Safety evaluation of Streptomyces murinus glucose isomerase.

A glucose isomerase enzyme, obtained from Streptomyces murinus, was produced by a fermentation process and subjected to a series of tests to investigate its safety in use and manufacture. It was not mutagenic (Ames test, using liquid culture) nor did it provoke chromosomal damage (rat bone marrow cytogenetics test). It did not contain (nor did the organism produce) antimicrobial activity or macrolidpolyene antibiotics. It had no teratogenic activity when administered to pregnant rats at 100,000 ppm in the diet. It was without effect upon rats when administered at this dietary concentration for 4 weeks. Dietary administration at 5000, 15,000 or 50,000 ppm to rats for 13 weeks resulted in nephrocalcinosis in females at all dosages (probably a physiological response to the altered calcium:phosphate ratio in the admixed diet) and status spongiosus in the brains of males receiving 50,000 ppm. As the finding of nephrocalcinosis in rats is generally agreed to be of no toxicological importance with regard to the use in man, the dietary concentration of 15,000 ppm was considered to be highest no-effect level. This level corresponds to an intake of some 1000 mg/kg/day, which represents approximately 8000 times the human intake based on a conservative estimation.

Safety evaluation of esperase.

Esperase is a proteolytic enzyme preparation that can be used as a processing aid in the food industry. The following studies were performed to establish safety for the consumer: oral toxicity study (13 wk) in the rat; teratogenicity study in the rat; gene mutation assays in Salmonella typhimurium and mammalian cells in vitro, and chromosome aberration assay in vitro. General toxicity was low; the effects seen were attributed to proteolytic activity and the loading with sodium chloride. Neither of these factors will be relevant to consumers of the processed food. There was no evidence of effects on pregnancy outcome or mutagenic potential. When these results are considered together with existing knowledge of the production organism and the chemical and microbiological characterization of the enzyme preparation, they indicate that Esperase will be safe for its intended application in food processing.

Pharmacokinetic and pharmacodynamic response after intranasal administration of diazepam to rabbits.

Nasal application of drugs might be an alternative to intravenous administration in acute situations such as epileptic or fever seizures. In the search for a nasal formulation leading to a peak plasma concentration of diazepam at a tmax < or = 5 min bioavailability in rabbits has been studied after intranasal administration of the drug in ten vehicles of different polarity. The animals were dosed with 3 mg diazepam, dissolved in 100 microL vehicle, the solution being administered into both nostrils. The bioavailability, measured during the first 30 min, because periods after this are not relevant for acute treatment, was found to be between 49 and 62% for the four most promising vehicles, pure glycofurol 75, tetraethyleneglycol, poly(ethylene glycol) 200 and 30% glycofurol in tetraethyleneglycol. The tmax for these vehicles was achieved after 5 min, and they induced a very rapid pharmacodynamic response after 1.5 to 3.5 min. The bioavailability was reduced when more polar liquids such as ethanol and tween 20, or lipid oils, e.g. vegetable oil and miglyol 840 were added to the glycofurol. There was a good correlation between tmax and the induction of pharmacodynamic response. These results suggest that nasal application of diazepam in a water-free low-molecular-weight glycol might be of clinical importance as an alternative to intravenous injection, especially in acute situations.

Single- and repeated-dose local toxicity in the nasal cavity of rabbits after intranasal administration of different glycols for formulations containing benzodiazepines.

To furnish a systemic effect after intranasal administration, a formulation must contain the therapeutic dose in no more than 150 L, the maximum volume that can be applied as a single administration in one nostril in man. The objectives of these studies were to examine the local toxicity of formulations containing benzodiazepines and to document the effects to support clinical trials in man. After stability, pharmacological and pharmacokinetic studies of several benzodiazepine formulations, we studied nasal toxicity after single and repeated administration to rabbits of poly(ethylene glycol) 200, tetra(ethylene glycol), glycofurolum and mixtures of these vehicles both with and without benzodiazepines. Single-dose studies with examinations 5 or 10min after application were undertaken with poly(ethylene glycol), tetra(ethylene glycol), glycofurolum and tetra(ethylene glycol)-glycofurolum in the ratio 95:5; the reactions were similar to that after physiological saline. A 14-day repeated-dose study was conducted with diazepam, lorazepam and flunitrazepam formulations in poly(ethylene glycol), and flunitrazepam in poly(ethylene glycol)-glycofurolum in the ratio 70:30; the two vehicles without any benzodiazepine were also examined. Microscopic study revealed mild changes only in the treated groups. A final four-week study was conducted with repeated administration of clonazepam formulated in tetra(ethylene glycol)-glycofurolum in the ratio 95:5; microscopy revealed mild changes after three 150-microL doses daily, but no abnormalities after one or three 100-microL doses daily. It was concluded that these three solvents individually or as mixtures resulted in only mild local toxicity and might be acceptable as vehicles in nasal preparations of benzodiazepines and other non-irritating drugs for short-term use in man.

