Document x1p37J6G8YkgmGqp8rpzDbLgE

IN VITRO MICROBIOLOGICAL @FLTTAGENICITY ASSAYS OF THREE 3M COMPANY COMPOUNDS Final Report April 1978 By: Vincent F. Simmon, Ph.D., Manager Microbial Genetics Program Gregory F. Shepherd, @ficrobiologist Prepared for: 3M CO@IPANY Medical Department General Offices 3M Center St. Paul,, Minnesota 55101 Attention: J. E. Long, Sc.D. Manager, Toxicology Services SRI Project LSC 4442-16 Approved: ir, Gordon W. New@ll, 6irector Department of Toxicology W. A. Skinner, Executive Director Life Sciences Division International 333RavenswoodAve.- MenloPark,Californ9i4a025 (4153)26-6200-Cable:STANRES, MenloPark-TWX: 910-373-,246 SLMIARY SRI International examined 3M Company compounds T-2lj6 CoC, T-2138 IM, and T-2140 I@l for mutagenic activity with strains TA1535, TA1537, TA1538, TA98, and TA100 of the bacterium Salmonella typhimurium in the standard Ames Salmonella/microsome assay and with the yeast Saccharomvces cerevisiae D3. Each assay was performed in the presence and in the absence of a metabolic activation system. None of the compounds was mutagenic in either the Salmonella/microsome assay or the S. cerevisiae assay. INTRODUCTION SRI International examined compounds T-2136 CoC, T-2138 IM, and T-2140 IM for mutagenicity by in vitro microbiological assays with Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 and with the yeast Saccharomyces cerevisiae D3. An Aroclor 1254-stimulated, rat liver homogenate metabolic activation system was included in the assay procedures to provide metabolic steps that the bacteria either are incapable of conducting or do not carry out under the assay conditions. The purpose of this study was to determine whether these compounds elicited a mutagenic response in microorganisms. The assay procedure with S. typhimuri-um has proven to be 80 to 90% reliable in detecting carcinogens as mutagens, and it has about the same reliability in identifying chemicals that are not carcinogenic. The assay procedure with S. cerevisiae is about 60% reliable in detecting carcinogens as agents that increase mitotic recombination. 2 The combination of the two assay procedures-significantly enhances the probability of detecting potentially hazardous chemicals. However, because the systems do not always provide 100% correlation with the results of carcinogenicity investigations in animals, neither a positive nor a negative response proves conclusively that a chemical is hazardous or nonhazardous to man. 2 .METHODS Salmonella tvvhi-Muri= Strains TA'L535, TA1537, TA1538, TA98, and TA100 The Salmonella _typhimurium strains used at SRI are all histidine auxotrophs by virtue of mutations in the histidine operon. Wien these histidine-dedendent cells are grown on minimal medium petri plates containing a trace of histidine, only those cells that revert to histidine independence (his+) are able to form colonies. The small amount of histidine allows all the plated bacteria to underaao a few divisions; in many cases, this growth is essential for mutagenesis to + occur. The his revertants are easily scored as colonies against the slight background growth. The spontaneous mutation frequenc-,7of each strain is relatively constant, but when a mutagen is added to the agar the mutation frequency is increased 2- to 100-fold. We obtained our S. tvphimurium strains-from Dr. Bruce Ames of the University of California at Berkeley. 