EPA-600/1-78-020
March 1978
Environmental Health Effects Research Series
STUDIES ON THE EFFECT OF AMMONIUM
SULFATE ON CARCINOGENESIS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-78-020
March 1978
STUDIES ON THE EFFECT OF AMMONIUM SULFATE
ON CARCINOGENESIS
by
John J. Godleski and Joseph Leighton
Department of Pathology
Medical College of Pennsylvania
Philadelphia, Pa. 19129
Grant No. R-802839
Project Officer
David L. Coffin
Office of the Director
Health Effects Research Laboratory
Research Triangle Park, NC 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, NC 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial
products consitute endorsement or recommendation for use.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation of
affadavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
Evidence exists that exposure to atmospheric "sulfate" may be associated
with increased pulmonary disease in people. Biological studies indicate that
the response to the various sulfates varies according to the specific
compound, some compounds of sulfate having more effect than sulfur dioxide
gas, others no effect whatever. Ammonium sulfate, which is numbered among
the more reactive compounds in these studies, is of interest because it is
generally believed to be one of the more common compounds of sulfur in
polluted air. Sulfur dioxide has been shown to enhance the effect of a
standard carcinogen (benzo(a)pyrene) when both compounds are given by
inhalation.
The following EPA study evaluates the influence of ammonium sulfate
as a cofactor in carcinogenesis studies employing benzopyrene as the prime
agent. Additional information concerning influence on a metabolic enzyme
aryl hydroxylase and the body's absorption and excretion of sulfate is
also included. These factors are relevant to the overall EPA program
since the agency is attempting to evaluate the importance of the sulfates
in relation to other atmospheric factors and their possible health effects.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
iii
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ABSTRACT
This project was designed to evaluate health effects of ammonium sulfate*
inhalation using experimental animals. The questions studied were:(l) Is
inhaled ammonium sulfate co-carcinogenic. (2) What are the deposition and
clearance patterns of inhaled ammonium sulfate? (3) What effect does am-
monium sulfate have on pulmonary defensive mechanisms?
The carcinogenesis studies utilized hamsters given 5 mg intratracheal
injections of Benzo(a)pyrene weekly for 15 weeks and 6 hour daily inhalation
of ammonium sulfate at concentrations in the range of 200 ug/m for 15 weeks.
Incidence of respiratory cancer was 1.4% in unexposed controls, 2.9% in ham-
sters exposed to ammonium sulfate alone, 14.4% in those given only Benzo(a)
pyrene injections and 11.8% in those given Benzo(a)pyrene injections and
exposed to ammonium sulfate. The increased incidence of cancer with Benzo(a)
pyrene was statistically significant (p < 0.005). Ammonium sulfate inhala-
tion had no effect on the development of cancer and no effect on the devel-
opment of other significant pulmonary diseases.
35
For deposition and clearance studies, S -labeled ammonium sulfate aero-
sols with high specific activity were used. A five minute exposure time and
a short, reproducible time period in which tissues were obtained for the
first analysis after exposure were necessary to determine deposition. Clear-
ance was then assessed at 1, 3 and 6 hours after exposures. Hamsters, guinea
pigs and rabbits were studied. Total respiratory tract deposition was greater
with the larger particle size in all studies. Clearance patterns were similar
for the three species regardless of particle size. The half time for clearance
of ammonium sulfate from the lung was 18 to 20 minutes. Inhaled and injected
sulfate was cleared via the urinary tract and by six hours after exposure
95% of the total collectable sulfate was present in the urine.
Pulmonary defensive parameters evaluated in this project were levels of
aryl hydrocarbon hydroxylase activity in hamster lungs and pulmonary macro-
phage numbers. Ammonium_sulfate exposure concentrations were in the range of
200 ug/m and 1,000 ug/m . Enzyme activity was studied after I, 3 and 10
weeks of exposure. Significant inducement of the enzyme was found with
Benzo(a)pyrene at all analysis periods. Ammonium sulfate inhalation had no
effect on this enzyme. Pulmonary macrophage number was not affected by
ammonium sulfate inhalation.
Overall, ammonium sulfate was not found to be a deleterious air pollutant
for the animal species assessed at the inhalation concentrations used.
* (Nh4)2
iv
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CONTENTS
Foreword iii
Abstract iv
F igures vi
Tables viii
1. Introduction 1
2. Cone lus ions 2
3. Recommendations 2
4. Studies
I. Goals and Objectives 3
II. Carcinogenesis Studies 4
General Plan 4
Technical Details 5
Results 9
Discussion 20
III. Deposition and Clearance Studies 20
Hamster Deposition Studies 20
Guinea Pig Deposition Studies 24
Rabbit Deposition Studies 26
Clearance Studies in Hamsters,
Guinea Pigs and Rabbits 31
IV. Pulmonary Defense Studies 40
V. References 43
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FIGURES
Number
1. Flow diagram of ammonium sulfate exposure system 6
2. Mean animal weights - Sulfate co-carcinogenesis study 10
3. Mortality of sulfate co-carcinogenesis study 11
4. Squamous carcinoma arising in the lung of hamster exposed
to BaP-sulfate 15
5. Poorly differentiated squamous carcinoma of the bronchus
in hamster exposed to BaP alone 16
6. Higher magnification of cancer seen in Figure 5 16
7. Papillary and invasive squamous carcinoma of the upper third
of the trachea in hamster exposed to BaP alone 17
8. Higher magnification of cancer seen in Figure 7 17
9. Undifferentiated giant cell malignancy in the lung of a hamster
exposed to BaP-sulfate 18
10. Benign cellular proliferation with squamous metaplasia seen
in a hamster exposed to BaP alone 18
11. Area of benign cellular proliferation and inspissation of hyaline-
like material in the lung of a hamster exposed to ammonium
sulfate 19
12. Vasculitis seen in the lung of a hamster exposed to sulfate alone... 19
13. Flow diagram of exposure system used with radioactive aerosol 21
14. Large particle deposition and clearance in hamsters 32
15. Small particle deposition and clearance in hamsters 33
16. Large particle deposition and clearance in guinea pigs 34
17. Small particle deposition and clearance in guinea pigs 35
18. Large particle deposition and clearance in rabbits from
whole body exposure 36
vi
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Number Page
19. Small particle deposition and clearance in rabbits from
whole body exposure 37
20. Macrophages obtained by lavage in control and ammonium sulfate
exposed hamsters. Mean _ S.E 41
21. Macrophage obtained by lavage in control and ammonium sulfate
exposed hamsters: Comparison of washout curves 42
vii
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TABLES
Number Page
1. Exposure parameters of carcinogenesis - studies I and II... 9
2. Incidence of all cancer and respiratory cancer.. 12
3. Chi square and p for compared treatment groups 12
4. Location of respiratory tract cancers 13
5. Histologic types of cancer in each exposure group 13
6. Percentage of animals with benign proliferation and percentage
of animals with respiratory benign proliferation 14
7. Percentage of animals with other significant pulmonary diseases 14
8. Percentage of animals with non-pulmonary significant diseases 14
9. Exposure parameters in hamster deposition and clearance studies 23
10. Deposition of S-"-labeled ammonium sulfate in hamsters... 23
11. Exposure parameters in guinea pig deposition and clearance studies.. 25
12. Deposition of S-"-labeled ammonium sulfate in guinea pigs 26
13. Exposure parameters in rabbit "nose only" deposition studies 27
14. Pulmonary versus nasal deposition in rabbits 28
15. Rabbit exposure parameters for whole body exposures 29
16- Deposition of S^^-labeled ammonium sulfate in rabbits using
whole body exposure 29
17. Second rabbit "nose only" exposure 30
18. Pulmonary versus nasal deposition in second "nose only" exposure.... 30
19. Pulmonary and nasal deposition of all deposition studies
(ng sulfate + S.D.) 30
20. Effect of ammonium sulfate aerosol on aryl hydrocarbon hydroxylase
activity in hamster lungs 38
21. Effect of ammonium sulfate aerosol on aryl hydrocarbon hydroxylase
activity in hamster lungs 39
viii
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SECTION 1
INTRODUCTION
An area of great concern in environmentally related research is the con-
sideration of multifactorial determinants of human lung cancer. Animal models
for induction of pulmonary neoplasia have employed both intratracheal instil-
lations and inhalational exposure to a variety of organic or inorganic agents,
alone or in combinations. Exposures to combinations of agents have been pro-
ductive of large amounts of important data, but have in addition raised vital
questions relevant to many environmental contaminants. Data already available
from animal exposures have been useful in postulating pathogenic mechanisms as
well as in elucidating specific human health hazards.
Presently, policy making and regulatory agencies are confronted with need
for data relevant to the potential health effects of exposure to airborne par-
ticulate sulfates. This research project evaluates the role of these airborne
particulates on the development of pulmonary neoplasia following exposure of
Syrian Golden Hamsters to the ubiquitous hydrocarbon carcinogen, benzo(a)py-
rene. This investigation combines exposure to aerosols of particulate sulfate
with an intratracheally administered suspension of benzo(a)pyrene. The end
points evaluated following the period of daily aerosol and weekly intratracheal
exposures are the frequency of development and morphologic characterization of
respiratory tract neoplasms in animals maintained for their natural life-time.
