rabbits)
    received vehicle alone. The positive control, estrone, was given s.c.
    at a dose of 0.001 (4 rabbits) and 0.002 (12 rabbits) mg/day. A
    decrease of the stimulated endometrial glandular proliferation
    observed in animals treated with 0.1 mg progesterone alone was used as
    one index of progesterone-antagonistic activity. After 5 daily doses
    (buccal administration) of 300 mg/day, neither aspartame nor DKP
    altered the glandular arborization induced by progesterone. Aspartame
    also failed to alter the carbonic anhydrase activity induced by
    progesterone. However, SC-19192 at 300 mg/day did reduce the
    concentration of carbonic anhydrase by 59% of control. The positive
    control, estrone, at a dose of 0.002 mg/day reduced both the degree of
    arborization and the concentration of carbonic anhydrase. Accordingly,
    in this test aspartame does not display progesterone-antagonistic
    activity. DKP does, however, exhibit some progesterone antagonism at a
    buccal dose of 300 mg/day for 5 days (Nutting, 1972).

    Androgenic-myotrophic activity

         White male rats were castrated at 22-24 days of age. After 19-21
    days recovery, test compounds in oil were given daily for 7 days
    (orally). Aspartame and DKP were given at doses of 50 (8 rats each)
    and 350 mg (6 rats) (total dose). Controls (20 rats) received oil
    alone. The positive control, methyl testosterone, was given orally at

    total doses of 10 and 60 mg (8 rats each). On the day after the last
    injection, the rats were sacrificed. Increases in the weights of the
    seminal vesicles and ventral prostate gland compared to oil control
    were used as a measure of androgenicity. An increase in levator ani
    muscle weight compared to oil controls served as an index of
    myotrophic activity. Methyl testosterone at a total dose of 10 or
    60 mg significantly increased the weights of the seminal vesicles,
    prostate gland, and levator ani muscles. Neither aspartame nor DKP
    significantly altered any of these parameters. Thus, they did not show
    either androgenic or myotrophic activity in this test (Nutting, 1972).

    Androgen antagonism

         The test was similar to that for androgen-myotrophic activity
    except that all groups were treated simultaneous to the test compounds
    with testosterone propionate, intramuscularly, at a total dose of
    0.5 mg. Aspartame and DKP were given orally doses of 50 (15 rats) and
    350 mg (7 rats each) daily for 7 days. Controls (20 rats) received
    oil. A decrease in weight of the seminal vesicles and ventral prostate
    glands compared to a group treated with 0.5 testosterone propionate
    alone was used as a measure of inhibition of the response of
    testosterone. A decrease in the weight of the levator ani muscle,
    compared to a group treated with testosterone alone, was used as an
    index of catabolic activity. No positive control was employed.
    Although neither test compound given orally at a total dose of 50 or
    350 mg/day produced a statistically significant change in the weights
    of the tissues examined, both aspartame and DKP at 350 mg/day did
    reduce the weight of the seminal vesicles by about 20% (Nutting,
    1972).

    Glucocorticoil activity

         The test employed the fact that in fasted, adrenalectomized rats
    the ability to store glycogen in the liver is impaired but that
    glucocorticoids reverse this. Adrenalectomized adult male rats,
    maintained on saline fluid and a high protein diet were fasted for
    24 h, 3 days after surgery. After fasting, test compounds were
    administered orally in oil in 4 equal doses at about 2 h intervals.
    Aspartame and DKP were given at a total dose of 45 mg (9 rats each).
    Controls (9 rats) received oil. Positive controls received cortisone
    acetate, s.c., at a total dose of 0.5 or 0.2 mg (10 rats each). The
    rats were sacrificed 6-8 h after the first injection and livers were
    analysed for total glycogen. An increase in liver glycogen above
    controls was the index of neoglycogenic or glucocorticoid activity.
    This activity was demonstrated in the animals receiving 0.5 mg (total
    dose) cortisone. Aspartame and DKP did not demonstrate glucocorticoid
    activity in this test (Nutting, 1972).

    General physiological effects

    Effects on fertility (implantation)

    Rats, post-ovulatory

         Sexually mature female rats were mated and treated for seven
    days, 60 mg/day aspartame or DKP (five rats each) (orally) beginning
    on the day sperm appeared in the vagina (conception). Controls (10
    rats) received vehicle (oil). A positive control, oestrone, was given
    s.c. at 2 or 4 g/day (10 rats each). On the 15th day post coitum, the
    animals were sacrificed. Aspartame and DKP failed to decrease the
    number of rats with normal implantation sites (Nutting, 1972).

    Hamsters, post-ovulatory

         Sexually mature female hamsters were mated and treated for five
    days with 30 mg/day aspartame or DKP (orally; five hamsters each)
    beginning on the day sperm were found in the vagina. Controls (15
    hamsters) received vehicle (oil) alone. The positive control,
    oestrone, was given s.c. at 10 or 20 g/day (15 hamsters). Animals
    were sacrificed six days post coitum. The corpora lutea and
    implantations were counted and their condition noted. Oestrone
    markedly attenuated the 1% of implantation, increased the 1% of
    abnormal corpora lutea and significantly decreased the implantation
    rate considering all sites with an ED50 of 12.4 g/day. Aspartame had
    no effect on any of the parameters. DKP did slightly reduce the 1%
    normal implantation sites and slightly increased the % abnormal
    corpora lutea but had no effect on the implantation rate value
    (Nutting, 1972).

    Inhibition of pituitary gonadotrophin (GTH)

         The test is based on the phenomenon of stimulation of pituitary
    GTH secretion in response to unilateral ovariectomy and subsequent
    hypertrophy of the intact ovary. Seventy-eight-day-old female rats
    were unilaterally ovariectomized. Oral administration of aspartame and
    DKP at 60 mg/day or buccal administration of aspartame at 10 and
    2 mg/day was carried out for 14 days beginning on the day of surgery
    (9-19 rats per group). Controls (10 rats) received vehicle (oil)
    orally. A positive control, northynodrel, was given s.c. at 20, 50 or
    100 g/day (29-30 rats per dose). On the day following the last
    treatment, the rats were sacrificed and the remaining ovary weighed.
    Norethynodral inhibited the compensatory ovarian hypertrophy at doses
    of 20 g/day or greater. None of the groups treated with aspartame or
    DKP exhibited a decrease in ovarian weight; pituitary GHT secretion
    was unaffected (Nutting, 1972).

    Anti-inflammatory activity

    Foot oedema test

         An inflammatory reaction (oedema of the hind feet measured a
    volume displacement) was induced in intact male rats (about 120 g) by
    injecting 0.1 mg of 1% carrageenan under the plantar surface of the
    hind feet. Aspartame or DKP in saline were administered orally to
    eight rats each at a total dose of 36 mg one hour before injection
    of the carrageenan. Controls received saline (63 rats). Volume
    displacements were measured five hours after carrageenan injection.
    The positive control, hydrocortisone, administered orally, reduced the
    carrageenan-induced oedema at a dose of 10 mg/rat. Neither aspartame
    nor DKP administered as a single oral dose of 36 mg reduced the oedema
    inflammatory response to carrageenan (Nutting, 1972).

    Cotton wad granuloma formation

         In response to subcutaneous implantation of cotton,
    adrenalectomized rats develop granulomas surrounding the cotton
    pellets. Hydrocortisone prevents this. Adrenalectomized male rats
    (200 g) were given four subcutaneous cotton pellet implantation and on
    the following day, aspartame was administered orally at 20 and
    65 mg/day (six rats each) and SC-19192 was given at 32 and 65 mg/day
    10 and 21 rats, respectively). Controls (57 rats) received saline
    alone. The positive control, hydrocortisone, was administered s.c. at
    1 mg/day. Hydrocortisone significantly reduced the granuloma
    formation. Aspartame at 20 and 65 mg/day had no effect, neither did
    DKP at 32 mg/day. However, at 65 mg/day SC-19192 did produce a
    significant, but small inhibition of granuloma formation (Nutting,
    1972).

    Chronic polyarthritis

         An inflammatory response resembling rheumatoid arthritis
    (assessed in rats on the basis of ankle volume) was induced in male
    rats (150 g). Aspartame or DKP in saline were administered, orally, at
    60 mg/day for 19 days (11-12 rats each). Negative controls (18 rats)
    received saline alone and positive controls (11-12 rats per dose)
    received hydrocortisone i.g. at 5.1 or 2 mg/day. Rats were sacrificed
    and volume displacement of rear ankle joints were determined 24 hours
    after the last injection. Hydrocortisone at all doses significantly
    decreased the ankle volume, whereas aspartame and DKP at 60 mg/day had
    no effect (i.e. no anti-inflammatory activity) (Nutting, 1972).

