A new 90-day drinking water study of sodium cyanide in rats to further evaluate National Toxicology Program findings and inform risk assessment.

BACKGROUND: The National Toxicology Program (NTP, 1993) reported male reproductive effects in a sodium cyanide (NaCN) drinking water study. The critical effect, decreased cauda epididymis weights, was used by U.S. Environmental Protection Agency for their hazard characterization and risk assessment of hydrogen cyanide and cyanide salts. To further investigate potential male reproductive effects, we conducted a new 90-day drinking water study. METHODS: Our study expanded evaluations of testes and thyroid. Male F344 rats received NaCN in drinking water at 0, 0 (water restricted; paired to top dose), 3, 10, 30, 100, and 300 ppm for 13 weeks, followed by 10-weeks recovery. RESULTS: Plasma thiocyanate increased dose-dependently but returned to baseline during recovery. NaCN caused neither effects on survival, body weight, food consumption, hematology, serum chemistry, urinalysis, thyroid hormones, testes or epididymides weights, sperm motility/viability, sperm morphology, or sperm production; nor clinical, ophthalmic, or histopathologic findings. Increased organ weights in thyroid/parathyroid and liver occurred at 300-ppm but were recoverable. No changes occurred in male reproductive organs. CONCLUSIONS: Absent adverse effects, the NOAEL was 300 ppm (21.66 mg/kg/day; highest dose tested). Based on organ weight increases at 300 ppm, the NOEL was 100 ppm (7.46 mg/kg/day).

A 90-day oral (dietary) toxicity and mass balance study of corn starch fiber in Sprague Dawley rats.

The potential toxicity of corn starch fiber was assessed and compared to polydextrose, a commonly used bulking agent with a long history of safe use in the food supply. Groups of male and female Crl:CD(SD) rats were fed 0 (control), 1,000, 3,000, or 10,000 mg/kg-bw/day corn starch fiber in the diet for 90 days. The polydextrose reference article was offered on a comparable regimen at 10,000 mg/kg-bw/day. Following a single gavage dose of [14C]-corn starch fiber on study day 13 or 90, the mass balance of the test article was assessed by analysis of excreta samples collected from 0 to 168 h post-dose. There were no toxicologically or biologically relevant findings in any of the test article-treated groups. The few minor differences observed between the corn starch fiber and polydextrose exposed groups were considered to be due to normal biological variation. Following [14C]-corn starch fiber dosing, nearly complete excretion of the administered dose occurred over 168 h post-dosing, with the majority excreted in the feces. The dietary no-observed-adverse-effect level of corn starch fiber after 90 days was 10,000 mg/kg-bw/day. Similar toxicity profiles for corn starch fiber and polydextrose were observed due to the structural and compositional similarities of these materials.

Pigs in Toxicology: Breed Differences in Metabolism and Background Findings.

Both a rodent and a nonrodent species are required for evaluation in nonclinical safety studies conducted to support human clinical trials. Historically, dogs and nonhuman primates have been the nonrodent species of choice. Swine, especially the miniature swine or minipigs, are increasingly being used in preclinical safety as an alternate nonrodent species. The pig is an appropriate option for these toxicology studies based on metabolic pathways utilized in xenobiotic biotransformation. Both similarities and differences exist in phase I and phase II biotransformation pathways between humans and pigs. There are numerous breeds of pigs, yet only a few of these breeds are characterized with regard to both xenobiotic-metabolizing enzymes and background pathology findings. Some specific differences in these enzymes based on breed and sex are known. Although swine have been used extensively in biomedical research, there is also a paucity of information in the current literature detailing the incidence of background lesions and differences between commonly used breeds. Here, the xenobiotic-metabolizing enzymes are compared between humans and pigs, and minipig background pathology changes are reviewed with emphasis on breed differences.

Background Pathological Changes in Minipigs: A Comparison of the Incidence and Nature among Different Breeds and Populations of Minipigs.

Swine, especially the miniature swine or minipigs, are increasingly being used in preclinical safety assessment of small molecules, biopharmaceutical agents, and medical devices as an alternate nonrodent species. Although swine have been used extensively in biomedical research, there is a paucity of information in the current literature detailing the incidence of background lesions and differences in incidence between commonly used breeds. This article is a collaborative effort between multiple organizations to define and document lesions found in the common breeds of minipigs used for toxicological risk assessment in North America (NA) and the European Union (EU). We retrospectively assessed 10 years of historical control data from several institutions located in NA and EU, covering the period of 2004-2015. Here we report the background lesions with consideration of breed and geographical location. To our knowledge, this is the first report documenting spontaneous background lesions in commonly used breeds of swine in both NA and EU. This report serves as a resource to pathologists and will aid in interpretation of findings and differentiation of background from test article-related changes.

A 90-day dietary study of a (2R,4R)-monatin salt in Beagle dogs.

(2R,4R)-Monatin salt (Na/K) [sodium/potassium (2R,4R)-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate, hereafter "R,R-monatin"] was administered in the diets of groups of Beagle dogs (4/sex/group) at concentrations of 0 (basal diet), 5000, 20,000, or 35,000 ppm for 13 weeks. There were no effects on survival, clinical observations, body weight and body weight gain, feed consumption and feed efficiency, functional observational battery, ophthalmic examination, and electrocardiographic evaluation. No adverse effects on hematology, serum chemistry, and urinalysis parameters were reported. A statistically significant decrease in testicular weights associated with germ cell hypocellularity and reduced luminal sperm in the epididymides was reported in all treated male groups. Based on these findings, the dietary no-observed-adverse-effect level (NOAEL) of R,R-monatin for 90 days was considered 35,000 ppm for female dogs (approximately 1101 mg/kg bw/day) and <5000 ppm for male dogs (approximately <151 mg/kg bw/day).

