Document NGeVVEXzKmjkxZoOr34bY4x7E

CHEMICAL MANUFACTURERS ASSOCIATION June 1,1995 Dear Vinyl Chloride Health Committee Members: A draft protocol for the vinyl chloride combined 2-generation and developmental toxicity study prepared by Bill Breslin of Dow is enclosed for your review. Please bring your comments on the draft to the next Committee meeting scheduled for June 7 from 10:00 a.m. to 3:30 p.m. at the CMA offices in Washington, D.C. If you are unable to attend the meeting, please send your written comments to me as soon as possible. Please call me at (202) 887-1192 if you have any questions. I look forward to seeing you next week. Sincerely, Enclosure Hasmukh C. Shah, Ph.D. Manager, Vinyl Chloride Panel BFG42852 2501 M Street, NW, Washington, DC 20037 Telephone 202-887-1100 Fax 202-887-1237 BFG 01887 Responsible Care APublic Commitment THE DOW CHEMICAL COMPANY HEALTH AND ENVIRONMENTAL SCIENCES 1803 Building Midland, Michigan 48674 FAX Number 517/638-9863 ___________ FAX TRANSMITTAL SHEET_________ Date 6/1795 To: Hr Hasmukh Shah Company Name: PM A _ Fax Number ?n2-fi87-'*l27 From: William T. Breslin Phone: S17-fi3fi.1311 Number of Pages: 23 (including this page) Material sent by: W.J. Brpslin Phone: 517-636-1311 Comments: Hasmukh, please End in the subsequent pages of this FAX the draft protocol for the vinyl chloride combined 2-generation and developmental toxicity study. I look forward to your comments following the Panel's review. At this time it is unlikely that I will be able to make the Panel meeting next week. I was also unable to reach J. Ramlow as he is still ill and out of the office. Bill If you do not receive all material being transmitted, please call the sender listed above. Updated S/89 qatton BFG42853 BFG 01888 Preliminary CopyJL. Final 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): FILE NUMBER: PROJECT NUMBER: TASK NUMBER: OSD NUMBER: COST CENTER: SPONSOR: CAS NUMBER: INHALATION SPECIALIST: STUDY PATHOLOGIST: --------------------------------------------------------- -- APPROVAL(S): -------------------------------------------------------------SPONSOR: ___ _____________________________________ DISTRIBUTION - - SEE ATTACHED LIST /DATE /DATE /DATE /DATE /DATE /DATE BFG42854 BFG 01889 -2- MA.TER.IALS AND METHODS The test material used for this study was obtained from X(name), X(dty), 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: Rash Point: Boiling Point: Specific Gravity: Conversion Factors: 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 exposure phase of this study. An additional group of male CD rats (Charles River Breeding Laboratory, Kingston, NY or Portage, MD approximately ten weeks of age will be purchased for breeding stock for the developmental portion of this study and will not be exposed to the test material. Animals DRAFT BPG42856 BFG 01891 -3- will be ordered to ensure that a sufficient 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 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, according to the Standard Operating Procedures of the Reproductive Toxicology Group. 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 1215 changes/hour. A feed erode and a pressure-activated stainless steel water 1Fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). DRAFT &FG42857 BFG 01892 -4- 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 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. 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 MIRAN 1A infrared spectrophotometer (Foxboro DRAFT BFG42858 BFG 01893 -5- 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 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, RDThe 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. * Trademark of The Dow Chemical Company * Trademark of SAGIAN Indianapolis, Indiana. DRAFT BFG42859 BFG 01894 -6- Exposure Concentrations. Rats will be exposed to target concentrations of 0, X, Y or Z ppm of the test material. These exposure 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, 100 percent absorption and a 6 hour/day exposure. The chosen concentrations were selected by the sponsor and were based upon the results of the inhalation toxidty studies previously discussed. . Reproduction - 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 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 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. DRAFT BFG42860 BFG 01895 -7- 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. Reproduction - Culling end 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 21 days after delivery. Weanlings not held for prospective generations or selected for necropsy will be examined grossly for abnormalities and euthanized by C02 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 DRAFT BFG42861 BFG 01896 -8- 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 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. Reproduction - 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. DRAFT BFG42862 BFG 01897 9- - 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 on day 35 (Korenbrot et al., 1977). If there is a treatmentrelated effect 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. Reproduction - 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. 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 die 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 DRAFT BFG42863 BFG 01898 -10- 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 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 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. 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 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 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. DRAFT BfG42864 BFG 01899 -11* Reproduction - Sperm Count, Motility and Morphology For all PI and P2 males at termination, samples of sperm from die 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 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. 