FAO Nutrition Meetings
Report Series No. 48A
WHO/FOOD ADD/70.39
TOXICOLOGICAL EVALUATION OF SOME
EXTRACTION SOLVENTS AND CERTAIN
OTHER SUBSTANCES
The content of this document is the
result of the deliberations of the Joint
FAO/WHO Expert Committee on Food Additives
which met in Geneva, 24 June -2 July 19701
Food and Agriculture Organization of the United Nations
World Health Organization
1 Fourteenth report of the Joint FAO/WHO Expert Committee on Food
Additives, FAO Nutrition Meetings Report Series in press; Wld Hlth
Org. techn. Rep. Ser., in press.
MONOSODIUM GLUTAMATE
Biological data
Biochemical aspects
L-glutamic acid occurs as a common constituent of proteins and
protein hydrolysates and can be synthesized by the rat and rabbit from
acetate fragments. Human plasma contains 4.4 - 4.5 mg/l of free
glutamic acid and 0.9 mg/100 ml, of bound glutamic acid. Human urine
contains 2.1 - 3.9 µg/mg creatinins of free glutamic acid and 200
µg-/mg creatinins 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). Human milk contains 1.2% protein of
which 20% is bound glutamic acid which is equivalent to 3g/1
calculated as sodium glutamate. The free glutamic acid concentration
is 300 mg/l. In contrast cows milk contains 3.5% protein equivalent to
8.8 g/1 calculated as MSG, but only 30 mg/l free glutamic acid (Maeda
et al., 1958; 1961). Strained infant foods provide 80 Cal/100g with
wide variations depending on the recipe, while human milk provides 70
Cal/g (Dept. Health & Soc. Sec., 1970).
Infant food may contain up to 0.4% added MSG, the natural content
depending on the basic constituents. Carrots contribute 0.32%. free
glutamic acid calculated as MSG, tomatoes, 0.45% and cheese 0.7%.
Substitution of some unprepared foods in equal weights for prepared
baby foods containing 0.3% added monosodium glutamate would also
result in an ingestion of greater amounts of glutamate than is
provided by mothers' milk an a calorie for calorie basis. The level of
0.3% in prepared foods appears to be an upper level since higher
concentrations impart an unpleasant flavour.
Infants aged 3 days and weighing 3kg consume 480 g mothers'
milk/day. This is equivalent to a daily intake of 1.104 g bound
glutamic acid, and 0.115 g free glutamic acid corresponding to 0.408
g/kg body-weight of glutamic acid/day. One-month old infants, weighing
3.8 kg consume 600 g mothers' milk/day. This is equivalent to a daily
intake of 1.37 g of bound glutamic acid and 0.144 g free glutamic acid
corresponding to 0.405 g/kg body-weight of glutamic acid/day.
Infants aged 5-6 months, weighing 7.5 kg, consume 500 g cows milk
and 2 jars of baby food/day. The respective daily intake of bound
glutamic acid amounts to 3.5 g and 0.5 g. The corresponding free
glutamic acid intake is 0.015 g and 0.060 g/day, which is equivalent
to 0.62 g/kg body-weight of glutamic acid/day. If the 2 jars
(200g/jar) contain 0.3% MSG, this increases the total intake of free
glutamic acid from 0.06 to 0.60 g. In a seven day survey of children
aged 9 to 12 months the intake of baby foods has been observed to
range from zero (in 20% of the surveyed cases) to a maximum of 250 g
daily in which up to 12 different preparations may be represented and
not all of which have monosodium glutamate added (Berry 1970).
There is evidence of rapid absorption of dietary glutamate since
in rats the glutamic acid level in portal blood rose within 1/2/3/4
hour to 250 per cent. in adults and 150 per cent. in young animals
over the testing level (Wheeler & Morgan, 1958). L-glutamic acid
absorption by the dog failed to increase noticeably the amino-N2 of
the peripheral blood but increased that of portal blood, possibly
because of increased uptake by tissue (Christenson et al., 1948).
Groups of 8 rats were given by gavage 200 mg/kg body-weight of
MSG alone or with 2000 mg/kg raw veal. Blood samples were taken at 10
minute intervals and after 30 minutes the animals were killed and free
glutamic acid determined in blood and brain. Plasma glutamic acid rose
rapidly to a peak in 20 minutes if monosodium glutamate was given
alone and more slowly to a peak in 30 minutes if given with veal.
