TOXICOLOGICAL SCIENCES 126(2), 317–324 (2012) doi:10.1093/toxsci/kfr348 Advance Access publication January 6, 2012
Pharmacokinetics of Melamine and Cyanuric Acid and Their Combinations in F344 Rats Cristina C. Jacob, Linda S. Von Tungeln, Michelle Vanlandingham, Frederick A. Beland, and Goncxalo Gamboa da Costa1 Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079 1
To whom correspondence should be addressed at Division of Biochemical Toxicology, National Center for Toxicological Research, HFT-233, 3900 NCTR Road, Jefferson, AR 72079. Fax: (870) 543-7136. E-mail:
[email protected]. Received September 28, 2011; accepted December 21, 2011
The intentional adulteration of pet food with melamine and cyanuric acid has been implicated in the kidney failure and death of a large number of cats and dogs in the United States. Although individually these compounds present low toxicity in a range of experimental animals, coexposure can lead to the formation of melamine cyanurate crystals in the nephrons and eventual kidney failure. Given this mode of action, a good understanding of the pharmacokinetic profiles of melamine and cyanuric acid and their combinations is essential to define properly the risk associated with different exposure scenarios. Previous studies have investigated the individual pharmacokinetic profiles of melamine and cyanuric acid. In this work, we report a comparison between the pharmacokinetic profiles of melamine and cyanuric acid administered individually, administered simultaneously as the individual compounds, and administered as a preformed melamine cyanurate complex. Although the oral coadministration of 1 mg/kg body weight of melamine and cyanuric acid did not alter significantly the pharmacokinetic profiles in relation to those determined upon individual oral administration of each compound, the administration of equal amounts of each triazine as the preformed melamine cyanurate complex significantly altered the pharmacokinetics, with reduced bioavailability of both compounds, lower observed maximum serum concentrations, delayed peak concentrations, and prolonged elimination half lives. These results indicate that in order to estimate properly the combined nephrotoxic potential of melamine and cyanuric acid, the experimental design of toxicological experiments and the evaluation of animal or human exposure scenarios should consider the detailed mode of exposure, with particular emphasis on any possible ex vivo formation of melamine cyanurate. Key Words: melamine; cyanuric acid; melamine cyanurate; pharmacokinetics.
In 2007, the intentional adulteration of ingredients used in the preparation of pet food with ‘‘scrap melamine’’ containing melamine and a number of oxytriazines, including cyanuric Disclaimer: The opinions expressed in this paper do not necessarily represent those of the U.S. Food and Drug Administration. Published by Oxford University Press 2012.
acid, was implicated in the kidney failure and death of a large number of cats and dogs in the United States. Although melamine and cyanuric acid, high production volume chemicals used in a range of industrial and household products, present low individual toxicity, recent animal studies revealed that the combined administration and systemic distribution of melamine and cyanuric acid can give rise to a remarkably insoluble complex of melamine cyanurate in the nephrons (Brown et al., 2007; Dobson et al., 2008; Puschner et al., 2007). The accumulation of this complex can eventually lead to the loss of renal function and death. Although the precise mechanism that leads to the formation of melamine cyanurate in the kidney (but not in other organs) is not entirely clear, it has been proposed that the concentration of the urine filtrate in the nephrons may be responsible for the nucleation of the melamine cyanurate complex and subsequent crystal growth (Dobson et al., 2008). Considering this mode of action, it is apparent that the pharmacokinetic parameters governing the absorption, distribution, and excretion of melamine and cyanuric acid should be considered when assessing the risks associated with different exposure scenarios, such as simultaneous exposure, staggered exposure, exposure to the individual compounds, or exposure to a preformed complex. Although previous studies have addressed the individual pharmacokinetics of melamine and cyanuric acid in a range of animal models, there are no reports in the literature of pharmacokinetic studies in mammals upon the coadministration of melamine and cyanuric acid. We report herein a comparison between the pharmacokinetic profiles of melamine and cyanuric acid when administered individually, simultaneously as the individual compounds, or as a preformed melamine cyanurate complex.
