Document 8V0EDm5KOg58938OREoEMBXed

Huntingdon 20 June 1997 Hasmukh C. Shah, Ph.D. Chemical Manufacturers Association Chemstar Department 1300 Wilson Boulevard Arlington, VA 22209 Re: Huntingdon Life Sciences Study No 1 96-4080 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats Dear Has, Enclosed is a copy of the amended protocol for the above referenced study as distributed at Huntingdon Life Sciences. Please retain this copy for your files. Feel free to call me with any questions or comments. Sincerely, Raymond n Schroeder, M.S., DABT Study Director RES/idb Enclosure Huntingdon Life Sciences Inc. PO Box 2360, Mettlers Road, East Millstone, NJ 08875-2360 USA. Tel: +1 908 873 2550 Fax: +1 908 873 3992 CMA 118219 Huntingdon life Sciences Protocol & Protocol Amendment Cover Page General Information Number of Pages: Sponsor Code: C-35 Study Number; 96-4080 Study Title: Vinv' "hloride Combined Inhalation Two-ge.ieration Reproduction and Developmental Toxicity Study in CD Rats Required Information Protocol Signedf'Dated: Test I.later,a! Info.: PUmane Treatment Sig: Prestudy (CAI Checklist: Pepcrt Sc redding Info: X X X X X Distribution List RES X Study Director GMH X Inhalation Toxicologist Sponsor DSM JBB GDT EW CL X Study Monitor Supervisor X In-Life Study Monitor X Inhalation Study Monitor X In-Life Supervisor X In-Life Primary Technician CJB X In-life Inhalation Supervisor Clinical Pathology MAV X Histology HJ X Necropsy DW X Pharmacy Diet Preparation (Diet Only) LVC RRL X Bioanalytical services X In-Life Manager MC X Quality Assurance X Contract Accountant Authorization to Distribute EX, '/ Amendments T^4_ No 1 Direr Approved By 2 n #&// yist> JL itOfiW Pt/^LSt^y_ 61-5*1 3,/y. S'S'-S-S' JU- ufStl Distribution: C^-L U Date Date Distributed Sf-eb 9*7 IjJBhSi 1^*07 i f ih^i s? IS Qf\97 ! <7 For Internal Use Only (Revised 7-Mar-96) CMA118220 Huntingdon I ;fp c;- -r HUNTINGDON LIFE SCIENCES VINYL CHLORIDE COMBINED INHALATION TWO-GENERATION REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDY IN CD RATS STUDY NO.: ISSUE NO.: 96-4080 3 SUBMITTED TO: Chemical Manufacturers Association Chemstar Department ATTENTION: Robert Venezia, Ph.D. DATE: October 3, 1996 Huntingdon Life Sciences Inc. PO Box 2360, Mettlers Road, East Millstone, NJ 08875-2360 USA. Tel: 908 873 2550 Fax. +1 908 873 3992 CMA 118221 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 2 Protocol 03 1 INTRODUCTION: 1.1 STUDY NO.: 96-4080 1.2 ISSUE NO.: 1.3 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats 1.4 TEST MATERIAL: Vinyl Chloride 1.5 SPONSOR: Chemical Manufacturers Association Chemstar Department 1300 Wilson Boulevard Arlington, Virginia 22209 1.6 SPONSOR REPRESENTATIVE: Robert A. Venezia, Ph.D. Phone - 703-741-5639 Fax - 703-741-6091 * MA 1.7 TESTING FACILITY: Huntingdon Life Sciences Mettlers Road P.O. Box 2360 East Millstone, NJ 08875-2360 1.8 PURPOSE: The objectives of the combined inhalation two-generation reproduction and developmental toxicity study outlined in this protocol are to evaluate the effects of the test material on parental toxicity, reproductive capability, in utero development, and neonatal growth and survival in rats. CMA 118222 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 3 Protocol 03 2 STUDY PERSONNEL: Study Director: Raymond E. Schroeder, M.S., DABT Alternate: Dean E. , 'dwell, M.S. Vice-President of Toxicology Director of Toxicology: Carol S. Auletta, B.A., DABT Inhalation Toxicologist: Gary M. Hoffman, B.A., DABT Supervisor Rodent Toxicology: Ellen Whiting, AALAS LAT Primary Technician: Vice President, Pathology: Study Pathologist: Ward R. Richter, D.V.M., M.S., ACVP ft Director, Quality Assurance & Regulatory Affairs: Michael Caulfield Additional personnel will be documented in the project file and presented in the final report. 3 REGULATORY REFERENCES: 3.1 TEST GUIDELINE: This study is designed to meet or exceed the guideline requirements of the following: EPA (Environmental Protection Agency: TSCA Test Guidelines (EPA, 1985); Organization for Economic Co-operation and Development (OECD, Guidelines for testing of chemicals. Section 4: Health Effects, (OECD, 1981); and European Economic Community (EEC), Methods for the Determination of Toxicity (EEC, 1988). #to be determined CMA118223 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 4 Protocol 03 3.2 GOOD LABORATORY PRACTICES: This study will be conducted in compliance with the following: FDA (Food and Drug Administration) Good Laboratory FVactice (GLP) Regulations for Nonclinical Studies (FDA, 1988 - Part 58 of 21 CFR); EPA TSCA Good Laboratory Practice Standards (EPA,1990 - Part 160 of 40 CFR): OECD Good Laboratory Practice Procedures (OECD,1982 - Annex 2 C [81] 30 [FINAL]); Standard operating procedures of the Testing Facility 3.3 FACILITIES MANAGEMENT/ANIMAL HUSBANDRY: Currently acceptable practices of good animal husbandry will be followed, e.g., Guide for the Care and Use of Laboratory Animals; DHHS Publication No, (NIH) 86-23, Revised 1985. Huntingdon Life Sciences (East Millstone, NJ) is fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). 3.4 ANIMAL WELFARE ACT COMPLIANCE: This study will comply with all appropriate parts of the Animal Welfare Act regulations: 9 CFR Parts 1 and 2 Final Rules, Federal Register, Volume 54, No. 1 68, August 31,1989, pp. 36112-36163 effective October 30, 1989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register, Volume 56, No. 32, February 15,1991, pp. 6426-6505 effective March 18, 1991. The Sponsor should make particular note of the following: 1. The Sponsor's signature on this protocol documents for the study described, there are no generally accepted non-animal alternatives and the study does not unnecessarily duplicate previous experiments. 2. All procedures used in this study have been designed to avoid discomfort, distress and pain to the animals. All methods are described in this study protocol or in written laboratory standard operating procedures. 3. Any aspects of this study which cause more than momentary or slight pain or distress to the animals will be performed with appropriate sedatives, analgesics or anesthetics unless the withholding of these agents is justified for scientific reasons, in writing by the Sponsor and the Study Director, in which case the procedure will continue for the minimum time necessary. CMA 118224 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 5 Protocol 03 3.4 ANIMAL WELFARE ACT COMPLIANCE: 4. Animals that experience severe or chronic pain or distress that cannot be relieved will be painlessly euthanatized as deemed appropriate by the Testing Facility's veterinary staff and the Study Director. The Spor. will be advised by the Study Director of all circumstances which could lead to this action in as timely a manner as possible. 5. Methods of euthanasia used during this study are in conformance with the above referenced regulations. 4 QUALITY ASSURANCE MONITORING: The Huntingdon Life Sciences Quality Assurance Unit (East Millstone, NJ) will monitor the facilities, equipment, personnel, methods, practices, records and controls used in this study to assure that they are in conformance with this protocol, company standard operating procedures, and the appropriate Good Laboratory Practice regulations. 5 ALTERATION OF DESIGN: Alterations of this protocol may be made as the study progresses. No changes in the protocol will be made without the consent of the Sponsor. In the event that the Sponsor authorizes a protocol change verbally, such changes will be honored by the Testing Facility and will be followed by a written verification. All protocol modifications will be signed by the Study Director and a Sponsor representative. Any modifications potentially affecting animal welfare will also be signed by two members of the Institutional Animal Care and Use Committee prior to the modification's implementation. CMA 118225 Huntingdon Life Sciences Study No. 96*4080 Chemical Manufacturers Assoc. Page 6 Protocol 03 6 PROPOSED STUDY DATES: Two-generation Reproduction Study: Initiation date: Date Study Director Signs protocol See Section 18.2. Receipt of test animals: (to be addressed by amendment) Initiation of exposures (P1): (Experimental start date) Termination of exposures (P^: Selection of the P2 parental animals: Termination of exposures (F2): Necropsy Experimental Termination (Date of last data collection): Submission of draft final report: Developmental Toxicity Study: Receipt of test animals: Initiation of mating: Initiation of exposures (first Day 6 gestation): Termination of exposures (last Day 19 gestation): Experimental termination: (date of last data collection) Submission of draft final report: Study completion date: * * * * * * * * Date final report is signed by Study Director. CMA118226 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. 7 EXPERIMENTAL DESIGN: 7.1 Two-generation Reproduction Study: Page 7 Protocol 03 Group Exposure Level (ppm)8 1 (control) II III IV 0C 10 100 1100 Number of Animals Mated Adults Microscopic Pathology Adult Generations*1 Pi MF P2 MF Pi MF P2 MF 30 30 30 30 30 30 30 30 30 30 30 30 A.R. A.R. A.R. A.R. 30 30 30 30 A.R. A.R. A.R. A.R. 30 30 30 30 30 30 30 30 8 Whole body exposure 5 days/week, 6 hrs/day during the premating periods (P1( F-,) and 7 days/week, 6 hrs/day for all other periods. Animals will be exposed.in a glass and stainless steel inhalation chamber. b Histologic examinations will be performed for tissues listed in Appendix A. c Control animals will be chamber-housed and sham-treated with clean room air for a comparable period of time as the test animals. Key: A R. = As Required: 1] tissues that demonstrate treatment-related histologic changes in Group IV (additional cost); 2) gross lesions from animals found dead or euthanatized in a moribund condition during the study (additional cost); and 3) gross lesions terminal animals (additional cost); M = Male; F = Female. CMA 118227 Huntingdon Life Sciences Chemical Manufacturers Assoc. Study No. 96-4080 Page 8 Protocol 03 7.2 Developmental Toxicity Study: The developmental toxicity study will be performed with a unique population of animals during the exposure period for the P., parental animals in the two-generation reproduction study. This will be during the mating, gestation and lactation periods for the F, pregnancies when animals are being exposed 7 dsys/week, 6 hrs/day. Animals in the developmental toxicity study will be exposed in the chamber along with the animals in the twogeneration reproduction study. Group 1 (chamber housed, sham air control) U Hi IV Exposure Levels (ppm) Treatment Schedule 6 hrs/day Mated Number of Animals Sacrificed* Gestation Day 20 Proportion of Gestation Day 20 Fetuses/Litter Malformation/Variation Evaluations External Soft Tissue Skeletal 0 Gestation 25 A.S. All % K Days 6-19 10 Gestation 25 A.S. All % S Days 6-19 100 Gestation 25 A.S. AH K % Days 6-19 1100 Gestation 25 A.S. All % >4 Days 6-19 * Gross postmortem examination, liver and kidneys will be weighed and preserved along with gross lesions. Microscopic examinations of these tissues will not be conducted unless deemed necessary to interpret other observations made during the study or requested by the Sponsor (additional cost). A.S. All Survivors CMA118228 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 9 Protocol 03 8 TEST MATERIAL: 8.1 TEST MATERIAL: Vinyl Chloride Description, lot number, storage, expiration date and handling procedures, as well as other pertinent information will be documented in the study data. Properties of the test material are presented in the Appendix B. 8.2 IDENTIFICATION OF TEST MATERIAL: Unless otherwise noted, the identity, strength, purity, composition, stability, and method of synthesis, fabrication and/or derivation of each batch of the test material will be documented by the Sponsor before its use in the study. 