1-5 In addition to having mutations in the hist4@d4-neoperon, all the indicator strains have a mutation (r-4a that leads to a defective lipopolysaccharide coat; they also have a deletion that covers ,e-iesinvolved in the synthesis of vitamin biotin (bio and in the repair of ultraviolet (uv)-induced D@':TdAamacre (uvrB-). The r-Fa-mutation makes the strains more oer.-,ieable co manv lar,,e aromatic molecules, thereby increasing the mutagenic effect of these molecules. The uvrb mutation decreases repair of some types of chemically or physically damaged DNA and thereby enhances the strains' sensitivity to some -nutagenic agents. Strain 'LA1335 is + reverted to his by many mutagens that cause base-pair substitutions. TAICO is derived from TA1535 by the introduction cf the resistance transfer factor plasmid pEZ1101. 'Lhis plasmid is believed to cause an increase in error-prone DNA repair that leads to --any more -utat4-ons 3 for a given dose of most mutagens-3 In addition, plasmid pRI101 confers resistance to the antibiotic ampicillin, which is a convenient marker to detect the presence of the plasmid in the cells. The presence of this plasmid also makes strain T.AIOO sensitive to some frameshift mutagens (e.g., ICR-191, benzo(a)pyrene, aflatoxin Bi, and 7,12dimethylbenz(a)anthracene). Strains T.4,1537and TA1538 are rezerted by many frameshift mutagens. TA1537 is more sensitive than -TAI-538to mutation by some acridines and benzanthrace-.ies,but the difference is quantitative rather than qualitative. Strain 7-A98is derived from TA1538 by the addition of the plasmid pIM01, which makes it more sensitive to some mutagenic agents. All indicator strains are kept at 4* C on minimal med4-= plates, supplemented with a trace of biotin, and an excess of histidine. The plates with the Dlasmid-carry-:-ng st=ains contain in addition a=icillin (25 @Lg/mi", to ensure stable maintenance of the plasmid piL'4-101. New stock culture plates are made every two months from single colony reisolates that were checked for their genotypic characteristics (his, r-4a.,uvrb, bio) and for the presence of the plasmid. Fo.each e.%-Deriment,an inocul= from the stock culture plates is grown overni,,-htat 37' C in nutrient broth (Oxoid 0167). A-40terstat4a-onarover-.iL.ghgtrowth, the cultures are shaken for 3 to 4 hours to ensure optimal growth. Arocior 1254-Stimulated Metabolic Activation Svstem Some carcinogenic chemicals, either of the aromatic am--'-.ti-o,@--opre polycvclic hydrocarbon t-@,pe,are L-iactive unless they are metabol4---ed to active forms. in animals and man, an enzyme system in the i-Lveror other organs (e.a., lung or kidney) is capable of metabolizing a lar,,e number c-6@these c-liem-i-catlos ca-rci.-,ogens."6-1 Some of these 4-.-.te=ed4-ate =etabol--;tesare very potent mutagens in the S. ty-phimuri= test. L-.es has described the liver metabolic activation system that we USe.6 In brief, adult male rats (250 to 300 g) are gi-7ena single 500-m,-,/k'T intraperitoneal inject4@on of a polychlor4-nated biphenyl, %--ocior 11-54. This treatment enhances the synthesis of enzymes involved ;--.ti*,-.meetaboljic 4 conversion of chemicals. Fourdays after the injection the animals' food is removed but drinking water is provided ad libitum. On the fifth day, the rats are killed, and the liver homogenate is prepared as follows. The livers are removed aseptically and placed in a preweiahed sterile glass beaker. The organ weight is determined, and all subsequent operations are