MATERIALS AND METHODS Chemicals. Melamine (Aldrich, stated purity 99%, CAS 108-78-1) and cyanuric acid (Fluka, anhydrous, stated purity > 98%, CAS 108-80-5) were obtained from Sigma-Aldrich (St Louis, MO). Carboxymethylcellulose (CMC,
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sodium salt, high viscosity) was obtained from MP Biomedicals (Solon, OH). Melamine cyanurate complex (CAS 37640-57-6) was prepared by coprecipitation of aqueous equimolar solutions of melamine and cyanuric acid. The precipitate thus obtained was filtered, thoroughly washed with hot water (80–90°C), and dried under reduced pressure. The purity and identity of the test articles were confirmed by high-performance liquid chromatography (HPLC) coupled with ultraviolet detection and electrospray mass spectrometry and by gas chromatography-mass spectrometry in electron impact mode. All solvents used for the dosing solution analyses were purchased from Sigma-Aldrich and met or exceeded American Chemical Society specifications. Preparation and certification of the dosing solutions. Intravenous (iv) dosing solutions were prepared by dissolving melamine or cyanuric acid at the concentration of 1 mg/ml in normal saline and filter sterilizing the solutions. The gavage solutions were prepared by dissolving the respective compound in a 0.1% CMC solution in water. These solutions were prepared at the following concentrations: individual gavage studies with melamine or cyanuric acid— 200 lg/ml; combined gavage studies with melamine and cyanuric acid— 400 lg/ml each of melamine or cyanuric acid. The gavage suspension of melamine cyanurate was prepared by adding finely pulverized melamine cyanurate at a concentration of 400 lg/ml to 0.1% CMC in water. All dosing formulations were analyzed with a Thermo Surveyor Plus HPLC (Thermo, Waltham, MA) using a Synergi Polar RP 80A, 4 lm, 2 3 250 mm column (Phenomenex, Torrance, CA) eluted isocratically at 400 ll/min with 95% 10mM ammonium phosphate and 5% acetonitrile. The eluate was monitored at 209 nm. All dosing solutions were determined to be between 99.6 and 104.9% of their nominal concentrations. The melamine cyanurate suspension was determined to be at 102.3 ± 1.4% of its nominal concentration, as determined by analysis of three independent samples drawn under simulated dosing conditions. Animals, dosing procedure, and blood collection schedule. All procedures involving the care and handling of animals were reviewed and approved by the Institutional Animal Care and Use Committee at the National Center for Toxicological Research (NCTR). F344 rats were obtained from the breeding colony at the NCTR at 6 weeks of age. The rats were weight-ranked (acceptable weight: ± 20% of the mean body weight [bw]), and randomly assigned to treatment groups (eight males and eight females per iv dose group; six males and six females per gavage dose group) that were fed ad libitum with NIH-41 irradiated feed. The animals were housed in individual polycarbonate cages with hardwood chip bedding and the room environmental controls were set to maintain a relative humidity of 40–70% and a 12-h light/day cycle. At 10 weeks of age, the animals were injected in the saphenous vein with 1 ml/kg bw of 1 mg/ml solution of melamine or cyanuric acid in saline (iv treatment groups), were gavaged with 5 ml/kg bw of a 200 lg/ml solution of melamine or cyanuric acid in 0.1% CMC (individual gavage groups), were gavaged with 2.5 ml/kg of a 400 lg/ml melamine solution in 0.1% CMC followed, within less than 1 min, by a separate gavage with 2.5 ml/kg of a 400 lg/ml cyanuric acid solution in 0.1% CMC (combined melamine and cyanuric acid gavage group), or were gavaged with 5 ml/kg bw of a 400 lg/ml suspension of melamine cyanurate in 0.1% CMC in water. The iv injections were performed with zero-dead volume disposable syringes equipped with 28 gauge ½ inch