Toxicological evaluation of some food
additives including anticaking agents,
antimicrobials, antioxidants, emulsifiers
and thickening agents
WHO FOOD ADDITIVES SERIES NO. 5
The evaluations contained in this publication
were prepared by the Joint FAO/WHO Expert
Committee on Food Additives which met in Geneva,
25 June - 4 July 19731
World Health Organization
Geneva
1974
1 Seventeenth Report of the Joint FAO/WHO Expert Committee on
Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539;
FAO Nutrition Meetings Report Series, 1974, No. 53.
L-GLUTAMIC ACID AND ITS AMMONIUM, CALCIUM, MONOSODIUM
AND POTASSIUM SALTS
Explanation
These substances have been evaluated for acceptable daily intake
by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
Ref. No. 23) in 1970.
Since the previous evaluation, additional data have become
available and are summarized and discussed in the following monograph.
The previously published monograph has been expanded and is reproduced
in its entirety below.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
There is evidence of rapid absorption of dietary glutamate since
in rats the glutamic acid level in portal blood rose within 1/2 to 3/4
hour to 250% in adults and 150% 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 (Christensen et al., 1948).
Groups of eight rats were given by gavage 200 mg/kg bw 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 bw 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 (0.5-4 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
alphaketoglutarate 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 aspartic acid labelled in the -COOH radicals and
glutamic acid labelled in position 1-C and 2-C. Intracaecally
administered 2-C14-labelled DL-glutamic acid is rapidly converted
to acetate, labelled in the methyl group, by the mesaconate and
citramalate cycle. After gastric intubation of 2-C14-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 oxidize 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 monosodium L-glutamate
(2 g/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 C14-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.,
1969).
Fifty patients with circulatory hypoxia received orally 1 g
three times a day of glutamic acid for one week. All patients
showed less blood lactic acid, a better alkali reserve and clinical
improvement (Gorbunova et al., 1960). 15 g equivalents of
un-neutralized L-glutamic acid, L-glutamic acid-HCl 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-HCl, but monosodium glutamate was well
absorbed, the blood level rising in one hour from 20 to 350 mg/l
(Himwich, 1954). Six to 10 male healthy children were given 1, 2 and
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% 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% 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 alanine occurs in the
liver. In man only two out of four subjects given 0.1 g/kg glutamic
acid as a 7% 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 (Bessman 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 fetal circulation
has a higher amino acid concentration than the maternal in the rhesus
monkey (Kerr & Waisman, 1967).
Monosodium glutamate (8 g/kg bw) was administered orally to
pregnant Wistar-Imamichi rats on day 19 of gestation. Plasma glutamic
acid was determined in mothers and fetuses, at 30, 60 and 120 minutes
after dosing. In the mothers' plasma, glutamic acid increased from
approximately 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 fetuses during this
period (approximately 50 µg/ml) (Ohara et al., 1970a).
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 (Fumiwake, 1957). A 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 four days and not observed with
L-glutamic acid (Ando, 1959) 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 gluconeogenesis (Marcus & Reaven, 1967).
Rats (Wistar-Imamichi), male adults (11-14 weeks of age) and male
neonates (two to three days of age), were dosed orally with monosodium
glutamate (0.5-8 g/kg bw for adults, and 0.5-4 g/kg bw for neonates).
Plasma glutamic acid was measured over a four-hour period. For adult
rats, the highest level tested showed maximum plasma glutamic acid,
1650 ug/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 g/kg dose level. Neonate
mice showed maximum level of plasma glutamic, 700 µg/ml 30 minutes
after treatment at the 2 g/kg dose level, and 2300 µg/ml, two hours
after treatment at the 4 g/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 bw monosodium glutamate. Measurements were made during the
period 1-100 days of age (Hashimoto et al., 1970).
When monosodium glutamate (1 g/kg bw) or monosodium glutamate
(1 g/kg bw) plus powdered milk (1.5 g/kg bw) or powdered milk
(1.5 g/kg bw) 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 (Ohara et
al., 1970b).
L-monosodium glutamate-3-4-14C was administered to pregnant
rhesus monkeys and serial maternal and fetal plasma samples analysed.
In maternal plasma, 68% of the radioactivity remained in association
with the glutamate, 22% was converted to glucose and smaller amounts
into lactate, aspartate, glutamine and ornithine. In fetal blood,
glucose and lactate accounted for more than 80% of the radioactivity
with less than 2% of the label found in glutamate. Although maternal
glutamate levels increased 25-fold, fetal levels were unchanged.
Labelled glutamate administered to the fetus was not transferred to
maternal circulation until fetal plasma glutamate levels reached
1000 µmoles/100 ml (normal 5 µmoles/100 ml) (Stegink et al., 1973).
Levels of glutamic acid averaged 1.3-1.6 mg/g in the brain and
0.02-0.03 mg/ml in plasma of adult rat, mouse, guinea-pig and rabbit.
