BIOLOGICAL CONFERSNCE Southampton 9-11th April 1984 Distribution: Dr. L.C.F. Blackman Dr-.P.J. Dunn Dr. F. Seehofer Mr. R.G. Nicholls Dr. C.J.P. de Siqueira Dr. N.H. Bilimoria Dr. S.R. Evelyn Ors. G.A. Road/W.W. Templeton Drs. G. Smith/P.M.M. Godden/E.D. Hassey Dr. R.E. Thornton Dr. K.-Westphal c::> CC, BATCo document for Province of British Columbia 8 November '1999 BIOLOGICAL CONFERENCE Southampton, 9-11th April, 1984 Delegates Dr. C.I. Ayres (Chairman) Southampton Dr. M. Bilimoria Montreal Dr. S.R. Evelyn Southampton Dr. P.M.N. Godden Southampton Dr. S.D. Massey Southampton Dr. F. Seehofer -Hamburg Dr. G. Smith Southampton Dr. R.E. Thornton Southampton K. Westphal Hamburg .J-. 7 Session V only Mr. G.A. Read Southampton Dr. W.W. Templeton Southampton c::) co BATCo document for Province of BritiSh Columbia 8 November 1999 BIOLOGICAL ODNFERENCE Southampton 9-11th April 1984 Monky Introduction, including a recap on the Ptino C.I. Ayres (Conference Chairman) SESSION I Project RIO .The approach to Project RIO Southampton 7 be story so far - in-vivo tests Southampton Mutagenicity and cigarette design Hamburg Mutagenicity of commercial cigarettes Southampton Montreal up-date including fractionation Montreal SESSION II Specific'topics relevant to RIO Deproteinisation. Southampton Vintage cigarettes Southampton SESSION III The RIO test requirements An inhalation carcinogenicity test: (see enclosed discussion paper) Southampton Differential enzyme induction Southampton The mutagenicity test battery Southampton SESSION IV Effective inter-laboratory activities Southampton A "RIO-style" project in action Tuesday SESSION V 7he-biological activity of nicotine Acute toxicity Hamburg Hutagenicity and carcinogenicity 1hunburg Other possible actions of nicotine Louisville/ Southanpton Piogramme-of the Genmn-Verband. 11amburg High nicotine tobaccos Montreal SESSION VI BAT needs in the future background (Z--*) Trends in legislation Southampton CD 4 :1. Trends in epidemiology Southampton c-n Synergism/antagonism in complex mixtures Hamburg CkD BATCo document for Province of British Columbia 8 November 1999 -2- Wednesday % SESSION VII The needs of BAT in the future In the light of Session VI and other background, what are the types of problem that will need BAT resources. Topics that come to mind include - Will the needs be dentred on mainstream smoke or is passive smoking the growth area? The use of additives? Maternal smoking and effects on progeny. Clearance mechanisms. co Q;q BATCo document for Province of British Columbia 8 November 1999 BIOLOGICAL CONFERENCE Southampton - 9-11th April 1984 ProJect RIO 1. A draft survey of the relevant biological results from mouse skin painting studies had been circulated prior to the Conference. It had been concluded in GR&DC that the main design parameters available to reduce specific activity were: - Incorporation of stem - Incorporation of sheet materials such as PCL, PRT, SRT and Gerlach - Reduction in circumference - Addition of nitrate this was not perceived as a viable commercial possibility in view of the increased deliveries of* -..n4.ProsaTiy.gs -and oxides.. of. nitrogen Increased flow rate' aecre ased activity Choice of tobacco, grades offered only limited advantages. Pu.ring - the. meeting,, two re made; _points we a) There is a small, but definite indication that finer cut tobacco leads to a reduction in activity. b) The increase in activity associated with filters, certain tobacco types and sheet materials should not be forgotten. If the reasons for the increase could be determined, then it might be feasible to reverse the trend in future. cigarette-designs. ' -- ' 7 ' ' T e " he creeni ~ es test is t -main ' ' say being used ng as . ' - " in Project RIO. From the results of cigarettes evaluated to date, it is clear that Cigarette brands c&n,.be readily distinguished. This in-contrast with.the earlier mouse skin painting ..results. . An. unfortunate side-effect is that the Sensitivity increases the probability of an Ames League Table appearing. A further unfortunate examination is that, to date, it is not uncommon for BAT brands to have a higher result than those from (ZD the opposition. CD co BATCo document for Province of British Columbia 8 November 1999 -2- b) Important fractionation work in Montreal indicates that the Ames activity is associated with the basic materials in cigarette condensate. Again, this is in contrast to the mouse skin painting results, where the bulk of the activity resides in the neutral fraction. Any response to this observation must wait until the initial work in Montreal has been completed. c) Initial results indicate that reduction in circumference reduces activity. d) Early results from Hamburg indicate that the addition of casings and flavours can increase the Ames activity. Observations from Montreal suggest that certain other casings bring about a reduction in Ames act4vity. e) Ventilation brings about an increase in mutagenicity which with Canadian cigarettes was not significant. German cigarettes however showed a significant increise. It could be important to evaluate changes in design features with a number of cigarette types as the interaction with the tobaccoblend could well be important.' '-fY:-:: -Montreal' is -'to -produce a -review examining variations in design parameters and mutagenicity. 3. 'Only very limited work has,:been -compf6ted -on Ideproteinisationl-of-tobacco. In view of th& sensitivity of the Ames test to nitrogenous materials and the current interest. in sugar-aminoweid' interactions in flavour generation-, this work will continue, albeit at a low level. 4. Professor Nicholas Wald has an interest in vintage cigarettes - he is searching for information to support the view that a 1980's cigarette has a lower specific activity than the cigarette available in 1950-1970. The inherent problems associated with the aging of vintage cigarettes were discussed and it was agreed that it would -worthwhile to examine any vintage cigarettes or simulated 1'.--vintage cigarettes -we o--eould -obtain-- It might lye feasible to 'influence 'the tone of -Wald's eventual publication. -Ames. 5. Clearly, we need tests in addition to the Currently, Southampton are,obtaining encouraging results from an enzyme induction_ assay. ess.ence, rats are exposed, rvia inhalation-, to- cigarette smoke for four days. The levels of two enzyme systems P448 and P450 - in the lungs are assayed. These enzymes are involved in the metabolism of materials. In general terms, the P450 system leads to non-toxic metabolites, whilst there are indications that the P448 system can lead to carcinogenic metabolites. There was general agreement that the work should be actively pursued. CD cc BATCO document for Province of BritiSh Columbia 8 November 1999 -3- 6. Yeast has a nucleus, cell organelles and DNA arranged in chromosomes from both the "mother" and "father". Such an arrangement is much closer to the mammalian cell than bacteria. Also the presence of its own enzyme system makes it a useful cell in measuring mutagenicity, thus supporting the Ames assay. Preliminary work suggests that an assay based on yeast can differentiate tobacco products and so support was given to the incorporation of a yeast system as part of the mutagenicity screen. 7. The eventual need for a long term bioassay, preferably based on inhalation, was considered. The significance of such a test to the tobacco industry was discussed, particu- larly if the animals used were pretreated with a known initiator, such as radon. No firm conclusions were reached. It was, however, agreed that Southampton should explore the feasibility of a long term assay for possible use in the future. 8. Several new methods for measuring smoke activity have been established both in GR&DC and other CAC countries. In order to -va-lidate in-house test procedures, a study four low tar cigarettes from Germany was initiated. Cigarettes are tested in both BAT laboratories and at -do nfideht7ial contract laboratbrie~ Initial.results. The_'d_o-'_ope'ratioii- biitween Hamburg and- GR&DC has ;been' very effective-. b) The Ames results from GR&DC, Hamburg and the contract house are the same. c) Sub-chronic inhalation studies are underway at both GR&DC and a contract house. The results should be available by the end of 1984. The biolozical activity of nicotine n he mutagenicity-bt nicotine plus minor "alkiloids-,' only the-E.Co_li "ind' SCE tests gav'e a positive n response. At normal dose levels, there is no problem with nicotine with regard to mutagenicity. ..A possible gap in our knowledge of n'icotine-may be related to chromosome aberrations. -It'was suggested tha -t a study e:'Famining chromosome.aberrationi; both in vitro and in vivo'was under- taken at"a contract laboratory. Nicotine, cotinine and N-I-'oxide should be evaluated.. The results would allow the Company to place existing data into perspective. A protocol will be prepared by Southampton and circulated to Hamburg and Montreal. (ZD CD CO BATCo document for Province of British Columbia 8 November 1999 -4- 10. The cocarcinogenic effect of nicotine is still unresolved, there being major problems in designing a study to clarify the situation. Using high/low nicotine tobaccos would introduce other variables whilst adding nicotine to condensate may not be realistic. There is evidence to suggest that material added to condensate does not behave the same as if the materials is "native" to the condensate. No further tests were considered necessary; the situation thus remaining unresolved. 11. In all assays relating to testing nicotine, pH effects must be considered, as there could be an increased response at higher PH values. Adding nicotine to condensate has no effect on mutagenicity. 12. The role of nicotine and cardiovascular disease was outlined, in particular the role of smoke in decreasing prostacyclin and increasing thromboxane levels. The significance of such markers in a "cardiovascular bioassay" requires further discussion. A review paper on the role of nicotine/smoke and cardiovascular disease is tq be prepared ~_by. Hamburg. Nicotine has been shown to be chemotactic thus attracting neutrophils; this could prove to be an important area of work. ~biiiding --,of,- nlcb~tifi76:1736- lymph.ocy.t.es-is of unknown 1.7TIfe s gn . ad-ci~--i,~--tlid--~r-~'ec-e-ptc-Ff'-s"ites may have no specific 3- ttc~ function. An awareness of this field of research is, z.,however..important. 14. The role of nicotine/smoke in cardiovascular disease is becoming less definite, but is still closely associated with peripheral vascular disease and cerebrovascular disease. The role of nicotine could be only a secondary factor to diet, lifestyle, etc. in all such cardiovascular conditions. 15. Possible effects of nicotine on the foetus include lowbirth weight and-perinatal-mortality. Such changes -7, -could be--.associated with-changes in blood flow. The role ---..-.-of carbon monoxide. was.di.scussed briefly. 