Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic humanized monoclonal antibody.

Recombinant humanized antivascular endothelial growth factor (rhuMAbVEGF) is a monoclonal IgG1 antibody that is being developed as an antiangiogenic agent for use in treating a variety of solid tumors. Preclinical safety studies included an immunohistochemical tissue cross-reactivity study, in vitro hemolytic potential and blood compatibility studies, and multiple dose toxicity studies. Toxicity studies were conducted in cynomolgus monkey because rhuMAbVEGF is pharmacologically active in this species and does not bind rat or mouse vascular endothelial growth factor (VEGF). Following twice weekly administration of rhuMAbVEGF for 4 or 13 wk, young adult cynomolgus monkeys exhibited physeal dysplasia characterized by a dose-related increase in hypertrophied chondrocytes, subchondral bony plate formation, and inhibition of vascular invasion of the growth plate. In addition, decreased ovarian and uterine weights and an absence of corpora lutea were observed in females receiving 10 and 50 mg/kg/dose in the 13-wk study. Both the physeal and ovarian changes were reversible with cessation of treatment. No other treatment-related effects were observed following rhuMAbVEGF administration at doses up to 50 mg/kg. These findings indicate that VEGF is required for longitudinal bone growth and corpora lutea formation and that rhuMAbVEGF can reversibly inhibit physiologic neovascularization at these sites.

Comparison of the toxicity profiles of ISIS 1082 and ISIS 2105, phosphorothioate oligonucleotides, following subacute intradermal administration in Sprague-Dawley rats.

The systemic toxicity of two phosphorothioate oligonucleotides specific for herpes simplex viruses (ISIS 1082) and human papiloma virus (ISIS 2105) were evaluated following repeated intradermal injections of vehicle control, 0.33, 2.17, or 21.7 mg/kg daily to Sprague-Dawley rats (10/sex/group) for 14 days. Animals were sacrificed 1 day after the last dose, except for a portion of the ISIS 1082-treated animals (5/sex/group) which were maintained for an additional 14-day recovery period. The profile of alterations noted for both compounds was very similar. Other than local signs of irritation at the site of injection, there were no clinical signs of toxicity or treatment-related mortality, but there was a slight decrease in body weight gain for the 21.7 mg/kg dose groups. Alterations in hematology parameters included dose-dependent thrombocytopenia and anemia. Alterations in serum chemistry parameters were suggestive of mild alterations in hepatic metabolism, with increases in liver transaminases and bilirubin, along with decreases in albumin and cholesterol. Both spleen and liver weights were significantly elevated in a dose-dependent fashion. Histopathological alterations noted in liver, kidney, lung, injection site skin, and spleen were characterized as perivascular and interstitial infiltrates of macrophages and monocytes. Additional microscopic alterations in the spleen included mild lymphoid hyperplasia (seen in lymph nodes as well), and extramedullary hematopoiesis. Treatment-related cytopenias were likely related to mild, focal hypocellularity in the bone marrow. Alterations in ISIS 1082-treated animals were only partially reversed following the 14-day treatment-free period. In conclusion, repeated intradermal administration of ISIS 1082 and ISIS 2105 produced a similar spectrum of toxicities, with liver, kidney, spleen, and bone marrow being identified as target tissues.

Influence of dietary protein, fat, and fiber on growth, blood chemistry, and tumor incidences in Fischer 344 rats.

Composition of diets may influence growth, diseases, tumor rates, and responses to chemical treatment. For two years, Fischer 344 rats were fed the NIH-07 open-formula nonpurified diet (approximately 23% protein, approximately 5% fat, and approximately 3.5% fiber) and nonpurified experimental diets (NTP-90, NTP-91, and NTP-92) containing lower protein and higher fat and fiber (14.6-15.3% protein, 7.2-8.5% fat, and 9.4-14% fiber) than the NIH-07 diet. Rats were evaluated for growth patterns, survival, hematology, serum chemistry, nephropathy, and tumor incidences. Growth patterns were similar in rats fed the experimental diets and in those fed the NIH-07 diet. However, in rats fed the experimental diets, the adult body weights were significantly (6-9%) lower and the survival at 110 weeks of age was significantly higher (15-20%) than in rats fed the NIH-07 diet. Lower protein content of experimental diets decreased the severity of nephropathy. Higher fat content of experimental diets appears to have decreased the incidence or delayed the development of leukemia and associated mortality in males. Higher fiber content of experimental diets appears to have delayed the development of mammary tumors and associated mortality in females. Higher fat and/or fiber of the experimental diets decreased the incidence of pheochromocytomas in males. The lower protein and higher fat and fiber contents of the experimental diets decreased the spontaneous tumor burden in two-year studies. These studies indicate that diets for rats in long-term studies could be modified to decrease the severity of nephropathy and to decrease/delay the development of spontaneous tumors.