The studies include use of radiolabeled airborne particles to evaluate the de-
position and clearance of sulfate in the animal respiratory tract, and studies
to evaluate pulmonary defense mechanisms operative in this model system.
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SECTIONS 2 and 3
CONCLUSIONS AND RECOMMENDATIONS
The results of these studies on the health effects of exposure to air-
borne particulate ammonium sulfate indicate no deleterious effect from inha-
lation of this pollutant by Syrian Golden Hamsters at concentrations ran-
ging from 200 /ig/nr to 1,000 .ug/m^. There was no effect of ammonium sulfate
inhalation on benzo(a)pyrene carcinogenesis. In addition, no other patho-
logic findings in the respiratory tract could be attributed to ammonium sul-
fate exposure. Pulmonary defensive parameters, including aryl hydrocarbon
hydroxylase enzyme levels and pulmonary macrophage numbers, were not affected
by inhalation of ammonium sulfate.
Deposition studies showed that airborne ammonium sulfate in particle
size distributions of 0.3 u and 0.6 u MMAD reached the lung. However, a sub-
stantial portion of the total respiratory deposition was in the nose. The
noses of the animals studied, therefore, acted as a protective mechanism
against these submicronic particles. Clearance studies showed a rapid removal
of sulfate from the lung with one half of the deposited amount cleared in 18
to 20 minutes. Clearance was^via the blood to the urinary tract with 95% of
the collectable sulfate in the urine within 6 hours of exposure. Therefore,
when sulfate was inhaled in this highly soluble form, it was cleared very
rapidly from the body.
This research project was part of a large program to evaluate the bio-
logic effects of airborne sulfate. Data derived from this project does not
support the contention that sulfate ion is a harmful pollutant. However, the
data must be interpreted only in terms of the chemical form of sulfate
studied, the concentrations studied and the animal species assessed. We
recommend that evaluation of the findings of the entire sulfate research pro-
gram should be done before proceeding to initiate additional research or de-
velop policy.
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SECTION 4
STUDIES ON THE EFFECT OF ATMOSPHERIC POLLUTANTS ON CARCINOGENESIS
I. GOALS AND OBJECTIVES OF THIS PROJECT
This project was initiated to determine the role of selected atmospheric
pollutants as co-factors in carcinogenesis. During the project period, the
goals and objectives of this program were reshaped from a broad based study
on particulate pollutants as co-factors in carcinogenesis to a more defini-
tive study designed to evaluate health effects of ammonium sulfate inhalation
using experimental animal model systems. The questions studied were: (1) can
inhaled ammonium sulfate act as a co-carcinogen? (2) What are the deposition
and clearance patterns of inhaled ammonium sulfate? (3) What effect does am-
monium sulfate have on pulmonary defensive mechanisms?
The rationale for testing ammonium sulfate as a co-carcinogen was based
on evidence from three studies. Findings from the EPA-CHESS program (1, 2)
indicated that levels of suspended atmospheric sulfates, much lower than sul-
fur dioxide levels, were associated with increased prevalence of pulmonary
diseases. Amdur and her associates (3) had used the parameter of increased
pulmonary air flow resistance of guinea pigs as an indicator of irritation.
They studied a number of compounds and found only those containing sulfate
ions produced an irritant response. No epidemiologic studies had been done
correlating lung cancer incidence and levels of suspended sulfate. However,
Laskin (4) had used sulfur dioxide as an irritant in carcinogenesis studies
and found it to be a promoting agent of carcinogenesis. It was, therefore,
quite possible that sulfate ion, a more potent irritant in the epidemiologic
and guinea pig studies, could be a promoting agent of carcinogenesis. The
choice of ammonium sulfate was based on Amdur's demonstration of the irrita-
tive effects of this compound and evidence that suggested this was a pollu-
tant likely to increase in ambient air in the future (2, 5).
Deposition and clearance studies were needed to determine if the size of
ammonium sulfate particles planned for use in the carcinogenesis studies
would reach the lungs of our animals. No data was available on deposition
and clearance of ammonium sulfate or other water soluble hygroscopic particles
in hamsters. These studies required development of methodology. Once this
was achieved, it became apparent that questions of deposition and clearance
had implications beyond our carcinogenesis studies. Since we had the method-
ology and facilities, we extended these studies to guinea pigs and rabbits,
animals frequently used in assessment of pollutant effects on health.
Pulmonary defensive studies were a major portion of the original pro-
posal. We were attempting to elucidate the mechanism of co-carcinogenic ac-
tivity of pollutants by assessment of their effect on aryl hydrocarbon hy-
droxylase enzyme and pulmonary macrophage activity. These studies were done
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using ammonium sulfate at several different concentrations.
II. CARCINOGENESIS STUDIES
A. General Plan
The carcinogenesis studies utilized Benzo(a)pyrene (BaP), a known carci-
nogen (6, 7), given intratracheally. This compound instilled in this manner
had been shown to result in a low incidence of pulmonary cancer (8, 9). Am-
monium sulfate was given by inhalation to determine if this irritant could in-
crease the incidence of cancer in this experimental model system. There were
four study groups: (1) BaP alone, (2) sulfate alone, (3) BaP-sulfate and (4),
unexposed control. In these studies four groups of 80 hamsters, all from the
same litter date, from Lakeview Hamster Colony, were used.
Hamsters in the group receiving BaP alone were given 5 mg doses of the
carcinogen by intratracheal injection on Wednesday of each week for 15 weeks.
Monday through Friday of each week these animals were placed in a chamber
without food or water at a negative pressure of 0.15 inches of water for 6
hours. Filtered room air passed through the chamber at a rate of one com-
plete air change per minute. In this way, these animals were held under the
same conditions as those in the sulfate exposure groups. The 5 mg dose was
selected on the basis of Saffiotti's findings (6).
The sulfate alone group was exposed for 6 hours each day, Monday through
Friday without food or water, in individual holding cages at a negative pres-
sure of 0.15 inches of water. Exposures lasted 15 weeks. Three sulfate
samples were taken each day, and samples for particle sizing were done inter-
mittently. Sulfate concentrations in the chamber were in the range of 200ug/
m-^. This level was selected as a conservative extrapolation of known sulfate
levels of major metropolitan areas (10).
The BaP-sulfate group was exposed to sulfate daily and BaP weekly. The
sulfate exposure followed the same protocol used for the animals exposed to
sulfate alone. The BaP injections followed the same procedure used for ani-
mals receiving BaP alone.
Unexposed control animals were maintained in the same animal facility
with the exposed groups.
All animals were earmarked for identification, facilitating rotation of
animals through different areas within the chamber each day, and for easy
identification when weighing the animals. Animals were weighed every month
during their lifetime. They were observed daily and moribund animals iso-
lated to prevent canabalism on death. Dead animals were necropsied. At ne-
cropsy, gross observations were made on all internal organs, including the
brain, nasal structures and larynx. Histologic sections were routinely done
on the noses, larynx, trachea, bronchi and all lobes of the lungs. All tis-
sues from animals were saved until the end of the study. Animals not dying
spontaneously were sacrificed 2 years after completion of exposure. Necropsy
evaluation was the same as for animals dying spontaneously. All necropsies
were done under the direction of a pathologist. The tissues were prepared
4
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for histologic study by routine methods. Hematoxylin and eosin-stained slides
were evaluated independently by two pathologists. Observations on all tissues
studied were recorded and then coded for computered evaluation of the findings.
We proposed two carcinogenesis studies to evaluate the effect of ammonium
sulfate as a co-carcinogen. The first was the study outlined above; the
second study was to use a concentration of sulfate determined by the prelimi-
nary findings of the first study.
In studies that attempt to relate experimental environmental exposure to
adverse health effects, a number of factors are important. Temperature and
relative humidity must be within normal ambient ranges. If particulates are
studied, these must be respirable by the test species. However, the most im-
portant parameter is the concentration level of the pollutant in the exposure
system. Studies using concentrations that could not be reasonably extrapo-
lated to the natural situation are only of value in showing that an effect
could take place. They are not useful in developing air quality standards
based on adverse health effects. Carcinogenesis and co-carcinogenesis studies
can be sensitive indicators of a most significant health effect. However, the
concentrations used in the experiments determine their relevance to air
quality standards. These studies are expensive and time consuming, so that
careful selection of concentration level is critical. In our first carcino-
genesis experiment, the exposure concentration was based upon a conservative
extrapolation of the ambient sulfate level in the Philadelphia area. The ex-
posure range of 200 jug/nr for 6 hours per day was used. In carcinogenesis ex-
periments, the number of animals is also important and must be selected so
that expected results will yield statistically significant data. The estima-
ted malignancy rate with BaP alone was in the range of 10%. Therefore, using
80 animals per group, a doubling of this rate in the BaP-sulfate groups would
be significant at the 5% level. Since both the rate of malignancy and the ex-
posure level were chosen empirically in the first study, a second carinogenesis
study was planned. The exposure parameters of this experiment were to be
based on the 6 month findings of the first experiment. At that point, malig-
nancies had been seen in the BaP group at a rate of 10%. No malignancies were
seen in the BaP-sulfate group. Obviously, there had been no enhancement by
sulfate. In fact, there was a statistically significant reduction of the
malignancy rate in this group. Since our original hypothesis predicted en-
hancement, it would have been unreasonable to go to a lower sulfate dose which
had been planned if enhancement had been seen. Significantly increasing the
concentration level would not have been in keeping with the objective of this
experiment, since we would not then be working with environmentally relatable
levels. We, therefore, decided to repeat the experiment at the same concen-
tration so that data from these experiments could be evaluated both indepen-
dently and in combination. The combined data would have twice the number of
animals in each group so that the significance of small differences among
treatment groups could be determined with greater statistical certainty.