    Immunosuppressive activity

         In the Jerne Plaque Test the number of specific antibody
    (haemolysin) producing cells in the spleens of mice sensitized to
    sheep erythrocytes (SRBCs) is measured. Immunosuppressive agents
    inhibit a sensitization injection of SRBCs. In the test system the
    number of in vitro "plaques" formed is proportional to the number of

    haemolysin producing spleen cells. Male mice (five to seven weeks old)
    were injected i.p. with SRBCs in saline. SC-18862 and SC-19192 in
    saline were administered once daily, orally for four days (beginning
    the same day as the SRBCs injection) to groups of 6-10 mice. Dose
    levels of 50, 125, 250, 500 and 750 mg/kg/day were employed. Several
    different replications were performed. Additionally, several
    experiments included administering phenylalanine and aspartic acid at
    these same daily dose levels. In each case, 24 hours after the final
    injection, the mice were sacrificed and plaque formation determined.
    In one set of experiments aspartame, DKP, phenylalanine and aspartic
    acid (oral) inhibited plaque formation (i.e., inhibited the immune
    response) at a dose of 500 mg/kg/day. Only SC-18862 was active at a
    lower (250 mg/kg/day) or at a higher (750 mg/kg/day) dose. Replicates
    of this experiment failed to confirm this activity (Nutting, 1972).

    Acute toxicity

                                                                        

                               LD50
    Animal       Route      (mg/kg/bw)             Reference
                                                                        

    Rat          Oral         >5 000          Andress et al., 1973b
                 i.p.         >2 033          Andress et al., 1973b

    Mouse        Oral         >5 000          Andress et al., 1973b
                 i.p.         >1 000          Andress et al., 1973b

    Rabbit       Oral         >5 000          Andress et al., 1973b
                                                                        

    Short-term studies

    Mouse

         Groups of five male and five female mice received 0, 3, 5 and
    13 mg/kg/day of aspartame in their diets for four weeks. No compound
    related differences between control and test groups in terms of
    physical, motor or behavioural effects were seen, nor were effects
    evident in terms of body weight or food consumption. On autopsy the
    mucosa of the stomach, duodenum, and jejunum of the high dose level
    animals were found to be coated with a clear moderately viscous fluid
    (Rao et al., 1972b).

    Rat

         Groups of 10 male and 10 female rats received 0, 5 and
    1250 mg/kg/day of aspartame in their diets for a two month period. No
    compound related effects were noted in terms of gross appearance,
    behaviour, gross eye appearance, mean body weights, food consumption
    and urinalysis. Haematological findings were within normal limits.

    Blood levels of glucose found were indicative of a dose related
    increase, while a dose related decrease of serum albumin and SGOT was
    noted particularly in the male rats. Gross and microscopic pathology
    did not reveal evidence of compound related lesions (Anonymous,
    1969a).

         Groups of five male and female rats received 0, 2, 4 and
    10 mg/kg/day of aspartame in their diets for a four-week period. No
    adverse behavioural or physical effects were noted in treated animals.
    A decrease in body weight noted in the high dose level animals was not
    statistically significant. At necropsy no treatment related gross
    alterations were noted other than a heavy coat of clear viscous
    material on the mucosa of the stomach, duodenum and jejunum of the
    high level rats (Rao et al., 1972c).

         Groups of five male and five female rats received a basal diet or
    basal diet with aspartame (100:9 w/w) or basal diet with phenylalanine
    (100:5 w/w) for nine weeks. Both aspartame and phenylalanine fed
    groups showed similar reductions in growth (11%) and food consumption
    (20%). Aspartame treated males showed significantly lower SGPT and
    plasma Ca++ and Cl- values. No treatment related effects were seen
    upon haematology findings, urinalysis, organ weights and gross and
    microscopic pathology (Hemm et al., 1972).

    Dog

         Groups of two male and three female beagle dogs received orally
    5 mg/kg or 125 mg/kg of aspartame daily by capsule for eight weeks.
    Two male and two female beagles served as controls. No treatment
    related changes were seen in body weights, food consumption,
    haematology, biochemistry, urinalysis, ophthalmoscopy. Small, dose-
    related increases in testicular weight were noted. Increases in organ
    to body weight ratios noted for heart, kidney and adrenal in both test
    groups were not dosage related. Gross autopsy and microscopic
    evaluation of tissues did not reveal any evidence of compound related
    effect (Anonymous, 1969b).

         Groups of five males and five female beagle dogs received 0,
    1000, 2000 and 4000 mg/kg/day of aspartame (DKP content less than 1%)
    incorporated into 200 g of powdered basal diet for 106 weeks. At all
    levels of aspartame fed, growth was depressed. Cataracts seen in one
    intermediate and one high-level dog (litter mate) were considered to
    be congenital based upon appearance of a cataract in one of two pups
    resulting from a remating of the parents. Consistent and statistically
    lowering of haemoglobin, haematocrit, and total red blood cells was
    noted in the male dogs at the high dose level. To a lesser frequency
    and degree it was noted in the intermediate dogs. This was not seen in
    the low dosage group. There were sporadic treatment-related changes in
    some clinical chemistry parameters, but no consistent trends in time
    or dose relationships. A large and statistically significant decrease
    in BSP values was seen in the male intermediate and high dose level

    males at 78 and 106 weeks, but no changes were seen in other liver
    function tests. Gross and microscopic pathological findings were not
    indicative of compound related changes (Rao et al., 1972d). A detailed
    histopathological study was made of the brains of two dogs on the high
    dose level. No neoplastic alternations were seen (Kommineni, 1973).

    Monkey

         Seven newborn rhesus monkeys were divided into three dosage
    groups. Aspartame was administered dissolved in a commercial milk
    preparation, first using a nursing bottle, later from a cup.
    Concentrations of aspartame were increased incrementally so as to
    approximate intended dosages. Treatment groups were as follows:

                                                                        

    Dosage                             Age at            Total days
    mg/kg/day           Sex         start (days)        on treatment
                                                                        

    1 000                M                6                  210
    1 000                F                3                  204

    3 000                M                3                  360
    3 000                M                3                  362
    3 000                F                2                  363

    4 000-6 000          M                9                  357
    4 000-6 000          M                1                  279
                                                                        

    Data from the experimental animals was compared to historical control
    data derived from 14 monkeys. Aspartame intake calculated for the low
    and intermediate dosage levels was within 5% of that planned. For the
    high level, intake was calculated as 1210 and 5330 mg/kg/day, mean for
    the entire study being 3600 mg/kg/day. Group mean intake of the
    diketopiperazine (DKP) conversion product of aspartame over the entire
    study was estimated as being 4.84, 15.07 and 18.12 mg/kg/day
    respectively for low, intermediate and high dose groups. Body weights
    of one of the two low dose group animals, two of the three
    intermediate dose group animals and one of the two high dose group
    animals were below normal limits based upon the historical controls.
    Growth rates however were comparable to the controls. While there was
    a decrease in the total volume of liquid formula ingested in all
    groups this was severe only in one of the two high treatment level
    monkeys. All animals in the intermediate and high dosage groups
    exhibited convulsions of the grand mal type observed for the first
    time following 218 days of treatment. These were described as similar
    to those induced by feeding L-phenylalanine to infant monkeys and were
    ascribed to the L-phenylalanine moiety of aspartame. One of the high

    dose level animals (calculated intake 1210 mg/kg/day) died after 279
    days on study, cause of death not determined. No haematological
    evidence of effect was noted. Clinical chemistry findings were
    similarly without evidence of effect aside from serum phenylalanine
    and tyrosine values for the intermediate and high dose level animals
    in which the values found were similar to those found in animals fed
    2000-2500 mg/kg/day of L-phenylalanine. Urinalyses were similarly not
    indicative of other than the consistent presence of phenylketone in
    the two higher dosage groups. No final gross or microscopic evaluation
    was made, the study being abruptly terminated due to the
    investigator's death (Rao et al., 1972e).

    Long-term studies

    Mouse

         Groups of 36 male and 36 female mice received 1000, 2000 and
    4000 mg/kg/day of aspartame in their diets for 110 weeks. A group of
    72 males and 72 females served as controls. No effects were reported
    in terms of appearance, behaviour or survival. Ophthalmological
    findings were also without evidence of effect. Mean body weights of
    treated animals were not markedly different from controls; food
    consumption however was decreased with increased dosage. Haematology
    and clinical chemistry findings though showing sporadic incidence of
    statistically significant differences between test and control animals
    gave no indication in trend or dose-relationship of being compound
    related. At termination scattered incidences of increased organ
    weights or organ to body weight ratios were noted. Among these were
    thyroid, heart, and prostate. Distribution and incidence were not
    indicative of compound relationship. Gross and microscopic pathology
    reported did not indicate the presence of tumorigenic or non-
    tumorigenic changes which would be attributed to the administration of
    aspartame (Anonymous, 1974d).

