Anne Landman's Collection
Integrating Report A 0500/3047 21-Day Smoke Inhalation Study with Mainstream and Sidestream Cigarette Smoke of Standard Reference Cigarette Type 2r1 on Rats
Abstract
This 1982 scientific paper from Philip Morris' biological research lab, INBIFO (Institut for biologlsche Forschung) in Cologne, Germany reports on experiments done on groups of rats who for 21 days were made to inhale either mainstream or secondhand smoke (called "sidestream" smoke in the report) from cigarettes obtained from Philip Morris (PM).
The study reports that secondhand smoke exposure was more irritating than mainstream smoke, and most particularly to the upper airways (nasal cavities, olfactory membranes, etc.):
(From Page 11 of the report):
"All of the examined sidestream-exposed rats showed slight to severe atrophic or necrotic lesions of the olfactory epithelium, in some cases together with reactive inflammation. The ciliated epithelium of all sidestream exposed rats showed squamous-cell metaplasia, with cornification in some cases."
"Generally spoken, sidestream exposure induced more frequent and more severe epithelial lesons in the olfactory and ciliated epithelium of the nasal cavity than mainstream of equal TPM [total particulate matter] concentration..."
"Sidestream exposure induced much stronger irritative changes in the mucosa of the nasal cavity than mainstream of equal TPM [total particulate matter] dose..."
and
"If one extrapolates from the experience of previous mainstream inhalation studies (b), the mainstream TPM concentration of this study would have to be increased by a factor of 3 to produce similar strong reactions than seen with sidestream exposure in this study."
The report was written by Wolf Reininghaus, general manager of contract research at INBIFO, to Ragnar Rylander, a Swiss scientist who contracted with Philip Morris). It shows that Philip Morris was well informed by 1982 that secondhand smoke had the propensity to inflict harm on nonsmokers.
Fields
- Quotes
"In the present 21-day inhalation study on rats the subacute toxicity of sidestream cigarette smoke has been compared to the subacute toxicity of mainstream smoke..."
[From page 8, Bates No. 2029190336]
"As commonly seen in cigarette inhalation studies of this type, the rats resisted to the daily loading into the exposure tubes and continued to struggle inside the tubes right after the beginning of the exposure. By and large, the rats of the sidestream groups reacted more vigorously than those of the mainstream group. All rats showed general signs of exhaustion after the end of the daily exposure. In contrast to the rats of the mainstream group, which recovered by the next morning, the rats of the sidestream groups continued to show shaggy fur and some pronounced respiratory symptoms characterized by whistling and rattling sounds..."
(From Pp 10-11: 2029190338/0339)
"The nasal cavity of 53 rats (b) and the larynx and lungs of 20 rats (c) killed at dissection after 21 days of exposure were examined histopathologically...The nasal cavities (d) of the rats of the control group were without histopathological alterations...All of the examined sidestream-exposed rats showed slight to severe atrophic or necrotic lesions of the olfactory epithelium, in some cases together with reactive inflammation. The ciliated epithelium of all sidestream exposed rats showed squamous-cell metaplasia, with cornification in some cases."
"Generally spoken, sidestream exposure induced more frequent and more severe epithelial lesons in the olfactory and ciliated epithelium of the nasal cavity than mainstream of equal TPM concentration..."
"...The ciliated epithelium in the dorsal parts of the larynx showed squamous-cell metaplasia without cornification in 50 percent of the mainstream-exposed rats whereas all rats of the sidestream-exposed groups showed squamous-cell metaplasia with cornification."
(From Page 12-13: 2029190340/0341)
"Sidestream exposure induced much stronger irritative changes in the mucosa of the nasal cavity than mainstream of equal TPM dose...Sidestream smoke exposure invoked atrophy of the olfactory and metaplasia of the ciliated epithelium. Mainstream smoke produced some metaplasia of the olfactory epithelium..."
"These findings may be interpreted as a stronger inflammatory response of the lung to sidestream-smoke than to mainstream of equal TPM concentration..."
(From Page 15-18, under "Discussion, Conclusion," Bates Nos. 2029190343/0346)
"If one extrapolates from the experience of previous mainstream inhalation studies (b), the mainstream TPM concentration of this study would have to be increased by a factor of 3 to produce similar strong reactions than seen with sidestream exposure in this study."
"The strong irritative changes with sidestream exposure, seen at the olfactory and ciliated nasal and laryngeal epithelium, are presumably caused by low molecular, fast- absorbed irritative sidestream components.
The alkaline reactivity, together with the high buffer capacity of the sidestream gas/vapor phase, as well as the known excess of ammonia in sidestream smoke (a), point to ammonia as a key substance for the irritative capacity of sidestream smoke...Histopathological changes similar to those seen with sidestream exposure in the nasal mucosa could be evoked in rats by exposure to pure ammonia under comparable experimental conditions (b)."
- Company
- Philip Morris
- Author
- Gugel, H.
- Reininghaus, Wolf (INBIFO, Gen. Mgr., Contract Research)
Institut fur Biologische Forschung, Cologne, Germany - (Philip Morris' offshore research lab).- Romer, E.
