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.     

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.     

Investigation of the Effects of Benomyl on Rat Nasal Mucosa

Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate,CAS Registry No. 17804-35-2] is a widely used agricultural fungicide.Previously, olfactory epithelial lesions were produced followinga 45-day inhalation exposure to 50 and 200 mg/m³ benomyl. Thepresent study, part of a range-finding study for a two-generationreproduction study, was conducted to determine if the previouslyreported effects on the nasal mucosa are the result of systemictoxicity or attributable to the inhalation route of exposure.Groups of 10 7-week-old male Crl:CD BR rats were fed diets containing0, 5000, 10000, or 15000 ppm benomyl for 32 days. Individualbody weights and food consumption were determined weekly andon the last day of the study. After 32 days on test, rats wereeuthanatized by pentobarbital anesthesia and exsanguinationand were examined for gross alterations. The nasal cavity wasprocessed for pathological examination. Mean body weight gainwas statistically significantly decreased during the first weekof treatment and the overall test period (Days 0–32) atthe two highest dose levels. A significant decrease in foodconsumption also was seen during test interval Days 0–7for the two highest dose groups. In addition, statisticallysignificant decreases in food consumption were observed at theDay 7–14 interval for the 15,000 ppm dose group and atthe 21–28 and 28–32 intervals for the two highestdose groups compared with controls. No histopathological lesionswere noted in the nasal epithelium of any of the control orbenomyl-treated rats. These results suggest that the nasal cavityis not a target following dietary administration of benomyland the olfactory epithelial damage reported following inhalationexposure is specific to the route of exposure.     

Identification of Stage-Specific Changes in Protein Secretion by Isolated Seminiferous Tubules from the Rat Following Exposure to Either m-Dinitrobenzene or Nitrobenzene

The objective of this study was to identify effects of two knownSertoli cell toxicants on the secretion of proteins by seminiferoustubules (ST) isolated from adult rats at different stages ofthe spermatogenic cycle and cultured in vitro for 24 hr with[³⁵S]methionine Adult rats received a single oral dose of 50mg/kg metadinitrobenzene (m-DNB) or 300 mg/kg nitrobenzene (NB).Long lengths of ST at stages II–V, VI–VII or IX–XIIwere then isolated from control and treated rats at 1 or 3 dayspost-treatment; selection of stages was based on the stage specificityof the early (24–72 hr) adverse effects of m-DNB and NBon spermatogenesis in vivo. In addition, ST at the same stageswere isolated from untreated rats and cultured in the presenceor absence of m-DNB or NB (10^–4 M). Incorporation of [³⁵S]methionneinto secreted proteins was assessed and the pattern of proteinsecretion evaluated using two-dimensional sodium dodecyl sulfate-polyacrylamidegel electrophoresis (2-D SDS-PAGE). ST isolated from rats pretreated24 hr earlier with NB in vivo showed a significant decreasein the overall incorporation of [³⁵S]methionne into secretedproteins at stages VI–VIII and IX–XII, whereas STat stages II–V showed no such change; comparable proteinchanges were observed when 10^–4M NB was added in vitrofor 24 hr to ST isolated at the same stages from untreated rats.Similar results were obtained when ST protein secretion wasevaluated 72 hr after treatment with NB in vitro or after theaddition of NB in vitro to isolated ST from untreated rats for24 or 72 hr. In general, the results obtained after exposureto m-DNB were comparable to those observed for NB, with theexception that the incorporation of [³⁵S] into secreted proteinsby ST at stages II–V tended to be increased by m-DNB exposure.Analysis of ST-secreted proteins by 2-D SDS-PAGE identified6 "marker proteins" (MP) which showed major reproducible changesin secretion following exposure to either m-DNB or NB. In mostcases (MP–1, –2, –3, –5, and –6)their secretion was reduced markedly; two of these proteins(MP–2 and MP–3) are secreted almost exclusivelyat stages VI–VIII and correspond in molecular size andpI to two recently reported androgen-regulated proteins whichare thought to be products of the Sertoli cell. Exposure toeither NB or m-DNB also resulted in the appearance of one protein(MP–4) which was not secreted in detectable amounts byST from control animals. Previous data has shown that by 24hr after administration of either m-DNB or NB to rats thereis massive loss/degeneration of pachytene spermato cytes atstages VI–XII, whereas stages II–V of the spermatogeniccycle are not affected until 72 hr. The presently observed proteinchanges therefore either precede (stages II–V) or accompany(stages VI–XII) germ cell degeneration. This suggeststhat they may have potential use as markers of early toxicant-induceddamage and/or that changes in these proteins may mediate someof the adverse testicular effects of m-DNB and NB.     

