Document ORY65b0Yo9jzv0z1qVxnB4xK

THE DEVELOPMENT SERVICES COMPANY AR6-0131 g~ y 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 1 of 25 001346 Covance 20784-0-449 QUALITY ASSURANCE STATEMENT Chromosomal Aberrations in Human Whole Blood Lymphocytes With PFOS The report has been reviewed by the Quality Assurance Unit of Covance Laboratories Inc., in accordance with the Good Laboratory Practice regulations as set forth in the Environmental Protection Agency (EPA - TSCA), Title 40 of the U.S. Code of 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 09/01/99 Auditor P. Cceres 10/13,14/99 Draft Report Review 10/14/99 P. Cceres 10/25/99 Final Report Review 10/25/99 P. Cceres Representative, Qi lity Assurance Unit Date -2 - 001S47 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), Title 40 of 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 integrity of the study or the interpretation of the 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 evaluation of the test article as presented herein represents an appropriate conclusion within the context of the 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: Hemalatha Murli, PhD Genetic and Cellular Toxicology Study Completion Date -3- 001848 Covance 20784-0-449 TABLE OF CONTENTS Page No. ABSTRACT.................................................................................................................................... 6 STUDY INFORMATION.............................................................................................................. 7 Sponsor Test Article Assay Information Study Dates Supervisory Personnel OBJECTIVE ................................................................................................................................... 7 TEST SYSTEM RATIONALE.......................................................................................................7 MATERIALS AND METHODS.....................................................................................................8 Test System Media and Cell Culture Conditions Test Article Control Articles S9 Metabolic Activation System Chromosomal Aberrations Assay DATA ........................................................................................................................................... 10 Data Presentation Assay Acceptance Criteria Assay Evaluation Criteria RESULTS ..................................................................................................................................... 12 Test Article Handling Chromosomal Aberrations Assay Without Metabolic Activation Chromosomal Aberrations Assay With Metabolic Activation CONCLUSION ............................................................................................................................. 14 PROTOCOL DEVIATIONS.........................................................................................................14 RECORDS TO BE MAINTAINED .............................................................................................14 -4- 001549 Covance 20784-0-449 TABLE OF CONTENTS (Continued) Page No. REFERENCES ............................................................................................................................. 14 DATA TABLES ........................................................................................................................... 15 HISTORICAL CONTROL DATA .............................................................................................. 22 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS .. 23 -5- C01S50 ABSTRACT Covance 20784-0-449 The objective of this in vitro assay was to evaluate the ability of PFOS to induce chromosomal aberrations in human whole blood lymphocytes with and without metabolic activation. Due to toxicity constraints, the highest concentration tested in the assay was 599 pg/mL without metabolic activation and 449 pg/mL with metabolic activation. The test article was dissolved in dimethylsulfoxide (DMSO) for the assay. The stock solution and its dilutions were dosed using a dosing volume of 1% (10.0 pL/mL) and the vehicle control cultures were treated with 10.0 pL/mL of DMSO. 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 initiation of treatment. 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 concentrations of 199,249,299, and 349 pg/mL without metabolic activation and 99.3, 149, 199, and 299 pg/mL with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. PFOS was considered negative for inducing chromosomal aberrations in human whole blood lymphocytes with and without metabolic activation. -6- 001851 STUDY INFORMATION Covance 20784-0-449 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 Type of Assay: Chromosomal Aberrations in Human Whole Blood Lymphocytes Protocol No..: 449, Edition 14 Covance Study No.: 20784-0-449 Study Dates Initiation Date: August 20,1999 Experimental Start Date: September 01,1999 Experimental Termination Date: October 01,1999 Supervisory Personnel Study Director: Hemalatha Murli, PhD Laboratory Supervisor: Emilie C. Beckelhimer, BS OBJECTIVE The objective of this 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 