Principles and Practices in Inhalation Toxicology

1 Principles and Practices in Inhalation Toxicology Jon A. Hotchkiss, Ph.D. Toxicology & Environmental Research and Consulting The Dow Chemical Company, Midland MI Outline 2 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 vs Absorbed Dose In Vitro Systems Why Conduct Inhalation Studies? 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 pathways 3 1

What is the Goal? 4 5 Gases Vapors Aerosols Gases and Vapors 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 10 6 /AtmP (mm Hg) mg/l (vapor or gas) = ppm x (molecular wt/24450) Liquids/solids with significant Vp are 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/LRT 6 2

Aerosols 7 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 (> 0.5 µm) Can be formed from molten test material Can be formed from a liquid aerosol that is dried Fiber Aerosols 8 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 µm and an aspect ratio (length/width) 3 Major differences in the nature and persistence of lung injury from fiber exposure Dependent on composition, durability, size, reactivity and exposure concentration Ultrafine and Nanoscale Aerosols Ultrafine particles (UPFs) Particles 1 µm 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 (e.g. 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? 9 3

Ultrafine and Nanoscale Aerosols Nanoscale materials 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 6.5 µm Specific surface area BET (Brunauer Emmett Teller) Surface charge Zeta potential Physical density Gas displacement Morphology/Primary Particle Size Range TEM, SEM, AFM 10 Aerosol Properties 11 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 clearance Aerosol Properties Mechanism of particle deposition is dependent on aerodynamic size 12 4

Aerosol Properties Aerodynamic size distribution Based on mass, number, or surface area What metric is most important for your material? 13 Particle Mass MMAD = 955 nm Particle Number CMD = 130 nm Aerosol Particle Deposition 14 Aerosol Dosimetry Concepts Inhalable Fraction The fraction of total airborne particles that enter the body through the nose and/or mouth during breathing (d ae 100 µm) Relevant to health effects anywhere in the respiratory tract and systemic effects Thoracic Fraction Subfraction of inhalable particles that can penetrate into the tracheo alveolar region (d ae < 30 µm) Important for asthma, bronchitis, and lung cancer Respirable Fraction Subfraction of inhalable particles that penetrate into the alveolar region (d ae 10 µm) Chronic respiratory diseases: pneumoconiosis, emphysema 15 5

Particle Dosimetry Models http://www.ara.com/products/mppd.htm 16 Human and rat airway particle dosimetry CIIT/Hamner Institute Bahman Asgharian Owen Price Fred Miller Dutch Nat. Inst. Pub. Health Flemming Cassee Renata de Winter Sorkina Nasal Airflow, Deposition & Absorption 17 Courtesy of Dr. Julia Kimbell, CIIT Centers for Health Research as presented by Dr. Jack Harkema, Michigan State University 18 Exposure Systems Test Atmosphere Generation and Analysis Testing Guidelines 6

Exposure Systems HEPA filtered 30 70% RH 20 24 C 10 15 changes/hour Air Supply System Generation System Dilution and Delivery System 19 Mass flow controllers Elutriation/mixing/aging Diluters Cyclones Exposure Chamber Sampling and Analytical System Stable and Redundant Remove test material Filter Adsorb/convert Oxidize Exhaust System Exposure Systems 20 Whole body Rochester, Hinners, Hazelton Top down laminar flow Side side laminar flow Top down turbulent flow Various sizes Single animal Walk in TERC: 125 L, 2 m 3, 4 m 3, 14 m 3 Gas or vapor studies Confounding effect of fur coat grooming with aerosols or sticky vapors Exposure Systems 21 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 metabolites 7

Exposure Systems: Nose Only 22 Flow Past Directed Flow Test Atmosphere Generation Gases Vapors 23 Pure or diluted gas in tanks Dilute and deliver to chamber Use mass flow controllers or other flow meters (rotometer) Potential explosive hazard Liquid or solid samples Vaporize in air or N 2 Heat as necessary Potential explosive hazard Test Atmosphere Generation 24 Liquid Aerosols Aerosol generators Jet nebulizer Ultrasonic Atomizers Is your aerosol representative of the test material? 8

Test Atmosphere Generation 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 µm Repeat: 1 3 µm Cyclone WDG Jet mill RBG 25 Test Atmosphere Sampling and Analysis 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 Isokinetic sampling 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 components 26 Test Atmosphere Sampling and Analysis 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 Analytical Concentration Measured concentration of exposure atmosphere Gravimetric Filter and/or sorbent tubes Mass or chemical analyses Analytical IR, GC, MS Total hydrocarbon 27 9

