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2227 i BUSHY RUN RESEARCH CENTER R. D. 4, Mellon Road, Export, Pennsylvania 15632 Talsphons (412) 327-1020 CONFIDENTIAL: Not to be released outside UCC without the written consent of the UCC-sponsorlng 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. Sleslnskl, 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 f battery of three in vitro tests, which were: the Chinese Hamster Ovary (CHO) 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 I dose-related effect in the SCE test with CHO 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 test concentrations in which the maximum concentration would allow survival of approximately 10X of the treated cells. A maximum concentration of 30 x 10~*Z (by weight) was chosen for the highest it- 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 10-4X was used as the highest dose level. CHO ttitatlon 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 classlfl'catlon of the test chemical as mutagenic or non-mutagenic. 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 2663 Report 43-129 Pag 2 SCE Test - Epoxy Resin ERL-2774 produced a dose-ralated and highly statistically significant effect on the 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 lO^X to 7.5 x 10~*^Z (by weight) was used. All five of the concentrations tested produced a highly statistically significant effect 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 Test - 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 Resin ERL-2774 was considered to be active in the present test with the hepatocyte test 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 the 3-test 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 (EHS), a known mutagen and carcinogen. Contradictory reports exist in the literature on the potential carcinogenicity of Epoxy Resin 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 test 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 In this study would be consistent with the identification of Epoxy Resin ERL-2774 as an active mutagen. SAMPLE Quantity: 8 ounces CHF Sample No.: 42-138 Submitted by: W. C. Kuryla, for Toxicology Advisory Group Date Received: March 15, 1979 Division: Specialty Chemicals and Plastics Union Carbide Corporation South Charleston, WV 25303 Identification: Tellow, viscous liquid 222 Report 43-129 Pag* 3 BAKELITE* Epoxy Reala ERL-2774 In Vitro Mutagenesis Studies; 3-Test Battery Sponsor: Union Carbide Corporation ***** OBJECTIVE The purpose 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 DNA (deoxyribonucleic acid) level of molecular organization* A battery of three in vitro, short-term tests which detect each of these genetic endpoints ues 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 111 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 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 development and validation of the in-house battery of mutagenicity assays at the Bushy Rtn 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 10Z 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~*Z to eliminate zeros in the lower concentration values (eg. 0.00075 Z * 7.5 x 10^Z). 2230 Report 43-129 Fag* 4 B. Mutation - CBO cells were exposed for 3 hours to a minimus of five concen trations of Epoxy Resin ERL-2774 both with and without the addition of an S9 metabolic activation system* Two separate experiments were performed to insure that an adequate range of doses of the test agent were testedDilutions 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 the 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 the top five concentrations which allowed sufficient cell survival for assessment of survival and induction of mutants are usually presented in the tables. The percentage of cells sur viving the treatment, the frequencies of mutant 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-2774 was studied in CHO cells without the incorporation of an S9 metabolic activation system. Selection of a maximum dose level which wuld 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-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 the 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 "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. A total of 15 cells/dose level and 5 dose levels, tested without metabolic activation, were examined. The 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. CDS Test (Detailed procedures in Appendix III): Induction of primary DNA damage In rat liver cells (hepatocytes) was studied with eight dose levels triiich spanned a 333-fold range of concentra tions. Cells were treated with Epoxy Resin ERL-2774 for 2 hours in culture medium containing ^H-thymidlne, hydroxyurea and appropriate dilutions of Epoxy Resin ERL-2774 prepared in DMSO. Determination of UDS activity was r1 Report 43-129 Pag* 3 2231 performed by analyses of Incorporation of ^H-thymidin* Into Isolated hepatocyte nuclei or In DNA (precipitated from allquota of the Isolated nuclei) using a Searle Analytic Model 81 or Packard Model 2630 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 test system and th* concurrence of the results to previous test performance. For the CHO and SCE assays, dlmethylnitrosamine (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. Deionized water, sterilized by membrane filtration, and glass-distilled dlmethylsulfoxide (DMSO) were used as the negative and solvent controls, respectively. In tha 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 1254induced, Sprague-Dawley male rats, was purchased from Litton Blonetics. The S9 preparation used for the CHO test in the first test contained 38.3 mg/ml protein and had a benzo(a)pyrene hydroxylase (BPH) 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 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 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 HarborBanbury 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. N6 superscript (or NS) indicates p > 0.05. 