I. I: l~u C ~s i ICEC~d 1~r~C~t j dblfrC' tlOU) REftAleH IYD OEVelOrYLlt CEltll I#nln.wl~e~~ al~eeo cour~wv uunED IEttlfdllR[ RO~R JOVfY~YITOr EY6L~IO, fd HE Ai·~l· 011 h~l 1781 ,,, 1, t. Thornton (Dr.) 1T/~/16D 21 July, 1981 :. Dr, C, PL~, Snuthanpton, 8esearch Conference llu9ust 1981: Biological Proprl I enclose four doantr ~fb incorporate points arising iron the Icctiql held at 80uthampton in May and Suly 1981, and cements made by our consultantJ in this area, liith kind regards, Yours sincerely, 1, P, TROIW~ in pi~ lt4 ~f'h"~' 13! ~V~L ~' ~F~i( glcxl II" C ~I* Il r·* r qr ~· mummn r~ u~u. R.hrYt rV ji- ~· ~4~s~ ClibPDF - v~~fas~o.soni : nETlLPP/46B 23 July, 1981 '8ov Best Ca we Ensure That Future Group products Have de lowest Posaible Biologie~ activity!" ~T~b~O~ This document was discussed in draft form at the Biological Meeting held at Southamptan in July 1981. This version has been Plaid in the light Of these dlOCPSIIOPO. 2 ~~ STRATFCy The strategy for meeting requirements within the Bg Group for reductions in biological activity should be on the see basis as that for meeting delivery targets for smoke components, i,c, a Cigarette Design Bm~~k approach in which measurements of biological ac~vi~ replace the deliveries of smoke components, On this basis, measurement Of biological activity would be related to major cigarette design parameters, ~C biological tests being analogous to ~e various different smoke components, One would then be in a position to 'design' cigarettes of reduced biological activity. Problems in the two aporoacbas are also a~flar, Just as a change in a cigarette design paracttr (sucn as filtration) will not necessarily reduce the delivery of all aPo~e coPPonents ~~oss·~e-bol:d, den vill aot nocoPslrily be fhii~ mangol in activity as defined by the various biological tests, The main criteria for the choice of biological tests are discussed later, The other coecn Ftob:eP is the posoibili:y of pgnprgistle or Mtagonlsiic effects occpr:fng when two or pre design factors are used in attempts to reduce delivery or to modify biological activity, In ~~ cases such effects can only be ~ra~elled after intensive etPdv. The use of additives s:ccld be viewed as mother cigarette design parameter; in fact, additives usually merit more attention for their possible biological Iftflitv than for their affect on delivery, elthcuqh b~ cu occr Ucr~i~). j C#~B~X FCR C~SaG ~S~ OP 9I013Gf~ ~IVrTI Whereas it is relatively easy to decide which smoke components one is iaterested:n, the importance or status of particular biological tests is tar harder to eot~lish, For this reason Tests are ebaractorisad as follows: Recommended: Reasonablv well established with adc~ate h~J!a; pf ~porye. ClibPDF - v~~fastlo.som w. ·2el Potentially useful: Promising but not yet fully evaluated, Atienticn is also drawn to areas where tests are not yet avail- able~ 3.1 EVACUATION OF E(AJOR C~CdRE~E DESIGN PARAMETERS (paper, filter design, tobacco type~ ventilation etc) Recommended (but currently net used on ground of cost-effectfveness] Skin-painting Well established, reliable (arthough relevance to man arguable), Recamoended Inhd~ation toxicity Useful in that it measures the general toxicity (short-term) of smoke in the respiratory tract of animals in a reasonable period of time, Potentially Use411 bg-term inhalation It is extremely difficult to produce rumours toxicity test by smoke inhalation, Without such an end-?oint this lest is an extremely expensive extension of the short-term inhalation study, However, it has been claimed that harnsters are the animals of choice for such studies, Short-term tests · related to rancer Ames Test Only limited studies on condensates as yet. Sebaceous gland test ColFJnents to be made by BaT Germany, NMFT Test Simple, but only fair correlation with o~et tests, Chromosome damage test Should complement Ames Test. Under development at Hamburg and GRE1DC, Test not yet available Tests related to: (a) At:erosele,rosis Various approaches will be evaluated at (b) Bronchitis GR8DC as potential model systems for these (C) m~physema diseases, If the work is successful t~e work will form the basis of tests. 3,2 ADDfTTV3S Recowended W General Toxicity Often available in the literature, but essential iC1 in establishing broad category of possible tCI usage levels~ p0 CI I:PDF -\:11II::! fasiio.;:om ~P -3- TeratDgenicity Essential if 'unknown' additive is to be considered, lnhalation toxicity Useful in demonstrating no increase in hazard can be associated with particular additive, Short-term tests · related to cancer: Bes Test Well established method of screening compounds for mutagenieity, Chromosome damage test Alternative and complementary method of screening additives for mutagenicitII. (Other tests depending on the additive) 4 CURSE:NT POSITI~N The current state of our knowledge of changes in biological activity in relation to the major cigarette design parameters is shown in the fellowing figure. The work an which our knowledge is based is not necessarily carried out solely within the BAT Group and outside studies have been considered, if judged to be rcliijie, Cigarette design Skin- Inhalation Ames Sebacsoust NMFIf Chromosome parameter painting toxicity test gland test test damage test Tobacco type I O I Poret: porosity O O Filter ventilation I O O d Filter type I O 1 Cig arette circumference O f Tobacco cuts per inch I Paper type O O Material (stem, lamlna) Cj Ex3anded tobacco o Synthetic materials (i Sidestream smoke O O hbacco, nicotine content Reconstitution I B O V1 I Studied in some depth O Partly studied IrJ O A little information B Placned studies r0 1 To be completed after discussion with BAT Germany. V1 03 C1 Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 No weh table can be given for additives and flavours since each and every additive mst be treated on its merits, In this rontext, we now have availtble advice on appropriate tests from external consultants of international reputation, Dr· F,J,C. poe and professor B~~,Bridgesl 5 nr~l~l?o~ This is considered under See time-scales: Iamediate: $pliation of existing methods short-term: Developent of additional tests LonSer-tenn: NecessarY research Immediate (u.o to 1 year) Using existing tests we shall systematically fill the important gaps in our knowledge indicated in t~e figure (page 3)~ we will also carry out any necessary work On additives or flavours which the company may wish to usel we are currently establishing two short-team tests at South~n~i~oton, the Ames Test and a chroclosome damage test. Both ate likelY to be reo~uired by re~ulatory act~btities in the future, particularly witi! regard to the evaluation of f!avou:s and additives. Should a~iticnal short-term tests be required by regulatory authorities, we would expect to use a contract research house, Short-term (cp to 2 years) Efforts will be made to develop short-term tests related to atherosc!erosis, broneltis and emphysema. We will also evaluating alternative inhalation tests, witn especial reference to stu~ing low delivery cigarettes and the use of alternative rodents which may be more sensitive. Longer-team ton-going) We ate eirryinq out or gonsoeing research related to current theories of the induction of cancer and athesosclerssis, As and when this research comes to fruition, the results should infl~ence the development of the test battery available at Southanatonl 6 re BIOLOGICFL S:LPfES GXO'JP COLtkaOPATrON The increasing environmental pressures to screen materials will ultimately be felt on a World-wide basis, although there are likely C to be sSstantia! local variations in re~uirementsl Consepwntly, i- there is merit in the Group maintaining ,,ie: of cen~res PI expertisli ~ at present these are Ea~burg, ~nfreal and Southanpmn. Close contact Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill _ ____ ~·_ should be maintained between these laboratories in developing standard tests e,g, the res' test in any associated research areas and in providing a fort for discussing [toqether with the Crslp's cpnsultants) the ~~iocs problem-areas related to biological tests. 