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MUTAGENICITY EVALUATION OF T-2014 CoC IN THE AMES -SALMORErU/MICROSOME PLATE T-EST FINAL REPORT SUBMITTED TO: 3M COMPANY 3M CENTER SAINT PAUL, MINNESOTA 55101 BIONETICS L'ttm SUBMITTED BY: LITTON BIONETICS, INC. 5Sl6 NICHOLSON LANE KENSINGTON, MARYLAND 20795 LBI PROJECT NO. 20838 FEBRUARY 1978 I ii. III. IV. V. Vi. SPONSOR: 3M Company MATERIAL* A. Identification: T-2014 CoC B. Date Received: December 20, 1977 C. Physical Description: White powder TYPE OF ASSAY: Ames Salmonella/Microsome Plate test PROTOCOL NO.: OMT-100 RESULTS The results of this assay are presented in Table 1. INTERPRETATION OF RESULTS AND CONCLUSIONS The test compound was examined for mutagenic activity in a series of in vitro microbial assays employing Salmonella and Saccharomyces indl'citor-organisms.The compound was tested diFectl@-and-inthe presence of liver microsomal enzyme preparations from Aroclor-induced rats. The compound was tested over a series of concentrations such that there was either quantitativeor qualitativeevidence of some chemically-inducedphysiologicaleffect at the highest dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluationof this compound was from 0.1 ug to 500 ug per plate. The results of the tests conducted on the compound in the absence ,of a metabolic activationsystem were all negative. The test with TA-100 was repeated at 100, 500 and 1000 ug's because of increased revertantsobserved at 500 ug dose level in the initial test. The repeat test was negative. The results of the tests conducted on the compound in the presence of a rat liver activationsystemwere all negative. The test with TA-100 was repeated at 500, 1000 and 2000 ug dose per plate, because of increasedrevertantsobserved at 500 ug dose level in the initial test. The repeat test was negative. *Informationwas supplied by the sponsor. If informationwas not indicated by the sponsor, N.I. was entered. [EBIONETICS Utton VI. INTERPRETATION OF RESULTS AND CONCLUSIONS (Continued) The test compound, T-2014 Coc did not demonstrate genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions. FRBIONETICS Utton Submitted by: Study Director "@T _x B.-R. Jagannath, Ph.D. Date Section Chief Submammalian Genetics Department of Genetics and Cell Biology Reviewed by: David B usick, Director Department of Genetics and Cell Biology eoaat'e 2 Vo RFSIILTS ----------------;---------------- TABLE I A. R. C. NOTE: NAME OR CODE DESIGNATION SNLVENT: nmso OF THE TEST compounns TFST INITIATION DATE: BECO 28* 1977 CONCENTRATIONS APE GIVEN IN MICROLITERS (UL) T-2014 CDC OR MICROGRAMS IUG) PER PLATE. TFST ---- NONACTIVATINN ------------- SPECIES TISSUE ------- ------ R E V E R T A N T S P E R PL A T E ------- ------- -T;:IS;S -----T;:1;3;-----TA:;;;e ------;;:9R TA:100 D;;- ---------- ---------- ---------- ---------- ---------- ------- 1 2 1 z 1 2 1 2 1 2 1 snLVFNT RONTROL PISTTIVF CONTROL** TFST COMPOUND 0.100000 IOG 1.000000 116 10.000000 t)G 104.00nooo un 500.000000 UG 1000.000000 un ACTIVATTNN ---------- ----- ------------- ----- ------------- 19 410 14 IR it 13 is in 524 14 In it A 10 19 1555 24 24 20 14 IFI 43 119A 46 41 31 35 37 lao 198 67 izsa 840 212 193 87 210 93 273 176 92 275 141 98 9z SOLVENT CONTROL POSITTVF CONTROL*** TFST COF4POUND RAT LIVER *0 12 24 46 277 167 13R RAT LIVER PAZ 233 963 8?2 1426 189 0.100000 Ila RAT LIVER 20 13 217 46 234 167 10000000 too PAT LIVER 30 16 zo 39 248 136 10.000000 to(; RAT LIVER 74 p;w 23 42 2AR 147 100.000000 UG RAT LIVER 19 14 24 49 271 148 ROO.000000 UG PAT LIVER is 13 27 34 291 IS? 164 1000.000000 UG RAT LIVER 79 PODO.000000 UG RAT LTVIEP To -------------------------------------------------------------------------------------------------------------- 0 TRY* CONVFRTANTS PER PLATE 0* TA-191S TA-IRIT TA-IS38 TA-9P TA-INO n4 QOLVFNT FMS OM NF NF FMS EMS NMSO 10 UL/PLATE 10 UG/PLATE 10 UO/PLATE 10 UC*/PLATE 10 UL/PLATE 10 UL/PLAYE 50 UL/PLATE so* TA-1535 TA-1537 TA-1538 TA-98 TA-100 04 SOLVENT ANTH ANTH ANTH ANTH ANTH OMNA DMSO 2-S 2orp 2.9; 2.5 2-S 100 50 Un/PLATE UG/PLATE UG/PLATE UG/PLATE UG/PLATE MICROMOLFS/PLATE UL/PLATE PROTOCOL 1. PURPOSE The purpose of this study was to evaluate the test material for genetic activity in a microbial assay with and without the addition of mammalian metabolic activationpreparations. 2. MATERIALS A. Indicator Microorganisms A descriptionof strain verificationis given in StandardOperating Procedure on page 10. Salmonella typhimurium TA-1535 TA-1537 TA-1538 TA-98 TA-100 Saccharomyces cerevisiae D4 B. Activation System 1. Reaction mixture Component Final Concentration/ml TPN (sodium salt) Glucose-6-phosphate Sodium phosphate (dibasic) MgCl2 KC1 Homogenate S9 fraction 4 limol 5 pmol 100 pmol 8 pmol 33 pmol 0.1 .05 ml 2. S9 homogenate A 9,000 x q supernatant was prepared from Sprague-Dawley adult male rat liver induced by Aroclor 1254 five days prior to kill according to the procedureof Ames et al. (1975). S9 samples were coded by lot number and assayed for milligrams protein per milliliterand relativeP448/P450activity by methods described in LBI Technical Data on Rat Liver S9 Product. BIONETICS 4 Ufton 2. MATERIALS (Continued) C. Positive Control Chemicals The chemicalsused for positivecontrolsin the nonactivationand activationassays are given in Table 1 of SectionV. Results. 