DISODIUM 5'-GUANYLATE AND DISODIUM 5'-INOSINATE
First draft prepared by
Dr K. Ekelman and Dr K. C. Raffaele, Additives Evaluation Branch
Division of Health Effects Evaluation
Center for Food Safety and Applied Nutrition
Food and Drug Administration, Washington, DC, USA
1. EXPLANATION
The disodium salts of 5'-guanylic acid and 5'-inosinic acid
were previously evaluated at the eighteenth meeting of the
Committee, when an ADI "not specified" was allocated (Annex 1,
reference 35). At that time, metabolism, teratogenicity, and acute,
short-term and long-term toxicity of the two substances, as well as
data on the reproductive toxicity of inosinic acid and its calcium
and sodium salts, were reviewed. The present Committee reviewed
these compounds together, as many of the new toxicological studies
had been performed on mixtures of the two salts. Data from the
earlier reviews have been incorporated into this consolidated
monograph.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
Disodium 5'-guanylate
Male and pregnant female (day 10 or 18 of gestation) rats were
given 25 mg/kg bw of 8-[14C] disodium 5'-guanylate by gavage.
Plasma radioactivity reached a maximum 30 minutes after ingestion
and decreased abruptly to near zero within 24 hours; t´ was about
one hour. Twenty-four hours after exposure, about 84% of total
activity appeared in urine, 0.2% in faeces, none in expired air,
between 0-0.6% remained in organs; about 12% of total activity
remained in organ-free carcass of males and pregnant females (18th
day of gestation) 24 hours after treatment. Fetuses contained about
0.01% of activity 24 hours after treatment (Ohara et al., 1973).
Disodium 5'-inosinate
Male and pregnant female (day 10 or 18 of gestation) rats were
given by gavage 25 mg/kg bw of 8-14C labelled disodium
5'-inosinate. Radioactivity in plasma reached maximum levels ´ to 2
hours after treatment and slowly decreased to practically zero after
24 hours; t´ was about 5 hours. About 70% of total activity
appeared in the urine, 6-7% in faeces, none in expired air, 0-2% in
organs, and 8-17% in the organ-free carcass of males and pregnant
females (18th day of gestation) 24 hours after treatment. Fetuses
contained about 0.77% of total activity 24 hours after treatment
(Ohara et al., 1973).
2.1.2 Biotransformation
Disodium 5'-guanylate
The greater portion of disodium 5'-guanylate in the body is
derived from de novo purine bio-synthesis and the rest is derived
from pre-formed dietary purines. Biosynthesized disodium
5'-inosinate is the precursor of disodium 5'-guanylate, to which it
is converted by oxidation to xanthosine-5-mono-phosphate and
amination. Dietary or endogenous purine bases and ribonucleosides
are converted to the 5'-ribonucleotides by phosphorylation.
Disodium 5'-guanylate is dephosphorylated to guanosine,
hydrolyzed to guanine, deaminated to xanthine and oxidized to uric
acid. In most mammals, uric acid is further oxidized by the liver
enzyme urate oxidase to allantoin. Primates, including humans, lack
the enzyme, however; for them, uric acid is the main end product of
purine metabolism. In humans, 2/3 of uric acid is excreted in the
urine; the rest is further broken down in the gut. Digestion of
disodium 5'-guanylate appears to take place mainly in the duodenum:
the nucleosides are probably absorbed actively, the purines probably
diffuse passively across the intestinal wall (Kojima, 1974).
Disodium 5'-inosinate
Disodium 5'-inosinate derives from dephosphorylation of ATP
(adenosine-5-triphosphate) to AMP and deamination. Further
conversion to hypoxanthine is slow compared to the conversion of ATP
to disodium 5'-inosinate. The greater portion of disodium
5'-inosinate in living tissue is derived from de novo purine
biosynthesis, and less is derived from ingested dietary purines or
nucleotides. Disodium 5'-inosinate is the first purine formed in
the complex biosynthesis of purine nucleotides (Kojima, 1974).
2.1.3 Effects on enzymes and other biochemical parameters
Groups of five male rats (control group size was 10) were given
0%, 1%, or 4% disodium 5'-guanylate or disodium 5'-inosinate for
five or 10 days in a purine-free basal diet. Levels of uric acid in
serum and urine were not significantly affected, and most of the
ingested disodium 5'-guanylate and disodium 5'-inosinate was rapidly
excreted in urine as allantoin. However, liver hypoxanthine-guanine
phosphoribosyl transferase and adenine phosphoribosyl transferase
activities were increased and the ratio of liver uricase/xanthine
oxidase activity was increased, suggesting metabolism of ingested
disodium 5'-guanylate and disodium 5'-inosinate by shunt pathways
(Hashimoto et al., 1973).
2.2 Toxicological studies
2.2.1 Acute toxicity studies
Disodium 5'-guanylate
Results of acute toxicity studies with disodium 5'-guanylate
are summarized in Table 1.
