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    L-GLUTANIC ACID AND ITS AMMONIUM, CALCIUM, MONOSODIUM AND
    POTASSIUM SALTS

    EXPLANATION

         These substances were evaluated at the fourteenth and seventeenth
    meetings of the Committee (Annex 1, references 22 and 32). The ADI of
    0-120 mg/kg b.w. allocated to L-glutamic acid encompassed the glutamic
    acid equivalents of the salts and was additional to glutamic acid
    intake from all non-additive dietary sources. The ADI did not apply to
    infants under 12 weeks of age and further work required if the use was
    to be extended to infant foods included the determination of oral
    non-adverse effect levels of glutamates in neonatal animals and age
    correlations between neonatal experimental animals and the human
    infant.

         Since the last review additional data have become available and
    are summarized and discussed in the following monograph. The
    previously-published monographs have been expanded and are
    incorporated into this monograph.

    BIOLOGICAL DATA

    Biochemical aspects

    Metabolism and pharmokinetics

         Glutamic acid is metabolized in the tissues by oxidative
    deamination (von Euler et al., 1938) or by transamination with
    pyruvate to yield oxaloacetic acid (Cohen, 1949) which, via
    alpha-ketoglutarate, enters the citric acid cycle (Meister, 1965).
    Quantitatively minor but physiologically important pathways of
    glutamate metabolism involve decarboxylation to gamma-aminobutyrate
    (GABA) and amidation to glutamine (Meister, 1979). Decarboxylation to
    GABA is dependent on pyridoxal phosphate, a coenzyme of glutamic acid
    decarboxylase (Perrault & Dry, 1961), as is glutamate transaminase.
    Vitamin B6-deficient rats have elevated serum glutamate levels and
    delayed glutamate clearance (Wen & Gershoff, 1972). A number of
    reviews on metabolism of glutamate that contain more comprehensive
    information have been published (Munro, 1979; Meister, 1979;
    Stegink, 1984).

         Glutamate is absorbed from the gut by an active transport system
    specific for amino acids. This process is saturable, can be
    competitively inhibited, and is dependent on sodium ion concentration
    (Schultz et al., 1970). During intestinal absorption, a large
    proportion of glutamic acid is transaminated and consequently alanine
    levels in portal blood are elevated. If large amounts of glutamate are
    ingested, portal glutamate levels increase (Stegink, 1984). This
    elevation results in increased hepatic metabolism of glutamate,
    leading to release of glucose, lactate, glutamine, and other amino
    acids, into systemic circulation (Stegink, 1983c).

         The pharmacokinetics of glutamate depend on whether it is free or
    incorporated into protein, and on the presence of other food
    components. Digestion of protein in the intestinal lumen and at the
    brush border produces a mixture of small peptides and amino acids;
    di-and tri-peptides may enter the absorptive cells where intracellular
    hydrolysis may occur, liberating further amino acids. Defects are
    known in both amino acid and peptide transport (Matthews, 1975, 1984).

         Glutamic acid in dietary protein, together with endogenous
    protein secreted into the gut, is digested to free amino acids and
    small peptides, both of which are absorbed into mucosal cells where
    peptides are hydrolyzed to free amino acids and some of the glutamate
    is metabolized. Excess glutamate and other amino acids appear in
    portal blood. As a consequence of the rapid metabolism of glutamate in
    intestinal mucosal cells and in the liver, systemic plasma levels are
    low, even after ingestion of large amounts of dietary protein
    (Munro, 1979; Meister, 1979; Stegink, 1984).

         Oral administration of pharmacologically high doses of glutamate
    results in elevated plasma levels. The peak plasma glutamate levels
    are both dose and concentration dependent (Stegink et al., 1973,
    1974, 1975b, 1979a, 1982, 1983b, 1983c, 1985b; Ohara et al., 1977;
    Bizzi et al., 1977; Airoldi et al., 1979a; Daabees et al., 1985;
    Heywood et al., 1978). When the same dose (1 g/kg b.w.) of
    monosodium glutamate (MSG) was administered by gavage in aqueous
    solution to neonatal rats, increasing the concentration from 2% to 10%
    caused a five-fold increase in the plasma area under curve; similar
    results were observed in mice (Bizzi et al., 1977). Conversely, when
    MSG (1.5 g/kg b.w.) was administered to 43-day-old mice by gavage at
    varying concentrations of 2 to 20% w/v, no correlation could be
    established between plasma levels and concentration (James et al.,
    1978).

         Administration of a standard dose of 1 g/kg b.w. MSG by garage as
    a 10% w/v solution resulted in a marked increase of plasma glutamate
    in all species studied. Peak plasma glutamate levels were lowest in
    adult monkeys (6 times fasting levels) and highest in mice (12-35
    times fasting levels). Age-related differences between neonares and
    adults were observed; in mice and rats, peak plasma levels and area
    under curve were higher in infants than in adults while in guinea pigs
    the converse was observed (Stegink et al., 1979a; Airoldi et al.,
    1979a; Ohara et al., 1977).

         Studies on the effects of food on glutamate absorption have been
    carried out in mice, pigs, and monkeys. When infant mice were given
    MSG with infant formula or when adults were given MSG with consomme by
    gastric intubation, peak plasma glutamate levels were markedly lower
    than when the same dose was given in water, and the time to reach peak
    levels was longer (Ohara et al., 1977). The simultaneous
    administration of metabolizable carbohydrate was found to increase
    glutamate metabolism in mice, pigs, and monkeys, leading to lowered
    peak plasma levels (Stegink et al., 1979a, 1983a, 1983b, 1985a;
    Daabees et al., 1984). In contrast to gastric intubation, ad lib
    feeding of MSG in the diet caused only slight elevation of plasma
    glutamate above basal levels (Ohara et al., 1977; Airoldi et al.,
    1979a; Heywood et al., 1977; Yonetani & Matsuzawa, 1978).

    Metabolic studies in humans

         Similar effects of food on glutamate absorption and plasma levels
    have been observed in man. Only slight rises in plasma glutamate
    followed ingestion of a dose of 150 mg MSG/kg b.w. to adults with a
    meal; human infants, including premature babies, have the capacity to
    metabolize similar doses given in infant formula (Tung & Tung, 1980).
    Human plasma glutamate levels were much lower when large doses of MSG
    were ingested with meals compared to ingestion in water; in studies in
    which MSG was given with tomato juice, sloppy joes, Sustagen,

    Polycose, starch, or sucrose, metabolizable carbohydrate significantly
    lowered peak plasma glutamate levels (Bizzi et al., 1977;
    Byun, 1980; Ghezzi et al., 1980, 1985; Marts et al., 1978;
    Stegink et al., 1979a, 1979b, 1982, 1983a, 1983b, 1983c, 1985a,
    1986).

         In general, foods providing metabolizable carbohydrate
    significantly attenuate peak plasma glutamate levels at doses up to
    150 mg MSG/kg b.w. Carbohydrate provides pyruvate as a substrate for
    transamination with glutamate in mucosal cells so that more alanine is
    formed and less glutamate reaches the portal circulation
    (Stegink et al., 1983b).

    Special studies on transplacental transport

         When MSG (8 g/kg b.w.) was administered orally to rats on day 19
    of gestation, maternal plasma levels rose from approximately 100 µg/ml
    to 1650 µg/ml, but no significant changes were observed in plasma
    glutamic acid of the fetuses (Ohara et al., 1970).

         Infusion of MSG into pregnant rhesus monkeys at a rate of 1 g/hr
    led to a 10-20-fold increase in maternal plasma glutamate, but fetal
    levels remained unchanged. Higher rates of infusion resulted in
    maternal plasma glutamate levels up to 70 times basal levels, but
    fetal levels increased less than 10 times (Stegink et al., 1975a;
    Pitkin et al., 1979).

         In vitro perfusion studies using human placenta indicated that
    the placenta served as an effective metabolic barrier to the transfer
    of glutamic acid (Schneider et al., 1979).

    Special studies on the blood brain barrier

         Glutamate levels are far higher in the brain than in plasma in
    mice, rats, guinea pigs, and rabbits (Garattini, 19711 Giacometti,
    1979; Bizzi et al., 1977).

         Efflux of glutamate from the brain has been reported to be seven
    times greater than influx, reflecting biosynthesis in the brain. The
    transport rate of glutamate from blood to the brain is much lower than
    for neutral or basic amino acids (Oldendoff, 1971). Normal plasma
    glutamate levels are nearly 4 times the Km of the transport rate to
    the brain, so that glutamate transport systems are virtually saturated
    under physiological conditions (Pardridge, 1979).

         In guinea pigs, rats, and mice, brain glutamic acid levels
    remained unchanged after administration of large oral doses of MSG
    which resulted in plasma levels increasing up to 18-fold
    (Peng et al., 1973; Liebschultz et al., 1977; Caccia et al.,
    1982; Airoldi et al., 1979a; Bizzi et al., 1977). Brain glutamate
    increased significantly only when plasma levels were about 20 times
    basal values following an oral dose of 2 g MSG/kg b.w.
    (Bizzi et al., 1977).

         The failure to observe changes in whole brain glutamate when
    plasma levels are elevated does not preclude the possibility that
    levels in small regions, such as the arcuate nucleus of the
    hypothalamus, may increase (Perez & Olney, 1972).

         Subcutaneous injection of high doses (2 g MSG/kg b.w.) to
    neonatal mice caused an increase in serum glutamate to 270 times basal
    values, while levels in the arcuate nucleus increased 4-7 fold
    (Price et al., 1981).

         No appreciable changes in glutamate concentrations were observed
    in the lateral thalamus and in the arcuate nucleus of adult or
    neonatal rats given 4 g MSG/kg b.w. or 2 g MSG/kg b.w., respectively,
    by garage. Peak plasma glutamate levels were 11-12 times normal after
    these doses (Airoldi et al., 1979b).

    Endocrinology studies

         Numerous studies have been carried out in which large doses of
    MSG were administered by subcutaneous or intraperitoneal injection to
    neonatal mice. A common effect of this treatment is a metabolic
    obesity without hyperphagia and stunted growth (Araujo & Mayer, 1973;
    Matsuyama et al., 1973; Nagasawa et al., 1974). The observed
    obesity in these studies was associated with decreased
    adrenaline-stimulated lipolysis. Decreased pituitary weight and
    impaired pituitary function resulted in atrophy of related target
    organs such as the gonads, accessory sexual organs, thyroids, and
    adrenals. Prolactin and growth hormone levels were depressed, but
    hypothalamic LHRF was reported to be unaffected (Lechan et al.,
    1976). Repressed ossification reported in one study was thought to be
    due to deranged PTH/calcitonin regulation (Dhindsa et al., 1978).

         Similar experiments in rats also resulted in stunting and
    obesity, with reduction in weights of the pituitary, adrenals, and
    gonads. Growth hormone levels were reduced in both sexes but LHRH,
    TRH, somatostatin, and norepinephrin levels were unaffected. Rats
    receiving 1 g MSG/kg b.w. subcutaneously showed elevated prolactin but
    reduced growth hormone and TSH levels (Nemeroff et al., 1975,
    1977a,b,c; Redding et al., 1971).

         The effects of treatment are age-dependent in both mice and rats.
    Neonatal rats show a permanent reduction in GH secretion without
    evidence of excessive prolactin secretion whereas acute administration
    of MSG to adults causes suppression of GH and PRL release by effects
    on the dopamine systems in the medial basal hypothalamus
    (Terry et al., 1977). Reduction in weight of the endocrine glands
    without obvious histological changes did not affect fertility
    (Trentini et al., 1974; Lengvari, 1977).

    Physiological role of MSG

         L-Glutamate and GABA supposedly act as excitatory and inhibitory
    transmitters, respectively, in the central nervous system. Glutamate
    is also involved in the synthesis of proteins (Krnjevic, 1970).

    Taste physiology

         Chemical senses (taste and olfaction) affect the cephalic phase
    of secretion of gastric acid, the exocrine pancreas, gastrin,
    glucagon, insulin, and pancreatic polypeptide hormone (Brand et al.,
    1982).

         MSG has a unique taste, called umami (Ikeda, 1908, 1909, 1912)
    and in addition glutamate has flavour-enhancing properties in some
    foods (Kirimura et al., 1969; Solms, 1969; Yamaguchi, 1979). A
    detailed review of glutamate and the umami taste has been published
    and deals with physiological and psychological aspects (Kawamura &
    Kare, 1987).

