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Philip Morris

Report B1033 / P 0500/3204

Date: 15 Nov 1993 (est.)
Length: 62 pages
2061990287-2061990348
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Author
Bachmayer, D.
Gomm, W.
Reininghaus, W.
Teredesai, A.
Terpstra, P.M.
Tewes, F.
Voncken, P.
Vonholt, K.
Walk, R.A.
Type
SCRT,
ABST, ABSTRACT
BIBL, BIBLIOGRAPHY
CHAR, CHART, GRAPH,
Area
INBIFO/FURNACE ROOM
Site
I48
Named Organization
Bundesgesetzblatt
Cage Group Control
Charles River US
Crc
Crl, Charles River Breeding Lab
Federal Register
Iarc
Inbifo, Institut Fur Biologische Forschu
Intl Org for Standardization
Iso
Systeme Intl Dunites
Univ of Ky
American Board of Toxicology
American Society of Toxicology
Named Person
Cochran
Coggins
Edwards
Finney
Haenszel
Koch
Lewis
Mantel
Smith
Walker
Characteristic
DRFT, DRAFT
ILLE, ILLEGIBLE
MISS, MISSING PAGES
Author (Organization)
Crc
Inbifo, Institut Fur Biologische Forschu
Litigation
Ppla/Produced
Date Loaded
29 May 2000
Brand
Ursus 35
1r4f
UCSF Legacy ID
pob55d00

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REPORT 51033 / P 0500/3204 931115 ABSTRACT
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REPORT B 1033 / P 0500/3204 931126 PAGE 4 ABSTRACT The biological activity of the mainstream smoke of the test cigarette URSUS-35 was deter- mined relative to that of the standard reference cigarette lR4F in a 21-day inhalation study on rats. The reversibility of findings was investigated following a 42-day postinhalation period. Male Sprague Dawley rats were head-only exposed to diluted mainstream smoke (MS) for 5 hours/day, 7 days/week. The smoke was generated in basic conformity with ISO standards, the major deviation being the conditioning of URSUS-35 at 50 % relative humidity. URSUS- 35 was puffed at 151oule with 1 s on-time. Its smoke was diluted with filtered and conditioned fresh air using fixed dilution factors, i.e. 110- and 50fold. The resulting smoke concentrations were expected to produce slight and moderate changes in the respiratory tract in rats. Smoke of the 1R4F was diluted 290- and 150fold to generate 2 concentrations , which were expected to produce biological changes similar to the respective dose of URSUS-35. For the comparison of the biological activities of URSUS-35 and 1R4F mean specific activities for the low and high dose groups of each of the 2 smoke types were calculated in relation to dose parameters relevant to cigarette smoke uptake in smokers, i.e., TPM, nicotine and smoke concentration (puffs/l). Chemical analyses of the URSUS-35-MS revealed the following concentrations of selected smoke components as compared with those at the respective dose level in the MS of the 1R4F: In the MS of URSUS-35 at-the-respectivedose_level the TPM concentrations and the calcu- lated smoke concentrations were less by half.-The TPM concentrations for the low and high dose groups of URSUS-35 were 40 and 80 µg TPM/1 respectively;-The nicotine concentrations were approx. equal, the formaldehyde and acrolein concentrations were higher by a factor of 6 and 1.3, respectiyely, and the carbon monoxide concentrations were lower by a factor of 6.The carboxyhaemoglobin concentrations determined in blood of MS-exposed rats where in line with the carbon monoxide concentrations, not exceeding 24%. Following biological effects, commonly seen in MS-inhalation studies, were also seen in the present study: There were no mortality, no unexpected in-life observations or differences between URSUS-35 and 1R4F-groups. Respiratory volumes were reduced by 20 and 25% in the high dose groups of URSUS-35 and 1R4F, respectively. Since the reduction was similar in both high dose groups, it is assumed that the inhaled dose was lower by roughly 20% for both cigarette types. In the URSUS•35-exposed groups a less pronounced, dose-dependent body ~ weight gain reduction was found than in the 1R4F-exposed ones (up to 14%, high dose group x of 1R4F). There were no gross pathological findings. The for both MS types similar reductions in liver weights, both absolute (up to 25 %) and relative to body weight (up to 13 %), were ~ more pronounced in the present study than in similar previous ones. For both MS-types, slight to moderate/marked histopathological changes were observed: hyperplasia, squamous,,-'\ , t E etaplasia, and atrophy in the nose, as well as hyperplasia and metaplasia in the larynx.~ ~ achea and lung.l~ Severities and incidences were more or less similar for both MS-types except for the atrophy. The incidence in the high dose group of URSUS-35 was slightly higher (3 out of 8 rats) than in the respective 1R4F-group (1 out of 8 rats). The ~ 5 spectrum as well as the severity and incidence of the histopathological findings obtained in ~~ the present study are sintilar to those obtained in the previous URSUS-35 study following 21 ~ days. Following the 42-d postinhalation period the rats completly recovered from all MS- ~' ':, YK L i w rt
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REPORT B1033/ P 0500/3204 931126 PAGE 5 The biological activity of URSUS-35-MS as compared with that of the 1R4F was dependent on the dose parameter used a factor of 2 higher, i.e. when related to the TPM concentration, equal, i.e. when related to the nicotine concentration, and less than half, i.e when related to the smoke concentration. These factors agree well with those gained from the smoke analyses of.{{ F URSUS-35-MS.
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REPORT B1033 / P 0500/3204 931125 PAGE ~ 1 OBJECTIVE The biological activity of the MAINSTREAM SMOKE of the test cigarette URSUS-35 was determined relative to that of a standard reference cigarette in this subchronic inhalation study. The re- versibility of findings was investigated. 2 EXPERIMENTAL DESIGN AND PROCEDURES 2.1 General Desian The study was performed as a 21-day inhalation study on male Sprague Dawley rats. The rats were head-only exposed to diluted mainstream smoke (MS) of the test cigarette URSUS-35 and of the standard reference cigarette 1R4F as well as to filtered and con- ditioned fresh air (sham control group) for 5 h/d, 7 d/week. The mainstream smoke was generated in basic conformity with ISO standards, with some defined deviations being necessary for tech- nical reasons. URSUS-35 was puffed at 15 J with a 1 s on-time„ as. URSUS-35 smoke was diluted with filtered, conditioned fresh air using fixed dilution factors, i.e., 110- and 50-fold, to produce histopathological changes in the respiratory tract, on the average slight for the low concentration and on the average moderate for the high concentration (TABLE 1). Smoke of the 1R4F was diluted 290- and 150-fold to produce biological changes similar to'the respective doses of URSUS-35 MS.
