Document 2JeLMdNG4R59dwMxrYVZ9j03a

187 BUSHY RUN RESEARCH CENTER R. D. 4, Mellon Rood, Export, Pennsylvania 1M32 Telephone (412) 327-1020 CONFIDENTIAL: Not to be released outside VCC without the written consent of the UCC component sponsoring the work. .Project -Report 43-112 17 Pages Tel: (412) 327-1020 December 12, 1980 BAKELITEe Epoxy Resin ERL - 4206 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-4206 was evaluated for potential mutagenic activity with a 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 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 CHO test. Although there was no positive effect la 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 cells. A maximum concentration of 0.01Z (by volume) was selected as the maximum concentration for testing both with and without metabolic activation. At slightly higher concentrations (eg. 0.03Z) fewer than 1Z of the treated cells were capable of producing colonies. CHO Mutation Test - Epoxy Resin ERL-4206 was active only at the highest dose level (10 x lO'-^Z) in producing a statistically significant increase of mutant cells when tested without as $9 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 4f the data and the test without S9 actlvati n was eon- Bushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation 063105 1872 Report 43-112 Page 2 sldered quastionably-to-weakly positive. The test with an S9 metabolic activa tion system did not result In a significant increase in the mutant frequency, which suggested that the metabolic activation system may have obscured the weak response obtained without S9. SCE Test - 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 metabolic activation provided a dear indication that Epoxy Resin ERL-4206 was active in stimulating SCE in CHO cells. The test without a metabolic activation system indicated that Epoxy Resin ERL-4206 did not require metabolic conversion for activation and the test incorporating an S9 metabolic activation system was not performed. OPS Teat - Epoxy Resin ERL-4206 failed to induce dose-related increases in the UDS detected with either nuclei or DNA. In evaluations over a relatively wide range of concentrations, no concentration of Epoxy Resin ERL-4206 produced statistically significant levels of DOS activity. Production of numerically in creased levels of UDS by a few of the concentrations of Epoxy Resin ERL-4206 tested for activity might be an indication of a low level of primary DNA damage. However, the lack of a statistically significant effect indicated that Epoxy Resin ERL-4206 was not active in the induction of UDS 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 in the UDS and CHO tests. Because only the highest concentration of Epoxy Resin ERL-4206 prdduced a significant effect in the 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 UDS test (which uses metabolically competent liver cells) and in the CHO test with liver S9 activation suggested that Epoxy Resin ERL-4206 may be metabolically converted to a non-mutagenic form by the liver activation systems. The production of positive response in mouse dermal carcinogenesis studies (CHF 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 CHF Sample No.: 42-137 Submitted by: tf. C. Kuryla, for UCC Toxicology Advisory Committee Date Received: March 15, 1979 Division: Specialty Chemicals and Plastics Identification: Yellow liquid CAS #: 106-87-6 UCC 063106 Report 43-112 Pag* 3 BAKELITE* Cycloaliphatic Epoxy Resin ERL-4206 In Vitro Mutagenesis Studies: 3-Test Battery Sponsor: Onion Carbida Corporation ***** OBJECTIVE The purpose of this study was to evaluate the potential of Epoxy Resin ERL4206 to induce genetic damage in mammalian cells at the gene, chromosome and/or DNA (deoxyribonucleic acid) level of molecular organization. A battery of three in vitro, short-term tests which detect each of these genetic endpoints was employed to evaluate Epoxy Resin ERL-4206 for potential mutagenic activity. A general description of the theoretical basis for each of these three tests Is presented in Appendices I, II and III attached to this report. SAMPLE CHARACTERISTICS A typical, commercial sample of Epoxy Resin ERL-4206 was received from UCC, South Charleston, WV for testing on March 15, 1979. The 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 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 0.5Z to 1 x 10"3Z (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 10Z of the treated cells. Glass-distilled dimethylsulfoxide (DMSO) was used as the solvent and solvent control; sterile, distilled water (H2O) was used as the negative control. To simplify tables and to allow comparisons between different tests, concentrations of Epoxy Resin ERL-4206 in the following sections of the report are given in terms of volume percentages x 10"3Z to eliminate zeros in the lower concentration values (eg. 0.000625Z0.625 x 10"3Z). UCC 063107 1874 Report 43-112 Page 4 B. Mutation - CHO cells were exposed for 3 hours to a minimum of five concen trations of Epoxy Resin ERL-4206 with the addition of an S9 metabolic activation system and to an identical range of concentrations for 16 hours without S9 activation. Dilutions of Epoxy Resin ERL-4206 for testing were prepared in DMSO and various aliquots of the test agent were pipetted into the cell culture media. The surviving fraction was determined at 20 to 24 hours after treatment and the mutant fraction was determined after a 7- to 9-day period to allow "expression" of the mutant phenotype. Only the top five concentrations which allowed sufficient cell survival were assessed for survival and induction of mutants. The percentage of cells surviving the treatment, the frequencies of 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 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-4206 for testing were prepared by direct addition into 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 S9 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 test agent. Bromodeoxyurldlne (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 medium 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/chromosome and the level of statistical significance of the increases above concurrent solvent control values are presented in tabular form. 3. DPS Test (Detailed procedures in Appendix III): Induction of primary DNA damage in rat liver cells (hepatocytes) was studied at a minimum of six dose levels which spanned a 1000-fold range of concentrations. Cells were treated with Epoxy Resin ERL-4206 for 2 hours in culture medium containing ^H-thymidine, hydroxyurea and appropriate dilu tions of the test agent prepared In DMSO. Determination of UDS 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. UCC 063108 Report 43-112 Page S 4. Controls - Positive, negative and solvent controls were tested concurrently with the test sample to assure the sensitivity of the test system and the concurrence of the results to previous test performance. For the CHO and SCE assays, dlmethylnltrosamlne (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-dlstllled dlmethylsulfoxlde (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^' DMSO was used as the solvent and the solvent control. 