Safety evaluation of a novel muramidase for feed application.

Safety evaluation of a muramidase produced by a Trichoderma reesei strain (safe lineage), expressing a muramidase gene isolated from Acremonium alcalophilum is presented. Intended use in feed of this enzyme is as digestive aid in broiler chickens. Muramidase 007, was non-mutagenic and non-clastogenic in vitro, and no adverse effects were observed in 90-day subchronic toxicity studies in rats at doses up to 1132 mg TOS/kg body weight/day. The enzyme did not exhibit, in vitro, skin, nor eye irritation potential. Acute aquatic toxicity evaluated on daphnia and algae showed absence of effect of the enzyme at the standard doses tested. Muramidase 007 was fully tolerated by broiler chickens in a 6-weeks tolerance study showing no adverse effects in any of the dietary treatments (0, 1×, 5× and 10× maximum recommended dose). In conclusion, Muramidase 007 is found to be toxicologically inert, and there are no worker's safety concerns if standard precautions are instituted and a non-dusty formulation is employed. Muramidase 007 is well tolerated by the target species (broiler chickens) and cause no harm to the environment. The beneficial safety evaluation of Muramidase 007 is in line with this type of enzyme that is found ubiquitously in nature.

A precise and efficient stereological method for determining murine lung metastasis volumes.

We have developed a computer-assisted stereological method based on unbiased principles for estimating metastasis volumes in mouse lungs. We evaluated this method using the transplantable Lewis lung carcinoma. Twenty-one days after subcutaneous inoculation of 10(6) Lewis lung cells into C57BL/6J mice, the mice had primary tumors with an average volume of 2300 mm(3). After perfusion fixation, the lungs were removed, embedded in OCT compound, snap-frozen, and processed for stereology. The metastasis volumes were estimated by application of the Cavalieri principle after evaluation of single sections from several evenly distributed tissue levels. The metastasis volume in a group of nine mice varied between 0.01 and 14.4 mm(3), with an average of 6.1 mm(3). The coefficient of variation was 0.9. The coefficient of error of the volume estimation was determined in five cases and varied from 0.08 to 0.23. Thus, the variation on the metastasis volumes that is achieved by this method contributes very little, 2.5%, to the total variance within the group of mice. In conclusion, we have developed an efficient and unbiased method to determine the metastasis burden in mouse lungs.

Sustained elevated plasma aprotinin concentration in mice following intraperitoneal injections of w/o emulsions incorporating aprotinin.

This study was initiated to test the feasibility of w/o emulsions as a sustained release system for aprotinin following intraperitoneal injection in mice. The emulsion was well tolerated in mice and sustained release was observed over a period of 96 h. The time for maximum plasma concentration of aprotinin was 10 min and 12 h after injection of a control solution and the emulsion dosage form, respectively. Furthermore, the hemolytic activity of the emulsion constituents was low indicating a low acute toxicological potential of the emulsion. The present study also showed that the lipolytic activity in peritoneal exudate from mice is important for the clearance of oily vehicles from the peritoneal cavity with lipolytic rate constants ranging from 50 to 130 nmol free fatty acid released/min/mg exudate protein at 37 degrees C, pH 8.5. It was concluded that the w/o emulsion was well suited to provide sustained elevated plasma aprotinin concentrations in mice.