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,
    Ref. No. 7) in 1963.

         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.



         The coefficients of digestibility of the fatty acid moieties of
    polyoxyethylene (20) sorbitan monooleate, monostearate and tristearate
    were found to be 100%, 98% and 84%, respectively, when fed at
    concentrations of 10% in the diet (Oser & Oser, 1957a).

         Studies with 14C-labelled polyoxyethylene sorbitan monostearate
    in rats showed that 6-10% was excreted in the urine, 2-7% was
    recovered in the expired air, and the remainder was excreted in the
    faeces. Labelling with 14C was confined to the sorbitol part of the
    molecule (Wick & Joseph, 1956).

         The amounts of 14C-containing compounds found in the urine and
    expired air were considerably smaller than in similar studies with
    14C-labelled sorbitan monostearate (Wick & Joseph, 1953).

         Studies in six human subjects with polyoxyethylene (20) sorbitan
    monooleate showed that the distribution of the polyoxyethylene moiety
    was 3.9-5.8% in the urine and 90.4-98.3% in the stools; total recovery
    of the polyoxyethylene in these investigations was 96.2-102.2% (Culver
    et al., 1951).

         Polyoxyethylene (20) sorbitan monooleate ("Tween 80") fed to rats
    for one week in amounts of 0.1% and 1% of the diet augments the
    absorption of fats when these are present in preparations of 10% to
    33% of the diet, but not when fats comprise less than 7% of the diet
    (Sergiel et al., 1971).

         Polysorbate (80) and polysorbate (20) [= polyoxyethylene (20)
    sorbitan monooleate and polyoxyethylene (20) sorbitan monolaurate] in
    concentrations of 0.01% or less increase the absorption of lipid
    soluble drugs (4-amino-antipyrine = ampyrone; secobarbitone),
    indicating that these surfactants affect the permeability of
    biological membranes (Levy & Anello, 1969; Anello & Levy, 1969).

         However, administration of 0.03% of polysorbate 80 to rats
    together with drugs (4-aminoantipyrine, salicylic acid, salicylamide)
    did not affect their absorption from the small intestine (Levy &
    Perälä, 1970).

         Metabolic studies were carried out in rats with polyoxyethylene
    (20) sorbitan monolaurate with 14C-label in the lauric acid moiety or
    the polyoxyethylene moiety. Doses administered were 1 g/kg orally and
    0.5 g/kg i.v. With the label in the fatty acid, the approximate
    proportions of radioactivity 24 hours after oral administration were:
    liver, 1.2%;urine, 2.5%; faeces and gastrointestinal contents, 4%;
    carcass, 12%; expired CO2, 80%; after i.v. injection, they were
    liver, 0.7%; urine. 5%; faeces and gastrointestinal contents, 2.5%;
    carcass, 22%; expired CO2, 68%. With the label in the polyoxyethylene
    moiety, the distribution of radioactivity 24 hours after oral
    administration was different: liver, nil; urine, 8%; faeces, 90%;
    carcass, nil; expired CO2, nil. After i.v. administration the
    distribution was: liver 0.15%; urine, 83%; faeces, 11%; carcass, 2%;
    expired CO2, nil. The findings indicate the fatty acid ester bond is
    hydrolyzed and the fatty acid is utilized metabolically; however, the
    polyoxyethylene moiety is poorly absorbed and is not subject to
    metabolism (Nelson et al., 1966).

         Metabolic studies were carried out in rats after oral
    administration of polyoxyethylene (20) sorbitan monolaurate ("Tween
    20") prepared with 14C-labelling of the polyoxyethylene or the lauryl
    moieties. The 14C-lauryl moiety was rapidly absorbed and oxidized,
    more so in fasted than non-fasted rats. After 24 hours, some 75% of
    the administered radioactivity was recovered in expired CO2, and 4%
    was not absorbed. The 14C-polyoxyethylene moiety was poorly absorbed,
    87% of radioactivity remaining unabsorbed, and about 8.5% was
    recovered in the urine.  After i.v. injection of the substance, the
    14C-lauryl moiety was metabolized as after oral administration.
    The 14C-polyoxyethylene moiety was not metabolized, since no
    radioactivity was recovered as 14C-CO2: most appeared in the urine,
    but some was present in the faeces indicating biliary excretion.
    Polyoxyethylene (20) sorbitan monooleate ("Tween 80") with a
    14C-sorbital label was also administered to rats. After oral
    administration, 91% of the radioactivity was recovered in the faeces,
    2.1% in the urine, 1.6% in the carcass and none in expired CO2,
    liver, kidney, spleen, adrenals, brain, gonads or fat (Treon et al.,

