Document 7OXOGRwNDxqmN00QkVEwr14qe

CHEMICAL MANUFACTURERS ASSOCIATION March 23,1995 Dear Vinyl Chloride Health Committee Members: A March 22,1995 letter to William Cibulus of ATSDR is enclosed for your review and files. I will contact Mr. Cibulus next week to schedule a meeting. If you have any questions, please call me at (202) 887-1192. Sincerely, It&X) --* Hasmukh C. Shah, Ph.D. Manager, Vinyl Chloride Panel Enclosures 2501 M Street, NW, Washington, DC 20037 Telephone 202-887-1100 Fax 202-887-1237 Responsible Care' A Public Commitment 3<*3> in OCO) CO CHEMICAL MANUFACTURERS ASSOCIATION March 22, 1995 William Cibulas, Ph.D. Chief, Research Implementation Branch Division of Toxicology Agency for Toxic Substances & Disease Registry 1600 Clifton Road, N.E. Mailstop E29 Atlanta, Georgia 30333 Dear Dr. Cibulas: On January 23, Dr. Christopher DeRosa responded to my letter of November 28, 1994 indicating the intent of the Chemical Manufacturers Association (CMA) Vinyl Chloride Panel to address certain data needs for vinyl chloride identified by the Agency for Toxic Substances and Disease Registry (ATSDR). Dr. DeRosa requested that the Panel take the lead role in developing the protocol for a two-generation reproductive toxicity study of vinyl chloride by the inhalation route. We have prepared the enclosed protocol of such a study for ATSDR review. Pursuant to your telephone conversation with our counsel, Caffey Norman, we have not attempted to identify dose levels at this time. Dose levels would be determined after a review of existing literature or, if necessary, following a range-finding study, in either event in consultation with ATSDR. You will note that in a few places information has been redacted from the enclosed protocol. The purpose of these deletions is to remove certain references that would limit the study to a particular laboratory. Such information will be included when a final protocol is submitted for review. As Mr. Norman discussed with you, selecting dose levels and otherwise providing a completely final protocol for your review would require several additional weeks. We believe that it would be more useful if this time were devoted to considering ways that the enclosed protocol might be enhanced to include measures of 2501 M Street, NW, Washington, DC 20037 Telephone 202-887-1100 Fax 202-887-1237 L J Responsible Care' 11 APublicCommitment } R&S146814 William Cibulas, March 22, 1995 Page 2 Ph.D. developmental toxicity. In this regard, Dr. DeRosa's letter requested clarification of the statement in my letter of November 28 that "[i]n light of the existing two-species inhalation developmental toxicity study, and other available data concerning the developmental toxicity of vinyl chloride, we would like to discuss how [the reproductive toxicity] study and other available data might be enhanced to include measures of developmental toxicity as an alternative to the two-species developmental toxicity study referred to EPA." This passage was unfortunately garbled during our internal review and the phrase "and other available data" was mistakenly repeated. The italicized words should not appear in the sentence. Our intent is to determine in consultation with ATSDR scientists whether it is possible to include measures of developmental toxicity of interest to ATSDR in the protocol for the two-generation reproductive toxicity study that is enclosed for your review. To facilitate this discussion, we also have enclosed a review of the available data on the developmental toxicity of vinyl chloride. We look forward to meeting with ATSDR scientists to discuss the strengths and weaknesses of the existing developmental toxicity data and options to obtain any needed additional data that do not require a full two-species developmental toxicity study. We look forward to a meeting between scientists from the Vinyl Chloride Panel member companies and ATSDR in the near future to discuss the enclosed protocol, the utility of the available developmental toxicity data, and the ways the enclosed protocol might be enhanced. Sincerely, R&S146815 Enclosures cc: W. Caffey Norman, Esq. Hasmukh C. Shah, Ph.D. Manager, Vinyl Chloride Panel 2 INTRODUCTION Prior Toxicity Data. (TO BE INSERTED) Objective, The objective of the two-generation inhalation reproduction study outlined in this protocol is to evaluate the effects of the test material on the reproductive capability 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 CoOperation 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 Practice. 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 . 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. Reproductive Toxicology 01 to this Animal Care and Use Activity. 9l99rVS'0O 3 MATERIALS AND METHODS The test material used for this study was obtained from X(name), X(city), X(state) and was identified as (Lot number of the test material). The purity of the test material was reported to be xx.x% (reference). 