Subchronic toxicity and mutagenicity/genotoxicity studies of Irvingia gabonensis extract (IGOB131)

African Bush Mango from Irvingia gabonensis is a West African culinary fruit and the mucilage from this fruit seed is used to make traditional soups and sauces. Extract from the kernel (IGOB131) has been claimed for its health benefits. In the present investigations, potential adverse effects, if any, of IGOB131 were investigated in dose–response 90-day study and genotoxicity studies. In the subchronic study, Sprague Dawley rats (20/sex/group) were gavaged with I. gabonensis extract (IGOB131) at dose levels of 0, 100, 1000 and 2500 mg/kg body weight (bw)/day for 90-days. No treatment-related changes in clinical signs, functional observations, mortality, ophthalmologic observations, body weights, body weight gain or feed consumption were noted. Similarly, hematological, clinical chemistry, urine analysis parameters, and organ weights did not reveal any toxicologically significant treatment-related changes. No treatment-related macroscopic and microscopic abnormalities were noted at the end of treatment period. The mutagenicity as evaluated by Ames assay, in vitro and in vivo chromosomal aberration test and in vivo micronucleus assay did not reveal any genotoxicity of IGOB131. The results of subchronic toxicity study suggest the no-observed-adverse-effect level (NOAEL) for I. gabonensis extract (IGOB131) as ?2500 mg/kg bw/day, the highest dose tested.     

Safety assessment of a solid lipid curcumin particle preparation: Acute and subchronic toxicity studies

Curcumin, a polyphenol, is obtained from turmeric, the ground rhizomes of Curcuma longa L. Extensive research over the past half century has revealed several health benefits of curcumin. The objective of the present study was to investigate potential adverse effects, if any, of a novel solid lipid curcumin particle (SLCP) preparation in rats following acute and subchronic administration. The oral LD₅₀ of the preparation in rats as well as in mice was found to be greater than 2000 mg/kg body weight (bw). In the subchronic toxicity study, Wistar rats (10/sex/group) were administered via oral gavage 0 (control), 180, 360, and 720 mg/kg bw/day of SLCP preparation for 90 days. Administration of the curcumin preparation did not result in any toxicologically significant treatment-related changes in clinical (including behavioral) observations, ophthalmic examinations, body weights, body weight gains, feed consumption, and organ weights. No adverse effects of the curcumin preparation were noted on the hematology, serum chemistry parameters, and urinalysis. Terminal necropsy did not reveal any treatment-related gross or histopathology findings. Based on the results of this study, the No Observed-Adverse-Effect Level (NOAEL) for this standardized novel curcumin preparation was determined as 720 mg/kg bw/day, the highest dose tested.     

Safety assessment of mushroom β-glucan: Subchronic toxicity in rodents and mutagenicity studies

Mushroom β-glucan, a polymer of β-(1,3/1,6)-glucan, has been claimed for its health benefits. The objective of this study was to assess the safety in-use of mushroom β-glucan as dietary supplement and food ingredient. Hence, a subchronic toxicity and mutagenicity studies were conducted. In the subchronic toxicity study, Sprague Dawley rats (12/sex/group) were administered (gavage) mushroom β-glucan at dose levels of 0, 500, 1000 and 2000 mg/kg body weight (bw)/day for 90 days. As compared to control group, administration of β-glucan did not result in any toxicologically significant treatment-related changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, and organ weights. No adverse effects of the β-glucan on the hematology, serum chemistry parameters, urinalysis or terminal necropsy (gross or histopathology findings) were noted. The results of mutagenicity studies as evaluated by gene mutations in Salmonella typhimurium, in vitro chromosome aberrations and in vivo micronucleus test in mouse did not reveal any genotoxicity of β-glucan. Based on the subchronic study, the no observed-adverse-effect level (NOAEL) for mushroom β-glucan was determined as 2000 mg/kg bw/day, the highest dose tested.     

