Using physiologically based pharmacokinetic modeling and benchmark dose methods to derive an occupational exposure limit for N-methylpyrrolidone.

The developmental effects of NMP are well studied in Sprague-Dawley rats following oral, inhalation, and dermal routes of exposure. Short-term and chronic occupational exposure limit (OEL) values were derived using an updated physiologically based pharmacokinetic (PBPK) model for NMP, along with benchmark dose modeling. Two suitable developmental endpoints were evaluated for human health risk assessment: (1) for acute exposures, the increased incidence of skeletal malformations, an effect noted only at oral doses that were toxic to the dam and fetus; and (2) for repeated exposures to NMP, changes in fetal/pup body weight. Where possible, data from multiple studies were pooled to increase the predictive power of the dose-response data sets. For the purposes of internal dose estimation, the window of susceptibility was estimated for each endpoint, and was used in the dose-response modeling. A point of departure value of 390 mg/L (in terms of peak NMP in blood) was calculated for skeletal malformations based on pooled data from oral and inhalation studies. Acceptable dose-response model fits were not obtained using the pooled data for fetal/pup body weight changes. These data sets were also assessed individually, from which the geometric mean value obtained from the inhalation studies (470 mg*hr/L), was used to derive the chronic OEL. A PBPK model for NMP in humans was used to calculate human equivalent concentrations corresponding to the internal dose point of departure values. Application of a net uncertainty factor of 20-21, which incorporates data-derived extrapolation factors, to the point of departure values yields short-term and chronic occupational exposure limit values of 86 and 24 ppm, respectively.

Chronic toxicity and oncogenicity of N-methylpyrrolidone (NMP) in rats and mice by dietary administration.

A two-year feeding study in rats and an 18-month feeding study in mice were conducted to evaluate the potential chronic toxicity and oncogenicity of NMP in Crl:CD (SD)BR rats and B6C3F1/CrlBR mice. Groups of 62 male and female rats were administered diets containing 0, 1600, 5000, or 15,000 ppm of NMP for approximately 2 years. Groups of 50 male and female mice were administered diets containing 0, 600, 1200, or 7200 ppm NMP for approximately 18 months. In vivo parameters were evaluated weekly during the first 3 months of the study, and every other week or monthly during the remainder of the study. For rats, an ophthalmoscopic examination was conducted prior to study start and near the end of the study. Periodically, blood samples were collected from rats and mice for determination of leukocyte differential counts, and from mice for red blood cell morphology. After approximately 2 years of dietary administration in rats and 18 months in mice, all surviving animals were sacrificed. Selected tissues were processed for morphological evaluation. Over the course of the two-year study in rats, test substance-related decrements in body weight and weight gain occurred in 15,000 ppm males and females, which correlated with decreased food consumption and food efficiency. A toxicologically significant, test substance-related increase in the incidence of severe chronic progressive nephropathy occurred in 15,000 ppm males. Several morphological changes noted grossly and/or microscopically were secondary to the increased severity of chronic progressive nephropathy. NMP was not oncogenic in male or female rats at dietary concentrations of 15,000 ppm and below. A test substance-related decrease in the percentage of 15,000 ppm males surviving to the end of the two-year study compared to the control group resulted from the higher incidence of severe chronic progressive nephropathy. However, a sufficient population of 15,000 ppm rats were at risk for potential oncogenicity, so the lower survival did not impair the ability to detect an oncogenic response in this study. There were no adverse, test substance-related effects on the incidences of clinical observations, ophthalmic observations, or differential leukocyte counts in males or females, or on survival of females at any dietary concentration. Male and female mice administered dietary concentrations of 7200 ppm had significantly increased liver weight, significantly increased incidence of hepatocellular adenoma, and significantly increased foci of cellular alteration in the liver. At 7200 ppm, male mice also had an increased incidence of hepatocellular carcinoma while the increased incidence of hepatocellular carcinoma in female mice fell within the historical control range. In addition, the incidence of hepatocellular hypertrophy was increased in 7200 ppm males. Liver weight and hepatocellular hypertrophy were also increased in 1200 ppm males. There were no adverse, test substance-related effects on the incidences of clinical observations, food consumption, body weight, differential leukocyte counts, red blood cell morphology, or survival in either males or females at any dietary concentration. Under the conditions of the study, the no-observed-effect level for NMP was 5000 ppm for male and female rats, 600 ppm for male mice, and 1200 ppm for female mice.

90-day subchronic toxicity study in rats and mice fed N-methylpyrrolidone (NMP) including neurotoxicity evaluation in rats.