Table 1: Acute toxicity studies with disodium 5'-guanylate
Species Sex Route LD50 Reference
(mg/kg bw)
Mouse M&F oral >10 000 Usui et al., 1971
Mouse M oral 15 000 Ichimura & Muroi, 1973
Mouse F oral 16 300 Ichimura & Muroi, 1973
Mouse M s.c. 5 050 Ichimura & Muroi, 1973
Mouse F s.c. 5 050 Ichimura & Muroi, 1973
Mouse M i.p. 6 800 Ichimura & Muroi, 1973
Mouse F i.p. 5 010 Ichimura & Muroi, 1973
Mouse M i.v. 3 580 Ichimura & Muroi, 1973
Mouse F i.v. 3 950 Ichimura & Muroi, 1973
Mouse M i.v. 3800 Shimamoto et al., 1974
Rat M&F oral >10 000 Usui et al., 1971
Rat M oral 17 300 Ichimura & Muroi, 1973
Rat F oral 17 300 Ichimura & Muroi, 1973
Rat M s.c. 3 550 Ichimura & Muroi, 1973
Rat F s.c. 3 400 Ichimura & Muroi, 1973
Rat M i.p. 4 750 Ichimura & Muroi, 1973
Rat F i.p. 3 880 Ichimura & Muroi, 1973
Rat M i.v. 2 720 Ichimura & Muroi, 1973
Rat F i.v. 2 850 Ichimura & Muroi, 1973
A series of experiments was performed to assess a variety of
acute effects of disodium 5'-guanylate. In mice, an i.v. dose of
500 mg/kg bw disodium 5'-guanylate produced abdominal postures,
slight respiratory depression and slight depression of avoidance
response to mechanical stimuli at 15 min (response had returned to
normal at 30 min). There was no muscular relaxation (defined as
failure to remain on a 2 mm diameter metal bar for 30 sec, measured
at 10 and 30 minutes after dosing) but spontaneous revolutions
(during placement in a rotating cage) were decreased (p<.05 vs.
control) at one hour and decreased (not statistically significant)
at 2 hr. There was no modification of electroshock convulsion after
an i.v. dose of 500 mg/kg bw, but there was a dose-dependent
decrease of the dose of metrazol required to produce convulsions
when infusion of metrazol was started immediately after i.v.
injection of 200 or 500 mg/kg bw disodium 5'-guanylate (this
decrease was significant only at 500 mg/kg bw). Doses of 125 mg/kg
bw and 500 mg/kg bw disodium 5'-guanylate, i.v., also caused
prolongation of the time during which there was loss of righting
reflex following methylhexabital anaesthesia (methylhexabital was
injected i.p. 10 min after dosing; the time of measurement was not
stated), but there was no effect on this response at 50 mg/kg bw
disodium 5'-guanylate. Oral administration of 500 mg/kg bw disodium
5'-guanylate to mice did not affect their analgesic response to
thermal stimuli (measured as pain threshold in seconds [the method
was not described but was presumably the latency of paw removal from
a hot plate]). Compared to controls, 100 mg/kg bw s.c. disodium
5'-guanylate in mice depressed salivary secretion following
stimulation by pilocarpine. Pilocarpine was administered 35 min
after disodium 5'-guanylate; secretion was measured for 20 minutes
starting immediately after pilocarpine injection. There was no
effect of an oral dose of 500 mg/kg bw disodium 5'-guanylate on
carrageenin-induced oedema in rats. Administration of 100 mg/kg bw
disodium 5'-guanylate s.c. to rats did not affect the secretion of
gastric juices but slightly increased their pH and depressed their
total acidity. Intragastric administration of 25 mg/kg bw disodium
5'-guanylate had no diuretic effect in rats. Concentrations of
disodium 5'-guanylate below 10-4g/ml had no effect on the
contractile response of isolated guinea-pig ilium to acetylcholine,
histamine, or barium chloride (Shimamoto et al., 1974).
Topical disodium 5'-guanylate (applied to the tongue acutely at
a concentration of 0.01% disodium 5'-guanylate with 0.3% MSG)
enhanced the electrical response of the chorda tympani to topical
MSG in the rat (Sato et al., 1971).
Disodium 5'-guanylate administered i.p. had no marked effect on
the conditioned avoidance response of the rat. Parenteral disodium
5'-guanylate produced transient changes in the electroencephalogram
(EEG) of rabbits (Hirayama, 1968).
Intravenous disodium 5'-guanylate had no significant effect on
the blood pressure, heart-rate, or electrocardiogram (ECG) of the
anaesthetized rabbit (Yabo, 1964).
Rapid intracarotid injection of disodium 5'-guanylate increased
the cerebral blood flow but did not affect oxygen consumption or
glucose uptake in the perfused cat brain (Otsuki et al., 1968).
Anaesthetized cats were exposed to disodium 5'-guanylate via a
tube inserted into the femoral vein or a tube inserted into the
medial sacral artery. Acute infusions of 10, 25, or 50 mg/kg bw
disodium 5'-guanylate caused transient, slight, dose-dependent
hypotension that was associated with tachycardia, and increased
blood flow to the hind limbs. Intra-arterial infusion of disodium
5'-guanylate at doses greater than 10 mg/kg caused transient
increases in blood flow to the hind limbs. There was no change in
ECG pattern with either method of dosing.
