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.     

Subchronic and Chronic Inhalation Toxicity of Antimony Trioxide in the Rat

Fischer 344 rats were exposed by inhalation to Sb₂O₃ (antimonytrioxide) dust at exposure levels of 0, 0.25, 1.08, 4.92, and23.46 mg/m³ for 6 hr/day, 5 days/week for 13 weeks followedby a 27-week observation period. Subsequently, an inhalationon-cogenicity study was conducted at exposure levels of 0, 0.06,0.51, and 4.50 mg/m³ for 12 months followed by a 12-month observationperiod. The Sb₂O₃ in the subchronic study had a mass medianaerodynamic diameter (MMAD) of 3.05 ± 0.21 microns (mean± SD) with a geometric standard deviation (GSD) of 1.57± 0.06. In the chronic study, the MMAD was 3.76 ±0.84 and the GSD was 1.79 ± 0.32. Except for the eyes,no adverse clinical observations were attributed to Sb₂O₃ ineither study. In the subchronic study, corneal irregularitieswere seen after about 2 weeks of exposure and did not abateduring the observation period. In the chronic study, ophthalmoscopicevaluation at 24 months revealed a dose-related increase incataracts of 11, 24, 28, and 32% (both sexes combined) for eachgroup, respectively. Body weights were significantly lower (6%)than the control group's weights in the 23.46 mg/m³ males inthe subchronic study. These rats did not recover this weightduring the 27-week observation period. Body weights of the femalesin both studies and males in the chronic study were unaffected.There were no Sb₂O₃ effects on clinical chemistry or he-matologyin either study. Mean absolute and relative lung weights weresignificantly increased in the 4.92 and 23.46 mg/m³ groups inthe subchronic study. The 23.46 mg/m³ group's lung weights didnot recover to control levels during the 27-week observationperiod. Lung weights for rats in the chronic study were unaffected.Microscopic changes in the lungs in the subchronic and chronicstudy were limited to subacute-chronic interstitial inflammation,increased numbers of alveolar-in-traalveolar macrophages, foreignmaterial in the alveolar-in-traalveolar macrophages in the peribronchialand perivascular (chronic study only) lymphoid aggregates andin the peribronchial lymph nodes, granulomatous inflammation/granulomas,and fibrosis. In the chronic study, any observed neoplasms occurredwith comparable incidence among all groups and were within thehistorical range for controls. Clearance of Sb₂O₃ from the lungwas burden dependent and was reduced by 80/ in the 4.50 mg/m³group in the chronic study. The previously reported studies,which found Sb₂O₃ to be a carcinogen, were run at higher lungburdens. Under the exposure conditions of the current study,Sb₂O₃ was not a carcinogen.     

Pulmonary Fibrosis Produced in F-344 Rats by Subchronic Inhalation of Aerosols of a 4000 Molecular Weight Ethylene Oxide/Propylene Oxide Polymer