5. Metabolic Activation - S9 liver homogenate, prepared from Arochlor 1254lnduced, Sprague-Dawley male rats, was purchased from Litton Bionetlcs. 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 rnln/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 Irr and Snee: (MF + 1)0*15 (jrr> j. p. *nd g. 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. 7. 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 ucc 063109 1876 Report 43-112 Page 6 B. Selection of Test Concentration (Data pot ahown In tablea) CHO cells were exposed to six concentrations of Epoxy Resin ERL-4206 which spanned a concentration range from 500 x lO'^Z to 1 x lO^Z by volume. The exposure period was 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 S9 metabolic activation system, was determined by counting the number of coloniespro duced by the survivors after a 5 to 7 day Incubation period. A concentra tion of 10 x 10~3z was selected as the maximum concentration for testing with and without S9 activation; a higher concentration (30 x 10~3z) allowed survival of fewer than 1Z of the cells treated with the testagent 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 cells 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 em ployed 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~^Z and 10 x 10~^Z. These data ucc 063110 1877 Report 43-112 Pag* 7 suggested the existence of a dose-related effect of exposure which progress ed to a 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 10Z of the exposed cell population (10 x 10~3z allowed only 8.5X cell survival as a percentage of the control). Data produced from testing of dose-levels which produce substantially more than 90Z 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 questlonable-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 S9 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 testa in our laboratory (Appendix II) 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 10~3z (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 ----------------- UCC 063111 Epoxy Resin ERL-4206 produced a questlonable-to-weak response as a mutagenic agent for CHO cells when tested for direct action without an S9 metabolic activation system. Statistically significant increases In the numerical frequency f mutants were obtained at only the highest Report 43-1X2 Page 8 concentration of Epoxy Resin ERL-4206 tested without S9 activation. Although a similar positive result was not observed at any other doses, an evaluation of these data in comparison to historical experiences 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 frequencies 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 10~3Z was chosen as the top dose levels for testing without S9 activation based on cytotoxicity data from the CHO mutation test. Higher concentrations were expected to produce delays in the mitotic cycle and to decrease the number of cells with SCE staining, A 16-fold range of Epoxy Resin ERL-4206 concentrations from 10 x 10~3g to 0.625 x 10-3j by volume was tested. No experiments with S9 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 DMSO controls were also in an acceptable range of values included in the variability encountered in our historical control values for this test. D. Deviations from Standard Procedures - None ucc 063112 Report 43-112 Page 9 1879 Conclusion# Epoxy Resin ERL-4206 produced a significant increase in the frequency of SCE in CBO cells in tests without addition of an S9 metabolic activation system. Evidence of highly statistically significant and dose-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 Test Dates - Initiated: August 10, 1979 Completed: August 25, 1980 Selection of Test Concentrations Standard procedures were followed and Epoxy Resin ERL-4206 was tested over a 3-log range of concentrations from 100 x 10~3j to 0.1 x 10~^Z by volume. The maximum dose-level was selected with consideration of the cyto toxicity data obtained in the CH0 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 ^H-thymldine into hepatocytes are generally even more sensitive to chemi cal effects than survival measurements with CH0 cells (in which cytotoxicity is measured after a 24 hour recovery period following treatment). Determination of UDS Induction 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 increaaing 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 detect weak mutagenic activity. Induction of "unscheduled" incorporation of radioactive thymidine into nuclei of hepatocytes exposed to Epoxy Resin ERL-4206 or to appropriate positive and negative controls is presented in Table 4. In hepatocytes treated with Epoxy Resin ERL-4206, no concentration tested for potential activity Induced a statistically significant increase in the amounts of ^H-thymldine incorporation. A few numerically elevated increases In the amounts of radioactive Incorporation, particularly for concentrations between 1 x 10~3% to 10 x 10~^Z, was a 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. ucc 063113 1880 Report 43-112 Page 10 2. DNA-Bound Radioactive Label (Data in Table 5) Analyses of DNA, from aliquots of hepatoeyte nuclei 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 hepatoeyte nuclei are presented in Table 5* For hepatocytes treated with Epoxy Resin HlL-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"^Z and 10 x 10'^Z 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 DMN 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 mutagenic agents. D. Deviations from Standard Procedures - Testing of Epoxy Resin EtL-4206 was performed as part of the in-house development and validation of the mutagenicity test 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. ucc 083114 Reviewed end Approved by: Report 43-112 Page 11 1881 Ronald S. Slesinski, Fh.D. Study Director Manager, Genetic Toxicology