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.     

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.     

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.     

Effects of Chlorpyrifos on High-Affinity Choline Uptake and [3H]Hemicholinium-3 Binding in Rat Brain

High, subcutaneous doses of the organophosphorus insecticidechlorpyrifos (CPF) in adult male rats can be well-tolerateddespite extensive and persistent acetylcholinesterase (AChE)inhibition. We propose that changes in acetylcholine synthesiscould modulate the toxicity associated with extensive AChE inhibitionfollowing CPF exposure. High-affinity choline uptake (HACU,the rate-limiting step in acetylcholine synthesis) and bindingto [³H]-hemicholinium-3 (HC-3, a specific ligand for the cholinetransporter) were chosen as indicators of acetylcholine synthesis.Female, Sprague-Dawley rats (220–280 g) were treated witheither vehicle (peanut oil, 2 ml/kg, sc) or CPF (280 mg/kg,2 ml/kg, sc), examined daily for clinical signs of toxicity,and sacrificed 1, 2, or 7 days later for neurochemical measurements{AChE inhibition, muscarinic receptor binding using [³H]quinuclidinylbenzilate (QNB) and [³H]cis-methyldioxolane (CD) as ligands,HACU and [³H]HC-3 binding} in frontal cortex. Despite extensiveAChE inhibition (90–93%) at all time points, relativelyminor degrees of overt toxicity were noted in CPF-treated rats.Binding to the non-selective muscarinic antagonist [³H]QNB wasreduced (10–34%), whereas binding to the putative m2-selectiveagonist [³H]CD was increased (15–23%) at all three timepoints. HACU was reduced (20%) in crude synaptosomes preparedfrom CPF-treated rats 1 day following exposure but no significantchanges were noted at 2 or 7 days after treatment. CPF-oxon,the active oxidative metabolite of CPF, was a weak inhibitorof HACU in vitro (IC₅₀>200 µM). Binding to [³H]HC-3was reduced in a dose-related manner 1 day after CPF exposure.Kinetic analyses of [³H]HC-3 binding 1 day after CPF (280 mg/kg)indicated a significant reduction in density {B_max: control,187±18 fmol/mg protein; CPF, 104±12 fmol/mg protein)with no apparent change in binding affinity (K_d: control, 25±3nM; CPF, 19±3 nM). These results suggest that a reductionin HACU/acetylcholine synthesis may contribute, along with compensatorychanges in cholinergic receptors, to the diminished toxicityfollowing extensive AChE inhibition by CPF.     

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.     

The Effects of Inhalation Exposure to Sulfuryl Fluoride on Fetal Development in Rats and Rabbits

Sulfuryl fluoride is a fumigant insecticide used for soils andpermanent structures. Pregnant Fischer 344 rats and New ZealandWhite rabbits were exposed to 0, 25, 75, or 225 ppm of sulfurylfluoride vapor via inhalation for 6 hr/day on Days 6–15and 6–18 of gestation, respectively. Among rats, maternalwater consumption was increased in the 225 ppm exposure group,but there were no indications of embryotoxicity, fetotoxicity,or tera-togenicity in any of the exposed groups. Among rabbits,maternal weight loss during the exposure period (Days 6–18)was observed in the 225 ppm group. Decreased fetal body weights,considered secondary to maternal weight loss, were also observedat 225 ppm. However, no evidence of embryotoxicity or teratogenicitywas observed among rabbits in any exposure group. Thus, inhalationexposure to sulfuryl fluoride was not teratogenic in eitherrats or rabbits exposed to levels of up to 225 ppm, and fetotoxiceffects (reduced body weights) were observed among fetal rabbitsonly at an exposure level that produced maternal weight loss.     