Pulmonary Fibrosis Produced in F-344 Rats by Subchronic Inhalationof Aerosols of a 4000 Molecular Weight Ethylene Oxide/PropyleneOxide Polymer. KLONNE, D. R., DODD, D. E., Losco, P. E., TROUP,C. M., AND TYLER, T. R. (1988), Fundam Appl. Toxicol 10, 682–690.Inhalation of aerosols of the ethylene oxide/propylene oxidepolymer (U-5100) evaluated in this study has previously beenshown in acute and 2-week studies to produce toxicologic effectson the lungs, with increased lung weights and microscopic findingsof congestion and hemorrhage of pulmonary alveolar capillariesand necrosis of alveolar epithelial cells (D. R. KLONNE, D.J. NACHREINER, D. E. DODD, P. E. Losco, AND T. R. TYLER, 1987,Fundam. Appl. Toxicol. 9, 7737–784). In the present studies,F-344 rats were exposed 6 hr/day, 5 day/week for 2 weeks toaerosols at mean concentrations of 0,0.9, or 5.0 mg/m³ or for13 weeks to mean concentrations of 0, 0.3, 1.1, or 5.2 mg/m³.Following the 2-week study, minimal multifocal hemorrhage andeosinophilic proteinaceous debris in alveoli were observed inthe 0.9 mg/m³ group; similar lesions plus alveolar cell necrosiswere found in the 5 mg/m³ group. In the 13-week study, the 5.2mg/m³ group had a slight decrease in body weight gain, whileincreases in absolute and/or relative lung weights occurredfor both the 1.1 and 5.2 mg/m³ groups at the end of the exposureregimen and at the end of a 5-week recovery period. Histologiclesions of the lungs occurred in all U-5100-exposed groups andconsisted of hemorrhage, alveolar histiocytosis, interstitialpneumonia, and multifocal fibrosis. The incidence and severityof the pulmonary lesions were concentration related. At theend of the 5-week recovery period, there was little change inthe severity or incidence of the pulmonary lesions in the 1and 5 mg/m³ groups when compared to rats killed the day afterthe termination of exposures. In conclusion, exposure to aerosolsof U-5100 for 13 weeks produced generally slight but biologicallysignificant pulmonary fibrosis in rats at all the exposure concentrationstested in this study.     

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.     

Embryotoxicity Study of Monomeric 4,4'-Methylenediphenyl Diisocyanate (MDI) Aerosol after Inhalation Exposure in Wistar Rats

One of the uses of MDI is as an alternative to formaldehydein the manufacture of furniture, its main route of exposureto humans being by inhalation. There have been no previous studieson the potential prenatal toxic effects of this compound. Toclose this gap in information, gravid Wistar rats, Crl:(WI)BR,were exposed by whole-body inhalation to clean air (control)and to 1, 3, and 9 mg/m3 MDI, respectively, for 6 hr per dayfrom Days 6 to 15 post conception (p.c). Rats were killed onDay 20 p.c. and the following results were obtained: Treatmentcaused a dose-dependent decrease in food consumption in allsubstance-treated groups during exposure, returning to normalvalues after cessation of treatment. The lung weights in thehigh-dose group were significantly increased compared to thesham-treated control animals. Treatment did not influence anyother maternal and/or fetal parameters investigated (maternalweight gain, number of corpora lutea, implantation sites, pre–and postimplantation loss, fetal and placental weights, grossand visceral anomalies, degree of ossification), although aslight but significant increase in litters with fetuses displayingasymmetric sternebra(e) was observed after treatment with thehighest dose of 9 mg/m³. Although the relevance of an increaseof this minor anomaly in doses which cause toxic effects indams (reduced food consumption, increased lung weights) is limitedand the number observed is within the limits of biological variability,a substance-induced effect in the high-dose group cannot beexcluded with certainty. Consequently, a no embryotoxic effectlevel of 3 mg/m³ was determined.     

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 paraaramidfibrils (subfibers) have been investigated as part of a subchronicand chronic inhalation toxicity testing program. Fibrils recoveredfrom lung tissue in para-aramid-exposed Sprague-Dawley ratswere microscopically counted and measured after exposures toairborne fibrils which were about 12 µm median length(ML) and <0.3 µm median diameter. In each of threestudies lung-recovered fibrils were progressively shorter withincreasing residence time in the lungs. Twenty-eight days aftera single 6-hr exposure at 400 respirable fibrils per cubic centimeter(f/cm³) the ML of recovered fibrils decreased to about 5 µm.Twenty-four months after a 3-week exposure to 25 or 400 (f/cm³)fibrils reached about 2 µm ML. After 2 years of continuousexposure at 2.5, 25, or 100 f/cm³ or 1 year exposure plus 1year recovery at 400 f/cm fibril ML approached 4 µm. Inthe 2-year study, the lung-fiber accumulation rate/exposureconcentration was similar for the three highest concentrationsand was about 3x greater than that seen at 2.5 f/cm³ indicatingthat concentrations of about 25 f/cm or more may overwhelm clearancemechanisms. Time required for fibrils to be reduced to <5µm in the lung was markedly less at lower exposure concentrationand shorter exposure time. The primary shortening mechanismis proposed to be long fibril cutting by enzymatic attack atfibril defects. However, length-selective fibril depositionand clearance may contribute to shortening in the first fewdays after exposure. The enzymatic cutting hypothesis is supportedby measured increases in numbers of short fibers following cessationof exposures, continued shortening of the fibril length distributionup to 2 years following exposure, and in vitro fibril shorteningafter 3 months in a proteolytic enzyme preparation. The conclusionis that para-aramid fibrils are less durable in the lungs ofrats than expected from the known chemical resistance of commercialyarn. These data suggest that at the low para-aramid fibrilexposures found in the workplace, this fibril-shortening mechanismmay limit the residence time of long fibers in the lungs ofexposed workers. In addition, associated cascade impactor aerodynamicmeasurements indicate that due to their ribbon shape and curlynature, para-aramid fibrils behave aerodynamically larger thanstraight fibers.     

