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INBIFO Institut fur biologische Forschung • Koln Dr.med. R. Rylander c/o FABRIQUES DE TABAC REUNIES S.A. Switzerland REPORT P 0500/3068 n imoru - 26.Oct.87 DKU/UBE COPY NO.: Skin Tumorigenicity of Mainstream and Sidestream Whole Smoke Condensate of Standard Reference Cigarette 2R1 80-Week Dermal Application Study with CD1(ICR)BR and B6C3F1 Mice INBIFO Irustiful fiir biologische Forschung GmbH, Fuggerslra6e 3, D-5000 Koln 90 Sitz der Gesellschafl: K&In HR B 367, 29. Oktober 1959 5085 Telefon: Porz (02203) 303-1, Telefax: (02203) 303362, Telex: 8874675 inbi d Institulsleiter und Geschaftsfiihrer: Dr. med. Ulrich Hackenberg

INBIFO Institut fiir biologische Forschung • Koln Dr.med. R. Rylander c/o FABRIQUES DE TABAC REUNIES S.A. Switzerland REPORT P 0500/3068 26.Oct.87 DKU/UBE COPY NO.: Skin Tumorigenicity of Mainstream and Sidestream Whole Smoke Condensate of Standard Reference Cigarette 2R1 80-Week Dermal Application Study with CD1(ICR)BR and B6C3F1 Mice Volume 1 INBIFO Institut tiir biologische Forschung GmbH, Fuggerstra0e 3, D-5000 Koln 90 Sitz der Geselischaft: Koln HR B 367, 29. Oktober 1959 5oas Teleton: Porz (02203) 303-1, Telefax: (02203) 303362, Telex: 8874875 inbi d Institutsleiter und Geschaftsfiihrer: Dr. med. Ulrich Hackenberg

HEPUK'1' P 0500/3068 UBE1:43RB18 7010 PREFACE This is a part (integrating report and subreport of team Animal Treatment) of the complete report. The complete report consists of an integrating report, which gives a general description of the complete project, and 4 subreports: (1) Analytical Chemistry (2) Animal Treatment (3) Microbiology (4) Pathology Each subreport had been worked out by the respective responsible team. Methods are described in detail in the respective sulbreport but not in the integrating report. Remarks: This part of the report (integrating report and subre- port of team Animal Treatment), including title page, contains 296 pages. 3587

INB'IFO Institut fur hiologische For~,chung - Kol'n 3 iriru DR•MED• R• RYLANDER 26•OCT•87 c/o FABRIGIUES DE TABAC REUNIES S•A• DKU/UBE UBE1'41RA3 SW'I TZERLANI) INTEGRATING REPORT ----------------------------------- P 0500/3068 SKIN TUMORIGENICITY OF MAINSTREAM AND SIDESTREAM WHOLE SMOKE CONDENSATE OF STANllNRD R'EFERENCE CIGARRETTE 2R1 8D-WEEK DERMAL APP'LICATION'STUDY WITH CD1(ICR')BR AND B6C3F1 MICE INBIfO kiatGtul fiir biolbyische Forschuny GmbH; FuQyerette8e 3, D•5000 Ktfin 90 Sifz der GeeellschaR: KSIn HR 8 367, 29. Oktober 1959 28.Kwe3 Telefon: Porz (02203) 303-1, Telefax:,(02203) 303362„ Telex:8874675 inbi d Institutsleiler und Gesch&ItBfOhrer•. Dr. med. Ulrie:IrHeckenberg

INTEGRATING REPORT P 0500/3068 UBE141RB11 7295 PAGE 0-1 CONTENTS PAGE ABBREVIATIONS 0-3 1 SUMMARY 1.1 Study Concept 1-1 1.2 Objectives 1-1 1.3 Method 1-3 1.4 Results 1-5 1.4.11 Condensates 1-5 1.4.2 General condition and behavior 1-6 1.4.3 Mortality 1-7 1.4.4 Body weight 1-8 1.4.5 Development of macroscopic skin tumors 1-9 ~ 1.4.6 Skin irritations 1-9 1.4.7 Prediction of the microscopic tumor probability in future studies 1-10 1.4.8 Microbiological findings 1-11 1.4.9 Pathological findings 1-12 1.4.10 Conclusions 1-15 FIGURE A: OBJECTIVES 1-2 2 RESPONSIBILITY 2-1 2.1 Project Management 2-1 2.2 Contributing Teams 2-1 't^g5

