Document G6OjNk5NvBa2ypy63mLKYEB8x

COVANCE> FINAL REPORT `CHROMOSOMAL ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES `WITH PFOS AUTHOR Hemalatha Murli, PhD PERFORMING LABORATORY Covance Laboratories Inc. (Covance) 9200 Leesburg Pike Vienna, Virginia 22182 LABORATORY PROJECT IDENTIFICATION Covance Study No.: 20784-0-449 3M Corporate Toxicology Study No.: T6295.18 SUBMITTED TO 3M Corporate Toxicology 3M Center Building 220-2E-02 St. Paul, Minnesota 55144-1000 STUDY COMPLETION DATE October 25, 1999 10f25 01816 Covance 20784-0449 QUALITY ASSURANCE STATEMENT Chromosomal Aberrations in Human Whole Blood Lymphocytes With PFOS `The report has been reviewed by the Quality Assurance Unitof Covance Laboratories Inc., in accordance with the Good Laboratory Practice regulations as set forth in the Environmental Protection Agency (EPA - TSCA), Title 40of the U.S. Codeof Federal Regulations Part 792, with any applicable amendments. The following inspections were conducted and the findings reported to the Study Director and Study Director Management. Written status reports of inspections and findings are issued to Covance Management according to standard operating procedures. Inspection Dates 09/01/99 Phase Dosing Dates Reported to Study Director and Study Director Management ___ Auditor 09/01/99 P. Ciceres 1013,14/99 Draft Report Review 10/14/99 P. Ciceres 1025199 Final Report Review 10725199 P. Ciceres Representative, QuAlity Assurance Unit Jofes) a9 Date 2 601847 Covance 20784-0-449 STUDY COMPLIANCE AND CERTIFICATION `The described study was conducted in compliance with the Good Laboratory Practice regulations as set forth in the Environmental Protection Agency (EPA - TSCA),Title40of the U.S. Code of Federal Regulations Part 792, with any applicable amendments. There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrityof the study or the interpretationofthe test results, except that the control substances were not fully characterized. The raw data have been reviewed by the Study Director, who. certifies that the evaluationofthe test article as presented herein represents an appropriate conclusion within the contextofthe study design and evaluation criteria. All test and control results in this report are supported by an experimental data record and this record has been reviewed by the Study Director. Study Director: Nowe Mus 'Hemalatha Murli, PhD Genetic and Cellular Toxicology Lo/ar(41 Study Completion Date a 001548 TAOFBCONLTENE TS Covance 20784-0-449 Page No. STUDYINFORMATION continence Sponsor Test Article. Assay Information Study Dates Supervisory Personnel GBIECTIVE cevviesorissssmsssstsmsirsasimessisssssssssirsasssssosassenasaessd TESTSYSTEMBATIONALR +r0sovcicesssvtrtesssnosrtrtommpporseeeesstonrsessd MATERIALS ANDMETHODS.........oeeeeieeeeieeeeeeeeeninee8e Test System Media and Cell Culture Conditions TestArticle Control Articles 59 Metabolic Activation System Chromosomal Aberrations Assay DATA cvs csscensscmnpsssnsssnsstenpissnsscansesesmesnssssessrsapasanis Data Presentation Assay Acceptance Criteria Assay Evaluation Criteria Test Article Handling Chromosomal Aberrations Assay Without Metabolic Activation Chromosomal Aberrations Assay With Metabolic Activation CONCLUBION .covsr evs rvs assess sessssssossiss cassarsonssesrs PROTOCOLDEVIATIONS..........oeaeieeaiiieiiseieeenieeeieeeieen 14 REC TOO BEMR AIND TAIS NED .........uoiiiieaiiineenennninnennnn1n4n "+ 01819 Covance 20784-0449 TABLE OF CONTENTS (Continued) PageNo. REFERENCES 10visiristesssrestsssmmmormmnnnnnnunnnssnnsssrormrsnnsinnnninsh DATATABLES 1vivrirssstirssrtrmmirsommpmmpunpisnimmmpimmpimimmiinnn1sS HISTORICAL CONTROL DATA... sss sais BE DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS ..23 + 01850 Covance 20784-0-449 ABSTRACT `The objective ofthis in vitro assay was to evaluate the ability of PFOS to induce chromosomal aberrations in human whole blood lymphocytes with and without metabolic activation. `Dmueetabtooltiocxiaccittiyvactoinosntraanindts4,49thpeghi/gmhLeswtictohnmceetnatbroatliiocnatcetsitveatdiionn.thTehaesstaeystwaarstic5l9e9wapsg/dmisLswolivtehdouitn dimethylsulfoxide (DMSO) for the assay. The stock solution and its dilutions were dosed using a dRoLs/imngLvooflDuMmeSOo.f 1% (10.0 pL/mL) and the vehicle control cultures were treated with 10.0 In the chromosomal aberrations assay, the treatment period was for 3.0 hours with and without `metabolic activation, and cultures were harvested 22.0 hours from the initiationoftreatment. Replicate cultures of human whole blood lymphocytes were incubated with 12.5, 24.9, 49.7, 99.3, 149, 199, 249, 299, 349, 449, and 599 pg/mL without metabolic activation and 12.5, 24.9, 49.7,99.3, 149, 199, 249, 299, 349, and 449 pg/mL with metabolic activation. Cultures treated `with concentrationsof 199, 249, 299, and 349 pg/mL without metabolic activation and 99.3, 149, s1i9g9n,ifaincdan2t9i9ncprgea/smeLinwictelhlsmewtiatbholcihcroacmtoisvoatmiaoln wabeerreraatniaonlsy,zepdolfyoprlocihdryo,moorsoenmdaolreadbueprrlaitciaotnis.on wNaos observed in the cultures analyzed. PlyFmOpShowcaystecsownisitdhearnedd nweigtahtoiuvtemfeotraibnodluicciancgticvhartioomno.somal aberrations in human whole blood -6- 01851 Covance 20784-0449 STUDY INFORMATION Sponsor 3M Corporate Toxicology Test Article Sponsor's Identification: FC-95 (PFOS), Lot #: 217 Date Received: August 19, 1999 Physical Description: White, crystalline powder Storage Conditions: Room temperature Assay Information TypeofAssay: Chromosomal Aberrations in Human Whole Blood Lymphocytes Protocol No.: 449, Edition 14 Covance Study No.: 20784-0449 Study Dates Initiation Date: August 20, 1999 Experimental Start Date: September 01, 1999 Experimental Termination Date:October01, 1999 Supervisory Personnel Study Director: Hemalatha Murli, PhD Laboratory Supervisor: Emilie C. Beckelhimer, BS OBJECTIVE `The objectiveofthis in vitro assay was to evaluate the ability of PFOS to induce chromosomal aberrations in human whole blood lymphocytes with and without metabolic activation. TEST SYSTEM RATIONALE Aberrations are a consequenceoffailure or mistakes in repair processes such that breaks either do not rejoin or rejoin in abnormal configurations (Evans, 1962, 1976). Descriptions of the types ofaberrations are provided in this report. Structural aberrations maybeof two types, chromosome or chromatid: + Chromosome aberration: structural chromosome damage expressed as breakage, or breakage followed by reunion, ofboth sister chromatids at an identical sie. 7 01852 Covance 20784-0-449 +Chromatidaberration: structural chromosome damage expressed as breakage of single. chromatids or breakage followed by reunion between chromatids. This is the most common typeofstructural aberration. Most known chemical clastogens (chromosome-breaking agents) require a period ofDNA synthesis to convert initial DNA damage into chromosome alterations that become visible at mitosis. The lymphocytes in blooddonot usually divide, but they were stimulated to divide in culture by exposure to phytohemagglutinin (PHA). At predetermined intervals after exposure to the