sample="quota" bates="508676946" isource="rjr" decade="1990" class="ui" date="19901230" The Effects of Sidestream Smoke on the Rat Middle Ear Experimental Protocol TOX-35 I. OBJECTIVE The primary objective of this study is to evaluate the effects of sidestream smoke or low humidity on the middle ear of rats exposed to sidestream smoke (SSS). In particular, the ear of the rat will be examined for serous effusion (fluid behind the ear drum) potentially induced by exposure to sidestream smoke or low humidity. A second objective is to evaluate the effect of SSS on the rate of clearance or resolution of an experimentally induced middle ear effusion. II. INTRODUCTION Controversy exists in the literature regarding the contribution of parental smoking in the development of acute otitis media in children (refs. 1-4). There have been numerous recent reports in the literature linking parental smoking with the middle ear effusions and acute otitis media in children. The majority of theses studies were composed of data collected from questionnaires mailed to parents regarding home environmental factors. The number of middle ear effusions was then correlated with parental smoking. No basic science research using animal models to test the effects of SSS on middle ear effusions have been reported. In addition, no animals studies have been performed to test the effects of low humidity on production of middle ear effusions. This study is designed to test whether SSS exposure induces a serous effusion in the rat middle ear, or influences the rate of clearance or resolution of an established effusion. The potential effect of an environmental factor of low humidity on induction of serous effusions will also be examined. III. FACILITIES AND ADMINISTRATION Facilities R. J. Reynolds Tobacco Company Building 630-2 Winston-Salem, NC 27102 Contractors Histopathology: Veritas Laboratories, Burlington, NC Animal Care: Program Resources Inc., Winston-Salem, NC Serology: Study Administration Study Director: Paul H. Ayres, Ph.D., D.A.B.T. R. J. Reynolds Research and Development, Inhalation Division Principal Investigator: Hugh Lovejoy, M.D. , Bowman Gray / Baptist Hospital Medical Center, ENT Department Inhalation Study Technicians: Jim Corn and Delma France IV. OVERVIEW This study will be composed of five groups of male rats with a total of 89 animals allocated to the study. All study groups will be housed in whole body exposure chambers during the exposure phase of this study. Two groups of rats, 23 rats per group, will be exposed to SSS in nose only exposure tubes at concentrations of 0.1 mg wet total particulate matter (WTPM) per cubic meter of air or 1 mg WTPM per cubic meter of air at 40-60 % RH for 6 hours per day for 5 consecutive days. A third group of rats, 23 in the group, exposed in nose only exposure tubes to air at 40 - 60 % relative humidity (% RH) for 6 hours a day for five consecutive days will serve as sham controls. The portion of the day in which the animals will not be in nose only exposure tubes, they will be housed in the whole body exposure chambers and exposed to purified air at 40 - 60% RH. One ear in each of the animals in the SSS and sham exposure groups will have a serous effusion (fluid behind the ear drum) experimentally induced by the use of cold air directed into the external auditory canal prior to beginning the exposure phase (Goldie and Hellstrom, 1986). Since each ear is an anatomically distinct organ system, each ear will be considered as an independent experimental unit. These groups will be used to evaluate 1) the effect of SSS on the clearance or resolution of an experimentally induced middle ear effusion in the treated ear; and 2) the effect of SSS on the induction of a middle ear effusion in the untreated ear. The fourth and fifth groups, to rats in each group (a total of 20 independent experimental units in each group), will be exposed to low humidity (0-10 % RH) or normal humidity (40-60 %RH) air, respectively, for 24 hours a day during the exposure phase of this study. These groups will not be exposed in nose only exposure tubes. These groups will be used to evaluate the effect of low humidity on the induction of middle ear effusions. B. Quarantine and Serological Evaluation: Upon delivery, animals will be housed individually in transparent cages in room #47 for 14 days prior to the first exposure. These animals will be assigned a pre-study identification number which will be indicated on a cage card. This 