8.3 ANALYSIS OF THE TEST MATERIAL: The purity of the lot of test material used on study will be determined at the start of study, approximately 6-months into the study and at termination. These analyses will be performed by the analytical laboratory of the Testing Facility. 8.4 ARCHIVAL SAMPLES: A small sample from the lot of test material used on study will be taken and stored in the Archives of the Testing Facility at East Millstone, NJ. 8.5 UNUSED TEST MATERIAL: At completion of the study unused test material in the one ton pressurized cylinder will be returned to the supplier (The GEON Company, Pedricktown, NJ 08067). 9 TEST ANIMALS: Albino Rats (Outbred) VAF/Plus* 9.1 STRAIN: Sprague Dawley - derived (CD*) [Crl: CD* BR] 9.2 SUPPLIER: Charles River Laboratories Portage, Michigan CMA 118229 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 10 Protocol 03 9.3 JUSTIFICATION FOR TEST SYSTEM SELECTION: The rat is a rodent animal model commonly utilized in reproduction and developmental toxicity studies as recommended in the referenced guidelines. In addition, a historical co; ml data base with this strain of animal and supplier facility is available for comparative evaluation. 9.4 ANIMAL REQUIREMENTS/SPECIFICATIONS: 9.4.1 Number: 9.4.1.1 Two-generation Reproduction Study (P^J: Placed on test Total 240 Males 120 Females 120 9.4.1.2 Developmental Toxicity Study: Placed on test - 100 mated females (25/group). 9.4.2 Age: 9.4,2.1 Two-generation Reproduction Study (P1): Males and females: approximately four weeks at receipt; approxi mately six weeks (males will be 160-210 grams and females will be 125 - 175 grams at initiation of treatment). Animals outside this weight range will be used at the discretion of the Study Director. 9.4.2.2 Developmental Toxicity Study: Females: eight weeks at receipt and at least 10 weeks (200-275 grams) at initiation of mating. Animals outside this weight range at mating will be used at the discretion of the Study Director. Females will be nulliparous and non-pregnant. Males: in-house breeding colony used only for mating. 9.5 ACCLIMATION PERIOD: Approximately two weeks; all animals will be checked for viability twice daily. Prior to assignment to study all animals will be examined to ascertain suitability for study. CMA 118230 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 11 Protocol 03 9.6 ANIMAL HUSBANDRY: 9.6.1 Housing: 9. '.i.l Two-generation Reproduction Stuuy (P1-): 1 Animals will be housed in suspended, stainless steel cages with wire mesh fronts and floors. For the first week of acclimation, animals will be housed two/sex/cage. Thereafter, animals will be housed individu ally except as follows: Mating: one male and one female co-housed nightly. Lactation: dam with litter. Fostweaning: two littermates/sex until selection for P2 parental generation. . During exposures, parental animals will be housed individually in suspended stainless steel wire mesh cages. Neonates will be housed 1-2/cage (littermates) until the formal initiation of the premating treatment period. 9.6.1.2 Developmental Toxicity Study: Animals will be housed in suspended, stainless steel cages with wire mesh fronts and floors except during mating when females will be co housed overnight with a male. During exposures, animals will be housed in suspended stainless steel wire mesh cages. 9.6.2 Food: Certified Rodent Diet, No. 5002; (Meal) (PMI Feeds, Inc., St. Louis, MO). Each animal's cage will be fitted to retain a glass feeder cup with a stainless steel lid. Feed will be available ad libitum during non exposure periods except on the evening prior to necropsy when parental animals (P1 and P2) will be fasted. During exposures, animals will not have access to feed. 9.6.3 Water: Facility water supply (Elizabethtown Water Company, Westfield, NJ); without restriction during exposures and non-exposure periods, via an automated water delivery system to individual animal cages. CMA 118231 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 12 Protocol 03 9.6.4 Bedding Material - Two-generation Reproduction Study: Hardwood shaving bedding (Lab Aspen Shavings, North Eastern d' Products Corporation, Warrensburg, NY) will be provided for each mated female on Day 20 of gestation. Fresh bedding will be provided as needed to Day 14 of lactation. 9.6.5 Feed Analysis: Analytical certification of batches of feed used during the study which are provided by the manufacturer, will be maintained on file at the Testing Facility. There are no known contaminants in the feed which are expected to interfere with the results of this study. 9.6.6 Water Analysis: Monthly water analyses, provided by the supplier, will be maintained on file at the Testing Facility. Biannual chemical and microbiological analyses of water samples collected from representative rooms in this facility will be conducted to assure that water being provided meets standards specified under the EPA National Primary Drinking Water Regulations (40 CFR Part 141). Results will be maintained on file. There are no known contaminants in the water which are expected to interfere with the results of this study. 9.6.7 Bedding Analyses: Analyses for each batch of bedding used on study provided by the supplier, will be maintained at the Testing Facility. There are no known contaminants in the bedding which are expected to interfere with the results of this study. 9.6.8 Veterinary Care: Animals will be monitored by the technical staff for any conditions requiring possible veterinary care. If any such conditions are identi fied, a staff veterinarian will be notified for an examination and evalua tion. Any medical veterinary intervention will be made only with approval of the staff veterinarian and the Study Director. The Sponsor will be consulted whenever possible. However, in emergency situations, decisions will be made as needed and the Sponsor will be advised as soon as possible. CMA 118232 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 13 Protocol 03 9.6.9 Environmental Conditions: 9.6.9.1 Light/Dark Cycle: Twelve hour lie'' ia h cycle daily. 9.6.9.2 Temperature: Monitored and recorded twice daily. Temperature in the animal room and exposure chambers will be maintained in the range of approxi mately 20-24C to the maximum extent possible. 9.6.9.3 Humidity: Humidity in the animal quarters will be monitored and recorded once daily. The desired humidity range in the animal room is 40-70% and 40-60% in the exposure chambers during exposures. Humidity will be maintained in this range to the maximum extent possible. 9.7 SELECTION FOR STUDY: 9.7.1 Two-generation Reproduction Study (P^) : More animals than required for the study will be purchased and accli mated. Animals considered suitable for study on the basis of pretest physical examinations, body weight data and any other pretest evalua tions will be randomly assigned to control or treated groups in an attempt to equalize mean group body weights. Individual weights of animals placed on test shall not exceed _+ 20% of the mean weight for each sex. Disposition of all animals not used in the study will be maintained in the study file. 9.7.2 Developmental Toxicity Study: More females than required for the study will be purchased and accli mated. Animals considered suitable for study on the basis of pretest physical examinations will included into the mating phase of the study. Females which mate will be assigned to groups daily in such a way as to most nearly equalize both the Day 0 mean body weights between groups and the distribution of animal into groups. CMA118233 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. 9.8 ANIMAL IDENTIFICATION: ^3 9.8.1 Two-generation Reproduction Study: / '^ Page 14 Protocol 03 Each animal will be assignci -mporary identification number upon receipt. After selection for study ^ generation), each animal will be ear-tagged with a number assigned by the Testing Facility. This number plus the study number will comprise the unique identification for each study animal. Following the selection procedure (postweaning) to identify the P2 parental animals from the F.j offspring, these selected animals will be ear-tagged with a unique number. If the tag is lost, it will be replaced or the animal will be tail tattooed for identification. Each animal's cage will be provided with a card which will be color-coded for dose level identification and will contain the study number and animal number. 9.8.2 Developmental Toxicity Study: Each female will be assigned a temporary identification number upon receipt. Mated females sorted into study groups will be ear-tagged with a number assigned by the Testing Facility that will be unique from the identification numbers used in the two-generation study. This number plus the study number will comprise the unique identi fication for each study animal. Females will be eartagged on Day 0 of gestation as they are sorted into groups. 10 MATING, GESTATION AND LACTATION PROCEDURES: 10.1 MATING PROCEDURE: 10.1.1 Two-generation Reproduction Study (P1, P2): After animals have been exposed to the test substance for the appro priate length of time (premating treatment period), one male and one female of equivalent dose levels will be caged together nightly (same animals) until a sign of mating (microscopic observation of sperm in the vaginal smear and/or a copulation plug in the vagina) is observed or for 14 consecutive days. The day evidence of mating is observed will be defined as Day 0 of gestation. If mating has not occurred after this interval, the animals will be separated without further opportunity for mating. During mating of the F1 generation cohabitation of male and female littermates will be avoided. CMA118234 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 15 Protocol 03 10.1.2 Developmental Toxicity Study: Females selected for mating will be placed with breeder males nightly in a 1:1 ratio. Vaginal smears will be taken early in the morning following nightly interval of co-housing anc iernales will be considered to have mated if sperm is observed microscopically in the vaginal smear and/or a vaginal plug is observed. The day on which evidence of mating is observed will be defined as Day 0 of gestation. PARTURITION AND LACTATION - TWO-GENERATION REPRODUCTION STUDY (P1 and P2): On Day 20 of gestation, several days prior to expected parturition, each mated female's cage will be fitted to retain a stainless steel floor pan and bedding material provided. Examination for signs of parturition will be made twice daily (morning and afternoon). Evidence of difficult or prolonged parturition, if observed, will be recorded. The day on which all pups have been delivered will be defined as Day 0 of lactation. For females which were caged with males but exhibited no evidence of mating, preparations for undetermined pregnancies (i.e., floor pan, bedding material) will be made when the first animals mated reach their day 20 of gestation. 