Document mb0oL8Mowkrp5ONKnmBvaq1O

ARMrcm^ MUTAGENICITY EVALUATION OF T-2015 CoC IN THE AMES SALMOREtn/MICROSOME ----------- F CTTE TEST------------- FINAL REPORT SUBMITTED TO: 3M COMPANY 3M CENTER SAINT PAUL, MINNESOTA 55101 DB BIONETICS Utton SUBMITTED BY: LITTON BIONETICS, INC. 5516 NICHOLSON LANE KENSINGTON, MARYLAND 20795 LBI PROJECT NO. 20838 FEBRUARY 1978 C00165 I. II. III. IV. V. VI. SPONSOR: 3M Company MATERIAL* A. Identification: T-2015 CoC B. Oate Received: December 20, 1977 C. Physical Description: White powder TYPE OF ASSAY: Ames Salmonella/Microsome Assay PROTOCOL NO.: DMT-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 1n a series of vitro microbial assays employing Salmonella and Saccharomyces indicator organisms. The compound was tested directly and in the presence of U v e r microsomal enzyme preparations from AroclorInduced rats. The compound was tested over a series of concentrations such that there wa either quantitative or qualitative evidence of some chemically-induced physiological 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 yg to 500 yg 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. Information was supplied by the sponsor. If Information was not Indicated by the sponsor, N.I. was entered. f f l BIONETICS Litton 0G0166 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. EQ BIONET1CS Litton Submitted by: Study Director D. R. Jagannath, Ph.D. Section Chief Submammalian Genetics Department of Genetics and Cell Biology Date Reviewed by: javi< Director Department of Genetics and Cell Biology r 000167 V. RESULTS TABLE 1 A. NiME OR CODE DESIGNATION OF THE TEST COMPOUND! T-20IS COC fl. SOLVENT: OMSO C. TFST INITIATION DATE! DEC. 28. 1977 NOTE! CONCENTRATIONS ARE GIVEN IN MICROLITERS (UL1 OR MICROGRAMS (UGI PER PLATE. REVERTANTS PER PLATE TFST .... nonactivaiion SPECIES TISSUE TA-153S 12 TA-1S37 1? TA-1538 12 TA-98 12 TA-100 12 SOLVFNT CONTROL POSITIVE CONTROL TFST COMPOUND 0.100000 UG 1.000000 UR 10.000000 UG 100.000000 UG SOO.000000 UG ACTIVATION A --- --- --- ... -- --- 12 *10 19 18 18 17 13 10 19 *5 180 52* 1555 119A 1258 12 23 5* 183 11 19 36 171 5 29 *3 193 n 28 32 1*3 6 23 39 211 SOLVENT CONTROL POSITIVF CONTROL TFST COMPOUND 0.100000 UG 1.000000 UG 10.000000 UG 100.000000 UG SOO.000000 UG RAT RAT RAT RAT RAT RAT RAT TRY c o n v e r t a n t s PER PLATE TA-1S35 TA-1S37 TA-1538 TA-9 TA-100 0* SOLVFNT FMS QM NF NF EMS FMS DM50 10 UL/PLATE 10 UG/PLATE 10 UG/PLATE 10 UG/PLATE 10 UL/PLATE 10 UL/PLATE 50 UL/PLATE LIVER 20 23 2* 46 LIVER 262 233 963 872 LIVER 29 6 23 38 LIVER 2* 11 29 36 LIVER 13 16 2* 3* LIVER 19 21 28 39 LIVER 19 9 29 30 Ta -1535 TA-1537 TA-1538 TA-98 TA-100 0* SOLVENT ANTH ANTH ANTH ANTH ANTH DMNA OMSO 2.5 UG/PLATE 2.5 UG/PLATE 2.5 UG/PLATE 2.5 UG/PLATE 2.5 UG/PLATE 100 MICROMOLES/PLATE 50 UL/PLATE 277 1*26 234 232 2*7 231 276 D* 1 87 8*0 50 83 80 86 86 138 189 173 128 170 13* 150 000168 00 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 mamma lian metabolic activation preparations. 2. MATERIALS A. Indicator Microorganisms A description of strain verification is given in Standard Operat ing Procedure on page 10. Salmonella typhimurium TA-1535 T A - 1537 TA-1538 TA-98 TA-100 Saccharomyces cerevisiae D4 B. Activation System 1. Reaction mixture ________ Component_______ Final Concentration/ml TPN (sodium sait) G1ucose-6-phosphate Sodium phosphate (dibasic) MgCl2 KC1 Homogenate S9 fraction 4 pmol 5 pmol 100 pmol 8 pmol 33 pmol 0.1 .05 ml 2. S9 homogenate A 9,000 x 2 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 assayed for milli grams protein per milliliter and relative P448/P450 activity by methods described in LBI Technical Oata on Rat Liver S9 Product. m BIONETICS Litton 000169 2. MATERIALS (Continued) C. Positive Control Chemicals The chemicals used for positive controls in the nonactivation and activation assays are given in Table 1 of Section V. Results. D. Solvent Either deionized water or dimethyl sulfoxide (DMSO) was used to prepare stock solutions of solid materials. All dilutions of test materials were made in either deionized water or DMSO. The sol vent employed and its concentration are recorded in Table 1 of Section V. Results. 3. EXPERIMENTAL DESIGN A. Plate Test (Agar Incorporation)* Approximately 10 cells from an overnight culture of each indica tor strain were added to separate test tubes containing 2.0 ml molten agar supplemented with biotin and a trace of histidine. For nonactivation tests, at least 4 dose levels of the test com pound were added to the contents of the appropriate tubes and poured over the surfaces of selective agar plates. In activation tests, at least 4 dose levels of the test chemical were added to the appropriate tubes with cells. Just prior to pouring, an aliquot of reaction mixture (0.5 ml containing the 9,000 x 3 liver homogenate) was added to each of the activation overlay tubes, which were then mixed, and the contents poured over the surface of a minimal agar plate and allowed to solidify. The plates were incubated for 48 hr at 37C and scored for the number of colonies growing on each plate. D4 yeast plates were incubated at 30C (nonactivation) and 37C (activation) for 3-5 days and then scored. The concentrations of all chemicals are given in Table 1 of Section V. Results. Positive and solvent controls using both directly active positive chemicals and those that require meta bolic activation were run with each assay. * Certain classes of chemicals known to be mutagens and carcinogens do not produce detectable responses using the standard Ames agar incorporation method. Some dialkyl nitrosamines and certain substituted hydrazines are mutagenic in suspension assays, but not in the plate assay. Chemicals of these classes should be screened in a suspension assay. OE BIONETiCS Litton 5 0001.70 3. EXPERIMENTAL DESIGN (Continued) B. Recording 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 reported on a printout. The results are presented as revertants (or convertants for D4) per plate for each indicator strain employed in the assay. The positive and solvent controls are pro vided as reference points. Other relevant data are provided on the computer printout. 