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 6-month 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: Test Species and Husbandry. Male and female CD rats (Charles River Breeding Laboratory, Kingston, NY) approximately four weeks of age will be purchased for the study. Animals will be ordered to ensure that a sufficient number of animals of acceptable health and R&S146817 4 weight are available to conduct the study as designed. This strain of rat has been selected because of its general acceptance and suitability for toxicity 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 for approximately two weeks . For the randomization procedure, the rats will be weighed and ranked according to body weight and those from the extremes of the distribution will be identified and removed from the population until only the number of animals required for the study remain. These rats 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 file. 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 the 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 deotized cage board to minimize odor and aid in maintaining a clean 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 1Fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). R&S146818 5 six hour per day exposures at which time feed will be withheld. Municipal drinking water will be available ad libitum 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 will be analyzed for chemical parameters and biological contaminants by . In addition, specific analyses for chemical contaminants will be conducted at periodic intervals . Exposure Chamber. Animals will be housed and exposed to test material vapors in a 14.5 m.3 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 J-tube method (Miller et al., 1980). Liquid test material will be metered into a glass Jtube assembly and vaporized by a preheated stream of compressed air (up to 100 1/min) passing through die 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 a total flow rate of 29001/min and the desired concentration of test material vapors. Chamber Monitoring. The concentration of the test material in each chamber will be measured at least once per hour using a MIRAN 1A infrared spectrophoto-meter (Foxboro Analytical, Norwalk, CT). Analytical equipment will be calibrated using standards having a known test material vapor concentration contained in 90 liter 6V89^VS18y 6 SARAN* film gas bags prior to the first exposure and at least monthly thereafter. Daily checks of the analytical equipment will be performed prior to each exposure period using a single the test material standard concentration. In addition, the amount of the test material used each day will be recorded and the nominal concentrations (amount of the test material used/total chamber airflow) of the 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. 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 presssure 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 (HMP112A, 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. * Trademark of The Dow Chemical Company * Trademark of SAGIAN Indianapolis, Indiana. R&S146820 7 Rats will be exposed to target concentrations of 0, X, Y or Z ppm of the test material. These dose levels correspond to an oral equivalent dose of approximately 0, X, Y and Z mg/kg/day assuming ventilation rates of 1 1/min/kg and 100 percent absorption. The chosen concentrations were selected by the sponsor and based upon the results of the inhalation toxicity studies previously discussed. Experimental Design. Groups of 30 male and 30 female rats will be exposed to 0, X, Y or Z 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 for this study is depicted in Table 1. The treatment of the first generation parental (PI) rats will begin at approximately 6 weeks of age. After approximately 10 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 tire 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. 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 the 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. Breeding Procedure R & S 146821 8 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 either three estrous cycles or two weeks has 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. 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 Beuthanasia-D Special (Schering Corporation, U.S.A., Kenilworth, NJ) into the oral cavity. Weaning of all litters will be done 21 days after delivery. Weanlings not held for prospective generations or selected for necropsy will be examined grossly for abnormalities and euthanized by CO2 inhalation. Physical Observations Each rat on study will be observed twice daily (a.m. and p.m.) for mortality, morbidity and moribundity as well as availability of feed and water. In addition, 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 R&S146822 9 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. Body Weights and Feed Consumption All PI animals will have body weights and feed consumption 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 females will be weighed on Days 0,7,14 and 21 of gestation. Females that deliver litters will 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. Following 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 dining the first and second week of lactation and at 2 - 3 day intervals during the last week of lactation. A similar schedule will be followed for the P2 generation. Litter Data All litters will be examined as soon as possible after delivery. The following parameters will be recorded for each litter: total litter size on the day of parturition (day 0), the number of live 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 abnormalities or demeanor changes in the neonates will be recorded during the lactation period. 