Document XzorrkDv8G9637Jq04dJ0MkMy

CLIENT PRIVATE AF26-OI6I fk -- PROTOCOL TITLE: Range finding Cytotoxicity Assay of T-6292, T-6293, T-6294, T-6295 in R at Hepatocytes SRI Study No. B010-95 RESEARCH CLIENT 3M Medical Department Medicine, Health Physics, Industrial Hygiene, Toxicology 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 Study M onitor: Steven C. Gordon, Ph.D ., D.A .B.T. TESTING LABORATORY SRI International Toxicology Laboratory 333 Ravenswood Avenue Menlo Park, CA 94025 Study D irector: Carol E. Green, Ph.D ., D.A .B.T. Telephone: (415) 859-4083 FAX: (415) 859-2889 APPROVALS: Research Client's Authorized Representative Date Date 004030 1 CLIENT PRIVATE I. STUDY OBJECTIVE The objective of this study is to evaluate the cytotoxicity of 4 different test articles as measured by MTT conversion. The ECsoand EC10values will be estimated for each test article and these data will be used to select a suitable concentration of each test article for subsequent in vitro metabolism studies. H. APPROACH Hepatocytes will be isolated from adult male Sprague-Dawley rats. The cells will be allowed to attach in monolayer culture in the wells of a 96 well plate. The cells will then be exposed to either test article, solvent, or positive control in 4 replicate cultures. In this experiment, the compounds will be tested in 6 different concentrations at a single time point (24 hours). The concentrations for each test article are described below in the Experimental Design. H I. TEST ARTICLE A. Test Article Identification Name: T-6292 Lot No.: Ifl.bg ,provided by-Rsseatflli.,!Client M olecular weight: 571.2 Name: T-6293 Lot No.: To be provided bv Research Client M olecular weight: 658.28 Name: T-6294 Lot No.: To be provided bv Research Client M olecular weight: 527.2 2 C04031 CLIENT PRIVATE Name: T-6295 Lot No.: To be provided bv Research Client M olecular weight: 538.1 B. Purity and Stability Documentation of the identity, strength, purity, and stability of the test articles will be the responsibility of the Research Client. The positive control, acetaminophen, and the solvent control, DMSO, will be obtained from Sigma Chemical Co. (St. Louis) and their documentation of the identity and purity of these chemicals will be used. C. Handling and Storage On receipt, the test articles will be placed in a secondary container (lightproof if required) and stored as recommended by the Research Client. A Material Safety Data Sheet (MSDS) or other comparable document specifying any hazards and identifying first aid and clean-up procedures in case of an accidental spill shall be provided by the Research Client and shall accompany the test articles. The test articles will be logged in and stored in Building L, Room LB286. D. Disposition of the Test Article Any remaining unused portion of the test articles will be returned to the Research Client after completion of the study. TV. TEST SYSTEM A. Background and Justification for Selection of the Test System The liver is the major site of metabolism of most organic chemicals, both endogenous and foreign. Species-related differences in the metabolism of xenobiotics are well known and have been documented to be correlated to the toxic effects of chemicals on certain species (Caldwell, 1980; Calabrese, 1983). The proposed colorimetric cytotoxicity assay has been used to determine mammalian cell survival and proliferation (Mosmann, 1983; Otoguro et al., 1991). This assay detects living, but not dead cells. Because the signal generated is 3 C04032 CLIENT PRIVATE dependent on the degree of activaiton of the cells, cytotoxicity, proliferation, and activation can all be measured. This assay