test article, the lymphocytes were treated with a metaphase-arresting substance, Colcemid, then were harvested and stained, and metaphase cells were analyzed microscopicallyforthe presenceof chromosomal aberrations. Many mutagenic chemicals do not act directly on DNA but do so after being converted to active intermediates by enzymes found in liver. Human lymphocytes have onlay limited capacity to `metabolize some test articles, so an exogenous metabolic activation system (rat liver S9 homogenate) is included with a seriesof treatments to enhance the degree ofconversion and the ability of the assay to detect clastogenic, metabolic intermediates. Numerical aberrations (a change in the number ofchromosomes from the normal number of 46 for human lymphocytes) were not determined. However, the occurrenceofpolyploidy or endoreduplication, which were scored, might indicate that the test article has the potential to induce numerical aberrations. MATERIALS AND METHODS Test System `Human venous blood from a healthy, male, adult donor (nonsmoker withouta history of radiotherapy, chemotherapy, or drug usage, and lacking current viral infections) was drawn into sterile, heparinized "vacutainers". Whole blood cultures were initiated in 15mLcentrifuge tubes by adding 0.6 mloffresh heparinized blood to 9.4 mLof culture medium. Media and Cell Culture Conditions Cultures in 15 mi tubes were incubated with loose caps at 37C 2C in a humidified atmosphereof 5% 1.5% CO in air. The medium was RPMI 1640 supplemented with approximately 15% fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 g/mL), L-glutamine (2 mM) and1% phytohemagglutinin M (PHA-M). The total volume. for each culture was 10 mL. Test Article `The test article was a white, crystalline powder that was stored at room temperature. The test article was dissolved in DMSO. 5 01855. 3 Covance 20784-0-449 Control Articles Negative and Vehicle Controls. In the nonactivation assays, vehicle controls were cultures containing the vehicle for the test article, DMSO, at the highest concentration used in test cultures, 10.0 wL/mL. In the activation assays, the vehicle controls were the same as described in the nonactivation assays but with the S9 activation mix included. Positive Control Agents. The positive control agents which were used in the assays were mitomycin C (MMC) for the nonactivation series and cyclophosphamide (CP) in the metabolic activation series. Mitomycin C (CAS# 50-07-7, Sigma, Lot# 88H2508) is a clastogen that does not require metabolic activation. Cyclophosphamide (CAS # 6055-19-2, Sigma, Lot# 73H0846) does not act directly but must be converted to active intermediates by microsomal enzymes. In the chromosomal aberrations assays, three concentrations of MMC (1.0, 1.5, and 2.0 pg/mL) and CP (25, 50, and 75 pg/mL) were used to induce chromosomal aberrations. Oneofthe concentrations was analyzed in eachofthe aberration assays based on the current information on the positive control response in the laboratory. Both MMC and CP were dissolved in water. $9 Metabolic Activation System `The in vitro metabolic activation system (Maron and Ames, 1983) consistedof a rat liver post`mitochondrial fraction (9) andan energy-producing system (NADP plus isocitric acid). Various hepatic P450 isoenzyme levels are increased by treatmentofthe rats with AroclorTM 1254 (single concentrationof 500 mg/kg and sacrificed 5 days later (Molecular Toxicology, Inc., Lot No. 929). The $9 fraction, prepared in sucrose or in potassium chloride, was retained frozen a1.<-70C until use. AliquotsofS9 were thawed immediately before use and added to the other `components to form the activation system described as follows: 9 Activation System Component "NADP (sodium salt) Tocitric acid Homogenate ($9 fraction) Concentration in Cultures 1.5 mg/mL (1.8 mM) 2.7 mg/mL (10.5 mM) __15.0 uLimL* (1.5%) * This concentrationofrat $9, obtained from Molecular Toxicology Inc., Boone, NC, has consistently caused CP to be highly clastogenic for many different lots. Chromosomal Aberrations Assay In the chromosomal aberrations assays, replicate cultures were used at each concentration and the vehicle controls and for eachofthree concentrationsofthe positive control. Inthe aberrations. assay without and with metabolic activation, 22.0- hour harvests were conducted. This harvest time corresponds to 1.5 times the cell cycle timeofapproximately 15 hours after the lymphocytes are induced to divide by the additionofPHA-M (Galloway et al, 1994). 9 01854 Covance 20784-0-449 Aberrations Assay Without and With Metabolic Activation. Cultures were initiated with 0.6mL of whole blood in 9.4 mL of fresh culture medium. For the nonactivation assay, 2 days afterculture initiation, the cells were incubated at =37C with the test article in fresh medium at `predetermined concentrations, for 3.0 hours. For the activation assay, 2 days after culture initiation, the cultures were incubated at 37Cfor3.0 hours in the presenceof the test article and the S9 reaction mixture in fresh RPMI 1640 culture medium without FBS. The cultures were then washed with buffered saline, refed with complete RPMI 1640 culture medium and incubated for the restofthe culture period up to the timeofharvest with 0.1 pg/mL Colcemid present during the last 2.0 hoursofincubation. The cultures were then harvested. Harvest Procedure. The cultures were centrifuged, the supematant discarded, and the cells were swollen with 75 mM KCl hypotonic solution. The cultures were then fixed with an absolute methanol: glacial acetic acid (3:1, Viv) fixative. Slide Preparation and Staining. Slides were prepared by dropping the harvested cultures on clean, glass slides and air-dried. The slides were stained with 5% Giemsa solution for the analysisofmitotic index and chromosomal aberrations. All slides were then air-dried, rinsed in xylene, and mounted permanently. DATA Cells were selected for good morphology and only cells with the number ofcentromeres equal to the modal number 46 were analyzed. `One hundred cells, ifpossible, from each replicate flask from four concentrationsofthe test article, and from the vehicle and one doseofthe positive control cultures were analyzed for the differenttypes of chromosomal aberrations (Evans, 1962, 1976). At least 25 cells were analyzed from those cultures that had greater than25%of cells with one or more aberrations. Mitotic: index was evaluated from the vehicle control and a rangeofconcentrations byanalyzingthe `numberofmitotic cells in at least 1000 cells per culture and the ratio expressed as apercentage ofmitotic cells. Percent polyploidy and endoreduplication were also analyzed by evaluating at least 100 metaphases per culture,if available, and tabulated. For control ofbias, all slides were coded prior to analysis, Cells with aberrations were recorded on the data sheets by the `microscope stage location. Data Presentation Data were summarized in tables showing mitotic index, percent polyploidy, percent endoreduplication, the numbersof cells analyzed, typesofaberrations found, frequencies of aberrations per cell, and