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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.


    CUPRIC SULFATE

    Explanation

         This compound has been evaluated for acceptable daily intake by
    the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
    Ref. No. 22) in 1970.

         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

         Copper is an essential trace element and is a constituent of
    plants and of animal and human tissues. The tissues containing the
    largest concentrations are liver with 0.30-0.91 mg/100 g and brain
    with 0.22-0.68 mg/100 g (Kehoe et al., 1940). The whole human body
    contains 100-150 mg (Browning, 1969). At subcellular level a number of
    enzymes, such as tyrosinase, contain Cu as part of their structure or
    require it for proper functioning, e.g. catalase (Dawson & Mallette,
    1945).

         About 3.2 mg Cu is consumed daily in food (mainly in meat, eggs,
    oils etc., oysters having the highest concentration with 27.4 mg/100
    cals). Water provides 40-500 pg. The total daily intake in soft water
    areas is calculated as: food 3200 pg, water 200 pg, beverages 300 pg,
    air 2 pg. Excretion is calculated as: urine 60 pg, faeces 3640 pg,
    sweat 2 pg/day (Schroeder et al., 1966).

         Somewhat controversial evidence suggests that the metal is an
    essential co-factor in haemoglobin synthesis and is involved in Fe
    metabolism. Some animal diseases, especially severe anaemias, are
    suspected to arise from nutritional copper deficiency. Copper
    intoxication may cause acute haemolysis in sheep (Anon., 1966). In man
    the average daily requirement for adults is estimated at 2 mg, and for
    infants and children at 0.05 mg/kg bw (Fd. Std. Cttee, 1956; Browning,
    1969). The copper content of various foods ranges from 20 to 400 ppm
    (0.002% to 0.04%) (Underwood, 1962). The average daily dietary intake

    for adults is estimated at 2 to 5 mg, of which up to 0.7 mg are
    excreted in the urine (Browning, 1969). 0.8 mg are retained mainly in
    the liver, kidney and intestine, while 1.40 mg are excreted in the
    faeces. Increased intake appears to have little effect on urinary
    output but faecal excretion may rise to 10 to 20 times the urinary
    excretion. Absorption from the g.i. tracts is limited. Normal human
    serum levels range from 68 to 90 mg/ml of which 95% is carried by the
    alpha-globulin copper oxidase ceruloplasmin. The remainder is bound to
    albumin or amino acids. In vitro studies on liver and kidney slices
    using 64Cu-acetate demonstrated intra-cellular transport by histidine
    and other amino acids (Neumann & Silverberg, 1966).

         Rats fed 2.5 mg/day copper sulfate and sacrificed 1, 3, 6 and 24
    hours later showed significant concentrations of Cu in kidneys, liver
    and plasma (up to 2.7 pg/g in kidney and 1.1 pg/g in liver) (Decker et
    al., 1972). The copper is attached to hepatic mitochondria and cell
    nuclei, more being found in the nuclei at concentrations above
    100 pg/g (Lal & Sourkes, 1971).

         Copper and molybdenum levels become most critical when one or the
    other is present in either deficient or toxic amounts. The level at
    which molybdenum becomes toxic depends on the amount of copper in the
    diet, and an excess of molybdenum can induce or intensify a deficiency
    of copper. In addition, sulfate ion can act either to modify or
    intensify the adverse effects of molybdenum. A similar but reverse
    pattern occurs when molybdenum is deficient and copper is in excess
    (Underwood, 1962; Gray & Daniel, 1964).

         Continued intake of high levels of copper in experimental animals
    leads to considerable accumulation in the liver. In the pig and the
    rat this has resulted in lowered iron levels in haemoglobin and liver
    and haemolytic jaundice in some stressed animals. Long-term
    administration of even low concentrations of copper results in some
    increased storage in the liver (O'Hara et al., 1960; Buntain, 1961;
    Bunch et al., 1965; Harrison et al., 1954).

         Effect on ascorbic acid availability was tested by giving guinea-
    pigs copper sulfate or copper gluconate in drinking-water at levels
    equivalent to 1600 ppm Cu (0.16% Cu) of the diet for 11 weeks. Animals
    were sacrificed and examined grossly for scurvy and serum ascorbic
    acid. No evidence of scurvy was found and serum levels of ascorbic
    acid were not affected (Harrison et al., 1954).