Elton R. Homan, Ph: Associate Director, Toxicology Contributors: Chinese Hamster Ovary test Sister Chromatid Exchange test Unscheduled DNA Synthesis Assay WPC/1055-5 Fred R. Frank, Director .D Peggy J. Guzzle, B.S. Master Technologist Michelle W. Gaunt, B.S. Master Technologist W. Christopher Bengler, M.S. Assistant Scientist ucc 063115 Table 1882 Report 43-112 Page 12 REFERENCES A previous study on 30* Epoxy Resin ERL-4206 in acetone (formerly termed 0N0XO Epoxide 206) showed that this material was carcinogenic and tumorigenlc following lifetime dermal exposures of mice (CHF Report No, 27-152). No other references on Epoxy Resin ERL-4206 were found in a search of the Environmental Mutagen Information Center data file. ucc 063116 Table 1 Chinese Hamster Ovary (CHO) Mutation Assay: Determination of Toxic Effects of Chemical Treatment Test Chemicals Total I Colonies Total 9 Cells Plated [Epoxy Resin (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 DMSO (20 ul/ml) - Solvent H2O (20 ul/ml) EMS (200 ug/ml) - 340 333 137 800 800 800 [Epoxy Resin (ERL-4206)) (Z, v/v) 10.0 x 10-3 301 5.0 x 10-3 424 2.5 x 10-3 501 1.25 x 10-3 509 0.625 x 10"3 483 With S9 Activation-5 hr exposure 800 800 800 800 800 Controls DMS0 <20 ul/ml) - Solvent H20 (20 ul/ml) DMN (3700 ug/ml) - 539 528 423 800 800 800 Abbreviations: H2O - water; S9 - liver homogenate; DMSO - dimethylsulfoxide EMS ~ ethylmethanesulfonate; DMN - dlmethylnitrosamlne 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 ucc 063717 Z of Solvent Control 8.5 43.2 117.6 120.6 101.8 -- 97.9 40.3 / 55.8 78.7 92.9 94.4 89.6 98.0 78.5 ffS? U4IN , P 00 CD Cd Test Chemicals Table 2 Chinese Hamster Ovary (CHO) Mutation Assay: Results on Evaluation of Mutant Induction by Epoxy Resin ERL-4206 Plating Efficiency Mutation Induction 00 GO Total I Colonies Total I Cells Plated Viable Fraction Total # Mutant Colonies Total # Cells Plated Mutants* 10* Viable Cells [Epoxy Resin (ERL-4206] (I, 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 Without 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 l X 10* 1 X 106 1 X 106 1 X IQ* 38.9 b 13.7 4.9 0 23.5 Controls: DMSO (20 ul/ml) - Solvent H20 (20 ul/ml) EMS (200 ug/ml) - 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 IO-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 l 400 0.740 1 1 X 10& 1 X 106 1 X 106 i 1 X 10^ 1 X 106 1 X 106 1 X to6 1 X 106 0 1.8 196.8 c 6.7 0 0 1.4 1.4 Controls: DMSO (20 ul/ml) - Solvent H20 (20 ul/ml) DMN (3700 ug/ml) - 314 272 299 400 0.785 2 300 0.907 0 400 0.748 39 1 X 106 1 X 106 l X 106 2.5 0 52.2 c 063118 *Total I mutant colonies per 10& cells plated divided by viable fraction. Statistical significance above solvent control: b: 0.01 > p > 0.001: c: p < 0.001. Ho superscript indicates p > 0.05. Data analyzed by Student's t-test. Abbreviations: H2O - water; S-9 - liver homogenate; DMSO - dlmethylsulfoxide; EMS - ethylmethanesulfonate; DMN - dlmethylnltrosamlne. WPC/1055-5 0m0d Tow1>$ Sister Chromatid Exchange (SCE) Assay: Induction of SCE*a by Epoxy Resin (ERL-4206) Without S9 Metabolic Activation 5-Hour Treatment Test Chemicals Total # of Chromosomes Total f of SCE SCE/Celll Mean Number SCE/Chromosome^ + S.D. .. Significance Above Solvent Control [Epoxy Resin (ERL-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 Controls DMSO (5 ul/ml) - Solvent H20 (5 ul/ml) EMS (100 ug/ml) - 299 289 294 300 294 288 296 292 833 55.53 2.766 + 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 NS - NS c ^Fifteen cells examined per dose level. ?Mean value of SCE/chromosome determined from the values of the Individual cells examined. 3statlstical significance above solvent control: c: p < 0. 001 NS: p > 0.05 Data analyzed by Student's t-test. Abbreviations: H2O - water; S9 - liver homogenate; DMSO - dlmethylsulfoxlde; EMS - ethylaethanesulfonate; S.D. - standard deviation WPC/1055-5 mV tmd 063119 uI . Table 4 Unscheduled 0M4 Synthesis in Hepatocytes from Rat Liver Nuclear-bound label! all DPM values are calculated froa nuclei per 10 viable hepatocytes. Each average Is calculated froa duplicate samples, except for DMSO which was done In quadruplicate. I Test Chemical Solvent - DMSO Positive Controls: 4 - NQO Concentration 2Z 3.0 ug/ml 1.0 ug/ml 0.3 ug/ml Radioactivity in Nuclei Avg. DPM + S.D. 2070 + 256 19743 + 1572 2951 + 430 3704 + 791 * of Solvent Control + S.D. 100.0* + 12.3* 953.8* + 75.9* 142.6* + 20.8* 179.0* + 38.2* Significance Above Solvent Control3 - c NS a DMN 1000 ug/ml 9079 + 1341 438.7* + 64.8* c 300 ug/al 2397 + 311 115.8* + 15.0* NS 100 ug/ml 2297 + 162 111.0* + 7.8* NS Test Chemical: [Epoxy Resin ERL-4206] ,(!E v/v) 100 x 10-3* 30 x 10"3Z 10 x 10*3* 3 x 10-3* 1 x 10"3* 0.1 x 10-3* 1670 + 2230 + 3004 + 2579 + 2688 + 2135 + 338 98 203 61 225 94 80.7* + 16.3* 107.7* + 4.7* 145.1*1+ 9.8* 124.6* + 2.9* 129.9* + 10.9* 103.2* + 4.5* NS NS NS NS NS NS ucc 063120 ^Statistical significance above solvent control: a: 0.05 > p > 0.01; c: p < 0.01; NS: p > 0.05. Data analyzed by Duncan's Multiple Range analysis. Abbreviations: DMSO - dimethylsulfoxide; 4-NQ0 - 4-nitroqulnollne oxide; DMN - dlmethylnltrosamlne; DPM - disintegrations per minut ; S.D. - standard deviation c ^ Table 5 Unscheduled DNA Synthesis In Hepatocytes from Bat Liver DNA-bound label: all DPM values are calculated from DNA precipitated per 10^ viable hepatocytes. Each average la calculated from duplicate samples, except for DMSO which was done in quadruplicate. Test Chemical Solvent - DMSO Positive Controls: 4-NQO Concentration 21 3.0 ug/ml 1.0 ug/ml 0.3 ug/ml Radioactivity in DNA Avg. DPM + S.D. 1487 + 152 19886 + 1510 8929 + 1076 2264 + 337 X of Solvent Control + S.D. 100.OX + 16.22 1337.OX +101.5Z 600.4Z + 72.3Z 152.2X + 22.6X Significance Above Solvent Control1 - c c NS DMN 1000 ug/ml 2908 + 783 195.51 + 52.6Z a 300 ug/ml 1770 + 366 119.0Z + 24.6Z NS 100 ug/ml 1897 + 79 127.51 + 5.3X NS Test Chemical: [Epoxy Resin ERL-4206J (X, v/v) 100 x 10"3X 30 x 10"3Z 10 x 10-3X 3 x 10"3Z l x 10~3Z 0.1 x 10-32 1241 + 1568 + 2049 + 1994 + 1735 + 1402 + 373 72 155 270 128 438 83.4Z + 25.IX 105.41 + 4.8X 137.7X + 10.5X 134.IX + 18.2X 116.7Z + 8.6X 94.21 + 29.4X NS NS NS NS NS NS Report 43-112 Page 17 1887 ucc 063121 ^Statistical significance above solvent control: a: 0.05 > p > 0.01; c: p < 0.001; NS: p > 0.05. Data analyzed by Duncan's Multiple Range analysis saseBxecB Abbreviations: DMSO - diaethylsulfoxide; 4-NQO - 4-nitroquinoline oxide; WIN - dlmethylnitrosaaine; DPM - disintegrations per ainute; S.D. - standard deviation WPC/1055-5 Fag* l of 