In suckling rats glutamic acid in the brain increased from 0.5 mg/g at
one day of age to 0.8 mg/g at seven days, 1.1 mg/g at 15 days and
1.3 mg/g at 21 days, whereas the levels in plasma decreased slightly
from 0.04 mg/ml at one day, 0.03 mg/ml at 7-15 days and 0.02 mg/ml at
21 days. In suckling guinea-pigs there was only a slight rise in brain
levels, from 1.3 mg/g at one day to 1.5 mg/g at 21 days, and no
change in plasma levels. In adult rats glutamic acid levels averaged
1.53 mg/g brain and 0.02 mg/ml in plasma after three days' starvation,
1.61 mg/g and 0.02 mg/ml respectively after three days' starvation and
one day normal feeding, 1.66 mg/g in brain and 0.54 mg/ml in plasma
after three days' starvation plus a glutamate load of 10 mg/kg bw
orally. Adult mice were given an oral load of MSG (10 g/kg bw). The
glutamate levels in the brain averaged 1.28-1.43 mg/g during the
subsequent 14 hours. The levels in plasma averaged 0.02 mg/ml at
0 hour, 1.45 mg/ml at one hour, 0.32 mg/ml at two hours, 0.05 mg/ml at
four hours, and 0.03 mg/ml at 14 hours after treatment. Sprague-Dawley
rats, body weight 175 g, were given an oral load of MSG, 10 g/kg bw.
Glutamic acid levels in brain averaged 1.37-1.50 mg/g during the
subsequent 14 hours, in plasma 0.02 mg/ml at 0 hour, 0.36 mg/ml at one
hour, 0.30 mg/ml at two hours, 0.26 mg/ml at four hours, 0.12 mg/ml at
six hours and 0.03 mg/ml at 14 hours after treatment. Rabbits, body
weight 2.5 kg, were given an oral load of MSG, 10 g/kg bw. Glutamic
acid levels in brain averaged 1.49-1.57 mg/kg during the subsequent
six hours, in plasma they averaged 0.03 mg/ml at 0 hours, 0.37 mg/ml
at one hour, 0.28 mg/ml at four hours and 0.16 mg/ml at six hours.
Rats were given a subcutaneous injection of MSG at five days of age,
5 g/kg bw. Levels of glutamic acid in the brain averaged 0.66 mg/g at
0 hour, 0.76 mg/g at one hour, 0.73 mg/g at three hours, 0.90 mg/g at
five hours and 0.96 mg/g at 14 hours, in plasma 0.03 mg/ml at 0 hours,
1.64 mg/ml at 1 hour, 0.41 mg/ml at three hours, 0.10 mg/ml at five
hours and 0.03 mg/ml at 14 hours (Garattini, 1971).
NUTRITIONAL 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 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). Human milk contains 1.2% protein of
which 20% is bound glutamic acid which is equivalent to 3 g/l
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/l calculated as MSG, but only 30 mg/l free glutamic acid (Maeda
et al., 1958; 1961). Strained infant foods provide 80 Cal/100 g with
wide variations depending on the recipe, while human milk provides
70 Cal/100 g (United Kingdom Dept. Health & Soc. Sec., 1970).
Free amino acids were analysed by GLC in fruits and fruit juices.
High levels of aspartic acid were found in figs (2.6 g/kg), nectarine
(2.0 g/kg), peaches (1.1 g/kg), yellow plums (1.8 g/kg), dry prunes
(1.9-5.2 g/kg). High levels of glutamic acid were found in cantaloupe
(0.5 g/kg), grapes (0.4 g/kg) (Fernandez-Flores et al., 1970).
Free glutamic acid was analysed in various foodstuffs by paper
electrophoresis. Fish and meat had less than 0.1 g/kg, sausage
0.1-1.5 g/kg, cheese 0.2-22 g/kg, "tomatenflocken" 15 g/kg and dried
mushrooms 17 g/kg (Müller, 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 on a calorie for calorie basis. The level of
0.3-0.5% in prepared foods appears to be the current level of use,
since higher concentrations impart an unpleasant flavour.
Infants aged three days and weighing 3 kg consume 480 g mothers'
milk per 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 bw of glutamic acid per day. One month old infants,
weighing 3.8 kg consume 600 g mothers' milk per 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 bw of glutamic
acid per day.
Infants aged five to six months, weighing 7.5 kg, consume 500 g
cows' milk and two jars of baby food per 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 per
day, which is equivalent to 0.62 g/kg bw of glutamic acid per day. If
the two jars (200 g/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 9-12 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).
Nutritional studies in the rat have shown glutamic acid to be a
non-essential 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 fetal life, is present in rat fetal liver on day 17
and reaches its maximum within two weeks after birth (Francesconi &
Villee, 1968).
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 (No. 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 the chick growth
was examined. The basal diet contained 26.8% 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 basal diet), or serine 8.1%. Of these, glutamic acid and aspartic
acid were found to be very useful nitrogen source, alanine was useful,
glycine and proline were insufficient, and serine was harmful for
chick growth. The chicks fed on the L-glutamic acid diet showed less
growth than those fed on the basal diet, though there was no
statistical significance between the groups (Sugahara & Ariyoski,
1967).