In isolated guinea-pig atria, doses of disodium 5'-guanylate up
to 10-5 g/ml did not affect contractile height or atrial rhythm;
disodium 5'-guanylate at 10-4 g/ml increased the rate and strength
of contractions; 10-3 g/ml further increased the rate and slightly
decreased the strength of contractions (Shimamoto et al., 1974).
Disodium 5'-inosinate
Results of acute toxicity studies with disodium 5'-inosinate
are summarized in Table 2.
A series of experiments was performed by Shimamoto and
coworkers to assess a variety of acute effects of disodium
5'-inosinate. In mice, i.v. administration of 500 mg/kg bw disodium
5'-inosinate caused behavioural excitement, increased reflex
response, no muscular relaxation (measured by the ability to remain
on a horizontal metal bar for 30 sec or by the ability to remain on
a screen inclined at 60°) and depressed rotating activity (measured
as the number of spontaneous rotations during placement in a
rotating cage) during the first hour following administration of
disodium 5'-inosinate. Administration of 500 mg/kg bw i.v. did not
modify electroshock convulsions in mice; however, disodium
5'-inosinate doses of 100 and 500 mg/kg bw decreased the dose of
metrazol that produced continuous convulsions. Administration of
50-500 mg disodium 5'-inosinate/kg bw i.v. to mice prolonged loss of
the righting reflex after anaesthesia with methylhexabital.
Administration of 10-50 mg disodium 5'-inosinate/kg bw i.v. to cats
had no effect on blood pressure, heart rate, ECG, or blood flow to
the hind limbs. Incubation with medium containing 10-5 g/ml
disodium 5'-inosinate did not affect the height and rhythm of
spontaneous movements of isolated guinea-pig atria; incubation with
10-3 g/ml disodium 5'-inosinate, however, depressed contractile
height by 16.5% and slightly increased the rate of contractions
(Shimamoto et al., 1974).
A 1:4 000 dilution of disodium 5'-inosinate first increased
motility and then decreased tone of an immersed, isolated guinea pig
uterus (Floessner, 1934).
Topical disodium 5'-inosinate enhanced the electrical response
of the chordotympani to topical MSG in rats and cats (Adachi, 1964;
Sato et al., 1965).
A 1% solution of disodium 5'-inosinate decreased the mobility
of isolated guinea-pig intestine (Hara et al., 1966).
In a series of studies, Shimamoto and coworkers reported that:
1) administration of 50 and 100 mg/kg bw disodium 5'-inosinate s.c.
to mice decreased the pilocarpine-induced increase in salivary
secretion; 2) administration of 100 mg/kg bw disodium 5'-inosinate
s.c. to mice had no effect on charcoal transport in the small
intestine; 3) incubation with medium containing 10-3 g/ml disodium
5'-inosinate did not affect the contractile response of isolated
guinea-pig ileum to acetylcholine, histamine, or barium chloride;
4) oral administration of 500 mg/kg bw disodium 5'-inosinate had no
effect on analgesic response to thermal stimuli in mice or
carrageenin-induced oedema in rats; 5) administration of 100 mg/kg
bw disodium 5'-inosinate s.c. had no effect on gastric juice volume
in the rat but slightly increased gastric pH; and 6) rats given 100
mg/kg bw intragastric disodium 5'-inosinate showed no diuresis
(Shimamoto et al., 1974).
Disodium 5'-inosinate caused no changes in renal function or
renal venous renin after arterial infusion at 50-500 ug/min in
anaesthetized, sodium-depleted dogs (Tagawa and Vander, 1970).
Disodium 5'-inosinate injected i.v. in anaesthetized rabbit had
no effect on blood pressure or respiration at 5-10 mg/kg bw, but 50
mg/kg bw and above caused a transient decrease in blood pressure and
potentiation of respiration (Hara, 1966).
Disodium 5'-inosinate injected i.v. caused hypotension in the
rabbit at 0.2 mg/kg bw and in the dog at 0.35 mg/kg bw (Floessner,
1934).
Pharmacological studies on disodium 5'-inosinate showed no
effect on the S-A or A-V nodes in the Langendorff preparation of rat
heart (Versprille, 1966).
