Safety of Elixinol Hemp Extract: In Vitro Genetic Toxicity and Subchronic Toxicity in Rats.

The results of safety studies performed with Elixinol Hemp Extract, a blend of hemp extract, cannabidiol (CBD) isolate, and copaiba containing approximately 65% total CBD, are described in this paper. In a 15-day range-finding study in rats, there were no effects of treatment with up to 101.4 mg/kg bw/day of the extract by gavage on any safety parameter measured in the study, with the exception that centrilobular hepatocellular hypertrophy occurred in all treatment groups, which correlated with increases in absolute liver weight in high-dose females and liver to terminal body weight ratio in mid-dose and high-dose females. A GLP-compliant 90-day OECD Guideline 408 study in rats that included a behavioral battery and a 28-day recovery phase was also conducted with Elixinol Hemp Extract administered by gavage. The doses used in the 90-day study were 0 (vehicle), 28.94, 50.64, and 86.81 mg/kg bw/day. The findings were similar to those observed in the range-finding study. There were no effects of the test material on any test parameter in the 90-day study other than findings related to the liver (increased liver weight in high-dose main study males and mid-dose and high-dose main study females and low incidences of hepatocellular hypertrophy and vacuolation in main study high-dose males). Similar findings were not observed in the recovery animals, and there were no alterations in the clinical chemistry suggestive of liver toxicity in any of the main study or recovery animals. Therefore, the liver outcomes observed in the main study were not considered adverse. The test material also tested negative for mutagenicity in bacterial reverse mutation assays (plate incorporation and preincubation) in the absence and presence of metabolic activation. The results indicate that the oral 90-day no observed adverse effect level (NOAEL) of Elixinol Hemp Extract in rats is 86.81 mg/kg bw/day (highest dose administered), and that the extract is not mutagenic.

Toxicity Investigations of (R)-3-Hydroxybutyrate Glycerides In Vitro and in Male and Female Rats.

TCN006, a formulation of (R)-3-Hydroxybutyrate glycerides, is a promising ingredient for enhancing ketone intake of humans. Ketones have been shown to have beneficial effects on human health. To be used by humans, TCN006 must be determined safe in appropriately designed safety studies. The results of a bacterial reverse mutation assay, an in vitro mammalian micronucleus study, and 14-and 90-day repeat dose toxicity studies in rats are reported herein. In the 14- and 90-day studies, male and female Wistar rats had free access to drinking water containing 0, 75,000, 125,000 or 200,000 ppm TCN006 for 92 and 93 days, respectively. TCN006 tested negative for genotoxicity and the no observed adverse effect level (NOAEL) for toxicity in the 14- and 90-day studies was 200,000 ppm, the highest dose administered. In the longer term study, the mean overall daily intake of TCN006 in the 200,000 ppm groups was 14,027.9 mg/kg bw/day for males and 20,507.0 mg/kg bw/day for females. At this concentration, palatability of water was likely affected, which led to a decrease in water consumption in both males and females compared to respective controls. This had no effect on the health of the animals. Although the rats were administered very high levels of (R)-3-Hydroxybutyrate glycerides, there were no signs of ketoacidosis.

Toxicological and safety evaluations of Weissella cibaria strain CMU in animal toxicity and genotoxicity.