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REPORT B1033 / P 0500/3204 931125 PAGE 5 CIGAREPSE TEST DOSE DILUTION EXPECTED CONCENTRATION GROUP TYPE ATTPOSPI= LEVEL FALZllR IN EXPOSURE CBAMBER TPM CO (Rg/1) (pFn) 1R4F URSUS - (sham exposure) MS " - (cage control ) - low high low high - - 290 150 110 50 - 0 83 172 38 84 - 0 94 198 16 32 - 0•-GR 1•-GR 2-GR 3--GR 4-GR 5-GR TABLE 1 . Local STUDY DESIGN effects on the respiratory tract were of major interest, but selected systemic effects, e.g., changes in body weight were also determined. To compare the biological activity of URSUS-35 and 1R4F, the responses for the low and high dose groups were used to calculate the mean specific response for each of the 2 smoke types in rela- tion to dose parameters relevant to cigarette smoke uptake in smokers, i.e., total particulate matter (TPM), nicotine, and smoke concentration (puffs/1). The study "was conducted in compliance with good laboratory prac- tice (GLP) and animal welfare (a) standards. (a) "Guiding Principles in the Use of Animals in Toxicology", adopted by the American Society of Toxicology
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REPORT B1033 / P 0500/3204 931125 PAGE 6 2.2 Animals and Treatment Male outbred Sprague Dawley rats (Cr.1:CDBR), bred under specified pathogen-free conditions, were obtained from Charles River U.S.A. Our extensive experience with Sprague Dawley rats in MS inhala- tion studies as well as the large amount of published data on this strain (e.g., IARC, 1986) recommended their use in the present study. The rats were kept in an animal laboratory unit (CRC modular laboratories C111 to C113) with restricted and controlled access and under defined and good hygienic conditions. The laboratory air (filtered fresh air) was conditioned. Positive pressure was maintained inside the laboratory unit. Room temperature and rela- tive humidity were maintained at 22.7 °C (SD: 0.7 °C) and 57.5 % (SD: 2.8 %). The light/dark cycle was 12 h : 12 h. Two rats per polycarbonate cage were housed together except during exposure. The bedding material was autoclaved softwood granulate. A sterilized, fortified pellet diet from cage lid racks and heat-treated tap water from water bottles with autoclaved sipper tubes were supplied ad libitum in each cage. Diet and bedding material were checked for aflatoxins, selected pesticides, heavy metals - bedding material also for polychlorinated phenols - before autoclave sterilization. Additionally, the nonautoclaved diet was checked for Salmonella species. No diet and drinking water were available to the rats during exposure. The rats were individually identified by subcutaneous transponders. One hundred and twenty rats were randomly allocated to 4 smoke-exposure groups, 1 sham-exposure group, and 1 cage- control group, 20 rats per group (TABLE 1). The age at the start of the inhalation period was approx. 9 weeks. The body weight at
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REPORT B1033 / P 0500/3204 931125 PAGE 7 that time was approx. ... g (RSD: .. ~). The acclimatization period was 5 d. The histopathological examination of the respiratory tracts of the rats performed on arrival revealed no findings. Serological screening revealed no antibodies to 12 rat-related viruses and to bacteria, such as Mycoplasma pulmonis, Bacillus piliformis, and cilia-associated respiratory bacillus as well as to the protozoon Encephalitozoon cuniculi performed on arrival, after 21 d of inhalation and at the end of the postinhalation period. Adequate hygienic conditions within the animal housing and ex- posure room were maintained as evidenced by the results of the bacteriological examinations of the rat diet, drinking water, and the laboratory air and surfaces. The rats were head-only exposed to the test atmospheres (TABLE 1) to minimize contamination of the fur with smoke depositions which could be absorbed dermally or ingested during grooming. INBIFO exposure chambers type B (glass, aluminum, and brass; dead volume: approx. ... 1), each equipped with 20 glass exposure tubes (FIGURE 1), were used. The rats were exposed 5 h/d, 7 d/week for 21 d. The tube size was constant throughout the 21-d inhalation period. The position of the rats within the exposure chamber was changed daily. Sham-exposed rats were exposed to fil- tered and conditioned fresh air, the exposure conditions being the same as those for the smoke-exposed rats. The inhalation period began with a dose adaptation period of 1/4, 1/2, and 3/4 of the final daily exposure duration on study days 1, 2, and 3, respectively. - N 0 m ~ ~ 0 N .0 47
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REPORT B1033 / P 0500/3204 931125 PAGE 8 Ten rats per group were kept for a 42-d postinhalation period. Complete recovery of possible findings was expected within this period of time. The inhalation period was from 27 Jul. to 16 Aug.93. The postin- halation period lasted until the 28 Sep.93.