needles (1 cc Micro-Fine iv Insulin Syringe; Becton Dickinson and Company, Franklin Lakes, NJ), and the gavages were conducted with syringes equipped with ball-tipped stainless steel gavage needles. Approximately 100-ll samples of blood were collected from the tail veins with zero-dead volume 1 cc disposable syringes with permanently attached 25 gauge 5/8 inch needles (Terumo Medical Corporation, Somerset, NJ) at 0 (pretreatment), 5, 15, and 30 min, and 1, 2, 3, 4, 5, and 6 h after treatment for the iv treatment groups or 0 (pretreatment), 0.5, 1, 2, 3, 4, 5, and 6 h for the gavage treatment groups. At 8-h past treatment, the animals were euthanized by deep anesthesia with carbon dioxide and terminal exsanguination, providing an 8-h blood sample. The blood samples were allowed to clot at room temperature and the serum samples, prepared by centrifugation, were stored at 80°C. Quantification of melamine and cyanuric acid in serum by UPLC-MS/ MS. The concentrations of melamine and cyanuric acid in the serum samples were determined by isotopic dilution mass spectrometry as described in Jacob
and Gamboa da Costa (2011). Briefly, the methodology incorporated 13C3labeled cyanuric acid and 15N3-labeled melamine as internal standards and was based on ion-exchange solid-phase extraction and ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry in multiple reaction monitoring mode (UPLC-ESI-MS/MS). The method used 15 ll samples of serum and afforded lower limits of quantification for melamine and cyanuric acid of, respectively, 5 and 10 ng/ml. Pharmacokinetic and statistical analyses. Plots of serial serum concentrations of melamine or cyanuric acid versus time after melamine, cyanuric acid, melamine and cyanuric acid, or melamine cyanurate oral or iv administration to individual rats were analyzed using model-independent pharmacokinetic analysis (PK Solutions 2.0 software, Summit Research Services, Montrose, CO). Natural log-linear plots were fit to one (elimination) or two (distribution/absorption) kinetic phases corresponding to elimination and distribution/absorption. The firstorder elimination rate constants (kElim) for melamine or cyanuric acid were determined from the terminal slope of the respective curves, and the first-order distribution/absorption rate constants (kD/A) were determined after subtracting the contribution from the terminal elimination phase of the respective curves (i.e., feathering). Internal exposures to melamine or cyanuric acid were determined from area-under-the-concentration (AUC) curve using the trapezoidal rule. The Cmax and Tmax after oral treatments were observed values. Half-lives (t1/2 [Elim] and t1/2 [D/A]) were determined from rate constants using the relationships: halflife ¼ ln 2/k (Elim) and ln 2/k (D/A), respectively. Cinitial after iv treatment was the extrapolated y-axis of the elimination curve. The volume of distribution (Vd) was calculated using iv administration data from the relationships: Vd ¼ dose/ (AUC 3 kelim) and Vd ¼ dose/(Cinitial). Both gave comparable values. Bioavailability (the fraction absorbed, F) was calculated as the ratio of AUCoral to AUCiv. The data are reported as the mean ± SD, with the exception of Tmax, which is reported as the median and range. The SD (rz) for bioavailability (F) was calculated from the following relationship: ðrz =ZÞ2 ¼ ðrz =AÞ2 þðrB =BÞ2 where Z ¼ A/B; A ¼ AUCoral; B ¼ AUCiv; (rA) ¼ SD of AUCoral; and (rB) ¼ SD of AUCiv Statistical analyses (with the exception of Tmax statistical analyses) were conducted by one-way ANOVA, with pairwise comparisons being conducted by the Student-Newman-Keuls method. Statistical analyses of Tmax values were conducted, as appropriate, by Kruskal-Wallis one-way ANOVA on Ranks, with pairwise comparisons being conducted by Dunn’s method or Mann-Whitney Rank Sum tests. p values < 0.05 were considered to be significant.