7 0~ OF msaa (a) drhnsll~ll; product dc*lolat teams can be advised of the likely eoasegPnees or any design changes or of use of additives in te~s of biological rcs~nlc, (b) offensively: by analogy with cigarette dasignlsoh chemistry procdPr~sl cigarettes of low biological activity can be designed. me main problem anticipated is ~lt of cigarettes which do not give uniformly low activity in all tests. TLrD is no easy answer to this problem; it is diffleult to weight arts against each o~or. It present there is no recourse but O use judgement, aldogh our position is greatly strengthened by the recent qpoia~nt oi consultants. 8 SUIBY OF OB~bI~S OP B~aOCT~ PFSEAFlll in addition to meeting rha question given It ~a baginnino of ~is no:e, "U~ boEt car. we ~r~n Ba~ fu:urc group producln havs the lowest possible biological activity!" the pmgrane outlined should also put bAT into a gtTOn( PCSltlQ 13 the following areas: Discussions with ~gPlc~~ ~;~oIi;les on protocols for the testing of tobacco products and dditi~l~ neetln( the rep~axn:s of Regulatory Al~rirics, ClibPDF - v~~fastio.soni I~/WPID 21 Jply, 1981 INTERI3l faTBS 01 BIOLOGICAL IglllG, 16 JULY PY POIN'IS It ru trcld that: 1 Tbe dDCldDt 'HoI belt can we enlurs tbat future group products have tbe lowelt possible biological IctivitfP" was an acceptable format for letting out tbs biological ltndiel Ilthough variouo alterations would be required in tbe light of dilcussion, a S~in~lnting There were lame obvious gaps in our ]rnowledge and the lasting recommended that consideration should be given to examining the following lireal, (a) low-tar modern prodpcts, Since there were many design options available for the design of low-tar products, I number should be examined to determine which of the de~iln options was bolt, Such studies ~hould also allay criticisms of modern cigarettes, which imply that the use of additives etc., may be associated with increased activity, Ib) Olds-strso, Knowledge of tbs biological activity of ~ida·ltrs~ smoke was sparse, Il~o~g~ it was pointed art that knowledge of the activity of 'amblent smoke' was a more realistic objective. (c) UPJOr additives, Occasionally it night be necessary to test such matters e,g, ii suspicion was cast on such a material from a external stud~, One example is cocoa, (d) Is the test on B1( series of cigarettes do not give 1 clo~·ePt answer on nicotine carcinogenicity I iurtber evaluation of nicotine is reconrpended, but only after preliminary studier based on the Amel test, 3 Inhalation toxicity (O) It lid recommended that Sopth·pton should carry apt an 8-wssk bamlter inhalation study for comparison rft~ the rat, (b) long-term hamster inhll~tion studies should not be carried out, st lellt In tbs i·adilto iatarl, Iltlolh this position should be rlril~d in tL lilht ai m~ yphlicltionl on nolv inion~tion. (c) There would be collaboration between Bnhrl and Southrpton, with a view to seeing if information on changes in ClibPDF - v~~fastio.soni (i) bone marrow cells (ii) blood lymphocytes (iii) sperm abnorm alities could be obtained from the lone-ten rat inhalation completed at Soutbampton, 4 Diseases. ether than lung cancer There was support for the Southampton progr~n~e looking at possible models for cardiovascular disease and bronchitis, The view that if was not sensible to allocate resources to explore the difficult area of emphpsePa model-systems ~ls edorsed (but sec Consultants' comments]. 5 Additives The programme for examining additives listed in RET's draft: document was accepted, note being taken of the importance of teratogenicicy studies, Furthermore it was agreed that Hamburg and lontreal, and subsequently Soutbampton would carry out collaborative studies with a view to standardising, as far as possible, the Ames test, 6 Human studies In some cases human studies as possible (effects of smoking in pre;rla:t women, measures of respiratory function), It was agreed that while such tests were important, potential studies in such areas, inevits~!? carried out under contract must Se treated on their merits, ? Foeta~ studies tin animals) As for emphysema-1Podels the potential pay-off did not appear to !us:iig allocating any effort at Southampton in this area, However, a; noter; in (5) straightforward teratogenicity studies were needed, 8 Epidemioiogical studies It was pointed out that the ultimate test was a study of the results of epideniological surveys. 9 Human smoking The importance of associated human smoking behaviour studies (e.g. to compliment the evaluation of 5 mg cigarettes) was agreed, 10 Questions for Dr, F,J,C, poe A list of specific questions to be put to EAT's Toxicological Consultant, Dr, F,J,C, poe, on Friday, 1'1 July, was agreed, These are listed sepanrely, Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 4~ -· ··s ny~lasP 17 July, 1981 Meeting on Biological Tests, SoP~qtoa~ 15 July 1981 Short-tan and Long-ten Tests (with special reference to the beo Test) PWSEIT Dr, C,I,Ayrss Mr, B,S, wade Dr, R,E,Thornton Dr, M,B~ BilLorfa Mr, 6,A, Bead Dr, f, Westphal Dr, 6. ,smith · L, 6. OndIacxek Dr, E,D,Masset Dr. B, P, Evelyn Mr,E,B, Yilkei Mr, a,s, Dagnall Mr, P,H, Lee (CcosaltaPt) The teeting reached the following conclusions: Leg Test 1,1 The keo test was seen as an essential feature in the evaluation of tobacco additivies, and evaluations by this test were likely to be required by some regulatory authorities in the near future, possibly late 1981, 1,2 With regard to the evaluation of tobacco products (~ comparison or condensates) the kco test should be scan in its own rights as a possible component of a test battery, measuring mPtagtnicitg. However, further work was needed to demonstrate the range and teprodo~ ibility of the method, To this end, ra Montreal and B1T Hamburg agreed to compare the condensates from ~ea cigarettes using as far as possible, stzndsrd ne~ds (including statistical analysis), mt study to be completed during 1981, BAT S6u~aDton to ~ll~=a~ as and when the Ames test it develaped The tltllte aim would be a high degree of standardisation within the 6rouo , 1,3 The kes test could not be regarded as a substitute for long-ten stin-pdntirp studies of smoke conCLnsate, mis is due to the following reasons: C la) diffeIiq dose-response and (b) probable reaction to differing smoke components: ClibPDF - v~~fastio.soni e2t Ames Skin-painting Initiators Initiators and promoters, 1,1 As we have been informed by a consultant (Professor B.A, Bridgesl that fractionaticn of motor-car exhaust has already led to the identification of a new-clasp of mutagens, the nitto-substituted aromatic hydrocarbons, fractionation