    Rat

         Groups of 40 male and 40 female rats received 1000, 2000, 4000 or
    8000 mg/kg/day of aspartame in their diets for 104 weeks. A group of
    60 male and 60 female rats served as controls. No evidence of compound
    related effects were noted in terms of physical appearance. While no
    effects were noted upon growth and food consumption at the two lower
    dose levels, these parameters were slightly decreased at the
    4000 mg/kg/day and markedly decreased at the 8000 mg/kg/day level. Two
    year survival, poor for all groups in both sexes and particularly for
    the male control group was attributed to spontaneous disease. However,
    survival for females of the 4000 and 8000 mg/kg/day groups was lower
    than that of the control groups, significantly so at the 8000
    mg/kg/day level where survival was 54% of the control level. No
    evidence of compound related changes was noted in terms of
    haematology, blood chemistry or eye examination findings. Increased
    red and white blood cells were seen persistently in the urine of the
    rats tested at the highest dietary level. Gross and microscopic

    pathology findings were in general not treatment or dosage related
    (McConnell, 1973; Anonymous, 1973c). However, astrocytomas were seen
    in all treated animals none being noted in the controls. Incidence of
    brain tumours found were as follows:

                                                                        

                                              Feeding level
        Tumours           Control                                       
                                     1 000    2 000    4 000    8 000
                                                                        

    Astrocytomas             0         4        1        4        1

    Oligodendroglioma        0         0        0        1        0

    (Hazleton Labs., 1973)
                                                                        

    Groups of 40 male and 40 female rats received 2000 and 4000 mg/kg/day
    of aspartame in their diets for 104 weeks. A group of 60 male and 60
    female rats served as controls. All animals were selected from the
    F1A litter of a multi-generation study in which the parents had been
    exposed to corresponding dietary levels of aspartame for 60 days prior
    to mating. No compound related effects were noted in terms of
    appearance, behaviour, results of ophthalmological findings or
    survival. Decreased weight gain and food consumption was noted for the
    higher dosage level animals. Sporadic, statistically significant
    variations were noted in both haematology and blood chemistry
    findings; there was not however any clear trend of dose or treatment
    relationship. Heart to body weight ratios were significantly decreased
    for males of both treatment groups and liver weights were increased
    for both female treatment groups. Grossly inconsistent treatment
    related differences were noted. Histopathologically several
    statistically significant alterations were noted for several organs.
    These were increased stomach ulceration and gastritis in high level
    females, increased incidence of liver hyperplastic nodules at both
    feeding levels in females, increased incidence of nodular hyperplasia
    of the adrenal cortex. Incidence of brain tumours found may be
    tabulated as follows:

                                                                        

                                                 Feeding level
       Tumour               Control                                     
                                              2 000        4 000
                                                                        

    Astrocytoma                4                3            1

    Meningoma                  0                0            1

    (Anonymous, 1974e)
                                                                        

    A detailed histopathological review of the brains, liver and pituitary
    glands of control rats, and rats fed aspartame was carried out. The
    incidence of intracranial neoplasm did not appear to predominate in
    any treated group nor did any particular type of neoplasm. The
    intracranial neoplasms appeared in both the untreated control and
    treated rats. Hyperplastic nodules from the liver were considered to
    be non-neoplastic and were not treatment related. Chromophobe adenomas
    occurred frequently in test and control animals. It was concluded that
    the neoplastic alterations in the rats were not treatment related and
    within "normal" limits (Kommineni, 1974). A detailed statistical
    analysis of the incidence of the hyperplastic nodules of the liver
    showed that for animals sacrificed at week 104, there was no
    significant increase for test animals over control animals (Springer,
    1974).

    Hamsters

         Basic groups each consisting of five male and five female
    hamsters were fed aspartame containing less than 1% DKP at dietary
    levels of 1000, 2000, 4000 and 12 000 mg/kg/day for 46 weeks. Groups
    of 10 male and 10 female hamsters served as controls. Each group was
    replicated seven times. Somewhat erratic decreases in body weights
    were seen in the high dosage level males. Transient food consumption
    decreases were noted for both sexes of the high feeding level group.
    No unequivocal compound related changes in physical, behavioural signs
    or mortality were noted for this study. However, there was a spread of
    an unidentified infection considered to be "wet tail" in both control
    and treated animals considered to be responsible for the high
    mortality rate (50%). It was because of this that the study was
    terminated at 46 weeks. No consistent treatment or dose-related
    effects were noted upon haematology, clinical chemistry and
    haematology. Gross pathology was not delineated by groups, but
    indicated that gross lesions were "uniformly recorded both in control
    and all treated groups and were attributed to the unidentified
    infection in the colony". Neoplastic changes were not reported.
    Microscopic lesions reported appeared to be scattered throughout the
    groups with no apparent treatment relationship (Rao et al., 1972b).

    OBSERVATIONS IN MAN

    Normal adults

    1.  Short-term tolerance

         Thirty-one normal men and 38 normal women (aged 21-45) were the
    subjects of this study. The study design was double blind, with the
    subjects randomly assigned to receive aspartame or matching placebo
    capsules. The aspartame dose was increased from 0.6 g/day during the
    first week to a final level of 8.1 g during the 6th week of the test.
    The total amount of aspartame consumed by each individual during the
    study was 160.7 g. The following laboratory tests were done one week
    before each subject entered the study, and then again at weeks 4 and 6
    of the test: complete blood count, complete urinalysis, partial
    thromboplastin, prothrombin time, BUN, thyroxine, bilirubin (direct
    and indirect), SGOT, alkaline phosphatase, uric acid, creatinine,
    cholesterol (total), triglycerides. Serum insulin and glucose levels
    were determined after a four-hour fast, and again after dosing the
    subject with 100 g glucose orally, during weeks 3 and 6 and follow-up
    week 7. Plasma phenylalanine and tyrosine values were determined twice
    a week, on blood samples following a four-hour fast. Phenylalanine
    tests of urine were done during weeks 3, 5 and 7. Methanol
    determinations were done on samples of serum and urine during weeks 4
    and 6. General physical examinations with special eye studies were
    also performed. No significant difference between the aspartame and
    the placebo groups were reported for any of the tests (Langlois,
    1972).

    2.  Long-term tolerance

         The subjects used in this study consisted of individuals who had
    participated in the short-term study, and who had agreed to continue
    in the study for an additional 21 weeks, and subjects who would follow
    the same study design and fulfil the same criteria for admission. A
    total of 76 persons (30 male, 37 female) were involved in the study.
    The daily intake of aspartame during the 21 weeks was 1.8 g/day. The
    laboratory tests described in the short-term study were carried out
    initially and at weeks 6, 12, 20 and 21 of the study. Serum insulin
    and serum glucose levels, plasma tyrosine and phenylalanine levels
    were measured at the commencement of the study and weeks 12, 16, 20
    and 21. Urine was tested for phenylpyruvic acid prior to and at weeks
    1, 10, 20 and 21 of the study. Clinically significant differences
    between those taking aspartame and the placebo group were not shown
    for either sex in the results of the tests performed (Frey, 1972a).

    Obese individuals

         The study group consisted of 95 men and women aged 21-70, whose
    weight exceeded more than 20% the mean normal weight for height, sex,
    body frame, and age as taken from the Metropolitan Life Insurance Co.
    Weight Tables (Four Steps to Weight Control, New York, Metropolitan
    Life Insurance Co., 1969). The study design and dose level of

    aspartame was the same as that described for the short-term study with
    normal adults. The laboratory tests were carried out over the same
    period. In addition to the tests previously described, weight and
    blood pressure were monitored throughout the study. Eighty-four of the
    subjects completed the test (44 on aspartame, 40 on placebo). Sixty-
    nine individuals who completed the first six weeks of the study,
    continued taking 1.8 g of aspartame/day for an additional 21 weeks. In
    addition, another 36 individuals took aspartame for 21 weeks only. The
    regime for laboratory tests was the same as that described for the
    long-term normal studies. No significant differences were reported
    between the aspartame and placebo groups (Hoffman et al., 1972;
    Hoffman & Romano, 1973).

    Normal children and adolescents

         Five age-groups were studied of age ranges, 2-3(13), 4-6(22),
    7-9(22), 10-12(24), 13-20(45). The figures in parenthesis represent
    number of individuals in the study. The study design was double blind
    with the subjects randomly assigned to receive foods containing
    aspartame or sucrose. The mean daily intake of aspartame ranged from
    39.5 to 58.1 mg/kg bw, for the 13 weeks of the study. The following
    laboratory determinations were made initially and during weeks 7 and
    13 on all subjects aged two through 12 years: plasma phenylalanine,
    plasma tyrosine, complete blood count, partial thromboplastin,
    prothrombin time, creatinine, bilirubin (direct, indirect and total),
    SGOT, urinalysis complete, urine test for phenylpyruvic acid. The
    following additional tests were done on subjects aged 13 through 20
    years; serum thyroxine, fasting glucose, alkaline phosphatase, uric
    acid, cholesterol (total and esters), triglycerides and BUN. No
    significant clinical differences were reported between the sucrose and
    aspartame groups (Frey, 1972b).

