Fourteen-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette: II. DNA Adducts and Alveolar Macrophage Cytogenetics

The chemical constituents of cigarette smoke are greatly dilutedin environmental tobacco smoke (ETS). In the typical indoorenvironment where cigarettes are smoked, the mean value of respirablesuspended particles is approximately 0.1 mg/m³. In this study,we used aged and diluted sidestream smoke (ADSS) of 1R4F Universityof Kentucky research cigarettes as a surrogate for ETS and exposedSprague-Dawley rats nose-only to 0, 0.1, 1.0, and 10 mg wettotal particulate matter (WTPM)/m³ for 6 hr per day for 14 consecutivedays. DNA from lung, heart, larynx, and liver was tested foradduct formation after 7 and 14 days of exposure and after 14days of recovery. In addition, alveolar macrophages from animalsexposed for 7 days were examined for chromosomal aberrations.Exposure-related DNA adducts were not observed in any of theanimals at 0.1 or 1.0 mg WTPM/ m³, which represent ambient and10-fold exaggerated ETS concentrations, respectively. Slightdiagonal radioactive zones, characteristic of adducts observedin human smokers and in animals exposed to mainstream smoke,were observed, but only in lung and heart DNA of animals exposedto the highest concentration of ADSS (10 mg WTPM/m³), a 100-foldexaggeration of typical field measurements of ETS. The meanrelative adduct labeling values (±SE) were 8.7 (±0.2)adducts per 10' nucleotides for lung DNA and 5.7 (±0.7)adducts per 10' nucleotides for heart DNA after 14 days of exposure.No elevation in chromosomal aberrations was observed in alveolarmacrophages. These results indicate a no-observed-effect-level(NOEL) of 1.0 mg/m³ for DNA adduct formation in lung and heartand a NOEL of at least 10 mg/m³ for the induction of chromosomeaberrations in alveolar macrophages under the conditions ofthis study.     

Replicative dna synthesis in tissues of the rat exposed to aged and diluted sidestream smoke

Abstract

Male Sprague-Dawley rats were exposed to aged and diluted sidestream smoke (ADSS) from Kentucky 1R4F reference cigarettes for 6 h/day, 5 days/wk, for a 13-wk period. Exposure concentrations were 0, 0.1, 1, and 10 mg ADSS/m³. Exposures were conducted in whole-body inhalation chambers. Rats were held in nose-only exposure tubes for the 6-h exposures to minimize pelt deposition and subsequent ingestion of ADSS. Groups of 10 rats from each exposure group were killed after 5, 28, and 90 d of exposure to examine the rates of replicative DNA synthesis; 6 rats from each exposure group were kept for a 90-day recovery period after termination of exposures to examine replicative DNA synthesis rates. Three days prior to each scheduled necropsy, an osmotic pump containing 5-bromo-2′-deoxyuridine (BrdU) was implanted subcutaneously. After necropsy, tissues were processed for examination of BrdU-containing cells at several sites. Incorporation of BrdU was assessed either by counting the number of labeled cells along a length of an epithelial surface or by counting the number of labeled cells in an area of tissue. Tissues examined were from the nasal cavity, ventral larynx, and trachea, in addition to bronchial, bronchiolar, and alveolar regions of the lung. Endocardium, myocardium, epicardium, and aortic smooth muscle sites were also examined. Increased replicative DNA synthesis occurred in some sites of the respiratory tract at the 5-day timepoint at the mid or high exposure concentrations, although at 28 and 90 days, these effects had diminished in intensity or were not present, indicating adaptation to the ADSS exposure. The only tissues with elevated rates of replicative DNA synthesis at 90 days were the cuboidal and respiratory epithelium at the most rostra! portion of the nasal cavity at the highest exposure concentration. Increased rates of replicative DNA synthesis were not noted in heart tissues or lung alveolar epithelium at any concentration at any time point. Examination of rats killed after the end of the 90-day recovery period indicated that the increase in replicative DNA synthesis was not sustained after termination of exposures. The no observed effect level (NOEL) for increased replicative DNA synthesis after subchronic exposure to ADSS in the rat is greater than 1 mg ADSS/m³.     

Effect of Chronic Cigarette Smoke Exposure on Lung Clearance of Tracer Particles Inhaled by Rats

Cigarette smoking can influence the pulmonary disposition ofother inhaled materials in humans and laboratory animals. Thisstudy was undertaken to investigate the influence of cigarettesmoke exposures of rats on the pulmonary clearance of inhaled,relatively insoluble radioactive tracer particles. Following13 weeks of whole-body exposure to air or mainstream cigarettesmoke for 6 hr/day, 5 days/week at concentrations of 0, 100,or 250 mg total particulate matter (TPM)/m³, rats were acutelyexposed pernasally to ⁸⁵Sr-labeled fused aluminosilicate (⁸⁵Sr-FAP)tracer particles, then air or smoke exposures were resumed.A separate group of rats was exposed to the ⁸⁵Sr-FAP then seriallyeuthanized through 6 months after exposure to confirm the relativeinsolubility of the tracer particles. We observed decreasedtracer particle clearance from the lungs that was smoke concentration-dependent.By 180 days after exposure to the tracer aerosol, about 14,20, and 40% of the initial activity of tracer was present incontrol, 100 mg TPM/m³, and 250 mg TPM/m³ groups, respectively.Body weight gains were less in smoke-exposed rats than in controls.Smoke exposure produced lung lesions which included increasednumbers of pigmented alveolar macrophages distributed throughoutthe parenchyma and focal collections of enlarged alveolar macrophageswith concomitant alveolar epithelial hyperplasia and neutro-philicalveolitis. The severity of the lesions increased with smokeexposure duration and concentration to include interstitialaggregates of pigmented macrophages and interstitial fibrosis.Our data confirm previous findings that exposure to cigarettesmoke decreases the ability of the lungs to clear inhaled materials.We further demonstrate an exposure-concentration related magnitudeof effect, suggesting that the cigarette smoke-exposed rat constitutesa useful model for studies of the effects of cigarette smokeon the disposition of inhaled particles.     

