Document wkR5yB0z1gDdnz9DkYvOOdvQ

Huntingdon 11 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 +1 908 873 2550 Fax: +1 908 873 3992 CMA 118439 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.: 3 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 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 118440 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. Rodwell, 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: Ward R. Richter, D.V.M., M.S., ACVP Study Pathologist: 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 CMA 118441 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 Practice (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 118442 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 Sponsor 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 118443 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 (P^: (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. CMA 118444 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)* 1 (control) II III IV 0e 10 100 1100 Number of Animals Mated Adults Microscopic Pathology Adult Generations1* Pi MF 30 30 30 30 30 30 30 30 P2 MF 30 30 30 30 30 30 30 30 Pi MF 30 30 A.R. A.R. A.R. A.R. 30 30 P2 MF 30 30 A.R. A.R. A.R. A.R. 30 30 `Whole body exposure 5 days/week, 6 hrs/day during the premating periods (Plt 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. e 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 118445 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 Pj parental animals in the two-generation reproduction study. This will be during the mating, gestation and lactation periods for the F1 pregnancies when animals are being exposed 7 days/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) II III 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 % % Days 6-19 10 Gestation 25 Days 6-19 A.S. All >4 100 Gestation 25 A.S. All W >4 Days 6-19 1100 Gestation 25 A.S. All J4 V4 Days 6-19 a 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 CMA 118446 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*) ICrl: CD* BR] 9.2 SUPPLIER: Charles River Laboratories Portage, Michigan CMA 118447 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 control 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 (P1): 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 (Pt ): 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 118448 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 11 Protocol 03 9.6 ANIMAL HUSBANDRY: 9.6.1 Housing: 9.6.1.1 Two-generation Reproduction Study (P.,): 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. Postweaning: 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 118449 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 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 118450 10. 96*4080 Page ._ Protocol 03 rk cycle daily. xded twice daily. Temperature in the animal room mbers will be maintained in the range of approxi.o the maximum extent possible. animal quarters will be monitored and recorded once red humidity range in the animal room is 40-70% and exposure chambers during exposures. Humidity will be this range to the maximum extent possible. UDY: don Reproduction Study (P.,) : ils than required for the study will be purchased and accliils considered suitable for study on the basis of pretest inations, body weight data and any other pretest evaluabe randomly assigned to control or treated groups in an o equalize mean group body weights. Individual weights of placed on test shall not exceed,+. 20% of the mean weight sex. Disposition of all animals not used in the study will be ted in the study file. pmental Toxicity Study: females than required for the study will be purchased and acclid. Animals considered suitable for study on the basis of pretest ical examinations will included into the mating phase of the study, ales which mate will be assigned to groups daily in such a way as nost nearly equalize both the Day 0 mean body weights between ups and the distribution of animal into groups. Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 14 Protocol 03 9.8 ANIMAL IDENTIFICATION: 9.8.1 Two-generation Reproduction Study: Each animal will be assigned a temporary identification number upon receipt. After selection for study (P, 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, 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 (P.,, 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. CMA 118452 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 and females 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. 10.2 PARTURITION AND LACTATION - TWO-GENERATION REPRODUCTION STUDY (Pn 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 F, litter, 30 F, 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 118453 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 P2 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 6-19 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 118454 tf^bes Study No. 96-4080 ;turers Assoc. Page 17 Protocol 03 <TION EXPOSURE GENERATION PROCEDURE: 0 liters per minute. The final airflow will be set to provide at least one mge in 5.0 minutes (12 air changes/hour) and a equilibrium time oroximately 23 minutes. This chamber size and airflow rate are Jered adequate to maintain the oxygen level above 19% and the animal ig factor below 5%. MBER MONITORING: ominal exposure concentration will be calculated, if possible. The flow lir through the chamber will be monitored using appropriate calibrated jipment. The test material consumed during the exposure will be divided the total volume of air passing through the chamber (volumetric flow rate nes total exposure time) to give the nominal concentration. uring each test material exposure, measurements of airborne concentrations /ill be made at least hourly using a MIRAN infrared spectrophotometer. Also jrior to initiating of animal exposures, additional samples will be taken to determine the distribution of the test material in the exposure chamber. ng each week of exposure, particle size determinations will be performed Eg an appropriate instrument to characterize the aerodynamic particle size ibution 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). CMA11B4Ss Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 18 Protocol 03 11.5 CHAMBER MONITORING: 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 0,10,100 and 1100 ppm. These exposure levels correspond to oral equivalent doses of approximately 0, 9.2, 92 and 1012 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 118456 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 (P^, 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 (P1 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 118457 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 20 Protocol 03 12.2 BODY WEIGHTS: 12.2.1 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, 1 5 and 20 of gestation. 12.3 FOOD CONSUMPTION: 12.3.1 Two-generation Reproduction Study: Males: P.,: one week pretest. P1 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: P^ 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 118458 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 DETERMINATIONS - TWO-GENERATION REPRODUCTION STUDY: Estrous cycle length and normality will be evaluated daily by vaginal lavage on the first 15 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 (Ft 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 (F1 pups only). CMA 118459 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 22 Protocol 03 12.5.4 Pup Body Weights and Sexing Data: 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 (F, pups only) of lactation. 12.5.5 Physical Maturation Landmarks Selected P2 Animals: All F1 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 F1 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 - Ft 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. CM4 118460 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 (P1r P2): P, and P2 males will be sacrificed after the last F, 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 P.| and P2 males. 13.1.2.3 Terminal Necropsy Females (P1# P2): All P, 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 118461 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 24 Protocol 03 13.1.2.4.2 Unselected F., 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, will 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. CMa 118462 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 25 Protocol 03 13.1.3.3 Parental Animals: The following organs will be weighed at terminal sacrifice from the first 15 P, and P2 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 1 5 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 118463 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 will be preserved for all parental animals. Tissues listed in Appendix A will be obtained at necropsy and preserved for all P1 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 P-, 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 seen 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). 118464 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 P, 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. 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 19 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 CMA118465 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 118466 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. ' Page 29 Protocol 03 13.2.3.3 Petal 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 fresh 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 CM* 118487 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 (F1 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 (Ft and F2 litters); Mean gestation length (Fn and F2 litters); Mean pup live birth indices (Ft 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 118468 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.Q1), 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. CMA118469 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 32 Protocol 03 15.1.3 STATISTICAL ANALYSES CONTINUOUS DATA - MULTIPLE GROU:P ANALYSES References for these techniques are Snedecor, G.W., Cochran, W.G., Statistical Methods, 6th edition, 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: 1096-1121 (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 & Wolfe Hollander & Wolfe Snedecor & Cochran Snedecor & Cochran Regression Analysis-Trend pp. 135-153 Lack of fit pp. 456-459 Jonckheere's Statistic pp. 120-123 Snedecor & Cochran Snedecor & Cochran Hollander & 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 118470 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 33 Protocol 03 15.2.3 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., Statisticalmethods, 6th 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 118471 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, BJ. 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: 17.1 STATUS REPORT: For the two-generation reproduction study, status reports will be issued monthly during the premating treatment period and after weaning of the Ft 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; CMMl8472 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 (F1# 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 118473 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 36 Protocol 03 17.2.1 General: Quality Assurance Statement. 17.2.2 Data tabulations for parental generations (Pv 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 (F., and F2): 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 - F, 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 118474 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 of litters containing fetuses with malformations/variations (external, soft tissue and skeletal) Mean fetal weights; Fetal sex distribution ratios; 17.2.5 Appendices: All 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 118475 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 38 Protocol 03 18 SIGNATURES: 18.1 INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC): The IACUC Protocol Review Subcommittee has reviewed this protocol and found it to be in compliance with all appropriate regulations. BY: DATE: \\ C'cA TITLE: Institutional Animal Carf Use Committee Member FOR: Huntingdon Life Sciences Institutional Animal Care and Use Committee BY: // Oc-h% TITLE: Institutional Animal Care and Use Commi ee Member FOR Huntingdon Life Sciences Institutional Animal Care and Use Committee 1 8.2 PROTOCOL REVIEWED AND ACCEPTED: BY: DATE: Raymond E. Schroeder, M.S., DABT TITLE: Study Director FOR: Huntingdon Life Sciences jfgUtj BY: TITLE: FOR: Robert A. Venezia, Ph.D. Sponsor Representative Chemical Manufacturers Association Chemstar Department CMA 118476 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 nodes 1 mediastinal tissues 1 mesenteric lymph nodes 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 Examination (Groups) 1. IV II, III X X X X X X CMA118477 Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 40 Protocol 03 APPENDIX A Tissues Preserved/Examined Microscopically No.* Tissue 1 mesenteric tissue* 4 nasal tissues (turbinates) 1 oral tissues 1 ovaryb 2 oviducts 1 pancreas 2 parathyroid glands 1 peripheral nerve 1 pituitary 1 prostate 1 rectum 2 salivary glands 2 seminal vesicles 1 skeletal muscle 1 skin 3 spinal cord (cervical, thoracic, lumbar) 1 spleen 1 stomach 2 testis 1 thymus 2 thyroid gland 1 tongue 1 trachea 1 urinary bladder 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 Examination (Groups) uv II. Ill X X X X X X X X CMA 118478 * Huntingdon Life Sciences Study No. 96-4080 Chemical Manufacturers Assoc. Page 41 Protocol 03 APPENDIX A Tissues Preserved/Examined Microscopically No.