Evaluation of circulating miR-216a and miR-217 as biomarkers of pancreatic damage in the L-arginine-induced acute pancreatitis mouse model.

We investigated the usefulness of circulating miR-216a-5p and miR-217-5p that are pancreas-enriched micro RNAs (miRNAs) as biomarkers of acute pancreatic damage, and compared them with conventional pancreatic biomarkers in L-arginine-induced acute pancreatitis mouse model. As the results, amylase and lipase levels apparently increased and peaked on Day 3 when acute pancreatitis including acinar cell degeneration/necrosis and inflammatory cell infiltration reached its peak. In contrast, miR-216a-5p and miR-217-5p increased from Day 1 when histopathological findings in the acinar cells were limited to decreased zymogen granules, and the increases in ratios were much higher than those of amylase and lipase. The miRNAs remained at high levels until Day 5 when the pseudo-tubular complex and replacement of inflammatory cells and fibrotic cells were apparent instead of necrosis, whereas amylase and lipase levels decreased to the control levels. Furthermore, we examined the relationship between biomarker levels and histopathological degeneration/necrosis scores in the acinar cells. miR-216a-5p and miR-217-5p levels increased depending on the score of degeneration/necrosis, and all individual miRNAs exceeded the control levels from a score of 2 (focal necrosis), whereas all individual amylase and lipase levels exceeded the control levels at scores of 4 (lobular necrosis) and 3 (sublobular necrosis), respectively. In conclusion, we demonstrated that circulating miR-216a-5p and miR-217-5p could detect pancreatic damage earlier with greater magnitude, and the sensitivity to detect acinar cell degeneration/necrosis was superior to that of conventional biomarkers in the L-arginine-induced acute pancreatitis mouse model.

13-week repeated dose toxicity study of l-tyrosine in rats by daily oral administration.

To evaluate the potential toxicity of l-tyrosine, 4 groups of Crl:CD(SD) rats of both sexes were administered l-tyrosine in water suspension by gavage once daily for 13 weeks at doses of 0 (vehicle), 200, 600 or 2000 mg/kg bw/day. Findings related to l-tyrosine administration were as follows. Edema of the cornified layer at the limiting ridge or forestomach was seen in 600 mg/kg bw/day female group and in both sexes of 2000 mg/kg bw/day group. In the liver, increased weight and hypertrophy of centrilobular hepatocytes were seen in both sexes at 2000 mg/kg bw/day, associated with slight increases in ALT and AST. Regarding the kidney morphology and function, increased hyaline droplets in the proximal tubules and increased urinary protein were seen in the 2000 mg/kg bw/day male group. In addition, increased kidney weight was also observed in both sexes of the 2000 mg/kg bw/day group, although the histological changes attributable to the weight increase remained unclear. As for blood chemistry, increases in triglycerides, total cholesterol, phospholipids, potassium ion, calcium, total protein, and α1 globulin were also seen in both sexes at 2000 mg/kg bw/day. Thus, in this study the no-observed-adverse-effect level (NOAEL) of l-tyrosine was considered to be 600 mg/kg bw/day for males and 200 mg/kg bw/day for females.

Study for collecting background data on Wistar Hannover [Crl:WI(Han)] rats in general toxicity studies--comparative data to Sprague Dawley rats.

The purpose of the present study was to collect background data from repeated dose toxicity studies in Wistar Hannover [Crl:WI(Han)] (hereafter Wistar Han) rats with dosing periods of 4, 13 and 26 weeks from four safety research facilities of pharmaceutical companies and contract research organizations participating in the International Genetic Standardization (IGS) rat forum supported by Charles River Laboratories Japan, Inc. The data from Wistar Han rats were compared with those from Sprague Dawley Crl:CD(SD) rats. In addition, the effects of restricted feeding of SD rats were also investigated by one facility. As a result, body weights and food consumption in Wistar Han rats were lower than those of SD rats. White blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were almost half of those noted for SD rats and platelet counts were almost 20% less than those in SD rats. Minimal strain differences were noted in several biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase, total cholesterol, triglyceride and phospholipids, and in thymus, ovary and testis weights. Ophthalmologic or histopathologic examinations revealed a higher incidence of corneal opacities or corneal mineralization in Wistar Han rats. Restricted feeding of SD rats resulted in intermediate values for body weights and food consumption between the ad libitum fed SD and Wistar Han rats, and WBC and AST were lower than those in the ad libitum fed SD rats. Based on these results, some strain differences might be ascribable to reduced food consumption and associated body weight changes in Wistar Han rats.