062667 2232 Report 43-129 Page 6 7. Raw Data Storage - Coplea of Che final report, statistical analyses, analytical data and data used to prepare the final report are scored in the BRRC Archives. Slides are scored in the Genetic Toxicology slide storage area. RESULTS SECTION I - CHO MUTATION TEST - BAKELITE* Epoxy Resin ERL-2774 A. Test Dates ~ Initiated: June 14, 1979 Completed: September 11, 1980 B. Selection of Test Concentration (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~^X to 3 x 10*^X by weight. The 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. A concentration of 30 x 10~^Z 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 10~^X) 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 QtL-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 S9 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 testa both with and without S9 metabolic activation. 2. Mutation Table 2 presents the data for induction of mutants by Epoxy Resin ERL-2774 and control agents. Epoxy Resin ERL-2774 did not produce a doserelated effect on the frequency of mutants/lO* 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. Nb 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 were 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 062683 Report 43-129 Pag* 7 2233 ' obtained with both positive controls and the absence of cytotoxicity at the highest dose level suggested that higher concentrations should be tested to assure the reproducibility of the data. In the repeat experiment, summarlzed 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 f 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. D. 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 #1 because a different dilution scries 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 CHO cells when tested with and without the incorporation of an S9 metabolic 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~^Z) 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. 2234 Report 43-129 Pag* 8 SECTION IX - SCE TEST - BAKELITE* Epoxy Resin ERL-2774 A. Test Dates - Iniciaced: December 5, 1979 Completed: March 24, 1980 B. Selection of Test Concentration* A maximum concentration of 120 x 10~*X of Epoxy Resin ERL-2774 was chosen as the top dos* level for testing with and without S9 activation respectively, 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. A 16-fold range of Epoxy Resin ERL-2774 concentrations from 120 x 10~^Z to 7.5 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 elderly 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 ERL-2774 or with positive, negative or solvent control agents without an S9 metabolic activation system are summarized in Table 5* 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 range of values included in the variability encountered in our historical control values for this test. i 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 range 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. ucc 062670 Report 43-129 Pag* 9 2235 SECTION III - OPS TEST - Epoxy Resin ERL-2774 A. Teat Datea - Initiated: August 23, 1979 Completed: March 31, 1980 B. Selection of Teat Concentrations Epoxy Resin ERL-2774 was tested over a wide range of concentrations from 1000 x 10~^Z to 3 x 10~^Z by weight. The maximum dose-level was selected with consideration of the cytotoxicity data obtained in the CHO (fetation test which indicated that higher values would result in excessive cell killing. C. Determination of UPS Induction Values for "unscheduled1* incorporation of ^H-thymidine into the DNA 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 ^Hthymidine 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 DNA in the lysates were determined and values were expressed as radioactivity per microgram 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-thymldine Incorporation at dose levels of 30 x 10~^Z or above are a probable indication of the cytotoxic effects of the test chemical* Both of the positive control agents, NQO 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 v. 