Bruce K. Bernard

Toxicology and carcinogenesis studies of dietary titanium dioxide‐coated mica in male and female Fischer 344 rats

Male and female Fischer 344 rats were fed diets containing 0, 1.0, 2.0, or 5.0% titanium dioxide (TiO2) coated mica for up to 130 wk. This dosage regimen produced no consistent or biologically important changes in survival, body weight gains, hematologic or clinical chemistry parameters or histopathology. Under the conditions of this 130 wk feeding study there was no evidence that TiO2-coated mica produced either toxicologic or carcinogenic effects at dietary concentrations as high as 5.0%. The results suggest that dietary exposure to TiO2-coated mica does not pose a significant human health hazard.

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-lsoleucyl-L-prolyl-L-proline): I. Executive summary

East Asian and European folklore has long held that the consumption of fermented milk assists in the maintenance of good health, including maintenance of normal blood pressure. Studies in rats and humans provide scientific support for this tradition and suggest that fermented milk has a normotensive effect in hypertensive animals, but no effect on normal blood pressure. Two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucylL-prolyl-L-proline (IPP), discovered to be constituents of sour milk, have been identified as possessing significant angiotensinconverting enzyme inhibitory activity and are believed to be the source of the normotensive effects. This review, although containing some of the available pharmacology and efficacy information, deals primarily with the evaluation of data related to the safety of these compounds. This review consists of nine chapters (I to IX), the first of which is this Executive Summary. The Executive Summary provides a short synopsis of the key findings that are discussed in detail in the remaining documents. It is provided to introduce and orient the reader to the wide variety of information covered herein, and to assist the reader in locating particular information of interest. The second chapter (II) serves as an introduction to these two tripeptides and provides background information. Early research suggested that the normotensive activity of fermented milk resided in two tripeptides, VPP and IPP. More recently, it has been determined that the mechanism by which normotensive effects are induced by these tripeptides is through inhibition of the angiotensin-converting enzyme, an important component of both the renin-angiotensin and kinin-kalikrein systems which are known to be associated with blood pressure control. Extensive work was required to create a method for increasing the concentrations of the tripeptides. Screening of a large number of organisms demonstrated that many species and some

First human studies with a high-molecular-weight iron chelator ☆ ☆☆

The release of free, reactive iron from cellular iron stores has been implicated as an important contributor to tissue damage in a variety of clinical situations, including ischemia and reperfusion injury, hemorrhagic shock, and burn injury. Deferoxamine mesylate (DFO), the only iron chelator currently approved for clinical use, is used for the treatment of iron overload, including acute iron poisoning and treatment of chronic iron overload in transfusion-dependent anemias such as beta-thalassemia. However, it is not suitable for acute care situations because of its toxicity, primarily hypotension when given at high intravenous doses, and its short plasma half-life. We have produced a high-molecular-weight iron chelator by chemically coupling DFO to hydroxyethyl starch. This novel chelator (HES-DFO) was administered to healthy male subjects by intravenous infusion over a 4-hour period. The drug was well tolerated, and signs of DFO acute toxicity were not observed. Maximum plasma chelator levels of approximately 3 mmol/L were achieved with HES-DFO, which is more than an order of magnitude higher than has been reported with injections of DFO. Drug residence time in plasma was markedly prolonged, with an initial half-life of 22 to 33 hours. Urinary iron excretion was 7.1 +/- 2.2 mg in 48 hours in the highest dose group, as compared with 0.06 +/- 0.15 mg in control subjects who received normal saline infusions. Intravenous infusion of HES-DFO is well tolerated, produces substantial and prolonged plasma chelator levels, and markedly stimulates urinary iron excretion.

An Evaluation of the Common Mechanism Approach to the Food Quality Protection Act: Captan and Four Related Fungicides, a Practical Example:

Under the Food Quality Protection Act (FQPA) of 1996 (Act), the United States Environmental Protection Agency (EPA) is mandated to conduct cumulative risk assessment on pesticides that act through a common mechanism of toxicity. Incumbent on the Agency is the development of sound scientific principles upon which to evaluate compounds for the presence of a common mechanism. Using the currently available draft guidance criteria, this paper employs five fungicides of the same general class, typified by captan, to evaluate both the criteria and the available scientific data. Captan and folpet are two chloroalkylthio fungicides currently registered with EPA under Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) for agricultural use. As such, these compounds are subject to the provisions of FQPA. Three additional fungicidal compounds—dichlofluanid, tolylfluanid, and captafol—are not registered for use in the United States; however, these five compounds have chemical structure and biological toxicity similarities and differences that permit their utility as test cases to determine what the EPA would conclude, with regard to common mechanism, if these draft guidelines were applied to these compounds. The results of the analyses are consistent and support the conclusion that captan and folpet share a common mode of toxicity for mouse duodenal tumors as defined in the Act. This common mode of toxicity is not shared by dichlofluanid, tolylfluanid, or captafol. The basis for concluding a common mechanism exists between captan and folpet. They include 1. Structural Similarity—The compounds are structural analogs having the identical biologically active moiety (i.e., the-SCCl3 side chain). 2. Mechanisms of Pesticidal Action—The compounds have the same mechanism of action. The overwhelming body of evidence suggests they are active because of their reactions with thiols. Both compounds, in reacting with thiols, produce similar degradates. Differences in rates of reaction are attributable to physical-chemical properties of the two compounds. 3. General Mechanisms of Mammalian Toxicity—The compounds induce mammalian toxicity through the same mechanism that is responsible for their pesticidal action, reactions with thiols. Another, albeit less likely, mechanism (for both compounds) is cross-Unking of proteins with DNA, although the extremely short half-lives of these compounds (seconds) argues against this possibility. 4. Sites of Action—Both compounds express their primary toxicity as local rather than systemic effects. 5. Common Toxic Endpoint—These two compounds induce gastrointestinal tumors (in mice only). 6. Mode of Action—Both compounds express their common toxic endpoint through a nongenotoxic, compensatory proliferation mechanism. 7. Specificity of Action—For both compounds, the majority of tumors appear in the duodenum. Furthermore, these tumors are induced only in mice. Repeated carcinogenicity testing suggests that rats are refractive to the effects of captan and folpet. The significantly faster hydrolytic rate for folpet at the lower pH values (e.g., increased 8-fold at pH 5) encountered in the stomach is believed to account for the tumors of the stomach observed with folpet and not captan. 8. Other Toxic Endpoints—For other toxic endpoints where comparative data are available, captan and folpet show similar patterns of toxicity (e.g., mutagenicity, skin sensitization, and acute toxicity).

Studies of the Toxicological Potential of Tripeptides (l-Valyl-l-prolyl-l-proline and l-Isoleucyl-l-prolyl-l-proline): III. Single-and/or Repeated-Dose Toxicity of Tripeptides-Containing Lactobacillus helveticus–Fermented Milk Powder and Casein Hydrolysate in Rats

The objective of these studies was to assess the toxicological potential of orally administered tripeptides in rats. The studies employed powdered L-valyl-L-prolyl-L-proline (VPP)- and L-isoleucyl-L-prolyl-L-proline (IPP)-containing test articles, including (1) powdered Lactobacillus helveticus-fermented milk (FM), (2) pasteurized casein hydrolysate (CH) generated by Aspergillus oryzae protease, and (3) synthesized VPP. All test articles were administered by oral gavage to male and female Sprague-Dawley rats. Specific goals of the single-dose and repeated-dose studies were to (1) identify doses that produce evidence of systemic and/or local (i.e., gastrointestinal) toxicity (e.g., lowest-observable-effect level [LOEL]); (2) estimate the maximally tolerated oral dose (MTD) ; and (3) identify specific target organs for toxicity of these tripeptides. Single doses of CH (2000 mg/kg), powdered FM (2000 or 4000 mg/kg), or VPP (40, 200, or 400 mg/kg) were administered 14 days prior to study termination. No treatment regimen caused either antemortem (gross observations, body weight, and food consumption parameters) or postmortem (necropsy) evidence of either systemic or local toxicity. In the repeated-dose study, powdered FM (0, 500, 1000, or 2000 mg/kg body weight [BW]/day) was administered by gastric gavage to male and female rats for 28 consecutive days. Antemortem evaluative parameters included gross observations, ophthalmic examinations, and clinical pathology (clinical chemistry, hematology, and urinalysis). Post mortem parameters included necropsy, determination of organ weights, and microscopic examination of major organs. There was neither in-life nor postmortem evidence that powdered FM administration caused physiological or toxicological changes. Under the conditions of these experiments, the single-dose LOEL of powdered FM, CH, and VPP were found to be greater than 4000,2000, and 400 mg/kg, respectively. The results of the repeated-dose study do not support identification of a target organ for powdered FM toxicity. Similarly, there was no evidence to support establishment of either the LOEL or MTD; both being greater than 2000 mg/kg/day for up to 28 consecutive days.