- Schnell, P.
- Speck, M.
- Teredesai, A.
- Tewes, F.
- Walk, R.A.
- Reininghaus, Wolf (INBIFO, Gen. Mgr., Contract Research)
- Recipient
- Rylander, Ragnar, M.D. (PM contractor, Environmental Hygienist, U of Gothenburg)Professor of Environmental Hygiene, University of Gothenburg, Sweden. Worked on contract to Philip Morris overseeing biological laboratory work being performed at INBIFO, PM's biological labs in Cologne, Germany. PM paid Rylander $150,000 per year (salary determined from Bliley PM doc 2022850392, from 1992)
- Region
- Switzerland
- Named Organization
- Fach Tierarzt Fur Pathologie
- INBIFO, Intitut Fur Biologische Forschung (Philip Morris' secret biological research lab in Europe)
"INBIFO" stands for Institut Fur Biologische Forschung, or Institute for Biological Research. It is located in Germany. Philip Morris acquired Inbifo on June 30, 1971. Its stated mission was "quantitative biological product evaluation" by using "comprehensive toxicological and physiological testing. Major activities are listed as: product evaluation and modifications, product ingredients and ETS-related technical knowledge and smoke components. Inhalation toxicology was a key feature of Inbifo. (Derived from Bates No. 2505235055/5088) - INBIFO, Intitut Fur Biologische Forschung (Philip Morris' secret biological research lab in Europe)
- Litigation
- STMN/Produced
- Named Person
- Corn
- Type
- SCRT, REPORT, SCIENTIFIC
- CHAR, CHART, GRAPH, TABLE, MAPS
- FOOT, FOOTNOTES
- CHAR, CHART, GRAPH, TABLE, MAPS
- Subject
- secondhand
- secondhand smoke
- secondhand smoke/health effects
- Secondhand Smoke/Toxicity
- secondhand smoke
Document Images
INBIFO Institut fur biologische Forschung Koln
111BiFu
DRMEU R. RYLANDER 29JUL82
c/o FABRIQUES DE TABAC REUNIES SA DRRS/RM
RM9 (R) Al
S W I T Z E R L A N D
I N T E G R A T I N G REPDRT
A o5oo/3o47
21-DAY SMOKE INHALATION STUDY WITH
MAINSTREAM AND SIDESTREAM CIGARETTE SMOKE OF
STANDARD REFERENCE CIGARETTE TYPE 2R1
ON RATS
COPY NO.:
INBIFO Institut fur biologische Forschung GmbH, Fuggerstraee 3, D-5000 K81n 90 Sitz der
Gesellschalt: KSln HR 8 367, 29. Oktober 1959
5t.Kw et Tele(on: Porz (02203) 303-1, telelax: (02203) 303362, Telex: 8874 675 lnbi d
lnstitutsleiter und GeschVsliihrer: Dr. med. Ulrioh Hecicenberg
1NB1FO Institut filr biologische Forschung K6ln
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A2 PAGE o-1
CONTENTS PAGE
1 SUMMARY 1-1
1.1 Introduction 1-1
1.2 Generation and Administration of smoke 1-2
1.3 Results 1-5
1.4 Discussion, Conclusion 1-13
2 RESPONSIBILITY 2-1
3 CIGARETTES 3-1
TABLE A: TYPES OF SIDESTREAM USED FOR INHALATION 3-2
TABLE B: SPECIFICATIONS OF CIGARETTE TYPES 2R1 3-3
TABLE C: PHYSICAL PROPERTIES AND CHEMICAL COMPOSITION
OF FILLER OF CIGARETTE TYPE 2R1
3-4
TABLE D: COMPOSITION OF SMOKE COMPONENTS OF CIGARETTE
TYPE 2R1
3-6
4 STO RAGE OF MATERIALS AND RECORDS 4-1
This integrating report contains, including front page, 26 pages.
2s.xwa2
INBIFO lnstitut ftir biologische Forschung K6ln
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A3 PAGE 1-1
,
SUMMARY
1.1 Introduction
In previous studies at INBIFO the biological activity of inhaled
mainstream cigarette smoke on small laboratory animals was inve-
stigated. In order to quantitate biological activity, systemic
responses, metabolic responses, effects indicative for mutageni-
city, histological responses of the respiratory tract as well as
free lung cell responses were studied (Bioassay program).
In addition to mainstream, which leaves the burning cigarette
through the mouth end or filter during puffing, smoke is also
released from the burning zone directly into the surrounding
atmosphere without passing through the filler and the mouth
end. Additional smoke diffuses through the cigarette paper. This
smoke types are termed sidestream smoke (a).
Since "open" smoking is performed, smoke leaks out of the mouth
end during puff intervals. This fraction is not included into the
side- or mainstream fraction (see FIGURE A, TYPES OF SIDESTREAM
USED FOR INHALATION).
Though qualitatively similar, the chemical composition and physical
properties of sidestream is known to be quantitatively different
from mainstream (b). This difference is also reflected in the
different biological activity of mainstream and sidestream cigar-
ette smoke condensate in the mouse skin painting model (c).
