Oral 28-day and developmental toxicity studies of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate.

(R)-3-Hydroxybutyl (R)-3-hydroxybutyrate (ketone monoester) has been developed as an oral source of ketones, which may be utilized for energy. In a 28-day toxicity study, Crl:WI (Wistar) rats received diets containing, as 30% of the calories, ketone monoester (12 and 15 g/kg body weight/day for male and female rats, respectively). Control groups received either carbohydrate- or fat-based diets. Rats in the test group consumed less feed and gained less weight than control animals; similar findings have been documented in studies of ketogenic diets. Between-group differences were noted in selected hematology, coagulation, and serum chemistry parameters; however, values were within normal physiological ranges and/or were not accompanied by other changes indicative of toxicity. Upon gross and microscopic evaluation, there were no findings associated with the ketone monoester. In a developmental toxicity study, pregnant Crl:WI (Han) rats were administered 2g/kg body weight/day ketone monoester or water (control) via gavage on days 6 through 20 of gestation. No Caesarean-sectioning or litter parameters were affected by the test article. The overall incidence of fetal alterations was higher in the test group; however, there were no specific alterations attributable to the test substance. The results of these studies support the safety of ketone monoester.

Safety evaluation of an enzymatically-synthesized glycogen (ESG).

An enzymatically-synthesized glycogen (ESG), intended for use as a food ingredient, was investigated for potential toxicity. ESG is synthesized in vitro from short-chain amylose by the co-operative action of branching enzyme and amylomaltase. In an acute toxicity study, oral administration of ESG to Sprague-Dawley rats at a dose of 2000 mg/kg body weight did not result in any signs of toxicity. ESG did not exhibit mutagenic activity in an in vitro bacterial reverse mutation assay. In a subchronic toxicity study, increased cecal weights noted in the mid- (10%) and high-dose (30%) animals are common findings in rodents fed excess amounts of carbohydrates that increase osmotic value of the cecal contents, and thus were considered a physiological rather than toxicological response. The hematological and histopathological effects observed in the high-dose groups were of no toxicological concern as they were secondary to the physiological responses resulting from the high carbohydrate levels in the test diets. The no-observed-adverse-effect level for ESG in rats was therefore established to be 30% in the diet (equivalent to approximately 18 and 21 g/kg body weight/day for male and female rats, respectively). These results support the safety of ESG as a food ingredient for human consumption.

Safety evaluation of amylomaltase from Thermus aquaticus.

A recombinant amylomaltase, MQ-01, obtained by cultivation of Bacillus subtilis expressing the amylomaltase gene from Thermus aquaticus is to be used in the production of enzymatically-synthesized glycogen; which is intended for use as a food ingredient. In order to establish the safety of MQ-01, the enzyme was subjected to standard toxicological testing. In a battery of standard Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537) and in Escherichia coli WP2 uvrA, both with and without metabolic activation, MQ-01 failed to exhibit mutagenic activity. Similarly, MQ-01 did not display clastogenic properties in Chinese hamster lung fibroblast cells (CHL/IU), in an in vitro chromosomal aberration assay. In a 13-week subchronic toxicity study in rats, oral administration of MQ-01 at doses of up to 15 mL/kg body weight/day (corresponding to approximately 1230 mg/kg body weight/day) did not produce compound-related clinical signs or toxicity, changes in body weight gain, food consumption, hematology, clinical chemistry, urinalysis, organ weights, or in any gross and microscopic findings. The results of this study support the safety of MQ-01 in food production.

Oxoguanine glycosylase 1 protects against methamphetamine-enhanced fetal brain oxidative DNA damage and neurodevelopmental deficits.

In utero methamphetamine (METH) exposure enhances the oxidative DNA lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in CD-1 fetal mouse brain, and causes long-term postnatal motor coordination deficits. Herein we used oxoguanine glycosylase 1 (ogg1) knock-out mice to determine the pathogenic roles of 8-oxoG and OGG1, which repairs 8-oxoG, in METH-initiated neurodevelopmental anomalies. Administration of METH (20 or 40 mg/kg) on gestational day 17 to pregnant +/- OGG1-deficient females caused a drug dose- and gene dose-dependent increase in 8-oxoG levels in OGG1-deficient fetal brains (p < 0.05). Female ogg1 knock-out offspring exposed in utero to high-dose METH exhibited gene dose-dependent enhanced motor coordination deficits for at least 12 weeks postnatally (p < 0.05). Contrary to METH-treated adult mice, METH-exposed CD-1 fetal brains did not exhibit altered apoptosis or DNA synthesis, and OGG1-deficient offspring exposed in utero to METH did not exhibit postnatal dopaminergic nerve terminal degeneration, suggesting different mechanisms. Enhanced 8-oxoG repair activity in fetal relative to adult organs suggests an important developmental protective role of OGG1 against in utero genotoxic stress. These observations provide the most direct evidence to date that 8-oxoG constitutes an embryopathic molecular lesion, and that functional fetal DNA repair protects against METH teratogenicity.