    Nutritional aspects

         L-Glutamic acid occurs as a common constituent of proteins and
    protein hydrolysates. Nutritional studies in the rat have shown
    glutamic acid to be a non-essential amino acid that is required in
    substantial amounts to ensure high growth rates in rats
    (Hepburn et al., 1960). Some interconversion between glutamic acid
    and arginine can occur to cover minor dietary deficiencies (Hepburn &
    Bradley, 1964).

         As an essential substrate in intermediary metabolism, glutamate
    is present in organs/tissues in the concentrations shown in Table 1
    (Giacometti, 1979).

         The free amino acid pools in the tissues constitute about 70 g in
    the adult, of which the major components are alanine, glutamic acid,
    glutamine, and glycine. The daily turnover of glutamic acid in a 70 kg
    man has been estimated as 4,800 mg (Munro, 1979). Human plasma

    contains 4.4-4.5 mg/l of free glutamic acid and 9 mg/l bound glutamic
    acid; human urine contains 2.1-3.9 µ/mg creatinine of free glutamic
    acid and 200 µg/mg creatinine of bound glutamic acid (Peters et al.,
    1969). Human spinal fluid contains 0.34-1.64 (mean 1,03) mg/l free
    glutamic acid (Dickinson & Hamilton, 1966).

    Table 1.  Free glutamate concentrations in organs and tissues
                                                                        

          Organ/tissue                Total Free Glutamate (mg)
                                                                        

          Muscle                              6,000
          Brain                               2,250
          Kidneys                               680
          Liver                                 670
          Blood plasma                           40

                           Total              9,640
                                                                        

         Premature and full-term infants hydrolyze any given protein in
    the stomach to very similar extents (Berfenstam et al., 1955).
    Hepatic glutamate dehydrogenase appears at 12 weeks of human fetal
    life; it is present in rat fetal liver on day 17 and reaches its
    maximum within 2 weeks after birth (Francesconi & Villee, 1968). Gouty
    patients have raised levels of plasma glutamate compared to normal
    and, following a protein meal, glutamate reaches excessive levels
    (Pagliari & Goodman, 1969).

         Glutamine and glutamic acid are the most abundant amino acids in
    the milk of all species; human milk contains 1.2% protein, of which
    20% is bound glutamic acid, equivalent to 3 g/l calculated as MSG. The
    free glutamic acid concentration is about 300 mg/l. In contrast, cow's
    milk contains 3.5% protein but only 30 mg/l free glutamic acid
    (Maeda et al., 1958, 1961). Later studies have indicated that human
    milk contains about 600 µmole glutamate/l from days 1-7 post-partum,
    rising to 1,300-1,500 µmole/l thereafter. Human or chimpanzee milk is
    10 times higher in free glutamate than is rodent milk (Rassin
    et al., 1978).

         Daily intake of free glutamic acid by the breast-fed infant has
    been estimated to be about 36 mg/kg b.w. (equivalent to 46 mg/kg b.w.
    as MSG) while daily intake of protein-bound glutamate was estimated as
    approximately 360 mg/kg b.w. The breast-fed infant in the USA ingests
    more glutamate, on a body-weight basis, than at any other time of life
    (Baker et al., 1979).

         High levels of free glutamic acid have been found in cantaloupe
    (0.5 g/kg) and grapes (0.4 g/kg), while high levels of aspartic acid
    were found in figs (2.6 g/kg), nectarines (2.0 g/kg), peaches
    (1.1 g/kg), yellow plums (1.8 g/kg), and dry prunes (1.95.2 g/kg)
    (Fernandez-Flores et al., 1970). Fish and meat had less than
    0.1 g/kg of free glutamic acid, sausage 0.1-1.5 g/kg, cheese
    0.222 g/kg, "tomatenflocken" 15 g/kg, and dried mushrooms 17 g/kg
    (Mueller, 1970).

         Daily intakes of free and bound glutamate by breast-fed infants
    at 3 days of age were 1.10 g bound and 0.115 g free, corresponding to
    0.408 g/kg b.w. At one month of age, intakes were 1.37 g bound and
    0.144 g free, corresponding to 0.405 g/kg b.w. Infants aged 5-6 months
    receiving 500 g cow's milk and 2 jars baby food per day would have a
    daily intake of 4.0 g bound and 0.075 g free glutamic acid, equivalent
    to 0.62 g/kg b.w. (Berry, 1970). The mean daily intake of MSG of
    individuals over 2 years of age has been estimated as 100-225 mg
    per capita, an increment of 3-7% over the glutamic acid supplied by
    dietary protein (GRAS, 1976).

         Consumption of MSG in various countries has been estimated
    (see Table 2) (Maga, 1983).

    Table 2.  MSG consumption in several countries
                                                              

    Country             MSG Consumption (g/day)
                                                              

    Taiwan                     3.0
    Korea                      2.3
    Japan                      1.6
    Italy                      0.4
    USA                        0.35
                                                              

         Male weanling rats were fed casein or purified amino acid diets
    for 14 or 21 days. The addition of 0.33% glycine or 1.14% glutamic
    acid to a diet with a protein equivalent (N x 6.25) of 10% essential
    amino acids increased food efficiency (g weight gain/g food) from 0.38
    ± 0.01 to 0.41 ± 0.01 (Adkins et al., 1967).

         The nutritional value of non-essential amino acids as the
    nitrogen source in a crystalline amino acid diet for chick growth was
    examined. The basal diet contained 27% of an essential amino acid
    mixture and an additional 24% of non-essential amino acids. In the
    test diets all non-essential amino acids were removed, and single
    amino acids were added as follows: glutamic acid 11.3%, aspartic acid
    10.2%, alanine, 6.8%, glycine 5.8%, proline 8.7% (additional to the 1%

    in the basal diet), or serine 8.1%. Of these, glutamic acid and
    aspartic acid were found to be very useful nitrogen sources, alanine
    was useful, glycine and proline were insufficient, and serine was
    harmful for chick growth. The chicks fed the L-glutamic acid diet
    showed less growth than those fed the basal diet, although the
    differences between the groups were not statistically significant
    (Sugahara & Ariyoshi, 1967).

         In an 80-day feeding study, young rats received a diet containing
    8% milk protein and 0, 2, 4, or 6% MSG. Body-weight gain and
    body-nitrogen content were significantly greater in the groups
    receiving 4 or 6% MSG relative to controls. No hypothalamic pathology
    was observed. When MSG was added to the diet of rats containing 12%
    milk protein at levels of 2 or 8% for six weeks, an effect on
    body-weight gain was observed (Huang et al., 1976).

    Toxicological studies

    Special studies on mutagenicity

         Cells (kangaroo rat cell line) were exposed continuously for 72
    hours to 0.1% monosodium glutamate without showing any toxic effects
    (US FDA, 1969).

         Groups of 12 male albino Charles-River mice received single oral
    doses, by gavage, of monosodium glutamate at levels of 0, 2.7, or
    5.4 g/kg b.w. The treated animals were mated with groups of 3
    untreated females for each of 6 consecutive weeks. Females were
    sacrificed at mid-term of pregnancy, and the uteri were examined for
    signs of early embryonic death. Females that had been mated with
    treated males showed no differences compared to controls in the number
    of implantations, resorptions, and embryos (Industrial Bio-Test,
    1973a).

         In a host-mediated assay using Salmonella typhimurium G46, the
    bacteria were administered intraperitoneally to the host rats which
    received 0.2 or 5.7 g MSG/kg b.w. orally for 14 days. No increase in
    revertants was seen relative to controls (Industrial Bio-Test, 1973b).

         Potassium and ammonium glutamate, L-glutamic acid, and L-glutamic
    acid HCl were not mutagenic when tested against S. typhimurium
    strains TA98, TA100, TA1535, TA1537, and TA1538 and Saccharomyces
    cereviseae in the presence or absence of S-9 mix (Litton Bionetics,
    1975a, 1975b, 1977a, 1977b).

    Special studies on neurotoxicity

    Mice and rats

         The effects of glutamate on cerebral metabolism were studied by
    intraventricular injection of L-glutamic acid into mice; 150 mg
    produced convulsions, uncoordinated grooming, or circling of the cage
    (Crawford, 1963).

         Two percent intraarterial sodium glutamate increased epileptic
    fits and intracisternal L-glutamic acid caused tonic-clonic
    convulsions in animals and man. High parenteral doses of L-glutamic
    acid caused EEG changes only in dogs with previous cerebral damage,
    and no rise was detected in the cerebral spinal fluid level of
    glutamate (Herbst et al., 1966).

         Monosodium glutamate injected i.p. at a level of 3.2 g/kg b.w.
    caused reversible blockage of beta waves in the electroretinogram in
    immature mice and rats, indicating retinotoxicity (Potts et al.,
    1960). The timing of treatment of mice was quite critical. After 10-11
    days postnatal it was difficult to produce significant lesions of the
    retina (Olney, 1969a). A study of the glutamate metabolizing enzymes
    of the retina of glutamate-treated rats indicated a decrease in
    glutaminase activity, an increase in glutamic aspartate transaminase,
    and no change in glutamyl synthetase and glutaminotransferase. The
    effects appear to be due to repression and induction of enzyme
    synthesis. Glutamate uptake by the retina, brain, and plasma decreases
    with age and is slower in 12-day old animals than in 5-day old animals
    (Freedman & Potts, 1962, 1963).

         Subcutaneous injection of L-monosodium glutamate at 4-8 g/kg into
    mice caused retinal damage with ganglion cell necrosis within a few
    hours. In very young animals there was extensive damage to the inner
    layers (Lucas & Newhouse, 1957).

         Neonatal mice aged 9-10 days were given single subcutaneous
    injections of 4 g/kg monosodium glutamate; the animals were killed
    from 30 minutes to 48 hours later. The retinas showed acute lesions on
    electron microscopy with swelling dendrites and early neuronal changes
    leading to necrosis followed by phagocytosis (Olney, 1969a).

         Mice aged 2 to 9 days were killed 1-48 hours after single
    subcutaneous injections of 0.5-4 g/kg monosodium glutamate. Lesions
    were seen in the preoptic and arcuate nuclei of the hypothalamic
    region on the roof and floor of the third ventricle and in scattered
    neurons in the nuclei tubercles. No pituitary lesions were seen, but
    subcommissural and subfornical organs exhibited intracellular oedema
    and neuronal necrosis. Adult mice given subcutaneously 5-7 g/kg
    monosodium L-glutamate showed similar lesions (Olney, 1969b).

         Degeneration of neonatal mouse retina has been reported following
    parenteral administration of MSG (10 subcutaneous injections of
    2.2-4.2 g/kg 1-10 days after birth) (Cohen, 1967).

         Sixty-five neonatal mice aged 10-12 days received single oral
    doses of monosodium glutamate at 0.5, 0.75, 1.0, or 2.0 g/kg b.w. by
    gavage. Ten were controls and 54 mice received other amounts. After 3
    to 6 hours all treated animals were killed by perfusion. Brain damage,
    as evidenced by necrotic neurons, was evident in arcuate nuclei of 51
    animals: 52% at 0.5 g/kg, 81% at 0.75 g/kg, 100% at 1 g/kg, and 100%
    at 2 g/kg. The lesions were identical by both light and electron
    microscopy to subcutaneous-produced lesions. No lesions were seen at
    0.25 g/kg. The number of necrotic neurons rose approximately with
    dose. Four animals tested with glutamic acid also developed the same
    lesions at 1 g/kg b.w. The effect was additive with aspartate
    (Olney, 1970).

         High subcutaneous doses of 1 or 4 g/kg of MSG caused hypothalamic
    changes in 42 and 60%, respectively, of treated 5 to 7-day old mice.
    Oral administration (1 or 4 g/kg) of a 4% aqueous solution elicited a
    predominantly glial reaction in 26-28% of the mice. The remainder were
    unaffected (Abraham et al., 1971).

         Six 9 to 10-day old mice, dosed orally with 10% monosodium
    glutamate (2 g/kg), showed characteristic brain lesions (Geil, 1970).