The glutamate ion has a specific flavour effect that is more
readily detected than NaCl when sodium chloride and MSG are added at
approximately equal sodium concentrations (3-6 ml) to chicken broth
containing normal amounts of NaCl (0.5-0.75%). MSG could readily be
detected in the chicken broth at 0.04%. The ease of detecting
glutamate is not greatly influenced by variation in levels of
thickening agent, fat content or seasonings that would normally be
encountered in foods. Added glutamate was much more difficult to
detect in foods normally containing very high levels of glutamate
(> 0.2%) than in those with very low levels of glutamate (> 0.02%).
The glutamate content of the foods must be considered in developing
recommendations for levels of added glutamate (Hanson et al., 1960).
TOXICOLOGICAL STUDIES
Special studies
Special studies on multigeneration, reproduction and teratology
Mouse
The 4CS strain and Swiss white strains were studied. Groups of
six mice (three male, three female), were maintained on diets
containing 0%, 2% (= 4 g/kg/day) or 4% (= 8 g/kg/day) monosodium
glutamate. Mice were mated after two to four 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
Six groups of 5-6 male and 5-10 female rats received by oral
intubation daily 25 mg/kg or 125 mg/kg bw of glutamic acid
monohydrochloride. 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% 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 one male fed for seven months on either 0%,
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% survived because of cannibalism. No
abnormalities regarding fertility were seen on mating other groups of
four females and one male at nine and 11 months. The F1 generation
was mated at 10 months and an F2 generation produced in most groups
but only the groups at 0.1 and the 0.4% L-glutamic acid produced an
F3 and F4 generation. No impairment of fertility was noted (Little,
1953a).
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
(Khera et al., 1970).
Two female rats received 4 g/kg bw of monosodium glutamate
commencing at day 1 of pregnancy. There was no effect 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 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).
Groups of female rats were maintained on diets containing 0.5%,
1% or 2% vitamin level. At each vitamin level diets also contained
monosodium glutamate at 0, 1% or 2%. Reproductive performance of the
rats (P) as well as 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 mothers (P) 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 P generation. The differences present at birth
disappear at weaning (Semprini et al., 1971).
Rabbit
In one group of 10 female and four male rabbits only the females
received orally 25 mg/kg bw of glutamic acid for 27 days. Two of the
females were pregnant and the others were not pregnant. A second group
of four female and two males received orally 25 mg/kg glutamic acid
with 25 mg/kg vitamin B6. A third group of six females and two males
received orally 25 mg/kg glutamic acid alone. A fourth group of
20 females and eight males served as controls. The test substance was
given by gavage. The first group showed two animals with delayed
pregnancy, the uterus containing degenerate 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 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 two
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 three 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 either
0, 0.1%, 0.825% or 8.25% of monosodium glutamate in their diet for two
to three 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 on
embryos, resorption and pups and all litter data were comparable among
test animals and negative controls (Hazelton Laboratories, 1966). The
brains of five female and five male pups at the 8.25% level were
subsequently checked for neuronal necrosis compared with controls, but
none was found (Hazelton, 1969a). Similar investigations on five male
and five female pups at the 0.1 and 0.825% levels were also negative
(Hazelton, 1969b; Ebert, 1970).
In another experiment on rabbits, these animals received
2.5 mg/kg bw, 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 fetuses was performed on the eleventh, seventeenth and thirtieth
day post coition in three different series. The corporal lutea, the
resorbed, 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 in a dose of 25 mg/kg bw 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 nine 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 fetuses, but these observations were made in
all groups, with an incidence of 21% in the glutamic acid
hydrochloride group, of 25% after administration of monosodium
glutamate, and of 27% in the controls. There were no external and
skeletal malformations in the aborted fetuses (Yonetani, 1967).
Chick embryo
Fertilized 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% 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 route of injection, dose and time of injection. No
obvious toxicity or teratogenicity was observed (United States Food &
Drug Administration, 1969).
Special studies on mutagenicity
Cells (kangaroo-rats cell line) were exposed continuously for
72 hours at 0.1% monosodium glutamate without showing any toxic effect
(United States Food & Drug Administration, 1969).
Groups of 12 male albino mice (Charles River strain), received a
single oral dose, by gavage, of monosodium glutamate at levels of
0, 2.7 and 5.4 g/kg bw. The treated animals were mated with groups of
three untreated females for each of six consecutive weeks. Females
were sacrificed at mid-term of pregnancy, and the uterus examined for
signs of early embryonic death. Females that had mated with treated
males had numbers of implantations, resorptions, and embryos similar
to those mated with controls (Industrial Bio-Test, 1973).
Special studies on neurotoxicity
L-glutamate and gamma-aminobutyric acid (GABA) supposedly act
respectively as excitatory and inhibitory transmitters in the central
nervous system. The glutamate-GABA metabolic pathway can use up a
substantial fraction of the total oxygen consumed by the brain, and
this may reflect the large amount of energy needed to maintain an
adequate supply of these essential transmitters. Thus although
cerebral glutamate first became well known because of its effects on
the oxygen consumption and the sodium and potassium contents of the
brain, these are probably secondary manifestations of transmitter
functions. Glutamate is also involved in the synthesis and breakdown
of cerebral proteins (Krnjevic, 1970)
The level of 30 ninhydrin reactive compounds was studied in the
brain of chick embryos (8, 14, 19, 20 and 21 days old) and chicks
(1, 4, 8, 15, 30 days old and adult). Only glycerophosphoethanolamine,
glutathione (GSH), aspartate, glutamine, glutamate and gamma-
aminobutyric acid (GABA) increase during development. In later stages
only the levels of GSH, aspartate, glutamate, glutamine and GABA
remain as high as on the first day after hatching (Levi & Morisi,
1971).