A 4-week Repeated Dose Toxicity Study of Glycine in Rats by Gavage Administration.

In order to examine the toxicity profile of glycine, an authorized food additive, a solution of glycine in water for injection was administered orally (via gavage) to male SD rats (Crl:CD(SD)) once daily for 4 weeks at doses of 500, 1000 and 2000 mg/kg/day in a volume of 10 mL/kg. Control animals received vehicle only. No animals died, and no glycine-related changes were observed in body weight, food consumption, water consumption, hematology, organ weight, gross pathological examination or histopathological examination. In urinalysis, daily urinary volume and urinary Cl excretion were significantly higher in the 2000 mg/kg/day dose group, and urine pH and urinary protein showed lower trends in the glycine-treated groups. However, these changes were considered to be of little toxicological significance, because there were no histopathological changes in the kidneys or urinary bladder and no changes in other urinary parameters. As regards blood chemistry, phospholipids were significantly higher in the 2000 mg/kg/day dose group. However, the increase was small and was not considered to be toxicologically significant. In conclusion, none of the animals in any of the glycine-treated groups showed changes that were considered toxicologically significant. Therefore, the no-observed-adverse-effect level of glycine was estimated to be at least 2000 mg/kg/day under the conditions of this study.

Studies of the toxicological potential of capsinoids XIV: a 26-week gavage toxicity study of dihydrocapsiate in rats.

To further evaluate the safety of dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate, CAS No. 205687-03-2), a 26-week gavage toxicity study was conducted in Sprague-Dawley rats (20/sex/group). Test animals received either dihydrocapsiate, 100, 300, or 1000 mg/kg/day, or vehicle (medium-chain triglyceride) by gavage and were observed for antemortem and postmortem signs of toxicity including changes in clinical signs, body weights, food consumption, water intake, ophthalmology, clinical pathology (clinical chemistry, hematology, urinalysis), tissue findings (macroscopic and microscopic examination), as well as organ weights. After the end of the dosing period, reversibility was assessed (10/sex/group for the control and 1000 mg/kg groups) following a 4-week recovery period. There were no adverse or toxicological changes observed in clinical signs, body weight, food consumption, water intake, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, or histopathology. It was concluded that the no observable adverse effect level (NOAEL) of dihydrocapsiate was 1000 mg/kg/day for both sexes in this 26-week gavage study.

Collaborative work on evaluation of ovarian toxicity. 4) Effects of fertility study of 4-vinylcyclohexene diepoxide in female rats.

As part of a collaborative project, 4-vinylcyclohexene diepoxide (VCD), an ovarian toxicant, was intraperitoneally administered to female Sprague-Dawley rats at 0, 5, 20 or 80 mg/kg from 2 weeks prior to mating to Day 7 of gestation. At necropsy, the number of implanted embryos, rate of implantation decreased and the rate of preimplantation loss showed an increasing tendency in the 80 mg/kg group. As for organ weight, decreases in absolute and relative ovary weight were observed in the 80 mg/kg group. Histopathologically, the ovaries showed a decrease in number of small follicles at 80 mg/kg

Studies of the toxicological potential of capsinoids: I. Single-dose toxicity study and genotoxicity studies of CH-19 Sweet extract.

A single-dose oral toxicity lethal-dose study was conducted to examine the toxicity of capsinoids contained in CH-19 Sweet extract. CH-19 Sweet extract was administered once by gavage to SPF (Crl:CD(SD)) Sprague-Dawley male and female rats at dose levels of 0 (vehicle), 5, 10, or 20 ml/kg of body weight (BW). The concentration of capsinoids in the CH-19 Sweet extract was 71.25 mg/ml; this resulted in administered dose levels of capsinoids of 356.25, 712.5, and 1425 mg/kg BW, respectively. The toxicity of CH-19 Sweet extract by single oral administration was low; only transient salivation or decreased spontaneous movement was observed on the day of administration at > or =10 ml/kg BW. It was concluded that the lethal dose of CH-19 Sweet extract was estimated to be higher than 20 ml/kg (1425 mg/kg as capsinoids) for both males and females since no deaths were observed at any dose in this study. A bacterial reverse mutation test of CH-19 Sweet extract was performed employing Salmonella typhimurium and Escherichia coli and using the preincubation method. Treatment with CH-19 Sweet extract did not increase the number of revertant colonies compared with negative controls either in the presence (+S9) or absence (-S9) of metabolic activation. An in vitro chromosome aberration test was conducted using Chinese hamster lung cultured cells (CHL/IU). Treatment with CH-19 Sweet extract failed to induce chromosome aberrations in either short-term or continuous treatment scenarios, with or without metabolic activation (-S9, +S9). In an in vivo micronucleus test using BDF(1) male mice, CH-19 Sweet extract failed to increase the incidence of micronucleated polychromatic erythrocytes (MNPCEs) or decrease the ratio of polychromatic erythrocytes (PCEs) in any of the treatment groups. These results suggest the absence of mutagenicity as well as in vitro and in vivo clastogenicity of capsinoids contained in CH-19 Sweet extract.