         Monosodium glutamate, monopotassium glutamate, sodium chloride,
    and sodium gluconate at 1 g/kg in a 10% w/v solution (and comparable
    volumes of distilled water), were administered orally and
    subcutaneously to mice and rats at 3 or 12 days of age and to dogs at
    3 or 35 days of age and the animals were killed within 24 hours of
    dosage. Examination of the eyes and of the preoptic and arcuate nuclei
    of the hypothalamus by two pathologists revealed no dose-related
    histomorphological effects in any of the test groups at either of the
    two ages selected to correspond to the periods before and at the
    beginning of solid food intake (Oser et al., 1971).

         Seventy-five infant Swiss albino CD-1 mice (3 to 10 days old)
    were given single subcutaneous injections of MSG at concentrations
    equal to 2 or 4 g/kg (0.1 ml in distilled water). Another group of 50
    adult CD-1 mice were injected either subcutaneously or
    intraperitoneally with MSG at doses varying from 6 to 10 g/kg (1 ml
    volume). Control animals were injected with sodium chloride. Brain
    tissue was examined by light and electron microscopy. Ninety-five
    percent of the animals injected with MSG developed brain lesions in
    the arcuate nucleus of the hypothalamus. Lesions involved primarily
    microglial cells, with no effects to the perikarya of neurons. Distal
    neuronal processes were only slightly affected (Arees & Mayer, 1971).

         Thirteen neonate CFl-JCL mice received single subcutaneous
    injections of 1 g/kg of MSG at days 2 and 4 after birth. Brains were
    removed 1, 3, 6, and 24 hours post-injection and examined by light
    microscopy. The common finding after 3 and 6 hours was necrosis of the
    neural element in the region of the hippocampus and hypothalamus. When
    pregnant mice of the same strain were injected subcutaneously with
    5 mg/g on days 17 and 18 of pregnancy, examination of fetal brains 3,
    6, and 24 hours after treatment showed cellular necrosis in both the
    ventromedial and arcuate nuclei (Murakami & Inouye, 1971).

         Groups of 3- and 12-day old C57BL/J6 mice, each containing 5
    animals, were given single intragastric or single subcutaneous doses
    of monosodium glutamate, sodium chloride, sodium gluconate, potassium
    glutamate (all 10% solutions, 10 ml/kg b.w.), or water. All animals
    were killed 24 hours after dosing. Microscopic examination of the
    brains, particularly the ventral hypothalamus, did not show any
    neuronal necrosis of the hypothalamic arcuate nuclei (Oser
    et al., 1973).

         Groups of 10-day old Swiss Webster albino mice, each containing
    10 animals, were given single subcutaneous doses of one of 24
    compounds structurally related to MSG. The dose level was either 12 or
    24 µmole/kg b.w. Five hours post-dosing the brains and retinas were
    processed for light or electron microscopy. Roughly quantifying the
    pathological reaction in the infant hypothalamus was used as a method
    for comparing the neurotoxic potency of the test compounds. Except for
    L-cysteine, all neurotoxic compounds were acidic amino acids known to
    excite neurons. The most potent neurotoxic compounds were those known
    to be powerful neuroexcitants (N-methyl-DL-aspartic and DL-homocystcic
    acids) (Olney et al., 1971).

         Groups of 10-12-day old Swiss Webster albino mice, each
    containing 7-23 animals, were given single oral doses of MSG at levels
    of 0.25, 0.50, 0.75, 1.0, or 2.0 g/kg. Groups of 2 or 4 mice of the
    same age were given single oral doses of either 1.0 or 3.0 g/kg
    L-glutamic acid or monosodium-L-aspartate or 3.0 g/kg
    L-glutamate-L-aspartate, monosodium glutamate, NaCl, L-glycine,
    L-serine, L-alanine, L-leucine, D,L-methionine, L-phenylalanine,
    L-proline, L-lysine, L-arginine, or L-cysteine. The animals were
    sacrificed after dosing and brains were examined by either light or
    electron microscopy. The severity of brain damage was estimated by
    quantifying the pathological changes in the hypothalamus. One g/kg of
    glutamic acid destroyed approximately the same number of hypothalamic
    neurons as a comparable dose of MSG. Of the amino acids tested, only
    aspartate and cysteine produced hypothalamic damage. These amino acids
    caused both retinal and hypothalamic lesions similar to those found
    after treatment with MSG (Olney & Ho, 1970).

         Infant litter mates of Swiss Webster mice were divided into two
    groups. The experimental group received single daily subcutaneous
    injections of MSG for 10 consecutive days. The control litter-mate
    group received injections of 0.9% saline. All injections were of
    0.02 ml volume. The dose of the first MSG injection, starting 24 hours
    after birth, was 2 g/kg b.w. Subsequent daily doses were increased by
    0.25 g/kg per day so that the final dose on the tenth day was
    4.25 g/kg. When the surviving MSG-treated and control mice attained
    20-28 g b.w., they were subjected to a battery of behavioural and
    pharmacological tests. The study period lasted until 50 days after
    birth. There were no significant or observable differences in response
    to behavioural tests or to selected drugs (Prabhu & Oester, 1971).

         A group of 10-day old mice were given single subcutaneous doses
    of 18 mmole MSG/kg b.w. and sacrificed from 15 minutes to 8 days
    subsequently. Further groups were given 2 g, rising to 4 g MSG/kg b.w.
    daily, from day 1 to day 10 and sacrificed 9 months later, or 4 g
    MSG/kg b.w. by gavage on day 10. There was quite irreversible damage
    to neurons in the arcuate nucleus and rapid cell necrosis; there were
    fewer cells 2-4 days later. An early, reversible glial and ependymal
    oedema was also seen (Olney, 1971).

         Neonatal mice were given 0, 2, or 4 g MSG/kg b.w. by gavage and
    sacrificed after 20 or 30 minutes or after 1, 2, 3, or 24 hours. At
    the higher-dose level, oedema and necrosis of the neurons of the
    arcuate nucleus were seen after 20 minutes and preoptic and arcuate
    nucleus lesions were observed after 30 minutes. The lesions spread
    wide with time affecting the tectum and other structures in 2-3 hours.
    Phagocytosis was seen in the arcuate nucleus after 24 hours. Primary
    lesions in neurons were seen at the electron microscope level after 30
    minutes (Lemkey-Johnston & Reynolds, 1972, 1974).

         Sodium chloride was administered to 3-9-day old mice as a 10%
    solution by gavage at doses of sodium equivalent to 1-10 g MSG/kg
    b.w.; glutamic acid HCl was administered at 2 and 4 g/kg b.w. as a 20%
    solution and sucrose (80% w/v) was given at dosages equivalent to 4,
    8, or 10 g MSG/kg b.w. Oedema and pyknotic nuclei were seen in sodium
    chloride-treated animals of 5 days of age; no lesions were seen in
    animals older than 6 days at the time of treatment, nor in mice given
    sucrose (Lemkey-Johnston et al., 1975).

         Forty male and 41 female mice were given daily subcutaneous
    injections of 2.5 g MSG/kg b.w. from 5-10 days of age and were
    subsequently reared to maturity. Adult animals had an 80% decrease in
    perikarya of the arcuate nucleus, endocrine deficits, reduced
    reproductive performance, stunted growth, obesity, and decreased
    weights of the pituitary, ovaries, and testes (Holzwarth-McBride et
    al., 1976).

         Monosodium glutamate was administered to 4 weanling mice of each
    sex ad libitum in the diet or drinking water at levels of 46 g/kg/
    day or 21 g/kg/day, respectively. No hypothalamic lesions were
    induced. Plasma glutamic acid levels were doubled by giving MSG at 10%
    w/v in the diet, but the threshold for neurotoxicity of MSG by dietary
    administration was not exceeded (Heywood et al., 1977).

         Groups of 24 mice were given single subcutaneous injections of 1,
    2, 3, or 4 g MSG/kg b.w. at age 10 days, or 2, 4, or 6 g MSG/kg b.w.
    at age 60 days. Examination of the brains 3-5 hours after treatment
    showed damage to the area postrema (Olney et al., 1977).

         Weanling mice were fasted and deprived of fluids overnight, then
    given as drinking fluid either 10% MSG; a mixture of 5% MSG, 4.5%
    monosodium aspartate and 1% aspartame; 5% MSG; 2.5% MSG and 2.5%
    aspartate; or a choice between 10% MSG and deionized water. The brains
    were examined about 4 hours after start of treatment. All animals
    ingesting MSG-containing solutions sustained hypothalamic injury
    (Olney et al., 1980).

         Neonatal mice aged 6-12 days were injected with single doses of
    0.25, 0.5, 1, 2, or 4 g MSG/kg b.w. Hypothalamic lesions were evident
    at doses of > 0.5 g/kg b.w. (Reynolds et al., 1976).

         A total of 52 mice, 10 days of age, were given single
    intraperitoneal doses of 4 g MSG/kg b.w. Two mice were killed every
    hour from 1 to 20, then at 24, 48, 72, 96, and 168 hours after
    injection and the brains were examined. Neuronal changes appeared 2
    hours after dosing, comprising somatic ballooning, nuclear chromatin
    clumping, pyknosis, and cytoplasmic vacuolation in the neurons of the
    arcuate nucleus. The histopathologic changes became more severe with
    time (Takasaki, 1978a).

         A total of 38 mice, 2-20 days old, were given single
    intraperitoneal doses of 4 g MSG/kg b.w.; the animals were killed 8
    hours after injection and the brains were examined. All treated
    animals showed necrotic changes, including pyknosis and karyorrhexis,
    of neurons of the arcuate nucleus; the number of necrotic neurons was
    maximal in mice dosed at 6-8 days of age and diminished in severity
    with age of dosing until 20 days of age, when few necrotic neurons
    were seen (Takasaki, 1978a).

         Four mice, 10 days old, were given single intraperitoneal doses
    of 4 g MSG/kg b.w., killed 8 hours after treatment, and the brains
    examined to determine the total extent of the lesions. Acute necrotic
    changes were noted in the subfornical organ, preoptic area, and area
    postrema, in addition to the arcuate nucleus; in 1 mouse pyknotic
    neurons were also found in the cerebral cortex (Takasaki, 1978a).

         Eighteen 1-day old mice were injected intraperitoneally daily
    with doses of 4 g MSG/kg b.w. for 1-10 days and killed 8 hours after
    the last dose. No neurons disappeared after a single injection but
    neurons that withstood a single injection were subsequently affected
    and disappeared almost completely by the fourth consecutive daily
    injection (Takasaki, 1978a).

         Twenty-two mice, 10 days old, were injected with 0.1-2 g MSG/kg
    b.w. intraperitoneally, killed 8 hours later, and the hypothalamic
    arcuate region examined. A further 120 mice received doses of
    0.1-4.0 g MSG/kg b.w. by gavage and were examined similarly. The
    threshold dose was 0.4 g MSG/kg b.w. i.p. or 0.7 g/kg b.w. by garage
    (Takasaki, 1978a).

         Pregnant mice received MSG at levels of 0, 5, 10, or 15% in the
    diet or 5% in drinking water on the eighteenth day of gestation. The
    animals were killed and fetal brains were examined. Groups of
    lactating mice were put on the same regimes for 1-4 days and the
    brains of the pups examined. Further groups of weanling mice were
    given similar diets for 1-4 days and killed 3 hours after feeding. No
    changes were seen in the arcuate nucleus of the fetuses, suckling
    pups, or weanlings (Takasaki, 1978b).

         The smallest neurotoxic dose increases with age and oral doses
    give lower plasma levels than parenterally-administered doses. Dietary
    feeding of MSG does not affect serum levels of LH or testosterone.
    Small subcutaneous doses given to neonatal and infant mice or large
    oral doses given to weanling mice produced no adverse effects
    (Takasaki et al., 1979a).

         Groups of 10-day old mice were given by gavage 2 or 4 g MSG/kg
    b.w. together with 0.63 g NaCl/kg b.w. or 1.93 g glucose/kg b.w.
    Sodium chloride did not potentiate MSG-induced brain lesions, while
    glucose reduced significantly the number of neurotic neurons in the
    arcuate nucleus. In another experiment, groups of 10-day old mice were
    given by gavage 2 g MSG/kg b.w. alone or together with 1.93 g/kg b.w.
    of glucose, fructose, galactose, or lactose, or 1.83 g/kg b.w. of
    sucrose. The mono- and disaccharides reduced significantly the number
    of neurons affected in the arcuate nucleus (Takasaki, 1979).