* Tissue 2 uterus (body/horns with cervix) 1 vagina gross lesions Prsserved X X X Microscopic Examination (Groups) 1. IV II, III X X X * Number of organs/sections preserved/examined. b Only the right ovary will be routinely processed for microscopic evaluation. The left ovary will be 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- and mid-dose groups may be evaluated if treatment-related changes are observed in the high-dose group. CMA 118479  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 CMA118480 Appendix A CMA118481 CURRICULUM VITAE NAME: Paul E. Newton, PhD. Diplomate, American Board of Toxicology PRESENT POSITION: (since November 1995) Project Manager Huntingdon Life Sciences Inc. East Millstone, NJ 08875-2360 Responsible for all aspects of conducting toxicity studies following GLPs including protocol design, development of test exposure/dose and monitoring systems, evaluation of data, preparation of final reports and client/auditor interface. EDUCATION: 1979 The Medical College of Wisconsin PhD. Respiratory Physiology Dissertation: "Control of Human Ventilation During C02 Breathing" 1968-1970 Kansas State University Engineering (Graduate courses in Convective Heat and Mass Transfer, Boundary Layer Theory, Radiation Heat Transfer, Fluid Mechanics, Thermodynamics, Control Theory, Engineering Analysis, Analog Computer and Statistics) 1968 Rose Polytechnic Institute M.S. Biological Engineering 1967 Rose Polytechnic Institute B.S. Mathematics PROFESSIONAL EXPERIENCE: 1986 - 1995 Director of Inhalation Toxicology Pbarmaco LSR Incu, Toxicology Services Worldwide East Millstone, NJ 08875 Responsible for the Department of Inhalation Toxicology, overseeing the activities of the twhwiral staff. As a Study Director, responsible for all aspects of the conduct of inhalation studies. Responsibilities include protocol design, development of test exposure and monitoring systems, evaluation of data, preparation of final reports and client/auditor interface. CMAH8482 2- - Paul E Newton, PhD. PROFESSIONAL EXPERIENCE-continued: 1985 - 1986 American Biogenics Corporation Decatur, Illinois Director, Inhalation Toxicology Director of Inhalation Toxicology and responsible for overall operation of inhalation toxicology facility. This included client-sponsored acute, subchronic and chronic studies of vapors and liquid or dust aerosols as well as pyrolysis testing following NBS guidelines. 1979 - 1985 University of California, Irvine Toxic Hazards Research Unit, Wright-Patterson AFB, OH Head. Respiratory Toxicology Department Responsible for design, implementation and operation of a pulmonary toxicology program including preparation of budgets, protocols and standard operating procedures, management of personnel, equipment specification and computer software development for high speed data acquisition and processing using GLP techniques. Research involved evaluation of pulmonary dynamics, gas exchange, distribution, pulmonary defense mechanisms, and the tccdcodynamics and toxicoldneucs of various chemical agents on rats, mice and dogs. Compounds studied included: ozone, carbon dioxide, propylene glycol dinitrate, jet fuels JP-8 and shale JP-4, papain, bleomycin, 4-ipomeanol and o-ethyl-o-(2-diisopro-pylamino-eth>1) methylpbosphonite. 1971 - 1979 The Medical College of Wisconsin Department of Environmental Medicine Senior Bioengineer Responsible for specification of instrumentation systems, writing computer programs and the collection, processing and statistical analysis of all pulmonary, cardiovascular, neurological and behavioral test data investigated during human inhalation toxicological experiments. Compounds included: carbon monoxide, methylene chloride, trichloroethylene, 1,1,1-trichloro-ethane, tetrachloroethylene, acetone, toluene, xylene, ethyl alcohol, fluorocaibon-11, fluorocarbon-12, isobutane, propane, propylene glycol dinitrate, methyl chloride, Halon 1211, Halon 2402 and Halon 1301. PROFESSIONAL ACCREDITATION: Diplotnate, American Board of Toxicology, 1984 Recertified in 1989 and 1993 CERTIFICATES/AWARDS: Best Poster Presentation, Mid-Atlantic Soc. of Toxicol (1992) Distinguished Service Award (1976-79) Milwaukee Fire Department CMA118483 -3- Paul E Newton, P1D., PROFESSIONAL MEMBERSHIPS: Sodety of Toxicology, Mid-Atlantic and Midwest Regional Society of Toxicology American College of Toxicology American Association of Pharmaceutical Scientists American Assoriation Aerosol Research American Thoracic Society Assodation of Inhalation Toxicologists European Society of Toxicology International Soriety of Aerosols in Medicine PUBLICATIONS (Book Chapters): Stewart, R.D., Hake, CL, Lebrun, AJ,, Kalbfleisch, JJL, Newton, PE., Peterson, JJL, Cohen, H.H., Struble, R. and Busch, KA 1974. "Effects of Trichloroethylene on Behavioral Performance Capabilities", Behavioral Toxicology. Xintaras, C, Johnson, B.L and deGroot, L, Eds. p. 96-129. NIOSH, Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402 Stewart, R.D., Hake, CL, Peterson, J.E, Forster, H.V,, Newton, P.E, Soto, RJ. and Lebrun AJ. 1974. "Development of Biological Standard for Trichloroethylene*, op. dL, p. 81-91. Stewart, R.D.. Newton, PE., Hosko. MJ., Peterson, JE. and Mellender, J.W. 1975. "The Effect of Carbon Monoxide on Time Perception, Manual Coordination, Inspection and Arithmetic", Behavioral Toxicology, Edited by Weiss & Laties. Chap. 2, p. 29-60. Plenum Press, New York Stewart, R.D.. Herrmann, AA., Baretta, ED., Forster, KV., Crespo, J.H., Newton, P.E and Soto, RJ. 1981. "Human Exposure to Aerosol Propellants", Aerosol, Airways and Asthma Trautlein, JJ., Ed. Spectrum PubL. Jamaica, New York. Newton, Paul E 1995. "Inhalation Toxicology', CRC Handbook of Toxicology, Edited by Derelanko, MJ. and Hollinger, MA, Chap. 5, CRC Press, Boca Raton, FL JOURNAL ARTICLES: Newton, P.E 1971. "The Effects of Sound on Plant Growth", J. And. Eng. Soc, 19 (3): 202*205 Stewart, R.D., Newton, P.E, Hosko, MJ. and Peterson, J.E 1973. "Effects of Carbon Monoxide on Time Perception", Arch. Env. Health, 27: 155-160 Stewart, R.D., Peterson, JE., Newton, P.E, Hake, CL Hosko, MJ., Lebrun, AJ. and Lawton, G.M. 1974. "Experimental Human Exposure to Propylene Glycol Dinitrate", Toxicol. AppL Pharmacol. 30: 377-395 CMA 118484 Paul E Newton, PhX>. JOURNAL ARTICLES - continued; Stewart, R.D., Herrmann, AA., Baretta, ED., Sikora, JJ-, Newton, P-E and Soto, RJ. 1977. "Acute and Repetitive Human Exposure to Isobutane*, Scand. J. Work, Environ. & Health 3: 234-243 Stewart, RJD., Newton, P.E, Barena, ED., Herrmann, AA., Forster, H.V. and Soto, RJ. 1978. "Phvsicological Response to Aerosol Propellants', Environmental Health Perspectives, 26: 275285' Erk, S.D., Jarboe, C.H., Newton, P.E and Pfledderer, C 1982. "Gas Chromatographic Determination of 1, 2-Propanediol Dinitrate in Blood, Journal of Chromatographic Science, 240: 117-113 Newton, P.E, Hamilton, EH. and Forster, H.V. 1983. "Measurement of Ventilation using Digitally Filtered Transthoracic Impedance", J. AppL Physiol: Respirat, Environ. Exercise Physiol. 54: 1161-1166 Newton, P.E, Latendresse, J.E, Mattie, D. and Pfledderer, G 1985. "Alterations in Alveolar Gearance after 4-lpomeanol Induced Necrosis of Clara Cell and CBiated Cells in the Terminal Bronchiole of the Rat", Toxicol AppL Phann. 80: 534-541 Newton, P.E and Pfledderer, C 1986. "Deposition and Clearance of Radio-labeled Particles Inhaled during C02 Exposures", J. AppL Toxicology, 6: 113-119 Newton, P.E, Becker, S.V. and Hixon, GJ. 1991. "Pulmonary Function and Particle Deposition and Gearance in Rats after a 90-Day Exposure to Shale Oil Derived Jet Fuel JP-4", Inhalation Toxicology 3: 195-210 Hoffman, G.M., Newton, PE^ Thomas, W.G, Bimbaum, HA. and Kennedy, Jr., G.L 1991. "Acute Inhalation Toxicity Studies in Several Animal Species of an Ethylene Oxide/Propylene Oxide Copolymer (UCON 50-HB-5100)", Drug and Chemical Toxicology 14: 243-256 Nair, R.S., Johannsen, F.R-, Bolte, HF., Newton, P.E and Rinehart, WE 1992. Toxidty of Calcium Sodium Metaphosphate Fiber; IL Chronic Inhalation and Oncogenicity Study", Fundamental and Applied Toxicology 19: 79-90 Newton, P.E, Schroeder, R.E, Sullivan, J.B., Busey, W.M., and Banas, DA. 1993. Inhalation Toxicity of Phosphine in the Rat: Acute, Subchronic and Developmental", Inhalation Toxicology, 5 (2): 233-239 CMA 118485 Paul E. Newton, Ph^. JOURNAL ARTICLES - continued: Newton, PE-, Bohe, H.F., Daly, I.W,, pffisbuiy, B.D., Ben-Dyke, R_, Drew, R.T. and Sheldon, AW. 1994. 'Subchronic and Chronic Inhalation Tccdchy Studies of Antimony Trioxide in the Rat', Fundamental and Applied Toxicology 22, 561-576 POSTERS AND CONFERENCE PROCEEDINGS: Stewart, R.D., Fisher, T.N,, Peterson, JE-, Hosko, MJn Baretta, EX>,, Dodd, H.C, Newton, PE. and Herrmann, AA 1970. Experimental Human Exposure to Carbon Monoxide', Proc. of First Annual Conference on Environmental Toxicology, AMRL-TR-70-102, Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, Ohio Newton, P.E, Forster, H.V. and Klein, JJP. 1977. "Ventilation, PaC02 and Arterial pH in Man while Breathing 1 and 2% C02', Fad. Proc, 36 (3): 446 Forster, H.V., Klein, J.P,, Newton, PE. and Hamilton, L 1979. "Effect of Increased FIC02 on Oxygen Consumption (Vo2), C02 Storage* (CC02), Fad. Proc 38: 1034 Klein. J.P., Forster, H.V., Newton, P.E. and Kampine, JJ. 1979. "Can Humans Maintain PaC02 Homeostases as FIC02 is Increased?', Fed. Proc, 38: 1033 Stewart, R.D., Newton, P.E, Wu, A, and Stewart, T.A 1979. The Milwaukee Program for Detecting Carboxyhemoglobin Levels in Firefighters Fifth Symposium on the Occupational Health and Hazards of the Ftre Service*, San Diego, International Association of Firefighters, Washington, D,C. Newton, P.E 1980. 'Elevated Carbaxyhemoglobins in Fire Fighters", Proc of Tenth Annual Conference on Environmental Toxicology, AMRL-TR-79-121, Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, Ohio Newton. P.E, Forster, H.V., Hamilton, L and Christman, N.T. 1980. "Ventilation Measurements made using Digitally Filtered Transthoracic Impedance*, Fed. Proc 39*. 576 Newton, P.E, Erk, S.D. and Pfledderer. C 1981. "Hypotensive Effect of Tween 80 in Dogs", Physiologist, 24: 14 CMA118486 Paul E Newton, PhD. ABSTRACTS, CONFERENCE PROCEEDINGS - continued: Newton, P.E, Pfledderer, C and Erie, SD. 1982. Tong and Short Term Pulmonary Clearance in Rats after Exposure to Ozone', Abstracts of Papers, AULA. Meeting, Cincinnati, OH. Newton, FJE, Latendxesse, J.R., Mattie, D.R. and Pfledderer, C 1985. 'Alterations in Alveolar Clearance after 4-ipomeanol Induced Necrosis of Clara and Ciliated Cells in the Terminal Brochiole of the Rat', The Toxicologist 5: 932 Hoffman, G.M., Newton, PE., Thomas, W.C and Kennedy, G.L. 1990. 'Multi-Species Acute Inhalation Toxicity Study of an EO/PO Copolymer (UCON-50-HB-5100)*, Toxicologist 10: 807 Newton, P.E, Bolte, HE. and Sheldon, AW. 1990. "Pulmonary Toxicity after Chronic Inhalation Exposure to Antimony Tricodde (St^Oj) in Rats, The Toxicologist 10: 624 Newton, P.E, Bolte, HE. and Sheldon, AW. 1990. "Pulmonary Tcodcity after Chronic Inhalation Exposure to Antimony Triaxide (SbjOj) in Rats, J. Aerosol Medicine 3: 78 Newton, P.E, Sullivan, J.B., Busey, W.M. and Banas, DA. 1991. "Acute and Subchronic Inhalation Toxicity of Phosphine, The Toxicologist 11: 210 Duffy, J., Newton, PE Cockrell, B., Soiefer, A, Kirwin, G and Daughtrey, W. 1991. "A Thirteen Week Inhalation Toxicity Study of Commercial Hexane in the Rat and Mouse, The Toxicologist 11: 1219 Nair, R.S., Johannsen, F.R., Bolte, H.F., Newton, PE. and Rinehart, WE 1991. "Chronic Inhalation and Oncogenicity Study with Calcium Sodium Metaphospbate (CSM) Fiber", The Toxicologist 11: 255 Newton. P.E, Lake, L.K., Bolte, HE. and Osimitz, T.G. 1992. "A Subchronic (4-Week) Inhalation Toxicity Stud}' of a High Molecular Weight Emulsion Polymer in the Rat", The Toxicologist 12: 828 Schroeder, RE, Newton. PE, Sullivan, J.B. and Busey, W.M. 1992. "An Inhalation Developmental Toxicity Study of Phosphine in Rats, The Toxicologist 12: 401 Knaak, J.B., Barfknecht, T.R_ Smith, L.W. and Newton, PE 1994. "Subchronic Inhalation Toxicity Study of p-Chlorobenzoatrifluoride (PCBTF) in the Rat via Whole-Body Exposures", The Toxicologist 14, No. 1213 CMA 118487 -7- Paul E Newton, PhD. ABSTRACTS, CONFERENCE PROCEEDINGS - continued; Newton, P.E, Wooding, WE and Rinehart, W 1994. "An Inhalation Oncogenicity Study in Mice of Methyletbyketomne", The Toxicologist 14: No. 1220 Duffy, J.S., Haddock, ES., Daughtrey, WD., Keenan, T.H. and Newton, PE. 1994. "Chronic Inhalation Study of Commercial Hexane in Rats", The Toxicologist 14: No. 1233 Hoffman, G.M., Newton, PE and Thomas W.C 1994. "Inhalation Toxicology Testing Methods of Fiber Finishes", American Fiber Manufacturers' Association Conference Knaak. J.B., Smith, LW. and Newton, PE 1995. "Subchronic (90-Day) Inhalation Toxicity and Neurobehavioral Study of PCBTF in the Rat", The Toxicologist, VoL 15, No. 444 Newton. P.E, Wooding, WE and Rinehart, WE 1995. "An Inhalation Oncogenicity Study in Rats of Methylethylketoodme", The Toxicologist, VoL 15, No. 978 TECHNICAL REPORTS: Stewart. R.D., Baretta, ED., Dodd, H.C, Donohoo, K., Graff, S,, Kalbfleisch, J,, Newton, PE Platte. L, Rimm, A., Stewart, E and VanYscrioo, B. National Carbaxyhcmoglobin Survey, COCOHb Symposium, Washington, D.C, March 1973. Stewart. R.D., Hake, CE, Lebrun, AJ., Kalbfleisch, J.H., Newton, PE, Peterson, JE, Cohen. H.H.. Struble. R. and Busch, K_A "Effects of Trichloroethylene on Behavioral Performance Capabilities". NIOSH Report #NIOSH-MCOW-ENVM-PCE-74-6, National Institute for Occupational Safety and Health, Cincmnati, OH (1974). Stewart. R.D., Hake, CE, Forster, H.V., Lebrun, AJ., Peterson, JE, Wu, A. and Staff; Tetrachloroethvlene: Development of a Biologic Standard for the Industrial Worker by Breath Analysis", NIOSH Report #NIOSH-MCOW-ENVM-PCE-74-6, National Institute for Occupational Safety and Health, Cincinnati, OH (1974). Stewart. R.D., Hake, CE, Forster, H.V., Lebrun, AJ., Peterson, JE, Wu, A. and Staff; Trichloroethylene: Development of a Biologic Standard for the Industrial Worker by Breath Analysis", NIOSH Report #NIOSH-MCOW-ENVM-PCE-74-8, National Institute for Occupational Safety and Health, Cincinnati, OH (1974). Stewart, R.D., Hake, CE, Forster, H.V., Lebrun, AJ., Peterson, JE Wu, A and Staff; "Methylene Chloride: Development of a Biologic Standard for the Industrial Worker by Breath Analysis", NIOSH Report #NIOSH-MCOW-ENVM-PCE-74-9, National Institute for Occupational Safety and Health, Cincinnati, OH (1974). CMA 118488 Paul E. Newton, Ph-D. TECHNICAL REPORTS: Stewart, R.D., Hake, CL., Wu, A, Graff, SA, Forster, H.V., Lebrun, AJ., Newton, PE and Soto, RJ.; "1,1,1-Trichloro-ethane: Development of a Biologic Standard for the Industrial Worker by Breath Analysis', NIOSH Report #NIOSH-MCOW-ENVM-l,l,l-T-75-4, National Institute for Occupational Safety and Health, Cincinnati, OH (1975). Stewart, R.D., Hake, CL., Forster, H.V., Lebrun, AJ., Peterson, JE, Wu, A., and Staff; Toluene: Development of a Biologic Standard for the Industrial Worker by Breath Analysis', NIOSH Report #N10SH-MC0W-ENVM-MC-75-3, National Institute for Occupational Safety and Health, Cincinnati, OH (1975). Stewart. R.D., Hake, CI_, Wu, A., Graff, SA-, Forster, H.V,, Keeler, WJL, Lebrun, AJ,, Peterson, JE, Newton, PE. and Soto, RJ.; "Acetone: Development of a Biologic Standard for the Industrial Worker by Breath Analysis", NIOSH Report #NIOSH-MCOW-ENVM-A-75-5, National Institute for Occupational Safety and Health, Cincinnati, OH (1975). Stewart. R.D., Hake, Cl_, Wu, A., Graff, S_A., Forster, H.V., Keeler, W.H.. Lebrun, AJ-, Peterson, JE. Newton, PE. and Soto, RJ.; "Methyl Chloride: Development of a Biologic Standard for the Industrial