Of the free amino acids found in extracts of cat spinal roots,
dorsal root ganglia and peripheral nerves, glutamate was present in
disproportionally high concentrations in the parts of the dorsal roots
between ganglia and spinal cord (Duggan & Johnston, 1970; Johnson &
Aprison, 1970).
The distribution of glutamate and total free amino acids in 13
specific regions of the cat was investigated. The highest levels of
free glutamate were found in different cortical areas and in the
candate nucleus. No such gradient was noted for total free amino acids
in the same grey areas. The glutamate and total free amino acid
content of the central grey tissues was always higher than that of the
central white areas (Johnson & Aprison, 1971).
The metabolism of glutamic acid in the brain is dependent on
glucose and oxygen. By formation of glutamine it may influence ammonia
levels in the brain. Most important is the formation of gamma-
aminobutyric acid (GABA) by decarboxylation, a reaction which is
dependent on pyridoxal phosphate, the coenzyme of glutamic acid
decarboxylase (Perrault & Dry, 1961).
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). 2% intra-arterial sodium glutamate increased
epileptic fits and 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 & Frankel, 1951; Perrault & Dry, 1961).
Rat
An experiment was conducted feeding 45 male and 45 female rats
with 1 g/kg or 250 mg/kg MSG daily from day 1 to 90 days of age at
which time the animals were killed. Comparable control group received
laboratory chow only over the same period of time. General clinical
observations, body weights, haematologic parameters and other
clinical chemical measurements were within the normal. At autopsy,
organ weights were within the normal range. Histochemical and
ultrastructural studies of hypothalamus and median eminence showed no
evidence of repair or replacement of neuronal cells by elements of
glial or ependymal cells (Golberg, 1973).
Guinea-pig
A single subcutaneous injection of 1 g/kg MSG was given to two or
three-day old guinea-pigs. Six animals were killed three 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, vacualization of cell in
the arcuate nuclei and some evidence of cell necrosis was 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).
Monkey
In a follow-up experiment, doses of 1 g or 250 mg/kg MSG daily
for 30 days were administered orally to two groups of three infant
rhesus monkeys starting at one day after birth. General clinical
observations over a period of 30 days revealed normal growth,
development and activity. No changes were observed also in the levels
of haemoglobin haematocrit, RBC, WBC count or reticulocytes. The
levels of glucose, urea nitrogen, serum potassium, calcium and sodium
were within the normal range. 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).
Acute toxicity
LD50
Animal Route (mg/kg bw) References
Mouse i.p. 6900 Yanagisawa et al.,
1961
p.o. 12 961 Izeki, 1964
p.o. 16200 (14 200-18 400) Ichimura & Kirimura,
1968
p.o. 19 200 (22 840- 16130) Pinto-Scognamiglio
et al., 1972
Mouse i.v. 30 000 Ajinomoto Co., 1970
Rat p.o. 19 900 (L MSG) International Minerals
& Chem. Corp., 1969
p.o. 10 000 (DL MSG) International Minerals
& Chem. Corp., 1969
p.o. > 30 000 (L-GA) International Minerals
& Chem. Corp., 1969
p.o. 16 600 (18 900-14 500) Pinto-Scognamiglio
et al., 1972
Guinea-pig i.p. 15 000 Ajinomoto Co., 1970
Rabbit p.o. > 2300 (L-GA) International Minerals
& Chem. Corp., 1969
Cat s.c. 8000 Ajinomoto Co., 1970
Mouse
Mice aged two to nine days were killed 1-48 hours after single
subcutaneous injection of monosodium glutamate at doses from
0.5-4 g/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 intra-
cellular oedema and neuronal necrosis. Adult mice given subcutaneously
5-7 g/kg monosodium L-glutamate showed similar lesions (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
loads of monosodium glutamate at 0.5, 0.75, 1.0 and 2.0 g/kg bw by
gavage. Ten were controls and 54 mice received other amounts. After
three to six 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 both by light and electron
microscopy to s.c. 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 bw. The effect was additive with aspartate (Olney,
1970b).
High s.c. doses (4 or 1 g/kg of MSG caused hypothalamic changes
in 60% and 42% respectively of treated five to seven day old mice.
Oral administration (1 or 4 g/kg) of a 4% aqueous solution elicited a
predominantly glial reaction to 26-28% of the mice. The remainder were
unaffected. Four monkeys receiving 4 g/kg (4% solution) s.c. or orally
at four days of age showed no difference in appearance of the arcuate,
median eminence or ependymal cells when compared with controls
(Abraham et al., 1971).