         Groups of infant mice, 10 days of age, were given by gavage 2 g
    MSG/kg b.w. alone or 2 g MSG/kg b.w. along with 2.3 g arginine HCl/ kg
    b.w., 0.2 g leucine/kg b.w., or 0.02 units of insulin (prior to
    gavage). All the additional treatments reduced the extent of injury to
    the arcuate nucleus relative to MSG alone (Takasaki & Yugari, 1980).

         Weanling mice were deprived of water for 14 hours overnight and
    then given solutions of MSG as the sole drinking fluid; 12-180 animals
    receiving > 3 g MSG/kg b.w. in drinking water developed lesions of
    the arcuate nucleus. Fewer lesions were observed if free choice was
    allowed between water and MSG solutions, if glucose or arginine were
    also added to the MSG solutions, or if water deprivation occurred
    during the day (Torii & Takasaki, 1983).

         Suckling mice were given by gavage 2, 6, or 9 mg MSG/kg b.w. on
    days 6-10 postnatally and were killed on day 11. No hypothalamic
    lesions were noted. Another group given 6 mg MSG/kg b.w. for 5 days
    were weaned and observed for 12 months. No hypophagia, obesity, or
    hyperactivity were noted (Wen et al., 1973).

         Casein and fibrin hydrolysates were administered to 9-11-day old
    mice at dose levels of 20, 50, or 100 µl/g b.w. and plasma amino acid
    levels were determined at intervals. No neuronal damage resulted when
    plasma glutamate levels were below 24 µmole/dl (normal, 6-10 µmole/
    dl); the minimal threshold plasma level for neuronal injury was
    estimated as about 50-52 µmole/dl. Older (25 days old) mice showed a
    markedly greater ability to metabolize glutamate, which probably
    accounts for their decreased sensitivity to glutamate (Stegink et al.,
    1974).

         MSG was administered at dose levels of 1 g/kg to infant mice and
    2 and 4 g/kg to infant rats. All the animals developed lesions in the
    arcuate area of the hypothalamus and median eminence. No evidence of
    cellular pathology was noted in controls (Burde et al., 1971).

    Rats

         Weanling Sprague-Dawley male rats were given 20 mmoles MSG
    intraperitoneally (equal to 3.4 g/kg b.w.). Marked somnolence was
    observed within 5 to 20 minutes. About one-third to one-half of the
    animals salivated copiously and had myocolonic jerking about 1 hour
    after injection, sometimes followed by vigorous running about the cage
    and stereotyped biting (Bhagavan et al., 1971).

         At birth, the rat brain glutamate concentration is about 4 mM and
    increases over a period of 20 days to the adult value of approximately
    10 mM. When a 4 g/kg dose was given intragastrically, convulsions were
    seldom observed, and then only after 90 minutes. Two g/kg MSG given
    intraperitoneally always caused convulsions. When young rats were
    given 4 g/kg MSG, monosodium aspartate, or glycine, the glutamine
    level was increased significantly in the brain in all cases, but only
    monosodium glutamate and aspartate caused convulsions. D-Glutamate
    (4 g/kg), which is not deaminated by the rat, also caused convulsions.
    These results suggest that the convulsions caused by MSG are not due
    to liberated ammonia, but rather to the amino acid anion. At 4 g/kg,
    MSG gave rise to serum concentrations of glutamate of about 70 mM,
    strongly suggesting osmotic problems (Mushahwar & Koeppe, 1971).

         An experiment was conducted in which 45 male and 45 female rats
    were fed 1 or 250 mg/kg MSG daily from 1 to 90 days of age, at which
    time the animals were killed. A comparable control group received only
    laboratory chow over the same period of time. General clinical
    observations, body weights, haematologic parameters, and other
    clinical chemical measurements were within the normal range. At
    autopsy, organ weights were within the normal range. Histochemical and
    ultrastructural studies of the hypothalamus and median eminence showed
    no evidence of repair or replacement of neuronal cells by elements of
    glial or ependymal cells (Golberg, 1973).

         Groups of 10-day old Charles-River rats (10 male and 10 female)
    were dosed orally with 0.2 ml of either strained baby food containing
    no monosodium glutamate, strained baby food containing monosodium
    glutamate up to 0.4%, or strained baby food containing monosodium
    glutamate equal to a dosage level of 0.5 g/kg, additional to that
    found in normal commercially distributed baby food (390 mg per jar).
    The rats were mated; half of the offspring were removed from parental
    females and sacrificed after 5 hours. Histological studies were made
    of brains in the area of the hypothalamus at the roof and the floor of
    the third ventricle. The remaining rats were returned to parental
    females and allowed to grow to maturity (90 days post-weaning), then
    sacrificed and histological studies made of the brain. No lesions were
    observed in the brains of animals sacrificed at either 5 hours
    post-treatment or after reaching maturity. Animals which were reared
    to maturity showed normal growth and food consumption (Geil, 1970).

         Male and female Wistar rats, 3 to 4 days old, received single
    subcutaneous injections of MSG. The total dose was 4 g/kg b.w. in a
    volume of 0.1 ml. Control rats were injected with an equal volume of
    saline. In experiments concerned with the acute effects of MSG on the
    brain, the infant rats were killed 3 hours after the injection of MSG.
    Light microscopic and electron microscopic examinations failed to
    reveal any effects upon the lateral preoptic nucleus, arcuate nucleus,
    or median eminence. To determine long-range effects of MSG, uniform
    litters (8 pups per mother) were kept in an environmentally-controlled
    room until weaned. The experiment was terminated at 68 days
    post-treatment for males and 88 days post-treatment for females. In
    the MSG-treated females, the relative ovarian weights were
    significantly less than in the controls (29.3 ± 1.4 mg/100 g versus
    34.7 ± 0.9 (P < 0.1)), but otherwise there were no significant
    differences in the weight of reproductive organs (ovaries, testes,
    seminal vesicles, prostate). The adult MSG-treated females cycled
    normally and were capable of mating and producing normal litters
    (Adamo & Ratner, 1970). In a letter to Science, it is pointed out
    that Adamo & Rather injected a 40% solution of MSG instead of the 10%
    solution used by Olney et al. (1971).

         Fifty weanling rats (Food and Drug Research Labs strain), 3 days
    of age, were divided into five groups. Each group was subdivided and
    the test animals given either single intragastric or single
    subcutaneous doses of monosodium glutamate, sodium chloride, sodium
    gluconate, potassium glutamate (all 10% solutions, 10 mg/kg b.w.), or
    water. Another group of 12-day old rats were treated in a similar
    manner. All animals in these groups were killed 24 hours after dosing.
    In addition, another group of 60 rats (3 days of age) was divided into
    subgroups and treated with the same test compounds at the same dose
    levels. Half of each group was sacrificed at 6 hours and the other
    half at 24 hours after dosing. Microscopic examination of the brain,
    particularly the ventral hypothalamus, did not show neuronal necrosis
    of the hypothalamic arcuate nuclei, except in 1 rat dosed with 1 g/kg
    monosodium glutamate at 3 days of age, and killed 24 hours later,
    which showed an area in the median eminence which contained cells with
    slight nuclear pyknosis and prominent vacuolation (Oser et al.,
    1973).

         Neonatal rats were given MSG by injection on days 1-14 and the
    retina, optic nerve, and optic tracts were examined 2-10 months later.
    Observed changes included destruction Of the ganglion cell layer and
    the inner nuclear layer together with a reduced number of myelinated
    axons (Hansson, 1970).

         Rats were given by injection 0.25-4 g MSG/kg b.w. from the
    sixteenth day of pregnancy (in utero exposure) to the twenty-first day
    post partum. The animals were killed 1, 2, or 3 hours after injection.
    Lesions were seen in the cerebral cortex, hippocampus, thalamus, and
    hypothalamus. The earliest changes were lysis of organelles followed
    by oedema of the parenchyma and finally neuronal pyknosis
    (Everly, 1971).

         Male rats aged 23 days were deprived of water or water and food
    overnight, and then given aqueous solutions of MSG at concentrations
    up to 10%. No hypothalamic lesions were found with intakes up to 5 g
    MSG/kg b.w. (Takasaki & Torii, 1983).

         Five groups of 6 weanling rats were given basal diet with added
    20% glucose, 20% MSG, 40% MSG, or 17% L-glutamic acid and sodium
    equivalent to 20% MSG for 5 weeks and then killed. No specific
    endocrine or neurological defects were seen (Wen et al., 1973).

         Lesions of the arcuate nucleus and retina were produced by
    intraperitoneal administration of 4 g MSG/kg b.w. to rats on days 2,
    4, 6, and 10 after birth followed by examination 3-5 months later.
    There was a reduction of cell bodies in the arcuate nucleus and
    degeneration of the visual system; reduced weights of the anterior
    pituitary and gonads were observed in adults (Clemens et al., 1978).

         No CNS damage was observed in rats which were fed 4% MSG in the
    diet for 2 years or in dogs which were fed 10% MSG (Heywood & Worden,
    1979).

    Hamsters

         Neonatal hamsters were given single subcutaneous injections of
    8 g MSG/kg b.w. on days 6, 7, 8, 9, or 10 post partum and the brains
    were examined 6-12 hours later. Lesions of the arcuate nucleus were
    produced on days 6-8; dosing on days 9 or 10 still caused prominent
    lesions, but they were less severe than on previous days (Tafelski &
    Lamperti, 1977).

         Groups of male and female hamsters aged 25 days were deprived of
    water or water and food overnight, then offered solutions containing
    0, 2, 4, 6, or 8% MSG for 30 minutes. Six hours later the animals were
    killed and the brains examined; no hypothalamic lesions were seen
    (Takasaki & Torii, 1983).

    Guinea pigs

         A single subcutaneous injection of 1 g/kg MSG was given to 2- or
    3-day old guinea pigs. Six animals were killed 3 hours after treatment
    and the hypothalamic area investigated by histochemical and
    histological techniques. No clear effect of MSG was discernible. With
    a dose of 4 g/kg, an increase in glial cells, vacuolization of cells
    in the arcuate nuclei, and some evidence of cell necrosis were
    observed.

         The severity of the lesions was in no way comparable to the
    effects seen in the hypothalami of mice treated with the same doses
    (Golberg, 1973).

    Chickens

         Groups of 6 chickens were given subcutaneous doses of 4 g MSG/kg
    b.w. at days 5, 70, or 120 post-hatching. A further group of 120-day
    old birds was given subcutaneous doses of 7 g MSG/kg b.w. At 140 days
    there was no damage in the brains of birds injected at 120 days
    post-hatching, but lesions were seen in the hypothalami of the birds
    injected 5 days post-hatching and, less markedly, 70 days post-
    hatching (Robinzon et al., 1974).

         Six groups of 14 birds were injected subcutaneously 5 days
    post-hatching with single doses of 1 or 4 g MSG/kg b.w. or with daily
    doses of 1 or 4 g MSG/kg b.w. on 10 consecutive days; controls
    received equivalent doses of saline. Forty days later, 3 birds from
    each group showed frequent necrotic neurons in the hypothalamus and a
    reduced-cell density (Snapir et al., 1971).

    Ducks

         Groups of 5-6 ducks aged 5 days were given either single
    subcutaneous injections of 1 or 4 g MSG/kg b.w. or 10 consecutive
    daily doses of 1 or 4 g MSG/kg b.w. and observed up to 235 days after
    injection. Brain lesions in the rotundus nuclei and the ventro-medial
    hypothalamic nuclei were noted in all birds; sperm mobility was low in
    birds with lesions in the mamillary bodies, but this probably was not
    due to MSG (Robinzon et al., 1975).

    Rabbits

         When rabbits were injected intraperitoneally with 0.25, 0.5, 1,
    or 2 g MSG/kg b.w. daily for 16 days, histology of the retinae showed
    degeneration of all layers. In the ERG the amplitudes of the a and b
    waves were decreased to less than half their normal values. The
    minimum effective dose for retinal degeneration was 0.25 g/kg b.w.
    (Hamatsu, 1964).

    Dogs

         Intravenous casein hydrolysate or synthetic amino acid mixture
    caused nausea and vomiting in dogs (Madden et al., 1944).

