Sixty male rats weighing 224-286 g and 60 female rats weighing 170-218 g were distributed to treatment groups stratified by body weight among the dose and control groups . For both the 14-day and the 90-day studies, body weights were recorded twice during the acclimation period and weekly for the duration of the study. Feed intake was determined at the same day body weights were determined. Filtered potable water and feed were provided ad libitum. Feed and water were assayed for detrimental substances and none were found at levels that would alter study results. In the 90-day study, sentinel rats were kept in the animal rooms. Serology done on samples collected at the end of the study from the sentinel rats were negative for Rat Parvovirus, Toolan’s Virus , Kilham Rat Virus, Rat Minute Virus, Parvovirus NS-1, Rat Coronavirus, Rat Theilovirus, and Pneumocystis carinii. The animals in the 14-day and the 90-day study were observed daily for clinical evidence of ill health and given physical exams weekly corresponding to body weight determinations. The physical exam included observing for changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity . The exam also included changes in gait, posture, and response to handling, as well as the presence of clonic or tonic movements, stereotypies , or bizarre behavior . All abnormal observations were recorded. Rats in the 90-day study received a Functional Observation Battery in an open field for excitability, autonomic function, gait and sensorimotor coordination , reactivity and sensitivity and other abnormal clinical signs including, but not limited to convulsions, tremors, unusual or bizarre behavior, emaciation, dehydration and general appearance. Additionally, during week 12 rats in the 90-day study underwent a Motor Activity Assessment using a Photobeam Activity System [San Diego Instruments, Inc ] following recommended procedures. Investigators doing the physical examinations, Functional Observation Battery, and Motor Activity Assessment were blind to the treatments the rats were receiving. The clinical chemistry parameters for the 14-day and 90-studies are given in Table 5.
For the 14-day study, blood,vertical grow after overnight fasting, was collected before necropsy from the inferior vena cava while the rats were anesthetized with isoflurane. For the 90-day study, blood was collected from all groups for hematology and clinical chemistry on study day 94 for males and study day 95 for females in Groups 1 to 4 and on study day 124 for Groups 5 to 8 . Blood samples for hematology and clinical chemistry were collected by sublingual bleeding after the rats were anesthetized with isoflurane. Approximately 500 μL of blood was collected for hematologic parameters in a pre-calibrated tube containing Potassium EDTA4 anticoagulant and 1000 μL of whole blood was collected in tubes for clinical chemistry parameters . Whole blood samples were kept cold until examined in the laboratory using standard hematology methods. For clinical chemistry, blood was allowed to coagulate, and the samples were centrifuged in a refrigerated centrifuge. The serum supernatant was harvested and placed in cryotubes, and frozen and stored at -80 ◦C until thawed and assayed. Hematology parameters were determined on an ADVIA 120 Hematology System and clinical chemistry parameters were determined on a COBAS C311 autoanalyzer . Blood samples used to determine the prothrombin time and activated partial thromboplastin time were collected immediately before terminal sacrifice by venipuncture of the inferior vena cava during anesthesia with isoflurane. Approximately 1.8 mL of blood was collected in a pre-calibrated tube containing anticoagulant . These samples were centrifuged in a refrigerated centrifuge and the plasma was transferred to labeled tubes. Plasma samples were frozen and stored in a -80 ◦C freezer until thawed and analyzed on a Sysmex CA620 . The day before collection of samples for the clinical chemistry evaluations, the animals were placed in metabolism cages. Food was withheld for at least 15 hours prior to blood collection, and voided urine was collected from each animal. Urine samples were refrigerated until analyzed . Urine volume was measured, the appearance was recorded, chemical parameters were measured by Multistix® 10 SG Reagent Strips and urine sediment was evaluated by light microscopy. A full necropsy was done on each study animal including animals removed from the studies. Included in the necropsy were examination of the external body surface, body orifices, and the thoracic, abdominal and cranial cavities inclusive of contents. All surviving animals were weighed, anesthetized with isoflurane and exsanguinated from the abdominal aorta. All gross lesions were recorded.The eyes, epididymides, optic nerve and testes were fixed in modified Davidson’s fixative and then stored in ethanol.