of cigarette smoke condensate has atrrzctl~ns in terms of identifying the compounds with mutagenic activity~ A correlation with mutagenic activity and nitrogen levels had been observed in Mo;ltreal, The meeting asked ITL Montrea! to consider whether they could cars cut such fractionation studies, 2 B14 Series This series of cigarettes involved adding varicus levels of nicc-.ine citrate to a tobacco blend with a low level of nicotine~ bltholr~ the results bo~ of long-term skin-painting and with t~e kes test indicated that a14/4l the cigarette with the greatest nicotine delivery, had the highest activity, other changer (i.e, the pres~ce of pyrolysis products from t~e added citrate) precluded a simple interpretation of the results, ?urthermore nicotine itself was without activity in the Ames test, It was recor;rnended that :urll:er studies should be carried out, initially using the Ames test to investigate various methpds of adding nicotine to cigarettes or 's condensate, 3 B15 Series This series investigated differing levels of ventilation, filter pressure droD and paper type, The cigarettes were examined by long-term sk:m-painting, in the ~oes test and, by short-ten inhalation toxicity studies, The results can he summarised as follows: Cigarette i\mes Tesr Inhalation Skin- Parameter Hont:eal Ha~olq Toxicltp e~P4~ Increase in p2eer Decrease Decrease Increase permeabilig lo Increase in filter Increase Decrease? Decrease pressure drop r Presence of Rn- Decrease NONE tilatic;a in filter NONE Effect of paper O additives I~OBE NONE NONE, L~I iij L~ z C\ F~ rV vl L~ t either alone or as added to condensate, Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill 4 rnte.rPretation of results Beyond noting the comments in section 1,3 of the note, no satisfactory explanation was offered for Yle fact that in the B14 series there was good agreement between the Ames' test and skin-painting, whereas for the B15 series the opposite occurred, Itovever, it was felt that the results obtained from the laboratories using Fnes' test would benefit if stnndardised procedures were used (see 1,2 above), ADDENDUM Subse~uent to this meeting, representatives from Hamburg, Montreal and Sout~am~ton met and agreed an a standard protocol for the Ames test, Tbey'further agreed to examine two cigarettes, to be provided by Hamburg, One, of high activity, would be manufactured from air-cured tobacco of European origin: the other, of low activity, would be manufactured from a sheet material. Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 II : 6L' ·` ·c~i· .. . L .·· I; :· ··~:~' · Y :I . ".' ·~·: AT, BRITISR·AMERXCAN TOBACCO COMPANY WMITED Td RE THORNTON ESQ I FRa~ L C F BLACKMAN R~' 6CFB/SM I D~rE 23 JULY 1981 PI1CFESSOR C G CARQ r have discussed your memo of 8 July with Alan Heard and we agree that you should go ahead to arrange for a consultancy, in the first instance for one year, at the level of e7000, Please prepare the paper work as previously for Roe and Bridges. sR'Y"; n a C81gZ ·; Z~JUL19%1 It,: b C F BLACKMAN CC R I Reard Esq L' Ch F Ch Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 al~l~/~s 23 Lly, 1981 HMg ~ I, I, L, IAIU) 1~~~9 ~ai~~~ for nxpOniOg r~n~ to eipsratta anoka mra L~lo~ for eb~,t~~ with a carbon no~dl~~~~b phnan (COlTPM) ratio of Isal than ona, eurrsot ti9aratta ~l~n has awlad in nmlll~ll l#r dalivariaa of TPM d~~ a propOrtional raductiOn in MI is shown in Or, IlacOaan'n bOOk '~~e9 and Scisntific Iapaota Of Snoking and ftaa2th", p19 frOO wbich tha ~~dng ratioa can bO ~~·d (ratio mlaa in pasna~li~) "Ivnrng(" COmporeial prodnotn bcfOra 1960 in 1980 avnilablain 1980 Ux 20 15 1,5 (0,61) titol (1,5) 33 15 i 1 23 17 2 (0.51) (1.13) (2) ~3 15 1 In our sxiotin9 sxpOsnrc regLI afh ~rgs~ in ratios rssultJ in unaoosptabia, possibly iathal, 1a1s1s of ~~a Oonoxida in tho eha&r, bocsusa oxposura Ooourn It oOnntant inttnis of PU~~I~ phw, It L for tbis a# that w bad O abandon tha nant Ixr~~~~ of 2Ow dalivarl e~u~t~~, In order to lok tba ptpblan ~~~~q uork LI baan initiatad On altarnatitra c~a~ Conditions Pliq l#t eOnoantfationn Of 91 B~~r, in ordar to produa I raapOnaa In tha rasplratOry traot lOnQar aXPosnra ~r~~~ maY be DIC111~1 and Pdlt bill eOIlditiOna CO tOxioity Oay atiilba a pr81~~, Tha outceol of this work will k C rapOrtad as soon as paasibia, ClibPDF - v~~fastio.soni -2- in the avant of this ~proach failing altarnatfva solutions will ha aouqhtt one radically difiarant approach would ha to aDoka tba eigarattu difi,nntly, sina it has baan s~g~at~d that the standard smoking t~gf~s tends to mawimiaa carbon wno~ids ronesntrationll, Altarnativsly, the nease of oeeaaxch aimed at reducing carbon nonoxlda leals (~roject IOCO) Ips) Kilos the problP for uo, G, SMITII S1 11 BEVEN Dr, C.r,Ayres, Dr, R,E,Tbornton, D,E, Conway, O Ln ~1 iV L7 L1 Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 _ ___ LII : ····i '··· ·-·i RETIWLP/Q6D 23 July, 1981 "8ow Best Can Oe Ensure That future Group Products Bave me Lowest Possible Biological Activity!" INTRODUCTION This document was discussed in draft form at the Biological Meetina held at Southampton in July 1981, This version has been amende;i in the light of t~ese discussions, 2 I3~NERi~ STXATEGI The strategy for meeting requirements within the BAT Grouo fo: reductions in biological activity should be on the sa~e basis as that for meeting delivery targets for smoke components, i,e, a Cigarette Design tianctbcok approach in which measurements of biological activity replace the deliveries of smoke comwnents, On t4is basis, measurement of biological aetivity would be related to major cigarette design parameters, the biological tests i~in~ analogous to the various different smoke components, One would then be in a position to 'der;ign' cigarettes of reduced biological acti~it~i, Problems in the two approaches ale also ;i~ilar, Just as a change in a cigarette design parameter (srch as filtration) will nor necessarily reduce the delivery of all smoke components across-the-board, there will not. necessarily be similar changes in activity as defined by the various biological tests, The main criteria for the choice of biological tests are discussed later. l'he other c3Fsnon problem is t~L~e possibility of synesFistic or antagonistic effects occurring widen two or more design factors are used in attem~'is to reduce delivery or to modify biological activity, m'both cases sue~ effects can only be unravelled after i.ntensive study, The use of additives should be viewed as another cigarette design parameter; in fzcr., additives usually merit more attention for their possible biological activity than for their effect on delivery, although both can occur (torbonite), 3 CRITERIA iY)P, QOOSINP TESTS OF BIOLOGIC~ ACTIVITY Whereas it is relatively easy to decide which s3oke components one is interested in, the importance or status of particular biolcgical tests is far harder to establish, For this reason Tests are chawecterised as follows: Recommended: Reasonably well established with a~efilate y· background of knowledge, L·~ Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill C~ Potentially useful: Promising but not yet fully evaluated, At'tentlon is also drawn to areas where tests are not yet a~vail- able, 3.1 EVAL,OATION OF ~V~JOR CI~WE~E DES~C~ PARAMETERS (paper, filter design, tobacco type, ventilation etc) Reccr~nen~ed (but currently not