Using 100, 500 and 2500 mg/kg orally produced a dose-related increase
in plasma level only at the two higher test levels and more if
monosodium glutamate was given alone. There was no effect on the brain
glutamate acid levels. Using s.c. 500 mg/kg body-weight produced the
same plasma levels as oral feeding. There was no adaptation. Brain
levels were not affected. 100 mg/kg monosodium glutamate was the
threshold dose before plasma levels rose. There was great variability
in the response (McLaughlan et al., 1970). Continuous infusion of dogs
with glutamic acid (05-4 mg/kg/hr) did not result in entry of glutamic
acid into liver and muscle cells, cerebrospinal fluid or brain. Kidney
cells appeared to be freely permeable. Metabolism of the infused
glutamic acid was limited (Kamin & Handler, 1950).
The intact rat as well as rat liver and rat tissues metabolize
glutamate by oxidative deamination (von Euler et al., 1938) or
transamination to oxaloacetic or pyruvic acid (Cohen, 1949) via
alpha-ketoglutarate to succinate (Meister, 1965). This was shown by
the use of 2-C14-labelled DL-glutamic acid given i.p. and resulting
in the production of aspartin acid labelled in the -COOH radical and
glutaric acid labelled in position 1-C and 2-C. Intracaecally
administered 2-C14-1abelled DL-glutamic acid is rapidly converted to
acetate, labelled in the methyl group, by the messaconate and
citramalate cycle. After gastric intubation of 2C14-labelled
DL-glutamic acid part is absorbed and metabolized to succinate, the
rest to methyl labelled acetate (Wilson & Koeppe, 1959). Rat tissue
has only a poor ability to oxidise D-glutamate. After i.p. or s.c.
administration conversion to D-pyrrolidone carboxylic acid occurs. Rat
liver and rat kidney also convert enzymatically D-glutamic acid to
D-pyrrolidone carboxylic acid (Wilson & Koeppe, 1961). The specific
enzyme was isolated from the liver and kidney of mice, rats and man
(Meister et al., 1963). Oral administration of C-monosodium
L-glutamate (2g/kg) to weanling rats caused a marked increase in the
specific activity of liver carbamyl phosphate synthesase. Prolonged
administration resulted in a return to control values, indicating an
adaptation to the administered substrate (Hutchinson & Labby, 1965).
Biochemical aspects are summarized in recent reports (Ajinomoto Co.,
1970).
I.v. injection of C 14-labelled glutamic acid into intact rats
and mice showed it to enter rapidly the brain, liver, kidney and
muscle as such (Lajtha et al., 1959). Glutamic acid was shown to be
distributed among more than one metabolic pool as animals mature
(Berl, 1965). Compartmentation of glutamate metabolism in the mouse
brain has been demonstrated by examining the time course of C14
incorporation into glutamine and glutamate (Van den Berg, et al.
1968).
I.v. injection of sodium P-glutamate produced a prolonged
increase in the amino-nitrogen content of blood and prolonged urinary
excretion in rabbits (Yamamura, 1960). Other effects observed were a
decrease in tryptophan and tyrosine metabolism of the liver following
daily injection of 1-4 g/kg glutamic acid into rats (Funiwake, 1957)
reduction in the activity of liver catalase in mice after single
injections of D-glutamic acid at 1.5 mg/g reverting to normal after 4
days and not observed with L-glutamic acid (Ando, 1958), enhanced
oxygen consumption by rats after injection of 1 mg/kg sodium glutamate
at low pO2, not observed at normal pO2 (Genkin & Udintsev, 1957)
and hyperglycaemia after i.p. glutamate in rats due to conversion to
glucose and additional stimulation of gluconeogensis (Marcus & Reaven,
1967). The effect on cerebral metabolism was studied by
intraventricular injection of L-glutamic acid into mice, when 150 mg
produced convulsions or only incoordinated grooming or circling of the
cage (Crawford, 1963). Two per cent. intra-arterial sodium glutamate
increased epileptic fits add intracisternal L-glutamic acid caused
tonic-clonic convulsions in animals and man. High parenteral dosage of
L-glutamic acid caused EEG changes only in dogs with previous cerebral
damage, and no rise was detected in the CSF level of glutamate (Herbst
et al., 1966). L-glutamic acid is oxidized by the brain to
alphaketoglutaric acid, NH3 and later CO2 and H2O and is the only
amino acid that on its own can maintain brain slice respiration
(Weil-Malherbe, 1936). Decarboxylation to gamma aminobutyric acid is
significant in the mammalian brain (Roberts, 1951; Perrault & Dry,
1961).
I.v. 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 cat
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).
