Food flavor cinnamaldehyde-induced biochemical and histological changes in the kidney of male albino wistar rat

Rats were given food flavor cinnamaldehyde (CNMA) orally by gavage at the dose of 2.14, 6.96, 22.62 and 73.5 mg/kg body weight/day for 10, 30 and 90 days. Only the group of rats treated with CNMA at the dose 73.5 mg/kg body weight/day for 90 days showed histological changes in the kidney followed by increased activities of renal, serum and urinary enzymes. CNMA-induced glucosuria in these rats was accompanied by marked proteinuria and creatinuria. Increased serum blood urea nitrogen and serum creatinine and decreased serum protein and glucose levels were observed in these rats. Thus, CNMA at the dose of 73.5 mg/kg body weight/day for 90 days exert its effect on kidney of male albino wistar rat and its effect is time and dose dependent.     

Teratogenic potential of purified pentachlorophenol and pentachloroanisole in subchronically exposed Sprague-Dawley rats

Male and female Sprague-Dawley (Spartan) rats were exposed to dietary levels of 0, 60, 200 or 600 ppm purified pentachlorophenol (PCP) or pentachloroanisole (PCA) for 181 days, through mating and pregnancy. The daily intakes of PCP were 0, 4, 13 or 43 mg/kg body weight and of PCA were 0, 4, 12 or 41 mg/kg body weight. Animals exposed to PCP generally consumed more food than control animals during pregnancy. Dams at the high-dose level of both compounds showed evidence of toxicity, weighing less on day 0 of gestation and gaining less throughout pregnancy than did the controls. Dams exposed to the high dose of PCP gained less weight during pregnancy (exclusive of the gravid uterus) than control dams. At the 43 mg/kg/day dose level PCP was embryolethal. Foetuses at the lower dose levels of PCP exhibited dose-related decreases in body weights. A reduction in crown-rump length and an increase in foetal skeletal variations were seen at 13 mg/kg/day in PCP animals only. An intake of 41 mg PCA/kg/day was associated with a decrease in the number of corpora lutea and in embryolethality. PCA exposure also resulted in reductions in foetal body weight and crown-rump lengths of males at 4 and 41 mg/kg/day. Female foetuses were unaffected.     

Evaluation of the Developmental Toxicity of Methacrylamide and N,N'-Methylenebisacrylamide in Swiss Mice

Timed-pregnant CD-1 outbred albino Swiss mice received eithermethacrylamlde (MAC; 0, 60, 120, or 180 mg/kg/day) or N,N'-methylenebisacrylamide(BAC; 0, 3, 10, or 30 mg/kg/day) po in distilled water on gestationaldays (GD) 6 through 17. Maternal clinical status was monitoreddaily. At termination (GD 17), confirmed-pregnant females (27–30per group, MAC; 24–25 per group, BAC) were evaluated forclinical status and gestational outcome; live fetuses were examinedfor external, visceral, and skeletal malformations. For MAC,no treatment-related maternal mortality was observed. Maternalbody weight on GD 17, maternal weight gain during treatmentand gestation, and corrected maternal weight gain were reducedat the high dose. Relative maternal food and water intake wasnot adversely affected; neurotoxicity was not observed. Relativematernal liver weight was increased at a 120 mg/kg/day; graviduterine weight was decreased at 180 mg/kg/day. The maternalno-observed adverse effect level (NOAEL) was 60 mg/kg/day. TheNOAEL for developmental toxicity was also 60 mg/kg/day. At 120 mg/kg/day, mean fetal body weight was reduced. At 180 mg/kg/day,increased postimplantation death per litter was observed. Morphologicaldevelopment was not affected. The maternal NOAEL for BAC was10 mg/kg/day. At 30 mg/kg/day, decreased maternal body weighton GD 17, maternal body weight change during treatment and gestation,corrected maternal body weight, and gravid uterine weight wereobserved. Relative maternal liver weight increased at 30 mg/kg/day.The developmental NOAEL was 3 mg/kg/day BAC. Mean fetal bodyweight was reduced at 30 mg/kg/day. At 10 mg/kg/day, an increasedincidence of fetal variations (extra rib) was observed, althoughfetal malformation rate was unaffected. MAC and BAC were notteratogenic to Swiss mice at the doses tested. BAC was morepotent than MAC in causing adverse maternal and developmentaleffects.     