16. There is a need for a ''teratolo'gy type" study examining ..,nicotine. Existing data is not satisfactory, perhaps due that this test area is new and only a limited -.amount of information is. available. A review.of smoking and teratogenicity is to be prepared by Southampton. The effect of nicotine may be evaluated under contract. CD CD 1-1 (JI BATCo document for Province of British Columbia 8 November 1999 -5- Needs in the future - Background 17. Guidelines from advisory committees require tests on significant alterations in cigarette design, such as use of non listed additives. Generally agreed that BAT could handle most of the tests necessary, either in house or under contract (if long-term studies were requested). The pressures from various countries restricting smoking in public places, transport, etc. suggest a continuing need for work on sidestream/ambient smoke. 18. In evaluating complex mixtures, great care must be exercised in carrying out the tests as so many variables have to be considered. Standardisation of techniques will give greater confidence in comparing between laboratories. Simple addition of toxicity of individual components is not the way to determine the toxicity of a complex mixture, as there could be synergism/antagonism. 19. The policy with regards to publications will be raised at the ~ne-xt Research Conference. The needs of. BAT in --.the future -- Discussions based on -observations of.-the..previous two days-... _ Cai-diovi-6i27ulai`Dfsdase_ _ -;Biological'models for cardiovascular disease_are not that numerous, so when something does come along, there is the tendency to immediately jump on the band wagon. In recent months, the measurement of prostacyclin and thromboxane in smokers and non-smokers has received considerable interest. However, it would be unwise to work on the fringe of what is a specialised area of research. It was recommended that the Company maintains a watching brief in this area and review the situation in 1985. By then. the initial surge -of -erLthusiasm, should have- s_et_tled._'ddwn" and-:.a. realistic .1. :;Lppra sa . jckh - bd -made on the _r6f6vi3.Ace' o'-f" the measurements -with regard to_c&rdidvascular.'dis.ease.' 'A review.of...the . iole-of smoking and-ca. irdiovascular disease-wi'll'be prepared by-Hambtrrg in particular the role of prostaglandins. Smokirig*and pregnancy ......Although.-& teratogenicity assay has shown that carbon monoxide has no effect, nicotine has not been evaluated. This gap in our knowledge should be filled and a study. under contract should be considered. A review of the role of nicotine and the effect on the foetus will be prepared by Southampton. UM BATCo document for Province of British Columbia 8 November 1999 Passive Smoking The passive smoking issue is used from a non-scientific standpoint to reduce smoking per se. Such shock tactics as pointing out health risks to other people, particularly children, make the smoker an "undesirable", even though the basis for a health risk is unfounded. The situation is complicated by smoke components being measurable in non-smokers. Although the demand sidestream, the Company dould toxicity for a product sidestream. Thus .cheating" simply Independent Scientific Committee does not data, it was thought prudent to ensure that show no adverse effects on sidestream designed to have a lower visible the Company could not be accused of by making the sidestream invisible. It was agreed that the current level of work on ambient/ sidestream should continue. By the end of 1985, a comparative inhalation assay for both sidestream and ambient should be available in GR&DC. "Additives Slight change. of "emphasis by--members of-the ISC has e'd' pressure"on-the 'u se 'of 'a creat dditives. Volatile e e' _f6d:-fiem`i6a_ll'y~b_' t ii 7 7subgtahd~s 'dan b - valua -C y r nsfer studies --.'Olus use 0"f -oublisihed data -ass'u-ming inclusion levels are low. For other additives, Hamburg will be increasing their -worJc-- _load- usi_ng,:-_Ames as a pre-screen. A positive in the Ames test may result in Southampton testing the material using the inhalation system. Assuming it is not a "commerical exercise", this will present no ethical problems in using animals. It was agreed that all work involving additives should, where possible, be carried out in BAT laboratories. Human Smoking Conditions It-may-be-a requirement to evaluate cigarette smoke/ condensate toxicity generated by human smoking patterns. It was considered important to be aware of the different ways a cigarette could be smoked and attempts made to evaluate the effect of puff volume, profile, etc. on toxicity should be initiated albeit within existing resources. Any observed differences could well be used to advantage in future product development. Chronic Obstructive Lung Disease (COLD) CD Recent evidence points to a clear relationship between CD smoking and COLD (measured by FEV). This potentially important area is to be reviewed by Southampton and discussed-n at the next Biology Conference. The possible animal models for fibrosis, emphysema, etc. can be considered and their relevance to the Company established. C.0 BATCo document for Province of British Columbia 8 November 1999 -7- Promotion The promoting effect of tobacco smoke appears to be gaining momentum. Perhaps the best approach for the Company is to examine the interaction of smoking and environmental hazards, i.e. examine any synergistic action of smoking and how it may affect progression in the multi stage development of cancer. Promotion tests are becoming available and the possible use of a yeast and a mammalian cell transformation assay should be considered. Southampton will prepare a review on initiators/promotors with reference to cigarette smoke prior to the next Biology Conference. The mechanism of carcinogenisis will be included to keep up to date on such areas as oncogenes. Hamburg are to contact Mohr re his epithelial cell line, which may form a basis for a promotion type bioassay. "Clearance The whole area of clearance/inflammation is important -enough to examine in more detail than at present. The exact methods of.evaluation were not considered and Southampton is ~tW-taxo-Lore-the options available. Susceptible Individuals If a "marker" for individuals susceptible to cancer was available (e.g. P448/P450 ratio), it could best be used by joining onto an existing survey. There is to be no immediate action in this area. C::) "C' (ZD BATCo document for Province of British Columbia 8 November 1999 C) BATCo document for Province of British Columbia 8 November 1999 The ApDroach to Project RIO The main objective of Project RIO is: "To produce an acceptable low activity cigarette (10 mg delivery). The target associated disease is cancer." The work is progressing along two lines examining (1) key commercial cigarettes from participating CAC countries, and (2) the evaluation of experimental cigarettes where chemical and physical parameters are altered. The initial screen should be the Ames test. Following the measurement of the relative mutagenicity of the co=ercial_ cigarettes fromAny one country, a confidential repart is isslwd to th.at'__*f_ountry_. At this stage, -no-attempt-is made to relate activity-to cigarette design. Wben al:C'- the ciga _~~ttes from all the countries have been examifi-ed, only then will mu.ta'genicity and design f ei~_t u res ITe l6oked at toget:her- By, then, further information orf -cigarette con'*~trti6tion 'Fm-y be available, '"thus allowing comparisons' using common design features. Finally, a report will be issued bringing all the observations together. Even as an early screen, it would be unwise to rely on any one test,. so all samples are also to be evaluated using the-enzyme induction assay= This is based on the whole smoke exposure of rats f or a 4 day period. For the experimental cigarettes, sub-chronic inhalation will also be used as appropriate. Beyond this initial battery of tests, all of which are "in-house", a number of further tests are available. BATCo document for Province of British Columbia 8 November 1999 Cr eroject Rio 1 . The evaluation of key national bands. 12. The evalwation" -ofi chemical and physical changes. Initial screening should be with the Ames test. d z 0 Cr Project Rio .pommercial Cigarettes Determine the relative mutagenicity of cigarettes from each of the OAC countries. A Issue a technical rebo*rt. W,hen the cigarettes from all the countries have been examined correlate mutagenicity against common design features. Issue a repqrt bringing together all the results. CO < M > cc cc (n T- C\j CY) U) U) U) BATCo document for Province of British Columbia 8 November 1999 0 0 CL 0 P 00ject Rfoo ~porporate - to q,,O,ysign features from the commercial cigarettes. into experimental f,Hrth'' S,~Mples for n g. 96)KLS~00[ Ui a) >:~ E x C CZ -' :3 4 :: LU C/) BATCo document for Province of British Columbia 8 November 1999 0 0 0 CD CD 0 Q0 z 0 CD CD 86 K-SV001 project Roo., Others? -hotagenicity. Warnmalian' ell ~t ainfing'! mouse skin 0 Inhalation arnstbt t Promotion te"St~'~--in vitro and in vivo. ciliostasis, etc. te Not cancer rel,4ted, ratology, Project RIO - The Story So Far A review of mouse skin painting studies from the Janus, TAC and Lokstedt experiments has shown that the carcino- genicity of condensate can be affected in a number of ways. How a cigarette is smoked may alter the products of combustion which, in turn, have different activities. As a general statement, increasing the linear flow rate through the cigarettes reduces the activity of the condensate. A _reduction in circumference has been shown to- reduce activity. _-The~_matiiiial-tUrnt-wil affect :the nature of the smoke. Burl ey.....tobacco-- is,--usually k~gher -.tn acr-tvity than f lue cured, whilst'_many.-_-of -the --rfflconstJ_tution- processes also ,7-bri n gabout AnoweAng -.~Ff..aAlvity.- Sevd-ral RTS products fn fact':~tncre"e-ad__tivit:y- -and-. under.-atandiag why this is. so may well lead t-o a revers"al providing a low activity product. Added nitrate to tobacco reduces activity. However, the elevated levels of nitrosamines and oxides of nitrogen make this a difficult area to pursue. In the immediate future, the work will involve examina- tion of novel cigarettes, comparison of stem and lamina, plus ways of increasing the linear flow through a cigarette. Although a great deal is known of several of these points from mouse skin painting, their relative activities in up to date "early screening assays" is not fully docu- C'D mented. BATCo document for Province of British Columbia 8 November 1999 Roo The story, po ifar~- ,668b, -akin painting studies Review Of a. carcinQ~'enicity` .b. promotion.,.,, C. hyperplmi~ fEvaluation of commercial cigarettes 'Evaluation of experimental cigarettes Vn 0 1 Ina (3) (J) cy) o U) Cz " L = -0 - - 03 C cz 0 CTJ 0 U- -j 0 0 BATCo document for Province of British Columbia 8 November 1999 Summary of mouse sidn,painting studiesj Puff volume Circumference Permeability 25 mli 31.5 MM, High 100%- 10 ml, Standard - 35 m! -25.3mm. Low. 75%- 1 OG ml 50 ml 19 mm CD 0 50%- E Cc CD 25%- 0%- BATCo document for Province of British Columbia 8 November 1999 of mouse skin painting studies: Tiobacco type Tobacco based* .Additives sheet materials, I I Indian sun-cured Arenco Potassium malate 1 009~-' Cigar tobacco AMF Potassium carbonate Flue-cured lamina Borgwald _75%- 0 E 50%- (D 25%- 0%_ Burley.larnina. Potassium nitrite P.C.L Potassium carbonate Flue-cured stem PRT with steam treatment SIRT Potassium nitrate Copper nitrate Burey stem :~EEthgdol -extracted-.