EPA-800/1-77-026
May 1977
Environmental Health Effects Research Series
INHALATION TOXICOLOGY OF AIRBORNE
PARTICULATE MANGANESE IN RHESUS
MONKEYS
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-600/1-77-026
May 1977
INHALATION TOXICOLOGY OF AIRBORNE PARTICULATE
MANGANESE IN RHESUS MONKEYS
by
Frederick Coulston Travis Griffin
Director, Institute of Comparative Director, International Center
and Human Toxicology of Environmental Safety
Institute of Comparative and Human Toxicology
International Center of Environmental Safety
Albany Medical College
Hoi1oman Air Force Base, New Mexico
Contract 68-02-0710
John H. Knelson and Gory J. Love
Health Effects Research Laboratory
Environmental Protection Agency
Research Triangle Park
North Carolina 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 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
constitute 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
affidavits 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.
The research on manganese which is described in this report was
undertaken because good information on the effects and toxicity of inhaled
manganese were not available for prolonged carefully measured exposures.
The metal is used as an additive in some distillate fuels, and is currently
being considered for broader application. This has caused increased
interest in the toxicology of this metal. Attempts are being made by
means of new research to correct deficiencies in our knowledge of manganese
metabolism and toxicology.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
iii
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CONTENTS
Disclaimer ii
Foreword iii
Figures v
Tables vi
1. Summary 1
2. Introduction 2
3. Methods and Materials 3
4. Results 9
5. Discussion 17
6. Conclusions 19
References 67
Pathology Appendix 68
iv
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FIGURES
Number Page
1 Manganese (Mn) generating system 4
2 Manganese delivery system 5
3 Manganese concentration changes during startup and shutdown ... 10
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TABLES
Number Page
1 Comparison of Air Samples Obtained Inside the Exposure Chamber
with Similar Samples Obtained Simultaneously at the Exhaust
Duct 20
2 Andersen Sample Studies 21
3 Weekly Mean Concentrations of Mn in Drinking Water 22
4 Monthly Means of Concentration of Mn in Drinking Water 23
5 Body Weights of Monkeys During Exposure to Mn 24
6-19 Clinical Chemistry Profiles 25-38
20-33 Hematology Profiles 39-52
34 Concentration of Mn in Urine of Monkeys Exposed to Airborne Mn . 53
35 Concentration of Mn in Feces of Monkeys Exposed to Airborne Mn . 54
36 Concentration of Mn in Selected Tissues of Monkeys Exposed to
Airborne Mn 55
37 Concentration of Mn in Tissues From Nervous System of Monkeys
Exposed to Airborne Mn 57
38 Excretion of Mn in Urine of Rats Exposed to Airborne Mn 58
39 Excretion of Mn in Feces of Rats Exposed to Airborne Mn 59
40 Concentration of Mn in tissues of Rats Exposed to Airborne Mn. . 60
41 Weekly Mean Concentrations of Mn in Air of High-Level Exposure
Chambers 61
42-43 Clinical Chemistry Profiles, High-Level Exposure 62-63
44-45 Hematology Profile, High-Level Exposure 64-65
46 Excretion of Mn by Monkeys Exposed to Airborne Mn at About
5000 yg/m3 66
vi
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SECTION 1
SUMMARY
Male and female rhesus monkeys were exposed to airborne particulate
manganese (Mn) at a concentration of 100 yg per cubic meter of air for periods
lasting up to 66 weeks. Observations of the animals, including visual obser-
vations for any signs of toxicity, and studies of clinical chemistry and
hematology did not indicate any effects due to exposure to manganese. Very
small increases in the excretion of manganese in the urine and feces were
observed signifying, principally, that the burden imposed by the airborne
manganese represented only a small fraction of the total daily intake of the
metal. In spite of the low level of exposure, a small but significant accu-
mulation of manganese was observed in -certain tissues of the animals examined
at autopsy. Manganese levels were increased in the lungs, liver, pancreas,
kidney, heart muscle, pallium, basal ganglia, cerebellum, and pons. In most
cases the degree of increase was two-fold or less, with the exception of the
lungs which showed a five-fold increase and the pons which showed a four-fold
increase. There were no changes in morphology, either macroscopic or micro-
scopic, which could be attributed to exposure to manganese.
Rats were also exposed to the airborne manganese at 100 yg per cubic
meter of air for periods up to eight weeks. Small increases in the excretion
of manganese were noted in the animals during exposure which continued for
periods up to eight weeks after the animals had been removed from the chamber.
Similarly, there was an increase in the concentration of Mn in the lung and
brain tissue of exposed rats but, in both cases, there was evidence of a
rapid return to normal levels within one week after animals were removed from
the exposure chamber.
Two rhesus monkeys were exposed to airborne manganese at about 5 mg per
cubic meter of air for a 23 week period of exposure. The principal goal of
this study was to determine whether exposure at that level would produce any
visible neurologic disorders in the animals. No consistent changes in the
clinical chemistry or hematology were observed during the exposure period.
The excretion of manganese was greatly increased in both the urine and the
feces. Significantly, however, there was no evidence of neurologic disorders
or behavioral changes either during the high level exposure, or later after
the animals had been removed from the chamber and observed for an additional
10 months.
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SECTION 2
INTRODUCTION
This is the final report of EPA Contract 68-02-0710, the objective of
which was to examine the potential toxicity of airborne particulate manganese
when inhaled by rhesus monkeys. Studies were designed to provide definitive
data on potential hazards involved in the use of methylcyclopentadienyl
manganese tricarbonyl (MMT) when used as a combustion improver in automotive
fuels. When used in automotive fuels, the organomanganese compound is burned
to produce an oxide of manganese (MnaOi*). It was this oxide, in the form of
solid particulates, to which the monkeys were exposed. The experiment was
designed to provide an almost continuous regimen of exposure of the monkeys
to the manganese particulates. The intended level of exposure (100 yg manga-
nese per cubic meter of air) was chosen as a level estimated to be far in
excess of that anticipated in ambient air as a result of motor fuel usage
and at the same time it was well below the threshold limit value* of manganese.
In the main experiment the animals were exposed for one year.
All animals in the study were examined daily for signs of toxicity. Also,
periodic clinical examinations of the animals determined the status of their
health and well-being. Exposure to manganese was monitored through measure-
ments not only of airborne manganese but also of the manganese content of the
diet and drinking water. Excretion of manganese was studied and the concen-
tration of the metal in the animal tissues was determined at autopsy which
included gross and histopathologic examination.
Two short ancillary studies of manganese inhalation were also conducted.
In one study, data were sought which would yield information on the dynamics
of manganese turnover in rats which were exposed to the airborne particulate
manganese. In the other study, an effort was made to develop the rhesus
monkey as a model for studying acute maixganism following exposure at or near
the threshold limit value.
* threshold limit value for Mn = 5000 Ug/m3, American Conference of
Governmental Industrial Hygienists.
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SECTION 3
MATERIALS AND METHODS
The system for exposure to manganese particulates was developed at the
International Center of Environmental Safety at Holloman AFB, New Mexico.
Although similar in basic design to the system used previously by researchers
at the Albany Medical College for exposure of animals to airborne particulate
lead (1), modifications were incorporated to permit more effective control of
the size of the particulate manganese produced by the system. In principle,
the system generates manganese oxide particulates through the combustion of
vaporized MMT in an air-natural gas flame. The MMT vapor was produced by
metering a portion of the air required for combustion through a thermostated
container of liquid MMT (Figure 1). Additional metered air was added to the
resulting MMT vapors and the mixture was burned in a torch type burner utilizing
natural gas as the fuel. The burner was mounted in a combustion chamber located
atop a 55-gallon drum holding chamber.
A safety shutoff system was provided to prevent the accumulation of
explosive gas mixtures in the holding and exposure chambers as a result of
flameout, and also to prevent inadvertent exposure of the animals to unburned
MMT. During operation the flame was constantly monitored by a UV detector,
and in the event that the flame became extinguished, a high voltage spark
ignition system was automatically activated to reignite the flame. This
system functioned automatically for approximately 15 seconds, or until the
flame was reignited. Failure of the system to reignite the flame resulted
in a total shut down of the burner by means of solenoid valves in both the
air and natural gas supply lines activated by the UV detector control
mechanism. When such a failure occurred it was necessary to reset the
system manually before the burner could again be ignited.
The generator system provided control of concentration of manganese
particulates in the air, and control of the size of the generated particulates.
Adjustment of the concentration of MMT vapors in the combustion mixture
maintained control over the final concentration of manganese oxide par-
ticulates. The concentration of MMT vapors was varied by altering the
temperature of the liquid MMT vessel or the flow rate of the air through
the vessel.
Products from the combustion chamber then entered a pair of 55-gallon-
drum holding chambers aligned in series (Figure 2). These holding chambers
served to delay passage of the particulate manganese into the exposure
chamber long enough to allow aggregation of the particles, thus yielding
particles of a size more likely to be found in the ambient air. A small
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TORCH
IGNITOR
f
ULTRA-
VIOLET
SENSOR
GAS
SOLENOID
VALVE
FLOWMETER
SAFETY
CONTROL
UNIT
THERMOSTATED
BUBBLER
FLOWMETER
o
AIR
{ PRESSURE
SOLENOID REGULATOR
VALVE
LIQUID
TRAP
FLOW-
METER
Figure 1. Manganese generating system.
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INHALATION
CHAMBER
i ^
FILTER
f
FAN
IGNITOR
\
ULTRAVIOLET
SENSOR
SAFETY
CONTROL
UNIT
ZI—• AIR INTAKE
HOLDING CHAMBERS
Figure 2. Manganese delivery system.
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blower mounted on the second holding chamber extracted its contents at a
constant rate and delivered the particulate material to the main exposure
chamber.
The exposure chamber itself consisted of a temperature-humidity controlled
room, 8 feet wide by 8 feet high and by 12 feet long, modified to permit intro-
duction of the particulate manganese. The effluent from the generator was
injected into the intake airduct of the main chamber. Mixing with intake
air occurred within the airduct and also within the chamber itself by the
action of large circulating fans. An exhaust blower removed air from the
chamber at a constant rate and conducted it to the outside of the building.
A second exposure system similar to the one described above and with an
identical manganese oxide generating system, but with smaller exposure
chambers, was utilized to expose monkeys to a concentration of about 5,000 ug
of manganese per cubic meter of air.
Aiz> Sampling
Air samples obtained from the chamber were collected on a daily routine
basis to monitor the concentration of manganese in the exposure chamber.
The sampling system consisted of a vacuum pump which removed air from the
chamber through a particulate filter. A limiting orifice located between
the pump and the filter provided control of the airflow rate. The particulate
manganese was trapped on a Gelman Type A, 47 mm, glass-fiber filter. The air
sampling system ran continuously during periods of exposure. Airflow through
the sampling system was frequently calibrated using a spirometer as the
standard. Manganese entrapped by the glass-fiber filter was analyzed by
atomic absorption spectrophotometry. (See below for details of manganese
analytical procedures.)
Prior to exposure of animals to the airborne particulate manganese,
several studies were conducted to establish the operating parameters of the
exposure chamber. These are briefly discussed below:
a. It was thought that a sampling port located in the exhaust
duct of the chamber would provide a suitably representative air
sample, and to substantiate this, a comparison was made between
air samples collected at the duct and similar samples collected by
a sampler located within the chamber itself.
b. In order to determine the speed with which the exposure
chamber would respond to the daily routine of shutdown and startup,
a series of short-term samples were collected during these operations.
c. Size determinations of the particulate manganese in the
exposure chamber were made with a modified Andersen Cascade Impactor
(2).
d. Measurements of carbon monoxide and carbon dioxide were
made periodically in the exposure chamber.
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The exposure chamber was routinely operated with the burner on for
23 hours a day, seven days a week. During the one-hour period each day in
which the burner was off, all necessary animal care and chamber maintenance
was accomplished. Routine air samples consisted of samples collected during
the 23 hour period in which the chamber was operating.
Determination of Mangan&se
Determinations of the manganese content of environmental and biological
samples were made using an atomic absorption spectrophotometer (AAS). In all
cases the instrument used was a Perkin-Elmer model 303 using conventional
air-acetylene flame techniques. The wavelength chosen for analysis was
279.5 tun. All glassware used for determinations of manganese was washed and
rinsed thoroughly, and then further soaked in 10% HNOa and finally rinsed with
deionized water. In most cases samples were either ashed dry or digested
in acids, a suitable dilution prepared, the solutions aspirated directly into
the AAS and compared with aqueous standards. Drinking water samples required
preliminary chelation and extraction to prepare solutions of sufficient con-
centration. Details of the various procedures are outlined below.
