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 compounds 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 monograph has been expanded and is reproduced
    in its entirety below.



         Sorbic acid did not act as an antimetabolite for essential fatty
    acids in the rat. The incorporation of sorbic acid into the diet of
    rats did not decrease the efficiency of utilization of calories.
    Sorbic acid is used by the animal organism as a source of calories
    (Deuel et al., 1954a). Sorbic acid is metabolized similarly to caproic
    acid (Cohen, 1937; Witter et al., 1950; Deuel et al., 1954b).

         In vitro studies using isolated rat liver particle systems show
    oxidation via 2-carbon atom fragments, which recondense to
    acetoacetate (Witter et al., 1950). Tissue homogenates or isolated
    mitochondria oxidize sorbic acid at the same speed as other fatty
    acids (Lang, 1960). The oxygen consumption of liver homogenates of
    parent rats fed for 120 days on diets containing 0% and 10% sorbic
    acid was similar in both groups. The female controls of the F1
    generation showed a significantly increased O2 consumption compared
    with male or female F1 rate on 10% sorbic acid (Demaree et al.,
    1955). Fasting female rats with exogenous ketonuria when fed sodium
    sorbate at various levels with or without additional glucose, produced
    similar amounts of ketone bodies in the urine or showed similar
    suppression of keto body formation as with caproic acid (Deuel et al.,
    1954b). Enzyme studies have shown that a concentration of 0.112% of
    sorbic acid inhibited catalase activity by 72 to 77% (Lück, 1957).
    Sulfhydryl enzymes in ficin and alcohol dehydrogenase were inhibited
    at a concentration of 10-4M. Aldolase and urease were not
    significantly inhibited by sorbic acid. Irradiated sorbic acid was a
    stronger inhibitor of ficin than sorbic acid (Whitaker, 1959). This
    inhibition of dehydrogenase is the main basis for the fungistatic
    activity of sorbic acid. It appears to be impossible to give
    sufficient sorbic acid to inhibit the dehydrogenase enzyme systems in
    the animal body (Melnick, et al., 1954a).

         When sorbic acid is incorporated into food it may undergo
    oxidation, with the formation of peroxides and secondary oxidation
    products. In the presence of sufficient metabolizable carbohydrates
    the end-products are carbon dioxide and water. If metabolizable
    carbohydrates are not present, acetoacetate and acetone are also
    produced (Melnick et al., 1954b).

         The metabolism of 1-14C-sorbic acid has been studied by
    Fingerhut et al. (1962a); 85% of the activity was found in the expired
    CO2, 0.4% in the faeces, 2% in the urine as urea and CO2, 3% in
    internal organs, 3% in the skeletal muscles and 6.6% in the other
    parts of the carcass. No glycogen was formed from sorbic acid. Most of
    the activity was found in the subcutaneous fat deposits and in the
    lipids of the organs. There was a linear relationship between dose and
    oxidation rate; the half-life of the oxidation was 40-110 minutes in
    the dose range from 60-1200 mg/kg bw. In a similar experiment on mice,
    also using 1-14C-sorbic acid, these results were confirmed; 81 ± 10%
    of the sorbic acid was oxidized to CO2, the dose given ranging from
    40-3000 mg/kg bw. About 7% of the activity was excreted as sorbic acid
    and 0.4% as trans, trans-muconic acid (Westöö, 1964).

         The studies on the metabolism of 14C-labelled sorbic acid were
    performed by using either sorbic acid neutralized with sodium
    hydroxide (Fingerhut et al., 1962) or the potassium salt (Westöö,


    Special studies on reproduction


         Mice fed for eight months 0 or 40 mg + 2 mg Nisin/kg/day sorbic
    acid were mated and allowed to produce F1 to F4 generations. Weight
    gains were compared at 3.5 months after weaning. Weight gain was
    better than controls in the F4 only (Shtenberg and Ignatev, 1970).