Worker by Breath Analysis", NIOSH Report #NIOSH-MCOWENVM-MCM*77-1, National Institute for Occupational Safety and Health, Cincinnati, OH (1977). Stewart. R.D., Hake, CL., Wu, A-, Graff, S.A., Forster, H.V,, Keeler, W.JL, Lebrun, AJ,, Peterson. JE. Newton, PE and Soto, RJ.; "Styrene: Development of a Biologic Standard for the Industrial Worker by Breath Analysis", NIOSH Report #NIOSH-MCOW-ENVM-STY-77-2, National Institute for Occupational Safety and Health, Cincinnati, OH (1977). Stewart. R.D.. Hake, CL., Wu, A., Kalbfleisch, J., Newton, PE, Marlow, S.K and VicicevicSalama. M.: 'Effects of Perchloro-ethylene/Drug Interaction on Behavior and Neurological Function", U.S. Department of Health, Education and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Division of Biomedical and Behavioral Science, Publication #77-191, April 1977. Stewart, R.D., Herrmann, A_A^ Baretta, ED., Forster, H.V., Crespo, J.H., Newton, PE and Soto, RJ.; "Acute and Repetitive Human Exposure to Difluorordichloromethane", Report #PB279204/LLC, U.S. Department of Commerce, The National Technical Information Service, Springfield, VA (1978). CMA 118489 Paul E. Newton, PhD. TECHNICAL REPORTS - continued: Stewart, R.D., Newton, P.E, Baretta, ED., Hemnann, AA, Forster, H.V, Sekora, JJ. and Soto, RJ.; "Acute and Repetitive Human Exposure to Fluorordichloromethane", Report #PB279203/LLC, U.S. Department of Commerce, The National Technical Information Service, Springfield, VA (1978). Stewart, R.D., Hermann, AA, Baretta, ED., Forster, H.V., Crespo, JJL, Newton, P.E and Soto, RJ.; "Acute and Repetitive Human Exposure to Isobutane and Propane", Report #PB279205/LLC, U.S. Department of Commerce, Tlie National Technical Information Service, Springfield, VA (1978). Stewart, R.D., Newton, P.E, Wu, A Kalbfleisch, J.H. and Stewart, TA; "Carbaxybemoglobin Trend irrSL Louis Blood Donors, 1971-1975", Report #CRC-AORAC-CAPM-8-68/MCOWENVM-CO-78-1, The Coordinating Research council, Inc., New York, NY (1978). Stewart, R.D., Newton, PE*, Kaufman, J., Forster, H.V., Klein, J.P., Keelen, MIL, Stewart, DJ., Wu, A and Hake, CL.; The Eflea of a Rapid 4% Saturation Increase in Carbaxybemoglobin on Maximal Treadmill Exercise," Report #CRC-APRAC-CAPM-22-75/MCOW-ENVM-CO-78-2, The Coordinating Research Council, Inc, New York, NY (1978). Stewart, R.D., Newton, P.E, Wu, A, Hake, CL and Krivanek, ND.; "Human Exposure to Halon 1301", El. DuPont de Nemours & Co., Inc^ Wilmington, DE (1978). Erk. S.D., Newton, P.E, MacEwen, JD. and Veraot, EH.; "Evaluation of the Tcoricokinetic Study of 1,2-propanediol Dinitrate (PGDN) in the Dog", AFAMRL-TR-82-27, Wright-Patterson Air Force Base, OH. Horton, J.R., Gaworski, CL, Newton. P.E et aL "Evaluation of the Acute Toxicity, Irritation, Sensitization and Subchronic Dermal Toxicity of Antimony Thioantimonate Lubricant", NTIS/ADA166 873/9 (1986). REPORTS ON PROPRIETARY COMPOUNDS SUBMITTED TO SPONSORS: Approximately 500 confidential reports on acute, sub-chronic, chronic, and reproduction/teratology inhalation studies in rodents and higher animal species. These reports were issued to the pharmaceutical, agricultural and industrial chemical, food additive and cosmetic industries. I certify that the above isa true and accurate account of my professional career. Signed: Date: --- ^ I CMA 118490 CURRICULUM VITAE NAME: Henry F. Bohe, D.V.M., Ph.D. PRESENT POSITION: Senior Staff Pathologist Huntingdon Life Sciences East Millstone, NJ 08875 Performs the duties of a pathologist. Conducts macroscopic and microscopic examinations of specimens of animal body tissues, fluids and secretions; diagnoses and dictates reports of findings. Reads and follows protocols in examining slides to determine initial requirements. Records all observations noting any abnormalities which might impact on the results of the study or lead to advanced or expanded explorations. EDUCATION: 1969 Purdue University Ph.D. Pathology Uresis: "Ultrasmxcture ofIsonicotmic Add Hydiazide Induced Encephalopathy in the Peking Duck" 1966 Purdue Univeisrty M.S. Pathology Thesis: "Uveitis, a Sequela to Experimentally Induced Leptospira Pomona Infection in the Shetland Pony" 1963 Iowa State University Dr. Veterinary Medicine PROFESSIONAL EXPERIENCE: 1/83-11/95 Associate Director of Pathology Phaimaco LSR East Millstone, NJ 08875 5/79-1/83 Staff Pathologist Bio/dynamics, Inc. East Millstone, NJ 08875 1970-1979 Group Leader Ledsiie Laboratories Department of Experimental Pathology CMA 118491 Henry F. Bohe, D.V.M., Ph.D. PROFESSIONAL EXPERIENCE - continued: 1968-1970 Administrative Assistant Puiriue University Graduate Housing 1967-1970 1964-1967 Special Post-Doctoial Fellow Graduate instructor Purdue University Veterinary Pathology PROFESSIONAL MEMBERSHIPS: International Academy of Pathology Society ofToxicologic Pathology Mid-Atlantic Chapter ofthe Society ofToxicology The New Yozk Academy ofScience The American. Association forthe Advancement of Science Phi 2eta Phi Kappa Phi Gamma Sigma Delta Sigma Xi PUBLICATIONS: Bolte. H.F., Koralek, AV., Traitor. C.E., Toxicology Studies ofFenbufen", Arzneim.-Forsdi/Dnig Res. 30 (I). Nk 4a 91980. 721-728. Rusch. G.M., Clary, John J., Rinehart. William E,, and Bohe, Henry F., "A Twenty-Six Week Inhalation Toxicity Study with Foimaldehyde in the Monkey, Rat and Hamster", Toxicology and Applied Pharmacology, 1982. Rusch. George M.. Bohe. Henry F.. and Rinehart, William E., 'A Twenty-Six Week Inhalation Toxicity Study with Formaldehyde in the Monkey, Rat and Hamster", Formaldehyde Toxidty, James . Gibson. Ed., Hemisphere Publishing Company. Andrews, L.S., Clary, John J., TenilL J.B., and Bohe. Henry F., "Subehronic inhalation Toxicity of Methanol", Journal of Toxicology and Environmental Health 20: 117-124 (1987). CMA118492 -3Henry F. Bolte, D.V.M., Ph.D. PUBLICATIONS - continued: Atkinson, John E.. Daly, Ira W., Bohe, Henry F., Morishima, Hidelri and Sasaki, Satoshi- 'One-Year Oral Gavage Toxicity Study ofLansoprazole (AG-1749) in Rats", Japanese Pharmacology and Therapeutics 18, Suppl. 10: 59 (S-2713)-91, (S-2745), 1990. Wood, F.E., Tierney, W.J., Knezevich, AJ... Bolte, RF., Maurer, JJC and Bruce, RD., Chronic Toxicity and Carcinogenicity Studies of Olesoa. in Fischer 344 Rats, Food Chemical Toxicology, Vol. 29, No. 4, pp. 223-230, 1991. Newton, PE, Bolt, RF., Daly, I.W.,'Pillsbury, BJD., Terrill, J.B., Drew, R.T. and Sheldon, AJW., "Inhalation Toxicity of Antimony Tiioxide", Toxicology and Applied Pharmacology fm press). Nair. R.S., Johannsen, R.F.. Bohe, RF_ Newton, P.E. and Rinehart, W.E., "Chronic Toxicity & Oncogenicity Study with CSM Fiber", Toxicology and Applied Pharmacology (in press). Sumio Minemaxsu, John E. Atkinson, Henry F. Bohe, Hiroshi Sakai and Yuichi Fujii; A Subchionic (3Month) Oral Toxicity Study ofTsumura Sho-saiko-to (TJ-9) in the Rat via Oral Gavage .Administration with a 4-Week Recovery Period. Ovo Yakuri/Pharmacometrics 43 (1) 19-42 (1992). Newton, P E., Bolte, RF., Daly, I.W., Pillsbuty, B.D., Ben-Dyke, R., Drew, R.T. and Sheldon. A.W. "Subchronic and Chronic Inhalation Toxicity Studies ofAntimony Tiioxide in the Rat", Fundamental and Applied Toxicology 22, 561-576 (1994). Atkinson, J.E.. Bolte. RF., Rubin, L.F. and Sonawane, Meena. "Assessment of Ocular Toxicity in Dogs During 6 Months Exposure id a Potent Oiganophosphate" Journal of Applied Toxicology, 14,145-152 (.1994). ABSTRACTS: R.D. McCabe. D.L. Reynolds. J. Schreuis, A. Childs, R_A. Braeckmaa. RJ. Zimmerman and RF. Bolte, "Toxicology of M-CSF in Cvnomoigus Monkeys by IV Infusion for 28-Days", Toxicologist 15, No. 1 (1995). Nair. RS., Johannsen, F.R. Bolte. H.F.. Newton, P.E. and Rinehart, W.E. "Toxicity of Calcium Sodium Metaphosphate Fiber, II. Chronic Inhalation and Oncogenicity Study", Fundamental and Applied Toxicology 19: 79-90 (1992). CMA 118493 Henry F. Bolte, D.V.M., Ph.D. ABSTRACTS - continued: Nair, R.S., Johannsen, F.R., Bohe, H.F., Newton, P.E. and Rinehart, W.H. "Chronic Inhalation and Oncogenicity Study with Calcium Sodium Mctaphosphatc (CSM) Fiber", Toxicologist 11: 255 (1991). Newton, P.E., Bohe, H.F. and Sheldon, A.W., "Pulmonary Toxicity after Chronic Inhalation Exposure to Antimony Ttioxide (Sb^) in Rats, Toxicologist 10: 624 (1990). Newton, P.E.. Bohe, H.F and Sheldon, A.W., "Pulmonary Toxicity after Chronic Inhalation Exposure id Antimony Tiioxide (St^Oj) in Rats, J. Aerosol Medicine 3: 78,1990. PRESENTATIONS: Newton, P.E., Lake, L.K., Bolte, H_F. and Osimitz, T.G. "A Subdmmic (4-Week) Inhalation Toxicitv Study of a High Molecular Weight Emulsion Polymer in the Rat" (presented at the 31st Annual Society ofToxicology Meeting, Seattle, WA: February 1992). "Evaluation of Reproductive Toxichy of Oitho-Dichlorobenzene (ODCB) in the Rat" (abstract): R Nair. J. Barter, H. Bohe, R. Schroeder and C. Stack (presented at the V International Congress ofToxicology. July 1989). CMA 118494 CURRICULUM VITAE NAME: PRESENT POSITION: (since November 1995) Raymond E. Schroeder, M.S. Project Manager Huntingdon Life Sciences Inc. East Millstone, NJ 08S75 Study Director responsibility for the performance of predinical toxicity and safety assessment studies in small animal species particularly in the areas of developmental toxicity and reproduction/fertflity. EDUCATION: 1967 _. University of Illinois Champaign/Urbana, Illinois M.S. Zoology 1965 Wheaton College Wheaton, Dlinois 6.S. Biology 1974 Medical Pharmacology Course Northwestern Medical School Chicago, Illinois PROFESSIONAL EXPERIENCE: 1990-1995 Sr. Toxicologist/Study Director Pharmaco LSR Inc., Toxicology Services Worldwide East Millstone, NJ 08875 1976-1990 Manager/Study Director Reproduction/Teratology Department Bio/dvnamics, Inc. East Millstone, NJ 08875 1975-1976 Manager L.B. Allen, Inc. Schiller Park, Illinois 1967-1975 Sr. Research Assistant (Teratology) Searle Laboratories Department of Pathology/Toxicology Skokie, Illinois CMA 118495 Raymond E. Schroeder, M. PROFESSIONAL ACCREDITATION: Diplomats, American Board of Toxicology PROFESSIONAL MEMBERSHIPS: Mid-Atlantic Reproduction Teratology Association (MARTA) -Member Steering Committee 1977-1981 -Member Nominating Committee 1984-1985 Mid-Atlantic Society of Toxicology Society of Toxicology Society of Toxicology Reproductive Specialty Section Teratology Society Metropolitan New York Brandi of AALAS (American Association for Laboratory Animal Science) Midwest Teratology Association (MTA) PUBLICATIONS: Carol S. Auletta, Raymond E. Schroeder, Walter I. Krasavage and Carol R. Stack, "Toxicology of Dietbylene Glycol Butyl Ether. 4. Dermal Subchronic/Repreduction Study in Rats", Journal of the American College of Toxicology, Volume 12, Number 2, 1993. Blacker, A.M., Schroeder, R.E., English, J.C., Murphy, SJ., Krasavage, WJ. and Simon, G.S. *A Two-Generation Reproduction Study with Hydroquinone in Rats", Fundamental and Applied Toxicology 21, 420-424 (1993). Newton, P.E., Schroeder, R.E., Sullivan, J.B., Busey, W.M. and Banas, D.A. 'Inhalation Toxicity of Phosphine in the Rat: Acute, Subchronic and Developmental', Inhalation Toxicology 5 (2): 233239 (1993). R. A. Kuna, MJ. Nicolich, RJE. Schroeder and G.M. Rusch, *A Female Rat Fertility Study with Inhaled Benzene", Journal of the American College of Toxicology, Volume 11, Number 3, 275-282 (1992). S. J. Murphy, R.E. Schroeder, A.M. Blacker, WJ. Krasavage and J.C. English, "A Study of Developmental Toxicity of Hydroquinone in the Rabbit', Fundamental and Applied Toxicology 19, 214-221 (1992). E.C. Robinson and R.E.Schroeder, 'Reproductive and Developmental Toxicity Studies of a Linear Alkylbenzene Mixture in Rats', Fundamental and Applied Toxicology 18, Number 4, 549-556 (1992). CMA 118496 Raymond E. Schroeder, M-S. PUBLICATIONS - continued: S.C. Lewis, R.E. Schroeder and G.L. Kennedy, Jr., 'Developmental Toxicity of Dimethylformamide in the Rat Following Inhalation Exposure', Drug and Chemical Toxicology, Vol. 15, Number 1,1* 14 (1992). F.R. Johannsen, GJ. Levinskas, G.M. Rusch and R.E. Schroeder, "Subchronic Inhalation Toxicity and Reproduaive Assessment in Rats of Three Chlorinated Propane*", J. Toxicol. Environ. Health Vol. Vol. 33 (No. 3), 291-302 (1991). R.S. Nair, C.S. Auletta, R.E. Schroeder and F_R. Jobannsen, 'Chronic Toxicity, Oncogenic Potential, and Reproductive Toxicity of p-Nitroanfline in Rats', Fundamental and Applied Toxicology, Vol. 15, 607-621 (1990). Biles, R.W., Schroeder, R.E. and C.E. Holdsworth; 'Methyl Tertiary Butyl Ether Inhalation in Rats: A Single Generation Reproduction Study', Toxicology and Industrial Health, Vol. 3, No. 4, p. 519534 (1987). R.S. Nair, J.A. Barter, R.E. Schroeder, A. Knezevich and C.R. Stack, 'A Two-Generation Reproduction Study with Monochlorobenzene Vapor in Rats', Fundamental and Applied Toxicology, Vol. 9, 678-686 (1987). Conaway. C.C., Schroeder, R.E. and Snyder, N.K.; 'Teratology Evaluation of Methyl Tertiary Butyl Ether in Rats and Mice', Journal of Toxicology and Environmental Health. Vol. 16, 797-809 (1985). Nair. R.S., Jobannsen, F.R. and Schroeder, R.E., 'Evaluation of Teratogenic Potential of Paranitroaniline and Para-nitrochloro-benzene in Rats and Rabbits'; Toxidty of Nitroaromatic Compounds, Ed. by D.E. Rickert, Hemisphere Publishing Corporation, Washington, 1985; pp. 6185. Homan, E.R., Schroeder, R.E. 'Inhalation Teratology Studies on Cyclohexanone', presented at 'A Symposium on an Industry Approach to Chemical Risk Assessment-Caprolactam and Related Compounds as a Case Study'; (Abstract) published in the Proceedings of a Symposium on An Industry Approach to Chemical Risk Assessment, May, 1984, pp. 206-218. Daly, I.W., Schroeder, R.E., and Killeen, J.C. *A Teratology Study of Topically Applied Linear Alkyibenzene Sulphonate in Rats'; Food Cosmetic Toxicology. Vol. 18, 55-58 (1980). Ranney, R.E., Radzialowski, R.M., Mares, S.E., Schroeder, R.E. and Hutsell, T.C. "The Phenylalanine and Tyrosine Content of Maternal and Fetal Body Fluids from Rabbits Fed SC-18862"; Toxicology and Applied Pharmacology. Vol 32, 339-346 (1975). CMA 118497 Raymond E. Schroeder, M.S. PRESENTATIONS; Hoffman, G.M. and R.E. Schroeder; *A nose-only inhalation developmental toxicity study in ms comparing naive 'untrained* to tube acclimated 'trained* animals." Presented by poster at the Sixteenth annual meeting of the American College of Toxicology, November, 1595) Carney, E.W., Schroeder, R. and WJf. Breslin; Developmental Toxicity Study in Rats with Fluroxypyr methylheptyl ester"; Teratology, Vol. 51, No. 3, March 1995. (Presented by poster at the 35th Annual Meeting of the Teratology Society, June, 1995). Camey, E.W., Schroeder, R. and WJ. Breslin; 'Developmental Toxicity Study in Rats with Nitrapyrin*; Teratology, Vol. 51, No. 3, March 1995. (Presented by pewter at the 35th Annual Meeting of the Teratology Society, June, 1995). Schroeder, R.E., Newton, P.E., Rusch, G.M., and Trochimowicz, H.F.; Inhalation Developmental Toxicity Studies with HCFC-123 and HCFC-124 in the Rabbit"; Teratology, Vol. 51, No. 3, March 1995. (Presented by poster at the 35th Annual Meeting of die Teratology Society, June, 1995). Stephens. E., Nair, R., Carlton, B., Weiner, M., Smith, L. and Schroeder, R.:"Association of Liver Necrosis with Maternal Deaths in a One Generation Reproduction Study of Elemental Phosphorus*. The Toxicologist, Vol. 12, No. 1, February 1992. (Presented by poster at the 31st Annual Meeting of the Society of Toxicology, February, 1992). Bannister, R.M., Brewster, D.W., Rodwell, D.E., Schroeder, R.E. and Barnett, J.W., Jr. 