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 on day 35 (Korenbrot et al, 1977). If there is a treatment-related effect R&S146823 10 observed on the FI sex ratio, age of vaginal opening or age of preputial separation, then anogenital distance will be measured on post natal day 4 for all F2 pups. Estrous Cycling Estrous cycle length and normality will be evaluated daily by vaginal lavage (Cooper et al., 1993) for all PI and P2 females starting three weeks prior to mating and continuing throughout cohabitation. 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 euthanatized. Adult females will be necropsied on day 2 of diestrus whenever possible. This will be accomplished by monitoring (by vaginal lavage) for the occurence 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 cohabitated females will be examined for the presence and number of implantation sites. Tissues routinely collected (Table 2) 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 Bourn'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 R &S 146824 11 the tissue. Moribund rats and those dying spontaneously will be necropsied in a similar manner. However, body and organ weights will not be recorded. Organ Weights - Adult Rats The following organs of all PI and P2 parental animals will be weighed: 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. Histology - Adult Rats Histologic examination of potential target organs and reproductive tissues (Table 2) will be performed on the control and high dose groups. Examination of tissues from the low and middle groups will be limited to those tissues which demonstrate treatment-related histologic changes in the high dose group. Only the right ovary will be routinely processed for standard microscopic examiantion. 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 2, will be prepared from all rats dying spontaneously or euthanized in a moribund condition and examined in an attempt to determine cause of death. Sperm Count, Motility and Morphology For all PI and P2 males at termination, samples of sperm from the distal cauda epididymis or the proximal vas deferens 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 R&S146825 12 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 (IVOS) motility analyzer (HamiltonThom Research, Beverly, Massachusetts). All samples for motility analyses will be videorecorded 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. 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 euthanatized. Terminal body weights will be recorded. Gross pathologic examination and preservation of tissue samples (Table 2) will be performed as described above for adults. Organ Weights - Weanling Rats For all 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. 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 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. Statistical Evaluation. Descriptive statistics (means and standard deviations) will be reported for feed consumption. Body weights, gestation/lactation body weight gains, organ R&S146826 13 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, 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 (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 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 die neonatal sex ratio will be performed by the binomial distribution test. Survival indices and other incidence data among neonates will be analyzed using the litter as the experimental unit by the Wilcoxon test as modified by Haseman and Hoel (1974). The nominal alpha levels to be used are as follows: Bartlett's Test a=0.01 (Winer, 1971) Parametric ANOVA a=0.10 (Steel and Torrie, 1960) Nonparametric ANOVA a=0.10 (Hollander and Wolfe, 1973) Dunnett's Test (Winer, 1971) a=0.05, two-sided Wilcoxon Rank-Sum Test a=0.05, two-sided with (Hollander and Wolfe, 1973) Bonferroni correction (Miller, 1966) Fisher's Test a=0.05, two-sided (Siegel, 1956) Censored Wilcoxon Test a=0.05, two-sided (Haseman and Hoel, 1974) Outlier Test a=0.02, two-sided RSS146827 14 (Grubbs, 1969) 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 interpretation of numerical data will consider statistical analyses along with other factors such as doseresponse 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 avaiable for inspection by .. All data generated including the protocol, addenda, and final report will be archived at R&S146828 15 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 Z, 579-586. Cooper, R. L., Goldman, J. M. and Vandenbergh, J. G. (1993). Monitoring of the estrous cycle 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. EEC (1988). European Economic Community. Methods for the Determination of Toxicity. Official Journal of the European Communities, Vol., 31, No. L 133, 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 3942639428. 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 Nonclinical Studies. 