         Polyoxyethylene (20) sorbitan monooleate ("Tween 80") in
    concentrations of 0.2 and 1 mM did not affect bile salt transport in
    isolated preparations of rat intestine; 4 mM slightly inhibited
    transport (Holt, 1964).

         Polyoxyethylene (20) sorbitan monooleate (polysorbate 80) in a
    concentration of 0.5% decreased the rates of absorption from rat small
    intestine perfused in situ of various drugs (sulfamethoxypyridazine,
    diphenhydramine, salicylic acid, p-hydroxybenzoic acid) (Aoki et al.,

         The absorption of aspirin from the gut into the blood in rats was
    facilitated by simultaneous administration of polyoxyethylene (20)
    sorbitan monooleate in a dose of 50 mg/kg, but not by 10 mg/kg (Cid et
    al., 1971).


    Acute toxicity

         The acute oral toxicity in the mouse, rat and hamster was shown
    to be extremely low (Eagle & Poling, 1956; Hopper et al., 1949).

         Polyoxyethylene (20) sorbitan monolaurate has an LD50 of
    3.75 g/kg bw when injected i.v. into mice (Hopper al., 1949).

         The accidental administration of a dose of 19.2 g of
    polyoxyethylene (20) sorbitan monooleate per kg bw on two successive
    days to an infant was followed by no ill effects, apart from purgation
    (Chusid & Diamond, 1955).


    "Tween 20" =   polyoryethylene (20) sorbitan monolaurate:
                   Oral LD50 > 39 000 mg/kg
                   i.v. LD50    1 450 mg/kg
                   (male, 1680; female, 860)

    "Tween 40" =   polyoxyethylene (20) sorbitan monopalmitate:
                   Oral LD50 > 38 400 mg/kg
                   i.v. LD50    1 580 mg/kg
                   (male, 2320; female; 1350)

    "Tween 60" =   polyoxyethylene (20) sorbitan monostearate:
                   Oral LD50 > 38 000 mg/kg
                   i.v. LD50    1 220 mg/kg
                   (male, 1750; female, 1180)

    "Tween 65" =   polyoxyethylene (20) sorbitan tristearate:
                   Oral LD50 > 40 000 mg/kg

    "Tween 80" =   polyoxyethylene (20) sorbitan monooleate:
                   Oral LD50 > 38 000 mg/kg
                   i.v. LD50    1 790 mg/kg
                   (male, 2180; female, 1720)

                                                      (see Brandner, 1973)

         A range of polyethylene sorbitan esters of fatty acids ("Tween
    20, 21, 40, 60, 61, 65, 80, 81, 85") failed to produce reactions on
    patch testing of each in 50 subjects. On instillation in the rabbit
    conjunctival sac, most failed to produce a reaction even when the eye
    was not subsequently washed and did not produce reaction when the eye
    was washed (Treon et al., 1967).

    Short-term studies


         Groups of 10 to 12 mice, studied over three to four months were
    found to tolerate 2.5%, 5% and 10% polyoxyethylene (20) sorbitan
    monostearate without showing any ill effects, but at the 15% level
    there was some gastrointestinal disturbance, with reduced food intake
    and some growth retardation (Brush et al., 1957).


         Groups of young rats fed 3% and 5% of polyoxyethylene (20)
    sorbitan monolaurate for eight weeks showed a significantly slower
    weight gain as compared with controls; this was attributed to mild
    diarrhoea. However, there were no apparent gross abnormalities nor any
    significant histopathological findings in the treated animals (Krantz,

         When groups of 30 rats were fed 2% of polyoxyethylene (20)
    sorbitan monooleate in their diet for three generations, no evidence
    of alteration in their fecundity and growth pattern was found, nor
    were there any histological findings in the livers and kidneys
    (Krantz, 1946).

