90-Day Inhalation Study with Cigarettes containing G7-25 Measurement of particle size will be made from the retro-orbital sinus, using anesthesia once every two weeks for each of the groups. with 70% CO 2 in air and heparinized Temperature and RH measurements will micropipettes (American Hospital Supply, be made once every two weeks. RH will be McGaw Park, IL), after 50-60 minutes of determined by comparing the dry bulb exposure. Blood samples will be collected in temperature and the dew point temperature, Eppendorff micro centrifuge vials (Brinkmann using an 100DP Dew Point Hygrometer Instruments, Westbury, NJ) containing the (General Eastern Instruments Company, sodium salt of ethylene diamine tetra acetic Waterstown, MA). The RH of the aerosols acid (Na2-EDTA) and stored on ice. will be controlled by regulating a portion of Blood COHb concentrations will be the air dilution stream through an air determined using a Model 482 CO-Oximeter humidifier and then joining a non-humidified (Instrumentation Laboratories, Hartford, CT). air stream prior to mixing with the smoke Each animal will be sampled once during the aerosol. experiment, with samples collected in exposure weeks 1, 4, 7, 10 and 13. IN-LIFE MEASUREMENTS AND Plasma nicotine and cotinine OBSERVATIONS At the same time as blood samples are Gross Observations obtained for CO-Oximetry (see above) an Animals will be observed for signs of additional blood sample will be taken from the toxicity on each exposure day as they are micropipette into Vacutainer tubes being transferred from their cages to the (Becton-Dickinson, Rutherford, NJ) containing restraint tubes, and when being transferred Na 2 -EDTA. Plasma samples will be obtained back to their cages. In addition, more detailed immediately by centrifugation, and samples clinical observations will be recorded on each stored at -70°C until subsequent analysis for animal once every four weeks, before each plasma nicotine and cotinine. Additional exposure and within 2 hours of the end of the plasma samples will be obtained from exposure (once per day for sentinels). plethysmograph animals: see below. Early Deaths / Viability Checks Either gas chromatography (Davis, Viability checks will be made twice 1986) or a newly-developed ELISA method daily at intervals of at least 4 hours (5 days per will be used to measure the plasma week, with only a single check each day at concentrations of nicotine and cotinine. weekends and holidays). Necropsies with Respiratory Physiology tissue collection for any subsequent Tidal volume, respiratory rate and histopathology will be performed by a minute ventilation will be measured in 4 veterinary pathologist (or experienced animals per sex per group, during exposure assistant) on animals found dead or in a weeks 2, 3, 5, 6, 8, 9, 11 and 12. The moribund condition. If necropsy cannot be measurements will be derived from respiratory performed immediately, the carcass will be flow measurements, using whole body stored at 4°C to minimize tissue autolysis. plethysmography (Coggins et al., 1981), a Animals will thus be necropsied no later than pulmonary mechanics analyzer (Buxco 16 hours after the discovery of death. Electronics Inc, Sharon, CT), and pulmonary Body Weights; Feed consumption physiology software (Branch Technologies, Individual non-fasted body weights will Dexter, MI). Measurements will be made be determined within 48 hours of receipt, at throughout the 60 minutes of exposure, plus group allocation, and weekly thereafter. Body 10-15 minutes pre-exposure. weight data will be acquired using Mettler PM At the end of the 60 minutes of exposure 2000 balances (Mettler Instrument the animals will have blood samples drawn for Corporation, Hightstown, NJ). Feed assays of plasma nicotine and cotinine (see consumption will be measured over 72 hours above). Each plethysmograph animal will be in exposure week 13, using the same sampled twice during the experiment, with equipment as described for body weights. maximized temporal separations between Blood CO-Oximetry samplings. Blood for determination of COHb and other CO-Oximetry parameters will be drawn DRAFT: DO NOT QUOTE. Page 5 Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0103