RESULTS
Intravenous Treatments Groups of male and female F344 rats were injected in the saphenous vein with a solution of melamine or cyanuric acid in saline to afford a dose of 1 mg/kg bw. Figures 1A and 1B depict the serum concentration profiles of melamine and cyanuric acid in the male (black line) and female (gray line) rats as determined by isotopic dilution UPLC-ESI-MS/MS at various intervals after dosing. Model-independent pharmacokinetic analysis revealed clear differences between the pharmacokinetic parameters of melamine and cyanuric acid for both sexes, with cyanuric acid showing a higher initial concentration (Cinitial), a smaller AUC, a more rapid elimination (t1/2 [Elim]), and a small volume of distribution (Vd) (Table 1). In addition, intersex differences were observed in the melamine-exposed rats, with the males presenting slightly higher, but statistically significant, values
PHARMACOKINETICS OF MELAMINE AND CYANURIC ACID
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FIG. 1. Time concentration profiles of melamine (MEL) and/or cyanuric acid (CYA) obtained upon analysis of serum samples from rats treated iv with 1 mg/kg bw of melamine alone (panel A), treated iv with 1 mg/kg bw of cyanuric acid alone (panel B), gavaged with 1 mg/kg bw of melamine alone (panel C), gavaged with 1 mg/kg bw of cyanuric acid alone (panel D), gavaged with 1 mg/kg bw of melamine and 1 mg/kg bw of cyanuric acid (panels E [melamine profile] and F [cyanuric acid profile]), or gavaged with 2 mg/kg bw of melamine cyanurate complex (panels G [melamine profile] and H [cyanuric acid profile]). The male rat traces are represented in black, and the female traces are represented in gray. The error bars represent ± SD (n ¼ 8 per sex in the iv treatments and n ¼ 6 per sex in the oral treatments).
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— 0.54 ± 0.10b
1.05 ± 0.16c —
— 0.56 ± 0.09
for Cinitial and AUC and slightly lower values for t1/2 (Elim) and Vd than those determined in the females (Table 1).
Data are reported as the mean ± SD; n ¼ 8. Significantly different (p < 0.001) from the comparable measurement obtained for melamine for the same sex of rats. c Significantly different (p 0.039) from the comparable measurement obtained in male rats. b
Male Male
Melamine Cyanuric acid Melamine Cyanuric acid
a
— 0.66 ± 0.17b 1.43 ± 0.12c —
Sex Treatment
Female Female
972 ± 153c —
— 1,833 ± 326b
2,023 ± 222c —
— 1,744 ± 293b
1.03 ± 0.13c —
— 0.49 ± 0.08b 0.84 ± 0.05 — — 0.47 ± 0.08b 0.82 ± 0.06 — — 0.62 ± 0.10b 1.31 ± 0.08 — 1,925 ± 135b — 2,101 ± 345b 1,189 ± 78 —
2,309 ± 191 —
Cyanuric acid Melamine Cyanuric acid Melamine Cyanuric acid Melamine Cyanuric acid Melamine Cyanuric acid Melamine
t1/2 (Elim) (h) AUC (ng-h/ml) Cinitial (ng/ml)
TABLE 1 Pharmacokinetic Parameters After iv Treatmenta
Vd (ml/g) (Dose/[kElim 3 AUC])
Vd (ml/g) (Dose/Cinitial)
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Oral Exposure to Melamine or Cyanuric Acid Male and female F344 rats were gavaged with 5 ml/kg bw of a 200 lg/ml solution of melamine or cyanuric acid to afford an exposure of, respectively, 1 mg/kg bw melamine or cyanuric acid. Figures 1C and 1D depict the concentration profiles of melamine and cyanuric acid in the male (black line) and female (gray line) rats as determined by isotopic dilution UPLC-ESIMS/MS for up to 8 h after treatment. With the exception of Cmax for cyanuric acid and t1/2 (Elim) for both compounds, there were no significant differences between the serum concentration profiles of male versus female rats (Table 2). There were clear differences between the profiles obtained upon oral versus iv exposure to the same doses of melamine or cyanuric acid, with the AUCoral being lower and the t1/2 (Elim)oral being longer. This reflects