All ,36r IMPERIAL TOBACCO LIHITEO RESEARCH LABORG~ORY HOH'FREAL The Detection of Mutagenic Actfvlty of Chemi- cals and Tobacco Smoke in a Bacterial Systa. Research laboratory Report No. 150 BATCo document for Legal SeNices : Health Canada 20 May 1999 Re~eareh Laboratory Report Nd. ~ Copy No. 13 IMPERIAL TOBACCD IIMITED RESEARCH lABORAmRY MONtREAL ME O~E[TIOI OF MUTAGENIC ACTIVIn bF CHEMICALS AIO TOBACCO SMOKE II A BACTERIAI S1SfM F""D t;CE[~ r.l""'' ~'f I . AUTHOR: M,H, Bllfmorlii ~I Project No,: T·7708 j ISSUED BY: Or, T,d, Smith DATE ISSUED: Deeember 23, ~97~, DISIRIBUTID~ i. Iibrary 13, Or, 0,6, Felton 2, Mr, R,M, Glbb 14, Dr, F, Haslam 3~ Mr.R,S,Wade 15, Or,l.W,Hughcs a. OT, T.I, Smith 16. Or, 9.A, Sanlord 5, Mr, S,M, Candllsh 17, Or, R.A, Sanford 6, Dr, M,H. Bfllmorla 1B, Mr, R,G, Nicholls 7, Or, S,J, Green 19, Mr, R,G, Nlcholls 6, Or, S,J, Green 20~ Or, P,J, Dunn 9. Dr. S,J, Green 21, Mr, A,F, El Naggar 10, Dr,S.J, Green 22, Mr, E,R. Freicsleben 11, Dr, 6,3, Green 23, Ms, 3, Johnson 12, Or, D,G. Felton 21~ Mr, R,1, Rice 25. Mr, M,P, Scherbak BA~Co document lor Legal S,nksl: Health Canada ii raY 119 The Detection of Yulageni: Activity of Chenicalr and Tobacco Smoke in a Bacterial System SUMMARY The bacterial test described by Rosenkranr's group (Cancer Res,, 31 (7), 970-13, 1971) and involving use of an Escherichia coli strain deficient in DNA polymerase, was used for the detection of mutagenic activity of tobacco smoke as well as of some lure com· pounds known to occur therein, The original method as described by the authors was found suitable for detecting mutagenic activity of pure chemicals, but failed to detect mutagenic activity of tobacco smoke, However a modified precedure, involving pre·ineuba· tion of tobacco smoke solutions with the bacterial cultures, enabled detection of mutagenic activity in cigarette, cigar and pipe smokes. The ph of the pre-incubation mixture markedly influenced the mu· tagenicity of the tobacco smoke condensate, which was more active at all:aline than at neutral pH. The bases nicotine, pyridine and pyrollidine were weakly mutagenic compared to the smoke condensate, and showed activity at pH 9.0 but not at neutral pH in the con- centrations tested. Cyanide, hydroxylamine formaldehyde, acrolein, hydroquinone and pyrogalloi were found to be mutagenic in this system, while caffeine did not inhibit growth of either the mutant or the parent culture, Metabolic activation using liver microsomal enzymes was not attempted. it is felt:hat this procedure ;nerits further investigation for routine monitoring of tobacco product modifications. BATCo document for Legal Services : Health Canada 20 May 1999 IBTROOUCTION Recent sears have seen a renewed interest in mutagenicity tests in mierobtlanisns as reliable predictors of carcinooenicity in mammals 11·3). The availability of such quick pre-screening procedures would take a considerable load off animal testing, and prove of great :,dlue to the chemical, food and drug industries. The tobacco indust~, too, would gain a valuable tool, both for studying product modification as well as for tracing mutagens and possible carcinogens in:ne comolex mixture that is tobacco smoke. Two recently described bacterial systems, described in the literature for :aDid detection of mutagenic and possible carcinogenic activity, are being used in this laboratory. These