Weissella cibaria belongs to the Lactobacillaceae family and has been isolated from traditional fermented foods and saliva of children with good oral health. Previous investigations have shown that W. cibaria CMU (Chonnam Medical University) is expected to be safe based on results of in silico and in vitro analyses. However, there is a lack of studies assessing its safety in vivo. A toxicological safety evaluation of W. cibaria CMU was performed using an acute oral safety study in rats, a 14-day oral range finding study, a subsequent 13-week oral toxicity study in rats and a genetic toxicity battery (in vitro bacterial reverse mutation, in vitro chromosome aberration in Chinese Hamster Ovary cells and in vivo micronucleus study in mice). The results of the studies in rats showed that the acute lethal dose of W. cibaria CMU is > 5000 mg/kg body weight (bw)/day (1.8 × 109 CFU/kg bw/day) and the 14-day or 13-week no observed adverse effect level (NOAEL) is 5000 mg/kg bw/day (1.8 × 109 CFU/kg bw/day), the highest dose administered. W. cibaria CMU was non-mutagenic in the bacterial reverse mutation test and non-clastogenic or aneugenic in vitro and in vivo. In conclusion, the toxicological studies performed demonstrated W. cibaria CMU to be a safe strain to consume. This study is the first study examining the potential of a W. cibaria strain to cause genetic toxicity and subchronic toxicity in rats according to the Organization for Economic Cooperation and Development guidelines.

Genotoxicity evaluation of magnesium salts of isobutyrate and 2-methylbutyrate.

Results of genotoxicity studies for magnesium salts of isobutyrate and 2-methylbutyrate, two candidate ingredients for inclusion in animal feed, are described in this manuscript. Both substances were tested for mutagenicity in a bacterial reverse mutation assay and clastogenicity/aneugenicity in an in vitro micronucleus study in human lymphocytes, conducted according to Organisation for Economic Co-operation and Development (OECD) Guidelines. The substances were tested up to the limits of solubility in the tests. The results showed that that magnesium salts of isobutyrate and 2-methylbutyrate are not mutagenic, clastogenic or aneugenic. The tests were valid, as the negative and positive controls produced expected responses.

A review of the evidence to support interim reference level for dietary lead exposure in adults.

FDA developed the interim reference level (IRL) for lead of 3 µg/day in children and 12.5 µg/day in women of childbearing age (WOCBA) to better protect the fetus from lead toxicity. These IRLs correspond to a blood lead level (BLL) of 0.5 µg/dL in both populations. The current investigation was performed to determine if the IRL for WOCBA should apply to the general population of adults. A literature review of epidemiological studies was conducted to determine whether a BLL of 0.5 µg/dL is associated with adverse effects in adults. Some studies reported adverse effects over a wide range of BLLs that included 0.5 µg/dL adding uncertainty to conclusions about effects at 0.5 µg/dL; however, no studies clearly identified this BLL as an adverse effect level. Results also showed that the previously developed PTTDI for adults of 75 µg/day lead may not be health protective, supporting use of a lower reference value for lead toxicity in this population group. Use of the 12.5 µg/day IRL as a benchmark for dietary lead intake is one way FDA will ensure that dietary lead intake in adults is reduced.

U.S. Food and Drug Administration's interim reference levels for dietary lead exposure in children and women of childbearing age.

Reducing lead exposure is a public health priority for the US Food and Drug Administration as well as other federal agencies. The goals of this research were to 1) update the maximum daily dietary intake of lead from food, termed an interim reference level (IRL), for children and for women of childbearing age (WOCBA) and 2) to confirm through a literature review that with the exception of neurodevelopment, which was not evaluated here, no adverse effects of lead consistently occur at the blood lead level (BLL) associated with the IRL. Because no safe level of lead exposure has yet been identified for children's health, the IRLs of 3 µg/day for children and 12.5 µg/day for WOCBA were derived from the Centers for Disease Control and Prevention reference value of 5 µg/dL BLL, the level at which public health actions should be initiated. The literature review showed that no adverse effects of lead consistently occurred at the BLL associated with the IRLs (0.5 µg/dL). The IRLs of 3 µg/day for children and 12.5 µg/day for WOCBA should serve as useful benchmarks in evaluating the potential for adverse effects of dietary lead.

Bioactive nutrients - Time for tolerable upper intake levels to address safety.

