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Protocol B6030 / P 0500/3080 Chronic Effects of Room-Aged Sidestream Smoke of the Standard Reference Cigarette 1r4f As A Model for Environmental Tobacco Smoke Long-Term Inhalation Study on Rats

Date: 04 Dec 1992
Length: 45 pages
2029252561-2029252605
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Author
Gomm, W.
Kindt, R.
Reininghaus, W.
Schepers, G.
Stinn, W.
Teredesai, A.
Tewes, F.
Voncken, P.
Vonholt, K.
Author (Organization)
Inbifo, Institut Fur Biologische Forschung
Area
VON HOLT,KLAUS/INBIFO OFFICE
Type
LPRO, LAB PROTOCOL
ABST, ABSTRACT
BIBL, BIBLIOGRAPHY
CHAR, CHART, GRAPH, TABLE, MAPS
DRAW, DRAWING
Named Organization
American Society of Toxicology
Bundesgesetzblatt
Charles River Wiga
Epa, Environmental Protection Agency
Inbifo, Institut Fur Biologische Forschung
Intl Org for Standardization
NIH, Natl Inst of Health
Ntp
Oecd, Office (Org) of Economic Cooperation & Development
Pag
Pj Murphy Forest Product
Univ of Ky
Volkswagen
Agway Country Foods
Named Person
Bomhard
Boorman
Cochran
Conover
Crain
Deerberg
Duncan
Edwards
Finney
Haenszel
Hallmann
Heinrich
Hollander
Kalbfleisch
Koch
Kolmer
Kroes
Kruskal
Lamb
Lewis
Lutzen
Maita
Mantel
Miyamoto
Mullink
Pappenheim
Pauluhn
Peto
Prentice
Rehm
Reid
Rick
Sachs
Takizawa
Til
Ueberberg
Vandenberghe
Wallis
Woutersen
Young
Holt, K.V.
Recipient (Organization)
Ftr, Fabriques De Tabac Reunies S.A.
Document File
2029252530/2029252606/Ets Lifetime
Request
Stmn/R2-038
Litigation
Stmn/Produced
Characteristic
MARG, MARGINALIA
Site
I22
Master ID
2029252531/2605

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1r4f
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yrg79e00

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INBIFO Institut fur biologische Forschung GmbH ~ kna 'u ~iPu FABRIQUES DE TABAC REUNIES S.A. 4 Dec.92 CH-2003 Neuchatel WST/CST Switzerland COPY NO.: PROTOCOL B6030 / P 0500/3080 Chronic Effects of Room-Aged Sidestream Smoke of the Standard Reference Cigareite 1 R4F as a Model for Environmental Tobacco Smoke Long-Term Inhalation Study on Rats INBIFO Institut Or biobyIsche Forschunp GmbH. Fuyperotr. 3, 5000 K81n 90 SIt[ der Gesellachaft: K61n HR B 367 's~ Talafon (02203) 303•1, Telefax (02203) 303•362, Telex 6874675 Inbl d Geach3flsl3hrer: Dr. WoH Reinfnphaus
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 1 CONTENTS PAGE ABSTRACT 3 1 OBJECTIVE 4 2 EXPERIMENTAL DESIGN AND PROCEDURES 4 2.1 General Design 4 2.2 Animals and Treatment 5 TABLE 1 HOUSING CONDITIONS 7 TABLE 2 HEALTH CHECK SCHEDULE 9 TABLE 3 PHYSICAL DETERMINATIONS OF TEST ATMOSPHERES WITHIN THE EXPOSURE CHAMBERS 11 TABLE 4 GROUPS AND CONCENTRATIONS 12 2.3 SS Generation and Room Aging 12 FIGURE 1 SS GENERATION, AGING, AND EXPOSURE 13 2.4 Generation of Diesel Engine Exhaust (DEE) 14 FIGURE 2 DEE GENERATION AND EXPOSURE 14 2.5 Test Atmosphere Characterization 15 2.6 Biological Assays and Determinations 15 2.6.1 Biomonitoring 15 2.6.2 General Observations 15 n-92
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I N B I FO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 2 CONTENTS PAGE TABLE 5 ANALYSIS OF TEST ATMOSPHERES 16 TABLE 6 BIOMONITORING 19 TABLE 7 GENERAL OBSERVATIONS 20 2.6.3 Hematology 22 2.6.4 Pathology 22 TABLE 8 HEMATOLOGICAL AND PATHOLOGICAL EXAMINATIONS 24 TABLE 9 ORGANS/TISSUES FOR ORGAN WEIGHT DETERMINATIONS AND HISTOPATHOLOGICAL EXAMINATIONS 26 2.7 Statistics and Evaluation 30 3 BACKGROUND 32 4 TEST MATERIAL 33 4.1 RASS 33 4.2 DEE 34 5 RESPONSIBILITIES 35 6 GOOD LABORATORY PRACTICE STATEMENT 37 7 STORAGE OF RECORDS AND MATERIALS 38 8 REFERENCES 39 9 ABBREVIATIONS 44 1562
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I N B I FO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 ABSTRACT PAGE 3 In the present inhalation study on rats, chronic effects of ROOM-AGED CIGARETTE SIDESTREAM SMOKE (RASS) will be investigated, the respiratory tract being of major interest. Sidestream smoke (SS) will be generated from the standard reference filter cigarette 1R4F smoked in basic conformity with ISO standards. RASS will be generated dynamically by aging SS for 0.5 hours in a ventilated room under controlled conditions. Diesel engine exhaust (DEE), as positive control, will be generated by running a conventional passenger car Diesel engine according to a defined city driving cycle. The test atmospheres will be characterized by comprehensive chemical and physical analyses. Male and female Wistar rats will be head-only exposed to RASS or DEE for 7 hours/day, 5 days/week, for 30 months. The study will be terminated if mortality in the low RASS groups or sham-exposed groups reaches 75 %. The RASS concentrations will be 0, 1, 3, and 10 µg total particulate matter (TPM)/l, that of DEE will be 10 µg TPM/I. Biological assays and determinations as required by OECD guideline 451 will be carried out to determine the chronic effects of RASS and DEE. Nonneoplastic and neoplastic lesions par- ticularly in the respiratory tract will be of major interest. Systemic effects, e.g., changes in body and organ weights, signs of intoxication, will also be determined. Biomonitoring will be performed. tf92
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I N B I FO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 4 1 OBJECTIVE In the present inhalation study on rats, chronic effects of ROOM- AGED CIGARETTE SIDESTREAM SMOKE (RASS) will be investigated, the respiratory tract being of major interest. The highest particle concentrations to be used exceed those determined for extreme human exposure to environmental tobacco smoke (ETS) by a factor of approx. 10. 2 EXPERIMENTAL DESIGN AND PROCEDURES ---------------------------------- ---------------------------------- 2.1 General Design The present study will be performed as a long-term inhalation study on male and female rats head-only exposed to 3 concentra- tions of RASS (1, 3, and 10 µg TPM/1) as well as to filtered air (sham control group) and diluted Diesel engine exhaust (DEE, 10 µg TPM/l, positive control) for 7 hours/day, 5 days/week. The inhala- tion period will be 30 months, but the study will be terminated if 75 % mortality in the low RASS or sham-exposed rats is exceeded. A group of sentinel rats will be kept for 24 months to monitor the health status of the rats. RASS will be used as an experimental model for ETS. Sidestream smoke (SS) will be generated from the standard reference filter cigarette 1R4F smoked in basic conformity with ISO standards. RASS will be generated dynamically by aging SS for 0.5 hours (mean age) in a ventilated room under controlled conditions. DEE will be gen- erated by running a conventional passenger car Diesel engine according to a defined city driving cycle. The test atmospheres will be characterized by comprehensive chemical and physical analyses. 1592
