Nitrapyrin: a scientific advisory group review of the mode of action and carcinogenicity in B6C3F1 mice.

Nitrapyrin has been registered as a nitrogen stabilizer in the United States for many years based on a robust set of regulatory data. These data demonstrated that nitrapyrin was not genotoxic and that there were no tumors elicited in rats or mice that were relevant for human risk assessment. A repeat carcinogenicity study in B6C3F1 mice, conducted at two substantially higher-dose levels (0, 125 or 250 mg/kg/day) than the original study (0, 5, 25 or 75 mg/kg/day) identified liver, stomach, epididymal and Harderian gland tumors. In order to assess the relevance of these findings for human risk assessment, a Scientific Advisory Group (SAG) examined relevant microscopic changes in these tissues and also evaluated genotoxicity and mechanistic data. The SAG determined that the maximum tolerated dose had been exceeded in mice given 125 or 250 mg/kg/day, based on 26-33% decreased body weight gains (males-250 mg/kg/day), hepatocellular necrosis and compensatory hepatocellular proliferation (males and females-125 and 250 mg/kg/day). The SAG believed that the increased incidences of hepatocellular foci of alteration and hepatocellular neoplasms represented an epigenetic response to hepatocellular necrosis and increased mitogenesis. Increased incidences of proliferative lesions in the forestomach mucosa were likely secondary to the irritant effects of nitrapyrin. Neither the liver nor forestomach effects were interpreted to be a direct carcinogenic effect. Higher incidences of Harderian gland adenomas (females) and undifferentiated sarcomas in the epididymis represented normal biological variations in incidence and were unrelated to nitrapyrin. Therefore, it was the SAG's opinion that nitrapyrin exposure that does not produce target organ toxicity in exposed individuals would not be expected to increase the risk of cancer.

Rapid uptake, metabolism, and elimination of inhaled sulfuryl fluoride fumigant by rats.

Sulfuryl fluoride (SO(2)F(2)) is a structural fumigant gas used to control drywood termites and wood-boring beetles. The pharmacokinetics and metabolism of inhaled SO(2)F(2) were evaluated in male Fischer-344 rats exposed to 30 or 300 ppm (35)S-labeled SO(2)F(2) for 4 h. Blood, urine and feces were collected during and after the exposures and analyzed for radioactivity, (35)S-labeled fluorosulfate and sulfate, and fluoride (urine and feces only). Selected tissues were collected 7 days post-exposure and analyzed for radioactivity. During and after unlabeled SO(2)F(2) exposures, blood, brain, and kidney were collected and analyzed for fluoride ion. SO(2)F(2) was rapidly absorbed, achieving maximum concentrations of radioactivity in both plasma and red blood cells (RBC) near the end of the 4-h exposure period. Radioactivity was rapidly excreted, mostly via the urine. Seven days post-exposure, small amounts of radioactivity were distributed among several tissues, with the highest concentration detected in respiratory tissues. Radioactivity associated with the RBC remained elevated 7 days post-exposure, and highly perfused tissues had higher levels of radioactivity than other non-respiratory tissues. Radioactivity cleared from plasma and RBC with initial half-lives of 2.5 h after 30 ppm and 1-2.5 h after 300 ppm exposures. The terminal half-life of radioactivity was 2.5-fold longer in RBC than plasma. Based on the radiochemical profiles, there was no evidence of parent (35)SO(2)F(2) in blood. Identification of fluorosulfate and sulfate in blood and urine suggests that SO(2)F(2) is hydrolyzed to fluorosulfate, with release of fluoride, followed by further hydrolysis to sulfate and release of the remaining fluoride.

Reanalysis with optimized power of red blood cell acetylcholinesterase activity from a 1-year dietary treatment of dogs to chlorpyrifos.