Quantitative Histology and Cytochrome P-450 Immunocytochemistry of the Lung Parenchyma Following 6 Months of Exposure of Strain A/J Mice to Cigarette Sidestream Smoke

Abstract

Male strain A/J mice were exposed for 6 h/day, 5 days/wk to aged and diluted cigarette sidestream smoke (ADSS) at a chamber concentration of 4 mg/m³ of total suspended particulate matter (TSP). After 6 mo, the lungs were examined for altered expression of cytochrome P-450 isozymes and for differences in total alveolar tissue volume or surface area, as well as changes in the numbers of epithelial type II cells and alveolar macrophages. Morphologic measurements showed no statistically significant differences for the air, alveolar tissue, or capillary volumes of the lungs or changes in the total number of epithelial type II cells or alveolar macrophages. In contrast, cytochrome P-4501A1 was elevated in the lungs of ADSS-exposed animals and localized in capillary endothelial cells. CYP2B1 was present in airway epithelial cells as well as in epithelial cells throughout the lung parenchyma, but its distribution was not changed by ADSS exposure. Isozyme CYP2E1 was also found in airway epithelial cells, but not in the lung parenchyma, with no differences noted between ADSS exposed animals and controls. CYP2F2 was found in the bronchiolar Clara cells and in type II cells located within the alveolar parenchyma, but was also unchanged. It is concluded that chronic exposure to cigarette ADSS at 4 mg/m³ of TSP produces no changes in alveolar macrophages or epithelial type II cells in mouse lung but increases the expression of cytochrome P4501A1.     

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 concentrationsof 0, 10, 50, and 150 mg/m³ for 6 hr/day, 5 days/week for 4weeks. Rats were killed after 4 weeks of exposure and 10 daysor 3 months post exposure (PE). The exposure concentration of10 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 significantlyin rats exposed to 50 and 150 mg/m³ Ludox CS, but lung weightswere similar to those of controls at 3 months PE. After 4 weeksexposure to 50 mg/m³ Ludox CS, a slight alveolar macrophageresponse, polymorphonuclear leukocytic infiltration, and TypeII pneumocyte hyperplasia in alveolar duct regions were present.After 3 months PE, these pulmonary lesions had almost disappearedwith removal of most dust-laden alveolar macrophages (AMs).The pulmonary response to 150 mg/m³ Ludox CS was similar incharacter but increased in magnitude from that seen at 50 mg/m³At 3 months PE, most particleladen AMs had disappeared and theremaining AMs were aggregated and sharply demarcated. A fewaggregates of particle-laden AMs appeared to transform intosilicotic nodules comprising macrophages, epithelioid cells,and lymphocytic infiltration in some animals. Some silicoticnodules showed reticular fiber networks with minute collagenfiber deposition. Tracheobronchial lymph nodes were enlargedwith aggregates of particle-laden AMs and hyperplastic histiocyticcells. Lung-deposited Ludox cleared rapidly from the lungs withhalf-times of approximately 40 and 50 days for the 50 and 150mg/m³ groups, respectively.     

A 90-Day Inhalation Toxicity Study with Benomyl in Rats

A 90-Day Inhalation Toxiaty Study with Benomyl in Rats. WARHEIT,D. B., KELLY, D. P., CARAKOSTAS, M. C., AND SINGER, A. W. (1989).Fundam Appl Toxicol./ 12, 333-345. Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate,CAS Registry No. 17804-35-2] is a fungicide and the possibilityfor inhalation exposure exists for field workers. To assessthe toxicity of benomyl, groups of 20 male and 20 female CDrats were exposed nose-only 6 hr a day, 5 days a week, to concentrationsof 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 exposureperiod, blood and urine samples for clinical evaluation werecollected from 10 rats/group/sex, and these animals were sacrificedfor pathological examination. Similar evaluations were performadon all remaining rats at the end of the 90-day test period.After approximately 45 days on test, compoundrelated degenerationof the olfactory epithelium was observed in all males and in8 of 10 female rats exposed to 200 mg/m³ benomyl. Two male ratsexposed to 50 mg/m³ had similar, although less severe, areasof olfactory epithelial degeneration. After approximately 90days of exposure, the remaining 10 rats/group/sex were sacrificedand examined. Of these rats, all of the males and females exposedto 200 mg/m³ had olfactory degeneration, along with 3 malesexposed to 50 mg/m³ of benomyl. No other observed lesions wereinterpreted to have been caused by the benomyl exposure. Inaddition, male rats exposed to 200 mg/m³ benomyl had depressedmean body weights compared to controls and this finding correlatedwith a reduction in food consumption. Based on pathologicalobservations, 10 mg/m³ represents the no-observable-effect level(NOEL) for the male rats, and 50 mg/m³ is the NOEL for the femalerats.     

