Document K6xNVpb16jDXYYmDB5de546V0

1ST BUSHY RUN RESEARCH CENTER R. 0. 4. Mellon Road, Export, Pennsylvania 15632 Taiaphona (412) 327-1020 CONFIDENTIAL; Not to be released outside UCC without the written consent of the UCC component sponsoring the work. Project Beport 43-105 (Amendment) Tel; (412) 327-1020 January 14, 1981 First Amendment to BAKELITEB Cycloaliphatic Epoxy Resin ERL-4221 In Vitro Mutagenesis Studies: 3-Test Battery Authors: R. S. Sleslnskl, M. W. Gunt, P. J. Guzzle, W. C. Hengler Sponsor: Union Carbide Corporation ***** On page 5, Section 2 (SCE Test), the unit of measure should have been ug/ml, therefore, the second to last sentence should read: ~ "Bromodeoxyuridine (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: /j, Ronald S. Sleslnskl, Fh.D. Study Director Manager, Genetic Toxicology -- Elton R. Roman, Ph.D. Associate Director, Toxicology WPC/1103-1 Fred R. Frank, Fh.D. Director Bushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation 11nr QW71 1803 BUSHY BUN RESEARCH CENTER R. D. 4, Mellon Road, Export, Pennsylvania 15632 Telephone (412) 327-1020 CONFIDENTIAL; Not to be released outside UCC without the written 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. Slesinskl, M. V. Gaunt, P. J. Guzzle, V. C. Hengler Sponsor: Union Carbide Corporation SUMMARY Epoxy Resin ERL-4221 was 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 DNA 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 questionableto-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 was selected for mutagenesis testing in part to develop and validate the sensitivity of our in-house battery of mutagenicity tests. Epoxy Resin ERL-4221 was 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 experiments were performed to select 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 maximtm dose level tested with S9 activation was Increased to 0.022. i irr mw2 Rushy Run Resssrch Csntsr A Joint Mellon Institute--Unton Carbide Corporation Operation 1804 Report 43-105 Page 2 CHO Mutation Tett - Epoxy Resin ERL-4221 was 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 was considered inactive as an agent for inducing mutation of CHO cells in culture. SCE Test - Epoxy Resin ERL-4221 was highly active in significantly stimulating the Induction of SCE ^n vitro at three of six of the concentrations tested. 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 was 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 was not required for activity of the test chemical. UPS 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 wide range of concentrations, the lowest three concentrations f Epoxy Resin ERL-4221 produced highly numerically elevated levels of UDS acti vity. Because these values were not consistently significant 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 questlonably-to-weakly 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 was not a potent mutagenic agent but that it appeared to possess a low level of activity In the UDS 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-mutagenlc. 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 (CHF Report #27-6), 1 tumor In 15 mice In the effective group was observed but this result was not significantly above the tumor incidence in the control group of animals. ucc 063073 Report 43-105 Page 3 SAMPLE Quantity: 8 ounces CHF Sample No.: 42-136 Submitted by: V. C. Kuryla, for UCC Date Received: March 15, 1979 Toxicology Advisory Group Division: Specialty Chemicals and Plastics Identification: light yellow. viscous liquid CAS #: 2386-87-0 ISOS ijOC 063074 18C6 Report 43-105 Page 4 BAKELITEa Cycloaliphatic Epoxy Resin ERL-4221 In Vitro Mutagenesis Studies; 3-Test Battery Sponsor: Onion 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 DNA (deoxyribonucleic acid) level of molecular organization. A battery of 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, II and III (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 IV. METHODS A description of the technical procedures used in the CHO test, the SCE test and the UDS assay are presented in greater detail in Appendices I, II 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 10X of the treated cells. Glass-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 10~4 to eliminate zeros in the lower concentration values (eg. 0.0003125Z - 3.125 x 10"*X). lu1 uAuA 063075 mm 1807 Report 43-105 Pag* 5 B. Mutation - In experiment #1, CHO cells were exposed for 16 hours to five concentrations of Epoxy Resin ERL-4221 from 100 x 10"*Z to 6.25 x 10~^Z (by volume) without the addition of an S9 metabolic activation system and for 5 hours to an Identical range of concentrations with S9 activation. A second, repeat experiment was performed at identical concentrations in the test without S9 but concentrations from 200 x lO^X to 12.5 x 10"^Z were tested with S9 activation. Dilutions of Epoxy Resin ERL-4221 were prepared by either direct addition of the test agent Into the cell culture media or by making sequential one-half dilutions from the stock solution f r the highest concentration using glass-distilled DMS0. The surviving fraction was determined at 20 to 24 hours after treatment and the mutant fraction was determined after a 7-to 9-day period to allow "expression" of the mutant phenotype. Only the top five concentrations which allowed sufficient cell survival were assessed for survival and induction of mutants. The percentage of cells surviving the treatment, the frequencies of mutanq colonies and the number of mutants/10^ viable cells are presented In tabular form. 2. SCE Test (Detailed procedures in Appendix II): Production of SCE'a following exposure to various concentrations of Epoxy Resin ERL-4221 was studied In CHO 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 Mutation test. Dilutions of Epoxy Resin ERL-4221 for testing, ranging from 100 x 10"^Z to 3.125 x 10"^Z (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 metabolic 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 agent. Bromodeoxyurldlne (BrdU) required to differentiate between the individual "sister" chromatids by SCE staining, was present at a concentra tion of 3 g/ml in the growth medium during treatment and during the culture period following exposure. A total of 15 cells/dose level and 5 dose levels, with or without metabolic activation were examined. The number of SCE/cell, mean # of SCE/chromosome and the level of statistical significance of the Increases above concurrent solvent control values are presented In tabular form. 3. UPS Test (Detailed procedures In Appendix III): Induction of primary DNA damage in rat liver cells (hepatocytes), was studied at a minimum of six dose levels which spanned a 1000-fold range of concentrations. Cells were treated with Epoxy Resin ERL-4221 for 2 hours in culture medium containing ^H-thymldlne, hydroxyurea and appropriate dilutions of Epoxy Resin ERL-4221 prepared in DMS0. Determination of DDS activity was performed by analyses of radioactive Incorporation into UCC nf.'WlS 1808 Report 43-103 Pag* 6 isolated hepatocyte nuclei and in DMA (precipitated from aliquot* of the Isolated nuclei) using a Searle Analytic Model SI or Packard Model 2650 scintillation spectrometer. Data are presented in tabular form with an indication of the level of statistical significance above the concurrent solvent 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, dlmethylnltroaamine (DMN) and ethylmethaneaulfonate (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-distilled dlmethylsulfoxlde (DMSO) were used as the negative and solvent controls, respectively. In the UDS assay, DMN and 4-nltroqulnollne oxide (4-NQ0) 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 1254lnduced, Sprague-Dawley male rats, was purchased from Litton Blonetics. 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 min/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 f r statistical analyses at the Bushy Run Research Center. Data from the CHO test do not follow a 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 Irr and Snee: (MF + 1)^*^ (Irr, J. D. and R. Snee, Proceedings of the Cold Spring Barbor-Banbury Conference, XI (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 c: p < 0.001. No superscript (or NS) Indicates p > 0.05. 7. Raw 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 Toxicology slide storage ares. ucc 063Q77 Report 43-105 Pag* 7 1809 RESULTS SECTION I - CHO MUTATION TEST - Epoxy Resin ERL-4221 4* Test Dates - Initiatedi April 26, 1979 Completed: May 16, 1980 B. Selection of Test Concentration (Data not shown In tables) In tests without S9 activation, CHO cells vere exposed Cor sixteen hours to six concentrations of Epoxy Resin ERL-4221 which spanned a concentration range Cron 0.5Z to 5 x 10~4z by volume. An identical range of concentrations was tested for five hours with S9 activation. Th percentage of cells which survived the exposure, both In the presence and absence of an S9 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, n 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 concentratl n 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"4X) In comparison to the markedly lower 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 cells. 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. ucc Q63Q78 1810 Report 43-105 Page 8 2. Mutation Table 2 presents the data for Induction of mutants 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 mutagenic action without the presence of an S9 metabolic activation system. Although no concentration of Epoxy Resin ERL-4221 produced a statistically significant Increase in the mutation 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, summarized on Table 4, data were consistent with the negative responses observed in experiment #1 (Table 2). No dose-level of Epoxy Resin ERL-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 system. 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 Procedures - 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 devlati ns 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 stated 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"*Z 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. ucc 063079 Report 43-105 Page 9 E. Conclusion* Epoxy Resin ERL-4221 was consistently Inactive as a mutagenic agent for CHO cells when tested with or without an S9 metabolic activation system over a 16-fold range of concentrations. Although small Increases In the numeri cal frequency of mutants were obtained at some test concentrations of Epoxy Sesln ERL-4221, these results appeared to be a random effect without statistical or probable biological significance. SECTION II - SCE TEST - Epoxy Resin ERL-4221 A. Test Dates - Initiated: December 10, 1979 Completed; March 24, 1980 B. Selection of Test Concentrations A maximum concentration of 100 x 10~*X 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"^Z to 3.125 x 10-4Z (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, testa 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 sunmarlzed in Table 5. Epoxy Resin 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 EKS positive control was highly statistically significant from the concurrent solvent control and these data indicated an appropriate sensitivity of the teat system comparable to our historical positive control data. The numbers of SCE obtained with the H2O solvent and DMS0 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. ucc 063080 Report 43-105 Page 10 D. Devlatlona from Standard Procedures The experiment wee repeated three times but only the final, successful study was 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 system. 