An evaluation of the genotoxicity and 90-day repeated-dose toxicity of a CBD-rich hemp oil
Corresponding Author
Amy Clewell
AIBMR Life Sciences, Inc., Seattle, Washington, 98122 USA
Correspondence
Amy Clewell, ND, DABT, AIBMR Life Sciences, Inc., 1425 Broadway, Suite 458, Seattle, WA 98122, USA.
Email: [email protected]
Search for more papers by this authorJohn R. Endres
AIBMR Life Sciences, Inc., Seattle, Washington, 98122 USA
Search for more papers by this authorTimothy S. Murbach
AIBMR Life Sciences, Inc., Seattle, Washington, 98122 USA
Search for more papers by this authorGábor Hirka
Toxi-Coop Zrt., Budapest, Hungary
Toxi-Coop Zrt., Balatonfüred, Hungary
Search for more papers by this authorCorresponding Author
Amy Clewell
AIBMR Life Sciences, Inc., Seattle, Washington, 98122 USA
Correspondence
Amy Clewell, ND, DABT, AIBMR Life Sciences, Inc., 1425 Broadway, Suite 458, Seattle, WA 98122, USA.
Email: [email protected]
Search for more papers by this authorJohn R. Endres
AIBMR Life Sciences, Inc., Seattle, Washington, 98122 USA
Search for more papers by this authorTimothy S. Murbach
AIBMR Life Sciences, Inc., Seattle, Washington, 98122 USA
Search for more papers by this authorGábor Hirka
Toxi-Coop Zrt., Budapest, Hungary
Toxi-Coop Zrt., Balatonfüred, Hungary
Search for more papers by this authorFunding information: The authors disclose that financial support for the research described herein was provided by Kazmira LLC, Watkins, Colorado, 80137, USA.
Adél Vértesi, Erzsébet Béres, Ilona Pasics Szakonyiné are co-senior authors.
Abstract
Currently, there is much interest in the sales and study of consumable Cannabis sativa L. products that contain relatively high levels of cannabidiol (CBD) and low levels of Δ-9-tetrahydrocannabinol. While there are published safety evaluations for extracts containing low concentrations of CBD, toxicological assessments for those with higher concentrations are still scant in the public domain. In this paper, genotoxicity tests and a 90-day repeated-dose toxicity study of an ethanolic extract of C. sativa containing ~85% CBD were performed following relevant OECD guidelines. No increased gene mutations were observed in a bacterial reverse mutation assay compared to controls up to the maximum recommended concentration of the guideline. An in vitro chromosomal aberration assay showed no positive findings in the short-term (3 h) treatment assays. Long-term treatment (20 h) showed an increased number of cells containing aberrations at the highest dose of 2 μg/mL, which was outside of historical control levels, but not statistically significantly different from the controls. An in vivo micronucleus study showed no genotoxic potential of the test item in mice. A 90-day repeated-dose gavage study using 0, 75, 125, and 175 mg/kg bw/day showed several slight findings that were considered likely to be related to an adaptive response to consumption of the extract by the animals but were not considered toxicologically relevant. These included increases in liver and adrenal weights compared to controls. The NOAEL was determined as 175 mg/kg bw/day, the highest dose tested (equivalent to approximately 150 mg/kg bw/day of CBD).
CONFLICTS OF INTEREST STATEMENT
The authors have no conflict of interest to report.
Open Research
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
REFERENCES
- Agriculture improvement act of 2018, H.R. 2, 115th Cong., 2nd Sess., (2018).
- Ames, B. N., McCann, J., & Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research, 31(6), 347–364. https://doi.org/10.1016/0165-1161(75)90046-1
- Aviello, G., Romano, B., Borrelli, F., Capasso, R., Gallo, L., Piscitelli, F., Di Marzo, V., & Izzo, A. A. (2012). Chemopreventive effect of the non-psychotropic phytocannabinoid cannabidiol on experimental colon cancer. Journal of Molecular Medicine (Berlin, Germany), 90(8), 925–934. https://doi.org/10.1007/s00109-011-0856-x
- Baldrick, P., Cosenza, M. E., Alapatt, T., Bolon, B., Rhodes, M., & Waterson, I. (2020). Toxicology paradise: Sorting out adverse and non-adverse findings in animal toxicity studies. International Journal of Toxicology, 39, 1091581820935089. https://doi.org/10.1177/1091581820935089
- Baron, E. P. (2018). Medicinal properties of cannabinoids, terpenes, and flavonoids in cannabis, and benefits in migraine, headache, and pain: An update on current evidence and cannabis science. Headache, 58(7), 1139–1186. https://doi.org/10.1111/head.13345
- Bergamaschi, M. M., Queiroz, R. H., Zuardi, A. W., & Crippa, J. A. (2011). Safety and side effects of cannabidiol, a Cannabis sativa constituent. Current Drug Safety, 6(4), 237–249. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/22129319, https://doi.org/10.2174/157488611798280924