(a) According to DIN 1o24o "Maschinelles Abrauchen von Zigaretten
und Bestimmung des Rauchkondensats", Apr.78, sidestream smoke
is defined as the whole amount of smoke, which leaves the cig-
arette by another way than through the cigarette mouth end.
(b) see De Bardeleben, M., An Overview of sidestream Smoke: its
components, its analysis, some influencing factors, PM inter-
office corresp. March 1981
(c) sidestream condensate: higher tumorigenic activity than main-
stream condensate (Wynder, E.L. and Hoffman, D., Tobacco and
tobacco smoke, Acad. press, N.Y., pp. 183 and 291, 1967 )
sa.Kws2
INBIFO lnstitut fGr biologische Forschung KtSIn
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A4 PAGE 1-2
These differences are related to the deviating burning- and distil-
lation processes during puffing and puff intervals. Sidestream
smoke will generally contain a higher proportion of oxidative
combustion products, but a lower proportion of distillation and
pyrolysis products as mainstream smoke. For normal cigarette
types the particle phase of sidestream contains smaller, highly
charged particles (Corn, 1974) and its gas phase relatively more
volatile components than mainstream.
In the present 21-day inhalation study on rats the subacute toxici-
ty of sidestream cigarette smoke has been compared to the subacute
toxicity of mainstream smoke. In addition to parameters of general
toxic effects, local histological changes of the epithelium in the
respiratory tract and cytotoxical effects on free lung cells were
analysed.
As a prerequisite, the methods for generation, administration and
for analytica3l monitoring of cigarette sidestream smoke had to be
acquired.
Cigarette type 2R1 was used to generate sidestream and mainstream
smoke. As in previous inhalation studies with mainstream, the TPM
concentration was used to relate administered "doses" of side- and
mainstream smoke.
1.2 Generation and Administration of Smoke
Mainstream smoke was generated as in previous studies using 1
automatic 3o-port positive pressure smoking machine.
Parallel to mainstream smoke generation, the resulting sidestream
smoke ("puffed sidestream smoke") was quantitatively collected by
a circular hood inside the smoking machine.
28. Kwa2
INBIFO lnstitut ftir biologische Forschung Mln
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A5 PAGE 1-3
In a 2nd smoking machine, the cigarettes were only lighted with 1
mainstream puff, and the sidestream smoke originating from the
nonpuffed smoldering cigarettes ("nonpuffed sidestream smoke")
was collected in the same way as far as the puffed sidestream
smoke.
By adjusting the sample flow rates of the sidestream smoke and
the flow rate of the diluting air for the mainstream smoke, the
TPM concentration of all 3 types of cigarette smoke was adjusted
to be equal (see IB TABLE A, GROUPS, EXPOSURE AND DOSES).
loo male rats with an initial mean body weight of 17o g were
randomly allocated to 5 groups of 2o rats each.
The cage control group was not treated and was kept in cages.
The tube control group was sham exposed to a similar flow of
fresh air as the dose groups.
The mainstream group was exposed on 42o min/day for 21 consecu-
tive days to o.5 : 21o diluted mainstream smoke resulting in a TPM
concentration of o.17 mg/3. (a).
The "puffed" sidestream group was simultaneously exposed to equal
TPM concentrations of sidestream smoke, generated in parallel.
The "nonpuffed" sidestream group was simultaneously exposed to
equal TPM-concentrations of sidestream smoke from nonpuffed
cigarettes.
(a) This represents about 1/3 of the highest TPM concentration in
previous 21-day inhalation studies. For the given cigarette
type and exposure duration this "daily TPM dose" is limited by
the relatively high CO concentration of the puffed sidestream
smoke.
28. KW 82
INBIFO Inst9tut fiar bfologische Forschung Kt51n
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A6 PAGE 1-4
I
The rats of the 3 dose groups were kept in glass tubes, sealed with
rubber stoppers at the back and ending in a stainless steal wire
head basket at the front end which extended into the aerosol duct
of the inhalation chamber (head only exposure).
The rats of the tube control group were kept in Makrolon tubes.
The flow of the aerosol (or air) inside the inhalation chambers was
adjusted to be approx. o.35 m/s for all groups. This resulted in a
constant pressure of about lo mbar inside the inhalation chamber
relative to the outside.
The mean temperature of the diluted smoke (or air) inside the
inhalation chamber was:
23.3 degrees centigrade for the tube control group,
25.2 degrees centigrade for the mainstream group,
33.5 degrees centigrade for the puffed sidestream group and
33.9 degrees centigrade for the nonpuffed sidestream group.
The remarkable decrease of body temperature (see 1.3 Results)
during exposure and its possible effect on the body weight develop-
ment of the rats was investigated by exchanging some of the Makro-
lon tubes with glass tubes (tube control group only) and some of
the glass tubes with temperature controlled glass tubes (nonpuffed
sidestream group only).
