Document QJMX2Mog8xQ8qeJMd5roE4BxE

AR226-2797 Du Font HLR 150-88 POP DO FONT OSB ONLY Study Title IN VmO^IOCOMPATAHLITY-EVALUAKOy____________________ WITH IMR-90 HUMAN DIPLOID FIBROBLASTS IN THE OTT COLORIMETRIC ASSAY" Author Ralph G. Stahl, Jr. Issue Date April 11, 1988 reforming Laboratory E. I* du Pone de Nemours and Coapany, Inc. Haskell Laboratory for Toxicology and Industrial Elffton Road, P. 0. Box 50 Netoark, Delaware 19714 Medicine Medical ^aaearch Ho. Laboratory Project ID Haskell Laboratory Report No. 150-88 Page 1 of 12 PBapany Sanitized. Does not contain TSCA CBI Du Font HLR 150-88 COQD LABORATORY PRACTICE STATgMENT SgulSSoL^l Thia study was conducted according to PDA Good laboratory Practice CFR 58). Any area. of nonco-pllance are documentedin the .Sidyrecords. No deviation, exiated that .ignifleantly affected the validity of the study* Subaltter Sponsor Study Director E.I. 4u Font de Nenioura & Co., Inc. Cheal^ale and Pignenta Department ^^p^ ^ Sch^^ '^r.-------- .^pariy Sanitize. Do9s not contain TSCA CR; Du Font HLR 150-88 COMPOUINNFDOBMATIOW Material Tested; Synonyms: Medical Research No.^1 Haskell No.: H-17.157 CAS Registry No.; Other Codes; |) Purity: Contaminants: Sponsor; Chemical and Pigments Department W^ Material Submitted by:|lCBheBm|i^caBls--an|dJ Pigments Department Jackson Laboratory Study Initiated/Completed: 2/15/88-2/19/88 Distribution:! There are 12 pages In this report* RGS/Aaea 18-12 - 3 - Company Sanitized. Does not contain TSCA CB? IN VITRO BIOCOHPATABILITY EVALUATION OF WITH IMR-90 HUMAN DIPLOIQ FIBROBLASTS IN Du Font HLR 150-88 Summary IBHI^IHBBB^8 ce6ted for ^ vitro biocompatabiUty with IMR-90 'human diplotd fibrobUsts. Under the condltiona of this assay, K18 cytotoxic. Work by: W^ 3//^ "OeffreyT^.^T^u--rne--r, ^S--r. -- ----H<too /Day/Yr Technician Study Director; RoJ^^ &-.^>VA VJg^^V. Ralph fe. Stahl, Jr., Ph.D. Research Genetic Toxicologlst siz-i]^ Mo/Day/Yr Approved -by: Ui^ Stu^f Awnl M. Sarrif Ph.D. Sedtlon Supervisor Molecular and Genetic Toxicology (/ /// /<^ Mo/Day/Yr - 4 eon-fflanyga^Sfead- Does no?eor^in TSCA CB? Du Font HLR 150-88 QUALITY ASSURANCE DOCUMENTATION STUOY:|,^H^^^RI -I--IN--V--ITR--O--B--IOC--O--HP--AT--AB--Hi--ITY--HEVIHMAIRLTU-H9A0TIOHNUMOANF 'uiPLQll^^V^^wSuW^vS THE MTT COLORIMETRIC ASSAY AUDITS: I tens Audited Protocol, records and final report ____Audit Pates 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. [\ Hanill'Da'te Reported by; /Ww^<u6y^^--^C- Wvi-K^^^M'i MJoJsoesepphh C. Haaill Quality Assurance AAuuditor '{/^/^ 5 - Company Sanitized. Does not cc;ifain TSCA CBt Du Font HLR 150-88 INTRODUCTION flstudy waa to evaluate the cytotoxic potential of In IMR-W primary huaffn diploid fibroblasts. :he growth of cella 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 Co metabolize 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-diaethylthiazol-2-yl 1-2, 5-^diph "nyltetrazolium bromide. or MTT, is dissolved in culture medium and added to the cells. MTT is metabolized by the mitochondria in viable respiring cells to an insoluble purple formazan salt (l-(4,5-diBethylthiazol-2-yl]-3,5-diphenylfoi-nazan). Fourth, after 2-3 hours* the insoluble foroazan trapped in the mitochondria is leached with dimethylsulfokide. The optical density of the formazan, measured spectrophotometricalfLy, is proportional to the number of viable, respiring cells. A measure of the test material's cytotoxicicy, and by Inference its potential biocompatabillty, is obtained by comparing the optical densities from created 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 Denlzot and Lang (1986). This study was conducted according to protocol Biocompatability-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 Prr<oOcceedduurres;: In Vitro Biocompatability Evaluation of Material with Human an< 1 Mammalian Cells: Cell Counting Method; In Vitro Biocompatabilicy Evaluation of Material with Human and Mammalian 6 - Company s a n f e s d. ". "Doo^-.'or ^o;-i.a t ? r- f . s C A C B i Du Pone HLR 150-88 B. TEST MATERIALS the conditiona of this a9say. observed during the study. Based on Information supplied fs assumed to be stable under No evidence of instability was 2. Negative and Positive Indicators Negative Control (noncytotoxic); RPMI-1640 culture medium (Gibco Lot fa 12N9273 and 43K8473). Positive Indicator (cytotoxic): Mitomycin-C (MMC) (Sigma Lot f 66F-0494) at a final concentration of 10 ug/mL. MMC was prepared in distilled deionlzed water* The negative control and poslclve Indicator were assumed to be stable under the conditions of this study; no evidence of instability was observed. C. CELLS AND CELL CULTURE IMR-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 fron the lungs of a 16-week female fetus (Nichols et al. Science 196: 60-63 1977). IMR-90 cells are cultured routinely in a 5Z C0 atmosphere at 37C without antibiotics using RPMI-1640 culture medium (Gibco Lot is 12N9273 and 43K8473) supplemented with L-glutamine (Gibco Lot f 10N0666) and 10Z heat inactivated fetal bovine serum (Gibco Lot ? 