- Dziwenka, M., Coppock, R., Alexander, M., Palumbo, E., Ramirez, C., & Lermer, S. (2020). Safety assessment of a hemp extract using genotoxicity and oral repeat-dose toxicity studies in Sprague-Dawley rats. Toxicology Reports, 7, 376–385. https://doi.org/10.1016/j.toxrep.2020.02.014
- Dziwenka, M., Dolan, L., & Mitchell, J. (2021). Toxicological safety of VOHO hemp oil; a supercritical fluid extract from the aerial parts of hemp. PLoS ONE, 16(12), e0261900. https://doi.org/10.1371/journal.pone.0261900
- ElSohly, M. A., Radwan, M. M., Gul, W., Chandra, S., & Galal, A. (2017). Phytochemistry of Cannabis sativa L. Progress in the Chemistry of Organic Natural Products, 103, 1–36. https://doi.org/10.1007/978-3-319-45541-9_1
- Elsohly, M. A., & Slade, D. (2005). Chemical constituents of marijuana: The complex mixture of natural cannabinoids. Life Sciences, 78(5), 539–548. https://doi.org/10.1016/j.lfs.2005.09.011
- Everds, N. E., Snyder, P. W., Bailey, K. L., Bolon, B., Creasy, D. M., Foley, G. L., Rosol, T. J., & Sellers, T. (2013). Interpreting stress responses during routine toxicity studies: A review of the biology, impact, and assessment. Toxicologic Pathology, 41(4), 560–614. https://doi.org/10.1177/0192623312466452
- FDA. (2023). FDA statement. FDA concludes that existing regulatory frameworks for foods and supplements are not appropriate for cannabidiol, will work with congress on a new way forward [press release]. Retrieved from https://www.fda.gov/news-events/press-announcements/fda-concludes-existing-regulatory-frameworks-foods-and-supplements-are-not-appropriate-cannabidiol
- Foster, B. C., Abramovici, H., & Harris, C. S. (2019). Cannabis and cannabinoids: Kinetics and interactions. American Journal of Medicine, 132(11), 1266–1270. https://doi.org/10.1016/j.amjmed.2019.05.017
- Greenwich Biosciences, & FDA. (2021). Epidiolex: Prescribing information.
- Hamden, D., Klinger, H., Jensen, J., & Kaelbling, M. (1985). ISCN 1985: An international system for human cytogenic nomenclature. Report of the Standing Committee on Human Cytogenic Nomenclature.
- Iffland, K., & Grotenhermen, F. (2017). An update on safety and side effects of cannabidiol: A review of clinical data and relevant animal studies. Cannabis Cannabinoid Research, 2(1), 139–154. https://doi.org/10.1089/can.2016.0034
- Jiang, R., Yamaori, S., Takeda, S., Yamamoto, I., & Watanabe, K. (2011). Identification of cytochrome P450 enzymes responsible for metabolism of cannabidiol by human liver microsomes. Life Sciences, 89(5–6), 165–170. https://doi.org/10.1016/j.lfs.2011.05.018
- Keller, D. A., Juberg, D. R., Catlin, N., Farland, W. H., Hess, F. G., Wolf, D. C., & Doerrer, N. G. (2012). Identification and characterization of adverse effects in 21st century toxicology. Toxicological Sciences, 126(2), 291–297. https://doi.org/10.1093/toxsci/kfr350
- Kier, L. D., Brusick, D. J., Auletta, A. E., Von Halle, E. S., Brown, M. M., Simmon, V. F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T. K., & Ray, V. (1986). The salmonella typhimurium/mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research, 168(2), 69–240. https://doi.org/10.1016/0165-1110(86)90002-3
- Lattanzi, S., Brigo, F., Trinka, E., Zaccara, G., Cagnetti, C., Del Giovane, C., & Silvestrini, M. (2018). Efficacy and safety of cannabidiol in epilepsy: A systematic review and meta-analysis. Drugs, 78(17), 1791–1804. https://doi.org/10.1007/s40265-018-0992-5
- Lucas, C. J., Galettis, P., & Schneider, J. (2018). The pharmacokinetics and the pharmacodynamics of cannabinoids. British Journal of Clinical Pharmacology, 84(11), 2477–2482. https://doi.org/10.1111/bcp.13710
- Maron, D. M., & Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research, 113(3–4), 173–215. https://doi.org/10.1016/0165-1161(83)90010-9
- Marx, T. K., Reddeman, R., Clewell, A. E., Endres, J. R., Beres, E., Vertesi, A., Glávits, R., Hirka, G., & Szakonyine, I. P. (2018). An assessment of the genotoxicity and subchronic toxicity of a supercritical fluid extract of the aerial parts of hemp. Journal of Toxicology, 2018(Jun 7), 8143582–26. https://doi.org/10.1155/2018/8143582
- Millar, S. A., Stone, N. L., Bellman, Z. D., Yates, A. S., England, T. J., & O'Sullivan, S. E. (2019). A systematic review of cannabidiol dosing in clinical populations. British Journal of Clinical Pharmacology, 85(9), 1888–1900. https://doi.org/10.1111/bcp.14038
- Millar, S. A., Stone, N. L., Yates, A. S., & O'Sullivan, S. E. (2018). A systematic review on the pharmacokinetics of cannabidiol in humans. Frontiers in Pharmacology, 9, 1365. https://doi.org/10.3389/fphar.2018.01365
- National Research Council. (2011). Guide for the care and use of laboratory animals. T. N. A. Press.