- tobacco Sodium nitrate Gerlach sheet from ethanol extracted tobacco rrr-~00 I- 1101 BATCo document for Province of British Columbia 8 November 1999 S : 15 I S '-f-S) E :3 CC . CID ic U) U) U) 0 0 0 CI) If) w w wz z z w w Cz C: E 0 Cn =3 0 Cz E Q) =3 cn a) > a) - CD 0 C: Ca _0 =3 . C+/-) U-1 - ) t3) >(I) EL < 0- CD BATCo document for Province of British Columbia 8 November 1999 0 0 0. 0 (D 0 0 0 CO Z 0 -3 Cr coqc, ~J t700 Project Rio Cdmmercial Cigarettes Australia* Germany Brazil "Canada All experimenlial w''o;'rk completed by end of April. 0 0 CL 0 0 CD eroject Rio - the immediate future 1 Novel cigarette, design 2. Lamina v Stem (burley/flue cured) 3. Heat treatment 4. Circumf6r;ence 5, Puff volume 6. Storage "conditions zo C4 CO) cc cc cc CD .(M cr) CD .IRT 04 CO CU (7) 41 C) =3 LO CO CU LL C-4 T- BATCo document for Province of British Columbia 8 November 1999 CD CD (D . CD CO G) Influences of design parameters. 1) The type of tobacco influences the biological activity. 2) Changes can be expected from expanded tobacco, filters and different puff volumes. 3) There is some evidence that compounds of flavour and casing material can increase the biological activity. BATCo document for Province of British Columbia 8 November 1999 OBJEKT VARIATION RESULT Burley. Tobacco Orient 0 Virgin .She*et M 11/7 M 20 thin Diameter- normal thick Paper double Additives K-acetate 0 (paper) .- 7--Na-z;-acet-aJe Na-cit rate - N a- acetate No-acetate + F' bre' No-acetate+ f i bre +succhrose + Tobacco cut width .0 RESEARCH& DEVELOPMENT AND PRODUCTION 1983 Ln C"D 1%0 BATCo document for Province of British Columbia 8 November 1999 CI% C*4 IA .IC C*4 90 U) w 0 w F- D F- w I.C CV w 0 > of z INC 0 xj < Cr IL Ui LLI a .0 in 0 Ln a In a in In Ln M tM SINY183AS8 d6 ON BATCo document for Province of British Columbia 8 November 1999 nr= cu Q) CD VD VA C) Lr,) M C) A C) LD C) OU C~ C) 0 CD < CD C:) C:) CD C) C:) C:) CD CD CD CD .-JCD C) C) C) C:) C=) C) (ZD C) T_ M M --i %--q CD C~ n CZ) C) CD C) CD j0 BATCo document for Province of British Columbia 8 November 1999 SCEs in CHO 23 - &Vanillin 20 Menthol -u,m" a r i n 10 75 z .7 0,2 tng/rnl lnkub.vol. SI-SI-00 I z I , BATCo document for Province of British Columbia 8 November 1999 Mutagenicity of Commercial Cigarettes The experimental work on Ames bacterial mutagenicity on condensate from commercial cigarettes from Australia, Brazil, Canada and Germany is now complete. The data from Australia has been analysed numerically indicating that BAT possesses the brand with the highest mutagenic activity. In Brazil, the company has brands at the upper and lower limit of activity. However, two cigarettes from this market were observed to have considerably variable activity over the 5 times the assay was conducted. This same phenomenon was found for two of the German cigarettes, whilst the remainder of the German series gave a consistently well separated ranking. The possibility of these 'variable' cigarettes having a widbr manufacturing. toleraaice was discussed and investigations -into this effect :slFould be pursued. From the CaCnadian- -brands, one -non-BAT- brand appeared to. most mutag,en-uc, whilst the remaiiider--of the group appeared indistinguishable In activity. To date, the consistent feature of the -cigarette producing the most high ranking condensate from each country is the possession of a ventilated filter. However, the full design characteristics of each cigarette is not yet known completely, therefore full comments on design features in relation-to mutagenic activity cannot Yet be made. The "kemain-inig"-datA be-numerically-;~-analysed; -individual reports issued to the coufi7tries conc;;rned, :then a combined report, drawing the data together, will be prepared. CD Url k_111 C-TI U-4 BATCo document for Province of British Columbia 8 November 1999 C? 0 CY CY 0 1- 0 a .J I- ca 0 < < w CO CL CO 0 ad z Cl) V 0 LU W 0 za . U1 Ui M x Op C%j Uj -CO CL 1-1 LL z CL L-CI4 Z LU CL 00" 0(j) Oct L Z CM Z_ CO CY BATCo document for Province of British Columbia 8 November 1999 0 TOBACCO PRO'CESSING 0 0 TA 98 MUTANTS/PLATt CD 400- 0 320- Control 1. 1 (D 'A 0 HHD 1.0 240- 0 Control 1.8 0 F 160- TSB 0 1 . 80- 0 6,0 lio 160 240 3bO ug PMWNF/PLATE AUSTRALIA Winfield B&H Marlboro HALLMARK Relative Mutagenic 1 1.3 2.0 2.3 Activity PMWNF mg cig-1 13.8 14.3 15.3 7.8 Filter Ventilation % 0 0 9 26 BATCo document for Province of British Columbia 8 November '1999 :r 0 0 E, c0 z 0 cr TA 98 MUTANTS/PLATE 420- 336- 252- 168.- s ~7 0 0 1 84 0 i 60 PROJECT RIO BRAZIL 0 COLUMBIA .00 A Marlboro 000 - 0 PLAZA 0 Galaxy 00, HOLLYWOOD ? ips ? 120 lio 2~O 3;0 mg PMWNF/PLATE fi I. BRAZIL PMWNF Relative Filter mg-cig-1 Mutagenic Activity Ventilation COLUMBIA 4.4 ++++ 60 Marlboro 15-5 ... 0 Galaxy + 22 PLAZA + 0 HOLLYWOOD ? 0 J.P.S. ? 0 C-M C1--,) BATCo document for Province of British Columbia 8 November 1999 0 PROJECT R10' 0 .'2 CL CANADA TA 98 MUTANTS/PLATE 420- 336- Craven A 252- *X + + *X + .168- 00 + X PLAYERS LIGHT z 0 Du AqRIER + MATINEE cr 84- *Mark 10 0 Export A 0 4 120 160 240 300 619c,-Sf,7001 f-19 PMWNF/PLATE CANADA Craven A Export A Mark Ten MATINEE DU MAURIER PLAYERS LIGHT PMWNF Filter Ventilation mg.cig-I % 13.3 17 12.3 0 16.8 0 10.7 0 13.6 0 15.0 0 Un BATCo document for Province of British Columbia 8 November 1999 P K 0: 1 E C-F P, 10 GE R M A N Y KF- IV -r z 0 cr 0 w t, o I ;o pmwNr PLA-M 3 ;0 ~40 .4- t-I Mutagenicity of cigarette smoke condensates and fractions Three brands, viz. Gauloises (dark, air-cured), Oriental (oriental) and Senior Service (flue-cured), selected for the studyat the 1983 biological meeting are being fractionated by the Swain et &I. and Elmenhorst and Grimmer procedures. The fractionation and mutagenicities procedures have been standardised employing Canadian Check cigarettes (flue-cured). Good reproducibility of the fractionation and mutagenicity procedures have been obtained for both fractionation,proceduret. Condensate f rom Gauloises alone, c. f . the three brands, has.been.fractionated by the method of Swain et al. An examination of these fractions has shown that the basic fractions had the highest activities in terms of both total and specificactivities. Considerable amounts of activities were al-so presenl.in.some -of -the. acidic fractions'. The XesiLlts...obtainedzugg.est.:that-het rocyc.lic.and nitrogen- containing polycyclic hydrocarbons are the important mutagens in smoke condensate. Essentially similar results have been obtained by other workers for the University of Kentucky "reference" and "low nicotine" cigarettes, albeit with some differences in the distribution of the mutagenic activities between some of the fractions. Recoveries with -Gaul-oises' as well as the Check cigarettes were below 50% of the activities of the condensates, suggesting either a loss of mutagenicity or removal of synergistic effects as a result of fractionation. Fractionation of condensates from Oriental and Senior Service is in progress. CD CZ) r-9 BATCo document for Province of British Columbia 8 November 1999 -2- Condensate from Gauloises has been fractionated by the Elmenhorst and Grimmer procedures only once, while condensates from Oriental and Senior Service has been done twice. Mutagenicity testing has shown that 80-90% of the detected mutagenicity was present in the aqueous methanol fraction in the case of all three brands. The specific activity of the aqueous methanol fraction from Gauloises was the highest and that of Senior Service the lowest, thus giving the same rankings as the whole condensates. Even in this relatively mild fractionation procedure, in which smoke condensates are partitioned between different solvent systems, total recovery was below 50% of the activity of the whole condensate, once again suggesting removal of synergistic effects. Once all three brands have been fractionated at least three times each, attempts will be made to see if .-.syne-r-gistic effects between fractions account for the loss of mutagenic activity on fractionation. It is suggested that these fractions also be examined for their ability to induce -drug metabolising enzymes in rodents. CD CD ~jl BATCo document for Province of British Columbia 8 November 1999 w t-- Uj U1 z LOISES 07 ZIENTA SENIOR SERVICE PLAYERS CHECK 2-7 CD BATCo document for Province of British Columbia 8 November 1999 CON.DENSATE- PER PLATE IL3 A CAPILLARY PRESS 750 p- Soo U- 0 X 250 ,T)zOSTATIC rip MATIoi\, RAL :)ENSrEx BATCo document for Province of British Columbia 8 November 1999 0 100 200 300 400 CONDENSATE PER PLATE Ft 0. + NaOH 2 CL 0 xt 0 2 HaOll H i 1 A pH 6.1 1 (D , + Et 0 2 I Il I rl~ FA ction DIA 11 Aq.j-s;i j, . . ._.. Fract ton A Fraction WA Aq Fraction WA Ppt. st 0 ppt. 2 ~d"21.0 T I + zt!O . 2 . .. .. . pH 1.0 + Et 0 0 j 2 Fraction BA Aq. Fraction 8 Fraction SA Aq. Fraction SA Ppt t 2 0 ,: (D t1 0 Evaporste , Evaporate, absolute 0 abs lute ztOH EtOH t Fraction 8 Baits , Fraction SA Salto -Filtrate W 0 Evaporate Cyclobox a + MOOH 0 fH 2 UGOH N 0 2 Cyclob Iexan'D CO NItromethane Fraction N CH Fraction N Cr (D Mitromethane- FIGURE 6 Tbe separation of whol e smoke condensate into neutral, basic and acidic fractions. '(Swata, Cooper and Stedman, 1969). CL _h 0 "1 Q 0 _h :r 0 M z (D Cr (D I Whole Smoke Condensate (100%) '1 Aq. MeOH Cyclohexaue 1 Stage 1 11 -Aqueous Uotbanol III Cyclohexame Fraction Fraction (76.3%) (25 9%) cyclobexane Nitromethans. Stage 2 IV Cyclohexane V Nitromethans Fraction Fraction cliromatography 2 nd slution let elution. iothanal Benzene/Cyclohexane Ptage 3 VII Residual VI Benzenel Fraction Cyclobexan Sub-Practlaq FIGURE 7 'Fractionation Involving simple s6lvan ;t ext.ractioA and silica gal chronat.ography. (Elmenhorst and Grimmer, 1907). S 0 0 1 TABLE MUTAGENICITY OF CONDENSATES FROM PLAYERS CHECK 26 CIGARETTES AND FRACTIONS OBTAINED BY THE PROCEDURE OF SWAIN ET AL. MUTACENICITY SPECIFIC ACTIVITY FRACTION EXPERIMENT NO. 2 3 Whole CSC 0.6183 0.6092 0.4558 BIa 1.4742 2.2883 1.3675 BIb 5.6100. 1.8642 BE 2.1383 3.6717 2.4542 BW 0.1792 0.3917 0.0758 WAI 0.8600 1.0583 0.1867 FAE --0.-0967 05 ~0-. 0.1408 SAI 0.1483 0.1775- 0.1767 SAE 0,0317 0.0125_ .0.0333 SAW 0.0333 0.0767 0.0633 NME 0.4400 0.2900 0.4458 NCH 0.6483 0.0808 0.1400 NNH 0.2553 0.2467 0.3392 C"D BATCo document for Province of British Columbia 8 November 1999 TABLE MUTAGENiC ACTIVITY OF CIGARETTE SMOKE CONDENSATES AND FRACTIONS OBTAINED BY THE PROCEDURE OF SWAIN ET AL. MUTAGENICITY SPECIFIC ACTIVITY C IGARETTE IDENTI TY Players cauloises Juiv. XY W Univ. KY IRI Univ. KY 2 Check (low nicotine) (reference cigt,' (low nicot Whole CSC 0.5611 3.6042 1.2400 0.688 0.510 Bla 1.7100 5.0850 4.84 2.784 4.272 Bib 4.5950 26.2372 16.48 6.900 17.552 BE 2.7547 15.8104 2.04 8.752 6.646 BW -0.2156 --L-0358 0-76 0.160 0.506 WA 0.7017 3.6425 2.40 1. 160~ 1.366 WAE 0.0975 0.6308 0.44 0.368 0.694 A1 .0.1675 O.L513 0.88 0.240 0.192 SAE 0.0258 0.1029 0.04 0.124 0.088 SAW 0.0578 0.3367 0.04 0.168 0.056 -NME 0.3919 .1.0005~,- 0 J6,' 0. 712--~-' 0.316 NCH 0.0897- 0.1379C 0 -04", O.W2-, 0.324 N NM 0.2804 0.7107 0 A4 0. 