         Groups of 3 dogs at 3 days or 35 days of age received
    subcutaneously or orally single acute doses of 1 g/kg b.w. monosodium
    glutamate, monopotassium glutamate, sodium chloride, sodium gluconate,
    or distilled water, and were sacrificed 3 or 24 hours after treatment.
    Preliminary light microscopic studies of the large midbrain area
    showed similar non-specific scattered tissue changes in all treated
    groups (Oser et al., 1971).

         Groups of 6 pups, 3 to 4 days of age, were dosed either orally or
    subcutaneously with 1 g/kg b.w. monosodium glutamate, sodium chloride,
    sodium gluconate, potassium glutamate, or water. Pups were sacrificed
    at either 3 hours, 24 hours, or 52 weeks after dosing. Other groups of
    dogs 35 days of age received single doses of test material, and were
    sacrificed at either 4 or 24 hours post-dosing. Body weights of dogs
    which were dosed once at 3 days of age and followed for a year, showed
    no evidence of effects of any treatment. Femur weight, as well as
    weight of the pituitary gland, ovaries, uterus, and mammary glands
    were similar to controls. Gross and microscopic examinations of these
    tissues failed to reveal any abnormalities. Extensive microscopic
    examination of brain tissue of all test animals did not show any
    treatment-related changes (Oser et al., 1973).

    Monkeys

         Doses of 250 mg or 1 g/kg MSG were administered orally daily for
    30 days to two groups of 3 infant rhesus monkeys starting at 1 day
    after birth. General clinical observations over a period of 30 days
    revealed normal growth, development, and activity. No changes were
    observed in the levels of haemoglobin, haematocrit, RBC or WBC counts,
    or reticulocytes. The levels of glucose, urea nitrogen, and serum
    potassium, calcium, and sodium were within the normal ranges. At
    autopsy, complete histological, histochemical, and ultrastructural
    investigations of the entire arcuate nuclei and median eminence region
    failed to reveal any necrotic or damaged neurons (Golberg, 1973).

         A newborn (8 hours old) rhesus infant, probably somewhat
    premature, was given subcutaneously 2.7 g/kg b.w. monosodium
    glutamate. After 3 hours (no abnormal behaviour noted) the monkey was
    killed and the brain perfused in situ for 20 minutes. A lesion was
    seen in the periventricular arcuate region of the hypothalamus
    identical to those seen in mice given similar treatment. Electron
    microscopic pathological changes were seen in dendrites and neuron
    cells but not in glia or vascular components (Olney & Sharpe, 1969).

         Monkeys, 4 days old, received single doses of monosodium
    glutamate (4 g/kg in phosphate buffer), either subcutaneously or
    orally. Animals receiving subcutaneous injections were sacrificed at
    3, 24, and 72 hours, the one receiving an oral dose at 24 hours. No
    brain lesions were observed (Abraham et al., 1971).

         Three infant monkeys, 5 days of age, received MSG by stomach tube
    at a dose of 2 g/kg. Two infant monkeys at 10 days of age, 2 at 20
    days of age, and 2 at 40 days of age, received the same treatment. Two
    animals at 80 days of age received 4 g/kg. One control monkey was
    included in each group. The animals were observed for 4 hours after
    dosing and then sacrificed. After a period of fixation, a block of
    tissue was removed from each brain which included the hypothalamus.
    Serial sections, 10 mm thick, were made in the horizontal plane and
    examined by light microscopy. No changes that were considered to be
    associated with the administration of MSG were observed in the
    hypothalamus of the monkeys (Huntington Research Centre, 1971).

         A group of 3 to 4 day-old cynomolgus monkeys received either
    subcutaneously or orally single doses of 1 g/kg b.w. monosodium
    glutamate, sodium chloride, sodium gluconate, or potassium glutamate
    and were sacrificed 3 or 24 hours post-dosing. Another group of
    monkeys (3 to 4 days old) received orally either 4 g/kg monosodium
    glutamate or sodium chloride, and were sacrificed at 3, 6, and 24
    hours post-dosing (3 and 24 hours in the case of sodium chloride-dosed
    monkeys). Detailed microscopic examination of the hypothalamus did not
    show any evidence of monosodium glutamate-induced necrosis or any

    differences between any of the groups. Examination of the eyes did not
    reveal any effects due to monosodium glutamate. Glutamate and
    glutamine blood levels showed considerable variation in individual
    values among the animals dosed orally and subcutaneously. Subcutaneous
    dosing resulted in values an order of magnitude higher than those
    observed by oral dosing (Oser et al., 1973).

         Monosodium glutamate was administered to 6 pregnant rhesus
    monkeys (Macaca mulatta) at a daily dosage equivalent to 4 g/kg b.w.
    during the last third of pregnancy. Four pregnant monkeys not
    receiving treatment were used as controls. Body weight and condition
    was unaffected throughout the gestation period. The duration of
    gestation was within the accepted range (156-178 days). There were no
    cases of delayed parturition or dystocia. Nursing, suckling, and
    behavioural patterns were normal except for one monkey which killed
    its infant at birth. Birth weights of the neonates were within the
    normal range. Infants, when removed from mothers, showed distress but
    no signs of abnormal behaviour. The hypothalamus region and related
    structure of the brain were examined by light microscopy. No
    abnormalities were observed (Heywood et al., 1972a).

         Monosodium glutamate was administered by intragastric intubation
    at dosage levels of 2 g/kg to 2 monkeys aged 2 days. Two monkeys of
    similar age were used as controls. Four hours after dosing, the
    animals were sacrificed. Examination of the hypothalamus (bordered
    rostrally by the optic chiasma and caudally by the pons) by light and
    electron microscopy did not show changes caused by administration of
    the test compound. Changes observed by electron microscopy occurred as
    frequently in control animals as in test animals, and appeared to be
    due to fixation artifacts (Heywood et al., 1972b).

         Rhesus monkeys (Macaca mulatta) aged between 4 and 80 days were
    divided into groups by age. Each group contained 3 test animals and 1
    control animal. Dosage levels were 2 g/kg for animals up to 44 days of
    age and 4 g/kg for animals up to 80 days of age. The animals were
    observed for 4 hours and then sacrificed. Immediately prior to dosing
    and prior to sacrifice, serum and plasma samples were obtained for
    measurement of SGPT, SGOT, and plasma glutamic acid. At sacrifice
    liver samples were obtained for measurement of GPT and GOT. SGPT and
    SGOT values did not show significant increases over the test period.
    Plasma glutamic acid was within the normal range. Liver GPT and GOT
    values were within the normal ranges. Examination of the hypothalamus
    region and associated structures by light microscopy did not reveal
    any compound-related effects (Heywood et al., 1971).

         Sixteen infant monkeys (M. mulatta or M. irus) were fasted
    for 4 hours before receiving by stomach tube single doses of a 50%
    solution of monosodium glutamate, equivalent to doses of 1, 2, or
    4 g/kg b.w. Control animals received distilled water. At 6 hours

    post-dosing the animals were sacrificed and the brains perfused for
    examination by light and electron microscopy. No morphological
    differences were observed in the hypothalamic regions of treated and
    control monkeys. Inadequately fixed tissue had the same appearance as
    that of a previously-reported brain lesion in a newborn monkey
    (Reynolds et al., 1971).

         Monosodium glutamate was administered into the circulation of
    primate fetuses (Macaca species) via the umbilical vein at dose
    levels of approximately 4 g/kg b.w. A total of 7 animals were treated.
    At time periods of 2 to 6 hours post-dosing the fetuses were delivered
    by caesarean section and the brains fixed for microscopic examination.
    The hypothalamic areas of the brain from all 7 fetuses were found to
    be completely normal. There was no evidence of pyknotic nuclei, tissue
    oedema, or neuronal loss in the arcuate region (Reynolds &
    Lemkey-Johnston, 1973).

         Ten infant squirrel monkeys were fed either a 0, 4.8, 9.1, or 17%
    (based on dry weight) MSG formula diet for 9 weeks. Three of the test
    monkeys died. Two died of effects not related to MSG. The third, which
    was on the 17% diet, developed convulsive seizures. However, the other
    2 animals in this group were unaffected. Clinical observations were
    made daily, and at the end of the test period the monkeys were
    sacrificed and the major organs examined microscopically. Sections of
    the retina and hypothalamus were examined by electron microscopy. No
    hypothalamic or retinal lesions were observed (Wen et al., 1973).

         In another study, an infant cynamologus monkey and an infant
    brush monkey were fed 0.1% MSG formula for one year. Daily
    observations revealed no behavioural abnormalities. Their weight
    gains, ERG, EEG, and plasma amino acids were similar to controls not
    consuming MSG. No evidence of gross obesity developed (Wen et al.,
    1973).

         Groups of 3 infant monkeys were dosed with a mixture of water and
    skimmed milk containing either added NaCl or MSG, on an equivalent
    mole/kg basis. Administration was via nasogastric tube. Other groups
    were injected subcutaneously with either a 25% aqueous solution of MSG
    or a 10% solution of NaCl. The doses ranged from 1-4 g/kg b.w. All
    animals were sacrificed after dosing and the brains examined by
    combined light and electron microscopy. Infants given relatively low
    oral doses of MSG (1 and 2 g/kg) sustained small focal lesions
    confined primarily to the rostro-ventral aspect of the infundibular
    nucleus. Those treated with high subcutaneous doses developed lesions
    which spread throughout, and sometimes beyond, the infundibular
    nucleus. At all doses tested, and by either route of administration,
    rapid necrosis of neurons (within 5 hours) was observed. Measurements
    of blood glutamate levels suggested that the threshold for lesion
    formation in 1-week old rhesus monkeys may be in the range of 200 mg/l
    (Olney et al., 1972).

         Neonatal primates given 1-4 g MSG/kg b.w. orally showed only
    elevated aspartate and glutamate blood levels; no hypothalamic lesions
    were noted (Boaz et al., 1974).

         Three monkeys were given orally 2 g MSG/kg b.w. at 3, 60, or 99
    days of age; a fourth monkey aged 180 days served as a control. After
    4 hours, examination of the brains revealed no abnormalities. A
    further 16 monkeys were divided into five groups; four groups received
    orally 2 g MSG/kg b.w. and one group was given 4 g/kg b.w. Examination
    of the brains and of serum GOT and GPT showed no abnormalities
    (Newman et al., 1973).

         Six pregnant monkeys were given 4 g MSG/kg b.w. for the last
    third of pregnancy and a further 4 untreated animals served as the
    source of control offspring. The offspring were killed 4 hours after
    birth, when no abnormalities in histopathology of the brains were
    seen. In another experiment, two monkeys aged 2 days were given 2 g
    MSG/kg b.w. and the brains were examined after 4 hours. No
    abnormalities were seen (Newman et al., 1973).

         No treatment-related hypothalamic lesions were observed in
    neonatal rhesus monkeys killed 3 or 24 hours or 8, 15, or 30 days
    following oral or subcutaneous administration of 0.25, 1, or 4 g
    MSG/kg b.w. The hypothalamus was examined by light and electron
    microscopy and the arcuate nuclei, median eminence, ependymal and
    glial cells were comparable to controls (Abraham et al., 1975).

         Neonatal monkeys aged 1-14 days were given 1-4 g MSG/kg b.w. by
    gavage. No lesions were detected in the hypothalamus (Reynolds et al.,
    1976). No evidence of hypothalamic lesions was seen following
    in vitro exposure in 1 embryonic and 7 fetal brains
    (Reynolds et al., 1979).

         Ten neonatal squirrel monkeys were divided into four groups of
    2-3 animals; 2 received infant formula, 3 received infant formula with
    5% added MSG, 2 were given infant formula with 10% added MSG, and 3
    were given infant formula with 20% added MSG from day 11 to day 21
    post partum followed by observation for 9-10 weeks. The animals were
    killed in the twelfth week. No abnormalities were observed in EEG or
    ERG scans and no retinal or hypothalamic lesions were detected
    (Wen et al., 1973).

         One cynomolgus monkey and 1 bushbaby were given 10% MSG in their
    diets from the first to the eleventh month of age. No adverse effects
    were noted on growth or on EEG or ERG scans. In another experiment, a
    3-week old cynomolgus monkey was injected intramuscularly with 2.7 g
    MSG/kg b.w. and observed for 2.5 years. No untoward effects were seen
    (Wen et al., 1973).

