Document 93gERyVaGvZ7jLob0wonZOmNp

150 ff AR 226- 0947 Telomer Research Program 2 Update and Status Report : Presented to U.S. EPA, OPPT 2 23 October, 2000 5 22:04 2- HOHE sis o oS = as TRP Member Companies Asahi Glass Atofina * Clariant * Daikin * DuPont 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 --July 11, 2000: Met with MITT -- 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 4 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 s 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 6 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 7 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 3 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-CgF,CH,CH,0H Purity >99%, and characterized Sample made, 20 kg, now in analysis Telomer Research Program Oct 2000 " Acute Oral Toxicity - Fixed Dose Method Objective: To assess the acute oral toxicity (lethality, clinical signs of response) by administration of gavage doses to rats Method: Rats, male and female + Single oral doses + Sighting Study: - 5-50-500-2000 mg/kg, | rat/dose - clinical signs; mortality to 14 days select first dose on available information, analogous chemicals + Main Study: - SM/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 Telomes Research Program Oct 2000 a 12 Repeated-Dose Oral Toxicity Gavage Range- Finding Study in Rats Objective: To determine dose levels for use in 90-day toxicity study which will enhance ability to achieve NOAEL, LOAEL Method: Rats, SM/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 ProgramOct2000 +H 13 | Previous Study with Telomer B Alcohol (C8-2) | 1bia . \-- 1" Subchronic Toxicity - 90-Day Gavage Study in Rats with Recovery Ojective: To evaluate the subchronic toxicity and the kinetics of uptake and clearance of this chemical in the blood. Methods: + Rats, 10M/10F/group, oral gavage, 90 days, control and 3 test levels; 10M/10F/group control and high 4 wk reversibility + Parameters: In vivo, clinical pathology, neurobehavior, pathology + SM/SF/group Blood Kinetics: "Results: + Determine target organ(s) and dose required to produce effect + Determine reversibility of change(s) produced + Determine uptake and clearance of organofluoride in blood Goal: Establish target site, potency, (NOAEL....LOAEL....) "Telomer Research Program Oct 2000 4 ig : Developmental Toxicity Study in Rats Oective: To evaluate the potential effect on the fetal rat following administration of the chemical to the maternal rat from implantation to the end of | gestation. Methods: | 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 of chemical to effect either the maternal or fetal rat (or both) and the types and severity of the induced changes + The potency of the chemical - maternal NOAEL....LOAEL - fetal NOAEL....LOAEL "Telomer Research Program Oct 2000 2 16 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 productionofat least I set of litters in each generation). Methods: + Rats, 30M/30F/group, graded dietary levels, control +3 test levels + Feed chemical 70 days pre-mating (P1 and F1) 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 asa target (yes or no) - functional (1st, 2nd generation) histologic (Ist, 2nd generation) ~ effects in offspring + Potencyofeffects in offspring + NOAEL....LOAEL.... `Telomer Research Program Oct 2000 2 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 ofS. typhimurium and at the tryptophane locusofE. coli (with and without S-9 activation) Methods: Tester strains - Salmonella typhimurium TA98; TA 100 TAIS35; TA 1537 E. Coli WP2uvrA 5 graded test concentrations - selected based on evidence oftoxicity 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 == 18 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: Exp(owsitehhpuomsiatnivleyamnpdhonceygtaetisvteocaotntlreoalsts)4 graded concentrationsofthe test agent | + Non-activated system treatment times - 4 and 20 hours | + S-9 activated system treatment time - 4 hours + ASrcroersetadimviindiinmgucmelolsf 2in00mestparpehaadsse/;dohsaervleesvteland evaluate at 20 hours (100/duplicate treatment tube) Result+s: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 9 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 Method: + Single IP injection of rats at 3 graded doses (from pilot study but not to exceed 2000 mg/kg) + SM/SF/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 Resul+tsP:roportion of micronucleated polychromatic erythrocytes/ polychromatic erythrocytes + Apply appropriate statistics versus controls + Valid test onlyif control incidenceofmicronucleated cells <0.3%; positive control statistically clevated Telomer Research Program Oct 2000 OECD 474 uses mice; consider oral gavage vs. IP a 20 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 bufferat 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 TelomerResearch Program Oct 2000 Note: Protocols still in draft form Pr 2 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 Telomes Research rogram 0st 2000 Note: Protocols still in draft form ~~ 34 2 TRP Pharmacokinetics Study Toxicokinetics of Telomer 8-2 Alcohol (TBA) in the Rat TObejleocmteivre:8-2Daeltceorhmoilnien tmhaelaebasonrdptfieomna,ledirsattrsibfuotliloonw,imnegtoarbaollainsdm daenrdmeaxlcrdeotsiionng.of Metho+d:Conduct pilot experiments ++++ Radiolabeled 2 male and 2 afnemdacloeldraTtsBA used as test material ++++ DDeetteerrmmiinnee epxlcarestmiokninaentdicmseftoalbloolwiinsgm foslilnoglweinhgigahsionrgalledhoisgehoofrTalBdAose of + ConducrtaadbisoolrabpetlieodnTexBpAeriments +++ 4TBmaAletesatnmad4tefreimaalle rats ++++ SSiinnggllee llooww aanndd ssiinnggllee hhiigghh dorearlmdaolseexposure (8 hrs) ++++ SFionlglloewlpolwaisnmtaracvoenncoeunstdraotsieons of test material Telomer Rescarch Program Oct 2000 Note: Protocols still in draft form 2 EH | TRP Pharmacokinetics Study Toxicokinetics of Telomer 8-2 Alcohol (TBA) in the Rat (Continued) * Distribut+i+onReadxipoelraibmeelnltesd 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 **+ DIdaeinltyidfyoseexocrfeTtiBoAn rwaittehs laansdt dmoesteabboeliincgprraodfiiollesabelled TBA Results: * Metabolic profiles of TBA * Qualitative and quantitative descriptionofbiodistribution 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 `Telomer Research Program Oct 2000 Note: Protocols still in draft form~~ 7 ap Radiolabeled Fluorotelomer Alcohol 1G Label in perfluoro-chain F(-- CF, CF,),-CH,CH,-OH 14C or 3H Label in ethylene `spacer' + Need two-labeled compounds: perfluoro-chain and ethylene `spacer' + Expect Fluorinated chain label to be difficult + Expect Ethylene `Spacer" Label to be Straightforward Telomer Research Program Oct 2000 a a5 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 26 pail TRP Environmental Fate Study + Objective: Laboratory Study of Atmospheric Degradation of n-CgF,,CH,CH,0OH -Determine persistence via reaction with OH | radical -Determine major oxidation products via reaction with O,and NO,(NO, NO,) -Determine reactivity of the oxidation products. Telomer Research Program Oct 2000 27 4 | 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 elomerRescarh Program Oct 2000 *Algae to be addressed in later work us a8 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 '4C 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 Telomer Research Program Oct 2000 Ar 2A TRP Toxicology, Pharmacokinetics, and Environmental Fate & Effect Timelines* Chronology ne pep a Tee eT - np me, i ea [sum eassTe PO ricer cee (TL paogn EERE on EEer roe r Sys me ESEe esomncaysn me [1 comm Fmeron - ay || i I amCSopmmelimeenn]] vormorta Fate ESommem, 8 801 II -- | Tetomer Research Progam oe 200 *TRP will present results as available on "01 43 20 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 = 3! Title: Acute Oral Toxicity - Fixed Dose Method Protocol Contain NO cB) : Jv 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, location, in accordance with all applicable Good Laboratory Practice standards." The study design is based on EDuervoepleoapnmeEncton(oOmEiCc DC)om,mtuenstitgiuiedsel(inEesE.C)Araenads Organization for Economic Cooperation and 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 Inc. The female address Crl:CD*(SD)IGS of thesupplier (ci BR rats ty/state) will will be be obtained from Charles River Laboratories, documented in the study records and final rtehpiosrts.traTinheanCdrlit:s CsDui*t(aSbiDl)itIyGwSitBhRrerastpehcats tboeleonngseevlietcyt,edhaornditnheessb,asseesnsoiftievxittye,nsainvde leoxwpeirniceindceencweitohf spontancous 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. Al animals will be provided tap water ad libitum. All animals will be fed PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 ad libitum 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 integrityofthe study: + Water samples are analyzed for total bacterial counts, and the presenceofcoliforms, 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 srpeeqcuiifrieemdehntesavayndmentoatlst,o aefxlcaetoexdins,tactheldormianaxtiemd uhmydcroonccaernbtornast,ioannsdoofrkgeaynocpohnotsapmhiantaen.ts,Tihnecluding 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 atiending laboratory animal veterinarian. Data are maintained separately from included in the final report at the discretionof the study director. study records and may be 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 atleast 3 times during quarantine. + observed with respect to weight gain and any gross signs of disease or injury. `oTnhethaenibmasailssowfilalccbeeptraeblleeasbedodfyrowmeiqguhatrsanatnidneclbiynitchale sliagbnos.ratory animal veterinarian or designee E. Assignment to Groups Animals of cach sex, selected on the bases of adequate body weight gain and freedom from any clinical signs of discase or injury, will be assigned to the study. After assignment to the study, aancihmaalnnimuamlbewirllanbde ahnouisneddiviindduiavlidiudaelnltyi.ficIantfioonrmnautmiobneronastshiegcneadgetolaebaeclsh wainlilmailn.cluTdheetlhaestun3ioqrue4 digits of the animal number will be marked on the tail of each animal. At study start (test day 0) 3 2% 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 docs 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 S00 mg/kg dose is used in the frst 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. Ifsevere effects, necessitating humane killing are seen at the initial dose (c.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 (0 use more than 5 animals in this procedure. 3. Main Sdy Atleast 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 mg/kg, the substance can be investigated at a lower dose level. a 35 In most cases it is likely that the data obtained from the sighting study will be aledveeqluathtaetoproadlluocwestheeviadpepnrtoptroixaitcietdyobsuetlneovemlorttoableitsy)e.lecHtoedweivn etrhiefmaeviindesnttudtyox(iici.tya is not level. scen at the IFanimals initial die at dose level, the the initial dose substance chosen, or will be retested at a severe reaction the next requires higher removal of be retested aatnithmealnsexftrolmowtehre dsotsuedylefvoerl.animal welfare reasons, the substance will 4. Dose Volume When practical, substances will liquid test substances will be suspended or dissolved be administered neat. Solid test in a vehicle. Variability in volume will dboesmeilneivmelisz.edThbey daodjsuestvionlgutmhee wciolnlcennottreatxicoenedto1enmsLu/r1e0a0cgonosftabnotdyvowleuigmhet,ateaxlclept in the cases of aqueous solutions where 2 mL/100 g dosing material administered to each animal will be may be used. based on the The volume of animal's fasted body weight determined on the day of dosing. 5. Frequency "pTohsesitbelset,stuhbestdaonsceemwailyl bbee gaidvmeinniisntsemraeldlienrafrsaicntgiloensdoosvee.r aIfpaersiiongdlenodtoesxeciesendoitng p2r4ohvoiudres.theWahneirmealasdwoisteh ifsoaoddm,idneispteenrdeidnginofnratchteiolnesn.gtithmoafythbeednoesciensgsaperryiotdo. 6. Vehicle c`Tohnesiudseeraotfioann oaqafuemoiuxstumriextiunrceosrhnoouilld abnedcothnesnidbeyrecdonfsrisdt,erfaotliloonowefdabypossible `mixture in other vehicles. Commonly used vehicles in the order of preference are awacteetro,nemeatnhdylwcaetlelru,loorsemiixntwuarteesr,ofMaazceotloane caonrdn coiolmoroile.quiCvoalmenoti,l,mmiextthuyrlecselolfulose, and acetone will be used as supplied: any contaminants that can reasonably be evxehpiecclteedusteodbweilplrebseendtocaruemenontteedxpiencttheedsotuadfyfercetcotrhdesinatnedgrfiitnyalofretphoerts.tudy. The 7. Observations and Body Weights for Sighting Study and Main Study Except animal where animals need to be removed from the study and welfare reasons, animals wil be observed for at least 7 humanely killed days (sighting for switluldyn)otorbe14fidxaeydsr(imgiadilny.stItudwyi)llafbteerddeotseirnmg.ineHdobwyevteher,totxhiecdrueraacttiioonnso,fraotbesetrovoantsieotn and length necessary. of recovery period, The times at which and may signs of thus be toxicity extended when considered appear and disappear are important, especially if there is a tendency for oxic signs to be delayed. All s - 3% 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 conditionofthe 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 timeofdeath will be recorded as precisely as possible. The rats will be checked twice daily for `mortality and signsofillness, 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 thepreviously determined body weight) occurs, all animals in the group will be weighed daily until there is no significant weight loss. Al 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, location>. Laboratory-specific or site-specific records such as personnel files and equipment records will be retained at the facility where the work was done. --errn--_-- 8 37 -_-- REFERENCES 1. EPAJFIFRA Good Laboratory Practice Standards (40 CFR 160). (1989). 2. OECD PrinciplesofGood Laboratory Practice ENV/MC/CHEM(98)17 (OCDE/GD(92)32). (as revised in 1997, published in 3. Official Journal of the of Toxicity. (1992) European Communities. Part Blbis: Methods for the Determination 4. OECD Guidelinesfor the Testingof Chemicals, No. 420. (1992). -_-- 7 38 SIGNATURE Approved by: Tw SDbeme Study Director Dae --_ am -- s - 39 - <Cmpd> Repeated-Dose Oral Toxicity Gavage Range-Finding Study in Rats Protocol Contain NO cpg; _----p <`GCarvapgde>RaRnegpee-aFtiendd-iDngosSetOurdayliTnoRxaitcsity TAOFBCOL NTEE NTS Page INTRODUCTION tis sss:3 OBIECTIVE ccm sms -- i. SPONSOR AND TEST FACILITY ccm: 3 STUDY DESIGN mms MATEARNDIMETAHOLDSS A TSE SUDSIANCrrr cc ts nn E ------nb I CATING] HUSDAY...rrrrrrssss ---0 ess ll D. PretestPeriod. FE ---------- I ---------- F. DosingSolution Preparationand Sampling G. AdminiofsDotsirngaSustpeinsioonns. w..........c..... -------- ---- I H1.. ClBiOniYcaWlEOIbEsReSrvatiocnsraendtMeOTaIYsv ss e rises -- cwmmm---- J. K. TAontaatloFmIiUcOalRNPEaLetVehloElVogAyI..U.AON...T.--..--c--rr--s--te--s--s---- 9 FE A 2. DOSE PIS rrr ------ mss 9 SAANF D HOE USET KEEY PING... s---- RECORDS AND SAMPLE RETENTION... mena} PROTOCOL APPENDIX... ----------------. SIGNATURES ccm sss sss wont 73) : . <GaCvmapgde>RaRnegpee-aFtiendd-iDnogsSetOurdayliTnoRxaitcsity INTRODUCTION The test substance, <Cmpd:, is a<compound> used as an < >. Dose levels for this study were selected based on <>. OBJECTIVE 9`T0h-edoabyjeocratlivseubocfhtrhoinsiocrasltugday.vagTeheraonrgael-frionudtienogfsatdumdiyniissttroadtieotnerwmaisnesetlheecdteodsebelcevaeulssef<o>r.use ina SPONSOR AND TEST FACILITY s"Tphoinsssotrudsyubimsistptoendstohreedwobryk.<spTohnesosrtundaymwei>l.l The sponsor's be conducted at approval was <laboratory, effective locations the date the 3 W& <C> Reoep rtTotney GuvageRangeFindingStodyinRass STUDYDESIGN "The dydesig is aFollows: wadGuFuemale MiNleofGFoewmi Mins SciaboMdelTisone po? Taman T A s swe -- Nims coAlliecmtison mghgi6dsy Cowt wVoowusossos WimSS shSoexx ve Anose Mon 3o mmo mcmeb <me 2b Ehe wi egea md oonyotn nad str. lon n isidf nit todo -- GaneRang FindingSoyin Rats I EE J [rr] Fest Day ts 0. o ttaimmentof steady satc. Vehicleanimalswillbedosed until lastdosegroupattains. ETa ---- -- -- -- ---- -- Ia BBhpi reEigaTin mCcesantt, eentae sCorte mrs : "Select Tissue/Sample Collection (Al rats inthe 0mg/kg/day (Control) and <> mg/kgiday <Conpd>-grouDpa)te. of sacrificewildepend on | es <GCaivnapgdesR:anRgeepFeinadtiengd-SDtOuodrsyaelinToRxatiscity MATERIALS AND METHODS A. Test Substance `The test substances will be supplied by the sponsor. The test substances) were assigned <> Laboratory Number(s) <>. B. Test Species MInacl.eTahned afdedmraelses Corflt:hCeDsu(ppSlDi)eIrG(ScitBy/Rstraattes) wwiillll bbee odbotcauimneendtferdominCthhaerlsetsudRyivreecroLradbsoraantdorfiiensal, rtehpsorstt.raTinheanCdrlit:sCsDuit(abSiDl)itIyGwSitBhRrreastpehcats tboeheanrdsienleescst,edloonngetvhietyb,asseesnosifteixvitteyn,sainvde leoxwpeirniceindceencweitohf spontaneous diseases. C. Animal Husbandry Arlolomrastswiwlilllbebemhaionutsaeidneind sattaaintleesmspesrteaetlu,rweiorfe-2m2e=sh3caCgeasndsausrpeelnatdievde ahbuomivdeictaygoefb5oa0rd2s.20%A.nimal Animal rooms will be artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. All rats will be provided tap water ad libitum. All ats will be fed PMI Nutrition International, Inc. Centified Rodent LabDiet" 5002 ad libitum. Afosllspoewciinfgiepdroicnetdhuere<s>aLreabpoerraftoorrmyeadnipmerailodhiecaalltlhyatnodaesnsuvrierotnhamtenctoanltammoinniatnotrilnegveplsroagrreabme,ltohwe 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 arc 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. Creerqtuiifrieemdeanntismaanldfenoetd tios uesxecde,edgusatraatnetdemedaxbyimthuemmcaonnucfeancttruarteirontsoomfekteyspceocnitfaimeidnannuttrsi,tiionncalluding sppreecsiefniceed ohfeathveysemectoanlts,amaifnlaatnotxsinb,eclholwortihneatmeadxhiymdruomcacrobnocnesn,traantdioonrgsatnatoepdhobsyphtahteemsa.nuTfahceturer would not be expected to impact the integrity of the study. 