Document 4M00X3g92vn870eZQXQonOEN

/t BUSHY RUN RESEARCH CENTER R. 0.4, Mellon Road, Export, Penneyhrsnla 16632 Telephone (412) 327-1020 CONFIDENTIAL: Not to be released outside UCC trlthout the written consent of the UCC component sponsoring the work. .Project Report 43-105 (Amendment) Tel: (412) 327-1020 January 14, 1981 First Amendment to BAXELITER Cycloaliphatic Epoxy Resin ERL-4221 In Vitro Mutagenesis Studies: 3-Test Battery Authors: R. S. Sleslnskl, M. V. Gaunt, P. J. Currie, V. C. Hengler Sponsor: Onion Carbide Corporation **a** On page 5, Section 2 (SCE Test), the unit of measure should have been ug/ml, therefore, the second to last sentence should read: "Bromodeoxyurldlne (BrdU) required to differentiate between the Individual "sister" chromatids by SCE staining, was present at a concentration of 3 ug/ml in the growth medium during treatment and during the culture period following exposure-" Reviewed and Approved by: Ronald S- Sleslnskl, Bi.D. Study Director Manager, Genetic Tbxlcology Elton R. Homan, Ph.D. Associate Director, Toxicology VPC/1103-1 Fred R. Frank, Ph.D Director Rushy Run Research Center A Joint Mellon Institute-Union Carbide Corporation Operation UCC 062599 BUSHY RUN RESEARCH CENTER R. 0. 4, Mellon Rood, Export, Pennsylvania 1S832 Telephone <4i2> 327-1020 CONFIDENTIAL: Not to be released outside UCC without the Witten consent of the UCC component sponsoring the work. Project Report 43-105 20 Pages Tel: (412) 327-1020 December 15, 1980 BAKELITE* Cycloaliphatic Epoxy Resin ERL-4221 In Vitro Mutagenesis Studies: 3-Test Battery Authors: R. S- Sleslnski, H. W. Gaunt, P. J. Guzzle, V. C. Hengler Sponsor: Onion Carbide Corporation e* * * * SUMMARY Epoxy Resin ERL-4221 mas evaluated for potential mutagenic activity with a battery of three In vitro tests, which were: the Chinese Hamster Ovary (CH0) Mutation test, the Sister Chromatid Exchange (SCE) test and an assay for induc tion of Unscheduled UNA Synthesis (UDS) In rat liver cells. The results indicated that Epoxy Resin ERL-4221 did not produce a strong mutagenic effect typical of known chemical mutagens but it appeared to possess significant mutagenic potential in the sister chromatid exchange test and questlonableto-weak activity in the UDS test. The lack of a definitive response in at least two of the three tests prevented an unequivocal classification of Epoxy Resin ERL-4221 as mutagenic or non-mutagenic However, the strongly positive and dose-related Increase In the SCE frequency in cells treated with Epoxy Resin ERL-4221 suggests that additional testing may be appropriate to investigate the possible biological significance of these results. RESULTS AND INTERPRETATION Epoxy Resin ERL-4221 mas selected for mutagenesis testing in part to develop and validate the sensitivity of our in-house battery of mutagenicity tests. Epoxy Resin ERL-4221 mas found to be Inactive in a previous lifetime dermal carcinogenesis study with mice performed at our laboratory (CHF Report #27-6, January, 1964) and it was used for testing the ability of our 3-test battery to discriminate between chemicals found to be either active or inactive in animal studies. Selection of Test Concentrations - Preliminary Mtperiments were performed to aelect an appropriate range of concentrations in which the maximum dose level would allow survival of approximately 102 of the treated cells. A maximum concentration of 0.012 (by volume) was chosen as the top dose level for tests both with and without an 89 metabolic activation system. In a second repeat test, necessitated by technical problems in the first test, the maximvis dose level tested with 89 activation mas Increased to 0.02Z. Rushy Run Research Center A Met Mellon Institute--Union Cerbloe Corporation Operation UCC 062600 1804 Report 43*105 Pag* 2 CHO Mutation Teat * Epoxy Resin ERL-4221 tras not active In stimulating a dose-related increase of mutant cells when tested either with or without the presence of an S9 metabolic activation system. Neither of two experiments provided any Indication of a statistically significant mutagenic effect of the test agent. Epoxy Resin ERL-4221 ms considered inactive as an agent for inducing mutation of CEO cells in culture. SCE Test - Epoxy Resin ERL-4221 ms highly active in significantly stimulating the induction of SCE in vitro at three of six of the concentrations t sted. The indication of a dose-related increase in the number of SCE In tests without S9 metabolic activation provided a convincing Indication that Epoxy Resin ERL-4221 ms active in stimulating SCE In CHO cells. Tests of SCE production with the addition of a metabolic activation system were not performed because the test without addition of liver homogenate indicated that metabolic conversion ms not required for activity of the test chemical. OPS Test - Epoxy Resin ERL-4221 did not produce dose-related increases in the amount of UDS detected with either nuclei or DNA. However, in evaluations over a relatively vide range of concentrations, the lowest three concentrations of Epoxy Resin ERL-4221 produced highly numerically elevated levels of (IPS acti vity. Because these values were not consistently slgniflcsnt in statistical comparisons to the concurrent solvent control, the results could not be defini tively labelled as either positive or negative. The data were considered to be suggestive of a low level of activity and Epoxy Resin ERL-4221 appeared to be questionably-to-wealtly active in the present test with the hepa.tocyte test system. Comparative Mutagenicity - The pattern of responses produced in the 3-test battery of mutagenicity tests indicated that Epoxy Resin ERL-4221 ms not a potent mutagenic agent but that it appeared to possess a low level of activity in the CPS and significant activity in the SCE test. The lack of definitively positive responses in at least two tests of the 3-test battery prevented an unequivocal classification of Epoxy Resin ERL-4221 as either mutagenic or non-mutagenic. However, significantly positive results related to the treatment dose observed in the SCE test should be considered as an indication of unconfirmed but potential biological activity. Additional testing of this chemical using other test systems may be warranted to determine the biological significance of the results of these in vitro studies. In the previous dermal carcinogenesis test of this chemical TCBt Report #27-6), 1 tumor in 15 mice in the effective group ms observed but this result ms not significantly above the tumor incidence in the control group of animals. ucc 062601 I Raport 43-105 Pag* 3 SAMPLE Quantity: 8 ounces CH7 Sample No.: 42-136 Submitted by: W. C. Kuryla, for PCC Date Received: March 15, 1979 Toxicology Advisory Group Division: Specialty Chemicals and Plastics Identification: light yellow. viscous liquid CAS #: 2386-87-0 1805 'l 1 i I \ 18C6 Report *3-105 Pag* * BAKELITE* Cycloaliphatic Epoxy Resin ERL-4221 la Vitro Mutagenesis Scudleti 3-T*at Battery Sponsor: Union Carbide Corporation OBJECTIVE The purpose of this study was to evaluate the potential of Epoxy Resin ERL-4221 to induce genetic damage In mammalian cells at the gene, chromosome and/or ON* (deoxyribonucleic acid) level of molecular organization. A battery f three In vitro, short-term tests which detect each of these genetic endpoints was employed to evaluate Epoxy Resin ERL-4221 for potential mutagenic activity. A general description of the theoretical bases of these three tests is presented in Appendices I, XI and XXI (attached to the complete report). SAMPLE CHARACTERISTICS A typical, commercial sample of Epoxy Resin ERL-4221 was received for testing on March 15, 1979. The available information from the Toxicology Data Bank or from "Material Safety Data Sheets" for this product is attached to this report as Appendix XV. METHODS A description of the technical procedures used in the CEO test, the SCE test and the CDS assay are presented in greater detail In Appellees X, XI and III, respectively (attached to the complete report). Testing was performed as part of the in-house development and validation of the mutagenicity test battery. A copy of the current procedures used for these tests at the Bushy Run Research Center are attached to this report and deviations from there procedures are noted in the individual test results. 