Safety of dietary nitrate supplementation by calcium nitrate for finishing pigs as measured by methemoglobin and serum and tissue nitrate levels.

Nitrate supplementation has been studied as a beneficial constituent of the human diet, particularly for its effects on vascular health through vasodilation. Recent studies have focused on the benefits of nitrate supplementation in animals, especially in swine. Up to 1,200 mg/kg dietary nitrate supplementation from Ca nitrate was beneficial in farrowing and lactating sows and their offspring, and up to 6,000 mg/kg supplemental nitrate showed no adverse health effects in sows or piglets. Controlled study data evaluating the safety of nitrate supplementation to growing swine of any weight class is scant. Therefore, an experiment was conducted to test the hypothesis that increased inclusion rates of dietary nitrate through the addition of Ca nitrate in diets would not influence concentrations of nitrate or nitrite in serum and tissue, nor blood hemoglobin and methemoglobin. Forty-eight individually housed pigs (initial weight 119.1 ±â€…5.3 kg) were randomly allotted to four dietary treatments containing 0, 500, 1,000, or 2,000 mg/kg dietary nitrate and fed experimental diets for 28 d. Growth performance was not influenced (P > 0.10) by dietary treatment. The most sensitive safety endpoint, methemoglobin, did not change (P > 0.10) with dietary nitrate exposure up to 2,000 mg/kg. Serum and tissue nitrate and nitrite levels, myoglobin, and hemoglobin were not adversely affected (P > 0.10). Total myoglobin in the loin linearly increased (P < 0.05) with greater dietary nitrate in the diet, which is correlated with the red color of meat. This work established the safety of up to 2,000 mg/kg dietary nitrate from Ca nitrate as an ingredient in food for finishing pigs.

Acute and sub-chronic oral toxicity studies of erythritol in Beagle dogs.

Polyols, also known as sugar alcohols, are widely used in the formulation of tooth-friendly and reduced-calorie foods. Considering the significant health benefits of polyols in products formulated for human use, there is increased interest in evaluating potential uses in companion animal applications. Erythritol and xylitol are two polyols which are currently widely used in products ranging from reduced-sugar foods to personal care and cosmetics. Published studies have shown that both of these compounds are well-tolerated in rodents. Their toxicity profiles differ when comparing canine safety data. Doses of xylitol as low as 0.15 g/kg-BW in dogs can result in life-threatening hypoglycemia and acute liver failure, whereas erythritol is well-tolerated in dogs with reported No Adverse Effect Levels upwards of 5 g/kg-BW/day in repeat-dose studies. While pivotal studies substantiating the safe use of erythritol in humans have been published, there are limited published studies to support the safe use of erythritol in dogs. Here we present the results of an acute oral and a sub-chronic oral toxicity study in Beagle dogs. Given the potential health benefits of oral products formulated with erythritol and the data presented herein substantiating the safe use in dogs, erythritol can be safely used in products for canines.

A 90-day oral (dietary) toxicity and mass balance study of corn starch fiber in Sprague Dawley rats.

The potential toxicity of corn starch fiber was assessed and compared to polydextrose, a commonly used bulking agent with a long history of safe use in the food supply. Groups of male and female Crl:CD(SD) rats were fed 0 (control), 1,000, 3,000, or 10,000 mg/kg-bw/day corn starch fiber in the diet for 90 days. The polydextrose reference article was offered on a comparable regimen at 10,000 mg/kg-bw/day. Following a single gavage dose of [14C]-corn starch fiber on study day 13 or 90, the mass balance of the test article was assessed by analysis of excreta samples collected from 0 to 168 h post-dose. There were no toxicologically or biologically relevant findings in any of the test article-treated groups. The few minor differences observed between the corn starch fiber and polydextrose exposed groups were considered to be due to normal biological variation. Following [14C]-corn starch fiber dosing, nearly complete excretion of the administered dose occurred over 168 h post-dosing, with the majority excreted in the feces. The dietary no-observed-adverse-effect level of corn starch fiber after 90 days was 10,000 mg/kg-bw/day. Similar toxicity profiles for corn starch fiber and polydextrose were observed due to the structural and compositional similarities of these materials.