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 types of aberrations are provided in this report. Structural aberrations may be of two types, chromosome or chromatid: Chromosome aberration: structural chromosome damage expressed as breakage, or breakage followed by reunion, of both sister chromatids at an identical site. -7- 001852 Covance 20784-0-449 Chromatid aberration: 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 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 microscopically for the presence of 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 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 of 46 for human lymphocytes) were not determined. However, the occurrence of polyploidy 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 without a history of radiotherapy, chemotherapy, or drug usage, and lacking current viral infections) was drawn into sterile, heparinized "vacutainers". Whole blood cultures were initiated in 15 mL centrifuge tubes by adding 0.6 ml of fresh heparinized blood to 9.4 mL of culture medium. Media and Cell Culture Conditions Cultures in 15 ml tubes were incubated with loose caps at 37C 2C in a humidified atmosphere of 5% 1.5% C 02in air. The medium was RPM I1640 supplemented with approximately 15% fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 /g/mL), L-glutamine (2 mM) and 1% 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. -8- 001S53 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 pL/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. One of the concentrations was analyzed in each of the aberration assays based on the current information on the positive control response in the laboratory. Both MMC and CP were dissolved in water. S9 Metabolic Activation System The in vitro metabolic activation system (Maron and Ames, 1983) consisted of a rat liver postmitochondrial fraction (S9) and an energy-producing system (NADP plus isocitric acid). Various hepatic P450 isoenzyme levels are increased by treatment of the rats with AroclorTM 1254 (single concentration of 500 mg/kg and sacrificed 5 days later (Molecular Toxicology, Inc., Lot No. 929). The S9 fraction, prepared in sucrose or in potassium chloride, was retained frozen at <-70C until use. Aliquots of S9 were thawed immediately before use and added to the other components to form the activation system described as follows: S9 Activation System Component NADP (sodium salt) Isocitric acid Homogenate CS9 fraction) Concentration in Cultures 1.5 mg/mL (1.8 mM) 2.7 mg/mL (10.5 mM) 15.0 uL/mL* (1.5%) * This concentration o f rat S9, obtained from M olecular T oxicology Inc., B oone, 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 each of three concentrations of the positive control. In the aberrations assay without and with metabolic activation, 22.0- hour harvests were conducted. This harvest time 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). -9- C01S54 Covance 20784-0-449 Aberrations Assay Without and With Metabolic Activation. Cultures were initiated with 0.6 mL of whole blood in 9.4 mL of fresh culture medium. For the nonactivation assay, 2 days after culture 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 =37 C for 3.0 hours in the presence of 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 rest of the culture period up to the time of harvest with 0.1 pg/mL Colcemid present during the last 2.0 hours of incubation. The cultures were then harvested. Harvest Procedure. The cultures were centrifuged, the supernatant discarded, and the cells were swollen with 75 mM KC1 hypotonic solution. The cultures were then fixed with an absolute methanol: glacial acetic acid (3:1, v/v) 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 analysis o f mitotic 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 of centromeres equal to the modal number 46 were analyzed. One hundred cells, if possible, from each replicate flask from four concentrations of the test article, and from the vehicle and one dose of the positive control cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962, 1976). At least 25 cells were analyzed from those cultures that had greater than 25% of cells with one or more aberrations. Mitotic index was evaluated from the vehicle control and a range of concentrations by analyzing the number of mitotic cells in at least 1000 cells per culture and the ratio expressed as a percentage of mitotic cells. Percent polyploidy and endoreduplication were also analyzed by evaluating at least 100 metaphases per culture, if available, and tabulated. For control of bias, 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 numbers of cells analyzed, types of aberrations found, frequencies of aberrations per cell, and percentages of 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. -1 0 - C01S55 Covance 20784-0-449 Assay Acceptance Criteria An assay was considered acceptable for evaluation of test results only if all of the following criteria were satisfied. The two treatment conditions are independent units 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. If the aberration results are negative and there