Assessment of the Developmental Toxicity and Placental Transfer of 1,2-Dichloroethane in Rats

This study evaluates the developmental toxicity and placentaltransfer of 1,2-dichloroethane (DCE) in rats. Sprague–Dawleyrats were given 0–2.4 mmol DCE kg^-1 day^-1 by gavage, orwere exposed for 6 hr per day to 0–300 ppm DCE by inhalation,from Day 6 to 20 of gestation. Maternal toxicity was observedafter inhalation exposure to 300 ppm DCE and oral administrationof 2.0 or 2.4 mmol DCE kg^-1 There was no evidence of alteredgrowth nor teratogenic effects after either inhalation or oraladministration of DCE at any concentration tested. The timecourse disposition of ¹⁴C was examined over a 48-hr period in12- and 18-day pregnant rats after a single oral dose of 1.6mmol [¹⁴C]DCE kg^-1. Peak concentrations of radiocarbon occurredbetween 2 and 4 hr postdose. Conceptus (Day 12) and fetal (Day18) tissues accounted for 0.06 and 0.4% of the administereddose, respectively. Up to 4 hr, levels of radiocarbon in placentaand fetus were slightly less than in maternal plasma of 18-daypregnant rats and were two to five times higher at later periods.At 2 hr, unchanged DCE accounted for most of radioactivity (78–86%)recovered in maternal plasma, placenta, and fetus. Acidic metabolitesand radioactivity bound to macromolecules increased up to 24hr (0.01 µmol-eq DCE g^-1)in either placental or fetaltissues. Thereafter, their levels declined more slowly thanthose in the maternal plasma. Results from this developmentaltoxicity study in rats confirm embryonic exposure to radiocarbonassociated with [¹⁴C]DCE and/or its metabolites and has demonstratedthe lack of observable teratogenic effects.     

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.     

Lung Toxicity after 13-Week Inhalation Exposure to Nickel Oxide, Nickel Subsulfide, or Nickel Sulfate Hexahydrate in F344/N Rats and B6C3F1 Mice

Lung Toxicity after 13-Week Inhalation Exposure to Nickel Oxide,Nickel Subsulfide, or Nickel Sulfate Hexahydrate in F344/N Ratsand B6C3F₁ Mice. DUNNICK, J. K., ELWELL, M. BENSON, J. M., HOBBS,C. H., HAHN, F. F., HALY, P. J., CHENG, Y. S., AND EIDSON, A.F. (1989). Fundam. Appl. Toxicol. 12, 584–594. The relativetoxicity of nickel oxide (NiO), nickel sulfate hexahydrate (NiSO₄6H₂O)and nickel subsulfide (Ni₃S₂) was studied in F344/N rats andB6C3F₁ mice after inhalation exposure for 6 hr/day, 5 days/week,for 13 weeks. Exposure concentrations used (as mg Ni/m³ were0.4–7.9 for NiO, 0.02–0.4 for NiSO₄ 6H₂O and 0.11–1.8for Ni₃S₂. No exposure-related effects on mortality and onlyminor effects on body weight gain were seen in rats or mice.The most sensitive parameter for nickel toxicity was histopathologic change in the lungs of exposed animals where chronic activeinflammation, fibrosis, and alveolar macrophage hyperplasiawerez associated with nickel exposure. There was an exposure-related increase in lung weight in rats and mice. Equilibriumlevels of nickel in the lung were reached by 13 weeks of nickelsulfate and nickel subsulfide exposure, whereas lung levelsof nickel continued to increase throughout exposure to nickeloxide. Additional exposure-related histopathologic lesions intreated animals included atrophy of the olfactory epitheliumafter nickel sulfate and nickel subsulfide exposure. No nasallesions were seen after nickel oxide expo sure. Lymphoid hyperplasiaof the bronchial lymph nodes developed in animals exposed toall three nickel compounds. The order oftoxicity correspondedto the water solubility of the nickel compounds, with nickelsulfate being most toxic, followed by nickel subsulfide andnickel oxide.     

