Safety evaluation of a ß-mannanase enzyme preparation produced with Thermothelomyces thermophilus expressing a protein-engineered ß-mannanase gene.

Mannanase 19287 enzyme is an engineered ß-mannanase that can be added to diets for animals raised for human consumption to hydrolyze ß-mannans. Established toxicological analyses were conducted with the enzyme preparation to ensure the safety of this product for the intended use. The mannanase 19287 preparation was produced with Thermothelomyces thermophilus strain DSM 33149. In vitro toxicity studies presented here used dosages of the mannanase 19287 test articles up to 5000 µg/plate. For in vivo toxicity studies in Wistar rats, test articles were administered at 5.1 mg/L for inhalation toxicity and up to 15,000 mg/kg rat feed for oral toxicity, based on the Total Organic Solids (TOS) content in each test article. No treatment related adverse effects were reported in any study. The No Observed Adverse Effect Levels in the high dose group of the subchronic oral toxicity study were calculated as 1117-1298 mg TOS/kg bw/day in rats. Comparing these values to an Estimated Daily Intake for poultry demonstrated safety factors larger than 5000. Our results confirm that T. thermophilus fulfills the recognized safety criteria for the manufacture of food enzyme preparations and represent the first peer-reviewed safety evaluation of an enzyme preparation by T. thermophilus. The results of the toxicity studies presented herein attest to the safety of the mannanase 19287 enzyme for its intended use.

Safety evaluation of xylanase 50316 enzyme preparation (also known as VR007), expressed in Pseudomonas fluorescens, intended for use in animal feed.

Xylanase 50316 enzyme (also known as VR007) can be added to the diets of monogastric animals to hydrolyze the glycosidic linkages of xylans that are found in these animal feeds. This enzyme was produced from a Pseudomonas fluorescens (P. fluorescens) production strain and was tested in acute, subchronic, and genotoxicity studies. Dosages of the test article preparation ranged from 5000 µg/plate for in vitro toxicity studies to 2000 mg/kg/day for in vivo toxicity studies. The highest oral dose tested in vivo (NOAEL of 2000 mg/kg/day) resulted in a safety margin of 7226 based on TOS and a conservative estimate of total poultry consumption at the highest inclusion rate. There was no toxicity reported in any of the studies conducted. A review of the public literature indicated that P. fluorescens fulfilled the recognized safety criteria pertinent to microbial production strains used in the manufacture of food/feed enzyme preparations. The results of the toxicity studies presented herein attest to the safety of the xylanase 50316 enzyme for its intended use.

Letter to the editor regarding "GRAS from the ground up: Review of the Interim Pilot Program for GRAS notification" by.

Present letter is aimed at clarifying some critical points highlighted by Hanlon et al. regarding the common knowledge element of the safety of food enzymes in support of their GRAS designation. Particularly, we outline the development of peer-reviewed, generally recognized safety evaluation methodology for microbial enzymes and its adoption by the enzyme industry, which provides the US FDA with a review framework for enzyme GRAS Notices. This approach may serve as a model to other food ingredient categories for a scientifically sound, rigorous, and transparent application of the GRAS concept.

Safety evaluation of phytase 50104 enzyme preparation (also known as VR003), expressed in Pseudomonas fluorescens, intended for increasing digestibility of phytate in monogastrics.

Phytase 50104 enzyme (also known as VR003) can be added to swine and poultry diets to catalyze the hydrolysis of phosphate from phytic acid, thereby increasing phosphorus bioavailability in these animals. This enzyme was produced from a Pseudomonas fluorescens (P. fluorescens) production strain and was tested in acute, subchronic and genotoxicity studies. Dosages of the test article preparation ranged from 5000µg/plate for in vitro toxicity studies to 2000mg/kg/day for in vivo toxicity studies. The highest oral dose tested in vivo (NOAEL of 2000mg/kg/day) resulted in a safety margin of 5870 based on TOS and a conservative estimate of total poultry consumption at the highest inclusion rate. There was no toxicity reported for any of these studies or in the following additional safety studies: eye irritation, dermal irritation, and delayed hypersensitivity studies. A review of the public literature indicated that P. fluorescens fulfilled the recognized safety criteria pertinent to microbial production strains used in the manufacture of food/feed enzyme preparations. The results of the toxicity studies presented herein attest to the safety of phytase 50104 enzyme for its intended use.