5 6 CGapvaage:RanRgeep-eFaitneddi-nDgosSetuOdryalinToRxaitcsity Tlahbeoraantiomraylahneiamlatlhvaentedriennavriirano.nmDeanttaalarmeonmiationrtianignepdrosegpraarmatieslyadfmrionmisstteurdeyd rbeycotrhdesaattnedndmianyg be included in the final report a the discretion of the study director. D. Pretest Period Upon arrival at <> Laboratory, al rats will be housed 1 per cage, sexes separate. in quarantine. "The rats will be quarantined foar minimum of $ 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 signsofall rats Rats that discarded are accidentally killed or without necropsy. Rats removed from study during the pretest period will be that are found dead or sacrificed in extremis during the pretest pdiesreiaosde.wilDlepbeensdeentnttoupPaotnhothleosgeyfainnddigngisv,enfuartghreorssdieaxganmoistniactipornoc1e0dcuhreesckmfaoyr tbheeepmrpelsoenyceedoafl the discretionofthe study will not be reported in director, the final a pathologist, or the laboratory animal report unless considered significant to veterinarian. The results the evaluation of the study. E Assignment to Groups Rclaitnsicoafl esaicghnssoefx,disseelaesceteodroinnjtuhreybwaislelsboefdaidsterqiubautteedbobdyycowmepiugthetrigzaiend,anstdraftrifeieeddormanfdroommizaantyion idnitfofesrteundcyesgraomuopnsgasgrdoeuspigbnoatdeydwienigthhetSmteuadnysDweistihginn, a50setxh.atTthheereweairgehntovsatraitaitsitoincaolflysseilgencitfeidcarnatts will not exceed = 20% of the mean weight for each scx. Awifltlerinacslsuidgentmheentuntioqgureouapnsi,maelacnhurmabtewrilalnbdethhoeuusneidquiendiinvdiidvuaildluya.l Information on the cage labels identification number assigned to cach rat. The last3 or 4 digits of the unique animal number will be tattooed on the tailof each at Austudy start body weights (test that day 0) are not the rats within will be 7 20% of weeks old. On test day 0, when possible, rats with 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 eriteria as the original rats). #6 1 Cpl:aRenpeeatrDosge Oyrt Toit Rats that have not been assigned to a test day 0, for out-of-range body weight, will group or which have be released for other been removed from laboratory purposes, study or be on test sacrificed by carbon dioxide asphyxiation and discarded without pathological evaluation, at the discretion of the study director. F Dosing Solution Preparation and Sampling Tphreepatersetds<udbasltya>n.ce will be <suspended> in <>. Dosing solutions of the test substance will be G. `Administration of Dosing Suspensions Tnhieltsestteasduybssttaatneceiswoiblltabienesdus(epsetnidmeadteidn t<o>.beT<h>edraaytss)wialtlabveodlousmeed obfy |inmtLra/g1as0t0rigcrianmtsuboaftiboondy wwielilghbte,bAasneidmaolnstwhiellbobdeydwoesiegdh7tsdcaoylsleacwteedekt.wicTehea awmeoeuknatndofttheestsosluubtsitoannscceonecaecnhtrraattiroenc.eives Control rats will be dosed with <> at a volume of 1 mL/100 grams of body weight. "<T>hemfgo/lklgo/wdianyg dsoussep;en<s>iomngc/omnlcefnotrrattheio<n>s wmigl/lkbge/duasyeddofsoer:eaacnhd d<o>sem:g/<m>lmfgor/mtlhef<or>tmhge/kg/day value of 1 dose. Control mraits10wi0llgrbaemdsosofedbowidtshwtehiegsha.me vehicle used to administer the test compounds at Ho Body Weights The rats will be weighed twice per week at 3 to 4 day intervals during the study. I Clinical Observations and Mortality bDeuhraivnigorthaentde/sotrpaerpipoeda,rcaangcee-saimteonexgarmaitnsawtiilolnbsetcoodnedtueccttemdoratibleuansdt towridceeaddairlaytstahnrdouagbhnooutrmtahle esatucdhy.ratMowirlilbbuendinrdaitvsidwuiallllbyehsaancdrliefdicaedndanedxadmisicnaerddefdorwaibthnoourtmanlecbreophsayv.iorAtanedvearpypewaeriagnhcien.g, Clinical signs noted will be recorded. Animals that be recorded as "no abnormality detected" (NAD) in do the not exhibit clinical study records. signs of toxicity will aPrnidoorrtostsaucdryifdiicreecotform,obrlioboudnsdarmaptsl,eisf pfoorsshiebmlaetaonldogayt the and dcilsicnriectailocnhoefmitshtercylimneicaaslupraetmheonlotgsismtay be collected for clinical pathology evaluations. rs - <`GCavpadge:RangRee:peFaitneddi-nDgasSetuOdryalinToRratiscity J. Total Fluorine Level Evaluation Pstraitoerftoordtoostailngfl(ueosrtindeaiyn--b4l)o,od1, iasnadtt7aidnaeyds, bafltoeords(tuadpyprstoaxrti,maantdelwyee0.k5 --ly1tmheLr)eafwiilelr buneticlolslteecatdeyd from the fluorine orbital sinusof each analysis. On the day animal while under of blood collection, light carbon dioxide anesthesia for total blood will be collected from the animals prior ttohedorseifnrgi.gerTathoer ubnltoioldthwielylabree sceonltletcote<d>inwigtlhaisns 2t4behsoucrosntfaorintiontaglEfDluTorAinwehainlaelyosins.iceFaonrdeasctohred in brleeaecdhiendg,fobrlaoopdarsthiocuulladrbdeosceolglreocutpe,d aatdmaipnpirsotxriamtiaotneloyftthheatsdaomseetoifmteesotfadratyi.cleOwniclleesntde.ad(yNsottaete--is the control group will continue to be dosed with vehicle until the last test article group achieves steady state). Bplosotoddoswiilnlg aflosroebaechcodlolseectgerdoufopr.toTthalefblluooordinesaamnpalleyssiscoaltle3c,t7e,d 1p4o,st30d,os4i5n,g6m0,ayanbde7a5nadlayyzsed as needed toanswer questions regarding distribution and metabolism of the test substances. A`dmdeittaiboonlailslmy,caognecsefaordcooslelegcrtoiuopn oafttfaiencsesstaenaddyursitnaete,fotrhe3 adanyism.alsUrwiinlel athnednfbeecepslwaicleldbien collected idnoc2u4mheonutredincarleomnegntwsi.thTthheeteoxtaalctvoilmuemepeorfioudrionfecoobltlaeicnteido.n oTfhueriunreinaendanfdecfeescewsilolbtnaeiendedtofbreom a fpoarrttioctuallarflduoosreingeraonualpyswiisl.l Tbeheposoalmepdle(ssewxielsl sbeepasreantte)t.o <B>loiond,couorlienres, caonndtafienciesngwiilcel. beAdsdeinttiotnoa<ll>y, 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 cAolllleracttsioinn otfheticsosnutesr.ol Raantdshwiiglhl dboeseeutghraonuaptsizweidllbbyecsaernbtotnodPiaotxhiodleoagnyesftorhegsrioassanedvaelxusaatnigouninaantdion. "The following tissues wil be groups sacrificed by design. collected and weighed from The tissues will be placed in rats in plastic the control and high-dose freezer bags and stored in the freezer until analysis of total fluorine levels. #8 5 B-i GaCvragpe:RanRegpeeFaitndeidn-gDosSetuOdryalinToRxaitcsity 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 Gproeopdarahtoiuosnekfaeceipliitnigesparnodcepdoutreentsiwailllhebaeltphrahcaztaircdesd. toTaovoaivdoicdonstkaimnicnoanttiaocnt,ogfldoovseisngwilslolbuetiwoonrn when handling the test handled in a chemical substance or dosing solutions. hood. Dosing solutions will be In addition, prepared in the test substance will be: properly ventilated areas. Animal carcasses and feces wil be incinerated. RECORDS AND SAMPLE RETENTION All original records will be retained at `personnel files and equipment records <>. will Laboratory-specific or site-specific be retained at the facility where the records such as work was done. 49 0 .. <GaCva>ge:RaRnegpeeaFtienddiDnogsSetuOdryalinToRxaiscity Study Function Study Director: PROTOCOL APPENDIX StudyPersonnel < <Senior Research Scientist> Study Dates Prebleed < Initiationof Test Substance Administration <> Scheduled Sacrifice Dferpoemntdheenbtlouopdonotfhteheelviamriinoautsiodnosreatgeroofupfsluorine 50 " :L <GCamvpadg>e:RanRgeep-eFaitneddi-nDgosSetuOdryalinToRxaitcsity Approved by: SIGNATURES < Primary Technician Management - << Study Director cc I se <CMPDs>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery Protocol Contain NO CBI 0000 50 . <CMPD>: Subchronic Toriciy: 9-Day Gavage Study in Rats with Recovery TABLE OF CONTENTS Page INTRODUCTION sss s------ 3 MATERIALS AND METHODS ..c.c smmm------y Ar B. TESUSUBSHCE sss TestSpecies -- esse s-------- wh 4 C. An1 imHaOlUHSuEsbandry. ---- wm cmsm--e--t 32.. CEangvieroRnamceknPtaOlSCOonNdNitEio.n.s....n -- s-- unis5 45.. WaAtniemraalnHdeFalotohdMonitoring. nm-- are - wm A5 D. Pretest Period... ------ wmm---- E. Assignto mGroeupns.t.. ---- ---- F. Dosing Solution Preparation and Sampling nm------ 7 G. AdminoifTsesttSUrbSaIANtCEion a----] He Body WEES cece snsm---- I. Food ConsanduFoomd EpFfiCtienicy.o ....n.........o- mad J. DetailedClinical Observaantd ions -- MOMlty.............c..c..o... m---------- 8 8 L. N1. euSreonbseohrayvFiunocrtaiolnEValUtion......ar.--............ ----n-- n oe M. C1.liniHceamlatPoalotghyology Evaluation.....w.m--... en-------- I 09 32.. CSoearguulmaCthieomnisPtarryameters. I a - Le10 4 Urinalysis. N.Blood Analysis.. ------------ si I" nsemmmm----_1 0. A1 natProemteisct Pathosloogy... a -------- MA 1| 23.. RRaatssDDeesisgffioonrr<SaguBbltcohonredoFndliaocrTiotdxeic>iAetnyaFldaysliustion s---- " ee3 4 Rots DesigonraREtCOeNEdY rrr " re SAFETY AND HOUSEKEEPING ..... s----------" RECORDS AND SAMPLE RETENTION... smsmm--3 PROTOCOL APPENDIX..... E-------- W-- 1 SIGNATURES... ---- 20 hyd 2 <CMPD>: Subchrone Toxiciy: 90-Day Gavage Study in Rats with Recovery INTRODUCTION The test substance, <CMPDS>, is under consideration for use as <use>. In a <duration> range finding study conducted with <CMPDs> in rats, there was <results> Dose levels for this 90-day gavage study were selected based on <results> observed in the rangefinding 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, location. "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 dGoocoudmeLnabtoerdatinortyhePrfaicntalicreepSotrta.ndards (59 NohSan No. 3850)" Areas of noncompliance will be The study design is based on European Economic Communities (EEC), U.S. Environmental Protection Agency (EPA) Office of Prevention, Pesticides, and Toxic Substances (OPTS), Organization for Economic Cooperation and Development (OECD), and the Ministry of Agriculture, Forestry and Fisheries (MAFF) Japan" test guidelines. 000 00% 0000000 . <CMPD>: Subchrone Toxicity: 9-Day Gavage Study in Rats with Recovery STUDY DESIGN The study design is as follows: Group Male Female 1 [0 m Vv v vi vi vm Number/Group Male Female 25 2s 15 15 1s 15 2s 25 Daily Dosage' 0 (Control) <> mylkg < mg kg < mg/kg a Winegirgdhiteinw)e.ight of test substance (adjuscd for sponsor-suppled purity of active Study Parameters Body Weight Food Consumption Clinical Observations Mortality/Moribundity Checks Clinical Pathology Neurobehavioral Evaluations Necropsy Gross Observations Organ Weights Frequency 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. TestSubstance "The test substanceofknown 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 LSaPbFormaatolreieasn,dInfce.maTlheeCeadld:rCeDss(oSfDt)hIeGsSupBpRlierrat(scwiityl/lstbaeteo)bwtialilnebde fdroocmumCehnartleedsiRnitvheerstudy reexctoerndssivaenedxpfienrailernecpeortw.ithThtheisCrstlr:aiCnDan(dSDi)tsIsGuSitBabRilirtayt whiatshbreeesnpescetletcoteldonognevtihtey,bahasredsionfess, sensitivity, and low incidence of spontaneous diseases. s : <CMPD>: Subchronc Toxicity: 9-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 ats will be provided tap water ad libitum. All rats will be fed PMI Nutrition International, Inc. Certified Rodent LabDiet" 5002 ad libitum. 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 integrityof the study: + Water samples are analyzed for total bacterial counts, and the presenceofcoliforms, 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 organophosphate. 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. $6 s <CMPD>: Subchronic Toriciy: 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 extremis 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 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 weeksofage. On test day 0, when possible, rats with body weights that are not within = 20% ofthe 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 . <CMPD>: Subshronic Toricity: 0-Day Gavage Studyin Rats with Recovery Rats that have not been assigned to atest 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: Type' EE Control DoCsoincnegnStorlautitoinosn | Storage Conditions Until Analysis i 0 mg/ml frozen rr --1 1 analyzed immediately or frozen 1 S-hour room temperature Al hours room temperature then 7-day and 14-day refrigerated | frozen 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. 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 preparation.> G. Administrationof Test Substance Dosing solutions will be administered daily by oral gavage at a dose volumeof <10/20> ml/kg, based on the most recently recorded weight. Dose solutions will be kept <refrigerated> until used. Dosing solutions stored beyond <duration> afier 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. ss 5 <CMPD>: Subchronic Toxicity: 0-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. IL 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 cach 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 andlor appearance among rats will be conducted at least twice daily throughout the study. Moribund rats will be sacrificed. Al 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, aulonNOMc 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 discretionof the clinical pathologist andor 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. $9 s <CMPD>: Subchronic Toricity: 90-Day Gavag 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 batery (FOB) and motor activity will be evaluated on all animals in the low and mid-dose group and in the rais 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 (0 exposure. 1. Sensory Function Prior to test substance administration and then during week 12of 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 albino (Chatillon Digital rats under ambient Force gauge). Due lighting conditions. ptuoptielcshinziecawlildlifbfeiceuvltayluoafteevdabluyaatsisngespsumpeinltsoizfe in 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 ofthe recovery period.ifeffects 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 oneof30 nominally identical, automated activity monitors (thCeouflubloluesrtne)x.tenGtrpoousspibalned. gTehnedeirnfwrialrlebdemcoonuinttoerribnagldaenvciecdeaecnraobslsetshememaosnuirtoermsenatnodfti2mdeeopfenddaeyntto variables, duration of movement and number of movements. A continuous movement is counted as | movement regardlessofduration. 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. Presenceofdefecation 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,ifeffects 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 0000 e nn > <CMPD>: Subchvonie Toxicity: 0-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 vena cava 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 extremis I. Hematology The following hematology parameters wil 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,ifrequired, 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 el 0 SCMPD>: 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 oH 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 $ 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 t0 90 days post-dosing, or until <fluoride levels> in exposed rats return to those of controls. Blood collection time points will be: day | (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 sinusofdesignated 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 endofthe recovery period. or sooner if <fluoride levels> return to control levels. 0. Anatomic Pathology Lo Pretest See Section D. Pretest Period. 62 u <CMPD>: Subchronic Tosicty: 0-Day Gavage Stady in Rats with Recovery 2. Rats Designated for Subchronic Toxicity Evaluation All rats found dead, accidentally killed, sacrificed in extremis, 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 aftemoon 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 wil be collected under carbon dioxide immobilization from the abdominal vena cava of each rat a 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 extreniis, removed from study (except out-of-range body weight on test day 0), or sacrificed by design. Digestive System liver esophagus stomach duodenum jejunum ileum cecum colon rectum salivary glands pancreas Urinary System Kidneys urinary bladder Respiratory System lungs trachea nasal tissue larynx pharynx Cardiovascular System heart aorta Hematopoietic System spleen thymus `mandibular lymph node: `mesenteric lymph node bone marrow Endocrine System pituitary gland thyroid gland parathyroid glands adrenal glands Nervous System brain (including cerebrum, cerebellum, `medulla/pons) spinal cord (cervical, `mid-thoracic, lumbar) sciatic nerve Musculoskeletal System skeletal muscle femur/knee joint sternum Reproductive System Male testes epididymides prostate seminal vesicles Female ovaries uterus mammary glands Miscellaneous skin eyes (including optic nerve) gross observations" ba GBroonsesmsaerrrvoawtiwiolnlsbmeacdoellacttneedcwriotphsythfeorwfehmiucrhanhdissttoerpnautmh,oloigsy not appropriate (eg. uid, ruffed fr, and missing anatomic parts) will generally not be collected 63 2 <CMPD>: Subchronic Toriciy: 90-Day Gavage Study in Ras 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 (percentoffinal 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 extremis, 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 wil 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 (issue integrity permitting), or are sacrificed in extremis, 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 (.2., osteoarthritis, pododermatits, ail chronic dermatitis, calculus, and deformities of the teeth, toe, tal, or ear pinna) wil 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 atthe discretion of the pathologist and will be documented in the final report. 3. Ras Designated for <Blood Fluoride>Analysis Any rats designated for <blood fluoride evaluations that are found dead, accidentally killed, or are sacrificed in extremis 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 extremis 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 O.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. oH 5 CMPD: Subchronic Tociy: 90-Day Gavage Study in Rts with Recovery STATISTICAL ANALYSES. Except for Bartlett's test (p < 0.005), significance was judged at p < 0.05. Parameter BBooddyy WWeeiigghhtt Gain Food Consumption Food Efficiency OrganWeight | | Meta petivi t ots OE play | FClluionirciadle Pathology Survival Incidence of Clinical Observationos ls Descriptive Parameters Incidence of MicroscopicLesions |Preliminary Test___| I preliminary testis | If preliminary test is not significant significant | Sequential `Test for lack of application" of the | Preliminary tests for trend Jtornencdkhteeestrie-yTerpsra rs ovens T or Toree a = e { hShoamponigHomaereosn-eWiietlykrtesat|n1%d)||| Tor vormality" Ovanrei-anwcaey!"yaanrafloysto lslioswoefd with Dunestest | Kruskal-Wallis test' ||| fesotllesorwed with Dunn's | TLevene's testfor |Repeated measures | Sequential Shhaopmiroog-eWinlekitteasynt!"d? || afnoalllyoswiesdobfyvariance |Joancpkphleeirce-aTteorifptoshtnrea for normality" Bartlett' testTM for Contrasts" | One-way analysis of | tKrreunsdkatle-sWta!l"lis testTM homogeneity of variances variance''" followed withDunnett' test"? | followed test" with Dunn's Levene's test for | Homogeneitayn"d analys | One-vay avisof 9 | lowe ithDens | Shapiro-Wilk esc! WEIS IONE ron fornormality" i, ste * None Cochran-Armitage tes't for trend!ox | None | a sPiganiwfiscanctomparisons and associated preliminary tests ar only conducted if the test for ack of rnd is IwFilhlebeShuaspeid.ro-Wilk tests not significant but Levene' sts significanta, bust version of Dunnetdesst 0 TWehsetndaaynainnddibvlidoucakl(o1b0s-emrivnauttieonEiPOrCecHo)rwdieldlasbebeusnegdlaesssretphaenatecde-rmteaaisnarvaelufea,ctcoarlsculations are performed on Hpaelrfftohremerdecwoirtdhedthvaatlubei.liFruobrinedxoatam.ple, ibilirubin is reported a <0.1, 0.05 is used for any calculations IBFoinhfeerinrcoindecnocmeecistinootn sisigunsiefdi.cant, but a significant lack offt occurs, the Fishers exact tei" witha 65 u . SCMPD>: Suchronic Toriciy: 9-Day Gavage Studyin 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 contaminationofdiet 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 wil be retained at <facility, location. 