Hepatotoxicity induced by the herbicide atrazine in the rat

The hepatotoxicity of atrazine was investigated by studying clinical parameters related to hepatic function and by electron microscopy. Three groups to male albino rats (Wistar strain) received 100, 200 and 400 mg of atrazine/per kg of body weight/per day, for 14 days. One group received 600 mg atrazine/per kg of body weight/per day, for 7 days. At termination of dosing, the animals were sacrificed and blood was drawn for the determination of serum total lipids, glucose, alanine aminotransferase (ALT) and alkaline phosphatase (SAP). A dose dependent decrease in serum glucose concentration was observed in all the groups. In contrast, a dose relate increase in total serum lipids, was apparent at all dose levels studied. Activity of serum ALT and SAP increased approximately 60% and 200% respectively in rats given 600 mg atrazine/kg bw for 7 days. The liver was examined grossly and microscopically. Electron microscopy disclosed no changes in the hepatocytes of rats treated with the low dose (100 mg/kg bw). At high doses, electron microscopy revealed hepatocytic proliferation and degeneration of smooth endoplasmic reticulum, lipid accumulation and alteration of bile canaliculi proportional to dose and duration of treatment.     

Short-term toxicity of 2,6-dimethylhept-5-en-1-al in rats

Groups of 15 male and 15 female rats were fed for at least 90 days on diets that provided 2,6-dimethylhept-5-en-1-al (DMH) at average intakes of 0 (control), 9, 37 and 150 mg/kg body weight/day. Steps were taken to limit loss of DMH during diet mixing, storage and feeding. No effects attributable to treatment were encountered in body weight, food intake, water intake, haematology (at wk 6 and 13) or the gross and microscopic pathological examinations. At the highest dose level there was a slight reduction in renal concentrating ability at wk 6 in males and wk 14 in females, together with a small increase in relative kidney weight and liver weight in females. The serum-glucose concentrations of both sexes on the highest dose were elevated compared with the controls. It was concluded that the intermediate dose, providing an intake of 37 mg/kg/day, was the no-untoward-effect level.     

Maleic acid dimethylester: evaluation of dermal toxicity and genotoxicity

Maleic acid dimethylester (MAD) was investigated in acute and subacute dermal toxicity studies, for sensitization potential, and for in vivo and in vitro genotoxicity. The acute dermal toxicity in rats was low (LD50 greater than 2000 mg/kg body weight). Only local effects, erythema and necrosis, occurred at the site of application. Corresponding dose-related effects were observed in a 28-day repeated dermal toxicity study in rats. Treatment-related systemic alterations were observed in feed consumption, body weights, haematology and clinical chemistry at 170 and 500 mg MAD/kg body weight. Based on the results of this study, the no-toxic-effect level of MAD was considered to be 60 mg/kg body weight/day. However, slight dermal irritative effects were also present at the lowest dose level (60 mg/kg body weight). The primary skin irritation test in rabbits showed only slight erythema and oedema. The results of the maximization test in guinea-pigs indicated a clear sensitizing potential of MAD. In the Ames test, with five strains of Salmonella typhimurium, MAD was not mutagenic up to the highest dose level of 5000 micrograms/plate. In the micronucleus test, in which mice were given 1000 mg MAD/kg body weight by gavage the compound revealed no clastogenic effects.     

Evaluation of the maternal and developmental toxicity of aluminum from high doses of aluminum hydroxide in rats

The potential of aluminum hydroxide [Al (OH)3] to induce developmental toxicity in rats was evaluated in the present study. Al (OH)3 was given by gavage at dose levels of 192, 384, and 768 mg/kg/day to groups of pregnant rats from day 6 through day 15 of gestation. Control animals received distilled water. Pregnant rats were evaluated for body weight, weight gain, food consumption, appearance, behavior and reproduction data. Cesarean sections were performed on gestation day 20, and the fetuses were removed for teratological evaluation. No significant maternal or developmental toxicity was observed at any Al (OH)3 dose level. Consequently, the no-observed-effect level (NOEL) for Al(OH)3 maternal or developmental toxicity would be greater than or equal to 768 mg/kg/day, which was the highest dose tested. This dose would be equivalent to a 60 kg person ingesting 16 g Al/day.     