408 0.438 BATCo document for Province of British Columbia 8 November 1999 M U T A G EN I C I TY O F P LAY E R S C H E c k 2 6 C I G A RE T T E C O N D E N S A T E AN D * F R A C T I O N S OB T A I N E D B Y T H E P R OC E D U RE O F EL ME N H O R S T AN D G RI M M E R ( W E I G H T l i~ T A G EN I C I T Y - S P E C A C T IV I TY C I F I F R A C T IO N : FR A O C F T I O N SA M P L E ? N O . W C h o l e s a t e 1 2 3 . - . 3 3 3 . . . 7 8 8 6 1 2 9 5 8 7 0 8 0 a . 7 8 6 6 I 0 b . 7 9 6 5 0 . 7 8 8 3 0 b .7 6 4 6 0 a . 7 1 5 0 0 b . 7 5 3 1 o n d e A M q e u t e h o a us no l 1 2 3 - - - 2 2 2 . . . 7 6 7 2 9 2 i 6 4 r 1 4 0 . 2 4 8 0 0 . 2 9 0 8 0 . 9 0 9 i 0 .7 4 9 1 0 . 3 8 5 0 0 . 4 4 2 3 C y c l o e x a n e 2 3 . . 0 0 0 ;' : . 9 6 6 2 9 8 / 6 3 9 * 8 3 4 0 . 0 9 5 4 0 . 1 5 0 3 0 . 1 1 5 8 0 0 7 5 3 0 . 0 8 6 6 0 . 0 8 6 7 l C B e y e t c n l z E 1 u t i a o n n e . 1 r - u 0 0 0 2 . . . ! 0 0 0 2 2 4 3 4 0 3 5 5 0 . 2 9 0 8 1 0 . 2 9 5 0 0 . 2 2 9 6 0 2 2 7 5 0 . 1 7 9 6 0 . 2 5 9 3 o e he ne x 2 M n e d t h E a 1u no t l i . n I 2 3 - - - 0 0 0 . . . 1 1 1 5 7 5 9 0 7 0 8 7 0 . 6 5 2 5 0 . 7 5 3 9 0 . 8 2 5 0 0 .7 2 2 5 0 . 7 5 7 5 0 . 9 8 5 4 0 0 1 CL 0 0 CD 0 0 0 Z 0 (D cr P : T AB L E H ~U T A G E N I C I T Y O F P L A Y E RS C HE C K - 26 C I G A RE TT E C O N D E N SA T E AN D F RA C T I O N S -O B T AI N E D B Y T HE P R O CE D U R E O F E L MEN H O R S T AN D G RI M ME R F R A C T I O N _ VE A ( IG OF CT gm H I ) T O , ' a M U T A G E N IC I T Y S - A P M E P R L C E E 2 N N T O. T O T A L b A C T I V I TY 3 b W C h o o n l d e e n s a t e 1 2 3 1- . - 3. 3. 3. 7 8 8 6 1 2 9 5 8 7 0 8 10 0 10 0 1 0 0 1 0 0 1 0 0 1 00 A M q e u t e h o a u n s o l 1 2 3' - - = 2. 2. 2. ,7 6 7 2 9 2 1 6 4 1 1 4.- 7 9. 8 0 7 8. 1 7 9 1 . 5 0 9 2 . 1 6 .0 5 . 0 9 82 .6 7. C y c l o h e x a n e 1 2 3. - - - 0. 0. M 6 6 2 9 q 6 3 1 8 3 4 7. 0 7 9. 3 1 2 . 9 9 2. 3 8 - 4 . 6 2 5 .9 5'. l C o y t c l E o l h u t i o n e - - be n z e n t 1 2 3 - - - 0. 0. 0. 0 0 0 2 2 4 3 4 0 3 5 5 0. 8 0 0 . 6 8 d . 2 1 0. 2 5 0 . 5 9 . 0.7 2 e x a n 2 M n e d t b E a l r u o t l i o n 1 2 3 = - = 0. 0. 0. 1 1 1 5 7 5 9 0 7 0 8 7 1 2. 1 3 1 1 1 .8 4 5 . 2 6 5. 2 0 9 . 6 8 1 0.6 6 q_ CIL ci 0 0 1 CL 0 0 < (D 0 00 0 < (D cr (D (o Q0 TABLE 0 0 MUTAGENICITY Of CONDENSATES FROM GALTLOISE (G) RIENTA'(0) AND SENIOR SERvrCE (ss) crGARETTES A ND CL 0 THEIR FRACTIONS OBUI NED BY THE PR: OCEDURE OF ELMENHORST AND GRI~MR (D WEIGHT OF MUTAGENICITY - SPECIFIC ACTIVITY- FPACTION FRACTION CIGARETTE IDENTITi ~AULOI 4 ORIENTA SEN IOR SERVICE ; Who 1e "';'G = 2.8184. < Condensate 0:!= 4.2852 3.0565! 1 2208 5 IS 3.7092 . a. a. 1.0367 b. 1.2155 b. 1.0122 (D 0 k Aqueous G 1.6980 Methanol 0 f-- 2.7241 ~ " 2.36331 a. 0.9530 0 3681 SS 2.4189 . b. 0.7416 b 0:4809 . 0 Cyclobexane G 0.6771 10 t- 0.9440 1832-'- a. 0.1635 a 0 0699 I;SS 0.8764 M f~: b. 0.2071 -b : 0:1022 00 Z - lot Elution G 0 0373 0 . Cyclohexane . 0 0 0303 0.3491 4621, a 0 (D Benzene SS 0:0336 . . a. -O.3822 b. 0.4830 b. 0.5842 cr 2 nd Elution C - 0.1540 to Methanol 0.- 0.1865 2.1730 a 1 3679 SS:- 0.1571 . . a 1.1215 b. 1. 1934 : b 1.0772 q S1700 1 A Nu tb ae ni ci ty of s m ok e con de ns a tes f ro T m AB cl LE aa -1 re tt e s be lo nql ~q to th e Ja n us 9 -1 5 s e ri es Co de We. U si S n g CO S CO S B ut te r- fA t Sp No of co ec . T nd ifi of A-9 ens c lil 8 &t 0u st pe e. t r * s4 ri p cn ve q icI rta ty nt - s 1 - 1 1 5 - 19 2 1 nl 1 fl - c 3 an 8 ce 6 o 4 f t 1 he - 5 dif 1 fe - re 6 nc 1 e 6 I 7 n Co 16 me de 16 a . ns No 8 . 8 by 6 t 9 he B St W ud O en 5 t- 6 lit 1 ai '8 n-X 6- CU) 2 t 1 e 6 s - t 1 1 6 1 - 8 3 V B 3 B 0 1 11 8 2 5 ` 3 8 )6- 16- 6- 16- )6- 16- 16- 16- 16- 16- 16- 16- - 16 16- 1 2 3 4 5 6 7 9 9 10 11 12 13 14' N 8 N 9 N 8 Il 8 N 8 N 8 i 8 i1 .7 il .7 i1 .6 i 1 .7 il .6 fl .7 1 .6 Ni Ni 0.4 0.3 0.8 0.7 0.4 0.3 0.8 0.7 0.4 0.3 .1 0.7 l l 1 7 2 5 1 7 2 4 1 7 ) 4 0 0 0 0 0 0 0 0 0 0 0 0 Ni Ni .0 .0 .1 .0 .0 .0 .1 .1 .1 .1 2 .2 l l 5 5 0 9 8 7 6 4 1 0 2 0 3. 3. 3. 3. 3. 3. 3. 3. 3. 2. 3. 2. . 2. 65 12 51 48 95 67 96 69 44 116 33 91 0 59 2 87 08 1 A 34 22 36 58 50 07 78 01 2 45 4 4 A I 4 : s 0 0 0 ! 1 1 0 0. . i 91 0 Q' O*. . . . 0. 0. . . 79 5 ? 6 i 69 5 77 85 88 115 93 94 99 8 67 , 1 N a 14 a a a 4 4 N a s S s S s s s s 5 $ S s s s u a a N . a a 'i K N a a s s s S S s s s s S S s s it it u It N a u : u Pi s s s s s S s s s s s s s - HS NS NS US as NS us :s s as as s S I N 14 a W N N a s H s s S s S s S S S s s S s s s It N u N N N N N a ' s s S s S S S S S s s s S s N N a a H N : N s s S s S s s S S s s S s s l NS as is NS as NS HS $ s NS s s as NS NS NS as as NS NS 's us :s 5 s us NS HS ss s ss s as as NS s KS N N " N u N a N a H a u N S S S S s S s S s S s s S s NS Its NS ss s ss s as as NS S NS a N N a a N ft N H a t s S S s s s s S s S S s s s li s s s s s N N u N s s s s s s s s s S S s S - U The S val not ues si g 9i ni v f en ica a n re tly M ea di n f t fe S. re D. nt .' e ca S lc a ul s at ig ed ni fr fic om an 4 t1 y re d p1 if ica fer ti en on t s a at t P S 4 c 0. on 05 cc ; nt S ra S ti a o s ns ig o ni f fl con can de tl n y sat di e. ff e er ac en h t c a on t ce V n c tr O. a 0 ti 1. on i n t ri pl ic at e. 0 0 1 (D 0 M "1 (D 0 0 0 E, 0 Cr (D FIC.. Bu-rTER-FAT CONTENT OF TOBACCO A1JD AM- ES 4--0- MUTAGENICITY NO CAW O~Vp- ff%~ P ^A ::,.NC) CASING' No COCOA LU 4- CASING NOCOCOA 3-0- w tn CD 0 0.05 0.10 0-15 0-20 PEP, CENT - BUTTER-FAT BATCo document for Province of British Columbia 8 November 1999 I SESSION TWO BATCo document for Province of British Columbia 8 November 1999 Deprote-inisation Pyrolysis products of amino acids and proteins form powerful mutagenic agents, hence the removal of these agents from tobacco may result in a product with reduced mutagenic activity. The work on tobacco deproteinisation has progressed steadily, but at a low level. Cut tobacco samples have been treated with proteolytic enzymes and by comparison s 1-- --triiited with buffer only, with amp es approximately a 40% reduction in tobacco protein ha been produced. This result S is currently subje0t- to confirmation, then batches of tobacco of a size --to manufactu-rg cigarettes will be processed. CD CD U-4 ON BATCo document for Province of British Columbia 8 November 1999 va I 0 0 CL 0 0 c 3 (D 0 "0 0 S' 0 0 0 ou 3. to 7 0 0 c 3 E 0) w z 0 (D 3 C7 (D (D (D CD Vintage Cigarettes An influencial UK epidemiologist, Professor N. Wald, has a collection of cigarettes dating back to the 1930's. In the light of decreasing lung cancer rates, Professor Wald is interested in examining the mutagenicity of these vintage products compared to modern cigarettes, then correlating this data with the epidemiological data. ha7e a limited supply of old-cigarettes (1950's) and it is planned to re-manufacture c1garettes to a 1930's -style specification, then to compare-Meir biological activity wtth modern products. These'experiments will be carried out-at the same time as those of Prnft-Rqnr Wald, but,to~tally unknown to him. Thus with the programme, it is intended to influe nce Wald's programme to pmovide the most balanced study. CZ) BATCo document for Province of British Columbia 8 November 1999 VINTAGE CIGARETTES 1980 1930 Paper 50 Coresta 8 Coresta. Length mm. .84'= 64 Tob. 70 Circumference m ~p. 24.75 25 Tobacco % 80 Lam. 20 W.T.S. 84 Lam. 16 CRS C.P.1. 35 160 60 180 Filling density 283 310 (m g /C, c) -Blend Nicotine 1.99 1.63 % d.W.bo* Filter type Cellulose Acetate Plain Filter Ventilation' 0-40 % PMWNF mg/cig. 10 33* CD C:D (-n C-I! Qn _r__ CZ) BATCo document for Province of British Columbia 8 November 1999 An Inhalation Carcinogenicity Test When considering the biological activ'ity of cigarette smoke, a great deal is achieved using Ln vitro and in vivo short term tests. However, at sometime, a product ;usT be evaluated using a long term assay, where the end point is a tumour. Mouse skin painting has been the assay of choice for many years, resulting in a considerable data base. With legislation asking for tests to be more realistic with regard to human exposure, the final test prior to human monitoring should be based on inhalation. Here the response to the smoke comes into play, for example, inhalability; also the material is in direct contact with the respiratory system. In the past, rodent inhalation models with cigarette smoke have not produced a significant number of lung tumours, significant changes being limited to laryngeal tumours in the hamster. Many arguments have been put forward to explain this including that cigarette smoke in isolation is a very weak carcinogen, but more potent as a promotor. Should then tobacco smoke be tested in association with a known tumour causing agent and, if so, how valid would such a test be to the Industry? ....One such test has been used for several years by the Atomic Energy Toxicology Laboratory in France and their work was recently presen.ted.at Erice. The results of 3 studies involving radon exposure to large numbers of rats were described. In the first study, involving no smoke exposure, increased lung cancer incidence could be shown all the way from a cumulative lifetime dose index of 20 WLM ("working level months") which produced 2.2% tumouirs (as against a background incidence of 0.8%) up to 5000 WLM, which produced an incidence of over 50%. Above this level incidence fell off, perhaps due to poorer survival, incidences apparently not being age standardised. 20-40 WLM is an exposure level that is not rare in many granitic countries. In the second study, groups of 30 to 50 rats were exposed as follows: Radon(WLM) Smoke* Lung Cancer Incidence 4000 No 35% 4000 Yes 68% 500 No 7% 500 Yes 28% 100 No 0% 100 Yes 4% (based on 0 Yes 0% only 1 tumour) *Exposed for 1 year 4days/week. CD CD BATCo document for Province of British Columbia 8 November 1999 As can be seen, though smoke alone prodUced no response, it was associated with quite a marked increase in incidence when given in conjunction with radon. In the third study, the effect of change in order of radon and smoke exposure was examined. The results are given below: Total tumour s Number of tumours Treatment Animals N % Squamous Other Controls 600 5 0.83 1 4 Radon alone 50 11 22 6 5 Smoke then radon 50 8 16 7 1 Radon then smoke 50 39 78 39 0 The conclusions made from these findings are: (i) smoke exposiLre, when given before radon exposure, had..7A-o effeKt on _the incidence of -e-quamouS. tumours (ii) smok& exposirre, when given a-f ter rj7don expbsure, caused a marked aFd- highly sUnif i-cant in=rease in the :Fneidenee of squamous tumours It w .aj.~ concluded that tabacm smokii--seemed to be acting as a proifib-tor rather than as an initiat67r of si-mamous tumours. Reference Chameaud J. , Perraud R. Chretin J. , Mass R. and Laf uma, J. Lung carcinogenisis during in vivo cigarette smoking and radon daughter exposure in rats. Recent Results in Cancer Research, Vol-82, Springer Verlag CD Berlin 1982. CD UM Z::. BATCo document for Province of British Columbia 8 November 1999 rX 0 0 C ,13 (D 0 0 0 z 0 Cr Longterm biological tests 13-1141("1 Mouse skin painting Chronic inhalation a. Rat lilt b. Hamster Promotion studies Intratra.cheal instillation C, Ip q,- q !.7 0 0-1 ~ I I Amtiv6-stage lp'roc,es.