    TOXICOLOGICAL STUDIES

    Acute toxicity

                                                                                       

    Substance     Animal        Route       LD50        LD100        Reference
                                         (mg/kg bw)   (mg/kg bw)
                                                                                   

    Copper
       chloride   Rat           Oral        140                      Spector, 1956
                  Guinea-pig    s.c.                     100         Spector, 1956
    Copper
      nitrate     Rat           Oral        940                      Spector, 1956

    Copper
      sulfate     Mouse         i.v.                      50         Spector, 1956
                  Rat           Oral        300                      Spector, 1956
                  Guinea-pig    i.v.                       2         Spector, 1956
                  Rabbit        i.v.                     4-5         Spector, 1956
                                                                                   
    
         In animals ingestion of three ounces of 1% CuSO4 solution
    produces intense g.i. tract inflammation (Browning, 1969). In mammals
    injection or inhalation of copper and its compounds leads to
    haemochromatosis, liver injury or lung injury (Browning, 1969).

    Short-term studies

    Rat

         Young rats (100-150 g) were injected daily with CuCl2 solutions
    at 0, 1, 2.5 and 4 mg/kg for 236 days. Controls showed no lesions.
    Weight loss was evident in all treated groups and deaths occurred at
    the two higher levels. Liver pathology showed necrotic cells in the
    periphery of lobules with inflammation and regeneration, periportal
    fibrosis, and nuclear hyperchromatism with large hyalinized cells.
    Kidney lesions described were sloughing and degeneration of epithelial
    cells of proximal convoluted tubules (Wolff, 1960).

         Young (21-days old) albino rats were fed ad libitum for four
    weeks on diets containing copper sulfate to give 0, 500, 1000, 2000
    and 4000 ppm (0%, 0.05%, 0.1%, 0.2% and 0.4%) of added copper. The
    daily food intake was less, the higher the copper content, the average
    copper intakes being about 5, 8, 11 and 8 mg/rat/day respectively. All
    the rats on the highest dose died in the first week; one out of eight
    in the second highest dosage group died in the fourth week. It was

    suggested that the deaths in the highest dosage group were due partly
    to reduced food intake. The growth rate in the lowest dosage group was
    slightly decreased, otherwise the rats appeared normal. There were
    slight increases in the copper contents of blood and spleen and a
    marked (14-fold) increase in copper content of the liver (Boyden et
    al., 1938).

         Copper sulfate at 0.135% and 0.406% (equivalent to 530 ppm and
    1600 ppm copper, respectively) and copper gluconate at 1.14%
    (equivalent to 1600 ppm Cu) were fed in the diet of rats for up to
    44 weeks. A negative control group was also maintained. Each group
    comprised around 25 male and 25 female rats.

         Significant growth retardation, discernible at the twenty-sixth
    week, occurred with the high level copper sulfate and the copper
    gluconate. Mortality was increased in the high level copper sulfate
    group and greatly increased (90% dead between four and eight months)
    in the copper gluconate group. Four high level copper sulfate, copper
    gluconate, and control rats were sacrificed between 30 to 35 weeks and
    all survivors were sacrificed between 40 to 44 weeks. Haematology and
    urine examinations were within normal limits except for high (83 mg%)
    blood nonprotein nitrogen (NPN) in males ingesting the high level
    copper sulfate and copper gluconate; the lower level copper sulfate
    was just above the expected range of 60-70 mg% NPN). Serum levels of
    ascorbic acid were not affected. Animals receiving copper gluconate
    had hypertrophied uteri, ovaries and seminal vesicles. High level
    copper sulfate and copper gluconate animals showed enlarged, distended
    and hypertrophied stomachs, occasional ulcers, some blood, bloody
    mucous in intestinal tract, and bronzed kidneys and livers.
    Histopathology of the higher test level animals showed toxic
    abnormalities in the liver and minor changes in the kidneys. Varying
    degrees of testicular damage were noted in both high and low levels of
    copper sulfate animals whereas control animals were normal. Liver,
    kidney and spleen tissue-stored copper was elevated in all test
    groups, liver being most pronounced. Liver-copper levels recorded per
    100 g wet tissue at 40 weeks were: <2 mg (controls), 12-32 mg
    (low copper sulfate), 38-46 mg (high copper sulfate) and at 30 weeks
    56-75 mg (copper gluconate). Also noted was a marked depression in
    tissue storage of iron in high level copper sulfate and copper
    gluconate animals.

         In conclusion, copper sulfate and copper gluconate at 1600 ppm
    copper were toxic while copper sulfate at 530 ppm copper caused only
    variable effects on testicular degeneration and tissue storage of
    copper (Harrison et al., 1954).