'Developmental Toxicity Studies in Rats with 4-Aminodiphenylamine (4-ADPA) and 4Nitrodiphenylamine (4-NDPA)". The Toxicologist, Vol. 12, No. 1, February 1992. (Presented by poster at the 31st Annual Meeting of the Society of Toxicology, February, 1992). 'R.E. Schroeder, P.E. Newton, J.B. Sullivan and W.M. Busey. "An Inhalation Developmental Toxicity Study of Phosphine in Rats'. The Toxicologist, Vol 12, No. 1, February 1992. (Presented by poster at the 31st Annual Meeting of the Society of Toxicology, February, 1992). Blacker, A.M., Schroeder, R.E., English, J.C., Muipby, SJ. and Krasavage, WJ. "A TwoGeneration Reproduction Study with Hydroquinone in Rats*. Teratology, Vol. 43, No. 5, June, 1991. (Presented by poster at the 31st Annual Meeting of the Teratology Society, June, 1991). Schroeder, R.E., Gerhart, J.M. and Kneiss, J. "Developmental Toxicity Studies of Tributyl Phosphate in the Rat and Rabbit". Teratology, Vol, 43, No. 5, June, 1991. (Presented by poster at the 31st Annual Meeting of the Teratology Society, June, 1991). T.R. Hanley Jr., R.E. Schroeder and W.J. Breslin. 'Developmental Studies on s Series of 2,4-D Salts and Esters in the Rat* The Toxicologist, Vol 11, No. 1, February 1991. (Presented by poster at the 30th Annual Meeting of the Society of Toxicology, February, 1991). CMA 118498 Raymond E. Scbroedff, mjl V PRESENTATIONS - continued: WJ. Breslin, R.E. Schroeder and T.R. Hanley Jr.'Developmental Toxicity of Pidoram Potassium (L) and Triisopropanolamine (TIPA) Salts in die Rat' The Toxicologist, Vol 11, No. 1, February 1991. (Presented by poster at die 30th Annual Meeting of the Society of Toxicology, February, 1991). C.S. Auletta, R.E. Schroeder, WJ. Kroavage and C. Stack Toxicology of Diethylene Glycol Butyl Ether: 3. Dermal Subchronic Toxicity/Fertility Study in Rats*. The Toxicologist, Vol. 11 No. 1, February 1991. (Presented by poster at the 30th Annual Meeting of the Society of Toxicology, February, 1991). S J. Murphy, R.E. Schroeder, A.M. Blacker, WJ. Krasavage and J.C. English 'Study of Developmental Toxicity of Hydroquinone (HQ) in the Rabbit*. The Toxicologist, Vol. 11, No. 1, February 1991. (Presented by poster at the 30th Annual Meeting of the Society of Toxicology, February, 1991). R. Nair, J. Baner, H. Bolte, R. Schroeder and C. Stack. Evaluation of Reproductive Toxicity of Ortho-Di chlorobenzene (ODCB) in tbe Rat' (abstract); (presented at die V International Congress of Toxicology, July 1989). R.S. Nair, F.R. Johannsen, C.S. Auletta and R.E. Schroeder. 'Chronic Toxicity and Potential Reproductive Effects of P-NitroanBine (PNA) in the Rat'. The Toxicologist, Vol. 7, No. 1, February 1987. (Presented by poster at the 26th Annual Meeting of tbe Society of Toxicology, February, 1987). R.S. Nair, F.R. Johannsen and RE. Schroeder. 'Absence of Teratogenic Response in Rats and Rabbits Given a Detergent BuQder'. The Toxicologist, Vol. 7, No. 1, February 1987. (Presented by poster at the 26th Annual Meeting of tbe Society of Toxicology, February, 1987). E.C. Robinson, R.D. Short, F.R. Jobannsen and R.E. Schroeder. *A Teratology Study of a Mixture of Decyldodecyl Benzenes". The Toxicologist, Vol. 7, No. 1, February 1987. (Presented by poster at the 26th Annual Meeting of the Society of Toxicology, February, 1987). R.E. Schroeder, l.W. Daly and VJ. Theodorides. "A Teratology Study in Rats with Virginiamycm". The Toxicologist, Vol. 7, No. 1, February 1987. (Presented by poster at the 26th Annual Meeting of the Society of Toxicology, February, 1987). Biles, R.W., Schroeder, R.E. and Holdsworth, C.E., 'Single Generation Reproduction Study of Methy!-T-Butyl Ether in Rats", The Toxicologist Vol. 6, No, 1, 1986. (Presented by poster at die 25th Annual Meeting of the Society of Toxicology, March, 198Q. Schroeder, R.E., Terrill, J.B., Lyon, J.P., Kaplan, A.M. and Kimmerle, G., "An Inhalation Teratology Study in the Rabbit with Nitrobenzene", The Toxicologist. Vol. 6, No. 1, 1986. (Presented by poster at the 25th Annual Meeting of the Society of Toxicology, March, 1986). CMA 118499 Raymond E. Schroeder, M.S. PRESENTATIONS - continued: Parker, J.A., MacGregor, J.A. and Schroeder, R.E., "Diet Restriction During Gestation in NZW Rabbits Does Not Adversely Affect Fetal Outcome", Teratology. Vol. 33, pp. 71 C 1986 (Presented by poster at the 26th Annual Meeting of the Teratology Society, July, 1986). Nair, R.S., Johannsen, FJL, Levinskas, GJ.t and Schroeder, 1LE., "A Rat Teratology Study with Tetrathal", presented at the Federation of American Societies for Experimental Biology (FASEB), April 1984. Lewis, S.C., Rinehart, W.E., Schroeder, R.E., Thackan, J.W. "Dominant Lethal Mutagenic Bioassay Dimethyl Formamide (DMF)*; Environmental Mutagen Society (abstract) (1979). Schroeder, R.E., Vondruska, JJ. and YoukOis, LJ. *Teratological Evaluations of a Copper Containing Intrauterine Device (CuIUD) in Rats and Rabbits"; (abstract); Teratology. Vol. 3, (1975). Bost, R.G., McConnell, R.G., Rao, K.S., Reno, F.E., Schroeder, R.E., and Vondruska, J.F. "An Evaluation of the Mutagenic Potential of Asparatame and SC-19192"; American College of Nutrition (abstract) (1974). McConnell, R.G., Rao, K.S., Reno, R.F., Schroeder, R., Trotter, J. and Vondruska, JJ*. 'Fertility, Reproduction and Teratology Studies with Aspartame and SC-19192"; American College of Nutrition (abstract) (1974). REPORTS ON PROPRIETARY COMPOUNDS SUBMITTED TO SPONSORS: Numerous confidential predinical toxicity reports in the areas of general toxicity, reproduction/fertility, developmental toxicity and mutagenicity (i.e., dominant-lethal studies). These reports are prepared for Pharmaceutical, Agricultural and Industrial Chemical, Food Additive and Cosmetic Industries for submission to regulatory agencies around the world. I CERTIFY THAT THE ABOVE IS A TRUE AND ACCURATE ACCOUNT OF MY PROFESSIONAL HISTORY TO DATE. SIGNED, '1 --------DATE: l CMA 118500 CURJJCULUM VITAE NAME: Ira W. Daly, PhD. Diplomate, American Board of Toxicology PRESENT POSITION: (since November 1995) Vice President, Agrochemical & Industrial Toxicology Huntingdon life Sciences East Millstone, NJ 06875 Acts as the client representative on site. Responsible for maintaining communication with client without impeding direct communication between client and study management. Acts as program director "for multidisciplinary projects to ensure that precise requirements of the client are fully understood and met and to be available to provide client with appropriate advice and consultancy. Develops appropriate protocols and programs to provide the most scientifically valid and cost* effective solution to the client's requirements which will be acceptable to regulatory agencies. Responsible to senior management for the quality of service provided to clients. EDUCATION: 1977 St John's University Jamaica, New York PhD. Pharmacology 1974 Sl John's University Jamaica, New York M. Pharmacology PROFESSIONAL EXPERIENCE: 1993-1995 1989-1993 19S5-1989 1982-1985 1980-1982 1978-1980 1979-1980 Vice President Business Development Operations Sr. Vice President <fc Director of Toxicology Pharmaco LSR Inc, Toxicology Services Worldwide East Millstone, NJ 08675 Vice President and Director of Toxicology Director of Toxicology Associate Director of Toxicology/Operations Staff ToxicologistyStudy Director Bio/dynamics, Inc. East Millstone, NJ 08875 Adjunct Assistant Professor Graduate School of Allied Health Professions St John's University, Jamaica, New York CMA 118501 -2- Ira W. Daly, PhJX PROFESSIONAL EXPERIENCE - continued: 1977-1978 Supervisor, Applied Pbannacology/Consultant Toxicologist Department of Biological Sciences and Clinical Affairs American Chicle Division of Warner Lambert Company Morris Plains, New Jersey Conducted in-house toxicity studies, monitored contract toxicological investigations. Managed laboratory in the areas of basic and applied Gastrointestinal and Respiratory Pharmacology. Conducted clinical investigations in Gastrointestinal Pharmacology. PROFESSIONAL ACCREDITATION: Diplomate, American Board of Toxicology (1980) Regulatory Affairs Certification (1992) PROFESSIONAL APPOINTMENTS: Technical Committee - Society of Toxicology (1964-1986) PROFESSIONAL MEMBERSHIPS: Society of Toxicology European Society of Toxicology Society of Toxicology Reproductive Specialty Section Mid-Atlantic Chapter of the Society of Toxicology Society of Environmental Toxicology and Chemistry PUBLICATIONS AND PRESENTATIONS: Carol S. Auletta, Lionel F. Rubin, Ira W. Daly, Ward R. Richter, Kazuhiro Hosoi, Hiroshi Suda and Toshimi Ikuse; "26-Week Ocular Toxicity Studies in Pigmented Rabbits Treated Topically with Bunazosin Hydrochloride Ophthalmic Solution"; Atarashil Ganka (JXye), Vol 12, No. 3,1995. Newton, P.E., Bolte, H.F, Daly, LWn Pillsbury, BJD., Ben-Dyke, It, Drew, R.T. and Sheldon, A.W. "Subchronic and Chronic Inhalation Toxicity Studies of Antimony Triaxide in the Rat", Fundamental and Applied Toxicology 22, 561-576 (1994). "Validation of a Neurotoxicity Screening Battery" (presented at the 33rd Annual Society ofToxicology Meeting, March 1994). "Validation of a Developmental Neurotoxicity Screening Battery: Effects of Hydroxyurea, Methylmercuric Chloride and Diphenylhydantoin". KLE. Sloan, WJL Richter, GS. Auletta and LW. Daly (presented at the 51st Annua! Society of Toxicology Meeting, February 1992). CMA118502 -3- Ira W. Daly, PhD. PUBLICATIONS AND PRESENTATIONS - continued: "Oncogenicity Study of AO-128 in Mice", Japanese Pharmacology and Therapeutics 19, 131-142 (1991). "One-Year Oral Gavage Toxicity Study of AO-128 in Rats", Japanese Pharmacology and Therapeutics 19, 233-260 (1991). "One-Year Oral Toxicity Study of AO-128 in Beagle Dogs*, Japanese Pharmacology and Therapeutics 19, 261-282 (1991). "Subchronic Oral Toxicity of Cellulose Acetate in Rats", Food and Chemical Toxicology 29, 453-458 (1991). "One-Year Oral Gavage Toxicity Study ofLansoprazole (AG-1749) in Rats", Japanese Pharmacology and Therapeutics 18, 59-91 (1990). "One-Year Oral Gavage Toxicity Study ofLansoprazole (AG-1749) in Dogs", Japanese Pharmacology and Therapeutics 18, 93-118 (1990). "Malignant Hyperthermia Induction in Susceptible Swine Following Exposure to Arduan" (presented at the International Anesthesia Research Society 64th Congress, March 1990, Honolulu, HI). "A Subchronic Oral Toxicity Study with Cellulose Acetate" (presented at the 29th Annual Meeting of the Society of Toxicology, February 1990). The Toxicologist 10, #670 (1990). "Subchronic Inhalation Toxicity of Ethylbenzene in Mice, Rats and Rabbits", Fundamental and Applied Toxicology 13. 399 (1989). "Subchronic Inhalation and Oral Toxicity of Hydrogenated Terphenyls in Rats", Fundamental and Applied Toxicology 13, 559 (1989). "Subchronic Inhalation Toxicity of Ethylbenzene in Mice, Rats and Rabbits" (presented at the 28th Annual Meeting of the Society of Toxicology, March 1989), The Toxicologist, 9 (1) 1989. "Human Safety Studies with Albendazole" (presented at the Proceedings of the American Association of Veterinary Parasitologists, 34th Annual Meeting, July 1989) Orlando, FL. "Cardiac Performance and Hemodynamics in Malignant Hyperthermia Susceptible and Normal ` Pigs During Sevoflurane Anesthesia" (presented at the 42nd Post Graduate Assembly in Anesthesiology, December 1988). "A Teratology Study in Rats with Virginiamycin" (presented at the 26th Annual Meeting of the Society of Toxicology, February 1987), The Toxicologist 7 (1) 1987. CMA 118503 Ira W. Daly, Ph.D. PUBLICATIONS AND PRESENTATIONS - continued: *A Two*Generation Reproduction Study in Rats with Virgmiatnycin* (presented at the 26tb Annual Meeting of the Society of Toxicology, Februaiy 1967); The Toxicologist 7 (1) 1987. 'Chronic Toxicity and Oncogenicity Studies of LASSO (Alachlor) Herbicide in Rodents* (presented Fourth Internationa] Congress of Toxicology, July 1986); Toxicology Letters. 31 (SuppL) 56 (1986). Dose-Dependent Clearance of Antimony from Rat Lungs' (presented Society of Toxicology, March-1986); The Toxicologist 6-(l) 36 (1986). "Ectopic Pupil in Mice*, Laboratory Animal Science, 32 (1) 1982. 'Subchronic Toxicity of Chloropropanol in Rats and Dogs' (presented Society of Toxicology, San Diego, 1981). *A Teratology Study of Topically Applied Linear Alkyibenzene Sulfonate in Rats', Food and Cosmetic Toxicology, VoL 18,1960. The Effect of Prostaglandins PGE2 and PGF2A on Spermatogenesis in Adult Male SpragueDawley Rats" (presented ASPET August 1977); International Journal of Fertility, 24 (3) 1979. Testicular Morphology Following Prostaglandin Administration' (presented ASPET, August 1977); The Pharmacologist, 19 (2) 523 (1977). "Effects of d-Ampbetamine Sulfate on Aggressive Behavior in Laboratory Mice*, Clinical Toxicology, 8 (3) 1975. REPORTS ON PROPRIETARY COMPOUNDS SUBMITTED TO SPONSORS: Approximately 1500 confidential reports on acute, sub-chronic, chronic, reproduction/teratology and inhalation studies in rodent and higher animal species. These reports were issued to the pharmaceutical, agricultural, industrial chemical and food additive industries. I CERTIFY THAT THE ABOVE IS A TRUE AND ACCURATE ACCOUNT OF MY PROFESSIONAL HISTORY TO DATE. CMA118504 CURRICULUM VITAE NAME: Teresa S. Kusznir, V.M.D. PRESENT POSITION: (since November 1995) Veterinarian, Toxicology Department Huntingdon Life Sciences Inc. East Millstone, NJ 08875 Responsibilities include tbe development, implementation and maintenance of animal care standards for animals utilized (non-human primates, dogs, rodents, rabbits, birds and others with an average monthly census of approximately 9,000). Responsibilities also include assurance of compliance with local and federal laboratory animal regulations as well as AAALAC standards. EDUCATION: 1993 University of Pennsylvania Philadelphia, PA V.M.D. 1982 Delaware Valley College of Science and Agriculture Doylestown, PA B.S. Animal Husbandry PROFESSIONAL LICENSING: Pennsylvania PROFESSIONAL EXPERIENCE: 1995 Veterinarian, Toxicology Department Pbarmaco LSR Inc., Toxicology Services Worldwide East Millstone, NJ 08875 1973-1995 Post Doctoral Fellow, Laboratory Animal Science SmithKline Beecham Pharmaceuticals Swedeland, PA Responsibilities included the complete medical and surgical care of various species of research animals within RAD, including non-human primates, dogs, rodents, swine and rabbits. Provided veterinary consultation and surgical services to investigators including, but not limited to, implantation of vascular and gastrointestinal access devices. Additional responsibilities included necropsy and clinical diagnostics. Facilitated technical training programs, including SB 1995 Investigator Training Seminar 'Post-Operative Care and Monitoring of Lab Animals', and "Techniques in Anesthesia*, L.A.S.T.S. Seminar. 