10.3 SELECTION OF P2 PARENTAL GENERATION: At weaning of each F1 litter, two pups/sex/litter will be chosen at random to become a pool of animals from which the P2 parental generation will be selected. Pups as chosen will be housed two littermates of the same sex/cage. After weaning of the last F1 litter, 30 Ft pups/sex/group will be selected from this pool of animals to become the P2 parental animals. In this selection procedure, each litter will contribute at least one pup/sex, when possible, so as to maximize the representation of animals from different litters in each group. After the last litter is weaned, the 30 Ft pups/sex/group designated for the P2 parental generation will formally initiate the premating treatment period. Thus, there may be a maximum of two weeks difference in age for the P2 animals within each treatment group at initiation of the premating growth period. During this postweaning period, the pool of Ft pups will be chambered and exposed (6 hrs/day) at the treatment level of the dam. In the selection procedure, grossly malformed or obviously-diseased animals within the litters will be excluded from the pool of animals eligible for selection if, in the judgement of the Study Director, their condition may adversely affect long-term survival. Runts, if otherwise normal, will not be excluded from the selection procedure. CMA 118235 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 16 Protocol 03 11 TEST MATERIAL ADMINISTRATION: 11.1 ROUTE OF ADMINISTRATION: Inhalation (whole body). 11.2 JUSTIFICATION FOR ROUTE OF ADMINISTRATION: The inhalation route is one of the potential routes of human exposure to this test material. 11.3 FREQUENCY OF ADMINISTRATION: 11.3.1 Two-generation Reproduction Study: P., and ?2 generation animals will be exposed 6 hrs/day, five days/week for at least 10 weeks prior to mating (designated the premating treatment period). Males will continue to be exposed daily, 6 hrs/day during the 14-day mating period and postmating period until sacrificed. Females will continue to be treated daily, 6 hrs/day during mating and mated females will continue to be treated, 6 hrs/day during the ensuing gestation period through to Day 20. Treatment of females will discontinue at this point to allowed them to deliver (Day 0 of lactation) and treatment will resume on Day 4 of lactation. Females will continue to be treated (6 hrs/day) thereafter for the remainder of lactation (litters weaned on lactation Day 25) and postweaning period until sacrificed. Unmated females will continue to be treated daily (6 hrs/day) until sacrificed. 11.3.2 Developmental Toxicity Study: Females will be treated 6 hrs/day over the Day 6-1 9 gestation interval. The exposure interval for these animals will coincide with the exposures for the mating/gestation/lactation periods for the F, litters in the two-generation reproduction study. 11.4 INHALATION EXPOSURE GENERATION PROCEDURE: The test material will be administered as a gas in the breathing air of the animals. The test atmospheres will be generated by an appropriate procedure to be developed at the Testing Facility. The generation method will be described in the final report and maintained with the data package for this study. The whole-body exposure chambers will have a volume of approximately 6000 liters (6 m3). Each chamber will be operated at a minimum flow rate CMA 118236 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 17 Protocol 03 11.4 INHALATION EXPOSURE GENERATION PROCEDURE: of 1 200 liters per minute. The final airflow will be set to provide at least one air change in 5.0 minutes (12 air changes/hour) and a T99 equilibrium time of approximately 23 minutes. This chamber size and airflow rate *. i3 considered adequate to maintain the oxygen level above 19% and the animal loading factor below 5%. 11.5 CHAMBER MONITORING: A nominal exposure concentration will be calculated, if possible. The flow of air through the chamber will be monitored using appropriate calibrated equipment. The test material consumed during the exposure will be divided by the total volume of air passing through the chamber (volumetric flow rate times total exposure time) to give the nominal concentration. During each test material exposure, measurements of airborne concentrations will be made at least hourly using a MIRAN infrared spectrophotometer. Also prior to initiating of animal exposures, additional samples will be taken to determine the distribution of the test material in the exposure chamber. During each week of exposure, particle size determinations will be performed using an appropriate instrument to characterize the aerodynamic particle size distribution of any aerosol present. If more than the normal amount of trialing is required because of test material generation or monitoring problems (two weeks or 150 technician hours), the Sponsor will be consulted prior to additional trialing (additional cost). CMA 118237 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 18 Protocol 03 11.5 CHAMBER MONITORING: .<S2r". The minimum frequency of chamber activity is summarized below: Activity Analytical Test Material Concentration Temperature Relative Humidity Airflow Rate Nominal Test Material Concentration (excluding the control chamber) Rotation Pattern of Exposure Cages Loading/Unloading Verification Frequency/chamber/day 6 times (hourly) 7 times 7 times 7 times once once once 11.6 EXPOSURE CONCENTRATIONS: For both the two-generation reproduction and developmental toxicity studies the targeted exposure concentrations of vinyl chloride will be 0,10,1 00 and 1100 ppm. These exposure levels correspond to oral equivalent doses of approximately 0,9.2,92and 101 2 mg/kg/day assuming ventilation rates of 1 L/min/kg, 100 percent absorption and a 6 hr/day exposure. The high concentration of vinyl chloride was selected based on the oral equivalent of the limit dose of 1000 mg/kg body weight/day. The high exposure level of 1100 ppm is also expected to produce effects on the liver and other organ systems (ATSDR, 1993). The middle and low exposure levels were selected to provide a dose response for the observed effects and a no-observed-effect level, respectively. CMA 118238 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 19 Protocol 03 12 EXPERIMENTAL EVALUATIONS: 12.1 OBSERVATIONS: 12.1.1 Viability Checks - Two-generation reproduction study and develop mental toxicity study: Observations for mortality, general appearance and signs of severe toxic or pharmacologic effects as well as availability of feed and water will be made at least twice daily (morning and afternoon). Animals in extremely poor health or in a possible moribund condition will be identified for further monitoring and possible euthanasia. All animals found dead will be submitted for a gross macroscopic examination. Parental animals found dead after normal working hours will be refrigerated until a necropsy can be performed. 12.1.2 Physical Examinations: 12.1.2.1 Two-Generation Reproduction Study: Each animal will be removed from its cage and examined at least once pretest (P1), at the study start (first day of treatment for the P! and formal initiation of the premating period for the P2) and weekly thereafter during the study period (P.| and P2). Examinations will include observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration, and palpation for tissue masses. These examinations will be performed postexposure when animals are being transferred to their home cages. 12.1.2.2 Developmental Toxicity Study: Mated animals will be given a detailed physical examination on Days 0 and 6-20 (daily) of gestation. Examinations will include observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration, and palpa tion for tissue masses. During the test period, these examinations will be performed postexposure when animals are being transferred to their home cages. CMA 118239 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Protocol 03 12.2 BODY WEIGHTS: Two-generation Reproduction Study: Males: P.,: at the time of sorting into groups, on the day treatment initiates, weekly throughout the study and at termination. P2: at the formal initiation of the premating treatment period and weekly throughout the study and at termination. Females: P.,: at the time of sorting into groups. P1 and P2: on the day treatment initiates (formal initiation of the premating period for the P2), weekly during the premating growth periods; gestation - Days 0, 7, 14 and 20; and lactation: Days 0, 4, 7, 14, 21 and 25 (Pn only); and at termination. 12.2.2 Developmental Toxicity Study: Females will be weighed on Days 0, 6,9,12,15 and 20 of gestation. 12.3 FOOD CONSUMPTION: Two-generation Reproduction Study: Males: P1; one week pretest. P-j and P2: weekly during the premating treatment period and from termination of mating through to sacrifice (postmating period). Food consumption will not be recorded during the mating periods. Females: P1; one week pretest. P., and P2: weekly during the premating period; during gestation Days 0-7, 7-14 and 14-20; during lactation - Days 1,4, 7, 10 and 14. Food consumption will not be recorded during the mating period or postweaning period prior to termination. CMA 118240 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 21 Protocol 03 12.3.2 Developmental Toxicity Study: Food consumption will be recorded for the following intervals during Gestation: Days 0-6, 6-9, 9-12, 12-15 and 15-20. 12.4 ESTROUS CYCLE DETERMINATI NS -TWO-GENERATION REPRODUCTION STUDY: -VS'. Sr rO V Estrous cycle length and normality will be evaluated daily by vaginal lavage on the first 1 5 P., and P2 females per group for the three week period prior to mating and continuing throughout the mating period or until the female is confirmed mated. 12.5 LITTER EVALUATIONS (Fv F2) - TWO-GENERATION REPRODUCTION STUDY: 12.5.1 Observations: Litters will be observed as soon as possible after delivery for the number of live and dead pups and pup abnormalities (Day 0 of lactation). Thereafter, litters will be observed twice daily (morning, afternoon) for the presence of dead pups. These pups and pups euthanized in a moribund condition will be examined to the extent possible for defects and/or cause of death and preserved in 10% neutral buffered formalin. Litter size will be recorded on Days 0, 4, 7, 14, 21 (F1 and F2) and 25 (F1 only) of lactation. 12.5.2 Culling: On Day 4 of lactation, each litter with more than eight pups will be culled to that number with sex distribution equalized (four/sex) when possible. Pups will be culled using a random number table. Preferen tial culling of runts will not be performed. Culled pups will be examined grossly for abnormalities and following euthanasia by an overdose of inhaled carbon dioxide (C02) will have sex confirmed by internal inspection of the gonads. Only culled pups with abnormalities will be preserved in 10% neutral buffered formalin. 