The toxicologic and oncogenic potential of JP-4 jet fuel vapors in rats and mice: 12-month intermittent inhalation exposures.

Three-hundred Fischer 344 rats and 300 C57BL/6 mice of each sex were divided into three treatment groups and exposed intermittently (6 hr/day, 5 days/week) to JP-4 jet fuel vapors at concentrations of 0, 1000, and 5000 mg/m3 for 12 months. At exposure termination, 10% of the animals were killed and those remaining were held for a 12-month postexposure tumorigenesis observation period. Pathologic findings in male rats revealed treatment-related renal toxicity and neoplasia consistent with the male rat unique alpha 2 mu-globulin nephropathy syndrome. Distinct JP-4-induced respiratory toxicity was not observed, and pulmonary neoplasms were not significantly increased in any treatment group. Benign hepatocellular adenomas were slightly increased in high-dose female mice, but the trend was reversed in male mice. Other pathologic findings were regarded as equivocal or compatible with expected biologic variation. The study did not demonstrate target organ toxicity or carcinogenesis which could be extrapolated to other species.

Spontaneous corneal dystrophy and generalized basement membrane changes in Fischer-344 rats.

Groups of young, sexually mature Fischer-344 rats (n = 25/sex) obtained from commercial breeders were examined ophthalmologically and histopathologically to determine the prevalence and severity of corneal basement membrane lesions (corneal dystrophy) and basement membrane changes in select nonocular tissues. Results disclosed a high incidence of corneal basement membrane dystrophy in rats of both sexes from all breeders; however, severity levels were significantly increased in rats obtained from one breeder when compared to others. Furthermore, rats that displayed the most advanced corneal lesions also exhibited more severe basement membrane changes in other organs, especially renal tubules and vascular internal laminae. These findings suggest that both ocular and nonocular dystrophic changes may have been linked through common physiologic (or genetic) mechanisms. Animals that displayed basement membrane lesions were not considered to represent compromised biologic test systems.

The pathology of chronic bovine fluorosis: a review.

Clinical, pathologic, and analytical records from 200 cattle were reviewed to determine if long-term exposures to elevated fluorides resulted in previously unrecognized or unreported pathologic changes, especially skeletal neoplasia. Animals were part of comprehensive field and laboratory investigations of bovine fluorosis conducted by the Utah State University Agricultural Experiment Station over a 25-year period. Records indicated that over 170 cattle included in this review were exposed to dietary fluorides levels in excess of 25 ppm (dry wt), for most of their life span, and these animals exhibited bone fluoride concentrations ranging between 2,000 and 12,500 ppm (dry wt). Although dental and/or skeletal changes were present in most animals, significant soft tissue damage or neoplasia was not observed in any organ system. Renal degeneration and mineralization were slightly more prevalent in range cattle ingesting high fluoride levels, but these changes were not recognized in animals that received high experimental fluoride doses. The absence of significant soft tissue damage or neoplasia in these cattle combined with results of an extensive literature review suggests that environmental fluorides are not significant factors in mammalian carcinogenesis.

Histopathology in hosts parasitized by Sarcoptes scabiei.

Histopathologic evaluation of nondermal tissue in rabbits infested with Sarcoptes scabiei var. canis was investigated. Severe infestation resulted in deviant serological and serum biochemical values. Histological study revealed structural changes in the tissues of specific organs. The most prominent histological finding was amyloidosis in the liver, glomerulus of the kidney, red pulp of the spleen, intestines, and tongue. Hosts treated for infestation exhibited no abnormal organ histology.

The determination of the repeated oral toxicity of halocarbon oil, series 27-S.

Halocarbon 27-S (HC 27-S), a polymer of chlorotrifluoroethylene (CTFE), is used as a lubricating oil for pumps in hyperbaric chambers. Although monomeric CTFE has been shown to produce renal lesions in rats, the toxicity of CTFE polymers have not been investigated. To assess the toxicity of repeated exposure to HC 27-S, three groups (N = 6/group) of male and female Fischer-344 rats were dosed with 2.5 g HC 27-S/kg for 7 or 21 consecutive days. Groups were sacrificed at 7, 21, and 35 days (14 days after the 21-day dosing). Corresponding control groups (N = 6) were dosed with deionized water. Decreased water consumption and urine output were apparent in all test groups. Statistically significant increases in fluoride excretion were noted in 24-hr urine samples assessed periodically during the study. Neurotoxic signs were observed in female rats but not in male rats. Significant increases in liver and kidney weights were seen in all rats, regardless of number of dosing days. The increased fluoride burden in treated animals appeared sufficient to alter bone calcium/phosphate ratios in male rats but not female rats. Gross liver enlargement and hepatocellular cytomegaly indicated that the liver was probably the primary target organ following repeated administration of HC 27-S.

