Toxicity and carcinogenicity of 2,3-dibromo-1-propanol in F344/N rats and B6C3F1 mice.

2,3-Dibromo-1-propanol is a metabolite of the flame retardant tris(2,3-dibromopropyl) phosphate, previously shown to be a mutagen and carcinogen in experimental animals. Toxicology and carcinogenesis studies of 2,3-dibromo-1-propanol were conducted by applying the chemical in 95% ethanol to the interscapular skin of male and female F344/N rats and B6C3F1 mice 5 days a week for 13 weeks in the prechronic study and 48-55 weeks (rats) or 36-42 weeks (mice) in the carcinogenicity study. In the 13-week study, 10 rats and 10 mice of each sex received doses of 0, 44, 88, 177, 375, or 750 mg/kg. Deaths associated with chemical application occurred only in the high-dose (750 mg/kg) male mice. Chemical-related lesions were seen in the kidney of male rats, liver of female rats, and liver and lung of both sexes of mice. Based on the toxicity observed in the 13-week study, 50 rats of each sex received doses of 0, 188, or 375 mg/kg and 50 mice of each sex received 0, 88, or 177 mg/kg in the carcinogenicity study. The planned 2-year study was terminated early because of reduced survival of rats related to chemical-induced neoplasia and because of the appearance of antibodies to lymphocytic choriomeningitis virus in sentinel mice. Nearly all dosed rats had malignant neoplasms at one or more sites, while only one control male and one control female had malignant neoplasms. In rats, neoplasms induced by 2,3-dibromo-1-propanol occurred in the skin, nasal mucosa, Zymbal's gland, oral mucosa, esophagus, forestomach, intestines, liver, kidney, mammary gland (females), clitoral gland (females), spleen (males), and mesothelium (males). In mice, chemical-induced neoplasms occurred in the skin, forestomach, liver (males), and lung (males).

Effect of oral dosing vehicles on the subchronic hepatotoxicity of carbon tetrachloride in the rat.

Previous studies in this laboratory have shown that corn oil delayed and prolonged the gastrointestinal absorption of carbon tetrachloride (CCl4) and reduced its acute hepatotoxicity in rats. The objective of the present study was to extend the duration of ingestion of CCl4 to assess vehicle effects on the subchronic oral toxicity of CCl4. Male Harlan Sprague-Dawley rats were given doses of 0, 25, or 100 mg CCl4/kg body weight by gavage in either corn oil or a 1% Emulphor aqueous emulsion 5 times a week for 13 wk. Blood was collected at 4, 8, and 13 wk for measurement of serum enzymes. Liver samples were also taken at 13 wk for measurement of triglyceride and microsomal enzyme levels, as well as for histopathological examination. Serum enzyme levels peaked at 8 wk in the high-dose groups, but not until 13 wk in the low-dose animals. Effects of CCl4 on serum and microsomal enzymes were of similar magnitude in the two vehicle groups. A comprehensive histopathological examination revealed no qualitative or quantitative differences between the corn oil and aqueous vehicle groups in hepatic lesions. Although CCl4 and chloroform have been reported by other investigators to be more hepatotoxic to mice when given for 90 d in corn oil, current findings indicate that corn oil does not significantly alter the subchronic hepatotoxicity of CCl4 in rats from that when the halocarbon is given in an aqueous medium.

Oral toxicity of 1,2-dichloropropane: acute, short-term, and long-term studies in rats.