In mice, there were no effects on body weight or food consumption. As observed in rats, mice fed 2,500 or 7,500 ppm exhibited a change in urine coloration which was not associated with morphological changes in cholesterol, triglycerides, calcium, and alkaline phosphatase occurred at 28 days but not 90 days. These changes are thus assessed as being of minor toxicological relevance. Liver weights were elevated in males fed 2,500 or 7,500 ppm and centrilobular hypertrophy was seen in both sexes fed 7,500 ppm. These changes may be regarded as an adaptation process but are clearly related to NMP exposure. Other toxicological endpoints examined were unaffected by NMP. The NOAEL was 3,000 ppm for both sexes of rats based on body weight effects and changes in 3 neurobehavioral parameters (males only) at higher feeding levels. In mice, the NOAEL was 1,000 ppm based on liver responses to higher concentrations.

Subchronic inhalation toxicity study of caprolactam (with a 4-week recovery) in the rat via whole-body exposures.

This study was designed to assess the potential subchronic inhalation toxicity of caprolactam when administered as a 3-micron aerosol from an aqueous solution to Sprague-Dawley CD rats (10/sex/group) via whole-body exposure. The study was enhanced with the inclusion of motor activity measurements and a functional observational battery to assess the neurotoxic potential of caprolactam. The rats were exposed at least 65 times over a 13-week period for 6 h per day, 5 days per week, to target concentrations (3 microns, mass median aerodynamic diameter) of 0, 25, 75, and 250 milligrams per cubic meter (mg/m3). An additional 10 animals/sex/group were similarly exposed and then held for a 4-week recovery period. Exposure levels were determined gravimetrically six times daily; one daily sample was analyzed by high-pressure liquid chromatography. No deaths were observed in the study during the exposure or recovery periods. Treatment-related responses such as labored breathing and nasal discharge were seen during many of the exposures. Similar responses as well as moist rales were seen during the nonexposure periods during the 13 weeks of exposure. However, these responses abated during the 4-week recovery period. There were no clearly treatment-related responses observed with ophthalmoscopic examinations, body weight measurements, food consumption measurements, neurobehavioral evaluations, clinical pathology evaluations, organ weight measurements, or macroscopic pathology examinations. Microscopic findings that were considered related to exposure to the test material were seen in the nasoturbinal tissues (hypertrophy/hyperplasia of goblet cells in the respiratory mucosa and intracytoplasmic eosinophilic material in epithelial cells of the olfactory mucosa) of the two higher-exposure group animals and in the laryngeal tissues (squamous/squamoid metaplasia/hyperplasia of the pseudostratified columnar epithelium covering the ventral seromucous gland) of all three exposure group animals. These changes were considered to be adaptive responses to an irritant (caprolactam). The keratinization of the metaplastic epithelium in the larynx was considered to be an adverse effect. By the end of the 4-week recovery period, there was complete regression of the keratinization in the larynx, but recovery of the adaptive nasoturbinal effects had not completely resolved. In conclusion, the whole-body exposure of Sprague-Dawley rats to caprolactam as a respirable aerosol for 6 h/day, 5 days/week, for 13 weeks at gravimetrically determined levels of 24, 70, and 243 mg/m3 resulted in respiratory tract effects (laryngeal) at the highest exposure level with complete recovery within 4 weeks postexposure. The results indicate that the no-observed-adverse-effect level for caprolactam is 70 mg/m3, based on upper respiratory effects, with 243 mg/m3 representing a no-observed-effect level for systemic toxicity, neurotoxicity, and lower respiratory tract effects.

Inhibition of immune opsonin-independent phagocytosis by antibody to a pulmonary macrophage cell surface antigen.

Unlike other hamster phagocytes, hamster pulmonary macrophages (PM) avidly ingest albumin-coated latex particles in the absence of serum. They also possess a highly specific cell surface antigen. To evaluate the relationship between these two characteristics, PM were incubated with mouse monoclonal antibody directed against the PM antigen. After unbound antibody was removed, the amount of bound antibody and the phagocytic capability of PM were measured by flow cytometry and fluorescence microscopy. Maximum antibody binding produced a 25% inhibition of ingestion. Particle attachment was not affected. This effect was antigen specific, since neither a nonspecific mouse myeloma protein of the same subclass nor a mouse antibody that bound to another hamster surface antigen had any effect on binding or ingestion. If antigen-specific F(ab')2 fragments were introduced both before and during the period of phagocytosis, the inhibition of particle ingestion approached 100%. Particle binding increased at low F(ab')2 concentrations but declined at higher concentrations. Because calcium may play a role in the ingestion process, the effect of antibody on 45Ca uptake was evaluated. It was observed that antigen-specific F(ab')2 fragments stimulated 45Ca uptake, whereas control antibodies did not. These results suggest that the antigen reacting with our anti-hamster PM monoclonal antibody is involved in immune opsonin-independent phagocytosis and that calcium participates in this phagocytic process.