Chronic Inhalation Toxicity and Carcinogenicity Study of Respirable Polymeric Methylene Diphenyl Diisocyanate (Polymeric MDI) Aerosol in Rats

Four groups of 60 Wistar rats of each sex were exposed by inhalationto 0, 0.2, 1.0, or 6.0 mg/m³ respirable polymeric methylenediphenyl diisocyanate (polymeric MDI) aerosol (93.5% < 4.2µm)for 6 hr a day, 5 days a week for up to 24 months. In addition,satellite groups of 10 rats/sex/group received the same treatmentfor 12 months. There was no adverse effect on general health,survival, body weight, or hematological or clinical chemistryparameters. Lung weights were increased in both males and femalesexposed to 6.0 mg polymeric MDI/m³ for 12 or 24 months. Grossexamination at autopsy of males exposed to 6.0 mg polymericMDI/m³ for 24 months revealed an increased incidence of spottedand discolored lungs. Increased incidences of degeneration andbasal cell hyperplasia of the nasal olfactory epithelium, oftenaccompanied by hyperplasia of Bowman's glands, were found inthe 1.0 and 6.0 mg/m³ groups. Light and electron microscopicstudies of the lungs revealed accumulations of alveolar macrophagescontaining polymeric MDI-associated refractile yellowish materialat the level of the alveolar duct in all exposed groups. Alveolarduct epithelialization as well as fibrosis of tissues surroundingthe macro phage accumulations occurred at the 1.0 and 6.0 mg/m³exposure levels. In addition, increased incidences of calcareousdeposits and localized alveolar bronchiolization were seen inthe 6.0 mg/m³ group. Moreover, eight pulmonary adenomas (sixin males and two in females) and one pulmonary adenocarcinoma(in a male) were observed in the 6.0 mg/m³ exposure group. Thetime sequence of the spectrum of pulmonary changes indicatesthat recurrent alveolar wall damage by polymeric MDI and/orpolymeric MDI-containing alveolar macrophages leads to alveolarbronchiolization and ultimately to bronchioloalveolar tumors.No lung tumors were found in the lower concentration groupsand in the control group. The incidence and distribution ofother types of tumors were not influenced by polymeric MDI.It was concluded that in the present study, the "no-observed-adverse-effectlevel" of polymeric MDI was 0.2 mg/m³ and that chronic exposureto polymeric MDI at a level of 6.0 mg/m³ was related to theoccurrence of pulmonary tumors. It was also concluded that exposureto polymeric MDI at concentrations not leading to recurrentlung tissue damage will not produce pulmonary tumors.     

Four-Week Inhalation Toxicity Study with Ludox Colloidal Silica in Rats: Pulmonary Cellular Responses

Four-Week Inhalation Toxicity Study with Ludox Colloidal Silicain Rats: Pulmonary Cellular Responses. WARHEIT, D. B., CARAKOSTAS,M. C, KELLY, D. P., AND HARTSKY, M. A. (1991). Fundam. Appl.Toxicol. 16, 590–601. This study was designed to complementa traditional subchronic inhalation toxicity study with Ludoxcolloidal silica. CD rats were exposed nose-only for 2 or 4weeks at concentrations of 0, 10, 50, and 150 mg/m³ Ludox (driedSiO₂). Additional groups of rats exposed for 4 weeks were givena 3-month recovery period. Following exposure and/or recovery,fluids and cells were recovered from the lungs by bronchoalveolarlavage (BAL) and measured for cellular and biochemical parameters.Additional groups of animals were processed for cell labelingstudies or lung deposition studies. Inhaled doses of Ludox colloidalsilica were measured after 4-week exposures and were found tobe 489 µg/lung (10 mg/m³ group), 2418 µg/lung (50mg/m³), and 7378 µg/lung (150 mg/m³), respectively. Resultsshowed that exposures to 150 mg/m³ Ludox for 2 or 4 weeks producedpulmonary inflammation along with increases in BAL protein,LDH, and alkaline phosphatase values (p<0.05) and reducedmacrophage phagocytosis. Inflammatory responses, evidenced byincreased numbers of neutrophils, were also measured in thelungs of the 50 mg/m³ group following 2 and/or 4 weeks of exposure.Most biochemical parameters for all groups returned to controlvalues following a 3-month recovery period. Autoradiographicstudies demonstrated that the labeling indices of terminal bronchiolarand lung parenchymal cells were generally increased in the 50and 150 mg/m³ groups after 2 and 4 weeks of exposure but, withone exception, returned to normal levels following a 3-monthpostexposure period. No significant alterations in any measuredparameters were detected in rats exposed to 10 mg/m³ Ludox atany time postexposure. The determination of a no-observable-effectlevel (NOEL) of 10 mg/m³ was consistent with results obtainedby conventional toxicology methods and affirms the utility ofthese biochemical, cellular, and autoradiographic techniquesfor providing a predictive screen to assess the toxicity ofinhaled particles.     