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REPORT B1033 / P 0500/3204 930720 PAGE 9 'n N N ~ W
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REPORT B1033 / P 0500/3204 931125 PAGE 11 PARP.MEPER SEPPING conditioning 45 to 50 (rel. humidity in %) smoking machine 30-port heater type 2 (10 mil) holes in sleeves - add. caxr]boaxrl sleeve - energy (J) 15 on-time (s) 1 power (W) 15 po-wer supply control voltage - puff frequency (1/min) 1 puff duration (s) 2 puff profile rectangular TABLE 2 CONDITIONS F17R URSUS-35 MAINSTREAM SNpKE GENERATION
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REPORT B1033 / P 0500/3204 931125 .... PAGE 12 FIGURE 2 ENERGY PER PUFF (J) Remarks: Error bars represent standard deviations. L6Z066i90Z
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REPORT B1033 / P 0500/3204 931125 PAGE 13 The MS was conveyed via a minimal length of glass tubing, approx. 1 m, from the smoking machines to the exposure chambers. The flow through the exposure chambers was approx. ... 1/min for the 1R4F groups and ... 1/min for the URSUS-35 groups, equivalent to ..., and ... l/rat and minute, respectively. The mean age of the MS was ... s in the exposure chambers. The temperature of the test atmospheres in the exposure chambers was maintained at 22.4 °C (SD: 0.5 °C) and the relative humidity of the conditioned air used for dilution and sham exposure at 54.7 % (SD: 3.4 %). For sham exposure, filtered and conditioned fresh air was conveyed through the same type of smoking machine used to generate the 1R4F MS. The MS and the air used for sham exposure were characterized at rat's breathing zone according to the analytical parameters listed in TABLE 3. The TPM concentrations for the low and high dose groups of URSUS-35 were 40 and 80 µg TPM/1, the corresponding ones of the 1R4F were 2 times higher (TABLE 4). Except for the adapta- tion period they were relatively stable (FIGURE 3). The carbon monoxide concentrations of URSUS-35 were a factor of 6 lower than those in the respective 1R4F group (FIGURE 4). The smoke con- centrations in the MS in the low and high dose groups of URSUS-35 were approx. 3 times higher than the corresponding ones in the respective 1R4F groups (TABLE 5). The nicotine concentrations in the respective dose groups of URSUS-35 and 1R4F were approx. equal (FIGURE 5).. l 3>~ ti~(~.S ~dr~s~hyd~ann ra ' on ..... +t nS-5: l.. °_(1
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REPORT B1033 / P 0500/3204 931125 PAGE 14 ~ 6 i ^'~ C, .cJ.. .- {rit.ec G-v -3 U Thelacrolein conce~trations determined in the MS of URSUS-35 were hiqhera~than those in the MS of the 1R4F (FIGURE 6) b ~! - ~ - - -- for the particle size of the smoke particles, no differences were seen between the 1R4F and the URSUS-35 groups, the MMAD being ap- prox. ... µm (GSD: ...) for the 1R4F groups and approx. .. µm (GSD: for the URSUS-35 groups. Results undiluted mainstream smoke
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REPORT B1033 / P 0500/3204 931125 PARAMETER ASSAY PRINCIPLE tM (b) gravinetry after trapping on Cambridge filters particle size fluoxometry after precipitation distribution in a spinning spiral duct (aerosol centrifuge) water in TPM Rarl-Fischer titration of water in TPM-loaded Cambridge filters carbon monoxide nondispersive infrared photometry of gas/vapor phase nicotine capillary gas chromatography after trapping on sulfuric acid- impregnated diatomite capillary gas chranatocgaphy after extraction from TPM-loaded Cambridge filter TABLE 3 ANALYSIS OF NIATNSZREBM SM7RFS PACE 15 DETERMINATION CONDITIONED MAINSTREAM SNBgKE SCHEDULE AIR ?1 t.ime/day + 1 time in the week after-the inhalation period 2 times/w2ek - continuously, daily + 2 timvs/wEek + UNDILUPED (a) DILUTED + + + + Ramarks: sample collection sites: FIQIRE 1 deto„a;nation of relative humidity before exposure chamber inlet and of tamperature at exposure chamber inlet and outlet (a) before start and after the end of the daily exposure, imnediately behind the pump (b) In addition, the particle concentrations was monitored on-line in the MS exposure groups using real-tine aerosol sen- sors. 00E066t90Z
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REPORT B1033 / P 0500/3204 931125 PdGE 16 PARAMETER formaldehyde, acetaldehyde, and acrolein glycerol temperature relative humidity BSSAY PRINCIPLE DEPERMINATIDN CONDITIDNED MAIPSIREAM SNDRE SCHEDULE AIR reversed phase high oerfonmance 2 times/wzek + liquid chir.matography of 2,4-d.in.itmphenylhydrazine (DNPH) derivatives after trapping in DNPH solution capillary gas chmaatography 2 times/wtiek + thenroelenent 2 times/day + psychrcmetzy continuously, daily - UNDILUTED (a) DILUi'ED + TABLE 3 (cont. ) ffiALYSIS OF MAINSTRF.AM SNDKES RaTiarks: sample collection sites: FIGURE 1 detprm;nation of relative humidity before exposure chamber inlet and of te1pP_rature at exposu.re chamber inlet and outlet (a) before start and after the end of the daily exposure, imnediately behind the pump '[06066I90Z
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REPORT B1033 / P 0500/3204 931125 PP.CCE 17 ~/ PhRAMETER ~f'iF~ UNIT OF NEASORE (gH8M / ~ 006E LEVEL !`. -t' 1H4F C7r7 IQi DR9US-35 lFw ICdi srtnke, diluted TPM ' µg/1 <1.4 83.4 ± 7.7 172.2 ± 12.4 38.1 ± 6.1 83.9 ± 8.0 water in TPM µg/1 ... ... ... ... ... carbon aunoxi.de ppn <1.5 94.0 ± 7.0 197.9 ± 9.9 15.5 ± 3.1 32.4 ± 4.6 nicotine µg/1 <0.01 4.75 ± 0.48 10.15 ± 0.88 5.64 ± 0.68 11.39 ± 0.77 glycerol µg/1 <2.74 5.95 ± 1.25 13.23 ± 1.15 8.30 ± 1.28 18.44 # 1.65 forn aldehyele ... ... . . acetaldehyde ~n <0.02 3.94 ± 0.46 8.05 ± 1.30 1.97 ± 0.18 4.29 ± 0.27 acrolein ppn <0.01 0.31 ± 0.04 0.60 ± 0.06 0.39 ± 0.03 0.80 ± 0.04 smoke, undiluted TPM mg/1 - 27.75 + .., 28.15 ± ... 6.20 ± ... 6.20 ± ... water in TPM mg/1 - 3.19 +... 3.14 +... 1.23 +... 1.17 ±..; nicotine ml/1 - 1.64 + ... 1.64 + ... 0.69 + ,.. 0.67 ± ... glycerol mg/1 - 2.07 ± ... 2.03 ± ... 1.07 ± ... 1.03 ± ... TABLE 4 CONC3ENII'RATIBIS OF SE •LF'CTEn SPDKE COMPONENTS Rararks: values represent means ± standard deviations or medians 4,.u~ C:,4c ]t0 vr~S` Z0E066190Z
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REPORT B1033 / P 0500/3204 931125 H02281 PAGE 18 N O T N 10 O w FIGURE 3 TPM CONCENTRATION, TIME COURSE o ~
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REPORT B1033 / P 0500/3204 931125 H02284 PAGE 19 FIGURE 4 CARBON MONOXIDE CONCENTRATION, 21-DAY MEANS
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REPORT B1033 / P 0500/3204 931125 H022BG PAGE 20 FIGURE 5 NICOTINE CONCENTRATION
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REPORT B1033 / P 0500/3204 931125 H02288 PAGE 21 CIGARETTE TYPE TEST ATMOSPHERE DOSE LEVEL DILUTION FACTOR FLOW, BATTELLE PUMP (1/min) SMOKE CONCENTRATION (puff:/1) (sham exposure) - - - - 1R4F MS low 294 1.07 0.10 ^ high 147 1.08 0.19 URSUS-35 " low 109 1.07 0.26 " - (cage control) high 52 1.08 0.54 TABLE 5 SMOKE CONCENTRATION
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REPORT B1033 / P 0500/3204 931125 H02288 PAGE 22 FIGURE 6 ACROLEIN CONCENTRATION
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REPORT B1033 / P 0500/3204 931125 PAGE 23 2.4 Biological Assays and Determinations In order to determine and compare the biological activity of the MS of URSUS-35 and 1R4F, reliable assays and determinations were carried cut (TABLE 6) that have shown to be sensitive in sub- chronic inhalation studies with 14S (Coggins et al., 1980; Lewis, 1981; Smith et al., 1978; Walker et al., 1978a,b) and capable of detecting differences in the biological activity of MS (CRC/INBIFO studies 131019 / P 0500/3165, 1991; B1032 / P 0500/3197). To provide an estimate of the smoke dose inhaled, selected respiratory parameters and the bloodd carboxyhemoglobin proportion were determined.