Safety assessment of Cissus quadrangularis extract (CQR-300): Subchronic toxicity and mutagenicity studies

Cissus quadrangularis has been used for centuries for therapeutic and culinary purposes. Extract from this plant (CQR-300) has been claimed for its health benefits. The objective of present investigation was to delineate adverse effects, if any, of CQR-300 in subchronic toxicity, and gentotoxicity studies. In the subchronic study, Sprague Dawley rats (20/sex/group) were administered (gavage) C. quadrangularis extract (CQR-300) at dose levels of 0, 100, 1000, and 2500 mg/kg body weight (bw)/day for 90 days. No treatment related clinical signs of toxicity, mortality or changes in body weights, body weight gain or food consumption were noted. Functional observation tests and ophthalmological examination did not reveal any changes. No toxicologically significant treatment related changes in hematological, clinical chemistry, urine analysis parameters, and organ weights were noted. No treatment related macroscopic and microscopic abnormalities were noted at the end of treatment period. The results of mutagenicity studies as evaluated by Ames assay, in vitro chromosomal aberration and in vivo micronucleus assay did not reveal any genotoxicity of CQR-300. Based on the subchronic study, the no-observed-adverse-effect level (NOAEL) for C. quadrangularis extract (CQR-300) determined as 2500 mg/kg bw/day, the highest dose tested.     

Toxicologic evaluation of Dichrostachys glomerata extract: Subchronic study in rats and genotoxicity tests

In western Cameroon, edible fruits and seeds from the plant Dichrostachys glomerata are commonly used as spices. Extract from the fruit pods has been reported as a good natural source of antioxidants and may provide health benefits. The objective of the present study was to investigate potential adverse effects, if any, of D. glomerata fruit pod extract (Dyglomera™) in a subchronic toxicity study and in genotoxicity studies. In the toxicity study, Sprague Dawley rats (20/sex/group) were gavaged with D. glomerata extract at dose levels of 0, 100, 1000 and 2500 mg/kg body weight (bw)/day for 90-days. Dyglomera™ administration did not result in mortality or show treatmentrelated changes in clinical signs of toxicity, body weights, body weight gain or feed consumption. Similarly, no toxicologically significant treatment-related changes in hematological, clinical chemistry, urine analysis parameters, and organ weights were noted. Macroscopic and microscopic examinations did not reveal treatment-related abnormalities. Mutagenic and clastogenic potentials as evaluated by Ames assay, in vitro and in vivo chromosomal aberration test and in vivo micronucleus test did not reveal any genotoxicity of the extract. The results of subchronic toxicity study supports the no-observed-adverse-effect level (NOAEL) for D. glomerata extract as 2500 mg/kg bw/day, the highest dose tested.     

Safety assessment of astaxanthin-rich microalgae biomass: Acute and subchronic toxicity studies in rats

Astaxanthin, a natural nutritional component, is marketed as a dietary supplement around the world. The primary commercial source for astaxanthin is Haematococcus pluvialis (microalgae). The objective of the present study was to investigate the acute and subchronic toxicity of an astaxanthin-rich biomass of H. pluvialis (AstaCarox((R))). The oral LD(50) of the biomass in rats was greater than 12g/kg body weight. In the subchronic study, Wistar rats (10/sex/group) were fed diets containing 0%, 1%, 5% and 20% of the biomass (weight/weight) for 90 days. trans-Astaxanthin was quantifiable in the plasma of the high-dose treated group only. Compared to the control group, no treatment-related biologically significant effects of astaxanthin were noted on body weight or body weight gain. Biomass feeding did not affect hematological parameters. In the high-dose group, slightly elevated alkaline phosphatase and changes in some urine parameters and an increase in kidney weight in both sexes were noted. Histopathology examinations did not reveal adverse effects except for a marginal increase in pigment in the straight proximal tubule of the kidney in 5/10 female rats treated with the high-dose. These changes were not considered as toxicologically significant. Although the rats in high-dose group received about 9% more fat, it is unlikely that this confounding factor significantly altered the outcome. The no-observed adverse-effect-levels (NOAEL) of the astaxanthin-rich biomass for male and female rats were determined as 14,161 and 17,076mg/kg body weight/day, or 465 and 557mg astaxanthin/kg/day, respectively, the highest dose tested.     

Safety assessment of pomegranate fruit extract: Acute and subchronic toxicity studies

Pomegranate (Punica granatum L.) fruit is widely consumed as fresh fruit and juice. Because of its potential for health benefits, pomegranate fruit extracts have been commonly marketed as dietary supplements in recent years. The objective of the present study was to investigate potential adverse effects, if any, of a standardized pomegranate fruit extract in rats following subchronic administration. The extract was standardized to 30% punicalagins, the active anomeric ellagitannins responsible for over 50% of the antioxidant potential of the juice. The oral LD(50) of the extract in rats and mice was found to be greater than 5 g/kg body weight. The intraperitoneal LD(50) in rats and mice was determined as 217 and 187 mg/kg body weight, respectively. In the subchronic study, Wistar strain rats (10/sex/group) were administered via gavage 0 (control), 60, 240 and 600 mg/kg body weight/day of the extract for 90 days. Two additional groups received 0 and 600 mg/kg/day of the extract for 90 days, followed by a 28 day recovery phase. Compared to the control group, administration of the extract did not result in any toxicologically significant treatment-related changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, clinical pathology evaluations and organ weights. The hematology and serum chemistry parameters that showed statistical significant changes compared to control group were within the normal laboratory limits and were considered as biological variations and not the toxic effect of the extract. Terminal necropsy did not reveal any treatment-related gross or histopathology findings. Based on the results of this study, the no observed-adverse-effect level (NOAEL) for this standardized pomegranate fruit extract was determined as 600 mg/kg body weight/day, the highest dose tested.     