Tropism and histopathology of the D, B, K, and MM variants of encephalomyocarditis virus.

Variants of encephalomyocarditis virus (EMCV), which are immunologically indistinguishable by hyperimmune serum, produce different disease syndromes in mice. For instance, in ICR Swiss male mice, EMCV-B produces no overt illness, EMCV-MM produces severe neurological signs followed by death, EMCV-D destroys pancreatic beta cells producing a disease syndrome resembling insulin-dependent diabetes mellitus, and EMCV-K is lethal but produces no overt signs of infection. The present study was done to determine the tissue tropism and histopathology of each of these EMCV variants in the ICR Swiss mouse model. The data show the highest concentrations in the following organs: EMCV-D in the pancreas, EMCV-B in the pancreas, EMCV-MM in the cerebrum, and EMCV-K in the medulla/brainstem. They also show that the pathological lesions produced by each variant correlate well with viral titers.

Pathological and hepatic ultrastructural effects of a single dose of perfluoro-n-decanoic acid in the rat, hamster, mouse, and guinea pig.

In rats, the liver is the primary target organ of perfluoro-n-decanoic acid (PFDA) toxicity. Therefore, the effects of PFDA on hepatic ultrastructure were studied in rats. Pathological changes induced by PFDA in hamsters, mice, and guinea pigs were also examined. PFDA caused a severe reduction in body weight in all four species studied. A reduction in food intake was observed in rats and hamsters. However, hamsters continued to consume food at a reduced level, while rats stopped eating for a 5- to 6-day period about 6 days after dosing. The PFDA-induced pathological changes in the hamsters, mice, and guinea pigs resembled those seen in rats to varying degrees. As in the rat, PFDA caused a marked liver enlargement in mice and hamsters and a moderate swelling in guinea pigs. This hepatomegaly was ascribed primarily to individual cell swelling. Thymic atrophy was noted in PFDA-treated hamsters, mice, and guinea pigs. Seminiferous tubular degeneration observed in hamsters and guinea pigs, but not in mice, was not as severe as in the rat, where in some cases frank necrosis has been seen. Ultrastructural changes in the livers of all PFDA-treated animals, regardless of species, included disruption of the rough endoplasmic reticulum, rounding and swelling of the mitochondria with related structural alterations, and mild to extensive proliferation of peroxisomes. This peroxisome proliferative response was greatest in mice and almost absent in guinea pigs. Accumulation of lipid droplets in liver cells due to PFDA treatment was more pronounced in hamsters and guinea pigs than in rats and mice. PFDA-induced hepatomegaly with a concomitant increase in peroxisomes in several rodent species may be associated with an impairment of normal lipid metabolism in the liver by PFDA.

A subchronic dermal exposure study of diethylene glycol monomethyl ether and ethylene glycol monomethyl ether in the male guinea pig.

Diethylene glycol monomethyl ether (DEGME) has been selected as a replacement anti-icing additive for ethylene glycol monomethyl ether (EGME) in Navy jet aircraft fuel. This experiment was performed to determine whether DEGME produced similar toxicity to EGME following dermal exposure. Male guinea pigs were dermally exposed to 1.00, 0.20, 0.04, or 0 (control) g/kg/day DEGME for 13 weeks, 5 days/week, 6 hr/day. Another group of animals was similarly exposed to 1.00 g/kg/day EGME. Body weights as well as testicular and splenic weights were reduced as a result of exposure to EGME, DEGME-exposed animals exhibited decreased splenic weight in the high- and medium-dose (1.00 and 0.20 g/kg/day) exposure groups only. Hematologic changes in EGME-exposed animals included mild anemia with increased erythrocytic mean corpuscular volumes and a lymphopenia with increased neutrophils. Similar hematological changes were not observed in any animals exposed to DEGME. Serum creatine kinase activity was increased in animals exposed to EGME, and serum lactate dehydrogenase activity was increased in EGME and 1.00 g/kg/day DEGME-exposed animals. In general, DEGME produced minimal toxicological changes following dermal exposure, whereas the toxicological changes observed following similar exposure to EGME were much more profound.

Long-term inhalation toxicity of hydrazine.

