COPPER

    Explanation

         Copper as cupric sulfate was evaluated for a maximum acceptable
    load by the Joint FAO/WHO Expert Committee on Food Additives in 1966,
    1970 and 1974 (see Annex I, Refs. 12, 22 and 32). A toxicological
    monograph was published in 1974 (see Annex I, Refs. 23 and 33). 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.

    Introduction

         The essential role of copper in maintaining normal health in both
    animals and humans has been recognized for many years. The average
    daily dietary requirement for copper in the adult human has been
    estimated at 2 mg and for infants and children at 0.05 mg/kg bw (Food
    Standards Committee, 1956; Browning, 1969; WHO, 1973). More recently
    the NRC (1980) reported "estimated safe and adequate" daily dietary
    intakes of copper ranging from 0.5 to 0.7 mg/day for infants 6 months
    of age or less up to 2-3 mg/day for adults.

         Dietary copper intake will vary considerably with the types of
    food consumed, the condition of the soils (e.g., copper content, pH,
    etc.) from which certain foods are produced and drinking-water
    characteristics. It is generally agreed that the average daily intake
    of copper is between 1-3 mg, or about 15-45 µg/kg bw in adults
    (Adelstein & Vallee, 1961; Robinson et al., 1973; Alexander et al.,
    1974; Klevay, 1975). More recently, Holden et al. (1979) used a
    duplicate diet analysis method to determine the dietary copper intake
    of 22 subjects, aged 14-64 years, consuming self-selected diets over a
    14-day period. A mean daily copper intake of 1.01 ± 0.4 mg was
    determined. Based on total diet studies (FDA, 1978) the average adult
    intake of copper in the United States of America was estimated to
    equal 1.6 mg/day from a 3000 calorie diet and 2.1 mg/day from a 3900
    calorie diet. In the United Kingdom the estimated average daily intake
    of copper calculated from total diet results is approximately
    1.8 mg (Ministry of Agriculture, Fisheries and Food, 1981). While
    ubiquitously distributed in foods, the richest sources of copper in
    the human diet are liver, seafood (especially shellfish and
    crustaceans), grains and cereal products as well as potatoes. It has
    been estimated (FDA, 1978) that these particular foods account for
    about 65% of the total dietary copper intake in adults. Drinking-water
    is known to contain from a few micrograms to more than 2 mg/litre
    (Karalekas et al., 1976) and as such may contribute a considerable
    amount to the total daily intake of copper, particularly in arid areas
    where intake of water may be high. The US EPA (1979) has reviewed its

    primary drinking-water standard for copper of 1 mg/litre and concluded
    that it is well below any minimum hazard level even for special risk
    groups.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

         The absorption of dietary copper has been studied in a number of
    animal species as well as in man. In most higher animals, the majority
    of copper absorption appears to take place in the duodenum and
    jejunum. However, based on studies in humans using radiolabelled
    copper, it is believed that the major absorption of copper occurs from
    the stomach (Bush et al., 1955; Jensen & Kamin, 1957; James & McMahon,
    1970). Quantitative measurements of the actual amount of copper
    absorbed from the gastrointestinal tract is complicated by the fact
    that there is considerable biliary secretion of copper which would
    tend to give lower estimates based on total copper analyses of faecal
    material. The abbreviated table below provides some indication of the
    variability in estimated copper absorption that has been reported in
    humans.

    Gastrointestinal absorption of copper in humans

                                                        

    Investigator(s)                         Absorption
                                                 %
                                                        

    Van Ravesteyn (1944)                        25
    Cartwright & Wintrobe (1964)                32
    Sternlieb (1967)                            40
    Weber et al. (1969)                     60 (15-97)
    Strickland et al. (1972)                56 (40-70)
    King et al. (1978)                          57
                                                        

         Total faecal excretion of copper represents the major means by
    which this metal is eliminated from the body, whereas urinary losses
    represent only about 0.5-3.0% of the daily intake of copper (Mason,
    1979). In normal individuals, increased intake of dietary copper
    appears to have only a slight effect on the amount of this metal
    voided in the urine, whereas faecal copper excretion will increase
    markedly. Based on studies such as those by Cartwright & Wintrobe
    (1964), it is estimated that as much as 25% or more of the faecal
    copper results from biliary secretion. Additional, although poorly
    quantified amounts of faecal copper occur as a result of salivary,
    gastric and duodenal secretion and from the sloughing-off of
    epithelial cells of the mucosal lining of the gastrointestinal tract.

         Copper absorption, like that of many other metals is affected by
    a number of factors including species (as indicated above), age,
    chemical form, physiological status (e.g., pregnancy) and various
    dietary components. The effect of age on copper absorption has been
    demonstrated in sheep (Suttle, 1975) and in the rat (Mistilis &
    Mearrick, 1969). In the former studies, suckling lambs were found to
    absorb between 47% and 71% of 64Cu added to their diet while only
    8-10% was absorbed at weaning. As much as 100% of an intragastric dose
    of 64Cu was absorbed in 7-10-day-old rat pups with subsequent
    decreases during the suckling period and even further decreases at
    weaning. Chemical form of ingested copper has been shown to exert a
    definite affect on its bioavailability in the rat, in swine and in
    cattle (Underwood, 1977). Pregnancy has been reported to result in
    greater retention of ingested copper due not only to decreased biliary
    secretion as shown by Terao & Owen (1977) but also by an increased
    efficiency of absorption. Davies & Williams (1976) reported that 54%
    of an intragastric dose of 64Cu was absorbed by the pregnant rat as
    compared to approximately 26% in the non-pregnant animal. In pregnant
    women, it has been postulated that increased levels of plasma copper
    are not due to a greater efficiency of intestinal absorption of copper
    but rather to increased biosynthesis of ceruloplasmin and mobilization
    of liver copper stores (Scheinberg et al., 1954; Markowitz et al.,
    1955; Henkin et al., 1971).

         The interactions between copper and various organic and inorganic
    components of the diet have been studied in several animal species.
    For example, the effect of copper 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) in 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). On the other hand, elevated levels of ascorbic acid ingestion
    have been shown to impair intestinal absorption of copper in several
    species (Evans, 1973). Imbalances between dietary nickel (Nielsen et
    al., 1980), cadmium (Evans et al., 1970), tin (Schroeder & Nason,
    1976; Greger & Johnson, 1981), lead (Petering, 1980) or molybdenum
    (Osterberg, 1980; Suttle, 1980) have all been shown to alter the
    absorption and/or metabolism of copper. The relationship between
    copper and molybdenum becomes 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 (Gray &
    Daniel, 1964; Underwood, 1977). In all likelihood, these interactions
    are mediated by some direct antagonism with copper at the intestinal
    site of absorption (e.g., competitive binding with metallothionein or

    other similar transport protein), by the formation of insoluble copper
    complexes in the gut or by inhibition of essential enzyme systems
    which either directly incorporate copper as a functional component or
    require it as an essential co-factor.

