Document oeNqeBpK8yY29NRJzDawDYo3r

3 PROTOCOL TITLE VINYL CHLORIDE; COMBINED INHALATION TWO-GENERATION REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDY IN CD RATS RESPONSE TO THE ATSDR REVIEW Prepared by William J. Breslin. Ph.D. The Dow Chemical Company October 10,1995 Reviewer. Dr. M. S. Christian The reviewer requested CMA provide further justification and clarification on certain areas of the protocol dealing with study design and methods. Although we have chosen not to reiterate each specific comment here, all comments are addressed in the following section. Route/Method of Exposure Whole body exposures were selected over nose only exposures for the following reasons. 1) Vinyl chloride is highly volatile with a vapor pressure of 2,530 mm Hg at 20 G Given the high volatility of vinyl chloride, the exposure concentrations to be used and the exposure conditions to be employed in this study, aerosol formation and deposition of vinyl chloride onto the fur or skin of the rats is expected to be very low and represent an insignificant source of both oral or dermal exposure. 2) Many of the reproductive and developmental endpoints to be evaluated in this study are highly sensitive to general stress. The stress associated with the confinement (immobilization) of nose only exposure is significant and represents an unnecessary confounder. Although the stress of nose only exposure can be controlled for, to a limited extent, through acclimating animals, the undesirable effects of this additional factor outweigh any potential benefits of reducing the limited amount of oral or dermal exposure due to whole body exposure. 3) The cost for a long term, large animal nose only exposure study is significantly greater than for a whole body exposure study due to extensive 1 BFG41023 BFG 00540 animal handling and monitoring requirements. Since there is little scientific merit in conducting a nose only study, the additional cost associated with a nose only study would provide little or no value. 4) Whole body exposure is more representative of human exposure conditions. The protocol states that animals will be exposed to the test material 5 days/week during the 10-week premating periods and 7 days/week during die breeding, gestation and lactation periods. The 5 day/week exposures were selected to simulate human exposures. Five days/week during a 10 week premating period is an adequate period of exposure to produce effects on male or female gonadal growth and function. The exposures were extended to 7 days/week following the premating period in view of the compressed time frame associated with the critical reproductive and developmental events of mating, gestation and lactation in the rodent, relative to humans. In rodents, embryonal/fetal (22 days) and neonatal (21 days prior to weaning) development are rapid, requiring daily exposure to assure the animals are exposed during all critical gestational and lactational events. Areas in the protocol identifying information regarding exposure, housing and duration of exposure. The following sentence in the protocol has been modified to provide greater clarification regarding animal housing procedures. The protocol modifications are in italics. Page 7, line 9. "All rats will be housed continuously in exposure chambers following the initial exposure to the test material, except during late gestation and throughout lactation periods when female rats will be housed outside of the exposure chambers during non exposure periods Exposure Levels Attained Pharmacokinetics is beyond the scope and objectives of this study, and therefore, have not been incorporated into the design of this study. Pharmacokinetics are best obtained under a separate protocol and study design. The rationale for housing females outside of the exposure chambers during late gestation and throughout lactation, during non exposure periods, is 2 BFG41024 logistical. Since the females will be held in the larger nesting boxes during late gestation and throughout lactation, following exposure, we chose to house the animals outside the actual exposure chambers to avoid crowding within the chambers. In addition, maintaining the nesting cages within the exposure chambers would necessitate exposing both the dam and pups during parturition and lactation. Exposing the dam during the process of parturition and the pups during lactation prior to weaning is unwarranted scientifically and not representative of human exposure. We are also not aware of any EPA Guideline inhalation studies designed for risk assessment that exposed the dam and pups during this period. It should also be noted that although the dam and pups will be housed outside the actual exposure chambers during late gestation and throughout lactation, following exposure, the rooms in which they will be housed will be identical unused exposure chambers. Since the choice of what room to house the animals in after exposure has no bearing on the study design, results, interpretation of the results or validity of the study, adding an extensive rationale to the protocol for this minor procedure is considered unnecessary. Dams are withheld from the exposure chamber during the late gestation and early lactation period to allow the dam to deliver their litters and care for their pups during this critical period. Exposing the dams late in the gestation period (days 20-22) risks allowing the dams to deliver in the inhalation chambers and the loss of pups through the caging floor. The cages designed for inhalation exposure are made of wire mesh to allow unimpeded air flow and air circulation. The design of the inhalation cages are not compatible with maintaining the dams and their litters. Following parturition, the dams are allowed to remain with their litters for the first four days of lactation to dean and care for the young at a time in which the young are highly susceptible to neglect. It is well documented that removing the dams during the 6-hour exposure periods during lactation days 5 through 21 has an effect on pup growth. It is also well documented that infant weight and mortality are inversely correlated. Although we are not aware of any published data on whether or not increased pup mortality occurs if you remove the dam from the litter during the first four days of lactation, common sense tells us that we would exacerbate the well known growth retardation observed with removal of the dam during later periods of lactation and likely affect early mortality. 