Studies of the toxicological potential of capsinoids: II. A 26-week daily gavage dosing toxicity study of CH-19 Sweet extract in rats.

A 26-week oral toxicity study of capsinoids-containing CH-19 Sweet extract was conducted in Sprague-Dawley rats (20 males and 20 females per group) at 6 weeks of age. The test substance was administered by gavage for 26 weeks at dose levels of 0 (vehicle), 1.25, 2.5, and 5.0 ml/kg/day. The concentration of capsinoids in the CH-19 Sweet extract employed was 71.25 to 73.15 mg/ml, resulting in dose levels of capsinoids of 89.06 to 91.44, 178.13 to 182.88, and 356.25 to 365.75 mg/kg, respectively. Adverse test article-related changes were only observed in males, not in females, and within the males, only at the high dose (5.0 ml/kg). Within that group (high-dose males), increases were observed in the numbers of segmented neutrophils, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities, liver weights, and in the incidence and severity of hepatocellular focal necrosis. No test substance-related changes were detected in clinical signs, body weight, food consumption, water intake, ophthalmology, or urinalysis. No adverse test article-related changes were observed in low- or mid-dose males or in females at any dose. Based on the results of this chronic gavage study, the target organ was the liver and the no observed adverse effect level (NOAEL) for CH-19 Sweet extract in the rat was 2.5 ml/kg/day in males and 5.0 ml/kg/day in females (178.13 to 182.88 mg/kg and 356.25 to 365.75 mg/kg as capsinoids, respectively).

Studies of the toxicological potential of capsinoids: III. A two-generation reproduction study of CH-19 Sweet extract in rats.

CH-19 Sweet extract, containing 66.5 to 75.05 mg/ml capsinoids, was administered once daily by gavage, to two generations of male and female Sprague-Dawley rats, at dose levels of 0 (vehicle), 1.25, 2.5, and 5.0 ml/kg/day (83.13 to 93.81, 166.25 to 187.63, and 332.50 to 375.25 mg/kg as capsinoids, respectively) in order to determine its potential reproductive effects. In the first generation (F(0)) males and females, there were no test substance-related deaths, toxic changes, gross pathological findings, or adverse findings in clinical signs, body weight, or food consumption. There were no test substance-related effects on estrous cycles, copulation index, days required for copulation, fertility index, number of implantations, gestation period, number of liveborn pups, delivery index, stillbirth index, livebirth index, or lactation or nursing. In the second generation (F(1)), there were no test substance-related changes observed in clinical signs, body weights, sex ratios at birth, external abnormalities, differences in survival at any point from birth to weaning, and no deaths after weaning. There were no changes suggestive of adverse test substance-induced effects on body weight, food consumption, or external differentiation after birth, and there was no test substance-related damage on sensory/reflex functions. As with the first generation, there were no test substance-related effects on reproductive indices, in the offspring, no untoward effects on development, viability during the lactation period, body weight, external differentiation, or sensory/reflex functions, and there were no gross morphological abnormalities. Based on these results, the no observed adverse effect level (NOAEL) of CH-19 Sweet extract on the reproductive function and growth of offspring in this two generation study was judged to be 5.0 ml/kg/day (332.50 to 375.25 mg/kg as capsinoids).

Studies of the toxicological potential of capsinoids: IV. Teratology studies of CH-19 Sweet extract in rats and rabbits.

