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 clinical chemistry parameters for the 14-day and 90-studies are given in Table 5 . For the 14-day study, blood, after overnight fasting, was collected before necropsy (study day 15) 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 (90-day sacrifice) and on study day 124 for Groups 5 to 8 (recovery sacrifice). Blood samples for hematology (except coagulation samples) 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 EDTA 4 anticoagulant and 1000 μL of whole blood was collected in tubes (no anticoagulant) for clinical chemistry parameters (Table 5) . 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 (Siemens, Erlangen, Germany) and clinical chemistry parameters were determined on a COBAS C311 autoanalyzer (Roche, Rotkreuz, Switzerland). 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 (3.2% sodium citrate). 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 (Siemens, Erlangen, Germany). 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 (Table 5 ). Urine volume was measured, the appearance was recorded, chemical parameters were measured by Multistix® 10 SG Reagent Strips (Siemens, Erlangen, Germany) and urine sediment was evaluated by light microscopy. Dose is mg test article/kg body weight/day. Dose is mg test article/kg body weight/day. Histopathology -14-day study. 4 Histopathology -90-day study, Groups 1 and 4. 5 All animals in 90-day study. 6 Gut associated lymphoid tissue. 7 All lesions observed during necropsy.