14 day period will serve as a quarantine period with limited personnel access. Within 2 days of delivery, 5 rats will be randomly selected by staff from the Animal Biology Division for serological evaluation of antibodies to disease. The rats will be euthanized with 70% carbon dioxide and serum collected. The serum will be tested for the following antibodies to disease: Reovirus Type 3, cilia associated respiratory bacillus, Kilham's rat virus, lymphocytic choriomeningitis virus, and Mycoplasma pulmonis. Lungs from these 5 rats will be collected and examined histopathologically to ascertain health status. The start of the exposure phase of the study is dependent upon negative serology data being obtained on the pre-study samples, and upon a histopathology statement on the animals killed at delivery, releasing the animals from the 14 day quarantine. C. Group Allocation: Within 7-10 days of delivery, rats will be allocated into experimental groups such that the body weights in the groups are as homogenous as possible. Surplus animals from the above procedure will be used by the Study Director for methods development projects approved by the animal care committee. D. Animal Identification: After allocation into experimental groups, animals will be tail-tattooed (Animal Identification and Marking Systems, Piscataway, NJ) with their permanent identification number by personnel trained by the Animal Identification and Marking Systems company. The animals will be returned to cages with cards attached recording the study number, animal number, sex, pre-study number, and study director. The following animal identification numbers will be used: Group 1, numbers 101-123; Group 2, numbers 201-223; Group 3, 301-323; Group 4, 401-410; and Group 5, numbers 501-510. E. Animal Husbandry: The animals will be housed and cared for in accordance with the Animal Welfare Act of 1970 and amendments (Public Law 91-579), as set forth in CFR Title 9, Part 3, Subpart E, "Specifications for the humane handling care treatment and transportation of warm-blooded animals other than dogs, cats, rabbits, hamsters, guinea pigs, and non-human primates." During the quarantine period, the animals will be housed in room # 47 in building 630-2. The room will have controlled lighting (12 hours of darkness, from 6:00 pm), temperature (20-24 C), and humidity (40-60 %RH). Seven day continuous recordings will be kept of %RH and temperature. After the quarantine period, the animals allocated into experimental groups will be housed in individual stainless steel wire mesh cages in Hazelton 2 cubic meter whole body inhalation exposure chambers (Moss et al., 1982). Rats housed in whole body exposure chambers will have ad libitium access to feed and water except during inhalation exposures for groups 1,2, and 3. Feed will be removed from the chamber in the morning prior to inhalation exposures for groups 1,2, and 3 and returned to the chambers after the completion of inhalation exposure. Paper lining the excreta collection pans in the whole body exposure chambers for all groups will be changed once daily in the morning as animals are prepared for inhalation exposure. This preparation involves an A.M. viability check for all groups and loading rats into exposure tubes for groups 1,2, and 3. This pre-exposure preparation will be conducted by the Animal Care staff. At the end of the daily inhalation exposures, the rats will be removed from the nose only exposure tubes (groups 1,2, and 3) by the inhalation technical staff. Feed will be returned to the animals in groups 1,2, and 3 after completion of daily exposures by the inhalation technical staff. Whole body exposure chambers will be operated at an air flow to maintain approximately 15 air changes per hour. Air for the whole body exposure chambers will be drawn from humidified HEPA filtered room air except for group 4. Group 4 (low humidity group) will be supplied non-humidified HEPA filtered air from the Delmonox air purifier system. The temperature will be maintained at 20-24 degrees C and the humidity will be maintained at 40-60 %RH except for group 4 which will be maintained at 0-10 %RH. On exposure days, animals in groups 1,2, and 3 will be taken from their cage in the whole body chamber, placed in a nose only exposure tube, and replaced into the same cage position. The ventilation tubes on the tubes will be covered with duct tape. The position of the racks within the chambers will be changed daily to minimize any