Principles and Practices in Inhalation Toxicology …

1 7/12/20121 Principles and Practices in Inhalation ToxicologyJon A. Hotchkiss, & Environmental Research and ConsultingThe Dow Chemical Company, Midland MI1 JAH 7/26/2012 Outline Classes and Properties of Inhaled Materials Gases, Vapors and Aerosols Inhalation Toxicity Testing Exposure Systems Atmosphere Generation and Analysis Testing Guidelines Dosimetry and Dose Regional Dosimetry Inhaled vsAbsorbed Dose In Vitro Systems2 JAH 7/26/2012 Why Conduct Inhalation Studies?3 Major route of human exposure Unique interface between environment and circulation Upper (URT) and lower (LRT) respiratory tract important Identify critical responses to inhaled materials Portal of entry effects Cells and tissues of the URT and LRT Systemic effects Internal organs and tissues Exposure response data for human risk assessment Integration of material properties, deposition, absorption, transport, metabolism and elimination Susceptibility Life stage, health status, species specific metabolic pathwaysJAH 7/26/20127/12/20122 What is the Goal?

2 4 JAH 7/26/2012 Gases Vapors Aerosols5 Gases and Vapors6 Gases Exist in the gaseous state at STP Easiest class of materials to use Vapors The gas phase fraction of volatile solids/liquids at STP Saturated vapor (ppm) = Vp(mm Hg) x 106/AtmP(mm Hg) mg/L (vapor or gas) = ppm x (molecular wt/24450) Liquids/solids with significant Vpare challenging Gas and aerosol fractions may both be present Impacts deposition, local dose, absorption, biologic response and analytical methods May be absorbed by both the URT/LRTJAH 7/26/20127/12/20123 Aerosols7 Suspensions of particles in air Quasi stable exposure atmospheres Size, composition, concentration vary with time Stable (hrs days) or unstable (sec min) Liquid Aerosols Suspension of liquid droplets Atomization of liquid test material or condensation of vapors Droplet size can change due to evaporation/condensation Solid Aerosols Suspension of solid particles Can be formed from mechanical processes Crushing, grinding, milling (> m) Can be formed from molten test material Can be formed from a liquid aerosol that is driedJAH 7/26/2012 Fiber Aerosols8 Special type of solid aerosol with potential for significant human health impact Naturally occurring or manmade fibers Association with development of pulmonary fibrosis and carcinogenesis in humans Fibers are particles with a length > 5 m and an aspect ratio (length/width)

3 3 Major differences in the nature and persistence of lung injury from fiber exposure Dependent on composition, durability, size, reactivity and exposure concentrationJAH 7/26/2012 Ultrafine and Nanoscale Aerosols9 Ultrafine particles (UPFs) Particles 1 m aerodynamic diameter May induce more severe pulmonary responses compared to larger particles of the composition Association with increased morbidity/mortality Engineered nanoparticles (EN) Particles 100 nm (at least one dimension) Relative paucity of data Some types ( SWNT) more toxic to lung than bulk material Is nanoness a risk factor? Are there toxicologically unique properties of EN? Is the current testing paradigm sufficient?JAH 7/26/20127/12/20124 Ultrafine and Nanoscale Aerosols10 Nanoscalematerials require unique study design Normal sized test material of same composition will be included Include points of common mass and surface area Detailed characterization of bulk test material Particle size distribution Dynamic light scattering (DLS) 3 nm to m Specific surface area BET (Brunauer Emmett Teller)

