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.

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.

Evaluation of a subchronic (13-week) oral toxicity study, preceded by an in utero exposure phase, with arachidonic acid oil derived from Mortierella alpina in rats.

Arachidonic acid oil (ARA-oil) derived from the fungus Mortierella alpina for use in infant nutrition was tested in a subchronic (13-week) oral toxicity study in rats, preceded by an in utero exposure phase. The ARA-oil was administered as admixture to the rodent diet at dose levels of 3000 ppm, 15,000 ppm and 75,000 ppm. An additional high-dose group received 75,000 ppm ARA-oil in combination with 55,000 ppm fish oil containing docosahexaenoic acid (DHA), at a ratio of ARA to DHA, comparable to the ratio in mother's milk of 2:1. The total levels of fat in each diet were kept constant by adding the appropriate amounts of corn oil. A concurrent control group received 130,000 ppm corn oil in the diet. An additional carrier control group was fed unsupplemented rodent diet. Administration of the test substances from 4 weeks prior to mating, throughout mating, gestation, lactation of parental (F(0)) animals and weaning of the F(1) pups did not affect fertility or reproductive performance, nor the general condition of pups, viability, sex ratio or number of pups. Pup weight gain in the ARA/DHA-oil group was lower than the controls administered equal amounts of corn oil. In the subsequent subchronic study survival, clinical signs, body weight gain and food consumption were not adversely affected by the test substances. Ophthalmoscopic examination did not reveal any treatment-related changes. There were no treatment-related effects observed up to dietary test substance concentrations of 15,000 ppm. The following statistically significant differences were found in the ARA high-dose group and /or in the ARA/DHA group compared to the corn oil control group: decreased alkaline phosphatase activity, decreases in cholesterol, triglycerides and phospholipids concentrations, increased creatinine and urea concentrations. Furthermore, these groups showed increased adrenal, spleen and liver weights. The incidence of hepatocellular vacuolation was increased in females of the ARA high-dose group and the ARA/DHA group. Oil droplets were observed in the mesenteric lymph nodes and in the intestinal villi in the ARA high-dose group and the ARA/DHA group. In addition, lipogranulomas were observed in the mesenteric lymph nodes in these groups. The observed changes in the high-dose groups may be effects of the high intake of high-fat levels, rather than specific effects of the ARA-oil. The no-observed-effect level in this study was placed at 15,000 ppm ARA-oil. This level is equivalent to approximately 970mg ARA-oil/kg body weight/day.

Effect of urinary pH on the progression of urinary bladder tumours.

Systemic alkalosis has been postulated to enhance tumorigenesis, whereas systemic acidosis has been implicated to exert a favourable influence on tumour control and regression. In the present study the urinary pH was influenced by feeding acid-forming or base-forming diets, and the effect of alkaline or acid urine on the early and late progression phase of urinary bladder carcinogenicity was investigated in male Wistar rats. Bladder lesions were initiated by N-butyl-N-(4-hydroxybutyl) nitrosamine (0.05% BBN in the drinking water during 4 weeks) and promoted by sodium bicarbonate (3.4% NaHCO3 in the diet during 15 or 25 weeks). After short- (15 week) and more long-term (25 week) promotion with NaHCO3, groups of 20 rats were fed a diet containing the acidifying salt ammonium chloride (2.1% NH4Cl) or the control diet. All surviving rats were killed after a total study duration of 52 weeks. Additional control groups were, after initiation, fed diets containing NaHCO3 and killed after 15 wk or 25 wk of promotion, or at the end of the study. In rats fed diets with added salts, water intake and the amount of urine produced were increased and the urinary density was decreased compared to rats fed control diet. During NaHCO3 feeding, urinary pH and sodium concentration were increased. During NH4Cl feeding, urinary pH was decreased and urinary chloride and calcium concentrations were increased. Initiation by BBN followed by treatment with NaHCO3 caused a high incidence of papillary/nodular hyperplasia, papillomas and carcinomas of the bladder epithelium. These lesions progressed with time or longer duration of NaHCO3 promotion. A tumour protective effect of urinary acidification by NH4Cl was not found. In fact, both acidification and prolonged alkalinization tended to aggravate the malignancy of bladder carcinomas.

Preliminary safety assessment of an arachidonic acid-enriched oil derived from Mortierella alpina: summary of toxicological data.