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 is <CMPD>: Subchronic Toriciy: 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 ENV/MC/CHEM(98)17 (OCDE/GDI(92)32). (as revised in 1997, published in 3. MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850). 4. Official Journalof the European Communities. Part B: Toxicity. (87/302/EEC). Methods for the Determination of 5. `UTnoixtiecdSSutbastteasncEensvi(rOoPnmPeTnSt)alHeParlotthecEtfifoenctAsgTeensctyG(uEidPeAl)i,neOsf,fiOcPe PofTSPr8ev7e0n.t3i1o0n,0 9Pe0s-tDicaiydeOsr,aland Toxicity in Rodents (AUG-1998). 6. Organisation for Economic Cooperation and Development. Chemicals, Section 4 (Part 408): Health Effects (1998). Guidelines for Testing of 7. The and Ministry of Agriculture, Forestry, and Fisheries, Regulations, Japan (ID), (59 NohSan No. 4200). Japan., (1985). Agricultural Chemicals Laws 8. DWrialpeeyr.,NNe.Rw. YaonrdkSmith, H. (1981). Applied Regression Analysis, 2" edition, pp 266-273. 9. Selwyn, MLR. (1995). animal safety studies. The use of trend tests to determine a no-observable-effect level in Journalof the American CollegeofToxicology 14(2), 158-168. 10. Jonckheere, A.R. (1954). A distribution-free K-sample test against ordered altematives. Biometrika 41, 133-145. 11. Levene, H. Statistics (J. (1960). Olkin, Robust ed.) pp test for equality of variances. 278-292. Stanford University Contributions to Press, Palo Alto. Probability and 12. Shapiro, S.S. and Wilk., M.B. (1965). samples). Biometrika 52, 591-611 An analofyvasriiancse test for normality (complete 13. S34n9e-3d5e2c.orT,heG.W.IoawnadCSotactherUanni,veWr.sGi.ty (P1r9e6s7s),. Slotwaat.istical Methods, 6" edition, pp 246-248 and 14. Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Statist. Assoc. 50, 1096-1121. 15. Kruskal, W.H. and Wallis, W.A. (1952). J. Amer. Statist. Assoc. 47, 583-621 Use of ranks in one-criterion analysis of variance. 16. Dunn, 0.J. (1964). Multiple contrasts using rank sums. Technometrics 6, 241-252. A . CMPD: Subvons Tovisty: 9Day va Soy in ats ith Resonry 17. Hocking, R.A. (1985). The Analysis of Linear Models. Brooks/Cole, Monterey. 18. Bartlett, M.S. (1937). Some examples of statistical methods of research in agriculture and applicd biology. J. Royal. Statis. Soc. Suppl. 4, 137-170. 19. Fisher, R.A. (1985). Statistical Methodsfor Research Workers, 13" edition. Haffner, New York. = e& 00000 on <SCMPD>: 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 69 = . CMPD: Subchronic Toiciy: 90-Day Gavage Study in Ras 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 weeksofexposure 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) 70 1 . <CMPD>: Subchronic Toxicity: 90-Day Gavage Study in Rats with Recovery SIGNATURES Approvedby: -- = bw PrimaryTechnician <~ Date AnalyticalChemist ~ <> Neurotoricologist owe eT bw Clinical Pathologist wee Pathologist Date = Manager Tw Study <Director Due ce: EE 7 } Titles: Multigencraton Reproduction Sudy in Rats Title: Multigeneration Reproduction Study in Rats Protocol Contain NO CB) 7x 7 i ME nT TABLE OF CONTENTS Page SPONSOR AND TEST FACILITY ovscuummnsmsimmsnsmsmirsmnsismimmonesmissssmmmonsmmsisionms 2. Purity ...... - TT --------------------l B. ATHDA wma" 1. Species (Strain). rm mmmm-- --------------------ell SOUR umm --------m ------ nonin 5. Identification inns nnn---------------------- 5 Co ATI FU1rrS rrrummmsmI smsnnN n G ------ .5 1. Environmental CORItioNs.............cococovreeererernenserenene m-- 5. Animal Health Monitoring.................... rimmmn----_-- 6. Quarantine rssercnnnnnanan 6 D. EXPEiMENtal DESIZN ....ocooevrerrersersersessessessnsnsnsnsns i E. Selectionof DoseLevels....................... mannnannausanl F. Randomization...........cccococec.. . 1. Py Generation... HIRE IGRI 8 G. Diet Adminisration. Peparaion. andSumiing E 1. Administration. anaaanaas------------ . 2. Prepaand r SAMaPHtNGi...o...n .c....ceeevrevevsvsnsiesine H. Safety Precautions and Disposal of Waste Material. SERS 8 _ [1] 1. Body Weight Measur foe rPamree ntanlRt atss... TTT 1. PLEMAtING FONG PETIOU cvrorerrnssrmonermesmmessssesessessssses 10 -- 2. Gesat nd La actt atioi nPero iodsn ........ mmmmmmmmm-- 0 J. Food Consumption and FoodEfficiencyfor Parental Rats...........ccccueeerremcsieee 10 1 Bemaing Poding Pod wwmmmmmmmemnommmnssensises ines S--T) 2, Gestion PEHod uunnuuuunuunuunnunuununnussnsmn SR K. Clinical Observationsfor ParentalRats .............. mmm 1 1. Start of Cohabitatio.n.. RA un = 000== OO ils: Moligenermion ReproSuoeyiinoRnt 2. Durationof Cohabitation Period ........................... TR 11 3. EvidenceofCopulation. TLE I IM. ESOS CYIe 4. Cohousing. ENGRONtts srere ser -------- msnsnmnins I11 N. GestationProcedures. inna" 1 1-Day 0 Postpartum O. Lactation Procedures trsetas seresen ers a ST . nmin J 2. Day 4 Postpartum - TT 3. Da7yansd14 POSIPATUM vcr 4.Day21 Postpartum (Weaning) EE ---- wwnmnmosonn 12 rrr 12 2 2Preputsl Separtion P. Developmental Landmarks. 1. VAINal PAENCY error cessreresas sere ------ I1 Fr --n 1I2H Q. Euthanasia and Postmortem Examinations HI -------------- ern 12 21: OPifaptrii)ngAoGeUS cornummmmmummmmmmmmisssmmmimmiin FT mmm 12 3. F, and F; Weanlings.. A ---- a5 14 R. Sperm Assessment. em---- EE -- mmmmmmmmori1n RECORDS AND SAMPLE STORAGE.....oocccnnsesssssssssssssssssssssmsssssssssesssssssssssssssssssossssssss 16 PROTOCOL APPENDIX L.cccnssenssmsssmssnmmmmmmsmsmmesmsmsmsssssses [9 - T=ow OO Titles: Muligeneration Reproduction Studiyn 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 setoflitters in each generation. `This study will conform with applicable Good Laboratory Practice Standards" and Test Guidelines. *" SPONSOR AND TEST FACILITY "This study is sponsored by (name, sponsor) and wil 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 Compounds 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 tbeesctaiunsgeasexrteecnsoimvmeebnadcekdgrboyutnhde guidelines. information iTshaevaCirllab:lCeDfr(oSmD)tIheGSlitBeRratsutrrea,inthweassupcphloiesre,n raenldatpirveevtiooulsonsgteuvdiiteys, whiatrhdiontehsesr, caonmdpionucniddse.nceTohfisspstornatianniesoaulssodicsoenassei.dered suitable - 75 Tiles: Muligeneration 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 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 hous) 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 Al rats will be housed as breeding pairs in stainless steel, wire-mesh cages. At the end of the cohabitation period, females without evidenceof copulation will be housed individually in polycarbonate pans. c. Gestation Period Days 0:20: cages. Females will be housed individually in stainless steel, wire-mesh : 76 "Tie 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 liters in polycarbonate pans. The stainless steel, wire-mesh cages will be suspended above cageboard. Polycarbonate pans will contain bedding. 3. Food PapMpIroNpurtiraitteidoonsIangteeronfatioCnaolm,pInoc.unCdertwiiflilebdeRaovdaeinltablLeabaDdielitbit5um0.02 containing the 4. Water Water from <> will be available ad libitum. 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 presenceofcoliforms, 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. Cnuetrrtiitfiioendalanriemqaulirfeemeedntissuasnedd,togubaerafnrteeeeodfbiymptuhreitmiaensuwfhacitcuhremrigthotmieneftlusepnecceiftiheedresults of the study. "The animal health monitoring program is administered by the Laboratory Animal Veterinarian. Data are maintained in the final report unless warranted separately from study by the results of these records and evaluations. will not be included 6. Quarantine Rats will approval be of quarantined for at least the Laboratory Animal six days, and then released Veterinarian or designee. for the study upon the --_-- = "Tiitleer::MMuullitiggeennecrraatvioonnRReepprroodduuccttiioonnSStdudyyiinnReatss Rats that die or are sacrificed in extremis during the pretest period will be necropsied to cphreoccekdfuorresthmeapyrebseenecmeplofoydeisdeaastet.heDdeipsecnrdeteinotn uopfotnhethsetsuedyfidnidriecntgosr,,ftuhrethpearthdoilaoggniossttic assigned to the study, or the laboratory veterinarian. D. Experimental Design `Treatment Groups and Dietary Concentrations Group Number Male Female Pn Fi on mo kl V0 Va VIO VILL Pp Fi oo I VO IVa VEO VE VIL VII #Group 30 30 30 30 Concentration (ppm) 0 (Control) < (Low) < (Intermediate) < (High) * Weighuweight concentration of Compounds, active ingredient, (adjusted for purity). Treatment Schedule Approximate Age at Start of Generation~~ Feeding(days| Py 6 Fi 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 PLE After siring litters (at discretion of study director) PLE On day of weaning liters PLE With pregnant females FiF: On day of weaning (except Fy's selected for parental rats) _--mmm 7 Title: Muliigeneration Reproduction Study in Rats E. Selectionof Dose Levels Dose levels selected for this study were based upon <>. F. Randomization 1. P, 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 inadistribution in which the mean body weights for all groups within a sex are not statistically different (p>0.05). Animals the startof dosing will be removed from that the lose excessive weight or that are study and replaced. if possible. ill prior to Rats not assigned 0a test group, at the discretion of the study director, may be released for use in other studies, or will be sacrificed evaluation. by carbon dioxide euthanasia and discarded without pathological 2. Fy Generation O(onnedaryat/2s1exp/loistttperarwthuem,n opfofssspirbilneg) itno tsheervFei alsitptearrsoenftseafocrhtthreeaFtmgeennterlaetvieoln.willFobregsreoluepctsed without sufficient ltrs, additional pups will be chosen from randomly selected litters within the group to achive the required group size. Selection of ras within liters will be random. G. Diet Administration, Preparation, and Sampling 1. Administration Compound will be administered During the test period, rats in each orally group as i will is be a potential route for human fed a diet of PMI Nutrition exposure. International, Inc. Certified Rodent LabDiet S002 that contains 0, <>, <>, or <> ppm of Compound. 2. Preparation and Sampling Compound will be added to PMI Nutrition International, Inc. Certified Rodent dLeatbeDrimeitnat5i0on0,2 iasnaddetqhuoartoeugtholeynmsuirxeedhofmorogaepneeroiouds odfisttirmiebutthiaotn, ibnytehxepdeireite.nc<eThore ptreesttest csounbcsetnatnrcaetiwoilnlobfeadciestsoonlevweidl/lsuaslspoenbdeedadidneadcetotocnoentprroilorditeotsm.ixiTnhge.manTuhfeacstaumree.r, 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 5 Titles: Muligeneration Reproduction Studiyn Rats nature of the contaminants nor the use ofacetone 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 Collect From Control Homogeneity:"" Top Middle Bottom Stability:* 7-day Room Temperature: Diet Mixer Diet Mixer Top Middle Bottom Feed Jars 14-day Room Temperawre ~~ Feed Jars 21-day Room Temperature ~~ Feed Jars 21-day Refrigerated Concentration Verification: Diet Mixer As Apportioned Storage Conditions Until Analysis Frozen Frozen Frozen Frozen Frozen 7 days at room temperature, then frozen 14 days at room temperature, then frozen 21 days at room temperature, then frozen 21 days refrigerated then frozen Analyzed immediately or frozen * "DTihcet mseaampnlevsalmueayofassaombpelescowlillelctbeedutsoeedvatloudaetteehromminoegecnoencietnytirfataionne.w method of diet preparation is used. DSitetasbamipollfiettshyewilflestbesucbosltlacntceednattlheeadsic3ttwiamsesesdtuarbilnigshtehdefsotu<dy>faonrdca<c>hpdipemtarry leaovel90t-modvaeyrifraytcfoenecdeinntgasttioundy. SitnasbialmiptlyeosfcolCloemctpeoduannddsfirnozfeenediwmmaesddieatteerlmyitnoedthbosyecsotmopraerdiantgrcooonmcetnetmrpaetriaotnusroefofCroefmrpigoeurantded. Extra diet samples may be taken at the discretion of the study dircetor. Whenever extra dict samples are preparation. taken,a sample of control diet will be collected and frozen the same day as dict --_-- -- <Talen: Muligencration Reproduction Study in Rats H. Safety Precautions and Disposal of Waste Material Good housekeeping procedures will be practiced to avoid potential health hazards and contaminationofdict preparation facilities. To avoid skin contact, gloves will be wor when handling cither 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. 1. Body Weight Measurements for Parental Rats 1. Premating Feeding Period AIP; and Fy 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 Py and F, females will be weighed on days 0, 7 14, and 21 of each period. Females without evidenceof copulation, that copulate and do not deliver a litte, 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 Py and F 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 Py and Fy parental rats will be individually handled and carefully examined for abnormal behavior and/or appearance. To 8 Tiles: Muligeneration 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 ofliters. M. Estrous Cycle Evaluation Vaginal smears will be collected daily from all Py and Fy female rats in order to determine the stagesofthe 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 Py and Fy 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. . 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 afer delivery is completed. Live pups in each litter will be individually weighed. Anogenital distance in i 82 Titles: Muligeneration Reproduction Study in Rats Fa pups will be recorded,iftreatment-related effects are observed in sex ratio or sexual maturationof 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 cight offspring or less will not be reduced. Litter counts and individual pup weights will be determined prior to and after culling 3. Days7 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 Fy litters will be randomly selected to serve as parents for the F generation. Randomly selected offspring from the liters of both the Fy and Fs 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. Pyand F; Adults a. Method of Euthanasia Carbon dioxide euthanasia and exsanguination o- 8> Tiles: Maliigeneraton Reproduction Sudy in Ras b. Postmortem Examinations All; and Fy parental generation rats will be subjected to gross pathological `examination, including those that die or are sacrificed in extremis prior (0 the end of feeding and thosefor 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 Piwitary Liver Kidneys Adrenal Glands Spleen Tissues Collected from Parental Rats" Male Testis Epididymis Prostate Seminal Vesicles Coagulating Gland Female Ovaries Uterus" (with oviducts) Vagina Cervix Both Sexes Adrenal Glands, Pituitary Gross Observations _ * Reproductive tissues collected from male parental rats for possible histopathological evaluation will be placed in Bouin's solution. Reproductive tissues collected from female parental ats and all non-reproductive tissue will be placed in formalin The uteri of all cohabited females will be examined for the presence and numberof implantation sites. 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 Py and Fy 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 -- = x Ties: Muliigeneration 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 cyelicity or sperm parameters) will be evaluated. Tn 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 um 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, caleulus, and deformities of the teeth, toe, ail, or car 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 F; Weanlings a. Method of Euthanasia Carbon dioxide euthanasia and exsanguination b. Postmortem Examinations Three Fy and three Fs weanlings/sex/littr, litter size permitting. will undergo gross pathological evaluation. One weanling/sex/liter 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. <. 