Six, nine to ten day old mice, dosed orally with 10% monosodium
glutamate (2 g/kg), showed characteristic brain lesions (Geil, 1970).
Monosodium (i.p., 3.2 g/kg bw) 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 10-11 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 five day old animals (Freedman & Potts, 1963).
Degeneration of neonatal mouse retina has been reported following
parenteral administration of MSG (10 s.c. injections 2.2-4.2 g/kg,
1-10 days after birth) (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).
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 first MSG injection, started 24 hours
after birth, was 2 g/kg bw. Subsequent daily doses were 2.25,
2.5-4.25 g/kg through the tenth day. When the surviving MSG treated
and control mice attained 20-28 g bw, 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).
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 three or 12 days of age and to dogs
at three 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 mice (CD-l) (three to 10 days
old) were given single subcutaneous injection of MSG in concentrations
equal to 2 and 4 g/kg (0.1 ml in distilled water). Another group
of 50 adult mice (CD-1) were injected either subcutaneously or
intraperitoneally with MSG in doses varying from 6 to 10 g/kg
(1 ml volume). Controls were injected with sodium chloride. Mice were
sacrificed between three and 72 hours post-dosing. Brain tissue was
examined by light and electron microscopy. 95% of the animals injected
with MSG developed brain lesions in the arcuate nucleus of the
hypothalamus. Lesions involved primarily microglial cells with no
effect to the perikarya of neurons. Distal neuronal processes were
only slightly affected (Arees & Mayer, 1971).
Thirteen neonate mice (CF # I-JCL) received a single subcutaneous
injection of 1 g/kg of MSG a day two and four after birth. Brains were
removed one, three, six and 24 hours post-injection and examined by
light microscopy. The common finding after three and six 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 day 17 and 18 of pregnancy, examination
of fetal brain three, six and 24 hours after treatment of mother
showed cellular necrosis in both the ventromedial and arcuate nuclei
(Murakami & Inouye, 1971). Groups each of five three-day old mice
C57BL/J6 strain, were given either a single intragastric or single
subcutaneous dose of monosodium glutamate, sodium chloride, sodium
gluconate, potassium glutamate (all 10% solutions, 10 ml/kg bw) or
water. Groups of 12-day-old mice were also treated in a similar
manner. All animals were killed 24 hours after dosing. Microscopic
examination of the brain, particularly the ventral hypothalamus did
not show any neuronal necrosis of the hypothalamic arcuate nuclei
(Oser et al., 1973).
Groups each of 10 10-day-old Webster Swiss albino mice were given
a single subcutaneous dose of one of 24 compounds structurally related
to MSG. The dose level was either 12 or 24 m moles/kg bw. Five hours
post-dosing 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 neurones. The most
potent neurotoxic compounds were those known to be powerful
neuroexcitants (N-methyl DL-aspartic and DL-homocysteic acids) (Olney
et al., 1971).
Groups each of 15 Webster Swiss albino mice 10-12 days old were
given a single oral dose of MSG at a level of 0.25, 0.5, 0.75, 1.0 and
2.0 g/kg. Groups of two and four mice of the same age were given a
single oral dose of either L-glutamic acid, monosodium-L-aspartate,
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, L-cysteine at a dose level of 3 g/kg.
The post-dosing animals were sacrificed and brains examined by either
light or electron microscopy. The severity of brain damage was
estimated by quantifying the pathological changes in the hypothalamus.
1 g/kg dose of glutamic acid destroyed approximately the same number
of hypothalamic neurones 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).
MSG was injected subcutaneously into four-day-old mice at a dose
level of 2 g/µg bw, administered as an aqueous solution. Mice were
sacrificed at 7.5, 15 and 30 minutes and one, three, six, nine, 12 and
18 hours post-injection. Brains were disected and glutamate levels
determined in arcuate nucleus, ventromedial hypothalamus and lateral
thalamus. Blood levels of glutamate were also determined at these time
intervals. Glutamate levels in the arcuate nucleus reached a maximum
three hours post-dosing (111 m mole/kg dry weight). The highest levels
were reached in the ventromedial hypothalamus and lateral thalamus
about nine hours post-dosing and were about 42 m mole/kg dry weight.
Glutamate levels in the three brain regions returned to control values
(about 25 m mole/kg dry weight) about 12-15 hours post-dosing. Blood
glutamate levels reached a maximum (40 mM) within 15 minutes from a
basal level of 0.12 mM but returned to baseline values (below 1 mM) in
the one to three hour interval after injection (Perez & Olney, 1972).
Rat
Groups each of 20 10-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 g/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 five hours.
Histological studies were made of brain 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 brain of
animals sacrificed at either five hours post-treatment or after
reaching maturity. Animals which were reared to maturity showed normal
growth and food consumption (Geil, 1970).
Male and female rats of the Wistar strain, three to four days
old, received a single subcutaneous injection of MSG. The total dose
was 4 g/kg bw 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 three hours
after the injection of MSG. Light microscopic and electron microscopic
examination failed to reveal any effects upon the lateral pre-optic
nucleus, arcuate nucleus, and median eminence. To determine long-range
effects of MSG, uniform litters (eight 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 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 & Ratner injected a 40% solution of MSG instead
of the 10% solution used by Olney et al. (Olney, 1971).