are (i) the method describei, by Ames and his group (4-6) employing histidine auxotraphr of Salmonella typhimurium, in which reversions to prO- totrophy are Scored, and (ii) the method described by Rosenkranz's group (7,8) emcldying a ONA polymerase deficient mutant of Escherichia eoli [Pol A') and its parent culture (Pol A'). In the latter, a greater inhibition of the mutant compared to the parent culture is used as a criterion for mutagenicity. This procendure has been used extensivei? in this laboratory to find out if tobacco smoke, as well as some ccrpounds known to occur in it, possess mutagenic activity. T~iS St~dyl and the results obtained, form the subject matter of this report, BATCb document for Legal Services : Health Canada 20 May 1999 MATERIALS ANO MEMOOS · The media eloloyed~'~at the maintenance of bacterial strains as well as for performing the tests by the dise and pre-inc~bllti0n procedures, have been adequately described by Rosenkran2 and his co-workers (1,9)l The procedure employing discs was essentially similar to the: described by Slater et i, (7), while the procedure involving pre-incubation of cultotes with the test substance was similar to that used by Garner etpl. (10) in studying the tpxicity of aflatoxin B to Sal~oneila ty~hirnuriul, as well Is to that des- cribed by Rosenkranz (li) for studying the effect of dichlorvos jlapona) on E.coli mutant deficient in ONA pol~nerase. For preparation of smoke condensates, comnercialiy available cigarettes, cigarillos, and cigars were smoked under standard ton- ditions originally selected to simulate human e'garette smoking (121, using 35 mi puffs, of 2·second duration, taken every minute using a suitable smoking n~ehine, in the care of pipe smoking, a 35 mi of 2·Eecond duration was Collected every 10 seconds, and the tobaccos were snaked in O gm quantities in stainless steel smoking pipes and employing a pipe tobacco smoking machine designed in this laboratory. She smoke was trapped in Spiral condensers coaled to ·fOOC and the condensate was dissolved out using aliquots of acetone~ The acetone Has evaporated off and the condensate dried to constant weight on a rotar:, evaporator, weighed and re·dirsolved in suitable amounts of acetone~ BATCo document for Legal Services : Health Canada 20 May 1999 RESULTS AND DISCUSSION That the plate method employing filter paper discs is suitable for pure compounds is shown in Table I. Chemicals such as ampicillin and chioromycetin, reported to be negative by Rosenkrant j7,13), show equal inhibition of both strains, while crystal violet, methylmethanesul phonate, reported to be positive, show a greater inhibition of tte ~oi A. Strain than the parent c;lture. From the taale,'lt wjjl b,,,,, that acrolein, which is known to occur in tobacco smoke, gave positive results. While the mutagenic effect of acrolein is known (1,14), these results suggest that this compound be re·evaluated in carcinogenic tests, bearing in mind its high reactivity, Caffeine, as will be seen from the table, did not inhibit either strain, although i: has been reported to give positive results in this system (2), Com- pounds such as hydroquinone, pyrogalloi, which are dvtwxidi~able, and potassium cyanide and hydroxylamine, which are unstable in presence of air, C02 or moisture, were also examined and these gave variable results. These have