Safety Assessment of a Hemp Extract using Genotoxicity and Oral Repeat-Dose Toxicity Studies in Sprague-Dawley Rats

Abstract

Keywords:Cannabis sativa cannabinoids cannabidiol CBD toxicology olive oil hemp extract CYP liver mutagenicity CW hemp Charlotte's Web, Inc. A B S T R A C TCannabinoids are extracted from Cannabis sativa L. and are used for a variety of medicinal purposes. Recently, there has been a focus on the cannabinoid Cannabidiol (CBD) and its potential benefits. This study investigated the safety of a proprietary extract of C. sativa, consisting of 9% hemp extract (of which 6.27% is CBD) and 91% olive oil. The mutagenic potential of the hemp extract was evaluated with the AMES assay inclusive of a hepatic drug metabolizing mix (S9) rich in CYP enzymes. The test article did not elicit evidence of bacterial mutagenicity. GLP compliant 14-day and a 90-day toxicity study were conducted. Olive oil was used as a control. The 90-day study had a 28-day recovery period. Treatments for the 14-day non-recovery range-finding study were 0, 1000, 2000 and 4000 mg test article/kg body weight (bw)/day for 14 days. There was a non-statistically significant (p > 0.05) decrease in body weights for the male and female rats receiving the test article. Hypoactivity, hyperactivity, reduced food consumption and piloerection were observed in the rats receiving 4000 mg test article/kg bw. Histopathology showed an increase in the size of liver cells (hypertrophy) around the central vein (centrilobular) in Groups 3 (3/10) and 4 (5/10) that correlated with increased liver weights. In the 90-day study, 8 groups of rats were dosed with 0, 200, 400 and 800 mg test article/kg bw/day. Groups 5 to 8 had a 28-day recovery. There were no test article-linked changes in clinical observations, physical examinations, Functional Observation Battery, ophthalmology, Motor Activity Assessment, hematology, clinical chemistries and macropathology (all groups). With the exception of the liver and adrenal gland, no test article-linked pathology was observed. For all rats receiving the test article, histopathology showed hypertrophy of liver cells around the central vein. The increase of liver weight is most likely caused by hypertrophy due to up-regulation of the hepatic drug metabolizing enzymes. The hepatocellular hypertrophy was completely reversed in 28 days and was not considered to be an adverse effect. Vacuolization of the adrenal zona fasciculata was observed in the control and 800 mg test article/kg bw groups. The vacuolization of the zona fasciculata was of the same incidence and severity in treatment and control male rats and correlated with an increased in the weights of the adrenal glands. In addition, a statistically significant increase (p < 0.05) in adrenal-to-body weight ratios was observed for females receiving 800 mg test article/kg bw. This increase in adrenal-to-body weight ratio did not correlate with any of the pathology findings. The NOAEL for the test article is 800 mg/kg bw/day for female and 400 mg/kg bw/day for male Sprague Dawley rats.

Toxicological safety of VOHO Hemp Oil; a supercritical fluid extract from the aerial parts of hemp

“…Similar to what is observed with other hemp extracts [5,6], increases in liver weight and fatty changes were observed in high dose animals; however, these changes were not accompanied by increases in ALT, AST, ALP or BIL and did not occur in the recovery group, indicating reversibility. These changes were therefore not considered to be adverse.…”

“…With the increase in consumer consumption of hemp extracts there has been an increased interest in and demand for determining the safety of these extracts. There are recently published studies in both humans and laboratory animals evaluating the safety of orally consumed hemp extracts, however with the variation in the composition of these extracts, comparison of the information must be conducted in tandem with a detailed evaluation of the extract composition [5][6][7]. The bioactivity or potential for hemp extracts to cause toxicity may be influenced by method of manufacture or slight differences in the chemical profile.…”

Evaluation of extracts of wild Cannabis sativa L. for genotoxicity and phytochemical composition

“…The toxicity of the extracts observed in the present study was therefore attributed to the presence of cytoxic secondary plant metabolites in the solvent extracts. In the Ames assay with extracts of C. sativa diluted with olive oil as well as the extracts produced with an isopropanol and supercritical CO 2 extraction method, toxicity was evident for strains TA 98, TA 1535, TA 1537 and E. coli WP2 uvrA at≥50 μg/plate, with and without S9, in the plate incorporation and/or pre-incubation tests (Dziwenka et al 2020). These results are similar to results of other researches that demonstrated cytotoxicity of plant extracts including betel and tobacco leaf extracts and some Nigerian folk medicines to root-tip cells of A. cepa (Sopova et al 1983;Abraham and Cherian 1978).…”

“…However a supercritical CO 2 extract of the aerial parts of the C. sativa, was not mutagenic in the Ames bacterial reverse mutation test, in vitro mammalian chromosomal aberration test, or in an in vivo mouse micronucleus study (Marx et al 2018). In another assessment of extracts of hemp (C. sativa) using the Ames reverse mutation assay, the extracts produced with an isopropanol and supercritical CO 2 extraction methods were diluted with olive oil and the undiluted extract formulated as a solution in DMSO; no mutagenic effect was observed in the four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of E. coli (WP2 uvrA) that were used (Dziwenka et al 2020). In the present study, methanol and ethyl acetate extracts of the areal parts of C. sativa dissolved in 2.5% acetone as solvent, induced genotoxiciy in the A. cepa root meristem cells.…”

Genotoxicity and repeated-dose toxicity evaluation of dried Wolffia globosa Mankai

“…The polyphenolic extract of clove buds was reported to be non-mutagenic and it exhibited antimutagenic potential against known mutagens such as tobacco and sodium azide [ 17 ]. Hemp extract was shown to be non-mutagenic in an Ames test conducted in accordance with U.S. FDA Redbook and ICH guidelines [ 18 ]. The seed oil of Helianthus annuus Linné (sunflower) was reported to be non-mutagenic in an Ames test performed according to the OECD guideline for testing of chemicals [ 19 ].…”