Air Samples—
Samples collected on fiber glass filters were placed in 50 ml plastic
centrifuge tubes and 25 ml of 10% (v/v) HC1 added. The tubes were then
placed in a boiling water bath for 10 minutes, cooled, and enough deionized
water added to restore the volume to 25 ml. Following centrifugation the
samples were ready for aspiration into the AAS.
Drinking Water—
A 50 ml volume of water collected from the automatic watering system
of the exposure chamber was placed in 100 ml glass bottles fitted with a
cap which utilized a plastic liner. To this was added 20 ml of 0.4 M TRIS
buffer, pH 8.0, and the solution mixed well. To this was added 10 ml of a
0.4M freshly prepared solution of ammonium pyrrolidine dithiocarbamate and
the mixture again shaken. Finally 10 ml of water-saturated methyl isobutyl
ketone was added, the mixture shaken for one minute, the layers allowed to
separate and the organic phase transferred to a clean glass tube. The
organic phase was then aspirated into the AAS and compared with standards
similarly chelated and extracted.
Animal Diets—
Purina Monkey Chow biscuits were ground to a fine powder and 1.0 gram
accurately weighed into a porcelain crucible. The sample was then ashed
overnight at 550°C. Four drops of concentrated HNOs and 10 drops of con-
centrated HCl was then added to the cooled crucible which was then heated
on a hot plate. A minimal volume of deionized water was used to transfer
the acid digest to a 25 ml volumetric flask. The acid/water transferring
procedure was repeated two more times, the flasks brought to volume with
deionized water, mixed well, and the samples aspiration into the AAS.
Urine Samples--
Urine was collected from each animal for 24 hours, and the volume
determined. To each sample, 10 ml of glacial acetic acid was then added,
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and the samples stored in plastic under refrigeration until ready for
analysis. Approximately 5 ml aliquots were filtered into a clean glass
tube, aspirated into the AAS and compared with aqueous standards.
Fecal Samples—
Feces were collected from each animal for 24 hours, weighed and dried,
and the dry weight determined. A 0.25g portion was weighed into a porcelain
crucible, ashed overnight at 550°C and then transferred with acid and dionized
water as for the diet samples (see above).
Tissue Samples—
Accurately weighed portions of tissue in the range of 2 to 4 grams
obtained at autopsy were placed in porcelain crucibles. The samples were
dried at 100°C for 3 hours, and then ashed at 550°C overnight. The residue
was transferred 2 or 3 times using 2 drops of concentrated HC1, 5 drops of
concentrated HNOa and suffienct deionized water for a final volume of 2 ml
or 10 ml depending on the tissue. The aqueous solutions were then aspirated
into the AAS.
Animal Studies
The animal exposures were initiated with a study of eight rhesus monkeys
(four male and four female Macaco, mulatta) with three additional animals of
each sex serving as nonexposed controls. The animals were allowed free access
to food and water throughout the experiment. Body weights of the animals were
determined at monthly intervals. Each animal was also observed daily for
signs of toxicity or for any other signs of exposure to airborne manganese.
Periodically, a series of clinical chemistry and hematology studies were
performed on the animals. Hematology studies included total erythrocyte and
leucocyte counts, packed cell volume and hemoglobin concentration. Serum
chemistry studies included determination of sodium, potassium, chloride,
calcium, creatinine, urea nitrogen, phosporous, bilirubin, glutamic-oxaloacetic
transaminase, glutamic-pyruvic transaminase, lactate dehydrogenase, and total
cholesterol.
Definitive manganese balance studies were not attempted, but the diet
and excreta were monitored on a routine basis for their manganese content.
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SECTION 4
RESULTS
EXPOSURE CONDITIONS
Results of the study comparing air sampling at the exhaust duct and
within the chamber itself are shown in Table 1. Although the samples collected
at the duct were slightly lower than those collected within the chamber, the
differences were very small and within the limits of experimental error.
This established the validity of using the exhaust-duct sampling port for
routine measurements. A similar series of tests showed that the concentration
determined by a single 24-hour sample was very close to the average of several
one-hour samples collected during the same 24-hour period. This established
the reliability of the 24-hour sample which also had the advantage of yielding
integrated results over the entire daily operating period.
Buildup of the proper concentration of manganese oxide in the chamber
during the daily routine of startup is shown in Figure 3. The results of
this study demonstrated that within less than 30 minutes after the burner was
ignited the proper manganese concentration was reached. Within a similar
period the chamber contents were exhausted of manganese after the burner was
turned off. This is also shown in Figure 3.
The results of our studies to determine the size of the generated manga-
nese oxide particulates using the modified Andersen Cascade Impactor are
shown in Table 2. In this table, the results of these studies are compared
with the lead particulate measured during our previous human studies (3) and
with lead particulate collected in ambient air near a roadway. These data
demonstrate the effectiveness of the 55-gallon-drum holding chambers in
increasing the size of the generated particulates.
Periodic measurements of carbon dioxide and carbon monoxide in the
exposure chambers established that the levels of these materials were well
within acceptable ranges. The concentration of carbon dioxide never exceeded
0.1% and the concentration of carbon monoxide was estimated to be about one
part per million.
The weekly means of concentrations of manganese in the air of the
exposure chamber is shown in Table 3. Each mean shown is the average of the
seven daily determinations. Also shown in the table is the overall mean for
52 weeks and, since some animals were exposed for an additional 14 weeks, this
66 week mean is also shown. These data demonstrate that the intended concen-
tration of 100 Wg of manganese per cubic meter.if air was very nearly achieved
although there were some weekly fluctuations.
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Mn Concentration — Micrograms per Cubic Meter of Air
8 8
Ul
o
Figure 3. Concentration changes during startup and shutdown.
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The diet and drinking water of the animals also contributed, to some
extent, to the exposure of manganese. The average concentration of manganese
in the Purina Monkey Chow was 47 yg per gram of diet. The concentration of
manganese in drinking water determined at monthly intervals is shown in
Table 4. During the study, the weekly average concentrations of manganese
varied only from 0.017 yg to 0.033 yg per ml of water.
Clinical Evaluations
Throughout the investigation, all of the animals were observed on a
daily basis for any signs of toxicity. At all times during the study the
animals were normal in appearance and did not display any behavior or any
other visual manifestations of toxicity attributable to exposure to manga-
nese at 100 yg per cubic meter of air. Growth was monitored by periodic
determinations of body weights, and these data are shown in Table 5. Body
weights and body weight gains were normal and did not show any effect of
exposure to manganese.
Blood samples were obtained on a periodic basis for clinical biochemistry
studies. Parameters measured were selected to show any changes in electrolyte
balance, kidney status, liver status, and general matabolic status. Profiles
for each animal are shown in Table 6 through 19. There was no evidence in
any of the animals exposed to 100 yg of manganese per cubic meter of air
which would indicate an alteration of status in any of the parameters chosen
for study.
In a similar fashion, hematology profiles of each animal were determined
periodically throughout the study. These data are shown in Tables 20 through
33. Again, there was no evidence of an effect on the parameters chosen for
investigation.
Escaretion of Manganese
At intervals during the investigation, the excretion of manganese in
the urine and feces of the animals exposed to airborne manganese was evaluated.
In order to obtain suitable samples, it was necessary to remove the animals
from the exposure chamber during a 24-hour period for collection of the
samples. This was necessary to avoid contamination of the urine and feces
samples collected in metabolism cages by the airborne manganese present in
the exposure chamber. The entire volume of urine and weight of feces samples
collected from each animal was recorded during these 24-hour periods.
Urinary excretion of manganese by the monkeys is shown in Table 34.
These data express excretion as the total amount of managanese excreted in
a 24-hour period. It is noteworthy that in all cases the amount of manga-
nese excreted was very small regardless of the condition or period of
exposure. The amount of manganese excreted under any of these conditions
was always less than 100 yg in the 24-hour period. Considerable variations
were also observed in the individual rates of excretion and also in the
amount of manganese excreted by any one animal at different times, regardless
of the condition of exposure. For example, in the control groups the amount
of manganese excreted ranged from less than 1 yg to as high as 41 yg for one
11
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particular animal. Similarly, in the exposed group the elimination of manga-
nese ranged from 1 to 77 yg excreted in a 24-hour period. Although it is
difficult to make generalizations from the data, there was a trend to greater
excretion of manganese during exposure than either during preexposure or among
control animals. This was not universally observed, however, and in at least
one instance the rate of excretion was greater during the baseline period
than after 8 or 12 months of exposure. The significant decrease in the rate
of excretion of manganese among the control animals after the 12th month of
the experiment is not understood. The analyses were performed by the same
technical staff using the same procedures and equipment utilized throughout
the study.
The excretion of manganese in the feces of the animals is shown in
Table 35. Based on the amount of manganese excreted in a 24-hour period, the
rate of excretion of the metal in the feces is in the range of three orders of
magnitude greater than in the urine samples obtained at the same time. Note
that the data in Table 35 are expressed as milligrams excreted in a 24-hour
period. Here again, however, there are variations in the excretion rate of
manganese from individual to individual and within the same individual from
time to time regardless of condition of exposure or duration of exposure to
manganese. Thus, at any one time, for example the four-month period of
exposure, the rate of excretion of manganese is greater in the control group
than in the exposed group. In fact, examination of the data throughout the
study indicated a somewhat greater level of excretion of manganese by the
control animals than by the exposed animals.
Studies by other investigators (4, 5, 6) have shown that excretion of
manganese is principally through the gastrointestinal tract. This is substan-
tiated in the present study in which the highest concentration of the metal
is found in the feces. It is not possible to speculate on the disposition of
the small amount of manganese to which the animals were exposed in the chamber
experiment. If it is accepted that the respiratory minute volume for rhesus
monkeys is about 0.86 liters per minute (7), then one can compute that the
animals were exposed to a little more than 100 yg manganese in a 23-hour
period in the chamber containing about 100 yg of manganese per cubic meter
of air. Even if one assumes 100% absorption of the manganese by the lungs,
which is unlikely, then it can be seen that the amount of manganese to which
the animals were exposed in the air is only a very small fraction of a total
amount of manganese excreted by the animals in a 24-hour period. These data
indicate that, even among the animals in the exposure chamber, the greatest
source of exposure to manganese was not the air but rather some other source.
Examination of the diet indicates this as that source. If the dietary con-
centration is on the order of 47, yg of manganese per gram of solid diet as
indicated by our studies, then a animal consuming approximately 100 g of the
dry diet would consume between 4 and 5 mg of manganese in a 24-hour period.
This is consistent with the rate of excretion of manganese that we observed
in this experiment.
12
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Accumulation of Manganese
Although exposure to airborne manganese could account for only a small
fraction of the total intake of manganese in a 24-hour period, this is not to
say that distribution of metal within tissues of the animals was not altered.
At autopsy a number of tissues obtained from the animals were examined for
their manganese content. The control animals for this phase of the study,
although not the same as those used in other phases of the study, were, none-
theless, in our colony for the same period of time as the animals in the main
manganese study, and were housed and otherwise maintained under identical
conditions to the controls used in the excretion studies and clinical investi-
gations. They were, in fact, housed in the same room in our animal quarters.
The concentration of manganese found in the animals following different con-
ditions of exposure to the metal in the air is shown on Tables 36 and 37.
Concentrations in the lung, liver, pancreas, spleen, kidney, bone, adrenal
gland, heart muscle, and skeletal muscle are shown in Table 36. Where suffi-
cient numbers were available, as was the case for those animals sacrificed
after 12 months of exposure, it was possible to compare the means using the
Students t-test. This indicated that levels of manganese were slightly but
significantly increased in the lungs, liver, pancreas, kidney, and heart
muscle. Among those tissues showing a significant increase, the degree of
increase was by a factor of two or less, with the exception of the lung in
which the concentration of manganese was on the order of five times the level
found in the nonexposed animals. The number of animals sacrificed at periods
other than 12 months was not sufficient to permit similar statistical com-
parisons. However, the trends reflected those found in the animals exposed
for 12 months.
It should be noted here that a considerable effort was made to monitor
levels of manganese in blood samples obtained from the monkeys. Although we
were not able to develop a procedure capable of yielding consistantly accept-
able data, we were able to determine that manganese levels in blood samples of
the rhesus monkeys was of the order of about 0.06 to 0.10 ug/g when measured
in either whole blood or serum. Comparable levels were encountered in both
control and exposed animals.