Immune opsonin-independent phagocytosis by pulmonary macrophages.

The uptake of albumin-coated latex particles by hamster pulmonary macrophages (PM) in vitro was investigated by using a new technique that combined flow cytometry and fluorescence microscopy to differentiate and quantitate bound vs ingested particles. In the absence of serum, PM avidly bound and ingested particles, whereas phagocytosis by hamster polymorphonuclear leukocytes (PMN) was less marked. In the presence of serum, phagocytosis by PM was slightly depressed, whereas phagocytosis by PMN was stimulated more than 10-fold. The binding of particles to PM in the absence of serum was pH, temperature, and trypsin sensitive and was dependent on the presence of extracellular Ca++ but not Mg++. The ingestion of particles by this immune opsonin-independent pathway was also temperature sensitive but was not affected by either pH or extracellular Ca++. Particle ingestion, but not binding, was inhibited by cytochalasin D and the divalent cation ionophore A23187.

Uptake of latex particles by macrophages: characterization using flow cytometry.

Flow cytometry can be used to characterize the uptake of particles by small samples of pulmonary macrophages both quickly and accurately. We found that there was a linear relationship between the number of fluorescent latex particles and the fluorescent intensity associated with each cell up to 47 particles/cell. Macrophages lavaged from the lungs of Syrian golden hamsters were metabolically and functionally stable for 3 h of incubation; the average intracellular concentrations of Na+ and K+ were 29 +/- 2 and 158 +/- 6 mM, respectively. Particle uptake was significantly inhibited by removing divalent cations with ethylenediaminetetraacetic acid and lowering the incubation temperature (37 degrees C) to 24 and 3 degrees C. The cell-to-cell variability in the uptake of particles was greater than the Poisson distribution would predict based on the mean number of particles per cell.

Uptake of latex particles by pulmonary macrophages: role of calcium.

The uptake of latex particles by both viable and fixed hamster pulmonary macrophages was calcium and trypsin sensitive. Particle uptake did not stimulate the uptake of 45Ca. However, when 45Ca uptake was stimulated with A23187, particle uptake was inhibited. When cobalt was added with A23187, the uptake of 45Ca was inhibited and particle uptake returned to control levels. A23187, cytochalasin B, and A23187 plus cytochalasin B all reduced particle uptake to the same extent. Although both A23187 and ouabain produced similar changes in the intracellular levels of Na+ and K+, only A23187 inhibited particle uptake. We conclude that extracellular Ca2+ promotes particle-cell binding through its interaction with a trypsin-sensitive receptor in the pulmonary macrophage membrane. In contrast, elevated intracellular Ca2+ levels inhibit particle ingestion but not attachment.

Stimulus-permeability coupling in rat lacrimal gland.

Incubation of isolated cells wtih 10(-2) M ethylene glycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid or cobalt inhibits 22Na and 45Ca uptake stimulated by carbachol. The artificial introduction of Ca into the cytosol by the cation ionophore A23187 also initiates the 22Na uptake. Amiloride (10(-5) M) partially inhibits 22Na uptake induced by carbachol, but has no effect on receptor-stimulated 45Ca uptake or 86Rb release. Tetrodotoxin (TTX) has no effect on 22Na uptake stimulated by carbachol, whereas methoxyverapamil (D 600) produces a small but significant decrease in both 22Na and 45Ca uptake. This effect of D 600 may be related to a block of receptor activation and not to a block of Ca channel activation. Incubation in high K (56 mM) does not prevent the change in membrane permeability to Ca, K, and Na initiated by carbachol. It is concluded that carbachol stimulates the influx of Ca; the rise in the cytosolic Ca concentration then couples receptor activation to a change in membrane permeability to K and Na. The permeability mechanisms for Ca, K, and Na that are activated by carbachol appear to be specific for each of the three cations and appear to be dissimilar to permeability mechanisms in excitable tissue that carry the same ions.

Muscarinic and alpha-adrenergic stimulation of Na and Ca uptake by dispersed lacrimal cells.

Rat lacrimal gland acinar cells were isolated and observed to be physiologically stable for several hours of incubation in vitro. With a double-isotope technique, it was found that carbachol and epinephrine stimulated the uptake of 22Na and 45Ca by lacrimal cells. These respnses were maximal at agonist concentrations of 10(-5) M and were blocked by atropine and phentolamine, respectively. It is concluded that muscarinic and alpha-adrenergic receptor activation increase the membrane permeability of the lacrimal gland acinar cell to Na and Ca, ions that may be important in the secretion of water by the lacrimal gland.

Membrane potential changes in lacrimal gland acinar cells elicited by carbachol and epinephrine.

