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



    WHO FOOD ADDITIVES SERIES NO. 5







    The evaluations contained in this publication
    were prepared by the Joint FAO/WHO Expert
    Committee on Food Additives which met in Geneva,
    25 June - 4 July 19731

    World Health Organization
    Geneva
    1974

              

    1    Seventeenth Report of the Joint FAO/WHO Expert Committee on
    Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539;
    FAO Nutrition Meetings Report Series, 1974, No. 53.

    PROPIONIC ACID AND ITS CALCIUM, POTASSIUM AND SODIUM SALTS

    Explanation

         These compounds have been evaluated for acceptable daily intake
    by the Joint FAO/WHO Expert Committee on Food Additives (see Annex I,
    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 monograph has been expanded and is reproduced
    in its entirety below.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

         At concentrations up to 0.004 mol/l the acid is fungistatic (Peck
    & Rosenfeld, 1938). Sodium and calcium propionate inhibit moulds and
    fungi at specific concentrations varying from 0.0125% to 1.25% at pH
    5.5 and sodium propionate at 1.6% to 6% inhibits various bacteria
    (Keeney & Boyles, 1943). In tests with a number of microorganisms it
    has been shown that the bacteriostatic and fungistatic activity of
    sodium propionate was greater in acid than in neutral or slightly
    alkaline solution, which suggests that the antimicrobial action is due
    to the undissociated acid (Heseltine, 1952b and Preservatives Report
    1959).

         Propionic acid is not a component of the edible fats and oils,
    but arises in the intermediary metabolism of the body as the terminal
    three-carbon fragment in form of propionyl coenzyme A in the oxidation
    of odd-number carbon fatty acids. Oxidation of the side-chain of
    cholesterol by rat liver mitochondria yields propionate as the
    immediate product of cleavage (Mitropoulos & Myant, 1965). Propionates
    are metabolized and utilized in the same way as normal fatty acids and
    even after large doses no significant amounts of propionic are
    excreted in the urine (Bässler, 1957). In vitro propionic acid is
    completely oxidized by liver preparations to CO2 and water
    (Huennekens et al., 1951).

         The metabolic fate of propionates varies in microorganisms. Some
    have enzyme systems converting succinate to propionyl-coenzyme A and
    by various further steps to propionate, CO2 or propionyl phosphate.

    Others convert propionic acid to B-alanine or directly to CO2. The
    inhibiting effect for microbials is probably related to competition
    with acetate in the acetokinase systems, to blockage of pyruvate
    conversion to acetyl-coenzyme A and to interference with B-alanine in
    pantothenic acid syntheses (Bässler, 1959). In mammals observations
    have shown easy absorption from the gastrointestinal tract (Dawson et
    al., 1964) and absence of any excretion in the urine whatever the mode
    of administration. Decomposition by bacteria in the gut also occurs
    (Hermann et al., 1938). In the mouse 14C-tagged propionate acts as
    a precursor of body fat (Kleiber et al., 1953) and is incorporated
    into the odd-numbered fatty acids of milk fat (James et al., 1956).
    1-14C-labelled propionate fed to fasted rats produced 50% of the
    activity as expired CO2 within two hours, the remainder being
    incorporated into glucose, glycogen, succinate, malate, fumarate and
    proteins (Buchanan et al., 1943; Lorber et al., 1950; Pritchard &
    Tove, 1960). Rabbits oxidize propionate without keto-intermediates
    (Mackay et al., 1940a; Mackay et al., 1940b). Dairy cows, when
    injected i.v. with propionic acid labelled with 1-C14, produce active
    lactose, casein and butter fat and convert 2-C14 acid to asparaginic
    acid and serine (Assoc. Food & Drug Officials). Propionate increases
    glucose output in phlorhidzinised animals (Rittenberg et al., 1937),
    and has a glycogenic effect in fasting rats without being involved
    in cholesterol synthesis (Buchanan et al., 1943). The in vitro
    antiketogenic action of propionate was not confirmed in vivo.
    Nine rabbits, made diabetic with alloxan, were given orally 1000 and
    3000 mg/kg bw sodium propionate daily without significant useful
    effect on their acetonuria and 30% of the animals died at the higher
    dosage level. 740 mg/kg bw oral sodium propionate did not alter the
    quantitative urinary excretion of volatile carboxylic acids but a
    higher proportion of acetic acid appeared in normal rabbits' urine and
    a proportionate rise in diabetic animals. Sugar excretion beyond that
    of the diabetic level was noted (Maurer & Lang, 1956). Studies on
    liver and kidney mitochondrial extracts or intact mitochondria and
    homogenates of cardiac or skeletal muscle tissue showed conversion of
    propionate to CO2 and water. The pathways involve combination with
    coenzyme A in the same way as acetic acid, becoming carboxylated to
    methylmalonyl-coenzyme A and undergoing a quantitative conversion to
    succinate which then enters the Krebs citric acid cycle (Beck et al.,
    1957; Flavin & Ochoa, 1957). Intact rat liver mitochondria fix
    14C-labelled bicarbonate to propionate with conversion to succinate
    in the presence of a cosubstrate while guinea-pig or beef liver
    mitochondria and pig heart enzymes act without cosubstrate (Friedberg
    et al., 1956). In vitro 1 g liver (rat) oxidize 3 mg/hom (Friedberg
    et al., 1956), 1 g heart muscle (dog) 0.23 mg/hom (Cavert & Johnson,
    1956). Skeletal muscle (dog) metabolizes 1-14C-labelled propionate,
    which contributes 9% to 13% of the CO2 produced (Lifson, 1957).