Safety evaluation of a lipase enzyme (BD29241 Palmitase) preparation, expressed in Pseudomonas fluorescens, intended for removing palmitic acid from triacylglycerol.

The lipase enzyme, BD29241 Palmitase, can be used as a processing aid for removing palmitic acid from triacylglycerol in the production of refined oil. This enzyme was produced from a Pseudomonas fluorescens (P. fluorescens) production strain and was tested in acute, inhalation, and subchronic toxicity studies. In addition, this enzyme was also tested for its potential to induce genotoxicity. Dosages of the test article preparation ranged from 5000µg/plate for in vitro toxicity studies to 2000mg/kg/day for in vivo toxicity studies. The highest oral dose tested in vivo (NOAEL of 2000mg/kg/day) resulted in a safety margin of 2.442×10(3) based on a conservative estimate of the total human consumption of BD29241 Palmitase of 0.819mg/kg/day. There was no toxicity reported for any of these studies including additional safety studies. A review of the literature indicates that P. fluorescens fulfills recognized safety criteria pertinent to microbial production strains used in the manufacture of food enzyme preparations. The results of the toxicity studies presented herein attest to the safety of BD29241 Palmitase for its above-stated intended use.

Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil.

BD16449 lipase is the product of a phospholipid-specific lipase gene expressed in the yeast Pichia pastoris strain DVSA-PLC-004. This type C phospholipid lipase (EC 3.1.4.3) is intended for use in the degumming of edible vegetable oil. BD16449 lipase was tested as a refined test article preparation (DV16449) for its effects on genotoxicity and in acute, inhalation, and subchronic toxicity studies. Dosages ranged from 5000 microg/plate for in vitro toxicity studies to 2000 mg/kg/day for in vivo toxicity studies. The highest oral dose tested in vivo (NOAEL of 2000 mg/kg/day) resulted in a safety margin of 133,000 based on the conservative estimate of the total human consumption of BD16449 lipase of 0.015 mg/kg/day. When adjusted for total organic solids (TOS), the highest oral dose tested in vivo (NOAEL of 1680 mg TOS/kg/day) resulted in a safety margin of 18,300 based on the conservative estimate of the total human consumption of BD16449 lipase of 0.092 mg TOS/kg/day [corrected] There was no toxicity reported for any of these studies including additional safety studies. A review of the literature indicates that P. pastoris fulfills recognized safety criteria pertinent to microbial production strains used in the manufacture of food enzyme preparations. The results of the toxicity studies presented herein attest to the safety of BD16449 lipase for use in the degumming of edible vegetable oil.

Safety evaluation of a phytase, expressed in Schizosaccharomyces pombe, intended for use in animal feed.

BD006 phytase is the product of the Escherichia coli B app A gene expressed in Schizosaccharomyces pombe strain ASP595-1. This enzyme preparation is intended for use in animal feed applications where it improves the bioavailability of phosphate for monogastric animals. BD006 phytase was tested as an unrefined (DV006U) and a refined (DV006R) preparation for its effects on genotoxicity and in acute, inhalation and subchronic toxicity studies. Dosages ranged from 5000 microg/plate for in vitro toxicity studies to a high of 2000X for oral in vivo toxicity studies. The highest oral dose tested (2000X) is 2000 times the highest expected consumption of product by poultry or swine (X=50 units of phytase per kg bwt/day). There was no genotoxicity or any in vivo toxicity reported which could be directly related to systemic effects of the product. Other additional safety studies conducted were devoid of any relevant toxicity. The historic safety profile of the production organism S. pombe, and the results of the toxicity studies presented herein, attest to the safety of BD006 phytase for use in animal feed applications.