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.



         Chemical methods are available for the determination of
    p-hydroxybenzoic acid and its methyl ester in tissues and body fluids.
    The alkyl esters of p-hydroxybenzoic acid are well absorbed after oral
    administration to dogs. Dogs were given either the free acid or the
    methyl ester in doses of 1000 mg/kg bw orally or 50 mg/kg bw i.v. and
    excretion in the urine was measured. The total material recovered
    (i.e., p-hydroxybenzoic acid, p-hydroxybenzoates and metabolic
    conjugates) represented from 60% to 95% of that ingested. Plasma ester
    concentrations rarely reached measurable levels, but high plasma
    levels and high urinary output of p-hydroxybenzoic acid and conjugated
    products indicated hydrolysis of the ester linkage. Enzymatic
    hydrolysis of the ester was demonstrated in vitro using preparations
    of liver and kidney. Studies on one man given 70 mg/kg orally
    suggested that metabolism in man is similar to that in the dog (Jones
    et al., 1956). In dogs the diglucuronide of p-hydroxybenzoic acid has
    been shown to be the main metabolite excreted in the urine. Man
    excreted free p-hydroxybenzoic acid and p-hydroxyhippuric acid in
    approximately equal proportions (Quick, 1932). The urine of rats
    receiving p-hydroxybenzoic acid or its methyl, ethyl or propyl esters
    contained the following metabolites: p-hydroxybenzoic acid 40%,
    p-hydroxyhippuric acid 23.5%, ether sulfate 5%, ester glucuronides
    23%, ether glucuronides 1.2% and 5% of an unidentified substance
    (Derache & Gourdon, 1963). Dogs given 50 mg/kg bw i.v. or orally
    excreted 80-89% in the urine within 48 hours. After i.v. injection
    significant levels were found in the plasma only immediately
    afterwards. When dogs were infused at the rate of 2 mg/kg/min until a
    total of 100 mg/kg bw was given, the levels in most organs were below
    the plasma level. Appreciable amounts were only present in liver and
    kidneys. No accumulation was noted in a dog given 1 mg/kg bw of the
    methyl ester orally every day for a period of one year. This dog
    excreted 96% of the daily dose within 24 hours in the urine (Sokol,

         Similar metabolic experiments in the dog to those described with
    the methyl ester have been reported for the propyl ester with
    essentially similar results (Jones et al., 1956).

         Dogs given 1000 mg/kg bw ethyl ester orally 50 mg/kg i.v.
    excreted 66-70% in the urine. Very low plasma levels, just detectable
    were found two hours after oral administration or at five and 15
    minutes after i.v. injection (Sokol, 1952).

         Results of further experiments in the dog were essentially
    similar to those using the methyl ester except that detectable but
    very low levels of the ethyl ester were found in the plasma two hours
    after oral administration, and at five and 15 minutes after i.v.
    administration (Jones et al., 1956).

         The metabolism of various alkyl-p-hydroxybenzoates have been
    investigated in rabbits and in rats.

         It was noted that metabolism in the rat of p-hydroxybenzoic acid
    and its methyl, ethyl and propyl esters resulted in the appearance in
    the urine first of free p-hydroxybenzoic acid, followed by the
    glucuronide and p-hydroxyhippuric acid, the concentration of which
    increased as that of the free p-hydroxybenzoic acid fell out (Derache
    & Gourdon, 1963).

         In the rat the propyl ester was eliminated as p-hydroxybenzoic
    acid, p-hydroxyhippuric acid, as ester and ether glucuronides, as an
    ethereal sulfate and an unidentified compound. No unhydrolyzed ester
    was detected in the urine (Derache & Gourdon, 1963).