been examined by the pre·incu· batton procedure, the results of which will follow, Our main interest In this test lay in applying it to tobacco smoke condensates. It is here that the disc procedure failed to work in our hands, even though the method has been applied to eig· arette sroke condensates by Rosenkranz (13, personal camrmnilation). The procedure initially gave highly variable and inconsistent results, and we thought this to be due to changes in the poi A strain. Consequently, we obtained fresh cu:tvres through the kind courtesy of professor Rosenkranl, as well as from another laboratory, These cultures were maintained as frozen permanents in glycerol. Examina:ion of cigarette smoke now gave consistent results and showed cigarette smoke to be negative as will be seen from Table II, BATCo document for legal Services : Health Canada 20 May 1999 Fu~her, the exalination Of condensat;s from cigarettes, cigars and cigarillos as well as from pipe smoking also gave negative results as shown in Table III. Since positive results have been reported for cigarette smoke by Rosenkranz and Anderson (13), and since the nature of tobacco smoke employed by these workers was unknown to us, we prepared smoke fractions by the procedures of Elmenhorst and Grinm~er (15) and Swain et al~ (15) and examined them for mutagenic activity by the disc procedure. Once again negative results were obtained. The negative results obtained with whole smoke as well as with smoke fractions are not surprising in view of the water·insolubie nature of these materials. Along with lack of diffusion of most of the smoke material, it would appear that pemeability into the bacterial Cells was also a problem. We now employ a procedure involving pre·incubation of suspensions of the bacterial strains with acetone-containing solutions of total condensates or with fractions obtained therefrom, Employing such a pre·ineubation step, we have now been able to show that tobacco smoke condensates obtained from cigarettes, cigars and pipe·sroking are distinctly mutagenic in the E~o~ system, This is clearly shown in fable IV, Cigarette smoke appears to be less mutagenic than cigar smoke, but more so than pipe smoke, however, more experimentation will be necessary before such a differentiation can be shown conclusively. One of the obstacles in these experiments was removal of water from the pipe condensate, not to mention the difficulty of working with highly insoluble material In an aqueous environment. Fractionation of cigarette smoke, as carried out for ob- taining nitromethane fractions of smoke (15), has shown activity in both the methanol and nitra~athane fractions (lable V). Since a larger proportion of the smoke fraction is concentrated in the methanol fraction (70-75: of total dry condensate as against 9-10% in the nitromethane fraction), it is higher in terms of total BA'FCo document for legal Services : Health Canada 20 May 1999 activity, whereas in terms of specific activ,itr the two fractions may not be different. Insufficient data do not permit such cal· ctllations involving specific activity~ It is also possible that the nitrontethane fraction, which should contain most of the polycyclic hydrocarbons of cigarette smoke, needs metabolic activation before its full mutagenic potential can be measured. Having shown that a pre-incubation step is necessary