5 y 1889 APPEHDIX I China** Hamster Ovary (CBO) Mutation Ass*y Theoretical Basle Mutation la a harltabla altaration la a call In which a gan* specifying tha geoaclc cod* for a apacific pro tala is aodlflad In structure and/or function. Mutations, Induced by chemical or physical aganta, of th* BCPRT (hypoxanthinequanln* phosphoribosyltransfarasa) gan* may b* dataetad by tha growth of colonies of "mutant" calls which ara resistant to tha purine analogs 6-thioguanina (TG) or 8-azaguanine. Normal calls contain a functional BGPRT enzyme which phosphorylatas TG and allows its incorporation into OKA causing tha calls to die. Mutant calls with a non-functional ffiPRT anzyoa ara unable to phesphorylata or incorporate TG, thus survive and grow in its presence. Tha CHO mutation test is an assay which detects "forward mutations" from TC^ensitivity to TB-reeistanc* caused by a direct loss of tha activity of the BGFRX anzyoa (HGFRT+ -* BGPRT*). An aasassoant of tha ability of several hundred agents to esusa gene mutations in vitro lndicatss that the CEO mutation assay provides a reasonable estimate of the potential genetie activity of the test chemical. Methods Cell Culture Procedures; ffiO cells used in these studies were obtained from Abrahaa Haie at Oak. Ridge National Laboratory with the designation CH0-K1-BH4-D1 (or simply CBO for report purposes). Cells are maintained in active growth by subculturing 2 to 3 timas/week in antibiotic-free, Barn's Modified F12 Medium supplemented with 10Z (v/v) heat-inactivated, fetal bovine sera (F12-10), and lacking in hypozanthina. For treatment of cells without metabolic activation, F12 medium with 50 unlts/ml of penicillin, 50 ug/ml streptomycin end 5Z (v/v) of dialyzed bovine serum (F12-DS) 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-thioguanina) 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 pre-countad suspension of latex beads. Presence of Mycoplasma cell contaminants is detarmined by a microscopic fluorescence assay employing Boechsc 33258 dye. All culture procedures and treatments with test chemicals are performed under aseptic conditions in s laminar-flow, biohazard hood'. Positive and Negative Controls: Stsrile water or glass-distilled dimethylsulfoxide (DMSO) are the usual solvents for test ehemicals snd the respective solvent is tested ss s control st the maximum concentration used to add the test agent. Dimethylnitrosemine (DMN) or tthylmechanesulfonste (SMS) are used as positive control mutagens for tests with or without *n 39 metabolic activation system, respectively. Mutation frequencies obtained with concurrent positive and negative controls art used as the basis for monitoring the sensitivity and stability of eh* CBO mutation east system. Comparison of concurrent control values with historical controls is used to della ate th* range of acceptable variations in eh* test system. UCC 063122 1890 Appendix I Page 2 of 5 Metabolic Activation: Rat liver, 89 homogenate prepared fro* Aroehlor-1254 induced, Sprague-Dawley, sale rata ia purchaaed froa Litton Bioaetica, Kensington, MD. Each lot of liver homogenate la preacreened for metabolic capability to activate DMN in our laboratory before uae in the testing program. The complete S9 metabolic activation ayatem contains the following: 8 uaolas/nl MgCl2i ^ umolea/ml KC1, 5 umoles/ml glucose*6-phosphate, 4 umolea/ml NADP-oxidized (nicotinamide adenine dinucleotide phosphate), 100 uaoles/ail Na2HP04, and between S00 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 902 cell killing. Cytotoxicity is determined by either of the following two methods: (1) Clonal assay - 200 to 400 (20 cells are exposed to a minimum of five dose levels of the teat agent at concentrations from 0.12 to 3 x 10 "*Z (by weight or volume, as appropriate) with and without the presence of a metabolic activation system. The number of calls 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 ere incubated for en additional 18 to 24 hours. Cytotoxicity is determined by compering the relative number of cells in control (untreated cells) and in cells treated with various concsntrations 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 e greeter number of treatment flasks starting at higher doee levels. If marked toxicity is evident even et the lowest dose, the cytotoxicity test Is repested et e concentration range of 3xl0*4 to 3x10*3 percent by weight or volume, as appropriate. Dose levels which are moderately toxic but permit survival of et least 10 to 20Z of the cells, in comparison to the solvant control, are selected as the maximum dose, end et least four additional one-half dilutions are tasted for induction of mutations. If cytotoxicity data are equivocal, a total of 5 to 8 one-half dilutions of the selected, maximum concentration ere used to treat cells; but only the highest five concentrations which permit survival of a sufficient number of cells are assessed for mutation induction. ucc 063123 Appendix I Page 3 of 5 1891 Chemical tempie* are sterilized by membrane filtration vhen microbiological teste indicate this is required to asaure sterility. Liquid test agents are tested on a percentage by volume basis. Solid chemicals are dissolved in an appropriate solvent by asking a 10 to 20Z stock solution (by weight) and subsequent dilutions are aade froa this stock on a volume/volume basis. Treataent with Test Chemicals; For tests of chemicals which may act directly without incorporation of an S9 metabolic activation system, 5xl05 cells are Inoculated 20 to 24 hours prior to treataent Into 23 cm* culture flasks containing F12-05 medium and Incubated at 37*C In a 3 to GZ COj atmosphere. Appropriate concentrations of the test agent or control chemicals are added to the cells and cultures are treated for 3 hr at 37*C. The medium and test agents are removed by suction, cells are rinsed once or twice and fresh F12-D5 medium is added. The cells are allowed a period of 20 to 24 hours of recovery from treatment before survival is determined. Treatment of cells for testing of chemicals which require metabolic activation for mutagenic capacity is performed Identically with the procedure above, with the exception chat F12 medium without serum and containing 1.0 ml of S9 activation mixture per 4.0 ml of medium Is employed. Determination of Cytotoxicity; The relative survival of treated cells, in comparrson "to 'solvent controls, is determined one day after the exposure to th 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 calls 