         The administration of MSG to a 2-day old rhesus monkey at a dose
    level of 4 g/kg b.w. in baby formula failed to induce any pathological
    changes in the hypothalamus (Heywood & James, 1979).

    Special studies on postnatal behaviour

    Mice

         Neonatal mice aged 2-11 days were injected with increasing doses
    of 2.2 to 4.4 g MSG/kg b.w. over 10 days. Examination at 2, 10, and 20
    weeks after weaning showed no increase in food consumption, but
    obesity was observed. Locomotor activity was depressed at all three
    time-points, and oxygen consumption was decreased at 10 and 20 weeks.
    There were no effects on plasma thyroxine levels (Poon & Cameron,
    1978).

    Rats

         Eight litter mates from each of 10 pregnant Holtzmann rats were
    divided into four groups consisting of two litters from each mother.
    Litters of these groups received water (control) and MSG solution
    (1.25, 2.5, or 5 g/kg) by stomach tube daily from days 5 to 10 of age.
    At day 21 the rats were placed in separate cages and at 3 months of
    age they were subjected to three different behavioural situations,
    namely, spontaneous motor activity, T-maze, and fixed-ratio food
    reinforcement. Rats in the high-dose group showed less spontaneous
    motor activity than the controls and a deficiency in discrimination
    learning in the T-maze study. However, learning of a fixed-ratio food
    reinforcement schedule was not affected. After 3 months weight gain of
    the treated animals was less than controls, the effect being greatest
    in the 5 g/kg dose group (Pradhan & Lynch, 1972).

         Rats given 4 g MSG/kg b.w. on days 1-10 of life and tested at 50
    days in a swimming maze were less able to learn the maze. Glutamate
    levels in the brain, liver, and blood were raised after 10 days, while
    other amino acid levels were also changed. Structural alterations were
    probably responsible for the permanent impairment of brain function
    (Berry et al., 1974).

         Rats given 10 g MSG/kg b.w. orally showed depressed avoidance
    acquisition and shuttlebox test performance. True pharmacological
    tolerance rapidly appeared (Pinto-Scognamilio et al., 1972).

         A group of 16 neonatal rats was given MSG between day 3 and day 8
    in progressively increasing subcutaneous doses of 2.5 to 4.2 g/kg
    b.w.; a further 16 animals acted as controls. At weaning, half of each
    group was given exercise wheels. The animals were killed at 110-112
    days post-weaning. No effects of MSG were observed on motor activity
    but both sexes were obese despite exercise and hypophagia.
    Disturbances of the female reproductive system and arrested skeletal
    development were noted (Nikoletseas, 1977).

         Rats (174 males and 196 females) were divided according to
    treatment as follows; three groups 2 days of age were injected
    subcutaneously with saline, 0.2 g MSG/kg b.w., or 4 g MSG/kg b.w. for
    10 days; one group 10 days of age was injected subcutaneously with 4 g
    MSG/kg b.w. for 10 days; two groups 10 days of age were given saline
    or 0.5 g MSG/kg b.w. by gavage; and one group 10 days of age was put
    on an ad libitum diet containing 10% MSG. The animals were observed
    for up to 9 months. Multiple injections of 4 g MSG/kg b.w. to neonates
    caused low grip strength, hypoactivity, changes in spontaneous motor
    activity, deficit of learning ability, and tail mutilation. The same
    treatment beginning at 10 days of age resulted in only slight
    behavioural abnormalities later in life or no detectable changes.
    Administration of subneurotoxic doses either by subcutaneous injection
    or gavage, or in high levels in the diet, were without behavioural
    effects. Adverse effects were not observed when the brains were free
    from histological evidence of injury (Iwata et al., 1979).

         Neonatal male rats were given 4 consecutive daily subcutaneous
    injections of 4 g MSG/kg b.w. on days 1 to 4 post partum. When adult
    studies of sleep patterns indicated an increase in total sleep
    duration with more pronounced effects on paradoxical sleep due to
    treatment, circadian rhythmicity tended to degenerate into ultradian
    (6, 8, 12 hours) harmonics. There was an almost complete disappearance
    of ACTH and alpha-MSH immunoreactive perikarya in the rostal part of
    the arcuate nucleus (Olivo et al., 1986).

    Chickens

         Applying 7.5 mg MSG/kg b.w. to forebrains of chicks once in the
    first week after hatching caused learning defects (Rogers, 1982).
    However, intercranial applications are irrelevant to the evaluation of
    orally-ingested MSG and learning in birds depends on motivation, which
    was not described in this experiment (Wurtman, 1983).

    Special studies on reproduction and teratogenicity

    Mice

         Groups of 6 4CS or Swiss white mice (3 males and 3 females) were
    maintained on diets containing O, 2 (equal to 4 g/kg b.w./day), or 4%
    (equal to 8 g/kg b.w./day) monosodium glutamate. Mice were mated after
    2 to 4 weeks on the test diets. F1 offspring were weaned at age 25
    days and fed the same diet as the parents. At age 90 days, selected
    F1 male and female mice from each group were allowed to produce a
    single F2 litter.

         Parent mice were maintained on test diets for 100 days after
    delivery and F1 mice for 130 days after delivery. F2 mice were
    reared until 20 days of age. No effects were observed on growth, feed
    intake, oestrous cycle, date of sexual maturation (F1 generation),
    organ weights, litter sizes, body weights of offspring, or
    histopathology of major organs (including brain and eyes) of the
    parent and F1 generations. Day of eye opening, general appearance,
    and roentgenographic skeletal examinations of F2 generation animals
    showed no abnormalities (Yonetani et al., 1970).

         Five groups of 24-30 mice received 0, 5.2, 24, 112, or 520 mg
    MSG/kg b.w. for 10 days during pregnancy. No clear adverse effects
    were seen on nidation or on maternal or fetal survival. There were no
    adverse effects on resorptions, fetal weights, or litter parameters,
    and no differences were noted in soft tissue or skeletal abnormalities
    (Food and Drug Research Laboratories, 1974a).

         Neonatal mice were injected with increasing daily doses of MSG
    from 2.2 to 4.2 g/kg b.w. over 10 days and observed subsequently for
    up to 302 days. Treated females had fewer pregnancies and smaller
    litters than the controls; males displayed reduced fertility. The body
    weights of both sexes were increased while organ weights of the
    pituitary, thyroid, and ovaries/testes were reduced (Pizzi et al.,
    1977).

         Six groups of mice were given O, 1, or 2% MSG in a diet
    supplemented with 1 or 2% vitamin mix. Females from the F1 (33) and
    F2 (29) generations were observed for reproductive function. Animals
    receiving MSG showed higher weaning weights, better survival rates,
    and no effects on brain cellularity (Semprini et al., 1974a).

         A multi-generation reproduction study on MSG was conducted on
    IVCS and Swiss albino mice. Groups of 3-5 60-day old male and female
    mice were maintained on diets containing O, 2, or 4% MSG from 2 weeks
    prior to mating until 100 days after parturition. Animals in the F1
    generation were maintained on the same diets and mated at 90 days of
    age. Animals in the F2 generation were killed on day 20. No
    significant abnormalities were observed on growth, food consumption,
    oestrus cycle, date of sexual maturation, organ weights, litter sizes,
    body weights of offspring, or histopathology of major organs
    (including brain and retina) of the parents and the F1 generation.
    Mice of the F2 generation showed normal date of eye-opening. No
    teratogenic effects were observed (Yonetani et al., 1979).

         In a 3-generation reproduction study, two groups of 17 male and
    51 female CD-1 COBS mice were given 1 or 4% MSG in the diet; a further
    control group of 33 male and 99 female animals received diets without
    MSG. Animals in the F1 and F2 generations were sacrificed at 27-36
    weeks of age, while some of the F3 generation animals were examined

    histopathologically at 0, 3, 14, and 21 days. Growth and food intake
    were similar in all groups. The actual MSG intakes were 1.5 and 6 g/kg
    b.w./day for males and 1.8 and 7.2 g/kg b.w./day for females in the 1
    and 4% treatment groups respectively. The MSG intake of dams rose
    markedly during lactation, rising to a maximum of 25 g/kg b.w./day. No
    adverse effects were noted on fertility, gestation, viability, or
    lactation indices of progeny of any generation, and no brain lesions
    or other treatment-related histopathology were observed (Anantharaman,
    1979).

    Rats

         Six groups of 5-6 male and 5-10 female rats received by oral
    intubation daily 25 or 125 mg/kg b.w. glutamic acid monohydrochloride.
    Males and females received the compound during days 5-19 of the first
    month, days 20-31 of the following month, and days 1-10 during the
    third month. No adverse effects were noted on weight gain, feed
    intake, or sexual cycles of females. No organ-weight changes were seen
    in females but males at the higher-dose level had enlarged spleens.
    Animals were mated at the end of the experiment and the pups were
    normal (Furuya, 1967).

         Rats were given thalidomide combined with 2% L-glutamic acid, and
    showed essentially the same defects in the pups as groups treated with
    thalidomide alone. A group receiving L-glutamic acid alone was not
    different from controls (McColl et al., 1965).

         Four females and 1 male fed for 7 months 0, 0.1, or 0.4%
    monosodium L-glutamate, monosodium DL-glutamate, or L-glutamic acid
    were mated. The number of pups per litter was similar in all groups.
    Only 15-20% survived because of cannibalism. No abnormalities
    regarding fertility were seen after mating other groups of 4 females
    and 1 male at 9 and 11 months. The F1 generation was mated at 10
    months and an F2 generation was produced in most groups, but only
    the groups fed 0.1 and 0.4% L-glutamic acid produced F3 and F4
    generations. No impairment of fertility was noted (Little, 1953a).

         Monosodium glutamate was administered orally at doses up to
    7 g/kg/day to pregnant rats on days 6-15 or 15-17 following
    conception. The substance produced no adverse effect in the progeny up
    to the period of weaning. Further physical development to maturity was
    also normal except that the progeny obtained from gravida treated on
    days 15-17 during gestation showed impaired ability to reproduce
    (Khera et al., 1970).

         Two female rats received 4 g/kg b.w. monosodium glutamate
    commencing at day 1 of pregnancy. There were no effects on pregnancy
    or lactation. Pups were divided into three groups. Two groups were
    nursed by parents receiving monosodium glutamate, and one group by
    untreated parents. At weaning (day 20), one group of pups that had

    been nursed by a parent receiving monosodium glutamate received
    approximately 5 g/kg monosodium glutamate daily for 220 days. Parents
    received 4 g/kg monosodium glutamate for 336 days. No effects were
    observed on growth or the oestrus cycle. All pups developed normally,
    and no abnormalities were noted in growth rate, time of sexual
    maturity, oestrus cycle, or fertility. For histological studies, the
    brain, hypophysis, and eye were fixed in 10% neutral buffered
    formalin. Sections were stained with haematoxylin-eosin and Luxol fast
    blue-cresyl violet. No differences were observed in the arcuate
    nuclei, medium eminence of the hypothalamus, or retina between control
    and monosodium glutamate-treated groups (Suzuki & Tagahashi, 1970;
    Shimizu & Aibara, 1970).

         Groups of female rats were maintained on diets containing 0.5, 1,
    or 2% vitamin mix. At each vitamin level diets also contained
    monosodium glutamate at 0, 1, or 2%. Reproductive performance of the
    parental rats as well as of the F1 offspring maintained on similar
    diets was studied. The addition of monosodium glutamate to the diet
    resulted in an increased fertility rate as well as increased survival
    at weaning of the offspring of the F1 generation. Addition of
    monosodium glutamate to the diet had no effect on growth rate in the
    neonatal period. Analysis of the brain tissue of first and second
    generation offspring at birth for RNA, DNA, protein, nucleus number,
    and cellular size showed that the brains of rats born of parental
    mothers on monosodium glutamate diets contained a smaller number of
    nuclei and larger cells than controls. In contrast, offspring of the
    F1 generation showed increased RNA, DNA, and nucleus numbers when
    compared with the offspring of the parental generation. The
    differences present at birth disappeared at weaning
    (Semprini et al., 1971).

         Parental rats were fed 0 or 10% MSG in the diet, mated, and F1
    pups were maintained for 100 days on the same diet as the parents,
    then mated. Of the F2 pups, 10 were sacrificed on each of days 1, 2,
    3, 5, 10, and 21 post partum. No effects on reproductive function were
    observed as indicated by conception rates and numbers of pups per
    litter, and no differences were noted in post-natal development, as
    measured by brain, liver, and body weights. Brain and liver glutamate,
    aspartate, protein, DNA, RNA, and the activity of glutamic acid
    decarboxylase were unaffected by treatment (Prosky & O'Dell, 1972).

