Document LggNMxYnaGvoo57RDDankOxxX

AR226-2797 Du Pont RLR 150-88 POR DP PONT OSE ORLY Study Title IN VITRO BIOCGMPATABILiITY EVALUA.'TION OF WITH IMR-90 HUMAN DIPLOIqD FIBBRROOBBLLAAISTS IN THE ifTT COLORIMETRIC ASSAY Author Ralph G. Stahl, Jr. Issue Date April 11, 1988 Peforalng Laboratory E. I. du Pont de Nemours and Company, Inc. Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P. 0. Box 50 Newark, Delaware 19714 Medical ssearch No. Laboratory Project ID Haskell Laboratory Report No. 150-88 Page 1 of 12 S^JH^any Sanitized. Does not contain TSCA CBI iM r n i iTlr IttigitfttttSM Du Pont HLR 150-88 COOP T-ABORATORY PRACTICE STATEMENT This study was conducted according to FDA Good laboratoryPractice Regulations (21 CFR 58). Any areas of noncoapllance are documented in the study records. Ho deviations existed that significantly affected the validity of the study. Submitter Sponsor E.I. 4u Pont de Nemours fi Co., Inc. Chemicals and Pigments Department Study Director ------- - 2- sparii" Sanizec. Doss not contain TSCA CR Du Pont HLR 150-88 COMPOUND INFORMATION Material Testedi Q Synonyms: Q Medical Research No.^l Haskell Ho.: H-17,157 CAS Registry No: Other Codes; j Purity; Contaminants; Sponsor: Chemical and Pigments Department Material Submitted `Chemicals and Pigments Department Jackson Laboratory Study Initlated/Compieted: 2/15/88-2/19/88 Notebook(s): Distribution: There are 12 pages In this report. RGS/Aaes 18-12 - 3- Company Sanitized. Does not contain TSCA CBf IN VITRO BIOCOMPATABILlTY EVALUATION OF] WITH IMR-90 HOMAN DIPLOIlj) FIBROBLASTS IN ! Du Pont HLR 150-88 ASSAY Summary was tested for In vitro bloeompatabillty with IMR-90 Insts. Under the conditions of this assay, M i s cytotoxic. Work by: "T rey T. Turner, Sr. Technician 3 h * l Si Mo /Day/Yr Study Director: RodLpi\ M<^V Ralph G. Stahl, Jr., Ph.D. Research Genetic Toxicologist ^ )S*b Mo/Day/Yr Approved by: Awni M. Sarrif Ph.D. Section Supervisor Molecular and Genetic Toxicology V !tt /<ff Mo/Day/Yr I 4 fioffiBany- Does no? ioniain TSCA CB/ Du P o n t HLR 150-88 QUALITY ASSURANCE DOCUMENTATION JIL-gITRO BIOCOMPATABILITY EVALUATION OP imr-9o human DIrLoI^^nBR0BBCT!^xw THE MTT COLORIMETRIC ASSAY AUDITS: Items Audited Protocol, records and final report ________A u d i t D a t e s 3/28,29/88; 4/4/88 SHORT-TERM AUDIT REPORT NUMBER: R-318 DATE FINDINGS REPORTED TO MANAGEMENT AND STUDY DIRECTOR: 4/5/88 In-life critical phases from a representative study of this test type are inspected quarterly. Since short-term studies are numerous and routine in nature, the in-life critical phases from one study exemplify the conduct of other studies from the same test type. Reported by J s e p h C. Hamill /ality Assurance Auditor - 5- Company Sanitized. Does not contain TSCA CBl Du Pont HLR 150-88 INTRODUCTION The-purpose ,af ^ h l ^ ^ t u d y was to evaluate the cytotoxic potential of in 3 M H 9 0 primary human diploid fibroblasts, toxicity decreases ~the growth of cells in culture and cell viability. The basis for this test is the alteration of normal cell morphology and a decrease in the cell's ability to metabolise a chemical substrate. First, cells are exposed to the test! material for 48-72 hours. Second, cells are then examined microscopically for morphological abnormalities. Third, a soluble dye, 3-[4,5-dimethylthlasol-2-yl 1-2.5-dlnb -nyltetrajsollum_brnjin, or MTT, Is dissolved in culture medium and added to the cells. MTT is metabolised by the mitochondria in viable respiring cells to an insoluble purple formasan salt (l-(4,5-iiimethylthiazol-2-ylJ-3,5-diphenylformazan). Fourth, after 2-3 hours, the insoluble formasan trapped in the mitochondria is leached with dimethylsulfokide. The optical density of the formasan, measured spectrophotometricaljLy, is proportional to the number of viable, respiring cells. A measure of the test material's cytotoxicity, and by inference its potential biocojnpatabillty, is obtained by comparing the optical densities from treated cells to those not exposed to the test material. MATERIALS/METHODS A. PROTOCOL The MTT assay is an adaptation of the method developed by Mossman (1983) and modified by Denlsot and Lang (1986). nils study was conducted according to protocol Biocompatabillty-1 filed with the Quality Assurance Section of Haskell Laboratory on October 3, 1986, and updated February 16, 1988. This protocol includes the Standard Operating Procedures: In Vitro Blocompatabillty Evaluation of Material with Human and Mammalian Cells; Cell Counting Method; In Vitro Blocompatabllity Evaluation of Material with Human and Mammalian Cells: MTT Colorimetric Method and Microscopic Examination; Preparation of Blomaterlals; and Using the IBM PC with the Titertek Hultlskan MC for the MTT Assay. - 6- ^ p a n y s a n f& e d . Do; inos cc tair? IS C A CBi Du Pone BLR 150-88 B. TEST MATERIALS 1 Test Compound ____ ______________________________ Based on Information supplied wiffi the test ftlfi compound ws assumed to be stable under the conditions of this a$say. No evidence of instability was observed during the study. 