Pulmonary and Pleural Responses in Fischer 344 Rats Following Short-Term Inhalation of a Synthetic Vitreous Fiber: I. Quantitation of Lung and Pleural Fiber Burdens

The pleura is an important target tissue of fiber-induced disease,although it is not known whether fibers must be in direct contactwith pleural cells to exert pathologic effects. In the presentstudy, we determined the kinetics of fiber movement into pleuraltissues of rats following inhalation of RCF-1, a ceramic fiberpreviously shown to induce neoplasms in the lung and pleuraof rats. Male Fischer 344 rats were exposed by nose-only inhalationto RCF-1 at 89 mg/m³ (2645 WHO fibers/cc), 6 hr/day for 5 consecutivedays. On Days 5 and 32, thoracic tissues were analyzed to determinepulmonary and pleural fiber burdens. Mean fiber counts were22x10⁶/lung (25x10³/pleura) at Day 5 and 18x10⁶ (16x10/pleura)at Day 32. Similar geometric mean lengths (GML) and diameters(GMD) of pulmonary fiber burdens were observed at both timepoints. Values were 5 µm for GML (geometric standard deviationGSD 2.3) and 0.3 µm for GMD (GSD 1.9), with correlationsbetween length and diameter () of 0.2–0.3. Size distributionsof pleural fiber burdens at both time points were approximately1.5 µm GML. (GSD 2.0) and 0.09 µm GMD (GSD 1.5; 0.2–0.5). Few fibers longer than 5 µm were observedat either time point. These findings demonstrate that fiberscan rapidly translocate to pleural tissues. However, only short,thin (<5 µm in length) fibers could be detected overthe 32-day time course of the experiment.     

Evaluation of the Developmental Toxicity of Ethylene Glycol Aerosol in the CD Rat and CD-1 Mouse by Whole-Body Exposure

