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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

IPJ"1'L•'lik/i'1'1Nli kEPUk'1" Y 0500/.iUbt3 UBE141RA6 7295 PAGE 1-4 lent to 51 micrograms) per mouse. Both CD1 and B6C3F1 mice re- ceived the same DMBA dose. 10 days later (a) WSC-I application was started. To CD1 mice either 60, or 90, or 120 milligrams/(mouse x week) of processed MWSC-I or SWSC-I prepared in acetone or acetone alone were applied on 5 consecutive days per week. The application volume was 100 microliters/(mouse x application). B6C3F1 mice showed an approx. 30 percent lower body weight than CD1 mice throughout their lifetime. Therefore, B6C3F1 mice received MWSC-I doses reduced by approx. 30 percent compared with the doses for CD1 mice (reduction of the applicatiom volume to 70 microliters/ (mouse x application)). 1 dose was equal for both strains (60 milligrams/(mouse x application)). Considering the strain-de- pendent reduced body weight, the B6C3F1 mice received the same doses (mg/kg body weight) as the CD1 mice. The mice were checked for general condition, behavior, and mor- tality. The body weight was determined at least fortnightly up to week 20 of the condensate application period and every 4th week thereafter. Visual examinations of the skin for macroscopic tumors and ulcers were performed weekly. All surviving mice were sacrificed 80 weeks after the 1st conden- sate application. All mice sacrificed at that time and those that died "spontaneously" were examined for skin lesions. The skin of the application area and those sites outside the application area with lesions were removed and fixed with formaldehyde solution. Each lesion and 50, skin samples from the application areas without any macroscopic finding were trimmed, processed, and individually stained with hematoxylin eosin. The slides were examined and evaluated histopathologically. Life table estimates of the histologically confirmed skin tumor rates (tumor probabilites) were calculated according to Armitage (1971). For the statistical evaluation of the tumor probability the chi-square test of life table-adjusted incidences according to Peto et al. (1980) was used. (a) no applications in between ..q~

_ _- -_ - - ---- ----- INTEGRA''1NG REPORT P 0500/3U68 UBE141RA7 --7295 P_AGE 1-5 1.4 Results 1.4.1 Condensates For this study 176 batches of MWSC-I and 175 batches of SWSC-I were prepared from approx. 320000 cigarettes (standard ref- erence cigarette 2R1 ), . For MS the yield of crude and processed condensate (a) was 48.5 and 39.6 milligrams per cigarette respectively, for SS 22.1 and 19.4 milligrams per cigarette respectively. Thus the yield of the processed condensate was twice as high for MS as for SS. The MS application solution was slightly more acidic than the SS application solution: the pH values were 6.0 and 6.5. The nicotine concentration for MS was 20.8 grams/liter, and thus about 20 percent lower than that of SS at 25.1 grams/liter. The catechol concentration was nearly identical: 1.21 grams/ liter for MS and 1.10 grams/liter for SS. The concentrations of the nitrosamines NNN, NATB, NNK, NAB, NPY, and DMNA were between 0.01 and 1.9 milligrams/liter for MS and between 0.08 and 6.1 millig,rams/liter for SS. The main dif- ferences between MS and SS were the higher concentrations of NNK, NPY, and DMNA for SS. Benzo(a)pyrene, dibenz(a,h)anthracene, and the benzofluoranthenes may be the most relevant PAH for the mouse skin carcinogenic activity. Their concentrations were 0.18, 0.01, and 0.17 milli- grams/liter for MS and 1.25, 0.07, and 1.42 milligrams/liter for SS. The sum of all PAH were 5-fold higher for SS than for MS. (a) defined by the procedure of preparation (see SUBREPORT AC FIGURE A)

INTEGRATING REPORT P 0500/3068 UBE141RA8 7295 PAGE 1-6 The comparison of volatile components in MS and SS (headspace chromatography) showed a higher number and concentration in the former MS than in: the latter SS as well as in the crude and in the processed condensate. Processing caused a reduction of the volatile components. The reproducibility of the condensate preparation was very good with a relative standard deviation for the processed condensate yield of 4.7 percent for MWSC-I and 6.1 percent for SWSC-I. No fluctuations of the concentration of condensate components over the whole time of condensate preparation (approx. 80 weeks) were observed. The crude condensate and nicotine yields for MS were in good accordance with the d ata provided by the client. For SS, data were not provided by the client. Concerning the relevance of all condensate data, it has to be mentioned that condensate characteristics depend on their pre- paration method. This is especially true for SS condensate as there are more variables in the generation process than that for MS. 1.4.2 General-condition-and behavior In CD1 mice, signs of intoxication were observed from approx. 5 weeks after the start of the application period up to approx. the 25th application week in the WSC-I treated groups. Only in the SWSC-I-treated groups did acute signs of intoxication occur up to the end of the study. The intensity of these signs decreased with time. The main signs of intoxication were spontaneous activity which increased immediately after application and later decreased, prone position, dyspnea, decreased body temperature, and either a partial or complete closing of the palpebral fissure. The signs of intoxication increased with ascending doses. Dose groups with strong signs of intoxication showed high mortality.

INTEGRATING REPORT P 0500/3068 UBE141RA9 7295 PAGE 1-7 The comparison of the general condition and behavior of CD1 mice treated with MWSC-I and those with SWSC-I showed differences in the proportion of signs of intoxication. The number of mice with signs of intoxication was higher in SWSC-I- than in MWSC-I-treated groups. No differences were seen in the general condition and behavior of both mouse strains which were pretreated with DMBA. Signs of intoxication were observed in CD1 but not in B6C3F1 mice. The comparison of the general condition and behavior of CD1 mice did not result in differences depending on whether MWSC-I was stored before application at 4 or at -75 degrees centigrad~e. 1.4.3 Mortality The comparison of the mortality of MWSC-I- and SWSC-I-treated CD1 mice showed a statistically significantly higher mortality in the SWSC-I-treated groups. This is considered to be biologically rele- vant. In CD1 mice the pretreatment with an initiating dose of DMBA re- sulted in a statistically significantly higher mortality in the SWSC-I dose groups. This statistical significance is considered to be biologically relevant. In B6C3F1 mice a slightly higher mortality was seen in the DMBA pretreated: groups which was sta- tistically significant in the medium- and high-dose groups. This statistical significance is considered to have no biological relevance. The comparison of the mortality of the MWSC-I-treated CD1 and B6C3F1 mice showed a statistically significantly higher mortality for CD1 mice. This is considered to be biological relevant.

1N'1'EGttAY'iNG REPUI2T P 0500/3068 UBE141'RA10 7295 PAGE 1-8 The comparison of the mortality for CD1 mice showed no difference depending on whether MWSC-I was stored before application, at 4 or at -75 degrees centigrade. The proportion of dead CD1 mice with macroscopic skin tumors did not generally exceed that without skin tumors. Also the overall mortality of mice with macroscopic skin tumors did not increase, i. e., the mortality did not increase in relation to: the appear- ance of skin tumors. Therefore, the reason for the higher mor- tality in the WSC-I-treated groups is not known. 1.4.4 B'ody-weiSht Beginning in the 1st third of the study the mean body weight of both mouse strains of all condensate-treated groups was slight- ly lower (in application week 80 a maximum of 13 percent in CD1 mice and 9 percent in B6C3F1 mice) than the body weight of the corresponding control groups. This is considered to be biological- ly relevant, indicating a slight toxic effect. The toxicity is thought to be caused by nicotine in the condensate, but other components may also be relevant. The body weight reduction indi- cates an appropriate WSC-I dose selection with the highest dose at the maximum tolerated dose (MTD:), i. e., a dose causing an approx. 10-percent reduction in body weight gain. The comparison of the body weights of CD1 mice with respect to MWSC-I and SWSC-I showed no differences. The pretreatment with an initiating dose of DMBA did not result in a difference in the body weight of both.mouse strains. 3285

INTEGRATING REPORT'P 0500/3068 UBE141RA11 7295 PAGE 1-9: The body weight of the CD1 mice is strain-dependently higher than that of the B6C3F1 mice. The relative body weight reduction in the MWSC-I-treated groups was the same in both mouse strains (-7 percent). The comparison of body weight in CD1 mice showed no difference depending on whether MWSC-I was stored before application at 4 or at -75 degrees centigrade. 1.4.5 Develoement_of_macroscopic_skin_tumors No macroscopic skin tumors were found in the acetone control groups. In WSC-I-treated groups the 1st macroscopic skin tumors were recorded in CD1 mice between WSC-I application weeks 14 and 54 and in B6C3F1 mice between WSC-I application weeks 18 and 66. At the time when a 10-percent macroscopic tumor rate (MTR) was reached, a strong dose dependency was observed in CD1 mice treated with SWSC-I and in B6C3F1 mice treated with DMBA plus MWSC-I. 1.4.6 Skin_irritations_imacroscoLic_diagnosis) ---- ----------- ----- No skin irritations were found in CD1 mice in the control groups. The 1st skin irritations (translucent and/or reddened skin): in CD1 mice were detected approx. 2 weeks after the start of the WSC-I application. The number of mice with skin irritations reached a maximum during the 4th and 5th week of WSC-I applica- tion, i. e., after approx. 15 to 25 applications. In the high-dose groups, skin irritations occurred earlier. Approx. 10!weeks after the beginning of the WSC-I applications, skin irritations were no longer observed. P~

INTEGRATING REPORT P 0500/3068 UBE:141RA12 7295 PAGE 11-10 The comparison of skin irritations in MWSC-I- and SWSC-I-treated CD1 mice showed a statistically significantly higher number of mice with skin irritations in the SWSC-i-treated groups. This is considered to be biologically relevant. In CD1 mice the pretreatment with an initiating dose of DMBA re- sulted in a statistically significantly lower number of mice with skin irritations both in the MWSC-I- and in the SWSC-I-treated groups. This effect is considered to be biologically relevant. The reduced number of mice with skin irritations in the DMBA-pre- treated WSC-I groups is unexplainable. In B6C3F1 mice no differ- ence was seen between the MWSC-I-treated groups without and with DMBA pretreatment. The comparison of skin irritations between the MWSC-I-treated CD1 and B6C3F1 mice showed a statistically significantly higher number of mice with skin irritations in the CD1 mouse strain. This, how- ever, is not considered to be biologically relevant, because the skin inspection and scoring of skin irritations were hindered by the brown pigmentation of the skin in B6C3F1 mice. The comparison of the number of mice with skin irritations in CD1 mice showed no difference depending on whether MWSC-I was stored before application at 4 or at -75 degrees centigrade. 1.4.7 Prediction of the microscopic tumor probability in future studies For the prediction of the microscopic probability at an early stage of the experiment, the microscopic tumor probability was compared with the macroscopic skin irritation, the week of the -pr.

INTEGRATING REPORT P 0500/3068 UBE141RB28 7295 PAGE 1-11 tumor onset, the week when 5-percent tumor probability was reached, and the macroscopic tumor probability of application weeks 30 and 40 in CD1 and B6C3F1 mice. In CD1 mice all macroscopic "short-term" parameters correlated well with the 80-week microscopic tumor pro- bability (correlation coefficients 0.93 to 0.68). The macroscopic parameters, such as the tumor onset, the 5-percent tumor proba- bility, and the tumor probability in application weeks 30 and 40 were superior to the skin irritation for the predictability of the microscopic tumor probability. Therefore, for this type of experi- ment and for comparable test materials, the microscopic tumor pro- bability can be predicted by the macroscopic tumor probability as early as after 30 weeks of WSC-I application or by the skin irrita- tion and the histopathological examination for "hyperkeratosis" (a) as early as after 5 weeks of WSC-I application. Also in B6C3F1 mice all macroscopic "short-term" parameters (b)~ correlated well with the 80-week microscopic probability (correlation coefficients 0.911 to 0.77). The macroscopic parameters, such as the 5-percent tumor probability and the tumor probability in application weeks 30 and 40 were superior to the tumor onset for the predictability of the microscopic tumor probability. 1.4.8 Microbialoqical_findincs The microbiological screening of the CD,1 and B6C3F1 mice showed significant antibody titers to 4 mouse-related viruses, but no influence on the results of the study was observed. In addition, (a) INBIFO study P 0500/3117 (1986) (b) In B6C3F1 mice skin irritations could not be used for the prediction, because due to the brown pigmentation of skin such changes were not observed. 1,8~

INTEGRATING REPORT P 0'50U/3-068' UBE141RA14 7295 PAGE 1-12 the results of the bacteriological analyses of the mice and the environment within the animal laboratory units were in agreement with the microbial flora acceptable for optimal hygienic condi- tions during long-term studies on mice. 1.4.9 Pathological_findings 50 skin samples from the application, area without any macroscopic findings were randomly selected f rom various groups and showed no histopathological findings. Macroscopic skin lesions consisting of ulcerated and/or nonulce- rated tumors, ulcers, and pachyd'ermia were observed in many CD1 and B6C3F1i mice in the WSC-I-treated groups, in few CD1 and many B6C3F1i mice in the DMBA pretreatment acetone control groups, and rarely in mice of both strains in the acetone control groups. Almost all of these lesions were confirmed histopathologically. With few exceptions, the incidence of mice with macroscopic tumors correlated well (approx. 80 percent) with the incidence of mice with histopathologically confirmed tumors in the various groups. Histopathologically, CD1 and B6C3F1 mice in the acetone control groups showed no skin neoplasms after 80 weeks of WSC-I applica- tion. In DMBA-pretreated acetone control groups benign as well as malignant epithelial tumors (papillomas, acanthomas, carcinomas) were observed in B6C3F1 and only benign epithelial skin tumors in CD1 mice. Therefore, the chosen DMBA dose was too high to be only initiating, but the general concept of this study is not inva- lidated by the DMBA overdosing. The incidence of epidermal hyper- plasia and of dermal inflammatory changes was low (1 to 3 percent) in both mouse strains. 1285

1... ., .. . ., -....1 iru. i ~.~....~~......J' tVUl11 INTEGRATING REPORT P 0500/3068 UBE191RA15 7296 PAGE 1-13' The mice of the WSC-I-treated groups without and with DMBA pre- treatment showed neoplastic and monneoplasttiic skin changes in the application area which are usually expected after repeated appli- cation of whole smoke condensate. The neoplastic changes consisted mainly of benign (papilloma, acanth:oma) and mal~ignant (carcinoma) epithelial neoplasms. In addition to these epithelial neoplasms sporadically benign (hemangioma) and a few malignant (fibrosarcoma) mesenchymal skin neoplasms mainly in SWSC-I groups without and with DMBA pretreatment were also observed. The incidence of these mesenchymal neoplasms was low (below 3 percent). A similar inci- d~ence was also observed in former skin tumorigenicity studies: The incidence of spontaneous mesenchymal skin tumors in CD1 mice as mentioned in the literature does not exceed 1 percent (Hombur- ger et al., 1975, Faccini et al., 1981). Cigarette smoke conden- sate (CSC) usually induces both benign and malignant tumors of epidermal origin on mouse skin. Induction of mesenchymal tumors, however, has not been reported in the literature. Though they are observed in WSC-I-treated groups in the present and previous INBIFO studies their association with WSC-I treatment is of doubt- ful biological relevance. Leukemic infiltrations were sporadically observed in CD1 mice in all WSC-I-treated groups. The incidence ranged between 1 to 5 percent in different groups. A dose-dependent increased incidence was observed in SWSC-I with DMBA pretreatment. Mouse skin painting studies with CSC reported to date have not shown a linkage between CSC application and incidence of leukemic infiltration. Such a linkage, however, was described between CSC application and the low but significant incidence of cutaneous mastocytomas and diffuse dermal mast-cell infiltration. This information may make a reevaluation necessary. With few exceptions, at comparable dose levels, numerically an earlier tumor onset, a higher skin tumor probability for total epithelial tumors as well as for benign and malignant epithelial tumors, a higher multiplicity and more malignant epithelial tumors p,

.------ INTEGRATING REPORT P 0500/3068 t'7BE141RA16 7295 PAGE 1-14 were seen for (1)', SWSC-I without and with DMBA pretreatment than in without and with DMBA pretreatment, MWSC-I (2) WSC-I with DMBA than without DMBA pretreatment in CD1 mice (MWSC-I and SWSC-I) and B6C3F11 mice (MWSC-I (a)), (3) in CD1 than in B6C3F1 mice, (4) MWSC-I stored at -75 degrees centigrade than at 4 degrees centigrade. The relative skin tumor rate (b)' was statiistically significantly higher in (1) SWSC-I without and with DMBA pretreatment than in MWSC-I without and with DMBA pretreatment, (2')' WSC-I with DMBA than without DMBA pretreatment in CD1 mice (MWSC-I and SWSC-I) and in B6C3F1 mice (MWSC-I (a)), and (3') MWSC-I without and with DMBA pretreatment in CD1 than in B6C3F1i mice. These effects are considered to be biologically relevant. No sta- tistically significant difference in the relative skin tumor rate was found between MWSC-I stored at -75 degrees centigrade and 4 degrees centigrade. The results of INBIF'O mutagenicity studies, however, have indicated a difference in the mutagenic activity of 2R1 MWSC-I, stored at 4 degrees centigrade (decrease in the acti- vity), and at -75 degrees centigrade (no change in the activity). Based on these results, the relative skin tumor rate was con- sidered to be probably biologically relevant. Among the nonneoplastic changes consisting of hyperplasia and cysts of sebaceous glands, hyperkeratosis, epidermal hyperplasia, and dermal inflammatory changes inside the application area, the latter 2 were the remarkable and frequently observed findings seen (a) SWSC-I not investigated (b) The tested hypothesis assumes that in spite of the different treatment of eacfi subpopulation (pools of differently treated groups) their skin tumor rate is equal to the mean skim tumor rate of the total; populations. The relative skin tumor rate of a subpopulation is calculated' as the ratio between the total observed and the total expected number of mice with skin tumor.

INTEGRATING REPORT P 0500/3068 UBE141RB7 7296 PAGE 1-15 after the application of the WSC-I onto mouse skin. All of the above mentioned nonneoplastic findings are consistent with those seen in previous INBIF0 studies. The spectrum of these findings did not vary with regard to the different condensates (MWSC-I and SWSC-I) and condensate doses. MWSC-I and SWSC-I without and with DMBA pretreatment showedd the same spectrum of neoplastic and nonneoplastic findings outside the application area. 1.4.10 Conclusions From the results obtained in the present study, the following is concluded for the skin tumorigenicity, i. e., tumor probability, tumor multiplicity, and malignancy of WSC-I of the standard refer- ence cigarette type 2R1: (1) SWSC-I assayed for complete tumorigenic activity, i. e., with- out DMBA pretreatment, showed a 2- to 6-fold higher skin tumor- igenicity than MWSC-I. SWSC-I with DMBA pretreatment showed a 2- to 3-fold higher skin tumorigenicity than MWSC-I with DMBA pretreatment indicating a higher promoting activity of the SWSC-I; (2) WSC-I with DMBA pretreatment showed up to 6-fold higher skin tumorigenicity than WSC-I without DMBA pretreatment; (3), CD1 mice were up (4) to 2-fold more sensitive than B6C3F1 mice to the skin tumorigenicity of MWSC-I; the skin tumorigenicity of MWSC-I stored at -75 degrees centigrade before application was up to 2-fold higher than that of MWSC-I stored at 4 degrees centigrade. I N B I F 0 Institut fur biologische Forschung GmbH 7^B5

INTEGRATING REPORT P 0500/3068 UBE141RA18 7138' M PAGE 2-1 2 RESPONSIBILITY 2.1 Project Management Study Director: D. Kuhn 2.2 Contributing Teams ~ s2 ; C4 .~S AL"., Analytical Chemistry: ...................... ~.......... Dr.rer.nat. B. Gerstenberg Food Chemist (Staatl. geprufter Lebensmittelchemiker) Animal Treatment: Microbiology: Gross Pathology: Histopathology: Dr.rer.nat. F. Tewes Biologist (Diplombiologe) . . .~1... . . ~.--: : :~: : :~ . . . . . . . . . . . . . . . . Dr.med.vet. A. Teredesai Pathologist (Fachtierarzt fur Patholog,ig ) Prof.Dr.med. C. Thomas Pathologist (Facharzt fur Pathologie) Director, Dept. of Pathology, University Marburg 3587

1tvY'l:aaKYiY'1Nli NE!'Ukr' Y U5UU/sueu u8Ei41HA19 7295 M PAGE 3-1 3 QUALITY ASSURANCE STATEMENT The study was conducted according to the Good Laboratory Practice Regulations (a). Inspections on this study were performed by the quality assur- ance unit on 3.Dec.82, 5.Jan.83, 7.Jan.83, 12.Jan.83, 30.Mar.83, 23.Nov.83, 9.Mar.84, and 2. to 4.Ju1.84. All findings were imme- d:iately reported to the study director and to the general mana- gement. This report accurately reflects the study carried out and the results obtained. Quality Assurance Manager E. Romer Biologist (Diplombiolog!e) (a) Federal Register (1978)

INTEGRATING REPORT P 0500/3068 UBE143RA1 7295 PAGE 4-1 4 INTRODUCTION _____=====3= Laboratory studies can contribute to a better understanding of the factors and mechanisms involved in the induction of disease by environmental agents. There have been numerous bioassays conducted on mainstream smoke (MS). In examining the effects of MS, many research workers have used condensates of the smoke painted on the shaved skin of mice. This contrasts with the human exposure that occurs mainly in the respiratory tract. Skin painting with WSC serves as a model for skin tumorigenicity of tobacco smoke. Nonetheless, these skin painting studies have been useful in examining the carcinogenicity of different tobacco constituents and thus increases knowledge of the actions of MS. Similar skin painting work has not been sufficiently done with sidestream smoke (SS)~ condensate and would be of value for assessing the differential tumorigenicity of SS condensate and MS condensate. The burning of tobacco products leads to the formation of MS and SS. MS from cigarettes is generated during puff-drawing in the burning cone and hot zones. It travels through the tobacco column and exits from the mouthpiece. SS is formed in between puff- drawing and is emitted freely from the smouldering tobacco product into the ambient air, and vapor phase components diffuse through the cigarette paper (a). SS is known to have a different composition than MS (see TABLES A and B) in its vapor phase as well as in its particulate phase. It was found that the level of carbon monoxide, carbon dioxide, pyri- dine, ammonia, nitrogen oxides, nitrosamines, phenols, amines, polycyclic aromatic hydrocarbons such as benzo(a)pyrene and benz(a)anthracene was much higher in SS than in MS (IARC Mono- graphs, 1986). These differences are related to the fundamentally (a) According to DIN 10240 "Maschinelles Abrauchen von Zigaretten und Bestimmung des Rauchkondensates", Apr. 78, sidestream smoke is d~efined as the whole amount of smoke, which leaves the cigarette by way other than through the cigarette mouth end. ^?ps

ILVPE(GRA'PILVu REPOirP P 0500/3068 UBE143RA2 7295 PAGE 4-2 UNIT OF MEASURE MS Ss Ms particulate matter mg 15 to 40 1.3 to 1.9 nicotine mg 1.7 to 3.3 1.8 to 3.3 anatabine Ug 2.4 to 20.1 0.1i to 0.5 phenol ug 60 to 140 1.6 to 3.0 catechol ug'g 100 to 360 0.6 to 0.9 hydroquinone ug 110 to 300 0.7 to 0.9 aniline ng 360 30 ortho-toluidine ng 160 19 2-naphthylamine ng 1.7 30 4-aminobiphenyl ng 4.6 31 benz(a)anthrancene ng 20 to 70 2.2 to 4 benzo(a)pyrene rig 20 to 40 2.5 to 3.5 cholesterol Ug 14.2 0.9 g&mna-butyrolactone ug 10 to 22 3.6 to 5.0 quinoline ug 0.5 to 2 8 to 11 harman ug 1.7 to 3.1 0.7 to 1.9 N'-nitrosonorniootine ng 200 to 3000 0..5 to 3 4-(methylnitrosamino)-1- ng 100 to 1000 1.4 (3-pyridyl)-1-butanone N-nitrosodiethanolamine ng 20 to 70 1.2 cadmium rig 100 3.6 to 7.2 nickel rx3 20 to 80, 0.2 to 30 zinc rx3 60 0.2 to 6.7 benzoic acid ug 14 to 28 0.67 to 0.95 lactic acid ug 63 to 174 0.5 to 0.7 glycolic acid ug 37 to 126 0.6 to 0.95 succinic acid ug 112 to 163 0.43 to 0.62 TABLE A CONCFTJPRATIONS OF SELECTED1C(MPOUND6 IN 1QDNFILTER CIGAREZTE MAINSTREAM SMOKE (MS) AND THE RATIO OF THEIR RELATIVE DISTRIBUTION IN SIDESTREAM SMOKE (SS/MS), PARTICULATE PBASE Remarks: from IARC Monographs (1986)

---- INPEGRATI[dG Rk:PORT P 0500/3068 UBE143RA3 7295 PAGE 4-3 CCMPOUND UNIT OF MS MEASURE carbon monoxide carbon dioxide carbonyl sulphide benzene toluene formaldehyde acrolein acetone pyridine 3-vinylpyridine hydrogen cyanide hydrazine aminonia methylamine dimethylaaine nitrogen oxides N-nitrosodimethylamine N-nitrosopyrrolidine formic acid acetic acid mg mg ug ug ug u9 ug ug ug ug ug ng ug ug ug ug ng ng ug ug 10 to 23 2.5 to 4.7 20 to 60 8 to 11 18 to 42 0.03 to 0.13 12 to 48 10 160 6 to 8 70 to 100 0.1 to 50 (a) 60 to 100 8 to 15 100 to 250 2 to 5 16 to 40 7 to 20 15 to 30 20 to 40 400 to 500 0.1 to 0.25 32 3.0 50 to 150 40 to 170 17.5 to 28.7 4.2 to 6.4 7.8 to 10 3.7 to 5.1' 100 to 600 4 to 110 10 to 40 20 to 100 6 to 30 6 to 30 210 to 478 1.4 to 1.6 330 to 810 1.9 to 3.9 TABLE B CONCENi4tATIONS OF SEIECTED1CCMPO[JNIDS IN NONFILTER CIGARE'ITE MAINSTREAM SMOKE (MS) AND THE RATIO OF THEIR RELATIVE DISTRIBiJPION IN SIDESTRF•AM SMOKE (SS/MS), VAPOR PHASE Remarks: from IARC t4ortiographs (1986)

INTEGRATING REPORT P 0500/3068 UBE143RA4 7295 PAGE 4-4 different burning and distillation processes during puffing and puffing intervals. An important cause of the differences between sidestream and mainstream smoke is that the peak temperature in the burning cone of a cigarette d uring puffing reaches 800 to 900 degrees centigrade (Wynder and Hoffmann, 1967), but that between puffs reaches only about 600 degrees centigrade (Hoffmann et al., 1983). It is thought that these differences in the smoke will also result in differences in the composition of SWSC and MWSC col- lected in an impaction trap. To date, only 1 study has been published on the carcinogenic po- tential of SWSC collected in a cool trap. The biological activity of this type of SWSC was found to be different from MWSC. SWSC was found to be more tumorigenic than MWSC in the mouse skin painting model (Wynder and Hoffmann, 1967). In contrast to the afore- mentioned study INBIFO developed a method for the collection of SWSC in an impaction trap to produce a higher amount of conden- sate (INBIFO study P 5007, 1984 and SUBREPORT AC P 0500/3068). Mouse skin is an experimental tissue which responds to repeated applications of cigarette smoke condensate by forming benign and malignant epithelial neoplasms. MWSC of cigarette 2R1 (standard reference cigarette) applied at a dose of 150 milligrams dry condensate/(mouse x week) was found to cause a histologically confirmed tumor rate of 24 percent in a previous INBIFO skin painting study using mouse strain CD1 (INBIFO study P 0500/3010, 1983}. This mouse strain has been used in experiments carried out over the last 20 years because of the low mortality, the suscepti- bility to carcinogens, the low rate of spontaneous tumors, and the resistance towards nicotine toxicity. For the differentiation of WSC' of several experimental cigarettes the response of CD1 mice appeared to be too small and models

INTEGRATING REPORT P 0500/3068 UBE143RA28 7295 PAGE 4-5 resulting in higher tumor rates with the reference cigarette type 2RI were envisaged. The low tumor rate may be related to the relatively weak tumor initiating activity of WSC or the relative insensitivity of the mouse strain used. Therefore, the pretreat- ment of several groups with a single dermal application of a tumor initiating dose of 200 micrograms DMBA was included in this study. Wynder and Hoffmann (1962) demonstrated the tumor promoting acti- vity of mainstream condensate by using 300 micrograms DMBA as an initiator, followed by repeated applications of tobacco smoke condensate. Since an appropriate number of mice of the highly sensitive SENCAR strain was not available, the B6C3F1i hybrid strain was chosen as a possibly more sensitive substitute for the CD1 strain. B6C3F1 is known to: be responsive to the tumorigenic activity of polycyclic aromatic hydrocarbons and related chemical compounds. This strain is presently used by the NTP in the USA carcinogenesis studies including skin painting of several com- pounds including tobacco condensate components. Hybrid strains in comparison to inbred strains (a)' combine the advantage of pheno- typic uniformity with that of greater longterm genetic stability. Before its use in the NTP, the B6C3F1 mouse strain ranked 4th in the "popularity" scale of F1 hybrid mice (Festing, 1979). The B6C3F1 strain was treated with MWSC-I without and with, DMBA pre- treatment. In skin painting studies female mice are preferred to males, because females are considerably less aggressive. It is important to minimize fighting among the test animals in skin painting studies, because bites and scratches may lead to false results when counting skin irritations (caused by test substances) and tumors. The time and the temperature of WSC storage is known to influence its biological activity. The mutagenic activity of 2R1-WSC-I Individuals are hieterozygous at all loci at which the parental strains differ genotypically. qr

_ _ - ---- - _ _----- ,~~_ ----- INTEGRATING REPORT P 0500/3068 UBEl43RA5 7295 PAGE 4-6 as determined in the Salmonella plate incorporation mutagenicity assay with tester strain TA98 iri' the presence of Aroclor 1254- induced rat liver S9 fraction was found to decrease between the 2nd and the 6th week after preparation of the condensates with a rate of approx. 15 percent per week, when the condensates were stored at 4 degrees centigrade (INBIFO study P 0268/2017, 1979). A rough approximation of the half-life of the activity from this experiment was 3 weeks. During the 1st 24 hours after WSC prepa- ration, no indications of a change of the mutagenicity to Salmo- nella typhimurium were observed (storage at 2.5 grams dry conden- sate per liter) (INBIFO study P 0268/2017, 1979). No decrease of the activity was observed after 1 year of storage at -75 degrees centigrade (storage at 40 grams dry condensate per liter) (INBIFO study P 0268/2054). Those changes at higher temperatures are thought to be related to secondary reactions of components in the condensate. There was an increasing amount of particles in acetone solutions of WSC collected in a cool trap during storage and with increasing temperatures (-70 to 20 degrees centigrade) (Schonherr et al., 1973j. The authors speculated that reactions among consti- tuents of the gaseous phase (approx. 7.5 percent) and between those of the gaseous and the particulate phase (.GT.90 percent) may be responsible for this increase. These secondary reactions appear to be prevented during storage at -75 degrees centigrade. Therefore, WSC (a)i stored at -75 degrees centigrade was used in this study. In addition the tumor rate of MWSC stored at -75 degrees centigrade and in the conventional way at 4 degrees cen- tigrade for at least 14 days and not longer than 6 weeks compared. was Indications for skin irritation have been observed after applica- tion of MWSC-C and MWSC-I of the standard reference cigarette type 2R1 as well as of TPA to the dorsal skin of female CDI and (a) diluted to its final concentration for application

IN'1'EGkAY'LNG REPUR'i' P 0500/3068 UBE143RA6 7295 PAGE 4-7 NMRI mice (INBIFO studies P 0500/3043, P 0500/3046, 1982, and P 0500/3058, 1982). The 1st lesions, i. e., reddening and translu- cent appearance of the epidermis, were detected approx. 5 days after the start of the application (a) and the proportion of mice with irritations reached a highpoint approx. 12 to 19 days after the list application. The intensity index and the size index indi- cated that at the applied doses MWSC-C caused a stronger response than TPA. The occurence of such lesions was found to be transient since less mice with irritations were found during the time fol- lowing continued condensate application. In the present study the development of skin irritation was followed during the initial weeks. Pullinger (1940) described an increase of thickness of the mouse epidermis after application of carcinogens. Lazar et al. (1966) and Chouroulinkov et al. (1969) ascertained a correlation between, the thickness of mouse epidermis in subchronic and tumor rate in chronic studies. In INBIFO study P 0500/3117 (1986)'~ a correlation between macroscopic skin irritation, the histopathological finding hyperkeratosis, and the final microscopic tumor rate was deter- mined. It was shown that in CD1 mice pretreated with DMBA a pre- diction of the final microscopic tumor rate of chronic skin tumor- igenicity studies in an early stage is possible. In mice without pretreatment with DMBA both parameters did not allow a prediction of the final tumor rate. The experimental design of the study is summarized in TABLE C and FIGURE B. (a) no reduced dose during initial weeks as in skin painting studies

llri•WRP;1'1AG RL;POR`i" P 0500/3068' UBE143RA7 7014 PAGE 4-8 GR7[JP MYJUSE APPLICATION P1mCESSED CCNDENSATE OF 2R1 APPLICATION' ViOLUmE b STRAIN MATERIAL (a) CONC. (g/1) DOSE (mg/(mouse x appl.)) (mg/(mouse x week)) ) ( (ul/(mouse x appl.)) 0.1-GR CD1 acetone - - - 100 0.2-GR CDI DMBA plus acetone - - - 100 1-GR CD1 MniSC-I 120 12 60 100 2-GR CD1 MWSC-I 18& 18 90 100 3-GR CD1 MWSC-I 240 24 120 100 4-GR CD1 MWSC-I (c) 120 12 60 100 5-GR CD1 MWSC-I (c) 1:80 18 90 100 6-GR Cfll. MWSC-I (c) 240 24 120 100 7-GR ®1 LY4BA plus MVi;SC-I 120 12 60 100 8-GR CD1 DMBA plus MWSC-I 180 18 90 100 9-GR W DMBA plus MWSC-I 240' 24 120 100 10-GR CD1 SWSC-I 120 12 60 100 11-GR CD1 SWSC-I 180 18 90 100 12-GR CD1 SWSC-I 240 24 120 100 TABLE C GROUPS AND DOSES Remarks: application cycle: 5 times per week, number of mice per group: 105 (a) DNBA dose: 200 nmol/mouse equiv. 51 ug/mouse, single application NWSC-I: mainstream condensate, collection in impaction trap SWSC-I: sidestream~condensate, collection in impaction trap (b) application solution: processed condensate plus acetone or acetone alone (c) stored at 4 degrees centigrar3e. Solutions have room temperature when applied. Condensate of all other groups stored at -75 degrees centigrade. ,IBS

INIEGRATING REPOFtT P 0500/3068 Uf3E 143RA8 7014 PAGE 4-9 GPOUP l+YJUSE APPLICATION PROCESSEU COIVTiENSA2E OF 2R1 APPLICATION STRP,IN, MATERIAL (a) WLUME (b) OOIC. DOSE (c) (g/1), (mg/(mouse (mg/(mouse (ul/(mouse x appl.)) x week)) x appl.)) 13-GR Cp 1 14-GR CD1 15-GR C{11 0.3-GR B6C3F1 0.4-GR B6C3F1 16-GR B6C3F'1i 17-GR B6C3F11 18-GR B6C3F1 19-GR B6C3F1 20-GR B6C3F1 21--GR B6C3F1 DMBA plus Sfti'SC-I DMBA plus SWSC-I DMBA plus SWSC-I acetone DMBA plus acetone MWSC-I MWSC-I MWSC-I DMBA plus MWSC-I WBA plus MWSC-I DMBA plus MWSC-I 120 180 240 - - 120 180 240 120 180 240 12 18 24 - - 8.40 12.6 16.8 8.40 12.6 16.8 60 90 120 - - 42 63 84 42 63 84 100 100 100 70 70 70 70 70 70 70 70 TABLE C ( cont i nuerl ) GE40UPS AND DOSES Remarks: application cycle: 5 times per week, number of mice per group: 105 (a) DMBA dose: 200 nmol/mouse equiv. 51 ug/mouse, single application MdSC-I: mainstream condensate, collection in impaction trap SWSC-I: sidestream condensate, collection in impaction trap (b) application solution: processed aondensate plus acetone or acetone alone (c) reduced dosage for B6C3F1 mice due to reduced body weight as compared to CD1 mice

INTEGRATING REPORT P 0500/3068 UBE143RA9 7294 PACE 4-10 date week of WSC appl. arrival of mice microbiol. screening of mice allocation to groups determination of body weight individual marking shaving DNIDA application irritation monitoring macroscopic exami- nation of skin WSC application reduced dose (adaptation) full dose final dissection 29. 20. 17. 14. 14. 11. 9. 6. 4. 1. // 11. 2. Nt7v. DEC. JAN. FEB. MAR. APR. MY JUN. JUL. AUG. // JUN. JUL. 82 82 83 83 83 83 83 83 83 83 // 84 84 I = = I = _ = I = _ = I = _ = I = _ = I = = = I = _ = I = _ = I = _ = I = _ // I = = I -2 1 5 9 13 17 21 25 29 33 // 78 81 X // X // X X // xxx X x x x x x X x x x x X x // x X // xxxxxXxxxxxxxxxxxXXxxxxxxxxxXXxxxxxxx // XXx x // X X X X X X X X X x X X xxxxxxXxXxxxxxxxxxXxXxxxxxxxxxXxxxx // X X X X x X X X X X X a a a X X X X X X X X X X X X X X X X X X X X X X X X X X X // X X X // X I ==I ===I ===I ===I ===I =__~ ===I ===I ===3 =_ // I ==I -2 1 5 9 13 17 21 25 29 33 // 78 81 FIGURE B CHHANUIC= Remarks: a: refers only to B6C3F1 mice ts,11sosrzoz

1N'1'Ehiitl'1'1N!u kEYCUR'1' P U500/3U6tf' 5 SUBSTANCES EXAMINED 5.1 Cigarette (a) Type (cigarette code): Source: Number of cigarettes: UBE'143RA10 7295 PAGE 5-1 2R1 (standard reference) Philip Morris, USA CIG. TYPE NO. OF CIGARETTES DELIVERED REQUIRED Packaging: Date of receipt at INBIFO: 2R1 from stock 320000 bundles with 200 cigarettes, 10 packs with 20 cigarettes/pack Nov.82 Storage Main storage: walk-in cold room R907, 1 to 3 degrees centigrade, relative humidity uncontrolled Laboratory storage: Analytical data: conditioning room R326, beginning at least 10 days prior to smoking storage in opened packs or bundles temperature: 22 + 1i degrees centi- grade, relative humidity: 60 + 3 0/0 see TABLES D to F (a) Actually used in this study is the MWSC-I and SWSC-I olf this cigarette type. For the preparation of MWSC-I and SWSC-I see SUBREPORT AC. 1195

INTEGRATING REPORT P 0500/3068 PARAMETER length (mm/cig.) weight (g/cig.) tobacco weight (g/cig.) puff resistance (Pa) puff count (N/cig.) butt length (mm) TABLE D PHYSICAL DATA OF CIGARETTE 2R1 UBE143RA11 7295 PAGE 5-2 DATA 85 } ) 1.17 ) determined 13. and 15.Oct.80 ) 1.10 ) 861 ) ) 11.5 ) calculated from values of ) INBIFO study P 0500/3030 (1984) 24 ) T'^5.

INTEGRATING REPORT P 0500/3068 UBE143RA12 7014 PAGE 5-3 COMPONENT CONCENTRATION IN FILLER (g/kg) tobacco moisture 132 total alkaloids 19.8 reducing sugars 106 total nitrogen 21.7 nitrate nitrogen 1.8 soluble ammonia 1.3 ash 148 hot water solubles 590 TABLE E CONCENTRATION'OF SELECTED COMPONENTS IN FILLER, CIGARETTE TYPE 2RI Remarks: data except tobacco moisture provided by the supplier and related to dry weight: Aug.77 tobacco moisture determined at INBIFO: on 24.Aug.84 q~

INTEGRATING REPORT'P 0500/3068 UBE143RA13 7014 PAGE 5-4 COMPONENT YIELD (mg/cig.) TPM 48.4 nicotine 3.32 catechol (a) 0.22 water in TPM 4.9 nitrogen oxide 0.39 carbon monoxide 25.4 hydrogen cyanide 0.45 total aldehydes 3.13 TABLE F YIELD OF SELECTED SMOKE COMPONENTS, CIGARETTE TYPE 2R1 Remarks: data except catechol provided by the supplier: Aug.77 (a) catechol determined in INBIFO study P 0500/3109 (1986)

INTEGRATING REPORT P' 0500/3068 UBE143RA14 7295 M PAGE' 5-5 5.2 7,12-Dimethylbenz(a)anthracene Synonyms: 9,10-dimethyl-1,2-benzanthracene, 1,4-dimethyl-2,3-benzphenanthrene Structure: CH3 1/ 11 l0 12 1212b \ I 10 9 CH3 Chemical Abstracts Registration Serial No.: 579!76 Data compilation: Windholz et al. (1983) Source: no. D3254, Sigma GmbH, D-8024 Deisenhofen Date of receipt at INBIFO: 10.Nov.82 Shipment container: brown glass bottle Description on container: a V s~ i M 61t1~ 'j~ I= s= 1.I OAYE1M7l 1'.Y ~OQJ~1R1r>~~~ a",ii= i N iS q. O. .r W IF b.1 ~ ~ ~ ~ r } ~ ~ OFrr Ip.IMH r f s~iS ~ ii= 7, s Amount: 1 g 1'dS

INTEGRATING RhPORT P 05D0/3068 UBE143RA15 7014 PAGE 5-6 Storage: INBIFO substance no.: RT, screened from light S 2859 A Physical and chemical properties Relative molecular mass: Appearance: 256.3 plates, lightly yellow Odor : Melting point (degrees centigrade): - 122 to 123 Solubility: benzene: acetone: alcohol: water: freely soluble moderately soluble slightly soluble insoluble Stability: Absorption maxima: 296 nm (log epsilon: 4.90) 345 nm (log epsilon: 3.8'3) 364 nm (log epsilon: 3.54) 384 nm (log epsilon: 3.83) maximum fluorescence at 440 nm Scientific versionr SOP QA 8/3 Text version: 15.Aug.85 ~^95

IN'i'EG1tA'1"iNG iZEPURI' P 050U/3U6t3 UBE143A16 7279 PAGE 6-1 6 STORAGE OF' MATERIALS AND RECORDS - 3-~ ---~ 5-------3 ~ TZ ------- 3 ----- Remains of tissues and organs in formaldehyde solution, slides, and records are stored in our archives for at least 5 years. They can be claimed by the client. Scientific version: SOP QA 10/2, QA 11/2 Text version: 25.Nov.85 °"BS~

INTEGRATING REPORT P 0500/3068 UBE143RA17 7296 PAGE 7-1 7 REFERENCES Armitage, P.: Statistical methods in medical research, Oxford: Blackwell Scientific Publications, 1971 Brunnemann, K.D., Yu, L., Hoffmann, D., Assessment of carcinogenic volatile N-nitrosoamines in tobacco and in mainstream and side- stream smoke and cigarettes, Cancer Res. 37 : 3218-3222 (1977) - (INBIFO reference: L092/C03) Chouroulinkov, I., Lazar, P., Izard, C., Libermann, C., Guerin, M., "Sebaceous glands" and "hyperplasia" tests and screening! method~s for tobacco tar carcinogenensis, J. Natl. Cancer Inst. 42 : 981-985 (1969) - (INBIFO reference: L149/A13) DIN Deutsches Institut fur Normung: Maschinelles Abrauchen von Zigaretten und Bestimmung des Rauchkondensats, DIN 10240, Berlin: Beuth Verlag, 1978 Faccini, J.M., Irisani, E., Monro, A.M., A carcinogenicity study in mice of a beta-adrenergic antagonist, primidolol, increased: total tumor incidence without tissue specificity, Toxicology 21 : 219-290 (1981) Federal Register, USA, Nonclinical laboratory studies: good labo- ratory practice regulation, 43 (248) ; 59986-60025 (1978) Festing, M.F.W., Inbred, strains in biochemical research, London: The Macmillan Press Ltd., 1979, pp. 99-103 Hoffmann, D., Hyley, N.J., Brunnemann, K.D., Adams, J.D., Wynder, E.L., Cigarette sidestream smoke: Formation, analysis and model studies on the uptake by nonsmokers. Presented at the US-Japan Meeting on 'New Etiology of Lung Cancer', Honolulu, March 21-23 (1983) Homburger, F., Russfield, A.B., Weisburger, J.HR, Lim, S., Chak, S.P., Weisburger, E.K., Aging changes in CD -1 HaM/ICR mice reared under standard laboratory condition, J. Natl. Cancer Inst. 55 : 37-43 ( 1i 975 ) IARC Monographs on the Evaluation of the carcinogenic risk of chemicals to humans, International Agency for Research on Cancer, Vol. 38 . , 1986 INBIFO study P 0268/2017, Plate incorporation mutagenicity study of whole smoke condensate of test cigarettes 2R1, 2R1iF and SMIM-5 on Salmonella typhimurium strain TA98, methodological study, influence of preparation and storage on the mutagenicity, Study director: Dr.rer.nat. R.-A. Walk Date of report: 5.Dec.79 - Rc

INTEGRATING REPORT P 0500/3068 UBE143RA18 7296 PAGE 7-2 INBIFO study P 0268/2054, Plate incorporation mutagenicity study of whole smoke condensate of standard reference cigarette 2R1 on Salmonella typhimurium strain TA98, methodological study, influence of storage of muta- genicity (PT), Study director: Dr.rer.nat. F. Tewes not yet reported INBIFO study P 0500/3010, Skin painting study on whole smoke condensates from test cigaret- tes xd9pz and xd9qa and standard reference cigarette 2R1 with CD1 mice, Study director: Dr.med.vet. A. Teredesai Date of report: 25.May 83 INBIFO study' P 0500/3030, Skin painting study on whole smoke condensates from test ciga- rettes X6-DO AQP, X6-DO AQQ, X6-DO AQR, X6-DO AQS, X6-DO AQT, X6-DO AQU, X6-DO! AQV, X6-DO AQW, X6-DO AQX and standard reference cigarette 2R1 with CD1 mice, Study director: Dr.med.vet. A. Teredesai Date of report: 25.May 84 INBIFO study P 0500/3043, 29-day dermal application of cool trap condensates of test ciga- rette type X6DOAQQ and standard reference cigarette type 2R1 on CD1 and NMRI mice (PT), Study director: Dr.rer.nat. R.-A. Walk not yet reported: INBIFO study P 0500/3046, 33-day dermal application of cool and impaction trap condensates of test cigarette types X6DOAQQ, X6DOAQU and standard reference cigarette type 2R1 on CD1 mice, Study director: Dr.rer.nat. R.-A. Walk Date of report: 4.Oct.82 IN'BIFO study P 0500/3058, 25-day dermal application of TPA and whole smoke condensate of standard reference cigarette type 2R1 on NMRI mice, determination of epidermal and dermal acid phosphatase and epidermal aryl hydro- carbon monooxygenase, Study'director: Dr.rer.nat. R.-A. Walk Date of report: 7.Jul.82 INBIFO study P 0500/3109, Chemical analysis of condensate of test cigarettes X6D3DAM, X6D3DFZ, X6D3DGA, X6D3DBF, X6D3DNY, X6D4CDL, X6D4CUC, X6D5RN and of standard reference cigarette 2R1, condensate preparation with an impaction trap, Study director: Dr.rer.nat. B. Gerstenberg Date of report: 20.Jun.86 - ,s

INTEGRATING REPORT P' 0500/3068 UBE'143RA19 7296 PAGE 7-3 INBIFO study P 0500/3117, Comparison, of macroscopical and histological evaluation of skin irritations.after dermal application of mainstream and sidestream whole smoke 'condensate of standard reference cigarette 2R1, con- densate collection by impaction trap, mouse strains: CRL:CD1 (ICR)~BR andB6C3FI, Study director: D. Kuhn Date of report: 9.Jul.86 INBIFO study P 5007, Technische Vorarbeiten fur P 0500/3106, Study director: Dr. rer. nat. M. Speck Date of documentation: 15.May 84 Lazar, P., Chouroulinkov, I., Liberman, C., Guerin, Benzo(a)- pyrene content and carcinogenicity of cigarette smoke condensate- results of short-term and long-term tests. J. Natl. Cancer Inst. 37 : 573'-579~ (1966) - (INBIFO reference: L767 C07) Peto, R., Pike, M.C., Day, N.E., Gray, R.G., Lee, P.N., Parish, S., Peto, J., Richards, S., Wahrendorf, J.: Guidelines for simple, sensitive significance tests for carcinogenic effects in long-term animal experiments, in: IARC: Long-term and short-term screening assays for carcinogens, a critical appraisal, Lyon: 1980, pp. 311-426 Pullinger, B.D., The first effects on mouse skin of some polycyc- lic hydrocarbons, J. Path. Bact. 50 : 463-471 (1940)- (INBIFO reference: L767/E08) Schonherr, H., Klimisch, H.J., Harke, H.P., Alterung von Cigaret- tenkondensat, Beitr. Tabakforsch. 7 : 18-23 (1973) - (INBIFO reference: L003/F28) Windholz, M., Budavari, S., Blumetti, R.F., Otterbein, E.S. (Eds.): The Merck index, 10th edition, Rahway, New Jersey: Merck u. Co., Inc., 1983 Wynder, E.L., Hoffmann, D., Studies with the gaseous and particu- late phase of tobacco smoke, Proc. Am. Assoc. Cancer Res. 3 : 373 (1962) Wynder, E.L., Hoffmann, D., Tobacco and tobacco smoke, New York: Academic Press, 1967, pp. 127-130, and pp. 183-184 END OF INTEGRATING REPORT

INBIFO Institut fuir biologische Forschung - Kolln Ifl!IPO- Uk•PIEll• R. RYLANllER 26•UCT•87 c/o FAISRIQUES UE TABAC REUNIES S.A. DKU/UBE UBE144RA1 S W I TIERLAN'll , SUfiRENURT NN II''IiAL T R E A T M E N T P 0500/3068 SKIN TUInURIGENPCITY OF MAI NSTREAM AND S I llESTfIEAM WHULE SMOKE CONDENSATE OF STANDARD REFERENCE CIGARETTE 2R1 80-WEEK UERMAL APPLICATION STUllY WTTH Cll1( ICR)8ft AND B6C3F1 MICE INBIFO Instilut liir biologische Forsehung GmbH, Fuggerstre88 3, D-5000 KSIn 90 Silz denGeselRschaR: K81n HR B 367, 29. Oktober 1959 28.KW83 Telefon: Porz (02203) 303-T, Telefax: (02203) 303362, Telex: 8874675 inbi'd Institutsleiter und Gesohgflslilhrer: Dr. med. Ubch Hackenberg

INBIFO Institut fur biologische Forschung • Kt3in SUBREPORT P 0500/3068 UBE144RA2 7293 AT PAGE 0-1 CONTENTS I AT PAGE ABBREVIATIONS 0-8 1 SUMMARY 1-1 1.1 Objective 1-1 1.2 Method 1-1 1.3 Results 1-3 1.3.1 General condition and behavior 1-3 1.3.2 Mortality 1-4 1.3.3 Body weight 1-5 1.3.4 Development of macroscopic skin tumors 1-6 1.3.5 Skin irritations (macroscopic diagnosis ) 1-7 1.3.6 Comparison of mortality between CD1 mic without and!with macroscopic skin tumor application area e s in 1-8 1.3.7 Prediction of the microscopic tumor pro in future studies bability 1-9 2 RESPONSIBILITY 2-1 3 INTRODUCTION 3-1 AT TABLE A: SPONTANEOUS SKIN TUMOR RATE, ACETONE CONTROL GROUPS'IN DIFFERENT INBIFO STUDIES, RESULTS AFTER 80 WEEKS OF APPLICATION, FEMALE CD1 MICE 3-5 AT FIGURE A: BODY WEIGHT, ACETONE CONTROL GROUPS'IN DIFFERENT INBIFO STUDIES 3-3 AT FIGURE B: MORTALITY, ACETONE CONTROL GROUPS IN DIFFERENT INBIFO STUDIES 3-4 4 METHOD 4-1 4.1 Chronology 4-1 4.2 Laboratory Hygiene 4-1 3285

INBIFO I'nstitut fur biologischie Forschung, • Mln SUBREPORT P 0500/3068 UBE144RA3 7293 AT PAGE 0-2 CONTENTS (continued) I AT PAGE 4.2.1 Chemical disinfection 4-1 4.2.2 Entrance of area B 4-3 4.2.3 Entrance laboratory inside area B 4-4 4.2.4 Cleaning of laboratory equipment and disposal of materials contaminated with hazardous materials 4-4 4.3 Animals and Animal Treatment 4-6 4.3.1 Animals 4-6 4.3.2 Animal housing 4-8 4.3.3 Diet and drinking water 4-10 4.3.4 Groups and doses 4-12 4.3.5 Shaving 4-15 4.3.6 Application 4-1:7 4.3.7 General condition, behavior and mortality 4-21 4.3.8 Macroscopic examination of skim 4-24 4.3.9 Determination of body weight 4-28 AT TABLE B: GROUPS AND DOSES 4-13 AT TABLE C:' SHAVING OF MICE 4-16 AT TABLE D: CHECKLIST "SIGNS OF INTOXICATION, MOUSE" 4-22 AT'TABLE E: EXAMINATION OF SKIN LESIONS (TUMORS AND ULCERS) 4-25 AT TABLE F: BODY WEIGHT DETERMINATION, CAGEWISE 4-30 AT FIGURE~C: CHRONOLOGY 4-2 5 EVALUATION 5-1 5.1 General Condition, Behavior and Mortality 5-1 5.2 Body Weight 5-1 5.3 Macroscopic Examination of Skin 5-1 5.3.1 Tumor rate based on macroscopic appearance 5.3.2 Skin tumors 5-2 5.3.3 Skin irritations 5-2 3285

INBIFO Institut fur biologische Forschung • Wn SUBREPORT P 0500/3068 UBE144RA4 7293 AT PAGE 0-3 CONTENTS (continued) I AT PAGE 6 RESULTS AND DISCUSSION 6-1 6.1 Text 6-1 6.1.1 General condition and behavior 6-1 6.1.2 Mortality 6-2 6.1.3 Body weight 6-4 6.1.4 Development of macroscopic skin tumors 6-5 6.1.5 Skin irritations (macroscopic diagnosis)' 6-7 6.1.6 Comparison of mortality between CD1 mice without and with macroscopic skimtumors in application area 6-9 6.1.7 Prediction of the microscopic tumor probability in future studies 6-9 6.2 Tables and Figures AT TABLE. MORTALITY, CD1 MICE 1.1 MORTALITY, B6C3F1 MICE 1.2 MORTALITY, WEEK 80 2 MORTALITY, NEGATIVE CONTROL GROUPS IN DIFFERENT INBIFO STUDIES 3' INFLUENCE OF SMOKE TYPE ON MORTALITY 4.1 INFLUENCE OF DMBA PRETREATMENT ON MORTALITY 4.2 INFLUENCE OF'MOUSE STRAINS (CD1 AND B6C3F1) ON MORTALITY 4.3 INFLUENCE OF CONDENSATE STORAGE TEMPERATURE ON MORTALITY 4.4 BODY WEIGHT (GRAMS), APPLICATION WEEK -2 TO 0 5 2 TO 8' 6 0 1,0~ TO 16 7 ~ 18 TO 28 8' 32 TO 44 9 N ~ 48 TO 60 10 CA 64 TO 76 11 80 12 3285

INBIIFO Institut fuir biolbgiische Forschung • K6In SUBREPORT P 0500/3068 UBE144RA5 7293 AT PAGE 0-4 CONTENTS (continued) AT TABLE BODY WEIGHT, WEEK 80 13 BODY WEIGHT, NEGATIVE CONTROL GROUPS IN DIFFERENT INBIFO STUDIES 14 INDIVIDUAL BODY WE'IGHT'AT FINAL DISSECTION', MALE MICE 15 INFLUENCE OF SMOKE TYPE'ON BODY WEIGHT AT FINAL DISSECTION 16.1 INFLUENCE OF DMBA PRETREATMENT ON BODY WEIGHT AT FINAL DISSECTION 16.2 INFLUENCE'OF CONDENSATE STORAGE TEMPERATURE ON BODY WEIGHT AT FINAL DISSECTION 16.3 SKIN TUMORS (BASED ON MACROSCOPIC APPEARANCE) MORTALITY AND BODY WEIGHT 17 APPLICATION WEEK FOR TUMOR ONSET, MACROSCOPIC TUMORS 18 MACROSCOPIC TUMOR RATE IN DEPENDENCE ON TIME AFTER 1ST'APPLICATION OF ACETONE OR CONDENSATE' 19 MACROSCOPIC TUMOR RATE, WEEK 80 OF APPLICATION PERIOD 20 MACROSCOPIC TUMOR RATE, SKIN PAINTING STUDIES WITH 2R1 STANDARD REFERENCE CIGARETTE, MWSC-C AND MWSC-I, WEEK 80 21 MICE WITH SKIN IRRITATION, WEEK 1 TO 13 22 SKIN IRRITATION, WEEK OF 1ST APPEARANCE, MAXIMUM AND LAST APPEARANCE, NUMBER OF'MICE WITH SKIN IRRITATION IN 0/0 23 NUMBER OF MICE WITH SKIN'IRRITATION, MAXIMUM PER WEEK 24 INFLUENCE OF SMOKE TYPE ON MICE WITH SKIN IRRITATION (N) 25.1 INFLUENCE OF DMBA PRETREATMENT ON MICE WITH' SKIN' IRRITATION (N) 25.2 INFLUENCE OF'MOUSE'STRAINS (CD1 AND B6C3F1) ON MICE WITH SKIN IRRITATION (N) 25.3 INFLUENCE OF'CONDENSATE STORAGE'TEMPERATURE ON MICE WITH'SKIN IRRITATION (N): 25.4 3285.

INBIFO Institut fur biologische Forschung, • NCt9In SUBREPORT P 0500/3068 UBE144RA6 7293 AT PAGE 0-5 CONTENTS (continued) AT TABLE SURVEY: COMPARISON OF DIFFERENT CONDENSATE TYPES WITHOUT AND WITH DMBA PRETREATMENT AND DIFFERENT MOUSE'STRAINS 26 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, 27 MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE, 1-GR 2-GR 28 3-GR 29 4-GR 30 5-GR 31 6-GR 32' 7-GR 33 8-GR 34 9-GR 35 10-GR 36 11I-GR 37 12-GR 38 13-GR 39 14-GR 40 15-GR 41 MACROSCOPIC AND HISTOPATHOLOGICAL FINDINGS, MOUSE STRAIN CD1 42 MACROSCOPIC AND HISTOPATHOLOGICAL FINDINGS, MOUS'E STRAIN B6C3F1 43 CORRELATION: MACROSCOPIC AND HISTOPATHOLOGICAL FINDINGS, MOUSE STRAIN CD1, WITHOUT AND WITH DMBA PRETREATMENT 44 CORRELATION: MACROSCOPIC AND HISTOPATHOLOGICAL FINDINGS, MOUSE STRAIN B6C3F:1, WITHOUT AND WITH DMBA PRETREATMENT 45 I ~ CORRELATION: MACROSCOPIC AND HISTOPATHOLOGICAL FINDINGS 46 ~ ~ 3285

INBIFO Institut fur biologische Fo'rschung • KSIn SUBREPORT P 0500/3068 UBE144RA7 7293 AT PAGE 0-6 CONTENTS (continued)' I AT FIGURE MORTALITY, CDI MICE 1 MORTALITY, B6C3F1 MICE 2 BODY WEIGHT DEVELOPMENT, CD1 MICE 3 ODY WEIGHT DEVELOPMENT, B6C3F1 MICE 4 TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, CD1 MICE 5 TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, B6C3F1 MICE 6 SKIN IRRITATION'DEVELOPMENT, CD1 MICE 7 SKIN IRRITATION DEVELOPMENT, B6C3F1 MICE 8 SKIN IRRITATION, MAXIMAL NUMBER OF MICE PER WEEK, CD1 MICE 9 SKIN IRRITATION, MAXIMAL NUMBER OF MICE PER WEEK, B6C3F1 MICE 10 MORTALITY OF MICE WITHOUT AND WITH MACROSCOPIC SKIN'TUMORS IN APPLICATION AREA 1'1 CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND APPLICATION WEEK OF TUMOR ONSET, MOUSE STRAIN CD1!, WITHOUT AND WITH DMBA PRETREATMENT 12 CORRELATION'BETWEEN MICROSCOPIC TUMOR PROBABILITY AND APPLICATION WEEK IN WHICH A TUMOR PROBABILITY OF 5 0/0 WAS REACHED, MOUSE STRAIN CD1, WITHOUT AND WITH DMBA PRETREATMENT 3 CORRELATION B'ETW'EEN MICROSCOPIC TUMOR PROBABILITY AND THE TUMOR PROBABILITY OF APPLICATION WEEK 30, MOUSE STRAIN'CD1, WITHOUT AND WITH DMBA PRETREATMENT 14 CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND THE TUMOR PROBABILITY OF APPLICATION WEEK 40, MOUSE STRAIN CD1, WITHOUT AND WITH DMBA PRETREATMENT 15 CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND SKIN IRRITATION'y MOUSE STRAIN CD1, WITHOUT DMBA PRETREATMENT 16 3285

INBIFO Ilnstitut fur biologische Forschung • KtSin SUBREPORT P 0500/3068 UBE144RA8 7293 AT PAGE 0-7 CONTENTS (continued) ~ AT FIGURE CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND SKIN'IRRITATION, MOUSE STRAIN CD1, WITH DMBA PRETREATMENT 17 CORRELATION BETWEENIMICROSCOPIC TUMOR PROBABILITY AND APPLICATION WEEK OF TUMOR ONSET, MOUSE STRAIN;B6G3F1, WITHOUT AND WITH DMBA PRETREATMENT 18 CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND APPLICATION WEEK IN WHICH A TUMOR PROBABILITY OF 5 0/0 WAS REACHED, MOUSE STRAIN B6C3F1, WITHOUT AND WITH DMBA PRETREATMENT 9 CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND THE TUMOR PROBABILITY OF APPLICATION~WEEK 30, MOUSE STRAIN B6C3F1, WITHOUT AND WITH DMBA PRETREATMENT 20 CORRELATION'.BETWEEN MICROSCOPIC TUMOR PROBABILITY AND THE TUMOR'PROBABILITY OF APPLICATION WEEK 40, MOUSE STRAIN B6C3F1, WITHOUT AND WITH'DMBA PRETREATMENT 21 AT PAGE 7 REFERENCES 7-1 Remarks: This subreport, including front page, contains 253 pages. 3285

INBIFO Institut fulr biologische Forschung • K6in SUBREPORT P 0500/3068 UBE144RA9 7293 AT PAGE 0-8 ~ ABBREVIATIONS (a,b) Appl. : application AT r teamiAnimal Treatment DI,N : Deutsches Institut f ur Normung (German Commitee of Standards) DMBA Ex : 7,12-dimethylbenz(a)anthracene . x as exponent to the base of 10, e. g- e 102 .GE'. GT. . greater than or equal to . greater than 1 ight light/darkness less than or equal to less than arithmetic mean relative molecular mass MTD : maximum tolerated dose MTR : macroscopic tumor rate MWSC-C: mainstrean whole smoke condensate collected with cool trap MWSC-I: mainstream whole smoke condensate collected with impaction trap N : number of individual values OHC : optimal hygiene conditions PCB : polychllorinated biphenyls PT : preliminary title PY : team Pathology RSD : relative standard deviation SOP : standard operating procedure sp. : species SPF : specified pathogen free ' SWSC-I: sidestream whole smoke condensate collected with impaction tra WSC : whole smoke condensate H W (a) in addition to those, which are explained immediately on the same2 page, table or figure in the text (b) Units of measure are given in accordance with SI-norms (Systeme International d'Unites). 3285

INBIFO Institut fOr biologische Forschung • Mn SUBREPORT P 0500/3068 UBE144RA10 7293' AT PAGE'0-9' Statistical Analysis p : probability of receiving the value of the test statistic or a more extreme value of the test statistic, if the null hypothesis holds _ : p .GT.0.05, statistically not significant + : p .LE.0.05 ++ : p .LE.0.01 +++ : p .LE.0.001 : not evaluated (e. g. no value available) : F-test (alpha = 0.05) indicates an inhomogeneity of variances of compared groups. In this case a modified t-test according to Welch is calculated. Remarks: Measured data are rounded and mostly tabulated with a fixed decimal point. In most tables, figures are printed: with 3 significant digits. For calculations, however, all available digits are evaluated. This results in some cases in minor but apparent discrepancies between tabu- latedd and calculated values, such as means and relative means. Unless otherwise specified the word "mean" in the text stands for "arithmetic mean". 3285

INBIFO Institut fuir biologische Forsclhung • K6in SUBREPORT P 0500/3068 UBE1i44RA11 7299 AT PAGE 1-1 SUMMARY 1.1 Objective In accordance with the objectives of this study specified in the SUMMARY of the INTEGRATING REPORT the team AnimaL Treatment was responsible for (1) animal housing, (2) acetone and test substance applications, (3) general condition, behavior and mortality checks, (4) body weight determinations, (5) visual skin examinations for tumors and ulcers. 1.2 Method 2625 mice, i. e., 1785 (a) outbred female CD1 mice from Charles River, France (St. Aubin les Elbeuf) and 840 female B6C3F1 mice from Charles River, USA (Portage, Michigan) reared under specificc pathogen free conditions and shipped in protective filter shipping boxes were kept in am animal area used only for subchronic and chronic studies with small laboratory rodents. Floors, walls and ceilings as well as fixtures of the windowless rooms are coated with epoxy resim. To ascertain optimal hygiene conditions person- nel and material entered this area only in accordance with strict specifications. The 2625 mice were randomly allocated to 25 groups (105 mice per group). 7 mice each were kept in polycarbonate cages with granulated, softwood bedding. Cages, bedding material and water were changed 3 times per week. The mice were fed with autoclaved fortified diet standard pellets and watered with tap water ad libitum. Air temperature was 22 + 2 degrees centigrade, the relative humidity was 55 + 10 percent and light cycle was 12 hours : 12 hours (L 06.00 to 18.00 standard time). The hair of the application area was shaved with electric clippers before 7,12'-dimethylbenz(a)anthracene (DMBA) and the 1st condensate (a) mice for microbiological analysis not included 3285

INBIFO Institut fur biologische Forschung • K6In, SUBREPORT P 0500/3068 UBE144RA12 7299 AT PAGE 1-2 application, subsequently 1 time per week and additionally for individual mice showing an increased hair growth prior to the application of the day. At the age of 36 + 2 days the mice of several groups received a single dermal application of 200 nanomols DMBA (equivalent to 51 micrograms) per mouse. Both CD1 and B6C3F1 mice received the same DMBA dose. 10 days later (a) whole smoke condensate (WSC) application was started. Application materials were applied with automatic pipettes. To CD1i mice either 60 or 90 or 120 milligrams/(mouse x week) of processed mainstream whole smoke condensate with impaction trap (MWSC-I) or sidestream whole smoke condensate collected with impaction trap (SWSC-I) prepared in acetone or acetone alone were applied on 5 consecutive days per week. The application volume was 100 microliters/(mouse x applica- tion). From the 1st to the 9th application week the application volume was increased from 20 microliters/(mouse x day) to 100 microliters/(mouse x day). Thereafter a constant application volume was applied. B6C3F1 mice showed an approx. 30 percent lower body weight than CD1 mice throughout their lifetime. Therefore, B6C3F1 mice received MWSC-I doses reduced by approx. 30 percent compared with the doses for CD1 mice (reduction of the application volume to 70 microliters/(mouse x application)). From the 1st to the 9th application week the application volume was increased'.from 15 microliters/(mouse x day) to 70 microliters/(mouse x day). Thereafter a constant application volume was applied. 1 dose was equal for both strains (60 milligrams/(mouse x application)). The mice were checked for general condition, behavior, and mor- tality 2 times per working day (Monday to Friday) and on weekends 1 time daily. The body weight was determined at least fortnightly up to week 20 of the application period and every 4th week there- after. Visual examinations of the skin for macroscopic tumors and ulcers were performed weekly. The macroscopic rate (percent) was weekly calculated according to Blanding et al. (1951). All changes (a) no applications in between 3285

INBIFO Institut fur biologische Forschung • K6In SUBREPORT -P 0500/3068 UBE144RA13 7299' AT PAGE: 1-3 . I of the macroscopic tumors and ulcers were checked again by 2 tech- nicians. in case of the 1st appearance of a tumor for an indiivi- dual mouse this mouse was checked by the group leader. Compu- terized data acquisition ensured the integrity and reliability of the data. All surviving mice were sacrificed after 80 weeks of application. 1st condensate application: 20.Dec.82 Last condensate application: 29.Jun.84 1.3 Results 1.3.1 General condition and behavior ------------------------------ In CD1 mice, signs of intoxication were observed from approx. 5 weeks after the start of the application period up to approx. the 25th application week in the WSC-I treated groups. Only in the SWSC-I-treated groups d'id' acute signs of intoxication occur up to the end of study. The intensity of these signs decreased with time. The main signs of intoxication were spontaneous activity which increased immediately after application and later decreased, prone position, dyspnea, decreased body temperature, and either a partial or complete closing of the palpebral fissure. The signs of intoxication increased with ascending doses. Dose groups with strong signs of intoxication showed high mortality. The comparison of the general condition and behavior of CD1 mice treated with MWSC-I and those with SWSC-I showed differences in the proportion of signs of intoxication. The number of mice with signs of intoxication was higher in SWSC-I- than in MWSC-I treated groups. 3285

INBIFO Institut fur biologische Forschung. • KSI'n SUBREPORT P 0500/3068 UBE144RA14 7279 AT PAGE 1-4 No differences were seen in the general condition and behavior of both mouse strains which were pretreated with DMBA. Signs of intoxication were observed in CD1 but not in B6C3F1 mice. The comparison of the general condition and behavior of CD1 mice did not result in differences depending on whether MWSC-I was stored before application at 4 or at minus 75 degrees centigrade. 1.3.2 Mortality The mortality in CD1 mice of the acetone and the DMBA plus acetone control groups was 33 and 19 percent respectively. The mortality in B6C3F1 mice of the acetone and: the DMBA plus acetone control group was 7 and 5 percent respectively. In CD1 mice the lowest mortality with 24 percent was found in the MWSC-I medium dose group (a) and the highest mortality with 86 percent in the DMBA plus SWSC-I high dose group. There was no dose dependency in the MWSC-I treated groups. A slightly dose dependent increase in the percentage of mortality was observed in the DMBA plus MWSC-I groups. Dose dependency was obtained in the SWSC-I and in the DMBA plus SWSC-I groups. In B6C3F1 mice the lowest mortality with 2 percent was found in the MWSC-I medium dose group and the highest mortality with 10 percent in the DMBA plus MWSC-I medium dose group. No dose dependency was observed. The comparison of the mortality of MWSC-I- and SWSC-I-treated CD1 mice showed a statistically significantly higher mortality in the SWSC-I-treated groups. This is considered to be biologically relevant. (a) condensate stored at 4 degrees centigrade 3285

INBIFO Institut flur biologische Forschung • K6In SUBREPORT P 0500/3068 UBE144RA15 7299 AT PAGE 1-5 In CDT mice the pretreatment with an initiating d'ose of DMBA resulted in a statistically significantly higher mortality in the SWSC-I dose groups. This statistical significance is considered to be biologically relevant. In B6C3F1 mice a slightly higher mor- tality was seen in the DMBA pretreated groups which was statisti- cally significant in the medium- and high-dose groups. This sta- tistical significance is considered to have no biological rele- vance. The comparison of the mortality of the MWSC-I-treated CD1 and B6C3F1 mice showed a statistically significantly higher mortality for CD1 mice. This is considered to be biological relevant. The comparison of the mortality for CD1 mice showed no difference depending on whether MWSC-I was stored before application at 4 or at minus 75 degrees centigrade. The proportion of dead CD11 mice with macroscopic skin tumors did not generally exceed that without skin tumors. Also the overall mortality of mice with macroscopic skin tumors did not increase, i. e. the mortality did not increase in relation to the appearance of skin tumors. Therefore, the reason for the higher mortality in the WSC-I-treated groups is not known. 1.3.3 Body_weight Beginning in the 1st third of the study the mean body weight of'both mouse strains of all condensate treated groups was slight- ly lower (in applicatiom week 80 a maximum of 13 percent in CD1 mice and 9 percent in B6C3F1 mice) than the body weight of the corresponding control groups. This is considered to be biological- ly relevant, indicating a slight toxic effect. The toxicity is thought to be caused by nicotine in the condensate, but other components may also be relevant. The body weight reduction 3285

INBIFO Institut fur biologische Forschung • K6in SUBREPORT P 0500/3068 UBE144RA16 7299 AT PAGE 1-6 indicates an appropriate WSC-I dose selection with the highest dose at the maximum tolerated dose (MTD), i. e., a dose causing an approx. 10-percent reduction in body weight gain. The comparison of the body weights of CD1 mice with respect to MWSC-I and SWSC-I showed no differences. The pretreatment with an initiating dose of DMBA did' not result in a difference in the body weight of both mouse strains. The body weight of the CD1 mice is strain-dependently higher than that of the B6C3F1 mice. The relative body weight reduction in the MWSC-I-treated groups was the same in both mouse strains (-7 percent). The comparison of body weight in CD1 mice showed no difference depending on whether MWSC-I was stored before application at 4 or at -75 degrees centigrade. 1.3.4 Development of macroscopic-skin-tumors ----- -- -------- ---- No macroscopic skin tumors were found in the acetone control groups in CD1 and B6C3F1' mice. The 6 percent macroscopic tumor rate (MTR) (a) in the UMBA pretreated CD1i acetone control group indicates that the DMBA d'ose of 200 nanomols for the single appli- cation may have been too high to represent initiator activity only. The DMBA plus acetone control group in B6C3F1 mice showed an unexpected and': unexplainably high MTR of 31 percent (b). (a) Corresponding to a histologically confirmed tumor rate of 4 percent (b) Corresponding to a histologically confirmed tumor rate of 13 percent 3285

INBIFO Institut fur biologische Forschung • KOIn SUBREPORT P 0500/3068 UBE144RA17 7299 AT PAGE 1-7 In WSC-I-treated groups the 1st macroscopic skin tumors were re- corded in CD1 mice between WSC-I application weeks 14 and 54 and in B6C3F1 mice between WSC-I application weeks 18 and 66. At the time when a 10-percent MTR was reached, a strong dose dependency was observed in CD1 mice treated with SWSC-I and in B6C3F1 mice treated with DMBA plus MWSC-I. In CD1 mice the lowest MTR of the WSC-I treated groups was ob- tained with 12 percent in the MWSC-I, medium-dose group (a) and the highest MTR with 99 percent in the DMBA plus SWSC-I, high-dose group. The MTR increase was dose-dependent in the DMBA plus MWSC-I, the SWSC-I and in the DMBA plus SWSC-I dose groups. In B6C3F1 mice the lowest MTR of the MWSC-I-treated groups was obtained with 5 percent in the MWSC-I, low-dose group and the highest MTR with 67 percent in the DMBA plus MWSC-I, high-dose group. The MTR increase was dose dependent in the MWSC-I and in the DMBA plus MWSC-I groups. The detailed evaluation of the tumorigenicity (tumor probability, tumor multiplicity and malignancy) is given in SUBREPaRT PY. 1.3.5 Skin_irritations_Smacroscopic_diaqnosis) ---- ----------- - No skin irritations were found in CD1 mice in the control groups. The 1st skin irritations (translucent and/or reddened skin) in CD1 mice were detected approx. 2 weeks after the start of the WSC-I application. The number of mice with skin irritations reached a maximum during the 4th and 5th week of WSC-I applica- tion, i. e., after approx. 15 to 25 applications. In the high-dose groups, skin irritations occurred earlier. Approx. 10 weeks after the beginning of the WSC-I applications, skin irritations were no longer observed. In B6C3F1 mice only a few mice with skin irri- tations were observed in control and MWSC-I-treated groups. (a) condensate stored at 4 degrees centigrade 3285

INBIFO Institut fiur biblogische Forschung Kt51n SUBREPORT P 0500/3068 UBE144RA18 7299 AT PAGE 1-8' The comparison of skin irritations in MWSC-I- and SWSC-I-treated CD1 mice showed a statistically significantly higher number of mice with skin irritations in' the SWSC-I-treated groups. This is considered to be biologically relevant. In CD1 mice the pretreatment with an initiating dose of DMBA resulted' in a statistically significantly lower number of mice with skin irritations both in the MWSC-I- and in the SWSC-I- treated' groups. This effect is considered to be biologically rellevant. The red'uced number of mice with skin irritatiions in the DMBA-pretreated WSC-I groups is unexplainable. In B6C3F1i mice no d'ifference was seen between the MWSC-I-treated groups without and with DMBA pretreatment.. The comparison of skin irritations between the MWSC-I-treated CD1 and B6C3F1 mice showed a statistically significantly higher number of mice with skin irritations in the CD1 mouse strain. This, however, is not considered to be biologically relevant, because the skin inspection and scoring of skin irritations were hindered by the brown pigmentation of the skin in B6C3F1 mice. The comparison of the number of mice with skin irritations in CD1 mice showed no d'ifference depending on whether MWSC-I was stored before applicatiion' at 4 or at -75 degrees centigrade. 1.3.6 Compariison of mortality between CD1 mice without and' with maeroscopic_skin_tumors_in_agplication_area -------- - ----------------- The percentage of dead mice without macroscopic skin tumors was compared to the percentage of dead mice with macroscopic skin tumors weekly from' begin up to the end of the study. 3285

INBIFO Institut fur biologische Forschung • KtSIn SUBREPORT P 0500/3068' UBE144RA19 7279 AT PAGE 1-9 The percentage of dead mice with skin tumors in the individual groups was practically the same or lower than those without skin tumors with exception of 2 groups. Also the overall mortality of mice with macroscopic skin tumors did not increase, i. e. the mortality did not increase in relation to the appearance of skin tumors. 1.3.7 Prediction of the microscopic tumor probability in future studies For the prediction of the microscopic tumor probability at an early stage of the experiment, the microscopic tumor probability was compared with the macroscopic skin irritation, the week of the tumor onset, the week when 5 percent tumor probability was reached, and the macroscopic tumor probability of applicationn weeks 30 and 40 in CD1 and B6C3F1 mice. In CD1 mice the comparison between the microscopic tumor probabi- lity and the macroscopic parameters (except skin irritation which showed only a weak correlation) showed correlation coefficients from 0.812 to 0.933. These correlation coefficients are statisti- cally significant with p=.LE.0.001 and therefore allow a predic- tion of the final tumor probability. This prediction is accom- panied by a standard deviation from + 12 to + 19 percent for the parameter microscopic tumor probability for week 80. In addition the parameter skin irritation can be used~ for the prediction, although the correlation is not so strict (correlation coeffi- cient: 0.716 and 0.677, statistical significance: p=.LE.0.01 and .LE.0.05), but the values are already available in applica- tion week 5. In B6C3F1 mice the comparison between the microscopic tumor pro- bability and the macroscopic parameters (except tumor onset which 3285

INBIFO I!nstitut flur biologische Forschung • K61n SUBREPORT'P 0500/3A68 UBE144RB30 7299 AT PAGE 1-10, showed only a weak correlation and skin irritation (a)) showed correlation coefficients from 0.859 to 0.911. These correlation coefficients are statistically significant between p=.LE.01.01. and .LE.0.001 and also allow a prediction of the final tumor probability. This prediction is accompanied by a standard devia- tion from + 9' to + 11 percent for the parameter microscopic tumor probability for week 80. In addition, the parameter tumor onset can be used for the prediction, although the correlation is not so strict (correlation coefficient: 0.768, statistical signifi- cance: p = .LE..0.05) (b). In addition to the above mentioned macroscopic parameters the histopathological finding, "hyperkeratosis", (investigated in INBTFO~ study P 0500/3117, 1986) allows a prediction of the final tumor probability in application week 5 for the strain CD1 as well as B6C3F1 pretreated with DMBA. I N B I F 0 Institut fur biologische Forschung GmbH' (a) In B6C3F1 mice practically no skin irritations were seen, which is probably due to the brown pigmentation of skin. (b) Due to the brown pigmentation of skin in B6C3F1 mice the tumor onset is the earliest macroscopic available parameter for the prediction. 3285

INBI'FO Institut fwr biofogiische Forschung • Koln SUBREPORT P 0500/3068 UBE144RA20 6311 2 RESPONSIBILITY AT PAGE 2-1 Quality Assurance: E. R6mer Biologist (Diplombiologe)

INBIFO Institut flur biologische Forschung • N<6in SUBREPORT P 0500/3068 UBE144RA20 6311 M AT PAGE 2-1 2 RESPONSIBILITY Quality Assurance: D. Kuhn E. Romer Biologist (Diplombiologe) 3285

SUBREPORT P 0500/3068 UBE144RA21 6311 3 INTRODUCTION AT PAGE'3-1 Carefully planned skin painting studies allow the quantitative evaluation of tumorigenic and/or cotumorigenic potency of ciga- rette smoke condensates or their fractions. The 1st skin painting study carried out at INBIFO was started in 1974. Ever since then, without interruption, 1 or more skin painting studies were in progress. Before 1978 CFLP mice, obtained from Anglia Laboratory Animals (formerly Carworth Europe) Alcon- bury Huntingdon/England, have been used for mouse skin painting studies at INBIFO. The use of that strain was discontinued, however, when it was faund that these mice were infected with Pasteurella pneumotropica at the breeder. From 1978 up to now CD1 mice obtained from the Charles River Breeding Laboratories were used. Because of the low mortality, the sensitivity to carcinogens and the ]low rate of spontaneous tumors the CD1 mouse strain is quali- fied for skin painting studies. The CD1 mouse strain descends from 2 male and 7 female mice derived from a noninbred stock in the laboratory of Dr. de Coulon of Lausanne, Switzerland (Eaton et al., 1980). These mice were imported 1926 into the United States by Dr. Clara Lynch (Lynch, 1969) of the Rockefeller Institute for use in cancer research. Over the next quarter of the century, descendants of the original 9 mice were distributed across the world to scientists and breeders, giving rise to a large number of socalled "Swiss" strains. 1 of these "Swiss" strains, called the Hauschka strain Ha/'ICR was initiated in 1948 at the Institute for Cancer Research in Philadelphia (Hauschka, 1973, Eaton et al., 1980). Hauschka attained the growth rate and high prod'uction characteristic of this mouse. A part of this colony was given to Dr. Edwin Mirand of Roswell Park Memorial Institute where they ^2RF

SUBREPORT P 0500:/3068 UBE144RA22 7279 AT PAGE 3-2 were designated as HaM/ICR. Some of these were passed on to Charles River in 1959. Finally, these randombred mice were deve- loped further by the Charles River Breeding Laboratories (Percy et al., 1971) and are described as "originated in 1959 from caesarean sections on HaM/ICR (Hauschka and Mirand - Roswell Park Memorial Institute - Swiss) mice." In skin painting studies female mice are much preferred over males, because females are considerably less aggressive. It is im- portant to minimize fighting among the test animals in skin pain- ting studies, because bites and scratches may lead to false re- sults in counting of skin irritations (caused by test substances) and tumors. Homburger et al. (1975), described the body weight gain and mor- tality of CD1 mice in a 2-year study. The body weight of female mice was approx. 38 grams at the age of 22' months. These results are in accordance with INBIFO data (see AT FIGURE A). 50 percent mortality of female mice occurred at approx. 18 months. At INBIFO the 50 percent mortality of CD1 mice was never reached in the acetone control groups of 80-week skin painting studies, the mor- tality varied from 15 to 33 percent at an age of 22 months (see FIGURE B). The spontaneous macroscopic tumor rate in the acetone control groups of CD1 mice determined at INBIFO from 1978 up to now was 0 percent (see AT TABLE A). The corresponding microscopic tumor rate was practically 0 percent, too. Only in 1 mouse 1 tumor (prickle cell papilloma) was detected. MWSC-I of cigarette 2R1 applied at a dose of 120 milligrams processed condensate/(mouse x week)~ was found to cause a histo- logically confirmed tumor rate of 2'1 percent in a previous skin painting study at INBIFO using mouse strain CD1 (a). For the dif- ferentiation of WSC of several experimental cigarettes this res- (a) INBIFO study P 0500/3030 (1984)

P 0500/-, H04490C, V21 F67 V96 F53 SUBREPORT P 0500/3068 UBE144RA23 H04490C 7271 M .. 50-1 Q1 40 --i 30 " ~ 20 --1 10-~ 0 AT PAGE 3-3 -0 --------- X;;' -fl , P -/3000 (a ) ~ P -/3010 (a ) ~ P -/3023 (a ) , P -/30 24 (a ) P -/30 30 (a ) , P -/3068 (a) _ P -/30 73 (b ) ~ P -/3111 (a) : P -/3115 (a) 0 10 20 30 40 50 60 70 80 90 AT FIGURE A AGE OF MICE (week) BODY WEIGHT, ACETONE CONTROL GROUPS IN DIFFERENT INBIFO STUDIES (mice strain: CD1, female) (a) breeder: Charles River France (b) breeder: Charles River Canada %Visoszoz

P 0500/-, H04491C, V20 F67 V19 F67 SUBREPORT P 0500/3068 UBE144RA23 ~ 40 -I 30 --1 20-1 10-~ 0 H04491C 7271 M -- p P 3 # -/ 000 (a) --~ : P -/3010 (a ) -fl o P -/30 23 ( a ) -~ , P -/30 24 (a ) -~ e P -/30 30 ( a ) --~ : P -/3068 (a) -~ ~ P -/30 73 ( b ) -4 ~ P -/3111 (a) -~ : P -/3115 (a) I 10 AT FIGURE B T 20 I 30 -F 40 T 50 MORTALITY, ACETONE CONTROL GROUPS IN DIFFERENT (a) breeder: Charles River France (b) breeder: Charles River Canada "TTso.9Wz AT PAGE 3-4 60 70 80 90 AGE OF MICE (week) INBIFO STUDIES (mice strain: CD1, female)

SUBREPORT P 0500/3068 UBE144RA24 7288 AT PAGE 3-5 STUDY INITIAL SPONTANEOUS SKIN NUMBER OF' TUMOR RATE (0/0) MICE (P 0500/-) MACROSCOPIC MICROSCOPIC 3000 105 0 0 3010 210 0 0 3023 105 0 0 302'4 105 0 0 3030 105 0 0 3068 105 0 1 (prickle cell papilloma) 3073 105 0 0: 3111 105 1 -( a): 3115 105 1 - (a) AT TABLE A SPONTANEOUS SKIN TUMOR RATE, ACETONE CONTROL GROUPS IN'DIFFERENT INBIFO STUDIES, RESULTS AFTER 80 WEEKS OF APPLICATION, FEMALE CD1 MICE Remarks: application material: acetone, 5 times per week, (a) data not yet available 3587

SUBREPORT P 0500/3068 UBE144RA25 6311 AT' PAGE 3-6 ponse appears to be too small and systems resultitig in higher tumor rates with the high biological activity reference cigarette type 2R1 would be more appropriate. The low tumor rate may be related to the relatively weak tumor initiating activity of WSC and/or the relative insensitivity of the mouse strain used. Therefore the pretreatment of several groups with a single dermal application of a tumor initiating dose of DMBA was included in this study. Since an appropriate number of mice of the highly sensitive SENCAR strain was not available, the B6C3F1 hybrid strain was chosen as a possible future substitute for th~e CD1 strain. B6C3F1 is known to be responsive to the tumorigenic acti- vity of polycyclic aromatic hydrocarbons and related chemical compounds. This strain is presently used by the National Toxico- logy Program (NTP) in the USA for skin painting carcinogenesis studies of several compounds including tobacco condensate compo- nents. Hybrid strains (a) combine the adventage of phenotypic uniformity with that of greater long-term genetic stability than inbred strains. Before its use in the NTP, the B6C3F1 mouse straim ranked 4th in the "popularity" scale of F1 hybrid mice (Festing, 1979). The B6C3F1 strain was exposed to MWSC-I without and with DMBA pretreatment. (a) Individuals are heterozygous at all loci at which, the parental strains differ genotypically.

