Toxicology of 3-epi-deoxynivalenol,a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8

Microbial detoxification of deoxynivalenol (DON) represents a new approach to treating DON-contaminated grains. A bacterium Devosia mutans 17-2-E-8 was capable of completely transforming DON into a major product 3-epi-DON and a minor product 3-keto-DON. Evaluation of toxicities of these DON-transformation products is an important part of hazard characterization prior to commercialization of the biotransformation application. Cytotoxicities of the products were demonstrated by two assays: a MTT bioassay assessing cell viability and a BrdU assay assessing DNA synthesis. Compared with DON, the IC₅₀ values of 3-epi-DON and 3-keto-DON were respectively 357 and 3.03 times higher in the MTT bioassay, and were respectively 1181 and 4.54 times higher in the BrdU bioassay. Toxicological effects of 14-day oral exposure of the B6C3F₁ mouse to DON and 3-epi-DON were also investigated. Overall, there were no differences between the control (free of toxin) and the 25 mg/kg bw/day or 100 mg/kg bw/day 3-epi-DON treatments in body and organ weights, hematology and organ histopathology. However, in mice exposed to DON (2 mg/kg bw/day), white blood cell numbers and serum immunoglobulin levels were altered relative to controls, and lesions were observed in adrenals, thymus, stomach, spleen and colon. Taken together, in vitro and in vivo studies indicate that 3-epi-DON is substantially less toxic than DON.     

In vitro and in vivo safety evaluation of Nephure™

Nephure™ is a proprietary oxalate decarboxylase (OxDC) enzyme being developed as a food ingredient. In this study, the safety of Nephure™ was evaluated in a bacterial mutagenicity assay and in a sub-chronic (13-week) oral toxicity study in rats. Nephure™ did not show any mutagenic properties in the mutagenicity assay. In the 13-week sub-chronic oral toxicity study in which 10 Sprague Dawley rats per sex were administered 0, 118, 235 and 475 mg/kg bw/day (8260, 16450 and 33,250 Units/kg bw/day, respectively) of Nephure™ by gavage, male and female rats did not show any test article-related clinical observations or effects on body weight, body weight gain, food consumption, food efficiency, ophthalmology, functional observational battery parameters or motor activity. Furthermore, there were no changes in coagulation, clinical chemistry, urinalysis or hematology parameters, macroscopic/microscopic findings or organ weights that could be attributed to the test article. Based on these results, Nephure™ was not mutagenic and the no-adverse-effect level (NOAEL) in the 13-week study was determined to be 475 mg/kg bw/day (33,250 Units/kg bw/day). Evaluation of the estimated consumption of Nephure™, generation of the metabolite formate, and the current safety studies resulted in a conclusion of a tolerable upper limit of 3450 Units of OxDC activity/day (57.5 Units activity/kg bw/day), when Nephure™ is added to food to decrease dietary oxalate.     

A subchronic toxicity study of Radix Dipsaci water extract by oral administration in F344 rats

Radix Dipsaci, the dried root of Dipsacus asperoides C.Y. Cheng & T.M.Ai, has therapeutic effects on various disorders, and in particular, bone and joint disease. Despite such ethnomedicinal benefits, there is very little information regarding its in vivo toxicity or adverse effects. This study was conducted to evaluate the potential toxicity of the Radix Dipsaci water Extract (RD-wE) by using F344 rats. The RD-wE was administered orally to rats at doses of 0, 125, 250, 500, 1000, and 2000 mg/kg body weight (bw)/day for 13 weeks. During the treatment period there were no mortalities attributed to RD-wE. Moreover, no toxic effects were observed with regard to body weight, clinical pathology (hematology, clinical biochemistry, and urinalysis), and anatomic pathology (gross findings, organ weight, and microscopic examination). The changes related to the treatment were excessive salivation at the mouth and soft feces, observed in male and female rats at 1000 or 2000 mg/kg bw/day, but these were not accompanied by any microscopic correlate or other pathophysiological changes. Based on these results, the oral no-observed-adverse-effect level of the RD-wE was considered to be 2000 mg/kg bw/day in both genders, although the target organs were not determined under the current experimental conditions.     

