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    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 51 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