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187 BUSHY RUN RESEARCH CENTER R. 0. 4, Mellon Road, Export, Pennsylvania 18632 Telephone (412) 327-1020 CONFIDENTIAL; Not to bo released outaide UCC without the written consent of the UCC component sponsoring the work* Project Report 43-112 17 Pages Tel; (412) 327-1020 December 12* 1980 RAKELITER Epoxy Resin ERL - 4206 In Vitro Mutagenesis Studies; 3-Test Battery Authors: R. S. Sleslnskl, M. W. Gaunt, P. J. Gurrie, V. C. Hcngler Sponsor: Onion Carbide Corporation SUMMARY Epoxy Resin ERL-4206 was evaluated for potential mutagenic activity with a battery of three In vitro tests, which were; the Chinese Hamster Ovary (CH0) Mutation test, the Sister Chromatid Exchange (SCE) test and an assay for Induc tion of Unscheduled DNA Synthesis (UDS) In rat liver cells. The data Indicated that Epoxy Resin ERL-4206 produced significantly positive and dose-related effects In the SCE test and a significantly positive response at only the high est dose level In the CH0 test. Although there was no positive effect in the UDS test at any concentration tested, several concentrations produced numerical increases above the values for the concurrent solvent control Indicating a low level of activity. Epoxy Resin ERL-4206 was considered an active mutagenic agent based primarily on the significantly positive response In the SCE test and the suggestive, statistically significant production of mutants at the top concentration in the CHO test. RESULTS AND INTERPRETATION Epoxy Resin ERL-4206 was selected for mutagenesis testing to validate the sensitivity of our in-house battery of mutagenicity tests for detecting chemicals found to produce a positive effect In lifetime skin carcinogenicity assays with mice (CHF Report #27-152). Selection of Test Concentrations - Preliminary experiments were performed to select an appropriate range of test concentrations which would allow the survi val of at least 10Z of the treated eells. A maximum concentration of 0.01Z (by volume) mas selected as the maximum concentration for testing both with and without metabolic activation. At slightly higher concentrations (eg. 0.03Z) fewer than 12 of the treated eells were capable of producing colonies. CHO Mutation Test - Epoxy Resin ERL-4206 was active only at the highest dose level (10 x 10"^Z) In producing a statistically significant increase of mutant eells when tested without an 89 metabolic activation system. Although we did not observe a dose-related Increase In mutants, production of highly statisti cally significant Increases in mutants at the highest dose level prompted a cautious interpretation -of the data and the test without S9 activation was con- Rushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation 0^33 1872 Report 43-112 Peg* 2 sldered quastlonably-to-weakly positive. The test with an S9 astabollc activa tion system did not result In a significant Increase In the autant frequency, which suggested that the astabollc activation system aay have obscured the weak response obtained without S9. SCE Teat - Epoxy Resin ERL-4206 produced a significant Increase In SCE at four of the five dose-levels tested. The observation of a dose-related effect In the tests without S9 astabollc activation provided a clear Indication that Epoxy Resin ERL-4206 was active In stimulating SCE In CHO cells. The test without a aetabollc activation system Indicated that Epoxy Resin ERL-4206 did not require metabolic conversion for activation and the test Incorporating an S9 aetabollc activation system was not performed. OPS Test - Epoxy Resin ERL-4206 failed to induce dose-related Increases In the DPS detected with either nuclei or PNA. In evaluations over a relatively wide range of concentrations, no concentration of Epoxy Resin ERL-4206 produced statistically significant levels of OPS activity. Production of numerically In creased levels of UPS by a few of the concentrations of Epoxy Resin ERL-4206 tested for activity might be an Indication of a low level of primary SNA damage. However, the lack of a statistically significant effect Indicated that Epoxy Resin ERL-4206 was not active In the Induction of UPS In the present test. Comparative Mutagenicity - The pattern of responses produced by Epoxy Resin ERL-4206 In the 3-test battery of mutagenicity assays Indicated that the test agent was highly active in the SCE test and questionably active la the OPS and CHO tests. Because only the highest concentration of Epoxy Resin ERL-4206 produced a significant effect In tha CHO test, additional testing of a narrower range of concentrations would be necessary for Identification of potential for induction of gene (point) mutations. The definitive production of SCE In a highly statistically significant, dose-related manner indicated that Epoxy Resin ERL-4206 could be considered an active mutagenic agent without additional in vitro testing. The lack of activity In the UPS test (which uses metabolically competent liver cells) and in the CHO test with liver S9 activation suggested that Epoxy Resin ERL-4206 aay be metabolically converted to a non-autagenlc form by the liver activation systems. The production of positive response In mouse dermal carcinogenesis studies (CHP Report #27-152) are consistent with the positive mutagenicity data observed In this study but show that mouse skin cells are affected by the test agent and do not inactivate It following In vivo exposures. SAMPLE Quantity: 8 ounces CHP Smaple No.: 42-137 Submitted by: W. C. Kuryla, for UCC Toxicology Advisory Committee Pete Received: March 15, 1979 Olvlslon: Specialty Chemicals and Plasties Identification: Tallow liquid CAS #: 106-87-6 01 Saporc 43-11 Paco 3 1873 BAKELXTE* Cycloaliphatic Epoxy Resin ERL-4206 In Vitro Mutagenesis Studlet; 3-Teat Battary Sponaor: Onion Carbide Corporation ***** OBJECTIVE The purpose of this study mi to evaluate the potential of Epoxy Resin ERL4206 to indue* genetic dosage in nansallan cells at the gene, chrososose and/or DNA (deoxyribonucleic acid) level of nolecular organization. A battery of three in vitro, short-tern tests which detect each of these genetic endpoints was employed to evaluate Epoxy Resin ERL-4206 for potential nutagenlc activity. A general description of tha theoretical basis for each of these three tests is presented in Appendices X, II and III attached to this report. SAMPLE CHARACTERISTICS A typical, commercial eanple of Epoxy Resin ERL-4206 was received froo UCC, South Charleston, UV for testing on March IS, 1979. The Information available froa the Toxicology Data Bank or froa "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 tests at the Bushy Run Research Center. Deviations from current Standard Operating Procedures and the appendices attached to this report are noted In the indivi dual test results. 1. CHO test (Detailed procedures In Appendix I): A. Dose Selection - Epoxy Resin ERL-4206 was tested for cytotoxicity to CHO cells at six concentrations from 0.52 to 1 x 10*3X (by volume) 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 dose levels which would permit survival of approximately 102 of the treated cells. Glass-dlstil'led dlmethylsulfoxlde (DMSO) was used as the solvent and solvent control; sterile, distilled water (R2O) was used as the negative c ntrol. To simplify tables and to allow comparisons between different tests, c neentratlous of Epoxy Resin ERL-4206 in the following sections of the report are given in terms of volume percentages x 10"3X to eliminate zeros in the lower concentration values (eg. 0.0006252*0.625 x 10~3X). 