28.KW82
INBIFO Inst9tut fiar biologische Forschung - K6in
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A7 PAGE 1-5
,
1.3 Results
Diluted mainstream and sidestream smoke from puffed and nonpuffed
cigarettes were sampled from the inhalation chambers. The following
parameters were analyzed: total particulate matter, nicotine,
carbon monoxide, carbon dioxide, nitric oxide, acetaldehyde,
hydrogen cyanide and the hydrogen-ion concentration as well as the
buffer capacity of an aqueous extract of the particle and the
gas/vapor phase (see AC TABLE 1, SURVEY OF SMOKE ANALYSIS).
Within the accuracy of these analyses, none of these parameters
showed remarkable differences between the chemical composition of
puffed and nonpuffed sidestream smoke.
The concentration of TPM and nicotine as well as the buffer capa-
city and the pH of the particle phase showed no remarkable differ-
ence between the diluted main- and sidestream smoke.
The following gas/vapor phase components were distinctly higher in
diluted sidestream smoke than in diluted mainstream smoke: carbon
monoxide (4 times) (a), carbon dioxide (3 to 4 times), nitric oxide
(6 to 7 times) and acetaldehyde (2 to 3 times).
Only the hydrogen cyanide concentration was higher in diluted
mainstream than in sidestream smoke. The factor was 2.
The gas/vapor phase of sidestream smoke was more alkaline (pH 8.7)
and had a much stronger buffer capacity (26-fold) than the main-
stream gas/vapor phase (b).
(a) The higher CO concentration in sidestream smoke was in accord-
ance with the higher HbCO concentration in the blood of the
sidestream exposed rats, which limited the maximum inhaled
daily smoke dose.
(b) Both effects may mainly be due to the known higher ammonia
concentration in sidestream smoke (which was not analyzed in
this study).
2a.Kwa2
lNBIFO Institut fiir biologische Forschung Min
INTEGRATING REPORT A o5oo/3o47 RM9 (R) AB PAGE 1-6
i
As commonly seen in cigarette inhalation studies of this type,
the rats resisted to the daily loading into the exposure tubes and
continued to struggle inside the tubes right after the beginning
of the exposure. By and large, the rats of the sidestream groups
reacted more vigorously than those of the mainstream group. All
rats showed general signs of exhaustion after the end of the
daily exposure. In contrast to the rats of the mainstream group,
which recovered by the next morning, the rats of the sidestream
groups continued to show shaggy fur and some pronounced respiratory
symptoms characterized by whistling and rattling sounds.
1 rat of the mainstream group, 9 rats of the puffed and 11 of the
nonpuffed sidestream group died "spontaneously". 2 rats of the
puffed sidestream group and 1 rat of the nonpuffed sidestream group
were killed in moribund state.
Additional 12 rats of all groups died for reasons which were
considered to be of primarily technical nature.
The rats of the sham and the cage control groups increased their
body weight during the .21 days of exposure steadily in a normail
way to approx. 15o percent of the initial body weight.
The body weight of the mainstream group increased to approx.
13o percent.
The sidestream groups showed a decrease to approx. 8o percent of
their initial body weight.
The rats of the sham and cage control groups consumed approx.
24 grams powdered diet per day.
28.KW82
INBIFO Institut ftir biologische Forschung Kt31n
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A9 PAGE 1-7
This food consumption is in the normal range and was approx.
constant during the 21 days of exposure.
The food consumption of all dose groups dropped sharply during
the first days of inhalation.
The food consumption of the mainstream group increased gradually
after day 4 and reached a total mean of 92 percent of the con-
Aumption oC the sham control group.
The food consumption of the sidestream groups remained low, with
a resulting total mean of 45 percent of the sham control group.
The water intake showed a similar pattern as the food consumption:
it was 39 g/day for the rats of the sham and the cage control
group, and 113 percent for the mainstream group and 59 percent for
the sidestream groups relative to the sham control.
The mean body temperature at the end of each daily exposure was
in the range of 36.5 and 37.4 degrees centigrade for the rats of
the cage control and the sham-exposed group regardless of whether
kept in glass or Makrolon tubes.
The mean body temperature of the mainstream exposed rats was
36.5 and that of both sidestream groups 32.o degrees centigrade.
The respiratory frequency of the dose groups at the end of each
daily exposure showed a consistent decrease similar to the body
temperature.
The mean respiratory frequency was 1o7/min for the sham and
cage control groups, 91/min for the mainstream and 86/min for
both sidestream-exposed groups.
28.KW82
INBIFO Institut fiir biologische 1=orschung K6In
INTEGRATING REPORT A o5oo/3o47 RM9 (R) Alo \,PAGE 1-8
Some rats of sham control group, which were exposed after day 14 to
nonpuffed sidestream smoke in temperature controlled exposure
tubes (a) showed a similar drop in body weight, food consumption
and water consumption as the other rats of the sidestream groups
at the beginning of the study.
The increased tube temperature and hence the decreased heat loss
of the rats, showed no influence on these parameters.