0. Solvent Either deionizedwater or dimethyslulfoxide (DMSO) was used to preparestocksolutionsof solidmaterials.All dilutionsof test materialswere made in either deionizedwater or DMSO. The solvent employedand its concentrationare recordedin Table I of SectionV. Results. 3. EXPERIMENTAL DESIGN A. Plate Test (Aqar Incorporation)* Approximately108 cells from an overnightcultureof each indicator strainwere added to separatetest tubes containing2.0 ml molten agar supplementedwith biotin and a trace of histidine. For nonactivationtests, at least4 dose levelsof the test compound were added to the contentsof the appropriatetubes and poured over the surfacesof selectiveagar plates. In activation tests, at least 4 dose levelsof the test chemicalwere added to the appropriatetubes with cells. Just prior to pouring,an aliquotof reactionmixture(0.5ml containingthe 9,000x q liver homogenate)was added to each of the activationoverlay tubes, which were then mixed, and the contentspoured over the surfaceof a minimal agar plate and allowedto solidify. The plates were incubatedfor 48 hr at 37*C and scoredfor the numberof colonies growing on each plate. 04 yeast plates were incubated at 300C (nonactivationa)nd 37*C (activation)for 3-5 days and then scored. The concentrationsof all chemicalsare given in Table I of Section V. Results. Positiveand solventcontrolsusing both directlyactive positivechemicalsand those that requiremetabolic activationwere run with each assay. Certain classes of chemicals known to be mutagens and carcinogensdo not produce detectableresponses using the standard Ames agar incorporation method. Some dialkyl nitrosaminesand certainsubstitutedhydrazinesare mutagenicin suspensionassays, but not in the plate assay. Chemicals of these classes should be screened in a suspensionassay. [B BIONETICS Utton 5 3. EXPERIMENTAL DESIGN (Continued) B. Recordina and Presenting Data The numbers of colonies on each plate were counted and recorded on printed forms. These raw data were analyzed in a computer program and reportedon a printout. The resultsare presentedas revertants (or convertantsfor 04) per platefor each indicatorstrain employedin the assay. The positiveand solventcontrolsare provided as referencepoints. Other relevant data are providedon the computerprintout. 4. EVALUATION CRITERIA Plate test data consistof directrevertantcolonycountsobtainedfrom a set of selectiveagar platesseededwith populationsof mutantcells suspended in a semisolidoverlay. Because the test chemical and the cells are incubatedin the overlayfor 2 days, and a few cell divisions occur during the incubationperiod,the test is semiquantitative in nature. Althoughthese featuresof the assay reduce the quantitation of results,they provide certain advantagesnot containedin a quantitative suspensiontest: The small numberof cell divisionspermitspotentialmutagens to act on replicatingDNA, which is often more sensitivethan nonreplicatingDNA. The combined incubationof the compoundand the cells in the overlaypermitsconstantexposureof the indicatorcells for 2 days. A. Survivinq Populations Plate test procedures'do not permit exact quantitationof the number of cells survivingchemicaltreatment. At low concentrations of the test chemical,the survivingpopulationon the treatment plates is essentiallythe same as that on the negative controlplate. At high concentrations,the survivingpopulation is usually reduced by some fraction. Our protocol normally employs several doses ranging over 2 or 3 log concentrations,the highestof these doses being selectedto show slighttoxicityas determinedby subjectivecriteria. B. Dose-Response Phenomena The demonstrationof dose-relatedincreasesin mutant counts is an importantcriterionin establishingmutagenicity.A factorthat might modify dose-responseresults for a mutagen would be the [13BIONETICS 6 Utton 4. EVALUATION CRITERIA (Continued) B. Dose-Response Phenomena selectionof doses that are too low (usually mutagenicityand toxicityare related). If the highestdose is far lower than a toxic concentration, no increases may be observed over the dose range selected. Conversely,if the lowest dose employed is highly cytotoxic, the test chemical may kill any mutants that are induced, and the compound will not appear to be mutagenic. C. Control Tests Positive and negative control assays are conducted with each experiment and consist of direct-actingmutagens for nonactivation assays and mutagens that require metabolic