Effects of Inhaled Chromium Dioxide Dust on Rats Exposed for Two Years

Effects of Inhaled Chromium Dioxide Dust on Rats Exposed forTwo Years. LEE, K. P., ULRICH, C. E., GEIL, R. G., AND TROCHIMOWICZ,H. J. (1988). Fundam. Appl. Toxicol 10, 125–145. Ratswere exposed by inhalation to chromium dioxide (C1O2) dust atdesign concentrations of 0, 0.5 mg/m³(stabilized and unstabilized,respectively), or 25 mg/m³ (stabilized) for 6 hr/day, 5 days/weekfor 2 years. No dust-exposure-related pathological changes otherthan lung lesions were observed in all exposed rats. There wereno significant differences in pulmonary responses between unstabilizedand stabilized CrC>2 at the 0.5 mg/m³ exposure level. Thelungs showed minute dust deposition in the alveoli adjacentto the alveolar ducts, but maintained an intact general architecture.The pulmonary responses satisfied the biological criteria fora nuisance dust. At 25 mg/m³, dust deposition was sharply confinedto the alveoli in the alveolar duct region. Alveolar walls enclosingdust-laden macrophage (dust cell) aggregates were thickenedwith hyperplastic Type II pneumocytes and slightly collagenizedfibrosis. Alveoli adjacent to the terminal bronchioles werelined with bronchiolar epithelium (alveolar bronchiolar-ization).In addition, lungs showed foamy macrophage response, cholesterolgranulomas, alveolar protoeinosis, and minute fibrotic pleurisy.These pulmonary lesions occurred predominantly in female rats.Of 108 female rats, 6 developed keratin cysts and 2 had cystickeratinizing squa-mous cell carcinoma (CKSCC). None of 106 malerats had either a keratin cyst or a CKSCC. The lung tumors developedfrom metaplastic squamous cells in the areas of alveolar bronchio-larizationat the alveolar duct region. The lung tumors were well differentiatedand devoid of characteristics of true malignancy. The CKSCCis an experimentally induced, unique tumor type and is differentfrom the type of spontaneous lung tumor seen in man or animals.The relevance to man of this type of lung tumor appears to benegligible.     

INHALATION TOXICITY STUDIES OF AQUEOUS DISPERSION RESIN

Aqueous dispersion resin (ADR) is a water-based acrylate copolymer designed to allow a range of ethanol concentrations for hair-spray formulations. A series of inhalation (whole-body) aerosol toxicity studies, including acute, 9-day, and 90-day exposures, was conducted to determine any toxicological effects for ADR. An acute study of ADR was conducted for 4 h with a 14-day observation period. The maximum ADR aerosol concentration was 1.07 (+/- 0.04) mg/L with a mass medium aerodynamic diameter (MMAD) value of 1.46 mu m and a geometric mean (sigma_g) of 1.42. No deaths or exposure-related lesions were observed. Thus, the LC50 value was greater than 1.07 mg/L. For the 9-day study, each group contained 10 animals/sex and was exposed for 2 h/day. The mean exposure concentrations (+/- standard deviation) were 244 (+/- 39.6), 543 (+/- 71.9), and 843 (+/- 186.2) mg/m³ for the 250-, 500-, and 1000-mg/m³ groups, respectively. The mean MMADs were 2.83, 2.90, and 4.09 mu m for the 250-, 500-, and 1000-mg/m³ groups, respectively, with sigma g values ranging from 3.15 to 6.22. Unkempt fur and alopecia in the males and females from the 1000-mg/m³ group at sacrifice were the only exposure-related clinical signs observed. Increased lung weights in the 500- and 1000-mg/m³ exposure groups were found. Histopathological effects, such as alveolar histiocytosis and interstitial pneumonitis, were also noted in these two exposure groups. For the 90-day study, 10 animals per sex were included in each group with a 6-wk recovery group of 5 female rats/group. Exposures were conducted for 2 h/day. The mean exposure concentrations were 30.4 (+/- 2.3), 102 (+/- 11.6), and 308 (+/- 19.3) mg/m³ with mean MMADs of 3.09, 2.64, and 2.67 mu m and sigma_g values ranging from 3.54 to 3.90 for exposure concentrations of 30, 100, and 300 mg/m³, respectively. The only findings at the 90-day sacrifice were increased lung weights for the males and females in the 300-mg/m³ group and males in the 100-mg/m³ group. For the 6- wk recovery sacrifice of the females, the lung weights for the 300- and 100-mg/m³ groups were increased. Histopathological effects at the 90-day sacrifice included alveolar histiocytosis and lymphadenitis in the mediastinal lymph nodes in the 100- and 300-mg/m³ exposure groups for males and females, while alveolar histiocytosis, intraalveolar cellular debris, lymphadenitis in the mediastinal lymph nodes, and/or interstitial pneumonitis were noted in these 2 exposure groups of the females after the 6-wk recovery period. Animals exposed for 90 days to 100 and 300 mg/m³ for 2 h had increased lung weights. However, no effects were observed in the 30-mg/m³ exposure group. Thus, under the conditions of the present 90-day study, a no-observable-effect level (NOEL) was found to be 30 mg/m³.     