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 Pag* 10 E Conclusion Resin ERL--2774 stimulated a hichlv S2ssl--.sis*s: -4*1. . considered to be .otic, in the ,,....t tS.'S^gi . *- M 2237 Report 43-129 Pag* 11 Reviewad and Approved by: A. A^iica^Ju; Ronald S> Sleslnski, Ph.D. Study Director Manager, Ganetlc Toxicology >// z 1 ptT3 Aaaoclata Director, Toxicology Fred R. Frank, Fh.D. Director Contributors: Chines* Hamster Ovary test Sister Chromatid Exchange test Unscheduled DNA Synthesis Assay Peggy J. Guzzle, B.S. Master Technologist Michelle tf. Gaunt, B.S. Master Technologist tf. Christopher Hengler, M.S. Assistant Scientist WPC/1125-4 ucc 062673 2238 Report 43-129 Pegs 12 REFERENCES A search of tha major computer date file* 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 848 (Shell Chemical"*), the diglycidylether of blsphenol A, was the "most active compound of the resins tested, and ... more mutagenic than the positive control, ECH (eplehlorohydrin)" In tests with the Salmonella Ames 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., Kiel, P, Larsen, H. and Maxlld, J. (1978). Mutagenic action of epoxy resins. Nature 276, 391-392. 2* Kotin, P. and Falk, B. L. (cited In reference above) (1963). Radlat. Res. Suppl 3, 193-211. 3. Wei1, C. S., Condra, N., Haun, C. and Strlegel, J. A. (cited In #1) (1963). Am. Ind. Hyg. Assoc. J., 24, 305-325 (from data in CHF Report #23-99 and #27-152). Holland, J. M., Gosslee, 0. G., Gipson, L. C. and Whitaker, M. J. Epidermal carcinogenicity of bisf2,3-epoxycyclopentyllether,2,2-bls (p-glycldyloxyphenyl)propane, and m-phenylenediamine in C3H and C57BL/6 male and female mice. Oak Ridge National Laboratory Report. March 1^78, Contract ?W=7405-eng-26. C hinese Ham ster O vary (CIIO) M u ta tio n A ssay: & 04 I umieiH! iiwaster uvary (CHU) HutaCion Assay; Determination of Toxic Effects of Chemical Treatment During S Hr Mutation Induction Period Experiment #1 Teat Chemicals Total I Colonies [Epoxy tesin ESL-2774J (X, w/v) 30.0 x 10"* 15.0 x 10"* 7.5 x UH 3.75 x 10"4 1.875 x 10-4 Controls DMS0 (20 ul/ml) - Solvent H20 (20 ul/ml) EMS (200 ug/ml) - 429 500 8 390 520 889 543 445 [Epoxy Bestn EBL-2774J (X, w/v) 30.0 x 10"4 15.0 x 10-4 7.5 x 10*4 3.75 x 10-4 1.875 x 10-4 Controls DMSO (20 ul/ml) - Solvent1 U20 (20 ul/ml) tt*N (3700 ug/ml) 393 534 425 470 502 461 368 Total f Cells Plated Without S9 Activation 800 600 800 800 600 600 800 BOO With S9 Activation 800 800 800 800 800 800 800 X Survival 53.6 62.5 1.0 48.8 65.0 111.1 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 79.6 *A dilution error was made at this step and values in last column were calculated as a percentage of the negative control. Abbreviations: H20 - water; S9 - liver homogenate; 0HS0 - dimethylsulfoxide EHS - ethylaethanesulfonate; DHN - dlmethylnitrosamiue Report 43-129 ? 13 , WPC/1125-2 W CO CO Table 3 Chinese Hamster Ovary (CilO) Mutation Assay: Determination of1 Toxic Effects of Chemical Treatment During 5 Hr Mutation Induction Period Experiment #2 Test Chemicals Total 1 Colonies (Epoxy Resin EBL-27M] (X, w/v) 100.0 x 10** 50.0 x 10-* 25.0 x 10-* 12.5 x 10~4 6.25 x 10"* 2 0 131 328 457 Total I Cells Plated Without S9 Activation 800 400 400 400 400 Controls DMSO (20 ul/ml) - Solvent H20 (20 ul/ml) EHS (200 ug/ml) 398 465 424 (Epoxy Basin ERL-27741 (X, w/v) 100.0 x 10-4 60.0 x 10-4 30.0 x 10"4 138 238 295 400 400 400 Uith S9 Activation 800 800 800 Controls DMSO (10 ul/ml) - Solvent H20 (10 ul/ml) DMN (3700 ug/ml) 221 222 105 400 400 400 Abbreviations: H20 - water; S9 - liver homogenate; DMSO - dimethylsulfoxlde EHS - ethylmethanesulfonate; DMN - dlmethylnitrosamlne 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 82.4 114.8 100.0 116.8 106.5 31.2 53.8 66.7 100.0 100.5 47.5 WPC/1125-2 Report 43*129 Pag* 13 u Test Chemicals Table 2 Chinese Haaster Ovary (CHO) Mutation Aaaay: Results on Evaluation of Mutant Induction by,Epoxy Resin ERL-2774 Experiment # 1 Plating Efficiency 1 U~" "r " " Mutation Induction Total f Colonies Total I Celia Plated Viable Fraction Total # Mutant Colonies Total # Cells Plated Mutants^ 10*> Viable Cells [Epoxy Resin ERL-2774) (X, w/v) 30.0 x 10-* 449 15.0 x 10~4 264 7.5 x 10-4 270 3.75 x 10~4 310 1.875 x 10-4 249 Without S9 Actival: Ion 400 1.122 3 400 0.660 0 400 0.675 1 400 0.775 0 400 0.622 0 t x 106 1 x 106 1 x 106 l x 106 1 x 10* 2.7 0 1.5 0 0 Controls: DHSO (20 ul/al) - Solvent U2O (20 ul/al) EMS (200 ug/al) 363 379 289 400 0.908 0 400 0.948 3 400 0.722 27 1 x 106 1 x 10* 1 x 106 0 3.2 37.4b [Epoxy 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 I x 106 1 x 106 1 x 106 1 x 106 l x 106 7.6 2.5 1.0 0 3.1 Controls: DHSO (20 ul/al) - Solvent H20 (20 ul/al) DMN (3700 ug/al) 410 336 227 400 1.025 0 400 0.840 2 400 0.568 9 1 x 106 l x 106 0.9 x 106 0 2.4 17.6a Report 43-129 Page 14 o !3o ^Total # autant colonies per I0b 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: 1^0 - water; S-9 - liver honogenate; OMSO - dlmethyisulfoxide; EMS - ethylmethanesulfonate; DMN - diaethylnitrosamlne. HVC/U25-1 idUl 3 Chinese Hamster Ovary (CilO) Mutation Aaaay: Determination of Toxic Effects of Chemical Treatment taring 5 Hr Mutation Induction Period Experiment #2 Teat Chemicals Total 1 Colonies Total # Cells Plated [Epoxy Resin ERL-2774) (X, w/v) 100.0 x 10-* 50.0 x 10~4 25.0 x 10"4 12.5 x 10~4 6.25 x 10~4 2 0 131 326 457 Without S9'Activation 800 400 400 400 400 Controls DKSO (20 ul/ml) - Solvent H20 (20 ul/ml) EMS (200 ug/ml) (Epoxy Resin ERL-2774) (X, w/v) 100.0 x 10-4 60.0 x 10-4 30.0 x 10*4 398 465 424 138 238 295 400 400 400 With S9 Activation 800 800 800 Controls DHS0 (10 ul/ml) - Solvent H20 (10 ul/ml) DHN (3700 ug/ml) 221 222 105 400 400 400 Abbreviationsi H20 - water; S9 -- liver homogenate; DHSO - dimethylsulfoxide EHS - ethylaethaneaulfonate; DHN - dimethylnltroaamine 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 82.4 114.8 100.0 116.8 106.5 31.2 53.8 66.7 100.0 100.5 47.5 mhmo g UPC/1125-2 LtIdN*6 062678 r.