depends on the fact that MTT (which is yellow in aqueous solution at neutral pH) is metabolized by mitochondria of living cells to a blue formazan dye. When used as a cytotoxicity assay, spectrophotometric quantitation of the formazan generated by cells after their incubation with the test article will give an indication of the number and metabolic activity of the surviving cells. B. Test Species R at. Adult male Sprague-Dawley rats will be used for the preparation of hepatocytes. They will be purchased from Charles River Laboratories and will weigh approximately 200250 g at the time of purchase. The rats to be used for this study will be housed in Building L, where the cell isolation will be performed. Rats will be taken from the shipping containers, weighed, examined for general health, and placed three per cage in 22 x 1214 x 8-inch suspended polycarbonate cages labeled with their quarantine number assignments. Animals will be quarantined in the same room in which they will be housed. Rats will be fed (ad libitum) Purina Certified Rodent Chow (#5002) and the feed supplier will provide SRI with an analytical report identifying the diet to be free of contaminants for each lot of feed received. A copy of each analytical report will be available as part of the study record. Rats will receive purified (deionized and UVtreated) drinking water ad libitum. Temperature will be maintained at 72 4F and relative humidity maintained between 35 and 65%. A light cycle of 12 hours light and 12 hours dark will be maintained, with light starting at 06:00. The laboratory species will be quarantined for at least 3 days before use in an experiment. The Laboratory Animal Medicine Department at SRI will issue documentation to the Study Director on the health of animals during the quarantine period. Before isolation of hepatocytes, the rats will be anesthetized with sodium pentobarbital (65 mg/kg). V. EXPERIM ENTAL DESIGN A. Hepatocyte Isolation Rat hepatocytes will be isolated by either whole liver or biopsy perfusion as convenient (Green et al., 1983). In brief, the whole liver or wedges of the organ will be perfused first with a Ca+2-free buffer, followed by a buffer containing collagenase. Hepatocytes will be combed free from the digested tissue and purified by simple differential centrifugation. 4 004033 CLIENT PRIVATE Isolated hepatocytes will be plated onto collagen-coated culture dishes (35 mm diameter) in a modified Waymouth's 752/1 culture medium (CM Hla) that contains 11.2 /g/ml alanine, 12.8 /xg/ml serine, 24.0 /xg/ml asparagine, 84.0 /xg/ml gentamicin sulfate, 0.168 /xg/ml aminolevulinic acid, 5.0 /xg/ml oleic acid, 5.0 /xg/ml linoleic acid, 1.0 /xg/ml D,Ltocopherol, 288 ng/ml testosterone, 272 ng/ml estradiol, 393 ng/ml dexamethasone, 7.9 /xg/ml thyroxin, 30 ng/ml glucagon, 0.02 U/ml insulin, 0.1% ITS (Collaborative Research, Inc.; final concentrations: 5 /xg/ml transferrin, 5 /xg/ml insulin, and 5 ng/ml selenium), 0.2 mM Lascorbic acid 2-phosphate, and 0.2% BSA. The initial plating medium will contain 10% fetal bovine serum. After 2 to 3 hr of incubation at 37C in an atmosphere of 95% air:5 % C 0 2, the medium will be aspirated to remove nonviable, unattached cells and replaced with CM H la containing the test article. B. Range Finding Cytotoxicity Experim ent (MTT Assay) 1. Preparation and A dm inistration of Test Articles and Controls. Concurrent solvent controls will be examined in the experiment. Solvent control samples will consist of culture medium and DMSO at the same concentration used to dissolve the test articles. DMSO will be used so that the final concentration does not exceed 10 /xl/ml (1%). Acetaminophen (10 mM) will be the positive control and will be prepared as a stock solution in culture medium. Immediately before the experiment, an aliquot of each test article (solid or pure liquid) will be diluted in DMSO to form a concentrated stock solution. Serial dilutions will be made from these initial stocks. 