before obtaining a positive reaction for tobacco smoke, we thought if worthwhile it to see how this procedure worked for pure chemicalS, particularly those, like hydroxylamine and cyanide, that decompose rapidly in prerenee of air, COZ or moisture, Fr0p Table VI it will be seen that acrolein, hydroquinone and pyrogallol were positive. in as much as all three inhibited strain poi A~ more than they did Poi A , Caffeine and nicotine, even though examined at fairly high concentrations, failed to inhibit either strains. The detection of mutagenic activity of pyrogallol is interesting in the light of a report by Andenon and Linney (17), stating that pyragallol was the mast active among phenolic compounds tested in producing tumours during early Nicotiana hybrid seedling development. while phenol and cateehol were in;ctive. It is also interesting because auto· oxidation of pyrogallol is known to result in the formation of superoxide anions (18), which in turn could result in the formation of hydrogen peroxide and the most potent oxidant known to chemistry, the hydroxyl radical (19). The results obtained with potassium cyanide and hydroxylamine are presented separately. From fable VII it will be seen that po· tassiua cyanide iohibib the mutant, Poi A', more than it does the parent, Poi A ,while Table YIII shows that hycroxylamine has the same effect. While the mutagenic activity of hydroxylanine is well·known, that of cyanide was a Surprise. It is possible that the mutagenicity of cyanide remained undetected on account of the extreme toxicj ty of thi s compound. However, Stier and Cast6r (20), BATCa document for Legal Services : Health Canada 20 Nlay 1999 as early as 1941, produced respiratory deficjent mutants of yeas by exposure to cyanide. Further studies have shown that the pH of the pre·incubation mixture has a marked effect on mutagenic activity Of tobacco smoke. Ihis is shown in Table IX. At pH 8,9 cigarette saioke condensate, at a concentration of 1 mg. per mi, reaction mixture, shows 3 distinct differential inhibition of the E.coli strains, while at OH 6,8 the inhibition is negligible. Such a ph effect would suggest that basic compounds in tobacco smoke are also involved in;~e mutagenicity of tobacco smoke. Thus, when nicotine was re·~xa~ined at alkaline pH values, it showed a differential inhibition of the E.coli strains, an inhibition not evident pt ph I,O at the csn- centrations used, This will be clear from Table X. From:i~uw i, which is taken from a paper by Morie (21), it will be seen that there is very little un-ionised nicotine at pH 7.0, at which pH most of the nicotine is monoprotonated. ·At pH 9 eost of~the hi: cotine will be un·ionised, which apparently is the form in which it is inhibitory to E,coli, It must be pointed out here that in contrast to cigarette sroke in which mutagenic activity is detected at 1 mg (or less) per mi reaction mixture, 5 mg nicotine ler mi reaction mixture and longer pre-incubation time were needed before mutagenic activity was detected. See Table XI. This is a large amount of nicotine when it is considered that 1 mg of condensate from regular flue·cured cigarettes does not contain more than 50 ug of nicotine, which is be far the major component of the basic fraction of tobacco smoke. Thus, the mutagenic effec: of nicotine is, at the most, very slight compared to that of whole