Comparison of Histopathological and Cell Viability Results from In Vitro Rat and Human Airway™ Modelsfor Translational Inhalation Toxicity TestingJ. Vinall, H. Simpson, J. Baily and C. Roper Charles River Laboratories Edinburgh Ltd, Tranent, EH33 2NE, UK

1 INTRODUCTIONAcute airway toxicity is an important consideration during the development of pharmaceuticals, chemicals, cosmetics and agrochemicals. Currently validated methods require the use of animals, yet it is widely acknowledged that these methods may not be appropriate for accurate prediction of human risk. To combat this, as part of our integrated toxicology and 3Rs (Reduce, Replace, Refine) program, an evaluation of in vitro 3Dhuman and animal derived inhalation models was performed.

Human EpiAirway™ (MatTek Corp., Ashland, MA, USA) is a commercially available functional model of the human airway. Rat EpiAirway™ (MatTek Corp., Ashland, MA, USA) has recently been generated in a similar manner to the human model using cells from the airway epithelium of Charles River rats. In this study the rat and human models were compared on three independent occasions using 14 test chemicals.

3 RESULTS AND DISCUSSIONLight microscopic examination of untreated in vitro human and rat 3D airway morphologies correlated well with in vivo primate and rat airway mucosae (Figure 3 and 4). Human airway tissues exhibited a relatively uniform epithelium of similar depth (4 to 5 cells deep, pseudostratified), although with lower numbers of ciliated and mucous cells and increased numbers of degenerate cells compared to their in vivo counterparts. Rat EpiAirway™ tissues were similar, although with more variable thickness and a moderate increase in morphologic variability was seen compared to the human.

In both rat and human EpiAirway™ tissues, the spectrum of microscopic changes observed following exposure to known toxicants was similar as with in vivo studies. The key injury related findings(Figure 5) were erosion, epithelial detachment, intercellular separation and an increase in necrotic cells. Key repair related findings noted were loss of ciliated cells, epithelial thinning, re-epithelialisation, cyst formation, increased mitoses, epithelial thinning and focal thickening. Using the key injury related findings, a composite scoring system was constructed to assign a single grade to each sample (results for a sample of the test items presented in Figure 6). Samples were scored in a blinded fashion and both human and rat airways were found to respond to toxicants in a similar manner with cleardose-relationships evident.

4 CONCLUSIONIn conclusion, the panel of 14 chemicals were successfully tested on human and rat EpiAirway™ tissues. The TEER and MTT viability results for both the rat and human EpiAirway™ tissues were highly reproducible, and were in agreement with the results of the histopathological evaluation. EpiAirway™ tissue responses from both species were generally similar, with the notable exception of methyl methacrylate and ethyl alcohol, for which the rat tissues were considerably more sensitive, as demonstrated by all the measures used. This data suggested that acute toxicity information from these in vitro models may be successfully used for translational toxicology, to inform safe use of products, procedures for accidental exposure and dose range selection for in vivo studies.

Overall, the data from the MTT and TEER assays were in good agreement predicting similar IC75 values for each test chemical (Figure 7). There was also good intra-run reproducibility in the predicted IC75 values showing consistency between batches for the two species. For most of the test items, the IC75 values calculated from the human and rat tissues were very similar, although the rat tissues were particularly sensitive to methyl methacrylate and ethyl alcohol. Broadly, the 14 test chemicals were ranked in the same order for each end point and for each species. Furthermore, MTT and TEER data was in agreement with the findings of the histopathological evaluations. UN GSH categorisations for inhalation risk based on in vivo data are available for 12 of the 14 test chemicals (Figure 8). The results of each endpoint measured in vitro here are in general agreement with the UN GHS categorisations, ranking the test chemicals in largely the same order each time.

The authors would like to thank Patrick Hayden (MatTek Corporation, 200 Homer Avenue, Ashland, Massachusetts 01721, USA) and his team for their collaboration, expertise and advice over the course of this project.

This work was generously supported by the Charles River Innovations Fund.

Application of dose (100 µL) (n=3 or 4) 3 h exposure Rinse with PBS

(3 x 500 µL)21 h recovery

TEEREndpoints: MTT

Histology

Figure 1: Capped EpiAirway Tissues

5 ACKNOWLEDGMENTS

TEER was then measured in all tissues. Following TEER, replicates were either fixed in 4% paraformaldehyde and embedded in paraffin then stained with H&E before cross sectioning, or transferred to MTT solution and incubated for 1.5 h. Formazan was extracted from MTT tissues in

Figure 2: Schematic of the Experimental Design

Figure 5: Scoring system used to assign a grade to each tissue

METHODSOn arrival at Charles River, rat (AIR-100-R) and human (AIR-100 DAY20) EpiAirway™ were equilibrated in culture for5 days prior to testing in a humidified incubator set to maintain 37oC, 5% CO2. Media was changed at 2-3 day intervals. On the testing day, mucous was removed by rinsing with phosphate buffered saline (PBS), and tissues were transferred to fresh media.14 test items were formulated in corn oil or ultrapure water at 4 concentrations each and tested in parallel to appropriate vehicle and positive (formaldehyde, 14.7 mg/mL) controls. Undosed ALI (Air Liquid Interface) controls were treated exactly the same as test item treated tissues with the exception that they were not dosed.

During the exposure period, Millicell caps were placed on top of each tissue to prevent evaporation of volatile test items (Figure 1).See Figure 2 for a schematic of the full experimental design. All incubations were conducted in a humidified incubator set tomaintain 37oC, 5% CO2 with the exception of the formazan extraction which was conducted at room temperature. After a 3 h exposure, the tissues were rinsed with PBS to remove the test items and transferred to fresh pre-warmed media for 21 h recovery incubation. Following recovery, the rat tissues (only) were rinsed with PBS to remove mucus.

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extractant solution for 2 h on a shaking platform. The extract samples were analysed in a plate reader at 570 nm with correction at 650 nm. All test items were tested on at least 3 independent occasions. From the MTT and TEER data, an IC75 value (the concentration required to reduce the viability to 75% of the appropriate vehicle control) was calculated.

Figure 3: In Vitro Human EpiAirway (Above) and Primate In Vivo Airway (Below) Figure 4: In Vitro Rat EpiAirway (Above) and Rat In Vivo Airway (Below)

Figure 7: Summary of IC75 concentrations (mean ± SD) for rat and human EpiAirway (above)

Figure 8: UN GHS categorisations for in vivo inhalation risk (above) ND = No Data

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Figure 5: Composite scoring system of epithelial injury (above)

Figure 6: Results of histopathological scoring (below)

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Acrolein 1 Morpholine 3 Methyl Methacrylate 5

Formaldehyde 3 Vinyl Acetate 4 Dimethyl acetamide 4

NaOH ND Ethyl Formate 4 Dimethylformamide 4Butyl Amine 3 2-Ethoxyethyl Acetate 4 Ethyl Alcohol 5

Oxalic Acid ND Methyl Methacrylate 5 p-Dichlorobenzene 5