12.5.3 Physical Examinations: Each pup will be given a gross physical examination on Days 0, 4, 7, 14, 21 and 24 (Ft pups only). CMA 118241 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 22 Protocol 03 12.5.4 Pup Body Weights and Sexing Data: jf*' f v Individual pup weights and pup sexing data will be recorded on Days 0, 4 (presented for both pre- and postcull intervals), 7, 14, 21 and 25 (!=1 pups only) of lactation. 12.5.5 Physical Maturation Landmarks - Selected P2 Animals: All F, weanlings selected to become the P2 parental animals (30 sex/group) will be observed daily for vaginal opening beginning on postnatal day 30 or preputial separation beginning on postnatal day 35. If a treatment-related effect is seen in F, sex ratios, age of vaginal opening or preputial separation, then ano-genital distance will be measured on postnatal day 4 for all F2 pups. 13 POSTMORTEM: 13.1 TWO-GENERATION REPRODUCTION STUDY: 13.1.1 GROSS POSTMORTEM EXAMINATION: 13.1.1.1 Parental animals: Complete macroscopic postmortem examinations will be performed on all adult animals, including animals euthanatized in a moribund condi tion or found dead. These evaluations will be performed under the direct supervision of a veterinary pathologist. The eyes of the parental animals will be examined in situ by gently pressing a moistened glass slide against the cornea and observing the eyes under fluorescent light. During the gross postmortem examination all abnormal observa tions will be recorded. The necropsy of the parental animals will include examination of the external surface and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass. Examination of all parental females which were exposed to males, will include a count of uterine implanta tion scars, if present. 13.1.1.2 Weanling Pups (F1 and F2): At weaning (Day 25 - F1 and Day 21 - F2) one pup/sex/litter/group will be selected at random for a complete gross postmortem examination. These examinations will be performed under the supervision of a veterinary pathologist. To control for variation in body weight and organ weight, all pups will be sacrificed at the same age. j CMA118242 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 23 Protocol 03 13.1.2 TIME OF NECROPSY: 13.1.2.1 Moribund Animals: Animals showing signs of severe debility, particularly if death appears imminent, will be euthanatized to prevent loss of tissues through autolysis. 13.1.2.2 Terminal Necropsy Males (Pv P2): P-, and P2 males will be sacrificed after the last F1 and F2 litters, respectively, have been delivered. This will permit some evaluation of fertility (i.e., number of litters delivered) prior to sacrifice. The Sponsor will be notified and authorize the sacrifice of the P1 and P2 males. 13.1.2.3 Terminal Necropsy Females (Pv P2): All P1 and P2 females, regardless of reproductive status (unmated, mated but not pregnant, or retaining litters to weaning) will be sacrificed as a group after the last litters have weaned for all groups. Parental females (P1 and P2) will be necropsied on day 2 of diestrus whenever possible. This will be identified by daily vaginal smearing of the females 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 the following day. The presence of diestrus will be confirmed by vaginal smearing the morning of necropsy. 13.1.2.4 Terminal Necropsy Pups (F1, F2): 13.1.2.4.1 Dead and Culled Pups: Pups found dead at birth or during the lactation period will be examined to the extent possible for defects and/or the cause of death and the presence or absence of milk in the stomach. Dead pups will not be eviscerated. Viscera will remain intact and the pup will be preserved in 10% neutral buffered formalin. Cannibalized pups will be examined to the extent possible and discarded. Culled pups will be examined for external irregularities, sexed internally to confirm external sexing and if unremarkable will be discarded. Culled pups with external irregularities will be preserved intact in 10% neutral buffered formalin. CMA 118243 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 24 Protocol 03 Unselected F1 Pups: Pups selected as a pool of animals from which the P2 parental animals will be selected but which were not identified to continue on study, wi" be given an external examination and if unremarkable, sacrificed and discarded. Pups with external findings will be given a gross internal examination and only abnormal tissues taken and preserved in 10% neutral buffered formalin. Remaining pups in each litter will be given a gross external examination at weaning (Day 25) and if unremarkable, sacrificed and discarded. Pups with external findings will be given a gross internal examination and only abnormal tissues taken and preserved in 10% neutral buffered formalin. Some control pups not selected into the pool of animals to become the P2 parental generation will be used in a satellite study with vinyl chloride (see Study No. 96-408J. 13.1.2.4.3 Unselected F2 Pups: At weaning, unselected F2 pups in each litter will be given a gross external examination and if unremarkable, sacrificed and discarded. Pups with external findings will be given a gross internal examination and only abnormal tissues taken and preserved in 10% neutral buffered formalin. 13.1.3 METHOD OF EUTHANASIA: 13.1.3.1 Parental Animals: Exsanguination following anesthesia with inhaled carbon dioxide. 13.1.3.2 Weanlings: Weanling pups selected for a complete gross postmortem examination will be sacrificed by exsanguination following anesthesia with inhaled carbon dioxide. Remaining pups will be sacrificed with an overdose of inhaled carbon dioxide. CMA118244 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 25 Protocol 03 13.1.3.3 Parental Animals: a^.szr. ^ u\ y The following organs will be weighed at terminal sacrifice from the first 1 5 Pi and P- animals in each group: uterus ovaries testes right epididymis (total and cauda) seminal vesicles (with coagulating prostate glands and their fluids) brain liver kidneys lungs adrenals spleen thymus Organ weight data will be presented as absolute values and relative to terminal body weight. Organ weights will not be recorded for animal dying spontaneously or sacrificed moribund. 13.1.3.4 Weanling Pups (Fn and F2): The following organs will be weighed from the first 15 male and female pups per group selected for complete macroscopic examination at weaning: ovaries testes brain liver kidneys adrenals spleen thymus Organ weight data will be presented as absolute values and relative to terminal body weight. CMA 118245 Huntingdon Life Sciences Study No. 96-4080 . Chemical Manufacturers Assoc. Page 26 Protocol 03 13.1.4 TISSUES PRESERVED: 13.1.4.1 Parental Animals: Grossly abnormal tissues '-.ill be preserved for all parental animals. Tissues listed in Appendix A will be obtained at necropsy and preserved for all P., and P2 parental animals. 13.1.4.2 Weanling Pups: Grossly abnormal tissues and tissues listed in Appendix A will be preserved for all F1 and F2 weanling pups selected for complete gross macroscopic examination. 13.1.5 Preservatives: All tissues - 10% neutral buffered formalin. Testes and epididymides of the parental animals will be fixed in Bouin's solution for at least 48 hrs prior to permanent storage in 10% neutral buffered formalin. 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. 13.1.6 MICROSCOPIC PATHOLOGY EVALUATIONS: Slides of tissues listed in Appendix A (under Microscopic Examination) will be prepared and examined microscopically for all P1 and P2 animals in the control and high-dose groups. If microscopic findings indicative of an effect of test material administration are seen in highdose animals, then examinations will be made of these tissues/organs for low- and mid-dose animals (additional cost). Additional examina tions will be made only after consultation with the Sponsor and receipt of authorization from the Sponsor. Note: any abnormalities not noted during macroscopic postmortem examinations which are seeri during histological processing will be recorded. Gross lesions will be examined at additional cost. 13.1.7 STAINS: Standard stains used, hematoxylin and eosin. Special stains may be employed on selected tissues to aid in making a diagnosis at the discretion of the Study Pathologist. Special stains may be employed at the request of the Sponsor (additional cost). CMA 118246 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 27 Protocol 03 13.1.8 SPERM COUNT, MOTILITY AND MORPHOLOGY ASSESSMENTS: ^ From the first 15 Pn and P2 parental males sacrificed at termination in each group, samples of sperm from the vas deferens (left) and distal cauda epididymis (left) will be collect. Motility will be assessed on sperm collected from the vas deferens and a count will be performed on the sperm sample collected from the cauda epididymis. A manual procedure developed in the Testing Facility will be used to assess motility and perform the count. Additionally, a slide of the sperm sample from the cauda epididymis will be prepared for morphological assessment if deemed necessary (Sponsor decision - additional cost item). & 13.2 DEVELOPMENTAL TOXICITY STUDY: 13.2.1 MATERNAL TERM SACRIFICES: Macroscopic postmortem examinations will be performed on all mated rats, including those dying spontaneously or killed in a moribund condition and on females sacrificed after aborting or premature delivery of a litter. The livers and kidneys will be weighed and preserved in 10% neutral buffered formalin for all females killed on Day 20 of gestation but microscopic examinations of these tissues will not be conducted unless deemed necessary to interpret other observations made during the study or as requested by the sponsor (additional cost item). Gross lesions identified during the macroscopic evaluations will be saved in 10% formalin. Dams showing signs of abortion or premature delivery (expulsion of concepti) will be killed (overdose of inhaled carbon dioxide) on the day such evidence is observed. Reproductive tracts will be examined and fetuses obtained 1 9 days or later will be given an external examination, eviscerated and processed for skeletal staining with Alizarin Red S. These fetuses will then be examined for skeletal malformations. Fetuses obtained earlier than Day 19 will be evaluated for external malformations and saved (10% neutral buffered formalin) at the discretion of the Study Director. Examination data for these fetuses (Day 19 of gestation or earlier) will not be analyzed with data for term Day 20 gestation fetuses. Data for these fetuses will be presented in a separate appendix to the final report. 