Document JJVXKK6agY1x0qOxdmR2vONZZ

T 3347* f BUSHY RUN RESEARCH R. 0. 4, Mellon Rood, Export, Pennsylvania IBS32 CENTER Telephone (412) 733-6200 CONFIDENTIAL j Not to be released outside UCC without the written consent of the UCC-sponsorlng Division HS&EA Manager. Project Report 47-183 10 Pages February 4, 1985 BAKELITE* Cycloaliphatic Epoxy Resin ERL-4206 Salmonella/Mlcrosome (Ames) Bacterial Mutagenicity Assay Sponsor: R. E. Plevan, HS&EA Manager Union Carbide Corporation Specialty Polymers & Composites Division Summary Epoxy Resin ERL-4206 was tested for potential mutagenic activity using the Salwonella/mlcrosome bacterial mutagenicity assay (Ames test). Epoxy Resin ERL-4206 was tested with and without metabolic activation in triplicate at five concentrations, ranging from 0.3 to 30 milligrams per plate. The highest concentration was cytotoxic in the mutagenicity test and In a preliminary teBt to choose appropriate doses. Positive, dose-related Increases of the number of colonies above the solvent control levels were observed with strains TA98, TA100, TA1535 and TA1537 without metabolic activation and with strains TA98, TA100, and TA1535 with activation. The results of this test indicate that the test chemical caused frameshlft and base-pair substitution mutations. Epoxy Resin ERL-4206 was an active mutagen In this In vitro screening test. Sample Chemical Name: BAKELITE* Cycloaliphatic Epoxy Resin ERL-4206 I.D. #: TF3-96521 3083 RKT BRRC Sample #: 47-202 BRRC Account #: 84-18-18050-46 CAS #: 106-87-6 Chemical Synonyms: 7-oxabicyclo[4.1.0]heptane,3-oxiranyl; UN0X 206; 4-vlnylcyclohexene dlepoxlde; EP-206 Molecular Formula: CgHi22 Submitted by: R. J. Cotter, Bound Brook, NJ Sponsor: Division: R. E. Plevan, Danbury, CT Specialty Polymers & Composites Bound Brook, NJ Bushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation UCC 062503 3348 Report 47-183 Page 2 BAKEL1TE* Cycloaliphatic Epoxy Resin BtI-4206 Salmonella/Microtome (Ames) Bacterial Mutagenicity Assay I. Introduction and Theory The Salmonella typhlmurlum/mlcrosome mutation assay Is a microbial screening test which detects the ability of chemicals to cause genetic alterations in the histidine gene of selected Indicator strains of Salmonella typhimurlum. The Indicator strains used for thiB test are all histidinerequiring (his-) bacteria which carry a mutation In the histidine locus. The strains were obtained from Dr. Bruce Ames, University of California, Berkeley, CA. Histidine-independent (hls-0 strains can arise either spontaneously or by the mutagenic action of a chemical or physical agent. Base pair mutagens cause a base change In the UNA molecule at the site of the original mutation or at a second site In the UNA which supresses the original mutation. Frameshift mutagens cause the addition or deletion of single or multiple base pairs In the UNA molecule. The relative frequency of Induced or spontaneous reversion to his+ can be quantltltated by plating the Indicator strains on minimal agar and counting the number of revertant colonies, representing bacteria that were able to grow and form colonies In the absence of histidine. To test the mutagenic potential of a test chemical, the bacterial strains are exposed to several concentrations of the chemical. Tests are done both with and without the addition of a mammalian liver homogenate (S9) because some chemicals require metabolic conversion for detection of their biological effects. Concurrent solvent and positive controls are also run with each assay to determine the responsiveness of the test system. After a suitable period of Incubation (48-72 hrB>, direct revertant colony counts are made and evaluated. Test chemicals which produce at least a 2-fold and dose-related increase In mutant colonies over the concurrent control value are considered to be bacterial mutagens and suspect mammalian mutagens. UCC 062504  ,3349 Report 47-183 Page 3 tt. Salmonella typhlmurlum Mutagenicity Assay ft A. Objective The objective was to assess the mutagenic potential of Epoxy Resin BU.-4206 as measured In the Salmonella typhlmurlum/ mlcrosome mutagenicity assay. A more complete discussion of the theoretical basis of this test Is presented In Appendix 1. B. Test Chemical (Available analytical and physical data Is attached as Appendix 2) 1. Sample name: BAKELITE* Cycloaliphatic Epoxy Resin ERL-4206 2. BRRC Sample Number: 47-202 3. CAS Number: 106-87-6 4. Purity: > 972; IR spectra consistent with structure (see Appendix 2) 5. Density: 1.0986 6. Date Received: July 25, 1984 7. Storage Conditions: Room Temperature 8. Storage Location: Chemical Hood/Genetic Toxicology Dept. C. Control Substances &1. Solvent: dimethylsulfoxide, Burdick Jackson, Muskegon, Ml; Lot #AD912; CAS #67-68-5 2. Positive Controls: i a) 4-nitro-o-phenylenediamine; BRRC #44-71; CAS #99-56-9 b) 9-aminoacridlne; BRRC #44-233; CAS #90-45-9 * c) sodium azide; BRRC #44-72; CAS #26628-22-8 d) 2-amlnoanthracene; BRRC #44-67; CAS #613-13-8 0. Metabolic Activation S9 liver homogenate, prepared from Arodor 1254-induced, Sprague-Davley male rats, was purchased from Microbiological Associates, Bethesda, MD. For tests with metabolic activation, 0. 