21 CFR Part 58 (April 1,1988 Edition). Grubbs, F. E. (1969). Procedures for Detecting Outlying Observations in Samples. Technometrics IX 1-21. 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. R&S 146829 16 Johanson, G. (1990). NEG and NIOSH Basis for an Occupational Health Standard: Propylene Glycol Ethers and Their Acetates. Arbete och Halsa, 32. Korenbrot, C. C., Huhtaniemi, IT. and Weiner, R. I. (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. Siegel, S. (1956). Non-Parametric Statistics for the Behavioral Sciences. McGrawHill, 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. Steel, R. G. D. and Torrie, J. H. (1960). Principles and Procedures of Statistics. McGraw-Hill, New York, NY. Winer, B. J. (1971). Statistical Principles in Experimental Design, 2nd edition. McGraw-Hill, New York, NY. R&S146830 17 TABLE 1 A TWO-GENERATION INHALATION REPRODUCTION STUDY IN CD RATS CHRONOLOGY OF EVENTS WEEKS ON -STUDYElF1/F2-------------------------------- & 1-10 Exposure of PI males and females prior to first mating. 11-12 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 35-36 pup/sex/dose/litter 37 Mating period of P2 for F2 litters. F2 bom and litters culled on day 4 post-parturn to 8 pups each. F2 litters weaned on day 21 post-partum; 1 F2 selected for necropsy; remaining pups euthanized. Necropsy P2 adults. R&S 146831 t 21 TABLE 2 Two-Generation Inhalation Reproduction Study in CD Rats TISSUES COLLECTED AND PRESERVED AT NECROPSY ADRENALS KIDNEYS AORTA LACRIMAL/HARDERIAN GLANDS AUDITORY SEBACEOUS GLANDS LARYNX BONE (INCLUDING JOINT) LIVER BONE MARROW BRAIN (CEREBRUM, BRAINSTEM, CEREBELLUM) LUNGS MAMMARY GLAND CECUM LUMBAR) MEDIASTINAL LYMPH NODE CERVIX* COAGULATING GLANDS* COLON DUODENUM EPIDIDYMIDES* ESOPHAGUS MEDIASTINAL TISSUES MESENTERIC LYMPH NODE MESENTERIC TISSUES NASAL TISSUES* ORAL TISSUES OVARIES EYES GROSS LESIONS* HEART OVIDUCTS PANCREAS PARATHYROID GLANDS ILEUM J JEJUNUM PERIPHERAL NERVE PITUITARY* *TISSUE SELECTED FOR HISTOPATHOLOGIC EVALUATION. PROSTATE RECTUM SALIVARY GLANDS SEMINAL VESICLES SKELETAL MUSCLE SKIN SPINAL CORD (CERVICAL, THORACIC, SPLEEN STOMACH TESTES THYMUS THYROID GLAND TONGUE TRACHEA URINARY BLADDER UTERUS VAGINA S89HS1SH REVIEW OF LITERATURE CONCERNING THE POTENTIAL DEVELOPMENTAL TOXICITY OF VINYL CHLORIDE Introduction A number of experimental studies of the possible developmental toxicity of vinyl chloride (VC) were reported in the 1970's, after VC had been shown to be a human and animal hepatocarcinogen. The number of such studies available for review in the open literature is small, and some are of questionable quality. On balance, however, the literature suggests that in utero exposure to VC does not lead to developmental toxicity in experimental animals. Summary of studies The largest teratology study of VC exposure was performed in the Toxicology Research Laboratory of The Dow Chemical Company, under the sponsorship of the Manufacturing Chemists Association. The results were reported by John et al. in 1977. (1) In this experiment, bred female Sprague-Dawley rats and New Zealand rabbits were exposed to air concentrations of 0,500, or 2500 parts per million (ppm) of VC for 7 hours per day during gestation. Bred female CF-1 mice were exposed to air concentrations of 0, 50, or 500 ppm of VC for 7 hours per day. Additional groups of all three species, along with corresponding levels of VC exposure, were given drinking water containing 15% ethanol (which was suspected at the time of being a potentiating agent for VC toxicity). The dams were examined for signs of maternal toxicity (decreased food consumption, decreased gestational weight gain, or change in absolute liver weight). Fetal offspring were examined for gross external, soft tissue, or skeletal anomalies. Signs of maternal toxicity were observed in all three species at the high level of VC exposure. In the mice, maternal toxicity appeared at 500 ppm VC with and without concomitant ethanol exposure. Rats exposed to 2500 ppm with and without ethanol experienced increased liver weight. Rats and rabbits exposed to 2500 ppm plus ethanol experienced decreased gestational weight gain, while those exposed to 2500 ppm without ethanol did not. The only l R&S146834 2 maternal effects observed in a lower-dose group were among the mice exposed to 50 ppm VC plus ethanol; these animals had decreased food consumption, gestational weight gain, and liver weight. No effects were seen among the mice exposed to 50 ppm VC without ethanol. Exposure to VC alone did not appear to be embryotoxic in any of the three species. The frequency of resorptions in mice exposed to 500 ppm VC was significantly higher than among the control group (13% vs. 7%), but this control frequency was unexpectedly low for the laboratory. Some decrease in fetal body weight or crown-rump length was observed in rats exposed to 500 ppm, but not in the group exposed to 2500 ppm. No effects on resorption frequency or fetal size was observed in either group of exposed rabbits. There was also no clear evidence of teratogenicity after exposure to VC alone in any of the three species at either high or low exposure concentration. No gross external or skeletal anomalies were observed among any of the three species