Ethylene glycol (EG) is a major industrial chemical, shown tobe teratogenic at high doses by gavage in rodents. Since oneroute of industrial exposure is to the aerosol at high concentrations,timed-pregnant CD rats and CD-1 mice were exposed, whole-body,to a respirable aerosol of EG (mass median aerodynamic diameter,2.3 µm) on Gestational Days (GD) 6 through 15 for 6 hrper day at target exposure concentrations of 0, 150, 1000, or2500 mg/m³ (analytical concentrations of 0, 119 ± 13,888 ± 149, and 2090 ± 244 mg/m³, respectively),with 25 plug-positive animals per species per group. Clinicalobservations and maternal body weights were documented throughoutgestation for both species. Maternal food and water consumptionwas measured in rats only throughout gestation. At schedulednecropsy (GD 21 for rats, GD 18 for mice), maternal animalswere evaluated for body weight, liver weight, kidney weight,gravid uterine weight, number of ovarian corpora lutea, andstatus of implantation sites, i.e., resorptions, dead fetuses,live fetuses. Fetuses were dissected from the uterus, counted,weighed, sexed, and examined for external, visceral, and skeletalmalformations and variations. All rat dams survived to scheduledtermination. Minimal maternal toxicity was indicated by a significantincrease in absolute and relative liver weight at 2500 mg/m³.Food and water consumption, maternal body weights and weightgain, and maternal organ weights (other than liver) were unaffectedby exposure. Gestational parameters were unaffected by exposure,including pre- and post-implantation loss, live fetuses/litter,sex ratio, and fetal body weight/litter. There was no treatment-relatedincrease in the incidence of any individual malformation, inthe incidence of pooled external, visceral, or skeletal malformations,or in the incidence of total malformations by fetus or by litter.There were no increases in the incidence of external or visceralvariations. Evidence of fetotoxicity, expressed as reduced ossificationin the humerus, the zygomatic arch, and the metatarsals andproximal phalanges of the hind-limb, was observed at 1000 and2500 mg/m³. All mouse dams survived to scheduled termination.One dam at 2500 mg/m³ was carrying a totally resorbed litterat termination. Maternal toxicity was observed at 1000 and 2500mg/m³, expressed as reduced body weight and weight gain duringand after the exposure period, and reduced gravid uterine weight.(Maternal effects may have been due, in part or in whole, toeffects on the conceptuses; see below.) Embryo/fetal toxicitywas also observed at 1000 and 2500 mg/m³, expressed as an increasein nonviable implantations/litter, a reduction in viable implantations/litter,and reduced fetal body weights (male, female, and total)/litter.The incidences of individual and pooled external, visceral,and skeletal malformations were increased at 1000 and 2500 mg/m³,as was the incidence of total malformations. Malformations werefound in the head (exencephaly), face (cleft palate, foreshortenedand abnormal face, and abnormal facial bones), and skeleton(vertebral fusions, and fused, forked, and missing ribs). Theincidences of many fetal variations were also increased at 1000and 2500 mg/m³ (and only a few at 150 mg/m³). The no observableadverse effect level (NOAEL) for maternal toxicity in rats was1000 mg/m³ (analytical concentration 888 mg/m³) and in micewas 150 mg/m³ (analytical concentration 119 mg/m³). The NOAELfor development toxicity in rats was 150 mg/m³ and in mice wasat or below 150 mg/m³, under the conditions of this study. Analysisof EG on the fur of rats and mice during and after the exposureperiod at 2500 mg/m³ indicated that much of the EG "dose" (65–95%)was potentially derived from ingestion after grooming and/orpercutaneous absorption. This contribution of the ingested and/orabsorbed chemical could have been sufficient, per se, to producethe teratogenic effects observed in mice. The definitive evaluationof the possible role of inhaled EG aerosol alone in teratogenesisrequires an exposure regimen which limits or precludes exposureby any other route.     

Acute and 2-Week Inhalation Toxicity Studies on Aerosols of Selected Ethylene Oxide/Propylene Oxide Polymers in Rats

The ethylene oxide/propylene oxide (EO/PO) polymers evaluatedin this study have previously been shown to have a low orderof toxicity and/or irritancy by ocular, dermal, or oral routesof administration. These studies evaluated the acute inhalationtoxicity of respirable aerosols of three EO/PO compounds (U-660,U-2000, and U-5100) that differ in chain length, molecular weight,and viscosity. The respective 4-hr LC50 values (95% confidencelimits) for U-660, U-2000, and U-S 100 in Wistar albino ratswere 4670 (4090–5320), 330 (227–480), and 106 (45–245)mg/m³. Occasionally, slight increases in respiration rate andslight hyperactivity were observed during the postexposure period.All deaths were delayed for 2–5 days postexposure. Bodyweight gains were transiently depressed in rats exposed to U-2000and U-5100. Discolored lungs and livers occurred in animalswhich died during the 14-day postexposure period. Subsequently,a repeated-exposure study was conducted on U-5100 in F-344 ratsexposed for 6 hr/day, 5 days/week, for 9 exposures at mean concentrationsof 0, 5, 26, and 50 mg/m³ Portions of the control and 50 mg/m³groups were maintained for an additional 2-week recovery period.Exposure-related effects included transient urogenital wetnessin 50 mg/m³ group females; decreased body weight gain (7–29%)in all U-5100 groups except the 5 mg/m³ group females; increasesin absolute (17–52%) and relative lung weights in allU-5100 groups; macroscopic red foci in the lungs; and microscopicfindings of congestion and hemorrhage of pulmonary alveolarcapillaries and necrosis of alveolar epithelial cells. Lungweights remained elevated after the 2-week recovery period,but the severity of the microscopic lesions was noticeably less,indicating partial reversibility of the lesions. In conclusion,EO/PO polymers have a higher order of toxicity by inhalationin comparison to other routes of administration, vary considerablyin their acute lethal toxicity as a function of chain length/molecularweight, and induce pulmonary hemorrhage, and possibly edema,following repeated aerosol exposures at concentrations as lowas 5 mg/m³.     

