David P. Kelly

Inhalation toxicity study on rats exposed to titanium tetrachloride atmospheric hydrolysis products for two years

Rats were exposed to TiCl4 hydrolysis products by inhalation exposure at aerosol concentrations of 0, 0.1, 1.0, and 10 mg/m3 for 6 hr/day, 5 days/week for 2 years. There were no abnormal clinical signs, body weight changes, or excess mortality in any exposed groups. No pathological changes other than a mild rhinitis were observed at 0.1 mg/m3. At 1.0 mg/m3, the incidence of mild rhinitis and tracheitis was increased. The lungs showed a minute dust-laden macrophage (dust cell) reaction with slight Type II pneumocyte hyperplasia in alveoli adjacent to the alveolar ducts. The pulmonary response at the 1.0 mg/m3 satisfied the biological criteria for a nuisance dust. At 10 mg/m3, extrapulmonary particle deposition occurred in the tracheobronchial lymph nodes, liver, and spleen without any tissue response. An increased incidence of rhinitis, tracheitis, and dust cell response with Type II pneumocyte hyperplasia, alveolar bronchiolarization, foamy dust cell accumulation, alveolar proteinosis, cholesterol granuloma, and focal pleurisy was also observed. The pulmonary lesions developed in the alveolar duct region where dust cells had accumulated and had provoked a chronic tissue response. In addition, a few well-differentiated, cystic keratinizing squamous carcinomas were developed from alveoli showing bronchiolarization with squamous metaplasia in the alveolar duct region. No tumor metastasis was found in other organs. The lung tumors were a unique type of experimentally induced tumor and have not been seen usually in man or animals. Therefore, the relevance to man of this type of lung tumor is highly questionable.

Degeneration and recovery of rat olfactory epithelium following inhalation of dibasic esters

Dibasic esters (DBE) are solvent mixtures used in the paint and coating industry. To evaluate the potential subchronic toxicity of DBE, groups of male and female rats were exposed for periods of up to 13 weeks to DBE concentrations of 0, 20, 76, or 390 mg/m3. After approximately 7 and 13 weeks of exposure, 10 rats per sex per group were subjected to clinical chemical, hematological, and urine analyses. Following 7 or 13 weeks of exposure, 10 or 20 rats per sex per group, respectively, were euthanized. An additional 10 rats were euthanized following a 6-week recovery period. A standard profile of tissues, including four levels of nasal cavity, was evaluated histopathologically. After 7 weeks of exposure, slight degeneration of the olfactory epithelium was observed in both male and female rats at 76 and 390 mg/m3. After 13 weeks, degeneration of the olfactory epithelium was present at all DBE concentrations in female rats, but only at the mid and high concentrations in male rats. The severity and incidence of the lesions were concentration related for both sexes with female rats being more sensitive than males. Following the recovery period, histological changes compatible with repair in the olfactory mucosa included an absence of degeneration, focal disorganization of the olfactory epithelium, and respiratory metaplasia. All other tissues were macroscopically normal. No other signs of toxicity were indicated by the other parameters evaluated. Inhalation studies of other esters demonstrate similar pathology in the olfactory epithelium. Since olfactory mucosa is rich in carboxylesterase activity, acids may be the toxic metabolites of these compounds. This hypothetical mechanism may explain the sensitivity of olfactory tissue to the effects of DBE.

The pulmonary response and clearance of Ludox colloidal silica after a 4-week inhalation exposure in rats

Rats were exposed to Ludox colloidal silica (CS) at concentrations of 0, 10, 50, and 150 mg/m3 for 6 hr/day, 5 days/week for 4 weeks. Rats were killed after 4 weeks of exposure and 10 days or 3 months postexposure (PE). The exposure concentration of 10 mg/m3 Ludox CS is considered to be the no-effect concentration. There were no exposure-related clinical signs in any group. After 4 weeks exposure, lung weights were increased significantly in rats exposed to 50 and 150 mg/m3 Ludox CS, but lung weights were similar to those of controls at 3 months PE. After 4 weeks exposure to 50 mg/m3 Ludox CS, a slight alveolar macrophage response, polymorphonuclear leukocytic infiltration, and Type II pneumocyte hyperplasia in alveolar duct regions were present. After 3 months PE, these pulmonary lesions had almost disappeared with removal of most dust-laden alveolar macrophages (AMs). The pulmonary response to 150 mg/m3 Ludox CS was similar in character but increased in magnitude from that seen at 50 mg/m3. At 3 months PE, most particle-laden AMs had disappeared and the remaining AMs were aggregated and sharply demarcated. A few aggregates of particle-laden AMs appeared to transform into silicotic nodules comprising macrophages, epithelioid cells, and lymphocytic infiltration in some animals. Some silicotic nodules showed reticular fiber networks with minute collagen fiber deposition. Tracheobronchial lymph nodes were enlarged with aggregates of particle-laden AMs and hyperplastic histiocytic cells. Lung-deposited Ludox cleared rapidly from the lungs with half-times of approximately 40 and 50 days for the 50 and 150 mg/m3 groups, respectively.