         Groups of 25 pregnant Wistar-derived rats were given 0, 4.5, 21,
    97, or 450 mg monopotassium glutamate/kg b.w. by oral intubation on
    days 6-15 of pregnancy. No effects were observed on nidation or on
    maternal of fetal survival and the number of abnormalities in the
    offspring in the test groups did not differ from those occurring
    spontaneously in controls (Food and Drug Research Laboratories,
    1974b).

         Rats were given daily s.c. doses of 2 or 4 g MSG/kg b.w. from
    days 2-10 post partum or a dose of 4 g/kg b.w. for 10 days from day
    10. Other groups of 10-day old rats received 0.5 g MSG/kg b.w. orally
    for 10 days or similar doses followed by weaning on to a diet
    containing 5% MSG. Females repeatedly treated as neonates showed
    precocious puberty, disturbed oestrus cycles, small ovaries and
    pituitaries, and a poor response to gonadotrophin. Females treated
    from day 10 developed normal puberty and oestrus cycles, but later
    cycling became irregular. Low doses of MSG given orally or by repeated
    injection had no effect (Matsuzawa et al., 1979).

    Hamsters

         Groups of neonatal hamsters were given subcutaneous injections of
    0, 4, or 8 g MSG/kg b.w. in saline on days 1-5, 6-10, or 1-10
    postnatally. The animals were sacrificed on day 60 (males and acyclic
    females) or on a day of ovulation near day 60 (regular cycling
    females). MSG-treated animals had significantly lower reproductive
    organ weights than controls. Lesions were detected in the arcuate
    nucleus only in hamsters receiving 8 g MSG/kg b.w. on days 6-10 or
    1-10; female hamsters were acyclic, and had ovaries with small
    follicles and no corpora lutea. Administration of 50 i.u. pregnant
    mare's serum to these animals caused follicular maturation, and
    ovulation occurred after administration of 10 i.u. human chorionic
    gonadotrophin (HCG). Hales had atrophic seminiferous tubules which
    recovered normal histology and steroid dehydrogenase activity after
    treatment with HCG. The evidence indicated that MSG had affected the
    hypothalamic centres controlling GSH and LH release (Lamperti & Blaha,
    1976).

         Male and female hamsters were given saline or 8 g MSG/kg b.w. by
    subcutaneous injection on days 7 and 8 postnatally. As adults, all
    MSG-treated females were acyclic, had significantly lower uterine and
    pituitary weights, and lower levels of FSH in the plasma and anterior
    pituitary from controls. The ovaries had small follicles and the
    interstitial cells were hypertrophied. It was reported that only 7.3%
    of the neurons of the arcuate nucleus were morphologically intact.
    Adult male hamsters that had been given MSG neonatally had a
    treatment-related reduction in testicular, seminal vesicle, and
    pituitary weights as well as lower FSH levels in the plasma relative
    to controls. Approximately 14% of the neurons in the arcuate nucleus
    were morphologically intact. The seminiferous tubules were
    histologically atrophic in only 3 out of 8 animals. These results were
    taken as supporting previous reports that MSG lesions of the arcuate
    nucleus result in alterations in FSH, but not LH, secretion in the
    hamster (Lamperti & Blaha, 1980).

    Chick embryos

         Fertilized hen eggs were incubated after single injections of
    0.01-0.1 mg glutamic acid into the yolk sac. The mortality of embryos
    was raised compared with controls (53% against 24%) and there was a
    higher incidence of developmental defects (24% against 3%), especially
    depression of development of the spine, pelvis, and lower limbs
    (Aleksandrov et al., 1965). In another study, many variables were
    studied such as the route of injection, dose, and time of injection.
    No obvious toxic or teratogenic effects were observed (US FDA, 1969).

    Rabbits

         In one group of 10 female and 4 male rabbits, only the females
    received 25 mg/kg b.w. glutamic acid for 27 days. Two of the females
    were pregnant and the others were not pregnant. A second group of 4
    females and 2 males received 25 mg/kg glutamic acid along with
    25 mg/kg vitamin B6. A third group of 6 females and 2 males received
    25 mg/kg glutamic acid alone. A fourth group of 20 females and 8 males
    served as controls. The test substance was given by gavage. The first
    group showed 2 animals with delayed pregnancy, the uterus containing
    degenerated fetuses. Two others had abortions of malformed fetuses.
    Two animals delivered at the normal time, but the pups had various
    limb malformations. Four animals did not conceive. The pups did not
    become pregnant before 7 months of age and showed limb deformities,
    decreased growth, and slow development compared with controls.
    Histopathological examination showed scattered atrophy or hypertrophy
    of different organs. The second group produced 2 pregnant females
    which delivered malformed pups. These died soon after birth and showed
    bony deformities as well as atrophic changes in various organs. The
    third group produced 3 pregnant females which delivered pups with limb
    deformities. All three groups showed testicular atrophy in parents and
    multiple changes in the pups (Tugrul, 1965).

         Four groups of rabbits (24 females and 16 males) received 0, 0.1,
    0.825, or 8.25% monosodium glutamate in their diet for 2 or 3 weeks
    before mating. A positive control group of 22 pregnant females
    received 100 mg/kg thalidomide from days 8 to 16 of pregnancy. All
    does were sacrificed on days 29 or 30 of gestation and the uteri and
    uterine contents were examined. All males were sacrificed and the
    gonads and any abnormal organs examined. No significant effects on
    body-weight gain, food consumption, general appearance, or behaviour
    were observed. Gross and histopathological examinations revealed no
    toxic effects on embryos or resorptions. Pups and all litter data were
    comparable among test animals and negative controls. The brains of 5
    female and 5 male pups at the 8.25% level were subsequently checked
    for neuronal necrosis compared with controls, but none was found.
    Similar investigations on 5 male and 5 female pups at the 0.1 and
    0.825% levels were also negative (Hazelton Laboratories, 1966,
    1969a,b).

         In another experiment in rabbits, animals received 2.5, 25, or
    250 mg/kg b.w. L-glutamic acid hydrochloride at 70 and 192 hours post
    coitum. Operative removal of fetuses was performed on the eleventh,
    seventeenth, and thirtieth day post coitum in three different series.
    The corpora lutea and the resorbed and implanted normal and deformed
    fetuses were examined. No significant effects due to L-glutamic acid
    were noted with respect to teratogenesis (Gottschewski, 1967).

         Glutamic acid hydrochloride at a dose of 25 mg/kg b.w. was given
    orally to 15 pregnant rabbits once a day for a period of 15 days after
    copulation; monosodium glutamate at the same dose and for the same
    period of time was given to 9 pregnant rabbits, and saline solution
    was administered to 11 pregnant rabbits which served as a control
    group. No differences were noted between the treated groups and the
    controls with regard to the rate of conception, mean litter size, or
    nursing rate. The average body weights of the young in the treated
    groups were slightly lower as compared with the control group, but the
    weights of testes, ovaries, and adrenal glands in the young and
    ovaries, adrenal glands, liver, kidneys, and spleen in the mothers
    were not different from those in the controls. In the young, no
    external or skeletal malformations were observed. There were some
    abnormal changes in gestation such as abortion or resoption of
    fetuses, but these observations were made in all groups, with
    incidences of 21% in the glutamic acid hydrochloride group, 25% after
    administration of monosodium glutamate, and 27% in the controls. There
    were no external or skeletal malformations in the aborted fetuses
    (Yonetani, 1967).

    Special studies on pharmacological effects

         Intravenous injection of large doses of glutamic acid in rabbits
    caused ECG changes that could be interpreted as symptoms of myocardial
    lesions. Arterial hypertension induced by glutamic acid preparations
    was demonstrated to be of central origin. Studies with isolated heart
    showed that large doses of glutamic acid slowed heart action,
    increased systolic amplitude, and constricted coronary vessels. Very
    large doses stopped cardiac action (Mazurowa et al., 1962).

         Ten male and 4 female subjects were given orally either 25 or
    250 mg MSG/kg b.w. alone, 250 mg MSG/kg b.w. together with atropine,
    or prostigmine, and plasma cholinesterase activity was measured. There
    was little effect with the lower dose or the higher dose plus
    atropine. The high-dose effect of MSG alone was similar to the effect
    of prostigmine. Hence, large doses may cause release of an
    acetylcholine-like substance acting on the parasympathetic system in
    so-called "Chinese restaurant syndrome" (Ghadimi et al., 1971).

    Acute toxicity

         The acute toxicity of glutamate by various routes of
    administration in several animal species is given in Table 3.

    Short-term studies

    Mice

         Thirty-eight neonatal mice were observed for 9 months. Twenty
    received monosodium glutamate by subcutaneous injection daily for 1 to
    10 days after birth at doses of 2.2-4.2 g/kg. Eighteen were used as
    controls. Even though the treated animals remained skeletally stunted
    and both males and females gained more weight than controls from 30 to
    150 days, the treated animals consumed less food than controls. The
    test animals were lethargic and the females failed to conceive, but
    male fertility was not affected. At autopsy, massive fat accumulation,
    fatty livers, and thin uteri were observed in test animals, and the
    adenohypophysis had fewer cells overall (Olney, 1969b).

         Ten test neonates received single subcutaneous injections of
    3 g/kg monosodium glutamate 2 days after birth. Thirteen neonates were
    used as controls. Test animals were heavier than controls after 9
    months, but less so than mice given repeated injections in the
    experiment described above. The author postulated that an endocrine
    disturbance could lead to skeletal stunting, adiposity, and female
    sterility. Lesions differed from those due to gold thioglucose or
    bipiperidyl mustard, which affect the ventro-medial nucleus and cause
    hyperphagia (Olney, 1969b).

    Rats

         Natural monosodium L-glutamate, synthetic monosodium L-glutamate,
    and synthetic monosodium D-glutamate in amounts of 20, 200, or
    2000 mg/kg b.w. were given orally to groups of 5 male rats once a day
    for a period of 90 days. No effects on body weight, growth, volume and
    weight of cerebrum, cerebellum, heart, stomach, liver, spleen, or
    kidneys in comparison with the control group were observed. No
    histological changes in internal organs were found by macroscopic and
    microscopic examinations (Hara et al., 1962).

         Male Sprague-Dawley albino rats were allowed water ad libitum
    and fed ground laboratory chow having 24% protein content. High levels
    of single amino acids in the diet of rats beginning at 21 days
    produced decreased food intake and a severe growth depression. These
    effects were dependent upon the kind and concentration of supplemented
    amino acid. L-Methionine caused the most severe growth depression
    while L-phenylalanine, L-tryptophan, and L-cysteine were also severely
    toxic. Less toxic were L-histidine, L-lysine, and L-tyrosine.

         All other amino acids tested, including glutamic acid, had only a
    slight effect on growth, or none at all. Growth depression was
    attributed both to depressed food intake and to specific toxic effects
    of the amino acids. A direct correlation was not found between the
    toxicity of any dietary amino acid and its concentration in the blood
    (Daniel & Waisman, 1968).

        Table 3.  Results of acute toxicity assays on glutamate
                                                                                              

    Species        Route       LD50                         Reference
                               (mg/kg b.w.)
                                                                                              

    Mouse          oral        12,961                       Izeki, 1964
                   oral        16,200 (14,200-18,400)       Ichimura & Kirimura, 1968
                   oral        19,200 (16,130-22,840)       Pinto-Scognamiglio et al., 1972
                   s.c.        8,2001                       Moriyuki & Ichimura, 1978
                   i.p.        6,900                        Yanagisawa et al., 1961
                   i.v.        30,000                       Ajinomoto Co., 1970

    Rat            oral        19,900 (L-MSG)               International Min. & Chem.
                                                              Corp., 1969
                   oral        10,000 (DL-MSG)              International Min. & Chem.
                                                              Corp., 1969
                   oral        16,600 (14,500-18,900)       Pinto-Scognamiglio et al., 1972
                   s.c.        8,2001                       Moriyuki & Ichimura, 1978

    Guinea-pig     i.p.        15,000                       Ajinomoto Co., 1970

    Rabbit         oral        > 2,300 (L-GA)               International Min. & Chem.
                                                              Corp., 1969

    Cat            s.c.        8,000                        Ajinomoto Co., 1970
                                                                                              

    1    No sex differences were noticeable in the toxic signs; high doses
         led to excitation.
    
         Nine groups of 20 rats were given 0.5 or 6% calcium glutamate in
    their diet. No effect was noted on maze learning or recovery from ECT
    shock (Porter & Griffin, 1950).