An arachidonic acid-enriched oil (AA-oil), derived from Mortierella alpina was subjected to a programme of studies to establish its preliminary safety for use in infant nutrition. This was addressed at two levels: (1) HPLC analysis of metabolites produced by the production strains at various conditions, and (2) an evaluation of the toxicity of the final product. The following studies were carried out on the AA-oil: gene mutation assays in bacteria and mammalian cells in vitro; chromosome aberration assays both in vitro and in vivo and acute and subacute (4-wk) oral toxicity in the rat. No known mycotoxins were produced by the production strains under the conditions tested. Further, the oil did not show mutagenic or clastogenic activity and the acute oral toxicity, expressed as the LD50 value, exceeded 20 ml/kg body weight, that is, 18.2 g/kg body weight. In the subacute oral toxicity study the AA-oil was tested as such and in combination with a docosahexaenoic-enriched oil (DHA-oil) derived from fish oil at a ratio of 2:1 (AA:DHA). This was done because high dose levels of AA may result in adverse effects; DHA can compensate for these effects. Furthermore, human milk contains both AA and DHA at a ratio of AA:DHA of 2 to 3:1. No obvious signs of toxicity were observed. Levels of phospholipids and triglycerides tended to be decreased in the highest dose groups. The no-observed-adverse-effect level of the AA-oil in the subacute 4-wk toxicity study was placed at the highest levels tested, namely 3000 mg AA-oil/kg body weight/day as such and in the combination of 3000 mg AA-oil and 1500 mg DHA-oil/kg body weight/day. This corresponds to an intake of 1000 mg AA/kg body weight/day, which represents approximately 37 times the infant intake of AA in human milk.

Chronic toxicity and carcinogenicity study of erythritol in rats.

The potential toxicity and carcinogenicity of erythritol, a low-calorie sugar substitute, were examined in Wistar Crl:(WI) WU BR rats. Groups of 50 rats of each sex consumed diets with 0, 2, 5, or 10% erythritol, or 10% mannitol, for a period of 104-107 weeks. To each of these main groups, two satellite groups of 20 males each were attached for interim kills after 52 and 78 weeks of treatment. At start of the study, the rats were 5-6 weeks old. The average intakes of erythritol in the 2, 5, and 10% groups were 0.9, 2.2, and 4.6 g/kg body wt/day for males and 1.0, 2.6, and 5.4 g/kg body wt/day for females, respectively. Mannitol intakes were 4.4 and 5.2 g/kg body wt/day in males and females, respectively. All treatments were well tolerated without diarrhea or other side effects. Body weights were significantly below control levels during most of the study in males of the 5% erythritol group and in males and females of the 10% erythritol and 10% mannitol groups. Survival of the animals was not adversely affected by the treatments. Hematological and clinicochemical examinations did not reveal noticeable changes which could be attributed to treatment. Analysis of urine samples collected during five 48-hr periods, from rats of the satellite groups in Weeks 26, 42, 50, and 78 and from rats of the main groups in Week 102, showed that about 60% of ingested erythritol was excreted unchanged. The urine volumes increased with increasing dietary erythritol levels. In line with previous observations on other polyols, erythritol and mannitol ingestion led to an increased excretion of urinary calcium and citrate. The urinary excretions of sodium, potassium, phosphate, N-acetylglucosaminidase (NAG), gamma-glutamyltransferase (GGT), low-molecular-weight protein (LMP), and total protein (TP) were slightly elevated in the 10% erythritol group. Increased GGT and NAG excretions also were seen occasionally at the 5% dose. Significantly increased relative cecum weights were seen in rats of either sex in the 10% mannitol and, somewhat less pronounced, 10% erythritol groups. Some cecal enlargement also was seen in the 5% erythritol group. The relative weight of the kidneys was highest in the 10% erythritol group, the difference from controls reaching statistical significance at interim kills (males) and termination (females). Except for more frequent pelvic nephrocalcinosis in female rats of all erythritol dose groups, the histopathological examinations did not reveal any nonneoplastic, preneoplastic, or neoplastic changes that could be attributed to the ingestion of erythritol. In male and female rats of the 10% mannitol group, pelvic nephrocalcinosis, which in females was associated occasionally with pelvic hyperplasia, was the only remarkable finding. The incidence and progression of nephrosis, which is commonly seen in aging rats of this strain, were not influenced by the treatments. In the absence of morphological alterations in the kidneys or other signs of nephrotoxicity, the increased excretions of NAG, GGT, LMP, and TP are regarded as innocuous, functional sequelae of the renal elimination of erythritol. In conclusion, the toxicological profile of erythritol in rats resembles that of other polyols in several respects. Except for nephrocalcinosis, which is commonly seen in polyol-fed rats, no other treatment-related, morphological changes were observed in the kidneys. Evidence for a tumor-inducing or tumor-promoting effect of erythritol was not seen.

Effects of a dietary load of acid or base on changes induced by lactose in rats.

