Zinc bis(dihydrogen phosphate)

Endpoint summary

Additional information

Non-human information

Absorption

In vitro dermal penetration studies

The dermal absorption of zinc 2-pyrrolidone 5-carboxylate, ZnO and ZnSO₄ (16 mg formulation/cm²; 0.02 – 5.62% zinc) in different formulations (3 emulsions and 2 ointments) using human abdominal skin was investigated. The receptor medium was 0.9% NaCl. After application for 72 hours, the skin was washed and stripped twice. The percutaneous absorption was determined as a percentage of the applied dose found in receptor medium and cutaneous bioavailability. It never exceeded 2%. The percentages for the absorption of zinc from ointments containing ZnO and ZnSO₄ were 0.36% and 0.34%, respectively. The percutaneous absorption of zinc from the emulsion containing zinc 2-pyrrolidone 5-carboxylate was 1.60% of the applied dose. Furthermore, the experiment showed a vehicle effect on absorption (Pirot et al., 1996a).

The dermal absorption of ZnSO₄ and ZnCl₂ (in 20 mg formulation/cm²) in petrolatum and hydrophilic gels using human breast or abdominal skin was also investigated. The receptor medium used was isotonic saline. After application for 72 hours, the skin was washed, and the epidermis was removed from the dermis. The result showed that the absorption was low (i.e., ≤ 2%) regardless of the choice of vehicle (Pirot et al., 1996b).

The utility of the data generated by Pirot et al., 1996a, 1996b, is limited due to the absence of membrane integrity measurements. Moreover, it is not clear whether the skin was occluded. The cutaneous bioavailability might be underestimated in the first study due to double stripping and in the second study, absorption is based on zinc in fresh dermis and receptor fluid, the fresh epidermis is not included.

An industry in vitro percutaneous absorption testing programme on two representative zinc compounds (ZnO and ZnSO₄) was conducted by (Grötsch, 1999). In this programme, a solution of ZnSO₄ monohydrate and a suspension of ZnO, each at concentration of 40 mg/mL in water, were tested for cutaneous penetration and absorption through pig skin in vitro. Skin preparations measuring 1 mm in thickness with stratum corneum, stratum germinativum and blood-vessel-containing parts of the dermis were obtained from pigs using a modified dermatome. In two independent experiments for each compound seven skin preparations were mounted in Teflon flow-through diffusion chambers which were continuously rinsed with physiological receptor fluid (0.9% NaCl in aqua bidest with antibiotics). Following an integrity check using the marker substance caffeine, each of the test formulations were applied to six skins at a dose of 1 mg/cm² for 8 hours without occlusion, and subsequently washed off with a neutral shampoo. After 0, 2, 4, 6, 8, 16, 24, 40, 48, 64 and 72 hours, the cutaneous permeation was determined by quantifying zinc with atomic absorption spectroscopic analysis (detection limit: 10 ng/mL) in the receptor fluid. The experiment was stopped at 72 hours. Zinc was analysed in the skin preparations and the rinsing fluids. In addition, blanks were measured in an unloaded control chamber. Results are summarized in Table below.

 

Table. Dermal absorption of Zn (% of dose) through pig skin in vitro within 72 hours

	Soluble ZnSO4^a	Slightly soluble ZnO^a
Receptor fluid	0.3 %	0.03 %
Horny layer	1.3 %	12.3 %
Residual skin	0 %	2.6 %
Potentially absorbed dose	1.6%	14.9%

^a Corrected for background levels of zinc in receptor fluid and skin.

 

Total recoveries of applied zinc in both experiments ranged from 82.0 to 109.6%. The results of the analysis of the receptor fluid used and of the blank chambers without topical application of zinc compounds indicated that both the receptor fluid and porcine skin contain an intrinsic level of zinc. The amounts of zinc detected in receptor fluid and different layers of the skin were therefore corrected for background levels. The authors concluded that dermal penetration of zinc was below 1% based on the cumulative amount recovered from the receptor fluid at 72 hours. However, the amount retained in the skin should be regarded as being absorbed because it may become available at a later stage. Hence, the rapporteur concluded that the dermal absorption of zinc from a solution of ZnSO₄ monohydrate and a suspension of ZnO in this in vitro system may amount to 1.6% and 14.9%, respectively (Grötsch, 1999).

Animal studies

Oral

Zinc acetate was added to the diet of Sprague-Dawley rats (9/group) to reach zinc concentrations of 58 (no zinc acetate added; normal zinc concentration in “control” feed), 117, 175, 293, 410 or 664 mg/kg via the feed, corresponding to ca. 3, 6, 9, 14.5, 20.5 or 33 mg Zn/kg bw. After 28 days, the unfasted animals were dosed with 1.2 mCi of ⁶⁵ZnCl₂ (ca. 0.15 ng). Whole-body radioactivity was determined at various time points up to 11 days post-dosing using a whole-body gamma counter. In the group which received the non-supplemented diet (i.e., 58 mg Zn/kg feed) ca. 20% of the administered radioactivity was retained at 24 h post-dosing which gradually decreased to about 9% at day 11. The amount of radioactivity retained at 24 h post-dosing declined with increasing dietary zinc levels to about 13% for the group with the highest dietary zinc. In this group after 11 days only ca. 2.3% of the administered radioactivity was left. The data indicated that low dietary zinc intake results in increased zinc retention and that at higher dietary zinc levels absorption of zinc is reduced (Furchner and Richmond, 1962).

After a pre-exposure period of 7 days, male Wistar rats, kept on a semi-synthetic diet, were dosed orally with 86 - 130 mg ⁶⁵Zn as ZnCl₂ (n=15), ZnCO₃ (n=15) or Zn₅(OH)₈Cl₂×H₂O (n=20) added to a test meal. It was assumed that during the first 5 days post-dosing non-absorbed zinc was excreted via the faeces. Absorption of labelled zinc was calculated from in vivo whole-body gamma counting results over the period 5-14 days post-dosing. The uptake was calculated to be 40, 45 or 48 % for Zn₅(OH)₈Cl₂×H₂O, ZnCl₂ and ZnCO₃, respectively (Galvez-Morros et al., 1992).