2. Negative and Positive Indicators Negative Control (noqcytotoxlc): RPMI-1640 culture medium (Glbco Lot #s 12N9273 and 43K8473). Positive Indicator (cytotoxic): Mitomycin-C (MHC) (Sigma Lot # 66F-0494) at a final concentration of 10 ug/mL. MMC was prepared in distilled deionlsd water. The negative control and positive indicator were assumed to be stable under the conditions of this study; no evidence of instability was observed. C. CELLS AND CELL CULTURE IHR-90 diploid human fibroblasts were obtained at passage 9 from the American Type Culture Collection (Cat. No. ATCC CCL 186), Rockville, MD. These cells were established in 1977 from the lungs of a 16-week female fetus (Nichols et al. Science 196: 60-63 1977). IMR-90 cells are cultured routinely in a 51 CO- atmosphere at 37C without antibiotics using RPMI-1640 culture medium (Gibco Lot #s 12N9273 and 43K8473) supplemented with L-glutamine (Glbco Lot t 10N0666) and 10Z heat inactivated fetal bovine serum (Gibco Lot # 28N2372). Cells were free of mycoplasma contamination (GenProbe). Because of senescence cells are not used after passage 30. TWenty-four hours before th test, cells were seeded into Corning 96^rell (half-well area) tissue culture dishes at a density of 60,000 cells/ea . Column 1 of the dish was left blank as a control for optical density measurements. Columns 2-12 recel i 100 uL of culture medlum/cell suspension. - 7- Company Sanitized. Does not contain TSCA CBI Du Pont HLR 150-88 D PREPARATION OF TEST MATERIALS The test material through a 0.22 u f: E. DOSE SELECTION (Millipore Clwas filter sterilized ex-GS) before use. Cells were plated in 100 uL of medium per well. Either 10, 25, 50 or 100 uL of test material was added directly to the medium (9%, 20%, 33%, or 50% final concentration of test material, respectively). Concentrations greater than 50% significantly dilute the culture medium, reduce cell growth, and therefore were not tested. The negative control received no test material. Approximately 10 uL of MMC, the positive indicator, stock solution was added to 100 uL of culture medium (final concentration was 10 ug/mL). Treatment wells were individually labeled with the appropriate concentration of test material. After the test material was added, dishes were placed in a 5% C02 , 37C incubator for 72 hours. G. CYTOTOXICITY DETERMINATIONS 1. Microscopic Examination All cells were examined under 250X phase contrast magnification for signs of toxicity after the 72 hour exposure. Observations for cell rounding, sloughing and detachment, and lysis were in accordance with guidelines of the American Society for Testing Materials (ASTM, 1983) and the National Reart, Lung and Blood Institute (1979). 2. Measurement of Effects On Growth After 72 hours of exposure, all 96-wall dishes were removed from the incubator. Each dish was Inverted to remove culture medium. Fifty microliters of a 1 mg/mL solution of MTT (Sigma Lot # 126F5054) prepared in RPMI-1640 culture medium (without phenol red; Gibco Lot # 14N0066) was added to each well. The dishes were returned to the 37"C incubator for about 3 hours. Afterwards, the dishes were removed and inverted to remove the MTT solution. Approximately 50 uL of dimethlysulfoxide (DMS0; Sigma Lot # 116F34861) was added to each well. The purple formazan color was allowed to develop at room temperature for about 30 minutes. Each dish was then placed in the reading chamber of A Titertek Multiskan MC connected to an IBM-FC. The optical density was measured at 560 (test wavelength) and 690 (reference wavelength) nanometers. Data were captured on an IBM-PC, stored on a data diskette, and printed. -8 emparty Sanilfeed. Dees not ,,tain T sc Du Pont HLR 150-88 H. STATISTICAL ANALYSES Optical densities from treated and control cultures were compared using RS/1 computerised statistical software (BBN Software Products Corporation, Book 2: Graphics and Statistics, Cambridge, MA, 1984). A student t-test, assuming unequal variances, compared each test