    Adult PKU heterozygotes

         The subjects of this study were the natural parents of
    phenylketonuric children. Sixty-five men and women (non-obese) between
    the ages of 21 and 45 were studied. The design of the study and the
    dose schedule was the same as that described for normal adults. In
    addition to the tests described, electroencephalograms were taken on a
    number of subjects before and after the study. Short-term tolerance
    studies were conducted for six weeks, 52 of the subjects continued the
    study, for a total period of 21 weeks. The design and dose schedule of
    this portion of the study is the same as that described for normal
    adults (long-term tolerance). There were no significant biochemical or
    physical changes during the course of the study in either the
    aspartame or placebo groups. There was no evidence that the
    phenylalanine content of aspartame caused any disturbance of the
    apparently normal phenylalanine metabolism (Koch et al., 1972; 1973).

    Tolerance of loading doses by normal adolescents

         A 12-year-old male and 15-year-old female were subjects of this
    study. The two subjects were given a loading dose of 34 g/kg bw
    aspartame in orange juice on one occasion and two weeks later they
    were given the molecular equivalent amount of L-phenylalanine
    (19 g/kg) in orange juice. A standardized diet was prescribed for
    each of the three 24-hour periods before the aspartame and the
    phenylalanine loads and for each of the three, 24-hour periods
    following each load. Urine was collected for three successive
    three-hour periods before each load, and then at 8, 16, 24-hour
    post-loading. Urine was analysed by chromatography for amino acids
    (phenylalanine and tyrosine), phenolic acids, phenylpyruvic acid and
    phenylacetylglutamine and methanol. Serum levels of phenylalanine and
    tyrosine were determined before the loading dose and at 1, 2, 4, 8,
    24, 48 and 72-hour post-loading. All parameters studied remained
    normal during the period of the test (Koch & Shaw, 1973).

    Tolerance of loading doses by phenylketonuric (PKU) homozygous children

         Two PKU homozygous boys, each approximately 14 years old were
    selected for this study. One was on a liberalized Lofenalac diet with
    an allowable phenylalanine dietary intake of 70 mg/kg/day. The other
    was on a well-controlled Lofenalac diet (PA intake 17 mg/kg/day). The
    two subjects were given a loading dose of 34 g/kg bw aspartame in
    orange juice and two weeks later were given a molecular equivalent
    amount of L-phenylalanine (19 g/kg) in orange juice. A standardized
    diet consistent with the subjects' clinical state was prescribed three
    days prior to and three days post-loading. Although one subject (1) on
    the liberalized diet was well within permitted phenylalanine range
    during the loading study (dietary intake 2539 mg, added PA from
    loading 689 mg), the other subject (2) for exceeding his dietary
    limitations (dietary intake 965 mg, added PA from loading, 1072 mg).
    Urine samples were collected at eight-hour intervals on the day prior
    to loading, then at eight-hour intervals for the first day post-
    loading, and then early morning samples on subsequent days 2 and 3.
    Urines were analysed for amino acids (phenylalanine and tyrosine),
    phenolic acids, phenylpyruvic acid and phenylacetylglutamine. Serum
    levels of phenylalanine and tyrosine were determined prior to and at
    1, 2, 4, 8, 24, 48 and 72-hour post-loading. One patient (1) at the
    time of the study was excreting large quantities of phenylalanine so
    any slight increase may not have been observed. The other subject (2)
    showed a slight increase in urinary excretion of phenylalanine and its
    metabolites. Analysis of serum did not show any significant increase
    in phenylalanine or tyrosine levels (Koch, 1972).

    Tolerance of aspartame by diabetic subjects

         Seventy-seven subjects (27 male, 50 female) aged 21-70 who were
    dependent on insulin for control of diabetes were studied. Prior to
    the study there was a complete physical examination and the following
    laboratory tests were carried out: complete blood count (CBC), partial

    thromboplastin time, prothrombin time, BUN, creatinine, T4, bilirubin
    (it was required that these values be within normal range), in
    addition the following tests were also carried out: SGOT, SGPT, LDH,
    alkaline phosphatase, glucose (fasting blood sugar), uric acid,
    cholesterol (total and esters), triglycerides. The study was double
    blind with subjects randomly assigned to receive aspartame or matching
    placebo capsules. The participants continued on their normal diets.
    The dose level of aspartame was 1.8 g/day. There was no evidence that
    either aspartame or placebo resulted in consistent changes in fasting
    blood sugar or any other parameters measured in this study (Bleicher &
    Stern, 1973).

    Effects of aspartame loading on plasma and erythrocyte free amino
    acids in normal adult subjects

         Twelve normal healthy subjects (six male and six female) were
    fasted overnight and administered either aspartame at 34 mg/kg bw or
    an equivalent amount of aspartic acid (13 mg/kg), dissolved in orange
    juice. The subjects received nothing by mouth for eight hours
    following the load, except for 240 ml of water at four and six hour
    post-dosing. Normal meals were allowed after that point. Plasma and
    erythrocyte amino acid levels were measured at 0, 0.25, 0.5, 0.75, 1,
    1.5, 2, 3, 4, 8 and 24 hours. The 24-hour sample was taken after an
    eight-hour fast. No significant changes were noted in plasma
    aspartate, asparagine or glutamin levels with either treatment. Plasma
    glutamate, alanine and proline levels increased, for both treatments.
    Plasma phenylalanine levels increased from fasting to normal
    postprandial levels. A small increase in the plasma tyrosine level was
    noted in the aspartame group. All other amino acid in the plasma
    decreased or remained unchanged. Erythrocyte glutamate, aspartate and
    asparagine levels were unchanged, after either treatment. Erythrocyte
    phenylalanine and tyrosine, alanine and proline levels were similar to
    those in the plasma. All other amino acids were unchanged or slightly
    elevated (Stegink et al., 1977a).

         In another series of tests six normal subjects (three male and
    three female) were administered aspartame in successive studies at
    dose levels equivalent to 100, 150 or 200 mg/kg bw. The subjects were
    fasted eight hours prior to dosing, and eight hours post dosing (with
    water allowed). Blood samples were taken at 0, 0.25, 0.5, 0.75, 1,
    1.5, 2, 3, 4, 5, 6, 7, 8 and 24-hour post-dosing for determination of
    plasma and erythrocyte amino acids. The 100 mg/kg dose of aspartame
    caused no increase in plasma aspartate levels. At the 150 mg/kg bw
    dose level, small increases in plasma aspartate and glutamate were
    noted. Increases in plasma phenylalanine and tyrosine also occurred.
    At the highest level tested (200 mg/kg) there was a small increase in
    plasma aspartate and glutamate levels. Plasma phenylalanine levels
    increased to a mean of 48.7  15 mol/dl after dosing and decreased
    rapidly to normal levels (Ca 7 mol/dl). Plasma tyrosine levels
    increased to a mean of 13.6  12.8 mol/dl (Ca 4 mol/dl normal

    level). Erythrocyte levels of glutamate and aspartate were unchanged,
    while phenylalanine and tyrosine increased to levels similar to those
    in plasma (Stegink et al., 1977a; 1979d; 1979c).

         In another study six healthy women with well-established
    lactation were administered either aspartame or lactose at 50 mg/kg
    bw. The order of administration was randomized in a crossover design,
    with an interval of at least two weeks between each segment of the
    study for each subject. Subjects were fasted eight hours prior to
    administration of the test compound and four hours after. Plasma and
    erythrocyte amino acid levels were measured at 0, 0.25, 0.5, 0.75, 1,
    1.5, 2, 3 and 4 hours post-dosing. Breastmilk samples were collected
    for amino acid analysis at 0, 1, 2, 3, 4, 8, 12 and 24 hours
    post-dosing. There was no significant effect on plasma aspartate,
    asparagine glutamine levels. Plasma glutamate and tyrosine levels were
    slightly increased after aspartame ingested, but were still within
    normal postprandial range. Plasma phenylalanine was also increased
    after aspartame ingestion but not after lactose ingestion. The level
    was higher than that generally observed postprandial. Plasma proline
    and alanine were increased after both aspartame and lactose ingestion.
    No significant effects were observed on other amino acids. Erythrocyte
    phenylalanine and tyrosine increased after aspartame ingestion, as did
    proline and alanine, but the increases were less than that in plasma.
    No significant differences were observed for the other amino acids.
    There was a small increase in tyrosine, phenylalanine and aspartate
    levels in breastmilk after aspartame loading as compared to lactose
    loading, e.g,, breastmilk phenylalanine increased from 0.5 mol/dl to
    2.2 mol/dl, and aspartate increased from 2.2 mol/dl to about
    4.5 mol/dl in the four-hour period post-dosing. At eight hours
    post-dosing the levels were in the normal postprandial range (Stegink
    et al., 1977a; 1979d; 1979e).

         Plasma methanol was determined in individuals administered
    aspartame at 100, 150 and 200 mg/kg bw. The peak levels of methanol
    were (in mg%) 1.27  0.48 (100 mg/kg group), 2.14  0.35 (150 mg/kg
    group) and 2.58  0.78 (200 mg/kg group). No formate was detected in
    the blood or urine of subjects receiving aspartame at 200 mg/kg bw
    (Stegink et al., 1977a; 1979d; 1979e).