Therefore, the protocol of the second carcinogenesis study was the same as the
first.
B. Technical Details
1. Inhalation Facility--
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The Medical College of Pennsylvania Inhalation Laboratory consists of
two 322 liter stainless steel and glass exposure chambers (11), a 42" x 30" x
30" stainless steel and glass glove box and a 6' x 3' x 3' plexiglass glove
box. All of these units are connected to exhaust ducts used exclusively for
this system. Each unit has two absolute filters (99% efficiency on all parti-
cles greater than O.lu MMD) connected in series to trap all particles emana-
ting from the system. The exhaust fan has an emergency back up fan connected
in parallel that can be activated by loss of the required pressure at strate-
gic points in the system. Each unit has a valve to control air flow into the
exhaust system. These units are connected to the sewer system for easy clea-
ning, but this drainage system can be sealed off from the units by valves so
that air cannot be drawn from the sewer system.
The stainless steel glove box was used for manipulations involving BaP.
The milling apparatus for grinding this carcinogen to fine particulate size
was housed here. The glass front of this glove box was covered with aluminum
foil to protect the BaP from photodecomposition. Operating pressure in this
unit is minus 0.50 inches of water with air flow through the unit set at 10
cubic feet per minute.
The flow diagram in Figure 1 outlines the parts and connections of the
aerosol generating and exposure system.
A. J
1
compressor—Refrigerated —^cooling —^pressure ?air line
air dryer coil regulating
valve
rotameters
/ \
nebulizer air dilution #1
9 L/min ,91 L/min
\
deionizer
4
O" x 3/4"
room air ^absolute filter y^Y fitting
225 L/min r J,
chamber
325 L
i
effl
uent
filter
exhaust , total flow <
system orifice
Figure 1. Flow diagram of ammonium sulfate exposure system.
The exposure chambers received air from two sources, one source providing
approximately 1/3 of the total air flow was a Cast model 7HDD oilless compres-
sor that ran continuously producing 200 liters of air at 35 PSIG. This source
6
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was used for aerosol production and one air dilution of the aerosol. Air from
this compressor passed through a Deltech refrigerated air dryer which removed
as much as 100 ml of water from 12 nr of air. At each chamber, an auxiliary
cooling coil (50* of V copper tubing coiled to a diameter of 3", 12" in
length) was present on this air line. This coil further lowered the tempera-
ture of the air when it was immersed in ice. Beyond the coils, each air line
had a pressure regulating valve to maintain constant pressure to the aerosol
nebulizer and a Watts coalescing air line filter that removed all solid parr
tides O.Olu and larger. The second source of air to the chamber was ambient
air from the room. Two thirds of the total chamber air flow was drawn
through an absolute filter into the chamber and used as a second air dilution
of the aerosol. An air flow of one air change per minute was found necessary
to assure endogenously produced ammonia levels below 100 ug/m^. Chamber pres-
sure in this system was negative and ranged between 0.14 and 0.16 inches of
water.
The aerosol generator used was a Collison nebulizer obtained from En-
vironmental Research Corporation, St. Paul, MN. At an incoming air pressure
of 35 PSIG, the output was 9 liters/min from this nebulizer. The deionizer
had a sealed Krypton 85 source of energy to neutralize static electrical
charge of the aerosol particles.
Preliminary studies over several weeks of operation showed that tempera-
ture and relative humidity were dependent on ambient conditions. Temperature
ranged from 22° - 27° C and relative humidity ranged from 28% to 62%. This
represented less control of these parameters than had been planned. However,
the higher air flow using room air was necessary to assure low ammonia levels
in the chamber.
*
2. Aerosol Sampling--
Sampling to determine sulfate concentration in the aerosol as well as
the size of the particles was done with a stainless steel V tube positioned
isokinetically within the chamber. For concentration sampling, the tube was
connected to a millipore filter unit for air sampling, containing a 47 mm di-
ameter 0.8u pore size millipore filter. A vacuum pump drew 14 liters of air
per minute through the filter. Air was sampled for 15 minutes. The filter
was then placed in a 60 mm diameter disposable Petri dish containing 2 ml of
deionized water and allowed to soak for 1 hour. The solution was then col-
lected into sealed tubes and stored until analyzed. Particle sizing was done
by connecting an Andersen Sampler to the tube and drawing air through the
sampler for 15 minutes. This sampled 424.5 liters of air in this period. The
plates of the sampler and the final filter were then placed in 100 mm dispo-
sable Petri dishes with 10 ml of deionized water and soaked for 1 hour. The
solution was then collected and stored in sealed tubes until analyzed.
Sulfate samples were analyzed by the method of Melnicoff et al (12).
This is an autoanalyzer method using sodium rhodizonate to colorimetrically
determine sulfate concentration. This technique was developed for analysis of
samples from this study. However, it also has broader applications in chemi-
cal analysis for sulfate.
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3. Benzo(a)pyrene Preparation—
Before we began the carcinogenesis studies, the BaP to be used had to be
characterized and prepared in an injectable form. BaP was obtained from
Aldrich Chemical Corp., Milwaukee, Wis. Purity was determined by spectral
analysis in benzene. The spectrum obtained matched that published by Sawicki
et al (13) and using the extinction coefficients determined by these authors,
concentration could be determined. Purity exceeded 99% by these methods.
BaP was given by intratracheal injection in a sterile saline gelatin sus-
pension with the BaP particles less than lu HMD. This suspension was prepared
in the following manner. A solution was prepared containing 0.5% gelatin
(Difco Laboratories, Detroit, Mich.) and 0.9% sodium chloride. This solution,
with 5 mm glass beads, was autoclaved in a Wheaton tissue culture roller bottle.
BaP was then added to make a 2.5% suspension. The bottle was then placed on a
Norton jar mill and turned continuously at 12 revolutions per minute for two
weeks in a darkened unit. After milling, the suspension was separated from
the glass beads and transferred sterilely in 10 ml aliquots into injection
vials.
Intratracheal injections of 0.2 ml of suspension were given through
blunt tipped needles at a 45° angle. The hamsters were suspended on a slant
board by the upper incisors, the tongue held outward with forceps, the larynx
visualized and injection made into the trachea.
4. Evaluation--
The following gross observations were made at necropsy of each animal:
a. Presence or absence of lung cancer.
b. Pathologic changes in all internal organs as well as the brain,
nasal structures and larynx.
c. Overall assessment of animal condition prior to death.
The following histologic observations were made on tissues of each
animal:
a. Larynx, trachea and bronchi.
(1) The presence or absence of cancer: if present, histopathologic
types.
(2) Condition of the epithelium: normal, atrophic, hyperplastic,
anaplastic, carcinomatous, ulcerated.
(3) Condition of mucous glands: normal, atrophic, hyperplastic.
(4) Condition of goblet cells: normal, increased, decreased.
(5) Presence or absence of inflammation - type.
b. Lungs:
(1) Presence or absence of cancer.
(2) Pattern of particulate accumulation, nodes, interstitial,
obliterative.
(3) Condition of bronchial, bronchiole and alveolar epithelium:
normal, ulcerated, atrophic, hyperplastic, anaplastic,
carcinomatous.
8
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(4) Presence of inflammation: peribronchiolar, intraalveolar,
interstitial - type.
(5) Maintenance of pulmonary architecture - presence of emphy-
sema or fibrosis.
c. Other structures:
(1) Presence or absence of cancer.
(2) Other pathologic changes.
Metastasis or primary?
Both these descriptive observations and the slides from which they were
made were then reviewed and the findings used to make specific diagnoses.
These included: the histologic types and locations of cancers, the presence
and location of benign cellular proliferations, the presence of bronchitis,
pneumonia, emphysema, fibrosis or other pulmonary diseases, and the presence
of other significant non-pulmonary diseases.
C. Results
Chi square analysis of mortality rates, body weights, development of
cancer and histologic types of cancer in the two carcinogenesis studies
showed no differences between these two studies. Review of protocols and ex-
posure parameters (see Table I) did not reveal differences between the two
studies. Therefore, all data from the two studies have been combined for pre-
sentation in this report.
Parameter
Exposure
Sulfate
concentration
Temperature
Relative
humidity
Particle
size MMAD
Carc|in,Q.genesis I Carcinogenesis II
Sulfate alone BaP t sulfate Sulfate alone BaP + sulfate
189 + 39 ug/m3 204 + 37ug/m3 181 + ug/m3 190 + 30 jug/m3
24.5 t 1.9° C 25.6 + 1.5°C 24.0 + 1.6°C 26.0 i 1.6°C
39.2 + 12.2% 37.2 t 9.0% 39.0 + 11.2% 43.8 + 15.1%
0.30u 0.30u 0.29;i
Table I. Exposure Parameters of Carcinogenesis - Studies I and II.