used on ground of cost-effectiveness] Skin-painting Well. established, reliable (altSaugh relevance to man arguable), Reconnnended rnhalalicn toxicity Useful in that it measures the general toxicity (short-term) of smoke in the respiratory tract of animals in a reasonable period of time. Poteo tially Usefu 1 Lang-te~n inhalation It is extremely difficult to produce turcccrs toxicity test by smoke inhalation, Without such an end·poin: this test is an extremely expensive ex:ensim of the short-term iohalation stcdl,~ Hoveler, it has been claimed that hsmsters are :1:? ar.il:.al.s of choice for S1:Et) Studies, Sbort· term tests - related to cancer Rmes Test Only limited studies on condensates as yet, Sebaceous gland test CoRlaents to be made by BAT C~many, NMFI Test Simple, but only fair correlation with other tests, Chromosome damage test Should complement bes Test, Under development at Bamburg and GRIDC, Test not yet available Tests related to: (aJ Atherosc!erosis Various approaches will be evaluated at (b) Bror,chitis GR&DC as potential model systems for these (C) nphysemd diseases, If the work is successful the work will form the basis of tests, 3,2 I~ Recommended W General Toxicity Often available in the literature, but esssntL1 in establishing broad category of possible Cn usage levels. IQ sl Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 -3- : Teratogenieity Essential if 'unknown' additive is to be considered, mhalation toxicity Useful in demonstrating no increase in hazard can be associated with particular additive , Short·term tests - related to cancer: Ames Test Well established method of screetling compounds for mutagenicity, Chromosome damage test Alternative and complementary method of screening additives for mutagenicity, (Other tests depending on the additive) 4 CURRENT POSI:TION The current state of our knowledge of changes in biological activity in relation to the major cigarette design parameters is shown in the following figure, The worje on which our knowledge is based is not necessarily carried out solely within ·jle BAT Group and outside studies have been considered, ii judged to be reliable, Cigarette design Skin· Inhalation Bes Sebaccous: NIIFI: Chromosome parameter painting toxicity test gland test test damage test Tobacco type O O Paper porosity O I litter vpatiii:ion ) O I g Filter T~e I O E Cigarette circumference O E; Tobacco cuts per inch I Paper type O s Baterlal (rt~, lamiaai I t~ Expanded tobacco Synthetic materials I Sidestream smoke O o Tobacco, nicotine 8 O content Reconstitution I B O Studied in some depth a Partly studied Q O A little information I Planned studies V1 LII t To be completed after discussion with 3AT Gennjny, TV VI Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 No ach table can be given far additives and flavcu:s since each and every additive mst be treated on its Writs. In this cOnteXtl we now have available advice on appropriate tests from external consultants of international repu~tion, Dr~ F.J,C. RPe and Professor B.A~Bridges, s NTURE WORK This is considered under three tise-scales: Immediate: Application of existing methods Short-tear: Developent of additional tests Longer-term: Necessary research Immediate (u,o to 1 pear) using exis~ng tests we shall systematically fill the i~asorrant gaes in our knowledge indicated in the figure (page 3)· We will also carry out any necessary vor~ on additives or fl;ivoun vi~ich the company may wish to usel We are currently establishing two short-term tests at Southa3lpton r 1~ Ames Test and a chroJlosomb damage test, aol;h are likely to be re5uired by regulatory authorities in the future, particularly wi~h regard 'co the evaluation of flavours and a~diti~tJ. Should a~ditional short-ten tests be required by regulatory authorities, we would expect to use a contract research house. Short-term (up to 2 years) Efforts will be made to develop short-term tests relaterl to atheroscle~osis, broncS,itis and cm~hysema, We will also evabatiq alternative inhalaticln tests, with esFecia! refersnce to studying low delivery cigarettes and tbe use of alternative rodents which may be nore sensitive. ~I~Anget-tP~n~ ton-going) We are carrying out or sponsoring research related to current theories of the Induction of cancer and atheroscleIosis. As and when this research cones to r'n~itioc, the results should influence the development of the test battery available at Soabaspton 1 6 GROUP COLI~BORAT~OIJ rl BIOWGIC~C *cTODIES _ .~~-~c~------- The increasing envi:ona,ental pressures to screen materials will ultinately be felt an a world-wide basis, although there are likely to be substantial local variations in requirements, Consequently, ~ n there is merit in the Group mainaining a number of centres of expertise; ;j at: p:er;ent t~ese are Ba~bu:F, bntraal and Southampton. Close contact CI I:PDF ·\:11II::, f3siio.;:om ~O sh3uld be maintained between these laboratories in developing standard tests e,g, the Paes' test. in any associated research arbas and in oroviding a forum for discussing (together with the Group's c~nss!tants) the various problem-areas related to biological tests, 7 OSE OF FESULTS (a) defensivelyl product development teams can be advised of ~e likely consequences of any design changes or of use of additives in terms of biological response, (b) offensively: by analogy with cigarette design/smoke chemistrl procedures, cigarettes of low biological activity can be designed, The main Droblem anticipated is that of cigarettes which do nor give unliomly low activity in all tests. There is no easy answer to this problem: it is difficult to weight tests against each other. At present there is no recourse but to use judgement, al:ijugh our position is greatly streng~ened by t~e recent appointment of consultants. 8 SUME~RY OF OBJEbIVES OF DIOlO~iC~RZ~SEAR~ In addition to meeting the question given at the beginning of this note, "Hsw best can we ensure that future group products have the lowest possible biological activity?" the programme outlined should also put BAT into a strong position in the following areas: Discussions with Regulatory Authorities on protocols for the testing of tobacco products and additives. Meeting the requirements of Regulatory Authorities, Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill 1~1 Br~/~6D 22 bly, 1981 Puestions for Dr, F,J,C, be 1 Is it reasonable to ignore model systems for cg$gena and 'foetal' o~laJ1 2' ~at ilould Se the best animal for a a~~oselt~sis model? 3 What are current ~ievs on the importance of %nological response in a~emsclemsis! 4 LJt is the impertance of the recently published studies on 'laay lungs' in bmas? 5 Could one use old rats rather ~~ young rats to improve model BYSt~S for cardiovascular disease! 