Studies of the Toxicological Potential of Tripeptides (l-Valyl-l-prolyl-l-proline and l-Isoleucyl-l-prolyl-l-proline): V. A 13-Week Toxicity Study of Tripeptides-Containing Casein Hydrolysate in Male and Female Rats

The objective of this multiple-dose toxicity study was to assess the toxicological potential of two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP), when administered once daily for 91 consecutive days to rats. The test article, powdered casein hydrolysate (CH) known to contain 0.6% VPP plus IPP, was prepared using Aspergillus oryzae protease. Prior to administration to the rats by oral gavage, the test article was suspended in sterile water. Groups of 12 male and 12 female Charles River rats were administered once daily doses of 0, 40, 200, or 1000 mg of CH (0, 0.2,1.2, or 6 mg VPP plus IPP/kg body weight [BW]). Antemortem evaluative parameters included gross observations of behavior and clinical signs; food consumption and body weight gains; ophthalmologic examinations; clinical pathology (hematology, clinical chemistry); and urinalysis. Postmortem parameters included determination of absolute and relative (to fasting body weight) organ weights and histopathological evaluation of approximately 50 organs and tissues from each animal. All rats survived until the scheduled termination of the study and no treatment-related clinical signs were observed. Food consumption was unaffected by administration of CH. All animals gained weight and there were no statistical differences between groups with respect to weight gains. There were no meaningful changes in hematological or coagulation parameters. Mid- and high-dose males (but not females) had slightly (<2%) increased mean serum chloride concentrations, but because the difference was so small and it was observed in only one sex, the authors considered its association with CH administration to be doubtful. Urinalysis revealed the occasional presence of crystals, leukocytes, and epithelial cells in animals from all experimental groups. Similarly, ophthalmic changes (lenticular clouding) were observed in both control and dosed animals. Mean relative (to body weight) kidney weight was decreased by 8 % in low-dose males and mean relative uterus weight was elevated 46 % in low-dose females. Absolute organ weights were not affected. Only naturally occurring microscopic changes were observed in all groups and none could be attributed to CH administration. It was concluded that, under the conditions of these experiments, the maximally tolerated dose (MTD) and the no-observable-effect level (NOEL) for powdered CH administered once daily for 13 weeks was greater than 1000 mg/kg BW/day or greater than 6 mg of VPP plus IPP/kg BW/day. There was no evidence of target organ toxicity associated with administration of the tripeptides. This corresponds to an margin of safety (MOS) of 60 based upon current thinking regarding incorporation in food.

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): VI. Effects of Lactobacillus helveticus-fermented milk powder on fertility and reproductive performance of rats.

The objective of these studies was to assess the effects of the tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP), on reproductive capabilities of male and female rats. The specific goals of the experiments were (1) to determine the effects of orally administered tripeptides on (a) fertility and reproductive behavior in both sexes of rats, (b) embryo-fetal development in pregnant rats, and (c) pre- and postnatal development of rats exposed to tripeptides in utero and during lactation; and (2) to estimate the no-observable-adverse-effect doses of tripeptides in maternal and fetal rats. During the conduct of these classical segment I, II, and III studies, the test material was powdered Lactobacillus helveticus-fermented milk (FM), which contains the tripeptides, VPP and IPP. FM (0, 500, 1000 or 2000 mg/kg body weight [BW]/day—equivalent to 0, 0.8, 1.6, or 3.3 mg/kg BW/day of VPP plus IPP) was administered to males by oral gavage from 4 weeks prior to mating until sacrifice, and to females from 2 weeks prior to mating through day 20 of lactation. Evaluative parameters included monitoring grossly observable clinical signs; food consumption and body weight gains; mating behavior and fertility indices of both sexes; implantation and maintenance of embryos; sex ratio of live pups ; fetal viability; incidences of external, visceral or skeletal variations; growth and behavioral development; as well as reproductive capabilities of Fi offspring exposed to FM during gestation and lactation. All animals were subjected to macroscopic examination at termination of their segment of the studies. Clinical signs, body weights, and food consumption were unaffected by administration of FM. During segment I, the test agent had no effect on estrus cycle, mating behavior, fertility index, or reproductive competence of either males or females. The results of segment II experiments revealed no effects of FM on postimplantation survival-loss, sex ratio or birth weights of live fetuses, and there was no evidence of treatment-associated developmental or teratological effects. During segment III, FM was without effect on pup viability, behavioral and sexual maturation, and reproductive capability of the F1 generation. Under the conditions of these experiments, the no-observable-adverse-effect level (NOAEL) of FM on reproductive performance in male and female rats is greater than 2000 mg/kg BW/day, the equivalent of 3.3 mg/kg BW/day of VPP plus IPP.