The mean weights of the hamsters in each treatment group throughout the
study period are illustrated in Figure 2. It can be seen that the animals
given BaP weighed less throughout the study than those animals that did not
receive this carcinogen. However, ammonium sulfate exposure did not have any
effect on body weight when comparisons were made between the unexposed control
group and sulfate alone or BaP alone and BaP-sulfate groups.
-------
MEAN ANIMAL WEIGHTS-SULFATE CO-CARCINOGENESIS STUDY
OQ
C
n
n>
ro
I
o
I
O
Ul
160-
150-
140-
130-
120
110
100
90 -I
S04
o BoP/so4
O BaP
A Control
I
2
i
4
i i i i i i i i i
8 10 12 14 16 18 20 22 24
MONTHS AFTER START OF EXPOSURE
-------
Cumulative mortality throughout the study period is illustrated in Figure
3. The BaP groups had higher mortality throughout the study. This can be
traced to higher mortality rates in the BaP alone group between the first and
fifth month after the start of exposure and in the BaP-sulfate groups in the
fifth through the eighth month. Many of the deaths were due to malignancy in
these time periods. We have no explanation for the earlier development of
cancer in the BaP alone group.
MORTALITY OF SULFATE CO-CARCINOGENESIS STUDIES
0
UJ
o
UJ
O
oc
UJ
CL
UJ
5
3
z
100 -1
90 -
80-
70-
60-
50-
40-
30-
• S04
O BaP/SQ, _ D
D BaP
A Control Q
D 0
0 °
f^
a
D
D °
a o A
n ° 0 A
n ° 0 0 •
D O
20-
lo^
a
a
O A
A A
gggii****
0 2 4 6 8 10 12 14 16 18 20 22 24
MONTHS AFTER START OF EXPOSURE
Figure 3.
11
-------
Table 2 illustrates the percentage of hamsters with cancer in each expo-
sure group, as well as the percentage of hamsters with cancer of the respira-
tory tract.
Table 3 lists the Chi square and p for comparisons between treatment
groups in the development of cancer.
TABLE 2. INCIDENCE OF ALL CANCER AND RESPIRATORY CANCER
Treatment
Control
Ammonium sulfate
BaP
BaP-sulfate
Cancer
5.9%
4.0%
34.6%
27.9%
Respiratory
1.4%
2.9%
14.4%
11.8%
TABLE 3. CHI SQUARE AND p FOR COMPARED TREATMENT GROUPS
Total Cancer
Comparison
Control-sulfate
Control-BaP
Control-BaP-
sulfate
Sulfate-BaP
Sulfate-BaP-
sulfate
BaP-BaP-sulfate
Chi Square
0.04
17.09
10.95
28.07
19.03
0.72
£
N.S.
< .001
< .001
< .001
< .001
N.S.
Respiratory Cancer
Comparison
Control-sulfate
Control-BaP
Control-BaP-
sulfate
Sulfate-BaP
Sulfate-BaP-
sulfate
BaP-BaP-sulfate
Chi Square
0.03
8.03
5.98
10.16
7.30
0.29
£
N.S.
< .005
<.02
<.005
<.01
N.S.
From Table 3 it can be seen that a statistically significant incidence
of cancer was found with the instillation of BaP. Ammonium sulfate inhalation
had no effect on the development of cancer.
Table 4 lists the percentage of cancers by location in the respiratory
tract in each exposure group. Table 5 lists the histologic types of cancer
found in each exposure group.
12
-------
TABLE 4. LOCATION OF RESPIRATORY TRACT CANCERS.
Treatment
Control
Sulfate
BaP
BaP-sulfate
Nasal
0
0
0
9
Trachea
0
50.0
40.0
45.5
Lung
100*
50.0
60.0
45.5
* Numbers indicate percentage of total respiratory cancers in each location.
TABLE 5. HISTOLOGIC TYPES OF CANCER IN EACH TREATMENT GROUP
Treatment Squamous Adenocarcinoma Und i fferent ia ted Lymphoma Other
Control 0 0 25.0* 75.0 0
Sulfate 50.0
BaP 38.9
BaP-sulfate 42.3
0
13.9
11.5
25.0
13.9
15.4
25.0
16.6
23.1
0
16.6
7.7
*Numbers indicate percentage of total cancers with each treatment.
The finding of nasal cancer in the BaP-sulfate groups is interesting.
These lesions included both squamous carcinomas and small cell undifferenti-
ated lesions. Cancer developed in the trachea of animals in the treatment
groups, but differences were not significant between those exposed and not
exposed to sulfate. The cancers of the respiratory tract were predominantly
squamous. Adenocarcinomas were in the gastrointestinal tract. Undifferenti-
ated tumors were often the large cell type of the lung. A larger than expec-
ted proportion of lymphomas was noted in all treatment groups. These usually
involved many nodes, the liver, spleen, and in some cases, the lung and skin.
Histologic types of lymphoma were predominantly the poorly differentiated
diffuse lymphocytic variety, but histiocytic types were also noted. Other
tumors included rhabdomyosarcomas, hepatomas and fibrosarcomas.
Table 6 compares benign cellular proliferation in the exposure groups.
In the respiratory tract, these benign lesions ranged from Type II hyper-
plasia associated with inflammatory lesions to squamous metaplasia and
hyperplasia in the bronchial tree. These lesions were seen in approximately
one third of the hamsters in all groups. In the "total" group, many of these
benign lesions were hemangiomas of the liver. Adrenal hyperplasia was also
noted in several animals.
13
-------
TABLE 6. PERCENTAGE OF ANIMALS WITH BENIGN PROLIFERATION AND PERCENTAGE
OF ANIMALS WITH RESPIRATORY BENIGN PROLIFERATION
Treatment
Control
Sulfate
BaP
BaP-sulfate
Total Percentage Present
25.4
32.3
33.6
33.3
Total Percentage Respiratory
19.4
22.2
30.8
31.2
Table 7 compares the non-neoplastic lung diseases found in the animals of
each exposure group.
Table 8. lists the percentages of other significant but non-neoplastic
and non-pulmonary diseases found in each exposure group.
TABLE 7. PERCENTAGE OF ANIMALS WITH OTHER SIGNIFICANT PULMONARY DISEASES
Treatment
Control
Sulfate
BaP
BaP-sulfate
Pneumonia
25.3
25.2
30.8
25.8
Emphysema
9.0
15.2
24.0
20.4
Fibres is
1.5
3.0
1.0
0
Other
1.5
4.0
1.0
0
TABLE 8. PERCENTAGE OF ANIMALS WITH NON-PULMONARY SIGNIFICANT DISEASES
Treatment
Control
Sulfate
BaP
BaP-sulfate
Percentage
38.8
36.4
30.8
30.1
Chi square analysis of these findings indicated that the development of
emphysema was statistically significant in the groups given intratracheal in-
14
-------
stillation of BaP. Ammonium sulfate exposure was not relatable to the devel-
opment of non-neoplastic pulmonary diseases. Other significant pulmonary di-
seases included cases of bronchitis and vasculitis of the pulmonary arteries
and arterioles. Bronchitis was found rarely in this study despite expecta-
tions of this being one of the major disease entities to be encountered.
Vasculitis was seen in control and sulfate alone groups. It occurred in a
group of animals which died about the same time so it may have been infectious,
However, no other evidence for infection was noted in these animals. Other
significant non-pulmonary diseases included infectious gastrointestinal and
renal disease, heart failure and trauma.
The following figures illustrate some of the typical pathologic findings
of this study.
Fig. 4. Squamous carcinoma arising in the lung of hamster
exposed to BaP-sulfate (160X).
15
-------
Fig. 5. Poorly differentiated squamous carcinoma of the bronchus in hamster
exposed to BaP alone (40X).
Fig. 6. Higher magnification of cancer seen in Fig. 5 (640X)
16
-------
Fig. 7. Papillary and invasive squamous carcinoma of the upper third of the
trachea in hamster exposed to BaP alone (40X).
Fig. 8. Higher magnification of cancer seen in Fig. 7 (160X).
17
-------
:*•
ysSfffaS^fr^'^ W%&*%ms»
** •i*?^-'*«' *^4^j»'^*?t 2t v >« >>*-'«4'i>'rf ;-*^KF^M*5>«^
'^^^fer^^^^S
SlfeM^lM^^i.^sSp9a*l^
Fig. 9. Undifferentiated giant cell malignancy in the lung of a hamster ex-
posed to BaP-sulfate (160X).
Fig. 10. Benign cellular proliferation with squamous metaplasia seen in a
hamster exposed to BaP alone (640X).
-------
Fig. 11. Area of benign cellular proliferation and inspissation of hyaline-like
material in the lung of a hamster exposed to ammonium sulfate alone (160X).
Fig, 12. Vasculitis seen in the lung of a hamster exposed to sulfate
alone (160X).
