Document g2zxGw04wGD0VJ2ee6VDX54mL

4R & S- 09th7 mo Oh < Telomer Research Program Update and Status Report Presented to U.S. EPA, OPPT 23 October, 2000 ZZ-ZttU AOHOOOZ -1u it id c lu G3AI303M Telom er Research Program Oct 2000 0I80 J.dd0 03A1333H o c2- TRP Member Companies Asahi Glass Atofina Clariant Daikin DuPont Telomer Research Program Oct 2000 2 Telomer Research Program (TRP) Industry-sponsored Research Program Focus on data gathering common to Telomer manufacturers Key Events Timeline - June 20, 2000 : Formal Organization Proposal - July 7, 2000 : Met with U.S. EPA -J u ly 11, 2000: Met with MITI - August 14-15, 2000 : First Meeting as Formal Consortium - October 23, 2000: Present Update to U.S. EPA - October 30, 2000: Present Update to MITI Telomer Research Program Oct 2000 3 TRP Guiding Principles Participation by all Telomer Manufacturers Expeditious and Effective Research of Publishable Quality Early and Full Disclosure of Results Equal Sharing of Costs Structure modeled on an earlier and very successful cooperative research program Structure: a Management Committee which acts on recommendations from a Research Committee Use independent third party to administer program Telomer Research Program Oct 2000 TRP Management Committee Oversee the affairs of the TRP One member designated by each participant company Appoint the Chair and other members of the Research Panel Approve projects for funding by TRP Have custody of the funds; determine financial needs of TRP and assess parties such amounts Formulate a policy for proper reporting of project results to the parties, to government agencies, and others Telomer Research Program Oct 2000 TRP Research Panel Comprised of representatives from each participant company Compile relevant existing studies Recommend research testing and develop study protocols Recommend how above can be executed and insure it gets accomplished Identify laboratory facilities that have the capabilities to conduct the recommended tests. Obtain bids or quotes from laboratories and present for approval (via Administrator) Telomer Research Program Oct 2000 TRP Research Quality Standards such as Good Laboratory Practices as established by the OECD and applicable country laws Encourage peer review and publication in the open literature Telomer Research Program Oct 2000 TRP Information Sharing Relevant telomer toxicology research information that has already been developed by a participant shall be shared with the TRP Testing results by or for the TRP shall be promptly disseminated to each participant A policy to govern disclosure and publication of research results will be developed Early and full disclosure to government and regulatory agencies Telomer Research Program Oct 2000 8 TRP Initial Meeting : 14-15 August, 2000 Information Handling and Sharing Chemicals to be Tested Toxicology Studies', acute, genetic toxicity, developmental toxicity; initial 45-day range-finder followed by 90-day subchronic with recovery, followed by multi-generation reproduction study Pharmacokinetics'. In Vitro Metabolism, Mass Balance and ADME (absorption, distribution, metabolism, elimination) Environmental Fate: atmospheric fate, acute aquatic toxicity, bioconcentration, and biodegradation Communications including meetings with regulatory authorities Telomer Research Program Oct 2000 TRP Information Handling & Sharing Condition of membership in TRP to share toxicology data on representative compounds from each participant Proposal to establish single repository for either final reports or summary data; all submissions due Oct 2000 -Summary data in standard format -Text in English -Identify key contact in each company Results from TRP work will be available to all members Initial repository will be Haskell Laboratory but this could transition to RAND at a later time Telomer Research Program Oct 2000 10 TRP Chemical to be Tested Telomer 8-2 Alcohol: n-C8F 17CH2CH2OH Purity >99%, and characterized Sample made, 20 kg, now in analysis Telomer Research Program Oct 2000 11 Acute Oral Toxicity - Fixed Dose Method Objective: To assess the acute oral toxicity (lethality, clinical signs o f response) by administration o f gavage doses to rats Method: Rats, male and female Single oral doses Sighting Study: - 5-50-500-2000 mg/kg, 1 rat/dose - clinical signs; mortality to 14 days - select first dose on available information, analogous chemicals Main Study: - 5M/5F/group - initial dose - toxicity response, no mortality in sighting study - test up/down dependent on results Results: - Lethal level(s) - Principal clinical signs - Recovery/time to recovery Telomer Research Program Oct 2000 Repeated-Dose Oral Toxicity Gavage RangeFinding Study in Rats Objective: To determine dose levels for use in 90-day toxicity study which w ill enhance ability to achieve NOAEL, LOAEL M ethod: Rats, 5M/5F/group, 1 control, 3 graded treatment levels, expect 3-8 week duration Endpoints Measured: - In vivo (body weight, clinical signs) - Blood organofluoride- pretest (day-4) day 1, weekly Post-dosing day 3, weekly - Collect urine and feces (to expand pharmacokinetics model if indicated) Results: In vivo response Blood organofluoride level to steady-state, define clearance profile Evaluate bio-response, bio-uptake and clearance versus dose Telomer Research Program Oct 2000 I? Previous Study with Telomer B Alcohol (C8-2) Normalized uM Equivalents of Telomer B Ale in Rat Plasma Resulting from a 10-day Oral Gavage (Norm) Telomer Research Program Oct 2000 AST* Subchronic Toxicity - 90-Day Gavage Study in Rats with Recovery Ojective: To evaluate the subchronic toxicity and the kinetics o f uptake and clearance o f this chemical in the blood. M ethods: Rats, lOM/lOF/group, oral gavage, 90 days, control and 3 test levels; lOM/lOF/group control and high 4 wk reversibility Parameters: In vivo, clinical pathology, neurobehavior, pathology 5M/5F/group Blood Kinetics: Results: Determine target organ(s) and dose required to produce effect Determine reversibility o f change(s) produced Determine uptake and clearance o f organofluoride in blood Goal: Establish target site, potency, (NOAEL... .LOAEL....) Telomer Research Program Oct 2000 -2T Developmental Toxicity Study in Rats Ojective: To evaluate the potential effect on the fetal rat following administration o f the chemical to the maternal rat from implantation to the end o f gestation. M ethods: Rats, oral gavage doses gestation day 6 through 21; control plus 3 graded dose levels Parameters - maternal - body weights, clinical signs, resorptions, implantations, gestation time - fetal - weight, structural integrity (external, internal, skeletal) Results: To evaluate the ability o f chemical to effect either the maternal or fetal rat (or both) and the types and severity o f the induced changes The potency o f the chemical - maternal NOAEL... .LOAEL - fetal NOAEL.....LOAEL Telomer Research Program Oct 2000 J2A-- \6 Multi-Generation Reproduction Study in Rats Objective: To evaluate the effect of the chemical on the gonadal function, conception, parturition, and the growth and development of offspring of rats over 2 generations (involving the production of at least 1 set of litters in each generation). Methods: Rats, 30M/30F/group, graded dietary levels, control + 3 test levels Feed chemical 70 days pre-mating (PI and FI) until sacrifice Parameters - body weight, clinical signs - reproductive function (mating, fertility, fecundity, gestation, implantations, pup survival and lactation, developmental landmarks, sperm assessments, estrous cycling) - pregnancy - growth, clinical signs, - selected histopathology (focus on reproductive organs) Results: Reproductive system as a target (yes or no) - functional (1st, 2nd generation) - histologic (1st, 2nd generation) - effects in offspring Potency of effects in offspring NOAEL.....LOAEL.... Telomer Research Program Oct 2000 2* r - 17 Genetic Toxicity - Bacterial Reverse Mutation Test Ojective: To evaluate the mutagenic potential by measuring the ability to induce reverse mutations at selected loci of several strains of S. typhimurium and at the tryptophane locus of E. coli (with and without S-9 activation) Methods: Tester strains - Salmonella typhimurium TA98; TA 100 TA1535; TA 1537 E. Coli WP2uvrA 5 graded test concentrations - selected based on evidence of toxicity and precipitate Incubate for 48/72 hours with and without S-9 mix Score revertant colonies/plate (all in triplicate) Score against negative + positive controls run concurrently Results: Control revertant rates must be within historical data Positive control rates at least 3x control Positive results - 3x control rate for TA1535/TA1537 - 2x control rate for TA98, TA100, WP2uvrA Telomer Research Program Oct 2000 is Genetic Toxicity - In Vitro Mammalian Chromosome Aberration Test Objective: To evaluate the clastogenic potential based on its ability to induce chromosome aberrations in human peripheral blood lymphocytes Method: Expose human lymphocytes to at least 4 graded concentrations of the test agent (with positive and negative controls) Non-activated system treatment times - 4 and 20 hours S-9 activated system treatment time - 4 hours Arrest dividing cells in metaphase; harvest and evaluate at 20 hours Score a minimum of 200 spreads/dose level (100/duplicate treatment tube) Results: Chromatid-type aberrations - chromatid/isochromatid breaks - exchange figures Chromosome-type aberrations - breaks - dicentrics/rings Fragments (can be chromatid or chromosome) Analyze % aberrant cells statistically (versus negative control) Telomer Research Program Oct 2000 13 Genetic Toxicity - Rat Bone Marrow Erythrocyte Micronucleus Test Objective: To evaluate the clastogenic potential as measured by its ability to induce micronucleated polychromatic erythrocytes in rat bone marrow M ethod: Single IP injection o f rats at 3 graded doses (from pilot study but not to exceed 2000 mg/kg) 5M/5F/group Negative and positive (cyclophosphamide) controls Sacrifice and collect bone marrow from 5/sex/group at 24 hours; 5/5 from high dose only at 48 hours. Examine bone marrow cells - 2000 micronucleated normocytes per rat Results: Proportion o f micronucleated polychromatic erythrocytes/ polychromatic erythrocytes Apply appropriate statistics versus controls Valid test only if control incidence o f micronucleated cells <0.3%; positive control statistically elevated Telomer Research Program Oct 2000 OECD 474 uses mice; consider oral gavage vs. IP - 2.0 TRP Pharmacokinetics Study Metabolism of Telomer 8-2 Alcohol by Mammalian Microsomes Objective: Determine the rate of test material metabolism and metabolic product identification in liver microsomes prepared from various mammalian species. Method: * Microsomes prepared for rat, rabbit, dog, monkey and human * Samples analyzed by HPLC-MS * Determine linearity of reaction rate and protein concentration * * Reaction media contains an NADP regeneration system, cofactors, microsomes, test compound and phosphate buffer at pH 7.4, 37C * Determine kinetics of test compound ** Reactions carried out at constant initial test material concentration and at a constant protein concentration ** Time course of metabolism by microsomes evaluated Results: * Rate of microsomal metabolism of test compound by each species tested * Identity of metabolic products * Estimates of test material half-life in-vitro and intrinsic clearance Telomer Research Program Oct 2000 Note: Protocols still in draft form <l TRP Pharmacokinetics Study Metabolism of Telomer 8-2 Alcohol by Mammalian Hepatocytes Objective: To evaluate the rate of metabolism and identification of metabolic products in cultured liver cells from several mammalian species. Method: * Prepare hepatocytes from rat, mouse, rabbit, monkey, dog and human * Negative (vehicle) and positive (ethoxycoumarin) controls included * Hepatocytes and compound incubated at 37C for 2 hrs * Samples analyzed by HPLC-MS for test compound and metabolites Results: * Qualitative and quantitative description of metabolic activity toward test material in several species * Description of metabolism in a metabolically "complete" system * Identity of test material metabolites in a whole cell system Telomer Research Program Oct 2000 Note: Protocols still in draft form -3* TRP Pharmacokinetics Study Toxicokinetics of Telomer 8-2 Alcohol (TEA) in the Rat Objective: Determine the absorption, distribution, metabolism and excretion of Telomer 8-2 alcohol in male and female rats following oral and dermal dosing. Method: * Conduct pilot experiments ** Radiolabeled and cold TBA used as test material ** 2 male and 2 female rats ** Determine plasma kinetics following a single high oral dose of TBA ** Determine excretion and metabolism following a single high oral dose of radiolabeled TBA * Conduct absorption experiments ** TBA test material ** 4 male and 4 female rats ** Single low and single high oral dose ** Single low and single high dermal exposure (8 hrs) ** Single low intravenous dose ** Follow plasma concentrations of test material Telomer Research Program Oct 2000 Note: Protocols still in draft form 36^ -*3 TRP Pharmacokinetics Study Toxicokinetics of Telomer 8-2 Alcohol (TBA) in the Rat (Continued) * Distribution experiments ** Radiolabelled TBA test material ** 4 male and 4 female rats ** Conduct oral and dermal exposures ** Evaluate tissue distribution of test material at Tmax and Tmax/2 * Excretion and metabolism experiments ** Radiolabelled TBA test material ** 4 male and 4 female rats ** Single low and high oral doses ** Single low and high dermal doses ** Daily dose of TBA with last dose being radiolabelled TBA ** Identify excretion rates and metabolic profiles Results: * Metabolic profiles of TBA * Qualitative and quantitative description of biodistribution of TBA * Description of absorption characteristics of TBA * Sex linked differences in TBA handling in rats * Description of TBA metabolism at varying concentrations and between sexes Telom er Research Program O ct 2000 Note: Protocols still in draft form Radiolabeled Fluorotelomer Alcohol 14C Label in perfiuoro-chain c _J\___v F(CF2CF2)4-CH2CH2-OH V_______ ! 14C o r 3H op at/G ii I'N /-s I i vs. +U i r! \r\ ^^ l-c iu c i il i c?u iy i d i t ; Need two-labeled compounds: perfiuoro-chain and ethylene `spacer' Expect Fluorinated chain label to be difficult Expect Ethylene `Spacer" Label to be Straightforward Telomer Research Program Oct 2000 X? -3*-- TRP Environmental Fate & Effect Study Initially focused on four areas: -Atmospheric Degradation -Biodegradation -Acute Aquatic Toxicity (Daphnia, Algae, Fish) -Chronic Aquatic Toxicity (Daphnia) -Bioconcentration and chronic fish (simultaneous study based on result from fish acute) Protocols still in draft form; Completion expected end of October Telomer Research Program Oct 2000 *Q> -39- TRP Environmental Fate Study Objective: Laboratory Study of Atmospheric Degradation of n-C8F17CH2CH2OH -Determine persistence via reaction with OH radical -Determine major oxidation products via reaction with 0 2and NOx(NO, N 0 2) -Determine reactivity of the oxidation products. Telomer Research Program Oct 2000 7 40--- TRP Environmental Effect Study Objective: Conduct Acute and Chronic Studies on Daphnia and Fish* -Acute: conduct simple limit test on Daphnia and fish in preparation for the chronic studies -Chronic studies: *Standard 21-day Daphnia reproduction test (OECD 211) carried out as a semi-static study *Modified 14-day fish sub-acute test (OECD 204) increased to maximum 56 day exposure with depuration step and whole body and tissue concentration measurements providing insights into bioconcentration Telom er Research Program O ct 2000 *Algae to be addressed in later work XZ .4+ TRP Environmental Fate Study Objective: determine the rate and/or amount of biodegradation and potential mineralization of the subject compound -Use Draft ISO standard test (ISO/DIS 14592 ptl), potentially with 14C label on ethylene to assess biodegradation of alkyl portion -Zahn-Wellens test with activated sludge to assess whether the subject compound is degradable or mineralizable; part of this test will also assess whether there is an inhibition of activity of bacteria under the test conditions and attempt to determine the fugacity of the substance or its degradation products by measurement of trapped volatiles Telom er Research Program Oct 2000 A TRP Toxicology, Pharmacokinetics, and Environmental Fate & Effect Timelines* Chronology Date Event 20-Jun-00 TRP Formal O rganization Proposal TRP Meets with U.S. EPAto Review 07-Jul-OO Proposed Testing Plans 11-JuWX) TRP Members Meet with MITI TRP Research Panel Meets; 14- AugC-OoOnsortium Meets for the First Time TRP Management Committee Meets; 15- AugT-R00P is O fficially Sanctioned________ Completion Date for Draft Protocols 18-Oct-OO for Toxicology and Kinetics TRP Meets with U.S. EPAto Review 23-Oct-OO Detailed Research and Testing Plans January-01 Acute O ral Toxicology Testing Repeated-Dose O ral Toxicity Range- January-01 Finding Study in Rats Subchronic Toxicity: 90-Day Gavage March-01 Study in Rats w ith Recovery M jltigeneration Reproduction Study in September-01 Rats Complete in '01 Developmental Toxicity Study in Rats Complete in '01 Bacterial Reverse Mutation Test In Vitro Mammalian Chromosome Complete in '01 Aberation Test Rat Bone Marrow Erythrocyte Complete in '01 KAcronucleus Test P harm acokinetics Environmental Fate & Effect Complete in '01 Atmospheric Degradation Complete in '01 Acute Aquatic Toxicity Complete in '01 Biodegradation Complete in '01 Bioconcentration 1Q 4Q Telom er Research Program 0 * 2 0 0 0 * T R P w il1 PreSent reSultS aS available On `01 30 43- TRP Path Forward Items Complete Respective Protocols Complete Information Database Submit and Obtain Competitive Bids on the Key Work Streams Complete the Telomer 8-2 Alcohol Characterization and Decision on Labeled Compounds Preparation Update MITI and other respective regulatory groups Meet as a Consortium to Finalize Remaining Details in Nov/Dec 2000 Begin Workstreams per Timeline Jan 2001 Telomer Research Program Oct 2000 Title: Acute Oral Toxicity - Fixed Dose Method Protocol Contain NO CBI INTRODUCTION This test is designed to assess the acute oral toxicity produced when a test substance is administered by the oral route (gavage) to rats. SPONSOR AND TEST FACILITY This study is sponsored by <facility, location>. The sponsor's approval was effective the date the sponsor authorized the work. The study will be conducted at <facility, locations in accordance with all applicable Good Laboratory Practice standards.11,2) The study design is based on European Economic Communities (EEC)<3>and Organization for Economic Cooperation and Development (OECD),<4) test guidelines. Areas of noncompliance will be documented in the final report. MATERIALS AND METHODS A. Test Substance The test substance will be supplied by the sponsor. The test substance will be assigned a unique number. B. Test Species Male and female Crl:CD(SD)IGS BR rats will be obtained from Charles River Laboratories, Inc. The address of the supplier (city/state) will be documented in the study records and final report. The Crl:CD(SD)IGS BR rat has been selected on the bases of extensive experience with this strain and its suitability with respect to longevity, hardiness, sensitivity, and low incidence of spontaneous diseases. C. Animal Husbandry All animals will be housed in stainless steel, wire-mesh cages suspended above cage boards. Animal rooms will be maintained at a temperature of 22 3C and a relative humidity of 50% 20%. Animal rooms will be artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. All animals will be provided tap water a d lib itu m . All animals will be fed PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 a d lib itu m except during fasting. 2 33 As specified in the <> Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to assure that contaminant levels are below those that would be expected to impact the scientific integrity of the study: Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Feed samples are analyzed for total bacterial, spore and fungal counts. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Data are maintained separately from study records and may be included in the final report at the discretion of the study director. D. Pretest Period Upon arrival at <> Laboratory, all animals will be housed 1 per cage, sexes separate in quarantine. The rats will be: quarantined for at least 5 days. identified by cage identification. weighed at least 3 times during quarantine. observed with respect to weight gain and any gross signs of disease or injury. The animals will be released from quarantine by the laboratory animal veterinarian or designee on the basis of acceptable body weights and clinical signs. E. Assignment to Groups Animals of each sex, selected on the bases of adequate body weight gain and freedom from any clinical signs of disease or injury, will be assigned to the study. After assignment to the study, each animal will be housed individually. Information on the cage labels will include the unique animal number and an individual identification number assigned to each animal. The last 3 or 4 digits of the animal number will be marked on the tail of each animal. At study start (test day 0) 3 3* the animals will be between 8 and 12 weeks of age. On test day 0, the body weights will be within 20% of the mean within a sex. F. Dosing and Evaluations 1. Fasting For both the sighting and main studies, the animals will be fasted prior to dosing by withholding food overnight (approximately 16-18 hours). Food will be returned to the animals approximately 3-4 hours after dosing. 2. Sighting Study The effects of various doses will be investigated in single animals. Normally female animals will be used in the absence of information derived from structureactivity relationships or other information indicating that males will be the more sensitive sex. Dosing will be sequential, allowing at least 24 hours before dosing the next animal. All animals will be carefully observed for signs of toxicity for at least 7 days; if signs of moderate toxicity persists at 7 days, the animal will be observed for up to an additional 7 days. The following initial dose levels will be considered: 5, 50, 500, and 2000 mg/kg. If the initial dose chosen does not produce severe toxicity, and the next higher level produces mortality, then it will be necessary to investigate one or more intermediate dose levels as appropriate. In this way it should be possible to build up information on the dose level(s) that produce(s) some signs of toxicity and the minimum dose level that produces mortality. An effort will be made to select the initial dose using evidence from related chemicals. In the absence of such information, it is suggested that the 500 mg/kg dose is used in the first instance. If no signs of toxicity are seen at the initial dose, then the next higher dose level will be investigated. If no mortality occurs at 2000 mg/kg, the sighting test is complete and the main study should be conducted at this dose level. If severe effects, necessitating humane killing are seen at the initial dose (e.g. 500 mg/kg), the next lower dose (e.g. 50 mg/kg) will be given to another animal. If this animal survives, further animals will then be dosed with the appropriate intermediate dose levels between the fixed doses. Normally, one would not expect to use more than 5 animals in this procedure. 3. Main Study At least 10 animals (5 male and 5 female) will be used for each dose level investigated. The dose level to be used in the main study is selected from one of four levels, 5, 50, 500, and 2000 mg/kg body weight and will be that which is judged on the basis of the results from the sighting study likely to produce evident toxicity but no mortality. If the data from the sighting study suggest that mortality will occur at 5 mg/kg, the substance can be investigated at a lower dose level. 4 35 In most cases it is likely that the data obtained from the sighting study will be adequate to allow the appropriate dose level to be selected in the main study (i.e. a level that produces evident toxicity but no mortality). However if evident toxicity is not seen at the initial dose level, the substance will be retested at the next higher level. If animals die at the initial dose chosen, or a severe reaction requires removal of animals from the study for animal welfare reasons, the substance will be retested at the next lower dose level. 4. Dose Volume When practical, liquid test substances will be administered neat. Solid test substances will be suspended or dissolved in a vehicle. Variability in volume will be minimized by adjusting the concentration to ensure a constant volume at all dose levels. The dose volume will not exceed 1 mL/100 g of body weight, except in the cases of aqueous solutions where 2 mL/100 g may be used. The volume of dosing material administered to each animal will be based on the animal's fasted body weight determined on the day of dosing. 5. Frequency The test substance will be administered in a single dose. If a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. Where a dose is administered in fractions, it may be necessary to provide the animals with food, depending on the length of the dosing period. 6. Vehicle The use of an aqueous mixture should be considered first, followed by consideration of a mixture in corn oil, and then by consideration of a possible mixture in other vehicles. Commonly used vehicles in the order of preference are water, methylcellulose in water, Mazola corn oil or equivalent, mixtures of acetone and water, or mixtures of acetone and corn oil. Corn oil, methylcellulose, and acetone will be used as supplied; any contaminants that can reasonably be expected to be present are not expected to affect the integrity of the study. The vehicle used will be documented in the study records and final report. 7. Observations and Body Weights for Sighting Study and Main Study Except where animals need to be removed from the study and humanely killed for animal welfare reasons, animals will be observed for at least 7 days (sighting study) or 14 days (main study) after dosing. However, the duration of observation will not be fixed rigidly. It will be determined by the toxic reactions, rate to onset and length of recovery period, and may thus be extended when considered necessary. The times at which signs of toxicity appear and disappear are important, especially if there is a tendency for toxic signs to be delayed. All 5 36 observations are systematically recorded with individual records being maintained for each animal. A careful clinical examination will be made at least twice on the day of dosing and once each day thereafter (weekends and holidays excluded except when warranted by the condition of the animals). Animals obviously in pain or showing severe signs of distress will be humanely killed. Additional observations may be necessary during the first few days after dosing so that the test may be terminated if it becomes apparent that the initial dose level chosen was too high. When animals are killed for humane reasons or are found dead, the time of death will be recorded as precisely as possible. The rats will be checked twice daily for mortality and signs of illness, injury, and abnormal behavior. Individual weights of animals will be determined at the time of fasting, shortly before the test substance is administered, daily for the next 3 days, and weekly thereafter. If significant weight loss (> 2% of the previously determined body weight) occurs, all animals in the group will be weighed daily until there is no significant weight loss. At the end of the test surviving animals are weighed and then sacrificed. STATISTICAL METHODS Descriptive statistics (e.g. mean, standard deviation, etc.) may be used. SAFETY AND HOUSEKEEPING Good housekeeping procedures will be practiced to avoid potential health hazards. To avoid skin contact, gloves will be worn when handling the test substance. Animal carcasses and feces will be incinerated. RECORDS AND SAMPLE RETENTION All original records will be retained at <facility, locations Laboratory-specific or site-specific records such as personnel files and equipment records will be retained at the facility where the work was done. 6 37 REFERENCES 1. EPA/FIFRA Good Laboratory Practice Standards (40 CFR 160). (1989). 