Intracellular microelectrode recordings of acinar cell membrane potentials were made from fragments of the rat lacrimal gland superfused in vitro. The average resting membrane potential was -45 mV. Carbachol and epinephrine produced virtually identical membrane potential changes consisting of an initial hyperpolarization (1 mV), lasting approximately 7 sec, followed by a depolarization of approximately 12 mV. The membrane potential generally returned to prestimulation levels after 2 min of exposure to agonist. The responses to carbachol and epinephrine were blocked by atropine and phentolamine, respectively. Superfusion with media lacking Ca or Cl reduced significantly both the resting membrane potential and the agonist-induced depolarization. The hyperpolarization was increased significantly in the absence of Ca and generally prolonged in the absence of Cl. Superfusion with 10 mM Co had no effect on either the resting membrane potential or the agonist-induced membrane potential changes. The hyperpolarization initiated by agonist was significantly enhanced during superfusion with low K, ouabain or amiloride while the depolarization was significantly reduced during superfusion with low K, amiloride or low Na. Resting membrane potentials during superfusion with low K, amiloride or low Na were not significantly different from control, whereas ouabain caused a small depolarization. It is concluded that muscarinic or alpha adrenergic receptor stimulation initiates a membrane potential change characterized by a hyperpolarization, due to an increased in membrane permeability to K, followed by a depolarization due to an increase in membrane permeability to Na.

An alpha-adrenergic receptor mechanism controlling potassium permeability in the rat lacrimal gland acinar cell.

1. Rat lacrimal gland slices, incubated in a balanced, buffered salt solution, were found to be physiologically stable for up to 2 hr with respect to O2 consumption, extracellular space, and water and ion content. 2. The release of 86Rb serves as a good substitute for 42K in monitoring the movement of K through the cell membrane. 3. Adrenaline appears to increase membrane permeability to K as evidenced by an increase in the rate of 86Rb efflux. 4. This response to adrenaline was blocked by phentolamine but not by propranolol and was mimicked by phenylephrine but not by isoprenaline. 5. The magnitude of the 86Rb release indicates that it is being released, at least in part, from the lacrimal gland acinar cell. 6. It is concluded that the lacrimal gland acinar cell has an alpha-adrenergic receptor, activation of which leads to an increase in membrane permeability to K.

The role of calcium in the receptor mediated control of potassium permeability in the rat lacrimal gland.

1. In the presence of extracellular Ca, adrenaline stimulated a large increase in the rate of K (86Rb) release from rat lacrimal slices, followed by a lower, more sustained rate. 2. In the absence of extracellular Ca, adrenaline elicited only a transient release of 86Rb. 3. The artificial introduction of Ca into the cytosol by the ionophore A-23187 could also initiate the release of 86Rb. 4. In a zero-Ca medium, if either adrenaline or carbachol produced a transient release of 86Rb, the tissue could not respond to the other agonist with a transient release unless Ca was momentarily reintroduced to the medium. 5. If Ca was present in a limiting concentration, the Ca-dependent rate of 86Rb release elicited from a lacrimal slice exposed simultaneously to carbachol and adrenaline was not significantly different from the release seen with carbachol alone. 6. It is concluded that the agonist-induced release of K from the lacrimal gland consists of both a Ca-independent phase which is initiated by the release of a limited pool of Ca, and a Ca-dependent phase which is mediated by the influx of extracellular Ca. 7. It is also concluded that both alpha-adrenergic and muscarinic receptor occupation activate a common, post-receptor mechanism which may be responsible for both phases of K release.

Calcium-mediated effects of carbachol on cation pumping and Na uptake in rat parotid gland.

Carbachol (10(-4) M), a cholinergic secretagogue, significantly increased the ouabain-sensitive uptake of 86Rb by rat parotid gland slices. This effect was blocked by the omission of Ca from the bathing medium. When extracellular Na was decreased from 125 to 5 mM, the effect of carbachol was reversed (86Rb uptake was significantly decreased). Increasing intracellular Na (by incubation in medium lacking K) produced a significant stimulation of 86Rb influx. A role for Na in the response was suspected, and so the metabolism, of 22Na by the slices was characterized. The 60-minute distribution of 22Na could be described by three kinetic components; one extracellular (0.315 ml/g, tau = 1.94 minutes) and two intracellular (0.075 ml/g, tau = 7.63 minutes and 0.017 ml/g, tau = 58.4 minutes). Carbachol enhanced the uptake of 22Na into the intracellular components (primarily the 7.63-minute component). The increased uptake of 22Na required Ca in the bathing medium. These observations could be explained by assuming that cholinergic receptor activation stimulates Ca influx which in turn enhances Na uptake. The resulting elevated intracellular Na acts to stimulate activity of the Na,K pump.