5 r ml of S9 mix containing 50 pi of S9 was added per plate. i E. TeBt Protocol The assay was performed according to BRRC Standard Operating Procedures 7.4.1A through 7.4.7A, 7.4.12A, and 7.4.13. A general description of the test methods is attached to this report as. Appendix 1. a \ i i i- i4 ucc 062505 33^0 Report 47-183 Page 4 P. Test Oates 1. Test Initiated: October 29, 1984 2. Test Completed: November 2, 1984 G. Storage Location of Materials 1. Raw Osta: BRRC Archives 2. Final Report: BRRC Archives H. Experimental Design (detailed procedures In Appendix 1) 1. Sample Preparation: The test substance was dissolved In dimethylsulfoxlde (DMSO) to a concentration of 300 mg/ml for doses of 30 mg/plate and below. All subsequent dilutions were made In the same solvent. Dilutions of the test substance were made fresh each day of testing. All dilutions for the mutagenicity tests were grsvlmetrlcally analyzed. 2. Dose Selection: A preliminary toxicity test was performed using strain TA100 to determine the level of toxicity of the test substance. Ten doses were tested for toxicity with a plate assay performed In the manner used for mutagenicity determinations. Toxicity was assessed at 24 to 48 hours after treatment by either growth Inhibition of the background lawn or a reduction In the number of spontaneous mutants. 3. Testing: The test chemical was tested In triplicate at five doses chosen to span a range which Included moderately toxic to relatively nontoxic concentrations. If the substance was nontoxic In the preliminary toxicity test, 100 pi pf liquid or 50 mg of solid was used as the maximum dose, unless limited by solubility. Testing was performed both with and without metabolic activation. Concurrent solvent and positive controls were run in each test. I. Quality Control Data from each test Is checked for accuracy and Integrity by a second Investigator not Involved with the study. Compliance of testing procedures with Good Laboratory Practices Is audited by the Independent Quality Assurance department at the Bushy Run Research Center. UCC 062506 3351 ' Report 47-183 Page 5 IH. Results Epoxy Resin ERL-4206 vss tested In a preliminary toxicity screen at 109.9, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 milligrams per plate vith strain TA100 only. The top dose of 109.9 milligrams per plate Inhibited all growth of the background lavn, while 30 milligrams per plate allowed only sparse growth of the background lawn. Based on these results, mutagenicity testing was done with 5 doses of 30, 10, 3, 1, and 0.3 milligrams per plate tested with triplicate cultures. The highest dose was expected to produce some degree of cytotoxicity based on preliminary toxicity test results, observed as either a reduction In the number of revertant colonies or an Inhibition of growth of the background lavn. The range of doses that was tested followed the exposure concentration recommendations of the tJ.S. E.P.A. Health Effects Test guidelines (HG Gene Muta-S. typhlmurlum. August 1982). Gravimetric analysis of the Initial dilutions of the test substance for both tests Indicated no more than a 0.6% error from the stated concentration. All subsequent dilutions had no more than a 5.8% error from the stated concentrations. All piste counts and the respective means and standard deviations are shown In Table 1 (without activation) and In Table 2 (with activation). Doserelated mutagenic activity was observed in both tests. In the test without metabolic activation, the ratios of the average number of revertant colonies to the average number of colonies observed In the solvent control cultures Increased with Increasing dose as follows: 1.0, 1.2, 1.3, and 2.5-fold increases with TA98; 1.4, 2.2, 4.3, and 12.7 with TA100; 1.6, 2.7, 4.1, and 10.2 with TA1535; and 1.2, 1.2, 1.2, and 2.8 with TA1537. No evidence of mutagenic activity was observed with TA1538. Strains TA98 and TA1537 detect frameshlft mutagens, while TA100 and TA1535 detect base-pair substitution mutagens. In the test with metabolic activation, the ratios of the average number of revertant colonies to the average number from the solvent control Increased vith Increasing dose as follows: 1.1, 1.1, 1.4, and 4.7-fold Increases with TA98; 1.6, 3.0, 6.3, and 15.9 with TA100; and 2.3, 6.7, 15.9, and 40.8 with TA1535. No evidence of