Thirteen-Week, Repeated Inhalation Exposure of F344/N Rats and B6C3F1 Mice to Ferrocene

Ferrocene (dicyclopentadienyl iron; CAS No. 102-54-5) is a relativelyvolatile compound used as a chemical intermediate, a catalyst,and an antiknock additive in gasoline. This organometallic chemicalis of particular interest because of its structural similaritiesto other metallocenes, some of which are carcinogenic. F344/Nrats and B6C3F₁ mice were exposed to 0, 3.0, 10, and 30 mg ferrocenevapor/m³, 6 hr/day, 5 days/week, for 13 weeks. During theseexposures, no rats or mice died, nor were any clinical signsof ferrocene-related toxicity observed. At the end of the exposures,male rats exposed to the lowest and highest level of ferrocenehad decreased body weight gains compared to filtered-air-exposedcontrol male rats, while body weight gains for all groups ofboth ferrocene- and filtered-air-exposed female rats were similar.Male mice exposed to ferrocene had no differences in body weightgains, compared to controls, but female mice had decreases inbody weight gains at the 10 and 30 mg/m³ exposure levels. Therewere exposure concentration- and exposure-time-related increasesin lung burdens of iron. The mean iron lung burden in rats exposedto 30 mg ferrocene vapor/m³ for 90 days was four times greaterthan the burden in control rats. No exposure-related changesin respiratory function, lung biochemistry, bronchoalveolarlavage cytology, total lung collagen, clinical chemistry, andhematology parameters were observed. This suggests that theaccumulations of iron in lung did not cause an inflammatoryresponse nor any functional impairment of the lung. There wereno indications of developing pulmonary fibrosis nor of any hematologictoxicity. No exposure-related gross lesions were seen in anyof the rats or mice at necropsy. Exposure-related histopathologicalterations, primarily pigment accumulations, were observedin the nose, larynx, trachea, lung, and liver of both species,and in the kidneys of mice. Lesions were most severe in thenasal olfactory epithelium where pigment accumulation, necrotizinginflammation, metaplasia, and epithelial regeneration occurred.Nasal lesions were observed in all ferrocene-exposed animalsand differed only in severity, which was dependent on the exposureconcentration. Histochernical stains of these target tissuesshowed the presence of iron ions. The results suggest that themechanism of ferrocene toxicity may be the intracellular releaseof ferrous ion through ferrocene metabolism, followed by eitheriron-catalyzed lipid peroxidation of cellular membranes or theiron-catalyzed Fenton reaction to form hydroxyl radicals thatdirectly react with other key cellular components, such as proteinor DNA.     

Pulmonary Retention of Inhaled Diesel Particles after Prolonged Exposures to Diesel Exhaust

Pulmonary Retention of Inhaled Diesel Particles after ProlongedExposures to Diesel Exhaust CHAN, T. L., LEE, P. S., AND HERING,W. E. (1984). Fundam. Appl. Toxicol. 4, 624–631. The effectof continuous exposure to diluted diesel exhaust on the pulmonaryretention of inhaled diesel particles was studied in male Fischer344 rats. Test animals were first exposed to clean air or diluteddiesel exhaust in exposure chambers at nominal particulate concentrationsof 250 µg/m³ or 6 mg/m³ for 20 hr/day, 7 days/week, forperiods lasting from 7 to 112 days, followed by a nose-onlyexposure to ^l4C-tagged diesel particles for 45 min. At preselectedtime intervals after the radioactive exposure, the ¹⁴C-activitiesin the lungs of groups of four animals were measured to determinethe clearance of the ^l4C-diesel particles up to 1 year. Thepulmonary retention of the radioactive diesel particles wasgreater in animals which had been preexposed to diesel exhaustThe slower alveolar clearance of particle-laden macrophagesand leukocytes can be described by a normal Diphasic clearancemodel. Since some of the macrophages were found sequesteredas aggregates in the pulmonary region, a slow-clearing residualcomponent was included in a modified lung retention model. Whenthese residual fractions were determined and excluded from theactive particulate transport within the lungs, normal alveolarclearance rates were calculated for the animals with a preexposurediesel particulate dose less than 0.8 mg. Slower clearance wasobserved at a dose of 6.5 mg and no clearance was evident ata dose of 11.8 mg in their lungs. In effect, the greater retentionof inhaled particles can be interpreted as-a sign of impairedlung clearance attributable to the prolonged exposures to highconcentrations of diesel exhaust gases and/or the presence ofaccumulated carbonaceous particles residing in sequestered macrophageaggregates in the lungs.     

