>A-600/3-75-i)10g
eptember 1975
Ecological Research Series
REPORT
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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
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series.
This series describes research on the effects of pollution on
humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investigations
include formation, transport, and pathway studies to determine the
fate of pollutants and their effects. This work provides the
technical basis for setting standards to minimize undesirable
changes in living organisms in the aquatic, terrestrial and
atmospheric environments.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.
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EPA-600/3-75-0109
September 1975
ANNUAL CATALYST RESEARCH PROGRAM REPORT APPENDICES
Volume VI
by
Criteria and Special Studies Office
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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CONTENTS
Page
CATALYST RESEARCH PROGRAM ANNUAL REPORT
EXECUTIVE SUMMARY 1
INTRODUCTION 5
PROGRAM SUMMARY 7
TECHNICAL CONCLUSIONS 17
DISCUSSION 22
REFERENCES 45
APPENDICES TO CATALYST RESEARCH PROGRAM ANNUAL REPORT
VOLUME 1
A. OFFICE OF AIR AND WASTE MANAGEMENT
Al. AUTOMOTIVE SULFATE EMISSIONS 1
A2. GASOLINE DE-SULFURIZATION - SUMMARY 53
A2.1 Control of Automotive Sulfate Emissions
through Fuel Modifications 55
A2.2 Production of Low-sulfur Gasoline 90
VOLUME 2
B. OFFICE OF RESEARCH AND DEVELOPMENT
B1. FUEL SURVEILLANCE
B1.1 Fuel Surveillance and Analysis 1
B1.2 The EPA National Fuels Surveillance
Network. I. Trace Constituents in Gasoline
and Commercial Gasoline Fuel Additives • • • 19
B2. EMISSIONS CHARACTERIZATION
B2.1 Emissions Characterization Summary 44
B2.2 Sulfate Emissions from Catalyst- and Non-
catalyst-equipped Automobiles 45
B2.3 Status Report: Characterize Particulate
Emissions - Prototype Catalyst Cars 68
B2.4 Status Report: Characterize Particuiate
Emissions from Production Catalyst Cars • • • 132
B2.5 Status Report: Survey Gaseous and Particu-
late Emissions - California 1975 Model Year
Vehicles 133
B2.6 Status Report: Characterization and Meas-
urement of Regulated, Sulfate, and Particu-
late Emissions from In-use Catalyst Vehicles -
1975 National Standard 134
B2.7 Gaseous Emissions Associated with Gasoline
Additives - Reciprocating Engines. Progress
Reports and Draft Final Report - "Effect of
Gasoline Additives on Gaseous Emissions" . . 135
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Page
B2.8 Characterization of Gaseous Emissions from
Rotary Engines using Additive Fuel -
Progress Reports 220
B2.9 Status Report: Exploratory Investigation of
the Toxic and Carcinogenic Partial Combus-
tion Products from Oxygen- and Sulfur-
containing Additives 232
B2.10 Status Report: Exploratory Investigation of
the Toxic and Carcinogenic Partial Combus-
tion Products from Various Nitrogen-
containing Additives 233
B2.11 Status Report: Characterize Diesel Gaseous
and Particulate Emissions with Paper "Light-
duty Diesel Exhaust Emissions" 234
B2.12 Status Report: Characterize Rotary Emissions
as a Function of Lubricant Composition and
Fuel/Lubricant Interaction 242
B2.13 Status Report: Characterize Particular
Emissions - Alternate Power Systems
(Rotary) 243
VOLUME 3
B.3 Emissions Measurement Methodology
B3.1 Emissions Measurement Methodology Summary 1
B3.2 Status Report: Develop Methods for Total
Sulfur, Sulfate, and other Sulfur Compounds
in Particulate Emissions from Mobile Sources 2
B3.3 Status Report: Adapt Methods for S<>2 and SO3
to Mobile Source Emissions Measurements 3
B3.4 Evaluation of the Adaption to Mobile Source
SC>2 and Sulfate Emission Measurements of
Stationary Source Manual Methods 4
B3.5 Sulfate Method Comparison Study. CRC APRAC
Project CAPI-8-74 17
B3.6 Determination of Soluble Sulfates in CVS
Diluted Exhausts: An Automated Method 19
B3.7 Engine Room Dilution Tube Flow Characteristics. ... 41
B3.8 An EPA Automobile Emissions Laboratory 52
B3.9 Status Report: Protocol to Characterize Gaseous
Emissions as a Function of Fuel and Additive
Composition - Prototype Vehicles 89
B3.10 Status Report: Protocol to Characterize Particu-
late Emissions as a Function of Fuel and Additive
Composition 90
B3.11 Interim Report and Subsequent Progress Reports:
Development of a Methodology for Determination
of the Effects of Diesel Fuel and Fuel Additives
on Particulate Emissions 192
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Page
B3.12 Monthly Progress Report #7: Protocol to
Characterize Caseous Emissions as a Function
of Fuel and Additive Composition 200
B3.13 Status Report: Validate Engine Dynoniometer Test
Protocol for Control System Performance 218
B3.14 Fuel Additive Protocol Development 221
B'3.15 Proposed EPA Protocol: Control System
Performance 231
VOLUME 4
B3.16 The Effect of Fuels and Fuel Additives on Mobile
Source Exhaust Particulate Emissions 1
VOLUME 5
B3.17 Development of Methodology to Determine the
Effect of Fuels and Fuel Additives on the Perform-
ance of Emission Control Devices 1
B3.18 Status of Mobile Source and Quality Assurance
Programs > • 260
VOLUME 6
B4. Toxicology
B4.1 Toxicology: Overview and Summary 1
B4.2 Sulfuric Acid Effect on Deposition of Radioactive
Aerosol in the Respiratory Tract of Guinea Pigs,
October 1974 38
B4.3 Sulfuric Acid Aerosol Effects on Clearance of
Streptococci from the Respiratory Tract of Mice.