-------
D. Discussion
These studies were designed to determine whether inhalation of ammonium
sulfate enhances the development of cancer. The studies utilized intratracheal
injections of BaP from which a low incidence of cancer was expected and inhala-
tion of ammonium sulfate for 6 hrs/day, 5 days/week, for 15 weeks, at a con-
centration of 200 ug/m3. BaP instillation resulted in both respiratory and
non-respiratory malignancy. Kobayashi (9) had reported an incidence of respi-
ratory tract cancer with BaP alone to be 36% using slightly lower total doses
than used in our studies. Others have reported incidences of 0% (14), 21%
(15) and 53% (16). Our overall incidence of 13% falls within this wide range.
Both the location and types of respiratory cancer observed in our studies were
similar to previously reported studies (6, 8, 9, 15). Our findings of a sub-
stantial incidence of non-respiratory cancers have not been reported by others.
However, published pulmonary carcinogenesis studies have not usually included
complete autopsy findings.
Ammonium sulfate inhalation did not result in enhanced carcinogenesis,
nor did it result in other significant pathologic changes. The concentration
of 200 ug/m3 used in these experiments can be viewed in two ways. First, in
terms of acute concentration, it is 20 times greater than usual ambient levels.
However, since the exposures were for 6 hours a day, 5 days per week, one can
compare this experimental exposure to ambient levels in terms of a time-
weighted average over one week. In this case, the experimental concentration
was only 3 times greater than ambient exposure. These levels viewed in
either way are easily relatable to air quality standards. Should higher con-
centrations be used in subsequent studies? With higher experimental concen-
tration levels, a positive effect would be useful only from a qualitative view-
point. Perhaps, this should be the first question raised in these kinds of
studies. Laskin's study (4), using sulfur dioxide inhalation as a co-carcino-
gen, was done at concentrations acutely 100 times higher than those in our
study. Levels this high are not quantitatively relatable to air quality stan-
dards. Therefore, in reporting our findings, we conclude that ammonium sul-
fate inhalation had no effect at concentrations that were acutely 20 times
greater than ambient levels, and an average of 3 times greater than ambient
levels.
III. DEPOSITION AND CLEARANCE STUDIES
A. Hamster Deposition Studies
Studies of deposition and clearance were proposed as preliminary experi-
ments to assure that the ammonium sulfate particle size we planned to use in
our carcinogenesis studies would reach the deep lung. These studies using ham-
sters were necessary since very little information was available on deposition
of water soluble hygroscopic aerosols in man or animal models. We expected
these deposition studies to be difficult since clearance occurs almost simul-
taneously with deposition. Our approach to the problem utilized an S^S-
labeled ammonium sulfate aerosol with high specific activity, a five minute
exposure time, and a short, reproducible time period after exposure in which
the animals were killed to obtain tissues for analysis.
20
-------
1. Methods--
The diagram in Fig. 13 outlines the exposure system used for hamster
studies.
Vacuum
-------
to make the aerosol. The aerosols were polydlsperse and all had distributions
with greater than 99% of the particles less than 2.1u. Aerosols with an MMD
of 0.3/i had 98% of the particles less than l.lji. Significant radioactivity
was never found in the scrubbers or cold trap (see Fig.13), but these were in
line as precautionary measures.
Immediately following a 5 minute exposure, the hamsters were killed with
an overdose of sodium pentobarbital injected intraperitoneally. The fur of
the animals was washed with a.decontaminating detergent (Contrad-70 (R)) to re-
move any radioactive sulfate, and final drippings were collected and analyzed
to be certain there was no external contamination of internal organs. Blood
was obtained from the heart. The lungs were removed and oven dried for 48
hours. The head was removed; the lower jaw, muscles and all fur were dissected
away. The nose was lavaged with water and then the nose and cranium were
placed in water to soak for 24 hours. Urine was collected and added directly
to scintillation cocktail for analysis. The esophagus, stomach, and small in-
testine were coarsely minced and placed in water to soak for 24 hours.
Sulfate was quantitated in the lung using a Thomas-Ogg combustor and the
method of Charles et al (17). Quantitation of sulfate in other areas was done
by adding trichloracetic acid to blood samples, nasal fluid and gastrointes-
tinal tract fluid, making a final TCA concentration of 5% which precipitated
protein. The precipitates were then washed three times and all supernates
were collected. A vacuum flask evaporator was then used to concentrate all
supernates to 5-10 ml. Two ml of this was then used to quantitate radioacti-
vity.
All radioactivity determinations were done in an Intertechnique scintil-
lation counter using Instabray (Yorktown Research) cocktail, into which up to
2 ml of sulfate solution could be placed for analysis. In analysis of biologic
materials, quench corrections were necessary and these were done using an in-
ternal standard. Preliminary studies using known amounts of sulfate added to
blood or injected directly into excised lungs showed a recovery of 97.9 i
3.66% with the technique outlined here. To be certain that the nasal lavage
was adequate for recovery of sulfate in the nose, an additional wash was col-
lected separately and quantitated for S35. in no case was any S35 found in
these samples.
To study deposition, hamsters were exposed for five minutes to two parti-
cle size distributions. S35 was quantitated in blood, lung, nose, urine and
GI tract immediately after exposure. Eight animals were exposed at one time.
Three exposures were used for each particle size distribution. Table 9 lists
the exposure parameters used.
Both particle size distributions were in the respirable range. The con-
centrations, temperatures and relative humidities were not significantly dif-
ferent .
22
-------
TABLE 9. EXPOSURE .PARAMETERS:IN-HAMSTER DEPOSITION AND CLEARANCE STUDIES
Parameter Group I Group II
Particle size, MMAD 0.65 u 0.36 u
Concentration (mean ± SD) 1.69 + 0.40 ug/1 1.32 + 0.11 ug/1
Temperature 22° C 22° C
Relative humidity (mean ± SD) 58•+ 2.2% 63.7+4.6%
Results and interpretation—
Table 10 outlines the deposition found in lung, nose, urine, blood and GI
tract for the two particle sizes.
TABLE 10. DEPOSITION OF S35-LABELED AMMONIUM SULFATE IN HAMSTERS
Particle size
Deposition in: lung
nose
GI tract
blood
urine
Total sulfate deposited
Expected sulfate deposited
% expected deposited
% deposited in lung
nose
GI tract
blood
urine
Group I
0.65 u
35.1 ng
21.8 ng
140.0 ng
76.5 ng
1.43 ng
274.8 ng
386.0 ng
71.1%
12.8%
7.9%
50.9%
27.8%
0.52%
Group II
0.36 u
24.0 ng
5.7 ng
35.3 ng
24.0 ng
0.7 ng
89.7 ng
356.0 ng
25.1%
26.7%
6.4%
39.4%
26.8%
0.8%
Mean total deposition in the nose and lung was greater with particles
0.65/1 than with particles 0.36 u MMD. An unusually large amount of sulfate
was found in the GI tract of hamsters exposed to both particle sizes. During
23
-------
exposure, the hamsters were actively chewing and licking the apparatus. This
was most likely the source of the sulfate found in the GI tract, The total
blood levels were extrapolated from blood concentrations using published data
(15) for total hamster blood volume. With our methods, animals were killed
immediately after exposure and all samples of tissue were collected within 5
minutes after the end of exposure. In less than 10 minutes from the first en-
counter with radiolabeled sulfate, the amount of sulfate found in the blood
was the second highest in the body. This indicated extremely rapid uptake
into this compartment regardless of whether the source was from the respira-
tory or gastrointestinal system. Also, the small amounts of sulfate in the
urine indicated clearance from the blood had begun within this short period.
The ratio of lung to nose deposition in the larger particles was 1.6 to 1.0.
With the smaller particles it was 4.2 to 1.0. This suggested that an ammonium
sulfate aerosol with an HMD of 0.65 ju can be trapped in the nose to a greater
extent than one with an HMD of 0.36 ju.
We decided to use the smaller particle distribution for our carcinogene-
sis studies based on the above findings. This also seemed to correlate with
the data of Amdur and Corn (19), who showed zinc ammonium sulfate particles at
0.3 M HMD to be 100 times more potent in increasing airway resistance in
guinea pigs than these particles in the same concentration at 0.7 u MMD.
B. Guinea Pig Deposition Studies;
Since we had developed the methodology, we subsequently proposed to study
deposition and clearance of s35-iabeled sulfate in guinea pigs so that our data
could be directly correlated with findings using the airway resistance model.
1. Methods—
The study of guinea pigs necessitated slight modifications of our methods.
These animals were exposed to S^^-labeled ammonium sulfate in our 322 liter
exposure chamber described earlier in this report. The animals were exposed
for five minutes in groups of 12 at a time. The animals were placed inside in-
dividual wire cages that allowed them to breathe without restraint and their
bodies were covered to the neck with cotton orthopedic stockings to minimize
fur contamination by 8^5.
Aerosols were generated from DeVilbis #40 nebulizers. A single nebulizer
containing 0.5% (NH4)2 804 was used to produce the large particle aerosol and
five nebulizers, each containing 0.05% (NH4)2S04, were used to produce an aero-
sol of the same concentration but with a smaller particle size. The aerosols
were monitored by Andersen sampling throughout the entire 5 minute exposure.
The sulfate concentration and particle size were measured as previously de-
scribed for the hamster studies.