SUBREPORT P 0500/3068 UBE144RA27 7288 AT PAGE 4-1 4 METHOD 4.1 Chronology (see AT FIGURE C) 4.2 Laboratory Hygiene 4.2.1 Chemical disinfection Principle: addition of chemical disinfectants to washing solutions Sample material and time: disinfection prior to study: (1) animal rooms (2) facilities (3) equipment Equipment: Chemicals: disinfection during experiment: (4) hands and forearms: several times daily (5) handling and application rooms: daily (6) animal room and facilities: weekly (7) clothing: daily (8) equipment (8.1) cage racks: every 10 weeks (8.2) others: if necessary formaldehyde disinfector, Gerhard Teck, D-4194 Hau high pressure cleaning machine, PIKO KW 50, Carl Platz GmbH, D-6710 Frankenthal/Pfalz washing machine Gir 112 APS, Miele und Cie. GmbH und Co., D-4830 GUtersloh formaldehyde, Baker Chemikalien, D-6080 Gross-Gerau Kohrsolin, no. 439, Sterillium, no. 669, Bacillolfabrik Dr. Bode, D-2000 Hamburg 54 3587

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SUBREPORT P 0500/3068 UBE144RA29 7288 ' AT PAGE 4-3 Dermasil, no. DE25, Henkel und Cie. GmbH, D-4000 Dusseldorf Procedure: (1), (2) and (3) disinfected with 30 ml Kohrsolin/1 and 370 ml formaldehyde/1 prior to start of INBIFO study P 0500/3068 (Dec.82) (4) and (8.2) washed with sterillium (5), (6) and (8.1) cleaned with 2.5 ml Kohrsolin/1 (7) washed with Dermasil solution Scientific version: SOP AT 80/2, AT 130/1 Text version: 3.Dec.85 4.2.2 Entrance of area B Animals: Personnel: Equipment: before entering area B disinfection of the exterior of animal trans- portation container (filter boxes) with 2.5 m1 Kohrsolin/1, care taken not to contaminate breathing zone (filters) of animals with disinfectant before entering the area, hand's disinfected with sterillium~and shoes changed, soles of laboratory shoes disinfected by wading through a matted depression, filled with~10 ml Kohrsolin/1 (a) in front of the door to the area chemical disinfection of surfaces with 2.5 ml Kohrsolin/l Scientific version: SOP AT 70/3 Text version: 14.Aug.87 (a) up to Apr.84 2.5 ml'/1 3587

SUBREPORT P 0500/3068 UBE144RA30 7226 AT PAGE 4-4 4.2.3 Entrance af_laboratory_inside_area_B Personnel: before entering the laboratory area, white laboratory coat as well as jewellery (rings, watch etc.) re- moved, hands and forearms disin- fected with sterillium. Soles of laboratory shoes disinfected by wading through a matted tub, filled with 2.5 ml Kohrsolin/l in front of the door, on entering, the laboratory Clothing During application of DMBA solution: disposable coat, mask, gloves (rubber), and green hair bonnet worn. Contami- nated coats replaced immediately. In addition gas mask with filter 88A/St worn during application and handling in animal room During application of condensate solution: green disinfected coat, mask, gloves (rubber) and green hair bonnet worn. Disinfected coat replaced every day. Scientific version: Text version: SOP AT 72/2 14.Aug.87 4.2.4 Cleaning of laboratory equipment and disposal of materials contaminated with hazardous materials Sample material: (1) polycarbonate cages (2) cage lids (3) sipper tubes (4) silicone plugs (5) , bottles (6) disposable cages Equipment: (1) cage washer: Hamo UP (2) bottle washer: Hamo TR and Newamatic 60

SUBREPORT P 0500/3068 UBE144RB1 7288 Chemicals: Procedure: acidifier SPF, Diversey GmbH, D-6000,Frankfurt/M. acetone, no. 14, E. Merck, D-6100 Darmstadt 1 AT PAGE 4-5 (1) automatic addition of acidifier to cage washer, 37 ml/min, final pH: approx. 2, temperature: approx. 60 degrees centigrade, final rinsing with tap water (2) after application of DMBA solution decontamination with acetone, then procedure (1) (3) and (4) after application of DMBA solution decontamination with acetone, then procedure (5) (5) Hamo TR: automatic addition of acidifier to bottle washer, 11 ml/washing run, 2 times 2 min at 70 degrees centigrade, pH approx. 3, final rinsing with tap water Newamatic 60: automatic addition of acidifier to bottle washer, 20 ml/washing run, 2 times 2 min at 80 degrees centigrade, pH approx. 3, final rinsing with tap water (6) destruction (a) Scientific version: SOP AT 1/3, AT 3/4, AT 3/5, AT 2.1/1, AT 2.2/1 Text version: 14.Aug.87 (a) to be burned by Fa. Widdig GmbH, D-5216 Niederkassel 3587

SUBREPORT P 0500/3068 UBE144RB2 5301 AT PAGE'4-6 4.3 Animals and Animal Treatment 4.3.1 Animals Species: mouse (mus musculus) Strain and designation: (1) Crl: CD1(ICR)BR (2) B6C3F1/Crl BR Color: (1) white (2) agouti Type of breeding: (1) outbred (2) hybrid, (F1 ) Sex : female Microbiological conditions of breeding: COBS (Caesarean-Originated, Barrier sustained)', Breeder: (1) Charles River France S.A., F-76410 St. Aubin les Elbeuf (2) Charles River USA, Portage, Michigan Transport containers: special filter boxes INBIFO animal supply number: (1) 301 (2) 300 INBIFO animal study approval number: (1) and (2) 27 Number of mice and animal treatment Acetone control: (1) 105 (2) 105 Acetone plus DMBA control: (1) 105 (2) 105

SUBREPORT'P 0500/3068 UBE144RB3 6311 AT' PAGE 4-7 Condensate-treated: Microbiological analyses: Total: (1) 1575 (2) 630 (1) 20 (2) 20 (1) 1805 (2) 860 Date of arrival: (1) 2.Dec.82 (2) 2.Dec.82 Age of mice (days) On arrival: 28 + 2 On application of DMBA solution: 36 + 2 On 1st application of condensate solution: 4 6 + 2' Mean body weight (grams) 1 day after arrival: (1) 18.9 (2) 13.0 On 1st application of condensate solution: (1) 22.1 (2) 16.6 Text version: 7.Nov.86

1! 41.i11. - 1..v.~lu. IUI UI,A-Ji,v.1IL1U. v... .......~ 1\V11.1' SUBREPORT P 0500/3068 UBE144RB4 7226 AT PAGE 4-8 4.3.2 Animal_housing Animal room: INBIFO main laboratory building, area B (a), room R201, R202 and R203 Construction and interior: windowless floors, walls and ceilings coated with epoxy resins Microbiological conditions: conventional under defined labora- tory conditions (b), disinfection of rooms with 30 ml Kohrsolin/1 and formaldehyde aerosol prior to start of study Conditioning and ventilation: 100 0/0 fresh air, delivered from a 50 m high air inlet stack, approx. 15 changes/h filter: fine filter class C Room temperature (degrees centigrade): 22 + 2 (c) Relative humidity (0/0): 55 + 10 (c) Light Time cycle: L/D 12 : 12, L 06.00 to 18.00 standard time Source: dampened "daylight" fluorescent lamps: Lumilux W11, Osram GmbH, D-8000 Munchen 1 Intensity in cages: approx. 20 to 100 Lux (a) area B only used for subcronic and long-term studies with small laboratory rodents, reared under SPF conditions and shipped in special filter boxes (b) specifications not as strict as those applied to INBIFO animal area A (SPF-barriered animal care) but above OHC level (c) At outside temperatures above 28 degrees centigrade and at ex- tremely high relative humitidy, these specifications may not al- ways be maintained. ,r-

SUBIZEYUkT P 0500/3Ubb UB'E144kB5 7288 Cages : AT PAGE'4-9 type 2 (a), polycarbonate (Makrolon), base area: 230 mmix 170 mm height: 140 mm Change of cages: 3 times/week Cage lids: stainless steel wire mesh with overhead hoppers Change of cage lids: every 10 weeks Bedding material: autoclaved granulated wood (type: T grob 2), dust free Supplier: Forsbach GmbH, D-5650 Solingen 11 Residue analysis: each batch screened for selected pesticides and PCB residues. Material used only when the tolerance levels given by the German Futtermittelver- ordnung (Jun.76) are not exceeded'. Sterilization: 15 min at 134 degrees centigrade pressure: 2.5E5 Pa Drying: 10 min routinely screened for bacteriological contaminates Replacement of bedding material: 3 times/week (Mo., Fve., Fr.) during application Animals per cage: 7 Scientific version: SOP AT 27/8 Text version: 7.Nov.86 (a) after application of DMBA solution until 1st application of con- densate solution disposable polystyrole cages inserted into poly- carbonate cages 3587

SUBREPORT P 0500/3068 UBE144RB6 7288 AT PAGE 4-10 4.3.3 Diet and drinkinq_water (a) Diet: autoclaved fortified diet, cylindri- cal pellets, 16 mm diameter, "HERILAN MRH-HALTUNG fiir Mause, Rat- ten und Hamster" (see AT PAGE'4-11) Supplier: H. Eggersmann KG, D-3260 Rinteln/'Weser Residue analysis: each batch screened for salmo- nella sp., selected pesticides and PCB residues, aflatoxins and heavy metals (arsenic, cadmium, iead'., mercury). Material used only when the tolerance levels given by the German Futtermittelverordnung (Jun.76) are not exceeded. Sterilization: Drying: Diet supply: Water: 5 min at 120 degrees centigrade pressure: 1.2E5 Pa 10 min ad libitum from~stainless steel hoppers in cage lid tap water, reduction of microorganisms by heating to approx. 60 degrees centi- grade Water supply: ad libitum from 250-ml DIN clear glass bottles, with stainless steel sipper tubes, water changed 3 times/week (Tu., Th., Sa.) Scientific version: SOP AT 20/10 Text version: 14.Aug.87 (a) random samples of all autoclaved batches of diet and of drinking ~ water microbiologically investigated 3587

SUBREPORT P 0500/3068 UBE144RB7 Specifications by supplier: 7292 U HerlLikh M Versuchstierdiaten AT PAGE 4-11 HERILAN MRH - HALTUNG (Fortified) Alleindiat fiir die Haltung von Maussn, Ratten_und Hamstern Umsetzbare Energie (Ratte): 11,73 Mj' (2.800 kcal) / kg Diat Gehalt an Rohnahrstoffen Gehalte an Spurenelementen (v. H.) (mg / kg Diat) Rohprotein Rohfett Rohfaser Rohasche Ca P Na K Mg 17,20 5,10 6,00 7,00 1,00 0,80 0,30 1,00 0,30 Fe 200 Mn 100 Cu 15 Zn 60 J 1,0 F 5,5 Gehalte an Aminosauren Zusatze an Vitaminen (g AS / kg Diat) (kg Diat) Lys 8,80 Vit. A I.E. 22.500 Meth 4,00. Vit. D3 I.E. 900 Me th + Cys 6,10 Vit. E mg 225 Try 2,30 Vit. K3 mg 15 Arg 10,20 Vit. C mg 300 H'i s 6,50 Vit. BL mg. 40 Ile 6,10 Vit. B2 mg_ 60 Leu 12,50 Vit. B6 mg 40 Thr 6,50 Vit. B12 mcg 60 Phe+ Tyr 12,00 Ca-Panto. mg 75 Val 8,60 Nikotins. mg 130 Cholinchl. mg 2.000 Folsaure mg 7 Inosit mg 7 Biotin (H) mcg 220

SUBREPORT P 0500/3068 UBE144RB8 7226 AT PAGE 4-1'2 4.3.4 Groups-and doses Group identification and doses: see AT TABLE B Group size: each group: 1'05 mice Allocation of animals to groups:: placing of 7 mice ima non marked cage, allocation of the marked shields to the cages according to a random number table, and then placing of cages in consecutive order in racks Identification of individuall animals: marking of individual mice and groups Text version: 14.Aug.87 1'A~

I~.u.... _ ,.......... ~~,. ......,... J,ti _..a. t vl.... . SUBREPORT P 0500/3068 UBE144RB9 7289 AT PAGE4-13 GRDUP MOUSE APPLICATION STRAIN MATERIAL (a) PRC)CESSED CONDENSATE OF 2R1 APPLICATION VOLI%IE (b) CDNC. (g/1) DOSE (mg/(mouse x appl.)) (mg/(imuse x week)) (ul/(mouse x appl.)) 0.1-GR CD1 acetone - - - 100! 0.2-GR CD1 uMBA plus acetone - - - 100 1-GR CD1 MWSC-I 120 12 60 100 2-GR CD1 MWSC-I 180 18 90 100 3-GR CD1 MWSC-I 240 24 120 100 4-GR CD1 MWSC-I (c) 120 12 60 100 5-GR CD1 MWSC-I (c) 180 18 90 100 6-GR CD1 MWSC-I (c) 240 24 120 100 7-GR CD1 DMBA plus MWSC-I 120 12 60 100 8-GR CD1 DMBA plus MWSC-I 180 18 90 100 9-GR CD1 DMBA plus MWSC-I 240 24 120 100 10-CR CD1 SWSC-I 120 12 60 100 11-GR CD1 SFASC-I 180 18 90 100 12-GR CD1 SWSC-I 240 24 120 100 AT TABLE B (RO[)PS AND DOSES Remarks: application cycle: 5 times per week, number of mice per group: 105 (a) DMBA dose: 200 nno1/mouse equiv. 51 ug/mpuse, single application NWSC-I mainstream'whole snake condensate, collection with impaction trap SWSC-I sidestream whole smoke condensate, collection with impaction trap (b) application solution: processed condensate plus acetone or acetone alone (c) stored at 4 degrees centigraae. Solutions have roem temperature when applied. Condensate of all other groups stored at -75 degrees centigrade..

SUBREPORT P 0500/3068 UBE144RB10 7289 AT PAGE 4-14 GROUP NIOUSE APPLICATION PROCESSED CONDENSATE OF 2R1 APPLICATION STRAIN MATERIAL (a) VOLUME (b) C(W. (g/1) DOSE ( c ) (mg/(mouse x appl.)) (ng/(mouse x week)) (ul/(mouse x appl.)) 13-GR CD1 DMBA plus SWSC-I 120 12 60 100 14-M CD1 DMBA plus SWSC-I 180 18 90 100 15-GR CD1 DNIBA plus SWSC-I 240 24 120 100 0.3-GR B6C3F1 acetone - - - 70 0.4-GR B6C3F1 DMBA plus acetone - - - 70 16-M B6C3F1 NWSC-I 120 8.40 42 70 17-GR B6C3F1 WSC-I 180 12.6 63 70 18-M B6C3F1 NWSC-I 240 16.8 84 70 19--GR B6C3F1 DMBA plus Mn1SC-I 120 8.40 42 70 20--GR B6C3F1 DMBA plus MWSC-I 180 12.6 63 70 21-M 66C3F1 DMBA plus KVX-I 240 16.8 84 70 AT TABLE B (continued) G1ifJilPS AND DOSES Remarks: application cycle: 5 times per week, number of mice per group: 105 (a) DMBA dose: 200 nmol/mouse equiv. 51 ug/mouse, single application NWSC-I mainstream whole smoke condensate, collection with impaction trap SWSC-I sidestream whole smoke condensate, collection with impaction trap (b) application solution: processed condensate plus acetone or acetone alone (c) reduced dosage for B6C3F1 mice due to reduced body weight as oanpared to (D1 mice

SUBkEPURT Y U500/3U6b UksE144kksll 7257 AT PAGE 4-15 4.3.5 Shaving Principle: Time: mechanical removal of fur on the back, around the sacrum on both sides of the spinal column, size of sheared area approx. 30 mm x 30 mm 1st shaving: 3 days prior to DMBA application 2nd shaving: 3 days prior to start of condensate and acetone application thereatter: 1 time weekly, 1 day before control for macroscopic skin lesions, before daily application Sample materiau and time: all mice (see TABLE C) Equipment: Procedure: (1) clipper for small animals: Favorita, no. GV 24, cutterhead: 1/20 mm, no. GH-700, Aesculap, D-7200 Tuttlingen (2) lightweight hair clipper (cord'less), Art. Nr.: 02600, cutterhead: 1/20 mm, A. Popp, D-7532'Niefern careful shaving to avoid skin lesions, blade of electric clipper changed reg u- larly to avoid overheating, hair remo- ving by hood ventilation Scientific version: SOP AT 86/5 Text version: 14.Sep.87 ,~8~

SUBREPORT P 0500/3068 LJBE144RB12 7288 AT PAGE 4-16 GRJiJP DAY OF WEEK NCkIDAY 7:L]ESDAY WEDNESDAY THURSDAY FRIDAY 4-GR to 9-GR 10-GR to 15-GR 16-GR to 21-GR - 0.1-GR to 0.4-GR 1 -GR to 3-GR AT TABLE C SHAVING OF MICE 1587

SUBREPORT P 0500/3068 UBE144RB13 6311 AT'PAGE 4-17 4.3.6 Application 4.3.6.1 DMBA solution Principle: application materials applied with automatic dosing apparatus onto sheared area Time of application: 10.Dec.82 Sample material and quantity Material: acetone plus DMBA control groups and treatment groups: solvent (acetone) with DMBA (see INTEGRATING REPORT) Dose of application material: Concentration of application material: Application volume: Equipment: 200 nmol/mouse equiv. 51 ug/mouse (single dose). 2 mmol DMBA/l acetone equiv. 0.51 g/l 100 ul/mouse dispenser Microlab P, no. 104000, with gastight syringe, 5 ml, no. 1005-PTLL, reproducibility: deviation .LT.0.2 0/0, accuracy: deviation .LT.1 0/0, Hamilton, D-6100 Darmstadt 1 Heidolph shaking apparatus, no. 302 DSG, via Faust GmbH, D-5000 Ko1n 90 laboratory hood, H'. Waldner GmbH und Co., D-7988 Wangen face mask with filter, 88 A/St, Auergesellschaft GmbH, D-1000 Berlin 65

SUBREPORT P 0500/30Ei8 UBE144RB14 7288 AT PAGE 4-18 Chemicals: Procedure: acetone (a), no. 14, E. Merck, D-6100 Darmstadt 1 DMBA, registration serial no. 57976 (see INTEGRATING REPORT): complete suspension of stored'DMBA application solutiomwith automatic shaking apparatus for 30 s prior to application during application special attention given to a uniform distribution of applied' material on the sheared area application starts when application materials are at room temperature Scientific version: SOP AT 131/1 Text version: 19.Nov.85 (a) diluted with water (final concentration 25 ml water/l acetone) 3587

SUBREPORT'P 0500/3068 UBE,144RB15 7288 AT PAGE 4-19 4.3.6.2 Condensate Principle: application materials applied with automatic dosing apparatus onto sheared area Time 1st application: 20.Dec.82 Last application: 29.Jun.84 Application cycle: 5 times/week Application period' (weeks): 80 Total number of applications: 400 Sample material and quantity Material: acetone control groups: solvent (acetone) treatment groups: application solutions (see AT TABLE B') 4-GR to 6-GR condensate application solutions used not earlier than 14 d, but not later than 60 d after preparation, stored at 4 degrees centigrade 1-GR to 3-GR and 7-GR to 21-GR condensate applications solutions used without limitations, but practically not earlier than 14 d, stored at -75 degrees centigrade Application volume Strain CD1: 100 ul/mouse Strain B6C3F1: 70 ul/mouse Rm~u

SUBREPORT P 0500/3068 UBE144RB16 7288 AT PAGE 4-20 Equipment: Chemicals: CD1 mice: from the 1st to the 9th application week increeae of application volume from 20 ul/(mouse x day) to 100 ul/(mouse x day) in all groups, weekly increase: 10 ul/(mouse x day), thereafter constant applica- tion volume applied B6C3F1 mice: from the 1st to the 9th application week increase of application volume from 1:5 ul/(mouse x day) to 70 ul/(mouse x day) in all groups. Approx. 30 percent of the application volume of the CD1 mice was applied. Thereafter constant application volume applied dispenser Microlab P, no. 104000, with gastight syringe, 5 ml, no. 1005-PTLL, reproducibility: d'eviation .L'i•.0.2 0/0, accuracy: deviation .LT.1 0/0, Hamilton, D-6100 Darmstadt 1: Heidolph shaking apparatus, no. 302 DSG, via Faust GmbH, D-5000 Koln 90 laboratory hood, H. Waldner GmbH und Co., D-7988 Wangen acetone (a), no. 14, E. Merck, D-6100 Darmstadt 1 (a) ) diluted with water (final concentration: 25 ml water/1 acetone) 3587

SUBREPORT P 0500/3068 Procedure: UBE144RB17 7226 AT PAGE 4-21 complete suspension of stored conden- sate application solution shaken with an automatic shaking apparatus for 30 s prior to application during application special attention given to a uniform distribution of applied material on the sheared area order of application changed from day to day to avoid any effect of staff training, beginning with cages 1, 2, 3, 4 etc. on dates with even numbers and with cages 15, 14, 13, 12 etc. on dates with odd numbers and so on application starts when application materials are at room temperature continued treatment of mice, which developed skin lesions during investigations Scientific version: Text version: SOP AT 105/1 14.Aug.87 4.3.7 Generall conditiont_behav_ior_and_mortality Time: checks 2 times per working day (after begin and before end), additional checking during handling (shearing, application, weighing etc.) on weekends U time daily Procedure: Scientific version: Text version: quantitative observation of morta- lity and qualitative observation of all mice for atypical behavior or excretions, classification made with the help of: CHECKLIST "SIGNS OF INTOXICATION, MOUSE" (see TABLE D) SOP AT 64/4 19.Nov.85 t7AF,

SUBREPORT P 0500/3068 UBE144RB18 63'11 AT' PAGE 4-22 PARAMETER INVESTIGATED DESCRIPTION OF OBSERVATION spontaneous activity increased, decreased; sleep irritability increased, decreased, nonexistent (reaction to sound and touch) posture and position of the body, neuromuscular symptoms long legged motion, humped posture, ataxia, prone, lateral and supine position, running motions whilst in lateral or supine position fascicular contractions, trembling of the entire body, tremor, clonic convulsion, tonic convulsion, clonicotonic convulsion, convulsive jumping, opisthotonos, Straub's phenomenon, compulsive biting, compulsive gnawing eyes palpebral fissure partially or completely closed or wide open, exophthalmia, opacity of the cornea condition of the fur bristled, rough, moist, discolored, loss of hair discharges (a) saliva: increased (salivation), foamy, amount urine: color, blood admixtures feces: consistency (formed, nonformed), admixtures (blood, mucus, parasites, test substance) amount, color excretions (a) liquid: from the eyes, from the audi- tory meatus, from the nostrils blood: from the eyes, from the audi- tory meatus, from the nostrils AT TABLE D CHECKLIST "SIGNS OF INTOXICATION, MOUSE" (a) In case of red or reddish discharge or excretions tests for erythrocytes and hemoglobin are carried out with "Sangur test" strips (Boehringer GmbH, Mannheim). If blood is suspected in the intestines microscopic slide checks for erythrocytes are carried out.

SUBREPORT P 0500/3068 UBE144RB19 6311 AT PAGE 4-23 PARAMETER INVESTIGATED DESCRIPTION OF OBSERVATION respiration dyspnea: frequency decreased (counting), gasping, rattling, irregular, jerky, labored color of the skin reddened, pale, reddish-blue (cyanotic)) muscle tonus increased, decreased (tonus of body and abdomen) reaction to pain increased, nonexistent reflexes corneal reflex: nonexistent auricular reflex: nonexistent roll-over reflex: diminished, non- existent body temperature (a) increased, decreased, degrees centigrade skin irritation translucent skin, erythema and eschar formation, edema formation mucous membranes of mouth redness, swelling, corrosion and anus further observations miscellaneous, e. g. aggregation pheno- menon, writhing, murmuring, contrac- tion of abdominal musculature, stereotype behavior (rocking of the head, circular motions of the head), rotating motion, backward motion, beating of the tail, licking of the snout, cannibalism AT TABLE D~ (continued) CHECKLIST "SIGNS OF INTOXICATION, MOUSE" Scientific version: SOP AT 60/7' Text version: 19.Nov.85 (a) In case of subjective impression of a rise or fall in (skin) temperature gained by the investigator from handling the animal rectal measurement with a resistance-thermometer with adequate probe attached (type H11, HSE, D-7801 Hug,stetten) is performed. , ,,I"

INBIFO Institut fur biologische Forschung • NColn SUBREPORT P 0500/3068' UBE14RB20 6311 AT PAGE 4-24 4.3.8 MacroscoPic-examination-of-skin -------- ----------- -- ~ 4.3.8.1 Skin lesions (tumors and ulcers) Principle: visual examination for macroscopic skin tumors and ulcers (see AT TABLE E) Time: (1) number of mice withitumors and (2) ulcers: number weekly of tumors and ulcers per mouse: in intervals of 10 weeks after the start of WSC application until the end of the study (3) all other observations: at their occurence (e. g. shearing or biting wounds). Sample material and quantity: total skin Results recorded in,: (1) number of mice with tumors and (2) ulcers number of tumors and' ulcers per mouse (3) in an index card separate recording: (1) inside the application area (2) outside the application area (3) inside and outside the application area Equipment:: data storage interface, INBIFO, D-5000 Koln 90 teletype: ASR 33, Vollwood, D-4000 D'usseld'orf teleprint: Basic ASR 43, Teletype Corporation, Skokie, Illinois 60077, USA 3285

INBIFO Institut fur laiologische Forschuing • K6In SUBREPORT P 0500/3068 UBE1i44RB2'1 7288 AT'PAGE 4-25 ~ GROUP DAY OF WEEK MCRNDAY ZtJESDAY WEDNESDAY ZHURSDAY FRIDAY 01.1-GR to 0.4-GR 4-GR to 9-GR 10-GR to 15-GR 16-GR to 21-GR - 1-GR to 3-GR AT TABLE E EXAMINATION'OF SKIN LESIONS (Z'UMOPS AND ULCERS). 3587

INBIFO Institut fur biologische Forschung • M1n SUBREPORT P 0500/3068 UBE144RB22 7226 AT PAGE 4-26 Proced!ure : all lesions of diameter .LT.2 mm excluded from counting recording of time of "1st observa- tion", when size of tumor or ulcer .GE.2 mm recording of all lesions others than tumors and ulcers (e. g. shearing: or biting wounds) in separate file skimevaluation performed: without relation to previous observations (1) counting inside or outside the application area, recording on paper tape with the aid of an input device. Keyboard input: cage no., number of mice per cage, individual number of mice, tumor (yes/no) and ulcer (yes/no). Recording of the appearance of the 1st tumor data on the tapes processed by team computer operations on the same day. Print-outs provided~ indicating the mice with changes occuring during the previous 3 checks. Based on these print-outs mice with~changes checked again by 2 trained technicians and the group leader independently. correcting of discrepancies, print-outs signed and dated, the actual and cumulated values printed out 1 time a week for each group (2) Iisting,on appropriate protocol forms with graphic descriptions of position and size of the finding, confluent tumors or ulcers assessed as single en- tities as long as they are clearly definable as a single entity, a new protocol form started for each period of inspection N Q N ~ 0 ~ N CA 3285

INBIFO Institut fur bioPogische Forschung • KtSIn SUBREPORT P 0500/3068 UBE144RB23 7226 AT PAGE 4-27 (3) listing in a,separate index card each mouse for which an index card has been made also ticked off in a list with mouse no. Scientific version: SOP AT 89/5 and SOP AT 89/6 Text version: 14.Aug.87 4.3.8.2 Skin irritations Principle: visual examination for macroscopic skin irritations Time: daily before application until 12 weeks after 1st condensate or ace- tone application Sample material and quantity: all mice individually, skin inside the application area Results expressed in: number of mice with skin irritations Equipment: Procedure: teletype: ASR 33, Vollwood, D-4000 D'usseldorf teleprint: Basic ASR 43, Teletype Corporation, Skokie, Illinois 60077, USA counting of sheared mice with trans- lucent and'/or reddened skin lesions obviously related to shearing, biiting,or scratching not recorded counting inside the application area, recording only after the appearance of 1st lesion 3285

INBIFO Institut fur biologische Forschung • Kt51n SUBREPORT P 0500/3068 UBE144RB24 7288 AT'PAGE 4-28' Scoring system: , appearance of translucent and/or red- dened skin COMPUTER TEXT CODE no change ch ang e Scientific version: Text Version: SOP'AT 88/4 19.Nov.8'5 4.3.9 Determination of body weight ---------------------------- Principle: Time: Material and quantity: Results record'ed in: Equipment: automatic gravimetric determination with time integration~to correct for motion of mice (1) ) 2 days after arrival (2) before DMBA application (3) before 1st condensate application (4) after 1st condensate application: every 2 weeks (5) after 20th week of application: every 4 weeks all mice cagewise (see TABLE F) 9 digital animal scales, Sartorius GmbH, D-3400 Gottingen teleprint: Basic ASR 43, Teletype Corporation, Skokie, Illinois 60077, USA data storage interface, INBIFO, D-5000 Koln 90 3587

INBIFO Institut fur biologische Forschung • K6In SUBREPORT P 0500/3068 UBE144RB25 6311 AT PAGE 4-29 Procedure: microprocessor-controlled data sto- rage interface connected to scales data with RSD .GT.20 0/0 checked for correctness by 2nd measurement Scientific version: SOP AT 36/2 Text version: 19.Nov.85 3285

INBIFO Institut fur biologische Forschung • K6In SUBREPORT'P 0500/3068 UBE144RB26 6311 AT PAGE 4-30 BODY WEIGHT DETEf44INATIOAi WEEK OF APPLICATION DAY OF THE WEEK G2OUP 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, Nbrx3ay 4-GR to 9-GR 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80 Tuesday 10-GR to 15-GR Wednesday Thursday 16-GR to 21-GR - Friday 1-GR to 3-GR AT TABLE F BODY WEIGHT DETERMINATION, CAGEWISE

INBI'FO Institut fur biologische Forschung • K6In SUBREPORT P 0500/3068 UBE144RB27 7289 AT PAGE 5-1 5 EVALUATION 5.1 General Condition, Behavior and Mortality General condition and behavior: verbal description Mortality: groupwise, absolute and relative to initial number of mice 5.2 Body Weight Body weight: groupwise 5.3 Macroscopic Examination of Skin 5..3..1 Tumor rate based on-macroscopic_apgearance_i0Z01 Tumor rate: calculation according to Blanding et al. (1951) tumor rate (0/0) _ NT effective number x 100 NT = number of mice (alive and dead) which have or had at least 1 skin tumor inside the applica- tion area effective number = (number of sur- viving mice) + (number of dead mice with skin tumors) 3285

INBIFO Institut fur biblogische Forschung • Kdln SUBREPORT P,0500/3068 UBE144RB28 5302 AT'PA6E 5-2 5.3.2 Skin tumors Skin tumors per animal and mean diameter of skin tumors: groupwise 5.3.3 Skin irritations Skin irritations:: groupwise 3285

INBIFO Institut fur biologilsche Forschung • K6In SUBREPORT P 0500/3068 UBE'145RA1 7299 AT PAGE 6-1 6 RESULTS AND DISCUSSION 6.1 Text 6.1.1 General condition and behavior ------------------------------ CD1 mice: No signs of intoxication were found in the control groups during the whole study. In WSC-I treated groups signs of intoxication were observed depending on the individual dose groups approx. 5 weeks after start of application period (a). They occurred imme- diately after application and were observed up to 5 hours. Except the SWSC-I dose groups from approx. the 25th application week up to the end of the study no remarkable signs of acute intoxication were observed. The main signs of' intoxication were: spontaneous activity increased immediately after application and later de- creased, prone position, dyspnea, decreased body temperature (minimum: approx. 25 degrees centigrad'e)' and either a partial or complete closing of the palpebral fissure. The signs of intoxi- cation increased with ascending doses. Im the SWSC-I groups a few number of mice with signs of intoxication was practically seen during the whole application period'.. Dose groups with strong signs of intoxication showed high mortality, too. B6C3F1 mice: The mice were generally in very good' condition. Signs of acute intoxication were not observed. In application week 63' in approx. 85 percent of the B6C3F1 mice pretreated with DM,BA (0.4-GR and 19-GR to 21-GR) several isolated blackish spots with a diameter (a) CD1 mice: from the 1st to the 9th application week increase of application volume from 20 ul/(mouse x day) to 100 ul/ (mouse x day) in all groups. Weekly increase: 10 ul/(mouse x day). Thereafter constant application volume applied B6C3F1 mice: from the 1st to the 9th application week in- crease of application volume from 15 ul/(mouse x day) to 7& ul/(mouse x day) in all groups. Approx. 30 percent of the application volume of CD1 mice was applied. Thereafter con- stant application volume applied' 3285

INBIFO Institut fur biologischle Forschung • KSI'n SUBREPORT P 0500/3068' UBE145RA2 7288 AT PAGE 6-2 up to 3 millimeters were observed in the application area region. Microscopic examination of the skin of 1 mouse (mouse no. 605 fron, 0.4-GR), which died "spontaneously" and showed the above mentioned changes, revealed an increased number of melanin containing cells in the dermis. This finding has been observed in pigmented Syrian hamsters and Mongolian gerbils following DMBA skin painting (Bock, 1983). The comparison of the general condition and behavior of CD1 mice treated with MWSC-I and those with SWSC-I showed differences in the proportion of signs of intoxication. The number of mice with signs of intoxication was higher in SWSC-I than in MWSC-I-treated groups. No differences were seen in the general condition and behavior of both mouse strains which were pretreated'with DMBA. Signs of intoxication were observed in CDI but not in B6C3F1 mice. The comparison of the general condition and behavior of CD1 mice did not result in differences depending on whether MWSC-I was stored before application at 4 or at minus 75 degrees centigrade. 6.1.2 Mortality (see AT TABLES 1 to 4, 1 7 and 26 and AT FIGURES 1 and 2) Of the 2625 female mice used, 787 (30 percent) died "spontane- ously" during the 80 weeks up to the time of final dissection. Considering the two mouse strains separately 743 CD1 mice of an initial number of 1785 (42 percent) and 44 B6C3F1 mice of 840 (5.2 percent) died. 3587

INBIFO Institut fOr biol'ogische Forschung • KtSIn SUBREPORT P 0500/3068 UBE145RA3 7288 AT PAGE 6-3 CD1 mice: • 1st deaths were recorded depending on the individual groups from: application week 1 (3-GR, MWSC-I, high dose and 11-GR, SWSC-I, medium dose) to week 38 (9-GR, DMBA plus MWSC-I). The mortality rate of the acetone control group was 33 percent. This value is in the range of comparable INBIFO studies (a). The mortality rate of the DMBA plus acetone control group was 19 percent. In WSC-I treated groups the lowest mortality with~ 24 percent was found in the 5-GR (MWBC-I, medium dose) (b) and the highest mortality with 86 percent in the 15-GR (DMBA plus SWSC-I). There was no dose de- pendency in the MWSC-I groups. A slightly dose dependent increase in the percentage of mortality was observed in the DMBA plus MWSC-I groups. Dose dependency was obtained in the SWSC-I and in the DMBA plus SWSC-I groups. B6C3F1 mice: 1st deaths were recorded' depending on the individual groups from application week 8 (18-GR, MWSC-I, high dose)~ to week 68 (17-GR, MWSC-I, medium dose). The mortality rate of the acetone and the DMBA plus acetone control groups was 7 and 5 percent respectively. In WSC-I-treated groups the lowest mortality with 2 percent was found in the 17-GR (MWSC-I, medium d'ose) and the highest mortality with 10 percent in the 20-GR (DMBA plus MWSC-I). There was no dose dependency in all groups. The comparison of the mortality of MWSC-I and SWSC-I-treated CD1 mice showed' a statistically significantly higher mortality in the SWSC-I-treated groups and is considered to be biologically rele- vant . In CD1 mice the pretreatment with an initiating dose of DMBA resulted in a statistically significantly higher mortality in the SWSC-I dose groups. This statistical significance is considered (a) P 0500/3000', 1982, P 0500/3010, 1983, P 0500/3023, 198'2, P 0500/3030, 1984, PART 2, and~P 0500/3073 (b) condensate stored at 4 degrees centigrade 3587

INBIFO Institut fur biologische Forschung • 14tSIn SUBREPORT P 0500/3068 UBE145RA4 7288 AT PAGE 6-4 to be biological relevant. In B6C3F1 mice a slightly higher mor- tality was seen in the DMBA pretreated groups which was statisti- cally significant in the medium- and hiigh-dose group. This statis- tical significance is considered to have no biological relevance. The comparison of the mortality of the MWSC-I-treated CD1 and' B6C3F1 mice showed a statistically significantly higher mortality for CD1 mice. This is considered to be biological relevant. The comparison of the mortality for CD1 mice showed no difference depending on whether MWSC-I was stored before application at 4 or at -75 degrees centigrade. 6.1.3 BodY_weiqht (see AT TABLES 5 to 1 7 and 26 and AT FIGURES 3 and 4) The body weight development was within the expected range and showed no dose dependency. Beginning imthe 1st third of the study the mean body weight of both mouse strains of all condensate treated groups was slightly lower (in application week 80 maximum 13 percent in CD1 mice and 9 percent in B6C3F1 mice) than the body weight of the corresponding control groups. This is considered to be biologically relevant, indicating a slightly toxic effect. The highest body weight was observed in the DMBA plus acetone control groups as well in the CD1 mice as im the B6C3F1 mice. At the beginning of the application period the mean body weight of all CD1 mice was 22.1' grams and of all B6C3F1 mice 16.6 grams. At the end of the application period the mean body weight of the acetone control groups was 36.7 grams for the CD1 mice and 31.0 grams for the B6C3F1' mice. The initially observed reduced body weight of B6C3F1 relative to CD1 mice was also seen at the end of the study, which justifies the reduced WSC-I doses administered to the former strain. The body weight of the CD1 mice in the nega- 3285

INBIFO Institut fur biologische Forschlung • KOIn SUBREPORT P 0500/3068 UBE145RA5 7299 AT PAGE 6-5 tive control group was at the end of the study slightly lower than in other comparable INBIFO studies (a) with exception in INBIFO study P 0500/3073. The comparison of the body weights of CD1 mice with respect to MWSC-I and SWSC-I showed no differences. The pretreatment with an initiating dose of DMBA did not result in a difference in the body weight of both mouse strains. The body weight of the CD1 mice is strain-dependently higher than that of the B6C3F1 mice. The relative body weight reduction in the MWSC-I-treated groups was the same in both mouse strains (-7 per- cent). The comparison of body weight in CD11 mice showed no difference, depending on whether MWSC-I was stored before application at 4 or at -75 degrees centigrade. 6.1.4 Develocment_of macroscopic_skin-tumors ------ - -------- ------ ( see AT TABLES 1 7 to 21, 27 to 41 and AT F IGURES 5 and 6). CD1 mice: 1st macroscopic skin tumors were recorded: depending on the indi- vidual groups from application week 13 (9-GR, DMBA plus MWSC-I, high dose), 10-GR and 11-GR (SWSC-I, low and medium dose):, and 13-GR to 15-GR (DMBA plus SWSC-I, low, medium and high dose) to week 54 (4-GR, MWSC-I, low dose). The time to reach a MTR of 10 percent showed strong dose dependency after SWSC-I treatment. No macroscopic skin tumor was found in the application area of the mice of the acetone control group. The 6 percent macroscopic tumor rate in the DMBA pretreated acetone control group indicates that the DMBA dose of 200 nanomols for the single application may have been too high to represent initiator activity only. In WSC-I- (a) P 0500/3'000, 1982, P 0500/3010, 1983, P 0500/3023, 1982, P 0500/3030, 1984, Part 2 3285

IN'BIFO Institut fur biologische Forschung • KOIn SUBREPORT P 0500/3068 UiBE145RA6 7288 AT PAGE 6-6 treated groups the lowest macroscopic tumor rate'was obtained'.with 12' percent in the 5-GR (MWSC-I, medium, dose) (a:) and the highest MTR with 99 percent in the 15-GR (DMBA plus SWSC-I, high d'ose). There was no dose dependency in the MWSC-I groups. The MTR in- crease was dose dependent in the DMBA plus MWSC-I, the SWSC-I and in the DMBA plus S4uSC-I dose groups. The MTR of 12 to 34 percent which were obtained in the 3 d'ose groups with CD1 mice and 2R1 MWSC-I without DMBA pretreatment is in general agreement with the MTR in the INBIFO study P 0500/3030 (1982'). B6C3F1 mice: 1st macroscopic skin tumors were recorded depending on the indi- vidual groups from application week 18 (21-GR, DMBA plus MWSC-I, high dose)~ to week 65 (18-GR, MWSC-I, high dose). The time to reach a MTR of 10 percent showed strong dose dependency after DMBA plus MWSC-I treatment. No macroscopic skimtumor was found in the application area of the mice of the acetone control group. The DMBA plus acetone control group showed an unexpected and unex- plainably high MTR of 31 percent. In WSC-I treated groups the lowest MTR was obtained with 5 percent in the 16-GR (MWSC-I, low dose) and the highest MTR with 67 per- cent in the 21-GR (DMBA plus MWSC-I, high~dose). The MTR increase was dose dependent in the MWSC-I and in the DMBA plus MWSC-I groups.. The number of mice with macroscopic skin tumors of comparable WSC-I treated groups is slightly higher (mean = 14 percent) than the number of mice with histologically confirmed tumors. The detailed evaluation of the tumor incidence, multiplicity and malignancy is given:in SUBRE,PORT PY. (a) condensate stored at 4' degrees centigrade 3587

INBIIFO Institut fur biologische Forschung • Wn SUBREPORT' P 0500/3068 UBE145RA7 7299 AT PAGE 6-7 6.1.5 Skin-irritations_(macroscopic_diagnosis), --- ----------- (see AT TABLES 22 to 26 and AT FIGURES 7 to 10) Skin inspection and scoring of skin irritations were hindered by the brown color of condensate on the skin and additionally by the brown pigmentation of skin in B6C3F1 mice. CD1 mice: The 1st skin irritations (translucent and or reddened skin) were detected approx. 2 weeks after start of the application. The num- ber of mice with skin irritations were maximal during, the 4th and 5th week of application after approx. 15 to 25 applications. Approx. 10 weeks after the beginning of the applications, skin irritations were no longer observed'.. In the high WSC-I treated groups mice with skin irritations occurred earlier. In the control groups no skin irritations were found. Except for the 7-GR (DMBA plus MWSC-I, low dose) in all WSC-I treated dose groups there were mice with skin irritations. The lowest maximal number of mice with skin irritations per week was obtained with 6 percent in the 4-GR and 8-GR (MWSC-I, low dose and DMBA plus MWSC-I medium dose) and the highest with 68 percent in the 12-GR (SWSC-I, high dose). The number of mice with skin, irritations was dose dependently in- creased in all dose groups. B6C3F1 mice: Only a few mice with skin irritations with a maximum of 2 percent in the 18-GR (MWSC-I, hig,h dose) were observed in control and MWSC-I treated groups. The comparison of skin irritations in MWSC-I- and SWSC-I-treated CD1 mice showed a statistically significantly higher number of mice with skin irritations in the SWSC-I-treated groups. This is considered to be biologically relevant. 3285 '

INBIFO Institut fur biologische Forschung • K6In SUBREPORT P-0500/3068 UBE145RA8 7299 AT PAGE 6-8 In CD1 mice the pretreatment with an initiatin~ dose of DMBA re- sulted in a statistically significantly lower number of mice with skin irritations both in the MWSC-I- and in the SWSC-I-treated groups. This effect is considered to be biologically relevant. The reduced number of mice with skin irritations in the DMBA pre- treated WSC-I groups is unexplainable. In B6C3F1 mice no differ- ence was seen between the MWSC-I-treated groups without and with DMBA pretreatment. The comparison of skin irritations between the MWSC-I-treated CD1 and B6C3F1 mice showed a statistically significantly higher number of mice with skin irritations in the CDU mouse strain. This, how- ever, is not considered to be biologically relevant, because the skin inspection and scoring of skin irritations were hindered by the brown pigmentation of the skin in B6C3F'1 mice. The comparison of the number of mice with skin irritations in CD1 mice showed no difference depending on whether MWSC-I was stored before application at 4 or at -75 degrees centigrade. 6.1.6 Comparison of mortality between CD1 mice without and with macroscogic_skin_tumors_in_application area (see AT TABLES 27 to 41 and AT FIGURE 11) The percentage of dead mice without macroscopic skin tumors was compared to the percentage of dead mice with macroscopic skin tumors weekly from begin up to the end of the study. The percentage of dead mice with skin tumors in the individual groups was practically the same or lower than those without skinn tumors with exception of 2 groups (mediumi and high MWSC-I dose groups with DMBA pretreatment, 8-GR and 9-GR). Also the overall mortality of mice with macroscopic skin tumors did not increase, i. e. the mortality did'not increase in relation to the appearance of skin tumors. 3285

INBIFO Institut fur biologische Forsctlung • KOIn SUBREPORT P 050'0/3068 UBE145RA9 7288 AT PAGE 6-9 6.1.7 Prediction of the microscopic tumor probability in future studies --------------------------------------------------------- (see AT TABLES 42 to 46 and AT FIGURES 12 to 21), For the prediction of the microscopic tumor probability, at an early stage of the experiment, the microscopic tumor probability of application week 80 was compared with the macroscopic skin irritation, the week of the tumor onset, the week when 5 percent tumor probability was reached, and the macroscopic tumor probabi- lity of application weeks 30 and 40 of this study in CD1 and B6C3F1 mice. In CD1 mice the comparison between the microscopic tumor probabi- lity and the macroscopic parameters (except skin irritation which showed' only a weak correlation) showed correlation coefficients from 0.812 to 0.933. These correlation coefficients are statisti- cally significant with p = .LE'.0.001 and therefore allow a pre- diction of the final tumor probability. This prediction is accom- panied by a standard' deviation from + 12 to + 19 percent for the the parameter microscopic tumor probability for week 80. In addition the parameter skin irritation can be used for the predic- tion, although the correlation is not so strict (correlation coefficient: 0.716 and 0.677, statistical significance: p = .LE.0.01 and .LE'.0.05), but the values are already available in application week 5. In B6C3F1 mice the comparison between the microscopic tumor pro- bability and the macroscopic parameters (except the tumor onset which showed only a weak correlation and skin irritation (a)) (a) In B6C3F1 mice no skin irritations were seen, which is prob- ably due to the brown pigmentation of skin. 3587

IINBIFO Institut fur biologische Forschung • K6I'n SUBREPORT P 0500/3068 UBE145RA10 7288 AT PAGE 6-10 ~ showed correlation coefficients from 0.859 to 0.911. This corre- lation coefficients are statistically significant between p = .LE.0.01 and .LE.0..001 and also allow a prediction of the final tumor probability. This prediction is accompanied by a standard deviation from + 9 to + 11 percent for the parameter microscopic tumor probability for week 80. In addition the parameter tumor onset can be used for the prediction, although the correlation is not so strict (correlation coefficient: 0.768', statistical signi- ficance: p = .LE.0.015). In B6C3F1 mice no skin irritations were seen, probably due to the brown pigmentation of skin, therefore the tumor onset is the earliest macroscopic available parameter for the prediction. Besides the above mentioned macroscopic parameters the histopa- thological finding "hyperkeratosis", (investigated in INBIFO study P 0500/3117, 1986) allow a prediction of the final tumor probability in application week 5 for the strain CD1 as well as B6C3F1 pretreated with DMBA. 3587

SUBREPORT P 0500/3068 UBE145RB1, B2 5302 M 6.2 Tables and Figures WEEK G[iOUP AID TR$A'IMQ7T 0.1-GR 0.2-{;Et 1--GR 2-GR 3-GR 4-GR 5-M 6-(;c't ACBTONE OMBd PEb9 !lWSC-I (ag/(wouae x week)) lWSC`I (a) (m3/(mouse x week)) (60) (90) (120) (60) (90) (120)' 0 0 0 0 0 1 0 0 0 2 0 0 0 0 1 0 0 0 4 0 0 0 0 1 0 0 2 6 0 0 0 0 2 0 1 3 8 0 0 0 0 2 0 1 3 10 0 0 0 0 2 0 1 3 12 0 0 0 0 2 0 1 3 14 0 0 1 0 2 0 1 3 16 0 0 1' 0 2 0 1 3 18 0 0 1 0 2 0 1 3 20 1 1 1 0 2 0 1 3 22 1: 1 1 0 2 0 1 3 24 2 1 1 0 2' 0 1 3 26 2 1 1 1 2 0 2 3 28 3 1 3 2 2' 0 2 3 30 4 1 3 3 2 2 3 3 32 4 1 4 4 2 3 4 3 34 5 3 5 4 3 4 4 3 36 5 4 5 5 3 4 4 3 38 6 4 5 5 3 4 4 3 40 7 6 5 5 4 5 5 3' 42 9' 7 5 6 5 5 6 3' 44 9 7 6 6 5 6 7 4 46 9 7 6 6 6 6 7 4 48 9 8 7 6 7 6 7 4 50 11 8 7' 6 7 7 9 4 52 11 8 8 6 7 7 10 6 54 11 9 10 7 7 7 10 7 56 11 10 12 7 7 7 10 8 58 13 10 13 7 8 8 10 10 60 16 10 13 7 9 8 12 13 62 17 11 13 9 10 9 12 14 64 19 11 13 10 11 12 13' 17 66 20 13 1'5 13 12 13 15 20 68 24 14 17 13 16 15 16 20 70 26 14 1s 14 19 19 16 26 72 27 14 19 19 20 21 17 26 74 27 15 20 23 21 26 17 26 76 28' 15 24 24 23 29 19 28 78 32 17 28 25 29 33 20 33 80 33 19 29 29 30 37 24 34 AT TABLE 1. 1 llOElTAGITY (0/0), CD1 MICE (a) stored at 4 degrees centigrade, oocdensate of all other groups stored at -75 degrees centigrade 3587

.1 .~~uj1' ._, .. ..,..•,.....~ i~au~. .: ~,,.i... J. _ .~- . Vi:... • REPORT P 0500/3068 UBE145RB3B3, B4 5302' M WEEIC GBOUP AbD, TRFA7MElJJ` 7-GR 8-m 9-Qt 10-M 11-Gt 12-GR 13-C,It 14-Qt 15-GR a48A PIM ls+ISC-I (mg,/(arorse x week) ) sWSC-I (ng/(,rouse x week))~ p4ep, Plals SW.SC-I (mgl(mouse x week) ) (60) (90)' (120) (60) (90) (120) (60) (90) (120) 0 0 0 0' 0 1 0 0 0 0 2 0 0 0 0 1 0 0 0 0 4 0 0 0 0 2 1 0 0 0 6 0 1 01 0 2 4 0 0 1' 8 0 1 01 0 4 9 0 1 1' 10 0 1 0 0 5 10~ 0 1 2 12 0' 2 0 0 5 10 0 1 2 14 0 2 0 0 5 10 0 1 2 16 01 2 0 0 5 10 0 1 2 18 0' 2 0 0 5 10 0 1 2 20 0~ 3 0 1 7: 11 0 1 2 22 1 4 0 1 7 11 2 1 2 24 1 4 0 1 7 11 3' 2 2 26 2 4 0 1 8 11 4 3 3 28 2 4 0 1 8 11 5 3 3 30 2 4 0 1 8 11 7 3 3 32 3 4 0 3 8 11 7 4 4 34 3 4 0 4 8 11 8 4 5 36 3 5 0~ 4 8 13 8 5 6 38 3 5 1 6 8 14 8 6 8 40 3 5 2 7 8 14 9 7 8 42 3 5 4 8 8 14 9 7 10 44' 3, 6 4 9 9 15 10 7 10 46 4 6 6 9 9 15 10 8 12 48 5 6 6 9 10 15 10 9 12 50 5 6 8 10 12' 17 11 10 13 52 5 7 9 10 14 18 12 11 17 54 6 9 9 10 115 20 13 11 22 56 6 10 lit 10 18 21 18 12 24 58 8 10 11 11 19 26 19 16 30, 60 8' 10 12 12 19 28 22 18 34 62 9 10 15 14 21 30 23 22 44 64 1l1 10 17 18 26 33 29 27 51 66 lit 11 19 21' 28 35 31 30 55 68 13 12 20 22 32 39 33' 30 59 70 13 17 22 25 34 43 34 37 63 72 13 19 25 29 36 44 43 44 67 74 14 19 29 30 44 48 47 49 69 76 16 22 30 33 47 51' 54 61 75 78 22 25 34 36 51 54 59 67 81 80 27 30 35 41 55 61 63 74 86 AT TAB[B 1.1 (oontimued). MOl2ThLITY (0/0), CD1 MICE

SUBREPORT P' 0500/3068 UBE145RB5, B6 5302 M WEEK GEi00P AND TRFA1M81•lP 0.3-(;Et' 0.4-GR 16-6R 17-GR 18-GR 19-M 20-GR 21-C;Et ACEPM DMBA PUS AC6T'QNE MNiSG-I (mg,/(mouee x week) ) L1MBh PI US' MWSC-I (ui9/('anuee x week) ) (42) (63) (84) (42) (63) (84) 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 8 0 0 0 0 1 0 0 0 10 0 0 0 0 1 0 0 0 12 0 0 0 0 1 0 0 0 14 0 0 0 0 1 0 0 0 16 0 0 0 0 1 0 0 0 18 0 0 0 0 1 0 0 0 20 0 1 0 0 1 0 0 0 22 0 1 0 0 1 1 0 0 24 0 1 0 0 1 1 0 0 26 0 1 0 0 1 1 0 0 28 0 1 0 0 1 1 0 0 30 0 1 0 0 1 1 0 0 32 0 1 0 0 1 1 0 0' 34 0 1 0 0 1 1 0 0 36 0 1 0' 0 1 1 0 0' 38 0' 1 0 0 1 1 0 0, 40 0' 1' 0 0. 1 1 0 0 42 0 1 0 0 1 1 0 0 44 1 1 0 0 1 1 0 1 46 1 1 1 0 1 2 0 1 48 1 1 1 0 1 2 0 1 50 1 1 1 0 1 2 0 1 52 1 1 2 0 1 2 0 1 54 1 1 2 0' 1 2 0 2 56 1 1 2 0 1 2 1 2 58 1 1 2 0 1 2 1 2 60 1 1 2 0 1 2 2' 2 62 1 2' 2 0 1 2 2 2 64 1 3 2 0 1 3 3 2 66 2 3 2 0 2 3 4 2 68 2 3 3 1 2 3 4 3 70 2 4 3 1 2 3 6 4 72' 2 4 3 2 2 3 6 4 74 3 4 5 2 2 3 8 4 76 3 5 5 2 2 4 9 7' 78 6 5 5 2 2 4' 10 7 80 7 5 5 2 3 4 10 8 AT TABLE 1.2 MORPA[.ITY (0/0), B6C3F1 MICE

SUBREPORT P 0500/3068 UBE145RB7 7289 APPLICATION MATERIAL MOUSE STRAIN 2R1 CONDENSATE DOSE (mg/(mouse x week)) MEAN 0 42 60 63 84 90 120 acetone CD1 33 DMBA plus acetone MWSC-I m m m 19 29 - - 29 30 29 MWSC-I (a) 37 - - 24 34 32 DMBA plus MWSC-I SWSC-I DMBA plus SWSC-I m h m 27 - 41 - 63 - - - - 30 55 74 35 61 86: 31 52 74 acetone B6C3F1 7 DMBA plus acetone m 5 MWSC-I m 5 - 2 3 3 DMBA plus MWSC-I n 4 - 10 8 7 AT TABLE 2 MORTALITY (0/0)I, WEEK 80 (a) stored at 4 degrees centigrade, condensate of all other groups stored at -75 degrees centigrade

SUBREPORT P 0500/3068 UBE145RB8 6316 APPL. TeIE E K STUDY P 0500/- 3000 30,10 3023 3024 3030 3068 3073' 75 17 11 13 22 17 28 23' 80 24 15 - - 29 33 33 AT TABLE 3 MORTALITY (0/0), NEGATIVE CONTROL GROUPS IN DIFFERENT INBIFO STUDIES mouse strain: CD1

SUBREPORT P 0500/3068 UBE145RB9 7289 TREATMENT 1 TREATMENT 2 GROUP SWSC-I SWSC-I PLUS DMBA GROUP 10-GR TO 12-GR 13-GR TO 15-GR MWSC-I 1-GR to 3-GR +++ +++ MWSC-I plus DMBA 7-GR to 9-GR +++ +++ AT TABLE 4.1 INFLUENCE OF SMOKE TYPE ON MORTALITY (week 80) (a),

.I4'L..ll - u.,+.., c.1 , e.U •YIVIIIJw." ~l.l VI,JI.., .......J. . "u11. SUBREPC)RT P 0500/3068 UBE145RB10 7289 TREAZMENP 1 TREAZMENP 2 GROiJP ACETONE MWSC-I Sh1SC-I MWSC-I GRO[lP 0. 1-m 1-GR Ta 3--GR 10-M Ta 12-M 16-M TO 18-GR acetone + plus DMBA 0.2'-GR MWSC-I e i-F+ plus DMBA 7-GR to 9-GR SWSC-I - +++ +++ plus DMBA 13-GR to 15-GR WSC-I plus DN1BA 19-GR to 21-GR + AT TABLE 4.2 INFLUENCE OF LYNBA PRETREATr1EDTP ON MORTALITF (week 80) ( a) (a) Chi2-test

SUBREPORT P 0500/3068 UBE145RB11 7289 STRP,IN CD1 TREAZMINJ? 1 STRAIN B6C3F1 TRFAIMFW r 2 GRO[7P ACETONE GRXJP 0.3-GR ACE'L'ONE PLQS DNIBA 0.4-GR MWSC-I 16-GR 10 18-GR @'&i1SC-I PLUS DMBA 19-GR TO 21-GR acetone +++ +++ 0.1 -GR acetone + ++ plus DMBA 0. 2'-GR 1rWSC-I - - +++ +++ 1-GR to 3-GR MnlSC-I - - +++ +++ plus DMBA 7-GR to 9-GR AT TABLE 4.3 INFLUENCE OF 1rI0USE STRAINS (CD1 AND B6C3F1) ON MORTALITY (week 80) (a) ~

SU'BREPORT'P 0500/3068 UBE145RB12 7289 TREATMENT li TREATMENT 2 GROUP MWSC-I (-75 degrees centigrade) GROUP 1-GR TO 3-GR MWSC-I (4 degrees centigrade) 4-GR to 6-GR e AT TABLE 4.4 INFLUENCE OF CONDENSATE STORAGE TEMPERATURE ON MORTALITY (week 80) (a)

A-/3068.00: GROUP DATE 4.DEC.82 9'.DEC.82 1D.DEC.82 16.DEC.82 0.10-GR N 105 105 105 105 M 19.6 21.9 21.9 22.8 RSD(%) 10.9 11.0 12.6 13.9 0.20-GR. N 105 105 105 105 M 20.4 22.2 22'.2 23.0 RSD(X) 21.5 11.4 10.6 10.5 0.30-GR N' 105 105 105 105 M •12.4 14.5 14.8 16.9' RSD(%) 27.2 18.7 18.0 10.8 0. 40-GR N 105 105 105 105 M 11.6 13.6 13.8 16.5 RSD('/.) 21.5 15.6 14.3 7.9 1.00-GR N 105 105 105 105 M •17.4 19.9 19.9 21.0' RSD(%) 13.9 10.5 8.1 8.8 2.00-GR N 10'5 105 105 105 M 18.5 20.7 20.7 22.0 RSD('/.) 30.1 22.5 20.8 19.3 3.00-GR N' 105 105 105 105 M 19.9 21.9 2•1.6 23.1 RSD('/.) 16.5 15.6 15.1 15.3 AT TABLE'5 BODY WEIGHT' (GRAMS), APPLICATION WEEK -2 TO 0 304.01JT304Nf3068.00

A-/3068.00 ----------------------------------------------------------------- GROUP DATE 4.DEC.82 9.DEC.82 10.DEC.82 16.DEC.82 ----------------------------------------------------------------- 4.00-GR N 105 105 105 105 M 20.1 21.4 21.3 22.6 RSDM 14.4 15.0 13.3 11.9 5.00-GR N 105 105 105 105 M 18.5 20.8 20.9 22.2 R S D('/. ) 15.8 14.7 14.5 -15 . 3 6.00-GR N 105 105 105 105 M 19.0 20.7 20.7 21.9 RSD ('J.) 25.0 12.7 13.1 11.6 7. 00-GR N 105 105 105 105 M 17.4 19.3 19.9 20.7 RSD(%? 19.1 10.6 10.7 11.6 8.00-GR N 105 105 105 105 M 17.9' 19.7 20.3 21.2 RSDM 17.9' 13.4 12.7 13.0 9.00-GR N 105 105 105 105 M 18.9 20.9 21.5 22.1 RSDM 20.1 9.7 8.6 10.6 AT TABLE 5 (continued) BODY WEIGHT (GRAMG)j, APPLICATION WEEK -2 TO 0: 304.01/T304N/3068,00

I ft - i i U 114:j 1 1 1 U I F'ULAc L.ULvlalzl.Ht F()KbI.HUi'dt7 11UtLN A-/3068.00 ----------------------------------------------------------------- GROUP DATE 4.DfC.82 9.DEC.82 10.DEC.82 16.DEC.82 ------------------------------------------=---------------------- 10.00-GR N 105 105 105 105 M 18.0 20.9 21.3 21.8 RSD(L) 201.7 16.0 15.3 14.1 11.00-GR N 105 105 105 105 M 19.5 21.4 21.6 22.5 RSD(%) 13.2 11.1 11.5 9.8 12. 00-GR N 105 105 105 105 M 18.5 20.6 20.6 22.0 RSD(7:) 25.2 14.0 13.0 13.2 13.00-GR' N 105 105 105 105: M 19.2 20.9 21.8 22.4 RSD(%) 5.8 4.9 6.3 6.3 14.001-GR.. ` N':;j 105 105 105 105 Ml 18.8 19.9 20.8 21.3 R.SD(%) 31.6 26.4 25.8 23.2 15.00-GR N 105 105 105 105 M 19.0 20.5 21.6 22.3 RSD(!) 6.7 7.2 8.1 6.7 ----------------------------------------------------------------- AT TA$LE 5 (continued) BODY WEIGHT (GRAMS), APPLICATION WEEK -2 TO 0 N ~ ~ W

A-A3068.00 GROUP DATE 4.DEC.82 9.DEC.82' 1G.DEC.82 16.DEC.82 ----------------------------------------------------------------- 16.00-GR N 105 105 105 105 M 12.3 13.7 14.2 16.2 RSD(%) 14.2' 13.0 13.2 9.4 17.00-GR N 105 105 105 105 M 13.7 14.7 15.1 16.7 RSD(%) 17.3 7.4 6.6 8.7 18.00-GR N 105 105 105 105 M 14.1 14.8 15.2 16.8 RSD(%) 25.7 14.8 18.2 •13.6 19.00-GR N 105 105 105 105 M 13.4 14.1 14.8 16.8 RS D('!. ) 43.5 26.0 22.8 11.9 20.00-GR N 105 105 105 105 M 13.7 14.6 15.2 16.8 RSD(l) 29.8 19.7 19.2 11.8 21.00-GR N 105 105 105 105 M 12.5 13.7 14.3 16.3 RSD ('/. ) 40.0 25.1 22.5 13.6 ----------------------------------------------------------------- AT TABLE 5 (continued) ~ BODY WEIGHT (GRAMS)'R APPLICATION WEEK -2 TO 0 0 ~ C?~ 0 304.01/T304N/3068.00

A-/3068.00 ----------------------------------------------------------------- GROUP DATE 31.DEC.82 14.JAN.83 28.J'AN.83 11.FEB.83 ----------------------------------------------------------------- 0.10-GR N 105 105 105 105 M 24.8 26.7 28.6 29.5 RSD(%) 13.0 12.0 10.6 10.6 0.20-GR N 105 105 105 105 M 25.5 27.3 29.2 30.2 RSD(y) 8.5 11.0. 11.9 11.2 0.30-GR N 105 105 105 105 M 18.6 19.8 21.4 22.5 RSD(%) 7.1 6.9 6.4 5.3 0.40-GR N 105 105 105 105 M 18.3 19.7 21.1 22.2 RSD(%) 9.9 7.4 7.7 6.5 1.00-GR N 105 10'S 105 105 M 22.9 24.8 26.5 27.5 RSD('1.) 8.8 10.2 13.2 10.8 2.00-GR N 105 105 105 105 M 23.8 25.5 27.3 28.1 RSD(X) 19.5 18.1 18.3 16.5 3.00-GR N 104 104 103 103 M 24.6 26.4 28.1 29.1 RSD(%) 15.5 15.2 15.2 12.6 AT TABLE 6 BODY WEIGHT (GRAMS), APPLICATION WEEK 2 TO 8 3014.01dT304Nl3068.00

A-/3068.00' ----------------------------------------------------------------- GROUP DATE 2'7.DEC.82' 10.JAN.83 24.JAN.83 7.FEB.83 ----------------------------------------------------------------- 4.00-GR'. N 10'5 105 105 105 M 24.0 26.3 27.9 29.2 RSD(%) 10.8 10.4 10.6 9.8 5.00-GR N. 105 105 104 104 M 23.2' 25.1 26.7 27.8 RSD('/.) 12'.9 13.1 13.3 11.8 6.00-GR N 105 105 102 102 M 23.1 25.0 26.6 27.5 RSD ('/.1 7.5 8.9 10. '2' 9.8 7.00-GR' N 105 105 105 105 M 22.4 24.8 26.4 27.6 RSD(%) 10.4 10.3 8.1 7.3 8.00-GR N 105 105 104 104 M 22.5 24.9' 26.6 27.9 RSD(X) 13.1 10.5 10.3 8.1 9.00-GR N 105 105 105 105 M 23.4 25.6 26.9 28.3 RSD(%)l 10.0 11.6 11.2 11.1 ---------------------------------------- ------------------------- AT TABLE 6 (continued) ~ BODY WEIGHT (GRAMS)R APPLICATION WEEK 2 TO 8 0 ~. ~ ~ ~ ~ 304.01fT304Nf3068.00l

A-/3068.00 ----------------------------------------------------------------- GRUUP' DATE 28.DEC.82 11.JAN.83 25.JAN.83 B.IFEB.83 ----------------------------------------------------------------- 10.00-GR N 10:5 105 105 105 M 23.8 25.6 27.6 28.5 RSDM 10.2 9.1 10.6 8.6 11.00-GR N' 104 103 103 101 M 24.0 25.8 26.7 28.0 RSDM 11.9 11.2 13.4 11.2 12. 00-GR N 105 104 102 97 M 23.2 24.7 26.1 27.7 RSD(X) 11.1 11.1 13.3 11.1 13.00-GR'. N 10:5 105 105 105 M 23.7 25.6 27.3 28.7 RSDM 7.2 6.7 7.4 7.0 14.00-GR N 105 105 105 10'4 M 23.0 25.3 26.7 28.2 RSDM 19'.5 16.3 18.2 15.2 15.00-GR N 105 105 105 104 M 23.5 25.1 27.4 28.8 RSDM 6.9 5.6 6.0 7.1 ----------------------------------------------------------------- AT TABLE'6 (continued) BODY WEIGHT (GR'AMIS), APPLICATION WEEK 2 TO 8 304.01/T304N/3068.010

A-l3068.00, ----------------------------------------------------------------- GROUP DATE 29.DEC.82 12.JAN.83 26.JAN.83 9.FEB.83 ----------------------------------------------------------------- 16.00-GR N 105 105 105 105 M 17.8 18.7 19'.9' 21.4 RSD(%) 8.7 8.6 11.7 9.6 17.00-GR N 105 10:5 1D5 105 M, 17.9' 18.8 20.3 21.7 RSDM 10.8 7.2. 8.1 7.3 18.00-GR N 105 105 105 1014 M 17.8 18.9' 20.1 21.8 RSD(y) 11.7 12.9 12.4 11.6 19.00-GR N 105 105 105 105 M 17.6 19.1 20.4 21.8 RSD(/) 5.9 8.0 7.2 6.4 20.00-GR N' 105 105 105 105 M 17.4 18.8 20.0 21.4 RBDl('/.) 7.3 9.2' 10.1 7.6 21.00-GR N 105 105 105' 105 M 17.2 18.8 20.1 21.5 RSD(%) 9.2 7.5 9.6 8.6 ----------------------------------------------------------------- AT TABLE 6 (continued) BODY WEIGHT (GRAMS)l, APPLICATION WEEK 2 TO 8 304.01/T304N/3068.00

A-/3068.00 GROUP DATE 2'5.FBB.83 11.MAR.83 25.MAR.83 8.APR.83 ----------------------------------------------------------------- 0.10-GR N' 105 105 105 105 M 30.6 3'1.7 32.1 33.2 RSD('l.) 9.7 10.6 11.2 11.2 0.20-GR N 105 105 105 105 M 31.3 32.2 32.7 33.9 RSD(%) 11.9 9.5 11.0 11.5 0.30-GR N 105 105 105 105 M 2'3.4 23.7 24.1 24.7 R'SD(%) 5.6 6.2 6.7 7.5 0.40-GR N 105 105 105' 105 M 23.0 23.5 24.2 24.6 RSD ('l.) 8.0 5.0 6.9' 7.5 1.00-GR N 10'5 105 105 104 M 28.7 29.5 29.6 30.4 RSD(%) 1'1.2 10.6 13.1 9.1 2.00-GR N 105 105 105 105 M. 29.6 30.5 30.5 31.3 RSD(%) 15.2 16.2 15.0 14.5 3.00-GR N 103 103 103 103 M 30.0 31.2 31.2 32.6 RSD(Y.) 13.4 12.2 12.0 12.7 AT TABLE 7 BODY WEIGHT (GRAMS), APPLICATTON WEEK 10 TO 16 304.01/T304N/3068.00

A-/3068.00 GROUP DATE 21.FEB.83 7.MAR.83 21.MAR.83 4.APR.83 ----------------------------------------------------------------- 4.00-GR N •105 105 105 105 M 29.9 30.7 30.9 32.1 R'SD('/.) 11.1 10.6 10.4 11.3 5.00-GR N 104 104 104 104 M 28.9 29.6 30.3 31.0 RSD('l.) 11.4 10.0 11.6 11.4 6.00-GR N •102 102 102 102 M :8.7 29.7 29.6 30.7 RSD(%) 9.7 9.1 8.6 9.8 7. 00-GR N 105 105 105 105 M 28.6 29.1 29.7 30.3 RSD(%) 7.0 7.1 7.0 7.2 8.00-GR N 104 103 103 103 M 29.1 29.6 30.0 30.7 RSD('!.) 10.0 7.6 7.8 7.8 9.00-GR N 105 105 105 105 M 28.7 29.9 30.7 30.9 RSD(V 12.8 11.1 11.0 11.4 AT TABLE 7 (continued) BODY WEIGHT (GRAMS), APPLICATION WEEK 10 TO 1:6 304.01/T304Nf3068.00

A-/30b8.00 ----------------------------------------------------------------- GRuUP' DATE 22.FEB.83 B.MAR.83 22.MAR.83 5.APR.83 ----------------------------------------------------------------- 10.00-GR N' 105 105 105 105 M 28.7 29.8 30.9 31.1 R'SD(%) 8.2' 9.0 8.9 9.4 1 I .00-GR N 101 100 100 '100 M 28.7 29.2 30.5 30.5 RSD('!.) 11.4 12.1 13.1 14.2 12. 00-GR'. N: 96 95 95 95 M 28.4 28.7 30.0 30.0 RSD(%) 10.3 9.5 9.1 9.4 13.00-GR N 105 105 105 105 M 29.5 30.0 30.2 30.7 RSD(%) 6.6 7.4 8.2 8.1 14.00-GR N 104 104 104 104 M 28.6 29.3 29.5 30.0 RSD(X) 14.9 14.7 14.0 13.4 15.00-GR N 103 103 103 103 M 29.3 30.2 30.1 31.1 RSDMl 5.9 7.3 7.3 6.6 AT TABLE 7 (continued) BODY WEIGHT (GRAMS) , APPLICATION WEEK 1'0 TO 16 ~ ~ ~ ~ 304.01/T304N/3068.00

A-/3068.00 GROUP DATE 23.FEB.83 9.MAR.83 23.MAR.83 6.APR.83 ----------------------------------------------------------------- 16.00:-GR N' •i05 105 105 105 M 22.3 22.4 23.2 23.2 R,SD(%) 8.7 7.8 8.7 7.7 17.00-GR N 105 105 105 105 M 22.5 22.7 23.3 23.2 RSD(X) 9.0 6.1 7.0 6.1 18.00-GR'. N 104 104 104 104 M 22.5 22.7 23.1 23.5 RSDM 13.2 10.0 11.2 11.0 19.00-GR N 105 105 105 105 M 22'. 5 22.8 23.2 23.6 RSDCY.) 7.5 6.7 6.6 7.1 20.00-GR N 105 105 105 •105 M 22.1 22.4 23.2 23.0 RSD(Y.) 8.2 6.6 6.4 5.9 21.00-GR N 105 105 105 105 M 22.2 22.5 23.1 23.1 RSD(%) 8.5 7.0 7.3 6.3 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - AT TABLE 7 (continued) BODY WEIGHT (GRAMS) , APPLICATION WEEK 10, TO 16 304.01/T304N'/3068.00

