Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 2 (2007)

Raghupathy Ramanathan, Ph.D. NASA-Johnson Space Center Toxicology Group Houston,Texas

Zinc (Zn) is a naturally occurring element found in the earth’s crust in most rock-forming minerals. It is also present in significant concentrations in soil near highways (because of emissions and tire wear) and industrial locations such as power plants and factories. In natural waters, zinc exists in several chemical forms. It usually occurs as zinc sulfide, zinc carbonate, zinc chromate, or zinc oxide (ZnO) (Merck Index 1989). Zinc compounds such as zinc chloride (ZnCl₂), zinc sulfate (ZnSO₄), ZnO, and zinc sulfide are found at hazardous waste sites, and the possibility that they could get into drinking water has been a concern. The acetates, chlorides, and sulfates of zinc are extensively used in the dyeing industry and in common consumer products such as ZnO skin ointments and shampoos. For chemical and physical properties, see Table 11-1.

The average zinc concentration in tap water across the United States is 0.245 milligrams per liter (mg/L) (NRC 1980). The highest mean value reported for tap water from galvanized pipes is about 2 mg/L (Sharrett et al. 1982).

Occupational exposure to zinc by means of inhalation occurs extensively in zinc mining, smelting, welding, and the manufacturing of galvanized metals, paints, tires, and certain personal consumer products. ZnCl₂ is primarily used in making batteries, zinc silicate in phosphors of cathode ray tubes, and ZnO in the rubber vulcanizing process. Exposure to zinc through drinking water can take place in areas near where these activities occur.

Zinc is an essential food element. Dairy products, grains, meats, fish, and poultry are the richest sources of zinc (Tanner and Friedman 1977). The Recommended Dietary Allowance (RDA) of zinc for nonpregnant women is 12 mg per day (d), and for men it is 15 mg/d. A typical mixed diet provides at least 65-80% of the daily RDA (Bowerman and Harrill 1983). According to the Food and Drug Administration (FDA) Total Diet Study (1991-1997), the mean intake of zinc from food by males between 31 and 50 years (y) of age is 13.38 ± 0.16 mg/d (n = 1,805). Daily intake by women of that age range is 8.51 ± 0.11 mg/d (n = 1,733) (see Appendix E in IOM 2001). Zinc occurs in all living cells as a constituent of metalloenzymes involved in major metabolic pathways (NRC 1989). Zinc controls several enzymes of intermediary metabolism, DNA and RNA synthesis, gene expression, and immunocompetence. Zinc can interact with almost all hormones and plays a significant role in homeostasis of hormones such as thyroid and steroid hormones, insulin, and pituitary hormones like prolactin (Brandao-Neto et al. 1995).

Summary reports on humidity condensates collected from the Mir space station and water recycled from Mir during the years 1995-1998 indicate that zinc was present at concentrations ranging from 1.26 mg/L to 5.3 mg/L in the humidity condensates and at concentrations ranging from 10.4 to 475.0 micrograms (µg) per liter in the processed (recycled) water (Pierre et al. 1999). Although the average concentrations did not exceed the U.S. Environmental Protection Agency’s (EPA’s) secondary maximum contaminant level (SMCL) of 5 mg/L, zinc was found very frequently in the recycled water. Concerns exist about potential system breakthroughs. This document will be limited to addressing the adverse effects of extraneous zinc that may leach into the drinking water through the water processing system (from distribution lines), the humidity condensate heat exchangers (through corrosion), or as a result of the failure of the ion-exchange resins to remove metal ions completely.

The existing studies strongly indicate that absorption of ingested zinc by humans varies widely depending on the type of diet and the presence of dietary components and minerals such as phytates, phosphates, sugars, amino acids, and iron, as well as other metals such as copper and cadmium.

Zinc in animal products has a higher coefficient of absorption than zinc from vegetables. Phytates (IP6 or inositol hexaphosphate) and fiber present in vegetables adversely affect the availability of zinc by reducing its absorption. For example, Sandstrom et al. (1987) determined the absorption of zinc in humans from 60 g meals based on rye, barley, oatmeal, triticale (a cross between wheat and rye), and whole wheat. It was lowest (8.4 ± 1.0%) from oatmeal porridge with a phytic acid content of 600 millimicromoles (mµmoles), and relatively highest (26.8 ± 7.4%) from rye bread meal with a phytic acid content of 100 mµmoles (Sandstrom et al. 1987); thus, absorption of zinc is negatively correlated to phytic acid content. Various numbers from 20% to 40% have been used for the absorption of zinc (NRC 1989). An average zinc intake of 8.6-17.2 mg/d from various diets has been reported (Tanner and Friedman 1977; Holden et al. 1979); this is below the RDA in most cases. After short-term exposures to zinc supplements in the diet, absorption ranged widely from 8% to 81% (Aamodt et al. 1983; Reinhold et al. 1991; Sandstrom 1992). Three days after individuals ingested zinc at 0.05 mg per kilogram (kg) from bread rolls containing different concentrations of proteins, fractional zinc absorption ranged from 8% from low-protein rolls to 26% from high-protein rolls (Hunt et al. 1991), indicating that dietary protein promotes zinc absorption. The fractional absorption of zinc seems to depend on zinc dose. For example, in a group of healthy young men on a constant daily dietary intake of 15 mg/d, when various test doses of zinc were administered, fractional absorption was 81% from a 4.52 mg dose, 67% from a 6.47 mg dose, and 61% from a 24.52 mg dose (Istfan et al. 1983; also see King et al. 2000). A similar phenomenon of changes in fractional zinc absorption in response to changes in the dietary zinc concentrations have been described by several investigators (Wada et al. 1985; Taylor et al. 1991; Lee et al. 1993). Absorption of zinc also seems to depend on the zinc status of the individual. Zinc was absorbed at a higher level by zinc-deficient individuals than by zinc-sufficient individuals (Spencer et al. 1985; Johnson et al. 1988). In a 63-d study conducted with young men, Turnlund et al. (1984) reported that

zinc absorption from the basal diet was 33.8% ± 2.9%, but it dropped to 17.5% ± 2.5% when 2.3 g of phytate as sodium phytate was added to the basal diet.

Absorption of zinc also seems to depend on the form of the salt. For example, Galvez-Morros et al. (1992) reported that male rats absorbed 40% of labeled zinc from a diet containing zinc as ZnCl₂ at 0.81 mg/kg or 8.4% from a zinc-carbonate-containing diet. Zinc uptake from inorganic salts was in the order of sulfate > acetate > chloride > citrate > phosphate (Seal and Heaton 1983). Nevertheless, the total excretion or retention was independent of the salt form. Studies using in vivo ligated intestine and ⁶⁵zinc indicate that absorption was significantly greater from the duodenum than from the distal portion of the small intestine. In an experiment using an everted sac of rat duodenum and ileum, zinc absorption was shown to be pH dependent. Reducing the pH of the medium from 7.3 to 6.4 decreased absorption from the duodenal sacs.

When a dose of radioactive ZnCl₂ was intubated to rats maintained on a diet containing zinc at 40 mg, the maximum radioactivity attained in the whole blood was about 0.09% at 30 minutes (min), 0.045% at 1 hour (h), and 0.01% at 24 h. Liver and pancreas preferentially took up a higher percentage of radioactivity, with a peak uptake at 8 h (Methfessel and Spencer 1973).

A variety of mechanisms have been proposed for the absorption of zinc. It is generally believed that zinc absorption is homeostatically controlled by the zinc content in the intestine and by circulating zinc (Davies 1980; Cousins 1985). Data also indicate that zinc uptake may be partly controlled by a carrier-mediated diffusion mechanism. Cysteine-rich intestinal protein (CRIP), a diffusible intracellular zinc carrier, binds zinc in the mucosa during absorption, a process that seems to be saturable (Hempe and Cousins 1992). Zinc transport in the intestinal lumen is also influenced by metallothionein, which can inhibit zinc absorption by competing with CRIP for zinc (Hempe and Cousins 1991, 1992). It is beyond the scope of this document to discuss the vast amount of literature on various approaches to understanding the absorption mechanism. The interaction of zinc with other metals and the influence of ligands on zinc absorption will be described later.

Zinc is present in almost all tissues and body fluids in humans. In blood, zinc is present in erythrocytes (92.4% as a cofactor for carbonic

anhydrase isoenzymes and superoxide dismutase [SOD]), leukocytes, and platelets. Zinc is found in diffusible and nondiffusible forms in the blood (NRC 1977). About 98% of serum zinc is nondiffusible and is bound to proteins (85% to albumin, most of the remainder complexed with β2-macroglobulin [β2-MG]). Diffusible zinc in blood is associated with albumin and amino acids and not with β2-MG (EPA 1987). Circulating zinc that is tightly bound to α2-macroglobulin in the plasma is nondiffusible and not freely exchangeable with other zinc ligands in serum (Cousins 1985). The range of normal plasma zinc concentrations is 85-110 µg per deciliter (dL). Plasma proteins provide a metabolically active transport compartment for zinc in which about 70% of circulating zinc is loosely bound to albumin in the diffusible form and is freely exchangeable (Cousins 1985). Zinc is also bound to amino acids (primarily histidine and cysteine) as a diffusible form (Henkin 1974). The zinc-amino-acid complex is transported passively across tissue membranes to bind to proteins. An important binding protein in the kidney and liver is metallothionein, although other tissue-binding proteins may be present. The body of a 70 kg normal human male contains 1.4-2.3 g of zinc (see Table 11-2).