INTEGRATING REPORT P 0500/3068 UBE141RB12 7295 PAGE 0-2' CONTENTS (continued) PAGE' 3 QUALITY ASSURANCE STATEMENT 3-1 INTRODUCTION 4-1 TABLE A: CONCENTRATIONS OF SELECTED COMPOUNDS IN NONFIL- TER CIGARETTE MAINSTREAM SMOKE AND THE RATIO OF THEIR RELATIVE DISTRIBUTION IN SIDESTREAM SMOKE, PARTICULATE PHASE -2 TABLE B: CONCENTRATIONS OF SELECTED COMPOUNDS IN NONFIL- TER CIGARETTE MAINSTREAM SMOKE AND THE RATIO OF THEIR RELATIVE DISTRIBUTION IN SIDESTREAM SMOKE, VAPOR PHASE 4-3 TABLE C: GROUPS AND DOSES 4-8 FIGURE B: CHRONOLOGY 4-10 5 SUBSTANCES EXAMINED 5-1 5.1 Cigarette 5-1 TABLE D: PHYSICAL DATA OF CIGARETTE 2R1 5-2 TABLE E: CONCENTRATION OF SELECTED COMPONENTS IN FILLER, CIGARETTE TYPE 2R1 5-3 TABLE F: YIELD OF SELECTED SMOKE COMPONENTS, CIGARETTE TYPE' 2R1 5-4 5.2 7,12-DimethyTbenz(a)anthracene 5-5 6 STORAGE OF MATERIALS AND RECORDS 6-1 7 REFERENCES 7-1 Remarks: This integrating report, including title page, contains 41 pages. ., q1

INTEGRATING REPORT P 0500/3068 UBE141RB10 7295 PAGE 0-3 ABBREVIATIONS (a,b) AC : team Analytical Chemistry DIN : Deutsches Institut fur Normung (German Committee of Standards) • DMBA : 7,1i2-dimethylbenz(a)anthracene DMNA : N-nitrosodimethylamine .GT. : greater than MS : mainstream smoke MTR : macroscopic tumor rate MWSC : mainstream whole smoke condensate MWSC-C : mainstream whole smoke condensate, collected with cool trap MWSC-I : mainstream whole smoke condensate, collected with impaction trap NAB : N-nitrosoanabasine NATB : N-nitrosoanatabine NNK : 4-(N-methyl-N-nitrosoamino)-1-(3-pyridyl),-1-butanone NNN : N-nitrosonornicotine NPY : N-nitrosopyrrolidine NTP : National Toxicology Program PAH : polycyclic aromatic hydrocarbon PT : preliminary title QA : Quality Assurance Unit RT : room temperature SOP : standard operating procedure SS : sidestream smoke SWSC : sidestream whole smoke condensate SWSC-I : sidestream whole smoke condensate, collected with impaction trap TPA : 12-O-tetradecanoylphorbol-13-acetate, synomym: 4-beta-phorbol-12-myristate-13-acetate TPM : total particulate matter, determined gravimetrically WS : whole smoke WSC : whole smoke condensate WSC-C : whole smoke condensate, collected with cool trap WSC-I : whole smoke condensate, collected with impaction trap (a) in addition to those, which are explained immediately on the same page (b) Units are given: in accordance with SI-norms (Systeme International d'Unites). T~ lc.