There is increasing interest by consumers, researchers, and regulators into the roles that certain bioactive compounds, derived from plants and other natural sources, can play in health maintenance and promotion, and even prolonging a productive quality of life. Research has rapidly emerged suggesting that a wide range of compounds and mixtures in and from plants (such as fruits and vegetables, tea and cocoa) and animals (such as fish and probiotics) may exert substantial health benefits. There is interest in exploring the possibility of establishing recommended intakes or dietary guidance for certain bioactive substances to help educate consumers. A key aspect of establishing dietary guidance is the assessment of safety/toxicity of these substances. Toxicologists need to be involved in both the development of the safety framework and in the evaluation of the science to establish maximum intake/upper limits.

Two new nontoxic, non-pathogenic strains of Sphingomonas elodea for gellan gum production.

Two new strains of Sphingomonas elodea (designated as PHP1 and PBAD1) were tested for toxicity and pathogenicity in healthy Sprague-Dawley CD(®) IGS rats in separate studies. In each study, twelve rats/sex were administered ≥10(8) viable cells/rat by oral gavage, and four untreated rats/sex served as controls. Blood, feces, and selected organs/tissues collected at various times over the course of the 22 day study were evaluated for the presence of PHP1 or PBAD1 (depending on the study) by a validated method, to determine the potential for survival, propagation, or infectivity of PHP1 and PBAD1 cells in the rat. No mortalities, test substance-related changes in clinical or macroscopic findings, body weight or body weight gain were observed in treated animals compared with controls, indicating a lack of toxicity. PHP1 or PBAD1 were not detected in the tissue, fecal or fluid samples collected from treated animals. Therefore, neither PHP1 nor PBAD1 were pathogenic or acutely toxic under the conditions of the studies.

Hydroxytyrosol: lack of clastogenicity in a bone marrow chromosome aberration study in rats.

BACKGROUND: Hydroxytyrosol is naturally found in olives, olive oil and wine, and is consumed as part of a normal diet. The substance may have utility as a preservative in a wide variety of foods due to its antioxidant, antimicrobial and amphipathic properties. The potential for hydroxytyrosol to cause chromosome aberrations in vitro had been tested previously, with positive results at high concentrations. An OECD Guideline 475 study (mammalian bone marrow chromosome aberration test) was conducted in rats with the oral limit dose of 2000 mg/kg bw to determine whether hydroxytyrosol is a clastogen in vivo. RESULTS: The oral limit dose of 2000 mg/kg hydroxytyrosol was well tolerated by most rats; however, some rats exhibited clinical signs that abated within 24 hours. Treatment with hydroxytyrosol did not significantly enhance the number of aberrant cells or the mitotic index 24 or 48 hours post-dose. The positive control (cyclophosphamide) induced the expected increase in chromosomal aberrations and a decrease in the mitotic index, confirming the validity of the assay. CONCLUSION: An oral limit dose of 2000 mg/kg hydroxytyrosol does not induce chromosome aberrations in bone marrow cells of the rat. Accordingly, hydroxytyrosol is not a clastogen in vivo.

Pre-clinical in vitro and in vivo safety evaluation of bovine whey derived osteopontin, Lacprodan® OPN-10.

Lacprodan® OPN-10 is a proprietary whey-based protein product that contains bovine-derived osteopontin (OPN), found in human milk and other bodily tissues. In vitro genotoxicity tests conducted according to accepted guidelines at up to 5000µg/plate OPN failed to induce genetic mutations in Salmonella typhimurium strains and Escherichia coli strain and did not induce chromosomal aberrations or cytotoxicity in human lymphocytes. Administration of an acute dose of Lacprodan® OPN-10 (2300mg/kg body weight) to male and female mice did not induce chromosomal damage or mitotic apparatus damage to erythroblasts from bone marrow. Lacprodan® OPN-10 was evaluated in a 13-week oral toxicity study in which rats were fed diets containing 0.5%, 1.0% and 2.0% Lacprodan® OPN-10. No test-article-related clinical observations or toxicological effects on body or organ weights, food consumption, ophthalmic effects, locomotor activity, hematology, clinical chemistry, urinalysis, or pathology were identified. In a teratogenicity study, administration of Lacprodan® OPN-10 up to 2500mg/kgbw/day via gavage to pregnant rats had no effect on dams or pups. The No Observed Adverse Effect Level (NOAEL) for Lacprodan® OPN-10 in the 13-week toxicity study was 2.0% of the diet (equivalent to 1208mg/kgbw/day in male rats and 1272mg/kgbw/day in female rats).

