Document MGyxEZQk8D9nbJbE73z888aXy

B10 -TEST 1810 FRONTAGE ROAD NORTHBROOK, TLLINOIS 60062 REPORT TO REVERSE IN FIVE ONE 3M COMPANY MUTATION STUDIES WITH T-1485 SALMONELLA STRAINS AND SACCHAROMYCES STRAIN P.O. NO. LP-003397-400 AUGUST 10, 1976 IBT NO. 8540-09238 B 10 -T ES T 1810 FRONTAGE ROAD NORTHBROOK, ILLINOIS 60062 August 10, 1976 Dr. James E Long Manager, Toxicology 3M Company 31N,iCenter St. Paul, Minnesota Services 55101 Dear Dr. Long: Re: IBT No. 8540-09238 - Reverse Mutation Studies with T-1485 in Five Salmonella Strains and One Saccharomyces Strain - P.O. No. LP-003397-400 We are submitting herewith our laboratory report prepared in connection with the above study. Very truly yours, a - ro. -ev a 0-M,@@ J. C. Calandra President JCC: b-P REPORT TO 3M COMPANY REVERSE IN FIVE ONE MUTATION T-1485 SALMONELLA SACCHAROMYCES STUDIES WITH STRAINS AND STRAIN P.O. NO. LP-003397-400 AUGUST 10, 1976 IBT NO. 8540-09238 I. Introduction A sample identified as T-1485 was received from 3M Company for the purpose of conductin- reverse mutation studies. The test material was evaluated for genetic activity in microbial assays with and without the addition of mammalian metabolic activation preparations. This report presents the results of the investigation. (D- T b-- 5 T II. Surnrnary The test material, T-1485, was examined for rnutagenic activity in a series of in vitro microbial assays using Salmonella and Saccharomyces indicator organisms. The test material was tested directly and in the presence of liver rnicrosornal enzyme preparations from Aroclor-induced rats. Dose levels of 0. 25, 0. 5, 5, and 50 4g of T-1485 per plate were used in both the nonactivation and the activation tests. Some physiological effect was observed at the highest dose level (50 4g/plate). The lower dose levels were below toxic levels. The results obtained with the positive control materials demonstrate that the test systems are functional with known mutagens. The results of the tests conducted with T-1485, both in the absence and in the presence of the rat liver activation system, were all negative. It was concluded that T-1485 did not exhibit genetic activity in any of the in vitro assays employed in this investigation. Respectfully subrnitted, LNDUSTRJ-AL BIO-TEST LABORATORIES, INC. Report prepared by: --r I Gerald L. Kennedy, T(rJ-@, B. -E. Section Head, Toxicology Report approved by: ,3. L. Kepling,,.7i,Ph, Manac,er, Toxicolog trm 3 III. Procedure A. IndicatorMicroorganisms The followinCDg strains of indicator microorganisms were used in the investigation: 1. Yeast Strain: Saccharomyces cerevisiae, strain D4 2. Bacteria Strains: Salmonella typhimurium, strains TA-1535 TA-1537 TA-1538 TA-98 TA-100 B. Preparation of Liver Homogenate and 9,000 x g Cell Fraction The tissue homogenate and 9,000 x g cell fraction were prepared from the livers of adult male Sprague-Dawley rats. The animals were treated with 500 mg/kc, of Aroclor 1254 five days before kill. A sufficient number of animals to provide the necessary quantitiesof liver were killed by cranial blow, decapitated, and exsanguinated. The liver was immediately dissected from each animal using aseptic techniques and placed in ice-cold 0.25 M sucrose buffered with Tris at a pH of 7.4. Upon collectionof the desired quantity of livers, they were washed twice with fresh buffe.-ed sucrose and completely homogenized with a motor-driven homocenizing unit at 4C. The liver homoaeCD nate obtained from this step was centrifutcn ed for 20 minutes at 9,000 x a in a refrigerated centrifuge. The supernatant from the centrifuged sample was retained and frozen at -80' C. Samples from this preparation were used for the activationtests. B 10 -T 'z5@T 4 C. Reaction NExture The followinc,reaction mixture was employed in the activation tests: Component Final Concentration /rni 1. TPN (sodium salt) 2. Isocitricacid 3. Tris buffer, pH 7. 4 4. Mg Cl? 5. Liver hornogenate fraction equivalent to 25 mg of wet tissue 6 4rnoles 35 @Lmoles 28 p-moles 2 @imoles D. Plate Test (Overlay Method) Appro,,dmately 109 cells from a loc,phase culture of each indicator strain were added to test tubes containing 2.0 ml of molten acrar supplemented with biotin and a trace of histidine. For nonactivation tests, the 4 dose levels of the test material were added to the contents of the appropriate tubes and poured over the surfaces of selective agar plates. In activation tests the 9, 000 x - tissue supernatant and required cofactors (reaction mixture) were added to the overlay tubes. Four dose levels of the test chemical were added to the appropriate tubes, which were then mixed and the contents poured over the surface of a minimal agar (selective medium) plate and allowed to solidify. The plates were incubated for 48 to 7L'hours at 37 0C, and scored for the number of colonies growing on each plate. Positive and solvent controls were run with each assay. 5 E. Positive Control Chemicals Positive controls using both directly active positive chemicals and those that require metabolic activation were run with each assay. The following chemicals were used for positive controls in the nonactiva- tion and activation tests: Test Chemical", Solvent Probable Mutagenic Specificity .N-onactiva tion Methylnitros oguanidine (MNNG) 2-Nitrofluorene (NF) Quinacrine mustard (QM) Water or Saline BPSO@"' Dimethylsulf oxide-_',,--- FS4'- Water or Saline FS'@'-- -Activation 2-Anthramine (ANTH) 2 -Acetylaminofluorene (AAF) 8-Amino quinoline (AMQ) Dimethylnitrosamine (DMNA) Dimethyls ulfoxide BPS*,, Dimethylsulf oxide'-"---6@ F S @@4-- Dimethyls ulfoxide-, Saline FS** BPS-@* Concentrations -iven in Results Section BPS = Base-pair substitution FS = Frameshift Previously shown to be nonmuta,-,enic I h.-J a IV. Results A summary of the platetestresultsis presented in Table I. The test material was evaluated at a series of dose levels such that the highest level (50.@Lg/plate)showed some physiologicaleffect. The lower dose levels (0.25, 0. 5, and 5 4g/plate)were below toxiclevels. The results obtainedwith the positivecontrolmaterials demonstrate that the test systems are functionalwith known mutagens. The resultsof the tests conducted with T-1485 were allnegative. B 10 - T E 5 T -Z7 77