is no significant reduction (approximately >50%) in mitotic index, the assay must include the highest applicable dose (10 mM or 5 mg/mL, whichever is lower) or a dose where a well-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 evaluation of the test article data: Percentage of cells with aberrations. Percentage of cells with more than one aberration. Evidence for increasing amounts of damage with increasing dose, i.e., dose related increase. The experimental unit is the cell, and therefore the percentage of cells with structural aberrations was the basis for evaluation. Statistical analysis employed a Cochran-Armitage test for linear trend and Fisher's Exact Test (Thakur et al., 1985) to compare the percentage of cells with aberrations (and, if applicable, the percentage of cells 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 indicators of possible induction of numerical aberrations; however, the test article was evaluated only for structural aberrations and not for numerical aberrations by this protocol. Evaluation of a 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 number of cells with chromosomal aberrations is observed at one or more concentrations. Statistical evaluation of the percentage of cells with more than one aberration provided an indication of the severity of the positive response observed. The linear trend test evaluated the dose responsiveness. If a significant increase was seen at one or more concentrations, a dose-response should have been observed. -11- C01S56 Covance 20784-0-449 Evaluation of a Negative Response. A test article was considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of cells with chromosomal aberrations at any of the 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 activity of the test article. Results might remain equivocal or questionable regardless of the number of times the assay is repeated. Although the evaluation criteria provided here normally was sufficient, the Study Director might use additional considerations to obtain a final evaluation of the test article based upon the Study Director's scientific judgment. RESULTS Test Article Handling Solubility and Dose Determination. PFOS was insoluble in water at concentrations of 50.0 to 500mg/mL. In DMSO, a 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 of cells using a 1% (10 pL/mL) dosing volume in RPM I1640 culture medium. At a dosed concentration of 4010 pg/mL, a translucent suspension with fine particles of precipitate was observed and the pH was 8.0 (pH of RPMI 1640 culture medium was 8.0). DMSO (Acros Organics, lot # A 12649701) was selected as the vehicle for this assay. 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 pL/mL) dilution of the DMSO stock solutions, and the vehicle control culture was treated with 10.0 pL/mL of DMSO. Concentrations of 27.2, 38.8, 55.4, 79.1,113,161,230, 329,470, 671, 959, 1370,1960, 2800, and 4000 pg/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 activation with a 3.0-hour treatment and a 22-hour harvest. The stability of the test article under the preparation and dosing conditions used in this assay is the responsibility of the sponsor. -1 2 - C01357 Covance 20784-0-449 Chromosomal Aberrations Assay Without Metabolic Activation A summary of the treatment times for the chromosomal aberrations assay is given below. Summary of Chromosomal Aberrations Assay Treatment Schedule in Hours Activation Test Article Condition Added - S9 0 Wash Colcemid Harvest Added Started 3.0 20.0 22.0 Reductions of 38%, 8%, 15%, 15%, 12%, 19%, 42%, 69% and 92% were observed in the mitotic indices of the cultures treated with 12.5, 24.9, 49.7, 99.3,149,249, 299, 349, and 449 pg/mL, respectively, as compared with the vehicle control cultures (Table 2). Chromosomal aberrations were analyzed from the cultures treated with 199, 249,299, and 349 pg/mL (Table 3). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed. The sensitivity of the cell culture for induction of chromosomal aberrations is shown by the increased frequency of aberrations 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 summary of the treatment times for the initial 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 3.0 20.0 22.0 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 pg/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- C01S58 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 copy of the final report generated at Covance as a result of this study will be archived in the storage facilities of Covance-Vienna for at least 1 year following submission of the final report to the Sponsor. After the 1-year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities of Covance-Vienna for an additional period of time, or sent to a storage facility designated by the Sponsor. REFERENCES Evans, H.J., "Chromosomal aberrations produced by ionizing radiation." International Review o f Cytology, 13:221-321 (1962). Evans, H.J., "Cytological Methods for Detecting Chemical Mutagens." Chemical Mutagens, Principles and Methodsfor their Detection, Hqllaender, A. (ed.), Voi. 