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REPORT B1033 / P 0500/3204 931125 PAGE 24 BIOIAGICAL PARAMEPER PRINCIPLE histopathology of: nose, laiynx, ' trachea, and lungs SCHEDULE NOMBER OF REMARKS (DAY) (a) RA75/GROUP light microscopical examina- 22, 23, 8 tion, 5 to 6-Vm sections, 64; 65 henatoxylin-eosin (all sections) and Alcian Blue/ periodic acid-Schiff's staining (trachea and lungs only) thiclmess of laryngeal morphcmetry; 5 to 6-ym epithelia . sections; hematoxylin-eosin staining TABLE 6 BIOIpGICAL ASSAYS AND DEPEFFIINATIONS (a) for performance of assay or sanple collection nose: formaldehyde fixation, 2 transverse sections, one imnediately posterior to the upper incisor teeth and one posterior to the incisive papilla; larynx, trachea, and lungs: intratracheal instillation with and subsequent sui:mersion in ethanol/acetic acid/ formaldehyde/saline; larynx: transverse sections at least at base of epiglottis and at arytenoid projections; trachea: at least 1 longitudinal section at bifurcation; lungs: at least 1 longitudinal section of the left lobe transverse section of larynx at arytenoid projections which includes ventral depression, floor of the larynx ("ventral lumen^), and vocal cords 60E066T90Z
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REPORT B1033 / P 0500/3204 931125 PAGGE 25 BIOLIJGICAL PARAMETER PRINCIPLE SCHEDULE NUNIDER OF REMARKS . . (DAY) (a) RATS/GRCiJP body weight gravimetry 1 day 20 until. individual rats after day 22, . ' arrival, 10 until on days day 64 -2 and 0 and >1 Einw/week starting on day 1 in-life observations observation daily observation of all rats at least 2 times/day, checklist exantina- tion on 3 rats/(group x day) mortality several times daily gross pathology external inspection of organs 22, 23, 10, after pentobarbital anesthesia in neck, ahdminal, thoracic, 64, 65 10 and exsanguination and pelvic cavities at ne- cropsy, internal if necessary TABIE 6 (cont. ) BIOLOGICAL ASSAYS APID DF'TF'RNiNATIpNS (a) for perfornance of assay or sanple collection OtB066t902
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REPORT B1033 / P 0500/3204 931125 PAGE 26 BIOLOGICAL PARA[+4:PER PRINCIPLE wEights of lungs with gravimetry trachea and larynx, heart, adrenal glands, testes, kidneys, and liver blood car`•oxyh~a-,;oglobl.z gas chrcanatography after reduction to methane of the CO released fran hennglobin respiratory frequency whole-body plethysmbgraphy and volwne T1,8tE 6 (cont. ) BIOLOGICAL ASSAYS AND DETERI•fINF1TIONS SCHEDULE (DAY) (a) NUMBER OF RA15/GROUP 22, 23, 10, 64,-65 10 , 16 3 14 to 18 10 (a) for perfonnance of assay or sample collection TIS066T90Z REMP,RICS sample collection fraa retrolnilbar venous plexus of diethylether-anesthetized rats, samples collected within 1 h before the end of the daily exposure
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REPORT B1033 / P 0500/3204 931125 PAGE 27 2.5 Statistics and Evaluation For the analytical assays, detection limits were based on the sensitivity/reproducibility specified by the suppliers for the analytical equipment (e.g., carbon monoxide and nitrogen oxides analyzers) and on the calibration curves (e.g. nicotine, aldehydes). As descriptive statistics, the number of values, a mean value (e.g., the arithmetic mean), and a measure of variance (e.g., the relative standard deviation) were given for all continuous data (e.g., body weight and thickness of laryngeal epithelium). For samples including at least 1 measured value below the detection limit, the median, 25 and 75% quartiles were given. Values below the detection limit were graphically presented as zero. For or- dinal data (e.g., histopathological data), the arithmetic mean, the standard error, and the incidences were given. The particle size distribution was calculated using linear regression analysis after probit transformation (Finney, 1971). The following statistical tests were performed on the biological data: Objective-related tests: Comparison of biological activity of URSUS-35 and 1R4F using the following procedure: - calculation of the mean response using low and high dose groups of each MS type, - calculation of the mean specific biological activity (slope)in relation to different dose parameters (TPM, nicotine, smoke concentration) with consideration of the mean response of the sham-exposed group' , r
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REPORT B1033 / P 0500/3204 931125 PAGE 28 - calculation of dose ratios between equal biological effects of URSUS-35 and 1R4F by dividing the mean specific biological activity values. Additional statistical tests: Comparison of sham-exposed group with the groups exposed to each of the 2 types of MS using the one-way analysis of variance followed by the Duncan test (Duncan, 1955) for con- tinuous data; for ordinal data, the generalized Cochran-Mantel- Haenszel test (Koch and Edwards, 1988) were applied followed by the generalized Cochran-Mantel-Haenszel test for the underlying 2 x r-contingency tables. - Comparison of all groups exposed to the different types of MS using the one-way analysis of variance followed by the Duncan test for continuous data; for ordinal data, the generalized Cochran-Mantel-Haenszel test was used. If the overall com- parison showed a significant difference between these groups, the Cochran-Mantel-Haenszel test was also applied for pairwise comparison between the groups. All tests were conducted at the nominal level of significance of alpha = 0.05, alpha = 0.01, and alpha = 0.001 (2-tailed). Results were considered statistically significant at p<0.05.
