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AN ASSAY OF CELL TRANSFORMP.TIO!L AND CYTOTOXICITY IN C3H IOT 1/2 CLO.$",ACLELL LINE FOR THE TEST CHFMICAL T-2942 CoC Submitted to the 3M.Corporation 3M Center St. Paul, Minnesota 55133 By the Environmental Pathology Laboratory Department of Laboratory Medicine and Pathology Stone Research Laboratories Room 110 421 S.E. 29th Avenue Minneapolis, Minnesota 55414 March 4, 1981 Report Submitted By: Vincent F. riarry,M tf. Date Study Director Richard L. @lelson Date Associate Scientist TEST ARTICLE SPECIFICATIONS STUDY NUMBER: TEST ARTICLE I.D.. T-2942 CoC TEST ARTICLE LOT NUMBER: DATE RECEIVED: 8/20/80 DATE STUDY DATE STUDY BEGINS: COMPLETED: 8/27/80 10/5/80 TEST ARTICLE STORAGE CONDITIONS: Room Temperature DILUENT(S)USED: DMSO TEST ARTICLE FORM: GAS LIQUID TEST ARTICLE QUANTITY RECIEVED: .10 grams SPECIAL HANDLING PRECAUTIONS: None HAZARD RATING: CHEMICAL 14 EXPLOSIVE .1 4 BIOLOC-ICAL 1 4 SOLID Y LD 50 ORAL ACUTE: 540 mg/kg SOLUBILITY OF TEST MATERIAL: (H2 0; CH3 OH; Lipid, etc.) H20; CH,04; Acetone Luali,@X_Ls_sy-r-a@n-ct-Statement The C3H 1OT-1/2 cell transformationassay is a,highly reproducible methodrequiringseveralinternalcontrols. Cells,media and serum are routiiielsycreenedfor growthpotentialand contaminantsprior to incorporationinto the test system. This functionhas been carriedout by Mr. Nelsonand reviewedby Dr. Garryin this initialstudy. The raw data and the final reporthas been reviewedby our QualityControlOfficer, Mr. Robert Kreiger. VincentF. Garry,M.Df/ Study Director Robert Kreiger,--4'4.S. QualityControlOfficer Executive Summary Assays for both cytotoxicity and cell transformation have been completed for test article T-2942 CoC, using C3H IOT-1/2 cell cultures in vitro. Cytotoxicity was measured by a reduction of cloning efficienc'y for treated cells. The data showed an LD50 of approximately 50 ug/ml. A dose range extending from 0 to 200 ug/ml was tested for cytotoxic effect. Comparison to a wide range of chemicals reported in the literature indicates that the chemical studied (T-2942 CoC) exhibits a low order of cytotoxicity, utilizing C3H IOT-1/2 cells. Transformation assays were conducted using the previously derived LO 50 as the median dose, with a dose range spread of four log units. Both the 14 day colony assay for transformation and a longer term foci transformation regimen (38 day) were completed for the test article T-2942 CoC. No evidence of transformation was observed for either the colony or foci assay methods using C3H IOT-1/2 cell cultures. Introduction This study was conducted for 3*rb'y. V. F. Garry, I-I.D,.M.S., R. Nelson, B.A., R. Krieger, @I.S.and M. Sinn, B.A., from 8/27/80 to 10/b/80 at the Environmental Pathology Laboratory of the University of Minnesota, Minneapolis, fiinnesota. The experimental procedures employed are the test methods described by Reznikoff, Brankow and Heidelberger for the determination of transformation and cytotoxicity in the C3H 1OT-1/2 cell line. Aside from this classical methodology, we have provided 3M an early reporting feature to give a preliminary evaluation of results, All procedures are described in detail in the protocol included in the appendix of the report. tiaterialsand Methods Cell Source Cryopreserved cells of the C3H 1OT-1/2 clone 8 (Lot Number 3Cl-l-4) utilized in these studies were derived from passage 11, The original cell stock was obtained from Dr. C. Heidelberger, Director for Basic Research of the University of Southern California. The cryopreserved lots were prepared from subcultures of the original cell line obtained at the 5th passage. Cell Culture Conditions Stock cultures for these studies were grown in Eagle Basal Medium (BME) containing 10% heat inactivated fetal bovi'neserum (Reheis Chem. Co., Lot Number T-48609). Prior to the study, the serum lot was tested for cloning