Nutritional studies in the rat have shown glutamic acid to be a
nonessential amino acid replaceable by others and to be 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). Gouty patients have raised levels of plasma glutamate compared
with normals and following a protein meal glutamate reaches excessive
levels (Pagliari & Goodman, 1969). Premature and full term infants
hydrolyse any given protein in the stomach to very similar extents
(Berfenstam et al., 1955). Hepatic glutamate dehydrogenase appears at
12 weeks of human foetal life, is present in rat foetal liver on day
17 and reaches its maximum within 2 weeks after birth (Francesconi &
Villee, 1968),
Fifty patients with circulatory hypoxia received orally 1 g three
times/day of glutamic acid for 1 week. All patients showed less blood
lactic acid, a better alkali reserve and clinical improvement
(Gorbunova et al., 1960). 15 g equivalents of unneutralized L-glutamic
acid, L-glutamic acid-HC1 and monosodium glutamate were given orally
to man. There was little absorption of the poorly soluble L-glutamic
acid with very slight elevation of blood levels within one hour from
20 to 80 mg/l. Little absorption occurred with L-glutamic and
acid-HC1, but monosodium glutamate was well absorbed, the blood level
rising in one hour from 20 to 350 mg/l. (Himwich, 1954). 6-10 male
healthy children were given 1, 2 a 4 g sodium glutamate. Total
creatinine excretion was not affected but the amino acid/creatinine
ratio increased much more than the glucuronic acid/creatinine ratio.
Values returned to normal within 36 hours after 1-2 g and within 69
hours after 4 g (Inoue, 1960). Sodium glutamate has been used
therapeutically in uraemia to reduce blood levels of ammonia. 8 g of
i.v. glutamic acid caused nausea and vomiting in 11 from 17
individuals (Smyth et al., 1947).
Introduction of 0.15 per cent. glutamate solution into the small
intestine of the dog did not cause a rise in glutamate concentration
in the blood draining the intestinal loop. Only at 0.5 per cent. did
the venous blood contain extra glutamate. However, alanine appears in
high concentration in the portal blood. When this mechanism is
overwhelmed then glutamate appears also over and above the arterial
blood level (Neame & Wiseman, 1957). In the cat and rabbit in vivo
the same phenomenon occurs (Neame & Wiseman, 1958) and in the rat
in vitro (Matthews & Wiseman, 1953) and in vivo (Peraino & Harper,
1962). Further removal of excess portal glutamate and alinine occurs
in the liver. In man only 2 out of 4 subjects given 0.1 g/kg glutamic
acid as a 7 per cent. solution orally showed an appreciable rise of
free glutamic acid in plasma. Hence a similar mechanism may operate.
Additional glutamic acid, e.g, 10-20 g, if given to man, may
raise the amount of glutamic acid absorbed from ingested protein.
(Bessmann et al., 1948). Bound glutamate from proteins and
polypeptides is released gradually during digestion and would be
absorbed as alanine into the portal blood (Wiseman, 1970). L-glutamic
acid and DL-glutamic acid are absorbed orally by the rat to nearly the
same extent, L - being a little better absorbed (Aroskar & Berg,
1962). The foetal circulation has a higher amino acid concentration
than the maternal in the rhesus monkey (Kerr & Waisman, 1967).
Monosodium glutamate (8g/kg body-weight) was administered orally
to pregnant Wistar-Imamichi rats on day 19 of gestation. Plasma
glutamic acid was determined in mothers and foetuses, at 30, 60 and
120 min. after dosing. In the mothers' plasma, glutamic acid increased
from approx. 100 µg/ml to 1650 µg/ml, in the first 30 minutes. At the
end of the test period the level was 1000 µg/ml. No significant
changes occurred in the plasma glutamic acid of foetuses during this
period (approx. 50 µg/ml) (O'hara et al., 1970a).
Rats (Wistar-Imamichi), male adults (11-14 weeks of age) and male
neonates (2-3 days of age), were dosed orally with monosodium
glutamate (0.5-8 g/kg body-weight for adults, and 0.5-4 g/kg
body-weight for neonates). Plasma glutamic acid was measured over a 4
hour period. For adult rats, the highest level tested showed maximum
plasma glutamic acid, 1650 µg/ml, after 30 minutes. At the other dose
levels there were no appreciable changes in plasma glutamic acid. In
the case of neonates, levels rose to a maximum of 350 µg/ml after 90
minutes at the 2 g/kg dose level, and 1850 µg/ml after 90 minutes at
the 4.0 g/kg dose level. In a similar study with mice (4CS strain)
plasma glutamic acid of adults rose to a maximum of 530 µg/ml after 30
minutes, at the 2 g/kg dose level, and 1050 µg/ml at the 4 mg/kg dose
level. Neonate mice showed maximum level of plasma glutamic, 700 µg/ml
30 minutes after treatment at the 2 mg/kg dose level, and 2300 µg/ml,
2 hours after treatment at the 4 mg/kg dose level, (Ichimura et al.,
1970c). Another study showed that there was a marked correlation
between liver SOT, SPT and plasma glutamic acid of rats and mice dosed
orally with 1 g/kg body weight monosodium glutamate. Measurements were
made during the period 1-100 days of age. (Hashimoto et al., 1970).