Safety assessment of N-acetyl-l-threonine

N-acetyl-l-threonine (NAT) is a dietary constituent that has been identified at low concentrations (<1 μg/g fresh weight) in numerous foods. The current paper reports the outcome of toxicology studies conducted to assess the effects of NAT. No evidence of mutagenicity or genotoxicity was observed in in vitro bacterial or in vivo mammalian studies. No mortalities or evidence of adverse effects were observed in Sprague–Dawley (SD) rats following acute oral administration of 2000 mg of NAT/kg of body weight (kg of bw). A 28-day repeated dose toxicity study was conducted in SD rats by incorporating NAT into diets at concentrations targeting up to 1000 mg of NAT/kg of bw/day. All rats survived until scheduled sacrifice and no biologically significant differences were observed in any of the NAT treatment groups for body weights, feed consumption, clinical signs, behavioral, ophthalmology, hematology, coagulation, clinical chemistry, organ weights, or gross or microscopic changes. Based on these results, NAT does not represent a risk for mutagenicity or genotoxicity, is not acutely toxic, and the no-observed-adverse-effect-level (NOAEL) for systemic toxicity from repeated dose dietary exposure to NAT is 848.5 and 913.6 mg/kg of bw/day for male and female SD rats, respectively.     

A subchronic oral toxicity study on pyrroloquinoline quinone (PQQ) disodium salt in rats

A subchronic oral toxicity study on pyrroloquinoline quinone (PQQ) disodium salt was performed in rats. Sprague-Dawley rats were randomly divided into four groups (10 rats/sex/group) and administered with PQQ disodium salt at doses of 0 (control), 100, 200 and 400 mg/kg bw/day by gavage for 13 weeks. Daily clinical observations and weekly measurement of body weights and food consumption were conducted. Blood samples were obtained on day 46 and day 91 for measurement of hematology and serum biochemical parameters. Animals were euthanized for necropsy, selected organs were weighted and recorded. Histological examination was performed on all tissues from animals in the control and PQQ disodium salt treatment groups. No mortality or toxicologically significant changes in clinical signs, body weight, food consumption, necropsy findings or organ weights was observed. Differences between treated and control groups in some hematological and serum biochemical examinations and histopathological examination were not considered treatment-related. The no-observed-adverse-effect-level (NOAEL) of PQQ disodium salt in rats was considered to be 400 mg/kg bw/day for both sexes, the highest dose tested.     

A 90-day oral (dietary) toxicity study of the 2R,4R-isomer of monatin salt in Sprague–Dawley rats

The root bark of Sclerochitin ilicifolius contains an intensely sweet substance analytically identified as isomers of 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid and generically coined “monatin.” Groups of male and female Crl:CD(SD) rats were fed 0 (control), 5000, 10,000, 20,000 or 35,000 ppm R,R-monatin salt in the diet for 90 days. There were no toxicologically relevant clinical or histopathological findings in any of the test article-treated groups. Significantly lower cumulative body weight gains were noted in the 35,000 ppm group. Mean body weights in the 35,000 ppm group males and females were 7% and 12% lower, respectively, than the control group at study week 13. In the absence of other observations associated with systemic toxicity and lower food consumption, the magnitude of the body weight difference in the 35,000 ppm group females relative to the control group exceeded 10%, which indicated attainment of a maximum tolerated dose (MTD) level. Based on the results of this study, and conservatively assuming the body weight observations at the MTD to be indicative of an adverse effect, the dietary no-observed-adverse-effect level (NOAEL) of R,R-monatin salt for 90 days was 20,000 ppm in female rats (approximately 1544 mg/kg bw/day) and 35,000 ppm in male rats (approximately 2368 mg/kg bw/day).     

