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2227 BUSHY RUN RESEARCH CENTER R. 0. 4, Mellon Road, Export, Pennsylvania 19032 Telephone (412) 327-1020 CONFIDENTIAL: Not to be released out aide TTCC without the written consent of the UCC-sponsoring HS&EA Division Manager. Project Report 43-129 19 Pages Tel: (412) 327-1020 February 26, 1981 BAKELITE* Epoxy Resin ERL-2774 In Vitro Mutagenesis Studies: 3-Test Battery Authors: R. S. Slesinskl, M. W. Gaunt, P. J. Guzzle, W. C. Hengler Sponsor: Onion Carbide Corporation ***** SUMMARY Epoxy Resin ERL-2774 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 induction of Unscheduled DNA Synthesis (UDS) in rat liver cells. The results indicated that Epoxy Resin ERL-2774 produced a statistically significant and dose-related effect in the SCE test with CH0 cells and additional (but less definitive) positive effects in the CHO mutation test and the UDS test with hepatocytes. Epoxy Resin ERL-2774 was considered a probable positive mutagenic agent based on the suggestive pattern of activity in the 3 tests. Because definitive positive mutagenic activity was observed in only the SCE test, additional studies would be necessary to confirm this result. RESULTS AND INTERPRETATION Selection of Test Concentrations - Preliminary experiments were performed to select an appropriate range of testsoncentrations in which the maximum concentration would allow survival of approximately 10% of the treated cells. A maximum concentration of 30 x 10"*% (by weight) was chosen for the highest dose-level in the first experiment and a total of five concentrations of Epoxy Resin ERL-2774 was tested. In a second repeat test with higher concentrations, 100 x 10"*Z was used as the highest dose level. CHO Mutation Test - Epoxy Resin ERL-2774 produced a statistically signifi cant effect on the frequency of mutations of CHO cells at only one concentration in tests with the incorporation of a liver S9 metabolic activation system. The lack of a dose-related effect on the mutation frequency complicated the definitive classification of the test chemical as mutagenic or non-mutagenlc. However, the highly statistically significant increase in mutants obtained In the test with metabolic activation was clearly outside our historical control range of variation for this test. Because the data were equivocal, and further testing would be necessary to establish the existence of a dose-response relationship, the results from this test could not be used to classify the test chemical as either active or inactive. However, literature references concerning similar studies suggest that this single positive value may be a biologically significant effect. Bushy Run Research Center A Joint Mellon institute--Union Carbide Corporation Operation 0626! 2228 Report 43-129 Pag* 2 SCE Teat - Epoxy Resin ERL-2774 produced a doaa-ralaced and highly statistically significant affect on tha frequency of SCE in CHO cells in tests without the incorporation of an S9 metabolic activation system. An overall range of concentrations between 120 x 10"^X to 7.5 x 10-1*! (by weight) was used. All five of the concentrations tested produced a highly statistically significant efface on the SCE frequency. This result prompted an unequivocal consideration of Epoxy Resin ERL-2774 as a highly active mutagenic chemical in this test. OPS Teat - Epoxy Resin ERL-2774 produced statistically significant effects on UDS activity at two of eight concentrations between 1000 x 10~*X to 3 x 10~*X (by weight). Epoxy Realn ERL-2774 was considered to be active in the present test with the hepatoeyte teat system because of the high degree of statistical significance of the increases in UDS produced by apparently non-cytotoxlc doses of the test chemical. Comparative Mutagenicity - The pattern of responses produced by Epoxy Resin ERL-2774 in tha 3-teat battery to determine potential mutagenicity indicated that Epoxy Resin ERL-2774 was an active mutagenic agent. In the SCE test. Epoxy Resin ERL-2774 was comparable in activity to the historical data obtained with the positive control (EMS), a known mutagen and carcinogen. Contradictory reports exlat in the literature on the potential carcinogenicity of Epoxy Realn ERL-2774 (references 2, 3 and 4) but there is consistent agreement that this chemical possesses mutagenic potential in in vitro tests (references 2, 4 and this report). A comparison of data for the CHO mutation teat from this study and a previous study performed at the Oak Ridge National Laboratory (reference 4) indicated that the weak positive result obtained in the (SO test with S9 metabolic activation was a reproducible effect in two laboratories. Thus, all the test results la this study would be consistent with the Identification of Epoxy Resin ERL-2774 as an active mutagen. Quantity: 8 ounces SAMPLE CHF Sample Ns.: 42-138 Submitted by: W. C. Kuryla, for Toxicology Advisory Croup Date Received: March 15, 1979 Division: Specialty Chemicals and Plastics union Carbide Corporation South Charleston, WV 25303 Identification: Tallow, viscous liquid CAS#: 1675-54-3 EHLand/ of t was A gei in A Marel chem: Safe i test XII part assaj operz l. ( A. I ~ c tt t s d c r e 0 2229 Report 43-129 Page 3 MjgLITCT Epoxy R>.ln EgL-2774 In Vitro Mutagenesis Studies; 3-Test Battery Sponsor: Union Carbide Corporation ***** OBJECTIVE Tha purposa of this study was to evaluate the potential of Epoxy Resin ERL-2774 to induce genetic damage in mammalian cells at the gene, chromosome and/or DMA (deoxyribonucleic acid) level of molecular organization. A battery of three _ln vitro, short-term tests which detect each of these genetic endpoints was employed to evaluate Epoxy Resin ERL-2774 for potential mutagenic activity. A general description of the theoretical basis of these three tests is presented in Appendices I, XI and III attached to the complete report. SAMPLE CHARACTERISTICS A commercial sample of Epoxy Resin ERL-2774 was received for testing on March 15, 1979 and it was assigned sample number 42-138. The physical and chemical information available from the Toxicology Data Bank or from "Material Safety Data Sheets" for this product are attached to this report as Appendix IV. METHODS A description of the technical procedures used in the CIO 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 development and validation of the lo-house battery of mutagenicity assays at the Bushy Rw Research Center and deviations from current standard operating 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-2774 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 a dose level which would permit survival of at least 10X of the treated cells. Class-distilled dlmethylsulfoxlde (DMSO) was used as the solvent and solvent control. To simplify tables and to allow comparisons between different tests, concentrations of Epoxy Resin ERL-2774 in the following sections of the report are given in terms of weight/volume percentages x 10"*% to eliminate zeros In the lower concentration values (eg. 0.00075 X 7.5 x lO-^Z). Report 43-129 Fag* 4 B. Mutation - CHO c*lla war* expotd for 5 hour* to a mlnimta of five concentracloaa of Epoxy Resin ERL-2774 both with and without th* addition of an S9 metabolic activation system. Two separate experiments were performed to Insure that an adequate range of doses of th* test agent were tested. Dilutions of Epoxy Resin ERL-2774 for testing were prepared by weighing the test material in a sterile tube, preparing a stock solution for the highest concentration using DMSO as a solvent and then by making sequential one-half dilutions from the stock solution