         Four female subjects known to be heterozygous for phenylketonuria
    (PKU) were administered aspartame at a dose level equivalent to
    34 mg/kg bw. The subjects were fed eight hours prior to administration
    of the test substances, and then for another eight hours post-dosing
    with water being allowed. Blood samples were taken at 0, 0.25, 0.5,
    0.75, 1, 1.5, 2, 3, 4 and 8 hour post-dosing. Plasma aspartate levels
    were similar to those in normal subjects. Plasma phenylalanine levels
    in the PKU individuals were higher than that in normal subjects
    (16 mol/dl compared to 12 mol/dl normal). No significant differences
    in plasma tyrosine were noted. Erythrocyte levels of amino acids were
    similar to those in normal subjects (Stegink et al., 1977a; 1979d;
    1979e).

         Three male subjects (aged 25-55 years) were fasted overnight
    prior to administration by mouth of phenylalanine 14C aspartame
    (500 mg in 120 ml water). Four-hour post-dosing normal diet was
    allowed. Blood and urine samples were taken prior to dosing and then
    at various intervals post-dosing up to 64 hours for blood and 48 hours
    for urine. The 14C levels in plasma rose rapidly during the first 15
    minutes and reached a maximum four to eight hours post-dosing. The
    initial disappearance rate of 14C from plasma was estimated to be
    47.5 hours. Four hours after administration of the test substance, the
    major 14C plasma radioactivity was associated with high molecular
    weight substances (87%), with trace amounts being present as
    phenylalanine, tyrosine, diketopiperazine and aspartylphenylalanine.
    Less than 1% of the administered 14C was excreted in urine during the
    test period (Anonymous, 1972a).

         The effects of aspartame ingestion on blood amino acids in normal
    adults and one-year-old infants were examined in individuals given
    single 34, 50 or 100 mg/kg bw oral doses (Stegink et al., 1977c;
    1979d; 1979e).

         Plasma concentrations of aspartate were unchanged from fasting
    levels in both infants and adults at all ingestion levels, indicative
    of rapid aspartate metabolism. Phenylalanine levels in plasma peaked
    at 45-90 minutes after dosing. The increase was dose but not age
    related. The data are summarized below:

    Peak plasma phenylalanine levels (mol/dl)

                                                                        

                                       Dose
    Subject                                                             
                    0        34 mg/kg       50 mg/kg      100 mg/kg
                                                                        

    Infants     6.1  1.2    9.7  2.7     11.5  3.1     22.5  11.6

    Adults      5.7  1.2    11.1  2.5    16.2  4.6     20.2  6.8
                                                                        

    Six normal adult subjects (three male, three female) were examined for
    glutamate and aspartate plasma levels up to six hours after ingesting
    a 1 g protein/kg bw meal or an identical meal containing MSG or APM at
    a dose of 34 mg/kg bw (Stegink et al., 1977b; 1979d; 1979e).

         Plasma glutamate and aspartate levels did not differ
    significantly between groups. In an identical study using MSG at 150
    mg/kg bw and aspartame at 23 mg/kg bw, plasma glutamate plus aspartate
    levels did not differ significantly between the subject groups fed the
    MSG alone or MSG plus aspartame (Stegink et al., 1979c; 1979e).

         In a similar study, six male and three female subjects were given
    a meal consisting of a clear soup and a beverage containing either no
    added compound, MSG at a dose of 50 mg/kg bw, or MSG at 50 mg/kg bw
    plus aspartame at 34 mg/kg bw. Plasma aspartate and glutamate were
    measured for up to four hours postprandially. Plasma aspartate levels
    were unchanged following ingestion of the soup-beverage meal without
    added MSG or aspartame. The MSG dose produced a significant increase
    in plasma aspartate values 15-30 minutes after loading. Addition of
    both APM and MSG produced a small but statistically significant
    (p = 0.01) increase in plasma aspartate levels above those noted from
    MSG alone 30-60 minutes after loading. The addition of APM alone did
    not significantly increase the mean peak values of plasma glutamate
    plus aspartate (Stegink et al., 1979b; 1979e).

         Six known MSG sensitive individuals (susceptible to "Chinese
    Restaurant Syndrome") were given 300 ml of cold orange juice
    containing either sucrose (1 g/kg bw) or aspartame (34 mg/kg bw) in a
    crossover design study (Searle Labs, 1979b). No symptoms were reported
    by any of the subjects and plasma aspartate levels were similar after
    aspartame and sucrose loading. Plasma glutamate levels remained within
    normal fasting levels (Stegink et al., 1979a; 1979e).

         Multiple doses of 34, 100 or 200 mg/kg bw of aspartame were used
    to investigate the average steady state levels of plasma phenylalanine
    after repeated doses of the compound. The following table summarizes
    the T 1/2, Ke and K1 from this study:

    Plasma phenylalanine half-life, Ke and K1

                                                                        

    Aspartame dose    Half-life (T 1/2)       Ka*              K1**
      (mg/kg bw)           (hours)         (hour -1)         (hour -1)
                                                                        

         34                 1.65             0.420             3.47

        100                 1.7              0.408             2.72

        200                 1.7              0.408             1.26
                                                                        

    *  Ke = first rate constant for the disappearance of phenylalanine
             from the plasma.

    ** K1= first order rate constant for input of phenylalanine in
             plasma.

    The following table summarizes the average steady state plasma
    phenylalanine concentrations:

    Plasma phenylalanine (mol/dl)

                                                                        

    Time interval between doses
              (hours)                    Aspartame dose (mg/kg bw)
                                                                        

                                       34          100           200

                 1                     12           67           156
                 2                      6           34            78
                 3                      4           22            52
                 4                      3           17            39
                 8                      -            8            19

                                           (Stegink et al., 1979e)
                                                                        

    Adult PKU heterozygotes

         Five female PKU heterozygotes were given a single 100 mg/kg bw
    oral dose of aspartame in cold orange juice. As shown in the table
    below, plasma aspartate levels were not affected. However, plasma
    phenylalanine levels were elevated.

    Plasma aspartate and phenylalanine levels mol/dl)

                                                                        

                              Time (h)
                                                         
            0     0.5    1.0    1.5    2.0    3.0    4.0    Reference
                                                                        

           0.5    0.8    0.5    0.6    0.4    0.5    0.4
    ASP                                        
           0.3    0.6    0.3    0.5    0.3    0.3    0.3    Stegink et
                                                            al., 1979f
           7.0    36     37     42     32     24     17
    PHE                                        
           0.7    7.6    8.0    2.3    5.3    3.2    2.7
                                                                        


         Plasma and erythrocyte amino acid levels were measured in eight
    female subjects known to be PKU heterozygotes and 12 normal subjects
    (six males and six females) after a single oral dose of aspartame at
    34 mg/kg bw. No changes in either plasma or erythrocyte aspartate

    levels were noted up to eight hours after dosing in either group. In
    the normal subjects, plasma phenylalanine levels increased from
    fasting values (5.66  1.21 M/100 ml) to normal post-prandial
    levels (11.11  2.49 M/100 ml) and returned to close to baseline
    levels at eight hours after dosing. In the PKU heterozygotes, mean
    peak plasma phenylalanine levels were 16.03  2.25 M/100 ml and the
    concentration curve was broader over time. However, maximum plasma
    phenylalanine levels in this group were only slightly above
    postprandial values in the normal human infant and adult. Erythrocyte
    phenylalanine levels showed similar but smaller patterns of change
    (Stegink et al., 1978).

    Comments

         An extensive array of toxicological studies have been carried out
    with aspartame (APM and its breakdown product DKP).

         Metabolic studies with APM in a variety of species suggest that
    aspartame is hydrolysed to its constituent amino acids prior to
    absorption from the GI tract, and its subsequent metabolism resembles
    that of phenylalanine, aspartic acid and methanol. The possible
    effects of subsequent increases in serum phenylalanine in neonates has
    been demonstrated in the 52-week study in the infant Rhesus monkey
    which showed that with APM at dose levels of 3 and 4-6 g/kg grand mal
    seizures were observed at 218 days of treatment. The effect was not
    observed at the low doses (ca 1 g/kg). Thereafter, sporadic
    convulsions occurred inconsistently at various times. The seizures
    occurred most frequently during physical handling of the animals and
    were of the grand mal type similar to those induced by feeding
    L-phenylalanine to infant monkeys.

         In an early study in which infant monkeys were dosed with
    aspartame, the monkeys from the two highest aspartame dose groups (3.0
    and 3.7 g/kg) underwent seizures. In another study none of these
    effects were observed in the two highest aspartame dose groups (2.0
    and 2.7 g/kg). It is important to note that in the early study monkeys
    were fed the total intake of milk formula in an eight-hour period,
    while in the second study they were provided 24-hour access to
    aspartame-in-milk formula. This would result in a less pronounced
    peaking of phenylalanine blood levels in the monkeys of the later
    study. It seems that in the early study ad libitum watering was not
    allowed while in the second study it was. There is also evidence that
    normal food intake and growth were impaired in the early study. These
    methodological variations may explain the differences in the results
    of the two studies, i.e. seizuring versus no seizuring. It appears
    that, if proper nutrition, hydration and growth are maintained, even
    extremely high intakes of aspartame (1-3 g/kg/day) do not produce
    untoward effects in neonatal monkeys.