2. OECD Principles of Good Laboratory Practice (as revised in 1997, published in ENV/MC/CHEM(98) 17 (OCDE/GD(92)32). 3. Official Journal of the European Communities. Part B Ibis: Methods for the Determination of Toxicity. (1992). 4. OECD Guidelines for the Testing of Chemicals, No. 420. (1992). 7 SIGNATURE Approved b y :_______________________________________________ sd, SDDegree Study Director ___________ Date cc: <> 8 2>e <Cmpd> Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats Protocol Contain No cbi <Cmpd> Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats TABLE OF CONTENTS Page INTRODUCTION........................................................................................................................................3 OBJECTIVE..................................................................................................................................................3 SPONSOR AND TEST FACILITY.......................................................................................................... 3 STUDY DESIGN.........................................................................................................................................4 MATERIALS AND METHODS.............................................................................................................. 6 A. Test Substance................................................................................................................................. 6 B. Test Species......................................................................................................................................6 C. Animal Husbandry...........................................................................................................................6 D. Pretest Period................................................................................................................................... 7 E. Assignment to Groups....................................................................................................................7 F. Dosing Solution Preparation and Sampling................................................................................8 G. Administration of Dosing Suspensions....................................................................................... 8 H. Body W eights.................................................................................................................................. 8 I. Clinical Observations and Mortality............................................................................................8 J. Total Fluorine Level Evaluation................................................................................................... 9 K. Anatomical Pathology.................................................................................................................... 9 1. Pretest.........................................................................................................................................9 2. Dosing Phase.............................................................................................................................9 SAFETY AND HOUSEKEEPING......................................................................................................... 10 RECORDS AND SAMPLE RETENTION............................................................................................ 10 PROTOCOL APPENDIX....................................................................................................................... 11 SIGNATURES............................................................................................................................................12 2 TP <Cmpd> Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats INTRODUCTION The test substance, <Cmpd>, is a <compound> used as an < >. Dose levels for this study were selected based on <>. OBJECTIVE The objective of this oral gavage range-finding study is to determine the dose levels for use in a 90-day oral subchronic study. The oral route of administration was selected because <>. SPONSOR AND TEST FACILITY This study is sponsored by <sponsor name>. The sponsor's approval was effective the date the sponsor submitted the work. The study will be conducted at claboratory, locationx 3 <Cmpd>: Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats STUDY DESIGN The study design is as follows: Group3 No ./Group Male Female Male Female I II 5 5 Main Study 5/sex Metabolism Serial bleed Tissue/sample Dosageb Treatment _____ collection____ mg/kg/day___________ All rats All rats 0 Control III IV V VI VII Vili 5 5 5 5 5 5 5/sex 5/sex 5/sex All rats All rats All rats All rats <> <Cmpd> <> <Cmpd> <> <Cmpd> a Each rat will be assigned its own animal number. Rats will have the last 3 digits of the unique animal number marked on their tails, b Weight/weight concentration of test substance. <Cmpd>: Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats Study Parameters Frequency Dose Administration Body Weight Test Day 0 - to attainment of steady state. Vehicle animals will be dosed until last dose group attains steady state for total fluorine in blood. Twice per week at 3-4 day intervals Clinical Observations At every weighing Mortality/Moribundity Checks At least twice daily Serial bleeding for fluorine analysis Predosing - Day -4 During Dosing - Day 1 and weekly thereafter Postdosing - Day 3, 1 week, 2 weeks, 1 month, 45 days, 60 days, and 75 days Collection of urine and feces Once steady state is obtained for 3 consecutive days Necropsy (Anatomical Pathology) Select Tissue/Sample Collection All rats in the 0 mg/kg/day (Control) and <> mg/kg/day <Cmpd>-group). Date of sacrifice will depend on the rate of elimination of fluorine from the blood after dosing is stopped. <Cmpd>: Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats MATERIALS AND METHODS A. Test Substance The test substances will be supplied by the sponsor. The test substance(s) were assigned <> Laboratory Number(s) <>. B. Test Species Male and female Crl:CD(SD)IGS BR rats will be obtained from Charles River Laboratories, Inc. The address of the supplier (city/state) will be documented in the study records and final report. The Crl:CD(SD)IGS BR rat has been selected on the bases of extensive experience with this strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous diseases. C. Animal Husbandry All rats will be housed in stainless steel, wire-mesh cages suspended above cage boards. Animal rooms will be maintained at a temperature of 22 3C and a relative humidity of 50 20%. Animal rooms will be artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. All rats will be provided tap water a d lib itu m . All rats will be fed PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 a d lib itu m . As specified in the <> Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to assure that contaminant levels are below those that would be expected to impact the scientific integrity of the study: Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Feed samples are analyzed for total bacterial, spore and fungal counts. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. 6 <Cmpd>: Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Data are maintained separately from study records and may be included in the final report at the discretion of the study director. D. Pretest Period Upon arrival at <> Laboratory, all rats will be housed 1 per cage, sexes separate, in quarantine. The rats will be: quarantined for a minimum of 5 days. identified temporarily by cage identification. weighed at least 3 times during quarantine. observed with respect to weight gain and any gross signs of disease or injury. The rats will be released from quarantine by the laboratory animal veterinarian or designee on the bases of body weights and clinical signs of all rats. Rats that are accidentally killed or removed from study during the pretest period will be discarded without necropsy. Rats that are found dead or sacrificed in e x tre m is during the pretest period will be sent to Pathology and given a gross examination to check for the presence of disease. Dependent upon these findings, further diagnostic procedures may be employed at the discretion of the study director, a pathologist, or the laboratory animal veterinarian. The results will not be reported in the final report unless considered significant to the evaluation of the study. E. Assignment to Groups Rats of each sex, selected on the bases of adequate body weight gain and freedom from any clinical signs of disease or injury will be distributed by computerized, stratified randomization into study groups as designated in the Study Design, so that there are no statistically significant differences among group body weight means within a sex. The weight variation of selected rats will not exceed 20% of the mean weight for each sex. After assignment to groups, each rat will be housed individually. Information on the cage labels will include the unique animal number and the unique individual identification number assigned to each rat. The last 3 or 4 digits of the unique animal number will be tattooed on the tail of each rat. At study start (test day 0) the rats will be 7 weeks old. On test day 0, when possible, rats with body weights that are not within 20% of the mean within a sex, will be removed from study and replaced with rats having body weights within that range (subject to the same selection criteria as the original rats). M <Cmpd>: Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats Rats that have not been assigned to a test group or which have been removed from study on test day 0, for out-of-range body weight, will be released for other laboratory purposes, or be sacrificed by carbon dioxide asphyxiation and discarded without pathological evaluation, at the discretion of the study director. F. Dosing Solution Preparation and Sampling The test substance will be <suspended> in <>. Dosing solutions of the test substance will be prepared <daily>. G. Administration of Dosing Suspensions The test substance will be suspended in <>. The rats will be dosed by intragastric intubation until steady state is obtained (estimated to be <> days) at a volume of 1 mL/100 grams of body weight. Animals will be dosed 7 days a week. The amount of test substance each rat receives will be based on the body weights collected twice a week and the solutions concentration. Control rats will be dosed with <> at a volume of 1 mL/100 grams of body weight. The following suspension concentrations will be used for each dose: <> mg/ml for the <> mg/kg/day dose; <> mg/ml for the <> mg/kg/day dose; and <> mg/ml for the <> mg/kg/day dose. Control rats will be dosed with the same vehicle used to administer the test compounds at a volume of 1 ml/100 grams of body weight. H. Body Weights The rats will be weighed twice per week at 3 to 4 day intervals during the study. I. Clinical Observations and Mortality During the test period, cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats will be conducted at least twice daily throughout the study. Moribund rats will be sacrificed and discarded without necropsy. At every weighing, each rat will be individually handled and examined for abnormal behavior and appearance. Clinical signs noted will be recorded. Animals that do not exhibit clinical signs of toxicity will be recorded as "no abnormality detected" (NAD) in the study records. Prior to sacrifice of moribund rats, if possible and at the discretion of the clinical pathologist and/or study director, blood samples for hematology and clinical chemistry measurements may be collected for clinical pathology evaluations. 8 <Cmpd>: Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats J. Total Fluorine Level Evaluation Prior to dosing (test day --4), 1, and 7 days after study start, and weekly thereafter until steady state for total fluorine in blood is attained, blood (approximately 0 . 5 - 1 mL) will be collected from the orbital sinus of each animal while under light carbon dioxide anesthesia for total fluorine analysis. On the day of blood collection, blood will be collected from the animals prior to dosing. The blood will be collected in glass tubes containing EDTA while on ice and stored in the refrigerator until they are sent to <> within 24 hours for total fluorine analysis. For each bleeding, blood should be collected at approximately the same time of day. Once steady state is reached for a particular dose group, administration of that dose of test article will end. (Note the control group will continue to be dosed with vehicle until the last test article group achieves steady state). Blood will also be collected for total fluorine analysis at 3, 7, 14, 30, 45, 60, and 75 days postdosing for each dose group. The blood samples collected postdosing may be analyzed as needed to answer questions regarding distribution and metabolism of the test substances. Additionally, once a dose group attains steady state, the animals will then be placed in metabolism cages for collection of feces and urine for 3 days. Urine and feces will be collected in 24 hour increments. The exact time period of collection of urine and feces will need to be documented along with the total volume of urine obtained. The urine and feces obtained from a particular dose group will be pooled (sexes separate). Blood, urine, and feces will be sent to <> for total fluorine analysis. The samples will be sent to <> in coolers containing ice. Additionally, urine samples may be provided to the Clinical Pathology Group for method development studies. K. Anatomical Pathology 1. Pretest See Materials and Methods, Section D. Pretest Period. 2. Dosing Phase All rats in the control and high dose groups will be sent to Pathology for gross evaluation and collection of tissues. Rats will be euthanatized by carbon dioxide anesthesia and exsanguination. The following tissues will be collected and weighed from rats in the control and high-dose groups sacrificed by design. The tissues will be placed in plastic freezer bags and stored in the freezer until analysis of total fluorine levels. <Cmpd>: Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats Digestive System liver Reproductive System Male testes Miscellaneous fat The liver, testes, and fat will be sent to Jackson Laboratory for total fluorine evaluation in coolers containing ice. SAFETY AND HOUSEKEEPING Good housekeeping procedures will be practiced to avoid contamination of dosing solution preparation facilities and potential health hazards. To avoid skin contact, gloves will be worn when handling the test substance or dosing solutions. In addition, the test substance will be handled in a chemical hood. Dosing solutions will be prepared in properly ventilated areas. Animal carcasses and feces will be incinerated. RECORDS AND SAMPLE RETENTION All original records will be retained at <>. Laboratory-specific or site-specific records such as personnel files and equipment records will be retained at the facility where the work was done. H-3 <Cmpd>: Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats Study Function PROTOCOL APPENDIX Study Personnel Study Director: <> <Senior Research S cien tist Study Dates Prebleed <> Initiation of Test Substance Administration <> Scheduled Sacrifice Dependent upon the elimination rate of fluorine from the blood of the various dose groups <Cmpd>: Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats SIGNATURES Approved by: __________________________ <> Primary Technician <> Management <> Study Director cc: 57 12 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery Protocol C ontain NO CBi <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery TABLE OF CONTENTS Page INTRODUCTION........................................................................................................................................3 MATERIALS AND METHODS.............................................................................................................. 4 A. Test Substance................................................................................................................................. 4 B. Test Species..................................................................................................................................... 4 C. Animal Husbandry...........................................................................................................................5 1. H ousing....................................................................................................................................................................... 5 2. Cage Rack Positioning.............................................................................................................................................. 5 3. Environmental Conditions....................................................................................................................................... 5 4. Water and F ood ......................................................................................................................................................... 5 5. Animal Health Monitoring....................................................................................................................................... 5 D. Pretest Period................................................................................................................................... 6 E. Assignment to Groups.................................................................................................................... 6 F. Dosing Solution Preparation and Sampling................................................................................7 G. Administration of Test Substance................................................................................................ 7 H. Body W eights.................................................................................................................................. 8 I. Food Consumption and Food Efficiency..................................................................................... 8 J. Detailed Clinical Observations and Mortality............................................................................ 8 K. Ophthalmology................................................................................................................................ 8 L. Neurobehavioral Evaluation.......................................................................................................... 9 1. Sensory Function...................................................................................................................................................... 9 M. Clinical Pathology Evaluation.......................................................................................................9 1. Hem atology...............................................................................................................................................................10 2. Coagulation Parameters.......................................................................................................................................... 10 3. Serum Chemistry......................................................................................................................................................10 4. Urinalysis................................................................................................................................................................... 11 N. Blood Analysis.............................................................................................................................. 11 O. Anatomic Pathology......................................................................................................................11 1. Pretest........................................................................................................................................................................11 2. Rats Designated for Subchronic Toxicity Evaluation....................................................................................... 12 3. Rats Designated for <B lood Fluoride>Analysis............................................................................................... 13 4. Rats Designated for Recovery............................................................................................................................... 13 SAFETY AND HOUSEKEEPING......................................................................................................... 15 RECORDS AND SAMPLE RETENTION............................................................................................ 15 PROTOCOL APPENDIX......................................................................................................................... 18 SIGNATURES........................................................................................................................................... 20 5? <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery INTRODUCTION The test substance, <CMPD>, is under consideration for use as <use>. In a <duration> range finding study conducted with <CMPD> in rats, there was <results> Dose levels for this 90-day gavage study were selected based on <results> observed in the range finding study. OBJECTIVE The objective of this study is to evaluate the potential subchronic toxicity and reversibility of toxicity of <CMPD> when administered by gavage to male and female rats. A supplemental group will also be used to determine <> in the blood during the course of the study. The oral route of administration was selected because it provides an effective means of compound delivery.. SPONSOR AND TEST FACILITY This study is sponsored by <name, address of sponsor>. The sponsor's approval was effective the date the sponsor authorized the work. The study will be conducted at <facility, locationx This study will be conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles of Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 and MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850)(1,2,3) Areas of noncompliance will be documented in the final report. The study design is based on European Economic Communities (EEC),(4) U.S. Environmental Protection Agency (EPA) Office of Prevention, Pesticides, and Toxic Substances (OPPTS),(5) Organization for Economic Cooperation and Development (OECD),(6) and the Ministry of Agriculture, Forestry and Fisheries (MAFF) Japan<7) test guidelines. 3 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery STUDY DESIGN The study design is as follows: Group Male Female I II m IV V VI vn vm Number/Group Male Female 25 25 15 15 15 15 25 25 Daily Dosage' 0 (Control <> mg/kg <> mg /kg <> mg/kg a Weight/weight of test substance (adjusted for sponsor-supplied purity of active ingredient). Study Parameters Frequency Body Weight Food Consumption Clinical Observations Mortality/Moribundity Checks Clinical Pathology Neurobehavioral Evaluations Necropsy Gross Observations Organ Weights Day 0 and weekly thereafter Day 0 and weekly thereafter Day 0 and weekly thereafter Twice daily Week 7 and 13; optional at end of recovery phase (week 17) Pretest and week 13; optional at end of recovery phase (week 17) Week 13 and 17 MATERIALS AND METHODS A. Test Substance The test substance of known purity will be supplied by the sponsor. The test substance was assigned <> Laboratory Number <>. The stability of <CMPD> will be confirmed by analyses near the beginning and end of the study. B. Test Species SPF male and female Crl:CD(SD)IGS BR rats will be obtained from Charles River Laboratories, Inc. The address of the supplier (city/state) will be documented in the study records and final report. The Crl:CD(SD)IGS BR rat has been selected on the bases of extensive experience with this strain and its suitability with respect to longevity, hardiness, sensitivity, and low incidence of spontaneous diseases. r-f 4 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery C. Animal Husbandry 1. Housing All rats will be housed in stainless steel, wire-mesh cages suspended above cage boards. 2. Cage Rack Positioning Cage racks will be relocated within the animal room at least every other week and cages on the racks will be repositioned every 2 weeks. 3. Environmental Conditions Animal rooms will be maintained at a temperature of 22 3C and a relative humidity of 50% 20%. Animal rooms will be artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. 4. Water and Food All rats will be provided tap water a d lib itu m . All rats will be fed PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 a d lib itu m . 5. Animal Health Monitoring As specified in the <> Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to assure that contaminant levels are below those that would be expected to impact the scientific integrity of the study: Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Feed samples are analyzed for total bacterial, spore and fungal counts. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Data are maintained separately from study records and may be included in the final report at the discretion of the study director. r6 5 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery D. Pretest Period Upon arrival at <> Laboratory, all rats will be housed 1 per cage, sexes separate, in quarantine. The rats will be: quarantined for at least 5 days. identified temporarily by cage identification. weighed at least 3 times during quarantine. observed with respect to weight gain and any gross signs of disease or injury. given an ophthalmology examination. The rats will be released from quarantine by the laboratory animal veterinarian or designee on the basis of acceptable body weights and clinical signs of all rats. Rats that are accidentally killed or removed from study during the pretest period will be discarded without necropsy. Rats that are found dead or sacrificed in e x tre m is during the pretest period will be sent to Pathology and given a gross examination to check for the presence of disease. Dependent upon these findings, further diagnostic procedures may be employed at the discretion of the study director, a pathologist, or the laboratory animal veterinarian. The results will not be reported in the final report unless considered significant to the evaluation of the study. E. Assignment to Groups Rats of each sex, selected on the basis of adequate body weight gain and freedom from any ophthalmological abnormalities or clinical signs of disease or injury will be distributed by computerized, stratified randomization into study groups as designated in the Study Design, so that there are no statistically significant differences among group body weight means within a sex. The weight variation of selected rats will not exceed 20% of the mean weight for each sex. Study animals will consist of three subgroups: 10 rats/sex/dose - subchronic toxicity, 5 rats/sex/dose - blood fluoride analysis, and an additional 10 rats/sex in control and high dose groups designated as recovery animals. After assignment to groups, each rat will be housed individually. Information on the cage labels will include the unique animal number and an individual identification number assigned to each rat. The last 3 digits of the animal number will be tattooed on the tail of each rat. At study start (test day 0) the rats will be between 6 and 8 weeks of age. On test day 0, when possible, rats with body weights that are not within 20% of the mean within a sex will be removed from study and replaced with rats having body weights within that range (subject to the same selection criteria as the original rats). S7 6 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery Rats that have not been assigned to a test group, or which have been removed from study on test day 0 for out-of-range body weights, will be released for other laboratory purposes or be sacrificed by carbon dioxide asphyxiation and discarded without pathological evaluation, at the discretion of the study director. F. Dosing Solution Preparation and Sampling Dosing solutions of <CMPD> will be prepared <frequency> with <solvent>. The method of preparing the dosing solutions will be documented in the study records. <Samples will be analyzed to verify concentration and stability of <CMPD> in the dose solutions, as follows: Typea Dosing Solution Concentrations Storage Conditions Until Analysis Control 0 mg/ml frozen Concentration verification6 All analyzed immediately or frozen Stability:"" 5-hour room temperature All 5 hours room temperature then frozen 7-day and 14-day refrigerated All 7 or 14 days refrigerated then frozen a Duplicate samples will be collected and analyzed, b Samples will be collected at least 3 times during the study. c Stability of <CMPD> in solution will be demonstrated by comparing concentrations of <CMPD> in baseline samples to those of the refrigerated samples and those stored at room temperature. Extra samples may be taken at the discretion of the study director. Whenever extra samples are taken, a control sample will be collected and frozen the same day as dose-solution preparations G. Administration of Test Substance Dosing solutions will be administered daily by oral gavage at a dose volume of <10/20> ml/kg, based on the most recently recorded weight. Dose solutions will be kept <refrigerated> until used. Dosing solutions stored beyond <duration> after preparation will not be administered to animals. Control animals will be dosed with <solvent> only. All animals will be dosed for 90 days. Rats designated for recovery or fluoride analysis will not receive any dosing for the remainder of the recovery period. ft <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery H. Body Weights During the study, all rats will be weighed once each week unless experimental findings or special scheduling situations warrant a change in the weighing schedule. In addition, rats undergoing neurobehavioral assessments will be weighed on the days of those observations. I. Food Consumption and Food Efficiency During the study, the amount of food consumed by each rat over the weighing interval will be determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. From these measurements, mean daily food consumption over the interval will be determined. From the food consumption and body weight data the mean daily food efficiency will be calculated. J. Detailed Clinical Observations and Mortality During the study, cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats will be conducted at least twice daily throughout the study. Moribund rats will be sacrificed. At every weighing, each rat will be individually handled and examined for abnormal behavior and appearance in a standardized arena. The detailed clinical observations will include (but are not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior. Any abnormal clinical signs noted will be recorded. Prior to sacrifice of moribund rats, if possible and at the discretion of the clinical pathologist and/or study director, blood samples for hematology and clinical chemistry measurements will be collected for clinical pathology evaluations. K. Ophthalmology Two ophthalmological examinations will be conducted by a veterinary ophthalmologist. The pretest examination will be performed on all rats received for the study, prior to assignment to groups. Any rats with preexisting ophthalmological abnormalities may be eliminated from consideration for use in the study. All surviving rats designated for subchronic toxicity evaluation and recovery will be examined at the end of the exposure phase of the study. Both eyes of each rat will be examined by focal illumination and indirect ophthalmoscopy. The eyes will be examined in subdued light after mydriasis has been produced. A third ophthalmological examination may be conducted on rats designated for recovery, at the end of the recovery phase, if test substance-related effects are observed at the end of the exposure phase. &8 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery A report of findings from each ophthalmology examination will be submitted by the examining veterinarian. L. Neurobehavioral Evaluation For all of the following assessments, the experimenter will be unaware of the group designations of the animal. The abbreviated functional observational battery (FOB) and motor activity will be evaluated on all animals in the low and mid-dose group and in the rats designated for recovery (10 animals/group/sex) in control and high-dose groups. Spare rats will also be evaluated for the abbreviated FOB and motor activity prior to exposure. 1. Sensory Function Prior to test substance administration and then during week 12 of test substance administration, simple assessments of sensory function will be made (grip strength, response to approach/touch, tail pinch, and sharp auditory stimulus [clicker]) and evaluation of motor activity (see Section L.2. Motor Activity). Fore- and hind-limb grip strength will be measured by a strain gauge device (Chatillon Digital Force gauge). Due to technical difficulty of evaluating pupil size in albino rats under ambient lighting conditions, pupil size will be evaluated by assessment of pupillary response. Pupillary response will be conducted following motor activity. Pupillary response to a beam of light will be measured immediately prior to removing the rats from the motor activity chambers. Animals designated for recovery will be evaluated for sensory function during the last week of the recovery period, if effects are observed at the end of the exposure period. 2. Motor Activity Motor activity (MA) will be assessed following the sensory function evaluation on the same day. Rats will be individually tested in one of 30 nominally identical, automated activity monitors (Coulbourn). Group and gender will be counterbalanced across the monitors and time of day to the fullest extent possible. The infrared monitoring device enables measurement of 2 dependent variables, duration of movement and number of movements. A continuous movement is counted as 1 movement regardless of duration. Each test session will be 60 minutes in duration, and the results will be expressed for the total session as well as for 6 successive 10-minute blocks. Presence of defecation and urination on the cageboards below the motor activity cages will also be evaluated following each motor activity session. Animals designated for recovery will be evaluated for motor activity during the last week of the recovery period, if effects are observed at the end of the exposure period. M. Clinical Pathology Evaluation A clinical pathology evaluation will be conducted on all rats designated for subchronic toxicity at approximately 45 days and prior to necropsy on the last day of study. The day before collection of blood samples for the clinical pathology evaluation, the rats will be placed in metabolism cages. These rats will be fasted overnight and urine will be collected from each rat. Blood 60 9 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery samples for hematology and clinical chemistry measurements will then be collected from the orbital sinus of each rat while the rat is under light carbon dioxide anesthesia. Blood samples for coagulation parameters will be collected under carbon dioxide immobilization from the abdominal ve n a c a v a at sacrifice. Blood samples will be evaluated for quality by visual examination prior to analysis. At the discretion of the study director or clinical pathologist, additional samples for selected clinical pathology tests may be collected from animals showing clinical evidence of toxicity or sacrificed in e x tre m is. 1. Hematology The following hematology parameters will be determined: red blood cell count hemoglobin hematocrit mean corpuscular volume mean corpuscular hemoglobin mean corpuscular hemoglobin concentration red cell distribution width absolute reticulocyte count white blood cell count differential white blood cell count platelet count microscopic blood smear examination New methylene-blue-stained blood smears will be prepared from all hematology samples and will be evaluated, if required, to substantiate or clarify the results of hematology findings. 2. Coagulation Parameters The following coagulation parameters will be determined: prothrombin time activated partial thromboplastin time 3. Serum Chemistry The following serum chemistry parameters will be determined: aspartate aminotransferase alanine aminotransferase sorbitol dehydrogenase alkaline phosphatase total bilirubin urea nitrogen creatinine cholesterol triglycerides glucose total protein albumin globulin calcium inorganic phosphorus sodium potassium chloride plasma fluoride Gl 10 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery 4. Urinalysis The following urinalysis parameters will be determined: quality color transparency volume osmolality specific gravity pH 5. Recovery Animals urobilinogen blood glucose protein bilirubin ketone microscopic urine examination urine fluroide At the discretion of the clinical pathologist and study director and if hematological or clinical pathology evaluations suggest systemic toxicity, clinical pathology evaluations including hematology, serum chemistry, and urinalysis, may be performed on animals designated for recovery (10 rats/sex in control and high dose groups) at the end of the recovery period. N. Blood Analysis The designated 5 animals/sex/group will be evaluated for <fluoride levels> during the 90-day dosing period. The same rats will be evaluated for <fluoride levels> during a recovery period, up to 90 days post-dosing, or until <fluoride levels> in exposed rats return to those of controls. Blood collection time points will be: day 1 (males only), and days 4, 10, 21, 35, 56, 77, 90, 93, 98, 105, 119, 133, 154, and 175 (males and females). Blood (approximately 0.5 - 1 mL) will be collected from the orbital sinus of designated animals while under light carbon dioxide anesthesia. On the day of blood collection, blood will be collected from the animals prior to dosing. The blood will be collected in glass tubes containing EDTA while on ice and stored in the <refrigerator> until they are sent to <> within <24 hours> for analysis. For each bleeding, blood should be collected at approximately the same time of day. The samples will be sent to <> in coolers containing ice. Animals will be sacrificed at the end of the recovery period, or sooner if <fluoride levels> return to control levels. O. Anatomic Pathology 1. Pretest See Section D. Pretest Period. <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery 2. Rats Designated for Subchronic Toxicity Evaluation All rats found dead, accidentally killed, sacrificed in e x tre m is, or sacrificed by design will undergo a gross and microscopic evaluation. All rats removed from study (except for out-ofrange body weight on test day 0) will be sent to Pathology for gross evaluation and collection of tissues. Rats will be euthanatized by carbon dioxide anesthesia and exsanguination. Rats sacrificed by design will be fasted after 3 p.m. on the afternoon before their scheduled sacrifice. A final sacrifice will be performed on surviving rats following the final clinical pathology evaluation. The order of sacrifice for scheduled deaths will be random among all treatment groups within a sex. Blood samples for coagulation parameters will be collected under carbon dioxide immobilization from the abdominal ve n a c a v a of each rat at sacrifice and will be evaluated by Clinical Pathology. The following tissues will be collected from rats which are found dead or accidentally killed (tissue integrity permitting), sacrificed in e x tre m is, removed from study (except out-of-range body weight on test day 0), or sacrificed by design. Digestive Svstem liver esophagus stomach duodenum jejunum ileum cecum colon rectum salivary glands pancreas Urinary Svstem kidneys urinary bladder Respiratory Svstem lungs trachea nasal tissue larynx pharynx Cardiovascular Svstem heart aorta Hematopoietic Svstem spleen thymus mandibular lymph node mesenteric lymph node bone marrow" Endocrine Svstem pituitary gland thyroid gland parathyroid glands adrenal glands Nervous Svstem brain (including cerebrum, cerebellum, medulla/pons) spinal cord (cervical, mid-thoracic, lumbar) sciatic nerve Musculoskeletal Svstem skeletal muscle femur/knee joint sternum Reproductive Svstem Male testes epididymides prostate seminal vesicles Female ovaries uterus mammary glands Miscellaneous skin eyes (including optic nerve) gross observations11 a Bone marrow will be collected with the femur and sternum, b Gross observations made at necropsy for which histopathology is not appropriate (e.g., fluid, ruffled fur, and missing anatomic parts) will generally not be collected. 62 12 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery All tissues will be placed in the appropriate fixative. Rats sacrificed by design will have the following organs weighed: liver, kidneys, adrenal glands, thymus, brain, spleen, heart, and ovaries and uterus (female) or testes and epididymides (male). Relative organ weights (percent of final body weight; ratio to brain weight) will be calculated. Final body weights determined just prior to necropsy will be used in the assessment of organ weight changes. Organs from rats found dead, sacrificed in e x tre m is, or accidentally killed may be weighed at the discretion of the pathologist or study director. Bone marrow smears will be prepared at the final sacrifice from all surviving rats and will be evaluated if warranted by experimental findings. All tissues collected from rats designated for subchronic toxicity evaluation in the highconcentration and control groups, and from rats that are found dead or accidentally killed (tissue integrity permitting), or are sacrificed in e x tre m is, will be further processed to slides, stained with hematoxylin and eosin, and examined microscopically. Most gross lesions and any additional target organs from rats in the low- and intermediate-concentration groups will be evaluated microscopically. Selected gross observations for which a microscopic diagnosis would not be additive (e.g., osteoarthritis, pododermatitis, tail chronic dermatitis, calculus, and deformities of the teeth, toe, tail, or ear pinna) will be saved, but will generally not be processed for microscopic evaluation. Tissues from rats removed from study will not be processed for microscopic evaluation unless considered necessary by the study director or pathologist. Additional procedures to identify and/or clarify histologic features of lesions may be performed at the discretion of the pathologist and will be documented in the final report. 3. Rats Designated for <Blood Fluoride>Analysis Any rats designated for <blood fluoride> evaluations that are found dead, accidentally killed, or are sacrificed in e x tre m is will be discarded without pathological evaluation. Rats designated for <blood fluoride> analysis that survive the exposure period will be sacrificed by carbon dioxide anesthesia and exsanguination approximately 90 days after the final dose of <CMPD>, or when plasma fluoride levels return to control values. These rats will be discarded without pathological evaluation. 4. Rats Designated for Recovery Any rats designated for recovery evaluation that are found dead, accidentally killed, or are sacrificed in e x tre m is will be sent to Pathology for a gross evaluation. Tissues will be collected and examined microscopically at the discretion of the study director or pathologist. The results will not be reported in the final report unless considered significant to the evaluation of the study. One month after the final dose, rats designated for recovery (10 rats/sex in control and high dose groups) will be sacrificed by carbon dioxide anesthesia and exsanguination, and will undergo a gross examination. Tissues (see section 0 .2 .) will be collected and saved in the appropriate fixative. Gross lesions and target organs identified in rats sacrificed at the end of the 90-day exposure period will be examined microscopically. 13 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery STATISTICAL ANALYSES. Except for Bartlett's test (p < 0.005), significance was judged at p < 0.05. Method of Statistical Analysis Parameter Preliminary Test Body Weight Body Weight Gain Food Consumption Food Efficiency Organ Weight Motor Activity0 Grip Strength Foot Splay Clinical Pathologyd Fluoride Survival Incidence of Clinical Observations Incidence of FOB Descriptive Parameters Incidence of Microscopic Lesions Test for lack of trend(8) Levene's test for homogeneity*111and Shapiro-Wilk test(l2) for normality13 Levene's test for homogeneity*111 and Shapiro-Wilk test*121 for normality13 Bartlett's test*181for homogeneity of variances Levene's test for homogeneity*111 and Shapiro-Wilk test*121 for normality13 None None If preliminary test is not significant Sequential application*91 of the Jonckheere-Terpstra trend test*101 ORa If preliminary test is significant Preliminary tests for pairwise comparison One-way analysis of Kruskal-Wallis test*151 variance*131 followed followed with Dunn's with Dunnett's test*141 test*161 Repeated measures analysis of variance followed by Contrasts*171 One-way analysis of variance*131 followed with Dunnett's test*141 Sequential application*91 of the Jonckheere-Terpstra trend test*101 Kruskal-Wallis test*151 followed with Dunn's test*161 One-way analysis of Kruskal-Wallis test*151 variance followed followed with Dunn's with Dunnett's test*141 test*161 Cochran-Armitage test for trend*1310 None a Pairwise comparisons and associated preliminary tests are only conducted if the test for lack of trend is significant. b If the Shapiro-Wilk test is not significant but Levene's test is significant, a robust version of Dunnett's test will be used. c Test day and block (10-minute EPOCH) will be used as repeated-measure factors. d When an individual observation is recorded as being less than a certain value, calculations are performed on half the recorded value. For example, if bilirubin is reported as < 0.1,0.05 is used for any calculations performed with that bilirubin data. e If the incidence is not significant, but a significant lack of fit occurs, then Fisher's exact test<l9) with a Bonferroni correction is used. 14 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery Other methods will be used, if appropriate, at the time of analysis. The statistical methods used will be described in the final report. SAFETY AND HOUSEKEEPING Good housekeeping procedures will be practiced to avoid contamination of diet preparation facilities and potential health hazards. To avoid skin contact, gloves will be worn when handling either the test substance or diets. In addition, the test substance will be handled in a chemical hood. Diets will be prepared in properly ventilated areas. Animal carcasses, feces, and unused diet will be incinerated. RECORDS AND SAMPLE RETENTION All original records will be retained at <facility, locations Laboratory-specific or site-specific records such as personnel files and equipment records will be retained at the facility where the work was done. Preserved wet tissues, embedded tissues, histological slides, blood smears and bone marrow smears will be retained at <facility, location;. A sample of the test substance will be collected for archive purposes and retained at <facility, location;. 66 15 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery REFERENCES 1. EPA/FIFRA Good Laboratory Practice Standards (40 CFR 160). (1989). 2. OECD Principles of Good Laboratory Practice (as revised in 1997, published in ENV/MC/CHEM(98)17 (OCDE/GD(92)32). 3. MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850). 4. Official Journal of the European Communities. Part B: Methods for the Determination of Toxicity. (87/302/EEC). 5. United States Environmental Protection Agency (EPA), Office of Prevention, Pesticides, and Toxic Substances (OPPTS) Health Effects Test Guidelines, OPPTS 870.3100 90-Day Oral Toxicity in Rodents (AUG-1998). 6. Organisation for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4 (Part 408): Health Effects (1998). 7. The Ministry of Agriculture, Forestry, and Fisheries, Japan., Agricultural Chemicals Laws and Regulations, Japan (II), (59 NohSan No. 4200). (1985). 8. Draper, N.R. and Smith, H. (1981). Applied Regression Analysis, 2nd edition, pp 266-273. Wiley, New York. 9. Selwyn, M.R. (1995). The use of trend tests to determine a no-observable-effect level in animal safety studies. J o u r n a l o f th e A m e r ic a n C o lle g e o f T o x ic o lo g y 14(2), 158-168. 10. Jonckheere, A.R. (1954). A distribution-free K-sample test against ordered alternatives. B io m e trik a 41, 133-145. 11. Levene, H. (1960). Robust test for equality of variances. C o n tr ib u tio n s to P r o b a b ility a n d S ta tis tic s (J. Olkin, ed.), pp 278-292. Stanford University Press, Palo Alto. 12. Shapiro, S.S. and Wilk., M.B. (1965). An analysis of variance test for normality (complete samples). B io m e tr ik a 52, 591-611. 13. Snedecor, G.W. and Cochran, W.G. (1967). S ta tis tic a l M e th o d s , 6thedition, pp 246-248 and 349-352. The Iowa State University Press, Iowa. 14. Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. A m e r. S ta tist. A sso c . 50, 1096-1121. 15. Kruskal, W.H. and Wallis, W.A. (1952). Use of ranks in one-criterion analysis of variance. J. A m e r. S ta tis t. A s s o c . 47, 583-621. 16. Dunn, O.J. (1964). Multiple contrasts using rank sums. T e c h n o m e tric s 6, 241-252. 16 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery 17. Hocking, R.A. (1985). T he A n a ly s is o f L in e a r M o d e ls . Brooks/Cole, Monterey. 18. Bartlett, M.S. (1937). Some examples of statistical methods of research in agriculture and applied biology. J. R o y a l. S ta tis. S oc. S u p p l. 4, 137-170. 19. Fisher, R.A. (1985). S ta tis tic a l M e th o d s f o r R e s e a r c h W o rk e rs, 13thedition. Haffner, New York. bS 17 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery PROTOCOL APPENDIX Study Function Study Personnel Study Director: <> <Senior> Research Scientist Analytical Chemistry Evaluation: <> Analytical Chemist Neurobehavior Evaluation: <> Neurotoxicologist Clinical Pathology Evaluation: <> Principal Research Scientist Anatomic Pathology Evaluation: PP Anatomical Pathologist 63 18 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery Study Dates PROTOCOL APPENDIX (Continued) Initiation of Test Substance Administration <Analytical Chemistry Evaluations> <Pretest> <90-Day> Ophthalmology Evaluations Pretest Week 13 <Week 17: optional> Neurobehavioral Evaluations Pretest Week 13 <Week 17: optional> Clinical Pathology Evaluations 90-Day Blood Fluoride Analyses Every 2 weeks of exposure phase Every 2 weeks of recovery phase or until values return to control values. Scheduled Sacrifice Week 13: exposure phase Week 17: recovery phase Week 26: total fluoride evaluation rats (unless ended sooner) 10 19 <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery SIGNATURES Approved by: <> Primary Technician <> Analytical Chemist <> Neurotoxicologist <> Clinical Pathologist p p Pathologist <> Manager <> Study Director cc: Date Date Date Date Date Date Date 7/ 20 Title: Multigeneration Reproduction Study in Rats Title: Multigeneration Reproduction Study in Rats Protocol Contain NO CBI 7A - 1- Title: Multigeneration Reproduction Study in Rats TABLE OF CONTENTS Page OBJECTIVE.......................................................................................................................................... 4 SPONSOR AND TEST FACILITY................................................................................................... 4 MATERIALS AND METHODS........................................................................................................ 4 A. Test Substance................................................................................................................................. 4 1. Identification...............................................................................................................................4 2. Purity............................................................................................................................................ 4 3. Test Substance Stability............................................................................................................ 4 B. Anim als............................................................................................................................................ 4 1. Species (Strain)............................................................................................................................4 2. Reason for Selection...................................................................................................................4 3. Source........................................................................................................................................... 5 4. Description.................................................................................................................................. 5 5. Identification............................................................................................................................... 5 C. Animal Husbandry...........................................................................................................................5 1. Environmental Conditions.........................................................................................................5 2. Caging.......................................................................................................................................... 5 3. Food.............................................................................................................................................. 6 4. Water............................................................................................................................................ 