using DMSO as th* diluent. The surviving fraction was determined at 20 to 24 hours after treatment and the mutant fraction was determined after a 7-day period to allow "expression'* of the mutant phenotype. Only the data from th* top five concentrations which allowed sufficient cell survival for assessment of survival and induction of mutants are usually presented in the tables. Th* percentage of cells sur viving the treatment, th* frequencies of mutant colonies and the number of mutants/lO^ viable cells are presented in tabular form. 2. SCg Teat (Detailed procedures in Appendix II): Production of SCE'a following exposure to various concentrations of Epoxy Resin ERL-2774 was studied In CHO cells without th* incorporation of an S9 metabolic activation system. Selection of a maximum dose level which would permit survival of at least 50Z at the treated cells was based on the prescreening test for cytotoxicity performed as pert of the CHO Mutation test. Dilutions of Epoxy Resin ERL-2774 for testing were prepared by weighing the test chemical In a sterile tube, making a stock solution for the highest concentration using DMSO as a solvent and then making sequential one-half dilutions of the stock solution using DMSO as the diluent. For determination of direct mutagenic action, CHO cells were exposed to Epoxy Resin ERL-2774 and appropriate controls for 5 hours without S9 activation. Indirect mutagenic action, requiring metabolic activation by liver S9 homogenate, was not studied because th* highly significant positive response obtained in the test without metabolic activation indicated that the test chemical was highly active without metabolic conversion. Bromodeoxyuridine (BrdU) required to differentiate between the Individual "slater" 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. A total of 15 cells/dose level and 5 dose levels, tested without metabolic activation, were examined. Th* number of SCE/eell, mean number of SCE/chromosome and the level of statistical significance of the increases above the 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 with eight dose levels which spanned a 333-fold range of concentra tions. Calls were treated with Epoxy Resin ERL-2774 for 2 hours in culture medium containing ^H-thymldlne, hydroxyurea and appropriate dilutions of Epoxy Resin ERL-2774 prepared in DMSO. Determination of UDS activity was O' Report 43-129 Page 5 2231 performed by analyses of Incorporation of ^H-thyaldlna into Isolated hepatocyte nuclei or In DNA (precipitated from aliquots of the isolated nuclei) using a Searle Analytic Model 81 or Packard Model 2650 scintillation spec trometer . 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 teat system and the concurrence of the results to previous teat performance. For the (SO and SCZ assays, dlmethylnltrosamine (DMN) and ethylmethanesulfonate (EMS) were used as positive control agents to assure the sensitivity of the test system for detecting indirect and direct-acting mutagens, respectively. Deionised 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 ethylmethanesulfonate (EMS) were used as positive controls representing indirect- and direct-acting mutagens, respectively. DMSO was used as the solvent and the solvent control. 5. Metabolic Activation - S9 liver homogenate, prepared from Arochlor 1254indueed, Spragua-Dawley male rats, was purchased from Litton Bionetics. The S9 preparation used for the CHO test in the first test contained 38.5 mg/ml protein and had a benzo(a)pyrene hydroxylase (BFH) activity of 21.6 nmol of hydroxybenzpyrene/20 mln/mg protein, (assayed by Litton). A final concentra tion of 480 ug/ml of S9 protein was used in the culture media. In experi ment #2, the same lot of S9 homogenate was used and 360 ug/ml of 9 protein was used as the final concentration in the culture media. For the SCE test, the same lot of S9 homogenate, was used at a final concentration of 360 ug/ml of S9 protein in the culture media. 6. Statistical Analyses - Data from the SCE and UDS tests were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Rim Research Center. Data from the CEO test do not follow a normal distribution according to experience with his torical controls. Thus, the Student's t-test was used after transformation of the mutation frequencies (MF) according to the method of Irr and Snee (MF + 1)0*15 (Irr, J. D. and R. Snee, Proceedings of the Cold Spring Harbor-- Banbury Conference, II (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. m4Z 'M Report 43-129 Page 6 7. Raw Data Storage - Copits of the final report, statistical analyses, analytical data and data used to prepare the final report are scored in the BRRC Archives. Slides are scored in cha Genetic Toxicology slide storage area. RESULTS SECTION I - CHO MUTATION TEST - BAKELITE* Epoxy Resin ERI-2774 A. Teat Dates - Initiated: June 14, 1979 Completed: September 11, 1980 B. Selection of Test Concencration (Data not shown in tables) CHO cells were exposed for five hours to concentrations of Epoxy Resin ERL-2774 which spanned a concentration range from 1000 x 10~^Z to 3 x 10~*X by wight. The percentage of cells trtiich 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. A concentration of 30 x 10"^X was selected as the maximum concentration for testing with and without S9 acti vation. A total of five concentrations were tested in the later mutation tests and the prescreening results indicated that moderately higher concen trations (100 x lO^Z) would produce excessive cytotoxicity. C. Determination of Mutation Induction 1. Survival (Cytotoxicity) Table 1 presents the cytotoxicity data for CHO cells treated with Epoxy Resin ERL-2774 in the presence and absence of a liver S9 metabolic activa tion system. Only a moderate cytotoxic effect with the test agent was observed following exposure in tests with or without 59 activation. A second test, at higher concentrations, was performed to assure that adequate dose levels of the test chemical were tested and these results are shown in Table 3. In this repeat experiment, dose-related cytotoxic effects were obtained for tests both with and without S9 metabolic activation. 2. Mutation Table 2 presents the data for induction of mutants by Epoxy Resin ESL-2774 and control agents. Epoxy Resin ERL-2774 did not produce a doserelated effect on the frequency of mutants/10^ viable cells over the 16-fold range of Epoxy Resin ERL-2774 concentrations tested both with and without the presence of an S9 metabolic activation system. NO concentration of Epoxy Resin ERL-2774 produced an increase In the mutation frequency which was statistically significant from the concurrent solvent control. Small numerical increases in the mutant frequency obtained at some dose levels wre not considered to be biologically significant because occasional increases of similar magnitude have been obtained in previous tests with solvent or negative controls. However, relatively low mutation values were UCC 082668 Report 43-129 Pag* 7 \ 2333 obtained with both positive control# and the absence of cytotoxicity at the highest dose level suggested that higher concentration# should be tested to assure the reproducibility of the data* In the repeat experiment, summariz ed on Table 4, a single, highly statistically significant increase in the mutation frequency was obtained in the test which Included addition of a metabolic activation system* This result was definitely outside the range of the historical control values for this test but no dose-related effect was observed* Also, the induction of mutants at this same concentration in experiment #1 (Table 2) did not