3 BFG41025 BFG 00541 Certainly, pup mortality would be affected if the dams were allowed to deliver their litters in the chambers and have their pups fall through the wire mesh floor. Comments regarding the ages of P2 animals at body weight and feed consumption measurement intervals. Mating of the PI animals occurs over a two-week period. Past experience indicates that the majority of animals (70 to 100%) mate in the first week of mating. Therefore, the majority of the animals will be of similar age (within one week). For the pups from dams that mated during die second week of mating, it is dear that they will be one week younger than animals from the first week of mating. However, barring a highly significant treatment related effect on the timing of mating, the age spread of the animals should have no impact on the growth curves as the control group will have the same age distribution. In addition, by the time the P2 animals are mated at 13 to 14 weeks of age, the one week differences in age will not be a significant factor in growth or sexual maturation/development. Day Weaning Occurs (page 8. culling and weaning) The protocol, page 8, paragraph 1, will be revised as follows (revision made in italics). "Weaning of all litters will be done art lactation day 21" This correction should be suffident to darify the procedures. The repetitive definition of days 0 and 1 postpartum was not deemed necessary. Culling (page 8. culling and weaning) Culling of litters is allowed by US EPA guidelines. Due to the practices of the animal suppliers, the CD rat widely used for reproduction studies has been genetically selected for large litter sizes. As a result of this selective pressure, the litter size of the CD rat has increased significantly, beyond what is typical for this species. Culling of litters on lactation day 4 reduces die variation in pup growth associated with differences in litter size. The reduced interlitter variation can improve our ability to pick up true treatment-related differences in pup growth. The majority of pup mortality occurs early in lactation during days 0 through 4. Culling on lactation day 4 does not limit our ability to detect effects on viability during this sensitive period. We are also not aware of any data that documents that culling of litters on lactation day 4 confounds pup mortality in later periods of lactation. Regarding the 4 BFG41026 culling and definition of runts, the protocol clearly states that preferential culling of runts will not be performed. Extensive definitions of all terminology were not included in the protocol as this level of detail was considered unwarranted. However, the definition of runt will be included in the final report. Since the protocol dearly states that pups to be culled are selected using a computer generated randomization procedure, the reviewer's comment on needing a definition of runt to determine if preferential culling of runts will not occur makes no sense and is difficult to address beyond what has already been stated. Feed Consumption (page 8) Measuring feed consumption does not necessitate having feed containers in the chambers during the exposure period. In fact, the procedure for obtaining feed consumption during an inhalation study is to remove the feed containers during exposure and to return them following exposure. This procedure eliminates the potential for oral exposure due to absorption of the test material into the feed. Body Weight (pages 8 and 9) Historically, the body weight of dams from reproduction and developmental toxicity studies conducted in this laboratory, including inhalation studies, have been collected on gestation day 21. Therefore, the historical data from this laboratory include gestation day 21 data. Body weights in inhalation studies are generally collected in the morning prior to exposure and prior to the onset of delivery (gestation day 21). So as to assure collection of as much data as possible, morning measurements of gestation day 21 body weights will be recommended. Utter Data (page 9) On the day of parturition, dams and litters are observed for the number of pups delivered and the presence of dead pups. In order to reduce handling stress on the day of parturition, disturbances are kept to a minimum and data collection limited. The critical parameter observed on gestation day 0 is the live-dead count. BfG41027 5 BFG 00542 Reproduction - Pathology - Adult Rats (page lOi The rats are to be euthanized after the last litter has been weaned. This necropsy schedule was chosen for logistical reasons and to allow for flexibility in the study design. Euthanizing the dam immediately following weaning of the litter would result in a two-week necropsy period (highly inefficient) and preclude breeding the dams for a second litter. A common necropsy date after the last litter has been weaned allows time to review all the litter data and make informed decisions as to the future direction of the study. Should the first litter produce equivocal results or results requiring follow-up investigations, additional litters (Fib or F2b) can be incorporated into the study protocol. Reproduction - Organ Weights- Adults Rats (page 10) The first 15 PI and P2 adults were selected for organ weight measurements. Fifteen animals per group provide adequate sample size and power to detect biologically significant treatment related effects. Since the animals are randomly assigned to treatment group, and randomly placed within treatment group, the first 15 animals are, in effect, randomly selected. The selection of animals is not based on mating or delivery sequence. Reproduction - Sperm Count. Motility and Morphology (page 11) The rationale for selecting the first 15 males for sperm analyses is the same rationale provided above for organ weights. The source of sperm for motility analyses will be changed in the protocol to specifically state only caudal epididymis. Statistical Evaluation (page 14) The authors feel it is unnecessary to provide extensive definitions of terminology throughout the protocol, especially in the statistical analyses section. All terms required to interpret the results of the study will be defined in the final report. Typographical Errors All typographical errors identified by the reviewer have been corrected. 