1991-1991 Associate Scientist, Department of Investigative Pathology Conducted hepatocellular proliferation studies in rats. Technical duties included dosing rats, surgical implantation of BrDU osmotic pumps, necropsy and terminal collection of liver samples. Further responsibilities included reading liver slides, assessing hepatic labeling indeces, correlating and graphing data and presenting results to senior staff. CMA 118505 -2- Teresa S. Kusznir, V.M.D. PROFESSIONAL EXPERIENCE; 1990-1990 Associate Scientist, Department of Immunology Conducted experiments on murine models of endotoxin shock and effects of various inhibitors of shock, cyclo-oxygenase and lipoxygenase pathways. Performed ELISA assays to measure TNF levels of these models. Duties included media and drug preparation, dosing and bleeding. Utilized computer graphics and word processing software for data summarization. Assisted in experiments on the inhibition of rheumatoid arthritis in rats. 1987-1989 Associate Toxicologist, Investigative Toxicology Unit Conducted pre-clinical in-vivo toxicity studies in various species of laboratory animals and evaluated the resulting data. Duties included drug preparation, delivery of test article to animal* by standard and novel routes, phlebotomy, assessing non-invasive blood pressures and EKGs in dogs and primates, performing necropsies and identifying gross pathology. Various computer skills utilized in report production word processing and generation of graphics by RSE. 1984-1986 Research Technician, Toxicology Wyeth Laboratories Great Valley, PA Responsible for carrying out drug safety evaluation studies in rats, mice, dogs and primates. Duties also included dosing, performing necropsies, maintaining accurate records, preparing drug and obtaining physiological specimens. Prepared Standard Operating Procedures for compliance with GLP regulations. Special projects included long term maintenance of drug induced diabetic monkeys and radiolabeled drug metabolism studies in dogs. 1983-1984 Veterinary Technician/Adoption Coordinator Morris Animal Refuge Philadelphia, PA Provided general veterinary care to all shelter animals, including physical exams, treatments, vaccination, grooming, euthanasia and kennel duties. Also interviewed pet adopters and educated same in responsible pet ownership. 1976-1982 Veterinary Technician/Receptionist Hopewell Veterinary Hospital Philadelphia, PA Responsible for assisting veterinarians with appointments, treatments, x-rays, surgeries and post-op care of animals. Performed routine lab work including heartworm tests, fecals, urinalyses, hematocrits and ultrasonic teeth cleaning. Additional responsibilities included clerical work, hiring, training and scheduling of part-time employees. CMA 118506 Teresa S. Kusznir, V.M.D. -3- I CERTIFY THAT THE ABOVE IS A TRUE AND ACCURATE ACCOUNT OF MY PROFESSIONAL HISTORY TO DATE. CMA118507 CURRICULUM VITAE NAME: Ward R. Richter, D.V.M., M.S. Diplomate, American College ofVeterinary Pathologists PRESENT POSITION: (since 11/93) Vice President & Scientific Director Hunringdoo Life Sciences Inc. East Millstone, NJ 08873 Responsible for all scientific and technical operations including toxicology, pathology, chemistry and computer operations. Interlace with clients on scientific issues. EDUCATION: - 1962 Iowa State University, IA M.S. Pathology 1955 Iowa State University, IA D.V_M. 1951 University of'Wisconsin, WI Pre-Veterinary VETERINARY LICENSE: Iowa Tllrnfrit inactive *** PROFESSIONAL EXPERIENCE: 7/91-11/93 Vice President & Director ofPathology Responsibilities included otganmns and directing the Department ofPathology (a department comprised of approximately 43 professional and technical staff) and bitwrfaehia with clients on matters of Pathology. 1990-7/91 President Kemdru International, Inc. (A Safety Assessment Consulting Finn) Evansville, IN 1989-1990 Coordinator, Pesticide Product Safety Chevron Environmental Health Center Richmond, CA 1983-1991 Vice President Toad Hall Systems, Inc. (A Software Development Firm specializing in Systems for Toxicology & Pathology Laboratories) Union Grove, WI CMA 118508 2- - Ward R. Richter, D.V.M., M.S. PROFESSIONAL EXPERIENCE continued: 1985-1989 1980-1985 Chiefof Pathology, Genetic Toxicology & Reproductive Toxicology Chevron Environmental Health Center Richmond, CA Director. Pathology Division International Research and Development Cotp. Masawan, MI 1977-1985 Professor (Collaborator Basis) Department ofVeterinary Pathology Iowa State University, IA 1973-1980 Director AJ. Carlson Animal Research Facility University of Chicago, Q, 1973-1980 Professor, Department ofPathology University of Chicago, IL 1973-1980 Professor. The College University of Chicago, IL 1969-1972 Deputy Director AJ. Carlson Animal Research Facility University of Chicago, IL 1968-1973 1964-1977 1967-1968 1963-1968 Associate Professor of Comparative Pathology Universny of Chicago, IL Associate Professor (Collaborator Basis) Department ofVeterinary Pathology Iowa Sate University, IA Clinical Associate Professor Department of Oral Biology Loyola University, Chicago, IL Head. Cellular Pathology and Election Microscopy (Pathology Department) Abbott Laboratories, North Chicago, IL CMA118509 Ward R. Richter, D.V.M., M.S. PROFESSIONAL EXPERIENCE - continued: 1961-1963 Research Associate (Pathology Department) University of Louisville School ofMedicine Louisville, KY 1960-1963 Chief Cellular Pathology Branch (Pathology Department), U.S. Army Medical Research Laboratory, Ft Knox, KY 1958-1960 Instructor, Veterinary Pathology Iowa State University, IA SOCIETY MEMBERSHIPS: American College ofVeteiinaxy Pathologists .Amencan College ofToxicology Society ofToxicological Pathologists United States & Canadian Academy ofPathology American Society for Investigative Pathology American Association for the Advancement ofScience American Veterinary Medical Association International Society for the Study ofXenobiotics HONORS AND HONOR SOCIETIES: SPECIALTY BOARD: Alumni Merit Award (Iowa State University Alumni Association, 1971) Alpha Zeta Phi Zeta Phi Kappa Phi Sigma Xi Outstanding Service Award (Charies Louis Davis, D.V.M. Foundation, 1981) Diplomats, American College ofVeterinary Pathologists (1966) SPECIAL CONSULTANTSHIPS AND COMMITTEE MEMBERSHIPS: 1957-1958 Faculty Advisor to Pre-Vet Club, Iowa Sate University CMA 118510 Ward R. Richter, D.V.M., M.S. SPECIAL CONSULTANTSHIPS AND COMMITTEE MEMBERSHIPS - continued: 1938-1959 Admissions Committee, College ofVeterinary Medicine. Iowa State University 1958-1959 Member Faculty Senior. Iowa State University 1965-1966 President. Midwest Society ofElectron Microscopists r966=1973 Special Lecturer and Consultant in Compara tive Pathology, Resident Investigator Program in Laboratory Animal Medicine, V.V Hospital, Hines, Illinois 1968-1969 Deputy Coroner, Lake County, Illinois 1969-1970 Director. Midwest Society ofElectron Miaoscopists 1972-1973 Member ofthe Scientific Advisory Panel to Assistant Secretary ofHEW on the safety ofNTA as a detergent builder 1973 Meeting Arrangements Chairman for 1974 Meeting ofAmerican College ofVeterinary Pathologists 1972-1973 Consultant to U.AREP on long-term low level toxicity program development for the National Center for Toxicologic Research, Pine Bluff, Arkansas 1973 Member. Committee on Education, Society of Pharmacological and Environmental Pathol ogists 1973-1974 Consultant Dental Research Institute Great Lakes Naval Training Station 1974-1975 Consultant National Institute of Environ mental Health Sciences 1974-1975 Consultant Graduate Review Committee, Department of Pathology, University of Chicago CMA 118511 Waid R. Richter, D.V.M., M.S. -5- SPECIAL CONSULTANTSHIPS AND COMMITTEE MEMBERSHIPS - continued: 1974-1975 Consultant to HHHB Hospital Management Consultants, Chicago and San Fiandsco 1971-1978 Consultant, Argonne National Laboratories Ajgonne, Illinois 1974-1976 Member, Senior Scientific Advisory Board, National Center for Toxicologic Research, Jefferson. Arkansas 1976 Consultant, Medical Examiner Cook County. Illinois 1973-1979 Chairman, Committee on Animal Resources University of Chicago, Illinois 1969-1980 Representative to Council ofNational Society for Medical Research 1971-1980 1973-1980 1974-1980 Consulting Pathologist, Lincoln Park Zoo Chicago. Illinois Midwest Program Director, Charles Louis Davis. D.V.M. Foundation Consulting Pathologist, Berwyn Veterinary Medical Center, Berwyn, Hlmni< 1974-1980 1977-1981 1979-1980 1979-1980 1979-1980 1973-1991 1983 Consultant, Carcinogenesis and Toxicology Illinois Institute ofTechnology Research Institute Member. Editorial Board, Journal of Environmental Pathology and Toxicology Vice President. Illinois Society for Medical Research Chicago. Illinois Consultant to L'AREP (FDA) for review and evaluation ofNitric Carcinogenicity Studies Consultant to Tiacor-Jitco (NCI) for review of carcinogenic potential of Dioxanes Faculty of Discussants, Charles Louis Davis, D.V.M. Foundation Advisory Board, Charles Loui* Davis, D.V.M. Foundation CMA 118512 6- Ward R. Richter, D.V.M., M.S. PUBLICATIONS: Richter, W.R.: Tubular Adenomata ofthe Adrenal ofthe Goat, Cornell VeL 47:558-577, 1957. Richter, W.R.: Adrenal Conical Adenomata in the Goat, American Jour. VeL Res. 19: 895-901, 1958. Richter, W.R_: Observations on the Penile Development ofthe Angora Goat, Am. Jour. Vet Res. 20: 603-606, 1959. Pearsou. P.T., Jensen, E.C. and Richter, WJL: Urinary Calculi in a 2-Month Old Pup, Jour. A-V.M-A. 135:329-331,1959. Thompson, J.H. and Richter. WJL: Heraatoxylin-Eosin Staining Adapted to Automatic Tissue Processing, Stain Technology 35: 145-148, 1960. Richter, W.R.: The Electron Microscope and its Application to Medical Research, Jour. A.VJVLA. 140: 1304-1309, 1962. Richter, W.R.: Electron Microscopic Observations on Transitional Epithelium ofDogs Infected with Canine Distemper. U.S. Army Res. Lab. Report #56, Fort Knox Kentucky', 1962. Richier. W.R.. Effects ofRF Energy an Tissue Cultures, U.S. Army Med. Res. Lab. Report #600, Fort Knox, Kentucky, 1962. Richter. W.R. and Moize, S.M.: Electron Microscopic Observations on the Collapsed and Distsided Mammalian Urinary Bladder, J. infrastructure Res. 9: 1963. Richter. W.R. and YosL D.H.: A Morphological Study of Rat Testicular Tissue Exposed to RF Energy, U.S. Army Med. Res. Lab. Report #601, Fort Knox, Kentucky, 1964. Richter, W.R.. Shipkowhz. N.L. and Rdzok, EJ.: Oral Papillomatosis ofthe Rabbit, An Electron Microscopic Study. Lab. Invest 13: 430-438.1964. Richter, W.R.. Rdzok, E.J. and Moize, S.M.: Electron Microscopy ofVirus-like Panicles Associated with Duck Viral Hepatitis. Virology 24: 1964. Cohen, A.I., Nicol, E.C. and Richter, W.R.: Nerve Growth Factor Requirement for Development of Dissociated Embryonic Sensory and Sympathetic Ganglia in Culture. Proc. Soc. Exp. Bio. Med. 116: 784-789, 1964. Margoliash, E., Schenck, J.R., Hargie, M.P., Burokas, S., Richter, W.R., Barlow, GJi. and Mascona, A.A.: Characterizarion of Specifc Cell Aggregating Materials foam Sponge Cells. Biochem. and Biophys. Res. Communications 20: 383-388, 1965. Richter, W.R.. Stein. R.J., Rdzok. EJ., Moize. S.M. and Bischoff M.B.: Ultrasuuctural Studies in Intranuclear Crystalline Inclusions in the Liver ofdie Dog. Amer. J. Path. 47: 587-599, 1965. CMA'HS5''3 -7- Waid R Richter, D.V.M.. M.S. PUBLICATIONS - continued: Smith, RJ., Richards, RK.. Richter, W.R_ Hylton, RR, McCabe, JJL and Collin, S.C.. Electrical Anesthesia Produced by Combining Direct and Alternating Currents: Electzonmiaoscopy ofdie Dog Brain. Anesthesiology 26: 607-611, 1965. Rdzok, E.J., Shipkownz. N.L. and Richter, W.R: Rabbit Oral Papillomatosis, Uhrastracture of Experimental Infection. Cancer Res. 26: 160-165, 1966. Richter, W.R, BischoC M.B. and Churchill, R_A_ An Observation of Fme Tubules Within the Endoplasmic Reticulum in a Doe Liver. Zeitschrift fur ZeUforschung 70: 180-184,1966. Stein. R.J.. Richter, WJL Zussman. RA. and Biynjolfeson, G.: Uhrastmctuni Charactenzarion of Daphnia Heart Muscle. Jour. Cell BioL 29: 168-170, 1966. Stem. RJ., Richter, W.R and Brynjolfeson. G.: Uhrastrocmral Phannacopathology" I. Comparative Morphology' ofthe Livers ofthe Normal Street Dog and Purebred Beagle. A Baseline Studv. Exp. and Mol. Path. 5: 195-224. 1966. Richter, W.R, Stem. RJ and Blockus. LX.: Experimental Production of Imramhochondrial Filaments in Dog Liver Proceed. 6th InL Cong, for Electron Microscopy: 615-616, Ky* oto. 1#966. BischofL M.B. and Richter. W.R: Some Uhrastruaural Features ofthe Pineal Organ in Japanese Quail. Proceed. 6th InL Cong, for Electron Microscopy: 523-524, Kyoto, 1966. Gerand. J., Richter. W.R. Fsnen. J. and Holper, J.C.: Biophysical Studies with Rhinoviruses Using Zonal Ultracentrifuge Systems. Jour. Virology 2: 937-943, 1968. Stem R.J., Richter. W.R. Rdzok. E.J.. Moire, S.M. and Brynjolfeson. G.: Compararivt Hepatic Ultrastructurc of Subhuman Primates: A Baseline Study of Rhesus, African Green and Squirrel Monkeys in: Use of Subhuman Primates m Drug Evaluation, ed. by Harold Vagtbeig, University of Texas Press, Austin, 1968. Bischoff, M.B.. Richter. W.R and Stein, RJ.: Ultrastructural Changes in Pig Hepatocytes During the Transitional Period riom Late Foetal to Eariy Neonatal Life. J. Cell Sri. 4: 381-395. 1969. Richter, W.R and Moire. S.M.: Ukrasmictural Nature oHcanine Distemper Ineiiiyions in rite Urinary Bladder. Path. VeL 7: 546-252.1970. Vesselinovitch. S.D.. Mihailovich. N. and Richter, W.R: The Induction of Malignant Melanomas in the Syrian White Hamster by Neonatal Exposure to Urcthan. Cancer Res. 30: 2543-2547. 1970. CMA 118514 Ward IL Richter, D.V.M., M.S. -8- PUBLICATIONS - continued: Port, C.D., Richter, W.R. and Moize. S.M.: An Uhiastmctuial Study ofTyzzer's Disease in the Mongolian Gerbil [Meriones unguiculatus], Lab. Invest. 