demonstrate this mutagenic effect. NO definitive classification of the test agent was possible following these equivocal responses and the results of the other mutagenicity tests were used to discern the existence of a possible pattern of mutagenic potential. In experiment #1, mutation frequencies for the solvent controls for tests both with and without S9 activation were in an acceptable and low range based upon experience with historical control values. In the repeat experiment (Table 4), the value for the DMSO solvent control was abnormally high in the test without S9 activation. The lack of significant numbers of mutants Induced by the test chemical indicated that this high result for the solvent control probably did not influence the statistical evaluation of the test chemical data. Statistically significant increases in the mutation frequencies were obtained for the DMN and EMS positive controls for both experiments #1 and #2 and these values were within the expected range of values observed in historical control data. 0. Deviations from Standard Procedures Experiment fl. A dilution error for the DMSO-treated cells was made during the survival determination and comparisons to the negative (H2O) control were used to calculate relative differences. Only 0.9 x 10$ cells (total) were plated for the determination of mutation induction by DMN because cells treated with DMN grew poorly and not enough cells were available to assess the usual number of 1.0 x 10* Experiment #2. Aliquots of the diluted test sample were reduced in this second experiment in comparison to experiment #i because a different dilution series was used. Thus, the volume of the solvent control was reduced from 20 ul/ml to 10 ul/ml. An extended 13-day expression period was used for this experiment to attempt to Improve the sensitivity of the assay when we noted that a high degree of cytotoxicity was retarding the growth rate of the treated cells. E. Conclusions Epoxy Resin ERL-2774 was apparently Inactive as a mutagenic agent for CH0 ceils when tested with and without the incorporation of an S9 metab lie activation system over a wide range of concentrations. Only one statisti cally significant Increase above the concurrent solvent control was produced at the lowest dose level (30 x 10"*%) tested with metabolic activation in the repeat test. The high degree of statistical significance of this single result, in comparison to the concurrent control, was also outside the range of historical control values for this test, and prevented a definitive classification of positive or negative mutagenic potential. *1234 Report 43-129 Pag* 8 SECTION II - SCE TEST - BAKELITE* Epoxy Retln ERL-2774 A. Teat Data* ~ Initiated: December 5, 1979 Completed: March 24, 1980 B. Selection of Teat Concentrations A maximum concentration of 120 x 10~*X of Epoxy Resin ESL-2774 vaa chosen as the top dose level for testing with and without S9 activation respectively, based on cytotoxicity data from the CHO mutation test. Higher concentrations wet* expected to produce delays in the mitotic cycle and to decrease the number of cells with SCE staining. A 16-fold range of Epoxy Resin ERL-2774 concentrations from 120 x 10-4* to 7.3 x 10"*X was examined in the SCE experiment without S9 activation. Testing with the addition of a metabolic activation was not performed because the results of this first study cldarly indicated that the test chemical was highly active without metabolic conversion. C. Determinations of SCE Induction The data for SCE Induction in CHO cells treated with various dose levels of Epoxy Resin EBL-2774 or with positive, negative or solvent control agents without an S9 metabolic activation system are summarized in Table 3. Highly statistically significant increases in the SCE frequency were produced by all five of five dose levels of Epoxy Resin ERL-2774 tested for direct action in the absence of a metabolic activation system. Also, the doserelated effect of the test chemical on the frequency of SCE was considered a definitive indication of a biologically significant effect and an indication of the direct action of Epoxy Resin ERL-2774. The number of SCE produced 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 compar able to our historical positive control data. The numbers of SCE obtained with the solvent and controls were also in an acceptable rang* of values included in the variability encountered in our historical control values for this test. : D. Deviations from Standard Procedures - Two prior experiments were performed but are not reported because cytotoxicity of the test chemical reduced the mitotic index and the chromosomes could not be scored for SCE. E. Conclusions Epoxy Resin ERL-2774 produced highly statistically significant increases in the frequency of SCE over the 16-fold rang* of concentrations tested without addition of an S9 metabolic activation system. Definite and dose-related effects of Epoxy Resin ERL-2774 exposure on the SCE frequency were evident and the test agent was considered to be highly active in the present in vitro assay. UOC 062670 2235 Report 43-129 Pag* 9 SECTION III ~ OPS TEST - Epoxy Resin ERL-2774 A. Test Dates - Initiated: August 23, 1979 Completed: March 31, 1980 B. Selection of Test Concentrations Epoxy Resin ERL-2774 was tested over a wide range of concentrations from 1000 x 10~^I to 3 x 10"^X by weight. The maximum dose-level was selected with consideration of the cytotoxicity data obtained in the CHO Mitatlon test which indicated that higher values would result in excessive cell killing. C. Determination of OPS Induction Values for "unscheduled" incorporation of ^H-thymidine into the DKA of hepatocytes exposed to Epoxy Resin ERL-2774 or to appropriate positive and negative controls are presented in Tables 6 and 7. In hepatocytes treated with Epoxy Resin ERL-2774, two concentrations tested for potential activity induced a statistically significant increase in the amount of thymidine incorporation when UDS was assessed by measuring incorporation in whole cell lysates (Table 6). The stimulation of the UDS values was also observed when the amounts of ENA in the lysates were determined and values were expressed as radioactivity per mlcrograa of DNA; but the increases were not statistically significant by this second method of measurement. The degree of statistical significance of the UDS Induced by the test chemical and the similarity of the Increases to values observed with the concurrent positive controls indicated that Epoxy Resin ERL-2774 produced a probable, biologically significant effect in the hepatocyte UDS test. Decreased values for ^H-thymidine incorporation at dose levels of 30 x 10"4 Z or above are a probable indication of the cytotoxic effects of the test chemical. Both of the positive control agents, NQ0 and DMN, induced numerically elevated and statistically significant Increases in UDS over values obtained with the solvent control. Recent Improvements in this test system have Increased the sensitivity of measurements but this study was not repeated because the results of this test were considered unequivocal for evaluating the significance of the data. D. Deviations from Standard Procedures - One previous test was performed but was not reported because the positive controls did not produce a significant response for an acceptable test. Testing of Epoxy Resin ERL-2774 was per formed during the development of this procedure and an older, less-sensitive procedure was employed which differed from the current method described in Appendix III. The new procedures (not used for the test reported) Involve incubating hepatocytes in suspension vs. in petrl plates and isolating nuclei prior to DNA precipitation. A repeat test with this newer method would presumably detect even higher levels of activity which would not affect the positive conclusions from these test results. 