July 1974 63
B4.4 Ammonium and Sulfate Ion Release of Histamine
from Lung Fragments • 89
B4.5 Toxicity of Palladium, Platinum and their
Compounds 105
B4.6 Method Development and Subsequent Survey
Analysis of Experimental Rat Tissue for PT, Mn,
and Pb Content, March 1974 128
B4.7 Assessment of Fuel Additives Emissions Toxicity
via Selected Assays of Nucleic Acid and Protein
Synthesis 157
B4.8 Determination of No-effect Levels of Pt-group
Base Metal Compounds Using Mouse Infectivity
Model, August 1974 and November 1974 (2
quarterly reports) 220
B4.9 Status Report: "Exposure of Tissue Culture
Systems to Air Pollutants under Conditions
Simulating Physiologic States of Lung and
Conjunctiva" 239
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Page
B4.10 A Comparative Study of the Effect of Inhalation of
Platinum, Lead, and Other Base Metal Compounds
Utilizing the Pulmonary Macrophage as an Indicator
of Toxicity 256
B4.11 Status Report: "Compare Pulmonary Carcinogenesis
of Platinum Croup Metal Compounds and Lead Com-
pounds in Association with Polynuclear Aromatics
Using [n_ vivo Hamster System 258
B4.12 Status Report: Methylation Chemistry of Platinum,
Palladium, Lead, and Manganese 263
VOLUME 7
B.5 Inhalation Toxicology
B5.1 Studies on Catalytic Components and Exhaust
Emissions 1
B.6 Meteorological Modelling
B6.1 Meteorological Modelling - Summary 149
B6.2 HIWAY: A Highway Air Pollution Model 151
B6.3 Line Source Modelling 209
B.7 Atmospheric Chemistry
B7.1 Status Report: A Development of Methodology to
Determine the Effects of Fuel and Additives on
Atmospheric Visibility 233
Monthly Progress Report: October 1974 255
B7.2 Status Report: Develop Laboratory Method for Collec-
tion and Analysis of Sulfuric Acid and Sulfates . . . 259
B7.3 Status Report: Develop Portable Device for Collection
of Sulfate and Sulfuric Acid 260
B7.4 Status Report: Personal Exposure Meters for
Suspended Sulfates 261
B7.5 Status Report: Smog Chamber Study of SO2
Photo-oxidation to SO^ under Roadway
Condition 262
B7.6 Status Report: Study of Scavenging of SO_ and
Sulfates by Surfaces near Roadways 263
B7.7 Status Report: Characterization of Roadside
Aerosols: St. Louis Roadway Sulfate Study 264
B7.8 Status Report: Characterization of Roadside
Aerosols: Los Angeles Roadway Sulfate Study. . . . 269
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Page
VOLUME 8
B.8 Monitoring
B8.1 Los Angeles Catalyst Study. Background Pre-
liminary Report
B8.2 Los Angeles Catalyst Study; Summary of Back-
ground Period (June, July, August 1974)
B8.3 Los Angeles Catalyst Study Operations Manual
(June 1974, amended August 1974)
B8.4 Collection and Analysis of Airborne Suspended
Particulate Matter Respirable to Humans for
Sulfates and Polycyclic Organics (October 8, 1974).
1
13
33
VOLUME 9
B.9 Human Studies
1974
-194
1
B9.1 Update of Health Effects of Sulfates, August 28,
B9.2 Development of Analytic Techniques to Measure
Human Exposure to Fuel Additives, March 1974 .... 7
B9.3 Design of Procedures for Monitoring Platinum
and Palladium, April 1974 .............. 166
B9.4 Trace Metals in Occupational and Non-occupation-
ally Exposed Individuals, April 1974 ......... 178
B9.5 Evaluation of Analytic Methods for Platinum and
Palladium ....................... 199
B9.6 Literature Search on the Use of Platinum and
Palladium ....................... 209
B9.7 Work Plan for Obtaining Baseline Levels of Pt
and Pd in Human Tissue ............... 254
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Appendix B4.1
TOXICOLOGY
Overview and Summary
Automotive exhaust emissions are a major source of air pollutants in the
United States. Exhaust emissions include various chemical and physical agents
that represent a substantial risk to public health and welfare. These agents
include carbon monoxide, oxides of sulfur, hydrocarbons, trace metal ligands,
respirable particulate matter in addition to various resultant photochemical
products such as olefins, peroxyacetyl nitrates, oxides of nitrogen and ozone.
In order to guarantee ambient air quality and to protect public health and welfare,
stringent standards of exhaust emissions were set forth in the amended Clean Air
Act of 1970. In lieu of available alternate engine designs, automotive manu-
facturers are committed to the use of oxidation catalytic reactors in auto exhaust
systems to comply with 1975-76 regulations for auto emissions prescribed in the Clean
Air Act. These catalysts are effective in oxidizing much of the hydrocarbons and
oxidizes the sulfur dioxide to sulfates and sulfuric acid mists. The primary
oxidation product is sulfuric acid, a corrosive and toxic irritant. Sulfuric
acid and sulfates are existing environmental pollutants ti , . are in the realm
of unregulated emissions for mobile sources. Platini- , and palladium metals
and their compounds may also be in' the exhaust. Tiir a. unregulated emissions
result from the use and attrition of the catalyst unit and represent new
environmental contaminants. There are, therefore, th-°c- components that are
of current concern in evaluating the potential hazards in fie impending use
of catalytic converters in automobiles.
4 sulfuric acid aerosols (sulfates)
4 particulates
9 noble metals (Pt and Pd)
Assessment of public health hazards of existing environmental agents relies
principally on an integrated program that involves environmental monitoring.
1
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meteorology, epidemiology, clinical analysis and aerometric chemistry. The
community health surveillance system (CHESS) is a national program established
by the Environmental Protection Agency to survey the effect of environmental
pollution on exposed populace. To assure protection of public: health and welfare
*• - -
from new chemical compounds, the following questions must be addressed prior to
their appearance and ultimate uncontrolled release to the environment.
• Is the compound toxic and produced in amounts that, could
adversely affect health of populations and/or the ecosystem?
9 What is its capacity for biodegradability and biotrans-
formation?
9 What is known of its toxic effects in terms of dose-response
relationships?
t What is known of its industrial-occupational hygiene history
that warrant particular concern?