Deposition of Cigarette Smoke Particles in the Rat

The fractional deposition of cigarette smoke particles in therespiratory tracts of rats was studied. Male and female ratswere conditioned in nose-only exposure tubes 25 min/day for2 days, exposed to cigarette smoke at mass concentrations of95 or 341 mg/m³ 25 min/day for 3 days, and then exposed to smokeat mass concentrations of 212 and 657 mg/m³, 25 min/day for5 days. Mainstream cigarette smoke was generated by a modifiedWalton smoking machine from two 1R3 research cigarettes burnedsequentially for each exposure. Deposition studies were conductedby placing the rats in plethysmograph tubes to allow respiratoryminute volume measurements during exposure, then exposing themto [¹⁴C] cigarette smoke at mass concentrations of 202 or 624mg/m³ for 25 min, using the same smoking machine. Size distribution,real-time concentration, and ¹⁴C activity of the smoke particleswere determined using a multijet Mercer impactor, a real-timeaerosol monitor, and filter samples, respectively. Immediatelyafter the exposure, the rats were terminated to determine thedistribution of the ¹⁴C. Individual lung lobes, trachea andlobar bronchi, head, larynx, kidneys, liver, gastrointestinal(GI) tract, blood, and depelted carcass of each rat were analyzedfor ¹⁴C content. Results showed that the GI tract contained16–31% of the total activity, indicating significant clearancefrom the large airways and nose to the GI tract during the exposureand during the 10–15 min between the cessation of theexposure and the removal ofthe organs. Total deposition of theinhaled ¹⁴C activity was 20.1 ? 1.6% for both exposure concentrations.The intrapulmonary deposition fractions (lung lobes plus airwaysbelow the lobar bronchi) were 12.4 ? 0.9 and 15.9 ? 1.4% forconcentrations of 202 and 624 mg/m³ respectively, suggestinga slight enhancement in upper airway deposition for animalsexposed to the higher smoke concentration.     

Mucous Cell Metaplasia in the Airways of Rats Exposed to Machining Fluids

Occupational exposure to microbial-contaminated machining fluidsis associated with a variety of adverse pulmonary effects includingchronic bronchitis and increased sputum production. We havepreviously demonstrated in F344 rats that inhaled endotoxincan increase the amount of stored intraepithelial mucosubstances(Vs) in the respiratory tract. The purpose of the present studywas to examine the effect of endotoxin-contaminated machiningfluid aerosols on mucous production. Rats were exposed to aerosolsof pyrogen-free water, 1 or 10 mg/m³ used machining fluid, or10 mg/m³ unused machining fluid for 3 hr/day for 3 days. Twenty-fourhours after the final exposure, right lung lobes were lavagedand the nasal cavity and left lung were fixed in formalin. Theamount of Alcian blue/periodic acid-Schiff-stained mucosubstanceswas determined by morphometry. Exposure to 10 mg/m³ used machiningfluid (equivalent to 0.8 µg/m³ endotoxin) produced a significantincrease in Vs in the epithelial lining of both the nasal septumand intrapulmonary airways. These changes in Vs were accompaniedby a significant increase in total cells and neutrophils inthe lavage fluid. No changes in stored mucosubstances or lavageparameters were found in animals exposed to 1 mg/m³ used machiningfluid aerosols. A significant increase in Vs was observed inthe nasal septum but not in the intrapulmonary airways of animalsexposed to 10 mg/m³ unused machining fluids (no measurable endotoxin).These results suggest that in addition to endotoxin, nonendotoxincomponents of machining fluids may contribute to the increasein sputum and chronic bronchitis reported for workers exposedto machining fluid aerosols.     

Chronic Inhalation Toxicity of Size-Separated Glass Fibers in Fischer 344 Rats

This study was initiated to determine the chronic biologicaleffects in Fisher 344 rats of inhaled size-separated respirablefractions of fibrous glass (FG) having compositions representativeof common building insulation wools. Rats were exposed usingnose-only inhalation chambers, 6 hr/day, 5 days/week, for 24months to three concentrations (3, 16, and 30 mg/m³) of twodifferent compositions of FG (designated MMVF 10 and MMVF 11),or to filtered air (negative control). Fibrous glass findingswere compared to those from a concurrent inhalation study ofchrysotile asbestos and refractory ceramic fiber (RCF). TheFGs used in this study were size selected to be largely respirablein the rat and the aerosol generation technique did not alterthe dimensions of the fibers. Interim euthanizations took placeat 3- to 6-month intervals to monitor progression of pulmonarychanges. Fibers were recovered from digested lung tissue fordetermination of changes in fiber number and morphology. Inanimals exposed to 30 mg/m³ of MMVF 10 or MMVF 11, 4.2±0.9x10⁵and 6.4±3.1x10⁵ fibers/mg dry lung tissue, respectively,were recovered after 24 months of exposure. Exposure to chrysotileasbestos (10 mg/m³) and to a lesser extent RCF (30 mg/m³) resultedin pulmonary fibrosis as well as mesothelioma and significantincreases in lung tumors. FG exposure was associated with anonspecific inflammatory response (macrophage response) in thelungs that did not appear to progress after 6–12 monthsof exposure. These cellular changes are reversible and are similarto the effects observed after inhalation of an inert dust. Nolung fibrosis was observed in the FG-exposed animals. Further,FG exposure resulted in no mesotheliomas and no statisticallysignificant increase in lung tumor incidence when compared tothat of the negative control group. These findings, along withprevious inhalation studies, suggest that respirable fibrousglass does not represent a significant hazard for fibrotic orneoplastic lung disease in humans.     