In order to evaluate the safety of CH-19 Sweet extract that contains capsinoids, teratology studies were conducted in pregnant Sprague-Dawley rats (20 rats per group) and pregnant New Zealand white rabbits (17 to 22 animals per group). The test substance was administered to rats by gavage for 11 days on gestation days 7 to 17 at doses of 0 (vehicle), 1.25, 2.5, and 5.0 ml/kg and to rabbits for 13 days on gestation days 6 to 18 at doses of 0 (vehicle), 0.25, 0.5, and 1.0 ml/kg. As the concentration of capsinoids in CH-19 Sweet extract was 72.2 to 75.05 mg/ml, the resulting dose of capsinoids administered to rats was 90.25, 180.5, and 361 mg/kg, and to rabbits was 18.76, 37.53, and 75.05 mg/kg in the vehicle, low-, mid-, and high-dose groups, respectively. In the rat study, no deaths occurred in any group and there were no test substance-related changes or abnormalities in clinical signs, body weight, food consumption, or gross pathological findings. There were no test substance-related changes in the number of corpora lutea, number or index of implantations, index of embryofetal deaths, number of live fetuses, sex ratio, fetal body weight at the end of the gestation period, or abnormalities in the placenta of live fetuses. There were no test substance-related abnormalities or variations in the external, skeletal, or visceral examinations of live fetuses. It was concluded that the test article caused neither teratogenic effects nor abnormalities in the progression of ossification. In the rabbit study, there were no test substance-related effects on clinical signs, body weight, food consumption, or necropsy findings. There were neither test substance-related abortions nor test substance-related effects on the number of corpora lutea, or number or index of implantations. There were no test substance-related effects on the number of dead embryos/fetuses, the number of live fetuses, sex ratio, body weight of live fetuses, or gross pathological finding in the placentas. There were no test substance-related external abnormalities or incidences of visceral or skeletal abnormalities or variations, and there were no test substance-related effects on the progress of ossification in any group. The authors concluded the no observed adverse effect level (NOAEL) of CH-19 Sweet extract containing capsinoids on pregnant animals and fetal development/growth was > 5.0 ml/kg/day (> 361 mg/kg/day as capsinoids) in rats and > 1.0 ml/kg/day (> 75.05 mg/kg/day as capsinoids) in rabbits.

Studies of the toxicological potential of capsinoids: V. Genotoxicity studies of dihydrocapsiate.

A series of studies was performed to evaluate the safety of dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate; CAS no. 205687-03-2). This study evaluated the potential genotoxicity of this compound using a variety of in vitro and in vivo test systems, including bacterial reverse mutation test, chromosomal aberration test, micronucleus test, gene mutation assay with transgenic rats, and single-cell gel (SCG) assay (Comet assay). In vitro tests (bacterial reverse mutation test and chromosomal aberration test) produced positive results in the absence of metabolic activation, but negative results in the presence of metabolic activation. The in vivo gene mutation assay (with transgenic rats) produced negative results, as did the in vivo mouse micronucleus assay, which failed to induce micronucleated polychromatic erythrocytes. Although the rat SCG assay produced statistically significant increases in the Olive tail moment and % tail DNA of the liver and intestine in the 2000 mg/kg group (compared with the negative-control group), a number of factors caused the authors to question the validity of these findings. Taken together, these results suggest that dihydrocapsiate has a low or extremely low likelihood of inducing genotoxicity.

Studies of the toxicological potential of capsinoids: VI. Single-dose toxicity study and micronucleus test of commercial-grade dihydrocapsiate.

A single-dose oral toxicity study was conducted to examine the qualitative and quantitative toxicity of a commercial-grade batch of dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate; CAS No. 205687-03-2). Dihydrocapsiate was administered once by gavage to ICR mice at dose levels of 0 (vehicle) or 5000 mg/kg/day. No mortality was observed during the 14 day observation period following test article administration. During the 2 h immediately following dosing, mice of both sexes treated with dihydrocapsiate were observed to exhibit one or more of the following: staggered gait, decreased spontaneous movement, increased time in the prone position, tremors, gasping, or red-brownish urine. All mice had completely recovered by the 6 h observation interval. No effects on body weights or necropsy findings were observed as a result of dihydrocapsiate administration. These results suggested that the lethal dose of dihydrocapsiate was >5000 mg/kg. In an in vivo micronucleus test using BDF(1) male mice, a commercial grade of dihydrocapsiate neither increased the incidence of micronucleated polychromatic erythrocytes (MNPCEs) nor decreased the ratio of polychromatic erythrocytes (PCEs) in any of the treatment groups. The results suggest that commercial-grade dihydrocapsiate is unlikely to be an in vivo clastogen.

Studies of the toxicological potential of capsinoids: VII. A 13-week toxicity study of dihydrocapsiate in rats.

