AW139d O O ill O V Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 A~1311 G Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ImperialTobacco LimitediLimitP:e RESEARCH AND DEVELOPMENT DIVISION MONtREAL Aw1311 THIS REPORT HUST NOt BE COPIED OR SHOWN TO UNAUTHORPED PERSONS. C~IE· 189Y Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Laboratory Report No, 116 Copy lo. 4 InPERfbL TOBACCO ITO, RESEARCH ~ OEYE1OPIENT OIYISION nOHtREAl RESTRIREP INVESTIGATIONS on THE EFt~er or HUHIW SIMIIHG EonoITIonS on nstaloe~a ccrIvlrv or roanceo srmKE cowoarer~ AUTHOR: C· nc8tlde n,H, Blllllorla Issued 8y: S.R. narsey Gate Issued: January 23, 1981 OIStRIBU~TION: Or, P,3, Ounn Copy No. 1 Or, S,R, narsey Z nr, A,L, Heard 3 Dr, A, Blnnr ~, S nr, E~E, Kohnhorst nr. A,G, Nlehollr 8, 9 nerr, E, Rltterrhaus 10 Or, E, ~ausch 11 Or. C,J,P, de Sl~uelra 12, 13 Hbrary 14, 15, 16 ·O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 LaboratorY RaDort No, 116_ Copy no, IIPLRI~ tlICeD ~P, RESEIRP h ONELOPIUIT OIIYfl~ ~HINRiEIIl RErmlCfED IBIn16niW WI TWt ImlT Oi W_Y~I~P~O~J~P?f a mterolPlro~ Imvm or loeclP~P~iw~ e, Ic8t1de n,H, B111Rorla [kte Ilruld: Jdnury t3, 1986 IsrJ BY: S,R· ns~sey OI5tRIBUTION: or. 1,3, Deer. eopy Io· 1 Or, S,R, nar~ry ~t. A,1l Htard 1, 5 Or, R, Blnn~ Hr. E.E, Kohnhorrt 6, t HI. R.rJ~ nlchollr 8, 9 Herr. E, Rlttenhaur Or, E, ~auscn Dr. C.J.P. de Sl~ue~ra 12, 13 Library G Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 TABLE OF CONTENTS PAGE Sumnarv and Conclusions tntroductla 2 i, Methods: J t,t Clsan~te Selection 3 1,2 Hurnan Smoting Behaviour 3 1,3 DuPile~tlon 3 t.l nutascnteltu TeJttnp I 1,5 Statistical Analysis 1 2, Results and Ol~currlon 5 3. References 7 i Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Swmarv and Conelu~to?r The objective of this study was to investigate the effect of human smoking conditions on the specific biological activity of tobacco smoke condensate. From a preliminary study, two human smoking parameters, puff duration and puff Interval, appeared to have some Influence. It was felt Important to investigate the apparent effects of these two parameters while controlling for puff number. Two comnerctal Canadian ventilated products, Brand A and Bland B (declared tar deliveries of I and 11 mg respectively) were selected for use In the studies, The results of the present Investigation can be sunaarl2ed as follows: No It~tlltleally Ilgntfleant dlfferenrer In the Ipeelfle Btolog~cal activities of either cigarette, as determined by the Ames Test, were found for the tobacco smoke condensate generated under different human smoking conditions relative to: 1) standard smoking conditions or 2) each other. II appears, therefore, that the standard procedure for the determination of the specific biological activity of cigarette smoke Is an accurate Indicator of the specific biological activity to which a smoker may be expored~ Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Introdustlon host studies of the #olPrllili activity of to~eeo smoke condensate have teen concerned ulth the relative differences Letueen tobacco and product types, The majority of these studies have used standard smoking conditions (35 mi puff, 2.0 s duration, 58.1 s interval) to gmaatt the smoke condensate, It is hMm, hauwer, that actual human smoking eondlttanr can vary from rtandlrd conditions. In Il~ht of this, It ul felt prudmt to assess the effect, If any, of human smoking conditions relative to standard conditions, on the ks biological setlvltl of smoke condensate, Tne IIIAer Tert ws considered to he the most appropriate short term mutagenic testing procedure to follow, A preliminary investigation, carried Put In 1984, indicated that human smoking conditions, specifically puff duration and puff Interval MY effort the specific biological activity of smoke condensate. [t was decided, at that paint, to focus attention on the effect of varying these tw smoking para~neterr, i Q Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 i, Method 1,1 C1Parette Selection Tuo comnercial canadian brands, Brand A ad Bland B (dcclared tar deliveries 4 and 11 nlJ, respectively), wore selected for investigation, mese two products are typical examples of Canadian Cigarettes representing the extremes of the usual delivery range associated with ventilated products, All cigarettes vere selected for uelght, pressure drop and ventilation level. Oetalls of the cigarette rplclfteatlonr are given In Table i, 1,2 Human SRlolrlns lehaviour Out of a pool of 95 previously recorded human profiles, 6 profiler uere selected for further investigation using the follwlng crtterla, 1) Group A: all smoking parameters except puff duration to remain constant, 2) Group a: all smoking parameters except puff interval to renuln constant, When necessary, profiles were truncated to control for puff number and butt lengthl details are given In Table 2. 