Safety assessment of gamma-cyclodextrin.

Gamma-cyclodextrin (gamma-CD) is a cyclic alpha-(1,4)-linked oligosaccharide consisting of eight glucose molecules. Like other cyclodextrins, gamma-CD can form inclusion complexes with a variety of organic molecules because the inner side of the torus-like molecule is less polar than the outer side. In foods, gamma-CD may be used as a carrier for flavors, vitamins, polyunsaturated fatty acids, and other ingredients. It also has useful properties as a stabilizer in different food systems. The daily intake from all its intended uses in food at highest feasible concentrations has been estimated at 4.1g/person/day for consumers of gamma-CD containing foods. The present review summarizes the safety data of gamma-CD. The toxicity studies consist of standard genotoxicity tests, subchronic rat studies with oral and intravenous administration of gamma-CD for up to 3 months, a subchronic (3-month) toxicity study in dogs, a (1-year) oral toxicity study in rats, and embryotoxicity/teratogenicity studies in rats and rabbits. In the studies with oral administration, gamma-CD was given at dietary concentrations of up to 20%. All these studies demonstrated that gamma-CD is well tolerated and elicits no toxicological effects. Metabolic studies in rats showed that gamma-CD is rapidly and essentially completely digested by salivary and pancreatic amylase. Therefore, the metabolism of gamma-CD closely resembles that of starch and linear dextrins. A human study with ingestion of single doses of 8 g gamma-CD or 8 g maltodextrin did not reveal a difference in gastrointestinal tolerance of these two products. An interaction of ingested gamma-CD with the absorption of fat-soluble vitamins or other lipophilic nutrients is not to be expected because the formation of inclusion complexes is a reversible process, gamma-CD is readily digested in the small intestine, and studies with beta-CD, a non-digestible cyclodextrin, have shown that the bioavailability of vitamins (A, D, and E) is not impaired. On basis of these studies it is concluded that gamma-CD is generally recognized as safe (GRAS) for its intended uses in food.

The FEMA GRAS assessment of pyrazine derivatives used as flavor ingredients. Flavor and Extract Manufacturers Association.

This is the fifth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually taking into account the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of pyrazine derivatives as flavoring ingredients is evaluated.

Treatment with soybean-derived Bowman Birk inhibitor increases serum prostate-specific antigen concentration while suppressing growth of human prostate cancer xenografts in nude mice.

BACKGROUND: Bowman Birk inhibitor (BBI) is an anticarcinogenic serine protease inhibitor that may inhibit the protease activity of prostate-specific antigen (PSA) and the growth of human prostate cancer xenografts in nude mice. METHODS: Human prostate cancer xenografts were established by implanting LNCaP cells into the prostate glands of NCRNU-M athymic nude mice. The animals with established tumors were maintained on a control diet or diets supplemented with 1% BBI or 1%, 2%, or 3% BBI concentrate (BBIC) for 6 weeks. The serum PSA concentrations were determined before and after the BBI or BBIC treatment period. The final tumor loads were determined at autopsy. RESULTS: Treatment with BBI or BBIC decreased the final tumor load and increased the tumor doubling time and PSA density in the nude mice bearing human prostate cancer xenografts. CONCLUSIONS: BBI and/or BBIC could be useful for prostate cancer treatment.

The FEMA GRAS assessment of trans-anethole used as a flavouring substance. Flavour and Extract Manufacturer's Association.