25: 81-87, 1971. Richter, W.R., Zouhar, R.L., Tatsuno, J., Smith, RJi. and Cullen, S.C.: Election Microscopy ofthe Macaca Mulatta Brain after Repeated Applications ofElectrical Current. Anesthesiology 36: 374-377, 1972. Cburg^A. and Richter, W.R.; Histechemical Distribution of Carbonic Anhydrase after Ligation ofthe Pancreatic Duct. Am. J. Path. 68: 23-28, 1972. Pine. JJi., Richter, W.R. and Esteriy, J.R.: Experimental Bacterial Pneumonia. UluastructuraL Autoradiographic and Histochemical Observations. Amer. Jour. Path. 73: 115-122,1973. Richter, W.R.: A Support Laboratory for Medical School Animal Research Facility. Proceedings of National Conference on Research Animals in Medicine. Washington, D.C., 1974. Levin. S. and Richter, W.R.: A Systematic Method for the Detection of Experimentally Hyperplasia and Eariy Neoplasia in the Urinary Bladder. Toxicology and Applied Pharmacology 27: 680-684, 1974. Port. C.D., Baxter, D.W. and Richter, W.R.: The Mongolian Gerbil as a Model for T.rad Toxicity I. Studies of Acute Poisoning. Amer. Jour. Path. 76: 79-94, 1974. Torkelson, T.R.. Leong, B.K.J., Kodba, R.R-J., Richter, W.R. and Gehring, P.J.: TjA of Manifestation of Toxicity in Rais Inhaling 111 ppm 1.4 Dioxane for Two Years. Toxicology and Applied Pharmacology 30: 1974. Levin. S. and Richter. W.R.: Uhrastructure of Cell Surface Coal (Glycocalyx) in Rax Urinary Bladder Epithelimn. Cell Tissue Research 158: 281-283, 1974. Baxter. D.W.. Port, C.D. and Richter, W.R_: Mongolian Gerbil as a Model for Chronic T Toxicity. Jour, of Comparative Path. 85: 1975. ShybuL G.T., Richter, W.R. and Schuster. C.R: Absence of Pathological Changes Following Intravenous Methamphetamine and InsaArterial Iothalamate Meglumine. Research Communications in Chem. Path, and Path. 15: 53-73, 1976. Port C.D. and Richter, W.1L: Eosinophilic Leukemia in a Svrian Hamster. Vet. Path. 14' 283-286 1977. Takaia. A.N., Zaneveid, L. and Richter, W.R.: Laser-Induced Thermal Damage of Skin. US.AF school of Aerospace Medicine, 1977. CMA 118515 Wanl R Richter, D.V.M- M.S. -9- PUBLICATIONS - continued: Churg, A. and Richter, WJL: Eariy Changes in the Exochne Pancreas ofthe Dog and die Rat after Ligation c: the Pancreatic Ducl A Light and Election Microscopic Study. Am. J. Path. 3: 521-534, 1971. Goyer, RO.. Falk. H., Feldman, Hogan. M. and Richter. WJL: Renal Tumors in Rats Given Tnsodium Nitrilotriacstic Add in Drinking Water for Two Years. JNCI66: 86-89,1981. Shefhex. AM.. Dooley-. L_ Picks. A.. Grubbs, CJ.. Rust, JJL and Richter, W.R: Carcinogenicity of Diesel EMaiist Parades by hmaerachael Instillation-Dose Range Study. Proceedings of international Symposium on the Health Effects of Diesel Engine Emissions. 1981. Holmes. PA, Rust, J.H.. Sturdier. A.M. and Richier, W.R: Long-Tenn Effects ofTCDD and HCDD in Mice and Xas. Health Erects ofHalogfnated .Aromatic Hydrocarbons, New York Academy of Sciences, 1981. Levine. B.S.. Henry, M.C_ Port. C.D.. Richter, W.R. and Urbanek, MA: Nephrotoxic Potential of dsDiaminechloiplarinum and Four Analogs in Male Fischer 344 Rats. JNCI, 67: 201-206, 1981. Schoenig. G.P.. Goldenthai. EL GeiL RG.. Frith. CJL. Richter. W.R and Cariborg. F.W.: Evaluation of the Dos; Response and in utero Exposure to Saccharin in the Rat. Food and Cheat. ToxicoL 23: 475-190, 1985. KuncL RW and Richter. W.R.: Prevalence and Utrastruamt of Sarcocvstis in Rhesus Monkey's. Jpn J Vet Sci. 50: f 19-527, 1988. Cushman. J.R.. Richter. W.R. and Duke. J.T.. Effects of Skin Sensitization Test Wrapping on Guinea Pigs Contact Dermatitis 21: 279-280. 1989. Magaw. R.I.. Richter, W.R and MacGregor. JA: A Reexamination of Liver Tumors in Mice Exposed to Wholly Vaporized Unleaded Gasoline. Jour. American College of Toxicology 12: 195-199, 1993. MacGregor. JA, Richier. W.R and Magaw. RI.. Uterine Changes in Female Mice Following Lifetime Inhalation :: Wholly Vaporized Unleaded Gasoline: A Possible Relationship to Observed Liver Turnon? Jour. American College of Toxicology 12: 119-126, 1993. Dauehtrey. W.D., Duff}'. J.S.. Haddock L.S.. Keenan. Til. and Richter. W.R: Chronic Inhalation Study of Crmmerdal Hexane in Mice. The Toxicologist 14.1234, 1994. CMA 118516 Ward R. Richter, D.V.M., M.S. -10- PUBLICATIONS - continued: Carol S. Auletta, Lionel F. Rubin, IraW. Daly, Ward R. Richter, Kazuhiro Hosoi, Hiroshi Suda and Toshimi Dcuse; "26-Week Ocular Toxicity Studies in Pigmented Rabbits Treated Topically with Bunazosin Hydrodiloride Ophthalmic Solution"; Atanshh Ganka (JXye), Vol. 12, No. 3, 1995. Richter, W.R.: Author or Co-author ofseveral hundred reports on predhncal safety studies (1RDC 1980-1985 and at CEHC, 1985-1987). I CERTIFY THAT THE ABOVE IS A TRUE AND ACCURATE ACCOUNT OF MY PROFESSIONAL HISTORY TO DATE. SIGNED:, DATE: ? CMA 118517 CURRICULUM VITAE NAME: T Bill Waggoner, PhD. PRESENT POSITION: (since 11/93) Senior Sriemis. Chemistry Huntingdon Lie Sciences East Millstone. NJ 08875 Overall responsibility for the technical aspecs of Department of Chemistry, overseeing the amvmes of over 9 professional and technical staff. Responsible for all of the Residue, Environmental. Analytical Chemistry and Metabolism/Pharmacokmencs work conducted by the oiganiatian- Responsible for the preparation ofprotocols and chemistry cost estimates, and forthe preparation of final reports for completed studies to support reguiaory submissions. Reviews analytical procedures submiced by Sponsors and the development of appropriate of analysis. Coordinates in-house studies between other departments and ainrrurtrng with clients and other laboratories. EDUCATION: 1964 Michigan Stare University East Lansing, NO Ph.D. Organic Chemistry 1959 Oklahoma Stare University Stillwater. OK M.S. Biochemistry 1957 Oklahoma Stare University Stillwater. OK B.S Chemistry PROFESSIONAL EXPERIENCE: 7.92-11/93 Director of Menbolism & Analytical Chemisoy Phaonaco LSR inc. Overall responsibility for die Department c: Metabolism and Analytical Chemistry, overseeing the activities of over 30 professional and technical staff. Responsible for all of the Residne, Environmental, .Analytical Chemistry and Metabolism.?haxxnarokinetics work conducted by the organization. CMA 118518 7. Bill Waggoner, Ph_D. PROFESSIONAL EXPERIENCE - comrowri- 1981-1984 Pesdodes Registration Manager Rime Paoiaic Ag Company (formerly Rhone Poulenc Inc.) MaBnomh Junction. XI Primary contact with the EPA for .afimacm ofpesticides and r*hii<htng crop tolerances. 1979-1981 VksPresitign Amy-ocal Development Corporation Coiorado Springs, CO nfttfhnieal VtxffnTm pmvir^ng nrrrmrf Tn "Tt.-imnTnwwal few god asalvtical sxansr-. agnvrhwwiral phawwrwrwal COmpasiSS 1966-1978 Manager Biochemical Research Mocav Ccporanoc. Agricultural Chemicals Division Kansas Csy, MO Had. staff of35 professional and technical safffor development of new pesticide acme mgrediems: tadiosyathesis, metabolism, residue foenusy - moderate contact with EPA. 196J-1966 Scfor Resarch Chenist Mill ChsmcaJs Incorporated, Electroplating Division Deoh, Ml ACADEMIC EXPERIENCE: 1980 Visang Assistant Professor Cjyi*f*An ^allege Coicrado Springs. CO Orpnic ebraiisny and inorganic course and laboratory course 1960 Vising Assistant Pm&ssor Oklahoma State University Stijwater. OK Advanced aiochenical techniques; a course for graduate indents sd veeriaary medicine students PROFESSIONAL MEMBERSHIPS: American Chemical Society CMA 118519 T. Bill Waggoner, PhD. PUBLICATIONS: "SpeetroflucromeBic Dsennhatian afBayVp 2674 Residues in Poultry Tissue", JjLOjlC., 70, 813* 818 (1987) with M.C. Bownmi. "Problems and Pit&lis a Biochesscal Studies for Pesidde Toxicology", The Pesticide Chemist and Modern Toxicology, SX Bands! a aL sons, ACS Symposium Series 160, ACS, Washington, D C (1981). _ "New Aspects of Organophosphorous Peorides; VH Meabofiism. Biochemical and Biological Aspects of Nemaeui* and Rriarrri Aospoonmiac Compounds', Residue Reviews, VoL 53(1974) PRESENTATIONS: "Radiolabded Compound Dosing Starnes a Laboratory and Food Use Species for Tissue Residue Evaluations". Presaged m The Assccaxkn ofOfficial Analytical Chemists Spring Workshop, Ottawa, Canada; Drug Residue Sessions Spesal Warksfaop (1981). PATENTS: Two patents from reseama at MT (TTmirah, lnc_, ^lemnplawng Division. Royal Oak, MI: topics were "plating on plastic materials" with EJ. Seyb (1967). I CERTIFY THAT THE ABOVE IS A TRUE AND ACCURATE ACCOUNT OF MY PROFESSIONAL HISTORY TO DATL SIGNED: t V ^ fys// `/'Si DATE: '/ CMA 118520 Huntingdon Life Sciences Summary of Relevant Publications Paul E. Newton, PhD., DABT J.B. Knaak, TJL Baiflcnedn, L.W. Smith and P.E. Newton, "Subchronic Inhalation Toxicity Study of pChlorobenzoatrifluoride (PCBTF) in die Rat via Whole-Body Exposures", The Toxicologist 14, No. 1213, 1994. P.E.- Newton, W.L. Wooding- and W.E. Rinehart, "An Inhalation Oncogmmty Study in Mice of Methylethyketoxiine", The Toxicologist 14: No. 1220,1994. J.S. Duffy, L.S. Haddock, WD. Daughtrey, Til Keenan and P.E. Newton, "Chronic Inhalation Study of Commercial Hexane in Rats", The Toxicologist 14: No. 1233, 1994. Newton, PJE., Schroeder, R-E., Sullivan, JH., Busey, WX, and Banas, DA. "Inhalation Toxicity of Phosphine in the Rat: Acute, Subchrouic and Developmental", Inhalation Toxicology, 5 (2): 233-239 (1993). Ward R. Richter, D.V.M., ACVP Daughtrey, WD., Duffy, J.S., Haddock, L.S., Keenan, TJL and Richter, WJC: Chronic Inhalation Study of Commercial Hexane in Mice. The Toxicologist 14, 1234,1994. Magaw, R.I., Richter, W.R. and MacGregor, JA.: A Reexamination of Liver Tumors in Mice Exposed to Wholly Vaporized Unleaded Gasoline. Jour. American College of Toxicology 12:195-199, 1993. MacGregor, J.A, Richter, W_R_ and Magaw, RJ.: Uterine Changes in Female Mice Following Lifetime Inhalation of Wholly Vaporized Unleaded Gasoline: A Possible Relationship to Observed Liver Tumors? Jour. American College of Toxicology 12:119-126,1993. Raymond E. Schroeder, M.S., DABT Biles, R.W., Schroeder, R_E. and C.E. Holdsworth; "Methyl Tertiary Butyl Ether Inhalation in Rats: A Single Generation Reproduction Study", Toxicology and Industrial Health, Vol. 3, No. 4, p. 519-534 (1987). R_S. Nair, IA. Barter, R_E. Schroeder, A. Knezevich and C.R. Stack, "A Two-Generation Reproduction Study with Monochlorobenzene Vapor in Rats", Fundamental and Applied Toxicology 9, 678-686 (1987). F.R. Johannsen, GJ. Levinskas, G.M. Rusch and R.E. Schroeder, "Subcfaronic Inhalation Toxicity and Reproductive Assessment in Rac ofThree Chlorinated Propanes", J. ToxicoL Environ. Health 33 (3), 291-302 (1991). CMA 118521 Huntingdon Life Sciences Raymond E. Schroeder - continued: R-A. Kima, MJ. Nkolkh, RE. Schroeder and G.M. Ruscfa, "A Female Rat Fertility Study with TnhaiwH Benzene", Journal of the American College ofToxicology, Volume 11, Number 3,275-282 (1992). Conaway, C.C., Schroeder, RE. and Snyder, NX; Teratology Evaluation of Methyl Tertiary Butyl Ether in Rats and Mice", Journal ofToxicology and Environmental 797-809 (1985). Homan, EJL, Schroeder, RE "Inhalation Teratology Studies an Cyclohocanooe", presented at "A Symposhim ah anlndustry Approach to Chemical Risk a apmiar^atn gj Rotated Canmpounds as a Caff Study"; (Abstract) published in the Proceedings of a Symposhim on An Industry Approach to Chemical Risk Assessment, May, 1984, pp. 206-218. CMA 118522 Appendix B CMA 118523 Huntingdon Life Sciences HUNTINGDON LIFE SCIENCES VINYL CHLORIDE COMBINED INHALATION TWO-GENERATION REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDY IN CD RATS STUDY NO.: ft ISSUE NO.: 1 SPONSOR STUDY NO: # SPONSOR: CHEMICAL MANUFACTURERS ASSOCIATION SPONSOR REPRESENTATIVE: Hasmukh C. Shah, Ph.D DATE: 5 December 1995 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 118524 Huntingdon Lif Science* Chsmicsl Manufacturer* Aaaociation 1 INTRODUCTION: 1.1 STUDY NO.: 1.2 ISSUE NO.: 1.3 STUDY TITLE: Study Numbar * Sponaor Numbar Page 2 Protocol 01 1 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats. 1.4 TEST MATERIAL: 1.5 SPONSOR: 1.6 SPONSOR REPRESENTATIVE: Vinyl Chloride Chemical Manufacturers Association 2501 M Street. NW Washington, DC 20037 Hasmukh C. Shah, Ph.D. Work * 202-887-1192 Fax - 202-887-4756 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 development toxicity study outlined in this protocol are to evaluate the effects of the test material on parental toxicity, reproductive capabili ty, in utero development, and neonatal growth and survival in rats. 2 REGULATORY REFERENCES: 2.1 TEST GUIDELINE: This study is designed to meet or exceed the guideline requirements of the EPA (Environmental Protection Agency: TSCA Test Guidelines (EPA, 1985); the Organization for Economic Co-operation and Development (OECD, Guidelines for testing of chemicals. Section 4: Health Effects, (OECD, 1981); and the European Economic Community (EEC), Methods for the Determination of Toxicity (EEC, 1988). CMA118525 Huntingdon Life Sciences Chemical Manufacturers Association Study Number * Sponsor Number 2.2 GOOD LABORATORY PRACTICES: Page 3 Protocol 01 This study will be conducted in compliance with the FDA (Food and Drug Administration) Good Laboratory Practice (GLP) Regulations for nonclimcal studies (FDA, 1988), the EPA TSCA Good Laboratory Practice Standards (EPA,1990), the OECD Good Laboratory Practice Procedures (OECD, 1982) and the standard operating procedures of the Testing Facility. 2.3 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 1 985. Huntingdon Life Sciences (East Mill stone, NJ) is fully accredited by the American Association for Accredita tion of Laboratory Animal Care (AAALAC). 2.4 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. 168, 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 discom fort, 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. 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 Sponsor will be advised by the Study Director of all circumstances which could lead to this action in as timely a manner as possible. CMA118526 Huntingdon Ufa Sciancas Chemical Manufacturers Association Study Number ' Sponsor Number Page 4 Protocol 01 2.4 ANIMAL WELFARE ACT COMPLIANCE: 5. Methods of euthanasia used during this study are in conformance with the above referenced regulations. 3 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. 4 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 affect ing animal welfare will also be signed by two members of the Institutional Animal Care and Use Committee prior to the modification's implementation. 