A no-observed-effect level (NOEL) of 0.1 mg/kg/day was reported for inhibition of red blood cell (RBC) acetylcholinesterase (AChE) in two groups of Beagle dogs fed chlorpyrifos (0, 0.01, 0.03, 0.1, 1 or 3 mg/kg/day) in the diet for 1 or 2 years (McCollister et al., Food Cosmet. Toxicol. 12 (1974) 45-61). The statistical analyses were by t-test that had low statistical power due to small sample sizes. Common time points for blood samples in both phases allowed a reanalysis of the grouped data over a 1-year time period. The reanalysis increased statistical power by increasing the sample size to n=14 from n=3 or 4, and decreasing the variance, by statistical step-by-step aggregation of the data from both phases, both sexes, and four sample periods. Factors retained in the ANOVA were dose, sex, and phase (sex-by-dose was not significant). Contrasts with one-sided t-tests indicated the 1 and 3 mg/kg/day groups had significantly inhibited RBC AChE (P<0.0001). At alpha=0.05, the uncorrected one-sided model had 80% power to detect a 12% decrease, 93% power for a 15% decrease, and 99.5% power for a 20% decrease in AChE activity. Overall, the reanalysis had high power to detect a clinically significant decrease in RBC AChE activity, and substantiated the original NOEL for chronic treatment of dogs to dietary chlorpyrifos at 0.1 mg/kg/day.

Evaluation of the neurotoxic potential of chemicals in animals.

This review provides a scientific view on how to evaluate effectively the neurotoxic potential of chemicals in order to provide adequate safeguards for human health. Detection of compounds that may cause direct, persistent, adverse effects on the nervous system should be given the most critical attention. Evaluation of the neurotoxic potential of a chemical should include descriptions of functional and morphological effects as well as the determination of the dose response, no-observed-effect level, time course and reversibility of effects. Evaluation of the nervous system in the context of standard toxicity studies that use a variety of species and study durations are appropriate screening tests (Tier 1) for the detection of potential neurotoxicity. Studies specifically designed to assess neurotoxicity (Tier 2) should be performed with chemicals for which there is an indication of neurotoxic potential and where the available data are inadequate for risk assessment. Tier 2 studies should evaluate the function and structure of the nervous system by comprehensive clinical examinations and neuropathological assessment. These studies may be conducted in conjunction with standard toxicity studies so that any potential neurotoxicity can be interpreted in the context of other systemic toxicity. More specific neurotoxicity tests (Tier 3) may be necessary for advanced characterization of discovered neurotoxicants.

Neurotoxicologic evaluation of rats after 13 weeks of inhalation exposure to dichloromethane or carbon monoxide.

Male and female Fischer 344 rats were exposed to dichloromethane (methylene chloride, DCM) or carbon monoxide (CO) for 6 hr/day, 5 days/week, for 13 weeks. Since oxidative metabolism of DCM to CO and CO2 is a saturable process, DCM exposure concentrations were selected clearly below saturation (50 ppm), just below saturation (200 ppm), and well above saturation (2000 ppm). At saturation of metabolism, metabolic CO causes about 10% carboxyhemoglobinemia (COHb). Therefore, as a control for CO effects, a separate group of rats was exposed to 135 ppm CO to induce approximately 10% COHb. Postexposure functional tests included an observational battery, hindlimb grip strength, and a battery of evoked potentials (flash, auditory brainstem, somatosensory, caudal nerve). After functional tests were completed, rats from all groups were perfused with fixative and a comprehensive set of nervous tissues from the high DCM exposure group and from controls were examined by light microscopy. Although some miscellaneous functional and morphologic variations were recorded, none were related to treatment. Thus, subchronic exposures as high as 2000 ppm DCM or 135 ppm CO had no deleterious effects on any of the measures of this study.

The improbable association between the herbicide 2,4-D and polyneuropathy.

Isolated case reports have circumstantially linked the use of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) to polyneuropathy. However, a critical review of the literature reveals numerous reasons for doubting a relationship of 2,4-D to polyneuropathy: (1) too few cases given the wide use of the chemical; (2) no valid toxicologic or epidemiologic evidence; (3) the diversity of antecedent illness; (4) an unlikely time sequence of antecedent illness to exposure (pharmacokinetics); (5) the lack of polyneuropathy in medical patients given repetitive doses of 2,4-D; (6) the lack of polyneuropathy in heavily exposed military personnel involved in operation Ranch Hand; (7) the biological properties of 2,4-D which minimize penetration of 2,4-D into the nervous system under normal exposure conditions; and (8) the lack of polyneuropathy in a variety of experimental animal species given 2,4-D by several routes of exposure and at dose levels and durations of exposure many times greater than human applicator exposure. Thus, the weight of evidence indicates that 2,4-D is an unlikely cause of polyneuropathy.

Spontaneous lesions in subchronic neurotoxicity testing of rats.