1874 Report 43-112 Pago 4 B. Mutation - CHO colls were axpoood for 5 hours to a minimum of flvo coneontrotions of Epoxy Rosin ERL-4206 with tho addition of an S9 natabollc activation system and to an Identical range of concentrations for 16 hours without S9 activation. Dilutions of Epoxy Rosin ERL-4206 for testing were prepared in DMSO and various aliquots of tho tost agent were pipetted into the cell culture media. Tho surviving fraction was determined at 20 to 24 hours after treatment and the mutant fraction was determined after a 7- to 9-day period to allow "expression" of the mutant phenotype. Only the top five concentrations which allowed sufficient cell survival were assessed for survival and induction of mutants. The percentage of cells surviving the treatment, the frequencies of 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's by exposure to various concentrations of Epoxy Resin ERL-4206 was studied In CHO cells without the Incorporation of an S9 metabolic activation system. Selection of dose levels which would permit survival of at least 50% 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-4206 for testing ware prepared by direct addition int the culture medium of various aliquots of the test sample diluted In DMSO. For determination of direct mutagenic action, CHO cells were exposed to Epoxy Resin ERL-4206 and appropriate controls for 5 hours without $9 acti vation. Indirect mutagenic action, requiring metabolic activation by liver S9 homogenate, was not studied because the highly significant responses obtained In the test without metabolic activation Indicated metabolic con version was not required to detect the action of the teat agent. Bromodeoxyuridine (BrdU) required to differentiate between the Individual "sis ter" chromatids by SCE staining, was present at a concentration of 3 ug/ml In the growth media during treatment and during the culture period follow ing exposure* A total of 15 cells/dose level and 5 dose levels, tested without metabolic activation, were examined. The number of SCE/cell, mean number of SCE/chromoeome and the level of statistical significance of the increases above concurrent solvent control values are presented in tabular form. 3. UPS Test (Detailed procedures In Appendix III): Induction of primary DNA damage In rat liver cells (hepatocytes) was studied at a minima of six dose levels which spanned a 1000-fold range of concentrations. Cells were treated with Epoxy Resin ERL-4206 for 2 hours In culture medium containing ^H-thymldine, hydroxyurea and appropriate dilu tions of the test agent prepared In DMSO. Determination of CDS activity was performed by analyses of radioactive incorporation 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 spectrometer. Data are presented in tabular form with an Indication of the level of sta tistical significance above the concurrent solvent control values. 1875 Report 43-112 Pago 5 4. C_ntrols - Positive, negative and solvent controls were tested concurrently with the test sample to assure the sensitivity of the test system and the concurrence of the results to previous test performance. For the CHO and SCE assays, dlmethylnltroaamlne (DMN) and ethylmethanesulfonate (EMS) were used as positive control agents to assure the sensitivity of the test system f r 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 the UDS assay, DMN and 4-nltroqulnollne oxide (4-NQO) were used as positive controls representing Indirect or direct-acting mutagens, respec tively v 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 Blonetlcs. The S9 preparation used for the CHO test contained 38.5 mg/ml protein and had a benzo(a)pyrene hydroxylase activity of 21.6 nmol hydroxybenzpyrene/20 min/mg protein, (assayed by Litton). A concentration of 2400 ug of S9 protein was added to 5 ml of culture media. 6. Statistical Analyses - Data from the SCE and UDS 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 historical controls. Thus, the Student's t-test was used after suitable transformation of the mutation frequencies (MF) following the procedure of Xrr and Snee: (MF + l)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. 1. Raw Data Storage - Copies of the final report, statistical analyses, avail able analytical data and data used to prepare the final report are stored in the BRRC Archives. Slides are stored in the Genetic Toxicology slide storage area. RESULTS SECTION I - CHO MUTATION TEST - Epoxy Resin ERL-4206 A. Test Dates - Initiated: May 11, 1979 Completed: April 28, 1980 1876 Report 43-112 Fag* 6 B. Selection of Taat Concentration (Data not ihown In tablaa) CHO cella were exposed to six concentration! of Epoxy Realn ERL-4206 which panned a concentration range from 500 x 10"3X to 1 x 10~3X by volume* The exposure period waa for 5 hours with metabolic activation and for 16 hours without metabolic activation. The percentage of cells which survived the exposure, either In the presence or absence of an 89 metabolic activation system, was determined by counting the number of colonies pro duced by the survivors after a 5 to 7 day Incubation period. A concentra tion of 10 x 10"3X was selected as the maximum concentration for tasting with and without S9 activation; a higher concentration (30 x 10"3X) allowed survival of fewer than IX of the cells trested with the test agent either in the presence or absence of a metabolic activation system. C. Determination of Mutation Induction 1. Survival (Cytotoxicity) Table 1 presents the cytotoxicity data for CHO calls treated with Epoxy Resin ERL-4206 In the presence or absence of a liver S9 metabolic activation system. A suggestive dose-related effect with the test agent was apparent for the cytotoxicity values observed for the tests both with or without S9 activation. Although the tests with S9 metabolic activation could have enployed slightly higher concentrations, It Is likely (based on the test with out S9 and from the prescreening data for dose selection) that the highest concentration tested was near the upper limit which would allow sufficient cell survival. Cytotoxicity of Epoxy Resin ERL-4206 was greater In the absence of the S9 metabolic activation system than the response obtained for Identical concentrations In the test with S9 activation. The greater toxi city In the absence of S9 was likely due to the longer period of exposure in the test without S9 activation in comparison to the test with S9 [16 hours (-S9) and 5 hours (+89)]; metabolic Inactivation or conversion of the test agent by the S9 homogenate Is a possibility which can not be excluded with the present data. 2. Mutation Table 2 presents the data for Induction of mutants by Epoxy Resin ERL-4206 and control agents. No definitive evidence was obtained to Indi cate the presence of a dose-related Increase in the frequency of mutants/ 10& viable cells over the 16-fold range of concentrations tested for potential mutagenic action either with or without the presence of an S9 metabolic activation system. However, a single, highly statistically significant Increase In the frequency of mutants was produced by 10 x 10-3X of Epoxy Resin ERL-4206, the highest concentration tested for direct activity without S9 activation. Also, there was a progressive increase In the numerical frequency of mutants produced by three of four of the test concentrations between 1.25 x 10_3X and 10 x 10"3X. These data m port 43-112 Pl|i 7 suggested che existence of e doee-releted effect of exposure which progress ed to e statistically significant level of response only at the top dose level. This single response in the test without S9 complicated the inter pretation of the results as clearly positive or negative. Further studies of the existence of a biologically significant effect would require testing at higher doses which would produce very high cytotoxicity levels and survival of substantially less than 10X of the exposed cell population (10 x 10~3X allowed only 8.5X cell survival as a percentage of the control). Data produced from testing of dose-levels which produce substantially more than 90X cell killing may also be equivocal unless a comprehensive study over a narrow, effective dose-range can verify the presence or absence of a response. In consideration of the equivocal and weak responses obtained in this test, the data suggested that this chemical produced a positive mutagenic effect only at high dose-levels which were highly cytotoxic to the target cells. Epoxy Resin ERL-4206 was classified, conservatively, as a queatlonable-to-weakly active mutagenic agent in the test for direct mutagenic action in the CHO test system. * Mutation frequencies for the solvent controls for tests both with and without S? activation were in an acceptable and low range based upon experience with historical control values. Highly