Test Atmosphere Sampling and Analysis Particle size determination (liquid/solid aerosols) Cascade impactor Mass based aerodynamic particle size determination 20 µm 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 µm 5 nm) LM, TEM, SEM, AFM 28 Inhalation Testing Guidelines Study Duration EPA (OPPTS) Acute 870.1300 OECD 403 (LC 50 & C x T 436 (ATC) Limit Conc. EPA: 2 mg/l OECD: 5 mg/l Aerosol 20 mg/l Vapor 20,000 ppm Gas Subacute 412 Acute or data based 29 Subchronic 870.3465 413 EPA: 1 mg/l or data based OECD: Acute or data based Chronic Chronic Toxicity 870.4100 452 Carcinogenicity 870.4200 451 Combined 870.4300 453 Combined Fibrous particles 870.8355 Modified Acute Protocol Fulfills guideline requirements 4 h exposure; 14 day recovery period LC 50 determination 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 30 10

31 Regional Dosimetry Inhaled versus Absorbed Dose In Vitro Systems Determinants of Dose Reactive Gas/Vapor Aerosols 32 Bronchial Airways 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 deposition is size dependent, the dose is Cxt dependent. Interspecies differences Rodents are obligate nose breathers Oral vs nasal breathing in humans alters deposition Activity related differences in respiration rate and mechanics Surface area differences between species Metabolic pathway differences between species Dose Estimates Inhaled dose Concentration x minute ventilation x duration Rat: (mg/l) x (0.78 L/min kg) x min = mg/kg Mouse: (mg/l) x (1.533 L/min kg) x min = mg/kg Human: (mg/l) x (0.089 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 populations 33 11

34 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? Nasal and Pulmonary Uptake 35 URT Cannula To Vacuum Pump Cannula To 0 ppm Air Source LRT Bronchoalveolar lavage (BALF) Nasal Airway Nasopharynx Larynx Trachea Heart Nasal Lavage Whole Blood (NLF) and Plasma Bronchus 300 ppm Parent (4 h) Parent: blood, NLF, BALF Metabolites: plasma, NLF, BALF Albumin adducts: plasma NLF, BALF Centriacinus Alveolus Terminal Bronchiole Enlargement of Pulmonary Acinus Nasal and Pulmonary Uptake 36 ~12.5% of inhaled Parent was absorbed by rats exposed to 300 ppm URT uptake = 4.9% LRT uptake = 7.6% No Parent LLQ detected in NLF, BALF or blood Equimolar levels of metabolites in NLF, BALF and plasma Parent is rapidly hydrolyzed to metabolites at the portal of entry Not present systemically 12

37 PBPK model based on principle route of exposure oral Inhalation is a secondary exposure route workers and bystanders Previous inhalation studies conducted with vapor Maximum systemic dose = no biologic effect Solid aerosol exposure conducted to increase systemic dose Are kinetics of absorption/metabolism similar for vapor and solid? Does the route of exposure impact exposure response? Absorbed Dose: Dry powder aerosol Single 6 hr nose only exposure 4 exposure concentrations Serial sacrifices Blood, tissue and urine Analyses Parent and metabolites (dose) Blood and urine Enzyme activity (response) Blood and tissue Cumulative Metabolite Recovered in Urine (nmoles) 10000 1000 100 10 0-6 0-12 12-24 24-48 48-72 Exposure Post-Exposure 3.7 mg/m 3 12.9 mg/m 3 22.1 mg/m 3 53.5 mg/m 3 Urine Collection Interval (hours) 38 3.7 mg/m 3 12.9 mg/m 3 22.1 mg/m 3 53.5 mg/m 3 Elimination Half life (hr) Total Urinary Metabolite (mg/kg bw) Absorbed Dose (mg/kg bw) 11.5 ± 1.4 11.6 ± 1.7 10.6 ± 1.8 10.7 ± 1.6 0.22 ± 0.03 1.21 ± 0.11 0.94 ± 0.17 2.41 ± 0.62 0.52 ± 1.4 2.86 ± 0.25 2.21 ± 0.41 5.70 ± 1.48 Investigating Exposure Response 39 Absorption and Clearance Exposure Response Blood Concentration (nmole/g blood) 100 10 1 0.1 0.01 0.001 0.0001 2 2 46 6 12 24 48 72 Exposure Post-Exposure Sample Time (hours) (3.7 mg/m 3 ) (12.9 mg/m 3 ) (22.1 mg/m 3 ) (53.5 mg/m 3) (3.7 mg/m 3 ) (12.9 mg/m 3 ) (22.1 mg/m3) (53.5 mg/m 3 ) (3.7 mg/m 3 ) (12.9 mg/m 3 ) (22.1 mg/m 3 ) (53.5 mg/m 3 ) Peak Blood Concentration (ng/g blood) 100 10 1 Parent Metabolite 0.1 < LLQ 1.04 3.62 6.21 15.02 [3.7] [12.9] [22.1] [53.5] Inhaled Dose (mg Parent inhaled/kg bw) [Exposure [Exposure Concentration Concentration (mg (mg Parent/m CPF/m 3 )] 3 )] 160 120 100% of Control 80 40 0 Percent Control Enzyme Activity (2 hr Post-Exposure) RBC Plasma Lung Brain 13