When monosodium glutamate (1 g/kg body-weight) or monosodium
glutamate (1 g/kg body-weight) plus powdered milk (1.5 g/kg
body-weight) or powdered milk (1.5 g/kg body-weight) was administered
orally to 10 day old rats, the maximum levels of plasma glutamic acid
were 425 µg/ml, 160 µg/ml and 105 µg/ml respectively. These levels
occurred 30 minutes after dosing (O'hara et al., 1970b).
Acute toxicity
Animal Route LD50 References
mg/body-weight
Mouse i.p. 6900 Yanagisawa et al., 1961
p.o. 12961 Izeki, 1964
p.o. 16200 (14200-18400) Ichimura & Kirimura, 1968
i.v. 30000 Ajinomoto Co., 1970
Rat p.o. 19900 (L MSG) International Minerals
& Chem. Corp., 1969
p.o. 10000 (DI, MSG) international Minerals &
Chem. Corp., 1969
p.o. > 30000 (L-GA) International Minerals
& Chem. Corp., 1969
Guinea-pig i.p. 15000 Ajinomoto Co., 1970
Rabbit p.o. > 2300 (L-GA) International Minerals
& Chem. Corp., 1969
Cat s.c. 8000 Ajinomoto Co., 1970
Mouse. Mice aged 2 to 9 days were killed 1 to 48 hours after single
subcutaneous injection of monosodium glutamate at doses from 0.5-4
µg/kg, lesions 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 tuberales. No pituitary lesions
were seen but sub-commissural and subfornical organs exhibited
intracellular oedema and neuronal necrosis. Adult mice given
subcutaneously 5-7 µg/kg monosodium L-glutamate showed similar
lesions. Similar lesions were seen in another strain of mouse and in
neonatal rats (Olney, 1969b).
After a single subcutaneous injection of monosodium glutamate at
4 g/kg into neonatal mice aged 9-10 days. the animals were killed from
30 minutes to 48 hours. The retinas showed an acute lesion on electron
microscopy with swelling dendrites and early neuronal changes leading
to necrosis followed by phagocytosis (Olney, 1969a).
Sixty-five neonatal mice aged 10-12 days received single oral
very high loads of monosodium glutamate at 0.5, 0.75, 1.0 and 2.0 g/kg
body-weight by gavage. 10 were controls and 54 mice received other
amounts. After 3-6 hours all treated animals were killed by perfusion.
Brain damage as evidenced by necrotic neurons was evident in arcuate
nuclei of 51 animals. 62 per cent. at 0.5 g/kg, 81 per cent. at 0.75
g/kg, 100 per cent. at 1 g/kg and 100 per cent. at 2 g/kg. The lesions
were identical both by light and electron microscopy to s.c. produced
lesions. The number of necrotic neurons rose approximately with dose.
Four animals tested with glutamic acid also developed the same lesions
at 1 g/kg body-weight. The effect was additive with aspartate (Olney,
1970b).
Groups of five 3-day and 12-day old mice receiving subcutaneously
or orally a single acute dose (1g/kg) monosodium glutamate,
monopotassium glutamate, sodium chloride, sodium gluconate or
distilled water, were sacrificed 3 hours and 24 hours after treatment.
Preliminary light microscopic studies of the large mid brain area,
showed similar non-specific scattered tissue changes in all treatment
groups. (Oser et al., 1970). In another study, mice, 5-9 days old,
received a single dose of monosodium glutamate (4 g/kg in phosphate
buffer), either subcutaneously or orally. Animals were sacrificed at
24 hours. Light microscopy of the hypothalmic area of the brain
indicated abnormal neuronal cells in 12/30 of the mice receiving a
subcutaneous injection of the test substance only 5/35 mice receiving
the oral dose showed some change (Coulston et al., 1970). Six, nine to
ten day old mice. dosed orally with 10% monosodium glutamate (2
gm/kg), showed characteristic brain lesions (Geil, 1970).
Monosodium glutamate caused reversible blockage of beta wave 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 to 11 days postnatal age, it was
difficult to produce significant lesions of the retina (Olney, 1969a).
A study of the glutamate metabolizing enzymes of the retina of the
glutamate treated rat indicated a decrease in glutaminase activity, an
increase in glutamic aspartate transaminase, and no change in glutamyl
synthetase and glutamotransference. The effects appear to be due to
repression and induction of enzyme synthesis (Freedman & Potts, 1962;
Freedman & Potts, 1963). Glutamate uptake by retina, brain and plasma
decreases with age and is slower at 12 days when compared with 50 day
old animals (Freedman & Potts, 1963).
