sample="quota" bates="88208744" isource="ll" decade="1980" class="ui" date="19870603" UNSCHEDULED DNA SYNTHESIS IN RAT PRIMARY HEPATOCYTES TEST ARTICLE B34 Lot No. 2nd Sub. FINAL REPORT MICROBIOLOGICAL ASSOCIATES INC. 5331 RIVER ROAD, BETHESDA, MARYLAND 20816 RECEIVED JUN - 9 1987 UNSCHEDULED DNA SYNTHESIS I N RAT PRIMARY HEPATOCYTES TEST ARTICLE B34 Lot No. 2nd Subm. FINAL REPORT Author Rodger D. Curren Study Completed On June 3, 1987 Performing Laboratory MICROBIOLOGICAL ASSOCIATES, INC. 5221 RIVER ROAD BETHESDA, MARYLAND 20816 Laboratory Study Number T 5227.380 QUALITY ASSURANCE STATEMENT Study Title: UNSCHEDULED DNA SYNTHESIS IN RAT PRIMARY HEPATOCYTES Study Number: T5227.380 Study Director: Rodger D. Curren, PH.D. Initiation Date: 86/10/23 Review Completed Date: 87/06/03 This study has been divided into a series of phases. Using a random sampling approach, Quality Assurance monitors each of these phases over a series of studies. Procedures, documentation, equipment, etc., are examined in order to assure that the study is performed in accordance with the U.S. FDA Good Laboratory Practice regulations (21CFR8), the U.S. EPA GLPs (40CFR792 and 40 CFR160), and the OECD guidelines and to assure that the study is conducted according to the protocol. The following are the inspection dates, phases inspected, and report dates of QA inspections of the study. INSPECT ON 86/10/06 - 86/10/06, TO STUDY DIR 86/10/06,TO MGMT 86/10/06 PHASES: PROTOCOL REVIEW INSPECT ON 86/12/19 - 86/12/19, TO STUDY DIR 86/12/19, TO MGMT 86/12/29 PHASES: DETERMINATION OF LDH LEVELS INSPECT ON 87/05/19 - 87/05/19, TO STUDY DIR 87/05/19,TO MGMT 87/06/03 PHASES: FINAL REPORT This report describes the methods and procedures used in the study and the reported results accurately reflect the raw data of the study. Quality Assurance RA/QA Department Date UNSCHEDULED DNA SYNTHESIS IN RAT PRIMARY HEPATOCYTES FINAL REPORT Test Article: B34 Lot: 2nd Subm. MA Study No.: T5227.380 Test Article Description: White Powder Storage Conditions: Refrigerated in an amber bottle Date Sample Received: August 23, 1986 Initiation Date: October 23, 1986 Initiation Date: October 23, 1986 Completion Date: June 3, 1987 Sponsor: Lorillard Research Center P.O. Box 21688 420 English Street Greensboro, NC 27420-1688 Sponsor's Investigator: J. Daniel Heck, Ph.D. Testing Facility: MICROBIOLOGICAL ASSOCIATES, INC. 5221 River Road Bethesda, Maryland 20816 Study Director: Rodger D. Curren, Ph.D. Date Laboratory Technician Katherine T. Ault, B.A. Date Laboratory Technician Nagasundarl Durvasula, M.S. Date Laboratory Technician: Barbara J. Head, A.S. Date Laboratory Technician: Kathleen Wallace, B.S. Date SUMMARY Lorillard Research Center's test article, B34, was tested in the Unscheduled DNA Synthesis Test using rat primary heptaocytes. The test article was tested at eight dose levels ranging from 2.5 ug/ml to 2500 ug/ml and was fully evaluated at five dose levels from 7.5 to 1250 ug/ml. The results of the UDS assay indicate that under the test conditions, the test article did not cause a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the control), at any dose level. Therefore, the test article is considered negative in this study. INTRODUCTION This study was conducted from October 23, 1986 to May 8, 1987 at Microbiological Associates, Inc. The experimental procedure employed was essentially that of Williams, G.M. (Cancer Research 37:1845-1851, 1977) and is described in detail in the specific protocol for this study (see Appendix). The purpose of the study was to evaluate the test article, B34, for its ability to induce unscheduled DNA synthesis in rat primary hepatocytes as measured by autoradiographic methods. MATERIALS AND METHODS Indicator Cells Primary rat liver cell cultures derived from the livers of normal adult male Sprague-Dawley rats were used in this study. The animals were