         Once absorbed, copper is loosely bound to serum albumin and
    rapidly transported to the liver, bone marrow and other tissues for
    storage and incorporation into cuproproteins. The distribution of this
    element has been shown to vary with species, age, copper status of
    the individual and to some extent with geographical area. Several
    estimates of the total copper content of the average adult human have
    been reported. For instance, Chou & Adolph (1935) estimated a range of
    100-150 mg with an average concentration of 116 mg, while Cartwright &
    Wintrobe (1964), Sass-Kortsak (1965) and Sumino et al. (1975) reported
    lower values of 80, 75 and 70 mg, respectively. Tissues containing the
    highest concentrations of copper include the liver, brain, heart and
    kidneys with intermediate levels in the pancreas, spleen, muscles,
    bones and skin. Low levels of copper are normally found in the
    pituitary, thymus, thyroid and prostate glands and in the testis and
    ovary (Gubler et al., 1957; Hamilton et al., 1973). The liver and
    brain have been shown to have the largest concentrations of
    copper (e.g., 3.0-9.1 ppm (0.0003-0.00091%) and 2.2-6.8 ppm
    (0.00022-0.00068%) (wet weight), respectively, Kehoe et al., 1940) and
    combined account for about one-third of the total copper in the body
    (Sumino et al., 1975). Unanaesthesized male rats weighing between 275
    and 350 g were given (per os) 2.5 mg cupric sulfate and sacrificed
    1, 3, 6 and 24 hours post-dosing (Decker et al., 1972). After 24
    hours, concentrations of copper up to 2.7 and 1.1 µg/g, respectively,
    were found in the kidneys and liver. Human whole blood contains
    approximately 1 ppm (0.0001%) copper which is equally distributed
    between the plasma and the erythrocytes (Li & Vallee, 1973).
    Approximately 90% of the plasma copper is associated with the
    metalloprotein, ceruloplasmin while in contrast most of the copper in
    the erythrocytes is associated with the protein erythrocuprein.
    Although whole blood and plasma copper levels are similar between
    males and females of most species, in humans, the female tends to have
    higher plasma copper levels than the males, e.g., 1.2 versus 1.1
    (Cartwright & Wintrobe, 1964).

    Metabolism

         Hepatic copper appears to be associated mainly with the
    mitochondria and cell nuclei (Lal & Sourkes, 1971). Prolonged 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 (Harrison et al., 1954; O'Hara et al., 1960; Buntain, 1961;
    Bunch et al., 1965). Distribution of copper in the foetus and newborn

    is quite different from that of the adult as reported by Widdowson et
    al. (1951), Widdowson & Spray (1951) and Shaw (1973). The percentage
    of copper in the body of the developing foetus increases to a point
    where approximately half is associated with the liver and spleen. It
    has been estimated (Widdowson & Spray, 1951) that the liver copper
    content of the newborn is about 6-10 times greater than that in the
    adult liver, although within a few months post-partum the levels
    decrease to those of the adult.

         At the subcellular level a number of important enzymes, such as
    tyrosinase, contain copper as part of their structure or require it
    for proper function, e.g., catalase (Dawson & Mallette, 1945). The
    cuproprotein ceruloplasmin, also known as ferroxidase I, plays a
    critical role in the haematopoietic process, by facilitating the
    mobilization of iron from the reticuloendothelial cells of the liver
    and spleen to the bone marrow cells and by catalysing the oxidation of
    ferrous ions during the formation of ferritransferrin (Osaki et al.,
    1966, 1971; Freiden & Hsieh, 1976). Other important cuproproteins
    involved in various oxidative reactions in the body include cytochrome
    oxidase, superoxide dismutase, dopamine-B-hydroxylase and monoamine
    oxidases such as lysyl oxidase. These and others have been discussed
    in detail elsewhere (NAS, 1977; Osterberg, 1980). In addition to its
    role in haematopoiesis, studies in a number of species indicate copper
    may be essential in preventing certain types of cardiovascular
    defects, bone abnormalities and possibly neonatal ataxia (Evans,
    1981).

    TOXICOLOGICAL STUDIES

    Special studies on carcinogenicity

    Mouse

         Dietary copper sulfate at levels of 0.05% and 0.1% was found to
    potentiate the antitumour activity of pyruvate bis(thiosemicarbazone)
    (PTS) in mice implanted with a number of tumour systems including
    Sarcoma 180, Taper Liver Tumour (solid and ascites), Carcinoma 1025,
    Sarcoma T241, Ridgway Osteogenic Sarcoma, Mecca Lymphosarcoma and
    Walker Rat Carcinosarcoma 256 (W256). At appropriate doses, PTS alone
    produced growth retardation of various tumours (Taper Liver Ascites
    and W256) while dietary CuSO4 had no effect. However, in combination
    these 2 chemicals provided even greater antitumour activity than the
    PTS alone. Also of interest was the fact that non-inhibitory levels of
    PTS in the presence of 0.05% or 0.1% dietary CuSO4 exhibited
    considerable antitumour activity (Cappuccino et al., 1967).

         The lack of effect of orally administered copper (a
    CuSO4-5H2O) on the incidence of 7,12-dimethylbenz(alpha) anthracene
    (DMBA)-induced ovarian tumours, tumours of the breast and lymphomas in
    C57BL/6J mice and pulmonary tumours in strain A mice has been reported
    (Burki & Okita, 1969). Copper sulfate was provided to test animals in

    their drinking-water at a concentration of 198 ppm (0.0198%) and
    the DMBA was given parenterally or by skin painting. Individual
    experiments were terminated at any time from 33 to 77 weeks after DMBA
    treatment. Results indicated that CuSO4 had no effect on the
    incidence of DMBA-induced adenomas of the lung, lymphomas and breast
    tumours. While CuSO4 did not prevent the induction of pre-cancerous
    lesions in the ovaries, the authors concluded that it may have delayed
    the development of granulosa cell tumours.

    Rat

         The effects of deficient (1 ppm (0.0001%)) and excess
    (800 ppm (0.08%)) levels of dietary copper on the incidence of
    acetylaminofluorene (AAF) and dimethylnitrosamine (DMN)-induced
    neoplasms in the rat were studied by Carlton & Price (1973). Six
    groups of between 50-102 male weanling Sprague-Dawley rats were
    utilized. Three groups received the copper-deficient diet and the
    other 3 the same diet supplemented with 800 ppm (0.08%) copper as
    cupric sulfate. Within each of these dietary regimens 1 group received
    AAF in the diet at a level of 600 ppm (0.06%) and 1 group was given
    DMN in their drinking-water at a level of 50 ppm (0.005%). The study
    lasted for 9 months. The following observations were reported. Excess
    copper with or without AAF or DMN was toxic, with poorest growth
    occurring in the excess copper AAF group and with greatest mortality
    (72%) in the excess copper DMN treatment. Wet weight copper levels in
    the liver of animals fed the copper-deficient diets did not differ
    greatly. However, in the excess copper groups hepatic copper levels
    averaged 244 ppm (0.0244%) in controls and 354 and 294 ppm (0.0354 and
    0.0294%), respectively, in the AAF- and DMN-treated rats, thus
    suggesting a possible influence of the carcinogenic agents on copper
    absorption or retention by the liver under conditions of surfeit
    dietary copper. (Note: Recent studies by Cohen et al. (1979) also
    suggest an alteration in copper absorption patterns in tumour-bearing
    rats.) The incidence of hepatic neoplasms in both AAF- and DMN-treated
    animals was not influenced by the level of dietary copper. However,
    there was some indication that the latency period was slightly
    extended in the excess copper AAF group as hepatocellular carcinomas
    and metastases occurred about 1 month later than in the copper
    deficient AAF rats. With respect to extrahepatic neoplasms, 57% of the
    rats from the copper deficient DMN group had renal neoplasms compared
    to 0.0% in the excess copper DMN rats. Extrahepatic neoplasms in
    AAF-treated rats occurred in the lung, spleen, skin, intestine,
    pancreas and muscle. The combined incidence of extrahepatic tumours
    was approximately 31% in the copper deficient AAF animals versus 23%
    in the excess copper AAF rats.