5 STUDY PERSONNEL: Study Director: * Director of Inhalation Toxicology: * Study Pathologist: * Additional personnel will be documented in the project file and presented in the final report. CMA 118527 Huntingdon Lit* Sciancs* Chemical Manufacturers Association Study Number ' Sponsor Number Page 5 Protocol 01 6 PROPOSED STUDY DATES Reproduction Study: Initiation date: Receipt of test animals: Initiation of exposures (P.,): (Experimental start date) Date Study Director Signs proto* col * See Section 18.2. (to be addressed by amendment) Termination of exposures (P^: Selection (P2 from the F,): Termination of exposures (P2): Necropsy 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 signedby Study Director. CMA 118528 Huntingdon Lite Sciences Chemical Manufacturers Association 7 EXPERIMENTAL DESIGN: 7.1 Two-generation Reproduction Study: Study Number Sponsor Number Page 0 Protocol 01 Group 1 (control) II HI IV Exposure Level (ppm) 0* 10 too 1100 Number of Animals Mated Adults Microscopic Pathology Adult Generations11 ?! M F ?2 MF ?t M F P* 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 * Control animals will be chambar-housed and sham-treated with clean room air for a comparable period of time ea the test animals. b Histologic examinatione will be performed for tissues listed In Appendix A. Key: A.R. As Required: 1) tissues that demonstrate treatmen!-related histologic changes in Group IV (additional costl; 21 gross lesions from animals found deed or euthanatized in a moribund condition during the study (additional costl; and 3| gross lesions tarminal animals (additional costl; M - Male; F _ Female. CMA118529 Huntingdon Life Sciences Chemical Manufacturers Association Study Number Sponsor Number Page 7 Protocol 01 7.2 Developmental Toxicity Study: The developmental toxicity study wilt be performed with a unique population of animals during the exposure period for the P1 parental animals. This will be during the mating and gestation periods for the Ft pregnancies when animals ere being exposed 7 days/week. Animals in the developmental toxicity study will be exposed in the chamber along with the animals in the two-generation reproduction study. ' Group 1 (chamber housed, sham air control) II III IV Exposure Levels Ippm) 0 10 ioo 1100 Treatment Schedule 1 Gestation Days 6-19 Gestation Days 0-19 Gestation Days 6-19 Gestation Days 6-19 Mated Number of Animals Sacrificed* Proportion of Geatation Day 20 Feluses/Litter Malformation/Varialion Evaluations Gestation Day 20 External Soft Titaua Skeletal 25 A.S. 25 A.S. 25 A.S. 25 A.S. All All All All W A 'A 'A 1A 'A 'A 'A * Gross postmortem examination, liver and kidneys will ba weighed and preaerved along with gross lesions. Microscopic examinations ol 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: CMA 118530 Huntingdon Lrfa SciancM Chemical Manufacturers Association 8 TEST MATERIAL: 8.1 TEST MATERIAL: Study Number Sponsor Number Vinyl Chloride Page 8 Protocol 01 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 test material at the start of study and at 6*month intervals will be determined and reported. These analyses will be per formed in the analytical laboratory of the Testing Facility. 8.4 ARCHIVAL SAMPLES: A sample (approximately 10 grams) from each 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, waste material and empty test material containers will be returned to the sponsor (information concerning where these materials should be returned will be provided by the Sponsor). 9 TEST ANIMALS: 9.1 STRAIN: Sprague Dawley - derived (CD*) ICrl: CD* BR] Albino Rats (Outbred) VAF/Plus* CMA 118531 Huntingdon Ufa Sciance* Chemical Manufacturer Association Study Number Sponsor Number Page 9 Protocol 01 9.2 SUPPLIER: Charles River Laboratories Portage, Michigan 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 guide lines. In addition, a historical control 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 (P1): 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 (and no more than eight weeks) at initiation of treatment. 9.4.2.2 Developmental Toxicity Study: Females: eight weeks at receipt and at least 10 weeks at initiation of mating. 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 ascer tain suitability for study. CMA 118532 Huntingdon Ufa Sciancat Chemical Manufacturer* Acaociation Study Number Sponec Number Page 10 Protocol 01 9.6 ANIMAL HUSBANDRY: 9.6.1 Housing: 9.6.1.1 Two-generation Reproduction Study (P.,): 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 individually except as follows: Mating: one male and one female co-housed nightly. Lactation: dam with litter. Postweaning: 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 (P., and P2) will be fasted. During exposures, ani mals will not have access to feed. 9.6.3 Water: Facility water supply (Elizabethtown Water Company, Westfield, NJ); without restriction, via an automated water delivery system to individual animal cages. CMA118533 Huntingdon Uf Scincs Chemical Manufacturers Association Study Number Sponsor Number Page 11 Protocol 01 9.6.4 Bedding Material - Two-generation Reproduction Study: Hardwood shaving bedding (Lab Aspen Shavings, Noah Eastern Products Corporation, Warrensburg, NY) will be provided for each mated female on Day 20 of gestation. Fresh bedding will be provid ed as needed to Day 14 of lactation. 9.6.5 Feed Analysis: Analytical ceaification 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 main tained on file at the Testing Facility. Biannual chemical and microbi ological 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 Patt 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 evaluation. 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 emer gency situations, decisions will be made as needed and. the Sponsor will be advised as soon as possible. CMA118534 Huntingdon Life Sciancaa Chemical Manufacturer* Asaociation Study Numbaf Soonaor Number Page 12 Protocol 01 9.6.9 Environmental Conditions: 9.6.9.1 Light/Dark Cycle: Twelve hour light/dark cycle daily. 9.6.9.2 Temperature: Monitored and recorded twice daily. Temperature in the animal room will be maintained in the range of approximately 20-24C to the maximum extent possible. 9.6.9.3 Humidity: Monitored and recorded once daily. The desired humidity range is 40-70%. Humidity will be maintained in this range to the maximum extent possible. 9.7 SELECTION FOR STUDY: 9.7.1 Two-generation Reproduction Study (P1): More animals than required for the study will be purchased and acclimated. Animals considered suitable for study on the basis of pretest physical examinations, body weight data and any other pretest evaluations will be randomly assigned to control or treated groups in an attempt to equalize mean group body weights. Individ ual 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 acclimated. 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. 9.8 ANIMAL IDENTIFICATION: 9.8.1 Two-generation Reproduction Study: Each animal will be assigned a temporary identification number upon receipt. After selection for study (P, generation), each animal will be ear-tagged with a number assigned by the Testing Facility. CMA 118535 Huntingdon Ufa Sciancas Chemical Manufacturers Association Study Number Sponsor Number Page 13 Protocol 01 9.8.1 Two-generation Reproduction Study: This number plus the study number will comprise the unique identi fication for each study animal. Following the procedure (postweaning) to select the P2 parental animals from the F1 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-genera tion study. This number plus the study number will comprise the unique identification for each study animal. Females will be eartagged on Day 0 of gestation. 10 MATING, GESTATION AND LACTATION PROCEDURES: 10.1 MATING PROCEDURE: 10.1.1 Two-generation Reproduction Study (P.,, P2): After animals have been exposed to the test substance for the appropriate length of time (premating treatment period), one male and one female of equivalent dose levels will be caged together nightly until a sign of mating (microscopic observation of sperm in the vaginal smear and/or a copulation plug in the vagina) is ob served 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 P2 generation cohabitation of male and female littermates will be avoided. 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 and females 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 CMA 118536 Huntingdon Lifa Sciences Chemical Manufacturen Association Study Number Sponsor Number Page 14 Protocol 01 10.1.2 Developmental Toxicity Study: which evidence of mating is observed will be defined as Day 0 of gestation. 10.2 PARTURITION AND LACTATION - TWO-GENERATION REPRODUCTION STUDY: 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 pro longed 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, bed ding 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 Fj litter, two pups/sex/Iitter 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. CMA118537 Huntingdon Ufa Sciancas Chamical Manufacturer* Asaociation Study Numbar Spona ir Numbar 11 TEST MATERIAL ADMINISTRATION: Rags 15 Protocol 01 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 hours/day, five days/week for at least 70 days (10*week) prior to mating (designat ed 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. Females will be allowed to deliver (Day 0 of lactation) and initiate 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 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 hours/day over the Day 6-19 gestation interval. The exposure interval for these animals will coincide with the exposures for the mating/gestation/lactation periods for the F1 litters in the two-generation reproduction study. 11.4 GENERATION PROCEDURE: The test material will be administered as a vapor/aerosol in the breathing air of the animals. The test atmospheres will be generated by an appropri ate 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 minimum volume of approximately 6000 liters (6 m3). Each chamber will be operated at a minimum flow rate of 1200 liters per minute. The final airflow will be set CMA118538 Huntingdon Lit* Sciancac Chemical Manufacturer* Association Study Number Sponsor Number Page 16 Protocol 01 11.4 GENERATION PROCEDURE: 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 are considered adequate to maintain the oxygen level above 19%. 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 concentra tions will be made at least hourly using a MIRAN infrared spectrophotome ter. Also prior to initiating of animal exposures, additional samples will be taken to determine the distribution of the test material in the exposure chamber. 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). The minimum frequency of chamber activity is summarized below: Activity Analytical Test Material Con centration Temperature Relative Humidity Airflow Rate Nominal Test Material Con centration (excluding the control chamber) Rotation Pattern of Exposure Cages Loading/Unloading Verification Frequency/chamber/day | 6 times (hourly) 12 times 12 times 12 times once once once | CMA 118539 Huntingdon Lit Scinc Chemical Manufacturer* Aaaociation Study Number Sponaor Number Page 17 Protocol 01 11.6 EXPOSURE CONCENTRATIONS: For both the two-generation reproduction and developmental toxicity studies the targeted exposure concentrations will be 0, 10, 100 and 1100 ppm of the test material. These exposure levels correspond to oral equivalent doses of approximately 0, 9.2, 92 and 1012 mg/kg/day assum ing 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. 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: 12.1.2.1Two-Generation Reproduction Study: Each animal will be removed from its cage and examined at least once pretest (P-,), at the study start (first day of treatment for the P,) 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. CMA 118540 Huntingdon Lift 5cinc* Chamicol Manufacturer* Aaaociation Study Number Sponsor Numbar Page IS Protocol 01 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 observa tions 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. During the test period, these examinations will be performed postexposure when animals are being transferred to their home cages. 12.2 BODY WEIGHTS: 12.2.1 Two-generation Reproduction Study: Males: P.j: 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: Pn: at the time of sorting into groups. Pi 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 iactation: days 0, 4, 7, 14, 21 and 25 (P1 only); and at termination. 12.2.2 Developmental Toxicity Study: Females will be weighed on Days 0, 6, 9, 12, 15 and 20 of gesta tion. CAM 118541 Huntingdon Ufa Sciancaa Chemical Manufacture's Association Study Number Sponsor Number 12.3 FOOD CONSUMPTION: Page 19 Protocol 01 12.3.1 Two-generation Reproduction Study: Males: P,: one week pretest. P., and P2: weekly during the premating treatment period and from termination of mating through to sacrifice (postmating period). Food consumption will npt be recorded during the mating periods. Females: P,: one week pretest. P-i 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. 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 DETERMINATIONS - TWO-GENERATION REPRODUC TION STUDY: 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. 12.5 LITTER EVALUATIONS (F1, 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 neutral, phosphate-buffered 10% formalin. Litter size will be recorded on Days 0, 4, 7, 14, 21 (F, and F2) and 24 (F1 only) of lactation. CMA 118s42 Huntingdon Life Scianca* Chemical Manufacturers Aaaociation 12.5.2 Culling: Study Number Sponae r Number Page 20 Protocol 01 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. Preferential 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 con firmed buy 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 (F1 pups only). 12.5.4 Pup Body Weights and Sexing Data: 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 24 (F1 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 anogenital 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 condition or found dead. These evaluations will be performed under the direct supervision of a veterinary pathologist. The eyes of the parental animais will be examined in situ by gently pressing a moist ened glass slide against the cornea and observing the eyes under fluorescent light. During the gross postmortem examination all abnormal observations will be recorded. The necropsy of the CMA 118543 Huntingdon Life Scianca* Chemical Manufacturer* Aeaociation Study Numbar Sponaor Numbar Page 21 Protocol 01 13.1.1.1 Parental animals: 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 implantation scars, if present. 13.1.1.2 Weanling Pups (Fn and F2): At weaning (Day 24 - F, and Day 21 - F2) one pup/sex/Iitter/group will be selected at random for a complete gross postmortem exami nation. 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. 