mutagenic activity was observed with strains TA1537 and TA1538. The data from the 30 mg/plate dose were not used In the above calculations because of the observed cytotoxicity. The mutagenic effects of Epoxy Resin ERL-4206 appear to be caused by both frameshifts and base-pair substitutions. A greater degree of mutagenic activity was observed with a metabolic activation system than without metabolic activation, although strain TA1537 was not reverted In the test vith activation. The Increased mutagenic activity with metabolic activation could be due either to. the production of active metabolites from the parent compound or to reduced cytotoxicity caused by metabolic conversion of the test agent, thus allowing more revertant bacteria to survive the tested doses. Dose selection appeared to be in a suitable range in the mutagenicity tests because some toxicity was evident In both tests. In both tests, toxicity was observed at the maximum dose of 30 mg/plate with all strains. occ 062501 3352 Report 47-183 Rage 6 All strains exhibited a positive mutagenic response with the positive controls tested both with and without S9 metabolic activation* Negative (solvent) controls were sIbo tested with each strain, and the spontaneous reversion rates were within the historical ranges at this laboratory (see Appendix 3). All positive and negative controls were run concurrently with the test chemical. Concurrently run sterility checks showed that the S9 nix, PBS, the test chemical and all solvents and controls were sterile. IV. Conclusion Epoxy Resin ERI-4206 produced a dose-dependent mutagenic response with the Salmonella typhlmurlum strains IA98, IA100, TA1535, and TA1537 without metabolic activation and with strains TA98, TA100, and TA1535 with metabolic activation. Under the conditions of this assay. Epoxy Resin DlL-4206 was an active mutagen In the Salmonella/mlcrosome mutagenicity assay. Ronald S. Sleslnskl, Ph.D. Bate Manager, Genetic,Biochemical & Acute Toxicology ________________ ibis' Fred R. Frank, Ph.D. Director Date Contributors: Technical Assistance: Eileen R. Morablt, A.S. WPC/rkk/0620B-2 11-16-84 ucc 062508 TABLE 1 RESULTS OF THE SALMONELLA MUTAGENICITY ASSAY < 3353 Report 47-1B3 Fege 7 CHEMICAL: BRRC #47-202, Epoxy Reels BU/-4206 EXPERIMENT BATE: October 29 through November 1, 1984 WITHOUT ACTIVATION SOLVENT: DMSO TEST AGENT DOSE PER PLATE "STRAIN TA98 SOLVENT 110 MG POS: 01 0.01 MG BRRC# 0.3 MG 47-202 1 MG 3 MG 10 MG 30 MG PLATE COUNTS 12 3 22 29 819 953 33 20 41 22 48 27 68 67 TT 27 884 26 31 26 62 T AVERAGE 26 885 26 31 34 66 T STANDARD DEVIATION 3.6 67.0 6.5 9.5 12.4 3.2 - STRAIN TA100 SOLVENT 110 MG POS: 02 0.01 MG BRRC# 0.3 MG 47-202 1 MG 3 MG 10 MG 30 MG 134 2112 184 245 629 1735 T 138 2340 186 287 542 1711 T 129 2179 184 341 563 1669 T 134 2210 185 291 578 1705 T 4.5 117.2 1.2 48.1 45.4 33.4 SIRAIN TA1535 SOLVENT 110 MG POS: 02 0.01 MG BRRC# 0.3 MG 47-202 1 MG 3 MG 10 MG 30 MG 46 2095 52 92 148 385 T 31 2383 64 85 172 356 T 28 2290 56 108 115 329 T 35 2256 57 95 145 357 T 9.6 147.0 6.1 11.8 28.6 28.0 * SIRAIN IA1537 SOLVENT 110 MG 47 4 5 1.7 POS: 03 0.06 MG 78 102 93 91 12.1 BRRC# 0.3 MG 73 7 6 2.3 47-202 . 1 MG 77 4 6 1.7 3 MG 39 5 6 3.1 10 MG 15 14 13 14 1.0 30 MG TT T T * SIRAIN TA1538 SOLVENT 110 MG POS: 01 0.01 MG BRRC# 0.3 MG 47-202 1 MG 3 MG 10 MG 30 MG 10 1016 14 13 15 7 T 7 1023 5 5 6 7 T 9 1058 16 9 9 9 T 9 1032 12 9 10 8 T 1.5 22.5 5.9 4.0 4.6 1.2 T: TOXIC POS: POSITIVE CONTROLS 01: 4-NITRO-O-PHENYLENEDIAMINE 02: SODIUM A2IDE 03: 9-AHINOACRIDINE 04: 2-AMINOANTHRACENE o9 3354 TABLE 2 RESULTS OF THE SALMONELLA MUTAGENICITY ASSAY Report 47-183 Page 8 CHEMICAL: BRRC #47-202, Epoxy Resin ElL-4206 EXPERIMENT RATE: October 30 through Noveaber 2, 1984 WITH ACTIVATION SOLVENT: DMSO TEST DOSE AGENT PER PLATE STRAIN TA98 SOLVENT 110 MG POS: 04 0.01 MG BRRC# 0.3 MG 47-202 1 MG 3 MG 10 MG 30 MG PLATE COUNTS 12 3 20 1314 26 16 26 98 T 22 1342 16 29 32 100 T 22 1814 27 26 30 100 160 AVERAGE 21 1490 24 24 30 99 160CT) standard- deviation 1.2 28D.9 4.9 6.8 2.0 1.2 STRAIN IA100 SOLVENT 110 MG POS: 04 0.01 MG BRRC# 0.3 MG 47-202 1 MG 3 MG 10 MG 30 MG 113 1268 169 237 716 1652 T 100 1217 174 315 617 1588 T 67 1183 149 353 567 1522 1967 100 1223 164 302 633 1587 1967CT) 13.0 42.8 13.2 59.1 75.8 65.0 * STRAIN TA1535 SOLVENT 110 MG POS: 04 0.01 MG BRRC# 0.3 MG 47-202 1 MG 3 MG 10 MG 30 MG 11 10 40 95 19 21 41 61 156 152 381 407 TT 7 89 22 78 120 313 213 9 75 21 60 143 367 213CT) 2.1 30.2 1.5 18.5 19.7 48.5 ** STRAIN IA1537 SOLVENT 110 MG POS: 04 0.01 MG BRRC# 0.3 MG 47-202 1 MG 3 MG 10 MG 30 MG 58 79 32 12 7 68 44 89 TT 6 79 3 5 2 7 17 6 63 7 6 3 8 17(T> 1.5 27.1 4.5 1.5 1.2 1.0 *" STRAIN TA1538 SOLVENT O.H MG POS: 04 0.01 MG BRRC# 0.3 MG 47-202 1 MG 3 MG 10 MG 30 MG 17 7 184 209 8 13 9 27 95 31 17 15 T 20 226 17 21 13 18 T 15 206 13 19 9 22 15(T) 6.8 21.1 4.5 9.2 4.0 7.8 T: TOXIC POS: POSITIVE CONTROLS 01: 4-NITRO-O-PHENYLENEDIAMINE 02: SODIUM AZIDE 03: 9-AMINQACRIDINE 04: 2-AMINOAKTHRACENE Report 47*163 Page 9 3355 BUSHY RUN RESEARCH 6. 