sample="quota" bates="2026009338" isource="pm" decade="1980" class="ui" date="19890325" CH2003 Neuchatel Switzerland 25.Mar.86 FTE/SSU COPY NO.: REPORT P 0268/2132 Mutagenicity of Mainstream and Sidestream Whole Smoke Condensate of Test Cigarettes SLOW-72 and SLOW-77 on Salmonella Typhimurium Strains TA98 and TA100 CH 2003 Neuchatel Switzerland 24.Mar.86 FTE/SSU COPY NO.: REPORT P 0268/2132 Mutagenicity of Mainstream and Sidestream Whole Smoke Condensate of Test Cigarettes SLOW-72 and SLOW-77 on Salmonella Typhimurium Strains TA98 and TA100 1 SUMMARY 1.1 Objective This study was designed to investigate the mutagenicity of MAINSTREAM and SIDESTREAM WHOLE SMOKE CONDENSATE collected with impaction traps (MWSC-I and SWSC-I) of 2 test cigarettes of the FTR project SLOW in the Salmonella typhimurium reverse mutation assay with meatbolic promutagen activation. Salmonella typhimurium strains TA98 and TA100 were used to detect frameshift mutagens and mutagens causing base-pair substitution respectively. 1.2 Cigarettes The cigarettes coded SLOW-72 and SLOW-77 are filter cigarettes belonging to the FTR project SLOW. SLOW-72 is the reference test cigarette (a.). 1.3 Experimental Mainstream and sidestream whole smokes of each test cigarette were generated simultaneously from approx. 300 cigarettes for each condensate preparation with 1 automatic INBIFO smoking machine under standard conditions. The condensates collected with glass impaction traps (WSC-I) were suspended in dimethyl sulfoxide. The mutagenicity of MWSC-I and SWSC-I of each test cigarette was assayed in the plate incorporation assay at the doses 0, 0.05, 0.10 and 0.15 milligrams dry condensate per plate following the (a) SLOW-77 (magnesium salt impregnated cigarette paper) cigarettes appearing to be "wet" after the conditioning contained approx. 10 times more acetic acid in the cigarette paper than not conditioned SLOW-77 cigarettes. INBIFO standard procedure. This means that with each tester strain 2 independent consecutive batches, 4 doses and 4 plates per dose in each substudy. A homogenate (S9 protein) from Aroclor 1254-induced rat liver was used for metabolic promutagen activation of WSC-I. Mutation events were detected in tester strain bacteria reverted from histidine auxotrophy to prototrophy by growth on histidine-deficient agar plates. The number of revertants was used to calculate the dose-response relationship. The specific mutagenicity was calculated from the dose-response curve as the extrapolated increase in the number of revertants per milligram dry condensate. The total mutagenicity was calculated from the specific mutagenicity and the dry condensate yield per cigarette was expressed as the total number of revertants induced by the dry condensate yield of 1 individual cigarette. 1.4 Results 1.4.1 Specific mutagenicity of mainstream whole smoke condensate The mutagenic activity of MWSC-I of both test cigarettes obtained in substudy 1 was statistically not different from that in substudy 2 with the exception of cigarette SLOW-72 with respect to base-pair substitution, which showed a relative difference greater than the limit of 0.25. The mean specific mutagenicity was: (a) For frameshift mutation as well as for base-pair substitution the specific mutagenicity of MWSC-I of cigarette SLOW-77 was numerically higher than that of the reference cigarette SLOW-72. In both cases the difference was statistically not significant but approached the borderline. The mean specific mutagenicity of MWSC-I of cigarette SLOW-72 which showed a significant difference between both substudied is shown in brackets and has to be taken with reservation. 1.4.2 Specific mutagenicity of sidestream whole smoke condensate The mutagenic activity of SWSC-I of both test cigarettes in substudy 1 was statistically not different from that in substudy 2. The mean specific mutagenicity was: (a) relative difference to the reference cigarette SLOW-72 .GT.0.16 (a) For frameshift mutation as well as for base-pair substitution the specific mutagenicty of SWSC-I of cigarette SLOW-77 was higher than that of the reference cigarette SLOW-72. In both cases the difference was statistically significant. 1.4.3 Total mutagenicity of mainstream and sidestream whole smoke condensate For MWSC-I the total mutagenicity was found to be practically the same for both test cigarettes with respect to frameshift mutation as well as to base-pair substitution. The dry condensate yield was approx. 15 percent lower for cigarette SLOW-77. The total mutagenicity of MWSC-I of cigarette SLOW-72 which showed a significant difference between both substudies with respect to base-pair substitution is shown in brackets and has to be taken with reservation. (a) relative difference to the reference cigarette SLOW-72 .GT.0.16 For SWSC-I, the total mutagenicity of cigarette SLOW-72 was found to be higher than that of cigarette SLOW-77 which showed an approx. 