Iodine Content of the Best-Selling United States Adult and Prenatal Multivitamin Preparations.

BACKGROUND: Iodine is essential for thyroid hormone production and fetal development. Even mild maternal iodine deficiency in gestation may be associated with impaired child neurodevelopment. Iodine requirements increase during pregnancy and lactation. Supplements containing 150 µg/day are recommended by the American Thyroid Association for all U.S. women who are pregnant, lactating, or planning pregnancy. The contribution of multivitamin supplements as an iodine source for pregnant and nonpregnant US adults has not been well studied. This study aimed to understand better the contribution of the top-selling adult multivitamins (AMV) and prenatal multivitamins (PMV) to iodine nutrition in the United States. METHODS: Product names, dollar sales, unit and volume sales, and recommended daily intakes of the top-selling 99 AMV and 60 PMV from July 2016 to July 2017 were obtained from Information Resources, Inc. Iodine content and source were determined from product labels. After excluding private-label brands and unavailable product labels, a final sample of 89 AMV and 59 PMV was analyzed. RESULTS: Of the 89 AMV, 74.2% contained iodine. The median (range) iodine content of AMV was 150 µg (38-150 µg) per daily serving. Over the study period, 8,924,371,955 AMV doses were sold, of which 84.8% contained iodine. Thirty-four (57.6%) of the 59 PMV contained iodine, with a median (range) iodine content of 150 µg (25-290 µg) per daily serving. Over the study period, 466,927,559 PMV doses were sold, of which 76.8% contained iodine. All iodine-containing AMV used potassium iodine as an iodine source. Of iodine-containing PMV, 73.5% used potassium iodide, 23.5% kelp, and 2.9% inactivated Saccharomyces cerevisiae as an iodine source. CONCLUSIONS: During the one-year study period, a higher proportion of AMV doses than PMV doses contained iodine. Some PMV but no AMV included sources of iodine known to be highly variable. The median iodine content of the top-selling AMV and PMV was at the recommended 150 µg daily intake, but the range remains wide. Twenty-four percent of PMV doses sold did not contain iodine. More effort is needed to ensure adequate iodine content in prenatal vitamins for women who are pregnant, lactating, or planning pregnancy.

Evaluation of magnolia bark extract in chromosomal aberration assays.

Magnolia bark extract (MBE) has been used historically in traditional Chinese and Japanese medicines, and more recently as a component of dietary supplements and cosmetic products. The genotoxic potential of MBE was studied in two in vitro chromosomal aberration assays. In Chinese hamster ovary (CHO) cells, exposure for 3 h to MBE at concentrations of 0-30 microg/ml in the absence of a metabolic activation system (S9) and 0-7 microg/ml with S9 did not induce chromosomal aberrations, whereas higher concentrations were cytotoxic and did not allow for analysis of aberrations. Extended exposure for 18 h without metabolic activation at concentrations up to 15 microg/ml also resulted in a negative response. In V79 cells derived from Chinese hamster lung tissue, treatment for 6h with concentrations up to 52 and 59 microg/ml in the absence and presence of S9, respectively, did not increase the incidence of chromosomal aberrations compared to negative controls. Furthermore, MBE exposure for 24 h without metabolic activation did not induce aberrations. The results of these studies demonstrate that MBE is not genotoxic under the conditions of the in vitro chromosomal aberration assays in CHO and V79 cells, and support the safety of MBE.

Methamphetamine-enhanced embryonic oxidative DNA damage and neurodevelopmental deficits.