0, 4, Mellon Road, Export, Pennsylvania 16632 CENTER Telephone ut2i 731-6200 Good laboratory Practice* Compliance This atudy m conducted In accordance vlth the U.5. Environmental protection Agency's Good Laboratory Practices (GLP) Standards (40 CFR Part 792, effective December 29, 1983) vlth the following deviations: 1. The sponsor Indicated that Epoxy Resin ERL-4206 was soluble and stable in dlaethylsulfoxlde and no additional analyses of stability In the test system were performed. 2. The content of the test substance and control mixtures were documented and analyzed by weight or by volume, and no additional analyses were performed. In the opinion of the study director, these deviations do not affect the validity or reliability of this study. Prepared by; V. Christopher Study Director VPC/rkk/0620B-l 11-08-84 Bushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation UCC 062511 3356 Report 47-183 Rage 10 BUSHY RUN RESEARCH R. D. 4, Mellon Road, Export, Pennsylvania 15632 CENTER Telephone 1412) 733-5200 Quality Assurance Unit Study Inspection Summary Test Substance: BAXELITE Cycloaliphatic Epoxy Resin ERL-42M Study: Ames Bacterial Mutagenicity Assay Study Director: V. C. Hengler, M.S., M.B.A. The Quality Assurance Unit of BRRC conducted the inspections listed below and reported the results to the study director and to sianagenent on the dates indicated. It is the practice of this Quality Assurance Unit to report the results of each Inspection to both the study director and management. Inspection Date Type 1-31-84 Standard Protocol 10- >84 Standard Protocol Form ' 10-30-84 Event-Preparing Agar 1-15 to 1-17-85 Final Data and Final Report Date QAU Report Issued To Study Director To Management 2-2-84 2-3-84 10-3-84 10-3-84 11-2-84 11-28-84 1-18-85 2-1-85 Linda J. Calistf, Croup Leader Date Good Laboratory Practices/Quality Assurance Bushy Run Rssssrth Contsr A Joint Mellon Institute--Union CarPide Corporation Operation 512 ,3357 Report 47-183 Rage 1 of 4 ATPIMIIX i Standard Procedures for the Salmonella/mlcrosome Mutagenicity Assay A. Introduction The strains used In this test are all Salmonella typhlmurlua histidine auxotrophs developed by Dr. Bruce Anes of the University of California at Berkeley. The test la designed to assay for reverse autatlons In the histidine locus by plating the bacteria on minimal agar with only a trace of histidine. Bacteria that have undergone a reverse mutation In this locus can form colonies 1b minimal agar and are counted as mutants. The trace of histidine In the ainlaal agar allows the bacteria to undergo a small number of cell divisions which then form the background lawn, visible as a cloudiness In the agar* Observation of the lawn permits an estimate of toxicity* Testing with growing cultures also allows potential mutagens to act on replicating DNA which Is often a more sensitive test for several classes of mutagens. B. Indicator Organisms Salmonella typhlmurium strains TA98, TA100, TA1535, TA1537, and TA1538 are used In this assay. They all lack the excision repair mechanism, which Increases their sensitivity to potential mutagens. They also have a mutation affecting their llpopolyeaccbarlde layer which allows large molecules to enter the bacteria more easily. Strains TA98 and TA100 were derived from strains XA1538 and TA1535 by the addition of an R factor which Imparts ampldllln resistance and also Increases sensitivity to several classes of mutagens. Indicator organisms are stored at -80*C. Working cultures are prepared monthly by Inoculating nutrient broth from the frozen cultures and Incubating with agitation overnight. Bacteria are then plated onto Vogel-Bonner Medium agar plates (master plates) with an excess of histidine and biotin (required because of the llpopolysaecharlde deficiency). After Incubation for 24 hours, the strains are checked for their genetic markers to verify their Identity and purity (SOP #7.4.2A). For testing, the broth cultures are prepared by Inoculating from the master plates Into nutrient broth and Incubated overnight with agitation. The broth cultures are then kept on Ice during the day of testing. Fresh cultures are made each day of testing. C. Toxicity Testing Sterile tubes are prepared containing 2 ml soft agar (6 g/1 agar and 5 g/l NaCl) with a final concentration of 0.05 mM L-hlstldlne and 0.05 mM D-blotln (the entire mixture Is called top agar). Dilutions of the test chemical are made In an epproprlate solvent so that the correct amount of chemical