https://doi.org/10.1007/s11626-020-00517-7

Xuefei Cao¹, Jayme P. Coyle², Rui Xiong¹, Yiying Wang¹, Robert H. Heflich¹, Baiping Ren¹, William M. Gwinn³, Patrick Hayden⁴, Liying Rojanasakul²

¹ Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, 3900 NCTR Rd., AR Jefferson, USA
² Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers forDisease Control and Prevention,Morgantown,WV, USA
³ Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Durham, NC, USA
⁴ BioSurfaces Inc., Ashland, MA, USA

One important element for validating any new assay for making regulatory decisions is determining its performance relative to an accepted standard. Conducting in vivo inhalation toxicity studies using whole-body or nose-only exposure systems is expensive and time-consuming and typically requires a large number of animals. The goal of using alternative methods, like human in vitro ALI airway cultures, ultimately is to replace inhalation toxicity testing in animals with in vitro approaches. Transition from animal- to human-based models is ultimately expected to lead to faster and better predictive toxicity assessments and therapeutic development at lower cost.  This study shows the development and validation of alternative in vitro methods for acute toxicity testing, including acute inhalation toxicity testing.
 

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DOI: https://doi.org/10.1016/j.mrgentox.2020.503230 

Authors
Thorne D¹, Hollings M², Kilford J², Clements J², Payne R², Ballantyne M², Dalrymple, A¹, Dillon, D¹, Meredith, C¹, Gaҫa, M^{1  
 
1} British American Tobacco, R&D, Southampton, Hampshire SO15 8TL, UK. 
² Covance Laboratories Ltd, Otely Road, Harrogate, HG1 PY, UK

Highlights

• An experimental concept of exposing L5178Y cells at the air-agar-interface
• Cells were lightly seeded on an agar surface to achieve exposure
• Flowing air, plating and recovery efficiencies from the agar-surface were investigated
• Positive controls were responsive, suggesting a clear cellular interaction with the agar-matrix 
• Cigarette smoke aerosol showed a dose-response relationship with increasing mutant frequencies

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Robert Leverette¹, Brian Keyser¹, Michael Hollings² and Adam Seymour^2
1RAI Services Company, Winston−Salem, NC 27101 USA
²Covance Laboratories Ltd., Harrogate, North Yorkshire HG3 1PY, UK

• Freshly generated whole smoke / aerosol from three different tobacco product types (3R4F, THP and ENDS) was consistently delivered within the AMES 48 and 6/48 exposure modules.
• Biological endpoints: 3R4F whole smoke induced increased revertant counts (Ames) and cytotoxicity (NRU) in the AMES 48 and 6/48 modules, respectively.
• Revertant counts (Ames) and cytotoxicity (IC50) values were comparable to those from the standard exposure modules when run under similar exposure conditions.
• Overall, the AMES 48 and 6/48 modules are deemed “Fit for Use" for the in vitrO evaluation of different tobacco product types (combustible, THP, ENDS).
 

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Adam Seymour, Michael Hollings, Joanne Larner and Julie Clements;

Covance Laboratories Ltd, Harrogate, UK

QCMs were used to assess the deposition of cigarette aerosol from a Vitrocell® VC10® in Vitrocell® Ames, 6/4, 12/4 and 24/4 exposure modules. The power to resolve difference between 8 and 1 SLPM is statistically significant in all module types assessed. Uniformity within each module was confirmed (P >0.98) in all module types, at all airflows. Repeatability was assessed and found to be consistent (no significant difference) in all module types at airflows of 10, 8 and 4 SLPM, however, at 1 SLPM a significant difference was noted in 9 out of 12 experiments – likely an artifact of high deposition.
These results show that exposures utilizing the Vitrocell® VC10® and associated modules are robust; but indicate the necessity of dosimetry measures to aid in the quantification of delivered dose.
 

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¹Leverette R, ¹Keyser B, ²Seymour A, ²Hollings M and ¹Bombick B
¹Scientific & Regulatory Affairs, RAI Services Company Winston-Salem, NC 27102
²Covance Laboratories Ltd Otley Road, Harrogate, North Yorkshire HG3 1PY, UK

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