Male and female Fischer 344 rats, 30 weeks of age, were examined for neuropathologic changes after a 13-week inhalation neurotoxicologic study. Tissues were preserved by whole-body perfusion with 1.5% glutaraldehyde/4% formaldehyde solution. An extensive set of neural tissues was embedded in paraffin, sectioned, and stained with hematoxylin and eosin, luxol fast blue/periodic acid-Schiff/hematoxylin, Sevier-Munger silver, and cresyl echt violet. Lesions in the central and peripheral nervous system were comparable between sexes and between control and treated animals. Bilateral swollen axons were present in the medial aspect of the nucleus gracilis adjacent to the area postrema. Occasional swollen axons also were observed in the dorsal and ventral funiculi of the spinal cord. Degeneration of individual nerve fibers was present in the trapezoid body, vestibular nerve root, trigeminal nerve, cerebellar peduncles, and the funiculi of the spinal cord. Individual nerve fiber degeneration also was present in the spinal nerve roots, sciatic and tibial nerves. Nerve fiber degeneration was characterized by myelin disruption and degeneration, vacuoles and axonal fragmentation. Similar spontaneous neuropathology may be encountered in rats from other subchronic neurotoxicologic studies and must be differentiated from treatment-related toxicity.

Screening for neurotoxicity: complementarity of functional and morphologic techniques.

Our philosophy is that screening tests should be applicable across species and emphasize complementarity to neuropathology. Within this context, electrophysiological tests comparable to those in human clinical neurology are powerful screening tools. For example, while histopathologic evaluation of the cochlea for ototoxicity is difficult, evoked potential audiometry is fast and easy. In this instance, one might routinely screen for deficits in auditory function, and reserve morphologic techniques for a characterization role rather than one of discovery. Lesions of neurons, axons and myelin are, however, readily assessed by light microscopy. A suitable combination of functional and morphologic screening tests, therefore, enhances the ability to discover neurotoxicity, and these data often are ideal for generation of refined hypotheses for subsequent characterization studies.

Inhalation toxicity of sulfuryl fluoride in rats and rabbits.

The inhalation toxicity of the structural fumigant sulfuryl fluoride (SO2F2) was evaluated in rats and rabbits. Exposures for a preliminary 2-week study were 6 hr/day, 5 days/week, to 0, 100, 300, or 600 ppm SO2F2. Nine of ten rats at 600 ppm died or were moribund between the second and sixth exposures. Extensive kidney lesions were present in all rats exposed to 600 ppm, whereas only minimal renal changes were noted in rats at 300 ppm. Upper and lower respiratory tissues were inflamed in the single rat that survived the 2-week exposure to 600 ppm. Rabbits exposed to 600 ppm SO2F2 were hyperactive and one animal had a convulsion. Exposure to 300 or 600 ppm for 2 weeks resulted in vacuolation and/or malacia in the cerebrum of all rabbits and most of these rabbits also had moderate inflammation of nasal tissues; a few rabbits at 600 ppm had inflammation of the trachea or bronchi. A subsequent 13-week study evaluated rats and rabbits exposed to 0, 30, 100, or 300 ppm SO2F2 (337 ppm TWA for rabbits). Rabbits initially were exposed to a high concentration of 600 ppm; however, convulsions were noted in two animals after nine exposures and the concentration subsequently was reduced to 300 ppm. Vacuolation and/or malacia were observed in the cerebrum of all rabbits at the highest concentration; one rabbit exposed to 100 ppm also had cerebral vacuolation. Rabbits at the highest concentration, as well as one rabbit exposed to 100 ppm, had inflammation of the nasal tissues. Rats exposed to 300 ppm SO2F2 for 13 weeks had mottled incisor teeth, minimal renal effects, pulmonary histiocytosis, inflammation of nasal tissues, and cerebral vacuolation. Also, rats exposed to 100 ppm SO2F2 for 13 weeks had mottled teeth. Fluoride toxicity was suggested by mottled teeth in rats as well as elevation of serum fluoride levels in rats and rabbits exposed to SO2F2 for 13 weeks. Although repeated exposure of rats and rabbits to 100-600 ppm SO2F2 resulted in toxicity of the kidneys (rats only), brain, and respiratory system, no effects were detected in animals exposed to 30 ppm for 13 weeks.

Methylene chloride: two-generation inhalation reproductive study in rats.