High concentrations of zinc are also found in the prostate (100 µg/g wet tissue), semen (100 to 350 picograms [pg] per L) (Hidiroglou and Knipfel 1984), and retina (Bentley and Grubb 1991; Ugarte and Osborne 2001). With age, zinc concentrations increase in the liver, pancreas, and prostate and decrease in the uterus and aorta (Schroeder et al. 1967). Only 3% of zinc was transferred across the perfused placenta and seemed to involve potassium/zinc transport (Aslam and McArdle 1992). Zinc does not accumulate with continued exposure, because the body burden is controlled by homeostatic mechanisms. Studies in animals and humans have shown that the whole-body content of zinc remains constant over a 10-fold range of intakes (Johnson et al. 1993). The homeostatic mechanism acts mainly to adjust the gastrointestinal (GI) absorption and endogenous excretion (Wastney et al. 1986; Walsh et al. 1994; King et al. 2000). Increasing dietary concentrations of zinc were associated with decreasing concentrations of iron in the livers of rats (Yadrick et al. 1989). In the liver, as well as elsewhere, zinc is bound to metallothionein. The greatest concentration of zinc in the body is in the prostate, probably because this organ is rich in the zinc-containing enzyme acid phosphatase (see Klaassen 1996). About 60% of the zinc in the body is located in the skeletal muscle and 30% in the bones (Wastney et al. 1986).

Fecal excretion is the predominant route of elimination of zinc after an oral bolus (Davies and Nightingale 1975; Wastney et al. 1986; Reinhold et al. 1991). Only 1-2% is excreted in the urine (Wastney et al. 1986). In normal adults, daily excretion is 300-600 µ g/d. A linear excretion of zinc in the feces as a function of dose has been noted (Spencer et al. 1985). Excretion of zinc in the urine also reflects zinc intake (Wastney et al. 1986). Rats receiving zinc as ZnCl₂, ZnSO₄, zinc phosphate, or zinc citrate at 2.65 mg/kg/d over a 4-d period excreted 87-98% of the intake. Fecal excretion, total excretion, and retention of zinc did not differ for these various zinc forms (Seal and Heaton 1983).

A small amount of zinc has also been shown to be excreted by way of bile as a complex with reduced glutathione, and the transfer from liver to bile occurs by a glutathione-dependent process (Alexander et al. 1981). Low dietary intake of zinc, starvation, and high-protein diets alter the excretion of zinc (Spencer et al. 1976); nevertheless, a homeostatic mechanism maintains the balance by a greater absorption of zinc (Henkin et al. 1975; Hunt et al. 1991).

Many biologic interactions between trace elements are known to occur, especially when they are present together in the diet. With zinc being an essential trace element in a wide variety of biologic systems,

interactions of other metals with zinc are very critical for physiologic and pathologic conditions. The trace element interaction of greatest practical significance in human nutrition is the negative effect of excess zinc on copper bioavailability (Festa et al. 1985). One of the most studied modifiers of zinc absorption is the co-absorption of copper. High concentrations of dietary zinc have an antagonistic effect on the absorption of copper, and this phenomenon has been demonstrated in humans and animals. Although dietary intakes of copper and zinc do not interfere with each other’s absorption as long their ratio is 1:5 (copper:zinc), higher zinc concentrations in the diet (as with zinc supplements) depress copper absorption, concentrations of tissue copper, and the activity of copper enzymes such as ceruloplasmin and cytochrome oxidase. In zinc-deficient animals, copper concentrations in bones and liver are increased (Roth and Kirchgessner 1977). Excess copper in the diet inhibits zinc absorption from a zinc-sufficient diet, but the effect is relatively minor compared to the effect of excess zinc on copper status (O’Dell 1989). Excess zinc stimulates an increase in the intestinal concentrations of metallothionein, which traps copper because of its high affinity to copper, leading to copper loss when the intestinal cells slough off. Hypocupremia and hypoceruloplasminemia in sickle cell anemia patients who ingest supplementary zinc are well known (Prasad et al. 1978). Similarly, L’Abbe and Fischer (1984a, b) reported that when copper status (as assessed by measuring the concentration of the copper-transport protein serum ceruloplasmin) and activity of copper-zinc SOD were determined in rats fed zinc at 15, 30, 60, 120, or 240 mg/kg in their diet for 6 weeks (wk), the number of animals with low ceruloplasmin increased with increasing doses of zinc. The control diet contained copper at 6 mg/kg and zinc at 30 mg/kg. At 120 and 240 mg/kg of zinc, copper-zinc SOD decreased significantly. The copper-zinc SOD reductions seen in animals fed a high-zinc diet were similar to those in animals fed a low-copper diet. Because the elimination of excess zinc is slow and the intestinal absorption of copper will be affected until excess zinc is eliminated, the slow elimination rate is an important aspect of zinc interactions with other trace elements.

Iron and calcium are important examples of other metals that interact with zinc, because these are frequently used as dietary supplements. Iron inhibited zinc absorption when both were given in inorganic form without food (Solomons and Jacob 1981; Valberg et al. 1984). Metallothionein seems to play an integral part in these interactions. Large amounts of ingested iron (such as from iron supplements) affect the absorption of zinc (Solomons et al. 1983) and may lead to zinc deficiency, which is associated with poor growth, loss of appetite, skin lesions, lack

of taste and smell, delayed wound healing, delayed sexual maturation, onset of night blindness, impaired memory performance, and impaired immune response. Calcium and zinc have an antagonistic relationship. Experiments in animals indicate that if the intake of calcium is high, the absorption of zinc is decreased, and vice versa (Hanson et al. 1958; Yamaguchi et al. 1983). In vivo, it has been documented that oral administration of zinc to sickle cell anemia patients reduced the number of irreversible sickle cells (Brewer 1979). In this instance, zinc seems to interact with calcium at the red cell membrane by suppressing the calciumregulating protein calmodulin (Baudier et al. 1983), and this suppresses the formation of irreversible sickle cells.

Interaction of zinc with cadmium results in an increase in the excretion of cadmium when the two elements are administered together. This has been proposed as a mechanism by which zinc protects against cadmium toxicity (Stowe 1976; NRC 1980). Because zinc and cadmium compete for a common transport mechanism, simultaneous administration of zinc and cadmium has beneficial effects on cadmium toxicity (Coogan et al. 1992). Zinc acetate has been shown to prevent cadmium carcinogenesis in the prostate and testes (Waalkes et al. 1989). Similarly, co-administration of zinc with cobalt resulted in a major reduction of cobalt-induced testis tubule damage and degeneration in mice (Anderson et al. 1993). Exposure to cadmium affects the distribution of zinc, leading to preferential accumulation of zinc in liver and kidney and negatively affecting zinc concentrations in other tissues. Because cadmium and zinc additively increase metallothionein induction, they may adversely affect the absorption of other metals.

Zinc plays an important role in growth and many physiologic functions. It is an essential nutrient, and the RDA values (the estimated amount of zinc required to maintain tissue and the growth and metabolism of the individual) range from 12 mg/d for nonlactating and nonpregnant women to 15 mg/d for adult men. Consumption of concentrations of zinc below the RDA has been reported to lead to loss of appetite, loss of taste and sense of smell, and slow healing of skin sores. Retarded growth and development of reproductive organs and retarded development of offspring have been noted in humans. Zinc is present in blood plasma, erythrocytes, leukocytes, and platelets but is chiefly localized within erythrocytes (where 87% of it is in carbonic anhydrase, the major

binding site) (Ohno et al. 1985). A vast amount of data pertaining to zinc toxicity is available, and a significant portion of the data is from human case reports and human subject experiments, mainly from zinc-supplementation studies. A significant amount of data pertaining to the oral administration of zinc to rodents is also available. Most studies have addressed changes in hematologic parameters and changes in serum high-density lipoprotein (HDL) cholesterol.

A review of the literature clearly indicates that ingestion of a large amount of soluble zinc compound by humans or animals for an intermediate-to-long duration results in a variety of adverse effects in the GI, hematologic, immunologic, and nervous systems. Some of the key effects reported are decreases in serum HDL cholesterol, hematocrit, hemoglobin, and serum ferritin concentrations, as well as an impact on copper balance, anemia, and lesions in the adrenals, pituitary, and pancreas. Severe GI distress and bleeding were found only in acute cases exposed to high doses of zinc. These effects were not seen with small doses in the long-duration experiments. Occasional renal and reproductive toxicity has also been observed.

