Toxicological evaluation of some food
    additives including anticaking agents,
    antimicrobials, antioxidants, emulsifiers
    and thickening agents


    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


    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.



         These substances have been evaluated for acceptable daily intake
    by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
    Refs No. 6 and No. 13) in 1961 and 1965.

         Since the previous evaluation, additional data have become
    available and are summarized and discussed in the following monograph.
    The previously published monographs have been expanded and are
    reproduced in their entirety below.



         The liver is the main site of conjugation with glycine in both
    man and most experimental animals (rabbits, rats) with the exception
    of the dog. Where the kidney is the main site of biosyntheses (Snapper
    et al., 1924; Friedmann & Tachau, 1911), sheep appear to have reduced
    capabilities of conjugating benzoic acid with glycine. Infusion of
    increasing amounts of benzoic acid into the rumen at levels up to
    1.8 g/kg led to progressive fall in conjugation and increasing
    excretion of free benzoic acid in the urine. Doses of 1.1 and 1.8 g/kg
    were toxic leading to death. Potassium deficiency also occurred as
    shown by the usual symptoms of severe muscular weakness and tremors
    (Martin, 1966). For many years a liver function test was used in man
    based on the urinary excretion of hippuric acid after a test dose of
    benzoic acid (6 g orally or 1.5-2.0 g intravenously). Hence there
    exists a large amount of experience on the excretion of benzoic acid
    and hippuric acid in man. In the blood benzoates exist in the free
    state and not bound to proteins (Knoefel & Huang, 1956). In the dog
    the kidney clearance was estimated to be 0.90-1.89 (Knoefel & Huang,

         Normal urinary excretion of hippuric acid in man was estimated to
    be 1.0-1.25 g/day, equivalent to 0.7-1.7 g of benzoic acid (Stein et
    al., 1954). Other determinations of the normal excretion in man and
    rat yield values lying between 1-3 mg/kg bw (Armstrong et al., 1955).
    The maximum rate of the hippuric acid excretion after ingestion of
    benzoic acid was observed to be 17 mg/min and for benzoyl glucuronic
    acid 0.57 mg/min equivalent to 24 g/day calculated as benzoic acid
    (Schachter, 1957). Up to 10 g of benzoate is quantitatively excreted
    by man (Barnes, 1959). At high intakes of sodium benzoate up to 3% is
    conjugated with glucuronic acid and all metabolites eliminated
    completely within 14 hours (Schachter, 1957). 75 to 80% of
    administered benzoic acid is eliminated by man in six hours (Quick,
    1931). Sodium benzoate also decreases uric acid excretion (Quick,
    1931), urea and ammonia excretion in man (Lewis, 1914).

         Precursors of endogenous benzoate are phenylalanine and tyrosine.
    Experiments with labelled phenylalanine showed that about 1 to 2% is
    metabolized by this pathway. Rabbits given 50-400 mg/kg bw per day of
    deuterio-phenylalanine for six to 12 days, humans given 14-28 mg/kg bw
    per day for four to six days and guinea-pigs given 300 mg/kg bw per
    day for 12 days were examined (Bernard et al., 1955). 1-14C acetate,
    however, did not produce labelled benzoic acid in rabbits and guinea-
    pigs (Bernard et al., 1955). 3-14C-dl phenylalanine given
    intraperitoneally to rats produced 0.6 to 1% of activity as urinary
    hippuric acid (Altman et al., 1954).

         1,3,4,5-tetrahydroxycyclohexanoic acid (quinic acid) may also
    serve as precursor of benzoic acid in intermediary metabolism (Dickens
    & Pearson, 1951). Several human subjects were given 6 g quinic acid
    orally or 250 g prunes and excreted hippuric acid in increased amounts
    during the following 24 hours (Quick, 1931). When deuterio-benzoic
    acid was administered to man and rats it was excreted with its
    deuterium content unchanged. Feeding guinea-pigs, with body fluids
    enriched in D2O, with hydro-aromatic compounds led to urinary
    excretion of deuterio-benzoic acid with high D content. A similarly
    prepared rat, when fed 750 mg hydroxy benzoic acid over five days,
    excreted urinary benzoic acid enriched in D. When human subjects and
    guinea-pigs were given quinic acid over several days, 47 to 72% was
    converted to benzoic acid and excreted in the urine (Bernard et al.,
    1955). Four rats irradiated with 700 roentgens and four controls were
    given intraperitoneally carboxyl-14C-labelled sodiumbenzoate and
    fasted. Irradiation had no effect on the conjugating ability but the
    irradiated rats excreted less labelled hippuric acid due to dilution
    by endogenously produced benzoic acid (Schreier et al., 1954).