Evaluation of the gastrointestinal tolerability of corn starch fiber, a novel dietary fiber, in two independent randomized, double-blind, crossover studies in healthy men and women.

Two independent clinical studies were conducted to compare the gastrointestinal (GI) tolerability of corn starch fiber, a novel dietary fiber, at up to 50 g/day (single-dose study) or 90 g/day (multiple-serving study) with a negative control (no fiber) and a positive control (50 or 90 g polydextrose, for single- and multiple-serving studies, respectively) in generally healthy study volunteers. Flatulence and borborygmus were the primary symptoms reported at the higher doses of corn starch fiber and for the positive control interventions. Bowel movements were increased over 48 h with corn starch fiber at 90 g. Thresholds for mild GI effects were established at 30 g as a single dose and 60 g as multiple servings spread over the day. Other than moderate abdominal pain and mild increased appetite in one subject at 90-g corn starch fiber, no test article-related adverse events were reported.

A two-year dietary carcinogenicity study of (2R,4R)-monatin salt in mice.

Groups of Crl:CD-1 (ICR) mice (60/group/sex) were fed 0 (2 control groups), 5000, 20,000, or 40,000 ppm of enzymatically sourced (2R,4R)-monatin salt ("R,R-monatin") in the diet for up to two years. There were no adverse effects on survival, incidence of palpable masses and tumors, feed consumption, hematology or serum chemistry parameters, organ weights, or ophthalmic, macroscopic, and microscopic examinations. The only notable effect was statistically significantly lower mean body weights and body weight gains in all treated groups, which generally occurred throughout the study and were most likely a result of caloric dilution of the test diets and not considered adverse. There were no test article-related changes in the incidence or occurrence of neoplastic diseases in mice on this study. The no-observed-effect-level (NOEL) for carcinogenicity of R,R-monatin fed to mice for 24 months was 40,000 ppm, the highest dietary concentration tested, which was equivalent to approximately 6502 and 7996 mg/kg bw/day in males and females, respectively.

Detection of ECG effects of (2R,4R)-monatin, a sweet flavored isomer of a component first identified in the root bark of the Sclerochitin ilicifolius plant.

Enzymatically-synthesized (2R,4R)-monatin has, due to its pure sweet taste, been evaluated for potential use in foods. Non-clinical studies have shown that (2R,4R)-monatin is well tolerated at high dietary concentrations, is not genotoxic/mutagenic, carcinogenic, or overtly toxic. In a pharmacokinetic and metabolism study involving 12 healthy males, consumption of a single oral dose (2 mg/kg) of (2R,4R)-monatin resulted in a small reduction of heart rate and prolongation of the QTcF interval of 20-24 ms, corresponding to the time of peak plasma levels (t(max)). These findings were evaluated in a cross-over thorough QT/QTc study with single doses of 150 mg (2R,4R)-monatin, placebo and positive control (moxifloxacin) in 56 healthy males. Peak (2R,4R)-monatin plasma concentration (1720 ± 538 ng/mL) was reached at 3.1 h (mean tmax). The placebo-corrected, change-from-baseline QTcF (ΔΔQTcF) reached 25 ms three hours after dosing, with ΔΔQTcF of 23 ms at two and four hours. Using exposure response (QTc) analysis, a significant slope of the relationship between (2R,4R)-monatin plasma levels and ΔΔQTcF was demonstrated with a predicted mean QT effect of 0.016 ms per ng/mL. While similarly high plasma levels are unlikely to be achieved by consumption of (2R,4R)-monatin in foods, QTc prolongation at this level is a significant finding.

A combined dietary chronic toxicity and two-year carcinogenicity study of (2R,4R)-monatin salt in Sprague-Dawley rats.