         Groups of five male and five female rats were kept for 120 days
    on either 0 or 10% sorbic acid. After 60 days they were mated and
    produced similar numbers of off-spring. Fourteen rats of the F1
    generation were kept on control diet, 19 on 10% sorbic acid for a
    further 70 days and mated. The number of off-spring were similar in
    both groups. Females on 10% had normal liver weight but males showed
    reduced growth (Demarce et al., 1955).

    Acute toxicity

                                         LD50     References
    Substance          Animal  Route  (mg/kg bw)

    Sorbic acid        rat     oral    10 500     Deuel et al., 1954a

                       rat     oral     7 400     Witter et al., 1950
                               oral     7 360     Smyth & Carpenter, 1948

    Sodium sorbate     rat     oral     7 160     Smyth & Carpenter, 1948

    Sodium sorbate     rat     oral     4 000 &   Deuel et al., 1954a
                                        5 940

    Na-sorbate         rat     oral     7 200     Witter et al., 1950

    Potassium sorbate  rat     oral     4 920     Mellon Institute, 1954
    (solid isomer)

    Potassium sorbate  rat     oral     6 170     Mellon Institute, 1954
    (mixed isomer)

    K-sorbate          rat     oral     4 200

    Sodium sorbate     mouse   i.p.     2 500     Rhône-Poulenc, 1965

    Potassium sorbate  mouse   i.p.     1 300     Rhône-Poulenc, 1965

    Short-term studies


         Groups of 25 male or 25 female mice received for two months by
    oral intubation daily 40 mg/kg/day sorbic acid. Survival, weight gain
    and food consumption were no different from controls: at the end of
    the study food was restricted by 50% for five days. Mortality and
    weight loss was less than in controls. Physical stress had no effect,
    mortality after giving 0.1 ml CC14 was less in sorbic acid treated
    mice than controls. Groups of 50 males and 50 females received by oral
    intubation 80 mg/kg sorbic acid for three months. Growth was somewhat
    restricted compared with controls. When stressed by 90% food
    restriction for 18 days there was no difference in mortality from
    controls. When given 0.1 ml CC14 mortality was nil compared to 30% in
    controls (Shtenberg & Ignatev, 1970).


          Feeding sorbic acid at 1% or 2% in the diet for 80 days had no
    adverse effect on the growth of rats nor did any histological
    abnormalities appear in internal organs. Only the liver was slightly
    enlarged compared with controls (Kramer & Tarjan, 1962). Feeding 1%
    sorbic acid in the diet for four months had no effect on blood
    cholesterol level while 10% raised the level together with fat
    deposition in the internal organs. Leucocytes number was depressed
    after two months and there was partial impairment of cholinesterase
    activity (Slavkov & Petrowa, 1964). When groups of five male and five
    female rats were kept on a diet with 0% and 10% sorbic acid for 120
    days appearance, behaviour and food consumption were normal but some
    animals at the 10% level showed increased liver/bw ratios (Demarce et
    al., 1955).

         Tests with two different strains of rats in two separate
    laboratories showed that ingestion of diets containing 4% and 8% of
    sorbic acid for a period of 90 days did not affect the rate of weight
    gain. The animals receiving 4% of sorbic acid showed no abnormality of
    renal, hepatic or other tissues. Rats on the 8% diet showed a slight
    but statistically significant increase in relative liver weight. The
    histopathological appearance of the liver was, however, normal (Deuel
    et al., 1954a).

         Sorbic acid was shown to produce local sarcomas in rats when
    given by repeated subcutaneous injection at the same site in either
    arachis oil or in aqueous solution. The potassium salt when
    administered under similar conditions failed to produce any tumours.
    Sorbic acid in the drinking-water (10 mg/100 ml) for 64 weeks and the
    potassium salt given either in the drinking-water (0.3%) or in the
    diet (0.1%) for 100 weeks failed to produce any tumours (Dickens et
    al., 1966, 1968).