efficiency and transformation efficiency in the presence of direct and indirect acting carcinogens, In addition, the entire cell system was tested for possible bacterial or mycoplasma contamination to insure the adequacy of the procedures. Test Chemicals The test chemical, T-?94? CoC war rectived on 8/20/30 rroiiVii.McCormick, an employee of 3M Corporation. Contained in a coded glass vial was approximately logm of white powder. The miterial was accompanied by a letter of authorizationand information regarding in vivo toxicity. The test chemical was stored at room temperature. Immediately before use, the test material was dissolved in spectral grade DMSO (Fisher Chemical Co. Lot Number 702398) and diluted to appropriate concentrations. Within 20 minutes the diluted chemical was added to the cultures under subdued yellow light to avoid photo-inactivation. lqomore than 20 microliters of the diluted chemical was added to the cultures to avoid solvent effects. The chemicals, Benzo(a)pyrene (Eastman Kodak Lot tiumber4941) and di-epoxybutane (Aldrich Chem. Co. Lot @.lumber082297) were used as positive controls. Materials and Methods (continued) Initial CYtotoxicity Determination Prior to performance of the transformation assay, dose range data was obtained in the form of cytotoxicity measurements as expressed by plating (cloning) efficiency, The test chemical T-2942 CoC was added to cultures seeded the day before at 300 cells per 6(kmnculture dish. After 24-hour exposure to the test chemicals in Sml BME media supplemented with 10% fetal calf serum; T-2942 CoC was removed with a complete change of media. The test chemical was applied through a 4 log scale dilutions ranging from 200 to 0.1 ug/ml. Six replicate cultures per dose point were employed and a total of six dose points were studied. The cultures were refed with fresh media every three days. After 7 days in culture, the plates were washed in PBS, fixed with absolute methanol and stained with giemsa stain. Positive controls, solvent controls and serum baseline controls were included in this study. The number of colonies per plate were counted and the plating efficiency was determined by the formula: PE = Average No. Colonies/plate x 100 No. of Cells seeded/plate Transformation-Assay The approximate in vitro LD 50 cytotoxicity dose was chosen as the median dose for the study of the transformationpotential of the test cheinical (T-2942 CoC). The test was conducted in two phases. In the first phase, cell transformation in the colony mode was assessed. In the second part, foci transformation potential was determined. Phase 1 - Colony Transformation Potential Six replicate plates seeded with 300 cells per plate were treated with 200, 100, 50, 10, 1.0 and 0.1 ug/ml to give appropriate two-fold and logarrithmic dose relationships for the test chemical T-2942 CoC. The positive control, benzo(a)pyrene(BP) was tested at 10, 5, 2.5, 1.0, and 0.1 ug/ml. A .solvent control (D@ISO)was included (IOug/ml). After removal of the chemical after 24 hours exposure, the cultures were refed fresh media and thereafter every 3 days for 14 to 17 days. At 14 days after removal of the test chemical, the plates were washed, fixed and stained as described before. Each colony was Materials and Ifiethodsc,ontinued carefully examined macroscopically and microscopically and scored for transformationaccording to criteria of Reznikoff.et al. Phase 2 - Foci Transformation Potential Remaining replicates at 6 replicates per dose were allowed to continue in culture for 38 days. Three dose increments were studied; 100, 10 and 1.0 ug/ml respectively. A positive control (butadieneepoxide) in dose incrementsof 0.01, 0.001, 0.005 and 0.0001 ug/ml was included.along with a DtlSOsolvent control. The cultures were refed according to the schedule mentioned above for the first fourteen days. For the remainderof time in