Metallothionein Induction and Pulmonary Responses to Inhaled Cadmium Chloride in Rats and Mice

Inhaled CdCl₂ is a pulmonary carcinogen in rats but not in mice.We hypothesized that pulmonary metallothionein (MT) inductionmay be different in both species and thereby may lead to differentlevels of protection from Cd-induced lung injury. Fisher-344rats and B6C3F₁ mice were exposed for 4 weeks to CdCl₂ aerosolsof 0, 30, 50, and 150 µg/m³ air or 0, 10, 30, and 100µg/m³ air, respectively. Animals from each exposure groupwere terminated at 1, 30, and 133 days after the end of exposure.The lungs were lavaged for cell and biochemical analyses. Cadmiumand MT in lavagate and lung tissue were measured. The retentionhall-time of pulmonary Cd was greater in mice (290 vs 90 days,p<0.05). Cd exposure provoked an inflammatory response whichwas dose-dependent in both species, and while it was only short-livedin rats, it persisted throughout the observation period in miceat the high exposure concentrations. Mice were found to havea greater baseline level of MT (18.046.96 vs 11.71.98 µgMT/g control lung, p<0.05). Mice showed greater inducibilityof MT for a given CdCl₂ exposure concentration; however, bothspecies had a similar relationship between retained pulmonaryCd and MT induction though mice maintained increased MT levelsfor a longer period of time. The greater pulmonary baselineMT together with the longer presence of Cd-induced pulmonaryMT may result in greater protection from Cd carcinogenicityin spite of the greater pulmonary Cd-induced inflammation inmice.     

Evaluation of the Developmental Toxicity of Ethylene Glycol Aerosol in CD-1 Mice by Nose-Only Exposure

Ethylene glycol (EG; CAS No. 107-21-1) is teratogenic to miceby whole-body (WB) exposure to aerosol (1000–2500 mg/m³The WB results were confounded by possible exposure from ingestionafter grooming andior from percutaneous absorption. Therefore,CD-1 mice were exposed to EG aerosol (MIMAD 2.6 ± 1.7µm) on Gestational Days (GD) 6 through 15, 6 hr/day, bynose-only (NO) (0, 500, 1000, or 2500 mg/rn or WB exposures(0 or 2100 mg/m³ as positive control), 30/group. Five additional"satellite" females each at 2500 mg/m³ NO and 2100mg/m³ WB wereexposed on GD 6 for measurement of EG on fur. Control environmentswere water aerosol (4200 mg/rn for NO; 2700 mg/rn for WB). Femaleswere weighed and evaluated for clinical signs and water consumptionthroughout gestation. On GD 18, maternal uterus, liver, andkidneys (2) were weighed, with kidneys examined microscopically.Corpora lutea and implantation sites were recorded. Live fetuseswere weighed, sexed, and examined for structural alterations.For NO dams, kidney weights were increased at 1000 and 2500mg/m³ no renal lesions and no other treatment-related maternaltoxicity were observed. There were no effects on pre- or postimplantationloss; fetal body weights/litter were reduced at 2500 mg/m³ At2500 mg/m³ incidences of fused ribs and skeletal variationswere increased. The 2500 mg/rn NO satellite animals had {smalltilde}330 mg/kg extractable EG. The WB group exhibited maternaland developmental toxicity including increased fetal skeletalmalformations and variations, confirming previous results, with1390 mg/kg extractable EG on fur. Therefore, exposure of CD-1mice to a respirable EG aerosol during organogenesis by NO inhalationresulted in minimal maternal toxicity at 1000 and 2500 mg/m³and developmental toxicity at 2500 mg/m³ The NOAEL was 500 mg/m³NO for maternal and 1000 mg/m³ NO for develop mental toxicity.This study supports the interpretation of the initial EG WBresults as due to systemic exposure from noninhala tion routessince limiting noninhalation routes prevented almost all ofthe effects (including teratogenicity) observed in mice afterWB exposure.     