We were also able to measure the concentration of manganese in selected
tissues from the nervous system. Manganese was measured in the pallium,
basal ganglia, cerebellum, pons, and medulla. Some increases in manganese
content was also observed here among the animals exposed to airborne manga-
nese, and we were able to utilize the same statistical treatment on the
animals exposed for 12 months. The manganese concentration was significantly
greater in the pallium, basal ganglia, cerebellum, and pons. The degree of
difference was again about two-fold with the exception of the pons which had
four times the amount of manganese in the exposed animals compared with the
control animals.
The concentrations of manganese observed in the rhesus monkeys in the
present study are remarkably similar to concentrations of manganese found in
similar tissues' from other animal species (8). A notable exception to this
is the level which we found in adrenal tissue. In the monkey, even among
the control animals, the amount of the metal in the adrenal seems to be about
10 times that of the amount found in man, and about twice the amount found in
13
-------
rabbits. Although-manganese was significantly increased in some tissues of
the exposed animals, the degree of this increase was not extremely large nor
was the biological significance readily apparent.
Morphological and Histopathologioal Studies
Sacrificed animals were subjected to a thorough gross and microscopic
examination. With one exception all of the changes observed constituted
nonspecific normal pathological observations of rhesus monkeys in our colony.
The only exception to this was the observation of monocytic and lymphocytic
infiltration of the myocardium. The degree in each case was only minimal
and the change was observed in only three of the exposed monkeys which were
autopsied. Although the changes were found only in animals from the exposure
chamber, there was insufficient evidence to implicate manganese as the causa-
tive agent.
The complete pathology report is included as an Appendix.
Exposure of Rats to Airborne Manganese
Following our observation of some increase in certain tissue levels of
manganese in the animal exposed for 6 months, we conducted a short experiment
with rats to substantiate this observation. We also hoped to obtain addi-
tional data on the dynamics of excretion of manganese. Two groups of animals
were studied. In the first group, the excretion of manganese in the urine
and feces of four rats was examined. These animals were then placed in the
chamber, and subsequently the excretion was measured after one, four, and
eight weeks of exposure. When eight weeks of exposure was completed, the
animals were then removed from the chamber and the excretion of manganese
in the urine and feces again was measured after one, four, and eight weeks
following the exposure period. The second group of 14 animals was used to
study the concentration of manganese in tissues. The concentration of manga-
nese in liver, kidney, lungs and whole brain was measured in two animals
prior to the exposure period. Animals were then placed in the chamber and
pairs of animals killed after one, four and eight weeks of exposure. The
remaining animals were then removed from the exposure chamber and pairs
killed after one, four, and eight weeks postexposure.
The excretion of manganese in the urine of the rats is shown in Table 38.
In this table the data are expressed both as Ug of manganese per milliliter
of urine and as yg of manganese excreted in a 24-hour period. The only
generalization that can be made from these data is that the excretion of
manganese apparently increased after the animals were placed in the chamber
and that this excretion at an elevated rate appeared to continue even after
the animals were removed from the chamber. A similar increase in the rate
of excretion of manganese following exposure was demonstrated in the fecal
data shown in Table 39. Again the increased rate of excretion apparently
continued even eight weeks after the animals were removed from the exposure
chamber.
14
-------
The concentration of manganese found in the tissues of rats exposed to
airborne manganese is shown in Table 40. There were definite increases
observed in the lung and brain of the exposed animals, but the liver and
kidney data were inconclusive. In both the lung and the brain there was evi-
dence of a rapid return to normal levels within one week after the animals
were moved from the chamber.
Because of the low level of exposure, the data obtained with these
studies in rats were insufficient to permit conclusions regarding the turnover
rate of manganese in the body. The results obtained were not inconsistent
with published results using radioactive manganese (9). These have shown
disappearance of manganese from the body with two exponential components, one
with half-time of 39 days and one with half-time of four days.
High-Level Exposure
Neurologic manifestations of manganese toxicity have been described in
rhesus monkeys exposed to very high levels of the metal (10). Epidemiologic
studies in humans also exposed to very high levels of manganese dust suggests
a neurologic disorder with a Parkinson-like syndrome (11). The threshold
limit value for an eight-hour exposure has been established at five mg per
cubic meter of air. We conducted a study with two rhesus monkeys exposed to
manganese at the threshold limit value except that the animals were exposed
for 23-hours per day rather than eight hours. It was the intent of this study
to determine whether exposure at this level would also produce any visible
neurologic disorders in rhesus monkeys.
The system for generating the high level of manganese was identical to
that previously described for the study at 100 yg per cubic meter of air.
The principal difference in the two systems was that, in the case of the high
level of exposure, the chambers were fed directly from the generator without
the addition of outside air. Each of the two chambers housed one rhesus
monkey and daily measurements were made to establish the concentration of
manganese within each chamber. These data are shown in Table 41. The mean
concentration of manganese over the 23-week exposure period was for 4.86 mg
per cubic meter of air in one chamber and 5.10 mg per cubic meter of air in
the second chamber. The animals were observed daily for any signs of toxicity,
and during the exposure phase of the study, periodic clinical chemistry and
hematology samples were obtained. Following the exposure period of the study
the animals were not sacrificed but were removed from the chamber and observed
for an additional 10 months.
Clinical chemistry data for the two animals are shown in Tables 42 and
43. The only changes observed were small, inconsistent increases in the
level in inorganic phosphate in the sera. Significantly, there were no
increases in either of the transaminases or LDH which might have signified
tissue damage. Hematology data shown in Tables 44 and 45 do not show any
effects attributable to the high level of manganese.
Excretion of 'manganese by the two monkeys is shown in Table 46. These
data demonstrated significant increases in the excretion of the metal both
by the urinary and by the gastrointestinal route. Of particular interest is
15
-------
the very significant increase in urinary excretion signifying the extent to
which the kidney was involved in removal of manganese from the body following
a very high level of exposure. During exposure, the excretion rates were
increased by one or two orders of magnitude over excretion rates during the
preexposure period.
No evidence of neurological disorders was observed in the animals during
exposure to the high level of airborne manganese. Neither did any such
symptoms become evident during the 10-month postexposure period.
16
-------
SECTION 5
DISCUSSION
The goal of these investigations was to provide safety evaluation data
on airborne manganese particulates which might be encountered in ambient air
resulting from the use of MMT as a combustion improver in automotive fuels.
In achieving this goal, monkeys were chronically exposed to the combustion
products of MMT at a level of about 100 lag per cubic meter of air in an
environmentally controlled exposure chamber. The level of exposure was far
in excess of the current levels of lead measured in the atmosphere over cities
in the United States. Although the manganese particulates generated by the
exposure system were not expected to be identical to manganese particulates
counted in ambient air (as a result of the use of MMT in motor fuels), the
size of particles was somewhat similar to the size of particulate lead found
in the ambient air. Efforts were made to match the size of lead particulates
found in the air but the size of the manganese particulates was somewhat
smaller. The observed differences in size were small, however, and the labo-
ratory system can be considered to be a suitable model for ambient conditions.
Similar statements probably could not be made with regard to the com-
position of the manganese oxide particulates from automobile exhaust systems.
We considered that our exposure systems generated a relatively pure form of
manganese oxide. The manganese oxide found in automotive exhaust fumes would
likely be associated with other particulate matter formed in the exhaust fumes
and with other components of the atmosphere as well. These differences
could be expected to have an effect on the amount of manganese absorbed from
the particulates by the lungs of the animals.
Clinical data and visual observations of the animals showed that they
tolerated the exposure well. In fact, these data and observations did not
divulge any effects which could be attributed to manganese. This was not
unexpected since the additional body burden of manganese imposed by inhala-
tion of the particulates did not contribute significantly to the total of
manganese taken in daily by the animals. It was necessary to seek out subtle
changes which reflected respiratory exposure to the airborne manganese
although not necessarily indicating a toxic effect on the animals.
Small but statistically signficant differences were found in the con-
centration of manganese in selected tissues of the exposed animals. The
largest increase, as would be. expected, was found in the lungs of the animals.
Lesser increases were found in the liver,, pancreas, kidney, heart muscle and
certain tissues of the nervous system. Increases in manganese content were
not correlated with any functional or morphologic effects on tissues or
organs.
17
-------
The only large changes in the rate of the manganese excretion observed
in these studies were those encountered in the animals exposed to 5 mg of
manganese per cubic meter of air. This is not surprising since the routes
and hence the daily intake of manganese by these animals was largely increased
over normal intake per day. Airborne exposure of as much as 5 mg of manganese
per day produced levels of manganese in the chamber which were extremely high,
it is not unlikely that additional manganese intake occurred through the ali-
mentary tract from manganese particulates settling on food, on the animals'
hair, and on other surfaces in the chamber. Thus, even grooming behavior
could increase the manganese intake by these animals. All of the excretion
data indicated that the largest fraction of manganese was excreted in the
feces. It is significant, however, that the excretion rate of manganese in
the urine of animals exposed 5 mg/m3 of airborne manganese was very largely
increased. This points out the significance of the role played by the kidneys
in eliminating manganese from the body during periods of high level exposure.
18
-------
SECTION 6
CONCLUSIONS
Monkeys Exposed to 100 vg of Manganese per Cubic Meter of Air
(1) Chronic exposure to airborne manganese does not induce any change
in appearance or behavior.
(2) Chronic exposure is not reflected in any changes in clinical
chemistry profiles or hematology profiles.
(3) Airborne manganese at 100 yg cubic meter of air does not contribute
significantly to the total intake of manganese although excretion rates are
slightly increased.
(4) Small but significant increases of manganese in lungs, liver,
pancreas, kidney, heart muscle, pallium, basal ganglia, cerebellum and
pons result from exposure to 100 yg per cubic meter of air.
(5) No gross or microscopic changes were observed in any of the tissues
or organs which could be attributed to manganese.
Rats Exposed to 100 \ig of Manganese per Cubic Meter of Air
(1) Eight weeks of exposure causes small increases in the excretion
of manganese which persist even eight weeks following exposure of the animals.
(2) Concentrations of manganese in the lungs and brain increases
following exposure for eight weeks but decrease to normal levels within one
week following exposure.
Exposure of Monkeys to Airborne Manganese at 5 mg per Cubic Meter of Air
(1) No significant changes in chemistry profiles or hematology are
observed in animals exposed for 23 weeks.
(2) Excretion of manganese in the urine and feces is very greatly
increased.
(3) No neurological disorders occur in the animals during exposure nor
for at least 10 months following the expdsure period.
19
-------
TABLE 1. COMPARISON OF AIR SAMPLES OBTAINED INSIDE THE EXPOSURE
CHAMBER WITH SIMILAR SAMPLES OBTAINED SIMULTANEOUSLY
AT THE EXHAUST DUCT*
Inside chamber Exhaust chamber
Mean
98
104
104
104
98
98
"TOT"
99
102
99
98
93
94
~~98~
*Data are expressed as micrograms of manganese per cubic meter
of air (ygMn/m3 air).
20
-------
TABLE 2. ANDERSEN SAMPLE STUDIES
Stage
1
2
3
4
5
6
7
50%
Cutoff,
microns
4
2.6
1.5
0.9
0.4
0.18
<0.18
Lead
400 Ft
7.3
6.5
7.3
6.9
17.6
20.2
34.9
near road
20 Ft.
6.4
7.2
7.2
6.0
10.4
15.6
46.3
Lead,
human
chamber
1.6
1.5
1.9
2.8
7.5
13.9
70.8
Manganese,
animal
chamber*
1.1
1.5
2.8
4.3
9.0
23.0
58.3
* 55 gallon drum chamber
21
-------
TABLE 3. WEEKLY-MEAN CONCENTRATIONS OF Mn IN AIR OF EXPOSURE CHAMBER*
Week
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Cone
92
85
171
103
71
57
90
63
107
116
94
103
106
94
109
104
113
Week
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Cone
80
95
100
104
no
98
103
146
131
109
96
97
104
93
97
108
105
Week
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
\
Cone
86
102
111
116
97
100
90
no
103
120
95
94
111
125
102
117
100
126
103 ± 181
Week
53
54
55
56
57
58
59
60
61
62
63
64
65
66
Cone
123
105
105
110
82
76
99
68
84'
77
70
76
117
139
101 ± 192
*Each listed value is the mean of seven daily samples. Data are expressed
as ygMn/m3 air.
*Mean and standard deviation of weeks 1-52
2Mean and standard deviation of weeks 1-66
22
-------
TABLE 4. MONTHLY MEANS OF CONCENTRATION OF MANGANESE IN DRINKING WATER
Month
1
2
3
4
5
6
7
8
Concentration
0.028
0.033
0.029
0.030
0.026
0.024
0.031
0.029
Month
9
10
11
12
13
14
15
Concentration
0.031
0.033
0.021
0.020
0.023
0.018
0.017
*Values are means of weekly determinations and are expressed as yg/ml water.