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REPORT B1033 / P 0500/3204 931125 PAGE 29 3 RESULTS AND DISCUSSION 3.1 Cage Control Grouo There were no findings in the cage control group. 3.2 Exposed Groups 3.2.1 Carboxyhemoglobin The mean steady state proportions of carboxyhemoglobin in the smoke-exposed groups did not exceed 24 %. They were in line with those expected on the basis of the carbon monoxide concentration.s c- in the test atmospheres (CRC/INBIFO studies B1019, P 0500/3165, 1991; 51018, P 0500/3157, 1991; 31022, P 0500/3136, 1993, and B1029, P 0500/3179). 3.2.2 Respiratory physiology_ 3.2.3 In-life observations and mortality Partially or fully closed palpebral fissures, rough coat, and/or yellow to brown discoloration of the fur were observed in all smoke-exposed groups but not in the sham-exposed group. N O ~ .0 o 0 w .~ A
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REPORT B1033 / P 0500/3204 931126 H02339 PAGE 30 N O .+ a a FIGURE 7 REDUCTION IN RESPIRATORY MINUTE VOLUME 0 ,.. Remarks: Values corrected for body weight. C^
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REPORT B1033 / P 0500/3204 931126 PAGE 31 In all groups including the sham-exposed group, observations were made related to exposure of rats in tubes such as partially or fully wet fur and reddish-brown secretion from the Harderian glands. In addition, decreased muscle tonus and gripping ability, changes in excitability and small wounds at the nose, tail, and feet were incidentally observed in all groups. Abnormal or labored breathing were incidentally observed in the high dose groups of URSUS-35 and 1R4F.r There were no differences between URSUS-35 and 1R4F groups on the respective dose level. A~ ~ - , - The smoke-related findings were similar to those observed in pre- vious MS inhalation studies (e.g., CRC/INBIFO study B1019 / P 0500/3165, ?.991). There was no mortalit.y du~ echniea-l- causes. During the 42-d postinhalation period the rats completely recovered from tube or smoke-related in-life observations. 3.2.4 Body weight The body weight of all groups increased during the 21 days of in- halation (FIGURE 8). The body weight gains of the MS-exposed rats as compared to the sham-exposed rats were statisticallly significantly reduced by max- imally 14 B(1R4F, high dose level, FIGURE 9). The reductions were numerically dose-dependent for both MS types, but statistically significant dose dependance was only found for the 1R4F MS.
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REPORT B1033 / P 0500/3204 931126 PAGE 32 The reductions were as expected from previous studies (e.g., CRC/INBIFO study B1030, P 0500/3187, 1992). At the end of the 42-d postinhalation period there was no dif- ference between the body weights of the sham- and MS-exposed groups. The body weights of the exposed groups were 4ese-.&A': to that of the cage control group (FIGURE 8).
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REPORT B1033 / P 0500/3204 931126 H02600 PAGE 33 FIGURE 8 BODY WEIGHT, TIME COURSE, 21-DAY INHALATION PLUS 42-DAY POSTINHALATION PERIODS Remarks: The bend between days 21 and 25 is explained by the doubled number of rats/group before the disNection on day 21 as compared with that from day 22 to day 63. BiE066I90Z
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REPORT B1033 / P 0500/3204 931126 H02325 PAGE 34 FIGURE 9 RELATIVE BODY WEIGHT GAIN REDUCTION
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REPORT B1033 / P 05`0/3204 931126 PAGE 35 l\{o There were no gross pathological findings. The liver weights, both ;0 u v J absolute (appi:ox_`~9~0;4) and relative tojbody weight (a t30 8~1, were similarly lower for both MS typesis. This effect was more pronounced in the present study than in similar studies. For both MS types, slight to moderate/marked histopathological changes were observed: hyperplasia, squamous metaplasia, and atrophy in the nose; hyperplasia and metaplasia in the larynx. Slight changes such as reserve an'tgoblet cell hyperplasiaLof the respiratory [,-- epithelia were observed in the trachea and lungs. Severities and incidences were similar for both MS types with 1 exception: The incidence of atrophy in the olfactory epithelium in the high dose group of URSUS-35 was slightly higher (3 out of 8 rats) than in the respective 1R4F group (1 out of 8 rats). This difference was not statistically significant. Except for the atrophy of the olfactory epithelium, the spectrum as well as the seuerities and incidences of the aforementioned findings have been commonly observed in previous MS inhalation studies (e.g., CRC/INBIFO study B1018 / P 0500/3157, 1991; B1019 / P 0500/3165, 1991; B1022 / P 0500/3136, B1030 / P 0500/3187). They have also been reported in literature for subchronic cigarette smoke inhalation studies (e.g., Coggins et al., 1980; Walker et al., 1978a,b). Concerning URSUS-35 MS the histopathological find- ings observed in the present study agree well with those obtained in the previous URSUS-35 study following 21-d exposure (CRC/INBIFO study B 1032 / P 0500/3197). At the end of the 42-d postinhalation period complete recovery of the MS-related findings was observed in the nose, larynx and trachea. In the lung goblet cell hyperplasia with minor degrees of severity and incidence being not statistically significant was found.
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REPORT B1033 / P 0500/3204 931130 PAGE 28 ORGAN ORGAN WEIGHT DIFFERENCE TO SHAM 1R4F DOSE LEVEL URSUS-35 LOW - STAT. SIGNIF- ICANCE HIGH STAT. SIGNIF- ICANCE LOW STAT. SIGNIF- ICANCE HIGH STAT. SIGNIF- ICANCE heart -6 n -6 n -3 n -9 y lungs with -1 n -2 n -8 y -3 n larynx and trachea liver -14 y -25 y -19 y -18 y kidney right -7 n -10 n -9 y -9 y left -7 n -10 n -7 n -10 y testis right 0 n 2 n -1 n -1 n left 0' n 0 n -1 n -1 n adrenal right 4 n 7 n -1 n 7 n left 4 n 3 n 1 n 6 n TABLE 7 OPRGAN WEIGHT; ABSOLUTE, DIFFERENCE TO SHAM, 21 DAYS Remarks: n: no statistically significant difference y: statistically significant difference, p<0.05 iZ8OSbI90Z