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 Py and Fy 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 Tike: Muliigeneration 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 list the indices of reproductive function that will be calculated for the Py and Fi adults Reproductive Function Calculations Mating Index (%) = Number Copulating x 100 Number Cohabited Fertility Index (%) = Number Bearing Litters" x 100 Number Cohabited Fecundity Index (%) = Number Bearing Litters" x 100 Number Copulating Number of Litters with Gestation Index (%) = at Least One Live Pup x 100 Number of Litters Implantation Efficiency (%) = Number of Pups Born x100 Numberof Implantation Sites Pups Bom Alive (%) = Number of Pups Born Alive x100 Number of Pups Bor Viability Index (%)* = NumobfPeuprs Alive Day4 Preculling x 100 Numberof pups born alive Numberof pups alive Lactation Index (%)* = at weaning (21 postpartum) x 100 Number of pups alive Da4y Postculling *+ IEnvcilduednicnegotfhcoospeufloautnidonde=acdoppruelgantaonrty dpluurgi.ngfoguenstdatdieoand. pregnordaelnivter,y ofa ler. ETxoclbueddientgerlimtienresdsafcorricfaiccehdlditueer.0Mdeeaatnhoafnddastmandduarridngdelvaicattaitoinonfor cach dose level will be calculated. Parameter BBooddyy WWeeiigghhtt Gain Food Consumption `Statistical Methods Method of Starsical Analy Owhneen-wsaiygniAfniaclaynst,iswiotfhVDaruinannectetTM:efsotl"lowed, Tiles: Muligeneraion ReproductionStudy in Rats Gestation Length Organ Weight Hematology IImmppllaannttaattiioonn ESftfeicNiuemncbyers PMeeracenntNuBmobmerAloifvPeups Pr Liter SVeixabRialtyioIndex LParcetcaotiiaoln IInntderevxal SEspterromusPCayrcalmeetLeernsgth VPraegpiuntailalPSaetpeanrcaytion AMneoagnenPiutplWDeiisgthatnsce(C(oCvoavrairaitaetse:: ipeurp sweiieg,hste)x raiio) IInncciiddeennccee ooffCMliicnriocsalcoOpbiscerLveastiioonnss MNautmibnegrIonfdeFxemales Cycling Normally FFeerctuilnidtiytyInIdnedxex Gestation Index BSaartmleetas atebsotvef;ororhonmoongpeanreaimteytroifcvatreisasncifes is significant alpha = 0.005) L`eSvaemnee'asstaebsotvef:ororhonmoongpearnaemiettyriacndteSshtaspiirfoW(pi<l0k.0t5)e.st"Iftohr nSohrampailriot-yWariekseigsntifiiscnaontt sirgonbifuisctanvterbsuitonLeovfeDncu'ns ntees'tistesitgnwiifllicbaentused. Jonckheere's est" Linear contrastofthe least square means" CoscrohurpasnarAercmoimtpaagreedt.estFifosrherresndExoarc,tifesnott'al 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 freetpuesaetsedpeurntliilttneroosrigtnhiefilciatnetr mtreeannd wisildletbeecutseedd."as Ftohre elixtpteerripmaernatmaetleurnsi,ttfhoer psrtaotpiosrtticiaoln of affected euvseadl,uaatnidoont.h"erTphaeralmeevtelerosfasniaglnyizfeidc,aincfedseeelmecetdedneiscepss<ary.0.05. Additional statistical tests will be Where the data are tied and the standard large sample version of Jonckheeres test is not applicable. exactp values will be calculated using permutation methodology." RECORDS AND SAMPLE STORAGE <> 5 87 Titles: Muligeneration Reproduction Study in Rats CRITICAL DATES In-Life Study Start Date: <> Py Adult Sacrifice: <> Fy Weanling Sacrifice: <> Fy Adult Sacrifice: <> F; Weanling Sacrifice: <> In-Life Completion Date: <> (Approximate) (Approximate) (Approximate) (Approximate) REFERENCES 1. EPAJFIFRA Good Laboratory Practice Standards (40 CER 160). (1989). 2. OECD Principles of Good Laboratory Practice (C(81)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. 31,30.5.58). 7. MAFF Japan Testing Guidelines for ToxicologyStudies (59 NohSan No. 4200). (1985). 8. 3S4n9e-d3e5c2o.r, TGh.eW.loawnad SCtoactherUanni,veWr.sGit.y (P1r9e6s7s),. ISotwaat.istical Methods, 6" edition, pp 246-248 and 9. Dunnett, C:W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Statist. Assoc. $0, 1096-1121. 10. Shapiro, S.5. andWilk., M.B. (1965). An analysis of variance test for normality (complete: samples). Biometrika 52, 591-611 11. Jonckheere, AR. (1954). "A Distribution-Free K-Sample Test Against Ordered Aliernatives," Biometrika 41, 133-145. 12. Hocking, R.A. (1985). The Analysisof Linear Models. Brooks/Cole, Monterey. 13. Fisher, R.A. (1985). Statistical Methodsfor Research Workers, 13" edition. Haffner, New York. 7 97 . ies Mligemion Reprdusion StnRys 14. Selwyn, M.R. (1995). "The Use of Trend Tests to Determine a No-Observable-Effect Level in Animal Safety Studies," Journalof the American College of Toxicology 14(2), 158-168. 15. Haseman, 1K. 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. " 89 2 Tiles: Muligencraton 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 Lo 10 1-0 v0 V-0 VIO viLo vio Pi GENERATION Tattoo Number Male Female 101-130 301-330 501-530 701730 201-230 401-430 601-630 801-830 Dietary Concentration of Compounds (ppm) 0 (Control) < (Low) < (Intermediate) < (High) Groups Male Female Ll 1 m1 V1 V-1 VII ILI VIII Fi GENERATION Tattoo Number* Male Female 101x-130x ~~ 201%-230x 301x330x 401x-430x 501x-530x 601x-630x S01x-530x 801x-830x Dietary Concentration of Compounds (ppm) 0 (Control) < (Low) <> (Intermediate) < (High) * Each rat will be assigned a unique Animal Number "Fy rats digit. FwoirllmbaeleaspsuipgsnetdheaniniItDialnudimgbiet rwiblalsbeed on their changed Py to dthaemso'ddIDnunmubmebrercofrorlelsopwoenddibnygowniethmore thferiormgaroliutper.numFboerre.xaTmhplee,finiaflodniegimtawliellanindditcwaotefiefmiatliesstahreefsireslteocrtesdcfcroonmd pduapmo4f0a1 stehxeisreIleDcted numbers will be 3011. 4011 and 4012, respectively. Each Fy pup selected will also receive its own animal identification number. > 90 . Tiler: Muliigeneration ReproductionStudy in Rats Title: Multigeneration Reproduction Study in Rats Protocol Prepared by: Approved by: ce SD, SDDegreen SDTitlen Study Director Chemist Co Staff Pathologist <> Management Date as Date Date ct 7 a1 Sponsor Project Number: Test Substance Name: Rat Bone Marrow Erythrocyte Micronucleus Test 10 PURPOSE marrow. 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 20 SPONSOR 21 Name: < 22 Address: < 23 Representative: < 24 SpomsorProject > 30 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 31 "Test Substance: < 32 Controls: Vehicle: Positive: Test substance vehicle Cyclophospham(iCdPe) 33 Test Substance Characterization Unless alterate armangements are made, the testing facility will no perform alysis 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 strengthofthe test substance in the solvent (or vehicle). 34 Test Substance Retention Sample The retention of a reserve sample of the test substance will be the responsibility of the Sponsor. 40 TESTING FACILITY AND KEY PERSONNEL. 41 Name < 42 Address: < 43 Study Director: < Protocol A820 Page of? Contain NO cp; 50 PROPOSED STUDY DATES 51 Experimental Start Date: 52 Experimental Termination Date: Sponsor Project Number: < <> 53 Draft Report Date: < 54 Final Report Date < 60 TESTSYSTEM `ClSopsreadg-uceo-lDoanwyl,eyrraantsdoamr-ebsreeldectreoddbeentcsausaereofacthceepatvaabilleabimliotdyeolfshifsotrorimcuatlacgoenntircoiltydatsat.udies. 61 Source: CHhaarrllaensSRpirvaegrueBrDeaewdlienyg,LaInbco.r,atForreideersi,cKk,inMgsDtoonr, NY or Raleigh, NC or other approved alemates 62 Ageatinitiaion of sudy: 6-8 weeks 70 EXPERIMENTAL DESIGN AND METHODOLOGY The assay will be conducted et al, 1990; Hayashi et al., 1a9c9c4)o.rdiFnogltloowesitnagbltihseheaddmpirnoicsetdrautrieosn(oHfedtdhlree,e 1973; Mavournin concentrations of test substance bone marrow as well as positive and cells will be collected at negative 24 and (vehicle) 48 hours controls to male and examined for and the female rats, presence of wmiilclrobneucmleeaastuedrepdolbyychirtosmaabtiilcityertyothrionccyrteeass.e The clastogenic micronucleated potential of the test substance 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 Afier Dose Administration 24 Hr 48 Hr 5 5 5 - 5 - 5 5 5 - Protocol < August 28,2000 Page 20f7 . Sponsor Project Number: 7.0 SelectionofTest Substance Vehicle Unless the Sponsor determination will be has. indicated the conducted to measure test the substance vehicle, a_ solubility maximum soluble concentration or workable suspension system, in order of of test substance in preference, include, vehicle. but are Vehicles compatible with this test not limited to, distilled water or Saline, be that 19% carboxymethyleellulose in water, and allowing preparationofdosing solutions com oil. required The vehicle of choice will to achieve targeted doses. 72 Dose Selection Selection of doses test substance but for will the not micronucleus exceed 2000 assay wil mg/kg. In be based on the absence the of toxicity toxicity of the data, a fpielmoatlseturadtys.wi"llThbreeepoerrfmoorrmeedloatwearddoosseeosfw2il0l00bemgte/sktgeduussiinnggutpwtoo mfiavleemraaltes eaancdh.fivIef dmoisceroandumcilneiusstrtaetstiownilplrboedu2c0e0s0nmog/tkrge.atmIenntt-hreeleavteendt moofrtmaolrittayl,ittyheinheixgchesdsosoef 5fo0r%thaet 2000 up to mg/kg in the pilot four test substance study, doses, an extensive toxicity each containing up to study will five male be and performed using five female rats. 3Radtasyswiflorbcelionbicsaelrvsiegdnasftoefrcdhoesmeicaadlmienfifsecttr.atiBoondayndweciagchhtswowrilklinbge draeycotrhdeerdeapfrtieorrftoor dose administration and at | and 3 days after dose administration. Unless will be specified 50% to otherwise by the Sponsor, the high dose 80% of the LDsor (the dose required to for kill the micronucleus test 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. 73 Route and Frequency of Administration Animals will be dosed maximize delivery of by intraperitoneal (IP) injection. the test substance to the target IP injection system. IP was selected injection is to an aancicmeapltsa.bleAnmiemtahlosdwfiolrl ardemcieniivsettrhaetitoesnt osfubtsetstanscuebsatsaancseincgolnecaednmtirnaitsitornatsiotno.laboratory 74 Controls 7.4.1 Vehicle control The solvent vehicle for the test substance will be used as the vehicle control 742 Positive control Cyclophosphamide. administered as the monohydrate (CP, positive control at CAS a dose number of 30-60 6055-192) mg/kg. CP will will be be administered by the same route as the test substance. Protocol<> Aust 28.2000 Page of7 oh . Sponsor Project Number: 7.5 Animal Receipt and Quarantine dVoisruesaadmnitniibsotdrya-tfiroene. (VTAhFe)arnaitmsawlisllwiblel qbuearoabnsteirnveeddfcoarcnhowloersskitnhganda5ydafoyrsspirginosr otfo illness, health. uAnlulsaunailmaflosodwiallndbewjautedrgecdontosubmepthieaolnt,hyanpdrioorthteorutgielniezraatlioncoinndittheiosntsudoyf. poor 76 Animal Care Animals will be housed environment of 50+ 20% in an relative AAALAC-accredited facility humidity and 72 = 3F with a with a 12 hour controlled light/dark cycle. Rats autoclavable of the cages. same sex will be housed up Heat-reated hardwood chips to three will be per cage used for in plastic bedding `Animals analyzed wfiolrlehnavvierofnrmeeenatcaclecsosntotamaicnearnttifsieadndlatboortaatpowrayterro.dent chow which has been 77 Randomization u"Tshienganiamarlasndwoimlilzbaetiaosnsipgrnoecdetdourseevebnasgerdouopns of five males equalization and five of group females mean each body weights. At exceed 20% the of time of the mean randomization, the weight. Additional weight animals variation of animals will not may be designated and dosed as replacement animals prior to the scheduled in the high dose group sacrifice. This will be to be done used in the event of mortality at the discretion of the Study Director after evaluation of the toxicity data. Each animal will be given a sequential `number and identifiedbyear tag. 78 Dose Preparation and Administration The will test substance-vehicle mixture, the be given as a single administration. vehicle alone and the positive The rate of administration will control (CP) be 10 ml/kg btaordgyetwedeidgohste.unAllelssratlsarginertvheoleuxmpeesr,imuepnttaol 2g0romupiskewi,llarbee rweequiigrheeddtaonddeltihveerdotshee Volume will be based on individual body weight. 79 Protocol Bone Marrow Collection cTawrebnotny-dfioouxridaenadsp4h8yxhioatuirosn.aftTerhedopsoesiatdimvienciosnttrraotliognr,oaunpiwmialllsbewilslacbriefiscaecdri2f4ichedoubrys after dose administration. exposed, cut just above the Immediately following sacrifice, knee and the bone marrow will the be femurs will be aspirated into a asycraipnpgeedcocnetnatirniifnuggefettuablebcoovnitnaeinsicnrguma.pprTohxeimbaotneelyma|rmrLo.wfectealllsbowvililnebesetrruamn.sferred to The bone marrow cells will drawn off, leaving a small be pelleted amount of by centrifugation and the fetal bovine serum with supernatant will be the remaining cell pellet. The cells will be resuspended by aspiration with a capillary pipette and a Aug 28.2000 Pagetof7 5 2 Sponsor Project Number: small Each drop slide of the bone marrow will be identified by suspension will the experiment be spread onto a clean and animal number. glass slide. Al least two Ssltiadiensedwiwlilthbenupcrleeipcaraecdidf-rspoemcicfaicchstaanini,maalc,riadiirnedroireadn,gef.ixed by dipping in methanol, 7.10 Scoring for Micronuclei Slides will be coded using with the scoring process. a random number table by an Using medium magnification, individual an arca not involved of acceptable quality will immersion, be selected such that 2000 polychromatic the cells are erythrocytes well will spread and be scored stained. Using per animal for oil the 2pr0e0s0enpcoeloyfchmriocrmoantuiccleie.rythTrhoecynteusmbweirllofalmsiocrboenucelneuamteerdatneod.rmocTyhtees in the field proportion of of polychromatic erythrocytes. Tehreytphrroopcoyrtteisontooftoptoallyecrhyrtohmraotciycteesrywtihlrlocayltseos be to recorded per 1000 total erythrocytes in {est substance-treated animals should not be less than 20% of the control value. 80 CRITERIA FOR DETERMINATION OF A VALID TEST The mean incidence of (6/2000) polychromatic meircyrthornouccylteeast(ed0.p3o%l)ycihnrtohmeanteigcateirvyeth(rvoechyitcelse)mcuosnttronlo.t exceed 3/1000 The incidence of micronucleated polychromatic significantly increased relative to erythrocytes the negative in the positive control group must be control (ps0.05, Kastenbaum-Bowman Tables). 90 EVALUATION OF TEST RESULTS eTrhyethrionccyitdeesncwiellobfe mpircersoennutceldefaotredeacpholayncihmraolmaatnidc treerayttmhernotcygtreosup.perSt2at0i0st0icaplolsyicgnhirfoimcaatnicce will be determined using the Kasienbaum-Bowman tables which are based on the binomial distribution. mInarorrodewrttooxicqiutayn,ttihfey ptrhoepotresttiosnubosftapnocleychefrfoemcattiocn eerryytthhrroocpyotieessitso, taostalaneryitnhdrioccaytotresowfilblonbee presented for cach animal and treatment group. iAnltlercpornectaltuisoinoonfs twhielldatbae, tbhaesetedstosnubsstoaunncde wsiclilenbtieficconjsuiddgeermeedn1t0;inhdouwceevearp,osaistivae gruesipdoenset.o oifnaedoosre-mroerspeondsoisveesinacrreeassteatiinstmiiccalrloynueclleevaatteedd porellyacthirveomattoictheerytvhehriocclyetescoinstorbosler(vpe<d0.a0n5d, sKiagsnitfeincabnatluyme-lBeovwatmeadnatToanbelessa)criafticeantyimseawmiptlhinngo etviimde.enceIfoafasdionsgel-eretsrpeoantsmeenotrifgtrhoeurpe iiss erevliadteinvecetoofthaedocosnetrroelspgornosuep,witthhenSopoenvisdoerncwielloafbesciognntifaicctaendt. inTchreeasteesitn saunbysttarnecateedwiglrloubpe judged negative if `erythrocytes above no the statistically significant increase concurrent vehicle control values in micronucleated and no evidence of polychromatic dose response are observed at anysampling time. Protocol Auust28.2000 Page Sof7 ` . Sponsor Project Number: 100 REPORT `Aaccruerpaotretlyodfetshcerirbeesuallltsmoefthtohdiss sutsueddyfwoirlglenbeeraptrieopnaraendd baynaltyhseisToefsttihnegdLataab.orTathoeryreaponrdt wwiillll include: Tkensotwns;ubpshtyasnciec:ocheidmeinctailfipcraotpieonrtiaensdreCleAvSantntoo.thife known: conduct physical nature and purity, if of the study. if known; stability of the test substance,if known. + SolvenuVeicle: justification for choice of vehicles solubility and stability of the test Substance in the solvenuvehicl, if known. Test animals: species and strain of animals used; number, age and sex of animals; source, housing conditions, diet, etc. + Test conditions: finding study, if cpoonsdiutcitveeda:ndratnieognaatlievefo(rvedhoiscele/lesvoellvensetl)ecctoinotnr;oldedtaatial:s data from rangeof test substance preparation; details of administration; methods the administration for verifying that the of test steustb.stsaunbcsetarnecaec;hedratthioenagleen.erafolrcirrocuutlea.tioonf osramtparlgientgtisscshuee,diuflaepsp:limceatbhleo;d doetfaisllsidoefpfroeopdaraantidown;atmeretqhuaoldisty:fodresmceraipstuiroenomfetnrteaotfmteonxticiatnyd: criteria animal; cfroirtesrcioarfionrgcmoincsridoenruicnlgeastteuddyiamsmpaotsuirtievee,ryntehgraotciyvteeso;r number of equivocal cells analyzed per o eRresyutlhtrso:cytess;ignnsumobfertoxiocfity:micprroonpuocrlteiaotnedof ppoollyycchhrroommaattiicc erythrocytes erythrocytes among total per animal; r`emsepaonnsssetarnedlaartidondsehviipa,tiwohneroef pmoiscsriobnleu;clsetaattiesdticpaollayncahlryosemsa;ticconecruyrtrhernotcynteegsatpievregcroonutpro;lddoastea-s historical negative control data with ranges, means and standard deviations; concurrent positive control data Discussionofresults. 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>. 120 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE TTohxiiscoplroogtyo:coMlamhmaaslibaenenErwyrtihtrtoecnyteto MiccormopnluyclewuisthTesOt)E,CaDdopGtueiddel19i9n7e, 474 and (Genetic with the Protocol <> Aupst28.2000 Page60r7 97 - `Sponsor Project Number: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997). This study will be OECD, Japanese performed in (MAFF) and coEmEplCianGcoeodwitLhabtoheraptroorvyisiPornasctiocfethReegEuPlAati(oTnSsCAf)o,r Nonclinical Laboratory Studies. oUfnlfeoslslaorwirnagngaedmmeinntisstarraetimoandetottohtehetecsotntsryasrtye,munaunsdedaldlorseisnigdusaollutteisotnssuwbisltlabnecediwsipllosbeed disposed of following finalization of the report. 130 REFERENCES Heddle, L.A. 1973. A rapid in vivo test for chromosomal damage. Mutation Res. 18:187190. OHlayeassohni,Jr.M,.F,. RPRa.cchTiiecreo,ttJ,.TF.. MRacogmraeggonra,H,D.. SAhnidmeardsao,n,S. DS.uHt.oBulaaknedy,B.M.VanDiierrs.ch-V1o9l94d.ersf,nEv.iGv.o rodent erythrocyte micronucleus assay. Mutation Res. 312: 293-304 Mvaivvooumrincirno,nuKc.lH.e,usD-asHs.ayBliankmeya.mmMa.Cl.iaCnimbionnoe,mMaFr.roSwalaanmdopneeriapnhderJa.lA.blHoeodd.dleA. 1r9e9p0o.rtTohfethien U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res. 239:29-80. sMi.gAn.ifiKcaanscteeonfbmauutmatiaonnd fKre.qOu.encBieosw.maMnu.tatio1n970R.es. Tables for 9:527-549. determining the statistical 140 APPROVAL `Sponsor Representative Date (Print or Type Name) Study Director, Dae Study Management Dae Protocol Aust 28.2000 Page 7of7 2 Sponsor Project Number: Test Substance Name: In Vitro Mammalian Chromosome AberrationTest. 1.0 PURPOSE `uTphoenpiutrspaobsielitoyf ttohiisnsdtuucdeycihsrtoomeovsaolumaeteabtehreractliaosntsogiennihcumpoatnentpiearlipohfearaltebstlosoudbsltyamnpcheocbyatseesd (HPBL). 20 SPONSOR 21 Name <> 22 Address: < 23 Representative: < 24 Sponsor Project #: < 30 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 31 `Test Substance Name: < 32 Controls: Solvent: Positive: "Test Substance Solvent or Vehicle Mitomycin C (MMC) Cyclophosphamide (CP) 3.3 Test Substance Characterization Unless altemate arrangements are made, the testing facility will not perform analysis of the dosing solutions. The Sponsor will and documentationof the analytical purity be and directly responsible composition of the for test determination substance and the stability and strengthofthe test substance in the solvent (or vehicle). 