         Rabbits fed methyl p-hydroxybenzoate (0.8 g/kg bw) for three
    days excreted p-carboxyphenyl-ß-O-glucopyranosiduronic acid,
    p-hydroxybenzoic acid, and p-hydroxyhippuric acid as major urinary
    metabolites with minor amounts of the ester-type glucuronide and
    p-carboxyphenylsulfate (Tsukamoto & Terada, 1962). In another study
    where esters of p-hydroxybenzoate (methyl, ethyl, propyl, butyl,
    isopropyl, isobutyl, and secbutyl-p-hydroxybenzoate) were administered
    by stomach tube to rabbits at dose rates equivalent to 0.4 or
    0.8 g/kg, the alkyl esters were excreted in the urine as free acid
    (25-39%), glycine conjugate (15-20%), ester-type glucuronide (5-8%),
    ether-type glucuronide (10-18%), and sulfate (7-12%). The rate of
    excretion was decreased with increasing of chain length of the alkyl
    group (Tsukamoto & Terada, 1964).

         At high doses, propyl p-hydroxybenzoate was found to be a useful
    anaesthetic for frogs and tadpoles (Kopsch, 1949).


    Special studies on carcinogenicity


         Groups each of 20 weanling female mice were administered 0.1 ml
    of a 1% methyl p-hydroxybenzoate solution in polyethylene glycol, or
    polyethylene glycol alone, twice weekly into the vagina, for a period
    of 18 months. Of the control animals 11 to 20 survived 12 months, and
    7 to 20 the 18 months. Of the test animals 16 to 20 survived 12 months
    and 8 to 20 the 18 months. No carcinomas were observed in the test
    animals (Boyland et al., 1961).

         One hundred male mice (C57BL/6), seven weeks old were injected
    with 2.5 mg of methyl p-hydroxybenzoate, and five weeks after
    injection, injection site was excised and minced tissue injected into
    secondary host. Twenty-three weeks after injection, no carcinogenic
    response was observed (Homberger, 1968). In another study, female mice
    (C57BL/6) were injected i.v. with either methyl p-hydroxybenzoate, or
    methyl-p-hydroxybenzoate plus 3, 4, 9, 10-dibenzpyrene. The methyl
    p-hydroxybenzoate did not cause an acceleration of appearance of lung
    adenomas (Homberger, 1968).

    Special studies on neurotoxicity

         The blocking effect of a 0.1% solution of the methyl ester on
    nervous conduction when applied directly to the spinal roots or to the
    cervical vagus and sympathetics was found to be similar to that of a
    0.05% solution of procaine (Nathan & Sears, 1961). The local
    anaesthetic effect of the esters rose with increasing number of
    C-atoms and the toxicity decreased (Alder-Hradecky & Kelentey, 1960).

    Special studies on teratogenicity

         See butyl p-hydroxybenzoate.

    Special studies on chick embryo

         When femora from 10-day-old chick embryos were cultured for two
    days in a medium containing 10-5 or 10-6 M methyl p-hydroxybenzoate
    or concentrations of propyl p-hydroxybenzoate in the 10-5-10-7M range
    their dry weight increased in comparison in each case with that of the
    control femur from the same embryo. A mixture of the two parabens did
    not produce an additive effect. The favoured hypothesis is that
    parabens stabilize lysosymes and so prevent the partial autolysis that
    occurs in their absence during the early stages of culture (White,

    Acute toxicity


                        LD50                   References
    Animal    Route     (mg/kg bw)

    Mouse     oral      8 000                    Sokol, 1952

    Mouse     oral      Na salt: approx. 2 500   Matthews et al., 1956

    Mouse     i.p.      Na salt:         520     Matthews et al., 1956

    Guinea-   oral      2 000 - 2 400            Anon, 1939

    Rabbit    oral      5 000                    Sabalitschka &

    Dog       oral      5 000                    Sabalitschka &

         Doses of 5 g/kg were lethal in dogs and rabbits and 4 g/kg caused
    harmful effects (Schübel & Manger, 1929).


    Animal    Route     (mg/kg bw)               References

    Mouse     oral      8 000                    Sokol, 1952

    Mouse     oral      free acid: >8 000        Matthews et al., 1956

                        Na salt:    2 000        Matthews et al., 1956

              i.p.      free acid:  960          Matthews et al., 1956
                        Na salt:  760            Matthews et al., 1956

              i.v.      Na salt:  170            Matthews et al., 1956



    Animal    Route     (mg/kg bw)               References

    Guinea-   oral      3 000 -3 600             Anon, 1939

    Rabbit    oral      6 000                    Sabalitschka &

    Dog       oral      6 000                    Sabalitschka &

         Oral doses of 3000 mg/kg bw are reported to be lethal in the dog
    and rabbit, and doses of 2000 mg/kg bw caused harmful effects (Schubel
    & Manger, 1929).