45 percent lower yield of dry condensate. With respect to base-pair substitution this difference was statistically significant: (a) 1.4.4 Comment Based upon the specific and total mutagenicity of their condensates, the cigarettes SLOW-72 and SLOW-77 are considered to be equal with regard to MWSC-I. With regard to SWSC-I the specific mutagenicity of SLOW-72 is considered to be lower, but the total mutagenicity of SLOW-72 is considered to be higher than that of SLOW-77. Elution solvents: solvent 1: 10mmol/l sodium dihydrogen-phosphate in water, adjusted to pH 2.8 with phosphoric acid solvent 2: methanol Elution: see TABLE A Flow rate: 1.5 ml/min Column switching: see Table A Oven temperature: ambient Wavelength: 280 mn Injection volume: 100 ul Time between the analyses: 3 min Computation: calibration by internal standard method mixing 1 ml standard solution with 4 ml internal standard solution and 3-fold analysis of the mixture, evaluation of the peak area ration by the integrator The 1st analysis in a series has to be cancelled. Scientific version: SOP AC 59/3 Text version: 30.Jan.86 5.2.4.4 Bacteriological examination of WSC-I/DMSO suspension Principle: determination of bacterial contamination of test substance assayed for mutagenicity in the plate incorporation assay detection limited to aerobic bacteria growing minimal-glucose agar plates Time: at the day of mutagenicity assay Sample material and quantity: WSC-I/DMSO suspension, highest dose/plate, 1 WSC-I batch of each test cigarette, condensate type and substudy Results expressed in: CFU/plate Equipment: incubator: no. 3916, Forma Scientific, cia Labotect, D-3400 Göttingen petri dishes: no. 1029, 100 mm x 15 mm, polystyrene, sterilized, Falcon, via Becton Dickinson, GmbH, D-6900 Heidelberg 1 colony counter: Colony Star 2, Funke-Gerber, D-1000 Berlin Chemicals: top agar and test substance mixed by rotation and poured on minimal-glucose agar 2 plates/sample incubation of plates at 37 degrees centigrade, manual counting of colonies after 2 d of incubation Scientific version: SOP MB 44/1 Text version: 4.Dec.85 5.3 Dosing of Test Substance Principle: dilution of test substance stock solution with DMSO to the final concentration used in the study. Time: at the day of mutagenicity assay Sample material and quantity: 4 WSC-I suspension batches/test cigarette Equipment: brown glass vials, 8 ml, no. 224814, screw caps, no. 240409, Wheaton Scientific via Zinsser, D-6000 Frankfurt/Main Chemicals: DMSO, no. 2950, E. Merck, D-6100 Darmstadt 1 Procedure Preparation of application suspension: see Tables B 2 application suspensions/condensate batch Storage: in dark airtight vials at RT Dosing: see Table C Scientific version: SOP 43/2 Text version: 17.Dec.85 Metabolic Promutagen Activation System (S9) 5.4.1 Animals Species: albino rat (Rattus norwegicus) Strain and designation: Spraque Dawley CRL:CD(SD)BR Color: white Type of breeding: outbred Sex: male Microbiological conditions of breeding: SPF until delivery Breeder: Charles River Wiga GmbH, D-8741 Sulzfeld 1 Transport containers: special filter crates INBIFO animal supply no.: 531 INBIFO animal study approval no.: 191 Number of animals Arrived: 21 Applied: 20 Date of shipment: 9.Nov.84 Body weight (grams) At order: 180 1 day after arrival: 176.0 ± 2.2 At administration 209.7 ±2.0 On sacrifice: 201.4 ± 2.9 Age of animals (days) On arrival: 44 ± 1 At administration 49 ± 1 On sacrifice: 54 ± 1 Acclimation period (days): 5 Text version: 27.Jun.85 5.4.2 Animal housing Animal room: INBIFO main laboratory building, area c , rooms R310 and R312 Construction and interior: windowless floors, walls and ceilings coated with epoxy resins Microbiological conditions: conventional under defined laboratory conditions, rooms disinfected with 30 ml/l Kohrsolin prior to introduction of rats Conditioning and ventilation: 100 0/0 fresh air, delivered from a 50 m high air inlet stack, approx. 15 changes/h mean ± SE area C only used for acute studies with small laboratory rodents filter: fine filter class C Room temperature (degrees centigrade): 22 ± 2 Relative humidity (0/0): 55 ± 10 Light Time cycle: L/D 12 : 12, L 06.00 to 18.00 standard time Source: "daylight" fluorescent lamps: Lumilux W11, Osram GmbH, D-8000 München 1 Intensity in cages: approx. 