• Is there a segment of the population that is sensitive or
susceptible to adverse health effects?
• Is there sufficient information regarding its metabolism,
critical organ sensitivity and tissue burden levels that
signifies a biological overload having potential irre-
versible pathology?
• Are there specific clinical and experimental parameters
that could be used in defining subclinical pathogenesis?
Many of these questions were discussed at an open conference on the Health Conse-
quences of Environmental Controls: Impact of Mobile Emissions Controls, co-sponsored
by EPA and the National Institute of Environmental Health Sciences, April 17-19,
in Durham, N.C.
USE DISTRIBUTION AND EXPOSURE TO NOBLE METALS
The use of catalytic reactors on new motor vehicles could introduce as much
as 36,000 kg of platinum on a national scale during the first two vehicle model
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years. These estimates would equal and exceed the present national consumption
of this metal used for all other purposes. Limited studies have reported emissions
of noble metal particulates from these devices. These potentially toxic pollutants
will be released at breathing level of our narrow highway corridors. Some concern
has been expressed that these roads will provide a temporary or more lasting
reservoir for these pollutants. The major concern is the percent of the populace
that are or may become sensitive to extremely low levels of noble metals and
their compounds.
METALLIC COMPOUNDS IN CATALYST-MODIFIED EXHAUST EMISSIONS
In addition to refractory materials that provide a mechanical support for
the catalysts, several metallic compounds are added to automotive fuels. The use
of tetraethyl lead to increase the octane level of gasoline has had a profound
impact on the environment and public health hazards have been defined. It is
important to note that the environmental concern and risks to public health
are associated with combustion products and not with the fuels and fuel additives
themselves. This refers specifically to the soluble and insoluble compounds of
platinum, palladium, manganese, and lead with particular concern to the toxic
properties of the oxides, sulfates, and halides. be j par-- 7 luting the relative
solubilities for metals and metal compounds of concern in exhaust emissions of
motor vehicles.
BIOGRADABILITY AND B ^TRANSFORMATION
It can be anticipated that noble metals associated with the use of catalytic
converters may not only be emitted into the air but may also be deposited on
the ground. Accumulation in streams, rivers and lakes and their sediments * ay
result from discarded, used converters as well as from ground water contamination
by rain-washed highways and vehicle junk yards. While the effect of these ions
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on the normal microbiological flora and plant life is of particular concern, the
biomodification by the sediment microbial species is of specific interest. This
refers to the formation of soluble metal compounds.
Mercury has chemical properties similar to noble metals in that it is also
largely inert in its reactions. The use and distribution of mercury has well
documented adverse effects on both public health and environmental welfare.
Methylation can occur under mild reaction conditions via a Vitamin B^ intermediary.
This reaction encompasses the entire biota of the ecosystem; i.e., it occurs in
microbial organisms, plants, and animals.
The initial clinical symptoms of methylmercury poisoning are insidious,
becoming progressively more severe and finally irreversible, following a latent
period. Methylmercury may be absorbed through the skin, lungs, and gastro-
intestinal tract and is clinically defined as a severe neurotoxin.
Whether a stable form of methylated platinum is formed by a reaction with
Vitamin B is as yet undetermined. Preliminary studies suggest that platinum
displaces cobalt in Vitamin B,?. If this is proven to be the case with a
depletion of this vitamin, there is a possibility of pernicious anemia develop-
ment. Research is presently underway on this subject at AEC Lawrence Laboratory
in Livermore, California.
In addition to mercury, arsenic, sellenium and tellurium, there have been
recent reports indicating that thallium, gold and platinum can be methylated by
the common methylcobalamin (Vitamin B^) reaction. Biomodifications of noble
metals need to be studied and the stability of methylated products determined to
assure protection against immediate and delayed potential health adversities.
An interagency agreement has been initiated to determine if noble metals
can be methylated and to assess the stability of the methylated analysis. A
Toxicological program will follow to assess the hazards of stable methyl compounds.
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LETHALITY AND TOXICITY
The primary concern for the adverse health effects from environmental con-
tamination by the use of catalyst converters is the localized exposure to sulfates
and the long term-low level exposure to noble metals. To obtain useful information
on the toxic properties of these pollutants in a reasonable time frame, it is
necessary to conduct, in the laboratory, animal exposure studies at levels that
will be lethal or cause observable adverse physiological effects.
Also, animal studies are conducted to obtain a no effect level in a
finite period. There are chronic low-level repetitive doses administered to rats (,r
guinea pigs of up to a 90-day duration to determine this no effect level.
Levels of these pollutants found in the environment may in some instances
approach within an order of magnitude the no-effect level but generally the
pollutant level is and should be at least several orders of magnitude lower than
the so-called no-effect level.
The procedures and protocols used to ascertain the adverse effects of pollu-
tants to populace living in and near urban, industrial and also in some contami-
nated rural environments is embraced in the technical disr' .line termed epidemiology.
Protocols employed include analyses of morbidity and mortality information obtained
through medical records and relating this to environmental stresses such as
pollutants.
A major effort that has accumulated extensive information on the effect of
environmental contamination on selected populations has been underway on the
program CHESS (Community Health and Environmental Surveillance System). Increased
prevalence of chronic bronchitis, respiratory infections, aggravation of cardio-
pulmonary symptoms and of asthma are health indicators used to evaluate effects
of environmental pollution. A more detailed discussion of this program is included
under the section HEALTH EFFECTS. Another study specifically directed to environ-
mental health problems of fuel additives and unregulated contaminants resulting
5
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from the use of oxidation catalysts as being conducted by the Southwest Research
Institute. The objectives and results of this program are more fully described
under the section HEALTH EFFECTS.
Several toxicological programs have been implemented to establish the no-
effect, chronic, acute and lethal toxic effects or exhaust products from catalytic
converters.
Metals used in oxidation catalysts and compounds of these metals have been
studied for their toxic and lethal effects. Doses of these pollutants were
administered to animals to obtain information on the following:
e No-Effect Level - No effects observed.
« Chronic Effects - Effects observed with prolonged repetitive
exposure
• Acute Exposure - High level, single or repetitive exposure
resulting in severe morbidity or mortality.