A-13068.00 GROUP DATE 22.APR.83 6.MAY 83 3.JUN.83 1.JUL.83 ----------------------------------------------------------------- 0.10-GR N 105 105 103 102 M 33.1 33.0 33.5 34.6 RSD(%) 11.9 11.3 13.9 13.0 0.20-GR N 105 105 104 104 M 34.0 33.9 34.3 35.4 RSD(Y'.) 10.9 10.7 9.6 10.3 0. 30-GR N' 105 105 •105 105 M 25.2 25.4 25.9 26.8 RSD(%) 8.7 8.1 9.0 7.6 0.40-GR N 105 104 104 10'4 M 25.0 25.4 26.0 26.7 RSD(%') 6.4 6.3 7.5 7.4 1.00-GR N 104 104 104 102 M 30.5 31.3 31.7 32.1 R S D(%) 9.7 9.3 9.1 8.9 2.00-GR N 105 105 105 103 M 31.4 32.2 32.6 33.0 RSD(%) 15.0 15.4 14.8 13.8 3.00-GR N 103 103 103 103 M 32.4 32.8 33.4 34.2' RSD(%) 11.9 12.6 11.1 11.3 AT TABLE 8 BODY WEIGHT (GRAMS), APPLICATION WEEK 18 TO 28 304.01/T304Nl3068.00

A-L3068.00 ----------------------------------------------------------------- GROUP DATE 18.APR.83 2.MAY 83 30.MAY 83 27.JUN.83 ----------------------------------------------------------------- 4 .0!0-GR N 105 105 105 105 M 32.4 32.3 32.7 34.0 RSD (Y. ) 9.9 10.5 11.4 101.9 5.00-GR N 104 104 104 103 M 31.2 31.2 31.9 32.8 RSD(7.) 12.5 11.4 11.5 11.9 6.00-GR N 102 10:2 102 102 M 30.9 30.8 31.7 32.5 RSD (%) 9.8 9.4 101.0 ' 9.4 7.00-GR N 105 105 104 103 M 30.5 30.7 30.9 31.9 RSD('l.) 8.0 7.8 6.2 7.8 8.00-GR N 103 102 101 101 M 301.7 30.9 31.4 32.3 RSD(%) 9.0. 9.3 8.0 8.7 9.00-GR N 105 105 105 105 M 31.2 31.4 32.4 33.0 RSD('l.) 11.6 11.9 11.5 10.8 ----------------------------------------------------------------- AT TABLE 8 (continued) BODY WEIGHT (GRAMS), APPLICATION WEEK 18 TO 28 30'4. 01 LT304N/3G68. 00

A-13068.00 ----------------------------------------------------------------- GROUP DATE 19.APR.83 3.MAY 83 31.MAY 83 28.JUN.83 10.00-GR N 105 104 104 104 M 31.1 31.8 32.8 32.6 RSD(%) 9.7 8.9 11.5 11.5 11.00-GR N 100 98 98 97 M 30.7 31.4 32.7 32.3 RSD(Z) 12.2 11.8 12.3 11.8 12.00-GR N 95 93 93 93 M 30.1 30.5 32.1 31.4 RSD(Y.)' 11.0 10.6 11.5 9.9 13.00-GR N 105 105 102 100 M 30.9 31.4 32.3 32.3 RSD(%) 8.9 8.1 7.7 8.6 14.00-GR N 104 104 103 102 M 30.1 30.5 31.3 31.5 RSD(%) 14.3 13.6 14.6 15.0 15.00-GR N 103 103 103 102 M 30.8 31.4 32.8 32.2 RSD(%) 6.3 7.9 7.4 7.2 ----------------------------------------- ----------------------- AT TABLE 8 (continued) BODY WEIGHT (GRAMS), APPLICATION WEEK 18 TO 28 304.01lT304N/3068.00

A-/3068.00 ----------------------------------------------------------------- GROUP DATE 20.APR.83 4.MAY 83 1.JUN.83 29.JUN.83 ----------------------------------------------------------------- 16.00-GR N' 105 105 105 105 M 23.2 24.1 24.5 25.0'RSD('/.) 9.7 7.4 8.6 7.7 17.00-GR N 105 105 105 105 M 23.0 24.1 24.7 25.0 RSD(%) 6.4 6.2 6.4 6.7 •18.00-GR N 104 104 104 104 M 23.5 24.2 25.0 25.1 R S D(%) 11. 1'11.2 11.9 10.6 19.00-GR N 105 105 104 104 M 23.7 24.3 25.1 25.3 RSD(7:) 8.0 7.1 6.6 7.2' 20.00-GR. N 105 105 105 105 M 23.5 23.9 24.8 25.1 RSD(X), 5.5 6.3 6.6 5.6 21.00-GR N 105 105 105 105 M 23.3 24.0 24.9 25.0 RSD(%) 6.3 7.9 6.1 5.4 ------------------------------------------- ------------------------ AT TABLE 8 (continued)l BODY WEIGHT (GRAMS), APPLICATION WEEK 18 TO 28 304.01/T304N/3068.00

A-/3068.00 ----------------------------------------------------------------- GROU'P DATE 29.JUL..83 26.AUG.83 23.SEP.83 21.OCT.83 ----------------------------------------------------------------- 0.10-GR N M RSD(%) 0.20-G'R N m R S D('/. ) 101 35.5 13.1 104 36.7 9.6 100 35.8 12.2 101 37.1 10.0 98 35.4 14.8 99 35.9 8.8 96 35.7 12.6 98 36.7 11.5 0.30-GR N 105 105 105 104 M 27.1 27.8 28.3 29.1 RSD ('/.) 8.8 9.6 10.9 12.5 0.40-GR N 104 104 104 104 M 27.4 28.3 28.6 29.4 RSD(%) 8.9 8.8 9.1 9.4 1.00-GR N 101 100 100 99' M 32.6 32.9 33.5 32'.8 RSD('/.) 8.4 7.9 7.5 8.1 2.00-GR ' N 101 100 100 99 M 33.4 33.6 34.1 33.4 RSD(%) 16.1 16.5 15.1 16.7 3.00-GR N 103 102 101 100 M 34.5 34.8 34.7 35.0 RSD(%) 11.6 12.8 11.7 12.7 AT TABLE 9' BODY' WEIGHT (GRAMS), APPLICATION WEEK 32 TO 44 304.01/T304N/3068.00

A-/30b8.0'0 ----------------------------------------------------------------- GROUP DATE 25:.JUL.83 2'2.AUG.83 19.SEP.83 20'.0CT.83 ----------------------------------------------------------------- 4 . 010-GR N 103 101 100' 99 M 34.1 34.3 33.9 34.8 RSD(%) 10.8 12.9 11.1 10.4 5.00-GR N 101 ! 0''1 100 98 M 33.5 33.5 32.9 33.7 RSD(X) 11.4 11.6 12.1 11.1 6.00-GR N' 102 102 102 101 M 33.2 33.1 32.2 32.6 RSD(%) 8.3 9.1 10.1 8.7 7.D0-GR N 102 102' 102 102 M 32.5 32.3 32.6 32.5 RSD(X) 7.6 8.7 8.3 8.4 B.00-GR N 101 100 100 100 M 33.0 32.6 32.9 32'.9' RSD(%) 7.4 8.6 8.8 10.4 9.00-GR N 105 105 104 101 M 33.6 33.6 32.9 33.0 RSD(%) 10.6 11.8 11.5 11.9 ----------------------------------------------------------------- AT TABLE 9 (continued) BODY WEIGHT (GRAMS), APPLICATION WEEK 32 TO 44 304.01/T3014N/3068.00

A-/3068.00 GROUP DATE 26.JUL.83 23.AU'b.83 20.SEP.83 18.OCT.83 ----------------------------------------------------------------- 10.0D-GR N 103 101 99' 97 M 33.5 33.5 33.2 33.7 RSD(Y.) 9.7 10.8 11.1 10.3 11.00-GR N 97 97 97 97 M 33.4 33.6 33.4 33.8 RSD(f) 12.1 10.5 11.5 11.5 12.00-GR N 93 91 90 89 M 32.8 32.4 32.2 32.9 RSD(Z) 10.8 12.2 12.2 10.1 13.00-GR N 98 97 96 95 M 32.9 33.4 32.9 32.2 RSD(X) 9.6 10.1 10.4 10.3 14.00-GR N 102 100 99' 98 M 32.2 32.4 32.0 31.7 RSD('/.) 15.2 13.4 12.3 13.6 15. 010-GR N 10:1 99 97 95 M 33.8 33.6 34.2 34.3 RSD(X) 7.9 11.3 12.9 14.5 ----------------------------------------------------------------- AT TABLE 9 (continued) BODY WEIGHT (GR'AMS). APPLICATION WEEK 32 TO 44 304.01/T304N/3068.00

A-f3068.00 ----------------------------------------------------------------- GROUP DATE 27.JUL.83 24.AUG.83 21.SEP.83 19.OCT.83 ----------------------------------------------------------------- 16.00-GR N' 105 105 105 105 M 25.9' 26.2 26.3 26.5 RSD~('/.) 8.8 8.4 9.9 11.1 17.00-GR N 105 105 105 105 M 25.8 26.2 26.2' 26.3 RSD(X) 6.8 7.3 7.1 7.5 18.00-GR N 104 104 104 104 M 26.1 26.5 27.0 26.7 RSD(X) 10.6 10.9 11.9 11.5 19.00-GR N 104 104 104 104 M 26.2' 26.7 27.3 27.4 RSD(%) 7.0 8.2 7.7 10.4 2'0.00 GR N' 105 105 105 10'5 M 25.8 26.1 26.8 26.4 RSD(X) 6.5 5.3 6.2 6.9 21.00-GR N 105 105 105 104 M 25.8 26.1 26.6 26.4 RSD(%) 6.1 5.8 6.2 6.7 ----------------------------------------------------------------- AT TABLE 9 (continued) BODY WEIGHT (GR'AMS), APPLICATION WEEK 32 TO 44 3014 . 01 /T 304 N L 30'68 . 010

A-t3068.00 GROUP DATE 18.NOV.83 16.DEC.83 13.JAN.84 10.FEB.84 0.10-GR N 96 93 93 89 M 35.8 35.8 36.2 36.6 RSD(%) 13.5 12.8 12.9 12.2' 0.20-GR N 97 97 95 95 M 37.0 36.6 37.2 37.9 RSD(X) 11.1 12.1 11.8 12.0 0.30-GR N 104 104 104 104 M 29.3 29.1 29.7 29.8 RSD(%) 11.6 12.1 11.4 12.9 0.40-GR N 104 104 104 104 M 29.4 29.5 29.6 30.0 RSD('L) 9.5 8.4 9.2 10.1 1.00-GR N 98 97 92 91 M 32.6 32.9 33.2 32.9 RS0(7.) 8.4 101.3 7.8 9.1 2..00-GR N' 99' 99 98 98 M 33.6 33.6 34.4 34.6 RSD(%) 14.9 15.8 16.1 16.5 3.00-GR N 98 98 98 97 M 34.6 34.9 34.9 35.2 RSD,(Y.) 13.3 11.9 12.1 10.6 AT TABLE 10 BODY WEIGHT (GRAMS), APPLICATION WEEK 48 TO 60 304.01/T30'4Nl3068.00

A-/3068.00 ----------------------------------------------------------------- GROUP DATE 14.NOV.83 12.DEC.83 9.JAN.84 6.FEB.84 ----------------------------------------------------------------- 4.00-GR'. N 99 98 98 97 M 34.9 (a) 34.5 35.5 36.01 RSD(X) i 12.2 12.4 10.1 12.4 5.00-GR N 98 96 94 94 M 33.9 34.2 34.8 34.9 RSD(%) 12.4 13.1 11.9 11.4 6.00-6R N 101 10'0 97 93 Mi 33.3 34.0 34.2 34.3 RSD(%) 8.3 8.5 8.5 12.6 7.00-GR N 100 100 99 97 M 32.5 33.5' 33.7 33.5 RSD(X) 7.7 10.8 11.1 9.6 8.00-GR N 98 98 95 95 M 33.3 33.6 34.2 34.3 RSD(%) 15.6 8.8 9.8 11.7 9.00-GR N 99 96 94 92 M 34.1 34.2 34.3 35.3 RSD('/.) 13.6 9.8 10.7 11.6 AT TABLE 10 (continued) BODY WEIGHT (GR'AMS), APPLICATION WEEK 48 TO 60 (a)j,KOERPERGEWICHTS-BESTIM.MUNG VOM 15.NOV.83 304.01/T304N/3068.00

A-/3068.00 ----------------------------------------------------------------- GROUP DATE 15. N'(1V'. 83 13 . DEC . 83 10 . J AN . 84 7. FEB . 84 ----------------------------------------------------------------- 10.00-GR N 96 95 95 9'2 M 34.2 34.0 34.2 35.0 RSDM 12.7 11.4 10.8 12.5 11. 010-G R N 95 90 88 85 M 34.1 34.4 34.6 34.0 RSDM 14.4 10.3 11.7 12.4 12. 0'0-GR N 89 86 83 77 M 33.5 33.1 33.7 33.3 RSDM 10.0 11.1 10.6 11.5 93.00-GR N 94 92 88 83 M 34.0 33.6 33.2' 34.4 RSD('l.) 9.2 8.1 8.4 8.3 14. 010-G R N 96 93 93 87 M 33.1 32.8 33.0 33.6 RSDM 13.7 16.8 21.4 17.2 15.0:0-GR N 92 90 80 70 M 34.5 34.5 34.4 34.9 RSD(Y.) 13.6 11.2 11.8 14.6 ----------------------------------------------------------------- AT TABLE 10 (continued) BODY WEIGHT (GRAMS), APPLICATION WEEK 48 TO 60 304.01/T304NA3068.00

A-/3068.00 ----------------------------------------------------------------- GROUP DATE 16.NOV.83 14.DEC.83 11.JAN.84 8.FEB.84 ----------------------------------------------------------------- 16 . 001-G R N M RSDM 104 27.3 9.6 104 27.5 9.3 103 27.8 9.4 103 28.5 9.2 17.00-GR. N 105 105 105 105 M 27.1 27.2 27.6 28.2 RSDM 7.8 7.3 6.3 6.3 18.00-GR N 104 104 104 104 M 27.4 27.8 28.3 28.9 RSDM 10.4 10.3 10.7 11.0 19.00-GR N' 103 103 103 103 M 27.9' 27.9 28.5 29'.01 RS D(%) 9.4 8.0 7.0 8.7 20. 00-GR N 105 105 104 103 M 27.0 27.8 27.8 28.1 RSDM 7.9 12.0 7.7 5.4 21.00-GR'. N 104 104 103 103 M 27.2 27.6 27.9 28.3 RSDM 5.6 7.5 7.3 5.7 AT TABLE 10 (continued) ~ BODY WEIGHT (GRAMS)~, APPLICATION WEEK 48 TO 60 ~ © N ~ ~ ~ 304.01/T30'4N/3068.00

A-/3068.00 ----------------------------------------------------------------- GROUP DATE 9.MAR.84 6.APR.84 4.MA1!' 84 1.J'UN.84 ----------------------------------------------------------------- 0.10-GR' N 85 81 77 76 M 36.2 36.5 36.3 37.1 RSD(%)' 12.9 16.0 13.0 14.4 01. 20-GR N 93 90 90 89 M 37.7 37.5 37.9 38.5 RSD('/.) 12.6 12.9 12.6 11.3 0.30-GR N 104 103 103 102 M 30.0 30.1 30.6 31.2 RSD(X) 14.1 12.6 12.5 11.9 0.40-GR N 102' 102 101 100 M 30.1 30.4 30.6 31.3 RSD,(Y.) 12'.7 10.1 14.0 13.0 1.00-GR N 91 87 85 80 M 33.9 33.7 33.7 34.6 RSD(%) 9.0 8.4 10.3 9.4 2. 0!0-GR N 95 91 86 80 M 35.2 35.0 35.0 36.2 RSD(X) 16.2 15.1 15.2 14.2 3.00-6R N 93 89 84 81 M 36.1 35.7 35.5 36.6 RSD(%) 9.3 10.9 10.9 14.1 AT' TABLE 11 BODY WEIGHT (GRAMS), APPLICATION WEEK 64 TO 76 304.01/T3D4N/3068.00

A-/3068.00 GROUP DATE 10.MAR.84 2.APR.84 30.APR.84 28.MAY 84 4. 0'D-GR N 92 89 85 75 M 34.5 36.2 35.7 36.G RSD(Y.) 11.5 12.2 10.5 10.5 5.00-GR N 91 88 87 85 M 33.8 35.4 35.0 34.7 RSD(K) 10.6 13.0 13.2 12.2' 6.DD-GR N 87 84 78 77 M 33.2 34.8 34.6 34.6 RSD(%) 9.8 12.0 10.4 11.6 7.00-GR N 93 92 91 89' M 32.6 34.0 33.6 33.6 RSD(%) 9.3 9.7 10.5 13.6 8.00-GR N 94 92 85 82 M 33.2 34.7 34.3 34.5 RSD(X) 10.2 8.6 8.6 8.2 9.00-GR' N 87 84 81 74 M 33.8 35.7 35.3 36.0 RSD(%) 10.7 11.5 12.2' 10.1 AT TABLE 11 (continued) BODY WEIGHT (GRAMS) , APPLICATION WEEK 64 TO 76 304.01/T304N/3068.00

A-/3068.00 ----------------------------------------------------------------- GRcJUP DATE 6.MAR.84 3.APR.84 1.MAY 84 29.MAY 84 ----------------------------------------------------------------- 10.00-GR N 87 82 76 73 M 35.0 35.1 35..4 35.9 RSD(X) 10.5 12.6 11.7 13.0 11.D0-GR N 78 72 67 56 M 34.3 33.5 34.2 35.3 RSD(%) 15.7 13.1 12.1 10.7 12.00-GR N 71 68 60 51 M 33.5 33.0 34.0 35.6 RSD('1.) 9.01 10.8 13.6 13.4 13.00-GR N 77 70 62 49 M 34.2 33.9' 33.8 35.2 RSD(%) 9.3 8.3 11.0 11.1 14.00-GR N' 81 73 61 46 M 32.6 33.7 33.9 34.2 R'SD('J.) 21.8 20.1 17.4 14.3 15. 00l-G R N 54 43 37 30 M 34.8 34.2 34.4 35.6 RSD(%) I0.6 8.7 6.7 6.7 ----------------------------------------------------------------- AT TABLE 11 (continued) BODY WEIGHT (GRAMS), APPLICATION WEEK 64 TO 76 304.01/T3014N/3068.00

A-/3068.00 ---------------------------------------------------- ------------ GROUP DATE 7.MAR.84 4.APR.84 2.MAY 84 30.MAY 84 ----------------------------------------------------------------- 16.00-GR N 103 103 102 100 M 28.1 28.2' 28.7 28.7 RSDM 10'.9 11.8 12.8 13.3 17.00-GR' N 105 104 103 103 M 27.6 27.6 28.4 28.1 RSDM 7.4 6.4 6.5 7.5 18.00-GR N 104 103 103 103 M 27.9 28.5 28.5 28.7 RSDM 12.5 11.9 11.1 12.5 19.00-GR N 102 102 102 101 M 28.5 29.0 29.5 29.5 RSDM 9.2 8.2 9.5 10.7 20.00:-GR N 102 101 99 96 M 27.6 28.1 28.6 28.6 RSDM 6.4 7.01 6.7 6.9 21.00-GR N 103 102 101 101 M 27.8 28.3 28.9 28.6 RSD(%) 6.1 7.7 8.2 7.6 AT TABLE 11 (continued) BODY WEIGHT (GRAMS), APPLICATION WEEK 64 TO 76 304.01/T304N'l3068.00

A-/ 30,68 . 00 GROUP DATE 29.JUN.84 0. 10-GR N 70 M 36.7 RSD(X) 15.9 0.20-GR N 85 M 38.5 RSD(x) 13.7 0.30-GR N 98 M 31.0 RSD(X) 11.1 0.40-GR N 100 M 31.9 R'SD(X) 13.4 i.00-GR N' 75, M 33.8 RSD(X) 11.0 2.00-GR N 75 M 35.5 RSD(Y.) 13.3 3. 010-GR N 75 M 36.6 RSD(%) 15.1 AT TABLE 12 BODY WEIGHT (GRAMS), APPLICATION WEEK 80 304 . 01 / T30'4N'/3068 . 00

A-/3068.00' ----------------------------------------------------------------- GROUP DATE 25. JUN'. 84 ----------------------------------------------------------------- 4.0'0-GR N 67 M 35.9 RSD(%) 8.9 5.00-GR N 82 M 35.4 RSD(Y.) 12.9 6.00-GR N 69 M 34.5 RSD(%) 8.4 7.00-GR N 79 M 34..0 RSD(Y.) 10.5 8.00-GR N 78 M 35.0 RSD(%) 10.5 9.00-GR N 68 M 35.8 RSD(Y.) 13.0 ----------------------------------------------------------------- AT TABLE 12 (continued) BODY WEIGHT (GRAMS) , APPLICATION WEEK 80 304.01/T304N/3068.00

A-13068.00 GROUP DATE 26. JU N. 8 4 10.00-GR'. N 64 M 36.3 RSD(%) 13.1 11.00-GR'. N 48 M 35.1 RSD(%) 15.2 12.00-GR' N 42 M 35.8 RSD(%) 13.3 13.00-GR N 40 M 34.4 RSD('/.) 13.4 14. 010-G R N 30 M. 34.5 RSD(%) 10.9 15.00-GR N 16 M 34.9' RSD(%) 9.4 AT TABLE 12 (continued) BODY WIEIGHT (GRAMS), APPLICATION WEEK 80 304.01/T304N/3068.001

A-/3068.-00 GROUP DATE 27.JUN.84 16.00-GR N 100 M 29' . 6 RSD(%) 12.6 17.00-GR N 103 M 28.9 RSD,(X) 7.3 18.00-GR N 102 M 29.3 RSD(%) 13.2 19.00-GR N 101 M 30.4 RSD(X) 9'.6 20.00-GR'. N 95 M 29.0 RSD(X) 6.4 21.00-GR N 97 M 29.0 RSD(Z) 9.5 AT TABLE 12 (con..inuQd) BODY WEIGHT (GRAMS), APPLICATION WEEK 80 304.01<T304N/3068.00

INBIFO Institut fur biologische Forschung •K6tn SUBREPORT P,0500/3068 UBE145RB13 7289 ~ APPLICATION MOUSE 2R1 CONDENSATE DOSE MEAN MATERIAL STRAIN (mg/(mouse x week)) 0 42 60 63 84 90 120 acetone CD1 37 DMBA plus acetone " 39 MWSC-I m 34 - - 36 37 36 MWSC-I (a) DMBA plus MWSC-I SWSC-I m u m 36 34 36 - - - - - - 35 35 35 35 36 36 35 35 36 DMBA plus SWSC-I m 34 - - 35 35 35 acetone B6C3F1 31 DMBA plus acetone m 32 MWSC-I m 30 - 29 29 - - 29 DMBA plus MWSC-I m 30 - 29 29' - - 29' AT TABLE 13 BODY WEIGHT (g), WEEK 80 (a) stored at 4 degrees centigrade, condensate of all other groups stored at -75 degrees centigrade 3285

INBIFO linstitut fur biologische Forschiung • Kbin SUBREPORT P 0500/3068 UBE145RB14 6316 APPL. STUDY P 0500/- WEEK 3000 3010 3023 3024 3030 3068 3073 74/75/76 40 43 4& 40 41 37 36 80 40 43 - - 41 37 36 AT' TABLE 14 BODY WEIGHT (g), NEGATIVE CONTROL GROUPS IN DIFFERENT INBIFO STUDIES mouse strain: CD1 3285

IMBIIFO Institut fur biollogische Forschung Koin SUBREPORT P 0500/3068 UBE'145RB15 7041 GROUP STATISTICAL PARAMETER DATE 2.JUL.84 1-GR to 3-GR N 222 M 34.7 MR 100. RSD(,0/0 ) 10.7 10-GR to 12-GR N 147 M 34.7 MR 100.=I RSD(0/0) 10.5 13-GR to 15-GR N 80 M 33.8 MR 97.= RSD(0/0) 10.2 TABLE 15 INDIVIDUAL BODY WEIGHT (g) AT'FINAL DISSECTION, MALE'MICE 3285

INBIFO Institut fur biologische Forschung • KtSIn SUBREPORT P,0500/30b8 UBE145RB16 7289 GROUP STATISTICAL PARAMETER DATE 2.JUL.84 7-GR to 9-GR N 216 M 34.1 MR 100. RSD(0/0) 9.7 10-GR to 12-GR N 147 M 34.7 MR 100.=I RSD(0/0) 10.5 13-GR to 15-GR N! 80 M 33.8 MR 99.= RSD 10.2 TABLE 15 (continued) INDIVIDUAL BOD~.' WEIGHT (g ) AT FINAL DISSECTION, MALE MICE 3285

IINBIFO Institut fur biologische Forschuing • KtSIn SUBREPORT P 0500/3068 UBE145RB17 7289 GROUP STATISTICAL PARAMETER DATE 2. JUL. 84 0.2-GR N 85 M 37. 5 MR 100. RSD(0/0) 13.0 0. 1-GR N 70 M 36.2 MR 96.= RSD(0/0) 12,0 1-GR to 3-GR N 222 M 34.7 MR 93.+++I RSD ( 0/01) 10.7 10-GR to 12-GR N 147 M 34.7 MR 93.+++I RSD(0/0) 10.5 TABLE 15 (continued) INDIVIDUAL BODY WEIGHT (g) AT FINAL DISSECTION, MALE MICE 3285

INBIFO Institut fur biologische Forschung • K6In SUBREPORT'P 0500/3068 UBE145RB18 7289 GROUP STATISTICAL PARAMETER DATE 2.JUL.84 7-GR to 9-GR N 216 M 34.1 MR 100. RSD(0/0) 9.7 0.1-GR N 70 M 36.2 MR 106.+++I RSD(0/0) 12.0 1-GR to 3-GR N 222 M 34.7 MR 102.=I RSD(0/0) 10.7 10-GR to 12'-GR N 147 M 34.7 MR 102.=I RSD(0/0) 10.5 TABLE 15 (continued) INDIVIDUAL BODY WE!IGHT'(g,) AT FINAL DISSECTION, MALE MICE 3285

INBIFO Institut fur biologische Forschung • Kofn INDEX REPORT P 0500/3068 BRA95DIVA27 CONTENTS INDEX CODE INTEGRATING REPORT: SUMMARY Conclusions INTRODUCTION TABLE C: GROUPS AND DOSES FIGURE B: CHRONOLOGY SUBREPORT ANALYTICAL CHEMISTRY SUBREPORT ANIMAL TREATMENT SUBREPORT MICROBIOLOGY SUBREPORT PATHOLOGY =G~~se fz~~a,~~ vvGc L~9y ~ 3285

INBIFO Institut fur biblogische Forschung • KtSln SUBREPORT P 0500/3068 UBE145RB19 7289 GROUP STATISTICAL PARAMETER DATE 2.JUL.84 13-GR to 15-GR N 80' M 33.8 MR 100. RSD(0/0) 10.2 0.1-GR N 70 M 36.2 MR 107.+++I RS D( 0/'0 )'. 12.0 1-GR to 3-GR N 222 M 34.7 MR 103.= RSD(0/0:) 10.7 10-GR to 12-GR N 147 M 34.7 MR 1i03.= RSD(0/0) 10.5 TABLE 15 (continued) INDIVIDUAL BODY WEIGHT (g) AT FINAL DISSECTION, MALE MICE 3285

IN!BIFO Institut fur biologische Forschung • K6ln SUBREPORT P 0500/3068 UBE145RB20 7289 GROUP STATISTICAL PARAMETER DATE 2'.JUL.84 4-GR to 6-GR N 214 M 35.0 MR 100. RSD(0/0) 8.7 1-GR to 3-GR N 222' M 34.7 MR 9 9 . =I RSD(0/0), 10.7 TABLE!15 (continued) INDIVIDUAL BODY WEIGHT (g) AT FINAL DISSECTION, MALE MICE 3285

INBIFO Institut fur bio0ogische Forschung • K6In SUBREPORT P 0500/3068 UBE145RB21 7289 GROUP STATISTICAL PARAMETER DATE 2,JUL.84 16-GR to 18-GR N 305 M 28.7 MR 100. RSD(0/0) 9.7 119-GR to 21-GR N, 292 M 29.2 MR 102.+I RSD(0/0) 9.1 TABLE' 15 (continued) INDIVIDUAL BODY WEIGHT (g) AT FINAL DISSECTION, MALE MICE 3285

INBIFO Institut fur biologische Forsclhung NCOln SUBREPORT P 0500/3068 UBE145RB22 7033 TREATMENT 1 TREATMENT 2 GROUP SWSC-I SWSC-I PLUS DMBA GROUP MWSC-I 1-GR to 3-GR MWSC-I plus DMBA 7-GR to 9-GR AT TABLE' 16.1 10-GR TO 12-GR 13-GR TO 15-GR =I = =I INFLUENCE OF SMOKE TYPE ON BODY WEIGHT AT FINAL DISSECTION (a) (a) F- and t-test 3285

INBIFO Institut fur biologische Forsch.ung • KtSln SUBREPORT P 050;0/3068 UBE145RB23 7289 TREATNIENTr 1 TREATMENT 2 GROUP ACETOTIE MWSC-I SWSC-I hb+1SC -I CROUP 0. 1 -GR 1-GR TC3 3-,GR 10-GR Ta 12-GR 16-M Ta 18-GR acetone plus uMBA 0. 2-GR MWSC-I plus DMBA 7-GR to 9-GR SWSC-I +++I plus DMBA 13-GR to 15--GR MWSC-I - - - +I plus DMBA 19-GR to 21-GR AT TABLE 16.2 INFLUENCE OF EMBA PRETREATME6TP ON BODY WEIGEiT AT FINAL DISSECTION (a) (a) F- and t-test 3285

INBIFO Institut fOr biol'ogische Forschung • K6In SUBREPORT P,0500/3068 UBE145RB24 7289 TREATMENT 1 TREATMENT 2 GROUP MWSC-I (-75 degrees centigrade) GROUP 1-GR TO 3-GR MWSC-I =I (4 degrees centigrade) 4-GR to 6-GR AT TABLE 16.3' INFLUENCE OF CONDENSATE STORAGE TEMPERATURE ON. BODY WEIGHT AT FINAL DISSECTION (a) ( a )' F- and t-test 3285

INBI'FQ Institut fur biol'ogische Forschung • Kt9Ih SU9REPORP'P 0500/3068, U13E148RA1, A2' 5302 M 7 9 10 11 12 13 14 1B ~ 19 20 WEEK SURVI- MDR+ NiR18BR OF ALIVE: AND DEAD MICE,, WNICH HAVE CR NAD EF'FEC- MACE>o- BODY WpS TAL- ITY MACROS(."OPIC SKIN 1U61qRS' (a) TIVE IdA4sER SCOPIC Tl,lYJR WEIGHT IN APPL.- OUPSIDE IN'AND (b) (c) AREA CNLY APPL. -IRFA CUISIDE C>r7LY APPL.-ARFA (d) RATE (e) (0/0) (0/0) (9) 0 105 0' 0 0 0 0 0 105 0 22.8 2 105 01 0 0 0 0 0 105 0 24.8 4 105 0 0 0 0 0 0 105 0 26.7 6 105' 0 0 0 0 0 0 105 0 28.6 8 105 0 0 0 0 0 0 105 0 29.5 10 105 0 0 0 0 0 0 105 0 30.6 12 105 0 0 0 0 0 0 105 0 31.7 14 105 0 0 0 0 0 0 105 0 32.1 16 105 0 0 0 0 01 0 105 0 33.2 18 105 0 0 0 0 0 0 105 0 33.1 20 104 1 0 0 0 0 0 104 0 33.0 22 104 1 0 0 0 0 01 104' 0 - 24 103 2 0 0 0 0 0 103 0 33.5 26 103 2' 0 0 0 0 01 103 0 - 28 102 3' 0 0 0 0 0 102 0 34.6 30 101' 4 0 0 0 0 0 101 0 - 32 ' 101 4 0 0 0 0 0 101 0 35.5 34 100 5 0 0 01 0 0 100 0 - 36 100 5 01 0 0 0 0 100 0 35.8 38 99 6 0' 0 0 0 0 99 0 - 40 98 7 0 0 0 0 0 98 0' 35.4 42 96 9 0 0 0 0' 0 96 0 - 44 96 9 0 0 0 0' 0 96 0 45.7 46 96 9 0 0' 0 0 0 96 0 - 48 96 9 0 0 0 0' 0 96 0 35.8 50 93 11 0 0 0 0 0 93 0 - 52 93 11 0 0 0 0 0 92' 0 35.8 54 93 11 0 0 0 0 0 93 0 - 56 93 11 0 0 0 0 0 93 0 36.2 58 91 13 0 0 0 0 0 91 0 - 60 88 16 0 0 0 0 0 88 0 36.6 62 87 17 0 0 0 0 0 87 0 - 64 85 19 0 0 0 0 0 85 0 36.2 66 84 20 0 0 0 0 0 84 0 - 68 80 24 0 0 0 0 0 80' 0 36.5 70 78 26 0 0 0 0 0 78 0 - 72 77 27 0 0 0 0 0 77 0 36.3 74 77 27 0 0 0 0 0 77 0 - 76 76 28 0 0 0 0 0 76 0 37.1 78 711 32 0 0 0 0 0 71 0 - 80 70 33 0 0 0 0 0 70 0 36.7 AT TABLE 17 SKIN 1LAIDRS (BASED CN MACICSCOPIC APPEl4RAME ),n MORTALITY AND BODY WEI(iiT 0.1-(M, ACE`TONECDi MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tuanrs are included in these values (b) sum of values of colunns 10 and 12 1. e. mice'with macroscopic skin tumors "in"'as well as "in and outside" application area (c) dead mice with macroscopic skin tumors (from colurm 10 and 12). (d) sum of values of columns 7 and 14 (e) value of columi 13 multiplied by 100 and divided by value of colimn 18, i. e. sum of coluims 7 and 14 3285

I'NBIFO Institut fur biologische Forschung • BColn SUBREPORT P 0500/3068 IBE148RA3, A4' 5302 M 9 10 11 12 13 14 A 18 19 20 WEEK SURVI- MOR- N(A49ER OF AL,IVE:F4^ID DEA[? MICE, WHICH HAVE CR HAD EFFDC- MACRD- BODY 4(k1S TAtr MACRO6COPIC SfCIN T1lXMLS (a) TIVE 90DPIC b1ElGflf' ITY NUMBER T[l13R RqTE IN APPL.- OUISIDE IN AND (b) (c) (d) (e) AREA CNLY APPL.-ARFA OUTSIDE QN[.Y APPL.-ARFA (0/0) (0/0) (9). 01 105 0 0 0 0 0 0 105 0 23.0 2' 105 0 0 0 0 0 0 105 0 25.5 4 105 0 0 0 0 0 0 105 0 27.3 6 105 0 0 0 0 0 0 105 0: 29.2 8 105 0 0 0 0 0 0 105 01 30.2 10 105 0 0 0 0 0' 0 105 0 31.3 12 105 0 0 0 0 0 0 105 0 32.2 14 105 0 0 0 0 01 0 105 0 32.7 16 105 0 0 0 0 01 0 105 0 33.9 18 105 0 0 0 0 0 0 105 0 34.0 20 104 1 0 0 0 0' 0 104 0 33.9 22 104 1 0 0 0 0 0 104 0 - 24 104 1 0 0 0 0 0 104 0 34.3 26 104 1 0 0 0 0 0 104 0 - 28 104 1 0 0' 0 0 01 104 0 35.4, 30 104 1 0 0 0 0 01 104 0 - 32 104 1 0 01 0 0 0 104 0 36.7 34 102 3 0 0 0 0 0 102' 0 - 36 101 4 0 0 0 0 0 101' 0 37.1 38' 101', 4 0 0 0 0 0 101 0 - 40 99 6 1 0 0 1 0 99 1.01 35.9 42 98 7 0 0 0: 11 0 98 1.02 - 44 98 7 1' 0 01 1' 0 98 1.02 -36.7 46 98 7 1' 0 01 1 0 98 1.02 - 48 97 8 1' 0 0 1 0 97 1.03 37.0 50 97 8 1 3 0 1 0' 97 1.03 - 52 97 8 2 4 0 2 0 97 2.06 36.6 54 96 9 2 4 0 2 0 96 2.08 - 56 95 10 2 4 0 2' 0 95 2.11 37.2 58 95 10 2 4' 0 2' 0 95' 2.11 - 60 95 10 2 5 0 2' 0 95 2.11 37.9 62 93 11 2 5 0 2 0 93 2.15 - 64 93 11 3 7' 0 3 0 93 3.23 37.7 66 91 13 3 7' 0 3 0 91 3.30 - 68 90 14 3 7 0 3 01 90 3.33 37.5 70 90 14 4 8 0 4 0 90 4.44 - 72 90 14 4 8 0 4 0 90 4.44' 37.9 74 89 15 4 9 0 4 0 89' 4.49 - 76 89 15 5 11 0 5 0 89 5.62 38.5 78 87 17 5 12 0 5 0 87 5.75 - 80 85 19 5 13 0 5 0 85 5.88' 38.5 AT TABLE 17 (continued) SKINT.A4C1FtS(SlMSED (NM1VCR0&CAPICAPPEARANCE), NpR11AL.ITY ANDBOOYWEIGKI' 0.2-GR, [MBh P[.US RCEPONE C01 MICE (a), only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tumors are included in these values (b) su® of values of coluans 10 and 12 i. e. mice with macroscopic skin tumors "in" as we11 as "in and outside" application area (c) dead mice with macroscopic skin tuwmrs (fran oolumn~1'0 and 12) (d) sum of values of columns 7 and 14 (e) value of column 13 multiplied by 100 and divided by value of column 18, i. e. sum of c.olumns 7'and 14 3285

INBIFO Institut fur biologische Forschung • K6In SWBREPORT P 0500/3068 (SE148RA5, A6 5302 M 7 9 10 11 12 13 14 10 19 20 WEEK SURVI- MO[t- NOt4BER OF ALIVE AM? DFAD MICE, WHICH E@1VE OR HAD EPFOC- MACFD- gJDY WttS TAL- ITY MACis06C.'OPIC SKIN, 1tlMORS (a) TIVE 143MBF.R SCOPIC 1VlDR WEIC;ffr IN APPL.- Cl115I0E: IN AND (b) (c) AREl1 CNLY APPL.-AREA QIfSIDE ONLY APPL.-ARPA (d) RATE (e) (0/0) (0/0) (g) 0 105 0 0' 0 0 0 0 105 0 21.0 2 105 0 0 0 0 0 0 105 01 22.9 4 105 0 0 0 0 0 0 105 0 24.8 6 105 0 0 0 0 01 0 105 0 26.5 8 105 0 0 0 0 0 0 105 0 27.5 10 105 0 0 0 0 0 0 105 0 28.7 12 105 0 0 01 0 0 0 105 0 29.5 14 104 1' 0 0 0 0 0' 104 0 29.6 16 104 1' 0 0 0 0 0 104 0 30.4 18 104 1 0 0 0 0 0 104 0 30.5 20' 104 1 0 0 0 0 0 104 0 311.3 22' 104 1 0 0 0 0 0 104 0 , - 24 104 1 0 0 0 0 0 104 0 31.7 26 104 1 0 0 0 0 0 104 0 - 28 102 3 0 0 0' 0 0 102 0 32.1 30 102 3' 0' 0 0 0 0 102 0 - 32 101' 4 0 0 0 0 0 101 0 32.6 34 100' 5 0 0 0 0 0 100 0 - 36 100 5 0 0 0 0 0 100 0, 32.9 38 100 5 0 0 0 0 0 100 0 - 40 100 5 0 0 0 01 0 100 0 33.5 42 100 5 0 0 0 0 0 100 0 - 44' 99 6 0 0 0 0 0 99 0 32.8 46 99 6 0 0' 0 0 0' 99 0 - 48 98 7 0 0 0 0 0 98 0 32.6 50 98 7 1 0 0 1 0 98' 1.02 - 52' 97 8 1 1 0 1 0 97 1.03 32.9 54 95 10 1 1 0' 1 0 95 1.04 - 56 92 12 1' 1 01 1 0 93 1.08 33.2 58 91 13 2' 1 01 2 0 92 2.17 ' - 60 91 13 3 11 0 3 0 92 3.26 32.9 62 91 13 3 1' 0 3 0 92 3:26 - 64 91 13 4 1 0 4 0 91 4.40 33.9 66 89 15 4 1 0 4 0 89 4.49 - 68 87 17 4 1 0 4 0 88 4.55 33.7 ' 70 86 18 4 1 0 4 1 87 4.60 - 72 85 19 6 2' 0 6 1 86 6.98 33.7 74 84 20 8 1' 1 i 9 1 85 10.59 - 76 80 24 8 2 1 9 2' 82 10.98 34.6 78 76 28 9 2 1 10 2 78' 12.82 - 80 75 29 9 2 1 10 3 78: 12.82 33.8 AT TABLE 17 (conti'nued) SKIN 7lNNORS (BASED CN MACROSCOPIC APPFARANCE), MCRTALITY AND BODY'WEIGHT 1-GR, MWSC-I: 60 (ng/(nouse x week)) CD1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tuoors are included in these values (b) sum of values of coluans 10 and 12 i'. e. mice with macroscopic skin tumrs "in" as well as "in and outside" application area (c) dead mice with macroscopic skin twnors (fraa colurtn 10 and 12) (d) sum of values of colums 7 and 14 (e) value of column 13 multiplied by 100 and divided by value of column 18, i. e. sum of colurtns 7 ard 14 3285

INBIFO Institut fur biologische Forschung • K6lh SUBREPORT'P 0500/3068 UBE148RA7, h8' 5302 M, 7 9 10 11', 12 13 14 ~ 18 19 20 WEEK S(]RVI- MOR- NOMBER OF ALIVE AND DFAD MICE, F)EfICH HAVE OR HAD EFFEC- MAC14)- BWY VOiiS TAL- ITY MAC1aDSC.`CQIC SKIN 'iUMqR.S (a) TIVE Ni1MBER SCCPIC 1(WR WEIC;EIT IN APPL.- C1Ti5IDE IN 14149 (b) (c) AREA CNLY APPL.-lABFA O[JFSID6 Q+&Y APPL.-ARFA (d) AA7E (e) (0/0) (0/0) (g) 0 105 0 0 0 0 0 0 105 0 22.0 2 105 0 0 0 0 0 0 105 0 23'.8 4 105 0 0 0 0 0 0 105 0 25.5 6 105 0 0 0 0 0 0 105 0 27.3 8 105 0 0 0 0 0 0 105 0 28.1 10 105 0 0 01 0 0 0 105 0 29.6 12 105 0 0 0 0 0 0' 105 0 30.5 14 105 0 1 0 0 0 0 01 105 0 30.5 16 105 0 0 0 0 0 0 105 0 31.3 18 105 0 0 0 0 0 0 105 0 31.4 20' 105 0 0 0 0 0 0 105 0 32.2 22 105 0 0 0 01 0 0 105 0 - 24 105 0 0' 0 0 0 0 105 0 32.6 26 104 1 0 0 0 0 0 104 0 - 28 103 2 0 0 0 0 0 103 0 33.0 30. 102 3 0 0 0 0' 0 102 0 - 32 101 4 0 0 0 0 0 101 01 33.4 34 101 4 0 0 0 0' 0 101 0 - 36 100' 5 2 0 0 2 0 100 2.00, 33.6 38 100, 5 2 0 0 2 0 100 2.00 - 40 100 5 5 0 0 5 0 100 5.00 34.1' 42 99 6 0 0 0 6 0 99 6.06 - 44 99 6 9 0 0 9 0 99 9.09 33.4 46 99 6 9 0' 0 9 0 99 9.09 - 48 99 6 9 0 0 9 0 99 9.09 33.6 50 99 6 9 0 0 9 0 99 9.09 - 52 99 6 10 0 0 1'0' 0 99 10.10 33:6 54 98 7 10 0 0 10 0 98 10.20 - 56 98 7 11 1 0 11 0 98 11.22 34.4 58 98 7' 11 1 0 11 0 98 11.22 - 60 98 7' 12 1 0 12 0 98 12.24 34.6 62 96 9' 13 1 0 13 1 97 13.40 - 64 95 10 14 1 0 14 1 96 14.58 35.2 66 91 13 15 1 0 15 1 92 16.30 - 68' 91 13 15 1 0 15 1 92 16.30 35.0 70: 90 14 15 1 0 15 1 91 16.48 - 72 85 19' 16 1 0 16 2 87 18.39' 35.0 74 81' 23 18 1 i 0 18 2 83' 21.69 - 76 80 24 22 1 0 22 2 82 26.83 36.2 78 79 25 25 1 0 25 2 81 30.86 - 80 75 29 26 2 0 26 2 77 33.77 35.5 AT TABLE 17 (continued) SKIN 7UMDBS (BASED QV MACiifIuCDPIC APPEARANCE), MORTALITY A231 BODY MEI(;t1T 2-GR, M'v7SC-I: 90 (ng/(mouse x week)) CD1 MICE (a) only preliminary diagnosis based on macrosoopic t+ppearance, mice whirh lost their macrosccpic tunors are i'ncluded in these values (b) sum of values of columns 10 and 12 i. e. mice'with macroscopic skin tunors "in" as well as "in and outside" application area (c) dead mice with macroscopic skin tumors (fran column10 and 12) (d) sum of values of columis 7 and 14 (e) value of column 13 multiplied by 100 and divided by value of eolimn 18, i. e. sum of colunns 7 and 14 3285

INBIFO Institut fur biologische Forschung • K6lo SUBREPORT P 0500/3068'' IBE148RA9, A10 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SURVI- MOR- N[R4BER OF ALIVE' AND DEAD MICE, WHICH EWVE QR' HAD EFFEC- MACi?G>- BODY WRS 7A[.- MACR0.S(:OPIC SKIN UMRS (a) TIVE 9COPIC WEIQl'I' ITY NiR4BER 'i~R47R IN APPL.- OUPSIDE IN AND (b) (c) AREA CNLY APPL.-AREA G11iSI0E ClII.Y APPL.-AREA (d) RATE (e) (0/0) (0/0) (9) 0 104 1 0 0 0 0' 0 104 0 23.1 2 104 1 0 0 0 0 0 104 0 24.6 4' 104 1' 0 01 0 0 0 104 0 26.4 6 103 2' 0 0 0 0 0 103 0 28. 1 8 103 2 0 0 0 0 0 103 0 29.1 10 103 2 0 0 0 0 0 103 0 30.0 12' 103 2 0 0 0 0 0 103 0 31.2 14 103 2 0 0 0 0 0 103 0 311.2 16 103 2 0 0 0 0 0 103 0 32.6 18 103 2 0 0 0 0 0 103 0 32.4 20 103 2 01 0 0 0 0 103 0 32.8 22 103 2 0 0 0 0 0 103 0 - 24 103 2 01 0 0 0 0 103 0 33.4 26 103 2 0 0 0 0 0 103 0 - 28 103 2 0 0 0 0 0 103 0 34.2 30 . 103 2 0 0 0 0 0 103 0 - 32 103 2 0 0 0 0' 0 103 0 34.5 34 102 3 0 0 0 01 0 102 0 - 36 102 3 0 0 0 0 0 102 0 34.8 38 102 3' 0 0 0 0 0 102 0 - 40 101 4 1 0 0 1 0 101 0.99 34.7 42' 100 5 0 0 0 2 0 to0 2.00 - 44 100 5 2 0 0 2 0 1'00' 2.00 35.0 46 99 6 4 0 01 4 0 99 4504' - 48 98 7 4 0 01 4 0 98 4.08 34.6 50 98 7 4 0 0 4 0 98 4.08 - 52 98 7 4 0 0 4 0 98 4.08 34.9 54 98 7 6 0' 0 6 0 98 6.12 - 56 98 7 9 01 0 9 0 98 9.18 34.9 58 97 8 10 0 0 10 0 97 10.31 - 60 % 9 11 0 0 11, 01 96 11.46 35.2 62 95 10 11 0 0 111 0 95 11.48 - 64 93 11 12 0 0 12 0 93 12.90 36.1 66 92 12 12 0 0 12 1 93 12.90 - 68 88' 16 12 0 0 12 2 90 1'3.33' 35.7 70 85 19 13 0 0 13 2 87 14.94 - 72 84 20 13 0 0 13 2 86 15.12 35.5 74 83 21 13 0 0 13 2 85 15.29 - 76 81 23 15 0 0 15 2 83 18.07 36.6 78 75 29' 15 0 0 15 3 78 19.23 - 80 73 30, 16 0 0 16 3 76 21.05 36.6 AT TABLE 17 (continued) SKIN' TOMORS (BASED Qd Mi1CFrlSCX)PIC APPEARANIE ), MORTALITY AND 80DY WEI(41T 3--4M, MWSC-I: 120 (mg/(jrouse x week)) CD1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tumors are included in these values (b) sum of values of coluims 10 and 12 1. e. mice with maacroscopic skin twmrs "in" as well as "in and outside" application area (c) dead mice with macroscopic skin turtors (from colum 10 and 12) (d) sum of values of oolwms 7 and 14 (e) value of column 13 multiplied by 100 and divided by value of column 18, i. e. sum of coiuwms 7 and 14 3285

INBIFO Institut fur biologische Forschung • KOIn SOHREP08T P 0500/3068 UOE1'48RA1i1, A12 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SORVI- MDR- NlMBER CF ALIVE At47 DEAD MICE, FIElICH HAVE OR HAD EFFEC'- MRCfYJ- BODY' WRS TAi.- ITY MAuCRC>E?COPIC SKIN 711MCY+S (a) TIVE NUMBE6t' 90DPIC TLMDR WEIOff 0/0) IN APPL.- AREA OMLY' C(lfSIDE APPL. ARFA, Q1GY IN AND OUTSIDE APPL.-ARFA (b) (c) (d) RATE' (e). (0/0) g ) 0 105 0 0 0 0 0 0 105 0 22.6 2 105 0 0 0 0 0 0 105 0 24.0 4 105 01 0 0 0 0 0 105 0 26.3 6 105 0 0 0 0 0 0 105 0 27.9 8 105 0 0 0 0 0 0 105 0 29.2 10 105 0 0 0 0 0 0 105 0 29.9 12 105 0 0 0 0 0 0 105 0 30.7 14 105 0 01 0 0 0 0 105 0 30.9 16 105 0 0 0 0 0 0 105 0 32.1 18 105 0 0 0 0 0 0 105 0 32.4 20 105 0 0 0 0 0 0 105 0 32.3 22' 105 0 0 0 0 0 0 105 0 - 24 105 0 0 0 0 0 0 105 0 32.7 26 105 0 0 0' 0 0 01 105 0 - 28 105 0 0 0 0 0 01 105 0 34.0 30 103 2' 0 0 0 0 0 103 0 - 32 102 3 0 0 0 0 0 102' 0 34.1. 34 101 4 0 0 01 0 0 101 0 - 36 101 4 0 0 0 0 0 101 0 34.3 38 101 4 0 0 0 0 0 101 0 1 - 40 100 5 01 0 0 0 0 100 0 33.9 42 100 5 0 0 0 0 0 100 0 - 44 99 6 0 0 0 0 0 99 0 34.8: 46 99 6 0 0 0 0 0 99 0 - 48 99 6 0 01 0 0 0; 99 0 34.9 50 98 7 0 0 0 0 01 98 0 - 52 98 7 0 0 0 0 0 98' 0 34.5 54 98 7 1 0 0 1: 0 98: 1.02 - 56 98 7 2 0 0 2 0 98 2.04 35.5 58 97 8 3 0 01 3 0 97 3.09 - 60 97 8 4 0 0' 4' 0 97 4.12' 36.0 62 96 9 4 0 0 4 0 96 4.17 - 64 92' 12 5 0 0 5 0 92 5.43 34.5 66 91 13 8 0 0 8' 0 91 8.79 - 68 89 15 9 0 0 9 0 89 10.11 36.2' 70 85 19 10 1 0 10 1 86 11.63 - 72 83 21 111 1 0 11 1 84 13.10 35.7 74 78 26 11' 1 0 11 1 79 13.92 - 76 75 29 13 1 0 13 1 76 17.11 36.0 78 70 33 13 1 0 13 2 72 18.06 - 80 66 37 13 1 0 13 4 70 18.57 35.9 AT'TAH1.E 17 (continued) SKIN' 1U61Q11S' (BASED CN MACA06C70PIC' APPEARJINCE) y MDRTALIThf A[aD BODY WEIQ-JT 4-GR, MW".SC-I ( f) r, 60 ( m3/ ( mouse. x week )) CDI MICE (a) only preliminary diagnosis based on macroseopic appearance, mice which lost their macrosoopic tunors are included in these values (b) sum of values of eolumns 10 and 12 1. e. mice with macroscopic skin tumors "in" as well, as "i'n and outside" application area (c) dead mice with macroscopic skin tuirors (from colurtn 10 and 12) (d)'sum,of values of columns 7 and 14 (e)'value of coluim, 13 multiplied by 100 and:divided by value of coluim 18, i. e. sum of colunns 7 and 14 (f),stored at 4 degrees centigrade. Condensate of all other groups stored at -75 degrees centigrar3e. 3285

IMBIIFO Institut fOr biologische Forschung • K6ln: SUBREPDRT P 0500/3068 OBE148RA13, A14 5302 M 7 9 10 111 12 1'3' 14 18 19 20 WEEK SURVI- l4OR- NOMBER OF'AiLIVE AND DEAD MICE, WHICH HAVE CY2'ItAD EFFEC- MACRO- BODY 4VRS 7riL- ITY MAC1O6C'OPIC SKIN T()FURS (a) TIVE N:]lBER SCOPIC T(IM`1R WEIC;KP' IN APPL.- OUISIDE IN AND (b) (c) AREA QiLY APPL.-ARFA OUISIOE ON[.Y APPL.-AREA (d) RATE (e) (0/0) (0/0) (g ) 0 105 0 0 0 0 0 0 105 0 22.2 2 1'05 0 0 0 0 0 0 105 0 23.2 4 105 0 0 0 0 0 0 105 0 25.1 6 104, 1 0 0 0 0 0 104' 0 26.7 8 104 1 0 0 0 0 0 104 0 27.8 10 104 1 0 0 0 0 0 104 0 28.9 12 104 1 0 0 0 0 0 104 0 29.6 14 104 1 0 0 0' 0 0 104 0 30.3 16 104 1 0 0 0 0 0 104 0 31.0 18 104 1 0' 0 0 0 0 104 01 31.2 20, 104 1 01 0 0 0 0 104 0' 31.2 22 104 1 0 0 0 0 0 104 0' - 24 104 1: 0 0 0 0 0 104 0 31.9' 26 103 2 0 0 0 0 0 103 0 - 28 103 2 0 0' 0 0 0 103 0 32.8 30 102 3 0 0! 0 0 0 102 0 - 32 101 4 0 01 0 0 0 101 0 33.5 34 101 4 0 0 0 0 0 101 0 - 36 101 4 0 0 0 0 0 101 0 33.5 38 101 4 0 0 0' 0 0 101 0 - 40 100 5 0 0 0 0 0 100 0 32.9 42' 99 6 0 0 0' 2 0 99 2:02' - 44 98 7 0 0 0 4 0 98 4.08 33.7 46 98' 7 4 0 0 4 0 98 4.08 - 48 98' 7 4, 0 0 4 0 98 4.08 33.9' 50 96 9 4 0 0 4, 0 96 4.17 - 52 95 10 4 0 0 4 0 95 4.21 34.2 54 95 10, 4 0' 0 4 0' 95 4.21 - 56 94 10 4 0 0 4 0 94 4.26 34.8 58 94 10 4 0 0 4 0 94 4.26 - 60 92 12 5 0 0 5 0 92 5.43 9 34.9 62' 92 12 5 0 0 5 0 92 5.43 - 64I 91 13 5 0 0 5 0 91 5.49 33:8 66 89 15 6 0 0 6 1 90 6.67 - 68 88 16 6 0 0 6 1 89 6.74 35.4 70 88 16 6 0 0 6 1 89 6.74 - 72 87 17 9 0 0 9 1' 88 10.23 35.0 74 87 17 10 01 0 10 1 88' 11.39 - 76 85 19 10 0 0 10 1 86 11.63 34.7. 78 84 20' 10 0 0 10 1 85 11.76 - 80 80 24 10 0 0 10 1 81 12.35 35.4 AT TABLE 17 (continued) SKIN'1S7•1CNtS' (NASBID CN MACIm6COPIC APPEARANCE), MDRTALITY AND BDDY WN:I(;ff 5-cdR, KiSC-I (f): 90 ( mg/ ( tuouse x week )) CD1i MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic twmrs are included in these values (b) sum of values of coluoms 10 and 12 1. e. mice with macroscopic skin tuimors "in"'as well as "in and outside" application area (c)'dead mice with macroscopic skin tunors (from column 10 and 12) (d) sum of values of columns 7 and 14 (e) value of coluwm 13 multiplied by 100 and divided by value of coL.am 18, i. e. sum of coTuanns 7 and 14 (f) stored at 4 degrees centigrade. Condensate of all other groups stored at -75 degrees centigrade. 3285

INBIFO Institut fuir biologische Forschung • h'<t9in SUBREPaRr P 0500/3068 [6E148RA15, A16 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SORVI- MOR~- NUPffiER OF ALIVE AND DF.AD MICE, AHICH' NAVE Qt HAD EFFEC- MACRO- BDDY 4O1LS TAL- ITY MAC%.~6C()PIC SKIN' 7UM3RS (a) TIVE NURSF.R SWPIC TUK3R WEIGHP IN APPL.- WiSIDE IN AND (ki)' (c) AREA (KLY APPL.-AREA 0[/I5IDE Q3LY APPL.-ARFA (d) RA1E (e) (0/0)' (0/0) (g) 0 105 0 0 0 0 0' 0 105 0 21.9' 2 105 0 0 0 0 0 0 105 0 23.1 4 103 2 0 0, 0 0 0 103 0 25.0 6 102 3' 0 0 0 0 0' 102 0 26.6 8 102 3 0 0 0 0 0 102 0 27.5 10 102 3 0 0 0 0 0 102 0 28.7 12' 102 3 0 0 0 0 0 102 0 29.7 14 102 3 0 0 0' 0 0 102 0 29.6 16 102 3 0: 0 0' 0 0 102 0 30.7 18 102 3 01 0 0 0 0 102 0 30.9 20 102 3 0 0 0 0 0 102 0 30.8 22 102 3 0 0 0 0 0 102 0 - 24 102' 3 0 0 0 0 0 102 0 31.7 26 102 3 0 0 0 0' 0 102 0 - 28 102 3 0 0 0 0 0 102 0 32.5 30 102 3' 1 0 0 1 0 102 0.98 - 32 102 3 1 0 0 1 01 102 0.98 33.2 34 102 3 1 0 0 1 0' 102 0.98 - 36 102 3 1 0 0 1, 0 102' 0.96 33.1 38: 102 3 2 0 0 2 0 102 1.96 - 40, 102 3 2 0 01 2 0 102 1.96 32.2 42 102 3 0 0 0 2 0 102 1.96 - 44 101 4 2 0 0 2 0 101 1.98 32.6 46 109' 4 2 0 0 2 0 101 1.98 - 48 101 4 2 0 0 2 0 101i 1.98 33.3' 50 101 4 2 0 0 2 0 101 1.98 - 52 99 6 2 0: 0 2 0 99 2.02 34.0 54 98 7 3 01 0 3 01 98 3.06 - 56 97 8' 3 0 0 3 0 97' 3.09 34.2 58 94 10 3 0 0 3 1 95 3.16 - 60 911 13 3 0 0 3 1 92' 3.26 34.3 62 90 14 4 0 0 4 1 91 4.40 - 64 87 17 4 0 01 4' 1 88 4.55 33.2 66 84 20 5 0 0 5 1 85 5.88 - 68 84 20 5 0 0 5 1 85 5.88 34.8 70 78 26 6 0 0 6 1 79 7.59 - 72 78: 26 8 0 0 8 1 79 10.13 34.6 74 78 26 9 0 0 9 1 79 11.39 - 76 76 28' 11 0 0 11 2' 78 14.10 34.6 78 70 33' 13 01 0 13 4 74 17.57 - 80 69 34 13 1 0 13' 4 73 17.81 34.5 AT TABLE 17 (continued) SKIN' 1SR40RS (llASED CN MACRLlS00PIC APPEARANCE)', MO4II'ALITY AND BODY WEI(HP 6-GR, M'ASC-I (f): 120 (pg/(mouse x week)) CU1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tumors are included in these values (b) sum of values of colunns TO and 12 i. e. mice with macroscopic skin twnors "in" as well as "'in and outside" application area (c) dead'mice with macroscopic skin tumrs (fran column 10,and 12) (d) sum of values of colunns 7 and 14 (e) value of ooTumn 13 multiplied by 100 and divided by value of oolumi 18, i. e. sum of columns 7 and 14 (f),stored at 4 degrees centigrade. Condensate of all other groups stored at -75 degrees centigrade. 3285

INBIFO Institut fWr biol'ogische Forschung • KSIn S[DBREPORT'P'0500/3068 1BE148RA17, A1'8 5302 M 7 9 10 11, 12 13 14 18 19 20 WEEK SORVI- 4OFS MOR- T1YL- ITY NU40ER OF'ALIVE AND OFAD MICE, WHICH HASdE: C1<t'HAD MACROBODPIC SKIN T()MORS (a) EFFEC- TIVE NikIBER MAC7t0+ SODPIC T(kpR BODY WEIGHT 0/0)' IN APPL.- ARElti CNLY CVISIaE APPL.-ARFA CNLY IN AND CUISIDE APPL.-ARFl\ (b), (c) (d) FATE (e) (0/0) g) 0 105 0 0 0 0 01 0 105 0 20:7' 2 105 0 0 0 0 01 0 105 0 22.4 4 105 0 0 0 0 0 0 105 0 24.8 6 105 0 0 01 0 0 0 105 0 26.4 8 105 0' 0 0 0 0 0' 105 0 27.6 10 105 01 0 0 0 0 01 105 0 28.6 12' 105 0 0 0 0 0 0 105 0 29.1 14 105 0 0 0 0 0 0 105 0 29.7 16 105 0 0 0 0 0 0 105 0 30.3 18 105 0 01 0 0 0 0 105 0 30.5 20 105 0 01 0 0 0 0 105 0 30.7 22 104 1' 1 0 0 1 0 104 0.96 - 24 104 1 1 0 0 1 0 104 0.96 30~9 26 103 2 2 0 0 2 0 103 1.94 - 28 103 2 2 0 0 2 0 103 1.94 31.9 30 103 2 2 0 0 2 0 103 1.94 - 32 102 3' 3 0 0 3 0' 102 2.94 32.5 34 102 3 4 0 0 4 0 102 3.92 - 36 102 3 5 0 0 5 0 102' 4.90 32.3 38 102 3 6 0 01 6 0 102 5.88 - 40 102 3 9' 0 0 9 0 102 8'.82' 32.6 42 102 3 0 0 0 11 0 102 10.78 - 44 102' 3 11 0 0 11 0 102 10.78 32.5 46 101 4 11 0 0 11 0 101 10.89 - 48 100 5 13 0 0 13 0 100 13.00 32.5 50 100 5 13 0 0' 13 0 100 13.00 - 52 100 5 16 1 0 16 0 100 16.00 33.5 54 99 6 19 1 0 19 0 99 19.19 - 56 99 6 20 2 0 20 0 99 20.20 33.7. 58 97 8 21 1 1 22 2 99 22.22 - 60' 97 8 23 1 1 24' 2 99 24.24 33.5 62 96 9 24 1 1 25 2 98 25.51 - 64, 93 11 26 1 2 28' 2 95 29.47 32.6 66 93' 111 27 1 3 30 2 95 31.58 - 68 91 13 28 1 3 31 4 95 32.63 34.0 70 91 13 32 2 3 35 4 95 36.84 - 72 91 13 34 3 3 37 4 95 38.95 33.6 74 90 14 34 3' 3 37 4 94 39.36 - 76 88 16 35 3 4 39 5 93 41.94 33.6 78 82 22 38 3 4 42 7 89 47.19 - 80 77 27 39 3 4 43 11 88 48.86 34.0 AT TABLE 17 (continued). SKIN 7Llt0ItS (BASED Qd MACR06QOPIC APPEAR1iNCE), MatTALITY AND BODY WEI(HI' 7-GR, 0lIDA P1[1S MWSC-Ir 60 (mg/(house x week)) CD1 MICE (a) only preliminary diagnosis based on macroseopic appearance, mice which lost their macroscopic tumrs are included in these values (b) sinm of values of eolumn.s 10 and 12 1. e. mice with macroscopic skin tunors "in"'as well as "in and outside" application area (c)~dead mice with macroscopic skin tumors (fran column 10 and 12) (d) sum:of values of columis 7 and 14. (e) value of column 13'multiplied by 100 and divided by value of column 18, 1. e. sum of cvlimns 7 and 14 3285

INBIFO Institut for biologische Forschung • KOin SUBREPORT P 0500/3068' OBE448RA19, A20 5302 M 7 9 1'0' 11 12 13 14 16 19 20 WEEK S(7RVI- MOR- NOM®ER CIF ALIVE AND DP.AD MICE, P1EiICH' NAVE (1R HAD EFFFx- MACRO- BODY 4r)R6 TAL- Mi1CR06COPIC SKIN 7t)r10RS (a) TIVE SCAPIC WEIGEHI' ITY NJMBER T[kqR IN APPL.- CUPSIOE IN AND (b) (c) AREA QJLY APP4. kRFA OUISIDE QH.Y APPL.-ARPA (d) RATE (e) (0/0) (0/0) (9). 0 105 0 0 0 0 0 0 105 0 21.2 2 105 0 0 0 0 0 0 105 0 22.5 4 105 0 0 0 0 0 0 105 0 24.9 6 104 1 0 0 0 0 0 104 0 26.6 8 104 1 0 0 0 0 0 104 0 27.9 10 104 1 0 0 0 0 0 104 0 29. 1 i 12 103 2 0 0 0 0 0 103 0 29.6 14 103 2 1 0 0 1 0 103 0.97 30.0 16 103 2 1 0 0 1' 0 103 0197 30.7 18 103' 2 3 0 0 3' 0 103 2.91 30.7 20 102' 3 3 0 0 3 0 102 2.94 30.9 22 101 4 4 0 0 4 0 101 3.96 - 24 101 4I 8 0 0 8 0 101 7.92 31.4 26 101 4' 8 0 0 8 0 101 7.92 - 28 101 4 9 0 0 9 0 101 8.91 32.3 30 101 4 11l 01 0 11 0 101 10.90 - 32 101 4 12 1 0 12 01 101 11.88 33.0 34 101 4 15 1 0 15 01 101 14.85 - 36 100 5 17 1 0 17 1 1'01 16.83 32.6 38' 100 5 18 1 0 18 1 101 17.82 - 40 100 5 21 2 0 21 1 100 20.79 32.9 42 100 5 23 2 0 23 1 101 22.77 - 44 99 6 26 2 0 26 1 100 26.00 32.9 46 99 6 29 2 0 29 1 100 29.00 - 48 99 6 32 2 0 32' 1 100 32.00 33.3 50 99 6 31 2 1 32' 11 100 32.00 - 52 98 7 33 2 1 34 2 100 34.00 33:6 54 96 9 33 2 1 34 4 100 34.00 - 56 95 10 35 2 1 36 5 100 36.00 34.2 58 95 10 35 2 2 37 5 100 37.00 - 60 95 10 37 2 2 39 5 100 39.00 34.3 62 95 10, 37 2 2 39 5 100 39.00 - 64' 94 10 41 2 2 43 6 100 43.00 33.2 66 93 11 43 2 3 46 7 100 46.00 - 68 92 12 43 2 3 46 8 100, 46.00 34.7 70' 87 17 44 2 3' 47 12 99 47.47 - 72' 85 19 45 2 5 50 14 99 50.51 34.3 74 85 19 45 3 6 51 14 99 51'.52' - 76 82 22 46 3 6 52 14 96 54.17 34.5 78 79 25 49 3 6 55 15 94 58.51 - E0 73' 30 49 3 6 55 19 92 59.78 35.0 AT TABLE 17 (continued). SKIN TiJMOAS (BASED ON MACRO6C(JPIC APPFARANCE), lY3RTALITY AND BODY WEICRIP' 8-GR, OMBA PLU3 MWSC-I: 90 (mg/(mouse x week)) CDI MICE (a) only preliminary diagnosis based an macroscopic appearance, mice which lost their macroscopic tuffors are included:in these values (b) sum of values of colurms 10 ard 12 i. e. mice with macroscopic skin tunmrs "in"'as well as "in and outside" application area (c) dead mice with macroscopic skin tumors (from colum 10 and 12) (d) sum of values of colinms 7 and 14. (e) value of colurtn 13'multiplied by 100 and divided by value of colum 18, i. e. sum of caIuaens 7 and 14 3285

I1NBIFO Institut fur biologische Forschung • K6I'n SUBREPORT P 0500/3068 U8E148RA21, A22 5302 M 9 10 1'1' 12 13 14 18 19 20 WEEK SURVI- MDR- NCR9BER OF ALIVE'AND DEAD MICE, WHICH HAVE OR HAD EFFEC- MFICIYO- BODY' VO[t4 1h[.- MACRaSCOPIC SKIN T[)r1DRS (a) TIVE SOOPIC WEIGHT ITY NUMBER R[lhDR 0/0) IN'APPL.- AREA Q1LY COTSIDE APPL.-ARFA QNII.Y IN'A1•D CJIASIDE APPL.-ARFA (b) (c) (d), BATE (e) (0/0), g) 0 105 0' 0 0 0 0 0! 105 0 22.1 2 105 0' 0 0 0 0 0' 105 0 23.4 4 105 0 0 0 0 0 0 105 0 25.6 6 105 0 0 0 0 0 0 105 0 26.9 8 105 0 0 0 01 0 0 105 0 28.3 10 105 0 0: 0 0 0 0 105 0 28.7 12 105 0 0' 0 0 0 0 105 01 29.9 14 105 0 1 0 0 1 0 105 0.95 30.7 16 105 0 2 0 0 2 0 105 1.90 30.9' 18 105 0 3, 0 0 3 0 105 2.86 31.2' 20 105 0 5 01 0 5 0 105 4.76 31.4 22 105 0 5 0' 0 5 0 105 4.76 - 24 105 0 7 0 0 7 0 105 6.67 32.4 26 105 0 8 0 0 8 0 105 7.62 - 28 105 0 8 0 0 8 0 105 7.62 33.0 30 105 0 11 0 0' 11 0 105 10.50 - 32 105 0 13 0 0 13' 0 105 12.38~ 33.6 34 105 0 15 0 0 15 0 105 14.29 - 36 105 0 16 0 0 16 0 105 15.24 33.6 38 104 1 16 0 0 16 0 104 15.38 - 40 103 2 20 0 0 20 1 103 19.23 32.9 42 101 4 24 0~ 0 24 2 103 23.30 - 44 101 4 27 01 0 27 2' 103 26.21 33.0 46 99 6 28 0 0 28 3 102 28.43 - 48' 99 6 28 0 2 30 3 102 29.41 34.1 50' 97 8 29 0 2 31 4 101 30.69 - 52 96 9 31' 0 2' 33 5 101 32'.67' 34.2 54, 96 9 33 0 2 35 5 101 34.65 - 56 93 11, 34 1, 2 36 6 99 36.36 34.3 58 93 17 36 1 3 39 6 99 39.39 - 60 92 12 37 1 3 40 7 99 40.40 35.3 62 89 15 35 2 5 40 9 98 40.82 - 64 87 17 37 1 6 43 11 98 43.88 33.8 66 85 19 39 1 6 45 13 98' 45.92 - 68 84 20 40 1 6 46 13 97 47.42 35.7 70 82 22 44 1 6 50 15 97 51.55 - 72 79 25 49 2 6 55 17 96 57.29 35.3 74 75 29 51 2 6 57' 20 95 60.00 - 76 73 30 52 2 7 59 22 95 62.11 36.0 78 69' 34 55 2 7 62 25 94 65.96 - 80 68 35 57 2 7 64 26 94 68.09 35.8' AT TABLE 17 (continued) SKIN RlM10RS (BASED CN'MACROBCiOPIC APPEAR74NCE),, MOFa'ALITY AND BODY WEIQIT 9-GR;. OMiAPLUS MWSC-I: 120(rtg/(mouse x week.)'.). CD1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroswpic tunors are included in these values (b) suo of values of colwms 10 and 12 i. e. mice with macroscopic skin tumors "in" as well as "in and outside" application area (c) dead~mice with macroscopic skin tumors (fran~colum 10 and 12) (d) sum of values of colunns 7 an&1'4 (e) value of column 13 multiplied by 100 and divided by value of coluim 18,, i. e. sino of columns 7 and 14 3285

INBIFO Institut fur biologische Forschung • Ktilni SUBREPORT P 0500/3068 1IBE148RA23, A24 5302 M 7 9 10 11 12 13 14 ~ 18: 19 20 WEEK SURVI- MDR- NUMBER'QF ALIVE AND DEAD MICE, NHICN HAVE CR HAD EFFEC- MAC1><}~- BODY WRS TAtr P19f MACRO6WPIC SKIN 7UI43RS (a) TIVE NUMBER SOOPIC T111OR WEIGKP IN APPL.- Oll15IDE IN AND (b) (c) ARE{4 CNLY APPt.. ARFh 01115IDE ONLY APPL. AREA (d) RATE (e) (0/0) (0/0) (9) 0 105 0 0 0 0 0 0 105 0 21.8' 2 105 0 0 0 0 0 0 105 0 23.8 4 105 0 0 0 0 0 0 105 0 25.6 6 105 0 0 0 0 0 01 105 0 27.6 8 105 0 0 0 0 0 0 105 0 28.5 1'0' 105 0 0 0 0 0 0 105 0 28.7 12 105 U 0 0 01 0 0 105 0 29.8 14 105 0 1 0 0 1 0 105 0.95 30.9 16 105 0 1 0 0 1 0 105 0.95 31.1 18 105 0 2 0 0 2' 0 105 1.90 31.1' 20 104 1 2 0' 0 2 0 104 1.92 31.8 22 104 1 4 01 0 4 0 104 3.85 - 24 104 1 6 0 0 6 0' 104 5.77 32.8 26 104 1 6 0 0 6 0 104 5.77 - 28 104' 1 7 0 0: 7 0 104 6.73' 32.6 30 104 1 9 0 0 9 0 104 8.65 - 32 102 3 9 0 0 9 0 102 8.82 33.5 34 101 4 12 0 0 12 0 101 11.88 - 36 101 4' 13 0 0 13 0 101 12.87 33.5 38 99 6 13 0 0 13 1 100 13.00 - 40 98 7' 14: 0 0 14 1 99 14.14 33.2 42 97 8 14' 0 0 14 1 98 14.29 - 44 96 9 18 0 0 18 1 97 18.56 -33.7 46 96 9 20 0 0 20 1 97 20.62 - 48 96 9 23 0 0 23 1 97 23.71 34.2 50 95 10 23 0 0 23' 1. 96 23.96 - 52 95 10 26 0 0 26 1 96 27.08 34.0 54 95 10 27 0 0 27 1 96 28.13 - 56 95 10 30 0 0 30 1 96 31.25 34.2 58 93 11 31 0 0 31 2' 95 32.63 - 60 92 12 37' 0 0 37 3 95 38.95 35.0 62' 90 14 38 0 0 38 3 93 40.86 - 64 86 18 38' 0 01 38 5 91 41.76 35.0 66 83 21 42 0 0 42 6 89 47.19 - 68 82' 22 45 0 0 45 7 89 50.56 35.1' 70 79 25 50 0 0 50 10 89 56.18 - 72 75 29 50 0 0 50 11 86 58.14 35.4 74 74 30 51 01 0 51 12 86 59.30 - 76 70 33 55 0 0 55 14. 84 65.48 35.9 78 67 36 59 0 0 59 15 82' 71.95 - 80 62 41 59 0 0 59 19 81 72.84 36.3 AT TABIE 17' (continued) SKIN 2lqNORS (BASED ON MhCR06COPIC APPEARfiNCE), MORTALITY AND BODY WEIOff 10-02, .,WSC-I: 60 (mg/(mouse x week)) CD1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tumrs are included in these values (b) sum of values of colLnms 10 and 12 i. e. mice with macroscopic skin twmrs "in" as well as "'in and outside" application area (c) dead mice with macroscopic skin tunors (fran colum 10 and 12) (d) sinn of values of colunns 7 and 14 (e)' value of colum 13 multiplied by 100 and divided by value of colwm 18, i. e. sum of colwms 7 ard 14 3285

INBIFO Institut fur bi©logische Forschung • KtSln SOBREPORT'P'0500/3068 1BE148RA25, A26 5302 M 7' 9 10 11 12 13 14 WEEK SOR\7I- MOR- N[l4BER CF ALIVE AND DEAD MICE, WHICH HAVE'OR'HAD VOR.S TAL- MACROBCOPIC SKIN 1UMORS (a) ITY IN APPL.- QIilSIDE IN AND (b)' (c) l1RFA CNLY APPL.-AREA CUPSIDE QNLY APPL.-ARFA 18 19 20 EFFEC- M1uCRO- BODY TIVE SOOPIC WEIGiff' NIlC1BER T[)rUR' BATE (d) (e) (0/0) (0/0) (9 ) 0 105 1 0 0 0 01 0 104 0 22.5 2 105 1 0 0 0 0 0 104 0 24.0 4 103 2 0 0 0 0 0 103 0 25.8 6 103 2' 0 01 0 0 0 103 0 26.7 8 101 4 0 0 0 0 0 101 0 28.0 10 100 5 0 0 0 0 0 100 0 28.7 12 100 5 0 0 0 0' 0 100, 0 29.2 14 100 5 1' 0 0 11 0 100 1.00 30.5 16 100 5 1' 0 0 1 0 100 1.00 30.5 18 1'00 5 1' 0 0 1 0 100 1.00' 30.7 20 98' 7 2 0 0 2 0 98 2.04 31.4 22 98 7 6 0 0 6 0 98 6.12 - 24 98 7 6 0 0 6 0 98 6.12 32.7 26 97 8 7 0 0 7 0 97 7.22 - 28 97 8 9 0 0 9 0 97 9.28 32.3 30 97 8 10 0 0 10 0 97 10.30 - 32' 97 8' 13 0' 0 13 0 97 13.40 33.4. 34 97 8 16 0 0 16 01 97' 16.49 - 36 97 8 18 0 0 18 0 97' 18.56 33.6 38' 97 8 19 0 0' 19 0 97 19.59 - 40, 97 8 25 0 01 25 2 97 25.77 33.4 42 97 B 29 0 0 29 0 97 29:90i - 44 96 9 35 0 0 35 0 96 36.46 33.8 46 96 9 38 0 0 38 0 96 39.58 - 48 94 10 41 0 0 41 1 95 43.16 34.1' 50 92 12 44 0 0 44 2 94 46.81 - 52 90 14 46 0 0 46 3 93 49.46 34.4 54 89 15 50 01 0 50 4 93 53.76 - 56 86 18 54' 0 0 54 7 93 58.06 34.6 58 85 19 57 0 0 57 8 93 61.29 - 60 85 19 57 0 0 57 8 93 61.29 34.0 62 83 21 60 0 0 60 10 93 64.52 - 64 78 26 68 0 0 68 15 93 73.12 34.3 66 76 28 70 0 01 70 16 92 76.09 - 68 71' 32 71 0 0 71' 20 91 78.02 33.5 70 69 34 72 0 0 72 21 90 80.00 - 72 67 36 72 0 0 72 22 89 80.90 34.2 74 59 44 74 0 0 74 29 88 84.09 - 76 56 47' 75 0 0 75 31' 87 86.21 35.3 78 51 51 78 01 0 78 36 87 89.66 - 80 47 55 79 0 0 79 40 87 ' 90.80 35.1 AT TABLE 17 (continued) SKIN "I[JNqRS ( BhSED (x1 MAICAOBCOPIC APPEARANCE)', MOKIAISTY AND BODY WEI(iiT 11-GR, ShlSC'-1e 90 (mg/(mcwse x week)) CD1 MICE (a) only preliminary diagnosis based on macrosoopic appearance, mice which lost their macroscopic tumors are included in these values (b) sum of values of colwms 10 and 12 i. e. mice with macroscopic skin tumors "in" as well as "in and outside" application area (c)'dead mice with macroscopic skin tumnrs (fram cvliam 10 and 12) (d) sum of values of coluwms 7 and' 14 (e)' value of column 13 imultiplied by 100 ' and divided by value of column 18, i. e. sum of colwms 7 and 14 3285