tested. The only soft-tissue anomaly observed at greater frequency was dilated ureter among the rats exposed to 2500 ppm VC alone. The significance of this finding was regarded as uncertain, however, because the frequency of dilated ureter was significantly lower among rats exposed to 2500 ppm of VC plus ethanol. The strength of this study lies in its conclusion that "exposure of pregnant mice, rats, or rabbits to vinyl chloride by inhalation at concentrations sufficiently high to cause maternal toxicity was not teratogenic in any of the three species." This result suggests that developmental toxicity, as defined in this study, is very unlikely to occur at exposure levels not leading to frank maternal toxicity. The study is limited by the absence of microscopic tissue examination of the offspring, which may have revealed more subtle effects of exposure if any had occurred. A teratogenicity study in rats was conducted by the State Institute of Occupational Health, Budapest, Hungary, and reported by Ungvary et al. in 1978. (2) Bred female CFY rats were exposed for 24 hours per day to either 0 or 1500 ppm of VC in air, during the 1st, 2nd, or 3rd trimester of gestation. The R&S 146835 3 rats exposed to VC during the 1st trimester were also administered subcutaneous injections of bypan blue, a known teratogenic agent. Only one group of exposed dams, those exposed during the 3rd week of gestation, showed evidence of decreased weight gain. This group did not experience a change in absolute or relative liver weight, however, while the rats exposed during the 1st or 2nd week did show changes in liver weight. No maternal mortality was observed. There was some increase in resorptions among the rats exposed during the 1st or 2nd week of pregnancy. A higher implantation and birth rate was suggested as an explanation for the increase seen in the group exposed during the 2nd week, but VC toxicity was considered a probable cause of increased resorption in the group exposed during the 1st week. A small increase in the frequency of skeletal retardation was reported among the offspring exposed to VC alone during the 1st week of gestation and among the offspring of control and exposed dams also given trypan blue injections. The offspring observed to have skeletal retardation did not differ from control offspring in mean body weight. No gross or soft-tissue malformations were observed more frequently among the offspring of exposed dams. The authors concluded that "VC exposure in itself has no teratogenic effect in CFY rats, but an embryotoxic effect of VC exposure during the early stages of pregnancy at high atmospheric concentrations should be taken into consideration." It should be noted that this study's observation of a possible early embryotoxic effect occurred in the absence of clear maternal toxicity. A strength of this study is that the animals were exposed for 24 hours per day. A potential weakness was that while the intent of the investigators may have been to study the effects of exposure over the entire period of gestation, the design employed did not expose any individual animals over the entire period. A substantial weakness of the report, on the other hand, is its lack of statistical analysis of the findings. A study conducted by Mirkova et al. at the Institute of Hygiene and Occupational Diseases, Sofia, Bulgaria, was reported in 1978. (3) Pregnant R&S146836 4 Wistar rats were exposed to approximately 2.4 ppm of VC for 24 hours per day throughout the period of gestation. Control "values" were referred to in the report of the study, but their source was not explicitly described. Total embryonic mortality was stated to be twice as great in exposed animals as in the control values. The overall increase was attributed to an 8-fold increase in early post-implantation mortality. Overall reduced fetal weight was reported in the exposed group, but it was suggested that this was secondary to maternal liver damage. The data supporting this suggestion of maternal toxicity were not described. Teratogenic effects, consisting of blood vessel damage manifesting as microscopic hemorrhages, encephalocele, hydrocephalus, and ossification defects, were reported among the exposed offspring. In addition, postnatal development was reported to be affected in second and third generation offspring; the effects reported were liver and nervous system dysfunction. The value of this report is seriously limited by the inadequate description of the conditions of the experiment, the absence of clear maternal toxicity evaluation, and the lack of numerical or statistical data. Moreover, the results reported must be considered of questionable plausibility because of the extremely low VC air concentration to which the animals were exposed. Finally, another teratogenicity study was reported by Salnikova & Kitovskaya of the Institute of Hygiene and Occupational Diseases, U.S.S.R. Academy of Medical Sciences, in 1980. (4) In the experiment, pregnant Wistar rats were exposed for 4 hours per day throughout the period of gestation to 0,1.9, or 13.9 ppm of VC in air. Outcomes evaluated in the study were maternal toxicity, embryotoxicity, teratogenicity, and post-natal development. The