The body temperature increased with higher environmental tempera-
ture of the tubes, but the difference between body and tube tempe-
rature, which can be considered as proportional to the metaboli-
cally generated heat, decreased from approximately 5 degrees
centigrade for the nonheated tubes to 1.5 degrees centigrade at
35 degrees centigrade tube temperature.
The nasal cavity of 53 rats (b) and the larynx and lungs of 2o rats
(c) killed at dissection af ter 21 days of exposure were examined
histopathologically.
The nasal cavities (d)- of the rats of the control group were
without histopathological alterations.
(a) The 4 temperature levels of the tubes compared were: nonheated
(equilibrium with room temperature of 2o degrees centigrade),
30, 33 and 35 degrees centigrade
(b) 27 rats of the control groups, 16 mainstream-exposed rats
and 6 and 4 rats, exposed to puffed and nonpuffed sidestream
smoke respectively.
(c) 9 rats of the control groups, 4 mainstream-exposed rats and
3 and 4 rats, exposed to puffed and nonpuffed sidestream smoke
respectively.
(d) 2 dorsoven a1 sections, 1 at the incisive papilla and 1
between the 1st and 2nd palatal ridge were histopathologi-
cally evaluat d.
2a.xwa2
INBIFO Institut #Gr biofogische Forschung KBin
INTEGRATING REPORT A o5oo/3o47 RM9 (R) All PAGE 1-9
1
5o percent of the mainstream exposed rats showed mainly focal
squamous-cell metaplasia of the olfactory epithelium limited to the
anterior part of the nasal cavity. Slight atrophic changes of the
olfactory epithelium were found in only 6 percent and slight basal
or goblet cell hyperplasia of the ciliated epithelium were found in
only 13 percent of these rats.
All of the examined sidestream-exposed rats showed slight to severe
atrophic or necrotic lesions of the olfactory epithelium, in some
cases together with reactive inflammation. The ciliated epithelium
of all sidestream exposed rats showed squamous-cell metaplasia,
with cornification in some cases.
Generally spoken, sidestream exposure induced more frequent and
more severe epithelial lesons in the olfactory and ciliated epi-
thelium of the nasal cavity than mainstream of equal TPM concen-
tration.
No histopathological changes were found in the larynx of control
rats.
Almost all rats of the main- and sidestream-exposed groups showed
epithelial thickening (mostly basal cell hyperplasia) at the
ventral depression and thickening with cornification of the
stratified epithelium in the ventrolateral parts. Differences
between all exposed groups were small.
The ciliated epithelium in the dorsal parts of the larynx showed
squamous-cell metaplasia without cornification in 5o percent of the
mainstream-exposed rats whereas all rats of the sidestream-exposed
groups showed squamous-cell metaplasia with cornification. Quanti-
tatively the findings in rats exposed to puffed sidestream were
slightly more pronounced than in rats exposed to nonpuffed side-
stream (a).
(a) The findings in the larynx are based on 3 and 4 rats of the
respective sidestream group only, and should be interpreted
with care.
28,lCW82
1NBIFO lnstitut fiir biologische Forschung K6in
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A12 PAGE 1-io
The morphometrical evaluation of the laryngeal epithelium at the
ventral depression, the ventrolateral lumen and the dorsal lumen
showed, depending on the site, a 2 to 4 fold thickening of the
epithelium of smoke-exposed rats relative to that of control
rats.
Between dose groups, the rats exposed to mainstream showed slightly
thinner epithelium than the rats exposed to nonpuffed sidestream
and the rats exposed to puffed sidestream displayed a slightly
thicker epithelium than the latter.
Almost all the lungs of control and dose groups showed pronounced
histopathological changes such as mononuclear infiltrates around
blood vessels and of the parenchyma, interstitial thickening with
enlarged cells and hyperplasia of the bronchial epithelium. These
changes are attributed to viral infections. They inhibit reliable
histopathological evaluation of smoke-related changes in the lung.
In summary, exposure to diluted side- and mainstream produced
histopathological changes in the nasal cavity and in the larynx.
Sidestream exposure induced much stronger irritative changes in the
mucosa of the nasal cavity than mainstream of equal TPM dose..
Sidestream smoke exposure invoked atrophy of the olfactory and
metaplasia of the ciliated epithelium. Mainstream smoke produced
some metaplasia of the olfactory epithelium.
The irritative changes, seen in the stratified or cuboidal laryn-
geal epithelium were of the same magnitude for side- and mainstream
exposure. In the ciliated epithelium however, sidestream smoke
invoked metaplasia with cornification and mainstream smoke produced
some metaplasia without cornification.
28. KWf32
INBIFO Institut fiGr biologische Forschung K61n
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A13 PAGE 1-11
,
After 21 days of inhalation, free lung cells of 3 rats each out of
the cage control group, the sham control group and the groups
exposed to mainstream and puffed sidestream smoke were lavaged and
analysed for cell type, cell concentration and viability.
Mainstream exposed rats showed a 3 to 5-fold increase, and side-
stream-exposed rats a 15-fold increase of the relative number of
granulocytes as compared to the control groups. The proportions of
macrophages and lymphocytes was reduced accordingly.