Callender and Gentzkow (1937) reported that 80% of the two army companies experienced diarrhea and GI distress after drinking limeade prepared in galvanized trash cans. The average dose of zinc ingested was estimated to be about 7 mg/kg. Within 24-48 h after ingesting zinc-contaminated food (2.4-6.8 mg/kg) from galvanized containers, 300-350 persons developed intestinal symptoms such as severe diarrhea with abdominal cramping. About 50% had gross blood in the feces (Brown et al. 1964). In another episode, individuals who had consumed zinc-contaminated alcoholic fruit punch developed a hot taste and dryness in the mouth, nausea, vomiting, and diarrhea between 20 and 90 min post-ingestion. The symptoms resolved in 24 h. In the postacute phase, the individuals reported general discomfort and muscular pain. The estimated dose was 4.6-9.2 mg/kg (Brown et al. 1964). Several studies have reported that zinc ingestion causes GI distress. An individual who had drunk 3 ounces of liquid ZnCl₂ (dose not known) immediately suffered throat pain, burning and pain in the mouth, and vomiting. Later, acute symptoms of pancreatitis were noted (Chobanian 1981). A schoolgirl suffered abdominal cramps and diarrhea after she ingested a zinc-sulfate-containing capsule (ZnSO₄ at 440 mg/d) prescribed for acne. She suf-

fered from epigastric discomfort and fainting and also showed serious signs of intestinal bleeding (Moore 1978).

The acute oral LD₅₀ (the dose lethal to 50% of test subjects) value for ZnSO₄ in rats was reported by Fabrizio (1974) to be 920 mg/kg; acute oral LD₅₀ values for ZnCl₂ in rats, mice, and guinea pigs are 350, 502, and 200 mg/kg, respectively (Calvery 1942; see also EPA 1990). In a study by Domingo et al. (1988), four zinc compounds (acetate, nitrate, chloride, and sulfate) were administered as a single gavage dose to rats and mice. The LD₅₀ values are summarized in Table 11-3.

The majority of deaths occurred during the first 2 d (Domingo et al. 1988). In general, mice seem to be more sensitive than rats to the lethal effects of zinc. In rats and mice, zinc acetate was the most lethal compound tested.

The adrenal cortex is rich in zinc. In animals, zinc deficiency increased plasma 11-hydroxy steroids, and excess zinc decreased the same steroids (Quarterman and Humphries 1979). To understand the effect of excess zinc on adrenal function in humans, 13 subjects (males and females, 20-27 y old) were orally administered zinc (as the sulfate hepta hydrate) at 0.25, 37.5, and 50 mg after a 12-h fast. Plasma cortisol was measured in serial blood samples collected from these subjects for up to 240 min. Each individual served as his or her own control. An acute inhibition of cortisol secretion was observed (Brandao-Neto et al. 1990). Hypothalamus and hypophysis are rich in zinc, and in earlier studies designed to understand the role of zinc in functions mediated by these areas, it had been shown that in vitro addition of zinc to bovine pituitary extracts inhibited the secretion of newly synthesized prolactin (Login et al. 1983; Judd et al. 1984). For this reason, Brandao-Neto et al. (1995) wanted to evaluate the adverse effects of excess zinc on the regulation of prolactin in humans. They reported an inhibition of basal prolactin secretion in 17 normal adult men and women given oral doses of zinc as the sulfate at 0, 25, 37.5, and 50 mg. Serum prolactin was measured at sev-

eral intervals over 2 h. However, in later studies from the same labortory (Castro et al. 1999, 2002), the authors concluded that in humans, supplementation of oral zinc at 25 mg/d for 3 months (mo) did not change the basal secretion of prolactin. A 2-y-old child who ingested ZnCl₂ solution (zinc at 1,000 mg/kg) developed lethargy (Potter 1981). A 17-y-old male ingested about 85 tablets, each with 4 g of zinc gluconate (elemental zinc at 570 mg). He experienced severe nausea and vomiting within 30 min of the ingestion but had no further serious effects such as diarrhea, gastric erosion, esophageal burns, shock, neurologic dysfunction, symptoms of anemia, or hepatic inflammation. His serum zinc concentration was 4.97 mg/dL about 5 h after ingestion (Lewis and Kokan 1998).

A 16-y-old boy who ingested 12 g of elemental zinc over a 2-d period (zinc at 86 mg/kg/d) experienced light-headedness, lethargy, staggering gait, and difficulty writing legibly but no apparent GI disturbances (Murphy 1970). Anemia secondary to GI hemorrhage was seen in a case report study of acute exposure to zinc as ZnSO₄ at 2.6 mg/kg/d (Moore 1978) given for 1 wk as a medically prescribed treatment for acne.

In rats administered oral doses of zinc at 0.1, 1, or 10 mg/100 g of body weight (zinc at 1, 10, or 100 mg/kg) for 3 d, a significant decrease in femoral calcium was seen with the 100 mg/kg dose (Yamaguchi et al. 1983). These effects were seen as early as 1 d. In addition, a significant decrease in acid phosphatase in the femoral epiphysis was seen in the zinc-treated group. The result that zinc causes a decrease in bone calcium may be very important because it indicates that zinc may trigger bone resorption. This observation is relevant to space missions for which bone resorption has been a concern.

In rats administered zinc as ZnO in their diet at 487 mg/kg/d for 10 d, minor neuron degeneration and proliferation of oligodendroglia and increased amounts of secretory material in the neurosecretory nuclei of the hypothalamus were observed, indicating possible effects of zinc on the central nervous system (Kozik et al. 1980, 1981).

Hooper et al. (1980) conducted a study in which 12 healthy nonobese adult males aged 23-35 y old received oral pharmacologic doses of zinc as ZnSO₄ at 160 mg/d in capsules (zinc at 2.46 mg/kg/d) for 5 wk. A

25% reduction (from 40.5 to 30.1 mg/dL) in serum HDL cholesterol at week 5 and at week 7 (2 wk after completion of the study) was noted. Total serum cholesterol levels were unchanged. This study may indicate a lowest-observed-adverse-effect level (LOAEL) of zinc at 2.46 mg/kg/d, although only one dose was used and the effects were seen even 2 wk after the study was completed (Hooper et al. 1980). HDL cholesterol levels returned to normal 11 wk after dosing ceased. There was only a weak relationship between the rise in plasma concentrations of zinc and the fall in HDL cholesterol levels. Similar results were not observed in the double-blinded crossover design study by Samman and Roberts (1988) in women (discussed below).

Chandra (1984) reported that 11 healthy adult males who ingested ZnSO₄ as tablets (containing zinc at 150 mg) twice a day for a dose of zinc at 4.4 mg/kg/d for 6 wk showed elevated (p < 0.05) serum low-density lipoprotein (LDL) cholesterol and a 32% reduction (p < 0.001) in HDL cholesterol (during weeks 4 and 6). Serum copper was not measured. Chemotaxis and phagocytosis of bacteria by polymorphonuclear leucocytes were impaired (Chandra 1984). In addition, the authors reported a decrease in the lymphocyte stimulation response to phytohemagglutinin (PHA). These adverse effects returned to baseline 10 wk after dosing ceased. This study indicates a LOAEL of zinc at 4.4 mg/kg/d for serum lipoprotein profile and immunologic response. In this instance, the changes in the immune system variables may be an important factor because the reversal response to dose cessation was very slow. Zinc plays an important role in the immune system as a cofactor of thymic thymulin and in the lymphocyte response to mitogens. In the abovementioned study, no effects were seen on the number of lymphocytes or the relative number of T cells. The National Research Council (NRC) committee on spacecraft water exposure guidelines (SWEGs) recognized the various weaknesses in the Chandra study such as a lack of information about volunteer history, the potential for other factors that can influence the observed effects, interpretation flaws, and small sample size. Fischer et al. (1984) reported that in 13 subjects who received two daily doses of zinc as zinc gluconate at 25 mg each (a total of 50 mg/d) for 6 wk, a significant decrease in the enzyme activity of erythrocyte superoxide dismutase (ESOD) activity, indicative of impaired copper status, was observed.

In another study (Samman and Roberts 1988), 47 healthy volunteers (26 women and 21 men) took part in a double-blind crossover trial that lasted 12 wk. The subjects ingested elemental zinc at 50 mg (ZnSO₄ at 220 mg) or placebo, three times per day (a total of 150 mg/d) for 6 wk.

Eighty-four percent of the women (mean age 27 y, n = 26) and 18% of the men (mean age 28 y, n = 21) reported symptoms, which included headaches, abdominal cramps, nausea, loss of appetite, and vomiting. Six female volunteers discontinued the trial, five due to gastric irritations. An average dose of zinc at 2.46 mg/kg/d seems to be a LOAEL for these symptoms. A 24% reduction in the antioxidant activity of ESOD was observed in treated female but not male subjects (Samman and Roberts 1988). The authors concluded that this difference in response might be caused simply by the difference in size between males and females, who received the same total amount of supplemental zinc. This supplementation also resulted in a decrease in LDL cholesterol and a slight decrease in HDL cholesterol in females (n = 26). No changes in hematocrit were observed. In a study by Freeland-Graves et al. (1982), eight healthy adult women per group ingested zinc acetate as a dietary supplement at 0, 15, 50, or 100 mg/d for a dose of 0.45, 1.03, or 1.86 mg/kg/d (plus daily dietary zinc intake at 0.2 mg/kg) for 8 wk. Significant differences in plasma HDL cholesterol were observed only in the group receiving the highest dose (Freeland-Graves et al. 1982). The HDL cholesterol level for the 1.86 mg/kg/d group transiently decreased by 8.4% at week 4 and returned to its normal level at week 6. The decrease in HDL cholesterol coincided with a peak in plasma zinc (at week 4), which declined toward the initial value thereafter. The slight decrease of 5.4% in plasma HDL cholesterol (65 to 59 mg/100 mL) at a does of zinc at 0.45 mg/kg/d was not statistically significant. Thus, the reduction in HDL cholesterol was transient and may not be related to dose. In a 10-wk zinc-supplementation study (Yadrick et al. 1989), 18 women ingested zinc as zinc gluconate (zinc at 50 mg/d [1 mg/kg/d]), and a significant decrease in hematocrit, serum ferritin, and ESOD activity was seen. By the end of 10 wk, the ESOD activity had decreased to 53% of control levels. A similar significant decrease in ESOD activity was also reported in men receiving zinc gluconate for 6 wk (50 mg/d). A decrease in ESOD, which is related to the status of copper, and a significant decrease in serum ferritin and hematocrit values indicated a significant health risk involving the iron status of women (Yadrick et al. 1989).