This publication is the fourth in a series of safety evaluations performed by the Expert Panel of the Flavour and Extract Manufacturers' Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavouring substances under conditions of intended use. In this review, scientific data relevant to the safety evaluation of trans-anethole (i.e. 4-methoxypropenylbenzene) as a flavouring substance is critically evaluated by the FEMA Expert Panel. The evaluation uses a mechanism-based approach in which production of the hepatotoxic metabolite anethole epoxide (AE) is used to interpret the pathological changes observed in different species and sexes of laboratory rodents in chronic and subchronic dietary studies. Female Sprague Dawley rats metabolize more trans-anethole to AE than mice or humans and, therefore, are the most conservative model for evaluating the potential for AE-induced hepatotoxicity in humans exposed to trans-anethole from use as a flavouring substance. At low levels of exposure, trans-anethole is efficiently detoxicated in rodents and humans primarily by O-demethylation and omega-oxidation, respectively, while epoxidation is only a minor pathway. At high dose levels in rats, particularly females, a metabolic shift occurs resulting in increased epoxidation and formation of AE. Lower activity of the "fast" acting detoxication enzyme epoxide hydrolase in the female is associated with more pronounced hepatotoxicity compared to that in the male. The continuous intake of high dose levels of trans-anethole (i.e. cumulative exposure) has been shown in dietary studies to induce a continuum of cytotoxicity, cell necrosis and cell proliferation. In chronic dietary studies in rats, hepatotoxicity was observed when the estimated daily hepatic production of AE exceeded 30 mg AE/kg body weight. In female rats, chronic hepatotoxicity and a low incidence of liver tumours were reported at a dietary intake of 550 mg trans-anethole/kg body weight/day. Under these conditions, daily hepatic production of AE exceeded 120 mg/kg body weight. Additionally, neither trans-anethole nor AE show any evidence of genotoxicity. Therefore, the weight of evidence supports the conclusion that hepatocarcinogenic effects in the female rat occur via a non-genotoxic mechanism and are secondary to hepatotoxicity caused by continuous exposure to high hepatocellular concentrations of AE. trans-Anethole was reaffirmed as GRAS (GRASr) based on (1) its low level of flavour intake (54 microg/kg body weight/day); (2) its metabolic detoxication pathway in humans at levels of exposure from use as a flavouring substance; (3) the lack of mutagenic or genotoxic potential; (4) the NOAEL of 120 mg trans-anethole/kg body weight/day in the female rat reported in a 2 + -year study which produces a level of AE (i.e. 22 mg AE/kg body weight/day) at least 10,000 times the level (0.002 mg AE/kg body weight day) produced from the intake of trans-anethole from use as a flavouring substance; and (5) the conclusion that a slight increase in the incidence of hepatocellular tumours in the high dose group (550 mg trans-anethole/kg body weight/day) of female rats was the only significant neoplastic finding in a 2+ -year dietary study. This finding is concluded to be secondary to hepatotoxicity induced by high hepatocellular concentrations of AE generated under conditions of the study. Because trans-anethole undergoes efficient metabolic detoxication in humans at low levels of exposure, the neoplastic effects in rats associated with dose-dependent hepatotoxicity are not indicative of any significant risk to human health from the use of trans-anethole as a flavouring substance.

Bowman-Birk inhibitor concentrate reduces colon inflammation in mice with dextran sulfate sodium-induced ulcerative colitis.

Bowman-Birk inhibitor concentrate (BBIC) is a soybean extract enriched in the Bowman-Birk inhibitor, a protein protease inhibitor. The Bowman-Birk inhibitor can inhibit proteases released from inflammation mediating cells and suppress superoxide anion radical secretion from immunocytes. This study investigates the ability of Bowman-Birk inhibitor concentrate to inhibit colon inflammation in the dextran sulfate sodium model of ulcerative colitis, an inflammatory bowel disease. When compared to mice on a standard diet, mice given food supplemented with 0.5% BBIC during and after dextran sulfate sodium treatment showed suppression of three of four scored histopathological inflammation criteria (P < 0.01), total histopathological score (P < 0.01), a 15% lower mortality rate (P < 0.01), and a delayed onset of mortality. We conclude that dietary Bowman-Birk inhibitor concentrate can beneficially affect dextran sulfate sodium-treated mice and may be useful in the treatment of human inflammatory bowel diseases, particularly ulcerative colitis.

The FEMA GRAS assessment of furfural used as a flavour ingredient. Flavor and Extract Manufacturers' Association.