LN1'EGkAY'ING REPORT' P 0500/3068 UBE141RA4 7295 PAGE 1-1 1 SUMMARY 1.1 Study Concept In the present chronic study the skin tumorigenicity of WHOLE SMOKE CONDENSATE (WSC), of MAINSTREAM and SIDESTREAM SMOKE (MS and SS), of the standard reference cigarette 2R1 was determined after applications to the dorsal skin of female CD1 mice for 80 weeks. In addition, mainstream condensate was assayed on female B6C3F1 mice (a). WSC was applied to both mouse strains without and with pretreatment of a single dose (200 nanomols per mouse) of 7,12- dimethylbenz(a)anthracene (DMBA). This dose is thought to initiate but not to promote all sites in the application area which can be promoted to produce skin tumors subsequently. Therefore the pro- moting activity of the 2 WSC types can be derived from the skin tumorigenicity after DMBA pretreatment. 1.2 Objectives The objectives (b) of the study were the following: (1), the comparison of the skin tumorigenicity (tumor probabili- ty, multiplicity, and malignancy) of MWSC-I and SWSC-I of the standard reference cigarette 2R1; (2) the influence of the pretreatment of mice with an initiating dose of DMBA on the skin tumorigenicity of MWSC-I and SWSC-I; (3) the comparison of CD1 and B6C3F1 mice with respect to their sensitivity to the skin tumorigenicity of MWSC-I; (4) the comparison of the skin tumorigenicity of MWSC-I stored at 4 and -75 degrees centigrade before application. (a) hybrid strain used by the National Toxicology Program (N'TP) (USA) for carcinog~enesis studies including dermal application (b) see FIGURE A 'i995

INTEGRAZ'ING REPORT P 0500/3120 UBE141RA17 72'95 M PAGE 1-2 CD1 (MWSC-I) (3) B6C3F1 (MWSC-I) 4 °C ~IWSC-I (CD1 ) (4) -75 ~C MWSC-I (CD1) FIGURE A <: ---/ (2). ~------~ (2) /-------~ MWSC-I plus DMBA (CD1), (1) SWSC-I plus DMBA (CDI) CD 1 (M'IcSC-I pl u5 DMBA ) (3) B6C3F1 (MWSC-I plus DMBA) OBJECTIVES

INTEGRATING REPORT P 0500/3068 UBE141RA5 7295 PAGE 1-3 1.3 Method For the preparation of the application solution, MWSC and SWSC were collected in impaction traps (MWSC-I and SWSC-I), processed, and suspended in acetone. Concentrations of nicotine, water, hydrogen-ions, selected nitrosamines, catechol, selected poly- cyclic aromatic hydrocarbons, and volatile components of the WSC-I were regularly determined in either the processed condensate or the application solution. The application solutions were stored at -75 degrees centigrade until application except for 3 groups. These groups received MWSC-I stored at 4 degrees centigrade as in all previous INBIFO skin painting studies. 2625 mice, i. e., 1785 (a) outbred female CD1 mice from Charles River, France and 840 female B6C3F1 mice from Charles River, USA reared under specific pathogen free conditions were kept in barriered animal laboratory units. 7 mice each were kept in polycarbonate cages with granulated, softwood bedding. The mice were fed an autoclaved, fortified diet and watered with tap water ad libitum. The room temperature was 22 + 2 degrees centigrade, the relative humidity 55 + 10 percent and the light cycle 12 hours : 12 hours. The hair of the application area was shaved prior to the application of DMBA, before the 1st application of WSC-I, and subsequently 1 time per week and additionally for individual mice which showed increased hair growth. Microbiological screening of the mice as well as bacteriological controls of the laboratory unit, animal diet, drinking water, cage bedding material, and laboratory staff were performed. Selected pesticides, aflatoxins, and heavy metals were determined in the diet and in the bedding, material. Diet and bedding material were only used when the residues were below the tolerance levels. The 2625 mice were randomly allocated to 25 groups (105 mice per group)'i. At the age of 36 + 2 days the mice of several groups re- ceived a single dermal application of 200 nanomols DMBA (equiva- (a) mice for microbiological analysis not included ,. jr