Use of Lung Toxicity and Lung Particle Clearance to Estimate the Maximum Tolerated Dose (MTD) for a Fiber Glass Chronic Inhalation Study in the Rat

Short–term toxicity and lung clearance were assessed inrats exposed by inhalation to size-selected fibrous glass (FG)for 13 weeks. Results from this study and from a recent FG chronicinhalation study are presented here as guidelines for the selectionof a maximum tolerated dose (MTD) for chronic inhalation studiesof fibers. Fischer 344 rats were exposed using nose–onlyinhalation chambers, 6 hr/day, 5 days/week, for 13 weeks toone of five concentrations of FG (36, 206, 316, 552, or 714fibers/cc; expressed gravirnetrically, 3, 16, 30, 45, or 60mg/m³) or to filtered air. Rats were then held for an additional10 weeks of postexposure recovery. Test fiber was size–selectedfrom glass wool having a chemical composition representativeof building insulation. Rats were terminated at 7, 13, 19, and23 weeks after the onset of exposure to evaluate pulmonary pathology,lung epithelium cell proliferation, lung fiber burden, and lunglavage cells and chemistry. The effect of fiber inhalation onlung clearance of innocuous microspheres was also evaluated:following fiber exposure, six rats/group were exposed to ⁸⁵Sr–labeled3.0-µm polystyrene microspheres by intratracheal inhalationand then monitored for whole–body radioactivity duringthe 10–week recovery period. Data from the short–termstudy support the choice of 30 mg/m³ as the MTD for the previouschronic FG study and also provide indicators of long–termlung toxicity and functional impairment that can be used toestimate the MTD for future chronic fiber inhalation studies.     

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.     

4,4'-Methylenebis(2-chloroaniline) (MOCA): The Effect of Multiple Oral Administration, Route, and Phenobarbital Induction on Macromolecular Adduct Formation in the Rat

The effect of multiple oral administration of MOCA, a suspecthuman carcinogen, was studied in the adult male rat. As manyas 28 consecutive daily doses of [¹⁴C]MOCA at 28.1µmol/kgbody wt (5 µC1/day) were administered and rats were euthanizedat weekly intervals for 7 weeks. MOCA adduct formation for globinand serum albumin was evaluated by determination of [¹⁴C]MOCAcovalent binding. The covalent binding associated with globinshowed a linear increase over the 28-day exposure period with342 fmol/mg globin 24 hr after the final dose. More extensivecovalent binding was detected for albumin with 443 fmol/mg albuminafter the final dose, but increases were not linear. After cessationof dosing, the albumin adduct levels decreased rapidly (t _1/2=4.6 days) in relation to globin adduct levels (t _1/2 =16.1days). The MOCA-globin adduct t _1/2 is consistent with thatdetermined after a single 281 µmol/kg oral dose of MOCA.Significant differences related to route of administration weredetected for 24-hr globin covalent binding with ip > po >dermal. Distribution of undifferentiated [¹⁴C]MOCA was highestin the liver at 24 hr with tissue levels for liver > kidney> lung > spleen > testes > urinary bladder. Inductionof cytochrome P450 enzymes by administration of phenobarbital(100 mg/kg/day/3 days) resulted in a significant (p < 0.05)increase in MOCA-globin adduct formation detected with 33.5pmol/ mg globin for induced rats versus 13.6 pmol/mg globinfor control rats. Although MOCA-globin and albumin adducts showdiffering stability, quantification of such MOCA adducts maybe useful for long-term industrial biomonitoring of MOCA.     