The Expert Panel of the Flavor and Extract Manufacturers' Association (FEMA) has assessed the safety of furfural for its continued use as a flavour ingredient. The safety assessment takes into account the current scientific information on exposure, metabolism, pharmacokinetics, toxicology, carcinogenicity and genotoxicity. Furfural was reaffirmed as GRAS (GRASr) as a flavour ingredient under conditions of intended use based on: (1) its mode of metabolic detoxication in humans; (2) its low level of flavour use compared with higher intake levels as a naturally occurring component of food; (3) the safety factor calculated from results of subchronic and chronic studies, (4) the lack of reactivity with DNA; and (5) the conclusion that the only statistically significant finding in the 2-year NTP bioassays, an increased incidence of hepatocellular adenomas and carcinomas in the high-dose group of male mice, was secondary to pronounced hepatotoxicity. Taken together, these data do not indicate any risk to human health under conditions of use as a flavour ingredient. This evidence of safety is supported by the occurrence of furfural as a natural component of traditional foods, at concentrations in the diet resulting in a 'natural intake' that is at least 100 times higher than the intake of furfural from use as a flavour ingredient.

Esophageal carcinogenesis in the rat: zinc deficiency and alcohol effects on tumor induction.

Sprague-Dawley male rats were fed zinc-deficient or supplemented diets for 2 weeks, administered a carcinogenic dose of methylbenzylnitrosamine and observed over 20 or more weeks for effects of superimposing excess zinc or alcohol on development of esophageal tumors. In three separate experiments it was shown that (1) excess zinc offered no protection, (2) switching diets during or after carcinogen exposure pointed toward involvement of zinc in both initiation and promotion, (3) neither ethanol nor 3-methyl butanol alone affected tumorigenesis but the two combined and superimposed on a zinc deficiency resulted in a significant enhancement of neoplasia. In one group of rats fed the zinc-deficient diet only, with no carcinogen, 4 rats developed neoplasms, one of which was malignant. Cell proliferation, an integral component of zinc deficiency, appears to be an important contribution to tumor induction in this model.

Esophageal carcinogenesis in the rat: zinc deficiency, DNA methylation and alkyltransferase activity.

Rats fed zinc-deficient diets and given an esophageal carcinogen, methylbenzylnitrosamine, develop tumors in greater incidence and with increased frequency compared to zinc-supplemented rats. This greater susceptibility is associated with a unique esophageal lesion, parakeratosis, with markedly increased epithelial necrosis and cell proliferation. Recent studies have shown that the increased susceptibility to tumorigenesis was further associated with a number of metabolic and biochemical alterations including increased binding of the carcinogen to DNA, shifts in O6-methylguanine (O6MeG)/7-methylguanine ratios and suggestions that the promutagen O6MeG lesion is not repaired effectively in the zinc-deficient esophagus; the latter was not reflected in the amount of O6-methyltransferase activity, however. The weight of evidence supports a presumption that zinc deficiency interferes with normal DNA repair mechanisms, the nature of which is not clear. An interesting additional finding was that zinc deficiency alone was associated with esophageal tumor induction, without carcinogen, which indicates that genetic material in the zinc-deficient esophageal epithelium is damaged sufficiently, without further chemical injury, to result in loss of control of cell proliferation. Manipulation of the time of exposure to zinc deficiency and carcinogen exposure defined the initiation period as most affected by the deficiency. Furthermore, reduced carcinogen exposure (and less toxicity), along with zinc deficiency, permits development of more tumors of the endophytic type, the form more relevant to human esophageal tumors. The groundwork, as described in this paper, has now been prepared to directly address the latter issue, endophytic tumors, and the putative relation of zinc deficiency to esophageal cancer in human populations.

Diet: the neglected variable in chemical safety evaluations.

The objectives of this review are to alert the community of toxicologic pathologists to the types and significance of various diets fed to experimental animals used in safety evaluations of drugs and other chemicals, to acquaint investigators with dietary nutrients and contaminants of significance to research animals, to review some of the consequences of improper or inadequate diets on results of safety evaluations, and, finally, to offer a brief introduction to a highly promising new digital specimen technology for evaluating histopathology of tissues from animals used in safety evaluations with the aid of computers. Results that form the basis for this review have accumulated for more than 4 decades during research conducted during appointments at the University of Missouri, Columbia; Auburn University; Massachusetts Institute of Technology; and Boston University School of Medicine. The methods used in these studies have generally been state-of-the-art at the time the studies were conducted and involved an integration of techniques and methods used in fundamental nutrition, toxicology, and experimental pathology. Some of these investigations have shown that contaminants, naturally occurring and man-made, intentional and nonintentional, do occur in commercial animal diets; that essential nutrients, including total calories, protein and fat, can be provided in less or more than adequate amounts for the species of animal used in safety evaluations; and that such variables can influence responses to chemicals through effects on xenobiotic metabolism, immunocompetence, secondary diseases, and other factors. Under such conditions, serious differences in final interpretation of human risk for a drug or other chemical can contribute to failure to gain approval for a potentially valuable therapeutic agent. This review discusses the significance of various dietary components with examples of their effects on response of the animal to chemicals and demonstrates why nutrients, new technologies for assessing their effects, and other components in the diet should be accorded greater consideration by investigators and regulators.