In a combined chronic toxicity/carcinogenicity study, groups of Crl:CD(SD) rats were fed 0 (2 control groups), 5000, 20,000, or 40,000 ppm (2R,4R)-monatin salt (hereafter "R,R-monatin") in the diet for up to one year in the chronic toxicity phase and up to two years in the carcinogenicity phase. There were no adverse effects on survival, incidence of palpable masses, neoplasms, organ weights, or ophthalmic examinations. The only notable effect was statistically significantly lower mean body weights and body weight gains in all treated groups generally throughout the study, which were most likely a result of caloric dilution of the test diets. Effects of long-term R,R-monatin ingestion by rats were predominantly focused on the urinary system (i.e., clinical pathology alterations indicative of electrolyte and pH imbalances, increased incidence of renal calculi, mineralization and bone hyperostosis, and increased severity of chronic progressive nephropathy). The no-observed-adverse-effect level (NOAEL) for R,R-monatin from the chronic toxicity phase was 20,000 ppm (equivalent to an exposure level of 1080 mg/kg bw/day for males and 1425 mg/kg/day for females) and from the carcinogenicity phase was 5000 ppm (equivalent to an exposure level of 238 and 302 mg/kg bw/day for males and females, respectively).

A dietary two-generation reproductive toxicity study of (2R,4R)-monatin salt in Crl:CD(SD) rats.

(2R,4R)-Monatin salt [sodium/potassium 2R,4R-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate] was fed at 5000, 15,000, or 35,000 ppm to Crl:CD(SD) rats over two generations. Reduced body weights were observed at all dose levels. Sustained effect on body weight gain at 35,000 ppm in the F0 and F1 parental animals was associated with lower feed efficiency, soft stool, and slightly lower numbers of implantation sites. Lower numbers of pups born and live litter size at 35,000 ppm were considered secondary to slightly lower numbers of former implantation sites in the dams. Spermatogenic endpoints, estrous cyclicity, reproductive performance, mean gestation length, and parturition were unaffected in the F0 and F1 generations. There were no effects on F1 and F2 generation postnatal survival. Reduced pre-weaning pup body weights at 35,000 ppm resulted in lower F1 and F2 body weights at study termination. Slight delays in pubertal landmarks in the F1 offspring were considered secondary to the reduced pup body weights. The no-observed-adverse-effect level (NOAEL) was 15,000 ppm for systemic, reproductive, and neonatal effects based on test article-related effects on body weight and food efficiency, slight decrease in maternal implantation sites and corresponding reduction in live litter size, and reductions in pre-weaning pup body weights at 35,000 ppm.

A 90-day dietary study of a (2R,4R)-monatin salt in Beagle dogs.

(2R,4R)-Monatin salt (Na/K) [sodium/potassium (2R,4R)-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate, hereafter "R,R-monatin"] was administered in the diets of groups of Beagle dogs (4/sex/group) at concentrations of 0 (basal diet), 5000, 20,000, or 35,000 ppm for 13 weeks. There were no effects on survival, clinical observations, body weight and body weight gain, feed consumption and feed efficiency, functional observational battery, ophthalmic examination, and electrocardiographic evaluation. No adverse effects on hematology, serum chemistry, and urinalysis parameters were reported. A statistically significant decrease in testicular weights associated with germ cell hypocellularity and reduced luminal sperm in the epididymides was reported in all treated male groups. Based on these findings, the dietary no-observed-adverse-effect level (NOAEL) of R,R-monatin for 90 days was considered 35,000 ppm for female dogs (approximately 1101 mg/kg bw/day) and <5000 ppm for male dogs (approximately <151 mg/kg bw/day).

Plasma Pharmacokinetics and Routes of Excretion of [14C]-Labeled Arruva, a High-Potency Sweetener, Following Oral Administration to Beagle Dogs.

