Comparisons of l-cysteine and d-cysteine toxicity in 4-week repeated-dose toxicity studies of rats receiving daily oral administration.

Two 4-week repeated-dose toxicity studies were conducted to evaluate the potential toxicity of l-cysteine and d-cysteine. In one study, three groups of 6 male rats were each administered l-cysteine once daily by gavage at doses of 500, 1,000, or 2,000 mg/kg/day for 28 consecutive days. The control group was administered a 0.5% methylcellulose vehicle solution. The other study followed a similar protocol except that the experimental groups received d-cysteine. Toxicological observations showed that the l-cysteine-treated groups exhibited renal injuries such as basophilic tubules with eosinophilic material in the lumen, and there were increased numbers of basophilic tubules in all treated groups. In 1,000 or 2,000 mg/kg/day-treated groups, salivation and necropsy findings indicative of focal erosion in the stomach mucosa were found. Increases in reticulocyte counts were observed in the 2,000 mg/kg/day-treated group. Toxicological findings obtained for the d-cysteine-treated groups included anemia and renal injuries such as basophilic tubules with eosinophilic material in the lumen, increased numbers of basophilic tubules, and crystal deposition in the medulla in the 2,000 mg/kg/day-treated group. Additional findings included sperm granuloma in the epididymis, necropsy findings suggestive of focal erosion in the stomach mucosa, and salivation in the 1,000 or 2,000 mg/kg/day-treated groups. One rat in the 2,000 mg/kg/day-treated group died due to renal failure. In conclusion, the no-observed-adverse-effect levels (NOAELs) were estimated to be less than 500 mg/kg/day for l-cysteine and 500 mg/kg/day for d-cysteine under our study conditions. The toxicological profiles were similar for l-cysteine and d-cysteine; however, there were slight differences in the dose responses. The mechanisms underlying these differences remain to be determined.

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.

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.