~ N O O N H Table 4 Chinese Hanster Ovary (Clio) Mutation Assay: Results on Evaluation of Mutant Induction by Epoxy Realn ERL-2774 Experiaent I 2 ---------,------------------- Plating Efficiency T Mutation Induction Test Qiealcals Total # Colonies 1 (Epoxy fcestn ERL-2774] (I, w/v) 100.0 x 10"* 406 50.0 x 10"* - 25.0 x 10-4 443 12.5 x 10"4 149 6.25 x 10-* 234 Total # Cells Plated Viable Fraction Total 1 Mutant Colonies Without: 89 Activation 400 1.015 0 - SEE FOOTNOTE #2 - 400 1.108 11 400 0.372 0 400 0.585 ' 0 Total 1 Cells Plated a 1 X 10* 1 X 10* 1 X 10* 1 X 10* Mutants^ 10* Viable Celia 0 - 9.9 0 0 Controls: DMSO (20 uI/mI) - Solvent H20 (20 uI/mI) EMS (200 ug/ol) 170 - 440 400 0.425 11 - SEE FOOTNOTE #2 - 1400 1.100 216 1 X 10* 1 X 10* 25.9 - 196.4C (Epoxy Resin ERL-2774] (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 1 400 0.748 9 400 0.932 77 t X 10* 1 X 10* 1 X 10* 2.2 12.0 82.6C C ntrola: DMSO (10 uI/mI) - Solvent H20 (10 ul/nl) DMN (3700 ug/ol) 189 232 129 400 0.472 3 400 0.580 10 400 0.322 1 78 1 X 10*t 1 X 10* 1 X 10* 6.3 17.2 241.9C lTotal I autant colonies per 10** cells plated divided by viable fraction. Statistical significance above solvent control: c: p < 0.001 NO superscript Indicates p > 0.05. Data analyzed by Student's t-test. ^Loot during expression period following incubator Malfunction during routine trypsin!zatIon step. Abbreviations:' WPC/l i m i 112O - water; S-9 -""liver hon'o'g'e'nate; DMS0 DMH - dtnethylnltrosanlne. dlmetbyleulfoxIde; EMS - ethylnethanesulfonate; i inn 062679 Table 5 Report 43-129 Page 16 -N N Sister Chromatid Ejtchange (SCE) Assay: Induction of SCE'a by Epoxy Resin (ERL-2774) Without S9 Metabolic Activation 5 Hour Treataent ' Test Chemicala Total 1 of Chronosoaes Total I of SCR SCE/Cell1 Mean Number SCE/Chroaosoae2 1- S.D. (Epoxy Resin ERL-2774] (2, w/v) 120 x 10"* 60 x 10"* 30 x 10"* 15 x 10"* 7.5 x 10"* Controls DHS0 (5 ul/al) -Solvent U20 (5 ul/al) EHS (100 ug/al) 309 299 302 299 300 291 294 298 869 57.93 2.793 -t- 1.010 613 40.87 2.049 + 0.256 448 29.87 1.490 -1- 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 -I- 0.169 423 28.20 1.417 + 0.242 1 Fifteen cells exanlned per dose level. ^Hean value of SCE/chroaoaoae determined from tie values of the Individual cells exanlned. ^Statistical algniflcance above solvent control: c: p < 0.001; NS; p > 0.05. Abbreviations: U2O - water; S9 - liver honogenate; DMS0 - dlnethy1sulfoxide; EHS - ethylaethanesulfonate; S.D. - standard deviation Significance Above Solvent Control^ c c c c c NS c Report 43-129 UPC/1125-2 c<yj>_C_O ClJ*' 1 Table 6 Unscheduled DMA Synthesis In llepatocytea froot Hat Liver DPH/1Q6 cells! all DPM values are calculated from DMA precipitated per 10^ viable hepatocytea. Each average la calculated from duplicate samples, except for DMSO which tua done In quadruplicate. Teat Chemical Solvent - DMSO Positive Controls: EHS DMN Teat Chemical: (Epoxy Realn ERL-2774] (*, u/v) Concentration 3.3Z 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"4X 600 x 10~4x 300 x 10~*X 100 x lO^X 60 x I0-*X 30 x lO-^X 10 x 10~4X 3 x I0~4x . Radioactivity In DNA Avg. DPM + S.D. 22848 + 926 26618 + 4161 28573 1 3735 32107 + 1083 26715 + 549 28753 + 3744 27290 + 1686 30930 + 859 24270 + 1468 4100 + 247 3290 + 27 5328 + 126 11825 + 1876 8956 1956 15371 + 2438 38488 240 35198 + 2793 X of Solvent Control + S.D. 100.OX + 4.ix 125.3X + 18.2X 125.IX + 16.3X 140.5X + 4.7X 116.9X + 2.4X 125.8X + 16.4X 119.4X 7.4X 135.4X + 3.8X 106.2X + 6.4X 17.9X + 1.1X 14.4X + 0.1X 23.3X + 0.6X 51.6X + 8.2X 39.21 + 8.6X 67.3X + 10. 7X 168.5X + 1.1X 154.IX + 12.2X Significance Above Solvent Control^ - b b c a b a c NS NS 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. Abbr vlations: DMSO - dimethylsulfoxide; EHS - ethyluethane sulfonate; DMN - dimethylnitrosamlne; DPM - disintegrations per minute; S.D. - standard deviation UPC/1125-2 6ZT-C9 3JodE WPC/1125-2 Table 7 Unscheduled DNA Synthesis In llepstocytes Cron Rat Liver DPH/ug DMA: all DIM values are calculated per DNA precipitated froa rat hepstoeytea. Each average is calculated froa duplicate aaaplea, except for DMSO which was done In quadruplicate. Test Chenlcal Solvent - DMSO Positive Controls: ms DMN Teat Chenical: (Epoxy Resin ERL-2774] (X, v/v) Concentration 3. JU 400 ug/nl 200 ug/al 100 ug/al 50 ug/al 80 ug/nl 40 ug/nl 20 ug/nl 10 ug/al 1000 x 10~*X 600 x IQ"** 300 x 10"*X 100 x 10-4% 60 x 10"4x 30 x 10"4X 10 x I0"4g 3 x 10"4X Radioactivity in DNA Avg. DPH + S.D. 847 + 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 16 135 3 52 434 597 X of Solvent Control + S.D. 100.OX + 23.7X 174.OX 38.9X 175.9X + 14.62 142.8X + 0.2X 136.5X + 15.92 147.8X + 12.2X 164.5X + 2.3X 197.4X + 41.42 193.41 + 34.42 18.6X + 3.2X 17.2X + 2.3X 24.31 2.IX 67.2X + 15.9X 56.IX + 0.3X 95.5X + 6. IX 151.2X + 51.2X 143.92 + 70.42 Significance Above Solvent Control1 - A A NS NS NS A C C NS NS NS NS NS NS NS NS ^Statistical aignlficance'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 - dlaethylsulfoxlde; EMS - ethylnethane sulfonate; DMN - dlnethyinltrosamlne; DPM - disintegrations per ainute; S.D. - standard