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): P7 males will be sacrificed after the last F1 litters have been deliv ered. This will permit some evaluation of fertility (i.e., number of litters delivered) prior to sacrifice. The P2 males will be sacrificed approximately one week after completion of the mating period to produce the F2 pregnancies. The Sponsor will be notified prior to sacrifice of the P1 and P2 males. 13.1.2.3 Terminal Necropsy Females [Pv P2): All Pt and P2 females, regardless of reproductive status (unmated, mated but not pregnant, or with litter) will be sacrificed as a group after the last litters have weaned for all groups. Parental females (Pi and P2) will be necropsied on day 2 of diestrus whenever possi ble. 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. CMA118544 Huntingdon Ufa Scianca* Chemical Manufacturers Association Study Number Sponsor Number Page 22 Protocol 01 13.1.2.4 Terminal Necropsy Pups (F^ 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, 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 these pups will be discarded. Culled pups with external irregularities will be preserved intact in 10% neutral buffered formalin. 13.1.2.4.2 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, will 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 and if unremarkable, sacrificed and discarded. Pups with external findings will be given a gross internal examination and only abnor mal tissues taken and preserved in 10% neutral buffered formalin. 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 exami nation and only abnormal tissues taken and preserved in 10% neutral buffered formalin. CMA 118545 Huntingdon Lif Scincas Chemical Manufacturers Association Study Number Sponsor Number 13.1.3 METHOD OF EUTHANASIA: Page 23 Protocol 01 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 examina tion will be sacrificed by exsanguination following anesthesia with inhaled carbon dioxide. Remaining pups will be sacrificed with an overdose of inhaled carbon dioxide. 13.1.4 Organ Weights: 13.1.4.1 Parental Animals: The following organs will be weighed at terminal sacrifice from the first 1S Pn and P2 animals in each group: | uterus testes seminal vesicles (with coagulat ing glands and their fluids brain kidneys adrenals thymus ovaries right epididymis (total and caudal prostate liver lungs spleen | | f | 2| | Ijj CMA 118546 Huntingdon Ufa Science* Chemical Manufacture!* Association Study Number Sponsor Number Page 24 Protocol 01 13.1.4.1 Parental Animals: 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. 13.1.4.2 Weanling Pups (F1 and F2): The following organs will be weighed from the first 15 male and female pups per group selected for complete macroscopic examina tion 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. 13.1.5 TISSUES PRESERVED: 13.1.5.1 Parental Animals: Grossly abnormal tissues will 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. 13.1.5.2 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. 13.1.6 Preservatives: All tissues - 10% neutral buffered formalin. Testes and epididymi des of the parental animals will be fixed in Bouin's solution for at least 48 hrs prior to permanent storage in formalin. Lungs will be infused with formalin to their approximate normal inspiratory vol ume. The nasal cavity will be flushed with formalin via the pharyn geal duct to ensure rapid fixation of the tissue. CMA 118547 Huntingdon Lift Scianca* Chemical Manufacturers Association Study Number Sponscr Number Page 25 Protocol 01 13.1.7 MICROSCOPIC PATHOLOGY EVALUATIONS: Slides of tissues listed in Appendix A (under Microscopic Examina tion) 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 high-dose animals, then examinations will be made of these tis sues/organs for low- and mid-dose animals (additional cost). Addi tional examinations will be made only after consultation with the Sponsor and receipt of authorization from the Sponsor. Note: any abnormalities not noted during macroscopic postmortem examina tions which are seen during histological processing will be recorded. Gross lesions will be examined at additional cost. 13.1.8 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 em ployed at the request of the Sponsor (additional cost). 13.1.9 SPERM COUNT, MOTILITY AND MORPHOLOGY ASSESSMENTS: From the first 15 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. Motility will be as sessed 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 pre pared 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 CMA 118548 Huntingdon Lif* SetancM Chemical Manufacturar* Aaaociation Study Numbar Sponaor Numbar Paga 26 Protocol 01 13.2.1 MATERNAL TERM SACRIFICES: 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 exam ined and fetuses obtained 19 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 evalu ated 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 ana lyzed 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 abdomi nal 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 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 calcula tion of uterine implantation data. CMA 118549 Huntingdon Ufa Sciences Chemical Manufacturers Association Study Number Sponsor Number 13.2.3 Fetal evaluations: Page 27 Protocol 01 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 observation of the palate. The sex of each fetus will be noted by observation of the anogenital 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 stain ing 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. 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 proce dure of Staples (1974) . The evaluations will be performed on the fresh 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 CMA 118550 Huntingdon lrffl Scianca* Chamical Manufacturer* Aaaociation Study Numbar Sponaor Numbar Page 28 Protocol 01 13.2.4 Resorptions: fetuses recovered on Day 20 of gestation. Only late resorptions with external malformations 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 obser vations 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 five 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 one year from the issuance of the final report 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) * Weeks 0*10; -over each weighing interval during the gestation and lactation periods to include Days 0-20 of gestation and Days 0-24 (F1 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); CMA 118551 Huntingdon LH Sciences Chemical Manufectu ere Association Study Number Sponsor Number Page 29 Protocol 01 15.1.1 Two-generation Reproduction Study: 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 (F., and F2 litters); Mean gestation length (F, and F2 litters); Mean pup live birth indices (F1 and F2 litters); Mean pup viability indices (Days 0-4) and weaning indices (Days 424 or 21 for the F1 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 gesta tion) and weight gain (between all weighing intervals to include Days 6-20 of gestation using both the actual and corrected Day 20 gestation weight); 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 appropriate 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 vari ances are equal (p>0.01), parametric procedures will be used; if not (p<0.01), nonparametric procedures will be used. The para metric 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 non parametric procedure for testing equality of means is needed, the Kruskal-Wallis test will be used, and if differences are indicated, a CMA 118552 Huntingdon Ufa Sciancaa Chemical Manufacturer* Aaaociation Study Number Spor tor Number Page 30 Protocol 01 15.1.3 STATISTICAL ANALYSES CONTINUOUS DATA - MULTIPLE GROU:P ANALYSES summed rank test (Dunn) will be used to determine which treat ments 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 pre sented untransformed. References for these techniques are Snedecor, G.W., Cochran, W.G., Statistical Methods, 6th edition, 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: 1096-1121 (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 & Wolfe Hollander & Wolfe Snedecor & Cochran Snedecor & Cochran Regression Analysis - Trend pp. 135-153 Lack of fit pp. 456-459 Jonckheere's Statistic pp. 120-123 Snedecor & Cochran Snedecor & Cochran Hollander & Wolfe CMA''''8553 Huntingdon Ufa SciancM Chemical Manufacturer* Acaociation Study Number Sponsor Number Page 31 Protocol 01 IS.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. 15.2.3 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 per formed to determine if the proportion of incidences differed be tween 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 per formed. 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. CMA 118554 Huntingdon Life Sciencaa Chemical Manufacturers Association Study Number Sponsor Number Page 32 Protocol 01 15.2.3 INCIDENCE DATA ANALYSIS: References for the techniques are Snedecor, G.W., and Cochran, W.G., Statistical methods, 6th ed., Iowa State University Press, Ames, Iowa (1971); Bradley, J.V., Distribution Free Statistical Tests, Prentice*Ha!l, 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 Determina tion 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. 39426-39433. 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. CMA 118555 Huntingdon Lif* Scincc Chemical Manufacturer* Aaaociation Study Numbar Sponaor Numbar Page 33 Protocol 01 16 REFERENCES: 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: 17.1 STATUS REPORT: For the two-generation reproduction study, status reports will be issued monthly during the premating treatment period and after weaning of the F1 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; 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 (Fv 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; CMA 118556 Huntingdon Ufa Sciancaa Chamicai Manufactu arc Association Study Numbar Sponsor Numbar Page 34 Protocol 01 17.1 STATUS REPORT: 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; Inhalation Exposure Report; References for experimental methodology; Senior personnel participating in the study; Quality Assurance Statement. 17.2.2 Data tabulations for parental generations (P1, P2): Mortality - termination history; Physical in-life observations (summarized and individual data pre sented 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; CMA 118557 Huntingdon Ufa Seianca* Chemical Manufacturers Association Study Number Sponsor Number Page 35 Protocol 01 17.2.2 Data tabulations for parental generations (P1f P2): Macroscopic postmortem observations (adults, weanlings); Microscopic pathology examinations; Organ weight data; Sperm assessment data. 17.2.3 Data tabulations for litters and offspring (F, and F2): Mean gestation length; Mean number of pups (live, dead and total) at birth and live pups at Days 4, 7, 14 and weaning (Day 24 - F1 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; Types of findings and incidence of fetuses with external, soft tissue and skeletal malformations/variations; Incidence of litters containing fetuses with malformations/variations (external, soft tissue and skeletal) Mean fetal weights; Fetal sex distribution ratios; 17.2.5 Appendices: All 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, fitter and uterine implantation data, organ weight data, sperm assessment data, estrous cycle data, gross CMA 118558 Huntingdon Lif* ScioncM Chemical Manufacturers Association Study Numbar Sponaor Numbar Page 36 Protocol 01 17.2.5 Appendices: postmortem findings, microscopic examination data, chamber exposure data. 18 SIGNATURES: 18.1 INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC): The IACUC Protocol Review Subcommittee has reviewed this protocol and found it to be in compliance with all appropriate regulations. BY: DATE: TITLE: Institutional Animal Care and Use Committee Member BY: DATE: TITLE: Institutional Animal Care and Use Committee Member FOR: Huntingdon Life Sciences East Millstone Facility 18.2 PROTOCOL REVIEWED AND ACCEPTED: BY: TITLE: Study Director FOR: Huntingdon Life Sciences DATE: BY: DATE: Hasmukh C. Shah, Ph.D. TITLE: Sponsor Representative FOR: Chemical Manufacturers Association CMA 118559 Huntingdon Lift Sciences Chemical Manufacturers Aaaociation Study Number Sponaor Numbar Paga 37 Protocol 0' APPENDIX A Tissues Preserved/Examined Microscopically No.* Tissue 2 adrenals 1 aorta 1 auditory sebaceous glands 1 bona (including joint) 3 brain (cerebrum, brainstem, cerebellum) 1 cecum 2 coagulating glands 1 colon 1 duodenum 2 epididymis 1 esophagus 2 eyas 1 heart 1 ileum 1 jejunum 2 kidneys 2 lacrimal/Hardarian glands 1 larynx 2 liver 2 lungs 1 mammary glands 1 mediastinal lymph nodes 1 mediastinal tissues 1 mesenteric lymph nodes 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 Examination (Groups) 1, IV II. Ill X X X X X X CMA 118560 Huntingdon Lrf* Sciences Chemical Manufacturer* Association Study Number Sponsor Number Page 38 Protocol 01 APPENDIX A Tissues Preserved/Examined Microscopically No.* Tissue 1 mesenteric tissue* 4 nasal tissues (turbinates) 1 oral tissues 1 ovary* 2 oviducts 1 pancreac 2 parathyroid glands 1 peripheral nerve 1 pituitary 1 prostate 1 rectum 2 salivary glands 2 seminal vesicles 1 skeletal muscle 1 skin 3 spinal cord (cervical, thoracic, lumber) 1 spleen 1 stomach 2 testis 1 thymus 2 thyroid gland 1 tongue 1 trachea 1 urinary bladder 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 Examinstion (Groups) 1, IV II, III X X X X X X X X CMA118561 Huntingdon Life Sciences Chemical Manufacturer* Association Study Number Sponaor Numbar Page 39 Protocol 01 APPENDIX A Tissues Preserved/Examined Microscopically No.* Tissue 2 uterus (body/homs with cervix) 1 vagina gross lesions Preserved X X X Microscopic Examination (Groups) 1, IV II. Ill X X X * Numbar of organa/aactiona preaerved/examined. b Only the right ovary will be routinely processed for microscopic evaluation. The left ovary will be saved for possible oocyte quantification. If deemed necessary by the sponsor (additional coat), oocyte quantification will include evaluation of a minimum of 10 sections randomly salectad from one completely sectioned ovary per female of the high-dose and control groups. Ovarian follicles wilt be placed into one of three categories as described by Plowehalk et al., (1993). The total number of follicles and the number of follicles in sach of the three categories will be evaluated. Ovaries from the low- and mid-dose groups may be evaluated if treatment-related changes are observed in the high-dose group. CMA 118562 Huntingdon Lit* Scianca* Chemical Manufacturers Association Study Number Sponsor Number APPENDIX-B TEST MATERIAL PROPERTIES Chemical name Vinyl chloride Synonyms Monochloroethyiene, VC, VCM, vinyl chloride monomer Molecular formula C2H3C! 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/mm3 = 0.39 ppm in air Page AO Protocol 01 CMA 118563