1. CHO Test (Detailed procedures In Appendix I): A. Dose Selection - Appropriate concentrations' of Epoxy Resin ERL-4221 for testing were determined by measurements of cytotoxicity to CHO cells of six concentrations tested both in the presence and absence of a liver S9 metabolic activation system. Selection of a maximum concentration for testing depended upon an estimate of the dose level which should permit survival of at least 10Z of the treated cells. Class-distilled dlmethylsulfoxide (DMSO) was used as the solvent and solvent control; sterile water (HjO) was used as the negative control. To simplify tables and to allow comparisons between different tests, concentrations of Epoxy Resin ERL-4221 In the following sections of the report are given In terms of volume percentages x 10"^ to eliminate zeros in the lover concentration values (eg. 0.0003125Z " 3.125 x 10"*X). o'gfefe I 1807 port 43-105 Pag* 5 B. Mutation - In experiment #1, CHO calls wars exposed for 16 hours to five concentrations of Epoxy Basin ERL-4221 from 100 x 10"*X to 6.25 x 10"*X (by volume) without tha addition of an S9 aetabollc activation systaa and for 5 hours to an identical range of concentrations with S9 activation. A second, repeat experiment was perforaed at identical concentrations in the test without S9 but concentrations froa 200 x 10*^2 to 12.5 x 10"*X were tested with S9 activation. Dilutions of Epoxy Besln ERL-4221 were prepared by either direct addition of tha test agent into the cell culture aedla or by asking sequential one-half dilutions froa the stock solution for the highest concentration using glass-distilled DMS0. The surviving fraction was detemlned at 20 to 24 hours after treataent and the autant fraction was deterained after a 7-to 9-day period to allow "expression" of the autant phenotype. Only tha top five concentrations which allowed sufficient cell survival were assesaed for survival and induction of autants. The percentage of cells surviving the treataent, the frequencies of autant colonies and the nuaber of autants/106 viable cells are presented in tabular fora. 2. SCE Test (Detailed procedures in Appendix II): Production of SCE's following exposure to various concentrations of Epoxy Basin ERL-4221 was studied in CB0 cells without the incorporation of an S9 metabolic activation system. Selection of a maximum dose level which would permit survival of at least 50Z of the treated cells was based on the prescreening test for cytotoxicity performed as part of the CHO Citation test. Dilutions of Epoxy Resin ERL-4221 for testing, ranging froa 100 x 10~4% to 3.125 x 10~4z (by volume), were prepared either by direct addition into the culture medium or by addition of various aliquots of a stock solution prepared in DMS0. For determination of direct mutagenic action, CHO cells were exposed to Epoxy Resin ERL-4221 and appropriate controls for 5 hours without S9 activation. Indirect mutagenic action, requiring aetabollc activation by liver S9 homogenate, was not studied because a highly significant positive response was obtained without metabolic activation which indicated a direct-acting mechanism for this test ag nt. Bromodeoxyurldlne (BrdU) required to differentiate between the individual "slater" ehroaatlds by SCE staining, was present at a concentra tion of 3 g/ml in the growth aedlua during treataent and during the culture period following exposure. A total of 15 cells/dose level and 5 dose levels, with or without aetabollc activation were examined. The number of SCE/eell, mean # of SCE/chroaoeoae and the level of statistical significance of the Increases above concurrent solvent control values are presented in tabular fora. 3. DPS Teat (Detailed procedures in Appendix III): Induction of primary ENA daaage in rat liver cells (hepatocytee), was studied at a minimus of six dose levels which spanned a 1000-fold range of concentrations. Cells were treated with Epoxy Basin ERL-4221 for 2 hours in culture aedlua containing ^H-thyaidlne, hydroxyurea and appropriate dilutions of Epoxy Resin ERL-4221 prepared in EHS0. Determination of CDS activity was perforaed by analyses of radioactive ineorporati n into 1808 Import 43-105 Page 6 Isolated hepatocyte nuclei and In DNA (precipitated fron aliquots of the isolated nuclei) using a Searle Analytic Model 81 or Packard Modal 2650 scintillation spectrometer. Data are presented In tabular form with an Indication of the level of statistical significance above the concurrent aolveut control values. 4. Controls - Positive, negative and solvent controls were tested concurrently with the test sample to assure the sensitivity of the test system and the concurrence of the results to previous test performance. For the CHO and SCE assays, dlmethylnltrosamine (DKN) and ethylnethanesulfonate (EMS) were used as positive control agents to assure the sensitivity of the test system for detecting Indirect- and direct-acting mutagens, respectively. Deionized water sterilized by membrane filtration and glass-dlstllled dimethylsulfoxide (DKSO) were used as the negative and solvent controls, respectively. In the UDS assay, EMN and 4-nltroquinollne oxide (4-NQO) were used as positive controls for indirect- or direct-acting mutagens, respectively. DMSO was used as the solvent and the solvent control. 5. Metabolic Activation - S9 liver homogenate, prepared from Arochlor 1254induced, Sprague-Dewley male rats, was purchased from Litton Blonetlcs. The S9 preparation used for the CHO test contained 38.5 mg/ml protein and had a benzo(a)pyrene hydroxylase activity of 21.6 nmol hydroxybenzpyrene/20 mln/mg protein, (assayed by Litton). A concentration of 2400 ug of S9 protein was added to 5 ml of culture media. 6. Statistical Analyses - Data from the SCE and UDS testa were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center. Data from the CHO test do not follow s normal distribution according to experience with his torical controls. Thus, the Student's t-test was used after suitable transformation of the mutation frequencies (MF) following the procedure of Xrr and Snee: (M7 + 1)0*15 (irr, J. D. and R. Snee, Proceedings of the Cold Spring Harbor-Banbury Conference, IX (1979), 263-274). Rounding of data to either two decimal places or to the appropriate number of significant figures was performed for presentation on tables* Although statistically significant decreases In mutation Indices can occur because of cytotoxic responses, only statistically significant Increases in responses above control values are Indicated on Tables for simplicity. The degree of statistical significance Is denoted by: a: 0.05 > p > 0.01, b: 0.01 > p > 0.001, or e: p < 0.001. No superscript (or MS) Indicates p > 0.05. 7. Rav Data Storage - Copies of the final report, statistical analyses, analytical data and data used to prepare the final report are stored In the BRRC Archives. Slides are stored In the Genetic Tbxlcology slide storage area. t 1809 laport 43-105 Pag* 7 RESULTS SECTION I - CHO MUTATION TEST - Epoxy Kaaln ERL-4221 A. Tait Dates - Initiated; April 26, 1979 Completed: May 16, 1980 B. Selection of Taat Concentration (Data not shown In tablaa) In taata without S9 activation, CHO ealla were axpoaad for alxteen hours to six concentrations of Epoxy Rasln ERL-4221 which spanned a concentration range from 0.5Z to 5 x 10"*Z by volume. An Identical range of concentrations was tested for five hours with S9 activation* The percentage of cells which survived the exposure, both In the presence and absence of an $9 metabolic activation system, was determined by counting the number of colonies produced by the survivors after a 5- to 7-day Incubation period. In this prescreening determination of cytotoxicity, no surviving cells produced colonies after treatment with 0.5Z of Epoxy Resin ERL-4221, the highest concentration tested. As a percentage of the control values, 3.5Z of the cells produced colonies after treatment with 50 x 10~4z of Epoxy Resin ERL-4221 In the presence of an S9 activation system; without S9 activation, 0.8Z of the cells treated at this same concentration formed colonies. A concentration of 100 x 10~*Z was selected as the maxi mum concentration for testing with and without S9 activation. In a second repeat experiment, the maximum concentration tested with S9 activation was Increased to 200 x 10~^Z to attain a higher level of cytotoxicity. C. Determination of Mutation Induction 1. Survival (Cytotoxicity) Table 1 presents the cytotoxicity data for CHO cells treated with Epoxy Resin ERL-4221 in the absence of a liver S9 metabolic activation system. A steep dose-response effect with the test agent was suggested from the high degree of cytotoxicity observed for the top concentrations (100 x 10"*Z) In comparison to the markedly lover cytotoxicity obtained at only one-half the top dose-level (Table 1). The cells treated with the test agent together with an S9 activation system were not assessed for mutant In duction because the <X>2 concentration In the incubator used for these plates was abnormally high (due to a malfunction) and this malfunction Inhibited or killed the cello. The survival results of the second, repeat experiment are shown on Table 3. These data Indicated a similar cytotoxic response at the highest dose level tested without S9 activation In comparison to the value obtained In experiment #1 (Table 1). Also, the slope of the dose-response relationship for the cytotoxicity data In this second experiment suggested a more usual response without the steep drop observed at the highest two doses In experiment #1. 