1,1 Ouolleatlon The Human Smoking Behaviour Research group carrled~out the duplication of each profile using the Ill smoke dugllcator'~ in order to satisfy the reoulrementr of the kcr lest In terms of condensate concentration, each profile var duplicated 10 timer. Concurrently, u\th duplication, each brand var smoked under Standard machine condlttonr to i Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 obtain a control. The duplicated deliveries for each profile are given In Table 3. 1.4 nllta9entcltv Testl_nq The smoke condensate for each profile and the companion control samples for the two groups of smoking conditions were parted to the nlcroblotoglcal grog for mutagentclty testing. The condensate on the pads was extracted with OnSO and tested for mutagentclty by the standard praadure fallowd In thlr IlorltoryZ'1, Pllpallt twhlmurb a·98 was used to compare the rautagenlcitler of the condensates, The microbiological procedures and preparation of the Sprague·Oawley rat liver port-rnltoehondtlal supernatant (S-9), were according to procedures described by Pmes et al. 1.5 Statistical Analysis The mutagen\cltles of the condensates were calculated in two ways: i. RpJrcrslon antlyrtr of log~tranrfor~nad (atl In whlrh rcqrcrrlon Ilner were checked for parallelism by an ANOVA test ', The intercepts, using a common slope, were employed to express mutagenlctttes. 2. Converting each dose point to a specific activity (number of revertants per ug condensate). Means and standard deviations were calculated for each condensate from a total of 20 points, An ANOYA was performed to determine whether the differences between means were significant or not. t Cj Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 2, Results and Olscusslon It can be seen from Table I and Figures 1 and 2, that for varying puff duration, there were no significant differences in the rpectfic biological activity of the human smoking profiler, not any differences between the control and the human smoking profiles. There were also no significant differences In biological activity when puff Interval was varied (Table 5, Figures 3 and I), The earlier study indicated that an Increase In puff duration · resulted In a decrease In biological activity, while an Increase In puff Interval (deerease In puff number) led to an increase in specific biological activity. However, thess preliminary results were obtained from single experiments, In the study reported here, puff duration and Interval were varied white puff number war controlled. Furthermore, ruffldent replicates were conducted for statlrtlcal analyser. In light of these points, the preliminary findings appear puestlonablc, Other researchers have reported that an increase in flowrate during a puff, decreased the spedfle biological activity (GR a OC Status Review Noter, November 1984 · april 1985 and the Biological conference, nontreal, Canada 1985)l It was not the intent of the study described here, to investigate the effect of flowrate on biological activity. However, It can be seen from Table 2 that flowrates did vary within Group A smoking condltlonr (varylng puff durations). However, the Information in Table O indicates that there were no differences In specific biological activity which could be attributed to differences In either puff duration, as discussed previously, or flowrate. The reasons for these different results are not clear and should perhaps be further investigated. i h, Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 The results of this study appear to Indicate that the bner ~1o1b~lni Irtlvttr of moire ~ondcBdh ii unaff,cted Ly Ihanps In the puff duration or tnterval of a Imo~lng regime and porrlbly unaffected by changer In flcwrate. Therefore there flndlngr support the are of standard rstloq conditions to monitor the ploioglal activity of tobacco products, It Should be noted, however, that the eigaretter used In thtr study were Canadian products which are manufaeturrd with flus·cured tobacco. Prevtous MTJ: has shown that the r#lst influential factor in · relative Amer biological activity II tobacco type ~ Ilue·eured tobacco is less biologically active than other tobacro typa, Furthernore the differences In biological activity between Canadian clc]lretter (flua·cund tobacco) are small. Therefore there nnltr My not he applicable to all tobacco types and a further study urlng other tobaccos may be warranted. Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 References: i, nc8rlde, C., Oes!gn and Operation of thel~aPet~al tobacco Ltd, Slrmice OuDltcator_, R b D Report Ho. 170. 2, B111Roorla, n.H., Iaplrlal Tobacco Ltd,, Research Laboratory Report No, 152, 1981, 3, Ids, imperial Tobacco Ltd., mrmo dated 18 October 1919, knes, B.H,, ncCann 3, and ranas~l, E,, nutat. Res,, U (1915) 3~f. 5. Brownlec, 1(.A,, Statistical theory and Methodology In Science and Engineering, J. Wlley h Sons, N,Y,, 1960, 6. B1Tlmorta, n,H,, A Review of OAT Studies of the nutasenlcttv of Clarette Smate in the Amcr Salmanellalnlcrosome test (In pre~aratlon)~ i O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 TABLE t CtGARlff~eECfflCATrOES Brand A Brand B OPn (mglclgt,) 3.7 11,1 Hlcotlne (mglclpt,) O.g 0.93 Water (ngklgt,) 0.37 1.11 CO (sglclgt,) 3.8 11 ,I P,O, (nn W,G,) open 75 W Dllutton (X) 63 37 Paper Pororlty (C,U,) 36,8 35,2 Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 re 41 *Ir 09111 00 Trr r, P)~0 ~nr ~nrs "L"r etc NNN nr AC Za u.r lA ~eY~ ~> ~00D L ~n91D eo moim snN Ay 9 r*r uC rr rC ~O NNn ge d Orr 1·I 1~1 Y rrr CA N mNr AC tO 001 Lr o~a 01 9 NON II~ :t r. su~o mNO r 'NC Cr ALL lu rCI XA 00~ r~nr NQII1 NmO Er Ir 3 lllOlD 09r O Cr YI Qms Nea u~ul~ Dr HC rD 9C C~~~ 00 mr~e J 000 m"" L vrv uuu gu em. 0r~ O IV Y~ O Y j( . IU O O m'N r;34 ~O 0 "' o dii O br NNr EY O mma 000 rr rY19 u~ c "h9 010'0 DC " 9tC* I~ ' vlvlvl VllnVI Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 TABLE 3 COIPIRISOI OF fHE OELIVERIES OBTPIINEO FROI THE OIFFIRENT SROKIWS COIOITIONS I _ Bland 1 _ L Brand 8_~ ~ Snw~tng ) far Nlcgtlna nd lu I 'p' "' CYL'"S Candlttan ,I Innllellt.j ,I (IlrlPt,) Group A - I Puff Ouratlon I Std. Condltlonr I 1·0 0.10 0.42 1 10.( 0,99 0.99 s P9·328-11 1 ~·B 0.45 0.29 3.9 ( 9.8 O,BJ 0.69 9,1 S #1-328-11 1 5,5 0.52 O.U 5.5 1 11.0 0,93 0,93 12,5 s p 6-919·12 1 7.3 0,67 1.0 1 13,0 1,13 1,21 15.5 Group 8 - I Puff Interval Std, Condttlons 1 4.8 0.41 0l10 0.98 0,10 S ~-128-5 1 3,1 0.27 0.18 3.3 1 5,9 0,(8 0,49 6,8 S #1-328·1 1 3.8 0.32 0.22 · 3,9 i 1,1 0.57 0,50 8,3 S ~·328·9 1 2~8 0.23 0.15 2.9 1 5,8 0.11 0,11 (,1 Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ( ,· "' 1IIHnl ylHu)l ii;' "" u;;i *HI ii' 00 := :: ii ~1 .r ,, jI ii ·* eE " 14 1 n·l I E JrrrJ ·ICH rNHJ FH·I 1 *~ *non 'N" I ; II . 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""~· r L " oooo ; ; 0·00 ~OI Y* Ilu ;;f ,v ,, J; 'r CL ·H*· ~;nn · " :f II1LR u u y; * i c ~g ,,r, iu III NNH 1 ,e i r'c*~ *~ unnn yr unnn :Z ···· g ···I re * HHYIH UL ~IY)YIH O N P O I· N V 9 Crl Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 FIGURE i: AHES EWfAGEBICITY OF S~KE CONDENSATE GENERATED UNDER DIFFERENT PUFF WRA~IONS .BRPJ(OA- ii 70D ii i 600 I ~,,, I :i o, ~oo I : X · Std, Conditions -2,05 o · S19-328418 · 3l05 ~ 300 0 · S17·328-11 ·2,15 1 a·S116·919·12 ·1,1S too C 400 S~61KE CONDENSATE ·pglPlate Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 FIGURE 2: AMES MUTRGU1:CITY OF SMOKE tON085ATE GENERATED UNOER OIFARENT PUFF OURRfrONS ,BggD B · 600 :"P 500 d 400~ X·Std. Conditions- 2.05 o·y9·328·18 ·3.05 ,300 0·51~·328-11 '2.15 ) o·Si16·919-lt 1M) SMOKE tON~~RTE.yg/plate t O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 FIGURE 3: AMES MUTAGENICITY OF SMOKE CONOENSATE GENER4'IED UNOER DIFFERENT PUFF INTERVALS BRAND A - soo 500 d Q00! 300 X-Std. Conditions ·S8,05 Y o - S#1·328-1 · 65,05 0 · 5#5·328·9 - 36.05 ~ · 5#6-328-5 · 28,55 too 200 300 J00 SMOKE CONOENS~TE ·pglplate Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 FIGURE 4: AHES IIL~TAGEHIClfl OF SMOKE CONOfNSA~E GENERATED UNDER OIFARENT PUFF INTERYALS .BFJ\HOB· 600 O 400 X · Std, Conditfons · 58.0S o - Sfl·328·1 · 65.05 Z 300 P·S15·328-9 .36~0S I /1~! ·~-5Y6·328·5 .28,55 100 200 300 SMOKE GONOENSATE -pglplate Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 1 IV h' Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 C ~ O IV \D C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111