Weanling Sprague-Dawley male rats were given MSG
intraperitoneally 20 mmol = 3.4 g/kg bw. Marked somnolence was
observed within five to 20 minutes. About 1/3 to 1/2 of the animals
salivated copiously and had myocolonic jerking about one hour after
injection, sometimes followed by vigorous running about the cage and
sterotyped biting (Bhagavan et al., 1971).
At birth rat brain glutamate 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. MSG given intraperitoneally
always caused convulsions with 2 g/kg. When young rats were given
large doses of MSG, monosodium aspartate or glycine at 4 g/kg, the
glutamine level was increased significantly in the brain by all amino
acids, but only monosodium glutamate and aspartate caused convulsions.
D-glutamate (4 g/kg) which is not deaminated by the rat also causes
convulsions. These results suggest that the convulsions of MSG are not
due to liberated ammonia but rather to the amino acid anions. At
4 g/kg MSG gave rise to serum concentrations of glutamate of about
70 mM, strongly suggesting osmotic problems (Mushahwar & Koeppe,
1971).
Fifty weanling rats (FDRL strain), three days of age, were
divided into five groups. Each group was subdivided and the test
animals given either a single intragastric or single subcutaneous dose
of monosodium glutamate, sodium chloride, sodium gluconate, potassium
glutamate (all 10% solutions, 10 ml/kg bw), 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 (three days of age) was divided into groups and treated
with the same test compounds at the same dose levels. Half of each
group was sacrificed at six hours and the other half at 24 hours after
dosing. Microscopic examination of brain, particularly the ventral
hypothalamus did not show neuronal necrosis of the hypothalamic
arcuate nuclei, except in one rat dosed with monosodium glutamate
1 g/kg at three days of age, and killed 24 hours later showed an area
in the median eminence which contained cells with slight nuclear
pyknosis and prominent vacuolation (Oser et al., 1973).
MSG was administered at dose levels of 1 g/µg to infant mice and
2 and 4 g/µg 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).
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 and 5 g/kg) by stomach tube daily from day 5 to 10 of age.
At day 21 rats were placed in separate cages and at three months of
age were subjected to three different behavioural situations, namely,
spontaneous motor activity, T-maze and fixed-ratio food reinforcement.
Rats of the high dose level showed less spontaneous motor activity and
deficiency in discrimination learning in the T-maze study. However,
learning of a fixed ratio food reinforcement schedule was not
affected* (Pradham & Lynch, 1972).
Rabbit
I.v. injection of sodium L-glutamate produced a prolonged
increase in the amino-nitrogen content of blood and prolonged urinary
excretion in rabbits (Yamamura, 1960).
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 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).
Dog
Intravenous casein hydrolysate or synthetic amino acid mixture
caused nausea and vomiting in dogs (Madden et al., 1944). Groups of
three dogs at three 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 three 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., 1971).
Groups each of six pups, three to four days of age were dosed
either orally or subcutaneously with monosodium glutamate, sodium
chloride, sodium gluconate, potassium glutamate (1 g/kg bw) or water.
Pups were sacrificed at either three hours, 24 hours or 52 weeks after
dosing. Other groups of dogs 35 days old received a single dose of
test material, and were sacrificed at either four hours or 24 hours
* After three months the treated animals had a weight gain which
was less than controls, the gain being less in the 5 g/kg dose group.
post-dosing. Body weights of dogs which were dosed once at three days
of age and followed for a year, showed no evidence of effect of any
treatment. Femur weight, as well as weight of pituitary gland,
ovaries, uterus and mammary glands were similar to controls. Gross and
microscopic examination 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).
Monkey
A newborn (eight hours old) rhesus infant, probably somewhat
premature was given subcutaneously 2.7 g/kg bw monosodium glutamate.
After three 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, four
days 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 three, 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, five days of age, received MSG by stomach
tubes at a dose of 2 g/kg. Two infant monkeys at 10 days of age,
two at 20 days of age and two at 40 days of age received the same
treatment. Two animals at 80 days of age received 4 g/kg. The animals
were observed for four hours after dosing and then sacrificed. After a
period of fixation a block of tissue was removed from each brain to
include the hypothalamus. Serial sections, 10 mm thick were made in
the horizontal plane and examined by light microscopy. No changes were
observed in the hypothalamus of the monkeys that were considered to be
associated with the administration of MSG. One control monkey was
included in each group (Huntington, 1971).
Two infant monkeys at two days of age were given monosodium
glutamate by intragastric incubation at the dose level of 2 g/kg and
two infant monkeys of the same ages were used as control. The brains
were studied by electron microscopy. No morphological differences were
observed between treated and control monkeys. The changes observed
appeared to be due to fixation artifact and cannot be attributed to
monosodium glutamate (Huntington, 1971).
