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.
- 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)
- Litigation
- STMN/Produced
- Named Person
- Corn
- Type
- SCRT, REPORT, SCIENTIFIC
- CHAR, CHART, GRAPH, TABLE, MAPS
- FOOT, FOOTNOTES
- Subject
- secondhand
- secondhand smoke
- secondhand smoke/health effects
- Secondhand Smoke/Toxicity
Document Images
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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
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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.
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,
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.
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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
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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
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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
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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
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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).
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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
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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