4 Surface charge Zeta potential Physical density Gas displacement Morphology/Primary Particle Size Range TEM, SEM, AFMJAH 7/26/2012 Aerosol Properties11 Particle size Physical properties Diameter, length, light scattering, surface area, volume, number, electrical charge Aerodynamic properties Size, shape, density Aerodynamic diameter Determines deposition pattern Impacts target cell population May influence uptake and clearanceJAH 7/26/2012 Aerosol Properties12 Mechanism of particle deposition is dependent on aerodynamic sizeJAH 7/26/20127/12/20125 CMD = 130 nmParticleNumberMMAD = 955 nmParticleMassAerosol Properties13 Aerodynamic size distribution Based on mass, number, or surface area What metric is most important for your material?JAH 7/26/2012 Aerosol Particle Deposition14 JAH 7/26/2012 Aerosol Dosimetry Concepts Inhalable Fraction The fraction of total airborne particles that enter the body through the nose and/or mouth during breathing (dae 100 m) Relevant to health effects anywhere in the respiratory tract and systemic effects Thoracic Fraction Subfractionof inhalable particles that can penetrate into the tracheo alveolar region (dae< 30 m) Important for asthma, bronchitis, and lung cancer Respirable Fraction Subfractionof inhalable particles that penetrate into the alveolar region (dae 10 m) Chronic respiratory diseases.

5 Pneumoconiosis, emphysema15 JAH 7/26/20127/12/20126 Particle Dosimetry Models16 Human and rat airway particle dosimetry CIIT/HamnerInstitute BahmanAsgharian Owen Price Fred Miller Dutch Nat. Inst. Pub. Health FlemmingCassee Renatade Winter 7/26/2012 Nasal Airflow, Deposition & Absorption17 Courtesy of Dr. Julia Kimbell, CIIT Centers for Health Research as presented by Dr. Jack Harkema, Michigan State UniversityJAH 7/26/2012 Exposure Systems Test Atmosphere Generation and Analysis Testing Guidelines187/12/20127 Exposure Systems19 Air SupplySystemExhaustSystemExhaustSystemDi lution and Delivery SystemDilution and Delivery SystemGenerationSystemGenerationSystemSa mpling and Analytical SystemSampling and Analytical SystemExposureChamber Stableand Redundant Removetest material Filter Adsorb/convert Oxidize Mass flow controllers Elutriation/mixing/aging Diluters CyclonesJAH 7/26/2012 HEPA filtered 30 70% RH 20 24 C 10 15 changes/hourExposure Systems20 Whole body Rochester, Hinners, Hazelton Top down laminar flow Side side laminar flow Top down turbulent flow Various sizes Single animal Walk in TERC.

6 125 L, 2 m3, 4 m3, 14 m3 Gas or vapor studies Confounding effect of fur coat grooming with aerosols or sticky vaporsJAH 7/26/2012 Exposure Systems21 Nose Only Best for aerosol studies Enhanced delivery of test aerosol Reduces body contamination Use less test material Used for acute, subacute and subchronic ADME studies: permit serial sampling of blood Potential for increased stress to animals Must train animals to accept tubes Must control temperature Two main types Flow past Good for high concentrations Simplified operation Directed flow Superior aerosol delivery No re breathing of test material or exhaled metabolitesJAH 7/26/20127/12/20128 Exposure Systems: Nose Only22 JAH 7/26/2012 Flow PastDirected FlowTe s t Atmosphere Generation23 GasesJAH 7/26/2012 Vapors Liquid or solid samples Vaporize in air or N2 Heat as necessary Potential explosive hazard Pure or diluted gas in tanks Dilute and deliver to chamber Use mass flow controllers or other flow meters (rotometer) Potential explosive hazardTe s t Atmosphere Generation24 Liquid Aerosols Aerosol generators Jet nebulizer Ultrasonic Atomizers Is your aerosol representative of the test material?