conducted in an ice bath. The livers are washed in an equal volume of cold, sterile 0.15 M KCI (1 ml/g of wet organ), minced with sterile surgical scissors in three volumes of 0.15 X KC1, and homogenized with a Potter-Elvehjem apparatus. The ho=genate is centrifuged for 10 minutes at 9000 x R, and the supernatant, refer-red to as the S-9 fractio-.i, is quickly frozen in dry 4-ceand stored at -800 C. The metabolic activation mixture for each experiment consists of, Igor10 mi: * 1.00 ml of S-9 Fraction * 0.20 =Ll Of MgCl2 (0.4 M) and KC1 (1-65M) * 0.05 ml of gluco@;e-6-phosphate (1 M) * 0.40 ml of INADP (0.1 M) * 5.00 ml of sodium phosphate (0.2 M, pH 7-4) * 3.35 ml of H20. 5 Assays in Agar To a sterile 13 x 100 mm test tube placed in a 43* Cheating block, we add in the following order: (1) 2.00 ml of 0.6% agar* (2) 0.05 ml of indicator organisms (3) 0.05 ml of a solution of the test chemical (4) 0.50 ml of metabolic activation mixture (optional). For negative controls, we use steps (1), (2), and (4) (optional) and 0.05 ml of the solvent used for the test chemical. Because the ma.Jor4ty of organic compounds are not sufficiently water soluble-particularly at the higher concentrations-we routinely use dimethylsulfoxide (DMSO). Other solvents that are occasionally used are water, ethanol, or benzene. For positive controls, we test each culture by specific mutagens known to revert each stra4Ln using steps (1), (2),'(3),and (4) (optional). This mixture is stirred gently and then poured onto minimal agar .4. plates.' @-fterthe top agar has set, the plates are incubated at 37*C for 2 days. + 1-he number of his revertant-colonies is counted and recorded. 0. 6 17.agar contains 0 .05 mM hist4-di--,,ie0.05 mLNfbiotin, and 0. 1 *1 'TaCl. Minimal agar plates consist of, per liter, 15 g of agar, 10 g of glucose, 0.2 g of NfgSO4-7H20, 2 g of citric acid monohydrate, 10 g of K2HP04, and 3.5 g of NalWH4PC4-4H20. 6 Saccharomyces cerevisiae D3 The yeast S. cerevisiae D3 is a diploid microoraani-sm heteroz7gOus for a mutation leading to a defective enzyme in the adenine-metabolizing pathway.9 When grown on medium containing adenine, cells homozygous for this mutation produce a red pigment. These homozygous mutants can be generated from the heterozygotes by mitotic recombination. The 4:requency of this recombinational event mav be increasedby incubating the oro-anisms with various mutagens. The degree or mutao,-enicitvor-a compound or of its metabolite is determined from the number c,L*redpigmented colonies appearing on the plates. " The S. cerevisiae tester strain is stored at -80* C. For each experiment, the tester strain is inoculated in l,"ltryptone and 0-5/', yeast extract and grown overnight at 37' C with aeration. The in vitro yeast mitotic recombination assay in susdensicn is conducted as follows. The overnight culture is centrifuged, and the cells are resuspended"at a concentration of 10a cells ml/in a 67 mm phosphate buffer (pH 7.4). To a sterile test tube are added: 0 1.30 ml- of the or,)-anisms 0 0.50 al of either the metabolic activation mixture or buffer 0.20 -=I of the test chemical. Because many or-,anic chemicals are not appreciably water soluble, dimethylsul,,@@ox-4-d(eDMSO) is used routinely as the solvent for the test chemical. Other solvents that are used occasionally are ethanol, benzene, or water. Several doses of the chemical (up to 5%, w/,.ror v/v) are tested in each experiment, and