         Benzoic acid inhibits pepsin digestion and sodium benzoate
    inhibits trypsin digestion of fibrin but they have no effect on
    amylase or lipase. Trypsin digestion of casein is only initially
    depressed by sodium benzoate (Kluge, 1933). Benzoic acid is a specific
    powerful inhibitor of the D-amino acid oxidase (50% inhibition by
    10-4M) (Klein & Kamin, 1941). Concentrations in the range of 10-3M
    exert some unspecific inhibitory effects on the metabolism of fatty
    acids, e.g. on acetoacetate formation (Avigan et al., 1955).

         Benzoic acid is rapidly absorbed (Schanker et al., 1958) and
    rapidly and completely excreted in the urine (Schachter, 1957; Barnes,
    1959). One healthy man given 6, 9, 13.9, 34.7 and 69.3 millimoles of
    sodium benzoate showed a complete elimination of the drug within 10 to
    14 hours (Schachter, 1957). Cumulation does not occur as shown by
    experiments on the distribution and elimination of sodium
    benzoate-l-C14 administered i.p., orally or s.c. to the rat.
    Practically quantitative excretion occurs in the urine within one to
    two days, less than 1% of radioactivity appears in the faeces, a few

    ppm appear in organs. All radioactivity was identified as labelled
    benzoic acid (Lang & Lang, 1956). Orally or s.c. administered labelled
    benzoic acid appeared to 90% in the urine as hippuric acid, 0.1% of
    radioactivity occurred in the expired CO2 and 2% remained in the
    carcass (Bernard et al., 1955).

         Two urinary metabolites of benzoic acid are known, namely
    hippuric acid and benzoyl-glucuronic acid. Conjugation with glycine
    and glucuronic acid occurs in preference to oxidation because benzoic
    acid strongly inhibits fatty oxidation in the liver. In man, rabbit
    and rat, benzoic acid is almost entirely excreted as hippuric acid,
    whereas dogs excrete more conjugated glucuronic acid than hippuric
    acid (Williams, 1959). Sheep are less able to excrete hippuric acid
    and excrete much free benzoic acid in their urine (Martin, 1966). The
    urine of man, pig, rabbit and sheep contains up to 10% of benzoyl-
    glucuronic acid.

         The maximum urinary excretory rate achieved depends on the dose
    of benzoate given. Limiting values of hippuric acid excretion were
    approached in man at a dose of 13.9 millimoles (Schachter, 1957).
    Limitations in availability of glycine account for this (Quick, 1933).
    In the rat the tolerance of large doses of benzoic acid depends on the
    addition of adequate amounts of glycine to the diet leaving sufficient
    glycine for protein synthesis. Normally preformed glycine is used
    though some is synthesized as well by the rat (Quick, 1931; Barnes,
    1959). When rats were fed 1.5% benzoic acid (as the sodium salt) in
    the diet, they excreted 95% and more of the drug as hippuric acid in
    the urine. As the benzoate in the diet was increased to 3.75% so the
    ratio of hippuric acid to total benzoic acid in the urine decreased.
    Additional glycine raised elimination to 86 to 99%. The only other
    derivative, found in significant amounts in the urine, was benzoyl
    glucuronide (Griffith, 1929). Dogs and rabbits excrete hippuric acid
    independent of the route of administration of benzoic acid (Quick,


    Special studies on carcinogenicity

         See under long-term studies (Hosino, 1951).

    Special studies on reproduction


         From the animals subjected to a 17-month study some were mated
    and reproduction studied over five generations. Only body weights are
    given in the results (Shtenberg & Ignat'ev, 1970).