34 Test Substance Retention Sample `tThheeSproetnesnotri.on of a reserve sampleof the test substance will be the responsibility of 40 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: < 42 Address < Protocol <> Aus 28.2000 Page of Contain NO cp; 99 43 Study Director <> Sponsor Project Number: 50 PROPOSED STUDY DATES 5.0 Proposed Experimental Start Date: < 52 Proposed Experimental Termination Date: <> 53 Proposed Report Date: < 54 Final Report Date: < 60 TESTSYSTEM ofPecriitphheerralrabdliootohderlaypmyp.hvoicryalteisnfweicltliobnes oobrttahieneaddmfirniosmtrhaetailotnhyofaddurlutgss.witThhoiustsaysrteecmenthahsisbteoerny demonstrated etal. 1981). to be sensitive to the clastogenic activity of a variety of chemicals (Preston 70 EXPERIMENTAL DESIGN AND METHODOLOGY m"Tihneitemsut mwilolfbfeoucroncdouncctenetdruatsiionngsstoafndtaherdtepsrtocseudbusrteasn,cebyaesxwpeolslinagshtuompaonsitliyvmephaoncdytseoslvteonta controls. In the the S9 activated non-activated test system, test system, treatment wil be exposure will be for 4 hours for4 hours and (Swierenga et for al., 20 hours: in 1991). The dividing cells chromosome awbielrlrabteioanrsreastteadpipnromxeitmaapthealsye and harvested 20 hours (1.5 nfoorrmmailcroceslclopciyccleevsa)luaafttierontohef initiation of (Galloway et treatment al, 1994). i`nTheorcdlearstotgoeneincsuproetenetviaalluoaftitohne of test first-division metaphase cells substance will be measured by its 0 ability to a control increase group. chromosome The 4 hour aberrations in non-activated a dose-responsive and S9-activated manner studies when will compared be scored einxiptoiaslulyr.e Innont-haecetvievnattoefdastpuodsyitmiaveyrneostpobnesescionretdh.e 4Thhoeurtenstons-uabcsttiavnatceedwisltludya,lstohebepraoslsoensgseedd for its ability to induce numerical chromosome aberrations, 7.1 Solubility Determination dUentleersmsinatthieonSwpiolnlsboercohnadsuctiendditocatdeedtertmheinetetshte ssoulbvsetnatncaend stohlevemnat,xiam. umsolsuobliulbiltey `concentration up system, in order to of a maximum preference, of 500 include mg/ml. but are nSootlvleinmtistecdomtpoatsitebrliele wwiattherthi(sCtAesSt (77C3A2S-168756)4,-1d)i.metThhyel ssuollfvoenxtidweil(lCbAeSt6h7e-6t8es-t5)s,ubestthaanncoel s(oClAveSnt6,4-s1e7l-e5c)te,dainndoarcdeetronoef preference, 500 mg/ml that permits preparation of the highest soluble stock concentration, up to Protocol Agus 28.2000 Page 20r9 100 72 73 Procol <> `SponsorProject Number: Preliminary Toxicity Assay to Select Dose Levels oSeflemcittioosnisofatfhre dtorseeatlmeevenlts afsordtehteecrymtiongeedneitnic4s acsystaoytowxiilclitbyesbtausdey.d upCoelnlsinhwiibliltiboen heixgphoessetdcotnocesnoltvreanttioanlobneeinagn5d0t0o0agt /lemastonrin1e0 mcoMncewnthriacthieovnesroifs tleoswters.ubsTthanecep,Htohef ntheecehsisgahreys,t itnestorsduebrstatnocemadionstianignsoalunteiuotnrawlillpHbe imneatshueretdr,eaatnmedntw.ilmebdeiaudmj.ustedT,heif aopspmloilcaalbiltey) oafndthethheighhiegshtesdtossionlgubsloelutdioosne, lleovweelst(wphreecriepiataptpilnigcadboles)e wlielvlela(lswoherbee fmeetaaslurebdo.vinePersipehreurma,l b2lomoMd celLl-sglwuitlalmibneec,ult1u0r0ed uinnitRsPMpIe-ni1c6il4l0incoantnadini1n0g0 15% tig esatrrleipetrowmiylclinb/emiexapnodse1d9foprhy4tohhoemuargsgliuntithneina.bseCnelcles asnededperdesaepnpcreoxoifma$t9elayn4d6fohrou2r0s hours in the absence of S9. After exposure the cultures wil be grown in complete nimne/itdmiia)tuimownifloolfr bte1r6eaadhtodmueerndst.tboytEhicegenhctturelitefunuregsha.toiuornCs,elatlfrsteeawrtieltdlrewbaiettmhceonlhtlyepiconttietodinaiattcio2nK0CCIoholsucorelsmutiaidfotner (at0nh.d1e sftiaxiendedw.itThhmeetphearncoeln-tgalgaecoifaclealclestiicn amciidt.osiMseptearp5h0as0eceplrlespasrcaotrieodns(miwitloltibceinmdaedxe) wainldl be determined for each treatment group. sWehleencteevder10 pgoisvsieblaet, the least h5ig0h%dotosxeiciftoyr t(hmeitocthircoimnohsiboimteionabreerlraattiivoen assay 10 the will be solvent wcoinltlrolb)e. evAatluleaatsetdtwino atdhdeitciohnraolmdoossoemelevealbse,rrdaetmioonnstarsastayi.ng mIinnitmhaeleovrennto ttohxeicitteys,t ssoulbvsetnatnc1e0 cbaenntootxicb,e tdhiesnsotlhveedhiagtheasthidgohseentooubgeh tceosntceedntwrialltiobne itnheanconacpepnrtorpartiiaotne wriestuhlttihneguinnaimdiendiemyeu.m pIrnectihpeietvaetniotntihnetteesstt mseubdsituamn.cePdreemcoinpisttartaitoenswaildlosbee-dreestpeornmsiinvee.d `icnocnrceeansteratiinonstotxhiactiteyxce(emditsootliucbiliinthyibiintitorneatrmeelnattimveedituom,thtehensotlhveenhtighceosnttrdools)e at to mbeineivmaluumatperdecfiopritacthironomionstoemste meabdeirurma.tionsIn will the ebveentthethactonnceeintthrearticoyntotroexsiuclittiyngnoirn aibnesrolruabtiilointyaisssoabysewirlvledbein Sthemgpr/emllimionrar1y0 temst,Mthewhhiicghheesvterdoisse in the lower. cIhf reoxmcoesssoimvee mpreedciipuimt,atoiornioftfhetheostemsot lsuablsitotafnyceth-esotlrveeanttmesnotlumtieodnioucmcuirssexucpeosnsiavded,ittihoenStpootnrseoartmweinllt be consulted. Frequency and Route of Administration h"Toaurrgseticneltlhsewailblsebnecetreoafte$d9f,orby4 ihnocuorrspoirnatthieonabosfetnhcee taenstd spurbessteannccee-osfo$l9v,enatndmifxotru2r0e `imnteothtohdeoftrdeeattemcetnitonomefdcihuemm.icaTlhcilsastteocghennisquien thhaiss tdesetmosnyssttreamt(eEdva1n0s,be19a75n),effective A820 Pagedors 01 74 75 7.6 Protocol Sponsor Project Number: If the Sponsor is aware will be utilized in the of specific preparation omfettahbeolsitcudryequdiesriegmne.nts,Vetrhiefincatthiisoninofforamactlieoanr Jpuossiiftiicvaetiroenspocnasne is not required. be provided. Negative Equivocal results will not be confirmed results may be confirmed, when upon cTohnissulgtuaitdiaonncweitishbtahseeSdpoonnsotrhe, OanEdCmDayGueimdepllionyea47m3odi(faidcoapttieodnJouflyth1e9s9t7u)dyanddesiIgCn.H `Guidance (1996). on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals Controls 74.1 Solvent (or Vehicle) Control s`Tohlevesnotlsveontthefror tthhaenteswtatseurb,staphnycseiowliollgibcaelusbeudffears,thoersomlevdenitumc,ontrtohle. For final concentration in treatment medium will not exceed 1%. 742 Positive Controls Mitomycin C ug/ml as the will be positive used at control two for tchoencneonnt-raacttiiovnastweidttheisnt the range system. of 0.1-0.50 For the S9- wacittihviantetdhesryasntgeem,ofcy1c0l-o7p5hoisg/pmhla.midOenewidllosebeleuvseeldofateatcwhopocsointcievnetrcaotnitornosl willbe evaluated microscopically for chromosome damage. Exogenous Metabolic Activation Aroclor The 9 1254-induced rat will be prepared lfivreormSm9awliellSbperaugsueed-aDsawthleeymertaatbsoliincduaccetdivawtiitohn system. a single Tinhtera$p9eriwtiolnleable ibnajtecchtipornepoafrAerdoacnldorst1o2r5e4d,f5ro0z0enmgat/kagp,prfoixviemadtaeylsyp-r7io0rCtounstaiclriufsiecde. Each batch metabolize preparation of S9 2-aminoanthracene will and be assayed for sterility and its 7,12-dimethylbenz(anthracene ability to to forms mutagenic to Salmonella typhimurium TA100. Immediately prior to use, "The final concentration of the the S9 will be thawed cofactors and S9 in and the mixed with a cofactor reaction vessel will be pool. 2 mM MgCl, 6 mM KCI, dinucleotide phosphate 1 mM and20 glucose-6-phosphate, | ul S9 per mi RPMI 1640 mM nicotinamide adenine serum-free medium. PreparationofTarget Cells Peripheral containing blood lymphocytes will 15% fetal bovine serum, 2bemcMulLi-ugrleudtaimnicnoe,mp1l0e0teunmitesdpieunimci(llRiPnMaIn-d1614000 ng strepiomycin/ml, blood t0 a centrifuge and tube 1% phytohemagglutinin) by containing 9.4 ml complete adding medium. 0.6 ml heparinized The tubes will be Ausi28,2000 Pagesof9 10v 77 78 79 7.10 Protocol `Sponsor Project Number: incubated upright 44-48 hours. at 37 + 1C in a humidified atmosphereof 5 19% COs in air for Test System Ideniification BUisoiRnegliaancpeersmtaudnyenntumbmearr,kitnrgeatpmeenn,t ctohneditteisotn asnydstdeatme. will be identified by the Treatment of Target Cells rFeofretdy-wfiotuhr atpopr4o8xihmoautreslyaf1t0ermlculctoumrpeleitnietimaetidoni,umdufpolrictahteencoenn-tarcitfiuvgaetetduebxepsoswiulrle boer o1f0dmoisiSn9g rseoalcuttiioonn omifxtteusrteorfocrontthreolacstuibvastteadnceexpionssuorlevetnot.whLiacrhgewrilvlobleumaedsdeodf d1o0s0inpgl, solution maybe used if water or medium is used as the solvent. aFnor$t9hereSa9c-taicotnivmaitxetduerex,powsausreh,edthefrceeellosf wil be treated chemical and fo4r hours in the presence cultured for an additional of 16 ahcotuirvsatewditehxpCooslucree,mitdreat(m0.e1ntuwgi/lmll)be present for for 4 hours the last 2 followed hours. by a 16 For hour the non- recovery lpaesrti2odhoaunrsd. for 20 hours continuously with Colcemid (0.1 ug/ml) present for the Collection of Metaphase Cells Cells 64-68 will be hours acfotlelreccutletduraeppirnoitxiiatmiaotn)e.ly 2Th0ishoutrismeafitserseilneicttieadtiotno of treatment (about represent the first- division harvest, metaphase Colcemid after will initiation of test be added to the substance cultures treatment. at a final Two hours prior to concentration of 0.1 ng/ml. washed The with tcwelolschwainllgebseofcoflilxeacttievde (bmyetcheanntorlifugglaatciiaoln,acterteiacteadciwdi,t3h:10.v/0v7),5McapKpCeld, wainldl sbteorceodlloevcetrendigbhytcoenrtrliofnuggeartiatonapapnrdoxriemsautsepleynd2e-d8iCn.frTesoh pfrixeaptairvee.sliAdnes,altihqeuoctelolfs fixed cells will be will be identified applied dropwise by the experiment onto a microscope number, treatment slide and air-dried. condition and date. The At slide least two with sGliiedemsswailalndbepeprrmeapnaernetdlyfrmooumnteeadc.h treatment tube. The slides will be stained Scoring for Metaphase Aberrations. Slides will be coded scoring process. At using least random 3 dose numbers levels in by an each individual not harvest will be involved with evaluated in the the aberration assay 7.2. The 4 hour annond-awicltlivbaetesdelaencdted9-aaccctoirvdaitnegd tsotutdhieescrwiitlelribaedessccorriebdediniitniaslleyc.tioInn the event extended of a positive non-activated response exposure in 4 may hnooturbenons-caocrteidv.ateMdetstaupdhya,seslicdeelslsfrwiolml the be examined under oil immersion without prior knowledge of treatment groups. Aus 28.2000 Page Sof 103 SponsorProject Number: ____ Whenever possible, a centromeres from each minimum dose level of 200 metaphase (100 per duplicate spreads treatment containing tube) will 46 be a`le. xa1m9i9n0e)d. a`nTdhescnourmebdefrorocfhrmoemtaatpihda-steypsepraenaddschthraotmwoislombee-teyxpaemianbeerdraatnidonssc(oSrceodttpeert dstuaptliisctaictaellyflsaisgknifmiacayntbleeverlebdeufcoerde1i0f0tcheellspearrceenstcoargeed.ofCharboemrartaintd-tcyelples abreerarcahteisonsa qiunacdlruidreadicahlrsom(astymimdetarincdalisaoncdhraosmyamtmiedtribcraelakisntearncdhangeexsc)h,antrgieradifailgsuraensd such as complex. rearrangements. exchange figures Chromosome-type such as dicentrics aberrations include chromosome and rings. Fragments (chromatid breaks and or acentric) oorbscehrrvoemdosinomthee).absFernacgemeonftasnoybesxecrhvaedngweitfhigaunreewxiclhlabnegescfoirgeudreuswiallbnroetakbe(cshcroormeadtiads `acnhraobmeorrsaotmieo(ns)b,utpuwlivlelribzeedconcselildseraendd psaervteroeflythdeaimnacgoemdplceetlelse(x2c1h0anagbee.rraPtiuolnvse)riwzielld ailnscoludbeed riencortdheed.analCyshirso.matTihdeanXdYiscooocrhdrionmaatteisd gaps will be for each cell recorded but not with a structural aibnedrerxatwiiolnl obregraepcowridleldbeasrethceorpdeerdceunstianggeaocfalciebllrsatiend mmiitcorsoisscpoepre5s0ta0gec.ellTshceoumnitteodti.c The percent polyploid metaphase cells for each and dose endoreduplicated level analyzed for cells will be evaluated structural aberrations. per 100 80 CRITERIA FOR DETERMINATION OF A VALID TEST 8.1 Solvent Controls Tcohnetroflrseqmuuesntcybeowfitcheilnlsthweirtahngsetroufcttuhrealhiscthorriocamlossoolmveentacboenrtrraotlions in the solvent 82 Positive Controls The percentage of cells Fisher' exact test) in the with aberrations must positive control relative btoe tshteatsiosltviecnatllcyonitnrcolr,eased (p<0.05, 90 EVALUATION OF TEST RESULTS Tthheectyotxoitcoxeifcfietcytsaonfdtcrheartommeonstoamree baabseerdrautipoonnsitnuhdyi.bitiTohneofnumimtboesirsaanndd twyiplelsboefraebpeorrrtaetdiofnosr. (structural population and of nceulmlesriecxaal)mifnoeudnd,(ptehrecepnetrcaebnetrargaentofcesltlrsu)c,turtahlelypedracmeangtaegde cells of in the total numerically damaged structural cells in the total aberrations per cell population of cells examined, (mean aberrations per cell) will and the average be calculated and number reported of for enoatchintcrleuadtemdenitn gtrhoeupt.otalChperrocmeanttiadgeanofdcieslolschwriotmhatoindegoarpsmoarree parbeesrreanttieodnsinorthien dtahteaabvuetraagree number of aberrations per cell Statistical exact test. aTnahleysFiisshoefr'tshetepsterwcielnltabgeeuosfedabteorrcaonmtpcaerlelspwaiilrlwibsee performed using the the percent aberrant Fisher's cells of Protocol Aust 28.2000 Page 6or9 104 i Sponsor Project Number: each exact treatment group test at any test with that of the substance dose solvent level, control. In the event the Cochran-Armitage of a test positive will be Fishers used to measuredose-responsiveness. All conclusions interpretation of will be the data, based the test on sound substance wsiclilenbteificconjsuiddgeermedentto;inhdouwceevearp,osaistivae guide to response: cifontcheentpreartcieonnts abbeeirnrgansttatciesltliscailslyisnicgrneiafsiecdantin(pa<0d.o0s5e)-.reAsproenpsriovdeucmiablnenesirgnwiiftihcanotneincorreamsoeraet the high dose only with no dose level other than the high dose response or a with no dose reproducible significant increase at one response will be considered positive. dose Test substances not demonstrating concluded to be negative. a statistically significant increase in aberrations will be 100 REPORT A report of the results of this accurately describe all methods study will be prepared used for generation and by the Testing Laboratory analysisof the data. and will Results presented will include, but not be limited to * Test substance: identification and CAS no., known: physicochemical properties relevant if to known; physical the conduct of nawre and purity, if the study, if known; stability of test substance, ifknown. + sSoulbvsetnatn/cVeehinicsloel:venjtu/svteihfiiccaltei,onif kfnoorwnc.hoice of vehicle: solubility and stability of test + nsouumrbceer)ofacnedllssuiatnadbiltiitmye otfhethceelclesllwteyrpee obtained, used. karyotype features (modal chromosome + test conditions: composition solvent selection rationale; of medium; CO concentration of concentration; test substance incubation time; solvent and and concentration selection rationale; composition and acceptability criteria for the metabolic activation (S9) system: mduertaatbioolnicofatcrteiavtamteinotn; dsuyrsatteimon uosfedt:reaptomseinttivweitahnadndscoolvnecnetntrcaotnitoronlos;f Cmoeltcheomidds;oftypseliodef cproenpsairdaetriionng;stnuudmiebserposoiftivcea,llnecgualttiurvees;orcerqiutievrioacalfo.r scoring aberrations and criteria. for + results: description inhibition relative of to precipitation; pH and the solvent control: omsimtooltailcitiyndoefxthaentdrenatummebnetrmeodfiumme;tampihtaosteisc eanaaclhyzterde;atetdypaendancdonnturomlbecrulotufrea;becrornacteionntsra(tsitornu-ctruersaplonasned rneulmaetiroincsahli)psgistvaetnistsiecpaalraatnaellyysifso:r historical control data 110 RECORDS AND ARCHIVES All raw data, the protocol and all Operating Procedure by the <facility, reports will address>. be maintained according to Standard Protocol Aus 28.2000 Page709 105" Sponsor Project Number: ______ 120 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE `This protocol has been written Chemicals (Genetic Toxicology: to comply with OECD Guideline 473 for Testing of In Vitro 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 OECD. Japanese performed in (MAFF) and compliance with the provisions of the EPA (TSCA), EEC Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. oUfnlfeoslsloawrirnagngaedmmeinntisstarraetimoandeto ttohtehetecsotntsryasrtye,munaunsdedalldorseisnigdusaollutteisotnssuwbisltlabnecediwsipllosbeed disposed of following finalizationof thereport. 13.0 REFERENCES Evans, HJ. and M.L. ORiordan. 1975. Human analysisof chromosome aberraintmiutoangesn tests. peripheral Mutation blood lymphocytes Res. 31:135-148, for the Galloway, S.M., Mosesso and T. M.J. Aardema, M. Ishidate Jr., Sofuni (1994) Report from LL. Ivew, DJ. working group Kirkland, T. Morita, P. on in vitro tests for chromosomal aberrations, Mutation Research 312(3):241-261. Registraion of Pharmaceuticals for Human Use. Guidance on Specine. Anpests of Intemational Conference on Harmonisation (ICH) of Technical Requirements for Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 of the April 24, 1996. ICH process on July 19, 1995. Federal Register 61:18198-18202, Intemational Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity 4 of the ICH Testing of Pharmaceuticals. process on July 16, 1997. S2B document recommended for adoption at step Federal Register 62:16026-16030, November 21, 1997. OECD Guideline for the Testing of Chromosome Aberration Test), Revised Chemicals, Guideline 473 (In Draft Document, Adopted July Vitro 1997. Mammalian Preston, RJ., W. Au, M.A. S. Wolffand 1.5. Wassom. Bender, J.G. Brewen, 1981. Mammalian in A.V. vivo Carano, J.A. Heddle, AF. McFee, and invitro cytogenetics assays: A reportof the US EPA's Gene-Tox Program. Mutation Res. 87: 143-188, Scott, D., N.D. Danford, B.J. Aberration Assays In Vitro. Dean and D.J. Kirkland. In: Basic Mutagenicity 1990, Tests: Metaphase Chromosome UKEMS Recommended Pose 2 Pagetars 106 : Sponsor Project Number: Procedures. D.J Kirkland (ed). Cambridge University Press, New York, NY. SawnideEr.eRn.gaNeSs.tHm.Ha.nn1(A1.991H)edRdelce,omEm.eA.ndSeigdalp,roLtPo.cWo.ls Gbialsemdano,nRaLs.urBvreiylolfingceurr,reGn.tR.praDcotuigcelaisn genotoxicity exchange in testing Chinese hlaambsortaetroroiveas,ry,IVV.79CChhrionmeosseolmueng aberration and human and. sister-chromatid lymphocyte cultures, Mutation Research 246:301-322. 140 APPROVAL Sponsor Representative (Print or Type Name) `Study Director Study Management Date TT Dae Date Procol Aus 28.2000 Page9or9 07 `Sponsor Project Number: Test Substance Name: Bacterial Reverse Mutation Test with an Independent Repeat Assay 10 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 $9 activation. 