2236 Report 43-129 Pago 10 E. Conclusion Epoxy Reala ERL-2774 stimulated a highly significant increase in the incorporation of radioactive thymidine in hepatocytes created with 2 of 8 test concentrations which did not produce overt cytotoxicity. Epoxy Resin ERL-2774 was considered to be active in the present test with the hepetocyte test system. 2237 Report 43-129 Pag* U Reviewed and Approved by: , s4$JU3A*xiJia^ Ronald S. Sleslnski, Ph.D. Study Director Manager, Genetic Toxicology t----Elton R. Honan, Ph.D. Associate Director, Toxicology Fred R. Frank, Ph.D. Director Contributors: Chinese Haaster Ovary tear Sister Chromatid Exchange test Unscheduled DMA Synthesis Assay Peggy J. Guzzle, B.S. Master Technologist Michelle W. Gaunt, B.S. Master Technologist W. Christopher Rangier, M.S. Assistant Scientist WPC/1123-4 2238 Report 43-129 Page 12 C hinese H am ster O vary (CHO) M u ta tio n A ssay: Determ ination o f Toxic E ffe c ts o f Chemical Treatment During 5 Ur M utntion In d u ctio n Period REFERENCES A search of Che major computer data files concerning potential mutagenicity/ carcinogenicity of Epoxy Resin ERL-2774 produced only one pertinent reference. Andersen, et al., (see reference #1 below) reported that Eplkote resin 843 (Shell Chemicals), the diglycldylether of blsphenol A, was the "most active compound of Che resins tested, and . . . more mutagenic than the positive control, ECH (epichlorohydrin)" in tests with the Salmonella Anes Test, in those studies, Eplkote resin 848 was mutagenic both with and without S9 metabolic activation, but mutagenic effects were more definitive and greater when S9 metabolic activation was present. This report quoted other studies demonstrating positive carcinogenic potential for additional epoxy resins (2) and a negative dermal carcinogenicity study (3) reported by our laboratory on Epoxy Resin ERL-2774. Positive dermal carcinogenicity and mutagenicity were observed in a study on this chemical performed at Oak Ridge National Laboratory (4). 1. Andersen, M., Riel, P, Larsen, H. and Maxlld, J. (1978). Mutagenic action of epoxy resins. Nature 276, 391-392. 2. Kotin, P. and Falk, H. L. (cited in reference above) (1963). Radlat. Res. Suppl. 3, 193-211. 3. Well, C. S., Condra, N., Haun, C. and Strlegel, J. A. (cited in #1) (1963). Am. Ind. Hyg. Assoc. J., 24, 305-323 (from data in CHF Report #23-99 and #27-152). 4. Holland, J. M., Gosalee, D. G., Gipson, L. C. and Whitaker, M. J. Epidermal carcinogenicity of bisf2,3-epoxycyclopentyl)ether,2,2-bis (p-glycldyloxyphenyl)propane, and m-phenylenediamine in C3H and C57BL/6 male and female mice. Oak Ridge National Laboratory Report, March 1976, Contract 7w=T405-eng-26. a iiiumstitf oamster ovary (C1IO) Mutation Assay: Determination of Toxic Effects of Chemical Treatment During S Ur Mutation Induction Period Experiment #1 Test Chemicals Total # Colonies (Epoxy Kesln BEL-27741 (I, w/v) 30.0 x 10"* 15.0 x 10"* 7.5 x 10~* 3.75 x 10"* 1.875 x 10~* Controls DMSO (20 ul/ml) - Solvent U20 (20 ul/ml) EMS (200 ug/ml) 429 500 8 390 520 889 543 445 (Epoxy Basin EEL-2774] (X, w/v) 30.0 x 10'* 15.0 x 10r* 7.5 x 10~* 3.75 x 10-* 1.875 x IQ-* Controls DMSO (20 ul/ml) - Solvent1 W20 (20 ul/ml) DHli (3700 ug/ml) 393 534 425 470 502 461 368 Total f Cells Plated Without S9 Activation 800 800 800 800 800 800 800 800 With S9 Activation 800 800 800 800 800 800 800 X Survival 53.8 62.5 1.0 46.8 65.0 lll.l 67.9 55.6 49.1 66.8 53.1 58.8 62.8 57.6 46.0 X of Solvent Control 48.3 56.2 0.9 43.9 58.5 100.0 61.1 50.1 See Footnote 1 85.2 115.8 92-2 102.0 108.9 -- 100.0 79.8 1A dilution error was made at this step and values in last column were calculated as a percentage of the negative control. Abbreviations: N2O ~ water; S9 - liver homogenate; DMSO - dimethylsulfoxide EMS - ethylmethaneaulfonate; DMM - dimethyinitrosamlue ia p o rt 43-129 ? . UPC/112S-2 *0 fO CO CD Table 3 Chinese Hamster Ovary (C1IO) Mutation Assay: Determination of Toxic Effects of Chemical Treatment During 5 Ur Mutation Induction Period Experiment 12 Test Chemicals Total I Colonies Total 1 Cells Plated (Epoxy Besin BEL-2774J (Z, w/v) 100.0 x 10"* 50.0 x IQ-* 25.0 x 10-* 12.5 x 10~* 6.25 x to-* 2 0 131 328 457 Without S9 'Activation 800 400 400 400 400 Controls DMSO (20 ul/ml) - Solvent U2O (20 ul/ml) EMS (200 ug/ml) (Epoxy Basin EBL-2774) (X, w/v) 100.0 x 10-4 60.0 x 10-4 30.0 x 10"4 398 465 424 138 23S 295 400 400 400 Uith S9 Activation 800 800 800 Controls DMSO (10 ul/ml) - Solvent H20 (10 ul/ml) DHN (3700 ug/ml) 221 222 105 400 400 400 Abbreviations: H2O - water; S9 - liver homogenate; DMSO - dimethylsulfoxide EHS - ethylmethanesulfonate; DHN - dimethylnitrosamlne X Survival 0.2 0 32.8 82.0 114.2 99.5 116.2 106.0 17.2 29.6 36.9 55.2 55.5 26.2 X of Solvent Control 0.3 0 32.9 82.4 114.8 100.0 116.8 106.5 31.2 53.8 66.7 100.0 100.5 47.5 Report 43-129 Pag* 15 UPC/1125-2 Table 2 Chinese Hamster Ovary (CHO) Nutation Assay: Results on Evaluation of Mutant Induction by,Epoxy Resin EEL-2774 Experiment # 1 Test Chemicals Plating Efficiency Total # Colonies Total f Cells Plated Viable Fraction Total # Mutant Colonies Hutstion Induction Total f Cells Plated Mutants^ 10*> Viable Cells (Epoxy Resin ERL-27741 (X, w/v) 30.0 x 10*4 449 15.0 * 10"4 264 7.5 x 10~4 270 3.75 x 10"4 310 1.875 x 10-4 249 Without S9 Activation 400 1.122 3 400 0.660 0 400 0.675 1 400 0.775 0 400 0.622 0 1 X 10* 1 X 106 1 X 1Q6 1 X 106 1 X 10* 2.7 0 1.5 0 0 Controls: SMS0 (20 ul/ml) - Solvent HyO (20 ul/ml) EMS (200 ug/ml) 363 379 289 400 0.908 0 400 0.948 3 400 0.722 27 1 X 106 1 X 10* 1 X 1Q6 0 3.2 37.4b fEpoxy Resin ERL-2774) (X, w/v) 30.0 x 10*4 314 15.0 x 10-4 325 7.5 x 10~4 395 3.75 x 10-4 277 1.875 x 10-4 259 With S9 Activation 400 0.785 400 0.812 400 0.988 400 0.692 400 0.648 6 2 1 0 2 1 X 106 1 X 106 1 X 106 l X 106 1 X 106 7.6 2.5 1.0 0 3.1 Controls: DKS0 (20 ul/ml) - Solvent ByO (20 ul/ml) OHM (3700 ug/ml) 410 336 227 400 1.025 0 400 0.840 2 400 0.568 9 1 X 106 l X 106 0.9 x 10* 0 2.4 17.6* Report 43-129 Page 14 Nq iTotal f mutant colonies per 10& cells plated divided by viable fraction. Statistical significance above solvent control: a: 0.05 > p > 0.01; b: 0.01 > p > 0.001; No superscript indicates p > 0.05. Abbreviations: HjO - water; S-9^-^1 iver 'homogenate; DHS0 - dim thyiaulfoxide; DB e t hylme"thanes*ulfiTatt; 0MM - dimethylnitrosamine. WVC/U25-1 laoie s Chinese Haaster Ovary (CUO) Mutation Assay: Deteralnation of Toxic Effects of Chemical Treatment During 5 Hr Mutation Induction Period Experiment 12 Test Chealcals Total 1 Colonies Total Celia Plated (Epoxy Besln EEL-2774) (Z, u/v) 100.0 x 10-* 50.0 x 10~* 25.0 x 10~* 12.5 x 10~* 6.25 x 10~* 2 0 131 326 457 Uithout S9 Activation 800 400 400 400 400 Controls DMSQ (20 ul/sl) - Solvent H20 (20 ul/al) EHS (200 ug/al) 396 465 424 400 400 400 (Epoxy Eesln EEL-2774) (X w/v) 100.0 x 10-4 60.0 x 10-4 30.0 x 10*4 . 