6 BFG41028 Reviewer: Dr. R. D. Hood Reviewer comment no. 1 regarding the typographical error on the limit dose of 1 mg/kg body weight. The 1 mg/kg value has been corrected to read 1000 mg/kg/day. Reviewer comment no. 2 regarding the calculated dose 1010 vs. 1012. Recalculation of the dose using the formula and data provided on page 6 of the protocol results in a value of 1012. The discrepancy between our numbers may be due to rounding differences. Reviewer: B. F. Hales No response, other than correcting typographical errors was required. Typographical errors were corrected. William J. Breslin, Ph.D. BFG41029 7 bfg 00543 Preliminary CopyJLFinal Copy___ HEALTH AND ENVIRONMENTAL SCIENCES THE DOW CHEMICAL COMPANY PROTOCOL THE TOXICOLOGY RESEARCH LABORATORY, 1803 BUILDING, MIDLAND, MICHIGAN 48674 TITLE: VINYL CHLORIDE: COMBINED INHALATION TWO-GENERATION REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDY IN CD RATS DATE: PROPOSED EXPERIMENTAL START DATE: PROPOSED EXPERIMENTAL TERMINATION DATE: ESTIMATED DATE FINAL REPORT: SIGNATURES: STUDY DIRECTOR: COINVESTIGATOR(S): INHALATION SPECIALIST: FILE NUMBER: PROJECT NUMBER: TASK NUMBER: OSD NUMBER: COST CENTER: SPONSOR: CAS NUMBER: STUDY PATHOLOGIST: APPROVAL(S): SPONSOR: DISTRIBUTION - - SEE ATTACHED LIST /DATE /DATE /DATE /DATE /DATE /DATE BFG41030 BFG 00544 INTRODUCTION Objective. The objectives of the combined inhalation two-generation reproduction and developmental toxicity study outlined in this protocol are to evaluate the effects of the test material on parental toxicity, reproductive capability, in utero development, and neonatal growth and survival in rats. This study will be conducted to meet the requirements of the Environmental Protection Agency (EPA): TSCA Test Guidelines (EPA, 1985), the Organisation for Economic Co-Operation and Development (OECD), Guidelines, for Testing of Chemicals, Section 4: Health Effects, (OECD, 1981), and the European Economic Community (EEC), Methods for the Determination of Toxicity (EEC, 1988). Statement of GLP Compliance. This study will be conducted in accordance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations for NonClinical Studies (FDA, 1988), the EPA TSCA Good Laboratory Practice Standards (EPA, 1990), the OECD Good Laboratory Practice Procedures (OECD, 1982), and the Standard Operating Procedures of The Toxicology Research Laboratory of The Dow Chemical Company. In addition, in response to the Final Rules amending the U.S. Animal Welfare Act that were promulgated by the U S. Department of Agriculture effective October 30,1989, the Animal Care and Use Activity (ACUA) that is required for the conduct of this study has been reviewed and given full approval by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activity is in full accordance with these Final Rules. The IACUC has assigned Activity No(s). Reproductive Toxicology 01 and 02 to this Animal Care and Use Activity. October 11,1995 BFG41031 DRAFT BFG 00545 -2- MATERIALS AND METHODS The test material used for this study will be obtained from a commercial supplier and identified by lot number. The purity of the test material will be determined and reported. A sample of the test material will be taken and stored in a manner consistent with the reference sample retention policy of this laboratory. The test material will be reanalyzed at approximately 6month intervals to confirm purity and stability. The test substance has the following properties: Chemical Name: Synonyms: Molecular Formula: Molecular Weight: Structures: Appearance: Vapor Pressure: Saturated Atmosphere: Vapor Density: Flash Point: Boiling Point Specific Gravity: Conversion Factors: Vinyl chloride Monochloroethylene, VC, VCM, vinyl chloride monomer C2H3CI 62.5 CH2=CHQ Colorless gas 2,530 mm Hg at 20 C Gas at room temperature 2.16 -77.75 C (open cup) -13.6 C 0.9121 1 ppm 3 2.60 mg/m3 in air 1 mg/mm3 = 0.39 ppm in air Test Species and Husbandry. Male and female CD rats (Charles River Breeding Laboratory, Kingston, NY or Portage, MI) approximately four weeks of age will be purchased for the reproduction phase of this study. An additional group of adult female timemated CD rats (Charles River Breeding Laboratory, Kingston, NY or Portage, MI) approximately ten weeks of age will be purchased for the developmental portion of this study. Animals will be ordered to ensure that a sufficient October 11,1995 DRAFT BFG41032 BFG 00546 -3- number of animals of acceptable health and weight are available to conduct the study as designed. This strain of rat has been selected because of its general acceptance and suitability for toxidty testing and the availability of a reliable commercial source. Upon arrival at the laboratory1, all rats will be examined for health status by a veterinarian and acclimated to the laboratory environment (approximately two weeks for the reproduction study; 5 days for the developmental study), according to the Standard Operating Procedures of the Reproductive Toxicology Group. The animals will be randomly assigned by weight to the treatment groups to increase the probability of uniform group mean weights and standard deviations at the initiation of the study. Rats not placed on test will be removed from the test room and the disposition of these animals will be documented in the study rile. Identification of all rats on test will be accomplished by inserting a uniquely coded alphanumeric metal tag in one ear of each rat In the event that an ear tag becomes dislodged during the course of the study, it will be replaced with one having die same alphanumeric code (i.e., a new alphanumeric code will not be assigned) and noted in the study file. Rats will be housed singly in wire mesh, stainless steel cages in racks provided with cage board to minimize odor and aid in maintaining a dean environment. Prior to and following daily exposures during late gestation and throughout lactation, females will be housed in plastic nesting boxes provided with ground com cob nesting material (further details provided in experimental design section). The animal rooms of the facility are designed to maintain humidity at approximately 40-60%, temperature at approximately 22 C, photoperiod at 12 hrs light: 12 hrs dark and air flow at 12-15 changes/hour. A feed crock and a pressure-activated stainless steel water nipple will be components of all cages. A basal diet of Purina Certified Rodent Chow No. 5002 (Purina Mills Inc, St. Louis, MO) will be provided ad libitum except during the six hour per day exposures, at which time feed will be withheld. Municipal drinking water will be available ad libitum 1 Fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). October 11,1995 DRAFT BFG41033 BFG 00547 -4- throughout the prestudy and study periods. Analysis of the chow will be performed by Purina Mills Inc. to confirm that the diet provides adequate nutrition, and to quantify the levels of selected contaminants associated with the formulation process. Drinking water obtained from the City of Midland, MI will be analyzed for chemical parameters and biological contaminants by the City of Midland Water Department. In addition, specific analyses for chemical contaminants will be conducted at periodic intervals as stated in the Standard Operating Procedures of The Toxicology Research Laboratory, The Dow Chemical Company. Exposure Chamber. Animals will be housed and exposed to test material vapors in a 14.5 m3 chamber (2.4 m wide x 2.4 m high x 2.4 m deep with a pyramidal top) under dynamic airflow conditions. Chamber airflows will be maintained at approximately 2900 liters/minute, which is sufficient to provide the normal concentration of oxygen to the animals. Vapor Generating System. The various concentrations of test material will be generated using a glass Jtube method (Miller et al., 1980). Liquid test material will be metered into a glass J-tube assembly and vaporized by a preheated stream of compressed air (up to 1001/min) passing through the J-tube. Compressed air will be heated to the minimum extent necessary to facilitate complete vaporization of the test material. The compressed air and test material vapors will be diluted and mixed with room air to achieve the desired concentration of test material vapors at a total flow rate of 2900 1/min. Chamber Monitoring. The concentration of the test material in each chamber will be measured at least once per hour using a MERAN 1A infrared spectrophotometer (Foxboro Analytical, Norwalk, CT). Analytical equipment will be calibrated using standards having a known test material vapor concentration contained in 90 liter SARAN* film gas bags prior to the first exposure and at least monthly * Trademark of The Dow Chemical Company October 11,1995 DRAFT BFG 00648 -5- thereafter. Daily checks of the analytical equipment will be performed prior to each exposure period using a single test material standard concentration. In addition, the amount of test material used each day will be recorded and the nominal concentrations (amount of the test material used/total chamber airflow) of test material will be calculated. Prior to the start of the study, each of the chambers to be used will be checked to ensure that a uniform distribution of vapors occurs within the breathing zone according to Standard Operating Procedures. Airflow through each chamber will be determined at hourly intervals using a differential pressure transducer (Model C264, Setra Systems, Inc., Acton, MA) or a Universal Venturi tube (Series 180, BIF, 345 Harris Ave., Providence, RI). The manometer and differential pressure transducer will be calibrated with a gas meter (Singer Aluminum Diaphragm Meter, Model AL-1400, American Meter Division, Philadelphia, PA) prior to the start of the study. The Universal Venturi tubes have been calibrated at the factory. Chamber temperatures will be measured with a thermometer or resistance temperature device (RTD) and relative humidities will be measured with relative humidity gauges or humidity sensors (HMP 112A, Vaisala, Helsinki, Finland) at least once each hour. Calibration of the hygrometers, RTD's and humidity sensors will be documented in the study file The temperature and relative humidity in each chamber will be controlled by a system designed to maintain temperature at approximately 222C and relative humidity at approximately 40-60%. Output from the differential pressure transducer, RTD, humidity sensor and infrared spectrophotometer will be collected by the CAMILE* Data Acquisition and Control System. Exposure Concentrations. Rats will be exposed to target concentrations of 0,10,100 or 1100 ppm of the test material. These exposure levels correspond to oral equivalent doses of * Trademark of SAGIAN Indianapolis, Indiana. October 11,1995 DRAFT BFG41035 BFG 00649 -6- approximately 0,9.2, 92 and 1012 mg/kg/day assuming ventilation rates of 1 1/min/kg, 100 percent absorption and a 6 hour/day exposure. The high concentration of vinyl chloride was selected based on the oral equivalent of the limit dose of 1000 mg/kg body weight/day. The high exposure of 1100 ppm is also expected to produce effects on the liver and other organ systems (ATSDR, 1993). The middle and low exposure levels were selected to provide a dose response for the observed effects and a no-observed-effect level, respectively. The calculations used to convert ppm to an oral dose equivalent are as follows: Dose (mg/kg/day) = exposure concentration (mg/1) X minute volume/kg body wt X minutes of exposure X absorbed dose Exposure concentration (mg/1) = (ppm X molecular wt)/24,450 Dose (mg/kg/day) = ppm X molecular wt X 1/24,450 X minute volume/kg body wt (11/min/kg) X minutes of exposure X absorbed dose Where: Dose = 1012,92 or 92 mg/kg/day Minute volume/kg body wt = 1 liter/min Minutes of exposure/day = 360 Molecular wt = 62.5 Proportion absorbed = 100% Conversion factor used in converting mg/1 to ppm = 1/24,450 Example: 1012 mg/kg/day = 1100 ppm X 625 (molecular wt.) X 1/24,450 X1 1/min/kg body wt X 360 min X 100% absorption Reproduction - Experimental Design. Groups of 30 male and 30 female rats will be exposed to 0,10,100 or 1100 ppm of the test material via inhalation, for 6 hours/day, 5 days/week prior to mating and 6 hours/day, 7 days/week during mating, gestation and lactation. The overall chronology of events and study design for this study are depicted in Tables 1 and 2, respectively. The treatment of the first generation parental (PI) rats will begin at approximately 6 weeks of age. After approximately 10 October 11,1995 DRAFT BFG41036 BFG 00550 -7- weeks of exposure (5 days/week, excluding holidays), PI rats will be mated (one male to one female of the respective treatment group) to produce the FI litters. Following weaning (3 weeks of age) of the FI litters, 30 males and 30 females from each treatment group will be randomly selected and assigned to the respective treatment group to become the second generation parents (P2). After approximately 10 weeks of treatment following weaning of the last FI litter, the P2 adults will be bred to produce the F2 litters. Exposures