culture, the cells were refed once weekly with BME supplemented with 10% fetal calf serum. At 38 days after removal of the test chemical, the culture plates were processed as mentioned above. Scoring for Transformation Focal areas of transformationare classified according to the criteria of Reznikoff as follows: Type 1. Foci composed of monolayer cells are more densely packed than the background cells. This type is not considered malignant and is not scored. Type Il. Foci show massive piling up into virtually opaque multilayers. The cells are only moderately polar, thus criss-crossing is not pronounced. Fifty percent of Type 11 foci have been shown to be malignantly transformed. Type III, Foci are composed of highly polar, fibroblastic,multilayerd,crisscrossed arrays of densely stained cells. Eighty-fivepercent of Type III foci have been shown to be malignantly transformed. Selected Type III foci will be retained from living cultures and subcultured twice, after which the cells will be cryopreservedfor one year. If, during that time, confirmatory in vivo tumorogenesis studies are requested, these cells will be made available for injection into a suitable host. Materials and Methods, Continued Special C3H IOT-1/2 Early Reporting (Phas@-I-) Although the classic methodology for the C3@lIOT-1/2 prescribed a 6-week course of study, recent data obtained by our Laboratoryshows that the assay time may be foreshortenedto 15 to 20 days. Detailedmicroscopicstudyof the colonies allows for quantitationin this somewhat shorter frame using the classicmorphologiccriteria previouslydescribed. This early reportingcapability is availableas preliminaryinformationand is includedin our report. Recording of Data and Re_ports All data from the cytotoxicityscreening,transformationassay and early reporting informationare recordedin tabularform (see enclosure). A coded access number will be providedto allow retrievalof cryopreservedmaterial, and the fixed and stainedcultures. All reportswill remain confidential, subject to disclosureby the Corporation. Althoughthere are no specificFDA guidelinesfor in vitro carcinogenesistesting,we will in future,adheremore closely to the Good LaboratoryPractices protocol by formal establishmentof a qualitycontrol unit in our laboratory. References 1, Reznikoff, C,, Brankow, D., Heidelberger,C. Cancer Research, 33:3231-3238, Dec. 1973. 2. Renzikoff, C., Brankow, D., Heidelberger, C. Cancer Research, 33:3239-3249, Dec, 1973, 3. Modal, S., Brankow, D., Heidelberger,C. Cancer Research, 36:7, Part 1, 2254-2260, July, 1976. 4. Bertram, J. S. Cancer Research, 17:51, 1976. 5. Bertram, J. S. Cancer Research, 37.-2,514-523, 1977. 6. Haber, D. A., Fox, D. A., Dynan, W. S., Thilly, W. G., Cancer Research, 37:6, 1644-1648, June 1977. 7. Bertram, J. S., Cancer Letters 7:5, 288-298, September, 1979. Resul ts In the dose ranging cytotoxicity test, the chemical T-2942 CoC showed a plating efficiency of 21.9% of control at 100 ug/mi (Table 1). To provide an efficient description of the transformationpotential of the chemical, both colony and foci were studied based on the initial cyto'toxicity screening. In the colony mode, eight dose points enclosing the in vitro LD50 were assessed in log scale and tvio-folddilutions. There was no morphologic evidence of transformation in the fourteen day colonies treated with the test chemical (Table 2) Longer term foci (38 days in culture) were studied in dose increments of 100, 10, and 1.0 ug/ml. No evidence of morphologic transformation were observed at any of the concentrationsstudied (Table 3). - The positive control, benzo(a)pyrene showed a dose related transformation frequency in the colony foming mode. In the foci mode, the positive control (butadiene epoxide) transformants were again observed in the dose increments studied. The combined number of type two and type three foci varied from 43.0 to 21.3/plate. No abnormalities'weredetected in the solvent controls. Conclusions The test chemical T-2942 CoC was studied in C3H 1OT-1/2 cells for morphologic evidenceof transformationin vitro. At the dose levels employed,no evidence of transformationwas observed. The positivecontrols, benzo(a)pyrene and diepoxybutane showed developmentof either Type II-and Type III colonies or Type Il or Type III foci. The solventcontrol showed normal morphology. 