Table 2: Acute toxicity studies with disodium 5'-inosinate
Species Sex Route LD50 (mg/kg bw) Reference
Mouse M&F oral >10 000 Usui et al., 1971
Mouse ? oral 12 000-14 000 Hara et al., 1966
Mouse M oral 17 600 Ichimura & Muroi, 1973
Mouse F oral 19 800 Ichimura & Muroi, 1973
Mouse F oral >20 000 Merck petition
Mouse ? s.c. 6 200-7 000 Hara et al., 1966
Mouse M s.c. 5 480 Ichimura & Muroi, 1973
Mouse F s.c. 5 630 Ichimura & Muroi, 1973
Mouse ? i.p. 5 400-5 600 Hara et al., 1966
Mouse M i.p. 6 300 Ichimura & Muroi, 1973
Mouse F i.p. 6 200 Ichimura & Muroi, 1973
Mouse ? i.v. 3 300-3 900 Hara et al., 1966
Mouse M i.v. 3 950 Ichimura & Muroi, 1973
Mouse F i.v. 4 600 Ichimura & Muroi, 1974
Mouse M i.v. 4 400 Shimamoto et al., 1974
Rat M&F oral >10 000 Usui et al., 1971
Rat M oral 17 100 Ichimura & Muroi, 1973
Rat F oral 15 900 Ichimura & Muroi, 1973
Rat M s.c. 3 900 Ichimura & Muroi, 1973
Rat F s.c. 4 340 Ichimura & Muroi, 1973
Rat M i.p. 5 400 Ichimura & Muroi, 1973
Rat F i.p. 4 850 Ichimura & Muroi, 1973
Rat M i.v. 2 730 Ichimura & Muroi, 1973
Rat F i.v. 2 870 Ichimura & Muroi, 1973
2.2.2 Short-term toxicity studies
2.2.2.1 Rats
Disodium 5'-guanylate
Groups of 10 male rats were given 0%, 0.1%, or 1% disodium
5'-guanylate in their diet daily for three and six months. The
authors stated that no significant abnormalities were noted as
regards spontaneous behaviour, body weight gain, food intake,
haematology, urinalysis, and macroscopic and histological
examination. However, data in tables included only a few subjects
in each group so the authors' conclusions could not be confirmed
(Usui et al., 1971).
Disodium 5'-inosinate
Groups of 10 male rats were fed diets containing either 0, 10,
100 or 1 000 mg/kg bw/dy naturally derived or synthetically prepared
disodium 5'-inosinate for 90 days. No adverse effects were noted on
weight gain, organ weights, haematological parameters or
histopathology (Hara et al., 1966).
Eight male and 8 female Sprague-Dawley rats were fed diets
containing 0%, 0.5%, 1.0%, 2.0% or 4.0% disodium 5'-inosinate for 12
weeks followed by levels of 0%, 0.75%, 1.5%, 3.0%, or 6.0% disodium
5'-inosinate during weeks 13-25. No significant abnormalities in
any treatment group with regard to behaviour, body weight gain, food
intake, haematology or urinalysis were reported. Some animals in
higher dosage groups showed renal medullary calcification; relative
mean weights of kidney and spleen in the 6% disodium 5'-inosinate
groups were significantly increased.
In a second experiment, 6 male and 6 female Sprague-Dawley rats
were given 0%, 1%, 4%, or 8% disodium 5'-inosinate in the diet for
52 weeks. The only adverse effects noted were slight depression of
body weight gain in the groups fed 8% disodium 5'-inosinate.
Increased renal calcifications was seen in the 4% and 8% females;
this was probably related to urine osmolarity. The 8% males and 2%
and 8% females showed more severe nephrosis than rats in other
groups (Yonetani et al., 1973).
Groups of 10 male rats were given 0%, 0.1%, or 1% of disodium
5'-inosinate in their diet daily for three and six months. Average
daily intake of disodium 5'-inosinate was reported to be 45.8 mg/kg
bw/dy for the 0.1% group and 496.5 mg/kg bw/dy for the 1% group.
Tables in the report included data on only a few animals in each
group and did not always indicate the number of animals for which
mean values were calculated. The authors stated that no significant
abnormalities were noted in spontaneous behaviour, body weight gain,
food intake, haematology, urinalysis, or macroscopic and
histological examination. However, absolute lung weight was
decreased in both disodium 5'-inosinate-fed groups when compared to
the control group (mean lung weight at six months: 1239 for
controls; 869 for 0.1% disodium 5'-inosinate group; 879 for 1%
disodium 5'-inosinate group [units not reported]). Although the
authors attributed this change to the presence of pneumonitis
leading to increased lung weight, which was most pronounced in
control rats, this reviewer was unable to discern any relationship
between degree of pneumonitis and lung weight (Usui et al., 1971).
The authors stated that some animals were excluded from the final
tabulation of results due to pulmonary infection, but the numbers
excluded from each group were not provided.
Disodium 5'-guanylate + disodium 5'-inosinate
Male rats (group size unspecified, but apparently 10 at the
beginning of the study) were given 0.2, 0.4, 0.8, or 2% of a 50:50
mixture of disodium 5'-guanylate and disodium 5'-inosinate (50:50
mix) in the diet for 6 months. Three rats in each group died during
the course of the study due to pulmonary infection. Mean ribotide
intake was 30.8 mg/day for the 0.2% group, 63 mg/day for the 0.4%
group, 123.5 mg/day for the 0.8% group, and 308.6 mg/day for the 2%
group. There were no changes in body weight, haemoglobin,
erythrocyte, leucocyte, or haematocrit (blood parameters measured
for 5 rats/group only) for rats in any dose group compared to
control rats. There were some scattered statistically significant
changes in organ weights, which did not appear dose-related; there
was a trend toward an increase in kidney weight with dose, but this
was not statistically significant. The Committee did not consider
the statistics to be reliable, since some statistical findings
reported were anomalous.
An additional study reported by the same group included
administration of 0.8% and 4% 50:50 mix for 3 months; although some
results from the 3-month study were discussed in the report on the
6-month study, data from the 3-month study were not reported
separately (Usui et al., 1971).