20 SPONSOR 21 Name: > 22 Address: < 23 Representative: < 24 Sponsor Project No.: < 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 31 `Test Substance Name: < 32 Test Substance LD: <> (10 be used in the report title and text) 33 Controls: NPoesgiattiivvee:: T9e-satmisnuobasctraindcienevehicle 2me-tahmyilnomaentthharnaecseunlefonate 2-nitrofluorene Sodium azide 34 Test Substance Characterization Unless altemate arrangements are made, the testing facility will not perform analysis of the dosing solutions. The Sponsor will be directly responsible for dteesttesrumbisntaatnicoen, aanndd dtohecusmtaebnitliattyiaonndofsttrheengatnhaolyfttihcealtepsutristuybsatnadncceoimnpotshietsioolnveonft t(hoer vehicle). Protocol A220 Bloll0 Contain NC cri hg . Sponsor Project Number: 35 Test Substance Retention Sample The retentionof a reserve sampleof the test substance will be the responsibility of the Sponsor. 4.0 "TESTING FACILITY AND KEY PERSONNEL Name: < Address: < Study Director: < 50 PROPOSED STUDY DATES 5.1 Experimental Start Date: < 52 Experimental Termination Date: < 54 Final Report Date: < 6.0 TEST SYSTEM ma 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 [aCronesTwco[|mows|| toe | ur0sJ|awenp | |r| r| CC ow| Tow |ww wT w a Tan |m]| 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 EE Ts excision-repair system. Tester strains TA98 and TAI00 also contain the pKM101 plasmid (carrying the R-factor). It has been suggested that the plasmid increases oe ony sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase pi J 109 Sponsor Project Number: TA98 and TAIS37 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TAI00 is reverted by both frameshift and base substitution mutagens and TA1S35 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 trp gene (Wilcox et al., 1990). Tester strain WP2 uvrA hasa deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due 1 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). 70 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, TAI00, TA535, TAI537 and WP2 uvrA with and without $9 activation. All dose levels of test substance, negative controls and positive controls will be plated in triplicate. 7.0 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 10: 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 0 500 mg/mL. 72 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 TA9S. TAI00, TAIS3S, TAIS37 and WP2urA to negative controls and 0 at least eight concentrations of test substance, one plate per dose level, in both the presence and absence of $9 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 Smg/plate. In selecting dose levels for the mutagenicity assay the following guidelines willbe employed. Doses will be selected such that precipitate Protocol Aug 28,2000 Page30f10 no . `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 me/plate. For freely soluble, nontoxic test substances, the highest dose level will be 5 me/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 S me/plate or (2) the maximum achievable test substance dose level is less than 5 mg/plate and this dose level is nontoxic. 73 Frequency and Route of Administration `The test system will be exposed to the test substance via the plate incorporation methodology originally described by Ames ef al. (1975) and updated by Maron and Ames (1983). This test system has been shown to detect a wide range of classesof chemical mutagens (McCann ef al. 1975; McCann and Ames, 1976). After the data generated in the firs 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) 74 Controls 74.1 Positive Controls All combinations of positive controls and tester strains plated concurrently with the assay are listed below. Protocol <> Augusi28.200 Page dof 10 1" 75 76 Protocol Sponsor Project Number: _______ 9 Concentration [SalmonellaStrain[s|ow 2aminoanthracene [ [h0 o7] [vom [0 | TAI. TAIS3S [sosmaiee[0 [mmeeththanesu|ti10o0n0 ae| 742 Negative Controls Appropriate negative controls will be plated for ach 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. 7.43 Sterility Controls "The most concentrated test substance dilution and the Sham and S9 mixes willbe checked for sterility. Exogenous Metabolic Activation Aroclor 125-induced rat liver $9 will be used as the metabolic activation system. The $9 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 baich prepared and stored frozen al 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 `mutagenictoS. typhimurium TA100. Immediately prior to use, the S9 will be thawed and mixed with a cofactor pool to contain 10% $9 homogenate, SmM glucose-6-phosphate, 4 mM prnicotinamide-adenine dinucleotide phosphate, mM MgCl: and 33 mM KCl 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. Preparation of Tester Strains. `Overnight cultures wil 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 endof 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 Aug 28,2000 Page Sof 10 na Sponsor Project Number: 77 78 79 7.00 7.11 -- 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 (0 some mutagens. Cultures will be removed from incubation at a density of approximately 10 cells/mL. 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. Test Substance Preparation Unless specified otherwise, test substance dilutions wil be prepared immediately prior 10 use. All test substance dosing will be at room temperature under yellow light Treatment of Test System One half milliliter (0.5 mL) of 9 mix or Sham mix, 100 uL of tester strain and SOUL of vehicle, test substance dilution or positive control will be added to 2.0m 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. Alter the overlay has solidified, the plates will be inverted and incubated for approximately 48 to 72 hours at 37 2C. Plates tha are not counted immediately following the incubation period will be stored at 2 - 8C. 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 10 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 afer the incubation period by visual examination without magnification. Tester Strain Verification On the day of use in the mutagenicity assay, all tester strain cultures wil be checked for the appropriate genetic markers cited in 6.0. A220 Pager 13 Sponsor Project Number: 80 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 rf mutation, all S. typhimurium tester strain cultures must exhibit sensitivity to crystal violet. To demonstrate the presence of the uvrB mutation, all 5. typhimurium tester strain cultures must exhibit sensitivity 10 ultraviolet light. To demonstrate the presence of the uvrA mutation, all E. coli tester strain cultures must exhibit sensitivity to ultraviolet light. To demonsirate the presence of the pKMIO1 plasmid R-factor, tester strain cultures of TA98 and TAI00 must exhibit resistance to ampicillin 82 Spontaneous Revertant Background Frequency Based on historical control data, all fester strain cultures must exhibit characteristic number of spontaneous revertants per plate in the negative controls (vehicle). The mean revertanis per plate must be within the following ranges (inclusive): TA98, 10-50; TAI0O, 80-240; TAIS3S, 5-45: TAIS37, 3-21; WP2 wrA, 1- 060. 83 TesterStrain Titers To ensure that appropriate numbers of bacteria are plated. al tester strain culture titers must be equal to or greater than 0.3x 10" cells per milliliter. 84 Positive Control Values Each mean positive control value must exhibi at least a three fold increase over the respective meannegative control value (vehicle) for each tester strain. 85 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 (his 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. 90 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 minimum of two increasing concentrations of test substance as specified below: Procol <> A 28.2000 Page Tof 10 14 ~ `Sponsor Project Number: ______ 9.1 Stains TAIS3S and TAIS37 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). 92 Strains TA98, TAI00 and WP2 ivrA Data sets will be judged positive if the increase in mean revertans at the peak of the dose response is equal to or greater than two times the mean negative control value (vehicle). 100 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 est substance, if known, SolvenuVehicle: justification for choice of vehicle: solubility and stability of test substance in solvent/vehicle, if known. Strains: strains used; numofbcelles/mrL.perculture; 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. 110 RECORDS AND ARCHIVES All raw data, the protocol and all reports will be maintained according to Standard Operating Procedure by the <facility name, address>. 120 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE `CThheismipcraoltsoc(oGlenheatsicbeTeonxicworiltotgeyn: tBoacctoermipallyRewvietrhseOEMuCtDatiGounidAeslsianye),47a1dopfioredTe1s9t9i7nganodf Protocol <> Aus 28.2000 Page Sof 10 15 ~ 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 130 REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonellalmammalian-microsome mutagenicity test. Mutation Research 31:347-364, Green, MH.L., and Muriel, W.J. (1976). Mutagen testing using tp" reversion in Escherichia coli. 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 sGteenpot4ooxfictihteyITCeHstpirnogceosfsPohnarJmualcyeu1t6i,ca1l9s9.7. SF2eBderdaolcuRemgeinstterre6c2o:m16m0e2n6d-e1d60f3o0r,aNdoopvteimobneart 21,1997. SMaclCmaonnne,llaJ.lmaincdrosAommees,tesBt:Na.ssa(1y97o6f).300Dectheecmtiicoanls:ofdcisacrucsisnioogn.ensPraosc. mNuattal.geAncsad.in Stcih.e USA 73:950-954. McCann, 1, Choi,E., YamasakiE,. and Ames, B.N. (1975). Detectionofcarcinogens as mutagens in the Salmonella/microsome Sci. USA 72:5135-5139. test: assay of 300 chemicals. Proc. Natl. Acad. Maron, DM. and Ames, B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test. Mutation Research 113:173-215. OECD Guideline Mutation Assay), A4d7o1ptfoerdTJeusltyin1g99o7f. Chemicals (Genetic Toxicology: Bacterial Reverse Protocol Augsi28.2000 Pager 10 16 " `Sponsor Project Number: SWaillmcoonxe,llPa.,tyNpahiidmouor,iuAm,TAW1e0dd2,wiDtJh. EsacnhderiGcahtieahocuolsie,WPD2G.tes(t1e9r9s0t)r.ainCs.omMpuatraigseonnesiosf 5285-291 140 APPROVAL Sponsor Representative Dae (Print or Type Name) Study Director Date Study Management Due Procol Aus 28.2000 Page 100710 ny Tilleer: DDeevveellooppmmeennttalTTooxxiiccity SStwuddyyiinnRkaatss Title: Developmental Toxicity Study in Rats Protocol o~Ontain Np CRI -_ Pare ToT ne oikee: DeDevveellooppmmeentnalTTooxriicciltyySSwtuddyyiinnRkatess . TAOFBCOL NTEE NTS Page OBJECTIVE mss 3 SPONSOR AND TEST FACILITY corso3 MATAERTIESASLUSBSAANNDCEMETHc ODS c omc nts s 33 Be VEhCIC Co ABS sss sss sees 3 3 DE..ExHpUeSrDiAmAeInYtalo Design........ s A t ---------- Fe Ge RSANCAOMIOBFUC DIOOSNCELEe UVISO. Ntree HL. SParfeeptayraPtrieocna,uAtiodns amndiDisnpoasianldAsOnf aWltaysstriesoafTtestiSUSoPEnnMsiAo,nEs]..........................5 J. KK. APniAmaIl EIQU1AN0NDaESSiaLAIE..T..S........oc .. srs s ------ 6 1. 2. IPno-lsitfmeoOrtbesm eObrsevrvaaottfioFinesmoaonfleFssemal..e..s...D..y. ing ri Prior to Scheduled Euthanasia. ---- .........6 3. 4. PoPsotstmmoorrtteemmOObsbervsatioensoroff DFvaemmasalDeesltiSuvreivriivniognEgantrolSysc.h.e.duled -- Euthanasia (DY 2G) 5. FetwsesofFemales Surviving to Scheduled -- wre Euthanasia...7 E L.. CONOFBIAS OI ce E rm-- ------ NN. O. AR DSOIC PIIOaNNCOSfATO MePStIEaM SnIdOCFS AOZNETOL SUBSIANCES.....crrr ss 8 8 CRITICAL DATEScss REFERENCESoss 8. -_-- 2 19 Tieler:DDoevvdelooppmneenntaslTToorrieclityySSwtuddyyiinnRkaeiss . OBJECTIVE TadhmeinpiusrtpeorseedobfytghiasvsatguedytoisprteogenvaanltuartatestfhreodmevtehelotpimmeenotfalimtpolxaicnittaytioofntToittlheee(ndCoomfpgoeusntdatsio)n, This study will conform with applicable Good Laboratory Practice Standards and Test Guidelines. ** SPONSOR AND TEST FACILITY TShpiosnssotrudayppisrosvpaolnsoofrtehde bsytu<dsypwoinlslobrenianmdiec>ataenddbwyililssbueanccoendoufcttheedwaotr<klaabuotrhaotroirzya,tilooncaftoiornms.. MATERIALS AND METHODS A. Test Substance TbyhethteesStasmupblsteaNncuembweirll.beThiedenteisitfiseudbisntatnhicsepwrioltlobcoelsaupnpdliinedthbeys<tsupdoynrseocro>r.ds<bSyponCsoomrp>oiusnd or r7e9s2p.oSnusbibplaertfoFr,csheacrtaicotner7i9z2a.t1i0o5n(ao)f.thAevtaeisltasbulbestiannfcoermiantciloundionngtthheepiutrietmys,cciotmepdoisnitCiFoRn,40, Part choanztaarmdionuasntmsat,esriyanlonclyamsss,ifCicAatSiorne(gsi)swtirlylnbuembperro,vibdaesdicbyph<ysspicoanlsoprro>peorntitehse, shaamzparldes,evaanlduation form. The original form will be retained in the official file for the study. B. Vehicle The vehicle wil be 0.5% methyl cellulose, U.P. 4000 centipoises, C.A.S. Reg. No. 9004-67-5. C. Animals d`stTerhvaeeilCnorwplam:seCncDtha(olSseDtno)xIibcGeicStayuBtsReestreiaxtntgewanasssirvseeeclbeoacmctmkeedgnrfodourendtdhibsiynsfttohuredmygautbiideoeclnaiunissesea.viaiTilshaaeb.lpeCrreflfre:orCmrDetdh(esSplDeic)tieIreGastSufrBoer,Rthe. rseulpaptliiveer,toanhdarpdrienveisosuasnsdtuidniceisdewnicteh other compounds. This of spontancous disease. strain is also considered suitable OChnaerlheusndRirveedrtLhairbtoyrantuolrliiepasr,ouInsc,. tiBmoed-ymawteeidghftesmaolnest,hewidlalybtehreercaetisvaerde on <> and <>, <year> from mated, day 0 of gestation (day 1.2, 0G) 03 will days be of supplied by the gestation upon vendor. receipt, The rats for and between this study will be 51 to 70 days old. requested to be at cither Each prior animal is assigned to shipping. Upon areuceniipqtu,ecnacuhmbaenrimaanldwiildlenbteifaiesdsiwgintehd an a ear puncvnotch by the unique animal number. supplier Both the alinstimoaflunniuqmubeeranainmdalthneuemabrerpusnacnhd/ncootrcrhesnpuomnbdeirngwiulnliqbueerceacrorpduendchoinnoetacchh ncaugmebecrarsd.wilAl bmeaster maintained with the study records. _--_-- #3 120 Tilmeer: DDeevcelhopemernttalTTooriieclityySSutduydyininkeRaetss . D. Husbandry aAntiamrgaelterdoroemlsatiwviellhbuemimdaiitnytaoifn5e0d %at 1a0n%a.ccepAtnaibmlaeltermopoemrsatwuirlel of be 2ar0t-i2fi6ciaCll(ytairlgleutmeidnaatte2d3C) and (bfeluhooruessecdenitnldiigvhitd)uoalnlya i1n2-shuosuprenldiegdh,t/wdiarrke-cmyecsleh,(satpapirnolxeissmastteeellyc0a6ge0s0.-18Ne0s0thionugrsm)a.terAinailmwaillsl nwoiltl be provided because the dams will be euthanized prior to parturition. International, Inc. Certified Rodent LabDiet 5002 will be available PMI Nutrition ad libitum. Water from <> will be available ad libitum. p<e>rfLoarbmoeradtpoerryiohdaiscaalnlya:nimal health monitoring program. The following procedures are Water samples are analyzed and other contaminanis. for total bacterial counts, and the presenceof coliforms, lead, Food samples are analyzed for the presence of bacteria and fungi. Samples from freshly washed cages sanitation by the cagewashers. and cage racks are analyzed to ensure adequate rCeerqtuiifrieemdenatnsimaanldftoeedbeisfurseeedo,fgiumapruarnitteieesd wbhyitchhe mmiagnhutfaicntfulrueerncteotmheeertessupelctifoifetdhenusttruitdiyo.nal TDahteaaanriemamlaihnetaalitnhedmosneiptaorraitneglypfrroogmrasmtuidsyadrmeicnoirsdstearnedd by the Laboratory Animal will not be included in the Veterinarian. final report unless warranted by the resultsofthese evaluations. LRaatbsorwaitlolrbyeAqnuiamraalntVienteedrifnoarraitanleaosrt 3 a days, and designee. then released for the study upon approval of the E. Experimental Design `The experimental design is shown below: _-- |[ Group i 1 nn mo v v Dose" (mg/kg/day Concentration' mg/mL) | o <> 00 > < <> | <> <> = = Mated Females 25 25 25 25 25 J * Commipokuends, administered once daily, by gavage, on days 6:20 ofgestion, at dosage volume of #s 12 Title: eDevceloopmnentalTTooxviceityySSwtuddyyiinkReates . * .Toachieve these concentrations of active ingredient the solutions il be adjustedfora purityof<> CVoephuilcaletioonnlyco(n0f.i5r%memdethyl cellulose) F. Selection of Dose Levels The dose levels for the current study were based on <>. G. Randomization Dams will be basis of day 0 assigned to lots gestation body waecicgohrtdsinagndtoatshseiigrngeedsttaotcioonntdraoyl. Then, they will and experimental be ranked on groups by. the arlalngdroomupssatmhpaltianrgefnrootmsttahteisrtaicnakleldy dliisft.feTrehne(dpi>s0t.r0ib5u)t.ioDnasmhosulthdatrelsouslet einxcmaessainvebowdeyigwhetigorhtasppfoerar ill prior to the start of dosing will be removed from the study. H. Preparation, Administration, and Analysis of Test Solutions mSaotleurtiiaolnswiotfhthtehetevsethimcalteerwiialll ibnetdheocvuemhiecnlteewdililn btehepsrteupdayrerdecdoaridlsy.. The method of mixing the test Compound recommended will be administered by by regulatory agencies gavage for this tbyepceauosfesttuhdey.oraTlhreouvtoeliusmteheadrmoiutneiswtheircedh is will be based on the most recent body weight tShaempbelgeisnn(i~n3g,mLtheeamcihd)dloef,eaancdh test the suspension will be taken end of the dosing period. tAhnraeleytsiimseosfdtuhreinfgirstthesasmtpuldyi:ngnweialrl waidldlreasdsdrheosmsocgoennceeinttrya.ticoonn.cenAtdrdaittiioonn,aalnsdasmtpalbielsitmy.ayFobre tthaekesnecaonndd/oarnadntahliyrzdesdamatpltihnegdsi,scarneatliyosnesof Othne dstauydsysdaimrepclteosr.arSeatmapklene.stwhiellsobleutsiuobnmsitrteemda,insihnorgtlayfteafrtdeorspirnegpawrialtliobne,ftrooztheen AfnoarlaydtdiictailonGarloup. `aTnhaelyasniasl,yitficraelqumireetdh.odFursoezednwislollubteiodnoscwuimllenbteeddisincatrhdeedstiufdnyoreacdodridtsi.onal analysis is necessary I Safety Precautions and Disposal of Waste Material cGoonotdamhionuasteikoneeopfinfgorpmruolcaetdiuornefsacwiilliltiebse. pTraoctaivcoeidd1s0kaivnociodntpaoctte,ntgilaolvheesalwitlhlhbaezawrodrsnanwdhen ihnaancdhleinmgiceiatlhehrotohde.teDstossiunbgstsaonlcuetioornseswtilslolbuetiponrse.parIendadindiptrioopne,rtlhye vteensttisluabtesdtaanrceaes.wilAlnbiemahlandled carcasses, feces, and unused dosing solutions will be incinerated. J. Animal Euthanasia pFreomcaeleedsinwgilwlibtehevuitshcearnailzeedxabmyincaatriboonns;ditohxoisdeetionbhaelavtiisocne.ralFleyteusxeasmiwinleldbweidtehcoauptidteactaepdibteaftoiroen will pbeentionbjeacrtbeidtawlitbhefsooredifuixmatpieonnt.obarbital. All other fetuses also will be injected with sodium --_-- Ss [2% Tike: nDevoeloppmeentalTTooxviceityySSwtduydyiinnkeRaetss K. Parameters to be Studied 1. In-life Observations of Females Body weights will quarantine release, be recorded and on days at least 6-21G. twFiocoedpcrioonrstuompdtosiionngwtilolpbreovmiedaesduarteadfoorn days 4, 6, d8o.s1i0n.g12t,o p1r4o,vi16d,e 1d8a,ta20f,oranqdua2r1aGn.tinCelirneilceaalses;isgingsnwsilwlilblebererceocrodreddedatolnecasetdtawiilcyeopnridoarytso 4-21G and twice daily recorded by exception. on days 6-20G. Clinical signs observed at other times will be 2. Postmortem Observations of Females Dying Prior to Scheduled Euthanasia A gross external and female is found dead. viRsactesralfoeuxnadmidneaatdiowinllwiblel breefrpiegrefroatremdeduntwiilthniencr2o4pshioeudrs after the pPorsesginblaen,cythsetasittuesswwiillllbbeedceotuenrtmeidneadndbycltahsesipfrieedseanscaepopraraebnstelnycealoivfeiomrplraenstoartbiionngsaitetsh.e If time of the dams death. 