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"^t to 1.0 x 10^*% by volume. The dose-levels were selected to span a range of cytotoxic to non-cytotoxic concentrations based upon data obtained in the CH0 cytotoxicity test. / C. Determination of UDS 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-thymldine 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 questlonableto-weafe activity for Epoxy Resin ERL-4221. Both of the positive control agents, NQ0 and DMN, lniuced numerically elevated Increases in UDS over values obtained with the solvent control. With DMN, howsver, 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 for detection of weakly-active mutagenic agents which require metab lie conversion (eg. DMN). ucc 063081 Report 43-103 Pag* 11 1813 2< DNA-Bound Radioactive Label (Data In Table 7) Analyses of DNA, from aliquots of hepatocyt* nuclei used for the UDS studies presented on Table 6, were performed as a second assessment of "unscheduled* Incorporation of ^H-thymldln*. Values for radioactivity Incorporated into the DNA of these hepatocyte nuclei are presented in Table 7. For hepatocytea treated with Epoxy Resin ERL-4221, two of the six test concentrations Induced levels of UDS which were statistically significant from the solvent control. He also observed a similar pattern of responses as obtained in the assessment of nuclei from cells treated with the same range of concentrations; three of the lowest six concentrations produced numerical elevations In UDS similar in magnitude to values obtained with the DMN positive control. Although there was no Indication of a dose-effect relationship due to treatments with the test agent, the UDS 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 DNA damage in the present test with the hepatocyte test system. The test employing precipitated DNA for measurement of incorporation of ^H-thymldlne 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 DNA and nuclei. Vlth DMN four of six of the tested concentrations produced a numerical elevation In UDS 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 produced which is considered to be a definitive 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 ln-house program of mutagenicity testing. Two prior experiments were performed with methods different from the final procedures 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 metabolizing such indirect-acting chemicals. E. Conclusion Epoxy Resin 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 hepatocytea. ucc 063082 1814 Reviewed and Approved by: Report 43-105 Page 12 Ronald S. Slesinaki, Ph.D. Study Director Manager, Genetic Toxicology Aaaoclate Director, Toxicology Fred R. Frank, Ph.D. Director Contributors: / Chinese Hamster Ovary test Sister Chromatid Exchange test Unscheduled, DMA Synthesis Assay Peggy J. Guzzle, B.S. Master Technologist Michelle V. Gaunt, B.S. Master Technologist W. Christopher Hengler, M.S. Assistant Scientist WPC/1103-5 UCC 063083 Report 43-103 Pag* 13 1815 REFERENCES A search of the major mutagenesis/carcinogenesis computer data flies failed to find any article* pertinent to mutagenlc/carclnogenle potential for this test agent. A previous study on dermal carcinogenic potential for mice Indicated .that Epoxy Resin ERL-4221 did not produce a statistically significant increase in skin tumors (CHF Project Report #27-6; CHF Sample #24-172). ucc 063084 Table 1 Chinese Hamster Ovary (CHO) Mutation Assay: Determination of Toxic Effects of Chemical Treatment During 16 Hr Mutation Induction Period Experiment #1 T st Chemicals Total f Colonies Total # Cells Plated [Epoxy Resin ERL-4221] (Z, v/v) 100.0 x 10"* 50.0 x 10"4 25.0 x 10"4 12.5 x 10"4 6.25 x IQ"4 42 255 419 565 354 Without S9 Activation 800 800 800 800 800 Controls DHSQ (20 ul/ml) - Solvent B20 (20 ul/al) EMS (200 ug/ml) - 502 512 578 800 800 800 Abbreviations: H20 - mater; S9 - liver homogenate; DMSO - dimethylsulfoxide EHS - ethylmethanesulfonate; DMN - dlmethylnitrosamlne 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.L WPC/1103-3 com C co O ccno o CoB  Table Chinese Hamster Ovary (CHO) Mutation Assay: Results on Evaluation of Mutant Induction by Epoxy Resin ERL-4221 Experiment #1 - Plating Efficiency Test Chemicals Total I Colonies Total 1 Cells Plated 1 Viable Fraction Total # Mutant Colonies (Epoxy Resin ERL-4221] (X, v/v) 100.0 x 10~4 49 50.0 z 10~4 82 25.0 x 10-4 144 12.5 x 10"4 144 6.25 x 10-4 152 Without S9 ActivalLion 300 0.163 0 300 0.273 1 300 0.480 0 300 0.480 2 300 0.507 0 Controls: SMS0 (20 ul/ml) - Solvent H20 (20 ul/ml) Medium EMS (200 ug/ml) - 270 203 255 180 300 0.900 1 300 0.677 X 300 0.850 0 300 0.600 22 Mutation Induction Total # Cells Plated Mutantsl 10 Viable Cells 1 x 106 1 x 106 1 x 106 1 x 106 1 x ID* 1 x 106 1 x 106 1 x IQ6 1 x IQ 0 3.7 0 4.2 0 * 1.1 1.5 0 36.7 iTotal # mutant colonies per 10& cells plated divided by viable fraction. Statistical significance above solvent control: Ho superscript indicates p > 0.05. Data analysed by Student's t-test. Abbreviations: H20 ~ water; S-9 - liver homogenate; DMSO - dlmethylsulfoxlde; EMS - ethylmettianesulfonate; DMN - dimethylnitrosamlne. UPC/l103-3 o (-- CcOo O Sco 0 CD oa fi? 8 H2 GO ucc 063087 Table 3 Chinese Hamster Ovary (CHO) Mutation Assay: Determination of Toxic Effects of Chemical Treatment Experiment #2 Test Chemicals Total # Colonies Total # Cells Plated [Epoxy Resin ERL-4221] (Z, v/v) 100.0 x 10~* 50.0 x 10~* 25.0 x 10-4 12.5 x 10"4 6.25 x 10-4 i Controls DMS0 (20 ul/ml) - Solvent H20 (20 ul/ml) EMS (200 ug/ml) - - ' 7 60 123 208 266 469 399 43 Without S9 Activation 400 800 800 800 800 800 800 600 [Epoxy Resin ERL-4221] (Z, v/v) 200.0 x 10-4 100.0 x 10-4 50.0 x 10-4 25.0 x 10-4 12.5 x 10"4 351 381 326 419 337 With S9 Activation 800 800 800 800 800 Controls DMS0 (26 ul/ml) - Solvent H20 (20 ul/ml) DMN (3700 ug/ml) DMN (740 ug/ml) - 207 297 103 231 800 800 800 800 r ~ 1 r r iir Abbreviations: 11 ----1-------------^ EMS - ethylmethanesulfonate; DMN - dimethyInitrosamlne 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 X f 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 TJ 90 Chinese Hamster Ovary (CHO) Mutation Assay: Results on Evaluation of Mutant Induction by Epoxy Resin ERL-4221 Experiment #2 Test Chemicals Plating Efficiency Total # Colonies Total # Cells Plated Viable Fraction Total I Mutant Colonies Mutation Induction Total I Cells Plated Mutants* 10 Viable Cells (Epoxy Resin ERL-4221] (Z, v/v) 100.0 x 10~* 76 50.0 x 10~4 87 25.0 x 10-4 137 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 106 1 x 106 1 x 10* 1 x 106 1 x 106 0 13.8 0 4.7 6.5 Controls: DHSO(20 ul/ml) - Solvent H2O (20 ul/ml) EMS (200 ug/ml) - 211 246 180 400 0.528 3 400 0.615 2 400 0.450 124 1 x 106 1 x 106 1 x 106 5.7 3.3 275.6C (Epoxy Resin ERL-4221] (Z, v/v) 200.0 x 10-4 312 100.0 x 10-4 333 50.0 x 10-4 313 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 106 1 x 106 1 x 106 l x 10* 1 x 106 2.6 18.0 19.2 4.2 15.9 Controls: DMSO (20 ul/ml) - Solvent H20 (20 ul/ml) OHM (3700 ug/ml) DHM (740 ug/ml) - 362 386 198 263 400 0.905 18 400 0.965 5 400 0.495 63 400 0.658 30 l x 106 1 x 106 1 x 106 1 x 106 19.9 5.2 127.3C 45.6* - " S0T-P? 3J0d*g ucc 063088 ^Total # mutant colonies per 10^ 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: H2O - water; S-9 - liver homogenate; DMSO - dimethylsulfoxide; EMS - ethylmethanesulfonate; DHN - dlmethylnitrosamlne. WPC/11G3 Table 5 Slater Chromatid Exchange (SCE) Assay: Induction'of SCE's by Epoxy ReBln (ERL-4221) Without S9 Metabolic Activation 5 Hour Treatment Test Chemicals Total # of Chromosomes Total I of SCE SCE/Cell1 Mean Number SCE/Chromosome2 + S.D. [Epoxy Besln (ERL-4221)] (Z, v/v) 100.0 x 10-4 50.0 x 10*4 25.0 x 10"4 12.5 x 10"4 6.25 x 10"4 3.125 x 10"4 Controls DMSO (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 -1- 0.212 572 38.13 1.869 + 0.301 415 27.67 1.379 4- 0.184 316 21.07 1.041 4- 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 4- 0.169 423 28.20 1.417 4- 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: HjO - water; S9 - liver homogenate; DMSO - dimethyIsulfoxide; EMS - ethylmethanesulfonate; S.D. - standard deviation WPC/1103-3 ucc 063089 r-- 00 8 Significance Above Solvent Control^ NS c c c NS NS NS c M7 9 h* 00 Report 43-105 Table 6 Unscheduled DNA Synthesis In Hepatocytes from Rat Liver Nuclear-bound label; all DPM values are calculated from nuclei per 10& viable hepatocytes. Each average la calculated from duplicate samples, except for DMSO which was done In quadruplicate* Test Chemical Solvent - DMSO Positive Controls: 4 - NQO DMN Concentration 3.0Z 3.0 ug/ml 1.0 ug/ml ug/ml 1000 300 100 30 10 1 ug/ ml ug/ml ug/ml ug/ml ug/ml ug/ml Radioactivity in Nuclei Avg. DPM + S.D. 6995 + 909 16791 + 1307 10913 4668 9623 + 456 9286 + 748 8234 + 877 12230 + 927 10570 + 2484 9390 + 3683 10679 + 474 Z of Solvent Control + S.D. 100.0Z + 13.0Z 240.1Z + 18.7Z 156.0Z + 66.71 137.6Z + 6.5Z 132.8Z + 10.7Z 117.7Z + 12.51 174.81 + 13.3Z 151.1Z + 35.5Z 134.3Z + 52.7Z 152.7Z + 6.8Z Significance Above Solvent Control* - c a NS NS NbS NS NS NS Test Chemical: {Epoxy Resin ERL-4221] {Z, v/v) 1000 x 10-*Z 300 x 10"*Z 100 x lO^Z 30 x 10~*Z 10 x 10~4Z 1.0 x lO^Z 578 + 207 6636 + 292 9458 + 334 10322 + 2777 10547 + 478 11600 + 1399 8.3Z + 3.0Z 94.9Z + 4.2Z 135.2Z + 4.8Z 147.61 + 39.7Z 150.8Z + 6.8Z 165.81 + 20.0Z NS NS NS NS NS a . R eport 4 J -lu 5 Page 19 ^Statistical significance above solvent control: a: 0.05 > p > 0.01; b: 0.01 > p > 0.001; c: p < 0.001; NS: p > 0.05. Data analyzed by Duncan's Multiple Range Analysis. Abbreviations: DMSO - dimethylsulfoxlde; 4-NQ0 - 4-nltroquinollne oxide; DMN - dimethylnitrosamine; DPM - disintegrations per minute; S.D. - standard deviation WPC/1103-3 ucc 063090 t 8 Table 7 Unscheduled DNA Synthesis In Hepatoeytea from Rat Liver DNA-bound label; all DPM valueB are calculated from DNA precipitated per 10& viable hepatocytes. Each average Is calculated from duplicate samples, except for DMSO vhich ms done in quadruplicate. it I-* 00 0 N Test Chemical Solvent - DMSO Concentration Radioactivity In DNA Avg. DPM + S.D. 370?----------------------- 8537 + 1379 Positive Controls: 4 - NQO 3.0 ug/ml 1.0 ug/ml 0.3 ug/ml 16720 + 940 12011 + 3654 10219 + 757 DMN 1000 ug/ml 9818 + 786 300 ug/ml 14608 + 1669 100 ug/ml 13195 + 681 30 ug/ml 13322 + 4554 10 ug/ml 12198 + 3117 1 ug/ml 16243 + 1690 Test Chemical: [Epoxy Resin ERL-4221] (X, v/v) ucc 063091 1000 x 10"*X 300 x lO^X 100 x 10"*X 10 x 10_4X 10 x 10-4X 1.0 x 10~4x 495 + 289 +7864 + 307 10882 1198 14760 + 1453 12172 + 242 14629 + 993 X of Solvent Control + S.D. 100.OX + 1532-------- 195.OX + 11.OX 140.7X + 42.8X 119.7X + 8.9X 115.OX + 9.2X 171.IX + 19.5X 154.6X + 8. OX 156.OX + 53.3X 142.9X + 36.5X 190.3X + 19.8X Significance Above Solvent Controll -- b NS NS NS a a a NS b 5.8X + 3.4X 92.IX + 3.6X 127.5X + 14.OX 172.9X + 17.OX +142.6X 2.8X +171.4X 11.6X NS NS NS a NS . a ^Statistical 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 dlaethylsulfoxide;^T-NQO - 4-nltroquinoline oxide; DMN - dlmethylnitrosaalne; DPM - disintegrations per minute; S.D. - standard deviation HPC/1103-3 ^ nji o lta-J Lon Page 1 of 5 1823 APPENDIX I Chloaaa Hamster Ovary (CHO) Mutation Aaaay Theoretical Baals Mutation is a heritable alteration in a call in which a gene specifying the genetic code for a specific protein is modified in structure and/or function. Mutations, Induced by chemical or physical agents, of the HGPRT (hypoxanthlnequanine phosphoriboeyltransferaee) gene say be detected by the growth of colonies of "mutant".cells which are resistant to the purine analogs 6-thioguanine (TG) or 8-azaguanlna. Normal cells contain a functional HGPRT enzyme which phosphorylates TG and allows Its incorporation Into DNA causing the calls to die. Mutant cells with a non-functional HGPRT enzyme are unable to phoaphorylate or Incorporate TG, thus survive and grow In its presence* The CHO nutation test is an assay which detecta "forward nutations" from TG-eensitivlty to TG-resistance caused by a direct lose of the activity of the HGPRT enzyme (HGPRT+ * HGPRT"}. An assessment of the ability of several hundred agents to cause gene nutations in vitro Indicates that the CHO nutation assay provides a reasonable estimate of the potential genetic activity of the test chemical. Methods Cell Culture Procedures; CHO cells used in these studies were obtained froa Abrahaa Bale at Oak Ridge National Laboratory with the designation CHO-K1-BH4-D1 (or simply CHO for report purposes). Cells are maintained In active growth by subculturing 2 to 3 timas/week In antibiotic-free, Han's Modified F12 Medina suppleaented with 10Z (v/v) beat-inactivated, fetal bovine sera (F12-10), and lacking In