6 5. Animal Health Monitoring........................................................................................................6 6. Quarantine................................................................................................................................... 6 D. Experimental D esign...................................................................................................................... 7 E. Selection of Dose Levels................................................................................................................8 F. Randomization................................................................................................................................ 8 1. Pi Generation............................................................................................................................... 8 2. Fi Generation............................................................................................................................... 8 G. Diet Administration, Preparation, and Sampling.......................................................................8 1. Administration.............................................................................................................................8 2. Preparation and Sam pling......................................................................................................... 8 H. Safety Precautions and Disposal of Waste Material.................................................................10 I. Body Weight Measurements for Parental Rats......................................................................... 10 1. Premating Feeding Period....................................................................................................... 10 2. Gestation and Lactation Periods............................................................................................. 10 J. Food Consumption and Food Efficiency for Parental Rats.................................................... 10 1. Premating Feeding Period....................................................................................................... 10 2. Gestation Period........................................................................................................................ 10 K. Clinical Observations for Parental R ats.................................................................................... 10 L. Breeding......................................................................................................................................... 11 1. Start of Cohabitation.............................................................................................................. 11 Title: Multigeneration Reproduction Study in Rats 2. Duration of Cohabitation Period............................................................................................ 11 3. Evidence of Copulation............................................................................................................11 4. Cohousing...................................................................................................................................11 M. Estrous Cycle Evaluation.............................................................................................................11 N. Gestation Procedures.................................................................................................................... 11 O. Lactation Procedures.................................................................................................................... 11 1. Day 0 Postpartum..................................................................................................................... 11 2. Day 4 Postpartum..................................................................................................................... 12 3. Days 7 and 14 Postpartum....................................................................................................... 12 4. Day 21 Postpartum (Weaning)............................................................................................... 12 P. Developmental Landmarks.......................................................................................................... 12 1. Vaginal Patency.........................................................................................................................12 2. Preputial Separation................................................................................................................. 12 Q. Euthanasia and Postmortem Examinations...............................................................................12 1. Pi and Fi Adults........................................................................................................................12 2. Offspring....................................................................................................................................14 3. F) and FtWeanlings................................................................................................................. 14 R. Sperm Assessment.........................................................................................................................14 DATA ANALYSES............................................................................................................................15 RECORDS AND SAMPLE STORAGE..........................................................................................16 CRITICAL DATES.............................................................................................................................17 REFERENCES.................................................................................................................................... 17 PROTOCOL APPENDIX 1............................................................................................................... 19 Title: Multigeneration Reproduction Study in Rats OBJECTIVE The objective of this study is to evaluate the effect of Title (Compound) on the gonadal function, conception, parturition, and the growth and development of offspring of male and female Crl:CD(SD)IGS BR rats over two generations involving the production of at least one set of litters in each generation. This study will conform with applicable Good Laboratory Practice Standards0 "3' and Test Guidelines/4"7' SPONSOR AND TEST FACILITY This study is sponsored by (name, sponsor) and will be conducted at <>. Sponsor approval of the study will be indicated by issuance of the work authorization form. MATERIALS AND METHODS A. Test Substance 1. Identification The test substance will be identified in this protocol and in the study records by Compound or by the Sample Number. 2. Purity <>% 3. Test Substance Stability The stability of Compound will be confirmed by purity analyses conducted quarterly. B. Animals 1. Species (Strain) Crl:CD(SD)IGS BR rats 2. Reason for Selection The rat was selected for this study as it is a preferred species for reproductive toxicity testing as recommended by the guidelines. The Crl:CD(SD)IGS BR strain was chosen because extensive background information is available from the literature, the supplier, and previous studies with other compounds. This strain is also considered suitable relative to longevity, hardiness, and incidence of spontaneous disease. 7^ -4 - Title: Multigeneration Reproduction Study in Rats 3. Source Charles River Laboratories, Inc. 4. Description Approximately 150 male and 150 female rats (no male-female siblings), requested to be approximately 49 days of age, and weigh approximately 180 to 220 grams and 151 to 175 grams, respectively. 5. Identification a. Pretest Temporary cage identification b. After Assignment to Treatment Groups Each rat will be assigned a unique six digit number and an identification number will be tattooed on the tail (see Appendix I). C. Animal Husbandry 1. Environmental Conditions Animal rooms will be maintained at an acceptable temperature of 20-26C (targeted at 23C) and relative humidity of 50 10%. Animal rooms will be artificially illuminated (fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours). 2. Caging All male rats will be housed individually during non-mating periods in stainless steel, wire-mesh cages. a. Pretest and Premating Periods All rats will be housed individually in stainless steel, wire-mesh cages. b. Cohabitation Period All rats will be housed as breeding pairs in stainless steel, wire-mesh cages. At the end of the cohabitation period, females without evidence of copulation will be housed individually in polycarbonate pans. c. Gestation Period Days 0-20: Females will be housed individually in stainless steel, wire-mesh cages. 76 -5- Title: Multigeneration Reproduction Study in Rats Day 20-Delivery: Females will be housed individually in polycarbonate pans, d. Lactation Period Adult females will be housed with their litters in polycarbonate pans. The stainless steel, wire-mesh cages will be suspended above cageboard. Polycarbonate pans will contain bedding. 3. Food PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 containing the appropriate dosage of Compound will be available a d lib itu m . 4. Water Water from <> will be available a d lib itu m . 5. Animal Health Monitoring <> Laboratory has an animal health monitoring program. The following procedures are performed periodically: Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Feed samples are analyzed for the presence of bacteria and fungi. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Blood sera taken from nonstudy animals (sentinels) housed in the study room are analyzed for antibodies against common rodent pathogens. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and to be free of impurities which might influence the results of the study. The animal health monitoring program is administered by the Laboratory Animal Veterinarian. Data are maintained separately from study records and will not be included in the final report unless warranted by the results of these evaluations. 6. Quarantine Rats will be quarantined for at least six days, and then released for the study upon the approval of the Laboratory Animal Veterinarian or designee. -6- Title: Multigeneration Reproduction Study in Rats Rats that die or are sacrificed in e x tre m is during the pretest period will be necropsied to check for the presence of disease. Dependent upon these findings, further diagnostic procedures may be employed at the discretion of the study director, the pathologist assigned to the study, or the laboratory veterinarian. D. Experimental Design Treatment Groups and Dietary Concentrations Group Number Male Female Pi F, Pi F, 1-0 III-O V-0 VII-0 1-1 III-l V-l VII-1 II-O IV-0 VI-0 VIII-0 II-1 IV-1 VI-1 VIII-1 #/Group 30 30 30 30 Concentration (ppm)3 0 (Control) <> (Low) <> (Intermediate) <> (High) d Weight/weight concentration of Compound, active ingredient, (adjusted for purity). Treatment Schedule Generation Pi F, Approximate Age at Start of Feeding(davs) 63 21 Approximate Number of Study Days Before Mating 70 70 Duration of Feeding Until sacrifice Until sacrifice Animals Adult Males Pregnant Females Nonpregnant Females Weanlings Sacrifice Schedule Generation Schedule Pi, F, Pi, F, P .,F , F[, F2 After siring litters (at discretion of study director) On day of weaning litters With pregnant females On day of weaning (except Fj's selected for parental rats) -7 - Title: Multigeneration Reproduction Study in Rats E. Selection of Dose Levels Dose levels selected for this study were based upon <>. F. Randomization 1. Pi Generation Rats of each sex will be ranked by their most recently recorded body weight and randomly assigned to control and experimental groups. The randomization should result in a distribution in which the mean body weights for all groups within a sex are not statistically different (p>0.05). Animals that lose excessive weight or that are ill prior to the start of dosing will be removed from the study and replaced, if possible. Rats not assigned to a test group, at the discretion of the study director, may be released for use in other studies, or will be sacrificed by carbon dioxide euthanasia and discarded without pathological evaluation. 2. Fi Generation On day 21 postpartum, offspring in the Fi litters of each treatment level will be selected (one rat/sex/litter when possible) to serve as parents for the F2 generation. For groups without sufficient litters, additional pups will be chosen from randomly selected litters within the group to achieve the required group size. Selection of rats within litters will be random. G. Diet Administration, Preparation, and Sampling 1. Administration Compound will be administered orally as it is a potential route for human exposure. During the test period, rats in each group will be fed a diet of PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 that contains 0, <>, <>, or <> ppm of Compound. 2. Preparation and Sampling Compound will be added to PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 and thoroughly mixed for a period of time that, by experience or pretest determination, is adequate to ensure homogeneous distribution in the diet. <The test substance will be dissolved/suspended in acetone prior to mixing. The same concentration of acetone will also be added to control diets. The manufacturer, lot number, and purity of the acetone used to dissolve the test substance prior to incorporation into the diet will be recorded in the study records. Neither the amount and _____________________________________________________________ ____________________________________ 8- - Title: Multigeneration Reproduction Study in Rats nature of the contaminants nor the use of acetone is expected to affect the integrity or validity of the study.> Diets will be prepared biweekly and refrigerated until used. The following dietary analysis parameters will be evaluated. Stability of the test substance in diets was verified previously at <> and <> ppm. Other samples will be collected in duplicate and analyzed from the initial diet preparation prior to study start to verify homogeneity and concentration. In addition, samples will be collected in duplicate and analyzed for concentration at approximately 3, 6, and 9 months from study start. Type Control Homogeneity :a,b Top Middle Bottom Stability:c 7-day Room Temperature 14-day Room Temperature 21-day Room Temperature 21 -day Refrigerated Concentration Verification:11 Collect From Storage Conditions Until Analysis Diet Mixer Diet Mixer Top Middle Bottom Frozen Frozen Frozen Frozen Frozen Feed Jars 7 days at room temperature, then frozen Feed Jars 14 days at room temperature, then frozen Feed Jars 21 days at room temperature, then frozen Diet Mixer 21 days refrigerated then frozen As Apportioned Analyzed immediately or frozen a The mean value of samples will be used to determine concentration. b Diet samples may also be collected to evaluate homogeneity if a new method of diet preparation is used. c Stability of the test substance in the diet was established for <> and <> ppm from a 90-day rat feeding study. d Diet samples will be collected at least 3 times during the study for each dietary level to verify concentration. Stability of Compound in feed was determined by comparing concentrations of Compound in samples collected and frozen immediately to those stored at room temperature or refrigerated. Extra diet samples may be taken at the discretion of the study director. Whenever extra diet samples are taken, a sample of control diet will be collected and frozen the same day as diet preparation. go 9- - Title: Multigeneration Reproduction Study in Rats H. Safety Precautions and Disposal of Waste Material Good housekeeping procedures will be practiced to avoid potential health hazards and contamination of diet preparation facilities. To avoid skin contact, gloves will be worn when handling either the test substance or diets. In addition, the test substance will be handled in a chemical hood. Diets will be prepared in properly ventilated areas. Animal carcasses, feces, and unused diet will be incinerated. I. Body Weight Measurements for Parental Rats 1. Premating Feeding Period All P[ and F] rats will be weighed once a week. All animals being evaluated for developmental landmarks (vaginal patency, preputial separation) will be weighed the day criteria are achieved. 2. Gestation and Lactation Periods Pi and Fi females will be weighed on days 0, 7, 14, and 21 of each period. Females without evidence of copulation, that copulate and do not deliver a litter, and males will continue to be weighed on a weekly schedule. J. Food Consumption and Food Efficiency for Parental Rats 1. Premating Feeding Period Individual food consumption will be determined weekly throughout the period, ending test day 70. 2. Gestation Period Individual food consumption of pregnant Pi and Fj females will be recorded on gestation days 0, 7, 14, and 21. From these determinations and body weight data, individual daily food consumption and food efficiency will be calculated. K. Clinical Observations for Parental Rats Cage-site examinations will be conducted at least once daily throughout the study. Moribund rats will be sacrificed. At least once weekly throughout the premating feeding, gestation, and lactation periods, each of the Pi and Fi parental rats will be individually handled and carefully examined for abnormal behavior and/or appearance. z\ - 10- Title: Multigeneration Reproduction Study in Rats L. Breeding 1. Start of Cohabitation After approximately 10 weeks of exposure to the test substance 2. Duration of Cohabitation Period Until evidence of copulation is observed (designated as day 0 of gestation), or two weeks have elapsed. 3. Evidence of Copulation Intravaginal or extruded copulation plug 4. Cohousing Each female will be continually housed on a 1:1 basis with a randomly selected, nonsibling male of the same dietary concentration level, in the male's cage. On the day copulation is confirmed, the female will be transferred back to individual cage housing. Based on the outcome of the first mating and at the discretion of the study director, the females may be cohoused again in the manner outlined above, except with different males, to produce a second set of litters. M. Estrous Cycle Evaluation Vaginal smears will be collected daily from all Pi and Fi female rats in order to determine the stages of the estrous cycle. Vaginal smears will be collected beginning three weeks prior to mating, and continuing until copulation is confirmed, or the cohabitation period has ended. Vaginal smears will also be collected from all P) and Fi parental female rats at the time of sacrifice to determine the stage of estrous cycle. N. Gestation Procedures After being transferred into polycarbonate pans (on day 20 of gestation for mated females, or at the end of the cohabitation period for females without evidence of copulation), female rats will be observed at least twice daily for signs of delivery and offspring. O. Lactation Procedures The day when delivery is complete is designated day 0 postpartum. 1. Day 0 Postpartum Live and dead pups in each litter will be counted as soon as possible after delivery is completed. Live pups in each litter will be individually weighed. Anogenital distance in sx -11 - Title: Multigeneration Reproduction Study in Rats F2 pups will be recorded, if treatment-related effects are observed in sex ratio or sexual maturation of Fi pups. 2. Day 4 Postpartum Litters will be culled randomly to eight (four/sex when possible). Extra offspring will be euthanized (by decapitation) and discarded without pathological examination. Litters of eight offspring or less will not be reduced. Litter counts and individual pup weights will be determined prior to and after culling. 3. Days 7 and 14 Postpartum Pups in each litter will be counted by sex and individually weighed. 4. Day 21 Postpartum (Weaning) Pups in each litter will be counted by sex and individually weighed. Offspring from the Fj litters will be randomly selected to serve as parents for the F2 generation. Randomly selected offspring from the litters of both the Fi and F2 generations will undergo gross pathological evaluation. Unselected weanlings will be euthanized by carbon dioxide euthanasia and discarded without pathological evaluation. At each examination period, offspring will be individually handled and examined for abnormal behavior and appearance; any dead, missing, or abnormal pups will be recorded. P. Developmental Landmarks Developmental landmarks in the Fi generation male and female rats (designated for mating) will be monitored on a daily basis until criterion is achieved. 1. Vaginal Patency Female rats will be examined beginning on test day 0 (Postnatal day 21). 2. Preputial Separation Male rats will be examined beginning on test day 14 (Postnatal day 35). Q. Euthanasia and Postmortem Examinations 1. Pi and Fi Adults a. Method of Euthanasia Carbon dioxide euthanasia and exsanguination tb - 12- Title: Multigeneration Reproduction Study in Rats b. Postmortem Examinations All Pi and F] parental generation rats will be subjected to gross pathological examination, including those that die or are sacrificed in e x tre m is prior to the end of feeding and those for which mating did not result in production of offspring. The following table lists tissues to be collected and/or weighed from all parental generation rats (tissue integrity permitting): Organ Weights from Parental Rats Male Testes Epididymides Seminal Vesicles (with coagulating glands and their fluids) Prostate Female Uterus (with oviducts and cervix) Ovaries Both Sexes Brain Pituitary Liver Kidneys Adrenal Glands Spleen Tissues Collected from Parental Ratsa Male Female Both Sexes Testis Ovaries Adrenal Glands Epididymis Uterusb (with oviducts) Pituitary Prostate Vagina Gross Observations0 Seminal Vesicles Cervix Coagulating Gland_____________________________________________________________ a Reproductive tissues collected from male parental rats for possible histopathological evaluation will be placed in Bouin's solution. Reproductive tissues collected from female parental rats and all non-reproductive tissue will be placed in formalin. b The uteri of all cohabited females will be examined for the presence and number of implantation sites. c Gross lesions observed at necropsy for which histopathology is not appropriate (e.g., fluid, ruffled fur, no milk spot, fetus in birth canal, and missing anatomic parts) will generally not be collected. c. Histopathological Examination Histopathological examination of the tissues will be conducted for ten randomly chosen high dose and control animals per sex from both the Pi and Fi generations. Target organs and organs demonstrating treatment-related changes will be examined for the remainder of the high-dose and control animals and for all parental animals in - 13 - Title: Multigeneration Reproduction Study in Rats the low- and mid-dose groups in order to further delineate or establish a "noobservable-adverse-effect level". Reproductive organs of animals in low and intermediate dose groups with impaired reproductive performance (e.g., failure to mate, conceive, sire, or deliver healthy offspring; or effects in estrous cyclicity or sperm parameters) will be evaluated. In addition, a quantitative evaluation of primordial and growing follicles will be conducted on the F| females selected for histopathological evaluation. Five ovarian cross sections (6 pm thick) will be taken at least 100 pm apart from the inner third of each ovary. Primordial and growing follicles (up to but not including antral follicles) will be enumerated for each of 10 ovarian sections per animal. Most gross lesions and target organs from low and intermediate levels will also be evaluated microscopically. Selected gross observations for which a microscopic diagnosis would not be additive (e.g., osteoarthritis, pododermititis, tail chronic dermatitis, calculus, and deformities of the teeth, toe, tail, or ear pinnae) will be saved, but will generally not be processed for microscopic evaluation. 2. Offspring Offspring that are found dead during the lactation period will undergo gross pathological evaluation. 3. Fi and F2 Weanlings a. Method of Euthanasia Carbon dioxide euthanasia and exsanguination b. Postmortem Examinations Three Fi and three F2 weanlings/sex/litter, litter size permitting, will undergo gross pathological evaluation. One weanling/sex/litter will be designated for organ weight and histopathological evaluation. The weanlings selected for organ weight and histopathological evaluation will have brain, spleen, and thymus weights recorded and all gross lesions and target organs (<>) will be preserved for possible future histopathological examination. c. Histopathological Examination Microscopic evaluation of the preserved gross lesions will be conducted at the discretion of the study director or pathologist. Tissues collected from both male and female weanlings for possible histopathological evaluation will be placed in formalin. R. Sperm Assessment Sperm parameters for all P[ and Fi parental males will be evaluated. The right epididymis will be removed, and the right cauda epididymis will be weighed. Sperm will be collected from the right cauda epididymis and percent motility and morphology will be determined. The left Title: Multigeneration Reproduction Study in Rats epididymis and testis will be frozen in liquid nitrogen and stored between -65C and -85C for sperm and spermatid counts, respectively. DATA ANALYSES The following table lists the indices of reproductive function that will be calculated for the Pi and Fi adults. Reproductive Function Calculations Mating Index (% ) = Fertility Index (%) = Fecundity Index (%) = Gestation Index (% ) = Implantation Efficiency (%)c = Pups Born Alive (%)c = Viability Index (%)c,d = Lactation Index (% )c,d - Number Copulating3 Number Cohabited Number Bearing Littersb Number Cohabited3 Number Bearing Littersb Number Copulating3 Number of Litters with at Least One Live P ud Number of Litters Number of Pups Born Number of Implantation Sites Number of P uds Born Alive Number of Pups Born Number of P uds Alive Dav 4 Preculling Number of pups born alive Number of pups alive at weaning (21 postpartum) Number of pups alive Day 4 Postculling x 100 x 100 x 100 x 100 x 100 x 100 x 100 x 100 a Evidence o f copulation = copulatory plug, found dead pregnant, or delivery of a litter. b Including those found dead pregnant during gestation. c To be determined for each litter. Mean and standard deviation for each dose level will be calculated. d Excluding litters sacrificed due to death of dam during lactation. Parameter Body Weight Body Weight Gain Food Consumption Statistical Methods Method of Statistical Analysis One-way Analysis of Variance(8); followed, when significant, with Dunnett's test<9) / 86 Title: Multigeneration Reproduction Study in Rats Gestation Length Organ Weight Hematology Implantation Site Numbers Implantation Efficiency Mean Number of Pups Per Litter Percent Born Alive Sex Ratio Viability Index Lactation Index Precoital Interval Estrous Cycle Length Sperm Parameters Vaginal Patency Preputial Separation Mean Pup Weights (Covariates: litter size, sex ratio) Anogenital Distance (Covariate: pup weight) Incidence of Microscopic Lesions Incidence of Clinical Observations Number of Females Cycling Normally Mating Index Fecundity Index Fertility Index Gestation Index Same as above; or nonparametric tests if Bartlett's test for homogeneity of variances is significant (alpha = 0.005) Same as above; or nonparametric tests if Levene's test for homogeneity and ShapiroWilk test"01 for normality are significant (p<0.05). If the Shapiro- Wilk test is not significant but Levene's test is significant, a robust version of Dunnett's test will be used. Jonckheere's test"11 Linear contrast of the least square means"21 Cochran-Armitage test for trend or, if not all groups are compared, Fisher's Exact test"31 For each parameter analyzed with a trend test, the test will be applied to the data sequentially. If a significant dose-response is detected, data from the top dose group will be excluded and the test repeated until no significant trend is detected.