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 ae the measure of treatment-Induced cytotoxicity. Survival values which indicate the cytotoxic effects of the test agents are included in reports in tabular form. Statistical analyses are not performed on these data, since they are only useful to assess whether appropriate doses were employed and are not used to circulate^mutation frequencies. Determination of Mutant Induction: On days 1, 3 and 6 (or alternatively 1, 4 and i) after treatment "with" the 'various test agents, approximately 5x10^ cells art subcultured in 100 on tissue culture dishes in F12-03 medium and incubated at 37*C in a 3 to 6Z CO2 atmosphere. After a total of 7 days to allow "expression" of the'mutant phenotype, cells are dissociated with 0.05 to 0.07SZ trypsin, counted and plated at a concentration of 2.5x 105/dish in four culture dishes (lx 10& total cells) which each contain 5 ml of F12-D5 (TG) selective medium. At this time, cells are diluted and 100 cells/dish are added to four culture platea containing F12-05 medium (without TG) to assess viability (plating efficiency) of the treated cell population and to determine the surviving fraction. All cultures are then incubated for an additional 6 to 8 days to allow growth of calls; medium is then discarded and colonies art fixed end stained for counting. The number of colonies in selection plates and In the viability test are counted by electronic methods, cheeked by manual counts and data are recorded both as total mutants, autanta/106 total calls and outants/106 viable cells. UCC 063124 1892 Appendix I Page 4 of 5 Statistical Analyses! Unifora statistical procedures Co evaluate in vitro mutation date have not been developed. The distribution of mutation frequencies from historical controls in at least two laboratories indicates that the frequeney distribution and variances encountered do not justify the use of para* metric analyses unless data is transformed before application of standard para metric tests. Analysis of mutation frequencies In the CHO test follow the pro cedure of Irr and Snee (Reference 4) which employe the Box-Cox Transformation (Reference 5) to transform data before parametric analyses. The mutation fre quency for each plate Is Increased by 1.0 (to eliminate zeros) and raised to the 0.15 power. Experience with historical negative control data in our laboratory Indicates that a normal probability distribution of the data suitable for para metric analyses la achieved by this transformation. Parametric analysis of mu tation data by the Student's t-test Is performed with the transformed data. The degree of statistical significance for the mutation values are Indicative of a difference from the concurrent solvent control, but these statistical indicators must be viewed conservatively until additional historical control data are available. Interpretation of Data: The criteria for Interpretation of the test results as a positive or negative response depend upon both the level of statistical significance from the concurrent control and the evidence of a dose-response following treatment. When a definite dose-response relationship is not evident but one or more marginally significant values are obtained, a careful examina tion of the data from the concurrent positive and negative controls and compari sons to historical control data are used to evaluate the possible significance of the responses. Historical control data indicate that a spontaneous mutation frequency in CHO cells of approximately 4 to 3 mutants/10* viable cells, with a range of 0 to 25 mutants/10* viable cells, can be obtained in the absence of mutagenic treatment. Statistical comparisons against unusually high or low spontaneous controls are subjectively scrutinized In respect to the above variability. UCC 063125 Appendix I Page S of 3 '1893 References l* Chu, E. H. T end B. V. Helling Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells in Vitro. Proc. Natl. Acad. Sci. U.S.A.. 719^)7 UM-iJU.--------------------------- 2. O'Neill, J. P., P. A. Brimar, R. Machanoff, 6. P. Hirsh, A. W. Hsle. A Quantitative Assay of Mutation Induction at the Hypoxanthine-Guanine ~ Phosphoribosyl Transferase Locus tn Chinese Hamster Ovary Cells (CHO'/HCPRT System); Development and Definition oj the System, Mutation Research, 45 (19777, 9I-I01. -- 3. O'Neill, J. P. and A* tf. Hale* Phenotypic Expression Time of MutagenInduced 6-Thioguaaine Resistance In Chinese Hamster Ovary Qclls (CHO/HCPRT System), ttatation Research, 59, (19?9), 109-118. 4. Irr, J. D. and R. D. Snee. Statistical Evaluation of Mutagenicity in the CHO/HCPRT System. Proceedings of the Cold Spring Harbor-Banbury Coherence H.(1575), 263*274. 5. Box, G. E. P. and 0. &. Cox. An Analysis of Transformations. J. of the Royal Statistical Society, B, 24(l964), 211-232. WPC/1033 ucc 063A26 1894 Pag* 1 of 4 APPENDIX II Determination of Sister Chromatid Exchange (SCE) Frequencies in Chinasa Hamatar Ovary (CHO) Calls In Vitro Thaoretical Baala Exchangaa of ganatle matarial batvaan tha individual arms of a chromoaoma (i.a. aiatar chromatids) ara thought to ariaa from breakage and physical interchanges in tha DKA of a call during call division. An increase in the frequency of such interchanges between sister chromatids can be observed in calls treated with physical or chemical mutagenic agents, or in calls exposed to many suspect or proven human carcinogens. Thus, analysis of SCE frequencies in cells treated with a test agent has been suggested as a sensitive screening test for potential mutagenic/carcinogenic chemicals The method used in our study to visualize SCE's in CHO cells grown in cul ture is based on the procedure described by Perry and Wolff (1974). A standard concentration of 3*0 ug/ml of bromodaoxyuridlne (BrdtJ) was used in the growth medium to allow a visualization of SCE's after two call 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 Call Culture Procedures; Chinese hamster ovary (CHO) cells were obtained from Abraham Hale at Oak Ridge National Laboratory with the designation CH0-K1SB4-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 10X (v/v) of heat-inactiveted fecal bovine serum and lacking hypoxanthine and thymidine. Call concentrations are determined routinely with a Coulter* Model-F electronic call counter calibrated with a precounted suspension of latex beads. All call culture procedures prior to final harvesting of cells for chromosome preparations are performed under aseptic conditions in a laminar flow, biohazard