Enema in the horse. Distribution and rehydrating effect.

In the first series of experiments enema containing a water soluble marker was given to two groups of horses fasted for 1 and 18 hours, respectively. The marker was only in 1 out of 14 experiments found orally of the pelvic flexure (Table II). Fasting seemed to facilitate the flow in oral direction. The results were discussed with the possible significance in the treatment of large intestinal impactions. In the second series the effect of enemas on water, electrolyte and acid-base status in the acute furosemide-dehydrated horse was studied. Moderate acidifying and hypokalemic effect was encountered, while no effect was demonstrated on the water balance. The choice of method is questioned, and no conclusion is given with relation to a rehydrating effect of enemas.

[Shock. A review (author's transl)]. / Shock. en oversight.

Shock is defined as a secondary condition constituting a complication to a primary disease of which more than 100 are recorded in the literature. Shock is characterized by prolonged circulatory inadequacy leading to insufficient tissue perfusion and cell death. According to etiology shock is classified into three main groups: hypovolemic, vasogenic and cardiogenic shock. Taking hypovolemic shock as a model the pathgenesis of shock is presented. Hypovolemia acts on the baroreceptors giving rise to a sympatho-adrenal response resulting in increased vasoconstriction, which again leads to viscerocutaneous ischemia. This phase is known as the ischemic anoxic or centralized shock phase. Without treatment this phase develops into the second socalled stagnant anoxic or paralytic shock phase. "Irreversible shock" is discussed. The pathogenesis of vasogenic and cardiogenic shock is mentioned and compared with hypovolemic shock. It is emphasized that the sympatho-adrenal response is the central and common feature in every shock development. Special reference is made to septic shock with its outstanding circulatory conditions (arteriovenous shunting). Lacticacidemia and metabolic acidosis are described as the most important metabolic alterations in shock. With reference to pathenesis the main clinical symptoms of shock are presented: increased heart rate, initially pale later hyperemic, congested and terminally cyanotic mucosae, increased capillary filling time, cold skin and low body temperature. All these signs are related to the sympatho-adrenal response. It is pointed out that the patient in shock is depressed. Inevitably the primary disease will modify the shock symptoms. Hyperemia with edema, hemorrhages and thrombosis in organs and tissues are morphological manifestations of shock. Later microscopically detectable degenerative and necrotic alterations develop, and there are signs of intravascular coagulation (hyaline thrombi and spheres). Due to the rather nonspecific macroscopic alterations a post mortem shock diagnosis necessitates for completion histology and/or a clinical shock diagnosis. Some of the most important shock-provoking primary diseases dealt with in veterinary practice are mentioned along with their possible shock pathogenesis. Referring to the shock pathogenesis the therapy is discussed. The first and indispensable therapeutical measure in treating shock per se is increasing the circulating blood volume, Balanced electrolyte solutions are preferred. Examples of composition, doses (up to 80--200 ml/kg body weight) and infusion rate (initially 15--30 ml/kg body weight during the first 10--20 min., then quantum satis) are given.

Simulated small intestinal volvulus in the anesthetized horse.

Experimental closed loop small intestinal volvulus was studied in the anesthetized horse. Volvulus was simulated by ligation of the mesenterial veins to a segment of small intestine. Physical signs and hemodynamic, hematologic, clinical chemical, bacteriologic and peritoneal fluid values were examined. Compared to conscious horses anesthesia highly delayed and modified the clinical signs of shock (changes in mucosal colour, dehydration, decreased skin temperature, elevated pulse rate, low blood pressures) and of small intestinal volvulus (altered peristalsis, gastric dilation). Plasma glucose response to shock was also modified by unconsciousness. However, a dose response relationship was indicated between the extent of small intestinal damage and clinical symptoms. The same was applicable to changes in blood pressures, blood acid-base balance, lactate, potassium, chloride, glucose, inorganic phosphorus, creatinine, creatine kinase, red blood cell and total white blood cell counts and serum total protein. The relationship was also indicated in the following peritoneal fluid values: volume, lactate, pH, total white cell counts, alkaline phosphatase and bacteriology. Changes related to shock (insufficient tissue perfusion) were low blood pressures and metabolic acidosis due to anaerobic glycolysis with accumulation of lactic acid. Also low plasma glucose and elevated plasma potassium, creatinine, inorganic phosphorus and creatine kinase were regarded as consequences of shock.