    Rabbit skeletal muscle extract markedly stimulates by its
    fluorophosphate-forming enzyme system carboxylative activity of pig
    heart enzymes (Flavin et al., 1957). Other pathways use direct
    oxidation to pyruvate or direct condensation with acetate to produce
    fatty acids with odd numbers of C-atoms in mouse adipose tissue and
    ruminants (Bässler, 1959). Oxidation of 1-14C-labelled propionate to
    CO2 proceeded in the same way and at the same rate in mouse adipose
    tissue as in liver, while 2-14C propionate was incorporated
    preferentially into newly synthesized fatty acids at much increased
    rate in mouse adipose tissue compared with liver. The minor direct
    oxidation of 2-14C propionate proceeded at equal rates in adipose and
    liver tissue. Direct condensation of propionate and CO2 to succinate
    or 4-carbon intermediates did not occur (Feller & Feist, 1957, Wakil,
    1962).  Conversion of propionic acid B-alanine was shown (Rendina &
    Coon, 1957). High propionate concentrations inhibit acetate and other
    metabolism in tissue cultures, homogenates or mitochondrial
    preparations by competition for coenzymes A, e.g. formation of
    ketobodies from pyruvate and acetate, activation and oxidation of
    fatty acids, acetate metabolism and acetoacetate formation are
    effected (Lang & Bässler, 1953). The production of 14CO2 from
    labelled acetate by rat liver homogenates was almost abolished by
    propionate in only one-tenth the concentration of acetate (Pennington
    & Appleton, 1958). Inhibition of catalase activity has been reported
    (Lück, 1957). In man and in animals these enzyme-inhibiting activities
    are of little significance in vivo, as the fast rate of metabolism
    prevents accumulation of the high concentrations that would be
    necessary. In human plasma propionic acid represents 0% to 4% of the
    total fatty acid and is a by-product of normal intermediate
    metabolism. Absorbed propionate is removed by the liver, kidneys,
    heart, muscle and adipose tissue. The liver can deal with 4.5 g free
    acid or 5.8 g sodium propionate per hour, the isolated dog's heart
    metabolizes 0.24 mol/hour per 100 g tissue and isolated dog's
    gastrocnemius metabolizes 1-14C-labelled propionate such that 9% to
    13% of CO2 produced is being contributed by the labelled substrate.
    The normal breakdown of various amino acids yields propionate of
    propionyl-coenzyme A and fatty acids with odd numbers of C-atoms yield
    also propionate (Bässler, 1959). Propionate occurs in human sweat
    through glycogenolysis in the sweat glands and from excess in tissue
    fluids after the needs of fatty acid metabolism have been met
    (Heseltine, 1952a).

    TOXICOLOGICAL STUDIES

    Acute toxicity

         No LD50 estimations are published in the literature because of
    the very low acute toxicity of propionates (Heseltine, 1952a;
    Sollmann, 1957). The CNS effect is a direct action of the propionate
    on nervous tissue (Samson et al., 1956).

                                                                                                   
                                            Other
                            ED50*   LD50    dosages
                                                       
    Animal    Route              mg/kg bw                  Effects             References
                                                                                               

    Frog      i.m.          -       -       100            Twitching,          Fodera, 1894
                                                           paresis for
                                                           24 hours

    Rat       i.p.          2 156   -       -              Unconscious         Samson et al.,
              (sod,salt)                                   in 18-20            1956
                                                           minutes

              i.v.          1 330   -       -              Unconscious         Samson et al.,
              (sod.salt)                                   in 5 seconds        1956

              s.c.          -       -       2 800          Tiredness           Samson et al.,
              (sod.salt)                                                       1956

              oral          -       -       5 600          Nil                 Samson et al.,
              (sod.salt)                                                       1956

              (free acid)   -       2 600   -              -                   U.S. Food & Drug