• LD (10, 50, 90) - Lethal doses resulting in 10%, 50% and
90% lethality after a 14-day period following administration
of dose.
Three major studies using experimental animals have been implemented by the
Office of Fuel and Fuels Additive Registration during the past year to establish
various toxic levels of exhaust pollutants:
* NERC/RTP - Experimental Biology Laboratory
Dr. D. Coffin
• NERC/Cincinnati - Environmental Toxicology Research Laboratory
Dr. J. Stara
« UNC/Medical School - Biochemistry Department
Dr. D. Hoi brook
In the following paragraphs the results of these toxicology studies are
summarized.
Following the oral administration to animals of salts of manganese, lead,
palladium and platinum, the relative acute toxicities (LD-50 doses) in decreasing
order was determined to be:
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1. Soluble Platinum Chloride (PtCl.)
2. Soluble Platinum Sulfate (Pt(S04)2 ' 4H20)
3. Less Soluble Palladium Chloride (PdCl2 * 2H20)
4. Less Soluble Ruthenium Chloride (RuCl~)
5. Less Soluble Manganese Chloride (MnCU * 4H20)
6. Less Soluble Palladium Sulfate (PdS04)
*7. Very Low Soluble Platinum Chloride (PtClJ
*8. Insoluble Platinum Oxide (Pt02)
*9. Insoluble Lead Oxide (PbO)
*10. Insoluble Lead Chloride (PbClJ
*11. Insoluble Manganese Oxide (MnCL)
*12. Insoluble Palladium Oxide (PdO)
Thus, the two tetravalent soluble platinum salts were found to be the most
toxic following oral administration.
As anticipated, the insoluble salts were least toxic. *The salts listed
above from 7-12 could not be administered to attain 50% lethality and still
maintain the volume administered to 7% or less of the body weight. In each of
these cases, the LD-50 dose is greater than the 5g/kg body weight which NIOSH
(National Institute for Occupational Safety and Health) uses as a criterion as
a toxic substance. In terms of acute LD-50, these insoluble salts would be
considered "non-toxic" by definitions of this standard.
Acute LD-50 toxicity observations on animals following interperitoneal
injection for the above salts showed a similar order with the soluble tetravalent
salts of platinum being the most toxic.
Dr. Holbrook developed tables from data obtain 'j~, using experimental animals
for LD-10, 50 and 90. See pages 1 and 2 of Appendix B4./.
Acute toxicity studies at the NERC/Cincinnati Toxicology Laboratory also
determined soluble PtCl, to be highly toxic to rats when administered intravenously.
The rapidity of death in non-surviving rats following oral administration
varied widely. For example, rats receiving approximately the oral LD-50 dose of
soluble PtCl^ survived for less than 1 day. For soluble Pt(SO.)2 ' 4H20 the
survival time ranged between 1 and 3 days.
7
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TISSUE RETENTION
Retention,distribution and excretion studies of a soluble radioactive platinum
chloride was determined in mature, pregnant and suckling rats.
Whole body retention of this radioactive iostope varied significantly by
route of administration. Percent of retention during the 28-day period following
dosage was significantly higher for intravenous (IV) followed by intratracheal and
191
then oral administration. These data, including the retention of Pt by suckling
rats is shown on page 3 in Appendix A2 of Appendix B5.
The excretion of approximately 50 percent of the initial dose during the
first 24 hours following intratracheal dosing is attributed to mucociliary and
alveolar clearance.
Following oral dosing the total net gastrointestinal excretion was high
resulting in a rapid decline in the retention curve to less than 1% at the end
of 3 days (see Appendix A2, page 5, of Appendix B5.
191
Whole body retention of Pt was the highest following intravenous dosing.
The short radioactive half-life precluded an accurate determination of the
biological half-life for this metal.
TISSUE DISTRIBUTION
191
The distribution and tissue concentration of Pt was determined for different
organs as a function of time following single IV dosing of rats. The kidney and
191
liver contained the highest concentration of Pt. The amount of radioactive
platinum determined to be present in other organs was not significantly higher than
background. These data on distribution of Pt is summarized on page 6 in the
Appendix of this report.
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Similar observations were made by Dr. Holbrook of UNC also using rats as the
4+
experimental animal. Dose was administered using soluble Pt in drinking water
The highest tissue concentrations of Pt occurred in the kidney and ranged from
4.5-5 yg Pt/g of wet tissue. High levels, ranging from 0.7-2.5 yg Pt/g also
occurred in the liver. Brain tissue showed only a very low level of Pt which is
in agreement with the statement of EPA's Toxicology Laboratory in Cincinnati that
soluble platinum salts do not significantly traverse the blood brain barrier, oee
Appendix A2, Table 1 of Apoe-dix 85.
PLACENTAL TRANSFER
1 91
Rats were sacrificed 24 hours after administering Pt to determine placental
I gl
transfer. The maternal kidney showed the highest level of Pt with a radioactive
mean count of 127,064/gm followed by 43,375 counts/gm in liver and 27,750 in the
placenta. Distribution is shown in other tissues in Appendix A2, Table 2, of
Appendix B5.
The placental barrier of rats is more readily traversed than most other
species of experimental animals having more complex placenta! barriers. It is to
be noted that death occurred very rapidly following administration of toxic levels
of palladium chloride administered by the intravenous route. Rapid death appeared
to be due to respiratory arrest since breathing ceased and the heart continued to
function. Cyanosis was not observed. These effects suggest that platinum and
palladium may have different biological modes of int >r-ction. Accordingly,
differences are noted in the tissue distribution profile of platinum and palladium
presented in Table I.
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Table I.
ORGAN DISTRIBUTION PROFILE OF SOLUBLE NOBLE METAL SALTS*
Platinum Palladium
Kidneys ^ Kidneys
Liver Spleen
Testes Liver
Heart _Lungs
Blood Heart
Brain Testes
Brain
Preliminary conclusions regarding acute and chronic exposure to various
noble metal by various routes of administration are as follows:
» Noble metal compounds elicit toxic effects when absorbed.
• The exposure route influences retention of absorbed
noble metals in the body.