Acute and subacute oral toxicity assessment of dry encapsulated and non-encapsulated green coffee fruit extracts

The coffee fruit is a high source of bioactive compounds such as phenolic acids and methylxanthines, comprising chlorogenic acids and caffeine, respectively. Extract from this matrix may be used as supplement or active ingredient of functional foods, energy drinks, cosmetics or drugs. Safety of caffeine- and chlorogenic acid-rich encapsulated and non-encapsulated hydroethanolic extracts from green coffee fruit (GCFE) was assessed by acute and subacute toxicity tests. In the acute test, oral single dosage until 1000 mg/kg per body weight (bw) did not show any adverse effect on both female and male mice according to the Hippocratic screening and clinical parameters for a period of 14 days. While the oral median lethal dose of non-encapsulated GCFE was 5000 mg/kg bw/day, that of encapsulated GCFE was not detectable likely due to the delayed release of caffeine and other compounds from GCFE. Non-encapsulated GCFE displayed a stimulating effect at a dose of 1000 mg/kg bw/day after 30 min of oral administration, but not after 60 min. Daily consumption of encapsulated GCFE for 30 days showed no adverse effect in male rats even at the highest dose. Extrapolating this value of no-observed-adverse-effect level (1000 mg/kg bw/day) to human consumption, a human equivalent dose of 189 mg/kg bw/day or 11.34 g/day could be estimated for encapsulated GCFE considering a 60 kg adult body weight.     

Developmental toxicity of Spinosad administered by gavage to CD® rats and New Zealand White rabbits

The insecticide Spinosad was administered by gavage to pregnant CD® rats at 0, 10, 50 or 200 mg/kg/day on gestation days (gd) 6–15 and to New Zealand White rabbits at 0, 2.5, 10 or 50 mg/kg/day on gd-7–19. Rats and rabbits were monitored for clinical signs of toxicity and body weight gains. At gd-21 (rats) or gd-28 (rabbits), maternal organ weights, reproductive parameters, fetal body weights, and fetal external, visceral and skeletal structures were evaluated. Rats given 200 mg/kg/day exhibited a 4% lower body weight on gd-12 and decreased body weight gains on gd-6–16 relative to controls. There was no maternal toxicity at 10 or 50 mg/kg/day, and no developmental toxicity in rats at any dose level. Rabbits given 50 mg/kg/day exhibited decreased feed consumption, reduced fecal output, body weight loss during the initial dosing period (gd-7–10) and a non-statistically significant decrease (31%) in body weight gain during the dosing period (gd-7–20). Two litters aborted due to maternal inanition. There were no maternal effects at lower doses, and no signs of developmental toxicity at any dose. Thus, the maternal no-observed-effect levels (NOEL) were 50 and 10 mg/kg/day in rats and rabbits, respectively, and the embryonal/fetal NOELs were 200 mg/kg/day in rats and 50 mg/kg/day in rabbits.     

Teratology study of Tween 60 in rats

The teratogenicity of Tween 60 was studied in Wistar rats. Pregnant rats were given Tween 60 at a dose of 0, 0.1, 1.0 or 10% in the diet from day 7 to day 14 of pregnancy. Daily intakes of Tween 60 were 99 mg/kg for the 0.1% group, 960 mg/kg for the 1.0% group and 7693 mg/kg for the 10% group. No change induced by Tween 60 was detected in the number, sex ratio and body weight of live fetuses. External, skeletal and internal examinations of the fetuses revealed no evidence of teratogenesis. It could be concluded that Tween 60 has no harmful effects on the prenatal development of the rat offspring at doses employed in the present study.     

Short term toxicity study in rats dosed with pulegone and menthol

Pulegone and menthol, components of peppermint oil, were investigated in rats. The substances were administered by gavage for 28 days at 0, 20, 80, 160 mg pulegone and 0, 200, 400, 800 mg menthol/kg body wt./day, respectively. At the two highest doses, pulegone induced atonia, decreased blood creatinine content, lowered terminal body weight and caused histopathological changes in the liver and in the white matter of cerebellum. For menthol at all dose levels a significant increase in absolute and relative liver weights and vacuolisation of hepatocytes was found. No sign of encephalopathy was observed in rats given menthol.The no effect level for pulegone was 20 mg/kg body wt./day and for menthol < 200 mg/kg body wt./day.     