Inhalation Toxicity of 1,6-Hexanediamine Dihydrochloride in F344/N Rats and B6C3F1 Mice

1,6-Hexanediamine (HDA) is a high production volume chemicalwhich is used as an intermediate in the synthesis of paints,resins, inks, and textiles and as a corrosion inhibitor in lubricants.Two- and 13-week studies of the toxicity of the dihydrochloridesalt of HDA (HDDC) were conducted in male and female Fischer344/N rats and B6C3F₁ mice using whole-body inhalation exposure.Both species were evaluated for histopatho-logic and reproductiveeffects, and rats were examined for clinical chemistry and hematologicchanges. In the 2-week inhalation studies, animals were exposedto 10–800 mg HDDC/m³, 6 hr per day. All rats, all femalemice, and two of five male mice in the high-exposure group diedbefore the end of the study. Surviving mice in this group hada dose-dependent depression in body weight gain. Clinical signswere primarily related to upper respiratory tract irritationand included dyspnea and nasal discharge in both species. Treatment-relatedhistopathologic lesions included inflammation and necrosis ofthe laryngeal epithelium of both species and the tracheal epitheliumof mice, as well as focal inflammation and ulceration of therespiratory and olfactory nasal mucosa. In the 13-week inhalationstudies, animals were exposed to HDDC at concentrations of 1.6–160mg/ m³ for 6 hr per day, 5 days per week. In addition to thebase study groups, a supplemental group of rats at each exposurelevel was included to assess the effect of HDDC on reproduction.No treatment-related changes in organ weights or organ-to-body-weightratios occurred in rats, and no treatment-related clinical signsor gross lesions were seen in either species. Chemical-relatedmicroscopic lesions were limited to the upper respiratory tract(larynx and nasal passages) in the two highest exposure groupsand were similar in both species. These lesions included minimalto mild focal erosion, ulceration, inflammation, and hyperplasiaof the laryngeal epithelium, in addition to degeneration ofthe olfactory and respiratory nasal epithelium. HDDC causedno significant changes in sperm morphology or vaginal cytologyand no significant adverse effects on reproduction in rats ormice. Hematologic and clinical chemistry changes in rats wereminor and sporadic and were not accompanied by related histologicfindings. HDDC did not increase the frequency of micronucleatederythrocytes in mice. In summary, the toxicity of HDDC to ratsand mice was a result of the irritant properties of the chemical,was limited primarily to the nasal passages and upper airways,and was consistent with the effects of other irritant chemicalsadministered by inhalation.     

In Vitro 2,3,7,8-Tetrachlorodibenzo-p-dioxin Interference with the Anterior Pituitary Hormone Adrenocorticortropin

Treatment of male Sprague-Dawley rats with a single oral doseof 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shownto increase serum adrenocorticotropin (ACTH) and decrease serumcorticosterone. The present in vitro study was designed to assesswhether TCDD has a direct effect on the anterior pituitary underbasal and stimulated conditions. Primary anterior pituitarycell cultures were prepared from normal 180- to 220-g male Sprague-Dawleyrats and the cultures treated with 10^–9–10^–19M TCDD. Maximal secretion of ACTH occurred between 10^–11and 10^–15 M TCDD for both medium (2-fold) and intracellular(1.5-fold) concentrations after 24 h TCDD exposure. TCDD treatmentalso caused an early (6 h) and persistent (10 days) increasein basal medium (1.4- to 2.8-fold) and intracellular (1.1- to1.7-fold) ACTH concentrations. However, while stimulation withcorticotropin-releasing hormone (CRH) increased intracellularACTH 1.5- to 1.7-fold in pituitary cells treated for 24 h with10^–9–10^–13 M TCDD, ACTH secreted into themedia was decreased by 30–50% compared with controls.Lastly, the secretagogue arginine-8-vaso-pressin (AVP), didnot increase the amount of ACTH secreted above levels observedwith basal TCDD exposure. From this study, it appears that TCDDstimulates in vitro synthesis and secretion of ACTH by the anteriorpituitary under basal conditions, but decreases the pituitary'sresponsiveness to CRH and AVP stimulation.     