Inhalation

The rate or percentage of absorption of zinc following inhalation exposure is not available, but there are several studies investigating the zinc retention in the lung. Male and female rats were exposed to 15 mg ZnO dust/m³ (particle size < 1 mm) for 4 hours/day, 5 days/week during 1 day or for 2, 4 or 8 weeks. Animals were killed 24 hours after the last exposure and the zinc content of the lungs, liver, kidneys, tibia and femur was measured. After 1 day of exposure the total zinc content of the lung in males and females were approximately 46 and 49 mg, respectively. In the lung, liver, kidney and bone only minimal differences in tissue zinc content was seen during the experiment. As tissue zinc levels in non-treated animals were not studied, it is not clear whether tissue zinc comes from the experimental or from dietary exposure. However, as the pulmonary zinc level did not rise throughout the study it can be assumed that pulmonary deposition is very low and/or that pulmonary clearance of zinc particles is very high (Pistorius et al., 1976).

In another experiment, following exposure to 4.3 mg (rat), 6.0 mg (rabbit), 11.3 mg (guinea pig) mg ZnO (aerosol)/m³ (aerosol mass median diameter was 0.17 mm) for 2-3 hours, the pulmonary retention in rats, rabbits and guinea pigs was determined to be 11.5%, 4.7% and 19.8%, respectively (Gordon et al., 1992).

The lung clearance rate of zinc aerosols was determined in male Wistar rats (8/group) 0, 2, 4, 8 and 24 hours after exposure to ZnO aerosol at a concentration of 12.8 mg/m³ (mean aerodynamic diameter of 1 mm) for 17 hours. The ZnO aerosol was created by pyrolysis of a micronized zinc acetate aerosol at 500^o C. Eight animals were kept in clean air and served as controls. The lungs and trachea of the animals were removed and their zinc content was determined by flame photometry. In comparison with the controls, the lungs of exposed rats were increased in weight (presumably because of oedema), of which the increase was significant at 8 hours and even more pronounced at 24 hours. The zinc content in the trachea was not uniform but was above control values except after 24 hours. The zinc content in the lungs decreased mono-exponentially and was 7% of the initial burden after 24 hours. According to the short half-life of 6.3 hours found in this study for the pulmonary zinc content, a fast dissolution of the particles must occur, as the alveolar clearance of an inert Fe₂O₃ aerosol occurred with a half-life of about 34 hours. It was not clear whether the clearance of Zn particles from the lungs was affected by the pathological condition of the lungs (Oberdörster et al., 1980).

Intratracheal instillation

In a time course experiment male Wistar rats (3/group) received a single intratracheal instillation of 0.4 ml ZnO suspension (i.e., ZnO particles < 2 mm; particles appeared to form aggregates of 10-20 particles) at a dose of 100 mg Zn/rat and the rats were killed 1, 2, 3, 5, 7, 14 and 21 days after administration. In a dose-response experiment 0.4 ml ZnO suspension (ZnO particles < 2 mm, but they appeared to form aggregates of 10-20 particles) was instilled in the lungs of male Wistar rats (3/group) at doses of 20, 50, 100, 200, 500 and 1000 mg Zn/rat. The rats were killed after 2 days. Control animals were included in the experiments. A significant increase in lung wet weight 1 day after instillation remained throughout the study. Only a limited portion of zinc could be retrieved in the bronchoalveolar lavage fluid (BALF). No measurable amount of exogenous zinc was observed after 5 days. The half-life of ZnO instilled in the lung was calculated to be 14 hours. In the dose-response experiment, the lung wet weight increased with increasing dose of ZnO, two days after instillation. The results indicated that the rat lung was able to clear ZnO particles up to a dose of 50 mg Zn/rat at least within two days. No measurable accumulation of zinc was observed in the liver and kidneys even at a dose of 1000 mg Zn/rat (Hirano et al., 1989).

Dermal

The percutaneous uptake of ⁶⁵ZnCl₂ by the dorsal skin of the guinea pig was estimated by monitoring the decline of radioactivity emitted by ⁶⁵ZnCl₂ in at least 10 trials for each concentration tested ranging from 0.8 to 4.87 M ZnCl₂ (pH 1.8-6.1). It appeared that the loss of radioactivity after 5 hours was less than 1% except for the trials with the lowest pH where it might have been between 1 and 2%. The study gives too little details to be used for risk assessment as cited in EU 2004, a, b, c, d, e, f (Skog and Wahlberg 1964).

Zinc oxide, zinc omadine, zinc sulphate and zinc undecylenate (131 mCi/mole of ⁶⁵Zn) were topically applied to shaved skin on the back of rabbits. Each application consisted of 2.5 mg zinc compound containing 5 mCi ⁶⁵Zn. Two animals received one application on four skin areas left of the spine, while the four skin areas on the right side received two applications, the second one 24 hours after the first one. The rabbits were killed 6 and 24 hours after the second application. One rabbit served as the control. No significant differences were found in the amount and location of ⁶⁵Zn in skin treated with 4 different zinc compounds. High concentrations of ⁶⁵Zn were observed in the cortical and cutical zones of the hair shaft, being the highest in the keratogenous zone. Accumulation of ⁶⁵Zn in epidermis was very low but heavy in the subdermal muscle layer. No difference in the rates of absorption and concentrations of zinc compounds with different oil/water solubility, pH, and molecular weight were seen. It was therefore suggested that the major mode of ⁶⁵Zn uptake in skin is by diffusion through the hair follicles due to the heavy localization of ⁶⁵Zn primarily in the hair shaft and hair follicles. According to the author, this emphasizes that chemical differences in the compounds may not play a very important role in the skin uptake of ⁶⁵Zn. No data were given on systemic absorption (Kapur et al., 1974).

The dermal absorption of ⁶⁵Zn from ZnCl₂ and ZnO was studied by applying zinc preparations under occlusion on the shaven intact skin on the back of male Sprague-Dawley rats. The zinc absorption, being the ratio between ⁶⁵Zn-activity in the carcass, liver and gastrointestinal tract, and the ⁶⁵Zn-activity in carcass, liver, gastrointestinal tract, skin and bandage, was reported to range from 1.6 to 6.1%. It should be noted that the higher percentages (3.6 to 6.1%) were achieved after application of ZnCl₂ in acidic solution (pH = 1). Less acidic solutions with ZnCl₂ or with ZnO resulted in a dermal absorption of less than 2%. In this study, only the absorption into the body, excluding the skin, was determined. No data were available as to the effect of ZnCl₂ solutions with pH = 1 on dermal integrity (Hallmans and Lidén, 1979).

