Genetic and Rat Toxicity Studies of Cyclodextrin Glucanotransferase

[a]pyrene, 2 μg (TA100) and 2-aminoanthracene, 2 μg (TA98); 2.5 μg (TA97a, TA1535); 20 μg (E. coli WP2) were used as the positive controls with metabolic activation. Following a preliminary incubation at 37 °C for 20 minutes prior to plating, three test plates per concentration were incubated at 37 °C for 48 hours and then counted using the Sorcerer/Ames Study Manager System (Perceptive Instruments, Ltd., Suffolk, UK). To ensure accuracy of the results, reagent sterility and automated scoring checks were conducted. Criteria for a positive response were a ≥ 2-fold increase in the average plate count compared to the solvent control for at least one concentration level and a dose response over the range of tested concentrations in at least one strain with or without metabolic activation. In addition, the average response should fall outside the laboratory historical vehicle control range for the strain/metabolic activation condition.

2.3. In vitro micronucleus (MN) assay

The GLP in vitro MN assay was conducted in compliance with OECD TG 487 (OECD 2014a). Human TK6 cells (ATCC, Manassas, VA) were cultured and maintained in RPMI 1640 medium containing 10% heat inactivated horse serum plus 1.0% Pluronic F-68, 0.5% sodium pyruvate, and antibiotics (penicillin at 20 Units/mL and streptomycin at 20 μg/mL) at 37 °C, with 6% CO₂. The normal cell cycle time of these cells is approximately 12 hours. Metabolic activation was provided using phenobarbital/benzoflavone-induced rat liver S9 (Moltox, Boone, NC) with added Regensys™ cofactors (Moltox, Boone, NC) at a final concentration of 1% S9. The composition of the S9 mix was: 10% S9, 8 mM MgCl₂, 32.6 mM KCl, 4.7 mM glucose-6-phosphate, 4 mM NADP, and 0.1 M phosphate buffer. Triplicate cultures of exponentially growing cells seeded at 0.40 × 10⁶ cells/mL in 12-well plates were exposed to CGTase, sodium sulfate, or controls for approximately four hours in the presence of metabolic activation (+S9) and approximately 24–29 hours in the absence of metabolic activation (-S9). Cyclophosphamide (Sigma-Aldrich, St. Louis, MO) and vinblastine (Sigma-Aldrich, St. Louis, MO) were used as the positive controls with and without metabolic activation, respectively. On the basis of preliminary tests, the concentrations of CGTase selected for testing were 6000, 5000, 4000, 3000, 2000, and 1000 μg/mL of CGTase for approximately 4 hours with S9 and 24 hours without S9. Concentrations of sodium sulfate selected were 5000, 1667, 556, 185, 61.7, and 20.6 μg/mL for 4 hours with S9 and 24 hours without S9. At the end of the culture period, cells were analyzed for cytotoxicity and micronucleus induction by flow cytometry using the In Vitro MicroFlow™ kit (Litron Laboratories, Rochester, NY) according to manufacturer’s instructions. Unless limited by cytotoxicity, 20,000 cells from each sample were analyzed for the frequency of micronuclei (MN) using a FACSCalibur™ dual-laser bench top system (Becton Dickinson Biosciences, San Jose, CA).

Cytotoxicity was measured as relative increase in cell count or as relative survival of cells in vehicle control cultures compared to cells in treated cultures using ratios of counted nuclei to counted beads (inert latex microspheres added to each sample). Higher nuclei to bead ratios correspond to greater cell survival. Greater emphasis was placed on relative survival since this flow cytometry-based measurement may provide a more sensitive assessment of cytotoxicity, enhancing assay specificity (Avlasevich et al., 2011). As recommended by OECD test guideline #487 (OECD, 2014a), concentrations inducing greater than 60% cytotoxicity were excluded from the analysis. An increase in MN frequency ≥3-fold over the mean MN frequency in the concurrent vehicle control was considered a positive response.