condensate. Pyridine and pyrollidine show a similar differential in· hibition of the E,coli strains, and this inhibition is observable at oil 9 but not at ph 7 as will be seen in Table XII, Again, like with nicotine, large amounts are needed to detect mutagenic activity, BATCa document for Legal Services : Hea#h Canada M lay 1999 and when amounts of these bases that occur in cigarette smoke are considered (tne amount of pyridine detected in cigarette noke is about one hundredth that of nicotine), it is clear why these bases are, at most, only weakly mutagenic compared to cig- arette sloke. Of course, these results suggest that other nitrogen- containing compounds known to occur in tobacco smoke should be examined for their mutagenic activity in the E~coli system, Recently, kes and his group (5,6,22) and HuttOn and Hackney (23) have found that S,~phimurium ~A·T538 is extremely sensitive to nitrogen-containing polycyclic compounds and relatively insensi- tive to aiicyl polycyclics, Further studies are in progress to make the procedure more sensitive and quantitative so that it should be possible to di ff erenti ate smoke from di fferent smoki ng materi al s, Metabolic activation using 9,000 g supernatants from lung and liver hcmogenates from different animal species Hill also be attempted, since tobacco sncke contains careinagens that need to be activated before they are detected as mutagens (5,6,22,23). Attempts are also being made to discover in tobacco smoke, other compounds possessing mutagenic activity, In conclusion, it may be stated that, presently, by em· ploying a pre·ineubation step in the Rosenkranr test, it is possible t3 demonrtrate' mutagenic activity in not only pure compounds, but ai~o in tobacco smoke from different kincs of srraking products, as well as in smoke fractions, and that this microbial procedure has potential for use in routine monitoring of tobacco product modification, BATCo document far legal Services : Health Canada 20 May 1999 REFERENCES i. A. HO11AENOER jed.) Chemical Mutagens: Principles and Methods for their Detection. Yols. 1-3. Plenum Press, New York, N,v, , 1971 , 1973. 2, L. GO1BEilG led.) Carei nogenesi s Testing of Cherni cal 5, CRC Press, inc., Cleveland, Ohio, 1974. 3. The Testjno of Cheaicalr for Carcinogenicity, Hutagenicity, Teratjgenicity, Health and Ilelhre Canada, 1973. 4. B,H. AMES. In "~tageoic Effects of Environment~l Contarinanti", L, SU~ON and M, HARRIS (eds.), Academi c Press, N,Y. 1972, 5, B.ll, I~ES,;,O. LEE and W,E. OURSION. Proc..Bat. Acad. Sci,, U.S., 70, 782, 1F73. 6, B.N. AIES, W.E. OURSfON, E, YAMRSAKI and F.O, LEE. Ibid, 70, 2281, 1973. 7. E.E. SUTER, M.O. ARDERSON and H.S. ROSENKRANZ Cancer Res., 31, 970, 1971, 8. H.S, ROSENXRANZ, H.S. CARR and t. MORGRN. Blachen. Biaphys, Res. Cocmun., QQ, 546, 1971. 9, H.5. ROSENKRANZ, H,5, CAAR and H.M. ROSE J, Bact,, 8r, 1356, 1965. 10. R.C, GARNER, E.C. MILLER and 3.A, MILLER. Cancer Res.,32, 2058, 1972. 11, H,S, ROSEHK~ANZ, Ibid, 33, 458, 1973, 12. M,iI, 811:MORIA and H.A, NISBET. Beitr, rur Tabal:forsch., 6, 27, 1971. 13, H.S, ROSENKRANZ and M.O. RNOERSON. J, Israel Biochern, and Microbiol ,, 1 , 70, 1973. 14. C. I1ARO and R, ~AUTHEPET, to~np, rent., 265, 1799, 1967, 15. H, ELLENHORET anl 6. GRIMMER. Z. KrebJforrch., 11, 66, 19SB. 16. A,P, SWR;N, J.E. COL)PER and R.L. STEOMN. Cancer Res., 29, 579, 1959, 17. R.A, P~OERSSB and L. LINREY, Cancer Res., ?1, 2450, 1973, BATCo document for Legal Services : Health Canada 20 May 1999 18, S, ~RKLUNO and 6, EV\RKLUNO, fur, 3, Biachem. 