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 µg copper per kg per day (7
subjects) or diet with a supplement providing an average of 173 µg
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
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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
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