can be added to each tube In 100 pi amounts. If less than 100 pi amounts of the test chemical Is used, phosphate-buffered saline Is used to adjust the total volume to 100 pi. 0 3338 geport 47*163 Appendix 1 Page 2 of 4 At leaet five doeea are teeted with maximum doees of 50 mg (for aollds) or 100 pi (for llqulde) per plate for nontoxic chemicals* unless limited by solubility^ Generally, a chemical cannot be considered to be monmutagenlc unless at least 5 mg/plate has been tested* unless limited by toxicity or solubility (deSerres and Shelby* 1979; BG-Gene Muta-S. typhlmurlum, August 1982), A 100 id- aliquot of an overnight broth culture of strain IA100 Is added followed by the appropriate amount of test chemical. The mixture is vortexed and poured onto the surface of a Vogel-Bonner Medium agar plate (VB-E plate). The top agar Is allowed to harden and the plates are Incubated at 37*C for 24 to 48 hours. The plates are examined for the condition of their background lawns and growth Is recorded as either confluent, sparse, or absent. Confluency Is considered an Indication of nontoxlclty, sparse growth Indicates moderate toxicity* and lack of growth Is recorded as toxic. D. Bose Selection For the mutsgenlelty test* a minimum of five half'log doses are tested within a range of moderately toxic to relatively nontoxic doses based on the results of the toxicity test. If all doses in the toxicity test were nostoxlc, then s maximum dose of 50 mg or 100 pi Is used In the mutagenicity test* unless limited by solubility. . Controls Concurrent solvent and positive controls are run with each test. The solvent control Is the maximum amount of solvent added with the diluted test chemical. For chemicals tested without dilution (l.e.* by direct addition), water Is used as the solvent control. Both activation-dependent and activation-independent positive controls are used. The activation-independent controls are 4-nltro-n-phenylenedlamlne for TA98 and 2A1538* sodium aside for TA100 and TA1535, and 9-aminoacrldlne for XA1537* The activation-dependent control in 2-amlnoanthracene (2-anthramlne) for all strains. The concentrations are determined from prior dose-response experiments on these chemicals and are listed In the tables of results in this report. F. Metabolic Activation Each lot of Aroclor-1254 Induced* rat-liver homogenate (S9) Is prescreened for activity with 7,12-dimethylbenzanthracene to determine the appropriate amount to use for testing. The S9 Is added to the "S9 mix" which contains, per ml, $9 (50-250 pi), MgCl2 (8 inoles), KC1 (33 psoles), glucose-ephosphate (5 isnoles), MADP (4 pmoles), and sodium phosphate, pH7.4 (100 IBoles). S9 mix is prepared fresh each day of testing and kept at 0-4*C. G. Test Chemicals and Solvents The solvents of choice are wster, DMS0 or ethanol. A minimum of five concentrations of the test chemical are diluted In the appropriate solvent and tested. The chemical is diluted so that 100 pi will deliver the required UCC 062514 Report 47-183 Appendix 1 Page 3 of 4 3359 dose* If Muller volumes ere used, phosphate-buffered saline Is Added to adjust the totel volume to 100 lil. Test chemical solutions sre prepared on the day of testing and sre gravlsetrically analysed. If the analysis Indicates >101 error from the Intended concentration, then either the dilution Is repeated or the actual concentration Is stated In the report* I. Dosing To a sterile tube containing 2 si of top agar, 100 yl aliquot of the appropriate bacterial culture is added followed by the addition of 100 vl of the appropriate solvent, control, or test chemieal solution* Either 0.5 al of S9 alx or 0.5 al of phosphate-buffered saline (PBS) Is added for tests with and without aetabollc activation, respectively. The top agar alxture Is then poured onto a VB-E plate. Each dos? Is tested in triplicate and with all five bacterial strains* Sterility checks are done on the PBS and/or S9 alx, all solvents, and the highest concentration of each test chemical. The plates are transferred to a darkened 37*C Incubator after hardening and Incubated for 46-72 hours. I. Data Collection An Artek Model #880 Colony Counter is used to count bacterial colonies or handcounts of the plates are done. The counter Is calibrated for each test to check the counting accuracy (SOP I7.4.5A). The numbers of colonies per plate are counted and recorded. An examination Is also made of the background lawn on each plate. If toxicity is observed as an inhibition of growth of the background lavn, the plate Is not counted, but Is recorded as toxic. If the background lavn is sparse and the colony count is still recorded, that number is not UBed In the calculation of the mean number of colonies and Its standard deviation. J. Criteria for Test