138 238 295 Uith S9 Activation 800 800 800 Controls DHS0 (10 ul/ml) - Solvent HjO (10 ul/al) DMN (3700 ug/al) 221 222 105 400 400 400 Abbreviations! H20 water; S9 - liver boaogenate; DKSO - diaetbylsulfoxide EHS etbylaethanesulfonate; DMN - diaethylnltrosaalne X Survival 0.2 0 32.8 82.0 114.2 99.5 116.2 106.0 17.2 29.8 36.9 55.2 55.5 26.2 X of Solvent Control 0.3 0 32.9 62.4 114.a 100.0 116.6 106.5 31.2 53.8 66.7 100.0 100.5 47.5 UPC/1125-2 Report 43-129 ucc 062678 * h Table 4 Chinese Hans ter Ovary (CI10) Nutation Assay: Results on Evaluation of Hutant Induction by Epoxy Resin ERL-2774 Experiment I 2 1 Plating Efficiency 1 Test Chemicals Total # Colonies 1 [Epoxy fees to EEL-2774| (2, "575* 100.0 x 10"4 406 50.0 x ur* - 25.0 x 10-4 443 12.5 x 10"4 149 6.25 x UP* 234 Total f Cells Plated Viable Fraction Total f Hutant Colonies Without: 89 Activation 400 1.015 0 SEE FOOTNOTE #2 - 400 1.100 11 400 0.372 0 400 0.505 * 0 Nutation Induction Total I Cells Plated % 1 x 106 - 1 X 10* 1 x 106 1 x 106 Mutants1 10*> Viable Cells 0 - 9.9 0 0 Controls: DMS0 (26 ul/ml) - Solvent H20 (20 ul/ml) EHS (200 ug/ml) 170 - 440 400 0.425 11 SEE FOOTNOTE 12 - 400 1.100 1 216 1 x 106 - 1 x 10* 25.9 - 196.4C [Epoxy Resin ERL-2774J (X, w/v) 100.0 x 10~4 185 60.0 x 10-4 299 30.0 x 10"4 373 With S9 Activation 400 0.462 l 400 0.748 9 400 0.932 77 1 x 106 1 x 10* 1 x 106 2.2 12.0 82.6C Controls: DMSO (10 ul/ml) - Solvent H2O (10 ul/ml) DMN (3700 ug/ml) 189 232 129 400 0.472 3 400 0.580 10 400 0.322 | 78 1 x 106i 1 x 106 1 x 106 6.3 17.2 241.9C *R>tal I nutant colonies per 10& cells plated divided by viable fraction. Statistical significance above solvent control: c: p < 0.001 Mb superscript Indicates p > 0.05. Oats analyzed by Student's t-test. ^Lost during expression period following Incubator malfunction during routine trypslnlzation step. Abbreviations: V*VC/ 1125 2 II2O * water; S-9 - liver homogenate; Wtso' - dimethylsulfoxide; EHS - ethylaethanesulfonate; DMN - dlaethylnltrosaalne. ucc 062679 Table 5 Report 43-129 Page 16 rs Sister Chroutid Exchange (SCE) Assay; Induction of SCE'a by Epoxy Resin (ERL-2774) Without S9 Metabolic Activation 5 Hour Treat*eat ' Teat Chemicals Total # of Chromosomes Total 1 of SCE SCE/Cell* Mean Number SCE/Chromo some2 + S.D. (Epoxy Resin ERL-2774) (X, w/v) 120 x 10"* 60 x 10~* 30 x IQ"* 15 x 1Q"* 7.5 x HP* Controls DHS0 (5 ul/nl) -Solvent d20 (5 ul/nl) EMS (100 ug/nl) 309 299 302 299 300 291 294 298 669 57.93 2.793 + 1.010 613 40.67 2.049 + 0.256 446 29.67 1.490 + 0.344 357 23.80 1.193 + 0.249 306 20.40 1.022 + 0.272 182 12.13 0.628 + 0.214 200 13.33 0.680 -V 0.169 423 28.20 1.417 + 0.242 1Fifteen cells examined per dose level. ^Mesn value of SCE/chromosome determined fro* tike values of the Individual cells examined ^Statistical significance above solvent control; c; p < 0..001; NS; p > 0.05. Abbreviations; H2O - water; S9 - liver homogenate; DMSO - dimethylsulfoxide; ENS - ethylmethanesulfonate; S.D. - standard deviation UPC/l125-2 Significance Above Solvent Control^ c c c c c - NS c & Report 43-129 l t c Sc OJ'^> Table 6 Unscheduled OKA Synthesis In llepatocytes froa Hat Liver DPH/1Q6 cells, all DPM values are calculated froa DNA precipitated per 106 viable hepstoeytes. Each average is calculated froa duplicate saaples, except for DHSO which was done In quadruplicate. Test Chemical Solvent - DHSO Positive Control#; EMS OHM Teat Cheaical: (Epoxy Reein ERL-2774) (Z, w/v) Concentration 3.3X 400 ug/al 200 ug/al 100 ug/al SO ug/al 60 ug/al 40 ug/al 20 ug/al 10 ug/al 1000 x 1Q"*X 600 x 10~*Z 300 x 1Q~*X 100 x 10-*X 60 x 10-*X 30 x 10-*X 10 x 10~4Z 3 x 10*X . Radioactivity in DMA Avg. DPM + S.D. 22848 + 926 28618 + 4161 28573 1- 3735 32107 + 1083 26715 + 549 28753 + 3744 27290 + 1686 30930 + 859 24270 + 1468 4100 3290 5328 11825 8956 15371 38488 35198 247 27 126 1876 1956 2438 240 2793 X of Solvent Control + S.D. 100.OZ + 4.1Z 125.3X + 18.2Z 125.1Z + 16.3Z 140.51 -1 4.7Z 116.91 + 2.4Z 125.8Z 1- 16.4X U9.4X + 7.4Z 135.4X + 3.8X 106.21 + 6.4Z 17.9Z + 1.1X 14.4X + 0.1Z 23.3Z + 0.6Z 51.81 + 8.22 39.2X + 8.6Z 67.31 + 10.7X 168.5X + 1.1Z 154.1Z + 12.22 Significance Above Solvent Control^ - b b c a b a c NS MS NS NS NS NS NS c c ^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. ------- 1 Abbreviations: DHSO - diaethylsulfoxide; EMS - ethyluethane sulfonate; DMN - dlnethylnltrosamlne; DPM - disintegrations per alnute; S.D. - standard deviation UPC/1125-2 i Page .18 Report 43-129 *-&* r- * UPC/1125-2 Table 7 Unscheduled OKA Synthesis in tfepatocytes fro* Rat Liver DPM/ug DMA: all DPH values are calculated per DNA precipitated from rat hepstoeytea. Each average la calculated fro* duplicate samples, except for DMSO which wea done In quadruplicate. Test Chemical Solvent - DMSO Positive Controls: ns DMN Test Chemical: (Epoxy Resin ERL-27741 (X, w/v) Concentration J3I 400 ug/ml 200 ug/ml 100 ug/ml 50 ug/ml 80 ug/ml 40 ug/ml 20 ug/ml 10 ug/ml 1000 x 10_*X 600 x 10"*X 300 x IQ-*X 100 x 10-*Z 60 x 10"4X 30 x 10-*X 10 x 10"*X 3 x 10"*X Radioactivity In DMA Avg. DPH + S.D. 84? + 201 1474 + 1491 + 1210 + 1157 + 330 124 2 134 1252 + 1394 + 1673 + 1639 + 103 20 351 292 157 + 146 + 206 + 570 + 476 + 809 + 1282 + 1220 + 27 20 18 135 3 52 434 597 X of Solvent Control + S.D. 100.OX + 23.7X 174.OX + 38.92 175.9X + 14.62 142.82 + 0.2X 136.5Z + 15.92 147.8X + 12.2Z 164.52 + 2.32 197.42 + 41.4X 193.42 + 34.42 18.62 + 3.22 17.22 + 2.32 24.3X + 2.IX 67.22 + 15.91 56.IX 4- 0.3X 95.52 + 6.IX 151.22 + 51.22 143.9X + 70.42 Significance Above Solvent Control1 - a a NS NS NS a c c NS NS NS NS NS NS NS NS ^Statistical significance'above solvent control: a: 0.05 > p > 0.01; c: p < 0.001; NS: p > 0.05. Data analyzed by Duncan's Multiple Range Analysis. Abbreviations: DMSO - dinethylsulfoxlde; EMS - ethylmethane sulfonateDMN~-"dimethyl'll ltro saw ine; DPH - disintegrations per minute; S.D. - standard deviation WPC/l125-2 ucc 062S82 Report 43-129 Page 19 fn 2246 Pag* 1 of 5 APPENDIX I Chinas* Haaatar Ovary (CHO) Mutation Aaaay The...o..r.e..ticai l Basis Mutation is a heritable alteration in a cell in which a gen* specifying the genetic cod* for a specific protein la modified in structure and/or function. Mutations, Induced by chemical or physical agents, of th HGPRT (hypoxanthinaquanta* phosphoribooyltransferase) gen* may be detected by th* growth of colonies of "mutant" cells which ar* resistant to th* purln* analogs 6-thloguanin* (TG) or 8-azaguanine. Normal cells contain a functional HGPRT enzym* which phoaphorylataa TG and allows its incorporation into DNA causing the cells to die. Mutant cells with a non-functional HGPRT enzyme are unable to phospborylat* or Incorporate TG, thus survive and grow in Its presence. Th* CHO mutation teat is an assay which detects "forward mutations" from TG-sensitivity to TG-realstanc* caused by a direct loss of th* activity of th* HGPRT enzym* (HGPRT* -* HGPRT*). An assessment of th* ability of several hundred agents to eaus* gen* mutations in vitro indicates that th* CHO mutation assay provides a reasonable estimate of th* potential genetic activity of the test chemical. Methods Cell Culture Procedures; CHO cells used in thes* studies wr obtained from Abraham Hsi* at Oak Ridge National Laboratory with th* designation CH0-K1-SH4-DI (or simply CHO for report purposes). Cells ar* maintained in active growth by subculturing 2 to 3 times/week in antiblotlc-fr**. Ham's Modified F12 Medium supplemented with lOt (v/v) heat-inactivated, fetal bovine sera (F12-10), and lacking in hypoxanthine. For treatment of cells without metabolic activation, F12 medium with 30 unlts/ml of penicillin, 50 ug/ml streptomycin and 5X (v/v) of dialyzed bovine serum (P12-05) la used. For treatments Incorporating an S9 metabolic activation system, identical medium, but without serum, is employed. For determination of mutant frequencies, F12-D5 medium containing 2.0 ug/ml TG (6-thioguanlne) is used as a "selective medium." Cell numbers ar* determined routinely with a Coulter Model F electronic cell counter which is standardized periodically with a pro-counted suspension of latex beads. Presence