The free lung cell preparations of sidestream exposed rats showed
purulent exudate clusters with bacteria, epithelial cells and
immature mononuclear cell types which were not normally observed in
lavages from mainstream-exposed rats.
These findings may be interpreted as a stronger inflammatory
response of the lung to sidestream-smoke than to mainstream of
equal TPM concentration. However, the mixed viral and bacterial
infections of the lung, detected by histopathological and micro-
biological investigations of the rats of this study, may have
influenced these results. Furthermore the small number of investi-
gated rats do not allow a statistical evaluation and valididation
of these findings in future studies is necessary.
In addition to calcium and magnesium free PBS, media with added
newborn calf serum or bovine serum albumin (BSA) were used for lung
lavages. In spite of the relatively small number of investigated
rats - these investigations were done with sham- or mainstream-
exposed rats only - the following trends were observed:
Compared to lung lavage with calcium and magnesium free PBS, the
addition of serum reduced the yield of alveolar macrophages.
Those harvested, appeared to be less viable. On the other hand,
addition of BSA increased the total number of macrophages harvested
and seemed to increase their viability.
28 KW82
INBIFO Institut fOr bioiogische Forschung Min
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A14 PAGE 1-12
Lavage cycles 4 to lo yielded a higher number and a higher propor-
tion of macrophages with better viability than cycles 1 to 3.
For this reason, the use of PBS with BSA, in order to harvest free
lung cells of cycles 4 to lo, seems to be advantageous for future
functional tests with alveolar macrophages.
During this study a microbiological quality control program was
performed. It included the search for viral and bacterial infec-
tions in rats and the regular screening for bacterial contamina-
tions of laboratory, laboratory staff, animal diet, drinking water
and cage bedding material.
At the beginning of the study 5 rats were serologically examined.
All were found to be free from antibodies against Sendai virus,
Reo virus type 3 and mycoplasm. 3 rats showed a low positive
antibody titer against pneumonia virus of mice (PVM). Low positive
antibody titer against R-1 virus and Kilham rat virus were found
only in 1 rat each. A repeated test at the end of the study
resulted in a somewhat increased PVM titer in 4 of the same
5 rats.
At the end of the inhalation period the organs of 2 rats were
examined bacteriologically. Liver, lung, kidney, spleen and
mesenterial lymph node were found to be free from bacteria or only
lowly contaminated. Facultative pathogenic bacteria such as
Staphylococcus aureus, Escherichia coli and in 1 case Klebsiella
pneumoniae were isolated from colon content of both rats. Staphylo-
coccus aureus was also isolated from the nasal cavity in high
concentration.
The surfaces and the conditioned input air of the laboratory
unit as well as the fingerprints and face masks of the laboratory
staff were found to be free from pathogenic bacteria. Samples from
the exhaust air and 1 face mask however were found to contain the
facultative pathogenic Staphylococcus aureus.
2$.KW82
INBlFO lnsti#u# fur b9ologische Forschung K6In
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A15 PAGE 1-13
The non-autoclaved diet was found to be free from Salmonella sp.
All samples of the autoclaved food, drinking water and bedding
material were found to be sterile with respect to pathogenic or
f-acultative pathogenic bacteria.
The detected antibody titer against pneumonia virus of mice
and the occurence of Staphylococcus aureus in the nasal cavity
may impair the interpretation of other bioassays in this inhalation
study, although the histopathological finding in the respective
organs did not correlate well.
1.4 Discussion, Conclusion
With this study, the series of standard 21-day inhalation studies
with diluted mainstream cigarette smoke on rats was extended to
sidestream smoke exposure. Based on the results of bioassays for
systemic and local responses of the exposed rats, the biological
activities of diluted mainstream and sidestream from puffed and
nonpuffed 2R1 cigarettes were compared.
The TPM concentrations of these 3 smoke types were adjusted
to be equal.
Chemical analyses showed no remarkable differences between puffed
and nonpuffed sidestream and no remarkable differences between
particle phase components (a) of mainstream and sidestream smoke.
However, all measured low molecular gas/vapor phase components,
with the exception of hydrogen cyanide, had a distinctly higher
concentration in both sidestream smoke types than in mainstream
smoke. Most importantly, the gas/vapor phase of sidestream smoke
was more alkaline (pH 8.7) and had a much stronger buffer capacity
(26-fold) than the mainstream gas/vapor phase.
(a) only nicotine, pH and buffer capacity
zs.xwaz
INB11=O lnstitut ftir biologische Forschung - KtSin
INTEGRATING REPORT A o500/3047 RM9 (R) A16 PAGE 1-14
,
The systemic toxicity of mainstream and sidestream smoke impaired
the body temperature, food and water uptake, body weight develop-
ment and increased mortality. All of these parameters correlated
closely, which may implicate a - yet unknown (a) - common reason to
have caused those reactions. Puffed and nonpuffed sidestream caused
almost identical reactions, but the reaction to mainstream was much
less pronounced than to sidestream exposure.