Animals were killed immediately following exposure by an overdose of
sodium pentobarbital. The fur of the face and chest was then washed to reduce
any possible chance of contamination and the final washings quantitated for
radioactivity. Twice background was considered an acceptable level. A sample
of blood was taken from the heart. The lungs were removed, lavaged with sa-
line 5 times followed by 3 water lavages. The whole lungs were then homogen-
24
-------
ized. The head was removed, the lower jaw separated and the nasal cavity
lavaged from both the pharynx and the external nares, first with saline, then
with water. Nasal lavage fluid usually totaled 500-700 ml. Urine was collec-
ted from the bladder, gastric contents from the stomach.
Trichloracetic acid was added to the pulmonary lavage fluid, lung homog-
enates, blood samples, nasal lavage fluid and gastic material, making a final
TCA concentration of 57o which precipitated protein. These samples were then
handled in the same manner as described for the hamsters.
2. Results and interpretation—
Table 11 lists the parameters used in the guinea pig exposures.
TABLE 11. EXPOSURE PARAMETERS IN GUINEA PIG DEPOSITION AND CLEARANCE STUDIES
Parameter Group I Group II
Particle size, MMAD 0.65 u 0.31 ju
Concentration 1.42 ug/1 2.01 jig/1
Temperature 22.2°.C 22.2° C
Relative humidity 53% 40%
Total respiratory tract deposition at each particle size in guinea pigs
was not significantly different, although there was slightly more sulfate found
in animals exposed to the larger particles. The large particle to small parti-
cle deposition ratio was 1.2 to 1.0. In comparing lung to nose ratios, the
larger particles were again trapped to a greater extent in the nose (L/N ratio
1.5), whereas the smaller particles had less nasal entrapment (L/N ratio 2.0).
Significance of the difference in nasal deposition at the two particle sizes is
p
-------
Table 12 outlines the deposition of sulfate in the lung, nose, GI tract,
blood and urine.
TABLE 12. DEPOSITION OF S35-LABELED AMMONIUM SULFATE IN GUINEA PIGS
Particle size
Deposition in
Total sulfate
: lung
nose
GI tract
blood
urine
deposition
Expected sulfate deposition
% expected deposition
% deposition
in lung
nose
GI tract
blood
urine
Group I
0.65 u
123 ng
79 ng
62.3 ng
53.7 ng
0.63 ng
318.6 ng
1136
28.0%
38 . 6%
24.8%
19.6%
16.9%
0.19%
Group II
0.31 u
107 ng
53 ng
32.3 ng
74.9 ng
0.35 ng
276.6 ng
1616
16.6%
40.0%
19.8%
12.19%
28.0%
0.1%
C. Rabbit Deposition Studies
We also proposed to study deposition in rabbits so that our findings
could be correlated with studies of pulmonary responses in rabbits underway in
other laboratories.
26
-------
1. Methods--
Deposition studies in rabbits were carried out using both nose only and
whole body exposure. The exposure chamber for nose only exposure consisted of
a central plexiglass tube (4" in diameter) with two rabbit-holding tubes
mounted on each side perpendicular to the central tube. Two inch in diameter
openings into the central tube provided the rabbits with nose only exposures,
while loose fitting collars held the rabbit's head and nose in place. Rabbits
were selected to fit in the apparatus so that their breathing was not restric-
ted.
Whole body exposures were done in the same way as described for guinea
pigs. Analysis of tissues for sulfate was also the same as described for
guinea pigs.
Table 13 lists the exposure parameters used for the nose only exposures.
TABLE 13. EXPOSURE PARAMETERS IN RABBIT "NOSE ONLY" DEPOSITION STUDIES
Parameter Group I Group II
Particle size 0.5 u MMD 0.3 u MMD
Concentration (mean t SE) 1.88 t 0.40 ug/1 2.27 1" 0.15 ug/1
Temperature (mean + SD) 23.0 t 1.38° C 23.5 t 1.73° C
Relative humidity (mean + SD) 52.5 t 4.39° C 51.5 t 2.12° C
Estimated amount of sulfate 9 4 + 1 99 ue 11 38 "*" 0 78 ug
entering respiratory system '
Amount of sulfate recovered
from nose, lung, blood, urine 2.03 t 1.03 ug 2.12 1" 1.48 ug
and stomach
Percent of expected recovered ^^ + ^^ 2Q 25 +
(mean -
Since rabbits were exposed individually, there was variability in concen-
trations but concentrations at the two particle sizes were not significantly
different. Temperature and relative humidity were held constant within a nar-
row range. The total amount of sulfate recovered from animals in each group
was not significantly different. The percent of expected recovered varied
widely as can be seen by the mean and standard error shown. This was probably
27
-------
related to variation in breathing patterns of the animals.
2. Results--
Table 14 outlines the findings of pulmonary versus nasal deposition at
the two particle sizes.
TABLE 14. PULMONARY VERSUS NASAL DEPOSITION IN RABBITS
Group I
(0.5 p. MMD)
ug/nose
ug/ lungs
% nose
% lungs
0.72
0.25
73
26
t 0.16
1" 0.05
t 4%
± 4%
Group
(0.3 u
0.44
0.61
43
57
+
+
+
+
II
MMD)
0.17
0.35
2.5%
2.5%
Significance: p
-------
Table 15 lists the exposure parameters for the whole body exposures.
TABLE 15. RABBIT EXPOSURE PARAMETERS FOR WHOLE BODY EXPOSURES
Parameters
Group I
Group II
Parameter size, MMAD
Concentration (mean _ SD)
Temperature (mean + SD)
Relative humidity (mean t SD)
0.60 ;i 0.32 u
1.12 + 0.08 ug/1 1.28 + 0.09 tog/1
22.2 + 1.9° C
56 t 0.717o
21.6 I 2.7° C
55 + 8.5%
Table 16 outlines the deposition of sulfate in the lung, nose, GI tract,
blood and urine.
TABLE 16. DEPOSITION OF S35-LABELED AMMONIUM SULFATE IN RABBITS USING WHOLE
BODY EXPOSURE
Particle size
Deposition in
Total sulfate
lung
nose
GI tract
blood
urine
deposited
Expected sulfate deposited
% expected deposited
7= deposition
in lung
nose
GI tract
blood
urine
Group I
0.60 u
456 ng
107 ng
1.05 ng
83.3 ng
46.4 ng
703 ng
5992 ng
11.7%
66.1%
15.2%
0.15%
11.8%
6.6%
Group II
0.32 p
127 ng
90 ng
41 ng
60.3 ng
1.8 ng
320 ng
6848 ng
4.7%
39.7%
28.1%
12.8%
18.8%
0.56%
The findings in rabbits by these two modes of exposure were so different
that we felt it was necessary to do another group of rabbit exposures. We
therefore repeated the nose only exposures. Table 17 lists the parameters of
29
-------
these exposures.
TABLE 17. SECOND RABBIT NOSE ONLY EXPOSURE
Parameter
Particle size MMAD
Concentration
Temperature
Relative humidity
Group I
0.63 p
2.88 ug/1
23.1° C
50.3%
Group II
0.34 vi
3.30 ug/1
23.3° C
49.1%
Table 18 outlines pulmonary and nasal deposition in these experiments,
TABLE 18. PULMONARY VERSUS NASAL DEPOSITION IN SECOND NOSE ONLY EXPOSURE
Parameter
Group I
Group II
Particle size
Deposition in lung
Deposition in nose
0.63 u
371 ng
389 ng
0.34 ji
255 ng
220 ng
This repeat study showed that the larger particle size had a larger total
respiratory tract deposition than the smaller particle size and nasal and pul-
monary deposition fractions were almost equal. These findings were different
from the two previous studies. We then examined all our deposition studies
from the viewpoint of animal to animal variation. Table 19 lists mean i" SD of
pulmonary and nasal deposition of the studies.
TABLE 19. PULMONARY AND NASAL DEPOSITION OF ALL DEPOSITION STUDIES
(ng SULFATE t SD)
Study
Second rabbit study
nose only
Rabbit chamber study
First rabbit study
Guinea pig study
Hamster study
Area
Lung
Nose
Lung
Nose
Lung
Nose
Lung
Nose
Lung
Nose
Large Particles
371 t 231
389 ± 222
465 + 352
107 t 62.3
250 ± 50
723 ± 165
122.8 + 36.5
79.0 + 12.8
35.1 ± 10.5
21.8 + 3.9
Small Particles
255 ± 79
220 ± 100
127 + 66.0
90.2 . ± 37.0
615 t 352
441 ±171
107 t 14.0
53.1 ± 8.6
24.0 ± 6.4
5.7 ± 1.9
30
-------
By looking at standard deviation it can be seen that much greater dif-
ferences were present from animal to animal in the rabbit studies than in
those of hamster and guinea pig. The first rabbit study seemed to have the
least variation, while subsequent studies had much more. These findings were
difficult to explain. It is possible that since the rabbits were larger ani-
mals, they were not as amenable to our methods of analysis as smaller animals
and the variation seen was a methodologic problem. In any case, we did not
further pursue the rabbit studies.
D. Clearance Studies in Hamsters, Guinea Pigs and Rabbits
To study clearance, animals were exposed for five minutes to two particle
size distributions. S-" was quantitated in blood, lung, nose, urine and the
GI tract immediately, one hour, three hours and six hours after exposure.