    Cat       s.c.          -       -       1 000          Sleep               Mayer, 1886
              (sod.salt)

    Rabbit    i.v.          -       -       1 320          Death               Hermann, 1930a
              (free acid)

              (sod.salt)    -       -       2 200          Dyspnoea,           Mayer, 1886
                                                           narcotic
                                                           paresis &
                                                           urination
                                                           (24 hours)

    Dog       s.c.          -       -       900            Nil                 Knoop & Jost,
              (free acid)                                                      1923


              i.v.          -       -       500            Dyspnoea,           Mayer, 1886
              (free acid)                                  narcotic
                                                           weakness

                            -       -       370            Temporary           Hermann, 1930a
                                                           BP fall

              (sod.salt)    -       -       570            Vomiting,           Knoop & Jost,
                                                           weakness            1923
                                                                                               
    *     ED50 - dose producing temporary unconsciousness in 50% of animals.
    
    Short-term studies

    Rat

         Four groups of one control and two weanling test rats were pair-
    fed for four to five weeks on diets containing 1% sodium or calcium
    propionate and 3% sodium or calcium propionate. No effect on growth
    was observed (Harshbarger, 1942). In another experiment 14 groups of
    15 male animals were each fed on diets containing 0.075% or 3.75%
    sodium propionate in combination with varying proportions of other
    additives at 50 times their commercial level of usage. Transient
    growth depression was noted with 3.75% sodium propionate which later
    became normal. Final weights of the 3.75% groups were significantly
    depressed. Food consumption was reduced in all diets compared with
    controls and feed efficiency was very poor with 3.75% propionate.
    Haematological findings were normal at 16 weeks. Histopathology of
    cerebellum, cerebrum, heart, trachea, oesophagus, thyroid, salivary
    gland, thymus, adrenals, pancreas, bladder, testes, kidneys, lymph-
    glands, lung, stomach, liver, spleen, small gut showed kidney
    abnormalities in diets containing chlorine dioxide-treated flour, but
    no consistent change related to propionate. Mortality was not
    adversely affected in any group and weights of liver, left kidney,
    heart and spleen were comparable with controls (Graham & Grice, 1955).
    Over a period of 39 days 5% sodium propionate in a rat free from
    biotin, relic acid or vitamin B12 reduced growth rate and food
    intake. The addition of biotin, relic acid or especially vitamin B12
    inhibited this effect. Diets containing 3.0 and 6.0% sodium propionate
    also produced a growth retardation which was overcome by the addition
    of vitamin B12 (25 µg/kg diet), more effectively with the 3.0%
    propionate diet (Hogue & Elliot, 1964).

    Rabbit

         Normal and alloxan-diabetic animals were fed daily on 1000 mg/kg
    bw of sodium propionate. Normal animals showed no adverse effects but
    a small amount of acetic acid appeared in their urine. Diabetic
    animals excreted unchanged amounts of ketone bodies but urinary
    concentrations of volatile fatty acids and glucose increased. No
    propionate appeared in tho urine of either normal or diabetic animals
    (Maurer & Lang, 1956).

    Long-term studies

    Rat

         Groups of 13 male and 13 female weanling rats were fed on diets
    containing 0.075% and 3.75% sodium propionate in a baked bread in
    combination with various proportions of other additives at 50 times
    their usual commercial level for one year. No effect on growth,

    mortality rate or body weight was seen although food consumption was
    slightly depressed on all diets compared with controls. No other
    toxicological effects were found as judged by histopathology of
    bladder, small gut, spleen, stomach, pancreas, adrenal, kidney, liver,
    heart, lung, thyroid, brain or gonads or weights of heart, liver,
    spleen and kidneys. The kidneys of female rats on diets containing
    high levels of chlorine-dioxide-treated flour showed tubular
    degeneration and minor glomerulonephritic changes (Graham et al.,
    1954).

    OBSERVATIONS IN MAN

         In an adult male daily oral doses of 6000 mg of sodium propionate
    rendered the urine faintly alkaline but had no other effect (Bässler,
    1959).

         Solutions of propionate applied to the eye in concentrations up
    to 15% in man and up to 20% in rabbits had no irritating effect
    (Theodore, 1950). Propionic acid is a moderate irritant of skin
    causing stinging pain and subsequent hyperpigmentation (Oettel, 1936).
    No sensitization from topical use has been reported, nor has it any
    anticoagulant effect (Heseltine, 1952a).

         Two male and two female volunteers were treated locally with 0.05
    or 0.1% histamine phosphate and inhibition of the reaction by 7.5% or
    15% sodium propionate was measured. A moderate potency of about 1/7.5
    of that diphenhydramine was found (Heseltine, 1952a).

    Comments;

         There are no toxicological studies of longer duration than one
    year. However, propionate is a normal intermediary metabolite, and a
    normal constituent of foods.