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 5 Nonneoplastic and neoplastic lesions particularly in the respiratory tract will be of major interest. Systemic effects (e.g., changes in body and organ weights, signs of intoxication) will also be determined. Effects will be compared to those seen in the sham-exposed negative and DEE-exposed positive control groups. Biomonitoring will be performed. The study will be performed in basic compliance with OECD guide- line 451 and the NTP specifications for carcinogenicity studies. It will also be performed in accordance with the "Guiding Principles in the Use of Animals in Toxicology" adopted by the American Society of Toxicology. It will comply with good laboratory practice standards. 2.2 Animals and Treatment Male and female outbred Wistar rats (Crl:(WI)WU BR) (a), bred un- der specified pathogen-free conditions (Charles River Wiga, Germany), will be used. The use of Wistar rats in the present study is recommended for the following reasons: low rate of spon- taneous tumors particularly in the respiratory tract (Bomhard et al., 1986; Boorman and Hollander, 1973; Crain, 1958; Deerberg et al., 1980, 1982; Kroes et al., 1988; Maita, 1979; Rehm et al., 1984; Takizawa and Miyamoto, 1976; Ueberberg and LUtzen, 1979; Vandenberghe, 1990), longevity (Mullink, 1981), sensitivity to respiratory tract carcinogens (Heinrich et al., 1986, 1992; Woutersen et al., 1986), and suitability for head-only exposure (personal communications from Dr. Pauluhn, Dr. Kolmer, Dr. Hein- rich) as well as the large amount of additional data on this strain (e.g., Mullink, 1980, 1981; Til, 1978). The rats will be kept in INBIFO rooms R612 and R614, barrier main- tained to protect the rats from possible contamination by staff or (a) not in accordance with NTP which recommends the Fischer 344 rat t552
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 6 materials. Incoming air will be filtered, and access by staff and materials controlled. The staff is required to wear special laboratory clothing. Microbiological checks will be carried out routinely to ensure optimal hygienic conditions. The climate in the animal room will be properly maintained and the results of measurements recorded on-line (TABLE 1). The air pressure inside the animal room will be positive. The animal room is windowless and uniformly lighted with fluorescent lamps. Two rats per polycarbonate cage will be housed together except during exposure (a). The cages will be positioned in racks within like treatment groups in a vertical column. Once every 2 weeks, the position of the cages in the racks will be rotated vertically within a treatment group. Furthermore, the cage racks within the animal housing room will be rotated clockwise every week. The bedding material will be autoclaved, hardwood bedding that meets the NIH standards for physical quality and the NTP standards for chemical contaminants. Analysis data will be obtained from the supplier for each shipment of bedding. The autoclaved NIH-07 open formula pellet diet will be supplied ad libitum from cage lid racks. Analysis data on each diet batch for contaminants, protein, fat, fiber, ash, moisture, and heat-labile nutrients such as vitamin A and thiamine will be obtained from the supplier. Autoclaved tap water from water bottles with sipper tubes will be supplied ad libitum in each cage. Drinking water will be analyzed at least once a year for totally dissolved solids, heavy metals, chlorinated hydrocarbons, organophosphates, nitrate, nitrite, and total trihalomethanes according to NTP. (a) not in accordance with NTP which recommends individual housing ,foz
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I N B I FO Institut fur biologische Forschung GmbH PF010COL B6030 / P 0500/3080 921204 PARAMETER ventilation air changes overpressure temperature SPECIFICATION >10 times/h 100 + 50 Pa between 22 + 2 °C relative humidity between 35 and 65 % light/dark cycle 12 : 12 h, L 6.00 to 18.00 CET animal housing bedding material diet supply water supply 2 rats/cage between exposures, changes of cages >2 times/week changes >2 times/ week ad libitum, but no diet during exposure ad libitum, but no water during exposure TABLE 1 HOUSING CONDITIONS 1592 PAGE 7 REMARK air-filtered by mechanical filter class S on-line recording of pressure • for at least 90 % of the time, below 19 or above 25 °C not more than 2 h, continuous recording, accuracy of thernxaneter checked >1 time/quarter for at least 90 % of the time, below 30 or above 70 % not more than 2 h, continuous recording, accuracy of hygirdneter checked <1 time/quarter between 430 and 540 Lux at 1.50 m from the floor, light cycle check, "daylight" fluorescent lamps, Lumilux, L36W/11 and L58W/11 polycarbonate (Malaolon) cages, standard type 3, base area: 0.39 m x 0.23 in, height: 0.15 in, supplier: PAG, D-4300 Essen 1 steam sterilization at 134 °C for 15 min, supplier: P.J. Murphy Forest Product Corp., P.O. Box 67, Nbntville, N.J. 07045, U.S.A. storage of diet at <27 °C terrperature and <70 % relative humidity for max. 120 days post milling, cleaning of cage lid racks >1 time/month, steam sterilization at 120 °C for 5 min, supplier: Agway Country Foods Inc., P.O. Box 4933, Syracuse, NY 13221, U.S.A. steam sterilization at 120 °C for 20 min, storage of autoclaved water for max. 3 days
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 8 To check the hygienic conditions within the rat housing area, samples of the autoclaved diet, autoclaved bedding material, autoclaved tap water, laboratory air as well as impression samples of laboratory surfaces after sanitation and fingerprint samples of the laboratory staff will be collected once a month during the in- halation period and the bacterial plate count determined. Impression samples will be taken prior to the introduction of the rats to check the effectiveness of the laboratory disinfection. Each nonautoclaved diet batch will be screened for the presence of coliform bacteria, E. coli, and Salmonella sp., and the total bac- terial plate count will be determined. Six hundred and eighty rats, 136 per group, 68 per sex will be randomly allocated to 3 RASS exposure groups, 1 sham exposure group (negative control), and 1 DEE exposure group (positive con- trol). It is planned to use sixty rats from each group and sex for the examination of nonneoplastic and neoplastic lesions in order to provide a reliable basis for statistical evaluation. Eight rats of each group and sex will be used for biomonitoring. The rats will be individually identified with subcutaneous transponders. To ensure maximum sensitivity and to maximize the duration of the in- halation period, the age of the rats at the start of the inhalation period will be approx. 42 days. The body weight at that time will be approx. 150 g for the male and 130 g for the female rats, the RSD being 510 %. There will be a quaran- tine/acclimatization period of <14 but >10 days. To assess the health status of the rats prior to the inhalation period, 5 male and 5 female rats, randomly selected, will be killed, necropsied, and macroscopically examined for disease and parasites (TABLE 2). Diagnosis of lesions seen at necropsy will be confirmed by histopathological examination and/or microbiological culture. Blood samples will be taken from these rats for serology profiles. Ten male and 10 female unexposed rats will be used as sentinels. At 6, 12, and 18 months after the start of the inhala- tion period, blood samples will be collected from 5 male and 5 1592 4 ,+.