19. i. FRIOOVICH. In "Hororons in Biochemistry ilnd Giophys;es, Yol. i, E, t)uagliariello et al, (eds,) Addison - Ile5ley 20, T, STIER and 3, CASfOR J, ten. Physiol., 25,299,1991, 21. G,P, MORIE, Tobacco Sci., 16, 167, 1972, 22, L,D, KIER, E, YA~SAKI and B,N, Ames. Proc, Nat, Ace. Sei,, U,S,, 21, 4195, 1974, 23. J.~ HUUON and C, HACKNEY, Cancer Res., 35, 2461, 1975, BATCo document for Legal Services : Health Canada 20 May 1999 TABLE i EFFECT OF CHEMICALS ON GRO1'ITil CIF PARENT P.HD DNA POLYPERASE-DEFICIENT MUTANT OF E,COLI DIAMETER OF ZLlNES OF CSERICdL AMOONT (NHtBlflO~ Prll P.' P~I n+ CiiYSfdi IIIOLET 100 US 19,2 O AMP ICiLIIB 10 UG 211,13 211,2 CHtOROilYtEii H 30 UG 2~,7 23,e FORMnlDEHYDE,37X 20 UL R,1 4h,I ACAOLEIN 0,66 UL 17,5 O - ~ IMESHY'J~EIHANE- 10 UL 41,7 31,0 SULPHONATE B-PROPIOUCTOEIE i E,3111 112~2 2?,2 MICOTINE 1,0 HG O N~IROI~!EIHANE 100 U1 3 PHENYLHY3RAZINE 4 UL '56,0 112,1 MALE IC HYORAZIDE 1~0 U6 O I c(~NAPIIT HYIAM INE 10 ~E 23,9 1 24,1 SODllln CYCLA~TE 21! ilri n o SAGG~~~IN 67 MG 17,9 1s,3 o-TO1UENE 3,3 ~5 9 1 O SULPHON~~IDE CAFFEIIIE 1,9 MG BEI·gEIE Ir)O UL 13,0 BEHZO~UIHME 2 M6 54,5 52,P CATEC~OL 33 MG 30,E 30,8 CHLO~OSENIC ACID 12,5 ~G O O RUTII1 5,0 MG O ~ COURARIN 7,3 ~G ?1,4 II1ETHYLCOUI1AA IN 2,0 Mt 13, O 13, n ANTH4ACEHE 1,5 MG 13,0 1!,0 BENZO(J·)PYRENE 2,5 Mt O o BATCo document for Legal Services : Health Canada 20 May 1999 QM mn a 3M 00 1 u 21N~( a O I· IU t-W I IQ~~I N M i-I I~o M N ocL ~IU ia ~O a a Na 22 0( w3 10I~QJI YL I IQNO N a WI~ ia E~ U oc !o OU i 0- 1 I~HC cPW I M w oa I IQQO N I I m w ~ 24 w I IQ IC h I ia a I Im to N 00 1 IQ ia WC rZ oi Z 3 3L WZ a a 0~ tr nd Wt I I N W Z (W Wh f~ WYW ~4 UOt WZ tEC Z~a s~w W~X O 4 =~ a a r Wv) = IU r U ~v h O BATCo doeulent br Legal 8snket : Health Clnlde 20 May 1911 TABLE III EFFECT 01 CHE;IICALS AHD TOBACCO SMOKE OH GRO#TH OF PARENT AR3 I]MA POLYI~ERASE-DEFICIENi MlliAHT OF E,tOlI D(A1~E'IER OF ZOHES OF AGENT I~OllllT IHIIALATION POL A~ POI A' CR~SIAL VIOLET 1(10 09 191'1 O RT~YLMETHA#E- 10 U1 4g,q 2S,8 SU1PHONATE CHL~ROMYCETI~ 30 UC· 23,5 23~5 A~PICILLI~ 10 UG 23,3 23,5 CIGARETTE 10,E MG 21,0 19,1 SMOKE 2112 MG 21,7 24,4 CIGAR 10,2 1116 17,7 17,0 SMOKE 20,rl MG 20,3 20,9 CIGARILLO 10,7 MG 17,9 16,4 SMOKE 21,4 me 20,3 20,a PIPE SMOKE 2!,2 MG 17,1 16,5 (SPIRALS) 22,2 MG 18,1 17,6 PIPE SMOKE 2011 MG 15,6 141 (FAD) 23,3 MG 16,0 15, BATCo document for Legal Services : Health Canada 20 May 1999 TABLE IV EFFECT OF [HVIICALS ABO TOERCCO SMOKE 01 GAONTII OF PARENT ABIi ONA ~OLYMERASE·DEF[CIEBT MUTANT OF E,COII AGENT AMOUNT PRE·INCUBA· 110, OF V[AB1E BACTERIA PER RL TIOH PER M1 IIME MIXTURE (HRI POL A~ POL A' SIREPTOIMCIII 2,5 UG B sa x loi 67 X 107 0,5 125 X 101 92 X 107 1,5 109 X 107 88 X:07 MEIHY1~EiHRE·I1,2 UL C 107 X !C7 97 X 107 SULPHOIIRTE 0,5 IB X 101 83 X 101 l,j 115 X 104 88 X 107 CIGARETTE 5,7 EIG O SE X 107 205 X 107 SI1OKE 0,5 1!6 X 103 177 X 107 8011077 X 10 90 X 10731 X 107 0,S 44 X 103 58 X 107 1~4 4;110 71 X 107 CIGAR SMOKE 11,3 MG O 33 X 107 118 X 107 0,5 nrl 133 X 105 1,5 Nit ~lOfi 3,3 116 O 05 X 107 111 X 107 0,5 54 X 106 99 X 101 1,5 102 X 1?4 70 X 107 PIPE SMOKE 7,3 MG O r115 X 107 107 X 107 0,5 25 X Id 9'1 X 101 1,5 18 X 107 88 X 107 ,6 MG O j 96 X 101 78 X 107 