dams exposed to the higher VC level were reported to have significantly decreased erythrocyte counts and urinary hippuric acid levels than control dams. No effects were observed in the dams exposed to the lower level of VC. No other evidence of maternal toxicity was described in the report. R&S146837 5 There was no evidence of effect on pre- or post-implantation embryonic mortality in either exposure group. There did appear to be increased frequency of hemorrhage in the microscopic examinations of the fetuses in both exposure groups compared to the controls, and of intumescence in the offspring in the higher exposure group. Exposure was not observed to have any effect on lactation in the dams allowed to give birth, nor on survival of offspring from these dams. The live pups from both exposure groups were reported to show adverse behavioral and physiological effects, such as blood count changes, differences in selected organ weights, and urinary hippuric acid levels. In this study, maternal exposure at the then-current allowable occupational level in the U.S.S.R. (about 14 ppm) was compared with exposure nearly 1 order of magnitude less (about 2 ppm). The authors concluded that significant embryotropic effects were observed at the allowable level and recommended that this level be re-evaluated. The strengths of this study were in the highly detailed examinations performed on both dams and pups. The study's significance is limited by the apparently subtle nature of the effects reported, the limited description of the experimental methods, and the cursory discussion of the clinical significance of the effects observed. Conclusion This brief review of the literature demonstrates that the database concerning developmental effects of in utero VC exposure is not large and is quite inconsistent in terms of the experimental methods used, air concentrations of VC, durations of exposure of animals to VC, and data quality. Nevertheless, the largest and best reported of these studies, conducted with three species of experimental animals and high air concentrations, supports a conclusion that inhalation exposure of VC does not produce significant embryotoxic or teratogenic effects at levels which do not also induce significant maternal toxicity. R&S 146838 6 References 1. John JA, Smith FA, Leong BKJ, Schwetz BA. The effects of maternally inhaled vinyl chloride on embryonal and fetal development in mice, rats, and rabbits. Toxicol Appl Pharmacol 1977;39:497-513. 2. Ungvary G, Hudak A, Tatrai E, Lorincz M, Folly G. Effects of vinyl chloride exposure alone and in combination with trypan blue--applied systematically during all thirds of pregnancy on the fetuses of CFY rats. Toxicology 1978;11:45-54. 3. Mirkova Y, Mikhaylova A, Nosko M. Embryotoxic and teratogenic action of vinyl chloride. Khig Zdraveopaz 1978;23:440-443. 4. Salnikova LS, Kitovskaya IA. Effect of vinyl chloride on embryogenesis in the rat. Gigiena Truda I Professional'nye Zabolevaniia 1980;3:46-7. R&S146839 2 INTRODUCTION Prior Toxicity Data. (TO BE INSERTED) Objective. The objective of the two-generation inhalation reproduction study outlined in this protocol is to evaluate the effects of the test material on the reproductive capability 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 CoOperation 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 Practice. 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 . 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. Reproductive Toxicology 01 to this Animal Care and Use Activity. R&S146850 3 MATERIALS AND METHODS The test material used for this study was obtained from X(name), X(city), X(state) and was identified as (Lot number of the test material). The purity of the test material was reported to be xx.x% (reference). 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 6-month 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: Test Species and Husbandry. Male and female CD rats (Charles River Breeding Laboratory, Kingston, NY) approximately four weeks of age will be purchased for the study. Animals will be ordered to ensure that a sufficient number of animals of acceptable health and DeO cn cocnon 4 weight are available to conduct the study as designed. This strain of rat has been selected because of its general acceptance and suitability for toxicity 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 for approximately two weeks . For the randomization procedure, the rats will be weighed and ranked according to body weight and those from the extremes of the distribution will be identified and removed from the population until only the number of animals required for the study remain. These rats 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 file. 