3',@C'o,rporation Client Study Number TOXICITY AND PLATIMR, FFFICIENCY IN CLONED C3H IOT 1/2 CL 8 CELLS AFTER EXPOSIIRE TO CHFMICAL T-2942 CoC V. F. Garry, M.D. Investigator T-2942 CoC Test Compounri I.@n.@:o. DMSO Solvent 200 ug/ml - 1.1 uQ/-,. Dose Range DOSE ug/ml Av. No. Col 300 Cells/Dish 6 Dishes/Dose P.E.**** Percent of Controls Transformed Colonies/ Cell*** I Transformed Colonies (Total Colonies Percent)** T-2942 CoC 200 0 0 100 9.0 21.96 50 22.0 53.66 10 33.66 81.99 1.0 37.16 90.04 NONE NONE 0.1 40.00 97.58 CONTROL S. CONTROL* 41.0 40.8 99.27 *10 ug/ml Solvent Control (DIISO) **Percent Transformation (T/Col.%) ***Transfomation ****P.E. Percent of Control LA-vtr-LqeL-:II-l.lo.T/Plate Average No Col./Plate Average Mo. T/Platc.Mo.rells Plated/-nish P.E. of nase x 100 P.E. (Control) Table 11 COLONY TR-k4SFOR.MTION AND CLO!IIMG EFFICIENCY IN CLONED C3H IOT-11-Z CL 8 CELLS AFTER EXPOSURF- TO CHEt.4ICALT-2942 CoC 3M Corporation Client 1 Study Number V. F. Garry, M.D. Investigator D!4SO Solvent T-2942 CoC Test '@'ompoundI.D. !!umber '200 ug/ml - I ug/mi Dose Range Dose ug/ml Av. No. Col. 300 Cells/Disi 5 Dishes/ Dose (e) (d) P.E. P.E. Percent of Percent Controls T-2942 CdC 200 100 so 20 10 5 2.5 1.0 CONTROL ta) S. CONTROL 0 27.61 32.67 35.0 37.5 38.67 40.5 41.0 43.0 41.33 0 9.05 10.89 11.67 12.5 12.89 13.5 13.67 14.33 13.77 0 63.16 79.99 81.44 87.23 89.95 94.21 95.39 95.09 10 5 2.5 1.0 0.1 CONTROL S. CONTROL 26.0 29.3 37.83 40.5 44.33 45.8 43.0 8.67 9,7 12.6 13.5 14.78 15.27 14.3 56.78 63.52 82.51 88.41 96.79 93.65 Combined Type II and Type 11 Colonies Transformed TransformedColonies(b) Colonies/Cell('cl(TotalColoniesPer--ent NONE NONE 11.1 x io-3 8.86 x io-3 7.7 X 10-3 Z.2 X 10-3 4.43 X 10-3 12.81 9.08 6.16 5.32 3.0 (a)10 ug/mi Solvent Control (D!OSO) (b)Percent Transformation(T/Col.Percent) (c)Transformation(T/Cell) (d)P. E. Percent (e)P. E. Percent of Control Avera e No. T/Plate Average -!Io--Col-./Ol-ate Average No. T/Plate No. Cells Plated/Dish Averarr eINS o.-Coatf-eCd-ofli@siii h x 100 P. E. of Dose P. E. (Control)X@--loo C3H IOT-1 /2 CELLS TRANSFORMER) FOCI FOFCIATION ASSAY AFRER EXPOSURE *rO CHEMICAL T-2942 CoC 3M Corporation Client I Study Number V. F. Garry P..D. Investig-a-ror DIISO Solvent T-2942 CoC Test Compound I.D. No. 100 ug/ml - 1.0 ug/mi Dose Range Combined Typg Ii and Type III Transformation oc Per Cell Plated Dose Number of Days Total No. Foci AV. No. toel" Percent per plate T-2942 CoC 100 38 10 38 1.0 38 CONTROL 38 S. CONTROL 38 0/6 0/6 0/6 NONE NONE 0/6 0/3 + Control Butadiene Diepoxide 0.01 36 0.001 36 0.0005 36 0.0001 36 0 129/3 84/3 64/3 0 43.0 28.0 21.3 0 14.33 9.33 7.1 Average No. of Type II and Type III Foci per Dish No. of Cells Plated per Dish x 100 C3H IOT 112 CLO-*IE8 TfLANSFORMATIOti ASSAY The C3H IOT 1/2 cell line is derived from inouseembryoniccells. In the early 1970's Reznikoff, Bankow and Heidelbergerl,2utilized clonal cells from the initial isolate to establishan in vitro measure of carcinogenesis,the transformation assay. In brief, the &-ssay-consistosf exposure of mammalian cells to a potential carcinogen In vitro for a precise period of time; followedby a cultivation of newly seedi!7d--ce=fsor a period of 30 to 35 days. During this time, cytotoxicityand morphologicevidenceof neoplastictransformationis obtained. Because the cells are morphologicallyhomogenousand sensitiveto post confluenceinhibition,abnormal cellularfoci