         No toxic symptoms were found when rats were fed for eight weeks
    on 2% and 5% polyoxyethylene (20) sorbitan monostearate in their daily
    diet (Krantz, 1943b).

         A 15-week feeding study using 25% polyoxyethylene (20) sorbitan
    monostearate in the daily diet of male rats resulted in retardation of
    growth as compared with control animals on the same basic diet. The
    rats receiving the diet containing polyoxyethylene (20) sorbitan
    monostearate exhibited transient diarrhoea, but the haematological
    findings were normal and no abnormalities were revealed on gross
    pathological and histological examination of the important viscera
    (Krantz, 1949).

         Polyoxyethylene (20) sorbitan monostearate was fed for 14 weeks
    to groups of 12 male and 12 female weanling rats as 5% and 15% of a
    soybean meal diet. There were no clinical manifestations of toxicity
    throughout the experimental period, and the histopathological
    examination of the animals at the end of the experiments showed no
    abnormalities. Neither were deleterious effects observed when 5%
    polyoxyethylene (20) sorbitan monostearate in the soybean basal diet
    was fed to older rats for a period of 14 weeks. On the other hand,
    polyoxyethylene (20) sorbitan monostearate fed to weanling rats as 5%
    of a basal purified casein diet caused diarrhoea and retardation of
    growth (Chow et al., 1951; Chow et al., 1953).


         Weanling hamsters (12 per group) were fed 1% and 5% of
    polyoxyethylene (20) sorbitan monostearate in the diet for one year.
    For the first six weeks growth and food efficiency were the same as in
    controls. During the 12-month feeding period, the mortality and body
    weights of the treated animals were not significantly different from
    those of the controls, but chronic diarrhoea was exhibited at the 5%
    level. When the animals were sacrificed at the end of the experiment,
    no significant differences were noted between the treated animals and
    the controls in regard to organ weights and pathological findings,
    except that in the kidneys of those at the 5% level, there was a
    significantly greater frequency of hyaline casts and chronic
    interstitial nephritis, believed to be associated with the water
    imbalance brought about by the chronic diarrhoea (Brush et al., 1957).

         In another experiment with hamsters (10 in each group), which had
    been fed 5%, 10% and 15% levels of polyoxyethylene (20) sorbitan
    monolaurate, fatalities and chronic diarrhoea were unusually frequent,
    and when the remaining animals were sacrificed at 28 to 39 weeks
    highly significant changes were found in a number of organs (Eagle &
    Poling, 1956).

         A high mortality was also found in hamsters (10 per group) fed
    diets containing 10% and 15% of polyoxyethylene (20) sorbitan
    monolaurate. With 5% in the diet, growth was significantly retarded
    and diarrhoea occurred (Poling et al., 1956).

         Similar results were obtained when groups of 36 hamsters were fed
    diets containing 5% and 15% of polyoxyethylene (20) sorbitan
    monolaurate for 68 days (Harris et al., 1951).


         Beagle puppies were fed for one year on diets containing 5% and
    10% of polyoxyethylene (20) sorbitan monostearate; no abnormalities
    were observed (Brush et al., 1957).


         When six monkeys were fed polyoxyethylene (20) sorbitan
    monolaurate or monooleate at the rate of 1 g a day for periods of up
    to 17 months there were no significant changes in weight gain or
    histopathology (Krantz, 1943a, 1947a).


         Polyoxyethylene (20) sorbitan monostearate and monolaurate fed to
    groups of 12 chicks at levels of 0.1%, 1% and 2% of the diet for seven
    weeks did not cause any adverse effects (Ringrose & Waller, 1959).

    Long-term studies


         Long-term feeding studies have been carried out on groups of
    15-30 rats with polyoxyethylene (20) sorbitan monolaurate (Oser &
    Oser, 1957b), at levels of 0.5% and 2% in the diet and with
    polyoxyethylene (20) sorbitan monopalmitate (Wick & Joseph, 1956),
    monostearate (Wick & Joseph, 1953), tristearate (Culver et al., 1951),
    and monooleate (Eagle & Poling, 1956) at a level of 2%.