         Feeding copper salts, such as basic cupric acetate or cupric
    sulfate, to rats was reported to affect hepatic metabolism of the
    carcinogenic aminoazo dye DAB (4-dimethylaminoazobenzene) (Yamane &
    Sakai, 1974). Female rats of the Wistar strain, weighing between

    100-120 g, were fed 0, 0.1, 0.25 or 0.5% cupric acetate in their diet.
    After 2 weeks of feeding the 0.5% cupric acetate diet, hepatic
    activity for azo reduction of DAB had doubled in comparison to
    controls, while DAB ring hydroxylation activity was increased by
    1.2-fold and N-demethylation of DAB decreased by approximately 40%.
    Hepatic copper content in the 0.5% group was about 26 times greater
    than in controls. Experiments with isolated microsomal preparations
    from livers of rats fed the copper diets showed that the increased
    activities for azo reduction and ring hydroxylation of DAB were
    localized primarily in the microsomes and closely related to increased
    copper levels. Cupric sulfate at a dietary level of 0.5% had an effect
    on hepatic DAB metabolism similar to that produced with 0.5% cupric
    acetate. Rats fed 0.1% and 0.25% cupric acetate showed no evidence of
    increased hepatic metabolism of DAB.

    Other special studies on carcinogenicity

         No tumour induction was observed in rabbits orally dosed with
    12.5 mg Cu/kg bw (as cupric sulfate) every second day during a 479-day
    study (Tachibana, 1952). A similar conclusion was reported in male and
    female beagle dogs fed diets containing up to 0.24% (2400 ppm) copper
    gluconate over a 1-year period (Shanaman et al., 1972).

         Cupric acetate injected i.p. into strain A/Strong male and female
    mice at total doses up to 180 mg/kg bw did not produce pulmonary
    tumours at the end of a 30-week investigation (Stoner et al., 1976).
    Based on these and other animal studies, it is generally agreed that
    copper (or its salts) is not an animal carcinogen (Furst & Radding,
    1979).

    Special studies on embryotoxicity and teratology

    Mouse

         The effect of copper (either CuCl2 or metallic copper) on the
    preimplantation mouse embryo was studied in vitro by Brinster &
    Cross (1972). At molar concentrations of CuCl2 of 2.5 × 10-5 and
    higher, 2-cell embryos were killed, whereas at lower concentrations
    the embryos developed into blastocysts. In addition to the lethality,
    the higher concentrations of CuCl2 appeared to dissolve the zona
    pellucida of a number of embryos. The mouse blastocyst was found to be
    about as sensitive to the toxic effect of CuCl2 as the 2-cell
    embryos. Metallic copper liberated from fine pieces of copper wire
    (0.01 mm diameter × 1-2 mm length) placed in the embryo culture medium
    was found to be quite toxic. As the surface area of the wire
    increased, a shorter period of time was necessary for embryonic death
    to occur.

         The embryotoxic and teratogenic potential of orally administered
    copper gluconate (CG) was studied in gravid Swiss mice. The mice were
    dosed with 0, 0.1, 3 or 30 mg CG/kg bw per day from days 6 to 14 of
    gestation. Weekly body weights and implantation data (corpora lutea,
    implantation sites, implantation loss) did not show any significant
    influence of copper at any level tested. The mean numbers of
    foetuses/litter as well as foetal viability and resorption sites in
    treated groups were not significantly different from controls. Average
    weight and length of foetuses were comparable among all groups and
    there was no significant effect of copper on the incidence studied.
    Under the conditions of this investigation, it was concluded that
    copper gluconate was neither embryotoxic nor teratogenic in the mouse
    (de la Iglesia et al., 1972b).

    Hamster

         Gravid golden hamsters received i.v. injections of either cupric
    citrate or cupric sulfate on day 8 of gestation. Dose levels ranged
    from 0 to 4 mg Cu/kg bw as cupric citrate and from 0 to 10 mg Cu/kg bw
    as cupric sulfate. Increased embryonic resorption as well as the
    appearance of developmental malformations in surviving offspring were
    noted in copper-treated groups. Malformations of the heart appeared as
    a specific result of the toxicity of these copper compounds. Cupric
    citrate was slightly more embryotoxic but considerably more
    teratogenic than cupric sulfate. Results indicated that cupric citrate
    was teratogenic in the range of 0.25-4.0 mg/kg bw and cupric sulfate
    in the range of 2-10 mg/kg bw (Ferm & Hanlon, 1974).

         Di Carlo (1979, 1980) reported a specific pattern of
    cardiovascular malformations in the embryos of pregnant golden
    hamsters injected either i.v. or i.p. with 2.7 mg Cu/kg bw (as cupric
    citrate) on the eighth day of gestation. The syndrome consisted of
    double outlet right ventricles, pulmonary trunk hypoplasia and a
    ventricular septal defect.

    Rat

         Copper gluconate (CG) was administered via stomach tube to gravid
    Wistar rats from days 5 to 15 of the gestation period at dosages of 0,
    0.1, 3 and 30 mg CG/kg bw per day. Weekly body weights and food intake
    were similar among all groups. Implantation data (corpora lutea,
    implantation sites, implantation loss) were not affected by copper
    treatment. The mean number of foetuses/litter, foetal viability and
    resorption sites in the treated groups did not differ from the control
    group. Measurements of foetal weight and length as well as the
    incidence of skeletal abnormalities and soft tissue abnormalities were
    not affected by copper treatment. Based on these results, it was
    concluded that copper gluconate at the dose levels tested was neither
    embryotoxic nor teratogenic in the rat (de la Iglesia et al., 1972).

         Gravid Sprague-Dawley rats were treated i.p. with 7.5 mg
    CuSO4/kg bw on day 3 of gestation. On day 5 of gestation, all animals
    received an injection of colchicine (1 mg/kg bw) 1 hour before
    sacrifice. Upon sacrifice, blastocysts were collected by flushing the
    uterine horns with buffered saline and observed for morphological
    alterations. The number of blastomeres per blastocyst was also
    determined. Results indicate that copper-treated blastocysts showed
    serious morphological alterations and signs of degeneration (absence
    of the blastocoele; little, vesicolous and irregular blastomeres). The
    number of blastomeres was significantly reduced in the copper-treated
    group compared to controls. The authors concluded that CuSO4 exerted
    an embryolethal effect leading to a reduced number of blastocysts able
    to implant later into the uterus. The reduced number of blastomeres
    was considered as evidence of a toxic effect of CuSO4 on
    preimplantation rat embryos (Giavini et al., 1980).

    Chicken

         The embryotoxic and teratogenic potential of various copper salts
    was investigated in the developing chick embryo by Verrett (1973,
    1974, 1976). Copper gluconate (CG) was tested under different
    conditions at dose levels ranging from 1 to 50 mg/kg. Even at the
    lowest level, CG was found to be quite embryotoxic. Although
    inconclusive, the data suggested a teratogenic effect as well
    (Verrett, 1973). Subsequently, Verrett (1974) confirmed a teratogenic
    effect of copper gluconate in the developing chick embryo.

         Cupric chloride at levels as low as 0.25 mg/kg was shown to
    exhibit an embryotoxic effect in the chick embryo. However, under the
    test conditions, cupric chloride was found to be non-teratogenic
    (Verrett, 1976).