23
-------
TABLE 5. BODY WEIGHTS OF MONKEYS DURING EXPOSURE TO MANGANESE EXPRESSED IN KILOGRAMS
NJ
Animal
number
CONTROL
1704M
1706M
1867M
1403F
1547F
1916F
EXPOSED
1549M
1869M
1870M
1871M
1917F
1919F
1920F
1922F
Baseline
weight
4.00
4.50
2.75
--
4.25
4.25
•m~- -^ '
•^.J*
4.25
3.00
2.75
3.75
4.75
4.25
4.50
3.75
1 mo
5.00
—
3.50
5.00
5.00
4.75
4.75
3.50
3.50
4.50
4.50
4.50
5.00
4.50
2 mo
4.50
4.50
3.25
4.75
4.50
4.50
5.25
4.00
3.50
4.75
5.00
4.75
5.25
4.50
3 mo 4 mo
5.00 5.00
5.00 5.75
3.50 4.00
5.25 5.75
4.75 5.25
4.75 4.50
— 6.00
— 4.50
— 4.00
-- 5.25
— 4.75
— 5.00
-- 5.50
-- 4.75
5 mo
6.35
5.80
4.30
6.30
6.40
4.60
6.50
4.30
4.10
5.50
6.00
5.20
5.50
--
6 mo
6.25
6.00
_i
6.00
6.35
--
_i
5.60
4.20
4.40
6.00
5.30
5.50
4.50
7 mo
5.30
6.30
5.70
5.80
4.10
4.40
4.40
5.70
5.10
5.20
5.30
4.50
8 mo
6.80
6.90
6.20
6.10
4.60
4.70
4.50
6.00
5.90
5.30
5.00
4.50
9 mo
7.00
7.50
6.10
6.20
4.50
4.90
4.80
6.10
5.90
5.50
5.10
4.50
10 mo
7.10
8.00
5.90
5.70
4.70
5.10
4.90
6.20
5.90
5.20
5.40
5.70
11 mo
7.50
8.30
5.50
6.40
4.50
5.30
5.20
6.20
5.80
5.70
5.50
4.70
12 mo
6.50
8.80
5.90
6.80
4.30
5.70
--
--
--
5. .60
6.00
--
Sacrificed after 6 months.
-------
TABLE 6. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1403 (FEMALE, CONTROL)
Is)
Na+ . K* Cl" Ca++ Creat. BUN Bill. SGOT S6PT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1) (mEq/1) (mg%) (mg%) (mg%) (U/ml) (U/ml) (Sigma U) (mg%) (mg/dl)
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
143
145
140
142
144
150
136
142
4.3
3.9
4.7
4.3
5.0
4.1
3.8
3.6
104
107
109
105
110
103
107
106
5.3
4.9
5.0
5.0
5.1
5.2
4.7
4.8
0.08
1.00
0.90
0.90
0.80
0.90
0.90
1.10
23
21
21
24
10
19
18
30
0.10
0.30
0.30
0.40
0.70
0.35
0.38
0.19
41.0
32.0
38.0
40.0
34.0
2?.0
36.0
31.0
34.0
22.0
21.0
13.0
14.0
19.0
11.0
12.0
1080
420
720
550
740
350
660
770
179
95
132
109
130
100
109
110
5.00
-
-
4.10
4.40
3.50
4.20
4.30
Note: There was a trace of hemolysis at one month only.
-------
TABLE 7. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1547 (FEMALE, CONTROL)
NJ
Na+ K+ Cl" Ca++ Great. BUN Bill. SGOT SGPT LDH Choi.
(mEq/1) (mEq/1) (mEq/1) (mEq/1) (mg%) (mg%) (mg.%) (U/ml) (U/ml) (Sigma U) (mgX)
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
137
131
_
143*
143
150
143
144
4.6
3.5
_
4.4
4.4
4.1
4.1
4.1
102
108
109
106
111
102
105
106
5.0
4.4
_
4.9
5.0
4.8
4.9
5.0
0.90
1.00
0.80
0.50
0.80
0.85
0.90
1.00
23
16
15
16
20
21
21
25
0.15
0.20
0.35
0.50
0.50
0.35
0.40
0.20
39.0
23.0
42.0
29.0
22.0
23.0
27.0
39.0
21.0
22.0
16.0
13.0
17.0
19.0
16.0
18.0
1490
400
870
530
590
460
535
1440
200
105
149
146
170
100
144
172
PO'3
(mg/dl )
4.10
-
_
4.70
4.90
5.00
5.30
6.00
Note: There was a trace of hemolysis at one month only.
-------
TABLE 8. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1704 (MALE, CONTROL)
to
-J
"*.' Na+ K+ Cl" Ca++ Great. BUN Bill. SGOT S6PT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1) (mEq/1 ) (mg%) (mg%) (mg%) (U/ml) (U/ml ) (Sigma U) (mg%) (mg/dl )
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
140
139
135
146
146
149
145
144
5.0
4.3
4.4
4.9
5.5
4.4
4.1
3.4
97
97
102
106
104
100
105
103
5.2
5.6
4.9
4.9
5.0
5.0
5.0
4.7
1.00
0.70
0.80
1.05
1.00
1.00
1.00
1.10
18
22
16
20
18
19
19
27
q.n.s.1
0.01
0.25
0.42
0.70
0.25
0.42
0.15
37.0
34.0
45.0
33.0
35.0
28.0
35.0
38.0
25.0
23.0
13.0
16.0
19.0
19.0
14.0
16.0
650
520
780
550
840
520
615
900
190
245
193
178
209
125
154
153
3.60
3.70
-
5.90
5.60
5.70
5.10
6.50
^.n.s. = quantity not sufficient.
Note: There was a trace of hemolysis at one month only.
-------
TABLE 9. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1706 (MALE, CONTROL)
ro
oo
Na+ K+ Cl" Ca++ Creat. BUN Bill. SGOT SGPT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1 ) (mEq/1) (mgX) (mgX) (mgX) (U/ml). (U/ml ) (Sigma U) (mg%) (mg/dl)
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
150
145
_*•-• .^*
146
146
151
143
145
4.5
4.6
5.3
4.5
4.3
4.4
3.7
97
102
106
111
102
105
104
5.7
5.4
5.9
5.5
5.4
4.9
4.9
1.20
0.70
1.00
0.85
0.85
0.90
1.10
15
15
16
14
18
15
23
0.01
0.01
0.90
0.50
0.42
0.63
0.25
31.0
36.0
45.0
26.0
26.0
21.0
32.0
25.0
31.0
21.0
14.0
13.0
13.0
14.0
610
420
1060
450
210
616
750
187
273
150
174
142
157
143
7.04
4.00
6.60
4.70
5.70
4.70
5.70
Note: There was no evidence of hemolysis.
-------
TABLE 10. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1867 (MALE, CONTROL)
NJ
VO
Na+ ~" K+ Cl" Ca** Great. BUN Bill. S60T SGPT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1) (mEq/1) (rag*) (mg%) (mg«j (U/ml) (U/ml) (Siqma U) (mg%) (mg/dl)
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
158
136
137
143
149
5.5
3.7
3.8
4.5
4.9
121
104
99
105
100
5.4
5.0
5.0
5.0
5.3
0.75
0.90
0.80
0.75
0.85
35
14
17
15
18
0.30
0.30
0.50
0.40
0.50
39.0
46.0
43.0
32.0
32.0
27.0
27.0
16.0
16.5
19.0
1100
690
610
530
540
209
202
168
204
150
4.60
-
5.50
5.70
6.00
Note: There was no evidence of hemolysis.
-------
TABLE 11. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1916 (FEMALE, CONTROL)
Na+ K+ Cl~ Ca"1"1" Creat. BUN Bill. SCOT S6PT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1) (mEq/1) (mg%) (mg%) (mg%) (U/ml) (U/ml ) (Sigma U) (mg%) (mg/dl )
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
166
151
136
140
142
143
142
145
6.8
4.5
4.4
4.0
4.8
4.5
3.6
3.8
102
99
104
103
106
96
100
105
6.3
5.2
5.4
4.8
5.0
5.2
5.0
4.7
1.10
1.00
0.08
0.85
0.70
0.90
0.80
0.90
16
15
16
16
19
23
29
23
q.n.s.1
0.05
0.35
0.38
0.45
0.55
0.55
0.20
41.0
28.0
37.0
28.0
28.0
33.0
25.0
40.0
41.0
39.0
29.0
29.0
33.0
46.0
21.0
32.0
1070
700
940
500
610
840
500
1240
191
155
206
124
174
125
135
143
3.30
-
-
-
3.45
2.40
2.20
3.70
^.n.s. = quantity not sufficient.
Note: There was a trace of hemolysis at one month only.
-------
TABLE 12. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1549 (MALE, EXPOSED)
Na+ K+ Cl" Ca++ Creat. BUN Bill. SGOT SGPT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1) (mEq/1 ) (mg%) (mg%) (mg%) (U/ml) (U/ml) (Sigma U) (mg%) (mg/dl)
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
148
146
140
144
147
150
4.4
4.2
4.0
4.7
5.3
4.5
97
103
108
104
107
99
5.4
5.5
5.1
5.3
5.3
5.4
1.10
0.90
0.90
0.95
0.80
0.95
15
19
12
14
15
18
0.35
0.14
0.20
0.45
0.52
0.42
33.0
41.0
34.0
35.0
26.0
30.0
25.0
39.0
18.0
16.0
16.0
19.0
330
400
580
570
420
520
169
200
167
183
191
167
4.35
3.10
-
6.00
4.80
5.90
Note: There was no evidence of hemolysis.
-------
TABLE 13. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1869 (MALE, EXPOSED)
CO
to
Na+ K+ Cl" Ca++ Great. BUN Bill. SGOT SGPT LDH Choi. PO^3 -
(mEq/1) (mEq/1) (mEq/1) (mEq/1 ) (mg%) (mg%) (mg%) (U/ml) (U/ml) (Sigma U) (mg%) (mg/dl)
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
158
151
141
149
149
150
147
152
5.8
5.6
4.0
4.9
4.1
4.4
4.5
4.4
111
105
105
106
107
99
106
109
5.4
5.3
4.9
5.1
5.1
5.2
5.0
5.2
0.80
1.10
0.80
0.96
1.00
1.05
1.10
1.40
30
15
19
18
21
23
21
26
q.n.s.1
0.05
0.25
0.55
0.45
0.40
0.42
0.20
44.0
38.0
38.0
36.0
65.0
32.0
35.0
32.0
41.0
34.0
38.0
21.0
40.0
28.0
21.0
21.0
700
1140
820
650
760
580
740
500
182
205
195
188
191
142
172
176
4.20
-
-
6.20
4.30
4.70
4.30
4.70
^.n.s. = quantity not sufficient.
Note: There was a trace of hemolysis at one and at two months.
-------
TABLE 14. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1870 (MALE, EXPOSED)
to
u>
Na+ K* Cl" Ca*"1" Great. BUN Bill. SGOT SGPT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1) (mEq/1) (mg%) (mgX) (mg55) (U/ml) (U/ml) (Sigma U) (mg%) (mg/dl)
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
148
142
139
135
144
148
137
150
5.4
5.3
3.7
3.7
5.2
4.6
4.3
5.0
101
98
100
103
102
95
103
105
5.7
5.3
5.2
4.9
5.4
5.5
5.0
5.3
0.70
0.80
0.70
0.85
0.80
0.90
1.00
1.20
12
15
15
16
16
16
16
22
q.n.s.1
0.20
0.35
0.55
0.60
0.65
0.55
0.35
68.0
42.0
35.0
33.0
30.0
47.0
50.0
34.0
83.0
33.0
10.0
37.0
40.0
22.0
11.0
12.0
1230
990
890
500
570
1170
1220
590
153
155
175
170
191
145
149
152
3.60
-
-
5.40
4.70
5.70
4.70
4.80
xq.n.s. = quantity not sufficient.
Note: There was a trace of hemolysis at one month only.
-------
TABLE 15. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1871 (MALE, EXPOSED)
CO
Na+ K+ Cl" Ca++ Great. BUN Bili. SGOT S6PT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1) (mEq/1) (mg%) (mg%) (mg%) (U/ml) (U/ml) (Sigma U) (mg%) (mg/dl )
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
172
142
140
142
147
144
141
146
7.0
4.6
4.3
4.6
5.4
4.8
4.3
4.6
108
99
101
104
108
98
102
103
6.3
5.1
5.1
5.1
5.3
5.6
5.0
5.4
0.90
1.10
0.90
0.95
0.80
1.10
1.10
1.40
10
12
13
13
14
16
17
20
0.01
0.20
0.30
0.45
0.40
0.42
0.40
0.20
72.0
43.0
51.0
34.0
29.0
22.0
72.0
44.0
27.0
26.0
13.0
16.0
16.0
40.0
18.0
1840
900
1280
500
410
1000
1360
1120
167
136
193
190
209
150
168
143
2.80
-
-
5.50
4.70
5.30
5.60
5.60
Note: There was no evidence of hemolysis.
