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    FAO Nutrition Meetings 
    Report Series No. 48A 
    WHO/FOOD ADD/70.39




    TOXICOLOGICAL EVALUATION OF SOME
    EXTRACTION SOLVENTS AND CERTAIN 
    OTHER SUBSTANCES




    The content of this document is the 
    result of the deliberations of the Joint 
    FAO/WHO Expert Committee on Food Additives 
    which met in Geneva, 24 June  -2 July 19701




    Food and Agriculture Organization of the United Nations
    World Health Organization


                   

    1 Fourteenth report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series in press; Wld Hlth
    Org. techn. Rep. Ser., in press.


    COPPER AND CUPRIC SULPHATE

    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 to 0.91 mg/100 g and brain
    with 0.22 to 0.68 mg/100 g (Kehoe, Cholak & Story, 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).

         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 bodyweight (Fd. Std. Cttee, 1956;
    Browning, 1969). The copper content of various foods ranges from 20 -
    400 ppm (Underwood, 1962). The average daily dietary intake for adults
    is estimated at 2 - 5 mg, of which up to 0.7 mg are excreted in the
    urine (FAO/WHO, 1967; Browning, 1969). 0.8 mg are retained mainly in
    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 10-20 times the urinary excretion.
    Absorption from the G.I. tracts is limited. Normal human serum levels
    range from 68-90 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 intracellular transport by histidine and
    other amino acids (Neumann & Silverberg, 1956).

         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, sulphate 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 sulphate or copper gluconate in drinking water at
    levels equivalent to 1600 ppm 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).

    Acute toxicity
                                                                                            

    Substance          Animal        Route     LD50         LD100         Reference
                                               mg/kg        mg/kg 
                                               bodyweight   bodyweight
                                                                                        

    Copper chloride    Rat           oral      140                        Spector, 1956

                       Guinea-pig    s.c.                   100           Spector, 1956

    Copper nitrate     Rat           oral      940                        Spector, 1956

    Copper sulphate    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 3 ounces of 1% CuSO4 solution produces
    intense G.I. tract inflammation (Browning, 1969).

    Fatal oral human doses:    Basic copper sulphate      200 mg/kg
                                                          bodyweight
                               Copper chloride            "
                               Copper carbonate           "
                               Copper hydroxide           "
                               Copper oxychloride         "

         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 sulphate are very toxic especially the cupric
    salts while cuprous chloride is the most toxic. Local skin corrosion
    with eczema and eye inflammation occur. Copper sulphate 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 way 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
    nonindustrial is debatable (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 gms) 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 day old) albino rats were fed ad libitum for 4 weeks
    on diets containing copper sulphate to give 0, 500, 1000, 2000 and
    4000 ppm 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 sulphate at 0.135% and 0.406% (equivalent to 530 ppm and
    1600 ppm and 1600 ppm Cu) 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 26th week,
    occurred with the high level copper sulphate and the copper gluconate.
    Mortality was increased in the high level copper sulphate group and
    greatly increased (90% dead between 4-8 months) in the copper
    gluconate group. Four high level copper sulphate, copper gluconate and
    control rats were sacrificed between 30-35 weeks and all survivors
    were sacrificed between 40-44 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
    sulphate and copper gluconate; the lower level copper sulphate 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 sulphate 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 sulphate 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 gm wet tissue at 40 weeks were: < 2 mg (controls), 12-32 mg (low
    copper sulphate), 38-46 mg (high copper sulphate) and at 30 weeks
    56-75 mg (copper gluconate). Also noted was a marked depression in
    tissue storage of iron in high level copper sulphate and copper
    gluconate animals.

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

         Rabbit

         Copper acetate at 2 mg/gm (2000 ppm) 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 gm of wet
    liver. A relationship was established in which a longer feeding period
    resulted in a greater incidence of cirrhosis of the liver (Wolff,
    1960).

         Human

         New-born premature infants of about 1.2 kg bodyweight were fed a
    milk diet providing an average of 14 g copper per kg per day (7
    subjects) or diet with a supplement providing an average of 173 g
    copper per kg per day (5 subjects). The duration of the period of
    observation was 7 to 15 weeks. There were no differences in growth
    rate, haemoglobin, serum protein or serum copper between the two
    groups (Wilson & Lahey, 1960).

    Long-term studies

    None available.

    Comments

         It has been well demonstrated that copper is an essential trace
    element in the human diet. The total human body content of copper is
    estimated to vary from 100-150 mg for an adult. The normal range of
    intakes of copper from food and other environmental sources does not
    lead to cumulation or toxicity in man, but excessive intakes may lead
    to adverse reactions. The copper content of food is known to range
    from 1 to 80 mg/kg or more. The available animal data point to an
    effect level in rats at 30 mg/kg bodyweight and a no-effect level has
    not been established.

    Evaluation

         The absence of a no-effect level in animal studies is not germane
    to 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 10th report of the
    Joint FAO/WHO Expert Committee are probably conservative and more
    recent food analyses suggest that the daily intake of 20 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 bodyweight. 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

    Anon (1966) Lancet, i, 1082

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

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

    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

    Dawson, C. R. & Mallette, M. F. (1945) "Advances in Protein
    Chemistry". Vol. 11, Academic Press

    Food Standards Committee (1956) Report on Copper, H.M.S.O. London

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

    Hall, E. M. & MacKay, E. M. (1931) Amer. J. Path., 7, 327

    Harman, D. (1965) J. Gerontology, 20, 151

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

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

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

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

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

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

    Underwood, E. J. (1962) "Trace Elements in Human and Animal
    Nutrition".  Academic Press, New York and London

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


    See Also:
       Toxicological Abbreviations