Genotoxicity,acute and subchronic toxicity evaluation of savory food ingredients

The potential toxicity of two savory food ingredients produced by fermentation of enzymatically hydrolyzed corn starch (Savory Base 100 and Savory Base 200) was evaluated individually in a bacterial reverse mutation assay, an in vitro mammalian cell gene mutation assay, an acute oral study and as a mixture in a 90-day dietary study. In the bacterial reverse mutation and in vitro mammalian cell gene mutation assays, neither ingredient was mutagenic at concentrations up to 5000 μg/plate and 5000 μg/mL, respectively in the presence and absence of metabolic activation. In the acute study, the no-observed-adverse-effect level (NOAEL) for each Savory Base 100 and Savory Base 200 in male and female rats was 2000 mg/kg body weight. In the 90-day study, the hematology and clinical chemistry findings and histopathological changes noted in the liver, heart and kidneys were deemed to be of no toxicological significance, as the mean values were within the historical control range, were not dose-dependent, occurred at a similar frequency in control groups, or only occurred in the control group. Considering these findings, the NOAEL for Savory Base 100 and Savory Base 200 was 2333 and 1167 mg/kg body weight, respectively, the highest dose tested in each case.     

Safety evaluation of AMP deaminase from Aspergillus oryzae

Adenosine-5′-monophosphate (AMP) deaminase is an enzyme used to increase concentrations of 5’-inosine monophosphate in certain foods and beverages for flavoring purposes. One commercial source of this enzyme is Aspergillus oryzae, a filamentous fungus with a history of safe use in Asia as a fermentation organism used in the production of miso sauce and sake liquors. Noting the use of the enzyme in food intended for human consumption and potential presence at trace levels in finished goods, a series of safety studies including an in vitro Ames test and chromosome aberration assay with Chinese hamster lung fibroblasts were conducted along with a 90-day oral toxicity study in rats. AMP deaminase showed no evidence of genotoxicity in the in vitro tests. Following gavage administration of Sprague–Dawley rats at dosages of 19.8, 198.4, or 1984 mg total organic solids (TOS)/kg body weight (bw)/day for 90 days, no adverse effects on body weight gain, food consumption, hematology, clinical chemistry, urinalysis, ophthalmological and histopathological examinations were observed. The no-observed-adverse-effect level was considered to be 1984 mg TOS/kg bw/day, the highest dose tested. Results of the genotoxicity studies and subchronic rat study support the safe use of AMP deaminase produced from A. oryzae in food production.     

Safety evaluation of oleic-rich triglyceride oil produced by a heterotrophic microalgal fermentation process

Numbers of macro- and microalgae have been used as food sources in various cultures for centuries. Several microalgae are currently being developed as modern food ingredients. The dietary safety of oleic-rich microalgal oil produced using a heterotrophic fermentation process was assessed in a 13-week feeding trial in rats with genotoxic potential evaluated using in vitro and in vivo assays. In the genotoxicity assays, the test oil was not mutagenic in Salmonella typhimurium or Escherichia coli tester strains (⩽5000 μg/plate) with or without metabolic activation. Further, no clastogenic response occurred in chromosome aberration assays in the bone marrow of mice administered a single intraperitoneal dose (2000 mg/kg). In the subchronic study, rats consumed feed containing 0, 25,000, 50,000 or 100,000 ppm oleic-rich oil for 90 days. No treatment-related mortalities or adverse effects occurred in general condition, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights or histopathology. Although several endpoints exhibited statistically significant effects, none were dose-related or considered adverse. Taking all studies into consideration, the NOAEL for the oleic-rich oil was 100,000 ppm, the highest concentration tested and equivalent to dietary NOAELs of 5200 mg/kg bw/day and 6419 mg/kg bw/day in male and female rats, respectively.     

A 28-day oral toxicity study of echimidine and lasiocarpine in Wistar rats

Pyrrolizidine alkaloids (PAs) are a class of naturally-occurring plant toxins. Echimidine is one of the predominant PAs found in honeys produced in Australia and New Zealand. There is a lack of information on the oral toxicity of echimidine on which to base regulatory decisions concerning the risk to humans of these honeys. This GLP study was conducted to assess the subchronic dietary toxicity of echimidine to rats compared to that of lasiocarpine as a positive control. Wistar rats, 10/sex, were fed diets containing 0, 0.6, 1.2 or 2.5 mg/kg bw echimidine. Positive control groups, 10/sex, were fed diets containing 0.6, 1.2 or 2.5 mg/kg bw lasiocarpine.Neither PA had any effect on survival, food consumption, clinical signs, gross lesions, or histopathology. Consumption of lasiocarpine, but not echimidine, decreased bodyweight gain in males at ≥ 1.2 mg/kg bw, and in females at 2.5 mg/kg bw. Slight alterations in white cell counts and serum ALT concentrations at 2.5 mg/kg bw of both PAs were not clinically significant, had no histological correlates, and were considered to be of equivocal relevance.In conclusion, the subchronic No Observed Adverse Effect Level (NOAEL) for echimidine is 2.5 mg/kg bw/day, whereas, on the basis of a treatment-related decrease in bodyweight gain in males at 1.2 mg/kg bodyweight, the NOAEL for lasiocarpine is 0.6 mg/kg bw/day.     