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I N BI FO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 9 female sentinel rats, randomly selected, for serology profiles. At 24 months, blood samples will be collected from all sentinel rats which then will be necropsied and examined as described above. At the end of the inhalation period, blood samples will be collected from 5 male and 5 female rats of the high RASS group for serology profiles. All serum samples will be screened extramurally for the presence of antibodies to 11 rat-related viruses (GD7, H-1, Hantaan, KRV, LCM, MAV, PVM, RCV/SDA, RE03, Sendai), to the bac- teria Mycoplasma pulmonis and cilia-associated respiratory bacillus as wellas to the protozoon Encephalitozoon cuniculi. SCHEDULE NU14BER OF RATS EXAMINATION prior to inhalation period 6 months 12 months 18 months 24 months at the end of study MALE FEMALE SEROLOGY PATHOLOGY 5 5 X X 5 5 X 5 5 X - 5 5 X - 10 10 X X 5 5 X - TABLE 2 HEALTH CHECK SCHEDULE Remark: blood sample collection from retrobulbar venous plexus during diethylether narcosis The cages with the sentinel rats will be positioned in the racks, following the procedure used for the cages of the exposed groups. Mortality, general condition and behavior (a), and body weight determinations of sentinel rats will be carried out according to the schedule used for the exposed rats. Blood samples of moribund (a) observation without checklist 1592 .,,
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 10 sentinel rats will be taken. Dead or moribund sentinel rats will be necropsied and examined to determine the cause of death. The rats will be head-only exposed to the test atmospheres in INBIFO exposure chamber type X (a) (glass, aluminum, stainless steel, and brass), equipped with 140 glass restraining tubes. In this chamber, the rat will be individually exposed to a continuous flow stream of fresh test atmosphere to minimize rebreathing of the exhaled test atmosphere. The flow rate through the exposure chambers will be 140 1/min to be determined daily (TABLE 3). This should result in a calculated oxygen content of >19 %. The uniform distribution in the exposure chamber will be confirmed before the inhalation period begins without rats and during the inhalation period with rats. The positions of the rats within the exposure chamber will be changed weekly. The tube size will be varied ac- cording to the body weight of the individual rats. The exposure will be 7 hours/day (b), 5 days/week, for 30 months. The study will be terminated if 75-% mortality is seen in the male or female rats of the low RASS or sham-exposed groups. One group of rats each will be exposed to a low, medium, or high concentration of RASS with a TPM concentration between 1 and 10 pg TPM/l (TABLE 4). One group will be exposed to DEE at a particle concentration equal to that of the high RASS-exposed group. Sham-exposed rats will be exposed to conditioned air, the exposure conditions being the same as those for the RASS- and DEE-exposed rats. In the exposure chamber, the temperature will be maintained at 22 + 2°C, not to go below 20 °C or above 30 °C, and the relative humidity between 30 and 70 % in order to maintain suitable and reproducible exposure conditions (TABLE 3)- The inhalation period will start in ... 1993. (a) A new exposure chamber will be developed for this purpose. (b) OECD guideline 451 (1986) recommends 6 hours/day.
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PROTOCQL B6030 / P 0500/3080 921207 PBR23ME.'IER PRINCIPLE hcmogeneity of detennination of test atnqs- test atarosphere phere (TPM) concentration within the at least at 8 positions exposure chamber within the exposure chamber and calculation of relative starKlarrl deviation (RSD) flow rate dete~znination of pressure drop across a calibrated orifice behind the exposure chamber tatpexature detennination with thermistor probe relative humidity determination with capacitive sensor PAGE 11 Ff2EQJPNCY GROIJP SFAM RASS FIPTDI >_1 time before X X the inhalation period 21 time/quarter - X X >_1 time/P.xposure X X X day daily, X X X continuously X -a~, ~~ •+~ SOP nettxx3 to be developed IH 99/1 it IH 64/4 OECD guideline IH 65/3 451 requires determination in all groups TABIE 3 PHYSICAL DE~'IDN5 OF TEST A`QMDSPFERFS WITHIN THE F.XPOSURE CHAMBERS Ranark: Determinations will be ppxformed in all RASS- and DEE-exposed groups, exceptions: relative humidity and quarterly determination of test atnnsphere hanogeneity within exposure chambers; the latter will be perfornied only in the high RASS-exposed group. zLszszszoz
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INBIFO Institut fiir bfologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 12 GROUP TEST TPM EXPECTED ATMOSPHERE TARGET CARBON MONOXIDE CONC. CONC. (µg/1) (ppm) 0-GR conditioned air (sham) 0 0 1-GR RASS (low) 1 4 2-GR " (medium) 3 12 3 -GR (high) 10 40 4-GR DEE 10 45 TABLE 4 GROUPS AND CONCENTRATIONS 2.3 SS Generation and Room Aging Sidestream smoke (SS) will be generated from the 1R4F cigarette, a modern blend standard reference filter cigarette. Conditioning and smoking of the cigarettes will be performed in basic conformity with ISO 3402 (1991), ISO 4387 (1991), and ISO 3308 (1991). Some minor defined deviations from the smoking standards, e.g., puff profile, are necessary for technical reasons. The SS will be gen- erated using 30-port INBIFO smoking machines (INBIFO type SM85) modified for SS collection (FIGURE 1). The smoking condi- tions will be checked by determining butt length, puff volume, and puff count at least every week during the inhalation period. 1592
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 13 SS will be conveyed via an epoxy-coated duet (area 0.2 m2) into an aging room (volume approx. 30 m3) with surfaces consisting mainly of painted wall paper (a) and PVC flooring. RASS will be obtained in this room by aging and surface contact, the mean age being approx. 0.5 h. The TPM concentration of the RASS will be adjusted to 10 µg/1 resulting in a carbon monoxide concentration of approx. 40 ppm (TABLE 4). From this room, RASS will be drawn through the 3 exposure chambers. It will be diluted with conditioned air to obtain the low and medium RASS concentrations. Room Aging Condhioned Air Conditioned Air Smoking Machine FIGURE 1 SS GENERATION, AGING, AND EXPOSURE (a) replaced every 6 months Y Exposure Chamber Exhaust Exhaust 1592