the delayed absorption of these triazines in the gastrointestinal tract upon oral administration. As was observed with the iv dosing, the pharmacokinetic analysis revealed clear differences between the pharmacokinetic parameters of melamine and cyanuric acid for both sexes, with cyanuric acid showing a higher maximum concentration (Cmax), a smaller AUC, and a more rapid elimination (t1/2 [Elim]) (Table 2). The bioavailabilities of melamine and cyanuric acid determined by the ratio of the AUC obtained upon oral gavage and the AUC obtained upon iv administration of each compound ranged from 74% (cyanuric acid in male animals) to 98% (melamine in female animals) (Table 3). Oral Coexposure to Melamine and Cyanuric Acid Groups of male and female F344 rats were gavaged with 2.5 ml/kg bw of a 400 lg/ml melamine solution and immediately (< 1 min) after were gavaged with 2.5 ml/kg bw of a 400 lg/ml cyanuric acid solution to afford an oral coexposure to 1 mg/kg bw of melamine and 1 mg/kg bw of cyanuric acid. Figures 1E and 1F depicts the concentration profile of melamine and cyanuric acid in the male (black line) and female (gray line) rats as determined by isotopic dilution UPLC-ESI-MS/MS for up to 8 h after treatment. As suggested by a comparison of the concentration curves of the individual oral exposure to melamine (Fig. 1C) or cyanuric acid (Fig. 1D) and the combined exposure curves (respectively Figs. 1E and 1F), the calculated pharmacokinetic parameters were essentially unchanged by the coadministration of the triazines as compared with the compounds individually (Table 2). Oral Exposure to Melamine Cyanurate Complex Male and female F344 rats were gavaged with 5 ml/kg bw of a 400 lg/ml suspension of melamine cyanurate. Figures 1G and 1H depict the concentration profile of melamine and cyanuric acid in the male (black line) and female (gray line) rats at various intervals for up to 8 h after dosing. As suggested
— 0.99 ± 0.09b,c,f 1.00 ± 0.21c
1.88 ± 0.52c,d,e
1.92 ± 0.19b,f — 1.86 ± 0.24
3.02 ± 1.12d,e
0.65 ± 0.13d,e
b
Data are reported as the mean ± SD, except for Tmax, which is reported as the median, with the range reported in parentheses. n ¼ 6. Significantly different (p < 0.001) from the comparable measurement obtained in the same sex of rats after iv treatment (Table 1). c Significantly different (p 0.048) from the comparable measurement obtained for melamine for the same sex of rats. d Significantly different (p < 0.05) from the same sex of rats treated with melamine or cyanuric acid. e Significantly different (p < 0.05) from the same sex of rats treated with melamine and cyanuric acid. f Significantly different (p 0.009) from the comparable measurement obtained in male rats.
DISCUSSION
a
702 ± 126c,d,e 992 ± 204d,e 1.0 (0.5–1.0)c,d,e 180 ± 39d,e 156 ± 33d,e Female
2.0 (2.0–3.0)d,e
0.25 ± 0.06 — 0.24 ± 0.08 — 1375 ± 117b,c 1353 ± 62c 1992 ± 199 — 1837 ± 99 — 0.5 (0.5) 0.5 (0.5)c — 861 ± 43c,f 822 ± 95c 571 ± 57 — 541 ± 27 Female Female Female
0.75 (0.5–1.0) — 1.0 (1.0)
0.46 ± 0.12d,e
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by a comparison of the concentration curves of the individual oral exposure to melamine (Fig. 1C) or cyanuric acid (Fig. 1D) and the corresponding curves for the oral exposure to melamine cyanurate (respectively Figs. 1G and 1H), major differences were observed in the calculated pharmacokinetic parameters (Table 2). In both male and female rats, the oral administration of melamine cyanurate complex afforded substantially lower Cmax and AUC values than those determined in the animals exposed orally to the nearly equimolar amounts of the triazines contained in the complex (Table 2). In addition, the Tmax occurred later and t1/2 values were longer in the rats administered melamine cyanurate.