-------
TABLE 16. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1917 (FEMALE, EXPOSED)
10
Ul
Na+ K+ Cl" Ca++ Great. BUN Bill. SCOT SGPT LDH Choi. PO^3
(mEq/D (mEq/1) (rnEq/1 ) (mEq/1) (mg%) (mg%) (mg%) (U/ml) (U/ml ) (Sigma U) (mg%) (mg/dl)
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
150
144
137
142
142
143
142
149
6.6
4.2
4.3
4.5
5.2
5.1
4.7
4.2
107
97
104
108
101
90
107
107
5.7
5.3
5.1
4.9
5.2
5.4
4.8
4.9
0.95
0.80
0.80
0.70
0.90
0.80
0.80
1.10
10
11
13
13
21
16
16
27
0.10
0.05
0.40
0.40
0.55
0.50
0.50
0.18
54.0
40.0
32.0
31.0
25.0
39.0
45.0
40.0
25.0
23.0
8.0
19.0
16.0
22.0
18.0
18.0
900
870
935
560
700
1040
1220
1290
153
145
129
130
200
142
135
125
3.30
-
_
3.70
4.20
5.40
5.00
4.40
Note: There was no evidence of hemolysis.
-------
TABLE 17. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1919 (FEMALE, EXPOSED)
U)
Na+ K+ Cl" Ca++ Great. BUN Bili. S60T SfiPT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1) (mEq/1) (mg%) (mgX) (mgX) (U/ml ). (U/ml) (Sigma U) (mgX) (mg/dl )
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
158
140
136
147
142
148
142
148
5.4
4.2
4.5
5.0
7.0
4.8
3.8
3.7
103
103
102
104
107
99
105
106
6.3
4.9
4.9
5.2
5.3
5.7
5,0
5.0
1.00
1.10
1.00
0.98
0.65
1.20
1.30
1.40
13
15
20
20
14
23
20
28
0.01
0.02
0.35
0.40
0.75
0.75
0.40
0.20
36.0
27.0
35.0
27.0
30.0
26.0
41.0
35.0
33.0
23.0
18.0
19.0
16.0
24.0
29.0
19.0
695
470
980
500
820
620
1115
960
149
145
195
191
170
158
161
167
2.40
-
-
5.20
3.40
2.40
4.30
3.90
Note: There was a trace of hemolysis at one month and at two months.
-------
TABLE 18. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1920 (FEMALE, EXPOSED)
Na+ K+ Cl" Ca++ Creat. BUN Bill. S60T SGPT LDH Choi. PO^3
(iiiEq/1) (mEq/D (mEq/1) (mEq/1) (mg«) (mg«) (mg%) (U/tnl) (U/ml) (Sigma U) (mgX) (mg/dl)
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
180+
149
139
149
146
139
148
141
7.2
5.8
4.1
4.4
5.5
4.1
4.5
3.7
106
107
104
106
108
102
108
108
6.8
5.0
4.7
5.0
5.0
4.9
4.8
5.1
1.15
1.00
0.80
0.95
0.95
1.00
1.20
1.20
10
10
14
19
17
18
14
20
0.10
0.15
0.35
0.50
0.46
0.40
0.40
0.20
24.0
26.0
34.0
28.0
23.0
28.0
24.0
30.0
19.0
18.0
16.0
13.0
14.0
24.0
14.0
14.0
395
470
810
440
410
600
470
510
163
164
209
200
191
175
170
176
4.30
-
-
4.70
3.60
4.20
3.80
4.30
Note: There was a trace of hemolysis at one month and at two months.
-------
TABLE 19. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1922 (FEMALE, EXPOSED)
UJ
00
Na+ K+ Cl" Ca++ Great. BUN Bill. SGOT SGPT LDH Choi. P0~3
(mEq/1) (mEq/1) (mEq/1 ) (mEq/1 ) (mg%) (mg.«) (mgX) (U/ml) (U/ml) (Sigma U) (mg«) (mg/dl )
Baseline I
Baseline II
1 Month
2 Months
4 Months
6 Months
8 Months
12 Months
148
144
139
143
148
145
140
146
6.6
5.0
~ ~"^;-*
4.1
4.7
5.2
4.5
4.5
4.1
104
101
101
105
106
98
105
107
5.7
5.1
4.8
4.7
5.0
5.3
4.8
5.0
1.20
0.90
0.70
0.80
0.90
1.00
0.90
1.20
14
17
19
20
25
25
19
23
0.15
0.05
0.30
0.40
0.48
0.58
0.40
0.20
40.0
25.0
46.0
37.0
36.0
48.0
44.0
31.0
27.0
17.0
35.0
21.0
34.0
50.0
24.0
19.0
700
530
1150
670
640
1020
1220
360
200
164
184
182
200
192
170
157
3.80
-
-
3.60
3.60
4.80
2.90
3.50
Note: There was a trace of hemolysis at two months only.
-------
TABLE 20. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1403 (FEMALE, CONTROL)
Hgb PCV RBC WBC
(gm«) (vol %) (x TO6) (x 103)
Baseline I 12.2 36.0 4.78 10.9
Baseline II 12.0 36.0
1 Month 12.2 37.0 4.97 8.8
2 Months 13.0 37.0 4.91 9.4
4 Months 13.2 41.0 5.35 8.6
6 Months 12.2 36.0 4.77 6.8
8 Months 12.7 36.0 4.80 8.0
12 Months 12.7 36.0 4.84 6.9
39
-------
TABLE 21. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1547 (FEMALE, CONTROL)
Hgb PCV RBC WBC
(gmX) (vol %) (x 106) (x 103)
Baseline I 15.0 44.0 5.61 8.4
Baseline II 13.8 41.0 5.24 7.6
1 Month 14.4 43.0 5.38 14.2
2 Months 15.2 44.0 5.47 12.2
4 Months 14.4 43.0 5.66 12.8
6 Months 14.0 42.0 5.32 14.1
8 Months 14.4 42.0 5.53 14.2
12 Months 15.9 47.0 6.01 8.8
40
-------
TABLE 22. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1704 (MALE, CONTROL)
Hgb PCV RBC WBC
(gm%) (vol %) (x 106) (x 103)
Baseline I 15.2 44.0 5.77 8.4
Baseline II 14.0 42.0 4.91 7.7
1 Month 14.4 44.0 5.71 9.3
2 Months 16.0 48.0 6.18 9.6
4 Months 15.6 48.0 6.10 8.7
6 Months 15.2 46.0 6.02 5.4
8 Months 15.2 43.0 5.85 7.7
12 Months 13.4 40.0 5.29 9.2
41
-------
TABLE 23. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1706 (MALE, CONTROL)
Hgb PCV RBC WBC
(gm%) (vol %) (x 106) (x TO3)
Baseline I 16.0 45.0 5.89 13.3
Baseline II 15.0 44.0 5.09 11.7
1 Month 14.4 42.0 5.23 9.5
2 Months 14.0 42.0 5.60 10.7
4 Months 15.2 46.0 5.52 10.6
6 Months 15.0 43.0 5.31 8.3
8 Months 15.7 43.0 5.71 9.3
12 Months 15.4 45.0 5.54 12.1
42
-------
TABLE 24. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1867 (MALE, CONTROL)
Hgb PCV RBC WBC
(gm%) (vol %) (x 106) (x 103)
Baseline I 14.0 43.0 5.35 17.1
Baseline II
1 Month
2 Months
4 Months
6 Months
13.8
13.8
13.2
13.8
40.0
42.0
41.0
42.0
5.20
5.16
5.42
5.30
11.1
11.7
12.3
14.4
43
-------
TABLE 25. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1916 (FEMALE, CONTROL)
Hgb PCV RBC WBC
(gm%) (vol %) (x 106) (x TO3)
Baseline I 18.0 52.0 6.18 10.7
Baseline II 15.2 46.0 5.18 7.8
1 Month 16.0 48.0 6.35 8.0
2 Months 13.8 43.0 5.37 7.4
4 Months 15.0 45.0 5.94 6.0
6 Months 14.0 45.0 5.74 7.4
8 Months 14.6 42.0 5.49 8.4
12 Months 15.4 45.0 5.91 8.0
44
-------
TABLE 26. HEMATOL06Y PROFILE OF ANIMAL NUMBER 1549 (MALE, EXPOSED)
Hgb PCV RBC WBC
(gnrt) (vol %) (x 106) (x TO3)
Baseline I 15.2 44.0 5.95 7.5
Baseline II 13.2 40.0 " 5.12 7.3
1 Month 15.6 45.0 5.50 6.8
2 Months 15.0 45.0 5.60 7.1
4 Months 15.0 42.0 5.88 6.8
6 Months 14.4 45.5 5.24 6.5
45
-------
TABLE 27. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1869 (MALE, EXPOSED)
Hgb PCV RBC WBC
(gm%) (vol %) (x 106) (x 103)
Baseline I 15.0 44.0 5.19 21.3
Baseline II 13.2 44.0 4.87 11.8
1 Month 15.0 44.0 5.96 8.2
2 Months 14.4 43.0 5.32 11.2
4 Months 15.1 44.0 5.80 9.9
6 Months 15.0 46.0 5.89 9.1
8 Months 15.0 42.0 5.85 7.9
12 Months 15.4 45.0 5.89 6.5
46
-------
TABLE 28. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1870 (MALE, EXPOSED)
Hgb PCV RBC WBC
(gm%) (vol %) (x TO6) (x 103)
Baseline I 16.0 49.0 6.06 15.4
Baseline II 16.4 49.0 5.67 11.7
1 Month 16.0 46.0 5.91 8.2
2 Months 15.0 44.0 5.50 8.6
4 Months 16.0 48.0 5.89 7.6
6 Months 15.2 42.0 5.69 8.7
8 Months 16.6 48.0 6.20 8.1
12 Months 15.4 42.0 5.42 16.7
47
-------
TABLE 29. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1871 (MALE, EXPOSED)
Hgb PCV RBC WBC
(gm%) (vol 35) (x 106) (x 103)
Baseline I 15.6 46.0 5.69 14.1
Baseline II 16.0 45.0 5.57 9.1
1 Month 16.0 45.0 5.83 10.3
2 Months 15.0 45.0 5.80 8.3
4 Months 14.4 43.0 5.85 8.6
6 Months 15.6 46.0 6.02 11.4
8 Months 16.4 45.0 5.83 11.7
12 Months 14.5 43.0 5.65 12.5
48
-------
TABLE 30. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1917 (FEMALE, EXPOSED)
Hgb PCV RBC WBC
(gm%) (vol %) (x 106) (x 103)
Baseline I 16.4 49.0 5.96 8.6
Baseline II 15.6 45.0 5.37 6.9
1 Month 16.4 47.0 6.23 9.7
2 Months 16.0 48.0 5.96 6.1
4 Months 15.2 46.0 5.69 8.4
6 Months 18.4 58.0 7.03 9.2
8 Months 16.8 46.0 6.04 8.9
12 Months 16.4 47.0 6.10 9.1
49
-------
TABLE 31. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1919 (FEMALE, EXPOSED)
Hgb PCV RBC WBC
(gm%) (vol %) (x 106) (x 103)
Baseline I 16.0 49.0 5.96 14.7
Baseline II 15.2 43.0 5.08 9.4
1 Month 15.2 44.0 5.70 12.4
2 Months 15.2 45.0 5.61 9.3
4 Months 15.2 45.0 5.83 8.1
6 Months 15.0 46.0 6.09 10.5
8 Months 15.2 43.0 5.87 7.2
12 Months 15.9 45.0 5.81 8.5
50
-------
TABLE 32. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1920 (FEMALE, EXPOSED)
Hgb PCV RBC WBC
(gm%) (vol %) (x 106) (x 103)
Baseline I 14.4 42.0 4.84 7.0
Baseline II 15.0 44.0 5.81 5.9
1 Month 15.0 44.0 5.81 5.9
2 Months 15.2 44.0 5.40 7.2
4 Months 15.2 46.0 5.81 7.2
6 Months 15.2 45.5 5.32 6.4
8 Months 15.2 42.0 5.66 8.2
12 Months 15.4 46.0 5.87 7.2
51
-------
TABLE 33. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1922 (FEMALE, EXPOSED)
Hgb PCV RBC WBC
(gm«) (vol %) (x 106) (x 103)
Baseline I 18.0 54.0 6.54 18.7
Baseline II 15.0 43.0 5.01 9.0
1 Month 14.0 42.0 5.23 9.4
2 Months 15.0 45.0 5.48 9.8
4 Months 15.0 44.0 5.42 6.7
6 Months 15.0 42.0 5.38 6.7
8 Months 14.4 40.0 5.19 6.0
12 Months 14.8 43.0 5.50 10.3
52
-------
TABLE 34. CONCENTRATION OF MANGANESE IN URINE OF MONKEYS EXPOSED TO AIRBORNE
MANGANESE*
Animal
Number
CONTROL
1704M
1867M
1547F
1916F
Mean
EXPOSED
1549M
1869M
1870M
1871M
1917F
1919F
1920F
1922F
Mean
Baseline
1
12.2
8.0
15.2
7.9
10.8
7.6
4.0
4.1
9.4
16.7
6.7
12.6
5.0
8.3
2
14.6
3.8
32.6
4.7
13.9
8.5
2.4
7.7
8.9
15.8
4.8
5.3
1.0
6.8
1
9.1
5.5
30.9
3.2
12.2
27.6
17.0
15.8
21.4
70.0
39.3
30.6
12.8
29.3
Month
2
7.0
22.3
40.9
9.3
19.8
10.7
10.6
14.6
15.6
20.6
12.9
21.0
9.9
14.5
of exposures
4
13.7
15.8
11.2
3.5
11.0
39.3
28.9
18.0
68.4
52.2
29.0
45.6
33.2
39.3
8
7.2
i
16..1
2.7
8.7
i
3.0
2.0
29.6
4.5
8.4
14.3
1.5
9.0
12
< 1.0
-
< 1.0
< 1.0
< 1.0
-
10.2
5.1
68.0
3.7
12.1
14.9
77.0
27.3
*Data are expressed in ug Mn excreted per 24 hours
Sacrificed after 6 months
53
-------
TABLE 35. MANGANESE IN FECES OF MONKEYS EXPOSED TO AIRBORNE MANGANESE*
Animal
Number
CONTROL
1704M
1867M
1547F
1916F
Mean
EXPOSED
1549M
1869M
1870M
1871M
1917F
1919F
1920F
1922F
Mean
Baseline
1
1.87
2.52
2.32
2.51
2.31
3.39
1.03
1.86
1.94
0.89
4.55
2.82
1.22
2.21
2
2.91
1.92
2.90
2.65
2.59
-
3.48
3.96
-
0.68
2.89
5.06
0.44
2.75
1
4.20
2.15
1.59
4.31
3.06
1.20
1.66
0.38
2.61
3.43
3.53
2.61
1.08
2.06
Month of exposures
2
5.92
_2
5.22
10.52
7.22
1.57
0.91
0.32
1.05
32.48
1.58
1.96
1.42
5.16
4
4.93
1.66
4.47
4.66
3.93
1.08
0.33
1.29
0.69
1.34
2.35
1.91
1.61
1.32
8
4.83
_i
_2
9.38
7.10
_i
3.87
_2
8.03
3.07
2.18
1.12
1.92
3.36
12
7.29
5.72
2.88
5.30
0.71
0.93
2.41
2.29
2.47
2.16
1.58
1.79
*Data are expressed in milligrams of Mn excreted per 24 hours.