of Mycoplasma cell contaminants is determined by a microscopic fluorescence assay employing Hbechst 33258 dye* All culture procedures and treatments with test chemicals ar* performed under aseptic conditions in a laminar-flow, biohazard hood. Poaitiv* and Negative Controlst Sterile water or glass-distilled dimethylsulfoxide (>ta&0) are the usual solvents for test chemicals and th* respective solvent is tested as a control at the maximum concentration used to add the test agent. Dimethylnitrosamine (DMH) or ethylmathaneeulfonate (EMS) are used aa positive control mutagens for testa with or without aa S9 metabolic activation systam, raapectlvely. Mutation frequencies obtained with concurrent positive and negative control* ar* used as th* besia for monitoring the sensitivity and stability of th* CHO mutation east systam. Comparison of concurrent control values with historical controls is used to delineate the range of acceptable variations in the test system. Appendix I Page 2 of J 2247 Metabolic Activationt Ret liver, 39 homogenate prepared from Arochlor-1254 induced, Sprague-Dawley, sale rat* is purchased froa Litton Bloaetlcs, Kensington, MD. Each lot of liver hooogenate Is prescreened for aetabolic capability to activate DMN In our laboratory before use in the testing program. The couplet* 39 aetabolic activation system contains the following: 8 uooles/al Mg&2 33 uaoles/ml KC1, 3 uaolea/ol glucose-6-phosphate, 4 umoles/ml KADP-oxldized (nicotinamide adenine dlnuclaotlde phosphate), 100 uaoles/ml NayHFO^ and between 300 to 4000 ug/al of S9 protein (depending on aetabolic 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 assessment of mutagenic potential to select doses which produce a maximum of 80 to 90Z cell killing. Cytotoxicity is determined by either of the 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 0.1X 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 la determined by counting the number of colonies produced after a 7- to 8-day Incubation period (37*0 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 23 cm^ culture flasks are treated for 5 hours with a minimum of five case concentrations both with and without S9 metabolic activation. Following treatment the cells are rinsed, fresh P12-D3 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 cells treated with various concentrations of the test agent. If no cytotoxicity is evident at the highest concentrations in the cytotoxicity teats, the test Is either repeated at higher concentrations, or mutation testing la 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 la 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 mutation*. If cytotoxicity data are equivocal, a total of 3 t 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 cell* are assessed for mutation Induction. 2248 Appendix Peg* 3 of 5 Chemical samples *r* 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 appropriate solvent by making a 10 to 20Z stock solution (by weight) and subsequent dilutions are mad* from this stock on a volume/volume basis Treatment with Test Chemicals: 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 23 c&2 culture flasks containing F12-D5 medium and Incubated at 37*C in a 3 to 6Z CO2 atmosphere. Appropriate concentrations of the test agent or control chemicals are added to the cells and cultures are treated for 3 hr at 37*C. The medium and test agents are removed by suction, cells are rinsed once or twice and fresh F12-D5 medium is added. The cells are allowed a period of 20 to 24 hours of recovery from treatment before survival is determined. Treatment of cells for testing of chemicals which require metabolic activation for mutagenic capacity is performed identically with the procedure above, with the exception that FI2 medium without serum and containing 1.0 ml of S9 activation mixture per 4.0 ml of medium is employed. Determination of Cytotoxicity; The relative survival of treated calls, in comparison to solvent controls, is determined one day after the exposure to the test agents. The level of cytotoxicity is oftsn correlated with the mutation frequencies induced by known chemical mutagens. Thus, excssslve cytotoxicity may kill both normal cells and mutants and may depress the actual mutation fre quencies; insufficient cytotoxicity may indlcata an insufficient concentration of the test agent was employed. The colonyforming potential of 100 to 200 treated cells is used as the measure of treatment-induced cytotoxicity. Survival value* which indicate the cytotoxic effects of the teat 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 teat aganta, approximately 5x10* cells ars subctxlcured in 100 bh tissue culture dishes in F12-D5 medium and incubated at 37*C in a 3 to 61 C0j atmosphara. After a total of 7 days to allow "expression" of the mutant phenotype, cells sre dissociated with 0.03 to 0.073Z trypsin, countsd and plated at a concentration of 2.5x lQ*/dish in four culture dishes (lx 10$ total cells) which each contain 5 ml of F12-D5 (TG) aelective medium. At this time, cells are diluted and 100 cells/dish are added to four culture plates containing F12-D5 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 calls; medium Is then discarded and colonies are fixed and stained for counting. The number of colonies in selection plates and in cha viability tsat are counted by electronic methods, chseked by msnual counts and data are recorded both aa total mutants, mutant#/10$ total cells and mutants/10$ viabls cells. Appendix I Pag* 4 of 5 l 2249 Statistical Analyses: Uniform statistical procedures to evaluate in vitro mutation 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 3) 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 0.13 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-eest 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 negativeresponse depend upon both the level of statistical significance from the concurrent control and the evidence of a dose-response fallowing treatment. When a definite dose-response relationship is not evident but on* 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 3 mutants/10^ viable cells, with a range of 0 to 23 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. I ! 2250 Appendix I Psg 3 of 5 References 1. Chu, E. H. ? end 8, V. Mailing* Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells In Vitro. 6i (iWT 1^06-1312. ------------------------------- Proc. Natl. Acad. Sci. U.s.A, > 2. O'Neill, J. P., P. A. Brlmer, 8. Machanoff, <S. P. Hirsh, A. W. Hsie. A Quantitative Assay of Mutation Induction at the Hypoxanthine-Guanine ~ Phoaphorlboayl Transferase Locus In Chinese Hamster Ovary Cells (CHO/HGPRT S^tan):^^Development and Definition of the System. Mutation feeaeamh. LT 3. O'Neill, J. P. and A. W. Hsie. Phenotypic Expression Time of Mutagen* Induced 6-Thloguanine Resistance in Chinese Hamster Ovary Cells (CHO/HGPRT System), tfatation Research, 59, (1979). 109-118. "" '----- A Irr, J 0 and 8. D Snee Statistical Evaluation of Mutagenicity la the CHO/HGPRT System. Proceedings of the Cold Spring Harbor-Banbury" Conference II (1^79), 263-274. 5. Box, C* E. P. and D. 8. Cox. An Analysis of Transformations. J. of the Royal Statistical Society, B, 2^(1964), ill-iil. WPC/1033 UCC 062687 Pag* 1 of 4 <c<cox APPENDIX II Determination of Sister Chromatid Exchange (SCE) Frequencies In Chinese Hamster Ovary (CHO) Calls In Vitro ;T,h--a--a--r-a--t-l-e--a--l---B--a--s--is- j Exchanges of genetic material batvaan the Individual arms of a chromosome j(i.