The FEMA GRAS assessment of alicyclic substances used as flavour ingredients.

For over 35 years, an independent panel of expert scientists has served as the primary body for evaluating the safety of flavour ingredients. This group, the Expert Panel of the Flavor and Extract Manufacturers' Association (FEMA), has achieved international recognition from the flavour industry, government regulatory bodies including the Food and Drug Administration, and the toxicology community for its unique contributions. To date, the Expert Panel has evaluated the safety of more than 1700 flavour ingredients and determined the vast majority to be "generally recognized as safe" (GRAS). Elements that are fundamental to the safety evaluation of flavour ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavour ingredients are evaluated individually taking into account the available scientific information on the group of structurally related substances. The elements of the GRAS assessment program as they have been applied by the Expert Panel to the group of 119 alicyclic substances used as flavour ingredients, and the relevant scientific data which provide the basis for the GRAS status of these substances, are described herein.

Suppression of carcinogenesis in the intestines of min mice by the soybean-derived Bowman-Birk inhibitor.

We have performed experiments to determine whether the soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), has the ability to affect intestinal carcinogenesis in Min mice. Min mice have an autosomally dominantly inherited predisposition to multiple intestinal neoplasms and are known to have a very high spontaneous rate of tumor development in both the small intestine and colon. BBI was administered in the diet as BBI Concentrate (BBIC), the form of BBI which is currently being evaluated in human trials as a cancer chemopreventive agent. We observed that 0.5% dietary BBIC led to a 42-50% reduction in the number of tumors/mouse in the small intestine and colon and a 41% reduction of tumorigenesis in the colon when the data are analyzed in terms of the fraction of mice bearing tumors. Thus, tumor-development in both the small intestine and colon of Min mice can be significantly suppressed by BBIC, despite the fact that the animals carry a predisposing mutation that leads to a markedly increased intestinal tumor incidence and mortality rate.

Zinc: health effects and research priorities for the 1990s.

This review critically summarizes the literature on the spectrum of health effects of zinc status, ranging from symptoms of zinc deficiency to excess exposure. Studies on zinc intake are reviewed in relation to optimum requirements as a function of age and sex. Current knowledge on the biochemical properties of zinc which are critical to the essential role of this metal in biological systems is summarized. Dietary and physiological factors influencing the bioavailability and utilization of zinc are considered with special attention to interactions with iron and copper status. The effects of zinc deficiency and toxicity are reviewed with respect to specific organs, immunological and reproductive function, and genotoxicity and carcinogenicity. Finally, key questions are identified where research is needed, such as the risks to human health of altered environmental distribution of zinc, assessment of zinc status in humans, effects of zinc status in relation to other essential metals on immune function, reproduction, neurological function, and the cardiovascular system, and mechanistic studies to further elucidate the biological effects of zinc at the molecular level.

Preparation and production of a cancer chemopreventive agent, Bowman-Birk inhibitor concentrate.

We describe our studies to produce an extract of soybeans with anticarcinogenic activity that we believe will be useful as a human cancer chemopreventive agent for several different organs. The anticarcinogenic activity of the extract is thought to be due to chymotrypsin inhibitor activity, which is due to the Bowman-Birk protease inhibitor (BBI) present in the extract, termed BBI concentrate (BBIC). We describe the contents of BBIC, the ability of BBIC to inhibit malignant transformation in vitro in terms of its chymotrypsin inhibitor activity, and the results of long-term toxicity studies in which mice and rats were exposed to high levels of BBIC for long periods of time.