Obesity and acute irreversible degeneration in liver and retina
of neonatal mice has been seen following parenteral administration of
monosodium glutamate (Cohen, 1967). S.c. 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).
Rat. Groups each of 20, ten day old rats, Charles River Strain,
(10 male, 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 mg/kg,
additional to that found in normal commercially distributed baby food
(390 mg per jar), The rats were mated and half of the offspring were
removed from parental females, and sacrificed after 5 hours.
Histological studies were made of brain in the area of the hypothalmus
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 brain of animals sacrificed
at either 5 hour post treatment, or after reaching maturity. Animals
which were reared to maturity showed normal growth and food
consumption (Geil, 1970).
Groups of 5 3-and 12-day old rats receiving subcutaneously or
orally a single acute dose (1g/kg) monosodium glutamate, mono
potassium glutamate, sodium chloride, sodium gluconate or distilled
water, were sacrificed 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., 1970).
Dog. 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 a single acute dose (1 g/kg) monosodium glutamate,
monopotassium glutamate, sodium chloride, sodium gluconate or
distilled water; and were sacrificed 3 hours and 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., 1970).
Monkey. A newborn (8 hours old) rhesus infant, probably
somewhat premature was given subcutaneously 2.2 g/kg body-weight
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 day old, received a single dose 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. (Coulston at al., 1970).
Man
Pharmacological effects were studied in 56 men given 1-12 g
monosodium L-glutamate orally on an empty stomach. Burning of the face
and trunk, facial pressure and chest pain were noted as well as
headache, the last sometimes as the only symptom. Amounts of 3 g or
less were effective in all. Similar effects were obtained by 3-5 g of
monopotassium glutamate L-glutamic acid and DL-glutamic acid but no
effects were seen with monosodium D-glutamate or other L-aminoacids.
Thirteen subjects received i.v. 25-125 mg sodium glutamate with
symptoms occurring within 20 seconds. The burning sensation is due to
a peripheral mechanism and no genetic predisposition was noted
(Schaumburg et al., 1969). A survey was made of 912 Japanese
individuals to determine if any of these symptoms were noted after
eating a Prepared Oriental Type Noodle, containing 0.61-1.36 g
monosodium glutamate/serving. In no case were any of the
characteristic symptoms reported (Ichimura et al., 1970a). In another
study, the effect of monosodium glutamate on 61 healthy men was
determined by the double blind method. The doses of monosodium
glutamate administered were 2.2 g, 4.4 g or 8.7 g.Intake was either on
a non-empty stomach (30 minutes after meal) or an empty stomach
(overnight fast). In experiments on the non-empty stomach conditions
the number of persons showing some symptoms were the same for the
Placebo and the others. In the case of the empty stomach conditions a
number of the test subjects on the highest level of monosodium
glutamate experienced two of the typical symptoms at the same time. No
individual experienced three of the symptoms. The effect of monosodium
glutamate intake (2.2 g, 4.4 g or 8.7 g) on changes in blood pressure,
pulse rate, ECG and sodium and glutamate levels in blood, was measured
in 5 persons who had not experienced any symptoms, and 9 who had
experienced some symptoms. There were no differences in increase in
glutamic acid in the blood in either group. Sodium content of the
blood and all other parameters measured showed no changes in either
group (Ichimura et al., 1970b).
The occurrence of nausea and vomiting following the i.v.
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 i.v.
administration. When the serum glutamic acid reached 12 to 15 mg/100
ml, nausea and vomiting occurred in half the subjects. Other amino
acids appear to potentiate the effect (Levey et al., 1949).
Intravenous glutamic acid (100 mg/kg) produces vomiting (Madden
et al., 1945).
Intravenous solutions of 2.9 per cent. monosodium glutamate in 5
per cent. dextrose are given in hepatic coma but too rapid injection
causes salivation, flushing and vomiting; afterwards oral doses of
5-20 g are given daily. High doses (3 g) are said to produce Kwok's
disease, pain in the chest, tingling sensations or temporary numbness
of back and arms, weakness and palpitation in susceptible people
(Kwok, 1968). 25 g have no effect in non-sensitives (Schaumburg &
Byck, 1968).
Arginine glutamate may be used in the treatment of ammonia
intoxication. It is given by intravenous infusion in doses of 25 to 50
g every 8 hours for 3-5 days in dextrose and infused at a rate of not
more than 25 g of arginine glutamate over 1-2 hours. More rapid
infusions may cause vomiting (Martindale, 1967).