         Daily s.c. injection of 0.26 mg Cu for 80 days produced elevated
    erythrocyte and plasma copper levels and raised caeruloplasmin levels
    after a total dose of 3.64 mg Cu. The rises levelled out at 15.6 mg Cu
    total though tissue levels continued to rise. Anaemia and diarrhoea

    developed and mean survival was 67 days. Histology showed liver and
    kidney damage and enlarged caeca. Survivors were mated and offsprings
    were given 0.26 mg Cu daily for four weeks, then 0.65 mg/day for 8.5
    months. Sixteen of the 37 offspring survived (Weedwanders et al.,
    1968).

    Rabbit

         Copper acetate at 2 mg/g (2000 ppm (0.2%)) of diet fed to 21
    rabbits through days 21 to 105 showed pigmentation in 17, cirrhosis in
    9 and necrosis of the liver in 5; those with cirrhosis did not show
    necrosis. Copper in the liver varied from 9.7-237 mg/100 g of wet
    liver. A relationship was established in which a longer feeding period
    resulted in a greater incidence of cirrhosis in the liver (Wolff,
    1960).

    Pig

         Three-week-old pigs fed 250 (0.025%), 600 (0.06%) or 750 (0.075%)
    ppm Cu in a fish meal diet showed depressed weight gain and feed
    consumption while the same concentration of copper in soybean meal had
    no effect. No gross pathological changes were seen in either group
    (Clyde et al., 1969).

    Sheep

         Six out of 17 lambs fed from six to 12 weeks of age on a ration
    containing 80 ppm (0.008%) copper developed spongy transformation of
    the CNS white matter particularly in the region of the mid-brain, pons
    and cerebellum with severe lesions in the superior cerebellar pedicles
    (Doherty et al., 1969). Copper toxicity was found in three out of 170
    housed lambs fed on a diet containing 20 ppm (0.002%) copper and 1 ppm
    (0.0001%) molybdenum. The dead animals were well nourished but
    jaundiced, with swollen, friable liver, metallic black kidneys and
    myocardial haemorrhage. Some intravascular haemolysis was seen in one
    lamb (Adamson et al., 1969). Sheep are highly susceptible to copper
    poisoning and with over-dosage the liver may contain up to 1000 ppm
    (0.1%) (Bull, 1949).

    Turkey

         Turkey poults can tolerate 676 ppm Cu (0.0676%) in the diet
    without ill effect but growth was suppressed by over 810 ppm (0.081%).
    These effects were counteracted by EDTA (Ouhra & Kratzer, 1968).

    Long-term Studies

         None available.

    OBSERVATIONS IN MAN

         Copper poisoning, with diarrhoea and vomiting, developed when
    20 workmen drank tea containing 25 ppm (0.0025%) copper (Nicholas,
    1968). Rashes were reported after drinking water containing 7.6 ppm
    (0.00076%) copper (Paine, 1968) whilst jaundice and severe haemolytic
    anaemia with elevations of serum SGOT, copper and caeruloplasmin were
    seen in a child following repeated applications of copper sulfate to
    extensive areas of severely burnt skin (Holtzman et al., 1966).

         Mineral abnormalities occur in patients undergoing haemodialysis
    when Cu levels may be raised (Mahler et al., 1971). With prolonged
    i.v. infusions copper deficiency may occur (James & MacMahon, 1970).

    Fatal oral human doses:  Basic copper sulfate     200 mg/kg bw

                             Copper chloride          200 mg/kg bw

                             Copper carbonate         200 mg/kg bw

                             Copper hydroxide         200 mg/kg bw

                             Copper oxychloride       200 mg/kg bw

         Large doses cause severe mucosal irritation and corrosion,
    widespread capillary damage, hepatic and renal damage, CNS irritation
    and depression. Sulphaemoglobinaemia and haemolytic anaemia have been
    seen. The acetate and sulfate are very toxic especially the cupric
    salts while cuprous chloride is the most toxic. Local skin corrosion
    with eczema and eye inflammation occur. Copper sulfate has been used
    in suicide attempts. Rapid transfer of absorbed Cu to red cells causes
    haemolysis. Hepatic necrosis and renal tubular oedema with necrosis
    are seen (Chuttani et al., 1965; Browning, 1969). Occupational copper
    poisoning causes greenish hair and urine in coppersmiths and copper
    colic. Inhalation of dust or vapour causes copper fume fever - brass
    chills (Bur. Mines, 1953). Contact of food or soft acid water with
    copper utensils may cause poisoning, but no haemochromatosis or liver
    disease (Bur. Mines, 1953; Hueper, 1965; Browning, 1969). The
    existence of chronic copper poisoning in man whether industrial or
    non-industrial is debatable (Browning, 1969).