0.40 ± 0.19
1.54 ± 0.31c,d,e 2.99 ± 0.80d,e 508 ± 109c,d,e 919 ± 229d,e 1.0 (0.5–1.0) 184 ± 28d,e 155 ± 21d,e Male
1.5 (1.0–2.0)
— — —
— — — 0.26 ± 0.05 — 0.31 ± 0.10 — 0.75 ± 0.12c 0.76 ± 0.13c 1.62 ± 0.10b — 1.43 ± 0.06 — 1431 ± 190b,c 1280 ± 164c 1913 ± 95b — 1906 ± 153 — 0.5 (0.5)c 0.5 (0.5) — 979 ± 79c 907 ± 152c 581 ± 28 — 613 ± 77
Melamine Cyanuric acid Melamine and cyanuric acid Melamine cyanurate Melamine Cyanuric acid Melamine and cyanuric acid Melamine cyanurate
Male Male Male
1.0 (0.5–1.0) — 1.0 (0.5–1.0)
—
Cyanuric acid, t1/2 (D/A) (h) Melamine, t1/2 (D/A) (h) Cyanuric acid, AUC (ng-h/ml) Melamine, AUC (ng-h/ml) Cyanuric acid, Tmax (h) Melamine, Tmax (h) Cyanuric acid, Cmax (ng/ml) Melamine, Cmax (ng/ml) Sex Treatment
TABLE 2 Pharmacokinetic Parameters After Oral Treatmenta
Melamine, t1/2 (Elim) (h)
Cyanuric acid, t1/2 (Elim) (h)
PHARMACOKINETICS OF MELAMINE AND CYANURIC ACID
The study of the combined toxicity of melamine and cyanuric acid constitutes a remarkable example of the complexities involved in the toxicological assessment of mixtures. Although individually melamine or cyanuric acid present low toxicities in a range of experimental animals, recent events involving the adulteration of pet food with ‘‘scrap melamine,’’ containing melamine and cyanuric acid, revealed that oral coexposure to these triazines can lead to acutely nephrotoxic effects (Brown et al., 2007; Dobson et al., 2008; Puschner et al., 2007; World Health Organization 2009). Although the detailed mechanism of nephrotoxicity remains to be clarified, the data currently available demonstrate that upon coexposure to melamine and cyanuric acid, a crystalline complex of melamine cyanurate forms in the lumen of the nephrons, impairing their function and potentially leading to organ failure. The formation and accumulation of the complex are consistent with the solubility differential of melamine (ca. 3200 mg/l) and cyanuric acid (ca. 2000 mg/l) versus that of melamine cyanurate (ca. 2.2 mg/l), and it is believed that the physiological concentration gradient in the nephron loop of Henle may facilitate the nucleation of the complex and subsequent crystal growth, justifying the organ specificity of the effects (Dobson et al., 2008; Tolleson, 2008). Given this mode of action, a good understanding of the pharmacokinetic profiles of melamine and cyanuric acid and their combination is essential to define properly the risk associated with different oral exposure scenarios. A number of previous studies have addressed various aspects of the melamine pharmacokinetics in rats (Mast et al., 1983; Sugita et al., 1991; Wu et al., 2010; Yang et al., 2009), pigs (Baynes et al., 2008; Buur et al., 2008), rhesus monkey (Liu et al., 2010), goats (Baynes et al., 2010), and fish (Andersen et al., 2011; Reimschuessel et al., 2010; Xue et al., 2011). A limited set of studies have described the pharmacokinetic behavior of cyanuric acid in the rat (Hammond et al., 1986), dog (Hammond et al., 1986), and humans (Allen et al., 1982); however, only a single study has been published addressing pharmacokinetic aspects of a combined exposure to melamine and cyanuric acid, and this was
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conducted in rainbow trout (Xue et al., 2011). Given the substantial anatomical and physiological differences between fish and mammalian kidneys and the fact that the only exposure studied was an oral coexposure to melamine and cyanuric acid, further studies on the combined pharmacokinetics of melamine and cyanuric acid and their combinations were warranted. In this study, we compared the pharmacokinetics of melamine and cyanuric acid as administered to F344 rats individually by iv route, individually per os, in combination per os, and as preformed melamine cyanurate complex per os. A dose of 1 mg/kg bw was selected