To evaluate the safety of dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate; CAS No. 205687-03-2), a 13-week gavage toxicity study was conducted in Sprague-Dawley rats (10/sex/group). Test subjects received either dihydrocapsiate, 100, 300, or 1000 mg/kg/day, or vehicle by gavage and were observed for antemortem and postmortem signs of toxicity, which included changes in clinical signs, body weights, food consumption, water intake, ophthalmology, clinical pathology (clinical chemistry, hematology, urinalysis), tissue findings (macroscopic and microscopic examination), as well as organ weights. No changes attributable to the test article were observed in clinical signs, body weights, food consumption, water intake, ophthalmology, urinalysis, hematology, or histopathology. A number of sporadic blood chemistry differences were observed at the high dose between treated and controls, but were not of toxicological significance and were not attributable to the test article. These included increased alanine aminotransferase (ALT) activity in males; increased total protein in males and females; increased calcium, percentage of albumin fraction, and A/G (albumin/globulin) ratio and decreased percentage of gamma-globulin fraction in female rats. An effect, which was attributable to the test article, was increases in both absolute and relative liver weights in the high dose (both sexes). In the absence of histopathological changes attributable to the test article, the liver weight changes were considered adaptive (physiological) in nature and not of toxicological significance. It was concluded that the no observed adverse effect level (NOAEL) of dihydrocapsiate was 1000 mg/kg/day for both male and female rats in this 13-week gavage study.

Studies of the toxicological potential of capsinoids: VIII. A 13-week toxicity study of commercial-grade dihydrocapsiate in rats.

Dihydrocapsiate, (4-hydroxy-3-methoxybenzyl 8-methylnona- noate; CAS No. 205687-03-2) is a naturally occurring capsinoid compound found in nonpungent chili peppers. Although the safety of synthetically produced dihydrocapsiate has been previously evaluated, the purpose of this 13-week gavage toxicity study is to evaluate dihydrocapsiate produced with a slightly modified manufacturing process. Sprague-Dawley rats, 10 rats/sex/group, 6 weeks of age at study initiation, were administered the dihydrocapsiate daily by gavage at dose levels of 0 (vehicle), 100, 300, or 1000 mg/kg/day. The rats were observed for antimortem and postmortem signs of toxicity, including changes in clinical signs, body weights, food consumption, water intake, ophthalmology, clinical pathology (clinical chemistry, hematology, urinalysis), tissue findings (macroscopic and microscopic examination), as well as organ weights. There were no changes observed in clinical signs, body weight, food consumption, water intake, ophthalmology, urinalysis, hematology, or blood chemistry that were attributable to the administration of dihydrocapsiate. The only change observed attributable to the dihydrocapsiate administration involved the liver and that change occurred only at the high dose (1000 mg/kg). Both sexes had an increase in organ weights, but this increase correlated with a change in histopathology (i.e., hepatocyte hypertrophy) only in the males. No dihydrocapsiate-related histopathological changes were observed in males at doses < or = 300 mg/kg or in females at any of the doses tested (< or = 1000 mg/kg). It was concluded that the no observed adverse effect level (NOAEL) of dihydrocapsiate was 300 mg/kg/day for male rats and 1000 mg/kg/day for female rats in this 13 week gavage study.

Studies of the toxicological potential of capsinoids: IX. Teratology studies of dihydrocapsiate in rats and rabbits.

In order to determine the safety of dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate; CAS no. 205687-03-2), teratology studies were conducted in pregnant Sprague-Dawley rats (18 to 20 animals per group) and pregnant New Zealand white rabbits (20 to 21 animals per group). The test substance was administered by gavage for 11 days, from days 7 to 17 of gestation in rats, and for 13 days from days 6 to 18 of gestation in rabbits, at dose levels of 0 (vehicle), 100, 300, or 1000 mg/kg/day. In the rat study, no deaths occurred in any group and there were no test substance-related changes or abnormalities in clinical signs, body weight, food consumption, or gross pathological findings. There were no test substance-related changes in the number of corpora lutea, number of implantations, index of implantations, index of embryofetal deaths, and number, sex ratio, or body weight of live fetuses at the end of the gestation period and there were no abnormalities in the placentae of live fetuses. There were no test substance-related abnormalities or variations in the external, skeletal, or visceral examinations of live fetuses. There were no abnormalities in ossification. En toto, it was concluded there were no teratogenic effects in the rat study. In the rabbit study, there were no test substance-related effects on clinical signs, body weight, food consumption, or necropsy findings in any group. There were neither test substance-related abortions nor test substance-related effects on the number of corpora lutea, number of implantations, or implantation index in any group. There were no test substance-related effects on the number of dead embryos/fetuses, the number, sex ratio, or body weight of live fetuses, or gross pathological finding of placentae. There were no test substance-related external abnormalities, or incidence of visceral or skeletal abnormalities or variations, and there were no test substance-related effects on the progress of ossification in any group. Based upon these data, the no observed adverse effect level (NOAEL) of dihydrocapsiate for general toxicity in dams, reproductive functions of dams, and embryofetal development was judged to be 1000 mg/kg/day both in rats and rabbits.