         Newborn premature infants of about 1.2 kg bw were fed a milk diet
    providing an average of 14 g copper per kg per day (seven subjects)
    or diet with a supplement providing an average of 173 g copper per kg
    per day (five subjects). The duration of the period of observation was
    seven to 15 weeks. There were no differences in growth rate,
    haemoglobin, serum protein or serum copper between the two groups
    (Wilson & Lahey, 1960).

    EVALUATION

         There are no animal studies providing a no-effect level. However,
    this does not preclude the evaluation of this essential trace element.
    Reliance is placed on human epidemiological and nutritional data
    related to background exposure to copper. The estimates quoted in the
    tenth report of the Joint FAO/WHO Expert Committee are probably
    conservative and more recent food analyses suggest that the daily
    intake of 2 to 3 mg is likely to be exceeded by significant sections
    of the population with no apparent deleterious effects. On this basis
    there appears to be no reason to change the tentative assessment of
    the maximum acceptable daily load of 0.5 mg/kg bw. This figure is
    suggested on the understanding that the dietary levels of those
    constituents which are known to affect copper metabolism, for example,
    molybdenum and zinc, lie within acceptable limits.

    REFERENCES

    Adamson et al. (1969) Vet. Rec., 85, 368

    Anon. (1966) Lancet, 1, 1082

    Boyden, R., Potter, U. R. & Elvehjem, C. A. (1938) J. Nutr., 15, 397

    Browning, E. (1969) Toxicity of Industrial Metals, II ed., London,
         Butterworths

    Bull, L. B. (1949) British Commonwealth Special Conference in
         Agriculture in Australia 1949, p. 300, London, Her Majesty's
         Stationery Office (1951)

    Bunch, R. J. et al. (1965) J. An. Sci., 24, 995

    Buntain, D. (1961) Vet. Rec., 73, 707

    Bureau of Mines (1953) Information circular, 7666

    Chuttani, H. K. et al. (1965) Amer. J. Med., 39, 849

    Clyde, Parris & McDonald (1969) Can. J. Anim. Sci., 49, 215

    Dawson, C. R. & Mallette, M. F. (1945) Advances in Protein
         Chemistry, Vol. II, Academic Press

    Decker, W. J. et al. (1972) Toxicol. appl. Pharmacol., 21, 331

    Doherty et al. (1969) Res. Vet. Sci., 10, 303

    Food Standards Committee (1956) Report on copper, London, Her
         Majesty's Stationery Office

    Gray, L. F. & Daniel, L. J. (1964) J. Nutr., 84, 31

    Harrison, J. W. E., Levin, S. E. & Trabin, B. (1954) J. Amer. Pharm.
         Ass., sci. Ed., 4, 722

    Holtzman, W. A., Elliot, D. A. & Heller, R. H. (1966) New Engl. J.
         Med., 275, 347

    Hueper, W. C. (1965) UICC Symposium, Paris, Nov. 1965

    James, B. E. & MacMahon, R. A. (1970) Med. J. Aust., 1161

    Kehoe, R. A., Cholak, J. & Story, R. V. (1940) J. Nutr., 20, 85

    Lal, S. & Sourkes, E. L. (1971) Toxicol. appl. Pharmacol., 18, 562

    Mahler, D. J., Walsh, J. R. & Haynie, G. D. (1971) Amer. J. clin.
         Path., 56, 17

    Neumann, P. F. & Silverbeorg, M. (1966) Nature, 210, 416

    Nicholas, P.O. (1968) Lancet, 11, 40

    O'Hara, P. J., Newman, A. P. & Jackson, R. (1960) Aust. vet. J., 36,
         225

    Paine, C. H. (1968) Lancet, 11, 520

    Schroeder, H. A. et al. (1966) J. Cron. Dis., 19, 1007

    Spector, W. S. (1956) Handbook of Toxicology, Vol. 1, W. B. Saunders
         Co.

    Underwood, E. J. (1962) Trace elements in human and animal
         nutrition, New York and London, Academic Press

    Vohra, P. & Kratzer, F. H. (1968) Poult Sci., 47, 699

    Weedwanders, R. E., Evans, A. W. & Nasdahl, W. W. (1968) I - Lancet,
         88, 286

    Wilson, J. F. & Lahey, M. E. (1960) Pediatrics, 25, 40

    Wolff, S. (1960) Archives of Pathology, 69, 217


    See Also:
       Toxicological Abbreviations
       CUPRIC SULFATE (JECFA Evaluation)