A comprehensive study on in vitro and in vivo toxicological evaluation of Artemisia capillaris

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.     

Absence of subchronic oral toxicity and genotoxicity of rice koji with Aspergillus terreus

Koji products have been considered as an effective fermented food consumed in East Asia with many health benefits. Particularly, rice koji with Aspergillus terreus (RAT) has been reported to be able to prevent hyperlipidemia and hepatic steatosis through regulating cholesterol synthesis. Despite its biological activities, there is a lack of comprehensive information to give an assurance of its safety. Therefore, the objective of this study was to perform a series of toxicological studies (repeated dose oral toxicity and genotoxicity) according to test guidelines published by the Organization for Economic Cooperation and Development. Along with acute toxicity study using rats and beagle dogs, a 13-week toxicity study revealed no clear RAT-related toxic changes, including body weight, mortality, hematology, serum biochemistry, organ weight, and histopathology after oral administration at doses of 500, 1000, and 2000 mg/kg BW. The no-observed-adverse-effect level of RAT was considered to be more than 2000 mg/kg BW/day in rats of both genders. In addition, potential genotoxicity was evaluated using a standard battery of tests (Ames test, chromosome aberration assay, and micronucleus assay) which revealed that RAT showed no genotoxicity. Accordingly, these results suggest that RAT is a safe and non-toxic functional food for human consumption at proper dose.     

Delamanid is not metabolized by Salmonella or human nitroreductases: A possible mechanism for the lack of mutagenicity

Nitro-containing compounds such as nitrofuran and nitroimidazole are drugs used for the treatment of infectious diseases. However, many of these nitro-containing drugs are positive in the bacterial reverse mutation assay (Ames test). The recently approved anti-multidrug-resistant tuberculosis (MDR-TB) drug, delamanid (Deltyba™; OPC-67683), a derivative of 4-nitroimidazole, was negative for mutagenicity in the Ames assay. In Salmonella typhimurium, mutagenicity of nitro compounds has been closely associated with the ability of nitroreductase to metabolize (degradation)these compounds. To explore the lack of mutagenicity for delamanid, we examined the initial metabolic rate and mutagenic-specific activity of a series of nitro compounds in S. typhimurium TA100. The order of maximum mutagenic-activity was nitrofuran > 2-nitroimidazole > 5-nitroimidazole ≥ 4-nitroimidazole, which is very similar to the order of initial metabolic rate, i.e., the Pearson's correlation coefficient (r = 0.85) showed a correlation between metabolic rate and mutagenic-activity. No metabolism of delamanid was detected even after 60 h of treatment. In addition, delamanid was not reduced by two human nitroreductases. These facts may explain the absence of genotoxicity of delamanid in both in vitro and in vivo tests.     

Acute and subacute toxicity studies of CMICE-013, a novel iodinated rotenone-based myocardial perfusion tracer,in Sprague Dawley rats and Gottingen minipigs

PurposeExtensive acute and subacute toxicities studies are required to evaluate the toxicological profile of the novel cardiac perfusion imaging tracer ¹²³I-CMICE-013 to support applications for clinical trials.MethodsSprague-Dawley rats and Gottingen minipigs received injections of non-radioactive 127I-CMICE-013 at two dosage levels of 1 and 5 μg/kg, and vehicle buffer as control. In the acute toxicity studies, each animal was injected on two occasions 24 h apart and then underwent a 14-day recovery period; in the subacute study, animals received daily injections for 14 days continuously. The health status and mortality of test animals were monitored daily and body weight, food consumption, physiological and biochemical parameters were measured at various time points during the study. Animals were euthanized at the end of the studies and dissected for pathologic examination of organs and tissues.ResultsThe acute and subacute administrations of injections of the non-radioactive CMICE-013 in rats and minipigs were well tolerated. Little to no dosing-related adverse effects were observed in animal body and organ weights, hematology, coagulation, clinical chemistry, urinalysis, ophthalmoscopy, electrocardiograms, heart rates, blood pressure, macroscopic and microscopic examination of the preserved animal tissues including the brain.ConclusionThe lack of adverse effects from acute and subacute dosing suggest that the CMICE-013 injection solution has a reasonable safety margin within the designed concentration range to be utilized in imaging applications. The dosage level of 5 μg/kg was considered the no adverse effect level for both rats and minipigs based on our acute and subacute studies.     