         Groups of 10 rats (five male and five female) were fed potassium
    sorbate (solid or mixed isomers) at levels of 0, 1, 2, 5 and 10% of
    the diet for three months. Body weight gain was initially depressed at
    10%, and, to a lesser degree, in the 5% female group. There are no
    figures given for the food consumption during this time. At the end of
    the experiment, the weights of the rats receiving 10% of either isomer
    were slightly depressed, but the food consumption of these animals was
    smaller. The food efficiency (weight gain/g food) was practically the
    same as in the control group. Relative liver weights were the same in
    all groups. Kidney weights were increased at the 10% level, probably
    due to the high potassium load. This was also noted to a lesser
    degree at the 5% level. Gross pathological examination showed no
    abnormalities, even in the 10% level groups (Mellon Institute, 1954).


         3.3 g sorbic acid daily/kg bw was tolerated by rabbits without
    adverse effects (Kuhn et al., 1937).


         Eight dogs received 1% and eight dogs 2% of the solid or mixed
    isomers of potassium sorbate in the diet for three months; four dogs
    were used as controls. There were no differences in weight gain. Gross
    examination on autopsy showed no evidence of a deleterious effect
    attributable to the sorbates (Mellon Institute, 1954).

         In dogs fed for three months on a diet containing 50% of cheddar
    cheese to which 4% of sorbic acid or 4% of caproic acid had been
    added, the response was similar to that in dogs on the same cheese
    diet without such supplements. No histopathological differences were
    observed in tissues obtained from any of the three groups (Deuel et
    al., 1954a).

    Long-term studies


         Groups of 33 male and female mice were injected s.c. up to 44
    times with sorbic acid in arachis oil to a total of 31 mg sorbic acid.
    Controls had arachis oil only and 33 controls were untreated. Animals
    survived on average 40 weeks (range 4-80 weeks). No animal developed
    malignant tumours at the site of injection. Spontaneous mammary
    carcinomas occurred in equal numbers in untreated and treated groups
    (Gericke, 1968). Groups of 50 male and 50 female mice were given
    40 mg/kg/day sorbic acid in their diet. No tumours were seen in
    control or sorbic acid group. General condition, behaviour, weight
    gain and survival were similar in control and test groups. The
    relative weights of liver, kidney and testes were lower in the test
    group than in controls. 100% starvation for 13 days at the end of the
    test increased mortality compared with controls (Shtenberg & Ignatev,


         Groups of 10 male and 10 female rats received 40 mg/kg/day sorbic
    acid in their diet for 18 months. After the end of the test, animals
    were starved for 13 days completely but the test groups survived
    longer than controls. Weight gain was not measured. Blood pH,
    C-reactive protein levels and blood morphology were comparable with
    controls and no significant changes were seen in serum ceruloplasmin,
    serum complement or phagocytic activity of leucocytes. Tumour results
    were not reported (Shtenberg & Ignatev, 1970).

         Groups of six rats were given 0.1% K-sorbate in food and 0.3%
    K-sorbate in drink for 60 weeks.  Survival and general conditions were
    satisfactory. No special features were seen at 65 weeks in exploratory
    laparotomy nor at 100 weeks when all animals had died. No tumours were
    detected (Dickens et al., 1968).

         Groups of six rats received by s.c. injection (light) sorbic acid
    in water or K-sorbate (light) in water and groups of 12 rats received
    SG/sorbic acid (Hoechst) in oil, and K-sorbate (Hoechst) in water. A
    control group of 12 received oil only. Dosage was 2 mg/0.5 ml twice
    weekly for 56-60 weeks. No local or distant tumours were seen in 12
    controls, three of which survived 108 weeks. Sorbic acid (light) in
    water produced two local fibrosarcomata at the injection site, all six
    animals being dead at 76 weeks. Sorbic acid (Hoechst) in oil produced
    no local tumours at all with two animals surviving 108 weeks.
    K-sorbate (light) in water produced no local tumours in six rats,
    three animals surviving 100 weeks and K-sorbate (Hoechst) in oil also
    produced no tumours in 12 rats with two surviving 108 weeks (Dickens
    et al., 1968).