INB'IFO Institut fur biologische Forschung • Kolln SfJBREPORT P 0500/3068 1BE148RA27, A28 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SURVI- MD1T- NUMBER OF ALIVE:AtD DEAD MICE, I4NICH HAVE CR HAD EFFEC- MNCRD BODY WRS 'ML- ITY MACR0S(~,'OPIC SKIN'1[&4aRS (a) TIVE N[1MBER SCaPIC 1WR WEIGHf' IN APPL.- Q715IDE IN AND (b) (c) AREA ONLY APPL.--ARFA OUISIDE QiLY APPL.-AREA (d) RATE (e) (0/0) (0/0) (g) 0 105 0 0 0 0 0 0 105 0 22.0 2 105 0 0' 0 0 0 0 105 0' 23.2 4 104 1 0 0 0 0 0 104 0' 24.7' 6 101 4 0 0 0 0 0 101 0 26.1 8 96 9 0 0 0 0 0 96 0 27.7 10 95 10 0 0 0 0' 0 95 0 28.4 12 95 10 0 0 0 0 0 95 0 28.7 14 95 10 0 0 0 0 0 95 0 30.0' 16 95 W 0 0, 0 0 0 95 0 30.0 18 95 W 4 0 0 4 0 95 4.21 30.1 20 93 11 5 0 0 5 01 93 5.38 30.5 22 93 11 5 0 0 5 01 93' 5.38 - 24 93 11 B 0 0 8 0 93 8.60 32.1 26 93 11 10 0 0 9 0 93 10.75 - 28 93 11 13 0 0 13 0 93 13.98 31.4 30, 93 11 18 0 0 18 0 93 19.35 - 32 93 11 21' 0 01 21 0 93 22.58 32.8 34 93 11 21 0 0 21 0 93 22.58' - 36 91 13 23 0 0 23 1 92 25.00: 32.4 38 90 14 27 0 0 27 1 91 29.67 - 40 90 14 34 0 0 34 1 90 37.36 32.2 42 90: 14 38 0 0 38 1 91, 41.76 - 44 89 15 46 0 0 46 1 90 51.11 32.9 46 89 15 51 0 0 51 1 90 56.67 - 48 50 89 87 15 17 52 57 0 0: 0 0 52 57 1 3 90 90 57.78 63.33 33.5 - 52 86 18 59 01 0 59 3 89 66.29 33.1 54 84 201 61 01 0 61 5 89 68.54 - 56 83 21 64 0 0 64 6 89 71.91i 33.7 58 78 26 67 0 0 67 10 88: 76.14 - 60 76 28 69 0 0 69 12 88: 78.41 33.3 62' 74 30 70 0 0 70 14 88 79.55 - 64 70 33 71 0 0 71 18 88 80.68 33.5 66 68 35 72' 0 0~ 72 20 88 81.82 - 68 64 39 73 0 0 73 23 87 83.91 33.0 70 60 43 76 0 0 76 27 87 87.36 - 72 59 44 77 0 1 78' 28 87 89.66 34.0 74 55 48 77 0 1 78: 31i 88 88.64 - 76 51 51 79 0 2 81: 36 87 93.10 35.6 78 48 54' 82 0 2 84 39 87 96.55 - 80 41 61' 82 0 2 84, 46 87 96.55 35.8: AT TABLE 17 (aontinued) SKIN 1Sk10RS (BASED 1 CN' MACAF76COPIC APPEARANCE ), MO1iT'ALITS( AND BODY' WEIGHT 12-GR, SWSC-I: 120 (ng/(mouse x week)) CD) MICE (a) only preliminary diagnosis based on macroscopic appearance,, mice wkiich lost their macroscopic tunorss are included in these values (b) sum of values of columns 10 and 12 i. e. miee'with macroscopic skin turtors "in" as well as "in and outside" application area (c) dead mice with macroscopic skin tumors (from aolufln 10 and 12) (d) sum~of values of oolunns 7 and 14 (e) value of cvlumn 13 muTtiplied by 100 and divided by value of colwm 18, i. e. sum of columis 7 and 14 3285

IINBIFO Institut fur biologische Forschung • N<6in SUBREPORT P 0500/306,8 OBS148RA29, A30 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SURVIi- MDR- NUIIDER OF ALIVE'AND UFliD MICE, NNICH HAVE OR HAD EFFDC- MACRo- BODY WRS 1111.- MhCR06COPIC SKIN'7VMORS (a) TIVE SQ7PIC WEIGKP ITY KIMBER 1LMR IN APPL.- CUP3IDE' IN AND (b) (c) AREA Cf1LY APPL. AREA CJ4T1`SIDE ONLY APPL. ARFA (d) RATE (e) (0/0) (0/0)' (9): 0 105 0 0; 0 0: 0 0 105 0 22.4 2 105 0 0 0 0' 0 0 105 0 23.7 4 105 0 0 0 0 0 0 105 0 25.6 6 105 0 0 0 0 0 0 105 0: 27.3 8 105 0 0 0 0 0 0 105 0' 28.7 10 105 0 0 0 0 0 0 105 01 29.5 12 105 0 0 0 0 0 0 105 01 30.0 14 105 0 1 0 0 1 0 105 0:95 30.2 16 105 0 1 0 0 1' 0 105 0:95 30.3 18 105 0 3 0 0 3 0 105 2.86 3069 20 105 0 3 0 0 3 0 105 2.86 31.4 22 103 2 3 0 0 3 0 103 2.91 - 24 102 3 5 0 0 5 0 102 4.90 32.3 26 101 4' 7, 0 0 7 0 101 6.93 - 28 100 5 10 0 0 10 1 101 9.90 32.3' 30 98 7' 12 0 0 12 1 99 12.12 - 32 98 7' 13 0 0 13 1 99 13.13 32.9 34 97 8' 14 01 0 14 1' 98 14.29 - 36 97 8: 14' 01 0 14 1 98 14.29 33.4 38 97 8 19 0' 0 19 1' 98 19.39 - 40 96 9 25 0 0 25 1' 97 25.77 32.9 42' 96 9 26 0 0 26 1 97 26.80 - 44 95 10 31 0 0 311 1 97 31.96 32.2 46 94 10 34 0 0 34 2 96 35.42 - 48 94' 10 40: 0 0 40 2 96 41.67 34.0 501 93 11 46 0 0 46 3 96 47.92 - 52 92 12 47 0 0' 47 3 95 49.47 33.6 54 91 13 51 0 0' 51 3 94 54.26 - 56 86 18 53 0 0 53 7 93 56.99 33.2 58 85 19 57 0 0 57' 7 92 61.69 - 60 82 22 63 0 0 63 9 91 69.23 34.4 62 81 23 64 0 1 65 10 91 71.43 - 64 75 29 66 0 1 67 14 89 75.28 34.2 66 72 31 66 0 1 67 16 88 76.14 - 68 70 33 69 0 1 70 18 88 79.55 33.9 70 69 34 72 0 1 73 18 87 87.36 - 72 60 43 73 0 3 76 24 84' 90.48 33.8 74 56 47 74 0 5 79 28 84' 94.05 - 76 48 54 74 0 5 79 36 84' 94.05 35.2 78 43 59 76 0 5 81 41 84 96.43 - 80 39 63 76 0' 5 81 45 84 96.43 34.4. AT TABLE 17 (continued) SKIN.IUM)1L4 (BASED ON MACfifJCiWPIC APPEARANCE), MC4i!I'ALITYAND BODY WEI(}fF 13-GR, OMBA PLOS SWSC-I: 60, (xg/(mouse x week)). CD1 MICE (a),only preliminary diagnosis based on macroscopic appearanoe, mice which lost their macroscopic turtors are included'in these values (b) sumiaf values of columis 10 and 12 i, e. mice with macroscopic skin tumors "in" as well as "in and outside" application area (c) dead mice with macroscopic skin tumors (from colimn 10 and 12) (d),sum of values of columns 7 and 14 (e): value of column 13 muitiplied by 100 and'd'ivided by value of column 18, i. e. sum of columns 7 and 14 3285

INBIFO Institut tor biologische Forsclhung. • KOIn SOOREPORT P 0500/3068 OBE148RB1, 82 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SURVI- lqR- N.RMBER aF ALIVE AND DEAD MICE, NHICH HAVE CR HAD EFFEC- MACRO- BDDY iARS TAL- ITY MWC1a0600PIC SKIN"lUMORS (a) TIVE NOMBER SODPIC TOMDR WEIC~iF' IN APPL.- ClHSIDE IN AND (b)', (c) AREA ONLY APPL. ARFA GTISIDE CHS.Y APPL. ARFA (d) PATE (e) (0/0) (0/0) (9) 0 105 0 0 0 0 0 0 105 0 21.3 2 105 0 0 0 0 0 0 105 01 23.0 4 105 0 0 0 0 0 0 105 0 25.3 6 105 0 0 0 0 0 0 105 0 26.7 8 104 1 0 0 0 0, 0 104 0 28.2 10 104 1 0 0 0 01 0 104 0 28.6 12 104 1 0 0 0 0 0 104 0 29.3' 14 104 1 2 0 0 2 0 104 1.92 29.5 16 104 1' 4 01 0 4 0 104 3.85 30.01 18 104 1 5 0, 0 5 0 104 4.81 30.1 20 104 1' 8 0 0 8 0 104 7.69 30.5 22 104 1 10 0 0 10 01 104 9.62 - 24 103 2 12 0 0 12 1 104 11.54 31.3 26 102 3 15 0 0 15 1 103 14.56 - 28' 102 3 18 0 0 18 1 103 17.48 31.5 30, 102 3 23' 0 0 23 1 103 22.33 - 32' 101 4 34 1 0 34 1 102 33.33 32.2 34 101 4 38 1 0' 38 1 102 37.25 - 36 100 5 41 1, 0 41' 2 102 40.20 32.4 38 99 6 42 1 0 42 2 101 41.58' - 40 98' 7 42 1 3 42' 2 101 42.57 32.0 42 98 7 53 1 0 53 3 101 52.48 - 44 98 7 60 1 1 601 3 101 59.41 31.7 46 97 8 63 0 1 64 4 101. 63.37 - 48 96 9 66 0 1 67 5 101 66.34 33.1 50 95 10 72 0 1 73 5 100 73.00 - 52 93 11 73 0 11 74 7 100 74.00 32.8 54 93 11 76 0: 11 77 7 100 77.00 - 56 92 12 77 0 1! 78 7 99 79.79 33.0' 58 88 16 77 0 1 70 11 99 78.79 - 60 86 18 82 0 1 83 13 99 83.84 33.6 62 82 22 83 0 1 84 17 99' 84.85 - 64 77 27 84 0 2 86 22 99' 86.87 32.6 66 74 30 85 0 2' 87 25 99 87.88 - 68' 73 30 86 0 2' 88 26 99 88.89 33.7 70 66 37 89 0 2' 91 33 99 91.92 - 72 59 44 89' 0 2' 91 38 97 93.81' 33.9 74 54' 49 89 0 2 91 43 97 93.81 - 76 41 61 91 0 2 93 56 97 95.88: 34.2 78 35 67 92 0 2 94 62 97 96.91 - 80 27' 74 92 1 3 95 70 97 97.94' 34.5 AT 17ABLE 17 (continued). SKIN 4lNqRS (BASED ON MACR(l6C.OPIC APPEARANCE), MORTALITY AND BODY WEI©fP' 14-GR, DPIDA PLUS SWSC-I: 90 (ryg/(mouse x week)) CD1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tuaors are included in these values (b) sum of values of colwms 10 arc1'12 i. e. mice with macroscopic skin twrors "in" as well as "in and outside"'application area (c) dead mice with macroscopic skin twmrs (from coiimn 10 and 12) (d) sum of values of columns 7 and 14 (e) value of column 13 multiplied by 100 and divided by value of eolumn.18, 1. e. sum; of columns 7 and 14 3285

INBIFO Institut fur biologische Forschung • Kdin SUBREPORT P 0500/3068 OBE148RB3, B4 5302' M 7 9 10 11' 12 13 14 18 19 20, WEEK SURVI- M3R- NlA4BER CF ALIVE'AND aFliD MICE, WHICH HAVE CR HAD EFFEC- MACR}- BODY WFS TAL- ITY MtsCHDSCOPIC SKIN 'iUkqRS (a) TIVE I1A4BER SWPIC TUMfJR WEIGIif' IN APPL.- OUPSIDE IN AND (h) (o) AREA aFII.Y APPL.-ARFA OUPSIOE CNLY APPL.-AREA (d) RATE (e) (0/0) (0/0) (9) 0 105 0 0 0 0 0 0 105 0 22.3 2 105 0 0 0 0 0 0 105 0 23.5. 4 105 0 0 0' 0 0 0 105 0 25.1' 6 104, 1 0 01 0 0 0 104 0 27.4 8 104 1 0 01 0 0 0 104 0 28.8 10 103 2 0 0 0 0 0 103 0 29.3 12 103 2 0 0 0 0 0 103 0 30.2 14 103 2 3 0 0 3 0 103' 2.91 30.1 16 103 2 3 0 0' 3 0 103 2.91 31.1 18 103 2 7 0 0 7 0 103 6.80 30.8 20 103 2 15 0 0 15 0 103 14.56 31.4 22 103 2 23 0 0 23' 0 103 22.33' - 24 103' 2 28 0 0 28 0 103 27.18 32.8 26 102 3 30 0 0 30 0 102 29.41 - 28 102 3 40 0 0 40 0 102 39.22 32.2 30 102 3 46 0 0 46 0 102 45.10 - 32 101 4 48 0: 0 48 0 101 47.52 33.8 34 100 5 51 0 0 51 0 100 51.00 - 36 99 6 57 0 0 57 01 99 57.58 33.6 38: 97 8 61 0 0 61i 0 99 61.62 - 40' 97 8 69 0 2 69 2 99 69.70 34.2 42' 95 10 72' 0 0 72 4 99 72.73 - 44 95 10 77' 0 0 77 4 99 77.78 34.3 46 92 12 81 0 0 81 7 99 81.82' - 48 92 12 86 0 0 86 7 99 86.87 34.5 50 91' 13 86 0 1 87 8 99 87.88 - 52 87 17 87 0 2 89 12 99 89.89 34.5 54 82 22 90 0 2 90 17 99 90.91 - 56 80 24 90 0 2 92 19 99 92.93 34.4 58 74 30 90 0 2 92 25 99 92.93 - 60 69 34 91 0 2 93 30 99 93.94 34.9 62 59 44 92 0 2 94 39 98 95.92 - 64 51 51 93 0 2 95 47 98 96.94 34.8 66 47 55 93 0 2 95 51 98' 96.94 - 68: 43 59 94 0 2 96 55 98 97.96 34.2 70, 39 63 94 0 2' 96 59 98 97.96 - 72 35 67 94 0 2' 96 63 98 97.96 34.4 74 33 69 93 0 3 96 65 98 97.96 - 76 26 75 93 0 3 96 72 98 97.96 35.6 78 20 81 92 0 4 96 77 97 98.97 - 80 15 86 92 0 4 96 82 97 98.97 34.9' AT TABLE 17 (continued) SKIN TiR40RS (BASED ON M7tiCROSCOPIC APPEARNdCE), MORTALITY AND BODY WEI(il'P' 15-GR,, Dl1BA PLUS SFiSC-I: 120 (mg/(mouse x week)) CD1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tunors are included in these values (b) sum of values of c.olumis 10 and 12 i. e. mice with macroscopic skih tumors "in" as well as "in and outside" application area (c) dead mice with macroscopic skin tuwmrs (from column 10 and 12) (d) sum of values of colwins 7 and 14 (e) value of column 13 multiplied by 100 and divided by value of column 18, i'. e. sum of colinms 7 and 14 3285

UNBIFO Institut fur biologische Forschung • KSIn SUBREPORT P'0500/3068 OBE148B5, B6 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SURVI- MOR- Nll49ER' CF ALIVE AND DEAD MICE, WNI(21 HAVE OR HitD EFFEC- MWCRI>- BODY 4UR,S 274L- M1uCli()SCCPIC SKIN 1UFDRS (a), TIVE SCbPIC WEIGNT ITY tS.ROfER 'A1wqR IN APPL.- AREA ONLY W15IDE APPL.-AREA CM,Y IN' AND OOTSIDE APPL.-AREA, (b) (c) (d) RATE (e), (0/0) (0/0) (g). 01 105 0 0 0 0 0 0 105 0 16.9 2 105 0 0 0 0 0 0 105 0 18.6 4 105 0 0 0 0 0 0 105 0 19.8 6 105 0 0 0 0 0 0 105 0 21.4 8 105 0 0 0 0 0 0 105 0 22.5' 10 105 0 0 0 0 0 0 105 0 23.4 12 105 0 0 0 0 0 0 105 0 23.7 14 105 0 0 0 0 01 0 105 0 24.1' 16 105 0 0 0 0 0 0 105 0 24.7 18 105 0 0 0 0 0 0 105 0 25.2 20 105 0 0 0 0 0 0 105 0 25.4 22 105 0 0 0 0 0 0 105 0 - 24 105 0 0 0 0 0 01 105 0 25.9 26 105 01 0 0 0 0 0 105 0 - 28 105 01 0 0 0 0 0 105 0 26.8 30 105 0 0 0 0 0 0 105 0 - 32 105 0; 0 0 0 0 01 105 0 27.1 34 105 0 0 0 0 0 0 105 0 - 36 105 0 0 0 0 0 0 105 0 27.8 38 105 0 0 0 0 0 0 105 0 - 40 105 01 0 0 0 0 0 105 0 28.3 42 105 0 0 0 0 0 0 105 0 - 44 104 1 0 0 0 0 0 104 0 29.1. 46 104 1 0 0 0i 0 0 104 0 - 48' 104 1 0 0 0 0 0 104 0 29.3 50 104 1 0 0' 0 0 0 104 0 - 52 104 1 0 0 0 0 0 104 0 29.1' 54 104 1 0 0 0 0 0 104 0 - 56 104 1 0 0 0 0 0 104 0 29.7 58 104 1 0 0 0 0 01 104 0 - 60 104 1 0 0 0 0 0 104 0 29.8 62 104 1 0 01 0 0 01 104 0 - 64 104 1 0 0 0 0 0 104 0 30.0 66 103 2' 0 0 0 0 0 103 0 - 68 103 2 0 0 0 0 0 103' 0 30.1 70 103 2 0 0 0 0 0 103 0 - 72 103 2 0 0 0 0 0 103 0 30.6 74 102 3 0 0 0 0 0 102 0 - 76 102 3 0! 0 01 0 0 102 0 31.2 78 99 6 01 0 0 0 0 99 0 - 80 98: 7 0 0 0 0 0 98 01 31.0 AT 77RBLE 17 (oontinued) SKIN 1UMfJRS (BASED Ct^1 MAICB9SCOPIC APPEARAW-E), MJRTALITY AND BODY WEIQIT 0.3fi,R, ACE7UI+1E B6C3F1 MICE' (a) only preliminary diagnosis based on macroscopic appearance, mice whiett lost their macroscopic tuffOrs are included in these values (b) sum of values of columns 10 and 12 i. e. mice with macroscopic skin tunors "in" as we11ias "in and cutside" application area (c) dead mice with macroscopic skin tumors (from,coluan 10 and 12) (d) sum of values of columns 7,and 14 (e) value of coluim 13 multiplied by 100 and divided by value of columi 18, i. e. sum of colwms 7 and 14 3285

INBIFO Institut fuir biologische Forschung • Mln SUBREPaRT P 0500/3068 08E148ii87, 88 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SURVI- M!JR- NU+BER CF ALIVE AND DE4D MICE, WHICN 11AVE OR 871D EFFF7C- MACRO- BODY VOR3 ThL- TTq F@rCIaQSCOPIC SKIN:1UM13RS (a) TIVE N:INBER SOOPIC TOMOR WEIGHT IN APPL.- 0015IDE IN AKD (b) (c)' AREA CNLY APPL.-ARF.A, CUi'6IDE QNLY APPL.:-ARFA (d) RATE (e) (0/0) (0/0) (9) 0 105 0 0 0 0 0 0 105 0 16.5 2 105 0 0 0 0 0 0 105 0 18.3 4 105 0 0 0 0 0 0' 105 0 19.7 6 105 0 0 0 0 0 0 105 0 21.1 8 105 0 0 0 0 0 0 105 0 22.2 10 105 0 0 0 0 0 0 105 0 23.0 12' 105 0 0 0 0 0 0 105 0 23.5 14 105 0 0i 0 0 0 0 105 0 24.2 16 105 0 0' 0 0' 0 0 105 0 24.6 18 105 0 01 0 0' 0 0 105 0 25.0 20 104 1 0 0 0 0 0 104 0 25.4 22 104 11 0 0 0 0 0 104 0 - 24 104 1 0 0 0 0 0 104 0 26.0 26 104 1 0 0 0 0 0 104 0 - 28 104 1 0 0 0 01 0 104 0 26.7 30 104 1 0 0 0 0' 0 104 0 - 32 104 1 1 0 0 1 0 104: 0.96 27.4 34 104 1 2 0' 0 2 0 104 1.92 - 36 104 1 2 01 0 2 01 104' 1.92 38.3 38 104 1' 2 0 0 2 01 104 1.92 - 40 104 1' 3 0 0 3 0' 104 2.88 28.6 42' 104 1 4 1 0 4 0 104 3.85 - 44 104 1 4 2 0 4 0 104 3.85 29.4 46 104' 1 5 4 0 5 0 104 4.81 - 48 104 1 7' 6 0 7 0 104 6.73 29.4 50 104 1 10 ' 7 0' 10 0 104 9.62 - 52 104 1 10, 9 01 10 0 104 9:62' 29.5 54 104 1, 10 9 0 10 0 104 9:62' - 56 104 1i 12 12 0 12 0 104 11.54 29.6 58 104 1 10 15 2 12 0 104 11.54 - 60 104 1 13 15 3 16 0 104 15.38 30:0. 62 103 2 14 16 4 18 1 104 17.31 - 64 102 3 15 17 4 19 1 103 18.45 30.1 66 102 3' 20 18 4 24 1 103 23.30 - 68 102 3' 20 18 4 24 1 103 23.30 30.4 70 101 4 17 19 7 24 2 103 23.30 - 72 101 4 16 19 10 26 2 103' 25.24 30.6 74 101l 4 16 20 12' 28 2 103 27.18 - 76 100 5 15 20 14 29 3 103 28.16 31.1 78 100 5 16 20 15 31 3 103 30.10 - 80 100 5 14 21 18 32 3 103 31'.07' 31.9 AT' T14BLE 17' (continued) SKIN 1[R10RS (BASED Ql MACIm6COPIC kPPEARA[CE),, MORTALITY AND 'BODY WEIGHT 0.4-GR, L"3A PLUS ACE'PDNE B6C3F'1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic turmrs are included in these values (b) sum of values of c.olumis 10 and 12 i. e. mice with macroscopic skin twmrs "in" as well as "in and outside"'applicaticn area (c) dead mice with macroscopic skin tumors (from colum 10 and 12) (d) sum of values of columns 7 and 14 (e) value of colwan 13 multiplied by 100 and divided by value of colwm 18, i. e. sum of columns 7 and 14 3285

ONBIFO Institut for biologische Forschung • KOln SUBREPO1tf P 0500/3068 UBE1i48RB9, B10 5302 M 9 10 11 12 13 14 18 19 20 WEEK SURVI- NOR- NUMBER CF ALIVE AND OEAD MICE, WMICH HhVE OR HAD EFFEC- MACIZa- BCOY UORS TAL- ITY MACAO6COPIC SKIN 1[R4DR5 (a) TIVE NUMBER SODPIC 1LHDR WEIGHC 0/0) IN APPL.- AREA QII.Y QU1'SIDE APP[:.-ARFA ONII..Y IN AND GQPSIDE APPL.-ARFA (b) (c) (d) RATE (e), (0/0) 9) . 0 105 0 0 0 0 0 0 105 0 16.2 2 105 0 0 0 0 0 0 105 0 17.6 4' 105 0 0 0 0 0 0 105 0 18.7 6 105 0 0 0 0 0 0 105 0 19.9 8 105 0 0 0 0 0 0 105 0 211.4 10' 105 0 0 0 0 0 0 105 0 22.3 12 105 0 0 0 0 0 0 105 0 22.4 14 105 0 0 0 0 0 0 105 0 23.2 16 105 0 0' 0 01 0 0 105 0 23.2 18 105 0 0 0 0 0 0 105 0 23.2 20 105 0 0 0 0 0 0 105 0 24.1 22 105 0 0 0 0 0 0 105 0 - 24 105 0 0 0 0 0 0 105 0 24.5 26 105 0 0 0 0 0' 0 105 0 - 28 105 0 0 0 0 01 0 105 0 25.0 30 105 0 0 0 0 01 0 105 0 - 32 105 0 0 0 0 0 0 105 0 25.9 34 105 0 0 0 0 0 0 105 0 - 36 105 0 0 0 0 0 0' 105 0 26.2 38 105 0 0 0 0 0 01 105 0 - 40 105 0 0 0 0 0 01 105 0 26.3 42 105 01 0 0 0 0 0' 105 0 - 44 105 01 0 0 0 0 0 105 0 26.5 46 104 1 0 0 0 0 0 104 0 - 48' 104 1 0 0 0 0 0 104 0 27.3 50' 104 1 0 0 0 0 0 104 0 - 52' 103 2 1' 0 0' 11 0 103 0.97 27.5 54 103 2 1' 0 01 1 0 103 0.97 - 56 103 2 1 0 0 1 0 103 0.97 27.8 58 103 2 1 0 0 1 0 103 0.97 - 60 103 2 1 0 0 1 0 103 0:97 28.5 62 103' 2 1 0 0 1 0 103 0:97 - 64 103' 2 1 0 0 1 0 103 0.97 28'.1' 66 103' 2 1 0 0 1 0 103 0.97 - 68 102' 3 1 0 0 1 0 102 0.98 28.2' 70 102 3 3 0 0 3 0 102 2.94 - 72 102 3 4 0 0 4 0 102 3.92 28.7 74 100 5 5 01 0 5 0 100 5.00 - 76 100 5 5 0 0 5 01 100 5.00 28.7 78 100 5 5 01 0 5 0 100 5.00 - 80 100 5 5 0 0 5 0 100 5.00 29.6 AT TABLE 17 (continued) SKIN TOMORS (BASED Qd MACROSCOPIC APPEARANCE), MDCQALITY'AND H0DY WEI(7TT 16-GR, Mn/SC-I: 42 (mg/(mouse x week)) B6C3F1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tumors are included in these values (b) sum of values of columns 10 ard 12 i. e. mice with macroscopic skin tumors "in" as well as "in and' outside" application area (c) dead mice with macroscropic skin tunors (from eoluon 10 and 12) (d) sum of values of colunms 7 and' 14 (e) value of' column~1'3 multiplied by, 100 and divided by value of collmn 18, i. e. sum of columns 7 and 14 3285

INBIFO Institut fOr biologische Forschung • Min SUBRk1POtP P 0500/3068 UBE148RB11, B12' 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SURVI- MOR- NfMBER OF ALIVE AND DF.AD MICE, FHICH HAVE Qt HAD EFFEC'- MACF1l}- BODY 4URS 1A1.- MACFO6COPIC SKIN 411MOF5 (a) TIVE 800PIC WEIGHT ITY NUMBER' ZWR IN APPL.- CUPSIDE IN AtdO (b) (c), AREA ONLY APPL.-AREA OUISIDE: QMY APPL.-IARFIk (d) PATE (e) (0/0)i (0/0) (9) 0 105 0 0 0 0 0 0 105 0 16.7' 2 105 0 0 0 0 0 0 105 0 17.9' 4 105 0 0 01 0 0 0 105 0 18.8 6 105 0 0 0 0 0 0 105 0 20.3 8 105 0 0 01 0 0 0 105 0 21.7 10 105 0 0 0 0 0 0 105 0 22.5 12 105 0 0 0 0 0 0 105 0 22.7 14' 105 0' 0 0 0 0 01 105 0 23.3 16 105 0' 0 0 0 0 0 105 0 23.2 18' 105 0' 0 0 0 0 0 105 0 23.0 20 105 01 0 0 0 0 0 105 0 24.1 22' 105 0 0 0 0 0 0 105 0 - 24 105 0 0 0 0 0 0 105 0 24.7 26 105 0 0 0 0 0 0 105 0 - 28~ 105 0 0 0 0 0 0 105 0 25.0 30, 105 0 0 0 0 0 0 105 0 - 32' 105 0 0 0 0 0 0 105 0 25.8 34 105 0 0 0 0 0 0 105 0 - 36 105' 0 0 0 0 0 0 105 0 26.2 38 105 0 0' 0 0 0 0 105 0 - 40 105 0 0 0 0 0 0 105 0' 26.2 42 105 0 0 0 0 0 0 105 0 - 44 105 0 1 0 0 1 0 105 0:95 26.3 46 155 0 1 0 0 1' 0 105 0.95 - 48 105 0 1 0 0 1' 0 105 0.95 27.1 50 105 0 1 0 0 1 0 105 0.95 - 52 105 0 1 0 0 1 0 105 0.95 27.2 54 105 0 1 0 0 1' 0 105 0.95 - 56 105 0 2 0 0 2' 0 105 1.90 27.6 58 105 0 2 0 0 2 0 105 1.90 - 60 105 0 2 0 0 2 0 105 1.90 28.2 62 105 0 2 0 0 2 0 105 1.90 - 64 105 0 3 0 0 3 0' 105 2.86 27.6 66 105 0 3 0 0 3 01 105 2.86 - 68 104 1' 3 0 1 4 01 104 3.85 27.6 70 104 1' 5 0 1 6 01 104 5.77 - 72 103 2' 6 0 1 7 0 103 6.80 28.4 74 103 2' 7 0 1 8 0 103' 7.77 - 76 103 2 7 0 1 8 0 1'03: 7.77 28.1 78' 103 2 6 0 2 8 0 103 7.77 - 80 103 2 6 0 2 8 0 103 7.77 28.9 AT T11BLE 17 (continued) SKIN' T1A+pRS(BASED ON MAC1106OOPIC APPEAR7INCE)., MOf2TALITY ANDBODYWEIQPP 17-GR, MN1SC-I: 63 (jng/(mouse x week)) B6C3F1 Mice (a)'only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tunnrs are included in these values (b) sum of values of colums 10 and 12 i. e. mice with macroscopic skin tumors "in" as well as "in and outside" application area (c)~dead mice with macroscopic skin twrors (from column 10 and 12) (d) sum of values of colunns 7 and 14 (e) value of column 13 muitiplied by 100 and divided by value of column 18, i. e. sum of c*olumns 7 and 14 3285

INB1F0 Institut fur biologische Forschung • KoIn St1BREPORT P 0500/3068 1BE148RB13, B14 5302 M 9 10 11 1'2' 13 14 18 19 20 WEEK SURVI- MOR- Nl1MdER OF ALIVE AND DEAD MICE, NhfICH HAVE OR HAD EFFEC- MFtCRO-- BODY HaRS ZA[.- MACROB00PIC SKIN TONqRS (a) TIVE' S03PIC wEIGHT ITY NR48ER T(k1qR RATE IN APPL.- OUISIDE IN AND (b) (c) (d) (e) AS2FA QII,Y APPL.-AREA , OUP6IDE CNLY APPL.-ARFA (0/0) (0/0) (9 ) 0 105 0 0 0 0 0 0 105 0 16.7 2 105 0 0 01 0 0 0 105 0 17.8 4 105 0 0 0 0 0 01 105 0 18.9 6 105 0' 0 0 0 0 0 105 0 20.1 8 104 1 0 0 0 0 0 104 0 21.8 10 104 1 0 0 0 0 0 104 0 22.5 12 104 1 0 0 0 0 0 104 0 22.7. 14 104 1 0 0 0 0 0 104 0 23.1 16 104 1 0 0 0 0 0 104 0 23.5 18 104 1 0 0 0; 0 0 104 0 23.5 20 104 1 0 0 0' 0 0 104 0 24.2 22 104 1 0 0 0 0 0 104 0 - 24 104 1 01 0 0 0 0 104 0 25.0 26 104 1 0 0 0 0 0 104 0' - 28 104 1 0 0 0 0 0 104 0 25.1 30 104 1 0 0 0 01 0 104 0 - 32 104 1 0 0 0 01 0 104 0 26.1' 34 104' 1 0 0 0 0 0 104 0 - 36 104 1 0 0 0 0 0 104 0 26.5 38 104 1 0 0 0 0 0 104 0 - 40 104 1 0 0 0 0 0 104 0 27.0 42 104 1 0 0 0 0 0 104 0 - 44 104 1 0 0 0 0 0 104 0 26.7 46 104 1 0 0 0 0 0 104 0 - 48 104 1 0 0 0 0 0 104 0 27.4 50 104 1 0 0 0 0 0 104 0 - 52' 104 1 0 0 0 0 0 104 0 27.8 54 104 1 0 0 0 0 0 1'04' 0 - 56 104 1 0 0 0 0 0 104 0 28.3 58 104' 1 0 0 0 0 0 104 0 - 60 104 1 0' 0 0 0 0 104 0 28.9 62 104 1 01 0 0 0 0 104 0 - 64 104 1I 0 0 0 0 0 104 0 27.9 66 103 2 3 0 0 3 0 103 2.91 - 68 103 2 4 0 0 4 0 103 3.88 28.5 70 103 2 4 0 0 4' 0 103 3.88 - 72 103 2 4 0 0 4 0 103 3.88 28.5 74 103 2 6 0 0 6 0' 103 5.83 - 76 103 2 7 0' 0 7 01 103 6.80 28.7 78 103 2 9 0 0 9 01 103 8.74 - 80 102 3' 13 0 0 13 0 1'02' 12.75 29.3 AT TABLE 17 (continued) SKIN 71R10RS (BASED QJ MAC%]6C)OPIC APPEARAN:.E), MOFTPALITY 11ND BODY WEIGt!' 18-GR, Mi3SC-I: 84 (rtg/(nouse x week)) B6C3F1 MICE (a)ionly preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tuwmrs are included in these values (b) sum of values of colutms 10 and 12 i. e. mice with macroscopic skin twinrs "in1" as well as "in and outside" application area (c) dead mice with macroscopic skin,timnrs (fran eolimn 10 and 12). (d) sum of values of columns 7 and 14 (e) value of column 13 multiplied by 100 and divided by value of colum 18, i'. e. sum of colurms 7 and 14 3285

INBIFO Institut fur biologische Forschung' • Kal'n SOBREPCx1T' P 0500/306,8 UBE148i3B15, B16 6050 M 9 10 11 12 13 14 18 19 20 WEEK SURVI- MOR- NUMBER CF ALIVE AND DEAD MICE, WHICH HAVE OR HAD EFFEC- MRC%)- BODY VpRS '1AL- MACRt78COPIC SKIN'TUMDRS (a) TIVE: SoOPIC WEIGHT ITY NIMBER TCM7R RATE IN APPL.- GQISIDE IN AND (b) (c) (d) (e) AREA CNLY APPL.-ARFA, CAISIDE CNLY APPL.-ARFA (0/0) (0/0) (9) 0 105 0 0 0 0 0 0 105 0 16.8 2 105 0 0 0 0 0 0 105 0 17.6 4 105 0 0 01 0 0 0 105 0 19.1 6 105 0 0 0; 0 0 0; 105 0 20.4 8 105 0! 0 0 0 0 01 105 0 21.8 10 105 0 0 0 0 0 0 105 0 22.5 12 105 01 0 0 0 0 0 105 0 22.8 14 105 0 0 0 0 0 0 105 0 23.2 16 105 0 0 0 0 0 0 105 0 23.6 18 105 0 0 0 0 0 0 105 0 23.7 20 105 0 0 0 01 0 0 105 0 24.3 22 104 1 0 0 0' 0 0 104 0 - 24 104 1 0 0 0 0 0 104 0 25.1. 26 104 1 0 0 0 0 0 104 0 - 28 104 1 0' 0 0 0 0 104 0' 25.3 30 104 1' 0 0 0 0 0 104 0 - 32 104 1' 0 0 0 0 0 104 0 26.2 34 104 1 0 0 0 0 0 104 0 - 36 104 1 0 0 0 01 0 104 0 26.7' 38 104 1 0 0 0 0 0 104 0 - 40 104 1 0 0 0 0 0 104 0.96 27.3' 42 104 1 4 1' 0 4, 0 104 0.96 - 44'. 104 1 4 1 0 4, 0' 104 0.96 27.4 46 103 2 11 4 0 1 0' 103 0.97 - 48 103 2' 2 4 0 1 0 103 2.91i 27.9 50 103 2 2 8 1 3 0 103 2.91 - 52' 103 2 3 9 1 4 0 103 3.88 27.9 54 103 2 3 9 1 4 0 103 3.88 - 56 103 2 3 9 1 4 0 103 3.88 28.5 58 103 2 4 11: 1 5 0 103 4.85 - 60, 103 2 4 11. 1' 5 0 103 4.85 29.0 62 103 2 7' 12 1 8 0 103 7.77 - 64 102 3 6 14 2 8 11 103 7.77 28.5 66 102 3 7 14 3 10 11 103 9.71 - 68 102 3 7 14 3 1'01 1 103 9.71 29.0 70 1'02' 3 7 16 3 10 1 103 9.71 - 72 1'02' 3 8 16 3 11 1 103 10.68 29.5 74 102 3 9 16 3 12 1 103 11.65 - 76 101 4' 11 18: 3 14 1 102 13.73 29.5 78 101 4 12 18' 4 16 1' 102 15.69 - 80 101 4 13 20, 4 17 1 102 16.67, 30.4 AT TABLE 17 (continued) SKIN TUM()RS (BASED CN'MlCRCSCOPIC APPEARANCE), MDIrfALITY AND BODY WEIGHT 19-GK,. OMBA.PLOS 4WSC-I:42'(mg/(muuse x week).). 86C3F1' MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tuwmrs are included:in these values (b): sum,of values of colurtns 10 and 12 i. e, mice with macroscopic skin tumors "in" as well as "in and outside" application area (c); dead mice with macroscopic skin tumors (from column 10 and 12) (d)~sum of values of c•olumns 7 and 14 (e) value of eoluim 13 multiplied by 100 and divided by value of column 18, i. e. sum of colwmns 7 and 14 3285

INB'1FO Institut fur biologische Forschuing • K6In SUBREPCJRI" P 0500/3068 1BE148RB17, B18 5302 M 7 9 10 11 12' 13 14 18 19 20 ~ WEEK SURVI- M3R- N(.IMBER (F ALIVE ANI) DEAD MICE, PhfICH HAVE OR HRD EFFEC- MACRO- BODY WRS TAL- ITY MACir]6COPIC SKIN ZUN[)RS (a) TIVE NOMBER' SODPIC Ti1M3R WEIGHT INIAPPL.- OAPSIDE IN At4D (b) (cY AREA CNLY APPL. AREA QTI5IDE ONLY APPL. ARF14 (d) BRTE (e) (0/0) (0/0) (9) 0 105 0 0' 0 0 0 0 105 0 16.8 2 105 0 0 0 01 0 0 105 0 17.4 4 105 0 0' 0 0; 0 0 105 0 18.8 6 105 0 0: 0 0' 0 0 105 a 20.0 8 105 0 0 0 01 0 0 105 0 21.4 10 105 0 0 0 01 0 0 105 0 22.1 12 105 0 0 0 0 0 0 105 0 22.4 14 105 0 0 0 01 0 0 105 0 23.2 16 105 0 0' 0 01 0 0 105 0 23.0 18 105 0 01 0 0 0 0 105 0 23.5 20 105 0 01 0 0 0 0 105 0' 23.9 22 105 0 01 0 0 0 0 105 0 - 24 105 0 1 0 0 1 0 105 0.95 24.8 26 105 0 1 0 0 1 0 105 0195 - 28 105 0 1 0 0 1 0 105 0.95 25.1 30 105 0 3 0 0 3 0 105 2.86 - 32 105 0 5 0 0 5 0 105 4.76 25.8 34 105 0 6 0 0 6 0 105 5.71 - 36 105 0 7 0 0 7 0 105 6.67 26.1 38 105 0 7 1 0 7 0 105 6.67 - 40 105 0 8 1 9 9 1 105 8.57 26.8' 42 105 0 10 4 0 10 0 105 9.52 - 44 105 0 10 6 0 10 0 105 9.52 26,4 46 105 0 13 7 0 13 0 105 12.38 - 48 105 0 10 8 0 14' 0 105 13.33 27.0 50 105 0 11 9 4 15 0 105 14.29 - 52 105 0 11 12' 4 15 0 105 14.29 27.8: 54 105 0 11 11 5 16 0 105 15.24 - 56 104 1 11 11 5 16 1 105 15.24, 27.8'. 58 104' 1 13 11 5 18 1 105 17.14 - 60 103 2 13 11 5 18 2 105 17.14 28.1 62 103 2 14 10 7 21 2' 105 20.00 - 64 102 3 14 10, 8 22 3 105 20.95 27.6 66 101 4 14 9 9 23 4 105 21.90 - 68 101 4 16 9 9 25 4 105 23.81 28.1 70 99 6 18 8 11 29 6 105 27.62 - 72 99 6 24 7 12 36 6 105 34.29 28.6 74 97 8 27, 8' 13 40 8' 105 38.10 - 76 96 9 29 8' 13 42 9 105 40.00 28.6 78 95 10 31 7 17 48 9 104 46.15 - 80 95 10 33 7 20 53 9 104' 50.96 29.0 AT TABLE 17 (oontinued)i SKIN T[A40RS (BASED CN MANCPASCDPIC APPEARANCE), MDRTALITY' ANp BODY N1EI(41T' 20-GR, OP1BA PLUS MWSC-I: 63 (IOg/(mouse x week) ) B6C3F1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tuirors are included in these values (b) sum of values of colunns 10'and 12 i. e. mice.with macroscopic skin tumrs "in" as well as "in and outside" application area (c) dead mice with macroscopic skin tumors (fran oolum 10 and 12) (d) sum of values of coluims 7 and 14 (e) value of column 13 multiplied by 100 and divided by value of colKmn 18, i. e. sum of columns 7 and 14 3285

INBIFO Institut fur biologische Forschung • KtSIn SUBREPCRT'P 0500/3068 1BE148RB19, B20 5302 M 7 9 10 11 12 13 14 18 19 20 WEEK SURVI- NqR- NUMBER OF ALIVE AND DEAD MICE, WflICH HAVE OR HAD EFFDC- MACR(Y- BODY WRS TAi.- ITY MACI1DSCOPIC SKIN T1)rtOltS (a) TIVE N:ANBER SODPIC T[)MDR WEIGHT IN APPL.- CllI5I0E IN AND (b) (c) AREA QWLY APPL.:-AREA QTI'SIDE CNLY APPL.-ARFA (d) RRTE (e) (0/0) (0/0) (9) 0, 105 0 0 0 0' 0 0 105 0 16.3 2 105 0 0 0 01 0 0 105 0 17.2 4 105 0 0 0 01 0 0 105 _ 0 18.8 6 105 0 01 0 0 0 0 105 0 20.1 8 105 0 01 0 0 0 0 105 0 21.5 10 105 0 01 0 0 0 0 105 0 22.2 12 105 0 01 0 0 0 0 105 0 22.5 14 105 0 01 0 0 0 0 105 0 23.1 16 105 0 0 0 0 0 0 105 0 23.1 18 105 0 1 0 0 1 0 105 0.95 23'.3 20 105 0 1' 0 0 1 0 105 0.95 24.0 22 105 0 1 0 0 1 0 105 0.95 - 24 105 0 2 0 0 2 0 105 1.90' 24.9 26 105 0 2 0 0 2 0 105 1.90 - 28 105 0 2 0 0 2 0 105 1.90 25.0 30 105 0 2 0 0 2 0 105 1.90, - 32 105 0 2 0 0 2 0 105 1.90 25.8 34 105 0 3 0 0 3' 0 105 2.86 - 36 105 0 3 0 0 3' 0 105 2.86 26.1 38 105 0 4 0 0 4 0 105 3.81 - 40 105 0 5 2 0 7 0 105 6.67 26.6 42 105 0 10 2 0 10 0 105 9.52 - 44 104 1 12 3 0 12 1 105 11.43 26.4 46 104 1 14 3 0 14 1 105 13.33 - 48 104 1 14 5 0 14 1' 105 14.29 27.2 50 104 1 16 7' 1 17 1' 105 16.19 - 52 104 1 16 7' 1 17 1' 105 16.19 27.6 54 103 2 18 7 1 19 1' 104 18.27 - 56 103 2 20 8' 3 23 1 104 22.12 27.9 58 103 2 22 6 6 28 1' 104' 26.92 - 60 103 2 22 6 6 28 1' 104' 26.92 28.3 62 103 2 24 6 6 30 1 104' 28.85 - 64 103 2' 29 5 7 36 1 104 34.62 27.8 66 103 2 29 5 8 37 1 104 35.58 - 68 102 3 28 8 9 37 2 104 35.58 28.3 70 101 4 33 11 9 42 3 104 40.38 - 72 101 4 33 9 11 44 3 104 42.31i 28.9 74 101 4 37 7 13 50 3 104 48.08 - 76 98 7 43 6 17 60 6 104 57.69 28.6 78 98 7 45 5 19 64 6 104 61.54 - 80 97 8 49 4 21 70 7 104 67.31 29.0 AT TABLE 17 (continued) SKIN. 1SA1ORS ( kIASED ON ~ MACRf.)6C()PIC APPE74PANCE )., MORTALITY AND BODY WEI(i1T' 21-(it, DMBA PLOS MWSC-I: 84 (mg/(mouse x week)) B6C3F1 MICE (a) only preliminary diagnosis based on macroscopic appearance, mice which lost their macroscopic tumors are included in these values (b) sum of values of coluims 10 and 12 i. e. mice with macroscopic skin kumors "in" as well as "in and outside" application area (c) dead mice with macroscopic skin tumors (from column 10 and 12) (d) sum of values of col.mos 7 and 14 (e) value of column 13 multiQlied by 100 and divided by value of colurm 18, i. e. sum of coltams 7 and 14 3285

SUBREPORT P 01500/3068 UBE145RB25 7292 APPL. NIATERIAL MOUSE STRAIN 2R1 CQVDENSATE DOSE (ng/(nouse x week)) 0 42 60 63 84 90 120 acetone CD1 .GT.80 - - - - - - DMBA plus acetone " 40 - - - - - - MdSC-I " - - 50 - - 35 39 MWSC-I (a) " - - 53 - - 41' 30 DMBA plus NWSC-I " - - 22 - - 14 13' S(+rTSC-I " - - 13 - - 13 17 DMBA plus SWSC-I " - - 13 - - 13 13' acetone B6C3F1 .GT.80 - - - - - DMBA plus acetone " 32' - - - - - MWSC-I " - 51 - 43 65 - DMBA plus MWSC-I " - 40 - 24 18 - AT TABLE'18' APPLICATION WEEK P'Cd2 TIJMOR ONSET, NIACROCSCOPIC TUMORS (a) stored at 4 degrees centigracle, condensate of all other groups stored at -75 degrees centigrade

SUBREPORT P 0500/3068 UBE145RB26 7292 G%JUP APPLICATION NATERIAL DOSE TIME ( vaeeks ) TO REACH A TU~'lOR RATE (0/0) (mg/(Ymuse x week)) .Gr.10 .Gr.20 .GT.30 .GP.40 .Gf.50 .GT.60 .Gr.70 ..6TT.80 .GT.90 0.1-GR (a) acetone - 0.2--GR (a) DMBA plus acetone - 1-{,Yt (a) NWSC-I 60 74 - - - - - - - - 2-GR (a) MWSC-I 90 52 74 - - - - - - - 3-GR ( a ) Mn1SC-I 120 57 79 78 - - - - - - 4-GR (a) MWSC-I (b) 60 68 - - - - - - - - 5-,GR (a) MWSC-I ( b ) 90 71 - - - - - - - - 6-GR (a) MWSC-I (b) 120 72 - - -. - - - - - 7-GR (a) DMBA plus NW'~.~C-I 60 42 56 65 75 -. - - - - 8-GR (a) DN1BA plus Nb1SC-I 90 29 40 47 63 72 - - - - 9--GR (a) DMBA plus MASC-I 120 30 41 49 60 70 75 - - - 10-Q2 ( a ) SWSC-I 60 34 46 56 62 68 75 78 - - 11-GR (a) SWSC-I 90 30 39 43 47 53 57 64 71 80 12-GR (a) SWSC-I 120 26 31 39 42 44 50 56 63 75 AT TABLE 19 MA:CROS©OPIC TUMOR RATE IN DEPENIDENCE ON TIME AE'PER 1ST APPLICATION OF ACEZONE OR CO6IDEN.SATE (cigarette type 2R1 ) (a) mouse strain CD1 (b) condensate stored at 4 degrees centigrade, condensate of all other groups stored at -75 degrees centigrade 2TeTSO9zoz

SUBREPORT P 0500/3068 UBE145RB27 7292 GROUP APPLICATION MATERIAL DOSE TIME (weeks) ZO REACH A T[MOR RATE (0/0) (mg/(mouse x week)) .GT.10 .GT.20 .GT.30 .GT.40 .GT.50 .GT.60 .GT.70 .GT.80 .GT.90 13-CR (a) DMBA plus SWSC-I 60 29 39 44 47 53 57 61 69 72 14-M (a) DMBA plus SWSC-I 90 23 30 32 36 42 45 50 59 70 15-GR (a) DMBA plus SWSC-I 120 20 22 27 29 33 38 41 45 53 0. 3--GR ( b ) acetone - - - - - - - . . - - 0.4-GR (b) DMEA plus acetone - 55 68 78 - - - - - - 16-M ( b ) NbiTSC-I 42 ( c ) - - - - - - - - - 17-Qt (b) MWSC-I 63 (c) -. - - - - - - - - 18-M (b) Mn1SC-I 84 (c) 80 - - - - - - - - 19-M ( b ) DMBA pl us MWSC-I 42 ( c ) 71 - - - - - - - - 20-M (b) DMBA plus Mn1''C-I 63 (c) 46 63 71 77 80 - - - - 21-GR (b) DMBA plus KqSC-I 84 (c) 44 55 63 70 75 78 - - - AT TABLE 19 (continued) MACROSO©PIC TUMOR RATE IN DEPENDENCE ON TIME AFTER 1ST APPLICATION OF ACETCNE OR CONDENSATE (cigarette type 2R1) (a) mouse strain CD1 (b) mouse strain B6C3F1 (c) reduced dosage for B6C3F1 mice due to reduced body weight as ccmpared to CD1 mice vTeTSO9zoz

suHHKR)1rr P U5U0/:iU6a UBE145RB28 7292 APPLICATION MATERIAL MOUSE STRAIN 2R1 CONDENSATE D06E (mg/(mouse x week)) MEAN 0 42 60 63 84 90 120 acetone CD1 0 - (1i.0) DMBA plus acetone " 6 - (1.7) NWSC-I m 13 - - 34 21 23 (0.8) (1.2) (1.2) (1.1) NWSC-I (a) m 19 - - 12 18 16 (1.1) (0.9) (1.2) (1.1) DMBA plus MWSC-I " - - 49 - - 60 68 59 (1.1) (1.1) (1.3) (1.1) SWSC-I m 73 - - 91 97 87 (1.0) (1.1) (1.3) (1.1) DMBA plus Sn1SC-I " - - 96 - - 98 99 98 (1.2). (1.2) (1.1) (1.2) acetone B6C3F1 0 - (1.0). DMBA plus acetone " 31 (2.3) [wWSC-I m 5 - 8 13 - - 9 (l.7), (1.1) (0.9) (1.2) DMBA plus N6B1SC -I m 17 - 51 67 - - 45 (1.1!) (1.1) (1.2) (1.1) AT TABLE 20 MACR06COPIC T[k1OR RATE (0/0), WEEK 80 OF'APPLICATION PERIOD Remarks: number in brackets give ratio: number of mice with macroscopic tumors divided by number of mice with microscopic tumors (a) condensate stored at 4 degrees centigrade, condensate of all other groups stored at -75 degrees centigrade 1^es

SUBREPORT P 0500/3068 UBE145RB29 7257 STUDY P 0500/- MOUSE STRAIN DOSE OF PROCESSEID MWSC-C (mg/(mouse x week)) D06E OF PPDCESSED MWSC-I (mg/(mouse x week)) 60 90 120 42 60 63 84 90 120 3030 CD1 11 26 29 - 10 - - 26 37 3068 CD1 - - - - 19 - - 12 18 3068 CD1 - - - - 13 - - 34 21 3068 B6C3F1 - - - 5 - 8 13 - - 3073 Cp1 21 38 40 - - -. -. - - 3111 CD1 - - -. - 15 - - 29 33 3115 CD1 - - - - 13 -. - 20 37 AT TABLE 21 12EMARICS aandensate stored at 4 degrees centigrar3e " condensate stored at -75 degrees centigrade m m © m MACROSCCPIC TiJMQR FiATE (0/0), SKIN PAINTIidG STUDIES WITH 2R1 STANDARD REFERETiCE CIGAF<EZTE, NLQSC-C AND MWSC-I, WEEK 80 I sTETSO9zoz

St)BREPORT P 0500/3068 TJBE145I2830 7029 (RCXJP M:7[JSE STRAIN APPI,ICATION MATERIAL WEEK OF BPPLICATION PERIM N)KBER OF MICE WI3fi IRRITATION (0/0) 1 2 3 4 5 6 7 8 9 10 11 12 13 3 D06E CROUPS 0.1--GR CD1 acetone 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0.2-GR " DPIDA plus acetone 0 0 0 0 0 0 0 0 0 0 0 0 0 - 1-GR " MWSC-I 0 0 2 7 9 5 0 2 0 0 0 0 0 2--!GR " " 0 0 3 11 19 7 7 6 4 1 0 0 0 3--M " " 0 0 18 41 32 16 21 11 7 3 0 0 0 10.9 4--GR " MaSC-I (a) 0 0 0 4 6 4 4 3 2 0 0 0 0 5--GR " " 0 0 8 18 20 9 10 7 6 2 0 0 0 6-CR ° " 0 0 11 32 28 16 16 13 8 4 0 0 0 10.8 7-GR " DN>QA plus NbVSC-I 0 0 0 0 0 0 0 0 0 0 0 0 0 8-GR " " 0 0 1 4 6 5 3 3 0 1 0 0 0 9-.M " " 0 0 2 8 12 8 2 3 5 2 0 0 0 2.9 10-GR " SWSC-I 0 0 8 16 20 15 11 5 6 0 0 0 0 11 -GR " " 0 0 25 47 39 33 34 22 14 12 6 2 0 12-M ° " 0 1 47 68 50 53 59 32 23 30 17 5 7 30 13-<;2 " LI48A plus SWSC-I 0 0 1 7 7 1 3 1 0 0 0 0 0 14-GR " " 0 0 13 11 11 12 6 4 7 1 0 0 0 15-GR " " 0 2 39 35 31 31 39 13 14 16 13 5 9 13.7 0.3-CR B6C3F1 acetone 0 0 0 0 1 1 0 0 0 0 0 0 0 - 0.4--GR " DrBA plus acetone 0 0 0 0 1 0 0 0 0 0 0 0 0 - 16-GR " NWSC-I 0 0 0 0 1 0 0 0 0 0 0 0 0 17-Gt " ° 0 0 0 0 0 0 0 0 0 0 0 0 0 18-GR " " 0 0 0 0 2 0 0 0 0 0 0 0 0 0 19-GR " DMBA plus MVJSC-I 0 0 0 0 0 0 0 0 0 0 0 0 0 20-M " " 0 0 0 0 0 0 0 0 0 0 0 0 0 21-Q2 " " 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AT TABLE 22 MICE WITF3 SKIN IRRITATION (0/0), WEEK 1 TO 13 (a) corr3ensate stored at 4 degrees centigrade, condensate of all other groups stored at -75 degrees centigrade 9TeTSOSzoz

St]BREPORT P 0500/3068 UBE145RA15 7292 GRO[7P APPLICATION MOUSE SKIN IRRITATION MATERIAL STRAIN WEEK OF' NUMBER OF MICE 1ST MAXI- LAST MAXIMUM~ APPEA- MUM APPEA- RANCE RANCE (0/0) 0.1-GR acetone CD1' . . . 0 0.2-GR L1MBA plus acetone " . . . G 1 -GR MWSC-I 2-GR " 3-GR " 11 u It 3 5 8 9 3' 5 10 19 3 4 10 41 4-GR ° (a) 5-GR " (a) 6-GR " (a) 7-GR 8-GR 9-GR CMBA plus MWSC-I g n 10-GR SWSC -I 1 1'171i " 12-,GR " 1i3-GR L1MBA plus SWSC-I 1i4-GR " 15-GR " n „ m 3 5 9 6 3 5 10 20 3 4 10 32 . . 0 3 5 10 6 3 5 10 12 3 5 9 20, 3 4 12 47 2 4 .GT.13 68 3 4 8 7 3 3 10 13 2 3 .GB.13' 39 0.3-GR acetone B6C3F1 5 5 5 1 0.4--GR DMBA plus acetone " 5 5 5 1 16-GR MWSC-I 17--GR " 18-GR ° 11 ~ 5 5 5 1 . 0 5 5 5 2 19-GR CMBA plus MWSC-I 20-GR " 21-QZ " TABLE 23 . . . 0 . . . 0 . . . 0 SKIN IRRITATION, WEEK OF 1ST APPEARANCE, MAXIMUM AND LAST APPEARANCE, NUMBER OF MICE WITH SKIN IRRITATION IN 0/0 (max./week) Remarks: .: no response (a) oondensate stored at 4 degrees centigrade, condensate of all other groups stored at -75 degrees centigrade 1res

SUBREPORT P 05D 0/3068 UBE145RA16 7292 APPL.-MATERIAL MOUSE STRAIN 2R1 CONDENSATE:DOSE (mg/(mouse x week)) MEAN 0 42 60 63 84 90 120 acetone CD1 0 - - - - - - - DMBA plus acetone " 0 - - - - - - - MWSC-I " - - 9 - - 19 41 23 MWSC-I (a) " - - 6 - - 20 32 19 DMBA plus MWSC-I " - - 0 - - 6 12 9 SWSC-I " - - 20 - - 47 68 45 DMBA plus SWSC-I " - - 7 - - 13 39 20 acetone B6C3F1 1 - - - - - - - DMBA plus acetone " 1 - - - - - - - MWSC-I " - 1 - 0 2 - - 1 DMBA plus MWSC-I " - 0 - 0 0 - - 0 AT TABLE'24 NUMBER OF MICE WITH SKIN IRRITATION (0/0), MAXIMUM PER WEEK (a) stored at 4 degrees centigrade, condensate of all other groups stored at -75 degrees centigrade

SUBREPORT P 0500/3068 UBE145RP,17 7292 TREATMEKT 1 TREAZINENr 2 GRApP SWSC-I SWSC-I PLUS DMBA Q~X7P 10-GR TO 12-GR 13-GR T0 15-GR NWSC-I +++ e 1-GR to 3-GR MWSC-I plus DMBA +++ +++ 7-GR to 9-GR AT TABLE 25.1'. INFLUENCE OF SMdKE TYPE t7N MICE' WITEI SKIN IRRITATIOt3 (N) (max. nuQnber of mice per week) (a) (a) thi2-test

SUBREPORT P 0500/3068 UBE145RB18 7292 TRFA'IMENP 1 TRFATr±1FNP 2 GROIJP ACETC)IJE MWSC-I SWSC-I M4VSC-I fROUP G.1-GR 1-GR TiO 3-GR 10-M TO 12-G2 16-GR ZO 18-GR acetone e i-F+ +++ plus DMBA 0. 2-GR MWSC-I + +++ +++ plus DMBA 7--GR to 9-GR SWSC-I plus DMBA 13-GR to 15-M MWSC-I plus DN1BA 1i 94R to 21 -M +++ i-h+ e AT TABLE 25.2 INFLUENCE OF f1NIBA PRETREAZMENr ON MICE WITH SKIN IRRITATIOTI' (N) (max. number of mice per week) (a)

~_------ SUBREPOFtT P 0500/3068 UBE145RA19 7292 STRAIN CD1 TREATMENT 1 STRAIN B6C3F1 TREATMENT 2 GROUP ACETONE GROUP 0.3--GR ACETGNE PLUS DMBA 0.4-GR MWSC-I 16-GR T0 18-GR NWSC-I PLUS DMBA 19-GR TO 21--GR acetone 0.1-GR acetone plus DMBA 0.2-GR MWSC-I +++ +++ +++ +++ 1--GR to 3-GR MWSC-I + + ++ +++ plus DMBA 7-GR to 9-GR AT TABLE 25.3 INFLpENCE OF MOUSE STRAINS (CD1 AND B6C3F1) ON MICE'WITL3 SKIN IRRITATION (N) (max. number of mice per week) (a)

SUBREPORT P 0500/3068 UBE145RA20 7292 TREATMENT 1 TREATMENT 2 GROUP MWSC-I (-75 degrees centigrade) GROUP 1-GR TO 3-GR MWSC-I (4 degrees centigrade) 4-GR to 6-GR AT TABLE 25.4 INFLUENCE OF CONDENSATE STORAGE TEMPERATURE ON MICE WITH SKIN IRRIATION (N) (max. number of mice per week) (a)

SUBREP'ORT P 0500/3068 t,IBE 145RA21i 7033 C()NIPARISQN PARAMETER WITH RELEVANTLY HIGHER RESPQNSE BEZWEEN PARAMETER GENERAL MDRTALITY HODY SKIN CIk]DITION WEIGHT IRRI- 1' 2 AND BEHA- TATIONS VIOR CD1 mice SWSC-I MKSC'-I 1 1 L1NBA MWSC-I = _ = 2 plus MWSC-I mNIDA SWSC-I = 1 = 2 plus SWSC-][ 9WSC-I IINBA 1 1 = 1 plus NWSC-I 1 1 TMBA L'MBA plus plus SWSC-I NWSC -I MWSC-I (a) MWSC-I (bl e e 1 B6C3F1 mice DMBA NWSC-I plus MWSC-I e 1 CD1 mice B6C3FU mice 1 1 -(c) 1 AT TABLE 26 SURVEY: OC'MPP,RI90N OF' DIFFERENT CONDENSATE TYPES WITHO[1T AND WITH uMBA PRETREATMENT AND DIFFERENT MOUSE STRAINS (a): stored at -75 degrees centigrade (b) stored at 4 degrees centigrade (c) dependent on mouse strain

REPORT P -/3068 NOMITUGR 7062 I WEEK NUMBER OF M WITH TUMORS ABS. (N) DEAD AND AL ICE REL. (0/0) IVE DEAD DEAD WITHOUT'TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 4 0 0 - 105 0 0.0 5 0 0 - 105 0 0.0 6 0 0 - 105 0 0.0 7 0 0 - 105 0 0.0 8 0 0 - 105 0 0.0 9 0 0 - 105 0 0.0 10 0 0 - 105 0 0.0 11 0 0 - 105 0 0.0 12 0 0 - 105 0 0.0 13 0 0 - 105 1 1.0 14 0 0 - 105 1 1.0 15 0 0 - 105 1 1.0 16 0 0 - 105 1 1.0 17 0 0 - 105 1 1.0 18 0 0 - 105 1 1.0 19 0 0 - 105 1 1.0 20 0 0 - 105 1 1.0 21 0 0 - 105 1 1.0 22 0 0 - 105 1 1.0 23 0 0 - 105 1 1.0 24 0 0 - 105 1 1.0 25 0 0 - 105 1 1.0 26 0 0 - 105 1 1.0 27 0 0 - 105 2 1.9 28 0 0 - 105 3 2.9 29 0 0 - 105 3 2.9 30 0 0 - 105 3 2.9 31 0 0 - 105 3 2.9 32 0 0 - 105 4 3.8 33 0 0 - 105 4 3.8 34 0 0 - 105 5 4.8 35 0 0 - 105 5 4.8 36 0 0 - 105 5 4.8 37 0 0 - 105 5 4.8 38 0 0 - 105 5 4.8 39 0 0 - 105 5 4.8' 40 0 0 - 105 5 4.8 41 0 0 - 105 5 4.8 TABLE 27 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 1 -GR N mPb 3-MAR-87 17:36:43 Page 1 File : DRA1:[MROJNOMITUGR 3068.LIS,1

REPORT P -/3068 NOMITUGR 7062 I WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE DEAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD: 42 0 0 - 105 5 4.8 43 0 0 - 105 5 4.8 44 0 0 - 105 6 5.7 45 0 0 - 105 6 5.7 46 0 0 - 105 6 5.7 47 0 0 - 105 6 5.7 48 0 0 - 105 7' 6.7 49 0 0 - 105 7' 6.7 50 1 0 0.0 104 7' 6.7 51 1 0 0.0 104 7 6.7 52 1 0 0.0 104' 8 7.7 53 1 0 0.0 104 9 8.7 54 1 0 0.0 104 10 9.6 55 1 0 0.0 104 11 10.6 56 1 0 0.0 104 13 12.5 57 2 0 0.0 103 14 13.6 58 2 0 0.0 103 14 13.6 59 3 0 0.0 102 14 13.7 60 3 0 0.0 102 14 13.7 61 3 0 0.0 102 14 13.7 62 3 0 0.0 102 14 13.7 63 4 0 0.0 101 14 13.9 64 4 0 0.0 101 14 13.9 65 4 0 0.0 101 15 14.9 66 4 0 0.0 101 16 15.8 67 4 0 0.0 101 17 16.8 68 4 1 25.0 101 17 16.8 69 4 1 25.0 101 18 17.8 70 4 1 25.0 101 18 17.8 71 6 1 16.7 99 19 19.2 72 6 1 16.7 99 19 19.2 73 7 1 14.3 98 20 20.4 74 9 1 11.1 96 20 20.8 75 9 1 11.1 96 22 22.9 76 9 2 22.2 96 23 24.0 77 9 2 22.2 96 24 25.0 78 10 2 20.0 95 27 28.4 79 10 3 30.0 95 27 28.4 80 10 3 30.0 95 27 28.4 81 12 3 25.0 93 27 29.0 TABLE 27 (continued) DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCER ABSOLUTE AND RELATIVE 1 -GR 3-MAR-87 17:36:43 Page 2 File : DRA1:(MRO]NOMITUGR 3068.LIS;1

REPORT P -/3068 NOMITUGR 7062 I WEEK NUMBER OF MICE WITH TUMORS ABS. (N) .DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (W DEAD AND ALIVE EAD REL. (0/0) DEAD 1 0 - 105 0 0.0 2 0 - 105 0 0.0 3 0 - 105 0 0.0 4 0 - 105 0 0.0 5 0 - 105 0 0.0 6 0 - 105 0 0.0 7 0 - 105 0 0.0 8 9 0 0 - - 105 105 0 0 0.0 0.0 10 0 - 105 0 0.0 11 0 - 105 0 0.0 12 0 - 105: 0 0.0 13 0 - 105 0 0.0 14 0 - 105 0 0.0 15 0 - 105 0 - 0.0 16 0 - 105 0 0.0 17 0 - 105 0 0.0 18 0 - 105 0 0.0 19 0 - 105 0 0.0 20 0 - 105 0 0.0 21 0 - 105 0 0.0 2:2 0 - 105 0 0.0 2' 3 0 - 105 0 0.0 24 0 - 105 0 0.0 25 0 - 105 1 1.0 26 0 - 105 1 1.0 27 ' 0 - 105 2 1.9 28 0 - 105 2 1.9 29 0 - 105 2 1.9 30 0 - 105 3 2.9 31 0 - 105 3 2..9 32 0 - 105 4 3.8' 33 0 .- 105 4 3.8' 34 0 - 105 4 3.8 35 0 0.0 104 5 4.8 36 0 0.0 103 5 4.9 37 0 0.0 103 5 4.9 38 0 0.0 103 5 4.9 39 0 0.0 102 5 4.9 40 0 0.0 100 5 5.0 41 0 0.0 99 6 6.1 TABLE 28 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 2 -GR 3-MAR-87 20:10:02 Page 1 File : DRA1:[MROJNOMITUGR 3068.LIS;1 '12A5

REPORT P -/306$ NOMITUGR 7062 WEEK NUMBER OF MICE' WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALSVE EAD REL. (0/0') DEAD 42 6 0 0.0 99 6 6.1 43 8 0 0.0 97 6 6.2 44 9 0 0.0 96 6 6.3 45 9 0 0.0 96 6 6.3 46 9 0 0.0 96 6 6.3 47 9' 0 0.0 96 6 6.3 48 9 0 0.0 96 6 6.3 49 9 0 0.0 96 6 6.3 50 9 0 0.0 96 6 6.3 51 9 0 0.0 96 6 6.3 52 10 0 0.0 95 6 6.3 53 10 0 0.0 95 6 6.3 54 10 0 0.0 95 7 7.4 55 10 0 0.0 95 7 7.4 56 11 0 0.0 94 7 7.4 57 11 0 0.0 94 7 7.4 58 11 0 0.0 94 7 7.4 59 11 0 0.0 94 7 7.4 60 12 0 0.0 93 7 7.5 61 13 1 7.7 92 8 8.7 62' 13 1 7.7 92' 8 8.7 63 14 1 7.1 91 8 8.8 64 14 1 7.1 91 9 9,9 65 14 1 7.1 91 10 11.0 66 15 1 6.7 90 13 14.4 67 15 1 6.7 90 13 14.4 68 15 1 6.7 90 13 14.4 69 15 1 6.7 90 14 15.6 70 15 1 6.7 90 14 15.6 71 16 1 6.3 89 17 19.1 72 16 2 12.5 89 18 20.2 73 17 2 11.8 88 18 20.5 74 18 2 15.1 87 22 25.3 75 20 2 10.0 85 23 27.1 76 22 2 9.1 83 23 27.7 77 23 2 8.7 82 24 29.3 78 25 2 8.0 80 24 30.0 79 26 2 7.7 79 27 34.2 80 26 2 7.7 79 28 35.4 81 28 2 7.1 77 2'8 36.4 TABLE 28 (continued) ~ ~ DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, ~ MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE ~ 2 -GR 3-MAR-87 20:10:02 Page 2 File : DRA1:[MRO]NOMITUGR 3068.LIS;1 ,~I4

REPORT P -/3068 NOMITUGR 7062 t WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N'). DEAD AND ALIVE EAD REL. (0/0) DEAD: 1 0 0 - 105 1 1.0 2 0 0 - 105 1 1.0 3 0 0 - 105 1 1.0 4 0 0 - 105 1 1.0 5 0 0 - 10:5 2 1.9 6 0 0 - 105 2 1.9 7 0 0 - 105 2 1.9 8 0 0 - 105 2 1.9 9 0 0 - 105 2 1.9 10 0 0 - 105 2 1.9 11 0 0 - 105 2 1.9 12 0 0 - 105 2 1.9 13 0 0 - 105 2 1.9 14 0 0 - 105 2 1.9 15 .0 0 - 105 2 1.9 16 0 0 - 105 2 1.9 17 0 0 - 105 2 1.9 18 0 0 - 105 2 1.9 19 0 0 - 105 2 1.9 20 0 0 - 105 2 1.9 21 0 0 - 105 2 1.9 22 0 0 - 105 2 1.9 23 0 0 - 105 2 1.9 24 0 0 - 105 2 1.9 25 0 0 - 105 2 1.9 26 0 0 - 105 2 1.9 27 0 0 - 105 2 1.9 28 0 0 - 105 2 1.9 29 0 0 - 105 2 1.9 30 0 0 - 105 2 1.9 31 0 0 - 105 2 1.9 32 0 0 - 105 2 1.9' 33 0 0 - 105 2 1.9' 34 0 0 - 105 3 2.9 35 0 0 - 105 3 2.9 36 0 0 - 105 3 2.9 37 0 0 - 105 3 2.9 38 0 0 - 105 3 2.9 39 1 0 0.0 104 4 3.8 40 1 0 0.0 104 4 3.8 41 1 0 0.0 104 5 4.8 TABLE 29 DEAD: MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 3 -GR 3-MAR-87 22:44:14 Page 1 File : DRA1:(MRO]NOMITUGR 3068.LIS;1 ooqg

REPORT P -/3068 NOMITUGR 7062 i WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 2 0 0.0 103 5 4.9 43 2 0 0.0 103 5 4.9 44 2 0 0.0 103 5 4.9 45 2 0 0.0 103 5 4.9 46 4 0 0.0 101 6 5.9 47 4 0 0.0 101 6 5.9 48 4 0 0.0 101 7 6.9 49 4 0 0.0 101 7 6.9 50 4 0 0.0 101 7 6.9 51 4 0 0.0 101 7 6.9 52 4 0 0.0 101 7 6.9 53 6 0 0.0 99 7 7.1 54 6 0 0.0 99 7 7.1 55 6 0 0.0 99 7 7.1 56 9 0 0.0 96 7 7.3 57 10 0 0.0 95 8 8,4 58 10 0 0.0 95 8 8,4 59 10 0 0.0 95 8 8.4 60 11 0 0.0 94 9 9.6 61 11 0 0.0 94 10 10.6 62 11 0 0.0 94 10 10.6 63 11 0 0.0 94 11 11.7 64 12 0 0.0 93 12 12.9 65 12 0 0.0 93 12 12.9 66 12 1 8.3 93 12 12.9 67 12' 1 8.3 93 14 15.1 68 12 2' 16.7 93 15 16.1 69 12 2 16.7 93 18 19.4 70 13 2 15.4 92 18 19.6 71 13 2 15.4 92 18 19.6 72 13 2 15.4 92' 19 20.7 73 13 2 15.4 92 19 20.7 74 13 2 15.4 92 20 21.7 75 13 2 15.4 92 21 22.8 76 15 2' 13.3 90 22 24.4 77 15 2 13.3 90 25 27.8 78 15 3 20.0 90 27 30.0 79 16 3 18.8 89 27 30.3 80 16 3 18.8 89 29 32.6 81 18 3 16.7 87 29 33.3 TABLE 29 ' (continued) DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 3 -GR 3-MAR-8'7 22:44:14 Page 2 File : DRA1:[MRO]NOMITUGR 3068.LIS;1 "95

REPORT P'-/3'068 NOMITUGR 7066 r WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE REL. (0/0) DEAD DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 41 0 0 - 105 0 0.0 5 0 0 - 105 0 0.0 6 0 0 - 105 0 0.-0 7 0 0 - 105 0 0.0 8 0 0 - 105 0 0.0 9 0 0 - 105 0 0.0 10 0 0 - 105 0 0.0 11 01 0 - 105 0 0.0 12' 0 0 - 105 0 0.0 13 0 0 - 105 0 0.0 14 0 0 - 105 0 0.0 15 0 0 - 105 0 0.01 16 0 0 - 105 01 0.0 17 0 0 - 105 0 0.0 18 0 0 - 105 0 0.0 19 0 0 - 105 0 0.0 20 0 0 - 10:5 0 0.0 21 0 0 - 105 0 0.0 22 0 0 - 105 0 0.0 23 0 0 - 105 0 0.0 24 0 0 - 105 0 0.0 25 0 0 - 105 0 0.0 26 0 0 - 105 0 0.0 27 0 0 - 105 0 0.0 28 0 0 - 105 0 0.0 29 0 0 - 105 0 0.0 30 0 0 - 105 2 1.9 31 0 0 - 105 2 1.9 32 0 0 - 105 3 2.9 33 0 0 - 105 3 2.9 34 0 0 - 105 4 3.8 35 0 0 - 105 4 3.8 36 0 0 - 105 4 3.8 37 0 0 - 105 4 3.8 38 0 0 - 105 4 3.8 39 0 0 - 105 5 4.8 40 0 0 - 105 5 4.8 41 0 0 - 105 5 4.8' TABLE 30 DEAD MICE WITHOUT ANDWITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 4 -GR 7-MAR-87 02:32:11 Page 1 File : DRA1:[MRO]NOMITUGR 3068.LIS;2 1295

REPORT P -/3068 NOMITUGR 7066 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 0 0 - 105 5 4.8' 43 0 0 - 105 5 4.8 44 0 0 - 105 6 5.7 45 0 0 - 105 6 5.7 46 0 0 - 105 6 5.7 47 0 0 - 1015 6 5.7 48 0 0 - 105 6 5.7 49 0 0 - 105 7 6.7 50 0 0 - 105 7 6.7 51 0 0 - 105 7 6.7 52' 0 0 - 105 7 6.7 53 1 0 0.0 104 7 6.7 54 1 0 0.0 104 7 6.7 55 1 0 0.0 104 7 6.7 56 2 0 0.0 103 7 6.8 57 3 0 0.0 102 7 6.9 58 3 0 0.0 102 8 7.8 59 4 0 0.0 101 8 7.9 60 4 0 0.0 101 8 7.9 61 4 0 0.0 101 9 8.9 62 4 0 0.0 101 9 8.9 63 5 0 0.0 100 10 10.0 64 5 0 0.0 100 13 13.0 65 7 0 0.0 98 14 14.3 66 8 0 0.0 97 14 14.4 67 8 0 0.0 97 16 16.5 68 9 0 0.0 96 16 16.7 69 10 0 0.0 95 16 16.8 70 10 1 10.0 95 19 20.0 71 10 1 10.0 95 19 20.0 72 11 1 9.1 94 21 22.3 73 11 1 9.1 94 23 2'4.5 74 11 1 9.1 94 26 27.7 75 11 1 9.1 94 29 30.9 76 13 1 7.7 92 29 31.5 77 13 1 7.7 92 30 32.6 78 13 2 15.4 92 33 35.9 79 13 4 30.8 92 34 37.0 80' 13 4 30.8 92 35 38.0 81 13 4 30.8 92 35 38.0 TABLE 30 ( contrinued ) DEAD M]fCE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 4 -GR 7-MAR-87' 02:32:11 Page 2' File : DRA1:[MRO]NOMITUGR. 3068.LIS;2

J.... .. I . - ~ I %. ~ ~.- ... J~ r\ ~., .. 6 REPORT P -/3068 NOMITUGR 7066 1 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) -DEAD 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 4 0 0 - 105 0 0.0 5 0 0 - 105 1 1.0 6 0 0 - 105 1 1.0 7 0 0 - 105 1 1.0 8 0 0 - 105 1 1.0 9 0 0 - 105 1 1.0 10 0 0 - 105 1 1.0 11 01 0 - 105 1 1.0 12 0 0 - 105 1 1.0 13 0 0 - 105 1 1.0 14 0 0 - 105 1 1.G 15 0 0 - 105 1 1.0' 16 0 0 - 105 1 1.0 17 0 0 - 105 1 1.0 18 0 0 - 105 1 1.0 19 0 0 - 105 1 1.0 20 0 0 - 1015 1 1.0 21 0 0 - 105 1 1.0 22 ' 0 0 - 105 1 1.0 23 0 0 - 105 1 1.0 24 0 0 - 105 1 1.0 25 0 0 - 105 1 1.0 26 0 0 - 105 2 1.9 27 0 0 - 105 2 1.9 28 0 0 - 105 2 1.9 29 0 0 - 105 3 2.9 30 0 0 - 105 3 2.9 31 0 0 - 105 4 3.8 32 0 0 - 105 4 3.8 33' 0 0 - 105 4 3.8 34 0 0 - 105 4 3.8 35 0 0 - 105 4 3.8 36 0 0 - 105 4 3.8 37 0 0 - 105 4 3.8 38 0 0 - 105 4 3.8 39 0 0 - 105 5 4.8 ~ 40 0 0 - 105 5 4.8 ~ 41. _ 2_ 0. 0.0 103 6 5.8 ~ ~ 0 TABLE 31 (f~ .. W DEAD: MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, ~ MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE (a 5 -GR N 7-MAR-87 05:010:23 Page 1 File : DRA1:[MRO]NOMITUGR.306S.LIS;2 9995