Reproductive parameters in Fischer 344 rats were evaluated following inhalation of methylene chloride (MeCl2) for two successive generations. Thirty male and female rats were exposed to 0, 100, 500, or 1500 ppm MeCl2 for 6 hr/day, 5 days/week for 14 weeks and then mated to produce f1 litters. After weaning, 30 randomly selected f1 pups/sex/group were exposed to MeCl2 for 17 weeks and subsequently mated to produce f2 litters. Reproductive parameters examined included fertility, litter size and neonatal growth, and survival. All adults and selected weanlings were examined for grossly visible lesions. Tissues from selected weanlings were examined histopathologically. No adverse effects on reproductive parameters, neonatal survival, or neonatal growth were noted in animals exposed to methylene chloride in either the f0 or f1 generations. Similarly, there were no treatment-related gross pathologic observations in f0 or f1 adults or f1 and f2 weanlings. Histopathologic examination of tissues from f1 and f2 weanlings did not reveal any lesions attributed to methylene chloride. Thus, exposure of rats to concentrations as high as 1500 ppm methylene chloride, which has been shown in a 2-year study to produce treatment-related effects, did not affect any reproductive parameters.

Subchronic neurotoxicity in rats of the structural fumigant, sulfuryl fluoride.

Inhalation exposure of male and female Fischer 344 rats to sulfuryl fluoride [Vikane (Dow Chemical Company) gas fumigant] at 300 ppm for 6 hr/day, 5 days week, for 13 weeks caused diminished weight gain, dental fluorosis, a slight decrease in grooming, decreased flicker fusion threshold, slowing of flash, auditory and somatosensory evoked potentials, mild nasal and pulmonary inflammation, mild kidney effects, and mild vacuolation in the brain. Auditory brainstem responses (ABRs) and brain histology were evaluated two months postexposure in 2 male and 2 female rats. Both the ABRs and brain histology were within normal limits at this time, indicating that these treatment effects were, to at least a great extent, reversible. Exposure to 100 ppm resulted in dental fluorosis and very minor slowing of some evoked responses; all other measures, including brain histology, were normal. No treatment effects were noted at 30 ppm.

Acute, pharmacokinetic, and subchronic toxicological studies of 2,4-dichlorophenoxyacetic acid.

The single-dose oral LD50 values in Fischer 344 rats for technical-grade, 2,4-dichlorophenoxyacetic acid (2,4-D), esters, and salts ranged from 553 mg/kg (isobutyl ester in females) to 1090 mg/kg (dimethylamine salt in males). The LD50 values for the acid, esters, or salts, when expressed as acid equivalents, were consistent which suggests that the acute toxicity was due to 2,4-D per se. Acute dermal LD50 values in rabbits for the acid, esters, and salts were greater than 2000 mg/kg. Overall, these results indicate that the acute oral and dermal toxicity of 2,4-D are low. Pharmacokinetics were evaluated in male Fischer 344 rats given single oral doses of 10, 25, 50, 100, or 150 mg 2,4-[14C]D/kg. The amount of 2,4-D in the plasma, kidney, and urine 6 hr postdosing indicated that the urinary elimination of 2,4-D was saturated in male rats given oral doses in excess of 50 mg/kg. Subchronic dietary studies in male and female Fischer 344 rats used dose levels of 0, 15, 60, 100, or 150 mg/kg/day of purified or technical-grade 2,4-D acid for 13 weeks. Body weight gains were decreased for both sexes at the higher dose levels of purified and technical-grade 2,4-D acid. Kidney weights were increased in all treated male rats and in females given the higher three dose levels of purified 2,4-D. Treatment-related cytoplasmic alterations were present in the renal proximal tubules of most rats given 60 mg/kg/day and higher of purified or technical-grade 2,4-D; a few females given 15 mg/kg/day also had slight alterations in the cytoplasm of the proximal tubules. A dose-related degenerative change was identified in the descending proximal renal tubules of all male rats given the highest three dose levels of either test material and some given 15 mg/kg/day. Dose levels of 100 or 150 mg/kg/day of either compound for both sexes produced minimal swelling and increased staining homogeneity in the liver cells and were associated with a slight elevation of liver weight and serum glutamic pyruvic transaminase activity. Higher dose levels of technical-grade and purified 2,4-D decreased total serum tetraiodothyronine levels in female rats, however, the morphology of the thyroid gland was normal. The no-observed-effect level (NOEL) was less than 15 mg/kg/day for both purified and technical-grade 2,4-D acid.