Single and double blind studies were done with single oral doses
of monosodium glutamate in human male volunteers on a fasting stomach
(18 hours after last meal). 98 received 5 grams of monosodium
glutamate in single blind studies, 6 received 8 grams and 5 received
12 grams in double blind studies. Physical examinations were done on
all subjects. Complaints were registered In all groups ranging from
23-80%6. There was a low incidence of most complaints except for
lightheadedness and tightness in the face. No subject reported or was
observed to have experienced the complete triad of symptoms as
described in the original Chinese-Restaurant syndrome (Kwok's
disease). In the double blind studies where clinical chemistry, blood
pressure and pulse were measured in addition to clinical examination,
no significant differences between monosodium glutamate and sodium
chloride were detected (Rosemblum et al., 1969).
Short-term studies
Mouse. 38 neonate mice were observed for 9 months. 20 received
subcutaneous monosodium glutamate daily for 1 to 10 days in doses of
0.5 g/kg to 4 g/kg. 18 were controls. Although treated animals
remained skeletally stunted and both males and females gained more
weight than controls from 30 to 150 days yet treated animals consumed
less food than controls. Test animals were lethargic, females failed
to conceive but male fertility was not affected. At autopsy of test
animals massive fat accumulation was seen in test mice, fatty livers,
thin uteri and adenohypophysis had overall fewer cells in the
adenohypophysis.
10 test neonates received a single subcutaneous injection of 3
gm/kg monosodium glutamate 2 days after birth, 13 neonates were
controls. Again test animals were heavier than controls after 9 months
but less so than mice given repeated injection treatment. It was
postulated that an endocrine disturbance would 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).
Rat. Natural monosodium L-glutamate, synthetic monosodium
L-glutamate, and synthetic monosodium D-glutamate in amounts of 20,
200 and 2000 mg/kg body-weight were given orally to groups of 5 male
rats each 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 and kidneys in comparison with the control
group were observed. No histological changes in internal organs were
found by macroscopic and microscopic examination (Hara et al., 1962).
Nine groups of 20 rats were given 0.5 per cent. and 6 per cent.
of 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 difference in their learning ability for maze trails
was noted (Stella & McElroy, 1948). 8 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).
Man. Monosodium glutamate has been used in the treatment of
mentally retarded children in doses up to 48 g daily but on average
10-15 g was given.
150 children aged 4-15 years were treated with glutamic acid for
six months and compared with 50 controls. There was a rise in verbal
intelligence quotient but was not statistically significant. 64 per
cent. showed improvement of behavioural traits (Zimmerman &
Burgemeister, 1950).
17 patients received up to 15 g monosodium glutamate three times
a day but showed a raised blood level for 12 hours only. No effect on
BMR, EEG, ECG, BP, heart rate, respiration rate, temperature and
weight was noted over 11 months (Himwich et al., 1954a). 15 g then 30
g monosodium glutamate were given per dose for one week each, followed
by 45 g for 12 weeks to 53 patients without any effect on basal plasma
levels of glutamic acid (Himwich et al., 1954b).
DL-glutamic acid HC1 was given in doses of 12, 16 and 20 g to 8
patients with petit mal and psychomotor epilepsy without adverse
effects (Price et al., 1943). Five episodes of hepatic coma in 3
patients treated with i.v. 23 g of monosodium glutamate with
improvement (Walshe, 1953). 10-12 g of L-glutamic acid given to
epileptics and mental defectives appeared to improve 9 out of 20 cases
(Waelsch, 1949).
Long-term studies
Mouse. 1 control group of 200 male mice and 6 test groups of
100 male mice received 0 per cent., 1 per cent. or 4 per cent. in
their diet of either L-glutamic acid, monosodium L-glutamate or
DL-monosodium glutamate. No malignant tumours appeared after 2 years
that could be related to the administration of test material. Growth
and haematology were normal, histopathology showed no abnormalities in
the test animals (Little, 1953a).
Rat. Groups of 75 male and 75 female rats received for 2 years
dietary levels of 0, 0.1 per cent. or 0.4 per cent. either monosodium
L glutamate, monosodium DL-glutamate or L-glutamic acid respectively.
No adverse effects were noted on body-weight, growth, food intake,
haematology, general behaviour, survival rate, gross and
histopathology or tumour incidence (Little, 1953b).
Special studies
Mouse. The 4CS strain and Swiss white strains were studied.