Estimation of in vivo and in vitro exposure to bisphenol A as food contaminant

The goal of this cross-sectional study was to examine the occurrence of bisphenol A (BPA) in the morning spot urine taken from 145 female volunteers of various ages. Total urine BPA concentration was detected in 38.6% samples in the 0.92–70.96 μg/g Cr range. The majority of BPA + women belonged to the 25 + body mass index (BMI) group (54.5% were overweight and 43.4% were obese women). Occurrence of BPA in the urine samples was higher at 40 + ages. The maximum BPA concentration of 70.96 μg/g Cr was detected in the urine sample of an obese woman. It is known that BPA is highly toxic in vitro. In this study BPA impaired significantly the growth of all investigated cell lines, i.e. the EC₅₀ values were reached at very low concentrations, in the range from 3.24 to 34.85 μg/mL. The obtained in vivo results suggest that a higher exposure to BPA could contribute to weight problems in women and the absence of the BPA in vitro selective toxicity studies indicates to its general toxic mode of action and raises awareness of the health risks associated with its ubiquitous presence in the environment.     

Chemical-specific adjustment factors (inter-species toxicokinetics) to establish the ADI for steviol glycosides

The acceptable daily intake (ADI) of commercially available steviol glycosides is currently 0–4 mg/kg body weight (bw)/day, based on application of a 100-fold uncertainty factor to a no-observed-adverse-effect-level value from a chronic rat study. Within the 100-fold uncertainty factor is a 10-fold uncertainty factor to account for inter-species differences in toxicokinetics (4-fold) and toxicodynamics (2.5-fold). Single dose pharmacokinetics of stevioside were studied in rats (40 and 1000 mg/kg bw) and in male human subjects (40 mg/kg bw) to generate a chemical-specific, inter-species toxicokinetic adjustment factor. T_max values for steviol were at ∼8 and ∼20 h after administration in rats and humans, respectively. Peak concentrations of steviol were similar in rats and humans, while steviol glucuronide concentrations were significantly higher in humans. Glucuronidation in rats was not saturated over the dose range 40–1000 mg/kg bw. The AUC_0-last for steviol was approximately 2.8-fold greater in humans compared to rats. Chemical-specific adjustment factors for extrapolating toxicokinetics from rat to human of 1 and 2.8 were established based on C_max and AUC_0-last data respectively. Because these factors are lower than the default value of 4.0, a higher ADI for steviol glycosides of between 6 and 16 mg/kg bw/d is justified.     

Assessment of oxidative stress responses and the cytotoxic and genotoxic potential of the herbicide tembotrione in HepG2 cells

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus “cytome” assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.     

Embryo-fetal development toxicity of honokiol microemulsion intravenously administered to pregnant rats

The aim of this study was to evaluate the embryo-fetal development toxicity of honokiol microemulsion. The drug was intravenously injected to pregnant SD rats at dose levels of 0, 200, 600 and 2000 μg/kg/day from day 6–15 of gestation. All the pregnant animals were observed for body weights and any abnormal changes and subjected to caesarean-section on gestation day (GD) 20; all fetuses obtained from caesarean-section were assessed by external inspection, visceral and skeletal examinations. No treatment-related external alterations as well as visceral and skeletal malformations were observed in honokiol microemulsion groups. There was no significant difference in the body weight gain of the pregnant rats, average number of corpora lutea, and the gravid uterus weight in the honokiol microemulsion groups compared with the vehicle control group. However, at a dose level of 2000 μg/kg/day, there was embryo-fetal developmental toxicity observed, including a decrease in the body length and tail length of fetuses. In conclusion, the no-observed–adverse-effect level (NOAEL) of honokiol microemulsion is 600 μg/kg/day, 75 times above the therapeutic dosage and it has embryo-fetal toxicity at a dose level of 2000 μg/kg/day, which is approximately 250 times above the therapeutic dosage.     