2.2.2.2 Dogs
Disodium 5'-inosinate
One male and one female beagle were fed diets containing 0%,
3.6-3.9% or 8% disodium 5'-inosinate for four to six weeks without
any adverse effect (Noel et al., 1971).
Four male and four female beagles were fed diets containing 0,
0.5, 1 or 2 g/kg disodium 5'-inosinate for two years. No
significant changes were reported in body weight gain, feed
consumption or ophthalmoscopy. Haematology, biochemistry and
urinalysis were normal. Dogs fed 2 g/kg bw/day disodium
5'-inosinate had significantly increased allantoin levels in the
serum but these were not dose-related. Histopathological
examination showed no significant abnormalities (Rivett et al.,
1973).
Disodium 5'-guanylate + disodium 5'-inosinate
Preliminary studies were conducted using groups of 1 male and 1
female beagle dog/dose, with feeding of 50:50 mix in the diet at
levels of 0, 2, 5, or 10% for four weeks (6 weeks at the 10% level).
Weight gain was slightly decreased in the 10% group during the 3-6
week period. All post mortem findings, including organ weights
and histological examinations, were normal for all groups (no data
were presented for the preliminary study).
Beagle dogs, 4 animals/sex/group (4 months of age at the start
of the study) were fed diets containing 0, 0.1 (mean daily intake
30-40 mg/kg bw), 1.0 (mean daily intake 26-48 mg/kg bw), or 2.0%
(mean daily intake 51-93 mg/kg bw) 50:50 mix for 2 years. Clinical
signs and feed intake were recorded daily; water consumption was
checked at monthly intervals. Ophthalmoscopic examinations,
urinalyses, haematological examinations, and blood biochemistry
studies, including the determination of serum electrolytes,
allantoin and uric acid, were conducted before dosing and at 1, 2,
4, 6, 9, 12, 15, 18, 21, and 24 months after initiation of
treatment. Serum allantoin and uric acid levels were raised,
sometimes in a dose-related manner, at several time points during
the study: serum allantoin (mg/100 ml) was raised at 8 weeks in all
dosed groups (control=2.11; 0.1%=2.42; 1%=2.58; 2%=2.67), at 16
weeks in the .1% and 1% groups (control=2.03; 0.1%=2.67; 1%=3.23),
and at 92 weeks in all groups (control=1.82; 0.1%=2.43; 1%=3.04;
2%=2.90); serum uric acid (mg/100 ml) was raised at 26 weeks in the
1% and 2% groups (control=0.70; 1%=0.96; 2%=1.19), and at week 103
in the 1% group (control=0.51; 1%=1.00). No other findings related
to intake of test substance were noted (Worden et al., 1975).
2.2.3 Long-term toxicity/carcinogenicity studies
2.2.3.1 Rats
Disodium 5'-inosinate
Fourteen male and 14 female Sprague-Dawley rats were fed diets
containing 0%, 1%, 2%, 4%, or 8% disodium 5'-inosinate for 95 weeks.
No significant changes were seen in behaviour, body weight gain,
feed intake, haematology, blood chemistry, urinalysis,
histopathology or mortality (Yonetani et al., 1973).
Disodium 5'-guanylate + disodium 5'-inosinate
Male and female rats (group size unspecified, but it may have
been 10 animals/sex/group) were fed 50:50 mix at 0%, 1% or 2% of the
diet for 24 months. Average daily intakes were 427 and 864 mg/kg
bw/day for males and 528 and 1026 mg/kg bw/day for females on the 1%
and 2% diets, respectively. The authors noted no differences in
body weight, food utilization, food intake, mortality, or general
health among the treatment groups. Due to the small number of
animals/sex/group (10), the pathology findings are difficult to
interpret. No increase in tumours associated with consumption of
diets containing a 50:50 mixture of disodium 5'-guanylate and
disodium 5'-inosinate was reported. There may have been a small
increase in testicular atrophy in male rats (3/9 in both 50:50
mix-fed groups, 1/9 in control) and an increase in adrenal
enlargement in high-dose female rats (6/10 in females fed 2% a 50:50
mixture of disodium 5'-guanylate and disodium 5'-inosinate, 2/10 in
females fed 1% a 50:50 mixture of disodium 5'-guanylate and disodium
5'-inosinate, 2/10 in control females). No effect of 50:50 mix
ingestion on tumour incidence, tumour type, or pathological lesions
of various organs in rats was seen under conditions of this study
(Usui et al., 1971).
2.2.4 Reproduction studies
2.2.4.1 Rats
Disodium 5'-inosinate
In a three-generation reproduction study, groups of ten male
and 20 female rats were fed diets containing 0%, 0.5%, 1%, or 2%
disodium 5'-inosinate. Animals were fed experimental or control
diets for 60 days before mating. No effects on mating performance,
pregnancy rate, or duration of gestation were noted. Body weight
gain in disodium 5'-inosinate-fed rats was larger than in controls
in all generations. Litter size, pup weight, pup mortality and
incidence of abnormalities were unaffected by treatment. The
authors reported that organ weight analysis, histopathology and
skeletal staining of F3B litters revealed no consistent pattern of
adverse effects related to disodium 5'-inosinate consumption (Palmer
et al., 1973).