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 inhalationtoxicity of caprolactam when administered as a 3-µm aerosolfrom an aqueous solution to Sprague-Dawley CD rats (10/sex/group)via whole-body exposure. The study was enhanced with the inclusionof motor activity measurements and a functional observationalbattery to assess the neurotoxic potential of caprolactam. Therats were exposed at least 65 times over a 13-week period for6 h per day, 5 days per week, to target concentrations (3 µm,mass median aerodynamic diameter) of 0, 25, 75, and 250 milligramsper cubic meter (mg/m³). An additional 10 animals/sex/groupwere 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 orrecovery periods. Treatment-related responses such as laboredbreathing and nasal discharge were seen during many of the exposures.Similar responses as well as moist rales were seen during thenonexposure periods during the 13 weeks of exposure. However,these responses abated during the 4-week recovery period. Therewere no clearly treatment-related responses observed with ophthalmoscopicexaminations, 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 exposureto the test material were seen in the nasoturbinal tissues (hypertrophy/hyperplasiaof goblet cells in the respiratory mucosa and intracytoplasmiceosinophilic material in epithelial cells of the olfactory mucosa)of the two higher-exposure group animals and in the laryngealtissues (squamous/squamoid meta-plasia/hyperplasia of the pseudostratifiedcolumnar epithelium covering the ventral seromucous gland) ofall three exposure group animals. These changes were consideredto be adaptive responses to an irritant (caprolactam). The keratinizationof the metaplastic epithelium in the larynx was considered tobe 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 completelyresolved. In conclusion, the whole-body exposure of Sprague-Dawleyrats 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 within4 weeks postex-posure. The results indicate that the no-observed-adverse-effectlevel for caprolactam is 70 mg/m³, based on upper respiratoryeffects, with 243 mg/m³ representing a no-observed-effect levelfor systemic toxicity, neurotoxicity, and lower respiratorytract effects.     

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.     

Evaluation of a Multitiered Inhalation Exposure Chamber

Evaluation of a Multitiered Inhalation Exposure Chamber. Griffis,L.C, Wolff, R.K., Beethe, R.L., Hobbs, C.H. and McClellan, R.O.(1981).Fundam. Appl. Toxicol. 1:8–12. A multitiered inhalationexposure chamber was evaluated for use in aerosol toxicity studiesby determining the uniformity of pulmonary deposition in 144rats simultaneously exposed to ^99mTc-CsCl aerosols. The activitymedian aerodynamic diameter and geometric standard deviationwere 1.7–2.1 µm and 1.8 respectively. In one experiment,lung deposition of ^99mTc in male and female rats was 728 and544 nCi, respectively, after 130 min exposure to an aerosolconcentration of 173 nCi/L. Analysis of variance revealed asignificant effect of several factors on lung deposition. Animalshoused on one side of the chamber had lung burdens 8–11%greater than those on the opposite side. Because an animal'slocation within the chamber had a slight effect on its lungburden of inhaled aerosol we recommend the rotation of animalsamong the chamber's six tiers during chronic aerosol toxicitystudies. The overall coefficient of variation in lung burdenwas only 21% which is less than the variability reported insmall rodents given a nose-only aerosol exposure.     