Pulmonary response to inhaled Kevlar aramid synthetic fibers in rats

Groups of male rats were exposed to specially prepared ultrafine Kevlar pulp fibers (du Ponts registered trademark for certain aramid fibers) at atmospheric concentrations of either 0.1, 0.5, 3.0, or 18 mg/m3 for 2 weeks. Rats were killed at 0 and 2 weeks and 3 and 6 months postexposure (PE) except the rats exposed to 18 mg/m3, which were killed 0, 4, and 14 days and 1, 3, and 6 months PE. Another group of male rats was exposed to 18 mg/m3 (respirable dust approximately 2.5 mg/m3) of commercial Kevlar fibers for 2 weeks and were killed at 0 and 2 weeks and 3 and 6 months PE. Inhaled ultrafine Kevlar fibers were mostly phagocytized by alveolar macrophages (dust cells) in the alveolar ducts and adjoining alveoli after exposure to either 0.1 or 0.5 micrograms/m3. Most dust cells had disappeared and lungs showed a normal appearance throughout 6 months PE. The pulmonary response almost satisfied the biological criteria for a nuisance dust. Rats exposed to 3 mg/m3 ultrafine Kevlar fibers revealed occasional patchy thickening of alveolar ducts with dust cells and inflammatory cells but with no collagen fibers deposited throughout 6 months PE. After exposure to 18 mg/m3 ultrafine Kevlar, the respiratory bronchioles, alveolar ducts, and adjoining alveoli showed granulomatous lesions with dust cells by 2 weeks PE. The granulomatous lesions converted to patchy fibrotic thickening with dust cells after 1 month PE. The fibrotic lesions were markedly reduced in cellularity, size, and numbers from 3 to 6 months PE but revealed networks of reticulum fibers with slight collagen fiber deposition.

Subchronic inhalation of high concentrations of low toxicity, low solubility participates produces sustained pulmonary inflammation and cellular proliferation

Long-term inhalation exposures to high dust burdens can produce tumors or proliferative keratin cysts in the lungs of exposed rats. We hypothesized that dust burdens which overwhelm lung clearance mechanisms are associated with sustained cellular proliferation responses and pulmonary inflammation. Male rats were exposed to titanium dioxide (TiO2) or carbonyl iron (CI) particles for 4 weeks at concentrations of 5, 50 and 250 mg/m3. Following completion of exposure, the lungs of sham and dust-exposed animals were lavaged or assessed for cell proliferation or particle clearance immediately after, as well as 1 week, 1, 3 and 6 months postexposure. Exposures to TiO2 or CI at 250 mg/m3 produced persistent pulmonary inflammatory responses and increased BrdU labeling of terminal airway and pulmonary parenchymal cells. The results of this study clearly demonstrate that exposure to excessive dust concentrations of two low toxicity, low solubility particle-types produced sustained pulmonary inflammation, enhanced pulmonary cell labeling, impairment of particle clearance, and the development of pulmonary lesions.

Deposition, Clearance, and Shortening of Kevlar Para-aramid Fibrils in Acute, Subchronic, and Chronic Inhalation Studies in Rats