hood. Presence of Mycoplasma cell contaminants is determined using a fluorescent microscopic assay employing Hoechst 33258 dye. For treatments with test chemicals without 59 metabolic activation, modified F12 medium is used with 50 units/ml of penicillin, 50 ug/ml streptomycin and 52 (v/v) of heat-inactivated, dialyzed fetal bovine serum (F12-05). Identical medium but without serum is used for treatments incorporating an 59 metabolic activation system. Positive and Negative Controls: Sterile water or glass-distilled dimethyl sulfoxide (DMS0r*re the usual solvents used for test chemicals and tha respective solvent is tested as a control at tha maximum concentration used to add tha test agent. Dlmethylnltrosamlne (DMN) and tthylmethanesulfonate (EMS) ara 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 aa a basis of comparls n and for demonstrating tha sensitivity and stability of tha SCE test system. Comparison of concurrent control values with historical controls la used to delineate tha range of acceptable variations in tha test system. ^ 063127 1895 Appendix II Pt(* 2 of 4 Metabolic Activation; Sac liver S9 homogenate (prepared froa Arocblor 1254 induced, Sprague-Dawley, male rate) la purehaaad froa Litton Blonaclcs, Kanalngton, MD. Each lot of liver homogenate la preaereened for activity in our lab ratory bafora uaa in tha caacing program. Tha complete S9 metabolic activation system containa tha following; 8 umoles/al MgClj, 33 umoles/ml KC1, 5 umoles/al KC1, 5 uaolaa/al glucoaa-6-phoaphata, 4 umoles/al NADP-oxldized fora (nicotlasaidt adenine dinuclaocida phosphate), 100 uaolaa/al Na2HP04 and boevaan 500 to 4000 ug/ml of S9 protaln (daponding on aatabolie activity). A voluoa of 1.0 ml of tha coaplata alxtura of tha abova raaganta ia addad to aach 4.0 al of eultura aadiua. Doaa Salection; Toxicity of tha taac chaoical la datarainad prior to aaaassacst of autaganie potastial to aalact doaaa which produce a maximum of 80 to 90Z call hilling. Cytotoxicity la datarainad by aithar of tha following two aetboda aa part of tha CBO autatioa teating procedure: (1) Clonal aaaay - 200 to 400 CHO ealla are axpoaed to a minimum of five doaa lavala of tha taat ag*nC at concantratlona froa 0.11 to 3 x 10-4* (by weight or voluoa, aa appropriate) with and without tha praaanca of a aatabolie activation ayataa. The number of cella which aurvlva tha treatment ia datarainad by counting the number of coloniea produced after a 7- to 8^iay incubation period (37*C) in comparison with the colonics formed by cells created only with appropriate eoacantrationa of solvent (generally 20 ul/ml). (2) Growth Inhibition - 5 x 10^ cells in 25 cm2 culture flasks are treated for 5 hours with a minimum of five test concentrations both with and without S9 metabolic activation. Following treatment the cella are rinsed, fresh F12-D5 aadiua ia added and the flasks are incubated for an additional 18 to 24 hours. Cytotoxicity is determined by comparing the relative number of calls in control (untreated cella) and in cells treated with various concentrations of tha test agent. If no cytotoxicity la evident at the highest concentretions in the cytotoxicity tests, tha test is aithar 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 tha lowest dose, tha cytotoxicity test is repeated at a concentration range of SxlO"4 to 3x10'* percent by volume. Doaa levels which are moderately toxic but permit survival of at least 40 to 502 of the cella, in comparison to tha solvent control, arc 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 tha selected, maximum concentration are used to treat cells; but only tha highest five concentrations which permit survival of a sufficient number of mitotic calls with SCE staining, are evaluated for SCZ induction. Chemical samples are sterilized by membrane filtration whan 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 t 202 stock solution (by weight) and subsequent dilutions era made from this stock on a volume/volume basis. UCC 063128 1886 Appendix II Pag* 3 of 4 Treatment With Test Chemicals: Testing of chemicals for direct mutagenic action (without S9 metabolic activation) la prforaad first. For chemicals with clearly positive mutagenic capabilities by direct action, testing with metabolic activation is generally not performed. For tasting direct acting chemicals for SCE Induction, between 1 to 2 x 10 cells are plated Into 73 cm* culture flasks In F12-D5 medium at least 20 hrs prior to treatment and Incubated at 37*C In a 3 to 6Z CO2 atmosphere. Appropriate concentrations of the test agent or control chemicals are added to the cells and 3 ug/ml BrdU Is added to all flasks. Cells are treated with test agents for 3 hrs, media Is then removed by suetlon, cells are rinsed with buffered, physiological salt solution and fresh medium containing 3 ug/ml BrdU Is added, for at least 24 hrs of additional incubation at 37*C to allow two rounds of cell division. Cells are harvested and chromosomes are prepared for SCE staining. Treatment of cells for testing of chemicals which require metabolic activation for mutagenic effectiveness is performed similarly as for treatments without activation, except for three modifications: 1. Before treatment with the test agents, F12-D5 medium is removed and F12 medium without serum is added. 2. S9 metabolic activation mixture is added to each flask (including solvent and positive controls) before addition of test agents. 3. Calls are treated for a total of 2 hrs (rather chan 5 hrs) and then incubated for 38 to 42 additional hours before harvest for chromosome preparation. Preparation of Chromosomes: Colcemid (0.1 ug/ml) or Colchicine (0.2 ug/ml) is added to culture flasks 1 to 2 hrs prior to harvesting to arrest cells in mitosis. Calls are then removed from flasks, after a brief incubation with Q.01X DIFCO trypsin, suspended in 0.075M KC1 (hypotonic) solution and incubated for IS to 20 min at 37*C. Cells are centrifuged, fixed with 3 or 4 changes of Camoy's fixative (3:1 methanol acetic acid) and chromosome spreads are prepared from cells suspended in a small volume of fixative. One slide/dose level is prepared, but fixed eells are saved if needed for preparation of additional slides. Chromosomes are stained for SCE's by treatment with 5.0 ug/ml of Hoechst 33258 dye for 20 min, rinsed in distilled water, immersed in Sorenson's buffer and exposed to a high intensity sunlamp for 15 to 30 min., as required. Irradiated chromosomes are stained In Curr's glemsa (diluted 1:25 with water), rinsed in water