concentration with the untreated control* The null hypothesis was that the optical density in the treated culture was less than that of the control. All comparisons were at the 952 level of confidence (alpha 0.05). I. CLASSIFICATION GUIDELINES The guidelines below are used to aid in the classification of a test sample along with sound scientific Judgement and experience. A test sample is classified as CYTOTOXIC when: A. The optical densities in exposed cultures are significantly less than untreated controls or negative control at the 0.05 level of significance as measured during the test period, #- B. Cell rounding, sloughing or lysis was observed. A test sample is classified as N0NCYT0T0XIC when: A. The optical densities in exposed cultures are not significantly less than untreated controls or negative control at the 0.05 level of significance as measured during the test period, AND B. Cell rounding, sloughing or lysis was not observed. A test sample is classified as EQUIVOCAL when: A. Neither of the criteria for a cytotoxic nor noncytotoxic dassifcation is satisfied. - 9- Company Sanitized. Does not contain TSCA CBf Du Pont HLR 150-88 G. ACCEPTABILITY CRITERIA An acceptable assay Is biased on a single trial in which the negative and positive Indicators respond in the appropriate manner. In addition, contaminated wjells are excluded from data analysis. There must be a minimum of 8 noncontaoinated wells, for each exposure, and the positive and negatlvle indicators. The trial is repeated if these criteria are not det. H. RETENTION OF RECORDS All raw data and the flnial report are stored in the archives of Haskell Laboratory for Tbxicology and Industrial Medicine, Newark, Delaware or in the Du Pont Records Management Center, E. I. du Pont de Nemours and Co., Inc., Wilmington, Delaware. RESULTS/DISCUSSION 4Results o: __ shorn in Table city to IMR-90 human cells are was toxic at concentrations of 20Z, 33Z and 50Z, causing widespread logy was not affected at the 10Z concentration of There were signi leant decreases at ll concentrations o: MMC, the positive indicator, sign! reduced optical density and caused widespread cell lysis. Under the conditions of this assay cytotoxic. 10 - Sempany BanRfee* Does no? contain TSCA OBP Du Pont HLR 150-88 REFERENCES American Society for Testing qnd Materials (1983) Designation F813-83 Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices American Society for Testing and Materials, Philadelphia* PA. Denizot, F. and R. Lang (1986) Rapid colorimetric assay for cell growth and survival: Modifications to the tetrasolium dye procedure giving improved sensitivity and reliability. 'Journal Immunological Methods 69: 271-277. Green, L.M., J.L. Reade and C.F. Vare (1984) Rapid colorimetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphoklnes, Journal Immunological Methods 70: 257-268. Mossman, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal Immunological Methods 65: 55-63. National Heart, Lung and Blood Institute (1979) Guidelines for Phy8iochemlcal Characterisation of Biomaterials. Devices and Technology Branch, National Heart, Lung end Blood Institute, NIH Publ< ation No. 80-2186, Washington, D.C. RS/1 (1984) R.S/1 User's Guide, Book 2, BBN Software Products Corp., Cambridge, MA. - 11 - Company Sanitized. Dees not contain TSCA CBI Du Pont HLR 150-88 Table 1. In Vitro BiocoapatabiHty of H-17,157 With IMR-90 Human Cells. Results of MTT Colotlnetrlc Assay After 72 Hour Exposure. Well # Control 1 0.221 2 0.256 3 0.270 4 0.267 5 0.256 6 0.273 7 0.259 8 0.226 9 0.248 10 0.267 11 0.274 12 0.254 13 0.275 14 0.251 15 0.290 16 0.226 Mean 0.257 Stdev 0.019 p value 50Z H-17157 33Z H-17157 20Z H-17157 0.005 0.005 0.008 0.010 0.007 0.010 0.013 0.013 0.013 0.012 0.009 0.004 0.013 0.011 0.014 0.016 0.010 0.003 0.000* 0.003 0.004 0.010 0.005 0.002 0.012 0.006 0.005 0.003 0.003 0.003 0.003 -0.001 0.007 0.006 0.001 0.005 0.003 0.000* -0.002 -0.004 -0.001 -0.004 -0.003 0.003 0.001 -0.002 -0.003 0.000 0.001 -0.003 -0.002 0.006 0.001 -0.003 -0.001 0.003 0.000* 9Z H-17157 0.272 0.221 0.202 0.160 0.237 0.179 0.170 0.235 0.252 0.005 0.224 0.220 0.241 0.238 0.226 0.255 0.209 0.060 0.004* MMC 10 ug/oL 0.001 -0.001 0.004 -0.005 -0.002 0.004 0.002 -0.001 -- - - mm - 0.001 0.001 0.000* Data are optical densities corrected for reagent blanks. Stdcv - Standard deviation. p value - Probability that the mean is less than that of the control. * - Significant at the 95Zlevel ofconfidence. - Indicates not tested. " H " *7157 12 - Sanflfeetf. no! eoMaFn TSC a ee?