    Special studies on mutagenicity

         Copper gluconate (LBI, 1975) and cuprous iodide (LBI, 1977) were
    evaluated for genetic activity in a series of in vitro microbial
    assays with and without metabolic activation. Salmonella typhimurium
    and Sacchromyces cereviseae were the indicator organisms used. Under
    the conditions of test, neither copper gluconate nor cuprous iodide
    were found to be mutagenic.

    Special studies on reproduction

    Rat

         The effect of orally administered copper gluconate (CG) on
    fertility was studied using male and female Wistar rats (de la Iglesia
    et al., 1973).

         Three groups of female rats (20 per group) received either 0, 3
    or 30 mg CG/kg bw per day from day 46 to day 21 post-partum of the
    study. Each of these groups of females were mated with groups of
    untreated male rats (10 per group). To assess the effects of CG on the
    male rat, 2 groups of males (10 per group) were treated with 3 mg
    CG/kg bw per day from day 1 to day 60 of the study. A third group of
    10 males served as controls. The CG-treated males were mated with
    groups of female rats (20 per group) that received either 0 or 3 mg
    CG/kg bw per day from day 1 to day 60 of the study. The group of
    10 untreated males was allowed to mate with a group of untreated
    females. Parameters studied included percentage of pregnancies, number
    and distribution of embryos in each uterine horn, presence of empty
    implantation sites and number of resorption sites, abnormal uterine
    conditions that may have contributed to embryonic death, length of
    gestation, litter size, number stillborn/number live born, gross
    anomalies in the offspring and pup sex and weight. There were no
    significant differences between treated and control groups in any of
    the parameters studied. Under the conditions of the study it was
    concluded that copper gluconate did not affect the fertility potential
    of either male or female rats.

    Acute toxicity

         Sensitivity to the toxic effects of excess dietary copper is
    influenced by several variables including animal species, chemical
    form and the relationship between copper and other dietary minerals
    such as zinc, iron and molybdenum.

         Most laboratory and domestic animals are reasonably tolerant to
    copper and dietary exposures in the order of 20-50 or more times above
    normal are often necessary in order to produce copper toxicosis
    (Bremner, 1979). As a general rule, ruminant species, especially
    sheep, have a much lower tolerance to copper than non-ruminants, while
    among non-ruminant species the dog tends to be less tolerant than the
    rat, pig and even humans (Osterberg, 1980). The influence of chemical
    form is readily apparent from the oral LD50 data in the table. As
    alluded to in an earlier section, trace metals such as zinc, iron and
    molybdenum have been shown to influence the absorption of ingested
    copper. When these elements are present at adequate or surfeit levels
    they may act to alleviate the toxic effect of excess copper by
    competing for available binding sites of transport proteins in the
    intestinal mucosa, thereby reducing copper absorption.

                                                                                                 

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

    Cupric chloride     Rat           Oral          140                   Spector, 1956
                        Guinea-pig    s.c.                      100       Spector, 1956

    Cupric sulfate      Mouse         i.v.                       50       Spector, 1956
    (anhydrous)         Rat           Oral          300                   Spector, 1956
                        Guinea-pig    i.v.                        2       Spector, 1956
                        Rabbit        i.v.                      4-5       Spector, 1956
                                      Oral                       50       Eden & Green, 1939
                        Dog           Oral                      165       Gubler et al., 1953
                        Sheep         Oral                     9-20       Buck et al., 1973
                        Horse         Oral                      125       Bauer, 1975

    Cupric sulfate      Rat           Oral          960                   Smyth et al., 1969
    (hydrated)

    Cupric nitrate      Rat           Oral          940                   Spector, 1956

    Cupric acetate      Rat           Oral          710                   Smyth et al., 1969

    Cupric carbonate    Rat           Oral          159                   Spector, 1956

    Cuprous oxide       Rat           Oral          470                   Smyth et al., 1969
                                                                                             
    
         A wide range of symptoms have been observed in cases of acute
    oral copper intoxication. These include ptyalism (excessive
    salivation), nausea, severe abdominal discomfort, emesis (in
    phylogenetically higher mammals), tachycardia, hypotension, haemolytic
    crisis, convulsions, paralysis, collapse and death. Organ pathology
    includes marked gastroenteritis, hepatic, splenic and renal congestion
    and hepatic necrosis. The haemolytic crisis that has been associated
    with acute copper toxicosis is characterized by the development of a
    haemolytic anaemia with intravascular lysis of the erythrocytes
    (Hochstein et al., 1978). In studies on the possible mechanisms by
    which copper produces destruction of the erythrocyte, Adams et al.
    (1979) observed a marked reduction in the deformability of the
    erythrocytes as well as marked increases in membrane permeability and
    osmotic fragility. More recently, Hochstein et al. (1980) reported
    that copper-induced formation and subsequent degradation of peroxides
    of the membrane lipids of the erythrocyte may be a critical factor in
    altering membrane integrity that leads to haemolysis.

    Short-term studies

    Mouse

         Male mice were exposed to copper sulfate in their drinking-water
    at levels ranging from 0.006% (1.52 mg/kg bw per day) to 1.6%
    (407 mg/kg bw per day) during a 15-day study. At levels of 0.2% or
    less no adverse effects were seen in any of the test animals. At
    levels of 0.4% (100 mg/kg bw per day) copper sulfate and higher,
    growth was markedly slower than in control animals. Significant weight
    losses occurred among mice in the 0.8% and 1.6% treatment groups and
    there was marked mortality (80%) in the 1.6% group. At levels of 0.04%
    (10 mg/kg bw per day) and greater, liver copper levels were increased.
    For example, in control mice, liver copper content averaged 4 ppm
    (0.0004%) (wet weight) as compared to 16.3 and 178.4 ppm (0.00163 and
    0.01784%), respectively, in animals from the 0.2% and 0.8% treatment
    groups (Kojima & Tanaka, 1973).

    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 2 higher levels. Liver pathology showed necrotic cells in the
    periphery of lobules with inflammation and regenerations, periportal
    fibrosis, and nuclear hyperchromatism with large hyalinized cells.
    Kidney lesions described were sloughing and degeneration of epithelial
    cells of the proximal convoluted tubules (Wolff, 1960).

         Daily s.c. injection of 0.26 mg Cu administered to 3-month-old
    male and female Wistar rats for 90 days produced elevated erythrocyte
    and plasma copper levels and ceruloplasmin values after a total dose
    of 3.64 mg Cu had been given. These increases levelled out at 15.6 mg
    total Cu, although tissue copper 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
    the offspring were given 0.26 mg Cu daily for 4 weeks, then 0.65
    mg/day for 8.5 months. Sixteen of the 37 offspring survived
    (Wiederanders et al., 1968).

         Weanling male Wistar rats received daily injections (i.p.) of
    1.5 mg Cu/kg bw (as copper lactate) during a 160-day study. Copper-
    treated animals had a lower rate of growth compared to saline-treated
    controls. Serum copper levels rose gradually then decreased slightly
    between days 60 and 90 and then sharply increased thereafter up to
    about 500 µg/dl at 160 days in the copper-treated animals. As serum
    copper levels increased, there was a concurrent decrease in
    ceruloplasmin diamine oxidase activity, while that of serum glutamic-
    oxaloacetic transaminase was significantly greater than in control
    animals. Marked proteinuria and aminoaciduria occurred in copper-

    exposed rats and upon necropsy these animals were found to have
    fibrotic peritonitis, cirrhotic livers and slightly enlarged kidneys.
    Histological examination revealed degeneration of liver parenchymal
    cells with marked fibrosis, tubular necrosis of the kidneys, nerve
    cell degeneration and swelling of the brain stem. Granular copper
    deposits were observed in liver parenchymal cells, in glia cells of
    the central nervous system and in the degenerated tubular epithelial
    cells of the kidneys. Slight splenomegaly occurred but without
    specific histological or histochemical change in copper-exposed rats
    (Narasaki, 1980).