Methamphetamine (METH) causes dopaminergic nerve terminal degeneration and functional deficits in adult mice, but its neurodevelopmental effects are unclear. We investigated METH-initiated oxidative DNA damage in brain during the embryonic and fetal periods, and the postnatal histological and functional consequences. Pregnant CD-1 mice were treated with a single dose of METH (20 or 40 mg/kg ip) or its saline vehicle on Gestational Day 14 or 17. METH enhanced conceptal DNA oxidation, determined by 8-oxoguanine formation, in brain and liver by at least 2-fold at 1 h (P < 0.05), and more so in some fetal brains at 4 h. After birth, motor coordination on the rotarod apparatus in the METH-exposed offspring was impaired for at least 12 weeks (P < 0.05). Unlike in adults, this postnatal functional deficit in offspring exposed in utero to METH was not associated with degeneration of striatal dopaminergic nerve terminals at 12 weeks of age determined by tyrosine hydroxylase staining, suggesting a novel pathological mechanism in utero. This is the first evidence of oxidative DNA damage in embryonic and fetal brain caused by amphetamines, leading to long-term postnatal neurodevelopmental deficits via a mechanism different from that underlying the neurodegeneration observed in METH-exposed adults.

Molecular and biochemical mechanisms in teratogenesis involving reactive oxygen species.

Developmental pathologies may result from endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS), which oxidatively damage cellular macromolecules and/or alter signal transduction. This minireview focuses upon several model drugs (phenytoin, thalidomide, methamphetamine), environmental chemicals (benzo[a]pyrene) and gamma irradiation to examine this hypothesis in vivo and in embryo culture using mouse, rat and rabbit models. Embryonic prostaglandin H synthases (PHSs) and lipoxygenases bioactivate xenobiotics to free radical intermediates that initiate ROS formation, resulting in oxidation of proteins, lipids and DNA. Oxidative DNA damage and embryopathies are reduced in PHS knockout mice, and in mice treated with PHS inhibitors, antioxidative enzymes, antioxidants and free radical trapping agents. Thalidomide causes embryonic DNA oxidation in susceptible (rabbit) but not resistant (mouse) species. Embryopathies are increased in mutant mice deficient in the antioxidative enzyme glucose-6-phosphate dehydrogenase (G6PD), or by glutathione (GSH) depletion, or inhibition of GSH peroxidase or GSH reductase. Inducible nitric oxide synthase knockout mice are partially protected. Inhibition of Ras or NF-kB pathways reduces embryopathies, implicating ROS-mediated signal transduction. Atm and p53 knockout mice deficient in DNA damage response/repair are more susceptible to xenobiotic or radiation embryopathies, suggesting a teratological role for DNA damage, consistent with enhanced susceptibility to methamphetamine in ogg1 knockout mice with deficient repair of oxidative DNA damage. Even endogenous embryonic oxidative stress carries a risk, since untreated G6PD- or ATM-deficient mice have increased embryopathies. Thus, embryonic processes regulating the balance of ROS formation, oxidative DNA damage and repair, and ROS-mediated signal transduction may be important determinants of teratological risk.

Cockayne syndrome B protects against methamphetamine-enhanced oxidative DNA damage in murine fetal brain and postnatal neurodevelopmental deficits.

Methamphetamine (METH) increases the oxidative DNA lesion 8-oxoguanine (8-oxoG) in fetal mouse brain, and causes postnatal motor coordination deficits after in utero exposure. Like oxoguanine glycosylase 1 (OGG1), the Cockayne syndrome B (CSB) protein is involved in the repair of oxidatively damaged DNA, although its function is unclear. Here we used CSB-deficient Csb(m/m) knockout mice to investigate the developmental role of DNA oxidation and CSB in METH-initiated neurodevelopmental deficits. METH (40 mg/kg intraperitoneally) administration to pregnant Csb females on gestational day 17 increased 8-oxoG levels in Csb(m/m) fetal brains (p < 0.05). CSB modulated 8-oxoG levels independent of OGG1 activity, as 8-oxoG incision activity in fetal nuclear extracts was identical in Csb(m/m) and Csb(+/+)mice. This CSB effect was evident despite 7.1-fold higher OGG1 activity in Csb(+/+) mice compared to outbred CD-1 mice. Female Csb(m/m) offspring exposed in utero to METH exhibited motor coordination deficits postnatally (p < 0.05). In utero METH exposure did not cause dopaminergic nerve terminal degeneration, in contrast to adult exposures. This is the first evidence that CSB protects the fetus from xenobiotic-enhanced DNA oxidation and postnatal functional deficits, suggesting that oxidatively damaged DNA is developmentally pathogenic, and that fetal CSB activity may modulate the risk of reactive oxygen species-mediated adverse developmental outcomes.