13.2.2 Reproductive System: The intact uteri (ovaries attached) will be removed from the abdominal cavity and weighed. This uterine weight data will then be used to calculate a corrected Day 20 gestation body weight for each animal. The corrected Day 20 gestation weight will be determined by CMA118247 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 28 Protocol 03 13.2.2 Reproductive System: subtracting the gravid uterine weight from the terminal Day 20 gestation weight. Each uterine horn will then be evaluated for the number and location of the following: * - live fetuses (movement in response to touch); - dead fetuses (lack of movement in response to touch but with no visible degeneration); - late resorptions (recognizable dead fetus undergoing degeneration, regardless of size); - early resorptions (evidence of implantation but no recognizable fetus); - implantation sites (total of fetuses plus resorptions). Ovaries: corpora lutea will be counted for each ovary. Uteri without grossly visible implantations will be stained with ammonium sulfate (Salewski, 1964). If stained foci are present, the female will be considered pregnant for purposes of calculating pregnancy rates. The number of foci will not be used in the calculation of uterine implantation data. 13.2.3 Fetal evaluations: 13.2.3.1 External Evaluations: All fetuses will be weighed and individually identified. Each fetus will be given a gross external examination for defects to include observa tion of the palate. The sex of each fetus will be noted by observation of the ano-genital distance. 13.2.3.2 Fetal Skeletal Evaluations: Approximately one-half of the fetuses in each litter (alternating fetuses within the litter) will be processed for Alizarin Red S staining of the skeletal structures. Prior to processing, the intact fetuses will be killed (overdose of inhaled carbon dioxide) and eviscerated (internal sex noted). Following staining, these fetuses will be evaluated for skeletal malformations and ossification variations. CMA 118248 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. ' Page 29 Protocol 03 13.2.3.3 Fetal Soft Tissue Evaluations: The remaining fetuses in each litter will be processed for soft tissue examination using a microdissection technique similar to the procedure of Staples (1974) . The evaluations will be performed on the. .'resit fetal specimens soon after removal from the uterus. The fetuses designated for soft tissue evaluation will be decapitated (head placed in Bouin's solution for later evaluation). The fetal specimens will then be secured beneath a dissecting microscope and dissected so as to permit evaluation of tissues in the thoracic, abdominal and pelvic cavities. At completion of the examination, the decapitated fetal specimens will be eviscerated and processed for Alizarin Red S staining. These fetuses will not be evaluated skeletally unless authorized by the sponsor (additional cost). Fetal heads, preserved in Bouin's solution, will be sectioned with a razor blade. The serial, transverse sections generated during this procedure will be evaluated for malformations of the palate, eyes and brain. Fetal soft tissue and skeletal evaluations (microdissection, stained specimens and head sections) will be performed under a dissecting microscope. 13.2.4 Resorptions: Late resorptions will be examined externally for malformations. Malformation data for late resorptions will be reported but not included in the analyses of malformation data for live or dead fetuses recovered on Day 20 of gestation. Only late resorptions with external malforma tions will be saved (10% neutral buffered formalin) for future possible examination. Early resorption will be discarded. 14 PRESERVATION OF RECORDS AND SPECIMENS: All data documenting experimental details and study procedures and observa tions will be recorded and maintained as raw data. At the completion of the study, all reports, raw data, preserved specimens and retained samples will be maintained in the Testing Facility's Archives for a period of 10 years after submission of the signed final report. The Sponsor will be contacted in order to determine the final disposition of these materials. The Sponsor is responsible for all costs associated with the storage of these materials beyond 10 years from the issuance of the final report CMA 118249 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 30 Protocol 03 14 PRESERVATION OF RECORDS AND SPECIMENS: and for any costs associated with the shipment of these materials to the Sponsor or to any other facility designated by the Sponsor. 15 STATISTICAL EVALUATIONS: The following items will be analyzed statistically in the final report: 15.1 CONTINUOUS DATA: 15.1.1 Two-generation Reproduction Study: Mean body weights (all recorded intervals - premating, gestation, lactation and postmating); Mean body weight change; -entire premating period (males and females) - entire premating period; -over each weighing interval during the gestation and lactation periods to include Days 0-20 of gestation and Days 0-25 (Ft litters) or Day 0-21 (F2) of lactation; -males during the postmating period (weekly and over the entire period). Mean food consumption values; -premating growth period (weekly); -postmating period (weekly for males); -gestation (Days 0-7, 7-14, 14-20); -lactation (all recorded intervals); Organ weight data (absolute and relative to the terminal body weight); Mean pup weights (all recorded intervals during lactation); Mean number of pups (live, dead, total) at birth (F1 and F2 litters); Mean gestation length (F, and F2 litters); Mean pup live birth indices (F, and F2 litters); Mean pup viability indices (Days 0-4) and weaning indices (Days 4-25 or 21 for the Ft and F2 litters, respectively); Mean age-to-criteria for vaginal opening and preputial separation (P2 animals); Mean sperm count and motility data. 15.1.2 Developmental Toxicity Study: Mean maternal body weights (all recorded intervals during gestation) and weight gain (between all weighing intervals to include Days 6-20 of gestation using both the actual and corrected Day 20 gestation weight); CMA 118250 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 31 Protocol 03 15.1.2 Developmental Toxicity Study: Mean organ weight data, absolute and relative to the corrected Day 20 gestation weight: Mean number of corpora lutea, implants, live and dead fetuses, resorptions per pregnant female; Mean pre- and post-implantation loss indices; Mean fetal weights (distinguished by sex and as a composite for both sexes); 15.1.3 STATISTICAL ANALYSES CONTINUOUS DATA - MULTIPLE GROUP ANALYSES: Data will be compared between the sham, chamber-housed control and the treated groups. Statistical evaluation of equality of means will be made by the appro priate one way analysis of variance technique, followed by a multiple comparison procedure if needed. Bartlett's test will be performed to determine if groups have equal variance. If the variances are equal (p>0.01), parametric procedures will be used; if not (p<0.01), nonparametric procedures will be used. The parametric procedures will be the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means are indicated, Dunnett's test will be used to determine which means are significantly different from the control. If a nonparametric procedure for testing equality of means is needed, the Kruskal-Wallis test will be used, and if differences are indicated, a summed rank test (Dunn) will be used to determine which treatments differ from control. A statistical test for trend in the dose levels will also be performed. In the parametric case (i.e., equal variance), standard regression techniques with a test for trend and lack of fit will be used. In the non-parametric case, Jonckheere's test for monotonic trend will be used. The test for equal variance (Bartlett's) will be conducted at the 1% two-sided risk level. All other statistical tests will be conducted at the 5% and 1%, two-sided risk levels. All ratios (pup survival indices, pre- and postimplantation loss indices) will be transformed via Bartlett's transformation followed by the arc sine transformation prior to analysis. Data will be presented untrans formed. CMA 118251 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 32 Protocol 03 1 5.1.3 STATISTICAL ANALYSES CONTINUOUS DATA - MULTIPLE GROU:P ANALYSES References for these techniques are Snedecor, G.W., Cochran, W.G., Statistical Methods, 6th .ition. Iowa State Univ. Press (1967); Hollander and Wolfe, Nonparametric Statistical Methods, John Wiley and Sons, New York (1973); Dunnett, C.W., J. Am. Sta. Assn. 50: 1096-1 121 (1955) and Biometrics 20: 482(1964). Bartlett's Test pp. 296-298 ANOVA PP- 277-279 Dunnett's Test pp. 1096-1121 pp. 482-491 Kruskal-Wallis pp. 114-116 Summed Rank Test (Dunn) P- 131 Arc Sine Transformation pp. 327-329 Bartlett's transformation P- 329 Snedecor & Cochran Snedecor & Cochran Dunnett Biometrics Hollander St Wolfe Hollander St Wolfe Snedecor St Cochran Snedecor St Cochran Regression Analysis-Trend pp. 135-153 Lack of fit PP- 456-459 Jonckheere's Statistic pp. 120-123 Snedecor St Cochran Snedecor St Cochran Hollander St Wolfe 15.2 INCIDENCE DATA: 15.2.1 Two-generation Reproduction Study: Mortality rates; Mating indices (male and female); Pregnancy rates; Male fertility indices; Litter survival indices; 15.2.2 Developmental Toxicity Study; Maternal Mortality Pregnancy rates Incidence of females with resorptions; Incidence of fetuses with malformations/variations - external, soft tissue and skeletal examinations; Incidence of litters containing fetuses with malformations/variations external, soft-tissue and skeletal. CMA 118252 Huntingdon Life Sciences Study No. 96*4080 Chemical Manufacturers Assoc. Page 33 Protocol 03 1 5.2.3 INCIDENCE DATA ANALYSIS: Data will be compared between the sham, chamber*housed control and the treated groups. Statistical analysis of incidence data will be performed using contin gency tables. First, a standard Chi-square analysis will be performed to determine if the proportion of incidences differed between the groups tested. Next, each treatment group will be compared to the control group using a 2 x 2 Fisher Exact Test; the significance level will be corrected via the Bonferroni inequality to assure an overall test of the stated significance level. Thirdly, Armitage's test for linear trend in the dosage groups will be performed. In keeping with standard statistical practice, if any one cell has an expected value less than 5, the Chi-square and Armitage's tests will not be reported. When this occurs, only the Fisher Exact test (corrected via Bonferroni inequality) will be performed and reported. All tests will be reported at the 5% and 1 % level of significance. References for the techniques are Snedecor, G.W., and Cochran, W.G., Statistical methods, 61h ed., Iowa State University Press, Ames, Iowa (1971); Bradley, J.V., Distribution Free Statistical Tests, Prentice-Hall, Englewood Cliffs, New Jersey (1968); Miller, R.G., Jr., Simultaneous Statistical Inference, McGraw-Hill Book Co., New York (1966); Armitage, P., "Tests for Linear Trends in Proportions and Frequencies", Biometrics, (Sept. 1955). Chi-square Fisher Exact Test Bonferroni Inequality Armitage's Test pp. 250-253 pp. 195-203 p. 15 pp. 375-386 Snedecor & Cochran Bradley Miller Armitage 16 REFERENCES: ATSDR, (1993). Toxicology Profile for Vinyl Chloride. U.S. Department of Health and Human Services. Public Health Services. Agency for Toxic Substances and Disease Registry. EEC (1988). European Economic Community. Methods for the Determination of Toxicity. Official Journal of the European Communities, Vol.31, No.. LI 33, 30 May 1988. ISSN 0378-6978. EPA (1985). Environmental Protection Agency Toxic Substances Control Act Test Guidelines, Final Rule. 40 CFR Part 798, 27 September 1985, pp. 3942639433. CMA 118253 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 34 Protocol 03 16 REFERENCES: EPA (1990). Environmental Protection Agency Toxic Substances Control Act; Good Laboratory Practice Standards. 