obtained from Frederick Cancer Research Facility and Charles River Laboratories, Inc. and were quarantined for at least one week prior to the initiation of the study. The animals were maintained on standard laboratory diet throughout the quarantine period. The procedure used for obtaining rat hepatocyte cultures (HPC) was essentially that of Williams, et al., (In Vitro 13:809-817, 1977). Each rat used was sacrificed by inhalation of metofane. The animal was dissected and perfused first with 0.5mM EGTA solution and then with a collagenase solution. The liver was removed from the animal and the cells were dissociated, counted, and seeded into 35 mm dishes containing coverslips (5 x 10 viable cells/dish). The cells were seeded in Williams Medium E (WME) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 unites of penicillin and 100 ug of streptomycin/ml or 50 ug/ml gentamicin. The cultures were incubated at 37 ± 1ºC in a humidified 5± 1% CO2 incubator for 90-120 minutes, washed and refed with serum-free medium and used in the test. Test and Control Articles The test article, B34, was received on August 23, 1986, and stored refrigerated in an amber bottle. The test article was dissolved and diluted in DMSO (Aldrich, Lot 63309IM) to make up the stock solutions. 7, 12-Dimethylbenzanthracene (DMBA) (Kodak, Lt C13) was dissolved in DMSO (Aldrich, Lot 63309IM) and used as a positive control in this study. The test article was diluted to appropriate concentrations immediately prior to use. Approximately 20 to 30 minutes elapsed between the time the test article was dissolved and the final treatment of cells. All test article and control treatments were done under subdued yellow lights to avoid possible problems of photoinactivation. Documentation of the stability, purity and the method of synthesis, fabrication or derivation of the test substance is the responsibility of the sponsor. Identification of Test System All culture plates were labeled with pen with a code system which clearly identifies the test article or control, test phase, and the experiment number. Slides were similarly labeled with pencil or pen. Initial Cytotoxicity Test A preliminary cytotoxicity test was performed to establish an appropriate dose range fro the test article. Ten doses ranging from 0.075 to 2500 ug/ml were tested. The test article was tested by treating replicate cultures of HPC 90-120 minutes after seeding. Eighteen to twenty hours later, an aliquot of culture fluid was removed, centrifuged, and the level of lactic acid dehydrogenase (LDH) activity determined. Two replicate plates were used for LDH measurement at each dose level. Two replicate plates were used for comparing the treated to untreated control cultures. The LDH Values for the single condition of lysed cells plus test article may not be reliable due to substrate exhaustion. Inadvertently the samples for this condition were not further diluted and reassayed. Unscheduled DNA Synthesis Test Based on the test results of the initial cytotoxicity test, the test article, B34, was tested at eight dose levels. Three replicate plates seeded with 5 x 10 HPC/plate were treated with 2.3 ug/ml to 2500 ug/ml of test article. DMSO, which was used to dissolve the test article was also used as the solvent control for the test article. DMBA, at 3 ug/ml and 10 ug/ml, was used as the positive control. DMSO was used as the solvent control for the positive control. Each test article and control dish received ³H-thymidine at a final concentration of 10uCi/ml. In parallel with the test plates, three cultures per dilution were treated with the same compound for a parallel toxicity test. The cells were treated for 18-20 hours as described earlier. The parallel toxicity plates were harvested by removal of a portion of the medium for LDH determinations as described in the initial cytotoxicity test to obtain the relative