• Toxic effects appear to be a function of solubility.
• There may be variations in noble metal distribution among
critical organs and critical organ sensitivity due to
differences in exposure route, biomodification of noble
metals, and pharmacodynamic influences.
• Absorbed noble metals can be transferred across the
placental barrier in pregnant experimental animals.
• Biochemical studies point to differences in relative
toxicities of platinum and palladium which may be
dependent upon the chemical form present in the body.
• Soluble noble metal salts are more toxic than certain
other exhaust metallic compounds (lead, manganese), as
is evident from the threshold level values for these
compounds.
*
Organs are listed in decreasing order of noble metal content
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t Preliminary cardiovascular studies indicate that palladium
acts as a non-specific cardiac irritant as well as a
peripheral vasoconstrictor.
t It appears from these preliminary studies, long-term chronic
exposures are required to fully assess the toxicity of noble
metals and to determine irreversible biological effects, i.e.
cancer, mutants, teratogens.
Although this information is far from sufficient to permit safety assessments,
it is enough to warrant caution and further study. As noted earlier, the threshold
3
limit value for soluble metal salts is 0.002 mg/m . Threshold limit values refer
to time-weighted averages of industrial exposure levels of a given chemical com-
pound. It represents work place index to concentrations by which nearly all workers
may undergo repeated exposure without adverse health effects. These values are often
incorrectly used to distinguish harmful concentrations from safe exposure levels.
They are intended only as guides for establishing prudent industrial hygiene con-
ditions. A table has been compiled to demonstrate the variation in LD™ values
as a function of exposure route and threshold limit values of metallic compounds
that are found in automobile exhausts. It is obvious that many of these compounds
have not been an industrial problem and that little information is available for
comparative purposes of this program (See page A.3 2, Appendix B5.1)
DERMAL IRRITANCY
Dermal irritancy of catalytic exhaust pollutants needs to be ascertained as
part of the general toxicological evaluations.
A series of tests were performed by ETRL on palladium, platinum, lead and a
manganese antiknock additive (MMT).
Using Federal test procedures used by NIOSH, skin reactions were evaluated
and scored at 24 hours and at 72 hours after application of the chemical.
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Three palladium compounds and the soluble chloride salt of platinum were
tested and found to be irritating and unsafe for human sikin contact. Rabbits
were used as the test animal. A summary of these results is presented on page A.6-5
of Appendix B5.1.
Sensitizations may be far more serious or chronic than direct irritation.
They may develop at lower and more common levels of exposure and opportunity for
development of sensitivity may be greater by virtue of extended or repeated exposure
by inaestion, inhalation and/nr cutaneous contact.
OCULAR IRRITATION
Tests on ocular irritation were performed at the NERC/Cincinnati, ETRL using
test procedures outlined in the Code of Federal Regulation, Title 21, Part 191.12
revised as of April 1, 1973.
All six animals receiving PdCl^ showed a severe corrosive type of lesion of
the conjunctiva with severe inflammation of the cornea and anterior chamber of
the eye. This was noted at 24 hours and persisted throughout the test period.
None of the animals receiving the platinum compounds showed any ocular
irritation. PdO also was not an irritant.
BIOMEDICAL ASPECTS OF CONCERN - Immune Response
Platinum allergies and platinosis are the principle adverse health effects
that have been reported for those compounds. Platinosis is defined as an allergic
reaction following occupational exposure to soluble platinum salts which elicits
a progressively more severe asthmatic response with continued exposure. Classical
allergic symptoms are observed with occasional severe ariaphylaxes in sensitive
individuals. Insensitive individuals may work in the presence of this allergen
for years without noticeable adversities, but the vast majority develop either
respiratory or dermatological problems upon continued exposure. The platinosis
12
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syndrome consists of sneezing, rhinorrhea, tightness of chest, shortness of
breath, cyanosis, wheezing and cough that resembles a mixture of hay fever and
asthma. Once disabling symptoms appear in any one case, that individual will
never again become asymptomatic in a platinum environment. In a platinum refinery
survey', 60% of the employees examined displayed varying degrees of severity of
platinosis. The remaining 40% were asymptomatic with respect to asthmatic
syndrome but displayed varying degrees of irritation of the upper respiratory
tract, conjunctivae and mucous membranes with a tendency toward enlargement of
lymphatic tissues accompanied by lymphocytesis.^ '
Platinum compounds may contribute to airborne allergens in the ambient air
with aggravation of individuals with pre-existing asthmatic or cardio-respiratory
deficiencies. There are approximately 10-20 million asthmatics in the United
States that represent a rather large susceptible subgroup of the population.
Furthermore, those compounds may prove to be additive or synergistic with co-
exposure to other ambient air pollutants.
There have been little or no investigations dealing w'th basic immunological
mechanisms of platinosis. With the impending wic!'j~ccale use of platinum and
palladium in automobiles, it is prudent to develop a clearer understanding of
how these metals enter into and interfere with impot i pt body reactions. No
physiological need has been found for platinum. The very low concentrations of
platinum in man's natural environment may explain his adverse reactions to its
exposure in pure and cationic form when inhaled, ingested or from cutaneous
contact. Bivalent platinum would be expected to react and compete with essentit ,
trace elements for binding sites of important biological molecules such as cobalt
in Vitamin B, meta11o-enzymes, DNA and DNA-ase. Additional and more varied
research is needed to better elucidate these biochemical actions that are basic
to the allergen-type effects observed. Research on these basic biological processes
13
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at the subcellular level can be undertaken both in vivo and in vitro. The
information obtained will provide a scientific basis for arriving at safe levels
of exposure, if any, for the various physical and chemical forms of platinum.
The delayed type of hypersensitivity that has been observed in cases of platinosis
suggests a cellular immune response to an uiitigenic stimulus. Circulating anti-
body systems do .not appear to be involved in its pathogenesis. Evidence for this
is the presence of pathological lesions in the lungs. This mechanism could explain
why only a limited number of exposed persons develop manifestations of platinos.is.
Needed is a physiologic indicator to determine whether a person is sensitive.
Patch-testing the skin with salts of platinum may, however, risk sensitization.