If one extrapolates from the experience of previous mainstream
inhalation studies (b), the mainstream TPM concentration of this
study would have to be increased by a factor of 3 to produce
similar strong reactions than seen with sidestream exposure in this
study. '
The pronounced drop in body temperature, as well as some of the
forementioned other reactions are considered to specific for small
laboratory animals (c).
(a) One possible hypothesis, which would be in agreement with these
findings, assumes a quite unspecific impairment of the respira-
tory gas exchange, either due to morphological, functional or
behavioral changes.
(b) i, e. A o5oo/3o16, 21 d aerosol inhalation study on male rats
with 2R1 cigarettes, paraffin and DOS (PT)
(c) Reactions on respiration and circulation are known to be
triggered by nasal inhalation of irritants in rabbits and dogs.
Mc Ritchie, R.J., White, W., Role of trigeminal, olfactory,
carotid sinus and aortic nerves in the respiratory and circula-
tory response to nasal inhalation of cigarette smoke and other
irritants in the rabbits. Aust. J. exp. Biol. med Sci 52 (1) :
127-140.
2e.xwa2
INBIFO Inst9tut ftir biologische Forschung K6In
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A17 PAGE 1-15
Mainstream irritated the mucosa of the nasal cavity and of the
larynx. In the nasal cavity, mainstream caused mainly focal
squamous cell metaplasia, limited to the anterior parts of the
olfactory epithelium. In the laryngeal epithelium, mainstream
caused basal cell hyperplasia of the cuboidal, thickening with
cornification of the stratified and some metaplasia without
cornification of the ciliated epithelium.
These histopathological leasons in the upper respiratory tract were
seen in previous standard 21-day inhalation studies with diluted
mainstream smoke (a).
Additionally to the changes, seen with mainstream, sidestream
- puffed or nonpu-ffed alike - caused more severe atrophic and
necrotic leasons of the olfactory epithelium and frequent squamous
cell metaplasia in the ciliated epithelium of the nasal cavity.
In the stratified and cuboidal laryngeal epithelium, sidestream
caused very similar changes, as seen with mainstream of the same
TPM concentration. The ciliated laryngeal epithelium however,
showed frequent metaplasia with cornification.
Due to superimposed histopathological changes, attributed to a
serologically confirmed viral infection of rats of all groups,
possible smoke-related changes of the lower respiratory tract could
not be evaluated.
The evaluation of lung lavages, revealed a marked relative increase
of granulocytes among free lung cells, which is thought to be indi-
cative for inflamatory processes in the lower respiratory tract.
(a) e. g. A-/3o15, olfactory epithelium (PT)
A-/3o16, 21 d aerosol inhalation study on male rats with 2R1
cigarettes, paraffin and DOS (PT)
28.KWB2
1NBIFO Ins#Itut fOr biologische Forschung KtSIn
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A18 PAGE 1-16
The strong irritative changes with sidestream exposure, seen at the
olfactory and ciliated nasal and laryngeal epithelium , are pre-
sumably caused by low molecular, fast absorbed irritative side-
stream components.
The alkaline reactivity, together with the high buffer capacity of
the sidestream gas/vapor phase, as well as the known excess of
ammonia in sidestream smoke (a), point to ammonia as a key sub-
stance for the irritative capacity of sidestream smoke.
Histopathological changes similar to those seen with sidestream
exposure in the nasal mucosa could be evoked in rats by exposure
to pure ammonia under comparable experimental conditions (b).
(a) Johnson, W. et al. The distribution of products between main-
stream and sidestream smoke. Tobacco Sci. 17 : 141 (1973)
(b) Richard, D. et al. Effects of ammonia gas continuously in-
haled by rats and mice. Bull. Eur. Physiopathol Respir 14
(5) : 573-582 (1978)
Broderson, J.R. et al. The Role of Environmental Ammonia
in Respiratory Mycoplasmosis of Rats, Am. J. Pathology 85
(1) : 115 (1978).
28.KW82
INBIFO Institut fiir biologische Forschung K6In
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A19 GD PAGE 2-1
2 RESPONSIBILITY
Study Director: .,.. ..
Dr.rer.nat. W. Reininghaus
Physicist (Diplomphysiker)
Analytical Chemistry:
Dr.rer.nat. M. Speck
Chemist (Diplomchemiker)
Inhalation, Barrier: .
P. Schnell
Engineer (Ing . grad. )
Biological Chemistry:
Dr.rer.nat. R.-A. Walk
Biologist (Diplombiologe) and
Biochemist
Biometry: ......»..................
H. Gugel
Mathematician (Diplommathe-
matiker)
Pathology:
............................
Dr.med.vet. A. Teredesai
Pathologist (Fach-Tierarzt
fur Pathologie)
Microbiology: ... .. .. .....
Dr.rer.nat. F. Tewes
Biologist (Diplombiologe)
Quality Assurance:
............................