Topical application of ZnCl₂ in an oil vehicle to pregnant Sprague-Dawley rats which were fed a zinc-deficient diet for 24 hours resulted in an increase in plasma concentration of zinc cations to normal or slightly above normal levels. The absorbed fraction was not determined therefore it can be concluded that dermal absorption is possible but no quantification can be given (Keen and Hurley, 1977).

The application of ZnO dressings (containing 250 mg Zn/cm²) to rats for 48 hours with full-thickness skin excision resulted in a 12% delivery of zinc ions from the dressing to each wound, while application of ZnSO₄ dressings (containing 66 mg Zn/cm²) resulted in a 65% delivery of ions to each wound. The data suggest that the application of ZnO resulted in sustained delivery of zinc cations causing constant wound-tissue zinc cation levels due to its slow dissociation rate, while the more water soluble ZnSO₄ delivers zinc ions more rapidly to the wound fluid with subsequent rapid transferral into the blood (Agren et al., 1991a).

Distribution

The highest levels of radioactivity were found in the small intestine followed by the kidney, liver and large intestine six hours after a single oral administration of 0.1 mCi of ⁶⁵Zn ²⁺ as ZnCl₂ to Wistar rats. Smaller amounts were found in the lungs and spleen. Fourteen days after administration, the highest levels of radioactivity were found in the hair, testicles, liver and large intestines (Kossakowski and Grosicki, 1983). 

Organs with high zinc concentrations (ranging from 20 to 60 mg/kg fresh weight) are liver, gut, kidney, skin, lung, brain, heart and pancreas as cited in EU RARs (Bentley and Grubb, 1991; He et al., 1991; Llobet et al., 1988). High concentrations of zinc were also detected in the retina and in sperm as cited in EU RARs (EU 2004, a, b, c, d, e, f; Bentley and Grubb, 1991).

The tissue uptake of ⁶⁵Zn (as zinc chloride) was determined in adult male Wistar rats after intraperitoneal injection of 15 mCi ⁶⁵Zn. The liver displayed the greatest uptake for zinc ions, followed by the kidney, pancreas, spleen, ileum, lung, heart, bone, testis, blood cells, muscle and brain. Additional data on Zn uptake by the brain indicates that the blood-brain barrier is minimally permeable to zinc cations (Pullen et al., 1990).

Eight hours following intravenous administration of ⁶⁵[Zn]-chloride to rabbits, tissue levels were highest in the liver, intestine and kidney with levels being ³ 10%/g in tissue (Lorber et al., 1970).

 

Metabolism

As described in EU RARs, zinc is primarily bound to organic ligands rather than existing free in solution as a cation (Gordon et al., 1981). It is found in diffusible and non-diffusible forms in the blood. About 66% of the diffusible form of zinc in the plasma is freely exchangeable and loosely bound to albumin (Cousins et al., 1985). A small amount of the non-diffusible form of zinc is tightly bound to a₂-macroglobulin in the plasma and is not freely exchangeable with other zinc ligands. Zinc is incorporated into and dissociated from a₂-macroglobulin only in the liver (Henkin et al., 1974).

Excretion

After a single oral dose of 86 – 130 mg of ⁶⁵Zn as ZnCl₂, ZnCO₃ or Zn₅(OH)₈Cl₂×H₂O, male rats eliminated ⁶⁵Zn from the body with a rate of about 1.7% of the absorbed dose during day 5 to 14 post-dosing as determined from stool, urinary and in vivo whole-body gamma counting results. Male rats who received 25 mg ZnCO₃/kg feed or 100 mg Zn₅(OH)₈Cl₂×H₂O/kg feed for 14 days, the radioactivity from a subcutaneous dose of 37 kBq of ⁶⁵ZnCl₂ disappeared from the body with a rate of approximately 1% during the period 5 to 14 days post dosing (Galvez-Morros et al., 1992).

As described in EU RARs (EU RAR, 2004a-f) within certain limits, mammals can maintain the total body zinc and the physiologically required levels of zinc in the various tissues, constant, both at low and high dietary zinc intakes. The sites of regulation of zinc metabolism are: absorption of zinc from the gastrointestinal tract, excretion of zinc in urine, exchange of zinc with erythrocytes, release of zinc from tissue, and secretion of zinc into the gastrointestinal tract. Regulation of gastrointestinal absorption and gastrointestinal secretion most likely contributes the most to zinc homeostasis. In spite of the mechanism for whole-body zinc homeostasis, a regular exogenous supply of zinc is necessary to sustain the physiological requirements because of the limited exchange of zinc between tissues ((EU RAR, 2004a-f). It has been hypothesized by Hempe and Cousins (1992) that zinc entering the luminal cells is associated with cysteine-rich intestinal protein (CRIP), a diffusible intracellular zinc carrier, and that a small amount is bound to metallothionein; however, as the luminal zinc concentration increases, the proportion of cytosolic zinc associated with CRIP is decreased and zinc binding to metallothionein is increased. CRIP binds 40% of radiolabelled zinc entering the intestinal cells of rats when zinc concentration is low; but only 14% when the concentration is high.

Zinc is initially concentrated in the liver after ingestion, and is subsequently distributed throughout the body. When plasma zinc levels are high, liver metallothionein synthesis is stimulated, which facilitates the retention of zinc by hepatocytes (EU RAR, 2004a-f).

 

Human information

Absorption

Oral

A wide range of absorption (8-80%) is observed in humans (EU RAR, 2004a-f). This is likely due to differences in eating habits (Hunt et al., 1991; Reinhold et al., 1991; Sandstrom and Sandberg, 1992). Persons with adequate nutritional levels of zinc absorb approximately 20-30% of all ingested zinc. Those who are zinc-deficient absorb greater proportions of administered zinc while persons with excessive zinc intake, gastrointestinal uptake can be less (Babcock et al., 1982; EU RAR, 2004a-f).

