Safety evaluation of Evesse EPC, an apple polyphenol extract rich in flavan-3-ols.

The safety of the apple polyphenol extract EvesseEPC, which is rich in flavan-3-ols, particularly epicatechin, was evaluated. Both in a bacterial reverse mutation test and a mouse lymphoma assay, EvesseEPC showed a positive response in vitro. In vivo studies (UDS test in hepatocytes, bone marrow micronucleus test and comet assay in intestinal cells) were all negative and hence Evesse EPC is considered not to have genotoxic properties in vivo. In a 90-day study in rats, EvesseEPC was administered at dietary levels of 0%, 1.25%, 2% and 3.25%. Body weights were decreased in the high-dose group in both sexes without effects on feed or water intake. In the high-dose group, thrombocytes (males) and creatinine (both sexes) were decreased, prothrombin time (males) was increased, and liver, kidneys and spleen weights were increased (males), without histological correlates. Diffuse acinar cell hypertrophy, observed in the parotid salivary glands in all treatment groups, was not considered as adverse and presumably reflected a local, reversible and adaptive response to direct contact with EvesseEPC. The NOAEL for EvesseEPC in rats was 2% in the diet, equivalent to an overall average intake of 1.3 and 1.5 g/kg body weight/day for males and females, respectively.

90-Day feeding and genotoxicity studies on a refined arachidonic acid-rich oil.

The safety of a refined arachidonic acid-rich oil (RAO) was evaluated for reverse mutation, chromosome aberration and gene mutation, and in a 90-day Wistar rat feeding study with in utero exposure. The results of the genotoxicity assays were all negative. The in utero phase of the 90-day study involved dietary exposure to 0.5%, 1.5% and 5% RAO and two controls diets, a standard feed low-fat diet and a high-fat diet supplemented with 5% corn oil. This exposure covered four-weeks prior to mating, through mating, gestation and lactation until offspring (F(1)) weaning. A subsequent 90-day feeding study in the F(1) rats evaluated the same test and control diets. Statistically significant effects were seen for selected histopathology, clinical chemistry and organ weight endpoints; however, other than increased absolute and relative monocytes seen in both sexes of high-dose rats, the observations were not attributed to treatment for one or more reasons. Based on these findings, no adverse treatment-related effects for RAO were seen at up to 5% in the diet, equivalent to an overall average RAO intake of 3170 mg/kg bwt/day. These and similar findings for other refined ARA-rich oils establish a strong body of evidence for the safety of this RAO.

Subchronic (13-week) oral toxicity study, preceded by an in utero exposure phase, with arachidonate-enriched triglyceride oil (SUNTGA40S) in rats.

Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA) and docosahexaenoic acid (DHA) are natural constituents found in human milk, fish oil or egg yolk. Until recently, infant formulas, though providing the essential fatty acid precursors for these PUFAs, did not contain preformed ARA or DHA. In this study the safety of SUNTGA40S as source of ARA, not only for use in infant formulas but also for nutritional products or food supplements, was evaluated in a subchronic study in Wistar rats, preceded by a 4-week pretreatment period of parental (F(0)) rats and exposure of the F(0) dams throughout mating, gestation and lactation. SUNTGA40S was administered at dietary levels of 0.5%, 1.5% and 5% (wt/wt) adjusted with corn oil to 5.76% added fat. An additional group received 3.65% (wt/wt) SUNTGA40S in conjunction with 2.11% (wt/wt) high DHA Tuna oil, providing an ARA:DHA ratio of 2.7:1. High-fat and low-fat controls received basal diet with or without 5.76% corn-oil supplement. The content, stability and homogeneous distribution of the test substances in the diet were confirmed under study conditions. The administration of SUNTGA40S, with or without DHA oil, did not affect health, growth, fertility or reproductive performance of the parental rats, nor pup characteristics (condition, weight gain, viability, number per litter or sex ratio). In the subchronic study with the offspring (F(1)) rats, no significant differences were found in condition, neurobehavioural observations, ophthalmoscopy, growth, urinalysis or macroscopic and microscopic findings between the test groups and the low-fat or the high-fat controls. In males of the 5% SUNTGA40S and the SUNTGA40S/DHA group, red blood cell counts, haemoglobin concentration and packed cell volume were lower and reticulocytes were slightly higher than in the high-fat and low-fat control groups. Cholesterol, triglycerides and phospholipids in plasma were lower than in the high-fat controls in both sexes in the 5% SUNTGA40S and the SUNTGA40S/DHA group and (for triglycerides only) in the 1.5% SUNTGA group. Due to the administration of extra dietary fat, food intake and prothrombin time (males only) were lower and alkaline phosphatase activity was higher in all the high-fat groups, including the corn-oil controls, as compared to the low-fat controls. The weight of the spleen was higher in males of the 5% SUNTGA40S and the SUNTGA40S/DHA group compared to both the low-fat and the high-fat controls. The effects noted in this study at high dose levels of SUNTGA40S are consistent with previously reported physiological responses to dietary intake of high PUFA containing oils. The present results provide evidence that SUNTGA40S is a safe source of arachidonic acid. Except during lactation when the intake in dams doubled, 5% Suntga40S in the diet was equivalent to an overall intake of approximately 3g/kg body weight/day in F(0) and F(1) animals.

