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

Dziwenka, Margitta; Coppock, Robert; Alexander, McCorkle; Palumbo, Eddie; Ramirez, Carlos; Lermer, Stephen

The mutagenicity potential of the test article as well as undiluted extracts were evaluated in the Bacterial Reverse Mutation Assay in accordance with FDA GLP (21 CFR Part 58, 1987) and US FDA Redbook 2000 (IV. C.1.a, 2007) and ICH guidelines [14, 24, 25] . Four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) were used. The studies were conducted in the presence and absence of a metabolic activation system from male Sprague-Dawley rats which had been induced with phenobarbital and benzoflavone (Moltox Inc, USA). The overlay agar and minimal glucose agar plates were purchased (Moltox Inc, USA). The fresh bacterial suspension cultures in the nutrient broth were prepared so that they were in the late exponential phase of growth when used. The test article in olive oil was formulated as a solution in dimethyl sulfoxide (DMSO) to provide the required dose levels of up to 76,335 μg/plate to account for the 6.55% of active ingredient (6.27% CBD). For the undiluted extract prepared by isopropanol or supercritical CO 2 extraction, the extract was formulated as a solution in DMSO to provide the required dose levels up to 5000 μg/plate. Positive controls were used, both in the presence and absence of a metabolic activation system. The positive control substances included were sodium azide, ICR 191, daunomycin and methyl methanesulfonate for S. typhimurium strains TA100 and TA1535, TA1537, TA98 and E. coli WP2 uvrA, respectively in the absence of metabolic activation and 2-aminoanthracene for all strains in the presence of metabolic activation. The initial test for all test articles utilized the plate incorporation method in which the following materials were mixed and poured onto the minimal agar plate; 100 μL of the prepared test substance solutions/negative control/positive control substance, 500 μL of S9 mix or substation buffer, 100 μL bacterial suspension or 2000 μL overlay agar (at 45°C). The plates were then incubated at 37°C until the growth was adequate for enumeration. A confirmatory test for all test articles was conducted utilizing the pre-incubation method. The test or control substances, bacterial suspensions and the S9 mix or substitution buffer were incubated under agitation for approximately 30 minutes at 37°C prior to mixing with the overlay agar and pouring onto the minimal agar plates and proceeding as for the initial test. The strains used and dose levels were the same as that in the initial test for all test articles. The plates for both tests were prepared in triplicate for each experimental point. The final doses utilized for the extract diluted in olive oil were 0. 24 Values are mean ± standard deviation. lawn; the mean revertant colony counts for each strain treated with vehicle was close to or within the expected laboratory historical control range or published values; and the positive controls should produce substantial increases in revertant colony numbers with the appropriate bacterial strain. The plates were also evaluated for cytotoxicity which is indicated by the partial or complete absence of a background lawn on non-revertant bacteria or a substantial dose-related reduction in revertant bacteria.