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Housz ~ e~c Z~ZZZ4~TS .RF.J'OILT NO, RD.]..524 UNCZ,ASSZFZ]ED 2~.10.1977 AIF£'HOl: E.B. Wt.Zkms ZSSUED BY: N.E. W£11£s rRo~;.._..____.~_.~: 11.o4.o2 s)xs~'~u:cxo.: De. S.J. Green Cop! Ho. 1 Dr. D.G. FelCon " " 2, 3 L£bcar,¥ '" " k, 5 OOFY NO : cJv3 Qo BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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EBWICAP/46D-2 Group hsea~ch ~ Development C~re, Br£clsh-Amer~can Tobacco Co. Led., SOUTHAMPTON. 24:h Oc~obe:, 1977 MOUSE SKIN PJ~qTING EXPERIMENTS • ~ i ~ COU~CT~0N OF TSE n~TA (Reporu No. ED.1524 Unclassified) s~Y This report describes the general form of nhe 3A~0S -~use skin painting experiments and the methods used in the collection and recordlnK of the daua. The praparac£on of cabulacions of Che dana in a fot'm su£~able for subsequent stac£stical anatysis ~s also dQscribed. References Co ~he compuver sys~-~ used a=e provided. BAT Co LTD - MINNESOTA TOBACCO LITIGATION O", ".-O

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-2- LNTRODUCT~ON This report is the first of s ser£es in which will be given an accou~tC o£ the statistical methods used et Che Group ~sear~t & Development Centre Ln the a~lys£s o£ the turnout-bearing animal data arising from the skin painti~ exper;menCs conducted under ~roject JA~JS. This f~cst report describes the sahara1 form of the experiments and the methods used in the collection and record£ng of the dACe. The preparation of cabulat£ou~ of ~he dace in a £orm su£table for subsequent numerical anmlysis is also described. Later reports v£11 descr£be the mathematical techniques used in the analysis of the tumour-bearing ax~imal data in detail, and a sec of hypothetical data will be used to deuwnstrate chase techniques. Ke2erenees to the orii~s of the methods used will be liven, sad referezlces to amy computer prosrams used will also be supplied. 2. THZ, NODE OF KXFER, .ZH~RTAT~ON ~be Beneral style o£ mouse skin peintinI experiments carried out under Pro~ecc JANUS is described in detail in (1). Unmated fale elbluo mice are used as experimental mz~amls, d~ter a per~od o£ quaxanC~e they ameer the a=cperJJae~: at 8 to 9 weeks of tie sad welsh on average about 20 I. Treated at;mils i.e, chose subjected to condensate applic~c£on have the dorsal ~caa of chair backs shaved eve~-y eve Co three weeks mad a fixed volume o£ psi=fLu4 soluC£oU 4s appl£ed to the shaved area three C/Juts each week. "Unpa~ced" control an/~als. when used, are subjmcced to the same shaving and palntin8 regime, buc in ChLs case the paiucinS solution contains no smoke condensate. BAT Co LTD - MINNESOTA TOBACCO LITIGATION m C7~

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-3- Occas£onally "cage" controls have also been 4ncluded. These animals are shaved, but receive no painting of any kind. The cobden•aces are prepared by cold-trapping the smoke from cigarettes on a machine using fixed puff-smoulder ~ntervals. Usually a 2-second puff is followed by • 58-second smoulder period, the pressures in the machine being adjusted so thac the firs-C 2-second puff has • volume of 35 el. The cigarettes are then smoked to • fixed butt length; commonly 23 mm or filter LenEuh ÷ 8 an. The cigarette smoke condensate so obtained £s dissolved in • 9:1 acetone/water solution. The concentration of the condensate in solution is varied so as Co obtain a range of effective dose. levels. • n the JANUS experiments the concennrations have frequannly bean adjusted so chat the 0.3 ml of painting solution contained (•) 20 ms of condensate (b) 28.3 mS of condensate (c) 40 mE of condensate. In a typical experiment each condensate would be painted at three dose levels three times weekly. Thus an experiment ~n which say Uhree d£fferent tundras•aces were used would require nine groups of animals (3 con~ensates x 3 dose levels). 3. THE COLLECTTON OF THE DATA It is the practice at Battelle (Frankfurt) to inspect e~=h animal once per reek. Skin lesions within the pa£nced uea dee recorded U turnouts if they have • dLmneter of at Least 2 am mad protz~da above the skin surface, or have • firmer couslstlmcy than the surronnd/zsg skin. Zf at any subsequent examination a Cumour is judged to have disappeared a rescess£on £s recocded. The general condition of the BAT Co LTD - MINNE..C~TA TOBACCO LITIGATION

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-4- animals is observed, and any qualitative and quan~iEative chanses recorded (e.g. ulceration, animal wmiKhC). All animals are subjected co a posc-morcem examination. The post-soften find~ss are aimed at describing the pathological condition of the animal at death, and diaKnosiuK the cause of death. These post- mort~ findings £all into threm sections covering skin ~mours in ~he painted a~e~, ocher turnouts (includLnK sk.~n outside the pa~ced ~cem), and Keneral pacholoKy. For skin t~mours in the pa~ced area, the posiCi0n o£ the turnout on the dorsal skin is recorded, toKether with the time o£ appearance (in exper~Jnene yacks), t/~ne o£ disappearance (i£ regression occurs), time o~ reappearance, and tLmes o£ any repeat disappearance/reappearance until the de~ch o£ the animal. At death, each tumou= is examined u~croscopicmlly and classi£ied according ~o the cell nype, character of Krowch, type of Krowth, and any eddicional in£ormarlou available from the exMuLnaclon of the microscope slides. For ocher cumours, the dace o£ detection is recorded, (for internal tumours, this is the dace o£ death of the an4m~l, and the tumou~ Ls described in terms of the orKan systms, functional part of orKan, and the cha~cacter and cype of Krowth attacking fihe orKan. The seneral patholoKical condition of the animal 4s described 4~ terms o£ the c~Kan and functional part o£ the orK~ in which any abnoz~mallcy is sQ~, along with • descztpciou o£ the &baormAliCy. Savages1 pacholosical abnocmalitio8 (or c~mo~s) may be peasant 4n a sluSle an/mal, and an e££orc is made co indicate which of chose was responsible ~or the death o£ the anL~al. All orKans and tissues ace exauLned, mud BAT Co LTD - MINNESOTA TOBACCO LITIGATION O c~r~

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