Zinc absorption in the gastrointestinal tract occurs throughout the entire small intestine with the highest rate in the jejunum and the rate of total absorption appears to be concentration-dependent (Lee et al., 1989) as cited EU RARs (EU RAR, 2004a-f).

The zinc absorption process in the intestines includes both passive diffusion and a carrier-mediated process (Tacnet et al., 1990). At low zinc concentrations CRIP is involved in this process. This protein binds zinc entering the intestinal cells from the lumen but this process appears to be saturable. Metallothionein, a metal-binding protein (also rich in cystein), may be involved at higher zinc concentrations (Gunshin et al., 1991; Hempe and Cousins et al., 1992; Sturniolo et al., 1991). Zinc cations can induce metallothionein production in intestinal mucosa cells (Richards and Cousins, 1975; EU 2004 a-f).

The intestinal absorption following a single oral administration of ⁶⁵ZnCl₂ to 6 groups of 5 healthy adult volunteers has been determined by comparison of whole body radioactivity counting and faecal excretion data. The individuals fasted overnight prior to dosing. Approximately 55% of the administered ⁶⁵ZnCl₂ was absorbed at doses of 18, 45 and 90 mmol of zinc (i.e., approximately 1.2, 2.9 or 5.8 mg Zn). The absorption was reduced with increasing dose, indicating that zinc absorption is saturable. At test dose levels of 180, 450 and 900 mmol (i.e., approximately 11.6, 29 or 58 mg Zn), only 51, 40 and 25% of the ⁶⁵Zn was absorbed, respectively. Additional studies in 15 human volunteers with various intestinal diseases indicated that absorption of zinc occurred mainly in the proximal parts of the intestine. This study suggests that uptake levels vary maximally by a factor of 2 in healthy persons with intake levels differing by a factor of 10 (Payton et al., 1982).

The absorption of orally administered ⁶⁵Zn was studied in 50 patients with taste and smell dysfunction. The study was conducted in three phases: Prior to the start of the study 10 patients were admitted to a metabolic ward and put on a fixed daily diet containing 8-13 mg Zn. In the first phase, all patients were studied for 21 days after receiving a single oral dose of 3-18 mCi of ⁶⁵Zn (i.e., approximately 0.4 to 1.2 ng Zn) as ZnCl₂ after an overnight fast. In the second phase, which started after 21 days and continued for 290 to 440 (mean 336) days, all 50 patients received a placebo. To study the effect of additional zinc intake on the elimination of previously sequestered radioactivity, in the third phase of the study 14 patients continued on the placebo while 36 received ZnSO₄ (100 mg Zn/day) for 112 to 440 (mean 307) days. Phases two and three were a controlled clinical trial of the effects of zinc on retention of the ⁶⁵Zn tracer. The results of phase two and three are described under excretion. Total body retention and activity in plasma and red cells were measured for all patients throughout the study. It was estimated that for the ten in-patients ca. 55% of the administered radioactivity was absorbed while for the whole group of 50 patients the absorption was approximately 60 %. From the study description it is not clear whether the radioactivity was administered as pure radioactive ZnCl₂ or whether it was diluted with unlabelled ZnCl₂. The authors stated that “patients were given 3 to 18 mCi carrier free ⁶⁵Zn”, therefore for the calculation of the dose of ⁶⁵Zn in terms of nanogram zinc, it has been assumed that all zinc administered was in fact radiolabelled zinc (Aamodt et al., 1982).

The absorption of zinc from soluble zinc acetate, zinc sulphate, zinc aminoate, zinc methionine and insoluble zinc oxide were compared in ten human volunteers who were dosed orally with 50 mg zinc in various forms separated by two week intervals. The bioavailability of zinc from the various forms was compared on the basis of plasma zinc levels and area under the plasma curve analysis. Plasma peak levels were observed after about 2.5 h for all forms, but maximal plasma zinc concentration amounted to 221 and 225 mg/dL for the acetate and the sulphate form while the peak plasma level for zinc from the oxide was only 159 mg/dL. When AUC values for the different zinc forms were compared, it appeared that the bioavailability of insoluble ZnO was about 60% of the bioavailability of the soluble forms. Information on absolute bioavailability was not obtained (Prasad et al., 1993).

A study to measure the absorption half-life of zinc as ZnSO₄ was performed. Gelatine capsules containing 45 mg zinc as ZnSO₄ was administered once to 10 healthy young men. Serum concentrations were measured frequently during a total investigation time of 8 hours. A mean maximum concentration of 8.2 mmol Zn/L serum was found after 2.3 hours (t_max). There was evidence of an enteral recirculation, the first rebound effect appeared after 1.4 hours during the absorption phase before t_max was reached and exhibited mean reabsorption rates of 70% of the dose given. The subsequent ones (max. of 5) appeared at regular intervals of 1.2 hours with a decrease of the quantity reabsorbed. The absorption half-life of zinc administered as ZnSO₄ was 0.4 hours (Nève et al., 1991).

Factors that influence the gastrointestinal absorption of zinc cations include ligands (for example a decreased zinc absorption may occur by intake of plant proteins, such as soy and phytate), by intake of alcohol, use of EDTA or other trace elements in the diet (EU 2004 a-f). Also the zinc status of the body, the endogenous zinc secretion into the intestinal lumen via epithelial cells, bile and pancreatic secretion, and the intracellular transport have an influence on the zinc absorption in the gastrointestinal tract (Cunnane, 1988); Flanagan et al., 1983). However, the mechanism by which zinc is transferred to or across the mucosal surface of the microvilli is unknown (Cousins, 1989).

Inhalation

Elevated zinc concentrations in blood and urine of persons occupationally exposed to ZnO fumes suggest that there is some pulmonary absorption, but no quantitative human data are available (Hamdi et al., 1969 and Trevisan et al., 1982 cited in EU RAR, 2004a-f).