Effects of various preparations of dietary protease inhibitors on oral carcinogenesis in hamsters induced by DMBA.

We studied the ability of a soybean extract containing the Bowman-Birk protease inhibitor (BBI), referred to as BBI concentrate (BBIC), purified BBI (PBBI), and the chymotrypsin inhibitor from potatoes to suppress oral carcinogenesis in hamsters induced by 7,12-dimethyl-benz[a]anthracene (DMBA). Application of 1% solutions of BBIC and PBBI five times per week to DMBA-treated hamster cheek pouches were highly effective in suppressing oral carcinogenesis, whereas a 1% solution of the chymotrypsin inhibitor from potatoes did not lead to a significant suppression of carcinogenesis. BBIC and PBBI suppressed carcinogenesis at concentrations ranging from 1% to 0.01% and were equally effective when given as a 1% solution five times per week, three times per week, or once per week. A 1% solution of BBIC suppressed oral carcinogenesis when given at the following times during the assay period: 0-180, 0-90, 14-90, and 45-135 days. Thus, protease inhibitor treatment could be started as late as 45 days after the beginning of the carcinogen exposure and have an irreversible suppressive effect on the carcinogenic process.

Dietary lipids and immune function.

The influence of dietary lipids on immune function has come under serious study only within the past two decades. It is clear from whole-animal studies that obesity and consumption of diets high in fat, particularly unsaturated fat, depress immunocompetence and enhance risk for serious infectious disease and cancer. In vitro systems, cell cultures and the tools of molecular biology are moving nutrition and immunology closer together with promise of significant benefits.

Inhibition of N-nitrosomethylbenzylamine-induced esophageal neoplasms by the Bowman-Birk protease inhibitor.

Model systems in which carcinogenesis by given agents can be prevented or reduced offer a means of gaining insight into the mechanism(s) of action of carcinogens and the feasibility of chemoprevention in humans. In the current study, the ability of the soy-bean derived Bowman-Birk protease (BBI) to suppress esophageal carcinogenesis induced by N-nitrosomethylbenylamine (NMBzA) was examined. Esophageal lesions were produced in male Sprague-Dawley rats by i.p. injection of 2 mg/kg NMBzA twice weekly for 3 weeks. Groups receiving BBI were fed three tablets a week containing 180 mg BBI each in a mixture of Witepsol H15 and peanut butter for the duration of the experiment. The frequency of papillomas and carcinomas was reduced 45% in groups receiving BBI. Furthermore, the frequency of appearance of five separate characteristics of preneoplastic lesions was significantly reduced in the esophagi of BBI-treated animals. The most significant reduction was in the total number of lesions with simple hyperplasia. Groups receiving NMBzA and placebo tablets, containing only Witepsol H15 and peanut butter, did not display statistically significant differences in the frequency of esophageal lesions as compared to animals receiving NMBzA alone. These results demonstrate that BBI can effectively inhibit NMBzA-induced esophageal tumors when given in tablet form separate from the regular diet.

Protease inhibitor suppression of colon and anal gland carcinogenesis induced by dimethylhydrazine.

In the present study, we examined the ability of chymostatin, a highly specific inhibitor of chymotrypsin, to suppress dimethylhydrazine-induced colon carcinogenesis, and the dose-response relationship for an extract of soybeans containing the Bowman-Birk inhibitor (BBI) to suppress dimethylhydrazine-induced colon carcinogenesis, when added to the diet of mice. Our results showed that: (i) diets containing 0.1% BBI reduced the incidence of adenocarcinomas of the colon approximately 50%, but had no effect on the incidence of squamous cell carcinomas of the anal gland; (ii) the suppressive effect requires protease inhibitor activity, as the autoclaved BBI, in which all protease inhibitory activity has been destroyed, was ineffective at suppressing the incidence of adenocarcinomas; (iii) chymostatin suppressed the incidence of squamous cell carcinomas of the anal gland, but not adenocarcinomas of the colon; and (iv) the growth rates of the animals were the same in each of the experimental groups. Our results indicate that the levels of anticarcinogenic protease inhibitors present in the diets of these animals do not have any adverse effects on the growth or general health of the animals.