         Young (21-day-old) albino rats were fed ad libitum for 4 weeks
    diets containing 0, 500, 1000, 2000 and 4000 ppm (0, 0.05, 0.1, 0.2
    and 0.4%) copper, as copper sulfate. Daily food intake was less as
    dietary copper increased, with average copper intakes being 5, 8, 11
    and 8 mg/rat/day, respectively. All the rats in the 4000 ppm (0.4%)
    treatment group died within the first week while 1 of 8 animals in the
    2000 ppm (0.2%) treatment group died during the fourth week. It was
    suggested that the deaths in the highest dosage groups were due partly
    to reduced food intake. The growth rate in the lowest dosage group was
    slightly decreased, otherwise the rats appeared normal with only
    slight increases in the copper content of their liver (Boyden et al.,
    1938).

         Male albino rats, 90 days old, weighing between 90 and 110 g were
    gavaged with 0 or 100 mg/kg bw per day of copper sulfate for a period
    of 20 days. After 20 days all animals were fasted for 24 hours, bled
    and sacrificed. A marked depression in body weight occurred in copper-
    treated animals mid-way through the study. Haemoglobin levels,
    haematocrits and erythrocyte counts were all significantly depressed
    in the test rats. Copper-induced histopathological changes included
    centrilobular necrosis and perilobular sclerosis with nuclear oedema
    of the liver, and tubular necrosis as well as nuclear pycnosis and
    cell proliferation in the medullary region of the kidneys. Heavy
    deposition of copper was found in the centrilobular parenchyma of the
    liver with lesser deposits in the perilobular zone. Retention of
    copper also occurred in the epithelium of the distal tubules,
    interstitium and medullary cells of the kidneys (Rana & Kumar, 1980).

         Copper sulfate at 0.135% and 0.406% (equivalent to 530 ppm
    (0.053%) and 1600 ppm (0.16%) copper, respectively) and copper
    gluconate at 1.14% (equivalent to 1600 ppm (0.16%) Cu) were fed in the
    diet of rats for up to 44 weeks. A control group was also maintained.
    Each treatment group consisted of approximately 25 male and 25 female
    rats. Significant growth retardation, which was discernible at the
    twenty-sixth week, occurred in the high level copper sulfate and the
    copper gluconate groups. Mortality which was elevated in the high
    level copper sulfate treatment group was up to 90% between the fourth
    and eighth month in the copper gluconate group. Haematology and urine
    components were within normal limits except for high (83 mg%) blood
    non-protein nitrogen (NPN) in males ingesting the high level copper

    sulfate and copper gluconate diets, while serum levels of ascorbic
    acid were not affected. Animals receiving copper gluconate had
    hypertrophied uteri, ovaries and seminal vesicles while both high
    level copper sulfate- and copper gluconate-fed animals showed
    enlarged, distended and hypertrophied stomachs, occasional ulcers,
    bloody mucous in their intestinal tract, and bronzed kidneys and
    livers. Histopathological examination of these animals showed abnormal
    hepatic and renal changes, as well as varying degrees of testicular
    damage. Copper in the liver, kidneys and spleen was elevated in all
    test groups with liver concentrations being most pronounced. The
    following wet weight copper levels were reported in the livers from
    male and female rats from the various treatment groups: after 40
    weeks - 11.6 and 17.8 ppm (0.00116 and 0.00178%) (controls), 124.7 and
    323.6 ppm (0.01247 and 0.03236%) (530 ppm (0.053%) CuSO4), 328.8 and
    457.7 ppm (0.03288 and 0.04577%) (1600 ppm (0.16%) CuSO4); and after
    30 weeks - 751.0 and 566.0 ppm (0.0751 and 0.0566%) (1600 ppm (0.16%)
    copper gluconate). A marked depression in tissue storage of iron in
    the high level copper sulfate and copper gluconate animals was also
    noted. It was concluded that 1600 ppm (0.16%) copper either as copper
    sulfate or copper gluconate was toxic while 530 ppm (0.053%) copper as
    the sulfate caused only variable effects on testicular degeneration
    and tissue storage of copper (Harrison et al., 1954).

         Male weanling rats were fed a diet containing 2000 ppm (0.2%)
    copper as copper sulfate for 15 weeks. Serial sacrifice of test and
    control animals was conducted after weeks 1, 2, 3, 6, 9 and 15. The
    effects of copper treatment on the liver and kidneys as well as plasma
    enzyme activities were evaluated (Haywood, 1980; Haywood & Comerford,
    1980). Changes in the liver and kidneys occurred in 3 phases. The
    first was characterized by a gradual build-up of copper with
    progressive signs of cellular disturbances. The second phase was
    associated with maximal liver and kidney copper values (3360 and
    1447 ppm (0.336 and 0.1447%) (dry weight), respectively, after
    week 6) and severe cellular disruption. The final phase was one of
    regeneration and healing and was associated with somewhat lower liver
    and kidney copper levels (2144 and 1114 ppm (0.2144 and 0.1114%),
    respectively), thus suggesting that at least in the rat some form of
    metabolic adaptation to continued high level copper intake may take
    place. The mean copper concentrations in the liver and kidneys of
    control animals were 18 and 34 ppm (0.0018 and 0.0034%), respectively
    (Haywood, 1980).

         A biphasic fluctuation in whole blood and plasma copper
    concentrations was observed during the study. Mean values for controls
    were 0.9 and 1.4 ppm (0.00009 and 0.00014%), respectively. While only
    slight fluctuations occurred during the first 3 weeks of the study,
    copper levels in both whole blood and plasma of copper-exposed rats
    increased significantly (P <0.001) at week 6 and thereafter.
    Ceruloplasmin activity in experimental animals was not affected during
    the first 3 weeks but, from the sixth week on, this activity was

    significantly greater than in controls. Plasma alanine amino
    transferase activity was greater (P <0.05) in copper-exposed rats
    after the first week and rose to maximal levels between weeks 6 and 9
    and remained at such levels throughout the study. Alkaline phosphatase
    activity and bilirubin concentrations were not affected by copper
    treatment. There was no rise in erythrocyte copper content or any
    haemolysis (Haywood & Comerford, 1980).

    Rabbit

         Rabbits were fed a diet containing 2000 ppm (0.2%) copper acetate
    during a 105-day study. Varying degrees of pigmentation, cirrhosis
    and necrosis of the liver were observed in the copper-exposed
    animals. Liver copper concentrations varied from 97 to 2370 ppm
    (0.0097-0.237%), wet weight. There was a greater incidence of
    cirrhotic livers with prolonged feeding of the copper diet (Wolff,
    1960).

    Pig

         Three-week-old pigs fed 250, 600 or 750 ppm (0.025, 0.06 or
    0.75%) Cu in a fish-meal diet showed depressed weight gain and feed
    consumption while the same concentration of copper in soybean meal-
    based diet had no effect. No gross pathological changes were seen in
    either group (Clyde et al., 1969).