1810 Report 43-105 Peg* 8 2* Mutation Table 2 preaenta the data for .induction of autanta by Epoxy Resin ERL4221 and control agents* Epoxy Resin ERL-4221 did not produce a doserelated Increase in the frequency of mutants/lO^ viable cells over the 16-fold range of concentrations tested for potential autagenlc action without the presence of an S9 metabolic activation systea* Although no concentration of Epoxy Resin ERL-4221 produced a statistically significant increase in the nutation frequency In the test without S9 activation, the test was repeated because the EMS positive control was also not signifi cantly above the solvent control values. In experiment #2, sumarlxed on Table 4, data were consistent with the negative responses observed In experiment #1 (Table 2). No dose-level of Epoxy Resin EKL-4221 produced an increase which was statistically significant from the solvent control, and there was no indication of a dose-related production of mutants. This second experiment was consistent with the classification of Epoxy Resin ERL-4221 as not active in producing a mutagenic effect detectable in the CHO test systea. Mutation frequencies for the solvent controls for tests in both experiment #1 and #2 without S9 activation were in an acceptable and low range based upon experience with historical control values. The mutation frequency for the solvent control in experiment #2 with S9 activation was numerically higher than our historical control values but similar small Increases in the frequency of mutants have been seen in previous experiments in which the S9 liver homogenate itself displays a weak mutagenic activity. Small numerical Increases at some treatment levels of Epoxy Resin ERL-4221 were within the range of historical variability encountered for negative and solvent controls using this test. Highly statistically significant mutation frequencies were obtained for the DMN and EMS positive controls in experiment #2 and these values were within the normally expected range of variation observed in historical control data. D. Deviations from Standard Procedure* - Testing of Epoxy Resin ERL-4221 was performed as part of the development and validation phase for the in-house battery of mutagenicity assays. There are numerous deviations from our current SOP in the present test on this chemical but none of these de viations are believed to decrease the sensitivity of the test. The major deviation involved the use of a 16-hour exposure period in the test without S9 rather than 5 hours as etated in Appendix I. A 16-hour exposure period would normally be expected to Improve rather than to decrease the sensiti vity of the assay; exposure periods between 2 to 24 hours are acceptable if suitable toxic to non-toxic dose ranges are tested. The 100 x 10~*X dose-level was allowed an expression period of 9 days (rather than 7 days as used with all other dose-levels) because this top concentration produced an extended depression of cell growth and a longer period is thought to be more reliable at such high cytotoxic doses. 1811 Report *3-105 Pi|i 9 E* Conclusions Epoxy Resin ERL-*221 vat consistently inactive as a mutagenic agent for CHO call* whan tested with or without an S9 metabolic activation system over a 16-fold range of concentrations. Although snail Increases In the numeri cal frequency of autants were obtained at sons test concentrations of Epoxy Resin ERL-4221, these results appeared to be a random effect without statistical or probable biological significance. SECTION II - SCE TEST - Epoxy Resin EEL-4221 A. Test Dates - Initiated: Decenber 10, 1979 Completed: March 24, 1980 B. Selection of Test Concentrations A maximum concentration of 100 x 10~*Z was chosen as the top dose levels for testing without S9 activation based on cytotoxicity data from the CHO mutation test. Higher concentrations were expected to produce delays in the mitotic cycle and to decrease the number of cells with SCE staining, based on experience In other studies. A 32-fold range of concentrations from 100 x 10~^X to 3.125 x 10*^X (by volume) was examined without S9 activation. Because we observed a highly statistically significant and dose-related indication of a direct mutagenic effect of the test agent In this experiment without S9, tests with an S9 activation system were not performed. C. Determinations of SCE Production The data for SCE production In CHO cells treated with various dose levels of Epoxy Resin ERL-4221 and with appropriate positive, negative or solvent control agents are suunarized In Table 5. Epoxy Restn ERL-4221 pro duced statistically significant increases in the SCE frequency at three of the six dose-levels tested for direct action In the absence of a metabolic activation system. Also, the increase In the numbers of SCE was dosedependent. The test without S9 activation was considered an Indication of a significant direct mutagenic action of Epoxy Resin ERL-4221. Induction of SCE by the concurrent EMS positive control was highly statistically significant from the concurrent solvent control and these data indicated an appropriate sensitivity of the test system comparable to our historical positive control data. The numbers of SCE obtained with the H2O solvent and DMSO controls were also in an acceptable range of values included In the variability encountered in our historical experience with this test. Testing of Epoxy Resin ERL-4221 with S9 metabolic activation was not performed because the highly positive results obtained without S9 indicated that metabolic activation was not required to express mutagenic activity. 1812 1 Report 43-105 Reg* 10 D. Deviations from Standard Procedure* The experiment waa repeated three time* but only the final, successful study vas reported. In the first two experiments cytotoxicity of the test agent reduced the number of mitotic cells and the chromosome preparations were not suitable for scoring. E. Conclusions Epoxy Resin ERL-4221 produced highly significant Increases in the frequency of SCE when tested over a 32-fold range of concentrations In tests without addition of an S9 metabolic activation systa. Evidence of a dose-related effect on the SCE frequency following exposure to Epoxy Resin ERL-4221 Indicated that the test agent should be considered significantly active In the present In vitro assay. SECTION III - UDS TEST - Epoxy Resin ERL-4221 A. Test Dates - Initiated: August 31, 1979 Completed: March 11, 1980 B. Selection of Test Concentrations Standard procedures were followed and Epoxy Resin ERL-4221 was tested over a 3-log range of concentrations from 1000 x 10"^X to 1.0 x 10"^Z by volume. The dose-levels were selected to span a range of cytotoxic to non-cytotoxic concentrations based upon data obtained In the CHO cytotoxicity test. t C. Determination of PDS Induction 1. Nuclear-Bound Radioactive Label (Data In Table 6) Values for "unscheduled" Incorporation of radioactive thymidine Into nuclei of hepatocytes exposed to Epoxy Resin ERL-4221 or to appropriate positive and negative controls are presented In Table 6. In hepatocytes treated with Epoxy Resin ERL-4221, only one concentration tested for potential activity Induced a statistically significant increase in the amounts of ^H-thymidine Incorporation. A gradual decrease in the amounts of radioactive incorporation, over the entire range of concentrations tested, was considered an Indication of the cytotoxicity of the test agent. The production of statistically significant levels of UDS at only the lowest concentration may suggest that even lower concentrations should be tested. These data were considered equivocal but suggestive of a questionableto-weak activity for Epoxy Resin ERL-4221. Both of the positive control agents, NQ0 and DMN, lrfuced numerically elevated Increases in UDS over values obtained with the solvent control. With DMN, however, only a single concentration produced a sufficiently high level of activity to produce a response which was statistically significant. These data with nuclei Indicated that the test system may be less sensitive than desirable f r detection of wekkly-aetlve mutagenic agents which require metabolic eonversl n (eg. DMN). .V Report 43-103 t Pag* 11 2. DNA-Bound Radioactive Label (Data In Table 7) Analyse* of DMA, from aliquot* of hepatocyta nuclei used for the UBS studies presented on Table 6, were performed a* a second assessment of 'unscheduled" Incorporation of ^H-thyaldlne. Values for radioactivity Inc rporated into the DNA of these hepatocyte nuclei are presented In Table 7. For hepatocytes treated with Epoxy Besln ERL-4221, two of the six test concentrations induced levels of UBS which were statistically significant from the solvent control* We also observed a similar pattern of responses ss obtained In the assessment of nuclei from calls treated with the same range of concentrations; three of the lowest six concentrations produced numerical elevations In UBS similar In magnitude to values obtained with the XMN positive control* Although there was no Indication of a dose-effect relationship due to treatments with the test agent, the UBS values were sufficiently elevated to suggest a very weak level of mutagenic activity. These several considerations were consistent In the classification of Epoxy Resin