deviation WPG/1125-2 ucc 06268 Report 43-129 Page 19 N fO fn 2246 Fag* 1 Of 3 APPENDIX I Chinese Hamater Ovary (CHO) Mutation Assay Theoretical Baals Mutation la a heritable alteration in a cell 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 phosphorlbosyltransferase) gene any be detected by the growth of colonies of "mutant" cells which are resistant to the purine analogs 6-thioguanine (TG) or 3-azagusnlne. Normal cells contain a functional SGPRT enzyme which phosphorylates TG and allows Its Incorporation into DNA causing the cells to die. Mutant cells with a non-functional SSPRT enzyme are unable to phosphorylate or incorporate TG, thus survive and grow in its presence. The CHO mutation test is an assay which detects "forward mutations" from TG-sensitlvlty to TG-resistance caused by a direct loss of the activity of the HGPRT enzyme (BGFRr*' HGPRT-). An aasesament of the ability of several hundred agents to cause gene mutations in vitro indicates that the CHO mutation 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 from Abraham Bsle 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 times/week in antibiotic-free. Ham's Modified F12 Medium supplemented with lot (v/v) heat-inactivated, fetal bovine sera (P12-10), and lacking in hypoxanthine. For treatment of cells without metabolic activation, F12 medium with 30 unlts/ml of penicillin, 30 ug/ml streptomycin and SZ (v/v) of dialyzed bovine serum (F12-05) is 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 are determined routinely with a Coulter Model F electronic cell counter which is standardized periodically with a pro-counted suspension of latax beads. Presence of Mycoplaama cell contaminants is determined by a microscopic fluorescence assay employing Hoeehst 33238 dye. All culture procedures and treatments with test chemicals are performed under aseptic conditions in a laminar-flow, biohazard hood. Positive and Negative Controls! Sterile water or glass-distilled dimethylsulfoxide (dmS6) 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. Dlmethylnitroaamin* (DMN) or ethylmethaneaulfonate (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 CHO mutation test system. Comparison of concurrent control values with historical controls is used to delineate the rang* of acceptable variations la the test system. Appendix I JPage 2 of 5 2247 Metabolic Activation; Rat liver, S9 homogenate prepared from Aroehlor-1254 induced, Sprague-Dawley, male rata la purchased from Litton Blonetlca, Kensington, MD. Each lot of liver homogenate is prescreened for metabolic capability to activate DMN in our laboratory before uee in the testing program. The complete S9 metabolic activation system contains the following: 3 umoles/ml MgCl2 33 umolea/ml KC1, 3 umoles/ml glucoae-6-phosphate, 4 umoles/ml NADP-oxidized (nicotinamide adenine dlnuclaotida phosphate), 100 umoles/al 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 assessment of mutagenic potential to select doses which produce a maximum of 80 to 90S cell killing. Cytotoxicity is determined by either of the following two methods: (1) Clonal assay - 200 to 400 (SO cells are exposed to a minimum of five dos levels of the test agent at concentrations from 0.1Z to 3 x 1Q"*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 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 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 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 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. 2248 Appendix I Page 3 of 5 Chemical sample* 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 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, 5x10^ cells are inoculated 20 to 24 hours prior to treatment into 25 a#2 culture flaska containing F12-D5 medium and incubated at 37*C in a 5 to 6Z CO2 atmosphere. Appropriate concentrations of the test agent or control chemicals are added to the cells and cultures are created for 5 hr at 37*C. The medium and test agents are removed by suction, cells are rinsed once or twice and fresh F12-D3 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 F12 medium without serum and containing 1.0 ml of S9 activation mixture per 4.0 ml of medium ia employed. Determination of Cytotoxicity; The relative survival of treated cells, in comparison to solvent controls, is determined one day after the exposure to the test agents. The level of cytotoxicity is often correlated with the mutation frequencies induced by known chemical mutagens. Thus, excessive cytotoxicity may 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 valuaa which indicate the cytotoxic effects of the test agents are included in reports is tabular form. Statistical analyses ars not performed on these deta, slnca they are only useful to assess whether appropriate doses were employed end are not used to calculate mutation fraquencias. Determination of Mutant Induction: On days 1, 3 and 6 (or alternatively 1, 4 and 6) after treatment with the various tast agents, approximately 5x10^ cells are subeultured in 100 on tissue culture dishes in F12-D5 medium and incubated at 37*C In a 5 to 6Z COj 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 lO^/dlsh in four culture dishes (lx 10 total calls) which each contain 5 ml of F12-D5 (TO) selective medium. At this