         A total of 400 out of 2000 growing swine died over a 10-1/2-month
    period as a result of consuming feed containing 700 ppm (0.07%) copper
    (as the sulfate). Normal levels of supplemental copper range between
    125 and 250 ppm (0.0125 and 0.025%) in swine rations. Pre-mortem
    symptoms included anorexia, weight loss or reduced growth rate,
    weakness and pallor. Necropsy and histological examinations revealed
    abnormal liver pigmentation (yellow-brown to orange coloration),
    hepatic centrilobular necrosis, ulcers of the gastric cardia, watery
    blood, reddened bone marrow and splenic myeloid metaplasia. Blood
    studies showed microcytic, hypochromic anaemia, elevated erythrocyte
    glutathione concentrations, increased iron-binding capacity of serum
    and decreased serum iron levels. Hepatic copper levels in the poisoned
    animals ranged between 100 and 170 ppm (0.01 and 0.017%) (wet weight)
    in contrast to levels of 0.8-6.3 ppm (0.00008-0.00063%) in normal
    swine livers (Hatch et al., 1979).

    Sheep

         Sheep are especially sensitive to the adverse effects of excess
    copper intake. In a study with 6-12-week-old lambs fed a ration
    containing 80 ppm (0.008%) copper, the lambs 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 3 out of 170 housed lambs fed on a diet containing 20 ppm

    (0.002%) copper and 1 ppm (0.0001%) molybdenum. The dead animals
    appeared well nourished but were jaundiced, with swollen, friable
    liver, metallic black kidneys and myocardial haemorrhage. Some
    intravascular haemolysis was seen in 1 lamb (Adamson et al., 1969).

         Further observations on the changes in the CNS of copper-poisoned
    sheep were reported by Howell et al. (1974) using cross-bred animals
    between 6 and 12 months of age. All animals received (ad libitum) a
    diet containing 7 ppm (0.0007%) (dry weight) copper. Thirteen of the
    sheep served as controls, while the other 29 were given daily oral
    doses of a 0.5% CuSO4.5H2O aqueous solution over a period of 37
    weeks. Twenty-two of the test animals were dosed at a rate of 5.05 mg
    Cu/kg bw per day and 7 at a rate of 7.58 mg Cu/kg bw per day. Nine of
    the treated animals were sacrificed prior to development of the
    haemolytic crisis of copper poisoning, 11 died during the crisis and 9
    after the crisis had past. The brains from both test and control
    animals were fixed either by perfusion or immersion using neutral
    formalin. No abnormal changes in brain morphology and histology were
    seen in any control animal or from copper-treated sheep that died or
    were sacrificed prior to the haemolytic crisis which occurred from 6
    to 27 weeks into the study. Status spongiosus was seen in 5 of the 11
    sheep that died or were sacrificed during the haemolytic crisis, but
    this condition was extensive only in 2 of these animals. It was
    observed in 7 of the 9 sheep that died or were killed in the post-
    haemolytic period and was extensive in 4 of the 7. Out of 6 animals
    known to have had multiple periods of haemolysis, 5 had status
    spongiosus which was extensive in 2 of the 5. The status spongiosus
    involved areas of white matter in the brain and the spinal cord and
    was best seen in the cerebellar white matter. Changes were seen in
    astrocytes in the brain tissues from haemolytic and post-haemolytic
    animals. A greater number of enlarged astrocytes was seen in the
    thalamus and at the junction of the grey and white matter of the
    cerebral tissue in 2 haemolytic and in all 4 post-haemolytic animals.
    These observations are in good agreement with those reported in
    6-month-old lambs that were copper poisoned and underwent haemolytic
    crisis (Morgan, 1973).

         Non-acute copper poisoning in sheep has been described in 2
    distinct phases (Todd et al., 1962; Todd & Thompson, 1963). The first
    phase or pre-haemolytic period is characterized by an accumulation of
    copper in the liver and other organs over a period of weeks or months
    with no significant clinical signs. The second phase is referred to as
    the haemolytic crisis and is characterized by the rapid onset of
    severe haemolysis, haemoglobinaemia, haemoglobinuria, jaundice and a
    number of enzymatic changes. Until the haemolytic phase is reached,
    morbidity may be quite low. However, once the crisis phase occurs,
    mortality can be quite extensive.

         In 1971, Tait et al. reported on a case of accidental copper
    poisoning in feeder lambs receiving a total dietary copper level of
    27 ppm (0.0027%) (1.08 mg Cu/kg bw per day). Within 16-18 weeks after
    initial feeding of this particular diet, 13 of 55 lambs had died and
    an additional 6 animals were terminated because of icterus throughout
    their carcasses. Post-mortem examinations revealed an abnormal "liver-
    like" appearance of the lungs with evidence of severe haemorrhaging,
    jaundice-like livers and enlarged, dark brown kidneys. Haemosiderin-
    laden macrophages were seen in alveolar lung tissue and a fatty
    degeneration of the hepatic tissue was evident with increased numbers
    of haemosiderin-containing reticuloendothelial cells. Tubular
    degeneration and occlusion with haemoglobin casts were observed in the
    kidneys from copper-poisoned lambs. Liver copper concentrations ranged
    from 1017 to 1538 ppm (0.1017-0.1538%) (dry weight) in the copper-
    exposed animals as compared to normal values of 100-400 ppm
    (0.01-0.04%) (dry weight), while serum copper levels averaged 2.5 ppm
    (0.00025%) compared to normal values of 0.6-1.5 ppm
    (0.00006-0.00015%).

         Ishmael et al. (1971, 1972) reported a number of physiological
    changes which occurred in chronically poisoned sheep. Six-month-old
    ewe lambs were given a standard diet containing 7 ppm (0.0007%) copper
    in the dry matter. Four animals served as controls while 8 received
    1 g amounts of CuSO4.5H2O as a drench, 5 days per week throughout
    the study. Haemolysis and jaundice developed in the copper-treated
    animals between 4 and 10 weeks after initiation of the study and
    several of the lambs experienced multiple haemolytic episodes.
    Elevated blood copper levels occurred immediately before or during the
    haemolytic crisis and both plasma copper and RBC copper fractions were
    increased. Haematocrit and haemoglobin values fell rapidly during the
    haemolytic crisis and it was estimated that about 50-75% of the RBCs
    were lysed. Serum activities of sorbitol dehydrogenase, glutamate
    dehydrogenase, GOT and arginase showed an initial phase of increase
    during the first 3 weeks of copper exposure followed by a gradual
    decrease over the next 3-week interval. Marked rises in activity were
    noted 2-7 days before the haemolytic crisis. During the crisis, slight
    decreases in activity occurred except for arginase which remained
    high. In the post-haemolytic phase, activities fell only to rise again
    in animals experiencing further haemolytic crisis. Serum bilirubin
    concentrations followed a similar pattern and like arginase activity
    were greatest during the period of haemolysis. Haemolysis was
    associated with neutrophilia, Heinz body formation and high blood urea
    levels. As a percentage of live weight, the liver and kidneys from
    copper-poisoned lambs were considerably larger than in controls, e.g.,
    1.35-2.59% versus 1.02-1.19% and 0.33-1.97% versus 0.23-0.26%,
    respectively. Liver colour varied from pale yellow to orange, while
    that of the kidneys from brown to black. Pre-haemolytic changes in
    liver included vacuolation and swelling of parenchymal cells and
    parenchymal cell nuclei, parenchymal cell necrosis and swelling of the

    Kupffer cells. During the haemolytic crisis, extensive focal necrosis
    of liver tissue was seen. The most striking features of hepatic
    biopsies from sheep that survived the haemolytic crisis were the large
    amounts of bile pigment in canaliculi and small bile ducts and the
    occurrence of periportal fibrosis. While parenchymal cells showed
    fatty change and nuclear enlargement and vacuolation, focal necrosis
    was no longer evident. Changes in hepatic enzyme activities were also
    quite pronounced and related to the phase of copper intoxication.
    Alkaline phosphatase and especially acid phosphatase showed gradual
    increases in activity during the pre-haemolytic phase and marked
    activities during the crisis that tended to remain elevated in the
    post-haemolytic period. Adenosine triphosphatase, non-specific
    esterase, glutamic dehydrogenase and succine tetrazolium reductase
    activities gradually decreased during the haemolytic crisis with only
    partial recoveries in the post-haemolytic period.