    Acute toxicity

                                   LD50            Reference
    Animal     Route               (mg/kg bw)

    Rat        Oral (Na salt)      2 700           Deuel et al., 1954
               i.v. (Na salt)      1 714 ± 124     Spector, 1956

    Rabbit     Oral (Na salt)      2 000           Spector, 1956
               s.c. (Na salt)      2 000           Spector, 1956

    Dog        Oral (Na salt)      2 000           Spector, 1956

         Data on the LD50 of potassium benzoate are not available.
    Benzoic acid is not acutely toxic to man (Lehman, 1908) or to test
    animals in moderate doses (Rost et al., 1913; Smyth & Carpenter,

         Outbreaks of poisoning affecting 28 cats have followed ingestion
    of meat containing 2.39% benzoic acid. The effects were nervousness,
    excitability and loss of balance and vision. Convulsions occurred and
    17 cats died or were killed. Autopsies showed damage to intestinal
    mucosa and liver. The sensitivity of the cat may be due to its failure
    to form benzoyl glucuronide and toxicity may develop with quantities
    greater than 0.45 g/kg single doses or 0.2 g/kg repeated doses
    (Bedford & Clarke, 1971).

    Short-term studies


         Mice fed 3 g sodium benzoate daily for 10 days showed a 10%
    reduction of their creatine output, probably due to depletion of the
    glycine pool (Polonowski, 1941). Groups of 50 male and 50 female mice
    were given benzoic acid at the rate of 80 mg/kg/day, sodium bisulfite
    at 160 mg/kg/day and a mixture of the two at the same levels by
    gavage. The highest mortality was observed in mice given the
    combination (60% of 32% benzoic acid alone) and weight gain was
    reduced. A five-day period of food restriction at 2.5 months produced
    an 85% mortality in both groups (Shtenberg & Ignat'ev, 1970).


         Groups of 10 rats (five males and five females) were fed sodium
    benzoate for 30 days at levels ranging from 16-1090 mg/kg bw. There
    were no observable effects on body weight, appetite or mortality nor
    any histological changes in the organs (Smyth & Carpenter, 1948).

    Groups of three male and three female rats were fed on 0%, 2% and 5%
    sodium benzoate for 28 days. All animals on the 5% level died during
    the first two weeks showing hyperexcitability, urinary incontinence
    and convulsions. At the 2% level male rats showed a significant
    decrease in body weight. The food intake of male and female animals
    was decreased at the 2% level compared with controls (Fanelli &
    Halliday, 1963). Four groups of 15 rats were given 0%, 5% sodium
    benzoate and 5% benzoate + 1% glycine in their diet for three weeks.
    Body weight was reduced at the 5% level but less so when 1% glycine
    was added. Total cholesterol content of the liver was unaffected but
    phospholipids significantly reduced in the liver at the 5% level.
    Potassium concentration of skeletal muscle at the 5% level was also
    low. Glycine corrected the potassium and phospho-lipid deficiencies
    (Kowalewski, 1960). Twenty-eight young rats were given a diet
    containing 5% sodium benzoate for three weeks. Nineteen animals died
    within two weeks. Food consumption was significantly reduced and most
    animals developed severe diarrhoea. Autopsy changes were gut
    haemorrhage, nasal blood crust but normal urine. Five adult rats died
    within five weeks with severe weight loss on a similar diet
    (Kieckebusch & Lang, 1960). Groups of four to 19 male rats were fed
    diets containing 0%, 1.5%, 2.0%, 2.5%, 3%, 3.25% and 3.75% sodium
    benzoate for 40 days. Average growth was less than in controls at all
    levels but above 3% mortality was high, food efficiency poor and
    growth severely depressed. Addition of glycine reduced the toxic
    effects. Animals died with incoordination, tremor or convulsions and
    had severe eye inflammation. Feeding other groups of 10 to 15 young
    male rats on restricted amounts of diet containing 0%, 1.5%, 2.0%,
    2.5% and 3% sodium benzoate revealed no difference in weight gain at
    the 3% level. Glycine addition again improved this weight loss
    (Griffith, 1929).

         Ninety-day feeding tests were carried out on groups of eight to
    10 rats on diets containing 1%, 2%, 4% and 8% of sodium benzoate. In
    the group on the 8% diet there were four deaths (average number of
    days to death 13). The weight gain of the four survivors was two-
    thirds of that of the controls on an identical food intake. Kidney and
    liver weights were significantly higher than those of the control
    group. At the lower levels no demonstrable effect was observed (Deuel
    et al., 1954).