of PI and P2 adults rats to the test material will continue until the adults are sent to necropsy. All rats will be housed continuously in exposure chambers following the initial exposure to the test material, except during late gestation and throughout lactation periods, when female rats will be housed outside of the exposure chambers during non-exposure periods.. Maternal rats will not be exposed to the test material after day 20 of gestation (as calculated from day 0 of gestation via sperm-positive vaginal lavage) through die fourth day postpartum, in order to allow for parturition and initiation of lactation. During the lactation period, pups will not be placed in the exposure chambers, but will remain in the nesting boxes separated from the dam for approximately 7 hours/day on lactation days 5 through 21. Reproduction - Breeding Procedure Breeding of the PI and P2 adults will commence after approximately 10 weeks of treatment. Each female will be placed with a single male from the same dose level (1:1 mating) until pregnancy occurs or two weeks have elapsed. During each breeding period, daily vaginal lavage samples will be evaluated for the presence of sperm as an indication of mating. The day on which sperm are detected or a vaginal plug is observed in situ will be considered day 0 of gestation. Sperm- and plug-positive females will then be separated and placed back into wire mesh, stainless steel cages. If mating has not occurred after two weeks, the animals will be separated without further opportunity for mating. For the P2 mating, cohabitation of male and female litter mates will be avoided. BFG41037 October 11,1995 DRAFT BFG 00551 -8- Reproduction - Culling and Weaning To reduce the variation in the growth of the pups, the FI and F2 litters with a total number of pups exceeding eight will be culled on day 4 postpartum. Culled litters will be reduced to a total of eight pups, four males and four females, if possible. Pups to be culled will be selected using a computer generated randomization procedure. Litters with eight or fewer pups will not be culled. Preferential culling of runts will not be performed. Culled pups will be examined grossly for abnormalities and euthanized by the deposition of sodium pentobarbital (Succumb Butler, Columbus, OH) into the oral cavity. Weaning of all litters will be done on lactation day 21. Weanlings not held for prospective generations or selected for necropsy will be examined grossly for abnormalities and euthanized by CO2 inhalation. Reproduction - Physical Observations Each rat on study will be observed twice daily (a.m. and pm.) for mortality, morbidity and moribundity as well as availability of feed and water. Changes in behavior or demeanor and indications of overt toxicity will be evaluated during the a.m. or p.m. observation. In addition, a thorough clinical examination will be conducted on all animals prior to the start of the study and weekly thereafter. This examination will include thorough evaluations of the skin and fur, mucous membranes, respiration, nervous system and behavior pattern. All adult rats found dead or in moribund condition will be submitted for a gross pathologic examination. Adult rats found dead after normal working hours will be refrigerated until a necropsy can be performed. All pups found dead or pups that are euthanized in moribund condition will be examined to the extent possible for defects and/or cause of death and preserved in neutral, phosphate-buffered 10% formalin. Cannibalized pups will be examined to the extent possible and discarded. Reproduction - Body Weights and Feed Consumption All PI animals will have body weights and feed consumption (optional) recorded weekly during the 10-week pre-breeding treatment period, beginning on or before the first week of the study. Body weights for males will be recorded weekly throughout the course of the study. Sperm and plug positive October 11,1995 DRAFT BFG41038 BFG 00552 -9- females will be weighed on Days 0,7,14 and 21 of gestation. Females that deliver Utters wiU be weighed on Days 1,4,7,14, and 21 of lactation. During breeding, feed consumption will not be measured in males or females due to cohousing. FoUowing completion of the breeding periods, weekly feed consumption again will be measured in males. During gestation, feed consumption will be measured at weekly intervals in sperm and plug positive females. After parturition, feed consumption will be measured twice during the first and second week of lactation and at 2 - 3 day intervals during the last week of lactation. A similar schedule will be foUowed for the P2 generation. Reproduction - Litter Data All Utters will be examined as soon as possible after deUvery. The foUowing parameters will be recorded for each Utter total Utter size on the day of parturition (day 0), the number of Uve and dead pups on days 0,1,4,7,14, and 21 postpartum, and the sex and the weight of each pup on days 1,4 (before and after culling), 7,14, and 21 of lactation. Any visible physical abnormaUties or demeanor changes in the neonates will be recorded during the lactation period. Reproduction - Physical Maturational Landmarks All FI weanlings selected for mating will be observed daily for vaginal opening beginning on postnatal day 30 (Adams et al, 1985) or preputial separation beginning cm day 35 (Korenbrot et al., 1977). If there is a treatment* related effect observed on the FI sex ratio, age of vaginal opening or age of preputial separation, then anogenital distance wUl be measured on post natal day 4 for all F2 pups. Reproduction * Estrous Cycling Estrous cycle length and normaUty will be evaluated daily by vaginal lavage (Cooper et al., 1993) on the first 15 PI and P2 females starting three weeks prior to mating and continuing throughout cohabitation. October 11,1995 BFG41039 DRAFT BFG 0055j -10- Reproduction - Pathology - Adult Rats A complete necropsy will be conducted by a team of trained individuals under the direct supervision of a veterinary pathologist on all PI and P2 adults. The scheduled necropsy will be performed after the last litter of the respective generation has been weaned. Adult males will be fasted overnight, anesthetized with methoxyflurane and euthanized. Adult females will be necropsied on day 2 of diestrus whenever possible. This will be accomplished by monitoring (by vaginal lavage) for the occurrence of at least one estrous cycle after which time females found to be in day