The deposition and clearance of lung-deposited Kevlar para-aramid fibrils (subfibers) have been investigated as part of a subchronic and chronic inhalation toxicity testing program. Fibrils recovered from lung tissue in para-aramid-exposed Sprague-Dawley rats were microscopically counted and measured after exposures to airborne fibrils which were about 12 microns median length (ML) and < 0.3 micron median diameter. In each of three studies lung-recovered fibrils were progressively shorter with increasing residence time in the lungs. Twenty-eight days after a single 6-hr exposure at 400 respirable fibrils per cubic centimeter (f/cm3) the ML of recovered fibrils decreased to about 5 microns. Twenty-four months after a 3-week exposure to 25 or 400 f/cm3, fibrils reached about 2 microns ML. After 2 years of continuous exposure at 2.5, 25, or 100 f/cm3 or 1 year exposure plus 1 year recovery at 400 f/cm3, fibril ML approached 4 microns. In the 2-year study, the lung-fiber accumulation rate/exposure concentration was similar for the three highest concentrations and was about 3 x greater than that seen at 2.5 f/cm3, indicating that concentrations of about 25 f/cm3 or more may overwhelm clearance mechanisms. Time required for fibrils to be reduced to < 5 microns in the lung was markedly less at lower exposure concentration and shorter exposure time. The primary shortening mechanism is proposed to be long fibril cutting by enzymatic attack at fibril defects. However, length-selective fibril deposition and clearance may contribute to shortening in the first few days after exposure. The enzymatic cutting hypothesis is supported by measured increases in numbers of short fibers following cessation of exposures, continued shortening of the fibril length distribution up to 2 years following exposure, and in vitro fibril shortening after 3 months in a proteolytic enzyme preparation. The conclusion is that para-aramid fibrils are less durable in the lungs of rats than expected from the known chemical resistance of commercial yarn. These data suggest that at the low para-aramid fibril exposures found in the workplace, this fibril-shortening mechanism may limit the residence time of long fibers in the lungs of exposed workers. In addition, associated cascade impactor aerodynamic measurements indicate that due to their ribbon shape and curly nature, para-aramid fibrils behave aerodynamically larger than straight fibers.

Four-week inhalation toxicity study with ludox colloidal silica in rats : pulmonary cellular responses

This study was designed to complement a traditional subchronic inhalation toxicity study with Ludox colloidal silica. CD rats were exposed nose-only for 2 or 4 weeks at concentrations of 0, 10, 50, and 150 mg/m3 Ludox (dried SiO2). Additional groups of rats exposed for 4 weeks were given a 3-month recovery period. Following exposure and/or recovery, fluids and cells were recovered from the lungs by bronchoalveolar lavage (BAL) and measured for cellular and biochemical parameters. Additional groups of animals were processed for cell labeling studies or lung deposition studies. Inhaled doses of Ludox colloidal silica were measured after 4-week exposures and were found to be 489 micrograms/lung (10 mg/m3 group), 2418 micrograms/lung (50 mg/m3), and 7378 micrograms/lung (150 mg/m3), respectively. Results showed that exposures to 150 mg/m3 Ludox for 2 or 4 weeks produced pulmonary inflammation along with increases in BAL protein, LDH, and alkaline phosphatase values (p less than 0.05) and reduced macrophage phagocytosis. Inflammatory responses, evidenced by increased numbers of neutrophils, were also measured in the lungs of the 50 mg/m3 group following 2 and/or 4 weeks of exposure. Most biochemical parameters for all groups returned to control values following a 3-month recovery period. Autoradiographic studies demonstrated that the labeling indices of terminal bronchiolar and lung parenchymal cells were generally increased in the 50 and 150 mg/m3 groups after 2 and 4 weeks of exposure but, with one exception, returned to normal levels following a 3-month postexposure period. No significant alterations in any measured parameters were detected in rats exposed to 10 mg/m3 Ludox at any time postexposure. The determination of a no-observable-effect level (NOEL) of 10 mg/m3 was consistent with results obtained by conventional toxicology methods and affirms the utility of these biochemical, cellular, and autoradiographic techniques for providing a predictive screen to assess the toxicity of inhaled particles.

A 90-Day Inhalation Toxicity Study with Benomyl in Rats

Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate, CAS Registry No. 17804-35-2] is a fungicide and the possibility for inhalation exposure exists for field workers. To assess the toxicity of benomyl, groups of 20 male and 20 female CD rats were exposed nose-only 6 hr a day, 5 days a week, to concentrations of 0, 10, 50 or 200 mg/m3 of a benomyl atmosphere. At the midpoint (approximately 45 days on test) and at the end of the exposure period, blood and urine samples for clinical evaluation were collected from 10 rats/group/sex, and these animals were sacrificed for pathological examination. Similar evaluations were performed on all remaining rats at the end of the 90-day test period. After approximately 45 days on test, compound-related degeneration of the olfactory epithelium was observed in all males and in 8 of 10 female rats exposed to 200 mg/m3 benomyl. Two male rats exposed to 50 mg/m3 had similar, although less severe, areas of olfactory epithelial degeneration. After approximately 90 days of exposure, the remaining 10 rats/group/sex were sacrificed and examined. Of these rats, all of the males and females exposed to 200 mg/m3 had olfactory degeneration, along with 3 males exposed to 50 mg/m3 of benomyl. No other observed lesions were interpreted to have been caused by the benomyl exposure. In addition, male rats exposed to 200 mg/m3 benomyl had depressed mean body weights compared to controls and this finding correlated with a reduction in food consumption. Based on pathological observations, 10 mg/m3 represents the no-observable-effect level (NOEL) for the male rats, and 50 mg/m3 is the NOEL for the female rats.