         Experiments on groups of four animals showed that doses of
    benzoate + benzoic acid of 150 mg/kg bw given daily up to 65 days had
    no adverse effects. When the same dose was fed to scorbutic animals a
    shortening of the life-span was observed (Kluge, 1933).


         Feeding tests on 17 dogs over 250 days with sodium benzoate or
    benzoic acid at the rate of 1000 mg/kg bw had no effect on growth,
    appetite and wellbeing. Above this level ataxia, epileptic convulsions
    and death occurred (Rost et al., 1913).

    Long-term studies


         Parenteral administration of benzoic acid has been shown not to
    cause tumour development (Hosino, 1951). Groups of 25 male and 25
    female mice were given benzoic acid in doses of 40 mg/kg/day, sodium
    bisulfite in doses of 80 mg/kg/day and a mixture of the two at the
    same levels for 17 months. Mortality was increased in the groups
    receiving the mixture (62%) compared with the individual substance
    groups (32%) at eight months. Mortality at 17 months is not given and
    pathology is not reported (Shtenberg & Ignat'ev, 1970).


         Three groups of 20 male and 20 female rats were pair fed for
    eight weeks on diets containing 0%, 0.5% and 1% benzoic acid and
    thereafter fed ad libitum over four generations. Two generations
    were fed for their whole life-span, the third and fourth generations
    were autopsied after 16 weeks. No harmful effects were observed on
    growth, fertility, lactation and life-span. The post-mortem
    examination showed no abnormalities (Kieckebusch & Lang, 1960). In
    another experiment 20 male and 30 female rats were fed on a diet
    containing 1.5% benzoic acid with 13 male and 12 female rats as
    controls for 18 months. Fifteen animals died in the test group, in the
    control three only. The test animals showed reduced body weight and
    food intake. Repeat experiments on groups of 20 test animals and 10
    controls taken from another strain showed similar findings (Marquardt,

         Groups of 10 rats, males and females, received benzoic acid at
    40 mg/kg/day or sodium bisulfite at 80 mg/kg/day or a mixture of the
    two in the diet for 18 months. The growth was slightly reduced and ESR
    was increased. Rats fed benzoic acid developed some tolerance to a
    lethal dose of the compound given terminally. No pathology is reported
    (Shtenberg & Ignat'ev, 1970).


         In man tolerance appears to vary, 5.7 g sodium benzoate causing
    marked gastrointestinal disturbances in some (Meissner & Shepard,
    1866) others tolerating 25 g to 40 g (Bignami, 1924). Up to 12 g daily

    have been given therapeutically without ill effects (Senator, 1879)
    yet over five days have produced gastric burning and anorexia in 30%
    of other subjects (Waldo et al., 1949). Toxic symptoms are local
    gastrointestinal mucosal irritation or CNS effects with convulsions in
    animals. Acute toxicity in man is readily reversible and probably due
    to the disturbance in acid-base equilibrium rather than associated
    with any tissue damage (Barnes, 1959).

         Six men were given 0.3 g to 0.4 g of benzoic acid in their diet
    for periods up to 62 days. No abnormalities were seen in blood
    picture, urine composition, nitrogen balance and wellbeing (Chittenden
    et al., 1909). Nine patients receiving penicillin treatment were given
    1200 mg of benzoic acid daily divided into eight doses over a period
    of five days in eight of the subjects and 14 days in one case. No
    effect was observed, particularly, in no case did the endogenous
    creatinine clearance show significant changes nor did routine urine
    analysis show any abnormality (Waldo, 1949).


         Considering the metabolic and toxicological data the margin of
    safety seems to be very large in animals and man. Benzoic acid is
    effectively and rapidly metabolized and eliminated by the body without
    apparent tissue injury. The rat seems closest to man as far as the
    metabolism of benzoate is concerned. There is no reason to believe
    that potassium benzoate differs toxicologically from benzoic acid and
    sodium benzoate when these are used as food additives. The cat seems
    to be much more sensitive than other species. Allergic responses to
    benzoic acid have been reported.


    Level causing no toxicological effect

         Rat: 10 000 ppm (1%) in the diet equivalent to 500 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-5 mg/kg bw.*


    *    As sum of benzoic acid and Na and K benzoate (expressed as
    benzoic acid).


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    See Also:
       Toxicological Abbreviations