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 calls treated iwith 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 test agent has been suggested as a sensitive screening test for potential mutagenlc/cardnogenle chemicals. The method used In our study to visualize SCE's In CSO cells grown In cul ture Is based on the procedure described by Perry and Volff (1974). A standard concentration of 3.0 ug/ml of bromodeoxyuridine (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 3.0 ug/ml of 33258-Eoechat fluore scent dye, exposure to light and Glemsa staining was used to differentiate chromatids for SCE analysis. Methods Call Culture Procedures: Chinese hamster ovary (CEO) cells were obtained from Abraham Hsle at Oak Ridge Natioaal Laboratory with the designation CEO-K1BH4-D1 (referred to simply as CEO for report purposes). CEO 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 fetal 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 Hoechst 33238 dye. For treatments with test chemicals without S9 metabolic activation, modified F12 medium is used with 50 unlts/ml of penicillin, 50 ug/ml streptomycin and 5Z (v/v) of heat-inactivated, dialyzed fecal bovine serum (F12-D5). Identical medium but without serum- is used for treatments Incorporating an S9 metabolic activation system. Positive and Negative Controlst 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. Dlmathylnitrosamlne (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 comparison and for demonstrating che sensitivity and stability of 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* Appendix II Page 2 of 4 Metabolic Activation; Sat livar S9 homogenate (prepared from Arochlor 1254 induced, Sprague-Dawlay, male rata) la purchaaed from Litton Bionetica, Kensington, MD. Each lot of liver homogenate ta prescreened for activity in our laboratory before use la the testing program. The complete S9 metabolic activation system contains the following: 8 uaoles/ml MgCl2, ^3 umoles/ml KC1, S umoles/al KC1, 5 uaoles/ml glucose-d-phosphate, 4 uaoles/ml MADP-oxidi2ed form (nicotinamide adenine dlnueleotlde phosphate), 100 umoles/ml and between 500 to 4000 ug/ml of S9 protein (depending on metabolic activity). A volume of 1.0 ml of the coaplete 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 assessment of mutagenic potential to select doses which produce a maximum of 80 to 90Z cell killing. Cytotoxicity la determined by either of the following two methods as part of the CHO mutation testing procedure: (1) Clonal assay - 200 to 400 CHO cells are exposed to a minimum of five dose levels of che test agent at concentrations from 0.1X to 3 x 10"*X (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 che number of colonies produced after a 7- to 8-day incubation period (37*C) in comparison with the colonies formed by cells created 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 P12-05 medium is added and the flasks are Incubated for an additional 18 to 24 hours. Cytotoxicity Is determined by compering the relative number of cells in control (uncreated cells) and in cells treated with various concentrations of the test agent. If no cytotoxicity Is evident at che highest concentrations in the cytotoxicity tests, the teat 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 test is repeated at a concentration range of 3x10"* to 3x10"^ percent by volume. Dose levels which era moderately toxic but permit survival of at least 40 to SOX of che cells, in comparison to cha solvent control, are selected as the maximum dose, and at least four additional one-half dilutions are tasted for induction of mutations. If cytotoxicity data are equivocal, a total of 5 to 3 one-half dilutions of che 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 SC8 staining, are evaluated for SCS 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 appropriate solvent by making a 10 to 20Z stock solution (by weight) and subsequent dilutions are made from this stock on a volume/volume basis. ucc 062689 ac cl ac 1C hr Ap th 8 bu is to SC ac wi is mi 0. fc Ca fi P' si 33 am Ir ri wi TH re ar si Appendix IX Page 3 f 4 225<- Treatment With Teat Chemicals; Testing of chemicals 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 a 10* cells are plated Into 73 cm2 culture flasks in F12-D5 medium at least20 hrs prior to treatment and Incubated at 37*C in a 5 to 6X CO2 atmosphere. Appropriate concentrations of the test agent or control chemicals are added to the cells and 3 ug/al Brdff la added to all flasks. Cells are treated with test agents for 3 hrs, media la then removed by suction, cells are rinsed with buffered, physiological salt solution and fresh medium containing 3 ug/ml BrdtT 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 without 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. Cells are treated for a total of 2 hrs (rather than 3 hrs) and then Incubated for 38 to 42 additional hours before harvest for chromosome preparation. Preparation of Chromosomes: Colcemld* (0.1 ug/ml) or Colchicine (0.2 ug/ml) Is added to culture flasks I to 2 hrs prior to harvesting to arrest cells In mitosis. Calls are then removed from flasks, after a brief Incubation with 0.012 DIFCO trypsin, suspended In 0.075M KC1 (hypotonic) solution and Incubated for 13 to 20 min at 37*C. Calls are eentrlfuged, fixed with 3 or 4 changes of Camoy'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 calls are saved If needed for preparation of additional slides. Chromosomes are stained for SCZ's by treatment with 3.0 ug/al of Boechst 33238 dye for 20 min, rinsed In distilled water. Immersed In Sorenson's buffer and exposed to a high intensity sunlamp for 13 to 30 min., as required. Irradiated chromosomes are stained In Gurr's giamsa (diluted 1:23 with water), rinsed in water and dried before application of coverslips. Examination of SOS's: All slides are coded and read In a blind fashion without indication of the specific treatment or concentration of the teat agent. The number of chromosomes and the number of SCE's la a minimum of 13 cells are recorded for each dose level. The mean number of SCZ/cell and SCE/chromosome are calculated and recorded. Slides are decoded only after examination of all slides In the experiment has bean completed. ucc 062690 ^254 Appendix II Psg% 4 of 4 Statistical Analyses Data are analyzed by appropriate parametric statisti cal procedure* which follow BUG standard operating procedure* for analyses of data. Significance values and the statistical test employed are shown for data summarized in tabular form. Interpretation of Data: The criteria for evaluation of a positive or negatlve response depend both on the level of statistical significance and subjec tive analyses of concurrent and historical control data. The key determinant is whether a dose-dependent increase la SCE* a Is Induced by the test agent. When no clear doae-reeponse relationship Is svldsnt and when one or more responses of marginal statistical slgnlflcanc* are obtained, a careful examination of the data in comparison to eh* concurrent controls and historical data base is neces sary. Testing osy bs rspeatsd to clarify unusual responaas. If data for the concurrent positive or negative control* suggsst a defect In the original ex periment. Overall assessment will also rsly on corroborating data from the other teste in the testing battery. Clearly positive responses will Include any of the following: (1) Doubling in the SCE frequency at a minimum of two of the five concentrations tasted; (11) Statistically significant responses of p < 0.0S at three coneentretlons or et 2 concentrations if p < 0.01; (ill) Induction of a statistically significant, dosa-related Increase In the number of SCE. General References 1. Perry, P. end S. Wolff. New gieaee method for differential staining of sister chromatids. Nature, *251 (l$fr4)^ 156-lid.2 2. Latt, S. A., J. V. Allan, W. E. Rogers and L. A. Jusrgsna. In vitro and In vivo analysis of slstar chromatid exchange formation, pp 27S-291 la Handbook of Mutagenicity Tast Procedures, ad.' Kllbey. B. J., *t al. Elsevier Pubi.' Co. < 19791. 