percentagesof cells bearing aberrations. Chromatid and isochromatid `gaps were noted but were not added into the totals for aberration assessment since they are not considered to represent true breaks. The historical control data are presented after the data tables. -10- ; 01855 Covance 20784-0-449 Assay Acceptance Criteria An assay was considered acceptable for evaluation oftest results onlyifallofthe following criteria were satisfied. The two treatment conditions are independent nits and would be repeated independently, as needed, to satisfy the acceptance criteria. Acceptable Controls. The vehicle control cultures must contain less than approximately 5% cells with aberrations. The positive control result must be significantly higher (p < 0.01) than the vehicle controls. Acceptable High Dose. Ifthe aberration resultsarenegative and there is no significant reduction (approximately 250%) in mitotic index, the assay must include the highest applicable dose (10 mM or 5 mg/mL, whichever is lower)or a dose where awell-dispersed suspension in culture medium was obtained that did not settle rapidly. Acceptable Number of Doses. The assay must include at least three analyzable concentrations. Assay Evaluation Criteria `The following factors are taken into account in evaluationofthe test article data: Percentageofcells with aberrations. + Percentageofcells with more than one aberration. + Evidence for increasing amountsofdamage with increasing dose, i.., dose related increase. `The experimental unit is the cell, and therefore the percentageofcells with structural aberrations `was the basis for evaluation. Statistical analysis employed a Cochran-Armitage test for linear trend and Fisher's Exact Test (Thakur ef al, 1985) to compare the percentageofcells with aberrations (and,ifapplicable, the percentageofcells with more than one aberration) in treated cells to the results obtained for the vehicle controls. Statistical analysis was also performed for cells exhibiting polyploidy and/or endoreduplication in order to indicate significant (p <0.01) increases in these events as indicatorsofpossible inductionof numerical aberrations; however, the test article was evaluated only for structural aberrations and not for numerical aberrations by this protocol. Evaluation oaf Positive Response.A test article was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when P<0.01) in the numberofcells with chromosomal aberrations is observed at one or more concentrations. Statistical evaluationof the percentageofcells with more than one aberration provided an indicationofthe severity of the positive response observed. The linear trend test evaluated the dose responsiveness. Ifa significant increase was seen at one or more: concentrations, a dose-response should have been observed. -- - 01856 Covance 20784-0449 Evaluation ofa Negative Response.A test article was considered negative for inducing chromosomal aberrations ifno significant increase was observed in the numberof cells with chromosomal aberrations at anyofthe concentrations. Equivocal Evaluation. Although most assays gave clearly positive or negative results, in rare. cases the data set would preclude making a definitive judgment about the activityofthe test article. Results might remain equivocal or questionable regardlessofthe numberof times the assay is repeated. Although the evaluation criteria provided here normally was sufficient, the Study Director might use additional considerations to obtaian final evaluationofthe test article based upon the Study Director's scientific judgment. RESULTS Test Article Handling Solubility and Dose Determination. PFOS was insoluble in water at concentrationsof 50.0 to 500mg/mL. In DMSO, 2 translucent, light-yellow suspension with some undissolved test article `was obtained at a concentration of 501 mg/mL after vortexing. A translucent, light-yellow suspension was obtained at 401 mg/mL. The suspension at 401 mg/mL was dosed in the absence ofcells using a 1% (10 L/L) dosing volume in RPMI 1640 culture medium. At a dosed concentration of4010 g/mL, a translucent suspension with fine particlesofprecipitate was observed and the pH was 8.0 (pH ofRPMI 1640 culture medium was 8.0). DMSO (Acros Organics, lot # A12649701) was selected as the vehicle forthisassay. After sonicating for =5 minutes, a translucent, light-yellow suspension was obtained at a concentration of 400 mg/mL. All dosing was achieved with a 1.0% (10.0 iL/mL) dilltionofthe DMSO stock solutions, and the vehicle control culture was treated with 10.0 uL/mL of DMSO. Concentrations of27.2, 38.8, 55.4, 79.1, 113, 161, 230, 329, 470, 671, 959, 1370, 1960, 2800, `and 4000 g/mL were tested with a 3.0-hour treatment and a 22-hour harvest in the chromosomal aberrations assays with and without metabolic activation. Due to the poor growth of cells (Tables 1 and 4), the assay was repeated testing concentrations of 12.5, 24.9, 49.7, 99.3, 149, 199, 249, 299, 349, 449, and 599 pg/mL without metabolic activation and 12.5, 24.9, 49.7, 99.3, 149, 199, 249, 299, 349, and 449 pg/mL with metabolic activationwith a 3.0-hour treatment and a 22-hour harvest. The stabilityofthe test article under the preparation and dosing conditions used in this assay is the responsibilityofthe sponsor. a2 01857 Covance 20784-0449 Chromosomal Aberrations Assay Without Metabolic Activation A summaryofthe treatment times for the chromosomal aberrations assay is given below. Summary of Chromosomal Aberrations Assay Treatment Schedule in Hours Activation Test Article Wash Colcemid Harvest Condition Added Added Started -s9 0 30 200 220 Reductionsof38%, 8%, 15%, 15%, 12%, 19%, 42%, 69% and 929% were observed in the mitotic riensdpieccetsoivfeltyh,eacsucltoumrpeasrterdeawteidthwitthhe v1e2.h5i,cl2e4.c9o,nt4r9o.l7,cu9l9t.u3r,es14(9T,ab2l4e9,2).299C,h3r4o9m,oasnodma44l9abpegr/rmatLi,ons `siwgenriefaincaanltyziendcrferasoemitnhececlullstwuiretsh tcrheratoemdowsiotmhal19a9b,er2r4a9t,io2n9s9,,paonlydp3lo4i9dyp,go/rmeLnd(oTraebdluep3l)i. caNtoion was observed at the concentrations analyzed. `The sensitivityofthe cell culture for induction ofchromosomal aberrations is shown by the increased frequencyofaberrations in the cells exposed to mitomycin C, the positive control agent. The test article is considered negative for inducing chromosomal aberrations, polyploidy, and endoreduplication under nonactivation conditions, Chromosomal Aberrations Assay With Metabolic Activation A summaryofthe treatment times for the initial chromosomal aberrations assay is given below. `Summaryof Chromosomal Aberrations Assay Treatment Schedule in Hours Activation Test Article Wash Colcemid Harvest Condition _ Added. Added Started +89 0 30 200 20 Reductions of 12%, 41%, 71%, and 53% in the mitotic indices as compared with the vehicle control cultures were observed in the cultures treated with 49.7, 199, 249, and 299 pg/mL, respectively (Table 5). Chromosomal aberrations were analyzed from the cultures treated with 99.3, 149, 199, and 299 ug/mL (Table 6). Due to severe toxicity, only 27 and 4 metaphases were available for analysis