Safety studies conducted on a sanitizing agent containing benzalkonium chloride.

UNLABELLED: Free N Clear is a sanitizing agent composed of United States Pharmacopeial Convention grade benzalkonium chloride (BAC), acetic acid, and methylparaben. Free N Clear is proposed for use as a sanitizing agent at a 1: 50 dilution (2% solution), which contains approximately 100 ppm BAC. As part of a program to assess its safety, a 2% solution of Free N Clear (diluted Free N Clear) was administered by gavage to Sprague-Dawley rats for 91d and tested for genetic toxicity in vitro and in vivo. In the 91d study, the no observable adverse-effect level of diluted Free N Clear in male and female Sprague-Dawley rats is 5000 mg/kg bw/day, the highest dose administered. Diluted Free N Clear was not mutagenic in a bacterial reverse mutation assay that tested concentrations extending into the toxic range, and did not increase the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female Sprague-Dawley rats when tested at the maximum permissible dose volume of 20 mL/kg bw. The results support safety of Free N Clear, when used at the concentration proposed for use. PRACTICAL APPLICATION: The significance of these findings will allow for the development of Free N Clear as a potential sanitizing agent for food.

Safety evaluation of fibermalt.

Fibermalt is a new soluble fiber food ingredient produced with the use of an alternansucrase enzyme from Leuconostoc mesenteroides expressed in a non-pathogenic strain of Escherichia coli. Fibermalt is predominantly composed of indigestible maltose alternan oligosaccharides (≥ 80%). Fibermalt was non-mutagenic in a bacterial reverse mutation test. In a 13-week dietary rat study, fibermalt was administered at 0 (control), 50,000, 100,000 or 150,000 ppm. Statistically significant increases in food consumption were generally observed throughout the study in males receiving 100,000 or 150,000 ppm and in females receiving 100,000 ppm. However, there was no effect of fibermalt on mean body weight, body weight gain or food efficiency. All animals survived to scheduled termination and no adverse clinical signs were attributed to administration of fibermalt. There were no toxicologically relevant changes in hematology, clinical chemistry or urinalysis parameters or organ weights in males or females ingesting any concentration of fibermalt. Any macroscopic or microscopic findings were considered incidental, of normal variation and/or of minimal magnitude for test substance association. Based on these results, fibermalt is not mutagenic as evaluated in a bacterial reverse mutation test and has an oral subchronic (13-week) no observable adverse effect level (NOAEL) of 150,000 ppm in rats.

Safety evaluation of Lactobacillus pentosus strain b240.

Lactobacillus pentosus has a long history of use in cooked and uncooked fermented foods. Viable and heat-killed nonviable preparations of L. pentosus strain b240 were evaluated for short term and subchronic toxicity and genotoxic potential. Dose levels were determined through acute oral toxicity tests with viable (LD(50)>2500 mg/kg) and nonviable (LD(50)>2000 mg/kg) b240. In the short term study, rats received 2500 mg/kg/day (∼1.7×10(11)cfu/kg/day) viable b240 for 28 days. In the subchronic study, rats received 500, 1000 or 2000 mg/kg/day (up to ∼3.0×10(12) cfu equivalents/kg/day) nonviable b240 for 91 days followed by a 28-day recovery. No mortalities occurred. No treatment-related effects were identified for general condition, body weight, food-water consumption, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, histopathology and gross pathology. Although statistically significant effects were noted for several endpoints in the short term and subchronic studies, none were related to the test materials. The NOAEL for nonviable b240 was 2000 mg/kg/day, the highest dose tested. Additionally, nonviable b240 (≤ 5000 µg/plate) was not mutagenic in Salmonella typhimurium or Escherichia coli tester strains nor did nonviable b240 orally administered to rats at levels ≤ 2000 mg/kg/day for two days, induce a clastogenic response.