for the individual exposures to melamine and cyanuric acid because it was expected to afford quantifiable serum levels at extended time periods using our recently developed UPLC isotopic dilution mass spectral method (Jacob and Gamboa da Costa, 2011) and was below the threshold of exposure at which we have observed significant nephrotoxic effects in subchronic studies with a combination of melamine and cyanuric acid in rats (manuscript under preparation). The absolute dose of each compound was maintained in the combination studies, in order to allow a direct comparison of the concentration-time profiles of each triazine in serum. Given the small difference between the molecular weights of melamine (126.1 g/mol) and cyanuric acid (129.1 g/mol), the doses were nearly equimolar for each triazine. As expected from previously published data, the pharmacokinetics of melamine and cyanuric acid were noticeably distinct both by iv (Figs. 1A and 1B; Table 1) or oral routes (Figs. 1C and 1D; Table 2), with melamine presenting longer elimination half lives upon iv (ca. 1.3–1.4 h) or oral dosing (ca. 1.6–1.9 h) compared with those determined for cyanuric acid upon iv (ca. 0.6–0.7 h) or oral administration (ca. 0.8–1.0 h). These values are comparable to those previously published for melamine (t1/2 1.3–4.9 h) (Mast et al., 1983; Wu et al., 2010; Yang et al., 2009) and cyanuric acid (0.5–1 h) in rats (Hammond et al., 1986). The bioavailability of melamine in the male (F ¼ 0.83 ± 0.08) and
female rats (F ¼ 0.98 ± 0.15) (Table 3) was also comparable with the values previously reported in the literature for rats (F 0.73–0.98) (Wu et al., 2010; Yang et al., 2009); however, to the best of our knowledge, the bioavailability determined for cyanuric acid in this study (F ¼ 0.74 ± 0.11 for male rats; F ¼ 0.79 ± 15 for female rats) constitutes the first report of this parameter for rats in the literature. Given the instantaneous formation of melamine cyanurate known to occur upon mixing solutions of melamine and cyanuric acid in vitro, we anticipated that the sequential gavage of melamine and cyanuric acid in the combined oral exposure experiment might result in the coprecipitation of the triazines in the stomachs of the rats, impairing to some degree their efficient absorption. Surprisingly, the individual concentration curves for melamine (Fig. 1E) and cyanuric acid (Fig. 1F) in the combined treatment were almost indistinguishable from those resulting from the individual administration of each triazine (respectively, Figs. 1C and 1D). A detailed analysis of the main pharmacokinetic parameters (Table 2) failed to reveal any noteworthy differences between the individual and combined exposures, with bioavailabilities in the combined treatment ranging from 0.83 to 0.91 for melamine and 0.66–0.78 for cyanuric acid (Table 3). In contrast, the administration of equal amounts of each triazine as a preformed melamine cyanurate complex revealed very substantial differences in the concentration curves in both sexes for melamine (Fig. 1G) and cyanuric acid (Fig. 1H). Treatment with preformed melamine cyanurate led to significantly lower bioavailabilities for both melamine (F 0.40–0.49) and cyanuric acid (F 0.26–0.40) (Table 3) and to substantially lower observed Cmax values, a later Tmax, and prolonged elimination half lives (Table 2). We hypothesize that with the combined treatment with melamine and cyanuric acid, the hydrochloric acid naturally present in the stomach of the rats efficiently prevents the coprecipitation of the triazines as a melamine cyanurate complex by protonating the amine groups of melamine and disrupting the formation of the hydrogen bonds that stabilize the lattice of
TABLE 3 Bioavailabilities of Melamine and Cyanuric Acid in Oral Treatmentsa Males Treatment Melamine Cyanuric acid Melamine and cyanuric acidb Melamine cyanurateb a