REPORT P -/3068 NOMITUGR 7066 I WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 2 0 0.0 103 6 5.8 43 2 0 0.0 103 7 6.8 44 4 0 0.0 101 7 6.9 45 4 0 0.0 101 7 6.9 46 4 0 0.0 101 7 6.9 47 4 0 0.0 101 7 6.9 48 4 0 0.0 101 7 6.9 49 4 0 0.0 101 8 7.9 50 4 0 0.0 101 9 8.9 51 4 0 0.0 101 9 8..9' 52 4 0 0.0 101 10 9.9 53 4 0 0.0 101 10 9.9 54 4 0 0.0 101 10 9.9' 55 4 0 0.0 101 11 10.9 56 4 0 0.0 101 11 10.9 5 7 4 0 0.0 101 11 10.9 ' 58 4 0 0.0 101 11 10.9 59 4 0 0.0 101 11 10.9 60 5 0 0.0 100 13 13.0 61 5 0 0.0 100 13 13.0 62 5 0 0.0 100 13 13.0 63 . 5 0 0.0 100 13 13.0 64 5 0 0.0 100 14 14.0 65 5 0 0.0 100 15 15.0 66 6 1 16.7 99 15 15.2 67 6 1 16.7 99 16 16.2 68 6 1 16.7 99 16 16.2 69 6 1 16.7 99 16 16.2 70 6 1 16.7 99 16 16.2 71 9 1 11.1 96 17 17.7 7 2 9 1 11. 1 9 6 17 17.7 7 3 10 1 10.0 95 5 17 17.9 74 10 1 10.0 95 17 17.9 75 10 1 10.0 95 19 20.0 76 10 1 10.0 95 19 20.0 77 10 1 10.0 95 20 21.1 78 10 1 10.0 95 20 21.1 79 10 1 10.0 95 22 23.2 80 10 1 10.0 95 24 25.3 81 11 1 9.1 94 24 25.5 ~' Q N TABLE 31 (continued) 0 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, CA MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE OA 5 -GR ~ ~ W 7-MAR-87 05:00:23 Page 2 File : DRA1:[MROjNOMITUGR 3068'.LIS;2 ";r

~~ .. ., ._ ,. _...... .u. . ~~.. J.... . ~V I UI... .. ...j Ivvi~ i REPORT P -/3068 NOMITUGR 7056 r WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE' EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 4 0 0 - 105 2 1.9 5 0 0 - 105 3 2.9 6 0 0 - 105 3 2.9 7 0 0 - 105 3 2.9 8 0 0 - 105 3 2.9 9 0 0 - 105 3 2.9 10 0 0 - 105 3 2.9 11 0 0 - 105 3 2'.9 12 0 0 - 105 3 2'.9 13 0 0 - 105 3 2'.9 14 0 0 - 105 3 2.9 15 0 0 - 105 3 2.9 16 0 0 - 105 3 2.9 17 0 0 - 105 3 2.9 18 0 0 - 105 3 2.9 19 0 0 - 105 3 2.9 2'0 0 0 - 105 3 2.9 2'1 0 0 - 105 3 2.9 22 0 0 - 105 3 2.9 23 0 0 - 105 3 2.9 24 0 0 - 105 3 2.9 25 0 0 - 105 3 2.9 26 0 0 - 105 3 2.9 27 0 0 - 105 3 2.9 28 0 0 - 105 3 2.9 29 0 0 - 105 3 2.9 30 1 0 0.0 104 3 2,9 31 1 0 0.0 104 3 2.9 32 1 0 0.0 104 3 2.9 33 1 0 0.0 104 3 2.9 34 1 0 0.0 104 3 2.9 35 1 0 0.0 104 3 2.9 36 1 0 0.0 104 3 2,,9 37 1 0 0.0 104 3 2.9 38 2 0 0.0 103 3' 2.9 39 2 0 0.0 103 3 2.9 40 2 0 0.0 103 3 2.9' 41 2 0 0.0 103 3 2.9 TABL E 3 2 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE'AND RELATIVE 6 -GR 7-MAR-87 07:28:51 Page 1 File : DRA1:[MROjNOMITUGR 3068.LIS,•2

REPORT P -/3068 NOMITUGR 7066 I WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 2 0 0.0 103 3 2.9 43 2 0 0.0 103 4 3'.9 44 2 0 0.0 103 4 3.9 45 2 0 0.0 103 4 3.9 46 2' 0 0.0 103 4 3.9 47 2' 0 0.0 103 4 3.9 48 2' 0 0.0 103 4 3.9 49 2 0 0.0 103 4 3.9 50 2 0 0.0 103 4 3.9 51 2 0 0.0 103 5 4.9 52 2 0 0.0 103 6 5.8' 53 3 0 0.0 102 6 5.9' 54 3 0 0.0 102 7 6.9' 55 3 0 0.0 102 7 6.9' 56 3 0 0.0 102 8 7.8 57 3 0 0.0 102 9 8.8 58 3 1 33.3 102 10 9.8 59 3' 1 33.3 102 11 10.8 60 3 1 33.3 102' 13 12.7 61 3 1 33.3 102' 14 13.7 62' 4 1 25.0 101 14 13.9 63 4 1 25.0 101 16 15.8 64 4 1 25.0 101 17 16.8 65 5 1 20.0 100 19 19.0 66 5 1 20.0 100 20 20.0 67 5 1 20.0 100 20 20.0 68 5 1 20.0 100 20 20.0 69 5 1 20.0 100 22 22.0 70 6 1 16.7 99 26 26.3 71 6 1 16.7 99 26 26.3 72 8' 1 12.5 97 26 26.8 73 8 1 12.5 97 26 26.8 74 9 1 11.1 96 26 27.1 75 9 1 11.1 96 26 27.1 76 11 2 18.2 94 27 28.7 77 11 3 27.3 94 29 30.9 78 13 4 30.8 92 31 33.7 79 13 4 30.8 92 32 34.8 80 13 4 30.8 92 32 34.8 81 13 4 30.8 92 33 35.9 TABLE 32 (continued) DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 6 -GR 7-MAR-87 07:28:52 Page 2 File : DRA1:[MRO]NOMITUGR 3068.LIS;2 3285

REPORT P -/3068 NOMITUGR 7063 i WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 4 0 0 - 105 0 0.0 5 0 0 - 105 0 0.0 6 0 0 - 105 0 0.0 7 0 0 - 105 0 0.0 8 0 0 - 105 0 0.0 9 0 0 - 105 0 0.0 10 0 0 - 105 0 0.0 11 0 0 - 105 0 0.0 12 0 0 - 105 0 0.0 13 0 0 - 105 0 0.0 14 0 0 - 105 0 0.0 15 0 0 - 105 0 0.0 16 0 0 - 105 0 0.0 17 0 0 - 105 0 0.0 18 0 0 - 105 0 0.0 19 0 0 - 105 0 0.0 20 0 0 - 105 0 0.0 21 0 0 - 105 1 1.0 22 1 0 ' 0.0 104 1 1.0 23 1 0 ' 0.0 104 1 1.0 24 1 0 0.0 104 1 1.0 25 2 0 0.0 103 2 1.9 26 2 0 0.0 103 2 1.9' 27 2 0 0.0 103 2 1.9 28 2 0 0.0 103 2 1.9 29 2 0 0.0 103 2 1.9 30 2 0 0.0 103 2 1.9 31 2 0 0.0 103 3 2.9 32 3 0 0.0 102 3 2.9 33 4 0 0.0 101 3 3.0 34 4 0 0.0 101 3 3.0 35 5 0 0.0 100 3 3.0 36 5 0 0.0 100 3 3.0 37 6 0 0.0 99 3 3.0 38 6 0 0.0 99 3 3.0 39 7 0 0.0 98 3 3.1 40 9 0 0.0 96 3 3.1 41 10 0 0.0 95 3 3.2 TABLE 33 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 7 -GR 4-MAR-87 01:18:53 Page 1 File : DRA1:(MRO)NOMITUGR 3068.LIS;1 ~~~5

REPORT'P -/3068 NOMITUGR 7063 11 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE DEAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 11 0 0.0 94 3 3.2 43 11 0 G.0 94 3 3.2 44 11 0 01.0 94 3 3.2 45 11 0 01.0 94 4 4.3 46 11 0 0.0 94 4 4.3 47 12 0 0.0 93 5 5.4 48' 13 0 0.0 92 5 5.4 49 13 0 0.0 92 5 5.4 50 13 0 0.0 92 5 5.4 51 14 0 0.0 91 5 5.5 52 16 0 0.0 89 5 5.6 53 19 0 0.0 86 5 5.8 54 19 0 0.0 86 6 7.0 55 19 0 0.0 86 6 7.0 56 20 0 0.0 85 6 7.1 57 21 1 4.8' 84 6 7.1 58 22 2 9.1 83 6 7.2 59 22 2 9.1 83 6 7.2 60 24 2 8.3 81 6 7.4 61 24 2 8.3 81 6 7.4 62 25 2 8.0 80 7 8.8 63 28 2 7.1 77 9 11.7 64 28 2 7.1 77 10 13.0 65 29 2 6.9 76 10 13.2 66 30 2 6.7 75 10 13.3 67 31 3' 9.7 74 10 13.5 68 31 4 12.9 74 10 13.5 69 32 4 12.5 73 10 13.7 70 35 4 11.4 70 10 14.3 71 36 4 11.1 69 10 14.5 72' 37 4 10.8 68 10 14.7 73 37 4 10.8 68 10 14.7 74 37 4 10.8 68 11 16.2 75 38 5 13.2 67 11 16.4 76 39 5 12.8 66 12 18.2 77 39 6 15.4 66 15 22.7 78 42' 7 16.7 63 16 25.4 79 43 10 23.3 62 16 25.8 80 43 11 25.6 62 17 27.4 ~ 81 48 11 22.9 57 17 29.8 Q N ~ TABLE 33 (continued) Q ~ DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, ~ MACROSCOP'IC APPEARANCE, ABSOLUTE AND RELATIVE {~ 7 -GR ~ 4-MAR-87 01:18:54 Page 2 File : DRA1:[MRO]NOMITUGR 3068'.LISr1

REPORT P -/3068 NOMITUGR 7063 1 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD:AND ALIVE EAD REL. (0/0) DEAD WITHOUT'TUMORS ABS. (N)' DEAD AND ALIVE EAD REL. (0/0) DEAD~ 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 4 0 0 - 105 0 0.0 5 0 0 - 105 1 1.0 6 0 0 - 105 1 1.0 7 0 0 - 105 1 1.0 8 0 0 - 105 1 1.0 9 0 0 - 105 1 1.0 10 0 0 - 105 1 1.0 11 0 0 - 105 2 1.9 12 0 0 - 105 2 1.9 13' 0 0 - 105 2 1.9 14 1 0 0.0 104 2 1.9 15 1 0 0.0 104 2 1.9 16 1 0 0.0 104 2 1.9 17 2 0 0.0 103 2 1.9 18 3 0 0.0 102 2 2.0 19 3 0 0.0 102 2 2.0 20 3 0 0.0 102 3 2.9 21 3 0 0.0 102 4 3.9 22 4 0 0.0 101 4 4.0 23 7 0 0.0 98 4 4.1 24 8 0 0.0 97 4 4.1 25 5 8 0 0. 0 9 7 4 4.1 26 8 0 0.0 97 4 4.1 2'7 8 0 0.0 97 4 4.1 2'8 9 0 0.0 96 4 4.2 29 11 0 0.0 9 4 4 4.3 30 11 0 0.0 94 4 4.3 31 11 0 0.0 94 4 4.3 32 12 0 0.0 93 4 4.3 33' 13 0 0.0 92 4 4.3 34 15 0 0.0 90 4 4.4 35 16 0 0.0 89 4 4.5 36 17 1 5.9 88 4 4.5 37 18 1 5.6 87 4 4.6 38 18 1 5.6 87 4 4.6 39 18 1 5.6 87 4 4.6 40 21 1 4.8 84 4 4.8 41 23 1 4.3 82 4 4.9 ~ 0 ~ Q TABLE 34 CA DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, w MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE W~ 8 -GR 4-MAR-87 03:56:50 Page 1 File : DRA1:[MRO]NOMITUGR_3'068.LIS•1

REPORT'P -/3068 NOMITUGR 7063' I WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 23 1 4.3' 82 4 4.9 43 23 1 4.3' 82 4 4.9 44 26 1 3.8' 79 5 6.3 45 27 1 3.7 78 5 6.4 46 29 1 3.4 76 5 6.6 47 32 1 3.1 73 5 6.8 48 32 1 3.1 73 5 6.8 49 32 1 3.1 73 5 6.8 50 32 1 3.1 73 5 6.8 51 33 1 3.0 72 5 6.9 52 34 2 5.9 71 5 7.0 53 34 4 11.8 71 5 7.0 54 34 4 11.8 71 5 7.0 55 35 5 14.3 70 5 7.1 56 36 5 13.9 69 5 7.2 57 36 5 13.9 69 5 7.2 58 37 5 13.5 68 5 7.4 59 37 5 13.5 68 5 7.4 60 39 5 12.8 66 5 7.6 61 39 5 12.8 66 5 7.6 62 39 5 12.8 66 5 7.6 63 41 6 14.6 64 5 7.8 64 43 6 14.0 62 5 8.1 65 45 6 13.3 60 5 8.3 66 46 7 15.2 59 5 8.5 67 46 8 17.4 59 5 8.5 68 46 8 17.4 59 5 8.5 69 47 10 21.3 58 6 10.3 70 47 12 25.5 58 6 10.3' 71 47 14 29.8 58 6 10.3' 72 50 14 28.0 55 6 10.9 73 50 14 28.0 55 6 10.9' 74 51 14 27.5 54 6 11.1 75 51 14 27.5 54 8 14.8 76 52 14 26.9 53 9 17.0 77 52 14 26.9 53 11 20.8 78 55 15 27.3 50 11 22'.0 79 55 15 27.3 50 12 24.0 80 55 19 34.5 50 13 26.0 81 61 19 31.1 44 13 29.5 TABLE 34 (continued) DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 8 -GR 4-MAR-87 03:56:50 Page 2 File : DRA1:[MROJNOMITUGR 3068.LIS;1 9195

REPORT P -/3068 NOMITUGR 7063 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/'0) DEAD WITHOUT TUMORS ABS. (N) DEAD: AND ALIVE EAD REL. (0/0) DEAD 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 4 0 0 - 105 0 0.0 5 0 0 - 105 0 0.0 6 0 0 - 105 0 0.0 7 0 0 - 105 0 0.0 8 0 0 - 105 0 0.0 9 0 0 - 105 0 0.0 10 0 0 - 105 0 0.0 11 0 0 - 105 0 0.0 12 0 0 - 105 0 0.0 13 1 0 0.0 104 0 0.0 14 1 0 0.0 104 0 0.0 15 2 0 0.0 103 0 0.0 16 2 0 0.0 103 0 0.0 17 3 0 0.0 102 0 0.0 18 3 0 0.0 102 0 0.0 19' 3 0 0.0 102 0 0.0 20 5 0 0.0 100 0 0.0 21 5 0 0.0 100 0 0.0 22 5 0 0.0 100 0 0.0 23 5 0 0.0 100 0 0.0 24 7 0 0.0 98 0 0.0 25 7 0 0.0 98 0 0.0 26 8 0 0.0 97 0 0.0 27 8 0 0.0 97 0 0.0 28 8 0 0.0 97 0 0.0 29 10 0 0.0 95 0 0.0 30 11 0 0.0 94 0 0.0 31 13 0 0.0 92 0 0.0 32 13 0 0.0 92 0 0.0 33 13 0 0.0 92 0 0.0 34 15 0 0.0 9-0 0 0.0 35 16 0 0.0 89 0 0.0 36 16 0 0.0 89 0 0.0 37 16 0 0.0 89 0 0.0 38 16 0 0.0 89 1 1.1 39 16 0 0.0 89 1 1.1 40 20 1 5.0 85 1 1.2 41 23. _1 _ 4.3 82 1 1.2 TABLE 35 0 N 8 CA DEADMICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, ~ MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE ~ 9 -GR 4-MAR-87 06:36:32 Page 1 File : DRA1:[MRO]NOMITUGR 3068.LIS;1

REPORT P -/3068 NOMITUGR 7063 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 24 2 8.3 81 2 2.5 43 26 2 7.7 79 2 2.5 44 27 2 7.4 78 2 2.6' 45 27 2 7.4 78 3 3.8 46 29 3 10.3 76 3 3.9 47 29 3 10.3 76 3 3.9 48 30 3 10.0 75 3 4.0 49 31 4 12.9 74 4 5.4 50 31 4 12.9 74 4 5.4 51 32 5 15.6 73 4 5.5 52 33 5 15.2 72 4 5.6 53 34 5 14.7 71 4 5.6 54 35 5 14.3 70 4 5.7 55 35 6 17.1 70 5 7.1 56 3'6 6 16.7 69 6 8.7 57 3'8 6 15.8 67 6 9.0 58 39 6 15.4 66 6 9.1 59 39 7 17.9 66 6 9.1 60 40 7 17.5 65 6 9.2 61 40 8 20.0 65 6 9.2 62 40 9 22.5 65 7 10.8 63 42 11 26.2 63 7 11.1 64 43 11 25.6 62 7 11.3 65 45 11 24.4 60 7 11.7 66 45 13 28.9 60 7 11.7 67 46 13 28.3 59 8 13.6 68 46 13' 28.3 59 8 13.6 69 47 15 31.9 58 8 13.8 70 50 15 30.0 55 8 14.5 71 53 16 30.2 52' 8 15.4 72 55 17 30.9 50 9 18.0 73' 56 19 33.9 419 9 18.4 74 57 20 35.1 48 10 20.8 75 58 21 36.2 47 10 21.3 76 59 22 37.3 46 10 21.7 77 59 24 40.7 46 10 21.7 78 62 25 40.3 43 11 25.6 79 64 26 40.6 41 11 26.8 80 64 26 40.6 41 11 26.8 81 65 27 41.5 40 11 27.5 TABLE 35 (continued) DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 9 -GR 4-MAR-87 06:36:32 Page 2' File : DRA1:[MRO]NOMITUGR 3068.LIS;1 - J'v

REPORT P -/3068 NOMITUGR 7066 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N') DEAD AND ALIVE EAD REL. (0/0) DEAD: 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 4 0 0 - 105 0 0.0 5 0 0 - 105 0 0.0 6 0 0 - 105 0 0.-0 7 0 0 - 105 0 0.0 8 0 0 - 105 0 0.0 9 0 0 - 105 0 0.0 10 0 0 - 105 0 0.0 11 0 0 - 105 0 0.0 12 0 0 - 105 0 0.0 13 1 0 0.0 104 0 0.0 14 1 0 0.0 104 0 0.0 15 1 0 0.0 104 0 0.0 16 1 0 0.0 104 0 0.0 17 2 0 0.0 103 0 0.0 18 2 0 0.0 103 0 0.0 19 2 0 0.0 103 0 0.0 20 2 0 0.0 103 1 1.0 21 3 0 0.0 102 1 1.0 22 4 0 0.0 101 1 1.0 23 5 0 0.0 100 1 1.0 24 6 0 0.0 99 1 1.0 25 6 0 0.0 99 1 1.0 26 6 0 0.0 99 1 1.0 27 7 0 0.0 98 1 1.0 28 7 0 0.0 98 1 1.0 29 7 0 0.0 98 1 1.0 30 9 0 0.0 96 1 1.0 31 9 0 0.0 96 2 2.1 32 9 0 0.0 96 3 3.1 33 9 0 0.0 96 3 3.1 34 12 0 0.0 93 4 4.3 35 12 0 0.0 93 4 4.3 36 13 0 0.0 92 4 4.3 37 13 1 7.7 92 5 5.4 38 13 1 7.7 92 5 5.4 39 13 1 7.7 92 5 5.4 40 14 1 7.1 91 6 6.6 41 14 1 7.1 91 7 7.7 TABLE 36 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 10 -GR 7-MAR-87 10:06:20 Page 1 File : DRA1:[MRO]NOMITUGR 3068.LIS;2

REPORT P -/3068 NOMITUGR 7066 0 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 14 1 7.1 91 7 7.7 43 17 1 5.9' 88 7 8.0 44 18 1 5.6 87 8 9.2 45 18 1 5.6 87 8 9.2 46 20 1 5.G 85 8 9.4 47 22 1 4.5 83 8 9.6 48 23 1 4.3 82 8 9.8 49 23 1 4.3 82 8 9.8 50 23 1 4.3 82 9 11.0 51 25 1 4.0 80 9 11.3 52 26 1 3.8 79 9 11.4 53 27 1 3.7 78 9 11.5 54 27 1 3.7 78 9 11.5 55 28' 1 3.6 77 9 11.7 56 30 1 3.3 75 9 12.0 57 31 1 3.2 74 10 13.5 58 31 2 6.5 74 10 13.5 59 33 2' 6.1 72 10 13.9 60 37 3. 8.1 68 10 14.7 61 37 3 8.1 68 11 16.2 62 38 3 7.9 67 12 17.9 63 38 4 10.5 67 13 19.4 64 38 5 13.2 67 14 20.9 65 39 6 15.4 66 14 21.2 66 42 6 14.3 63 16 25.4 67 44 7 15.9 61 16 2'6.2 68 45 7 15.6 60 16 2'6.7 69 46 8 17.4 59 16 27.1 70 50 10 20.0 55 16 29.1 71 50 11 22.0 55 17 30.9' 72 50 11 22.0 55 19 34.5 73 51 12 23.5 54 19 35.2 74 51 12 23.5 54 19 35.2 75 52 13 25.0 53 19 35.8 76 55 14 25.5 50 21 42.0 77 55 14 25.5 50 21 42.0 78 59 15 25.4 46 23 50.0 79 59 17 28.8 46 24 52.2 N 80 59 19 32.2 46 24 52.2 Q 81 61 20 32.8 44 24 54.5 TABLE 36 (continued) CA N DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, w ~ MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 10 -GR ~ 7-MAR-87 10:06:20 Page 2 File : DRA1c.[MRO]NOMITUGR 3068.LIS;2

REPORT P -/3068 NOMITUGR 7066 i WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 1 0 0 - 105 1 1.0 2 0 0 - 105 1 1.0 3 0 0 - 105 2 1.9 4 0 0 - 105 2 1.9 5 0 0 - 105 2 1.9 6 0 0 - 105 2 1.9 7 0 0 - 105 4 3.8 8 0 0 - 105 4 3.8 9 0 0 - 105 4 3.8 10 0 0 - 105 5 4.8 11 0 0 - 105 5 4.8 12 0 0 - 105 5 4.8 13 1 0 0.0 104 5 4.8 14 1 0 0.0 104 5 4.8 15 1 0 0.0 104 5 4.8 16 1 0 0.0 104 5 4.8 17 1 0 0.0 104 5 4.8 18 1 0 0.0 104 5 4.8 19 1 0 0.0 104 6 5.8 20 2 0 0.0 103 7 6.8 21 5 0 0.0 100 7 7.0 22 6 0 0.0 99 7 7.1 23 6 0 0.0 99 7 7.1 24 6 0 0.0 99 7 7.1 25 6 0 0.0 99 7 7.1 26 7 0 0.0 98 8 8.2 27 9 0 0.0 96 8 8.3 28 9 0 0.0 96 8 8.3 29 9 0 0.0 96 8 8.3 30 10 0 0.0 95 8 8.4 31 12 0 0.0 93 8 8.6 32 13 0 0.0 92 8 8.7 33 15 0 0.0 90 8 8.9 34 16 0 0.0 89 8 9.0 35 16 0 0.0 89 8 9.0 36 18 0 0.0 87 8 9.2 37 18 0 0.0 87 8 9.2 38 19 0 0.0 86 8 9.3 39 23 0 0.0 82 8 9.8 40 25 0 0.0 80 8 10.0 41 28 0_ 0.0 77 8 10.4 N 0' ~' ~ ~ TABLE 37 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, W MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE ~ 11 -GR ~ 7-MAR-87 12:48:49 Page 1 File : DRA1:[MROjNOMITUGR_3068.LIS;2

REPORT P -/3068 NOMITUGR 7066 I WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE DEAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 29 0 0.0 76 8 10.5 43 33 0 0.0 72 8 11.1 44 35 0 0.0 70 9 12.9 45 37 0 0.0 68 9 13.2 46 38 0 0.0 67 9 13.4 47 40 0 0.0 65 9 13.8 48 41 1 2.4 64 10 15.6 49 41 1 2.4 64 11 17.2 50 44 2' 4.5 61 11 18.0 51 44 3 6.8 61 11 18.0 52 46 3 6.5 59 12 20.3 53 47 3 6.4 58 12 20.7 54 50 4 8.0 55 12 21.8 55 51 5 9.8 54 12 22.2 56 54 7 13.0 51 12 23.5 57 56 8 14.3 49 12 2'4.5 58 57 8 14.0 48 12 2'5.0 59 57 8 14.0 48 12 25.0 60 57 8 14.0 48 12 25.0 61 59 9 15...3' 46 12 26.1 62 60 10 16.7 45 12 26.7 63 64 14 21.9 41 12 29.3 64 68 15 22.1 37 12 32.4 65 68 16 23.5 37 12 32.4 66 70 16 22.9 35 13 37.1 67 70 18 25.7 35 14 40.0 68 71 20 28.2 34 14 41.2 69 71 20 28.2 34 15 44.1 70 72 21 29.2 33 15 45.5 71 72 22 30.6 33 16 48.5 72 72 22 30.6 33 16 48.5 73 72' 26 36.1 33 16 48.5 74 74 29 39.2 31 17 54.8 75 74 30 40.5 31 17 54.8 76 75 31 41.3 30 18 60.0 77 76 34 44.7 29 18 62.1 78 78 36 46.2 27 18 66.7 79 78 38 48.7 27 18 66.7 80 79 40 50.6 26 18 69.2 81 82 41 50.0 23 18 78.3 TABLE 37 (continued) DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 11 -GR 7-MAR-87 12:48:49 Page 2 File : DRA1:[MRO]NOMITUGR 3068.LIS;2

REPORT P -/3068' NOMITUGR 7066 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0). DEAD 1 0 0 - 105 0 0.0 2 0' 0 - 105 0 0.0 3' 01 0 - 105 0 0.0 4 0 0 - 105 1 1.0 5 0 0 - 105 2 1.9 6 0 0 - 105 4 3.8 7 0 0 - 105 7 6.7 8 0 0 - 105 9 8.6 9 0 0 - 105 9 8.6 10 0 0 - 105 10 9.5 11 0 0 - 105 10 9.5 12 0 0 - 105 10 9.5 13 0 0 - 105 10 9.5 14 0 0 - 105 10 9.5 15 0 0 - 105 10 9.5 16 0 0 - 105 10 9.5 17 2 0 0.0 103 10 9.7 18 4 0 0.0 101 10 9.9 19 4 0 0.0 101 12 11.9 20 5 0 0.0 100 12 12.0 21 5 0 0.0 100 12 12.0 22 5 0 0.0 100 12' 12.0 23 7 0 0.0 98 12 12.2 24 8 0 0.0 97 12 12.4 25 9 0 0.0: 96 12 12.5 26 10 0 0.0 95 12 12' . 6 2'7 11 0 0.0 94 12 12.8 28 13 0 0.0 92 12 13.0 29 15 0 0.0 90 12 13.3 30 18 0 0.0 87 12 13.8 31 20 0 0.0 85 12 14.1 32 21 0 0.0 84 12 14.3 33 21 0 0.0 84 12 14.3 34 21 0 0.0 84 12 14.3 35 21 1 4.8 84 13 15.5 36 23 1 4.3 82 13 15.9 37 26 1 3.8 79 14 17.7 38 27 1 3.7 78 14 17.9 39 33 1 3.0 72 14 19.4 40 3'4 1 2.9 71 14 19.7 41 36 1 2.8 69 14 20.3 TABLE 38 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 12 -GR 7-MAR-87 15:32:32 Page 1 File : DRA1:[MRO]NOMITUGR 3068.LIS;2 N 0 N ~ 0 C11 N cj ~ ~ 1 ?95

REPORT P -/3068 NOMITUGR 7066 I WEEK NUMBER OF MICE~ WITH TUMORS ABS. (N) DEAD AND ALIVE DEAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 38 1 2.6 67 14 20.9 43 40 1 2.5 65 15 23.1 44 46 1 2.2' 59 15 25.4 45 50 1 2.0 55 15 27.3 46 51 1 2.0 54 15 27.8 47 52 1 1..9' 53 15 28.3 48 52 1 1.9 53 15 28.3 49 53 3 5.7 52 15 28.8 50 57 3 5.3 48 15 31.3 51 59 3 5.1 46 16 34.8 52 59 3 5.1 46 16 34.8 53 59 5 8.5 46 16 34.8 54 61 5 8.2 44 16 36.4 55 62 5 8.1 43 16 37.2 56 64 6 9.4 41 16 39.0 57 66 8 12.1 39 17 43.6 58 67 10 14.9 38 17 4'4.7 59 68 10 14.7 37 17 45.9 60 69 12 17.4 36 17 47.2 61 69 13 18.8 36 17 47.2 62 70 14 20.0 35 17 48.6 63 71 16 22.5 34 17 50.0 64 71 18 25.4 34 17 50.0 65 72 18 25.0 33 17 51.5 66 72 20 27.8 33 17 51.5 67' 72 20 27.8 33 17 51.5 68 73 24 32.9 32 17 53.1 69 76 24 31.6 29 17 58.6 70 76 28 36.8 29 17 58.6 71 77 28 36.4 28 17 60.7 72' 78 29' 37.2 27 17 63.0 73' 78 31 39.7 27 17 63.0 74 78 33 42.3 27 17 63.0 75 79 36 45.6 26 18 69.2 76 81 36 44.4 24 18 75.0 77 82 38 46.3 23 18 78.3 78 84 39 46.4 21 18 85.7 79 84 42 50.0 21 18 85.7 80 84 46 54.8 21 18 85.7 ~ 81 85 46 54.1 20 18 90.0 ~ TABLE 38 (continued) ~ ~ DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, M+ MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE Gr~ 12 -GR ~ 7-MAR-87 15:32:33 Page 2 File : DRA1:[MRO]NOMITUGR 30$8.LIS;2

REPORT P -/3068 NOMITUGR 7063 i WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0! 3 0 0 - 105 0 0.0 4 0 0 - 105 0 0.0 5 0 0 - 105 0 0.0 6 0 0 - 105 0 0.0 7 0 0 - 105 0 0.0 8 0 0 - 105 0 0.0 9 0 0 - 105 0 0.0 10 0 0 - 105 0 0.0 11 0 0 - 105 0 0.0 12' 0 0 - 105 0 0.0 13 1 0 0.0 104 0 0.0 14 1 0 0.0 104 0 0.0 15 1 0 0.0 104 0 0.0 16 1 0 0.0 104 0 0.0 17 2 0 0.0 103 0 0.0 18 3 0 0.G 102 0 0.0 19 3 0 0A 102 0 0.0 20 3 0 0.0 102 0 0.0 21 3 0 0.0 102 1 1.0 2'2 3 0 0.0 102 2 2.0 23 4 0 0.0 101 3 3.0 24 5 0' 0.0 100 3 3.0 25 5 0 0.0 100 4 4.0 26 7 0 0.0 98 4 4.1 27 8 1 12.5 97 4 4.1 28 10 1 10.0 95 4 4.2 29 11 1 9.1 94 6 6.4 30 12 1 8.3 93 6 6.5 31 13 1 7.7 92 6 6.5 32 13 1 7.7 92 6 6.5 33 13 1 7.7 92 7 7.6 34 14 1 7.1 91 7 7.7 35 14 1 7.1 91 7 7.7 36 14 1 7.1 91 7 7.7 37 16 1 6.3 89 7 7.9 38 19 1 5.3 86 7 ~ 8.1 39 22 1 4.5 83 7 8.4 ~ 40 25 1 4.0 80 8 10.0 ~ 41 26 1 3.8 79 8 10.1 0 U1 TABLE 39 R"+ ' cj DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, ~P MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE ~ 13 -GR 4-MAR-87 10:00:45 Page 1 File : DRA1:(MRO)NOMITUGR 3068.LIS;1 "qs

REPORT P -/3068 NOMITUGR 7063 I WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE DEAD REL. (0/0) DEAD WITHOUT'TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 26 1 3.8 79 8 10.1 43 27 1 3.7 78 8 10.3 44 31 2 6.5 74 8 10.8 45 32 2 6.3 73 8 11.0 46 34 2 5.9 71 9 12.7 47 39 2 5.1 66 9 13.6 48 40 2 5.0 65 9 13.8 49 41 3 7.3 64 9 14.1 50 46 3 6.5 59 9 15.3 51 46 3 6.5 59 9 15.3 52 47 3 6.4 58 10 17.2 53 50 3 6.0 55 11 20.0 54 51 3 5.9 54 11 20.4 55 52 5 9.6 53 11 20.8 56 53 7 13.2 52 12 23.1 57 56 7 12.5 49 13 26.5 58 57 7 12.3 48 13 27.1 59 6:0 8 13.3 45 14 31.1 60 63 9 14.3 42 14 33.3 61 64 10 15.6 41 14 34.1 62 65 10 15.4 40 14 35.0 63 66 13 19.7 39 15 38.5 64 67 14 20.9 38 16 42.1 65 67 15 22.4 38 16 42.1 66 67' 16 23.9 38 17 44.7 67 69 18 26.1 36 17 47.2 68 70 18 25.7 35 17 48.6 69 73 18 24.7 32 17 53.1 70 73 18 24.7 32 18 56.3 71 75 21 28.0 30 20 66.7 72 76 24 31.6 29 21 72.4 73 77 28 36.4 28 21 75.0 74 79 28 35.4 26 2'1 80.8 75 79 34 43.0 26 2'1 80.8 76 79 36 45.6 26 21 80.8 77 79 37 46.8 26 21 80.8 78 81 41 50.6 24 21 87.5 79 81 44 54.3 24 21 87.5 80 81 45 55.6 24 21 87.5 81 81 45 55.6 24 21 87.5 TABLE 39. (continued) N 0' ~ ~ ~ DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, CAS MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE iA 13 -GR ~ 4-MAR-87 10:00:45 Page 2 File : DRA1:[MRO]NOMITUGR_3068.LIS;1

REPORT P -/3068 NOMITUGR 7063 I WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 4 0 0 - 105 0 0.0 5 0 0 - 105 0 0.0 6 0 0 - 105 0 0.0 7 0 0 - 105 1 1.0 8 0 0 - 105 1 1.0 9 0 0 - 105 1 1.0 10 0 0 - 105 1 1.0 11 0 0 - 105 1 1.0 12 0 0 - 105 1 1.0 13 2 0 0.0 103 1 1.0 14 2 0 0.0 103 1 1.0 15 2 0 0.0 103 1 1.0 16 4 0 0.0 101 1 1.0 17 4 0 0.0 101 1 1.0 18 5 0 0.0 100 1 1.0 19 6 0 0.0 99 1 1.0 2'0 8 0 0.0 97 1 1.0 21 9 0 0.0 96 1 1.0 22 10 0 0.0 95 1 1.1 23 11 1 9.1 94 1 1.1 24 12' 1 8.3 93 1 1.1 25 12 1 8.3 93' 1 1.1 26 15 1 6.7 90 2 2.2 27 16 1 6.3 89 2 2.2 28 18 1 5.6 87 2 2.3 29 20 1 5.0 85 2 2.4 30 23 1 4.3 82 2 2.4 31 27 1 3.7 78 2 2.6 32 34 1 2.9 71 3 4.2 33 35 1 2.9 70 3 4.3' 34 38 1 2.6 67 3 4.5 35 40 1 2.5 65 3 4.6 36 41 2 4.9 64 3 4.7 37 41 2 4.9 64 4 6.3' 38 42 2 4.8 63 4 6.3 39 42 2 4.8 63 4 6.3 40 43 3 7.0 62 4 6.5 41 46 3 6.5 59 4 6.8 TABLE 40 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION'AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 14 -GR. 4-MAR-87 18:49:15 Page 1 File : DRA1:[MRO]NOMITUGR 3068.LIS,1

REPORT P -/3068 NOMITUGR 7063 / WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 53 3 5.7 52 4 7.7 43 56 3 5.4 49 4 8.2 44 60 3 5.0 45 4 8.9 45 63 4 6.3 42 4 9.5 46 64 4 6.3 41 4 9.8 47 64 5 7.8 41 4 9.8 48 67 5 7.5 38 4 10.5 49 67 5 7.5 38 5 13.2 50 73 5 6.8 32 5 15.6 51 74 7 9.5 31 5 16.1 52' 74 7 9.5 31 5 16.1 53' 75 7 9.3 30 5 16.7 54 77 7 9.1 28 5 17.9 55 78 7 9.0 27 5 18.5 56 78 7 9.0 27 6 22.2 57 78 11 14.1 27 6 22.2 58 78 11 14.1 27 6 22.2 59 81 12 14.8 24 6 25.0 60 83 13 15.7 22 6 27.3 61 84 15 17.9 21 6 28.6 62 84 17 20.2 21 6 28.6 63' 86 19' 22.1 19 6 31.6 64 86 22 25.6 19 6 31.6 65 86 25 29.1 19 6 31.6 66 87 25 28.7 18 6 33.3 67 87 26 29.9 18 6 33.3 68 88 26 29.5 17 6 35.3 69 89 30 33.7 16 6 37.5 70 91 33 36.3 14 6 42.9 71 91 37 40.7 14 7 50.0 72' 91 38 41.8 14 8 57.1 73' 91 40 44.0 14 8 57.1 74 91 43 47.3 14 8 57.1 75 91 48 52.7 14 8 57.1 76 93 56 60.2 12 8 66.7 77 93 60 64.5 12 8 66.7 78 94 62 66.0 11 8 72.7 79 94 63 67.0 11 8 72.7 80 95 70 73.7 10 8 80.0 81 97 71 73.2 8 8 100.0 TABLEqp (continued) DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 14 -GR 4-MAR-87 18:49:15 Page 2' File : DRA1:[MROJNOMITUGR_3068.LIS',1 .,1 fl.

REPORT'P -/3068 NOMITUGR 7063 i WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD WITHOUT TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 1 0 0 - 105 0 0.0 2 0 0 - 105 0 0.0 3 0 0 - 105 0 0.0 4 0 0 - 105 0 0.0 5 0 0 - 105 0 0.0 6 0 0 - 105 1 1.0 7 0 0 - 105 1 . 1.0 8 0 0 - 105 1 1.0 9 0 0 - 105 2 1.9 10 0 0 - 105 2 1.9 11 0 0 - 105 2 1.9 12 0 0 - 105 2 1.9 13 3 0 0.0 102 2 2.0 14 3 0 0.0 102 2 2.0 15 3 0 0.0 102 2 2.0 16 3 0 0.0 102 2 2.0 17 6 0 0.0 99 2 2.0 18 7 0 0.0 98 2' 2.0 19 8 0 0.0 97 2 2.1 20 15 0 0.0 90 2 2.2 21 20 0 0.0 85 2 2.4 22 23 0 0.0 82 2 2.4 23 25 0 0.0 80 2 2.5 24 28 0 0.0 77 2 2.6 25 29 0 0.0 76 3 3.9 26 30 0 0.0 75 3 4.0 27 35 0 0.0 70 3 4.3 28 40 0 0.0 65 3 4.6 29 45 0 0.0 60 3 5.0 30 46 0 0.0 59 3 5.1 31 47 0 0.0 58 4 6.9 32 48 0 0.0 57 4 7.0 33 51 0 0.0 54 5 9.3 34 51 0 0.0 54 5 9.3 35 56 0 0.0 49 6 12.2 36 57 0 0.0 48 6 12.5 37 57 2 3.5 48 6 12.5 38 61 2 3.3 44 6 13.6 39 62 2 3.2 43 6 14.0 40 69 2 2.9 36 6 16.7 41 71 3 4.2 34 6 17.6 TABLE 41 DEAD MICE WITHOUT AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 15 -GR 4-MAR-87 21:40:03 Page 1 File : DRA1:[MROJNOMITUGR 3068.LIS'r1 ?P~

REPORT P -/3068 NOMITUGR 7063 WEEK NUMBER OF MICE WITH TUMORS ABS. (N) DEAD AND ALIVE EAD REL. (0/'0) DEAD WITHOUT TUMOR'S ABS. (N) DEAD AND ALIVE EAD REL. (0/0) DEAD 42 72 4 5.6 33 6 18.2 43 73 4 5.5 32 6 18.8 44 77 4 5.2' 28 6 21.4 45 80 5 6.3 25 6 24.0 46 81 7 8.6 24 6 25.0 47 84 7 8.3 21 6 28.6 48 86 7 8.1 19 6 31.6 49 86 7 8.1 19 6 31.6 50 87 8 9.2 18 6 33'.3 51 87 8 9.2 18 6 33.3 52 89 12 13.5 16 6 37.5 53 90 14 15.6 15 6 40.0 54 90 17 18.9 15 6 40.0 55 90 19 21.1 15 6 40.-0 56 92 19 20.7 13 6 46.2 57 92 23 25.0 13 6 46.2 58 92 25 27.2 13 6 46.2 59 92 29 31.5 13 6 46.2 60 93 30 32.3 12 6 50.-0 61 93 34 36.6 12 6 50.0 62 94 39 41.5 11 7 63.6 63 95 43 45.3 10 7 70.0 64 95 47 49.5 10 7 70.0 65 95 49' 51.6 10 7 70.0 66 95 51 53.7 10 7 700 . 67 96 55 57.3 9 7 77 ,8 68 96 55 57.3 9' 7 77,8 69 96 56 58.3 9 7 77,8 70 96 59 61.5 9 7 77,8 71 96 61 63.5 9 7 77,8 72 96 63 65.6 9 7 77,8 73' 96 65 67.7 9 7 77.8 74 96 65 67.7 9 7 77,8 75 96 66 68.8 9 7 77.8 76 96 72 75.0 9 7 77.8 77 96 75 78.1 9 8 88.9 78 96 77 80.2 9 8 88.9 79' 96 79' 82.3 9 8 88.9 84 96 82 85.4 9 8 88.9 81 96 82 85.4 9 8 88.9 TABLE 41 (continued) DEAD MICE WITHOUT'AND WITH TUMORS IN APPLICATION AREA, MACROSCOPIC APPEARANCE, ABSOLUTE AND RELATIVE 15 -GR 4-MAR-87 21:40s03 Page 2 File : DRAla~MRO]NOMITUGR 3068.LIS;1

P 0500/3068 UBE145RA22 7292 APPLICATION 2R1 MACR06COPIC FINDINGS (0/0) HISTOPATHOL. FINDINGS ATERIAL C M ( a ) M . 9 DOSE SKIN IRRITATION NUMBER OF MICE TI3MOR ONSET 5 0/0 TUMOR PROBABILITY (b) APPLICATION WEEK APPL. WEEK 80 MAXIMUM 30 40 (mg/mouse (0/0) (appl.. week) (appl. week) (tunor prob. (0/0)) (b) (microscopic tumor x week) prob. (0/0)) (b) acetone - 0 .Gr.80 .GT.80 0 0 0 MWSC-I 60 9 50 71 0 0 14 90 19 35 40 0 5 24 120 41 39 53 0 1 15 MWSC-I (d) 60 6 53 63 0 0 12 (d) 90 20 41 60 0 0 11 (d) 120 32 30 65 1 2 12 SWSC-I 60 20 13 24 9 14 62 90 47 13 21 10 26 81 120 68 17 20 19 37 77 AT TABLE 42 MA(,ROBCOPIC AND HISTOPATHOIOGICAL FINDINGS, MOUSE STRAIN CD 1 (a) DMHA dose: 200 nmol/mouse, single application (b) calculation according to Armitage (1971) (c) weeks 1 to 5 (see AT TABLE 23) (d) condensate stored in daily portions at 4 degrees centigrade, condensate of all other groups stored at -75 degrees centigrade Vseisoszoz

P 0500/3068 UBE145RA23 7292 Z co T O APPLICATIQN 2R1 MACR06COPIC FINDINGS (0/0) HISZC?PATHOL. FINDINGS ~ ~ IAL TE CON = R MA (a) D. DOSE SKIN IRRITATION NUMBER OF MICE TUMOR ONSET 5 0/0 TUMOR P%JBABILITY (b) APPLICATION WEEK APPL. WEEK 80 c ~ C° (mg/mouse MAXIMUM (0/0) (appl. week) (appl. week) 30 40 (tumor prob. (0/0)) (b) (microscopic tumor ~ 0 0 x week) prob. (0/0)) (b) sn re, n DMBA plus - 0 40 75 0 1 =r CD acetone 0 ~ ~ DMBA plus NWSC-I 60 0 22_ 37 2 9 41 C) ~ c 3 90 6 14 23 11 21 50 (C 120 12 13 24 11 19 52 ~ DMBA plus 60 7 13 26 12 25 75 ~ SWSC-I 90 13 13 19 22 42 90 120 39 13 17 45 69 88 AT TABLE 42 (continued) MACROSCOPIC AND HISTOPATHOLOGICAL FINDINGS, MOUSE STRAIN CD1 (a) DMBA dose: 200 runol/nause, single application (b) calculation according to Armitage (1971) (c) weeks 1 to 5 (see AT TABLE 23) ssETSO9zoz

W P 0500/3068 UBE145RA24 7292 0 APPLICATION MATERIAL ) 2R1 CaND MACROSCOPIC FINDINGS (0/0) HISTOPATHOL. FINDINGS (a , DOSE (mg/nquse x week) SKIN IRRITATION NUMBER OF MICE MAXIMUM (0/0) TUMOR ONSET 5 0/0 TOMOR PROBABILITY (b) (appl. week) (appl. week) APPLICATION WEEK 30 40 (tumor prob. (0/0)) (b) APPL. WEEK 80 (microscopic tumor prob. (0/0)) (b) acetone - 1 .GT.80 .GT.80 0 0 Nbn1SC-I 42 1 51 .GT.80 0 0 2 63 0 43 69 0 0 7 84 2 65 74 0 0 14 DMBA plus acetone 1 32 47 0 3 13 hMBA plus NbJSC-I 42 0 40 61 1 15 63 24 33 3 45 84 18 39 2 59 AT TABLE 43 MAC1m6COPIC AND HISTOPATHOIDGICAL FINDINGS, MOUSE STRAIN B6C3F1 (a) DMBA dose: 200 nmol/mouse, single application (b) calculation according to Armitage (1971) (c) weeks 1 to 5 (see AT TABLE 23) 9SCjS09Z0z

P 0500/3068 UBE145RA25 7292 CORRELATION PARAMETER 1 2 microscopic tunor tumor onset (appl. week) (b) probability (0/0) (appl. week 80) 5 0/0 tumor probability (appl. week) (b) application week 30 (timar probability 0/0) (b) application week 40 (tLUnor probability 0/0) (b) skin irritation, number of mice, maximum (0/0) (c) skin irritation, number of mice, maximum (0/0) (d) AT TABLE 44 z ca ~ O EVALUATION OF CORRELATION ~ ~ PARAMETER 2 = ~ AVAILABLE COEFFICIENT STATISTICAL STANDARD DEVIATION c ~ SfQNIFICANCE (a) - OF RESIDUALS OF c PARAMETER 1 ~ Cr 0 (week) (r) 0 ~ ~ 13 to 53 0.8337 +++ 17.8 n 17 to 71 0.9328 +++ 11.6 (D 0 ~ 0 30 0.8116 +++ 18.9 =r c ~ ~ 40 0.8851 +++ 15.0 3 5 0.7155 ++ 21.2 5 0.6771 + 22.5 CORRELATION: MACROSCOPIC AND HISTOPATHOIDGICAL FINDINGS, MOUSE STRAIN CD1, WITHO[1T AND WITH DMBA PRETRFATMEDTT (a) 1-sided test (b) without and with DMBA pretreatment (c) without DMBA pretreatment (d) with nN1BA pretreatment 4S€isoszoz

P 0500/3068 UBE145RA26 7292 CORRELATION PARAMETER 1 2 microscopic tumar timior onset (appl. week) (b) probability (0/0) (app1. week 80) 5 0/0 twnor probability (appl. week) (b) application week 30 (tumror probability 0/0) (b) application week 40 (tumor probability 0/0) (b) skin irritation, number of mice, maximum (0/0) skin irritation, number of mice, maximum (0/0) z W ~ O ~ EVALUATION OF PARAMETER 2 AVAILABLE CORRELATION COEFFICIENT TATISTICAL SIQQIFICANCE (a) TANDARD DEVIATION OF RE.SIDIIAIS OF ~ rt C ~ c Cr PARAMETER 1 0 0 (week) (r) ca 5' ~ CD 18 to 65 0.7680 + 13.3 ~ 0 33 to .GT.80 0.8586 ++ 10.9 ~ C) ~ C ~ 30 0.8858 ++ 9.84 co a 40 0.9105 +++ 8.76 ~ AT TABLE 45 CORRELATION: MACRpS(.'OPIC AND HISTOPATHOUJGICAL FINDINGS, MOUSE STRAIN B6C3F1, WITHOUT AND WITH DMBA PRETREAZMENP (a) 1-sided test (b) without and with DMBA pretreatment ssETso9zoz

P 0500/3068 UBE145RA27 7292 CORRELATION PARAMETER 1 2 number of mice microscopic with skin irri- tumor rate tations (0/0) (0/0), (mean of weeks week 80 2 to 4) hyperkeratosis microscopic tumor rate (0/0) week 80 AT TABLE 46 MOUSE DMBA EVALUATION OF CORRELATION STRAIN PRE- PARAMETER 1 TREAT- AVAILABLE COEFFICIENT STATISTICAL STANH]ARD DEVIATION MENT SIGNIFICANCE (a) OF RFSIDUAIS OF PARAMETER 2 (week) (r) CD1 - 4 0.578 + 26.5 yes 4 0.845 ++ 17.3 B6C3F1 - 4 yes 4 CD1 - 4 0.578 + 26.5 yes 4 0.959 +++ 9.21 B6C3F1 - 4 0.879 = 3.5 yes 4 0.997 ++ 1.5 CORRELATION: iNACROSCOPIC AND HISZOPATHOIAGICAL FINDINGS (b) (a) 1-sided test (b) sunnarized values from INBIFO study P 0500/3117 sSEtSO9zoz

SUBRElO O P 0500/3068 UBE148RB21 H02505 7145 M ! V 60 -~ 4 0 -~~ 20 -~ 0 ----- 0 &I -Q> r~ r-s~r-~. .~r- ...r~- s 0. 1- G R s 1-GR : 2-GR : 3-GR 0 f- 20 AT FIGURE 1 MORTALITY, CD1 MICE a 40 50 80 WEEK OF APPLICATION PERIOD o9Ejso9zaz

- uu SUBREPORT P 0500/3068 UBE148RB21 H03049 7145 M „ 1007 0 --e u 80 --~ 60 -I 40 i ------ 0 : 1-GR ~ : 4-GR 5-GR : -m : 6-GR fc. y ..... . . 20 -i 1 20 _nn 40 60 80 WEEK OF APPLICATION PERIOD AT FIGURE 1 (continued) MORTALITY, CD1 MICE T9ETSO9zoz

SUBREPORT P 0500/3068 UBE148RB21 H02512 7145 M r, 100 ~ ~ ., -----4C7 : 0 . 2 - G R ~- --~ : 7-GR 8 0 -' ~ J --~7 : 8-GR cr_ --p : 9-GR ~ ~ 0 z 60 -1 40 -~ 20 -r a ® 0 I 1 AT FIGURE 1 (continued) MORTALITY, CD1 MICE F 20 40 60 80 WEEK OF APPLICATIOM PERIOD z9cTSO9zoz

SUBREPORT P 0500/3068 UBE148RB21 H02509 7145 M ,-*, 100 -1 -----0 : 0. 1- G R ~ -~ ~ 10-GR ~ 80 -~ --g : 11-GR cl: -~ : 12-GR ~ ~ 0 z GO -~ 40- , eN 20 ~ 0 ( 1 AT FIGURE 1 (continued) MORTALITY, CD1 MICE V ,-, 0 40 60 80 I~JEEK OF APPLICATIOM PERIOD rgE'TSO9zoz

SUBREPORT P 0500/3068 /,\ 100-1 ~ ~ -y 80 m ow m A <2p em 4 0 -~~ : 0.2-GR AlF m 20 - 0 f 1 AT FIGURE 1 (continued) MORTALITY, CD1 MICE UBE148RB21 H02513 7145 M T 20 m m m 1 40 60 80 GJEEK OF APPLICATION PERIOD 0 v9mIS09zm

SUBREPORT P 0500/3068 UBE148RB21 H02515 7145 M ,,-*, 100 -1 1° 80 -I 60 -l 4 0 ~; ~ 2 0 --1 ( 1 ------ 0 -----o : 0.1-GR : 0.2-GR -~ 40 60 80 WEEK OF APPLICATION PERIOD AT FIGURE 1 (continued) MORTALITY, CD1 MICE S9CTS09;!m

SUBREPORT P 0500/3068 UBE148RB21 H03052 7145 M ,-11 100 7 I ON01 1%, 80 -1 50 --1 40 -{ 20 -{ 0 I 1 AT FIGURE 1 (continued) MORTALITY, CD1 MICE ------ 0 : 0. 1- G R -----o : 0. 2 - G R 40 60 80 WEEK OF APPLICATION PERIOD 99ETSO9zoz

SUBREPORT P 0500/3068 UBE148RB21 H02514 ,~ 100 -~ a~ l ~ ~ ~ ~ 80 -~ ~ ~ ~- ci~ 0 z 60 -I 40 -~ 20 -~ f 1 AT FIGURE 1 (continued) MORTALITY, CD1 MICE -V 20 7145 M 40 60 80 WEEK OF APPLICATION PERIOD 4sElso9zoz

SUBREPORT P 0500/3068 UBE148RB22 H03050 7145 „ 100 o\° " ------ : 0.3-GR r ~ -~ : 16-GR ~ 80 -{ J ~ -~ : 17-GR -~ : 18-GR ~ ~ 0 ~ ~ 60- 40 -- 20-, 1 20 40 60 80 WEEK OF APPLICATION PERIOD AT FIGURE 2 MORTALITY, B6C3F1 MICE 89ETso9zoz

SUBREPORT P 0500/306 8 UBE148RB22 H03051 7145 M 100-1 ., ------ D : 0 . 4 - G R } = 19-GR : 80 -{ -~ 20 -GR ~ : 21-GR T- ~ ~ 0 ~ 50 -I 40 -I 2 0 - ~ 20 AT FIGURE 2 (continued) MORTALITY, B6C3F1 MICE 40 60 80 WEEK OF APPLICATION PERIOD s9ETSO9zoz

-3 1 -1 20 40 60 80 WEEK OF APPLICATION PERIOD AT FIGURE 3 BODY WEIGHT DEVELOPMENT, CD1 MICE oZxj-So9zoz

SUBREPORT P 0500/3068 UBE148RB22 H03043 7145 M r. S o ~ O) W 3 20H -----0 : 10 H 1-GR : 4-GR : S-GR : 6-GR r- -3 1 20 40 60 80 aEEK OF APPLICATIOM PERIOD AT FIGURE 3 (continued) BODY WEIGHT DEVELOPMENT, CD1 MICE U.EIsosZoz

SUBREPORT P 0500/3068 UBE148RB22 ^ SO ~ O) u ~ I L40 ~ w 3 ~-~-~--~ 20H 10 H 0 1102500 7145 M T 20 - --0 ~a--a---~' , .- 8 ~ a- _a~_@-------~--~,- I 40 -----o : 0 . 2 - G R -~ : ?-GR -p : 8-GR -~ : 9-GR 1 80 WEEK OF fAPPLICATIOM PERIOD AT FIGURE 3 (continued) BODY WEIGHT DEVELOPMENT, CD1 MICE z2•EIso9zoz

SUBREPORT P 0500/3068 UBE148RB22 H02501 7145 60 -, r% O) .1 r-- _ lD 40 -i ..-4 W 3 } Q 0 30 -I w 0 -~ lw~ K~ Q~° ~ ® 10-I : 0.1-GR : 10-GR : 11-GR : 12-GR r- -3 1 20 40 60 80 WEEK OF APPLICATION PERIOD AT FIGURE 3 (continued) BODY WEIGHT DEVELOPMENT, CD1 MICE ~8--6--e-_-6--A-- --8--ry-- --v-- -- MEisoszoz

rl uJUUl'-:)uvd, nuc5uc. Ih, vzj5 r15G vbS AT FIGURE 3 (continued) BODY WEIGHT DEVELOPMENT, CD1 MICE -F 20 40 60 80 WEEK OF APPLICATION PERIOD UCjsoszoz

SUBREPORT P 0500/3068 UBE148RB22 H02504 7145 M r, 50 -1 v ~ Q 30- aa ~ 20 -~ 10-~. : 1-GR : 7-GR : 10-GR : 13-GR r- -3 1 20 40 60 80 WEEK OF APPLICATION PERIOD AT FIGURE 3 (continued) BODY WEIGHT DEVELOPMENT, CD1 MICE s4CTsoszoz

I 40 60 80 WEEK OF APPLICATION PERIOD AT FIGURE 3 (continued) BODY WEIGHT DEVELOPMENT, CD1 MICE s4E'ls0szoz

INBIFO Institut fur biologische Forschung • Kblm INDEX REPORT P 0500/3068 BRA95DIVA27 CONTENTS' INTEGRATING REPORT: SUMMARY Conclusions INTRODUCTION TABLE C: GROUPS AND DOSES FIGURE B: CHRONOLOGY SUBREPORT ANALYTICAL CHEMISTRY SUBREPORT ANIMAL TREATMENT SUBREPORT MICROBIOLOGY SUBREPORT PATHOLOGY INDEX CODE ~c'v~St"..~Zcrc?(2 Uotf 3285

SUBREPORT P 0500/3068 UBE148RB23 H02503 7145 M /N ..1 } 0 30 CA 20-'~ 10-j 0 -W-j : 3-GR -~ , 9-GR A 2-GR - : 1 _ 0 5-GR W 6 : 1 r- -3 1 20 40 60 80 WEEK OF APPLICATI0M PERIOD AT FIGURE 3 (continued) BODY WEIGHT DEVELOPMENT, CD1 MICE SLCTso9zoz

SUBREPORT P 0500/3068 UBE148RB23 H03044 7145 M „ SO C) ~ .-1 w 3 ~ 0 30 - aa I 20 -I 10--~ -3 1 ~ : 16-GR 17-GR : 18-GR 20 40 60 80 WEEK OF APPLICATION PERIOD AT FIGURE 4 BODY WEIGHT DEVELOPMENT, B6C3F1 MICE 6LETSOSZOz

AT FIGURE 4 (continued) -r 20 BODY WEIGHT DEVELOPMENT, B6C3F1 MICE 40 60 80 WEEK OF APPLICATION PERIOD oeCts©gzoz

SUBREPORT P 0500/3068 UBE148RB24 H02496 7145 M ~-. 100 --1 ~ ------ o : 0. 1- G R .~ w -~ _ 1-GR ~ -~ : 2-GR ~ 80 3-GR cy- ey- 0 ~ 60 -~ 40 -I 20 -1 0 I 1 AT FIGURE 5 -T 20 -~ -~"" =---------------------o . 40 60 80 WEEK OF flPPL I CfiT I OfV PER I OD TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, CD1 MICE jge-rsoszoz

SUBREPORT P 0500/3068 UBE148RB24 H02735 7145 M „ ~ u fy- 0 ~ ~ 50 40 -i 2 0 -~~ ------ 0 : 0.1-GR -GR : 4 S-GR : -a : 6-GR ~ Wrihi r !'A e 0 0 ~-r i 20 bd A 40 -T 60 -1 80 WEEK OF APPLICATION PERIOD AT FIGURE 5 (continued) TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, CD1 MICE z1gEltso9zoz

SUBREPORT P 0500/3068 UBE148RB24 H02497 7145 M /_1 aH ., w ~ ~ 100-1 -----v : 0.2-GR -~ : 7-GR -~ 8-GR 80 -~ --s : 9-GR <Z 40 -~ 2 0 - I 1 AT FIGURE 5 (continued) -- 20 m crw d4 ® r a as& 40 60 80 WEEK OF RPPLICATION PERIOD TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, CD1 MICE esclso9zoz

f 1 tseTso9zoz ~V- ~ .~~p1Y J 0 9 v VI m a r. . 2PE AT FIGURE 5 (continued) TUMOR RATE BASED ON MACRSOCOPIC APPEARANCE, CD1 MICE 40 60 80 WEEK OF APPLICATION PERIOD

SUBREPORT P 0500/3068 w ~ ~ 80-~ ~ a 0 ~ ~ ~ 60- ' 40 -I 20 -~ 0 ( 1 AT FIGURE 5 (continued) -i 20 H02495 7145 M 40 60 80 WEEK OF APPLICATIQM PERIOD TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, CD1 MICE UBE148RB24 sEelsomoz

~-~- i 20 AT FIGURE 5 (continued) I 40 60 80 WEEK OF APPLI6ATION PERIOD TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, CD1 MICE 99Elso9zoz

SUBREPORT P 0500/3068 r, 100 -~ ~ ~ 60-i 40 -I 20 -I ® e~e a 0 a a e ., 1 AT FIGURE 5 (continued) LeeTsoszoz UBE148RB24 H02734 20 7145 TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, CD1 MICE 40 60 80 WEEK OF APPLICATION PERIOD

SUBREPORT P 0500/3068 ~ o\° L 100 ~ ~ 60 40 -~ 20 -I 1 eeCTso9zoz UBE148RB214 H42487 m m m m m ~., a e A-4 IQ" _ ---------------- 0 40 60 80 WEEK OF APPLICATIOM PERIOD AT FIGURE 5 (continued) TUMOR RATE BASED ON MACRSOCOPIC APPEARANCE, CD1 MICE T 20 7145 M

SUBREPORT P 0500/3068 UBE148RB25 H03047 7145 ,~ 100 - ~ ~ u -----o : &I 0 . ~ - G R . : 16-GR : 17-GR ~ 80 -~ ly- ---~ : 18-GR 0 ~ ,~- 60 40 -j 20 -I 0 -F 20 AT FIGURE 6 I 1 1 40 60 80 WEEK OF APPLICATION PERIOD TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, B6C3F1 MICE seETsoszoz

SUBREPORT P 0500/3068 UBE148RB25 H03048 7145 /-,, 100 7 ------p : 0. 4-GR : 19-GR : 20 -GR : 21-GR w 0 ~ 6 0 --~ V1 40 --I v r wap E 20-y 0 40 60 80 WEEK OF APPLICATION PERIOD AT FIGURE 6 (continued) TUMOR RATE BASED ON MACROSCOPIC APPEARANCE, B6C3F1 MICE 06CjS09zOZ

SUBREPORT P 0500/3068 UBE148RB26 H02118 7145 M ~ 100 Q = 0.1-GR 0.2-GR 80-I 60 -~ 40 -1 2 0 -1 ~ 1 4 AT FIGURE 7 SKIN IRRITATION DEVELOPMENT, CD1 MICE 6 VJV---40 ~ I 8 @ VE 10 12 13 WEEK OF STUDY t6eTSo9zoz

r UaUu: ~puuG, o r- ~, ~ , , lr:G r l;~ U11 t l5 SUBREPORT P 0500/3068 UBE148RB26 H02021 7145 M /1, 100 -7 ., 80--'~ 60-i 40 -I W ~ ~0JI y- -=~ s 1-GR ---© s 2-GR --~ s 3-GR --r- 2 I 4 T 6 T 10 T---l 12 13 WEEK OF STUDY AT FIGURE 7 (continued) SKIN IRRITATION DEVELOPMENT, CD1 MICE zsETsa9zoz

SUBREPORT P 0500/3068 UBE1438RB26 H02023 7145 M /-, 100 4-GR " --p ~ 6-GR 7- ----0 : 6-GR 0 40 -~ 0 I 6 AT FIGURE 7 (continued) SKIN IRRITATION DEVELOPMENT, CD1 MICE 1 8 10 12 13 WEEK OF STUDY rseTsoszoz

...v..c -ul.v. I1v...u~i~, V1:. t l- Y.`F r 1J SUBREPORT P 0500/3068 UBE148RB26 H02027 /-, 100 -1 2 --~ : -GR --fl ~ B-GR --~ ~ 9-GR 80-+'~ 60 -I 40 --l 20 -~ 0 I 1 T 2 AT FIGURE 7 (continued) I 4 SKIN IRRITATION DEVELOPMENT, CD1 MICE T 6 1 8 10 12 13 WEEK OF STUDY tseTsoszoz

r' USUuI--Uad. NucuEy. olb r16 V15 F15 SUBREPORT P 0500/3068 UBE148RB26 H02029 7145 M ,~ 100 .. 7- ~ ~ U) 80--~ 60 -~ 40 -~ ~ 20- 0 u -D : 10 -GR --~ : 11-GR -~ i 12-GR 1 1 1 4 I 8 T 10 7---l 12 13 WEEK OF STUDY AT FIGURE 7 (continued) SKIN IRRITATION DEVELOPMENT, CD1 MICE sseTso9zoz

SUBFtEPOkT P 0500/3068 UBE148RB26 H02031 7145 ~r. 100 13-GR ~ ----~1 , 14 - G R -= ~ S-GR z - , 1 0 - 80 - ~ ~ c 60 z ~ ~ Cl) 40H 20 -h 0 T 2 I 6 AT FIGURE 7 (continued) SKIN IRRITATION DEVELOPMENT, CD1 MICE 1 8 I 1--~ 10 12 13 WEEK OF STUDY 9sEIso9zoz

1 V-vul...N-u, itu~J..J, v.0 fi~+ VA/ t'jJ SUBREPORT P 0500/3068 UBE148RB27 H02039 7145 M ~, 100 0.3-GR 0.4-GR 3 g0 --~ 60 -i W U 20 .7 4-~ I= 0 f 1 AT FIGURE 8 I 2 SKIN IRRITATION DEVELOPMENT, B6C3F1 MICE I g 10 12 13 WEEK OF STUDY L6ETso9zoz

. . - . .,.. 'y. v. I l-wo SUBREPORT P 0500/3068 UBE148RB27 H02033 7145 M ,\ 100 -, a` ~ z ~ Y U) ~ ~ 3 80 -r 60 --I 40 -I W U 20 0 --~ , 16-GR --~ ~ 17-GR -o : 18-GR 1 2 4 6 6 10 12 13 WEEK OF STUDY AT FIGURE 8 (continued) SKIN IRRITATION DEVELOPMENT, B6C3F1 MICE e6ETso9zoz

SUBREPORT P 0500/3068 UBE148RB27 U02037 7145 M f, 100 -1 s 19-GR " -~ e 20 -GR -o 1 21-GR 80-i 60-- y U) ~ ~ 3 W U 40 -I 207 ® I 1 I 2 AT FIGURE 8 (continued) I 4 SKIN IRRITATION DEVELOPMENT, B6C3F1 MICE T 6 l E 7 10 12 13 WEEK OF STUDY 6sETs©9zoz-

I ..:.w u,c.c,< r-w.~:jzj/, vi r 1.) Vo:: r l-r. k:111 , h'tK 1•IEEK SUBREPORT P 0500/3068 UB1148RB28 H03557 6315 M 100 -7 80-1 60 -,' 40 -r 20 -1 0 AT FIGURE 9 r 1 e low dose 2 : medium dose 3 : high dose 1 3 a r1WSC- I 1 2 3 1 2 3 P1WSC- I (b) Df1BA + f1WSC-I SKIN IRRITATION, MAXIMAL NUMBER OF MICE PER WEEK, CD1 MICE (a) no response (b) condensate stored at 4 degrees centigrade. Condensate of all other groups stored at -75 degrees centigrade oovjso9zoz

I LI% wLl_h SUBREPORT P 0500/3068 UBE148RB28 H03558 5311 M F, 100 a>~ 1: 1ow dose u 2 : medium dose ~ 3: h i gh dose 0 ~ 80- ~ Cc F- ~ ~ ~ ~ 60-7 7 - - ~ ~ U) 40 ~ 20--~ 0 f 1 2 3 SWSC-I I AT FIGURE 9 (continued) SKIN IRRITATION, MAXIMAL NUMBER OF MICE PER WEEK, CD1 MICE TorZSO9zoZ 1 2 3 , Df1BA + SWSC- I 1 I

SUBREPORT P 0500/3068 UBE148RB28 H03559 5337 M ~ .. 100 -, 80 -I 60-i' 40 -! 20-~ 0 1 2 3 I'1WSC- I AT FIGURE 10 I 1 2 3 DIrIBA + f1WSC- I SKIN IRRITATION, MAXIMAL NUMBER OF MICE PER WEEK, B6C3F1 MICE (a) no response 1 : low dose 2 : medium dose 3 : high dose 1 zoivjs©gzoz

SUBREPORT P 0500/3068 UBE142RB11 7293 M N-+ X 1 00 -~ W-I aos.' ao .p n16t-1 W ~ . .ica rlth I..spr.s. 7-.0R > rllhpyl t.A.o's. $-iR .~. ' J 60 M 1 70 14 fi 06 I/EER w ST/DY tfEEIC Oi 51101T 100" 00 7 , . 100. des.. 60 .0 INSC-1 ta) ` tlos. I'/ . 90 .p u115C-I {a! _ . . . ~i .iG ath Iu.Of•.i. 4-6R ~-~ .•iCG r7th Ir.ur•s, y_~g F -+ . i Ilrirl 1u.m•s, 4-0 ~ t - .su..... .._..... ...{{{ M ra~ 1 AT FIGURE 11 N -~ x-~ OJ uo n My 4 ~ i N-I Y so -~ I N 1 aw.. 170 .p MtfiC-1 -o . .ic. rtth t..ar.s. }i.R . i1hWl lu.m's. 0-.itt pus.. 120 .0 ArSC-1 t.! ~ . .iG rilh lu.Ors. L-OR . ri/hout ts.et•s, i-M OJ f-------- =---4 ~ ~ N 4 K YEEK OF STi1CY YEpC a Sh0]T MORPALITY OF MICE WITHQTf AND WITfi MACROSCOPIC SKIN TUMORS IN APPLICATION AREA (a) stored at 4 degrees centigrade, oandensate of all other groups stored at minus 75 degrees centigrade VOtiTS09zOZ

SUBREPORT P 0500/3068 UBE142RB12 7293 AT FIQJRE 11 ( rontinued ) IIEFx oF trinr MOEM.LITY OF MICE WITHOITP AND WITH MP,CRO6C.'OPIC SKIN T[AMORS IN APPLICATION ARFA us W - , w1 20 a J dws. 126 .q /MfC-I plus OrA -0 . •i[u ill. Irm+s, !-iR • ritiwyl Iu.ars. 9-0 IfEFK OF fTIDY YEEY. OF filOT tObTs09zOZ

SUBREPORT P 0500/3068 UBE142RB13 7293 M ® tui-1 _ uo-1 _ too-1 a f0 LKC-1 t 010~ - eea.. io .p iriC 1 plus Or~ ---~ . .ics ritn te.xs. 1J-GR iteoul twrs. 73-BR I Wd f I YEEL OF fT1aqT AT FIQIRE 11 ( caontinued ) ~ } W1 Nd 20 _~ a... p us p ~i . •iq .itn pmors. ts-6R riOwVI tr•nrs. t4-i! 1 T 20 T-7 W ll0 afFC OF sTla4T T 48 MOTAI,ITY OF MICE WIT4iOITr AND WIZgi MACROSCXJPIC SKIN.TUMORS IN APPLICATION ARFA fA ~ 40 1 sorlso9zoz

SUBREPORT P 0500/3068 UBE142RB1 H06257 7146 M .. 100 -~ IK .. 8 80 -~ ~ 60 -~I :D f- 401 -~~ 20-~ 0 1 20 r = 0.8337 n = 17 y = 1.39x + 82.5 O 40 60 80 TUMOR ONSET (appt. week) AT FIGURE 12 Ql '"i CORRELATION B'ETWEEN' MICROSCOPIC TUMOR PROBABILITY AND APPLICATION ph WEEK OF TUMOR ONSET, MOUSE STRAIN CD1, Q ' WITHOUT AND WITH DMBA PRETREATMENT (7)

IIVUIl v U.-tut 1ua uw~.vJw..,:~W c I ..,,.: SUBREPORT P 0500/3068 UBE'142RB2 H06255 7146 M ~. 10 0 -, x 1-0 2 0 -I ~- v U ~ ~ ~ 0 ...J > D .? L a . ~ -,. 0 20 40 60 -I 80 5`/. TUMOR PROBAB I L I TY ( app l. week ) O N O ~ = AT FIGURE 13 ~ n A ~ ~ . ~ ~ ~ CORRELATION BETWEEN'MICROSCOPIC TUMOR PROBABILITY AND APPLICATION ~"' WEEK IN'WHICH A TUMOR PROBABILITY OF 5 0/0 WAS REACHED, MOUSE STRAIN CD1,q WITHOUT AND WITH DMBA PRETREATMENT

SUBREPORT P 0500/3068 UBE142RB3 H06261 7147 M ~ ... 0 100-1 r = 0.8116 n = 17 y = 2.21x + 23.1 U 60-r 40 20--~ t D L .~ > D ~ > 0 © 11 0 10 20 30 40 50 60 70 v = TUMOR PROBAB I L I TY (%) .V O a = AT FIGURE 14 ~ CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND THE TUMOR D ~ PROBABILITY OF APPLICATION WEEK 30, MOUSE STRAIN CD:1, . WITHOUT AND WITH DMBA PRETREATMENT ~ ~ ~ ~ 1. ~1e"

SUBREPORT P 050D/3068 UBE142RB4 H06262 7147 M ... ~ 10 0 -, v ~ F" H ~ F -i ~ ~ m 0 m CL 0 - ~ 0 60 ~ ~ ~- U H Q. ° Q 0 (n 0 C14 U H ~ 20-I 0 © 01 10 20 30 40 510, 60 70 TUMOR PROBABILITY (Z) AT FIGURE 15 CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND THE TUMOR PROBABILITY OF APPLICATION WEEK 40, MOUSE STRAIN CD1, WITHOUT AND WITH DMBA PRETREATMENT

SUBREPORT P 0500/3068 UBE142RB5 ~ 100 --~ ~ z 20-~ 0 Q1 > 0^ N lt: m' > ~ D '.D = AT FIGURE 16 H06266B 7146 M © 0 0 01 10, 20 30 40 50 60 70 MICE WITH MAX. SKIN IRRITATION (%) CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND SKINlIRRITATION MOUSE STRAIN CD1, WITHOUT DMBA PRETREATMENT

SUBREPORT P 0500/3068 UBE142RB6 H06267B 7146 M .-. 100 "1 ~ ... p1 60 ~ ~ F-- u z a U 0 .~ \ a .~ .D ~ AT FIGURE 17 L 0 0 10 20 310 40 50 60'70 MICE WITH MAX. SKIN IRRITATION (%) p CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND SKIN IRRITATION, D MOUSE STRAIN CD1, WITH DMBA PRETREATMENT ~ ~ ~ ~ n a z:

SUBREPORT P 0500/3068 UBE142RB7 H06258 7146 M ,. 100-, x 80 -~ r = 0.7680 n = 8 y = -0.78x + 53.9 pp 6 0 '~ X: :D F-- O ~. D a 4 0 -~~ 0 A ~ O ~ AT FIGURE 18 a O 20 40 60 80 TUMOR ONSET (app l. week ) m CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND APPLICATION ~ WEEK OF TUMOR ONSET, MOUSE STRAIN'B6C3F1i, ~ "~ WITHOUT AND WITH DMBA PRETREATMENT . ~ ~ .'7 ~

SUBREPORT P 0500/3068 UBE142RB8 H06254 7146 .. 100-1 ... 80 --l r = 0.8586 n = 8 y = 0.98x + 78.5 U ~ a.. 0 U (fi 0 04 U ~ ~ G0' -~ 40-I U. 20 ~ co m 0 0 20 40 60 1 80 ~ 5% TUMOR PROBAB I LI TY ( app l. week ) n ,v . L AT FIGURE 19 ~ CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND APPLICATION ~ WEEK IN WHICH A TUMOR PROBABILITY OF 5 0/0 WAS REACHED, ~ MOUSE STRAIN B6C3F1, WITHOUT AND WITH DMBA PRETREATMENT ~ ~ n ~

SUBREPORT P 0500/3068 UBE142RB9 H06259 7147 M .. 1100-, ~ .. I.. . - 80-~ m ~ m 0 r = 0.8858 OC n = 8 CL y = 15.8x + 9.49 0 60 -1 ~ ~ o cl) F- V ~ w w OL 0 40-I U ~ . 0 J (]~ na'. U < .-, X: 20 --~ 0 n 1) ~ D ~ AT FIGURE 20 i 8 0 10 20 30 40 501 60 70 TUMOR PROB'AB I L I TY ( Y. a D CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND THE TUMOR ~ PROBABILITY OF APPLICATION WEEK 30, MOUSE STRAIN B6C3F1, '~ WITHOUT AND WITH DMBA PRETREATMENT ~ ~ .~ ~.