from the cultures treated with 299 pg/mL. No significant increase in cells `with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed. The successful activation by the metabolic system is illustrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive 13 C01858 Covance 20784-0-449 control agent. The test article is considered negative for inducing chromosomal aberrations, polyploidy, and endoreduplication under activation conditions. CONCLUSION PFOS was considered negative for inducing chromosomal aberrations in human whole blood lymphocytes with and without metabolic activation. PROTOCOL DEVIATIONS Negative control cultures were set up for the first trial. This had no impact on the study. Polyploidy and endoreduplication were evaluated from >100 cells from MMC- and CP-treated cultures. This had no impact on the study. RECORDS TO BE MAINTAINED All raw data, documentation, records, protocol and a copyofthe final report generated at (Covance as a resultofthis study will be archived in the storage facilities of Covance-Vienna for atleast 1 year following submissionofthe final report to the Sponsor. Afierthe 1-year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities of SCpoovnasnocre.-Vienna for an additional periodoftime, or sent to a storage facility designated by the REFERENCES Evans, H.J., "Chromosomal aberrations produced by ionizing radiation." International Review of Cytology, 13:221-321 (1962). Evans, H.J., "Cytological Methods for Detecting Chemical Mutagens." Chemical Mutagens, Principles and Methodsfortheir Detection, Hollaender, A. (ed.), Vol. 4, pp. 1-29, Plenum Press: New York and London (1976). Galloway, S.M., Aardema, M.J., Ishidate, M., Jr., Ivett, J.L., Kirkland, D.J., Morita, T., Mosesso, P., and Sofuni, T., "Report from working group on in vitro tests for chromosomal aberrations." Mutation Research, 312(3):241-261 (1994) . Maron, D.M., and Ames, B.N., "Revised methods for the Salmonella mutagenicity test." `Mutation Research, 113:173-215 (1983). `Thakur, A.J., Berry, K.J., and Mielke, P.W., Jr., "A FORTRAN program for testing trend and `homogeneity in proportions." Computer Programs in Biomedicine, 19:229-233 (1985). He 01859 Covance 20784-0-449 DATA TABLES 1s 01360 Covance 20784-0-449 TABLE1 ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY. AssayNo.2078 TralNo:1 Date: 09/0199 LabNo: CY8249 Compound: PFOS Metabolic Activation: -59 3.0 hour treatment, 22.0 hour harvest wwoee[]5 | Mimic Treatment J icontduerxe] [iMnidesxe||ReIdnodecxion VEHICLECONTROL DMSOloouUml 07 06 07 TEST ARTICLE nagml 08 06 07 0 Maugml 04 06 0s ssapgml 05 0s 0s Plwml 05 06 06 1 MigmL 04 07 06 1 logml 04 03 0s [gm 07 06 07 0 sgn 04 06 0s 2 Sougmi_19 16 18 0 DMSO = Dimethylsulfoxide -1601861 Covance 20784-0-449 TABL2E ASSESSMENT OF TOXICITY FAOSRSCAHYROMOSOMAL ABERRATIONS AsyNo:20784 TralNo:IA Dai091599 Labo CYSTS Compound: PFOS Metabolic Acvrion: 59 50hourwes2m20ehone htarv,est aTse |wse[| s=e ToncimcomoL wo wan Test arcs usm wom omen sown own wn wnt man wn wm DMSO Dimeylonds 22 0 26 0 15 13 16 ow 2s 2s 22 2 ms 22 was 2s 0 Bn 37 38 3s 0 16 2s uw 14 6 1s a 12 0 0s ow 020202 = ar 015862 L armsW moors res SM) arco ie ei51 rr ---- gl dy din 2. 2 [Trno trer be RS ome weve RELL gn, 4, mEog EE rs m3 woo ou ow ie 33 mon on ou os F mE mE onouog -- em AAA-- 0 A imiPT -- , m-- owraomeou oa o 2: o g B22g t Covance 20784-0-449 TABLE4 ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY AssayNo.20764 TrialNo:l Date:09/0199 LabNo: CY8249 Compound: PFOS Metabolic Activation: +9 Treament VEHICLE CONTROL DMSO TEST ARTICLE *DNMoScOell=s pDirmetehysolnseulnlfiotdxeide 3.0 hour treatment, 22.0 hourharvest MiW te |wMote |"f%ens|] Mitotic In icnoderxe|]ciundrexe| [dMeitxo |[Reitnodcesxion 100uUmL 06 05 06 0 napmt 02 02 02 @ samt 02 02 02 @ sami 02 03 03 2 mugm 02 04 03s Mem 03 04 04 wpm 01 01 0B dogm. 02 00 01 ow agml 01 04 03% dopgml + + . -19- 01864 Covance 20784-0-449 TABLES ASSESSMENTOF TOXICITY FOR CHROMOASBEORRMATIAONLS. ASSAY AssayNo.20784 TrialNoiIA Date: 09/15/99 LabNo.: CY9079 Compound:PFOS Metabolic Activation: +59 Trestment VEHICLECONTROL DMSO TESTARTICLE DMSO = Dimethylsulfoxide. 3.0hourtreatment, 22.0hour harvest MitW otic|wMioic Mit0otie Icnudlexe|] Icnudrexe] Recduectxion loouUmL 16 17 17 0 nSgm. 20 19 20 0 u9ugml 25 26 26 0 wgnl 14 16 1s on wpm 18 32 25 0 WogmL 20 24 22 0 Wgml 10 10 10 a 29upmi 05 05 0s 7M Zum 09 07 08 C01365 -20- recto Cop is SeoreT-- es nn eon cv si eE r --_----waela weal 3 wo me | we 23, --" am 34 J - T hea y T E tptE oeT cntit, o22g8 nano wor nono SE Es wonon aE i 31 Covance 20784-0449 HISTORICAL CONTROL DATA CONTROL DATA OF CHROMOS1/O9M7 ETHARBOEURRGAHT9I/O98NS IN HUMAN LYMPHOCYTES = 20-24 HOUR HARVEST Activation %ofWCiells W%oifhC>ellls %Pplooldy- %dEunpdliocrsee:d Mit%otic e 3Hour reTseeent Widow MN Aber0si t0ions Aberr0a0tions _ Ce0l0ls Ce0l0ls In1d2ex MAAVXG o2u5s 0005 o1nsl o0w0 228 oNw 1o9w o129 i0% 010 i1s Ye3tHioclu CTornetsrmoeln(tPooled) Wibos MIN 00 00 00 00 1 MAAGX o1ast 0o0w 0005s o0so 261 sNw o1s o1w o1l9s 1on aims 3PoHsoituirveTrCoanmtreonlt- MMC Wibow MN 200 13 00 00 - MAAVGX dGo20m n20g 0oosk o0m0 -: EN o1s 61 iooi o1w :: NCeognattiinvueouCsoTnreament Wibow MN 00 00 00 0 os MAAGX o1ks o00w o1x5 oows 30m DNE 02 020 02% 02 16 CVoenntiicnueoCuosntTrorlei(mPaonol!ed) Wibou MIN 00 00 00 00 1 AMVAGX a2s8e 005 oIio o00w 361s oNe ona o5z o3m o5w 15g PCoonstiivneuoCusoTrnei-mMenMtC Wibow MN 11S 15 00 00 In MAVAGX 361006 2790% o0sm o00w a43p DN@ I3ss s5n o3s o6m 133 NHegoatrivTerCoenatmreonlt win MN 00 00 00 0 or MAVAGX o2a0 0o5on 2o0m ooos 392 soN@ 0is8 oisss i04ss oPm 1is8 3 HiocurkCTonetrsolt(Pooled) wie MN 00 00 00 00 os MAAVXG o2u8 jorm) 0osm 0osm 268% oNw 0"s Porl o"n o4n 1"2 Po3 sitiveTCeosnmtren:CP wih MN 50 00 00 00 os MAAGX 2620 n24n0 o1m6 o0w0 2553 oNw 1r0s3 sai 0iw2 0i0 18s NMM=CNu=mMboerrmopefivinaCl-s. CP Cyckphasphanice 01867 2 Covance 20784-0449 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS Simple TB Chromatid Break: An achromatic region in one chromatid, larger than the width ofa chromatid. The associated fragment may be partially or completely displaced. SB Chromosome Break: Chromosome has a clear break, forming an abnormal (deleted) chromosome with an acentric fragment that is dislocated. This classification now includes the acentric fragment (AF). An AF is different from 2 SB only in that it can not be related to any specific chromosome. DM "Double Minute" Fragment: Theseare small double dots, which may represent terminal or interstitial deletions, or even small rings. These possible origins are not distinguishable. Complex ID Interstitial Deletion: Lengthof chromatid "cut out" from midregion ofa chromatid, resulting in a small fragment or ring lying beside a shortened chromatid or a gap in the chromatid. TR Triradial: An exchange between two chromosomes, or one chromosome and an acentric fragment, which results in a three-armed configuration. QR Quadriradial: An exchange like a trradial, but resulting in a four-armed configuration. CR Complex Rearrangement: An exchange among more than two chromosomes or fragments, caused by the inductionof several breaks. D Dicentric: An exchange between two chromosomes which results in a chromosome with two centromeres. This is often associated with an acentric fragment in which case it is classified as DF. DF Dicentric with fragment. 