         An evaluation may be made based on the metabolic information.

    EVALUATION

    Estimate of acceptable daily intake for man

         Not limited*

              

    *    See relevant paragraph in the seventeenth report (pages 10-11).

    REFERENCES

    Association of Food and Drug Officials, Quarterly Report

    Bässler, K. H. (1959) Z. Lebensmittel. Unters Forsch., 110, 28

    Beck, W. S., Flavin, M. & Ochoa, S. (1957) J. biol. Chem., 229, 997

    Buchanan, J. M., Hastings, A. B. & Nesbett, F. B. (1943) J. biol.
         Chem., 150, 413

    Cavert, H. M. & Johnson, J. A. (1956) Amer. J. Physiol., 184, 582

    Dawson, A. M., Holdsworth, C. D. & Webb, J. (1964) Proc. Soc. exp.
         Biol., 117, 97

    Feller, D. D. & Feist, A. (1957) J. biol. Chem., 228, 275

    Flavin, M., Castro-Mendoza, H. & Ochoa S. (1957) J. biol. Chem., 229,
         981

    Flavin, M. & Ochoa, S. (1957) J. biol. Chem., 229, 965

    Fodera, F. A. (1894) Arch. farm. sper., 2, 417

    Friedberg, F., Alder, H. & Lardy, H. A. (1956) J. biol. Chem., 219,
         943

    Graham, W. D. & Grice, H. C. (1955) J. Pharm. (Lond.), 7, 126

    Graham, W. D., Teed, H. & Grice, H. C. (1954) J. Pharm. (Lond.), 6,
         534

    Hermann, S., Neiger, R. & Zenter, M. (1938) Arch. exp. Path. Pharmak.,
         189, 539

    Harshbarger, K. E. (1942) J. Dairy Sci., 256, 169

    Hermann, S. (1930a) Arch. exp. Path. Pharmak., 154, 143

    Heseltine, W. W. (1952a) J. Pharm. Pharmacol., 4, 120

    Heseltine, W. W. (1952b) J. Pharm. Pharmacol., 4, 577

    Hogue, D. E. & Elliot, J. M. (1964) J. Nutr., 83, 171

    Huennekens, F. M., Mahler, H. R. & Nordmann, J. (1951) Arch. Biochem.,
         30, 66

    James, A. T., Pecters, G. & Lauryssen, M. (1956) Biochem. J., 64, 726

    Keeney, E. L. & Broyles, E. N. (1943) Bull. Johns. Hopkins Hosp., 73,
         479

    Kleiber, M. et al. (1953) J. biol. Chem., 203, 339

    Knoop, F. & Jost, H. (1923) Z. phys. Chem., 130, 338

    Lang, K. & Bässler, K. H. (1953) Biochem. Z., 324, 401

    Lifson, N. (1957) Amer. J. Physiol., 188, 227

    Lorber, V. et al. (1950) J. biol. Chem., 183, 531

    Lück, H. (1957) Biochem. Z., 328, 411

    Mackay, E. M., Wick, A. N. & Bernum, C. P. (1940a) J. biol. Chem.,
         135, 183

    Mackay, E. M., Wick, A. N. & Bernum, C. P. (1940b) J. biol. Chem.,
         136, 503

    Maurer, H. & Lang, K. (1956) Klin. Wschr., 34, 862

    Mayer, H. (1886) Arch. exp. Path. Pharmak., 21, 119

    Mitropoulos, K. A. & Myant, N. B. (1965) Biochem. J., 97, 26c

    Oettel, H. (1936) Arch. exp. Path. Pharmak., 183, 641

    Peck, S. M. & Rosenfeld, H. (1938) J. Indust. Dermat., 1, 237

    Pennington, R. J. & Appleton, J. M. (1958) Biochem. J., 69, 119

    Pritchard, G. J. & Tove, S. B. (1960) Biochem. biophys. Acta, 41, 130

    Rendina, G. & Coon, M. J. (1957) J. biol. Chem., 225, 523

    Report on Preservatives (1959) Food Standards Committee S.O. Code
         No. 24-280

    Rittenberg, D., Schoenhamma, R. & Evans, E. A. jr (1937) J. biol.
         Chem., 120, 503

    Samson, F. E. jr, Dahl, N. & Dahl, D. R (1956) J. clin. Invest.,
         35, 129

    Sollmann, T. (1957) A manual of pharmacology, 8th ed., Philadelphia &
         London, Saunders

    Theodore, J. (1950) J.A.M.A., 143, 226

    U.S. Food and Drug Administration, Unpublished data

    Wakil, S. J. (1962) Ann. Rev. Biochem., 31, 369


    See Also:
       Toxicological Abbreviations