Pharmacokinetics of the neuroprotective glutamate antagonist NBQX (6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione) in mice, rats, and dogs. Interactions with probenecid.

NBQX [6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione] has proven effective in protecting against cerebral ischemic insult in rodents. The preclinical development included pharmacokinetic and toxicological investigations in mice, rats, and dogs. For these purposes, NBQX was given as an intravenous bolus dose (in mice, rats, and dogs) or as a constant infusion for up to 4 weeks in rats and dogs. In NMRI mice t1/2, CL, and V2 were 1-4 hr, 0.6-1 liter/kg/hr, and 1-4 liters/kg following 3, 10, or 30 mg/kg. In Wistar and Sprague-Dawley rats, the mean +/- SD values of t1/2, CL, and Vz were 0.8 +/- 0.35 hr, 3.2 +/- 1.0 liters/kg/hr, and 4.0 +/- 1.1 liters/kg, respectively. About 33 +/- 5.2% of the dose was excreted unchanged in urine. The CLR was 0.90 +/- 0.20 liter/kg/hr. The pH of the urine samples ranged from pH 6.2 to 8.8, with a mean of 7.9 +/- 0.72. The plasma concentrations were proportional to the dose rate in the dose range 0.3-10 mg/kg/hr, independent of sex, and did not change during 4 weeks of infusion. CL and CLR were decreased to half their value when NBQX was administered in combination with probenecid. In beagle dogs, t1/2 and Vz were 1-3 hr and 1-3 liters/kg, respectively. The CL was determined to be 1.5 +/- 0.4 liters/kg/hr (N = 18) following 2 days of infusion (0.2-1 mg/kg/hr), but after 1 month CL had decreased significantly (p < 0.0001) to 1.0 +/- 0.1 liter/kg/hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Colic in the horse. A clinical and clinical chemical study of 42 cases.

42 horses were examined. The physical signs with relation to circulatory insufficiency and the abdominal disease were registered following a two-phased examination procedure. Great prognostic value was found in the degree of circulatory insufficiency judged by pulse rate and character, filling of the jugular vein, skin temperature, colour of mucous membranes, capillary refill time, sweating, depression, skin turgor and degree of enophthalmus. In making a causal diagnosis the abdomen was examined for shape, tenderness, peristaltic sounds, gastric dilation by siphoning, abnormal rectal findings and macroscopic changes in peritoneal fluid. Greatest diagnostic difficulties were encountered in cases of intestinal atonia, acute enteritis and torsion of the colon. In selected (severe) cases laboratory tests were obtained. Blood samples were examined for packed cell volume, hemoglobin, red and white blood cell counts, differential white blood cell count, blood gases and acid-base status, lactate, serum total protein and albumin, plasma sodium, potassium, chloride, calcium, magnesium, inorganic phosphorus, glucose, creatinine, BUN, total bilirubin, ASAT, CK, BASP and GGT. Peritoneal fluid was examined for red blood and white cell counts, total protein, specific gravity, pH and lactate, and enzymes as in blood. Laboratory results generally confirmed the clinical signs of shock, and packed cell volume and blood lactate were regarded to be of greatest prognostic interest. Although the performed laboratory information, macroscopic evaluation was thought to reveal sufficient information in most cases. It was concluded that supervening shock is of decisive importance in severe forms of colic, and that a careful and repeated evaluation of the circulatory insufficiency often provides one with a tentative prognosis although the final diagnosis is not obtained. In spite of therapy fatal outcome was found in all seriously shocked horses.

Glucagon produced by recombinant DNA technology: repeated dose toxicity studies, intravenous administration to CD rats and beagle dogs for four weeks.