2 . 01868 Complex (continued) TC Tricentric: QC Quadricentric: PC Pentacentric: HC Hexacentric: R Ring: RC Ring Chromatid: RF CI Chromosome Intrachange: T Translocation: AB Abnormal Covance 20784-0-449 `An exchange involving three chromosomes and resulting in a chromosome with three centromeres. Often associated with two to three AF. Such exchanges can involve many chromosomes andarenamed as follows: four centromeres, up to four AF five centromeres, up to five AF six centromeres, up to six AF A chromosome which formsa circle containing a centromere. This is often associated with an acentric fragment, in which case itis classed as RF. Single chromatid ring (acentric). Ring with associated acentric fragment. Exchange within a chromosome; e.g, a ing that does not include the entire chromosome. `Obvious transfer of material between two chromosomes resulting in two abnormal chromosomes. When identifiable, scored as "T" not "2AB". Abnormal monocentric chromosome. This is a chromosome `whose morphology is abnormal for the Karyotype, and often the result ofa translocation, pericentric inversion, etc. Classification used ifabnormality cannot be ascribed to, e.g., a reciprocal translocation. 24- 018G69 Other GT Greaterthan Five: PP Polyploid Cell: E Endoreduplication: Abs >14b Not Computed TG Chromatid Gap: SG Chromosome Gap: UC Uncoiled Chromosome: Covance 20784-0-449 Greater than aberrations: A cell which contains more than 5 aberrations. Heavily damaged cells will be analyzed to identify the typesofaberrations because multiple fragments, such as those found associated with atricentric,do not count as independent aberrations. A cell containing multiple copiesofthe haploid number (n) ofchromosomes. A failureofchromosomes to separate, resulting in a 4n cell. Cells with chromosomal aberrations. Cells with > 1 chromosomal aberrations ("tid gap"). An achromatic (unstained) region in one chromatid, the sizeof which is equal to or smaller than the width oaf chromatid. These are noted but not usually included in final totalsof aberrations asthey may not al be true breaks. (`isochromatid gap, IG"). Same as chromatid gap but at the same locus in both sister chromatids, Failureof chromatin packing. Probably not a true aberration. 25- rr C01870 COVANCTETM AMENDMENT TO THE STUDY PROTOCOL Page lof1 STUDY TITLE: Chromosomal Aberrations in Human Whole Blood Lymphocytes PROTOCOL NO.: 449, Edition 14 COVANCE STUDY NO.: 20784-0449 Amendment #3 `The Sponsor will be identified as 3M Corporate Toxicology and not as 3M Corporation. Reason: Sponsor's request. SyDon Ne 2LoTl0 M LE Hemalatha Mur, Ph.D. Mammalian Cytogenetics DepartmentofCellular and Genetic Toxicology 10/35/49 Date 041571 COVAANNCCEE. CPORVOATNOCCEOLSNTOU.D4Y49NOE.DI2T0IO7N8144 -0-44F CHROMOSOMAL ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES Covance Laboratories Inc. (Covance) will conduct this study in compliance with Good Lpraobgorreastso,raynPdratchteicfeina(lGLrePp)orrtegwuillaltbioenss.ubjTehcitstporaoutdoictolb,yatQulaelasittyonAesscurirtaincaclepihnaascecoofrdtahencweowrikthin S2t1a9n2d0a0rdLeOepsebruartignPgikPer,ocVeideunrneas, mVaiirngitnaiiane2d2a1t8C2o(vCaonvcaen.ceT-hVieesntnuad)y.will be conducted by Covance PART 1. SPONSOR INFORMATION AND APPROVALS 10 SPONSOR IDENTIFICATION ACddormepsasn:yName: 33MM CCoernptoerration BStu.iPladuiln,g M22N0-25E5-10424-1000 20 TEST ARTICLE IDENTIFICATION PFS ImSid, No Tlec i& 30 TEST ARTICLE ANALYSIS "The Sponsor will be responsible fo the determination and documentation of the analytical purity and composition of the test article and the stability and strengthof the dosing solutions. 40 NOTIFICATION OF REGULATORY SUBMISSION IpanrotrodferatsotucdoympisliynwtietnhdeGdLfPorrreegguullaattioornys,sucobnmsiuslstiionng.lCaboovraatnocreiemsaminutsatinbse anomtaisftieedrif all or schedule of studies which fall under regulatory review. Please indicate which agency, if any, might receive the results of this study: Oundeiemined Opa Mepazsca Oeearrra Omarr Omonw Doro Domer 298 . Lofls 01872 COVANCE> PROTOCOL NO. 449 EDITION 14 50 STUDY DATES Proposed Experimental Start Date: ple 127% Proposed Experimental Termination Date: __ Tic luge 1% %7 60 APPROVAL OF STUDY PROTOCOL Study Director: HowatNina, Mews Hemalatha Murl, PhD. pute: __elarioa Testing Facility Management: io CM Brian C. Myhr, PhD. Associate Director Dae: 8/20/59 Sponsor's Authorized Representative: Ml artine T. Caser , D.V.M., Ph.D. Date: /B4uguotgg 298 20f15 01873 COVANCE> PROTOCOL NO. 449 EDITION 14 PART 2. STUDY PROTOCOL CHROMOSOMAL ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES 10 OBJECTIVE `The objective of this in vitro assay is 10 evaluate the ability of a test article to induce chromosomal aberrations in cultured human lymphocytes with and without an exogenous metabolic activation system. 20 TEST SYSTEM RATIONALE Aberrations are a consequence of failure or mistakes in repair processes such that breaks either do not rejoin or rejoin in abnormal configurations (Evans, 1962, 1976). Descriptions of the typesof aberrations are provided in Section 10.0. Structural aberrations may be of two types, chromosome or chromatid: + Chromosomeaberration: structural chromosome damage expressed as breakage, or breakage followed by reunion, of both sister chromatids at an identical ite. + Chromatidaberration: structural chromosome damage expressed as breakage of single chromatids or breakage followed by reunion between chromatids. This is the most common type of structural aberration. Most known chemical clastogens (chromosome-breaking agents) require a period of DNA. synthesis to convert initial DNA damage into chromosome alterations that become visible: at mitosis. The lymphocytes in blood do not usually divide, but theyare stimulated to divide in culture by exposure to phytohemagglutinin (PHA). At predetermined intervals after exposure to the test article, the lymphocytes are treated with a metaphase-arresting `msiucbrsotsacncoep,icCaolllcyefmoirdth,etphreensearnecehoafrvcehstreodmoasnodmsatlaianbeedr,raantidonmse.taphase cells are analyzed Many mutagenic chemicals do not act directly on DNA but do so after being converted to active intermediates by enzymes found in liver. Human lymphocytes have only a limited capacity to metabolize some test articles, so an exogenous metabolic activation system (rat liver S9 homogenate) is included with a series of treatments to enhance the degree of conversion and the ability of the assay to detect clastogenic, metabolic intermediates. Numerical aberrations (a change in the number of chromosomes from the normal number of46 for human lymphocytes) are not determined by this protocol. However, the 298 3of15 01874 COVANCE> PROTOCOL NO. 449 EDITION 14 tohcecutresrtenacrteicolfehpaolsytphleoipdoyteonrtieanldotroeidnudpulciceantuimoen,riwchailcahbewrirlaltiboenss.cored, may indicate that : 30 MATERIALS 31 TestSystem `"VHaucmuatnaivneernso"u.s bWlhoooldefbrloomoad hceualtlutrheysdwoinlolrbewililnibtieatderdabwynaidntdoisntegri0l.6e,mlheopfarfirneiszhed heparinized blood to 9.4 ml of culture medium in each 15 ml centrifuge tube. 31.1 Media and Cell Culture Conditions hCuulmtiudriefsiewdilinbcuebiatnocru,baitneadnwaittmholsopohseerceaopfs aatbosubtou5t%371C.5%2COC,iinnaair. `The fetal medium will be RPMI 1640 supplemented with bovine serum (FBS), penicillin (100 units/ml), approximately streptomycin 15% `(T1h0e0tgot/almc)u,ltLu-reglvuotlaummienewi(l2lmbMe)10amnld.1% phytohemagglutinin (PHA-M). 