can easily be detected. The number of foci detected are proportionalto the dose of the carcinogen applied. Similarly,the numberof coloniesof cells detectedwithin the first 7-10 days of culture provide a quantitativebasis for the determinationof cytotoxicity. To confirm the carcinogenicpotentialof the transformedfoci, the abnormal cells are injected into a susceptiblehost. The developmentof macroscopic tumors within 90 days constitutesconfirmationof the neoplastic characterof injected,morphologicallyabnormal cells. - The assay has good reproducibilityin our hands as well as in those of our colleaguesin the field. Each assay to be performedincludesa positivecontrol, solvent control and five dose points for the test chemical. Six separatecultures are used to establish each dose point. 1. Procedures A. Cell Source Low passage cryopreserved cells of the C3H IOT-1/2 clone 8 were obtained froniDr. fleidelberger.From the initialculture, a low passagestock of cells has been established. For long-tem storage,the cellsare first suspended in 8 percent DMSO plus media and are preserved in 'liquidnitrogen. For short-tem storage,we routinelymaintaincells in a -800 centigradefreezer. Periodically,sample culturesare tested for mycoplasma contamination,cloning efficiency and postconfluence inhibition. In the past sixteenmonths, cloning efficiencyhas varied from 12 to 16 percent. There is no evidence of "spontaneoustransformation"in any of our culturestested to date. B. Cell Culture Conditions Routinely, stock cultures are grolviinn Eagle Basal Medium (BME) containing 10 percent heat-inactivatedfetal bovine serum without the use of antibiotics. The media is made from powdered form in high puri'6vwater obtained from certified commercialsources. Fetal calf serum lots are periodicallytested for their effect on cloning efficienc,a,n,d selected on this basis. To insure continuityof testing sufficien#q'uan'%,.ities of serum are held on reserve. C.. Test Chemicals Chemicpls to be tested should be identifiedas preciselyas possible. Informationregardingthe physicalcharacteristics(eg) solubility, in vivo toxicity (LD50).and sourceare importantconsiderations. Ca6h-chemicalreseived is coded and duplicaterecords of the test procedure are maintained by the Laboratory. Chemicals to be studied will be tested on a uglml basis in an appropriate solvent (eg) DMSO. Additionof the chemicals to culture is accomplished by media dilutionsunder photographicsafety light. Subsequent media changes are carried out under gold fluorescentligiit. 2. Cytotoxicity Prior to performanceof the transformationassay, dose range data is obtained in the form of cytotoxicitymeasurements. In accordance with the protocol established by Reznikoff, Bertram, Brankow, and Heidelbergerlgcytotoxicity is measured in terms of plating efficiency. A fixed number of cells (approximately 300/dish) are placed in culture and allowed to grow for 7 to 10 days. The percentageof survivin coloniesdivided by the number of cells initially plated is then determinedlie) the cloning efficiency. Routinely,chemicals to be tested are added to the cultures24 hoursafter plating. After a standard 24-hour exposure period, the chemicals are removed with a change of media. The cells are refed fresh media every three days. The test chemicals are applied through a 4-log dose scale using 10 dose points. Three replicate cultures per point are employed. Positivecontrol, solvent controland serum baseline controls are includedwitlieach study. All chemical or solven'additions to the media are not allowed to exceed 0.5% of the total media volume. At the end of 7 to 10 days, the culturesare fixed, stained and the number of colonies are counted macroscopicallyand microscopically. 