Validation of physiologically relevant in vitro human inhalation toxicity tests for volatile liquids, mists, and sprays

In vivo animal models are currently accepted by regulatory authorities for acute respiratory toxicity (ART) testing. However, animal tests have been discredited as predictors of human responses on physiological and ethical grounds. The goal of this work was to develop physiologically relevant ART tests utilizing the EpiAirway™ tissue model, to demonstrate correlation  to  OECD  accepted  GHS  categorization,  and  investigate  interlaboratory reproducibility.

Test articles (TA, n=53) were applied to EpiAirway tissues produced at MatTek (USA) and IVLSL (Slovakia) with two ART protocols, the Direct Application Protocol (DAP) for exposure to mists/sprays, and the Vapor Cap Protocol (VCP) for exposure to vapors/volatile liquids. In both protocols, tissues were exposed for 4h to 4 fixed doses (diluted in corn oil or water) to mimic in vivo rat exposure; followed by 20h post incubation. The effects on tissue viability (MTT assay) and barrier properties (Transepithelial Electrical Resistance, TEER) were determined. The effective doses which reduced tissue viability by 25% (ED-25) or by 50% (ED-50) were mathematically interpolated for the DAP and VCP methods, respectively, and correlated to the GHS categories.

In the DAP, TAs were applied to the apical surface. Using the MTT assay, the DAP discriminated between GHS Cat.1&2/3&4/5&NC with a Sensitivity/Specificity/Accuracy (S/S/A) of 63.5/76.1/69.8% (MatTek) and 63.8/76.1/70.0% (IVLSL). Utilizing the changes in TEER, the DAP discriminated between GHS categories with a S/S/A of 65.9/76.7/71.3% (MatTek) and 64.1/76.6/70.3% (IVLSL). The correlation coefficient between the two laboratories was R2=0.91 for MTT and 0.76 for TEER.

In the VCP, TAs were applied to an absorbent material in a special cap that forms a tight seal above the tissue allowing exposure to TA vapor. Using the MTT assay, the VCP discriminated  between  GHS  categories  with  S/S/A of  70.8/83.2/77.0  (MatTek)  and 71.9/83.2/77.5% (IVLSL). Utilizing the changes in TEER, the VCP discriminated between GHS categories with a S/S/A of 64.4/78.5/71.5 (MatTek) and 67.1/80.1/73.6 (IVLSL). The correlation coefficient between the laboratories was R2 =0.96 for MTT and 0.93 for TEER.

Note: Additional data was obtained after the abstract submission.

EpiAirway (AIR-100-Day20), respiratory toxicity, direct application, vapor cap protocol, MTT, TEER, GHS hazard classification, prediction model, interlab validation, Acrolein, Chloroacetaldehyde, Bis (2-chloroethyl) Ether, Crotonaldehyde, Allyl alcohol, Formaldehyde (37%), Ethyl acrylate, Acetaldehyde, Ethylenediamine, Acetic acid, Dichlovos, Diisopropylamine, Cyclohexanone, 2 pentanone, (Methyl propyl ketone), Isoamyl acetate, Cyclohexene, 1-Butanol, 2-heptanone (Methyl amyl ketone), Toluene, Styrene, Xylene, Ethylidene norbornene, 1,3-Dichloropropane, Ethylene glycol, Isobutanol, Methyl Methacrylate, 2-chloroethanol, Methyl isobutyl ketone, 2-Ethoxyethanol, ethylene glycol monoethyl ether, 1,4-Dioxane, Methanol, Diisobutyl Ketone, 1-Hexene, Trichloroethylene, Ethyl acetate, Perchloroethylene, Ethyl formate, 2-Butoxyethanol, ethylene glycole butyl ether, 2-Ethoxyethyl acetate, n-Nonane, Acetonitrile, 2-Butanone, Hexane, Heptane, Acetone, Isopropyl alcohol, Ethanol, Piperonyl butoxide, Dioctyl phthalate, Propylene glycol, Isoflurane, Triethylene glycol, Glycerol

Acrolein, Chloroacetaldehyde, Bis (2-chloroethyl) Ether, Crotonaldehyde, Allyl alcohol, Formaldehyde (37%), Ethyl acrylate, Acetaldehyde, Ethylenediamine, Acetic acid, Dichlovos, Diisopropylamine, Cyclohexanone, 2 pentanone, (Methyl propyl ketone), Isoamyl acetate, Cyclohexene, 1-Butanol, 2-heptanone (Methyl amyl ketone), Toluene, Styrene, Xylene, Ethylidene norbornene, 1,3-Dichloropropane, Ethylene glycol, Isobutanol, Methyl Methacrylate, 2-chloroethanol, Methyl isobutyl ketone, 2-Ethoxyethanol, ethylene glycol monoethyl ether, 1,4-Dioxane, Methanol, Diisobutyl Ketone, 1-Hexene, Trichloroethylene, Ethyl acetate, Perchloroethylene, Ethyl formate, 2-Butoxyethanol, ethylene glycole butyl ether, 2-Ethoxyethyl acetate, n-Nonane, Acetonitrile, 2-Butanone, Hexane, Heptane, Acetone, Isopropyl alcohol, Ethanol, Piperonyl butoxide, Dioctyl phthalate, Propylene glycol, Isoflurane, Triethylene glycol, Glycerol