[14C]-Labeled arruva [sodium/potassium (2R,4R)-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate] was administered as a single gavage dose (10 mg/kg bw) to male and female Beagle dogs and 1 bile duct-cannulated male. The mean peak arruva plasma concentration equivalent of 1.2 µg/g occurred at first sampling time point of 1 hour postdosing. The mean area under the concentration versus time curve from 0 hour postdosing to the last time point was approximately 20 µg·h/g and the mean terminal plasma elimination half-life ranged from 15 hours in females to 21 hours in males. Over 168 hours postdosing, 35% to 50% of the administered arruva was eliminated in the urine with 44% to 53% eliminated in feces; 1.3% of the administered dose was recovered in bile. Arruva and its derivatives were identified using tandem mass spectrometry, and the relative percentage of each substance was quantified via radio high-performance liquid chromatography. Over a 168-hour collection period, combined urine and feces extract data from the 6 noncannulated dogs showed that approximately 91% of the dose was excreted as unchanged parent arruva (41% in urine and 50% in feces). In the cannulated male, 95.3% was excreted as unchanged parent arruva; 50.2% in urine, 43.9% in feces, and 1.3% in bile. Lactone and lactam derivatives of arruva and 1 unidentified substance were detected in urine only during the first 24 hours postdosing with the greatest amounts detected during the first 6 hours of collection; up to 1% of lactone or lactam derivatives were detected in bile samples. Plasma pharmacokinetics data indicated rapid absorption of arruva with the majority of radioactivity located in the feces collected in the first 48 hours.

Mutagenicity and genotoxicity studies of arruva, an R,R-monatin salt isomer.

Arruva, the R,R-isomer of monatin (sodium/potassium 2R,4R-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate), an intense sweetener originally identified in root bark of the South African shrub Schlerochitin ilicifolius, was examined for its mutagenic and genotoxic potential via bacterial reverse mutation, mouse lymphoma and in vivo mouse micronucleus assays, all accomplished in the presence and absence of S9 metabolic activation. In the bacterial reverse mutation assay, arruva was determined to not cause reverse mutations in four Salmonella typhimurium strains and one Escherichia coli strain at concentrations up-cells did not exhibit concentration-related increases in mutant frequency at test concentrations up to 3200µg/ml. In the in vivo micronucleus test, arruva was administered to male mice via single gavage doses at 500, 1000 or 2000mg/kg bw. At 24 or 48h post-dose, the mice were euthanized and femoral bone marrow cells were collected for evaluation of micronucleated polychromatic erythrocyte (MPCE) presence. No statistically significant increases of MPEs were observed relative to the respective vehicle control groups. Under the conditions of these studies, arruva was concluded to be negative in all three assays, thereby indicating the absence of its potential mutagenicity or genotoxicity under the conditions tested.

Effect of structural modifications on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced hepatotoxicity in Fischer 344 rats.

Glitazones, used for type II diabetes, have been associated with liver damage in humans. A structural feature known as a 2,4-thiazolidinedione (TZD) ring may contribute to this toxicity. TZD rings are of interest since continued human exposure via the glitazones and various prototype drugs is possible. Previously, we found that 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT) was hepatotoxic in rats. To evaluate the importance of structure on DCPT toxicity, we therefore studied two series of analogs. The TZD ring was replaced with: a mercaptoacetic acid group {[[[(3,5-dichlorophenyl)amino]carbonyl]thio]acetic acid, DCTA}; a methylated TZD ring [3-(3,5-dichlorophenyl)-5-methyl-2,4-thiazolidinedione, DPMT]; and isomeric thiazolidinone rings [3-(3,5-dichlorophenyl)-2- and 3-(3,5-dichlorophenyl)-4-thiazolidinone, 2-DCTD and 4-DCTD, respectively]. The following phenyl ring-modified analogs were also tested: 3-phenyl-, 3-(4-chlorophenyl)-, 3-(3,5-dimethylphenyl)- and 3-[3,5-bis(trifluoromethyl)phenyl]-2,4-thiazolidinedione (PTZD, CPTD, DMPT and DFMPT, respectively). Toxicity was assessed in male Fischer 344 rats 24 h after administration of the compounds. In the TZD series only DPMT produced liver damage, as evidenced by elevated serum alanine aminotransferase (ALT) activities at 0.6 and 1.0 mmol kg(-1) (298.6 ± 176.1 and 327.3 ± 102.9 Sigma-Frankel units ml(-1) , respectively) vs corn oil controls (36.0 ± 11.3) and morphological changes in liver sections. Among the phenyl analogs, hepatotoxicity was observed in rats administered PTZD, CPTD and DMPT; with ALT values of 1196.2 ± 133.6, 1622.5 ± 218.5 and 2071.9 ± 217.8, respectively (1.0 mmol kg(-1) doses). Morphological examination revealed severe hepatic necrosis in these animals. Our results suggest that hepatotoxicity of these compounds is critically dependent on the presence of a TZD ring and also the phenyl substituents.