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 the 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 deotized cage board to minimize odor and aid in maintaining a clean 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 1Fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). R&S 146852 5 six hour per day exposures at which time feed will be withheld. Municipal drinking water will be available ad libitum 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 will be analyzed for chemical parameters and biological contaminants by . In addition, specific analyses for chemical contaminants will be conducted at periodic intervals... _ Exposure Chamber. Animals will be housed and exposed to test material vapors in a 14.5 m^ 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 J-tube method (Miller et al., 1980). Liquid test material will be metered into a glass Jtube assembly and vaporized by a preheated stream of compressed air (up to 100 1/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 a total flow rate of 29001/min and the desired concentration of test material vapors. Chamber Monitoring. The concentration of the test material in each chamber will be measured at least once per hour using a MIRAN 1A infrared spectrophoto-meter (Foxboro Analytical, Norwalk, CT). Analytical equipment will be calibrated using standards having a known test material vapor concentration contained in 90 liter R&S146853 6 SARAN* film gas bags prior to the first exposure and at least monthly thereafter. Daily checks of the analytical equipment will be performed prior to each exposure period using a single the test material standard concentration. In addition, the amount of the test material used each day will be recorded and the nominal concentrations (amount of the test material used/total chamber airflow) of the 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. 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 presssure 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 (HMP112A, 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. * Trademark of The Dow Chemical Company * Trademark of SAGIAN Indianapolis, Indiana. R&S146854 7 Rats will be exposed to target concentrations of 0, X, Y or Z ppm of the test material. These dose levels correspond to an oral equivalent dose of approximately 0, X, Y and Z mg/kg/day assuming ventilation rates of 1 i/min/kg and 100 percent absorption. The chosen concentrations were selected by the sponsor and based upon the results of the inhalation toxicity studies previously discussed. Experimental Design. Groups of 30 male and 30 female rats will be exposed to 0, X, Y or Z 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 for this study is depicted in Table 1. The treatment of the first generation parental (PI) rats will begin at approximately 6 weeks of age. After approximately 10 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. 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 the 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. Breeding Procedure R&S146855 8 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 either three estrous cycles or two weeks has 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. 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 Beuthanasia-D Special (Schering Corporation, U.S.A., Kenilworth, NJ) into the oral cavity. Weaning of all litters will be done 21 days after delivery. Weanlings not held for prospective generations or selected for necropsy will be examined grossly for abnormalities and euthanized by CO2 inhalation. Physical Observations Each rat on study will be observed twice daily (a.m. and p.m.) for mortality, morbidity and moribundity as well as availability of feed and water. In addition, 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 R&S146856 9 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. Body Weights and Feed Consumption All PI animals will have body weights and feed consumption 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 females will be weighed on Days 0,7,14 and 21 of gestation. Females that deliver litters will 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. Following 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 followed for the P2 generation. Litter Data All litters will be examined as soon as possible after delivery. The following parameters will be recorded for each litter: total litter size on the day of parturition (day 0), the number of live 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 abnormalities or demeanor changes in the neonates will be recorded dining the lactation period. Physical Maturational Landmarks All FI weanlings selected for mating will be observed daily for vaginal opening beginning on postnatal day 30 (Adams et al.r 1985) or preputial separation beginning on day 35 (Korenbrot et al, 1977). If there is a treatment-related effect R&S146857 10 observed on the FI sex ratio, age of vaginal opening or age of preputial separation, then anogenital distance will be measured on post natal day 4 for all F2 pups. Estrous Cycling Estrous cycle length and normality will be evaluated daily by vaginal lavage (Cooper et al., 1993) for all FI and P2 females starting three weeks prior to mating and continuing throughout cohabitation. 