         Post-mortem chemical analysis of the liver, kidneys and spinal
    cord from copper-poisoned sheep showed mean copper levels of 3153, 371
    and 6.6 ppm (0.3153, 0.0371 and 0.00066%) (dry weight), respectively,
    in comparison to control values of 176, 9.6 and 3.6 ppm (0.0176,
    0.00096 and 0.00036%), respectively. Additional observations reported
    in the test animals included congestion and oedematous lungs,
    extensive epicardial and endocardial haemorrhages, moderate to severe
    haemorrhage of the adomasal mucosa and submucosa of the jejunum and
    ileum, splenomegaly with large accumulations of haemosiderin and
    status spongiosus of the white matter of the brain and spinal cord.

         In sheep receiving a daily drench of copper sulfate at a rate of
    20 mg CuSO4.5H2O/kg bw, copper levels in the liver and copper and
    iron levels in the kidneys increased significantly during the pre-
    haemolytic phase without signs of impaired renal function. During the
    haemolytic crisis, degeneration, necrosis, decreased enzyme activities
    and reduced function of the proximal convoluted tubules occurred.
    These renal changes were accompanied by an increase in blood urea
    levels. The tubules contained large amounts of haemoglobin, iron and
    copper. In the post-haemolytic period, markedly elevated levels of
    iron and copper were still found as well as degenerative, necrotic
    tubular epithelial cells. There was some indication of a regenerative
    process in the damaged renal tissue based on the slight recovery of
    certain enzyme activities that were markedly reduced during the
    haemolytic crisis (e.g., glutamate dehydrogenase, succinic tetrazolium
    reductase) and the appearance of a number of groups of small cells
    without the cytoplasmic granules characteristic of degenerative cells
    (Gopinath et al., 1974). Despite this suggestion of recovery in
    animals that have survived the haemolytic crisis, Gopinath & Howell
    (1975) caution that further, progressive and fatal tissue damage may
    occur even after the source of copper exposure had been eliminated for
    some time.

         Additional investigations on chronic copper toxicity in sheep
    have been conducted in recent years which confirm and/or extend the
    findings reported above. These include the studies of Thompson & Todd
    (1974) and Gooneratine & Howell (1980) that show a sudden and dramatic
    increase in serum creatine phosphokinase (CPK) levels at the time of
    haemolytic crisis followed by a subsequent return to normal levels in
    the post-haemolytic period. Without evidence of muscular lesions or
    degeneration either during the pre-haemolytic phase or at haemolysis,
    it is postulated that the rise in CPK is associated with a transient
    increase in the permeability of muscle membranes. These observations
    as well as others reported by Norheim & Soli (1977) and Bremner &
    Young (1977) on the distribution and character of soluble copper
    binding proteins from the liver and kidneys of copper-poisoned sheep
    are considered in a recent paper by Soli (1980).

    Long-term studies

    Rabbit

         Rabbits were orally dosed, every second day, with 10 cc or a
    1% cupric sulfate solution for a period of 479 days. The dose
    administered was equivalent to approximately 12.5 mg Cu/kg bw. Hepatic
    damage, somewhat like that of liver cirrhosis in humans, was reported
    in the copper-dosed animals (Tachibana, 1952).

    Dog

         A 1-year chronic study was conducted with male and female beagle
    dogs to evaluate the potential oral toxicity of copper gluconate
    administered at levels of 0.012, 0.06 and 0.24% of the diet. These
    levels were equivalent to 3, 15 and 60 mg/kg bw per day. After 6
    months of ingesting such diets, 2 animals of each sex were sacrificed
    and necropsied. Weight gains and food consumption values were similar
    for the control and treated groups. Overall health, haematology and
    urinalysis were comparable to controls. After 1 year, minimal liver
    function changes were observed in 1 of 12 dogs receiving the 0.24%
    copper gluconate diet, a change that was reversed following a 12-week
    withdrawal period. Accumulation of copper in liver, kidneys and spleen
    was seen at the high dose. No compound-related effects were seen at
    the lowest dose and there were no compound-related deaths or gross or
    microscopic pathological lesions in any dog (Shanaman et al., 1972).

    OBSERVATIONS IN MAN

         Occupational copper poisoning causes greenish hair and urine in
    copper-smiths and copper colic. Inhalation of dust or vapour causes
    copper-fume fever/brass chill (Bureau of Mines, 1953). Jaundice and
    severe haemolytic anaemia with elevations in serum GOT, copper and
    ceruloplasmin levels were seen in a child following repeated

    applications of copper sulfate to extensive areas of severely burned
    skin (Holtzman et al., 1966). The occurrence of copper poisoning in
    patients during recurrent haemodialysis has been addressed in reports
    by Lyle (1967), Blomfield et al. (1971), Mahler et al. (1971) and
    Klein et al. (1972). A syndrome of headache, chills, nausea,
    diaphoresis and exhaustion during and after haemodialysis was reported
    in a patient on a home-dialysis unit. The system was carefully
    evaluated and upon removal of a 5 m copper tube and replacement with
    PVC tubing the patient experienced no further attacks of this
    "haemodialysis chills" syndrome except when dialysed on 2 separate
    occasions away from her home. In each case, the dialysis equipment was
    found to have copper containing parts (Lyle et al., 1976).

         With respect to oral toxicity, a number of studies have been
    concerned with either accidental or deliberate ingestion of large
    doses of copper salts, most notably, copper sulfate. Chuttani et al.
    (1965) investigated 53 cases of acute copper intoxication, 48
    involving subjects who were hospitalized for emergency treatment and 5
    from autopsy materials and records from individuals who died from
    copper poisoning. Of the hospitalized cases, 71% were between 16 and
    25 years of age and 67% were males. Reliable data on the exact
    quantities of copper sulfate that were consumed were unavailable.
    Based on patient information, the amount varied between 1 and 112 g.
    Clinical features included a metallic taste, a burning sensation in
    the epigastrium, nausea and repeated emesis of greenish material in
    100% of the cases. Diarrhoea and haemoglobinuria and/or haematuria
    occurred in about 30% of the cases while jaundice, oliguria and anuria
    were frequently reported. Hypotension and coma were seen in about 8%
    of the cases. Of the 48 hospitalized patients, 7 died within 24 hours
    after ingestion as a result of shock or at a later stage due to
    hepatic and/or renal complications. Whole blood copper levels were
    related to the degree of severity of poisoning, e.g., mild,
    287 ± 126.8 µg/dl; severe, 798 ± 396 µg/dl. Histopathological
    evaluations revealed superficial or deep ulcerations of gastric and
    intestinal mucosa, dilation of central veins in the liver with varying
    degrees of cell necrosis and bile thrombi. Renal changes included
    glomerular congestion, swelling or necrosis of tubular epithelial
    cells and haemoglobin casts.