ERL-4221 as a questlonable-to-weakly active agent In the induction of BNA damage In the present test with the hepatocyte test system. The test employing precipitated BNA for measurement of Incorporation of ^H-thymidine was apparently more sensitive than the assay with nuclei, following a comparison of the level of responses produced by the positive control agents NQO and DMN with data from BNA and nuclei. With DMN four of six of the tested concentrations produced a numerical elevation in UBS which was also statistically significant from the solvent control (Table 7). With NQO, only the highest concentration was statistically above the solvent control but a dose-related response was producad which Is considered to be a d finltlve biological Indication of a positive mutagenic effect. D. Deviations from Standard Procedures Testing of Epoxy Resin ERL-4221 was performed during the development and validation phase of our in-house program of mutagenicity testing. Two prl r experiments were performed with methods different from the final pr cedures developed and described In Appendix III. These previous experiments are not reported because insufficient responses with the positive control agents Invalidated the results for an acceptable test. The final experiment, which employed our current procedures, tested several concentrations of DMN (rather than three as stated In the SOP) because we wished to monitor the appropriate response of the cellular activation system for metabolising such Indirect-acting chemical*. E* Conclusion Epoxy Rasln ERL-4221 appeared to produce a very weak response in the present test with cells treated over a 1000-fold rang* of test concen trations. Epoxy Resin ERL-4221 was considered questlonable-to-weakly active In producing DNA damage In the tests with hepatocytes. 1814 Reviewed and Approved by: Report 43-105 Page 12 Ronald S. Slealnskl, Fh.D. Study Director Manager, Genetic Toxicology Associate Director, Toxicology luni Fred R. Frank, Fh.D. Director Contributors: Chinese Hamster Ovary test Sister Chromatid Exchange test Unscheduled, DMA Synthesis Assay Peggy J. Guzzle, B.S. Master Technologist Michelle W. Gaunt, B.S. Master Technologist W. Christopher Hengler, M.S. Assistant Scientist WPC/1103-5 J i ! 1 UCC 062611 I Report 43*103 Page 13 1815 REFERENCES A search of the major mutagenesis/carcinogenesis computer data flies failed to find any articles pertinent to mutagenlc/csrclnogenlc potential for this test agent. A previous study on dermal carcinogenic potential for alee indicated .that Epoxy Resin ERL-4221 did not produce a statistically significant Increase ,ln skin tumors (CHF Project Report #27-6; CHF Sample #24-172). Table 1 Chinese Hamster Ovary (CHO) Mutation Assay: Determination of Toxic Effects of Chemical Treatment During 16 Hr Mutation Induction Period Experiment #1 Test Chemicals Total I ColonleB Total # Cells Plated [Epoxy Benin EBL-4221J (Z, v/v) 100.0 x 10~* 50.0 x 10"* 25.0 x 10-* 12.5 x 10"* 6.25 x 10"* 42 255 419 565 354 Without S9 Activation 800 800 800 800 800 Controls DHS0 (20 ul/ml) - Solvent HjO (20 ul/ml) BHS (200 ug/ml) - 502 512 578 800 800 800 Abbreviations: H2O - water; S9 - liver homogenate; DMS0 - dimethylsulfoxide EMS - ethylnethanesulfonate; DMN - dimethylnitrosamine Z Survival 5.2 31.9 52.4 70.6 44.2 62.8 64.0 72.2 Z of Solvent Control 8.4 50.8 83.5 112.5 70.5 102.0 115.1 WPC/1103-3 oC 3:o <j0 "8 mUHoI* Table 2 Chinese Baaater Ovary (CHO) Mutation Aaaay: Results on Evaluation of Mutant Induction by Epoxy Resin ERL-4221 Experiment #1 Teat Chemicals Plating Efficiency Total # Colonies Total # Cells Plated Viable Fraction Total I Mutant Colonies Mutation Induction Total # Cells Plated Hutantsl 10* Viable Cells (Epoxy Resin ERL-4221) (Z, v/v) 100.0 x 10-* 49 50.0 x 10~* 82 25.0 x 10-* 144 12.5 x lO-4 144 6.25 x 10-* 152 Controls: DMSO (20 ul/ml) - Solvent H20 (20 ul/ml) Medium EMS (200 ug/ml) - 270 203 255 180 Without S9 Activat:ion 300 0.163 0 300 0.273 1 300 0.480 0 300 0.480 2 300 0.507 0 - 300 0.900 1 300 0.677 1 300 0.850 0 300 0.600 22 1 x 10* I x 10* 1 x 10* 1 x 10* 1 x 10* l x 10* 1 x 10* 1 x 10* 1 x 10* 0 3.7 0 4.2 0 * 1.1 1.5 0 36.7 literal # mutant colonies pet 10** cells plated divided by viable fraction. Statiatlcal significance above solvent control: No superscript indicates p > 0.05. Data analysed by Student's t-test. Abbreviations: H2O - water; *S-9 - liver honogenate; DMSo"dimethylsulfoxide; EMS - ethylnethaneaulfonate; DHN - dimethylnitrosamlne. WPC/1103-3 Report 43<*i05 Page 15 oo?_C cd:; o Table 3 Chinese Hanater Ovary (CHO) Nutation Assay: Determination of Toxic Effects of Chemical Treatment Experiment #2 Teat Chemicals Total # Colonies Total # Cells Plated [Epoxy Resin ERL-4221J (X, v/v) 100.0 x 10"* 50.0 x 10"* 25.0 x 10"* 12.5 x 10"* 6.25 x 10"* 7 60 123 208 266 Without S9 Activation 400 800 800 800 800 Controls OMSO (20 ul/nl) - Solvent H20 (20 ul/ml) EHS (200 ug/ml) - . 469 399 43 800 800 600 [Epoxy Resin ERL-4221) (X, v/v) 200.0 x 10~* 100.0 x 10"* 50.0 x 10"* 25.0 x 10"* 12.5 x 10"* 351 381 326 419 337 With S9 Activation 800 800 800 800 600 Controls DHSO (2(5 ul/ml) - Solvent B20 (20 ul/nl) OHM (3700 ug/ml) - DHN (740 ug/ml) - 207 297 103 231 800 800 800 800 Abbreviations: H2O - water i S9 - liver homogenate; DMS0 - dlmethylaulfoxlde EHS - ethylmethanesulfonate; DMN - dlmethylnitrosamlne X Survival 1.8 7.5 15.4 26.0 33.2 58.6 49.9 7.2 43.9 47.6 40.8 52.4 42.1 25.9 37.1 12.9 28.9 WPC/1103-3 Z of Solvent Control 3.0 12.8 26.2 44.3 56.7 85.1 12.2 169.6 184.1 157.5 202.4 162.8 . 143.5 49.8 111.6 af *5 *8 O' wi o Of Chinese Hamster Ovary^pmO) Mutation Assay} Result* on Evaluation of Mutant Induction by Epoxy Resin ERL-4221 Experiment #2 Test Chemicals Plating Efficiency Total # Colonies Total f Cells Plated Viable Pr action Total # Mutant Colonies Mutation Induction Total # Cells Plated Mutants* 104 Viable Cells (Epoxy Resin ERL-4221) (2, v/v) 100.0 x 10-4 76 50.0 x lO"4 87 25.0 x HT4 137 12.5 x 10"4 172 6.25 x 10**4 186 Without S9 Activation 400 0.190 0 400 0.218 3 400 0.342 0 400 0.430 2 400 0.465 3 0.74 x 10* 1 x 104 1 x 104 1 x 10* l x 10* 0 13.8 0 4.7 6.5 Controls: DMS0 (20 ul/nl) - Solvent H20 (20 ul/ml) EMS (200 ug/nl) - 211 246 180 400 400 400 0.528 0.615 0.450 3 2 124 l x 10^ 1 x 10* l x 104 5.7 3.3 275.6C (Epoxy Resin ERL-4221] (X, v/v) 200.0 x 10~4 312 100.0 x 10"4 333 50.0 x 10-* 313 25.0 x 10-4 289 12.5 x 10"4 353 With S9 Activation 400 0.780 2 400 0.832 15 400 0.782 15 400 0.722 3 400 0.882 14 1 x 10* l x 106 1 x 104 1 x 10* 1 x 10* 2.6 18.0 19.2 4.2 15.9 Controls: DHSO (20 ul/ml) - Solvent H20 (20 ul/ml) - OKU (3700 ug/nl) OKM (740 ug/ml) - 362 386 198 263 400 0.905 18 400 0.965 5 400 0.495 63 400 0.658 30 1 x 104 1 x 104 1 x 104 1 x 104 19.9 5.2 127.3 45.6* f! *Total f mutant colonies per 104 cells plated divided by viable fraction. Statistical significance above solvent control: a: 0.05 > p > 0.01; c: p < 0.001. No superscript indicates p > 0.05. Abbreviations: H20 - water; S-9 - liver homogenate; DMS0 - dinethylsulfoxide; EMS - ethylaethanesulfonate; DHN - diaethylnitrosamlne. WPC/1103 in t (.V GO & 1 Table 5 Slater Chromatid Exchange (SCE) Assay: Inductionof SCE*a by Epoxy Resin (ERL-4221) Without S9 Metabolic Activation 5 Hour Treatment Test Chemicals Total I of Chromosomes Total # of SCE SCE/Cell^ Mean Huaber SCE/Chromosome^ + S.D. [Epoxy Resin (ERL-4221)] (I, v/v) 100.0 x 10-4 50.0 x 10** 25.0 x 10-* 12.5 x 10"4 6.25 x 10-4 3.125 x 10-4 Controls OMSO (5 ul/ml) - Solvent H2O (5 ul/ml) EMS (100 ug/ml) - 280 307 301 303 297 299 291 294 298 174 11.60 0.626 + 0.212 572 38.13 1.869 + 0.301 415 27.67 1.379 + 0.184 316 21.07 1.041 + 0.316 220 14.67 0.743 + 0.233 233 15.53 0.779 + 0.238 182 12.13 0.628 + 0.214 200 13.33 0.680 + 0.169 423 28.20 1.417 + 0.242 ^Fifteen cells examined per dose level. ^Mean value of SCE/chromosome determined from the values of the Individual cells examined. ^Statistical significance above solvent control: c: p < 0.001 NS: p > 0.05. Data analyzed by Student's t-test. Abbreviations: H2O - water; S9 - liver homogenate; DMSO - dlmethylaulfoxide; EMS - ethylmethanesulfonate; S.D. - standard deviation WPC/1103-3 00 8 Significance Above Solvent Control^ NS c c c NS NS __ NS c * ut oUl 617 Table 6 Unscheduled DMA Synthesis in Hepatocytea Iron Rat Liver Huclear-bound label? all DPM values are calculated fro* nuclei per 10& viable hepatocytes. Each average la calculated fro* duplicate saaples, except for DHSO which waa done in quadruplicate. That Chealcal Solvent - DHSO Positive Controls; * - NQO DMN Concentration 3.0Z 3.0 ug/al 1.0 ug/al ug/al 1000 300 100 30 10 1 ug/al ug/al ug/al ug/al ug/al ug/al Radioactivity in Nuclei Avg. DPM 4- S.D. 6995 + 909 16791 + 1307 10913 + 4668 9623 + 456 9286 + 748 8234 + 877 12230 + 927 10570 + 2484 9390 4- 3683 10679 + 474 X of Solvent Control + S.D. 100.0X + 13.OX 240.IX 4- 18.7Z 156.OX + 66.7Z 137.6X + 6.52 132.8Z 4- 10.71 117.7X + 12.5Z 174.ex + 13.3X 151.IX 4- 35.5X 134.3X + 52.7X 152.7X + 6.82 Significance Above Solvent Contr 1* - c a MS NS NS b NS MS NS Test Chealcal: [Epoxy Resin ERL-4221] (Z, v/v) 1000 x io-*x 300 x 10"*X 100 x 10~*X 30 x 10~4X 10 x 10~*X 1.0 x io-*x 578 + 207 6636 + 292 9458 + 334 10322 4- 2777 10547 + 478 11600 + 1399 8.3Z 4- 3.OX 94.9X 4- 4.2X 135.2X 4- 4.8X 147.62 4- 39.7X 150.8X 4- 6.8Z 165.82 4- 20.0X MS NS MS NS MS a Report 44-ios Page 19 ^Statistical significance above solvent control: a: 0.05 > p > 0.01; b: 0.01 > p > 0.001; c: p < 0.001; MS: p > 0.05. Data analyzed by Duncan's Multiple Range Analysis. Abbreviations: DHSO - dl*ethylsulfoxide; 4-NQ0 - 4-nitroquinoline oxide; DMN - diaethylnitrosaalne; Dm - disintegrations per ainute; S.D. - standard deviation NFC/1103-3 tv a 0^8 162 Table 7 Unscheduled DMA Synthesis In lie pa toe ytea froa Rat Liver DHA-bound label; all DPM values are calculated froa DNA precipitated per 10** viable hepatocytea. Each average la calculated froa duplicate saaples, except for DMSO which was done In quadruplicate. II Test Chealcal Solvent - ntSO Concentration ----- J-QJ----------------------- Radioactivity X of Solvent in DNA Control Avg. DPM + S.D. + S.D. 5537" + 3379----------- ----------ioo.oX + 33761 Positive Controls: A - NQO 3.0 ug/al 1.0 ug/al 0.3 ug/al 16720 + 940 12011 3654 10219 + 757 195.OX + 11.OX 140.7X + 42.8X 119.7X + 8.9X DMN 1000 ug/al 9818 -f 786 115.OX + 9.2X 300 ug/al 14608 + 1669 171.IX + 19.5X 100 ug/al 13195 + 661 154.6X + 8.0X 30 ug/al 13322 + 4554 156.OX + 53.3X 10 ug/al 12198 + 3117 142.9X + 36.5X 1 ug/al 16243 + 1690 190.3X + 19.8X Test Chealcal: ^Epoxy Resin ERL-4221] (X, v/v) 1000 x io-*x 300 x 10"*X 100 x lO^X 10 x lO-^X 10 x lO-^X 1.0 x 10"^ X 495 + 289 7864 + 307 10882 + 1198 14760 + 1453 12172 + 242 14629 + 993 5.8X + 3.4X 92.IX + 3.6X 127.5X + 14.OX 172.9X + 17.OX 142.6X + 2.8X 171.4X + 11.6X Significance Above Solvent Control* -- -b NS NS NS a a a NS b NS NS NS a NS . a 1Statistical significance above solvent control: a: 0.05 > P > 0.01; b: 0.01 > p > 0.001; NS: p > 0.05. Data analyzed by Duncan's Multiple Range Analysis. Abbreviations: DMSO - diaethylaulfoxide; 4-NQ0 - 4-nitroquinollne oxide; DMN - dlaethylnitrosaalne; DIM - disintegrations per ainute; S.D. - standard deviation WPC/1103-3 offig Page 20 i t ((. APPENDIX I Fa|i 1 of 5 1823 .i Chlpsi Hamster Ovary (CHO) Hutatlon Aaiay Theoretical Basis Mutation la a heritable alcaradon In a call in which a gaoa specifying tha ganatic coda for a spaclfle protaln la aodlflad la structure and/or funcclon. Mutations, lnducad by chemical or physical agaaca, of tha BCPRT (hypoxanthinequanlna phosphorlbosyltransfaraaa) gana aay ba dataetad by tha growth of eolonlas of "mutant",calls which ara raalstant to tha purlna analogs 6-thloguanlne (TC) or 8-azaguanloa. Normal calls contain a functional BCPRT enzyme which phoaphorylatas TC and allows its incorporation Into DNA causing tha ealls to die. Mutant calls with a non-functional BGPKT enzyme are unabla to phospborylata or Incorporate TG, thus survive and grow In Its presence. Tha CHO nutation test Is an assay which dstaets "forward nutations" frost TC-aansitivlty to TC-realstanca caused by a direct loss of ths activity of the HCFRX enzyme (HGPRX*" BCPRT*)* An assessment of tha ability of several hundred agents to causa gana nutations In vitro lndleatas that tha (SO nutation assay provides a reasonable estinate of"the potential genetic activity of the test chemical. Methods Call Culture Procedurest CHO ealls used in these studies were obtained fron Abrahsn Bala at Oak Ridge National Laboratory with tha designation CH0-R1-BH4-Dl (or sinply CHO for report purposes). Calls are aalntained In active growth by subculturing 2 to 3 tlaas/veak In antibiotic-free, Han's Modified P12 Medina suppleaented with 10Z (v/v) heat-inactivated, fatal bovine sera (712-10), and lacking In hypoxanthine. For traatnant of calls without metabolic activation, 712 nadlua with 30 unlts/nl of penicillin, 50 ug/al straptoaycln and 5X (v/v) of dialyzed bovine sarua (712-03) is used. For treataants incorporating an S9 aatabolic activation syatea. Identical nadlua, but without sarua, is employed, for d termination of nutant frequencies, 712-DS nadlua containing 2.0 ug/al TG (6-thloguanlna) la used as a "selective nadlua." Call numbers ara detemlned routinely with a Coulter Model 7 electronic call counter which Is standardized periodically with a pro-counted suspension of latex beads. Prasanca of Mycoplasma call contaminants is detemlned by a microscopic fluorescence assay employing Boechst 33258 dye. All culture procedures and treataants with test chemicals ara perforaed under aseptic conditions in a laminar-flow, biohazard hood. Positive and Negative Controls t Sterile water or glass-dlstllled dimethylsulfoxide (DM&o) are tha usual solvents for test ehaaleals and the respective solvent is tested as a control at tha naxlaun concentration used to add the test agent* Dlaatbylnltrosaalns (DMH) or ethylaathanesulfonate (ZMS) are used as positive control outagens for taste with or without an S9 aatabolic activation system, respectively. Mutation frequencies obtained with concurrent positive and negative controls ara used as tha basis for aonltorlng tha sensitivity and stability f ths CEO nutation test system. Comparison of concurrent control values with historical controls la used to dallnaata tha range f acceptable variations la tha test system. UCC 062620 1824 Appendix I Page 2 of 5 Metabolic Activation; Rat liver, S9 homogenate prepared from Arochlor-1254 Induced, Sprague-Dawley, sale rats la purchased froa Litton Blonetles, Kensington, KD. Each lot of liver hoaogenate la prescreened for metabolic capability to activate DMN In our laboratory before use In the testing program. The eoaplete S9 aetabollc activation systea contains the following: 8 uaoles/ol MgCl2> 33 uaoles/al KC1, 3 uaoles/al glucose-6-phosphate, 4 uaoles/al NADP-oxidlzed (nicotinamide adenine dinucleotide phosphate), 100 uaoles/al NS2BP04, and between 300 to 4000 ug/al of S9 protein (depending on aetabolle activity); a volume of 1*0 al of the complete alxture of the above reagents Is added to each 4.0 ml of culture medium. Dose Selection: Toxicity of the test chemical Is determined`prior to assessment of mutagenic potential to select doses which produce a maximum of 80 to 90Z call killing. Cytotoxicity Is determined by either of the following two methods: (1) Clonal assay - 200 to 400 CB0 cells are exposed to a minimum of five dose levels of the test agent at concentrations froa 0.1Z to 3 x 10"*Z (by weight or volume, as appropriate) with and without the presence of a metabolic activation system. The number of cells which survive the treatment is determined by counting the number of colonies produced after a 7- to 8-day incubation period (37*C) In comparison with the colonies formed by cells treated only with appropriate concentrations of solvent (generally 20 nl/al). (2) Growth Inhibition - 5 x 10s cells In 25 cm? culture flasks are treated for 5 hours with a minimum of five test concentrations both with and without S9 metabolic activation. Following treatment the cells are rinsed, fresh F12-D5 medium Is added and the flasks are Incubated for an additional 18 to 24 hours. Cytotoxicity Is determined by comparing the relative number of cells 1a control (untreated cells) and In cells treated with vsrloua concentrations of the test agent. If no cytotoxicity is evident at the highest concentrations In the cytotoxicity tests, the test Is either repeated at higher concentrations, or mutation tasting Is performed with a greater number of treatment flasks starting at higher dose levels. If marked toxicity is evident even at the lowest dose, the cytotoxicity test Is repeated at a concentration range of 3x10** to 3x10*3 percent by weight or volume, as appropriate. Dose levels idtlch are moderately toxic but permit survival of at least 10 to 20Z of the cells, In comparison to the solvent control, are selected as the maximum dose, and at least four additional one-half dilutions are tested for induction of mutations. If cytotoxicity data are equivocal, a total of 5 to 8 one-half dilutions of the selected, maximum concentration are used to treat cells; but only the highest five concentrations which permit survival of a sufficient number of cells are assessed for mutation induction. 