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I N B I FO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 14 2.4 Generation of Diesel Engine Exhaust (DEE) DEE will be generated by passenger car engines (Volkswagen engines, 1.6 1, 40 kW, Type W 230) mounted on test beds to allow for microprocessor-controlled duty cycles according to US test protocol 72 (a) (FIGURE 2). They will be connected via automatic transmissions to dynamometers (asynchronous motor with frequency converter) that allow the simulation of resistive and inertial passenger car loads. Periodic maintenance will be performed according to supplier's specifications. European standard fuel (RF-03-A80) and engine lubricant will be used and changed after intervals recommended by the manufacturer. Diesel emissions will be routed through a standard automotive muffler without catalytic converter. Subsequently, the emissions will be diluted (EPA, 1984) with conditioned air to obtain the final concentration of 10 µg TPM/l. CondNioned ~ Air ' Dilution Tunnel FIGURE 2 DEE GENERATION AND EXPOSURE (a) reference not yet available DEE ->- Exhaust Exposure Chamber It Exhaust ,.42
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 2.5 Test Atmosphere Characterization PAGE 15 To characterize the inhaled RASS and DEE as well as to check the reproducibility of their generation and the quality of the air used for the sham exposure, relevant analytical and physical parameters will be determined at appropriate intervals in the test atmospheres at the breathing zone of the rats (TABLE 5). The parameters chosen will be used to characterize the particle phase as well as the gas/vapor phase. In all exposed groups, key analytes (nicotine for RASS, nitropyrene for DEE, PAHs for RASS and DEE) will be determined that serve to indicate a contamination of the conditioned air or a cross-contamination of the test atmos- pheres. 2.6 Biological Assays and Determinations 2.6.1 Biomonitoring To estimate the inhaled dose of RASS and DEE, the respiratory minute volume will be determined (TABLE 6). The steady-state proportion of blood carboxyhemoglobin will be determined in order to confirm test atmosphere exposure. The concentrations of nicotine and various nicotine metabolites in urine will be deter- mined in order to estimate nicotine uptake. Lung clearance for inhaled tracer particles will be determined. Additionally, the particle mass retained in the lungs will be determined to estimate the accumulated particle burden. 2.6.2 General Observations Mortality, general condition and behavior will be observed and body weight, food consumption, and ophthalmological status deter- mined before and during the inhalation period (TABLE 7). The decision to kill moribund rats will be made only by the veteri- narian or his deputy. 1592
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PRC7IOCM B6030 / P 0500/3080 921204 PAGE 16 PARAMETF.R TPM (a) TPM (UV) soot particle size distribution gravimetry after trapping on Cambridge filters UV detection after dichloro- methane extraction of TPM- loaded Cambridge filters photanetry of organic carbon fluorcmetry after precipita- tion in a spinning spiral duct (aerosol centrifuge) carbon monoxide nondispersive infrared photo- metry of gas/vapor phase carbon dioxide it TABLE 5 ANALYSIS OF TEST ATMYJSPHMES FREVJEWY TEST A7M7SPBERE REMARKS SOP RASS DEE CONIDITICINED AIR 21 time/day + + + IH 15/3 z1 time/week + + + method being 1, + + established method to be ?1 time/month + + established determination IH 45/2 without rats AC 72/3 daily, continuously + + + in the exposure chamber CS DA2/2 IH 16/3 ?1 time/week + + determination IH 42/1 before passing into exposure chambers Remarks: sanple collection sites for all parameters at the breathing zone of rats (a) in addition, on-line estimate of particle mass concentration by a tapered element oscillating microbalance (TEOM) Z.Lszszszoz
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PRC11= B6030 / P 0500/3080 921204 PARAMti.'I'ER MEi'E)JD nicotine gas chromatography (GC) 3-etheny1-pyridine nitrogen oxides chemolumuiescence of gas/va- por phase after reaction with ozone foanaldehyde and reversed phase high perform- acrolein ance liquid chromatography ( HPLC ) of 2,4-dinitrophenyl- hydrazine (DNPH) derivatives after trapping in DNPH solution solanesol liquid chranatography benzene, isoprene, GC/mass spectrcmetry (MS) and 1,3-butadiene after trapping in methanol at -78 °C pAGE 17 FRFQUENCY TEST AZN37SPFg:RE RASS DEE COTIDITICNED AIR RENfi9RKS SOP z1 time/week + + + AC 119/3 + - + method being established + + 1 time/month + + + - AC 135/1 I + method being established It + + AC 133/1 TASLE 5 (cont.) ANALYSIS OF TEST ATT3JSPFERES Ranarks: sample collection sites for all parameters at the breathing zone of rats s~szszszoz
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P1C710COL B6030 / P 0500/3080 921204 PAGE 18 PAPAMUM MEIIHOD FREQUE61CY TEST P.TIrD6PHM REM1RKS SOP RASS DEE COTIDITIONED AIR 4-(N-methyl-N-nitros- CaC/thenno energy 1 time/quarter + anuno)-1-(3-pyridyl)- analyzer 1-butarbne (NNK) N-nitirosodimethylamine " 1. catechal IPLC or 03 1 tine/nonth + + selected PAHs includ- GC/MS ing benzo(a)pyrene 0. method to be established + + + method to be modified for DEE anitine GC + + - method to be established nitropyi.~ene GC/MS or M5/MS + + + method being established zinc, nickel, lead, inductively coupled 2 times/year + + - performed extra- iron, and cadmium plasina spe~troscopy nnirally, sample collection on quartz fiber filters sulphur dioxide 1 time/month + + total hydrocarbons flane ionization detector " + TABLE 5 (cont.) ANALYSIS OF TEST AIT-JSPHERFS Ranarks: sample collection sites for all parameters at the breathing zone of rats method to be established " AC 99/2 AC 107/4 sLszszszDz
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PFiOIOCCQ, B6030 / P 0500/3080 921204 PAGE 19 PARAMETER respiratory minute volume carboxyhemoglobin of the blood nicotine and nico- tine metabolites including cotinine, trans-3-hydivxy- cotinine, nornico- tine, nicotine-N- oxide in the urine particle clearance frm the lungs lung particle burden plethysmographic detennina- tion of respiratory frequency and tidal volwne gas chranatography after reduction to methane of the CO released from hetroglobin HPIC after derivatization with diethylthiobarbituric acid (DElBA,) ? ? TABLE 6 BIOMQNIMRIlVG FRDQUf3QCY NUMBER OF REMARKS RA25/ (EMKSM GRfJITP AAID SEX) every 6 nnnths 6 o 3 6 >1 time/ study 8 >2 times/ 8 study SOP between_1 h after exposure IH 112/3 starts and end of exposure sample collection from, retrobulbar IH 31/4 venous plexus of rats during PAC 11/3 diethyl ether narcosis within 1 h before the end of exposure excluding DEE-exposed group, urine IH 91/4 sample collection for 24 h IH 92/2 BC 334/1 method to be established method to be developed o9szszszoz