Diethylene glycol monomethyl ether 13-week vapor inhalation toxicity study in rats.

Male and female Fischer 344 rats were exposed to 0, 30, 100, or 216 ppm diethylene glycol monomethyl ether (DEGME) vapors (0, 0.15, 0.49, or 1.06 mg/liter) 6 hr/day, 5 days/week, for 13 weeks. The 216-ppm exposure level was the maximum practically attainable concentration, and it was more than 60% of the theoretical maximum vapor concentration for DEGME at 25 degrees C and 1 atm pressure. Body weights, organ weights, hematological analyses, clinical chemistry analyses, urinalyses, and gross and histopathological examinations revealed no indication of a treatment effect in either male or female rats. Based on the absence of treatment-related effects in this study and the low vapor pressure of the material, DEGME should not present the same degree of inhalation hazard as its structural homolog, ethylene glycol monomethyl ether.

Fungal infections of ventriculi in captive birds.

Ventricular candidiasis was the apparent cause of death in 14 of 21 captive birds, and incriminated in six others. No clinical signs were recognized in 14 of the 21 birds prior to death, and in four of the 21, no other gross or microscopic lesions were documented. Microscopic lesions included colonization of the koilin layer of the ventriculi by fungi with occasional penetration into the glandular epithelium. No conclusive pattern of infection by family of birds was detected.

Tyzzer's disease in snow leopards.

Tyzzer's disease was diagnosed histologically in 2 litters of newborn snow leopard kittens. The gross and histological lesions were similar to those reported in domestic cats and other animals. No signs of illness was noted in either of the snow leopard dams.

A subclinical epizootic of sialodacryoadenitis in rats.

An epizootic of sialodacryoadenitis was identified in 10-week-old rats 5 days after they were housed with a group of older rats. The young rats generally did not develop the characteristic clinical signs of sialodacryoadenitis, but they did have typical histologic lesions of sialodacryoadenitis in Harderian, lacrimal, and salivary glands. The lesions were variable in distribution and severity, and chronic lesions persisted in the Harderian glands of the rats.

Counting renal corpuscles in tissue sections.

Two stereologic methods of counting renal corpuscles in tissue sections were evaluated. Weibel's stereologic method was found to be an accurate and efficient means of determining the number of corpuscles in normal and diseased kidneys. Weibel's method was preferred over the stereologic method of Elias and Hennig because the former is independent of tissue section thickness and is sensitive to variation in corpuscular size. A modification of Weibel's method eliminated the need for an additional point-counting procedure to determine the volumetric fraction of corpuscles in a kidney. The modification made use of previously completed measurements to calculate the area fraction of corpuscles and then substituted this value for the volumetric fraction of renal corpuscles.

Postnatal development of the canine kidney: quantitative and qualitative morphology.

The normal postnatal development of the canine kidney was investigated utilizing qualitative and quantitative histologic methods. Kidneys were examined at 2, 4, 8, 14, 22, 70, and 200 days of age. A subcapsular nephrogenic zone was present in the kidneys until approximately eight days of age. This zone contained tissues which interacted to produce new nephrons and interstitial tissues. Several developmental stages of forming nephrons were identified in this zone. Beneath the nephrogenic zone, renal corpuscles of increasing maturity were located at successively deeper cortical levels. The total number of nephrons was estimated to be 445,000 per kidney. This number did not vary significantly during growth. The corpuscular volume per nephron increased 249% from 14 to 200 days of age. During the same period there was a 303% increase in the tubular volume per nephron. Although the developing kidney differed anatomically from the adult kidney, the individual nephrons maintained volumetric corpuscular-tubular balance during growth.

Scanning electron microscopy of Spironucleus (Hexamita) muris infection in mice.

An epizootic disease characterized by failure to gain weight, lethargy, enteropathy, and death developed in weanlings from a production colony of NIH/Nmri CV mice. Light microscopic and scanning electron microscopic examination of the intestine revealed large numbers of protozoan parasites identified as Spironucleus (Hexamita) muris. The extensive, entangled mass of organisms in the intestinal lumen enveloped the villi and continued deep within the crypts of Lieberkühn. Inflammation and significant intestinal lesions were not observed. The scanning electron microscopic appearance of the small intestine in spironucleosis gives credence to a pathogenesis of malabsorption with subsequent, severe malnutrition and death in weanling mice.