*14) For litter parameters, the proportion of affected fetuses per litter or the litter mean will be used as the experimental unit for statistical evaluation.1<li) The level of significance selected is p < 0.05. Additional statistical tests will be used, and other parameters analyzed, if deemed necessary. Where the data are tied and the standard large sample version of Jonckheere's test is not applicable, exact p values will be calculated using permutation methodology/16) RECORDS AND SAMPLE STORAGE Title: Multigeneration Reproduction Study in Rats CRITICAL DATES In-Life Study Start Date: Pi Adult Sacrifice: F| Weanling Sacrifice: Fi Adult Sacrifice: F2 Weanling Sacrifice: In-Life Completion Date: <> <> <> <> <> <> (Approximate) (Approximate) (Approximate) (Approximate) REFERENCES 1. EPA/FIFRA Good Laboratory Practice Standards (40 CFR 160). (1989). 2. OECD Principles of Good Laboratory Practice (C(8 l)30(Final), Annex 2). 3. MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850). 4. EPA/FIFRA Pesticide Assessment Guidelines, Subdivision F; Hazard Evaluation: Human and Domestic Animals. Series 83-4 (PB 86-108958). (1984). 5. OECD Guidelines for Testing of Chemicals, Section 4, No. 416, adopted 26 May 1983. 6. Commission Directive 87/302/EEC (Official Journal of the European Communities No L 133, Vol. 31,30.5.88). 7. MAFF Japan Testing Guidelines for Toxicology Studies (59 NohSan No. 4200). (1985). 8. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th edition, pp 246-248 and 349-352. The Iowa State University Press, Iowa. 9. Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Statist. Assoc. 50, 1096-1121. 10. Shapiro, S.S. and Wilk., M.B. (1965). An analysis of variance test for normality (complete samples). Biometrika 52, 591-611.123 11. Jonckheere, A.R. (1954). "A Distribution-Free K-Sample Test Against Ordered Alternatives," Biometrika 41, 133-145. 12. Hocking, R.A. (1985). The Analysis o f Linear Models. Brooks/Cole, Monterey. 13. Fisher, R.A. (1985). Statistical Methods fo r Research Workers, 13thedition. Haffner, New York. Title: Multigeneration Reproduction Study in Rats 14. Selwyn, M.R. (1995). "The Use of Trend Tests to Determine a No-Observable-Effect Level in Animal Safety Studies," Journal of the American College o f Toxicology 14(2), 158-168. 15. Haseman, J.K. and Hogan, M.D. (1975). "Selection of the Experimental Unit in Teratology Studies," Teratology, 12, 165-171. 16. Patefield, W. (1982). "Exact Tests for Trends in Ordered Contingency Tables," Applied Statistics 31, 32-43. 83- jr - Title: Multigeneration Reproduction Study in Rats PROTOCOL APPENDIX I Animal Identification Marking System (AIMS) Rats will be identified by a tail tattoo number according to the following numbering system: Groups Male Female 1-0 m -o V -0 VII-0 II-O IV-0 VI-0 VIII-0 Pi GENERATION Tattoo Number" Male Female 101-130 301-330 501-530 701-730 201-230 401-430 601-630 801-830 Dietary Concentration of Compound (ppm) 0 (Control) <> (Low) <> (Intermediate) <> (H ig h ) Groups Male Female 1-1 III-l V-l VII-1 II-1 IV-1 VI-1 VIII-1 F, GENERATION Tattoo Numbera,b Male Female 101x-130x 301x-330x 501x-530x 501x-530x 201x-230x 401x-430x 601x-630x 801x-830x Dietary Concentration of Compound (ppm) 0 (Control) <> (Low) <> (Intermediate) <> (High) a Each rat will be assigned a unique Animal Number. b Fi rats will be assigned an ID number based on their Pi dams' ID number followed by one more digit. For male pups the initial digit will be changed to the odd number corresponding with their group number. The final digit will indicate if it is the first or second pup of a sex selected from a litter. For example, if one male and two females are selected from dam 401 their ID numbers will be 3011, 4011 and 4012, respectively. Each F| pup selected will also receive its own animal identification number. 60- y - Title: Multigeneration Reproduction Study in Rats Title: Multigeneration Reproduction Study in Rats Protocol Prepared by: Approved by: cc: SD, SDDegree SDTitle Study Director <> Chemist <> Staff Pathologist <> Management Date Date Date Date ~y 91 Sponsor Project Number:___________ Test Substance Name'. Rat Bone Marrow Erythrocyte Micronucleus Test 1.0 PURPOSE The purpose of this study is to evaluate the clastogenic potential of the test substance as measured by its ability to induce micronucleated polychromatic erythrocytes in rat bone marrow. SPONSOR 2.1 Name: <> 2.2 Address: <> 2.3 Representative: <> 2.4 Sponsor Project #: <> 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance: <> 3.2 Controls: Vehicle: Test substance vehicle Positive: Cyclophosphamide (CP) 3.3 Test Substance Characterization Unless alternate arrangements are made, the testing facility will not perform analysis of the dosing solutions. The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition of the test substance, and the stability and strength of the test substance in the solvent (or vehicle). 3.4 Test Substance Retention Sample The retention of a reserve sample of the test substance will be the responsibility of the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: <> 4.2 Address: <> 4.3 Study Director: <> Protocol <> August 28, 2000 Page 1 of 7 9i Contain 5.0 PROPOSED STUDY DATES Sponsor Project Number: 5.1 Experimental Start Date: <> 5.2 Experimental Termination Date: <> 5.3 Draft Report Date: <> 5.4 Final Report Date <> 6.0 TEST SYSTEM Closed-colony, random-bred rodents are acceptable models for mutagenicity studies. Sprague-Dawley rats are selected because of the availability of historical control data. 6.1 Source: Harlan Sprague Dawley, Inc., Frederick, MD or Charles River Breeding Laboratories, Kingston, NY or Raleigh, NC or other approved alternates 6.2 Age at initiation of study: 6-8 weeks 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The assay will be conducted according to established procedures (Heddle, 1973; Mavoumin e t a l., 1990; Hayashi e t a l , 1994). Following the administration of three concentrations of test substance as well as positive and negative (vehicle) controls to male and female rats, bone marrow cells will be collected at 24 and 48 hours and examined for the presence of micronucleated polychromatic erythrocytes. The clastogenic potential of the test substance will be measured by its ability to increase micronucleated polychromatic erythrocytes in treated animals as compared to vehicle control animals. The study design will be as follows: Vehicle Control Vehicle alone Test Substance Low Dose Mid Dose High Dose Positive Control Cyclophosphamide Number per Sex to be Sacrificed After Dose Administration 24 Hr 48 Hr 55 5555 5- Protocol <> August 28, 2000 Page 2 of 7 93 7.1 Selection of Test Substance Vehicle Sponsor Project Number:__________ Unless the Sponsor has indicated the test substance vehicle, a solubility determination will be conducted to measure the maximum soluble concentration or workable suspension of test substance in vehicle. Vehicles compatible with this test system, in order of preference, include, but are not limited to, distilled water or saline, 1% carboxymethylcellulose in water, and com oil. The vehicle of choice will be that allowing preparation of dosing solutions required to achieve targeted doses. 7.2 Dose Selection Selection of doses for the micronucleus assay will be based on the toxicity of the test substance but will not exceed 2000 mg/kg. In the absence of toxicity data, a pilot study will be performed at a dose of 2000 mg/kg using up to five male and five female rats. Three or more lower doses will be tested using two male rats each. If dose administration produces no treatment-related mortality, the high dose for the micronucleus test will be 2000 mg/kg. In the event of mortality in excess of 50% at 2000 mg/kg in the pilot study, an extensive toxicity study will be performed using up to four test substance doses, each containing up to five male and five female rats. Rats will be observed after dose administration and each working day thereafter for 3 days for clinical signs of chemical effect. Body weights will be recorded prior to dose administration and at 1 and 3 days after dose administration. Unless specified otherwise by the Sponsor, the high dose for the micronucleus test will be 50% to 80% of the LD50/3 (the dose required to kill 50% of the animals within 3 days after administration) but will not exceed 2000 mg/kg. Two additional doses will be tested, one-half and one-fourth of the high dose. 7.3 Route and Frequency of Administration Animals will be dosed by intraperitoneal (IP) injection. IP injection was selected to maximize delivery of the test substance to the target system. IP injection is an acceptable method for administration of test substance concentrations to laboratory animals. Animals will receive the test substance as a single administration. 7.4 Controls 7.4.1 Vehicle control The solvent vehicle for the test substance will be used as the vehicle control. 7.4.2 Positive control Cyclophosphamide monohydrate (CP, CAS number 6055-19-2) will be administered as the positive control at a dose of 30-60 mg/kg. CP will be administered by the same route as the test substance. Protocol <> August 28, 2000 Page 3 of 7 7.5 Animal Receipt and Quarantine Sponsor Project Number:__________ Virus antibody-free (VAF) rats will be quarantined for no less than 5 days prior to dose administration. The animals will be observed each working day for signs of illness, unusual food and water consumption, and other general conditions of poor health. All animals will be judged to be healthy prior to utilization in the study. 7.6 Animal Care Animals will be housed in an AAALAC-accredited facility with a controlled environment of 50 20% relative humidity and 72 3F with a 12 hour light/dark cycle. Rats of the same sex will be housed up to three per cage in plastic autoclavable cages. Heat-treated hardwood chips will be used for bedding. Animals will have free access to a certified laboratory rodent chow which has been analyzed for environmental contaminants and to tap water. 7.7 Randomization The animals will be assigned to seven groups of five males and five females each using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals will not exceed 20% of the mean weight. Additional animals may be designated and dosed as replacement animals in the high dose group to be used in the event of mortality prior to the scheduled sacrifice. This will be done at the discretion of the Study Director after evaluation of the toxicity data. Each animal will be given a sequential number and identified by ear tag. 7.8 Dose Preparation and Administration The test substance-vehicle mixture, the vehicle alone and the positive control (CP) will be given as a single administration. The rate of administration will be 10 ml/kg body weight unless larger volumes, up to 20 mL/kg, are required to deliver the targeted dose. All rats in the experimental groups will be weighed and the dose volume will be based on individual body weight. 7.9 Bone Marrow Collection Twenty-four and 48 hours after dose administration, animals will be sacrificed by carbon dioxide asphyxiation. The positive control group will be sacrificed 24 hours after dose administration. Immediately following sacrifice, the femurs will be exposed, cut just above the knee and the bone marrow will be aspirated into a syringe containing fetal bovine serum. The bone marrow cells will be transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells will be pelleted by centrifugation and the supernatant will be drawn off, leaving a small amount o f fetal bovine serum with the remaining cell pellet. The cells will be resuspended by aspiration with a capillary pipette and a Protocol <> August 28, 2000 Page 4 of 7 95 Sponsor Project Number:___________ small drop of the bone marrow suspension will be spread onto a clean glass slide. Each slide will be identified by the experiment and animal number. At least two slides will be prepared from each animal, air dried, fixed by dipping in methanol, stained with nucleic acid-specific stain, acridine orange. 7.10 Scoring for Micronuclei Slides will be coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality will be selected such that the cells are well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes will be scored per animal for the presence of micronuclei. The number of micronucleated normocytes in the field of 2000 polychromatic erythrocytes will also be enumerated. The proportion of polychromatic erythrocytes to total erythrocytes will also be recorded per 1000 erythrocytes. The proportion of polychromatic erythrocytes to total erythrocytes in test substance-treated animals should not be less than 20% of the control value. 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST The mean incidence of micronucleated polychromatic erythrocytes must not exceed 3/1000 (6/2000) polychromatic erythrocytes (0.3%) in the negative (vehicle) control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the negative control (p<0.05, Kastenbaum-Bowman Tables). 9.0 EVALUATION OF TEST RESULTS The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes will be presented for each animal and treatment group. Statistical significance will be determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. In order to quantify the test substance effect on erythropoiesis, as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes will be presented for each animal and treatment group. All conclusions will be based on sound scientific judgement; however, as a guide to interpretation of the data, the test substance will be considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes is observed and one or more doses are statistically elevated relative to the vehicle control (p<0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group is significantly elevated at one sacrifice time with no evidence of a dose-response or if there is evidence of a dose response with no evidence of a significant increase in any treated group relative to the control group, the Sponsor will be contacted. The test substance will be judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response are observed at any sampling time. Protocol <> August 28, 2000 Page 5 o f 7 Sponsor Project Number: 10.0 REPORT A report of the results of this study will be prepared by the Testing Laboratory and will accurately describe all methods used for generation and analysis of the data. The report will include: Test substance: identification and CAS no., if known; physical nature and purity, if known; physicochemical properties relevant to the conduct of the study, if known; stability of the test substance, if known. Solvent/Vehicle: justification for choice of vehicle; solubility and stability of the test substance in the solvent/vehicle, if known. Test animals: species and strain of animals used; number, age and sex of animals; source, housing conditions, diet, etc. Test conditions: positive and negative (vehicle/solvent) control data; data from range finding study, if conducted; rationale for dose level selection; details of test substance preparation; details of the administration of test substance; rationale for route of administration; methods for verifying that the test substance reached the general circulation or target tissue, if applicable; details of food and water quality; description of treatment and sampling schedules; method of slide preparation; methods for measurement of toxicity; criteria for scoring micronucleated immature erythrocytes; number of cells analyzed per animal; criteria for considering study as positive, negative or equivocal. Results: signs of toxicity; proportion of polychromatic erythrocytes among total erythrocytes; number of micronucleated polychromatic erythrocytes per animal; meanstandard deviation of micronucleated polychromatic erythrocytes per group; doseresponse relationship, where possible; statistical analyses; concurrent negative control data; historical negative control data with ranges, means and standard deviations; concurrent positive control data. Discussion of results. Conclusion. 11.0 RECORDS AND ARCHIVES All raw data, the protocol and all reports will be maintained according to Standard Operating Procedure by <facility, address>. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply with OECD Guideline 474 (Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test), adopted 1997, and with the Protocol <> August 28, 2000 Page 6 of 7 37 Sponsor Project Number:___________ International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997). This study will be performed in compliance with the provisions of the EPA (TSCA), OECD, Japanese (MAFF) and EEC Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. Unless arrangements are made to the contrary, unused dosing solutions will be disposed of following administration to the test system and all residual test substance will be disposed of following finalization of the report. 13.0 REFERENCES Heddle, J.A. 1973. A rapid in vivo test for chromosomal damage. Mutation Res. 18:187190. Hayashi, M., R.R. Tice, J.T. Macgregor, D. Anderson, D.H. Blakey, M. Dirsch-Volders, F.G. Oleson Jr., F. Pacchierotti, F. Romagna, H. Shimada, S. Sutou and B. Vannier. 1994. In vivo rodent erythrocyte micronucleus assay. Mutation Res. 312: 293-304. Mavoumin, K.H., D.H. Blakey, M.C. Cimino, M.F. Salamone and J.A. Heddle. 1990. The in v iv o micronucleus assay in mammalian bone marrow and peripheral blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res. 239:29-80. M.A. Kastenbaum and K.O. Bowman. 1970. Tables for determining the statistical significance of mutation frequencies. Mutation Res. 9:527-549. 14.0 APPROVAL Sponsor Representative (Print or Type Name) Study Director Study Management Date Date Date Protocol <> August 28, 2000 Page 7 of 7 Sponsor Project Number: Test Substance Name: In Vitro Mammalian Chromosome Aberration Test 1.0 PURPOSE The purpose of this study is to evaluate the clastogenic potential of a test substance based upon its ability to induce chromosome aberrations in human peripheral blood lymphocytes (HPBL). 2.0 SPONSOR 2.1 Name: <> 2.2 Address: <> 2.3 Representative: <> 2.4 Sponsor Project #: <> 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance Name: <> 3.2 Controls: Solvent: Positive: Test Substance Solvent or Vehicle Mitomycin C (MMC) Cyclophosphamide (CP) 3.3 Test Substance Characterization Unless alternate arrangements are made, the testing facility will not perform analysis of the dosing solutions. The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition of the test substance and the stability and strength of the test substance in the solvent (or vehicle). 3.4 Test Substance Retention Sample The retention of a reserve sample of the test substance will be the responsibility of the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: <> 4.2 Address: <> C ontain NO OBI Protocol <> August 28, 2000 Page 1 of 9 93 4.3 Study Director: <> Sponsor Project Number: PROPOSED STUDY DATES 5.1 Proposed Experimental Start Date: <> 5.2 Proposed Experimental Termination Date: <> 5.3 Proposed Report Date: <> 5.4 Final Report Date: <> 6.0 TEST SYSTEM Peripheral blood lymphocytes will be obtained from healthy adults without a recent history of either radiotherapy, viral infections or the administration of drugs. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals (Preston e t a l., 1981). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The test will be conducted using standard procedures, by exposing human lymphocytes to a minimum of four concentrations of the test substance as well as to positive and solvent controls. In the non-activated test system, treatment will be for 4 hours and for 20 hours; in the S9 activated test system, exposure will be for 4 hours (Swierenga et al., 1991). The dividing cells will be arrested in metaphase and harvested for microscopic evaluation of chromosome aberrations at approximately 20 hours (1.5 normal cell cycles) after the initiation of treatment in order to ensure evaluation of first-division metaphase cells (Galloway et al., 1994). The clastogenic potential of the test substance will be measured by its ability to increase chromosome aberrations in a dose-responsive manner when compared to a control group. The 4 hour non-activated and S9-activated studies will be scored initially. In the event of a positive response in the 4 hour non-activated study, the prolonged exposure non-activated study may not be scored. The test substance will also be assessed for its ability to induce numerical chromosome aberrations. 7.1 Solubility Determination Unless the Sponsor has indicated the test substance solvent, a solubility determination will be conducted to determine the solvent and the maximum soluble concentration up to a maximum of 500 mg/ml. Solvents compatible with this test system, in order of preference, include but are not limited to sterile water (CAS 7732-18-5), dimethyl sulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5), and acetone (CAS 67-64-1). The solvent will be the test substance solvent, selected in order of preference, that permits preparation of the highest soluble stock concentration, up to 500 mg/ml. Protocol <> August 28, 2000 Page 2 of 9 10V Sponsor Project Number:__________ 7.2 Preliminary Toxicity Assay to Select Dose Levels Selection of the dose levels for the cytogenetics assay will be based upon inhibition of mitosis after treatment as determined in a cytotoxicity study. Cells will be exposed to solvent alone and to at least nine concentrations of test substance, the highest concentration being 5000 Pg/ml or 10 mM whichever is lower. The pH of the highest test substance dosing solution will be measured, and will be adjusted, if necessary, in order to maintain a neutral pH in the treatment medium. The osmolality of the highest dosing solution, lowest precipitating dose level (where applicable) and the highest soluble dose level (where applicable) will also be measured. Peripheral blood cells will be cultured in RPMI-1640 containing 15% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin and 100 Pg streptomycin/ml and 1% phytohemagglutinin. Cells seeded approximately 46 hours earlier will be exposed for 4 hours in the absence and presence of S9 and for 20 hours in the absence of S9. After exposure the cultures will be grown in complete medium for 16 hours. Eighteen hours after treatment initiation Colcemid (0.1 pg/ml) will be added to the cultures. Cells will be collected at 20 hours after the initiation of treatment by centrifugation, treated with hypotonic KC1 solution and fixed with methanol-glacial acetic acid. Metaphase preparations will be made and stained. The percentage of cells in mitosis per 500 cells scored (mitotic index) will be determined for each treatment group. Whenever possible, the high dose for the chromosome aberration assay will be selected to give at least 50% toxicity (mitotic inhibition relative to the solvent control). At least two additional dose levels, demonstrating minimal or no toxicity, will be evaluated in the chromosome aberration assay. In the event the test substance cannot be dissolved at a high enough concentration in an appropriate solvent to be toxic, then the highest dose to be tested will be the concentration resulting in minimum precipitation in test medium. Precipitation will be determined with the unaided eye. In the event the test substance demonstrates a dose-responsive increase in toxicity (mitotic inhibition relative to the solvent control) at concentrations that exceed solubility in treatment medium, then the highest dose to be evaluated for chromosome aberrations will be the concentration resulting in minimum precipitation in test medium. In the event that neither cytotoxicity nor insolubility is observed in the preliminary test, the highest dose in the chromosome aberration assay will be 5 mg/ml or 10 mM whichever is lower. If excessive precipitation of the test substance-solvent solution occurs upon addition to treatment medium, or if the osmolality of the treatment medium is excessive, the Sponsor will be consulted. 7.3 Frequency and Route of Administration Target cells will be treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence of S9, by incorporation of the test substance-solvent mixture into the treatment medium. This technique has demonstrated to be an effective method of detection of chemical clastogens in this test system (Evans, 1975). Protocol <> August 28, 2000 Page 3 of 9 101 Sponsor Project Number: If the Sponsor is aware of specific metabolic requirements, then this information will be utilized in the preparation of the study design. Verification of a clear positive response is not required. Negative results will not be confirmed when justification can be provided. Equivocal results may be confirmed, upon consultation with the Sponsor, and may employ a modification of the study design. This guidance is based on the OECD Guideline 473 (adopted July 1997) and ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1996). 7.4 Controls 7.4.1 Solvent (or Vehicle) Control The solvent for the test substance will be used as the solvent control. For solvents other than water, physiological buffer, or medium, the final concentration in treatment medium will not exceed 1%. 7.4.2 Positive Controls Mitomycin C will be used at two concentrations within the range of 0.1-0.50 Pg/ml as the positive control for the non-activated test system. For the S9activated system, cyclophosphamide will be used at two concentrations within the range of 10-75 Pg/ml. One dose level of each positive control will be evaluated microscopically for chromosome damage. 7.5 Exogenous Metabolic Activation Aroclor 1254-induced rat liver S9 will be used as the metabolic activation system. The S9 will be prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 will be batch prepared and stored frozen at approximately -70C until used. Each batch preparation of S9 will be assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(oQanthracene to forms mutagenic to S a lm o n e lla typ h im u riu m TA 100. Immediately prior to use, the S9 will be thawed and mixed with a cofactor pool. The final concentration of the cofactors and S9 in the reaction vessel will be 2 mM MgCk, 6 mM KC1, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate and 20 pi S9 per ml RPMI-1640 serum-free medium. 7.6 Preparation of Target Cells Protocol <> Peripheral blood lymphocytes will be cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin and 100 pg streptomycin/ml, and 1% phytohemagglutinin) by adding 0.6 ml heparinized blood to a centrifuge tube containing 9.4 ml complete medium. The tubes will be August 28, 2000 Page 4 of 9 \0XJ Sponsor Project Number:___________ incubated upright at 37 1C in a humidified atmosphere of 5 1% CO2 in air for 44-48 hours. 