Oral subchronic and genotoxicity studies conducted with the amino acid, L-glutamine.

L-Glutamine is an abundantly occurring amino acid that serves numerous nutritional and physiological functions. It has current and potential applications as a therapeutic agent, dietary supplement, food ingredient, and in animal nutrition. To assess the safety of supplemental L-glutamine, a bacterial reverse mutation assay, in vitro chromosomal aberration assay, and a 13-week toxicity study were conducted. L-Glutamine showed no mutagenic activity in the bacterial reverse mutation assay, and did not induce chromosomal aberrations in Chinese hamster lung fibroblast cells in the in vitro chromosomal aberration assay. In the 13-week toxicity study, Sprague-Dawley rats (10/sex/group) were fed diets containing 0, 0.5, 2.5, or 5.0% L-glutamine. No deaths occurred, and no significant differences in body weights, body weight gains, ophthalmological findings, urinalysis parameters, or organ weights were observed between L-glutamine-fed rats and their respective controls. No toxicologically relevant effects on hematological or blood biochemical parameters were observed. Macroscopic and microscopic effects occurred at low frequency but were not associated with a dose-response relationship. Based on the results of the study, the no-observed-adverse-effect-level was determined to be 5.0% L-glutamine in the diet, the highest concentration tested (equivalent to 3832 and 4515 mg/kg body weight/day in male and female rats, respectively).

Influence of observational study design on the interpretation of cancer risk reduction by carotenoids.

Recently published literature has been reviewed to determine whether lycopene, beta-carotene, alpha-carotene, and beta-cryptoxanthin are associated with reductions in cancer risk and whether study findings differ by study design. A total of 57 publications meeting pre-defined inclusion and exclusion criteria were identified, with the majority (55) being observational studies. None of the intervention studies supported a significant reduction in cancer risk with carotenoid (beta-carotene) supplementation. The majority of observational studies did not support significant reductions in cancer risk with increased carotenoid dietary intakes/circulating levels. A larger percentage of case-control studies supported significant associations between increased dietary intakes/circulating levels of carotenoids relative to prospective (cohort and nested case-control) studies. Compared to prospective studies, case-control studies cannot be used to establish temporality and may be more susceptible to selection and recall biases. Thus, diet-disease relationships suggested by case-control studies should ideally be confirmed by additional evidence from prospective studies.

Oxidative stress in developmental origins of disease: teratogenesis, neurodevelopmental deficits, and cancer.

In the developing embryo and fetus, endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS) like hydroxyl radicals may adversely alter development by oxidatively damaging cellular lipids, proteins and DNA, and/or by altering signal transduction. The postnatal consequences may include an array of birth defects (teratogenesis), postnatal functional deficits, and diseases. In animal models, the adverse developmental consequences of in utero exposure to agents like thalidomide, methamphetamine, phenytoin, benzo[a]pyrene, and ionizing radiation can be modulated by altering pathways that control the embryonic ROS balance, including enzymes that bioactivate endogenous substrates and xenobiotics to free radical intermediates, antioxidative enzymes that detoxify ROS, and enzymes that repair oxidative DNA damage. ROS-mediated signaling via Ras, nuclear factor kappa B and related transducers also may contribute to altered development. Embryopathies can be reduced by free radical spin trapping agents and antioxidants, and enhanced by glutathione depletion. Further modulatory approaches to evaluate such mechanisms in vivo and/or in embryo culture have included the use of knockout mice, transgenic knock-ins and mutant deficient mice with altered enzyme activities, as well as antisense oligonucleotides, protein therapy with antioxidative enzymes, dietary depletion of essential cofactors and chemical enzyme inhibitors. In a few cases, measures anticipated to be protective have conversely enhanced the risk of adverse developmental outcomes, indicating the complexity of development and need for caution in testing therapeutic strategies in humans. A better understanding of the developmental effects of ROS may provide insights for risk assessment and the reduction of adverse postnatal consequences.

Genotoxicity and subchronic toxicity studies of DHA-rich oil in rats.