statistically significant mutation frequencies obtained for the DMN and EMS positive controls for both experiments were within the normally expected range of values observed in historical control data and were indicative of an acceptable test. D. Deviations from Standard Procedures - Epoxy resin ERL-4206 was tested as a part of the in-house program for development and validation of the battery of mutagenicity assays at our laboratory. Although several minor deviations from the current procedures used for these tests in our laboratory (Appendix 11) exist in the Methods section for this test, the single major deviation was the 16-hour exposure period for the test of direct action without S9, rather than 5 hour as stated In the Appendix. A 16-hour exposure was used in the early stages of development of this assay and exposure periods from 2 to 24 hours appear to be acceptable if proper dose ranges are selected for testing. Unusual differences in the values for the plating efficiencies in single plates from the 5 x 10"3X and 0.625 x 10-3X (without S9 acti vation; Table 2) were not included in the calculation of average plating efficiency. The lack of growth in only one of the four petrl dishes was considered atypical and its inclusion would have biased the results toward greater mutants/10^ survivors after correction for plating efficiency. Inclusion of this bias would make the results even more highly significant* thus a conservative interpretation of the results was employed to Indicate a questionable, but positive effect. E. Conclusions Epoxy Basin ERL-4206 produced a questlonable-t -weak response as a mutagenic agent f r CHO cells when tested for direct aetl n without an S9 aetab lie aetivati n system. Statistically significant increases in the numerical frequency f mutants were btalned at only the highest Report 43-112 Pag* 8 concentration of Epoxy Raaln ERL-4206 tasted without S9 activation. Although a siailar positive result was not observed at any other doses, an evaluation of these data In comparison to historical experlances with this test Indicated that the data should be considered as a suggestive, weakpositive effect. Other mutation values were not statistically significant from the concurrent solvent control and values were within the expected variation In mutant frequandaa observed In historical control data. SECTION II - SCE TEST - Epoxy Resin ERL-4206 A. Test Dates - Initiated: January 3, 1980 Completed: March 24, 1980 B. Selection of Test Concentrations A maximum concentration of 10 x 10"3X was chosen as the top dose levele for testing without S9 activation based on cytotoxicity data from the CHO nutation 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-4206 concentrations from 10 x 10~3Z to 0.625 x 10"32 by volume was tested. No experiments with $9 activation were performed because the highly statistically significant responses produced in the test for direct action of the test agent Indicated that metabolic conversion was not required for expression of mutagenicity. C. Determinations of SCE Induction The data for SCE Induction In CHO cells treated with various dose levels of Epoxy Resin ERL-4206 or with appropriate positive, negative or solvent control agents are summarized in Table 3. A highly statistically significant increase in the SCE frequency was produced by four of the five dose-levels of Epoxy Resin ERL-4206 tested for direct action In the absence of a metabolic activation system. Also, the production of SCE by Epoxy Resin ERL-4206 was related in a direct manner to the treatment dose, which is generally considered a definitive indication of mutagenic potential. The test without S9 activation was considered a significant Indication of potential direct mutagenic action of the test agent and Epoxy Resin ERL-4206 was classified as a positive mutagenic agent in the SCE test. Induction of SCE by the concurrent EMS positive control was highly statistically significant from the concurrent solvent control and these data indicated an appropriate sensitivity of the test system comparable to our historical positive control data. The numbers of SCE obtained with the H2O solvent and DMS0 controls were also In an acceptable range of values Included In the variability encountered In our historical control values for this test. D. Peviations from Standard Procedures - None Import 43-112 Pag* 9 1879 E. Conclusions Epoxy Resin ERL-4206 produced a significant Increase In the frequency of SCE In CHO cells In tests without addition of an S9 metabolic activation system. Evidence of highly statistically significant and doae-related effects of Epoxy Resin ERL-4206 exposure on the SCE frequency Indicated that the test agent was highly active In the present In vitro assay* SECTION III - UDS TEST - Epoxy Resin ERL-4206 A. Test Dates - Initiated: August 10, 1979 Coapleted: August 23, 1980 B. Selection of Test Concentrations Standard procedures were followed and Epoxy Resin ERL-4206 was tested over a 3-log range of concentrations frow 100 x 10-3Z to 0*1 x 10"3Z by volune. The maximum dose-level was selected with consideration of the cyto toxicity data obtained In the CHO Mutation test which Indicated that higher values would result In excessive cell killing. These dose levels were con sidered to be appropriate for testing because uptake and Incorporation of 3H-thymidine Into hepatoeytes are generally even more sensitive to chemi cal effects than survival measurements with CHO cells (In which cytotoxicity Is measured after a 24 hour recovery period following treatment). C* Determination of OPS Induction 1* Nuclear-Bound Radioactive Label (Data In Table 4) The positive control agents, NQ0 and DMN, Induced numerically elevated and highly statistically significant Increases In UDS over values obtained with the solvent control* With both positive controls an Increase in' the amount of UDS In relation to Increasing concentration Indicated the respon siveness of the test system with measurements using nuclei. However, this response was not linear and not all concentrations of the positive controls produced a response which was statistically significant from the concurrent solvent control. This finding may suggest that the present test with nuclei was less sensitive than necessary to datect weak mutagenic activity. Induction of "unscheduled" Incorporation of radioactive thymidine into nuclei of hepatoeytes exposed to Epoxy Resin ERL-4206 or to appropriate positive and negative controls la presented In Table 4. In hepatoeytes , treated with Epoxy Resin ERL-4206, no concentration tested for potential activity Induced a statistically significant Increase in the amounts of 3H-thymidine Incorporation. A few numerically elevated Increases In the amounts of radioactive incorporation, particularly for concentrations between 1 x 10~3Z to 10 x 10~3X, was s possible Indication for a weak level of mutagenic action of the test agent. However, these responses were not statistically above the solvent control values and the test was considered a negative Indication of mutagenic action. 