Data on the particle size distribution of zinc aerosol in three different industry sectors, i.e. the galvanising sector (three plants, 4 samples each), the brass casting sector (two plants, 3 and 4 samples respectively) and the zinc oxide production sector (one plant, 10 samples), has been provided using personal cascade impactors with cut-off diameters of 0.52, 0.93, 1.55, 3.5, 6.0 and 21.3 mm, and a final filter diameter of 0.3 mm (Groat et al., 1999; EU 2004, a-f). These data served as input for the Multiple Path Particle Deposition Model (MPPD version V1.11; Freijer et al., 1999) in order to estimate the airway deposition (in head, tracheobronchial and pulmonary region) for workers, by using:

• The human – five lobar lung model;


• A polydisperse particle distribution (i.e. this distribution contains a wide range of particle sizes), by taking the mean size distribution of the 10 samples for zinc oxide production (MMAD 15.2 mm, GSD 4.0). Using this MMAD and GSD for the total polydisperse distribution is preferred above treating the polydisperse particles on individual impactor stages (with given cut-off diameters) as being monodisperse particles, also because the maximum particle size in the MPPD model (20 mm) is lower than the largest size fraction of the cascade impactor (21.3 mm).


• Both the oral breathing and the oronasal (normal augmenter) mode, but not the nasal breathing mode. The latter is considered to present an underestimate because (1) many people are oronasal or oral breathers, independent of their activities, (2) people with a cold will not normally breath nasally and (3) with heavy exercise, short-term deep oral breathing will occur, resulting in increased deep pulmonary deposition.


• The possibility of inhalability adjustment for the oronasal augmenter. Inhalability is defined as that fraction of particles in an aerosol that can enter the nose or mouth upon inhalation. It must be noted that inhalability is different from respirability (which relates to the deposition of particles after making their entrance inside the airways). If “inhalability adjustment” is “off”, the calculations start by assuming that the airflow is in line with the direction of the nasal entrance. However, in reality this will not be the case because the airflow has to make turns to enter the nose. This results in losses that are larger with increasing particle size. Ménache et al., (1995) described the relationship between exposure concentration and concentration at the entrance of the airways for laboratory animals and humans as cited in EU RARs (EU RAR, 2004a-f).


• A tidal volume and breathing frequency corresponding to the default breathing rate of 10 m³ for an 8-hr shift (1100 mL and 20 breaths/min, respectively). This breathing rate is more representative for light exercise activities than for more moderate or heavy exercise activities (EPA, 1997), which can be expected to take place in the zinc industry. Therefore, also a non-default tidal volume and breathing frequency corresponding to a breathing rate of 19 m³ for an 8-hr shift have been taken (1700 mL and 23 breaths/min, respectively, based on a breathing volume of 40 L/min for moderate exercise activities (EPA, 1997)). It must be noted that at a minute volume <35.3 L/min for normal augmenters breathing is only through the nose, while at a minute volume ³3 mL/min there is combined nose and mouth breathing. For oral breathers, breathing is always only through the mouth, independent of the minute volume used.

The results of the MPPD modelling are given in Table below. It must be noted that the MPPD only models deposition, not clearance and retention.

 

Table. Deposition fractions for oral breathers and for oronasal augmenters, using a polydisperse particle distribution (MMAD 15.2 mm, GSD 4.0)

	Inhalability Adjustment	Tidal volume (mL)	Breaths (min^-1)	Deposition region
				Head	Tracheo-bronchial	Pulmonary	Total
Oral	Off	1100 1700	20 23	0.638 0.676	0.071 0.100	0.139 0.101	0.848 0.877
Oronasal	Off	1100 1700	20 23	0.927 0.804	0.011 0.064	0.021 0.064	0.960 0.932
Oronasal	On	1100 1700	20 23	0.519 0.585	0.011 0.063	0.021 0.064	0.551 0.713

 

From Table 23 it can be seen that for oral as well as for oronasal breathers the largest part of the deposition takes place in the head region when inhalability adjustment is “off”, irrespective of the breathing rate. When inhalability adjustment is “on” the head region deposition is reduced. However, as stated above, the corrections for inhalability of particles is based on relationships derived by Ménache et al., (1995). For humans this is based on experiments with 4 healthy adult volunteers. From the available data it is not possible to conclude that this correction is valid for all human subjects in all situations (children, elderly, exercise activity, etc). Therefore it is reasonable to estimate the deposition with the inhalability adjustment “off” which leads to a worst case scenario and therefore the inhalability adjustment “on” will not be considered further.

The fate and uptake of deposited particles depends on the clearance mechanisms present in the different parts of the airway. In the head region, most material will be cleared rapidly, either by expulsion (not the case for oral breathers) or by translocation to the gastrointestinal tract (half-life 10 min). A small fraction will be subjected to more prolonged retention, which can result in direct local absorption. This is concluded to be almost the same for the tracheobronchial region, where the largest part of the deposited material will be cleared to the pharynx (mainly by mucociliary clearance (half-life 100 min)) followed by clearance to the gastrointestinal tract, and only a small fraction will be retained (ICRP, 1994). Higher uptake rates may be assumed for the pulmonary region than for the head and tracheobronchial region.

Once translocated to the gastrointestinal tract, uptake will be in accordance with oral uptake kinetics. Hence, for the part of the material deposited in head and tracheobronchial region that is cleared to the gastrointestinal tract, the oral absorption figures 20% for soluble zinc compounds and 12% for slightly soluble and insoluble zinc compounds can be estimated. However, there are no data available on zinc to estimate the part that is cleared to the gastrointestinal tract and the part that is absorbed locally in the different airway regions. With respect to the latter, there are some data available for radionuclides. After instillation of small volumes (2-3 mL for rats, 10 mL for hamsters, 0.3 mL for dogs) of solutions or suspensions of radionuclides into each region of the respiratory tract, retention and absorption into blood was measured. For the more soluble chemical forms (a.o. citrate and nitrate) absorption values of 4.8-17.6% in the nasopharynx, 12.5-48% in the tracheobronchial region and up to 100% in the pulmonary region was found. For the slightly soluble chemical forms (i.e. oxide) retention and absorption in the nasopharynx and tracheobronchial region was negligible (ICRP, 1994). There are no data on how the solubility of the different chemical forms of the radionuclides compares to the solubility of the soluble zinc compounds. Although the applicability of the radionuclide figures to the zinc compounds is not quite clear, it is probably a reasonable worst case scenario to take the upper values found (i.e. 20, 50 and 100% in head, trachebronchial and pulmonary region, respectively) for local absorption of the soluble zinc compounds (zinc chloride and zinc sulphate). For the slightly soluble and insoluble zinc compounds (zinc oxide, zinc phosphate and zinc metal) it is probably safe to assume negligible absorption for the head and tracheobronchial region and 100% absorption for the pulmonary region. This is supported by the findings in the study by Oberdörster et al., (1980), where the dissolution half-life of 1 mm diameter zinc oxide particles in the deep lung was approximately 6 hrs. Given that the clearance to the gastrointestinal tract occurs within a time frame of minutes (10-100 min in head and tracheobronchial region), there will be no significant dissolution in these areas. Furthermore, most of the particles in these areas will have a diameter >1 mm, thus dissolution half-lives for these larger particles will be longer. Based on the above, Table below describes the assumptions used in estimating the absorption by inhalation.