Studies are needed to develop a test that can reliably determine the percent
of populace that are hypersensitive to platinum and possibly palladium. Infor-
mation from these studies is basic to our understanding of possible adverse health
effects from a general overall increase in mining, processing, use, and disposal of
platinum and products made from platinum.
Dose-response is a questionable area for study if the adverse effects of
platinum and its compounds is primarily an immune response action.
More information is needed to better elucidate response or time-recovery
rates to exposure. Certainly the -severity of effect must be related to air con-
centrations of this metal as well as time of onset of platinosis and other observed
effects.
In vitro protein binding studies have been made by EiTRL, NERC/Cincinnati.
They have performed in vitro protein binding studies with Pd and Pt chlorides
(PdCl?and PtCl,) using the Toribara ultracentrifugation technique at concentrations
up to 200 yg of compounds/ml whole blood plasma or plasma equivalent albumin.
Protein binding was greater than 99 percent at all concentrations. Temperature
(2)
and pH were found not to affect binding. '
-------
(3 4)
Several recent studiesv ' ' have been made on the potential merits of
chloro-platinum (II) complexes as tumor inhibiting agents. One of these studies
reported that almost constant concentrations of free platinum complexes could
be maintained in animals by injecting these complexes that are bound to sulfur
containing amino acids. These chelates would slowly decompose to replace consumed
free platinum. -Investigators also speculated that it should be possible to
purge an animal of free platinum by the injection of suitable chelating agents.
An EPA funded program to estimate the no-effect levels of immediate and delayed
allergenic response in animals exposed to soluble noble metal salts, Pt(SGh)2 '
4H20 and PdS04 was initiated at UNC Medical School on April 16, 1974.
GENETICS
A contractural program is planned to assess the cytogenetic and mutagenetic
potential of soluble salts of platinum and palladium. These investigations will
determine possible deleterious genetic effects of the noble metals. Genetic
effects can occur in the absence of a toxicological/pathological response. This
is consistent with the basic nature of genetic material which is the only biological
material that registers, accumulates and transfers information regarding environ-
mental stress. Genetic disturbances can occur in somatic cells of the body as
well as in gonadal germinal tissue. Somatic effects registered in the immediate
generation can result in pathology in tissues undergoing growth (children) or
tissues that have a high frequency of cell division in the adult (e.g. the
gastrointestinal tract). Since there is characteristically a prolonged latent
period, there are often no discernible signs of pathological onset. A particular
potential end-point resulting from "non-lethal" genetic damage in somatic cells
is carcinogenic, mutagenic, and teratogenic effects. A significant portion of
15
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genetic and teratogenic damage incurred by expos,, to both chemical and physical
environmental agents is manifested by numerical and structural alterations of
chromosomes.
Prevention of hereditary disease has become a growing concern due to expansion
of susceptible subgroups in the population with a- resultant increase in medicare
costs. Approximately 250,000 genetically abnormal children are born in this
country each year with defects ranging from chromosomal anomalies (trisomy and
mongolism) to subtle inborne errors of metabolism (enzymatic defects) leading
to clinical treatment, e.g. Lesch-Nyhan syndrome. It has been estimated that
each individual carries between 5-10 genetically harmful genes. Therefore, it is
prudent not only to protect and ascertain environmental quality but to minimize
excessive risks to future biological vitality.
CARCINOGENESIS
An urban factor of pulmonary carcinogenesis has been described by epide-
miological methods with a gradient existing between rural-low and urban-high
pollution levels. These factors persist following corrections made for cigarette
smoking. Carcinogenic compounds are present in benzene-soluble fractions obtained
from particulate matter in the ambient air, i.e. the polynuclear aromatic hydro-
carbons. This fraction has been shown to be carcinogenic in the laboratory. It
is well established that combustion of fuels and fuel additives in the internal
combustion engine is a ubiquitous source of those materials.
In subchronic feeding studies in which low levels of palladium chloride
(5.0 ppm) was administered to laboratory animals in their drinking water, palladium
had a selective effect in the growth of males. During the first six months, palla-
dium suppressed growth in males but not females. However., the longevity in treated
males exceeded that of control animals of both sexes. These animals were charac-
terized by an increase in tumorgenesis (see Table II). While this may at first be
attributed to differences in longevity, the treated animals displayed a tumor
profile different from that of controls.
16
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Table II.
TUMOR PROFILES OF MALE MICE ADMINISTERED 5.0 ppm OF PALLADOUS
CHLORIDE OVER THEIR ENTIRE LIFETIME
Controls Treated
Total Tumor Percentage
Lymphoma-Leukenria Type
Adenocarcinoma
Gross Tumors
not sectioned
13.8%
2
1
5
27.2%
10
6
9
The conclusion drawn from these studies is that palladium is slightly carcino-
genic in mice.
It is important to realize that approximately 80% of all human tumors are
associated with chemical induction. Furthermore, it is now recognized that various
chemical and physical agents can act as co-carcinogens in the role of activator and
promoter. Investigations into the mechanisms of pulmonary co-carcinogenesis has
described the role of both metallic oxides particle (ferric oxide), and benzo-
pyrene for tumor induction in the bronchial-tree of experimental animals. Benzo-
pyrene is found in particulate matter collected from automobile exhausts. In
this regard, a study is planned using this model of particulate - noble metal
compounds to assess their potential carcinogenicity.
Although one may speculate that noble metals such as platinum or its compounds
may initiate cancer, some of our most interesting data on the biochemistry of this
metal and associated complexes is from research directed to its tumor inhibiting
(3 4)
characteristics. ' ' Frequently, chemical agents that affect cellular metabolism
may either inhibit or initiate tumor growths depending upon the subtle biological
conditions, species differences, etc.
The work described in this paper suggests that a key factor in this tumor
inhibition could be the ability of uncharged complexes to bind weakly and reversibly
with proteins. This, in effect, slows down the metabolic processes and tumor growth.