A group of three to four day old cynomolgus monkeys received
either subcutaneously or orally a single dose of monosodium glutamate,
sodium chloride, sodium gluconate, potassium glutamate (1 g/kg bw) and
were sacrificed three or 24 hours post-dosing. Another group of
monkeys (three to four days old) received orally either 4 g/kg
monosodium glutamate or sodium chloride, and were sacrificed at three,
six and 24 hours post-dosing (three and 24 hours in case of sodium
chloride dosed monkeys). Detailed microscopic examination of the
hypothalamus did not show any features of monosodium glutamate induced
necrosis or any differences between any of the groups. Examination of
the eyes did not reveal any monosodium glutamate effect. Glutamate and
glutamine blood levels of monkeys showed considerable variation in
individual values between oral and subcutaneous doses. Subcutaneous
dosing resulted in values of an order of magnitude higher than those
observed by oral dosing (Oser et al., 1973).
Monosodium glutamate was administered to six pregnant rhesus
monkeys (Macaca mulatta) at a daily dosage equivalent to 4 g/kg bw
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 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 weight of neonates were within the normal
range. Infants, when removed from mothers, showed distress but no
signs of abnormal behaviour. About four hours after birth, infant
monkeys were sacrificed. The hypothalamus region and related structure
of the brain was 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 two monkeys aged two days. Two monkeys
of similar age were used for control. Four hours after dosing, 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 with the administration of the
test compound. Changes observed by electron microscopy occurred as
frequently in control animals as in test animals, and appear to be due
to fixation artifacts (Heywood et al., 1972b).
Rhesus monkeys (Macaca mulatta) aged between four and 80 days
were divided into groups by age. Each group contained three test and
one control animal. Dosage was 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 four 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 glutamic pyruvic transaminase
and glutamic oxalacetic transaminase. SGPT and SGOT values did not
show any significant increase over test period. Plasma glutamic acid
was within the normal range. Liver GPT and GOT were within the normal
range. Examination of 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 four hours before receiving by stomach tube a single dose of 50%
solution of monosodium glutamate, equivalent to a dose of 1, 2 or
4 g/kg bw. Control animals received distilled water. At six 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 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 approximately 4 g/kg bw. A total of seven animals were treated.
At time periods of two to six hours post-dosing the fetus was
delivered by Caesarean section and the brain fixed for microscopic
examination. The hypothalamic areas of the brain from all seven
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% and
17% (based on dry weight) MSG formula diet for nine weeks. Three of
the test monkeys died. Two died of effects not related to MSG. The
third on the 17% diet developed convulsive seizures. However, the
other two 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. In another study an
infant cynomologus monkey and an infant brush monkey were fed 9.1% MSG
formula for one year. Daily observations revealed no behavioural
abnormalities. Their weight gains, ERG, EEG and plasma amino acid were
similar to controls not consuming MSG. No evidence of gross obesity
developed (Wen et al., 1973).
Groups of three infant monkeys were dosed with a mixture of water
and skim 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 tested ranged from 1-4 g/kg bw.
All animals were sacrificed post-dosing and 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 five hours) was observed. Measurements
of blood glutamate levels, suggested that the threshold for lesion
formation in one week old rhesus monkeys may be in the range of
200 mg/l (Olney et al., 1972).
Short-term studies
Mouse
Thirty-eight neonate mice were observed for nine months. Twenty
received subcutaneous monosodium glutamate daily for one to 10 days
after birth in doses of 2.2-4.2 g/kg. Eighteen 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.
Ten test neonates received a single subcutaneous injection of
3 g/kg monosodium glutamate two days after birth, 13 neonates were
controls. Again test animals were heavier than controls after nine
months but less so than mice given repeated injection treatment. For
the latter 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 bw were given orally to groups of five 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).
Male albino rats (Sprague-Dawley) were allowed water
ad libitum and fed ground laboratory chow, 24% protein content. High
levels of single amino acids in the diet of rats beginning at 21 days
produced a decreased food intake and a severe growth depression. These
effects were dependent upon the kind and the concentration of amino
acid supplemented. L-methionine caused the most severe growth
depression while L-phenylalanine, L-tryptophan and L-cystine 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 a depressed food intake and to a specific toxic effect of the amino
acid. A direct correlation was found between the toxicity of any
dietary amino acid and its concentration in the blood (Daniel &
Waisman, 1968).
Nine groups of 20 rats were given 0.5% and 6% 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 trials was 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
Mouse
One control group of 200 male mice and six test groups of 100
male mice received 0%, 1% or 4% in their diet of either L-glutamic
acid, monosodium L-glutamate or DL-monosodium glutamate. No malignant
tumours appeared after two 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 two years
dietary levels of 0, 0.1% or 0.4% 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).
OBSERVATIONS IN MAN
Pharmacological effects were studied in 56 normal subjects
(30 male and 26 female) after oral administration of L-glutamate on an
empty stomach. Symptoms occurred in all but one subject. Three
categories of symptoms could be elicited by monosodium glutamate:
burning, facial and chest pressure. Headaches were a consistent
complaint of a minority of individuals. Oral administration of
monosodium glutamate caused symptoms after 15-25 minutes. The oral
threshold range for minimum symptoms in 36 subjects was from 1.5-12 g
(Schaumburg et al., 1969).