Developmental toxicity of diethanolamine applied cutaneously to CD rats and New Zealand White rabbits

Diethanolamine (DEA) was administered cutaneously to pregnant CD rats and New Zealand White rabbits during the periods of major organogenesis, Gestation Days 6-15 for rats and 6-18 for rabbits. Doses employed were 0, 150, 500, and 1500 mg/kg/day for rats and 0, 35, 100, and 350 mg/kg/day for rabbits. Rat dams exhibited reduced body weight at 1500 mg/kg/day, skin irritation and increased kidney weights at 500 and 1500 mg/kg/day, and a slight microcytic anemia with abnormal red blood cell morphology at all dose levels. Rat fetuses had increased incidences of six skeletal variations at 1500 mg/kg/day. Lower doses were without effect on the fetuses. Rabbit dams administered 350 mg/kg/day exhibited various skin lesions, reduced food consumption, and color changes in the kidneys but no hematological changes. Body weight gain was reduced at >/=100 mg/kg/day. There was no evidence of maternal toxicity at 35 mg/kg/day and no evidence of developmental toxicity in rabbits at any dose level. Developmental toxicity was observed only in the rat and only at doses causing significant maternal toxicity, including hematological effects. Due to a dose discrepancy, the no observable effect level (NOEL) for DEA developmental toxicity in rats was adjusted to 380 mg/kg/day. In rabbits, the embryonal/fetal NOEL was 350 mg/kg/day.     

The development of methods for assessing the in vivo oestrogen-like effects of xenobiotics in CD-1 mice.

The increasing awareness and concern about the potential health risks posed to the ecosystem and to man by endocrine disrupting chemicals with oestrogen-like activity in the environment has focused attention on the need for developing sensitive and specific methods for identifying these xenobiotics and to evaluate their degrees of toxic effects. We have conducted dose response studies in immature (21 days old) CD-1 female mice treated with four compounds, diethylstilboestrol (DES) (0.1 microg to 25 mg/kg body weight), alpha-zearalanol (0.5 mg to 25 mg/kg body weight), methoxychlor (0.5 mg to 500 mg/kg body weight) and bisphenol A (10 microg to 100 mg/kg body weight) administered subcutaneously daily for 3 days, and measured a number of uterine markers in treated and control (vehicle treated) mice. These were, in addition to the commonly measured changes in relative uterus weight and histopathological examination of uterine tissue, three other markers indicative of uterotrophic effects, namely, uterine luminal epithelium BrdU labelling index over the last 24 hr, peroxidase activity and lactoferrin expression. All of these markers showed clear dose-related increases in DES- and methoxychlor-treated animals. In the case of alpha-zearalanol treatment, relative uterine weight, peroxidase activity and lactoferrin expression showed dose-related increases at all the doses investigated. BrdU incorporation (an index of cell proliferation) also progressively increased at dose levels ranging from 0.1 mg to 5.0 mg/kg body weight, but apparently decreased at 25 mg/kg body weight. In contrast to these findings, bisphenol-A treatment showed no consistent changes in any of the four markers at the dose levels investigated. Additionally, studies were also conducted on a number of chemicals in CD-1 mice at one dose level. The chemicals investigated were: bisphenol A (1 g/kg body weight/day), naringenin (1 g/kg body weight/day) o,p'-DDT (500 mg/kg body weight/day), genistein (1 g/kg/day), coumestrol (0.5 mg/kg/day) and chlordecone (20 mg/kg/day) administered subcutaneously daily for 3 days. There was some variability in response of the markers perhaps indicating that the chemicals did not all act in the same way. The findings of our exploratory in vivo studies in CD-1 mice suggest that the measurement of a range of uterine markers, in addition to organ weight and histopathology, would provide useful information on the potential oestrogenicity of chemicals.     

Teratogenic and fetal toxicity following intravenous theophylline administration in pregnant rabbits is related to maternal drug plasma levels

This study investigated the teratogenic and fetal toxicity of i.v. theophylline and its relationship to maternal plasma levels in pregnant rabbits. From days 6-18 of gestation, each dose of theophylline (15, 30 and 60 mg/kg/day at a rate of 0.5 ml/kg/min) was administered i.v. to pregnant rabbits using an automatic infusion pump. Theophylline showed reversible toxicity: accelerated respiration, sluggish startle reactions, dilation of the auricular vessels and polyuria were observed in dams treated with 60 mg/kg/day but not in animals given 15 or 30 mg/kg/day. Fetuses from the dam group treated with 60 mg/kg/day exhibited teratogenic toxicity such as cleft palate and skeletal variation of the 13th rib. Fetal toxicity was also observed including abortion, increased number of late deaths and decreased body weight appearing on day 29 of gestation. No toxicity was observed in fetuses from the dam group treated with 15 or 30 mg/kg/day. However, in the 30 and 60 mg/kg/day theophylline-treated groups, maternal plasma concentrations (Cmax) during the treatment period were approximately 56 and 106 micrograms/ml, respectively. It is therefore suggested that the risk of teratogenic and fetal toxicity caused by theophylline is dependent on its dosage. In conclusion, caution should be taken when administering theophylline or aminophylline to pregnant individuals at doses that could result in high neonate peak blood levels.     