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). -14- 001S59 Covance 20784-0-449 DATA TABLES -15- C01S60 Covance 20784-0-449 TABLE 1 ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY Assay No.: 20784 Trial No.: I Date: 09/01/99 Lab No.: CY8249 Compound: PFOS__________________________________________________ Metabolic Activation: -S9 3.0 hour treatment, 22.0 hour harvest Treatment % % Average % Mitotic Mitotic % Mitotic Index Index Mitotic Index A culture B culture Index Reduction VEHICLE CONTROL DMSO 10.0 gDmL 0.7 0.6 0.7 0 TEST ARTICLE 27.2 (ig/mL 0.8 0.6 0.7 0 38.8 ng/mL 0.4 0.6 0.5 29 55.4 (tg/mL 0.5 0.5 0.5 29 79.1 (tg/mL 0.5 0.6 0.6 14 113 ng/mL 0.4 0.7 0.6 14 161 ng/mL 0.4 0.5 0.5 29 230 |ig/mL 0.7 0.6 0.7 0 329 jig/mL 0.4 0.6 0.5 29 470 |tg/mL 1.9 1.6 1.8 0 DMSO = Dimethylsulfoxide -16- CG13G1 Covance 20784-0-449 TABLE 2 ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY Assay No.: 20784 Trial No.: IA Date: 09/15/99 Lab No.: CY9079 Compound: PFOS__________________________________________________ Metabolic Activation: -S9 3.0 hour treatment, 22.0 hour harvest Treatment % % Average % Mitotic Mitotic % Mitotic Index Index Mitotic Index A culture B culture Index Reduction VEHICLE CONTROL DMSO lO-OgLAnL 2.2 3.0 2.6 0 TEST ARTICLE 12.5 gg/mL 1.9 1.3 1.6 38 24.9 gg/mL 2.5 2.3 2.4 8 49.7 gg/mL 2.2 2.2 2.2 15 99.3 gg/mL 2.2 2.1 2.2 15 149 gg/mL 2.5 2.0 2.3 12 199 gg/mL 3.7 3.8 3.8 0 249 gg/mL 1.6 2.5 2.1 19 299 gg/mL 1.4 1.6 1.5 42 349 gg/mL 1.2 0.4 0.8 69 449 gg/mL 0.2 0.2 0.2 92 DMSO = Dimethylsulfoxide -17- C013S2 TABLE 3 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES Cells Fixed 22.0 Hours After Initiation o f Treatment, 3.0 Hour Treatment Assay No.: 20784 Trial #: IA Date: 09/15/99 Lab#:CY9079 Metabolic Activation:-S9 Compound: PFOS CONTROLS VEHICLE: - POSITIVE: DMSO MMC TEST ARTICLE lO.OgL/mL 1.50 gg/mL 199 gg/mL 249 gg/mL 299 gg/mL 349 gg/mL CELLS SCORED NOT COMPUTED TG ! SG ; UC # OF % % % NUMBER AND TYPE OF ABERRATION ABERRA- CELLS CELLS % ENDO- TIONS WITH WITH >1 POLY REDUPLI SIMPLE COMPLEX O THER PER ABERRA ABERRA PLOID CATED TB [ SB ID ; TR ; OR CR ; D I R ; Cl 1DF GT CELL TIONS TIONS CELLS CELLS A 100 2 B 100 2 A+B 200 2 2 A 75 B 50 13 51 A+B 125 6 4 A 100 2 B 100 2 A+B 200 4 A 100 2 B 100 2 A+B 200 4 A 100 2 1 B 100 A+B 200 2 1 A 100 3 3 B 100 1 A+B 200 4 3 6 10 4 11 10 21 3 3 2 2 1 10 16 2 16 1 1 2 2 11 11 0.00 0.0 0.02 1.0 0.0 0.0 1.0 0.0 0.01 0.5 0.5 0.0 0.36 29.3 0.52 36.0 6.7 0.0 12.0 0.0 0.42 32.0* 8.8* 0.0 0.00 0.0 0.00 0.0 0.0 0.0 0.0 0.0 0.00 0.0 0.0 0.0 0.00 0.0 0.00 0.0 0.0 0.0 0.0 0.0 0.00 0.0 0.0 0.0 0.00 0.0 0.02 2.0 0.0 0.0 0.0 0.0 0.01 1.0 0.0 0.0 0.00 0.0 0.02 2.0 0.0 0.0 0.0 1.0 0.01 1.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 DMSO = Dimethylsulfoxide MMC = Mitomycin * Significantly greater than the solvent controls, psO.Ol. Covance 20784-0-449 C01S6 CO Covance 20784-0-449 TABLE 4 ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY Assay No.: 20784 Trial No.: I Date: 09/01/99 Lab No.: CY8249 Compound: PFOS__________________________________________________ Metabolic Activation: +S9 3.0 hour treatment, 22.0 hour harvest Treatment % % Average % Mitotic Mitotic % Mitotic Index Index Mitotic Index A culture B culture Index Reduction VEHICLE CONTROL DMSO lO.OpL/mL 0.6 0.5 0.6 0 TEST ARTICLE 27.2 pg/mL 0.2 0.2 0.2 67 38.8 pg/mL 0.2 0.2 0.2 67 55.4 pg/mL 0.2 0.3 0.3 50 79.1 pg/mL 0.2 0.4 0.3 50 113pg/mL 0.3 0.4 0.4 33 161 pg/mL 0.1 0.1 0.1 83 230 pg/mL 0.2 0.0 0.1 83 329 pg/mL 0.1 0.4 0.3 50 470 pg/mL a ___ a -- -- No cells present on slide DMSO = Dimethylsulfoxide C01S64 -19- Covance 20784-0-449 TABLE 5 ASSESSMENT OF TOXICITY FOR CHROMOSOMAL ABERRATIONS ASSAY Assay No.: 20784 Trial No.: IA Date: 09/15/99 Lab No.: CY9079 Compound: PFOS__________________________________________________ Metabolic Activation: +S9 3.0 hour treatment, 22.0 hour harvest Treatment % % Average % Mitotic Mitotic % Mitotic Index Index Mitotic Index A culture B culture Index Reduction VEHICLE CONTROL DMSO 10.0nL/mL 1.6 1.7 1.7 0 TEST ARTICLE 12.5 ng/mL 2.0 1.9 2.0 0 24.9 ttg/tnL 2.5 2.6 2.6 0 49.7 ng/mL 1.4 1.6 1.5 12 99.3 (ig/mL 1.8 3.2 2.5 0 149ng/mL 2.0 2.4 2.2 0 199jjg/mL 1.0 1.0 1.0 41 249 ng/mL 0.5 0.5 0.5 71 299 ng/mL 0.9 0.7 0.8 53 DMSO = Dimethylsulfoxide CG1365 TABLE 6 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES Cells Fixed 22.0 Hours After Initiation o f Treatment, 3.0 Hour Treatment Assay No.: 20784 Trial #: IA Date: 09/15/99 Lab#:CY9079 Metabolic Activation:+S9 npound: PFOS CONTROLS VEHICLE: DMSO POSITIVE: CP TEST ARTICLE to T CELLS SCORED NOT COMPUTED TG : SG ; UC # OF % % % NUMBER AND TYPE OF ABERRATION ABERRA- CELLS CELLS % ENDO- TONS WITH WITH >1 POLY REDUPLI- SIMPLE COMPLEX O TH E R PER ABERRA ABERRA PLOID CATED TB ; SB D I TR 1 QR 1 CR 1 D 1 R I CI I DF GT CELL TIONS TIONS CELLS CELLS 10.0nL/mL A 100 2 B 100 6 A+B 200 8 50.0 pg/mL' A 25 B 25 32 33 A+B 50 6 5 99.3 ng/mL A 100 B 100 A+B 200 3 149 ng/mL A 100 1 B 100 3 1 A+B 200 4 1 199 pg/mL A 100 4 B 100 2 A+B 200 6 299 ng/mL1' A 27 B4 A+B 31 2 2 8 9 17 3 3 6 3 1 4 2 2 0.00 0.0 0.02 2.0 0.0 0.0 0.0 0.0 0.01 1.0 0.0 0.0 0.40 28.0 0.36 32.0 12.0 0.0 4.0 0.0 0.38 30.0* 0.00 0.0 0.00 0.0 8.0* 0.0 0.0 0.0 0.0 1.0 0.00 0.0 0.0 0.5 0.03 3.0 0.03 