Safety evaluation of a high lipid Whole Algalin Flour (WAF) from Chlorella protothecoides

Microalgae such as Chlorella spp. have a long history of use in human food. A high lipid Whole Algalin Flour (WAF) composed of dried milled Chlorella protothecoides was evaluated for subchronic toxicity and genotoxic potential. Likelihood of food allergy potential was also evaluated by human repeat-insult patch test. In the subchronic study, rats were fed dietary levels of 25,000, 50,000 or 100,000 ppm WAF in feed for 93–94 days. No mortalities occurred. No treatment-related effects were identified for general condition, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights, and histopathology. Although statistically significant effects were noted for several endpoints, none was test-substance related. The no-observed-adverse-effect level (NOAEL) for WAF was based on consumption of the 100,000 ppm diet, the highest dietary concentration tested, and was 4807 mg/kg bw/d in male rats and 5366 mg/kg bw/d in female rats. Additionally, WAF (?5000 μg/plate) was not mutagenic in Salmonella typhimurium or Escherichia coli tester strains nor did WAF induce a clastogenic response in bone marrow from mice given a single oral dose (2000 mg/kg bw). Further, WAF did not elicit skin sensitization in a repeat-insult dermal patch test which indicates little potential for food allergy.     

Acute and subchronic toxicity studies of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™) in rats

The potential use of pyrroloquinoline quinone disodium salt (BioPQQ™), as a supplemental food ingredient, was evaluated in a range of oral toxicity studies in rats including an acute study, a 14-day preliminary and a 28-day repeated-dose study, and a 13-week subchronic study. The median lethal dose of BioPQQ™ was shown to be 1000–2000 mg/kg body weight (bw) in male and 500–1000 mg/kg bw in female rats. In the 14-day study, high doses of BioPQQ™ resulted in increases in relative kidney weights with associated histopathology in female rats only, while a follow-up 28-day study in female animals resulted in increases in urinary protein and crystals. These findings were reversible, and resolved during the recovery period. In the 13-week study, a number of clinical chemistry findings and histopathological changes were noted, which were deemed to be of no toxicological significance, as the levels 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. Based on these findings, a no-observed-adverse-effect level of 100 mg/kg bw/day was determined for BioPQQ™ in rats, the highest dose tested in the 13-week study.     

Safety evaluation of DHA-rich Algal Oil from Schizochytrium sp.

The safety of DHA-rich Algal Oil from Schizochytrium sp. containing 40–45 wt% DHA and up to 10 wt% EPA was evaluated by testing for gene mutations, clastogenicity and aneugenicity, and in a subchronic 90-day Sprague–Dawley rat dietary study with in utero exposure, followed by a 4-week recovery phase. The results of all genotoxicity tests were negative. In the 90-day study, DHA-rich Algal Oil was administered at dietary levels of 0.5, 1.5, and 5 wt% along with two control diets: a standard low-fat basal diet and a basal diet supplemented with 5 wt% of concentrated Fish Oil. There were no treatment-related effects of DHA-rich Algal Oil on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, coagulation, or urinalysis. Increases in absolute and relative weights of the liver, kidney, spleen and adrenals (adrenals and spleen with histological correlates) were observed in both the Fish Oil- and the high-dose of DHA-rich Algal Oil-treated females and were not considered to be adverse. The no observed adverse effect level (NOAEL) for DHA-rich Algal Oil under the conditions of this study was 5 wt% in the diet, equivalent to an overall average DHA-rich Algal Oil intake of 4260 mg/kg bw/day for male and female rats.     

Subchronic oral toxicity and cardiovascular safety pharmacology studies of resveratrol, a naturally occurring polyphenol with cancer preventive activity

To characterize the subchronic oral toxicity of resveratrol, CD rats received daily gavage doses of 0, 200, 400, or 1000 mg resveratrol/kg/day, and beagle dogs received daily capsule doses of 0, 200, 600, or 1200 mg resveratrol/kg/day for 90 days. Resveratrol induced only minimal toxicity, consisting of dose-related reductions in body weight gain in female rats and both sexes of dogs, and a statistically significant increase in bilirubin levels in rats at the 1000 mg/kg/day dose. Clinical observations, hematology, ophthalmology, neurotoxicity evaluations (functional observational batteries), organ weights, and gross pathology provided no biologically significant evidence of resveratrol toxicity in either species. In rats, the high dose of resveratrol reduced the incidence of cardiomyopathy; no other microscopic changes were seen. Histopathologic changes in dogs were limited to minimal inflammatory infiltrates in the kidney and urinary bladder, which were not considered toxicologically significant. A cardiovascular safety pharmacology (telemetry) study in dogs revealed no evidence of resveratrol toxicity. Based on body weight effects, the No Observed Adverse Effect Level (NOAEL) for resveratrol was 200 mg/kg/day in rats and 600 mg/kg/day in dogs. The apparent cardioprotective activity of resveratrol in rats demonstrates that its potentially beneficial activities may extend beyond efficacy in cancer prevention.     