062W1 iaou , Report 43*112 Page 10 2. DNA-Bound Radioactive Label (Data la Tabla 3) Analyse* of DU, from aliquots of hepatocyta auclsl used for the UDS studies presented on Table 4, were performed as a second assessment of "unscheduled" Incorporation of radioactive thymidine. Values for radio activity incorporated into the DNA of these hepatocyte nuclei are presented in Table 5. For hepatocytes treated with Epoxy Resin ERL-4206, none of the test concentrations Induced levels of UDS which were statistically significant from the solvent control. More critically, we did not observe a doserelated Increase in the levels of UDS; a result consistent with the pattern of negative responses obtained in the assessment of nuclei from cells treated with the same range of concentrations. Moderate numerical increases in the levels of UDS at the 3 x 10"3X and 10 x 10"3X dose levels suggested the possibility of a positive response, but these values were below the levels necessary to demonstrate statistical significance. These several considerations were consistent In the classification of Epoxy Resin ERL-4206 as an inactive agent in the induction of DNA damage discernable by UDS activity. For the positive control agents NQO and DMN, relatively large numerical increases above the concurrent solvent control were produced by the highest concentration of both agents. However, the lowest concentration of NQO and two of three concentrations of CNN did not produce a sufficient response for statistical significance from the solvent control. This result Indicated the responsiveness of the test system but suggested that the level of sensitivity of this particular test could be less than necessary to detect weak mutagealc agents* D. Deviations from Standard Procedures - Testing of Epoxy Resin ERL-4206 was performed as part of the in-house development and validation of the mutagenicity tast battery. One previous experiment using an older, less-sensitive method was performed but was not reported because the level of response with the positive controls was not acceptable for a valid test. E. Conclusion Epoxy Resin ERL-4206 failed to stimulate significantly the incorporation of radioactive thymidine in cells treated over a 1000-fold range of test concentrations. Although values for the positive controls were low, in the assessment of UDS with precipitated DNA and nuclei, the highest concentrations demonstrated the activity of the positive control agents. Epoxy Resin ERL-4206 was considered inactive in the tests with hepatocytes. Reviewed end Approved by; \ Report 43-112 Pago 11 J.001 SyfT7UiM A. Ronald S. Sleslnskl, Ph.D. Study Director Manager, Genetic Toxicology Aeeoclete Director, Toxicology Contributors: Chinese Haaster Ovary teat Sister Chromatid Exchange test Unscheduled DNA Synthesis Assay VPC/1055-5 --if Fred R. Frank, ftj.D. Director Peggy J* Guzzle> B.S. Hester Technologist Michelle tf. Gaunt, B.S. Master Technologist W. Christopher Rangier, M.S. Assistant Scientist 188* Report 43-112 Page 12 REFERENCES 'I ---------------A previous study on 302 Epoxy Rosin ERL-4206 In scstono (formerly termed UNOX* Epoxlds 206) shoved that chls material ms cmreinogenlc and tumorlgenic following lifetime domol oxposuros of ales (CHF Rsport No. 27-152). No other references on Epoxy Resin ERL-4206 were found In o search of the Environmental Mutagen Information Canter data file. ucc 062644 Table 1 Chinese Hamster Ovary (CHO) Hutation Assay: Determination of Tbxlc Effects of Chemical Treatment Test Chemicals Total f Colonies Total I Cells Plated [Epoxy Hesin (ERL-4206] (Z, v/v) 10.0 x 10-3 29 5.0 x 10-3 147 2.5 x 10-3 400 1.25 x 10-3 410 0.625 x 10-3 346 Without S9 Activation-16 hr exposure 800 800 800 800 800 Controls DHSO (20 ul/ml) - Solvent H20 (20 ul/ml) EMS (200 ug/nl) - 340 333 137 800 800 800 [Epoxy Resin (EEL-4206)] (Z, v/v) 10.0 x 10-3 301 5.0 x 10-3 424 2.5 x 10-3 SOI 1.25 x 10-3 509 0.625 x 10-3 483 With S9 Activation-5 hr exposure 800 800 800 800 800 Controls DMSO (20 ul/ml) - Solvent H20 (20 ul/ml) DMN (3700 ug/nl) - 539 528 423 800 800 800 Abbrevlati na: EM8 - ethylmethanesulfonate; DMN - dlmethylnltroaamlne Z Survival 3.6 18.4 50.0 51.2 43.2 42.5 41.6 17.1 37.6 53.0 62.6 63.6 60.4 67.4 66.0 52.9 WPC/1055-5 Z of Solvent Control 8.5 43.2 117.6 120.6 101.8 - 97.9 40.3 t 55.8 78.7 92.9 94.4 89.6 | - 98.0 78.5 If r-/ Table 2 Chinese Haaster Ovary (CHO) Mutation Assay; Results on Evaluation of Mutant Induction by Epoxy Resin ERL-4206 Test Cheaicals Plating Efficiency Total # Colonies Total 1 Cells Plated Viable Fraction Total # Mutant Colonies Mutation Induction Total f Cells Plated Mutantsl 10* Viable Cells [Epoxy Resin (ERL-4206] (X, v/v) 10.0 x 10~3 144 5.0 x 10-3 73 2.5 x 10-3 136 1.25 x 10-3 210 0.625 x 10-3 153 tflthout S9 Activation 400 0.360 14 200 0.365 5 400 0.408 2 400 0.525 0 300 0.510 12 1 X 106 1 X 106 1 X 10* 1 X 10* 1 X 1(|6 38.9 b 13.7 4.9 0 23.5 Controls: DMSO (20 ul/al) - Solvent H20 (20 ul/al) EMS (200 ug/al) - 238 225 187 (Epoxy Resin (ERL-4206)] (X, v/v) 10.0 x 10-3 358 5.0 x 10-3 405 2.5 x 10-3 378 1.25 x 10-3 296 0.625 x 10-3 296 400 0.595 0 400 0.562 1 400 0.468 92 With S9 Activation 400 0.895 6 400 1.012 0 400 0.945 0 400 0.740 1 400 0.740 1 1 X 106 1 X 106 1 X 106 J 1 X 106 1 X 106 1 X 10* 1 X 106 i X 10* . 0 1.8 196.8 c 6.7 0 0 1.4 1.4 Controls: DMSO (20 ul/al) - Solvent H20 (20 ul/al) DMH (3700 ug/al) - 314 272 299 400 0.785 2 300 0.907 0 400 0.748 39 1 X 106 1 X 106 1 X 106 2.5 0 52.2 c 1Total f autsnt colonies per 10* cells plated divided by viable fraction. Statistical significance above solvent control: b: 0.01 > p > 0.001: c: p < 0.001. No superscript indicates p > 0.05. Data analyzed by Student*s t-teat. Abbreviations: H2O - water; S-9 - liver hoaogenate; DMSO - dlaethylsulfoxide; EMS - ethylaethanesulfonate; DMH - diaethylnltrosaalne. WPC/1055-5 oo <no0 I Test Chealcals Table 3 Slater Chraaatld Exchange (SCE) Aaeay: Induction of SCE'a by Epoxy Resin (ERL-4206) Without S9 Metabolic Activation 5-Hour Treataent Total # of Chroaosoaes Total # of SCE SCE/Cell* Mean Nuaber SCB/Chroaosoae2 + S.D. Significance Above Solvent Contr l3 [Epoxy teain (EEL-4206)] (X, v/v) 10.0 x 10-3 5.0 x 10"3 2.5 x 10-3 1.25 x 10-3 0.625 x 10-3 Contr la DMS0 (5 ul/al) - Solvent 820 (5 ul/al) EMS (100 ug/al) - 299 289 294 300 294 288 296 292 833 55.53 2.786 + 0.633 639 42.60 2.206 + 0.492 387 25.80 1.329 + 0.418 293 19.53 0.975 0.283 227 15.13 0.768 + 0.207 190 12.67 0.665 + 0.212 143 9.53 0.485 + 0.186 395 26.33 1.347 + 0.284 c c c b MS MS c 1Fifteen celle exaained per doae level. ?Hesn value of SCE/chroaoeoae deteralned froa the values of the Individual cells exaained. 3statistlcal significance above solvent control: c: p < 0.001 MS: p > 0.05. Data analysed by Student's t-test. Abbreviation: R2O - water; S9 - liver hoaogenate; DMS0 - dlaethylaulfoxide; EMS - ethylaethanesulfonate; S.D. - standard deviation MFC/1055-S Table 4 Unscheduled DNA Synthesis In Hepatocytes from Rat Liver Nuclear-bound label; all DPM values are calculated froa nuclei per 106 viable hepatocytes. Each average la calculated froa duplicate saaples, except for DMSO vhlch was done la quadruplicate. Teat Cheaical Solvent - DMSO Positive Controls: 4 - NQO Concentration it 3.0 ug/al 1.0 ug/al 0.3 ug/al Radioactivity In Nuclei Avg. DPM + S.D. 2070 + 256 19743 + 1572 2951 + 430 3704 + 791 Z of Solvent Control + S.D. 100.0Z + 12.3Z 953.8Z + 75.9Z 142.6Z + 20.8Z 179.0Z + 38.2Z Significance Above Solvent! Control3 -- c NS a' DMN 1000 ug/al 9079 + 1341 438.7Z + 64.8Z c 300 ug/al 2397 + 311 115.8Z + 15.0Z NS 100 ug/al 2297 + 162 111.0Z + 7.8Z US Test Cheaical: [Epoxy Realn ESL-4206](X v/v) ii 100 x 10"3Z 30 x 10"3Z 10 x 10"3Z 3 x 10~3Z l x 10~3Z 0.1 x 10"3Z 1670 + 2230 + 3004 + 2579 + 2688 + 2135 + 338 98 203 61 225 94 80.7Z + 16.3Z 107.7Z + 4.7Z 145.1X1+ 9.8Z 124.6Z + 2.9Z 129.9Z + 10.9Z 103.2Z + 4.5Z NS NS NS NS NS NS ^Statistical significance above solvent control: a: 0.05 > p > 0.01; c: p < 0.01; NS: p > 0.05. Data analysed by Duncan's Multiple Range analysis. Abbreviations: DMSO - dlaethylsulfoxide; 4-NQO - 4-nitroquinollne oxide; DMN - dlaethylnltrosaaine; DPM - disintegrations per ainute; S.D. - standard deviation VPC/lOSS-b Table 5 Unscheduled DNA Synthesis In Hepstocytee from Bat Liver DMA-bound label; all Dm values are calculated froa DNA precipitated per 10& viable hepatocytes. Each average is calculated froa duplicate samples, except for DMSO which was done in quadruplicate. Test Chealcal Solvent - DHSO Positive Controls: 4 - NQO Concentration ix 3.0 ug/al 1.0 ug/al 0.3 ug/al Radioactivity in DNA Avg. ora 4- S.D. 1*87 + T52 19886 4- 1510 8929 + 1076 2264 + 337 X of Solvent Control + S.D. iob.o* + 10.2X 1337.OX 4-101.5X 600.4X 4- 72.3X 152.2X + 22.6X Significance Above Solvent Control1 - c c NS DHN 1000 ug/al 2908 4- 783 195.5X 4- 52.6X a 300 ug/al 1770 4- 366 119.OX 4- 24.6X NS 100 ug/al 1897 4- 79 127.5X 4- 5.3X NS Test Chealcal: (Epoxy Resin EEL-4206) (I, v/v) 100 x 10~3X 30 x 10"3X 10 x 10"3X 3 x 10"3X 1 x 10"3X 0.1 x 10"3X 1241 41568 42049 41994 41735 41402 + 373 72 155 270 128 438 83.4X 4- 25.IX 105.4X 4- 4.8X 137.7X 4- 10.51 134. IX 4- 18.2X 116.7X 4- 8.6X 94.21 + 29.4X NS NS NS NS NS NS aaporc m ^Statistical significance above solvent control: a: 0.05 > p > 0.01; c: p < 0.001; NS: p > 