Animal sacrificed after 6 months.
2Sample lost.
54
-------
TABLE 36. MANGANESE IN SELECTED TISSUES OF MONKEYS EXPOSED TO AIRBORNE
MANGANESE*
Animal
Number
Control
1867
1479
1731
1765
1781
Mean
S.D.
Lung
0.42
0.14
0.12
0.10
0.09
0.17
±0.14
Liver
2.40
2.10
0.74
0.74
1.31
1.46
±0.77
Pancreas
1.51
1.56
1.39
1.40
1.18
1.41
±0.15
Spleen
0.39
0.41
0.21
0.18
0.16
0.27
±0.12
Ki dney
0.82
1.00
1.05
0.91
0.80
0.92
±0.11
Exposed 6 months
1549
Exposed 12
1870
1871
1917
1922
Mean
S.D.
Exposed 15
1869
1919
Mean
0.77
months
0.39
0.59
1.21
1.10
0.82**
±0.39
months
0.26
0.56
0.41
2.87
3.00
2.25
2.35
2.26
2.46§
±0.36
1.75
1.25
1.50
2.21
2.17
2.32
2.22
2.93
2.41**
±0.35
2.02
1.29
1.66
0.48
0.37
0.52
0.36
0.43
0.42
±0.07
0.77
0.41
0.59
1.82
2.00
2.00
1.93
1.70
1.91**
±0.14
1.16
1.08
1.12
* Data are expressed as up/g of wet tissue.
** Mean significantly different from control at 0.01 level of probability.
§ Mean significantly different from control at 0.05 level of probability.
(continued)
55
-------
TABLE 36. (continued)
Animal Bone
number (Femur)
Control
1867
1479
1731
1765
1781
0.12
0.60
0.62
0.82
0.68
Mean 0.57
S.D. ±0.26
Exposed 6 months
1549
Exposed 12 months
1870
1871
1917
1922
Mean
S.D.
Exposed 15 months
1869
1919
Mean
0.13
0.35
0.11
0.35
0.34
0.29
±0.12
0.39
0.47
0.43
Adrenal
2.00
1.78
1.02
2.16
1.20
1.63
±0.50
1.20
2.43
3.04
2.11
1.60
2.30
±0.60
4.43
, 3.37
3.90
Heart
muscle
0.48
0.39
0.27
0.24
0.35
±0.11
0.64
0.63
0.64
0.64
0.64**
±0.01
0.52
0.61
0.56
Skeletal
muscle
0.16
0.17
0.13
0.30
0.19
±0.07
0.24
0.47
0.54
0.28
0.38
±0.15
0.28
0.25
0.26
** Mean significantly different from control at 0.01 level of
probability.
56
-------
TABLE 37. MANGANESE IN TISSUES FROM NERVOUS SYSTEM OF MONKEYS EXPOSED TO
AIRBORNE MANGANESE*
Animal
number
Control
1867
1479
1731
1765
1781
Mean
S.D.
Pal
0.
0.
0.
0.
0.
0.
±0.
lium
50
61
41
37
45
47
09
Basal
ganglia
0.
0.
0.
0.
0.
0.
±0.
60
50
44
41
54
50
08
Cerebel 1 urn
0.
0.
0.
0.
0.
0.
±0.
77
44
55
50
57
57
12
Pons
0
0
0
0
0
±0
.31
.10
.10
.17 .
.17
.10
Medulla
0.
0.
0.
0.
1.
0.
±0.
79
39
13
17
43
58
54
Exposed 6 months
1549
Exposed 12
1870
1871
1917
1922
Mean
S.D.
Exposed 15
1869
1919
Mean
0.
months
0.
0.
0.
0.
0.
±0.
months
80
83
63
90
94
83**
14
0.37
0.44
0.
41
1.
1.
1.
1.
1.
1.
±0.
1.
0.
1.
00
15
08
11
67
25**
28
29
93
11
0.
0.
0.
1.
1.
1.
±0.
0.
1.
0.
96
94
96
29
04
06**
16
93
03
98
0
0
0
0
0
0
±0
1
0
0
.44
.81
.72
.89
.79
.80**
.07
.34
.52
.93
0.
0.
0.
0.
1.
96
80
53
89
92
1.04
±0.61
0.
0.
0.
86
68
77
* Data are expressed as a yg/g of wet tissue.
** Mean significaritly different from controls at 0.01 level of probability.
57
-------
TABLE 38. EXCRETION OF MANGANESE IN URINE OF RATS EXPOSED TO AIRBORNE
MANGANESE
Exposure
condition
Pre-
exposure
1 Week
4 Weeks
8 Weeks
1 Week
post-exp.
4 Weeks
post-exp.
8 Weeks
post-exp.
Animal
number
1
2
3
4
Mean ± S.D.
1
2
3
4
Mean ± S.D.
1
2
3
4 .
Mean ± S.D.
1
2
3
4
Mean ± S.D.
1
2
3
4
Mean ± S.D.
1
2
3
4
Mean ± S.D.
1
2
3
4
Mean ± S.D.
Concentration
yg/ml
0.04
0.09
0.11
0.06
U70T± 0.03
0.10
0.15
0.35
0.13
0.18 ± 0.11
0.09
0.09
0.15
0.07
0.10 ± 0.03
0.07
0.48
0.10
0.04
0.17 ± 0.20
0.10
0.04
0.37
0.09
0.15 ± 0.15
0.11
0.40
0.20
0.14
0.21 ± 0.13
0.08
0.08
0.12
0.15
O.T1 ± 0.03
24 Hour
excretion, yg
0.32
0.20
0.61
0.39
0^8 ± 0.
0.90
1.52
5.43
1.25
n7 ± 2.
0.90
1.26
2.68
0.98
1.45 ± 0.
0.98
0.96
1.00
0.44
0.84 ± 0.
2.10
1.04
5.90
0.90
TM ± 2.
2.64
7.20
1.60
2.52
3.49 ± 2.
0.67
0.57
0.85
0.83
0.73 ± 0.
17
12
83
27
34
52
13
58
-------
TABLE 39. EXCRETION OF MANGANESE IN FECES OF RATS EXPOSED TO
AIRBORNE MANGANESE
Exposure
condition
Pre-
exposure
1 Week
4 Weeks
8 Weeks
1 Week
post-exp.
4 Weeks
post-exp.
8 Weeks
post-exp.
Animal
number
1
2
3
4
Mean ± S.D.
1
2
3
4
Mean ± S.D.
v 1
2
3
4
Mean ± S.D.
1
2
3
4
Mean ± S.D.
1
2
3
4
Mean ± S.D.
1
2
3
4
Mean ± S.D.
1
2
3
4
"Mean ± S.D.
Concentration
mg/g
0.66
0.36
0.49
0.43
0.48 ± 0.13
0.65
0.54
0.75
0.70
0.66 ± 0.09
0.48
0.56
0.62
0.43
0.52 ± 0.08
0.60
0.80
0.61
0.61
0.65 ± 0.10
0.57
0.61
0.55
0.56
0.57 ± 0.03
0.37
0.33
0.35
0.35
0.35 ± 0.02
0.93
0.98
1.08
1.12
tT03"± 0.08
24 Hour
excretion, mg
1.06
0.48
0.94
0.18
0.66 ±
2.97
2.78
5.29
2.57
3~40 ±
2.45
3.10
1.82
1.88
OT±
2.99
5.60
3.06
3.06
3.67 ±
2.83
11.55
3.31
2.82
~5ToT±
1.13
0.91
0.28
0.85
077¥±
6.05
4.41
4.81
4.94
5.05 ±
0.41
1.26
0.60
1.28
4.37
0.36
0.70
59
-------
TABLE 40. MANGANESE IN TISSUES OF RATS EXPOSED TO AIRBORNE MANGANESE*
Exposure
condition
Pre-
exposure
1 Week
4 Weeks
8 Weeks
1 Week
post-exp.
4 Weeks
post-exp.
8 Weeks
post-exp.
Animal
sex
M
F
M
F
M
F
M
F
M
F
M
F
M
F
Liver
1.82
1.75
1.61
1.80
1.46
1.65
2.16
1.82
1.81
1.39
1.25
1.83
1.32
1.87
Kidney
0.63
0.82
0.79
1.53
0.73
0.67
1.06
0.71
0.82
0.62
0.65
0.83
0.71
0.64
Lung
0.36
0.27
0.58
0.96
0.73
0.89
0.97
0.97
0.30
0.21
0.31
0.48
0.47
0.30
Brain
0.54
0.59
0.67
0.85
0.68
0.83
0.97
0.83
0.50
0.44
0.74
0.93
0.60
0.64
* Data are expressed in yg Mn/g of wet tissue.
60
-------
TABLE 41. WEEKLY MEAN LEVELS OF MANGANESE IN AIR OF HIGH-LEVEL EXPOSURE
CHAMBERS*
Week
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Mean
Chamber A
2620
2900
1630
1380
2440
2100
2460
3860
4670
4300
5890
12940
9160
5580
5900
6900
7380
6450
3940
3170
3460
7290
5540
4860
Chamber B
3240
4630
3760
1480
1450
1830
2120
3840
3880
5840
7260
11550
11340
6860
5910
6820
6930
5610
3670
3280
3230
6460
6430
5100
*Data are expresed as yg Mn/m3 of air.