         Phenylketonuria (PKU) and resulting brain damage and mental
    retardation can be experimentally induced in the monkey by feeding
    large doses of phenylalanine (PA) beginning shortly after birth. The
    large dosing results in suppression of phenylalanine hydroxylase
    activity in the liver, the enzyme required for conversion of PA to
    tyrosine. The mid and high doses (ca 3 and 4 g/kg/day) of APM resulted
    in a significant increase in serum PA and tyrosine levels, while the
    low dose (ca 1 g/mg/day) showed no appreciable change in serum PA and
    tyrosine levels.

         A comparison of the serum PA and tyrosine levels of positive
    control monkeys fed 2-2.5 g PA/kg/day with the serum PA levels from
    the animals dosed with 3 or 4 g/kg/day APM shows that the PA levels
    were similar. PA and APM at these dose levels cause similar effects in
    neonate monkeys. It is reasonable to conclude that the effect is due
    to the PA moiety of the aspartame, and that this problem related to
    neonate infants at an age where susceptibility to phenylalanine and
    the effects of PKU are critical. Long-term feeding studies with rats,
    mice and dogs, as well as a bladder implant study with mice, provide
    adequate data to assess the carcinogenic potential of APM as well as
    other long-term effects.

         In the two-year feeding studies in rats, effects at the highest
    dose levels (4 and 8 g/kg bw/day) fed included decreased growth,
    kidney weight increases, decreased thyroid weight, seminal vesicle
    atrophy, changes in the pancreas (fibrosis, mild atrophy and nodular
    hyperplasia) and gastritis and stomach ulceration. These effects were
    not observed in animals on the lower dose levels (1 and 2 g/kg/day).

         Neonate rats (two weeks old) which had been exposed to APM
    in utero showed subtle changes in the kidney (minimal to slight
    hypertrophy and vacuolation of nuclei in cells of tubules in the inner
    cortex). However, in the long-term feeding study in which rats were
    exposed in utero to aspartame, these effects were not observed in
    the mature animals, nor did they affect the well being of the animals.
    Both control and test animals showed an incidence of brain tumours,
    nodular hyperplasia of the adrenal cortex, and liver hyperplastic
    nodules. The brain lesions (tumours) seen in some test animals of the
    first two-year rat study were investigated by a detailed pathology
    evaluation of brain sections of the second, lifetime study. These same
    brain lesions were seen in untreated controls as well as in test
    animals in the second study. The fact that these lesions were not seen
    in control animals of the first two-year study could be attributed to
    biological variation and not treatment related. The statistically
    significant increase of adrenal nodular hyperplasia in survivor males
    of the second lifetime rat study was not borne out in non-survivor
    males, in females, nor in all groups of the first two-year rat study.
    The gastritis and stomach ulceration increase in female rats at high
    test levels of the second lifetime rat study, although possibly
    related to compound, was not seen consistently in all groups nor in
    any test group of the first two-year rat study.

         The incidence of hyperplastic nodules in the livers of animals of
    the two-year rat study was subjected to a detailed statistical
    analysis; there did not appear to be a significant increase of these
    lesions in test animals over controls.

         In the case of the 104-week mouse study, a detailed pathological
    study did not indicate the presence of compound-related tumorigenic or
    non-tumorigenic changes.

         In the two-year feeding study with dogs, at the highest dose
    level fed (4 g/kg/day) effects observed included decreased
    haemoglobin, haematocrit and total red blood cells. BSP values were
    also decreased. The effects were not noted at lower dietary levels.
    Gross microscopic pathological findings did not indicate any compound-
    related effect. A detailed study of the brain of test dogs did not
    show any neoplastic changes.

         The 56-week urinary bladder impact study in the mouse showed no
    significant differences in the incidence of neoplasms in the urinary
    bladder of negative control (cholesterol) and test groups (cholesterol
    plus APM).

         The general lack of carcinogenic response in these test systems
    provides an adequate data base for the non-carcinogenicity of APM.

         Studies of the carcinogenic potential of DKP include a long-term
    study in the rat and mouse, as well as a bladder implant study in the
    mouse. In the rat study there was a significant decrease in weight
    gain at the highest dose levels. At autopsy the only compound-related
    effect was a significant increase in the incidence of uterine polyps.
    The significance of this effect was carefully assessed. The lesions
    were considered to be non-neoplastic benign proliferations that are
    known to occur spontaneously in aging rats. In the mouse study the
    only compound-related effect was an increase in absolute relative
    thyroid/body weight. However, there were no pathological changes. The
    56-week urinary bladder implant study in the mouse showed no
    significant differences in the incidence of neoplasm in the urinary
    bladder of negative control (cholesterol) and test groups (cholesterol
    plus DKP).

         Metabolic studies with DKP in a number of animal species indicate
    that some DKP may be observed unchanged, or as its metabolite,
    phenylacetyl-glutamine. Other metabolic products have not been
    identified. Detailed investigations on the possible nitrosation of
    DKP show that n'-nitroso compounds were not formed in vitro or
    in vivo.

         A two-generation rat reproduction study with aspartame showed
    that the only effect observed at the highest dose level tested
    (4 g/kg bw) was that the body weights of F1A and F2A weanlings were
    significantly reduced. This effect was not observed in the lower dose
    levels (2 g/kg bw). All other parameters were normal. A single

    generation reproduction study with DKP at dose levels up to
    1.8 g/kg/day showed no compound-related effects. Teratological studies
    with the rat and rabbit with either AMP or AMP/DKP (3:1) mixture of
    DKP at dose levels up to 4 g/kg/day, administered either by gavage or
    in the diet (rabbit) or in the diet (rat), showed no significant
    compound-related effects. Mutagenicity studies, which included host-
    mediated assay, dominant lethal and cytogenetic studies with AMP or
    DKP, did not indicate that either AMP or DKP are mutagenic.

         AMP and DKP were subjected to a large nurser of biological tests
    to screen for any possible pharmacological, endocrinological or
    behavioural effects. A positive effect was only observed in one test,
    namely, that high levels of DKP exhibit some progesterone antagonism.

         Aspartame was tested in human subjects of various population
    types - normal adults, "healthy" obese adults, normal children and
    adolescents, PKU heterozygotes (natural parents of PKU children), PKU
    and normal adolescents, and insulin and non-insulin dependent
    diabetics. Various doses of aspartame and various treatment periods
    (from acute, large dose loading to prolonged reasonable dietary dosing
    up to 90 days) were used. Comparisons were made with phenylalanine,
    with sucrose controls, and normal diet controls (double-blind
    studies). The data indicated no significant toxicological problems
    within limitations of the studies.

         Studies on aspartame loading of normal individuals showed that
    this resulted in only minor changes in the levels of serum or
    erythrocyte amino acids. At the highest dose studied, the peak levels
    of phenylalanine were within the range tolerated in phenylketonurics
    subjected to high doses of aspartame who, however, showed somewhat
    higher levels of phenylalanine than normal subjects. The elevations
    were transient, and well below levels that could be expected to
    produce toxic effects. Loading with aspartame did not result in
    significant levels of methanol in the blood, or formate in blood or
    urine. Additional studies have been carried out on the effect of
    aspartame ingestion on blood amino acids in infants, normal adults and
    female PKU heterozygotes. The results of these studies confirm the
    previous observations. Aspartame loading has also been shown not to
    increase plasma glutamate levels in MSG-sensitive individuals.
    