The toxicity of glucagon produced by recombinant DNA technology (Glucagon (ge)) was studied by daily intravenous administration to rats and dogs for 4 weeks. Pancreatic glucagon of bovine or porcine origin (Glucagon Novo) was used as a reference control in the dogs. Glucagon (ge) has the same sequence of the 29 amino acids as pancreatic glucagon of humans, cows, pigs, rats and dogs. The dosages were 0 (control), 0.2, 1.0 and 5.0 mg Glucagon (ge)/kg/day in the rats, and 0 (control), 1.0 and 5.0 mg Glucagon (ge) and 5.0 mg Glucagon (Novo)/kg/day in the dogs. The studies complied with current EEC, US and Japanese guidelines for 4 week toxicity studies of drugs. All dose levels were well tolerated. The plasma glucose and cardiovascular responses to dosing were monitored in the dogs and found to be in agreement with well-known effects of pancreatic glucagon. The most consistent finding in both species was an increase in liver weight. This change was without concomitant pathological deviations in the other parameters examined. There were no differences in the reaction of dogs following treatment with Glucagon (ge) or Glucagon (Novo). A dose of 1 mg Glucagon (ge)/kg/day was regarded as a clear no-toxic-effect-level in both species.

Preclinical studies on human insulin. I. Acute and subacute toxicity studies.

Human insulin (prepared from porcine insulin) and porcine insulin were tested for acute toxicity by subcutaneous administration to unfasted mice and rats and fasted mice. The animals were observed for signs of reaction and post mortem examination was performed. LD50 values were calculated when possible. The LD50 values in the unfasted animals were several times higher than in the fasted mice, but similar for the two insulin preparations. The deaths and the signs observed were most probably caused by hypoglycaemia. No dose-related macroscopic organ changes were found. In a 28 day toxicity study rats were given human or porcine insulin subcutaneously at dosages of 2, 20 or 200 U/kg/day. The criteria examined included mortality, body-weight change, food consumption and utilization, haematology, blood chemistry, urinalysis, ophthalmoscopy, organ weights, gross- and histopathology. A few deaths occurred because of hypoglycaemia. In surviving rats from dosage groups 20 and 200 U/kg/day of either insulin preparation higher plasma glucose levels than in the control were observed 24 hours after dosing. Higher food intake and body-weight gains, lower plasma protein concentrations and higher urinary volumes with associated low specific gravity were observed in animals given either human or porcine insulin at 200 U/kg/day. No other adverse effects were registered, and no overt difference was found between the effect of human and porcine insulin.

Toxicological Safety Evaluation of a Bacillus acidopullulyticus Pullulanase.

Promozyme®, an amylopectin debranching enzyme produced by Bacillus acidopullulyticus , was studied to evaluate its safety in the food industry. A dietary subchronic toxicity study incorporating fertility and teratogenicity studies was performed in 1-month-old rats at concentrations of 0.5, 1.5 and 5% Promozyme. No adverse effects were seen at the 0.5 and 1.5% dose levels, and at the 5% dose level only minor or equivocal signs of toxicity were recorded. With the exception of a moderate reduction in body weight gain the FIA litters at the 5% dose level, no effects were found in the fertility study, and Promozyme was not teratogenic. In a 13-wk oral toxicity study in dogs, no adverse effects resulted from 0.5 g/kg/d, whereas mild gastrointestinal disturbances were seen clinically at 1.5 and 5.0 g/kg/d. In dogs given 5.0 g/kg/d, terminal investigations showed increased kidney weights and mineralized casts in renal cortical tubules. This was probably due to the high content of ash (phosphorus) in the test material. Lack of mutagenic potential was confirmed in bacterial mutagenic assays with Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and in an in vivo cytogenetic study in rat bone marrow cells after a single dose and daily dosing for 5 d of up to 8 g/kg/d. In an acute inhalation study with 4 h of exposure of rats, no death occurred at the highest dose level used, i.e., 2 mg/L. The test material was non-irritating to skin and did not produce eye injury in rabbits. A skin sensitization study in guinea pigs revealed no indication that the enzyme is a sensitizer. The pathogenic potential of the enzyme-producing B. acidopullulyticus was investigated by single intraperitoneal and subcutaneous administrations to rats and mice; the microorganism was found to be nonpathogenic (LD50>1010 cells/kg). Tests of culture broths revealed that the microorganism does not produce antibiotics. Results indicated that production and the intended use of Promozyme can be regarded as safe for plant workers and consumers.