3. Postmortem Observationsof Dams Delivering Early dDeatmecstitohnatadnedliavgerrobsesfeoxrteesrcnahledaunldedviesuctehraalnaseixaamwiinllatbieoneuwtihlalnbiezepdewriftorhmiend2.4 hLoeusrisonosf dneostiegdneweil.l Cboerrpeotraainleudtefaorcofuurntthseranexdatmhienantuimobneratotfhevidsiisbclreetiimopnloafntattheiosntusidtyesdiwrielcltobreor a recorded. pFoestsuisbelse,rebmutaitnhiencgolilnetchteedutdeartuaswainlld btehorseecdoerldievderweidtheatrhley mwaitlelrbnealexgaromsisnepdosttomtohreteexmtent findings 50 as live fetuses. to easily permit exclusion of the data from all calculations performed for 4. Postmortem Observations of Females Surviving to Scheduled Euthanasia (Day 21G) rVeitsacienreadwfiolrlfbuertehxerameixnaemidngartoisosnlyatitmhmeeddiisactreeltyioanftoefrtehuethsatnuadsyiad.ireLcetsoiroonrsanodteseidgnweilel. beThe 0intpaectrmaintdctahlecuelmaptitoyn uotfemrautseorfneaalcbhoddaymwehiagvhitngadajtulsetaesdt tooneexvcilaubdlee tfheetupsrwoidlulctbse owfeighed conception. recorded. The corpora lutea count for each ovary of females with viable fetuses will be fFeotrusceasc,hanfdemraelseorwpittihovni)siabnlde implantation their relative sites, the positions types of implants will be recorded. (live The and dead uterus of each caaprplayrernetsloyrp"tinoonnsp;recgonuanntts"wifelmbaelerewciolrldbede.staDiantead fwriothm afemmmaolensiduemtesrumlifnieddetotobdeeptercetgnvaenrty fbeymastlaeisniwnigthwiltoltable used only to resorptions. calculate the incidenceof pregnancy and the number of _--_-- o 123 Tiler: cDevmelopementalTTooxviceiytySSutuddyyiinnkRaetss. 5. Fetuses of Females Surviving to Scheduled Euthanasia `fTohteusiwnitlrlaubteerrineecolrodceadt.ioTnohfe eeaxtcehrnfaeltuasltweirlaltiboensredceotredecdt.ed Tfohreesaecxh alinvde bfoetduys wwielilghbte of cach recorded. For each litter, the fist Jive fetus and every other live fetus thereafter will be aetthxeeaxmsticenhreenadmlefeooxrfavmWiisoncoaertaailonnadlwHtieolrlaarbtei.o"enxsaI.nm"iaRnddeeitdtairfoodnre,dsaolrfletnltaiilvsesdufeeevteaullsteoespramwteiintotnhs;wmiadllelfcoabrpeimctalatatisiosoinnfsoiefvdistuihsbeilsneeg fetuses will be at Bouin's fixative, the the hdiesacdrsetoifondeocfaptihteastteuddfyedtiurseesctwoirlolrbea designee. examined After fixation in and alterations will be raelccoorhdoeld.fixEatxiaomni,ntahteiaolnisz awriilnl-sbteaibnaesdesdkoelnettohne method s will be of ex Barrow amined and and Tskaeyleltoarl .a"lteArfatteirons will be recorded for each live fetus. TDhatoasefofrettuhseesse cfleatsussiefsiewdilalsbdeeaedxcwliulldebdeferxoammialnlecdalecxutleartniaolnlys,pveirsfcoerramleldy,faorndlisvkeelfeettuaslelsy.. dFieatgalnoosrigs.ansBiolratteirsaslueosrgwailnlsbweilrlebtaeinreedtaiifndedeewmheedn noencleyssoanreyifsoarffoebcttaeid.ninSgufafdiecfiiennittitvisesues tfirsosmuecsownitlrlolbefedtiussceasrwdieldl ibfenoftixneededteodp.rovide comparison with suspect tissues,if needed: L. Control of Bias cIuntahdadniatsiioanatnodrwainldlormemaasisnigcnomdeendt dtuorgirnoguptsh,e calolllfeecmtailoensofwipllosbtemcoordteedm baenfdorfeetaslchdeatdau.led M. Statistics Sbeeqluoewn.tiIafla tswiog-ntiafiilceadnttrdeonsde-treesstpionngs"e wiisldletbeectaepdp.lideadtatofrthoemdtahteatfoopr dcoasceh gpraoruapmewtielrl abseteaxbculluadteedd aafnfdecttheedtfesetturseepseapteerdliutnetrilornothseiglniitfteircamnetatnrewnidllisbedetuescetdeda.s tFhoerelxiptteerripmaernatmaelteurnsit, ftohre sptraotpisotritcialon of euvseadl,uaatnidoont.h"erTphaeralmeevteleorfssainganliyfziecda,nicfedseeelmecetdedneiscepss<ar0.y0.5. Additional statistical tests may be `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.' [ Parameter Maternal Matemal weight weight change | Maternal food consumption Trend Test Linear contrast of means''" | | Dead fetuses. -_-- | 4 124 ee litmlee: DeDevvcellooppmeennttaalTToovriielctiyySSwtduydyininReRaetss . Resorptions Implantations Incidence of fetal alterations Incidence of pregnancy Clinical observations Cochran-Armitage test" | Maternal mortality Females with total resorption Early deliveries |i | Fetal weight sex ratio) (Covariates: litter size, Sex ratio (Covariate: liter size) Linear contrast of least square means'" | N. Absorption of Test and Control Substances nDoettenremciensastairoyntoofacthheiedveegrtehee of absorption objectives of of the the test study. and control substances by the test system is 0. Records and Sample Storage Specimens (ifapplicable), raw data, and the final report will be retained at <>. CRITICALDATES Study Start (first day 66): [ <> [ Completion (ast fetal evaluation): Final Report: | | <> <> (approximate) REFERENCES 1. RGeogoidsteLrab5o4r,aNtoo.ry1P5r8ac(tTiocxeiSctaSnudbasrtdasn,ceUs.SC.onEtnrvoilr;o4n0meCnFtaRlPParrotte7c9t2i)o,npApg3e4n0c4y3-(E3P4A0)5,0 F(e1d9e8r9a)l. 2. UI.nSs.ectEincvidier,onFmuenngtiacildPeraotnedctRioodnenAtgiecnicdey (EPA), Good Act), 40 CFR Laboratory 160 (1989). Practice Standards (Federal 3. Organisation for Standards. ISBN E9c2o-6n4o-m1i2c36C7o-o9p,erPaatriison(1a9n8d2)D.evelopment, Good Laboratory Practice 4. MSitnainsdtarrydsoffoArgTriocxuilctoulroeg,icFaolreSstturdyieasndonFiAsghreirciuelst(urMaAlFCFh)e,miOcnalGso,o5d9 LNaobhoSraatnorNyo.Pr3a8c5t0ic,e Tokyo, Japan (1984) 5. U.S. EPA Health Study (1998) Effects Test Guidelines OPPTS 870.3700 Prenatal Developmental Toxicity _--_--m + 125 . ike: DecveolompmeentalTToorviceiyySSutuddyyiinnRRaatss 6. OorfgCahneimsiactailosn,fSoercEtcioonno4m,iNco.Co4o1p4e,raPatriiosn(a1n9d81D)evelopment (OECD), Guidelines for Testing 7. DMiantiasftorryAopfpAlgirciactuilotnuroafl,AgFroirceuslttruyraalndChFeimsihecrailesRe(gMiAstFrFa)ti,onG,ui5d9elNionheSsaonn NToo.xi4c2o0l0o,gyToSktyuod,y Japan (1985). 8. CL1o3m3m,isVosli.on31,Di3r0e.c5t.i8ve8)87/302/EEC (Official Journal of the European Communities No. 9. ISmaplleawnstkait,ioEn. st(e1l9l6e4n).amFaUrtbeermuestdheordeRatztue.m makreoskopischen Archiv. Path. Exp. Nachweis von Pharmakol, 247:367. 10. Staples, R.E. (1974). 9(3):AIT-A38 Detectionof Visceral Alterations in Mammalian Fetuses. Teratology 11. Woo, D.C. and Hoar, Development in Late R.M. (1972). Apparent Gestationof Rats: The Hydronephrosis Effect of Methyl SaasliacyNloartem.alTAesrpateocltoogfy Renal 6:191- 196. 12. FBeatrwrsoews,."M.J.V.Moarnpdh.Tay1l2o7r(,3)W:.2J9.1-(310966.9). A Rapid Method for Detecting Malformations in Rat 13. SinelAwnyinm,alMRS.afe(t1y99S5t)u.dieTsh.e" UJsoeuronfalTroefntdheTeAsmtesrtiocaDnetCeorlmliengeeaofNToo-xOibcsoelrovagbyl1e4-(E2f)f:e1c5t8-L1e6v8e,l 14. SHtausdieemsa.n,TJerKa.toalnodgyHo1g2a:n1,65M-.1D7.1 (1975). Selection of the Experimental Unit in Teratology 15. Patefield, W. (1982). Statistics 31:32-43. Exact Tests for Trends in Ordered Contingency Tables. Applied 16. SUnnievdeercsoirt,yGP.rWe.ss,anlodwCao,cphpr.an2,46W-.2G4.8,(3149967-)3.52.Statistical Methods, 6th ed. The Iowa State 17. BJoinoemkehtercirkea,4A1:R1.33(-1194554.). A Distribution-Free K-Sample Test Against Ordered Alternatives, 18. Dempster, A.P., Computational ASseplewcytnso,fM.MRi.x,edPatMeol,deC.lM.A,naalnysdisR.otTh,heA.JJo.ur(n19a8l4)o.f Statistical and the Royal Statistical Society, Series C (Applied Statistics) 33(2):203-214. ES Re . Tim tlen:eDevee lopme entalTTooxviceityySSetddyyiinn kRaetes . Title: Developmental Toxicity Study in Rats Protocol Prepared by; SD, SDDegreer SDTitle Study Director -- Date Approved by: cc < - Chemist Date < <Director> Date - T0g 127 Modified Approach for Describing Telomer B Alcohol Kinetics | # | SOQ i|neprlminMaoyde x Y & v EKixnoentsMoodenletonRaeldioce. sep00 Joins with "B" wg I. I a} Joins with "C" pn 129 "on N,| iinntcoorMpoodraetle Define Metabolism) PBrioncdairnt?gisms TDaertgeermtiKnieneEtviecsn,t, Species Specific Joins with "D" AKbnsoowrpntDieonrmKailnetics > N_,| oPbaariiinopneCmoeefafbiciilenyts Incorporate 1950 130 FeomcpaN v a on'. Coefficients NeCompiare Cioeen Ny! Disuibuiion m[ootae | orioa RKEoporreootm 131 ign In Vitro Microsomal Metabolism Study Objective: `iTn hmeiocbrjoesctoimveesopfrtehpiasrsetdudfyrotmypleiviesrtsoodfaetenrummibneerthoef rsapteecioefsm(entoarbmoallilsymoraft, aratbebsittc, odmogp,ound monkey, and human) and to identify the metabolites produced in each species. Methods: Fdiorstthii,tirseancetcieosnssararyetorudnetweirthmipnreotleiinncacrointyceonftrraetaictoinosnirnattehewirtahngperooftein0.c5o-3ncmegn/trmaLtiporno.teTion Rwehaiclteihoonlsdairneg ctahreristeadrotiuntgacto3n7cenCt.raTtihoen raenadctiroenacmtieodnituimmecocnosnissttasnotffaorneNacAhDsPpeHcies. rceogmepneoruantdioins saydsdteedm,tocotfhaectroerasc,timoincmroesdoimuems,toanadchpiheovsepahfaitnealbucfofnecre,nptrHat7.i4o.n oTfes1t-100 uM, dientcuebramtiendedfobryapciolnotstsatnutditeism.eT,hdeetreeramctiinoendiisnsptialrottedstbuyditehse. aTdhdeitrieoanctoifonNAisDtPermsiolnuattieodnbaynd tpuhreiafdideidtbiyoncoefnetirtihfuegratoirogna,niscolsidolphvaesnoertexsttrraocntgioanc,ido.r bTohthe. tTeshtessaammpplleessaraerethtehnenpaarntailalylzyed by HPLCMS, Ncoenxstt,anttheikniitnaelticcosnocfentthreatteisotncoofmtpeostusnudbsatraendceetearnmdinaedc.onsTtaontdoprtohties,inrecaocntcieonntsraatrieonr.un at a Rreegaecnteiroantsioanresycasrtreime,dcoouftacattor3s7, mCi.crTohseomreeasc,tiaonndmpehdosipuhmatceonbsuifsftesr,ofpaHn7N.4.ADTPesHt dceotmepromuinneddibsyadpidleodt tsotutdhieesr.eTachteiorneamcetdioinuims tstoaarctehdiebvyetahefianadldictoinocnenotfrNatAiDonPofso1lu-t1i0o0nuaMn,d trheeacrteiaocntiisontseramrientaetremdinbayttehdeaatdvdairtiioounsotfiemiethpeorinotrsg,andiectesromlivnenetd oirn spitlrootngstaucdiide.s.TThheetest samples samples are are then then partially purified by centrifugation, analyzed by HPLC-MS. solid phase extraction, or both, The `rTehseulctoinncgecnotnrcaetnitornatoifoenasc(hinununkintsowonf WsMam)plareeipslcoatltceudloatnead usesmiin-glaogsatraintdhairmdiccuprlvoet.agTahienst ctiimsethienymiinnutteersc.eptAnanedxbpoinsenthteiaflirsrtegorredsesriorantleicnoenwsittahntthfeorgdeinesraaplpeeaqruaantcieoonfyth=ecetesTMtwhere cInotmripnosiucncdleisarfainttceed to the (CLiy) data. was Jn vitro half calculated by --life two was calculated as different methods. t7 = The 0.693/b. first method crueqruviere(sAdUiCv,idiinngnmtoheleinmiitina/l maLm)oufnrtomofa tpelsottcoofmcpoonucenndtr(aintinomnovle)rsbuystthiemet.otaTl haereqauoutnideenrt tihse utnhietns doifvimdLe/dmbiyn/thmegtoptraolteainm.ouMnutlotfipplryoitneginth(iisn vmagl)ueinbythteheregarctaimosnovfilali,vegrivwienigghCtLipyerinkg of body weight and mg of protein per gram of liver gives CLiy in units of mL/min/ke. 132 TthheepsreotceoinndcmoenctehnotdraotfiocnalicnulnaittisngoCfLmigy/imnLv,olwvheis cdhivgiidviensg CtLhieyfiirnsttoerrdmesr oaftmeLc/omnsitna/nmt gby pbroodtyeiwn.eiAghstiannmdemthgoodf1p,rmoutletinippleyringgrathmisovfalliuveerbgyivtehse CgrLamsinofunliitvserofwemiUgmhitnp/ekrgg.ram of `sTohmeeCLiinfyerveanlcueess twoebree mfuardteherrecgoamrdpianrgefdirwsittphasessteilmiamtiensatoifohneepfafteicctsblionotdheflvoawritoousalslpoewcies. Metabolites standards,if are identified available. by HPLC-MS, and their identity is confirmed with authentic 132 ee METABOLISM OF TELOMER B BY MAMMALIAN HEPATOCYTES Psroimnecwiphlaet: gIrseoaltaetrebdihoelopgaitcoaclytceomspulsepxeintsyiocnosmppraorveiddetoa shuibg-hceclalpualcairtfyraicntivointsr.o mHeetpaabtoolciyctessysetxepmreosfs galelneorfatlhleyepnrzovyimdeesaarnedalciostfiacctaoprpsroasxsiomcaitaitoend owfitahgniovremnaclhheempiactailcschinemviicvaolmemteatbaobloiltiespmroafnidl.e TTeesltomSpeercBiesm:etHaebpoaltioscmytsetsudfireso:m males and females of the following species will be used for Sprague-Dawley Rat CD-1 Mouse CNyenwomZoelalgaunsdMWohnikteeyRabbit Beagle Dog Human mHeeptahtoodc.ytCersyofprroemserrovdeendtnsopne-crioedseanrtehperpeaptaorceydteisn ahroeusaveaiblyabtlhee ctowmom-esrtcaigaellcyo.llagenase perfusion iSstuildlyusDterastiegdni:nTthheefboalsliocwdiensgitganblfe:oar hepatocyte metabolism experiment for a given species/sex. GE roTupoExpReNroiElmeental 2 Pos. Control 3 Neg Control s*oHeam4l inactNiNeveaggteCdCoonnttrrooll tN5 et Cells 5(x110)) TSSeuilbosbmtriearteB 5 5 Ethoxycoumarin 5 5 Vehicle 5s S5t TTeelloommeerrBB a`nGarloyutpic2alasbsaucrkesgrtohuenmdetsaabmoplliec aancdtiGvirtoyuopf 4thceohnetrpoaltsocfyotrensouns-peennzsyimoants,icGdreogurpad3atpiroonviodfetshaentest compound. Hepatocytes are suspended in Licbovitz L-1S medium ataconcentration of ecaxoptoprlraiocnxtgsi,maaarcteiedalinyfaicl|ayxtzieo1dn0,bocyreLlalCdsd//imMtLiS.onfRooerfapcoartrgieaonnntiscaansrdeolmivenetcnautbboaalstieatdpepsartop3r7iatCe.foF2orllhrosw,itnhgesnatmeprlmeincalteeadn-buyp, 134 _-- DRAFT PROTOCOL Toxicokinetics of Telomer B Alcohol in the Rat Project Number: DuPont-<> `Work Request Number: <> Service Code Number: <> DuPont Animal Welfare Commitee No. <> Page Tol 15 135" DL TraOftNPIo rCoOKtu IWoORNFcEXc TXoTEXLIlOXMCEo SR BMAd LoCOnHOw oL ImNTHen ERrAT oorTaovmB TAOFBCOL NTEE NTS ToxiocfToelokmeriB AnICOeRO!itfthie Rcals... | SOPbOESCIOVTEASNc TES FACHo Y v rs se e t 3 3 MRBEUGEATTAU ESIMtEGIAUNIOIEASTISNOESo FY ssr n m 3 Ar B. TRESUaSUBdSIAiNGEoTec sltSUaBSIbo ANCeESv lm ec d e or se 4 Di E. ADA ADIMa HHeUaSlDtAhNMALYOBw IONGo cc..e . s 6 F. DosePreparation, ANalayndSRiAEsS........euvemsomrrvrososos 6 He 1.PHOPUlSaWsAmIaEkSi.no etics follows ing oral dosie ng with non-radiolabeled TBA... 7 Lo 2. MAIExNcSrIetUiYonc and metabolismo following oraln dosing with rads iolabeled TBA..e .............87 1. 2. DAiBSSUObPUHLIOONNEEXXPPEFEMIENMIIr CNIS.o .cccrs smsse meeess eoee 8 J. 3. QExucreatinonaotnfdRiaMdeittoaabcaotliivtsemRiEEXSPoUESMn.EN.t.............uower cores 10 12 Le PIS tse 12 ET 5. CASESE corse 1 12 6 FERTCSIAUC..osnnememsmsomsor eo 12 Ke8. MBSIaETmipRlleBs AfrIomAdeNrmCalOeX.P.EToIMrENrS.r.s..sc.m.rsroermsesmsemesoomrsorn 13 13 L. Mo SClUinSicEal]OAbNSBeIrYvSaanEtdSMiO.ToAcnIirsYre..n..e.m..s.oo.omosmweeenrsemerneenremss 13 13 N. Safety aMd EHOUaSEKSEEPoUIfNGREAc dImONCMHVILEY s nt v o core 13 14 RECTUSaNd SAMPIC SIORAZE CHCA DAES. verses 14 14 SIREMFAEUTERNECSESo s nn se en er 15 15 _-- P13y 6 JoTDOrwaXcfItCoPOxrKonItNeoEcnToIlcCWsSORoFrXTTXEoLXOXwMER BbAAlLcCOoHnOoLuImNmTHeErRaArT INTRODUCTION sTuerlfaocmteanrtsB. alTcBohAolis(sTiBmAil)arisian fstlruuocrtiunraetetdo aplecrofhlouloruosoecdtainnotihceamcaidnu(fPaFcOtAur)e, oafnr8e-pcealrlebnotns,asntrdaight 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. fofolPlFowOiAn,g oarsaolndloyspianrge.n"t wTahsisdseetxecdtiefdfeinreenxccerewtaasaanpdpatrisesnutelsy, nboutt drautehetro tdoiftfheerepnrtieaslenmceetaobfolism Bteisntdoisntegrotonea, mwahliec-hspwecaisficcortreesltaotsetderwointeh-ianrdeucdiubclteiopnroitnetihneienlsimeirnuamtioorntriastseueofmaPyFOexAplianimnatlhee rats. excretion increased differences observed; renal elimination, via female rats the organic with acid tlreassnsPpoFrOt Asybsitnedmi,nwghpircohteiisnaacptpivaereinntflyemhaalvees but not male rats. P`Tehreflmueocrhoadenciasnmotioc aecxipdla(iPnFtDhAe)o,base1r0v-ecdarsbeoxndipfefrefrleunocreisnamtaedycahlesmoibceals,trwuacstuerlei-amcitniavtietdy mbaosrede. slowly than the sex differences shorter in renal 8e-xccarrebtoionnPaFnOd Atoxiincifteym. ale compared to male rats, and lacks the observed (OBJECTIVES The objectives `metabolism of of this study TBA in male are to determine and female rats the absorption, distribution, excretion following oral and dermal dosing, and SPONSOR AND TEST FACILITY t`ThheisdastteudtyheisspsopnosnosroraeudthboyritzheedTtehleowmoerrkRoensetahrechWoGrrkouApu.thTohriezsaptoinosnoFro'srma.pproval was effective `ET.heI. sdtuudPyonwtildlebNeecmoonudurcstaenddatCHoamspkaenlyl,LaNbeowraartko,ryDfeolrawTaorxei,coilnoagcycoarnddaInncdeuswtirtihalalMledaippcliincea,ble Good Laboratory final report. Practice Standards. >" Areas of noncompliance will be documented in the REGULATORY TEST GUIDELINES `TTehsitsisntguodfy Chhaesmbiecealnsd,eTsoixginceodkitnoemteicest,t4he17te(sAtpirniglre1q98u4i)r,emaenndts(2o)fU(S1) EOPEACHDeaGlutihdeElfifneecstsfoTrest Guidelines, OPPTS 70.7485, Metabolism and Pharmacokinetics. - 137 TDTOrNosIOPRwrMoNtoEccToIlCoWSRomFXTE0mL0OgVERrB AcLCOsHOoL TrEtATmoenBMconorwmener MATERIALS AND METHODS A. Test Substance The test substance, Telomer B alcohol purity will be supplied by the sponsor (TBA), and will and an analytical standard of TBA be assigned a Haskell Laboratory of known Number, which will be documented in the study records. CAS Registry Number: <> Molecular Weight: 464 Molecular Formula: CiHsF;; 0 Structure: oR E EN OH F F CF E F F F FF Purity: >95% -_-- 138 JTDTrOoaXxfItcCOPorKxoIntNoeEcTnoIlcCWsSORoFrXTTEXeLLXOoXMwEeRn BbAAtLcCOoHnOoLuINwTmHEeRrAaTr 0 B. Radiolabeled Test Substances Specific Activity: Radiochemical Purity: To be determined 595% [1-C"] Telomer B alcohol [10-C*) Telomer B alcohol LR K E , OH K yF R FF RR F RB E E OH LA E yF K R AS <% F [] denotes the position of the radiolabel C. Test Species MLaabloeraatnordifese,maIlnce.,CrRall:eCiDg*h,(SNDo)rItGhSCaBrRolirantas. will Th be used e rat was and sel obtained ected for from use in Charles River the study, as i t is the principal toxicology animal model. The Sprague-Dawley rat has been used for toxicokinetic testing of other fluorinated test materials.' At20th%e otfimteheomfedoasninwge,igrhatts bsyhoduolsdebgerosuepx.ualElaycmhaatnuirmeaalnwdiltlhebeweaisgshitgnveadriaatniioqnusehoiudledntniofticeaxtcioened number to be used will be marked on throughout the the tail of each study. animal The last 3 digits in indelible ink. of the animal identification number D. Animal Husbandry Upon arrival at Haskell Laboratory, ras appropriate cages, according to Standard wOilplerbaetirnegmoPvroecdedfurroemssLhAipQp0i3n-gPc-a0r0t6onsunalnedsshsopuesceifdieind in tahpeprsotvudeyd roetchoerrdwsi.seAnbiymtahlessiwtiellvebteermianianritaani,nehdavuendtehrreqeuraercaonrtdiende wfoerigahttlegaasitnssixanddaynsounless abnormalities detected. After the quarantine period, rats will be selected for study. _ 1 = JoTDOrxaXcfItCoPOxrKonItNeoEcmToIleCsWSoROrFXTTXEeLiXOoXMwEsReBBAAvLCcOoHnOoLmINmTeHErRaArT 0 Animal Animal rooms rooms are are targeted at artificially ailtleummpineartaetdur(efloufor2e3sc1entCliagnhdt)aornelaat1i2ve-hhouumridliigthyt/odfar4k0-cy6c0le%.. `Throughout appropriate the dosing period with cages according to the test SOP. compound, the rats will be housed individually in PadMIlibNiutturmi.tioAnniImntaelrsnawtiilolnanlo,tIbnce. fCaesrtteidfbieedfoRroeddeonstinLgabwDiithettes5t0s0u2bsatnandcet.ap water will be provided E. Animal Health Monitoring pHeasrkfeolrlmeLdabpoerraitodoircyalhlaystaonaasnsiurmealthhaetacltohntmaomniintaonrtisngarperonogtrapmr.eseTnhteatfollelveolwsintghaptrwooceudludrebse are expected to impact the scientific integrity of the study. + Water samples are analyzed other contaminants. for total bacterial counts and the presence of coliforms, lead, and Feed samples are analyzed for the presence of bacteria and fungi. + Samples from freshly by the cagewashers. washed cages and cage racks are analyzed to assure adequate sanitation HmaesektelslpeLcaifbioerdatnuotrryituisoensalcerretqiufiierdemaennitmsalanfdeetdo. bTehferefeeoefdaislgiusaorfanstpeeceidfbieydtchoenmtaamniunfaancttsu.rer to Creerqtuiifrieemdeanntismaanldfteoedbeisfurseeedo,fgiumapruarnitteieesd wbhyitchhe mmiagnhutfaicntfulrueerncteotmheeertessupletcsifoifetdhenusttruidtyi.onal D`Tahteaaanriemamlaihnetaalitnhemdosneiptaorraitneglypfrroogmrasmtuidsyadrmeicnoirsdstearnedd wbiyllthneotlabbeoriantcolruydaedniimnatlheveftienrailnarreipaonr.t at the discretion of the study director. F. Dose Preparation, Analysis and Rates `TWealtoemreorrBotahlecroshoollveanntd/moaryrabdeioaldadbeedleadsTrBeqAuiwrieldl tboeamcihixeevdewtihtehtaarngeatpplroowprainatdehdiogshidnogsveehicle. concentrationsof Telomer B alcohol. Dthoesdeapyreopfaursaet.ioDnsosfeorstohleutsiionnglfeordotshee reexppeeartidmoesnetsexmpaeyribmeenptrsepmaareydboenptrheepadraeydoifnubsuelko,rsptroiroerdto appropriately, and used as needed. 