Document G6MEgr90ZE2RzKaLKRkd2r5Jq

MUTAGENICITY EVALUATION OF T-2015 CoC IN THE AMES SALMMEM/MICROSOME PLATE itbi FINAL REPORT SUBMITTED TO: 3M COMPANY 3M CENTER SAINT PAUL, MINNESOTA 55101 113BIONETICS Utton SUBMITTED BY: LITTON BIONETICS, INC. 5516 NILHOLSON LANE KENSINGTON, MARYLAND 20795 LBI PROJECT NO. 20838 FEBRUARY 1978 I. ii. Ill. rv. V. -sponsok: 3M Company MATERIAL* A. Identification: T-2015 CoC B. Date Received: December 20, 1977 C. Physical Description: White powder TYPE OF ASSAY: Ames SalmonellalMicrosome Assay PROTOCOL NO.: 014T-100 ' RESULTS The results of this assay are presented in Table 1. Vi. 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 indtc-ito-orrganisms. The compound was tested-di-rectlia-nd in the presence of liver microsomal enzyme preparations from Aroclorinduced rats. The compound was tested over a series of concentrations such that there wag either quantitative or qualitative evidence of some chemically-inducedphysiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this compound was from 0.1 mg to 500 ug per plate. The results of the tests conducted on the compound in the absence of a metabolic activation system were all negative. The results of the tests conducted on the compound in the presence of a rat liver activation system were all negative. *Informationwas supplied by the sponsor. If informationwas not indicated by the sponsor, N.I. was entered. 10 SIONETICS Uttan VI. INTERPRETATION OF RESULTS AND CONCLUSIONS (Continued) The test compound, T-2015 CoC did not demonstrate genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions. 03BIONENCS Utton Submitted by: Study Director @agannath, Ph.USection Chief Submammalian Genetics Department of Genetics and Cell Biology 2- 20- Date Reviewed by: avi u i g@Di Daivriesctlocru@ - Department of Genetics and Cell Biology V. PFSOOLTS --------------------------------- TABLE I A. A. C. NOTET NAMF OR CODE DEStriNATION snl-VFNTI Dt4so OF THE TEST COMPOIJND: TFST INITIATION DATEI DEC. 28* 197? CONCENTRATIONS ARE O[VEN IN MICROLITERS (ULI T-201,; coc OR MICROGRAMS (UGI PER PLATE. TFR,T ---- NONACTIVAIION ------------- SPECIES TISSUF ------- ------ RE VE P TAN tS pE P PL A TE ----------------------------- -------- ---- ------- TA-153ri TA-ISIT TA:1538 ------;;-;R ;A-10; 4 ---------- ---------- ---------- ---------- ---------- -------- 1 2 1 p 1 2 1 2 1 r,nLVFNT CONTROL PNSITIVF CONTROL** 12 ... --- 410 TFST COmPOOINf) 0.100000 un ... --- 19 1.000000 1)" --- --- in 10.000000 tie --- --- is 100.000000 tics --- --- 17 -,00.000000 IJG --- --- 13 ACTIVATION ---------- in Iq 45 ISO al 5?4 isse) 119A IPSM 840 12 23 It 19 r. 29 11 26 6 23 54 1$)3 so 36 171 03 43 193 so 3? 143 06 39 211 86 SOLVFNT CONTROL PnglylVF CONTROLO*O TFST CoLoPOUNO PAT LIVER 20 23 24 4ft 2?? 138 PAT LIVER P.62 233 963 872 1426 169 0.100000 un PAT LIVER 29 23 38 234 1?3 1.000000 un PAT LIVER 24 11 Z9 36 232 1201 10.000000 uo PAT LIVER 13 16 24 34 24? 170 100.000000 I)o PAT LIVER 19 21 28 39 231 134 500.000000 un PAT LIVER 19 9 29 30 276 ISO -------------------------------------------------------------------------------------------------------------- 4 TRY* CONVERTANTS PEP PLATE 00 TA-ir,3s TA-1r,37 TA-lr3g TA-90 TA-INO n4 %OLVFNT FMS OM NF "F EMS Fms R)MSO 10 UL/PLATE 10 Uri/PLATE 14 UG/PLATE 10 UG/PLATE 10 UL/PLATE 10 UL/PLAIE SO UL/PLATE Off TA-1535 TA-1537 TA-1538 TA-98 TA-100 n4 SOLVENT ANTH ANTH ANTH ANTH ANTH DMNA OMSO 2.r,,UO/PLATE 2-5 UO/PLATE 2-rp UO/PLATE 2.5 Ufl/PLATE P.ri Utl/PLATE 100 HICROMOLFS/PLATE SO 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 activation preparations. 2. MATERIALS A. Indicator Microorganisms A description of strain verification is given in Standard Operating Procedure on page 10. Salmonella typhimurium TA-1535 TA-1537 TA-1538 TA-98 TA-100 Saccharomyces cerevisiae D4 B. Activation System I. Reaction mixture Component TPN (sodium salt) Glucose-6-phosphate Sodium phosphate (dibasic) MgC'2 KC1 Nomogenate S9 fraction 2. S9 homogenate Final Concentration/mi 4 pmol 5 pmol 100 pmol 8 pmol 33 pmol 0.1 .05 ml 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 procedure of Ames et al. (1975). S9 samples were coded by lot number and ass7y-erfor milligrams protein per milliliter and relative P448/P450 activity by methods described in LBI Technical Data on Rat Liver S9 Product. BIONETICS Utton 2. MATERIALS (Continued) C. Positive Control Chemicals The chemicalsused for positivecontrolsin the nonactivationand activationassays are given in Table 1 of Section V. Results. D. Solvent Either deionized water or dimethylsulfoxide (DMSO) was used to preparestocksolutionsof solidmaterials. All dilutionsof test materials were made in either deianized water or DMSO. The solvent employed and its concentrationare recorded in Table 1 of SectionV. Results. 