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 all FI and F2 pups that are examined macroscopically (one/sex/litter), die 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 groups will be limited to those tissues DRAFT BFG42865 BFG 01900 12- 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. Additional groups of 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 and gestation. The overall chronology and study design for the developmental portion of this study is depicted in Tables 1 and 2, respectively. Treatment of the rats will begin at approximately 6 weeks of age. After approximately 10 weeks of exposure (5 days/week, excluding holidays), the females will be mated with unexposed stock males, approximately 12 weeks of age. On gestation day 21 the females will be euthanized and maternal and developmental parameters evaluated. Developmental - Breeding Procedure Breeding of the adults will commence after approximately 10 weeks of treatment and continue until pregnancy occurs or two weeks has elapsed. Each female will be placed with a single unexposed stock male following the daily exposure period and left with the male until the following morning. In the morning prior to exposure, each female will be evaluated for the presence of sperm via vaginal lavage, and returned to her exposure cage. 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 be separated from the male and will remain in their exposure cage. If mating has not occurred after two weeks, the animals will be separated without further opportunity for mating. Developmental - Maternal Observations All animals will be observed daily during the study for alterations in behavior or demeanor as previously described under the 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 DRAFT BFG42866 BFG 01901 -13- submitted for a complete necropsy as described for the Reproduction Study. Body weights will be recorded weekly during premating and gestation as described for the Reproduction Study. 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. 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, 31 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 a 10% aqueous 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. DRAFT BFG42867 BFG 01902 -14- 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. Utter 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 outUers 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 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 the 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 die fetal population will be performed using a censored Wilcoxon test with Bonferroni's correction. Nonpregnant females, females pregnant following staining or females having totally resorbed Utters will be excluded from the appropriate analyses. The nominal alpha levels to be used are as follows: Bartlett's Test (Winer, 1971) a0.01 Parametric ANOVA (Steel and Torrie, 1960) a=0-10 Nonparametric ANOVA (Hollander and Wolfe, 1973) a=0.10 DRAFT BpG42868 BFG 01903 -15- Dunnett's Test (Winer, 1971) Wilcoxon Rank-Sum Test (Hollander and Wolfe, 1973) Fisher's Test (Siegel, 1956) Censored Wilcoxon Test (Haseman and Hoel, 1974) Outlier Test (Grubbs, 1969) Binomial Distribution Test (Steel and Tome, 1960) 0=0.05, two-sided a=0.05, two-sided with Bonferroni correction (Miller, 1966) a=0.05, two-sided a*0.05, two-sided a=0.02, two-sided 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 dted alpha levels would suggest. Thus, the final 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 Preaates-. 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 die 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. DRAFT BPG42869 BFG 01904 -16- 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. 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. Dawson, A. B. (1926). A Note on the Staining of the Skeletons of Geared Specimens with Alizarin Red-S. Stain Tech. 1:123-124. EEC (1988). European Economic Community. Methods for the Determination of Toxidty. 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 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 H, 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. DRAFT BFG42870 BFG 01905 -17- 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 Sakwski, E. (1964). [The Effect of Thalidomide on die 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. 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 toxidty of the ovary using follicle quantitation and morphometries. In DRAFT BpG4287i BFG 01906 -18- 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. DRAFT BFG 01907 -19- TABLE 1 VINYL CHLORIDE: COMBINED INHALATION TWO-GENERATION REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDY IN CD RATS CHRONOLOGY OF EVENTS WEEKS ON STUDYElF1/P2BL MO Exposure of PI males and females prior to first mating. 11-12 PI mating period for FI litters. Mating period for developmental toxicity evaluation 14-15 PI bom and litters culled on day 4 post-partum to 8 pups each. Necropsy of Developmental toxicity dams and fetal evaluations 17-18 FI Utters weaned on day 21 post-partum; offspring selected for P2 adults; 1 FI pup/sex/dose/Utter selected for necropsy; remaining pups euthanized. 19-28 Necropsy PI adults. Exposure of P2 males and females prior to first mating. 29-30 Mating period of P2 for F2 litters. 32-33 F2 bom and Utters culled on day 4 post-partum to 8 pups each. 35-36 F2 Utters weaned on day 21 post-partum; 1F2 pup/sex/dose/littcr setected for necropsy; remaining pups euthanized. 37 Necropsy P2 adults. DRAFT BFG42873 BFG 01908 -20- 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 30 Development X 30 30 Reproductive 30 Development Y 30 30 Reproductive 30 Development Z 30 30 Reproductive 30 Development TOTAL= 120 240 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 Examine External, Visceral and Skeletal Examination DRAFT BFG42874 BFG 01909 01610 IGa SLSZPDdZ <N t*> S itr> P i > iO FOR HISTOPATHOLOGIC EVALUATION. Sc s Z 3 & < S Z < g 3z g a 8 EgQSg ;2<0B6 z uwUP 2u 2 5 g 18- s f2U 5 8 OU 53 IE Q bu. aws n a3 & B 8X z pz p w C "?