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REPORT B1033 / P 0500/3204 931126 P%E 44 f, \\ 2ZE066I902
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2061990323
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REPORT B1033 / P 0500/3204 931126 PaGE 38 LOCATION EPITHE- FIMDING STAT. CONPROL 1R4F ORSUS-35 E3RSSN' ISUM PARP.P4aER (~ DOSE LEVEL SHAM LpW HIGH LOW HIGH level 1 resp. res. c. hypexpl M 0.0 1.4 2.1 1.5 2.0 SE 0.0 0.3 0.1 0.2 0.0 TEST i-+ t+-t +++ ~+-f incidence 0/8 6/8 8/8 8/8 8/8 squ. metapl. M 0.0 0.5 1.6 1.1 1.6 SE 0.0 0.2 0.3 0.1 0.2 TESp + +++ +++ +++ incidence 0/8 4/8 8/8 8/8 8/8 TIaBIE 9 HIS7.UPATIiOLOGICAL FIPIDIMG4, NDSE, 21 DAYS Renarks: cmh-test followed liy anh-test, shem versus 1H4F versus ORSOS-35 930913 08:37:06 P. 1 DSER DISK:[HM]H1ST Cm412 3204.LIS;3 4ZE066I90Z
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REPORT B1033 / P 0500/3204 931126 PeIGE 39 IqC.ATION EPITHE- FINDING S`IAT. CONTROL 1R4F URSUS-35 9AJIN_ LIUM • PARFiMEl'ER A~UOHP LOSE L.EVF~ SHAM LOW HIGH LOW HIGH level 2 olf. atr. M 0.0 0.0 0.5 0.0 1.4 SE 0.0 0.0 0.5 0.0 0.7 TEST incidence 0/8 0/8 1/8 0/8 3/8 res. c. hyparpl M 0.0 0.0 0.0 0.0 0.1 SE 0.0 0.0 0.0 0.0 0.1 TEST = _ _ _ incidence 0/8 0/8 0/8 0/8 1/8 TABLE 9 (cont.) HISTOPd1Tfi0LOGICAL FINDING4, NC6E, 21 DAYS ~~- Raoarks: cYnh-test follaaed by cmh-test, sham versus 1R4F, versus URSUS (-3-ER;9-(Qt) 930913 08:44:46 P. 1 USER DISK:[WGO]HISP CIt12 3204.LIS;3 BZE066190Z
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REPORT B1033 / P 0500/3204 931126 PAGE 40 IOCATION EPITHE- FINDING STP,T. CONPROL 1R4F UPSUS-35 LIUM . PARAMETER C328T3F"- DQSE LEVEL SHAM I(7W HIQ3 IArJ HIQ3 be pseudo. squ. metapl. M 0.0 3.5 4.0 3.6 4.3 SE 0.0 0.5 0.3 0.2 0.2 TEST +++ ±-t-f i-t+ +.+ incidence 0/8 7/8 8/8 8/8 8/8 sqn. hyperpl. M 0.0 2.8 3.1 3.0 3.8 SE 0.0 0.5 0.4 0.2 0.3 TEST +F+ +F+ +F+ i++ incidence 0/8 7/8 8/8 8/8 8/8 ap-fl pseudo. squ. metapl. M 0.0 2.7 2.6 2.0 2.7 SE 0.0 0.2 0.4 0.5 0.2 TEST +++ +H- ++ +4+ incidence 0/8 7/7 7/8 7/8 6/6 ap-vcl squ. hyperpl. M 0.0 1.6 1.6 1.1 1.8 SE 0.0 0.5 0.4 0.4 0.3 TEST ++ ++ ++ +a-f inciderce 0/8 5/7 7/8 6/8 6/6 TABLB 10 HISRUPAII-LSIAGICAL FIIIDINGS, LARYNX, 21 DAYS Fan;rks: cmh-test followed by cmh-test, sham versus 1R4F, versus URSOS (3=Gt;4-GR) 930913 08:53:06 P. 1 USER DISK:[AL,O]HIST CYIl12 3204.LIS;3 9ZE066Z90Z
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REPORT s1033 / P 0500/3204 931126 PAGE 41 SITE THIQNESS OF EPITIIELIUM CONPRCIS 1R4F URSUS-35 DOSE LEVEL I A -/ I I SHAM L(N1 TEST HIGH LqV7 TEST HI(3H 1 ABS. 3[J ABS. REC. TO I ABS. REC~ 3C7 TEST A&4. REL. TTI ~ ABS. REL '10 TES (wR) t~~ (~) s~lM {{{,,, (wm) SHAM(%) (pm) s1W M (pn) klAk (%) ventral depression 8.26 100 9.90 120 + 11.19 135 ++ 10.06 122 + 10.74 130 ++ floor of the larynx 10.07 100 30.04 298 +++ 34.31 341 +++ 21.57 214 +++ 26.10 259 +++ vocal cords 21.18 100 27.86 132 + 32.61 154 +++ 26.27 124 ++ 32.73 155 +++ TABLE 11 THIC[QJESS OF LARYNC•AL EPITHELIUM, 21 DAYS 17marks:.,analysis of variance followed by Duncan test: sham versus 1R4F, versus URSOS-35 (,,/O V [iI L/i C > 4NC) [ .1-~ "F "j"(.J LzE0s6190z
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REPORT B1033 / P 0500/3204 931126 PAGE 42 LOCA4TON EPITfE- FINDING STAT. C0DIIR0L 1R4F URSUS-35 LIUM • PARA[4E:PER CQSE LEVEL SHAM L(7+7 HIQ-I L17W HI(3i trach. bif. resp. res. c. hyperpl M 0.0 0.6 0.9 0.5 0.5 SE 0.0 0.3 0.1 0.2 0.2 TEST + +++ _ _ incidence 0/8 3/7 7/8 4/8 4/8 " gob. c. hypexpl M 0.0 0.1 1.1 0.3 0.1 SE 0.0 0.1 0.3 0.2 0.1 TEST ° +++ _ _ incidence 0/8 1/7 7/8 2/8 1/8 TABT,E 12 HIS7OPA4fi0ICGICIsL FIFIDIIdG.S, TRACf1EP: _ Raoarks: cmh-test follaxed by cmh-test, sham versus 1R4F, versus URSUS 931001 13:22:53 P. 1 USER OISK:[MhR+.]HIST Ct4Q3204.LIS;2 8ZEO66L90Z
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REPORT B1033 / P 0500/3204 931126 PAGE 43 IIX'ATIQN EPI743E- LIOM , FINDING STAT. PARAMt~:PER CONTROL 1R4F DQSE LEVEL UR.SUS-35 SHAM LCNJ HIQ-I IqPI HIQ-I lung 1 resp. res. c. hypeipl M 0.0, 0.3 0.8 0.4 0.3 SE 0.0 0.2 0.3 0.2 0.2 TEST - ++ _ _ incidence 0/8 2/8 5/8 3/8 2/8 gob. c. hyperpl M 0.3 1.8 1.9 1.3 1.9 SE 0.2 0.4 0.4 0.5 0.5 TE$P ++ _ + incidence 2/8 7/8 7/8 5/8 6/8 TABTE 13 HISTOPAI230LOGICAL FIDIDINCS, LUNG 3s` Ranarks: cmh-test followed by cmh-test, sham versus 1R4F, versus URSUS (J-QY4~GR) 931001 13:34:37 P. 1 USER DISK:[NM]FIIST (ZII2_3204.LIS;2 6ZE066I90Z
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REPORT B1033 / P 0500/3204 931126 PAGE 45 IACATION EPITHE- FINDING STAT. CONTROL 1R4F URSUS-35 LIOM . PARAM`:'ER DO.SE LEVEL SHAM LCN7 HIGH LCFA HIGH lung 1 resp. gob. c. hyperpl M 0.1 0.1 0.8 0.6 0.6 SE 0.1 0.1 0.4 0.3 0.3 TEST - _ _ _ incidence 1/8 1/7 4/8 3/8 3/8 alv. macroph. M 0.1 0.0 0.0 0.0 0.0 SE 0.1 0.0 0.0 0.0 0.0 TEST incidence 1/8 0/7 0/8 0/8 0/8 TABLE 14 HIS70PATEA7LOGICAL FINDINGS, LUNG, FQId.,OWING POSTINHALATION PERIOD Rsnarks: ©oh-test fo1lo..ed by cmh-test, shan versus 1R4F, versus URSUS 931111 15:37:50 P. 1 USER DISK:[MMA]HIST CF4IIt 3204.LIS;3 i OE6066190Z
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report b1033 / p 0500/3204 931126 PAGE 46 3.3 Comparison URSUS-35 with 1R4F As compared with the 1R4F, URSUS••35 showed, dependent on the dose parameter used for the body weight gain reduction and almost all pathological determinations, consistent factors to obtain equal effects by both MS-types (TABLE 15). For the TPM concentration, a factor of 2 was found (FIGURES 1A to ]k), for the nicotine concentration, it1 (FIGURES. 61. to 21) and for the smoke , concentration, it was less ~th~ae-ne-half (FIGURES 23 to FG). There were 2 exceptions: 1 was the atrophy of the olfactory epithelium in the nose, where the URSUS-35 MS showed higher factors than the above given means, the other being goblet cell hyperplasia in the trachea showing factors considerably lower than the above given means. For both exceptions, this holded true for all dose parameters used.