         Feeding 5% of sorbic acid in the diet to groups of 100 rats for
    1000 days in two generations had no effect on weight gain or
    reproduction. No ill effects were observed, and no sorbic acid could
    be found in the urine (Lang et al., 1967).

         The feeding experiment on groups of 100 rats (50 males and 50
    females) given 0 and 5% sorbic acid was extended to the whole life-
    span of the first generation. The average life-span of the group
    receiving sorbic acid was 811 days for the males and 789 days for the
    females. In the control group the life-span of the males was 709 days
    and for the females 804 days, possibly suggesting protection by sorbic
    acid against lung infection. Autopsies were performed on all rats of
    the first generation that died during the experiment. There were no
    differences in the organ weight of the individual groups nor in the
    distribution of the causes of death. In each group (5% sorbic acid and
    controls) only two tumours were found. The animals of the second
    generation were sacrificed after 250 days of feeding sorbic acid.
    Examination of liver, kidney, heart and testes showed no abnormalities
    (Lang, 1960: Lang et al., 1967).


         Sorbic acid is readily metabolized. Both man and rat appear to
    utilize identical metabolic mechanisms for oxidation of sorbate. The
    long-term studies suggest that the same no-effect level applies to the
    salts as to the free acid.  Sorbic acid and K-sorbate corresponding to
    the specifications do not cause tumours when administered orally or
    subcutaneously. The earlier results of s.c. injection with an

    unidentified sample remain unexplained. Long-term studies on
    parasorbic acid which, it has been claimed, may be produced from
    sorbic acid, also produce no evidence of carcinogenic potential when
    given orally.


    Level causing no toxicological effect

         Rat: 50 000 ppm (5%) in the diet, equivalent to 2500 mg/kg bw per

    Estimate of acceptable daily intake for man

         mg/kg bw 


    Cohen, P. P. (1937) J. biol. Chem., 119, 333

    Demarce, G. E. et al. (1965) J. Amer. Pharm. Ass. Sci., 44, 619

    Deuel, H. J. et al. (1954a) Food Res., 19, 1

    Deuel, J. J. et al. (1954b) Food Res., 19, 113

    Dickens, F., Jones, H. E. H. & Waynforth, H. B. (1966) Brit. J.
         Cancer, 20, 134

    Dickens, F., Jones, H. E. H. & Waynforth, H. B. (1968) Brit. J.
         Cancer, 22, 762

    Fingerhut, M., Schmidt, B. & Lang, K. (1962) Biochem. Z., 335, 118

    Gericke, D. (1968) Z. Ernahrungswiss., Suppl. 7, 29

    Kramer, M. & Tarjan, R. (1962) Int. Z. Vitaminforsch., 32, 149

    Kuhn, R., Köhlei, F. & Köhlei, L. (1937) Hoppe Seyler's Z. Physiol.
         Chem., 247, 197

    Lang, K. (1960) Arzneimittel-Forsch., 10, 997


    *    As sum of sorbic acid and calcium, potassium and sodium sorbate
    (expressed as sorbic acid).

    Lang, K. et al. (1967) Unpublished report

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

    Mellon Institute (1954) Unpublished report

    Melnick, D., Luckmann, F. H. & Gooding, C. M. (1954a) Food Res., 19,

    Melnick, D., Luckmann, F. H. & Gooding, C. M. (1954b) Food Res., 19,

    Rhône-Poulenc (1965) Unpublished report

    Shtenberg, A. J. & Ignatev, A. D. (1970) Fd. Cosmet. Toxicol., 8, 369

    Slavkov, B. & Petrowa, A. (1964) Higiena (Sofia) 7, 4, 52

    Smyth, H. F. jr & Carpenter, C. P. (1948) J. industr. Hyg. Toxicol.,
         30, 63

    Westöö, E. (1964) Acta chem. scand., 18, 1373

    Whitaker, J. R. (1959) Food Res., 24, 37

    Witter, R. F., Newcomb, E. H. & Stotz, E. (1950) J. biol. Chem.,
         185, 537

    See Also:
       Toxicological Abbreviations