The objective of this investigation was to characterize the acute and short- and long-term toxic potency of orally administered 1,2-dichloropropane (DCP). In the acute and short-term studies, male rats of 250-300 g were gavaged with 0, 100, 250, 500, or 1000 mg DCP/kg in corn oil once daily for up to 10 consecutive days. Although ingestion of DCP caused body weight loss and CNS depression, few other toxic effects were manifest 24 hr after a single dose of the chemical. Morphological changes were limited to liver centrilobular cells in 500 and 1000 mg/kg rats. Similarly, elevated activity of some serum enzymes occurred only at these two highest dose levels. Hepatic nonprotein sulfhydryl (NPS) levels were decreased and renal NPS levels increased at 24 hr. In the short-term study resistance developed to DCP hepatotoxicity over the 10 consecutive days of exposure, as reflected by progressively lower serum enzyme levels and by decreases in the severity and incidence of toxic hepatitis and periportal vacuolization. Nucleolar enlargement in hepatocytes, however, was observed at all dosage levels at 5 and 10 days. There were a number of manifestations of hemolytic anemia, including erythrophagocytosis in the liver, splenic hemosiderosis and hyperplasia of erythropoietic elements of the red pulp, renal tubular cell hemosiderosis, and hyperbilirubinemia. Urinalyses and histopathology revealed no evidence of nephrotoxicity. In the long-term study, male rats initially weighing 180-200 g were gavaged five times weekly for up to 13 weeks with 0, 100, 250, 500, or 750 mg DCP/kg. As over one-half the 750 mg/kg group died within 10 days, the survivors were sacrificed. Histopathological changes in the 750 mg/kg animals included mild hepatitis and splenic hemosiderosis, as well as adrenal medullary vacuolization and cortical lipidosis, testicular degeneration and a reduction in sperm, and increased number of degenerate spermatogonia in the epididymis in some members of the group. Similar testicular and epididymal degenerative change also were observed in some 500 mg/kg animals after 13 weeks of dosing. There was a progressive increase in the number of deaths in the 500 mg/kg group, such that more than 50% were dead by 13 weeks. No deaths occurred in the 100 or 250 mg/kg groups. The DCP dosage regimen also produced a dose-dependent decrease in body weight gain. DCP exhibited very limited hepatotoxic potential and no apparent nephrotoxic potential in the long-term study. Slight elevations in serum ornithine-carbamyltransferase activity, periportal vacuolization, and active fibroplasia in the liver were seen in the 500 mg/kg animals.

Oral toxicity of carbon tetrachloride: acute, subacute, and subchronic studies in rats.

This investigation was conducted to characterize the acute, subacute, and subchronic toxic potency of ingested carbon tetrachloride (CCl4). In the first acute and subacute toxicity study, male Sprague-Dawley rats of 300-350 g were gavaged with 0, 20, 40, or 80 mg CCl4/kg once daily for 5 consecutive days, rested for 2 days, and dosed once daily for 4 additional days. Rats of 200-250 g were gavaged with 0, 20, 80, or 160 mg CCl4/kg according to the same dosage regimen in the second acute and subacute study. In the first and second studies one group of rats at each dosage level was sacrificed for clinical chemistry and histopathological evaluation at 24 hr, 4 days, and 11 days after initiation of dosing. Single 20- and 40-mg/kg doses had no apparent toxic effect at 24 hr, although 80 mg/kg caused mild hepatic centrilobular vacuolization and significant increases in some serum enzyme levels. In general, there was progressively severe hepatic injury at each dosage level over the 11-day period. CCl4 was more hepatotoxic to the 200-250-g rats than to the 300-350-g rats. In the subchronic study, rats initially 200-250 g were gavaged 5 times weekly for 12 weeks with 0, 1, 10, or 33 mg CCl4/kg. Body weight and clinical chemistry indices were monitored during the 12 weeks of dosing and 2 weeks after cessation of dosing. A dose of 1 mg/kg had no apparent adverse effect; 10 mg/kg produced slight, but statistically significant increases in sorbitol dehydrogenase activity and mild hepatic centrilobular vacuolization; 33 mg/kg caused marked hepatotoxicity. Serum enzyme levels remained elevated during the 12-week dosing period, but returned toward normal within 13 days of cessation of CCl4 exposure. Microscopic examination of livers of the 33-mg/kg rats revealed cirrhosis, characterized by bile duct proliferation, fibrosis, lobular distortion, parenchymal regeneration, hyperplastic nodules, and single-cell necrosis. The fibrosis was not reversed within the 13-day recovery period.

Contagious ecthyma in lambs and laboratory personnel.

Contagious ecthyma, diagnosed in three lambs, was transmitted to two researchers having direct contact with oral secretions from these lambs. Intracytoplasmic viral particles were demonstrated by electron microscopy in gingival biopsies from one lamb. Lamb to lamb transmission was most likely caused by use of a contaminated gavage feeding tube. Concern for the effects of this disease and Q fever on patients having contact with contaminated medical researchers prompted the formulation of safety guidelines to prevent potentially disastrous zoonotic disease.

