Inhalation Toxicity Study
Materials Required
Principle
Inhalation toxicity studies expose rodents to a controlled aerosol, vapor, gas, or smoke atmosphere and assess respiratory and systemic toxicity using exposure-atmosphere characterization, clinical observations, body and organ weights, bronchoalveolar lavage fluid, histopathology, blood chemistry, hematology, and, when included, molecular endpoints such as transcriptomics, proteomics, lipidomics, or tissue burden analysis. The primary biological readouts are airway irritation, pulmonary inflammation, cytotoxicity, altered surfactant or lipid homeostasis, impaired particle clearance, and tissue remodeling, reflected by BALF cell differentials, BALF protein, LDH, phosphatase activities, cytokines, lung weight, microscopic respiratory-tract lesions, and retained lung burden.
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use the test article as an aerosol, vapor, gas, smoke, or particle atmosphere;
• Filtered air is used as the sham or negative-control atmosphere, and vehicle aerosols such as saline or propylene glycol/glycerol mixtures are used when the test article requires a vehicle.
• BALF assays reported in inhalation studies include total and differential cell counting, total protein, LDH, alkaline phosphatase, acid phosphatase, gamma-glutamyltransferase, beta-N-acetylglucosaminidase, phosphatidylcholine, cytokines, and soluble collagen;
• Optional molecular assays include lung transcriptomics, proteomics, lipidomics, comet assay, and micronucleus assay.
• The protocol requires a nose-only or whole-body inhalation exposure system, aerosol-generation and monitoring devices, particle-size characterization when particles or aerosols are tested, BAL collection equipment, clinical pathology instrumentation, histopathology processing equipment, and analytical instruments for lung burden such as ICP-MS when metal nanoparticle retention is measured.
• Filtered air is used as the sham or negative-control atmosphere, and vehicle aerosols such as saline or propylene glycol/glycerol mixtures are used when the test article requires a vehicle.
• BALF assays reported in inhalation studies include total and differential cell counting, total protein, LDH, alkaline phosphatase, acid phosphatase, gamma-glutamyltransferase, beta-N-acetylglucosaminidase, phosphatidylcholine, cytokines, and soluble collagen;
• Optional molecular assays include lung transcriptomics, proteomics, lipidomics, comet assay, and micronucleus assay.
• The protocol requires a nose-only or whole-body inhalation exposure system, aerosol-generation and monitoring devices, particle-size characterization when particles or aerosols are tested, BAL collection equipment, clinical pathology instrumentation, histopathology processing equipment, and analytical instruments for lung burden such as ICP-MS when metal nanoparticle retention is measured.
Experimental Procedure
• Select nose-only or whole-body exposure before the study because both systems can detect respiratory endpoints, but they can differ in exposure atmosphere composition, stress response, and systemic outcomes.
• Prepare randomized rodent groups including filtered-air controls and, when needed, vehicle controls;
• Published repeated-exposure studies commonly used 6 h/day exposures over 14 days, 28 days, or 13 weeks, usually with 5 days/week schedules for subacute or subchronic studies.
• Characterize the exposure atmosphere during the study, including measured concentration and particle-size metrics when aerosols or particles are used, because published nanotube studies reported target and measured concentrations and parameters such as MMAD and GSD as part of dose interpretation.
• Place animals in the inhalation exposure system and expose them to filtered air, vehicle, or test atmosphere at literature-supported schedules such as 6 h/day for single-day, 2-week, 28-day, or 13-week designs, with 5 days/week used in multiple repeated-dose studies.
• Monitor animals during and after exposure for clinical signs, body weight change, food consumption when reported, and terminal organ weights, because these endpoints were used to distinguish respiratory-local effects from systemic toxicity in repeated inhalation studies.
• At necropsy, collect BALF for inflammatory and cytotoxicity endpoints, collect respiratory-tract tissues for histopathology, and collect blood or other organs when systemic toxicity, genotoxicity, or molecular endpoints are part of the study design.
• For particle or nanoparticle studies, measure retained lung burden or lung-associated lymph node burden when feasible, because published studies used these measurements to assess deposition, retention, delayed clearance, translocation, and recovery after exposure.
• Interpret toxicity by comparing exposed groups with filtered-air and vehicle controls across exposure concentration, BALF inflammation or cytotoxicity markers, organ weights, clinical pathology, histopathology, and retained burden;
• Concentration-related increases in neutrophils, BALF protein, LDH, lung weight, granulomatous inflammation, fibrosis, or delayed clearance support an adverse pulmonary response.
• When multiple post-exposure time points are included, evaluate persistence or recovery, because nanotube studies reported delayed clearance and persistent inflammatory or fibrotic changes, whereas some lower-concentration findings recovered after post-exposure observation.
• Prepare randomized rodent groups including filtered-air controls and, when needed, vehicle controls;
• Published repeated-exposure studies commonly used 6 h/day exposures over 14 days, 28 days, or 13 weeks, usually with 5 days/week schedules for subacute or subchronic studies.
• Characterize the exposure atmosphere during the study, including measured concentration and particle-size metrics when aerosols or particles are used, because published nanotube studies reported target and measured concentrations and parameters such as MMAD and GSD as part of dose interpretation.
• Place animals in the inhalation exposure system and expose them to filtered air, vehicle, or test atmosphere at literature-supported schedules such as 6 h/day for single-day, 2-week, 28-day, or 13-week designs, with 5 days/week used in multiple repeated-dose studies.
• Monitor animals during and after exposure for clinical signs, body weight change, food consumption when reported, and terminal organ weights, because these endpoints were used to distinguish respiratory-local effects from systemic toxicity in repeated inhalation studies.