Safety assessment of kola nut extract as a food ingredient.

Kola nut extract is used in the food industry as a flavoring ingredient. Kola nut extract is derived from the seeds of primarily two tropical Cola species (Cola nitida (Vent.) Schott et Endl. or Cola acuminata (Beauv.) Schott et Endl.) of the Family, Sterculiaceae. Present day consumption of kola nut extract is 0.69 mg/kg/day. Caffeine and theobromine are two important constituents of kola nuts. Although limited biological data are available for kola nut extract specifically, the published data of the major constituents of kola nuts suggest the pharmacological/toxicological properties of kola nut extract, parallel to those of a roughly equivalent dose of caffeine. Frank developmental/reproductive effects have not been reported and changes in offspring cannot be extrapolated to humans. A NOEL/NOAEL cannot be defined for repeated oral exposure to kola nut extract from available data. Notwithstanding the foregoing, U.S. consumers have a history of safe consumption of cola-type beverages containing kola nut extract that dates at least to the late 19th Century, with a significant global history of exposure to the intact kola nuts that date centuries longer.

Role of biotransformation in 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced hepatotoxicity in Fischer 344 rats.

Cytochrome P450 (CYP)-mediated metabolism in the thiazolidinedione (TZD) ring may contribute to the hepatotoxicity of the insulin-sensitizing agents such as troglitazone. We were interested in determining if biotransformation could also be a factor in the liver damage associated with another TZD ring containing compound, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT). Therefore, hepatotoxic doses of DCPT (0.6 or 1.0 mmol/kg, i.p.) were administered to male Fischer 344 rats after pretreatment with vehicle, 1-aminobenzotriazole (ABT, non-selective CYP inhibitor) and troleandomycin (TAO, CYP3A inhibitor). Alternatively, rats were pretreated with vehicle or the CYP3A inducer dexamethasone (DEX) prior to a non-toxic DCPT dose (0.2 mmol/kg, i.p.). Vehicle-, ABT-, TAO- and DEX-only control groups were also run. Toxicity was assessed 24 h after DCPT administration. Both hepatotoxic doses of DCPT induced elevations in serum alanine aminotransferase (ALT) levels that were attenuated by ABT or TAO pretreatment. Liver sections from rats that received vehicle+DCPT revealed areas of gross necrosis and neutrophil invasion, whereas sections from ABT+DCPT and TAO+DCPT rats showed minor changes compared to controls. DEX pretreatment potentiated ALT levels associated with the non-toxic DCPT dose. Furthermore, DEX+DCPT rat liver sections exhibited hepatic injury when compared against rats that received vehicle+DCPT. Blood urea nitrogen levels, urinalysis and kidney morphology were not markedly altered by any combination of pretreatments or treatments. Enzyme activity and Western blotting experiments with rat liver microsomes confirmed the effects of the various pretreatments. Our results suggest that hepatic CYP3A isozymes may be involved in DCPT-induced liver damage in male rats. We believe this is the first report demonstrating that modulation of the biotransformation of a TZD ring-containing compound can alter hepatotoxicity in a common animal model.