2.0 0.0 0.0 1.0 1.0 0.03 2.5 0.5 0.5 0.03 2.0 0.01 1.0 1.0 0.0 0.0 0.0 0.02 1.5 0.5 0.0 0.00 0.0 0.00 0.0 0.0 0.0 0.00 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.5 0.0 0.0 0.0 *Only 70 metaphase cells from the B culture were scored for polyploidy and endoreduplication. bToo few metaphase cells available for analysis of polyploidy and endoreduplication. DMSO = Dimethylsulfoxide CP = Cyclophosphamide * Significantly greater than the solvent controls, p<0.01. Covance 20784-0-449 oo h* ta a a Covance 20784-0-449 HISTORICAL CONTROL DATA CONTROL DATA OF CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES 1/97 THROUGH 9/98 = 2 0 - 2 4 HOUR HARVEST Negative Control 3 Hour Treatment Vehicle Control (Pooled) 3 Hour Treatment Positive Control - MMC 3 Hour Treatment Negative Control Continuous Treatment Vehicle Control (Pooled) Continuous Treatment Positive Control - MMC Continuous Treatment Negative Control 3 Hour Treatment Vehicle Control (Pooled) 3 Hour Treatment Positive Control - CP 3 Hour Treatment N = Number of trials. MMC = Mitomycin C CP = Cyclophosphamide Activation % of Cells % of Cells % Poly- % Endore- % With With >1 ploid duplicated Mitotic Aberrations Aberrations Cells Cells Index Without MIN MAX AVG SD() N 0.0 2.5 0.45 0.60 19 0.0 0.0 0.0 1.2 0.5 1.5 0.0 6.2 0.03 0.11 0.00 2.86 0.12 0.36 0.00 1.54 19 19 19 19 Without MIN MAX AVG SD () N 0.0 1.5 0.61 0.54 19 0.0 0.0 0.0 1.1 0.0 0.5 0.5 6.9 0.00 0.05 0.08 2.79 0.00 0.16 0.19 1.77 19 19 19 19 Without MIN MAX AVG SD () N 20.0 62.0 40.97 10.64 19 1.3 0.0 0.0 26.0 0.5 0.0 12.87 0.08 0.00 6.85 0.19 0.00 19 19 19 - - Without MIN MAX AVG SD() N 0.0 1.5 0.42 0.40 32 0.0 0.0 0.0 0.9 0.0 1.5 0.5 6.7 0.00 0.20 0.02 3.28 0.00 0.38 0.09 1.63 32 32 32 32 Without MIN MAX AVG SD() N 0.0 2.5 0.56 0.62 33 0.0 0.0 0.0 1.1 0.5 1.0 0.0 6.7 0.03 0.14 0.00 3.15 0.12 0.29 0.00 1.67 33 33 33 33 Without MIN MAX AVG SD() N 11.5 64.0 31.06 11.66 33 1.5 0.0 0.0 1.8 28.0 0.5 0.0 4.9 7.93 0.08 0.00 3.12 5.72 0.18 0.00 1.58 33 33 33 3 With MIN MAX AVG SD() N 0.0 2.0 0.48 .0.59 45 0.0 0.0 0.0 0.7 0.5 2.0 0.5 9.4 0.01 0.22 0.01 3.22 0.08 0.43 0.08 1.83 45 45 45 45 With MIN MAX AVG SD () N 0.0 2.5 0.44 0.56 46 0.0 0.0 0.0 0.5 1.0 0.5 0.5 6.8 0.03 0.07 0.02 2.90 0.16 0.17 0.10 1.52 46 46 46 46 With MIN 9.0 MAX 54.0 AVG 32.52 SD () 10.39 N 46 0.0 0.0 0.0 0.4 34.0 1.6 0.0 5.9 11.71 0.07 0.00 2.53 8.11 0.26 0.00 1.95 46 46 46 6 001.867 -2 2 - Covance 20784-0-449 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS Simple TB Chromatid Break: An achromatic region in one chromatid, larger than the width of a 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 a SB only in that it can not be related to any specific chromosome. DM "Double Minute" Fragment: 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: Length of chromatid "cut out" from midregion of a 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 triradial, but resulting in a four-armed configuration. CR Complex Rearrangement: An exchange among more than two chromosomes or fragments, caused by the induction of 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. -23- CQ136S Complex (continued) Covance 20784-0-449 TC Tricentric: 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 and are named as follows: QC Quadricentric: PC Pentacentric: HC Hexacentric: four centromeres, up to four AF five centromeres, up to five AF six centromeres, up to six AF R Ring: 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. RC Ring Chromatid: Single chromatid ring (acentric). RF Ring with associated acentric fragment. Cl Chromosome Intrachange: Exchange within a chromosome; e.g., a ring that does not include the entire chromosome. T Translocation: Obvious transfer of material between two chromosomes resulting in two abnormal chromosomes. When identifiable, scored as "T" not "2AB". AB Abnormal: 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. -24- C01SS9 Other GT Greater than Five: PP Polyploid Cell: E Endoreduplication: Abs >1 Ab Not Computed TG Chromatid Gap: SG Chromosome Gap: UC Uncoiled Chromosome: Covance 20784-0-449 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 (n) of chromosomes. A failure of chromosomes 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 size of which is equal to or smaller than the width of a chromatid. These are noted but not usually included in final totals of aberrations as 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 not a true aberration. CQ1S70 THE DEVELOPMENT SERVICES COMPANY AMENDMENT TO THE STUDY PROTOCOL Page 1 of 1 STUDY TITLE: Chromosomal Aberrations in Human Whole Blood Lymphocytes PROTOCOL NO.: 449, Edition 14 COVANCE STUDY NO. : 20784-0-449 Amendment #3 The Sponsor will be identified as 3M Corporate Toxicology and not as 3M Corporation. Reason: Sponsor's request. Study Director: Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department o f Cellular and Genetic Toxicology Date C01571 THE DEVELOPMENT SERVICES COMPANY COVANCE STUDY NO. & 0 9 8 4 - - Q - J - j - 9 PROTOCOL NO. 449 EDITION 14 CHROMOSOMAL ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES Covance Laboratories Inc. (Covance) will conduct this study in compliance with Good Laboratory Practice (GLP) regulations. This protocol, at least one critical phase of the work in progress, and the final report will be subject to audit by Quality Assurance in accordance with Standard Operating Procedures maintained at Covance. The study will be conducted by Covance at 9200 Leesburg Pike, Vienna, Virginia 22182 (Covance-Vienna). PART 1. SPONSOR INFORMATION AND APPROVALS 1.0 SPONSOR IDENTIFICATION Company Name: Address: 3M Corporation 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 2.0 TEST ARTICLE IDENTIFICATION __________ P f o s ___________________________ ___________ `2/h d L J f AJ6 T i* ?