2. Test A rticle Concentrations. Six test article concentrations will be studied: 0.01, 0.05, 0.10, 0.50, 1.0, 5.0, 10.0 mM. 3. Incubation of Hepatocytes with Test A rticle. Four replicate monolayer cultures of isolated rat hepatocytes will be incubated at 37C for 24 hours with the test article or control. A negative control (no cells) will also be measured for determination of background staining. 4. MTT Assay. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay will be performed as described by Scudiero et al. (1988) as modified by this laboratory (Otoguro et al., 1990). MTT (Sigma) will be dissolved at 5 mg/ml in calcium and magnesium free phosphate buffered saline (CMF-PBS) and sterile-filtered with a 0.2 /xm filter unit. The MTT solution will be diluted 1:5 in CM Hla, and 150 /xl will be added to each of the 24 wells in the dish. After a 2 hour incubation at 37C, the incubation medium will be 5 004034 CLIENT PRIVATE aspirated and 450 /xl of DMSO will be added to solubilize the MTT-formazan product. After mixing, the absorbance at 540 nm will be measured. 5. D ata Collection. The net change (%) relative to the solvent control will be plotted against the chemical concentration. The EC50 values will be determined by a computer statistical program designed for simultaneous fitting of families of sigmoidal dose response curves using the four-parameter logistic equation (De Lean et al., 1988). VI. REPORTS A summary report containing the results of this experiment will be issued to the Study Monitor. The test article concentrations for the metabolism study will be selected based on these results.- LX. RECORDS TO BE MAINTAINED The laboratory notebooks, all raw data, relevant communications, and the original copy of the Final Report will be transferred to the SRI Records Center, Building B, after completion of the study. These materials will be maintained for 10 years; then the Research Client will be contacted concerning future disposition of the records. X. REFERENCES Calabrese, E. J. John Wiley & Sons, New York, pp. 203-282, 1983. Caldwell, J. In Enzymatic Basis o f Detoxication, Vol. I. William B. Jakoby (Ed.), Academic Press, New York, pp. 85-114, 980. DeLean, A., P. J. Munson, and D. Rodbard. 1978. Am. J. Physiol 235, E97-E102. Green, C. E ., J. E. Dabbs, and C. A. Tyson. Anal. Biochem. 129, 269-276, 1983. Mosman, T. J. Immunological. Meth. 65, 55-63, 1983. Otoguro, K ., K. Komiyama, S. Omura, and C.A. Tyson. ATLA 19, 356-360 (1991). 6 004035 4150592889 0C7.2e.l995 12:28PM SRI TOX LAB FAX # 415 859 2889 NO.939 P.l FACSIMILE TRANSMISSION M essage to: Dr. Steve Gordon Company: 3M FAX Number: 612/733-1773 Date: October 26,1995 R E : Cytotoxicity assay Number o f Pages: 5 From: Carol Green Direct Phone: (415) 859-4083 SRI International Toxicology Laboratory 333 Ravenswood Avenue M enlo Park, CA 94025 FAX (415)859-2889 (415)326-5512(att) CC: V. W eizer C. Tyson * * * * * *****+++++*+***+4<***************************#*** Enclosed are copies of the data from the MTT assay. You will also find some notes prepared by the technician on the solubility o f the compounds in the medium. After reviewing the raw data (absorbance values) for T-6292,1 found that the controls for that plate were low. As a result, I think the assay for that chemical may need to be repeated. Based on the appearance of the cells treated with T-6292, severe toxicity occurred between 0,1 and 0.5 mM, Please review the results and call me so we can discuss this further. 