one of diestrus will be fasted overnight and necropsied on the following morning. The expected, subsequent stage of the estrous cycle (day 2 of diestrus) will be confirmed by vaginal lavage on the day of necropsy, prior to euthanasia. Based upon the results of these smears, exclusion of appropriate data parameters used for statistics will be performed for females not found to be in the appropriate stage of the estrous cycle (day 2 of diestrus) on the day of necropsy. The fasted females will be euthanized as described for the males. The eyes of both males and females will be examined in situ by gently pressing a moistened glass slide against the cornea and observing the eyes under fluorescent light. The uteri of all cohabited females will be examined for the presence and number of implantation sites. Tissues routinely collected (Table 3) will be saved from these rats and preserved in neutral, phosphate-buffered 10% formalin, with the following exceptions. The testes and epididymides will be preserved in Boilin's fixative. The lungs will be infused with formalin to their approximate normal inspiratory volume. The nasal cavity will be flushed with formalin via the pharyngeal duct to ensure rapid fixation of the tissue. Moribund rats and those dying spontaneously will be necropsied in a similar manner. However, body and organ weights will not be recorded. Reproduction - Organ Weights - Adult Rats The following organs on the first 15 PI and P2 parental animals will be weighed at the scheduled terminal necropsy: uterus, ovaries, testes, single epididymis (total and cauda), seminal vesicles (with coagulating glands and their fluids), prostate, brain, liver, kidneys, lungs, adrenal glands, spleen, and thymus, and the organ-to-body weight ratios calculated. October 11,1995 DRAFT BFG41040 BFG 00554 -11- Reproduction - Histology - Adult Rats Histologic examination of potential target organs and reproductive tissues (Table 3) will be performed on the control and high dose groups. Examination of tissues from the low and middle dose groups will be limited to those tissues that demonstrate treatment-related histologic changes in the high dose group. Only the right ovary will be routinely processed for standard microscopic examination. The left ovary will be saved for possible oocyte quantification. If deemed necessary by the study sponsor, oocyte quantification will include evaluation of a minimum of ten sections, randomly selected from one completely sectioned ovary per female of the high-dose and control groups. Ovarian follicles will be placed into one of three categories as described by Plowchalk et al., (1993). The total number of follicles and the number of follicles in each of the three categories will be evaluated. Ovaries from the low and middle dose groups may be evaluated if treatment-related changes are observed in the high dose group. A complete set of tissues (excluding the left ovary for females), encompassing all organs listed in Table 3, will be prepared from all rats dying spontaneously or euthanized in a moribund condition and examined in an attempt to determine cause of death. Reproduction - Sperm Count, Motility and Morphology For the first 15 PI and P2 males at termination, samples of sperm from the distal cauda epididymis will be collected for evaluation of percent progressively motile sperm and possible evaluation of sperm morphology. The entire right cauda epididymis will be weighed and then minced in saline to enumerate the total number of sperm (cauda reserves). Sperm motility and count will be determined with the use of the Hamilton-Thom (HTM) Integrated Visual Optical System (TVOS) motility analyzer (Hamilton-Thom Research, Beverly, Massachusetts). All samples for motility analyses will be video recorded or recorded digitally on disk and the recording kept as raw data. Sperm samples will be prepared for morphological evaluation and saved, but will not be evaluated unless deemed necessary by the study sponsor. October 11,1995 BFG41041 DRAFT BFG 00555 -12- Reproduction - Pathology - Weanling Rats At the time of weaning, 1 pup/sex/litter/dose from the FI and F2 litters will be randomly selected for a complete necropsy by a team of trained individuals under the direct supervision of a veterinary pathologist In order to control for variation in body and organ weight, all FI and F2 pups selected for a complete necropsy will be euthanized at the same age. Pups will be anesthetized with methoxyflurane and euthanized. Terminal body weights will be recorded. Gross pathologic examination and preservation of tissue samples (Table 3) will be performed as described above for adults. Reproduction - Organ Weights - Weanling Rats For the first 15 male and female FI and F2 pups that are examined macroscopically (one/sex/litter), the following organs will be weighed: ovaries, testes, brain, liver, kidneys, adrenal glands, spleen and thymus. Reproduction - Histology - Weanling Rats Organs that demonstrate treatment-related effects in weanlings will be examined microscopically in the control and high dose groups. Examination of tissues from the low and middle dose groups will be limited to those tissues which demonstrate treatment-related histologic changes in the high dose group. Microscopic examination will also be made of all tissues showing gross pathologic changes. Developmental - Experimental Design. Groups of 25 adult time-mated female rats will be exposed to 0,10,100 or 1100 ppm of the test material via inhalation, for 6 hours/day, on gestation days 6 through 20. These exposure levels correspond to oral equivalent doses of approximately 0,9.2,92 and 1012 mg/kg/day assuming ventilation rates of 1 1/min/kg, 100 percent absorption and a 6 hour/day exposure The high concentration of vinyl chloride was selected based on the oral equivalent of the limit dose of 1 mg/kg body weight/day. For further discussion of dose level selection refer to the "Exposure Concentrations" section of this protocol. October 11,1995 DRAFT BFG41042 BFG 00556 -13- The overall chronology and study design for the developmental portion of this study is depicted in Tables 1 and 2, respectively. Developmental - Breeding Procedure Sexually mature, adult virgin females, approximately 10 weeks of age and weighing approximately 200 - 250 grams, will be naturally mated with male rats (one male/female) of the same strain at the Charles River Breeding Laboratory. Females will be checked for plugs die following