hypoxanthine. For treatment of cells without metabolic activation, F12 medium with 30 unlts/al of penicillin, 30 ug/al streptomycin and 5Z (v/v) of dialyzed bovine serua (F12-D5) is used. For treatments incorporating an S9 metabolic activation system. Identical medium, but without serua, Is employed. For determination of mutant frequencies, F12-D5 medium containing 2.0 ug/al TG (6-thioguanine) la used as a "selective medium." Cell numbers are determined routinely with a Coulter Model F electronic cell counter which is standardized periodically with a pre-counted suspension of latex beads. Presence of Mycoplasma cell contaminants la determined by a microscopic fluorescence assay employing Boeehst 33258 dye. All culture procedures and treatments with test chemicals are performed under aseptic conditions in a laminar-flow, biohazard hood. Positive and Negative Controls t Sterile water or glass-dlstUled dimethylsulfoxide (DMSO) are the usual solvents for test chemicals and the respective solvent is tested as a control at the maximum concentration used to add the test agent. Dlmethylnltrosamlne (DMN) or ethylmethanesulfonate (EMS) are used as positive control mutagens for tests with or without an S9 metabolic activation system, respectively. Mutation frequencies obtained with concurrent positive and negative controls are used as the basis for monitoring the sensitivity and stability of the CEO mutation test system. Comparison of concurrent control values with historical controls Is used to delineate the range of acceptable variations in th test system. UCC 063092 1824 Appendix I Page 2 of 5 Metabolic Activation: Rat liver, S9 homogenate prepared from Arochlor-1254 Induced, Sprague-Dawley, male rata la purchaaed from Litton Blonetlca, Kensington, MD. Each lot of liver homogenate la preacreened for metabolic capability to activate DMN in our laboratory before uae In the teating program. The complete S9 metabolic activation ayatem contalna the following: 8 umoles/ml MgCl2, 33 umolea/ml KC1, 3 umolea/ml glucoae-6-phosphate, 4 umoles/ml NADP-oxidized (nicotinamide adenine dlnucleotidc phosphate), 100 umolea/ml Na2HP04, and between 500 to 4000 ug/ml of S9 protein (depending on metabolic activity); a volume of 1.0 ml of the complete mixture 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 aasessment of mutagenic potential to select doses which produce a maximum of 80 to 90Z call killing. Cytotoxicity is determined by either of Che following two methods: (1) Clonal assay - 200 to 400 CHO cells are exposed to a minimum of five dose levels of the test agent at concentrations from 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 ul/ml). (2) Growth Inhibition - 3 x 10^ 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 In control (untreated cells) and In calls treated with various concentrations of the test agent. If no cytotoxicity la evident at the highest concentrations In the cytotoxicity tests, the test is either repeated at higher concentrations, or mutation testing 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 teat is repeated at a concentration range of 3x10"^ to 3x10" percent by weight or volume, as appropriate. Dose levels which 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. UCC 063093 Appendix I Page 3 of 5 1825 Chemical tuples ere sterilized by membrane filtration when microbiological testa indicate this is required to assure sterility* Liquid teat agents are tested on a percentage by volume basis. Solid chemicals are dissolved in an appropriate solvent by making a 10 to 20X stock solution (by weight) and subsequent dilutions are made from this stock on a volume/volume basis. Treatment with Test Chemicalsi For tests of chemicals which may act directly without Incorporation of an S9 metabolic activation system, 5xlQ5 cells are inoculated 20 to 24 hours prior to treatment into 25 cm2 culture flasks containing F12-D5 medium and incubated at 37*C in a 5 to 6% CO2 atmosphere. Appropriate concentrations ox the test agent or control chemicals are added to the cells and cultures are treated for 5 hr at 37*C. The medium and test agents are removed by suction, cells are rinsed once or twice and fresh P12-D5 medium is added. The cells are allowed a period of 20 to 24 hours of r covery from treatment before survival is determined. Treatment of cells for testing of chemicals which require metabolic activation for mutagenic capacity la performed identically with the procedure above, with the exception that P12 medium without serum and containing 1.0 ml of S9 activation mixture per 4.0 ml f medium la employed. Determination of Cytotoxicity: The relative survival of created cells. In comparison to solvent controls, la 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 kill both normal cells and mutants and may depress the actual mutation fre quencies; insufficient cytotoxicity may indicate an insufficient concentration of the test agent was employed. The colony-forming potential of 100 to 200 treated cells 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 5x10^ cells are subcultured in 100 mm tissue culture dishes in F12-D5 medium and incubated at 37 aC in a 5 to 6% CO2 atmosphere. After a total of 7 days to allow "expression* of the mutant phenotype, cells are dissociated with 0.05 to 0.075Z trypsin, counted and plated at a concentration of 2.5x 10^/dish in four culture dishes (lx 10* total cells) which each contain 5 ml of F12-D5 (TG) 'selective medium. At this time, cells are diluted and 100 cells/dish are added to four culture plates containing F12-05 medium (without TG) to assess viability (plating efficiency) of the treated cell population and to determine the surviving fraction. All cultures are then incubated for an additional 6 to 8 days to allow growth of cells; 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, checked by manual counts and data are recorded both as total mutants, mutants/106 total cells and autants/106 viable cells. ucc 063094 1826 Appendix I Page 4 of 5 / Statistical Analyses: Uniform statistical procedures to evaluate is vitro nutation data have not been developed. The distribution of mutation frequencies from historical controls in at least two laboratories indicates that 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 CHO 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 zeros) 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 la 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 CHO cells of approximately 4 to 5 mutants/10^ viable cells, with a range of 0 to 25 mutaats/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. 