40 CFR Part 792 (1 July 1990). FDA (1988). Food and Drug Administration Good Laboratory Practice for Nonclinical Studies. 21 CFR Part 58 (1 April 1988). OECD (1981). Organisation for Economic Co-Operation and Development Guidelines for Testing of Chemicals, Section 4 - Health Effects, Paris. OECD (1982). Organisation for Economic Co-Operation and Development, Principles of Good Laboratory Practice, ISBN 92-64-12367-9, Paris. 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. Vol.3. Part B. Female Reproductive Toxicology. (J. J. Heindel and R.E. Chapin, Eds.). Academic Press, Inc., New York, NY. Salewski, E. (1964). Farbemethode zum makroskopischen machweis von implantationsstellen am uterus der ratte. Archiv. Path. Exp. Pharmakol., 247:367 Staples, R. E. (1974). Detection of Visceral Alterations in Mammalian Fetuses. Teratology, 9:37 (Abstract). 17 REPORT; 17.1STATUS REPORT: For the two-generation reproduction study, status reports will be issued monthly during the premating treatment period and after weaning of the F.j and F2 litters. In the developmental toxicity study a status report will be submitted after the last Day 20 gestation maternal sacrifices. These reports will include: Two-generation Reproduction Study: Mortality rates Mean weekly body weight and weight gain data; Mean weekly food consumption premating period; Summary of detailed physical examinations; Mating indices (males and females); Male fertility indices; Pregnancy rates; Gestation length; CMA 118254 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 35 Protocol 03 17.1 STATUS REPORT: Number of pups at birth (live, dead and total) and number of live pups surviving during lactation; Mean pup weights (lactation): Individual female litter data (F1f F2); Maternal gestation body weights and weight gains; Maternal food consumption - gestation/lactation; Summary of gross postmortem evaluations (adults, weanlings) Developmental Toxicity Study: Mortality rates: Mean body weight and weight gain data during gestation; Mean food consumption data; Summary of detailed physical examination data; Mean corpora lutea and uterine implantation data; Mean number of live and dead fetuses and resorptions per pregnant female; Mean pre- and post-implantation loss indices; Incidence of females with resorptions; Mean fetal weights; Fetal external examination data; Individual maternal Day 20 gestation sacrifice data; Maternal organ weight data; Summary of maternal gross postmortem examination data. 17.2 FINAL REPORT: One copy of a draft report will be submitted following termination of the study. After receipt and review of the Sponsor's comments, appropriate changes will be made and two copies of a signed, final report will be issued. (Additional copies will be provided at additional cost). The report will include but not be limited to the following: 17.2.1 General: Compliance Statement; Abstract; Introduction; Experimental Design; Materials and Methods; Discussion of study results; Conclusion and No Observed Effect Level (NOEL) statement, if applicable; Inhalation Exposure Report; References for experimental methodology; Senior personnel participating in the study; CMA 118255 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 36 Protocol 03 17.2.1 General: Quality Assurance Statement. 17.2.2 lata tabulations for parental generations {P-(, P2): Mortality - termination history; Physical in-life observations (summarized and individual data presented monthly throughout the study); Mating indices; Pregnancy rates; Male fertility indices; Mean body weight data (all interval); Mean food consumption data (all intervals); Mean weight gain data (premating, postmating [males!, gestation and lactation intervals); Statement on estrous cycle data; Macroscopic postmortem observations (adults, weanlings); Microscopic pathology examinations; Organ weight data: Sperm assessment data. 17.2.3 Data tabulations for litters and offspring (F1 and F-,): Mean gestation length; Mean number of pups (live, dead and total) at birth and live pups at Days 4, 7, 14 and weaning (Day 25 - Ft and Day 21 - F2); Litter survival indices; Pup live birth index; Pup viability and weaning indices; Mean pup weights (all recorded intervals during lactation); Pup sex ratio at birth, Day 4 (pre- and postcull) and weaning; Pup gross postmortem observations; Individual female litter data. 17.2.4 Data tabulation for the developmental toxicity study: Maternal mortality Maternal body weight and weight gain data; Maternal food consumption; Physical observation data: Pregnancy rates; Mean number of corpora lutea and uterine implantations; Mean number of live and dead fetuses; Mean pre- and post-implantation loss indices; Mean number of resorptions; Incidence of females with resorptions; CMA 118256 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 37 Protocol 03 17.2.4 Data tabulation for the developmental toxicity study: Types of findings and incidence of fetuses with external, soft tissue and skeletal malformations/variations; Incidence or litters containing fetuses with malformations/variations (external, soft tissue and skeletal) Mean fetal weights; Fetal sex distribution ratios; 17.2.5 Appendices: Ail individual animal data (adults, pups, weanlings and fetuses) including but not limited to the following will be presented in the appendices: body weight and weight gain, food consumption, physical observation data, litter and uterine implantation data, organ weight data, sperm assessment data, estrous cycle data, gross postmortem findings, microscopic examination data, chamber exposure data. CMA 118257 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 38 Protocol 03 18 SIGNATURES: 18.1 INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUCJ: The IACUC Protocol Review Subcommittee has reviewed this protocol and found it to be in compliance with appropriate regulations. BY: DATE: w Ccktt TITLE: Institutional Animal Use Committee Member FOR: Huntingdon Life Sciences Institutional Animal Care and Use Committee BY: TITLE: Institutional Animal Care and Use Committee Member FOR Huntingdon Life Sciences Institutional Animal Care and Use Committee 18.2 PROTOCOL REVIEWED AND ACCEPTED: BY: 'P, J f f.2- _ OATE: iSOUu Raymond E. Schroeder, M.S., DABT TITLE: Study Director FOR: Huntingdon Life Sciences BY: ^^^lDATE: TITLE: FOR: Robert A. Venezia, Ph.D. Sponsor Representative Chemical Manufacturers Association Chemstar Department CMA 118258 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 39 Protocol 03 APPENDIX A Tissues Preserved/Examined Microscopically No.* Tissue 2 adrenals 1 aorta 1 auditory sebaceous glands 1 bone (including joint) 3 brain (cerebrum, brainstem, cerebellum) 1 cecum 2 coagulating glands 1 colon 1 duodenum 2 epididymis 1 esophagus 2 eyes 1 heart 1 ileum 1 jejunum 2 kidneys 2 lacrimal/Hardarian glands 1 larynx 2 liver 2 lungs 1 mammary glands 1 mediastinal lymph node* 1 mediastinal tissues 1 mesenteric lymph node* Preserved X X X X X X X X X X X X X X X X X X X X X X X X Microscopic Examinetion (Groups) 1, iv II, III X X X X X X CMA118259 Huntingdon Life Sciences Study No. 96*4080 Chemical Manufacturers Assoc. Page 40 Protocol 03 APPENDIX A ^ Tissues Preserved/Examined Microscopically No.* Tissue 1 mesenteric tissues Preserved X ^ Microscopic Examination- (Groups) 1, IV II. Ill 4 nasal tissues (turbinates) 1 oral tissues XX X 1 ovaryb 2 oviducts 1 pancreas XX XX X 2 parathyroid glands X 1 peripheral nerve X 1 pituitary 1 prostate 1 rectum XX XX X 2 salivary glands X 2 seminal vesicles 1 skeletal muscle XX X 1 skin X 3 spinal cord (cervical, thoracic, lumbar) X 1 spleen X 1 stomach X 2 testis 1 thymus XX X 2 thyroid gland X 1 tongue X 1 trachea 1 urinary bladder XX X CMA 118260 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 41 Protocol 03 No.* Tissue APPENDIX A x// Tissues Preserved/Examined Microscopically ______ Mic -pi . Examination (Groups) Preserved 1. IV II, III 2 uterus (body/horns with cervix) XX 1 vagina XX gross lesions XX * Number of organs/sections preserved/examined. b Only the right ovary will be routinely processed for microscopic evaluation. The left ovary will ba saved for possible oocyte quantification. If deemed necessary by the Sponsor (additional cost), oocyte quantification will include evaluation of a minimum of 10 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- end mid-dose groups may be evaluated if treatment-related changes are observed in the high-dose group. CMA 118261 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. APPENDIX B TEST MATERIAL PROPERTIES Page 42 Protocol 03 Chemical name Vinyl chloride Synonyms Monochloroethylene, VC, VCM, vinyl chloride monomer Molecular formula C2H3CI Molecular weight 62.5 Structures ch2=chci Appearance Colorless gas Vapor pressure 2,530 mm Hg at 20 degrees C Saturated atmosphere Gas at room temperature Vapor density 2.16 Flash point -77.75 degrees C (open cup) Boiling point -13.6 degrees C Specific gravity 0.9121 Conversion factors 1 mg/m3 = 0.39 ppm in air 1 ppm = 2.60 mg/m3 in air CMA 118262 PROTOCOL AMENDMENT Page 1 of 6 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 1 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Chanaes______________________________________________________________________ 1. Pages 1 and 2, 1 INTRODUCTION: 1.6 SPONSOR REPRESENTATIVE: Change from "Robert A. Venezia, Ph.D." to "Hasmukh C. Shah, Ph.D." and change phone number from "703-741-5639" to "703-741-5637" 2. Page 3, 2 STUDY PERSONNEL: Add the following: Primary Technician: Cynthia Lyskowski Study Pathologist: Henry F. Bolte, D.V.M., Ph.D. 3. Page 4, 3 REGULATORY REFERENCES: 3.3 FACILITIES MANAGEMENT/ANIMAL HUSBANDRY: Change this section as follows: Currently acceptable practices of good animal husbandry will be followed, e.g., Guide for the Care and Use of Laboratory Animals; National Research Council, 1996. Huntingdon Life Sciences is fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC)." 