-P. 3 Cr w Ciig a~'r'ette smoke Bronchial epithelium d ..'ditio~fbxic agents Epitheli I amage' Pahidulate matter Squamous epithelium darcinogenic' Dysplasia etabolite Carcinoma in situ Invasive carcinoma Differential Enzyme Induction The biological activity of smoke is dependent on the species of animal studied, the organ, cell. type within an organ and the presence of drug metabolising enzymes within those cells. Smoke induces drug metabolising enzymes in man and in experimental animals in a two phase system. In phase one, compounds of cigarette smoke are activated by enzymes such as cytochrome P448 to reactive compounds which may attack cells. Reactive intermediates may be detoxified by enzymes of the second phase of metabolism. The balance of activation and detoxification will determine the potential toxicity and damage the smoke may cause. One enzyme, EROD, is a measure of cytochrome P448. Induction of EROD in rat lung is dependent on the chamber concentration of smoke. Rat liver EROD is induded only by very high concentrations of smoke. Series'of 'enzymes related to EROD were studied in rat ung. A hon-carainogen '(phen'bbirbi tone) and a caf"6inogen (3- s_bf activities methyi'c'holanthrene) produced., different "profile of -thise - enzyries.- Cigarette - smoke 'produced _k_ profile of - "enzyme - ac tivity which suggested it contained carcinogens and non-carcinogens. --The effects of smoke from cigarettes from Canada and Germany have been studied within the framework of Project RIO. BAT brands from Canada fell within the range produced by competitors' brands. Mark 10 produced the lowest induction of EROD and Export A the highest induction. The results could not be related to physical characteristics of the cigarettes or the "kields'-of smoke components. Smoke 'from-Ger~an- _Cija~rettes -Inau-ced'-~ EROD- 'in rat lungs in the order Sdlecta 0 0 CL Z 0 0 a Cr (D _L a (oicins'and presence of d,r,.Yg metabolizing. enzymes in cells of lungs Bro'nchiolar epithelial,cells Ciliated Unciliated (Clara)jI 11, ,111 Alveolar epithelial cells Type I i1 .1 ivil ()I Damage Presence by toxins of -drug requiring metabolizing activation enzymes low low high high low low. high high FREIt",'RADICA S~ort lived species may not reachlungs 1 14, ;j React with other compounds to'produce long lived radicals eg O"r Destroyed by i I Rea superoxide dismutaset cells to cause DAMAGE Reduced by glutathione peroxidase -8 -m-o,- -k - e--c-6- -n-t a i n s f * r- e- e--r a--d i -6a- - I s Smoke induces activity of: superoxide dismutase glutathione peroxidase 10 0 'W C) w LL rr x iLlj 'W 0 w w 2 'Aim iCL 0 0 :1 C~ BATCo document for Province of British Columbia 8 November 1999 0 0 0 0 C T 0 0 CO Z 0 Cr Effect 'of slmo he on enzymes of activation and detoxification.' Activation Detoxif ication Cytochrorne P-448 Cytochrome, P-450 Aryl hydrocarbon] Cytochrorne, P-450 Epoxide hydrolase, hydroxylase Biphenyl 2 Biphenyl 4- UDP-glucuronyl hydroxylase hydroxylase, transferases resor'ufin Bemphetarnine, Ethoxy lutathione S- no effect 0-deethyla'se N-dernethylase transferase, Sulphotransferases 999c'-CP7001 r ZZZ c ) CL t4-= fi= CD IC'4 (JI e. q CC k4 tin (0 LO Itt 0 0 I -1 - E 9 '0" a I d Cj . cr E E C::) CD BATCo document for Province of British Columbia 8 November 1999 CIO CL F- CY) 0 O, 1 r= CD 0 CD -0 E CU 5 N, Nt. ci I cm t c) : I C) E E . BATCo docu ment for Province of British Columbia 8 November 1999 IC= CO 414 0 0 0 4 =2 12 Or r 0 182m E 0*0 q. co a.. cn .4 El e000, ca too 16 'C" : i a) 04 ;Z, BX= me= 9 :=V tq 'D IC4 7:c0 c%i 0 0 C3 0.q q 6 T CL E.9 cn I I . CD E E (-j7. BATCo document for Province of British Columbia 8 November 1999 ~M a 0 s100 W CD 0 S ca N Ile 0 . -- a = . r 0 on ca E 00, Cn (L Ch E El .2 CU &IWA 0 0 lop CU CO -0 :E k\j : =3 .C ig LO '0 to !LO Icn ;CO icm 'N Id. . 1.;~ IN L E-9 E E BATCo document for Province of British Columbia 8 November 1999 Pk -;.I 0 qM-I; Nj -aj LQO---"M qIiIi!,f f d~5 C~ 1-61 I t` 7 --f C !CO IC) Cn OZT CL .cc E.S; cn ILU c EF= --jTbEj-LNOO-f 110 CO CM C) BATCo document for Province of British Columbia 8 November 1999 OT T 11 ig rp 1- Lt L-M am= 1 CO Ci IN C) C) IC) 0) L .01 0 T CL Irl E G. a) :W1 r- E E ~7 p c::> Uri ON BATCo document for Province of British Columbia 8 November 1999 C5, 10-- 1 H-- I N-- 0 A- LO. cr) C\j : C) (D C) I d C:) I ploid Ipwosojowl6w Old 15u): C1083' I(L-U U1. ON BATCo document for Province of British Columbia 8 November 1999 tj) mum 41%I 13, to= r-3 77 7-Z ~40 C)- 1(,-uj9jOjd jewOSOJOI.W Ow ~-.U!w CD CD ON BATCo document for Province of British Columbia 8 November 1999 MOU.C~'qt Skin PaIntin 0 ov6rall,ttmeasure Of qqpIn .qqqn~~ity of smoke condensate in mouse skin Enzyme inductlip'", q 1 (kfl:~;! ~ Individual aspects of carcinog'e"n'icity (initiation, pr&n-otion, cocarc 'inogepicily), ~ Detokification potentlaO, if It 1("I, i Z Use to build up profile of biological activity of whole 0 smoke on rat liver and lungs. Cr CD C 0 j /,-I/ q ~00 I Z Cr CD .'Drug', metabolizing enzyme. studies in rats DO.N.OT measure hum" an'lung carcinogenesis DO.. 'indicate. relative amounts of bioactivation and detoxification in, rat cells critical f: ctor in det the a ermining a ccumulation;I of cytotoxic materials -Thi8 -system may- be used to rank the biological activity of different products. - I j, .1 99 The Mutagenicity Test Battery The fundamental assay of mutagenicity, the Ames Salmonella bacterial mutagenicity assay, is now established as a standard system in BAT for monitoring the biological activity of smoke condensate. International authorities recommend that bacterial mutagenicity should be complemented by a similar assay using higher organisms, preferably mammalian cells. Such an assay system using a mouse cell line is currently under investigation for BAT via a UK contract house. However, the above assays can only be directed at monitoring the activity of carcinogenic initiating agents in the particulate phase of smoke. Hence, carcinogenic promotor agents and components present in the vapour phase cannot be assayed,-- for these agents are known to be inactive in these point mutation assays. Data in the literature indicates that yeast systems are capable of detecting DNA interactive agents in both particulateand va.pour phase of _-cigarette smoke. Thus a yeast system should immediately complement the Ames test in terms of smoke components that can be investigated. Furthermore, the yeast system will also form a balanced portfolio in terms of supplying BAT with an effective mutagenicity screen. For yeast are eukaryotic organisms, thereby possessing similar characteristics to mammalian cells, i.e. they have-structured chromosomes, a nucleus and cell ~organelles, w.hereas bacteria..are more primitive, having 'naked' DNA 'floating' in the cell cytoplasm. The enzymes used to metabolically activate some mutagens/carcinogens are aiso naturaly present in yeasts, which thereby forms a physiologically robust system for investigating mutagenesis in adverse condifions such & smoke. The bacterial mutagen- icity system relies on enzymes added in the form of a rat liver fraction which is, therefore, unphysiological and is also labile to adverse environments during exposure to test compounds. BATCo document for Province of British Columbia 8 November 1999 -2- The yeasts also have an added advantage in that one strain appears to be sensitive to carcinogenic promotor agents and may complement the detection of carcinogenic initiating agents in the Ames test. A further mammalian cell transformation assay is also known to detect promotors, but this assay would take in excess of a year to develop. Therefore, though the mechanism of promotor detection in yeast is more empirical than in a cell transformation assay, it is proposed to use the yeast promotor system at GR&DC as this will naturally follow from the yeast mutagenicity system currently under installation. C) c::) 4 i- U-1 BATCo document for Province of British Columbia 8 November 1999 CD M MUTAGENICFY TEST SAII-TERY ..SMOKE CARCINOGENIC END P C MPONENT PHASE Pmk;~' Particulate Initiator P.M. Particulate/? Initiator (;f 101f v 11 Multbvs,-,:~, iParticulate Initiator'' Vapour Promotor BACTERIA MAMMALIAN CELLS I ~WIC ; ' YEAST-i: 6 9r-,1 (iS 0 0 1 > 0 0 CL cr AMES S. typhimurium7stratins 89 4 vOOO M. Whole- smoke Whole -smoke Va,p6ue Wase Va'pour phase Cells in PBS 00 0 DMSO:H Conventional 20 1 :1 Ames test > I 0 0 0 0 0 :r 0 0 E, 3 CO Z 0 < (D 3 Cr (D Yr-AST Saccharomyces cerevisiae Eukaryote Diploid Enzymes - Intqrna.1- Sq: DNA~ Alteration b'bkqentional:.' utation Abnormal mov'emLnts of DNA Cy t9 plasmic WitochoQVrial) DNA Mutation Cells with an abnormal chromosome n lumber - PROMOTORS 0 CL 0 cr E A s.cerevis iad D7 RELATIVE ACTIVITY P~,NE Al MUTATION CONVERSION FLUE-CURE D 0~.9 0.77 iBURLEY 0.5 0.6 ca 0 0 0. YFAST, cerevisiae' D 7 "RELATIVE ACTIVITY 6 ENE MUTATION CONYERSION CONTROL CIGARETTE CAMBRIDGE ~111,).-,10124' 0.4 cr (D FILTER -Mol 0 U) "Z LIJ CO LU C CO > M LLI D Li- a F > 0 LLJ CIO 04 V C14 C43 Z LLI U) 114 04 D BATCo document for Province of British Columbia 8 November 1999 0 Co Z 0 CD 47 Rat liver SALMONELLA Activation DNA x h'~i 'y'' m e s Target P3 J. Detoxification Internal YEAST activating enzymes cu cl) 9 z ss !7 0 0 1 CELL CELL. YEAST Initiator/Promotor TRANSFORMATION SCORE 1. CELL TRANSFORMED CELL Initiator Promotor 0 0 MITOTIC ANELIPLOIDY 35 .100 ANEUPLOID. "34 CELL DIVISION DAUGHTER CELL8 NORMAL PARENT co 000' z 0 CD 33 cr Saccharomyces cerevisiae strain D6 0 CM M M + + + 0 cc 0 0 0 0 er) CM z CO C/) w Cp (.) W 0 CZ . Z- a', CO BATCo document for Province of British Columbia 8 November 1999 0 0 CL 0 < 0 0 0 z 0 < Cr 0 6 /"q 0 0 1 Saccharomyces cerevisme D6 Aneuploidic Promotor 6 colonies/ 10 Control 3.7 TPA 41.5 PA 9.41 TPA=12-0- tetradecanoylphorbol-13-acetateI PA phorbol- 1 3-acet a*te Parry et al 1981 0 0 a 0 0 C 19 0 (D 0 iviAMMALIAN CELL TRANSFORMATION C3H/10T1/2 % Transformation -PAH 1.7 TPA 0 A 4 TPA + PAH PAH + TPA .47 TPA= 1 2-0-tetradecanoylphorbol- 1 3-acetate PAH = Polycyclic aromatic hydrocarbon Mondal et. al. oaqrl~j IM0 I SESSION FOUR CD -L-- BATCo document for Province of British columbia 8 November 1999 Several new methods for measuring smoke activity have been established both in GR&DC and other CAC countries. In order to validate in-house test procedures, a study with four low tar cigarettes from Germany was initiated. Cigarettes are tested in both BAT laboratories* and at confidential contract laboratories. Initial results: a) The-co-operation between Hamburg and GR&DC has been very effective. b) The Ames resid'ts"from GR&DC, Hamburg and the contract house are the same. C) Sub-chronic inhalation studies are underway at both GR&M%ind a con-tract house. The results should be available by the end of 1984. CD Uri CIO IN-) BATCo document for Province of British Columbia 8 November 1999 SESSION FIVE (=0 CD -.ti- C-n U-1 cc (-;Ii BATCo document for Province of British Columbia 8 November 1999 Nicotine 1) Nicotine has the well known acute toxicity. 2) Nicotine and its minor alkaloids have in physiological relevant dosis no effect on mutagenicity. 3) A cancer relation can not be concluded from existing data in literature. 