17
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CARDIOVASCULAR EFFECTS
Palladium chloride (PdCl,,) has been shown by Oresteno^ ' to be extremely toxic
when given intravenously to rabbits. Rapid death followed injection with 0.6/mg/kg
with damage inflicted chiefly to the heart. Further investigations were undertaken
to measure the effects of palladium solutions on heart rate, EC6 pattern, blood
pressure, cardiac contractility (dp/dt) and breathing for one hour following IV
injection. PdSCL was administered in doses ranging between 0.5-2 mg Pd /Kg body
weight. ECG changes, like those described with PdCl,,, were observed during the
injection period. Initial interuption of the cardiac cycle occurred at 0.5 mg Pd
Kg body weight.
Results from these preliminary experiments indicate that PdCl and PdSO^, when
injected IV, act as non-specific cardiac muscle irritants as well as a peripheral
vasoconstrictor. Since the chloride ion strongly dissociates in solution the Pd
ion itself, it is thought to be the irritant.
INHALATION TOXICOLOGY
Definitive toxicological investigations were conducted at the Toxicology
Laboratory at NERC/Cincinnati using laboratory animals and placing emphasis on
exposure routes (inhalation, oral-and dermal) of environmental significance. Other
routes, such as intravenous, are used to obtain a comparative toxicologic matrix,
geared to investigate the biological effects resulting from inhalation of the
individual catalytic components and the whole exhaust emissions after passage
through the catalytic converter.
AUTO EXHAUST EMISSION STUDIES
EPA has available at ETRL (Environmental Toxicology Research Laboratory in
NERC/Cincinnati) an auto exhaust generating system for the production of irradiated
and non-irradiated gasoline engine exhaust-air mixtures. By irradiation is meant
18
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exhaust gases exposed to artificial sunlight to generate photochemical reactions
with exhaust products. This system has been utilized in a series of acute studies
for evaluating the biological effects of exposure in various experimental animal
species to whole automotive exhaust emissions with fuel additives and with or
without a catalytic converter.
The system-has been used to determine the effects of acute exposure to diluted
automotive engine exhaust. Of specific interest were: (1) conversion of organic
sulfur compounds present in gasoline into sulfuric acid mist and sulfates; (2)
platinum and palladium emission due to catalyst attrition; and (3) alteration of
the emissions with the production of different quantities and/or new species of
pollutants.
Emission studies have indicated that the oxidation-type catalyst in the
exhaust system has resulted in the following changes in exhaust emissions: (1)
large reduction of carbon monoxide, total hydrocarbons, and various organic compounds
such as acetylene; (2) a nearly total elimination of aldehydes; (3) less photochemical
reactions of hydrocarbons (HC/NO ratio changed); (4) the presence of sulfuric acid
/\
as a major component in the particulate; and (5) emission of platinum and palladium
particulates*
The bio-effect studies were undertaken to determine the general toxicologic
effects of an acute exposure to exhaust from engines equipped with catalytic con-
verters (catalytic exhaust) vs exposure to exhaust from engines without catalytic
converters (non-catalytic exhaust). The results indicate that animals exposed to
non-catalytic exhaust exhibited profound changes in weight of lactating female and
infant rats as well as a decreasing survival rate of infant rats. There were no
apparent effects on the rats exposed to catalytic exhaust with the possible
exception of weight loss in lactating female rats.
19
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The clinical data indicates that the only acute exposure effect in the
catalytic exhaust was an increase in total serum proteins. In the non-catalytic
exhaust acute exposure, there were significant effects on the following: total
protein, platelet count, red blood cell and white blood cell count, white cell
differential, alkaline phosphatase, hemoglobin, hematocrit, partial ^iromuopla
levels, serum glutamic oxalaoacetate transmin^e, and serum glutamic pyruvate
transaminase levels. Lung pathology studies showed a"~greater incidence of
pathological conditions after non-catalytic exposure than after catalytic exposure.
These effects, during the short 1-4 week acute exposures, are likely due to the
dramatic decrease in CO, oxidant (photochemical reaction product) and organic
particulates as a result of the use of the oxidation catalyst. Weighing the
benefits of reducing these toxic compounds with the risks of generating new
pollutants such as sulfates, particulates and toxic metal attrition products
is the purpose of future studies.
The effect of catalytic and non-catalytic exhaust was tested in several
uio-chemical systems. Lung aryl hydrocarbon hydroxylase (AHH) (an enzyme
which is responsible for the biotransformation of various carcinogens) activity
is depressed with exposure to non-catalytic exhaust. The catalytic converter
significantly reduced lung aryl hydrocarbon hydroxylase depression, but did
not eliminate the irradiated exhaust AHH depression. Serum lactate dehydroge-
nase was also greatly elevated with non-catalyst exposure but was not signifi-
cantly changed with exposure to catalyst exhaust.
Plant damage occurred in both catalytic and non-catalytic exhaust. However,
damage appeared to be somewhat less in the catalytic exposure. Since healthy
plants were damaged in both types of exposure, then it appears that vegetation
is inherently more susceptible to exhaust damage than aminals and that the damage
threshold was exceeded in both types of studies.
20
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In summary, the acute whole emission studies have demonstrated that exposure
to automotive exhaust without a catalytic converter elicits profound effects on
physiological and biochemical function as well as producing histo-pathological
lesions. In contrast, exposure to exhaust from a system with a catalytic converter
did not result in such demonstratable physiological or biochemical dysfunction.
One may conclude that the introduction of the catalytic converter has reduced the
levels of certain exhaust adverse constituents. This has resulted in a decreased
biological impact of these exhaust emission products. However, it should be noted
that catalyst-modified emissions produce changes in viability of vegetation. In
addition, it is now known that catalyst-modified emissions are highly acidic. As
a result, longer term studies are needed and planned for the future using whole
animal exposure systems which are sensitive to the acid aerosol generated by the
catalytic devices.
TOXICOLOGICAL STUDIES OF CATALYST METAL COMPONENTS
Acute studies were undertaken to ascertain the relative toxicity of platinum
and palladium to obtain information that is presently inadequate.