“Simulated gastric hydrolysis and developmental toxicity of dioctyltin bis(2-Ethylhexylthioglycolate) [DOTE] in rabbits and mice”

Based on previous studies, dioctyltin dichloride [DOTC] was a putative toxophore for dioctyltin thioesters. Our results, generated with the use of ¹¹⁹Sn-NMR spectroscopy demonstrated that dioctyltin bis(2-ethylhexyl thioglycolate) [DOTE] hydrolyzed to form dioctyltin chloro-(2-ethylhexyl thioglycolate) [DOTCE] under simulated gastric conditions, but no DOTC was formed. DOTE was administered orally at 4, 20, and 80 mg/kg/day [GD6-GD28; rabbits] or at 15, 30, and 60 mg/kg/day [GD5-GD17; mice]. There were no maternal deaths, treatment-related statistically significant reductions in maternal body weight or weight gain, or adverse gestational outcomes in either species. Maternal thymus weight was significantly reduced in mice at 30 and 60 mg/kg. There were no effects on fetal growth, no dose-dependent pattern of external, visceral or skeletal malformations and no increase in anatomical variations in either species. We conclude that DOTE likely forms DOTCE, not DOTC, in the stomach and DOTE was not teratogenic or fetotoxic in rabbits or mice. The rabbit maternal NOAEL was 80 mg/kg/day. The rabbit developmental NOAEL was 80 mg/kg/day. The mouse maternal LOAEL was 30 mg/kg/day based on reduced thymus weight and a dose-dependent effect on maternal weight at 60 mg/kg. The mouse developmental NOAEL was 60 mg/kg bw/day, the high dose.     

Prenatal or postnatal exposure to bis(tri-n-butyltin)oxide in the rat: postnatal evaluation of teratology and behavior

The results of a series of screening tests to determine the potential teratogenicity and neurotoxicity of developmental exposure to TBTO in rats are presented in this paper. For prenatal exposure, pregnant Long Evans rats were intubated with 0-16 mg/kg/day bis(tri-n-butyltin)oxide TBTO from Days 6 to 20 of gestation (GD 6-20). For postnatal exposure, rat pups were intubated with 0-60 mg/kg TBTO on Postnatal Day 5 (PND 5). Following prenatal exposure, dams were allowed to litter and pups were evaluated using a postnatal teratology screen. Postnatal evaluation for both exposures included motor activity (PND 13-64), the acoustic startle response (PND 22-78), growth, and brain weight. The maximally tolerated dose (MTD) in pregnant rats was 5 mg/kg/day, which is one-third the MTD in nonpregnant rats. There were decreased numbers of live births, and decreased growth and viability at dosages greater than or equal to 10 mg/kg/day. Cleft palate was found in 3% of the 12 mg/kg/day group. There was mortality following postnatal exposure to 60 mg/kg and all prenatal dosages greater than or equal to 10 mg/kg/day. Preweaning body weight was significantly decreased for all postnatal dosages, and all prenatal dosages greater than 2.5 mg/kg/day. Body weight reductions persisted to the postweaning period only in the high dose groups (10 mg/kg/day and 60 mg/kg). Behavioral evaluation demonstrated transient alterations in motor activity development (prenatal exposure only) and the acoustic startle response (postnatal exposure only). Persistent behavioral effects were observed only at dosages that produced overt maternal toxicity and/or postnatal mortality. The demonstration of the teratogenic and neurotoxic potential of TBTO in rats is confounded by associated maternal toxicity and/or pup mortality.     