A 12-wk double-blind study was conducted on white males (Black et al. 1988) to determine the effect of zinc supplementation given orally (zinc as zinc gluconate at 50 mg/d [n = 13] or zinc at 75 mg/d [n = 9]) on serum lipid fractions. Serum was fractionated and cholesterol was determined in various fractions at week 0 and at the end of 2, 4, 6, 8, 10, and 12 wk. Zinc supplements had no more effect on serum total cholesterol, LDL cholesterol, very-low-density-lipoprotein (VLDL) cholesterol, or triglycerides than did the placebo. One-way analysis of variance with

repeated measures indicated that a significant time-by-treatment effect existed for serum HDL cholesterol: serum HDL cholesterol levels of subjects in the 75 mg/d group were significantly lower (about 15%) at weeks 6 and 12 than the HDL cholesterol concentrations of the placebo group and lower than their own baseline levels. The subjects in the 50 mg/d group had lower serum HDL cholesterol concentrations than the placebo group at week 12. However, statistical analysis of the ratio of HDL cholesterol to total cholesterol or HDL to LDL cholesterol did not show significance for treatment or interaction. For changes in HDL cholesterol, a LOAEL of zinc at 75 mg/d (1.09 mg/kg/d) and a NOAEL of zinc at 50 mg/d (0.85 mg/kg/d) can be identified in this study.

Bonham et al. (2003) evaluated short-term (2 wk) and subchronic (14 wk) effects of zinc supplements (30 mg/d as zinc Chelazome, a zinc glycine chelate) on the immune function and copper status of male subjects (n = 19) with a mean age of 35.6 y. The effect on immune function was evaluated by measuring circulating concentrations of peripheral blood leucocytes (total and differential) and lymphocyte subsets (CD3+, CD19+, and CD3−) in blood samples collected at 0, 2, and 14 wk. The dietary intake of zinc was about 10 mg/d. Copper status was determined by measuring plasma ceruloplasmin oxidase activity and ceruloplasmin concentration in addition to serum copper. Activity of SOD was determined in whole blood. No effect of zinc supplementation on circulating concentrations of leukocytes or lymphocyte subsets or on copper status could be observed. It must be noted that markers of immune status such as phagocytic activity of neutrophils and blastogenic response of cell types to T cells were not measured in this study. The authors also stated that the changes that were evident in the concentrations (number of cells/L) of the lymphocyte subset populations were independent of zinc supplementation. The authors concluded that a total of zinc at 40 mg/d (10 mg from diet and 30 mg from zinc supplement) can be estimated to be without any effect, a NOAEL, in agreement with the tolerance level for zinc recommended by the Institute of Medicine (IOM), Food and Nutrition Board, 2001. But the treatment was for a short duration of only 14 wk, and therefore such a conclusion may be premature.

Death was reported in mice that consumed zinc as ZnSO₄ at 1,110 mg/kg/d in their diet intermittently for 13 wk (Maita et al. 1981). Mortality was also observed in 20% of rats ingesting zinc as zinc acetate at 191 mg/kg/d in drinking water for 3 mo (Llobet et al. 1988).

Aughey et al. (1977) studied the effects of ingestion of zinc via drinking water on the histology and ultrastructural alterations of the pancreas, adrenal cortex, and the adenocorticotrophic hormone (ACTH)-secreting cells of the anterior pituitary gland in mice. Male and female C3H mice (total n = 150) drank water containing ZnSO₄ (0.5 g/L) ad libitum for up to 12 mo. Treated animals were as healthy as controls throughout the study (Aughey et al. 1977). Dietary zinc supplementation did not affect plasma insulin and glucose concentrations. However, histologic examination showed that the zinc-supplemented groups had ultrastructural changes in the individual beta cells of the pancreas, and the cells appeared larger. In the zinc-supplemented mice, the adrenal cortex was thicker than in controls. At 3 mo, hypertrophy of the adrenal cortex and highly positive lipid staining (histochemistry for cholesterol reaction) were seen; at 6 mo or longer, the glomerulosa and reticularis zones of the adrenals also gave strongly positive reactions for lipid staining. Zona fasciculata cells are responsible for the secretion of glucocorticoid hormones such as cortisol. A decrease in cortisol secretion indicates that this area has been damaged. This observation seems to be very similar to that reported in humans by Brandao-Neto et al. (1990). Morphologic changes consistent with hyperactivity were seen in the pituitary in groups that ingested zinc. No significant difference in zinc content between the control and zinc-supplemented groups was observed for the liver, spleen, or skin over a 6-mo observation period (no sex difference was observed with respect to the zinc content of these tissues). Only one dose was used in the mouse study. The dose can be estimated to be zinc at 70 mg/kg/d. This is the only study available that indicates serious adverse effects on beta cells of pancreatic islets, hypertrophy of the adrenal cortex, and hypertrophy of the pituitary. No effects on the pancreas were noted in the 13-wk Maita et al. (1981) study in Wistar rats and ICR mice, even at much higher doses of zinc at 565 mg/kg/d in rats and 1,110 mg/kg in mice. An increase in serum amylase and lipase was observed in C3H mice administered ZnSO₄ in drinking water, indicating damage to pancreatic acinar cells (Aughey et al. 1977), which produce amylase, lipase, and other enzymes.

In the 13-wk study of mice that received zinc as ZnSO₄ at 1,110 mg/kg/d in the diet, Maita et al. (1981) also reported increased relative and absolute kidney weights in female but not in male mice. The authors identified a LOAEL of 1,110 mg/kg for the hematologic effects (decreased hemoglobin, hematocrit, and erythrocyte concentrations) and a NOAEL of 104 mg/kg/d for both mice and rats for these effects. In an old study, Smith and Larsen (1946) reported that when rats were given zinc carbonate in drinking water for either 5 wk at a dose of 350 mg/kg/d

or for 6 wk at 500 mg/kg/d, reduction in hemoglobin, hematocrit, mean corpuscular hemoglobin (MCH, hemoglobin amount/red blood cells) and mean corpuscular hemoglobin concentration (MCHC) were reduced. A NOAEL was not identified in these studies.

In a study by Zaporowska and Wasilewski (1992), 2-mo-old Wistar rats of both sexes received ZnCl₂ in drinking water at a concentration of 0.12 mg/mL (12 mg/kg/d) for 4 wk. Statistically significant differences were found for less food and water consumption and lower body weight in the treated groups than in the untreated group. Also, a statistically significant decrease in the erythrocyte count and hemoglobin concentration in the peripheral blood was recorded. When these rats received zinc as ZnCl₂ at 12 mg/kg/d in drinking water, the percentage of reticulocytes and polychromatophilic erythrocytes increased in the peripheral blood after 4 wk of exposure. No change was observed in the composition of the bone marrow cells (Zaporowska and Wasilewski 1992).

A subchronic study was carried out with young female Sprague-Dawley rats by administering zinc acetate in their drinking water at 0, 47.5, 95, or 190 mg/kg body weight for 3 mo (Llobet et al. 1988). The volume of water consumed and urine excreted by animals in the group dosed with zinc at 190 mg/kg was always lower than these volumes for the other treatment groups and the control group. At this dose, 2 out of 10 rats died. Concentrations of urea and creatinine in plasma were significantly higher for animals in that group. At the end of the study, abnormal histology of the kidneys was seen. It included lesions in the glomerular Bowman’s capsule that consisted of flattened epithelial cells as well as proximal convoluted tubules exhibiting desquamation of tubular epithelial cells and pyknotic nuclei. The contribution of decreased water intake to abnormal renal function was noted. A NOAEL of 95 mg/kg/d for this effect in female rats was identified. The hematocrit and hemoglobin concentrations of rats given the highest dose were lower than, but not significantly different from, those of controls. No clear trends of effects as a function of dose were observed for hematocrit or hemoglobin or for the significant increases in serum urea and creatinine at the highest dose.