4. EVALUATION CRITERIA Plate test data consist of direct revertant colony counts obtained from a set of selective agar plates seeded with populations of mutant cells suspended in a semi sol id overlay. Because the test chemical and the cells are incubated in the overlay for 2 days, and a few cell divisions occur during the incubation period, the test is semiquantitative in nature. Although these features of the assay reduce the quantitation of results, they provide certain advantages not contained in a quanti tative suspension test: The small number of cell divisions permits potential mutagens to act on replicating DNA, which is often more sensitive than nonreplicating DNA. The combined incubation of the compound and the cells in the overlay permits constant exposure of the indicator cells for 2 days. A. Surviving Populations Plate test procedures do not permit exact quantitation of the number of cells surviving chemical treatment. At low concentra tions of the test chemical, the surviving population on the treat ment plates is essentially the same as that on the negative control plate. At high concentrations, the surviving population is usually reduced by some fraction. Our protocol normally employs several doses ranging over 2 or 3 log concentrations, the highest of these doses being selected to show slight toxicity as determined by subjective criteria. B. Dose-Response Phenomena The demonstration of dose-related increases in mutant counts is an important criterion in establishing mutagenicity. A factor that might modify dose-response results for a mutagen would be the f f l BIONET1CS Litton 6 000171 4. EVALUATION CRITERIA (Continued) B. Dose-Response Phenomena selection of doses that are too low (usually mutagenicity and tox icity are related). If the highest dose 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 cyto toxic, 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 biotransformation in activation assays. Negative controls consist of the test compound solvent in the overlay agar together with the other essential com ponents. The negative control plate for each strain gives a reference point 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 semi quantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical data base. Most data sets are evaluated using the following criteria: 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 concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic. 2. Strains TA-98, TA-100, and 04 If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with 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 04 is considered to be mutagenic. For these strains, the dose-response increase should start at approximately the solvent control value. D 3 BIONETICS Litton 7 00172 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 anticipated that if a given strain, e.g., TA-1537, responds to a mutagen in nonactivation tests, it will generally do so in activation tests (The converse of this relationship is not expected.). While similar response patterns are not required for all mutagens, they can be used to enhance the reliability of 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 initial positive test 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 situations and are presented to aid those individuals not familiar with this pro cedure. As the data base is increased, the criteria for evalu ation can be more firmly established. E. Relationship between Mutagenicity and Carcinogenicity It must be emphasized that the Ames Salmonella/Microsome Plate Test is not a definitive test for chemical carcinogens. It 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 results of microbial mutagenesis tests and in vivo rodent carcinogenesis assays. All evaluations and interpretation of the data presented in this report are based only on the demonstration, or lack, of mutagenic activity. m BIONETICS 000173 REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome muta genicity test. Mutation Res. 31, 347-354. McCann, J . , Choi, E., Yamasaki, E. and Ames, B.N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. 72. 5135-5139. Lti BIONET1CS Litton 9 000174 5. STANDARD OPERATING PROCEDURE All data will be entered jn 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 laboratory operations will be written out in standard protocol manuals. These manuals will be present in each laboratory area. Deviations from any established protocol will be described and justi fied. Data will be stored in bound form (notebooks or 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 identification will be recorded and main tained such that each animal can be traced to the supplier and ship ment. 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 descriptions will be maintained on all personnel involved in the study. Salmonella strains will be routinely checked for the his. uvrB. rfa, and pKM 101 phenotypes. Only appropriately screened stock cultures will be used in chemical evaluations. BIONETICS Litton 10 OC 03.75