Evaluation of Preclinical in vitro Cytotoxicity, Genotoxicity, and Cardiac-Toxicity Screenings of Hydrogenated Cannabidiol
Authors: Tesfatsion Tesfay T. , Ramirez Giovanni A. , Docampo-Palacios Maite L. , Collins Arianna C. , Ray Kyle P. , Cruces Westley 
Published: September 2023

Introduction Cannabidiol (CBD) is one of many naturally biosynthesized compounds produced by Cannabis sativa. There is limited information available in the literature on hydrogenated CBD (tetrahydro cannabidiol or H4CBD) ( Adams et al., 1940b ). As hydrogenated derivatives of tetrahydrocannabinol (THC) and CBD become increasingly popular in consumer markets, toxicological assessments are vital in identifying toxic characteristics, if any, caused by hydrogenated cannabinoids. Objectives Assessment of the preclinical toxicology of hydrogenated CBD is provided through the in vitro safety study of racemic H4CBD in hepatocytes, normal human lung fibroblasts (NHLF), and primary human neural progenitor (NPC) cell lines. The importance of these cell lines is related to major organs and is the primary focus in determining any major toxic characteristics when consuming products. The inclusion of the human ether-a-go-go related gene (hERG) patch clamp test, observes any inhibition of sodium and potassium ion channels related to the arrhythmia of the heart. Also, the AMES test was conducted to determine any carcinogenic characteristics that H4CBD might impose. Materials and Methods Plated NHLF, hepatocytes, and NPC were used in a preclinical 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay for cytotoxicity observations with the visible color change of cellular use of formazan, while a plated AMES test was conducted to monitor any visible mutations within Escherichia coli for carcinogenic activity. Plated cloned HEK293 cells were given set voltages to determine ion channel activity to determine if H4CBD causes inhibition within these pathways, which would mimic any arrhythmia potential in cardiomyocytes. Results Screening of the MTT assay had a median calculated 3.25 micromolar concentration where cell viability remained high in NHLF and NPC, with higher concentrations leading to decreased cell viability. A 3.25 micromolar concentration is also the median for hepatocytes, with a discrepancy in some of the data that could be accounted for by miscounting colonies. The hERG patch clamp test provided a zero net inhibition with values adding up to zero, determining that the compound did not inhibit normal processes within the ion channels of the plated HEK293 cells. The analysis of the different cell types revealed varying responses to H4CBD. NHLF exhibited a concentration-dependent reduction in cell viability, with sustained concentrations over 24 h at 6.25 µM resulting in a significant loss of viability. Conversely, hepatocytes showed a trend of decreased viability at longer exposure times and higher concentrations, but severe cytotoxicity was not observed. This suggests that hepatocytes are less susceptible to the cytotoxic effects of H4CBD compared to NHLF. In the hERG assay, H4CBD did not inhibit the action potentials within cardiomyocytes, indicating no inhibition of ion channels involved in cardiac function. This finding is important for assessing the potential cardiovascular effects of H4CBD. The AMES test results were negative, indicating that H4CBD did not demonstrate mutagenic activity in the tested strains. Conclusion This supports the conclusion that H4CBD does not possess carcinogenic potential based on this assay. Both human NPC and NHLF showed a median concentration of 3.25 µM before exhibiting a significant reduction in cell viability. This information is valuable for determining research or consumer usage limits for H4CBD. It is important to note that this study represents a preclinical assessment, and further studies are required. The experimental design followed protocols typically used for preclinical assessments of novel pharmaceuticals. These findings provide insight into the cytotoxic effects of H4CBD and contribute to establishing research and safety parameters as these compounds continue to gain attention.