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REPORT B1033 / P 0500/3204 931126 PAGE 47 EFFSCP DOSE PARAME7.'ER TPN NICCfPINE SAY31E body weight gain reduction organ weight liver 1.9 0.8 0.32 abs. 2.0 0.8 0.33 rel. to body weight nose level 1 1.8 0.8 0.31 reserve cell hypexplasia 2.1 0.9 0.36 squarous metaplasia level 2 2.7 1.1 0.46 atrophy larynx base of epiglottis (5.8 2.4 0.99) squamous metaplasia 2.2 0.9 0.38 hyperplasia • arytenoid projections floor of the larynx 2.4 1.0 0.41 squamous netaplasia vocal cords lower medial region 1.9 0.8 0.33 hyperplasia larynx morphometry 2.0 0.8 0.34 ventral depression 2.0 0.8 0.34 floor of the larynx 1.3 0.6 0.23 vocal cords trachea 1.9 0.8 0.33 reserve cell hyperplasia 1.5 0.6 0.25 goblet cell hyperplasia lung (0.6 0.3 0.10) reserve cell hyperplasia 1.3 0.5 0.22 goblet cell hyperplasia 1.8 0.7 0.30 ------------------------------- ----------- ------------ ---__-_-_ N O N tnean .• 1.9 0.8 0.33 0 ~ TABLE 15 W N D(.LSE RATIOS BEIWEEN Ef,RIAL BIOUJGII:AL EFFEG75 OF URSUS-35 AND 1R4F DE4 Renarks: Values in brackets omitted for calculation of nean because of considerable deviation from other values.
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REPORT B1033 / P 0500/3204 931126 H02339 PAGE 49 '1~ ( N O T r 10 FIGURE 1~ ... , L V o 4I w w w
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REPORT B1033 / P 0500/3204 931126 H02339 PAGE 50 FIGURE 11 ...
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REPORT B1033 / P 0500/3204 931126 H02339 PAGE 51 FIGURE 12 ... N 0 P r ~ d O ~ W N
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REPORT B1033 / P 0500/3204 931126 H02339 PAGE 52 FIGURE 13 ...
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REPORT B1033 / P 0500/3204 931126 FI02339 PAGE 53 N O P FIGURE 14 ... . ~ O m w J
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REPORT B1033 / P 0500/3204 931126 H02339 PAGE 54 N 0 a FIGURE 15 ... ~ 10 0 w m ro
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REPORT B1033 / P 0500/320 4931126 PAGE 48 4 CONCLUSION Compared with the MS of 1R4F, in the MS of URSUS-35 at the respec- tive dose level the TPM concentrations and the calculated smoke concentrations were less by half, the nicotine concentrations were approx. equal, the carbon monoxide concentrations were lower by a factor of 6 and the formaldehyde and acrolein concentrations were higher by a factor of 6 and 1.3, respectively. The biological activity of URSUS-35 MS as compared with that of the 1R4F was, dependent on the dose parameter used, a factor of 2 higher, i.e., when related to the TPM concentration, equal, i.e., when related to the nicotine concentration, and less than half, i.e., when related to the smoke concentration. These factors agree well with those gained from the smoke analyses of URSUS-35 MS.
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REPORT P 0500/1033 931126 PAGE 55 5 TEST MATERIAL (a) (1) Test cigarette: (2) Standard reference cigarette: URSUS-35 (lot ...) 1R4F Source: (1) PMR (2) University of Kentucky Number: (1) ... (2) ... Packaging: (1) approx. 1500/box (2) 20 cigarettes/pack, 10 packs/carton, 25 cartons/box Date of receipt at CRC: (1) 14 and 22 Jul.93 (2) 1 Feb.93 Storage: modular lab unit C122, room temperature and relative humidity controlled Conditioning Location: modular lab unit C119/120, room temperature and relative humidity controlled Duration: for at least 8, but no longer than 10 days prior to smoking Temperature: 22 i_ 1 °C Relative humidity: (1) 50 ± 2 % (2) 66 ± 2 % (b) Selection: (1) lot 4.1 N (2) no selection a .. (a) The test material is the MS of the test or standard reference cigarette. (b) The relative humidity was adjusted using a saturated sodium nitrite solution.