morning and those found with a vaginal plug will be removed from the male's cage. The day on which a vaginal plug is detected will be considered Day 0 of gestation. Day 0 body weights will be provided by Charles River Breeding Laboratory, and maintained in the study record. Rats will be shipped on Day 0 or 1 of gestation and will arrive at our laboratory on Day 1 or 2 of gestation. Developmental - Maternal Observations All animals will be observed daily during the study for alterations in behavior or demeanor as previously described under die Reproduction Study "Physical Observation" section, with the exception that a thorough weekly clinical examination will not be conducted. Any animal which dies, appears moribund or shows indications of early termination of pregnancy will be submitted for a complete necropsy as described for the Reproduction Study. Body weights will be recorded on gestation days 0,6,14 and 21. On Day 21 of gestation, all surviving animals assigned to the developmental study will be euthanized by carbon dioxide inhalation and given a limited necropsy. Any obvious structural or pathologic changes noted in the adult will be recorded and the weight of the liver, kidneys and gravid uteri will be recorded. Liver, kidneys and gross lesions will be preserved in neutral, phosphate-buffered 10% formalin, but microscopic examination of tissues will not be conducted unless deemed necessary to interpret other observations made during the study or requested by the sponsors. October 11,1995 5FG4l043 DRAFT BFG 0055 -14- Developmental - Fetal Observations At necropsy, the uterine horns will be exteriorized through an abdominal incision and the following data recorded: 1) the number and position of fetuses in utero, 2) the number of live and dead fetuses, 3) the number and position of resorptions, 4) the number of corpora lutea, 5) the sex and body weight of each fetus, and 6) any gross external alteration. The uteri of apparently non-pregnant animals will be stained with an aqueous 10% solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of early resorptions. Corpora lutea will not be recorded for females that are not visibly pregnant at C-section or for females that are submitted for necropsy prior to Day 21. At least one-half of the fetuses in each litter, selected using a table of random numbers, will be examined immediately by dissection under a low power stereo-microscope for evidence of visceral alterations (Staples, 1974). The heads of rat fetuses examined by dissection will be removed, placed in Bouin's fixative and examined by the serial sectioning technique of Wilson (1965). All fetuses will then be preserved in alcohol, eviscerated and stained with alizarin red-S (Dawson, 1926). Skeletal examination will be conducted on all fetuses that were not given visceral examinations. Statistical Evaluation. Descriptive statistics (means and standard deviations) will be reported for feed consumption. Body weights, gestation/lactation body weight gains, organ weights, and sperm count per gram cauda epididymis and percent motile sperm will first be evaluated by Bartlett's test for equality of variances. Based upon the outcome of Bartlett's test, either a parametric or nonparametric analysis of variance (ANOVA) will be performed. If the ANOVA is significant, a Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction will be performed. Gestation length, average time to mating, number of corpora lutea, number of implants, litter size, age at vaginal opening and age at preputial separation will be analyzed using a nonparametric ANOVA. If the ANOVA is significant, the Wilcoxon Rank-Sum test with Bonferroni's correction will be performed. Statistical outliers will be identified by the method of Grubbs October 11,1995 DRAFT BFG41044 BFG 00558 -15- (1969) and will be routinely excluded from analysis for feed consumption only. Outliers for other endpoints will only be excluded from analysis for documented, scientifically sound reasons. Fertility indices and pregnancy rate will be analyzed by the Fisher exact probability test and Bonferroni's correction will be used for multiple testing of groups in comparison to a single control. Evaluation of the neonatal sex ratio will be performed by the binomial distribution test. Survival indices will be analyzed using die litter as the experimental unit by the Wilcoxon test as modified by Haseman and Hoel (1974). Statistical evaluation of the frequency of pre-implantation loss, resorptions and fetal alterations among Utters and the fetal population will be performed using a censored Wilcoxon test with Bonferroni's correction. Nonpregnant females, females pregnant foUowing staining or females having totally resorbed Utters will be excluded from the appropriate analyses. The nominal alpha levels to be used are as foUows: Bartlett's Test (Winer, 1971) a=0.01 Parametric ANOVA (Steel and Torrie, 1960) a=0.10 Nonparametric ANOVA a=0.10 (HoUander and Wolfe, 1973) Dunnett's Test (Winer, 1971) Wilcoxon Rank-Sum Test (Hollander and Wolfe, 1973) a=0.05, two-sided a=0.05, two-sided with Bonferroni correction (Miller, 1966) Fisher's Test (Siegel, 1956) a=0.05, two-sided Censored Wilcoxon Test (Haseman and Hoel, 1974) a=0.05, two-sided Outlier Test (Grubbs, 1969) a=0.02, two-sided Binomial Distribution Test (Steel and Torrie, 1960) a=0.05, two-sided Because numerous measurements are statistically compared in the same group of animals, the overall false positive rate (Type I errors) will be much greater than the cited alpha levels would suggest. Thus, the final October 11,1995 DRAFT BFG41045 BFG 00559 -16- interpretation of numerical data will consider statistical analyses along with other factors such as dose-response relationships and whether the results are significant in the light of other biologic and pathologic findings. Safety Precautions. Standard safety precautions will be followed during the conduct of this study. Quality Assurance. Permanent records of all data generated during the course of this study, the protocol, any addenda to the protocol, and the final report will be available for inspection by the Quality Assurance Unit. All data generated including the protocol, addenda, and final report will be archived at Health and Environmental Sciences, The Dow Chemical Company, Midland, Michigan. October 11,1995 DRAFT BFG41046 BFG 00560 -17- REFERENCES Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J. (1985) Collaborative Behavioral