7.7 Test System Identification Using a permanent marking pen, the test system will be identified by the BioReliance study number, treatment condition and date. 7.8 Treatment of Target Cells Forty-four to 48 hours after culture initiation, duplicate centrifuge tubes will be refed with approximately 10 ml complete medium for the non-activated exposure or 10 ml S9 reaction mixture for the activated exposure to which will be added 100 pi of dosing solution of test or control substance in solvent. Larger volumes of dosing solution may be used if water or medium is used as the solvent. For the S9-activated exposure, the cells will be treated for 4 hours in the presence of an S9 reaction mixture, washed free of chemical and cultured for an additional 16 hours with Colcemid (0.1 pg/ml) present for the last 2 hours. For the nonactivated exposure, treatment will be for 4 hours followed by a 16 hour recovery period and for 20 hours continuously with Colcemid (0.1 pg/ml) present for the last 2 hours. 7.9 Collection of Metaphase Cells Cells will be collected approximately 20 hours after initiation of treatment (about 64-68 hours after culture initiation). This time is selected to represent the firstdivision metaphase after initiation of test substance treatment. Two hours prior to harvest, Colcemid will be added to the cultures at a final concentration of 0.1 pg/ml. The cells will be collected by centrifugation, treated with 0.075M KC1, washed with two changes of fixative (methanol:glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at approximately 2-8C. To prepare slides, the cells will be collected by centrifugation and resuspended in fresh fixative. An aliquot of fixed cells will be applied dropwise onto a microscope slide and air-dried. The slide will be identified by the experiment number, treatment condition and date. At least two slides will be prepared from each treatment tube. The slides will be stained with Giemsa and permanently mounted. 7.10 Scoring for Metaphase Aberrations Slides will be coded using random numbers by an individual not involved with the scoring process. At least 3 dose levels in each harvest will be evaluated in the aberration assay and will be selected according to the criteria described in section 7.2. The 4 hour non-activated and S9-activated studies will be scored initially. In the event of a positive response in 4 hour non-activated study, slides from the extended non-activated exposure may not be scored. Metaphase cells will be examined under oil immersion without prior knowledge of treatment groups. Protocol <> August 28, 2000 Page 5 of 9 103 Sponsor Project Number:___________ Whenever possible, a minimum of 200 metaphase spreads containing 46 centromeres from each dose level (100 per duplicate treatment tube) will be examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that will be examined and scored per duplicate flask may be reduced if the percentage of aberrant cells reaches a statistically significant level before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure will be scored as a break (chromatid or chromosome). Fragments observed with an exchange figure will not be scored as an aberration but will be considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (>10 aberrations) will also be recorded. Chromatid and isochromatid gaps will be recorded but not included in the analysis. The XY coordinates for each cell with a structural aberration or gap will be recorded using a calibrated microscope stage. The mitotic index will be recorded as the percentage of cells in mitosis per 500 cells counted. The percent polyploid and endoreduplicated cells will be evaluated per 100 metaphase cells for each dose level analyzed for structural aberrations. 8.0 CRITERIA FOR DETERMINATION OF A VALIDTEST 8.1 Solvent Controls The frequency of cells with structural chromosome aberrations in the solvent controls must be within the range of the historical solvent control. 8.2 Positive Controls The percentage of cells with aberrationsmust be statistically increased (p<0.05, Fisher's exact test) in the positive control relative to the solvent control. 9.0 EVALUATION OF TEST RESULTS The toxic effects of treatment are based upon inhibition of mitosis and will be reported for the cytotoxicity and chromosome aberration study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) will be calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell. Statistical analysis of the percentage of aberrant cells will be performed using the Fisher's exact test. The Fisher's test will be used to compare pairwise the percent aberrant cells of Protocol <> August 28, 2000 Page 6 of 9 lOlf- Sponsor Project Number: each treatment group with that of the solvent control. In the event of a positive Fisher's exact test at any test substance dose level, the Cochran-Armitage test will be used to measure dose-responsiveness. All conclusions will be based on sound scientific judgement; however, as a guide to interpretation of the data, the test substance will be considered to induce a positive response if the percent aberrant cells is increased in a dose-responsive manner with one or more concentrations being statistically significant (p<0.05). A reproducible significant increase at the high dose only with no dose response or a reproducible significant increase at one dose level other than the high dose with no dose response will be considered positive. Test substances not demonstrating a statistically significant increase in aberrations will be concluded to be negative. 10.0 REPORT A report of the results of this study will be prepared by the Testing Laboratory and will accurately describe all methods used for generation and analysis of the data. Results presented will include, but not be limited to: Test substance: identification and CAS no., if known; physical nature and purity, if known; physicochemical properties relevant to the conduct of the study, if known; stability of test substance, if known. Solvent/Vehicle: justification for choice of vehicle; solubility and stability of test substance in solvent/vehicle, if known. source of cells and time the cells were obtained, karyotype features (modal chromosome number) and suitability of the cell type used. test conditions: composition of medium; CO2 concentration; incubation time; solvent and solvent selection rationale; concentration of test substance and concentration selection rationale; composition and acceptability criteria for the metabolic activation (S9) system; duration of treatment; duration of treatment with and concentration of Colcemid; type of metabolic activation system used; positive and solvent controls; methods of slide preparation; number of cell cultures; criteria for scoring aberrations and criteria for considering studies positive, negative or equivocal. results: description of precipitation; pH and osmolality of the treatment medium; mitotic inhibition relative to the solvent control; mitotic index and number of metaphases analyzed; type and number of aberrations (structural and numerical) given separately for each treated and control culture; concentration-response relationship; statistical analysis; historical control data 11.0 RECORDS AND ARCHIVES All raw data, the protocol and all reports will be maintained according to Standard Operating Procedure by the cfacility, address>. Protocol <> August 28, 2000 Page 7 of 9 Sponsor Project Number: 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply with OECD Guideline 473 for Testing of Chemicals (Genetic Toxicology: In V itro Mammalian Chromosome Aberration Test), adopted 1997, and with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997). This study will be performed in compliance with the provisions of the EPA (TSCA), OECD, Japanese (MAFF) and EEC Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. Unless arrangements are made to the contrary, unused dosing solutions will be disposed of following administration to the test system and all residual test substance will be disposed of following finalization of the report. 13.0 REFERENCES Evans, H.J. and M.L. ORiordan. 1975. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Res. 31:135-148. Galloway, S.M., M.J. Aardema, M. Ishidate Jr., J.L. Ivett, D.J. Kirkland, T. Morita, P. Mosesso and T. Sofuni (1994) Report from working group on in vitro tests for chromosomal aberrations, Mutation Research 312(3):241-261. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 of the ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24, 1996. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21, 1997. OECD Guideline for the Testing of Chemicals, Guideline 473 (In V itro Mammalian Chromosome Aberration Test), Revised Draft Document, Adopted July 1997. Preston, R.J., W. Au, M.A. Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F. McFee, S. Wolff and J.S. Wassom. 1981. Mammalian in v iv o and in vitro cytogenetics assays: A report of the US EPA's Gene-Tox Program. Mutation Res. 87:143-188. Scott, D., N.D. Danford, B.J. Dean and D.J. Kirkland. 1990. Metaphase Chromosome Aberration Assays In Vitro. In: Basic Mutagenicity Tests: UKEMS Recommended Protocol <> August 28, 2000 Page 8 of 9 106 Sponsor Project Number:___________ Procedures. D.J Kirkland (ed). Cambridge University Press, New York, NY. Swierenga S.H.H., J.A. Heddle, E.A. Sigal, J.P.W. Gilman, R.L. Brillinger, G.R. Douglas and E.R. Nestmann (1991) Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese lung and human lymphocyte cultures, Mutation Research 246:301-322. 14.0 APPROVAL Sponsor Representative (Print or Type Name) Study Director Study Management Date Date Date Protocol <> August 28, 2000 Page 9 of 9 107 Sponsor Project Number: Test Substance Name: Bacterial Reverse Mutation Test with an Independent Repeat Assay 1.0 PURPOSE The purpose of this study is to evaluate the mutagenic potential of the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA in the presence and absence of S9 activation. 2.0 SPONSOR 2.1 Name: <> 2.2 Address: <> 2.3 Representative: <> 2.4 Sponsor Project No.: <> IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance Name: <> 3.2 Test Substance I.D.: <> (to be used in the report title and text) 3.3 Controls: Negative: Positive: Test substance vehicle 9-aminoacridine 2-aminoanthracene methyl methanesulfonate 2-nitrofluorene sodium azide 3.4 Test Substance Characterization Unless alternate arrangements are made, the testing facility will not perform analysis of the dosing solutions. The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition of the test substance, and the stability and strength of the test substance in the solvent (or vehicle). Protocol <> August 28, 2000 Page 1 of 10 C ontain N O C B i lOg Sponsor Project Number: 3.5 Test Substance Retention Sample The retention of a reserve sample of the test substance will be the responsibility of the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL Name: <> Address: <> Study Director: <> 5.0 PROPOSED STUDY DATES 5.1 Experimental Start Date: <> 5.2 Experimental Termination Date: <> 5.3 Draft Report Date: <> 5.4 Final Report Date: <> 6.0 TEST SYSTEM The tester strains will include the S. typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and the E. coli tester strain WP2 uvrA as described by Green and Muriel (1976).______________________________ Genotype of the Strains Used for Mutagen Testing Histidine Mutation Tryptophan Mutation Additional Mutations hisG46 hisC3076 HisD3052 TrpE LPS Repair R-factor TA1535 TA 100 TA1537 - TA98 - Rfa AuvrB Rfa AuvrB +R - - - WP2 uvrA - AuvrA - Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98 and TA100 also contain the pKMIOl plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. Protocol <> August 28, 2000 Page 2 of 10 Sponsor Project Number: TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions. The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2 uvrA has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base-pair substitution mutations (Green and Muriel, 1976). The S. typhimurium tester strains were received directly from Dr. Bruce Ames, University of California, Berkeley. The E. coli tester strain was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland (United Kingdom). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The test substance will be tested at a minimum of five dose levels along with appropriate negative and positive controls with tester strains TA98, TA100, TA1535, TA1537 and WP2 uvrA with and without S9 activation. All dose levels of test substance, negative controls and positive controls will be plated in triplicate. 7.1 Solubility Determination A solubility determination will be conducted to determine the maximum soluble concentration or workable suspension up to a maximum of 500 mg/mL. Vehicles compatible with this test system, in order of preference, include but are not limited to: deionized water (CAS 7732-18-5), dimethyl sulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5) and acetone (CAS 67-64-1). The vehicle of choice will be the solvent, selected in order of preference, which permits preparation of the highest workable/soluble stock concentration, up to 500 mg/mL. 7.2 Preliminary Toxicity Assay to Select Dose Levels Selection of dose levels for the mutagenicity assay will be based upon the toxicity and precipitation profile of the test substance assessed in a preliminary toxicity assay. This preliminary assay will be conducted by exposing TA98, TA100, TA1535, TA1537 and WP2 u v rA to negative controls and to at least eight concentrations of test substance, one plate per dose level, in both the presence and absence of S9 activation. Unless indicated otherwise by the Sponsor, the highest dose will be the highest workable concentration in the vehicle of choice but not to exceed 5 mg/plate. In selecting dose levels for the mutagenicity assay the following guidelines will be employed. Doses will be selected such that precipitate Protocol <> August 28, 2000 Page 3 of 10 HO Sponsor Project Number: does not interfere with manual scoring. Whenever possible, the highest dose for the mutagenicity assay will be selected to give some indication of toxicity without exceeding 5 mg/plate. For freely soluble, nontoxic test substances, the highest dose level will be 5 mg/plate. For precipitating, nontoxic test substances, the highest dose level will be selected in an attempt to yield precipitate at only the top one or two dose levels. The Sponsor will be consulted regarding dose selection if (1) the maximum dose level is selected based on precipitation and this dose level is less than 5 mg/plate or (2) the maximum achievable test substance dose level is less than 5 mg/plate and this dose level is nontoxic. 7.3 Frequency and Route of Administration The test system will be exposed to the test substance via the plate incorporation methodology originally described by Ames e t al. (1975) and updated by Maron and Ames (1983). This test system has been shown to detect a wide range of classes of chemical mutagens (McCann e t a l , 1975; McCann and Ames, 1976). After the data generated in the first assay have been evaluated, the mutagenicity assay will be repeated. The dose levels used in the second assay will be the same as those used in the first assay unless the Study Director determines that the dose levels should be changed due to an equivocal response, excessive cytotoxicity or excessive precipitate. If the Sponsor is aware of specific metabolic requirements (e.g., azo compounds), this information will be utilized in designing the assay, (e.g., activation system or treatment method). This guidance is based on the OECD Guideline 471 (adopted July 1997) and ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997). 7.4 Controls 7.4.1 Positive Controls All combinations of positive controls and tester strains plated concurrently with the assay are listed below: Protocol <> August 28, 2000 Page 4 of 10 III Sponsor Project Number: Strain Salmonella Strains WP2 uvrA TA98 TA100, TA1535 TA1537 WP2 uvrA S9 Activation Rat None Positive Control 2-aminoanthracene 2-nitrofluorene sodium azide 9-aminoacridine methyl methanesulfonate Concentration (pg/plate) 1.0 10 1.0 1.0 75 1,000 7.4.2 Negative Controls Appropriate negative controls will be plated for each tester strain with and without S9 activation. The negative control will be the vehicle alone, unless there is no historical basis for use of the selected vehicle. In the latter case, both untreated and vehicle controls will be used. 7.4.3 Sterility Controls The most concentrated test substance dilution and the Sham and S9 mixes will be checked for sterility. 7.5 Exogenous Metabolic Activation Aroclor 1254-induced rat liver S9 will be used as the metabolic activation system. The S9 homogenate will be prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 will be batch prepared and stored frozen at approximately -70C until used. Each batch of S9 homogenate will be assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenzanthracene to forms mutagenic to S. typ h im u riu m TA100. Immediately prior to use, the S9 will be thawed and mixed with a cofactor pool to contain 10% S9 homogenate, 5 mM glucose-6-phosphate, 4 mM (3-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCb and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. This mixture is referred to as S9 mix. Sham mix will be 100 mM phosphate buffer at pH 7.4. 7.6 Preparation of Tester Strains Overnight cultures will be inoculated from the appropriate master plate or from the appropriate frozen stock. To ensure that cultures are harvested in late log phase, the length of incubation will be controlled and monitored. At the end of the working day, each inoculated flask will be placed in a resting shaker/incubator at room temperature. The shaker/incubator will be programmed to begin shaking at Protocol <> August 28, 2000 Page 5 of 10 U3j Sponsor Project Number: approximately 125 rpm at 372C approximately 12 hours before the anticipated time of harvest. All cultures will be harvested by spectrophotometric monitoring of culture turbidity rather than by duration of incubation since overgrowth of cultures can cause loss of sensitivity to some mutagens. Cultures will be removed from incubation at a density of approximately 109 cells/mL. 7.7 Test System Identification Each plate will be labeled with a code system that identifies the test substance, test phase, dose level, tester strain and activation type as described in standard operating procedures. 7.8 Test Substance Preparation Unless specified otherwise, test substance dilutions will be prepared immediately prior to use. All test substance dosing will be at room temperature under yellow light. 7.9 Treatment of Test System One half milliliter (0.5 mL) of S9 mix or Sham mix, 100 pL of tester strain and 50 pL of vehicle, test substance dilution or positive control will be added to 2.0 mL of molten selective top agar at 45 2C. When necessary to achieve the target concentration or eliminate toxic vehicle effects, aliquots of other than 50 pL of test substance/vehicle/positive control will be plated. The mixture will be vortex mixed and overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay has solidified, the plates will be inverted and incubated for approximately 48 to 72 hours at 37 2C. Plates that are not counted immediately following the incubation period will be stored at 2 - 8C. 7.10 Scoring The condition of the bacterial background lawn will be evaluated for evidence of test substance toxicity and precipitate. Evidence of toxicity will be scored relative to the negative control plate and recorded along with the revertant count for that plate. Toxicity will be evaluated as a decrease in the number of revertant colonies per plate and/or a thinning or disappearance of the bacterial background lawn. Precipitation will be evaluated after the incubation period by visual examination without magnification. 7.11 Tester Strain Verification On the day of use in the mutagenicity assay, all tester strain cultures will be checked for the appropriate genetic markers cited in 6.0. Protocol <> August 28, 2000 Page 6 of 10 //3 Sponsor Project Number: 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST The following criteria must be met for the mutagenicity assay to be considered valid: 8.1 Tester Strain Integrity To demonstrate the presence of the rfa mutation, all S. typh im u riu m tester strain cultures must exhibit sensitivity to crystal violet. To demonstrate the presence of the u vrB mutation, all S. typ h im u riu m tester strain cultures must exhibit sensitivity to ultraviolet light. To demonstrate the presence of the u v rA mutation, all E. c o li tester strain cultures must exhibit sensitivity to ultraviolet light. To demonstrate the presence of the pKMIOl plasmid R-factor, tester strain cultures of TA98 and TA100 must exhibit resistance to ampicillin. 8.2 Spontaneous Revertant Background Frequency Based on historical control data, all tester strain cultures must exhibit characteristic number of spontaneous revertants per plate in the negative controls (vehicle). The mean revertants per plate must be within the following ranges (inclusive): TA98, 10-50; TA100, 80 - 240; TA1535, 5 -45; TA1537, 3 -21; WP2 u v rA , 10 - 60. 8.3 Tester Strain Titers To ensure that appropriate numbers of bacteria are plated, all tester strain culture titers must be equal to or greater than 0.3xl09 cells per milliliter. 8.4 Positive Control Values Each mean positive control value must exhibit at least a three fold increase over the respective mean negative control value (vehicle) for each tester strain. 8.5 Toxicity A minimum of three non-toxic dose levels will be required to evaluate assay data. A dose level is considered toxic if it causes a >50% reduction in the mean number of revertants per plate relative to the mean negative control value (this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in the background lawn. In the event that less than three non-toxic dose levels are achieved, the affected portion of the assay will be repeated with an appropriate change in dose levels. 9.0 EVALUATION OF TEST RESULTS For a test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below: Protocol <> August 28, 2000 Page 7 of 10 Sponsor Project Number: 9.1 Strains TA1535 and TA1537 Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean negative control value (vehicle). 9.2 Strains TA98, TA100 and WP2 u v rA Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean negative control value (vehicle). 10.0 REPORT A report of the results of this study will be prepared by the Testing Laboratory and will accurately describe all methods used for generation and analysis of the data. The report will include: Test Substance: identification and CAS no., if known; physical nature and purity, if known; physicochemical properties relevant to the conduct of the study, if known; stability of test substance, if known. Solvent/Vehicle: justification for choice of vehicle; solubility and stability of test substance in solvent/vehicle, if known. Strains: strains used; number of cells/mL per culture; strain characteristics. Test conditions: amount of test substance per plate with rationale for dose selection and number of plates per concentration; media used; type and composition of metabolic activation system, including acceptability criteria; treatment procedures. Results: signs of toxicity; signs of precipitation; individual plate counts; the mean number of revertant colonies per plate and standard deviation; dose-response relationship, where possible; statistical analysis, if any; concurrent negative and positive control data means and standard deviations; historical negative and positive control data with ranges, means and standard deviation. Discussion of results. Conclusion. 11.0 RECORDS AND ARCHIVES All raw data, the protocol and all reports will be maintained according to Standard Operating Procedure by the <facility name, address>. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply with OECD Guideline 471 for Testing of Chemicals (Genetic Toxicology: Bacterial Reverse Mutation Assay), adopted 1997 and Protocol <> August 28, 2000 Page 8 of 10 Sponsor Project Number: with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997). This study will be performed in compliance with the provisions of the EPA (TSCA), OECD, Japanese (MAFF) and EEC Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. Unless arrangements are made to the contrary, unused dosing solutions will be disposed of following administration to the test system and all residual test substance will be disposed of following finalization of the report. 13.0 REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the 5a/mone//a/mammalian-microsome mutagenicity test. Mutation Research 31:347-364. Green, M.H.L., and Muriel, W.J. (1976). Mutagen testing using trp+ reversion in E s c h e r ic h ia c o li. Mutation Research 38:3-32. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 of the ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24, 1996. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21, 1997. McCann, J. and Ames, B.N. (1976). Detection of carcinogens as mutagens in the S a lm o n e lla lm ic r o s o m t test: assay of 300 chemicals: discussion. Proc. Natl. Acad. Sci. USA 73:950-954. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975). Detection of carcinogens as mutagens in the S a lm o n e lla !microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. USA 72:5135-5139. Marn, D.M. and Ames, B.N. (1983). Revised Methods for the S a lm o n e lla Mutagenicity Test. Mutation Research 113:173-215. OECD Guideline 471 for Testing of Chemicals (Genetic Toxicology: Bacterial Reverse Mutation Assay), Adopted July 1997. Protocol <> August 28, 2000 Page 9 of 10 II & Sponsor Project Number: Wilcox, P., Naidoo, A., Wedd, D.J. and Gatehouse, D.G. (1990). Comparison of S a lm o n e lla ty p h im u riu m TA102 with E s c h e r ic h ia c o li WP2 tester strains. Mutagenesis 5:285-291. 14.0 APPROVAL Sponsor Representative Date (Print or Type Name) Study Director Date Study Management Date Protocol <> August 28, 2000 Page 10 of 10 H7 Title: Developmental Toxicity Study in Rats Title: Developmental Toxicity Study in Rats Protocol c-coonnttaaiin N O C B fi Page 1 of <> Ul / ?f Title: Developmental Toxicity Study in Rats TABLE OF CONTENTS Page OBJECTIVE...........................................................................................................................................3 SPONSOR AND TEST FACILITY................................................................................................... 3 MATERIALS AND METHODS........................................................................................................ 3 A. Test Substance................................................................................................................................. 3 B. Vehicle.............................................................................................................................................. 3 C. Anim als............................................................................................................................................ 3 D. Husbandry........................................................................................................................................4 E. Experimental D esign...................................................................................................................... 