'chHePmLiCca-lMSconocreanntroatthieorn.suiDtaobslee asnoalluyttiiocnaslcmoenttahiondinwgirlaldbieoluasbeedletdoTanBalAyzweildlosbee assolsuatyieodnsinfor tpruirpiltiycawtiellfobrerdadeitoearcmtiinveedcobnyceanptprraotpiroinatbeymleiaqnuidussicningtirlaldaitoimoentcroiucndteitnegct(ioLnS.C), and radiochemical _-- =o 140 JDTrOaXfItCoOPrKoItNou EcTolICWScROFXTXEoLXOXMEnR BAAncLCoOwHeOoLuIn NwTmHEec RRATesorTeiowenB Oral doses will be administered at a rate of4 mL/kg. Dermal doses will be administered ata rate of 10 pL/em?. G. Sacrifice Rats will be sacrificed by CO; asphyxiation and exsanguinated by cardiac puncture. H. Pilot Studies TrehseolfvoellhoowwinTgBpiAloitssetluidmiiensawtieldlfboellcoownidnugctoeradltaoddmientiesrtmrianteiotnh.e pTlhaesdmaatakifnretoimcsthoefsTeBeAxpearnidmetnots mouatyesbeanusdefruatlesinosfeetltiinmgintahteiownh,oalnedbelsotoadblsiesrhiailngcotlhleencteieodn ftoirmeo-npeoionrtst,wodertaedrimoilnaibnegletdhefoprrimnsciopfal TBA to describe elimination/metabolism. 1. Plasma kinetics following oral dosing with non-radiolabeled TBA `aTlwloowmeadlteoarnedcotvweor ffoermaatleleraasttsownielldhaayvbeecfaonrenudloasisnugr.gicRaaltlsy wiimlpllbaentaeddmiinnitshteejruegdultaher tveesitn and `compound (non-radiolabeled) by the oral route and housed in suspended wire-mesh cages. FinotloloMwicirnogtdaoisnienrgt,usbeersiaclonbtlaoiondisngamEpDleTsA.wilSlerbiealdrsaawmnplfirnogmttihmee-jpuogiunltasr wvielilnbceandnoucluameanndtepdlaicnetdhe study record. Altematively, whole blood may be collected from the tail vein, c`eWnhtorliefubglatoioodn.samPpllaessmwailwlilblebmeasitnotraeidnferdoozennwaettaipcper.oxPilmaastmealywi~l1l5beCseunptairlatperdocfersosmedrefdorcells by analysis 2. Excretion and metabolism following oral dosing with radiolabeled TBA In be saedpmairnaitseteexrpeedrtihmeetnetsst for cach material fraodrimoullaabteeldedwiftohrmraodfioTlBabAe,letdwToBmAalaet and the two high fdeomsaelelervaetls will by oral tghaevacgoel.lecFtoilolnoowfiunrgindeo,sifnegc,es,raetsxpwiirlledbeairhoaunsdedvoliantdiilveidouraglalnyicisn.glass metabolism units suitable for Udorsiinnegaunndtiflescaecsriwfiilcle.be collected (on dry ice) at approximately 12 hours and every 24 hours after sEexcpoinreddcoainrtawiilnlinbgeeptahsysleednethgrlyocuoglh otrwootshecrrusbubiitanbgletoswoelrvse,nto.neSccrounbtabiinnigngto2weNrsNwailOlHbeanrdeptlhaeced every 24 hours, post-dose, until sacrifice. Aafntierma7ldsawyisllpobset-sdaocsriinfigc,ewdhaifctehrevaterletasatke9s5p%laocef afdismtinistered radioactivity has been excreted or BE Li] JoDTxrOaXcfItoCOPnrKnoIteNoEncToeIlCsWSRoOrFTXTEXoLXOwXMeErR BbAAvLCcOoHnOLoIwNTmHeEkRaATr `The following tissues will be collected at sacrifice: [Lung 7 Testes [Bone Brin [OLtractandcontents|Adrenals | Ovariewutens | [Spleen | | Skinsample | Aanfatleyrsicso.lle`cTthieonr,emtaisisnuiengsacmaprlceassswiwlilllbbeeshtoormeodgaetnaipzperdoxainmdataeslsyay-e1d5soC tuhnattiltoptraolcmeastseirnigalanbdalance can be determined. vIfollaetsislethoarnga2ni%cso,fctohleletcottailondoosfeexispierleidmianiartweidlldunroitnbgetpheerdfuorramtieodnionftthheemsatiundystausdeyitehxeprer"iCmOen;tso.r I greater than 20% study experiments, of the dose is eliminated via the feces, bile will be collected in the main IafnrdesmuelttasbsoulgigsemsotfthTatBeAi,thoenrerafdoiromlawbiellledbefosrelmeoctfeTd BanAd will suffice used in the in establishing the excretion main study distribution, excretion and metabolism experiments. 1. Main Study 1. Absorption Experiments `The principal absorption of objective of the TBA following absorption experiments is to oral and dermal dosing using determine the rate plasma Kinetics and extent of Dose Routes and Frequency `The absorption experiments will be comprisedoffive dose groups. Low dose, single oral gavage High dose, single oral gavage Low dose, single 8-hour dermal exposure High Low dose, dose, single 8-hour intravenous dermal exposure Each dose group non-radiolabeled will test be composed compound. of4 male and 4 female rats. Each dose group will receive a`Tnhdedienrtmraavleenxoupsosduorseerwoiultlesp.rovide a reference for estimating bioavailability of TBA by the oral eee c-- = en J TDOrXaItCo rOoKtIoNu cEoTlICWSc ROFXTXEoLXOXMEuR BMn ALoCOne HOoL Imn N TeHEre RAsT sorTeioweRD Experimental Design Rats will have a cannula surgically implanted in the jugular vein one or two days prior to dosing. Rfraetesofdehsaiigrnattheeddtaoyrpercieoirvteoaddoosisneg.viaAfttheerdsehramvailnrgotuhtee swiitlelwhilalvebethweadsohresdo-wtihtohraacidcilruetgeisonoasphaved fsiotltuetdiownitahnadncEolviezraebdetwhiatnh-sgtayulzeecoalnldaratboopdryevwernatpgrtooopmreivnegntofcothnetaampipnlaitciaotni.onRsaittes. Twihlel cboelltahrenwill be wom for the duration of the experiment. On the day of dosing, the protective body wrap and g10autzhee schoavveerdwidlolrsboe-trheomroavciecdraengdioansuisliincgonaenOa-drhiensgivwei.thFaonlliontweirnngaldoasrienago,ft1he0 Oc-mr?inwgilwlilble bgelued pceorvieorde,dtwheitnhona-roicgcildunsyilvoencmoveesrhwailnldbae nroenm-oocveeldusainvde tbhoedsyitwerawpa.sheAdt,trhienseendd aonfdtdhreieexdpwoistuhre natural sponges. MFioclrlootwaiinngerdotsuibnegs,csoenrtiaalinwihnoglEeDbTlAoo.d sSaammpplleisngwitlilmbees dwirlalwbnefdroocmutmheenctaendnuinlathaendstpuldaycreedcoirndtso. Altematively, `groups, whole whole blood blood may be collected will be collected during from after the the til vein. exposure For rats phase (8 in the dermal hours). The exposure application site will be washed free of excess test material at 8 hours. `Whole frozen blood will be placed on at approximately ~15C wet ce, and plasma until analyzed. will be separated by centrifugation and 2. Distribution Experiments `cTlheearparnicneciopfalToBbAjecitnisveeleocfttehdetdiissstureisbuatnidonoregxapnesriamtepnltassimsatoTidsestearnmdiTnesuthze fcoorncTeBntAraftoilolnoawnindg oral and dermal dosing. Tissues should results of the pilot excretion/metabolism be selected based experiment. upon previous toxicology data or Dose Routes and Frequency `fTohremdsiosftrTibBuAt.ionTehxepenreiemdenttosuwsiellbobtehcroamdpiorliasbeedleodfffoorurmsdoofseTgBrAoupwsi,llfboreeeascthabolfisthwedo irnadpiiololtabeled experiments. Low dose, single oral gavage High dose, single oral gavage Low dose, single 8-hourdermal exposure High dose, single 8-hour dermal exposure Each dose group will dose formulated with be composed of4 maleand radiolabeled TBA. 4 female rats. Each dose group will receive _-- 5 DTrOaXfItCOPrKoItNoEcToIlCWSROFTXEXLXOXMER B ALCOOLINTHERAT Experimental Design SSS, pRraet-sdwoislilnbgeprdoocseeddurveisa tfhoer roartasl ianndthdeedremramlalexepxopsousruererogurtoesupast twihlellboewaasnddehsicgrhibdeodsuenldeevreltsh.e The absorption Following experiments. The dosing rats will be exposure period killed at plasma TfoorssthaenddeTrmaaluefxoproTsuBrAe.groups will be hours. aTniaslsyuseiss.wiTlolbtaelcroaldlieocatcetdivaittysaicnritfiiscseueasnwdilsltboreeddetaterampiprnoexdi.maSteelleyct-e1d5tiCssuunetsilmparyocbeesseixntgraacntded and analyzed to determine the metabolite profile. Methods will be documented in the study records, 3. Excretion and Metabolism Experiments `fTohreeloibmjeicntaitvieosn oofftThBe Aexcarnedt/ioorniatsndmemteabtoalbiotleis,smtoeexsptearbilmieshnttshearperitnocidpeatlerpmaitnhewathyesrfaotresmeatnadbrooluitsems omfetTaBboAliasnmdotfo TdeBtAe.rmiAnemiafsdsaiblayla(nrceepefaotr)tlootawlorraadliodaocsteiveixtpyoasdumrieniasltteerrsetdhweiellxcarlestoiobnekinetics or determined. Dose Routes and Frequency Trahdeioelxacbreelteidofno/mremtsaobfolTiBsAm.exTpehreimneenetds1w0iulslebeboctohmrpardiisoeldaboeflefdivfeodromsseogfrTouBpsA, wfiolrlebaechesotfabtlwioshed in pilot experiments. Low dose, single oral gavage HLiogwhddoossee,,ssiinnggllee8o-rhalougradveargmeal exposure High dose, single 8-hourdermal exposure * Low dose, daily (repeat) oral gavage Each dose group will group (single dose) will receive dose formulated be composedof 4 with radiolabeled male TBA. rats and 4 female rats. Each dose aFdomritnhiestdearielyd (nroenp-eraatd)iloloawb-edloesdetoersatlcgoamvpaoguenedxpaetrtihmeenlto,wfdoousremraaltee faonrdaftooutralfnemuamlbeerrastsofwidlalybse necessary to experiments achieve steady-state plasma levels will be 8 hours. Once steady state of TBA. has been The exposure time for the dermal achieved, as determined by periodic sthaemptelsitnmgaotefrwihalolfeorbmluoloadtefdorwTitBhAraadnido/loarbesleeldecTtBeAd.metabolites, rats will be administered a dose of Experimental Design Pre-dose procedures for absorption experiments. rFatosllinowtihnegdaedrmmianlisetxrpaotsiuorneogfrtohueprsawdiilollabbeelaesdddeosscer,ibuerdinfeorantdhefeces will be collected (on dry ice) at approximately 12 hours and 24 hours following dosing, and each 24. _-- Too JoTDxrOaXcfIotCkPOrKnoIetNorEcTioIlcCWsSoROfFXTTXeEiLXoOXMuEgRR BAAcLoCnOHoOuLiIwNmTHeEeRaArT 0 ahdomuirnsipsotsetr-eddorsaidnigoaucnttiilvistaycrhiafsicbe.eeAnneixmcarlestewdilals bdeetsearcrmiifniecdedinafptielrotatexlepaesrti9me5n%tso.fTthhee excreta `may be analyzed to determine the metabolite profile. If significant amounts of CO; or volatile organics were observed in the pilot study, traps will be uaisrewdiltlo bcoellpeacstsaendyth'r*oCuOg;h atnwdo/otrravposlactoinleneocrtgeadniicns stehraitesm,atyhebfeirlstibceorantteadi.nsI2f rNeqNuairOedH, atnhed etxhpeired second containing replaced every 24 ethylene glycol or hours post-dosing other suitable throughout the solvent. study. Solvent traps will be Tn the case of the Low collected and Dose Multiple dose groups, expired air will only be collected following administration of the radiolabeled dose, `"Tehxepetriismseunetsscoalnlde/cotredpialtotsasctruidfiiecse. wAifltlerbecodleltecetrimoinn,etdisbsyuethseamrpesluelstswiolflpbreevsitooursedtoatxiacpoplroogxyimately -15C until processing and analysis. The remaining that total material balance can be determined. carcass will be homogenized and assayed so Selected tissues may be analyzed to determine the metabolite profile. Awatttehre(c5o0n:c5l0usvi/vo)n,owfatteher raanddioalcaebteolneed. dCoasgeeesxpmearyimbenetwsi,pce-atgeesstewidlltobdeertienrsmeidnewietfhfidceiteenrcgyenotf caangde tinse. A second cage rinse may be stored at room temperature. be carried out at the study director's discretion. Cage wash will If the feces contains greater experiments, two additional than 20% groups of of the administered four male and four dose, as determined female rats (oral and in pilot dermal) will be uimspeldatnoteesdtainblainshd tbhieleodriugcitnaonfdmpartoerviiadledinwtihteh faecbeisl.e mRaaitnst,ewniallncheavderiankcianngnuslolautsuirogni(ciasloltyonic dFeoxltlroowsien/gKdCoIs/iNnagC,lu)rianse,thfeescoelseasnodurbcileeowfildlribnekicnolglfelcutieddf(oorntdheryduircaet)iaotnaopfprtohxeiemxapteerliyme1n2t.hours and 24 hours. determine the Animals will be sacrificed metabolite profile. after 24 hours. The excreta may be analyzed to - The hs J TDrOaXfItCPOo rKoItNoEcTow IlCWSROFc XTXELXOXo MER Bu MAcLoCnOn oHmOImLNem TrHnE sRAe yT sormonemB J. Quantitationof Radioactive Residues 1. Plasma cPoluanstmiangsa(mLpSlCe)s. wPilllabsemathmaawyeda,lsaondbealainqauloytzseadnfaolryzpeadreinntdcuoplmipcoautendfoarn'd*CmebtyabloilqiutidesscbiyntLilCl-atMiSon. 2. Urine Urine samples from each collection interval will be thawed, and aliquots analyzed in triplicate for ""C by LSC. Urine may also be analyzed for parent compound and metabolites by LC-MS. 3. Feces Fceocmebsusftreodm. eTahche cCoOl;lecltiiboenraitnetderfvraolmwiclolmbbeushtoimoongewinlilzbeedtirnapwpaetderanadndanaalilqyuzoetds iwniltlribpelicate for aHnCd baynaLlySzCedtofodretpearremnitnecotomtpalofuencdalarnaddimoeatcatbioviltiytefsorbiynLdiCv-iMdSua.l rats. Feces may also be extracted 4. Bile bBiylLeSfCr.omBielaechmacoyllaelcstoiobne peexrtiroadctweidllanbde atnhaalwyezdedanfdoraplairqeuonttscwoimllpobuenadnaalnydzemdetianbtorliiptleicsatbeyfor "*C LC-MS. 5. Cage rinses Aliquots of cage rinses will be analyzed in triplicate for "C by LSC. 6. Feed residue Feed residue will be homogenized and aliquots will be combusted. The CO,liberated from combustion will be trapped and analyzed in triplicate for '*C by LSC. 7. Carcass Ienxdcirveitdauaanldcaprlcaassmsaesfowriltlhabteaanniamlaylzeisd<f9o8r%r.esidCuaarlca"s*sCesonwliyllifbtehegrroeucnodveorny dorfytoitcael arnaddioactivity in homogenized, and aliquots of the resulting homogenate will be combusted. The CO liberated from combustion will be trapped and analyzed in triplicate for "CO; by LSC. - 146 i DTraOftNPIroCtoOcoKl WIORNFEXTEXTLXIOXMCESR 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 willbe conducted at least once daily throughout the study. Moribund rats will be sacrificed. M. Statistical Analyses Gifrnoeucpesdsaatray.will be represented as a mean + SD. Other statistical evaluations may be performed, N. Measurement of Radioactivity Radioactivity solid samples in selected liquid samples will be assayed by LSC. Total radioactive will be determined by combustion, trapping liberated "CO; followed residues by LSC. in Samples for LSC will be analyzed for 10 minutes, or until 160,000 disintegrations (0.5% 20) are accumulated, whichever comes first. --_-- EB 7 J TDOraXfItCo POrKoItNoEcu ToIlCWSRc OFXTXEo LXOXMERrBMALn oCOnHOoe LmINeTm HrE RsAe T sorTeiowsB SAFETY AND HOUSEKEEPING All chemicals used during this study will be Chemical Catalog and/or the accompanying handled according to the procedures MSDS and disposed of according to specified the Stine- in the dHiasspkeolsledWoafstineaDcicsoprodsaanlcGeuwiidtehlitnheesg(uPirdoelciendeusreou1t0l0i)n.edRiandithoechSetmiinec-aHlasskweillll bReadhiaantdiloendSaanfedty Manual and Radioactivity Users Guide. RECORDS AND SAMPLE STORAGE SNpeewcairmke,nDsel(iafwaaprpel,icoarblaet),IrronawModautnat,aainnd RtehceofridnaslMraenpoargtewmielnl tb,e Wrieltmaiinnegdtoatn,HaDseklealwlarLea.boratory, CRITICAL DATES Date of Approval Proposed Experimental Start: Proposed Experimental Termination: Proposed Study Completion: Date Study Director signed Protocol <first date system> test substance is applied to test <last from date on which the study> data are collected directly <date final report wil issuc>. - 148 TrDOraXfIotCOPrKodItNoEcToeIlCWSoROFXmTXEoLXOvXMEeRBB AoLChOROeL ImNTeHE RAT REFERENCES 1. PVearnfdleuonroHoecutvaenloiJcPA,cDiadviinsMJaWl,e SRaotmsm:eIrnshibRi,toarnydEPfefteecrtsoofnRTesEt.os(t1e9r9o2n)e.. Renal Excretion of J Biochem Toxicology, 7, 1, 31-36. 2. Hanhijarvi H, Ophaug R H, and perfluorooctanoate excretion in Singer the rat. L. (1982). Proc. Soc. The sex-related difference in Exp. Biol. Med., 171, S1-55. 3. pVearnfdleuonroHdeeucvaanloiJcP,acKiudslinikmiaslBe 1, Rafelghem M and female rats. J, and Peterson Toxicol. Appl. R E. (1991). Pharmacol. Disposition of 107, 450-45. 4. pOelrsfolnuoCr'oTd,eacanndoAicndaecridssoninMmaEl.e(r1a9t8s3)a.ndTehffeecatcsutoenttoixsiscuietyfaotftypeacrifdlsu.orToooxcitcaonlo.icApapnld. 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) SIGNATURES Issued by Study Director: <name, credentials> <title> Date Approved by: <name, credentials> <iitle> Date cc oa Te ls TOXICOKImNEToICoSOmFToELoOMER B vALcCoOHmOeLrINwTHmEeRnATy Summary ofstudy experiments 1). Pilot experiments ) b) NTeusmtbmeatreroifala:niTmeallsompeerr Btraelactomheonlt (TBA) group: 2anmdal[e"CanJdT BA 2 fe male ) iT)reaPtlmaesnmtagkrionueptsi:cs following a single high oral dose of TBA i) Excretion and metabolism (1) [10-C*)TBA following a single high oral dose of ("citBA @) [1-C*|TBA 2) Absorption experiments ) b) Test material: TBA Number of animals per treatment group: 4 male and 4 female ) Treatment groups: i) ii) Single low oral dose Single high oral dose iii) iv) Single Single low high dermal dermal exposure exposure (8 ( hours) hours) v) Single low intravenous dose. 3) Distribution experiments @) b) ) NTeusmtbmearteorifaaln:i[ma"lCspIeTrBAtreatment group: Treatment groups: 4 maleand 4 female. i) ii) SSiinnggllee llooww oorraall ddoossee,, ssaacc aatt TTaoyu, iii) iv) Single Single high high oral oral dose, dose, sac sac at at Tou, Trays V). vi) Single Single low low dermal dermal exposure exposure (8 (8 hours), hours), sac sac at at Tox Trg vivii)iiS)ingSlienghlieghhidgehrmdaelrmeaxlpoesxuproesu(r8eh(o8urhso)u,rss)a,c satacToatusTsu 4) Excretion and metabolism experiments @) b) NTeusmtbmearteorifaaln:i[maClIsTpeBrAtreatment group: 4 male and 4 female ) Treatment groups i) ii) SSiinnggllee lhiogwhoorraallddoossee iiivi)) SSiinnggllee hliogwhddeerrmmaalleexxppoossuurree((88hhoouurrss)) V) Single low daily oral dosing with TBA, last dose with ["CITBA _--_-- I1s50e