3. Carrano, A. V., L. H. Thompson, P. A. Lindl and J. L. Miakler. Sister 331-551: 4. Galloway, 3. M. and S. Wolff. The relation between chemically Induced 3. Snedecor, G. W. end W. G. Cochran. Statistical Methods, 6th Ed., low* Scat* Haiv. Preae, tees, low* (1967). WPC/1033 Page 1 n 2255 APPENDIX III Unscheduled DNA Synthesis (UPS) la Hepatocytes from Rat Liver Th oretlcal Basis Chemicals nay Interact with both tha cellular components and the genetic material of a cell (a.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, theoretically, 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 mettods of Seglen (1973) and Williams (1976). Bepatocytee 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 Metafana(H.!). The abdominal cavity is surgically exposed and 1230 units of heparin is injected intravenously. A catheter is Inserted into th portal vein and warm Hanka Balanced Salt Solution (HBSS) is pumped into the vein and through the liver. This first solution contains heparin and EGTA, (ethylene glycol-bis-(beta-sminoethyl-ether)N,N-tetracetic 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 lov centrifugation speed. After resuspension la 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 i ml medium 199 containing 10 mM hydroxyurea and 30 mM Hepas (N-2-hydroxysthy1 piperaslne-N-Z 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 In orporated into the nucl 1 Is expected t result from repair or unscheduled DNA synthesis. 225b Appendix III Pag* 2 of 3 Selection of Doses of Teat Chemical: Initially, eh* following eoneentracionax 1Q*JT (by' voiuaa) ara castad: 100, 30, 10, 3, l, and 0.1. If these concentration* prove to be cytotoxic, or if additional information la available 'i from other In vitro teata aa to the proper doae levela, then an appropriate aerie* of concentration* la uaed over a 3-log rang* of concentration*. Treatment of Hepatocytes: After preincubation, 23 olcroCurle* of tritiaced thymidine (26 Curiaa/mlilimole) la added to each tube. The teat chemical and poaltive control* are diluted In an appropriate aolvent and they are then added to each labeled cube. Generally, at leaat six concentration* of the teat chemical over a 3-log range of concentration* are coated and each concentration ia run in duplicate. The tube* are returned to the rocker platform for a 2-hour expoatire at 37*C. Poaitlve and Negative Control*: 4-aicroqulnolln* oxide (NQO), a directacting"mutagen,' which induce* UV-cyp* DMA repair and dlmethylnltroaamln* (DMN), which require* metabolic activation by mieroaomal enzyme* for activity, are run in duplicate aa poaitlve control chemical*. The solvent control 1* run in quadruplicate and consists of 100 to 130 microlitera (concentration specified in individual report*) of the solvent used to dilute the sample. Dimethyl-, sulfoxide (DMSO) or water are the usual solvents for test chemical*. Harvesti 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 0.23Z Triton X-100, 5Z citric acid and 3 mM MgCl2 * lysing solution which liberate* the nuclei. The nuclei are rinsed once in this solution and resuspended in 0.23 M sucrose, 2.3Z citric acid and 3 oM MgClj. The nuclei are then centrifuged at 600 x g for 10 min at S*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 HC3 tissue solubilizer in a scintillation vial. Ten ml of Dimilurn** scintillation cocktail Is added and the radioactive disintegrations per minute (DPM) 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 cables. Determination of DHA-Bound Label: The amount of radioactive thymidine in corporated into DMAris quantitated in DMA Isolated and precipitated from 1.00 co 1.25 x 10-5 viable hepatocytes. To 1.25 ml of the nuclear suspension, 2.75 ml of IZ sodium dodecyl sulfate (SDS) and 5 mM Ethylenediamlnetetraacetlc Acid (EDTA) 1* added to lyse the nuclei. The DMA is 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 minute* 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 is rinsed once with methanol, dried and placed in a scintillation vial. The filters are Incubated at 50*C for l hour with 1 ml of a diluted solution of MCS tissue solubilizer; prepared by adding 1 part solubilizer to 2 parts of Diailume# cocktail. Dlailumae is then added to each vial and the vials are counted twice in a scintillation countsr for tan minutes. Appendix Hi Page 3 of 3 2357 Statistical Analysis: The average DPM la calculated tor each dose level and the controls and final results are expressed as DPM/106 viable hepatocytes. Data are also expressed as a percent of the solvent control for purposes of comparison. The original data are statistically analyzed by the appropriate parametric test, following the BRRC standard procedures for statistical analyses and the test(s) eaployed Is Indicated on the respective tables. Coaparlson between the aean for each dose level with the 95X confidence limits of the historical solvent control may also be used in some cases to assess the potential biological significance of the data. Testing oay 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 CDS activity. If a definite dose-response relationship is not evident, or when a few Increases with marginal statistical significance are obtained, coaparlson 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 UD3 data is th 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 UV-Induced Unscheduled DNA Synthesis by Hydroxyurea. Experimental'Cell Research. 110. (TWf, 4t-6. -------- ------ 2. Mjramatsu, M. Isolation of Nuclei and Nucleoli. In: Methods in Cell Physiology, Vol. 17. 1970* Editor: D. M. Prescott. Academic Press, Mew York. 3. Seglen, P. 0. Preparation of Rat Liver Cells. III. Ensymatic Requirements for Tissue Dispersion. Experimental Cell Research, 82, (1973), 391-398. -- 4. 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, (1^76). 6. Williams, G* M. and M. 7. Laspla. The Detection of Various Nitroaaminas in the Hepatocyte Primary Culture/DNA Repair Test. Cancer Letters. 6. (197$). 7. Snedecor, G. W. and W. G. Cochran. Statistical Methods, 6th edition, Iowa State University Press, Ames, Iowa (15(47^. WPC/1033 2258 Report 43*129 APPENDIX IV Physical and Chemical Characteristics of Teat Material BRRC Chemical Nb: CAS No.: Chemical Name: Trade Name and/or Synonyms: Molecular Weight: Formula: Specific Gravity ( 25"C): Boiling Point: Solubility in HyO (Z by vt): Purity Vapor Pressure (# 20*C): pH: Flash Point: Stability: Incompatibility: Appearance and Odor: Disposal: 42-138 1675-54-3 BAKELITE* Liquid Epoxy Resin ERL-2774 Dlglycldyl Ether of Blsphenol A 340 621H24O4 1.15 to 1.17 Not applicable Insoluble Not available Not available Not available 450'F (Cleveland open cup) Stable Avoid high temperatures > 450*F and contamination with acids, amines and H2O. Clear liquid Incinerate. Spills should be absorbed and burned. Protective Measures: Health Hazard: Use rubber or plastic gloves, safety glasses and mechanical ventilation Appeared to be relatively non*toxic following acute animal exposure tests. Repeated or prolonged contact may cause skin rash. Avoid prolonged or repeated skin contact and breathing of vapors. ga* rac exc res das per adm hig In chr adn. pro 40che pen Acu to s vary be tv toxi the conc< patti furtl toxii perle Id or level