Quantitation methods were the same as used for the animals in deposition
studies. In fact, clearance and deposition were usually studied in the same
group of animals so that exposure parameters for these studies were those
listed earlier for each animal group.
The patterns of sulfate clearance in the three species studies are de-
scribed by Figures 14 through 19. In contrast to deposition, the clearance
patterns of sulfate were similar in all three species tested. All animals
showed rapid sulfate clearance from the lung. The rate of clearance, as ex-
pressed by the slope of the lung graph from 0 to 1 hour, was the same for the
three species. T% was 18-20 minutes. The rate of clearance from the lung was
the same for large and small particles. Within species, the clearance pattern
for each tissue studied was almost identical for large and small particles.
In all animals studied, the sulfate cleared from the lung, nose and GI tract,
and then ultimately appeared in the urine. In preliminary studies, using 24
hour urine collection, it was found that greater than 95% of sulfate that ap-
peared in urine was there by six hours. There was a difference in the clea-
rance from the GI tract of the hamster and the two larger species studied.
The hamster had a large initial GI deposition, as explained earlier, which was
followed by rapid clearance, almost parallel to that of the lung. The rabbit
and guinea pig showed a maximum in GI at one hour after exposure. This pattern
was possibly due to clearance into the GI from the nose and lungs, followed by
the clearance of sulfate from the GI to the urine. This clearance pattern is
consistent with the clearance model used by the ICRP for soluble inhaled sub-
stances (Task Group in Lung Dynamics) (20). This model predicts that 50% of
inspired soluble particles rapidly pass into the gastrointestinal tract and
25% of these particles promptly enter the circulation.
The blood concentration of sulfate varied somewhat throughout all the de-
position and clearance studies, but without any definitive pattern. The varia-
tion in blood was much less than for any other tissue studied. This mild flux
was most likely due to blood being the route of sulfate transport from the lung,
nose and GI tract to the urine.
31
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DEPOSITION AND CLEARANCE OF AMMONIUM SULFATE IN HAMSTERS
(Concentration-1.69 >/g per liter; Particle size-0.65//MMAO)
1000-
UJ
g
CO
o
<
100-
10-
I
URINE
BLOOD
10 ml Trial HomtMr
'• NOSE
LUNG
I
6
TIME-HOURS
Fig. 14. Large particle deposition and clearance in hamsters,
32
-------
DEPOSITION AND CLEARANCE OF AMMONIUM SULFATE IN HAMSTERS
(Concentration -1.32 //g per liter; Particle size - 0.36//M MAD)
iu
co 100-
C0
10-
I
URINE
BLOOD
Total Hamttr
Blood Volurn*
0.1. TRACT
NOSE
•»A LUNG
013 6
TIME- HOURS
Fig. 15. Small particle deposition and clearance in hamsters,
33
-------
DEPOSITION AND CLEARANCE OF AMMONIUM SULFATE IN GUINEA PIGS
(Concentration- 1.42 >/g per liter; Particle size - 0.65// MMAD)
1000-
Id
£
CO
100-
o
I
URINE
BLOOD
30 ml Totql Guinto Pig
Blood VoliMM
•—•• NOSE
0.1. TRACT
^••A LUNG
I
TIME-HOURS
Fig. 16. Large particle deposition and clearance in guinea pigs,
34
-------
DEPOSITION AND CLEARANCE OF AMMONIUM SULFATE IN GUINEA PIGS
(Concentration- 2.02 //g per liter; Particle Size-0.31 jj MM AD)
1000-
UJ
co
100-
CO
a:
o
o
10-
I
O URINE
• BLOOD
30 ml Total GuinM Pig
Blood Volum*
NOSE
"••O G.I. TRACT
LUNG
013 6
TIME-HOURS
Fig. 17. Small particle deposition and clearance in guinea pigs,
35
-------
DEPOSITION AND CLEARANCE OF AMMONIUM SULFATE IN RABBITS
(Concentration - 1.12 j/q per liter; Particle Size - 0.60 // MM AD)
1000-
Iti
€0
100-
e
o
10-
URINE
• BLOOD
100 ml. Total Rabbit
Blood Volumo
0.1. TRACT
NOSE
6
TIME-HOURS
Fig. 18. Large particle deposition and clearance in rabbits
from whole body exposure.
36
-------
DEPOSITION AND CLEARANCE OF AMMONIUM SULFATE IN RABBITS
(Concentration-1.28>/g per liter; Particle size-0.32 jj MMAD)
1000 j
111
CO
100-
co
K 10-
o v
o
URINE
BLOOD
100 ml. Total RobbH
Blood Volume
G.I. TRACT
LUNG
NOSE
I
3
TIME- HOURS
6
Fig. 19. Small particle deposition and clearance in rabbits from
whole body exposure.
37
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IV. PULMONARY DEFENSE STUDIES
A. Aryl Hydrocarbon Hydroxylase Studies
Aryl hydrocarbon hydroxylase is an enzyme that acts in the metabolism of
BaP and other carcinogens. Its role is to render these organic substances
more hydrophilic and thereby to aid in excretion. It is an inducible enzyme,
and has been shown to be inhibited in the lung by oxidant air pollutants (21).
Our purpose was to determine whether ammonium sulfate had an inhibiting influ-
ence on this enzyme, which could be important in the clearance of carcinogen
and consequently in carcinogenesis.
Animals were exposed using basically the same protocol as in the carcino-
genesis experiments. Four groups were studied: control, sulfate, BaP and
BaP-sulfate. Animals were exposed to ammonium sulfate at a concentration of
189 i" 30 ug of sulfate per cubic meter for 6 hours per day, five days per week.
Animals received BaP intratracheal injections on Wednesday of each week, using
a 5 mg dose in saline-gelatin suspension. Exposure temperature and relative
humidity for animals exposed to sulfate were 24.3 1" 1.1 C and 48.1 - 6.6%.
These parameters for BaP only and control animals were 23.3 jf 0.75° C and
39.0 * 9.8%. Animals were killed for enzyme assay at the end of the full
week's exposure. Six animals per group per time period were studied. Sodium
pentobarbital overdose was used to kill the animals. This had no effect on
enzyme levels in preliminary studies. The lungs were removed en bloc and
quick frozen with dry ice for subsequent analysis.
Aryl hydrocarbon hydroxylase assays were performed by the method of
Okamoto et al (22). Six determinations were done on each animal. Three were
0 time assay controls without reduced triphosphopyridine nucleotide (TPNH),
and three were determinations at 20 minutes with TPNH. Preliminary studies
with this enzyme assay showed the amount of product produced with time was
linear from 10 to 25 minutes.
Table 20 outlines the results of these studies.
TABLE 20. EFFECT OF AMMONIUM SULFATE AEROSOL ON ARYL HYDROCARBON
HYDROXYLASE ACTIVITY IN HAMSTER LUNGS
_ AHH Activity AHH Activity
Treatment ,^ , , ,. 0 ,
after 1 week after 3 weeks
Control 1.33 t 0.737 units1 1.60 + 0.801 units
Sulfate 1.38 ± 0.980 units 1.59 t 0.879 units
BaP 11.0 ± 1.34 units 13.2 + 4.04 units
BaP-sulfate 11.8 t 3.39 units 10.9 j" 3.85 units
1. One unit is equivalent to the fluorescence of 50 ug of quinine sulfate/ml
0.1 N H2S04 when converted to 3-OH BaP by 10 mg lung tissue/20 min. (Okamoto,
T., P. Ahan and B. So, Life Sciences _11, II, 733-741, 1972).
38
-------
As expected, benzo(a)pyrene injection induced significant increases in
aryl hydrocarbon hydroxylase activity. However, exposure to ammonium sulfate
at the levels used did not affect this enzyme in stimulated or unstimulated
animals.
Studies were then done exposing hamsters to concentrations of sulfate
five times greater and continuing the exposures for 10 weeks. Actual exposure
parameters were: sulfate concentration - 1.35 1 0.15 mg/nr; temperature -
23.9 t 2.4° C; relative humidity - 34.7 + 4.7% and particle size - 0.04 y. -
MMAD. Assays were done in the same way as the previous study.
TABLE 21. EFFECT OF AMMONIUM SULFATE AEROSOL ON ARYL HYDROCARBON HYDROXYLASE
ACTIVITY IN HAMSTER LUNGS
Treatment AHH Activity AHH Activity
after 1 week after 10 weeks
Control
Sulfate
BaP
BaP-sulfate
0.471 t 0.264*
0.583 + 0.309
7.53 + 2.52
4.62 + 0.904
n=6 for each group
0.246 ± 0.0530
0.319 t 0.413
8.50 + 1.02
8.20 + 1.40
*Units are the same as in Table 20.
Exposures to ammonium sulfate at a higher concentration and for a longer
time also had no effect on aryl hydrocarbon hydroxylase activity.