004036 OCT.26.1995 12:29PM SRI TOX LAB FAX # 415 859 2889 Project 7565 Rat M TT Assay NO.939 P.2 Project 7565 | MTT Assay using Rat hepatocytes ________ I________ I Hepatocytes isolated on 10/16/95. Cells exposed to test articles for 24 hours. MTT assay performed on 10/17/95. At the 24 hour time point the media containing test article was aspirated from the plate and replaced with MTT assay solution. MTT Assay solution was incubated on the cells for 2 hr MTT assay solution was removed after 2 hr and the cells were lysed with DMSO releasing into the DMSO the Formazan Blue Dye. The plates were read on a Molecular Devioes Kinetic Plate reader at 540nm. Data calcu ations were performed on the Softmax operating program for the plate reader. T-6295 (mM) 0.1 0.05 0.1 0.5 1.0 5.0 10.0 . %of control cells 127.3 127.3 113.8 8.318 8.796 6.13 14.58 SD 19.68 24.42 16.92 3.492 .5.443 5.184 17.89 - T-6294 (mM) 0.01 0.05 0.1 0.5 1.0 5.0 10.0 %of control cells 104.7 92.73 48.43 5.366 4.983 6.54 6.807 SD 8.301 6.273 2.411 1.669 3.694 0.919 0.883 Pagel 004037 OCT.26.1995 12:29PM SRI TOX LAB FAX # 415 859 2889 Project 7565 Rat MTT Assay NO.939 P.3 T-6293 (mM) F C-teT 0.01 0.05 0.1 0.5 1.0 5.0 10.0 %of control cells 107.4 95.22 103.5 109.6 121.6 36.71 2.969 8D 23.8 19.66 17.53 24.2 32.13 14.49 1.586 , T-6292 (mM) 0.01 0.05 0.1 0.5 1.0 5.0 10.0 %of control cells 149.8 128.1 404.5 25.99 17.31 227.1 13.59 SD 52.49 38.64 . 52.7 17.9 4.426 60.39 4.414 APAP (mM) Positive control 10 %of control cells . 36.45 SD 8.027 Page 2 1 004038 OCT.26.1995 12:29PM SRI TOX LflB FAX # 415 B59 2899 NO.939 P.4 Cytotoxicity to Rat Hpatocytes 004039 OCT.26.1995 12:30PM SRI TOX LAB FAX # 415 859 2889 NO.939 P.5 Notes on solubility o f compound during MTT experiment o f O ct 1647. 1 T-6292 Visibly precipitated out at 10,5, and 1 mM concentrations T-6293 Not all solubilized at 10,5, and 1 mM concentrations in the DMSO added CM H la and after agitation appeared to have gone into solution. T-6294 Visibly precipitated out at 10 and 5 mM concentrations. No visible precipitate at 1 mM concentration. T-6295 Visibly precipitated out at the 10 mM concentration. No visible precipitate at the 5 and 1 mM concentration. Notes on appearance o f cells after incubation with test articles for approximately 22 hours. T-6292 Rows 3-5 some cells spread out some cells rounded. Rows 6-9 very few cells spread out most rounded. Row 9 crystals present dark colored media. 0 .6' >o . o r , o . t * * 6 .1^ \ . 0 > *<*-* T-6293 Rows 3-6 no visible abnormalities U o i - o . ^ i * Rows 7-10 have a lot o f debris, the cells are rounded or unattached, t-o } t T-6294 Rows 3-5 no visible abnormalities. 0 . o \ - o \ Rows 6-9 cells not spread out, rounded. . o * T-6295 APAP Rows 3-5 cells mostly spread out w ith some rounded. 0">< - o *- k Rows 6-7 cells not spread out with some debris. ,x - t.c Rows 8-9 a lot o f debris, the cells were rounded. ^ ,o I rows 2-3 no visible abnormalities. Rows 4-6 few to no spread out cells. A** Appearance of T6295 after 2 hour incubation with MTT solution Wells 8-9 had a heavy yellow precipitate. Well 7 had a medium yellow precipitate. Well 6 had a light yellow precipitate. Wells 1-5 no visible precipitate. The other Plates had no remarkable abnormalities. For plate T-6293 one row of wells was repeated. When read by plate'reader and by visible observation o f color change rows 8 and 9 were 5.0 mM concentration. This corresponds w ith an interruption during the application o f test articles and time I noticed the error. vlw 10/24/95 J 004040 4158592889 DE''. 