Disodium 5'-guanylate + disodium 5'-inosinate
Rats were fed diets containing 0, 0.1, 1.0, or 2.0% 50:50 mix
for 3 generations (20 female and 10 male rats per generation). Two
litters were produced per generation and the parent group for the
next generation was selected from the second litter. The parent
group was apparently not randomly chosen, but pups were chosen from
as many litters as possible and selected so that pup weights were as
close as possible to the mean pup weight at weaning. In parent
animals, mortality, bodyweight change, feed consumption, mating
performance, pregnancy rate, and gestation period were assessed.
All offspring were examined for external abnormalities within 12
hours of birth. At 21 days of age, all pups from the first litter
of each generation and surplus pups from the second litter were
killed and examined internally and externally for evidence of
abnormality. Ten males and 10 females from the third generation
(control and 2% 50:50 mix groups only) were subjected to detailed
histological examination of the pancreas, urinary bladder, a long
bone, stomach, small and large intestines, and bone marrow smears.
Brain, liver, heart, pituitary, spleen, thyroid, kidneys, thymus,
adrenals, lungs, and gonads were weighed. An additional 10 males
and 10 females from the third generation of each exposure group
underwent skeletal examination.
The author concluded that there was no evidence of any
treatment-related effect at any dose level (few data were included
in the article). However, there was a tendency for the litter size
to be larger for 50:50 mix-fed rats than for control rats for the
first litters and smaller than controls for the second litters.
Similarly, pup mortality tended to be lower than controls in the
first litters of 50:50 mix-fed rats and higher than controls in the
second litters. These tendencies reached statistical significance
for the 1.0% treatment group in the second and third generations,
but were not dose-related.
The author reported a trend toward increased skeletal variants
with increasing dose of 50:50 mix (variants included bipartite
thoracic centrum, seven sternebra, and extra ribs); however, only 10
animals of each sex were examined, so the relevance of these
findings is unclear (controls: 20% of males and 20% of females
exhibited some skeletal variations; 0.1% 50:50 mix group: 10% of
males and 30% of females exhibited skeletal variations; 1% 50:50 mix
group: 50% of males and 30% of females exhibited skeletal
variations; 2% 50:50 mix group: 80% of males and 40% of females
exhibited skeletal variations). The author concluded that the
findings in this study had no toxicological significance (Palmer,
1975).
2.2.5 Special studies on embryotoxicity/teratogenicity
2.2.5.1 Mice
Disodium 5'-guanylate
Pregnant female mice were treated with 0, 750 or 1 000 mg/kg bw
guanosine, injected i.p. on day 10 or day 13 of gestation. Mice
were sacrificed on day 19 of gestation, and examined for number of
implantation sites and signs of fetal death in situ. Live fetuses
were removed, weighed, and examined for external deformities and
skeletal malformations. Control values were: body weight - 1.40 g,
dead offspring - 6.5%; skeletal malformations - 0%; external
malformations - 0.5%; subcutaneous haematoma - 0%. Fetuses from
mothers injected on day 10 showed a decrease in mean body weight
(mean=1.32 g) and an increase in percent live offspring with
skeletal malformation (7.7%) at 750 mg/kg bw; there was an increase
in percent of dead offspring (26.2%), decrease in mean body weight
(mean=1.34 g), and an increase in percent live offspring with
skeletal malformation (14.2%) at 1 000 mg/kg bw. Injection on day
13 caused an increase in percent dead offspring (21.3%) and decrease
in mean body weight (mean=1.33 g) at 750 mg/kg bw; there was an
increase in percent dead offspring (23.1%), decrease in mean body
weight (mean=1.35 g), and increase in percent live offspring with
external malformation (7.8%) and subcutaneous haematoma (5.3%) after
injection of 1 000 mg/kg bw (Fujii et al., 1972).
Disodium 5'-inosinate
Pregnant female mice were treated with 0, 250, 500 or 1 000
mg/kg bw inosine, injected i.p. on day 10 or 500 or 1 000 mg/kg bw
injected i.p. on day 13 of gestation. Mice were sacrificed on day
19 of gestation, and examined for number of implantation sites and
signs of fetal death in situ. Live fetuses were removed, weighed,
and examined for external deformities and skeletal malformations.
Control values were: body weight - 1.40, percent dead offspring -
6.5; percent skeletal malformations - 0; percent external
malformations - 0.5; percent subcutaneous haematoma - 0. Fetuses
from mothers injected on day 10 showed an increase in percent of
dead offspring (16.5%), decrease in mean body weight (mean=1.35),
and an increase in percent live offspring with skeletal malformation
(14.0%) at doses of 1 000 mg/kg bw; decrease in mean body weight
(mean=1.33) and an increase in percent live offspring with skeletal
malformation (15.3%) at 500 mg/kg bw; decrease in mean body weight
(mean=1.35) and an increase in percent live offspring with skeletal
malformation (12.4%) at 250 mg/kg bw. Injection on day 13 caused an
increase in dead offspring (24.6%) and decrease in mean body weight
(mean=1.31) at 1 000 mg/kg bw; increase in dead offspring (12.4%)
and decrease in mean body weight (mean=1.37) at 500 mg/kg bw, with
no increase in skeletal malformations at either dose (Fujii et al.,
1972).