'JCQ 063Qq$ Appendix I Fi|i 5 of 3 1837 References l Chu, E. H. T end H. V. Mailing. Chemical Induction of Specific Locus Mutations in Chinese Hamster Calls In Vitro.' Proc. Nati. Acad. Sci. U.S.A.. 5T<WMj", 13GS-i3ti. 2. O'Neill, J. P., P. A. Brlaer, R. Maehanoff, G. P. Hirsh, A. W. Hsie. A Quantitative Assay of Mutation Induction at the Hypoxanthlae-Guanina ~ Phoapho'rlboayl Transferase locus In Chinese Hamster dvary Cells (CHO/HGPRT System!): Development and Definition of the System, Mutation Research. 45 (1 7 7), 91-loi. -- 3. O'Neill, J. P. and A. W. Hale. Phenotypic Expression Time of Mutagen* Induced 6-Thiogttanine Resistance in Chinese hamster Ovary bells (CHO/HGPRT System), Mutation Research,*59, (1979), 109-II8. 4. Irr, J. D. and R. 0. Snee. Statistical Evaluation of Mutagenicity in the CHO/BGPRT System. Proceedings of the Cold Spring Harbor-Banbury Conference II (197&), 263-274. 5. Box, G. E. P. and D. R. Cox. An Analysis of Transformations. J. of the Royal Statistical Society, B, id(l964)7 211-252. WPC/1033 UCC 063096 1828 m Pag* 1 of 4 APPENDIX II Determination of Sister Chromatid Exchange (SCE) Frequencies in Chinese Hamster Ovary (CHO) Cells In Vitro Theoretical Baals Exchanges of genetic material between the individual arms of a chromosome (l.e. sister chromatids) are thought to arise from breakage and physical inter changes in the DNA of a cell during cell division. An increase in the frequency of such interchanges between sister chromatids can be observed in cells treated with physical or chemical mutagenic agents, or in cells exposed to many suspect or proven human carcinogens. Thus, analysis of SCE frequencies in cells treated with a teat agent has been suggested as a sensitive screening test for potential mutagenic/carcinogenic chemicals * The method used in our study to visualize SCE's in CHO cells grown in cul ture is based on the procedure described by Perry and Wolff (1974). A standard concentration of 3.0 ug/ml of bromodeoxyurldlne (BrdU) was used in the growth medium to allow a visualization of SCE's after two cell divisions in the pre sence of BrdU. Staining of chromosomes with 5.0 ug/ml of 33258-Hoechst fluore scent dye. exposure to light and Giemsa staining was used to differentiate chromatids for SCE analysis. Methods Cell Culture Procedures; Chinese hamster ovary (CHO) cells were obtained from Abraham Hale at Oak Ridge National Laboratory with the designation CH0-K1- BH4-D1 (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. Ham's F12 (modified) medium fortified with 10Z (v/v) of heat-inactivated fecal bovine serum and lacking hypoxanthlne and thymidine. Cell concentrations are determined routinely with a Coulter* Model-P electronic cell 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 Hoechat 33258 dye. For treatments with test chemicals without S9 metabolic activation, modified F12 medium is used with 30 units/ml of penicillin, 50 ug/ml streptomycin and 5Z (v/v) of heat-inactivated, dialyzed fetal bovine serum (F12-D5). 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 chemicals and the respective solvent is tested as a control at the maximum concentration used to add the test agent. Dlmethylnitrosamlne (DMN) and ethylmethanesulfonate (EMS) are used as positive control mutagens for tests with or without the addition of an S9 metabolic activation system, respectively. Results from treatments with concurrent control agents are used as a basis of comparis n and for demonstrating the sensitivity and stability f the SCE test system. Comparison of concurrent control values with historical controls is used to delineate the range of acceptable variations in the test system. jjqq 063097 .J 1829 Appendix II Page 2 of A Metabolic Activation: Rat liver 59 homogenate (prepared from Arochlor 1254 Induced, Sprague-Dawley, male rata) la purchased from Litton Blonetica, Renalogton, MD. Each lot of liver homogenate la prescreened for activity In our laboratory before use in the testing program. The complete S9 metabolic activation system contains the following! 8 umoles/ml MgCl2, 33 umolea/ml RC1, 5 umoles/ml KCl, 5 umoles/ml glucose-6phosphate, 4 umoles/ml NADP*oxldi2ed fora (nicotinamide adenine dinucleotide phosphate), 100 umoles/ml NS2HP04 and between 500 to 4000 ug/al of S9 protein (depending on metabolic activity). A volume of 1.0 ml of the complete mixture of the above reagents is added to each 4.0 ml of culture medium* Dose Selection! Toxicity of the test chemical la determined prior to assessment of mutagenic potential to select doses which produce a <!of 80 to 90Z cell killing. Cytotoxicity is determined by either of the following two methods as part of the CH0 mutation testing procedure: (1) Clonal assay - 200 to 400 CBO cells ere exposed to a'minimum of five dose levels of the test agent at concentrations from 0.12 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 ul/ml). (2) Growth Inhibition 5 x 10^ cells in 25 em^ 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 in control (untreated calls) and in cells treated with various 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 testing is performed with a greater number of treatment flasks starting at high r dose levels. If marked toxicity is evident even at the lowest dose, the cytotoxicity test is repeated at a concentration range of 3x10to 3x10" percent by volume. Dose levels which are moderately toxic but permit survival of at least 40 to 50Z 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 mitotic cells with SCZ staining, are evaluated for SCE induction. Chemical samples are sterilized by membrane