impact of rack position. Cages on the upper racks will be moved to the next lower rack on subsequent days of exposure and those on the bottom rack will be moved to the top position. Pre-Exposure Characterization Before the animal exposures begin, satisfactory achievement of uniformly distributed concentrations at or near the target concentrations will be confirmed. Daily Characterization of Inhalation Exposures Groups 1,2, and 3. During animal exposures probes inserted into the whole body exposure chambers will be used to monitor the aerosol presented. Gravimetric estimates of WTPM concentration will be determined with 25 mm Teflon membrane filters. On line monitoring of WTPM concentration will be accomplished with a RAM-1 aerosol monitor. Measurement of aerosol particle size in group 3 will be determined with a cascade impactor (In-Tox Products, Albuquerque, NM) twice during the study. Concentration of aerosol in group 2 is too low to permit particle size analysis by the cascade impactor. Groups 1,2,3,4 and 5. Carbon monoxide and carbon dioxide concentrations will be analyzed with instruments (Horiba PIR-2000, Horiba Instruments, Irvine, CA.) calibrated daily with certified gases. Oxygen concentration will be determined with a Horiba PMA-200 instrument, also calibrated daily with a certified gas. Ammonia will be determined with a handheld monitor ( GasTech, NH-275, Gas Tech, Newark, CA.) connected to a stainless steel probe inserted into the chamber. Very low concentrations of CO will be analyzed with daily calibrated Miran 80 gas analyzer (Foxboro Instrument, S. Norwalk, CT.). Data from on-line instruments will be logged manually every 60 minutes. IX. NECROPSY AND HISTOPATHOLOGY Animals from all groups will be killed within 24 hours after termination of the fifth day of exposure. At necropsy, animals will be weighed and then killed by first anesthetizing with 70 % CO2 in air and then exsanguination via the vena cava. Following euthanasia, tympanocentesis will be performed through the tympanic bulla to assess the viscosity of any middle ear effusion. The heads will be removed and the brain tissue removed as well. The temporal bones will then be harvested and fixed in 20 % formalin solution. The temporal bones will then be processed and cut in a manner to permit visualization of the mucosa. The tissues will be stained with hematoxylin and eosin and duplicate slides will be stained with periodic-acid-schiff (PAS) stain to facilitate evaluation of mucous secreting cells. Scanning electron microscopy will be conducted on the eustachian tube mucosa on three animal in group 1, 2, and 3 by RJR staff. In addition to collection of tissues from the middle ear, the larynx from each animal of each group will be taken for histopathological examination. The slides will be reviewed by Veritas Laboratories and the principal investigator in addition to a pathologist on staff at Wake Forest University Medical Center. X. STATISTICAL ANALYSIS Appropriate statistical analyses on the induction of effusion, rate of clearance of effusion, qualitative effusion differences, as well as histopathological changes will be performed using appropriate statistical methods by Dr. Tim Morgan, a statistician at Wake Forest University Medical Center. XI. QUALITY ASSURANCE All data pertinent to this experiment will be recorded and kept for review so that the experiment could be reproduced. XII. REPORTING An interim report will be prepared following necropsy. This will include data on micro ear exams and tympanograms that were obtained during the study. The final report will be prepared in draft form following completion of the review of histopathology. The final report will include: - objectives and procedures as stated in the protocol - description of the inhalation chambers and their operating conditions and performance - tabulation of response data - a separate pathology report, including tabulated gross and microscopic pathology - statistical analyses and any conclusions developed from these data. XIII. PROJECTED TIMING Attempts will be made to achieve the following target dates (1991): Delivery of animals: 17 January Quarantine: 17 January - 31 January Induction of middle ear effusions: 1 February - 5 February Exposure of starting dates: Group 1: 6 February Group 2: 7 February Group 3: 8 February Group 4: 9 February Group 5: 10 February Necropsies: Group 1: 11 February Group 2: 12 February Group 3: 13 February Group 4: 14 February Group 5: 15 February