61
-------
TABLE 42. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1881*
NJ
Na+ K* Cl" Ca++ Great BUN Bill. SGOT SGPT LDH Choi. PO^3
(mEq/1) (mEq/1) (mEq/1) (mEq/1) (mgX) (mgX) (mgX) (U/ml ) (U/ml) (Sigma U) (mg%) (mg/dl )
Baseline I
Baseline II
1 Month
2 Months
3 Months
4 Months
5 Months
138
142
145
147
150
146
151
4.4
4.5
4.6
4.6
5.5
5.0
5.3
103
105
104
112
101
115
109
4.8
4.8
5.4
5.0
5.1
5.5
4.7
1.2
0.8
1.2
1.7
1.3
1.5
1.5
32
20
17
25
25
16
20
0.50
0.50
0.40
0.44
0.18
0.14
0.58
46
31
14
22
13
20
21
18
14
13
18
11
13
20
1300
520
188
184
125
560
211
200
177
178
150
150
200
171
6.9
4.5
8.2
8.6
-
7.4
2.9
Rhesus monkey (male, exposed)
-------
TABLE 43. CLINICAL CHEMISTRY PROFILE OF ANIMAL NUMBER 1884*
Co
Na+ K+ Cl" Ca++ Great BUN Bill. S60T SGPT LDH Choi. P0~3
(mEq/1) (mEq/1) (mEq/1) (mEq/1 ) (mg%) (mgX) (mg%) (U/ml) (U/ml) (Sigma U) (mg%) (mg/dl )
Baseline I
Baseline II
1 Month
2 Months
3 Months
4 Months
5 Months
143
144
147
148
144
146
149
3.8
4.5
4.4
4.4
4.3
4.2
4.8
.08
107
106
114
105
112
110
5.0
5.2
5.3
5.1
5.8
5.5
4.7
1.0
0.8
1.2
1.4
1.1
1.1
1.0
42
12
12
19
16
13
12
0.38
0.40
0.20
0.42
0.03
0.70
0.42
25
25
11
18
16
27
16
18
14
14
18
16
14
21
400
440
102
240
99
353
125
141
154
133
125
-
180
162
4.3
5.1
7.2
8.6
-
5.9
9.8
Rhesus monkey (male, exposed)
-------
TABLE 44. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1881*
Baseline I
Baseline II
1 Month
2 Months
3 Months
4 Months
5 Months
Hgb
(gn«0
13.6
12.7
15.4
14.0
-
18.4
15.9
PCV
(vol X)
39.0
35.0
46.0
42.0
-
42.0
48.0
RBC
(x 106)
4.29
4.96
5.91
5.49
-
5.43
5.96
WBC
(x 103)
5.9
8.4
5.7
4.0
-
9.4
8.7
* Rhesus monkey (male, exposed)
64
-------
TABLE 45. HEMATOLOGY PROFILE OF ANIMAL NUMBER 1884*
Baseline I
Baseline II
1 Month
2 Months
3 Months
4 Months
5 Months
Hgb
(gm%)
14.4
14.2
17.5
14.7
12.9
15.0
15.5
PCV
(vol %)
42.0
41.0
54.0
48.0
41.0
46.0
47.0
RBC
(x 106)
4.45
5.54
6.63
6.29
5.43
5.94
5.55
WBC
(x 103)
8.1
8.1
9.2
5.9
13.0
12.3
10.7
* Rhesus monkey (male, exposed)
65
-------
TABLE 46. EXCRETION OF MANGANESE BY MONKEYS EXPOSED TO AIRBORNE MANGANESE*
Baseline
1
2
Exposed
1 Month
2 Months
3 Months
4 Months
5 Months
Monkey
Urine
ygMn/24hr
17
40
246
249
198
109
310
#1881
Feces
yg/Mn/g
200
215
400
850
415
400
355
Monkey
Urine
ygMn/24hr
9
15
425
193
323
857
465
#1884
Feces
yg/Mn/g
275
285
465
585
555
450
510
* Exposure at about 5000 yg/m:
66
-------
REFERENCES
1. Griffin, T. B., F. Coulston, H. Wills and J. C. Russel. Biologic Effects
of Airborne Particulate Lead on Continuously Exposed Rats and Rhesus
Monkeys. Environ. Qual. and Saf., Suppl. 2: 202, 1975.
2. Hu, J. N. An Improved Impactor for Aerosol Studies - Modified Andersen
Sampler. Environ. Sci. Tech. 5^ 251, 1971.
3. Griffin, T. B., F. Coulston, H. Wills and J. C. Russell. Clinical
Studies on Men Continuously Exposed to Airborne Particulate Lead.
Environ. Qual. and Saf., Supp. JZ: 221, 1975.
4. Greenberg, D. M., D. H. Copp, and E. M. Cuthbertson. Studies in
Mineral Metabolism with the Aid of Artificial Radioactive Isotopes,
VII. The Distribution and Excretion, Particularly by Way of the
Bile, of Iron, Cobalt, and Manganese. J_. Biol. Chem. 147; 749, 1943.
5. Betinchamps, A. J., S. T. Miller, and G. C. Cotzias. Interdependence
of Routes Excreting Manganese. Am. J^. Physiol. 211: 217, 1966.
6. Papavasiliou, P. S., S. T. Miller and G. C. Cotzias. Role of Liver in
Regulating Distribution and Excretion of Manganese. Am. J^. Physiol.
211: 211, 1966.
7. Altman, P. L. and D. S. Dittmer (eds.) Biology Data Book, FASEB,
Washington, D.C. 1964, pp. 220.
8. Underwood, E. J. Trace Elements in Human and Animal Nutrition (3rd ed.)
Academic Press, New York, 1971, pp. 180.
9. Mahoney, J. P. and W. J. Small. Studies on Manganese III The Biological
Half-life of Radiomanganese in Man and Factors Which Affect the Half-life.
j;. Clin. Invest. 47.: 643, 1968.
10. National Research Council - National Academy of Sciences. Manganese.
A Report Prepared by the Committee on Biological Effects of Atmospheric
Pollutants of the Division of Medical Sciences, 1973, pp. 121.
11. Cotzias, G. C., P. S. Papavasilious, J. Ginor, A. Steck, and S. Duby.
Metabolic Modification of Parkinson's Disease and of Chronic Manganese
Poisoning. Ann. Rev. Med. 22: 305, 1971.
67
-------
PATHOLOGY APPENDIX
TO THE CHRONIC INHALATION STUDY
ON AIRBORNE PARTICULATE MANGANESE BY RHESUS MONKEYS
M.J. latropoulos
I.C.E.S. Chief Pathologist
68
-------
CONTENTS OF PATHOLOGY APPENDIX
Figures 70
Tables 71
A. Material and Methods 72
B. Results 72
Body and Organ Weights 72
Gross Observations 73
Microscopic Observations 74
C. Discussion 76
D. Conclusions 77
69
-------
FIGURES
Page
Figure A-l 78
Figure A-2 79
Figure A-3 80
Figure A-4 81
70
-------
TABLES
Page
Table A-l 82
Table A-2 83
Table A-3 84
71
-------
PATHOLOGY APPENDIX
A. MATERIAL AND METHODS
A total of twelve male and female rhesus monkeys were used in the
present study (Table A-l).
Complete necropsies were performed on all animals with special attention
given to the lips, tongue, oral cavity, external ears, eyes, limbs, vagina,
and anus. Each organ was examined in situ and after removal under low
power (lOx) magnification.
Representative samples from salivary glands, mammary tissue, liver,
stomach, duodenum, jejunum, colon, pancreas, adrenals, kidneys, urinary
bladder, mesenteric lymph nodes, esophagus, trachea, lungs, heart, thymus,
thyroids, parathyroids, uterus and tongue were fixed in 10% buffered
formalin. The eyes were fixed in Zenker's acetic acid solution, the
ovaries in Bouin's solution, and the central nervous system in 37 - 40%
buffered formalin. Calcified structures were decalcified in a mixture of
45% aqueous formic acid and a 20% aqueous solution of Na citrate 1/1 (v/v).
Regions of the central nervous system were sampled at the following
levels: The first frontal section was through the precentral gyrus, the
second through the Ammon's horn, and the third through the calcarine sulcus;
the first transverse section was through the mesencephalon, the second through
the pons and part of cerebellum, the third through the medulla and part of
the cerebellum and the fourth through the cervical spinal cord.
All sampled tissues were routinely dehydrated and embedded in paraffin.
Sections were cut at 6u and stained with hematoxylin (Harris's) and eosin
(Eosin Y). One kidney was also stained with periodic acid-Schiff technique
(PAS). Body weights and organ weights from liver, kidney and brain (including
cerebellum, pons and medulla) were recorded and the relative organ weights
calculated.
B. RESULTS
1. Body and Organ Weights
Final body weights, absolute and relative organ weights, obtained at
necropsy are listed in Table A-2.
72
-------
2. Gross Observations on Rhesus Monkeys
No. 1867, male (control) 6 months:
Mesentric lymph nodes (MLN) - Extensive enlargement, upon cutting, small
cystic structures became visible.
The rest of organs and systems - No other pathologic changes evident.
No. 1549, Male (100 yg/m3) 6 months:
Lungs - Mites were present bilaterally.
The rest of organs and systems - No other pathologic change was evident.
No. 1479, female (control) 12 months:
No pathologic change was evident in any of the organs and systems.
No. 1731, female (control) 12 months:
Lungs - changes consistent with pneumonia were present bilaterally.
Kidneys - Both were pale.
The rest of organs and systems - No pathologic change was evident.
No. 1765, male (control) 12 months:
Lungs - changes consistent with pneumonia were present bilaterally.
The rest of organs and systems - No other pathologic change was evident.
No. 1781, male (control) 12 months:
Spleen - Granular.
The rest of organs and systems - No other pathologic change was evident.
No. 1870, male (100 yg/m3) 12 months:
Lungs - Mites were present bilaterally.
The rest of organs and systems - No other pathologic change was evident.
No. 1871, male (100 yg/m3) 12 months:
Lungs - Mites were present bilaterally.
The rest of organs and systems - No other pathologic change was evident.
No. 1917, female (100 Ug/m3) 12 months:
Lungs - Mites were present bilaterally.
Abdominal cavity - The lower pole of the left kidney was adhered to the
lateral peritoneum where a parasitic abscess was visible.
The rest of- organs and systems - No,other pathologic change was evident.
73
-------
No. 1922, female (100 yg/m3) 12 months:
Lungs - Mites were present bilaterally.
The rest of organs and systems - No other pathologic change was evident.
No. 1869, male (100 yg/m3) 15.5 months:
Lungs - Mites were present bilaterally.
The rest of organs and systems - No other pathologic change was evident.
No. 1919, female (100 yg/m3) 15.5 months:
No pathologic change was evident in any of the organs and systems.
3. Microscopic Observations
A summary of the microscopic changes is given on Table A-3. Below, only
the organs and tissues with pathologic changes will be listed in detail, the
rest will be understood to have been within normal limits.
No. 1867, male (control) 6 months:
Liver - Few, focal single cell necroses of mild intensity.
Kidney - Slight, occasional, interstitial mononuclear infiltrates.
Lung - Mild form of acariasis with peribronchiolitis and pneumonitis and
a tan to yellow-brown granular pigment within the macrophages.
MLN - Multiple parasitic abscesses with giant cells and pigment.
No. 1549, male (100 yg/m3) 6 months:
Liver - Few, focal, single cell necroses of mild intensity; mild
lymphocytic and monocytic infiltration in one periportal field.
Lung - Mild form of acariasis with peribronchiolitis and pneumonitis
and a tan to yellow-brown granular pigment within the macrophages.
No. 1479, female (control) 12 months:
Liver - Mild centrolobular hepatocellular hypertrophy with fine
vacuolation; few, focal, single cell necroses of mild intensity with
few mononuclear infiltrates.
Lung - Mild form of acariasis with peribronchiolitis and pneumonitis and
a tan to yellow-browjn granular pigment with the macrophages.
Spleen - Congestion and lymphoid depletion.
No. 1731, female (control) 12 months:
Liver - Mild centrolobular hepatocellular hypertrophy with fine
vacuolation; few focal, single cell necroses of mild intensity with
few mononuclear infiltrates.
Lung - Edema, atelectasis and peribronchiolitis.
74
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No. 1765, male (control) 12 months:
Liver - Mild centrolobular hepatocellular hypertrophy with fine
vacuolation; few, focal, single cell necroses of mild intensity with
new mononuclear infiltrates.
Lung - Edema, atelectasis and peribronchiolitis.