4) There are no experimental data available on cocancerogeneti6-ictivity- CD CO BATCo document for Province of British Columbia 8 November 1999 Acu~a Toxi'c;tj -of NicoHne- Mouse oral 14 ng /k_g C rnin Au~hor 'Rouen 1% C 4 Eb r5 1kq (min) i V 1A r 9/kg S r- 33.5 n3/kq LD (,o iv A ng I lig (rn*n) r,u;maa Sc 45-4o n .9 1 kg (m;n) IV h3 ~q min) -'Ra6b;~ SC .1o .9/kS n (leVhal)-- IV 9.4 r-9 k-9 IV 5 nq1kq Men So h,3 Oelrhol) Child cc~ Qn BATCo document for Province of British Columbia 8 November 1999 E. col i 1~apair FesF. (Ro-.s an kran-L). Substanz aufgetropfte Hemmhofdurchmesser * B/A Ergebnis Menge '(mg) + (MM) pol A pol Al JAI (B) 9-Aminoacridin 0,05 14,-5 21,0 1,45 + MO (20,ul) - 7,0 7,0 1,00 Rikotin 10 13,0 18,3 1,41 + Nikotyrin 5 11.3 16,0 1,42 + Anabasin..'.- 7- 10 21,3 22,5 1,06 - Anatabxn 10 14,5 .22,3 1,54 + MYOSMIn is 0 -19, 5 1,30 + Korn ikzcTtin, 5 17, 9 1,28 + cc BATCo document for Province of British Columbia 8 November 1999 Arne's t-TesF CO 30. 0- L 2- C 10- Qj 0 ib 20 ImMI Nikotin q~200. 7 Z 7 IOC 0 Ib I.MMI ....Anabasin 10 CL 5 U qp CD cc .0 1.6 -3~2 ImMI NikoNrin CC BATCo document for Province of British Columbia 8 November 1999 A Substanz Konzentration S 9- his+-Revertanten/Platte t S tandard (MM) Extrakt abweichung TA 98 TA 100 TA 1537 -Nikotin 20 + 18 4 214 t 60 7 2 10 17 1136- 57 6 2 5 + 18 8 173 48 6 2 2 21 6 185 44 7 3 1 + 21 6 196 47 6 3 0 18' 8 1b9 46 6 2 20 12 3 177 67 7 2 10 .11 2 191 '60 6 2 5 - 12 2 185 6 2 2-. - 12 3 203 61 6 . 3 1 - 12 4 207 63 5. + 2 0 12 2 182 65 + 3 Nikotyrin 3,2 + 19 4 138 24 7 2 1,6 + IS -4 135 37 6 + 2 O's + id 6 13-7 28 6 I 0,32 + -18 4 135-~ 25 6 t 2 0 16- + 7 4 t36' 22 7 2 -0. + -22 ' 2 123 29 6 1 3,2.- -18 3 11.5 37 10- 4 1,6 13 4 A4'* "'3 7 5 ---8 5 O'S. 14 4 142 47 8 2 132 3.4 3 153 40 8 4 0,16 1 2 6 i51 + 22. 7 + 4 0 16 3 126 35 8 2 Anabasin 10* 31- . 1 + 147'- 52 19 9 5 4 ~ 31 14" '56 7 -20 9 25 + -3 6' % 1 f.53'. 52 17 :t 3. + .- 7 + 34 -- 0 + 148 69 17 3 015, Z : L .52'- 0 -77 14 8 2 0 + -33 - --- 4 - - - 157. 71 19 6 10. 17' 6 107 34 20 7 2,. 127 '5 3 22 7 25 + -- 14, 2 134 49 23 -9 . C::) 12 + 8 .135 58 25 9 4:%- . 0 5 17 2 JL37 57 26 8 . # 0 14 2 118 32 22 6 CC cr-I BATCo document for Province of British Columbia 8 November 1999 Anatabin .3 7- 2 2 .135 '5 .12 2 23 .3 -135 .t7 13 1 0, 75 26 4 122' 54 '12 1 .0,3 + .23 .4 :'123 '46 12 3 0.1s + 32 4: .144 "6 5 12 1 .0 + 35 4 .141 57 12 2 3 14 S. 78 32 9 2 -1 5 13 2: 99 -.45 12 4 0,75 16 6 . 112 42 12 2 0 ; 3 1:5 1 13.8 -37 3.3. 0 D,15 13 3 118 52 .10 1 0 15 4 124 45 12 2 Myosmin 30 + 18 4 98 63 14 3 + 22 7 118 57 17 4- 6 :7.5 .+ 22 4 .59 14 5 3 + 23 6 133 49 .14 6 1,5 + 23 -7 -44 .14-7 IZ + 6 0 + 27 9 3.4.3 63 -15 6 -.3 p 8 .1 G2 1-4-4 -5 1 116 58 16 -12 + -5 118 + 48 15 6 3 12 4 123 -90 15 5 1,5 13 4 120 46 1 C- 8 0 15. 5 130 55 1 G. 6 Vornikotin 5 17 4 98 29 12 1 2,5 IB 4...-- -104 38 11 3 1,25- i8 -3 '109 '54 14 5 .20 3 -.121. -.-46.. 13 4 18 .12'7 45 12 2. 0 22 5 109 +.44 12. 5 1 9-3 37 13 6 Z; S 11 6 99 44 13 .7 17,25 is 5 113 -58 3.3 6 O's 11 2 120 31 12 2 C:D 0,25 14 2 .124 29 14 7 0 i2 4 100 20 13 6 BATCo document for Province of British Columbia 8 November 1999 CHO [SCE Substanz Konzentration S 9-Mix Anzahl der SCEs/Metap hase Ergebnis.:! (=H) 5 % ausgewerteten + Standard- Metaphasen* abweichung I 1kotin 30,8- 98 3.1, 7 0,'3 15,4 119 11,5 0,3 7,7 119 4- + 10,3 9;3 0 1119 9;-5 0,3 30,8 60. 9,2. 0,4 3.5,4 28 . 8,6 0,5 7,7 + 29 9,9 C), 5 0 + 31 8,2 0"2 a kMIG 0,003 59 16,3 0,G b BaP 0,05 + 16 I 16,9 1,2 N'.hotyrin 3,2 0 ILI, 3 0, 1, 6 79 ILI, 0 0.4 O's so -10,9 '0, 4 0 41 16,9 3,2 + 38 8,3 0,4 39 9 7, 0,7 0,8 + 39 8,3 0,5 0 A 0 7,9 0, a MUG 0,003 40 17,4 0,11 b BaP 0.05 + . 39 1 El, 3 3_2 A-abasin I's .127 11,5 0,4 8 121 -11-6 0,4 0.4 120 3 0.4 0 )L16 0,3 + 41 10,9 O'S O's -43 9,8. 0", S Qi-4 43 10,3 0.5 D 66 9,4 -0, 3 a G' 12 M 003 0, 59 -16,3 0,6 Nap 47 17, r.,. o,e C C C 0 :D -, 7- 0 1 BATCo document for Province of BritiSh Columbia 8 November 1999 Anatabin 3'1 58-- 15-5 0,5 1,6 55 .14,4 0,5 PIS .65. 12-,0 0,5 _0 58 11j4 0,4 41 10,.7 0,5 1,6 + 42 11,9 -0, r. 0.8 + 49 9,1 0,4 0 + 56 9,8 0,4 aMNNG 0,003 38 .15,3 0,6 b BaP. JD,-0,5 + 4 i 3.8,2. 1,0 Myosmin 1;7 99 11,1 0,4 0,9 90 10 *.S' 0,4 0,4 87 9,8 0,4 0 110 10,5 0,3 ~I, 7 -7 7 8 C), -P.9 2 ~9, 3 17 0,4 .0-4 + ."43 0,4 aMOM. 15, 4 O,E p 0-05 --26 -15.6 0, 8 Nornikotin 16,9 61 12,5 0,5 8,4 .133 1.1,8 .0,3 4,2 L34 3-1,9. 0,3 + 0' 148 11,0 + 0.3 8,4 + 60 10,6 0,4 .,?0 0,9 0 + -30 lif-0. 8 0,6 aFINNG 03 0,0 59 3.6,3 0,6 bBaP . .0,05 20 17,2 1 2 BATCo document for Province of British Columbia 8 November 1999 Humcin Y=-~cy I-e 'Substanz on Konzentrati *PRI '24ikrontiklel/1000 Lymphocyten Versuch 2 .Nikotin 36 1#6 5 13 3 24 I's a 7 5 12 1,8 8 6 5 . 0 1.9 3 8 6 Aflatoxin -0,1 1,9 -16 -15 14 Nikotyrin 3 1,7 7 4 3 5 1,5 1,9 5 4 0,35 2,0 7 4 6 0 2,1 6 4 4 Aflatoxin . B 0.1 I . 1,9 19 14 14 . ,Anabasin 1 is . 1.8 4 5 7 7,5 1,9 :-1 6 -- :3 2,1 3 A ; . .- --0 -v --r- ..2 F '-.:. .- ;z"t .. *3 .: 7 A.Elatoxin B! 2 ;0 15 ..14 19 A n"i t ~5 b i n i 3 2 7 6 5 - , 1 8 9 ; 7 .9 0 2,0 4 4 5 iji'~toxin .; 0, 1 8 19 i 'Z . -5 1 9 J. A 5 -7 n kirosm 11 2 5 1,9 .. . 6 3 .6 # 1 2,1. 8 .5 0 2,0 4 5 Aflatoxin B 0,1 2id 15 20 Nornikoti n 7,5 117 5 7 5 3 4 ".9 8 3 . . 4 2,5 2;0 5 -3 -- - --. Aflalcoxin -B ----O-j-I 7=- " 3 :5 a9 X- BATCo document for Province of BritiSh Columbia 8 November 1999 -5000- UV (4J) 3000- 1000- 1 k 5000- CI) DMSO 3000- -:: V-7. 1000- "7! i v a a' ps C 'E E I', -Ei 1 Ak It --~000- 2 Nikotyrin -3 ` M Af) Cx 82 !2.3000- CL A .1 9 S E 15000- Nikatyrin c., (1, 6-aM) 3 00 0 1000, rU QTI 0-0- e r I Fraktion BATCo document for Province of British Columbia 8 November 1999 UDS -Substanz Konzentration DNA-Menge 3 IVM H. (MM) Vg) pg DNA Nikotin 42,2 5,5 I 2S,8 5,5 O's 21,9 2,7 0 .20,7 10,0 UV (4 J) 5!;, 1 157,3 Nikotyrin 3,2 56,2 1.9 I'S 60,2 0,32 411,6 -4,4 0 38,2 -.2,9 UV (4 T) - 213,5 Anabasin 3 -25,3 313 1 5 31,4. .3.4 0 :5 27,4 3,9 0 29iO 3,8 UV (4 J) 43,0 180,1 Anatabi n 5 21,4 5,9 . . 1 31,5 5,7 22.9 5,2 0 .28,7 6,5 UV J) - 42,8 152,9 Myosmin 4 24,3 1,7 2 28.0 1,3 0,4 27,0 1,4 0 31,2 1,9 UV (4 J) 1- '14 0 8 Nornikotin -2 25-3 0.7 1 28:8 O's 0,2 27,8 0,7 0 27,7 0,4 UV J) - 48,1 155,0 (-n BATCo document for Province of British Colu mbia 8 November 1999 ico P no- and Caricer Zock LI SA Cocanceroq enaH'r_* cc.~;on in c2 n;rnal e-xpzr;mp-4s Hoff mann USA -?rp-r-UrSor - of tobacco sper-ific Wirosam.;nes Grimrner T Trp-cursor cf N -PAH Z -A T S 44. &r;as tw C=) (-n BATCo document for Province of British Columbia 8 November 1999 0 06 0 C (D c o rv e. Coronary hp-ar~ dQSQQ3eS T V A.It I il I. I 0 o1, IIIE 'fit A I* r.) IIIIAt A I'l MI (D lass harmful 0 :r 0 0 00 a ss Ive mo 14 t*nq General resen're-h z 0 +H) (D S B 1. A M Cancer 9 6 q- 0 0 0 0 N I C O..T, 1) NE lf PREORSOR EFFECT HISTOLOGICAL EFFECT -HEART /VASCULAR EFFECT NtCOTINE INTAKE *.N-PAH KIN CONST/VARIADLE NICOTINE-INHALATION LiPID STUDY 0 k TS-NA t - ( MUNICH) F~F(NiCOTINE NITRATE,*,' E-INHALATION NICOTIN BY INTAKS MECHANISM ISt PRESTUDY OTHER N-comPOUNDSo . INHALING/NOT-INHALING 2ND. PRESTODYI OTHER ALCALOIDS. 11 (PLANNING) CIGARETTE j PIPE AND 0 0 AND'TOBACCO SPECA) CIGAR SMOKE. INTAKE MECHANISM F=(-ItIHALATION) -3 z .SF-tONDARY EFFECT 0 OR-,MOTIVATIONS THAN NICOTH OTHER 17 L E SS BLAME D C IGARETTE t6 S Sj M 0 f. > 0 0 CL 0 CD 0 cr ro, co cr c0 C,O-T,I,N VDC FELDVERSUCH..OSCH.IEVELBEIN i AU,,F:,N A miko'U.'L A~i 6N. SECUNDARE RAUC*H*VER'STARKER BAM NICOTINMETABo* rsmus LUCxE PFETFENRAUCHERSTUDIE SCHIEVELBEIN NIOTIN-ROTININWERTE ONTRA EDIDEMIOLOGISCHE DAM --- -1XR HEAZ EEISLAUFSCHADEN IL UITZ 4 1-14 > 0 0 19 0 NICOTINE RESEARCH CD 0 EFFECTS OF;-SMOKING AND NICOTINE DEPRIVATION WHILE DRIVING A CAR# kASUREMENT OF. HEMODYNAMIC PARAMETERS IN PERIPHERAL BLOOD VESSELS OF SMOKERS,AND NON-SMOKER$# WITH AND WITHOUTo(- BLOCKADE, 0 COTININE FORKATION IN SMOKERS, EFFECTS OF CHRONIC NICOTINE APPLICATION ON RENIN-ANGIOTENSIN SYSTEM# z 0 O-DEPENDENCY ON NICOTINE OF THE N-PAH FORMATION 14 TOBACCO SMOKE, TR OSA04'.Ngi.F(.)Rh4TION N HUt4ANS, co 66 SHW I 0 CL 0 0 0 % 0 cr z 0 Q 3 CD T 14.)i Allgcmcinc Pharmakol.gic u nd ToxilcolOgic dis Nikotins Forschunginchmer;i :1 -'4 Them's 4 Dr. Altmann, Wisn' Ein'flull von Nikotin suf den DNA-Staftwechici Prof. Gerlach, MUnchca Tiorcx porimcn tell# Studien Ober skuts und dironisdis Virkunject von Nikotin out himodynamische und motabolisdis Parameter des Homons und Jai Korananystems Prof, Grabockirl Tiorox Perim on% ells Unsersudiung Ober skuts und dironiiche Vir- V; Rojon4urg toren und molskulars Media- ,junsen voh Nikosin out Nikotiarsatop w4 mcn der Katedioliminfreisettung &us yrnpathisdion Nervon des Hcrz-/KrcisIaufiysccms und des Ncbennicrenmarks PrIv.-Dox. Dr. Habs. Der Einflud van Nikotin suf die chcmische Cancerojeness van N- Hsidelborl NitrogumethylharnsLoff (MNU) bei der Spragus-DawNy-Ratte Plot. Horstor, Wnchca Epithrlialco Zellwadistum, lonentransporic und Enzymaksivititcn ;dcfini;rter Nephroncpithclia in der Langzcitkultur unter der Wir- kunS von Nikogin Prof. Thurau und 0ber die Wirkunj dironisLhcr NikosinSabc auf -adrenocorLiCalS Dr. Seifert, hionchon Funktionen hci normotoncn und liypcrtoncn Rattan 009.1?'S 0 rl 0 cl c CD T 0 CD 0 cr Z 0 cr (D Fors~ungsnshmer T I Prof, Ballonk Froiburl qu3insifisis rung und Anslyso der Modilikation von Akuton Nikocin- wirku'n*gea ;uf Kreislauf unJ Koronarsystern nach chronisdier Mko- prly: .Doz. Dr. Dim, Wlrkii~ 'Zioarattonrauchgns suf kardiovoskulilro'Funknonta son be! Gllundfl~,- ~nJ PWIAICA mit Linksheraintuffixienz und koro- nartr H#rafrkrankunj Prof. GogharAg.Hoidelbers SOROS riph ildiii MJiweie ortarioskleratisdisr Argerienwandvir- ladoryngon bei Raudiern, und Niditraudiern inj Vorolsidl Prot Hoarldt.Wraburl Untsriu'444 ;ikrozirkulatorisdier Elfokso van Nikatin, Noben. ~ und Dr. brunif, Hamburg alkaloiJen und'Mccaboligen &us dem Zilarettenrluch in v#rsd%icJs- Den i4 vitro und in vivo Modellen Prof. Muller, m4allor Alvdalarmakro' hagan und Luagonemphyiern in AbhIngilktit van p den Roudipwohnhaiten Prof. Sirauer, Madien Untorsudiungen- aur slubalen und regionalen Funktion Jet linken Vontrikels in Abhingigkcit von Jer $sruninikotinkonxentration 1 091Z-S~100-1 0 CL 0 0 CID 0 < CD 0 0 3 Cr z 0 < Cr 0 0 .0 w I 31 ~;. cr Einflu4 dit Rauchina~'161 n N i Idl ~4 Th, onto Prof. Assmann. negir iiinua do# Zigarettinraudicit; suf don Stoffwc~hsrl der High- M D.11114y'Lipoproctin4 4, Priv.-Dox. Dr. August 61 ~r:V-04 J#1 RAUChOnt 34f Jon Lipoprolcinstollfwodiscl Hcidslbof g Prof. Wdol G861604"' 11444UNcin Zi oam top ri Lidion und Nikatin suf die 131nJung und ~:x 't , , q i~ah'R* yon Li Au 0 ~s yon Upoprmin(raligionon in Zollon unil Jon sollullron, b 'QmA Yon Nikotin unJ soinons flatiptnistaboliton Kotinln auf l 1,1 Praia Prof. We er Ym4 1 Heidelberg,' Dr. Spohr 'hdi~10601# SylC11114. Aul das caMiovaik4lirl SYSIOM unJ suf don . , i $i64#Lfisol nuh allginigen Nikotinapplikationan in Abgrgniung zu Nik6tiq'~Yirkungcn, wic sic durdi R-14dien von nikotinhaltigen ?ijil if it hurvorgartifca werJen ar C" IF Prof. Weber und'~' rau Der"influR volt Zigarettenraudion au( die Fibrinbilduns in vivo, go- Dr. HarenberS, HciJC1b4r6: ~iojidn Ahand! volt FibrinopwptiJ A-Spiveln 0 9 17 0 0 1 IL :11 Other Possible actions of Nicotine The role of nicotine was described in the context of a series of smoking associated diseases. Cardiovascular disease probably causes the greatest concern. The evidence for an association between smoking and coronary heart disease is less strong than was the case a few years ago. Nicotine in cigarette smoke increases heart rate and blood pressure, but these changes are within normal physiological variation found in healthy subjects. The high incidence of peripheral vascular disease and cerebrovascular disease amongst smokers still causes a lot of concern. Nicotine may have a role in precipitating these diseases by disturbing the prostaglandin balance so increasing platelet aggregation, and via chemo- tactic effects on thickening arterial walls. -ulcer&tion-, --amorkgst. -smokers, may be Pept c~ -more--common related to Ahe effect. _A-i!