Loss of leucine-rich repeat kinase 2 (LRRK2) in rats leads to progressive abnormal phenotypes in peripheral organs.

The objective of this study was to evaluate the pathology time course of the LRRK2 knockout rat model of Parkinson's disease at 1-, 2-, 4-, 8-, 12-, and 16-months of age. The evaluation consisted of histopathology and ultrastructure examination of selected organs, including the kidneys, lungs, spleen, heart, and liver, as well as hematology, serum, and urine analysis. The LRRK2 knockout rat, starting at 2-months of age, displayed abnormal kidney staining patterns and/or morphologic changes that were associated with higher serum phosphorous, creatinine, cholesterol, and sorbitol dehydrogenase, and lower serum sodium and chloride compared to the LRRK2 wild-type rat. Urinalysis indicated pronounced changes in LRRK2 knockout rats in urine specific gravity, total volume, urine potassium, creatinine, sodium, and chloride that started as early as 1- to 2-months of age. Electron microscopy of 16-month old LRRK2 knockout rats displayed an abnormal kidney, lung, and liver phenotype. In contrast, there were equivocal or no differences in the heart and spleen of LRRK2 wild-type and knockout rats. These findings partially replicate data from a recent study in 4-month old LRRK2 knockout rats and expand the analysis to demonstrate that the renal and possibly lung and liver abnormalities progress with age. The characterization of LRRK2 knockout rats may prove to be extremely valuable in understanding potential safety liabilities of LRRK2 kinase inhibitor therapeutics for treating Parkinson's disease.

Airway trefoil factor expression during naphthalene injury and repair.

While the role of trefoil factors (TFF) in the maintenance of epithelial integrity in the gastrointestinal tract is well known, their involvement in wound healing in the conducting airway is less well understood. We defined the pattern of expression of TFF1, TFF2, and TFF3 in the airways of mice during repair of both severe (300 mg/kg) and moderate (200 mg/kg) naphthalene-induced Clara cell injury. Quantitative real-time PCR for tff messenger RNA expression and immunohistochemistry for protein expression were applied to airway samples obtained by microdissection of airway trees or to fixed lung tissue from mice at 6 and 24 h and 4 and 7 days after exposure to either naphthalene or an oil (vehicle) control. All three TFF were expressed in normal whole lung and airways. TFF2 was the most abundant and was enriched in airways. Injury of the airway epithelium by 300 mg/kg naphthalene caused a significant induction of tff1 gene expression at 24 h, 4 days, and 7 days. In contrast, tff2 was decreased in the high-dose group at 24 h and 4 days but returned to baseline levels by 7 days. tff3 gene expression was not significantly changed at any time point. Protein localization via immunohistochemistry did not directly correlate with the gene expression measurements. TFF1 and TFF2 expression was most intense in the degenerating Clara cells in the injury target zone at 6 and 24 h. Following the acute injury phase, TFF1 and TFF2 were localized to the luminal apices of repairing epithelial cells and to the adjacent mesenchyme in focal regions that correlated with bifurcations and the bronchoalveolar duct junction. The temporal pattern of increases in TFF1, TFF2, and TFF3 indicate a role in cell death as well as proliferation, migration, and differentiation phases of airway epithelial repair.

Intra-coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs.

Mesenchymal stromal cells (MSCs) have the potential to treat many myocardial diseases. We investigated whether these multipotent stem cells derived from bone marrow could be administered safely into the coronary circulation of healthy dogs. We injected about 0.5 million cells per kg bodyweight of early passage autologous MSCs into the left circumflex coronary artery of anaesthetised dogs. During administration, we noted ST segment elevation and T wave changes characteristic of acute myocardial ischaemia. 7 days later, macroscopic and microscopic evidence of myocardial infarction was noted. Histological sections of myocardium showed several scattered regions of dense fibroplasia accompanied by macrophage infiltrates only in areas where the MSCs were observed. We also noted raised plasma concentrations of cardiac troponin I and collagen fibril deposition in the lesions. These findings show acute myocardial ischaemia and subacute myocardial microinfarction after intracoronary arterial injection of MSCs into dogs.

Abdominal fluid from a dog.

A 2-year-old intact female mixed-breed dog with a 1-month history of lethargy and anorexia was evaluated for abdominal distension and an abdominal mass. The dog's last heat cycle, her third, was 1 month prior to presentation, and no reproductive cycle abnormalities were noted at any time. Hematologic and serum biochemical abnormalities were consistent with hemorrhage and inflammation. Ultrasonographic examination confirmed a large midabdominal mass and a moderate amount of abdominal fluid. Cytologically, the fluid showed evidence of pyogranulomatous inflammation, hemorrhage, and mesothelial reactivity, as well as ciliated columnar cells and free cilia that were interpreted as likely of oviductal origin. The mass was removed surgically, and the histopathologic interpretation was oviductal hamartoma with marked stroma formation and acute hemorrhage. To the authors' knowledge, this is the first reported case of oviductal hamartoma in any species and the first reported case detailing the finding of ciliated columnar epithelial cells in the abdominal fluid of a dog. Ciliated columnar epithelial cells in abdominal fluid should be considered indicative of a likely underlying oviductal lesion.