A case report (Hoffman et al. 1988) of a woman patient who had three intestinal surgeries for gastric ulcer and ingested a total (as a zinc supplement and zinc in a multivitamin supplement) of ZnSO₄ at 520-680 mg/d (zinc at 3.9 mg/kg/d) for 10 mo had decreased concentrations of

hemoglobin and hematocrit after this chronic oral exposure. Various symptoms related to copper deficiency such as reduction in ceruloplasmin (copper transport protein), hypochromic and microcytic anemia, leucopenia, and neutropenia (Hoffman et al. 1988) were also observed. Similar signs of copper deficiency, including decreased concentrations of plasma ceruloplasmin, were also seen in sickle cell anemia patients given zinc supplements at 150-200 mg/d for 2 y (Prasad et al. 1978). Gyorffy and Chan (1992) reported a case history in which a 57-y-old man who was taking zinc as zinc amino acid chelate at 810 mg for 18 mo presented with hypochromic-microcytic anemia. Serum copper and serum ceruloplasmin were significantly low. When the patient discontinued zinc supplements, his hemoglobin rose to normal levels in about 8 wk. Simon et al. (1988) reported that a 44-y-old man who had been taking zinc as zinc gluconate at 200-300 mg/d for at least 2 y developed anemia and neutropenia and ringed sideroblasts. These effects were secondary to zinc-induced hypocupremia and hypoceruloplasminemia. Similarly, Patterson et al. (1985) reported that a 57-y-old woman who consumed zinc at 450 mg/d for 2 y developed severe anemia, and Hoogenraad et al. (1985) reported that a 23-y-old man who was treated with high doses of ZnSO₄ orally for 12 mo had normocytic anemia with granulocytopenia, hypocupremia, and decreased ceruloplasmin; the doses were Zn²⁺ at 90 mg, three times per day for 90 d and 405 mg/d for 3 mo, respectively. Porea et al. (2000) reported that a 17-y-old male developed anemia after taking a zinc preparation to control acne. Although the number of subjects was small and the supplemental doses varied, these studies clearly documented that excess zinc led to serious anemia secondary to copper deficiency. Although these strong data could have been used to derive an acceptable concentration (AC) for long-term exposure, a NOAEL could not be identified for this serious adverse effect.

Because of the small numbers of subjects and dose concentrations and the fact that the subjects were not considered healthy, these data cannot be used to determine a 1,000-d AC even though the resulting adverse effects have been seen in many animal and some human short-term studies.

Mice that received zinc as zinc oleate at 5,000 ppm (parts per million) in their diet developed severe anemia (based on the postmortem examination) during a 45-wk study. In blood from the orbital sinus of randomly selected mice in the dosed groups, the hemoglobin content was

only 40% of that of controls. The packed cell volume was also low. The authors did not provide any quantitative data. Furthermore, the investigators reduced the concentration of zinc in the diet every 3 mo to avoid losses from mortality. When ZnSO₄ was added to the animals’ drinking water at 1,000 ppm (zinc at 190 mg/kg/d) or 5,000 ppm (950 mg/kg/d), no increase occurred in the incidence of hepatoma, malignant lymphoma, or lung adenoma at the end of 45 wk (Walters and Roe 1965). The data are not useful for deriving an AC, because of the uncertainty of the dose and high mortality.

In the study of Aughey et al. (1977) described earlier, C3H mice (male and female) were also exposed to zinc as ZnSO₄ at 500 ppm in drinking water for 12 mo (zinc at 80 mg/kg). Between 9 and 12 mo after exposure, the pancreatic islets were found to be larger, and individual cells had a vacuolated appearance and were separated by wide vascular channels. These effects were not seen at 3 mo (Aughey et al. 1977). In addition, in the zinc-supplemented mice, the adrenal cortex was thicker (hypertrophy of the adrenal cortex) compared to that of control animals. Most of the fasciculate cells were hypertrophied, vacuolated, and distended. The histochemistry was weaker at 3 and 6 mo. The ACTH-secreting cells of the anterior lobe of the pituitary gland were also reported to manifest abnormal changes (increase in size and number of granules, with some evidence of increased synthetic and secretory activity). Thus, histologic changes were observed in the pancreas, adrenal cortex, and pituitary of mice given zinc at 80 mg/kg/d in drinking water. Similar pancreatic lesions were reported in rats and mice fed a diet containing ZnSO₄ for 13 wk at doses of 450-475 mg/kg/d for rats and 235-245 mg/kg/d for mice (Maita et al. 1981).

Feeding male New Zealand white rabbits (n = 7-8) a diet containing zinc carbonate (zinc at 5,000 µg/g diet; the estimated dose was 175 mg/kg/d) for 22 wk resulted in statistically significant differences in blood hemoglobin (13.55 ± 0.125 g/100 mL in controls and 11.51 ± 0.24 mg/100 mL in the treated group) and in serum copper concentration (0.793 ± 0.121 mg/mL in the control group and 0.274 ± 0.012 mg/mL in the treated group). No changes were reported in the growth rate of these young, recently weaned rabbits (Bentley and Grubb 1991).

The only study on the effect of zinc on human reproduction reported that when pregnant women in the United Kingdom received cap-

sules containing zinc as ZnSO₄ at 0.3 mg/kg/d (elemental zinc at 25 mg/d) during the last trimester, they did not show any change in maternal body weight gain, blood pressure, hemorrhage, or infection (Mahomed et al. 1989). Kumar (1976) orally administered zinc as 2% ZnSO₄ at 150 ppm daily to 13 pregnant rats. The diet that these rats received also contained zinc at 30 ppm. Compared with untreated pregnant animals, treated rats had increased resorption of fetuses. In another study of the relationship between development and maternal dietary zinc during gestation and lactation, groups of 10 female Sprague-Dawley rats were maintained on diets containing ZnO at 2,000 ppm (200 mg/kg/d) or 5,000 ppm for 35-38 d (from day 1 of gestation to day 14 of lactation). No malformations were observed, but fetal mortality was seen even in the 2,000 ppm group (Ketcheson et al. 1969). When female rats were exposed to zinc as zinc carbonate at 250 mg/kg/d in their diet for 150 d, there was no reproduction. The NOAEL for this effect was zinc at 50 mg/kg/d (Sutton and Nelson 1937).

Zinc seems to be an important factor in epididymal maturation and in the stabilization of sperm chromatic structure. High concentrations of zinc have been found in the semen and the prostatic fluid of humans, and zinc deficiency results in reduced testicular weight and, in severe cases, sterility. Evenson et al. (1993) studied the effect of excess zinc on sperm chromatin. They fed 3-wk-old male Sprague-Dawley rats (n = 10) experimental diets containing zinc as ZnCl₂ at 500 mg/kg/d for 8 wk and observed abnormal structure of the caudal epididymal sperm chromatin. As cell ploidy during spermatogenesis is known to proceed from diploid to tetraploid to haploid, alteration of this progression indicates impaired cell maturation and proliferation (Evenson et al. 1993). However, Maita et al. (1981) found no changes in the testes or ovaries of mice fed zinc as ZnSO₄ at 1,100 mg/kg/d for 13 wk.

There were no effects on implantation and no other adverse reproductive effects noted when rats were fed zinc as ZnSO₄ at 200 mg/kg/d for 21 d prior to mating, whereas exposure for 21 d during gestational day 0 to the end of the gestational period increased implantation losses (Pal and Pal 1987).

The developmental effects of excess zinc have been studied in animals exposed to zinc by oral ingestion before and during gestation and measuring the associated implantations, fetal resorptions, fetal weights, and growth in the offspring.

Administration of zinc as ZnO at 200 mg/kg/d in the diet to rats 21 d before mating and during gestation resulted in resorption of all fetuses, whereas only 4-25% of fetuses were resorbed if the rats were treated only during gestation (Schlicker and Cox 1968). Depressed growth of the fetal rats was noted at this dose, but zinc at 100 mg/kg/d had no adverse effect on their development. Administration of diet containing zinc as zinc carbonate at 250 mg/kg/d to female rats for 150 d resulted in increased still births. At 50 mg/kg/d there was no effect. In another study (Mulhern et al. 1986), reduced fetal weight, decreased hematocrit, and copper deficiency in the offspring of mice exposed to zinc at 260 mg/kg/d in their diet were observed.

Zinc compounds were generally negative in in vitro bacterial reverse-mutation assays with Salmonella typhimurium (Thompson et al. 1989) and in in vivo mouse-mediated assays such as the frequency of micronucleated polychromatic erythrocytes in the mouse bone marrow and dominant lethal test (Fabrizio 1974), the Escherichia coli assay (Nishioka 1975), the mouse lymphoma forward mutation assay (Amacher and Paillet 1980), and the in vivo rodent somatic and germinal cell cytogenetic assays (Vilkina et al. 1978). Although zinc acetate gave negative results in the S. typhimurium mutation assay and in induction of unscheduled DNA synthesis in primary rat hepatocytes, it was positive in the mouse lymphoma assay and in an in vitro cytogenetic assay with Chinese hamster ovary (CHO) cells (Thompson et al. 1989). Human lymphocyte cultures exposed to ZnCl₂ at 3 × 10⁻³ molar (M) to 3 × 10⁻⁵ M demonstrated only a weak clastogenic response (Deknudt and Deminatti 1978).