32 Test Article Suosleiddionrcluilqtuuirdestewsittahrttiicglhetslyarcelossuietdacbalpesf.orTthheistaessstaayr.ticVloelaitsiildeentteisftiaerdtiicnlPesarmtay1 bofe this protocol. Storage conditions will be as specified by the Sponsor. 33 Control Articles 33.1 Vehicle Control Article "The vehicle control article will be the vehicle selected for the test article. `The vehicle may be specified by the Sponsor or selected by the process described in Section 4.1 (Test Article Solubility Testing). The vehicle concentration in the vehicle control cultures will be equivalent to the vehicle concentration in the test articl-treated cultures. 33.2 Positive Control Articles Known clastogenic (chromosome-breking) agents will be used for structural aberrations analysis. 298 40f15 CO1875 COVANTCE> PROTOCOL NO. 449 EDITION 14 `Without 9 activation: Mitomycin C (MMC; CAS #50-07-7), a clastogen that does not require metabolic activation, will be dissolved in water and used at a final concentration between 0.025 t0 3.0 pg/ml. `With $9 activation: Many clastogens do not act directly on DNA but `must be metabolically converted to clastogenic intermediates by a cofactor-supplemented post-mitochondrial fraction (S9) prepared from liver. An example is cyclophosphamide (CP; CAS #6055-19-2), used here as the positive control to demonstrate the activity of the S9 metabolic activation system. CP will be dissolved in water and used at a final concentration between 10 to 300 pg/ml. 34 $9 Metabolic Activation System `The in vitro metabolic activation system (Maron and Ames, 1983) consistsofarat liver post-mitochondrial fraction (S9) and an energy-producing system (NADP plus isocitric acid). Prior to collecting livers from male Sprague-Dawley rats, various P450 isoenzyme levels will be increased by an acute treatment of the rats with AroclorTM 1254 at 500 mg/kg. The S9 fraction willbeprepared approximately 5 days later. The S9 fraction, prepared in sucroseor in potassium chloride, is purchased from a commercial supplier and is retained frozen at <70C until use. Aliquots of 9 will be thawed immediately before use and added 10 the other components to form the activation system described as follows: `CNoAmDpPon(esnotdium sal) Isocitric acid Homogenate (S9 fraction) C1.o5 mngc/meln(t1.i8rnmaCMutl)tiuorens 2.7 mg/ml (10.5 mM) 15.0 ulml* (1.5%) * This concentration of rat S9, obtained from Molecular Toxicology Inc., Boone, NC, has consistently caused CP to be highly clastogenic for many different lots. 40 EXPERIMENTAL DESIGN 41 Test Article Solubility Testing If solubility information is not provided by the Sponsor, a preliminary solubility test willbecarried out with sterile deionized water, serum-free culture medium, dimethyl sulfoxide (DMSO; CAS #67-68-5), ethanol (CAS #64-17-5), or acetone (CAS #67-64-1). The vehicles of choice are water or serum-free culture medium. Test article solutions in DMSO or ethanol wil be diluted 100-fold in culture 2098 Sof 15 01376 cov CE. PROTOCOL NO. 449 EDITION 14 `medium, and test article solutions in acetone will be diluted 1:200 in culture `medium. The solubility and dispersion characteristics of the test article will be determined, and the vehicle selected will be the one which gives the best solubility or dispersion after dilution in medium. In some cases, test articles are apparently insoluble in vehicles that are compatible with tissue culture. In such cases, the vehicle chosen and the highest dose tested will be those giving an evenly dispersed suspension. For liquid test articles, the dosing stocks may be prepared by weight or be calculated from the specific gravity. `The top dose tested for non-toxic test articles, where the molecular weight is unknown, will be 5 mg/ml.If the molecular weight is provided by the Sponsor and the test article has a purity of 95% or greater, the maximum dose will be. 10 mM or5mg/ml, whichever is lower. 42 Dose Rangefinding Dose rangefinding will be conducted as an integral part of the chromosomal aberrations assays. A range of doses will be applied, as appropriate for each treatment condition, to achieve a wide toxicity range, a range of soluble and insoluble doses, or a maximum dose of 10 mM or 5 mg/ml (whichever is lower) for nontoxic test articles. 43 Test Article Handling Test article concentrations will be expressed as weight per milliliter of culture `medium in the cell cultures. For test articles that are known tobe markedly volatile, the cultures will be incubated with tightly closed caps until the test article is washed off. To compensateforthe lack of CO, circulation, HEPES buffer (25 mM final concentration) will be included in the medium to control the pH. If marked pH changes are noticed (i.., color changes in medium) during the solubility test, HEPES buffer will be included in the medium to attempt to counteract the pH change. Further neutralization (.g., with HCI or NaOH) will be performed to maintain a normal culture pH range (approximately 6.8 t0 7.4) only after consultation with the Sponsor. 44 Chromosomal Aberrations Assay `The chromosomal aberrations assay will be conducted with about 3-hour 298 6of 15 01877 COVANCE> PROTOCOL NO. 449 EDITION 14 exposures to test article in the absence and presence of the S9 metabolic activation system. In the absence of any toxicity information, a wide range of test article concentrations will be applied, starting above the observed solubility limit in culture medium, or at approximately 10 mM or 5 mg/ml (whichever is lower) for soluble test articles. Successively lower doses will usually be determined by. using 70% of the preceding dose. A single harvest time of approximately 22 hours will be used. Thisharvesttime corresponds to 1.5 times the cell cycle time of approximately 15 hours after the lymphocytes are induced to divide by the addition of PHA-M (Galloway et al., 1994). Lymphocyte cultures will be initiated with 0.6 ml of whole blood in 9.4 ml of fresh culture medium. Duplicate flasks will be used for each dose level, vehicle, and for each of three doses of the positive control compounds. Vehicle control cultures willbe dosed with the vehicle for the test article at the sameor the `maximum concentration used in the test cultures. 