4 F. Selection of Dose Levels................................................................................................................5 G. Randomization................................................................................................................................ 5 H. Preparation, Administration, and Analysis of Test Suspensions............................................ 5 I. Safety Precautions and Disposal of Waste Material................................................................. 5 J. Animal Euthanasia..........................................................................................................................5 K. Parameters to be Studied................................................................................................................6 1. In-life Observations of Females............................................................................................. 6 2. Postmortem Observations of Females Dying Prior to Scheduled Euthanasia.................6 3. Postmortem Observations of Dams Delivering Early........................................................ 6 4. Postmortem Observations of Females Surviving to Scheduled Euthanasia (Day 21G).................................................................................................................................. 6 5. Fetuses of Females Surviving to Scheduled Euthanasia.................................................... 7 L. Control of B ias................................................................................................................................ 7 M. Statistics........................................................................................................................................... 7 N. Absorption of Test and Control Substances................................................................................8 O. Records and Sample Storage......................................................................................................... 8 CRITICAL DATES...............................................................................................................................8 REFERENCES...................................................................................................................................... 8 1132- - Title: Developmental Toxicity Study in Rats OBJECTIVE The purpose of this study is to evaluate the developmental toxicity of Title (Compound), administered by gavage to pregnant rats from the time of implantation to the end of gestation. This study will conform with applicable Good Laboratory Practice Standards'1'4' and Test G u id e lin e s/5'8' SPONSOR AND TEST FACILITY This study is sponsored by <sponsor name> and will be conducted at laboratory, locations Sponsor approval of the study will be indicated by issuance of the work authorization form. MATERIALS AND METHODS A. Test Substance The test substance will be identified in this protocol and in the study records by Compound or by the Sample Number. The test substance will be supplied by <sponsor>. <Sponsor> is responsible for characterization of the test substance including the items cited in CFR 40, Part 792, Subpart F, section 792.105(a). Available information on the purity, composition, contaminants, synonyms, CAS registry number, basic physical properties, hazards, and hazardous material classification(s) will be provided by <sponsor> on the sample evaluation form. The original form will be retained in the official file for the study. B. Vehicle The vehicle will be 0.5% methyl cellulose, U.S.P., 4000 centipoises, C.A.S. Reg. No. 9004-67-5. C. Animals The Crl:CD(SD)IGS BR rat was selected for this study because it is a preferred species for developmental toxicity testing as recommended by the guidelines. The Crl:CD(SD)IGS BR strain was chosen because extensive background information is available from the literature, the supplier, and previous studies with other compounds. This strain is also considered suitable relative to hardiness and incidence of spontaneous disease. One hundred thirty nulliparous, time-mated females, will be received on <> and <>, <year> from Charles River Laboratories, Inc. Body weights on the day the rats are mated, day 0 of gestation (day 0G) will be supplied by the vendor. The rats for this study will be requested to be at either 1, 2, or 3 days of gestation upon receipt, and between 51 to 70 days old. Each animal is assigned a unique number and identified with an ear punch/notch by the supplier prior to shipping. Upon receipt, each animal will be assigned a unique animal number. Both the animal number and the ear punch/notch number will be recorded on each cage card. A master list of unique animal numbers and corresponding unique ear punch/notch numbers will be maintained with the study records. \&0 Title: Developmental Toxicity Study in Rats D. Husbandry Animal rooms will be maintained at an acceptable temperature of 20-26C (targeted at 23C) and a targeted relative humidity of 50% 10%. Animal rooms will be artificially illuminated (fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours). Animals will be housed individually in suspended, wire-mesh, stainless steel cages. Nesting material will not be provided because the dams will be euthanized prior to parturition. PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 will be available a d lib itu m . Water from <> will be available a d lib itu m . o Laboratory has an animal health monitoring program. The following procedures are performed periodically: Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Food samples are analyzed for the presence of bacteria and fungi. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and to be free of impurities which might influence the results of the study. The animal health monitoring program is administered by the Laboratory Animal Veterinarian. Data are maintained separately from study records and will not be included in the final report unless warranted by the results of these evaluations. Rats will be quarantined for at least 3 days, and then released for the study upon approval of the Laboratory Animal Veterinarian or a designee. E. Experimental Design The experimental design is shown below: Group Dose3 (mg/kg/day) Concentration17 (mg/mL) Mated Females0 I 0d 0.0 25 II <> <> 25 III <> <> 25 IV <> <> 25 V <> <> 25 a Compound, administered once daily, by gavage, on days 6-20 o f gestation, at a dosage volume of <> mL/kg Ul4- - Title: Developmental Toxicity Study in Rats b To achieve these concentrations of active ingredient, the solutions will be adjusted for a purity of <> %. c Copulation confirmed d Vehicle only (0.5% methyl cellulose) F. Selection of Dose Levels The dose levels for the current study were based on <>. G. Randomization Dams will be assigned to lots according to their gestation day. Then, they will be ranked on the basis of day 0 gestation body weights and assigned to control and experimental groups by random sampling from the ranked list. The distribution should result in mean body weights for all groups that are not statistically different (p>0.05). Dams that lose excessive weight or appear ill prior to the start of dosing will be removed from the study. H. Preparation, Administration, and Analysis of Test Solutions Solutions of the test material in the vehicle will be prepared daily. The method of mixing the test material with the vehicle will be documented in the study records. Compound will be administered by gavage because the oral route is the route which is recommended by regulatory agencies for this type of study. The volume administered will be based on the most recent body weight. Samples (~3 mL each) of each test suspension will be taken three times during the study: near the beginning, the middle, and the end of the dosing period. Analysis of the first sampling will address homogeneity, concentration, and stability. For the second and third samplings, analyses will address concentration. Additional samples may be taken and/or analyzed at the discretion of the study director. Samples will be submitted, shortly after preparation, to the Analytical Group. On days samples are taken, the solutions remaining after dosing will be frozen for additional analysis, if required. Frozen solutions will be discarded if no additional analysis is necessary. The analytical method used will be documented in the study records. I. Safety Precautions and Disposal of Waste Material Good housekeeping procedures will be practiced to avoid potential health hazards and contamination of formulation facilities. To avoid skin contact, gloves will be worn when handling either the test substance or test solutions. In addition, the test substance will be handled in a chemical hood. Dosing solutions will be prepared in properly ventilated areas. Animal carcasses, feces, and unused dosing solutions will be incinerated. J. Animal Euthanasia Females will be euthanized by carbon dioxide inhalation. Fetuses will be decapitated before proceeding with visceral examinations; those to be viscerally examined without decapitation will be injected with sodium pentobarbital. All other fetuses also will be injected with sodium pentobarbital before fixation. 5 IZL Title: Developmental Toxicity Study in Rats K. Parameters to be Studied 1. In-life Observations of Females Body weights will be recorded at least twice prior to dosing to provide data for quarantine release, and on days 6-21G. Food consumption will be measured on days 4, 6, 8, 10, 12, 14, 16, 18, 20, and 21G. Clinical signs will be recorded at least twice prior to dosing to provide data for quarantine release; signs will be recorded once daily on days 4-21G and twice daily on days 6-20G. Clinical signs observed at other times will be recorded by exception. 2. Postmortem Observations of Females Dying Prior to Scheduled Euthanasia A gross external and visceral examination will be performed within 24 hours after the female is found dead. Rats found dead will be refrigerated until necropsied. Pregnancy status will be determined by the presence or absence of implantation sites. If possible, the sites will be counted and classified as apparently alive or resorbing at the time of the dam's death. 3. Postmortem Observations of Dams Delivering Early Dams that deliver before scheduled euthanasia will be euthanized within 24 hours of detection and a gross external and visceral examination will be performed. Lesions noted will be retained for further examination at the discretion of the study director or a designee. Corpora lutea counts and the number of visible implantation sites will be recorded. Fetuses remaining in the uterus and those delivered early will be examined to the extent possible, but the collected data will be recorded with the maternal gross postmortem findings so as to easily permit exclusion of the data from all calculations performed for live fetuses. 4. Postmortem Observations of Females Surviving to Scheduled Euthanasia (Day 21G) Viscera will be examined grossly immediately after euthanasia. Lesions noted will be retained for further examination at the discretion of the study director or a designee. The intact and the empty uterus of each dam having at least one viable fetus will be weighed to permit calculation of maternal body weight adjusted to exclude the products of conception. The corpora lutea count for each ovary of females with viable fetuses will be recorded. For each female with visible implantation sites, the types of implants (live and dead fetuses, and resorptions) and their relative positions will be recorded. The uterus of each apparently "nonpregnant" female will be stained with ammonium sulfide(9) to detect very early resorptions; counts will be recorded. Data from females determined to be pregnant by staining will be used only to calculate the incidence of pregnancy and the number of females with total resorptions. -6 - <s.b Title: Developmental Toxicity Study in Rats 5. Fetuses of Females Surviving to Scheduled Euthanasia The intrauterine location of each fetus will be recorded. The sex and body weight of each fetus will be recorded. The external alterations detected for each live fetus will be recorded. For each litter, the first live fetus and every other live fetus thereafter will be examined for visceral alterations.(10) Retarded renal development will be classified using the scheme of Woo and Hoar.<ll) In addition, all live fetuses with malformations visible at external examination will be examined for soft tissue alterations; decapitation of these fetuses will be at the discretion of the study director or a designee. After fixation in Bouin's fixative, the heads of decapitated fetuses will be examined and alterations will be recorded. Examinations will be based on the method of Barrow and Taylor.<12) After alcohol fixation, the alizarin-stained skeletons will be examined and skeletal alterations will be recorded for each live fetus. Those fetuses classified as dead will be examined externally, viscerally, and skeletally. Data for these fetuses will be excluded from all calculations performed for live fetuses. Fetal organs or tissues will be retained if deemed necessary for obtaining a definitive diagnosis. Bilateral organs will be retained when only one is affected. Sufficient tissues from control fetuses will be fixed to provide comparison with suspect tissues, if needed; tissues will be discarded if not needed. L. Control of Bias In addition to random assignment to groups, all females will be coded before scheduled euthanasia and will remain coded during the collection of postmortem and fetal data. M. Statistics Sequential two-tailed trend testing0 3>will be applied to the data for each parameter as tabulated below. If a significant dose-response is detected, data from the top dose group will be excluded and the test repeated until no significant trend is detected. For litter parameters, the proportion of affected fetuses per litter or the litter mean will be used as the experimental unit for statistical evaluation/l4) The level of significance selected is p < 0.05. Additional statistical tests may be used, and other parameters analyzed, if deemed necessary. Where the data are tied and the standard large sample version of Jonckheere's test is not applicable, exact p values will be calculated using permutation methodology,5) Parameter Maternal weight Maternal weight change Maternal food consumption Live fetuses Dead fetuses Trend Test Linear contrast of means06* Jonckheere's test07* -7 - Title: Developmental Toxicity Study in Rats Resorptions Implantations Incidence of fetal alterations Incidence of pregnancy Clinical observations Maternal mortality Females with total resorptions Early deliveries Cochran-Armitage test(16) Fetal weight (Covariates: litter size, Linear contrast of least square means(18) sex ratio) Sex ratio (Covariate: litter size)_____________________________________________ N. Absorption of Test and Control Substances Determination of the degree of absorption of the test and control substances by the test system is not necessary to achieve the objectives of the study. O, Records and Sample Storage Specimens (if applicable), raw data, and the final report will be retained at <>. CRITICAL DATES Study Start (first day 6G): <> Completion (last fetal evaluation): <> (approximate) Final Report: <> REFERENCES 1. Good Laboratory Practice Standards, U.S. Environmental Protection Agency (EPA), Federal Register 54, No. 158 (Toxic Substances Control; 40 CFR Part 792), pp 34043-34050 (1989). 2. U.S. Environmental Protection Agency (EPA), Good Laboratory Practice Standards (Federal Insecticide, Fungicide and Rodenticide Act), 40 CFR 160 (1989). 3. Organisation for Economic Cooperation and Development, Good Laboratory Practice Standards. ISBN 92-64-12367-9, Paris (1982). 4. Ministry of Agriculture, Forestry and Fisheries (MAFF), On Good Laboratory Practice Standards for Toxicological Studies on Agricultural Chemicals, 59 NohSan No. 3850, Tokyo, Japan (1984). 5. U.S. EPA Health Effects Test Guidelines OPPTS 870.3700 Prenatal Developmental Toxicity Study (1998). 8- - IS S Title: Developmental Toxicity Study in Rats 6. Organisation for Economic Cooperation and Development (OECD), Guidelines for Testing of Chemicals, Section 4, No. 414, Paris (1981). 7. Ministry of Agricultural, Forestry and Fisheries (MAFF), Guidelines on Toxicology Study Data for Application of Agricultural Chemical Registration, 59 NohSan No. 4200, Tokyo, Japan (1985). 8. Commission Directive 87/302/EEC (Official Journal of the European Communities No. L133, Vol. 31, 30.5.88). 9. Salewski, E. (1964). Farbemethode zum makreoskopischen Nachweis von Implantationstellen am Uterus der Ratte. A rc h iv . P a th . E xp. P h a rm a k o l. 247:367. 10. Staples, R.E. (1974). Detection of Visceral Alterations in Mammalian Fetuses. T e r a to lo g y 9(3):A37-A38. 11. Woo, D.C. and Hoar, R.M. (1972). Apparent Hydronephrosis as a Normal Aspect of Renal Development in Late Gestation of Rats: The Effect of Methyl Salicylate. T e r a to lo g y 6:191196. 12. Barrow, M.V. and Taylor, W.J. (1969). A Rapid Method for Detecting Malformations in Rat Fetuses." J. M o rp h . 127(3):291-306. 13. Selwyn, M.R. (1995). The Use of Trend Tests to Determine a No-Observable-Effect Level in Animal Safety Studies." J o u r n a l o f th e A m e r ic a n C o lle g e o f T o x ic o lo g y 14(2): 158-168. 14. Haseman, J.K. and Hogan, M.D. (1975). Selection of the Experimental Unit in Teratology Studies. T e r a to lo g y 1 2 :1 6 5 -1 1 \. 15. Patefield, W. (1982). Exact Tests for Trends in Ordered Contingency Tables. A p p lie d S ta tistic s 31:32-43. 16. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th ed. The Iowa State University Press, Iowa, pp. 246-248, 349-352. 17. Jonckheere, A.R. (1954). A Distribution-Free K-Sample Test Against Ordered Alternatives. B io m e tr ik a 41:133-145.18 18. Dempster, A.P., Selwyn, M.R., Patel, C.M., and Roth, A.J. (1984). Statistical and Computational Aspects of Mixed Model Analysis. The J o u r n a l o f th e R o y a l S ta tis tic a l S o ciety, S eries C (A p p lie d S ta tistic s) 33(2):203-214. US9- - Title: Developmental Toxicity Study in Rats Title: Developm ental Toxicity Study in Rats Protocol Prepared by: SD, SDDegree SDTitle Study Director Date Approved by: cc: <> Chemist <> <Director> Date Date -10- Modified Approach for Describing Telomer B Alcohol Kinetics ontain NO CB o N Y Refine and Validate Kinetic Model to Relate Exgosur^^ntemaMose Joins with "B " 2 19 Sep 2000 "B " 4 r i.v./oral doses H 3 Dose Levels 14C > Male and Female v. J 4 Joins with "C" ia.0 19 Sep 2000 N > Incorporate into Model ---------------------------------------------------------------------- v Quantitatively Define Metabolism v_____ ___________ J . Determine Extent, Target, Kinetics, Species Specific Binding____________ , Joins with "D " HO 19 Sep 2000 D ( ------------------A > Incorporate Into Model s_________ ________ * HI 19 Sep 2000 In V itro Microsomal Metabolism Study Objective: The objective of this study type is to determine the rate of metabolism of a test compound in microsomes prepared from livers of a number of species (normally rat, rabbit, dog, monkey, and human) and to identify the metabolites produced in each species. Methods: First, it is necessary to determine linearity of reaction rate with protein concentration. To do this, reactions are run with protein concentrations in the range of 0.5-3 mg/mL protein while holding the starting concentration and reaction time constant for each species. Reactions are carried out at 37 C. The reaction medium consists of an NADPH regeneration system, cofactors, microsomes, and phosphate buffer, pH 7.4. Test compound is added to the reaction medium to achieve a final concentration of 1-100 uM, determined by pilot studies. The reaction is started by the addition of NADP solution and incubated for a constant time, determined in pilot studies. The reaction is terminated by the addition of either organic solvent or strong acid. The test samples are then partially purified by centrifugation, solid phase extraction, or both. The samples are then analyzed by HPLC-MS. Next, the kinetics of the test compound are determined. To do this, reactions are run at a constant initial concentration of test substance and a constant protein concentration. Reactions are carried out at 37 C. The reaction medium consists of an NADPH regeneration system, cofactors, microsomes, and phosphate buffer, pH 7.4. Test compound is added to the reaction medium to achieve a final concentration of 1-100 uM, determined by pilot studies. The reaction is started by the addition of NADP solution and the reactions are terminated at various time points, determined in pilot studies. The reaction is terminated by the addition of either organic solvent or strong acid. The test samples are then partially purified by centrifugation, solid phase extraction, or both. The samples are then analyzed by HPLC-MS. The concentration of each unknown sample is calculated using a standard curve. The resulting concentrations (in units of pM) are plotted on a semi-logarithimic plot against time in minutes. An exponential regression line with the general equation y = cebx where c is the y intercept and b is the first order rate constant for disappearance of the test compound is fitted to the data. In v itr o half -life was calculated as ti/2 = 0.693/b. Intrinsic clearance (CLjnt) was calculated by two different methods. The first method requires dividing the initial amount of test compound (in nmol) by the total area under the curve (AUC, in nmolmin/mL) from a plot of concentration versus time. The quotient is then divided by the total amount of protein (in mg) in the reaction vial, giving CLmt in units of mL/min/mg protein. Multiplying this value by the grams of liver weight per kg of body weight and mg of protein per gram of liver gives CLnt in units of mL/min/kg. The second method of calculating CLjnt involves dividing the first order rate constant by the protein concentration in units of mg/mL, which gives CLjnt in terms of mL/min/mg protein. As in method 1, multiplying this value by the grams of liver weight per gram of body weight and mg of protein per gram of liver gives CLint in units of mL/min/kg. The CLint values were further compared with estimates of hepatic blood flow to allow some inferences to be made regarding first pass elimination effects in the various species. Metabolites are identified by HPLC-MS, and their identity is confirmed with authentic standards, if available. Metabolism of Telomer B by Mammalian Hpatocytes P r in c ip le : Isolated hepatocyte suspensions provide a high capacity in v itr o metabolic system of somewhat greater biological complexity compared to sub-cellular fractions. Hepatocytes express all of the enzymes and cofactors associated with normal hepatic chemical metabolism and generally provide a realistic approximation of a given chemical's in v iv o metabolite profile. T e s t S p e c ie s : Hepatocytes from males and females of the following species will be used for Telomer B metabolism studies: Sprague-Dawley Rat CD-I Mouse New Zealand White Rabbit Cynomolgus Monkey Beagle Dog Human Hepatocytes from rodent species are prepared in house by the two-stage collagenase perfusion method. Cryopreserved non-rodent hepatocytes are available commercially. S tu d y D e s ig n : The basic design for a hepatocyte metabolism experiment for a given species/sex is illustrated in the following table: Group Role 1 Experimental 2 Pos. Control 3 Neg. Control 4 Neg. Control * Heat inactivated N 5 5 5 5 Cells (x 106) Substrate 5 Telomer B 5 Ethoxycoumarin 5 Vehicle 5* Telomer B Group 2 assures the metabolic activity of the hepatocyte suspensions, Group 3 provides an analytical background sample and Group 4 controls for non-enzymatic degradation of the test compound. Hepatocytes are suspended in Liebovitz L-15 medium at a concentration of approximately 1 x 106 cells/mL. Reactions are incubated at 37 C for 2 hrs, then terminated by cooling, acidification, or addition of organic solvent as appropriate. Following sample clean-up, extracts are analyzed by LC/MS for parent and metabolites. DRAFT PROTOCOL Toxicokinetics of Telomer B Alcohol in the Rat Project Number: DuPont-<> Work Request Number: <> Service Code Number: <> DuPont Animal Welfare Committee No. <> Page 1 o f 15 \vr Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat Table of Contents Toxicokinetics of Telomer B Alcohol in the Rat.................................................................................... 1 Introduction.................................................................................................................................................. 3 Objectives......................................................................................................................................................3 Sponsor and Test Facility........................................................................................................................... 3 Regulatory Test Guidelines........................................................................................................................ 3 Materials and Methods................................................................................................................................4 A. Test Substance.................................................................................................................................4 B. Radiolabeled Test Substances...................................................................................................... 5 C. Test Species..................................................................................................................................... 5 D. Animal Husbandry......................................................................................................................... 5 E. Animal Health Monitoring............................................................................................................ 6 F. Dose Preparation, Analysis and Rates......................................................................................... 6 G. Sacrifice............................................................................................................................................7 H. Pilot Studies..................................................................................................................................... 7 1. Plasma kinetics following oral dosing with non-radiolabeled TBA ...................................7 2. Excretion and metabolism following oral dosing with radiolabeled TBA........................ 7 I. Main Study...................................................................................................................................... 8 1. Absorption Experiments............................................................................................................ 8 2. Distribution Experiments......................................................................................................... 