Melamine F [AUCoral/AUCiv]
Females
Cyanuric acid F [AUCoral/AUCiv]
Melamine F [AUCoral/AUCiv]
Cyanuric acid F [AUCoral/AUCiv]
0.83 ± 0.08 ––– 0.83 ± 0.10
––– 0.74 ± 0.11 0.66 ± 0.10
0.98 ± 0.15 ––– 0.91 ± 0.11
––– 0.79 ± 0.15 0.78 ± 0.14
0.40 ± 0.10c,d
0.26 ± 0.06c,d
0.49 ± 0.11c,d
0.40 ± 0.10c,d
Data are presented as the mean ± SD. Bioavailability was calculated as the ratio of AUCoral for melamine or cyanuric acid to, respectively, the AUCiv for melamine or cyanuric acid individually. c Significantly different (p < 0.001) from the same sex of rats treated with melamine or cyanuric acid. d Significantly different (p < 0.001) from the same sex of rats treated with melamine and cyanuric acid. b
PHARMACOKINETICS OF MELAMINE AND CYANURIC ACID
melamine cyanurate; however, with the preformed melamine cyanurate treatment, the acidic environment only slowly and partially decomposed the complex to the individual triazines. The slow and partial decomposition are in agreement with the lower bioavailabilities, lower Cmax, delayed Tmax, and longer elimination half lives and is consistent with the outcome of acute toxicity studies conducted in rats gavaged with melamine cyanurate complex (Babayan and Aleksandryan, 1985). In these studies, the authors determined that the LD50 for melamine cyanurate in rats was 4110 mg/kg bw, a value more than two orders of magnitude above that at which acute renal failure and lethality has been reported for a combination of melamine and cyanuric acid in rats (Jacob et al., 2011). Considering the mode of action of a combination of melamine and cyanuric acid, the threshold of toxicity is expected to be ultimately determined by the minimum coexposure capable of yielding a serum concentration and subsequent urine filtrate concentration of melamine and cyanuric acid that exceeds the value required for nucleation and initial crystal formation in the nephron. Moreover, it is probable that the subsequent crystal growth is governed by factors such as the absolute AUC attained upon exposure that is above the limit of solubility of melamine cyanurate. In light of the results presented herein, it is clear that the detailed mode of administration of melamine and cyanuric acid may have a profound impact on the nephrotoxic potential. These observations suggest that experimental study designs that allow the ex vivo formation of melamine cyanurate (e.g., gavaging with preblended melamine and cyanuric acid formulations) may underestimate the nephrotoxic potential of the mixture. Our results also highlight that a proper definition of the relative risks associated with animal or human exposure scenarios should take into consideration the detailed mode of coexposure to melamine and cyanuric acid and emphasize the importance of internal exposure measurements in toxicological studies.
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FUNDING
Puschner, B., Poppenga, R. H., Lowenstine, L. J., Filigenzi, M. S., and Pesavento, P. A. (2007). Assessment of melamine and cyanuric acid toxicity in cats. J. Vet. Diagn. Invest. 19, 616–624.
This study was supported through an interagency agreement between the Food and Drug Administration and the National Toxicology Program at the National Institute of Environmental Health Sciences (FDA IAG:224-07-0007; NIH Y1ES1027).
Reimschuessel, R., Evans, E., Andersen, W. C., Turnipseed, S. B., Karbiwnyk, C. M., Mayer, T. D., Nochetto, C., Rummel, N. G., and Gieseker, C. M. (2010). Residue depletion of melamine and cyanuric acid in catfish and rainbow trout following oral administration. J. Vet. Pharmacol. Ther. 33, 172–182. Sugita, T., Ishiwata, J., and Maekawa, A. (1991). Intestinal absorption and urinary excretion of melamine in male Wistar rats. J. Food Hyg. Soc. Japan 32, 439–443.
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