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N P1OrIC)COL B6030 / P 0500/3080 921204 PAGE 20 PARAME.TER MEIIM FREQUENCY NUMBER OF RAZS/ ( FnKsIRE GROUP AND SEX) nnrtality observation 2:1 tine/day 68 general condi- observation ?2 times/exposure day 68 tion behavior without checklist observation 1 time/exposure day 4 according to check list check for 1 time/day 68 body weight moribund rats gravinetry 1 day after arrival and on days 68 -6, 0, and 1; week 2 to 13: 2:1 time/week; week 14 to end of inhalation period: 21 time/rronth TAHLE 7 GENERAL OBSERVATIONS REMARKS SOP - IH 18/2 before start, during, and after end IH 89/4 of exposure after exposure, special attention paid to ttumr develognent, e.g:, recording tine of onset, location, dimensions, and appearance of each visible or palpable tiumr Rats considered morilxuxl by the IH 18/2 Pxa,f, i n i ng veterinarian or his deputy (based on set criteria). will be killed. individually IH 13/5 (a) not according to NPP which recoimiends 2 observation time points per day teszszGzOz
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PRaIOCOL B6030 / P 0500/3080 921204 PAGE 21 PARAQ49= MEP= FRDQIIENCY NUMBER OF RP,ZS/ (EUOSURE GROLJP AND SEX) food consump- gravimetry week 1 to 13: 1 time/week 68 tion week 14 to end of inhalation period: 21 time/3 months ophthalnqlogy external examination and ophthal- imscopy 2 times/study 6 TABLE 7 (cont.) GEfRAL OBSERVATIONS REMARKS detenni.necl per group and sex Before inhalation period and during the 24th month of inhalation period; if at the latter time point ophthal- mological effects are seen, the eyes (with optic nerve) of all surviving rats will be removecl during final dissection and fixed for histopatho- logical examinations. SOP IH 1/2 zaszsz~z~z
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 2.6.3 Hematology PAGE 22 For the differential blood cell count, blood smears will be fixed by air-drying and stained according to Pappenheim (Hallmann, 1980) and subsequently evaluated according to Rick (1990) (TABLE 8). 2.6.4 Pathology Rats that die spontaneously or are killed in moribund state as well as those surviving at the end of the inhalation period will be necropsied. The order in which the rats are necropsied will be randomized. The necropsy will be conducted by technicians under the supervision of a pathologist. Rats that die spontaneously or are killed in moribund state will be examined in the same way as those scheduled for killing at the end of the inhalation period (TABLE 8). However, no organs will be weighed. Due to the rapid onset of postmortal changes in rats found dead, these rats will be dissected as soon as possible after discovery. Killing will be performed by transsecting the abdominal aorta and Vena cava under deep pentobarbital anesthesia. The rats will be examined macroscopically for changes with special attention paid to the respiratory tract. All gross lesions will be recorded in narrative, descriptive terms, including localization, size, shape, number, color, and texture. Selected organs will be weighed (TABLES 8 and 9). Organ weights and the body weight recorded after exsanguination will be used to calculate relative organ weights. To avoid drying and the resulting loss in weight, the time from removal of organ till weighing will be minimized and the tissues will be kept in saline. 1592
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P%7ICC0L B6030 / P 0500/3080 921204 PARAME,TER METHOD PAGE 23 SCHEDULE NIIl4BER OF RMVARRS RATS/ (EXPOSURE GtdJUPS ADID SEX) differential light microscopic evalua- at months 18 60 blood cell count tion of fixed and stained and 24 as well blood smeai-s to differen- as prior to tiate and count leu)ccyte final dissec- tYpes tion (a) (b) gross necropsy ccmplete gross examination; final dissec- 60 external examinations of tion (c) the carcass including body orifices; examination and fixation of all organs/ tissues according to OECD guideline 451 and all grossly visible timiors or lesions suspected of being tiunors organ weights gravimetty final dissec- 60 tion TABLE 8 FEMATt'1LlJGICAL ADID PA=AGICAL EXAMINATIONS SOP sample collection from retrobulbar PY 105/2 venous plexus of rats during diethyl ether narcosis; evaluation of those rats in the high RASS, DEE, and sham control groups; in case of effects in the high RASS group, the differential blood cell count will be extended to the next lower RA.SS group(s) as well after deep pentobarbital anesthesia PY 18/2 and exsanguination; period between PY 89/3 tine of death and necropsy will be PY 119/1 IIllll]IliiZed see Mffi,E 9 PY 16/4 (a) including rats killed in a wribund state (b) not in accordance with OECD guideline 451 which reconmends performing this examination at 12 months, 18 months, and prior to sacrifice (c) or when found dead during the inhalation period or killed in wribund state teszszszoz
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PRC71OCCJL B6030 / P 0500/3080 921204 PAGE 24 PARANlti'PER METE]OD SCHEDULE NUMBER OF REMARKS FtA'I5/ (EXP06URE GEifJtJP AND SEX) SOP histopathology light microscopic examina- at the end of 60 see TABLE 9, tion of histological the study (a) fixation of organs in 10-% neutral PY 10/4 slides buffered formaldehyde solution PY 52/3 except lungs with larynx and tra- PY 53/3 chea to be fixed in etharol PY 54/3 acetic acid/fornnal.dehyde/saline PY 56/2 (EAFS) solution, and testes to PY 57/4 be fixed in bouin's solution; PY 60/6 sectioning of nose at defined levels PY 61/5 according to Young (1981), PY 62/3 larynx according to Lewis (1980), PY 69/2 and lungs according to the nethod of PY 79/3 Iamb and Reid (1969) mod.ified at PY 84/4 INBIFO PY 90/2 thickness of norphaaetry 60 5- to 6-Wn sections, HE staining, PY 93/3 laryngeal determination at aryte.mid projec- epithelia tions includir-g ventral depression, ventral lwnen, and vocal cords TABLE 8 (cont.) HRQ=A~,'ICAL AND PAMiX)GICAL EMDLATIONS (a) including rats which die during the inhalation period or are killed in a moribund state serszszszoz
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PR0'IOGOL B6030 / P 0500/3080 921204 PAGE 25 ORGAN/TISSI]E ORGAN WEIQiT nEIT.u41NnmrCN HISTOPATHOLOGICAL EXP.MINATICN ALL GEtOJPS accessory genital organs prostate se„i na1 vesicles coagulation glands clitoral glands Pxeputial glands adrenals, right left aorta (thoracic) brain (cerebral cortex, X cerebellar cortex, pons/ medul].a oblongata) caectan colon duodentnn epididymis eyes with optic nerve ALL GROUPS ONLY SHAM-F:XPO6ED GROUP, HIQi RASS ( a ) CONCENfl'RATION C~iOtJP AND DEE GiWP X X X X X TABLZ 9 ORGAKS/TISSUES FOR ORGAN WEICHT DErErtnarnrnTrONS AND HIS'IUPATFmLaGICAL EXAPaNATIONS frontal section of ante- rior part of left seni- nal vesicles including coagulation glands cross section of clitoral and preputial glands only if indicated by the ophthalmological examination (a) In case effects are seen in high RA,.S,S group, the examination will be extended to the next lower RASS group(s). 98szszszflz