All other tissues were fixed in 10% neutral buffered formalin. Specified tissues were embedded in wax, thin sections cut and stained with hematoxylin and eosin, and examined by light microscopy for histopathology . For the 14-day study, liver and adrenal glands from all treatment and control animals, and the kidneys from Groups 1 and 4 were examined by histopathology. For the 90-day study, tissues from all animals removed from the study, tissues from Groups 1 and 4 and the livers from Groups 2 and 3 and groups 5 to 8 were examined for histopathologic changes by light microscopy . All gross lesions observed were described, the tissues taken and examined by histopathology. All pathology procedures were under the supervision of aveterinary pathologist5 . Mean and standard deviations were calculated for all quantitative data. For all in-life endpoints that were identified as multiple measurements of continuous data over time , treatment and control groups were compared using a two-way analysis of variance , testing the effects of both time and treatment, with methods accounting for repeated measures in one independent variable. Significant interactions observed between treatment and time, as well as main effects, were further analyzed by a post hoc multiple comparisons test; e.g. Dunnett’s test of the individual treated groups to control. When warranted by sufficient group sizes, all endpoints with single measurements of continuous data within groups were evaluated for homogeneity of variances and normality. Where homogeneous variances and normal distribution was observed, treated and control groups were compared using a one-way ANOVA. When one-way ANOVA was significant, a comparison of the treated groups to control was performed with a multiple comparisons test, e.g., Dunnett’s test . Where variance was considered significantly different, groups were compared using a nonparametric method, e.g., Kruskal-Wallis non-parametric analysis of variance. When non-parametric analysis of variance was significant, a comparison of treated groups to control was performed, e.g., Dunn’s test. Significance was a probability value of p < 0.05. For hematology and clinical chemistry, the data from male and female rats were analyzed separately. Means and standard deviations were calculated for all quantitative clinical pathology parameters using Pristima® version 7 . These data were analyzed in a sequential manner. First, Bartlett’s test for homogeneity and Shapiro-Wilk test for normality was done.
If the Bartlett’s test for homogeneity and Shapiro-Wilk test for normality were not significant, a one-way analysis of variance followed with Dunnett’s test was performed. If the Bartlett’s test for homogeneity and Shapiro-Wilk test for normality were significant then data transformations to achieve normality and variance homogeneity were done. The order of transformations attempted was log, square root, and rankorder. If the log and square root transformations fail, the rank-order was used. When an individual observation was recorded as being less than a certain value, e.g., below the lower limit of quantitation, calculations were performed on one-half of the recorded value. For example, if bilirubin was reported as <0.1 or ≤0.1, then 0.05 was used for all calculations performed with that bilirubin data. When an individual observation was recorded as being greater than a certain value, e.g., above the upper limit of quantitation, then a greater value was used in place of the recorded value. For example, if specific gravity was reported as >1.100 or ≥1.100, then 1.100 was used for all calculation performed using that specific gravity value. For all statistical testing, significance was a probability value of p < 0.05. 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 and US FDA Redbook 2000 and ICH guidelines. Four strains of Salmonella typhimurium and one strain of Escherichia coli 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 . The overlay agar and minimal glucose agar plates were purchased . 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 to provide the required dose levels of up to 76,335 μg/plate to account for the 6.55% of active ingredient . For the undiluted extract prepared by isopropanol or supercritical CO2 extraction, indoor growers 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.
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. For the undiluted isopropanol extract, the final doses utilized for both the initial and confirmatory tests were 1.58, 5.0, 15.8, 50, 158, 500, 1580 and 5000 μg/plate. For the undiluted supercritical CO2 extract, the final doses utilized were 1.58, 5.0, 15.8, 50, 158, 500, 1580 and 5000 μg/plate for the initial test and 0.5, 2.5 and 25 μg/plate for the confirmatory test. Due to toxicity noted for strains TA100 and TA1537 with the supercritical CO2 extract, a supplemental test was conducted to ensure five concentrations could be assessed without toxicity. Both the plate incorporation and pre-incubation methods were used as previously described at final doses of 0.5, 2.5 and 25 μg/plate. Following incubation, the number of colonies per plate was counted manually and/or with the aid of a plate counter. The mean and standard deviation were calculated for each set of triplicate plates. The test was considered valid if the control plates had normal background 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. Recently, there has been an increasing interest regarding the health benefits of CBD and other phytocannabinoids and with this increased interest, more research is also being conducted to assess the safety of these compounds for human consumption. The current studies were performed to better understand the toxicological profile of a CBD rich proprietary hemp extract and to assess the results in tandem with information currently available regarding the toxicity and safety of CBD. Marx et al. reports on a battery of GLP compliant toxicological studies which were conducted on a supercritical CO2 extract of the aerial parts of the C. sativa plant.