Clearance of Diesel Soot Particles from Rat Lung after a Subchronic Diesel Exhaust Exposure

Clearance of Diesel Soot Particles from Rat Lung after a ChronicDiesel Exhaust Exposure. Griffis, L.C., Wolff, R.K., Henderson,R.F., Griffith, W.C., Mokler, B.V. and McClellan, R.O. (1983).Fundam. Appl. Toxicol. 3:99-103. The participate exhaust ofdiesel engines consists of 0.1–0.2 µm mass mediandiameter particles composed of a carbonaceous core and adsorbedorganic compounds. A technique was needed to determine accumulatedlung burdens of particles in animals exposed to diesel exhaustas a determinant of dose. A method was developed for determininglung burdens of diesel soot particles in rats at 1 day, andat 1, 5, 15, 33 and 52 weeks after cessation of a subchronicexposure to diluted diesel exhaust. Lung tissue was dissolvedin tetramethylammonium hydroxide and the diesel soot separatedby centrifugation. The soot was suspended in water by sonicationand the light absorption of samples was compared to standardsuspensions of diesel soot. Recovery from lungs spiked with50–1000 µg of soot was 89±5%. Rats exposedover a period of 18 weeks to diluted diesel exhaust at averagenet diesel particle concentrations of 150, 940 and 4100 µg/m³had lung burdens of 35,220 and 1890 µg/g lung, respectively,one day after the last exposure. The long-term clearance ratesof soot had estimated half-times of 87±28, 99±4days, for the low and medium exposure groups, respectively.The clearance half-time for the high level exposure group of165 ± 8 days was significantly longer (P < 0.0001)than those of the other two groups.     

The Effects of in Vitro and Aerosol Exposures to Cadmium on Phagocytosis by Rat Pulmonary Macrophages

The Effects of in Vitro and Aerosol Exposures to Cadmium onPhagocytosis by Rat Pulmonary Macrophages. GREENSPAN, B. J.,and MORROW, P. E (1984). Fundam. Appl. Toxicol. 4, 48–57.Aerosol exposures of rats were performed to assess the in vivoeffects of cadmium on the phagocytosis of latex particles bypulmonary macrophages. An in vitro assay for particle uptakewas devised which allowed quantification of phagocytosis byadhering and nonadhering macrophages. In vitro exposure to CdCl₂caused dose-dependent decreases in viability (trypan blue exclusion),percentage cells with particles, total number of particles phagocytized,and the ability of the macrophages to adhere to a siliconizedglass surface. In vivo effects were studied following 30-minaerosol exposures to 1.5 mg/m³ Cd (MMAD = 0.35 µm, _g =1.45) or 5.0 mg/m³ Cd (MMAD = 0.45 µm, _g = 1.60) as CdCl₂.Phagocytic activity in the in vitro test system was increasedimmediately and at Day 1 in the low exposure group. However,following exposure to 5.0 mg/m³ Cd, phagocytic activity wasdepressed until 8 days postexposure. The results show that cadmiumis capable of modifying the phagocytic ability of macrophagesin vivo as well as in vitro. Determinations of the total numberof particles phagocytized were found to be more sensitive thanpercentage cells phagocytizing in detecting the effects of cadmiumexposure.     

Subchronic Inhalation Toxicity of Hexamethylenediamine in Rats

Four groups of 15 male and 15 female Sprague-Dawley-derived(CD) rats each were exposed to aqueous hexamethylenediamine(HMD) aerosols for 6 hr/thy, 5 days/week for 13 weeks at meananalytical concentrations of 0, 12.8, or 51 mg/m³ Because ofexposure-related deaths in a group of male and female rats similarlyexposed to 215 mg/m³ HMD, this group was terminated during theseventh week of the study. Signs of respiratory and conjunctivalirritation were observed in rats at both the 51 and 215 mg/m³HMD test levels. Body weight gain was significantly reducedin both sexes exposed to 215 mg/m³ HMD. At the 5-week studyinterval, slight hemopoietic stimulation of peripheral bloodparameters was observed in rats of both sexes exposed to 215mg/m³ HMD. Treatment-related microscopic lesions were seen onlyin rats exposed to 215 mg/m³ MD and were confined to the trachea,nasal passages, and lungs. The noeffect level in this studyis considered to be 12.8 mg/m³ HMD.