0.05. Data analysed by Duncan's Multiple Range analysis WPC/1055-5 Dm - disintegrations per ainute; S.D. - standard deviation J? Pag* 1 of 3 1889 moon i Chinese Hamster Ovary (CHO) Mutation Aasay Theoretical Basis Mueaclea la a harlcabla alteration la a call la which a gana specifying cha gaaaelc coda for a specific protein la modified la structure and/or fuacfeloa. Mutations, Induced by chemical or physical agents, of the HGPST (hypoxanthlnaquanlne phoaphorlbosyltranaferaae) gana nay be detected by the growth of colonies of "mutant" calls which are resistant to the purine analogs 6-thioguaninm (TG) or 8-*aaaguanina. Koraal calls contaia a functional HGPBX snzyma which phosphorylstes TG and allows Its incorporation into DNA causing the calls to die. Mutant colls with a nonfunctional BGPftT enzyat are unable to ph sphorylata or incorporate TG, thus survive and grow In Its presence. The CHO mutation teat la an assay which detects "forward nutations" fron TG-e nsltlvley to TG-reaiatancm caused by a direct loss of the activity of the HGPST snzyma (HGPST4, - BCPBX"). An assessaent of the ability of several htadred agents to cause gene nutations la vitro Indicates that the CHO nutation assay provides a reasonable estimate of the potential genetic activity of the test chemical. Methods Call Culture Procedures: CHO cells used In these studies were obtained fron Abrahan Hsle at Oak Sidge National Laboratory with tha daslgnatlon CHO-K1-BH4-D1 (or simply CHO for report purposes). Cells are maintained la active growth by subculturlag 2 to 3 clmes/week In antibiotle-frem, Ham's Modified F12 Medium supplemented with 102 (v/v) heat-Inactivated, fetal bovine sera (P12-10), and lacking In hyposanthina. For treatment of calls without metabolic activation, ?12 eediun with 30 ualts/al of penicillin, SO ug/al streptomycin and 32 (v/v) of dialyzed bovine serum (F12-05) la used* For treatments Incorporating an S9 metabolic activation system, Identical medium, but without serum, is employed. Tot determination of mutant frequencies, F12-03 medium containing 2.0 ug/al TG(6-thloguanlne) is used as a "selective medium." Cell numbers are determined routinely with a Coulter Modal F electronic cell counter which la standardized periodically with a pre-couatad suspension of latex beads. Presence of Mycoplasma cell contaminants Is determined by a microscopic fluorescence assay aployiag Hoeehst 33238 dye. All culture procedures and treatments with test chemicals are performed under aseptic conditions in e laminar-flow, biohazard hood; Positive and Negative Controls! Sterile water or glass-distilled dimethylsulfoxide (0MS0) are the usual solvents for tsst chemicals and tha respective solvent Is tested as a control at tha maximum concentration used to add the east sgent. Dlnethylaleresamine (DMH) or ethylmethanesulfonate (SMS) are used aa positive control mutagens for tests with or without an 89 aatabollc activation system, respectively. Mueatlon frequencies obtained with concurrent positive ad negative controls are used as the besla for monitoring the sensitivity and stability of the CHO mutation tost system. Comparison of concurrent control values with historical controls la used to delineate she range of acceptable variations in the test system. 062' Appendix I Page 2 f 5 Metabolic Activation: lie Uvir, S9 homogenate prepared fro* Arochlor-1254 induced, Sprague-Dawley, sale rats la purchased fro* Lieton Bionetlcs, Kensington, HD. Each lae of llvor hoaogonaea la praacraanad for aaeabollc capability to activate DMN la our laboratory bafora uaa In tha tatting progra*. Tha complete S9 aaeabollc activation ayataa containa tha following: 8 uaolaa/al MgClj, 33 uaolaa/al K&, S uaolaa/al glueose-6-phosphete, 4 uaolaa/al NADP^xidlsad (nleotlnaalda adenine dlnucleotlda phosphate), 100 uaolaa/al NajHPOt, and batman 500 to 4000 ug/al of 39 protein (depending on aaeabollc activity); a volume of 1.0 al of tha complete mixture of tha above reaganta la added to each 4.0 ml of culture aedlua. Doaa Selection: Toxicity of the teat chemical la determined prior eo assessment of mutagenic potential to select doses which produce a maximum of 80 to 902 cell killing. Cytotoxleley la determined by either of the following two methods: (1) Clonal assay - 200 to 400 (SO ealls are exposed to a minimum of five dose levels of the teat agent at concentrations from 0.12 to 3 x 10 "*2 (by weight or volume, as appropriate) with and without tha presence of e 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/al). (2) Growth Inhibition - 5 x 10$ cells In 25 cm2 culture flasks are created for i hours with a minimum of five teat 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 la determined by comparing the relative number of cells In control (untreated cells) and In cells treated with various concentrations of the test agant* t no cytotoxicity Is evident at the highest concentrations in the cytotoxicity tests, the test la either repeatad 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 cytotoxleley test Is repeated at a concentration range of 3x10** to 3x10" percent by weight or volume, as appropriate. * Dose levels t*ich are moderately toxic but permit survival of at least 10 to 202 of the cells. In comparison to the solvent control, are selected as the maximum dose, end 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 era used to treat cells; but only the highest five concentrations which permit survival of a sufficient number of cells are assessed for mutation ixuluctlon* Appendix I Pa** 3 of 5 '1891 Chemical aamplaa ar* sterilised by membrane filtration whan alerobiolo*lcal caata lndlcata thia la raqulrad to aaaur* atarllity. Liquid ttac aganta ara taatad on a pareanta*a by volume baala. Solid ehaaleala ar* dissolved in an approprlata aolvaat by asking a 10 to 20X acock solutloa (by weight) and subaaquane dilution* ara aada froa thla atock on a volume/voluae baala. Traataaat with Taat Chemicals! For taata of ehaaleala which say ace directly without incorporation of an S9 aatabolle activation system, SxlO^ call* ar* iaoculatad 20 to 24 hour* prior to traataaat Into 23 cm* eultura flaak* eoatalala* 712-D5 aadlua and Incubated at 37*C la a 5 to 6X COj ataoaphara. Approprlata eoaeantratlona of tha taat agent or control ehaaleala ara added to the call* and eultura* ara treated for 3 hr at 37 *C. Tha aadlua and taat aganta ar* removed by auction, cell* ara rlnaad one* or tvlea and freah 712-D3 aadlua la added. Tha call* ar* allowed a period of 20 to 24 hour* f recovery froa eraataeat before survival la determined. Traataaat of call* for teatlag of ehaaleala which require aatabolle activation for autagenle capacity ia performed identically with the procedure above, with the exception ehat 712 aadlua withoue aerua and containing 1.0 al of 39 aetlvaeloa mixture per 4.0 al of aadlua la aaployad. Determination of Cytotoxicity: The relative aurvlval of treated call*. In eoaparlaott to solvent-controls, ia determined on* day after the expoaur* to the teat agent*. Tha level of eytotoxlelty ia often correlated with eh* autatloa frequencies induced by (mown chemical mutagen*. Thu*, axcasalv* cytotoxicity aay kill both normal call* and autant* and aay dapraaa the actual autaelon fre quencies; lnaufflelent cytotojd.city aay lndlcata an lnaufflelent concentration of tha taat agent waa aaployad* The colony-forming potential of 100 to 200 created cell* 1* used aa tha asaaura of treatment-induced eytotoxlelty. Survival valuas which Indicate the cytotoxic effeeta of tha teat aganta ara included In raporta la tabular form. Statistical analyse* ara not performed n these data, since they ara only uaaful to assaas whether appropriate dose* were aaployad and ara not used to calculate^autaelon frequencies. Determination of Mutant Induction; On day* l, 3 and 6 (or alcamaeivsly l, 4 and 6) after treatment with the various tost agents, approxlaacely 5x10^ cells are subculcured la 100 am tissue eultura dish** In 712*03 aadlua and incubated at 37-C la a 3 to 6Z COj ataoaphara. After a total of 7 days to allow "expression" of the*autant phenotype, eells are dissociated with 0.05 to 0.073X trypsin, counted and plated at a concentration of 2.5x 10^/dlah In four culture dishes (lx 10* total calls) which aaeh contain 3 al of 712-DS (TG) elective medium. At this tlaa, cell* are diluted end 100 cells/dlah are added to four culture plates containing 712-D5 medium (without TG) to assoss viability (plating efficiency) of the treated cell population and to determine the surviving fraction. All eultura* ar* than Incubated for aa additional 6 to 8 days to allow growth of calls; aadlua la then discarded and colonies ar* fixed and stained for counting. The number of colonies la selection plates and in tha viability taat ar* counted by electronic mathods, checked by annual counts and data ara recorded both as total autant*, mutant*/10^ total calls and autanta/lO* viable calls. 