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 five persons who had not experienced any symptoms, and
nine 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., 1944).
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 eight hours for three to five days in dextrose and infused
at a rate of not more than 25 g of arginine glutamate over one to two
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). Ninety-eight received 5 g of monosodium
glutamate in single blind studies, six received 8 g and five received
12 g in double blind studies. Physical examinations were done on all
subjects. Complaints were registered in all groups ranging from
23-80%. 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 (Rosenblum et al., 1971),
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.
One hundred and fifty 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% showed improvement of behavioural traits (Zimmerman &
Burgemeister, 1959).
Seventeen 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). Fifteen
grams then 30 g monosodium glutamate were given per dose for one week
each, followed by 48 g for 12 weeks to 53 patients without any effect
on basal plasma levels of glutamic acid (Himwich et al., 1954b).
DL-glutamic acid HCl was given in doses of 12, 16 and 20 g to
eight patients with petit mal and psychomotor epilepsy without
adverse effects (Price et al., 1943). Five episodes of hepatic coma in
three patients were treated with i.v. 23 g of monosodium glutamate
with improvement (Walshe, 1953). Ten to 12 g of L-glutamic acid given
to epileptics and mental defectives appeared to improve nine out of
20 cases (Waelsch, 1949).
In a study with double blind and crossover techniques, MSG was
administered to 24 healthy subjects aged 18 to 24 years, MSG was given
"at lunch time in doses of 3 g/subject in 150 ml of beef broth". No
differences in subjective symptoms were observed between the subjects
given MSG and those who had normal beef broth. Nobody, either in the
control or in the MSG group, experienced the burning feeling which is
typical of the Chinese restaurant syndrome (Morselli & Garattini,
1970).
Six women with well established lactation patterns were fasted
overnight and received a single oral dose of 6 g monosodium glutamate
in water and liquid diet formulation and four controls received
lactose. Milk samples were obtained one, two, three, four, six and
12 hours after administration. Blood samples were taken 0, 30, 60, 120
and 180 minutes after administration of MSG in water, and 0, 60, 90,
150 and 210 minutes after administration of MSG in a liquid diet
formulation or the lactose. Small increases of plasma glutamate
aspartate and alanine levels were noted. Little change was observed in
breast milk amino acid levels (Stegink et al., 1972).
One hundred and twelve human subjects in an unselected population
received doses of MSG ranging from 1 to 20 g dissolved in water. 32%
of the persons tested responded at the 5 g level when challenged by a
single placebo controlled exposure. The subjects showed dose-response
relationship, particularly marked in the case of stiffness and
tightness symptoms and somewhat less marked in the case of pressure,
warmth, and tingling sensation which exhibited a clear threshold for
their appearance at the 2 to 3 g level (Kenney & Tidball, 1972).
Comments:
Glutamic acid is a component of proteins and comprises some 20%
of ingested protein. During gastrointestinal absorption transamination
to alanine occurs, consequently there is only a slight rise in
glutamate levels in the portal blood of animals and probably also of
man. However, if the capacity of this mechanism and the further
conversion of glutamate in the liver is overwhelmed, or if a glutamate
is administered parenterally in large doses, it is possible to obtain
high blood levels. It has been demonstrated that, at least in the rat
and rhesus monkey, glutamate does not readily cross the placental
barrier or the blood-brain barrier, nor is it secreted in human
mother's milk.
Numerous reproduction studies in mice, rats, rabbits and monkeys,
revealed no deleterious effects on the offspring if tho parent
generation was fed glutamate in high doses, suggesting that the fetus
or young infant are not exposed to high levels of glutamate through
the mother's diet. The earlier claim of teratogenic effects in the
rabbit was probably not related to glutamate administration.
Some investigations have demonstrated a vulnerability of the
developing mouse, rat or primate CNS to high levels of glutamate in
addition to other amino acids following subcutaneous administration.
Neonatal mice are relatively susceptible to the development of a
highly localized lesion in the arcuate nuclei of the hypothalamus with
incomplete myelination. Oral administration of high doses failed to
reproduce these lesions in neonatal rats, guinea-pigs or monkeys,
which had been reported by previous investigators.
Acute reactions reported after ingestion of glutamate are
probably due to the rapid absorption of large amounts of the substance
(greater than 3 g): this syndrome has been termed Kwok's disease.
On the data provided it is possible to arrive at an acceptable
daily intake for glutamate and its salts making allowance for the fact
that glutamate is a normal constituent of protein and free glutamate
also occurs in the diet. 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 to avoid adding glutamate to
foods intended for infants under 12 weeks of age. Neither the fetus
nor the suckling infant are at risk from maternal ingestion of
glutamate. There is no reason to suspect that the potassium, sodium,
calcium and ammonium salts exert effects other than those due to the
respective cations.
EVALUATION
Estimate of acceptable daily intake for man
0-120* ag/kg bw.
FURTHER WORK REQUIRED IF USE IS TO BE EXTENDED TO INFANT FOODS
1. Oral non-adverse effects levels of glutamates in neonatal
animals.
2. Age correlations between neonatal experimental animals and the
human infant.
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See Also:
Toxicological Abbreviations