Subchronic oral toxicity studies with alpha-cyclodextrin in rats.

The toxicity of alpha-cyclodextrin (alpha-CD), a cyclic polymer of six alpha-1,4-linked glucopyranosyl units with potential applications as a food ingredient, more specifically a water-soluble dietary fiber, was examined in a 4-week range finding study and a 13-week oral toxicity study in rats. In the 4-week study, the test substance was administered to groups of Bor:WISW(SPF;Cpb) rats at dietary levels of 0, 1, 5, and 15% (5 rats/sex/group). An additional group received a diet with 5% beta-CD. In the 13-week study, groups of Crl:(WI)WU Br rats received diets with 0, 1.5, 5, or 20% alpha-CD. An additional group received a diet with 20% lactose (20 rats/sex/group). Satellite groups of 10 rats/sex were attached to the control, 20% alpha-CD and 20% lactose group. Following the 13-week treatment period, these satellite groups were kept on a standard, cereal-based rodent diet for a 4-week recovery period. Parameters measured during the two studies included clinical signs, body weights, food and water intake, hematological and clinicochemical parameters, and organ weights as well as gross and histopathological observations at necropsy. In the 13-week study, ophthalmoscopic examinations as well as urine and feces analyses were also conducted. There were no treatment-related mortalities in either study. In the 4-week study, persistent diarrhea was the most prominent, treatment-related effect observed in the animals of the 15% alpha-CD group especially in the male animals. In association with this effect, food consumption and food conversion efficiency were decreased. In line with observations from studies with other low-digestible, yet fermentable carbohydrates, the weight of the full and empty cecum was increased significantly in the 5% alpha-CD, 5% beta-CD, and 15% alpha-CD group. The reduced relative liver weights (in males and females) and the significantly increased relative testes weight which were observed in the 15% alpha-CD group, were attributed to the impaired nutritional condition (due to diarrhea) and the reduced body weight of the animals of this group, respectively. Microscopic examination of the main organs did not reveal pathological alterations that could be attributed to the alpha-CD treatment. In the 13-week study, soft stool and infrequent mild diarrhea were observed only during the first 2-3 weeks in the 20% alpha-CD and 20% lactose group (mainly male animals). Accordingly, body weights were reduced in males of the 20% lactose group throughout the study and in the 20% alpha-CD group during the last week of the study. Food intakes were slightly increased in the 20% alpha-CD group and the food conversion efficiency, was significantly reduced in males, but not females, of the 20% alpha-CD and 20% lactose group. There were no treatment-related changes of hematological parameters. In line with similar observations from studies on other low-digestible carbohydrates, the urinary pH was decreased and urinary calcium levels increased in the 20% alpha-CD and 20% lactose group. Similarly, the fecal dry weight and nitrogen output was increased in these groups. At termination of the treatment, significantly in creased cecum weights (full and empty) were observed in the 5 and 20% alpha-CD groups and the 20% lactose group. The relative (not absolute) weight of the spleen was significantly increased in males of the 20% alpha-CD group. In the 20% lactose group, the relative weights of the spleen and liver (females) and the testes, brain, and adrenals (males) were significantly increased. The histopathological examination of these and all other organs and tissues did not reveal any abnormalities that could be attributed to the alpha-CD or lactose treatment. In conclusion, the ingestion of alpha-CD for 13-weeks at dietary levels of up to 20% (corresponding to intakes of 12.6 and 13.9 g/kg bodyweight/d in male and female rats, respectively) did not produce any signs of toxicity or adverse effects.