1892 Appendix I Page A of 3 Statistical Anilymi Uniform statistical procedures to evaluate In vitro mutation data have not been developed* Tha dlatrlbueloa of mutation frequencies froa hlatorlcal eontrola In at laaat two laboratorlaa lndicataa chat tha fra* quency dlatrlbueloa and variances aneounearad do not justify tha uaa of para* metric analysta talaaa data la cranaforaad bafora application of atandard para* aatrlc tasca. Analysis of mutation fraquanclaa In tha CHO east follow tha pro* cadura of Irr and Snaa (Rafareacs 4) which employs tha Box-Cox Transformation (Reference S) to tranafon data bafora parametric analyses. Tha autaeion fre quency for each plate la lncreaaad by 1.0 (to alialnate xaroa) and ralaed to the 0.15 power. Experience with hlatorlcal negative control data In our laboratory lndicataa that a normal probability distribution of tha data suitable for pars* aatrlc analyses la achieved by this transformation. Parametric analysis of au* tatlon data by the Student's t-teat is performed with the transformed data. The d gree 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 era available. Interpretation of Data; The criteria for Interpretation of the test results as a positive or negative response depend upon both the level of statistical significance from the concurrent control and the evidence of a dose-response following treatment. When a definite dose-response relationship is not evident but one or more marginally significant values are obtained, a careful asaminati n of the data froa the concurrent positive and negative controls and coaparl* sons to historical control data are used to evaluate the possible significance of the responses* Historical control data indicate that a spontaneous mutation frequency In CHO cells of approximately 4 to 5 mutants/10^ viable cells, with a range of 0 to 25 mutants/106 viable cells, can be obtained in the absence of mutagenic treatment. Statistical comparisons against unusually high or low spontaneous controls are subjectively scrutinised In respect to the above variability. Appendix I Pa*e 3 of 3 1333 References 1 Chu, E. H* Y end H. V. Malllnff* Cheaical Induction of Soeelfle Loru* IfT^.-ubtinii.1^1" *** , .2 O'Neill, J. P., P. A. Brlaer, R. Meehsnoff, 6. P. Hirsh, . W. Bale. A J MuC*tTl0n Infuctlon at the Bypoxan chine -Guanine " hsphorlboayl Tranaferase locua in Chinese Haaster Ovary Celia (CHO/BCPB.T Sgtsm^Pe^opnent end Deration o? the Sene-. iF 3. O'Neill, J. p, and A. W. Bale. Phenotypic Expree*l TfiM Mra*nTi- InduceA A-Thiamin*, ReelstancelT'ghlneae faaane'er (Wirt'Ml. tAxnt**** Syaten), tatacioa Research. 3$, (1979).' 1Q9-11R. ~` ' *' ^1*^/2^^*** 8' S"* logistical gysluatlon of Mueagealclcv in aug^TS Ulm. mSh^ th* "id iptl"* 5- E* s^r;H.ir,torMClo--J-- - WPC/1033 1894 Pat* 1 of 4 APPENDIX II Determination of Sister Chromatid Exchange (SCE) Frequencies In Chinese Haastar Ovary (CHO) Call* In Vitro Thaoradeal Basis Exchanges of t*natlc material batwaao tho Individual am* of a chromosome (i.a. siaear chromatids) ara thoufht to aria* from breakage and physical interehangas in tha DNA of a call during call division. An incraaao in tha frequency of such intarchangas batuaan sistar chromatids can bo observed in calls created with physical or chaaical mutagenic agents, or in calls exposed to aany suapa t or proven human carcinogans. Thus, analysis of SCE frequencies in cells traatad with a test agent has baen suggested as a sensitive screening cast for potential outaganic/carcinogenic chaaical*. The mthod used in our study to visualise SCE's in CHO calls grovn in cul ture is based on tha procedure described by Parry and Wolff (1974). A standard concentration of 3.0 ug/al of bromodeoxyurldlaa (BrdtJ) was used in tha grouth medium to allow a visualisation of SCE's after two call divisions in the pre sence of BrdU. Staining of chroaoseaes with 3.0 ug/al of 33258-Soechst fluore scent dye, exposure to light and Glaasa staining was used to dlfferantlat* chroaatid* for SCE analysis. Methods Call Culture Procedures: Chinese haastsr ovary (CHO) calls vara obtained froa Abraham isle at 6afc "Edge National Laboratory with the designation CEO-KlBH4-DI (referred to slaply as CHO for report purposes). CHO cells era maintain ed in active growth by 2 to 3 weekly subcultures into fresh antibiotic-free, Han's F12 (nodifled) aadiua fortified with 10Z (v/v) of heat-inactivated fatal bovine serum and lacking hypoxanthine 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 calls for ehroaosoae 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 Hoeehst 33238 dye. For treatments with test chemicals without 59 metabolic activation, modified F12 medium is used with 50 units/ml of penicillin, SO ug/al streptomycin and 3Z (v/v) of heat-inactivated, dialyzed fetal bovine serum (F12-D5). Identical medium but without serum is used for treatments incorporating an S9 metabolic activation system. Positive and Negative Controls; Sterile water or glass-distilled dimethyl sulfoxide (DMSO) are tha usual solvents used for test chemicals and tha respective solvent is tasted as a control at the maximum concentration used to add the test agent. Dlmethylnltrosamin* (DMN) and ethylmathanasulfonate (EMS) are used as positive control mutagens for tests with or without the addition of an 59 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 test system. Comparison of concurrent control values with historical controls is used to delineate the range of acceptable variations in the test system. 062655 18SJ5 Appendix II Vage 2 of 4 Metabolic Activation: lac liver 39 homogenate (prepared from Arochlor 1234 induead, Sprague-Dawley, male raca) la purehaaad froa Liecon Blonetlcs, Kensington, HD. Each lot of llvar homogenate la prescreened for activity la our lab retory bafora uaa la cha caaeln* program. Tha complete 89 aatabolle acelraeloa system cooealoa tha following: g umolee/ml MgClg, 33 umoles/ml KCl, 3 uaolaa/al KCl, 5 uaolaa/al glucoae-6-phosphate, 4 uaolaa/al NADP-oxidizad fora (oleoelaaalda adaniaa dlauclaoclda phosphate), 100 uaolaa/al NejHPOa aad baevaaa $00 to 4000 ug/al of 39 proeoln (dapaodlog on aatabolle activity). 4 vo1uaa of 1.0 al of tha coaplota mixture of tha above raaganta la addad to aach 4.0 al of eultura aadlua. Doaa Salaction: Toxicity of tha eaat chaalcal la dataraiaad prior to aaaaasaane of autagaaic potantlal to aalact doaaa which produce a aaxiaua of 80 to 90Z call killiog. Cytotoxicity la dataraiaad by either of the following two methods aa part of tha CEO mutation teating procedure: (1) Clonal assay 200 to 400 CEO celia are axpoaad to a minimum of five doaa lavala of tha teat ageat at concentrationa froa 0.1Z to 3 x 10"*Z (by weight or volume, aa appropriate) with aad without tha pretence of a aatabolle activation ayatea. The number of calla which aurvlva tha treatment la deceralaad by couaclag tha number of coloniea produced aftar 7- to 8-day Incubation period (37*C) la comparlaoa with the coloolea formed by calla treated only with appropriate eonceneraelona of aolveat (ganarilly 20 ul/al). (2) Growth Inhibition - 3 x 10^ calla in 23 ca^ eultura flasks are treated for hours with a minimum of five east concentrations both with and without S9 aatabolle activation. Following treatment the cells arc rinsed, frash F12-D5 medium la addad aad tha flasks arc Incubated for an additional 18 to 24 hours* Cytotoxleley la determined by comparing cha relative number of calla la control (untreated cells) and