    Animal    Route     (mg/kg bw)               References

    Mouse     oral      8 000                    Sokol, 1952

    Mouse     i.p.      400                      Sokol, 1952

    Mouse     oral      free acid: >8 000        Matthews et al., 1956

    Mouse     oral      Na salt: 3 700           Matthews et al., 1956

    Mouse     i.p.      free acid: 640           Matthews et al., 1956
                        Na salt: 490             Matthews et al., 1956

    Mouse     i.v.      Na salt: 180             Matthews et al., 1956

         Doses of 6 g/kg were lethal in dogs and rabbits, and 3-4 g/kg
    caused harmful effects (Schuebel & Manger, 1929).

    Short-term studies


         Daily doses of 11-100 mg methyl p-hydroxybenzoate for 120 days
    showed no effect. Forty animals were given i.d. injections of 0.1%
    solution in physiological saline, three times weekly up to 10
    injections. There was no sensitivity reaction two weeks later
    (Matthews et al., 1956).

         Mixed propyl and methyl esters were fed to animals on scorbutic
    diets. No additional pathological effects were noted (Cremer, 1935).
    For details see the methyl ester. Similar experiments on skin
    sensitivity to those done with the methyl ester revealed no skin
    sensitivity (Matthews et al., 1956).


         0.5-5.0 mg methyl p-hydroxybenzoate daily for 80 days had no
    effect. The blood picture was not influenced (Cremer, 1935). Groups of
    10 animals on a vitamin A deficient diet given 7.5-75 mg/kg bw of
    mixed methyl and propyl ester for 30 days showed no additional
    pathological changes (Cremer, 1935).

         Feeding experiments similar to those reported for the methyl and
    propyl esters were done at the 2% and 8% levels, but the duration was
    only 12 weeks. Concentrations of 2% were without effect, but there was
    reduced growth rate and evidence of toxicity at 8% (Matthews et al.,


         Suspensions of 0.5 and 7.5% ethyl p-hydroxybenzoate had the same
    local anaesthetic effect on the cornea as 0.12 and 0.27% solutions of
    cocaine hydrochloride. Therefore, the local anaesthetic activity of
    the ester is three to four times less than that of cocaine and twice
    that of procaine (Truhaut, 1962a). Using the same method (Regner &
    Quevauviller, 1939; Alder-Hradecky & Kelentey, 1960) found no local
    anaesthetic effect on the cornea by 0.25-0.30% solutions of methyl,
    ethyl, propyl or butyl p-hydroxybenzoates.


         Dogs were fed 0.7 g/kg bw propyl p-hydroxybenzoate for 90 days
    without ill effects or macroscopic changes (Ghirardi, 1940).

         Three mongrel puppies were fed 1000 mg/kg bw methyl
    p-hydroxybenzoate daily and two were fed 500 mg/kg daily for six days
    a week. Those in the lower dose group were on test for 313 days, and
    those on higher dose levels for periods longer than a year. At the end

    of the experimental period all the animals appeared in excellent
    condition with reasonable gains in weight. One animal receiving
    500 mg/kg produced a normal litter of puppies near the end of the
    experimental period. At a late stage of the experiment, blood samples
    were analysed for presence of the drug and for metabolic end-products,
    but there was no evidence of cumulation. Blood counts and urine
    examination were normal. All dogs were killed and autopsied; no
    abnormalities were found on microscopic or macroscopic examination of
    the organs (Matthews et al., 1956).