Assessment of functional, morphological, and enzymatic tests for acute nephrotoxicity induced by mercuric chloride.

The relative merits of a comprehensive series of contemporary methods for detection of acute nephrotoxicity were evaluated. Male Sprague-Dawley rats were given 0, 0.25, 0.5, 1.0, or 3.0 mg mercuric chloride (HgCl2)/kg body weight by ip injection. Indices of nephrotoxicity were examined 8, 24, 48, 72, and 96 h later. Alterations in urine osmolality, volume, and protein levels were seen within 24 h in response to 1 mg/kg or more of HgCl2. Administration of 0.5-3.0 mg/kg produced dose-dependent increases in urinary excretion of maltase activity and glucose by 24 h, the period of peak effect. There was no increase in maltase or alkaline phosphatase (AP) activity in the serum of these animals. Enzymuria was not apparent in rats that had marked elevations in serum AP, argininosuccinate lyase, and ornithine carbamyl transferase activities as a result of physical (i.e., dichlorodifluoromethane-frozen) or chemical (carbon tetrachloride-induced) damage of the liver. Morphological alterations, in the proximal tubular epithelium of perfusion-fixed kidneys from HgCl2-dosed rats, paralleled the changes in enzyme excretion with respect to time of onset and dose-effect. There was a dose-dependent inhibition of tetraethylammonium (TEA) and p-aminohippurate (PAH) uptake by renal cortical slices at 24 h. Interestingly, increases in uptake of TEA and PAH were seen 8 h after a 1-mg/kg dose. Clearance of inulin and PAH in vivo were altered at 8 h by 0.5 and 1 mg/kg. Marked depression of these functional indices was seen at 24 h, by which time blood urea nitrogen (BUN) levels were increased. The 0.5- and 1.0-mg/kg doses also produced time- and dose-dependent increases in intracellular Na+ content which were maximal at 24 h. These results illustrate the importance of using a combination of biochemical and functional tests to elucidate the sequence of events in the kidney following toxic insult. Nevertheless, some of the simpler, traditional techniques (e.g., histopathology, urinalyses, BUN) were sensitive and organ-specific, and should continue to be very useful in nephrotoxicity testing/screening.

Inner ear disease characterized by rolling in C3H mice.

A neurologic disease characterized by rolling developed in C3H mice. Pseudomonas aeruginosa was isolated from the middle and internal ears or meninges of affected mice. The principal pathologic finding was aggressive, primary, purulent otitis media, with extension to the inner ear, acoustic nerve, and meninges. Stresses that may have contributed to induction of the disease were not delineated; however, acidification of the drinking water resulted in near elimination of the disease.

Longevity of exercising obese hypertensive rats.

The purpose of this study was to determine whether daily running lengthens the life-span of animals dying prematurely due to cardiovascular disease. We used a strain of rat that is genetically hypertensive and obese and is reported to develop atherosclerosis (Exp. Mol. Pathol. 19: 53--60, 1973). These animals were divided into three groups consisting of runners exercised daily on treadmills from an early age life, food-restricted sedentary rats, and libitum eaters that were sedentary. This latter group had significantly higher average daily food intakes and body weights than either of the other two groups. The average life-span of both sedentary groups was significantly longer than the running group. Runners had a greater frequency of focal myocardial necrosis, but atherosclerosis was absent in all three groups. We speculate that daily running may have accentuated the development of factor s that may have contributed to the early death of runners.

Effects of hyperparathyroidism and dietary calcium supplementation on bone healing.

Effects of nutritional secondary hyperparathyroidism and dietary calcium supplementation on bone healing were determined. Groups (n = 4) of 5 mature male dogs each were fed the following diets: group 1, control diet (0.48% Ca, 0.43% P); group 2, test diet (0.12% Ca, 1.14% P): group 3, control diet plus calcium; group 4, test diet plus calcium. The dietary calcium supplementation was calcium gluconate. Lesions were induced in the right tibial cortex by trephinization. Within the time limitations of this study, it was determined that nutritional secondary hyperparathyroidism does not inhibit bone healing and that dietary calcium supplementation does not aid bone healing.