Disodium 5'-guanylate + disodium 5'-inosinate
Groups of 14 pregnant mice were given 0 or 2 000 mg/kg bw/dy
50:50 mix orally via gastric tube from days 8-13 of pregnancy.
Fetuses were removed by Caesarean section on day 19. Parameters
measured were: number of embryonal implantations and fetal deaths,
weight, sex differentiation, gross external and visceral
malformations, and skeletal malformations; the authors reported no
effects of treatment on any measured parameters (Kaziwara et al.,
1971).
2.2.5.2 Rats
Disodium 5'-guanylate
Groups of 9 pregnant rats were given 0 or 100 mg/kg bw/dy
disodium 5'-guanylate orally via gastric tube on days 9-15 of
pregnancy. Fetuses were removed by Caesarean section on day 21.
Parameters measured were: number of embryo implantations and fetal
deaths, weight, sex differentiation, gross external and visceral
malformations, and skeletal malformations; the authors reported no
effects of treatment on any measured parameters. The authors
reported no effects of treatment on any measured parameters
(Kaziwara et al., 1971).
Disodium 5'-guanylate + disodium 5'-inosinate
A group of 9 pregnant rats were given 2 000 mg/kg bw/dy 50:50
mix orally via gastric tube from days 9-15 of pregnancy. Fetuses
were removed by Caesarean section on day 21. Parameters were
measured as above. There was a change in the sex ratio of the
fetuses (M:F ratio was 1.11 in control litters, 0.64 in treated
litters); the significance of this change was not commented on by
the authors. No other changes were found in any measured parameters
(Kaziwara et al., 1971).
2.2.5.3 Rabbits
Disodium 5'-guanylate
Pregnant rabbits were fed either a normal diet (12 animals) or
diets containing 0.2 g disodium 5'-guanylate/kg bw/dy or 2.0 g
disodium 5'-guanylate/kg bw/dy from days 6-18 of gestation (9-10
animals/group). All except four dams in each group were sacrificed
on day 29; remaining dams were allowed to litter spontaneously and
their pups were observed until 30 days of age. No adverse effects
on body weight were noted; rabbits fed diets containing 2 g/kg bw/dy
had reduced feed consumption. Implantation numbers did not differ
from controls but mortality of fetuses in the 2 g/kg bw/dy group was
lower than in controls. All disodium 5'-guanylate-treated groups
showed some delay in ossification, but no treatment-specific
skeletal abnormalities. There were no effects on number of
delivered fetuses, and survival rate of the 0.2 g/kg group was
greater than controls at weaning. Mean pup body weights were
reported to be normal and no significant malformations were observed
in pups of either dose group (Jojima et al., 1973).
Disodium 5'-inosinate
Groups of 13-18 pregnant female Japanese white rabbits received
0, 200 or 2 000 mg/kg bw/dy disodium 5'-inosinate in their diet
during days 6-18 of gestation. Four to five females of each group
were allowed to deliver spontaneously and pups were observed to day
30. All other dams were killed on day 29 of gestation. The authors
reported that no significant effects were observed on implantation
sites, number of live or dead fetuses, body weights of live fetuses
nor external abnormalities. The mortality of fetuses in the 0.2
g/kg bw/dy group was lower than that of other groups. All disodium
5'-inosinate-treated groups showed some delay in ossification but no
specific skeletal abnormalities were found that appeared to be due
to disodium 5'-inosinate. The authors concluded that daily
administration of 2 000 mg/kg bw disodium 5'-inosinate had no
adverse effect on pup development (Jojima et al., 1973).
2.2.5.4 Chickens
Disodium 5'-guanylate
Chick embryos were injected with guanosine into the yolk sac at
4 days, with doses ranging from 2-12 mg/egg. Of 65 embryos
injected, 49 remained alive at 10 days and 45 remained alive at 18
days. 6 embryos showed abnormalities at 11-18 days (12%). The
approximate LD50 for guanosine in this system was estimated to be 8
mg/egg. Guanosine was neither highly toxic nor teratogenic in this
system (Karnofsky et al., 1961).
2.2.5.5 Monkeys
Disodium 5'-guanylate + disodium 5'-inosinate
Pregnant Cynomolgus monkeys were given 0 (2 monkeys), 500
mg/kg bw/dy 50:50 mix (2 monkeys) or 1 000 mg/kg bw/dy 50:50 mix
(3 monkeys) orally via gastric tube from day 21 to day 30 of
pregnancy. Fetuses were removed by Caesarean section on day 100.