~RtjpS: -.~~ct,_3~educe_ paj~qre_atic bica:~bonate`_"se'cret.ion. which- Is-_"sential for. .-neutralising gastric acid. i ;~A-40 Smoking has been associated wii.6 four main'.". respiratory -a z gdf6i6lins't s--.. fn ~ the '0--f - -"I-u-n'g cancer.7-7, there is limi ted evidence that nicotine may act as a co-careinogen. There is either poor quality data, or no data at all, to relate nicotine to chronic obstructive lung disease, chronic bronchitis or emphysema. Smokers have low birthweight babies with a debatably increased incidence of perinatal mortality.; . Nicotine can reduce uteri e' blood flow and so *reduce theF: supply of T nutrients to _.~he fetus. Nicotine and its metabolites can pass through-..the placenta -and into the fetus. Fetal tissues can metabolise nicotine. The effect of.-'-nicotine on fetal growth and the incidence of-congenital malformations is unclear. Nicotine reduces the production of milk and may thus moderate neonatal nutrition. BATCo document for Province of BritiSh Columbia 8 November 1999 Discussion on 'Other possible actions of nicotine' Evidence on the weakening association between coronary heart disease and smoke was considered with reservations. The debate concerning the contribution of hype rcholes te rolaemia smoking/hypertension and personality type in risk of coronary heart disease continues. Nicotine may not play a strong role in coronary heart disease, but it could be a precipitating factor in peripheral vascular disease and cardiovascular disease by its actions, indirectly on the prostaglandins and chemotactic effects on platelet aggregation and thickening of the arterial walls. Although-'nicdfi'.n'e 'may have some effedt' tqCredu~ uterine blood nutrien flow the supply of , ts- to the fetus e , - - duri:fig pregnsC~cy, carbon monoxide is-likel" y t4i;::be more impc~-itant in-alteritig oxygen availability..'--. There i inedoclusive evidence for any role olt nicoti ne;~:in smoke affecting congenital-malformations.'-, --4 BATCo document for Province of British Columbia 8 November 1999 CL 0 C (D :r 0 z (D Cr M 'QQLAR DISEASES C A R D IQV , Coronary heart dlsbases.:~'- myocardial infarction- angina poctoris, Ischemic heart disease b1 vascula"I' k e r Cerebr'ov'ascu lar disea se. Does smoking increase the incidence of these diseases ? ' ~Does nicotine hav'e a specific role in these diseases ? V091LSWI > Cr aCULAR PARAMETERS DIOVA OTHER CAR , 'N SMOKERS At TEM2D II r)"oI f~,.~,'h'aematocflt thrombosis ..,platelet adhesiveness WNkening ollirWHAI 'WhIls.0 irregular M.'aWt bt a t s' io fr! i II I that nicotine may affect any of these parameters 9 M-Wol EFFECTS OF NICOTINE Ldw'do.~es.to central nervous system 9/kg ais P01-rs!"Us and Van. Zwlete 97-8) c n rats'. ..Sutton and Isaac (1973) ,"OA 11 heart rate 'J:..:,arterial blood pi"essure Ai7 Main effects of nicotine,.are via release*of c~`dlamlnese.. renalin e:'doses -100 Ajg/kg cat6 g., nora-d heart rate b'loo'd pressure cardiac output coronary, blood flow ..+.:pe.ripheral vasocon'striction NICOTINE IN THE CONTEXT OF CIGARETTE SMOKE Nicotine yield -4 2.0 mg per cigarette, spread over the normal period of smoicing, InIdependent Scientific Committee produce at most only minor changes In blood pressure and heart rate of an order well within the normal daily physiological variation'. Small doses of nicotine may, however be harmful to people with certain disease states, most particularly ischaernic heart.disease and'peripheral va.,;cular disease". 0 0 1 > 0 0 CL 0 Cr 0 Wald et al (1 9P 1) .;.. ; I .I .; Urinary cotinine similar in,,,cigarette and pipe smokers although coronary heart disease risk higher in cigarette, smokers Kauf man et al (1983) Risk of'first (non-fatal) myocardial infarction in men un er 55 years of age was unrelated to nicotine yield of cigarettes smoked IQ091_1q'1001 0 9 0 0 1 Straler showed there was no difference in the increases In heart rate and blood pressure after smoking a cigarette between healthy subjects and patients with circulatory disf unctions. Holmer demonstrated that the pathology of stenosis was not associated with cigarette smoking. (Symposium on Smoking and Cardiovascular Function, 1984) > 0 0 Q RAULTIPLE RISK ErkC OR INTERVENTION TRIALS OAR FIT) 0 Iva -Oslo Nermann et al 1981) dietary cholesterol CD U.S.A. (MR FIT group 1982) smoking 0 U.K. (Rose et al .1983) #body weight 0 0 Belgium (Kornitzer et al 1983) #physical exercise North Karelia (Puska et al 1983) control of hypertension z 0 Russia (Glazunov et al 1983) (D Cr W Effects on cardiovascular disease INCONCLUSIVE 0 9r;,, 0 0 1 (D 0 3 z 0 DIS;=ASES OF TH- R`*-SP1R/-`7 ORY TRACT ASSOCIfil"D WITH SMOKING: POSSIBLE ROLE OF NICOTINE Disease' Effect of nicotine' 19 0 0 1 Cancer Chronic obstructive lung disease c S.' C Wr` 6'n: I' bronchit'j" Emphysema Cocarcinogen ? None None None z 0 Cr W W 19 0 1 SMOKING AND,PEPTIC ULCERATION Smoking Increases the incidence of peptic ulceration Dependent on number of cigarettes smoked length of smoking history (Harrison et al 1978, 19791 Clee and Clark, 1982) Ratb -of healing of ulcers Is slower in smokers JElsahoff and Goodman.- 1980) 0 0 SMOKING AND PEPTIC ULCERATION ACID produced by stomach neutrallsed i/ by SICA0130NATE secreted by pancreas into duodenum ACID BICARBONATE Cr w and/or Ulcers form if Effect of nicotine Inconsistent 0 0 FFECTS OF ICOTIM ON GASTRIC ACID SE-CRETION rl 0 Generally Inc onsistent ef f ects Dose Species Effect Reference 1 t m Th o (1970) 00 pg/kg ra o n ps 0 0 E, 00 Ajg1kq r hompson et al ('1970) 3 xdally,15d z 100 tig/kg dog no change Konturek et al (1971) 0 Cr 200 jug/kg cat no. change Radeckiet al (1972) (D 400 *ug/kg cot Radecklet al (1972) 001 W > 0 0 CL 0 (D 0 (D 0 :r 0 0 z 0 (D Cr THE EFFECTS OF SMOKING DURING PREGNANCY % 9!.-L -Cj 0 a .1 birth weight perinatal mortality ? Incidence of malformations IS NICOTINE INVOLVED ? CO Z 0 (D Cr (D 9191~~j 1700 1 Nicotine, cotinine and nicotine - N oxide cross the d a6imals. placenta in humans an (Hibberd et al, 1978) Nicotine Is distributed and. metabolized by fetal tissues. (Stalha'ndske et al 1969, Martin 1982) NICY IN HUMAN SMOK'ERS DURING PFlEGNAIN Variable changes in ute*ro-placental blood flow Cloeren et al 1974 Morphometric changes in fetal vessels of. placenta Van der Velde et al 1983 IZ fetal blood viscosity z Buchan, 1983 0 < IS NICOTINE RESPONSIBLE ? 19 q ~7 00 1 > 0 0 Nicotine Release of catecholamines Constriction of uterine blood vessels 0 0 Reduced availability of oxygen and substrates z Important before and after implantation. 0 1.e. In early and late pregnancy 0 0 0 0 z 0 Cr EFFECT 0r N!C01 IN" ON UTERIN" BLOOD FLOVY, Rats No off ect 1.0 or 1 0.0,pg/min 5 mins Bruce and Parkinson 100 Ng/mln' 5 mins, (1979) 5.0 mg Mitchell et al 0 983) Sheep 1- 1.5 mg/min Resnick et al (1978) Kirschbaum et al (1970) Nicotine Injection to dogs damaged placental capillaries (Fischer 1957) r 17 61 ~:',; 00 1 RELATIONSHIP BETWEEN SMOKING DURING PREGNAMI-CY AND CONGENITAL MALFORMATIONS Inconclusive epidemiologic' evidence 0 0 F z 0 Kelsey et al* (1978) No risk with e. 20 cigs/d Some risk with > 20 cigs*/ d - this was specific for smoking during rather than before pregnancy.. Animal studies. Inconclusive. Some. Indication ~f malformation at high doses.* 0/,q~-C!7001 I.. .~ .-I ~: NICOTINE ADMINISTRAT-10N TO PRE'%'.-'A%NANT ANIMALS q,1,7,4,0 mg/d fe'ta'l,.weight, ratp,, (Becker at al 1968) fetal and placental weight, mice (Rowell -and Clark,1982) 1.5 - 2.0 mg/d No effect on fetal or placental weights In mice (Wang at al 1983) neonatal death, rats (Becker at al 1968) Cr No reduction In litter size (Rowell and Clark.1982) 9'1~ Sib;: 0 0 1 00 . . > I 0 0 CL 0 LACTATION 19 0 Nicotine, cotinine and nicotine-N '-oxide are transferred 0 to'breast milk in human smokers. p, (Ferguson et al 1976, Hibberd et al 1978) 0 0 z Nicotine -may suppre.ss.:production of milk but not the 0 suckling Induced release of milk once formed. Cr CD W (Blake and Sawyer, 1972 rats) 00 > I 0 0 Q 0 -h 0 0 0 0 0 2 : a) Co z 0 CD 3 Cr 11 RETARDATION OF NEONATAL GROWTH Reduced Milk Supply Effect of smoke components transferred to milk on t1 h eneonate Effect of smoke In atmosphere breathed by neonate, 9 Mutagenicity of condensates from high nicotine tobacco cigarettes Information on the biological activity of nicotine is of paramount importance to the tobacco industry. Cigarette smoke condensates examined by the National Cancer Institute, USA, have suggested that increased nicotine content of smoke condensate correlated with increased skin painting activity. However, it was also suggested that this association might be due to other chemicals whose concentrations changed along with nicotine yields. These results prompted BAT, Southampton, to produce the JANUS B-14 cigarettes in order to confirm or reject the NCI conclusions. Imperial Tob acco's interest in the biological activity of nicotine led to its testing in the Rosenkranz E.coli test in which it was found to be positive and that pH had a marked effect on its activity just as it affected the activity of smoke condensate. With the availability of the Ames Salmonella microsome test, nicotine was systematically investigated in this assay system and found to be negative. These studies were carrried out by employing metabolic activation systems prepared from the following tissues: (a) Aroclor-treated from the rat liver (b) 3-Methyleholanthrene-treated rat and guinea pig livers (c) Nicotine-treated rat liver (d) Cigarette smoke-treated rat kidney An examination of the JANUS B-14 cigarettes in the Ames test showed that higher nicotine content of tobacco and smoke correlated positively with mutagenicity. Similarly, the mouse skin test carried out by BAT Southampton gave a similar positive correlation between nicotine and C__ carcinogenicity. However, doubt was cast concerning the production of appropriate control cigarettes for this set of cigarettes. BATCO document for Province of British Columbia 8 November 1999 -2- Consequently, when cigarettes made from certain strains of high nicotine tobacco grown in Canada were available, it was considered of great interest to examine them in the Ames test. Thus, cigarettes produced from these high nicotine strain tobaccos grown in 1980 and 1981, when examined for Ames mutagenicity, showed that the nicotine content of the tobacco or smoke had no relationship with their Ames activity. These results suggest that the positive correlation observed in the JANUS B-14 series was an artifact of nicotine applicaiton to the tobacco. An examination of these high nicotine tobacco -cigarettes in the mouse skin carcinogenicity test would provide information of great value to.BAT. CD BATCo document for Province of British Columbia 8 November 1999 TABLE I EFFECT OF CHEM ICALS ON GROWTH OF PARENT AND DNA POLYPIERASE-DLFI CI ENT MUTANT OF E-.COLI DIAMETER OF ZONES OF - CHEMICAL AMOUNT INHIBITION POL A- POL A+ CRYSTAL VIOLET 100 UG 19.2 0 AMP I C I LLI N 10 UG 24.0 24.2 CH LOROHYCET INI 30 UG 23.7 23.9 FORMALDEHYDE, 37~ 20 UL 52.1 463 ACROLEIN 0.66 UL 17.5 0 MET11YU-1.ETHANE- 10 UL 48. 