Validity and for Interpretation of Results The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen If the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related Increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced in a second test, the test result will be considered negative. If there is no evidence of a dose-related increase in the number of reveTtant colonies and the number of revertant colonies is not twice the solvent control, then the test chealcal is not considered to be a bacterial mutagen. X* Data Storage and Retrieval Copies of the final report, statistical analyses, analytical data and raw data are stored in the BRRC Archives. uce 062515 <3360 Report 47-183 Appendix 1 Peg* 4 of 4 Reference* 1. Ames, B.N,, J. McCann and P. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammallani&lcrosone mutagenicity teat. Mutation Research, 31: 147-364 (1973)* 2. Vogel, B.J., and D.M. Bonner, Acetylornithaae of Escherichia coll; partial purification and some properties. Journal of Biological Chemistry, 218: 97-106 (1956). 3. deSerres, F.D., and M.D. Shelby, Recommendations on data production and analysis using the Salmonella/mlcrosome mutagenicity assay, Mutation Research, 64: 159-165 tl979). 4. Environmental Protection Agency, Health Effects Test Guidelines, BG-Gene Muta-. typimurlum. EPA Report No. 560/6-82-001, August, 1982. VPC/rkk/06203-1 11-08-84 i UCn 062516 mk REOUEST FOR STANbARp TOXfCOLOGYSTUDtESl wt It v-: AT BUSHY RUN RESEARCH CENTER foEnl Page 1 of 9 wwO* vnto-'t BUSINESS CONFIDENTIAL If Mr. R.C. Myers Bushy Run Research Centar RD 4 Mellon Road Export, PA 1S632 ftpm; N*m fo Sponsoring Oept Ktt /itOfi&Zf/ ltMAlt/7Ef Location (city) Charge to Aeeeunt No. xr-m-Wo Technology Manager </77 ZcU/tfTgUl$,/ il T.fa ffj U k' .JUftorization: ) ^ 7/if/a Wenatur* Oat* COMPLETE COPIES OF THIS POBM MUST BE SENT (BY THE SUBMITTER! TO: A Dr. Bryan Bailantyna Technical Center (3005) South Charleston. WV 25303 B. Dr. W.C. Kuryta Technical Center (3005) South Charleston, WV 25303 C. Dr. E R. Homan Bushy Run Research Center RD 4 Mellon Road Export PA 15632 D. Sponsoring Division HS & EA Manager Name: Loc.: TESTS REQUESTED (Standard procedures; other tests require specific protocols. Please contact the Corporate ' Applied Toxicology Dept lor advice and guidance.) 1 MNGE FINDING: 3. INVITRO: ^ RECEIVED 0 Acute Oral D Acute Percutaneous 0 Acute Inhalation 0 Skin Irritancy D Eye Irritancy D Other________ & Ames (Salmonella) Test AU G 2 4 1984 D Gene Mutation (CHO) -- W. C. HENGLFP Chromosome Exchange (SCE) P Chromosome Damage (Micronucleus) P DNA Damage (UDS) WOWN PROCEDURAL REQUIREMENT (circle): P Macrophage (RAM) test K:d. FHSA, DOT. FIFRA, TSCA. RCRA. Other. P Other :3DUCT/COMPOSITION NAME: 'ffrt i:S NOMENCLATURE: I'OXj&CyCJc fa 4 *)/ J rn 106-87-6 IlEMICAL SYNONYMS/ALTERNATE/TRADE NAMES: '^UP/U) i;U/Otx Uft) aJCTURAL FORMULA OR COMPOSITION OF MIXTURES: O' CM-0*1 H ,2 i Tib I^IBUTIQN OF REPORT: A standard divisional distribution list will be used unless otherwise requested (see page 4). IX*C USE) Received ACKNOWLEDGEMENT OF SAMPLE RECEIPT f>y ucc 062517 3362 Page 2 f 9 | CHEMICAL INFORMATION 4 1. SAMPLE PURITY/COMPOSmON: &Uu*u*L ^udjtSh^, ,, LO, rMluVfii ftU&WAt. Analytical taat method employed* I------------------- L-------- l tecot/.'^1 /t. --___________ ___ . (Send eopy ofavailable data, aourca and location of analyses.) Percent Composition Amounts and identities of known impurities: Sfe--_ Amount of unidentified impurities: ** Test material: _____ % 2. REQUIRED SAMPLE QUANTITY: Acute Tests: in vitro Tests: 4 ounces 1. 32 ounces for full series of tests 2. 16 ounces for chemicals with known high toxicity 3. 2 ounces for irritancy tests only NOTE. To reduce problems involving disposal of unused amounts of the sample, supply only the amount specified above. 3. PHYSICAL CHARACTERISTICS: (Give all which ere known): Gas. Liquid. </ . Solid. ____ PowderGranular Molecular Wl ____ Density (liquids) W0.I? Vapor Pressure (@ 3 temp between 15* and 150'C): f.dP -I-10 ( U f U* ^ 1. _______ 0.1 mm @ 2* *' fc' *C pH (aqueous sol.) Freezing point--Tf *CBoiling point IWC*{ ly* /mm*. 2. #c 3. *c mm @ *C (Si *C Flash pointt-P"7 *C (Tag, closed cup) 4. SOLUBILITY: Solvent Watsir^jbj kI Z?C Acetone_______________ Ethanol________________ Dimethylsulfoxide______ Vegetable Oil__________ . Other (specify)__________ Percent Solution LZ. 5. STABILITY: (Check aoDrooriateiv): Shwlf 1 if* Light sensitive //_ < 6 mo: . >6 mo. . no Temp, sensitive VM / no Oxidizes .... ...... * Polymerizes . v/ ViSfi ,/ yes; _ no _ no Known solvent Incompatibility (specify) urfy UtJ' !