B. Pulmonary Macrophage Studies
The pulmonary macrophage is an important defensive cell in the lung. Its
role in bacterial clearance is well known, but its role in chemical carcino-
genesis is debatable. It undoubtedly plays a major role in clearing deposited
carcinogen from the lung. However, there has been speculation that perhaps
pulmonary macrophages, in clearing carcinogens, concentrate these chemicals and
thereby produce an undesirable situation in which a more concentrated carcino-
gen is redeposited on pulmonary epithelium. The effect of several known co-
carcinogens on pulmonary macrophage number has been studied. Using standardized
methods of quantitation, Brain et al (23) have shown that ferric oxide decreases
macrophage numbers in the lung in some species. Our studies were designed to
determine whether ammonium sulfate inhalation had any effect on macrophage num-
ber using a change in number as an indication of pulmonary irritation or toxic-
ity.
Hamsters were exposed to 860 jig/m sulfate as ammonium sulfate for 12
hours. Mean relative humidity was 437o and temperature was 22.7° C. The parti-
cle size was 0.3 ^u MMAD as determined by Andersen sampling. To achieve this
particle size at this concentration, the aerosol was generated by four Collison
atomizers arranged in parallel. Control hamsters were held in the same en-
vironmental conditions except that their air contained no detectable sulfate.
39
-------
Six sulfate-exposed animals were killed by sodium pentobarbital overdose im-
mediately after exposure, and six, 24 hours later. Control animals were
studied in the same manner. Pulmonary macrophages were collected and counted,
and are reported (see Figs. 20 and 21) by the standardized methods of Brain
and Frank (24).
No difference could be seen between the shape of the washout curve of
controls and those of the post-exposure groups. Similarly, no significant
differences were seen in the macrophage number of control and post-exposure
hamsters. Data points in Figs. 20 and 21 represent the mean of six animals
1" standard error. Although there was statistically no difference between
the three groups, the standard error was greater in the exposed groups than
in controls. The significance of this change is speculative. The study was
repeated with similar parameters and the same results were obtained. There-
fore, based on these data, it can be concluded that ammonium sulfate in-
halation at this level had no significant effect on pulmonary macrophage
numbers.
40
-------
PULMONARY MACROPHAOE RESPONSE TO AMMONIUM SULFATE INHALATION
H£AN± S.E.
II
KH
| 9-
J 8
6
K
w .
o. 5
M .
O 4
" 3
CO
J 2
iij
CONTROLS
IMMEDIATELY AFTER EXPOSURE
24 HOURS AFTER EXPOSURE
Hi
4
*
10
WASH NUMBER
Fig. 20. Macrophages obtained by lavage in control and ammonium sulfate exposed hamsters.
Mean + S.E.
-------
N>
ac
ui
o.
K
CO
_J
_J
Ul
o
CONTROLS
IMMEDIATELY AFTER EXPOSURE
24 HOURS AFTER EXPOSURE
PULMONARY MACROPHAQE RESPONSE TO AMMONIUM SULFATE INHALATION
WASH-OUT CURVE
II -
10-
9n
8
7-j
6
5-
4-
3-
2-
I-
456
WASH NUMBER
8
9
10
- 21. Macrophages obtained by lavage in control and ammonium sulfate exposed hamsters:
Comparison of washout curves.
-------
REFERENCES
1. French,J.G., G. Lawrence, W.C.Nelson, J.F.Finklea, T.English and M.Hertz,
The Effect of Sulfur Dioxide and Suspended Sulfates on Acute Respiratory
Disease., Arch. Environ. Health, 27:129-133, 1973.
2. Chapman,R.S., C.M.Shy, J.F.Finklea, D.E.House,H.E.Goldberg and C.H.Hayes,
Chronic Respiratory Disease, Arch. Environ. Health, 27:138-142, 1973.
3. Amdur,M.D. and D.W.Underbill, The Effect of Various Aerosols on the Re-
sponse of Guinea Pigs to Sulfur Dioxide, Arch. Environ. Health, 16:460-468,
1968.
4. Laskin,S., M.Kuschner and R.T.Drew, Studies of Pulmonary Carcinogenesis,
U.S.Atomic Energy Commission, AEC Symposium, 18:321-350, 1970.
5. Likens,G.E., Acid Rain: A Serious Regional Environmental Problem. Science,
184:1176-1179, 1974.
6. Saffiotti,U., R. Montesano, A. Sellakumar, F.Cefis and D.G.Kaufman, Respira-
tory Tract Carcinogenesis in Hamsters Induced by Different Numbers of Ad-
ministrations of Banzo(a)pyrene and Ferric Oxide, Cancer Res. 32:1073-1081,
1972.
7. Pyley,L.N. Induction of lung cancer in rats by intratracheal insufflation
of cancerogenic hydrocarbons, Acta Und. Int. Cancer, 19:688-691, 1962.
8. Delia Porta.G., L.H. Kolb and P.Shubik, Induction of Tracheobronchial Car-
cinomas in the Syrian Golden Hamster, Cancer Res. 18:592-598, 1958.
9. Kobayashi,N., Production of Respiratory tract tumors in hamsters by Benzo(a)
pyrene, Gann, 66:311-315, 1975.
10. EPA Publication from Human Studies Laboratory. Health Consequences of Sul-
fur Oxides: A Report from CHESS, 1975.
11. Hinners,R.G., J.K.Burkart and C.L.Punte, Animal Inhalation Exposure Chambers,
Arch. Environ. Health, 16:194-206, 1968.
12. Melnicoff,M.J., J.J.Godleski and J.P.Bercz, An Automated Method for the De-
termination of Sulfate, Res. Comm. in Chem. Path, and Pharm. 14:377-386,
1976.
13. Sawicik,E., T.R.Hauser and T.W.Stanley, Ultraviolet, Visible and Fluores-
cence Spectral Analysis of Polynuclear Hydrocarbons, Int. J. Air Poll., 2:
253-272, 1960.
43
-------
14. Kuschner,M., The Causes of Lung Cancer, Am. Rev. Respir. Dis., 98:573-590,
1968.
15. Henry,M.C., C.P.Port, R.R.Bates and D.G.Kaufman, Respiratory Tract Tumors
in Hamsters Induced by Benzo(a)pyrene, Cancer Res., 33:1585-1592, 1973.
16. Feron,V.J., Respiratory Tract Tumors in Hamsters after Intratracheal In-
stillations of Benzo(a)pyrene Alone and with Furfural, Cancer Res. 32:
28-36, 1972.
17. Charles,J.M. and D.B.Menzel, Absorption of Sulfate Ions in the Rat Lung,
Res. Commun. Chem. Pathol. Pharmacol., 12:389-396, 1975.
18. Charles River Digest, Some Physiological Parameters of Small Animals,
Charles River Breeding Labs., 10: 1971.
19. Amdur,M.O. and M.Corn, The Irritant Potency of Zinc Ammonium Sulfate of
Different Particle Sizes, Amer. Ind. Hyg. Assoc. J., 24:326-333, 1963.
20. Deposition and Retention Models for Internal Dosimetry of the Human Res-
piratory tract, Task Group on Lung Dynamics, Health Physics, 12:173-207,
1966.
21. Palmer,M.S., D.H.Swanson and D.L.Coffin, Effect of Ozone in Benzo(a)
pyrene Hydroxylase Activity in the Syrian Golden Hamster, Cancer Res.
3:730-733, 1971.
22. Okamoto,T., P.Chan and B.T.So, Effect of Tobacco, Marijuana and Benzo(a)
pyrene on Aryl Hydrocarbon Hydroxylase in Hamster Lung, Life Sci., 11:
733-741, 1972.
23. Sorokin,S.P. and J.D.Brain, Pathways of Clearance in Mouse Lungs Exposed
to Iron Oxide Aerosols, Anat. Rec., 181:581-626, 1975.
24. Brain,J.D. and N.R.Frank, Recovery of Free Cells from Rat Lungs by Re-
peated Washings. J. Appl. Physiol., 34:75-80, 1973
44
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
2.
4. TITLE AND SUBTITLE
STUDIES ON THE EFFECT OF AMMONIUM SULFATE
•ON CARCINOGENESIS
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
John J. Godleski and Joseph Leighton
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Pathology
Medical College of Pennsylvania
Philadelphia, PA 19129
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
R-802839
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park. NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP.NC
14. SPONSORING AGENCY CODE
EPA-600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This project was designed to evaluate the health effects of ammonium sulfate
(Nh4)2 SO* inhalation using experimental animals. The questions studied were: (1)
Is inhaled ammonium sulfate co-carcinogenic. (2) Mhat are the deposition and
clearance patterns of inhaled ammonium sulfate? (3) What effect does ammonium
sulfate have on pulmonary defensive mechanisms?
The study showed that ammonium sulfate inhalation had no effect on the develop-
ment of cancer and no effect on the development of other significant pulmonary
diseases in hamsters.
Hamsters, guinea pigs and rabbits were studied for deposition and clearance of
inhaled ammonium sulfate. Total respiratory tract deposition was greater with the
larger particle size in all studies. Clearance patterns were similar for the three
species regardless of particle size. The half time for clearance of ammonium
sulfate from the lung was 18 to 20 minutes. Inhaled and injected sulfate was cleared
via the urinary tract and by six hours after esposure 95% of the total collectable
sulfate was present in the urine. Pulmonary macrophage number was not affected by
ammonium sulfate inhalation.
17.
a.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
ammonium sulfate
air pollution
toxicity
respiration
carcinogens
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
06 F, T
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
53
20. SEC
ifapage)
22. PRICE
EPA Form 2220-1 (9-73)
45
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