         As compared with control animals, no abnormalities were found
    that could be attributed to the experimental diets. In addition to
    growth measurements, mortality statistics and studies of blood
    chemistry, gross and histological examinations were made of the
    following organs: brain, spleen, pancreas, thyroid, parathyroid,
    prostate, pituitary, salivary and adrenal glands, bladder, liver,
    kidney, bone marrow, heart, lung, testis, lymph nodes and muscle (Oser
    & Oser, 1957a; Wick & Joseph, 1956; Wick & Joseph, 1953; Culver et
    al., 1951; Eagle & Poling, 1956; Krantz, 1943c, 1947b, 1947c, 1947d,

         Four groups of rats, each containing 12 males and 12 females,
    were fed for the whole life-span with diets containing 2%, 5%, 10% and
    25% of polyoxyethylene (20) sorbitan monostearate. This study revealed
    no effect of the ester at the 2% and 5% levels. However, the compound
    produced marked diarrhoea at the 10% and 25% levels, as well as
    enlargement of the caecum (slight to moderate at the 25% level, less
    at the 10% level), and a questionable fatty change of a very slight
    degree in the livers of the rats fed the 25% level (Fitzhugh et al.,

         In another experiment, the effects of feeding polyoxyethylene
    (20) sorbitan monostearate, tristearate and monooleate to groups of
    12 males and 20 females over the whole life-span at dosage levels of
    5%, 10% and 20% of the diet were studied. Observations were also made
    on three successive generations. This extensive study included tests

    on gestation and fertility, mortality, blood and urine constituents,
    and histopathology. No abnormalities were found at the 5% level. At
    the 10% and 20% levels, many of the animals, particularly the males,
    had diarrhoea. The 20% level had some adverse effect on postnatal
    survival, lactation efficiency, and duration of breeding activity.
    Growth rate in the males and caloric utilization efficiencies were
    slightly diminished (Oser & Oser, 1957a; Oser & Oser, 1956a, 1956b,

         Two colours which produce tumours when given subcutaneously,
    Brilliant Blue FCF and Solid Green FCF (? le Vert solide FCF?) were
    given orally in a concentration of 1% together with a surface active
    agent, polyoxyethylene (20) sorbitan monostearate ("Tween 60") or
    polyoxyethylene (20) sorbitan monooleate ("Tween 80"), in a
    concentration of 5% to rats for two years. The presence of the
    surfactants did not affect the incidence of tumours. In another
    experiment, it was shown that they did not affect the absorption of
    the dyes (Truhaut, 1970).


         4.5-6 g of polyoxyethylene (20) sorbitan monooleate were taken
    daily by 100 adults - 10 for three to four years, 17 for two to three
    years, 19 for one to two years, 54 for less than one year. No
    deleterious effects could be demonstrated (Krantz, 1951).

         These results have been confirmed by other authors (Steigmann,
    1953; Waldstein et al., 1954).


         Some of the early short-term studies with these polyoxyethylene
    sorbitan esters in rats and hamsters showed deleterious effects.
    Subsequent work suggests that these were largely due to diarrhoea
    resulting from a large amount of unabsorbed polyglycol, possibly
    aggravated in some experiments by the use of an unsuitable basal diet.
    Since that time there has been considerable improvement in testing
    procedures, and more extensive long-term studies have been carried
    out. It seems reasonable therefore to base the evaluation of these
    substances on the levels causing no adverse effects indicated by the
    results of the more recent investigations.

         The significance of the local tumours which were produced by
    injection has been discussed at the meeting of the Scientific Group
    (1966). No increase in tumour incidence has followed the oral intake
    of polyoxyethylene sorbitan esters. Furthermore, large doses of the
    oleate and stearate have been well tolerated by human subjects.