and dried before application of coverallps. Examination of SCE's: All slides are coded and read in a blind fashion without indication oi the specific treatment or concentration of the test agent. The number of chromosomes and the number of SCE's in a minimum of 15 cells are recorded for each dose level. The mean number of SCE/cell and SCE/chrom some are calculated and recorded. Slides are decoded only after examination of all slides in the experiment has been completed. UCC 063129 Appendix II Page 4 of 4 Statistical Analysts: Data ara analyzed by appropriaca parametric statistieal procedures which follow BBRC standard operating procedures for analyaas of data. Significant valuaa and cha acaclstlcal ease aaployad ara shown for data sunarlzad in cabular fora. Interpretation of Data: Tha crlearla for evaluation of a positive or naga* tive response depend both on cha level of seaeisclcal signlficanea and subjec tive anaiysas of coneurrane and historical control daea. Tha key daearainane is whathar a dose-dependent incraasa in SCE's is Induced by tha tast agane. Whan no claar dosa-rasponsa ralationship is evident and whan ona or more rasponsas of marginal statistical signlficanea ara obtained, a careful examination of tha data in comparison to tha concurrent controls and historical data base is neces sary. Tasting may be repeated to clarify unusual responses, if data for tha concurrent positive or negative controls suggest a defect in tha original ex periment. Overall assessment will also rely on corroborating data from the other teats in the tasting battery. Clearly positive rasponsas will include any of tha following: (1) Doubling in the SCE frequency at a minimum of two of tha five concentrations tastad; (il) Statistically significant responses of p < 0.03 at three concentrations or at 2 concentrations if p < 0.01; (ill) Induction of a statistically significant, dose-related Incraasa in tha number of SCE. General References 1. Perry, P. and S. Wolff. New giamsa method for differential staining of sister chromatids. Mature, 231 (L9M), 156-138. 2. Lett, S. A., J. W. Allan, W. E. Rogers and L. A. Juergens. In vitro and in vivo analysis of sister chromatid exchange formation, pp 27j-i9l in'.. Handbook of Mutagenicity Test Procedures, ad. Kilbey, B. J., at al. Elsevier Publ. Co. (1$79)'. 3. Carrano, A. 7., L. B. Thompson, ?. A. Llndl and J. L. Minkler. Sister chromatid exchange as an indicator of mutagenesis. Mature, 271,(!l97fe). nrafl.------------- 8-- -------------------------- 8---------- -- 4. Galloway, S. M. and S. Wblff. Tha relation between chemically induced jmwnrrwr.sister-chromatid exchanges and chromatid breakage. Mutation Res.. 61. ------------ ------------------------------------- -- 3. Snedeeor, G. W. and W. G. Cochran. Statistical Methods, 6th Ed., Iowa State Bniv. Press, 4mes, Iowa (1967). ------------ WPC/1033 ucc 063130 1898 Pag* 1 of 3 APPENDIX III Unachedulsd DNA Synthesis (UPS) in Bapstocytss from Rac Liver TheoraticalBaais Chemicals nay interact with both th* cellular components and ch* ganetlc aatarlal of a call (e.g. DNA and RNA) bacauaa of chair alaccrophlllc nature or by conversion Into reactive electrophiles by th* metabolic enzymes of the cell. Damage to th* DNA of a call can result in call death, mutation or, theoretically, carcinogenic transformation. Studies of agents which are capable of reacting and damaging tha cellular DNA have suggested chat such methods may be useful as a sensitive screening test for detecting potential mutagenic/ carcinogenic chemical properties. Detection of the relatively small amounts of DNA damage induced by chemical treatment requires a cellular system in which normal, semi-conservative DNA rep lication, which occurs during cell division, is inhibited. The system employed for the present study uses e suspension culture of primary hspatocyte cells iso lated from rat liver according to the general mathods of Seglen (1973) and Williams (1976). Hepetoeytes do not normally divide in tha minimal culture me dium employed and stimulation of "unscheduled" incorporation of radioactive DNA precursors can be detected by scintillation spectrometry. Th* stimulation of incorporation of crltlated thymidine into both purified hepatoeyte 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 ss wall as with historical data for similar tests. Methods Preparation of Bepatocytt Suspensions: Hilltop-Wistar albino rats are anesthetized with Matafane(R). The abdominal cavity is surgically exposed and 1230 units of heparin is lnjactad intravenously. A catheter is inserted into the portal vain and warm Hanks Balanced Salt Solution (HBSS) is pumped into the vein and through tha livar. This first solution contains heparin and EGTA, [ethylene glycol-bia-(beta-amiaoethyl-eth*r)N,N-tstr*c*tic acid], which preferentially chelates calcium; the solution contains no magnesium or calcium. After the liver is blanched, a second solution of HBSS containing 60 unlts/ml of collagenase is perfused. This solution is pumped through the liver until the liver is digested. Tha livar is than removed and the cells are freed in cold medium 199 by combing through the lobes with a sterile metal comb. The cell suspension is passed through two nylon meshes to remove cell clumps and the cells are weshed once at low centrifugation speed. After resuspension in medium 199, equal volumes of calls and 0.4Z trypan blue arc mixed together end the cell viability and number of viable calls per ml is datsrmlncd microscopically. Praincubatlon of Hepatocytes; Approximately 2 x 10$ viable hepatocytes are added to 5 ml medium 199 containing 10 mM hydroxyurea and 30 mM Hepes (N-2-hydroxyechyl piperazlne-N-2 ethane sulfonic acid) buffer. After the cells are dispensed into the tubes, they ere placed on a rocker platform and are incubated at 37*C for 1 hour. Although hepatocytes do not normally divide in culture, medium 199 which lacks serum and contains hydroxyurea is used to further block semi-conservative DNA synthesis. Thus, any radioactive thymidine incorporated into the nuclei is expected to result from repair or unscheduled DNA synthesis. oon Appendix III Pag* 2 of 3 1899 Selection of Doses of Teat Chemical! Initially, tha following eoncentratlona x 101JI (by volum*) art caatad: 100, 30, 10, 3, i, and 0.1. If these concentrations prow* to be cytotoxic, or if additional information la availabl* from othar in vitro taata aa to th* proper doa* levels, than an appropriate aariea of concentrations la uaad over a 3-log range of