0,5 31 X 107 60 X 107 1,5 80 X 103 61 x 105 WHEI APPROPRIATE CONTROLS VERE RUH SIMULTANEOUSLY, THEY SHOWED NO INHIBITION OF EIMER POL A- OR POL A' BATCo document for Legal Services : Health Canada 20 May 1999 TABLE i' IRHIBITION OF PAREEfT AND O~ POLYMERASE·DEilCIE~T MUTAHT OF E,COLI BY SMOKE AMD SMOKE FR4CT:ONS FROM FLUE-CURED TOBACCO CIGARETTES AMOUNT NO, OF VIABLE BACTERIA I #HOLE SMOKE PER ML PP,E-INCUBA- P8 ML OR REACTlOn TION TIRE SMOKE FRACTIOB MIXTURE (HR) (MG) POL A' POL At O 80 X 107 31 X 107 VHOLE SMOKE 5,6 0,5 44 X 103 58 X 107 1,5 40 17 X 107 8,9 O 12 X 107 84 X 107 0,5 26 X 101 78 X 107 j METHABO1 1,5 Nit 38 X 107 FRACT IDN 0 15 X 107 68 X 107 8,8 0,5 1 X 104 78 X 107 1,5 ?0 39 X 107 O 30 X:07 59 X 107 1,5 0,5 32 X 106 85 X 107 HITRO~ETHANE 1,5 31 X 105 76 X 107 FRACTIDN O 8 X 107 E7 X 107 2,3 O,Z 17 X 107 126 X 107 1,5 44 X 105 70 X !07 THE CYCLOHEXAIIE FRACTION AS IrlELL AS ACE'TONE CONTROLS RUN SIMULTANEOUSLY, SHOWED NO I~HIBITIOH OF EITHER P01 A' OR P01 A' BATCo document for Legal Services : Health Canada 20 May 1999 TAB~ vl · INHIBITIOR OF PARENT AND ONA POLYMERASE-OEFICIENT MU~AH~ OF E,GOLI BY SOME CHEMICALS RO, OF VIABLE SABERIA AMOUNT PRE-IHGUBA- PER ML CHEMICAL PER ML TION REACTION MIXTURE REACTIOI TfR POL A' P01 A' MIXTURE (HR) 2,5 0! 0 39 X 107 74 X 107 SULPHONATE 0,5 1145 X 105 12 X E7 1,5 23 X 101 14 X 107 SI3EPTCMYC[N 2 5 06 O 21 X 107 11 X 107 0,5 55 X 107 61 X 107 1,5 23 x lol 82 x !07 FORMA~ESYDE 6,25 UL 0 /174 X 106 !10 X 101 0,5 /210 X 102 57 X 107 1,5 59 X 102 33 X 101 ACRO1EIN 0,02 UL O (131 X 104 77 X 10E 0,5 118T X 102 is X 105 1,5 1202 X 10 71 X 106 HY3R0031NOE 6,f MG 0 40 X 101 107 X !117 0,5 1?10 X 104 87 X 107 1,5 NI1 112 X 107 CATECHOL 8,4 MG O ( 45 X 107 111 X 107 0,5 85 X 106 61 X iOf 1,5 NI1 III PYROGALLOL 6,3~ F1G O 67 X 107 107 X 10! 0,5 47 X 106 79 X 10' 1,5 In x 102 J3 x 136 CAFFEINE 9,7 MG O 64 X 107 94 X 107 0,5 rl~ X 101 IC~ X 10! 1,5 60 x 107 95 x 10' NICOTINE 20 ~G O !3 X 107 80 X 101 0,5 10 X 107 84 X 107 1,5 8 X 107 70 X 117 BATCo document for Legal Services : Health Canada 20 May 1999 TABLE VII INHIBITION OF PARENT AND DNA POLYMEilASE- 31FiCIEBT MUiAWT OF E,COLI aY POTASSIUM CYAIIIDE COlliENTilATION j PRE-INCUBA- NO, OF VIABLE BACTERIA PER ML 1 Oi KCN iiON TIR REACilON MIXTURE IN REACTION (HR) iliXTUfiE POL R~ PI A' 215 O NI1 PULL GRO#IH Ifil 0,5 111 201 1,5 Nit NI1 1,0 O 148 FUL1 GROWIH 101 Nit FULL GROWTH 11; 1,5 NI1 FCLL GROllTli 10i O (A) 196 X 104 54 X Idj 0,5 136 X 114 209 X 105 1~Z 22 ~O X 10% O iBi '10G FUL! GROXTH 101 0,5 g~ FCLL GROYTH 101 1~5 ( 52 FU11 GROIITH 101 O 99 X 107 133 X 107 015 j04 X12X 173 X 107 1,5 119 X:07 I i I - - - BATCo document for legal Services : Health Canada 20 May 1999 TABLE VIII INHIBIIIOW OF PARE~I O~A AND POLY6ERASE-OEFICIE~T MUTANT OF E,CO1I BY HYDROXYIAMI~JE HYI)ROCHLORIDE AMOUNT OF HO, OF VIABLE BACTERIA PER HYOROXYWIIIE-HC1 PRE-IFICUBA- R1 REACTION MIXTUB PER M1 REACTION TIOEI TIRE MIXTURE (MG) (HR) POL A~ POL A) j18 O BE X 107 93 X 107 0,5 32 X 107 132 X 107 1,5 81 X 107 96 X !07 8,7 O 52 X 107 103 X 107 0,5 !08 X 106 77 X 107 1,5 59 X 107 92 X 107 O 300 X 102 FU11 CROI~H 102 0,5 501 X 1P2 FULL GROIIIII 102 1,5 1000 X 102 FULIGROWIH 102 8~7 O 2! X 105 68 X 107 0,5 111 Y, 105 7'1 X !07 1,5 121 X 105 81 X 107 0 NO GROI~H 102 FULL GPO#TH 102 0,5 22 X 112 FULL GROWTH 102 1,5 500 X 102 Fllli GROXTH 102 BATCo document for legal Services : Health Canada 20 May 1999 TABLE IX EFFECT OF PH ON IIIHIBITIOH OF PAREEIT PIPID ~#A PO1YMERASE - DEFIClmT ~UTAHT OF E, COLI BY CIGARE~E SMOI(E CO'I~ENSATE A:IOI]RT CF RM IN 3H OF PRE-I~CUBA~IONI f101 I]F VILE BACTERIA PER SEACTIOH mlxmRe I pRi·I4cu- TIME I?i (MG) aATIO;I (~~R) REACTIOIII i~lXIL;RE F1IXTURE POL R~ POL AS 3 51 x 107 72 x 107 6,5 · I i ·1 19 x 1I1 52 x 1 2 11 Wxl 3 81 x 116 62 x 1?7 T 9,'? 