In calla treated with various concentrations of the teat agent. If no cytotoxicity la evident at the highest concentrations In the cytotoxicity teats, tha taat 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 la evident even at the lowest dose, the eytotoxieity teat la repeated at a concentration range of 3x10"* to 3x10-0 percent by volume. Dose levels which are moderately toxic but permit survival of at least 40 to 30Z of the cells, la comparison to the solvent control, are selected as the eexiaum dose, end et least four additional one-half dilation* ere tested for induction of mutations. If eytotoxieity data are equivocal, a total of 5 to 8 on*-half dilutions of the selected, aav-tann concentration art used to treat calla; but only the highest five concentrationa which permit survive! of e sufficient numbar of mitotic cells with SCE staining, art evaluated for SCE induction. Chemical samples are sterilized by membrane filtration when microbiological tests indicate ehls is required to assure sterility. Liquid teat agents are teatad on e percentage by volume basis. Solid chemicals era dissolved In an appropriate solvent by asking e 10 to Z0Z stock solution (by weight) end subsequent dilutions art mads from ehls stock n a volume/volume bests. ucc 062656 18S6 Appendix XI Page 3 of 4 Treatment With Teat Chaaleala : Teaelnf of chaaleala for dlraee autagenlc action (without S9 metabolic acclvaclon) la parforaad flrat. For chaaleala with clearly positive autagenlc capabllltlaa bp direct action, taatlng with aatabolle activation la generally not parforaad. For taatlng direct acting eheolcala for SCE induction, between l to 2 x 10 ealla are plated Into 73 ca* culture flaaka In F12-D3 aadlua at leant20 hra prior to traatoant and Incubated at 37#C In a 3 to 6X CO2 ataoaphara. Appropriate coneentratlona of the teat agent or control chaaleala are added to the eella and 3 ug/al BrdU la added to all flaaka* Celia era treated with teat aganta for 3 hra, oedla la than removed bp auction, cella are rlnaed with buffered, phpalologlcal aalt eolation end fresh aadlua containing 3 ug/al BrdU la added for at least 24 bra of additional Incubation at 37*C to allow two rounds of cell division. Calls are harvested and chroooaoaaa era prepared for SCE staining. Traatoant of calls for tasting of chaaleala which require aatabolle activation for autsgenlc effectiveness la parforaad slollarlp aa for treataents without activation, except for three aodlficatlona: 1. Before treatoent with the teat agents, F12-D5 aadlua Is reaoved and F12 aadlua without serua la added. 2. S9 aatabolle activation alxture is added to each flask (Including solvene and positive controls) before addition of test agents. 3. Calls are treated for a eotal of 2 hra (rather than 3 hra) ud then incubated for 38 to 42 additional hours before harvest for chromosome preparation. Preparation of Chromosomes: Colceoid* (0.1 ug/al) or Colchicine (0.2 ug/al) la a^dad to culture flasks 1 to 2 hra prior to harvesting to arrest cells In mitosis. Calls are then reaoved from flasks, after a brief Incubation with 0.012 DIFCO trypsin, suspended in 0.075M KC1 (hypotonic) solution and Incubated f r 13 to 20 oln at 37*C. Calls are centrifuged, f^ed with 3 or 4 changes of Carnop's fixative (3:1 aethanol acetic add) and chrooosoae spreads are prepared froa cells suspended la a small volume of fixative. One slide/dose level is prepared, but fixed cella are saved if needed for preparation of additional slides. * Chromosomes are stained for SCS's bp treatoent with 3.0 ug/al of Boechst 33238 dpe for 20 oln, rinsed In distilled water, Immersed In Sorenson's buffer and exposed to a high intensity sunlaap for 13 to 30 oln., as required. Irradiated chromosomes are stained In Gurr'a glemsa (diluted 1:25 with water), rinsed in water and dried before application of coveralips. Examination of SCE*a: All slides are eoded and read la a blind fashion without indication of the specific treataent or concentration of the teat agent. The noaber of chroaosoaes and the nuaber of SCE's in a alnlaum of 15 cella are recorded for eseh dose level. The a an nuaber of SCE/cell and SCE/chrooosoae ere calculated and recorded. Slides are decoded nip after examination of all slides in the experiment has been completed. UCC 062657 AppaodiX IX Pag* A of 4 -1897 Statistical Analyse*: Data art analysed by appropriaca paraaetrle statisti cal procedure* which follow BHftC standard operating procaduras for analysts of data. Slgnlflease* value* and eh* ataeiatlcal t*e employed ar* ahova for data auanarlzad la tabular fora. Interpretation of Data: The critarla for avaluatloa of a poaltlva or nagatlva response depend both on tha laval of tatla deal algalfleaaea and eubj ac tive analyse* of cooeurrant aad hlatorleal eoatrol data. Tha key determinant la whether a doee-dependent laereaae la SCE'a la induced by the teat agent. When ao clear doae-raapoaae ralatlooahlp la *vld*at aad when on* or aor* responses of oerglnel atatlatleal aigniflease* ar* obtained, a careful eocaalaatloa of the data la coatparlaon to th concurrent control* aad hiatorleal data baa* la neeaseery. Testing nay be repeated to clarify unusual responses, If date for the c ncurrant positive or negative controls suggest a defeet in the original ex* periaent. Overall assessment will also r*ly on corroborating data from the other tests In the testing battery. Clearly positive responses will laelude any of the following: (1) Doubling In the SCE frequency at a alnlaua of two of the five concentrations tested; (11) Statistically significant responses of p < 0.03 at three concentrations or at 2 concentrations If p < 0.01; (111) Induction of a statistically aignifleant, dose-related Increase in the nuaber of SCE. General References 1. Perry, P. and S. Wolff. Sew glams* method for differential staining of sister chromatid*. Nature, ill(!l9H), 154-158. 2. Lett, S. A., J. W. Allen, W. E. Rogers aad L. A. Juergens. In vitro and in vivo analysis of sister chromatid exchange formation, pp 275-191 in Handbook of Mutagenicity test Procedures. *d. Kilbey, B. J*, et el. Elsevier Publ. 3. Carraso, A. 7., 1. B. Thompson, P* A. lindl and J. 1. Hinkler. Sister chromatid exchange as as indicator of autagenasls. Secure, 271, (l97S), nran:------------ 1--s---------- 88-------------------- 1---------- -- A. Galloway, S. M. and S. Wolff. The relation between chealcally Induced sistar-chromatld exchanges and chroaatld breakage. Mutation Sea., 61, 'tunr, wjgr.--^ 3. Snedecor, G. W. and W. G. Cochran. Statistical Mathods, 6th Ed., Iowa Stat* Univ. Pvesa, imes, Iowa (1967). " WPC/1033 UCC 062658 Page 1 of 3 APPENDIX III Pnachadulsd DNA Synthesis (UPS) In Hapatocytss from Roc Liver Theoretical Bails Chemicals may interact with both tho cellular components and tha gsnstic material of a coll (a.g. DNA and SNA) bocauaa of thalr electrophilic atcurt or by contortion into reactive electrophiles by tho aotabollc enzyme* of tho cell. Damage to tho DNA of a coll can result la coll death, oueatlon or, theoreti cally, carcinogenic transformation. Studios of agents which are capable of reacting and damaging tho callular DNA hate suggested that such maebeda may be useful as a sensitive screening test for detecting potential nutagenic/ carcinogenic chemical properties* Detection of the relatively snail amounts of DNA damage Induced by chemical treatment requires a cellular system in which normal, semiconservative DNA rep lication, which occurs during cell division, la Inhibited. The system employed for the present study uses a suspension culture of primary hepatocyte cells iso lated from rat liver according to the general methods of Seglen (1973) end Villisms (1976). Hepatocytes do not normally divide In the minimal culture ae- dlum 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 poeitive end negative controls es well es with historical data for similar tests. Methods * Prspsrscion of Hspetocyts Suspensions: Hilltop-Vistar albino rats srs anesthetized with Mataisne(k). The abdominal cavity Is surgically exposed end 1230 units of heparin is injected lntrsvenously. A catheter Is inserted into tha portal vain and warm Hanks Balanced Salt Solution (HBSS) Is pumped into the vain end through tha livor. This first solution contains heparin sad EGTA, [ethylene glycol-bia-(bet-eaioo*thyl-eth*r)N,N-tetr*cetlc add], which preferentially chelates calcium; the solution contains no magnesium or cslelun. After tha liver is blanched, e second solution of H3SS containing 60 units/ml of collagenase Is perfused. This solution is pumped through the Uver until the liver is digested. The liver le then removed and the cells ere freed in cold medium 199 by combing through the lobes with e sterile metsl comb. The cell suspension is passed through ewo nylon meshes to remove eell dumps end the cells ere washed once et low centrifugation speed. After resuspension in medium 199, equal volumes of cells sad 0.4X trypan blue ere mixed together and the cell viability and number of viable cells per ml Is determined microscopically. I Prelncubetlon of Hepatocytes: Approximately 2 x 10* viable hepatocytes are added to 5 ml medium 1$9 containing 10 mM hydroxyurea and 30 mM Hepes (N-2-hydroxysthy1 pipere*lne-N-2 ethane sulfonic add) buffer. After the cell* ere dispensed Into the tubes, they ere pieced on e rocker platform and ere Incubated et 37*C for 1 hour. Although 'hepatocytes do not normally divide in culture, medium 199 whleh lacks serum end contains hydroxyurea is used to further bl ek semi-conservative DNA synthesis. Thus, any radioactive thymidine Incorporated into the nuclei Is expected to result fro repair or unscheduled DNA synthesis. On ~o I Appendix III Fags 2 of 3 1603 Selection of Doses of Teat Chemical; Initially, the following concentretlona a iO'^l (oyvolume) ere coated! 100, 30, 10, 3, 1, end 0.1. If these concentrations prove to be cytotoxic, or If additional Information Is available from other in vitro tests as to the proper dose levels, then an appropriate serias of concentrationa is used over a 3-log range of concentrations* Treatment of Hapatocytas; After prelnenbation, 23 uieroCuries of trlclated thymidine (20 Curies/aliiLaois) Is added to each tube* The test chemical and positive controls are diluted In an appropriate solvent and they are then added to each labeled tube. Generally, at least six concentrations of the test chemical over a 3-log range of concentrationa are tested and each concentration Is run in dupllcats* The tubes are returned to the rocker platform for a 2-hour exposure at 37*C. Positive and Negative Controlsi 4-nltroqulnollna oxide (HQO), a directacting mutagen, which induces U^-type DMA repair and dimethylaleroeamlna (DMH), vhich requires metabolic activation by microsomal enzymes for activity, are run la duplicate as positive control chemicals. The solvent control la run in quadruplicate and consists of 100 to 130 mleroliters (concentration specified la Individual reports) of the solvent used to dilute the sample. Dimethylsulfoxide (DMSQ) 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 3 ml of cold medium 199 and are resuspended In 0.232 Triton X-100, SZ citric add and 3 mtl MgClj, * lysing solution which liberates the nuclei. The nuclei are rinsed once la this solution and resuspended In 0.23 M sucrose, 2.3Z citric acid and 3 aH M$Cl2* 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 nuelear suspension is mixed with 1*0 mg of NC3 tissue solubilizer in a scintillation vial. Ten ml of Dlmlluma* scintillation cocktail la added and the radioactive disintegrations per minute (DFM) are determined by counting twice In a scintillation counter for tea minutes. The measured DFM are then used to calculate the DPM/IO* viable hspatoeytes presented on tables* Determination of DMA-Bound Label: The amount of radioactive thymidine incorporated into DMA is quantltated^n DMA isolated and precipitated from 1.00 to 1.23 x 103 viable hepatocytes* To 1*23 ml of the nuclear suspeaelou, 2.73 ml of 1Z sodium dodecyl sulfate (SDS) and 3 mM Ethylenadlamlnetetraacetlc Acid (EDTA) is added to lyes the nuclei. The DMA Is precipitated from this solution with 4 ml of ice^old 10Z trichloroacetic add (TCA) and the sample tubes are incubated at 0*C for at least 30 minutes* Tbs 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 30*C for 1 hour with l ml of a diluted solution of 5CS tissue solubiliser; prepared by adding 1 part solubiliser eo 2 ports of Dlallume# cocktail. Dtmllumee is then added to each vial and the vials are counted twice in a scintillation counter for ten minutes. Appendix III Page 3 of 3 Statistical Analysis: The avaraga DPM if calculated for each doso level ad the control* and final results ora expressed oa DPM/106 viable hapatocytea. Data art alao expressed aa a percent of the solvent control for purpoaea of comparison. The original data are statistically analyzed by the appropriate parametric teat, following the BRRC atandard procedurea for atatiatical analyaea and the taat(a) employed la indicated on the reapective tablea. Coaparlaon between the aeon for each doae level with the 9JZ confidence lialta of the hlatorical solvent control aay alao be uaed in aoae caaea to aaaeaa the potential biological algniflcance of the data* Teating aay be repeated to clarify unuaual responses, if data with the concurrent controla auggeat a defect in the original experiment. Interpretation of Results: The elaaalfleatlon of a chemical aa a poaltive, active agent dependa upon eke production of a atatiatically aignifleant, doae-ralatad lncreaae in the aaount of CDS activity. If a definite dose-response relatlonahlp la not evident, or when a few increaaea with marginal atatiatical aignlfleance are obtained, coaparlaon of the reaponaea to hlatorical control data provider a meaningful aaaeaament of the poaalbllity for random varlatlone which may be atatiatically significant only la relation to the concurrent control* A key determinant of the reliability of the UDS data la the detection of a similar response with both DMA and isolated nuclei determined at two or three consecutive concentrations. General References 2. ifaraoatsu, M. Isolation of Nuclei and Nucleoli. In: Methods in Cell Physiology, Vol. 'tv. H76. Editor: D. M. PTeacott. Academic Press, Mew York. * 3. Seglen, P. 0. Preparation of Rat Liver Cells. III. Earymatle Acquirements for Tissue Dispersion. Experimental Cell Research, 82, (1973), 391-398. ~~ 4. Williams, 0. M. Detection of Chemical Carcinogens by Unscheduled DMA 3. Williams, G. M. The Use of Liver Epithelial Cultures for the Study of Chemical Carcinogenesis. American Journal oi Pathology, 8$, 11978), 737=733: 8. Williams, G. M. and K. F. Laspla. The Detection of Various Mitroaamines la the Hepatocyte Primary Culture/DWA Repair Test. Cancer Letters, 4, (1979)7 7. Snedeeor, G. W. and W. G. Cochran. Statistical Methods, 6th edition, Iowa State Ohiveralcy Prase, Ames, lows (Hi?). WPC/1033 Report #43-112 Appendix IV Physical and Chemical Charactertitles of Tent Material CHP Sample No: CAS No.: Chemical Name: Trade Naae and/or Synonyms: Molecular Weight: Formula: Molecular Structure: 42-137 106-87-6 3-(epoxyethyl)-7-oxabtcyclo(4.1.0)heptene BakellteV Cycloaliphatic Epoxy Rexla ERL-4206; vlnyl-3-eyclohexene diepoxide; Epoxyethyl-3, 4-epoxy cyclohexane 140.18 c8a122 Specific Gravity (8 20*C): Bolling Point: Solubility In B2O (I by vt): Purity: 1.0986 227 *C 9 760m Bg 18.3 9 20*C Approx. 1001 Vapor Preaaure (8 20*C): pH: Plaah Point: St blllty: Incompatibility: Appearancea and Odor: Dlapoeal: <0.1m Bg Not Available 225*7 (eloeed cup) Unstable, avoid temperaturee > 100*7 Avoid amines, aclda, alkallee Low vlecoalty liquid; characterlatlc odor Small epllls can be flushed with water Larger quantltlee can be diluted with an lnaert aolvent and burned. Protective Meaauree: Use rubber glove*, goggle* and protective creme on arms and face. An eye bath and eafety ahower should be available. A local exhauat ventilation ayatem la preferable to reduce expoaure. In caae of contact fluah yea or akin with plenty of water. Health Baxard: Prolonged and repeated contact with liquid or breathing of vapora or mlata nay cause delayed and serious injury. Absorption through akin is harmful and the liquid causa* eye injury. Skin painting teats in animals caused skin cancers, but no cancers have been reported in humans.