Aspartame was first evaluated by JECFA in 1975 (see Annex,
    Ref. 37). 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 (see Annex,
    Ref. 40).

         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 (see Annex, Ref. 43).

         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 (see Annex, Ref. 51). In 1980
    JECFA evaluated additional toxicity animal studies and several human
    studies and an ADI of 0-40 mg/kg bw for aspertame and an ADI of
    0-7.5 mg/kg bw for its breakdown, diketopiperazine, were established
    (see Annex, Refs. 54 and 56).

         Since that evaluation, additional studies have become available
    and are summarized and discussed in the following monograph addendum.



    Long-term studies


         In a study designed to evaluate and characterize the effects
    of long-term administration of aspartame or aspartame and
    diketopiperazine in Wistar rats, groups of 86 male and 86 female rats
    were fed a powdered basal diet containing 0, 1, 2, 4 g/kg/day of
    aspartame or 4 g/kg/day of aspartame and diketopiperazine (3:1). Each
    group was divided into a main and a satellite group for interim


    *    Monograph addendum to the monograph appearing in Ref. 56 (Annex).

    clinical and post mortem examination. In satellite groups, 10 males
    and 10 females were examined after 26 weeks and 16 males and 16
    females of each group after 52 weeks. The remaining survivors were
    killed at 104 weeks. No spontaneous deaths were observed at 26 and 52
    weeks. Mortality rate of the various test groups was comparable at 104
    weeks. A significant increase in urinary specific gravity and a
    decrease in urinary pH were noted in the 4 g/kg aspartame and 4 g/kg
    aspartame plus diketopiperazine groups. Urinary calcium excretion was
    increased in both male and female at 2 and 4 g/kg aspartame and 4 g/kg
    aspartame plus diketopiperazine throughout the study. Relative kidney
    weights were increased at the higher dose levels in both sexes at 26
    and 52 weeks. Histopathology of the kidneys revealed a high incidence,
    over 95%, of chronic nephropathy in all groups including control. The
    incidence of nephrocalcinosis including pelvic and medullary and
    metastic mineralization appeared to be increased mainly in the females
    of the aspartame treated groups when compared to the control group.
    The spontaneous incidence of nephrocalcinosis in the controls was
    relatively high, particularly in females (Ishii et al., 1981). The
    incidence of brain tumours in this study was reported separately. No
    brain tumours were detected at 26 or 52 weeks. The incidence of brain
    tumours in rats exposed to the test material for more than one year
    was as follows:

         Control          - 1/119 (0.8%) - 1 female astrocytoma
                                           atypical at 99th week

         1 g/kg           - 1/119 (0.8%) - 1 male oligodendroglioma
                                           at 75th week

         2 g/kg           - 2/120 (1.7%) - 1 female astrocytoma and
                                           1 female ependymoma at
                                           terminal sacrifice

         4 g/kg           - 1/120 (0.8%) - 1 male astrocytoma at
                                           93rd week

         4 g/kg           - 1/120 (0.8%) - 1 female oligodendroglioma
         (APM plus DKP)                    at 51st week

                                                         (Ishii, 1981)


         Four normal subjects and four normal obligate PKU heterozygotes
    received 34 mg/kg aspartame in 8 oz of orange juice in a fasting
    state. The normal controls were healthy young women varying in age
    from 20 to 28 years. The PKU heterozygote mothers were selected to
    match the age of the controls. Blood samples were obtained by
    venipuncture at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4 and 8 hours after
    ingestion and analysed for amino acid content of plasma and

    erythrocytes. The data on normals revealed that plasma and
    erythrocyte phenylalanine levels remained within the normal range
    (6.06-18.18 Ám/dl). The values for PKU heterozygotes were higher than
    for the controls, but were generally within the range considered
    normal. Peak values occurred between 0.5 and 2 hours and returned to
    near pretest values during the period of testing. This was generally
    the case for all the amino acids analysed (Koch & Blaskovics, 1978).

         Six normal healthy adults (three male and three female) and five
    female subjects heterozygous for phenylketonuria were administered
    aspartame at 100 mg/kg bw dissolved in 500 ml of orange juice.
    Aspartame was administered to subjects in a fasting state. Plasma and
    erythrocyte amino acid levels were measured at 0, 0.25, 0.5, 0.75, 1,
    1.5, 2, 3, 4, 5, 6, 7 and 8 hours after test load. Plasma levels of
    aspartate were not significantly affected in either group. Similarly,
    levels of glutamate, asparagine and glutamine, which are readily
    derived from aspartate, were essentially unchanged. Plasma
    phenylalanine levels were significantly increased after aspartame load
    in both groups. Mean maximum phenylalanine levels observed in normal
    subjects were approximately 20 Ámol/dl at 30-90 minutes after loading
    while those noted for heterozygous PKU subjects were twice as large,
    ranging from 36.5 Ámol/dl at 30 minutes to 41.7 Ámol/dl at 90 minutes.
    Plasma tyrosine levels increased in both groups after loading, with
    higher levels noted in normal subjects. This is to be expected since
    heterozygous PKU subjects have a decreased ability to convert
    phenylalanine to tyrosine. Erythrocyte levels of amino acids followed
    the same pattern as those reported for plasma (Stegink et al., 1978).