1 NI1 9r3 x 1~ 2 PIIL !IIIL 1 58 x 117 80 x 107 1,S Ei,6 I 44 x 17!! 57 x 107 49 x 112 51 x II' n 1!G x 116 7'1 x 1'17 2,5 ~,7 1 YIL 84 x 111 2 BIL 115 x a5 9 34 x 10! 132 x g7 !,O 5,8 i 21 x 11' 97 x 1~7 2 36 x ~E l~a x 107 1 144 x 116 121x 107 3. ~O 819 1 31 ic Iii? 7~ x 101 2 37 x 113' 59 x !01 ~CETO~E CO1TROLS P\UII SIMULTAHEOUSLY SHO#Erl FIO SIG"IIFICANT INHIBITION OF EITHER '01 R~ OR POIA+ BATCo document for Legal Services : Health Canada 20 May 1999 GABLE X EFFECT OF PH ON INHIBITION OF PARENT ANO DNA POLYMEW\SE-OEFICIENT MUTANT OF E,COLI BY NICOTINE AF1OUNT OF PH OF PRE-INCUBR- ND, DF VIABLE BACTERIA NICOTINE [N REACTION TION TIME PER ML REACTION MIXTURE MIXTURE (HR) (MG) POL A~ POL R' 20 J,O O 5 X 107 82 X 107 0,5 26 X 106 55 X !07 1,5 5 X 107 55 X 107 BUFFER . 1 7~0 · · 0 3E X 107 5~ X 107 0,5 ?E X 107 73 X 107 1,5 15 X 107 101 X 107 20 8,0 O 5 X 107 32 X !07 0,5 16 X 104 5 X 107 1,5 8 X 10~ 4 X 107 BUFFER 8,0 O 32 X 107 65 X 107 0,5 27 X 107 73 X 107 1,5 39 X 107 81 X 101 20 9,0 O 5 X 104 13 X !06 0,5 NI1 2 X 104 1,5 NIL 25 X ~O BUFFER 9,0 0 44 X 107 107 X 107 0,5 30 X 107 83 X 107 1,5 32 X 107 95 X 107 FIATCo document for legal Services : Health Canada 20 May 1999 FIGURE I *HOip101On,trd SBBMdrwp~b~OMl~d +yU"p"lO~Ul~d 'i Fraction el Earh Spccire 1 2 34 I 1 ~crcw 1,0 r·~, ,d ii o.a ii i 34 pH ol Cilpr Smaia1.2 pH oi Cicgrrtle Smoke 0.0 0.4L ,,i ....; . - 0 2.0 4.0 0,0 8.0 10.0 12,0 pH of Soivlion FiqurF i, Dirhibut~h af go~nll~d ~nd uqtd~nottd nico8ne in lobl~o rmabr ~~ varipu~ ph InCrcmenrr, BATCo document for Legal Services : Health Canada 20 May 1999 TABLE Xi EFFECT OF NICOTINE CONCEnTRATIO~ OH INEIBITIO~ Oi PAFSNT AHD DBA POLY~ERASE·DEFICIE~ I~U'IANI OF E,COLI AT PH 9 10, OF VIABLE BACTERIA I AnOllNT OF PRE-IIGtlBAiION PER FI A!COIINE PER 1?1 TIME REACTION MIXTURE (HR) POL 4' POL A' O 41 X 10! 80 X 1 36 X 10' 71 X 0,69 2 30 X lo! 7E X 245 22 X 10:41 X 10' 76 X16~ X1yl O 27 X 10~ ;rO X 1Q~ 1 62 X 10' 59 X !O' 215 2 51 X i 44 X i 5 311 X 10: 58 X 10' 211 / IX10' 4X107 O 34 X 10i, 84 X 10! 1 3 X 10' 5E X 10' 5,0 2 7X lo~ 24 X1 5 2 X 10" 31 X 100 24 ;fX 101 8X 106 BATCo document for Legal Services : Health Canada 20 May 1999 TABLE XII EFFECT OF P~~OH IEIHIBITI[IN~OF PAeEHT AHC ~i~A POI~1ERASE-DEFI GIENT PIUTANT'3F E,COLI BY PYR[DIHE AND PYROLIIDINE A~UilT 110, DF VIABLE BACTERIA PEI Hi PH O~ P4E- IICUBA- PER FL REACIIOEI REACTION TID~ TIRE RIXTURE RIKIURE [HRI P01 R. POL i~ (MG) PYAIDINE O 18 X 1C,7 13 X 107 20 6,95 1 10 X 10! 36 X 10! 2 6 X 10' 45 X 10' PYROLLI~IIIE i i 0 4 X 107 22 X 107 20 6,8 1 6 X 10! 27 X 10! 2 5 X 10' 25 X 10' PYRIDIHE O 18 X 10~ 7X107 20 8,65 1 27 X 113q 16 X 10! 2 ~IL .25 X 1~~ PYRDLLIDINE O ~7 X 9X107 20 8165 1 r3 X 30 X 106 2 17 X 104 11 X 106 APPROPRIATE COFITROIS RUN SIM1I1TANEOUSLY SHO~O NO INHIBITIOH OF EITHER POI Al OR POI AS BATCo document for Legal Services : Health Canada 20 May 1999 r~J \D ca BATCo document for Legal Services : Health Canada 20 May 1999