Use of vectorcardiography for the detection of +Gz-related cardiac pathology in miniature swine.

Vectorcardiograms were recorded from anesthetized, adult miniature swine 1-2 weeks before high sustained +Gz exposure and 2-6 h after exposure. Each +Gz run consisted of one 60-s exposure, respectively, to 3, 5, 7, and 9 +Gz, with 3 min rest between each +Gz plateau. The full range, from severe to minor, of +Gz-induced cardiac pathology was observed in this group of miniature swine. In spite of the large variation in the amount and degree of cardiac pathology, there were no post-exposure vectorcardiographic changes which might be diagnostic of +Gz-induced cardiac pathology. The results of this study indicate that vectorcardiography, performed after +Gz exposure, is not a reliable technique for detecting the presence of +Gz-induced cardiac pathology in miniature swine.

Cardiac pathology associated with high sustained +Gz: I. Subendocardial hemorrhage.

Adult miniature swine were exposed to various levels and durations of +Gz. After exposure, all swine were euthanized and necropsied. Gross, histologic, and electronmicroscopic observations were made on the heart tissue. Subendocaridal hemorrhage (SEH) was commonly found in the left ventricle, rarely in the right ventricle, and its severity was directly related to : a) level and duration of G exposure, b) heart rate, and c) catecholamine activity. SEH was made more severe with i.v. atropine 4 mg, and prevented with i.v. propranolol 20 mg. Heart hemorrhage was usually limited to the immediate subendocardial region and frequently surrounded Purkinje's fibers. In severe cases, however, hemorrhages penetrated several millimeters into the heart muscle and sometimes penetrated Purkinje's fibers. Restraint of unanesthetized swine in the centrifuge couch, low G-levels, and/or i.v. injections of atropine or epinephrine produced minimal SEH lesions.

Cardiac pathology associated with high sustained +Gz: II. Stress cardiomyopathy.

The myocardial pathology of 14 pigs exposed to HSGz stress of 9 and 15, or 3, 7, and 9 Gz was studied; six control pigs were used as comparisons. Four pigs received propranolol prior to centrifugation and four pigs received atropine. Hearts were studied by light and electron microscopy. Myocardium from stressed pigs showed myofibrillar degeneration, pooling of mitochondria, and cell death. Lesions occurred in random cells of the subendocardium and papillary muscles. Purkinje fibers were also involved. Pretreatment with atropine increased the number of dead cells found and propranolol increased the number of cells showing myofibrillar degeneration. It is postulated that this is a pluricausal cardiomyopathy similar to several experimental conditions. Significance to aerospace medicine is briefly discussed.

Heart pathology associated with exposure to high sustained +Gz.

The pathology of +Gz acceleration was examined using unanesthetized adult miniature and immature "farm-type" swine, with and without anti-G suit inflation. Following single exposures of +8 or 9 Gz for 45 to 90 s--acceleration exposures that have been shown "tolerable" to man--swine were sacrificed and a detailed necropsy performed. Considering only the adult miniature swine, the endocardial area of the left ventricles showed evidence grossly of recent hemorrhage of varying severity involving both the wall and papillary muscles. The degree and location of the subendocardial hemorrhage were quantitated by grading the area of ventricle involved--1 (slight) to 4 (extensive). Of the 23 adult miniature pigs autopsied, the scores for papillary muscle hemorrhage, after one exposure to +9 Gz (45 to 90 s) ranged from a mean of 2.3 to 3.3 and the extent of ventricular wall involvement was 2.5 to 3.3. Histologically, heart hemorrhage was limited to the subendocardial area, primarily involving the space between heart muscle and the endocardium and was particularly evident surrounding Pukinje's fibers. Similar studies using immature farm-type swine (not miniature pigs) found these younger swine (4 to 5 months of age) to be less susceptible to such endocardial hemorrhage. Heart tissue recovery in these pigs following one exposure to +9 Gz for 45 s, required approximately 14 d. It appears that this lesion is similar, although less severe, than heart muscle lesions associated with loss blood volume (hemorrhagic shock) studies and may have similar physiologic bases. It was concluded that particular attention should be made of the endocardium of victims of high-performance aircraft accidents.