    EVALUATION

    Aspartame:

    Estimated level causing no toxicological effect in the rat

    4 g/kg bw

    Estimate of acceptable daily intake for man

    40 mg/kg bw

    Diketo piperazine:

    Estimated level causing no toxicological effect in the rat

    750 mg/kg bw

    Estimate of acceptable daily intake for man

    7.5 mg/kg bw
    

    REFERENCES

    Andress, J., Martinez, T. & Youkilis, G. SC-19192: Acute toxicity
         studies in the rat, mouse and rabbit. Unpublished report from
         Department of Pathology-Toxicology, submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1973a

    Andress, J., Martinez, T. & Youkilis, G. SC-18862: Acute toxicity
         studies in the rat, mouse and rabbit. Unpublished report from
         Department of Pathology-Toxicology of Searle Laboratories,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1973b

    Anonymous. SC-18862: Two-month oral administration - rats. Unpublished
         report from Hazleton Laboratories, Inc., submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1969a

    Anonymous. SC-18862: Two-month oral toxicity - dogs. Unpublished
         report from Hazleton Laboratories, Inc., submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1969b

    Anonymous. Chicken embryo study: SC-18862, calcium cyclamate, sucrose.
         Unpublished report from Hazleton Laboratories, Inc., submitted to
         the World Health Organization by G. D. Searle & Co., Skokie,
         Ill., USA, 1970a

    Anonymous. SC-18862: Mutagenic study in rats. Unpublished report from
         Hazleton Laboratories, Inc., submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1970b

    Anonymous. SC-18862: Two generation reproduction study in rats.
         Unpublished report from Hazleton Laboratories, Inc., submitted to
         the World Health Organization by G. D. Searle & Co., Skokie,
         Ill., USA, 1971

    Anonymous. The metabolism of Aspartame, Vol. I, Parts I-XIV.
         Unpublished report from the Department of Biochemical Research of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972a

    Anonymous. The metabolism of Aspartame, Vol. III, Parts XX-XXIII.
         Unpublished report from Huntington Research Center, submitted to
         the World Health Organization by G. D. Searle & Co., Skokie,
         Ill., USA, 1972b

    Anonymous. The metabolism of Aspartame, Vol. II, Parts XV-XIX.
         Unpublished report from the Department of Biochemical Research of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972c

    Anonymous. SC-18862 and 19192 (3:1 ratio): Segment II teratology study
         in the rat. Unpublished report from Hazleton Laboratories, Inc.,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1972d

    Anonymous. SC-18862: An evaluation of mutagenic potential employing
         the host-mediated assay in the rat. Unpublished report from
         Hazleton Laboratories, Inc., submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1972e

    Anonymous. SC-19192: Segment II teratology study in the rabbit.
         Unpublished report from Hazleton Laboratories, Inc., submitted to
         the World Health Organization by G. D. Searle & Co., Skokie,
         Ill., USA, 1972f

    Anonymous. SC-19192: An evaluation of mutagenic potential employing
         the in vivo cytogenetics method in the rat. Unpublished report
         from Hazleton Laboratories, Inc., submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1972g

    Anonymous. A study of the possible reaction of DKP with aqueous
         nitrous acid. Unpublished report from Searle Laboratories,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1972h

    Anonymous. The metabolism of Aspartame, Vol. II, Parts XV-XIX.
         Unpublished report from the Department of Biochemical Research of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972i

    Anonymous. SC-18862: A sweetening agent. Pharmacological studies.
         Unpublished report from the Department of Biological Research of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972j

    Anonymous. SC-18862: An evaluation of mutagenic potential employing
         the in vivo cytogenetics method in the rat. Unpublished report
         from Hazelton Laboratories, Inc., submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1972k

    Anonymous. SC-19192: An evaluation of mutagenic potential employing
         the host-mediated assay in the rat. Unpublished report from
         Hazleton Laboratories, Inc., submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1972l

    Anonymous. SC-18862 and 19192: (3:1 ratio): Segment II teratology
         study in the rabbit. Unpublished report from Hazleton
         Laboratories, Inc., submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1973a

    Anonymous. SC-18862: Segment II - An evaluation of the teratogenic
         potential in the rabbit. Unpublished report from Hazleton
         Laboratories, Inc., submitted to the World Health Organization by
         G. D. Searle & Co., Skokie, Ill., USA, 1973b

    Anonymous. SC-18862 (Aspartame): A supplemental study of rat brains
         from two tumorigenicity studies. Unpublished report from
         Department of Pathology-Toxicology of Searle Laboratories,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1973c

    Anonymous. The metabolism of Aspartame, Vol. IV, Parts XXIV-XXXI.
         Unpublished report from the Department of Drug Metabolism and
         Radiochemistry of Searle Laboratories, submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1974a

    Anonymous. SC-18862: Segment II - An evaluation of the teratogenic
         potential in the rabbit. Unpublished report from Hazleton
         Laboratories, Inc., submitted to the World Health Organization by
         G. D. Searle & Co., Skokie, Ill., USA, 1974b

    Anonymous. SC-19192: 110-week toxicity study in the mouse. Unpublished
         report from Hazleton Laboratories, Inc., submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1974c

    Anonymous. SC-18862: 104-week toxicity study in the mouse. Unpublished
         report from Hazleton Laboratories, Inc., submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1974d

    Anonymous. SC-18862: Lifetime toxicity study in the rat. Unpublished
         report from Hazleton Laboratories, Inc,, submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1974e

    Bleicher, S. J. & Stern, S. B. Tolerance of Aspartame by diabetic
         subjects. Unpublished report from Tulane University School of
         Medicine, submitted to the World Health Organization by G. D.
         Searle & Co., Skokie, Ill., USA, 1973

    Bryan, G. T. SC-18862: A 56 week urinary bladder tumorigenicity study
         in the mouse by the intravesical pellet implant technique.
         Unpublished report from University of Wisconsin School for Health
         Sciences, submitted to the World Health Organization by G. D.
         Searle & Co., Skokie, Ill., USA, 1974a

    Bryan, G. T. SC-19192: A 56 week urinary bladder tumorigenicity study
         in the mouse by the intravesical pellet implant technique.
         Unpublished report from University of Wisconsin School for Health
         Sciences, submitted to the World Health Organization by G. D.
         Searle & Co., Skokie, Ill., USA, 1974b

    Frey, G. H. Long-term tolerance of Aspartame by normal adults.
         Unpublished report from Hill Top Research, Inc., submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1972a

    Frey, G. H. Long-term tolerance of Aspartame by normal children and
         adolescents. Unpublished report from Hill Top Research, Inc.,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1972b

    Hemm, R. D., Rao, K. S., Martinez, T. B., Calhoun, D. W. & Mayer,
         J. E. SC-18862: Nine week oral toxicity study in the rat.
         Unpublished report from Department of Pathology-Toxicology of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972

    Hoffman, R. S. & Romano, F. Long term tolerance of Aspartame by obese
         adults. Unpublished report from Staten Island Hospital, submitted
         to the World Health Organization by G. D. Searle & Co., Skokie,
         Ill., USA, 1973

    Hoffman, R. S., McGinn, T. & Romano, F. Short term tolerance of
         Aspartame by obese adults. Unpublished report from Staten Island
         Hospital, submitted to the World Health Organization by G. D.
         Searle & Co., Skokie, Ill., USA, 1972

    Koch, R. Tolerance of loading doses of Aspartame by phenylketonuric
         (PKU) homozygous children. Unpublished report from Children's
         Hospital of Los Angeles, submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1972

    Koch, R. & Shaw, K. Tolerance of loading doses of Aspartame by normal
         adolescents. Unpublished report from the Children's Hospital of
         Los Angeles, submitted to the World Health Organization by G. D.
         Searle & Co., Skokie, Ill., USA, 1973

    Koch, R., Peterson, R. M. & Scriver, C. R. Short term tolerance of
         Aspartame by adult PKU heterozygotes. Unpublished report from the
         Children's Hospital of Los Angeles, submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1972

    Koch, R., Peterson, R. M. & Wolfinger, H. L. Long term tolerance of
         Aspartame by adult PKU heterozygotes. Unpublished report from the
         Children's Hospital of Los Angeles, submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1973

    Kommineni, C. Personal communication to C. J. Kokoski of the United
         States Food and Drug Administration, submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1973

    Kommineni, C. Personal communication to C. J. Kokoski of the United
         States Food and Drug Administration, submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1974

    Langlois, K. J. Short term tolerance of Aspartame by normal adults.
         Unpublished report from Hill Top Research., Inc., submitted to
         the World Health Organization by G. D. Searle & Co., Skokie,
         Ill., USA, 1972

    Lemkey-Johnston, N., Reynolds, W. A. & Kulikowski, H. Damage in the
         neonatal mouse brain following ingestion of Aspartame.
         Unpublished report from the Illinois State Institute for
         Developmental Disabilities, and the Department of Anatomy,
         University of Illinois College of Medicine, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1977

    McConnell, R. G. SC-18862: Two-year toxicity study in the rat.
         Unpublished report from Hazleton Laboratories, Inc., submitted to
         the World Health Organization by G. D. Searle & Co., Skokie,
         Ill., USA, 1973

    Nutting, E. F. SC-18862: A sweetening agent. Endocrine studies.
         Unpublished report from Department of Biological Research of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972

    Potts, W. J. SC-18862: A sweetening agent. Pharmacological studies.
         Unpublished report from Department of Biological Research of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972

    Rao, K. S., Martinez, T. B., Hemm, R. D. & McConnell, R. G. SC-19192:
         Two week oral toxicity study in the mouse. Unpublished report
         from Department of Pathology-Toxicology of Searle Laboratories,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1971a

    Rao, K. S., Mauro, J. & McConnell, R. G. SC-19192: Two week oral
         toxicity study in the rat. Unpublished report from Department of
         Pathology-Toxicology of Searle Laboratories, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1971b

    Rao, K. S. Staunton, C. & McConnell, R. G. SC-19192: Five-week oral
         toxicity study in the rat. Unpublished report from Department of
         Pathology-Toxicology of Searle Laboratories, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1972a