• At necropsy, collect BALF for inflammatory and cytotoxicity endpoints, collect respiratory-tract tissues for histopathology, and collect blood or other organs when systemic toxicity, genotoxicity, or molecular endpoints are part of the study design.
• For particle or nanoparticle studies, measure retained lung burden or lung-associated lymph node burden when feasible, because published studies used these measurements to assess deposition, retention, delayed clearance, translocation, and recovery after exposure.
• Interpret toxicity by comparing exposed groups with filtered-air and vehicle controls across exposure concentration, BALF inflammation or cytotoxicity markers, organ weights, clinical pathology, histopathology, and retained burden;
• Concentration-related increases in neutrophils, BALF protein, LDH, lung weight, granulomatous inflammation, fibrosis, or delayed clearance support an adverse pulmonary response.
• When multiple post-exposure time points are included, evaluate persistence or recovery, because nanotube studies reported delayed clearance and persistent inflammatory or fibrotic changes, whereas some lower-concentration findings recovered after post-exposure observation.
Troubleshooting
High BALF protein or LDH occurs after exposure:
Possible CauseThe exposure atmosphere may be irritating or cytotoxic to the lower respiratory tract.
Solution
Confirm exposure concentration, compare against filtered-air and vehicle controls, and interpret BALF protein, LDH, neutrophils, and histopathology together rather than as isolated endpoints.
Particle toxicity appears stronger than expected at high concentrations:
Possible CausePoorly soluble particles or nanotube agglomerates may produce overload-related delayed lung clearance and persistent inflammation.
Solution
Include lung burden, lung-associated lymph node assessment, post-exposure observation, BALF analysis, and histopathology to distinguish clearance impairment from acute inflammatory response.
Nose-only and whole-body studies produce different systemic outcomes:
Possible CauseExposure-system design can change breathing-zone composition, animal stress, and systemic responses.
Solution
Report the exposure system explicitly and avoid pooling nose-only and whole-body data as directly equivalent without system-specific interpretation.
Referencias:
- [1]. Park JD, Kim JK, Jo MS, Kim YH, Jeon KS, Lee JH, et al. Lobar evenness of deposition/retention in rat lungs of inhaled silver nanoparticles: an approach for reducing animal use while maximizing endpoints. Part Fibre Toxicol. 2019;16(1):2. [Content Brief]
- [2]. Kogel U, Wong ET, Szostak J, Tan W, Lucci F, Leroy P, et al. Impact of whole-body versus nose-only inhalation exposure systems on systemic, respiratory, and cardiovascular endpoints in a 2-month cigarette smoke exposure study in the ApoE-/- mouse model. J Appl Toxicol. 2021;41(10):1598-1619. [Content Brief]
- [3]. Pauluhn J, et al. Two-week inhalation toxicity of polymeric diphenylmethane-4,4'-diisocyanate (PMDI) in rats: analysis of biochemical and morphological markers of early pulmonary response. Inhal Toxicol. 1999;11(12):1143-1163. [Content Brief]
- [4]. Pauluhn J. Subchronic 13-week inhalation exposure of rats to multiwalled carbon nanotubes: toxic effects are determined by density of agglomerate structures, not fibrillar structures. Toxicol Sci. 2010;113(1):226-242. [Content Brief]
- [5]. Ma-Hock L, Treumann S, Strauss V, Brill S, Luizi F, Mertler M, et al. Inhalation toxicity of multiwall carbon nanotubes in rats exposed for 3 months. Toxicol Sci. 2009;112(2):468-481. [Content Brief]
- [6]. Kasai T, Umeda Y, Ohnishi M, Kondo H, Takeuchi T, Aiso S, et al. Thirteen-week study of toxicity of fiber-like multi-walled carbon nanotubes with whole-body inhalation exposure in rats. Nanotoxicology. 2015;9(4):413-422. [Content Brief]
- [7]. Pauluhn J. Short-term inhalation toxicity of polyisocyanate aerosols in rats: comparative assessment of irritant-threshold concentrations by bronchoalveolar lavage. Inhal Toxicol. 2002;14(3):287-301. [Content Brief]
- [8]. Phillips B, Esposito M, Verbeeck J, Boué S, Iskandar A, Vuillaume G, et al. Toxicity of aerosols of nicotine and pyruvic acid (separate and combined) in Sprague-Dawley rats in a 28-day OECD 412 inhalation study and assessment of systems toxicology. Inhal Toxicol. 2015;27(8):405-431. [Content Brief]
- [9]. Pothmann D, Simar S, Schuler D, Dony E, Gaering S, Le Net JL, et al. Lung inflammation and lack of genotoxicity in the comet and micronucleus assays of industrial multiwalled carbon nanotubes Graphistrength C100 after a 90-day nose-only inhalation exposure of rats. Part Fibre Toxicol. 2015;12:21. [Content Brief]
- [10]. Joseph P, Umbright C, Roberts JR, Cumpston JL, Orandle M, McKinney W, et al. Lung toxicity and gene expression changes in response to whole-body inhalation exposure to cellulose nanocrystal in rats. Inhal Toxicol. 2021;33(2):66-80. [Content Brief]
- [11]. Phillips B, Titz B, Kogel U, Sharma D, Leroy P, Xiang Y, et al. Toxicity of the main electronic cigarette components, propylene glycol, glycerin, and nicotine, in Sprague-Dawley rats in a 90-day OECD inhalation study complemented by molecular endpoints. Food Chem Toxicol. 2017;109(Pt 1):315-332. [Content Brief]
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