6 Any viavs on the results of the 814 series of ei~arc:tsg! 7 ~at tests were essential to an adequate battery of tests? ~P POIX~ I~ BP DB, P,J,C, IP ~SPX To Eos~as 1 - 7, I Pqbyscna was of greater importance than broacMtis, particularly as nitrogen oxides lay be ~lic~~8 in its deral6plest. Pc shoeld ~srafo:e uo:X in this area, re would Se desirable to study an ePphpsena mole!.. In this context, work carried out by Passey ct al ape time ago csuld Se a useful starting PSiDt, Coen allowing for the criticisms aC this 3LldC=1~ hr bm~1~4 ~ CoF Ir i C~1~~ Dr ndLd by Yette~~L~) al~c~h oSviously precluded from use new, It might ~eIe~ore be ~r~ examining the effect of esoie on s~-acoaal glands al~o~h ~n!Bi;a~ results ton rats) It GBLtC were non too encouraSinrj, Baw ~stal studies, of the type proposed by professor Cppe, were ~li~alp to be a~am!. 2 Dr, Roe pointed out ~at ~e epidcdclog!cal data relating a~crosc!ercsis is poor: i; was a disease ~c#iilr to 'Wes~~ pan', ~~ was essential to analyse for separate li~3ratela fractions in zx~aricental studies, In the absence o p=3ate studies ~e aite Ca3eaJ pigeon had been found to be useful, e,g, by TBC: the rat was u3lnsl~ to be a good ~d?l. ClibPDF - v~~fastio.soni 3 The importance of imnunological response in atherosclerosis was :still, open to debate: more importantly, mueh of the dz:a on rabbits, which had been studied in detail, was probably irrelevant to the human situation, (Subsequentlv Dr, Roe has suggested Dr. David Bowyer of Cambridge as an expert in this area should a lone-of:' opinion be required), 4 Dr, Roe thought that this could be a useful test; it might be feasible to use it to show that smoking certain cigaretss resulted in a move in the direction of non-speking~ Nevertheless its current status was perhaps that of a 'technique chasing a meaning", 5 Not usefully, although other ways of priming animals e,g, through Vitamin A deficiency were possibly useful although re~!at6ry bodies would need to be convinced of their merit. 6 Or, Roe said that in his view the increase in activity associated with added nicotine was not causally related. He thought it much more likely that the effect was mediated by a non·s~eclfic epigenetic medianism i,e, nicotine was an irritant and Yis mag have an effect on subse~3en'e processes of tumour develc~mant, ~ltexnatively, as discussed earlier, other explanations are possible e,g, citrate pyrolysis products or solvent effects, 7 From both a toxicological and a regulatory point of view, the Ames test and a cl(togenic test (e.cl~ chromosome-damage) were seen as a good combination, However, the Ames test should be considered on its own merits and not as a substitute for other tests, such as skin-painting. There were biological objections to the Ilmes test, quite apart from problems arising from statistically based comparisons of Fmes test data with that from other tests. In this context Dr, Roe said that: there were fewer biological objections to mouse skin-painting, The Anes test and cytogenic test should be regarded as telling one whether or not to do further tests, B OTHEFI EYITIERS The 'inhalabi!ity' of cigarette smoke had neither been quantified nor applied to the development of cigarettes. mis was important (pipe smokers v, cigarette smokers in relacjon to smoking-associated diseasesl , 2 The interaction of smoke with other environmental agents might became increasingly important, Ideally, reasearch should be discussed with specific industries; this may prevent the matter from ending up as a 'slanging-match'. 3 Dr, Roe endorsed the importance of our current smoke i:.halation studies, although he specifically recommended their e:r=asion to 90-days, because of E,P,A, requirements for 90-day tests. This O test was oarticular!y important for additives where t~e effects V1 were not ~nowc. Sats wore the anisals of choice beet-se they had Ccl been more extensively sr~died than ether rodents. on ~ie question of hamsters, Dr. Rce said t~at the Homburger strain was not Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill IV -3- necessarily better than any other (reqardless of the extravagEnt claims made), but it would be sensible to carry out a reasona~ly shar;, feasibility study vith hamsters. Q V1 I~ IV O C~l Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 .....r s . i.. :.· . PRIVATE AND ~ONPtDENTIBL h. Y, a, lelcb, R, ii, Tbornton Public Bfiaira, YlllbaPlr, I~TILdPIQj3 21 July, 1981 Thank gau very cuch for secdinn se tSe document about product Ifs5lllty, ivhicb I have read witb interest~ IP return I t~f;uglt you G,i~l 'Ue interested is a confidential report on tht~ ·:L'j.j jt~~~:l-li~:l jO i~FSt~ri0ilSlr 3~~e8;~~ in t~L? I~t!i~ Eiv~ ~J~ ;z~;a o~i- ;r t~·J, drift of our bco2, I leave yac to jui;;~ ;;.lzt:ier ii ~aj I.l~oted or clijqcated, finally, you nay be interested to knoa that "aedical and Scientific As3ects of P~o~in~ and iiealt~'' -~z; launched Ene O C17 Ck rU O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ~ r:n? ~''' L, .· ."~'? pET/Lhp/48O 21 Jell, 1981 I~~BIX AOTES OI BIO~GIC~ laEETlNO, ThuRsOIv, 16 mlr KEY POIITS it was agreed that: 1 The doc·ent "Bow best can we ensure that future group products bPva the lowest possible biolopic?l Ictiviryl" ~ls an IcceptJble fornat ior sattin( out the blologioP1 studies Ilthoueh vuious alterations would he repuired in the light oi discussion, 2 O~in-plintf~ There were some obvious gaps in our knowledge and the meeting recommerded tbat consideration should be given to examining the iollcwing areas, Low-tar modern products, Since there were many design options available ior the design oi low-tar products, a number shoald be examined to determine which oi the design options was best, Such studies should also allay criticisms oi modern cigarettes, ~hich imply that the use Oi additives etc,, may be associated with increased activity, (b) Side-stream, Knowledge oi the biological activity oi side-stream smoke was sparse, ~lthoueh if was pointed out that ~nowledac oi the activity oi 'rbfcpt smoke' was a moro realistic objective. (c) ~ajor additives, Occasionally it might be necessary to test such matters ii suspicion tls cast on such 1 material iron an Irternll st~dy, One example is cocoa, (d) II the test on pi,! series oi cigarettes do not give a clean-cut answer on nicotine earcinogenicity a further evaluation of nicotine is reco~cnded, but only alter preliminary studies based an tbs Iws test. S Inhalation toxicity (n) It was recolonended that ~~thlpton shoald carry opt an 8-~0~ hamster inhalation study ior comparison with the rat. (b) ~ng·rcn hamster inhalation studies should pot be carried oPr, at Icast is the i~edilre iuturc, olthouph this position should be reviewed in the light oi any publications on new inionotiPn, W (C) Bers would he eolllontion between He3hurg and Southlnpton, vith ;via to secin~ ii Iniormntioo on chpnge~ in ClibPDF - v~~fastio.soni ·2· (i) bone marrow cells (il) blood lymphocytes (iii) sperm abnorm all ties could be obtained from the long-term rat inhalation completed at Southampton, 4 Diseases other than lung cancer There was support for the Southampton progran~ne looking st possible models for cardiovascular disease and bronchitis, The view that it was not sensible to allocate resources to explore the difficult area of emphyselpa model-systems was endorsed (but see Consultants' comments), 5 Additives The programme for examining additives listed in RET's draft document was accepted, note being taken of the importance of teratogenicity studies, Furthermore it was agreed that hamburg and Ilontreal, and subsequently Southampton would carry out collaborative studies with a view to standardising, as far as possible, the Ames test, 6 Human studies In some cases human studies as possible (eilects of smoking in pregnant women, measures of respiratory function), It was agreed that while such tests were important, potential studies in such areas, inevitably carried out under contract must be treated on their merits, 'I Foctal studies tin animals) As far cnphysema-models the potential pay-off did not appear to justify allocating any effort at Southampton in this area, Ilswevcr, as noted in (5) straightforward teratogenicity studies were needed, 8 Epidemiologica~ studies It was pointed out that the ultimate test was a study of the results of epidemiological surveys, 9 Human smoking The importance of associated human smoking behaviour studies (e,g, to compliment the evaluation of 5 mg cigarettes) was agreed, 10 Questions for Dr, P.J.C. poe A list of specific questions to be put to BAT's Toxicological Consultant, Dr, F,J,C, poe, an Friday, 1? 