44.1 Assay without S9 activation `Two days after culture initiation, the culture medium will be replaced with fresh medium and the cells will be treated with the test, vehicle control, and positive control articles. The cultures will be returned to the incubator at 37 22C for an exposure period of about 3 hours. The cultures then will be washed with phosphate buffered saline (PBS)toremove the test article, and fresh medium will be added. Incubation will be continued for about 18 hours, with Colcemid (0.1 pg/ml) added for the last 2.0.5 hours of incubation. 442 Assay with S9 activation `Two days after culture initiation, culture medium will be replaced with fresh medium without fetal bovine serum (FBS) and the cells will be: treated with the test, vehicle control, and positive control articles. The S9 `metabolic activation system also will be included in all cultures. The cultures will be retumed to the incubator at 37 2 C for an exposure period of about 3 hours. The short incubation time is used because prolonged exposure to the $9 mixture is toxic 10 cells, and enzyme activity is lost rapidly at 37C. Serum is omitted to avoid possible inactivation (by. binding to serum proteins) of short-lived, highly reactive intermediates produced by the S9 system. After exposure, the cells will be washed at 298 : Tof 15 01878 COVANCE> PROTOCOL NO. 449 EDITION 14 least twice with PBS, and normal culture medium containing 15% FBS will be added. Incubation will be continued for about 18 hours, with Colcemid (0.1 pg/ml) added for the last 2 0.5 hours of incubation. 443 Summary of assay schedule Schedule (hours)" Test TestAticle Exposure Wash Colcemid Harvest Condition Added Completed Completed Added _ Started $9 0 3 4 20 2 +59 0 3 4 20 2 * Times given are approximate and may vary with the sizeofthe assay. 444 Assay termination `The cultures will be centrifuged, the supematant discarded, and the cells will be swollen with 75 mM KCl, fixed in methanol: glacial acetic acid (3:1, vv), dropped onto glass slides, and air dried. The slides will be stained with 5% Giemsa andairdried. After drying, the slides will be rinsed in xylene and mounted permanently. For controlofbias, the slides `will be coded for analysis. One thousand (1000) cells will be analyzed for `mitotic index (percentage of cells in mitosis) from each culture in the dose range inclusive of the toxicity range obtained as well as the negative and vehicle control cultures. `The doses selected for analysis will be based on the observations of mitotic index reduction, and the maximum dose selected will be the highest dose at which a sufficient number of analyzable metaphase cells are expected. If possible, the highest dose selected would suppress the: `mitotic index by about 50%, but not greater than 60% reduction. Cells will be selected for scoring on the basis ofgood chromosome `morphology, and only cells with the number of centromeres equal to the `modal number46 will be analyzed. Normally, 200 metaphases per dose level (100 from each of the duplicate cultures), if available, will be read. 1 200 metaphases are not available from cach of these doses, lower doses maybe evaluated. `The complete number of 100 metaphases per culture `may not be available due to toxicity or quality of preparation. At least 25 298 8of 15 041879 COVANTCE> PROTOCOL NO. 449 EDITION 14 2me5t%apohfacseelslswwililthbeonaenaolryzmeodrefraobmertrhatoisoencsu.ltPuerrecsetnhtatpohlayvpelogirdeyataenrdthan endoreduplication will also be analyzed culture, if available, and tabulated. by evaluating 100 metaphases per Standard forms will be used to record analyses. For each cell bearing an aberration, the microscope stage location (vemier readings) will be orfecaobredrerdatsioontshastctohreedcealnldmtahyeibrederfeilnoictaitoendsiafrneepcreossvairdye.d iTnhSeecctoimopnle1t0e.0.list Gaps are recorded separately total aberration frequency. and reported but will not be included in the 50 DATA 51 Data Presentation Data will be summarized in tables showing mitotic index, percent polyploidy, fpeorucnedn,tfernedqoureendcuipelsiocaftaiboenr,ratthieonnsumpberercesllo,facnedllspearncaelnytzaegde,sotfypceesllosf babeearrriantgions aberrations. Chromatidand isochromatid gaps will be noted but will not be added tirnuteo btrheeatkost.als for aberration assessment since they are not considered to represent 52 Assay Acceptance Criteria Athne afsoslalyowwiinlgl cbreitceorniasiadreerseadtiasfciceedp.taTbhleefmoerteavbaolluiactiaoctniovfattieosnt arensdulntosnoanctliyvifaatlilonof sections of the aberrations assays are independent units and will be repeated independently, as needed, to satisfy the acceptance criteria. 521 Acceptable Controls `The vehicle control cultures must contain less than approximately 5% cells with aberrations. `The positive control result must be significantly higher (p < 0.01) than the vehicle controls. If the positive control result in the test with 9 is adequate in an assay where activation and nonactivation assays are run concurrently, but the positive control in the nonactivation assay fai, the test need not be repeated because the S activation positive control demonstratehse sensitivity of the cells. 298 9of 15 01880 COVANCE> PROTOCOL NO. 449 EDITION 14 522 Acceptable High Dose If the aberration results are negative and there is no significant reduction (approximately 250%) in mitotic index, the assay must include the highest applicable dose (a target dose of 10 mM or 5 mg/ml, whichever is lower) or a dose exceeding the solubility limit in culture medium. Testing will be conducted at insoluble dose levels when a well-dispersed suspension in culture medium is obtained that does not settle rapidly. 523 Acceptable NumberofDoses Each assay treatment condition must include at least three analyzable dose levels. 53 Assay Evaluation Criteria After completion of microscopic analysis, data will be decoded. The following factorsaretaken into account in evaluation of the test article data: Percentage of cells with structural aberrations. Percentageofcells with more than one structural aberration. Evidence for increasing amounts of damage with increasing dose, i.., a doserelated increase in aberrations. `The experimental unit is the cell, and therefore the percentageofcells with structural aberrations will be the basis for evaluation. Statistical analysis will employ a Cochran-Armitage test for linear trend and Fisher's Exact Test (Thakur et al., 1985) 10 compare the percentage of cells with aberrations (and, if applicable, the percentage of cells with more than one aberration) in treated cells 10 the results obtained for the vehicle controls. Statistical analysis also will be performed for cells exhibiting polyploidy and/or endoreduplication in order to indicate significant (p 5 0.01) increases in these: events as indicators of possible induction of numerical aberrations; however, the test article will be evaluated only for structural aberrations and not for numerical aberrations by this protocol. 