Several in vivo studies have shown positive results indicating genotoxicity. Chromosomal aberrations in bone marrow cells have been observed in rats exposed to zinc as zinc chlorate at 14.8 mg/kg/d in drinking water (Kowalska-Wochana et al. 1988). It has been suggested that the calcium-depleting action of zinc may be responsible for these aberrations (Deknudt and Gerber 1979) because the effects were not seen in mice fed a standard calcium diet. Also, in rats exposed to zinc chlorate at 17.5 mg/kg/d in drinking water, sister chromatid exchanges were observed in the bone marrow (Kowalska-Wochana et al. 1988). Thus, in the bone marrow of both mouse and rat, sister chromatid exchanges and chromosomal aberrations were observed as a result of in vivo exposure

to zinc salts. Using alkaline single-cell gel electrophoresis (the Comet assay), Banu et al. (2001) reported that single-stranded breaks were induced in the peripheral blood lymphocytes from mice orally administered various doses of ZnSO₄ and studied after 24, 48, 72, 96 h, and the first wk posttreatment.

No human data are available that relate cancer to zinc administered orally as zinc supplements or otherwise. When rats were provided with ZnCl₂ in their drinking water at 450 ppm (zinc at 13.4 mg/kg/d) for 25 wk and tested for renal lesions, no renal tumors were seen (Kurokawa et al. 1985). Zinc as ZnSO₄ was added to drinking water at 1,000 ppm (190 mg/kg/d) or 5,000 ppm (950 mg/kg/d), and mice ingested this for 45 wk. No increase in the incidence of tumors at any of the sites (hepatoma, malignant lymphoma, lung adenoma, or testicular tumors) was noted (Walters and Roe 1965). There was also no evidence of forestomach or glandular epithelial tumors in the treated groups.

There have been numerous human and animal studies on zinc deficiency and effects on immune functions (Shankar and Prasad 1998). There are only a few reports on the effect of excess zinc ingestion on the immune system. Several studies indicate that zinc supplementation has beneficial effects on susceptibility of human populations to infectious diseases. These effects include reduction of the incidence and duration of acute and chronic diarrhea, incidence of lower respiratory infections, and even the incidence of malaria. Zinc lozenges were shown to decrease the duration of the common cold. It has also been reported that zinc-deficient animals have suppressed immune responses and are highly susceptible to various viral (for example, herpes simplex and Semliki Forest viruses) and bacterial infections such as Salmonella enteritidis and mycobacterium tuberculosis (Shankar and Prasad 1998). In general, zinc seems to play a paramount role in normal development and in many effectors of immunity. The effects of zinc deficiency and zinc excess on the immune system and the immune systems relation to plasma zinc concentration has been reviewed by Chandra (1991).

In a study using a ground-based spaceflight analog, head-down bedrest, Krebs et al. (1993) found increased fecal excretion and decreased zinc balance 17 wk after bedrest even though the dietary concentration of zinc was sufficient (zinc at 13.8 ± 0.7 mg/d). In an animal study using simulated weightlessness, a decrease in femoral-diaphyseal zinc concentration was reported (Yamaguchi et al. 1991). This decrease may represent the loss of zinc from muscle and bone, where the most zinc is stored. Yamada et al. (1997) reported a significant decrease in zinc concentrations in weight-bearing and non-weight-bearing bones of growing rats after 14 d in space. These investigators also reported abnormal thickening of the endosteal surface of the cortical bone. But on longer spaceflights, zinc excretion seemed to return to normal concentrations (Volpe et al. 2000).

A general overview of the effects of zinc salts is provided in Table 11-4.

For each exposure duration, the SWEG value will be listed based on the lowest values among the ACs for all the significant adverse effects at that exposure duration. AC values were determined by following the methods for the development of SWEGs (NRC 2000). ACs were calculated assuming a nominal potable water use of 2.8 L (including 0.8 L of water used for the reconstitution of food), in contrast to EPA’s reference volume of 2 L of water per day as drinking water. When the end points are adverse hematologic effects or reduction in water consumption, a spaceflight factor of 3 has been used. The most significant hematologic changes resulting from spaceflight are reductions in plasma volume and red blood cell mass—the greatest decreases in hematocrit having been up to 15% after 18- and 63-d Soviet flights and the 28-d Skylab mission (Huntoon et al. 1994). The greatest decreases in hemoglobin concentration were about 25% after a Salyut 4 flight of 96 d. After a Mir flight of 365 d, a 12% decrease was reported, and hemoglobin decreased after 60 d on two Skylab missions (Huntoon et al. 1994). Reduced fluid intake is one of the factors for increased risk for the formation of renal stones during spaceflights (Whitson et al. 1999).

The Office of Drinking Water (ODW) of EPA has not established a primary maximum contaminant level (MCL) for soluble zinc salts. However, EPA determined a SMCL of zinc at 5 mg/L for U.S. drinking water based on taste. EPA established an oral reference dose (RfD) of 0.3 mg/kg/d for soluble zinc salts based on a LOAEL of zinc at 59.72 mg/d (corresponding to about 1 mg/kg/d based on the human diet-supplement study). The LOAEL was based on the study by Yadrick et al. (1989), who reported a 47% decrease in the activity of ESOD in adult females after 10 wk of oral ingestion of zinc gluconate capsules. The decrease in ESOD activity in humans seems to reflect the turnover of erythrocytes and the status of copper. The data were also based on decreased hematocrit and serum ferritin. EPA applied a modifying factor of only 3 to the LOAEL of 60 mg/d to arrive at a NOAEL, and they used 60 kg as a nominal body weight for a woman to arrive at an RfD of 0.3 mg/kg/d. EPA stated that a factor of only 3 was used, because of the minimal LOAEL probably meaning that no clinical manifestation occurred at such a dose.

The Agency for Toxic Substances and Disease Registry (ATSDR) did not derive an oral minimal risk level (MRL) for acute or chronic durations, but an MRL of zinc at 0.3 mg/kg/d was established for intermediate-duration exposure by the oral route (ATSDR 1994). This MRL was based on reduced concentrations of ferritin and hemoglobin and reduced ESOD activity reported by Yadrick et al. (1989). This value (zinc at 0.3 mg/kg/d) was accepted as a MRL for chronic duration without applying any time factor.

Recently, IOM’s Food and Nutrition Board (of the National Academy of Sciences) derived dietary reference intakes (DRI) for some vitamins and minerals. The board reviewed the existing recommended dietary intakes to derive a tolerable upper intake level (UL) that this board defined as “the highest average daily nutrient intake level that is likely to pose no risk of adverse health effects to almost all individuals in the general population. As intake increases above the UL, the potential risk of adverse effects may increase.” zinc was one of the micronutrients evaluated. The RDA for zinc has been set at 11 mg/d for men and 8 mg/d for women (IOM 2001). In deriving the UL for zinc, the board considered the data on the effect of exogenous zinc on reduced copper status in humans (using the change in activity of copper-zinc ESOD as a sensitive indicator of copper status) similarly noted in the studies by Fischer et al. (1984), Samman and Roberts (1988), and Yadrick et al. (1989). Decreased circulating copper concentrations were documented in other studies (IOM 2001). Because of consistent observations in several stud-

ies, this board used a change in erythrocyte copper-zinc ESOD activity as a credible adverse end point rather than using hematocrit from the Yadrick study, which the National Aeronautics and Space Administration (NASA) has used because of its clinical significance. Using the Yadrick data, the board set 60 mg/d (supplemented zinc at 50 mg and the mean daily dietary intake at 10 mg) as the LOAEL for reduced copper status in female subjects. Similar results were noted by Fischer et al. (1984) were seen in 12 human subjects who were supplemented with zinc at 50 mg/d. Also, Samman and Roberts (1988) showed that ESOD decreased (25%) in women (n = 26) ingesting zinc as ZnSO₄ at 150 mg for 12 wk but not in men (n = 21).

Using a LOAEL of 60 mg/d, the board applied an uncertainty factor (UF) of only 1.5 to arrive at a NOAEL. This factor was also used to accommodate interindividual variability. The board justified the use of a UF of only 1.5 by saying that reduced copper status is rare in humans (IOM 2001). A UL was derived by dividing the LOAEL by 1.5, giving a value of 40 mg/d (60 mg/d ÷ 1.5 = 40 mg/d). This included contributions from all sources (diet, water, air, supplements, etc.). See Table 11-5 for a few other agencies’ guidelines.

If NASA were to use the value of 40 mg/d for the UL and extend it to calculate an AC for NASA with a nominal volume of water consumption of 2.8 L, then after subtracting a nominal value of 12 mg/d from food (old RDA), the corresponding AC (for 1,000 d) would be

IOM used data from a subacute study and derived a long-term value without any time factor. The LOAEL-to-NOAEL factor is very low. A prolonged decrease in copper status can result in anemia, as indicated in several studies. Changes in hematocrit concentration, a clinically significant result from the Yadrick study, were not considered. See Table 11-6 for SWEG values.