Feeding lactose or other slowly digestible carbohydrates to adult mammals may induce a variety of effects including hyperplasia and neoplasia. The most fundamental effect probably is the increased production in the large intestine of short-chain fatty acids (SCFA) resulting from increased fermentation of carbohydrate residues. To find out whether the increased production of these acidic compounds is involved in the induction of certain alterations caused by low-digestibility carbohydrates, the modifying effects of an acidifying (NH4Cl) or an alkalizing (KHCO3) diet supplement on lactose-induced changes in rats were studied. Three groups of 50 rats per sex were fed a 20% lactose diet unsupplemented or supplemented with 1% NH4Cl or 2% KHCO3, for at most 2.5 yr. One control group was fed the basal diet which contained wheat starch instead of lactose. Feeding lactose resulted in wet faecal pellets, reduced pH of the faeces, higher intake of food and water, lower body weights, increased caecal weights and fewer deaths. These effects were not significantly modified by NH4Cl or KHCO3. Feeding lactose increased urinary calcium levels, the effect being enhanced by NH4Cl and reduced by KHCO3. Lactose also tended to increase blood values of alkaline phosphatase and to decrease those for bicarbonate and base excess. These tendencies were generally more marked with NH4Cl, and less marked or absent with KHCO3. In addition, rats fed lactose showed decreased severity of nephrosis, increased mineralization and hyperplasia of the renal pelvic epithelium, and relatively high incidences of Leydig cell hyperplasia and neoplasia. NH4Cl supplementation was associated with a relatively small number of single and multiple tumours, with decreased incidences of hyperplasia and mineralization of the renal pelvis epithelium and with a markedly reduced incidence of proliferative changes in the adrenal medulla. With the KHCO3 supplement the incidences of Leydig cell proliferation and of bladder tumours were relatively high. These findings, in particular the differences between the diet groups in urinary calcium levels and possibly also the variations in blood levels of alkaline phosphatase, bicarbonate and base excess, suggest that the acidic end products of carbohydrate fermentation (SCFA) act as an acid load on the body.

The role of alkalizing and neutral potassium salts in urinary bladder carcinogenesis in rats.

Using an initiation-promotion rat model, we have previously shown that the alkalizing salt KHCO3 is a strong and the neutral salt KCl a weak promoter of urinary bladder carcinogenesis. We have now studied the effects of these salts on rat urinary bladder epithelium without prior exposure to a bladder tumour initiator. In four studies ranging in duration from 4 to 130 weeks, (equimolar) amounts of K+ were administered in the diet to male and female rats (85 rats/sex/group) as KHCO3 or KCl. Comparable increases in urinary volume and potassium levels were found with both KHCO3 and KCl, but only KHCO3 induced an elevated urinary pH. The feeding of KHCO3 resulted in simple epithelial hyperplasia and, after prolonged administration, in papillary/nodular hyperplasia, papillomas and transitional cell carcinomas of the urinary bladder. With KCl, only a slight increase in proliferative urothelial lesions was found; one male showed papillary hyperplasia and one female exhibited nodular hyperplasia and a papilloma. Our results allow the conclusion that KHCO3, a strong promoter of bladder carcinogenesis, is capable of inducing urinary bladder cancer in rats without prior application of an initiator, whereas KCl, a weak tumour promoter, induced only a few (pre)neoplastic lesions.

Effect of coffee drinking on cell proliferation in rat urinary bladder epithelium.

A possible effect of freshly brewed drip coffee on urinary bladder carcinogenesis was investigated in male Wistar rats using cell proliferation in urinary bladder epithelium as the indicator of tumour promotion. Male rats were given either undiluted coffee brew (100% coffee), coffee diluted 10 times (10% coffee) or tap water (controls), as their only source of drinking fluid for 2 or 6 wk. Uracil, known to induce cell proliferation in urinary bladder epithelium, was included in the study as a positive control. In rats receiving 100% coffee, body weights, liquid intake and urinary volume were decreased. Neither histopathological examination of urinary bladder tissue nor the bromodeoxyuridine labelling index revealed biologically significant differences between rats receiving coffee and the tap water controls. Uracil increased the labelling index and induced hyperplasia of the urinary bladder epithelium, as expected. It was concluded that these results produced no evidence that drinking coffee predisposes to tumour development in the urinary bladder.

Effects of urinary potassium and sodium ion concentrations and pH on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in rats.

The promoting activities of low and high sodium or potassium ion concentrations, under conditions of neutral as well as elevated urinary pH, in urinary bladder carcinogenesis, were investigated in rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Male Wistar rats were given 0.05% BBN in their drinking water for 4 weeks and then treated for 32 weeks with either control diet (group 1) or this diet supplemented with equimolar amounts of the following minerals: 2.34% NaCl (group 2), 2.98% KCl (group 3), 3.36% NaHCO3 (group 4), 1.68% NaHCO3 + 2% KHCO3 (group 5), or 4% KHCO3 (group 6). The alkalizing salts NaHCO3 and KHCO3 induced comparable increases in urinary pH and elevated urinary sodium or potassium ion concentrations respectively. The combination of NaHCO3 + KHCO3 similarly caused an elevation of the urinary pH and less increased sodium and potassium ion concentrations. In the groups fed NaHCO3 and KHCO3 either alone or in combination, the incidences of papillary/nodular hyperplasia, papillomas and carcinomas in the urinary bladder had increased as compared to controls. NaCl and KCl also induced high urinary sodium or potassium ion concentrations without alteration of urinary pH. This was accompanied by increased incidences of simple hyperplasia, papillary/nodular hyperplasia, and/or papillomas but no carcinomas. The present results indicate that the potassium ion is as potent as the sodium ion in promoting urinary bladder carcinogenesis under conditions of elevated urinary pH, and that both the sodium and potassium ions may exert weak promoting activity under conditions of neutral urinary pH.