         Two groups of 14 rats received 200 mg L-monosodium glutamate per
    animal for 35 days. No differences in their learning ability for maze
    trials were noted (Stellar & McElroy, 1948).

         Eight male rats fed 5% dietary DL-glutamic acid in a low protein
    diet (6% protein) showed little or no depression of growth, when
    compared to low protein controls. There was a 50% increase in the free
    glutamic acid in the plasma (Sauberlich, 1961).

    Long-term studies

    Mice

         One control group of 200 male mice and six test groups of 100
    male mice received 1 or 4% L-glutamic acid, monosodium L-glutamate, or
    DL-monosodium glutamate in their diet. No malignant tumours appeared
    after 2 years that could be related to the administration of test
    material. Growth and haematology were normal, and histopathological
    examination showed no abnormalities in the test animals (Little,
    1953a).

         Six groups of C57B1 mice, comprising 50 males and 50 females,
    were given diets containing 1 or 4% L-glutamic acid, L-MSG, or DL-MSG
    for 715 days. A further control group of 100 animals of each sex
    received basal diet. No treatment-related differences were seen in
    mortality, body-weight gain, incidence of concurrent disease,
    haematology, or tumour incidence (Ebert, 1979a).

    Rats

         Groups of 75 male and 75 female rats received for 2 years dietary
    levels of 0, 0.1, or 0.4% monosodium L-glutamate, monosodium
    DL-glutamate, or L-glutamic acid. No adverse effects were noted on
    body weight, growth, food intake, haematology, general behaviour,
    survival rate, gross or histopathology, or rumour incidence (Little,
    1953b).

         Six groups of Sprague-Dawley rats, comprising 35 animals of each
    sex, were fed diets containing 0.4 or 4% L-glutamic acid, L-MSG, or
    DL-MSG from 12 weeks to 2 years of age; a control group of 61 males
    and 69 females received basal diets. The protocol included a
    reproduction phase. No adverse effects were noted on behaviour,
    body-weight gain, food consumption, motor activity, clinical
    observations, haematology, or tumour incidence. Fertility, survival of
    the young, organ weights, and histopathology were comparable between
    controls and test animals (Ebert, 1979b).

         Groups of 40 rats of each sex were given diets containing 0, 1,
    2, or 4% MSG or 2.5% sodium propionate (positive control) for 104
    weeks. Animals of each sex per group were examined at 12 weeks with
    full histopathology. Ophthalmological examinations every 13 weeks were
    negative and no adverse effects were noted on body weight, food
    consumption, haematology, blood chemistry, terminal organ weights, or
    survival rates. Tissues from 25 organs were examined histologically.
    Water consumption, urinary volume, and sodium excretion were increased
    at the 4% MSG level and sub-epithelial basophil deposits were observed
    in the renal pelvis. Focal mineral deposits from the renal
    corticomedullary junction were equally distributed in all groups
    (Owen et al., 1978a).

    Dogs

         Beagle dogs were fed diets containing 0, 2.5, 5, or 10% MSG or
    5.13% sodium propionate (control) for 104 weeks. There were no adverse
    effects on body-weight gain, food consumption, behaviour, ECG,
    ophthalmology, haematology, blood chemistry, organ weights, or
    mortality due to treatment. Urinary volume and sodium excretion were
    slightly elevated in animals receiving MSG or sodium propionate but
    kidney function was unimpaired. No treatment-related histological
    changes were observed (Owen et al., 1978b).

    Observations in man

         Intravenous glutamic acid (100 mg/kg b.w.) produced vomiting
    (Madden et al., 1944). The occurrence of nausea and vomiting
    following the intravenous administration of various preparations in a
    series of 57 human subjects was found to parallel the free glutamic
    acid content of the mixture. There was a direct relationship between
    free serum glutamic acid and the occurrence of toxic effects following
    intravenous administration. When serum glutamic acid reached
    12-15 mg/100 ml, nausea and vomiting occurred in half the subjects.
    Other amino acids appeared to potentiate the effect (Levey et al.,
    1949).

         Arginine glutamate may be used in the treatment of ammonia
    intoxication. It is given by intravenous infusion at doses of 25 to 50
    g every 8 hours for 3 to 5 days in dextrose and infused at a rate of
    no more than 25 g over 1 or 2 hours; a more rapid infusion may cause
    vomiting (Martindale, 1967).

         Monosodium glutamate has been used in the treatment of
    mentally-retarded children at doses up to 48 g daily, but on average
    at doses of 10-15 g. One hundred and fifty children aged 4-15 years
    were treated with glutamic acid for 6 months and compared with 50
    controls. No significant rise in intelligence quotient was observed,
    but 64% were claimed to show improved behavioural traits (Zimmerman &
    Burgemeister, 1959).

         Seventeen patients received up to 15 g monosodium glutamate 3
    times a day, but they showed a raised blood level for 12 hours only.

         No effects were noted on BMR, EEC, ECG, blood pressure, heart
    rate, respiration rate, temperature, or weight over a period of 11
    months (Himwich, 1954).

         Fifteen grams, then 30 g, monosodium glutamate were given for one
    week each, followed by 45 g for 12 weeks, to 53 patients. There were
    no effects on basal plasma levels of glutamic acid (Himwich et al.,
    1954).

         DL-Glutamic acid * HCl was given at doses of 12, 16, or 20 g to
    eight patients with petit mal and psychomotor epilepsy without
    adverse effects (Price et al., 1943). Five episodes of hepatic coma
    in 3 patients were treated with 23 g monosodium glutamate i.v. with
    improvement (Walshe, 1953). L-Glutamic acid, 10 to 12 g given to
    epileptics and mental defectives, appeared to improve 9 out of 20
    cases (Waelsch, 1949).

         In studies of long-term health effects of MSG, no increased
    neurological symptoms and less myocardial infarction and stroke were
    seen. Blood glucose, cholesterol, and obesity were unrelated to MSG
    intake (Go et al., 1973).

         Six women with well-established lactation patterns were fasted
    overnight and given single oral doses of 6 g MSG in water or in liquid
    diet; 4 controls received lactose. Milk samples were obtained 1, 2, 3,
    4, 6, and 12 hours after administration; blood samples were collected
    0, 30, 60, 120, and 180 minutes after administration of MSG or
    lactose. Small increases in plasma glutamate, aspartate, and alanine
    levels were noted, but little change was observed in breast milk amino
    acid levels (Stegink et al., 1972).

         Following early anecdotal reports of subjective symptoms after
    ingestion of Chinese meals (Kwok, 1968) and experiments involving
    administration of MSG (Schaumberg et al., 1969; Kandall, 1968;
    Beron, 1968), MSG was implicated as the causative agent of "Chinese
    restaurant syndrome". Extensive studies in human volunteers have been
    carried out subsequently and have been reviewed recently (Kenney,
    1986). These studies have failed to demonstrate that MSG is the causal
    agent in provoking the full range of symptoms of Chinese restaurant
    syndrome. Properly-conducted double-blind studies among individuals
    who claimed to suffer from the syndrome did not confirm MSG as the
    causal agent. Food symptom surveys have been considered technically
    flawed because of questionnaire design (Kerr et al., 1977, 1979a,
    1979b).

         Twenty-four subjects, including 18 who had a history of
    subjective flushing symptoms after eating Chinese restaurant food,
    were challenged with 3-18.5 g MSG. No one reported flushing
    sensations. Six subjects, 3 with a history of flushing, were
    challenged with 3-18.5 mg MSG/kg b.w. or 7.1-7.4 mg pyroglutamate/kg
    b.w. None reported flushing sensations and significant changes in
    facial cutaneous blood flow were not recorded (Wilkin, 1986).

    COMMENTS

         Food additive uses of MSG include its incorporation into food and
    its use as a condiment. Glutamic acid is a component of proteins and
    comprises some 20% of ingested protein. Bound glutamate is released
    during digestion in the lumen of the gut and in the mucosa.
    Transamination to alanine occurs during gastrointestinal absorption
    with concomitant formation of alpha-ketoglutarate; glutamine and
    glutathione are other metabolic products. After deamination, excess
    glutamic acid may be utilised in gluconeogenesis. As a consequence of
    transamination, there is only a small rise in portal glutamate levels
    unless very large doses are administered. Further metabolism occurs in
    the liver and only when this system is overwhelmed does the level of
    glutamate in systemic circulation rise significantly. Human infants,
    including premature infants metabolize glutamate similarly to adults.

         High oral doses of MSG by gavage (in excess of about 30 mg/kg
    b.w.) or parenteral administration of MSG may lead to elevated blood
    levels. Administration with food, particularly metabolizable
    carbohydrate, lowers the peak plasma levels attained; peak plasma
    levels are also concentration-dependent and limited by unpalatability
    at high concentrations.

         Oral ingestion of large amounts of glutamate does not increase
    concentrations of glutamic acid in maternal milk and, at least in rats
    and monkeys, glutamate does not readily cross the placental barrier.

         Reproducible lesions of the CNS have been produced in rodents,
    lagomorphs, and primates following parenteral administration of
    glutamate or after forced intubation of very high doses. CNS lesions
    have never been observed after ad libitum administration of high
    concentrations of MSG in the diet or drinking water, except following
    dehydration by water deprivation in mice.

         There are species, strain, and age-dependent differences in
    sensitivity to neuronal injury, neonatal mice being most sensitive and
    rats, guinea pigs, and primates less so. The threshold plasma levels
    for neuronal damage in the mouse, the most sensitive species, are
    100-130, 380, and > 630 µmoles/dl in infant, weanling, and adult
    animals, respectively. In human studies, plasma levels of this
    magnitude have not been recorded even after ingestion of a single dose
    of 150 mg MSG/kg b.w. in water.

         The oral ED50 for production of hypothalamic lesions in
    neonatal mice is approximately 500 mg MSG/kg b.w., while the largest
    palatable dose is about 60 mg MSG/kg b.w.

         Acute, short-term, and chronic toxicity studies on MSG in the
    diet of several species have not shown specific toxic effects and
    there is no evidence of carcinogenicity or mutagenicity. Reproduction
    and teratologic studies using the oral route have been uneventful,
    even when the parental generation is fed glutamate at high doses,
    suggesting that the fetus or suckling neonate is not exposed to toxic
    levels through the maternal diet.

         Controlled, double-blind crossover trials have failed to
    demonstrate an unequivocal relationship between "Chinese restaurant
    syndrome" and consumption of MSG. MSG has not been shown to provoke
    bronchoconstriction in asthmatics.

         Caution should be used when ingesting MSG as a large single dose
    rather than divided between several meals because high plasma levels
    may be reached under the former conditions.

         In its previous evaluation, the Committee concluded that it would
    be prudent not to apply the ADI for glutamate to infants under 12
    weeks of age (Annex 1, reference 32). In view of the finding that
    infants metabolize MSG in a similar way to adults, no additional
    hazard to infants was indicated. However, the present Committee
    expressed the general opinion that the use of any food additives in
    infants foods should be approached with caution.

    EVALUATION

    Estimate of acceptable daily intake for man

         ADI "not specified".

         1. This is a group ADI for L-glutamic acid and its ammonium,
    calcium, magnesium, monosodium, and potassium salts.

         2. Substances given an ADI "not specified", such as glutamate
    salts in this instance, are of low toxicity. On the basis of the
    available data (chemical, biochemical, toxicological, and other), the
    total dietary intake of glutamates arising from their use at the
    levels necessary to achieve the desired technological effect and from
    their acceptable background in food do not, in the opinion of the
    Committee, represent a hazard to health. For that reason, the
    establishment of an acceptable daily intake expressed in numerical
    form is not deemed necessary. The Committee reiterated the general
    principle expressed in its first report (Annex 1, reference 1) that
    the amount of an authorized additive used in food should be the
    minimum necessary to produce the desired effect.

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    See Also:
       Toxicological Abbreviations