filtration when microbiological tests Indicate this is required to assure sterility. Liquid test agents are tested on a percentage by volume basis. Solid chemicals are dissolved in an approprlat solvent by making a 10 t 202 stock solutl n (by weight) and subsequent dilutions are made from this stock on a volume/volume basis. UCC 063098 e -ii i 1830 Appendix II Page 3 of 4 Treatment With Teat Chemicals: Testing of chaaicals for direct mutagenic action (without S9 metabolic activation) la 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 x 106 cells are plated into 75 cm^ culture flasks in F12-D5 medium at least 20 hrs prior to treatment and incubated at 37*C in a 5 to 62 CO2 atmosphere. Appropriate concentrations of the teat agent or control chemicals are added to the cells and 3 ug/ml BrdUla added to all flasks. Calls are treated with test agents for 5 hrs, media la then removed by suction, 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 cell 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 vlthout activation, except for three modifications: 1. Before treatment with the test agents, F12-D5 medium is removed and F12 medium without serum Is added. 2. S9 metabolic activation mixture Is added to each flask (Including solvent and positive controls) before addition of test agents. 3. Celia 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 Colchicine (0.2 ug/ml) Is added to culture flasks 1 to 2 hrs prior to harvesting to arrest cells In mitosis. Calls are then removed from flasks, after a brief Incubation with 0.01Z DIFCO trypeln, suspended in 0.075M KC1 (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 cells 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 are stained for SCE's by treatment with 3.0 ug/ml of Hoechst 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 Gurr's glemsa (diluted 1:25 with water), rinsed In water and dried before application of coversllpa. Examination of SCE's: All slides are coded and read In a blind fashion without Indication of the 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 dose level. The mean number f SCE/cell and SCE/chromosome are calculated and recorded. Slides are decoded only after examination of all slides in the experiment has been completed. UCC 063099 Appendix IX Peg* 4 of 4 1831 Statistical Analyses; Data ara analyzed by appropriate parametric statiatieal procedures which follow BRRC standard operating procedures for analyses of data* Significance values and the atatiatlcal test employed are shown for data summarized In tabular fora* Interpretation of Data; The criteria for evaluation of a positive or negafive 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-dependent increase in SCE'a is Induced by the test agent. When no dear dose-response relationship is evident and when one or more 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 ther testa in the testing battery. Clearly positive responses will include any f the following: (1) Doubling in the SCE frequency at a minimum of two of the five concentrations tested; (11) Statistically significant responses of p < 0.0S at three concentrations or at 2 concentrations if p < 0.01; (ill) Induction of a statistically significant, dose-related increase in the number of SCE. General References 1. Perry, P. and S* Wolff. New glamsa method for differential staining of sister chromatids. Batura, 251 (1974). 156-13^. 2. Lett, S. A., J. W* Allan, W. E. Rogers and L. A. Juergens. In vitro and in vivo analysis of slater chromatid exchange formation, pp 271-291 in Handbook of Mutagenicity Test Procedures, ad. Kilbey, B* J., et al. Elsevier Publ. Co. (19?9). 3. Carrano, A. 7., L. H. Thompson, P. A. Lindl and J. L. Hinkler. Sister chromatid exchange as an indicator of mutagenesis. Batura, 271. (1978). UT-TM.-------------- --=------------ -----------------------8----------- -- 4. Galloway, S. M. and S. Wolff. The relation between chemically induced sister-chromatid exchanges and chromatid breakage. Mutation Res., 61, (1979), 297-307. ^ 5. Snedecor, 6. W. and W. 6* Cochran. Statistical Methods, 6th Ed., Iowa State Unlv. Press, 4mes, Iowa (1967). WPC/1033 IJCC 063^ 1832 f / I APPENDIX III fw 1 of 3 Unscheduled DNA Synthesis (UPS) In Hepatocytes from Rat Liver / Theoretical Basis Chemicals may Interact with both the cellular components and the genetic material of a cell (.g. DNA and SNA) because of their 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) and Williams (1976). Hepatocytes 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 trltlated 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 Suspensions; Hilltop-Wistar albino rats are anesthetized with Metafane(R). The abdominal cavity is surgically exposed and 1250 units of heparin is injected intravenously. A catheter is inserted into the portal vein and warm Hanks Balanced Salt Solution (HBSS) is pumped into the vein and through the liver. This first solution contains heparin and EGTA, [ethylene glycol-bis-(bata-aminoethyl-ether)N,N-tetraeetie acid], which preferentially chelates calcium; the solution contains no magnesium or calcium. After the liver Is blanched, a second solution of HBSS containing 60 units/ml of collagenase 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 cell suspension is passed through two nylon meshes to remove cell clumps and the cells are washed once at low centrifugation speed. After rasuspenslon 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 Hepatocytes? Approximately 2 x 10* viable hepatocytes are added to 5 ml medium 199 containing 10 mti hydroxyurea and 30 mM Hepes (N-2-hydroxyethyl piperazine-N-2 ethane sulfonic acid) buffer. After the cells are dispensed into the tubes, they are placed on a rocker platform and are Incubated at 37*C for 1 hour. Although hepatocytes 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 is expected to result from repair or unscheduled DNA synthesis. i irr Vwv 063101 Appendix III Page 2 of 3 1833 Selection of Doses of Test Chemical: Initially, the following concentratlona x [o^i (by volume) are tee ted: 100, 30, 10, 3, 1, and 0.1. If thaaa concentrations