3ulg, was agreed, These ore listed separately, Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill ~1 1~JC~I IPntll~CIIC~ C;e~ RETIL~PI16D 1? July, 1981 questions for Dr. F,S,C, Roe -- 1 IS it reasonable to ignore model systems for emphysema and 'foetal' studiesl a Phat would be the best animal for an athero- sclerosis modell 3 Ihat are current views on the importance of immunological response in atherosclerosisl 4 Pihat is the importance of the recently published studies on 'leaky lungs'l 5 Could one use old rats rather than young rats to improve model systems for cardiovascular disease7 6 Any views on the results of the B14 series of cigarettes? Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 nr "'F L.; ,.j m~/46D 17 July, 1981 Meeting an Biological Tests, Oon~~too~ 15 July 1981 Short-tea and Long-term Tests (uith special reference to the res Test) PBSEBT Dr. C, f,ly~es Mr, B,S, Yade Dr, B, g. Thornton Dr, a. 8, Bilimoria Mr, 6~~~ Bead Dr, X, Yes~h~ Dr, O, Smith Mr, 6, Oadraczek Dr, E,D.Massey Dr, S, B, Bvelyn Mr.E,B, glgas B,B, Dagnall Mr. P,1. Lee (Con9dta:t) The meeting reached the following conclusions: Ames Test 1,i The Enes test was seen as an essential feature in the evaluation of tobacco dditivies, ad evaluations by this test were likely to be required by some regulatory authorities in the near future, possibly late 1961~ l~a with regard to the cvaluat!ca of tobacco products (by comparison of condensates) the Lts test should be seen in its own riqb$ as a possible comoonent of a test battery, measuring nPtagEnici~, However, further work was needed to demonstrate the range and repxo~c- ibility of the method, To ~ik end, ITL bnaeal and BAT Hamburg agreed to compare the condensates from ~:aa cigarettes using as far as possible, standard methods (fnclding statistical analysis), The study to be copletcd during 1981, B;T Sou~so$n to collaborate a and when the Yes test is dsvalaocd thera. aim would be a high degree of s~dardfsaticn within the Groua, 1,3 The ~es test could not Se regarded as a substitute for loog·ton s~i~-;inti~ studies of smoke condensate, O This Is due to ~a following reasons: (a) differing dose-response c~as (b) probabie nlction to diifcring smoke components: ClibPDF - v~~fastio.soni _· -2· Rmes ' Skin-painting Initiators and promoters~ Initiators 1~4 Rs we have been informed by a consultant (Professor B.A~ Bridges) that fractiPnation of motor-car exhaust has already led to the identification of a new-class of mutace"s, the niuo-substigted arottatie hydrocarbons, fractionatlan of cigarette smoke condensate has attractlas in terms or rbntifying the campauls with mutagenic ,ctivity~ A correlation with mutagenic activity and nitrogen levels had been observed in MontIeal· The meeting asited ITL Montreal to consider whether they could carry out such fractionation studies. 2 B14 Se~s This series of cigarettes involved adding various levels of nicotine citrate to a tobacco blend with a low level of nicotine· Althp~4h the results both of long-tenn skin-painting and with :4 Ames test indicated that B!i1/4, the cigarette with the greatest nicotine delivery, had ~e highest activityl other changes (i,e· the presence of p~'0lysis products from the added citrate) precluded a Eimple inte-?~etatial of t~e results~ FurthefiJars nicotine"itself was without activity in t,heAEeS test. It was reecnmended that furtze: studies should be carried out~ initially using the 12mes test to investigate varia,s methods of adding nicotine to cigarettes or to condensate. B1S Series This series investigated differing levels of voatilation, filter pressure drop and Taper type~ The cigarettes were examined by long-term sltin-pzintin9, in the ;Voes test and by short-term inhalation toxicity studies. The results can be sunnatised as follows: Ames Test Inha!ation Skin- cigarette paietina par~-.eter HO"tIeal Bc~b~r~ Toxi~i~e z Decrease I"erease Increase in Taper Decrease o perPeabilitY w Increase in filter Increase Decrease! Decrease pressure drop Presence of Ven- E Decrease NONE tilation in filter NONE a G Effect of pacer ~ NaE Na~E cn additives NONE ~h' g 0\ Z IV F1 O\ a C t ai-~er alone or as added to condensate, Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill c31. 4 Interpretation of results Beyond noting the comments in section 1.3 of the note, no satisfactory explanation was offered for the fact that in the B14 series there was good agreement between the Rmes' test and skin·painting, whereas for the B15 series the opDosite occurred, However, it was felt that the results obtained ,,, the laboratories using Ames' test would benefit if standardised procedures were used (see 1,2 above). ADDENDUM Subsequent to this meeting, representatives from Hamburg, Montreal and Southampton met and agreed on a standard protocol for the Ames test, They further agreed to examine two cigarettes, to be provided by Hamburg. One, of high activity, would be manufactured from air-cured tobacco of European origin: the other, of low activity, would be manufactured from a sheet material, Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Ilr, N, R, Page R, E, Thdmtoa RgTILAPI4BD 14 July, 1981 Flex-time Rack - life Sciences As gou ale nare I have some resenrati6ns about the listing oi the names a~ Biological Sciences staff ~p the ~lex-tipe rack in the entrance of the building, Tt can be seen very easily by all casual Pisltora (sewice engineers etc,,) to the site, I would, thereiore, be grateiul ii the names could be removed iarthtofth and r am sure that the staii hen would not m~nd ~i thep were ananomised to the extent oi haP~ng numbers instead, Numbers would correspond to those on the accounts degartment monthly return, R, E, THORNTON Mr, A, ~, Heard, Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 '·: ...: *,.1 · r. ·, .r. 'I '· I. L .. ·' C "'"'' ·· I J' · r :i ·.I c· c ·' '~·.' r· · : '· I· · · Memo Iram L E F BLACI(MAN ( L .I ·. ·) "' .··.~.C ,2 'I ' /r; .r· ~jl\CCI~·l(t\h "' 1. 