<;. i f ________________ 3.0 TEST ARTICLE ANALYSIS The Sponsor will be responsible for the determination and documentation of the analytical purity and composition of the test article and the stability and strength of the dosing solutions. 4.0 NOTIFICATION OF REGULATORY SUBMISSION In order to comply with GLP regulations, consulting laboratories must be notified if all or part of a study is intended for regulatory submission. Covance maintains a master schedule of studies which fall under regulatory review. Please indicate which agency, if any, might receive the results of this study: undetermined FDA p $ EPA-TSCA EPA-FIFRA MAFF MOHW OECD OTHER __________ 2/98 l o f 15 C01372 CO VM C> THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 5.0 STUDY DATES Proposed Experimental Start Date:________ ^ \ Proposed Experimental Termination Date: 'y '-UJma 1- * " 6.0 APPROVAL OF STUDY PROTOCOL Study Director: / ' - i t i X '___________________ Date: Hemalatha Murli, Ph.D. - i % Testing Facility Management: c\ Brian C. Myhr, Ph.D Associate Director Date: Sponsor's Authorized Representative: Date: / L A Ufa.**- f a f f 2/98 2 of 15 C01S73 THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 PART 2. STUDY PROTOCOL CHROMOSOMAL ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES 1.0 OBJECTIVE The objective of this in vitro assay is to evaluate the ability of a test article to induce chromosomal aberrations in cultured human lymphocytes with and without an exogenous metabolic activation system. 2.0 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 types of aberrations are provided in Section 10.0. Structural aberrations may be of two types, chromosome or chromatid: Chromosome aberration: structural chromosome damage expressed as breakage, or breakage followed by reunion, of both sister chromatids at an identical site. Chromatid aberration: 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 they are 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 substance, Colcemid, then are harvested and stained, and metaphase cells are analyzed microscopically for the presence of 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 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 of 46 for human lymphocytes) are not determined by this protocol. However, the 2/98 3 of 15 C01S74 C O V /H C ^ THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 occurrence of polyploidy or endoreduplication, which will be scored, may indicate that the test article has the potential to induce numerical aberrations. 3.0 MATERIALS 3.1 Test System Human venous blood from a healthy donor will be drawn into sterile, heparinized "Vacutainers". Whole blood cultures will be initiated by adding 0.6 ml of fresh heparinized blood to 9.4 ml of culture medium in each 15 ml centrifuge tube. 3.1.1 Media and Cell Culture Conditions Cultures will be incubated with loose caps at about 37C 2C in a humidified incubator, in an atmosphere of about 5% 1.5% C 02in air. The medium will be R PM I1640 supplemented with approximately 15% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 /g/ml), L-glutamine (2 mM) and 1% phytohemagglutinin (PHA-M). The total culture volume will be 10 ml. 3.2 Test Article Solid or liquid test articles are suitable for this assay. Volatile test articles may be used in cultures with tightly closed caps. The test article is identified in Part 1 of this protocol. Storage conditions will be as specified by the Sponsor. 3.3 Control Articles 3.3.1 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 article-treated cultures. 3.3.2 Positive Control Articles Known clastogenic (chromosome-breaking) agents will be used for structural aberrations analysis. 2/98 4 o f 15 C01S75 THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 W ithout S9 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 to 3.0 jLig/ml. W ith S9 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 /ig/ml. 3.4 S9 Metabolic Activation System The in vitro metabolic activation system (Maron and Ames, 1983) consists of a rat 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 will be prepared approximately 5 days later. The S9 fraction, prepared in sucrose or in potassium chloride, is purchased from a commercial supplier and is retained frozen at <70C until use. Aliquots of S9 will be thawed immediately before use and added to the other components to form the activation system described as follows: Component NADP (sodium salt) Isocitric acid Homogenate (S9 fraction) Concentration in Cultures 1.5 mg/ml (1.8 mM) 2.7 mg/ml (10.5 mM) 15.0 il/ml* (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. 