survivals and relative toxicities. After eighteen to twenty hours of exposure, the cells in the Unscheduled DNA Synthesis assay plates were washed in serum-free WME, swelled in 1% sodium citrate and fixed in ethanol-acetic acid fixative. The coverslips were air-dried, mounted cell side up on glass slides, and allowed to dry. The slides were coated with Kodak NTB emulsion and stored for ten days at 4°C in light tight boxes with desiccant. The slides were then developed in Kodak D-19 developer, fixed in Kodak fixer and stained in hematoxylin-sodium acetate-eosin stain. Scoring The slides were read "blind" on an Artek Colony Counter. Nuclear grains were counted in 25 cells in random areas on each of three coverslips per treatment where possible. The net nuclear counts were determined by counting three nucleus-sized areas adjacent to each nucleus and subtracting the average cytoplasmic count from the nuclear count. Replicative synthesis was identified by nuclei completely blackened with grains and such cells were not counted. Nuclei exhibiting toxic effects of treatment, such as dark staining, disrupted membranes or irregular shape, were not counted. Presentation of Data For each treatment slide, the net nuclear counts were averaged and the standard deviation (S.D.) determined and recorded on a summary form. Also reported are the grand mean and S.D. for each dose level as well as the percent of cells in repair (cells with 5 net nuclear grains). Means, standard deviations and percent survivals were computed using a LOTUS 1-2-3 program on IBM DS or compatible computer. Criteria for Evaluation of Test Results The results of this study were evaluated according to the criteria described below. If the mean net nuclear count was increased by at least five counts over the control, the results for a particular dose level were considered significant. A test article was judged positive if it induced a dose-related response and at least one dose produced a significant increase in the average net nuclear grains when compared to that of a control. In the absence of the dose response, a test article which showed a significant increase in the mean net nuclear grain count in at least two successive doses was considered positive. If a test article showed a significant increase in the net nuclear grain count at one dose level without any dose response, the test article was considered to have a marginal positive activity. The test article was considered negative if no significant increase in the net nuclear grain counts at any dose level was observed. Records All raw data, final report and stained slides of this study are maintained in the archives of Microbiological Associates, Inc. located at 5221 River Road, Bethesda, Maryland 20816. RESULTS AND DISCUSSION The results of the preliminary cytotoxicity assay are recorded in Table 1. Toxicity was present at the highest concentration of test article, 2500 ug/ml. The remaining dose levels were non-toxic. The highest concentration of test article selected for Unscheduled DNA Synthesis assay was 2500 ug/ml. The results of the parallel cytotoxicity assay are recorded in Table 2. Microscopic examination of the hepatocyte cultures indicated high toxicity through 250 ug/ml and a slight toxicity at 75 and 25 ug/ml. LDH determination showed a 12% toxicity at the highest concentration, 2,500 ug/ml, and 5% toxicity at 1,250 ug/ml. However, when the test article was added directly to lysed cells, the measured LDH levels were much lower than when lysed cells alone were measured (corrected LDH of 150.0 and 383.8, respectively; Table 2). This indicates that the test article interferes with the LDH assay. Microscopic inspection of the slides showed at 2,500 ug/ml the nuclei could not be evaluated since cytoplasmic definition was poor. Dose levels of 1,250, 750, 250, 75 and 7.5 ug/ml were fully evaluated for UDS. The