>**" UCC 062518 Report 47-183 Appendix 2 XmxlJ* of |HANDUNG/STORAGE/D)SPOSAHNFORMAHON:| 3363 . .crnUMENDED PRECAUTIONS fOR HANDLING*: t&S tPWf ftltH St'l/J AM! EUZsT Enclose UCC (or other company) Materia' Safety Data Sheet * If available. Any unusual fire or explosion hazards: --tL________________ Any chemical incompatibilities (materials to avoid): SjfbitJfy /ft/if AfJ?s lSPECIAL PERSONNEL PROTECTIVE EQUIPMENT NEEDED: "T t'l 3RECOMMENDED INACTIVATION/DECONTAMINATION FROCEDURE(S): UfirS4 Mrm fix! ujAi&Z RECOMMENDED STORAGE CONDITIONS: Temperature (Check appropriately): Room Temp. iS n'! Refrigerate_______________ freeze . < 20eC < O'C 1 j SAMPLE DISPOSAL: (Check appropriately): * "I D Return unused sample to: (attach DOT shipping instructions) Special Conditions Required (Cheek appropriately) Under N* gas Dessieator Other______ ___________________ ___________ Disposal by BRRC is authorized in compliance with applicable regulations UCC 062519 I jiTtteR <*flnNEKT iMf&RMATtPNl Report 47*183 Appendix 2 Page 4 of 9 The fotlowinp information will be useful in determining or confirming the necessity of specific toxicity imt 1. Proposed use of material (circle): Industrial Product Food Drug Other use (specify): Intermediate Home Use Experimental Cosmetic Packaging (specify Food, Drug. Cosmetic) 2. Potential hazard route(s) (circle): Other (specify): 3. Form shipped in commerce (circle): Solvent or Vehicle composition: Inhalation Gas Suspension or Slurry ATTACHMENTS (TO BE PROVIDED BY SUBMITTER): * Analytical data for sample. * - Material Safety Data Sheet of sample material (if available). * * DOT shipping information (if applicable). DISTRIBUTION LIST: Unless indicated below, the standard distribution list for the sponsoring division will be used. Please use the following divisional distribution list for the final study report WCK/Mar S3 UCC 062520 mMicniMk nrcii whim ncci KmwiWI bv UA. Debarment or lobar *?aanuaifv.CmMar" ta Pawn LSaOOtai teport 47-183 Appendix 2 uubo' Page 5 of 9 ^JcAL NAME: SAKE LITE CYCLOALIPHATIC EPOXY ItESIN ERL4206 ___ *** 1 (jgJlA: Vinyl-3*Cyclohaxana Diepoxide; Eooxvethyl-3,4-Epoxycyelohxne. C|H,,0 CHEMICAL FAMILY: Epoxy Resins MOLECULAR WEIGHT: 140.1B UOE NAME AND SYNONYMS: BAKE LITE Cydeeliphatic Epoxy Rain ERL4206 V-> *: YV'-'-'*:'* f- ('ING POINT. 760 mm. H9 pjf 1C GRAVITY (HjO * 1) tfK DENSITY (ait 11 :jnt volatiles r VOLUME 227*C. (440.6* F.) iJDQeer 20/20'C. 4JB6 Nil FREEZING POINT VAPOR PRESSURE AT 20*C. SOLUBILITY IN WATER, % by wt. at 20*C. EVAPORATION RATE (Butyl Acetate - 1) Sett to glatt below-55*C. <0.1 mm. Hg V8.3 0.01 rtlRANCE AND ODOR Low viscosity liquid; characteristic odor. . .M-v*v-w-.'XW^aTj-r^TV'Rtr'RKrr^"------- *-- wm^m -- "*rv-vam'tnbfa;V^TV^WTT, L. V V 1 .. *.: :>s JB. HAZARDOUS INGREDIENTS*t#'< art- a a * -* - m >.fJL roavr* - ' - , . -Ittt eXX^-Ven w _* MATERIAL Vinyl Cyclohexane Diepoxide % TLV (Unit*) 100 Not fttiblithed by ACGIHor OSH A (Sat Soctioni III through VIII) 1 - V* YT'h e^vtjjna.adi, a . V|a V * apt, ` p* * W` . <W 1 III. FIRE AND EXPLOSION HAZARD DATA 1 '.WPOINT 1 225* F,, Pensky-Mertens doted cup ASTM D 63 | vrethodl 1 230*F., Cleveland open cup ASTM D 92 | AUTOIGNITION 1 TEMPERATURE .ON ,, 1 ,,_ee 1 747^F* f MBMABLE LIMITS IN AIR. % by volume Not determined (Nonvolatile material) 1 2 frCuiSHING fjCiA Ute carbon dioxide or dry chemical for amall fires. Um alcohol-type foam for targe firet. *1 FIRE FIGHTING ."rCEDURES |- ------------------------- Weter in fine tpray can be utad. Do not ute aolid ttream of water; h may cauK frothing. &sual fire and jif.OSION HAZARDS * 1 -- _______________, 4 vi Hazardous polymerization may occur, due to heat ganaratad by exposure to a firt. none 304/744 3487 Th number araMab** dayi. mps, weekends and holiday!. bob Union Corbida Corporation beiovoi that th dm eonuirwd herein *T tactual end th# eomiont acornied err thpw o< oueiifito *0*rt> ^'dm* Tt* raauta of tt* watt conduced. tha da are not to fa* taken at a warranty or rpr*Mnteiion lot wnicn Union Cerbidt Corporation ** ta' ratoonilbility. They n offtrad totaly for your centidoration, and verification. Any uw of thaw data and information muii bt bbmiinad by Via utar to be In accordance with applicable Federal, Stt. end local lowt and regulation!. i UNION CARBIDE CORPORATION a CHEMICALS AND PLASTICS 270 PARK AVENUE. NEW YORK. N Y. 10017 ycc ~ ~t*i-. Vda THRESHOLD LIMIT VALUE EFFECTS OF OVEREXPOSURE v* *. 10 ppm. ACGIH notice of intended changes (1B7S). : Appendix 2 Pa*ge 6 of 9 Contact with akin or eves may be irritating, with ayes most tavartly affected. Absorption through skin may causa nai/sea, vomiting, and othtr signs of illneu. Skin painting tasts in animals caused skin cancers, but no cancers have been reported in humans. EMERGENCY AND FIRST AID PROCEDURES In case of contact immediately flush ayes with plenty of watar for at least IS minutes and wash skin thoroughly with plenty of soap and water. Call a phytieien for ayes. Remove contaminated clothing at one* and wash underlying skin. Wash clothing before reuse. J * "J | , .' * 'W* ... * .A * * w -- - y^.REACTfVITY DATA** y^'C** t !