12.11995 12:59PM SRI TOX LAB FAX # 415 859 2889 NO.804 p . l SRI INTERNATIONAL Toxicology Laboratory 333 Ravenswood Avenue, Bldg 205 Menlo Park, C A 94025-3493 Fax No. (415) 859-2889 Alternate Fax No. (415) 859-3342 Facsim ile Transmittal From: Direct Phone: H iC- `i o T S M essage To: &*% S+c*Company: 3 ^ Fax Number:^*1 * Date: December 12,1995 T h is M essage Transmitted from: Fax No. (415) 859-2889 J Number of Pages: 4 including this transmittal sheet M ESSAGE: ^ - K . r * - * * * ,V T T (jit re> /s*.V W $ % Vea. |>. h- i-- -+U * 3c -V- ^ U o ^ SJip v SO ^ ^ ItKYU. ( U tf c J l c L '\O c ,'v u l^ h - C . fw ^ rKyU | ^ u \ + * * * ,. ^ . Please call the sender directly if you have any problem with the reception of this message. 004041 DEC.12.1995 12:59PM SRI TOX LAB FAX # 415 859 2089 NO.804 P .2 Project 7665 Rat MTT Assay i Project 7566 MTT Assay using Rat hepatocytes 1 Hepatocytes isolated on 12/6/95. Cells exposed to test articles for 24 hours. MTT assay performed on 12/7/96. At the 24 hour time point the media containing test article was aspirated from the plate and replaced with MTT assay solution. MTT Assay solution was incubated on the cells for 2 hr MTT assay solution was removed after 2 hr and the cells were lysed with DMSO releasing into the DMSO the Formazan Blue Dye. The plates were read on a Molecular Devices Kinetic Plate reader at 540nm. Data caicu ations were performed on the Softmax operating program for the plate reader. T-6293 (mM) 0.01 0.05 0.1 0.5 1.0 6.0 10.0 %of control ceils 84.01 104.7 108.2 45.28 6.739 55.96 97.4 SD 4.495 23.85 14.95 15.69 1.440 18.52 10.36 . T-6292 (mM) FC'fO 0.01 0.05 0.1 0.5 1.0 5.0 10.0 %of control cells 106.1 110.0 59,52 7.882 9.208 24.44 93.92 SD 24.85 41.77 33.77 2.914 2.199 9.503 23.96 SCto - 0 . 0 eC so - APAP (mM) Positive control 10 %of control cells 32.34 SD . 12.80 Pagel ii 004042 DEC.12.1995 1:00PM SRI TOX LAB FAX # 415 859 2889 NO.804 P.3 Notes on solubility o f compound during MTT experiment o f Dec.6-7 T-6292 Compound was not all solubilized at 10,5, and 1 ffiM concentrations. The 1 mM concentration appeared to be the most solubilized in the DMSO and C M H la and after agitation appeared to have gone into solution. T-6293 Compound was not all solubilized at 10,5, and 1 mM concentrations. The 1 mM concentration appeared to be the most solubilized in the DMSO and CM Hl a and after agitation appeared to have gone into solution. Sonicated 10,5 and 1 mM concentrations for 10 minutes after addition o f CM Hla to increase solubility. Notes on appearance o f cells after incubation with test artioles for approximately 22 hours. T-6292 Rows 8-9 Some cells spread out some cells rounded. Visible crystals of compound. (5,10 mM) Rows 6-7 Few to no cell-cell attachments m ost all cells appear Visible crystals o f compound. (1.0,0.5 mM) Row 5 Some flattened cells w ith cell-cell attachment. (0.1 mM) Row 4 More cell-cell attachment. Few to no crystals. Some rounded cells. (0.05 mM) Row 3 Cells slightly better than row 4, (0.01 mM) Row 2 A couple o f rounded cells otherwise good. (Normal media) T-6293 APAP Row 9 Cells visible many rounded with some spread having cell-cell attachm ent (10.0 mM) Rows 6-8 Cells not visible due to grainy debris. (5.0,1.0,0,5 mM) Row 5 Very grainy cells rounded. (0.1 mM) Row 4 Some cells are clumped together growing on top o f each other, grainy debris present (0.05 mM) Row 3 Good w ith grainy appearance. (0.01 mM) Row 2 A couple o f rounded cells otherwise good. (Normal media) rows 2-5 no visible abnormalities. (Normal media) Rows 6-9 few to no spread out cells.