 

Table. Assumptions used for estimating the inhalation absorption

	Soluble zinc compounds   (e.g., ZnCl2, ZnSO4)	Slightly soluble to insoluble zinc compounds (e.g., Zn; ZnO; Zn3(PO4)2)
Fraction absorbed in airway region	20% head 50% tracheobronchial 100% pulmonary	0% head 0% tracheobronchial 100% pulmonary
Fraction cleared to GI tract, followed by oral uptake kinetics	80% head  x 20% 50% tracheobronchial  x 20% 0% pulmonary	100% head  x 12% 100% tracheobronchial  x 12% 0% pulmonary

 

By applying the above assumptions to the deposition fractions, the % of inhalatory absorption of the soluble zinc compounds (zinc chloride and zinc sulphate) and slightly soluble to insoluble zinc compounds (zinc oxide, zinc phosphate and zinc metal) can be estimated as described in Table below.

Table. Percentage estimations for inhalation absorption of soluble, slightly soluble and insoluble zinc compounds

	Inhalability	Tidal volume (mL)	Breaths   (min^-1)	Soluble zinc compounds   (e.g., ZnCl2, ZnSO4)	Slightly soluble to insoluble zinc compounds (e.g., Zn; ZnO; Zn3(PO4)2)
Oral	off	1100 1700	20 23	41.1 40.4	22.4 19.4
Oronasal	off	1100 1700	20 23	36.1 39.2	13.4 16.8

Inhalation absorption for the soluble zinc compounds (zinc chloride and zinc sulphate) is at maximum 40%, while for the slightly soluble and insoluble zinc compounds (zinc oxide, zinc phosphate and zinc metal) inhalation absorption is at maximum 20%. These values are assumed to be a reasonable worst case and are thought to cover existing differences between the different zinc industry sectors with respect to the type of activities (therefore breathing rate) and the particle size distribution.

Dermal

Zinc has been reported to be absorbed through damaged or burned skin however in the absence of quantitative data it is difficult to assume that zinc can be absorbed through intact skin (EHC, 1996).

An increase in serum zinc levels was observed in 8 patients suffering from second and third degree burns, who were treated with adhesive zinc-tape (ca. 7.5 g ZnO/100 g dry weight). The maximum value (up to 28.3 mmol/litre) was reached within 3-18 days during treatment. It is noted that the absorption through intact skin could not be assessed (Hallmans, 1977).

The systemic absorption from topical application of 40% zinc oxide ointment (with petrolatum) was investigated in 6 healthy subjects in a cross-over study. On two separate days, one week apart the subjects received a topical application of 100 g of the 40% zinc oxide ointment or 60 g of control ointment (100% white petrolatum base) to the chest, upper legs and lower legs (exposed skin area: not specified; occlusion: not specified) for 3 hours. Each subject fasted for 12 hours before treatment started (only water ad libitum). During the study no food or water was consumed. Blood samples were taken after the 12 hr-fast (baseline value), and at 1, 2 and 3 hours after the start of the topical application. Mean serum zinc concentrations at these time points were 107.3, 116.1, 105.3 and 112.6 mg/dL for the zinc ointment and 115.2, 103.5, 105.5 and 110.5 for the control ointment, respectively. Normal serum zinc concentrations were considered to be in the range of 68 to 136 mg/dL. An increase in serum zinc over the baseline value was observed in 4/6 subjects. In 3 of them, the rise was most pronounced after 1 hr. In 2/6 no increase was observed throughout the treatment. Overall, 1 hour after application, there was a mean serum zinc increase of 8.8 mg/dL over the baseline. This represented an 8.2% rise in serum zinc which was not statistically significant (Derry et al., 1983).

The systemic absorption was also investigated in patients receiving total parenteral nutrition (TPN) for a minimum of 3 days prior to the start of the experiment. TPN is known to result in zinc deficiency (mean decrease 6.6 mg/dL/week), and the longer the period of TPN without zinc supplementation, the greater the decrease in serum zinc concentration. In a controlled, cross-over study (on two separate days, one week apart) 6 patients received a topical application of 15 g of the 40% zinc oxide ointment onto the upper legs (10x15 cm) once daily for 8 consecutive days under occlusion. Blood samples were taken before treatment (baseline value), at 4, 6 and 8 days (just prior to application), and at day 10. The mean baseline level of the patients (88.6 mg/dL) differed significantly from the mean baseline level of the healthy subjects. The mean zinc concentration in the 3 patients that completed the study remained relatively stable over the 10 day period (78-93 mg/dL) (Derry et al., 1983).

It can be concluded that topical applications of 40% zinc oxide ointment did not result in a significant increase in serum zinc concentration in healthy human subjects over a 3-hr period nor in TPN-patients over 10 days. The authors suggested that after topical application, zinc is locally absorbed and stored in the hair follicles where it is relatively unavailable for immediate systemic absorption in subjects with normal serum zinc concentrations. In subjects that are hypozincemic, there is absorption from the storage depot at a rate sufficient to prevent a decline in serum zinc concentration. The authors concluded that the 3-hr sampling time in normal subjects may have been insufficient to allow for appreciable systemic absorption from the storage depot (Derry et al., 1983).

When ZnO-mediated occlusive dressings (25% w/w; 4x5 cm) were applied to the lower arm of 10 healthy volunteers for 48 hours it appeared that the mean release rate of zinc to normal skin was 5 mg/cm²/hour. After treatment of 5 other volunteers with the ZnO dressings for 48 hours the zinc content was significantly increased in the epidermis and in the accumulated blister fluid (to model percutaneous absorption, suction blisters were used).  It should be noted, however, that the zinc penetration was enhanced during the formation of blisters, indicating that the barrier function was impaired (Agren, 1990).