SUBREPORT P 0500/3068 UBE142RB10 H06260 7147 M 100 -, r = 0.9105 n = 8 y = 5.38x + 5.92 °LO 6 0 -1 ~ H-- u- 20 ~ a a4 0 .J Q 0 10 20 30 40 50 60 70 TUMOR PROBAB I L I TY ('l. ) CORRELATION BETWEEN MICROSCOPIC TUMOR PROBABILITY AND THE TUMOR PROBABILITY OF APPLICATION WEEK 401, MOUSE STRAIN B6C3F1, WITHOUT AND WITH DMBA PRETREATMENT

SUBREPORT P 0500/3068 UBE145RA28 7292 AT PAGE 7-1. 7 REFERENCES Armitage, P.: Statistical methods in medical research, Oxford: Blackwell Scientific Publications, 1971: Blanding, F.H., King, W.H., Priestley, W., Rehner, J., Linden, N.J., Properties of high-boiling petroleum products. Quantita- tive analysis of tumor-response data obtained from the appli- cation of refinery products to the skin of mice. Arch. Ind. Hyg. Occupat. Med. 4 : 335-337 (1951) Bock, F.G.,: Progress in Experimental Tumor Research, Vol. 26, Basel: Karger, 1983, pp. 5-17 Clauss, G., Ebner, H. (Eds.): Grundlagen der Statistik fur Psycho- logen, Padagogen und Soziologen, Frankfurt: Harri Deutsch Verlag, 1970 Eaton, G.J., Johnson, F.N., Custer, R.P., Crane, A.R., The Icr:HA(ICR) mouse: a current account of breeding, mutations, diseases and mortality. Lab. Anim. 14 : 17-24 (1980) Festing, M.F.W.: Inbred Strains in biomedical research, London: The Macmillan Press Ltd., 1979, pp. 99-103 Hauschka, T.S., Mirand, E.A., The "Breeder: Ha(ICR)" swiss mouse, a multipurpose stock selected for fecundity. In: Perspectives in cancer resesarch and treatment, New York: Allan R. Liss, Inc. 1973, PP• pp 319-331 Homburger, F., Russfeld, A.B., Weisbu~rger, J.H., Lim, S., Chak, S.P., Weisburger, E.K., Aging changes in CD1 HaM/ICR mice reared under standard laboratory conditions. J. Nat. Cancer Inst. 55 : 37-43 (1975)~ INBIFO study P 0500/3000~, Skin painting study on whole smoke condensates from cigarettes X6D7BVW, X6D7BVX and X6D7BVY with CD-1 mice, Part II: tables and figures, Study director: Dr.med.U. Hackenberg Date of report: 28.May 82 INBIFO study P 0500/3010, Skin painting study on whole smoke condensates from test cigarette XD9PZ and XD9QA and standard reference cigarette 2R1 with CD1 mice, part II: Tables and figures Study director: Dr.med.vet. A. Teredesai Date of report: 25.May 83

SUBREPORT P 0500/3068 UBE145RA29 7292 AT PAGE 7-2 INBIFO study P 0500/3023 Comparative study on whole smoke condensates from cigarettes with and without addition of cocoa in the mouse skin painting assay, Part II: tables and figures, Study director: Dr.med.vet. A. Teredesai Date of report: 1.Mar.82 INBIFO study P 0500/3030, Skin painting study on whole smoke condensates from test ciga- rettes X6-DO AQP, X6DO-AQQ, X6-D:O AQR, X6-DO AQS, X6-DO AQT, X6-DO AQU, X6-DO AQV, X6-DO AQW, X6-DO AQX and standard reference cigarette 2RI with CD-1 mice, Part II: Tables and figures Study director: Dr.med.vet.A. Teredesai Date of report: 25.May 84 INBIFO study P 0500/3073, 80-Week dermal application of mainstream whole smoke condensate of cigarette types X6D2DJV, X6D3AMD, X6D3MS, X6D3MQ, X6D3QK, X6D3MU and the standard reference cigarette type 2R1, condensate collec- tion in a cool trap, mouse strain: CrL:CD1(ICR)ZR (PT), Study director: D. Kuhn, not yet reported INBIFO study P 0500/3111, Tumorigenicity of mainstream whole smoke condensate of test cigarettes X6D3DAM, X6D3DFZ, X6D3DGA, X6D3DBF, X6D3DNY, X6D4CDL, X6D4CUC and of standard reference cigarette 2R1, 80-week dermal application study on CD1 mice (PT), Study director: D. Kuhn not yet reported INBIFO study P 0500/3115, Tumorigenicity of mainstream whole smoke condensate of test cigarette X6D5RAN and standard reference cigarette 2R1 and of catechol: 80-week dermal application study on CD1 mice (PT), Study director: D. Kuhn not yet reported - INBIFO study P 0500/3117, Comparison of macroscopical and histological evaluation of skin irritations after dermal application of mainstream and sidestream whole smoke condensate of standard reference cigarette 2R1, con- densate collection by impaction trap, mouse strains: CRL:CD1 (ICR)BR and B6C3F1, Study director: D. Kuhn Date of report: 9.Jul.86 Lynch, C.J., The so-called swiss mouse, Lab. Anim. Care 19 : 214-220 (1969) Percy, D.H., Jonas, A.M., Incidence of spontaneous tumors in CD1 HaM/ICR mice, J. Nat. Cancer Inst. 46 : 1045-1065 (1971) END OF SUBREPORT AT V°S

INBIFO Institut fiir biollogische Forschung • Koln Dr.med. R. Rylander c/o FABRI~QUES DE TABAC REUNIES S.A. Switzerland REPORT P 0500/3068 II131ri71 26.Oct.87 DKU/UBE COPY NO.: Skin Tumo:rigenicity of Mainstream and Sidestream Whole Smoke Cond~ensate of Standard Reference Cigarette 2R1 8~0-Week Dermal Application Study with CD1(ICR)BR and B6C3F1i Mice Volume 2 INBIFO Inslitut fnr biologiache Forschurp GmhW, Fuggeostra9e 3, D-5000 KtSln 90, Sitz der GeseNschaft: KtNn HR B 367, 29. Oktober 1959 5085 Tele(on: Porz (02203) 303-1, Telelax: (02203) 303362, Telex; 8874675 inbi d Institutsleiter und GeschAllsluhrenDr. med. Ulrich Hackenberg

INBIFO Institut fu~r biologische IForschung • Koln DR-MED• R'• RYLANIIER 26•OCT•87 c/o FA'BRIQUES DE TABAC REUNIES S.A. BGE/IJK/MNO IJK3 (R) Al SWITZERLAND SUBREPORT ANALYTICALCHEMISTRY P US0U/3U68 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 Instituf fiir biologische Forschung GmbH, FuggerstraBe 3,,D-5000 Kbin 90 Sitz der Gesellbchatt: K81n HR 8 367, 29. Oktoben1959 28.KW83 Talefon: Porz (02203) 303-1, Telefax: (02203), 303362, Telex: 8874675 inbf d' Inslifutsleiler und GeschAflsfilhrer. Dr. med. Ulrich Hadtenberg

INBIFO Institut fur biologische Forschung • KoIn SUBREPORT P 0500/3068 IJK3 (R) A2 6199 AC PAGE 0-1 CONTENTS AC PAGE ABBREVIATIONS 0-6 1' SUMMARY 1-1 2 RESPONSIBILITY 2-1 3 INTRODUCTION 3-1 4 METHOD 4-1 4.1 Condensate Preparation and Storage 4-1 4.1.1 Preparation of mainstream smoke crude condensate 4-1. 4.1.2 Preparation of sidestream smoke crude condensate 4-8 4.1.3 Preparation and storage of application solution 4-11 4.2 Condensate Analyses 4-14 4.2.1 Determination of water concentration~ 4-14 4.2.2 Determination of nicotine concentration 4-15 4.2.3 Determination of catechol 4-17 4.2.4 Determination of hydrogen-ion concentration 4-21i 4.2.5 Determination of nitrosamines 4-22 4.2.6 Determination of polycyclic aromatic hydrocarbons 4-25 4.2.7 Determination of volatile components 4-29' 4.3 Residue Analysis in Diet and Bedding Material 4 -31 4.3.1 Determination of pesticide and PCB residues 4-31 4.3.2 Determination of aflatoxins and heavy metals 4-34 AC TABLE A: PHYSICAL DATA OF CIGARETTE 2R1 _ 4-5 AC TABLE'B: PREPARATION OF' APPLICATION SOLUTION 4-13 AC TABLE C: CONDITIONS OF THE HPLC DETERMINATION 4-20 AC TABLE D: RESIDUE ANALYSIS OF PESTICIDE AND PCB, DETECTION LIMIT AND TOLERANCE LEVEL 4-32 3285

INBIFO Institut fur bi'OI©gische Forschung • K6In SUBREPORT P 0500/3068 IJK3 (R) A3 7097 AC PAGE 0-2' CONTENTS (continued) ~ AC PAGE AC TABLE E:. RESIDUE ANALYSIS OF AFLATOXINS AND HEAVY METALS, DETECTION LIMIT AND TOLERANCE LEVEL 4-35 AC FIGURE A: SCHEME FOR CONDENSATE PREPARATION, ANALYSES AND STORAGE 4-2 AC FIGURE B: SETUP FOR THE SIMULTANEOUS PREPARATION OF MAINSTREAM AND SIDESTREAM SMOKE CDNDENSATE' 4-3 AC FIGURE C: IMPACTION TRAP, MAINSTREAM SMOKE 4-7 AC FIGURE D: IMPACTION TRAP, SIDESTREAM SMOKE 4-10 5 RESULTS' 5-1 5.1 Text 5-1 5.1.1 Condensate analysis 5-1. 5.1.1.1 Condensate yield 5-1 5.1.1.2 Water 5-3 5.1.1.3 Hydrogen-ion concentration 5-3 5.1.1.4 Nicotine 5-4 5.1.1.5 Catechol 5-5 5.1.1.6 Nitrosamines 5-5 5.1.1.7 PAH 5-7 5.1.1.8 Volatile components 5-8 5.1.2 Evaluation of smoke condensate preparation 5-9 5.1.3 Storage of the application solution 5-9 5.1.4 Residue analysis 5-10 5.2' Tables and Figures AC TABLE MWSC-I YIELDS AND WATER CONCENTRATION IN THE MWS_C-I SUSPENSIONS, SINGLE VALUES SWSC-I YIELDS AND WATER CONCENTRATION IN THE SWSC-I SUSPENSIONS, SINGLE VALUES 2 pH', NICOTINE AND CATECHOL CONCENTRATIONS'IN THE MWSC-I APPLICATI=SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES 3 3285

INBIFO Ilnstitut fur biologische Forschung • NCSIn SUBREPORT P 0500/3068 IJK3 (RY A4 7103' AC PAGE 0-3 CONTENTS (continued) % AC TABLE pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE SWSC-I APPLICATION S'DLUTION,OF THE HIGHEST CONCENTRATION, SINGLE VALUES 4 NITROSAMINE CONCENTRATIONS IN PROCESSED MWSC-I, SINGLE VALUES 5 NITROSAMINE CONCENTRATIONS IN PROCESSED SWSC-I, SINGLE VALUES 6 NUMBERING OF PAH FOR THE AC TABLES 8 TO 11 7 CONCENTRATIONS OF:SOME PAH (1 TO 7) IN PROCESSED MWSC-I, SINGLE'VALUES CONCENTRATIONS OF SOME PAH (8' TO 15) IN PROCESSED MWSC-I, SINGLE VALUES 9 CONCENTRATIONS OF SOME PAH (1 TO 7) IN PROCESSED SWSC-I, SINGLE' VALUES 10 CONCENTRATIONS OF SOME PAH (8 TO 15) IN PROCESSED SWSC-I, SINGLE VALUES 11 PARAMETERS OF CONDENSATE'PREPARATION 12 PARAMETERS OF THE MWSC-I APPLICATION SOLUTION OF' THE'HIGHEST CONCENTRATION, MEAN VALUES 13 PARAMETERS OF THE SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION,, MEAN VALUES 14 PARAMETERS OF MWSC-I OF CIGARETTE 2R1, CONCENTRATIONS, MEAN VALUES 15 PARAMETERS OF SWSC-I OF CIGARETTE 2R1, CONCENTRATIONS, MEAN VALUES 16 PARAMETERS OF MWSC-I OF CIGARETTE 2R1, YIELDS, MEAN VALUES 17 ~ 0 PARAMETERS OF SWSC-I OF CIGARETTE 2R1, ~ YIELDS, MEAN VALUES _ 18 (~ ~ DETERMINED NITROSAMINE YIELDS AND LITERATURE DATA FOR CJ1 NONFILTER CIGARETTES 19 M~ ~ DETERMINED PAH YIELDS AND LITERATURE DATA ON,PAH 20 tV' 3285

INBIFO Institut fur biologische Forschung • KOIn SUBREPORT P 0500/3068 IJK3 (R) B29 71,03 AC PAGE 0-4 CONTENTS (continued) AC FIGURE . CRUDE MWSC-I YIELD 1 CRUDE SWSC-I YIELD 2 PROCESSED MWSC-I YIELD 3' PROCESSED SWSC-I YIELD 4 WATER CONCENTRATION IN CRUDE MWSC-I SUSPENSION 5 WATER CONCENTRATION IN CRUDE SWSC-I SUSPENSION 6 WATER CONCENTRATION IN'PROCESSED MWSC-I SUSPENSION 7 WATER CONCENTRATION IN PROCESSED SWSC-I SUSPENSION 8 NICOTINE CONCENTRATION IN THE MWSC-I APPLICATION' SOLUTION OF THE HIGHEST CONCENTRATION 9 NICOTINE CONCENTRATION IN THE SWSC-I APPLICATION 10 SOLUTION OF THE HIGHEST CONCENTRATION CATECHOL CONCENTRATION IN THE MWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION 11 CATECHOL CONCENTRATION IN THE SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION 12 CONCENTRATION OF TOBACCO SPECIFIC NITROSAMINES IN PROCESSED MWSC-I 13 CONCENTRATION OF TOBACCO SPECIFIC NITROSAMINES IN PROCESSED SWSC-I 14 CONCENTRATION OF THE VOLATILE NITROSAMINES IN PROCESSED MWSC-I 15 CONCENTRATION OF THE VOLATILE NITROSAMINES IN PROCESSED SWSC-I 16 CONCENTRATION OF BENZO(-)FLUORANTHENES IN PROCESSED MWSC-I 17 CONCENTRATION OF BENZO(-)FLUORANTHENES IN - PROCESSED SWSC-I 18 CONCENTRATION OF BENZO(a)PYRENE'AND DIBENZ(-) ANTHRACENES IN PROCESSED MWSC-I 19 3285

INB'IF©1 Institut fur biologische Forschung • KbIn SUBREPORT P 0500/30,68 IJK3 (R) B30 7289 AC PAGE 0-5 CONTENTS (continued) AC FIGURE CONCENTRATION OF BENZO(a)PYRENE AND DIBENZ(-) ANTHRACENES'IN PROCESSED SWSC-I 20 HEADS'PACE CHROMATOGRAM OF CRUDE MWSC-I 21 HEADSPACE CHROMATOGRAM OF PROCESSED MWSC-I 22 HEADSPACE CHROMATOGRAM OF'CRUDE SWSC-I 23 HEADSPACE CHROMATOGRAM OF PROCESSED SWSC-I 24 AC' PAGE 6 REFERENCES 6-1 Remarks: This subreport, including title page, contains 132 pages. 3285

INBIFO Institut fur biologische Forschung - K6in SUBREPORT P 0500/3068 IJK3 (R) A5 7289 AC PAGE 0-6 \ ABBREVIATIONS (a,b) AC AW DDD DDE DDT DDVP DMCS DMNA DMSO DPM Ex : team Analytical Chemistry : acid-washed : d'.ichlorophenyl-dichloroethane : dichlorophenyl-dichloroethylene : trichlorophenyl-dichloroethane : dichlorvos : dimethyld'ichlorosilane : N-nitrosodimethylamine : dimethyl sulfoxide : dry particulate matter : x as exponent to the base 1&, e. g• E2 = 2 F FID : factor : flame ionization detector x g - centrifugal force in terms of the constant of gravitation (1 x g = 9.81 m/s2) HCB HCH HPLC M MS' . hexachlorobenzene . hexachlorocylohexane . high performance liquid chromatography . arithmetic mean . mainstream smoke MWSC-I: N . NAB . NATB . NNK . NNN . NPY . PAH . mainstream whole smoke condensate collected with impaction trap number of individual values N-nitrosoanabasine N-nitrosoanatabine 4-(N-methyl-N-nitrosoamino)-1-(3-pyridyl)-1-butanone N-nitrosonornicotine N-nitrosopyrrolidine polycyclic aromatic hydrocarbons (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). 3285

INBIF0 Institut fur Ibioiogische Forschung • Kt51n SUBREPORT P 0500/3068 IJK3 (R B21 7097 AC PAGE 0-7 ABBREVIATIONS (continued) PCB : polychlorinated biphenyls PT : preliminary title PY : team Pathology QA : Quality Assurance Unit RSD : relative standard deviation SE : standard error SOP : standard operating procedure SS : sidestream smoke 4 SWSC-I: sidestream whole smoke condensate collected with impaction trap TEA : thermal energy analyzer TPM : total particulate matter (determined gravimetrically) TSNA : tobacco specific nitrosamines UV : ultra violet VNA : volatile nitrosamines x : individual value 3285

INBIFO Institut fur biologische Forschung • HCt>In SUBREPORT P 0500/3068 IJK R A - 1 SUMMARY The team Analytical Chemistry was responsible for (1) the preparation of MAINSTREAM WHOLE SMOKE CONDENSATE (MWSC-I) and': SIDESTREAM WHOLE SMOKE CONDENSATE (SWSC-I), (2) the preparation of the skin painting application solutions of different concentrations from!the condensates, (3) the chemical characterization of the condensates or appli- cation solutions, (4) the storage of the application~solutions and (5) the residue analysis of the diet and bedd'ing material. In the following the results are presented according to this division. (1) The mainstream (MS) and' sidestream (SS) condensates were prepared simultaneously on a 30-port smoking machine equ~ipped with a circular hood above the cigarettes for SS collection. Both condensates were trapped in impaction traps. For this study 176 batches of MWSC-I and 175 batches of SWSC-I were prepared from approx. 320000 cigarettes (standard ref- erence cigarette 2R1). (2) For the preparation of the application solution the crude condensate, for MS as well as for SS, had to be dried because of its high water concentration. For this purpose the crude condensate was suspended in acetone and' the suspension evapo- rated by a rotary evaporator at 40 degrees centigrade down~to a vapor pressure of approx. 100 Pa. This processed condensate was suspended in acetone to a concentratiom of 240 grams/ liter. For preparing the application solution of the highest concentration the water concentration was determined in the suspension and adjusted to 25 grams/liter. The concentrations of the other 2 application solutions of 180 and 120 grams 3285

INB'IFO Institut fur biologische Forschung • K6Cn SUBREPORT P' 0500,/3068 IJK3 (R) B27 7289 AC PAGE 1-2 processed condensate/liter were prepared by dilution of solution of the highest concentration with acetone containing, 25 grams water/liter. For MS the yield of crude and processed condensate was 48.5 and 39.6 milligrams per cigarette respectively, for SS 22.1 and 19.4 milligrams per cigarette respectively. Thus the yield of the processed condensate was twice as high for MS as for SS. (3) The condensates or the application solutions were ch~arac- terized by the following parameters: concentration of hydro- gen-ion, nicotine, catechol, nitrosamines, polycyclic aromatic hydrocarbons (PAH) and volatile components. In the following, the results are given only for the application solution of the highest concentration.. The MS application solution was slightly more acidic than the SS application solution. The pH values were 6.0 and 6.5. The nicotine concentration was with 20.8 g:rams/liter about 20 percent lower for MS than for SS with 25.1 grams/liter. The catechol concentration was nearly identical, 1.21' grams/ liter for MS and 1.10 grams/liter for SS. The determined nitrosamines were N-nitrosonornicotine (NNN), N-nitrosoanatabine (NATB), 4-(N-methyl-N-nitrosoamino)-1-(3- pyridyl)-1-butanone (NNK), N-nitrosoanabasine (NAB), N-nitro- sopyrrolidine (NPY) and N-nitrosodimethylamine (DMNA). For MS the concentrations of NNN, NATB, NNK, NAB, NPY and DMNA were 1.9, 1.7, 1.5, 0.5, 0.07 and 0.01 milligrams/Iiter respectively, whereas for SS they were 1.7, 0.8, 6.1, 0.4, 0.32 and 0.08 milligrams/'liter respectively. The main dif- ferences between MS and SS' were the higher concentrations of NNK, NPY and DMNA for SS. 3285

INBIFO Institut fur biologische Forschung • Wn SUBREPORT P 0500/3068 IJK3 (R) B2'8 7125 AC PAGE 1-3' ~ 14 PAH were determined, of which benzo(a)pyrene, dibenz(a,h) anthracene and the benzofluoranthenes may be most relevant for mouse skin carcinogenic activity. Their concentrations were 0.18, 0.01 and 0.17 milligrams/liter for MS and 1.25, 0.07 and 1.42 milligrams/liter for SS. The sum of all PAH were 5-fold higher for SS than for MS. The comparison of volatile components found in headspace chromatography showed a higher number and concentration of these components in MS than in SS, especially in the crude condensate. By processing of the condensate these components were markedly reduced for MS and slightly for' SS and the difference between MS and SS became smaller. As the components were not identified no evaluation of these findings can be given. The reproducibility of the condensate preparation was very good with a relative standard deviation for the processed condensate yield of 4.7 percent for MWSC-I and 6.1 percent for SWSC-I and' no tendencies over the whole time of condensate preparation. The crude condensate and nicotine yields for MS were in good accordance with the data provided by the client. For SS there are no data available. (4) The storage time of the application solutions was between 14 and 29 days with a mean value of 20 days. There was no analy- sis performed after storage. Therefore no information on chemical changes dependent on storage time and conditions are available. Concerning biological activity the tumorigenicity of MWSC-I stored at minus 75 degrees centigrade before appli- cation was higher (numerically) than MWSC-I stored at 4 degrees centigrade, however, statistically not significant (see SUBREPORT PY). 3285.

IN'BIFO Institut fur biologische Forschung • KtSln SUBPREPORT P 0500/3068 IJK3 (R), B17 7289 AC PAGE 1-4 (5) Diet and bedding material were checked fbr pesticides, poly- chlorinated biphenyls, aflatoxins and heavy metals. Batches with concentrations higher than the tolerance levels according to the German Futtermittelverordnung were not accepted for use. I N B I F O Institut fur biologische Forschung GmbH 3285

INBIFO Institut f~r bi'ologische Forschung BC81n SUBREPORT P 0500/3068 IJK3 (R) A7 6171 M AC PAGE 2-1 2 RESPONSIBILITY -------------- Quality Assurance: ....... .... .... .. .... Dr.rer.nat. B. Gerstenberg Food Chemist (Staatl. gepruf- ter Lebensmittelcliemiker) £-• --i----. E. Romer Biologist (Diplombiiologe) N O N ~ ® C1t ~ W 3285

INBIFO Institut fur biologische Forschung • K6Ini SUBREPORT P 0500/3068 IJK3 (R) A7 6171 M AC PAGE 2-1 2 RESPONSIBILITY Quality Assurance: Dr.rer.nat. B. Gerstenberg Food Chemist (Staatl. gepruf- ter Lebensmittelchemiker) E. Romer Biologist (Diplombiologe) 3587

INBIFO Institut fuir biol!ogische Forschung • KtSin SUBREPORT P 01500/3068 MN065RA29 7125 AC PAGE 3-1 3 INTRODUCTION . The objective of this study was among other things the comparison of MS and SS condensate in a dermal tumorigenicity study. For this purpose SS condensate had to be prepared in a large scale and over a long period for the 1st time. Therefore a standardized method for the reproducible generation and collection had to be developed. This was realized by the special equipment consisting of a 30-port smoking machine with a circular hood and an impaction trap. The hood was made of stainless steel containing a stainless steel wire mesh. For SS condensate collection only an impaction trap could be used as high volumes were to be dealt with. A special impaction trap was designed in which SS was passed through a circular nozzle which impacted the condensate on the wall of a cooled glass bottle. For reasons of comparison MS condensate was also collected with an impaction trap. But the MS trap was equipped with a capillary instead of the circular nozzle, because it was passed only by low volumes. The comparison of MWSC-I and SWSC-I and the influence of the impaction trap on condensate collection is discussed in detail in the INBIFO studies P 0500/ 5007 (1984) and P 0500/3106. The recovery of the condensate preparation was approx. 90 percent for MWSC-I (losses of 1 percent in the impaction trap and 8 percent in the smoking machine and tubes) and 80 percent for SWSC-I (losses of 5 percent in the impaction trap and 15 percent in the hood and tubes) (INBI FO study I 49 presented in poster show 1983). For characterization of the condensate the determination of some chemical parameters were of special interest. For several of them methods of determination had to be developed or at least estab- lished. 3285

INBIFO Institut fur biologische Forschung • Kt51n For catechol a new and very selective method has been developed which adapts the liquid-liquid extraction step with borate, performed by several authors, to liquid-solid extraction which can be automated in an HPLC procedure. For the nitrosamines a method f rom FTR could be used with only minor changes. For the development of the method for PAH determination the isolation: steps were taken over from Levins (1978) and Grimmer (1979). The determination was performed by GC because with GC compared to HPLC also the PAH with lower molecular weight, e. g. naphtalene and anthracene, could be detected sensitively. For the volatile components a headspace GC method: had been devel- oped which provides a chromatogram which characterizes the condensate like a finger print and gives information about the drying process. The identification of the compounds was not intended as a mass spectrometer was not at disposal. Only for a few components, e. g. nicotine a comparison to reference sub- stances was performed. Thus for all desired parameters adequate methods for determination became available during the study or were already available before as for nicotine, water and pH. 3285.

INBIFO Institut fuir biologische Forschung • NC6In A - 4 METHOD 4.1' Condensate Preparation and Storage (see AC FIGURE: A) Mainstream and sidestream smoke condensates were always prepared simultaneously with the same smoking machine. 4.1.1 Pre2aration-of mainstream smoke crude condensate (see AC FIGURE B) Principle: mechanical open-end smoking to a defined: butt length in automatic negative pressure (vacuum pump) smoking machine, condensate col- lection with impaction trap Sample material and quantity: cigarettes (see INTEGRATING REPORT) Cigarette type: 2R1 (standard'reference) Source: Philip Morris, USA Number of cigarettes: 320 000 (a) Packaging: in bundles with 200 cigarettes, 10 packs with 20 cigarettes each Date of receipt at INBIFO: 6.Nov.82 St or age Main storage: walk-in cold room R907, 1 to 3 degrees centigrade, relative humidity uncontrolled Laboratory storage: conditioning room R326, at least 10, days prior to condensate preparation (a) inclusive 10 0/0 loss plus 10 0/0 reserve 3285

SUBPREPORT P 0500/3068 IJK3 (R) A9 6148 SUPPLIER - » RY.CrIPT OP CIGARETTrS AT INHIPO, STOHAi:E I N COLO ROOM UNCONUITIONED CICAHrTTES CONDITIONING IN I CONDITIONED LAHORATONY STOHAGE DCPERMINATION OP MATER, ADJUSTMENT OP NATEN CONCCNTRATION APPL/CATION •~ STORAG6 -75 DEGREES CENTIGRADE APPLICATION SOLUTION(S) PNEPARATION OP APPLICATION SOLUTION(S), CHEMICAL ANALYS6S_, part 2 CIGAHETTCS PROCESSED CONDENSATE SUSPCNSION AC FI GURE A SCHEME FOR CONDENSATE PREPARATION, ANALYSES AND STORAGE PHCPARATION OP CNUDC CONDENSATE BY IMPACTION TRAP hs CHEMICAL ANA- LYSES, part 1 NEIGHING, SUSPrNDING IN ACETONE AC PAGE 4-2 » CRUDC CONDENSATE WP.IGHING, SUSPCNUING IN ACETONE CRUOB CONDENSATE SUSPENSION OPf ERM I NAT I(1N OF NATEN, hNYI MITH VAL'UUM NOTARY EVAPORA PROCESSED CONDENSATE G TO R gCVIS09Z0~

INBIFO Institut fuir biollogische Forschung • KtSln SUBREPORT P 0500/3068 IJK3 (R) B18' H0306&M 6148 AC PAGE 4-3 . Q- ROOM TEMPERATURE 1.05 I /min 000000 . ~60 DEGREES CENTIGRADE !_J---- 4- 120 I/min AC FIGURE B ICE WATER SETUP FOR THE SIMULTANEOUS PREPARATION OF MAINSTREAhi:AND S I EUE;STREAM SMOKE CONDENSATE

INBIFO Institut fiir biologische Forschung • K6In SUBREPORT P 0500/3068 IJK3 (R) A10 Physical and chemical data: Equipment Smoking,machine Type : Number of machines: Machine numbers: Load'ing of cigarettes: Lighting of cigarettes: Ejection of cigarettes: Vacuum pump: Flowmeter: 71013 AC PAGE 4-4 temperature: 22 + 1 degrees centigrade, relative humidity: 60 ± 3' percent, cigarettes in vertical positionn in opened packages see AC TABLE A automatic INBIFO smoking machine 2 0035, 0036 automatically automatically or manually with an iodine spot lamp iodine spot lamp: Halogen-Bellaphot, 15 V, 150 W, gold-plated reflector, Osram, no. 64635, R. Schahl, D-80010 MUnchen 71 automatically at butt length of 23 mm for filterless standard ref- erence cigarette (automatic scanning with infrared photodiode) membrane vacuum pump N 0135/ANE, K. Neuberger KG, D-7800 Freiburg-Munzingen rotameter, L 4/160, Rota, Dr. Henning KG, D-7867 Wehr/Baden soap-film flowmeter, Faust GmbH, D-5000 Ko1n 90 3285

IN'BIFO Institut fur biologische Forschung • KtSIn SUBREPORT P 0500/3068 IJK3 (R) All 7103 M AC PAGE 4-5 PARAMETER DATA length (mm/cig.)' weight (g/cig.) tobacco weight (g/cig,.) puff resistance (Pa) puff count (N/cig.) butt length (mm) 85 1.17 1.10 determined 13. and 15.Oct.80 861 11.5 calculated from values of INBIFO study P 0500/3030 (1984) 24 AC TABLE A PHYSICAL DATA OF CIGARETTE 2R1 3285

INBIFO Institut fur biologische Forschung • KtSln SUBREPORT P 0500/3068 IJK3 (R) A12 6175 AC PAGE 4-6 Impaction trap 'rype : glass "impaction trap for cigarette smoke condensate collection" according to Philip Morris (see AC FIGURE C), Faust GmbH, D-5000 KSln 90 Capillary: length: 5 mm, bore: 0.4 mm Mode of installation of the impaction trap insert: Installation of impaction trap: distance of 0.5 mm between capillary tip and wall of flask calibrated with 0.5 mm thick teflon sheet spacer impaction trap lies horizontally below smoking machine connected via glass tubes dimension of glass tubes: length: 57 cm~, outer diameter: 17 mm, inner diameter: 10 mm~ Procedure Puffs/cigarette: 11.5 Puff frequency/cigarette: 1 puff/min Puff duration: approx. 2 s minus time for change of position Puff volume: 35 ml parameter checked and regulated during condensation with a rota- meter or soap-film flowmeter scientific version: SOP AC 41/1 Text version: 24.Jun.86 3285

IINBIFO Institut f[1r biologische Forschung • Min SUBREPORT P 0500/306H' IJK3 (R) A13 H05 M 6148 AC PAGE 4-7 lX ( --SPHERIICAL 1QINT I S 12~5 i I ~GLASS TUBING 8 mm OUTER DIAMETER I 5 mm INNER DIAMETER ( -SCHOTT GL 18 1 i ROUND BOTTOM , FLASK I /~~ 500 ml SPACING 0.5 mm ` CAPILLARY WITH BEVELED END LENGTH 5 mm BORE 0.4 mm AC h.IGURL, C IMPACTION TRAP, MAINSTREAM SMOKE 3285

INBIFO Institut fur biologische Forschung • KOIn SUBREPORT,P 0500/3068 IJK3 (R) A14 7292 AC PAGE 4-8 4.1.2 Preparation of sidestream smoke crude condensate ------------------------------------- Principle: mechanical open-end smoking to a defined butt length in automatic negative pressure (vacuum pump)) smoking machine, condensate col- lection by means of a circular hood and a special impaction trap Sample material and quantity: Equipment Smoking machine Type: Number of machines: Machine number: Loading of cigarettes: Lighting of cigarettes: Ejection of cigarettes: Vacuum pump: Hood for sid'estream smoke collection: see 4. 1.1 automatic INBIFO smoking machine 2 0035, 0036 automatically automatically or manually with an iodine spot lamp iodine spot lamp: Halog.en-Bellaphot, 15 V, 150 W, gold-plated reflector, Osram., no. 64635, R. Schahl, D-8000 Munchen 71 automatically at butt length of 23 mm for filterless standard ref- erence cigarette (automatic scanning with infrared photodiode) water ringpump LRKA 10603, SIHI Halberg, via Hartmann und Essen GmbH und Co., D-5000 Koln 91 inner diameter: 310 mm, height: 100 mm plus 40 mm wire nettin~ width: 95 mm, flow: approx. 120 1/min circular stainless steel hood with wire netting installed above the cigarettes, outer diameter: 500 mm, 3285

INBIFO Institut fOr biologisEhe Forschung • K6In SUBREPORT P 0500/3068 IJK3 (R) A15 6317 AC PAGE 4-9 Impaction trap Type: stainless steel impactor for side- stream smoke condensate collection (see AC FIGURE D), INBIFO, D-5000 Koln 90 Outlet nozzle: Distance of impaction plate from outlet nozzle: Installation of impaction trap: Procedure Puffs/cigarette: Puff frequency/cigarette: Puff duration: Puff volume: Suction volume for sidestream collection: Pressure in impaction trap: Scientific version: Text version: annular fissure of 100 mm length and 0.1 mm width 0.1 mm in vertical position in an ice water bath below smoking machine connected with collection hood via copper tubes dimension of tubes: length: 70 cm, outer diameter: 35 mm, inner diameter: 30 mm 11.5 1 puff/min approx. 2 s minus time for change of position 35 ml approx. 120 1/min approx.. 5E4 Pa SOP AC 42/1 23.Dec.85 3285

INBIFO Institut fur biologische Forschung • K6ln SUi13RF.PORT P 0500/306$' IJK3 (R) A16 H01662 M 6148 AC PAGE 4-10 STAINLESS STEEL TUBING 40 mm WTER DIAMETER 34 mm INNER DIAMETER AC FIGUttE D IMPACTION,TRAP, SIDESTREAM SMOKE Remarks: schematic view 3285

INBIFa Institut Mr biologische Forschung • Kt3In SUBREPORT P 0500/3068 IJK3 (R) A17 6171 AC PAGE 4-11 4.1.3 Preparation-and storage-of-applicationysolution Principle: processed condensate prepared from crude condensate by reduced pressure evaporation, application solutions prepared by resuspension of processed condensate in acetone Time: on the day after preparation of crude condensate Sample material and quantity: total condensate prepared with 1 or 2 smoking machines from 1 day Equipment: rotary evaporator: Rota Vapor R/A, Buchi GmbH, D-7332 Esslingem vacuum pump: Trivac D2A, Leybold-Heraeus AG, D-5000 K61n 51 pressure gauge: Diavac-N, Tele Diavac S, Leybold-Heraeus AG, D-5000 K61n 51 recorder: Servogor 210, Metrawatt GmbH, D-8500 Nurnberg deep freezer: no. 8218, Forma Scientific, via Labotect, D-3400 Gottingen Chemicals: acetone, no. 14, E. Merck, D-6100 Darmstadt 1 3285

INBIFO Iinstitut fOr biologische Forschung • IftSi'n SUBREPORT P 0500.30 IJK3 R A 18 7103 A - Procedure ,. Processed condensate: crude condensate washed out of impac- tion trap with acetone (1 g crude condensate plus 3 ml acetone) into an evaporation flask, determination of water (see 4.2.1i), acetone and other low evaporating, substances removed by rotary evaporation (pressure approx. 35 kPa, water bath temperature 40 degrees centigrade). Receiver flask cooled in a dry ice/acetone mixture, pressure reduced stepwise (pressure reduction controlled with a recorder) to approx. 100 Pa and residue allowed to dry for 5 min. Amount of processed condensate calculated from weight of evaporation flask before and imme- diately after preparation of processed condensate Application solutions: processed condensate suspended in acetone to a concentration of 240 g,/1 after determination of water concen- tration (see 4.2.1), bidistilled water added to a final water concentration of 25 g/1 (a) (application solution of the highest concentration) preparation of the other application solutions by dilution with acetone containing 25 g/l water (see AC TABLE B) Storage of the application solutions: d'aily portions for groups with equal concentration filled in brown glass bottles with teflon-lined screw caps and immediately stored in deep freezer at -75 degrees centigrade (1-GR to 3-GR and 7-GR to 15-GR) or refrigerator at 4 degrees centigrade (4-GR to 6-GR) storage time: minimum 14 days, maximum unlimited ~ 0 Scientific version: SOP AC 43/1 N Text version: 28.May 86 0 ~ N ~ 4th (a) condensate concentration not corrected for volume changes by ~ water addition, max. fault 2 0/0 3285

I'NBIF0 Institut fur Ibiologische Forschung • KOln SUBREPORT P 0500/3068 IJK3 (R) B15 6171 AC PAGE 4-13 PROCESSED CONDENSATE PREPARATION CONCENTRATION IN APPLICATION SOLUTZON (g/1) 240 180: 120 AC TABLE B 24 g: processed condensate plus 76 ml acetone, water determination and addition of water to a final concentration of 25 g water/1 75 ml application solution of 240 g processed condensate/1 plus 25 ml acetone containing 25 g water/l 50 ml application solution of 240 g prooessed condensate/l plus 50 ml acetone containing 25 g water/'1 PREPARATION OF APPLICATION SOLUTION 3285

SUBREPORT P 0500/3068 IJK3 (R) A19 6148 AC PAGE' 4-14 4.2' Condensate Analyses 4.2.1 Determination of water conc.entration Principle: Time: titration according to Karl Fi~schex, back-titration on the day of the preparation of the application solution Sample material and quantity: crude or processed condensate suspension, 1 ml, 2 determina- tions/suspension Results expressed in: g/l crude or processed conden- sate suspensiom Equipment: Chemicals: Procedure Karl Fischer Titrator E452, Deutsche Metrohm GmbH, D-7024 Filderstadt Karl Fischer solution, no. 9248, methanol, no. 6012, acetone, no. 14, E. Merck, D-6100 Darmstadt 1 Titration: a known excess of Karl Fischer solution (e. g. 3 ml) added to 1 ml application solution in the reaction vessel of the titrator and titrated with methanol with a known amount of water (approx. 3.5 g/1) Computation: for computation titer of the Karl Fischer solution determined by back-titration of 1 ml acetone with a known amount of water e. q. 10 mg, water content of acetone determined accordincaly Detection limit: 0.5 n/l -g5

SUBREBORT P 0500/3068 IJK.3 (k) 816 7226 AC PAGE 4-115 Reproducibility (relative standard deviation): 2.8 0/0 (10 g/1 water, Scientific version: SOP AC 44/1 Text version: 25.Oct.85 4.2.2 Determination of nicotine concentration --------------------------------------- Sample material and quantity: Results expressed in: Equipment: = 8' ) gas chromatography after extraction with dichloromethane computer integration of peaks on the day of the preparation of the application solution or within 2 days, at 4 degrees centigrade application solution of the highest condensate concentration, diluted 50-fold with acetone, 1 ml, 2 determinations/solution g/1 appliication solution, g/kg condensate, mg/cig. gas chromatograph: HP 5730 A, detector: FID, automatic sampler: HP 7671 A, laboratory data system: HP 3351 A, Hewlett-Packard GmbH, D-6000 Frankfurt/Main recorder: Servogor 210, Metrawatt GmbH, D-8500 Nurnbergg centrifuges model J6, rotor: JS-4.2, Beckman Tnstruments GmbH, D-8000 Munchen 40 >~RF

SUBREPORT P 0500/3068 IJK3 (R) A21 6171 AC PAGE' 4-16 Chemicals: nicotine, no. 77635, Serva Feinbiochemica GmbH und Co. KG, D-6900 Heidelberg 1 quinoline, no. 802407, dichloromethane, no. 822271, acetone, no. 1:4, sodium hydroxide (200 g/1), no. 5594, sulfuric acid, no. 9074, E. Merck, D-6100 Darmstadt 1 nitrogen, hydrogen, air (synthetic), Linde AG, D-5000 Koln 50 internal stand ard solution: 0.5 g quinoline/l (0.1 mol/1) sulfuric acid, standard solution: 1.0 g nicotine and 0.5 g quino- line/1 (0.1 mol/1) sulf uric acid Procedure Extraction: addition of 1 ml of the internal standard solution, 1 ml sodium hydroxide and 10 ml dichloromethane to 1 ml diluted application solution, after agitation (5 min) and centrifugation (approx. 7.8E3 m/s2 (= 800 x g), 5 min, approx. 10 degrees centigrade), injection of 1 ul of the lower organic phase into the gas chromato- graph Gas chromatography Column: 2 m x 1/8 inch outer diameter, glass ~ Column packing: 100 g/kg Apiezon L and 100 g/kg KOH ~ on Chromosorb W-AW DMCS (a), ~ 80 to 100 mesh ~ N Carrier gas and ,A flow rate: nitrogen, 30 ml/min (,/t 0 (a) AW: acid-washed, DMCS: treated with dimethyldichlorosilane

SUBREPORT P 050-0/30,68 IJK3 (R) A22 7103 AC PAGE'4-17 Oven temperature: 175 degrees centigrade Injection port temperature: 200 degrees centigrade Detector temperature: 200 degrees centigrade Computation: 1 ml of a standard solution diluted with 1 ml acetone and extracted as described above determination of calibration factor Detection limit: 0.02 g/1 Recovery: 98.5 0/0 Reproducibility (relative standard deviation): 0.5 0/0 (1 g nicotine/1 (0.1 mo1/1) sulfuric acid, N = 10 ) Scientific version: SOP AC 9/2 Text version: 24.Oct 85 4.2.3 Determination_of_catechol Principle: direct HPLC determination of catechol in the application solution by combi- nation of 2 different columns and column switching (1) prepurification by adsorption of catechol on a precolumn with selec- tivity for compounds with o-dihydroxy function, while washing the main com- pounds of condensate into the waste (2) switching the precolumn on-line to the main column (RP-18) and elution of the compounds adsorbed on the precolumn onto the main column (3) separation of these compounds on the main column and determination of the catechol by UV absorption ~A.

SUBREPORT P 0500/3068 Time: IJK3 (R) A23 Sample material and quantity: 6317 AC PAGE' 4-18 within 2 weeks after preparation of the application solution, storage at 4 degrees centigrade 100 ul application solution of the highest concentration, 2 determina- tions/solution Results expressed in: g/l application solution, g/kg condensate, mg/cig. Equipment: Chemicals: high performance liquid chromatograph with automatic sampler, variable wave- length detector and integrator, HP 1084 B column: RP-18, 10 um, 200 mm x 4.6 mm, no. 79916 B, Hewlett-Packard GmbH, D-6000 Frankfurt/Main precolumn: 40 mm x 4.6 mm filled with dihydroxyboryl silica gel, 30 um, no. -, Serva, D-6900 Heidelberg 1 centrifuge, model J-6 B, Beckman Instruments, D-8000 Munchen 40, methanol, no. 60017, phosphoric acid, no. 573, sodium dihydrogenphosphate, no. 6346, E. Merck, D-6100 Darmstadt catechol, no. 15880, Fluka, D-7 910 IVe u-Ulm standard solution: 100 mg/1 catechol in acetone

-----RE_ __ - __ SUBPORT P 0500/306$ IJK3 (R) A24 7226 AC PAGE'4-19 Procedure Sample preparation: application solution diluted 10-fold with methanol and centrifugation (3 min, 2000 g), supernatant ready for HPLC HPLC Determination Precolumn: dihydroxyboryl silica gel, 40 mm x 4.6 mm Column: E'lution solvents: Elution: " Flow rate: Column switching: Oven temperature: Wavelength: Injection volume: Time between the analyses: Computation: Scientific version: Text version: RP-18, 200 mm x 4.6 mm A = 10 mmol sodium dihydrogenphos- phate/l water, adjusted to pH = 2.9 with phosphoric acid, B = methanol see AC TABLE C 1.5 ml/minn see AC TABLE C ambient 280 nm 20 ul 3 min calibration by external standard method, 3-fold analysis of the standard solution and evaluation of the peak areas by.the integrator, the 1st analysis in a series has to be canceled SOP AC 59'/1. 24.Oct.85

SUiikEYUk'1' !' 05U(J/3Ub8 TIME (min) IJK'J (it) A25 /'1O'3 AC PAGE' 4-20 EVENT 0 1.0 to 2.0 B: 100 B t 100 0/0 0/0 t o: 5 0/0 2.1 column switching: precolumn on-line to the main column 3.5 to 13.5 13.5 to 15.0 B: B: 5 70 0/0 to 70 0/0 to 100 0/0 0/0 14.9 17.0 columm switching: precolumn off-line to the main column end of the analysis AC TABLE C CONDITIONS OF THE HPLC DETERMINATION

SUBREPORT P 0500/3068 IJK3 (R)', A27 6171 AC PAGE 4-21 4.2.4 Determination_of hydroqen_ion_concentration ------------- -- Principle: Time: electrochemical determination on the day of processed condensate preparation Sample material and quantity: applic ation solution of the highest condensate concentration, 2 ml Results expressed in: pH Equipment: Chemicals: pH meter: PW 9409, glass electrode; CA 1-S, Phil ips GmbH, D-3500 Kassel calibration buffer: pH 7.00, no. 9887, pH 4.00, no. 9884, E. Merck, D-6100 Darmstadt 1. Procedure: determination at room temperature after calibration of pH meter with 2 standard buffers Scientific version: SOP AC 45/1 Text version: 24.Oct.85

SUBREPORT P 0500/3068 IJK3 (R) A28 7103 AC PAGE: 4-22 4.2.5 Determination_of_nitrosamines (a) -- --------- -- ---------- - Principle: extraction of condensate with dichloro- methane, column chromatography with basic alumina and gas chromatography of eluates with thermal energy analyzer (TEA) as detector Time Sampling: immediately after preparation of the processed condensate Determination: immediately after sampling or samples stored at -75 degrees centigrade till determination Sample material and quantity: Results expressed in: Equipment: processed condensate, 11.0 g mainstream smoke and 0.5 g sidestream smoke condensate ug/1 or mg/1 application stock solu- tion, ug/kg or mg/kg condensate, ng/cig. gas chromatograph: HP 5710 A, automatic sampler: H~P 7671 A, laboratory data system: HP 3351 A, Hewlett-Packard GmbH, D-6000 Frankfurt/Main detector: thermal energy analyzer, TEA 502A, Thermo Electron Corporation, Waltham, USA rotary evaporator: rota vapor R, Buchi GmbH, D-7332 Esslingen mechanical shaker, INBIFO, D-5000 Koln 90 100: ml flask with screw cap, 500 ml separating funnel, 1 1, 500 ml and 100 ml round bottom flask, according to an unpublished method of Fabriques de Tabac Reunies S.A., Neuchatel

SUBREPORT P 0500/3'068 IJK3 (R) A29 7103 Chemicals: AC PAGE 4-23 5 ml and 2 ml volumetric flask, chromatographic column with teflon stopcock, 30 cm x 2 cm inner diameter N-nitrosodimethylamine (DMNA), N-nitrosodiisobutylamine (DIBNA), N-nitrosopyrrolidine (NPY), N-nitrosodihexylamine (DH~NA), N-nitrosonornicotine (NNN), N-nitrosoanatabine (NATB), N-nitrosoanbasine (NAB), 4-(N-methyl-N-nitrosoamino)-1- (3-pyridyl)-1-butanone (NNK), ISCONLAB, D-6900 Heidelberg acetone, no. 13, dichloromethane, no. 822271, alumina, basic, no. 1067, ascorbic acid, no. 127, citric acid, no. 244, sodium hydroxide, no 5594, disodium hydrogen phosphate, no. 6580, sodi=sulfate, no. 6649, E. Merck, D-6100 Darmstadt 1 helium, oxygen, Linde AG, D-5000 Ko1n 50 buffer solution: 11.6 g citric acid, 16 g disodium hydrogenphosphate (dihydrate) and 3.5 g of ascorbic acid/1 alumina: alumina with an activity of III prepared according to the description of the supplier preparation of chromatographic column: 65 g alumina (activity III) added with tapping to a column filled with di- chloromethane, which is allowed to empty dropwise. Afterwards column washed with 150 ml dlichloromethane

SUBREPORT P 0500/3068 IJK3 (R) A30 7226 AC PAGE 4-24 Procedure: Gas chromatography extraction: 1 g mainstream smoke or 0.5 g side- stream smoke processed condensate solved in 80 ml of buffer solution with addition of approx. 3 ml di- chloromethane, buffer/condensate mixture extracted 3 times with 100 ml dichloromethane each after addition of 2 ml sodium hydroxide solution (2 mo1/1)~. Organic phase filtered over sodium sulfate into a 1 1 round bottom flask and filtrate concentrated with a rotary eva- porator to approx. 5 ml. Concen- trate transferred to a chromato- graphic column filled with basic alumina (activity III) and elution with 250 ml dichloromethane. Eluate of volatile nitrosamines (VNA) concen- trated with slow destillation (no vacuum applied) after add~ition of the internal standard solution (a), concentrate transferred to a 5 ml volumetric flask and filled up to volume. 5 ul of the VNA extract injected into the gas chromatograph after elution with dichloromethane elution of the column with 350 ml dichloromethane/acetone (9 parts plus 1 part), eluate of the tobacco specific nitrosamines (TSNA) concentrated as described above after addition of the internal standard solution (b), concen- trate transferred to a 2 ml volumetric flask and filled up to volume. 2 ul of the TSNA extract injected into the gas chromatograph Volatile nitrosamines N 0 ~ Column: 2 m x 2 mm inner diameter, glass ~ © Column packing: 150 g/kg Carbowax 20M on Chromosorb ~ W-HP, 100 to 120 mesh N ~ Carrier gas and flow rate: helium, 30 ml/min ~ ~ (a) N-nitrosodiisobutylamine approx. 300 g/1 CH2CL2 (b) N-nitrosodihexylamine approx. 1 mg/1 CH2CL2 "R.

SUBREPORT P 0500/3068 Oven temperature: Injection port temperature: TEA interface temperature: TEA pyrolizer temperature: temperature program: initial temperature 120 degrees centigrade, rate 8 degrees centigrade/min, end temperature 190 degrees centigrade for 4 min 250 degrees centigrade 250 degrees centigrade 475 degrees centigrade Tobacco specific nitrosamines Column: 3 m x 2 mm inner diameter, glass Column packing: 10 0/0 SP-2100 on Supelcoport, 100 to 120 mesh Carrier gas and flow rate: Oven temperature: Injection port temperature: TEA pyrolizer temperature: helium, 30 ml/min 210 degrees centigrade 250 degrees centigrade 450 degrees centigrade Computation: injection of 5 ul (VNA) or 2 ul (TSNA) of the calibration solution and calculation of the peak height ratios with the internal standard Scientific versionr. SOP AC 35/2 Text version: 25.Oct.85 4.2.6 Determination_of_polycyelic_aromatic_hydrocarbons (a) Principle: ~ 0 ~ ~ ~ thin-layer chromatographic separa- 0 tion of polycyclic aromatic hydro- '"& (a) Some samples in the beginning of PAH determination were analyzed with a method which was different in some details. IJK3 (R) B1 7226 AC PAGE 4-25 4A ~ ~ -Ar,

SUBREPORT P 0500/3068 IJK3 (R) B2 7226 AC PAGE 4-26 Time carbons from processed condensate (a), extraction and concentration of crude aromatic fraction followed by high resolution gas chromatography after cold on-column injection digital integration of peaks, com- parison of retention times with pure reference components, determination of response factors and calculation of quantities with internal standard method Sampling: randomly from 1i batch per week, directly after preparation of processed condensate, storage at -75 degrees centigrade Determination: approx. 1 day after preparation of the processed condensate Sample material and quantity: processed condensate, 0.2 g Results expressed in: mg/1 application solution, mg/kg condensate, ug/cig. Equipment: gas chromatograph: Packard 438, detector: FID, on-column injector: Chrompack II, Packard Instrument GmbH, D-6000 Frankfurt/Main on-column syringe: SGE~5 A RN GP CE, capillary column, Chrompack Deutschland GmbH, D-1000 Berlin integrator/plotter: Shimadzu, Chromatopac C-RIB, Shimadzu GmbH, D-4000 Dusseldorf rotary evaporator: IKA-Dest RV-05, Janke und Kunkel KG, D-7813 Staufen (a) according to: Levins, R.J., Isolation of polyaromatic hydrocarbons from whole smoke condensate: A simple two-step procedure, Chromato- graphia 11 : 736 (1978)

SUBREPORT P 0500/3068 IJK3 (R) B3' 7289 AC PAGE 4-27 Chemicals: Procedure thin-layer plates: Whatman PLK 5, Hermle Laborgerate, D-6837 St. Leon-Rot universal UV-lamp: 29200, Camag GmbH, D-1000 Berlin polycyclic aromatic hydrocarbon kit, LAO-08378, ICT Handels GmbH, D-6230 Frankfurt 80 methanol, no. 6009, n-hexane, no. 4391, toluene, no. 8325, cyclohexane, no. 9666, E. Merck, D-6100 Darmstadt 1 n-dotriacontane, no. D 4634, Sigma Chemie GmbH, D-8024 Deisenhofen nitrogen, hydrogen, air (synthetic), Linde AG, D-5000 Ko1n 501 Clean up: 2.0 ml n-hexane plus 1.0 ml methanol/ water (7/3 v/v) added to 0.200 mg processed condensate and briefly sonicated to dissolve the sample completely after streaking, mixture on thin-layer plate, solvent evaporated by gently blowing with nitrogen th~in-layer plate developed once using ~ cyclohexane/toluene (3 ml plus 2 ml) in ~ unsaturated tank O„) polycyclic aromatic hydrocarbon d zone marked after detection under (11 UV-light (366 nm), scraped onto N glassine paper, transferred to frit ~ filter and extracted with n-hexane (4 Q7 portions, 5 ml each) under reduced M+ pressure solvent removed under vacuum on rotary evaporator at 30 degrees centigrade, residue redissolved in

SUBREPORT P 0500/3068 Analysis: Gas chromatography IJK3 (R) B4 7289 AC PAGE 4-28 Column: Column packing: Carrier gas and column head pressure: Purge gas and flow rate: Oven temperature: Injection port temperature: Detector temperature: Computation: approx. 200 ul to 500 ul internal standard solution (9.19 mg n-dotria- contane/l n-hexane) and stored at -30 degrees centigrade until analysis direct daylight exposure eliminated by covering glassware with aluminum foil and performing operations in semi-darkened room under yellow light approx. 1 ul injected directly into capillary column at low temperature 30 m x 0.32 mm inner diameter, fused silica, deactivated with polysiloxane wall coated, DB-5 (a) film thickness: 0.25 um hydrogen, 0.8 bar (corresponding linear velocity: 55 cm/s) nitrogen, 30 ml/min temperature program: 0.5 min 55 degrees centigrade, rate 20 degrees centigrade/min up to 150 degrees centigrade, rate 4 degrees centigrade up to 255 degrees centigrade, 20 min 255 degrees centigrade ambient 325 degrees centigrade gas chromatographic determination of a standard solution of several parent ~ polycyclic aromatic hydrocarbons, 0 determination of relative retention times and relative response factors byM~ using the internal standard method Q Scientific version: Text version: SOP AC 51/1 16.Oct.87 Cr1 OA ~i ~ N (a) non extractable stationary phase: cross linked and chemically bonded silicone containing 5 0/0 phenyl and 95 0/0 methyl groups

SUBREPORT P 0500/3068 IJK3 (R) B19 7103 AC PAGE 4-29 4.2.7 Determination_of_volatile_comporrents Principle: headspace capillary gas chromato- graphic analysis of diluted con- densate Time: approx. 1 day after condensate preparation, storage at -75 degrees centig rade Sample material and quantity: condensate, 0.5 g Equipment: Chemicals: gas chromatograph: Fractovap 2900, detector: FID, headspace sampler: HS-250, cryogenic unit: Lauda Kryoflex KF-40, gastight vials: CA 960035, Erba Science, D-6230 Hofheim/Taunus integrator/plotter: Shimadzu, Chromatopac C-RIB, Axel Semrau Analysen Systeme, D-5600 Wuppertal sonication water bath: Sonorex RK100, Bandelin KG, D-1000 Berlin capillary column: FS-OV-1-df-1, vial caps: N 20 TB, Macherey/Nagel, D-5160 Duren n-decane, no. 9603, dimethylsulfoxide (DMSO), no. 2931, E. Merck, D-6100 Darmstadt 1 nitrogen, hydrogen, air (synthetic), Linde AG, D-5000 Koln 50 internal standard solution: 45 mg n-decane/1 DMSO

SUBREPORT P 0500/3068 IJK3 (R) B20 7289: AC PAGE 4-30 Procedure Sample preparation: 50 ul internal standard solution, 2.5 ml DMSO and 250 mg sodium chloride added to 500 mg processed condensate in a 5 ml gastight vial, mixture soni- cated briefly and sample stored at -75 degrees centigrade until analysis Analysis: after conditioning (90 min at 90 degrees centigrade) approx. 1 ml of the headspace vapor phase injected into the gas chromatograph Gas chromatography Column: 50 m x 0.32 mm inner diameter, fused silic, wall coated with OV-1, film thickness: 1.0 um Carrier gas and column head pressure: hydrogen, 1.1 bar (corresponding linear velocity: 40 cm/s) Split ratio: 1' : 80 (nonlinear, at 30 degrees centigrade) Make-up gas and: flow rate: nitrogen, 30 ml/min Septum flush: 2 ml/min Oven temperature: temperature program: 1i min 30 degrees centigrade, rate 4 degrees centigrade/min up to 160 degrees centigrade, 20 min 160 degrees centigrade Injection port temperature: 200 degrees centigrade Syringe temperature: 100 degrees centigrade ~ ~ Detector temperature: 250 degrees centigrade ~ ~ © Scientific version: SOP AC 50/2 ~ Text version: 24.Jun.86 N ^'>p'

SUBkEPORT P 0500/3068 IJK3 (R) B5 6171 AC PAGE 4-31 4.3 Residue Analysis in Diet and Bedding Material 4.3.1 Determination_of_pesticide_and_PCB_residues --------- -- - - --- Principle: gas chromatographic determinationn after extraction with cyclohexane Type of pesticides and PCB: see AC TABLE D Time: each batch of diet and bedding material after delivery and before usage Sample material and quantity: diet: autoclaved and ground, 5 g out of a sample of approx. 300 g bedding material: autoclaved, 5 g out of a sample of approx. 100 g Results expressed in: mg/kg Equipment: Chemicals: gas chromatograph: HP 5710 A, detector: ECD (Ni-63), gas chrom~atograph: HP 5730 A, detector: FPD, P-Filter, automatic sampler: HP 7671 A, Hewlett-Packard GmbH, D-6000 Frankfurt recorder: Servogor 210, Metrawatt GmbH, D-8500 Nurnberg rotary evaporator: Rota Vapor R, Buchi GmbH, D-7332 Esslingen modified soxleth extractor, Faust GmbH, D-5000 K©ln 90 pesticide standards, Dr. S. Ehrenstorfer, D-8900 Augsburg

SUBR&tRI" h U500/3068 1JK3-1R) Bb 7103 M- - AC PAGE-4--31 SUBSPANCE DETDCTION LIMIT (mg/kg) TDLERANCE IEVEL (mg/kg) aldrin/dieldrin 0.005 0.02 chlordane approx. 0.04 0.05 o,p'-DDD 0.005 p,p'-DDD o,p'-DDE 0.005 0.005 0.05 (calculated as DDT) (a) p,p'-DDE 0.005 o,p' -DDT 0.005 p,p'-DDT 0.010 alpha-endosulfan 0.005 beta-endosulfan 0.010 endosulfan Sulfate 0.020 endrin 0.005 0.02 HCB 0.001 0.03 alpha-HCH 0.002 - beta-HCH 0.005 - gaRana-HCH (lindane), 0.002 0.10 delta:HCH 0.005 - heptachlor 0.002 ? 01 03 (calculated as heptachlor) heptachlor epoxide 0.002 . (b) methoxychlor 0.050 mirex 0.010 ethyl parathion 0.025 diazinon 0.020 DDi7P (dichlorvos) 0.020 dimethoate 0.020 fenchlorphos (Ronnel) 0.020 malathion 0.050 methyl parathion 0.020 mevinphos aroclor 1254 0.020 approx. 0.04 AC TABLE D jy 0 RESIDUE ANALYSIS OF PESTICIDE AND PCB, DETECTION LIMIT AND TOLERANCE LEVEL (c) N (a) tolerance level for the sun of all DDT-like pesticides (b) tolerance level for the sun of both heptachlor and heptachlor epoxide (c) tolerance levels according to the German Futtermittelverordnung (Jun.76)

SUBREPORT P 0500/3068 IJK3 (R) B7 7226 AC PAGE 4-33 cyclohexane, no. 2817, Florisil, 60 to 100 mesh, no. 12518, E. Merck, D-6100 Darmstadt I argon/methane (95/5), nitrogen, hydrogen, air (synthetic), Linde AG, D-5000 Koln 50 Procedure: Gas chromatography extraction of 5 g ground diet or 5 g bed~ding material mixed with 5 g Florisil (50 ml water/kg) and filled over another 5 g Florisil (50 ml water/kg) in a modified soxleth extractor with approx. 80 ml cyclohexane for 4 h, concentration of extract to 5 ml with a rotary evaporator andinjection of 11 ul of the concentrated extract into the gas chromatograph Organochlorine pesticides and PCB Column: 4 m x 2 mm inner diameter, glass Column packing: 15 g/kg SP-2250 plus 19.5 g/kg SP-2401 on Supelcoport, 100 to 120 mesh Carrier gas and flow rate: argon/methane, 35 ml/min Oven temperature: 215 degrees centigrade Injection port N temperature: 250 degrees centigrade O Detector temperature: 300 degrees centigrade ~ ~ N

SUttxEa'Uh'1' P USUU/3UbN IJKa (R) F3li' 6153 AC PAGE 4-34 Organophosphorus pesticid'es Column: 2 m x 2 mm inner diameter, glass Column packing: 100 g/kq SP-2100 on Supelcoport, 100 to 120 mesh Carrier gas and flow rate: nitrogen, 40 m1/min Oven temperature: temperature programm: 2 min 135 degrees centigrade, rate 4 degrees centigrade/min to 240 degrees centigrade, 8 min 240 deqrees centigrade Injection port temperature: 200 degrees centigrade Detector temperature: 200 decrees centiqrade Computation:: identification of pesticides by comFarison of standard chromatogram with sample chromatogram and cal- culation of amounts of identified pesticides with peak heights detection limit: see AC TABLE D Scientific version: SOP AC 6/2 Text version: 25.Oct.85 4.3.2 Ueterminatioii-of_aflatoxins_and-heavy_metal-, ------------- -- ---------- --- Time: each batch of diet after delivery and before usage Sample material and quantity: autoclaved diet, approx. 5 kg ~. 0 Type of aflatoxins and heavy ~ metals: see AC TABLE E' 0 Ln 06 Performance: Landwirtschaftliche Untersuchungs- und' -Ph Forschunqsanstalt Kiel, D-2300 Kiel 2 ~ Scientific version: SOP AC 7/1 Text version: 25.Oct.85 1,85

SUBREPORT P 0500/3068 IJK3 (R) B9 7103 AC PAGE 4-35 SUBSTANCE DETECTION LIMIT (mg/kg') TOLERANCE LEVEL (mg/kg) aflatoxin B1 0.0025 0.01 af latoxin B2 0.0025 - aflatoxin G1 0.0025 - aflatoxin G2 0.0025 - arsenic 0.2 2.0 cadmium 0.02' 0.5 lead 0.2 5.0 mercury 0.01 0.1 AC TABLE E RESIDUE ANALYSIS OF AFLATOXINS AND HEAVY METALS, DETECTION LIMIT AND TOLERANCE LEVEL (a) (a) tolerance levels except cadmium according to the German Futtermittelverordnung (Jun.76), tolerance level of cadmium according to Rossbach, W. (Hrsg.): Einsatz von Futter- und Einstreumitteln bei nichtklinischen Laboruntersuch~ungen, in: Veroffentlichung der Gesellschaft fur Versuchstierkunde, Nr. 9, Basel: 1980

SUBREPORT P 050'0/3-068 IJK3 (R) B11 7096 AC PAGE 5-1 5 RESULTS maa=aam 5.1 Text 5.1.1 Qondensate_analysis 5.1.1.1, Condensate yield (see AC TABLE 12 and AC FIGURES 1 to 4) In this study 176 batches of MWSC-I and 175 batches of SWSC-I were prepared from approx. 320000 cigarettes standard reference cigarette 2R1. Both kinds of condensate were prepared simulta- neously. The yield of the crude MWSC-I was 48.5 milligrams per cigarette (N: 176, RSD: 5.4 percent) and of the processed MWSC-I 39.6 milli- grams per cigarette (N: 176, RSD: 4.7 percent). The yield of the crude SWSC-I was 22.1 milligrams per cigarette (N: 175, RSD: 8.3 percent) and of the processed SWSC-I 19.4 milligrams per cigarette (N: 175, RSD: 6.1 percent). The processed condensate yield was nearly twice as high for MS as for SS. For MS a comparison can be made to PM data (a) on the standard reference cigarette 2R1. The crude condensate yield was nearly identical with the TPM yield of 48.4 milligrams per cigarette given by PM. Whereas the processed condensate yield was with 39.66 milligrams per cigarette lower than the DPM yield of 43.5 milli- grams per cigarette given by PM. This may be due to the fact that the drying process does not'remove only water but also other compounds to an extent of about 8percent of the crude condensate. (a) see CHARACTERISTICS OF EXPERIMENTAL CIGARETTES

SUBREPORT P 0500/3068 IJK3 (R) B26 6162' AC PAGE 5-2 LIST NO.: DATE : Ana-trccA 4..a"1.. 43 J«NN[A/y Te t SMOKE Units 2-01, -A 2Rri T: I i Puf f count number I ~A b. 3~ ± TPM mg/cig DPM mg/cig Tar mg/cig - o. 2 21. S - ' ! Water in TPM * mg/cig ! 3.3 ~ Smoke nicotine mg/cig. 3.32,_ Phsnols &g/cig 298 0A rg/cig 3ST ' Total aldehydes mg/cig 3.13 a.OQ• CO mg/cig ZS. S/ 2.2.3 ~ NO . mg/c ig 0.33 .3 9- ~ IsH ,41 ZSH 3u Gg . .• . 38- . • • . ' • ~ - FILLER .Tobacco -- - Clrilibriun: moisture S ~2, 6 'Ji1.'9 - ' Tob, weight e:t 12% MC mg/cig: ~1p $1V • .ii~_ - Filler density at - - ' • 12$MC - ' mg/m-l• 2S6 2S/I? - Total alkaloids -A ~O - Reducing sugars . Q. TL Nitrate lt ' 0.89 0.9~• . . Ammonia , , $ Q. (3 e. ~.f , - ~ • ' • " --- ----- ~ CODE : KeN T 4 C Ic y CHARACTERISTICS OF EXPERIMENTAL CIGARETTES (I)! REMARKS : Kef e re"ce Gs a.vt* eS

SUBREPORT P 0500/3068 IJK3 (R) B22 7096 AC PAGE 5-3 5.1.1.2 Water The water concentration was determined in the crude conden- sate suspension for characterization of the condensate preparation and in the processed condensate suspension to allow subsequent water adjustment to 25 grams/liter. In the crude condensate suspension it was performed since Apr.83, in the processed con- densate suspension during the whole study. The water concentration in the crude condensate suspension was with 42.4 grams/liter (N: 117, RSD 19.7 percent) for MS and 29.3 grams/liter (N: 115, RSD: 44.9 percent) for SS too high for skin painting application (see AC TABLE: 12 and AC FIGURES 5 and 6). Therefore the condensate was processed. In the processed condensate suspension the water concentration was 2.8 grams/liter (N: 174, RSD: 54.9 percent) for MS and 4.1 grams/liter (N: 174, RSD: 50.5 percent) for SS (see AC TABLE 12 and AC FIGURES 7 and 8). The high relative standard deviation showed that in spite of continuous vapor pressure control the drying process was difficult to perform regularly. The exact volume of the crude condensate suspension was not measured. Therefore dry condensate yield, calculated as crude condensate minus water, water concentration in crude conden- sate and yield of water per cigarette could not be determined. For processed condensate the water concentration in the condensate and the water yield were not of interest. .1.1.3 Hydrogen-ion concentration N C N ~ © C~'1 The hydrogen-ion concentration was determined in the application solutions of the highest concentration and was expressed by its N ~ negative logarithm (pH value). N ~~AS

SUBREPORT P 0500/3068 IJK3 (R) B13 7096 AC PAGE 5-4 The pH value in the application solution was 6.0 (N: 175, RSD: 1.6 percent) for MS and 6.5 (N: 175, RSD: 1.3 percent) for SS (see AC TABLES 13 and 14). It has to be mentioned that the value was not measured in an aqueous medium but in acetone. That means, that both application solutions were slightly acidic, the MS application solution a little more than the SS application solution. 5.1.1.4 Nicotine Nicotine was determined in the application solution of the highest concentration. The nicotine concentration in the solution was 20.8 grams/ liter (N: 174, RSD: 5.9 percent) for MS and 25.1 grams/liter (N: 174, RSD: 8.3' percent), for SS (see AC TABLES 13 and 14 and AC FIGURES 9 and 10). That means that the nicotine concentration in the MS application solution was about 20 percent lower than in the SS application solution. From the nicotine concentration in the application solution the nicotine concentration in processed condensate (see AC TABLES 15 and 16) and the nicotine yield per cigarette (see AC TABLES 17 and 18) were derived. For the calculation of the nicotine yield the mean processed condensate yield was taken as a basis. The nicotine concentration in processed condensate was 86.7 grams/kilogram for MWSC-I and 104.6 grams/kilogram for SWSC-I. The nicotine yield was

INDEX REPORT P 0500/3068 BRA95DIVA27 CONTENTS INTEGRATING REPORT: SUMMARY Conclusions INTRODUCTTON. TABLE C: GROUPS AND DOSES FIGURE B: CHRONOLOGY SUBREPORT ANALYTICAL CHEMISTRY Tbv{-SeizuN.0 IfDN 'f 93 SUBREPORT ANIMAL TREATMENT SUBREPORT'MICROBIOLOGY SUBREPORT PATHOLOGY INDEX CODE A4 F9 ~o~rlsef~~ J~ 6'~"4a5

.~I . ~ ... ............. ....... ,~ ...~ ,..~ VI .. . . .. SDBREPORT P 0500/3068 IJK3 (R) B12 7103 AC PAGE 5-5 3.43 and 2.02 milligrams per cigarette for MWSC-I and SWSC-I. For comparison, the MS smoke nicotine yield given by PK (a) is 3.3 milligrams per cigarette. 5.1.1.5 Catechol Catechol was determined in the application solution of the highest concentration. The determination was only performed since Aug.83, when the method had been developed and established. The catechol concentration in the solution was 1.21 grams/ liter W: 61, RSD: 6.0) for MS and 1.10 grams/liter (N: 61, RSD: 6.3) for SS (see AC TABLES 13 and 14 and AC FIGURES 11 and 12). Thus the catechol concentration in MS and SS application solutions was nearly identical. From the catechol concentration in the application solution the catechol concentration in processed condensate (see AC TABLES 15 and 16) and the catechol yield per cigarette (see AC TABLES 17 and 18) was derived. For the calculation of' the catechol yield the mean processed condensate yield was taken as a basis. The catechol concentration in processed condensate was 5.04 grams/kilogram for MWSC-I and 4.58 grams/kilogram for SWSC-I. The catechol yield was 0.200 and 0.089 milligrams per cigarette for MWSC-I and SWSC-I. 5.1.1.6 Nitrosamines Nitrosamines were determined in nearly every second batch of processed condensate since May 83, when the method had been established at INBIFO. (a) see CHARACTERISTICS OF EXPERIMENTAL CIGARETTES

SUBREPORT P' 0500/3068 IJK3 (R) B14 7103 AC PAGE 5-6 The following nitrosamines were under investigation: N-nitrosonornicotine (NNN), NLnitrosoanatabine (NATB), 4-(N-methyl-N-nitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanbasine (NAB), N-nitrosopyrrolidine (NPY), N-nitrosodimethylamine (DMNA) NNN, NATB, NNK and NAB are tobacco specific nitrosamines, whereas NPY and DMNA are generally found volatile nitrosamines. In processed MWSC-I the main~ components of the nitrosamines were NNN, NATB and NNK with 8.1 (NI: 46, RSD: 13.2 percent), 7.0 (N: 31, RSD: 18.1 percent) and 6.3 milligrams/kilogram (N: 46, RSD: 32.3 percent) respectively. The concentrations of NAB, NPY and DMNA were 1.9 (N: 31, RSD: 22.0 percent), 0.29 (N: 46, RSD: 16.4 percent) and 0.06 milligrams/kilogram (N: 46, RSD: 27.3 percent) respectively (see AC TABLE 15 and'AC FIGURES 13 and 15). In processed SWSC-I the main component of the nitrosamines was NNK with 25.5 milligrams/kilogram (N: 44, RSD: 18.1 percent), which was nearly the 4-fold of the concentration in MWSC-I. The components NNN, NATB and NAB were found in similar amounts as in MWSC-I with 7.2 (N: 44, RSD: 14.4 percent), 3.3 (N: 30, RSD: 14.7 percent) and 1.6 milligrams/kilogram (N: 30, RSD: 22.3 percent) respectively. The concentrations of NPY and DMNA were with 1.34 (N: 45, RSD: 38.6 percent) and 0.33 milligrams/kilogram (N: 45, RSD: 90.0 percent) nearly 5-fold higher than in MWSC-I (see AC TABLE 16 and AC FIGURES 14 and 16). Comparing the sum of all nitrosamines determined, it was nearly twice as high for SS as for MS. From the nitrosamine concentration in the condensate the concen- tration in the application solution (see AC TABLES 13 and 14)

SUBREPORT P 0500/3068 IJK3 (R) B23 7117 AC PAGE 5-7 and the yield per cigarette (see AC TABLES 17 and~ 18) were de- rived. For the calculation of the yield the mean processed con- densate yield was taken as a basis. A comparison between MS and SS for the concentration in the application solution shows the same as the concentration in the condensate, whereas for the yields per cigarette it has to be taken into account that the condensate yield was 2 times higher for MS than for SS. The yields of the nitrosamines found in this study for MWSC-I of the standard reference cigarette 2R1 were all within the wide range given in literature for tobacco smoke (see AC TABLE 19). Whereas for SS the yields were partly lower than the few data reported in literature. The greatest difference was found for DMNA which was lower by a factor of 0.01 to 0.04 (see AC TABLE 19). This was supposed to be due to evaporation during the proces- sing of the condensate. 5.1i.1.7 PAH The substances with the highest carcinogenic activity on mouse skin are the benzofluoranthenes, benzo(a)pyrene, 5-methylchrysene and dibenz(a,h)anthracene (a). These substances, with the excep- tion of 5-methylchrysene which is only very low concentrated in cigarette condensate, and 11 other PAH were determined in 36 batches of processed MWSC-I and 24 batches of processed SWSC-I. The concentrations of benzo(b)!fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene and dibenz(-)antracenes in processed condensate were 0.47 (N: 34, RSD: 16,2 percent), 0.26 (N: 23, RSD: 31.6 percent), 0.77 (N: 32, RSD: 16.9 percent) and 0.06 milligrams/ kilogram (N: 34, RSD: 105.3 percent) for MWSC-i and 3.4 (N: 21, RSD: 34.3 percent), 2.5 (N: 23, RSD: 63.7 percent), 5.2 (N: 24, (a) Hoffmann et al. (1978) ~?r'

SUBREPORT P' 0500/3068 IJK3 (R) B24 7125 AC PAGE 5-8 RSD: 17.4 percent) and 01.3 milligrams/kilogram (N: 20, RSD: 601.9 percent) for SWSC-I (see AC TABLES 15 and 16 and AC FIGURES 17 to 20). That means that the concentrations of PAH of the highest activity as well as the sum of all PAH were 5- to 10-fold higher in SWSC-I than MWSC-I. From the PAH concentration in the condensate the concentration in the application solution (see AC TABLES 13 and 14) and the yield per cigarette (see AC TABLES 17 and 18) were derived. For the calculation of the yield the mean processed condensate yield was taken as a basis. A comparison between MS' and SS for the concen- tration in the application solution shows the same as the concen- tration in the condensate, whereas for the yields per cigarette it has to be taken into account that the condensate yield is 2 times higher for MS than for SS. In literature for most of the PAH in MS but only for 2 in SS enough data are available to give a range for the yields. For these components the yields determined in this study were within the range with the exception of perylen in MWSC-I, which was higher (F = 12 to 19) (see AC TABLE 20). For the other components there were differences up to a factor of 8 which was smaller than the factors of the ranges (up to 39). 5.1.1.8 Volatile components The volatile components in condensate were characterized by headspace chromatography (see AC FIGURES 21 to 24). For MS the number and the concentration of the volatile components were much higher than for SS in the crude and in the processed condensate. By processing of the condensate the volatile components are markedly reduced for MS and slightly for SS. As the components were not identified no evaluation of these findings can be given.

SUBREPORT P 0500/3068 IJK3 (R) B25 7289 AC PAGE 5-9 5.1.2 Evaluatiion_of_smoke_condensate_preparation ---------- -- ----- The preparation of MWSC-I can be checked by the comparison with data provided by the client for crude condensate, nicotine and DPM. The crude condensate and nicotine yields were the same showing that smoke generation and collection was correctly performed. The processed condensate yield was lower (F = 0.9) than the DPM yield. As DPM is calculated as total particulate matter (TPM) minus water and TPM is comparable to crude condensate, this difference may indicate that processing does not only remove water but also other components. For SS condensate preparation no data for comparison are available as there are no generally accepted standard for SS generation and condensate collection. The reproducibility of the condensate preparation was very good with a relative standard deviation (RSD) of approx. 5 percent. For processed MWSC-I the RSD was 4.7 percent, for SWSC-I it was 6.1 percent. For crude the RSD was somewhat higher with 5.4 percent and 8.3 percent respectively, mainly because of the variations in water concentration. Nicotine as a major component, showed also comparable RSD, 5.9 percent for MWSC-I and 8.3 percent for SWSC-I. For none of the parameters a tendency can be seen over the time of approx. 80 weeks (see AC FIGURES 1 to 20). 5.1.3 Storaqe of_the_apPlication_solution The storage time of the application solutions was between 14 and 29 days with a mean value of 20 days. There was no analysis

SUBttEPUk'1' P 0500/3068 MN065KB3U 7~25 ~ AC A E - performed after storage. Therefore no information on chemical changes dependent on storage time and conditions are available. Concerning biological activity the tumorigenicity of MWSC-I stored at minus 75 degrees centigrade before application was higher (numerically) than MWSC-I stored at 4 degrees centigrade, however, statistically not significant (see SUBREPORT PY). 5.1.4 Residue_analysis All batches of diet and bedding material were checked for pesti- cides, PCB, aflatoxins and heavy metals. Batches with concentra- tions higher than the tolerance levels (a) were not accepted for use. In general diet and bedding material were of good quality with concentrations below the detection limits. (a) tolerance levels according to the German Futtermittelver- ordnung (Jun.76) and to Rossbach (1980), see AC PAGE 4-32 and 4-35 12A5

INBIF© Institut fur biologische Forschung HGOln SUBREPORT P 0500/3'068 MN065RB1 6171 5.2 Tables and Figures BATC'H' WEEK CONDENSATE YIELD WATER CONCENTRATION NO OF' . STUDY C'RUDE' PROCESSED COND. COND. (mg/cig.) (mg/cig.) CRUDE PROCESSED COND. COND. (g/1) (g/1) 50 -2 47.89 39.61 - 5.51 51 -2 48.69 40.62 - 3.45 52 -2 48.12 40.09 - 5.11 53 -2 46.86 39.48 - 5.13 54 -1 46.48 38.70 - 2'.24 55 -1 48.08 40.01 - 3.37 56 -li 44.77 38.35 - 6.64 58 1i 43.58 37.30 - 3.31 59' 1 44.37 37.67 - 1.60 60 1 44.74 38.06 - 4.06 61 1 47.08 38.99 - 1.19 62 2 48.98 41.03 - 4.82 63 2 47.32 38.96 - 3.98' 64 2 47.35 39.75 - 3.42' 65 2' 46.33 39.16 - 2.8'S 66 3 44.22 38.13 - 7.19 67 3 43.89 37.30 - 5.91 68 4 44.53 38.60 - 1.18 69 4 42.41 36.23 - 4.31. 71 5 45.13 38.64 - 2.83' 72 5 45.04 38.56 - 2.34 73 5 42.46 36.55 - 2.48 74 5 46.25 40.20 - 2.86 75 6 45.54 39.27 - 3.20 76 6 42.19 36.65 - 1.60 77 6 47.92 40.76 - 3.68 78 6 45.95 3'9.2b - 4.09 79 7 47.57 40.91 - 5.40 80 7 46.26 39.99 - 5.12 81 7 46.32 39.73 - 3.30 82 7 48.35 41.64 - 4.41 83 8 48.35 41.73 - 3.30 ~ 84 8 45.46 39.14 - 2.21 0 85 8 45.51 38.84 - 1.67 ~ - 0 U1 AC TABLE 1 ~ ~ MWSC-I YIELDS AND WATER CONCENTRATION IN THE MWSC-I SUSPENSIONS, ~ SINGLE VALUES 3285

INBIFO Institut fur biologische Forschung • Mn SUBREPORT P 0500/3068 MNO65RB2 6157 BATCH NO WEEK OF CONDENSATE YIELD WATER CONCENTRATION . STUDY CRUDE COND. (mg/cig.) PROCESSED COND. (mg/cig.) CRUDE COND. (g/1) PROCESSED COND. (g/l) 86 9 47.09 40.53 - 4.56 87 9 45.56 39.94 - 5.38 88 9 48.18 40.42 - 2. 15 89 10` 47.29 40.46 - 2.36 90 10 46.78 39.81 - 5.49 91 10 46.65 38.98 - 1.98 92 10 48.33 41.19 - 4.43 93 11 49.77 41.19 - 1.58 94 11 48.52 40.00 - 3.80 95 12 52.64 42.47 - 4.59 96 12 51.85 41.84 - 8.32 97 12 50.51 42.04 - 6.14 98 13 46.36 37.82 - 5.89 99 13 50.56 41.61 - 5.31 100 14 49.17 39.41 - 4.29 101 14 52.34 42.24 - 1.64 102 15 51.04 41.50 - 1.78 105 15 50.01 39.76 - 1.79 106 16 52.44 43.62 - 3'.20 107 16 48.49 40.54 - 3.38 108 17 53.30 43.69 - 2.49 109 17 48.48 41.00 - 4.67 110 17 51.36 42.64 - 4.00 111 17 53.14 42.52 - 5.01 112 18 48.93 40.59 - 3.115 116 19' 47.74 38.72 31.20 3.85 117 20 51.72 40.77 48.92 3.97 118 20 49.32 39.89 40.00 2.26 119 21 49.78 40.25 39.30 1.94 12& 22 51.12 40.67 49.90 2.59 121 22 51.16 45.56 50.60 6.38 122 23 48.47 39.60 32.40 2.73 124 23 46.67 37.70 50.50 3.61 125 23 49.15 38.96 44.10, 1.60 126 24 48.68 38.43 46.401 1.45 127 24 47.05 37.73 49.70 3. 32 AC TABLE 1 (continued) MWSC-I YIELDS AND WATER CONCENTRATION IN THE MWSC-I SUSPENSIONS, SINGLE VALUES 3285

INBIFO Institut fur biologische Forschung • Kdln SUBREPORT P 0500/3068 MNO65RB3 6157 . BATCH NO WEEK OF CONDENSATE YIELD WATER CONCENTRATION . STUDY CRUDE! COND. (mg/cig.) PROCESSED COND. (mg/cig.) CRUDE COND. (g/1) PROCESSED COND. (g/l) 128 25 46.73' 36.96 64.50 2.85 129 25 44.90 35.45 31.40 1.88 130 26 46.67 37.48 49.20 2.72 131 26 48.42' 39.21 40.30 2.78 132 27 43.18 39.12 45.60 4.13 133 27 49.73 40.13 53.70 4.15 134 28 49.35 38.78 50.60 1.41 135 28 47.12 38.75 40.60 3.26 136 28 48.19 3'8.78 43.00 1.16 137 29 48.86 40.01 43.90 1.71 138 29 49.32 38.97 46.10 1.47 139 30 50.09 40.08 50.70 4.32 140 30 48.73 38.55 43.10 1.42 141 31 47.67 38.69 23.20 2.76 142 31 44.31 35.18 - 1.79 143 32 50.36 40.20 51.00 3.66 144 32 51.81 41.02 47.50 1.70 145 33' 49.53 39'.63 44.50 1.86 1146 33 49.88 39'.87 43.60 1'.24 147 34 50.88 40.03 55.90 2.96 150 35 48.87 38.25 63.30 4.74 151 35 49.10 39.31 49.30 4.08 153 36 49.72 38.98 53.00 4.96 154 36 54.12 40.06 38.70 6.88 156 37 49.65 38.33 47.10 3.93 157 37 50.27 40.08 53.30 4.90 160 38 47.64 38.47 39.20 3.59 161 38 49.88 40.23' 65.80 4.31 162 39 47.33 37.02 46.50 1.31 163' 39 45.19 36.24 45.00 1.72 164 40 46.30 36.81 45.20 4.35 165 40 48.92 39.43 52.00 5.69 166 41 45.76 36.81 45.30 2.90 167 41 49.77 36.35 45.60 1.18 168 41 44.86 35.53 45.50 2.87 169 41 49.73 38.00 49.40 2.53 AC TABLE 1 (continued) MWSC-I YIELDS AND WATER CONCENTRATION IN THE MWSC-I SUSPENSIONS, SINGLE VALUES 3285.