- Nayak, N. C., Sathar, S. A., Mughal, S., Duttagupta, S., Mathur, M., & Chopra, P. (1996). The nature and significance of liver cell vacuolation following hepatocellular injury—An analysis based on observations on rats rendered tolerant to hepatotoxic damage. Virchows Archiv, 428(6), 353–365. https://doi.org/10.1007/BF00202202
- OECD. (1997). Test no. 471: Bacterial reverse mutation test, OECD guidelines for the testing of chemicals, section 4.
-
OECD. (1998). OECD principles of good laboratory practice. OECD Publishing. https://doi.org/10.1787/9789264078536-en
10.1787/9789264078536-en Google Scholar
- OECD. (2008). Test no. 407: Repeated dose 28-day oral toxicity study in rodents, OECD guidelines for the testing of chemicals, section 4. OECD Publishing.
- OECD. (2016a). Test no. 473: In vitro mammalian chromosomal aberration test, OECD guidelines for the testing of chemicals. OECD Publishing.
- OECD. (2016b). Test no. 474: Mammalian erythrocyte micronucleus test, OECD guidelines for the testing of chemicals. OECD Publishing.
- OECD. (2018). Test no. 408: Repeated dose 90-day oral toxicity study in rodents, OECD guidelines for the testing of chemicals, section 4. OECD Publishing.
- OECD. (2020). Test no. 471: Bacterial reverse mutation test, OECD guidelines for the testing of chemicals, section 4. OECD Publishing.
- Pandiri, A., Kerlin, R., Mann, P., Everds, N., Sharma, A., Payton Meyers, L., & Steinbach, T. (2017). Is it adverse, nonadverse, adaptive, or artifact? Toxicologic Pathology, 45(1), 238–247. https://doi.org/10.1177/0192623316672352
- Perucca, E., & Bialer, M. (2020). Critical aspects affecting cannabidiol oral bioavailability and metabolic elimination, and related clinical implications. CNS Drugs, 34(8), 795–800. https://doi.org/10.1007/s40263-020-00741-5
- Radwan, M. M., Elsohly, M. A., Slade, D., Ahmed, S. A., Wilson, L., El-Alfy, A. T., Khan, I. A., & Ross, S. A. (2008). Non-cannabinoid constituents from a high potency Cannabis sativa variety. Phytochemistry, 69(14), 2627–2633. https://doi.org/10.1016/j.phytochem.2008.07.010
- Ramaiah, L., Hinrichs, M., Skuba, E., Iverson, W., & Ennulat, D. (2017). Interpreting and integrating clinical and anatomic pathology results: Pulling it all together. Toxicologic Pathology, 45(1), 223–237. https://doi.org/10.1177/0192623316677068
- Saldanha, L. G., Dwyer, J. T., & Bailen, R. A. (2021). Modernization of the National Institutes of Health dietary supplement label database. Journal of Food Composition and Analysis, 102, 104058. https://doi.org/10.1016/j.jfca.2021.104058
- Savage, J. R. (1976). Classification and relationships of induced chromosomal structural changes. Journal of Medical Genetics, 13(2), 103–122. https://doi.org/10.1136/jmg.13.2.103
- Savage, J. (1983). Some practical notes on chromosomal aberrations. Clinical Cytogen Bulletin, 1, 64–76.
- Schmitz, S. M., Lopez, H. L., & Marinotti, O. (2020). Post marketing safety of plus CBD products, a full spectrum hemp extract: A 2-year experience. Journal of Dietary Supplements, 17(5), 587–598. https://doi.org/10.1080/19390211.2020.1767255
- Smith, T., Gillespie, M., Eckl, V., Knepper, J., & Morton Reynolds, C. (2019). Herbal supplement sales in US increase by 9.4% in 2018. HerbalGram, 123, 62–73.
- Smith, T., May, G., Eckl, V., & Reynolds, C. (2020). US sales of herbal supplements increase by 8.6% in 2019. HerbalGram, 127, 54–69.
- A. Suttie, J. Leininger, & A. Bradley (Eds.). (2018). Boorman's pathology of the rat: Reference and atlas ( Second ed.). Academic Press-Elsevier.
- van Berlo, D., Woutersen, M., Muller, A., Pronk, M., Vriend, J., & Hakkert, B. (2022). 10% body weight (gain) change as criterion for the maximum tolerated dose: A critical analysis. Regulatory Toxicology and Pharmacology, 134, 105235. https://doi.org/10.1016/j.yrtph.2022.105235
- Venitt, S., & Parry, J. (1984). Mutagenicity testing, a practical approach. IRL Press Limited.
- WHO, & Expert Committee on Drug Dependence. (2017). Cannabidiol (CBD). Pre-Review Report.