Pulmonary Toxicity of Inhaled Polypropylene Fibers in Rats

This study was initiated to assess the pulmonary toxicity ofpolyolefin fiber composed of polypropylene in male Fischer 344rats after 90 days of inhalation exposure. To increase fiberrespirability in the rodent, polypropylene fibers were size-selectedbefore aerosolization to have a geometric mean diameter of 1.6m (46% <1 µm) and a geometric mean length of 30.3 µm.Three groups of animals were exposed in nose-only inhalationchambers, 6 hr/day, 5 days/week, for 90 days to 15, 30, or 60mg/m³ of polypropylene, or filtered air (negative control).Microscopic examination of the polypropylene fiber-exposed lungsrevealed that, at all time points examined in the study, therewas a dose-dependent increase in pulmonary macrophages. Theseminimal or mild increases in cellularity appeared to be reversible,especially at the lower doses 30 days post exposure. No fibrosiswas observed in any of the groups. A strong correlation wasfound between the external exposure concentration, the timeexposure, and the lung fiber burden. The number of partiallydegraded (segmented) fibers within the lung increased with theexposure concentration and period of exposure, as well as withthe period of recovery after termination of exposure at 90 days.Fibers were recovered from exposed lungs using a hypochloritedigestion technique.     

The Effect of Exposure Pattern on the Accumulation of Particles and the Response of the Lung to Inhaled Particles

We hypothesized that a rapid rate of delivery of particles tothe lung would overwhelm the normal clearance mechanisms ofthe lung and result in a higher lung burden of particles anda greater inflammatory response than a slower rate of particlede livery. F344/N rats were exposed over a 12-week period tothe same weekly concentration times time product of carbon black(CB) particles, but at three different exposure rates: 3.5 mg/m³16 hr/day, 7 days/week; 13 mg/m³ 6 hr/day, 5 days/week or 98mg/rn 4 hr/day, 1 day/week. The intermediate exposure rate waschosen to mimic an occupational work week and to give an 8-br,time-weighted average exposure equal to the threshold limitvalue (TLV) for nuisance dusts of the American College of GovernmentalIndustrial Hygienists (10 mg/rn Pure CB has a lower TLV, 3.5mg/m³ than nuisance dusts, but this is based on avoidance ofexcessive dirtiness in the workplace, not on the toxicity ofCS. Lung burdens of CB were measured after 3, 6, 9, and 12 weeksof exposure and at 4,8, 12, 16, and 24 weeks after the exposureended. The inflammatory response was quantified by analysisof bronchoalveolar lavage fluid (BALF) after 6 and 12 weeksof exposure and at 1, 12, and 24 weeks after exposure. The histopathologyof the lung was evaluated at the end of the exposure and at24 weeks after the exposure. Acquired lung burdens were between3 and 4 mg/lung at the end of the exposure. There was an approximately20% higher lung burden in the rats exposed at the lowest rateof delivery, but this did not result in any differences amongthe three groups in the inflammatory response or the lesionsin the lung. By 6 weeks, there was a mild inflammatory responseto the particles and this response was increased by 12 weeksof exposure as indicated by an influx of neutrophils, two- tothreefold increases in protein and in lactate dehydrogenaseactivity, and up to sixfold increases in ß-glueuronidaseactivity in the BALF. Morphologic changes were minimal, consistingof slight thickening of the alveolar septa with hypertrophiedepithelial cells, scant collagen fibers, and occasional inflammatorycells adjacent to accumulations of particles. The data supportthe use of time-weighted averages in regulating exposures tonuisance dusts. The results of the study indicate that exposuresto a particle of relatively low toxicity at the current TLVfor nuisance dusts led to pulmonary inflammation and sufficientaccumulation of particles to slow lung clearance. The resultssuggest that the TLV for nuisance dusts may be too high.     

Comparative Inhalation Toxicity of Nickel Subsulfide to F344/N Rats and B6C3F1 Mice Exposed for 12 Days

Comparative Inhalation Toxicity of Nickel Subsulfide to F344/NRats and B6C3F₁ Mice Exposed for 12 Days. BENSON, J. M., CARPENTER,R. L., HAHN, F. F., HALEY, P. J. HANSON, R. L., HOBBS, C. H.,PICKRELL, J. A., AND DUNNICK, J. K. (1987). Fundam Appl. Toxicol.9, 251–265. Groups of F344/N rats and B6C3F₁ mice wereexposed to aerosols of nickel subsulfide (Ni₃S₂) 6 hr/day for12 days not including weekends. Actual exposure concentrationswere within 3% of target (target 10.0, 5.0, 2.5, 1.2, 0.6,and 0.0 mg Ni₃S₂/m³). Nickel lung burdens of exposed rats andmice increased linearly with exposure concentration. Two malerats and all mice exposed to 10.0 mg Ni₃S₂/m³ died before theend of the exposures. Exposure to Ni₃S₂ had no elfect on thenatural killer cell activity of mouse spleen cells. Lesionsin rats and mice related to inhalation of Ni₃S₂ were found inthe nasal epithelium, lung, and bronchial lymph nodes. The mostextensive lesions were found in the lung and included necrotizingpneumonia. Emphysema developed in rats exposed to 5.0 or 10.0mg Ni₃S₂/m³ while fibrosis developed in mice exposed to 5.0mg Ni₃S₂/m³ Degeneration of the respiratory epithelium and atrophyof the olfactory epithelium of the nose occurred in rats exposedto as low as 0.6 mg Ni₃S₂/m³ and mice exposed to 1.2 mg/m³ Resultsindicate that inhalation exposure of rats and mice to Ni₃S₂/aerosol concentrations near the current threshold limit value(TLV) for nickel compounds (1 mg/m³ for Ni metal and roastingfume and dust and 0.1 mg/m³ as Ni for soluble compounds) canproduce lesions in the respiratory tract. Atrophy of lymphoidtissues (spleen, thymus, and bronchial lymph nodes) was foundin animals of the highest exposure concentration. Degenerationof the testicular germinal epithelium was also observed in miceand rats that survived 5.0 or 10.0 mg/m³ exposure concentrations.     