A dietary two-generation reproductive toxicity study of (2R,4R)-monatin salt in Crl:CD(SD) rats

(2R,4R)-Monatin salt [sodium/potassium 2R,4R-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate] was fed at 5000, 15,000, or 35,000 ppm to Crl:CD(SD) rats over two generations. Reduced body weights were observed at all dose levels. Sustained effect on body weight gain at 35,000 ppm in the F₀ and F₁ parental animals was associated with lower feed efficiency, soft stool, and slightly lower numbers of implantation sites. Lower numbers of pups born and live litter size at 35,000 ppm were considered secondary to slightly lower numbers of former implantation sites in the dams. Spermatogenic endpoints, estrous cyclicity, reproductive performance, mean gestation length, and parturition were unaffected in the F₀ and F₁ generations. There were no effects on F₁ and F₂ generation postnatal survival. Reduced pre-weaning pup body weights at 35,000 ppm resulted in lower F₁ and F₂ body weights at study termination. Slight delays in pubertal landmarks in the F₁ offspring were considered secondary to the reduced pup body weights. The no-observed-adverse-effect level (NOAEL) was 15,000 ppm for systemic, reproductive, and neonatal effects based on test article-related effects on body weight and food efficiency, slight decrease in maternal implantation sites and corresponding reduction in live litter size, and reductions in pre-weaning pup body weights at 35,000 ppm.     

Acute and subchronic toxicity studies of seabuckthorn (Hippophae rhamnoides L.) oil in rodents

Seabuckthorn (Hippophae rhamnoides L.) has been traditionally used as medicine and nutritional supplement for a long period of time. However, information on the systemic toxicity and safety evaluation of seabuckthorn and its extracts is still scarce. The purpose of this study was to evaluate the potential toxicity of seabuckthorn oil by an acute oral toxicity study in mice and a 90-day repeated oral toxicity study in rats. No mortality or signs of toxicity was observed in mice treated with 20 mL/kg body weight seabuckthorn oil in the acute toxicity study. In the subchronic toxicity study, 80 Sprague-Dawley rats (10 animals per sex per treatment group) were administrated with 10, 5, 2.5 and 0 (control) mL/kg body weight of seabuckthorn oil daily for 90 days by gavage. There were no signs of toxicity and treatment-related changes in rats treated with seabuckthorn oil on mortality, body and organ weights, food consumption, blood biochemistry and hematology, gross necropsy and histopathological examinations. Based on the finding of this study, the maximum tolerated dose of seabuckthorn oil was >20 mL/kg for mice for acute toxicity study, and the no-observed-adverse-effect level was 10 mL/kg body weight for both male and female rats for 90-day toxicity study.     

Cyto-genotoxicity and oxidative stress induced by zinc oxide nanoparticle in human lymphocyte cells in vitro and Swiss albino male mice in vivo

ZnO-np has immense potential and application in cosmetic and health care sectors. Hence it was imperative to assess the toxicity/safety of these nanoparticles. In this study, we have evaluated the effects of ZnO-np in human peripheral blood mononuclear cells (PBMCs) in vitro and in Swiss albino male mice in vivo for cyto-genotoxicity and oxidative damage. In vitro results showed that ZnO-nps were weakly genotoxic, induced significant decrease in mitochondrial membrane potential and was capable of ROS generation, leading to apoptosis. In bone marrow cells in vivo, reduction of mitochondrial membrane potential (MMP), increased oxidative stress and G0/G1 cell cycle arrest was observed along with chromosome aberrations and micronuclei formation. In liver cells DNA damage and induction of oxidative stress with concurrent decrease in inhibition of antioxidant enzymes were noted. These in vitro and in vivo results demonstrated that ZnO-np induced genotoxic response and ROS production leading to apoptotic cell death and established a good co-relation between the two biological systems. More importantly, the results stress on the need of multiple endpoint assay-approaches, with an in vitro-in vivo study design to assess nanoparticle toxicology.     

Genotoxicity and subchronic toxicity evaluation of dried Euglena gracilis ATCC PTA-123017

Euglena gracilis is a microalga capable of synthesizing various nutrients of interest in human and animal nutrition. When cultivated aerobically in the dark, Euglena synthesize paramylon, a storage polysaccharide comprised of high molecular weight beta-1,3-D-glucose polymers organized in cytoplasmic granules. Beta-glucans have been shown to have immune modulation effects, including anti-microbial, anti-tumor, and anti-oxidant properties, and metabolic effects, such as regulation of cholesterol and blood sugar levels. Preparations of E. gracilis and paramylon may therefore have potential utility as functional food ingredients for human and animal nutrition. A battery of toxicological studies was conducted on a dried preparation of E. gracilis and paramylon to support their safe food use. The dried alga was not genotoxic in a bacterial reverse mutation test and mammalian micronucleus test. In the subchronic toxicity study, rats were provided E. gracilis in the diet at levels of 0, 12,500, 25,000 or 50,000 ppm. Paramylon was provided at a concentration of 50,000 ppm. No effects that could be attributable to treatment were observed in clinical observations, body weight, food consumption, ophthalmology, hematology and clinical chemistry, urinalysis, and macroscopic and microscopic findings. A NOAEL of 50,000 ppm in the diet was determined for both ingredients.