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REPORT P 0500/1033 931126 PAGE 56 6 STUDY ORGANIZATION AND RESPONSIBILITIES 6.1 Study Organization The present study was a collaborative study between CRC Contract Research Center (Zaventem, Belgium) and INBIFO Institut fur bio- logische Forschung (Cologne, F.R.G.). CRC was the initiator of the project and its responsibility in- cluded the animal experimentation part. INBIFO was the subcon- tractor performing analyses of and assays on sample materials received from CRC. 6.2 Responsibilities 6.2.1 CRC Study Director: ................ Date... ............................... Dr.Lic. P.M. Terpstra Veterinarian General Manager: ..............e.... ............................... Date Dr.rer.nat. R.-A. Walk Biologist (Diplombiologe), Biochemist and Toxicologist (ABT)
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REPORT P 0500/1033 931126 PAGE 57 6.2.2 INBIFO Coordinator: ................... ............................... Date D. Bachmayer Biologist (Diplombiologe) Analytical Chemistry: ................... ............................... Date Dr.rer.nat. P. Voncken Chemist (Diplomchemiker) Microbiology: ................... Date ............................... Dr.rer.nat. F. Tewes Biologist (Diplombiologe) Pathology: ................... ............................... Date Dr.med.vet. A. Teredesai Veterinary Pathologist (Fachtierarzt fiir Pathologie) Information Systems Management: ................... Date ............................... Dr.rer.nat. W; Gomm Mathematician (Diplommathematiker) General Manager: ..........•. Date ~ ............................... ~ Dr.rer.nat. W. Reininghaus o Physicist (Diplomphysiker) ~ N
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REPORT P 0500/1033 931126 PAGE 58 7 GOOD LABORATORY PRACTICE STATEMENT 7.1 Studv Director This study was conducted according to Good Laboratory Practice Regulations. For CRC the compulsory regulations are given in the Federal Register (1987); for INBIFO in the Bundesgesetzblatt (1990, 1991). The study director confirms the validity of the results obtained in this study. Date 7.2 Quality Assurance ` ~.. Inspections on this study were performed by the quality assurance S unit for the following: ;L. PARAMETER INSPECTED DATE ... ... . ~~` i ~All findings were reported to the study director and to the general management within at least 2 working days following the inspection. This report accurately reflects the study carried out and the results obtained (SOP QA 4, QA 6). ............. ....................e........... Date Quality Assurance Manager
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REPORT B.... / P.... /.... 931126 PAGE 59 Dr.med. Dr.rer.nat. K. von Holt Physician and Physicist (Arzt und Diplomphysiker)
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REPORT P 0500/1033 931126 PAGE 60 8 STORAGE OF MATERIALS AND RECORDS Records are ~y stored in our archives for at least 30 years after delivery of the final report to the client. Samples of the cigarette URSUS-35 and the standard reference cigarette and all specimens are bcs~stored at CRC or INBIPO 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.
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STUDY PLAN B6034 / P 0500/3204 931126 PAGE 61 9 REFERENCES 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) Coggins, C.R.E., Fouillet, X.L.M., Lam, R., Morgan, K.T., Cigarette smoke induced pathology of the rat respiratory tract: a comparison of the effects of the particulate and vapour phases, Toxicology 16: 83-101 (1980) -(INBIFO reference: L0016 H20) CRC/INBIFO study B1019 / P 0500/3165, Biological activity of the mainstream smoke of the research cigarettes X6DOCRN, X6DOCRO, X6DOCRP, X6DOCRQ, X6DOCRR, and X6DDCRS, 90-day inhalation study on rats, Study directorn Dr. H.-J. Haussmann Coordinator: Dr.M. Kaegler Date of report: 28.Aug.91 CRC/INBIFO study B1032 / P 0500/3197, Biological activity of the mainstream smoke of the test cigarette URSUS and the standard reference cigarette 1R4F, 90-day inhalation study on rats, Study director: Dr.Lic. P.M. Terpstra Coordinator: D. Bachmayer not yet reported DIN Deutsches Institut fur Normung: Chemische Analytik, Nachweis-, Erfassungs- und Bestimmungsgrenze, DIN 32645, Berlin, Beuth Verlag, 1991 Duncan, D.B., Multiple range and multiple F-tests. Biometrics 11: 1-42 (1955) - (INBIFO reference C196) Finney, D.J.: Probit analysis, Cambridge: Cambridge University Press, 1971 International Agency for Research on Cancer, IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans, Tobacco smoking, Vol. 38, Lyon: IARC, 1986 International Organization for Standardization: International Standard ISO 3308, Cigarettes - Routine analytical cigarette- smoking machine - Definitions and standard conditions, 3rd ed., 1991
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STUDY PLAN B6034 / P 0500/3204 931126 PAGE 62 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 total and nicotine-free dry particulate matter using a routine analytical smoking machine, 2nd ed., 1991 Koch, G.G. and Edwards, E., Clinical efficiency trials with categorical data, in: Pearce, K.E. (Ed.) Biopharmaceutical statistics for drug development, New York, Marcel Dekker, 1988, pp. 403-457. Lewis, D.J., Factors affecting the distribution of tobacco smoke-induced le- sions in the rodent larynx, Toxicol. Lett. 9: 189-194 (1981) -(INBIFO reference: L1590 C12) OECD Organization for Economic Co-operation and Development, Beschlup des Rates vom 28. Juli 1989 uber die Annahme einer Entscheidung/Empfehlung der OECD durch die Europaische Wirtschaftsgemeinschaft uber die Einhaltung der Grundsdtze der Guten Laborpraxis, ISSN 0376-9453, L 315, 28 October 1989, 88/320/EWG Smith, G., Wilton, L.V., Binns, R., Sequential changes in the structure of the rat respiratory system during and after exposure to cigarette smoke, Toxicol. Appl. Pharmacol. 46: 579-591 (1978) - (INBIFO reference: L0035 A01) Walker, D., Wilton, L.V., Binns, R., Inhalation toxicity studies on cigarette smoke (6), 6-week com- parative experiments using modified flue-cured cigarettes: histopathology of the lung, Toxicology 10: 229-240 (1978a) - (INBIFO reference: L0006 N24) Walker, D., Wilton, L.V., Binns, R., Inhalation toxicity studies on cigarette smoke (7), 6-week com- parative ekperiments using modified flue-cured cigarettes: histopathology of the conducting airways, Toxicology 10: 241-259 (1978b) - (INBIFO reference: L0006 0008)
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STUDY PLAN B6034 / P 0500/3204 937.126 PAGE 63 ABBREVIATIONS (a)(b) DABT: Diplomate American Board of Toxicology ISO : International Organization for Standardization MS : diluted mainstream smoke p : probability of receiving the value of the test statistic a more extreme value of the test statistic, if the null hypothesis holds or ppm : parts per million (10E-6), used only with ratios of gaseous volumes RSD : relative standard deviation(s) SOP : standard operating procedure(s) TPM : total particulate matter _ ~`..~ ~ END OF STUD~PLAN (a) in addition to those explained immediately on the same page (b) Units are given in accordance with SI units (Systeme Inter- national d'UnitAs).

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