Year-long intermittent exposures of rats, mice, hamsters, and dogs to hydrazine were conducted using concentrations of 0.05, 0.25, 1.0, and 5.0 ppm. Rats were held 18 months postexposure; hamsters, 1 year postexposure; mice, 15 months postexposure; and dogs, 38 months postexposure. Male and female rats exhibited dose-dependent incidences of benign nasal adenomatous polyps and smaller numbers of malignant nasal epithelial tumors after 1 year of exposure to hydrazine and 18 months postexposure holding. Nasal tumors were often associated with chronic irritation and were most frequent in male rats, with an incidence of greater than 50% in the highest exposure group. Hamsters exposed to 0.25-ppm and higher concentrations showed pathologic changes characteristic of degenerative disease, including amyloidosis. After exposure to 0.5 ppm hydrazine, hamsters developed a 10% incidence of benign nasal polyps compared to 0.5% in controls. Small numbers of colon neoplasms and thyroid parafollicular cell adenomas were found in hamsters, but only in the highest concentrations tested. Lung adenomas appeared to be marginally increased in mice exposed to 1.0 ppm hydrazine, the highest concentration tested in this species. No consistent clinical or pathological effects were seen in dogs during or after exposure to hydrazine at any concentration. Using amyloidosis as a criterion, a no-effect level was not achieved in hamsters. In rats, there appeared to be a marginal production of nasal tumors at 0.05 ppm, while mice showed no effects at 0.25 ppm. This study has demonstrated that the nasal respiratory epithelia of rats and hamsters are the most sensitive tissues to the tumorigenic action of hydrazine following inhalation exposures. This is similar to the reaction of rats to formaldehyde, another highly reactive water-soluble compound.

A 90-day vapor inhalation toxicity study of decalin.

A subchronic 90-day inhalation study was conducted to determine the toxic effects of decalin, a commonly used industrial solvent. Experimental groups consisting of male and female beagle dogs, male and female Fischer-344 rats, and female C57BL/6 mice were continuously exposed to decalin concentrations of 5 or 50 ppm. An unexposed control group was also maintained. All dogs and a portion of each rodent group were sacrificed and examined at exposure termination, while the remaining rodents were held for observation up to 21 months postexposure. No distinct exposure-related lesions were noted in dogs. Dog body weights, organ weights, and blood clinical pathology were also normal. At exposure termination hepatocellular cytoplasmic vacuolization was noted in female mice exposed to both concentrations. This liver tissue change was reversible and was not a significant finding in female mice examined during the 21-month postexposure observation period. In male rats, decalin exposure produced nephropathy characterized by hyaline droplets, necrosis, and intratubular casts. Accentuated tubular degeneration and medullary mineralization were noted in exposed rats held for long-term postexposure observation. There was no associated abnormal increase in mortality nor alterations in serum, blood urea nitrogen, or creatinine levels. Female rats were free of decalin-induced renal damage. There was a slightly greater incidence of commonly occurring pituitary tumors in both mice and rats; however, the tumor incidence was not dose related. The results of this study suggest that the toxic effects of decalin are similar to those previously described for other hydrocarbon solvents and fuels.

Carcinogenesis, benzo[a]pyrene metabolism, and sister chromatid exchanges in lungs of rats after intratracheal 3-methylcholanthrene.

Induction of squamous cell carcinomas in F344 rats by the intratracheal instillation of carrier-free 3-methylcholanthrene [(MCA) CAS: 56-49-5] was dependent on the total dose and on the size of the crystals. With the use of MCA particles of about 1 micron in mean diameter in doses up to 25 mg, delivered as 5 doses of 5 mg each at 2-week intervals, no tumors were produced. With particles in the range of 10-300 microns, all rats receiving 25 mg developed squamous cell carcinoma of the lung. At lower doses, the tumor incidence was dose-dependent. Metabolism of [3H]benzo[a]pyrene [CAS: 50-32-8] or [3H]MCA by microsomes from lungs and livers of treated rats was increased over that of controls and remained elevated for more than 6 weeks in the lung and 2 weeks in the liver. Repeated treatment of rats did not increase the levels of enzyme activity beyond that seen after a single treatment, nor did the increased activity persist longer than was seen after a single treatment. Sister chromatid exchanges (SCE) were measured in primary cultures of lung cells and in peripheral and spleen lymphocytes from treated rats. Elevated frequencies of SCE were found in lung cells up to 6 weeks following a single treatment with MCA. Repeated treatment did not increase the frequency or increase the persistence of the SCE. No increase in exchange frequency was found in lymphocytes of any treated rats.