Groups of 6 mice (3 male, 3 female), were maintained on diets
containing 0 per cent., 2% (=4 g/kg/day) or 4 per cent. (= 8 g/kg/day)
monosodium glutamate. Mice were mated after 2 to 4 weeks on the test
diet. Offspring (F1) were weaned at age 25 days, and fed the same
diet as parents. At age 90 days, selected (F1) male and female mice
from each group were allowed to produce a single litter (F2). Parent
mice were maintained on test diets, for 100 days after delivery and
F1, mice for 130 days of age. F2 mice were reared until 20 days of
age. No effects were observed on growth, feed intake, estrous cycle,
date of sexual maturation (F1 generation), organ weight, litter size
and body-weight of offspring, and histopathology of major organs
(including brain and eyes) of parent and F1 generation. Day of eye
opening, general appearance and roentgenographic skeletal examination
of F2 generation showed no abnormalities (Yonetani et al., 1970).
Rat. 6 groups of 5-6 male and 5-10 female rats received by oral
incubation daily 25 mg/kg or 125 mg/kg body-weight of glutamic acid
mono-hydrochloride. Males and females received the compound during
days 5-19 of one 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 on the higher dose level had enlarged
spleens. Animals were mated at the end of the experiment and pups were
normal (Furuya, 1967).
Rats were given thalidomide combined with 2 per cent. 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 no different from controls (McColl et al., 1965).
Four females and 1 male fed for 7 months on either 0 per cent.,
0.1%, 0.4% of monosodium L-glutamate, monosodium DL-glutamate or
L-glutamic acid were mated and number of pups per litter was similar
in all groups. Only 15-20 per cent. survived because of cannibalism.
No abnormalities regarding fertility were seen on mating other groups
of 4 females and 1 male at nine and eleven months. The F1 generation
was mated at 10 months and an F2 generation produced in most groups
but only the groups as 0.1 and the 0.4% L-glutamic acid produced an
F3 and F4 generation. No impairment of fertility was noted (Little,
1953).
Monosodium glutamate. was administered orally in doses up to 7
g/kg/day to pregnant rats on 6-15 or 15-17 days following conception,
it 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 the 15-17
days during gestation showed impaired ability to reproduce (Kbera et
al., 1970).
Two female rats received 4 g/kg body weight of monosodium
glutamate commencing at day one of pregnancy. There was no effect on
pregnancy or lactation. Pups were divided into 3 groups. Two groups
were nursed by parents receiving monosodium glutamate, and one group
by untreated parent. At weaning (day 20), one group of pups that had
been nursed by a parent receiving monosodium glutamate received
approx. 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 oestrus cycle. All pups developed normally, and
no abnormalities were noted in growth rate, time of sexual maturity,
oestrus cycle and fertility. (Suzuki & Tagahashi, 1970). For
histological studies, brain, hypophysis and eye were fixed in 10%
neutral buffered formalin. Sections were stained with
Hematoxylin-Eosin and Luxol fast blue-cresyl echt violet. No
differences were observed between arcuate nuclei, medium eminence of
hypothalmus and retina of control and monosodium glutamate treated
groups. (Shimizu & Aibara, 1970).
Rabbit. In one group of 10 female and 4 male rabbits only the
females received orally 25 mg/kg body-weight of glutamic acid for 27
days. Two of the females were pregnant and the others were not
pregnant, A second group of 4 female and 2 males received orally 25
mg/kg glutamic acid with 25 mg/kg vitamin B6. A third group of 6
females and 2 males received orally 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 two animals with
delayed pregnancy, the uterus containing degenerate foetuses. Two
others had abortions of malformed foetuses. 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 during
seven months and showed limb deformities, decreased growth and
development compared with controls. The histopathology 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 3 groups showed
testicular atrophy in parents and multiple changes in the pups
(Turgrul, 1965).
4 groups of rabbits (24 females and 16 males) received either 0,
0.1 per cent., 0.825 per cent. or 8.25 per cent. of monosodium
glutamate in their diet for 2-3 weeks before mating. A positive
control group of 22 pregnant females received 100 mg/kg thalidomide
from day 8 to 16 of pregnancy. All does were sacrificed on day 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 effect on body-weight gain or food consumption was
seen, nor on general appearance and behaviour. Gross and
histopathology revealed no toxic effects an embryos, resorption and
pups and all litter data were comparable among test animals and
negative controls (Hazleton Laboratories, 1966). The brains of 5
female and 5 male pups at the 8.25 per cent. level were subsequently
checked for neuronal necrosis compared with controls, but none was
found (Hazleton, 1969a). Similar investigations on 5 male and 5 female
pups at the 0.1 and 0.825 per cent. levels were also negative
(Hazleton, 1969b).
In another experiment on rabbits, these animals received 2.5
mg/kg bodyweight, 25 mg/kg and 250 mg/kg of L-glutamic acid
hydrochloride at 70 hours post coition and 192 hours post coition.