1. i~~b~b~i~go~t. 1~ ~Cv~ i~~.4" ai~~Kk ~s;s '' (~ yre~u ~ h~BU ~u~di~q~ q 1. I~nhar~g~j lua~ Z.Q~g~ C ~ · LT Cr OI · Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ~lh b~cli~:~P4: Ilrtrim RIIO':. Liv II, i~B1 ~i-5/~t Six samples If cigarettes Ye:( received fram the ADA)R~ Three : i bright tobacco and reconstituted sheer prepared ve:e composed of a mixturr o levels of nicotine vere added~ Nicotine bright tobacco to uhich varying tcl;tntr. Di oppr~ximarely 0·21, I·Ollnd 1.R vere desired in the final iii':;'"' m, strond icr ai rhral tiBa~''~L' Yr'( PI1P1IPI in 1 limilaI uz~ iroo buriey tobacco to have similar ~icorino conienrr~ The ciga?et:er arrived uirh coded ijealiiicarion· The:ode has not yet been broken se the r;-,:le designation is :hsr srovided by A00flR~ :he cigaier:rs vere smokei in a maliiold·mo~ing machine using lbe i:i~da:d conditions of a ?j mi. puff d:avn over 2 seconds once each ~inu:r, :-i ~ondal~arl vri Y"hld hOl IjlP LDnd~lllIl Filh l(1l0nl and ~p a~~rlnl :irorad Under reduced pressure. The rciu!ting toodlnsate var veigiied and il:L:ei to 30 per cen: ~eneenrration, the Eicc:ine level of rtis s~lutian :I, irre:.iled by s~el' dii;il:a:im ant OV abi:rbance. The~ solution var rher. :ii:tl !1~ rejatePur gland sUpPBrliO" d[tiVi:YI airer dilution Firi acelose :: ...E:13US COC.eCtTETiOSS ~ for spjaceous gland suppression 'I"yf, groups of 10 ie~ale ICR Sviss r::e ~i·bj d~?r oi age ulrl painted twe limes daily for three successive days ieer the first ap~lica~ion, the animals ,::, 0,2 e.l~ of test solution~ Six days a :·i:t sacr!il:e4 and tie Siir renoved by ~rea;ro~ Fith a depilat3ry cream. d ritin senile uell ui~hin tile treated area uas lelovlE, mounted :. a :::;I ard all~vre to irdRd in ~a!iae for three to six hours. It vas then :l;:ei in jjhh ier~a:in overnight. The iolloritl dsyl the subcutaneous far var ----, CI I:PCF · ~ ~:·~::: lasi 2.. o n . ~i~gsl i': .2- c;:lped !rom:hl skin and rh~ ra3lle var stained for four hours in 0.031 h'ile Biue sulfate at 780C, It Fai Tlm"Yed from the dye and rinsed veil in tap water, then placed in G acetic se;d for three hours, 50t glycerol for tvo hours and iOCZ glycerol ior one hour. ThP stained Skins veri examined i~mediartly and the presence or absence of sebaceous glands var scored in representative microscopic fields, A sebtceo~r gland was scored as present if any cells containing lipid Pe:e seen in the appropriate position along the hair falliele, Thus, a gland ~~r scored as present even though it was largely destroyed by the test solution, 'i~e normal full complement of sebaceous glands is tuo for each pair of hairs. fhe origlaal namber of gla?ds was considered equal to the number of hair icl!icler teach ceplel of two hair follicles is associated with two glands), li;e proportion 31 glands re&aitling after treatment var therefore equal to rie:c:jer of cSselved glands divided by the nurher of observed hair isllicler. I~is value is no; aiiec~ed by artefacts of his~rolbgjc~l ~aci7uia:ion, In tvo types of situations, glands vere not "caunted" because rep:ese·:ltive fields were nor readily alaareet, When no damage pr almost no d;ra;e was observed, on!? a iec glands were destroyed in several fields and the "ccoor" uas estimated as 90·1001. Where damage was so severe that only occarionai Ela3ds ue:e seen, the entire slide uar c~;i~ted to have from 0-101 of the normal cc;ple~e~~ of glands present. in other cares, the number of observed glands ccu.red was divided b? the arsesed capleaen; of tuo glands per pair of hairs to ~::iee at the per cent of se~acecu.L glands resnining afre: treatment, Each condensate was tested first at a 101, concentration and subsequently ,~ Ir~e: concentrations as indicated by the first result, Serial studies were O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 .3· than a factor of ~15 uere continued until e3r.ceaca:ions differing 4y no mere ialmd such that ane destroyed more than, and the other lass than, Im of the gia~ll in ttl:.a:ld I:ra. at 11": 01' Sf.a r"plt rl~Li:ld (" Il't'oY linear interpPlatian between these two blf of the glands was estimated by (onren'.ratiDnr- O"g'"i' of rebirPous gland rupprlrlion atrivitg is defined ii rh;t'moun: i! D"L:idl thil Yill ~11:r0r till ii th( Ilindi :a dr mar~d area of one mouse under the conditions of the assay. i,, lang·~e!n carcinogenesis assays, the s meke condensates were ueig'ned ,ions, Nicotine levels of the 107, and diluted virh acercne to 10~ or 51, concentra and W absorbance~ The solutions solutions ue:e determined by steam distillation ,,!, then rested for mouse skin carcinogenic dclivify· I'le ~s!p~ianr uir subdivided into three pa"i iIablP I)~ vhLh uere r:artod at i~:,rralr over a period of 8 months DiYiding path anpe:lmpar into data concerning tar delivery irom several batcher ol parts per~ni~red Fs '3 USe cigarettes to pr:jec; the total condensate available for the experiment· we a,d iarli i?rv?i,l la;l I: ditijmili c: ntld~ 131 LOhPII:( jl I1Li 1X)lli3mll! f anirals that could be treated e:oup. our goal "i fC'$!O!':he op:illum ~uier o i?r the time re::ired to acpieve d ~O'h rumor incidence in each group, In all, 1:jg n;:e E1:,;la~ll 31 alplriment, icr trtar~lnt pr'isdr pi 31 to 10 uaekr. ted 10 times weekly vith 0,2 ml~ female ICa Swiss mice are being pain ~bey are examined weekly ci me ace;oae I~lu~ionr oi the rei)ective c~ndlnsates~ ,,~ tte nu*j,r and dis:riktion of tuners is nc~ed~ Moribund animals are sacrificed as dicy ap)ear. Cpcr, dea~n or ra~:iiio, the n,iot are autopsied and the largest sin tumor i:p" any moose as veil IS Iny skin tunPrs suspected of being malignant will be classified as malignant 2:e taken ier his:lloeica! '~'-i"d~io"' Skin tumors a:eO through the pl""i[ulul carlacus laye' of ii aettrtarer ii r~e tumor cells pene are coniirsed eie:oscapicllly, I··__ .h· Benzotc]pyrene assays vcre conducted on 3-5 samples of each condensate 5y isolation using: cclu~m~ chroparography follwed by three thin layer chromatographs and measurement of fluorescence, After isolation, the excitation and emission spectre of the isolates were "crean'', Recovery was estimated by the addition of Clt BP to the crude sample prior to isolation, I~SULTS-- Tar and nicotine and 83 vields: The smoke condensates from the six cigarette types fell inta two clearly distinguishable series, Samples 16, 26 and 36 delivered 22 to 26 mg, of smoke condensate per cigarette, whereas the cigarettes :i Groups 1,6, 56 and 66 delivered from 9 to 10 mg, of condensate (Table 2), I~e average nicotine content of the condensates ranged from 0,6 to 17,, In 3Eneral, the nicotine concentration of 36 was similar to that of 66, and the c:ncecr:atter, of nicotine in condensate 26 was similar to that of 56, The ::ncenr:a~ion of condensate ~b was about three times chat of condensate ib, but l:th were relatively low, BP levels were not greatly different among the samples (:a'3le 8), Survival: The survi~a! of mice treated for 36-50 weeks with either :~e 5t or 10% condensate soi:Jtions was not greatly different from the survival ci the acetone controls (TaSle 3)~ It is noteworthy that mice treated with 10% solutions of condensates 36 and i~6··those with the highe~~ nicotine foncentratioos-· .t:rvived about as well as the ?I'ner mice, There is, therefore, no evidence of E~~s;actial mortality caused t~:icocine, Inasmuch as nicotine inhibition of carcinogenesis is associated Fi:~ esCeSSive mereality, survival is important, ;: Would appear that we will have good comparative data from the mice painted both the 10% and. the 5~ condensate solutions, I Clit; PDF -!::!!::!!::!.f3 StlC.i: nill IMIILLlli 5 · Sebaeeour Eland suoprerriPn acrivitv: The sebaceous gland suppression activity of plnpler 16, 26 and 37 did not differ significantly from each ether, :alging trolr 1,7 UIG to 9/4 U/C Bable I)~ In contrast, condensate 46 var about !01, less active than condensate 66· In our experience, these differences are reproducible and significant. Condensate Sb shoved activity inleraediare jctveen those extremes, Tumor incidence: The LumbI incidences, corrected for competing cause :i death, are indicated in Charts 1 and 2 and Table i. Cigarettes 16, 26 and 36 itiive: ca~eeosarer