Spleen - Congestion and lymphoid depletion.
No. 1781, male (control) 12 months:
Kidney - Slight, occasional, interstitial mononuclear infiltrates.
Liver - Mild centrolobular hepatocellular hypertrophy with fine
vacuolation; few, focal, single cell necroses of mild intensity with
few mononuclear infiltrates.
Lung - Congestion with focal areas of atelectasis.
Spleen - Slight reactive hyperplasia.
No. 1870, male (100 yg/m3) 12 months:
Liver - Centrolobular distension of sinusoids.
Lung - Extensive form of acariasis with atelectasis, peribronchiolitis,
pneumonitis, multinucleated giant cells, abundant eosinophils and a tan
to yellow-brown granular pigment within the macrophages.
No. 1871, male (100 yg/m3) 12 months:
Liver - Mild centrolobular hepatocellular hypertrophy with fine
vacuolation.
Lung - Extensive form of acariasis with atelectasis, peribronchiolitis,
pneumonitis, giant cells and a tan to yellow-brown granular pigment
within the macrophages.
Heart - Focal area of infiltration consisting of monocytes and small
and large lymphocytes; focal necrosis of muscle fibers (Fig. A-l).
No. 1917, female (100 yg/m3) 12 months:
Liver - Centrolbular distension of sinusoids; few, focal, single cell
necroses of mild intensity.
Kidney - Few, focal, mild, interstitial infiltrates.
Lung - Severe form of acariasis with etelectasis, peribronchiolitis,
pneumonitis and a tan to yellow-brown granular pigment within the
macrophages.
MesencephaIon - Behind the area of the collicular commissure and
within the gray matter, palisading was present; this consisted of
proliferation of astrocytes in a concentrated fashion (Fig. A-2).
No. 1922, female (100 yg/m3) 12 months:
Liver - Mild centrolobular hepatocellular hypertrophy with fine
vacuolation; few, focal single cell necroses of moderate intensity
with few mononuclear infiltrates.
75
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No. 1922 (continu'ed)
Lung - Moderate degree of acariasis with atelectasis, peribronchiolitis,
pneumonitis and a tan to yellow-brown granular pigment within the
inacrophages.
Heart - Focal areas of infiltration consisting of monocytes and small
and large lymphocytes; focal necrosis of muscle fibers. (Fig. A-3)
Internal capsule - Mild, focal, perivascular mononuclear cuffing.
No. 1869, male (100 yg/m3) 15.5 months:
Liver - Mild centrolobular hepatocellular hypertrophy with fine
vacuolation.
Kidney - Solid cortical adenoma; the lumens of the involved tubules
were not patent; towards the center of these solid tubules a PAS
positive material was present; focal areas of interstitial infiltration
was present; focal areas of interstitial infiltration within the adenoma.
Lung - Moderate degree of acariasis with atelectasis, peribronchiolitis
and a tan to yellow-brown granular pigment within the machrophages.
No. 1919, female (100 ug/m3) 15.5 months:
Liver - Mild centrolobular hepatocellular hypertrophy with fine
vacuolation.
Kidney - Slight, occasional, interstitial mononuclear infiltrates.
Heart - Focal areas of infiltration consisting of monocytes and small
and large lymphocytes; focal necrosis of muscle fibers (Fig. A-4).
Muscle (rectus femoris) - Granuloma with multinucleated giant cells
and eosinophils surrounding parasitic nidi.
C. DISCUSSION
The following changes which were observed constitute reactive changes to
microbial agents (Table A-3): pulmonary acariasis, in eight out of twelve
animals; parasitic granulomas and abscesses (2 out of 12); pulmonary atelec-
tases (3 out of 12); interstitial renal infiltrates (4 out of .12); splenic
lymphoid depletion or reactive hyperplasia; and finally necroses in the liver
(8 out of 12).
The mild centrolobular hepatocellular hypertrophy with the accompanying
fine vacuolation (vesiculation), which were observed in both control and
exposed monkeys, are probably responses to physiologic stimuli.
The astrocytic proliferation within the gray matter of the mesencephalon,
which was observed in only one animal (No. 1917 F); and the perivascular
cuffing within the internal capsule, which was observed in another animal
(No. 1922 F), of the 12 month time interval; are probably coincidental. They
were observed only in female monkeys of the 12 month interval, and not in
animals of longer exposure. These two reactive changes, because of their
location, are probably (but not conclusively) due to a transient anpxic
anoxia.
76
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The only growth observed was the renal adenoma in one male rhesus of the
15.5 month interval. This spontaneous tumor (renal tumor) is according to
the literature (O'Gara and Adamson, 1972*) among the most frequent spontaneous
growth of subhuman primates.
The minimal monocytic and lymphocytic infiltration of cardiac tissue,
which was observed in two 12 month (No. 1871M and 1922F) and one 15.5 month
rhesus monkeys, although it cannot be attributed to the inhalation of manga-
nese at this time, deserves further investigation. Maybe a specific followup
will be indicated.
D. CONCLUSIONS
1. No gross or microscopic changes were observed in any of the tissues
or organs of rhesus monkeys attributable to inhalation of 100 yg/m3
manganese for up to 15.5 months.
2. However, in three monkeys (No. 1871 M, 12 months; No. 1922 F, 12 months;
and No. 1919 F, 15.5 months) out of seven monkeys (Table A-3) minimal
monocytic and lymphocytic infiltration of cardiac tissue was observed.
The significance of this observation cannot at this time be attri-
butable to the inhalation of manganese.
* R.W. O'Gara and R. H. Adamson 1972. Spontaneous and Induced Neoplasms
in Nonhuman Primates, in Pathology of Simian Primates Part I, pp. 214.
77
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Figure A-l. Cardiac muscle from adult male rhesus (No. 1871), which received
100 yg/m3 manganese for 12 months. Focal area of monocytic and lymphocytic
infiltration. Hematoxylin and eosin, X376.
78
-------
'• ° - «.' 5\^
C-H;%'#^#ro •
i .. *%;rtVfi^ssas ••vv«.V:
k;<>* > i.^L'^1^'1 "• \ **^^?
Figure A-2. Mesencephalon from adult female rhesus (No. 1917), which received
100 yg/m3 manganese for 12 months. In the area behind the collicular commis-
sure and within the white matter astrocytic proliferation is present.
Hematoxylin and eosin, X372.
79
-------
mmJm
':* I L *
* A 4fff* *
3. '•*<'
^d .-:'^
/•V
M/ «'.:) • *
•: ;V &«* vi.
^*f *J * 09^ ,A -^ N» '
Figure A-3. Cardiac muscle from adult, female rhesus (No. 1922) which
received 100 yg/m3 manganese for 12 months. Monocytic and lymphocytic
infiltration and muscle fiber necroses are present (arrow). Hematoxylin
and eosin, X364.
80
-------
r*
,!"|V * &•
» . f
V.» \ •« A »\* •:
f > , ^ *«
'• • * V
'' * "' -«M
1 - r> W V <*k
( I i ». W
i \ *^* 1J^*
i.-l * * T»Ti
Figure A-4. Cardiac muscle from adult female rhesus (No. 1919), which
received 100 yg/m3 manganese for 15.5 months. Focal areas of monocytic
and lymphocytic infiltration are visible. Hematoxylin and eosin, X372.
81
-------
00
Ni
TABLE A-l. EXPERIMENTAL DESIGN OF THE CHRONIC* INHALATION STUDY OF AIRBORNE
PARTICULATE MANGANESE
Animal Number
Dose (yg/m3)
No. of animals
per group
Sex of animals
Time (months)
of exposure
1867
Control
1
M
6
1549
100
1
M
6
1765
1781
Control
2
M
12
1479
1731
Control
2
F
12
1870
1871
100
2
M
12
1917
•1922
100
2
F
12
1869
100
1
M
15.5
1919
100
1
F
15.5
* 6 to 15.5 months, given to male (M) and female (F) rhesus monkeys
-------
TABLE A-2. WEIGHTS* OF RHESUS MONKEYS EXPOSED TO CHRONIC INHALATION OF AIRBORNE PARTICULATE
MANGANESE
00
Animal no. Body
and sex weight
Control
1479 F
1731 F
1765 M
1781 M
1867 M
Exposed
1549 M
Exposed
1870 M
1871 M
1917 F
1922 F
Exposed
1869 M
1919 F
3.70
2.90
2.50
2.80
3.80
6 Months
6.30
12 Months
5.20
5.90
5.80
4.70
15.5 Months
6.1
6.7
Absolute
liver
weight
104.0
118.5
107.0
93.0
75.6
103.0
84.0
107.5
83.5
79.5
96.0
119.0
Relative
liver
weight
2.81
4.10
4.28
3.32
1.98
1.62
1.61
1.82
1.44
1.69
1.57
1.77
Absolute
kidney
weight
8.50
9.00
6.00
6.50
8.25
9.00
8.50
9.10
9.50
8.75
10.60
10.80
Relative
kidney
weight
0.23
0.31
0.24
0.23
0.22
0.14
0.16
0.15
0.16
0.18
0.17
0.16
Absolute
brain
weight
89.0
93.5
78.0
86.3
98.0
90.0
92.0
101.0
80.0
74.5
85.0
91.0
Relative
brain
weight
2.40
3.22
3.12
3.08
2.58
1.43
1.77
1.71
1.40
1.60
1.39
1.35
* Body weights (kg), absolute (g), and relative (%) organ weights.
-------
TABLE A-3. SUMMARY OF MICROSCOPIC CHANGES IN RHESUS MONKEYS EXPOSED TO
CHRONIC* INHALATION OF AIRBORNE PARTICULATE MANGANESE
Group
identification
Control ,
6 months
100 ug/m3,
6 months
Control ,
12 months
100 yg/m3,
12 months
100 ug/m3,
15.5 months
Organ
Liver
Lung
Ki dney
MLN§
Liver
Lung'
Liver
Lung
Kidney
Spleen
Liver
Lung
Ki dney
Heart
Mesencephalon
Internal capsule
Liver
Lung
Kidney
Heart
Muscle
Change
Single cell necroses
Acariasis
Interstitial infiltration
Parasitic abscesses
Single cell necroses
Peri portal infiltration
Acariasis
Single cell necroses
Centre! obular hyper-
trophy and vacuolation
Acariasis
Atelectasis
Interstitial infiltration
Lymphoid depletion
Reactive hyperplasia
Single cell necroses
Centre! obular hyper-
trophy and vacuolation
Acariasis
Interstitial infiltration
Single fiber necroses
with infiltration
Palisading
Peri vascular cuffing
Centre! obular hyper-
trophy and vacuolation
Acariasis
Interstitial infiltration
Adenoma
* Single fiber necrosis
with infiltration
Parasitic granuloma
Incidence*11
1/1
1/1
1/1
1/1
1/1
1/1
1/1
4/4
4/4
1/4
3/4
1/4
2/4
1/4
2/4
2/4
4/4
1/4
2/4
1/4
1/4
2/2
1/2
1/2
1/2
1/2
* Chronic exposure was for 6 to 15.5 months
** Expressed in number of occurrence over the number of animals in the group
§ Mesenteric lymph node
84
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-77-026
3. RECIPIENT'S ACCESSION>NO.
4. TITLE AND SUBTITLE
INHALATION TOXICOLOGY OF AIRBORNE PARTICULATE
MANGANESE IN RHESUS MONKEYS
5. REPORT DATE
May 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Frederick Coulston and Travis Griffin
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Institute of Comparative and Human Toxicology
International Center of Environmental Safety
Albany Medical College
Hoi 1oman Air Force Base, New Mexico
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02-0710
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP, NC
14. SPONSORING AGENCY CODE
EPA/600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Four male and four female rhesus monkeys were exposed to manganese oxide
(1^1304) aerosol at 100 yg/m3 in an exposure chamber for periods up to 66 weeks. Three
n)ale and three female monkeys were maintained as unexposed controls. Observation
and clinical chemistry during the experiment showed minor changes in excretion, and
no evidence of toxic effects. Manganese tissue levels showed small incrases except
in lung (five-fold) and pons (four-fold). No macro- or microscopic pathology was
observed. Two rhesus monkeys were exposed to 5 mg/m3 of the same material for
23 weeks, and observed for 10 months thereafter. Large increases in excretion of
manganese were observed in both urine and feces, but no evidence of neurologic or
other toxicity was seen. Two groups of 4 and of 14 rats were exposed in the same
manner to 100 yg/m3 of the same compound for 8 weeks. These experiments were done
to examine changes in excretion and tissue levels during and after exposure.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
Monkeys
Manganese
Toxicology
Respiration
06, T
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RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
91
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
85
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