4.0 EXPERIMENTAL DESIGN 4.1 Test Article Solubility Testing If solubility information is not provided by the Sponsor, a preliminary solubility test will be carried 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 will be diluted 100-fold in culture 2/98 5 of 15 001376 CO V A *fC> THE DEVELOPMENT SERVICES COMPANY 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 or 5 mg/ml, whichever is lower. 4.2 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. 4.3 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 to be markedly volatile, the cultures will be incubated with tightly closed caps until the test article is washed off. To compensate for the lack of C 0 2circulation, HEPES buffer (25 mM final concentration) will be included in the medium to control the pH. If marked pH changes are noticed (i.e., 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 (e.g., with HC1 or NaOH) will be performed to maintain a normal culture pH range (approximately 6.8 to 7.4) only after consultation with the Sponsor. 4.4 Chromosomal Aberrations Assay The chromosomal aberrations assay will be conducted with about 3-hour 2/98 6 of 15 C01S77 THE DEVELOPMENT SERVICES COMPANY 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. This harvest time 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 will be dosed with the vehicle for the test article at the same or the maximum concentration used in the test cultures. 4.4.1 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 2 C for an exposure period of about 3 hours. The cultures then will be washed with phosphate buffered saline (PBS) to remove the test article, and fresh medium will be added. Incubation will be continued for about 18 hours, with Colcemid (0.1 /xg/ml) added for the last 2 0.5 hours of incubation. 4.4.2 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 returned 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 S9 mixture is toxic to 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 2/98 7 of 15 C01S73 C O V /H tfC l> THE DEVELOPMENT SERVICES COMPANY 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 pig/ml) added for the last 2 0.5 hours of incubation. 4.4.3 Summary of assay schedule Schedule (hours)* Test Test Article Exposure Wash Colcemid Harvest Condition Added Completed Completed Added Started -S9 0 +S9 0 3 4 20 22 3 4 20 22 * Times given are approximate and may vary with the size of the assay. 4.4.4 Assay termination The cultures will be centrifuged, the supernatant discarded, and the cells will be swollen with 75 mM KC1, fixed in methanol: glacial acetic acid (3:1, v/v), dropped onto glass slides, and air dried. The slides will be stained with 5% Giemsa and air dried. After drying, the slides will be rinsed in xylene and mounted permanently. For control of bias, 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 of good chromosome morphology, and only cells with the number of centromeres equal to the modal number 46 will be analyzed. Normally, 200 metaphases per dose level (100 from each of the duplicate cultures), if available, will be read. If 200 metaphases are not available from each of these doses, lower doses may be evaluated. The complete number of 100 metaphases per culture may not be available due to toxicity or quality of preparation. At least 25 2/98 8 o f 15 001379 THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 metaphases will be analyzed from those cultures that have greater than 25% of cells with one or more aberrations. Percent polyploidy and endoreduplication will also be analyzed by evaluating 100 metaphases per culture, if available, and tabulated. Standard forms will be used to record analyses. For each cell bearing an aberration, the microscope stage location (vernier readings) will be recorded so that the cell may be relocated if necessary. The complete list of aberrations scored and their definitions are provided in Section 10.0. Gaps are recorded separately and reported but will not be included in the total aberration frequency. 5.0 DATA 5.1 Data Presentation Data will be summarized in tables showing mitotic index, percent polyploidy, percent endoreduplication, the numbers of cells analyzed, types of aberrations found, frequencies of aberrations per cell, and percentages of cells bearing aberrations. Chromatid and isochromatid gaps will be noted but will not be added into the totals for aberration assessment since they are not considered to represent true breaks. 5.2 Assay Acceptance Criteria An assay will be considered acceptable for evaluation of test results only if all of the following criteria are satisfied. The metabolic activation and nonactivation sections of the aberrations assays are independent units and will be repeated independently, as needed, to satisfy the acceptance criteria. 5.2.1 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 S9 is adequate in an assay where activation and nonactivation assays are ran concurrently, but the positive control in the nonactivation assay fails, the test need not be repeated because the S9 activation positive control demonstrates the sensitivity of the cells. 