7 JAH 7/26/20127/12/20129 CycloneTe s t Atmosphere Generation25 Solid Aerosols Delivery of bulk TM Wright Dust Generator Rotating Brush Generator Delivery and size reduction Jet mill Use cyclones to reduce particle size Acute: 1 4 m Repeat: 1 3 mJAH 7/26/2012 WDGRBGJet millTe s t Atmosphere Sampling and Analysis26 Exposure atmosphere must be uniform Always test prior to exposure Variation < 5% within chamber or between ports Rotate animals for repeat exposure studies Always sample within breathing zone Special considerations for aerosols Isokineticsampling to provide representative samples for gravimetric or particle size analyses Gravimetric sampling of complex aerosols may require capture/analysis of solid and vapor/gas phase componentsJAH 7/26/2012Te s t Atmosphere Sampling and Analysis27 Reporting test atmosphere concentration Nominal Concentration Mass of test material used/total airflow Only accurate for gases and some vapors Always reported represents maximum concentration

8 Analytical Concentration Measured concentration of exposure atmosphere Gravimetric Filter and/or sorbent tubes Mass or chemical analyses Analytical IR, GC, MS Total hydrocarbonJAH 7/26/20127/12/201210Te s t Atmosphere Sampling and Analysis28 Particle size determination (liquid/solid aerosols) Cascade impactor Mass based aerodynamic particle size determination 20 m 10 nm Low concentrations a problem Require long and/or high volume sampling May require analytical measurement of test material Quartz crystal micro balance cascade impactor real time Aerodynamic particle counters and sizers Particle scatter, intensity, and time of flight (solid aerosols) Laser diffraction (solid or liquid aerosols) Scanning mobility particle sizers Differential mobility analyzer + condensation particle counter (1 m 5 nm) LM, TEM, SEM, AFMJAH 7/26/2012 Inhalation Te s t i n g Guidelines29 Study DurationEPA (OPPTS)OECDL imit (LC50& C x TEPA: 2 mg/LOECD:5 mg/L Aerosol20 mg/L Vapor20,000 ppm Gas436(ATC)Subacute412 Acute or data : 1 mg/L or data basedOECD: Acute or data basedChronicChronic Fibrous 7/26/2012 Modified Acute Protocol30 Fulfills guideline requirements 4 h exposure.)

9 14 day recovery period LC50determination Provides more exposure response information Interim sacrifice groups Histopathology Upper and lower respiratory tract Other organs/tissues identified during necropsy Bronchoalveolar lavage Total and differential inflammatory cell counts Total protein, LDH, other enzymes or cytokines Cell proliferation BrdU labeling or Ki 67 JAH 7/26/20127/12/201211 Regional Dosimetry Inhaled versus Absorbed Dose In Vitro Systems31 Determinants of Dose32 Reactive, water soluble gases/vapors The degree of penetration into lower airways is concentration dependent. This means, C determines both the dose and the depth of penetration into the lower airways. Aerosols Aerosol depositionis size dependent, the doseis Cxt dependent. Interspecies differences Rodents are obligate nose breathers Oral vsnasal breathing in humans alters deposition Activity related differences in respiration rate and mechanics Surface area differences between species Metabolic pathway differences between speciesBronchial AirwaysAerosolsJAH 7/26/2012 ReactiveGas/Vapor Dose Estimates33 Inhaled dose Concentration x minute ventilation x duration Rat: (mg/L) x ( L/min kg) x min = mg/kg Mouse: (mg/L) x ( L/min kg) x min = mg/kg Human.

10 (mg/L) x ( L/min kg) x min = mg/kg Assumes 100% deposition and absorption Deposited dose Fractional Deposition x Inhaled Dose Better often quite good for particles Absorbed dose Mass transport (flux) x Deposited Dose Even better requires knowledge of regional deposition, mass transport Response modified by local metabolism and or sensitivity of cell populationsJAH 7/26/20127/12/201212 Goal: PBPK model for improved human risk assessment Extensive series of toxicokinetic studies Questions: How much is absorbed? Where is it absorbed? How and where is it metabolized? What is the basis of differential species sensitivity?34 Nasal and Pulmonary Uptake35 Nasal Airw ayLary nxTr a c h e aHeartBronchusAlveolusCentriacinusTermin al BronchioleNasopharynxEnlargement of Pulmonary AcinusNasal Lavage (NLF)Bronchoalveolar lavage (BALF)CannulaTo Vacuum PumpCannula To 0 ppm Air S