*r. . !._.*? "y *' ' t STABILITY UNSTABLE y/ STABLE CONDITIONS TO AVOID Avoid storage at tamparatures above 100*F. INCOMPATIBILITY (materials to avoid) Avoid amines, acids, alkalies. HAZARDOUS DECOMPOSITION PRODUCTS HAZARDOUS POLYMERIZATION May Occur Will not Occur y/ -- Thermal decomposition or burning may produce carbon monoxide and/or carbon dioxide. CONDITIONS TO AVOID Avoid amines, acids, alkalias. *vL SPCMlEAkPROCEDURESl - wr-uu^Ai^iaJ^T. STEPS TO BE TAKEN IF MATERIAL IS RELEASED OR SPILLED Smsll spilt* can far flushed with large quantities of water. Larger spills should be collected for disposal. 0>` WASTE DISPOSAL METHOD Dilute with an inert solvent, and incinerate in a furnace where permitted under appropriate Federal, State, and local regulations. * *$MRATO*Y protection ^ I I(stpoetceiiffyy type) Air-supplied mask in confined antes Page 7 of 9 wi)b7 ItffTILATlON LOCAL EXHAUST MECHANICAL (general) SPECIAL OTHER ACTIVE CLOVES Rubber EVE PROTECTION Coverall goggles OTHER protective fjyiPMENT r:.*V<Pl>l.e^CoVw Protective creams on arms and face; aye bath, and safety shower. SPECIAL PRECAUTIONS. . .. Hr****'* HPC7 * ****% "* .-tfc.. . 'I ' #*>' .. ` a. . * +, 4. rlcautionary labeling -4 SAKE LITE CYCLOALIPHATIC EPOXY RESIN ERL4206 WARNING) PROLONGED AND REPEATED CONTACT OF LIQUID OR BREATHING OF VAPORS OR MISTS MAY CAUSE DELAYED AND SERIOUS INJURY. ABSORPTION THROUGH SKIN HARMFUL. LIOUID CAUSES EYE INJURY. Do not get on skin, in eyes, or on dothing. Avoid inhalation of vapors or mitts. FIRST AID: In case of contact, immediately flush eyes or skin with plenty of water for at least 15 minutes. For eyes, call a physician. Remove contaminated dothing and shoes at once. Wash thoroughly befort reuse. FOR INDUSTRY USE ONLY OTHER HANDLING AND H5RAGE CONDITIONS Avoid storage tampersture above tOO*F. ( 3368 aeporc /-x3 Appendix 2 Pege 8 of 9 UNION CARBIDE CORPORATION eeniLTvpa.vMiEeECiWoarapriwo^ RECEIVED SEP 121984 .W.C HENGLER woiiJW-ecnc September 6, 1984 Mr. W. C. Hengler Bushy Run Technical Center RD4, Mellon Road Export, Pennsylvania 15632 Pear Mr. Henglert Infrared apeetra have been run on the samples of cycloaliphatic epoxide that you returned to me recently. The spectra are enclosed for your files. The spectra that were obtained on these samples were compared with standard spectre for these products. In two cases, 4205 and 4299, comparisons were made with et samples of the these products that are being utilised in current laboratory progrt Comparison of these various spectra by the Infrared experts in our Analytical Group (Dr. Clark and Mr. Lewis) led to the conclusion that the sample that you have are authentic samples of the UCC cycloaliphatic epoxides. Thus, th mutagenicity testing will be performed on valid samples of 4221, 4299, 4234, 4206 and 4205. ends. Very truly yours. RJCimjk cc;Dr. . M. Clark, Mr. J. Lewis, Dr. R. E. Pleven - DB 062524 r *I 07. nvf 1< ON 'i l l ABSCISSA WANStON *____ . . . . . __ _ s*mpu )>t VJO& , nWMM. /t-( -------- /utfl--- (.fv_ OPC'MATE WANSWN . * r.................. ABS . PCMAPKS j V_AM HMt . .. iMClfBUEB _ ______ . _ | Slrt PtOGBAM....................... .. jsoivem . .. j COMCtNIBAflON .. . fTP. SCAN TtMt 0rvf TJtBAtQt SIMGIF BEAM * cm PATH........... ............... ErPBfMTF.................. m f \ 1 j j> ii ' j i i i > l APPENDIX 3 Report 47-283 Page 1 of 1 A. Historical Data on Spontaneous Reversion Rates at BRRC As of June 30. 1984 Strain +/-S9 N Mean Standard Deviation Median Range 1A98 -S9 98 18.2 +S9 98 25.6 5.02 7.40 18 7, 33 26 14, 44 TA100 -S9 98 123.1 +S9 98 101.5 26.89 17.86 120 61. 187 100 65, 137 TA1535 -S9 +59 98 32.5 98 13.4 13.61 7.34 32 7, 60 14 2. 35 TA1537 -S9 98 4.3 +59 98 5.0 1.90 2.61 4 1. 9 5 1, 13 TA1538 -59 98 7.2 +59 98 15.0 3.72 5.41 7 2, 19 16 4, 29 j j j Strain B. Historical Data on Postive Control Values at BRRC Aa of January 26. 1984 Positive Control +/-S9 N Mean Deviation Median Range 1A98 NPD,10 vg -S9 84 877.1 166.73 870 2AA,10 Ug +S9 84 1434.2 327.54 1457 332, 1301 604, 2037 IA100 nz, io yg -S9 84 1621.8 277.37 1696 2AA.10 VC +S9 84 1181.7 323.54 1201 869, 2069 475, 2225 IA1535 NZ, 10 ic -59 84 1622.0 253.42 1666 2AA.10 VC +S9 84 99.3 30.84 98 837, 2050 37, 182 IA1537 9AA.60 ic -S9 84 140.6 130.70 2AA,10 Ug +S9 84 122.5 45.52 82 120 28, 489 36, 230 TA1538 NPD,10 ic -S9 84 1124.2 196.66 1168 2AA.10 l 459 84 532.0 230.61 486 573, 1582 146,. 1249 S9- rat liver homogenate; NPD- 4-nitrophenylenedlamine; 2AA- 2-ainoanthracene; NZ- sodium azide; 9AA- 9-aminoacrldine. VPC/rkk/0620B-l 11-08-84