         A total of 12 infants, aged eight to 12 months, were administered
    aspartame dissolved in Kool-Aid at 34 and 50 mg aspartame/kg bw (six
    at each dose level). Blood samples were obtained by heel stick at 0,
    30, 45, 60, 90, 120 and 150 minutes. A total of four samples was
    obtained from each infant (a fasting sample and three subsequent

         Each blood sample was analysed for plasma and erythrocyte free
    amino acid levels and blood methanol concentration. Plasma aspartate
    levels were higher in the infants than previously observed in adults.
    There was, however, no increase in plasma aspartate after loading with
    aspartame. No significant changes were noted in erythrocyte aspartate
    levels. Plasma phenylalanine levels increased slightly when 34 mg
    aspartame/kg bw was administered, rising from a mean of 6.3 Ámol/dl at
    zero time to 9.7 Ámol/dl at 30 minutes. Erythrocyte phenylalanine
    levels showed a similar, but smaller response. When a 50 mg/kg bw dose
    was administered plasma phenylalanine rose from 5.7 at zero time to
    11.6 Ámol/dl at 60 minutes. Erythrocyte phenylalanine values increased
    but response as lower. Blood methanol levels increased at both loading
    doses from 0.07 to 0.19 and 0.3 mg/dl respectively, 45 to 90 minutes
    after loading, followed by a decrease to baseline values. These data
    show that a one-year-old infant handles aspartame as well as the

    normal adult at these dosage levels. The failure to increase plasma
    aspartate, phenylalanine and methanol above post prandial levels would
    indicate little hazard to the infant from aspartame at the dosage
    levels studied. Since the infant metabolized aspartame as well as an
    adult and in previous studies no adverse effects were seen in the
    adult when given 100, 150 and 200 mg/kg bw of aspartame, this study
    was extended to giving a loading dose of 100 mg/kg bw of aspartame in
    Kool-Aid to a total of eight infants, eight to 12 months of age. A
    fasting and three subsequent blood samples were obtained from each
    infant. Plasma and erythrocyte aspartate levels were unchanged after
    aspartame loading. Plasma phenylalanine levels increased from 4.8 to
    21.4 Ámol/dl at 45 minutes. Erythrocyte phenylalanine showed a similar
    but somewhat lower response. Blood methanol levels increased from
    0.11 mg/dl to 1.02 mg/dl at 90 minutes (Stegink et al., 1977).


         The Committee evaluated an additional long-term study in rats of
    aspartame and diketopiperazine impurity and further biochemical
    studies of aspartame in man. It appears that the increased urinary
    excretion of calcium as well as the nephrocalcinosis are due probably
    to the consequence of a protein overload induced by the high intake of
    aspartame (2-4% in the diet). In accordance with other reported
    studies, the rat, especially the female, was more prone to the
    development of both functional and anatomical renal changes
    attributable to slight imbalance in calcium metabolism. Since neither
    hypercalciuria nor nephrocalcinosis was observed in mice or dogs with
    chronic administration of aspartame, the effect in the rat would
    appear to be species and sex specific. The probability of human renal
    changes due to aspartame consumption within the limits of the proposed
    ADI (40 mg/kg) would appear to be remote since this amount would not
    significantly increase the daily amino acid or protein intake. The
    incidence of brain tumours between the control and treated groups
    was comparable. It was concluded that neither aspartame or
    diketopiperazine caused brain tumours in rats in this study.

         The evidence available to the Committee when it established an
    ADI for aspartame at its twenty-fourth meeting was substantial. The
    additional data summarized in this working paper serves to confirm the
    previously established ADI.


    Estimated level causing no toxicological effect in the rat

    Aspartame: 4 g/kg bw

    Diketo piperazine: 750 mg/kg bw

    Estimate of acceptable daily intake for man

    Aspartame: 40 mg/kg bw

    Diketo piperazine: 7.5 mg/kg bw


    Ishii, H. (1981) Incidence of brain tumours in rats fed aspartame,
         Toxicology Letters (In press)

    Ishii, H. et al. (1981) Toxicity of aspartame and its
         diketo-piperazine for Wistar rats in dietary administration for
         104 weeks, Toxicology (In press)

    Koch, R. & Blaskovics, M. (1978) Effect of aspartame on plasma and red
         cell amino acids of apparently healthy female adults and on
         presume phenylketonuric heterozygotes. Unpublished report from
         the Departments of Pediatrics at the Children's Hospital of Los
         Angeles, and the University of Southern California School of
         Medicine, Los Angeles, submitted to WHO by G. D. Searle & Co.

    Stegink, L. D. et al. (1977) Effect of aspartame loading upon plasma
         and erythrocyte free amino acid levels and blood methanol levels
         in normal one-year-old children. Unpublished report from the
         Departments of Pediatrics and Biochemistry, University of Iowa
         College of Medicine, Iowa City, Submitted to WHO by G. D. Searle
         & Co.

    Stegink, L. D. et al. (1978) Effect of aspartame loading at
         100 mg/kg body weight upon plasma and erythrocyte levels of free
         amino acids in normal subject and subjects presumed to be
         heterozygous for phenylketonuria. Unpublished report from the
         University of Iowa College of Medicine, Iowa City, submitted to
         WHO by G. D. Searle & Co.

    See Also:
       Toxicological Abbreviations
       Aspartame (WHO Food Additives Series 15)
       ASPARTAME (JECFA Evaluation)