The use of human induced pluripotent stem cells to screen for developmental toxicity potential indicates reduced potential for non-combusted products, when compared to cigarettes

Abstract

devTOX quickPredict (devTOX ^qP ) is a metabolomics biomarker-based assay that utilises human induced pluripotent stem (iPS) cells to screen for potential early stage embryonic developmental toxicity in vitro. Developmental toxicity potential is assessed based on the assay endpoint of the alteration in the ratio of key unrelated biomarkers, ornithine and cystine (o/c). This work aimed to compare the developmental toxicity potential of tobacco-containing and tobacco-free non-combustible nicotine products to cigarette smoke. Smoke and aerosol from test articles were produced using a Vitrocell VC10 smoke/aerosol exposure system and bubbled into phosphate buffered saline (bPBS). iPS cells were exposed to concentrations of up to 10% bPBS. Assay sensitivity was assessed through a spiking study with a known developmental toxicant, all-trans-retinoic acid (ATRA), in combination with cigarette smoke extract. The bPBS extracts of reference cigarettes (1R6F and 3R4F) and a heated tobacco product (HTP) were predicted to have the potential to induce developmental toxicity, in this screening assay. The bPBS concentration at which these extracts exceeded the developmental toxicity threshold was 0.6% (1R6F), 1.3% (3R4F), and 4.3% (HTP) added to the cell media. Effects from cigarette smoke and HTP aerosol were driven largely by cytotoxicity, with the cell viability and o/c ratio dose-response curves crossing the developmental toxicity thresholds at very similar concentrations of added bPBS. The hybrid product and all the electronic cigarette (e-cigarette) aerosols were not predicted to be potential early developmental toxicants, under the conditions of this screening assay.

Keywords: ATRA, All-trans-retinoic acid; CDC, Centers for Disease Control and Prevention; COT, United Kingdom Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment; CV, coefficient of variation; Cigarettes; DART, developmental and reproductive toxicity; DNPH, 2,4-dinitrophenylhydrazine; Developmental toxicity; E-cigarettes; ECVAM, European Center for the Validation of Alternative Methods; EPA, United States Environmental Protection Agency; EVP, electronic vapour product; FDR, false discovery rate; HPHCs, Harmful and Potentially Harmful Constituents; HPLC-DAD, high-performance liquid chromatography with a diode-array detector; HTP, heated tobacco product; HYB, hybrid product; Human induced pluripotent stem cells; ISO, International Organization for Standardisation; ISTD, internal standard; In vitro reproduction assay; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LOQ, limit of quantification; ND, No effect was detected within the exposure range tested; NHS, United Kingdom National Health Service; NICE, National Institute for Health and Care Excellence; Nicotine; ODC, ornithine decarboxylase; OECD, Organisation for Economic Co-operation and Development; PBS, phosphate buffered saline; PG/VG, propylene glycol/vegetable glycerine; POD, point of difference; Q-TOF, Quadrupole Time-of-Flight; ROS, reactive oxygen species; TP, cell viability toxicity potential concentration; TT21C, toxicity testing in the 21st century; UPLC-HRMS, ultra-high performance liquid chromatography coupled high resolution mass spectrometry; bPBS, bubbled phosphate buffered saline; dTP, developmental toxicity potential concentration; dTT, developmental toxicity threshold; devTOXqP, devTOX quickPredict; e-cigarettes, electronic cigarettes; iPS cells, induced pluripotent stem cells; nAChRs, nicotinic acetylcholine receptors; o/c, ornithine/cystine ratio.