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 Utter of the respective generation has been weaned. Adult males will be fasted overnight, anesthetized with methoxyflurane and euthanatized. Adult females will be necropsied on day 2 of diestrus whenever possible. This will be accompUshed by monitoring (by vaginal lavage) for the occurence 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 cohabitated females will be examined for the presence and number of implantation sites. Tissues routinely collected (Table 2) 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 Bouin'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 R&S146858 11 the tissue. Moribund rats and those dying spontaneously will be necropsied in a similar manner. However, body and organ weights will not be recorded. Organ Weights - Adult Rats The following organs of all PI and P2 parental animals will be weighed: 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. Histology - Adult Rats Histologic examination of potential target organs and reproductive tissues (Table 2) will be performed on the control and high dose groups. Examination of tissues from the low and middle groups will be limited to those tissues which demonstrate treatment-related histologic changes in the high dose group. Only the right ovary will be routinely processed for standard microscopic examiantion. 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 2, will be prepared from all rats dying spontaneously or euthanized in a moribund condition and examined in an attempt to determine cause of death. Sperm Count, Motility and Morphology For all PI and P2 males at termination, samples of sperm from the distal cauda epididymis or the proximal vas deferens 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 R&S146859 12 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 (IVOS) motility analyzer (HamiltonThom Research, Beverly, Massachusetts). All samples for motility analyses will be videorecorded 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. 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 euthanatized. Terminal body weights will be recorded. Gross pathologic examination and preservation of tissue samples (Table 2) will be performed as described above for adults. Organ Weights - Weanling Rats For all 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. 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 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. Statistical Evaluation. Descriptive statistics (means and standard deviations) will be reported for feed consumption. Body weights, gestation/lactation body weight gains, organ R&S146860 13 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, 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 (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 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 and other incidence data among neonates will be analyzed using the litter as the experimental unit by the Wilcoxon test as modified by Haseman and Hoel (1974). The nominal alpha levels to be used are as follows: Bartlett's Test a=0.01 (Winer, 1971) Parametric ANOVA a=0.10 (Steel and Torrie, 1960) Nonparametric ANOVA a=0.10 (Hollander and Wolfe, 1973) Dunnett's Test (Winer, 1971) a=0.05, two-sided Wilcoxon Rank-Sum Test a=0.05, two-sided with (Hollander and Wolfe, 1973) Bonferroni correction (Miller, 1966) Fisher's Test a=0.05, two-sided (Siegel, 1956) Censored Wilcoxon Test a=0.05, two-sided (Haseman and Hoel, 1974) Outlier Test a=0.02, two-sided XeC>/) CD cGoi 14 (Grubbs, 1969) 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 interpretation of numerical data will consider statistical analyses along with other factors such as doseresponse 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 avaiable for inspection by .. All data generated including the protocol, addenda, and final report will be archived at R&S146862 15 REFERENCES Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, CJ. (1985) Collaborative Behavioral Teratology Study: Protocol Design and Testing Procedures. Neurobehavioral Toxicology and Teratology Z, 579-586. Cooper, R. L., Goldman, J. M. and Vandenbergh, J. G. (1993). Monitoring of the estrous cycle in the laboratory Rodent by vaginal lavage. 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R&S146864 17 TABLE 1 A TWO-GENERATION INHALATION REPRODUCTION STUDY IN CD RATS CHRONOLOGY OF EVENTS WEEKS ON STUDYElF1/P2E2. 1-10 Exposure of PI males and females prior to first mating. 11-12 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 35-36 pup/sex/dose/litter 37 Mating period of P2 for F2 litters. F2 bom and litters culled on day 4 post-partum to 8 pups each. F2 litters weaned on day 21 post-partum; 1 F2 selected for necropsy; remaining pups euthanized. Necropsy P2 adults. R&S146865 21 TABLE 2 Two-Generation Inhalation Reproduction Study in CD Rats TISSUES COLLECTED AND PRESERVED AT NECROPSY ADRENALS AORTA KIDNEYS LACRIMAL/HARDERIAN GLANDS AUDITORY SEBACEOUS GLANDS LARYNX BONE (INCLUDING JOINT) BONE MARROW LIVER LUNGS BRAIN (CEREBRUM, BRAINSTEM, CEREBELLUM) CECUM LUMBAR) MAMMARY GLAND MEDIASTINAL LYMPH NODE CERVIX* COAGULATING GLANDS* COLON DUODENUM EPIDIDYMIDES* ESOPHAGUS EYES GROSS LESIONS* HEART ILEUM MEDIASTINAL TISSUES MESENTERIC LYMPH NODE MESENTERIC TISSUES NASAL TISSUES* ORAL TISSUES OVARIES OVIDUCTS PANCREAS PARATHYROID GLANDS PERIPHERAL NERVE JEJUNUM PITUITARY* TISSUE SELECTED FOR HISTOPATHOLOGIC EVALUATION. PROSTATE RECTUM SALIVARY GLANDS SEMINAL VESICLES SKELETAL MUSCLE SKIN SPINAL CORD (CERVICAL, THORACIC, SPLEEN STOMACH TESTES THYMUS THYROID GLAND TONGUE TRACHEA URINARY BLADDER UTERUS VAGINA 9989WSSH