28N2372). Cells were free of oycoplasaa contamination (GenProbe). Because of senescence cells are not used after passage 30. Twenty-four hours before the test, cells were seeded into Corning 96-well (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 recei 1 100 uL of culture medium/cell suspension. Company San,t;zed. Does not contain TSCA CBI Du Font HLR 150-88 D. PREPARATION OF TEST MATBRIAJLS j|UB||^|^IIBj The test material was filter sterilized through a 0.22 u filter (Milllpore MilI'ex-GS) before use. E. DOSE SELECTION 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 (9X, 20Z, 33Z, or 502 final concentration of test material, respectively). Concentrations greater than 50Z 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 52 CO., 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 Heart, Lung and Blood Institute (1979). 2. Measurement of Effects On Growth After 72 hours of exposure, all 96-Ball dishes were removed from the incubator. Each dish was inverted to remove culture medium. Fifty nicroliters of a 1 mg/mL solution of MTT (Sigma Lot f 126F5054) prepared in RPMI-1640 culture medium (without phenol red; Gibco Lot f 14N0066) was added to each well. The dishes were returned to the 37C incubator for about 3 hours. Afterwards, the dishes were removed and inverted to remove the MTT solution. Approximately 50 uL of dimethlysulfoxide (DMSO; Sigma Lot 9 116F34861) was added to each well. The purple formazan color was allowed to develop at room temperature for about 30 ralnutea. Each dish was then placed in the reading chamber of 6 Titertek Multlskan MC connected to an IBM-PC. 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. ^"^"''^"'""^^rsMcs, Du Font HLR 150-88 H. STATISTICAL ANALYSES Optical densities from created and control cultures were compared using RS/1 computerized 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 CTTOTOXIC when; A. The optical densities in exposed cultures are significantly less than uncreated 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 observed. A test sample is classified as NONCYTOTOXIC 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, AMD 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 classifcation is satisfied. - 9 - Company Sanitized. Does not contain TSCA CBf Du Font HLR 150-88 G* ACCEPTABILITY CRITERIA : An acceptable assay Is teased on a single trial In which the negative and positive Indicators jrespond in the Appropriate manner* In addition, contaminated wells are excluded from data analysis* There oust be a oiniauo of 8 nbncontaoinated vails, for each exposure, and the positive and negative indicators* Tlie trial is repeated if these criteria are not let* H. RETENTION OF RECORDS All raw data and the final report are stored in the archives of Haskell Laboratory for Toxicology and Industrial Medicine, 1,'ewark, Delaware or in the Du Font Records Management Center, E. I* du Font de Neoours and Co., Inc., Wiloington, Delaware. RESULTS/DISCUSS ION Results on | shown in Tab: 20Z, 33Z and affec-ed at the 10Z concentration of signx leant decreases concentrations onl----________ indicator, signlflxancTy reduced optica: lysis. city to Dffl-90 human cells are was toxic at concentrations of ested. MMC, the positive density and caused widespread eel; Under the conditions Is cytotoxic. 10 ^fflpanyganiM Does no!eon?a,n TSCA CBF Du Font HLR 150-88 REFERENCES i American Society for Testing ^nd Materials (1983) Designation F813-83 Standard Practice for Direct Contact Cell Culture Evaluation of Material* for Medical DBvices AmericanSociety for Testing and Materials, Philadelphia, PA. Denixot, F. and R. Ling (1986) Rapid colorioetric assay for cell growth and survival: Modiflcacions to the tetraxoliua dye procedure giving Improved senaitivity and reliability* Journal Inmmological Methods 89: 271-277. Green, L.M.. J.L. Reade and C,F. Ware (1984) Rapid colorioetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lyaphokines. Journal laaunological Methods 70s 257-268. Mossoan, T. (1983) Rapid coloirinetric asay for ceP.'ilar growth and survival: Application to proliferation and eytotoxieity assays. Journal lamunological Methods 65: 55-63. National Heart, Lung and Blood Institute (1979) Guidelines for Physlocheaical Characterisation of Blooaterials. Devices and Technology Branch, National Heart, Lung and Blood Institute, NIH Publ' atlon No. 80-2186, Washington, D.C. RS/l (1984) RS/1 User's Guide, Book 2, BBN Software Products Corp., Cambridge, HA. 11 Company Sanitized. Does not contain TSCA CBI Du Font HLR 150-88 Table I. In Vitro Biocoapatabiliity of H-17,157 With IMR-90 Huaan Cells. Results of MTT Colorioetric Assay After 72 Hour Exposure. Well f Control I 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 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* 33Z H-17157 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* 20Z H-17157 -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 - - - - - - - -- 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 95Z level of confidence. Indicates-npt tested. H-17,157 6 12 Semssswi ^anRfeetf, Rnng nof eon^n T8C& cm