    Rao, K. S. Martinez, T. B. & McConnell, R. G. SC-18862: Four week oral
         tolerance study in the mouse. Unpublished report from Department
         of Pathology-Toxicology of Searle Laboratories, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1972b

    Rao, K. S. Martinez, T. B. & McConnell, R. G. SC-18862: Four week oral
         tolerance study in the rat. Unpublished report from Department of
         Pathology-Toxicology of Searle Laboratories, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1972c

    Rao, K. S. Mauro, J. & McConnell, R. G. SC-18862: 106 week oral
         toxicity study in the dog. Unpublished report from Department of
         Pathology-Toxicology of Searle Laboratories, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1972d

    Rao, K. S. Mauro, J. & McConnell, R. G. SC-18862: 46 week oral
         toxicity study in the hamster. Unpublished report from Department
         of Pathology-Toxicology of Searle Laboratories, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1972f

    Rao, K. S. McConnell, R. G. & Waisman, H. A. SC-18862: 52 week oral
         toxicity study in the infant monkey. Unpublished report from
         Department of Pathology-Toxicology of Searle Laboratories,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1972e

    Rao, K. S., Stejskal, R. & McConnell, R. G. SC-19192: 115 week oral
         tumorigenicity study in the rat. Unpublished report from
         Department of Pathology-Toxicology of Searle Laboratories,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1974

    Reynolds, W. A., Stegink, L. D., Filer, L. J., jr & Renn, E. Aspartame
         administration to the infant monkey: Hypothalamic morphology and
         blood amino acid levels. Unpublished report from the Department
         of Anatomy of the University of Illinois at the Medical Center,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1979a (Also submitted for publication to
         Anat. Rec., 1980)

    Reynolds, W. A., Bauman, A. F., Stegink, L. D., Renn, E. & Filer, L.
         J., jr. Developmental assessment of infant macaques receiving
         dietary Aspartame or phenylalanine. Unpublished report from the
         Department of Anatomy of the University of Illinois at the
         Medical Center, submitted to the World Health Organization by G.
         D. Searle & Co., Skokie, Ill., USA, 1979b

    Rozek, L. F. SC-18862: A sweetening agent. Pharmacological studies.
         Unpublished report from Department of Biological Research of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972

    Saunders, R. N. SC-18862: A sweetening agent. Pharmacological studies.
         Unpublished report from Department of Biological Research of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972

    Schroeder, R. E. & McConnell, R. G. Evaluation of embryotoxic and
         teratogenic potential in the rat. Unpublished report from
         Department of Pathology-Toxicology of Searle Laboratories,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1970

    Schroeder, R. E. & McConnell, R. G. SC-18862: An evaluation of the
         embryotoxic and teratogenic potential in the rabbit. Unpublished
         report from Department of Pathology-Toxicology of Searle
         Laboratories, submitted to the World Health Organization by G. D.
         Searle & Co., Skokie, Ill., USA, 1973

    Schroeder, R. E., Rao, K. S. & McConnell, R. G. Toxicological
         evaluation of SC-18862: Evaluation of reproductive performance.
         Unpublished report from Department of Pathology-Toxicology of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1972

    Schroeder, R. E., Rao, K. S., McConnell, R. G. & Sammeta, K. SC-18862:
         An evaluation of the mutagenic potential in the rat employing
         dominant lethal assay. Unpublished report from Department of
         Pathology-Toxicology of Searle Laboratories, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1973a

    Schroeder, R. E., Mitchell, A., Rao, K. S., McConnell, R. G. &
         Sammeta, K. SC-19192: An evaluation of the mutagenic potential in
         the rat employing the dominant lethal assay. Unpublished report
         from Department of Pathology-Toxicology of Searle Laboratories,
         submitted to the World Health Organization by G. D. Searle & Co.,
         Skokie, Ill., USA, 1973b

    Schroeder, R. E., Mitchell, A., Rao, K. S. & McConnell, R. G.
         SC-19192: Evaluation of reproductive performance in the rat.
         Unpublished report from Department of Pathology-Toxicology of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1973c

    Schroeder, R. E., Mitchell, A., Rao, K. S. & McConnell, R. G.
         SC-19192: An evaluation of the embryotoxic and teratogenic
         potential in the rat. Unpublished report from Department of
         Pathology-Toxicology of Searle Laboratories, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1973d

    Schroeder, R. E., Mitchell, A., Rao, K. S. & McConnell, R. G.
         SC-18862: An evaluation of the embryotoxic and teratogenic
         potential in the rabbit. Unpublished report from Department of
         Pathology-Toxicology of Searle Laboratories, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1973g

    Schroeder, R. E., Rao, K. S. & McConnell, R. G. SC-18862: A study of
         the pregnant and lactating rat and of her offspring. Unpublished
         report from Department of Pathology-Toxicology of Searle
         Laboratories, submitted to the World Health Organization by G. D.
         Searle & Co., Skokie, Ill., USA, 1973e

    Schroeder, R. E., Rao, K. S. & McConnell, R.G. SC-18862: An evaluation
         of the embryotoxic and teratogenic potential in the rabbit.
         Unpublished report from Department of Pathology-Toxicology of
         Searle Laboratories, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1973f

    Simmon, V. F. & Shan, H. G. An evaluation of the mutagenic potential
         of SC-18862 employing Ames Salmonella/microsome assay; S.A.
         1385. Unpublished report from SRI International, submitted to the
         World Health Organization by G. D. Searle & Co., Skokie, Ill.,
         USA, 1978

    Springer, J. Personal communication to C. J. Kokoski of the United
         States Food and Drug Administration, submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1974

    Stegink, L. D., Filer, L. J., jr & Baker, G. L. Effect of Aspartame
         loading upon plasma and erythrocyte free amino acid levels in
         normal adult subjects. Unpublished report from the University of
         Iowa College of Medicine, submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1977a

    Stegink, L. D., Filer, L. J., jr & Baker, G. L. Metabolic studies of
         Aspartame and MSG ingested as a meal component. Unpublished
         report from the University of Iowa College of Medicine, submitted
         to the World Health Organization by G. D. Searle & Co., Skokie,
         Ill., USA, 1977b

    Stegink, L. D., Filer, L. J., jr & Baker, G. L. Effect of Aspartame
         upon plasma and erythrocyte free amino acid levels and blood
         methanol levels in normal one-year-old children. Unpublished
         report from the University of Iowa College of Medicine, submitted
         to the World Health Organization by G. D. Searle & Co., Skokie,
         Ill., USA, 1977c

    Stegink, L. D., Filer, L. J., jr, Baker, G. L. & McConnell, J. E.
         Effect of Aspartame loading at 100 mg per kg body weight upon
         plasma and erythrocyte levels of free amino acids in normal
         subjects and subjects presumed to be heterozygous for
         phenylketonuria. Unpublished report from the University of Iowa
         College of Medicine, submitted to the World Health Organization
         by G. D. Searle & Co., Skokie, Ill., USA, 1978

    Stegink, L. D., Filer, L. J., jr & Baker, G. L. Effect of Aspartame
         loading in subjects who report symptoms of Chinese restaurant
         syndrome after glutamate ingestion. Unpublished report from the
         University of Iowa College of Medicine, submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1979a

    Stegink, L. D., Filer, L. J., jr & Baker, G. L. Metabolic studies of
         Aspartame and monosodium glutamate when ingested together as part
         of a soup-beverage meal. Unpublished report from the University
         of Iowa College of Medicine, submitted to the World Health
         Organization by G. D. Searle & Co., Skokie, Ill., USA, 1979b

    Stegink, L. D., Filer, L. J., jr & Baker, G. L. Metabolic studies of
         Aspartame and monosodium glutamate ingested as components of a
         hamburger-milk shake meal system in normal adult subjects.
         Unpublished report from the University of Iowa College of
         Medicine, submitted to the World Health Organization by G. D.
         Searle & Co., Skokie, Ill., USA, 1979c

    Stegink, L. D., Filer, L. J., jr, Baker, G. L., Brummel, M. C. &
         Tephly, T. R. Aspartame metabolism in human subjects. In:
         Guggenheim, B. Health and sugar substitutes. Basle, Munich,
         Paris, London, New York, Sydney, S. Karger, pp. 160-165, 1979d

    Stegink, L. D., Filer, L. J., jr & Baker, G. L. Personal communication
         to I. C. Winter of G. D. Searle & Co., submitted to the World
         Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
         1979e

    Stegink, L. D., Filer, L. J., jr, Baker, G. L. & McConnell, J. E.
         Effect of Aspartame loading upon plasma and erythrocyte amino
         acid levels in phenylketonuric heterozygotes and normal subjects.
         J. Nutr., 109: 708-717, 1979f
    


    See Also:
       Toxicological Abbreviations
       Aspartame (WHO Food Additives Series 16)
       ASPARTAME (JECFA Evaluation)