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PRC7IUGQL B6030 / P 0500/3080 921204 ORGAN/TISSUE fenair including joint Harderian glands heart ileum jejunim- kidney, right left liver lymph nodes (ma„d; rn, l ar, roesenteric, bronchial, and med.iastinal ) lazynx/laryngopharynx lungs mmwary glands nausculature (thigh) nose/nasopharynx PAGE 26 ORGAN WEIGEIl' DETERMINATION HISIUPA7.HOLOGICAL EXAMnvA'rrON ALL GRQ(7PS X (b) mid-longibadinally through the entire cortex and medulla section with largest surface area step sections, 4 levels, laryngopharynx at the base of epiglottis 5- to 6-M coronal sections every 1 mm cross and longitudinal sections step sections, 4 levels, nasopharynx at level 4 TAffiE 9 (cont.) ORGANS/TISS'ik'S FOR ORGAN WEIC33T DETERMIWIONS AND HISZUPAnCLAGICAL DWMINP=ONS (a) In case effects are seen in high RASS group, the examination will be extended to the next lower RASS group(s). (b) with larynx and trachea ALL GROJPS ONLY SfiAM-FXP06ED GROUP, HIQi RASS (a) COIVCENTRATION GROUP AND DEE GROUP Z.sszszsZoz
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PROZ= B6030 / P 0500/3080 921204 PAGE 27 ORGAN/TISSUE ORGAN WEICHT DEIERNmVATION HISNPATHQIrOGICAL EXANIDVATION REMARKS ALL GEmUPS ALL GROJPS ONLY SHAM-EXPOtSED CROUP, HIQi RASS (a) CONCENfl'RATION GROUP AND DEE GROUP oral cavity - - X ovaries (mesovaries), right - - X left - - X pancreas - - X parathyroid glarrls - - X peripheral nerve (sciatic) - - X pituitary - - X rectum - - X salivary vesicles (submarui.ib- - - X ular and sublingual) skin - - X spinal cord (cervical, thora- - - X cic, and lumbar) spleen X - X sternum with bone marrow - - X stomach (forestanach and - - X glandular stanach) TASLE 9 (cont.) ORGANS/TISSUES FOR ORGAN WEICHT DETERMINAmIONS AND HISZLUPATHOIAGICAL EXANII24ATIONS (a) In case effects are seen in high RASS group, the examination will be extended to the next lower RASS group(s). ssszszszoz
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PRC7ICJCOL B6030 / P 0500/3080 921204 PP,OE 28 ORGAN/TISSfIE testis (b), right left thymus thyroid tissue masses tongue trachea urinary bladder uterus zymba.l glands all grossly visible tiumrs or lesions suspected being ttumrs ORGAN WEIQ3T DF'-ERMTNA'rTON HISZOPA'IIHOLOGICAL EXANINATION REK,RKS ALL QmiJPS ALL C~TOiJPS CNII,Y SHAM-F.XPOSED GtOiJP, HIQi RASS (a) OONCIIVTRPTICN GROUP AND DEE GROUP 2 levels TASLE 9 (cont.) ORC.ANS/TISSUES kUR OROAN WEICHT DETEi2NINP,TIONS AND HIS'IOPATHOLOGICAL MAMTNn'r?ONS (a) In case effects are seen in high RASS group, the examination will be extended to the next lower RASS group(s). (b) excluding Tunica vaginalis, exception to NI'P reccmrnndations ssszszszoz
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I N B I FO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 29 The organs/tissues listed in TABLES 8 and 9 as well as gross le- sions will be removed from each rat and fixed. Lungs with trachea and larynx will be removed, weighed and thereafter distended and fixed by intratracheal instillation with EAFS at a pressure of 20 cm of water. The trachea will be ligated after instillation to ensure trapping of fixative in airways and alveoli. Nose will be flushed gently via nasopharyngeal duct to ensure rapid fixation of the mucosa. The representative parts of the gastrointestinal tract will be flushed first with water to remove ingesta and then with 10 % for- maldehyde to ensure rapid fixation of the mucosa; the remaining part will be incised for internal examination. Hollow organs will be trimmed and blocked to allow a cross section from mucosa to serosa. Representative sections of all major organs/tissues and visible or palpable tumors/lesions will be processed in the standard manner, embedded in Paraplast, sectioned at 5 to 6 µm, and stained routinely with hematoxylin and eosin. Sections of lungs and trachea will be stained in addition with al- cian blue/periodic acid Schiff's reagent (AB/PAS) to demonstrate goblet cells. Special staining procedures will be used as required by the pathologist to diagnose a specific tumor type or non- neoplastic change. The slides will be examined histologically by a veterinarian with knowledge of the treatment groups. Test substance-related effects will be verified by blind reading (a). (a) Tumors found in sentinels or rats allocated to biomonitoring will not be used for the statistical evaluation. 1592
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INBIFO Institut fOr biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 30 In case the survival of the rats in"the high RASS group is sub- stantially less than the sham-exposed group or results show undesirable effects altering tumor development, then the next lower RASS dose level will be examined. The histopathological examination of tumors of rats found dead or or of rats killed in a moribund state should clarify whether the cause of death was tumor-related (fatal tumor) or nontumor-related (incidental tumor). 2.7 Statistics and Evaluation As descriptive statistics for continuous data (e.g., CO concentra- tions, body weight, or thickness of laryngeal epithelium), the number of values, their arithmetic mean, relative mean, standard deviation (for analytical data) or standard error of mean (for biological data), and relative standard reference deviation will be given. For samples including at least 1 value below the detec- tion limit, the median, 25 and 75 % quartiles will be given. As descriptive statistics for ordinal data (e.g., histopathology), the arithmetic means, the standard error of the mean, as well as the incidences will be given. The particle size distribution will be calculated using linear regression analysis after probit transformation (Finney, 1971). ,!oz
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 31 With regard to the results of the biological assays, the following comparisons will be made: - comparison of sham- and RASS-exposed groups - comparison of sham- and DEE-exposed group - comparison of high concentration RASS- and DEE-exposed groups For continuous data, the aforementioned comparisons will be per- formed using: - Bartlett test (Sachs, 1982) for homogeneity of variances only if no value is below the detection limit, - either one-way analysis of variance (Sachs, 1982) followed by the Duncan test (Duncan, 1955) in case of homogeneous variances or the nonparametric analysis of variance of Kruskal-Wallis fol- lowed by multiple pairwise comparisons (Conover, 1980) in case of heterogeneous variances or samples with at least 1 value below the detection limit. For ordinal data except tumor incidences, the generalized Cochran- Mantel-Haenszel test (Koch and Edwards, 1988) will be applied followed by (chi)2 tests on the underlying 2 x r contingency tables. For mortality data, life-table estimates using the log rank test (Kalbfleisch and Prentice, 1980) will be performed. The (chi)2 test according to Peto (Peto et al., 1980) will be ap- plied for the tumor incidences and their classifications as either fatal or incidental. All tests will be conducted at the nominal level of significance of alpha = 0.05, alpha = 0.01, and alpha = 0.001 (2-tailed). Results will be considered statistically significant at p<0.05.