( lOmM APAP) vlw 12/7/95 i c 04043 Concentration (mM) Concentration (mM) 004044 \ V 0.01 1 10 Concentration (mM) M M T Response (% o f Control) MMT R esponse (% of Control) 4158592889 JPN.16.1996 6:12PM SRI TOX LPB FAX * 415 859 2889 ISO.233 P . l FACSIMILE TRANSMISSION M essage to : D r. Steve Gordon Com pany: 3M FAX N um ber; 612/733-1773 Date: January 16,1996 RE: Rat hepatocyte study Num ber of Pages: 3 From: C ard Green D irect Phone: (415) 859-4083 SRI Tntpmaftnirtiql Toxicology Laboratory 333 Ravenswood Avenue M enlo Park, C 94025 FAX (415)859-2889 (415)326-5512(ah) C C : V . Weizer C. Tyson ft********************************************************************* Enclosed are copies o f the data from the LDH assay that we performed on rat hepatocytes incubated with the test articles following the protocol for the metabolism study. W e used 100 M of each as discussed. No significant toxicity is evident .except wi(ft6294 ^ h ic h did increase LDH release. I suggest that we send the other samples from 6292, 6293, and 6295 to ABS for analysis. Do you agree? We can discuss these results after you have a chance to review them. 1 would also like to discuss the project budget because the LDH assay was not included in the original protocol and we now may need to repeat the incubation for 6294 metabolism. I will talk to you soon about these issues. 004045 004046 NO.233 P.2 . JAN.16.1996 6:12PM SRI TOX LAB FAX # 415 859 2889 B011-96 Rat 1 LDH 1/12/96 1/16/96 VLW LDH assay for 7565/ B011-R1 incubation performed on 1/11/96 Samples stored 0-5C untill analysis * LDH aflofeed on Gemeni on 1/ 2/96 _______ I_______ I________ Cells in 35 mm dish were scraped info 1.0 ml of PBS after washing with PBS Cete were diluted/tysed w8h 1.0 %triton X-100 in PBS 1.0 ml] I Cell sampleswere further,,diluted as necessary | 2X =100 piceli mix+100pi PBS 6294 c +d, 6293 c, 3X =100 picei! mix+200pi PBS normal, 6292 c+d, 6293 d,62 S c+ d I Media samples were aspirated into microfuge tubes and stored untiEIanalysis at 0-5C Normal ceHs were incubated with 2 ltd of medium Test articles were mcubated with 1 ml of medium i ii Media samples were furtherdiluted as necessary 2X = 100 pi medium +100 pi PBS 6294 c+d remaining sampleswere notdiluted normal ceils were in 2 ml of media so 2X t Calculations: Final LDH C =dilution C X LDH/Cell Final LDH M =dilution M X LDH/medta Total =Final LDHC +Final LDH M %released into media = Final LD 1M/Total expressed as It>) + S> tldM 1 in 1 111*1^ SAMPLE normal a normal a normal b normal b LDH/CELL dilution C final LDH C LDH/MEDIA Dilution M final LDH M Total %released Into media 384 6 2304 207 2 414 2718 15.2% 360 6 2160 211 2 422 2562 16.3% 438 6 ^ 2628 242 2 484 3112 15.6% 466 6 2736 248 2 496 3232 15.3% Average 15.6% Pagel 004047 NO.233 P.3 jpas.16.1986 6! 13PM SRI TOX LPlB FP1X # 415 859 2889 SAMPLE 8282 c 6282 c 6282 d 6282 d 6293 c 6293 c 6293 d 6293 d 6284c 6284c 6294 d 8284 d 8295 c 6295 c 6295 d 6295 d B011-86 Rat 1 L D H 1/12/96 LDH/CELL dilution C final LD H C LDH/MEDIA Dilution M final LDH M Total %releasee into media 779 6 4674 808 1 806 5656 14.3% 745 6 4470 815 1 815 5285 15.4% 749 6 4494 717 1 717 5211 13.8% 750 6 4600 732 1 732 5232 14.0% Average 14.4% o.T* 909 4 969 4 627 6 649 6 3636 3876 3762 3894 464 1 509 1 552 1 560 1 484 4120 509 4385 552 4314 560 4454 Average 11.7% 11.6% 12.8% 12.6% 12.2% 712 4 722 4 760 4 763 4 2848 2868 3040 3052 722 2 704 2 532 2 544 2 1444 1408 1064 108B 4292 4296 4104 4140 Average 336% 32.8% 25.9% 26.3% 28.7% 451 6 458 6 709 6 653 6 2706 2754 4254 3916 547 574 286 284 1 1 1 1 547 3253 574 3326 286 4540 284 4202 Average 16.6% 17.2% 6.3% 6.8% 11.6% * Jr , W Ii , 4 4 ') * k i l l K. 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