Operative removal of foetuses was performed on the 11th, 17th and 30th
day post coition in 3 different series. The corpora lutea, the
resorbed, implanted, normal and deformed foetuses were examined. No
significant effects due to L-glutamic acid were noted with respect to
teratogenesis (Gottschewski, 1968).
Glutamic acid hydrochloride in a dose of 25 mg/kg body-weight was
given orally to 15 pregnant rabbits once a day for a period of 15 days
after copulation, monosodium glutamate in the same dose and for the
same period of time to 9 pregnant rabbits and saline solution to 11
pregnant rabbits which served as control group. No differences were
noted between the treated groups and the controls as to rate of
conception. mean litter size. and nursing rate. The average
body-weight of the young in the treated groups was 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 and skeletal malformations were
observed. There were some abnormal changes in gestation such as
abortion or resorption of foetuses, but these observations were made
in all groups, with an incidence of 21 per cent. in the glutamic acid
hydrochloride group, of 25 per cent. after administration of
monosodium glutamate, and of 27 per cent. in the controls. There were
no external and skeletal malformations in the aborted foetuses
(Yonetani, 1967).
Chick embryo. Fertilised hen eggs were incubated after a single
injection of 0.01-0.1 mg glutamic acid into the yolk sac. The
mortality of embryos was raised compared with controls (53 per cent.
against 24 per cent.) and there was a higher incidence of
developmental defects (24 per cent. against 3 per cent.) especially
depression of development of the spine, pelvis and lower limbs
(Aleksandrov et al., 1965). In another study many variables were
studied such as route of injection, dose and time of injection. No
obvious toxicity or teratogenicity was observed (US Food & Drug
Administration, 1969).
Tests on tissue cultures
Cells (kangaroo-rats cell line) were exposed continuously for 72
hours at 0.1% monosodium glutamate without showing any toxic effect
(US Food & Drug Administration, 1969).
Comments
Glutamic acid is a component of proteins and comprises some 20
per cent. of ingested protein. Much is known about its metabolism in
various animal species. During gastrointestinal absorption
transanimation to alanine occurs. As a consequence there is only a
slight rise in glutamate levels in the portal blood. A similar
mechanism probably also occurs in man. However, if the capacity of
this mechanism and the further conversion of glutamate in the liver is
overwhelmed, or if monosodium glutamate is administered parenterally
in large doses, it is possible to obtain significantly high blood
levels. For primates and man it has been demonstrated that blood
levels of glutamate are higher in the foetus compared with the mother,
particularly during the early phases of foetal development. Recent
data show that after glutamate loading of the mother, the full term
prenatal rat foetus has less glutamate in its circulation than exists
in the maternal circulation.
Numerous reproduction studies in mice, rats and rabbits revealed
no deleterious effects on the offspring if the parent generation was
fed glutamate in high doses, suggesting that an earlier claim of
teratogenic effects in the rabbits was not related to glutamate
administration. There is evidence that glutamate administered
parenterally or orally is retinotoxic but only during a brief period
of neonatal life and not in utero or after weaning.
Work using subcutaneous injection suggests a vulnerability of the
developing mouse, rat and primate central nervous system to high
levels of glutamate in addition to other amino acids. Attempts at
reproducing these effects after oral administration were successful
only in mouse by the use of high doses.
Acute reactions reported after ingestion of glutamate as food
additive are probably due to the rapid absorption of large mounts of
the substance. These occur fairly frequently, and particularly
sensitive persons develop Kwok's disease.
Evaluation
On the data provided it is possible to arrive at a formal
acceptable daily intake making allowance for the fact that glutamate
is a normal constituent of protein. In arriving at the ADI the acute
reactions due to rapid absorption have been taken into consideration.
In view of the uncertainty regarding the possible susceptibility of
the very early human neonate to high oral intakes of glutamate, it
would be prudent not to add monosodium glutamate to foods specifically
intended for infants under one year of age. When the further work on
this aspect has become available, it may be possible to arrive at an
acceptable daily intake for these infants as well.
Level causing no toxicological effect in the mouse
4 per cent. in the diet equivalent to 6000 mg/kg body-weight
Estimate of acceptable daily intake in man
mg/kg body-weight
Unconditional acceptance* 0-120
*Except for infants under one year. This figure is additional to
intake from all dietary sources.
Further work required
1. Oral no-effect level of monosodium glutamate in neonatal mice.
2. Determination of the period of susceptibility to monosodium
glutamate in neonatal mammals.
3. Age correlation between neonatal experimental animals and the human
infants.
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See Also:
Toxicological Abbreviations