    Long-term studies


         Forty rats were fed 15 mg/kg bw, 20 animals 150 mg/kg bw and 20
    animals 1500 mg/kg bw of a mixture of 40% ethyl ester and 60% propyl
    ester as the sodium salts for 18 months in the diet. The rate of
    weight gain showed some growth stimulation at the 15 and 150 mg/kg bw
    levels. The group on 1500 mg/kg bw showed initial retardation followed
    later by normal growth. Mortality rate and pathological examination of
    the major organs in all treated groups showed no significant
    difference from the control group (Anon, 1940; Anon, 1942).

         When the methyl and propyl esters were fed to rats over an
    18-month period at a level of 150 mg/kg bw, no ill effects were
    observed. There was some evidence of growth stimulation. When fed at
    1600 mg/kg bw, there was a decrease in growth rate, but no
    pathological changes could be found (Sokol, 1952).

         Groups of 12 male and 12 female rats received 2% and 8% methyl
    p-hydroxybenzoate in the diet respectively and were compared with
    equal numbers of control animals over a period of 96 weeks. At the 2%
    level, the animals did not show significant differences from the
    controls, but at the 8% level there was a reduction in growth rate
    during the earlier part of the experiment, with a tendency to return
    to normal later. Food intake remained fairly constant throughout the
    experiment. All animals dying during the course of the experiment or
    killed at the end were autopsied and examined macroscopically and
    microscopically; no significant changes were found in the organs
    (Matthews et al., 1956).

         Experiments were done with groups of 12 animals, similar to those
    done with the methyl ester. No harmful effect was observed with a diet
    containing 2% of propyl p-hydroxybenzoate, but the growth rate was
    depressed at the 8% level. The feeding lasted for 96 weeks. There were
    no significant pathological findings (Matthews et al., 1956).

         A group of 65 rats (35 males and 30 females) was fed a diet
    containing 2% of the ethyl ester for life span, with 50 animals as the
    control group. All animals were autopsied on death. No adverse effects
    could be detected on weight gain, except a small retardation during
    the first month, and the mortality rate, haematology, tumour incidence
    and histopathology of major organs did not differ from the controls
    (Truhaut, 1962b).

         A group of 39 rats (19 males and 20 females) was injected weekly
    with 1 ml of an aqueous solution of 10% sodium ethyl p-hydroxy-
    benzoate for their life span. The 27 controls (16 males and 11
    females) were injected with 1 ml of a 3% sodium chloride solution.
    Because of irritation by the high pH of the ester solution, the
    frequency of injection had to be reduced to one in two weeks from the
    fourth to the tenth month and later to one injection a month up to the
    end of the experiment. No effect on mortality and tumour incidence
    could be detected (Truhaut, 1962).


         Sensitivity: see under the butyl ester (Observations in man).

         Solutions of methyl p-hydroxybenzoate in propylene glycol, in
    concentrations up to 5%, were applied to the skin of 50 human subjects
    for four to eight hours every other day up to 10 applications without
    evidence of irritation. Higher concentrations than this produced some
    irritation. There was no evidence of development of sensitization
    (Matthews et al., 1956).

         Tests for skin irritation and sensitivity were made similar to
    those done with the methyl ester. Ethyl p-hydroxybenzoate at a
    concentration of 7%, no evidence of irritation or sensitivity was
    observed, but irritation occurred at higher concentrations. Ingestion
    of 0.05% aqueous solutions caused local anaesthesia of the buccal
    mucosa (Bubnoff et al., 1957).

         A local anaesthetic effect on the buccal mucosa has been reported
    after ingestion of 0.1% aqueous solution of methyl p-hydroxybenzoate
    (Bubnoff et al., 1957). Two grams of the ester taken daily for one
    month produced no ill effects (Sabalitschka & Dietrich, 1924).

         The propyl ester has been used therapeutically at doses of
    800 mg/kg bw over three days for the treatment of moniliasis (Rossier
    & Wegmann, 1953). Tests for skin irritation and sensitivity were made
    similar to those done with the methyl ester. At a concentration of
    12%, no evidence of irritation or sensitivity was observed, but
    irritation occurred at higher concentrations (Matthews et al., 1956).
    Ingestion of 0.03% aqueous solutions caused local anaesthesia of the
    buccal mucosa (Bubnoff et al., 1957).