2.4. Animal husbandry

For the combined in vivo MN and comet assays male and female B6C3F1 mice (Charles River Laboratories International, Inc., Raleigh, NC) were 8–10 weeks of age at the time of treatment. Animals were housed in polycarbonate cages with absorbent hardwood bedding in an AAALAC-accredited specific pathogen free facility with a 12 hour light/12 hour dark cycle. Certified Purina Pico Chow No. 5002 (Ralston Purina Co., St. Louis, MO) and water were provided ad libitum. For the 90-day toxicity study male and female Hsd:Sprague Dawley rats (Harlan Laboratories, Inc., Frederick, MD) were housed one per cage in polycarbonate cages with heat-treated hardwood bedding (Northeastern Products Corp., Warrensburg, NY) and fed Purina Certified 5002 Meal Diet (Ralston Purina Co., St. Louis, MO) ad libitum. These studies were approved by the ILS, Inc. Institutional Animal Care and Use Committee, and all procedures were completed in compliance with the Animal Welfare Act Regulations, 9CFR 1–4, and theGuide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 2011).

2.5. In vivo MN/Comet assay experimental design

GLP studies were conducted in compliance with OECD TG 474 (OECD, 2014b) and in general accordance with OECD TG 489 (OECD, 2014c). Dose range finding studies were conducted to identify appropriate dose levels. Male and female mice were administered CGTase or sodium sulfate via oral gavage up to and including dose levels of 2000 mg/kg/day (test guideline limit dose) for three consecutive days. No marked changes in body weights were measured nor adverse clinical observations found in mice administered either chemical and there were no abnormal gross necropsy organ observations. Therefore, for the definitive studies, male and female B6C3F1 mice (5 animals/dose group) were administered CGTase or sodium sulfate at 1000, 1500, or 2000 mg/kg/day, vehicle (deionized water), or the positive control compound, ethyl methanesulfonate (EMS) (Sigma-Aldrich, St. Louis, MO) in 0.9% saline (Ricca Chemical Company, Arlington, TX) at 150 mg/kg/day, daily for three days by oral gavage. Three hours after the final dose, peripheral blood was collected for flow cytometric analysis of MN, and liver, duodenum, and stomach tissues were collected, frozen in liquid nitrogen, and stored at ‐80 °C until analysis by the comet assay (Recio et al., 2012).

2.6. Erythrocyte micronucleus assay

Peripheral blood samples were processed for flow cytometric evaluation of micronucleated reticulocytes (MN-RET) as described previously (Witt et al., 2008). Briefly, cells were fixed and labeled using a MicroFlow^PLUS Kit (Litron Laboratories, Rochester, NY) according to manufacturer’s directions and analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Sunnyvale, CA). For each peripheral blood sample, 20,000 RET were analyzed to determine the frequency of MN-RET. More than 10⁶ mature normochromatic erythrocytes were enumerated concurrently during MN-RET analysis, and the percentage of RET (%RET) among total erythrocytes was calculated as a measure of bone marrow toxicity.

2.7. Comet assay

For each animal, a portion of the left lobe of the liver was placed into a tube containing cold mincing solution [Mg⁺⁺ and Ca⁺⁺ free Hank’s Balanced Salt Solution (Invitrogen, Carlsbad, CA) containing 10% v/v dimethylsulfoxide (DMSO), and 20 mM disodium ethylenediaminetetraacetic acid (Na₂EDTA) pH 7.4–7.7] and rapidly minced. A portion of the duodenum proximal to the stomach was removed, flushed with mincing solution, and kept cold and moist with mincing solution. The tissue was trimmed and a small section placed in a microcentrifuge tube containing mincing solution and rapidly minced. The stomach was cut open and washed free from food using cold mincing buffer. The glandular stomach was placed into cold mincing buffer and incubated on ice for 15–30 minutes, then the surface epithelium was gently scraped two times using a scalpel blade. This layer was discarded and the gastric mucosa rinsed with cold mincing buffer. The stomach epithelium was carefully scraped 4–5 times in mincing solution with a scalpel blade to release the cells. The mincing solution containing released epithelial cells was transferred to microfuge tubes. Tissue samples were flash frozen in liquid nitrogen and stored at −80 °C until processed (Recio et al., 2012). Additional portions of each tissue were fixed in 10% neutral buffered formalin (NBF) for at least 24 hours, trimmed, and paraffin embedded for possible histopathology assessment of cytotoxicity in the case of a positive response in the comet assay.