A review of the literature on the adverse effects of ingesting exogenous zinc (zinc supplements) indicates that data are available from a sufficient number of human studies except for chronic exposures. In certain cases, zinc supplements have been administered as medical interventions. No epidemiologic study has been done to relate the concentration of zinc in drinking water to any adverse effects. Furthermore, the absorption of zinc supplements given as part of the diet is known to vary significantly depending on the nature of the diet (see section on absorption).

The accidental ingestion of a very high concentration of zinc-containing food and fluids by humans has resulted in serious GI dis-

tress with severe nausea, vomiting, and diarrhea (Brown et al. 1964). The doses were estimated to be metallic zinc at 2.4-6.0 mg/kg in one case and 2.4-9.2 mg/kg in another case. The victims recovered from the adverse effects within 24 h. A LOAEL could not be estimated. Because of the uncertainties in the dose, these data were not used. In a case report, a 16-y-old girl was reported to have suffered from epigastric symptoms after exposure to zinc at 2.6 mg/kg/d for 1 wk (Moore 1978). Because only one individual was involved, the data could not be used to derive a 1-d AC. One of the problems in deriving an AC based on GI distress, an important adverse effect, is that none of the short-term or subchronic studies has reported this effect. Brandao-Neto et al. (1990), who studied acute human exposure to zinc, reported that secretion of cortisol and prolactin was inhibited within 4 h after ingestion of zinc as ZnSO₄ at 0.5 mg/kg/d. This change indicates that the adrenals and pituitary may be target organs for zinc toxicity. This belief is supported by subchronic animal studies (Aughey et al. 1977; Kozik et al. 1980, 1981). However, later studies reported from the same laboratory could not confirm the effects from similarly designed studies. The effects may have been transient, and peripheral adverse effects resulting from such changes may have been documented. Therefore, for the 1-d AC derivation, the data that can be extracted from short-term zinc supplementation studies in humans can be used as described in the rest of this section.

In a study in which females took zinc as zinc gluconate at 50 mg/d, ESOD, hematocrit, and hemoglobin were measured at 6 and 10 wk. Yadrick et al. (1989) found no statistically significant changes in any of these variables at 6 wk. Effects were seen only 10 wk after supplementation. A

dose rate of zinc at 0.833 mg/kg/d can be identified as a NOAEL for 1 d or 10 d, because no effects were reported even at 4 wk.

A 1-d AC can be derived as follows:

where

0.833 mg/kg/d = NOAEL;

70 kg = nominal body weight; and

2.8 L/d = nominal water consumption.

The species factor was not needed because this is a human study, and a time factor is not applied because the 2-wk NOAEL will be protective of the 1-d AC.

The second short-term exposure study resulting in data that can be used to identify a NOAEL for 1 d is that of Bonham et al. (2003a, b). In this double-blinded study, groups of 19 healthy men were given an oral supplement of zinc as zinc Chelazome at 30 mg. The authors found no statistically significant differences in the indices of immune status or markers of copper status at the end of 2 wk or 14 wk between these subjects and placebo controls. Before the trial, subjects were extensively screened for blood diseases, liver function, and lipid profiles, and their responses to a lifestyle questionnaire were obtained to help determine their eligibility. A NOAEL of 30 mg/d (zinc at 0.43 mg/kg/d) can be identified.

A 1-d AC can be derived as follows:

where

0.43 mg/kg/d = NOAEL;

70 kg = nominal body weight; and

2.8 L/d = nominal water consumption.

No species or time extrapolation factors are needed. The species factor was not needed, because this is a human study, and a time factor is not applied, because the 2-wk NOAEL will be protective of the 1-d AC. Because 0.43 mg/kg/d is a NOAEL for up to 14 wk and used for 1-d AC derivation, no “small n” factor was applied, because it would be overly conservative.

Hooper et al. (1980) administered ZnSO₄ gelatin capsules (ZnSO₄ at 440 mg; with meals, elemental zinc at 80 mg/d or 1.14 mg/kg/d) to males for 5 wk, which resulted in a significant decrease (25%) in serum HDL cholesterol, a potential risk factor for arteriosclerosis. The values returned to baseline levels 11 wk after cessation of the dose. Such a short-term change is not likely to be a significant risk factor for a chronic cardiac disease although it took 11 wk for the values to return to baseline. Although a LOAEL (1.4 mg/kg/d) can be identified without a NOAEL, these data were not used for deriving an AC for 10 d, because the change in HDL cholesterol would not be a significant risk factor for chronic cardiac disease.

The second study considered for a 10-d AC was that of Chandra (1984), in which humans (11 males) were given ZnSO₄ tablets (zinc as ZnSO₄ at 150 mg twice a day, or 4.4 mg/kg/d) for 6 wk. Effects on serum HDL cholesterol (31% decrease in HDL cholesterol and 11% increase in LDL cholesterol) were observed that were very similar to those reported by others. The LDL-HDL cholesterol ratio (the atherogenic index) rose significantly from 2.6 to 4.2 at the end of 6 wk of supplementation. Immune system function, as measured by chemotactic migration and phagocytosis, was also found to be impaired in this study. A dose of zinc at 4.43 mg/kg/d was identified as a LOAEL in this study for both the lipoprotein profile effects and immune system effects—but primarily for the latter. Even though only one dose was used in this study, the effects were significant and supported by other similar investigations.

Thus, a 10-d AC for impaired immunologic variables is derived as follows:

where

4.4 mg/kg/d = LOAEL,

70 kg = nominal body weight;

10 = LOAEL to NOAEL extrapolation factor; and

2.8 L/d = nominal water consumption.

The AC calculated from the 4-wk LOAEL will be protective for a 10-d exposure. Therefore, no time factor has been applied.

It must be pointed out that the National Research Council (NRC) committee on SWEGs identified certain weaknesses in the design of the

study, such as lack of information on volunteer history, other factors that may potentially influence the observed effects, and small sample size. However, in the absence of stronger data, NASA has decided to use the study for 10-d AC derivation.

The second short-term exposure study with data that can be used to identify a NOAEL for 10 d is that of Bonham et al. (2003a, b). When these authors gave groups of 19 healthy men oral supplements of zinc as zinc Chelazome at 30 mg, they found no significant differences between the supplemented groups and placebo controls in the indices of immune status or in the markers of copper status at the end of 2 wk or 14 wk. A NOAEL of 30 mg/d (or zinc at 0.43 mg/kg/d) can be identified for 2 wk. As the NOAEL is valid for up to 14 wk, it is unnecessary to apply a modifying factor of “small n” on this NOAEL while deriving an AC for 10 d.

A 10-d AC can be derived as follows:

Three human-subject studies were identified as candidates for deriving a 100-d AC with similar toxicity end points. In a study reported by Yadrick et al. (1989), 18 female volunteers ingested zinc gluconate capsules for 10 wk (dose of zinc at 0.83 mg/kg/d), and hematocrit, serum ferritin, and copper-zinc ESOD activity were measured at the end of 6 and 10 wk. After 10 wk of supplementation, hematocrit, serum ferritin, and ESOD activity were significantly lower than their pretreatment levels, indicating that iron and copper status can be affected by zinc supplementation. Data were analyzed by the investigators for statistically significant differences using a repeated-measures design. The least significant difference (LSD) was used to compare the effects of time within a treatment.

The data are substantiated by the Fisher et al. (1984) study, in which ingestion of zinc gluconate at 50 mg by healthy men significantly decreased ESOD. Samman and Roberts (1988) showed a decrease in ESOD at 6 wk, but the dose they used was three times higher than that used by Yadrick et al. (1989), who at 6 wk reported a NOAEL of 50 mg/d (or 50 mg/60 kg/d = 0.833 mg/kg/d). Zinc and copper are key elements of the membrane-bound enzyme ESOD. The activity of this enzyme depends on copper status, and therefore, ESOD has been exten-

sively used in studies concerning effects of micronutrients as a marker for copper status. Several studies have documented that (chronically) reduced copper status has been associated with anemia. In the 10-wk zinc-supplementation study by Yadrick et al. (1989), a dose of zinc at 0.833 mg/kg/d was identified as a LOAEL for changes in hematocrit, a significant toxicologic effect. Although no change in hematocrit was observed at 6 wk (NOAEL for 6 wk), the same dose was identified as a LOAEL at 10 wk.

The 100-d AC was calculated using the 10-wk LOAEL and from extrapolating the data to 100 d using a time-extrapolation factor of 100 d/70 d. In addition, because the experiment data showed no effect at 6 wk, a factor of 3 instead of a default factor of 10 was applied to the LOAEL to get a NOAEL, and this factor will be sufficiently protective. This is also consistent with the lower LOAEL-to-NOAEL factors (3 and below) used by EPA and IOM, as mentioned earlier.

Thus, a 100-d AC based on the change in hematocrit concentrations can be calculated as follows:

where

0.833 mg/kg/d = LOAEL;

70 = nominal body weight;

3 = LOAEL to NOAEL extrapolation factor;

2.8 L/d = nominal water consumption;

100 d/70 d or 1.43 = time extrapolation factor; and

3 = spaceflight safety factor for anemia (changes in hemoglobin).

A 100-d AC based on the change in ESOD activity can be calculated as follows:

where

0.833 mg/kg/d = LOAEL;

70 kg = nominal body weight;

3 = LOAEL to NOAEL extrapolation factor;

2.8 L/d = nominal water consumption; and

100 d/70 d or 1.43 = time extrapolation factor.