3. Transformation Assay The transformationassay is performed in accord with the methodology developed by Reznikoff,et all,2 and modified by Bertram,et al4. In line with this classicalapproach, approximately300 cells are plated in each of six separate culture dishes to achieve six replicatesper dose point, once the desired concentration range has been determined by measurements of cloning efficiency. Routinely,aminimum of four dose points are studied in increasingdilutions from a dose which causes 50 to 75 percent decrease in cloning efficiency. Dilutionswill be made two-foldor more, dependingon the slope of the cytotoxicity curve. The proceduresfor chemicaladditions to the culturesare identicalto that employedfor cytotoxicity. All cultures are refed with Basal Eaglesmedia with 10 percent fetalcalf serum. Routinely,re-feedingis done every three days until a confluent monolayer of cells is acheived;usually 15 to 20 days. Thereafter,the cultures are refed weekly. -----Transformation Assay Continued At 35 days after removal of the compound, all culture dishes are fixed, stained, and examined macroscopically and microscopically, and scored for transforma.tion. All of the fixed and stained cultures are retained for one year as a semi-permanent record of the study. Scoring of morphological trans rmation is in accordance with the types described by Reznikoff, et alf! A. Scoring of Transformation Focal areas gf transformationare classified according to the criteria of Reznikofflas follows: Type 1. Foci composed of monolayer cells are more densely packed than the background cells. This type is not considered malignant and is not scored. TVI)EII. Foci show massive piling up into virtually opaque multilayers. rh; cell are only moderately polar, thus criss-crossing is not pronounced. Fifty percent of Type 11 foci have been shown to be malignantly transformed. Type III-. Foci are composed of highly polar, fibroblastic,multilayered, criss-crossed arrays of densely stained cells. Eighty-five percent of Type III foci have been shown to be malignantly transformed. Selected Type III foci will be retained from living cultures and subcultured twice, after which the cells will be cryopreserved for one year. If, during that time, confirmatory in v"vo tumorogenesis studies are requested, these cells will be made7-av-aTFablfeor injection into a suitable host. 4. Speci al C3H io,r1/2 Early Reporting Although the classic methodology for the C3H IOT 1/2 prescribes a 6-vieek course of study, recent data obtained by our Laboratory shows that the assay time may be foreshortened to 15 to 20 days. Detailed microscopic study of the colonies allows for quantitation in this somewhat shorter frame using the classic morphologic criteria previously described. This early reporting capability is available as preliminary informationand is included in our report. 5. Recording of Data and Reports All data from the cytotoxicity screening, transformation assay and early reporting information are recorded in tabular form (see enclosure). A coded access number will be provided to allow retr*@lvalof cryopreserved material, and the fixed and stained cultures. All reports will remain Confidential, 4 -----Recording of Data and Reports,Continged subject to disclosure by the Corporation. Although there are no specific FDA guidelines for in vitro carcinagenesis testing, we adhere to the Good Laboratory.Practicie7s-Frot-ocpbult forward by this Federal Agency. 6. References 1. Reznikoff, C., Brankow, D., Heidelberger, C. Cancer Research, 33:3231-3238, Dec.-1973. 2. Reznikoff, C., Brankow, D., heidelberger, C. Cancer Research, 33:3239-3249, Dec. 1973. 3. Mondal, S., Brankow, D., Heidelberger, C. Cancer Research, 36:7, Part 1, 2254-2260, July, 1976. 4. Bertram J.S. Cancer Research, 17:51, 1975.* 5. Bertram, J.S. Cancer Research, 37:2, 514-523, 1977. 6. Haber, D.A., Fox, D.A. Dynan, W.S., Thilly, W.G., Cancer Research, 37:6. 1644-1648, June i977. 7. Bertram, J.S., Cancer Letters 7:5, 288-298, September, 1979.