0' Appendix I Fill 3 of 3 1825 Chemical tuples ere sterilized by membrane fll tree Ion when nleroblologlesl tests lndleste this is required to sssure sterility* Liquid test sgents are tested on a percentage by volume basis* Solid chuieals are dissolved In an appropriate solvent by asking a 10 to 20Z stock solution (by weight) and subsequent dilutions are aada from this stock on a volume/volume basis* Treatment with Test Chemicalst For tests of chuieals which may act directly without incorporation of an S9 metabolic activation system, 5xl05 cells are Inoculated 20 to 24 hours prior to treatment Into 25 cm* culture flasks containing F12-D5 medium and Incubated at 37*C In a 5 to 6Z CO2 atmosphere. Appropriate concentrations Ox the test agent or control chemicals are added to the cells and cultures are treated for 5 hr at 37aC. The medium and test agents are ruoved by suction, cells are rinsed once or twice and fresh F12-D5 medium Is added. The calls are allowed a period of 20 to 24 hours of recovery from treatment before survival Is determined. Treatment of cells for testing of chemleals which require metabolic activation for mutagenic capacity Is performed Identically with the procedure above, with the exception that F12 medium without serum and containing 1.0 ml of S9 activation mixture per 4.0 ml f medium Is uployed. Determination of Cytotoxicity; The relative survival of treated cells. In comparison to solvent controls, is determined one day after the exposure to the test agents. The level of cytotoxicity is often correlated with the mutation frequencies induced by known chemical mutagens. Thus, excessive cytotoxicity may Idll both normal calls and mutants and may depress the actual mutation frequencies; insufficient cytotoxicity may Indicate an insufficient concentration f the test agent was employed. The colony-forming potential of 100 to 200 treated calls is used as the measure of treatment-Induced cytotoxicity. Survival values which Indicate the cytotoxic effects of the test agents are included In reports in tabular form. Statistical analyses are not performed on these data, since they are only useful to assess whether appropriate doses were employed and are not used to calculate mutation frequencies. Determination of Mutant Induction: On days 1, 3 and 6 (or alternatively 1, 4 and 6)after treatment with the various test agents, approximately SxlO5 cells are subeultured in 100 an tissue culture dishes in F12-D5 medium and incubated at 37'C In a 3 to 6Z CO2 atmosphere. After a total of 7 days to allow "expression" of the mutant phenotype, eells are dissociated with 0.05 to 0.075Z trypsin, counted and plated at a concentration of 2.5x loVdlsh in four culture dishes (lx 10* total eells) which each contain 5 ml of F12-D5 (TG) 'selective medium. At this time, eells are diluted and 100 cells/dish are added to four culture plates containing 712-D5 medium (without TG) to aaaess viability (plating efficiency) of the treated cell population and to determine the surviving fraction. All cultures are then ineubated for an additional 6 to 8 days to allow growth of eells; medium is then discarded and colonies are fixed and stained for counting. The number of colonies In selection plates and In the viability test are counted by electronic methods, cheeked by manual counts and data are recorded both as total mutants, autanta/106 total eells and mutants/lO* viable cells. 1826 Appendix I Fag* 4 of 5 Statistic*! Analyses; Uniform statistical procaduraa Co evaluate in vitro utatlon data have not been developed. The distribution of mutation frequencies from historical controls in at least two laboratories Indicates Chat the fre quency distribution and variances encountered do not justify the use of para metric analyses unless data is transformed before application of standard para metric tests* Analysis of mutation frequencies in the CBO test follow the pro cedure of Irr and Snee (Reference 4) which employs the Box-Cox Transformation (Reference 5) to transform data before parametric analyses. The mutation fre quency for each plate is increased by 1.0 (to eliminate xaros) and raised to the 0.15 power. Experience with historical negative control data in our laboratory indicates that a normal probability distribution of the data suitable for para metric analyses is achieved by this transformation. Parametric analysis of mu tation data by the Student's t-teat is performed with the transformed data. The degree of statistical significance for the mutation values are indicative of a difference from the concurrent solvent control, but these statistical indicators must be viewed conservatively until additional historical control data are available. Interpretation of Data; The criteria for Interpretation of the test results as a positive or negative response depend upon both the level of statistical significance from the concurrent control and the evidence of a dose-response following treatment. When a definite dose-response relationship is not evident but one or more marginally significant values are obtained, a careful examina tion of the data from the concurrent positive and negative controls and compari sons to historical control data are used to evaluate the possible significance of the responses. Historical control data indicate that a spontaneous mutation frequency in CEO cells of approximately 4 to 5 mutants/10 viable cells, with a range of 0 to 25 mutants/10 viable cells, can be obtained in the absence of mutagenic treatment. Statistical comparisons against unusually high or low spontaneous controls are subjectively scrutinized in respect to the above variability. UCC 062623 Appendix I Pt 5 of 3 1827 " jr;tT'iiTt'r--' .......... ** t *? *; mm.. *.. a,i.. A 4* ** So*` lutlxtlcal EvaXuxtion of Mu^-enleltv m vm^^S (Urn; &?hFv ui tk* ^ ^'-^r WC/1033 ucc 062624 1828 Page 1 of 4 AfFENSIX XI Determination of Sister Chromatid Exchange (SCE) Frequencies In Chlnaaa Hamster Ovary (CBO) Calla Xn Vitro Thaoratlcal Baala Exchanges of ganatle notarial between tha individual arna of a chromosome (l.a. alatar chromatids) ara thought to arlaa from braakaga and phyalcal Interehangaa in tha DN4 of a call during call division. An ineraasa in tha frequency of auch intarehangaa batwaan alatar chromatids can bo obaarvad in ealla traatad with phyalcal or chemical mutagenic agents, or In ealla axpoaad to aany auapect or provan human carclnogana. Thua, analysis of SCE fraquanciaa In ealla traatad with a taat agent haa baan auggaatod aa a aanaltlvo aereanlng teat for potential autaganle/carcinogenic chealeala Tha oethod used In our atudy to visualize SCE'a in CHO ealla grown In cul ture la baaed on the procedure described by Perry and ffolff (1974). A standard concentration of 3.0 ug/al of bromodeoxyurldlne (BrdU) was used In the growth medium to allow a visualization of SCE'a after two eall divisions in the pre sence of BrdU. Staining of chromosomes with 3.0 ug/al of 33258-Boeehst fluore scent dye, exposure to light and Glemsa staining was used to differentiate chromatids for SCE analysis. Methods Cell Culture Procedures; Chinese hamster ovary (GSO) cells were obtained from Abraham Bale at Oak Ridge National Laboratory with the designation CHO-El BB4-01 (referred to simply as CHO for report purposes). CHO cells are maintain ed In active growth by 2 to 3 weekly subcultures into fresh antibiotic-free. Baa's F12 (modified) medium fortified with 10Z (v/v) of heat-inactivated fetal bovine serum and lacking hypoxanthlna and thymidine. Cell concentrations are determined routinely with a Coulter* Model-F electronic call counter calibrated with a precounted suspension of latex beads. All cell culture procedures prior to final harvesting of cells for chromosome preparations are performed under aseptic conditions In a laminar flow, biohazard hood. Presence of Mycoplasma cell contaminants Is determined using a fluorescent microscopic assay employing Boechst 33238 dye. For treatments with test chemicals without S9 metabolic activation, modified F12 medium is used with 30 units/ml of penicillin, 30 ug/al streptomycin end 5Z (v/v) of heat-inactivated, dialysed fetal bovine serum (712-D3). Identical medium but without serum is used for treatments incorporating an S9 metabolic activation system. * Positive and Negative Controls; Sterile water or glass-distilled dimethyl sulfoxide (DMSO) are the usual solvents used for test ehemleals and the respective solvent la tested as a control at the maximum concentration used to add the test agent. Olaathylnltrosamine (DMN) and ethylmethanesulfonata (EMS) are used as positive control mutagens for testa with or without the addition of aa 59 metabolic activation system, respectively. Basults from treatments with concurrent control agents are used as a basis f comparison and for demonstrating the sensitivity and stability of the SCE test system. Comparison of concurrent control values with hist rlcal controls is used to delineate the range of acceptable variations In the test system. Appendix II Pag* 2 of 4 Metabolic Activation* Sat llvat S9 homogenate (prepared fro Arochlor 1254 Induced, Sprague-Dawiey, sale rata) la purchaaed froa Litton Blonetlce, Kensington, MD. Each lot of liver hooogenate la preacreened for activity In our laboratory before itae In the teatlng program. The complete S9 metabolic activation ayetaa containg the following* 8 umolss/ml MgCl2, 33 umolea/ml KC1, 3 uoolea/al SCI, 5 uoolea/ol glucosa-6-phosphete, 4 uoolea/ol NADPoxidized fora (nicotinamide adenine dinucleotide phoaphate), 100 umolea/ol NejHPt^ and between 500 to 4000 ug/al of 39 protein (dapending on metabolic activity)* A volume of 1.0 al of the complete mixture of the above raagantt 1* added to each 4.0 ml of culture medium. Doae Selection: Toxicity of the teat chemical la determined prior to eaaeaament of mutagenic potential to ealect doaaa