INBIFO Institut fur biologische Forschung • KbIn SUBREPORT •P' 0500/3068 MNO65R'B4 6157 BATCH NO WEEK OF CONDENSATE YIELD WATER CONCENTRATION . STUDY CRUDE COND. (mg/cig.) PROCESSED COND. (mg/cig.) CRUDE COND. (g/1) PROCESSED COND. (g/1) 170 42 47.93 36.52' 49.50 4.25 171 42 46.82 37.06 54.001 3.07 172' 43 46.711 37.64 46.50 3.10 173' 43 51.48 41.04 46.70 2.74 174 44 53.94 43.19 42.90 2.19 175 44 54.78 43.86 48.30 2.69' 176 45 53.64 42.46 43.50 1.16 177 45 54.54 42.45 43.80 1.24 178 46 51.34 41.83 46.80 4.56 179 46 53.14 41.77 37.20 1.03 18'0 47 50.29 39.31 46.70 1.82 181 47 48.30' 38.38 47.00 1.42 182 48 48.15 38.26 42.80 2.07 183 48 47.65 37.60 47.70 1.97 184 49 47.03 37.92 43.00 1.54 185 49 49.39 39.06 401..00 1.36 186 50 48.64 40.20 40.60 1.75 187 50 48.21 39.09 43.00 1.41 188 51 50.33 40.45 30.00 1.58 189 51 44.15 36.56 41.00 1.53 190 52 49.23 39'.60 33.80 1.30 191 52 43.52 35.85 38.30 1.13 192 53 50.41 40.40 44.80 0.71 193 53 51.10 41.10 32.10 11.35 194 54 47.67 38.35 50.60 1.14 195 54 45.65 37.38 34.90 1.16 196 55 45.81 37.66 31.80 1.07 197 55 47.13 38.03 43.50 1.42 198' 56 45.86 37.26 38.70 1.03 199 56 46.93 38.42 43.60 0.88 200 57 50.64 40.81 48.60 11.35 201 57 45.84 37.511 53.70 1.11. 202 58 - 37.33 45.90 1.48 203 58 50.59 40.40 37.3'0, 1.20 204 59 46.38 38.14 46.40 2.08 205 59 49.29 40.10 31.90 1.42 206 60 50.20 40.53 43.50 1.27 207 60 50.50 40.76 39.90 - 1.36 AC TABLE 1 (continued). MWSC-I YIELDS AND WATER CONGENTRATION IN THE MWSC-I SUSPENSIONS, SINGLE VALUES 3285

INBIFO Institut fiir hiologische Forschung • KtSln SUBREPORT P 0500/3068 MNO65RB5 6157 BATCH' WEEK CONDENSATE YIELD WATER CONCENTRATION NO OF . STUDY CRUDE' COND. (mg/cig.) PROCESSED COND. (mg/cig.) CRUDE COND. (g/1) PROCESSED COND. (g/1) 208 61 51.09 41.53 42.90 1.94 209 61 47.01 38.56 31.80 1.03 210 62 48.41 39.12 38.50 1.32 211 62 49.63 39.95 32.50 1.41 212 63 49.67 40.74 30.50 3.03 213 63 49.38 39.96 36.10 1.03 214 64 50.14 41.61 24.00 3.04' 215 64 49.11 40.13' 38.60 2.96 216 65 47.20 38.74 46.90 0.89 217 65 5D.04 40.36 37.60 1.25 218 66 50.56 41.66 40.70 0.99 219' 67 48.48 40.53 34.20 2.27 220, 68 45.62 38.22 29.80 2.56 221 68 49.07 40.29 32.50 2.63 222 69' 51.62 41.27 37.40 2.07 223 69 47.16 38.45 35.00 2.37 224 70 45.06 36.96 32.30 3.79 225 70 46.94 38.75 30.30 2.75 226 71 47.64 38.74 39.50 0.61' 227 71 50.20 40.84 40.80 3.74 228 73 46.85 37.54 40.30 2.12 229 73 49.76 40.06 41.30 2.05 230 73 48.86 38.88 42.30 1.85 231 73 49.53 38.17 41.00 1.97 232 74 47.37 38.37 44.8'0 3.29 233 74 51.79 41.33 46.20 1.75 234 75 54.00 42.69 42.10 1.46 235 75 53.60 42.86 43.70 1.28 236 76 48.87 40.37 34.20 1.65 237 76 53.26 42.66 42.50 1.17 238 77 52.38 44.75 35.20 1.17 239 77 52.14 42.38 37.30 1.88 AC TABLE 1 (continued) MWSC-I YIELDS AND WATER CONCENTRATION IN THE MWSC-I SUSPENSIONS, SINGLE VALUES ~a. ,0 3285

INBIFO Institut fur biologische Forschung • K6In SUB'REPORT,P 0500/3068 MNO65RB6 6157 BATCH NO WEEK OF' CONDENSATE YIELD WATER CONCENTRATION .. STUDY CRUDE! COND. (mg/cig.) PROCESSED COND. (mg/cig.) CRUDE COND. (g/1) PROCESSED COND. (g/1) 50 -2 27.40 21.13 - 5.25 51 -2 23.41 21.06 - 1.88 52 -2 24.13 21.18 - 4.87 53 -2 22.64 20.23 - 2.83 54 -1 22.07 19.78 - 2.76 55 -1 21.87 20.82 - 4.66 56 -1 23.09 19.74 - 2.97 58 1 20.70 19.72 - 3.25 59 1 18.42 17.09 - 2.26 60 1 19.03 17.19 - 1.96 61 1 18.80 17.38 - 1.03 62 2 20.76 19.56 - 3.55 63 2 18.07 17.10 - 1.62 64 2 20.24 19.08 - 1.98 65 2' 21.93 20.60 - 4.99 66 3 22.82 21.59 - 4.45 67 3 24.93 20.76 - 3.03 68 4 21.67 20.15 - 6.59 69 4 23.40 19.82 - 1.21 71 5 20.09 18.58 - 2.10 72 5 19.87 18.51 - 4.92 73 5 21.67 19.08' - 2.69 74 5 21.18 19.63' - 4.08 75 6 20.97 18.73 - 1.76 76 6 18.04 17.21 - 3.17 77 6 20.66 18.63 - 7.43 78 6 20.67' 18.68 - 7.14 79 7 18.9'1 17.48 - 6.73 80 7 21.96 20.05 - 6.73 81 7 19.71 18.35 - 2.55 82 7 20.75 17.59 - 2.00 83 8 21.57 20.02 - 2.33 84 8 21.00 18.96 - 2.29 85 8 21.30 20.21 - 2.88 AC TABLE 2 SWSC-I YIELDS AND WATER CONCENTRATION IN THE SWSC-I SUSPENSIONS, SINGLE VALUES 3285

INBIFO Institut flur biologische Forschung • K6Ib SUBREPORT P 0500/3068 MNO65RB7 6157 BATCH NO WEEK OF' CONDENSATE YIELD WATER CONCENTRATION . STUDY CRUDE' COND. (mg/cig.) PROCESSED COND. (mg/cig.) CRUDE COND. (g/1) PROCESSED COND. (g/1) 86 9 21.35 20.07 - 2.15 87 9 21.06 19.76 - 3.37 88 9 21.12 19.63 - 2.68 89 10 19.60 18.80 - 2.54 90 10 21.01 19.52 - 5.62 91 10 20.05 19.34 - 2.95 92 10 21.75 20.3'1. - 3.64 93 11 21.63 20.61 - 5.62 94 11 19.89 18.85 - 2.87 95 12 20.70 19.47 - 5.07 96 12 22.46 20.09 - 10.81 97 12 22.03 18.95 - 2.72 98 13 21.24 16.67 - 6.28 99 13 23.27 20.58~ - 7.19 100 14 22.44 18.99 - 1.38' 101 14 22.56 18.24 - 3.00 102 15 20.21' 17.99 - 5.07 105 15 24.16 20.64 - 7.71 106 16 22.82 21.04 - 6.96 107 16 23.88 20.44 - 1.56 108 17 22.62 19.43 - 7.79 109 17 19.69 17.40 - 5.50 110 17 21.77 18.83 - 3.83 111 17 22.84 19.58 - 4.04 112 18' 21.41 19.36 - 3.75 116 19 23.92 1!8.68 44.80 6.13 117 20, 21.98 20.18 16.14 5.12 118 20 22.17 20.08 19.50 5.36 119 21 22.48 19.66 28.90 3.01 120 22 21.15 19.43 22.60 4.91 121 22 21.90 21.97 22.80 2.43' 122 23 24.26 20.03 28.10 2.12 124 23 21.22 18.97 35.10 7.05 125 23 21.56 18.59 28.90 2.09 AC TABLE 2 (continued) SWSC-I YIELDS'AND WATER CONCENTRATION IN THE SWSC-I SUSPENSIONS, SINGLE VALUES 3285

IINBIFO1 Institut fur biolog,ische Forschuing • K6In SUBREPORT P 0500/3068 MNO65RB8 6157 ,. BATCH NO WEEK O CONDENSATE YIELD WATER CONCENTRATION . F STUDY CRUDE COND. (mg/cig.) PROCESSED COND. (mg/cig.) CRUDE PROCESSED COND. COND. (g/1) (g/1) 126 24 20.64 18.53 37.50 3.13 1,27 24 21.99 19.00 41.00 4.26 128 25 22.88 18.75 43.80 2.34 129 25 22.22 18.87 37.40 2.49 130 26 22.20 19.07 35.90 3.49 131 26 21.79 19.10 36.10 7.39 132 27 21.55 18.77 38.90 4.25 133 27 22.68 20.27 39.10 7.39 134 28 23.16 19.61 37.90 3.35 135 28 21.38 21.14 21.80 5.21 136 28 . 24.03 19.54 45.40 7.68 137 29 25.09 20.47 58.60 7.23 138 29 24.55 19.90 50.10 3.29 139' 30 28.79 19.00 95.60 8.22 140 30 24.69 20.30 33.70 5.31 141 3'1 20.92 18.69 30.10 7.45 142 31 26.93 20.3'6 67.206.69' 143 32' 22.01 18.95 36.80 8.74 144 32 27.91 23.32 47.50 7.42 145 33 22.52 20.20 23.10 5.72 146 33 21.47 19.72 26.80 6.71 147 34 22.35 19.06 35.30 6.61 150 35 22.14 18.50 35.70 8.45 151 35 25.09 20.41 59.90 8.14 153 36 23.79 19.21 44.30 9.18 154 36 25.44 20.78 44.70 7.21 156 37 25.55 19'.85 49.90 9.12 157 37 20.33 17.85 21.90 5.89 160 38 24.25 20.45 32.00 7.42 161 38 20.75 19.50 26.10 4.88 162 39 22.56 18.50 38.40 8.12 163 39 20.36 18.18 27.70 4.86 164 40 22.27 17.65 45.20 7.99 165 40 19.43 21.11 17.30 2.98 166 41 20.93 18.16 21.60 2.76 167 41 22.26 18.48 34.40 2.99 AC TABLE 2 (continued) SWSC-I YIELDS AND WATER CONCENTRATION IN THE SWSC-I SUSPENSIONS, SINGLE VALUES 3285

INBIFO Institut fur biologische Forschung • MIn SUBREPORT P 0500/3068 MNO65RB9 6157 V BATCH NO WEEK OF CONDENSATE YIELD WATER CONCENTRATION . STUDY CRUDE COND. (mg/cig.) PROCESSED COND. (mg/cig.) CRUDE COND. (9/1) PROCESSED COND. (9/1) 170 42 20.85 17.01 27.90 3.18 171 42 20.61 16.55 51.70 4.08 172 43 19.48 16.99 31.30 3.62 173 43 23.99 20.87 39.30 5.82 174 44 23.59 19.39' 36.50 6.63 175 44 24.72 20.22 46.40 4.66 176 45 21.58 19.72 16.90 4.611 177 45 22.62 18.43 3'6.90 2.63 178 46 21.90 19.27 26.60 3.04 179' 46 25.22 18.59 25.90 2.12 180 47 22.46 19.76 26.60 3.71 181 47 21.88 18.74 25.70 2.64 182 48 20.31 17.90 25.90 2.51 183 48 18.43 16.82 17.40 2.57 184 49 22.06 19.68 23.70 2.01. 185 49 20.43 18.35 19.90' 2.93 186 50 21.16 19.18 24.30 2.88 187 50 22.01' 19.09 30.30 2.38 188 51 21.83 19.41 19.20 2.26 189 51 22.18 19.83 22.20 3.00 190 52 20.03 17.59 22.30 2.55 191 52 18.62 17.26 17.70 2.74 192 53 21.21 19.17 57.90 2.53 193 53 23.08 20.71 28.50 2.25 194 54 22.33 18.83 28.80 1.36 195 54 22.21 19.25 31.40 6.59 196 55 21.55 19.66 17.40 1.59 197 55 20.53 18.50 15.30 2.82 198 56 20.81 18.68 18.90 2.78 199 56 20.94 19.39 15.50 2.72 200 57 20.79 17.57 23.20 2.88 201 57 21.94 18.44 27.80 3.34 202 58 21.95 19 . 26 23.00 2.79 203 58 22.26 19.71 23.60 3.75 204 59 21.02 18.99 20.20 2.04 205 59 22.27 20.00 19.70 2.42 206 60 22'.26 19.25 19.10 2.09 207 60 22.60 19.52' 22.20 3.62 AC TABLE 2 (continued) SWSC-I YIELDS AND WATER CONCENTRATION IN THE SWSC-I SUSPENSIONS, SINGLE VALUES 3285

INBIFO Institut tur biologische Forschung; • K6in SUBREPORT P 0500/3068: 6157 BATCH NO WEEK OF CONDENSATE YIELD WATER CONCENTRATION . STUDY CRUDE COND. ('mg/cig'•) PROCESSED COND. (mg/cig.) CRUDE COND. (g/1) PROCESSED COND. (g/1) 208 61 23.04 20.33 22.60 3.66 209 61 21.88 20.66 13.20 4.00 210 62 20.92 18.98 13.50 3.55 211 62 21.87 19.42 17.00 4.42 212 63 23.00 20.62 18.80 2.14 213 1' 3 63 22.16 19.911 19.10 2.25 214 64 21.86 19.80 14.50 4.59 215 64 21.50 20.14 17.2'0 4.55 216 65 21.02 19.11 17.80 5.23 217' 65 22.23 19.83 25.90 5.01 218' 66 22.17 19.98 17.50 1.98 219 67 21.80 20.16 9.80 2.05 220 68 20.14 18.53' 9.90 1.74 221 68' 20.05 18.59 21.20 2.41 222' 69 20.48 18.87 15.50 2.12' 223' 69 21.70 19.41 24.90 5.71 224 7-0 23.19 20.64 24.10 4.33 225 70 22.97 19.62 27.50 1.83' 226 71 19.66 17.86 12.90 0.99' 227 71 22.26 18.85 23.00 4.17 228 73 24.20 20.66 27.50 3.47 229 73 22.48 19.10 24.30 3.10 230 73 21.28 18.34 30.00 5.14 231 74 21.99 18.77 23.60 5.42 232 74 24.36 20.77 32.90 5.31 233' 74 26.06 21.53 44.40 3.54 234 75 23.41 20.63 20.80 2.34 235 75 24.92 21.13 32.10 2.49 236 76 23.12 19.46 32.30 2.44 237 76 24.10 20.40 34.70 2.10 238 77 26.61 22.21 18.10 1.60 239 77 24.97 21.44 29.80 1.85 AC TABLE 2 (continued) SWSC-I YIELDS AND WATER CONCENTRATION IN THE SWSC-I SUSPENSIONS, SINGLE VALUES 3285

INBIFO Institut fur biologische Forschung • NCSIn SUBREPORT P 0500/3068 MN065RA7 7103 BATCH NO. WEEK OF STUDY pH NICOTINE CATECHOL CONC. CONC. ( g/7-) (g/1) 50 -2 6.0 19.50 51 -2 6.0 19.73 52 -2 6.1 20.38 53 -2 6.1 20.43 54 -1 6.1 19.68 55 -1 6.1 19.88 56 -1 6.2 19.30 58 1 6.1 19.83 59 1 6.0 20.33' 6 0 1 6.0 2' 0. 0 0 61 1 6.0 19.60 62 2 6.0 20.85 63 2 6.0 20.98 64 2 5.9 20.70 65 2 6.1 20.08 66 3 6.1 1i9.55 67 3 6.0 19.50 68 4 6.1 20.35 69 4 6.0 19.95 71 5 6.1 20.1'5 72 5 6.1 20.59 73 5 6.1 21.10 74 5 6.1 20.48 75 6 5.9 20.38 76 6 5.9 21.25 7 7 6 6.0 23.48 78 6 5.9 20.68 79 7 6.0 19.85 80 7 6.1 20.38 81 7 6.0 20.53 82 7 6.0 21.08 83 8 6.0 22.88 84 8 5.9 22'.90 85 8 6.1 22.68 AC TABLE 3 pH, NICDTINE'AND CATECHOL CONCENTRATIONS IN THE MWSC-I APPLICATION SOLUTION OF TH'E HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/l 3285

INBIFO Institut fur biologische Forschung • Kt91n SUBREPORT P' 0500/3068 MNO65RA8 6157 BATCH WEEK pH NICOTINE CATECHOL NO. OF CONC. CONC. STUDY (g/1) (g/1) 86 9 6.1 21.73 87 9 5.9 21.85 88 9 6.0 21.25 8 9 10 6.1 21. 5 3 90 10 6.1 22.03 91 10 6.2 22.80 92 10 6.0 22.35 93' 11 5.9 21.75 94 11 5.9 21.45 95 12 5.9 21.05 96 12 5.8 21.55 97 12 5.9 19. 83 98 13 5.9 20.68 99 13 5.8 22.25 100 14 5.9 20.15 101 14 5.9 21!.70 102 15 5.9 19.45 105 15 5.9 19.58 106 16 6.0 20.43 107 16 6.1 21.25 108 17 6.0 20.65 109 17 5.9 20.80 110 17 6.01 21.45 111 17 6.0 21.60 112 18' 6.0 201.93 116 19' 6.0 20.58 117 20 6.0 20.33 118 20 6.1 19.48 119 21 6.1 20.43 120 22 6.3 20.13 121 22 6.0 20.50 122 23 6.1 18.45 124 23 6.1 - 125 23 6.1 18.60 AC'TABLE 3 (continued) pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE MWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/1 3285

IN!BiFO Institut fur biologische Forschung - K6In, SUBREPORT P 0500/3068 MNO65RA9 6157 I BATCH WEEK pH NICOTINE CATECHOL NO. OF CONC. CONC. STUDY (g/1) (g/1) 126 24 6.1 18.38 127 24 6.1 19.40 128 25 6.1 18.35 129 25 5.8 19.63 130 26 5.8 21.10 131 26 5.8 20.80 132 27 6.2 20.54 133 27 6.2 21.32 134 28' 6.1 22.00 135 28 6.0 20. 10 136 28 6.1 19.53 137 29 6.1 20.67 138 29 6.1 21.13 139 30 6.0 19.47 140 30 6.0 19.62 141 31 6.0 21.73 142 31 6.1 19.42' 143 32 6.0 19.1G 144 32 6.0 19.37 145 33 6.0 19.80 146 33 6.0 20.32 147 34 5.9 18.60 150 35 5.9 19.35 151 35 5.8 19.80 153 36 6.0 17.82 154 36 6.0 20.82 1:56 37 6.0 19. 57 157 37 6.0 20.45 160, 38 6.1 20.25 161 38 6.0 19.65 162 39 6.1 20.05 163 39 6.0 24.30 164 40 6.1 20.15 165 4& 6.1 20.35 AC TABLE 3 (continued ) t pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE'MWSC-I APPLICATION CA SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES ~ Remarks: application solution of the highest concentration: ~ 240 g processed condensate/l 3285

INBIFO Institut fur biologische Forschung • K6In SUBREPORT P 0500/3068 MNO65RA10 6157 BATCH NO. WEEK OF STUDY pH NICOTINE CONC. (g/1) CATECHOL CONC. (g/1)' 166 41 6.1 20.32 - 167 41 6.0 21.90 - 168 41 6.1 20.32 - 169 41 6.0 20.05 - 170 42 6.1 21.60 - 1'71' 42 6.0 22.00 - 172 43 6.0 23.58' - 173 43 5.8 24.48 - 174 44 6.1 23.73 - 175 44 6.1 24.05 - 176 45 6.0 22.22 - 177 45 5.8 20.60 - 178 46 5.9 20.45 1.15 179 46 5.9 20.60 1.18 180 47 6.0 20.70 1.18 181 47 5.9 20.33 1.19 182 48 6.0 21.53 1.22 183 48 5.9 20.98 1.22 184 49 6.0 20.92 1.21 185 49 6.1 21.05 1.12 186 50 6.1 21.72 - 187 50 6.1 21.75 1.16 188 51 6.0 19.85 1.20 189 51 5.8 21.35 1.26 190 52 5.9 20.18 1.25 191 52 5.9 20.28 1.23 192 53 5.9 17.85 1.44 193 53 6.0 19.28' 1.19 194 54 5.9 19.68 1.17 195 54 6.0 20.90 1.22 196 55 5.8 19.92 1.15 197 55 5.7 19.80 1.19 198 56 5.8 17.80 1.10 199 56 5.8 19.85 1.18 AC TABLE 3' (continued) pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE MWSC-I APPLICATION. SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/l 3285

INBIFO Institut fur biologische Forschung • K6In SUBREPORT P 0500/3068 MNO65RA11 6157 BATCH' NO. WEEK OF STUDY pH NICOTINE CONC. (g/1) CATECHOL CONC. (g/1) 200 57 6.0: 21. 10 1.35 201 57 5.9 21.15 1.21 202 58 6.0 20.68 1.26 203 58 5.9' 20.30 1.13 204 59 6.0 21.45 1.18 205 59 6.0 21.35 1.14 206 60 6.0 20.82 1.16 207 60 6.0 21.10 1.12 208 61 6.0 21.38 1.12 ' 209 61 5.9 21.48 1.12 210 62 6.0 21.48 1.16 211 62 6.0 21.68 1.15 212 63 6.1 21.98 1.24 213 63 6.1 21.12 1.28 214 64 6.1 20.98' 1.20 215 64 6.0 21.50, 1.25 216 65 6.1 20.12 1.2D 217 65 6.1, 22.90 1.23 218' 66 6.11 23.25 1.30 219 67 6.1 22.78 1.26 220 68 6.0 21.65 1.30 221 68 6.1 21.55 11.27 222 69 6.1 23.90 1.29 223 69 5.9 22.20 1.20 224 70 5.9 19.98 1.05 225 70 6.0 19.20 1.17 226 71 6.1 21.22 1.15 227 71 6.0 21.12 1.14 228 73 6.0 20.35 1.26 229 73' 6.1 21.65 1.31 230 73' 6.0 20'.88 1.27 231 73 6.1 21.82 1.31 232 74 6.0 21.65 1.25 233 74 6.1 20.98' 1.26 AC TABLE 3 (continued) pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE MWSC-I APPLICATION SOLUTION OF TH'E'HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/1 3285

INBIFO I'nstitut flur biologische Forschung • IK6ln SUBREPORT P 0500/3068 MNO65RA12 6157 BATCH NO. WEEK OF STUDY pH NICOTINE CONC. (g/1) CATECHOL CONC. (g/1) 234 75 6.0 22.48 1.15 235 75 6.0 23.60 1.28 236 76 6.0 21.08 1.19 237 76 6.0 22.40 1.33 238 77 6.0 20.98 1.27 239 77 6.2 18.65 1.06 AC TABLE 3 (continued) pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE MWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/I 3285

INBIFO Instifuf fur biologische Forschung • Kt9Vn SUBREPORT'P 0500/3068 MN065R'A13 7103 BATCH NO. WEEK OF STUDY pH NICOTINE CATECHOL CONC. CONC. (g/1)' (g/1) 50 -2 6.6 28.13 51 -2 6.5 27.75 52 -2 6.5 27.40. 53 -2 6.6 27.85 54 -1 6.5 2'5.10' 55 -1 6.5 25.08 56 -1 6.6 26.20 58 1 6.4 22.48 5 9 11 6.3 ' 21' . 9 8 60 1' 6.3 21.48 61 1 6.4 22.88 62 2 6.3 24.35 63 2 6.3 22.50 64 2 6.3 23.45 65 2 6.4 23.53 66 3 6.5 23.25 67 3 6.5 28.10 68 4 6.5 23.70 69 4 6.4 26.28 71 5 6.5 26.83 72 5 6.6 26.33 73 5 6.4 27.33 74 5 6.5 25.68 75 6 6.2 24.95 76 6 6.2 24.25 77 6 6.4 22.53 78 6 6.5 24.85 79 7 6.4 25.43 80 7 6.5 25.75 81 7 6.4 26.13 82 7 6.5 25.28 83 8 6.5 27.80 84 8 6.4 27.10 85 8 6.4 23.60 AC TABLE 4 pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed,condensate/1 3285

INBIFO Institut fur biologische Forschung • B(6in SUBREPORT P 0500/3068 MNO65RA14 6157 - ~ BATCH WEEK pH NICOTINE CATECHOL NO. OF CONC. CONC. STUDY (g/1) (9/1) 86 9 6.5 23.30 87 9 6.4 24.05 88 9 6.5 25.55 89 10 6.5 21.98 90 10 6.4 23.75 91 10 6.5 24.20 92 10 6.5 24.95 93 11 6.5' 25.55 94 11 6.4 24.55 95 12 6.4 23.80 96 12 6.4 23.70 97 12 6.5 25.25 98 13 6.6 25.70 99 13 6.5 29.25 100 1:4 6.4 27.18 101 14 6.4 26.88' 102 15 6.4 24.58'. 105 15 6.4 24.05 106 16 6.4 24.45 107 16 6.5 26.83 108 ' 17 6.4 23. 10 109' 17 6.4 23.15 110 17 6.4 28.65 111 17 6.5 25.15 112 18 6.5 25.13 116 19 6.4 23.70 117 20 6.4 22.83 118 20 6.5 23.95 119 21 6.4 23.60 120 22 6.7 23.38 121 22 6.5 23.68 122 23 6.6 27.83' 124 23 6.6 25.95 125 23 6.6 26.75 AC TABLE 4 (continued) pH, NI!COTINE'AND CATECHOL CONCENTRATIONS IN THE-SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/l 3285

INBIFO Institut fur biologische Forschung • KtSIn SUBREPORT P 0500/3068 MN065RA15 61,57 I BATCH WEEK pH NICOTINE' CATECHOL NO. OF CONC. CONC. STUDY (g/1) (g/1) 126 24 6.5 26.23 127 24 6.5 25.20. 128 25 6.6 29.05 129 25 6.3 28.58 130 26 6.4 28.55 131 26 6.3 28.30 132' 27 6.6 32.32 133 27 6.6 27.30 134 28 6.6 27.60 135 28 6.5 27.90 136 28 6.6 26.45 137 29 6.6 26.70 138 29 6.5 28.43 1'39 30 6.5 28.02 140: 30 6.5 26.20 141 311 6.5 27.25 142 311 6.6 25.60 143 32 6.5 22.35 144 32 6.5 25.07 145 33 6.5 27.35 146 33 6.5 26.62 147 34 6.5 25.82 150 35 6.3 23'.3'5 151 35 6.4 23.42 153 53 36 6.6 22.82 154 36 6.7 26.02 156 37 6.5 25.95 157 37 6.5 24.67 160 38 6.6 24.00 161 38 6.5 23.15 162' 39 6.5 24.80 163 39 6.6 28.10 164 40 6.5 27.42 165 40 6.5 20.97 AC TABLE 4 (continued) pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/1 3285

INBIFO Institut fur biologische Forschung • KOln SUBREPORT P 0500/3068 MN065RA16 6157 . BATCH NO. WEEK OF STUDY pH NICOTINE CONC. (g/1 ) CATECHOL CONC. ( 9/1 ) 166 41 6.5 24.65 - 167 41 6.5 21.78 - 1,70 42 6.6 23.90 - 171 42 6.6 23.30 - 172 43 6.5 27.70 - 173 43 6.5 29.13 - 174 44 6.5 28.98 - 175 44 6.5 26.90 - 176 45 6.4 25.77 - 177 45 6.3 26.22 - 178 46 6.5 25.017 1.15 179 46 6.4 27.42 1.08 180 47 6.5 24.75 1.11 181 47 6.4 23.38' 1.14 182 48 6.5 26..301 1.10 183' 48 6.4 22.65 1.22 184 49 6.5 25.44 1.24 185 49 6.5 25.38 1.21 186 50 6.5 26.20 - 187 50 6.5 25.75 1.09 188 51 6.5 24.30 11.09 189 51 6.4 23.38 1.20 190 52 6.4 24.52 1.16 191 52' 6.4 22.32 1.14 192 53 6.4 21.85 1.12 193 53 6.5 22.90 1.07 194 54 6.4 21.60 1.15 195 54 6.4 24.42 1.19 196 55 6.3 20.38 1.07 197 55 6.3 201.95 1. 111 198 56 6.4 23.42 1.05 1199 56 6.4 22.40 1.05 200 57 6.4 21.15 1.02 201 57 6.4 21.20 1.08 AC' TABLE'4 (continued) pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/1 3285

INBIFO Institut fuir biol'ogische Forschung • K6In SUBREPORT P 0500/3068' MNO65RA17 6157 BATCH NO. WEEK OF STUDY pH NICOTINE CONC. (g/1) CATECHOL CONC. (g/l) 202 58 6.4 20.90 0.98 203 58 6.4 21.40 1.01 204 59 6.4 24.35 1.03 205 59 6.4 24.15 1.03 206 60 6.4 25.58 1.05 207 60 6.5 25.00 0.99 208 61 6.4 26.22 1.00 209 61 6.4 25.40 0.94 210 62 6.5 23.95 1.12 211 62 6.4 24.03 1.01 212 63 6.5 24.20 1.16 213 63 6.5 24.3'8' 1.18' 214 64 6.5 24.02 1.12 215 64 6.5 23.95 1.09 216 65 6.5 21.70 1.10 217 65 6.5 23.78' 1.04 218 66 6.5 25.70 1.17 219 67 6.5 23.70 1.13 220 68 6.5 25.45 1.22 221 68 6.6 26.05 1.20 222 69 6.5 27.00 1.23 223 69 6.4 25.10 1.14 224 70 6.4 26.20 1.01 225 70 6.4 25.35 1.10 226 71 6.5 21.12 1.03 227 71 6.5 26.12' 1.15 228 73 6.4 25.20 1.12 229 73 6.5 25.28 1.11 230 73 6.4 27.20 1.03 231 74 6.5 26.60 1.09 232 74 6.4 24.75 1.01 233 74 6.5 27.38 1.14 234 75 6.5 26.35 1.10 235 75 6.5 26.38 1.15 AC TABLE 4 (continued) pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE-SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/1 3285

INBIFO Insfitut fur biblogische Forschiung • KOln SUBREPORT P 0500/3068 MN065RA18 6157 BATCH' NO. WEEK OF STUDY pH NICOTINE CONC. (g/1) CATECHOL CONC. (g/1) 236 76 6.5 26.93 1'.05 237 76 6.5 26.30 1.16 238 77 6.5 26.13 1.13 239 77 6.4 22.90 1.03 AC TABLE 4 (continued) pH, NICOTINE AND CATECHOL CONCENTRATIONS IN THE SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION, SINGLE VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/l 3285

I~NBIFO Institut fur biologische Forschung, • NCt51n SUBREPORT P 0500/3068 M06bRA1 7103 BATCH NO. WEEK OF STUDY NITROSAMINE TOBACCO SPECIFIC (mg/kg) VOLATILE (ug/kg) NNN NATB NAB NATB PLUS NAB NNK DMNA NPY 123 23 8.8 9.0 3.6 12.6 14.3 63' 353 126 24 7.8 6.2 1.8 8.0 6.0 42 285 128' 25 7.3 6.1 1.8 7.9 5.6 42' 305 130 26 7.0 5.4 1.5 6.9 5.4 46 297 132 27 7.4 6.1 1.7 7.8 5.6 62 292 134 28 8.7 7.1 1.9 9.0 6.6 43 283 139 30 8.8 7.4 1.9 9.3' 6.9 76 316 141 31 9.2 6.8 1.7 8.5 7.0 44 247 143 32 9.5 7.6 1.9 9.5 7.6 64 281 145 33 8.5 7.1 1.8 8.9 6.3 98 302 147 34 8.G 6.7 1.9 8.6 5.8 51 306 150 35 7.9 6.5 1.7 8.2 4.6 63 325 151 35 9'.1' 7.5 2.0 9.5 5.5 69 290 154 36 8.8 7.5 2.0 9.5 5.7 56 410 160 38 7.6 7.6 2.1 9.7 5.8 68 345 162 39 6.5 5.7 1.5 7.2 4.1 41 305 164 40 8.2 7.1 1.4 8.5 5.8 49 246 166 41 9.5 7.5 1.9 9.4 10.7 59 284 170 42' 6.2 5.1 1.4 6.5 3.6 39 199 AC TABLE 5 NITROSAMINE CONCENTI'RATIC,RVS IN PROCESSED MWSC-I, SINGLE VALUES 3285

INBIFO Institut fur biologische Forschuing • KtSIn SUBREPORT P 0500/3068 NN065RA2 7103 1~ BATCH NO. WEEK OF STUDY NITROSAMINE TOBACCO SPECIFIC (mg/kg) VOLATILE (u9Ag) NNN MTB NAB NATB PLUS NAB NNK uNAIA NPY 172 43 11.3 8.9 2.3' 11.2 13.2 54 249 174 44 5.9 4.6 1.4 6.0 3.7 41 277 176 45 6.3 4.9 1.4 6.3 3.7 37 241 178 46 6.1 5.1 1.5 6.6 3.9 50 284 180 47 7.3 - - 9.9 5.4 37 313 182 48 7.5 7.7 2.0 9.7 6.1 51 87 186 50, 8.7 8.4 1.7 10.1 6.2 75 302 188 51 6.5 5.3 1.4 6.7 4.3 41 263 190 52 6.6 7.6 2.1 9.7 4.9 55 299 192' 53' 8.5 - - 11.0 6.1 48 269 194 54 7.7 8.9 2.0 10.9 7.0 56 324 196 55 9.4 - - 10.2 6.9 72' 314 198 56 9.1 - - 10.0 6.0 53 300 200 57 7.7 - - 8.6 5.3 41 269 202 58 8.4 - - 8.7 5.9 40 255 204 59 8.0 - - 8.5 5.4 82 324 206 60 7.9 - - 8.3 5.5 43 305 208 61 8.3 - - 9.6 6.7 72 274 210 62 9.2 - - 7.2 7.2 67 314 AC TABLE 5 ( continued ) NITROSAMIINE CONCEMf2ATIONS IN PROCESSED MWSC-I, SINGLE VALUES 3285

INBIFO Institut fuir biologische Forschung • KOIn SUBREPORT P,0500/3068 M065RA3 7103 . BATCH NO. WEEK OF STUDY NITROSAMINE TOBACCO SPFCIFIC (mg/kg) VOLATILE (ug/kg) NNN NATB NAB NATB' PLUS NAB'. NNK DhIlJA NPY 212 63 8.7 - - 6.7 7.0 99 314 215 64 8.5 - - 8.9' 6.6 69 274 216 65 8.8 - - 8.9 7.0 71 335 218 66 7.9 - - 7.9 5.3 71 351 224 70 8.2 8.1 2.1 10.2 7.0 76 292 232 74 7.7 8.7 2.3 11.0 7.0 89 335 236 76 8.3 - - 10.4 6.8 67 302 238 77 7.5 8.0 1.8 9.8 6.8 60 368 AC TABLE 5 (continued) NITROSAMINE CONCEhIIRATIONS IN PROCESSED MWSC-I, SINGLE VALUES 3285

INBIFO I'nstitut tur biologische Forschung • K6ln SUBREPORT P 0500/3068 M065RA4 7103 RATCH NO. WEEK OF SrUDY NITROSAMINE TOBACCO SPECIFIC (mg/kg) VOLATILE (ug/kg) NNN NATB NAB NATB PLUS NAB NNK DMNA NPY 126 24 8.1 3.5 1.5 5.0 30.7 272 1215 128 25 8.5 3.3' 1.7 5.0 35.3 169 1225 130 26 8.6 3.4 1.8 5.2 32.8 235 1153 132 27 9.1 3.6 1.7 5.3 39.6 210 1375 134 28 8.1 3.3 1.7 5.0 30.2 165 1070 139' 30 6.9 2.8 1.3 4.1 25.3 147 1297 141 31 8.4 3.7 1.4 5.1 28.0 163 854 143 32 7.9 3.2' 1.5 4.7 28.8' 167 866 145 33 7.9 3.2 1.3 4.5 32.8 157 1114 147 34 7.0 3.0 1.6 4.6 27.1 171 1124 150 35 7.1 3.0 1.5 4.5 22.0 164 1474 154 36 7.5 3.4 1.6 5.0 25.6 269 1558 156 37 7.2 3.1 1.4 4.5 24.0 190 1316 160 38 8.6 3.6 1.7 5.3 28.2' 208 1022 162 39 7.1 2.8 1.5 4.3 23.9 129 853' 164 40 6.6 3.0 1.3 4.3' 20.8' 111 1053 166 41 6.4 3.0 1.2 4.2' 20.8 149 828 170 42 9.9 4.7 2.3 7.0 32.6 154 796 172 43 7.1 3.3 1.4 4.7 23.5 175 829 AC TABLE!6 NITROSANIINE CONCEN IRATIONS IN PROCESSED SWSC-I, SINGLE VALUES 3285

INBIFO Institut fur biologische Forschulng • Wn SUBREPORT P 0500/3068 M065RA5 7103 l. BATCH NO. Tn1EEK OF STUDY NITROSAMINE TOBACCO SPECIFIC (mg/kg) VOLATILE (ug/kg) NNN NATB NAB NATB PLUS NAB NNK DP'IldA NPY 174 44 - - - - - 87 793 176 45 6.8 3.9 2.2 6.1 24.1 728 2310 178 46 5.9 2.6 1.2' 3.8 20.0 91 899 180 47 6.9 3.4 1.9 5.3 26.8 498 2196 182 48 5.6 3.2 1.5 4.7 19.5 153 997 186 50 5.7 2.7 1.5 4.2 17.4 63 800 188' 51 6.9 2.9 2.7 5.6 23.4 120 946 190 52' 5.6 2.5 1.6 4.1 20.4 132 895 192 53 7.7 4.3 2.0 6.3 24.1 216 1062 194 54 7.1 3.6 1.1 4.7 23.4 187 1282 196 55 8.8 3.5 31.5 170 1074 198 56 7.4 5.4 25.2 491' 1809 200 57 5.8 4.8 22.3 456 1669 202 58 7.8 4.4 26.7 308 1125 204 59 6.2 6.0 22.5 957 2581 206 60 5.2 4.7 21.8 815 1912 208 61 7.0 - - 4.6 22.7 771 1824 210 62 8.7 - - 4.0 29.3 538 1192 212' 63 7.1 - - 4.8 21.6 588 1401 AC TABLE'6 (continued) NITROSAMINE CONCENIRATIONS IN PROCESSED SWSC-I, SINGLE VALUES 3285

INBIFO Institut fur bi4logische Forschung • K6in SUBREPORT P 0500/3068 NYV065RA6 71'D3 BATCH N0. WEEK OF STUDY NITROSAMINE TaBACCO SPECIFIC (mg/kg) VOIATILE (ug/kg) NNN NATB NAB NATB PLUS NAB NNK DNA1A NPY 215 64 7.3 - - 6.5 24.5 964 2314 216 65 7.1 - - 6.3 23.1 496 1775 218 66 6.4 - - 3.7 24.4 191 1155 224 70 7.0 - - 4.7 27.8 612 2191 232 74 6.5 3.9 1.8 5.7 20.8 173' 1037 236 76 6.6 3.6 1.1 4.7 22.5 145 1274 238 77 6.3 - - 5.5 24.0 1467 2836 AC TABLE 6 (continued) NITROSAhIINE CONCELaIRATIONS IN PROCESSED SWSC-I, SINGLE VALUES 3285

INBIFO Institut fur biologische Forschung • NGt91n SUBREPORT P 0500/3068 MN065RB23 7097 NO. PA H'~ 1 phenanthrene 2 anthracene 3 fluoranthene 4 pyrene 5 benzo(b)fluorene 6 benzo(a)anthracene 7 chrysene plus triphenylene 8 benzo(b)fluoranthene 9 benzo(k)fluoranthene 10 benzo(e)pyrene 11 benzo(a)pyrene 1 2 pe ryI en 13 dibenz( -) anthracenes 14 benzo(ghi}perylene AC TABLE 7 NUMBERING OF PAH FOR THE AC TABLES 8 TO 11 3285

I'NB1F©' Institut fur biologische Forschung • K6I'n SUBREPORT P 0500/3068 MN065RB24 7103 BATCH NO WEEK OF PAH (mg/kg) . STUDY 1 2 3 4 5 6 7 130 28 15.55 4.81 3.92 4.92 2.13 1.65 1.84 132 29 20.03 6.52' 5.66 5.90 5.36 1.41 2.36 134 30 15.27 5.21 3.78 4.77 4.05 0.90 2.01 137 31 18.46 5.81 4.63 6.44 4.64 1.47 2.32 139 32 18.97 5.87 6.33 7.33 4.84 1.47 2.62 141 33 16.27 4.77 6.67 4.93 3.48 1.25 2.73 143 34 14.29 4.27 4.87 4.58 3.87 1.14 1.86 147 36 17.50 5.83 5.37 6.99 3.78 1.50 2.29 150 37 15.89 4.77 4.44 5.36 4.93 1.27 2.02 156 39 16.06 4.45 4.30 6.14 4.03 1.60 2.01 160 40 15.74 5.00 5.69 4.17 3.62 1.19 2.12 162 41, 13.86 4.48' 5.49 3.82 2.67 1.22 1.85 164 42 15.21' 5.16 5.36 3.99 3.11 1.13 2.46 166 43 16.24 5.35 6.08 3.97 2.46 1.05 1.75 170 44 18.61 7.64 5.33 4.18 4.01 1.80 2.22 172 45 18.80 6.78 3.94 6.47 3.51 0.70, 2.12 174 46 19.67 7.011. 5.08 5.72 4.36 1.18 2.06 176 47 18.89 7.03 6.31 5.70 4.18 1.27 2.27 178 48 19.17 6.48 4.49 6.07 4.41 1.21 2.27 AC TABLE 8 CONCENTRATIONS'OF SOME PAH (1 TO 7) IN PROCESSED MWSC-I, SINGLE VALUES Remarks: for the numbering of the PAH see AC TABLE 7 3285

INBIFO Ilnstitut fur biologische Forschung • KOin SUBREPORT P 0500/3068 MN065RB25 7103 BATCH NO WEEK OF PAH (mg/kg) . STUDY 1 2 3 4 5 6 7 18'0 49 14.18 4.56 4.59 6.01 3.46 1.32 1.87 182 50 11.80 3.75 5.75 3.16 3.01 1.29 2.07 184 51 12.36 3.85 5.55 3.49 2.91 1.14 2.11 186 52 14.89 4.79 4.93 3.93 3.35 1.23' 2.56 188 53 14.62' 4.85 4.62 4.83 3.97 1.44 1.97 190 54 12.84 3.83' 3.63 5.01 3.12 1.30 2.35 192 55 13.26 3.98' 4.48 5.37 3.06 0.91 1.98' 194 56 11.68 3.73 4.95 3.15 2.39 1.24 2.22 196 57 10.57 3.28 4.01 2.84 2.82 0.94 2.02 198 58 14.54 4.88 5.42' 3.80 3.23 1.06 2.36 200 59 10.93 3.24 4.85 2.76 2.70 0.94 1.63 202 60 14.28 4.39 5.38 3.80 2.52 1.38 2.19 204 61: 15.29 4.84 5.48 4.11 2.16 1.06 2.45 206 62 14.78' 5.11 4.49' 3.72 2.97 0.83 1.56 208' 63 15.28 5.37 4.69 4.02 2.30 0.92 2.52 212 65 14.79 4.84 5.23 3.59 2.51 1.14 2.12 215 66 15.34 4.96 5.88 3.65 2.41 0.97 2.26 AC TABLE 8 (continued) CONCENTRATIONS OF'SOME PAI1 (1 TO 7) IN PROCESSED MWSC-I, SINGLE VALUES Remarks: for the numbering of the PAH see AC TABLE 7 3285

IINBIF© Institut fur biologische Forschung • KtSln SU'BREPORT P 0500/3068 MN065RB27 7103 . BATCH NO WEEK OF' PAH (mg/kg) . STUDY 8 9 10 11' 12 13 14 130 28' 0.43 - 0.49 0.93 1.74 0.18 0.20' 132 29 0.55 - 0.32 0.44 - 0.14 0.14 134 30 0.38 - - - - 0.09 0.10 137 31 0.63 - 0.61 0.64 2.00 0.16 0.11 139 32 0.53 - 0.56 01.75 1.53 0.12 0.17 141 33 0.46 - - 0.63 1.67 0 0 143 34 0.40 - 0.40, 0.51 1.52 0 0.10 147 36 0.48 - 0.55 0.71 1.93 0 0.23 150 37 0.56 - 0.46 0.67' 1.89 0 0 156 39 0.62 - 0.39 0.63 1.53 0 0 160 40 0.57' 01.30 0.45 0.57 0 0 0 162 41 0.40 0.22 0.50 0.76 1.50 0 01 164 42 0.48 0.25 0.72 0.86 1.86 0.16 0 166 43 0.42' 0'.2'5 0.56 0.84 1.67 0.12 0.12 170 414 0.57 - 0.76 0.86 - - - 172 45 0.35 0.22' - - - - - 174 46 - - 0.69 0.78 1.05 0..10, 0.11 176 47 0.41 0.32 0.73 0.86 1.64 0 0 178 48 0.32 0.39 0'.77 0.80 1.56 0 0.09 AC TABLE 9 CONCENTRATIONS OF SOME PAH (8 TO 15) IN PROCESSED MWSC-I, SINGLE VALUES Remarks: for the numbering of the PAH see AC TABLE 7 0: below detection limit of 01.06 mg/kg 3285

IINBIFO Institut fur biolog:ische Forschung • K81n SUBREPORT'P 0500/3068 MN065RB28 7103 BATCH' NO WEEK OF PAH (mg/kg) . STUDY 8 9 10 11 12 1,3 14 180 49 0.41 0.39 - - 1.68 0 0.11 182 50 0.40 0.25 0.65 0.81 1.64 0.09' 0.12 184 51 - - 0.82 0.84 1.51 0.09 0.12 186 52 0.58 0.31 0.71 0.88 1.41 0.10 0.13 188 53 0.51 - 0.71 0.81 - 0 0.11 190 54 0.55 0.23 0.49 0.51 - 0 0 192 55 0.42 0.38 - - 1.2'4 0 0.09 194 56 0.42 0.24 0.72 0.81 1.64 0.06 0.06 196 57 0.41! 01.21 0.66 0.92 1.55 0 0 198 58 0.47 0.30 0.54 0.77 - 0.10 0.10 200 59 0.43 0.23 0.78 0.94 - 0.06 0.08' 202 60 0.44 0.24 0.71 0.87 0.95 0.07 0.10 204 61 0.47 0.23 0.60 0.87 0.90 0 0 206 62' 0.42 0.30 0.68 0.79 1.22 0.09 0.08 208 63 0.45 0.26 0.65 0'.86 0.83 0.09' 0.07 212 65 0.55 0.23 0.64 0.76 0.79 0.09 0.09 215 66 0.49 0.17 0.67 0.85 1.00 0 0 AC TABLE 9 (continued) CONCENTRATIONS OF SOME PAH (8 TO 15) IN PROCESSED MWSC-I, SINGLE VALUES Remarks: for the numbering of the PAH see AC TABLE 7 0: below detection limit of 0.06 mg/kg 3285

INBIFO Institut flur biologische Forsch~ung • K6in SUBREPORT,P 0500/3068 MN065RB11 7103 , BATCH NO WEEK O F PAH (mg/kg) . STUDY 1 2 3 4 5 6 7 130 26 69.5 18.4 31.3 22.4 7.6 8.0 22.2 132 27 53.5 11.7 28.6 18.0 5.3 8.0 21.7 134 28 68.7 17.2 33.8' 21.2 7.0 9.7 26.0 137 29 82.0 22.6 34.9 25.9 7.7 8.8 22.6 139 30 54.8 13.3 29.9 18.3 6.8 9.4 23.9 145 33 89.4 29.9 39.0 26.3 8.4 110.7 26.1 147 34 15.6 3.2 13.0 4.3 0.8 5.2 14.8 150 35 92.2 36.6 43.3 36.0 10.4 8.5 25.3 154 36 95.5 30.0 41.4 27.4 15.4 9.1 20.7 156 37 81.5 23.0 36.2 29.1 9.8 9.7 25.6 160 38 116.9 33.6 44.7 37.5 11.9 10.7 27.1 162 39 65.4 16.3' 34.7 22.6 7.7 9.1 23.4 166 41 57.5 21.1 35.5 21.2 11.1 8.1 23.2 170 42 76.2' 28.6 41.1 30.3 21.9 10.2 25.1 172 43 38.8 8.5 24.8 13.1 7.3 6.4 19.7' 184 49 83.1 20.8' 32.4 119.8 8.5 7.8 22.4 186 50 109.3 28.8 40.1 25.1 11.4 10.4 27.1 188 51 111.1 30.8 41.2 27.4 10.4 8.5 26.5 192 53 77.3 17.7' 28.7 19.3 3.7 8.9 22.9 194 54 75.7 17.5 3.1 18.6 2.3 9.8 24.4 AC TABLE 10 CONCENTRATIONS OF SOME PAH (1 TO 7) IN PROCESSED SWSC-I, SINGLE VALUES Remarks: for the numbering of the PAH see AC TABLE 7 3285

IN'BIFO Institut fur biologische Forschuing • KtSin SUBREPORT,P 0500/3068 MN065RB12 7103 . BATCH NO WEEK OF PAH (mg/kg) . STUDY 1 2 3 4 5 6 196 55 75.9 17.2 33.4 25.1 2.8 10.4 26.1 210 62 83.6 19.9 37.1 20.4 8'.41 8.4 23.2 215 64 84.2 19.6 34.4 20.7 3.7' 9.4 24.8 216 65 112.3 27.9 - - 3.5 10.8 27.4 AC TABLE 10 (continued) CONCENTRATIONB OF SOME PAHI(1 TO 7) IN PROCESSED SWSC-I, SINGLE VALUES Remarks: for the numbering of the PAH see AC TABLE 7 3285

INBIFO Institut flur biologische Forschung • KbIn SUBREPORT P 0500/3068 MN065RB13' 7103 , BATCH NO WEEK OF PAH (mg/kg) . STUDY 8 9 10 11 12 13 14 130 26 - 7.2 1.9 5.3 2.4 0.2 0.5 132 27 3.9 3.3 2.7 5.6 1.8 0.5 0.6 134 28 - - 2.7 6.0 2.2 0.2 0.7 137 29 3.0 2.5 2.4 4.5 5.1 0.3 0.6 139 30 0.7 4.7 2.8 5.9 3.7 - 0.8 145 33 4.6 1.8 3.3 6.8 5.0 0.5 0.3 147 34 3.4 2.3 1.8 4.0 2.7 - 0.5 150 35 - 5.9 2.9 4.3 3.6 0.7 0.1 154 36 1.1 2.3 1.7 3.8 3.8 0.2 0.3 156 37 3.7 2.6 2.9 5.8 3.3' 0.4 0.6 160 38 4.1 2.0 3.3 5.0 4.3 0.5 0.6 162 39 3.3' 1.3 2.9 5.8 3.9 0.3 0.7 166 41 3.2' 1.3 2.8 6.8 4.0 0.1 0.5 170 42 1.4 4.0 2.6 6.4 3.8 0.3' 0.8 172' 43 4.5 0.8 2.0 4.0 1.1 - 0.6 184 49 3.8' 1.3' 2.3 4.3 1.1 0.1 0.8 186 50 3.4 2.0' 2.7 5.7 3.8 0.2 0.6 188 51 3.7 2.3 2.2 5.2 3.8 0.2 0.9 192 53 3.0 1.9 1.8 5.5 4.1 0.1 0.8 N 0 194 54 5.0 1.0 2.2 4.5 2.6 0.1 0.7 ~ AC TABLE 11 CA CONCENTRATIONS OF SOME PAH (8 TO 15) IN PROCESSED SWSC-I, SINGLE VALUES~ Remarks: for the numbering of the PAH see AC TABLE 10 3285

INBIFO Institut fur biologi'sche Forschung • KbIn SUBREPORT P 05010/3068 MNOb5RB14 7103 . BATCH NO WEEK OF PAH (mg/kg) . STUDY 8' 9 10 11 12 13 14 196 55 3.7 2.0 2.6 5.9 2.9 0.2 0.9 210 62 3.7 1.5 2.1 4. 1 1.6 0.1 0.6 215 64 5.4 1.2' 2.4 4.4 0.7 - 0.7 216 65 3.2 1.8 2.4 5.5 4.0 0.4 0.6 AC TABLE 11 (continued) CONCENTRATIONS OF SOME PAH (8 TO 15) IN PROCESSED SWSC-I, SINGLE VALUES Remarks: for the numbering of the PAH see AC TABLE 10 3285

SUBREPORT P 0500/3068 MN065RA20 7126 PARAMETER TYPE OF CONDEN ATE YIELD OR CONCENTRATION SE N RSD (0/0) S M x min. x max. condensate yield (mg/cig.) MWSC-I, crude 48.5 42.2 54.8 0.2 176 5.4 MWSC-I, processed 39.6 35.2 44.8 0.1 176 4.7 SWSC-I, crude 22.1 18.0 28.8 0.1 175 8.3 SWSC-I, processed 19.4 16.6 23.3 0.1 175 6.1 water conc. MWSC-I, 42.4 30.0 65.8 0.8 117 19.7 (g/1) crude MWSC-I, processed 2.8 0.6 7.2 0.1 174 54.9 SWSC-I, crude 29.3 9.8 95.6 1.2 115 44.9 SWSC-I, processed 4.1 1.0 10.8 0.2 174 50.5 AC TABLE 12 PARAMETERS OF CONDENSATE PREPARATION Remarks: water concentration determined in the condensate suspensions cigarette 2R1 eirslsowaz

SUBREPOkl`r P 0500/3068 MN065RA21 %126 PARAMEPER C1rNCENTrRATION SE N RSD (0/0) M x min. x max. hydrogen-ion (pH) 6.0 5.7 6.3 0.0 175 1.6 nicotine (g/1) 20.8 17.8 24.5 0.1 174 5.9 catechol (g/1) 1.21 1.05 1.44 0.01 61 6.0 nitrosamines (mg/1) NND1 1.9 1.4 2.7 0.0 46 13.2 NATB 1.7 1.1 2.2 0.0 31: 18.1 NNK 1.5 0.9 3.4 0.0 46 32.3 NAB 0.5 0.3 0.9 0.0 31 22.0 NPY 0.07 0.02 0..10 01.00 46 16.4 DMNA 0.01 0.01 0.02 0.00 46 27.3 sum 5.68 3.73 9.32 PAH (mg/1) phenanthrene 3.71 2.54 4.811 0.10 36 16.0 anthracene 1.21 0.78 1.83 0.04 36 21.2 fluoranthene 1.21 0.87 1.60 0.03 36 15.0 pyrene 1.13 0.66 1.76 0.05 36 25.7 benzo(b)fluorene 0.82 0.51 1.29 0.03 36 25.1 benz(a)anthracene 0.30 0.17 0.43 0.01 36 21.1 chrysene plus trighenylene 0.52 0.37 0.66 0.01 36 12.8 benzo(b)fluoranthene 0.11 0.08 0.15 0.00 34 16.2 benzo(k)fluoranthene 0.06 0.00 (a) 0.09 0.10 23 31.6 benzo(e)pyrene 0.15 0.08 0.20 0.10 31 20.9 benzo(a)pyrene 0.18 0.11 0.22 0.10 32 16.9 perylene 0.35 0.19 0.48 0.02 27 23.7 dibenz(-)anthracenes 0.01 0.00 (a) 0.04 0.00 34 105.3 benzo(ghi)perylene 0.02 0.00 (a) 0.06 0.00 34 76.9 sum 9.78 6.36 13.62 AC TABLE 13 PARAMETERS OF THE MWSC-I APPLICATION SOLUTION OF THE HIGHEST'(xNCENTRATION, MEAN VALUES Remarks: application solution of the highest concentration: 240 g processed condensate/1, cigarette 2R1 (a) below detection limit

SUtaEtE:'PCJkrt' P U5U0/3U68 MIJt)65FtA22 /126 PARAMETER 0CNCENZRATION ~ SE N RSD (0/0) M x min. x max. hydrogen-ion (pH) 6.5 6.2 6.7 0.0 175 1.3 nicotine (g/1) 25.1 20.4 32.3 0.2 174 8.3 catechol (g/1) 1.10 0.94 1.24 0.01 61 6.3 nitrosamines (mg/1) NNN' 1.7 1.2 2.4 0.0 44 14.4 NATB 0.8 0.6 1.1 0.0 30 14.7 NNK 6.1 4.2 9.5 0.2 44 18.11 NAB 0.4 0.3 0.6 0.0 30 22.3 NPY 0.32 0.19 0.68 0.02 45 38.6 DMNA 0.08 0.01 0.35 0.01 45 90.0 sum 9.40 6.50 14.63 - - - PA4i (mg/1) phenanthrene 18.70 3.74 28.06 1.16 24 30.3 anthracene 5.14 0.77 8.78 0.40 24 4.7 fluoranthene 7.97 0.74 10.73 0.48 23' 28.6 pyrene 5.52 1.03 9.00 0.35 23 30.7 benzo(b)flworene 1.94 0.19 5.26 0.22 24 56.3 benz(a)lanthracene 2.16 1.25 2.56 0.07 24 15.1 chrysene plus triphenylene 5.71 3.55 6.58 0.14 24 11.9 benzo(b)fluoranthene 0.82 0.17 1.30 0.06 21 34.3 benzo(k)fluoranthene 0.60 0.19 1.73 0.08 23 63.7 benzol(e)pyrene 0.60 0.41 0.79 0.02 24 18.1 benzo(a)pyrene 1.25 0.91 1.63 0.05 24 17.4 perylene 0.74 0.17 1.20 0.06 24 39.4 dibenz(-)anthracenes 0.07 0.02 0.17 0.01 20 60.9 benzo(ghi)perylene 0.14 0.02 0.22 0.01 24 30.7 swn 51.36 13.16 78.01 AC TABLE 14 PARAMETERS OF THE SWSC-I APPLICATION SOLUTICN OF THE HIGHEST CONCE3'I'RATICN 1, MEAN VALUES Remarks: application solution of the highest concentration: 240 g processed'd condensate/1 cigarette 2R1

LIV.U{1~.~ .: ..................... . ~.....,J._~_.., ..... v/a" SUBREPORT P 0500/3068 MN065RA23 7165 PARAMETER (ICNCENTRATION SE N RSD (0/0) M x min. x max. nicotine (g/kg) 86.7 74.2 102. 1 0.4 174 5.9 catechal (g/kg) 5.04 4.38 6.00 0.04 61 6.0 nitrosamines (mg/kg) NNN 8.1 5.9 11.3 0.2 46 13.2 NATB 7.0 4.6 9.0 0.2 31 18'.1 NNK 6.3 3.7 14.3 0.8 46 32.3 NAB 1.9 1.4 3.6 0.8 31' 22.0 NPY 0.29 0.09 0.41 0.01 46 16.4 DMNA 0.06 0.04 0.1.0 0.00 46 27.3' sum 23.65 15.73 38.71 PAH (mg/kg) phenanthrene 15.5 10.6 20.0 0.4 36 16.0 anthracene 5.0 3.2 7.6 0.2 36 21.2 fluoranthene 5.1 3.6 6.7 0.1 36 15.0 pyrene 4.7 2.8 7.3 0.2 36 25.7 benzo(b)fluorene 3.4 2.1 5.4 0.1 36 25.1 benz(a)anthracene 1.2 0.7 1.8 0.0 36 21.1 chrysene plus triphenylene 2.2 1.6 2.7 0.1 36 12.8 benzo(b)fluoranthene 0.47 0.32 0.63 0.01 34 16.2 benzo(k)fluoranthene 0.26 0.00 (a) 0.39 0.02 23 31.6 benzo(e)pyrene 01.61 0.32 0.82 0.02 31 20.9 benzo(a)pyrene 0.77 0.44 0.93 0.02 32 16.9 perylene 1.46 0.79 2.00 0.07 27 23.7 dibenz(-)anthracenes 0.06 0.00 (a) 0.18 0.01 34 105.3 benzo{ghi)perylene 0.08 0.00 (a) 0.23 0.01 34 76.9 sum 40.81 26.47 56.68 AC TABLE 15 PARAMEi'ERS OF PFCCESSED MWSC-I OF CIGARETTE 2R1, CONCENTRATIOI+]S, MEAN VALUES (a) below detection limit

SUBd2EPORT P 0500/3068 NA1065RA24 7126 PARAMETiER OONCENTRATION SE N RSD (0/0) M x min. x max. nicotine (g/kg) 104.6 85.0 134.6 0.7 174 8.3 catechol (g/kg) 4.58 3.92 5.17 0.04 61 6.3 nitrosamines (mg/kg) NNN 7.2 5.2 9.9 0.2 44 14.4 NATB 3.3 2.5 4.7 0.1 30 14.7 NNK 25.5 17.4 39.6 0.7 44 18.1 NAB 1.6 1.1 2.7 0.1 30 22.3 NPY 1.34 0.79 2.84 0.08 45 38.6 DIYIIVA 0.33 0.06 1.47 0.18 45 90.0 sum 40.27 27.05 61.21 PAH (mg/kg) phenanthrene 77.9 15.6 116.9 4.8' 24 30.3 anthracene 21.4 3.2 36.6 1.7 24 4.7 fluoranthene 33.2' 3.1 44.7 2.0 23 28.6 pyrene 23.0 4.3 37.5 1.5 23 30.7 benzo(b)fluorene 8.1 0.8 21.9 0.9 24 56.3 benz(a)anthracene 9.0 5.2 10.8 0.3 24 15.1 chrysene plus triphenylene 23.8 1:4.8 27.4 0.6 24 11.9 benzo(b)fluoranthene 3.4 0.7 5.4 0.3 21 34.3 benzo(k)fluoranthene 2.5 0.8 7.2 0.3 23 63.7 benzo(e)pyrene 2.5 1.7 3.3 0.1 24 18.1 benzo(a)pyrene 5.2 3.8 6.8 0.2 24 17.4 perylene 3.1 0.7 5.0 0.3 24 39.4 dibenz(-)anthracenes 0.3 0.1 0.7 0.0 20 60.9 benzo(ghi)perylene 0.6 0.1 0.9 0.0 24 30.7 sum 214.0 54.9 325.1 AC TABLE 16 PARAMETERS OF'PImCESSED SWSC-I OF CIGARETTE 2R1, CONCENTRATIONS, MEAN VALUES

SUfjREPORT P 0500/3068 MN065RA25 7126 PARAMETER' YIELD SE N, gSD (0/0) M x min. x max. condensate (mg/cig.) crude 48.5 42.2 54.6 0.2 176 5.4 processed 39.6 35.2 44.8 0.1 176 4.7 nicotine (mg/cig.) 3.43 2.94 4.04 0.02 174 5.9 catechol (mg/cig.) 0.200 0.173 0.238 0.002 61 6.0 nitrosamines (ng/cig.) NNN 320 230 450 20 46 13.2 NATB 280 180 360 10, 31 18.1 NNK 250 150 570 10 46 32.1 NAB 80 60 140 0 31 22.0 NPY 12 3 16 0 46 16.4 DNNA 2 1 4 0 46 27.3 sum 944 624 1540 PAH (ng/cig.) phenanthrene 613 419 793 16 36 16.0 anthracene 200 128 303 7 36 21.2 fluoranthene 200 144 264 5 36 15.0 pyrene 186 109 290 8 36 25.7 benzo(b)fluorene 135 84 212 6 36 25. 1 benz(a)anthracene 49 28 71 2 36 21.1 chrysene plus triphenylene 85 62 108 2 36 12.8 benzo(b)fluoranthene 19 13 25 0 34 16.2 benzo(k)fluoranthene 10 0 (a) 15 1 23 31.6 benzo(e)pyrene 24 13 32 1 31 20.9 benzo(a)pyrene 30 17 37 1 32 16.9 perylene 58 31 79 3 27 23.7 dibenz(-)anthracenes 2 0 (a) 7 0 34 105.3 benzo(ghi)perylene 3' 0 (a) 9 0 34 76.9 (a) below detection limit ,,,Ag

SUBREPORT P 0500/3068 MNOfi5RA26 7126 PARNMETER CONCENTRA~Z'iON' SE N RSD (0/0) M x min, x max. condensate (ng/cig.) crude 22.1 18.0 28.8 0..1 175 8.3 processed 19.4 16.6 23.3 0.1 175 6.1 nicotine (mg/cig.) 2.02 1.64 2.60 0.01 174 8.3' catechol (ng/cig.) 0.069 0.076 O.1i00 0.000 61 6.3 nitrosamines (ng/cig.) I~1N 140 100 190 0 44 14.4 NATB 60 50 90 0 30 14.7 NNK 490 340 760 10 44 18.1 NAB 30 21 50 0 30 22.3 NPY 26 15 55 2 45 38'.6 Dt4I1 6 1 28' 0 45 90.0 sum 752 527 1173 PAH (ng/cig) phenanthrene 1511 303 2268 93 24 30.3 anthracene 415 62 710 33 24 4.7 fluoranthene 644 60 867 39 23 28.6 pyrene 446 83 728 29 23 30.7 benzo(b)fluorene 157 16 303 17 24 56.3 benz(a)anthracene 175 101 210 6 24 15.1 chrysene plus triphenylene 462 287 532 12 24 11.9 benma(b)fluoranthene 66 14 105 6 21 34.3 benzo(k)fluoranthene 49 16 140 6 23 63.7 benzo(e)pyrene 49 33 64 2 24 18.1 benzo(a)pyrene 101 1 74 132 4 24 17.4 perylene 60 14 97 6 24 39.4 dibenz(-)anthracenes 6 2 14 0 20 60.9 benzo (ghi ) perylene 12 2 17 0 24 30.7 sum 4153 1067 6187 AC TABLE 18 PARAMEI'ERS OF SVYSC-I OF CIGARETPE' 2R1, YIELDS, MEAN VALUES

SUBREPORT P 0500/3068 t4N065RA27 7090 NITROSAMINE YIELD (ng/cig.) MAINSPREAM LITERATURE INBIFtD STUDY P 0500/3068' SIDESTREAM LITERAZURE INBIFO STUDY P 0500/3068 NNN 120 to 3700 320 1700 140 NATB 140 to 4600 280 270 60 NNK 80 to 770 250 410 490 NAB n. d. to 150 80 - 30 NPY n, d. to 110 12 84 to 510 26 L1MNA 0.5 to 180 2' 156 to 823 6 AC TABLE 19 DETERMINBD NITI~)SAMINE YIEL0.S AND LITEiiATUftS DATA F~2 NO1+IFILTII2 CIGAREiTFS Remar}ts: from IAFaC monographs, 1986 n. d.: not detectable

S[)BREPORT P 0500/3068 1wYd065RA28 7098' POLYCYCLIC ARLiMATIC HYDROCARBON YIELD ( ng/cig. ) T[hlORI- GENIC' REFERFNCE MS LITERATC>E2E INBIF0 STUDY P 0500/ SS LIlERATORE INBIFC} ST[DY P 0500/ ACTNITY 3068 3068 phenanthrene 85 to 624 613 - 1511 - - anthracene 23 to 235 200 - 415 0 D fluoranthene 10 to 272 200 126 644 0 A pyrene 50 to 270 186 390 to 1010 446 0 A benzo(b)fluorene 20 135 - 157 - - benz(a)anthracene 4 to 76 49 - 175 + A, C, E chrysene 6 to 96 - - - +? A, E chrysene + triphenylen - 85 - 462 - - benzo(b)fluoranthene 4 to 22 119 - 66 ++ A, E benzo(k)fluoranthene 6 to 12 10 - 49 - - benzo(e)pyrene 2 to 25 24 135 49 +? A, E benzo(a)'.pyrene 2 to 78 30 25 to 199 101 +++ A, D, E perylene 3 to 5 58 39 60 0 A dibenz(a,c)anthhraoene present + C, E dibenz(:a,h)anthracene 4 ++ A, D, E dibenz(a,j,)anthraoene 11 41 dibenz(-)anthracene 2 6 benzo(ghi)perylene 3 to 39 3 98 12 + A AC 'l41BLE 20: DETERMINBD PAEI YIELM AND LITERATU,tE DATA ON PAH Remarks: from IARC monographs, 1986, extended, reference A: Lse et al., 1981, " B: Van Duuren, 1980, " C: Van Duuren and Orris, 1965 and Van Duuren et al., 1970, " D: Selkirk, 1980', " E: Hoffinenn et al., 1978

P 0500/3068, H05264, V20 F30 V62 F16, MAINSTREAM SUBREPORT P 60-n 5 0 ---i ~ 40 - w H 0500/3068 I,. af'q~IrI7 7__ -2 1 20 40 60 80 AC FIGURE 1 WEEK OF STUDY CRUDE MWSC-I YIELD Remarks: lowest and highest value of 2 to 4 batches per week 0 MN065RB16 6161 H05264 M ZZ91saszoz

P 0500/3068, H05271, V39 F30 V20 F30, SIDESTREAM SU BREPORT P 0500/3068 MN065RB16 6161 H05271 M 60-, 50 -I 0 ~ 40 w 0-6 ~ 30-I 20 -~ , 10-1 r -- - -- -2 1 20 40 AC FIGURE 2 CRUDE SWSC-I YIELD Remarks:: lowest and highest value of 2 to 4 batches per week 60 80 WEEK OF STUDY ~x~

P 0500/3068, H05265, V42 F30 V21 F30, MAINSTREAM SUBREPORT P 0500/3068 MN065RB16 6161 H05265 M O1 60 -, U -' `- 50 01 E v °z 0 U 0 w ~ U) w U 0 W CL 30-~ 20 ~-i 10-i 0 I~ - ~i`~I I ~TI ~ 2F`-~T r- -- -2 1 20 40 60 80 AC FIGURE 3 WEEK OF STUDY PROCESSED MWSC-I YIELD Remarks: lowest and highest value of 2 to 4 batches per week 6=TS0wOZ

P 0500/3068, H05272, V41 F30 V21 F30, SIQESTREAM SUBREPORT P 0500/3068 MN065RB16 6161 H05272 M O1 _~ 0 30 z 0 v 20 H 10H 0 r- - - -2 1 20 40 SO 80 AC FIGURE 4 WEEK OF STUDY PROCESSED SWSC-I YIELD Remarks: lowest and highest value of 2 to 4 batches per week ~~~~~

P 0500/3068, H05266, V23 F30 V22 F30, MAINSTREAM, CRUDE SUBREPORT P 0500/3068 MN065RB16 6161 H05266 M ~ rn z 0 100--' 20-I 0 0 r- -2 1 20 40 60 80 AC FIGURE 5 WATER CONCENTRATION IN CRUDE MWSC-I SUSPENSION Remarks: lowest and highest value of 2 to 4 batches per week WEEK OF STUDY imisoszoz

r 0500/3068, H05273A, V74 F30 V43 F30, SIUESTREAM, CRUDE SUBREPORT P 0500/3068 MNQ65RB16 6161 H05273A M .~. ~ ~ 01 ..r 120-1 oc w ~ 407 20-~ I 20 40 AC FIGURE 6 WATER CONCENTRATION IN CRUDE SWSC-I SUSPENSION Remarks: lowest and highest value of 2 to 4 batches per week 60 80 WEEK OF STUDY zMTsMoz

P 0500/3068, H04745, V25 F30 VX F30, MAINSTREAM, PROCESSED SUBREPORT P 0500/3068 MN065RB16 6161 H04745 M ~ ~ ~ rn v I _ ol 0 r Ili ~lI r- - -2 1 20 40 AC FIGURE 7 WATER CONCENTRATTON IN PROCESSED MWSC-I SUSPENSION Remarks: lowest and highest value of 2 to 4 batches per week 12--, 0 -1 60 80 WEEK OF STUDY CESisoszOz

P 0500/3068, H04746A, V44 F30 VX F30, SIDESfiREAM, PROCESSED SUBREPORT P 0500/3068 MN065RB17 6161 H04746A M ~ ~ ~ 01 ~ z 0 ~ 12-1 10-~ TM T y 2 0 x I I I --- -2 1 20 40 60 80 AC FIGURE 8 WATER CONCENTRATION IN PROCESSED SWSC-I SUSPENSION Remarks: lowest and highest value of 2 to 4 batches per week WEEK OF STUDY tmiSO9zoz

P 0500/3068, H04744A, V45 F30 VX F30, MAINSTREAM SUBREPORT P 0500/3068 MN065RB17 6161 H04744A M 9 .. 35-n ~ ~ ~ z O H ~"- ~ t-- z w U Z O U w Z .-, F-- 30-~ 25 -t ~ 20-I 15 - ~ 5 r- -2 1 20 40 60 80 AC FIGURE 9 WEEK OF STUDY NICOTINE CONCENTRATION IN THE MWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION Remarks: lowest and highest value of 2 to 4 batches per week sMtsQ9zoz

P 0500/3068, H04743A, V47 F30 VX F30, SIDESTREAM SUBREPORT P 0500/3068 MN065RB17 6161 H04743A ~ ~ ~ rn V z 0 H 30- . 0 10 - U 1~" z 0 r - -- --- -2 1 20 40 50 80 WEEK OF STUDY AC FIGURE 10 NICOTINE CONCENTRATION IN THE SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION Remarks: lowest and highest value of 2 to 4 batches per week 9stsWzoz

P 0500/3068, H04741A, V46 F30 V88 F19, MAINSTREAM SUBREPORT P 0500/3068 MN065RB17 6161 H04741A ... z 0 ~ ~-- ~ az F- z w U z 0 U 1.2-~ 0.9-I 0 M r- -- -2 1 20 40 60 80 WEEK OF STUDY AC FIGURE 11 CATECHOL CONCENTRATION IN THE MWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION Remarks: lowest and highest value of 2 to 4 batches per week i i zx'sTSa9zoz

P 0500/3068, H04742A, V48 F30 V89 F19, SIDESTREAM SUBREPORT P 0500/3068 MN065RB17 6161 H04742A w.I 1.2-~ 0.9-- 0 M T d~= =~I~~iI iI']II r -- -2 '! 20 40 60 80 WEEK OF STUDY AC FIGURE 12 CATECHOL CONCENTRATION IN THE SWSC-I APPLICATION SOLUTION OF THE HIGHEST CONCENTRATION Remarks: lowest and highest value of 2 to 4 batches per week sESTSO9zoz

P 0500/3068, H04738A, V49 F30 VX F30, MAINSTREAM SUBREPORT P 0500/3068 MN065RB17 6161 H04738A M 40 --1 -----~ ~ NNN -~ ~ NATB plus NAB -Q ~ NNK 30-i 20-~ 10--; r- -- --- -2 1 20 AC FIGURE 13 _T 40 I 1 60 80 WEEK OF STUDY CONCENTRATION OF TOBACCO SPECIFIC NITROSAMINES IN PROCESSED MWSC-I

P 0500/3068, H04734A, V51 F30 VX F30, SIDESTREAM SUBREPORT P 0500/3068 MN065RB17 6161 H04734A M r. 40 rn v ~ rn E ~ zo ~ D0-I f-- ~ ac ~- z w U 20-~ z © U w z .-, ~ 10-~ ~ V) CQ CY F- 0 F- - - -2 1 20 40 AC FIGURE 14 4 v Lal m 60 80 WEEK OF STUDY CONCENTRATION OF TOBACCO SPECIFIC NITROSAMINES IN PROCESSED SWSC-I obs9sowoz

P 0500/3068, H04709A, V50 F30 VX F30, MAINSTREAM SUBREPORT P 0500/3068 MN065RB18 ~,. 3000 --'--' i DMNA ~ ~ --E> : NP Y ? 2500 - z 0 ~ ~- 2000 ~ oc E-- z w u 50 0 z 0 U w z ~ 1000-I z e Lo 00 -I F-- 0 6161 H04739A a W&AGE-ACNAC r- -2 1 20 40 AC FIGURE 15 CONCENTRATION OF THE VOLATILE NITROSAMINES IN PROCESSED MWSC-i ?N--- -QQ~ GXN.c92ssw e 60 80 WEEK OF STUDY c jtsTSO9zoz

P 05U0/306E3, H05274, V53 F30 V52 F30, SIDESTREAM SUBREPORT P 0500/3068 MN065RB18 6161 H05274 M --, 3000-, ~t rn ? 2500 -~i z 0 ~ ~ ~ oc I-- z w U z 0 U 2000- ' 15 0 0 -+ 1000-~ 50 0 I 0 AC FI GURE 16 -----'v a DMNA -f> 1 NP Y --2 1 20 40 CONCENTRATION OF THE VOLATILE NITROSAMINES IN PROCESSED SWSC-I 60 80 WEEK OF STUDY ztsTSO9zoz

P 0500/3068, H04740A, V77 F30 V59 F30, MAINSTREAM SUBREPORT P 0500/3068 MNO65RB18 6318 H04740A M :v 5 rn ---b~ i Benzo ( a) f l uoran t hene -m ~ Benzo ( k) f l uoran t hene rn e I., 4-I z ~ ~ 3 z w U z 0 U 2 4 Q. 1 ~-, 0 ~ IL ;-'GLI ( r.j C6 C)b -F 40 -1 80 WEEK OF STUDY AC FIGURE 17 CONCENTRATION OF BENZO(-)FLUORANTHENES IN PROCESSED MWSC-I vvsTso9zoz

° R 0500/3068, HQ5275, V75 F30 V58 F30, SIDESTREAM SUBREPORT P 0500/3068 MN065RB18 6318 H05275 M 10 -i -~ : Benzo(b)ftuoranthene ~ ~ -----~ s Benzo ( k) f l uoran t hene a) E ~ 8 z O 4, H m ® u it 1 r- - - -2 1 20 40 AC FIGURE 18 CONCENTRATION OF BENZO(-)FLUORANTHENES IN PROCESSED SWSC-I tmsmoz m 60 80 WEEK OF STUDY

P 0500/ 3068, H05277A, V84 F30 V78 F30, MAINSTREAM SUBREPORT P 0500/3068 MN065RB18 6161 H05277A M .. 5-, ~ ~ -O : ----- ~ Benzo ( a ) pyrene D i benz (- ) an t hracenes rn E 4 z 1 cib -Gu "' `~ t.) -.. ~ w w /-%wltb6 16/ -ca W ® AC FIGURE 19 1 40 F- 1 E0 80 WEEK O(" STUDY CONCENTRATION OF BENZO(a)PYRENE AND DIBENZ(-)ANTHRACENES IN PROCESSED MWSC-I s",tso9zoz

V" 0500f3068, H05276, V76 F30 V58 F30, SIDESTREAM SUBREPORT P 0500/3068 MN065RB18 6161 H05276 10-1 ~ ~ -A s ------~ : Benzo ( a ) pyrene Dibenz(-)anthracenes rn E v z D(L m [I] ® (Z ~e7 m ® 0 F- -2 1 20 40 60 80 AC FIGURE 20 WEEK OF STUDY CONCENTRATION OF BENZO(a)PYRENE AND DIBENZ(-)ANTHRACENES IN PROCESSED SWSC-I sbsisowoz

SUBRr:YUkT P U5UU/3U68 MN065HB18 6161 M HEADSPACE CHROMATOGRAM OF CRUDE MWSC-I Remarks: 1i = DMSO~, 2 = nicotine