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I N B I FO Institut fOr biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 32 3 BACKGROUND ki
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 33 4 TEST MATERIAL 4.1 RASS Cigarette types: 1R4F Source: University of Kentucky Number: approx. 100000 Packaging: 4000 cigarettes/carton Date of receipt at INBIFO: 15 May 92 Storage Conditioning Duration: room R922, 4 °C temperature, relative humidity uncon- trolled for at least 8 days prior to smoking Temperature: 22 ± 1 °C Relative humidity: 66 ± 2 % Selection: no selection
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INBIFO Institut fOr biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 4.2 DEE PAGE 34 The test substance is generated DEE which will be cooled down to 22 + 2°C by diluting with conditioned air. Passenger car Diesel engine: Volkswagen Passat engine, 1.6 1, 40 kW, type W230 Standard load cycle: computer-controlled according to U.S. 72 Federal test protocol Fuel: European standard fuel, type RF-03-A80 Engine lubricant: - (a) (a) will be given in the report, recommendation by the Volkswagen AG
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I N BI FO Institut fitr biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 35 5 RESPONSIBILITIES ---------------- ---------------- The study as outlined in the objective, general design, and back- ground was conceived by INBIFO management in agreement with the sponsor; INBIFO management bears the responsibility for it. The study will be performed at INBIFO Institut fiir biologische Forschung, Fuggerstraf3e 3, D-5000 Koln 90, F.R.G. General Manager: ................. Date ............................... Dr.rer.nat. W. Reininghaus Physicist (Diplomphysiker) Study Director: Date ............................... W. Stinn Biologist (Diplombiologe) Manager Analytical Chemistry: ................ Date ........................... Dr.rer.nat. P. Voncken Chemist (Diplomchemiker) Manager Biochemistry: .................. Date ............................. Dr.rer.nat. G. Schepers Chemist (Diplomchemiker)
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 36 Manager Cytometry: ................... Date Manager Information Systems: .............................. Dr.rer.nat. R. Kindt Biologist (Diplombiologe) ................... ........................... Date Dr.rer.nat. W. Gomm Mathematician (Diplommathematiker) Manager Inhalation: ................. Date Manager Microbiology: ................. Date ................................ W. Stinn Biologist (Diplombiologe) .............................. Dr.rer.nat. F. Tewes Biologist (Diplombiologe) Manager Pathology: ................... ............................... Date Dr.med.vet. A. Teredesai Veterinarian (Fachtierarzt ftir Pathologie)
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INBIFO Institut fUr bioiogische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 37 6 GOOD LABORATORY PRACTICE STATEMENT This protocol contains all the necessary information needed to perform this study according to the Good Laboratory Practice Regulations (Bundesgesetzblatt 1990, 1991). All qualified personnel, resources, facilities, equipment, materials, and methodologies needed to perform this study accord- ing to GLP are available or are scheduled to be available on time. Inspections on this study will be performed by the INBIFO quality assurance unit at appropriate intervals according to the quality assurance audit schedule for this study. The report will be reviewed to assure that it accurately reflects the study carried out and the results obtained. .................... .......................... Date Quality Assurance Manager Dr.med. Dr.rer.nat. K. von Holt Physician and Physicist (Arzt und Diplomphysiker)
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 38 7 STORAGE OF RECORDS AND MATERIALS -------------------------------- -------------------------------- Records will be stored in our archives for at least 30 years after delivery of the final report to the client. Samples of the cigarettes and all specimens will be stored for as long as their quality under state-of-the-art storage conditions allows further evaluation, but not longer than 30 years. All records, samples, and specimens can be claimed by the client. 1592
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 39 8 REFERENCES Bomhard, E., Karbe, E., Loeser, E., Spontaneous tumors of 2000 Wistar TNO/W. 70 rats in two-year car- cinogenicity studies, JEPTO 7: 35-52 (1986) Boorman, G.A., Hollander, C.F., Spontaneous lesions in the female WAG/Rij (Wistar) rat, Journal of Gerontology 28: 152-158 (1973) Bundesgesetzblatt I (13): Grundsatze der Guten Laborpraxis (GLP), Anhang 1(zu Paragraph 19a Abs. 1) der Neufassung des Chemikalien- gesetzes vom 14. Marz 1990: 521-548 (1990) Bundesgesetzblatt I (35): Grundsatze der Guten Laborpraxis (GLP), Dritte Verordnung zur Anderung der Gefahrstoffverordnung vom 5. Juni 1991, Artikel 2: 1219 (1991) Conover, W.J.: Practical nonparametric statistics, 2nd ed., New York: Wiley, 1980 Crain, R.C., Spontaneous tumors in the Rochester strain of the Wistar rat, American Journal of Pathology 34: 311-323 (1958) Deerberg, F., Rapp, K.G., Pittermann, W., Rehm, S., Zum Tumorspektrum der Han; Wist-Ratte, Z. Versuchstierkd. 22: 267-280 (1980) Deerberg F., Rapp K.G., Rehm, S., Mortality and pathology of Han: Wist rats depending on age and genetics, Experimental Biology and Medicine 7: 63-71 (1982) Duncan, D.B., Multiple range and multiple F-tests, Biometries 11: 1-42 (1955) - (INBIFO reference: C196) US Environmental Protection Agency (EPA), Federal Register 49: No. 200 (1984) Finney, D.J., Probit analysis, Cambridge University Press, Cambridge, 1971 Hallmann, L., Klinische Chemie und Mikroskopie, Stuttgart: Thieme Verlag, 1980 t:92