time, cells are diluted and 100 eells/dlsh are added to four culture plates containing F12-D5 medium (without TG) to assess viability (plating affielaney) of the treated cell population and to determine the surviving fraction. All cultures arc then incubated for en additional 6 to 3 days to allow growth of calls; medium Is then dlscsrded and colonies are fixed and stained for counting. The number of colonies In selection plates and in the viability tast are counted by electronic methods, cheeked by manual counts and data are recorded both *s total mutants, mutanta/106 total calls and mutants/lO6 viable cells. Appendix I Pag* 4 of 5 2249 Statistical Analyses? Uniform statistical procaduraa to evaluate in vitro mutation data hava not been developed. The distribution of mutation frequencies froa historical controls In at least two laboratories Indicates that the fra* 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 Snea (Reference 4) which employs th* 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 th 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* cation 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 th* concurrent solvent control, but these statistical indicators must be viewed conservatively until additional historical control data are available. Interpretation of Data? Th* criteria for interpretation of the test results as a positive or negative response depend upon both the level of statistical significance from th* concurrent control and the evidence of a dose-response fqllowlag 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 Che 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/106 viable cells, with a range of 0 to 25 mutants/10 viable cells, can be obtained in th* absence of mutagenic treatment. Statistical comparisons against unusually high or low spontaneous controls are subjectively scrutinized In respect to the above variability. UCC 062636 2250 Appendix I Pag* 5 of 5 References 1. Chu, E. H. Y and H. V. Mailing. Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells In Vitro. Proe. Natl. Acad. Sci. U.S.A. 41 1306-L312. 2. O'Neill, J. P., P. A. Brimer, R. Machanoff, G. P. Hirsh, A. W. Hsle. A Quantitative Assay of Mutation Induction at the Bypoxanthlne-Guanine ~ Phosphoribosyl Transferase Locus In Chinese Hamster Ovary Cells (CHO/HGPRI S<1y3s7te7m),): 51D-1e6v1e.lopm--e-n-t- and De--fi-n-i-t-io-n---o:-f--t-h-e- Sy-s-t-e-m, Mutation Research. --45 3. O'Neill, J. P. and A. W. Hsie. Phenotypic Expression Time of MutagenInduced 6-Thloguanine Resistance~~In Chinese Hamster Ovary Cells (CHO/HGPRI System), Mutation Research, 59, (1979), 109-118. 4. Irr, J. 0. and R. D. Snee. Statistical Evaluation of Mutagenicity in the CHO/HGPRY System. Proceedings of the Cold Spring Harbor-Banbury Conference II (1979)7 263-274. 5. Box, G. E. F. and D. R. Cox. An Analysis of Transformations. J. of the Royal Statistical Society, B, 26(1964), 211-231. WPC/1033 ucc 062687 Fag* l of 4 2251 APPENDIX II Determination of Sister Chromatid Exchange (SCZ) Frequencies in Chinees Hamster Ovary (CHO) Cells In Vitro ;Th--e--o---r-e--t-i-c--a--l---B--a--s--i-s- I Exchanges of genetic material between 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 cells treated !with physical or chemical mutagenic agents, or in cells exposed to many suspect or proven human carcinogens. Thus, analysis of SCZ frequenelas in cells treated with a test agent has been suggested as a sensitive screening test for potential mutagenic/carcinogenic chemicals* The method used In our study to visualize SCE'e in CHO cells grown In cul ture is based on the procedure described by Ferry and Volff (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 Glemsa 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-K1BH4-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 fetal bovine serum and lacking hypoxanthlne and thymidine. Cell concentrations are determined routinely with a Coulter* Model-F 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 Mycoplasme 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 50 units/ml of penicillin, 50 ug/ml streptomycin and 5Z (v/v) f heat-inactivated, dialyzed fetal bovine serum (F12-D5). Identical medium but without serum- la used for treatments incorporating an S9 metabolic activation system. Positive and Negative Controls; Sterile water or glaas-dlstilled 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 comparison and for demonstrating the sensitivity and stability of the SCE tast system. Comparison of concurrent control values with historical controls is used to delineate the range of acceptable variations in the test system. 