In another study, five human volunteers were exposed to different occlusive ZnO dressings (with hydrocolloid vehicle or gum rosin). After 48 hours, suction blisters on treated skin were raised and zinc concentration in blister fluid was determined. Furthermore, the zinc concentration in the stratum corneum (10 successive tape strippings) was determined. The absorbed amount could not be determined from the data presented but it appeared that the vehicle is an important factor for zinc penetration (Agren, 1991b).

Distribution

After absorption from the gastrointestinal tract, the zinc is bound in plasma primarily to albumin and then transported to the liver and subsequently throughout the body. The normal plasma zinc concentration is ca. 1 mg/L, the total zinc content of the human body (70 kg) is in the range of 1.5-2 g (ATSDR, 2005).

Zinc is found in all tissues and tissue fluids and it is a co-factor in over 200 enzyme systems. In humans, the major part of total body zinc is found in muscle and bone, approximately 60% and 30%, respectively (Wastney et al., 1986). Under normal conditions, the highest zinc concentration per tissue weight is found in bone, hair and in the prostate (Cleven et al., 1993).

The distribution of zinc in humans appears to be influenced by age. The zinc concentration levels increase in the liver, pancreas and prostate and decreases in the uterus and aorta with age. Levels in kidneys and heart peak at approximately 40-50 years of age and then declines. Levels in the aorta decline after 30 years of age (Schroeder et al., 1967).

Metabolism

Zinc is mostly bound to organic ligands rather than existing free in solution (Gordon et al., 1981). Zinc is found in diffusible and non-diffusible forms in the blood and about 66% of the diffusible form of zinc in the plasma is freely exchangeable and loosely bound to albumin (Cousins, 1985). A small amount of the non-diffusible form of zinc is tightly bound to a₂-macroglobulin in the plasma and is not freely exchangeable with other zinc ligands. Zinc is incorporated into and dissociated from a₂-macroglobulin only in the liver (Henkin, 1974).

 

Excretion

In humans, the faecal zinc consists of un-absorbed dietary zinc and endogenous zinc from bile, pancreatic juice and other secretions. About 70-80% of the ingested amount of zinc is excreted via faeces (5 to 10 mg/day depending upon the dietary zinc concentration) (Spencer et al., 1976; Venugopal and Lucky, 1978; Reinhold et al., 1991; Wastney et al., 1986). In humans, of the amount of zinc consumed, about 10% is lost through urine (approximately 200 to 600 mg zinc/day). The urinary zinc excretion appears to be sensitive to alterations in the zinc status (Babcock et al., 1982; Aamodt et al., 1982).

Saliva, hair loss, mother’s milk and sweat appear to be minor routes for zinc excretion. In tropical climates about 2-3 mg Zn/day may be lost in sweat (Venugopal and Lucky, 1978; Rivlin, 1983; Prasad et al., 1963; Rossowka and Nakamoto, 1992; Henkin et al., 1975).

In humans with no excessive intake of zinc, the half-life of absorbed radio-labelled zinc ranges from 162 to 500 days. After parenteral administration of ⁶⁵Zn, half-lives ranged from 100 to 500 days (Elinder, 1986).

Sixteen healthy adult human volunteers were given oral administration of 92 mmol of ⁶⁵Zn (as ZnCl₂) to investigate the body retention of zinc at 7 to 10 days after dosing. The results showed that about 10% of the initially absorbed amount of zinc was excreted during the first 10 days post dosing. Thirty other volunteers were dosed with 18 to 900 mmoles of ⁶⁵Zn. Table below shows the elimination data following 10 to 60 days post-dosing.

Table. Elimination data obtained following thirty humans dosed with 18 to 900 mmoles of ⁶⁵Zn

Dose group (mmoles; (mg))	Excretion rate (% of remaining Zn/day)	Biological half-life (days)
18 (1.2)	0.44	157
45 (2.9)	0.62	111
90 (5.8)	0.37	186
180 (11.6)	0.49	141
450 (29)	0.37	186
900 (58)	0.74^a	93

^a significantly different from the 18 mmoles group

The excretion rates for the 18 to 450 mmoles dose groups were not significantly different. The 900 mmole dose group showed a significant increase in elimination rate (Payton et al., 1982).

The effect on excretion following oral administration of radiolabelled zinc as zinc chloride in 50 patients with taste and smell dysfunction was investigated. The study was conducted in three phases. In the first phase all patients were studied for 21 days after receiving a single oral dose of 3-18 mCi of ⁶⁵Zn (i.e., approximately 0.4 to 1.2 ng zinc) as ZnCl₂ after an overnight fast. In the second phase, which started after 21 days and continued for 290 to 440 (mean 336) days, all 50 patients received placebo. To study the effect of additional zinc intake on elimination of previously sequestered radioactivity, in the third phase of the study 14 patients continued on placebo while 36 received ZnSO₄ (100 mg Zn/day) for 112 to 440 (mean 307) days. In the controlled clinical trial of phases two and three, observations were made to see the effects of zinc on retention of the ⁶⁵Zn tracer. The results from the first phase of the study are described under absorption section. Total body retention and activity in plasma and red cells were measured for all patients throughout the study. About one-third of the absorbed radioactivity was eliminated from the body with a half-life of ca. 19 days, while after about 100 days post dosing the remainder of the absorbed dose was eliminated with a biological half-life of 380 days (i.e. phase two of the study). During the third phase patients receiving ZnSO₄ showed an accelerated loss of total body ⁶⁵Zn (half-life ca. 230 days) which was significantly different (P>0.001) from half-life values during placebo treatment. Accelerated loss of ⁶⁵Zn from the thigh was apparent immediately while that from the liver began after a mean delay of 107 days. There was no apparent effect of zinc on loss of mean ⁶⁵Zn activity from red blood cells (Aamodt et al., 1982). From the study description it is not clear whether the radioactivity was administered as pure radioactive zinc chloride or whether it was diluted with unlabelled zinc chloride. As the authors stated that “patients were given 3 to 18 mCi carrier free ⁶⁵Zn” for the calculation of the dose of ⁶⁵Zn in terms of nanogram zinc, it has been assumed that all zinc administered was ⁶⁵Zn (Aamodt et al. 1982).