40 Absorption and metabolism Isolated perfused lung Explants (nasal, bronchioles, tissue slices) High throughput toxicity screens Submerged cultures (A549, BEAS 2B, etc.) Dose response and epithelial transport Isolated nasal mucosa Organotypic cultures Air Liquid Interface (ALI) Models 41 Human tracheal/bronchial and nasal cells Normal, smokers, COPD, asthma Stratified fully differentiated mucociliary phenotype Actively secrete mucins Maintain normal complement of metabolizing enzymes Electrogenic Tight junctions Ion Transport Inhalation toxicology, nasal drug drug delivery, basic research Exposure Response/Risk Assessment 42 In Vivo Response (rat) Human Response In Vitro....... Response(rat) In Vitro....... Response (human) 14

43 Review Classes and Properties of Inhaled Materials Gases, vapors and liquid/solid aerosols Overview of Inhalation Toxicity Testing Exposure systems and generation/analysis methods Concepts of regional dosimetry and systemic dose Robust and predictive risk assessment models Final Thoughts. Every good study starts with a good exposure Healthy, well acclimated animals Well characterized test atmosphere Detailed in life and post mortem analyses Test guidelines are just the beginning Resist box checking advocate for the best science Inhalation studies are $$$ combine studies Generate toxicokinetic data as soon as possible Know the guideline requirements for your market Use these basic tools to help choose a good CRO or review inhalation studies 44 Suggested Reference Sources Inhalation Toxicology (2006). Second edition. H. Salem and S. A. Katz, eds. CRC Taylor & Francis Group, Boca Raton, FL. Concepts in Inhalation Toxicology. (1989). R. O. McClellan and R. F. Henderson, eds. Hemisphere Publishing Corp, Member of the Taylor & Francis Group. New York, NY. Particle Toxicology. (2007). K. Donaldson and P. Borm eds., CRC Press, Taylor & Francis Group. Boca Raton, FL. Hinds, W. C. (1999) Aerosol Technology: Properties, behavior, and measurement of airborne particles. John Wiley & sons, Inc. Aerosol Measurement: Principles, techniques and applications. (1993). K. Willeke and P. A. Baron eds. Van Nostrand Reinhold, New York, NY. Valentine, R. and Kennedy, G. L. (2007). Inhalation Toxicology. In Principles and Methods of Toxicology. A. W. Hayes, ed. Informa Healthcare USA, Inc. New York, NY Wong, Brian A. (2007). Inhalation Exposure Systems: Design, Methods and Operation. Toxicol Pathol 35: 3 13. GUIDELINES EPA (OPPTS) Acute (870.1300): http://www.regulations.gov/#!documentdetail;d=epa HQ OPPT 2009 0156 0054 Subchronic 870.3465): http://www.regulations.gov/#!documentdetail;d=epa HQ OPPT 2009 0156 0014 Chronic Toxicity (870.4100): http://www.regulations.gov/#!documentdetail;d=epa HQ OPP 2002 0302 0129 Carcinogenicity (870.4200): http://www.regulations.gov/#!documentdetail;d=epa HQ OPPT 2009 0156 0020 Combined Chronic Toxicity/Carcinogenicity (870.4300): http://www.regulations.gov/#!documentdetail;d=epa HQ OPPT 2009 0156 0021 Combined Chronic Toxicity/Carcinogenicity Testing of Respirable Fibrous Particles (870.8355): http://www.regulations.gov/#!documentdetail;d=epa HQ OPPT 2009 0156 0050 OECD Acute (403): http://www.oecdbookshop.org/oecd/display.asp?k=5lmqcr2k7pr5&ds=test No. 403 Acute Inhalation Toxicity Acute (436): http://www.oecdbookshop.org/oecd/display.asp?k=5lmqcr2k7pr5&ds=test No. 403 Acute Inhalation Toxicityt no 436 acute inhalationtoxicity acute toxic class method_9789264076037 en Subacute (412): http://www.oecdbookshop.org/oecd/display.asp?lang=en&sf1=di&st1=5lmqcr2k7nxv Subchronic (413): http://www.oecdbookshop.org/oecd/display.asp?k=5lmqcr2k7nvf&ds=test No. 413 Subchronic Inhalation Toxicity 90 day Study Chronic Toxicity (452): http://www.oecdbookshop.org/oecd/display.asp?k=5lmqcr2k7mlw&ds=test No. 452 Chronic Toxicity Studies Carcinogenicity (451): http://www.oecdbookshop.org/oecd/display.asp?k=5lmqcr2k7mq4&ds=test No. 451 Carcinogenicity Studies Combined Chronic Toxicity/Carcinogenicity (453): http://www.oecdbookshop.org/oecd/display.asp?lang=en&sf1=di&st1=5lmqcr2k7mjg Guidance Document on Acute Inhalation Toxicity Testing (GD 39): http://iccvam.niehs.nih.gov/suppdocs/feddocs/oecd/oecd GD39.pdf Guidance Document on the Design and Conduct of Chronic Toxicity and Carcinogenicity Studies, Supporting TG 451, 452, 453: http://www.oecd.org/dataoecd/24/49/46766792.pdf 45 15