4. Page 5, 4 QUALITY ASSURANCE MONITORING: Add the following sentence: "The only exception will be the sperm assessments, this will be audited by the Quality Assurance Unit of Pathology Associates International (PAD." CMA118263 -vy- PROTOCOL AMENDMENT Paae 2 of 6 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 . . AMENDMENT NO.: 1 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Changes 5. Page 6, 6 PROPOSED STUDY DATES: Add the following study dates: Two-generation Reproduction Study: Receipt of test animals: Initiation of exposures (Pi) (Experimental start date): Termination of exposures (P^): Selection of the P2 parental animals: Termination of exposures (F2): Necropsy: Pv P2: Experimental Termination: (Date of last data collection) Submission of draft final report: 23 December 1 996 07 January 1997 20 May 1997 04 May 1997 24 September 1997 28 April 1997 (Males) 19 May 1997 (Females) 15 September 1997 (Males) 24 September 1997 (Females) 09 January 1 998 Week of 23 March 1998 Developmental Toxicity Study: Receipt of test animals: Initiation of mating: Initiation of exposures (first Day 6 of gestation): Termination of exposures (last Day 19 gestation): Experimental termination: (date of last data collection) Submission of draft final report: 24 February 1 997 1 2 March 1997 19 March 1997 21 April 1997 21 July 1997 Week of 25 August 1997 6. Page 17, 11 TEST MATERIAL ADMINISTRATION: 11.5 CHAMBER MONITORING: Add the following statement: "One day per week for the first four weeks of study and for the first and third week of each month thereafter for the remainder of the study, GC (gas chromatography) analyses of the chamber atmospheres will be performed along with the IR (MIRAN infrared spectrophotometer) analyses in each exposure group at each hourly interval." CMA 118264 - vs~PROTOCOL AMENDMENT Paoe 3 of 6 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 1 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Chanoes__________________________________________________________ _ 7. Page 21,12 EXPERIMENTAL EVALUATIONS: 12.4 ESTROUS CYCLE DETERMINATIONS - TWO-GENERATION REPRODUCTION STUDY: Change the following sections as follows: From: "Estrous cycle length and normality will be evaluated daily by vaginal lavage on the first 15 P1 and P2 females per group for the three week period prior to mating and continuing throughout the mating period or until the female is confirmed mated." To: "Estrous cycle length and normality will be evaluated by microscopic evaluation of vaginal smears. These evaluations will be performed for the first 1 5 P-| and ?2 females per group for the three week period prior to mating and continuing throughout the mating period or until the female is confirmed mated." 8. Page 21, 12 EXPERIMENTAL EVALUATIONS: 12.5 Litter Evaluations (F.,, F2) - Two-Generation Reproduction Study: 12.5.3 Physical Examinations: Concerning the examination intervals for the F1 pups change from "Day 24" to "Day 25". 9. Page 22, 12 EXPERIMENTAL EVALUATIONS: 12.5 Litter Evaluations (Fv F2) - Two-Generation Reproduction Study: 12.5.5 Physical Maturation Landmarks - Selected P2 Animals: Change the interval for starting observations for vaginal opening of the P2 parental animals from "Postnatal Day 30" to "Postnatal Day 28" and for preputial separation from "Postnatal Day 35" to "Postnatal Day 40". CMA 118265 -y*- PROTOCOL AMENDMENT Page 4 of 6 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 1 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Changes______________________________________________________ __________ _______ _ 10. Page 24, 13 POSTMORTEM: 13.1 TWO-GENERATION REPRODUCTION STUDY: 13.1.1 GROSS POSTMORTEM EXAMINATION: 13.1.2.4 Terminal Necropsy Pups (F1. F2): 13.1.2.4.2 Unselected F1 Pups: In the last sentence of the section change the study number of the satellite study from "96-4080" to "96-4092". 11. Page 27, 13 POSTMORTEM: 13.1 TWO-GENERATION REPRODUCTION STUDY: 13.1.8 SPERM COUNT, MOTILITY AND MORPHOLOGY ASSESSMENT: a) Provides for Pathology Associates International (PAI) to perform these analyses. b) Revise this section as follows: "From the first 15 P1 and P2 parental males sacrificed at termination in each group, the right epididymis will be removed intact, weighed, and frozen on dry ice for transport to Pathology Associates International, 1 5 Worman's Mill Ct., Suite 1, Frederick, MD 21701. The epididymides will be stored frozen at -70C until evaluation for a caudal epididymal sperm count. Each epididymis will be thawed and the caudal portion removed and weighed. A homogenized sample of the caudal epididymis will be stained and examined using the Hamilton Thorne IVOS sperm analyzer. For each stained preparation, 20 fields will be counted. The total number of sperm in the caudal epididymis will be calculated and reported adjusted for the caudal epididymal weight. Additionally, for each male two sperm morphology slides will be prepared, stained with Eosin for morphological development if deemed necessary (Sponsor decision - additional cost item). The right vas deferens will be excised and placed in a prewarmed solution of phosphate buffered saline and 1% Bovine Serum Albumin. After a minimum three minute "swimout" period, a sample will be placed in a Hamilton Thorne IVOS sperm analyzer and five fields will be stored on an optical disk. These fields will subsequently be analyzed for percent motility. CMA 118266 PROTOCOL AMENDMENT . / il (l.V Page 5 of 6 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 1 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Reasons for Changes_____________ 1. Confirms change in Sponsor Representative. 2. Confirms the "Primary Technician" and "Study Pathologist" for this study. 3. We are presently using the recently revised Guide for the Care and Use of Laboratory Animals: National Research Council, 1996 as a guide for animal husbandry practices within our facility. 4. The auditing of the sperm assessments will be performed by the Quality Assurance Unit of PAI. 5. Confirms study dates. 6. Provides for the comparative GC to IR analyses at the hourly intervals for each exposure group one day/week for the first four weeks of study and for two days per month thereafter for the remainder of the study. This GC/IR comparative work and analytical schedule was requested in our 25 November 1 996 phone conversation. The actual schedule for these comparative analyses was discussed further in subsequent conversations. 7. We've recently changed our standard operating procedure for collecting vaginal tissues for evaluating estrous cycling. The procedure was changed from lavage to use of a swab with an inoculating loop and distilled water. The vaginal tissues collected on the loop and in the water are spread on a glass slide and evaluated microscopically. 8. The F1 pups wean on Day 25 and final examination of these pups should occur at that time point. The protocol incorrectly stated Day 24 of lactation for this last evaluation interval. 9. Review of data from previous studies conducted at this laboratory have indicated that these are the more appropriate intervals for scoring of these particular developmental landmarks in this strain of rat. 10. Confirms the correct Huntingdon Life Sciences study number for the satellite studies. I la. We a presently utilizing the expertise of Pathology Associates International (PAI) for performing sperm assessments in rats within our laboratory. They use the Hamilton Thorne IVOS sperm analyzer to assess motility (video-taped and data retained on disc for archiving with the study data) and to perform the caudal epididymal sperm counts. The use of PAI for this work was discussed with Dr. James Knaak in our 1 5 January 1 997 phone conversation. PAI will prepare a report on the sperm assessments for this study and this will be amended to the final report for the two-generation study. II b. Describes exactly what PAI will be doing in regard to the sperm assessments (motility and caudal epididymal sperm count). The scope of work is similar to that which is already in the protocol. CMA 118267 PROTOCOL AMENDMENT Page 6 of 6_ HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 1 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NJ.: 3 Additional Cost Required: X Yes (GC analyses of the chamber atmosphere) Cost: See attached letter Amendment approved by: Title: Study Director For: Huntingdon Life Sciences Hasmukh C. Shah, Ph.D. Title: Sponsor Representative For: Chemical Manufacturers Association Date / -2 7-97 Date For: Occidental Chemical Corporation CMA118268 PROTOCOL ------------------ Page 1 of 2 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 2 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Changes 1. Page 17,11 TEST MATERIAL ADMINISTRATION: 11.5 CHAMBER MONITORING: Change the following statement - From: "One day per week for the first four weeks of study and for the first and third week of each month thereafter for the remainder of the study, GC (gas chromatography) analyses of the chamber atmospheres will be performed along with the IR (MIRAN infrared spectrophotometer) analyses in each exposure group at each hourly interval." To: "One day per week for the first four weeks of study and twice per month with approximately two weeks between analysis for the remainder of the study, GC (gas chromatography) analyses of the chamber atmospheres will be performed along with the IR (MIRAN infrared spectrophotometer) analyses in each exposure group at each hourly interval." 2. Pages 40 and 41; Appendix A Tissues Preserved/Examined Microscopically a! Change the following: From: To: auditory sebaceous glands auditory sebaceous glands/Zymbal glands bone (including joint) bone - femur/tibia joint (right) mesenteric Lymph nodes mesenteric tissues/mesenteric lymph nodes oral tissues oral cavity/buccal mucosa peripheral nerve peripheral nerve (sciatic) skeletal muscle skeletal muscle (biceps femoris) thymus thymus/mediastinal tissue b) delete "mediastinal tissues" and "mesenteric tissues". CMA118269 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 2 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Reasons for Chancres ________ 1. I was incorrect in indicating that th; comparative GC/IR evaluations would be performed the first and third week each month. These monthly analyses are scheduled for twice each month with at least two weeks between analysis following the first four weeks of evaluation. This change was discussed and accepted by Dr. James Knaak in our conversation of 4 February, 1997. 2a. As per our recent conversation the Pathology/Necropsy Unit wanted to clarify some of the tissues on this list to be more specific. 2b. Mediastinal t ssue will be collected with the thymus and mesenteric tissues will be collected with the mesenteric lymph nodes as noted in 2a above. Additional Cost Required: X No Amendment approved by: naymo^fl E Schrceder, M.S., DABT Tele: Study Director For: Huntingdon Life Sciences Hasmukh C. Shah, Ph.D. T itle Sponsor Representative For: Chemical Manufacturers Association JJLPAlLi Date JL - - y 7 Date Jam^S B. Knaak,, h.D. Title'S-SPday Monitor For: Occidental Chemical Corporation flfDate CMA 118270 PROTOCOL AMENDMENT Page 1 of 4 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Changes 1. Page 12, 9 TEST ANIMALS: 9.6 Animal Husbandry: 9.6.4 Bedding Material - Two-generation Reproduction Study: Change the interval for providing bedding material from "as needed to Day 14 of lactation" to "as needed throughout the lactation period." 2. Page 14, 9 TEST ANIMALS: 9.8 Animal Identification: 9.8.1 Two-generation Reproduction Study: Add the following statement: ''At parturition the Ft and F2 pups will be individually identified in their litters using a paw tattooing procedure." 