For processing, cells were partially thawed in a warm water bath and placed on ice until slide preparation. Cell samples were empirically diluted with 0.5% low melting point agarose (Lonza, Walkersville, MD) dissolved in Dulbecco’s phosphate buffer (Ca⁺⁺, Mg⁺⁺ and phenol free) at 37 °C, layered onto each well of a 2-well CometSlide™ (Trevigen, Gaithersburg, MD), and immersed in cold lysing solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris, pH 10, containing freshly added 10% DMSO and 1% Triton X–100) overnight. After rinsing in 0.4 M Trizma base (pH 7.5), slides were treated with alkali (300 mM NaOH, 1 mM Na₂EDTA, pH > 13) for 20 minutes, then electrophoresed at 4 °C for 20 minutes at ∼1.0 V/cm, 300 mA. After electrophoresis, slides were neutralized with 0.4 M Trizma base (pH 7.5) for 5 minutes, incubated for 5 minutes in ice-cold 100% ethanol (Pharmco-AAPER, Shelbyville, KY) and allowed to air-dry. Slides were stored at room temperature in a desiccator until stained and scored. After staining slides with SYBR Gold™ (Molecular Probes, Invitrogen, Carlsbad, CA), 100 cells were scored per sample at 200x magnification without knowledge of sample identity using Comet Assay IV Imaging Software¸ Version 4.3.1 (Perceptive Instruments, Ltd., Suffolk, UK). The extent of DNA migration was characterized using the % tail DNA endpoint measurement (intensity of all tail pixels divided by the total intensity of all pixels in the comet, expressed as a percentage). NaCl, Na₂EDTA, Triton X–100, and Trizma base were purchased from Sigma-Aldrich (St. Louis, MO); NaOH and DMSO were purchased from Fisher Scientific (Pittsburgh, PA).

2.8. 90-day repeat dose toxicity study

This GLP-compliant 90-day rat study was conducted following OECD TG 408 (OECD, 1998). Ten males and ten females were allocated to each of four designated dose groups. The animals were administered one of three dose levels (250, 500, or 1000 mg/kg/day) of CGTase or deionized water for 90 days via oral gavage. Cage-side observations were performed daily and body weight measurements and clinical observations performed weekly. Ophthalmological examinations were performed prior to exposure to CGTase and again within a week of termination. Following 11 weeks of exposure, neurotoxicity screening was performed including measurement of motor activity and a functional observation battery as defined in OECD TG 424 (OECD, 1997b).

After 90 days of CGTase administration, animals were humanely euthanized. Prior to termination, urine was collected and submitted for analysis. A full screen necropsy was performed and tissue weights were collected for adrenals, brain, epididymides, heart, kidneys, liver, lungs, ovaries, pituitary, prostate, salivary glands, seminal vesicles, spleen, testes, thymus, thyroids with parathyroids and uterus with cervix. The following tissues were fixed in 10% NBF (with the exception of eyes and testes that were fixed in modified Davidson’s fixative), embedded in paraffin, sectioned, and microscopically evaluated: adrenals, aorta, bone (sternum and femur), bone/bone marrow (sternum and femur), brain (cerebrum, cerebellum, and medulla/pons), cecum and colon, corpus and cervix uteri, epididymides, esophagus, eyes, heart, kidneys, liver, intestines (small and large including Peyer’s patches), lungs (with main-stem bronchi), lymph nodes (mesenteric and mandibular), mammary glands, muscle (skeletal), nasal cavity, ovaries, oviducts, pancreas, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles, skin, spinal cord (3 locations: cervical, mid-thoracic, lumbar), spleen, stomach, testes, thymus (thymic region), thyroids/parathyroid(s), tongue, trachea, uterus, urinary bladder, vagina, Zymbal’s glands. Blood, collected by vena cava puncture immediately following termination, was submitted for hematology, clinical chemistry, and hormone determinations (See Tables 5–7 for lists of measurements).