that are core than twice as active as the condensates iIOP :i~c:e::es lE, 56 and 6L, The 5: condensate of the first groups of cigarettes .3 311e active thdn the 1.Oil condensates al the latter groups, In general, there appears to be a satisfactory dose-response effect :i:~ee~ the j~ and 10:i condensates of the various sa;lples of cigarettes, These ii:; ~2:;t no; jeen subjected to statistical ar~iyris bur there is little docb; :'.lt for ~os; ro;pl'i5'"S the dose ei!ecr vosl~ be sig;iiiiednr· Within groups 16, 26 and 36, consisting of the rae typer of tobacco, i.: Fi[: diiierer.t levels of nicotine, there is no discernible effect of nicotine :5:·ei liable 6), ::SCCSSIOS-- I: is clear irorr~ Be yields of condE7Eate and BP concentrations, that ::;E:e:re Codes Ib, ZE and 36 are of one :rle (Iype A), whereas Codes hbl ii and 66 ire :i ~?e second (lyp 8), mt tumor incidence of the Type B cigarettes fits the :I~;:i~: a Fould have prelic:ei on the baris ci parr experience, mt nore dilute ilr:!es rboueC a sig~iiicanr trend i~ilh increasing iumcr incidence proportional C U: 0\ Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ·6· to increased nicotine conrent~ In animals treated vith 101, condensates, the sa;pie uirh the least nicotine gave the iovesr tumor yield, The sample uirh the most nicotine ~ar less active than the sample vith the intermediate level Jf nicotine, particularly during the early stage of the experiment. This is similar to results ue have seep vith condensates and vith pure compounds, Llkeuise, in the first series of ei5dTeites tested by the National Cancer institute, there var a clear relationship betueen dose·rerponse effect and li:crine caneetlt:e~ian, Samples Fi~h high levels of nicotine frequently rholied a,s-alous dose-response behavio:, Ihac ist more tumors veTe seen in groups ::e;:ed with dilu:e condensate rather than concentrated condensate, Ue have found that i01t O[ OUT observations fit an equation involving -:;·nicotine fcnde~sate (T) nicotine (~) and the square of the nicotine dose The preliminary data have been applied to that efua~ian even though ;te licired nueber of groups and ;j, correlatio~ berveen nicotine and tar doses 3 ~i:h (;je Eceisae controls ·e:e included as one group). In contrast to the results l;ith Type B cigarettes, ~there ~ar no '·· iti:earidn iarroever that the carcinogenic activity of the Type A condensates Fir riiec:ed by the nicotine level. I:is uzs true in mice treated ~ith both i:ces of condensate. These results are diiierenr iras those ve expected bared F: studies o: pure eclpoc"ds E~d p:e"i0"s experiments conducted by the Carte: T,Jt'.tutt, "~e seba:eous gland suppression data for each type of cigarette :;~Cenrare S'tdic:ed tie Ireai of:a:c:aagenic activity quire c~e!l. ?here Fas :: rigni:icalr di:ie:ence in;:tirit! among the Type A cigarettes and there :II a trend ci increasing sebeceo::s Q!and ~upF:esjion activi:? Fi;~ increasing Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 .7· :icotine levels among the Type B cclndensafes, Our previous experience led _r to expect that nicotine concentration would be positively correlated with rebaeeous gland suppression ae~iviiy, On the ether hand, there vas no general : difference in sebaceous gland activity that would have predicted the much i higher carcinogenic activity of the Type A condensates in comparison with the jl:pP B c~ailnlater. Our larr rcpaiea:l his rhwp dlar II)ICOWI $hnd suppression is a better predictor of carcinogenic activity than is the content ci anv individual chemical co~stituenr of the condensates, The devia tion ~3se:~el in the study is, therefore, of great interest, BP levels had no r·lationship with either sebaceous gland suppression ~:~ivi~r or carcinogenic activity, The data thus fat available leads me to conclude that the Type A :;~~re::es are providing the anomalous results that deserve further study, ~ :i such unusual samples might help to identify important cocarcinogenic 3rVrIaI YL'LCIII:~I: rn~lCII~I~U113 ~VI L11L) effect might be i~::::I in the smoke~ These could include the absence of a nicotine-activated cocarcinogen ;~ ~~:r one particular batch of tobacco, One could also postulate that the ::rse-:o of nicotine in the tobacco before smoking night give rise to products C .:·. C3*Linn Ik~l ;~b;~i~s~ rJir:nnoan.r;e ~~rl ~~a hrnrlii~~in~ nC rk.ra ··-··I J*IV~~L16 LII~L ~IIIIIY~LSU C~I~·LIVSSIILJ~J ~1IU LII~L LIIF ~IVVV~~~VII VI ~LISJF ::ducts is different in the ti·o types of cigarette , This la tter poss ibi ii t~ :::: -e checked by smokifig Code It cigarettes in quantity and adding nicotine to to provide three batches of condensate with different nicotine :~ese "spllicd" condenJaies vill ~Sen be assayed for sebaceous gland i,~;:Essicn and carcinogenic acriv;t?~ Ln 0\ Glib TABIE 2 Tar and Nicotine Yield of LxperLentat Cigateetes Average Nicotine Average Tar Yield dverage Nicotine Yield Concentration mg,lcig, i 5i mg,/cig, ~ SE mg,/g~~ ~ SE Cisarcra ~ Ib 22,ai 1,0 .141~01 a.] _i .3 2~ zs.l~ l.o 1·lai ·O' 1''1 -i '07 IO,i t_ l.d id 10.5 f ,L ,22 i .01 ~ 21.1+ 1.0 ii ~·1~ ·' 52.0I1,L ab 10.3 i .f .66 i .04 b~,3 i 1,2 Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 TABLE 3 Survival of Mice on Experiment Mice alive in part b~ B (50 vks,l C (36 uks.) Cigarerre Dcse Numbe! 2 Number 2 16 loll, lol 96 26 11 9h 90 " 91 89 46 " 65 87 8 80 56 " · 79 90 9 9f a6 " ~2 loci Total 10% 545 91 22 88 16 j% 100 95 26 a 95 90 36 " 99 91, 46 " 90 92 50 100 Sb " 80 91 24 9E 66 " 41 Total 5; 561 94 120 9i Bcerone con:ro;s iZ 90 30 165 O Cr: OC h' h` Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 Table 4 Stltclpu~ Cbnl Sapprlla!pn letivi~y o[ S~h Candln~ater Sebaecous CIand Suppression Sl~p!e Aetivi (un!tsl ~---L--------LL, 8,5 7,7 5,9 5C 7,6 10,5 ? CII ceil of ~lbl~rara $ileC rupprer~ion atl:viry !r i~i:?i a, sil: ~sdn: o: atc·ial tL: u!ll dailr~y l'ali I~: ~i.g13n(l In Ile rrcsrcd arcJ oi ons moat Udpl :!le conCl::onJ of ti~l allay. 0\ TV 0\ TABP 5 'lumor Incidence of Mice no Experiment (36· 70 vks,)n tgare tte Dose Hiee with Tumors Corrected Tumor incidence (Z) 16 101, If 55 26 II bb 57 36 " 42 50 46 a 21 26 56 11 29 41 66 11 29 37 16 5% 38 45 26 11 26 29 3t " 36 38 116 II 11 14 jb " 15 2h 66 " 22 30 Iceeone c3nt:nls 0 O ~ Only a few raDrs have bees seen or expected in Series C, w'nich has been on experiment only 36 Weeks. O Ln 0\ FU 0\ FU C~J Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ~BU 6 Seatiaticll In~lyJia of 'lomor IPeidence Dau 60-4 7 Yccb Groaps frcnd CDIPPICd Chi 5601rC SE 10~ 16, 26, 36 ~05 ·56 303 46, 56, 66 O 114 163 .la 5~ 16, 26, 36 j,l ·66 130 jI ~6, 56, 66 253 127 ,024 i T' ttcs~ st~tisric (i); ~ ~osirive Jign Df T indicatcf ~hl: inc:rased nico~!ne is assoc!ared vith inc!cised tumor incidcnce~ ~Si~Ple tail, O k a ClibPDF - v~~fastio.soni TABP 7 Regression Egua tions Codes 46, 56, 66, aeetone control T N N2 9' C~~1AV,T ·t A2 AV,N i A3 Av~N2 C A~ A2 A3 R ,06 ,20 -- -- 173 ,08 -- ,17 -- 185 ,15 ·· ·- ,09 161 ,04 ,087 ,119 -- ,91 ,06 ,162 -· 1047 .8? ,004 ·· ,389 ·~146 ,98 -,005 ,013 ,331 ·~127 ,99 Ioro: Rb,Si' .i4 RalH2); ·61 Q Cri Cnl 0\ IV Ln Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 a Bentopyrene levels of Condensates BP level lunber ~glgm~+ S~) Code of assays iC 3 l,oo~ ,07 26 3 1.05 i ,ol 36 i 1.13~ ,10 !b 3 1,61 , .oa 16 3 1.14 i ,06 06 3 1.s3i ·02 - Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111