2/98 9 of 15 COISSO THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 5.2.2 Acceptable High Dose If the aberration results are negative and there is no significant reduction (approximately >50%) 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. 5.2.3 Acceptable Number of Doses Each assay treatment condition must include at least three analyzable dose levels. 5.3 Assay Evaluation Criteria After completion of microscopic analysis, data will be decoded. The following factors are taken into account in evaluation of the test article data: Percentage of cells with structural aberrations. Percentage of cells with more than one structural aberration. Evidence for increasing amounts of damage with increasing dose, i.e., a dose- related increase in aberrations. The experimental unit is the cell, and therefore the percentage of cells 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) to compare the percentage of cells with aberrations (and, if applicable, the percentage of cells with more than one aberration) in treated cells to 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 s 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. 5.3.1 Evaluation of a Positive Response A test article will be considered positive for inducing chromosomal aberrations if a significant increase (the difference will be considered 2/98 10 of 15 C01331 covAtrc> THE DEVELOPMENT SERVICES COMPANY PROTOCOL 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. If a significant increase is seen at one or more dose levels, a dose-response should be observed. 5.3.2 Evaluation of a Negative Response A test 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. 5.3.3 Equivocal Evaluation Although most assays will 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 evaluation of the test article based upon the study director's scientific judgment. 6.0 REFERENCES Evans, H.J. (1962) Chromosomal aberrations produced by ionizing radiation, International Review of Cytology, 13: 221-321. Evans, H.J. (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, M.J., Ishidate, M., Jr., 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, D.M., and Ames, B.N. (1983) Revised methods for the Salmonella mutagenicity test, Mutation Research, 113:173-215. 2/98 11 of 15 001.352 THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 Thakur, A.J., Berry, K.J., and Mielke, P.W., Jr. (1985) A FORTRAN program for testing trend and homogeneity in proportions, Computer Programs in Biomedicine, 19: 229-233. 7.0 REPORT FORMAT Covance employs a standard report 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. Dates of study initiation and completion. Identification of study director and senior technician. Methods. Evaluation criteria. Interpretation of results. Conclusions. References. Test results presented in tabular form. Historical control data. 8.0 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. 9.0 RECORDS TO BE MAINTAINED All raw data, documentation, records, protocol, and final reports generated as a result of this study will be archived in the storage facilities of Covance-Vienna for at least one year following submission of the final report to the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities of Covance-Vienna for an additional period of time or sent to a storage facility designated by the Sponsor. 2/98 12 o f 15 C013S3 THE DEVELOPMENT SERVICES COMPANY PROTOCOL NO. 449 EDITION 14 10.0 DEFINITIONS OF CHROMOSOMAL ABERRATIONS FOR GIEMSA STAINED CELLS SIMPLE TB Chromatid Break: An achromatic region in one chromatid, larger than the width of a 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 a SB only in that it can not be related to any specific chromosome. DM "Double Minute" Fragment: 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: Length of chromatid "cut out" from midregion of a 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 triradial, but resulting in a four armed configuration. CR Complex Rearrangement: An exchange among more than two chromosomes or fragments, caused by the induction of 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. 2/98 13 of 15 C01334 C O V A 1iC i> THE DEVELOPMENT SERVICES COMPANY COMPI P.X (Continued) TC Tricentric: QC Quadricentric: PC Pentacentric: HC Hexacentric: R Ring: RC Ring Chromatid: RF Cl Chromosome Intrachange: T Translocation: AB Abnormal: PROTOCOL NO. 449 EDITION 14 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 and are named as follows: four centromeres, up to four AF five centromeres, up to five AF 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., a ring 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. 2/98 14 of 15 C013S5 THE DEVELOPMENT SERVICES COMPANY OTHER GT Greater Than five: PP Polyploid Cell: E Endoreduplication: NOT COMPUTED 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 (n) 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 equal to or smaller than the width of a chromatid. These are noted but not usually included in final totals of aberrations as 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 not a true aberration. 2/98 15 of 15 C01SSG