Effect of chronic administration of an oral contraceptive on the blood pressure of rats

Dietary administration of the oral contraceptive, Enovid, to female rats at 7.5 mg/kg of food (420 mg/kg body weight/day) for 20 weeks was accompanied by an elevation of systolic blood pressure and an increase in the ratios of both heart weight to body weight and renal weight to body weight. A second study, in which Enovid was administered at the same dose for 24 weeks revealed significant increases in systolic, diastolic and mean blood pressures of treated female rats measured by direct cannulation of the carotid artery. A third study tested the effect of the same dose of Enovid on systolic blood pressure of female rats receiving 0.15 m NaCl solution as their sole drinking fluid. The first significant elevation of blood pressure in these rats occurred during week 6 of drug treatment and continued for the remainder of the 12-week experiment. The ratio of heart weight to body weight was also increased significantly in the treated group. Thus, chronic administration of Enovid at the dosage used was accompanied by hypertension in rats.     

Evaluation of the oral toxicity of acetaldehyde and formaldehyde in a 4-week drinking-water study in rats

A subacute oral toxicity study of acetaldehyde and formaldehyde was carried out in rats. Groups of ten male and ten female 5-wk-old rats received one of the aldehydes in the drinking-water for a period of 4 wk, acetaldehyde being given at dose levels of 25, 125 and 675 mg/kg body weight/day and formaldehyde at dose levels of 5, 25 and 125 mg/kg body weight/day. A group of 20 males and 20 females served as controls and received unsupplemented drinking-water ad lib. An additional group of ten males and ten females was given unsupplemented drinking-water in an amount equal to the amount of liquid consumed by the group given the top dose of formaldehyde. Food and liquid intake were decreased in the groups on the top dose of both acetaldehyde and formaldehyde. Hyperkeratosis of the forestomach, observed only in the top-dose rats, was the only adverse effect of acetaldehyde detected. Effects of formaldehyde, also observed only in the top-dose group, were yellow discoloration of the fur, decreased protein and albumin levels in the blood plasma, thickening of the limiting ridge and hyperkeratosis in the forestomach, and focal gastritis in the glandular stomach. It was concluded that in this study the no-observed-adverse-effect levels of acetaldehyde and formaldehyde were 125 and 25 mg/kg body weight/day, respectively.     

Studies on the prenatal toxicity of N,N-dimethylformamide in mice, rats and rabbits.

Prenatal toxicity studies with N,N-dimethylformamide (DMF) in rabbits, rats and mice were carried out using the oral (gavage), dermal, inhalation and ip injection routes of administration. Administration of DMF by gavage led to an increase in malformations in rats and mice in the absence of overt maternal toxicity. The lowest-observable-effect level was 182 mg/kg body weight/day in mice and 166 mg/kg body weight/day in rats. After dermal administration a dose-dependent incidence of teratogenicity was observed in rats at 94-944 mg/kg/body weight/day in the absence of overt maternal toxicity. In rabbits dermal administration led to a steeper increase in the dose-response relationship and at 400 mg/kg body weight/day to a clear teratogenic effect in the presence of slight maternal toxicity. The 200 mg/kg body weight/day dose appeared to be the no-adverse-effect level. Inhalation in rats caused foetotoxicity and embryolethality at 287 ppm. A clear teratogenic effect was shown in rabbits at 450 ppm and a marginal effect at 150 ppm. The no-effect level for does and foetuses was 50 ppm. Ip injection in mice caused clear teratogenicity at 944 mg/kg body weight/day and slight embryotoxicity at 378 mg/kg body weight/day. The rabbit appears to be more sensitive than the rat to DMF-related prenatal toxicity and should, therefore, be used as the basis for the evaluation of teratogenic risk in humans.     

Subacute toxicity assessment of carotenoids extracted from citrus peel (Nanfengmiju, Citrus reticulata Blanco) in rats

The mixture of carotenoids extracted from citrus peel (Nanfengmiju, Citrus reticulata Blanco) was tested for subacute oral toxicity. In this study, dose levels of 0, 200, 500 and 2000 mg/kg body weight/day were administered by gavage to 10 Wistar rats/sex/group for 28 days. No statistically significant, dose-related effect on food consumption, food efficiency, body weight gain, clinical signs or ophthalmoscopic parameters was observed in any treatment group. Urinalysis, hematological, blood coagulation and serum biochemical examination as well as necropsy or histopathology showed that no observed adverse effect was found. These findings suggested that the No-Observed-Adverse-Effect Level for the mixture of carotenoids extracted from citrus peel was at least 2000 mg/kg body weight/day.