appropriate controls are ircluded. The susdersion mixture is incubated at 300 C for 4 hours on a roller drum. Tae sample is diluted serially in sterile t)h7s4co"c,4cal saline, and a volume of 0.2 ml of the 10-5 and 10-3 d-;-7-ut4-oniss spread on ti7ptone-yeast agar plates; five plates are used for the 10-3 dilution and three plates are used for the 10-5 dilut4-on. '@-"%e plates are i-rcibated for 2 days at 30* C, followed by 2 days at 460C to enhance the development of the red pigment indicative of aden4-ne- 7 deficient homozygosity. Plates of the 10-3 dilution are scanned with a dissecting microscope at 10 X magnificatioa, and the number of red colonies or red sectors (mitotic recombinants) is recorded. The survivina fraction of organisms is determined from the number of colonies appearing on the plates of the 10-5 dilution. The number of mitotic recombinants is calculated per 10" survivors. A positive response in this assay is indicated by a dose-related increase of more than threefold in the absolute number of mitotic recombinants per milliliter as well as in the relative number of mitotic recombinants per 103 survivors. 8 RESULTS AND DISCUSSION Tables 1 through 3 present the results of testing T-2138 IM, and T-2140 IM in the Ames Salmonella/microsome with the yeast S. cerevisiae D3. T-2136 CoC, assay and T-2136 CoC was dissolved in water. T-2138 IM and T-2140 IM were extracted in DMSO overnight; various volumes of the extract were used in the bacterial assay procedure and made up to 100 ul with DMSO (Tables 2 and 3). The negative control used for these two samples was 1001;1 of DMSO. When known mutagenic compounds were added to the top agar of the positive control plates, the number of mutants above the background count was increased. When various concentrations of T-2138 IM, or T-21'.0 I@lwere added to the top agar, no dose-related increase in the number of mutants over the background count was observed, either with or without metabolic activation. These results lead to the conclusion that T-2136 CoC, T-2138 IM, and T-2140 IM are not mutagenic in the Salmonella/microsome assay. Tables 4 and 5 present the results of testing T-2136 CoC with S. cerevisiae D3. At various concentrations ranging from 0.1% to 5%, this compound did not cause a reproducible, dose-related increase in the number of mitotic recombinants. Therefore, we conclude that this compound is not mutagenic in S. cerevisiae D3. This assay was not conducted on T-2138 IM or T-2140 IM because onlv limited quant4-ties 0,1_: these compounds were available. 9 IN VITRO ASSAYS Table 1 WITII SALMONELLA T-2136 CoC TYPI]IMURIUM Compound Negative control (11,,0) Positive controls Sodiuto azide 9-Aminoacridine 2-Nitrofluorene 0 2-Anthramine T-2136 CoC @letabolic Activation + @ticrograms of Compound Added per Plate 50&000 501060 Average Hiatid@ne )Rev TA1535 TA1537 TA153 31 7 23 9 0,5 279 50 623 5 84 2,5 25 5 + 2,5 236 185 1,0 1 + 1,0 37 10 50 100 500 1000 5000 + 10 + 50 + 100 + 500 + 1000 + 5000 27 6 31 5 1 31 7 1 31 8 1. 