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INBIFO Institut for biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 Heinrich, U., Department of Experimental Hygiene, Fraunhofer Institute of Toxicology and Aerosol Research, D-3000 Hannover 61 PAGE 40 Heinrich, U., Muhle, H., Takenaka, S., Ernst, H., Fuhst, R., Mohr, U., Pott, F., Stober, W., Chronic effects on the respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions, J. Appl. Tox. 6 (6): 383-395 (1986) Heinrich, U., Fuhst, R., Mohr, U., Tierexperimentelle Inhalationsstudien zur Frage der tumorin- duzierenden Wirkung von Dieselmotorabgasen und zwei Teststauben, in: Bundesminister fur Forschung und Technologie (Ed.): Auswirkungen von Dieselmotorabgasen auf die Gesundheit, Arbeitsschwerpunkt im Rahmen des Programmes "Umweltforschung und Umwelttechnologie", Forderschwerpunkt "Umweltbelastung und Gesundheit", 1992 International Organization for Standardization: International Standard ISO 3308, Cigarettes - Routine analytical cigarette smoking machine - Definitions and standard conditions, 3rd ed., 1991 International Organization for Standardization: International Standard ISO 3402, Tobacco and tobacco products - atmospheres for conditioning and testing, 3rd ed., 1991 International Organization for Standardization: International Standard ISO 4387, Cigarettes - Determination of to- tal and nicotine-free dry particulate matter using a routine analytical smoking machine, 2nd ed., 1991 Kalbfleisch, J.D., Prentice, R.L.: The statistical analysis of failure time data, New York: Wiley, 1980 Koch, G.G., Edwards, E., Clinical efficiency trials with categorial data, in: Pearce, K.E. (Ed.): Biopharmaceutical statistics for drug development, New York, Marcel Dekker, 1988, pp. 403-457 t592
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 41 Kt3lmer, V. , Boehringer, D-6507 Ingelheim Kroes, R., Garbis-Berkvens, J.M., de Vries, T., van Nesselrooy, J.H.J., Histopathological profile of a Wistar rat stock including a sur- vey of the literature, Journal of Gerontology 36: 259-279 (1988) Lamb, D., Reid, L., Goblet cell increase in rat bronchial epithelium after exposure to cigarette and cigar tobacco smoke, Brit. Med. J.: 33-35 (1969) Lewis, D.J., Experimental pathology of the rat larynx following exposure to tobacco smoke, Surrey: University of Surrey; 1980. Dissertation Maita, K., The age related tumor incidence in Wistar Imamichi rat, Exp. Anim. 28: 555-560, 1979 - (INBIFO reference: D7551) Mullink, J.W.M.A., Achtergrondpathologie Cpb/WU rat, Verslag 13 weeks proef (CPB-TNO), Intern rapport, Central Institute for the Breeding of Laboratory Animals - TNO Mullink, J.W.M.A., Backgroundpathology of the Cpb:WU rat, Part II: 12 months study (CPB), Intern rapport 003, Central Institute for the Breeding of Laboratory Animals - TNO (1980) Mullink, J.W.M.A., Backgroundpathology of the Cpb:WU rat, Part III: 24 months study (CPB), Pathological findings in diseased rats and in rats which died spontaneously before the age of 25 months, Intern rapport 004, Central Institute for the Breeding of Laboratory Animals - TNO ,M
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I N B I FO Institut fur biotogische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 42 Mullink, J.W.M.A., - Backgroundpathology of the Cpb:WU rat, Part IV: 24 months study (CPB), Intern rapport 1004, Central Institute for the Breeding of Laboratory Animals - TNO (1981) National Toxicology Program (NTP), Specifications for the conduct of studies to evaluate the toxic and carcinogenic potential of chemical, biological and physical agents in laboratory animals for the national toxicology program, Research Triangle Park, NC: National Toxicology Program, 1991 OECD Guideline 451, Carcinogenicity studies, in: OECD guidelines for testing of chemicals, Paris: Organization for Economic Co-operation and Development, 1986 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 car- cinogenic effects in long-term animal experiments, in: IARC: Long-term and short-term screening assays for carcinogens: a critical appraisal, IARC monographs on the evaluation of the car- cinogenic risk of chemicals to humans, Supplement 2, Lyon: IARC, 1980, pp. 311-426 Pauluhn, J., Bayer AG, Department of Toxicology, Institute of Toxicology Agriculture, P.O. Box 101701, D-5600 Wuppertal 1 Rehm, S., Deerberg, F., Rapp K.G., A comparison of life-span and spontaneous tumor incidences of male and female Han: WIST virgin and retired breeder rats, Lab. Anim. Sci. 34: 458-464 (1984) Rick, W., Klinische Chemie und Mikroskopie, Berlin: Springer Verlag, 1990 Sachs, L., Applied statistics, Springer Verlag, New York, 1982 Takizawa, S., Miyamoto, M., Observations on spontaneous tumors in Wistar Furth strain rats, Hiroshima Journal of Medical Sciences 25: 89-98 (1976) 1592
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I N S I FO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 921204 PAGE 43 Til, H.P., Background pathology of the Cpb:WU (Wistar Random) rat, 12- and 24-month and life-span study: hematology and clinical chemistry, Report no. V81.038/080151, Project no. V0151, Institute CIVO-Toxicology and Nutrition - TNO (1978) Ueberberg, H., Lf3tzen, L., The spontaneous rate of tumors in the laboratory rat: Strain Chbb: THOM (SPF), Arzneim. Forsch./Drug Res. 29: 1976-1979 (1979) Vandenberghe, J.,' Life-span data and historical data in carcinogenicity testing in Wistar rats Crl:(WI)BR, Charles River Deutschland (1990) Woutersen, R.A., Appelman, L.M., Van Garderen-Hoetmer, A., Feron, V.J., Inhalation toxicity of acetaldehyde in rats, 3. carcinogenicity study, Toxicology 41: 213-231 (1986) - (INBIFO reference: C1927) Young, J.T., Histopathologic examination of the rat nasal cavity, Fundam. Appl. Toxicol. 1: 309-312 (1981)
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INBIFO Institut fur biologische Forschung GmbH PROTOCOL B6030 / P 0500/3080 9 ABBREVIATIONS (a) 921204 PAGE 44 CET : Central European Time DEE : Diesel engine exhaust EAFS: ethanol/acetic acid/formaldehyde/saline GC : gas chromatography HE : hematoxylin-eosin HPLC: high performance liquid chromatography ISO : International Organization for Standardization L : light MS : mass spectrometry NIH : National Institute of Health NTP : national toxicology program OECD: Organization for Economic Cooperation and Development p : probability of receiving the value of the test statistic or a more extreme value of the test statistic, if the null hypothesis holds PAHs: polycyclic aromatic hydrocarbons ppm : parts per million (10E-6), used only with ratios of gaseous volumes PVC : polyvinyl chloride RASS: room-aged sidestream smoke RSD : relative standard deviation SOP : standard operating procedure(s) SS : sidestream smoke TPM : total particulate matter UV : ultraviolet END OF PROTOCOL (a) in addition to those explained immediately on the same page 1592

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