concentrations. Treatment of Hepatocytesi After pralncubatlon, 25 microCuries of trltlatcd thymidine (20 Curiea/millimole) is added to each tub*. Th* test chemical and positive controls are diluted is an appropriate solvent and they are then added to each labeled tub*. Generally, at least six concentrations of tha 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 Hegatlv* Controls: 4-nitroquinolin* oxide (NQO), a directacting mutagen, which Induces UV-type DMA repair and dlaethylnitrosaoina (DMN), which requires metabolic activation by microsomal enzymes for activity, are run in duplicate as positive control chemicals. Th* solvent control is run in quadruplicate and consists of 100 to 150 microliters (concentration specified in Individual reports) of th* solvent used to dilute the sample. Dimethylsulfoxide dM5Q) or water era the usual solvents for teat chemicals. Harvestt At the end of incubation with th* test agent, the cells are centrifuged from th* 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 mM MgCl2, a lysing solution which liberates th* nuclei. The nuclei are rinsed once in this solution and resuspended in 0.25 M sucrose, 2.5Z citric acid and 3 mM HgCl2* The nuclei are then centrifuged at 600 x g for 10 min at 5*0 and resuspended in 2 ml of th* lysing solution. Determination of Nuclear-Bound Label; To measure th* amount of radioactive thymidine incorporated into th* nuclei, 0.25 ml of the nuclear suspension is mixed with 1.0 mg of NCS tissue solubilizer in a scintillation vial. Ten ml of Dlmilume* scintillation cocktail is added and th* radioactive disintegrations per minute (DPM) are determined by counting twice in a scintillation counter for ten minutes. The measured DPM are then used to calculate the DPM/10 viable hepatocytes presented on tables. Determination of DNA-Bound Label; Th* amount of radioactive thymidine incorporated into DMA is quantitated in DNA Isolated and precipitated from 1.00 to 1.25 x 10^ viable hepatocytas. To 1.25 ml of th* nuclear suspension, 2.75 ml of 1Z sodium dodecyl sulfate (SDS) and 5 mM Ethylenedlaminetetraacetlc Acid (EDTA) is added to lyse th* nuclei. Th* DMA is precipitated from this solution with 4 ml of ice-cold 10Z trichloroacetic acid (TCA) and tha sample tubes are incubated at 0*C for at least 30 minutas. The solution is then poured onto Whatman glass fiber filters under vacuum and th* tubes and filters are washed twice with cold 5* TCA. Finally, each filter is rinsed once with methanol, dried and placed in a scintillation vial. The filters are incubated at 50*C for 1 hour with 1 ml of a diluted solution of NCS tissue solubilizer; prepared by adding l part solubilizer to 2 parts of Dimilua* cocktail. Dimilume# is then added to each vial and the vials are counted twice In a scintillation counter for ten minutes. UCC 063132 Appendix III Pag* 3 of 3 Statistical Analysis: Th* average DPM Is calculated for each do** level and the controls and final reault* ara expressed as DPM/IO viable hepatocytes. Data are also expressed as a percent of th* solvent control for purposes of comparison* The original data are statistically analyzed by the appropriate parametric test, following th* BSRC standard procedures for statistical analyses and th* test(s) employed la Indicated on the respective tables. Comparison between the mean for each dose level with the 932 confidence limits of th* 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; Th* classification of a chemical as a positive, active agent depends upon che production of a statistically significant, dose-related Increase in the amount of CDS activity. If a definite dose-response relationship is not evident, or when a few increases with marginal statistical significance are obtained, comparison of the responses to historical control data provides a meaningful assessment of th* possibility for random variations which may be statistically significant only in relation to the concurrent control. A key determinant of th* reliability of the CDS data is the detection of a similar response with both DMA and isolated nuclei determined at two or three consecutive concentrations. General References 1. Lampidis, T. J. and J. B. Little. The Enhancement of PV-Induced Pnscheduled DHA Synthesis by Hydroxyurea. Experimental Cell Research. 110, (197?), 41-46. 2. Muramatsu, M. Isolation of Nuclei and Nucleoli. In: Methods in Cell Physiology, Vol. 17. 1^76. 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. William*, G. M. Detection of Chemical Carcinogens by Pnscheduled DMA Synthesis In Rat Liver Primary Cell Cultures. Cancer Research. 37. <i577),1B45'-iaii. ----- ------------------- " 3. Williams, G. M. The Use of Liver Epithelial Cultures for the Study of Chemical Carcinogenesis. American Journal of Pathology. 85. (1976). 73$-753. -- 6. Williams, G. M. and M. F. Laspla. The Detection of Various Nitrosamlnes In the Hepatocyta Primary Culture/DNA Repair Test. Cancer Letters, 6. (1979). 199-206. 7. Snedacor, G. W. and W. G. Cochran. Statistical Methods. 6th edition, Iowa State Pnivarsity Press, Ames, Iowa (1967). VPC/1033 UCC 063133 1901 Report #43-112 Appendix IV Physical and Chemical Characteristics of Test Material CHF Sample No: CAS No.: Chemical Name: Trade Name and/or Synonyms: Molecular Height: Formula: Molecular Structure: 42-137 106-87-6 3-(epoxyethyl)-7-oxabicyelo(4.1.0)heptane Bakelitem Cycloaliphatic Epoxy Resin ERL-4206; vlny1-3-cyclohexene diepoxide; Epoxyethyl-3 4-epoxy cyclohexane 140.18 c8h122 Specific Gravity (0 20*C): Bolling Point: Solubility In H2O (Z by vt): Purity: 227 *C 0 760mm Hg 18.3 0 20#C Approx. 1002 Vapor Pressure (0 208C): pH: Flash Point: Stabillcy: Incompatibility: Appearances and Odor: Disposal: <0.1mm Hg Not Available 225*F (closed cup) Onstabie, avoid temperatures > 100#F Avoid amines, acids, alkalies Low viscosity liquid; characteristic odor Small spills can be flushed with water Larger quantities can be diluted with an insert solvent and burned. Protective Measures: Use rubber gloves, goggles and protective creams on arms and face. An eye bath and safety shower should be available. A local exhaust ventilation system is preferable to reduce exposure. In case of contact flush eyes or skin with plenty of water. Health Hazard: Prolonged and repeated contact with liquid or breathing of vapors or mists may cause delayed and serious injury. Absorption through skin is harmful and the liquid causes eye injury. Skin painting tests in animals caused skin cancers, but no cancers have been reported in humans. UCC 063134