28 6 1 22 8 1 21 9 1 27 6 1 26 8 1 27 8 1 18 7 2 25 9 2 IN VITIZO ASSAYS Table 2 WITII SAI,@IONEI,I,ATYPIIIMURIU@L T-2li8 IM Compound Negative control (D@ISO) Positive controls Sodium azide 9-Aminoacridine 2-Nitrofluorene 2-Anthramine T-2138 im Metabolic Activation Amount of Compound Added per Plate 100 PI + 100 0.5 pg 50 5 2.5 + 2.5 1.0 + 1.0 - 2 - 5 - 10 - 25 - 50 - 100 + 2 + 5 + la + 25 + 50 + 100 Average of Lliree experiments. Av rage Histidine Reve YA-1535 TA1537 TA15 21 4 1 17 6 21 322 654 16 8 285 194 22 5 21 4 26* 7 26* 6 29* 5 30* 4 12 5 17 6 12 4 19* 8 23* 5 25* 7 1197 17 780 11 11 13 14 15 10 21 15 28 21 17 26 Couipoun(i Negative control (DMSO) Positive controls Sodium azide 9-Aminoacridine 2-Nitrofluorene 2-Anthramine T-2140 IM IN VITRO ASSAYS Table 3 WITII SALMONELLA T-2140 III TYPIII14URIUM @letabolic Activation + Amount of Compound Added Per Plate 100 pi 100 0.5 50 5 2.5 + 2.5 1.0 + 1.0 - 2 - 5 - 10 - 25 - 50 - 100 + 2 + 5 + 10 + 25 + 50 + 100 Average Histidine Rev TA1535 TA1537 TA15 18 4 16 6 322 654 16 8 285 194 71 14 6 16 5 1 17 8 1 19 4 1 15 4 1 15 5 1 17 6 2 13 9 1 16 7 1 11 5 1 14 6 1 12 6 1 Compound Negativecontrol (tl2o) Positive control 1,2,3,4-Diepoxybutane T-2136 COC Table 4 IN VITRO ASSAYS WITI] SACCILAROHYCES T-2136 CoC Experiment I CEREVISIAE Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells per mi (x 10-')--Percent Mitotic Rec Per ml (x io-3) 7.9 100 5.0 + 7.4 100 5.0 0.025 5.9 75 880.0 + 0.025 5.0 68 807.5 0.1 6.8 86 6.0 0.5 7.0 89 2,0 1.0 6.8 86 4.0 5.0 5.7 72 5.0 + 0.1 6.7 91 9,0 + 0,.5 8.3 112 3.0 + 1.0 6.5 88 3.0 + 5.0 5.8 78 5.0 Comoound Negativceontrol(H20) Positive control l92p3,4-Diepoxybutane T-2136 CoC Table 5 IN VITRO ASSAYS WITH SACCILAROMYCES T-2136 EoC Experiment 2 CEREVISIAE Metabolic Activation Percent Concentration (w/v or /-@V /V) Survivors Cells per mi (x 10-') Percent Mitotic Ree Per ml (x 1.0-3) 6.8 100 9.0 + 9.6 100 5.0 0.025 6.9 101 677.0 + 0.025 6.5 68 605.0 - 1.0 7.0 103 2.0 - 2.0 6.9 101 810 - 4.o 10.0 147 7.0 - 5.0 8.5 125 3.0 + 1,0 10,8 113 5.0 + 2.0 8.8 92 2.0 + 4.o 9.5 99 7.0 + 5.0 10.6 110 7.0 R.EFERE.XCES 1. J. McCann, E. Choi, E. Yamasaki, and B. N. Ames. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA, 72, 31355139 (1975). 2. B. N. Ames, E. G. Gur-,ieyJ,. A. Xiller, and H. Bartsch. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylam.,@nofluorenaend other aromatic amine carcinogens. 1.1rocN.at. Acad. Sci. USA, 69, 31283132 (1972). 3. B. N. Ames, F. D. Lee, and W. E. Durston. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nat. Acad. Sci. USA, 70, 782-.786 (1973). 4. B. N. Ames, W. E.*Durston, E. Y;;masaki,and F. D. Lee. Carcinogens are mutagens: A simple test system combinina liver homagenates for activation and bacteria for detection. 0Proc. @.Tat.Acad. Sci. USA, 70, 2281-2285 (1973). 5. J. McCann, N. E. Spingarn. J. Kobori, and B. N. Ames. Detection of carcinogens as mucagens: 3acteria7-cester strains with R factor plasmids. Proc. Nat. Acad. Sci. USA, 7-77 979-983 (1975) . 6. L. A. Poir4-erand V. F. Si=on. lfutaaenic-caricnogenic relationships and the role of mucagen37 .cscreening tests for carcinogenicity. Clin. Toc:Lcol , 9 (5), 761-7/"-(1976)- 7. L. D. Kier, E. Yamasaki, and B. N. Ames. Detection of mucagenic activity in cigarette smoke condensates. Proc. -Nat. Acad. Sc-i-. USA, 71-P4139-'4163 (1974). 8. B. LN. kmes, J. McCann, and E. Yamsaki. Methods for detecting carcinogens and mutagens with the Sai@monella/.ma=alian-raicrosome mutagenicit7 test. Mutation Res., 31, 347-364 (1975). 9. F. K. Zi=e--mann and R. Schwaier. Induction of mitotic gene conversion with nitrous acid, 1-methyl-3-nitro-lnitrosoguanidine and other alkylat4-n-agents 4-nSaccharomvces cereiiisiae. 4ol. Gen. Genet., loo, 63-69 (1967). 15 10. D. J. Brusick and V. W. Mayer. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspectives, 6, 83-96 (1973). 16 SRI