biotransfomation in activation assays. Negative controls consist of the test compound solvent in the overlay agar together with the other essential components. The negative control plate for each strain gives a referencepoint to which the test data are compared. The positive control assay is conducted to demonstrate that the test systems are functional with known mutagens. D. Evaluation Criteria for Ames Assay Because the procedures used to evaluate the mutagenicity of the test chemical are semiquantitative,the criteria used to determine positive effects are inherentlysubjectiveand are based primarily on a historicaldata base. Most data sets are evaluated using the followingcriteria: 1. Strains TA-1535, TA-1537, and TA-1538 If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrationswith the lowest increase equal to twice tFe solvent control value is considered to be mutagenic. 2. Strains TA-98, TA-100,.and D4 If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrationswith the highest increase equal to twicft-he solvent control value for TA-100 and 2-3 times the solvent control value for strains TA-98 and D4 is consideredto be mutagenic. For these strains, the dose-response increase should start at approximatelythe solvent control value. 113SIONETICS Utton 7 4. EVALUATION CRITERIA (Continued) D. Evaluation Criteria for Ames Assay 3. Pattern Because TA-1535 and TA-100 are both derived from the same parental strain (G-46) and because TA-1538 and TA-98 are both derived from the same parental strain (D3052), there is a built-in redundancy in the microbial assay. In general the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipatedthat if a given strain, e.g., TA-1537, responds to a mutagen in nonactivation tests, it will generallydo so in activationtests (The converse of this relationshipis not expected.). While similar response patterns are not required for all mutagens, they can be used to enhance the reliabilityof an evaluation decision. 4. Reproducibility If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initialpositivetest data lose significance. The preceding criteria are not absolute,and other extenuating factors may enter into a final evaluation decision. However, these criteria are applied to the majority of situationsand are presented to aid those individualsnot familiarwith this procedure. As the data base is increased,the criteriafor evaluation can be more firmly established. E. Relationship between Mutagenicity and Carcinogenicity It must be emphasized that the Ames Salmonella/MicrosomePlate Test is not a definitivetest for chemical carcinogens. It is recognized,however, that correlativeand functionalrelationships have been demonstrated between these two endpoints. The results of comparative tests on 300 chemicalsby McCann et al. (1975) show an extremely good coi-*relatiobnetween results7-of-microbial mutagenesis tests and in vivo rodent carcinogenesisassays. All evaluationsand interpretationof the data presentedin this report are based only on the demonstration,or lack, of mutagenic activity. IBBIONETICS utton 8 REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mamalian-microsomemutagenicitytest. MutationRes. 31, 3Z7--T6T.- McCann,J., Choi, E., Yamasaki,E. and Ames, B.N. (1975). Detectionof carcinogensas mutagens in the Salmonella/microsometest: Assay of 300 chemicals. Proc. Nat. Acad. SLi. 72, 5135-5139. 113BIONETICS Utton 9 5. STANDARD OPERATING PROCEOUPS All data will be enteredin ink (no pencil). All changes or correctionsin entries will be made with a single line through the change, and an explanation for the change must be written. All calculations(weights,dilutions,dose calculations,etc.) will be shown on data records. All data entrieswill be dated and initialed. All laboratoryoperationswill be written out in standardprotocol manuals. These manuals will be present in each laboratory area. Deviations from any establishedprotocol will be described and justified. Data will be stored in bound form (notebooksor binders). These bound data books will be reviewed by the appropriate Section Heads. Chemicals submitted for testing will have date of receipt and initials of entering person. Lot numbers for all referencemutagens, solvent, or other materials used in assays will be recorded. Animal orders, receipts, and identificationwill be recorded and maintained such that each animal can be traced to the supplier and shipment. All animalson study will be properly identified. A copy of the final report plus all raw data and support documentswill be permanently stored in the archival system of Litton Bionetics,Inc. Current curricula vitae and job descriptionswill be maintainedon all personnel involved in the study. .Salmonellastrains will be routinely checked for the his, uvrb, rfa, and pKM 101 phenotypes. Only 'appropriatelyscreened stock cultures will be used in chemical evaluations. SIONEncS 10