Evidence-based review on the effect of normal dietary consumption of fructose on blood lipids and body weight of overweight and obese individuals.

Although some investigators have hypothesized that ingestion of fructose from foods and beverages is responsible for the development of hyperlipidemia or obesity, a recent evidence-based review demonstrated that there was no relationship between the consumption of fructose in a normal dietary manner and the development of hyperlipidemia or increased weight in normal weight individuals. Because overweight and obese individuals may exhibit metabolic abnormalities such as insulin resistance, impaired glucose tolerance, hyperlipedemia, and/or alterations in gut hormones involved in appetite regulation, the findings of fructose studies performed in normal weight subjects may not be particularly relevant for overweight or obese subjects. A systematic assessment of the strength and quality of the studies and their relevance for overweight or obese humans ingesting fructose in a normal dietary manner has not been performed. The purpose of this review was to critically evaluate the existing database for a causal relationship between the ingestion of fructose in a normal, dietary manner and the development of hyperlipidemia or increased body weight in overweight or obese humans, using an evidence-based approach. The results of the analysis indicate that there is no evidence which shows that the consumption of fructose at normal levels of intake causes biologically relevant changes in triglycerides (TG) or body weight in overweight or obese individuals.

Evidence-based review on the effect of normal dietary consumption of fructose on development of hyperlipidemia and obesity in healthy, normal weight individuals.

In recent years, there has been episodic speculation that an increase in consumption of fructose from foods and beverages is an underlying factor responsible for the relatively recent increase in obesity and obesity-related diseases such as diabetes. Reports in support of this hypothesis have been published, showing that concentrations of triglycerides (TG) are higher and concentrations of insulin and hormones associated with satiety are lower in animals following the ingestion of fairly large quantities of fructose, compared to other carbohydrates. However, results from human studies are inconsistent. A possible reason for the inconsistent results is that they are dependent on the particular study population, the design of the studies, and/or the amount of fructose administered. A systematic assessment of the strength and quality of the studies and their relevance for healthy, normal weight humans ingesting fructose in a normal dietary manner has not been performed. The purpose of this review was to critically evaluate the existing database for a causal relationship between the ingestion of fructose in a normal, dietary manner and the development of hyperlipidemia or increased body weight in healthy, normal weight humans, using an evidence-based approach. The results of the analysis indicate that fructose does not cause biologically relevant changes in TG or body weight when consumed at levels approaching 95th percentile estimates of intake.

Naturally occurring food toxins.

Although many foods contain toxins as a naturally-occurring constituent or, are formed as the result of handling or processing, the incidence of adverse reactions to food is relatively low. The low incidence of adverse effects is the result of some pragmatic solutions by the US Food and Drug Administration (FDA) and other regulatory agencies through the creative use of specifications, action levels, tolerances, warning labels and prohibitions. Manufacturers have also played a role by setting limits on certain substances and developing mitigation procedures for process-induced toxins. Regardless of measures taken by regulators and food producers to protect consumers from natural food toxins, consumption of small levels of these materials is unavoidable. Although the risk for toxicity due to consumption of food toxins is fairly low, there is always the possibility of toxicity due to contamination, overconsumption, allergy or an unpredictable idiosyncratic response. The purpose of this review is to provide a toxicological and regulatory overview of some of the toxins present in some commonly consumed foods, and where possible, discuss the steps that have been taken to reduce consumer exposure, many of which are possible because of the unique process of food regulation in the United States.