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.     

Safety evaluation of alpha-lipoic acid (ALA)

The safety of the antioxidant alpha-lipoic acid (racemic form) (ALA), also called thioctic acid (CAS RN 1077-28-7) was assessed in acute and subchronic toxicity studies as well as in in vitro and in vivo mutagenicity/genotoxicity studies. ALA was not acutely toxic to rats (LD(50)>2000mg/kg bw, OECD method 425). Administration of 31.6 or 61.9mg ALA/kg bw/day for 4 weeks to male/female Wistar rats did not show any adverse effects. Specifically, there was no significant difference between control and treated animals at 31.6 or 61.9mg ALA/kg bw with regard to body weight gain, feed consumption, animal behaviour, or haematological and clinical chemistry parameters. Only the high-dose of 121mg ALA/kg bw was associated with slight alterations in liver enzymes as well as histopathological effects on the liver and mammary gland. ALA did not possess any mutagenic activity in the Ames assays conducted with various bacterial strains of Salmonella typhimurium. Moreover, there was no evidence of genotoxic activity in a mouse micronucleus assay. The results of these studies support the safety of ALA. The no-observed-adverse-effect level (NOAEL) is considered to be 61.9mg/kg bw/day.     

Toxicological evaluation of an olive extract,H35: Subchronic toxicity in the rat

The safety of olive extract H35 containing 35% hydroxytyrosol (HT) was tested in a 90-day oral gavage study in Wistar rats. H35 was administered at 0, 345, 691 and 1381 mg/kg bw/day, equivalent to 0, 125, 250 and 500 mg HT/kg bw/day. Reductions in terminal body weight (9%), and a statistically significant reduction in body weight gain (17%, P < 0.05) at week 13 were observed in high dose males, as well as a statistically significant increase in relative weights of the liver, heart, and kidneys of high dose males and females. These changes were not accompanied by pathological or clinical observations and a trend towards reversal was observed in the recovery phase. H35 was well-tolerated and no toxicologically significant treatment-related changes were observed in condition and appearance of rats, neurobehavioral outcomes, motor activity assessments, functional observational battery (FOB), food intake, ophthalmoscopic examinations, hematology, clinical chemistry, urinalysis, necropsy findings, sperm parameters or estrus cycle. The lowest observed adverse effect level (LOAEL) was the 500 mg HT/kg bw/day based on statistically significant reductions in body weight gain and decreased body weight in males. The no observed adverse effect level (NOAEL) was 250 mg HT/kg bw/day, equivalent to 691 mg/kg bw/day of H35 extract.     

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.     

Acute, subchronic and genotoxicity studies conducted with Oligonol, an oligomerized polyphenol formulated from lychee and green tea extracts

Oligonol is a phenolic product derived from lychee fruit extract and green tea extract, containing catechin-type monomers and oligomers of proanthocyanidins, produced by a manufacturing process which converts polyphenol polymers into oligomers. The safety of Oligonol was assessed in acute and subchronic studies and genotoxicity assays. In a single dose acute study of Oligonol, male and female rats were administered 2000mg/kg body weight (bw) Oligonol in water by gavage. Oligonol caused no adverse effects and body weight gain and food consumption were within normal range, thus the LD(50) of Oligonol was determined to be greater than 2000mg/kg. A 90 day subchronic study (100, 300 and 1000mg/kgbw/day, oral gavage) in male and female rats reported no significant adverse effects in food consumption, body weight, mortality, clinical chemistry, haematology, gross pathology and histopathology. Similarly, no adverse effects were observed in mice fed diets providing 2, 20 or 200mg/kgbw Oligonol or 200mg/kgbw lychee polyphenol for 90 days. Oligonol did not show any potential to induce gene mutations in reverse mutation tests using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA strains. Oligonol did not induce chromosomal aberrations in cultured Chinese hamster lung cells, but it showed increased polyploidy. In a micronucleus assay in mice, Oligonol did not induce any micronuclei or suppress bone marrow, indicating it does not cause chromosome aberrations. The results from these safety studies and previous reports support the safety of Oligonol for human consumption.     

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.