531 Evaluation ofa Positive Response Atest article will be considered positive for inducing chromosomal abermations ifa significant increase (the difference will be considered 298 100f15 01381 COVANCE> PROTOCOOCLOL NO.NO. 449 EDITION 14 significant when p < 0.01) in the number of cells with chromosomal aberrations is observed at one or more dose levels. Statistical evaluation of the percentage of cells with more than one aberration provides an indication of the severity of the positive response observed. The linear trend test evaluates the dose-responsiveness. Iaf significant increase is seen atoneor more dose levels, a dose-response should be observed. 532 Evaluation ofa Negative Response Atest article will be considered negative for inducing chromosomal aberrations if no significant increase is observed in the number of cells `with chromosomal aberrations for any of the dose levels. 533 Equivocal Evaluation Although most assays wil give clearly positive or negative results, in rare. cases the data set will preclude making a definitive judgment about the activity of the test article. Results may remain equivocal or questionable regardless of the number of times the assay is repeated. Although the evaluation criteria provided here normally will be sufficient, the study director may use additional considerations to obtain a final evaluationofthe test article based upon the study director's scientific judgment. 60 REFERENCES Evans, HJ. (1962) Chromosomal aberrations produced by ionizing radiation, Intemational Review of Cytology, 13: 221-321. Evans, HJ. (1976) Cytological Methods for Detecting Chemical Mutagens. In: Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. (ed.), Plenum Press, New York and London, pp. 1-29. Galloway, S.M., Aardema, MJ, Ishidate, M., J., Ivett, J.L., Kirkland, D.J., Morita, T., Mosesso, P., and Sofuni, T. (1994) Report from working group on in vitro tests for chromosomal aberrations, Mutation Research, 312 (3): 241-261 Maron, DM., and Ames, B.N. (1983) Revised methods for the Salmonella mutagenicity test, Mutation Research, 113: 173-215. 298 : 1of15 01882 COVANCE> PROTOCOL NO. 449 EDITION 14 `Thakur, A.J., Berry, K.J., and Mielke, PW., Jr. (1985) A FORTRAN program for testing trend and homogeneity in proportions, Computer Programs in Biomedicine, 19: 229-233. 70 REPORT FORMAT Covance employs a standardreport format for each assay design. The final report will `provide the following information. + Sponsor identification. * Quality Assurance statement. + Statement of GLP compliance. * Signature of study director. * Test article identification and Covance Study Number. A physical description of the test article and date of receipt will be included in this section. + Type of assay and protocol number. * Datesof study initiation and completion. * Identificationof study director and senior technician. * Methods. Evaluation criteria. + Interpretation of results. Conclusions. * References. + Test results presented intabularform. Historical control data. 80 CHANGES OR REVISIONS Any changes to or revisions of this approved protocol will be documented, signed by the study director, dated, and maintained with this protocol. 90 RECORDS TO BE MAINTAINED All raw data, documentation, records, protocol, and final reports generated as aresult of this study will be archived in the storage facilities of Covance-Vienna for at least one year following submissionof the final report to the Sponsor. After theoneyearperiod, the `Sponsor may elect to have the aforementioned materials retained in the storage facilities of Covance-Vienna for an additional periodoftime or sent to a storage facility designated by the Sponsor. 298 . 120f15 01883 COVANTCE> PROTOCOL NO. 449 EDITION 14 100 DEFINITIONS OF CHROMOSOMAL ABERRATIONS FOR GIEMSA STAINED CELLS SIMPLE TB Chromatid Break: SB Chromosome Break: DM "Double Minute" Fragment: An achromatic region in one chromatid, larger than the `width of a chromatid. The associated fragment may be partially or completely displaced. Chromosome has a clear break, forming an abnormal (deleted) chromosome with an acentric fragment that is dislocated. This classification now includes the acentric fragment (AF). AnAFis different from a SB only in that it can not be related to any specific chromosome. These are small double dots, which may represent terminal or interstitial deletions, or even small rings. `These possible origins are not distinguishable. COMPLEX ID Interstitial Deletion: TR Triradial: QR Quadriradial: CR Complex Reamangement: D Dicentric: DF Lengthof chromatid "cut out" from midregion ofa chromatid, resulting in a small fragmentor ring lying beside a shortened chromatidor a gap in the chromatid. An exchange between two chromosomes,orone chromosome and an acentric fragment, which results in a three-armed configuration. An exchange like a riradial, but resulting inafourarmed configuration. An exchange among more than two chromosomes or fragments, caused by the induction of several breaks. An exchange between two chromosomes which results in a chromosome with two centromeres. This is often associated with an acentric fragment in which case it is classified as DF. Dicentric with fragment. 298 130f15 01884 COVANCE.> COMPLEX (Continued) TC Tricentric: QC PC Quadicentric: Pentacentric: HC Hexacentric: R Ring: RC Ring Chromatid: RE Cl Chromosome Intrachange: T Translocation: AB Abnormal: PROTOCOL NO. 449 EDITION 14 Ainna ecxhcrhoamnogesoimnevowlivtihngtthhrreeeeccenhtrroommoerseosm.esOfatnednresulting associated with two to three AF. Such exchanges can involve many chromosomes and are named as follows: ffiovuer cceennttrroommeerreess,, uuppttoofifovuerAAFF six centromeres, up to six AF A chromosome which forms a circle containing a centromere. `This is often associated with an acentric fragment, in which case it is classed as RF. Single chromatid ring (acentric). Ring with associated acentric fragment. Exchange within a chromosome; e.g.aing that does not include the entire chromosome. Obvious transfer of material between two chromosomes resulting in two abnormal chromosomes. When identifiable, scored as "T" not "2AB". Abnormal monocentric chromosome. This is a chromosome whose morphology is abnormal for the Karyotype, and often the result of a translocation, pericentric inversion, etc. Classification used if abnormality cannot be ascribed to, e.g.a reciprocal translocation. 298 140f15 01885 COVANCE> OTHER GT GreaterThan five: PP Polyploid Cell: E Endoreduplication: NOTCOMPUTED TG Chromatid Gap: SG Chromosome Gap: UC Uncoiled Chromosome: PROTOCOL NO. 449 EDITION 14 Greater than 5 aberrations: A cell which contains more than 5 aberrations. Heavily damaged cells will be analyzed to identify the types of aberrations because: multiple fragments, such as those found associated with a tricentric, do not count as independent aberrations. A cell containing multiple copies of the haploid number (a) of chromosomes. A failure of chromosomes to separate, resulting in a 4n cell. ("tid gap"). An achromatic (unstained) region in one. chromatid, the size of which is equaltoor smaller than the widthof a chromatid. These are noted but not usually included in final totals of aberrationsas they may not all be true breaks. (`isochromatid gap, IG"). Same as chromatid gap but at the same locus in both sister chromatids. Failure of chromatin packing. Probably nota true aberration. 2098 : 150f15 01836