Subchronic (13-week) oral toxicity study of alpha-cyclodextrin in dogs.

The oral toxicity of alpha-cyclodextrin (alpha-CD) was examined in a 13-week feeding study in which groups of Beagle dogs received alpha-CD in the diet at concentrations of 0 (control), 5, 10, or 20% (4 dogs/sex/group). No treatment-related changes were noted in behavior or appearance of the dogs and no mortalities occurred. Diarrhea occurred in all alpha-CD groups. The incidence and severity of diarrhea increased with increasing dietary levels of alpha-CD and was more pronounced in males than females. Nonetheless, all dogs remained in good health and gained weight. Food intake was slightly increased and food efficiency was slightly decreased in the 20% alpha-CD group. However, these changes did not reach statistical significance. No treatment-related differences were observed with respect to ophthalmoscopic examinations, hematological parameters, clinicochemical analyses of the plasma, and semiquantitative urine analyses. Only the urinary pH was slightly below control levels in males (p > 0.05) and females (p < 0.05) of the 20% alpha-CD group. No abnormalities were seen at necropsy that could be attributed to the treatment. The organ weight data revealed cecal enlargement in the 10 and 20% alpha-CD groups (significant only in males). The relative weight of the colon was also slightly increased in the 10 and 20% alpha-CD groups (significant only in females of the 10% alpha-CD group). On microscopic examination, no treatment-related alterations were observed in any of the various organs and tissues. In conclusion, transient diarrhoea, enlargement of the cecum and colon and a slightly increased acidity of the urine were the only treatment-related effects. These changes are well-known physiological responses to the presence of high amounts of not digested, fermentable carbohydrates in the lower gut. They are known to be reversible on cessation of the treatment and are not associated with histological alterations of the intestinal tissues. It is concluded, therefore, that the high dose level, at which the male and female dogs consumed about 9.8 and 10.4 g alpha-CD/kg bw/d, respectively, is the NOAEL of this 13-week toxicity study.

Toxicity and carcinogenicity of acidogenic or alkalogenic diets in rats; effects of feeding NH(4)Cl, KHCO(3) or KCl.