2252 Appendix II Pag* 2 of 4 Metabolic Activation; Rat liver S9 homogenate (prepared from Arachlov 1254 induced, Sprague-Dawley, male rata) ia purchased from Litton Blonetlca, Kensington, MD. Each lot of liver homogenate ia prescreened for activity in our laboratory before us* in the testing program. The complete S9 metabolic activation system contains the following: 8 umoles/ml MgCl2, 33 umoles/ml KC1, 5 umoles/ml KC1, 5 umoles/ml glucose-6-phosphate, 4 umoles/ml NADP-oxidized form (nicotinamide adenine dinucleotlde phosphate), 100 umoles/ml Na2&P04 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 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 as part of the CH0 mutation testing procedure: (1) Clonal assay - 200 to 400 CEO cells are exposed to a minimum of five dose levels of the test agent at concentrations from 0.1Z to 3 x lO*4! (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 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 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 higher dose levels. If marked toxicity is evident even at the lowest dose, the cytotoxicity test is repeated at a concentration rang* of 3x10s4 to 3x10* percent by volume. Dos* 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 anT-tjimc 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 SCE staining, are evaluated for SCE induction. Chemical samples are sterilised 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. a< c. ai It b: A; t! ai b' i: ri St a> w. i; m: 0 f< c. fl p si 3: ai Ii r. ft 2 a fl ucc 062689 Appendix II Pag* 3 of 4 > 22b Treataent With Teat Chemicals: Tasting of chemicals for diraet mutagenic action (without S9 metabolicactivation) is parforaad first* For chaalcals with clearly positive autaganlc capabilities by direct action, testing with aetaboilc 1 activation is generally not perforated. For testing direct acting chaalcals for SCZ induction, between 1 to 2 x 10 cells are plated into 73 ca^ culture flasks in F12-D5 aedlua at least 20 hrs prior to treataent and Incubated at 37*C in a 5 to 6Z CO2 ataosphere* Appropriate concentrations of the test agent or control chaalcals are added to the cells and 3 ug/al BrdO is added to all flasks* Cells are treated with test agents for 3 hrs, aedla is then removed by suction, cells are rinsed with buffered, physiological salt solution and fresh medium containing 3 ug/al 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 SCZ 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 treataent with the test agents, F12-D5 aedlua 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* Calls 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; Colcemid* (0*1 ug/al) or Colchicine (0.2 ug/al) is added to culture flasks 1 to 2 hrs prior to harvesting to arrest cells in mitosis. Cells are then removed from flasks, after a brief incubation with 0.01X DIFCO trypsin, suspended in 0.075M KC1 (hypotonic) solution and Incubated for 13 to 20 min at 37aC* Calls 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/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 gieasa (diluted 1:23 with water), rinsed in water and dried before application of coversllps. Examination of SCE'a: All slides are coded and read in a blind fashion without indication of the specific treataent or concentration of the test agent. The number of chromosomes and the number of SCE's in a minimum of 13 cells are recorded for each dose level. The mean number of SCZ/cell and SCE/chromosome are calculated end recorded* Slides are decoded only after examination of all slides in the experiment has been completed. ucc 062690 2254 Appendix IZ Peg* 4 of 4 Statistical Analyses: Data are analyzed by appropriate parametric statisti cal procedure* which follow BK&C standard operating procedures 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 nega tive response depend both on the level of statistical significance and subjec tive analyse* of concurrent and historical control data. The key determinant is whether a dose-dependent increase in SCE's la Induced by the test agent. When no clear 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 other tests 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 tested; (ii) Statistically significant responses of p < 0.05 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 giemsa method for differential staining of sister chromatids. Nhturn, 251 (1974), 154-158. 2. Lett, S. A., J. W. Allen, V. E. Rogers and L. A. Juergena. In vitro and in vivo analysis of sister chromatid exchange formation, pp 275-2^1 in Handbook of Mutagenicity Test Procedures, ad". Kllbey, B. J., et al. Elsevier Publ. Co. (1979). 3. Carrano, A. V., L. H. Thompson, P. A. Lindl and J. L. Hinkler. Sister chromatid exchange as an indicator of mutagenesis. Nature, 271, ~(1~975), 551-551 4. Galloway, S. K. and S. Wolff. The relation between chemically Induced sister-chromatid exchanges and chromatid breakage. Mutation Ras., 61, (19l9), 29l-30>. 5. Snedeeor, G. W. and W. G. Cochran. Statistical Methods, 6th Ed., Iowa State Univ. Press, Ames, Iowa (1967). WPC/1033 Pag* 1 of 3 2255 APPENDIX III Unscheduled DNA Synthesis (UPS) In Hepatocyte* from Rat Liver Theoretical Basil Chemicals say interact with both tha cellular components and th* genetic material of a call (e.g. DNA and RNA) 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 th* 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. Th* system employed for the present study uses a suspension culture of primary hapatocyt* cells iso lated from rat liver according to the general methods of Seglen (1973) aul 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 Suspensionsi Hilltop-Wistar albino rats are anesthetized with Metafane(R) The abdominal cavity is surgically exposed and 1230 units of heparin is injected intravenously. A catheter is Inserted into the portal vein and warm Hanks Balanced Salt Solution (HBSS) is pumped into the vein and through the liver. This