In this study, as a result of zinc ingestion, a significant decrease (about 23%) occurred in serum ferritin concentrations, the iron storage protein. Concentrations of serum ferritin have been used clinically to indicate iron status and type of anemia. An AC based on changes in iron status is the same as that calculated for ESOD changes—that is, 5 mg/L.

A second study considered is that of Chandra (1984), in which 300 mg ZnSO₄ tablets were given to 11 adult males for 6 wk, and changes were studied at 2, 4, and 6 wk. A significant decrease (32%) in serum HDL cholesterol was reported at 6 wk. LDL cholesterol was significantly increased at 6 wk. The HDL percentile value fell from 43rd to 6th percentile based on population percentile normal values for that age group. Because zinc plays an important role in the maintenance of the immune system, the impaired immune status (as judged by decreased mitogenic response to PHA stimulation) and impaired inflammatory responses (as indicated by the decreased polymorphonuclear migration response to chemotactic migration and impaired phagocytosis of opsonized bacteria) were also documented in this study. Because of its adverse effects on these immune parameters, a dose of zinc at 4.4 mg/kg/d was identified as a LOAEL. The immunologic changes were seen starting at 4 wk.

A 100-d AC can be calculated as follows using a LOAEL of 4.4 mg/kg/d:

where

4.4 mg/kg/d = LOAEL;

70 kg = nominal body weight;

10 = LOAEL to NOAEL extrapolation factor;

2.8 L/d = nominal water consumption; and

100 d/28 d or 3.57 = time extrapolation factor.

Another study considered is that of Black et al. (1988), in which groups of young, healthy men were given zinc gluconate capsules at 0, 50, and 75 mg/d. A significant decrease in serum HDL cholesterol was reported as early as 6 wk and continued to be observed until the end of the study (12 wk). This variable usually represents a risk factor for cardiovascular disease. Coronary heart disease (CHD) risk can be estimated using the Framingham CHD risk calculator from the National Heart, Lung and Blood Institute of the National Institutes of Health. Estimation of a 10-y CHD risk, using the worst-case value of HDL cholesterol at the highest dose of zinc at 75 mg/d for 12 wk, did not indicate a significantly

greater risk for the zinc-supplemented subjects than for subjects who received placebo. Thus, the observed change in serum HDL cholesterol, especially after the relatively short duration of a 100-d zinc supplementation, is not likely to present a significant risk for the development of chronic arteriosclerosis. In addition, a review of data indicated that the direction of the time-response data for the observed changes was not clear. Therefore, the data were not used for deriving an AC for 100 d.

An additional study that can be used to derive a 100-d AC is that of Bonham et al. (2003a, b), described in detail earlier, in which the authors reported no adverse effects on copper status (as determined by ceruloplasmin oxidase activity, whole blood SOD activity, serum ceruloplasmin concentration, and serum copper concentrations) or on two indices of immune status (circulating levels of peripheral blood leucocytes [full blood profile] and lymphocyte subsets) when 19 healthy adult men ingested zinc at 30 mg daily for 14 wk (close to 100 d). The authors did not measure functional markers of immune function status, such as blastogenic response of cell types or phagocytic activity of neutrophils, as was done in the Chandra study. Before they could be included in the study, the subjects underwent extensive screening that included obtaining their responses to a lifestyle questionnaire. The authors were aware of seasonal variations in lymphocyte subsets, and in this study, the changes that were evident were independent of zinc supplementation.

A 100-d AC can be calculated using a NOAEL of zinc at 0.43 mg/kg/d as follows:

where

0.43 mg/kg/d = NOAEL;

70 kg = nominal body weight;

2.8 L/d = nominal water consumption;

10/√19 = uncertainty factor applied to the NOAEL when sample size in a

human study is low (NRC 1994, 2000).

No time or species factors are needed.

Very few long-term human studies with robust data that can be used to derive a 1,000-d AC are available. One case report describes a

woman who underwent three GI surgeries for gastric ulcer and was prescribed zinc at 2.9-3.9 mg/kg/d for 10 mo. She developed hypocupremia, hyperchromic and microcytic anemia, and leukopenia (Hoffman et al. 1988). According to several published case reports (see Chronic Exposure >100 d in this document), ingestion of supplemental zinc over a period of 1-2 y resulted in severe anemia (Hoogenraad et al. 1985; Patterson et al. 1985; Simon et al. 1988; Broun et al. 1990; Gyorffy and Chan 1992). Although anemia seems to be a very well-documented adverse effect of chronic zinc ingestion, and although the effects were predicted by indicative observations in subacute studies on animals, these studies could not be used, because of small sample size and lack of a clear NOAEL for this serious adverse effect. These were case studies, and the subjects had no medical conditions before their exposure to zinc.

Data from short-term studies by Black et al. (1988) and the 6-wk study by Chandra (1984), described in detail in earlier sections, could not be extrapolated to 1,000 d with a large time extrapolation factor. Such an extrapolation would result in a very low, unrealistic value that would be far lower than the amount of zinc present in multivitamin tablets used by the public. The study by Black et al. did not identify a clear NOAEL because, although some differences between treated and placebo groups were statistically significant, the effects of placebos varied greatly. An attempt to evaluate a 10-y CHD risk (using the Framingham CHD risk calculator) using the worst-case value of HDL cholesterol at the highest dose of zinc at 75 mg/d for 12 wk did not indicate a risk significantly different from that of the placebo group. One might assume that the risk, if any, of CHD for 1,000 d must then be much lower than the 10-y risk. For this reason, the effect on serum HDL cholesterol was not considered a significant end point for a 1,000-d AC. A 1,000-d value also could not be derived from existing human data that document anemia in subjects therapeutically treated with zinc compounds and in subjects who used zinc supplements (Porter et al. 1977; Hoffman et al. 1988; Prasad 1988; Simon et al. 1988).

Therefore, data from some long-term animal studies were evaluated. In a study by Aughey et al. (1977), male and female C3H mice that ingested zinc as ZnSO₄ at 70 mg/kg/d in their drinking water ad libitum for 12 mo exhibited hypertrophy and vacuolation of islet cells of the pancreas, hypertrophy of the zona fasciculata of the adrenal cortex, and histopathologic evidence of increased synthetic and secretory activity of adrenals. Although these effects were prominent at 12-14 mo, they were not seen at 3 mo. Because histochemistry was weakly positive at 6 and 9 mo, a dose of zinc at 70 mg/kg/d can be identified as a LOAEL for 6 mo.

The limitation of the study is that there are no dose-response data. However, the time-response data were used to derive a 1,000-d AC. A 1,000-d AC was calculated from the 6-mo data as a LOAEL after using a time factor of 1,000 d/180 d. Because tissue damage was demonstrated histopathologically, a factor of 10 was used to derive a NOAEL from a LOAEL.

A 1,000-d AC for hypertrophy of the adrenal cortex and pancreatic and islets was calculated as follows:

where

70 mg/kg/d = LOAEL;

70 kg = nominal body weight;

10 = LOAEL to NOAEL extrapolation factor;

10 = species extrapolation factor;

2.8 L/d = nominal water consumption; and

1,000 d/180 d or 5.555 = time extrapolation factor.

Bentley and Grubb (1991) reported that feeding rabbits a diet containing zinc carbonate at 5,000 µg/g (estimated 175 mg/kg/d) for 22 wk resulted in a significant decline in blood hemoglobin and copper concentration. Anemia, possibly secondary to hypocupremia, was also seen. Although the authors had used a 1,000 µg/g diet (studied for only 8 wk) and a 5,000 µg/g diet (fed for 22 wk), they reported the changes in the hemoglobin and copper concentrations only for the highest dose for 22 wk. Although no dose-response data were available, this effect on hemoglobin has been reported by several studies in humans and animals. Therefore, a 1,000-d AC was derived using a LOAEL of 175 mg/kg/d for 22 wk (155 d).

Thus, a 1,000-d AC can be calculated as follows:

where

175 mg/kg/d = LOAEL;

70 kg = nominal body weight;

10 = LOAEL to NOAEL extrapolation factor;

10 = species extrapolation factor;

2.8 L/d = nominal water consumption;

1,000 d/154 d or 6.5 = time extrapolation factor; and

3 = spaceflight safety factor for anemia (changes in hemoglobin).

EPA determined that an oral RfD of 0.3 mg/kg/d is equivalent to 21 mg/d for a 70 kg adult human. This dose includes sources such as drinking water and diet. If one subtracts 11 mg as the contribution from diet, an acceptable amount from water is 10 mg/d. Using the EPA nominal water volume of 2 L, the ACs will be 5 mg/L (for a human lifetime). A 1,000-d AC of 2 mg/L has been derived by NASA. Although the 1,000-d AC is lower than the EPA guideline, given the fact that NASA’s 1,000-d AC also incorporates a safety factor that protects against any spaceflight-induced hematologic effects, the value for spacecraft water derived by NASA from animal studies is consistent with that of EPA.

A summary of ACs and SWEGs for various durations is listed in Table 11-7.

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