Polyunsaturated fatty acids, including docosahexaenoic acid (DHA), are natural constituents of the human diet. DHA-algal oil is produced through the use of the non-toxigenic and non-pathogenic marine protist, Ulkenia sp. The safety of DHA-algal oil was assessed in a subchronic toxicity study and in genotoxicity studies. In a 90-day study, rats were orally administered water or DHA-algal oil at concentrations of 0, 500, 1000, and 2000 mg/kg in combination with 2000, 1500, 1000 or 0 mg/kg DHA-containing fish oil, respectively. Additional animals were administered water, 2000 mg/kg DHA-algal oil, or 2000 mg/kg fish oil for 90 days, followed by a 4-week recovery phase. No treatment-related effects were observed in clinical observations, food and water consumption, mortality, gross pathology, and histopathology. Increased body weights and liver weights in oil-treated groups were attributed to the large lipid load and were not regarded as toxicologically significant. Furthermore, no treatment-related differences in the measured parameters between the DHA-algal oil and fish oil groups were detected. In genotoxicity experiments, DHA-algal oil exerted no mutagenic activity in various bacterial strains, nor did it induce chromosomal aberrations in Chinese hamster fibroblast cells. These results support the safety of DHA-algal oil as a dietary source of DHA.

One-generation reproductive toxicity study of DHA-rich oil in rats.

Polyunsaturated fatty acids, including docosahexaenoic acid (DHA), are natural constituents of the human diet. DHA-algal oil is produced through the use of the marine protist, Ulkenia sp. The reproductive toxicity of DHA-algal oil was assessed in a one-generation study. Rats were provided diets containing DHA-algal oil at concentrations of 1.5, 3.0, or 7.5%, and the control group received a diet containing 7.5% corn oil. Males and females were treated for 10 weeks prior to mating and during mating. Females continued to receive test diets during gestation and lactation. In parental animals, clinical observations, mortality, fertility, and reproductive performance were unaffected by treatment. Differences in food consumption, body weight, and liver weight in the treated groups were not considered to be due to an adverse effect of DHA-algal oil. Spleen weight increases in treated animals were associated with extramedullary hematopoiesis. Yellow discoloration of abdominal adipose tissue was observed in rats from the high-dose group, and histological examination revealed steatitis in all treated parental groups. Exposure to DHA-algal oil did not influence the physical development of F(1) animals. These results demonstrate that DHA-algal oil at dietary concentrations of up to 7.5% in rats does not affect reproductive capacity or pup development.

Evaluation of the in vitro and in vivo genotoxicity of magnolia bark extract.

Magnolia bark extract (MBE) is an extract of the dried stem, root, or branch bark of magnolia trees that has been used historically in traditional Chinese and Japanese medicines, and more recently as a component of dietary supplements and cosmetic products. To study the genotoxic potential of MBE, a bacterial reverse mutation assay and an in vivo micronucleus test were conducted. Compositional analysis of the test substance revealed that MBE contains 94% magnolol and 1.5% honokiol. MBE exerted no mutagenic activity in various bacterial strains of Salmonella typhimurium and in Escherichia coli WP2 uvrA, either in the absence or presence of metabolic activation at all doses tested. In the micronucleus test, various doses of MBE did not affect the proportions of immature to total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of Swiss albino mice. The results of these studies demonstrate that MBE is not genotoxic under the conditions of the in vitro bacterial reverse mutation assay and the in vivo micronucleus test, and support the safety of MBE for dietary consumption.

Evaluation of short-term and subchronic toxicity of magnolia bark extract in rats.

Magnolia bark has been traditionally used in Chinese and Japanese medicines, and its extract is a constituent of currently marketed dietary supplements and cosmetic products. The safety of magnolia bark extract (MBE) was assessed in short-term and subchronic studies. In a 21-day pilot study, rats were administered MBE at levels of 0, 60, 120, 240 or 480 mg/kg body weight (bw)/day in the diet. There were no treatment-related effects in clinical observations, macroscopic or microscopic findings, hematological, clinical chemistry, urinalysis, or organ weight measurements, and there were no deaths or significant differences in body weight and weight gain. In the 90-day study, rats were administered 0, 60, 120 or 240 mg MBE/kg bw/day in the diet. No mortality, ophthalmic abnormalities or treatment-related findings in clinical observations, hematology, coagulation or organ weight measurements were observed. There were no treatment-related macroscopic or microscopic findings. Differences between treated and control groups in body weight, weight gain, food consumption and utilization, clinical chemistry and urinalysis parameters were not considered toxicologically significant as they were not dose-related and/or because values remained within historical control ranges. These results support the safety of MBE for oral consumption.