Inhalation toxicity study of disk-shaped potassium octatitanate particles (Terracess TF) in rats following 90 days of aerosol exposure

Since fibrous particles such as asbestos and some man-made fibers (MMF) have been known to produce carcinogenic or fibrogenic effects, disk-shaped potassium octatitanate (POT) particles (trade name: Terracess TF) were manufactured as nonfibrous particles. A 90-day inhalation toxicity study of Terracess TF was performed to evaluate comparative inhalation toxicity of the disk shape with a fibrous shape that was previously evaluated. Four groups of 20 male and 15 female rats each were exposed to Terracess TF aerosols at concentrations of 0, 2, 10, or 50?mg/m³ for 90 days. Ten male and 10 female rats per group were sacrificed at 90 days of exposure. After 90 days of exposure, 5 male rats per group were sacrificed at 3 wk of recovery period and 4–5 male rats per group or 5 female rats per group were sacrificed at 15?wk of recovery for lung clearance and histopathology. The mass median aerodynamic equivalent diameter (MMAED) of the aerosols of test materials ranged from 2.5 to 2.9?μm. There were no test-substance-related adverse effects on clinical observations. At the end of the 90-day exposure, a slight increase in lung-to-body weight ratios was observed at 50?mg/m³ in male but not in female rats. However, lung weights were within normal limits after 3- or 15-wk recovery periods. Microscopically, inhaled Terracess TF particles were mostly phagocytized by free alveolar macrophages (AMs) in the alveolar airspaces and alveolar walls maintained normal structure at 2 and 10?mg/m³. At 50?mg/m³, some alveoli were distended and filled with aggregates of particle-laden AMs. The alveolar walls showed slight type II pneumocyte hyperplasia, but neither proliferative inflammation nor alveolar fibrosis was present at 50?mg/m³. The clearance half-times for Terracess TF were estimated to be in the order of 6 to 9?mo for the 50-mg/m³ group and 2 to 3?mo for the 10- and 2-mg/m³ groups. The lung responses and lung clearance rate were comparable to those of “nuisance” type dusts at these concentrations. Based on interpretation that aggregated particle-laden AMs in alveoli was considered to be an early histopathological sign of lung overloading, an effect level was considered to be 50?mg/m³ and no-observed-adverse-effect level (NOAEL) was 10?mg/m³. This experiment clearly demonstrated that particle morphology was considered to be an important factor to determine inhaled particle toxicity.     

Toxicity of Calcium Sodium Metaphosphate Fiber: II. Chronic Inhalation and Oncogenicity Study

Male and female Fischer 344 rats (80/sex/group) were exposedto CSM fiber 6 hr/day, 5 days/week at target-exposure levelsof 0, 1, 5, or 25 mg/m³ for 24 months, corresponding to 0, 27,80, and 513 fibers/cc, respectively. Number and size of theairborne fibers were determined during the course of the study.At 3 and 12 months, 10 rats/sex/group were euthanized and at18 and 24 months 5 rats/sex/group were euthanized. In addition,5 rats/ sex/group were removed from exposure at 18 months andmaintained for a 6-month recovery period. All animals survivingat the completion of the exposure period were maintained ina clean environment for up to 5 additional months. Clinicallaboratory examinations were performed on 10 animals/sex/groupat 3, 12, and 24 months. The number of fibers in the lung werealso determined at 3, 12, 18, and 24 months. Body weight andsurvival did not appear to be affected by treatment. There wereno biologically significant effects on clinical parameters.There was a dose-related increase in lung weight during theexposure period which was generally reversible during the recoveryperiods. There also was a dose-related increase in the numberof fibers/ milligram of lung, but no increase in lung fiberburden after the first 3 months. The number of fibers in thelungs of animals exposed to CSM fiber for 18 months and allowed6-month recovery period showed a decrease especially at thehigh dose. No increase in tumors (benign or malignant) was observedin this study. Microscopic changes considered reflective ofan irritant response were observed in the nasal turbinates notablyat the 5 and 25 mg/m³ levels. Histological changes were alsoobserved in the lungs at the 5 and 25 mg/m³ levels. The incidenceand/ or severity of histopathological changes in the 1 mg/m³group was considered to be essentially comparable to controls.