         Singh & Singh (1968) evaluated the biochemical changes in the
    blood of 40 patients suffering from acute copper sulfate poisoning.
    Elevated as well as persistent levels of whole blood copper were
    determined. The appearance of haemolysis was positively correlated
    with whole blood copper levels and occurred in 18 of the 40 subjects
    (40%). Three of 4 mortalities in this particular study were associated
    with severe intravascular haemolysis. Subsequent reports on acute
    copper sulfate intoxication in humans by Deodhar & Deshpande (1968),
    Mittal (1972) and Wahal et al. (1976) confirm the findings above.

         Stein et al. (1976) reported on a fatal case of copper sulfate
    poisoning in a 44-year-old female who was hospitalized for alcohol-
    diazepam intoxication. A 10% cupric sulfate solution was administered
    as an emetic in 2, 10 cc doses (for a total of 2 g cupric sulfate).
    Autopsy revealed acute haemorrhagic necrosis of the entire small
    bowel, confluent areas of opaque yellow mottling of the liver with a
    hepatic copper content of 75 ppm (0.075%), wet weight (normal, 8 ppm
    (0.0008%), wet weight). Renal damage included acute tubular necrosis
    with many of the tubules containing casts.

         Acute renal failure was diagnosed in 11 of 29 patients treated
    for acute copper sulfate intoxication (Chugh et al., 1977). The
    amounts of copper sulfate ingested ranged from 1 to 50 g.
    Symptomatologies were similar to those previously described. Severe
    intravascular haemolysis was present in all 11 subjects and is
    believed to have been the chief factor responsible for the renal
    lesions in these patients. Such lesions varied from those of mild
    shock to well-established acute tubular necrosis. The tubules showed
    loss of epithelial cell lining and the presence of haemoglobin cells.
    Others showed proliferation of cells indicating regeneration,
    interstitial oedema and scattered inflammatory cells. For those
    subjects showing recovery, renal biopsies revealed uniformly dilated
    tubules with flattened epithelial lining.

         The World Health Organization (1974) concluded that the fatal
    oral human dose of various copper salts, including basic copper
    sulfate, copper chloride, -carbonate, -hydroxide and -oxychloride, is
    about 200 mg/kg bw. It should be clear that there is considerable
    variability in individual sensitivity to this metal.

         Ingestion of copper-contaminated foods and beverages including
    drinking-water has been responsible for occasional cases of human
    copper intoxication. For example, 20 workmen became ill following the
    ingestion of their morning tea. Five individuals vomited within
    minutes after ingesting the tea and 1 about 2 hours later. Four of the
    5 had diarrhoea 3-5 hours later, while 5 others had diarrhoea but no
    emesis. The remaining workers had nausea without any other symptoms.
    These symptoms were not severe except in 1 individual with a history
    of gastric problems. Investigations finally revealed that a gas-heated
    hot-water geyser had been used to brew the tea and had contributed a
    considerable amount of copper from corrosion products to the tea.
    Levels of copper up to 30 ppm (0.003%) were found in the tea
    (Nicholas, 1968).

         McMullen (1971) reported on an incident in which at least 10
    individuals became nauseated and vomited following ingestion of soft
    drinks (orange squash and lime juice cordial) dispensed from bottles
    stoppered with pourers having tubes made of chromium-plated copper.
    Examination of the tubes showed they were badly discoloured and had a
    greenish tinge. Analyses of the drinks revealed 190 and 222 ppm (0.019

    and 0.0222%) copper, respectively, in the orange squash and lime juice
    cordial. The acidic nature of the juices was believed to have
    contributed to migration of copper from the tubes. This association
    between acidic beverages in contact with copper tubing in beverage
    dispensers has been identified as a cause of copper-induced
    gastroenteritis in more recent times (Witherell et al., 1980).

         The number of confirmed cases of chronic copper poisoning in
    humans is limited. In 1971, Salmon & Wright described a possible case
    of chronic copper poisoning in a 15-month-old male infant. Prior to
    hospitalization, the child underwent a 5-week period of behavioural
    change, diarrhoea and progressive marasmus. Clinical

    [Note:  page 285 is blank in original book]

    Comments

         Copper is an essential trace element in both animals and humans.
    It plays a vital role in a number of critical enzyme systems and is
    closely linked with normal haematopoiesis and cellular metabolism. The
    metabolism of copper has been studied in experimental animals and man.
    Copper absorption in man ranges from 25 to 60% of that ingested and
    has been shown to vary with diet. Copper absorption may be reduced by
    other metals, such as zinc or cadmium, and by organic materials, such
    as ascorbic acid. Faecal excretion is the main route of elimination,
    with only minor amounts being excreted in the urine. Total body copper
    in adult humans has been estimated to range from 70 to 150 mg with
    highest concentrations in the liver, brain, heart and kidneys. In
    humans, an adequate daily dietary intake of copper has been estimated
    to range from 0.5 to 0.7 mg/day for infants of 6 months of age or less
    up to 2-3 mg/day for adults. In general the levels of copper in the
    diet are adequate to meet nutritional requirements.

         Although copper is an essential trace element, high levels of
    intake can cause symptoms of acute toxicity. Accidental or deliberate
    ingestion of large quantities of copper salts, notably copper sulfate,
    has been responsible for a number of human deaths. An oral dose of
    about 200 mg/kg bw is generally considered fatal in humans. However,
    high levels of copper in food and water adversely affect its
    palatability.

         Chronic copper intoxication has been demonstrated in experimental
    animals, especially sheep, a species particularly sensitive to copper.
    Monogastric species have a high tolerance for copper. In a 1-year
    feeding study in the dog, the no-effect level of copper was
    approximately 5 mg/kg. Copper salts (gluconate, iodide) were not
    embryotoxic in the mouse and the rat. There is no evidence that copper
    is carcinogenic to either animals or humans.

         There are a limited number of reports of chronic copper toxicity
    in human infants, but none in adults. In general, copper does not
    appear to be a cumulative toxic hazard for man, except for individuals
    suffering from Wilson's disease.

    EVALUATION

         Nutritional data related to background exposure to copper from
    the diet indicate that the level of copper in food meets the
    nutritional requirements (2-3 mg/day). However, it is recognized that
    this level of intake is likely to be significantly exceeded by
    sections of the population, particularly in arid areas where there may
    be a high intake of water containing high levels of copper. At this
    time there is no information that indicates that such populations are
    adversely affected. In addition, at this time copper does not appear
    to be a cumulative toxic hazard for man, except for individuals with
    Wilson's disease. On this basis the previous tentative evaluation of a
    maximum daily load of 0.5 mg/kg bw was reaffirmed as a provisional
    value for a maximum tolerable intake of 0.5 mg/kg bw per day from all
    sources.

    Estimate for provisional maximum tolerable daily intake for man

    0.05-0.5 mg/kg bw.

    FURTHER WORK OR INFORMATION

    Desirable

    (1) Information be collected about the ranges of intake of copper from
    all sources by selected samples of people.

    (2) Epidemiological survey of high intake groups that may be detected
    to determine whether or not there is any evidence of copper-induced
    ill-health.

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    See Also:
       Toxicological Abbreviations
       Copper (EHC 200, 1998)
       Copper (ICSC)
       COPPER (JECFA Evaluation)
       Copper (UKPID)