20 to 100 Lux Cages: type 3, poycarbonate (Makrolon), base area: 39 cm x 23 cm, height: 15 cm Cage lids: stainless steel, wire mesh with overhead hoppers Change of cages: 3 times/week Litter: wire grid, autoclaved granulated dust free wood underneath each batch screened for pesticides, PCB residues, aflatoxins and heavy metals (arsenic, cadmium, lead, mercury) and used only when the tolerance levels given by the German Futtermittelverordnung (Jun.76) not exceeded sterilization: 15 min at 134 degrees centigrade, 2.5E5 Pa(equiv. to 2.4 kg/cm2) At outside temperatures above 28 degrees centigrade and at extremely high relative humidity, these specifications may not always be maintained replacement of bedding material: 3 times/week (Mo., We., Fr.) rats set on double wire grids when diet removed Number of animals per cage: 2 rats, except 1 cage with 1 rat 8.1.4.2 Reversion assay 180 out of 180 (100 percent) colonies of strain TA98 or of strain TA100 from mutagenicity assay plates, to which the highest dose of MWSC-I or SWSC-I has been added, were found to be histidine prototrophs (revertants) in the subsequent reversion assay on plates without histidine (a) (see TABLES 11). 8.1.4.3 Reproducibility The mutagenic activity of WSC-I of the cigarettes obtained in substudy 1 was statistically not different from that obtained in substudy 2 with the exception of MWSC-I of cigarette SLOW-72 for base-pair substitution. The mutagenicity of MWSC-I of this cigarette showed a relative difference greater than the limit of 0.25 between both substudies (see TABLES 12 to 15). (a) A trace amount of histidine was added tot he top agar in the plate incorporation assay to allow the bacteria on the plate to undergo several divisions which are in many cases necessary for mutagenesis to occur. In case of massive cell death during exposure of the bacteria to a test substance more histidine is available to the individual surviving bacteria, and they undergo more cell divisions forming small colonies ("false revertants") which can be mistaken for revertants (Ames et. al., 1975).a The specific mutagenicity of MWSC-I and SWSC-I of the standard reference cigarette 2R1 was found to be in the expected range for inducting frameshift mutation as well as base-pair substitution when compared with all previous INBIFO data obtained with 2RI-WSC-I. 8.1.4.4 Specific mutagenicity of mainstream whole smoke condensate The mean activity of MWSC-I to induce frameshift mutation in strain TA98 was found to be 1752 revertants per milligram dry condensate for the cigarette SLOW-72 and 2046 for SLOW-77 (see TABLE 16.1 and FIGURE 3). The mutagenicity of MWSC-I of SLOW-77 was higher than that of the reference cigarette SLOW-72. The difference was statistically not significant but approached the borderline (see TABLE 18). The mean activity of MWSC-I to induce base-pair substitution in strain TA-100 was found to be 1060 revertants per milligram dry condensate for the cigarette SLOW-72 and 1250 for SLOW-77 (See TABLE 16.2 and FIGURE 3). The mutagenicity of MWSC-I of SLOW-77 was higher than that of the reference cigarette SLOW-72. Due to the difference between substudies 1 and 2, the evaluation of MWSC-I of cigarette SLOW-72 has to be taken with reservation (see TABLE 18). 8.1.4.5 Specific mutagenicity of sidestream whole smoke condensate The mean activity of SWSC-I to induce frameshift mutation in strain TA98 was found to be 1367 revertants per milligram dry condensate for the cigarette SLOW-72 and 2156 for SLOW-77 (see TABLE 17.1 and FIGURE 4). The mutagenicity of SWSC-I of SLOW-77 was higher than that of the reference cigarette SLOW-72 and this difference was statistically significant (see TABLE 18 and FIGURE 4). The mean activity of SWSC-I to induce base-pair substitution in strain TA100 was found to be 1312 revertants per milligram dry condensate for the cigarettee SLOW-72 and 2043 for SLOW-77 (see TABLE 17.2 and FIGURE 4). The mutagenicity of SWSC-I of SLOW-77 was higher than that of the reference cigarette SLOW-72 and this difference was statistically significant (see TABLE 18 and FIGURE 4). 8.1.4.6 Total mutagenicity of mainstream and sidestream whole smoke condensate For MWSC-I, the total mutagenicity was found to be practically the same for both test cigarettes with respect to frameshift mutation as well as to base-pair substitution (see TABLES 19 and 21). The dry condensate yield was approx. 15 percent lower for cigarette SLOW-77. Due to the difference between substudies 1 and 2 with respect to base-pair substitution, the evaluation of MWSC-I of cigarette SLOW-72 has to be taken with reservation. For SWSC-I, the total mutagenicity of cigarette SLOW-72 was found to be higher than that of cigarette SLOW-77 which showed an approx. 45 percent lower yield of dry condensate (see TABLE 20). With respect to frameshift mutation the difference approached the significance limit, and with respect to base-pair substitution the difference was statistically significant (see TABLE 21). 8.1.5 Comment Based upon the specific and total mutagenicity of their condensates, the cigarettes SLOW-72 and SLOW-77 are considered to be equal with regard to MWSC-I. With regard to SWSC-I the specific mutagenicity of SLOW-72 is considered to be lower, but the total mutagenicity of SLOW-72 is considered to be higher than that of SLOW-77.