Developmental Toxicity Evaluation of Inhaled 2-Ethoxyethanol Acetate in Ficher 344 Rats and New Zealand White Rabbits

Developmental Toxicity Evaluation of Inhaled 2–EthoxyethanolAcetate in Fischer 344 Rats and New Zealand White Rabbits. Tyl,R. W., Pritts, I. M, France, K. A., Fisher, L. C, and Tyler,T. R. (1988). Fundam. Appl. Toxicol. 10, 20–39. PregnantFischer 344 rats and New Zealand white rabbits were exposedto 2–ethoxyethanol acetate (EEA; CAS No. 111–15–9)vapor by inhalation on Gestational Days 6 through 15 (rats)or 6 through 18 (rabbits) at concentrations of 0, 50, 100,200,or 300 ppm, 6 hr/day. The animals were terminated on Gesta–tionalDay 21 (rats) or 29 (rabbits) and fetuses were examined forexternal, visceral, and skeletal malformations and variations.In rabbits, exposure to 100–300 ppm resulted in maternaltoxic–ity: decreased weight gain at 100–300 ppm,clinical signs at 200–300 ppm, alterations in hema–tologyat 100–300 ppm, reduced gravid uterine weight at terminationat 200–300 ppm, and elevated absolute liver weight at300 ppm. Developmental toxicity was observed at 100–300ppm: an increased incidence of totally resorbed litters at 200–300ppm, an increase in nonviable fetuses at 300 ppm, and a decreasein viable implants (live fetuses) per litter at 200–300ppm. The incidence of fetal malformations (external, visceral,and skeletal) was increased at 200–300 ppm. The incidenceof total malformations was 100% at 300 ppm and significantlyincreased at 200 ppm. Reduced fetal ossification was observedat 100–300 ppm. In rats, exposure to 100–300 ppmalso resulted in maternal toxicity: reduced weight gain andreduced food consumption at 200–300 ppm and elevated relativeliver weight and alterations in hematology at 100–300ppm. Absolute maternal liver weight was increased at all EEAexposure concentrations; relative liver weight was increasedat 100–300 ppm. Developmental toxicity was observed at100–300 ppm: increased nonviable implantations/litter(300 ppm), reduced fetal body weight/litter (200–300 ppm),and increased incidence of external (300 ppm), visceral, andskeletal (100–300 ppm) variations indicative of toxicity.The incidence of visceral, skeletal, and total malformationswas increased at 200–300 ppm. In conclusion, in both species,inhalation exposure to EEA during organogenesis produced maternaltoxicity at 100–300 ppm and developmental toxicity at100–300 ppm, including teratogenicity at 200–300ppm. At 50 ppm in both species, there was no evidence of maternalor developmental toxicity, including teratogenicity.     

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.     

Teratology of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in a Complex Environmental Mixture from the Love Canal

The organic phase of a leachate (OPL) from the Love Canal chemicaldump site contains more than 100 organic compounds including2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The teratogenicpotential of OPL was determined in two inbred and one hybridmouse strain which differ in their sensitivity to aromatic hydrocarbon(Ah) receptor-mediated toxicity. OPL was orally administeredin corn oil on Days 6–15 of gestation to C57BL/6J mice(Ah^b/ Ah^b) at doses of 0, 0.1, 0.3, 0.5, and 0.7 g kg^–1day^–1 and to DBA/ZJ (Ah^d/Ah^d) females, which were matedwith either DBA/2J or C57BL/6J males, at 0, 0.5, 1, and 2.0g kg^–1 day^–1. In C57BL/6J mice, which express ahigh-affinity Ah receptor that avidly binds TCDD, the ED50'sof OPL for cleft palate and hydronephrosis were 0.44 and 0.11g OPL kg^–1 day^–1, respectively. Maternal mortalitywas 5% at the highest dose. In DBA/2J fetuses, which expressa low-affinity receptor, neither treatment-related cleft palatenor hydronephrosis was induced by dose levels that caused 36%maternal mortality. In hybrid D2B6F1 fetuses, the incidenceof cleft palate reached only 8% at 2 g OPL kg^–1 day^–1but the ED50 for hydronephrosis was 0.76 g OPL kg^–1 day^–1.TCDD was similarly administered to pregnant C57BL/6J mice at0, 0.5, 1, 2, and 4 µg kg^–1 day^–1 and to DBA/2Jmice at 0, 0.5, 2, 4, and 8 µg kg^–1 day^–1.In C57BL/6J fetuses, the ED50's for cleft palate and hydronephrosiswere 4.6 and 0.73 µg TCDD kg^–1 day^–1, respectively.In DBA/2J fetuses the ED50's for cleft palate and hydronephrosiswere 15.0 and 6.4 µg TCDD kg^–1 day^–1, respectively.Both the OPL and TCDD caused maternal hepatomegaly and thymicatrophy in all strains, but increased only C57BL/6J fetal weights.OPL decreased the number of fetuses per C57BL/6J dam at thetwo highest doses but there were no other reproductive effectsin any of the groups. It was concluded that the OPL is teratogenicand that hydronephrosis is a sensitive measure of TCDD toxicityin a complex organic mixture. Based on the ED50's of OPL- andTCDD-induced cleft palate and hydronephrosis in the C57BL/6Jstrain, the OPL had TCDD equivalence of 6.6 and 10.5 ppm, respectively.These values compare closely with the chemical analysis of 3ppm. The results suggest that the teratologic effects are dueprimarily to the TCDD in the OPL and that these effects aremediated through the Ah receptor, but that the maternal thymicatrophy and hepatomegaly were due primarily to the non-TCDDcomponents of the OPL.     