Teratology Study: Protocol Design and Testing Procedures. Neurobehavioral Toxicology and Teratology 7, 579-586. ATSDR, (1993). Toxicology Profile for Vinyl Chloride U5. Department of Health and Human Services. Public Health Service. Agendy for Toxic Substances and Disease Registry. Cooper, R. L., Goldman, J. M. and Vandenbergh, J. G. (1993). Monitoring of the estrous cyde in the laboratory Rodent by vaginal lavage. In Methods in Toxicology. Volume 3. Part B. Female Reproductive Toxicology. (J. J. Heindel and R. E. Chapin and, Eds.). Academic Press, Inc, New York. Dawson, A. B. (1926). A Note on the Staining of the Skeletons of Cleared Specimens with Alizarin Red-S. Stain Tech. 1:123*124. EEC (1988). European Economic Community. Methods for the Determination of Toxicity. Official Journal of the European Communities, Vol., 31, No. L133, May 30,1988. ISSN 0378-6978. EPA (1985). Environmental Protection Agency Toxic Substances Control Act Test Guidelines, Final Rule. 40 CFR Part 798, September 27,1985, pp 3942639433. EPA (1990). Environmental Protection Agency. Toxic Substances Control Act; Good Laboratory Practice Standards. 40 CFR Part 792 (1 July 1990 Edition). FDA (1988). Food and Drug Administration Good Laboratory Practice for Nondinical Studies. 21 CFR Part 58 (April 1,1988 Edition). Grubbs, F. E. (1969). Procedures for Detecting Outlying Observations in Samples. Technometrics 11,1-21. October 11,1995 DRAFT BFG41047 BFG 00561 -18- Haseman, J. K. and Hoel, D. G. (1974). Tables of Gehan's Generalized Wilcoxon Test with Fixed Point Sensoring. J. Statis. Comput. Simul. Vol. 3, 117-135. Hollander, M. and Wolfe, D. A. (1973). Nonparametric Statistical Methods, John Wiley, New York, NY. Johanson, G. (1990). NEG and NIOSH Basis for an Occupational Health Standard: Propylene Glycol Ethers and Their Acetates. Arbete och Halsa, 32. Kopf, R, Lorenz, D. and Salewski, E. (1964). [The Effect of Thalidomide on the Fertility of Rats: In an Examination of Two Generations.) - Containing the Procedure for Staining Implantation Sites of Fresh Rat Uteri. NaunynSchmiedebergs Arch. Exp. Path. Pharmacol. 247:121-135. Korenbrot, C. C., Huhtaniemi, IT. and Weiner, R L (1977) Preputial separation as an external sign of pubertal development in the male rat. Biology of Reproduction 17,298-303. Miller, R G., Jr. (1966). Simultaneous Statistical Inference. McGraw-Hill, New York, NY. Miller, R R, Letts, R L., Potts, W. J. and McKenna, M. J. (1980). Improved Methodology for Generating Controlled Test Atmospheres. Am. Ind. Hyg. Assoc. J. 4,844-846 OECD (1981). Organisation for Economic Co-Operation and DevelopmentGuidelines for Testing of Chemicals, Section 4-Health Effects, Paris. OECD (1982). Organisation for Economic Co-Operation and DevelopmentPrinciples of Good Laboratory Practice, ISBN 92-64-12367-9, Paris. October 11,1995 DRAFT BFG41048 BFG 00562 -19- Siegel, S. (1956). Non-Parametric Statistics for the Behavioral Sciences. McGraw-Hill, New York, NY. Plowchalk, D. R., Smith, B. J. and Mattison, D. R. (1993). Assessment of toxicity of the ovary using follicle quantitation and morphometries. In Methods in Toxicology. Volume 3. Part B. Female Reproductive Toxicology. (J. J. Heindel and R. E Chapin and, Eds.). Academic Press, Inc, New York. Staples, R. E. (1974). Detection of Visceral Alterations in Mammalian Fetuses. Teratology 9:37 (Abstract). Steel, R. G. D. and Torrie, J. H. (1960). Principles and Procedures of Statistics. McGraw-Hill, New York, NY. Wilson, J. G. (1965). Method for Administering Agents and Detecting Malformations in Experimental Animals. In: Teratology: Principles and Techniques (J.G. Wilson and J. Warkany, eds.). University of Chicago Press, Chicago. Winer, B. J. (1971). Statistical Principles in Experimental Design, 2nd edition. McGraw-Hill, New York, NY. October 11,1995 DRAFT BFG 00563 -20- TABLE 1 VINYL CHLORIDE: COMBINED INHALATION TWO-GENERATION REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDY IN CD RATS CHRONOLOGY OF EVENTS WEEKS ON STUDYPI/Developmental_________________ F1ZE22__________________________ I- 10 Exposure of PI males and females prior to first mating. Exposure (gestation days 6-20) of the dams for developmental toxicity evaluation Necropsy of developmental toxicity dams and fetal evaluations II- 12 PI mating period for FI litters. 14-15 FI bom and litters culled on day 4 post-partum to 8 pups each. 17-18 FI litters weaned on day 21 post-partum; offspring selected for P2 adults; 1 FI pup/sex/dose/litter selected for necropsy; remaining pups euthanized. 19-28 Necropsy PI adults. Exposure of P2 males and females prior to first mating. 29-30 32-33 Mating period of P2 for F2 litters. F2 bom and litters culled on day 4 post-partum to 8 pups each. 35-36 BFG41050 F2 litters weaned on day 21 post-partum; 1F2 pup/sex/dose/litter selected for necropsy; remaining pups euthanized. 37 Necropsy P2 adults. October 11,1995 DRAFT BFG 00564 -21- TABLE 2 VINYL CHLORIDE: COMBINED INHALATION TWO-GENERATION REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDY IN CD RATS STUDY DESIGN EXPOSURE LEVELS (PPM) 0 NO. NO. MALES 30 NO. FEMALES 30 Reproductive 25 Development 10 30 30 Reproductive 25 Development 100 30 30 Reproductive 25 Development 1000 30 TOTAL= 120 30 Reproductive 25 DeveloDment 220 PARAMETERS REPRODUCTION STUDY Animal Observations Body Weights (parental and neonatal) Feed Consumption (optional) Necropsy (parental and weanlings) Organ Weights (parental and weanlings) Gross Pathology (parental and weanlings) Histopathology (parental and weanling) Fertility Indices Estrous Cycling and Sperm Analyses Developmental Landmarks Neonatal Survival DEVELOPMENTAL STUDY Animal Observations Body Weights (maternal and fetal) Feed Consumption (optional) Necropsy Limited Organ Weights Gross Pathology Uterine Examination External, Visceral and Skeletal Examination BFG41051 October 11,1995 DRAFT BFG 00565 > Q D Ct--O u >< 2 j < H Z ui 2 cO_i ui > ui O Q > sc i y d o Q H < g a. <n Sj Qui >06 UV1I N -N Ul gss aQ. ss 2 Ul 9 O 2 O Q Z < Q ui S J J O <J c/) <on0 -<J X ss z < _j u z < X X u uUZil < a<oJ. U06l Q Z_<i aa<.j u . i *(A 3 2 > X H a 5 U3l 0 1 <> . j &3> S sf- 3 5 | 3 2 Ul Q i &z Xa. < y< Z < 2< 25 < Ul 22 2 3 mU1 Qui 8 CQ 2 O u5 8u CO 9 8s z Si 3 o Q5 o xi-i u S3 < zui 0a6 < < u<i > i < 33 y 4 Ul Z S s06 < 2 Ul Z 8 z i co 2 3 UUl u z H < X > U06l u <5 zoo-1 Uu zo 3 8 2 | SS 2 3 xd PITUITARY* JEJUNUM BFG41052 BFG 00566 TISSUE SELECTED FOR HISTOPATH< OC1C EVALUATION. Hb < aftS