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-lsoleucyl-L-prolyl-L-proline): II. Introduction

The consumption of fermented milk to maintain good health, including the maintance of normal blood pressure, is an ancient tradition in a number of areas of the world (e.g., East Asia, France). Recent studies have suggested that fermented milk has a normotensive effect in hypertensive rats and humans, but no effect on blood pressure in normotensive rats and humans. Two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP), have been identified as possessing significant angiotensin-converting enzyme inhibitory activity and are therefore believed to be the source of the normotensive effects. This document, the second of nine chapters, provides information on these two tripeptides, including physical/chemical properties, molecular weights, chemical structures, normal consumption in the diet, manufacturing information, regulatory approval in Japan, and Japanese consumption of food containing enhanced levels of VPP plus IPP. In addition, the results of studies in rats and humans conducted to evaluate the effect of these substances on blood pressure are presented. The research suggests that in adult normotensive volunteers, consumption of up to 7.92 mg of VPP and 4.52 mg IPP daily for 2 weeks causes neither clinical signs nor biologically meaningful effects on systolic or diastolic blood pressure, pulse rate, or clinical pathology (serum chemistry or hematology). However, when a similar study was performed using mildly and moderately hypertensive adults as subjects and they consumed 2.52 mg of VPP and 1.64 mg of IPP per day, a significant drop in systolic blood pressure was detected for a prolonged time interval. This chapter also introduces the issue of safety testing for these substances and describes the information to be found in the subsequent seven chapters.

Studies of the Toxicological Potential of Capsinoids: X. Safety Assessment and Pharmacokinetics of Capsinoids in Healthy Male Volunteers after a Single Oral Ingestion of CH-19 Sweet Extract

The safety and pharmacokinetics of capsinoids, physiologically active ingredients of CH-19 Sweet extract, were investigated in 16 healthy male volunteers following a single oral ingestion of CH-19 Sweet extract. The study subjects consumed soft gel capsules containing either capsinoids (15 or 30 mg/person) or placebo. Capsinoids were well tolerated, and no clinically significant changes in physical examinations, blood pressure, heart rate, body temperature, electrocardiogram, hematology, blood chemistry, and urinalysis were observed at either the 15 or 30 mg dose. Body temperature tended to increase after the ingestion of capsinoids, but remained within the normal range. Plasma levels of capsinoids and their metabolite, vanillyl alcohol, were below the lower limit of quantitation. In addition, some study subjects showed increases in urinary excretion of 3-methoxy-4-hydroxyphenylglycol that, when compared to the group receiving the placebo, did not achieve statistical significance.

Studies of the Toxicological Potential of Tripeptides (l-Valyl-l-prolyl-l-proline and l-Isoleucyl-l-prolyl-l-proline): IV. Assessment of the Repeated-Dose Toxicological Potential of Synthesized l-Valyl-l-prolyl-l-proline in Male and Female Rats and Dogs

The objective of these repeated-dose, 8-week studies was to assess the toxicological potential of a synthetic tripeptide, L-valyl-L-prolyl-L-proline (VPP), when administered to Charles River rats and Beagle dogs. Groups of 20 male and 20 female rats were fed powdered diets containing sufficient VPP to afford daily doses of 0, 2, 8, or 16 mg/kg body weight (BW)/day. Groups of five male and five female dogs were administered 0, 2, 8, or 16 mg/kg BW/day in hard gelatin capsules. Antemortem evaluative parameters for both species included grossly observable clinical signs, body weight and food consumption, clinical pathology (hematology, clinical chemistry, urinalysis), and ophthalmological examinations. Dogs also received electrocardiographic examinations. Postmortem evaluations in both species included complete necropsy, determination of major organ weights, and histopathological examination of specimens from approximately 50 organs and tissues. All rats and dogs survived to the scheduled termination of the studies and neither species exhibited evidence of VPP effects on appetite or body weight gain/maintenance. Ophthalmic examinations revealed occasional lens clouding in rats, but this occurred in all groups and was not attributable to VPP. Some clinical pathology parameters in both species were occasionally altered, but there was no evidence that this was dose-related. Electrocardiographic examinations in dogs revealed no VPP-associated changes. Mid- and high-dose male rats (but not females) had slightly reduced mean pituitary and kidney weight parameters, whereas mid- and high-dose females had slightly increased mean uterus:body weight ratios. There were no microscopic correlates for these minor changes. Ten percent to 20 % of all female rats (but not males) exhibited corticomedullary mineralization of the kidney and gliosis of the optic nerve, and 10% to 20% of males (but not females) had thymic hemorrhage. Postmortem evaluations of dogs revealed no VPP-related effects on organ weights or either macro- or microscopic appearances of organs. The results of these studies provided no evidence of either local or systemic toxicity. Similarly, there was no evidence of neurotoxicity that might have been detected by the appearance of physical or behavioral changes during gross observations of animals. Although these results do not identify target organs for VPP toxicity, the no-observable-effect level and maximally tolerated dose are both greater than 16 mg/kg/day when administered to male and female rats and dogs for 8 consecutive weeks. Based upon food enhancement levels of VPP currently being evaluated, the resultant margin of safety (160) is substantial.