first solution contains heparin and EGTA, [ethylene glycol-bls-(beta-amino*thyl-ether)N,N*tetracetlc add], which preferentially chelates calcium; the solution contains no magnesium or calcium. .After the liver is blanched, a second solution of HBSS containing 60 units/ml of collagenase is perfused. This solution is pumped through the liver until the liver is digested. Th* 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 dumps and the cells are washed once at low 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 Hepatocyte*: Approximately 2 x 10 viable hepatocytes are added to 3 ml medium 199 containing 10 mM hydroxyurea and 30 mM Hepes (N-2-hydroxy*thy1 plperazlne-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 th* nuclei is expected to result from repair or unscheduled DNA synthesis. Appendix III ?ag 2 of 3 Selection of Doses of Teat Chemical: Initially, tha following concentra tions x 10*3i (by volume) are tasted: 100, 30, 10, 3, l, and 0.1. If chesa coacentrationa prove to ba cytotoxic, or if additional information is available froa other in vitro tests as to tha proper dose levels, then an appropriate series of concentrations is used over a 3-log range of concentrations. Treatment of Hepatocytes: After prelncubeelon, 23 mlcroCurles of tritlated thymidine (o Curias/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 cube. 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-nitroquinoline oxide (NQO), a directacting mutagen, which induces UV-type DMA repair and dimethylnltrosamine (DMH), 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 microliters (concentration specified in individual reports) of the solvent used to dilute the sample. Dimethyl-, sulfoxide (DM50) 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, 51 citric acid and 3 toM MgCl2> 4 l73iaS solution which liberates the nuclei. The nuclei are rinsed once in this solution and resuspended in 0.25 M sucrose, 2.5Z citric acid and 3 oM MgClj. The nuclei are then centrifuged at 600 x g for 10 min at 5*C and resuspended in 2 ml of the lysing solution. Determination of 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 og of DCS tissue solubilizer in a scintillation vial. Ten ml of Dlmllume* 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 thee used to calculate the DPM/10 viable hepatocytes presented on tables. Determination of DWA-Bound Label; The amount of radioactive thymidine in corporated into DMA is quantitated in DMA isolated and precipitated from 1.00 to 1.25 x 105 viable hepatocytes. To 1.25 ml of the nuclear suspension, 2.75 ml of 1Z sodium dodecyl sulfate (SDS) and 5 mM Ethylenedlamlnatetraaeetic Acid (EDTA) la added to lyse the nuclei. The DNA 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 minutes. The solution is 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 5Q*C for 1 hour with l ml of a diluted solution of MCS tissue solubilizer; prepared by adding 1 part solubilizer to 2 parts of Dimllume* cocktail. Dinllumed is then added to each vial and the vials ara counted twice in a scintillation counter for tan minutes. i irr" 062&&3 Appendix 111 Page 3 of 3 2257 Statistical Analyala: The average DPM ia calculated for each dose level and the controls and final results are expressed as DPH/10 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 i appropriate parametric test, following the BRRC standard procedures for statistical analyses and the test(s) employed is indicated on the respective tables* 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 the production of a statistically significant, dose*related increase in the amount of OSS 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 OSS 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, 110, (19?7), 41-46. 2. Muramatsu, M. Isolation of Nuclei and Nucleoli. In: Methods ia Cell Physiology, Vol. IV. 1970. Editor: D. M. Prescott. Academic Press, Mew York. 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 Synthesis in Rat liver Primary Cell Cultures. Cancer Research. 37. (1^71^3-rssn' ----------------------------- -- 5. Williams, G. M. The Dae of liver Epithelial Cultures for the Study of Chemical Carcinogenesis. American Journal of Pathology. &5, (1^76). 739-753. 6. Williams, G< M. and M. F. laspla. The Detection of Parlous Mitroaamines In the Hepatocyta Primary Culture/DMA Repair Test. Cancer letters. 6. (1979), id9-206. ~ 7. Snedecor, G. W. and W. G. Cochran. Statistical Methods, 6th edition, Iowa State University Press, Ames, Iowa (1^6?). WPC/1033 2258 Report 43-129 APPENDIX IV Physical and Chemical Characteristics of Teat Material BRRC Chemical No: CAS No.: Chemical Name: Trade Name and/or Synonyms: Molecular Weight: Formula: Specific Gravity (8 25*0: Boiling Point: Solubility in H2O (2 by wt): Purity Vapor Pressure (8 20C): pH: Flash Point: Stability: Incompatibility: Appearance and Odor: Disposal: 42-138 1675-54-3 BAKELITEO Liquid Epoxy Resin ERL-2774 Dlglycidyl Ether of Bisphenol A 340 C21H24O4 1.15 to 1.17 Not applicable Insoluble Not available Not available Not available 450*F (Cleveland open cup) Stable Avoid high temperatures > 450F and contaaminatlon with acids, amines and H?0. 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. pro 40che pn Acu to ! var beD tox: the cone patt furt t xl peri id o leve