In ten of the patients from the study described above (Aamodt et al. 1982), the kinetics of ⁶⁵Zn was studied in more detail by Babcock et al. (1982). These patients received a fixed diet containing 8 – 13 mg Zn per day for 4 to 7 days before and after the single ⁶⁵Zn dose, followed by 290-440 (mean 336) days of non-restricted diet, followed by an additional intake of 100 mg/day of non-radioactive zinc ion (as ZnSO₄) over the next 112-440 days (mean 307). The overall kinetic parameters of these 10 patients did not differ from those of the other patients (Aamodt et al., 1982). The authors further submitted retention-time curve data for whole body, plasma, red blood cells, liver and thigh to a multi-compartment kinetic model. From this model analysis it could be demonstrated that the increase in elimination of Zn during the third phase of the study by Aamodt et al. (1982) can be ascribed entirely to the change in parameters: reduction in absorption in the gastrointestinal tract (5-fold: from 43% absorption at the beginning of the study to 9% during the period in which patients were dosed with ZnSO₄) and to an increase in the urinary elimination rate (about 2-fold upon administration of ZnSO₄ during phase three of the study). Michaelis-Menten type saturation mechanisms were adequate to explain the observed parameter changes. These changes also accounted for the observed mean plasma zinc mass increase of only 37% above pre-load levels in face of an 11-fold increase in zinc intake (from ca. 10 mg/day to  ca. 110 mg/d) (Babcock et al., 1982). From this model analysis it was estimated that the total body Zn contents of these 10 patients at the start of the study was 1.4 g. Babcock et al. (1982) indicated that normally the body contents of zinc are in the range of 2.1 to 2.5 g. This may indicate that the patients studied by Babcock et al. (1982) and possibly by Aamodt et al. (1982) were deficient in total body zinc.

 

Summary

Zinc compounds release, depending on their solubility, zinc cations which determine the biological activity of the respective zinc compounds.

Sufficient data is available on the soluble zinc compounds zinc chloride and zinc sulphate and on the slightly soluble zinc compounds ZnO and ZnCO₃.

Zinc is an essential trace element which is regulated and maintained in the various tissues mainly by the gastrointestinal absorption and secretion during high and low dietary zinc intake and because of the limited exchange of zinc between tissues, a constant supply of zinc is required to sustain the physiological requirements. The zinc absorption process in the intestines includes both passive diffusion and a carrier-mediated process. The absorption can be influenced by several factors such as ligands in the diet and the zinc status. Persons with adequate nutritional levels absorb 20-30 % and animals absorb 40-50 %. Persons that are zinc deficient absorb more, while persons with excessive zinc intake absorb less.

For the soluble zinc compounds, the available information suggests an oral absorption value of 20 %. This value can be considered as the lower bound range at adequate nutritional levels. The oral absorption of the slightly soluble zinc oxide has been shown to be 60 % of that of the soluble zinc compounds. This corresponds to approximately 12-18 %. No oral absorption information is available for the remaining slightly soluble and insoluble zinc compounds (i.e. Zn(OH)₂, Zn₃(PO₄)₂, ZnCO₃, Zn, ZnS). However, considering that these substances have lower water solubility than ZnO, it can be conservatively assumed that the oral absorption of these compounds is ≤ 12 %.

Animal data suggests that there is pulmonary absorption following inhalation exposure. Half-life values of 14 and 6.3 hours were reported for dissolution of zinc oxide. The absorption of inhaled zinc depends on the particle size and the deposition of these particles therefore data was provided on the particle size distribution of zinc aerosol from three different industry sectors. The particle size distribution data was evaluated by using a multiple path particle deposition (MPPD) model. This model revealed that for zinc aerosols the largest part of the deposition is in the head region and much less in the tracheobronchial and pulmonary region. Although most of the material deposited in the head and tracheobronchial region is rapidly translocated to the gastrointestinal tract, a part will also be absorbed locally.

Based on data for local absorption of radionuclides in the different airway regions, it can be assumed that the local absorption of the soluble zinc compounds will be approximately 20 % of the material deposited in the head region, 50% of the material deposited in the tracheobronchial region and 100% of the material deposited in the pulmonary region. For the slightly soluble and insoluble zinc compounds a negligible absorption can be assumed for materials deposited in the head and the tracheobronchial region. 100 % of the deposited slightly or insoluble zinc compounds are assumed to be absorbed in the pulmonary tract. The deposited material will be cleared via the lung clearance mechanisms into the gastrointestinal tract where it will follow oral absorption kinetics. Therefore, the inhalation absorption for the soluble zinc compounds is a maximum of 40 % and for the slightly soluble and insoluble zinc compounds inhalation absorption is at a maximum of 20 %. These values can be assumed as a reasonable worst case because they are considered to cover existing differences between the different zinc industry sectors with respect to the type of exercise activities (and thus breathing rate) and particle size distribution.

The available information from in vivo as well as the in vitro studies suggests the dermal absorption of zinc compounds through intact skin to be less than 2 %. In vitro studies that estimated dermal absorption values only on the basis of the zinc levels in the receptor medium without taking into account the zinc present in the stratum corneum appear to underestimate absorption values derived from in vivo studies. Such zinc trapped in the skin layers may become systemically available at a later stage. Quantitative data to evaluate the relevance of this skin depot are however lacking. Given the efficient homeostatic mechanisms of mammals to maintain the total body zinc and the physiologically required levels of zinc in the various tissues to be constant, the anticipated slow release of zinc from the skin is not expected to disturb the homeostatic zinc balance of the body. Considering the available information on dermal absorption, the default for dermal absorption of all zinc compounds (solutions or suspensions) is 2 %. Based on the physical appearance, for dust exposure to zinc and zinc compounds a 10-fold lower default value of 0.2 % is a reasonable assumption.

Zinc appears to be distributed to all tissues and tissue fluids and it is a cofactor in over 200 enzyme systems. The excretion of zinc is primarily via faeces, but also via urine, saliva, hair loss, sweat and mothers-milk.