which produce a maximum of 80 to 90Z cell killing. Cytotoxicity la determined by either of the following two methoda aa pert of the CB0 mutation teatlng procedure: (1) Clonal a***y - 200 to 400 CEO cella are axpoeed to a'minimum of five done level* of tne teat agent at eoncentratlona froa 0.1Z to 3 x 10"*X (by weight or volume, aa appropriate) with and without the pretence of a metabolic activation ayatam. The number of cell* which aurvlve the treatment la determined by counting the number of colonies produced after a 7- to 8-day incubation period (37*C) In comparison with the colonies formed by cella treated only with appropriate concentrations of solvent (generally 20 ul/ml). (2) Growth Inhibition 5 x 105 cells in 25 cm? culture flasks era treated for 5 hours with a minimum of five test concentrations both with and without S9 metabolic activation, following treatment the cella ars rinsed, fresh t12-05 medium la added end the flasks are Incubated for an additional 18 to 24 hours. Cytotoxicity la determined by compering the relative number of cells In control (untreated cells) and in cells treated with various concentrations of the test agent. If no cytotoxicity la evident at the highest concentrations in the cytotoxicity tests, the test Is elthar repeated at higher concentrations, or mutation tasting is performed with a greater number of traatment flasks starting at higher dose levels. If marked toxicity is evident even at the lowest dose, tha cytotoxicity test Is repaated at a concentration range of 3x10"* to 3x10*3 percent by volume. Dose levels which are moderately toxic but permit survival of at least 40 to 50Z of the calls, In comparison to the solvent control, are selected as tha maximum dose, and at least four additional one-half dilutions are tested for induction of mutations. If cytotoxicity data are equivocal, a total of 5 to 8 one-half dilutions of tha selected, maximum concentration are used to treat cells; but only the highest five concentrations which permit survival of a sufficient number of mitotic ealls with SCS staining, are evaluated for SCE Induction. Chemical samples are sterilised by membrane filtration when microbiological testa Indicate this Is required to assure sterility. Liquid test agents are tested on a percentage by volume basis. Solid chamleals are dissolved In an appropriate solvent by making a 10 to 20Z stock golutl n (by weight) and subsequent dilutions are made froa this stock on a voluma/voluae basis. 1830 Appendix II Page 3 of A Treatment With Teat Chemical*; Testing of chemicals for direct mutagenic action (without sd metabolic activation) is performed first. For chemicals with clearly positive mutagenic capabilities by direct action, testing with metabolic activation is generally not performed. For testing direct acting chemicals for SCE induction, between 1 to 2 a 10 cells are plated into 75 cm* culture flasks in F12-D5 medium at laaat 20 hrs prior to treatment andincubatad at 37aC In a 5 to 6Z CO2 atmosphere. Appropriate concentrations of the test agent or control chemicals are added to the cells and 3 ug/ml BrdU is added to all flasks. Calls are treated with test agents for 5 hrs, madia la then removed by auction, cells are rinsed with buffered, physiological salt solution and fresh medium containing 3 ug/ml BrdU is added for at least 24 hrs of additional incubation at 37*C to allow two rounds of call division. Cells are harvested and chromosomes are prepared for SCE staining. Treatment of cells for testing of chemicals which require metabolic activation for mutagenic effectiveness is performed similarly as for treatments without activation, except for three modifications: 1. Before treatment with the test agents, P12*D5 medium is removed and F12 medium without sarum is added. 2. S9 metabolic activation mixture is added to each flask (including solvent and positive controls) before addition of test agents. 3. Cells are treated for a total of 2 hrs (rather than 5 hrs) and then incubated for 38 to 42 additional hours before harvest for chromosome preparation. Preparation of Chromosomes: Coleemid* (0.1 ug/ml) or Colehicine (0.2 ug/ml) Is added to culture flasks 1 to 2 hrs prior to harvesting to arrest calls in mitosis. Celia are then removed from flasks, after a brief Incubation with 0.012 DUCO trypsin, suspended in 0.075M EC1 (hypotonic) solution and incubated f r 15 to 20 min at 37*C. Cells are centrifuged, fixed with 3 or 4 changes of Carnoy's fixative (3:1 methanol acetic acid) and chromosome spreads are prepared from calls suspended In a small volume of fixative. One slide/dose level is prepared, but fixed cells are saved if needed for preparation of additional slides. Chromosomes era stained for SCE's by treatment with 5.0 ug/ml of Boeehst 33258 dye for 20 min, rinsed in distilled water, immersed in Sorenson's buffer and exposed to a high intensity sunlamp for 15 to 30 min., as required. Irradiated chromosomes are stained in Curr's glemsa (diluted 1:25 with water), rinsed in water and dried before application of eovarslips. Examination of SCE's: All slides are coded and raad in a blind fashion without indication of tha specific treatment or concentration of the test agent. The number of chromosomes and the number of SCE's in a minimum of 15 cells are recorded for each d se level. The mean number of SCI/cell and SCE/chromosome are calculated and recorded. Slides are decoded only fter examination of all slides in the experiment has been completed. Appendix II Pag* 4 of 4 1831 Statistical Analyses: Data art analysed by appropriate parametric atatietlcal procedures which follow BRRC standard operating procedures for analyses of data* Significance values and the statistical teat employed are shoim for data summarized in tabular fora* Interpretation of Data: The criteria for evaluation of a positive or negetlve response depend both on the level of statistical significance and subjec tive analyses of concurrent and historical control data* The key determinant la whether a dose-depandent Increase In SCE's la Induced by the test agent* When no dear dose-response relationship la evident and when one or wore responses of marginal statistical significance are obtained, a careful examination of the data In comparison to the concurrent controls and historical data base Is neces sary. Testing may be repeated to clarify unusual responses. If data for the concurrent positive or negative controls suggest a defect In the original ex periment. Overall assessment will also rely on corroborating data from the other tests In the testing battery. Clearly positive responses will Include any of the following: (1) Doubling in the SGE frequency at a minimus of two of the five concentrations tested; (11) Statistically significant responses of p < 0.05 at three concentrations or at 2 concentrations if p < 0.01; (111) Induction of a statistically significant, dose-related increase In the number of SCZ. General References 1. Perry, P. and S. Nolff. Hew glemsa method for differential staining of sister chromatids. Batura, 251 (1974), 156-158. 2. Lett, S. A., J. V. Allen, V. E. Rogers and L. A. Juergens. In vitro and in vivo analysis of slater chromatid exchange formation, pp 275-291 in Handbook of Mutagenicity Test Procedures, ed. KUbay, B. J., et al. Elsevier Publ. Co. (1979). 3. Carrano, A. V., L. H. Thompson, P. A. Uadi and J. I. Mlnkler. Slater chromatid exchange as an Indicator of mutagenesis. Nature, 271, (1$)5), 511-55*. 4. Galloway, S. M. and S. NoIff. The relation between chemically Induced 5. Snedecor, G. N. and V. 6. Cochran. Statistical Methods, 6th Ed., Iowa State Uhlv. Press, dues, lows (1967). WPC/1033 1832 I I 4 APPENDIX III Page 1 of 3 Unscheduled DNA Synthesis (OPS) In Hapetocytas from Rat Liver / TheoreticalBella Chemicals may-Interact with both the cellular components and the genetic material of a cell (e.g. DNA and SNA) because of thalr electrophilic nature or by conversion Into reactive electrophiles by the metabolic enzymes of the cell. Damage to the DNA of a cell can result in cell death* mutation or, theoreti cally, carcinogenic transformation* Studies of agents which are capable of reacting and damaging the cellular DNA have suggested that such methods may be useful as a sensitive screening test for detecting potential mutagenic/ carcinogenic chemical properties* Detection of the relatively small amounts of DNA damage Induced by chemical treatment requires a cellular system in which normal* semi-conservative DNA rep lication, which occurs during cell division. Is inhibited. The system employed for the present study uses a suspension culture of primary hepatocyte cells Iso lated from rat liver according to the general methods of Seglen (1973) end Williams (1976). Bepatocytes do not normally divide in the minimal culture me dium employed and stimulation of "unscheduled" incorporation of radioactive DNA precursors can be detected by scintillation spectrometry. The stimulation of incorporation of erltlated thymidine Into both purified hepatocyte nuclei and DNA Is used as the