results of the UDS assay are summarized in Table 3. Slides treated with B34 or DMBA were compared to the appropriate negative control. According to the criteria set for evaluating the test results, both doses of the positive control compound, DMBA, induced a significant increase in the average net nuclear count of silver grains. None of the test article doses caused a significant increase (5 counts above the solvent control) in the mean net nuclear counts. The highest concentration of test article, 1250 ug/ml, gave a grand mean of 2.3 net nuclear counts with 24% in repair. While these values are somewhat elevated, they do not meet our criteria for a positive response. All criteria for a valid test were met. CONCLUSION Lorillard Research Center's test article B34, was tested in the Rat Hepatocyte Unscheduled DNA Synthesis Assay. The test article was tested at eight dose levels ranging from 2.5 ug/ml to 2500 ug/ml and was fully evaluated at five dose levels, 1250, 750, 250, 75 and 7.5 ug/ml. The results of the UDS assay indicate that under the test conditions, the test article did not cause a significant increase in the Unscheduled DNA Synthesis as measured by the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the control), at any dose level. In this study the positive control, 7,12-Dimethylbenzantracene (DMBA), induced significant increases in the mean number of net nuclear gain counts over that in the solvent control. APPENDIX Received by RA/QA 10/3/86 APPROVED UNSCHEDULED DNA SYNTHESIS IN RAT PRIMARY HEPATOCYTES 1.0 PURPOSE The purpose of this study is to evaluate the potential of the test article to induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. 2.0 TEST ARTICLE 2.1 Identification: B34 2.2 Analysis: The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition of the test article (See attached Test Article Characterization form) and the stability and strength of the dosing solutions. 2.3 MA Study Number: T5227.380 3.0 SPONSOR 3.1 Name: Lorillard Research 3.2 Address: P. O. Box 21688 420 English St Greensboro, NC 27420 3.3 Authorized Representative: Connie J. Stone, Ph.D. J. Daniel Heck, Ph.D. 4.0 TESTING FACILITY 4.1 Name: Division of Genetic Toxicology Microbiological Associates 4.2 Address: 5221 River Road Bethseda, Maryland 20816 4.3 Study Director: Rodger D. Curren, Ph.D. 5.0 TEST SYSTEM Primary hepatocytes obtained from Sprague-Dawley or Fischer rats will be used in this study. Monitoring unscheduled DNA synthesis (UDS) in rat hepatocyte primary cultures (HPC) presents several advantages over other cell types used to monitor possible interactions between the test article and DNA. First, the target cells posses the ability to metabolize many promutagens/procarcinogens to their active from, thus eliminating the need for an exogenous source of metabolic activation. Secondly, HPC's are nearly 100% non-dividing, so no metabolic blocks are needed to inhibit replicative DNA synthesis. Thirdly, the target cells, HPC, are epithelial in origin. Since most human cancers are carcinomas, an assay using epithelial cells to monitor genetic damage may be more relevant to the in vivo situation than a similar assay using fibroblasts. Finally, the test is relatively short-term, requiring less than 20 days to evaluate a chemical. 6.0 EXPERIMENTAL DESIGN The experimental design of this study consists of a solubility of miscibility test to select a suitable solvent for the test article, a preliminary toxicity test, and then the UDS assay which includes a simultaneous toxicity test. The UDS assay is evaluated on the basis of incorporation of tritiated-thymidine (³H-TdR) into the HPC DNA, presumably as a consequence of DNA repair. This incorporation is evidenced by the presence of silver grains over the nuclei of cells that were coated with a photographic emulsion 8-12 days preceding development. The cells are stained, and the number of grains over the nucleus and adjacent nuclear-sized cytoplasmic areas are counted on an automated colony counter. UDS data are present as net nuclear grains (the number of nuclear grains minus the number of cytoplasmic grains) per cell. The toxicity data are presented as relative survival, based on viable cell counts after approximately 18-20 hours exposure to test articles. 