    Level causing no toxicological effect

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

    Estimate of acceptable daily intake for man

         0-25 mg/kg bw.*


    Anello, J. A. & Levy, G. (1969) J. Pharm. Sci., 58, 721

    Aoki, M. et al. (1969) Chem. Pharm. Bull., 17, 1109

    Brandner, J. D. (1973) Unpublished report submitted by ICI America

    Brush, M. K. et al. (1957) J. Nutr., 62, 601

    Chow, B. F. et al. (1951) Fed. Proc., 10, 378

    Chow, B. F. et al. (1953) J. Nutr., 49, 563

    Chusid, E. & Diamond, J. (1955) J. Pediat., 46, 222

    Cid, E., Dresse, A. & Jaminet, Fr. (1971) Pharm. Acta Helv., 46, 377

    Culver, P. J. et al. (1951) J. Pharmacol. exp. Ther., 103, 377

    Eagle, E. & Poling, C. E. (1956) Food Res., 21, 348

    Fitzhugh, O. G. et al. (1959) Toxicol. appl. Pharmacol., 1, 315

    Harris, R. S., Sherman, H. & Jeter, W. W. (1951) Arch. Biochem., 34,

    Holt, P. R. (1964) Proc. Soc. exp. Biol. Med., 117, 230

    Hopper, S. H., Hulpieu, H. R. & Cole, V. V. (1949) J. Amer. pharm.
         Ass., Sci. Ed., 38, 428

    Jaminet, Fr. (1971) Pharm. Acta Helv., 46, 377


    *    As total polyoxyethylene (20) sorbitan esters.

    Krantz J. C. jr (1943a) Unpublished report No. WER-124-88 to the Atlas
         Chemical Co.

    Krantz J. C. jr (1943b) Unpublished report No. WER-149-76 to the Atlas
         Chemical Co.

    Krantz J. C. jr (1943c) Unpublished report No. WER-149-76/160/188/A to
         the Atlas Chemical Co.

    Krantz J. C. jr (1946) Unpublished reports Nos. WER-149-130/123/A/B to
         the Atlas Chemical Co.

    Krantz J. C. jr (1947a) Unpublished report No. WER-149-123 to the
         Atlas Chemical Co.

    Krantz J. C. jr (1947b) Unpublished reports Nos. WEE-149-164/193/235/
         A/B to the Atlas Chemical Co.

    Krantz, J. C. jr (1947e) Unpublished reports Nos. WER-149-165/195/
         237/A/B to the Atlas Chemical Co.

    Krantz, J. C. jr (1947d) Unpublished reports Nos. WER-149-175/198/
         234/A/B to the Atlas Chemical Co.

    Krantz, J. C. jr (1947e) Unpublished reports Nos. WER-149-133/161/
         189/228 to the Atlas Chemical Co.

    Krantz, J. C. jr (1949) Unpublished report No. WER-149-270/A/B to the
         Atlas Chemical Co.

    Krantz, J. C. jr et al. (1951) Bull. Sch. Med. Maryland, 36, 48

    Levy, G. & Anello, J. A. (1969) J. Pharm. Sci., 58, 494

    Levy, G. & Perälä, A. (1970) J. Pharm. Sci., 59, 874

    Nelson, M. F. et al. (1966) J. Food Sci., 31, 253

    Oser, B. L. & Oser, M. (1956a) J. Nutr., 60, 367

    Oser, B. L. & Oser, M. (1956b) J. Nutr., 60, 489

    Oser, B. L. & Oser, M. (1957a) J. Nutr., 61, 149

    Oser, B. L. & Oser, M. (1957b) J. Nutr., 61, 235

    Poling, C. E., Eagle, E. & Rice, E. E. (1956) Food Res., 21, 337

    Ringrose, A. T. & Waller, E. F. (1959) Toxicol. appl. Pharmacol., 1,

    Sergiel, J.-P., Paris, R. & Clement, J. (1971) Cah. Nut. Diet, 5, 33

    Steigmann, F., Goldberg, E. M. & Schoolman, H. M. (1953) Amer. J. dig.
         Dis., 20, 380

    Treon, J. F. et al. (1967) Page 381 in Application of surface active
         Substances, Vol. III of Chemistry, Physics and Application of
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    Truhaut, R. (1970) Bull. Acad. Nat. Med., 154, 789

    Waldstein, S. S., Schoolman, H. M. & Popper, H. (1954) Amer. J. dig.
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    Wick, A. N. & Joseph, L. (1953) Food Res., 18, 79

    Wick, A. N. & Joseph, L. (1956) Food Res., 21, 250

    See Also:
       Toxicological Abbreviations