7 31.0 SULPHO14ATE P-PROPIOLACTONE 6.3 UL 42.2 29.2 NICOTINE 1.0 Mul 0 p N 1 T ROMETHANi-E 100 UL 0 0 PHERYI-HYDRAZ I NIE 4 UL 56.0 42.1 K4 LEIC HYDRAZIDE 100 UG 0 0 0(-NAPHTHYLAr-,I11E 10 11% 23.9 24. 1 SODIUM CYCLAtWE 20 MG 0 0 SACCHARIN 67 I-IG 17.9 19.3 o-TOLUENE 3.3 MG 0 0 SULPHONAMIDE CAFFEINE 1.9 MG 0 0, BENZENE 100 UL 111.3 13.0 BENZODUI14011E 2 MG 54.5 52.? CATECHOL 33 MG 30.6 30.8 CHLOROGENIC ACID 12.5 MG 0 0 RUTIN 5.0 MG 0 0 COUIMIARIN 7.3 M!G 20.6 21.4 METHYLCOUMARIN 2.0 I-IG 13.0 13.0. HD NT "ACENE A .5 G 3.0 3.0 BE14ZO(a)PYRENE 2.5 115 0 .0 BATCo document for Province of British Columbia 8 November 1999 CD C;\ TABLE. IX EFFECT OF PH ON INHIBITION OF'PARENT AND DNA POLYMERASE - DEFICIE-NT MUTAW OF E. COLI BY CIGARETTE SMOIK'E CONDENSATE MOUNT OF TPM IN - PH OF PRE-INCUBATION NO. OF VIABLE ~ACTERI.A PER -EACTION MIXTURE PRE-INCU- T I ME ML ....(,MG) BATION WR) REACTION MIXTURE MIXTURE POL A Pcr- A+ 0 x 107 51 72 x 107. 6.6 1 . 19 x 10 52 x 197 1.9 7. 38 x 19 81 x 196 62 197...,. 5 8.9 1 NIL 90 10 2 NIL --- 511-11 L 58 x 1.97 - 7 10 2.5- 6.6 1 k4 x 194 107 57 , 40 x 102 7 -51 19 0 7 5 116 x 19 7 7 n, i9 2.5 8.7 1 HI L 10 2 HIL U5 10E) 0 34 x 107 132 107 1.0 6.8 1. 21 x 107 97 107 2 36 x 106 log x 107 9 6 144 x 19 x In, 7 .1.0 8.9 1 ~ .9 x 10 ? 79 x 107 - 2 37 x. 101 -. 59 x rETO*'E CONTROLS RUN SIMULTANEOUSLY SHO'KD 110 SIGNIFIC.4NT 11141BITIO,%,' OF EITHER 'OL A- OR POL A + BATCo document for Province of British Columbia 8 November 1999 TABLE X EFFECT- OF PH ON INHIBITION OF PARENT AHD DRA PQLYMERASE-DEFICIE NT MUTANT OF E.COLI BY KICOTINE AMOUNT OF PH OF PRE-INCUBA- NO. OF VIABLE BACTERIA NICOTINE IN REACTION TION TIME PER M L REACTION MIXTURE MIXTURE (HR) RFACTION MUM~RF (MG) POL A- UOL A" 20 7.0 0 5 X ]07 12 X 107 0.5 26 x 106 X X -:-,7 1.5 5 X 107 %x 1r; BUFFER 7.0 0 36 X j07 01; X ]07 0.5 26 X 1117 j3, X 1,)7 . 5 15 X 107. X - r7 20 8.0 0 5 X 107 V x i 0.5 16 x loll 5 x Ij-'7 1.5 8 x 1011 q X 1,,7 BUFFER 8.0 0 32 X 107 -0 x lc~ 0.5 27 X 107 J'3 X K~ 1.5. 39 X 107 -a X j,07 20 9.0 5 x loll 03 X 105 0.5 NIL 2 X 104 1.5 NIL 25 X 10 BUFFER 9.0 0 44 X 107 307 X 107 o.5 30 X.107 -SB X 107 34 X 107 S5 X 107 BATCo document for Province of British Columb ia 8 November 1999 .INHIB[TION OF PARENT' AND DNA-I)EFICIENT MUTANT OF F- COLI ?5y NICOTINE'- E7FFECT OF Teh-r-a7,t Auxvivaf, cf E-. COL pH of 'Rot A.'~ TU A+ 8-0 0 58 -.10 175 0 C7 1.0 0-051 6-85 1-5 . 0 0-011 1-9-1 Cl- 0 1-0 0-0000 0- 002* 1.5 0-0000 0 - 00002 G in &eactzu 71"~r'y -9,0 hemed- a6 Cent CDn-koIL.- CD r\3 BATCo document for Province of British Columbia 8 November 1999 TABLE AMES MUTAGENICITY. OF SMOKE CO'NDENSATrS FPOM TANUS B14- CIGARETTES U3 ~ At-nes t-nutc9eni T CLty Cmtculat,~a . tj-~Iy, J c0 6 r-ar,4ormect dz~-tcL Untransforrnect- ao 0. ' c LosaritA- Siuare Poirit keelgression I;Kes . ryl;c root C0tCLLt0-- Sep.cui-v * Sfr, VonS es curv BM-4- o-5571 1-5953 2-05t 0-31 2-38t 0-39 2-4-0 Bf4--2- .0-5709 I-G105 2-07 0-40 2-50 0-2-9 2-5D B14-3 O-GI30 I-G630 B14-4- O-G910 1-7451 PL CK O-SG77 I - ro 10 G ,20 . 2-29 0416 2.92 0-4-7 2- %- 2-58 0-S9 3-48 0-G9 3-47 2-11 0-~-O 2--34- 0-22- 2-34- B14-4- siSnificcLntly higber than t~q other 4 ciqcxrettes (p Z- 0-0s) B14-3 C'UicL B14-4- sLqm;-F!caAt1y ~jqAer than t,~e otkef' 3 c' 1. , hi kel- hx-rettes (p<0-01). B14-4 sL n;fir-artj 3 t n B14-3 CD BATCo document for Province of British Columbia 8 November 1999 Zeflab*ons botimeem Amcs-Muto -Ilicity (calculatect usl~~13 umtf-Qhs-r, Ca dcLv--) total 1-iicotiric mika-loic6 in tke amd ,/Ctrius ?414 sel-les 3.0 B14,* B14-3 B14-3 ~/SlAro,2. 14 J B14-1 814-2 PLCK .2 8 B14-1 PMO-99 pa 0.99 0 0 TotnL rore d Ln -Y .0 C OD Qj 4 J .0 C4 00 C 0 4-J Ip kq! U -a &or w Saw BATCo document for Province of British Columbia 8 November 1999 Im > 0 TABLE 0 A I ES -M-e-'IGAR F-7--rEs 0 M2S MLTrA E N CIT[ Dj~_P_r_n N T) ems AT C-9 PRO ;--C-QNTAlNfN&'! -M-26/ A HIGH N.ICOTINE 7-n73ACCO CD A/,' 0 D e,6 eAy> t)', co Ole-, datcL cj t 0 CD ox!,M-2r. 0 to c G8 262. 1007. M-2G vet&/at~dcj, 004zt~ ZI-51 0 0 IoS 207.; M-26 q.9z 0'/2 0.11 0.1295 z 0 E,, ATWIVE t H- z ... . ........... . ........... . ........... + ... ......... .. ........ . ........ . tL U Ld oh I00='- CD 4 ~. SMOKE CONDENSATE PLATE CIN 4 >- BATCo document for Province of British Columbia 8 November 1999 ......... . ..... . ......... 'CONDENSATES: FROM MES*HMUTAGENIC[Tlr=S::-'OF~SMO) ........ . .................... 77- ------------ + 7-n- =7: I 77. z;--:2oO ........... SMOKE CONDENSATE.- /PIATE BATCo document for Province of British Columbia 8 November 1999 Trends in Epidemiology With the advent of improved statistical methods for examining the massive quantities of epidemiological data available, the incidence of malignant diseases in successive generations can be estimated. The bulk of such work has been carried'out in the UK by the Medical Research Unit at their Epidemiology Unit in Southampton. Such analyses, usually referred to as "cohort analyses" demonstrate a declining incidence in all cancers for generations of men born from 1900 onwards and for women born from 1930 onwards. Of particular interest, declining lung cancer incidence is found in men born from about 1900 onwards and in women born from about 1925 onwards. Cigarette consumpti.o.n f igVes on a cohort basis have been published in the UK_by the Tobacco Advisory Council, and on such a basis, comparison of the time trends in lung cancer with the time trends in cigarette consumption show quite remarkable differences. Likewise, the time trends for the individual cancers associated with smoking are different. Such trends are clearly inconsistent with the causal model of the relationship between smoking and diseases and further analysis of these time-trends is now.being undertalcon. BATCO document for Province of BritiSh Columbia 8 November 1999 Smoke condensate in short term test. An inhouse study with short term test was'done on cigarette smoke in R&D Hamburg. The short term tests which are used can be divided in three groups: 1) No response to CSC: E. coli test (Mohn) Bone marrow of mice 2) Qualitative answer only:E. coli repair test SCE in CHO - - -7 -MN .-In-humarr lymphocytes - SCE in human - lymphocytes UDS in human fibroblasts z 3)__QuantItati-v-e-a-nswer-can be Ames Test Sabaceus gland test mouse skin painting From the results it could be demonstrated that there must be some groups of active compounds in CSC because i f f ex-erLt.-genetic--endpoints-are-formed. By synergistic-antagonistic experiments it could be shown that the presence of chemical groups like PAH is not an indication for the final activity of the --ii-hole - mixture. CD C__ BATCO document for Province of British Columbia 8 November 1999 lRe F arence Cornpou'nds Te, s f c4 qjo ZC'i P phosphorn4k AF MNNG Conden-sole Arnes + + + + Te S S9 + E. col i Tas~ + + + CHO S9 + + SCE S9 + 4 + ct~ mph MN + + + c5 les SGE + + + Zone rna.rMw + C m. ice) 'Rapair E. coli + UD5 SQ6. ancl -j IN BATCo document for Province of British Columbia 8 November 1999 Ames TasF 4. Spon6naus 'Raver~o4s 2. ToxCci[s / Survi'vals I S9 JnducJ;on Specia-S PreinGubaH*on Cond an s a ~e 'PraparaHon .'Prer-ip;~aHon Ct'3areffe btArn;d; 1~ 'PufF volurne C igo re ifa cles;9riL -7 7 C-D C, N BATCo document for Province of BritISh Columbia 8 November 1999 SESSION SEVEN CD C::) _r~- I U. .! C'N Q.;-~ BATCo document for Province of British Columbia 8 November 1999 Biological research into smoking associated'diseases has often been hindered by the lack of good animal models, for instance, there is no biological model for the cardiovascular diseases. However, more modern techniques can be used to monitor biochemical changes which occur in human smoke'rs, both diseased and healthy. In many cases, the same biochemical changes can be detected in animals exposed to cigarette smoke. Areas where biochemical changes in animals reflect those found in man include: Cardiovascular system: The prostaglandin:thromboxane balance affects dilation and constriction of blood vessels and platelet aggregation. Respiratory tract.diseases: (a) Studies of drug metabolising enzymes may indicate the potential harm.smoke may have on lung tissues. (b) Emphysema is difficult to produce in rats by smoke. However, smoke exposure of rats causes similar changes in the protease:elastase ratio which is found in human smokers. (c) Mucociliary clearance is altered in human smokers and smoke exposed rats. Changes in the biochemical and physical properties of mucus are similar and can be measured in animal systems. Outcome of pregnancy: Smoking of either parent before conception and maternal smoking during pregnancy or breast feeding may affect growth and developmeni of the fetus and neonate. The question of increased congenital malformations in children of smokers remains unresolved. Studies of drug metabolising enzymes of maternal, placental and fetal (:D tissues could indicate biochemical potential to protect the fetus. BATCO doCUrnent for Province of British COIUMbia 8 NoveMber 1999 Efficacy of therapeutic drugs: Smoking alters the metabolism and clearance of drugs prescribed for therapeutic use. This can require modification of doses administered. Changes in the clearance of a drug could be used to monitor the development of cancer and the intake of carcinogens from various environmental sources including diet. Smoke produces irritancy at various sites. Measures of irritancy exist for eacb site and could be compounded into an overall estimate of the irritancy of a product. Each of -the "bio'logidal 4reas outline'd above can be studied using sensiti.ve-biodhemical technIques. Such techniques cin be- applied to, studying th6-effects of individual snwke77comii~nents -E~nd the relative activity of vapour versus, paiieulate-_phases of smoke and mainstream, sidestream and ambien-t smoke. The effects of changes in tobacco used or'i"he physical characteristics of cigarettes designed to meet market demands or alter the biological activity of smoke could now be investigated in animals and related to smoking associated diseases. BATCo doCUMent for Province of BrItISh Colurnbia 8 Novernber 1999 -6 kii 'n'- g--M-16 ed-dUce_th_"e__ eff icacy--of -drugs W eg ineffective use of propoxyphene to relieve I.. i headaches,,,., i 10. 1 %,qon-.,s mpkers 15.0% smokers < 20 cigs/day. Nwangwu 20.3% smokers > 20 cigs/day. eta/ (1982) ISmoking may,rp,09pe unwanted side effects eg drowsines'sw'ith use of chlorpromazine t I : ,I (6anquilliz&VA 22% n6h 1~ryibkeis 14% lldkftokers Jick (1974) 4% heavy smokers 0 0 1. I Clearance of drugs Activity of druq .i(,! 11~ --, iio t, ii metabolizing enzyMest. (cytochrome P448),,, Is the increased, QxJdation rate amongst cancer patients a genetic factor? .rwant A test compound., that, pan be given to subjects to get ameasure of cytochrome P448/P450 as a predictor of cancer. Relate to: Susceptibility to cancer Diet, meat, fat, vitamins. Smoking. Cancer patients Sm'oke'rs 1 ; 1/ 0 0 1 CD 0 < 0 0 0 CD 3 Cr Q0 0 r r- Assess physi 0'109i' ell modifications to protect the foetus by measuring drug metabolizing enzymes of a b" tivation and- deto~