9 3. Excretion and Metabolism Experiments...............................................................................10 J. Quantitation of Radioactive Residues........................................................................................12 1. Plasm a........................................................................................................................................12 2. Urine...........................................................................................................................................12 3. Feces........................................................................................................................................... 12 4. B ile ............................................................................................................................................. 12 5. Cage rinses................................................................................................................................ 12 6. Feed residue...............................................................................................................................12 7. Carcass........................................................................................................................................12 8. Samples from dermal experiments.........................................................................................13 K. Material Balance............................................................................................................................13 L. Clinical Observationsand Mortality........................................................................................... 13 M. Statistical Analyses....................................................................................................................... 13 N. Measurement of Radioactivity....................................................................................................13 Safety and Housekeeping......................................................................................................................... 14 Records and Sample Storage....................................................................................................................14 Critical Dates.............................................................................................................................................. 14 References................................................................................................................................................... 15 Signatures.................................................................................................................................................... 15 / 2>6 -2 - Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat In t r o d u c t io n Telomer B alcohol (TBA) is a fluorinated alcohol used in the manufacture of repellents and surfactants. TBA is similar in structure to perfluorooctanoic acid (PFOA), an 8-carbon, straight chain fatty acid analogue in which the aliphatic carbons are saturated with fluorine. In metabolism studies with PFOA, excretion was found to be more rapid in female than male rats /1 \ following oral dosing. This sex difference was apparently not due to differential metabolism of PFOA, as only parent was detected in excreta and tissues, but rather to the presence of testosterone, which was correlated with a reduction in the elimination rate of PFOA in male rats. Binding to a male-specific testosterone-inducible protein in serum or tissue may explain the excretion differences observed; female rats with less PFOA binding protein apparently have increased renal elimination, via the organic acid transport system, which is active in females but not male rats.(2) The mechanism to explain the observed sex differences may also be structure-activity based. Perfluorodecanoic acid (PFDA), a 10-carbon perfluorinated chemical, was eliminated more slowly than the shorter 8-carbon PFOA in female compared to male rats, and lacks the observed sex differences in renal excretion and toxicity.(3,4) O b je c t iv e s The objectives of this study are to determine the absorption, distribution, excretion and metabolism of TBA in male and female rats following oral and dermal dosing. Spo nsor and T est F acility This study is sponsored by the Telomer Research Group. The sponsor's approval was effective the date the sponsor authorized the work on the Work Authorization Form. The study will be conducted at Haskell Laboratory for Toxicology and Industrial Medicine, E. I. du Pont de Nemours and Company, Newark, Delaware, in accordance with all applicable Good Laboratory Practice Standards/5'6,7* Areas of noncompliance will be documented in the final report. R eg ulatory T est G uidelines This study has been designed to meet the testing requirements of (1) OECD Guidelines for Testing of Chemicals, Toxicokinetics, 417 (April 1984), and (2) US EPA Health Effects Test Guidelines, OPPTS 870.7485, Metabolism and Pharmacokinetics. 137 -3- Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat M aterials and M ethods A. Test Substance The test substance, Telomer B alcohol (TBA), and an analytical standard of TBA of known purity will be supplied by the sponsor and will be assigned a Haskell Laboratory Number, which will be documented in the study records. CAS Registry Number: Molecular Weight: Molecular Formula: Structure: <> 464 C 1 0 H 5 f 17o F F Wv OH Purity: fV v f >95% HP 4- - Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat B. Radiolabeled Test Substances Specific Activity: To be determined Radiochemical Purity: >95% [1-C14] Telomer B alcohol [10-C14] Telomer B alcohol [*] denotes the position of the radiolabel C. Test Species Male and female Crl:CD(SD)IGS BR rats will be used and obtained from Charles River Laboratories, Inc., Raleigh, North Carolina. The rat was selected for use in the study, as it is the principal toxicology animal model. The Sprague-Dawley rat has been used for toxicokinetic testing of other fluorinated test materials.(1,3) At the time of dosing, rats should be sexually mature and the weight variation should not exceed 20% of the mean weight by dose group. Each animal will be assigned a unique identification number to be used throughout the study. The last 3 digits of the animal identification number will be marked on the tail of each animal in indelible ink. D. Animal Husbandry Upon arrival at Haskell Laboratory, rats will be removed from shipping cartons and housed in appropriate cages, according to Standard Operating Procedures LA003-P-006 unless specified in the study records. Animals will be maintained under quarantine for at least six days unless approved otherwise by the site veterinarian, have three recorded weight gains and no abnormalities detected. After the quarantine period, rats will be selected for study. \2d____________________________________________________________ ___________________ -5- Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat Animal rooms are targeted at a temperature of 231C and a relative humidity of 40-60%. Animal rooms are artificially illuminated (fluorescent light) on a 12-hour light/dark cycle. Throughout the dosing period with test compound, the rats will be housed individually in appropriate cages according to the SOP. PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 and tap water will be provided a d lib itu m . Animals will not be fasted before dosing with test substance. E. Animal Health Monitoring Haskell Laboratory has an animal health monitoring program. The following procedures are performed periodically to assure that contaminants are not present at levels that would be expected to impact the scientific integrity of the study. Water samples are analyzed for total bacterial counts and the presence of coliforms, lead, and other contaminants. Feed samples are analyzed for the presence of bacteria and fungi. Samples from freshly washed cages and cage racks are analyzed to assure adequate sanitation by the cagewashers. Haskell Laboratory uses certified animal feed. The feed is guaranteed by the manufacturer to meet specified nutritional requirements and to be free of a list of specified contaminants. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and to be free of impurities which might influence the results of the study. The animal health monitoring program is administered by the laboratory animal veterinarian. Data are maintained separately from study records and will not be included in the final report at the discretion of the study director. F. Dose Preparation, Analysis and Rates Telomer B alcohol and/or radiolabeled TBA will be mixed with an appropriate dosing vehicle. Water or other solvent may be added as required to achieve the target low and high dose concentrations of Telomer B alcohol. Dose preparations for the single dose experiments may be prepared on the day of use or prior to the day of use. Dose solution for the repeat dose experiments may be prepared in bulk, stored appropriately, and used as needed. HPLC-MS or another suitable analytical method will be used to analyze dose solutions for chemical concentration. Dose solutions containing radiolabeled TBA will be assayed in triplicate for radioactive concentration by liquid scintillation counting (LSC), and radiochemical purity will be determined by appropriate mean using radiometric detection. \W -6- Draft Protocol WR XXXX Toxicokinetics o fTelomer B Alcohol in the rat Oral doses will be administered at a rate of 4 mL/kg. Dermal doses will be administered at a rate of 10 (xL/cm2. G. Sacrifice Rats will be sacrificed by CO2 asphyxiation and exsanguinated by cardiac puncture. H. Pilot Studies The following pilot studies will be conducted to determine the plasma kinetics of TBA and to resolve how TBA is eliminated following oral administration. The data from these experiments may be useful in setting the whole blood serial collection time-points, determining the principal routes and rates of elimination, and establishing the need for one or two radiolabeled forms of TBA to describe elimination/metabolism. 1. Plasma kinetics following oral dosing with non-radiolabeled TBA Two male and two female rats will have cannula surgically implanted in the jugular vein and allowed to recover for at least one day before dosing. Rats will be administered the test compound (non-radiolabeled) by the oral route and housed in suspended wire-mesh cages. Following dosing, serial blood samples will be drawn from the jugular vein cannula and placed into Microtainer tubes containing EDTA. Serial sampling time-points will be documented in the study record. Alternatively, whole blood may be collected from the tail vein. Whole blood samples will be maintained on wet ice. Plasma will be separated from red cells by centrifugation. Plasma will be stored frozen at approximately -15C until processed for analysis. 2. Excretion and metabolism following oral dosing with radiolabeled TBA In separate experiments for each radiolabeled form of TBA, two male and two female rats will be administered the test material formulated with radiolabeled TBA at the high dose level by oral gavage. Following dosing, rats will be housed individually in glass metabolism units suitable for the collection of urine, feces, expired air and volatile organics. Urine and feces will be collected (on dry ice) at approximately 12 hours and every 24 hours after dosing until sacrifice. Expired air will be passed through two scrubbing towers, one containing 2 N NaOH and the second containing ethylene glycol or other suitable solvent. Scrubbing towers will be replaced every 24 hours, post-dose, until sacrifice. Animals will be sacrificed after at least 95% of administered radioactivity has been excreted or after 7 days post-dosing, whichever takes place first. Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat The following tissues will be collected at sacrifice: Blood Kidney Lung Bone G.I. tract and contents Fat Muscle Testes Brain Adrenals Liver heart Ovaries/uterus Spleen Skin sample After collection, tissue samples will be stored at approximately -15C until processing and analysis. The remaining carcass will be homogenized and assayed so that total material balance can be determined. If less than 2% of the total dose is eliminated during the duration of the study as either 14C02 or volatile organics, collection of expired air will not be performed in the main study experiments. If greater than 20% of the dose is eliminated via the feces, bile will be collected in the main study experiments. If results suggest that either radiolabeled form of TB A will suffice in establishing the excretion and metabolism of TBA, one form will be selected and used in the main study distribution, excretion and metabolism experiments. I. Main Study 1. Absorption Experiments The principal objective of the absorption experiments is to determine the rate and extent of absorption of TBA following oral and dermal dosing using plasma kinetics. Dose Routes and Frequency The absorption experiments will be comprised of five dose groups. Low dose, single oral gavage High dose, single oral gavage Low dose, single 8-hour dermal exposure High dose, single 8-hour dermal exposure Low dose, intravenous Each dose group will be composed of 4 male and 4 female rats. Each dose group will receive non-radiolabeled test compound. The intravenous dose will provide a reference for estimating bioavailability of TBA by the oral and dermal exposure routes. )//-# -8 - Draft Protocol WR XXXX Toxicokinetics o fTelomer B Alcohol in the rat Experimental Design Rats will have a cannula surgically implanted in the jugular vein one or two days prior to dosing. Rats designated to receive a dose via the dermal route will have the dorso-thoracic region shaved free of hair the day prior to dosing. After shaving the site will be washed with a dilute soap solution and covered with gauze and a body wrap to prevent contamination. Rats will be then fitted with an Elizabethan-style collar to prevent grooming of the application site. The collar will be worn for the duration of the experiment. On the day of dosing, the protective body wrap and gauze cover will be removed and a silicone O-ring with an internal area of 10 cm2 will be glued to the shaved dorso-thoracic region using an adhesive. Following dosing, the O-ring will be covered with a rigid nylon mesh and a non-occlusive body wrap. At the end of the exposure period, the non-occlusive cover will be removed and the site washed, rinsed and dried with natural sponges. Following dosing, serial whole blood samples will be drawn from the cannula and placed into Microtainer tubes containing EDTA. Sampling times will be documented in the study records. Alternatively, whole blood may be collected from the tail vein. For rats in the dermal exposure groups, whole blood will be collected during after the exposure phase (8 hours). The application site will be washed free of excess test material at 8 hours. Whole blood will be placed on wet ice, and plasma will be separated by centrifugation and frozen at approximately -15C until analyzed. 2. Distribution Experiments The principal objective of the distribution experiments is to determine the concentration and clearance of TB A in selected tissues and organs at plasma Tmax and Tm ax/2 for TBA following oral and dermal dosing. Tissues should be selected based upon previous toxicology data or results of the pilot excretion/metabolism experiment. Dose Routes and Frequency The distribution experiments will be comprised of four dose groups, for each of two radiolabeled forms of TBA. The need to use both radiolabeled forms of TBA will be established in pilot experiments. Low dose, single oral gavage High dose, single oral gavage Low dose, single 8-hour dermal exposure High dose, single 8-hour dermal exposure Each dose group will be composed of 4 male and 4 female rats. Each dose group will receive dose formulated with radiolabeled TBA. Utl 9- - Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat Experimental Design Rats will be dosed via the oral and dermal exposure routes at the low and high dose levels. The pre-dosing procedures for rats in the dermal exposure groups will be as described under the absorption experiments. The exposure period for the dermal exposure groups will be 8 hours. Following dosing rats will be killed at plasma Tmax and Tmax/2 for TBA. Tissues will be collected at sacrifice and stored at approximately -15C until processing and analysis. Total radioactivity in tissues will be determined. Selected tissues may be extracted and analyzed to determine the metabolite profile. Methods will be documented in the study records. 3. Excretion and Metabolism Experiments The objectives of the excretion and metabolism experiments are to determine the rates and routes for elimination of TBA and/or its metabolites, to establish the principal pathways for metabolism of TBA and to determine if daily (repeat) low oral dose exposure alters the excretion kinetics or metabolism of TBA. A mass balance for total radioactivity administered will also be determined. Dose Routes and Frequency The excretion/metabolism experiments will be comprised of five dose groups, for each of two radiolabeled forms of TBA. The need to use both radiolabeled forms of TBA will be established in pilot experiments. Low dose, single oral gavage High dose, single oral gavage Low dose, single 8-hour dermal exposure High dose, single 8-hour dermal exposure Low dose, daily (repeat) oral gavage Each dose group (single dose) will be composed of 4 male rats and 4 female rats. Each dose group will receive dose formulated with radiolabeled TBA. For the daily (repeat) low-dose oral gavage experiment, four male and four female rats will be administered non-radiolabeled test compound at the low dose rate for a total numbers of days necessary to achieve steady-state plasma levels of TBA. The exposure time for the dermal experiments will be 8 hours. Once steady state has been achieved, as determined by periodic sampling of whole blood for TBA and/or selected metabolites, rats will be administered a dose of the test material formulated with radiolabeled TBA. Experimental Design Pre-dose procedures for rats in the dermal exposure groups will be as described for the absorption experiments. Following administration of the radiolabeled dose, urine and feces will be collected (on dry ice) at approximately 12 hours and 24 hours following dosing, and each 24 uat___________________________________________________________ _____________________ - 10- Draft Protocol WR XXXX Toxicokinetics o f Telomer B Alcohol in the rat hours post-dosing until sacrifice. Animals will be sacrificed after at least 95% o f the administered radioactivity has been excreted as determined in pilot experiments. The excreta may be analyzed to determine the metabolite profile. If significant amounts of 14CC>2 or volatile organics were observed in the pilot study, traps will be used to collect any 14CC>2 and/or volatile organics that may be liberated. If required, the expired air will be passed through two traps connected in series, the first contains 2N NaOH and the second containing ethylene glycol or other suitable solvent. Solvent traps will be collected and replaced every 24 hours post-dosing throughout the study. In the case of the Low Dose Multiple dose groups, expired air will only be collected following administration of the radiolabeled dose. The tissues collected at sacrifice will be determined by the results of previous toxicology experiments and/or pilot studies. After collection, tissue samples will be stored at approximately -15C until processing and analysis. The remaining carcass will be homogenized and assayed so that total material balance can be determined. Selected tissues may be analyzed to determine the metabolite profile. At the conclusion of the radiolabeled dose experiments, cages will be rinsed with detergent and water (50:50 v/v), water and acetone. Cages may be wipe-tested to determine efficiency of cage rinse. A second cage rinse may be carried out at the study director's discretion. Cage wash will be stored at room temperature. If the feces contains greater than 20% of the administered dose, as determined in pilot experiments, two additional groups of four male and four female rats (oral and dermal) will be used to establish the origin of material in the feces. Rats, will have a cannula surgically implanted in and bile duct and provided with a bile maintenance drinking solution (isotonic dextrose/KCl/NaCl) as the sole source of drinking fluid for the duration of the experiment. Following dosing, urine, feces and bile will be collected (on dry ice) at approximately 12 hours and 24 hours. Animals will be sacrificed after 24 hours. The excreta may be analyzed to determine the metabolite profile. -11 - Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat J. Quantitation of Radioactive Residues 1. Plasma Plasma samples will be thawed, and aliquots analyzed in duplicate for 14C by liquid scintillation counting (LSC). Plasma may also be analyzed for parent compound and metabolites by LC-MS. 2. Urine Urine samples from each collection interval will be thawed, and aliquots analyzed in triplicate for 14C by LSC. Urine may also be analyzed for parent compound and metabolites by LC-MS. 3. Feces Feces from each collection interval will be homogenized in water and aliquots will be combusted. The CO2 liberated from combustion will be trapped and analyzed in triplicate for 14C by LSC to determine total fecal radioactivity for individual rats. Feces may also be extracted and analyzed for parent compound and metabolites by LC-MS. 4. Bile Bile from each collection period will be thawed and aliquots will be analyzed in triplicate for 14C by LSC. Bile may also be extracted and analyzed for parent compound and metabolites by LC-MS. 5. Cage rinses Aliquots of cage rinses will be analyzed in triplicate for l4C by LSC. 6. Feed residue Feed residue will be homogenized and aliquots will be combusted. The CO2 liberated from combustion will be trapped and analyzed in triplicate for 14C by LSC. 7. Carcass Individual carcasses will be analyzed for residual l4C only if the recovery of total radioactivity in excreta and plasma for that animal is <98%. Carcasses will be ground on dry ice and homogenized, and aliquots of the resulting homogenate will be combusted. The CO2 liberated from combustion will be trapped and analyzed in triplicate for 14CC>2 by LSC. m-6 - 12- Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat 8. Samples from dermal experiments In addition to the samples normally collected and analyzed from experiments using radiolabeled TBA, the following samples unique to dermal experiments will also be collected and analyzed. The application skin site and sponges used for washing and drying the application skin site will be digested in IN potassium hydroxide and aliquots analyzed by LSC. The protective appliance used to contain and protect the dose site will be soaked in an appropriate solvent. Aliquots will be taken and analyzed by LSC. K. Material Balance Total recovery for rats receiving radioactive TBA should average 100 10% of the amount administered. L. Clinical Observations and Mortality Cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats will be conducted at least once daily throughout the study. Moribund rats will be sacrificed. M. Statistical Analyses Group data will be represented as a mean SD. Other statistical evaluations may be performed, if necessary. N. Measurement of Radioactivity Radioactivity in selected liquid samples will be assayed by LSC. Total radioactive residues in solid samples will be determined by combustion, trapping liberated 14C02 followed by LSC. Samples for LSC will be analyzed for 10 minutes, or until 160,000 disintegrations (0.5% 2 a ) are accumulated, whichever comes first. -13- Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat Safety and H ousekeeping All chemicals used during this study will be handled according to the procedures specified in the Chemical Catalog and/or the accompanying MSDS and disposed of according to the StineHaskell Waste Disposal Guidelines (Procedure 100). Radiochemicals will be handled and disposed of in accordance with the guidelines outlined in the Stine-Haskell Radiation Safety Manual and Radioactivity Users Guide. Records and Sample Storage Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. Critical D ates Date of Approval: Proposed Experimental Start: Proposed Experimental Termination: Proposed Study Completion: Date Study Director signed Protocol <first date test substance is applied to test system> <last date on which data are collected directly from the study> <date final report will issue> lit# - 14- Draft Protocol WR XXXX Toxicokinetics of Telomer B Alcohol in the rat References 1. Vanden Heuvel J P, Davis J W, Sommers R, and Peterson R E. (1992). Renal Excretion of Perfluorooctanoic Acid in Male Rats: Inhibitory Effect of Testosterone. J Biochem Toxicology, 7, 1, 31-36. 2. Hanhijarvi H, Ophaug R H, and Singer L. (1982). The sex-related difference in perfluorooctanoate excretion in the rat. Proc. Soc. Exp. Biol. Med., 171, 51-55. 3. Vanden Heuval J P, Kuslikis B I, Rafelghem M J, and Peterson R E. (1991). Disposition of perfluorodecanoic acid in male and female rats. Toxicol. Appl. Pharmacol., 107, 450-459. 4. Olson C T, and Anderson M E. (1983). The acute toxicity of perfluorooctanoic and perfluorodecanoic acids in male rats and effects on tissue fatty acids. Toxicol. Appl. Pharmacol., 70, 362-372. 5. EPA/FIFRA Good Laboratory Practice Standard (40 CFR 160). (1989). 6. OECD Principles of Good Laboratory Practice (OCDE/GD(92)32). (1987) 7. MAFF Japan Good Laboratory Practice Standards (59 NOHSan No. 3850). (1985) S ig n a t u r e s Issued by Study Director: ___________________________________________ cname, credentials> <title> _________ Date Approved b y : ___________________________________________ cname, credentials> <title> _________ Date CC: QA m - 15- Toxicokinetics of Telomer B Alcohol in the rat Summary of study experiments 1) Pilot experiments a) Test material: Telomer B alcohol (TBA) and [14C]TBA b) Number of animals per treatment group: 2 male and 2 female c) Treatment groups: i) Plasma kinetics following a single high oral dose of TBA ii) Excretion and metabolism following a single high oral dose of [14C]TBA (1) [10-C14]TBA (2) [1-C14]TBA 2) Absorption experiments a) Test material: TBA b) Number of animals per treatment group: 4 male and 4 female c) Treatment groups: i) Single low oral dose ii) Single high oral dose iii) Single low dermal exposure (8 hours) iv) Single high dermal exposure (8 hours) v) Single low intravenous dose 3) Distribution experiments a) Test material: [14C]TBA b) Number of animals per treatment group: 4 male and 4 female c) Treatment groups: i) Single low oral dose, sac at Tmax ii) Single low oral dose, sac at Tmax/2 iii) Single high oral dose, sac at Tmax iv) Single high oral dose, sac at Tmax/2 v) Single low dermal exposure (8 hours), sac at Tmax vi) Single low dermal exposure (8 hours), sac at Tmax/2 vii) Single high dermal exposure (8 hours), sac at Tmax viii) Single high dermal exposure (8 hours), sac at Tmax/2 4) Excretion and metabolism experiments a) Test material: [l4C]TBA b) Number of animals per treatment group: 4 male and 4 female c) Treatment groups: i) Single low oral dose ii) Single high oral dose iii) Single low dermal exposure (8 hours) iv) Single high dermal exposure (8 hours) v) Single low daily oral dosing with TBA, last dose with [14C]TBA