Measured parameters were weight, sex differentiation, gross external
and visceral malformations and skeletal malformations. All treated
females were reported to show some effects of treatment: monkeys
receiving 500 mg/kg bw/dy 50:50 mix evacuated soft faeces for 4 or 9
days during treatment; all monkeys receiving 1 000 mg/kg bw/dy 50:50
mix exhibited diarrhoea, one of the three exhibited profuse
diarrhoea and vaginal bleeding so that administration was stopped
after the 6th day of treatment. Body weight gain of treated animals
was similar to that of control animals, except for one animal
receiving 500 mg/kg bw/dy who showed no weight gain during the
study. The authors reported that one fetus from a treated monkey
(500 mg/kg bw) had a hypertrophic spleen which manifested blood
stagnation upon histological examination. No gross visceral nor
skeletal malformations were observed in treated or control fetuses.
A cervical rib was present in 1 control fetus, 2 fetuses from
females treated with 500 mg/kg bw/dy and 1 fetus from a female
treated with 1 000 mg/kg bw/dy (Kaziwara et al., 1971).
2.2.6 Special studies on genotoxicity
Sodium 5' guanylate, sodium 5' inosinate, and a 50:50 mix were
tested for genotoxicity in the Salmonella/microsome test (with and
without metabolic activation) and chromosomal aberration test
in vitro using a Chinese hamster fibroblast cell line (without
metabolic activation). All substances were negative in the
Salmonella/microsome test. Results of the chromosomal aberration
test were positive for all substances. For disodium 5'-guanylate,
the D20 (dose at which structural aberrations were detected in 20%
of the metaphases observed) was .024 mg/ml and the TR (frequency of
cells with exchange type aberrations per unit dose (mg/ml)) was 576.
For disodium 5'-inosinate, the D20 (dose at which structural
aberrations were detected in 20% of the metaphases observed) was
15.2 mg/ml, the TR (frequency of cells with exchange type
aberrations per unit dose (mg/ml)) was 0.8. For 50:50 mix, the D20
was 1.99 mg/ml and the TR was 4.75 (Ishidate et al., 1984).
2.3 Observations in humans
Disodium 5'-inosinate
Three healthy volunteers were given 0, 1, 1.5, 2, and 2.5 g
disodium 5'-inosinate for seven consecutive days (diets had equal
amounts of purines). Serum uric acid and urinary uric acid
excretion doubled without signs of ill effects. The author reported
that uric acid is the major endpoint of disodium 5'-inosinate
metabolism in humans; 2/3 of uric acid appears in the urine, with
the remainder excreted via the gut, where it was further degraded.
(Kojima, 1974).
Disodium 5'-guanylate + disodium 5'-inosinate
Three healthy men were fed 0, 250, 500, 1 000, 2 000, or 4 000
mg/day of a mixture of 50% disodium 5'-inosinate and 50% disodium
5'-guanylate. Equal divided doses were given with three daily
meals. Doses were given in an escalating pattern, from 250 to 4 000
mg/day, with 5 days at each dose; uric acid levels in serum and
urine were measured before dosing and on the final two days at each
dose. Uric acid levels in serum and urine were not significantly
increased at doses up to 1 000 mg/day, but doses of 2 000 and 4 000
mg/day caused significant increases in both measures (p<.005). The
authors stated that the measured values at 2 000 mg/day (serum uric
acid: 6.9 mg/100 ml; urinary uric acid: 0.82 g/day) were increased
above baseline (serum uric acid: 6.3 mg/100 ml; urinary uric acid:
0.6 g/day) but were within the normal range of these values; however
values at 4 000 mg/day (serum uric acid: 8.6 mg/100 ml; urinary acid
1.1 g/day) were elevated above the normal range (Mitoma et al.,
1972). (The normal ranges were not given in this article, but Kojima
[1974] reported that normal serum urate levels were 5±1 mg/100 ml in
969 normal men and mean urinary uric acid was 8.1 mg/kg bw/24 h in
normal men.).
3. COMMENTS
Disodium 5'-guanylate and disodium 5'-inosinate are widely
distributed in all animal and plant tissues. Their role in purine
metabolism as well as their breakdown to uric acid and to allantoin
(in most mammals, but not humans) is well documented. Data
presented at the 18th meeting as well as new data on the
metabolism, reproductive effects, genotoxicity, and short-term and
long-term toxicity of guanylate and inosinate were evaluated at the
present meeting. No evidence of carcinogenicity, teratogenicity, or
adverse effects on reproduction has been observed.
Changes in dietary purine intake over the past decade resulting
from the use of guanylate and inosinate as flavour enhancers are no
greater than those due to variability in the consumption of the
major dietary contributors of purines. Naturally occurring
nucleotides in the diet (calculated to be up to 2 g/person/day)
greatly exceeds their intake resulting from use as flavour enhancers
(approximately 4 mg/person/day).
4. EVALUATION
The Committee concluded that, on the basis of the available
data, the combined total daily intake of disodium 5'-guanylate and
disodium 5'-inosinate is not of toxicological significance, and
re-confirmed the ADI "not specified" that was previously
established. Because exposure to these substances from their use as
flavour enhancers is low compared with daily intake of naturally
occurring nucleotides in the diet, the Committee found no reason to
recommend that foods to which these substances have been added
should be labelled on the basis of safety, and withdrew its previous
recommendation for labelling.
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See Also:
Toxicological Abbreviations