7.0 METHODS The methods used are modifications of the procedures used by G. M. Williams ( , ) and J. Bradlaw ( ). 7.1 Media and Reagents 7.1.1 Plating Medium: Williams Medium E (WME) buffered with 0.01M HEPES, adjusted to pH 7.3 and supplemented with 10% fetal bovine serum APPENDIX (FBS), 2mM L-glutamine and 50 ug gentamycin/ml or 100 units of penicillin and 100 ug of streptomycin/ml. 7.1.2 Perfusion (collagenase) solution: Serum-free WME with HEPES, L-glutamine and gentamycin or penicillin and streptomycin at the same concentration as in the plating medium, pH adjusted to 7.3 and containing 100 units of collagenase (Type I)/ml. 7.1.3 Treatment medium: Serum-free WME plus HEPES, L-glutamine and gentamycin or penicillin and streptomycin at the same concentrations as in the plating medium, adjusted to pH 7.3 and containing 10uCi ³H-TdR/ml. 7.1.4 EGTA solution: 0.5mM EGTA in Ca++, Mg++, free Hanks' Balanced Salt Solution, buffered with 0.01M HEPES, pH adjusted to 7.3. 7.1.5 Metofane (Methoxyflurane). 7.2 Preparation and Delivery of Test Article The test article will be dissolved in deionized distilled water, WME, ethanol (CAS #64-17-05), dimethylsulfoxide (DMSO) (CAS #67-68-5), acetone (CAS #67-64-1), or other appropriate solvent. A 100X concentrate of test article in appropriate solvent will be prepared. If WME is the solvent, the 100X concentrate is not required. PROTOCOL AMENDMENT DATE: April 10, 1986 SPONSOR: Blanket for Many Sponsors SPONSOR'S TEST ARTICLE DESIGNATION: Blanket for Many Test Articles MA STUDY NO: Blanket for Many Sponsors PROTOCOL NO: SPGT380 PROTOCOL TITLE: Unscheduled DNA Synthesis in Rat Primary Hepatocytes AMENDMENT(S): (INCLUDE LOCATION IN PROTOCOL, AMENDMENT, AND REASON) Protocol SPGT380 Section 7.6 Dose Selection Line 18 and Section 7.7.3 UDS Assay Line 8 Alternatively, the supernatant will be removed from the cell cultures and tested for its level of Lactic Acid Dehydrogenase (LDH). Leakage of LDH from the cytoplasm into the culture medium is also a function of cell plasma membrane integrity. Measurement of LDH leakage, although of comparative sensitivity with the trypan blue vital dye staining, offers a less subjective alternative to cell counting procedures. APPROVAL: SPONSOR REPRESENTATIVE/INVESTIGATOR DATE STUDY DIRECTOR DATE PROTOCOL AMENDMENT DATE: Date: December 1, 1986 SPONSOR: Lorillard Research Center SPONSOR'S TEST ARTICLE DESIGNATION: B34 MA STUDY NO: T5227.380 PROTOCOL NO: SPGT380 PROTOCOL TITLE: Unscheduled DNA Synthesis in Rat Primary Hepatocytes AMENDMENT(S): (INCLUDE LOCATION IN PROTOCOL, AMENDMENT, AND REASON) Protocol SPGT380 Section 7.5.4 Preparation of Hepatocytes Line 4 and Section 7.5.6 Preparation of Hepatocytes Line 3 Change "approximately 20 ml/minute" to "...20 to 40 ml/minute" Reason for Change: To increase the number of usable hepatocyte preparations APPROVAL: SPONSOR REPRESENTATIVE/INVESTIGATOR DATE STUDY DIRECTOR DATE PROTOCOL AMENDMENT DATE: April 23, 1987 SPONSOR: Lorillard Research Center SPONSOR'S TEST ARTICLE DESIGNATION: B34 MA STUDY NO: T5227.380 PROTOCOL NO: SPGT380 PROTOCOL TITLE: Unscheduled DNA Synthesis in Rat Primary Hepatocytes AMENDMENT(S): (INCLUDE LOCATION IN PROTOCOL, AMENDMENT, AND REASON) Protocol SPGT380 1) Location: Section 7.1.2 Media and Reagents Line 5 Amendment: Change "...containing 100 units" to "containing 80 to 100 units" Reason: To increase the number of usable hepatocyte preparations APPROVAL: SPONSOR REPRESENTATIVE/INVESTIGATOR DATE STUDY DIRECTOR DATE