3. EXPERIMENTAL DESIGN A. Plate Test (Aaar IncorDoration)* Approximately108 cells from an overnightcultureof each indicator strain were added to separatetest tubes containing2.0 ml molten agar supplementedwith biotin and a trace of histidine. For nonactivationtests, at least 4 dose levels of the test compound were added to the contents of 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 contents poured over the surface of a minimal agar plate and allowed to solidify. The plates were incubatedfor 48 hr at 370C and scored for the number of colonies growing on each plate. D4 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 1 of Section V. Results. Positive and solvent controls using both directlyactive positive chemicalsand those that requiremetabolic activationwere run with each assay. Certain classes of chemicals known to be mutagens and carcinogensdo not produce detectable responses using the standard Ames agar incorporation method. Some dialkyl nitrosaminesand certain substitutedhydrazinesare mutagenic in suspensionassays, but not in the plate assay. Chemicalsof these classes should be screened in a suspension assay. 18 BIONETICS Ufton 5 3. EXPERIMENTAL DESIGN (Continued) B. RecordinQ 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 D4) per plate for each indicatorstrain employed in the assay. The positiveand solvent controlsare provided as referencepoints. Other relevant data are provided on the computer printout. 4. EVALUATION CRITERIA Plate test data consistof direct revertantcolony countsobtainedfrom a set of selectiveagar platesseededwith populationsof mutant cells suspended in a semisolid overlay. 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 number of cell divisionspermitspotential mutagens to act on replicatingDNA, which is often more sensitivethan nonreplicating DNA. The combined incubation of the compound and the cells in the overlay permits constantexposureof the indicatorcells for 2 days. A. Surviving Populations Plate test proceduresdo not permit exact quantitationof the number of cells survivingchemical treatment. 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 slight toxicityas determinedby subjectivecriteria. B. Dose-Response Phenomena The demonstrationof dose-relatedincreases in mutant counts is an importantcriterionin establishingmutagenicity. A factor that might modify dose-responseresults for a mutagen would be the 113BIONETICS Utton 6 4. EVALUATION CRITERIA (Continued) B. Dose-Response Phenomena selection of doses that are too low (usuallymutagenicity and toxicity are 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-acting mutagens for nonactivation assays and mutagens that require metabolic biatransfomation 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 the 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 twice the solvent control value for TA-100 and 2-3 times the solvent control value for strains TA-98 and D4 is considered to be mutagenic. For these strains, the dose-response increase should start at approximatelythe solvent control value. 113SIONEMCS Litbon 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 familiar with this procedure. As the data base is increased,the criteria for evaluation can be more firmly established. E. Relationship between MutMenicity and Carcinogenicity It must be emphasized that the Ames Salmonella/Microsome Plate Test is not a definitive test for che-mi'-ca@car@cinogensI.t is recognized, however, that correlative and functional relationships have been demonstrated between these two endpoints. The results of comparative tests on 300 chemicals by McCann et al. (1975) show an extremely good correlation between results7--of-microbial mutagenesis tests and in vivo rodent carcinogenesis assays. All evaluationsand interpretationof the data presented in this report are based only on the demonstration,or lack, of mutagenic activity. 113BIONETICS Utton 8 REFERENCES Ames, B.N., McCann, J. and Yamasaki,E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsomemuta- genicity test. Mutation Res. 31, 3Z7--SUT.- 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. Scl. 72, 5135-5139. 113BIONETICS Utton 9 5. STANDARD OPERATING PROCEDURE All data will be enteredin ink (no pencil). All changes or corrections in 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 entries will be dated and initialed. All laboratoryoperations will be written out in standard protocol 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 reference mutagens, 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 animals on study will be properly identified. A copy of the final report plus all raw data and support documents will be permanently stored in the archival system of Litton Bionetics, Inc. Current curricula vitae and job descriptionswill be maintained an all personnel involved in the study. Salmonellastrains will be routinelychecked for the his, uvre, rfa, and pKM 101 phenotypes. Only appropriately screened-stock cultures will be used in chemical evaluations. 113BIONETICS Utton 10