Short-Term Effects of Sidestream Smoke on Respiratory Epithelium in Mice: Cell Kinetics

Male strain A/J and C57BL/6 mice were exposed on five consecutivedays, for 6 hr a day, to sidestream smoke generated from Kentucky1R4F reference cigarettes. Chamber concentrations were 1 mg/m³of total suspended particulate matter and 528 to 549 µg/m³of nicotine. Cumulative labeling indices in the airways andin the pulmonary parenchyma were measured following 1, 3, or5 days exposure to unfiltered or filtered sides tream smoke.A significantly increased labeling index was found in A/J micein the epithelium lining large intrapulmonary airways and terminalbronchioles after 3 and 5 days exposure to unfiltered smoke,whereas following exposure to filtered smoke labeling indicesremained normal. The alveolar labeling index was not increasedfollowing smoke exposure. In C57BL/6 mice, sidestream smokedid not produce signs of increased cell proliferation in therespiratory tract. It is concluded that the response to sidestreamsmoke inhalation in mice may depend upon the strain of miceexamined.     

The Ototoxicity of Trichloroethylene: Extrapolation and Relevance of High-Concentration, Short-Duration Animal Exposure Data

Inhalation exposure to high concentrations of 1,1,2-trichloroethylene(TCE) has been shown to damage hearing in the mid-frequencyrange in the rat. The present study directly evaluated the adequacyof high-concentration, short-term exposures to TCE for predictingthe neurotoxicity produced by longer duration exposures. Adultmale Long-Evans rats (n = 10–12 per group) were exposedto TCE via inhalation (whole body) in 1-m³ stainless steel flow-throughchambers for 6 hr/day, 5 days/week. The following exposureswere used: 1 day (4000–8000 ppm), 1 week (1000–4000ppm), 4 weeks (800–3200 ppm), and 13 weeks (800–3200ppm). Air-only exposed animals served as controls. Auditorythresholds were determined for a 16-kHz tone 3–5 weeksafter exposure using reflex modification audiometry. Resultsreplicated previous findings of a hearing loss at 16 kHz forall exposure durations. The dBl5 concentrations (concentrationthat increases thresholds by 15 dB) for 16-kHz thresholds were6218, 2992, 2592, and 2160 ppm for the 1-day, 1-week, 4-weekand 13-week exposures, respectively. These data demonstratethat the ototoxicity of TCE was less than that predicted bya strict concentration time relationship. These data alsodemonstrate that simple models of extrapolation (i.e., C t= k, Haber's Law) overestimate the potency of TCE when extrapolatingfrom short-duration to longer-duration exposures. Furthermore,these data suggest that, relative to ambient or occupationalexposures, the ototoxicity of TCE in the rat is a high-concentrationeffect.