ACUTE INHALATION EXPOSURE TO CYCLOHEXANE AND SCHEDULE-CONTROLLED OPERANT PERFORMANCE IN RATS: COMPARISON TO D-AMPHETAMINE AND CHLORPROMAZINE

Adult male rats pressed a lever on a multiple fixed ratio-fixed interval (FR20-FI120 sec) schedule of food presentation, and after attaining a stable baseline subjects received an acute inhalation exposure to cyclohexane vapor (0 ppm, 500 ppm, 2000 ppm, or 7000 ppm) for 6 hr. During the operant session that began 30 min after termination of exposure, FR running rate for the 7000 ppm group decreased 11% relative to performance on the previous day. FR post-reinforcement pause duration and the rate and pattern of FI performance were unaffected. Cyclohexane exposures of 500 or 2000 ppm had no detectable effects. No enduring effects of cyclohexane occurred up to 2 weeks after exposure. An independent set of rats, trained under nominally identical conditions, received various doses (i.p.) of d-amphetamine (AMPH) or chlorpromazine (CPZ) at 1–2 week intervals. Effective doses of AMPH decreased FR running rate, decreased FR post-reinforcement pause duration and increased FI rate of response. AMPH also decreased the FI index of curvature, indicating a change from an accelerating rate during the FI to a more constant rate. Effective doses of CPZ decreased FR rate, increased FR pause duration, decreased FI rate, and decreased FI index of curvature. Thus, schedule-controlled operant procedures that were sensitive to the effects of psychoactive drugs were able to identify only a minor and transient effect of the highest concentration (7000 ppm) of cyclohexane vapor on operant performance.

Inhalation developmental toxicity and reproduction studies with cyclohexane

The reproductive and developmental toxicity of cyclohexane was assessed in a two-generation reproduction study with Crl:CD® BR rats and in developmental toxicity studies with Crl:CD®BR rats and Hra:(NZW)SPF rabbits. The animals were exposed whole-body to atmospheric concentrations of 0, 500, 2000, or 7000 ppm cyclohexane. In the two-generation reproduction study, parental effects included statistically significantly lower mean body weight, overall mean body weight gain, and overall mean food efficiency for P1 and F1 females of the 7000 ppm level and statistically significantly lower mean body weight for F1 males of that level. Adult rats exposed to 2000 ppm cyclohexane and above exhibited a transient diminished or absent response to a sound stimulus while in the chambers during exposure. Mean pup weight was statistically significantly lower than control from lactation day 7 throughout the remainder of the 25-day lactation period for both F1 and F2 7000 ppm litters. Changes observed at 500 ppm were either considered not to be compound related or not adverse. Therefore, the systemic-toxicity no-observed-effect level (NOEL) was 500 ppm and the reproductive NOEL was 2000 ppm. The reproductive NOEL was based solely on the decreased pup weights in both the F1 and F2 generations observed at 7000 ppm. In the developmental toxicity studies, only the rats showed evidence of maternal toxicity. For rats in the 7000 ppm group, statistically significant reductions were observed in overall maternal body weight gain and overall maternal food consumption for the treatment period. Rats exposed to 2000 ppm cyclohexane and above again exhibited a transient diminished or absent response to a sound stimulus while in the chambers during exposure. Therefore, for rats, the maternal no-observed-effect level (NOEL) was 500 ppm. In the rabbit developmental toxicity study, no compound-related maternal effects were observed at concentration levels of 7000 ppm and below. Therefore, the maternal NOEL for rabbits was 7000 ppm. No compound-related evidence of developmental toxicity was observed at any test concentration in either species. Therefore, the developmental NOEL for both species was 7000 ppm, the highest concentration tested.