The effects of diet-induced acid-base disturbances were examined in 4-week, 13-week and 18-month toxicity studies, and in a 30-month carcinogenicity study. Rats were fed a natural ingredient diet (controls), supplemented with 2% or 4% KHCO(3) (base-forming diets), or with 1% or 2.1% NH(4)Cl (acid-forming diets). Additional controls were fed 3% KCl (neutral diet providing K(+) and Cl(-) in amounts equimolar to those in the 4% KHCO(3) diet and the 2.1% NH(4)Cl diet, respectively). NH(4)Cl induced the expected metabolic acidosis, as shown by decreased base excess in blood, decreased urinary pH and increased urinary net acid excretion. KHCO(3) induced the opposite effects. KCl did not affect the acid-base balance. Clinical condition and death rate were not affected. The feeding of high levels of each salt resulted in growth retardation and increased water intake and urinary volume. Plasma potassium and urinary potassium excretion were increased with KHCO(3) and KCl. Plasma chloride was increased with NH(4)Cl, but not with KCl. Urinary calcium and phosphate excretion were increased with NH(4)Cl, but there were no indications that bone minerals were involved (weight, calcium content and fat free solid of the femur were not affected). Standard haematological and clinical chemistry parameters were not affected. Kidney weights were increased with 2.1% NH(4)Cl. Hypertrophy of the adrenal zona glomerulosa occurred with KHCO(3), KCl and NH(4)Cl, due to chronic stimulation of the adrenal cortex by either K(+) or by NH(4)Cl-induced acidosis. An early onset (from week 13) of oncocytic tubules was noted in the kidneys of rats fed KHCO(3) and, after 30 months, the incidence of this lesion was much higher than the background incidence in ageing controls. No progression to oncocytomas was noted. KCl showed only slight effects on the early onset of oncocytic tubules (from 18 months). In contrast, the severity of nephrosis and the incidence of oncocytic tubules were decreased with 2.1% NH(4)Cl, suggesting a protective effect of acidosis. The feeding of KHCO(3) resulted in hyperplasia, papillomas and carcinomas of the urinary bladder. With KCl only a slight increase in proliferative urothelial lesions was noted. Apart from these (pre-)neoplastic lesions in the urinary bladder there were no treatment-related differences in tumour response among the groups. We concluded that most of the observed changes represent physiological adaptations to the feeding of acid- or base-forming salts. Remarkable effects noted with KHCO(3), and to a far lesser extent with KCl, consisted of renal oncocytic tubules and (pre-)neoplastic lesions of the urinary bladder epithelium. NH(4)Cl-induced chronic metabolic acidosis was not associated with dissolution of alkaline bone salts in rats. Finally, a protective effect of chronic acidosis on tumour development was not found.

Isomaltulose (Palatinose): a review of biological and toxicological studies.

Isomaltulose is a natural occurring disaccharide composed of alpha-1,6-linked glucose and fructose. Commercial isomaltulose is produced from sucrose by enzymatic rearrangement and has been used as a sugar in Japan since 1985. It is particularly suitable as a non-cariogenic sucrose replacement and is favorable in products for diabetics and prediabetic dispositions. In vivo studies with rats and pigs indicate that isomaltulose is completely hydrolyzed and absorbed in the small intestine. This is supported by in vitro studies showing that intestinal disaccharidases from various species (including man) can hydrolyze isomaltulose. The rate of hydrolysis, however, is very slow compared with sucrose and maltose. Thus, blood glucose and insulin levels in humans after oral administration rise slower and reach lower maxima than after sucrose administration. After absorption, fructose and glucose are metabolized as typical for these monosaccharides. From intravenous studies it can be assumed that any systemic isomaltulose would be hydrolyzed as well, or excreted in urine. In several subchronic toxicity studies, the administration of large doses (up to 7.0 and 8.1 g/kg body weight/day in male and female rats, respectively) of isomaltulose, did not result in adverse effects. Isomaltulose induced neither embryotoxic or teratogenic effects in rat foetuses, nor maternal toxicity at levels up to 7 g/kg body weight/day. Isomaltulose was non-mutagenic in the Ames test. As hydrolysis in the small intestine is complete, even high levels of isomaltulose are well tolerated in animals and humans. In studies with healthy as well as diabetic subjects high doses up to 50 g were tolerated without signs of intestinal discomfort. On the basis of the data reviewed it is concluded that the use of isomaltulose as an alternative sugar is as safe as the use of other digestible sugars consisting of glucose and fructose.

13-Week oral toxicity study with isomaltulose (Palatinose) in rats.

The potential subchronic oral toxicity of isomaltulose (Palatinose) was examined by administering this substance in the diet to groups of 20 male and 20 female Wistar rats at levels of 0, 2.5, 5 and 10% for 13 consecutive weeks. Daily clinical observations, body weight, food conversion efficiency, food and water consumption were not affected at any stage of the study. Ophthalmoscopy, haematology, clinical chemistry, urinalysis, organ weights, gross and histopathological examination, neurobehavioural observations, motor activity assessment and the results of an immunotoxicity screen did not reveal any abnormalities related to the ingestion of the test substance. In conclusion, the administration of isomaltulose at dietary levels up to 10% for 13 consecutive weeks was well tolerated without any signs of toxicity. The overall intake at this level corresponded to 7.0 and 8.1 g/kg body weight/day in male and female rats, respectively.