         Two grams propyl ester was given daily to human volunteers for
    50 days. No unhydrolyzed ester could be detected in the urine
    (Sabalitschka & Neufeld-Crzellitzer, 1954).

         Human studies on six subjects involved administration of 10 or
    20 mg/kg bw of the propyl ester orally. After 60, 135 and 255 minutes,
    p-hydroxybenzoate but not the ester, was detectable in serum. The
    maximum serum level attained was 4.5 µg/ml (Heim, 1960-61).


         The long-term studies in rats are adequate for an assessment when
    taken in conjunction with the evidence from the feeding experiments
    lasting for a year with the dogs. Biochemical studies in animals have
    provided detailed information on metabolism. However, additional
    studies in man for methyl p-hydroxybenzoate and further biochemical
    studies in man and animal for the ethyl ester would be desirable.
    Allergic responses to the ethyl and methyl esters have been reported.


         Applicable to the methyl, ethyl and propyl esters.

    Level causing no toxicological effect


         20 000 ppm (2%) in the diet, equivalent to 1000 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-10 mg/kg bw.*


         p-hydroxybenzoate, ethyl: further biochemical studies in man and

         p-hydroxybenzoate, methyl: additional studies in man.


    *    Sum of ethyl, methyl and propyl esters of p-hydroxybenzoic acid.


    Alder-Hradecky, C. & Kelentey, B. (1960) Arch. int. Pharmacodyn.,
         128, 135

    Boyland, E., Charles, R. T. & Gowing, N. F. C. (1961) Brit. J. Cancer,
         15, 252

    Bubnoff, M. von., Schnell, D. & Vogt-Moykoff, J. (1957) Arzneimitt
         Forsch., 7, 340

    Cremer, H. (1935) Z. Lebensmitt. Untersuch., 70, 136

    Derache, R. & Gourdon, J. (1963) Food Cosmet. Toxicol., 1, 189

    Ghirardi, G. E. (1940) Arch. ital. Sci. farmacol., 9, 282

    Heim, F. (1960-1961) Sitzber, Physik-Med. Soz. Erlangen, 81, 14

    Anon. (1939) Unpublished report from Applied Research Inc. MX-124

    Anon. (1940) Unpublished report from Applied Research Inc. MX-185

    Anon. (1942) Unpublished report from Applied Research Inc. MX-185

    Homberger, F. (1968) PB-183207

    National Technical Information Service, Springfield, Va., USA.

    Jones, P.S. et al. (1956) J. Amer. pharm. Ass., Sci. Ed., 45, 268

    Kopsch, F. (1949) Anat. Anz., 97, 158

    Matthew, C. et al. (1956) J. Amer. pharm. Ass., Sci. Ed., 45, 260

    Nathan, P. W. & Sears, T. A. (1961) Nature (Lond.), 192, 668

    Quick, A. J. (1932) J. biol. Chem., 97, 403

    Regnier, M. & Quevauviller, A. (1939) Arch. Exp. Path. Pharm., 193, 48

    Rossier, P. H. & Wegmann, T. (1953) Wien. Med. Wschr., 19/20, 358

    Sabalitschka, T. & Dietrich, K. R. (1924) Pharmaz. Monatshefte, 5, 235

    Sabalitschka, T. & Neufeld-Crzellitzer, R. (1954) Arzneimitt-Forsch,
         4, 575

    Schuebel, K. & Manger, J. (1929) Arch. exp. Path. Pharmak., 146, 208

    Sokol, H. (1952) Drug Stand., 20, 89

    Truhaut, R. (1962a) Estratto dai Rendiconti dell-Instituto Superiore
         di Sanità (Document submitted to WHO in 1964)

    Truhaut, R. (1962b) Ann. pharm, franz., 20

    Tsukamoto, H. & Terada, S. (1962) Chem. Pharm. Bull. (Tokyo), 10, 86

    Tsukamoto, H. & Terada, S. (1964) Chem. Pharm. Bull. (Tokyo), 12, 765

    White, A. A. (1967) Proc. Soc. exp. Biol. Med., 126, 588

    See Also:
       Toxicological Abbreviations