prove to be cytotoxic, or If additional information la available from other in vitro teats as to the proper dose levels, then an appropriate series of concentrations la used over a 3-log range of concentrations. Treatment of Hepatocytes: After prelncubatlon, 25 microCuries of tritiated thymidine (20 Curies/millimole) is added to each tube. The test chemical and positive controls are diluted in an appropriate solvent and they are then added to each labeled tube. Generally, at least six concentrations of the test chemical over a 3-log range of concentrations are tested and each concentration is run in duplicate. The tubes are returned to the rocker platform for a 2-hour exposure at 37*C. Positive and Negative Controls: 4-nltroqulnollea oxide (NQO), a directacting' mutagen, which"induces UV-type DKA repair and dimethyloitrosaalne (DMN), which requires metabolic activation by microsomal enzymes for activity, are run in duplicate as positive control chemicals. The solvent control is run in quadruplicate and consists of 100 to 150 micro11tars (concentration specified in individual reports) of the solvent used to dilute the sample. Dimethylsulfoxide (DMSO) or water are the usual solvents for test chemicals. Harvest: At the end of incubation with the test agent, the cells are 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, 5Z citric acid and 3 mM MgCl2 a lysing solution which liberates the nuclei. The nuclei are rlnaed once in this solution and resuspended in 0.25 M sucrose, 2.5Z citric acid and 3 mM Mg&2* 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 Nuclear-Bound 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 solubilizer in a scintillation vial. Ten ml of DtmiluasO scintillation cocktail is added and the radioactive disintegrations per minute (DFM) are determined by counting twice in a scintillation counter for ten minutes. The measured DPM are then used to calculate the DPM/10^ viable hepatocytes presented on tables. Determination of DMA-Bound Label: The amount of radioactive thymidine in corporated into DNA is quantitated in DKA Isolated and precipitated from 1.00 to 1.25 x 10-5 viable hepatocytes. To 1.25 ml of the nuclear suspension, 2.75 ml of IX sodium dodecyl sulfate (SDS) and 5 mM Ethylenediaminetetraacetic Acid (EDTA) is added to lyse the nuclei. The DKA la precipitated from this solutl n with 4 ml of ice-cold 10Z trichloroacetic add (TCA) and the sample tubes are Incubated at 0*C for at laast 30 minutes. The solution is then poured onto Whatman glass fiber filters under vacuum and the tubes and filters are washed twice with cold 5X TCA. Finally, each filter is rinsed once with methanol, dried and placed in a scintillation vial. The filters are incubated at 50*C for 1 hour with 1 ml of a diluted solution of KCS tissue solubilizer; prepared by adding 1 part solubilizer to 2 parts of Diailuae* cocktail. Dimilume* is then added to each vial and the vials are counted twice in a scintillation c utter for ten minutes. ucc 063102 1834 Appendix III Pag* 3 of 3 Statistical Analysis: The averag* DPH la calculated for each do** level and the control* and final reaults are expressed as DPM/10* viable hepatocytes. Data are also expressed as a percent of the solvent control for purposes of coaparlaon. The original data are statistically analysed by the appropriate parametric test, following the BRRC standard procedures for statistical analyses and the test(s) employed la Indicated on the respective cables. Comparison between the mean for each dose level with the 95? confidence limits of the historical solvent control may also be used in some cases 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 tk* production of a statistically significant, dose-related increase in the amount of UDS 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 the detection of a similar response with both DNA and Isolated nuclei determined at two or three consecutive concentrations. General References 1. Lampldis, T. J. and J. B. Little. The Enhancement of PV-Induced Unscheduled DNA Synthesis by Hydroxyurea. Experimental Cell Research, 1X0, (197?), 41-46. 2* Ifaramatsu, M. Isolation of Nuclei and Nucleoli. In: Methods in Cell Physiology, Vol. IV. 1970* Editor': D. M. Prescott. Academic Press, New Tort:* 3. Seglen, P. 0. Preparation of Rat Liver Cells* III. Enzymatic Requirements for Tissue Dispersion. Experimental Cell Research, 82, (1973), 391-398. 4. Williams, G. M. Detection of Chemical Carcinogens by Unscheduled DNA mSay$nithrre;sist&i4nraRsa3t iL. iver Primary Cell Cultures. ------------------ Cancer Research. 37. -- 5* Williams, G. M. The Use of Liver Epithelial Cultures for the Study of Chemical Carcinogenesis. American Journal of Pathology, 85, (1976). 739-753. 6. Williams, G* M. and M. F. Laspia* The Detection of Various Nltrosamines in the Hepatocyte Primary Culture/DNA Repair Test. Cancer Letters. 6. (1979), 199-106. " 7. Snedecor, G. W. and W. G. Cochran. Statistical Methods. 6th edition, Iowa State University Press, Ames, Iowa (1961). WPC/1033 UCC 063103 APPENDIX XV - 1835 Report 43*103 Physical end Chemical Characteristics of the Teat Semple car No.: CAS No.: Chemical Neme: Trade Name ead/or Synonym*: Molecular Weight: Formula: 42*136 2386-87-0 3, 4-Epoxycydohaxylmathyl-3, 4 Epoxyeydohexylcarboxylate BAKELITE* Cycloaliphatic resin ERL-4221 (previously EF-221) 232.30 Ci4H20O4 Molecular Structure: <^1 Specific Gravity (0 20*C): Bolling Point: Solubility In H2O (X by vt): Purity: Vapor Pressure (@ 20*C): pH: Flash Point: Stability: lacompatibility: Appearances and Odor: Disposal: Protective Measures: Health Hazard: 1.1723 354*C (669.2'F) 0.03 (at 23*C) Not available; commercial sample tested < 0.01 mm Hg Not available 245*F Closed cup Stable, avoid heating over 100*F Avoid adds, amines, strong bases Low viscosity liquid; characteristic odor Dilute with Inert solvent and Incinerate. Small spills may be flushed with water; larger spills absorbed and burned Ose goggles, plastic gloves and exhaust ventilation. Avoid skin and eye contact. No effects of overexposure are currently known. O'CC 06310^