E. Romer
Biologist (Diplombiologe)
aa.Kwa2
INBIFO Institut fi]r biologische Forschung - Kdln
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A2o PAGE 3-1
3 CIGARETTES
Type (cigarette code):
Source:
Number of cigarettes:
Pnckncl i.nr1:
2R1, standard reference cigarette
Philip Morris
approx. 3oooo
cnrtons with 2oo c,i.gnretten,
io packages with 2o cigarettes/
package
Date of receipt at INBIFO:
7.May 76 and lo.Oct.81
Storage
Main storage:
walk-in cold room R911, 1 to
3 degrees centigrade, relative
humidity uncontrolled
Laboratory storage:
Selection:
Specifications of
cigarette (a):
Physica3l properties and
chemica3l composition of
filler of cigarette (a):
Composition of smoke
components of cigarette (a):
conditioning room R326, at least
8 days prior to smoking
storage in opened packages
temperature: 22 + 1 degrees centi-
grade, relative humidity: 6o + 3 o/o
no selection
see TABLE B
see TABLES C.1 and C.2
see TABLE D
(a) specification provided by the supplier
28. KW82
H0=.14
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A21 GD M H0414 PAGE 3-2
SIDESTREAM TYPE OF SMOKING STREAM TYPE (SCHEME) COMPOSITION OF SIDE-
TYPE STREAM FOR INHALATION
A puffing
ES
puff intervals
GS2
B static burning
TABLE A
TYPES OF SIDESTREAM USED FOR INHALATION
Remarks: GS1: glowstream, type 1
GS2: " , type 2
MS : -mainstream
ES : effusion stream
DS : diffusion stream
SMS: smoulder mainstream
(nomenclature according to Hoegg et al., 1972)
SMS
GS1 +GS2 +E5 +DS
GS 2
V v V V W V V
~
~
f
W
N Il~'PEGRATING RFPORT A o50o/3o47 1;N19 (R) A22 PAGE 3-3
Il7I'AL FILTII2 TCJB1CC0 IUI'AL 17IAM1±.TE'k.2 PUFF CCMPRESSI- STATIC PAPER
CIG. AND PAPER WEIGiT CIG. RESIS- BILITY BURNING TIME PC)I2OSITY
WEIGHT WEIGFT (a) IENGTii TANCE (b)
(mg/cig) (mg/cig) (mg/cig) (mm) (nm) (kPa) (mm) (min/4o mn) (ml/(an2 x min))
1194 110 (c) 1o84 85 7.96 o.79 -- 13.7
TABLE B
SPECIFICATIMS OF CIGAREZiE TYPE 2R1
specifications provided by the supplier
(a) at tobacco moisture determined (see TABLE C. 2)
(b) at 12 % moisture
(c) no filter with 2R1 cigarettes
.~r.. a...
~~t~U~i ` ~~
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A23 PAGE 3-4
TOTAL REDUCING NITRATE AMMONIA NITROGEN
ALKALOIDS SUGARS NITRITE KJELDAHL PROTEIN TOTAL
(o/o) (o/o) (0/0) (o/o) (o/o) (a/o) (0/0) (o/o)
1.98 10.5 0.89 o.13 - - - 2.17
TABLE C.1
PHYSICAL PROPERTIES AND CHEMICAL COMPOSITION OF FILLER OF CIGARETTE TYPE 2R1
specifications provided by the supplier
zqCoGTszoz
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A24 PAGE 3-5
CHLORIDE ASHES POTASSIUM CALCIUM MAGNESIUM HOT WATER TOBACCO TOBACCO EQUILIBRIUM
SOLUBLES pH MOISTURE MOISTURE
(0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0)
o.66 14.8 - - - 59 5.5 - 12.6
TAB LE C.2
PHYSICAL PROPERTIES AND CHEMICAL COMPOSITION OF FILLER OF CIGARETTE TYPE 2R1
specifications provided by the supplier
zsEasisZoz
INTEY3RATING REPORT A o5ao/3o47 RM9 (R) A25 PACE 3-6
PUFF SMOKF COMPONENTS ( a )
Nt7.
TPM WIATER IIPAi
IN TPM
NICO-
TINE
TAR
CO
NO
IiCN
ATIE-
HYDES
ISH
(1 /cig ) (mJ/ (mg/ (mg/ (M/ (mg/ (mg/ (mg/ ( ug/ (mg/ (% )
cig.) cig.) cig.) cig.) cig.) cig.) cig.) cig.) cig.)
13.1 48.4 4.9 43.5 3.32 4o.2 25.4 o.39 453 3.13 71
TABLE D
CCMPOSITION OF SKM COMPQNENTS OF CICARL"iIE TYPE 2R1
specifications provided by the supplier
(a) given for c.~letel.y smoked cigarettes, not corrected for minimum butt length (32 mn)
UsCaSTszoz
INBIFO Institut ftir biologische Forschung K6ln
INTEGRATING REPORT A o5oo/3o47 RM9 (R) A26 PAGE 4-1
4 STORAGE OF MATERIALS AND RECORDS
Remains of organs and tissues in formaldehyde solution, slides
and records are stored in our archives for at least 5 years, they
can be claimed by the client.
END OF INTEGRATING REPORT
28 KW82