3. Page 14, 10 MATING, GESTATION AND LACTATION PROCEDURES: 10.1 Mating Procedure: 10.1.1 Two-generation Reproduction Study (P1, P2): Add the following statement: "During the mating of the Pj parental animals, all unmated females following completion of the initial 14-day mating period will be reassigned to a different male in the group and co-housed nightly with this male for an additional six days. The males used for this re-assignment will have demonstrated sexual activity (mated a female during the first 14 days of the mating period) and the selection will be random." 4. Page 15, 10 MATING, GESTATION AND LACTATION PROCEDURES: 10.2 Parturition and Lactation - Two-generation Reproduction Study (P1 and P2): a) Change the following sentence - From: "On Day 20 of gestation, several days prior to expected parturition, each mated female's cage will be fitted to retain a stainless steel floor pan and bedding material provided." To: "On the first Day 20 of gestation, all females (mated and unmated) will be transferred to solid plastic cages and bedding material provided. Females with litters will remain in these cages throughout lactation." CMA 118271 PROTOCOL AMENDMENT HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 Page 2 of 4. -5a- AMENDMENT NO.: 3 /^ ^ STUDY TITLE: Vinyl Chloride Combined Inhalation Two*Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Changes _________ 4. b) Change the following statement From: "For females which were caged with males but exhibited no evidence of mating, preparations for undetermined pregnancies (i.e., floor pan, bedding material) will be made when the first animals mated reach their Day 20 of gestation." To: "For females which were caged with males but exhibited no evidence of mating, preparations for undetermined pregnancies (i.e., transfer to solid plastic cages and bedding material provided) will be made when the first animals mated reach their Day 20 of gestation." 5. Page 20, 12 EXPERIMENTAL EVALUATIONS: 12.2 Body Weights: 12.2.1 Two-generation Reproduction Study: Females: Change the following weighing intervals during lactation - From: "lactation: Days 0, 4, 7, 14, 21 and 25 (P-, only); and at termination." To: "lactation: Days 0, 4, 7, 10, 14, 21 and 25 (P, only); and at termination." 6. Page 20, 12 EXPERIMENTAL EVALUATIONS: 12.3 Food Consumption: 12.3.1 Two-generation Reproduction Study: Females: Change the following intervals for food consumption during lactation From: "during lactation - Days 1, 4, 7, 10 and 14" To: "during lactation - Days 0-4, 4-7, 7-10 and 10-14" Reasons for Changes _____ 1. Bedding material will be provided throughout the lactation period. 2. Our computerized data management system for reproduction and developmental toxicity data allows for the tracking of data for individual pups within the litter. The decision to individually identify pups within the litter at parturition was discussed and authorized in our 21 March 1997 conversation. CMA 118272 PROTOCOL AMENDMENT HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 Paoe 3 of 4 -53- V, AMENDMENT NO.: 3 N STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Reasons for Changes 3. Discussed and authorized in our 1 April 1997 conversation. I had summarized mating performance of the P1 after completion of the 14 day mating period. To maximize the number of mated females in each group it was decided to extend the mating period for six more days. During this period the unmated females were to be paired with another male in the same group but one that had mated a female during the initial 14 day period. This re-assignment of males and extending of the mating period initiated the evening of 1 April 1997. 4 a & b. We have changed our caging for parturition from use of the solid metal bottom floor inserts to the suspended metal cages to use of the molded plastic solid cages. Females with their litters will be housed in these cages throughout lactation. Due to space limitations in the room I'm recommending transfer of all females to these cages when the first mated animals reach their Day 20 of gestation. 5. Our new data management system in reproduction and developmental toxicity requires the intervals of weighing be consistent with the food consumption intervals. 6. Provides for recording of food consumption over the entire Day 0-14 lactation period. The intervals for recording of the food consumption are consistent with the intervals for recording body weights. Additional Cost Required: X No Amendment approved by: Title: Study Director For: Huntingdon Life Sciences Hasmukh C. Shah, Ph.D. Title: Sponsor Representative For: Chemical Manufacturers Association Date CMA 118273 PROTOCOL AMENDMENT HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 Page 4 of 4 -5*/- AMENDMENT NO.: 3 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Amendment approved by: > rnes~B. Kn^ak, Ph.D. tudy Monitor Occidental Chemical Corporation 1 a:jl n Date The IACUC Frotocol Review Subcommittee has reviewed this protocol and found it to be in compliance with all appropriate regulations: By: \---^ ^ <n Title: Institutional Animal Care and Use Committee Member Date: Ou ^ ^ 'j bY; Title: Institutional Animal Care and Use Committee Member CMA 118274 PROTOCC Page 1 of 4 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 4 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Chfnaes. 1. Page 3, 2 Study Personnel: (Protocol Amendment No. 1) Change "Primary Technician" from: "Cynthia Lyskowski" to "Linda Reed, B.S.". Effective: 1 March 1997 2. Page 14, 9.8 Animal Identification: Change the following sentence - From: "Following the selection procedure (postweaningl to identify the P2 parental animals from the F., offspring, these selected animals will be eartagged with a unique number." "Following the selection procedure (postweaningl to identify the P2 parental animals from the F, offspring, these selected animals will be tail tattooed with a unique number." Effective: 05 May 1997 3. Page 18, 11.5 Chamber Monitoring: Add the following activity to the chamber monitoring table: Activity: "In-chamber observation of study animals during exposures" Frequency/chamber/day: "once - mid-way into the exposures" Effective: 21 March 1997 4. Page 22, 1 2.5.4 Pup Body Weights and Sexing Data: Add the following: "All Ft generation animals retained as the P2 parental generation will be weighed on Postnatal Days 32 and 39." Effective; 12 May 1997 5. Page 24, 13.1.2.4.2 Unselected F., Pups: Delete the following sentence from this section: "Some control pups not selected into the pool of animals to become the P2 parental generation will be used in a satellite study with vinyl chloride (see Study No. 9640921." Page 25, 13.1.3.3 Parental Animals: In the list of tissues to be weighed change "right epididymis (total and caudal" to "left epididymis (total)". CMA 118275 PROTOCOL AMENDMENT Page 2 of 4 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 4 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Changes___________ ____________________________________ _______ . 7. Page 27, 13.1.8 Sperm Count, Motility and Morphology Assessments: Change the following sentence From: "From the first 15 P1 and P2 parental males sacrificed at termination in each group, samples of sperm from the vas deferens (left) and distal cauda epididymis (left) will be collected." To: "In the P., and P2 parental generation, sperm evaluations (count, motility and morphology) will be performed for a total of 15 males per group. Included in this group will be all males in each group that did not sire a litter. The remaining males identified for sperm assessment will be the first animals in the group until a total of 15 males per group have been evaluated." Effective: 24 April 1997 Reasons for Changes_______ _____________ ________ _____ ___________ _____________ 1. Cynthia Lyskowski left the company and Linda took over responsibility as "Primary Technician" for the study. Linda had been working with Cynthia on this study. 2. Because the weanling F1 animals are being handled so much with daily transporting back and forth between the home cage and exposure cages, we recommended identification by tattooing the animal's number on the tail as opposed to eartags. It was our opinion that the animals would be more likely to lose the eartags with such frequent handling and change in caging. 3. In-chamber observation of the animals during exposures was not in the protocol. It was decided in our 21 March phone conversation to start conducting these evaluations. This corresponded to early in the mating period of the P, parental animals. 4. In our 12 May 1997 conversation, I recommended that we weigh the F^ parental animals on Postnatal Days 32 and 39 since animals were being treated. Animals will be weighed again at the formal initiation of the premating treatment period and weekly thereafter. 5. The protocol for the satellite study (96-4092) provides for weanlings to come from a separate population of animals. The original intent was to use the excess F1 control pups for the satellite study but due to scheduling difficulties and the staggered weaning dates that would be encountered, it was decided not to use these animals. CMA 118276 -S7- V^>' PROTOCOL AMENDMENT Page 3 of 4 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 4 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 Reasons for Changes__________________________________________ 6. Both epididymides will be weighed intact. Since the right epididymis was being weighed and processed for sperm assessment (Section 13.1.8), the table in the protocol should have specified weighing of the left epididymis. The left epididymis was weighed and preserved intact. The most recent harmonized OPPTS guidelines (No. 870.3800 - Reproduction and fertility Effects, February 1996) recommends that the epididymis be processed intact as a longitudinal section that would include portions of the caput, corpus and cauda. To weigh the cauda segment of the epididymis would require cutting the organ. 7. The protocol had specified the first 15 P., and P2 males per group for sperm assessment. Following review of the reproductive performance of the P.| parental males it was decided (conversation with Dean Rodwell 24 April 1997) to include in this number the males that did not sire a litter (i.e., males that did not mate, males that mated but did not successfully impregnate a female). The remaining males identified for sperm assessment in each group were the first animals in the group until a total of 1 5 had been selected. Amendment approved by: fL---- ----- Raymond E, Schroeder, M.S., DABT Title: Study Director For: Huntingdon Life Sciences ^^17 Date Hasmukh C. Shah, Ph.D. Title: Sponsor Representative For: Chemical Manufacturers Association n, mg Date jL James B. Knaak, P^.D. udy Monitor Occidental Chemical Corporation Date CMA 118277  r PROTOCOL AMENDMENT^ Page 4 of 4 HUNTINGDON LIFE SCIENCES STUDY NO.: 96-4080 AMENDMENT NO.: 4 STUDY TITLE: Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats PROTOCOL ISSUE NO.: 3 The IACUC Protocol Reviev. Subcommittee has reviewed this protocol and found it to be in compliance with all appropriate regulations: . IS^u. Date: 11 Title: Institutional Animal Can and Use Committee Member By: =Date /*&* ``I Title: nstitutional Animal Care and Use Committee Member CMA 118278