Indicator of chemically induced DNA damage. The amount of unscheduled DNA synthesis (UDS) following treatment is compared with both con current positive and negative controls as well as with historical data for similar tests. Methods Preparation of Hepatocyte Suspensionst Hilltop-Wistar albino rats are anesthetized with Metafane(R). The abdominal cavity Is surgically exposed and 1230 units of heparin Is Injected intravenously. A catheter is Inserted Into the portal vein and warm Hanks Balanced Salt Solution (HBSS) la pumped Into the vein and through the liver. This first solution contains heparin and EGTA, [ethylene glycol-bla-(beta-amlnoethyl-ether)N,N-tetracetlc add], which preferentially chelates calcium; the solution contains no magnesium or calcium. After the liver Is blanched, a second solution of HBSS containing 60 unlts/ml of collagenasa is perfused. This solution Is pumped through the liver until the liver Is digested. The liver is then removed and the cells are freed in cold medium 199 by combing through the lobes with a sterile metal comb. The eell suspension Is passed through two nylon meshes to remove eell dumps and the cells are washed once at low centrifugation speed. After resuspension in medium 199* equal volumes of cells and 0.4Z trypan blue are mixed together and the cell viability and number of viable cells per ml is determined microscopically. Preincubation of Bepatocytes; Approximately 2 x 10$ viable bepatocytes are added to 3 ml medium 199 containing 10 mM hydroxyurea and 30 mM Hepes (N-2-hydroxysthy1 piperazlne-N-2 ethane sulfonic acid) buffer. After the eells are dispensed Into the tubes* they are placed on a rocker platform and are Incubated t 37*C for 1 hour. Although bepatocytes do not normally divide In culture, medium 199 which lacks serum and contains hydroxyurea Is used to further block semi-conservative DNA synthesis. Thus, any radioactive thymidine Incorporated Into the nuclei la expected to result fr m repair r unscheduled DNA synthesis. 1835 i Appendix III t Fag* 2 of 3 / Selection of Doses of Teat Chemical Initially, the following concentre* tlonax i0"3jT(6y volume) are teatedi 100, 30, 10, 3, 1, and 0.1. If thcae concentratlona prove to be cytotoxic, or If additional Information la available fro* ocher In vitro teata aa to the proper doae levela, then an appropriate aerlea of concentratlona la uaad over a 3-log range of concentratlona. Treatment of Hepatocytes After prelneubation, 25 mlcroCurles of trltlated thymidine <20 Curies/aliliaole) la added to each tube. The teat chealcal and poaltlva controla are diluted In an appropriate eolvent and they are than added to each labeled tube. Generally, at leaac alx concentratlona of the teat chemical over a 3-log range of concentratlona are teated and each concentration la run In duplicate. The tubes are returned to the rocker platform for a 2-hour exposure at 37#C. Positive and Negative Controla; 4-nitroquinolioe oxide (NQ0), a direct* acting mutagen, which' lnducesr 0V-"cype DNA repair and dlmethylnltrosamlne (DM0, which requlrea metabolic activation by microsomal enxymea for activity, are run in duplicate as positive control chemicals. The solvent control Is run In quadruplicate and consists of 100 to 150 mlcrollters (concentration specified In Individual reports) of the solvent used to dilute the sample. Dimethyl* sulfoxide (DMS0) or water are the usual solvents for test chemicals. Harvestt At the end of Incubation with the test agent, the cells arc centrifuged from the medium at 200 x g at 5*C. The cells are rinsed once in 5 ml of cold medium 199 and are resuspended In 0.25Z Triton X-100, 51 citric acid and 3 mK MgClj, a lysing solution which liberates the nuclei. The nuclei are rinsed once In this solution and resuspended In 0.25 M sucrose, 2.5Z citric acid and 3 aM MgClj. The nuclei are then centrifuged at 600 x g for 10 min at 5*C and resuspended In 2 ml of the lysing solution. Determination of NuclearBound Label: To measure the amount of radioactive thymidine Incorporated Into'the nuclei, 0.25 ml of the nuclear suspension Is mixed with 1.0 mg of DCS tissue solubiliser la a scintillation vial. Ten ml of Dlmllume* scintillation cocktail la added and the radioactive disintegrations per minute (DPM) are determined by counting twice In a scintillation counter for ten minutes. The measured DFM are then used to calculate the DPM/10 viable hepatocytes presented on tables. Determination of DMA-Bound Label? The amount of radioactive thymidine In* corporate! into DNA is quantitated in DNA Isolated and precipitated from 1.00 to 1*25 x 10* viable hepatocytes. To 1.25 ml of the nuclear suspension, 2.75 ml of 1Z sodium dodeeyl sulfate (SD8) and 5 mK Ethylenedlamlnetetraaeetle Acid (EDIA) la added to lyse the nuclei. The DNA la precipitated from this solution with 4 ml of Ice-cold 10Z trichloroacetic add (TCA) and the sample tubes are Incubated at 0#C for at least 30 minutes. The solution la then poured onto Whatman glass fiber filters under vacuum and the tubes and filters are washed twice with cold 5Z TCA. Finally, each filter la rinsed once with methanol, dried and placed In a scintillation vial. The filters are Incubated at 50aC for 1 hour with 1 ml of a diluted solution of DCS tissue solubilizer; prepared by adding 1 part solubiliser to 2 parts of Dlailuaee cocktail. Diuilume* Is then added to each vial and the vlala are counted twice la a scintillation counter for tea minutes 1834 Appendix III Pigi 3 of 3 Statistical Analysis; The average OPM la calculated for each dote level and the controla end final reaulta are expreaaed aa DPM/10 viable hepatocytes. Data are alao expreaaed aa a percent of the advent control for purpoaea of comparison. The original data are atatiatieally analyzed by the appropriate paraaetric teat, following the BRRC atandard procedure* for atatlatlcal analyaea and the teat(a) employed ia indicated on the reapectlve tables* Conparlaon between the nean for each dose level with the 9SZ confidence limits of the historical solvent control may also be used In some case* to assess the potential biological significance of the data* ^Testing may be repeated to clarify unusual responses. If data with the concurrent controls suggest a defect In the original experiment. Interpretation of Results: The classification of a chemical as a positive, active agent depends upon the production of a statistically significant, dose-related increase In the amount of DOS activity. If a definite dose-response relationship is not evident, or when a few Increases with marginal statistical significance are obtained, comparison of the responses to historical control data provides a meaningful assessment of the possibility for random variations which may be statistically significant only in relation to the concurrent control* A key determinant of the reliability of the UDS data Is tbs detection of a similar response with both DMA and Isolated nuclei determined at two or three consecutive concentrations* General References X. Lampidls, T. J. and J* B. little* The Enhancement of DV-Induced Unscheduled DMA Synthesis by Hydroxyurea. Experimental Cell Research. 110, (1977), 41-46. ----- 2. Mnramatsu, M. Isolation of Nuclei and Nucleoli. In: Methods In Call Physiology, Vol* IV. 1970. Editor: 0* M* Prescott. Academic Press, New York. 3. Seglan, P. 0. Preparation of Rat Liver Cells. III. Enzymatic Requirements for Tissue Dispersion. Experimental Cell Research, 82, (1973), 391-398. " A. Williams, G. M. Detection of Chemical Carcinogens by Unscheduled DNA 3. Williams, G. M. The Use of Liver Epithelial Cultures for the Study of Chemical Carcinogenesis. American Journal of Pathology, 85, (l76), 739-753. 6. Williams, G. M. and M. P. Laspla. The Detection of Various Nitrosamlnes la the Hepatocyt* Primary Culture/DNA Repair Test. Cancer Letters. 6. (1979)7 l99-i06. *" 7. Snedecor, G. W. and W. G. Cochran. Statistical Methods. 6th edition, Iowa State University Press, Ames, Iowa (16/'). WPC/1033 APPENDIX IP "4.0*30 Import *3-105 Physical >nd Chemical Charactsristics of the Teat Sample CEP No CAS No.: Chemical Name: Trad* Name and/or Synonyms: Molecular Weight: Formula: *2-136 2386-87-0 3, *-Epoxyeyclohaxylaethyl-3, 4 Epoxyeyelohexylcarboxylate BAJOELITE* Cycloaliphatic resin EXL-4221 (previously EP-221) 252.30 c1*h20* i Molecular Structure: .-Q"' *' -"Q> h< c Specific Gravity (9 20*C): Bolling Point: Solubility In H2O (Z by vt): 1.1725 35**C (669.2*P) 0.03 (at 25*C) Purity: Hot available; commercial sample tested Vapor Pressure (9 20*C): pB: Plash Point: Stability: Incompatlblllty: Appearances and Odor: < 0.01 am Hg Not available 2*5*P Closed eup Stable, avoid heating over 100*? Avoid adds, amines, strong bases low viscosity liquid; characteristic odor Disposal: Dilute with Inert solvent and Incinerate. Small spills may be flushed with water; larger spills absorbed and burned Protective Measures: Use goggles, plastic gloves and exhaust ventilation. Bealth Bacard: Avoid skin and eye contact. No effects of overexposure are currently known. in ). ucc 062632