INBIFO Institut fur biologische Forschung • Min SUBREPORT P 0500/3068 MN065RB19 6161! M' y~!..;. wvw.~.....rw.. ~ . BEADSP'ACE CHROMATOGRAM OF PROCESSED MWSC-I Remarks: 1 = DMSO, 2 = nicotine 3285

INBIIFO Institut fiir biologische Forschung • KtSIn SUBREPORT P 0500/30'68 MN065RB19 6161 M AC FIGURE 23 HEADSPACE CHROMATOGRAM OF CRUDE SWSC-I Remarks: 1 = DMSO, 2 = nicotine 3285

INBIFO Institut for biologische Forschung • K6ln SUBREPORT P0MNO RB' M ..~.~M~. •._.-~.. AC FIGURE 24 HEADSPACE CHROMATOGRAM OF PROCESSED SWSC-I Remarks: 1 = DMSO, 2 = nicotine 2 3285

SUBREPORT P 0500/3068 MN065RB21 7292 AC PAGE 6-1 6 REFERENCES Grimmer, G., Fraktionierung von Kondensaten aus verschiedenen Versuchszigaretten und Vergleich der PAH-Profile, Rauchen und Gesundheit 4 : 1-9 (1979) Hoffmann, D., Schmeltz, I., Hecht, S.S., Wynder, E.L.: Tobacco carcinogenesis, in: Gelboin, H.V., TS'o:, P.O. (Eds.): Polycyclic hydrocarbons and cancer, New York: Academic Press, Vol. 1, 1978 INBIFO study I 49 Sidestream-Kondensatgewinnung (PT), Study director: Dr.rer.nat. W. Reininghaus Data compilation filed on 19.Sep.83 INBIFO study P 0500/303'0 Skin painting study on whole smoke condensates from test cig- arettes X6-DO AQ~P, X6-DO AQQ, X6-DO AQR, X6-DO AQS, X6-DO AQT, X6-DO AQU, X6-DO AQV, X6-DO AQW, X6-DO AQX and standard reference cigarette 2R1 with CD-1 mice, part II: tables and figures, Study director: Dr.med.vet. A. Teredesai Date of report: 25.May 84 INBIFO study P 0500/3106 Determination of chemical parameters in mainstream and sidestream cigarette smoke condensate of standard reference cigarette 2R1 (PT) Study director: Dr.rer.nat M. Speck not yet reported INBIFO study P 0500/5007 Technische Vorarbeiten fur P 0500/3106, Study director: Dr.rer.nat M. Speck Date of documentation: 15.May 84 IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans, International Agency for Research on Cancer, Vol. 38, 1986 Lee, M.L., Novotny, M.V., Bartle, K.D., Analytical chemistry of polycyclic aromatic compounds, New York, Academic Press, 1981, pp. 441-449 Levins, R.J., Isolation of polyaromatic hydrocarbons from whole smoke condensate: A simple two-step procedure, Chromatographia 11 : 736 (197'8) Rossbach, W. (Hrsg.): Einsatz von Futter- und Einstreumitteln bei nichtklinischen Laboruntersuchungen, in: Veroffentlichung der Gesellschaft fur Versuchstierkunde, Nr. 9, Basel: 1980 ,", -

SUBREPORT P 0500/3068 MNO65RB22 7291 AC PAGE 6-2 Selkirk, J.K., Chemical carcinogenesis: a brief overview of the mechanism of action of polycyclic hydrocarbons, aromatic amines, nitrosamines, and aflatoxins, Carcinog. Compr. Surv. 5 : 1-31 (1980) Van Duuren, B.L., Orris, L., The tumor-enhancing principles of Croton tiglium L, Cancer Res. 25 : 1871 (1965) Van Duuren, B.L., Sivak, A., Goldschmidt, B.M., Katz, C., Melchi- onne, S., Initiating activity of aromatic hydrocarbons in two- stage carcinogenesis, J. Natl. Cancer Inst. 44 : 1167 (1970) Van Duuren, B.L., in: Gori, G.B., Bock, F.G. (Eds.): Banbury Report 3, A safe cigarette?: carcinogens, cocarcinogens, and tumor inhibitors in cigarette smoke condensate, 1980, pp. 105-112 END OF SUBREPORT AC 12R5

INBIFO Institut fur biologiscbe Forschung • Koln Dr.med. R. Rylander cLo FABRIQUES DE TABAC REUNUES S.A. Switzerland R E PQ RT P 0500/3068 .r rr.r.~r.r rrr.r.d~ HI5IFU 26.Oct.8Z DKU/UBE COPY NO.: Skin Tumorigenicity of Mainstream and Sid'estream Whole Smoke Condensate of Standard Reference Cigarette 2R1 80-Week Dermal Application. Study with CD1(IICR)1BR and B6C3F1 Mice Volume 3 INBIFO Instilut fur bidbgisahe Forsdsunp GmbH, Fugyerstre6e 3, D-fi000 Ktiln 90 Sitz der Gesellsc:hatl: KtSln HR B 367, 29. Oktober 1959 5065 Telefon: Porz (02203) 303-1, Telefex: (02203) 303362, Telex: 8874675 Inbi d Instilutsleifer und GesdhiiftBfiihrer: Dr. med. Ulrich Hddcenbere

INBIFO Institut fur biologische Forschung • Koln UR•MEU• R. RYLANDER 26•OCT.87 c/o FABR'IQUES llE TABAC REUNIES S.A. FTE/SSU SSU52 (R) B23 SWITZERLANU S U B R E P U R T Ih' I C R U B I 0 L U G Y P 050U/3068 SKIN TUfrl'URIbENICITY OF \ hIAINSTREAhI AND SIDESTREAM WHOLE SMOKE CONDENSATE OF STANDARU REFERENCE CIGARETTE 2R1 80-WEEK UERMAL APPLICATIUN STUDY W'ITH CI)1( ICR)BR AND B6C3F1 MICE INBIFO Institul /iii biologesche Forschung GmbH, Fuggerstraf3e 3, D-5000 KolA 90 Sitz der GesellsehaR: KSIn HR B 367, 29. Oktbber 1959 28.KW83 Tetebn: Porz (02203) 303-1, Teletax: (02203),303362, Telex: 8 874 675 inbi d Institutsltiler und Geschi;ttsluhrer, Dr. med. Ukich Hadcenberg

INBIFO Institut fur biologische Forschuing • K6ln SUBREPORT P,0500/3068 SSU52RB28 6136 MB PAGE 0-1 CONTENTS MB PAGE' ABBREVIATIONS 0-4 1 SUMMARY 1.1 Objective 1-1 1.2 Experimental 1-1 1.3 Results 1-2 1.4 Discussion 1-3 1.5 Comment 1-4 2 RESPONSIBILITY 2-1 3 INTRODUCTION 3-1 4 METHOD 4-1 4.1 Chronology of Microbiological Screening 4-1 4.2 Microbiological Screening of Mice 4-1. 4.3 Bacteriological Screening of Environment within the Animal Quarters 4-4 4.4 Identification of Bacteria According to INBIFO Standard Operating Procedures 4-7 MB FIGURE A: CHRONOLOGY OF MICROBIOLOGICAL SCREENING 4-2 5 RESULTS AND DISCUSSION 5-1 5.1 Text 5-1 5.1.1 Laboratory mice 5-1 5.1.2 Laboratory unit - 5-2 5.1.3 Diet, beddding material and drinking water 5-2 5.1.4 Laboratory staff 5-2 5.1.5 Discussion 5-3 5.1.6 Comment 5-4 3285

INBIFO Institut fur biologische Forschung • K6In SUBREPORT P 0500/3068 SU52RB29 6310 MB PAGE 0-2 CONTENTS (continued) MB TABLE 5.2 Tables SEROLOGICAL SCREENING FOR THE PRESENCE OF VIRUSES AND MYCOPLASMS FROM CD1 MICE (RECIPROCAL ANTIBODY TITER) SEROLOGICAL SCREENING FOR THE'PRESENCE'OF VIRUSES AND MYCOPLASMS FROM B6 MICE (RECIPROCAL ANTIBODY TITER) 1.2 MICROBIOLOGICAL AND PARASITOLOGICAL SCREENING, CD1 MICE 2.1 MICROBIOLOGICAL AND PARASITOLOGICAL SCREENING, B6 MICE 2.2 BACTERIOLOGICAL SCREENING OF ORGANS, CD1 MICE 3.1 BACTERIOLOGICAL SCREENING OF ORGANS, B6 MICE 3.2 BACTERIOLOGICAL SCREENING OF URINARY BLADDER WASHOUT, CD1 MICE 4.1 BACTERIOLOGICAL SCREENING OF URINARY BLADDER WASHOUT, B6 MICE 4.2 BACTERIOLOGICAL SCREENING OF LABORATORY SURFACES 5 BACTERIOLOGICAL SCREENING OF'LABORATORY AIR 6 BACTERIOLOGICAL SCREENING OF DIET FOR SALMONELLA SP. 7 BACTERIOLOGICAL SCREENING OF DIET 8 BACTERIOLOGICAL SCREENING OF DRINKING WATER, TAP WATER 9.1 BACTERIOLOGICAL SCREENING OF DRINKING WATER, WATER FROM BOTTLES WITH SIPPER TUBES 9.2 BACTERIOLOGICAL SCREENING OF DRINKING WATER, WATER FROM BOTTLES WITHOUT SIPPER TUBES 9.3 BACTERIOLOGICAL SCREENING OF CAGE: BEDDING'MATERIAL 10 BACTERIOLOGICAL SCREENING OF LABORATORY STAFF, FINGERTIPS 111 RESULTS OF SEROLOGICAL ASSAYS ON MICE 12 RESULTS OF BACTERIOLOGICAL, MYCOLOGICAL AND PARASI- TOLOGICAL ASSAYS PERFORMED BY EXTERNAL SPECIALISTS 13 . 2284

INBIFO Institut fur biologische Forschung • Kt51'n S D68 SU52RB 92 MB PAGE 0-3 CONTENTS (continued) MB TABLE RESULTS OF BACTERIOLOGICAL ASSAYS ON MICE 14 RESULTS OF BACTERIOLOGICAL ASSAYS ON THE ENVIRONMENT 15 FREQUENCY OF BACTERIAL SPECIES IN SAMPLES FROM THE ENVIRONMENT 16 MB PAGE 6 REFERENCES 6-1 Remarks: This subreport, including title page, contains 64 pages. 3285

INBIFO Institut fur biologische Forschung • Kt91n SUBREPORT P 0500/3068 SSU52 (R) B24 7292 MB PAGE 0-4 ABBREVIATIONS (a,b) ADENO : adenovirus of mice B6 : mouse strain B6C3F1 CD1 : mouse strain CRL:CD1(ICR)BR CFU : colony forming unit DMSO : d~imethyl sulfoxide ECTRO : ectromelia virus of mice ELISA : enzyme-linked iinmunosorbent assay EX : x as exponent to the base 10, e. g. E2 = 102 GD 7' : mouse encephalomyelitis virus strain GD 7 .GE. : greater than or equal to .GT. : greater than H'I : hemagglutination inhibition assay IF : immunofluorescence assay K : K virus LCM : lymphocytic choriomeningitis virus .LE'. : less than or equal to .LT. : less than MB : team Microbiology MHV : mouse hepatitis virus M. PULM.: Mycoplasma pulmonis MVM : minute virus of mice N : number of individual values OF : oxid'ation-fermentation POLY : polyoma virus PT : preliminary title PVM : pneumonia virus of mice KEO3 : reovirus type 3 RT : room temperature SOP : standard operating procedure sp. : species (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). 3285

INBIFO Institut fur biologische Forschung: • HCt9In SUBREPORT P 0500/3068 SSU79RB4 6127 MB PAGE. 0'-5 ABBREVIATIONS (continued) St. : Staphylococcus Strept.: Streptococcus 0 : no response + : response - : not assayed

INBIFO Institut fur biologische Forschung, • BCt9I'n SUBREPORT P 0500/3068 SSU52 (R) B19 6311 MB PAGE 1-1 1 SUMMARY Objective In accordance with the objectives of this study specified in the SUMMARY of the INTEGRATING REPORT the team Microbiology was responsible for (1) the virological and bacteriological screening of the mice and (2) the routine surveillance of the environment within the animal quarters. 1.2 Experimental In order to determine the hygienic status of the mouse strains CRL:CD1(ICR)BR (CD1) and B6C3F1 (B6), a microbiological screening program was performed. 20 untreated mice of each strain were screened microbiologically, 10 mice on arrival at INBIFO and 10 at the end of the study. Serological examinations were performed extramurally to detect antibodies against pathogenic viruses and the pneumotropic bacterium Mycoplasma pulmonis. Bacteriological, mycological and parasitological examinations of the mice were performed intramurally and/or extramurally. A routine surveillance program of the environment within the animal quarters was performed to examine periodically the labora- tory unit, animal diet, drinking water and cage bedding material as well as the laboratory staff for satisfactory hygienic condi- tions. The identification of the bacteria isolated after growth on nutrient agar under aerobic conditions was carried out accord- ing to their growth on selective and differential media, their stainability and their biochemical characteristics. 3285

INBIFO Institut fur biologische Forschung • KbIn SUBREPORT P 0500/3068 SSU52 (R) B20 7292 MB PAGE 1-2 1.3 Results All 5 CD1 and all 5 B6 mice examined on arrival at INBIFO for the presence of antibodies to 11 mouse-related viruses were found to be free from them with the exception of 4 out of 5 CD1 mice, which showed a low to moderate antibody level against to the pneumonia virus of mice. At the end of the study, antibodies to pneumonia virus were also detected in B6 mice. From both mouse strains, significant positive antibody levels against the mouse encephalo- myelitis virus strain GD 7 and mouse hepatitis virus were ob- tained. Antibodies against the minute virus of mice were detected in CD1 mouse, too. All mice also examined for the presence of serological activity to Mycoplasma pulmonis were found to be free from this pathogen. All 10 CD1 and all 10 B6 mice examined extramurally for the presence of pathogenic bacteria, fungi, endo and ectoparasites were found to be free from these pathogens. The organs heart, kidney, liver, lung and~ spleen as well as the urine of all mice examined intramurally were found to be sterile. The laboratory unit examined after disinfection but prior to the introduction of mice was found to be sterile. The low amount of .LT.300 colony forming units (CFU) per cubic meter and the pre- sence of potential pathogenic bacteria such as Staphylococcus aureus and Escherichia coli detected in the laboratory room air were referred to the mice present in that room. The incoming air was found to be sterile. The nonautoclaved diet was free from Salmonella. All samples of the autoclaved' diet and cage bedding material were found to be sterile. Samples of nonautoclaved drinking water were slightly contaminated with nonpathogenic bacteria or - only in few cases - 3285

INBIFO Institut fiir biologische Forschung • Mln SUBREPORT P 0500/3068 SSU52 (R) B21 7292 MB PAGE 1-3' with the potential pathogenic bacterium Pseudoinonas aeruginosa. The maximum amount of 120 CFU per liter tap water and 4700 CFU per liter drinking water taken from the bottles with special sipper tubes was within the acceptable level for drinking water for human consumption (a). Neither Escherichia coli nor coliforms were detectable in any water samples. The fingertips of the laboratory staff mainly contaminated with nonpathogenic bacteria were found to be also contaminated with potential pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Proteus mirabilis during animal handling. 1.4 Discussion The antibody titer against the pneumonia virus of mice detected in the serum of CD1 mice on arrival and in CD1 and B6 mice at the end of the study refers to the endemicity of this pathogen in the stock colony, which was already known to the breeder. The pneu- cnonia virus is known to appear strictly pneumotropic in mice without any clinical signs and seems to be not relevant for the present dermal application study. The mouse hepatitis virus detected at the end' of the study may have affected the gastrointestinal tract of the CD1 and B6 mice, but the influence of this pathogen on the results of the present study is not yet known. It should be noted, that CD1 mice derived from the same breeder 1 month later, showed a significant titer to this pathogen (INBIFO study P 0500/3071). The influence of the most prevalent mouse pathogen, the ence- phalomyelitis virus, and the influence of the- common endemic minute virus on mice in d'ermal application studies is not yet known. (a)' Federal Republic of Germany, Bundesgesetzblatt, (1975), limit: .LE..1E5 CFU per liter Teil 1: 453 3285

INBIFO Insfifutfiir bi'ologisehe Forschung • K6In SUBREPORT P 0500/3068 SSU79RB3 7132 MB PAGE 1-4 1.5 Comment The microbiological screening of the CD1 and B6 mouse strains showed significant antibody titers to 4 mouse-related viruses, but no influence on the results of the study was observed. Beside this, the results of the microbiological analyses of the environ- ment within the animal quarters were in agreement with the micro- bial flora acceptable for optimal hygienic conditions during long,term studies on mice. It should be noted that this is the 1st INBIFO study on 80-week dermal application with virological examinations reported. I N B I F 0 Institut fur biologische Forschung GmbH KK2

INBIFa Institut fiir biologische Forschung • Mn SUBREPORT P 0500/3068 SSU52 (R) Al M MB PAGE 2-1 2 RESPONSIBILITY Quality Assurance: ~:j .................. Dr.rer.nat. F. Tewes Biologist (Diplombiologe) E. Romer Biologist (Diplombiologe)' 3587

INBIFO Institut fur biologische Forschung • Kdln SUBREPORT P 0500/3068 SSU52 (R) Al l+4 2' RESPONSIBILITY Quality Assurance: MB PAGE 2-1 , Dr.rer.nat. F. Tewes Biologist (Diplombiologe). ... ......................... E. Romer Biologist (Diplombiologe) 2284

INBIFO Institut fur biologische Forschung • K6Iln SUBREPORT P 0500/3068 SSU52 (R) B22 6127 MB PAGE 3-1 3 INTRODUCTION Mice in conventional or barrier-maintained housing units are some- times affected by infectious viruses, bacteria, fungi, protozoa or parasites. Because of the impact on interpretation of experimental research, it is important to recognize those diseases and their effect on the laboratory mouse (Foster et al., 1982). In the present tumorigenicity study, microbiological controls of infectious contaminants in the laboratory mice as well as in their environment within the animal quarters were performed. Untreated mice were screened for pathogenic viruses such as the reovirus type 3 and~mouse hepatitis virus, which are able to cause diseases in the digestive system, for the parainfluenza virus type 1 murine, pneumonia virus of mice and K virus, which cause diseases in the respiratory system, for ectromelia of mice, the causative organism of mouse pox, for the lymphocytic choriomeningitis virus, which causes disease in the central nervous system not only in mice but also in man, for the oncogenic polyoma virus and for common endemic and potential covert contaminants such as the minute virus of mice, mouse adenovirus and mouse encephalomyelitis virus strain GD~ 7. The mice were also screened for the presence of serolog,ical activity to the pathogenic bacterium Mycoplasma pulmonis, for the presence of endoparasitic protozoa and hel- minthes as well as for ectoparasitic arthropoda. In order to detect infectious bacteria as soon as possible, the laboratory unit, diet, drinking water, cage bedding material as well as the laboratory staff were screened routinely, so that preventive steps can be taken to exclude entry of those bacteria to the mice. The routine detection of bacteria- were limited to gram-positive and~ gram-negative rods and~ cocci growing under aerobic conditions on blood or nutrient agar plates. This is the 1st INBIFO study on 80-week dermal application with virological examinations reported. 3285

INBIFO Institut fur biotogi'sche Forschung • KtSln SUBREPORT P 0500/3068 SSU52 (R) A2 7292 MB PAGE 4-1 4' METHOD \ 4.1 Chronology of Microbiological Screening (see MB FIGURE A) 4.2 Microbiological Screening of Mice Serological screening for the presence of viruses and mycoplasms Sample material and quantity: approx. 0.5 ml blood from the retro- bulbar venous plexus of'20 mice (a), 5 mice of each strain and time serum preparation see SOP MB 4/3, analysis performed by CRBS Charles River Biotechnical Services, Inc., via Charles River Wiga GmbH, w-8'741 Sulzfeld 1 1'ime : on arrival at INBIFO and at the end of the study, serum stored at -20 degrees centigrade until shipment to CRBS Results expressed in: species and antibody titer of viruses and' of Mycoplasma pulmonis Bacteriological, mycological and parasitological screening by external specialist Sample material and quantity: 20 living mice shipped in special filter crates, 5 mice of each strain and time Time: expert institute: Staatliches Veterinar-Untersuchungsamt, Dr.med.vet. R. Horter, Fachtierarzt fur Bakteriologie und Serologie, D-4930 Detmold on arrival at INBIFO and at the end of the study Results expressed im: species of organism detected (a) mice used for all serological and bacteriological screenings 3285

SUBREPORT P 0500/3068 SU52 (R) A3 7294 MB PAGE 4-2 date 29. 20. 17. 14. 14. 11. 9. 6. 4. 1. 29. 26. NOV. DEC. JAN. FEB. MAR. APR. MAY_ JUN. JUL AUG. AUG. SEP. 82 82 83 83 83 83 83 83 83 83 83 83 = = = I = = = I = = = I = = =. I = = = I = = = I = = = = I = = = I = = = I =_ __ = I = = = week of application -2 1 5 9 13 17 21 25 29 33 37 41 mice XX laboratory unit surfaces x air X X X X x x x x X X x fingertips X X x x x x x x x x diet autoclaved x x x x x x x x x x x nonautoclaved X(a)X X X X X X drinking water x x x x x x x x x X X cage bedding material x x x X X x x x x x x = = = I = = = I = = = I = = = I = = = I = = = I = = =. I = = = I = = = I = = =. I = = = I` = = = 2 1 5 9 13 17 21 25 29 33 37 41 MB FIGURE A GEIRON(S,OGY OF MICROBIOLOGICAL SCREIIJING (a) swiple was taken on 26.Oct.82 sssTso.qMz

SUBREPORT P 0500/3068 SU52 (R) A4 7294 MB PAGE 4-3 date 24. 21. 19. 16. 13. 12. 9. 7. 4. 2. OCT. NOV. DEC. JAN. FEB. MAR. APR. MAY JUN. JUL. 83 83 83 84 84 84 84 84 84 84 week of application mice laboratory unit surfaces air x x X X x x x x fingertips X X X x x x x x d iet autoclaved X X x x x x x x nonautoclaved X X x x drinking water X X cage bedding material I = _ = I = _ = I = _ = I = _ = I = _ = I = _ = I = _ = I = _ = I = _ = I 45 49 53 57 61 65 69 73 77 81 I =. _ = I = = = I = _ = I = = = I = _ = I = = = I =. _ = I = = = I = = = I 45 49 53 57 61 65 69 73 77 81 MB FIGURE A (continued) CHRONOLOGY OF MICROBIOLOGICAL SCREENING s~svsa9zoz

INBIFO Institut for biologische Forschuing • KSIn SUBREPORT P,0500/3068 SSU52 (R) A5 7226 MB PAGE 4-4 Bacteriological screening . Sample material and quantity: heart, kidney, liver, lung and spleen of 20 mice (a), 5 mice of each strain and time Time: urinary bladder washout of T0 mice (a) at the end~of the study, 5 mice of each strain on arrival at INBIFO and at the end of the study Results expressed in: bacterial species and number of CFU/ sample Scientific version: SOP MB 3/1, MB 4/3, MB 5/5, M.B 69/2 Text version: 14.Mar.86 4.3 Bacteriological Screening of Environment within the Animal Quarters Surfaces of laboratory unit Sample material and quantity: impressions from surface of floor, wall, rack, worktable and door handle, 1 sample each Time: after disinfection and prior to introduction of mice into the laboratory unit Results expressed in: bacterial species and number of CFU/sample Air conditioning system Sample material and quantity: Time: (1) incoming air, 500 1 (2) room air, 5001 each sample (1) after disinfection and prior to introduction of mice into the laboratory unit (2) 1 time/month, unannounced (a) mice used for all serological and bacteriological screenings 3285

INBIFO Institut fur biologische Forschung • KtSIn SUBREPORT P-0500/3068 SSU52 (R) A6 6134 MB PAGE 4-5 Results expressed in: bacterial species and number of CFU/m3 Diet Nonautoclaved Sample material and: quantity: nonautoclaved diet of each batch, 1 sample of approx. 10 g/batch Time: before feeding Results expressed in: absence or presence of Salmonella sp. Autoclaved Samples material and quantity: autoclaved diet, 2 samples of approx. 10 g/batch Time: immediately after sterilization, 1 time/month, unannounced Results expressed in: absence or presence of bacteria Drinking water Sample material and quantity: tap water and drinking water from drinking bottles with sipper tubes, approx. 250 ml, 3 samples each Time: 1 time/month, unannounced Results expressed in: bacterial species and number of CFU/1 Cage bedding material Sample material and quantity: autoclaved bedding material, 2 samples of approx. 0.5 g each N Time: immediately after sterilization, 0 1 time/month, unannounced N Results expressed~ in: absence or presence of bacteria ~ CA N ~ 3285

IMBIFO Institut fWir biol'ogische Forschung - MIn SUBREPORT P 0500/3068 SSU52 (R) B18 6063 MB PAGE 4-6 Laboratory staff Sample material and quantity: 5 fingertips of each person working within the laboratory unit Time: 1 time/month, unannounced Results expressed in: bacterial species and number of CFU/5 fingers Scientific version: SOP MB 13/3, MB 15/2, MB 16/4, MB 18/3, MB 19/5, MB 20/5, MB 22/2 Text version: 3.Mar.86 3285

INBIFO Institut fOr biologische Forschuing • K6In SUBREPORT P 0500/3068 SSU52 (R) A7 6063 M MB PAGE 4-7 4.4 Identification of Bacteria According,to INBIFO Standard Operating Procedures Aerobic and facultative anaerobic rods and cocci: identification of bacteria by micro- scopical determination, stainability, biochemical reactions, growth behavior on selective and differential media as well as susceptibility towards antibiotics Standard operating procedures: see attached microfiche no. E454 Scientific version: SOP MB 25/6, MB 26/1, MB 27/1, MB 28/2 MB 29/1, MB 30/3, MB 31/1, MB 35/2, MB 36/2, MB 37/1, MB 38/1, MB 39/1, MB 40/3, MB 41/2' Text version: 1.Feb.85

SUBREPORT P 0500/3068 SSU52RB25 6310 MB PAGE 5-1 5 RESULTS AND DISCUSSION 5.1 Text 5.1.1 Laboratory_mice On arrival at INBIFO, the serum of CD1 mice (4 out of 5) yielded a low to moderate titer of HI antibodies against the pneumonia virus of mice (see TABLES 1). At the end of the study, the antibody titer was found to be increased and a low to moderate titer was also detected in B6 mice (4 out of 5). CD1 as well as B6 mice showed significant ELISA antibody titer to the mouse encephalo- myelitis virus strain GD 7. Significant ELISA antibody titers to the mouse hepatitis virus detected in CD1 (1 out of 5) and B6 mice (2 out of 5) were confirmed by the presence of IF: antibodies. Significant ELISA antibody titer to the minute virus of mice was detected in 1 CD1 mouse and was confirmed by the presence of IF antibodies, too. All mice also examined for the presence of serological activity to Mycoplasma pulmonis were found to be free f rom this pathogen. All 10 CD1 and all 10 B6 mice examined extramurally for the presence of pathogenic bacteria, f ungi, endo and ectoparasites were found to be free from these pathogens on arrival and at the end of the study (see MB TABLES 2). The organs heart, kidney, liver, lung and spleen of all 10 CD1 and all 10 B6 mice examined intramurally were found to be sterile (see MB TABLES 3). The urine of 5 CD1 and 5 B6 mice examined at the end of the study was also sterile (see MB TABLES 4). -Ae

SUBREPORT P 0500/3068 SSU52Rh26 7292 MB PAGE 5-2 5.1.2 Laboratory_unit The laboratory unit examined after disinfection but prior to the introduction of mice was found to be sterile (see MB TABLE 5). The low amount of .LT.300 colony forming units per cubic meter and the presence of potential pathogenic bacteria such as Staphy- lococcus aureus and Escherichia coli detected in the laboratory room air were referred to the mice present in that room (see MB TABLE 6). The incoming air was found to be sterile. 5.1.3 Diet~_bedding_material_and_drinking_water The nonautoclaved diet was free from Salmonella (see MB TABLE 7). All samples of the autoclaved diet and cage bedding material were found to be sterile (see MB TABLES 8' and 10). Samples of nonautoclaved drinking water were slightly contaminated with nonpathogenic bacteria or in 7 out of 135 water samples with the potential pathogenic bacterium Pseudomonas aeruginosa (see MB TABLES 9). The maximum amount of 120CFU per liter tap water and 4700 CFU per liter drinking water taken from the bottles with special sipper tubes was within the acceptable level for drinking water for human consumption (a). Neither Escherichia coli nor coliforms were detectable in any water samples. 5.1.4 Laboratory_staff The fingertips of the laboratory staff mainly contaminated with nonpathogenic bacteria were found to be also contaminated, with potential pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Proteus mirabilis during animal handling (see MB TABLE 11).. (a) Federal Republic of Germany, Bundesgesetzblatt, Teil 1: 453 (1975), limit: .LE.1E5 CFU per liter

SUBKEPUR`1' P 05U0/3Uti8 SSU52RB27 7292 - MB PAGE 5-3 5.1.5 Discussion The antibody titer against the pneumonia virus of mice detected in the serum of CD1 mice on arrival at INBIFO refers to the endemi- city of this pathogen in the stock colony, which was already known to the breeder. During the present study the pneumonia virus was distributed to the B6 mice held in the same laboratory unit. This pathogen is known to appear strictly pneumotropic in mice without any clinical signs (Parker and Richter, 1982) and seems to be not relevant for present dermal application study. Antibody titers against the mouse hepatitis virus were detected in CD1 and B6 mice at the end of the study. Mouse hepatitis virus infections are reported to be associated with clinical signs in the range from none to paralysis etc. varying with virus strain, tissue tropism, mouse strain and age, and with the absence or presence of enhancing or inhibitory f actors (Kraft, 1982). The gastrointestinal tract with liver, stomach and' intestines may be affected. It should be noted, that CD1 mice derived from the same breeder 1 month later, showed a significant titer to this pathogen (INBIFO study P 0500/3071). The influence of this virus on mice in dermal application studies is not yet known. The most prevalent and ubiquitous of the mouse pathogens, the encephalomyelitis virus strain GD 7, was.detected in all CD1 and B6 mice at the end of the study. Natural infections with this pathogen are associated with flaccid paralysis of the hind leg with a frequency of paralyzed mice of 1E-4 (Downs, 1982). Apart from this, mice appear healthy. Mortality is seen only after intracerebral inoculation in mice. The encephalomyelitis virus in this study seemed to be distributed from a colony of SENCAR mice housed in the same environmental quarters (INBIFO study P 0500/ 3082). The influence of this pathogen on mice in dermal appli- cation studies is not yet known.

------- 5UBkEPUK'P P U500'/3U6ti S'SU79KB1 7292 - MB PAGE 5-4 Antibody titer against the minute virus of mice was detected in 1 out of 10 mice (CD1 and B6) at the end of the study. Infections with this highly contagious but mildly pathogenic agent is re- ported to be usually asymptomatic in laboratory mice (Ward and Tattersall, 1982). Because of the low observed prevalence of this pathogen no relevance to mice in the present study is assumed. 5.1.6 Comment The microbiological screening of the CD1 and B6 mouse strains showed significant antibody titers to 4 mouse-related viruses, but no influence on the results of the study was observed. Beside this, the results of the microbiological analyses of the environ- ment within the animal quarters were in agreement with the micro- bial flora acceptable for optimal hygienic conditions during long-term studies on mice (see MB TABLES 12 to 16). V

S[n3REPOR<r P 0500/3068 SSU52 (R) A8 6127 5.2 Tables PATHOGEN ME!'HOD (a) adenovirus of mice (adeno) CF ectromelia virus of mice ELISA (ectro) K virus (K) HI lynphocytic chorio- IF meningitis virus (LCM) minute virus of mice (MVM) ELISA mouse enoephalomyelitis virus ELISA strain GD 7 (GD 7) mouse hepatitis virus (MHV)', ELISA parainfluenza virus type 1 ELISA murine (sendai) pneumonia virus of mice (PVM) HI polyoma virus (poly) HI reovirus type 3 (reo3) HI Mycoplasma pulmonis (M. pulm.) ELISA MOUSE NO. SIGNIFI- CANCE MB-1 MB-2 MB 3 MB-4 MB-5 GE. 0 0 0 0 0 10 0 0 0 0 0 + 0 0 0 0 0 10 0 0 0 0 0! + 0 0 0 0 01 + 0 0 0 0 0 + 0 0 0 0 0 + 0 0 0 0 0 + 20 0 20 40 20 10 0 0 0 0 0 10 0 0 0 0 0 20 0 0 0 0 0 + MB TABLE 1.1 SERQIAGICAL SCREENING FOR THE: PRESENCE OF VIRUSES AN) MYCOPLASMS FROM CDU MICE (RECIPROCAL AbTPIBODY TITER) Remarks: date of serum sampling: 3.Dec.82, on arrival at INIDIFO date of shipment to expert institute: 31.Ju1.84 (a) CF: canplement fixation assay IF: immunofluorescence assay HI: hemagglutination inhibition assay ELISA: enzyme-linked immunosorbent assay

SUBREPORT P 0500/3068 SSU52 (R) A9 6127 PATHOGEN ME'1"HOD MO[]SE NO. (a) MB-6 MB-7 adenovirus of mice (adeno) CF 0 0 IF - - ectranelia virus of mice ELISA 0 0 (ectro) K virus (K) HI 0 0 lymphocytic chorio- IF - (b) 0 meningitis virus (LCNt) minute virus of mice (MVM) ELISA 0 0 IF - - mouse enoephalo:nyelitis virus ELISA + + strain GD 7 (GD:7) mouse hepatitis virus (MHV) ELISA 0 0 IF - - parainfluenza virus type 1 ELISA 0 0 murine (sendai) pneumonia virus of mice (PVM) HI .GT.80 10 polyoma virus (poly) HI 0 0 reovirus type 3 (reo3) HI 10 0 Mycoplasma pulmonis (M. pulm.) ELISA 0 0 MB TABLE 1.1 (continued) MB-8 MB-9' MB-10 SIGNIFI- CANCE GE. 0 - (b) 0 10 - - (b) - + 0 0 0 + 0 0 0 10 - (b) - (b) 0 + 0 0 + + - - + + + + + + 0 0 + + - - + + 0 00 + 0 201.GT.80 10 0 0 0 10 10 0 0 20 0 0 0 + SEROIDGICAL SCREENING FOR THE PRESENCE OF VIRUSES AND MYCOPLASMS FROM CD1 MICE (RECIPROCAL ANTIBODY TITER) Remarks: date of serum sanQling: 2.Jul.84, at the end of the study date of snipment to expert institute: 4.Ju1.84 (a). CF: camplement fixation assay IF: innwnofluorescence assay HI: henagglutination inhibition assay ELISA: enzyme-linked iimiunosorbent assay (b) nonspecific reaction

SUBl2FPORT P 0500/3068 SSU52 (R) A10 PATHOGEN METHOD ( a ) adenovirus of mice (adeno), CF ectranelia virus of mice ELISA (ectro) K virus (K) HI lymptiocytic chorio- IF meningitis virus (LCM) minute virus of mice (MVM) ET.ISA mouse enoephalomyelitis virus ELISA strain GD 7 (GD 7) :nouse hepatitis virus (MHV) ELISA parainfluenza virus type 1 ELISA murine (sendai) pneumonia virus of mice (PVM)' HI polyoma virus (poly) HI reovirus type 3: HI Mycoplasma pulmonis (M. pulm.) ELISA MB TABLE 1.2 6127 MOUSE NO. SIGNIFI- CANCE MB-1 MB-2 MB-3 MB-4 MB-5 GE. 0 0 0 0 0 10 0 0 0 0 0 + 0 0 0 0 0 10 0 0 0 0 0 + 0 0 0 0 0 + 0 0 0 0 0 + 0 0 0 0 0 + 0 0 0 0 0 + 0 0 0 0 0 10 0 0 0 0 0 10 0 0 0 0 0 20 0 0 0 0 0 + SEE20IAGICAL SCREENING FOR THE PRESENCE OF VIRUSES AND MYCOPLASMS FROM B6 MICE (RECIPROCAL AAirIBODY TITER), Remarks: date of serum sanpling: 2.Dec.82, on arrival at INBIFO date of shipment to expert institute: 31.Jul.84 (a) CF: compleroent fixation assay IF: imnunofluorescence assay HI: hemagglutination inhibition assay ELISA: enzyme-linked immunosorbent assay

SCBREPOFrr P 0500/3068 SSU52 (12) All 6127 PATHOGEN METHOD (a) adenovirus of mice (adeno) CF ectrcmelia virus of mice ELISA (ectro) K virus (K) HI lymphocytic chorio- IF meningitis virus (LCM) minute virus of mice (MVM) ELISA mouse encephalanyelitis virus ELISA strain GD 7 (GD 7) mouse hepatitis virus (MHV) ELISA IF CF parainfluenza virus type 1 ELISA murine (sendai) pneumonia virus of mice (PVM) HI polyoma virus (poly) HI reovirus type 3 (reo3) HI IF Mycoplasma pulmonis (M. pulm.) ELISA MB TABLE 1.2 (continued) MOUSE NO. MB-6 MB-7 MB-8 MB-9 MB-10 SIGNIFI- GANCE GE. 0 0 0 0 0 10 0 0 0 0 0 + 0 0 0 0 0 10 - (b) - (b) - (b), - (b) - (b) + 0 0 0 0 0 + + + + + + + + + (c) 0/+ 0/+ (c) 0 + + - (b) + - (b) - + - 0 - 0 - 10 0 0 0 0 0 + 20 0~ 10 10 40 10 0 01 0 0 0 10 0 0 0 0 20 (d) 20 - - - - 0 + 0 0 0 0 0 + SEftOirOGICAL SCREENINIG FdR THE PRE.SENCE OF VIRUSES AND MYCOPLASMS FR(M B6 MICE (RECIPROCAL ANTIBOllY TITER) Remarks: date of serum sampling: 2.Ju1.84, at the end of the study date of shipment to expert institute: 4.Ju1.84 (a)' CF: complement fixation assay IF: immunofluorescence assay HI: henagglutination inhibition assay ELISA: enzyme-linked immunosorbent assay (b) nonspecific reaction (c) not confirmed by CF (d) not confirmed by IF

SUB[tEPOFt`1' P 0500/3068 SSU52 (R) A12 7292 PATfIOCEN SAMPLE METHOD bacteria Leptospira sp. (australis, autun- blood agglutination- nalis, bataviae, gxippotyphosa, lysis test icterohaemorrhagiae, pomona) Haemobartonella sp. animal animal test Bacillus piliformis organs (a) cultivation and Bordetella bronchiseptica It " bacteriological Erysipelothrix rhusiopathiae u " identification Klebsiella pneunoniae Is ~ Listeria monocytogenes Mycoplasma sp. ~ n Pasteureila multocida ll i It Pasteure a pseudatuberculosis Streptocoocus pneumoniae 11 No Salmonella sp. organs (a), enrichment and: feces bact. ident. fungi Trichophyton mentagrophytes skin macroscopical, microscopical and cultivation test protozoa Noselna cuniculi brain, histological, Toxoplasma sp. " lung, liver, " toxoplasma dye kidney, and complement spleen fixation test RESULT 0 0 0 0 Eimeria sp. feces microscopical 0 test after en- ricfiment in sodiun chloride nematodes cestodes feces IN macroscopical and " microscopical test with enrichment in sodium chloride arthr d opo a acar i skin macroscopical 0 roallophaga n " and microscop- 0 pediculidae IN i ical test 0 siphonaptera It 01 0 MB TABLE 2.1 M'ICR()BIOLOGICAL AND PARASITOLOGICAL SCREENING, CD1 MICE ttenarks: date of shipment to expert institute: 7.Dec.82 and 3.Jul.84 10 mice, 5 mice on arrival and 5 mice at the end of the study (a) heart, liver, lung, spleen, kidney, intestine and pharynx

S'IJBREPOkT P 0500/3068 SSU5-27Lt) A1 a 7292 PATHO(EN SAMPLE METH00 bacteria Leptospira sp. (australis, autun- blood agglutination- nalis, bataviae, grippotyphosa, lysis test icterohasnorrhagiae, pomona) Haenobartonella sp. animal animal test Bacillus pilifocmis organs (a) cultivation and Bordetella bronchiseptica " bacteriological Erysipelothrix rhusiopathiae o identification Klebsiella pneumoniae ~~ Listeria monocytogenes ~ Mycoplasma sp. Pasteurella multocida ~ Pasteurella pseudotuberculosis 5treptocoocus pneunoniae ~ Salmonella sp. organs (a), enrichment and feces bact. ident. fungi Trichophyton mentagxophytes skin macroscopical, microscopical and cultivation test protozoa Nosema cuniculi brain, histological, Toxoplasma sp. " lung, liver, " toxoplasma dye kidney, and ccxnplenent spleen fixation test RESULT 0 0 0 0 0 0 0 Eimeria sp. feces microscopical 0 test after en- riahrnent in sodium chloride nematodes cestodes feces u macroscopical and " microscopical test with enridment in sodium chloride th d ar ropo a acari skin macroscopical 0 mallophaga n " and microscop- 0 pedioil idae n " ical test 0 siphonaptera u m 0 MB TABLE 2.2 MICl2OBIOUOGICAL AND PARASITOLOGICAL SC[tEENIW, B6 MICE Ranarks: date of shipment to expert institute: 7.Dec.82 and 3.Jul.84 10 mice, 5 mice on arrival and 5 mice at the end of the study (a) heart, liver, lung, spleen, kidney, intestine and pharynx

SUBREPUfrP P 0500/3068 SSU52 (R) A14 6063 DATE MOUSE NA. SAMPLE N[1MBEE2 OF ORGANISM IDEIdPIFIED BACTERIA (CFU/sample) 3.Dec.82 MB-1 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-2 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-3 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-4 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-5 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB TABLE 3.1 BACTERIOIUGICAL SCREERiIING OF ORGAINS, CD1 MICE Remarks: on arrival at INBIFO

SUBREPORT P 0500/3068 SSU52 (R) A15 6063 L1ATE MOUSE N3. SAMPLE Nf1HIBER OF ORGANISM IDEIJPIFIED BACTERIA ( CFU/sample ) 2.Jul.84 MB-6 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-7 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-8 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-9 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-10 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB TABLE 3.1 (continued) BACTERIOIAGICAL SCREENING OF ORiGAM, CD 1 MICE Renarks: at the end of the study

SUBREPURr P 0500/3068 SSU52 (R) A16 6063 DATE MOUSE RU. SAMPLE NUMBER OF BACTERIA (CFU/sample) ORGANISM IDEBTPIFIED 2.Dec.82 NIB-1 heart 0 - kidney 0 - liver 1(a), Streptococcus sp. group D lung 0 without hemolysis - spleen 0 - MB-2 heart 0 kidney 0 liver 0 lung 0 spleen 0 mB-3 heart 0 kidney liver lung 1 (a) 1 (a) 0 Streptococcus sp. group D without hemolysis Staphylocoecus epidermidis spleen 0 mB-4 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-5 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB TABLE 3.2 BACTERIOiLAGICAL SCREENING OF OR(3ANS, B6 MICE Remarks: on arrival at INBIFO (a) The nimber and species of bacteria detected does not indicate any bacterial infection. •?p,

SUBItN:YOlCl' P 0500/3068 SSU52 (it) !il /' DATE MOUSE ND. SAMPLE N[ANBER OF ORGANISM IDENI'IFIED BACTERIA (CFU/sample) 2.Jul.84 MB-6 heart 0 kidney 0 liver 0 lung 0 spleen 0 [4s-7 heart 0 kidney 0 liver 0 lung 0 spleen 0 mB-8 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-9 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB-10 heart 0 kidney 0 liver 0 lung 0 spleen 0 MB TABLE 3.2 (continued) BACTERIOLOGICAL SCREEIdING OF ORGAM, B6 MICE 1Remarks: at the end of the study

SUBREPORT P 0500/3068 SSU52 (R) A18 DATE MOUSE NUMBER OF ORGANISM IDENTIFIED NO. BACTERIA (CFU/sample) 2.Jul.84 MB-6 0 MB-7 0 MB-8 G MB-9 0 MB-10 0 MB TABLE 4.1 BACTERIOLOGICAL SCREENING OF URINARY BLADDER WASHOUT, CD1 MICE Remarks: at the end of the study

-- SUBREPORT P 0500/3068 SSU52 (R) A19 6063 DATE MOU'SE' NUMBER OF ORGANISM IDENTIFIED N0. BACT'ERIA (CFU/sample) 2.Ju1.84 MB-6 0 MB-7 0 MB-8 0 MB-9 0 MB-10 0 MB TABLE 4.2 BACT'ERIOLOGICAL SCREENING OF URINARY BLADDER WASHDUT, B6 MICE Remarks: at the end of the study

---- SUBREPORT P 0500/3068 SSU52 (R) A20 mATE HfJCM SAMPLE NUMBER OF ORGANISM IDENTIFIED NO. BACTERIA ( CFfJ/sample ) 30.Nov.82 201 floor 0 wall 0 rack 0 workttable 0 door handle 0 202 floor 0 wall 0 worktable 0 door handIle 0 203 floor 0 wal1 0 worktable 0 door handle 0 MB TABLE 5 BACTERIOUOGICAL SCREENING OF LABORATORY SURFACES 2294

------- SUBREPORT P 0500/3068 SSU52 (R) A21 DATE FR00E4 ND. AIR NUANBER OF BACTERIA (CFU/m3 ) ORGANISM IDENPIFIED 1.Dec.82 202 incoming 0 30.Dec.82 202 room 22 Staphylococcus epidermidis 14.Jan.83 202 room 12 gram-positive rods other gram-negative rods (a) Acinetobacter anitratus 7.Feb.83 202 roem 10 Staphylococcus saprophyticus Staphylococcus saprophyticus 1.Mar.83 202 room 14 gram-positive rods Staphylococcus epidermidis 8.Apr.83 202 room 14 gram-positive rods Pseudamonas-like group 5E-2 gram-positive rods 16.May 83 202 room 280 Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus 7.May 83 02 oom 0 gram-positive rods Acinetobacter anitratus Streptococcus sp. group D with alpha-heznolysis 60 6.Jun.83 202 room 28 Staphylococcus epidermidis 5.Ju1.83 02 oom 0 gram-positive rods Acinetobacter anitratus Pseuc3anonas-like, group 5E-2 Streptocoacus sp. group D without hemolysis Staphylococcus saprophyticus gram-positive rods Staphylococcus epidermidis Acinetobacter anitratus MB TABLE 6 BACTERIOIOGICAL SCREENING OF LAHORATORY AIR (a) swarming with round pseudocoloniies 2~l@,

SUBREeolrr P 0500/3068 SSU52 (R) A22 6063 DATE ROOM NO. AIR NUMBER OF BACTERIA ( CFU/m3 ) ORGANISM IDENTIFIED 23.Aug.83 202 roan 60 Staphylococcus saprophyticus P,ahromobacter sp. 2.Sep.83 202 rocm 40 Staphylococcus saprophyticus gram-positive rods Aerococcus viridans 10.Oct.83 202 room 60 Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis 18.Nov.83 202 rocan 46 gram-positive rods Staphylococcus saprophyticus Moraxella sp. Pseudomonas-like group:2K-1 2.Dec.83 202 room 20 Streptococcus sp. group D without hemolysis Staphylococcus saprophyticus Acinetobacter anitratus Streptococcus sp. group D with alpha-hemolysis 13.Jan.84 202 room 42 (a) gram-positive rods Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis 3.Feb.84 202 roan 130 Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis Staphylococcus aureus MB TABLE 6 (continued) BAC.'TEL2IOIAGICAL SCREENING OF LABORATORY AIR N 0 N ~ 0 ~ N ~ ~ N (a) add. 4 CFU of fungi

SUHREPOR`t' P 0500/3068 SSU52 (R) A23 6063 DATE ROOM AU. AIR NUMBER OF BACTERIA (CFU/m3) ORGANISM IDELJTIFIED 9.Feb.84 202 room 22 (a) Staphylococcus saprophyEicus 02 oom 6 (b) gram-positive rods Escherichia coli Streptococcus sp. with alpha-hemolysis Staphylococcus saprophyticus .Mar.84 02 oom 0 gram-positive rods Streptococcus sp. group D without hemolysis Streptococcus sp. group D with alpha-hemolysis Staphylococcus aureus Staphylococcus saprophyticus 3.Apr.84 02 oan 0: Streptococcus sp. gxoup D with alpha-hemolysis gram-positive rods Staphylococcus epidermidis Acinetobacter anitratus Staphylococcus saprophyticus .May 84 02 oan 8 gram-positive rods Staphylococcus epidermidis Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus 12.Jun.84 202 room 130 gram-positive rods Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus aureus Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis Escherichia coli MB TABLE 6 (continued) BACTERIOLOGICAL SCREENING OF IABOItATORY AIR (a) on blood agar (b) on nutrient agar

SUBREPORT P 0500/3068' SSU52 (R) A29 6073 DATE SAMPLE NO. SAMPLE SALMONELLA IDENTIFIED SIZE (g) 26.Oct.82 999 11 no t4.Dec.82 11111 11 " 3.Mar.83 1480 15 " 5.May 83 1917 11 " 16.Jun.83 2188 14 " 11.Aug.83 2469 110 " 30.Sep.83 3023 14 " 21.Nov.83 3400 16 " 3401 18 " 23.Jan.84 3724 10 " 14.Mar.84 3982 10 " 7.May 84 4303 15 " MB TABLE 7 BACTERIOLOGICAL SCREENING OF DIET FOR SALMONELLA SP. Remarks: nonautoclaved diet "Herilan MRH Haltung" batch no.: 4221, 4911, 0921, 1825, 2361, 3152, 3921, 4611, 4612, 0212, 1122, 1847 date of suipply: 25.Oct.82, 14.Dec.82, 2.Mar.83, 5.May 83, 15.Jun.83, 10.Aug.83, 29.Sep.83, 17.Nov.83, 19.Jan.84, 13.Mar.84, 4.May 84

INBIFO Institut fur biblogische Forschung • MIn SUBREPORT P 0500/3068 SSU:52 (R) A30 6073 DATE SAMPLE SAMPLE ORGANISM IDENTIFIED: NO. SIZE (g' ) 6.Dec.82 1076 11 no 1077 9 " 4.Jan.83 1158 9 " 1159 11 " B.Feb.83 1390 13 " 1391 11 " 2.Mar.83 1478 11 " 1:479 15 " 12.Apr.83 1795 22 " 1796 15 " 2.May 83 1882 9 " 1883 12 " 6.Jun.83 2158 13 " 2159 14 " 5.Jul.83 2243 13 " 2244 10 " 8.Aug.83 2455 14 " 2456 10 " 6.Sep.83 2690 8 " 2691 8 " 5.Oct.83 3075 9 " 3076 10: " 22.Nov.83 3450 14 " 3451 13 " MB TABLE 8 BACTERIOLOGICAL SCREENING OF DIET Remarks:: autoclaved diet batch no.: 4221, 4911, 0921, 1825, 2361, 3152, 3921 date of supply: 25.Oct.82, 14.Dec.82, 2.Mar.83, 5.May 83, 15.Jun.83, 10.Aug.83, 29.Sep.83 3285

INBIFO Institut fur biologische Forschung • 4(tSln SUBREPORT P 0500/3068 SSU52 (R) B1 6073 DATE SAMPLE NO:. SAMPLE SIZE, ORGANISM IDENTIFIED (9) 6.Dec.83 3502 14 no 3503 9 I 17.Jan.84 3696 13 o 3697 11 i 7.Feb.84 3851 10 n 3852 12 ~ 14.Mar.84 3983 10 i 3984 8 t 10.Apr.84 4158 10 ~ 4159 11 ~ 2.May 84 4273 9 1 4274 9 01 6.Jun.84 4450 11 ~ 4451 11 MB TABLE 8 (continued) BACTERIOLOGICAL SCREENING'OF DIET Remarks: autoclaved diet batch no.: 3921, 4611, 4612, 0212, 1122, 1847 date of supply: 29.Sep.83, 17.Nov.83, 19.Jan.84, 13.Mar.84, 4.May 84 KH2

INBIF4 lnstitut fur biologische Forschung • Kt5ln SUBREPORT P 0500/3068 SSU52 (R) B2 6073 DATE, SAMPLE NO. NUMBER OF BACTERIA (CFU/1). ORGANISM IDENTIFIED 7.Dec.82 1086 120 Acinetobacter anitratus 1087 0 - 1088 0 - 1089 12 other gram-negative rods 1090 0 - 1091 0 - 10.Jan.83 1178 0 1179 0 1180 0 1181 0 1182 0 1183 0 2'.Feb.83 1358' 0 - 1359 4 gram-positive rods 136o 4 Pseudomonas sp. 1361 0 - 1362 0 - 1363 0 (a) - 4.Mar.83 1484 4 Pseudomonas maltophilia 1485 0 - 1486 0 - 6.Apr.83 1735 0 1736 0 1737 0 9.May 83 1944 8 gram-positive rods 1945 4 Pseudomonas sp. 1946 60 Pseudomonas-like group 2K-11 gram-positive rods Pseudomonas sp. 8.Jun.83 2173 40 Pseudomonas-like group 2K-1 gram-positive rods 2174 0 - 2175 24 Pseudomonas-like group 2K-1 MB TABLE 9.1 BACTERIOLOGICAL SCREENING OF DRINKING WATER, TAP WATER (a) add. 4 CFU/1 of fungi 3285

INBIFO !'nstitut fur biologische Forschung • NCSIn SUBREPORT P 0500/3068 SSU52 (R) B3 6073 DATE' SAMPLE NO. NUMBER OF BACTERIA ORGANISM IDENTIFIED (CFU/1 )' 6.Jul.83 2257 48 Pseudomonas maltophilia 2258 0 - 2259 44 Pseudomonas maltophilia gram-positive rods 3.Aug;.83 2427 12 Pseudomonas maltophilia 2428 0 - 2429 0 - 21.Sep.83 2857 0 - 2858 4 gram-positive rods 2859 12 gram-positive rods 5.Oct.83: 3089 0 309'0 0 3091 0 2.Nov.83 3377 0( 3378 0 3379 0 7.Dec.83 3472 4 gram-positive rods 3473 0 - 3474 0 - 11.Jan.84 3641 4 (a) Staphylococcus epidermidis 3642 4 Acinetobacter lwoffii 3643 01 - 8.Feb.84 3853 0 - 3854 4 gram-positive rods 3855 0 - 7.Mar.84 3953 0 - 3954 0 - 3955 4 gram-positive rods 4.Apr.84 4128 0 4129 0 4130 0 MB TABLE 9.1 (continued) BACTERIOLOGICAL SCREENIN'G'OF DRINKING WATER, TAP WATER (a) add. 4 CFU/l of fungi 3285

INBIFO Institut fur biolbgische Forschung • Kt51n SUBREPORT P 0500/3068 SSU52 (R) B4 6073 .. DATE SAMPLE' NO. NUMBER OF ORGANISM IDENTIFIED BACTERIA (;CFU/1) 2.May 84 4265 0 4266 0 4267 0 6.Jun.84 4442 0 4443 0 4444 & MB TABLE 9.1 (continued) BACTERIOLOGICAL SCREENING OF DRINKING WATER, TAP WATER 3285

INBIFO Institut fur biotogisch~e Forschung • K6Iin SUBREPORT P 0500/3068 SSU52 (R) B5 6073 DATE SAMPLE NO. NUMBER OF BACTERIA (CFU/1). ORGANISM IDENTIFIED 7.Dec.82 1092 16 gram-positive rods 1093 32 gram-positive rods 1094 32 Acinetobacter Acinetobacter anitratus anitratus 1095 110 Staphylococcus Acinetobacter epidermidis anitratus Staphylococcus epidermidis Pseudomonas aeruginosa 1096 260 Acinetobacter anitratus gram-positive rods Staphylococcus epidermidis 1097 50 Pseudomonas aeruginosa Staphylococcus epidermidis 10.Jan.83 1184 100 Acinetobacter anitratus Staphylococcus epidermidis 1185 360 other gram-negative rods Staphylococcus epidermidis 186 5 Staphylococcus saprophyticus Pseudomonas aeruginosa Acinetobacter lwoffii Staphylococcus epidermidis other gram-negative rods 1187 280 Acinetobacter lwoffii Staphylococcus epidermidis Acinetobacter lwoffii 1188 140 other gram-negative rods Acinetobacter anitratus StaphyTococcus epidermidis 189 40 other gram-negative rods Pseudomonas-like group 2K-1 Pseudomonas maltophilia Staphylococcus epidermidis Acinetobacter anitratus Pseudomonas aeruginosa MB TABLE 9.2 BACTERIOLOGICAL SCREENING OF DRINKING WATER, WATER FROM BOTTLES WITH SIPPER TUBES 3285

INBIFO Institut fur biologische Forschung - K6ln SUBREPORT P 0500/3068 SSU52 (R) B6 6073 . DATE SAMPLE NO. NUMBER OF BACTERIA (CFU/1) ORGANISM IDENTIFIED 2.Feb.83 1364 520 (a) Pseudomonas sp. Pseudomonas-like group 2K-1 Acinetobacter lwoffii Pseudomonas aeruginosa 1365 90 Pseudomonas-like group 2K-1 Acinetobacter lwoffii Pseudomonas maltophilia Pseudomonas sp. gram-positive rods 1366 670 (b) Pseudomonas maltophilia Acinetobacter lwoffii Pseudomonas sp. Pseudomonas-like group 2K-1 1367 390 Acinetobacter lwoffii Pseudomonas-like group 2K-1 Pseudomonas sp. Pseudomonas maltophilia Staphylococcus epidermidis Staphylococcus saprophyticus Pseudomonas aeruginosa Acinetobacter anitratus 1368 220 Acinetobacter lwoffii Pseudomonas-like group 2K-1 Pseudomonas maltophilia Pseudomonas sp. Staphylococcus saprophyticus Staphylococcus epidermidis 1369 130 Pseudomonas maltophilia Pseudomonas sp. Acinetobacter lwoffii Pseudomonas-like group 2K-1 Acinetobacter anitratus Staphylococcus saprophyticus MB TABLE 9.2 (continued) BACTERIOLOGICAL SCREENING OF DRINKING'WATER, WATER FROM BOTTLES WITH SIPPER TUBES - (a) add. 4 CFU/l of yeast (b) add. 8 CFU/1 of fungi 3285

INBIFO Institut filr biologische Forschung • Kt91n SUBREPORT P 0500/3068 SSU52 (R) B7 6073' ~ DATE SAMPLE NO. NUMBER OF BACTERIA (CFU/1) ORGANISM IDENTIFIED 4.Mar.83 1487 140 Staphylococcu~s epidermidis Pseudomonas maltophilia 1488 70 Staphylococcus epidermidis 1489 24 Staphylococcus epid~ermidis 6.Apr.83' 1738 0 1739 0 1740 0 9.May 83 1947 1400 Staphylococcus epidermids Pseudomonas sp. 1948 440 Aerococcus viridans Staphylococcus epidermidis Pseudomonas sp. 1949 36 Staphylococcus epidermidis Pseudomonas sp. gram-positive rods B.Jun.83 2176 90 Staphylococcus epidermidis 2177 28 Staphylococcus epidermidis Pseudomonas paucimobilis 2178 130 Staphylococcus epidermidis gram-positive rods 6.Jul.83 2260 70 Staphylococcus epidlermidis Pseudomonas maltophilia 2261 60 Staphylococcus epidermidis Pseudomonas sp. 2262 190 Staphylococcus epidermidis Pseudomonas maltophilia Acinetobacter lwoffii 3.Aug.83 2430 48 Pseudomonas maltophilia 2431 55 Staphylococcus epidermidis 2432 130 Staphylococcus epidermidis MB TABLE 9.2 (continued) BACTERIOLOGICAL SCREENING OF DRINKING WATER, WATER FROM BOTTLES WITH SIPPER TUBES 3285

INBIFO Institut fur biologische Forschung • KtSin SUBREPORT P 0500/3068 SSU52 (R) B8 6073 ~ DATE SAMPLE NO. NUMBER OF BACTERIA (CFU/1) ORGANISM IDENTIFIED 21.Sep.83 2860 490 Staphylococcus epidermidis 2861 1300 gram-positive rods Acinetobacter anitratus Staphylococcus epidermidis 2862 230 Staphylococcus epidermidis 5.Oct.83 3092 20 Acinetobacter anitratus Staphylococcus epidermidis 3093 40 gramrpositive rod~s Staphylococcus epidermidis 3094 8 Acinetobacter anitratus Staphylococcus epidermidis Acinetobacter lwoffii 2.Nov.83 3380 0 - 3381 4 gram-positive rods 3382 0 - 7.Dec.83 3469 9 gram-positive rods 3'470 0 Staphylococcus epidermidis - 3471 34 Staphylococcus epidermidis 11.Jan.84 3644 4 Pseudomonas-like group 2K-1 gram-positive rods Staphylococcus epidermidis 3645 12 Staphylococcus epidermidis 3646 0 - B.Feb.84 3856 0 - 3857 0 - 3858 160 Staphylococcus epidermidis MB TABLE 9.2 (continued) BACTERIOLOGICAL SCREENING OF DRINKING WATER, WATER FROM BOTTLES WITH SIPPER TUBES Remarks: sample size 250 ml 500 ml (2.Nov.83) 470 ml (7.Dec.83) KKR

INBIFO Institut fur bialogische Forschung • KtSIn SUBREPORT P 0500/3068 SSU52 (R) B9 6073 DATE SAMPLE NUMBER OF ORGANISM IDENTIFIED NO. BACTERIA (CFU/1) 7.Mar.84 3956 4700 StaphyTococcus epidermidis 3957 60 Acinetobacter lwoffii Staphylococcus epidermid~is 3958 3100 gram-positive rods Staphylococcus epidermidis .Apr.84 131 50 Aerococcus viridans Acinetobacter lwoffii Pseudomonas aeruginosa Staphylococcus epidermidis 4132 13 Aerococcus viridans 41,33 230 Staphylococcus epidermidis Aerococcus viridans. 2.May 84 4268 24 Staphylococcus epidermidis Staphylococcus epidermidis 4270 16 Staphylococcus epidermidis 6.Jun.84 4445 0 Micrococcus sp. 4446 0 4447 0 MB TABLE 9.2 (continued) BACTERIOLOGICAL SCREENING OF DRINKING WATER, WATER FROM BOTTLES WITH SIPPER TUBES Remarks: sample size 250 ml KKM

INBIFO Institu.rt fur biologische Forschuing • KBIn SUBREPORT P 0500/3068 SSU52 (R) B10 6073 DATE SAMPLE NO. NUMBER OF BACTERIA (CFU/1) ORGANISM IDENTIFIED 9.May 83 1950 28 Pseudomonas sp. 1951 20 Pseudomonas-like group 2K-1 gram-positive rods Pseudomonas-like group 2K-1 1952 32 gram-positive rods Staphylococcus epidermidis Pseudomonas sp. gram-positive rods Moraxella sp. MB TABLE 9.3 BACTERIOLOGICAL SCREENING OF DRINKING'WATER, WATER FROM BOTTLES WITHOUT SIPPER TUBES Remarks: sample size 250 ml 3285

INBIF0 Institut fur biologische Forschung • K6In SUBREPORT P' 0500/3068 SSU52 (R) B11 6073 DATE' SAMPLE NO. SAMPLE SIZE (g) ORGANISM IDENTIFIED 6. Dec. 82' 1078 0.6 no 1079 0.7 s 1 1i . Jani. 83 1201 0.5 t 1202 0.6 i 7.Feb.83 1371 0.7 ~ 1372 0.6 ~ 21.Mar.83 1605 0.5 1 1606 0.5 T 5.Apr.83 1719 0.4 1 1720 0.3 t 9.May 83 1958 0.5 , 1959 0.4 ~ 7.Jun.83 2170 0.4 t 2171 0.7 g 18.Jul.83 2302 0.4 m 2303 0.4 1 11'.Aug,.83 2482 0.5 I 2483 01.5 t 6.Sep.83 2688 0.3 m 2689 0.3 1 4.Oct.83 3073 0.4 1 3074 0.4 n 22.Nov.83 3452 0.5 3453: 0.5 ~ 7.Dec.83 3476 0.6 n 3477 0.6 MB TABLE 10 BACTERIOLOGICAL SCREENING OF CAGE'BEDDING MATERIAL Remarks: autoclaved cage bedding material 3285

INBIFO Institut fiar biologische Forschung • K6I'n SUBREPORT P 0500/3068 SSU52 (R) B12 6073 DATE SAMPLE NO. SAMPLE ORGANISM IDENTIFIED SIZE' (g) 3.Jan.84 3564 0.6 no 3565 0.4 " 23.Feb.84 3'909 0.7 " 3910 0.6 " 8.Mar.84 3959 0.4 " 3960 0.4 " 16.Apr.84 4205 0.5 " 4206 0.5 " 2.May 84 4261 0.6 " 4262 0.5 " 5.Jun.84 4431 0.4 " 4432 0.4 " MB TABLE 10 (continued)' BACTERIOLOGICAL SCREENING OF CAGE BEDDING MATERIAL Remarks: autoclaved cage bedding material 3285

INBIF(7lnstitut fi]r biologische Forschung • Kbln SUBREPORT P 0500/3068 SSU52 (R) A24 6073 DATE SAMPLE NUMBER OF ORGANISM IDENTIFIED NO. BACTERIA (CFU/5 fingers)' 20.Dec.82 1116 700 1117 8 1118 500 1119 500 11120 500 3.Jan.83 1154 250 1155 200 1156 300 1157 28 16.Feb.83 1458 70 1459 39 MB TABLE li 1 Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus Proteus mirabilis Acinetobacter anitratus Staphylococcus epidermidis Streptococcus sp. group D with alpha-hemolysis Acinetobacter anitratus Streptococcus sp. group D with alpha-hemolysis Streptococcus sp. group D with alpha-hemolysis Micrococcus sp. Proteus mirabilis Staphylococcus epidermidis gram-positive rods Escherichia coli Acinetobacter anitratus Staphylococcus saprophyticus gram-positive rods Staphylococcus saprophyticus Acinetobacter lwoffii giram-positive rods Escherichia coli Staphylococcus epidermidis Staphylococcus saprophyticus gram-positive rods Micrococcus sp. Staphylococcus saprophyticus gram-positive rods Proteus mirabilis Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis BACTERIOLOGICAL SCREENING OF LABORATORY STAFF, FINGERTIPS 3285

INBIF4 Institut fi7r biol'ogische Forschung • KtSIn SUBREPORT P 0500/3068 SSU52 (R) A25 6073: DATE SAMPLE NO. NUMBER OF BACTERIA (CFU/5 fingers) 7.Mar.83 1495 250 1496 350 18.Apr.83 1797 600 1798 250 1799 800 2.May 83 1884 300 1885 150 1886 200 1887 1000 6.Jun.83 2160 70 2161 800 4.Jul.83 2239 180 MB TABLE 11 (continued) t ORGANISM IDENTIFIED Staphylococcus saprophyticus Acinetobacter lwoffii Pseudomonas maltophilia Staphylococcus saprophyticus Pseudomonas vesicularis Staphylococcus epidermidis Aerococcus viridans Proteus mirabilis Staphylococcus saprophyticus Aerococcus viridans Staphylococcus saprophyticus Aerococcus viridans Streptococcus sp. group D with alpha-hemolysis gram-positive rods Flavobacterium sp. Staphylococcus epidermidis Acinetobacter lwoffii Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus gram-positive rods Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis gram-positive rods Streptococcus sp. group D with alpha-hemolysis gram-positive rods BACTERIOLOGICAL SCREENING OF LABORATORY STAFF, FINGERTIPS Mip

I'NB1Ffa Institut fur biologische Forschung • Kbl'n SUBREPORT P 0500/3068 SSU52 (R) A26 6073 DATE SAMPLE NO. NUMBER OF BACTERIA (CFU/5 fingers) ORGANISM IDENTIFIED 1.Aug.83 2422 800 Staphylococcus saprophyticus 423 5 Streptococcus sp. group D with alpha-hemolysis gram-positive rods Staphylococcus saprophyticus 15.Sep.83 2815 65 Streptococcus sp. group D 2816 45 with alpha-hemolysis Staphylococcus saprophyticus Streptococcus sp. group D 10.Oct.83 3117 1000 with alpha-hemolysis Escherichia coli Acinetobacter anitratus .Nov.83 385 00 Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus Proteus mirabilis Staphylococcus saprophyticus 386 5 Streptococcus sp. group D with alpha-hemolysis Proteus mirabilis Streptococcus sp. group D 387 000 with alpha-hemolysis gram-positive rods Acinetobacter lwoffii Staphylococus saprophyticus 5.Dec.83 3461 200 Streptococcus sp. group D with alpha-hemolysis Proteus mirabilis 3462 500 Staphylococus saprophyticus Acinetobacter anitratus Staphylococcus aureus Acinetobacter anitratus Strepotococcus sp. group D with alpha-hemolysis MB TABLE 11 (continued) BACTERIOLOGICAL SCREENING OF LABORATORY STAFF, FINGERTIPS 3285

INBIFO Institut fur biologische Forschung • K61n SUBREPORT P 0500/3068 SSU52 (R) A27 6073 ~ DATE SAMPLE NUMBER OF ORGANISM IDENTIFIED NO. BACTERIA (CFU/5 fingers) 2.Jan.84 3559 250 3560 50 20.Feb.84 3893 1000 3894 800 12.Mar.84 3977 800 3978 500 2.Apr.84 4095 150 4096 1000 4097 200 MB TABLE 11 (continued) Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus gram-positive rods Staphylococcus saprophyticus Acinetobacter anitratus gram-positive rods Escherichia coli Proteus mirabilis Streptococcus sp. group D with alpha-hemolysis Staphylococcus aureus Acinetobacter anitratus Escherichia coli Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus Acinetobacter lwoffii Citrobacter amalonaticus Staphylococcus epidermidis Staphylococcus saprophyticus Acinetobacter anitratus Proteus mirabilis Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus Proteus mirabilis Staphylococcus saprophyticus Streptococcus sp. group D with alpha-h.emolysis Acinetobacter anitratus Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis Proteus mirabilis BACTERIOLOGICAL SCREENING OF'LABORATORY STAFF, FINGERTIPS 3285

INBIFO Institut fur biologische Forschung • Koin INDEX REPORT P 0500/3068 BRA95DIVA27 CONTENTS INTEGRATING REPORT: SUMMARY Conclusions INTRODUCTION TABLE C: GROUPS AND DOSES FIGURE B: CHRONOLOGY SUBREPORT ANALYTICAL CHEMISTRY SUBREPORT ANIMAL TREATMENT SUBREPORT MICROBIOLOGY SUBREPORT PATHOLOGY . INDEX CODE 643 . '6,-Ise!-zutts CWf' 4 c.6' 32b6

I'NB1Fd Institut fur biologische Forschung • K6ln SUBREPORT P 0500/3068 SSU52 (R) A28 6073 DATE SAMPLE NUMBER OF ORGANISM IDENTIFIED NO. BACTERIA (CFU/5 fingers) 14.May 84 4368 500 4369 100 4.Jun.84 4420 4 4421 70 MB TABLE 11 (continued) Staphylococcus saprophyticus Streptococcus sp. group D with alpha-hemolysis Staphylococcus aureus Streptococcus sp. group D with alpha-hemolysis Staphylococcus epidermidis Staphylococcus saprophyticus Acinetobacter anitratus gram-positive rods Staphylococcus epidermidis Streptococcus sp. group D with alpha-hemolysis Staphylococcus saprophyticus Proteus mirabilis BACTERIOLOGICAL SCREENING OF LABORATORY STAFF, FINGERTIPS 3285

SUBREPORT P 0500/3068 SSU52 (R) B13 6127 DATE NWBER OF MICE (N) ANALYZED WITfi POSITIVE RESULT MICROORGANISM ADENO ECTRO K on arrival 5 CD1 0 0 0 at INBIFO 5 B6 0 0 0 at the end 5_ CD1 0 0 0 of study 5 B6 0 0 0 MB TABLE 12 Z tW ~ O ~ ~ ~ c ~ ~ ~ M M 7 V NDAI M LY 03 . LM. Q O O EE3 N c) =r CD O 0 0 0 0 0 4 0 0 0 ~ 0 0 0 0 0 0 0 0 0 =r e ~ co ~ 0 1 5 1 0 4 0 0 a 3 0 0 5 2 0 4 0 0 0 RESULTS OF SEROLOGICAL ASSAYS ON MICE Remarks: expert institute: Charles River Biotechnical Services, Inc., via Charles River Wiga GmbH, D-8741 Sulzfeld 1 tT9TS09?A)z

INBIFO Institut fur biblogische Forschung • K6In SUBRE'PORT P 0500/3068 SSU52 (R) B14 DATE NUMBER OF MICE (N) on arrival at INBIFO at the end of the study MB TABLE 13 ANALYZED WITH POSITIVE' RESULT BACTERIA FUNGI PARASITES 5 CD1 0 0 5 B6 0 0 5 CD1 0 0 0 5 B6 0 0 0 RESULTS OF BACTERIOLOGICAL, MYCOLOGICAL AND PARASITOLOGICAL ASSAYS PERFORMED BY EXTERNAL SPECIALISTS 2284;

INBIiFO Institut fOr biologische Forschuang • KtNn SUBREPORT P 0500/3068 SSU52 (R) B15 BACTERIUM N[IMBER OF SAMPLES WITH POSITIVE RESULT (N) ON ARRIVAL AT AT THE EiJD OF:THE Si'UDY INBIFO STRAIN C'D 1 B6 STRAIN CD1 B6 Streptococcaceae Streptococcus sp. without fenolysis 0 0 0 0 with alpha-hemolysis 0 0 0 0 with beta-hemolysis 0 0 0 0 group D without hemolysis 0 0 0 0 Aerococcus viridans 0 0 0 0 Micrococcaceae Staphylococcus aureus 0 0 0 0 Staphylococcus epidermidis 0 0 0 0 Staphylococcus saprophyticus 0 0 0 0 Micrococcus sp. 0 0 0 0 gram-positive rods 0 0 0 0 Neisseriaceae Neisseria sp., nonpathogenic 0 0 0 0 Acinetobacter sp. 0 0 0 0 Pseudomonadaceae Pseudanonas aeruginosa 0 0 0 0 Pseudomonas sp. 0 0 0 0 Enterobacteriaceae Escherichia coli 0 0 0 0 Salmonella sp. 0 0 0 0 Citrobacter freundii 0 0 0 0 Klebsiella pneLUnoniae 0 0 0: 0 EQebsiella aerogenes 0 0 0 0 Enterobacter agglomerans 0 0 0 0 Proteus vulgaris 0 0 0 0 Proteus mirabilis 0 0 0 0 other gram-negative rods 0 0 0 0 number of samples analyzed 25 25 30 30 MB TABLE 14 RESULTS OF BACTERIOWGICAi, ASSAYS ON MICE 2284

INBIFO Institut fur biologische Forschung • IKSln S[7BREPORT P 0500/3068 SSU52 (R) B16 6310 BACTE2IUM NUMBER OF SAMPLE,S' WITH POSITIVE RESULT (N) LS LA DN DA WN' BA FT V Streptococcaceae Streptococcus sp. without hemolysis 0 1 - 0 0 0 group D without hemolysis 0 3 - 0 0 0 0 group D with alpha-hetnolysis 0 9 - 0 0 0 28 Aerococcus viridans 0 1 - 0 4 3 Micrococcaceae Staphylococcus aureus 0 4 - 0 0 0 3 Staphylococcus epidermidis 0 9 - 0 47. 0 8 Staphylococcus saprophyticus 0 17 - 0 4 0 32 Micrococcus sp. 0 0 - 0 1 0 2 gram-positive rods 0 15 - 0 25 0 14 Neisseriaceae Neisseria sp., nonpathogenic 0 0 - 0 0 0 0 Acinetobacter anitratus 0 6 - 0 13 0 11 Acinetobacter lwoffii 0 0 - 0 14 0 5 Moraxella sp. 0 1 - 0 1 0 01 Pseudomonadaceae Pseudanonas sp. 0 0 - 0 15 0 0 Pseudomonas aeruginosa 0 0 - 0 7 0 0 Pseudomonas maltophilia 0 0 - 0 14 0 1 Pseudomonas paucimobilis 0 0 - 0 1 0 0 Pseudomonas vesicularis 0 0 - 0 0 0 1 Pseudomonas-like group 2K-1 0 1 - 0 13 0 0 Pseudomonas-like group 5E-2 0 2 - 0 0 0 0 Achromobacter sp. 0 1 - 0 0 0 0 Flavobacterium sp. 0 0 - 0 0 0 1 Enterobacteriaceae Escherichia coli 0 2 - 0 0 0 5 Salmonella sp. 0 0 0 0 0 0 0 Citrobacter amalonaticus 0 0 - 0 0 0 1 Proteus mirabilis 0 0 - 0 0 0 12 other gram-negative rods 0 1 - 0 5 0 number of sanples analyzed 13 23 12 38 135 38 46 MB TABLE' 15 RESULTS OF BACTERIOLfGICAL ASSAYS ON Tf3E ENVIRON+lEPTP Remarks: LS: laboratory surfaces (after disinfection) LA: laboratory air DN: diet, nonautoclaved DA: diet, autoclaved WN: drinking water, nonautoclaved BA: cage bedding material, autoclaved FT: fingertips 2284

INBIFO Institut tur biologische Forschung • NCt51'n SUBREPORT P 0500/3068 SSU52 (R)' B17 6127 BACTERIUM NUMBER OF SAMPLES WITH POSITIVE RESULT (a) FREQUENCY (b) (0/0) Staphylococcus epidermidis 64 20.98 gram-positive rods 54 17.70 Staphylococcus saprophyticus 53 17.38 Streptococcus sp. group D with alpha-hemolysis 37 12.13 Acinetobacter anitratus 30 9.84 Acinetobacter lwoffii 19 6.23 Pseudomonas maltophilia 15 4.92 Pseudamonas sp. 15 4.92 Pseudcmonas-like group 2K-1 14 4.59 Proteus mirabilis 12 3.93 Aerococcus viridans 8 2.62 Staphylococcus aureus 7' 2.30 Pseudomonas aeruginosa 7 2.30 Escherichia coli 7 2.30 other gram-negative rods 6 1.97 Streptococcus sp. group D without hemolysis 3 0.98 Micrococcus sp. 3 0.98 Moraxella sp. 2 0.66 Pseudomonas-liike group 5E-2 2 0.66 Streptococcus sp. without hemolysis 1 0.33' Pseudanonas paucimobilis 1 0.33 Pseudomonas vesicularis 1 0.33 Achromobacter sp. 1 0.33 Flavobacterium sp. 1 0.33 Citrobacter amalonaticus 1 0.33 MB TABLE 16 FREQUENCY OF BACTERIAL SPECIES IN' SAMPLES FROM THE ENVIRONMENT (a) total number of sacples analyzed = 305 (b) relative to total number of sanples analyzed 3285

IN~BIFO~ Institut fur biologische Forschung • KSin SUBREPORT P 0500/3068 SSU79RB2 7292 MB PAGE 6-1 6 REFERENCES . Downs, W.G.: Mouse encephalomyelitis virus, in: Foster, H.L., Small, J.D., Fox, J.G. (Eds.): The mouse in biomedical research, New York: Academic Press, 1982, pp. 341-352 Foster, H.L., Small, J.D., Fox, J.G. (Eds.): The mouse in bio- medical research, New York: Academic Press, 1982 INBIFO study P 0500/3071, 20-Day dermal application of impaction trap mainstream and side- stream condensates of standard reference cigarette 2R1 on CRL: CD1(ICR)BR and'. B6C3F1 mice, determination of d'ecarboxylase and aryl hydrocarbon monooxygenase (PT), Study director: D. Kuhn not yet reported INBIFO study P 0500/3082, 20-Week dermal application of mainstream condensate of the standard reference cigarette 2'R1, condensate preparation by impaction trap, mouse strain SENCAR (PT), Study director: Dr.rer.nat. R.-A. Walk not yet reported Kraft, L.M.: Viral diseases of the digestive system, in: Foster, H.L., Small, J.D., Fox, J.G. (Eds.): The mouse in biomedical research, Naw York: Academic Press, 1982, pp. 173-191 Parker, J.G., Richter, C.B.: Viral diseases of the respiratory system, in: Foster, H.L., Small, J.D., Fox, J.G. (Eds.): The mouse in biomedical research, New York: Academic Press, 1982, pp. 134-141 Ward, D.C., Tattersall, P.J.: Minute virus of mice, in: Foster, H.L., Small, J.D., Fox, J.G. (Eds.): The mouse in biomedical research, New York: Academic Press, 1982, pp. 313-334 END OF SUBREPORT MB