Intratrachael administration of platinum or palladium results in greater body
retention than oral dosage. Furthermore, the differences among the intravenous,
intraperitoneal, and oral toxic dosages of palladium indicates a difference in
distribution of palladium depending on the route of administration. Biochemical
studies have shown that the relative toxicity of platinui,i or palladium on glutamic
oxalo-acetate transaminase and lactate dehydrogenase is dependent upon the chemical
form of the metal. In addition, palladium elicits an increase in C-leucine incor-
poration into heart and blood serum. Platinum produced a dose-response change in
C-leucine incorporation in the lung. Dermal irritancy, dermal absorption, and
ocular irritation tests are being utilized as screening techniques to determine
the relative toxicity of various types of platinum and palladium compounds.
21
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Preliminary neurophysiological screening tests have shown that palladium elicits
short-term changes at higher dosages and that these changes may be indirect effects
produced by changes in other systems such as the cardiovascular system. Preliminary
cardiovascular studies have indicated that palladium acts as a non-specific cardiac
irritant as well as a peripheral vasoconstrictor.
TOXICITY OF HHOLE AUTO EXHAUST
A facility has been established at the Environmental Toxicology Research
Laboratory in NERC/Cincinnati to assess animal toxicity of whole exhaust emissions
from vehicle engines equipped with catalytic converters. This program is known
under the acronym TAME (Toxicity Assessment of Mobile Emissions) and involves a
series of animal experiments directed to determining the toxic nature of whole
exhaust emissions on animals. Mixtures of these irradiated and non-irradiated
exhaust gases flow into inhalation animal exposure chambers.
A schematic of the facility used including the irradiation and inhalation
exposure chambers is shown in Appendix 155.1
For this research appropriate mammalian species of different ages (from
embryonic to maturity) are being used to obtain information on organ and tissue
effects comparable to human exposure to these pollutants.
From the standpoint of potential health effects, the emission components of
greatest concern following the use of the catalyst, is in the increased amount of
particulates, especially sulfates, and the potential release of catalytic attrition
products containing noble metals platinum and palladium into the breathing atmosphere
on highway corridors.
The acidity of these particulates were measured to be 65 times higher in cata-
lytic exhaust than exhausts without catalysts; when additional sulfur was added
to the gasoline (total 1000 ppm), the acidity increased 260 times (see Table V).
22
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Table III.
PARTICULATE AT 24 km/hr (15 mph)
Operating Condition
No Catalytic Converter
High-Sulfur Indolene Fuel
With Catalytic Converter
Regular High-Sulfur
Indolene Indolene
Relative Acidity
of Particulate
Total Particulate
7.5:l3Dilution
1
65
260
2.7
7.2
25.6
Two examples of studies on the toxic effects of platinum and palladium are
given in Table VI (showing neurotoxicological data). In a series of acute exposure
of animals to whole emissions from engines equipped with and without catalysts,
severe effects were observed in the neonatal animals and their dams after an
exposure to non-catalytic exhaust; however, thus far, toxic effects could not be
ascertained in catalyst-treated emissions in neonatal animal exposure test series
of one week to one month duration.
Table IV.
VISUAL-EVOKED POTENTIAL SCREEN
Metal Compounds
Reproducible Dose-Effect
Threshold (mg/kg)
Cobalt
Cadmium
Chronium
Palladium
Barium
Manganese
Platinum
0.010
0.10
0.40
0.40
2.0
2.0
Minimal effect
23
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Table V.
SURVIVAL OF SUCKLING RATS FOLLOWING EXPOSURE TO
WHOLE AUTOMOBILE EXHAUST
Clean Air Control
Non irradiated Exhaust
Irradiated Exhaust
Carbon Monoxide Control
TAME I*
100%
?^%
0%
96%
TAME J**
98%
100%
100%
TAME K**
100%
100%
1 00%
^Without Catalyst
**With Catalyst
TAME (Toxicity Assessment of Mobile Emissions)
Summary and conclusions from these experiments:
t Catalyst significantly reduced carbon monoxide, total hydrocarbons,
and various individual hydrocarbons such as acetylene.
3
• Concentration of 0.029 yg/m platinum was measured in the diluted
emissions of the animal exposure chamber. The possibility that
some Pt compounds deposited on exposure chamber walls (electrostic
attraction) needs to be assessed.
Aldehydes are virtually eliminated except for crotonaldehyde by
using catalysts.
Photochemical reactions in non-catalyst exhaust are significantly
greater than exhaust from motor vehicles using catalysts due to the
change in HC/NO ratio. A factor that needs to be considered is that
s\
these tests were run under constant air dilution. This would lower
total 0 and NO levels and adverse biological effects from these
A X
pollutants generated by oxidation catalysts.
Sulfuric acid is a major component in particulate exhausts of
catalyst equipped motor vehicles.
-------
e Sulfuric acid particulate component is in direct proportion to sulfur
contained in fuel. Conversion of sulfur to sulfates ranges from 22%
to 41% depending on efficiency.
e No adverse effects due to exposure to sulfuric acid aerosol were
observed. Previous studies, reviewed in reference 6, however, indi-
cate that no adverse effects would be expected at these exposure
levels and short exposure durations to these particular animal species,
Acid aerosol sensitive exposure studies are planned for FY 75.
HUMAN STUDIES
Several studies are being conducted to ascertain the health impact of various
pollutants on man. Many of these programs, while focusing specifically on certain
pollutant effects, are providing methodology development of significance to the
public health protocol discussed earlier in relation to toxicology and inhalation
sections of this report. Obviously, studies of pollutant effects on man himself,
is the final step in the assessment of potential public health risk.
The programs being conducted in this area of study are as follows:
• epidemiological studies to assess effects of SCL, total
suspended particulate, and suspended sulfates (CHESS);
0 development of techniques to assess human exposures to
metal pollutants;
• development of techniques to assess levels of noble metals
in animal and human tissues;
• comprehensive study of biochemical, physiological, immunological,
and mutagenic effects induced by exposure to platinum and
palladium compounds.
The relationship of adverse health effects of exposure to suspended sulfate
air pollutants has been studied in depth as part of the CHESS program. The indi-
vidual research reports developed within the Human Studies Laboratory, NERC/RTP,
have suggested pollutant-disease associations but left a number of problems
25
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unanswered including the relative contribution of various air pollutants, especially
sulfur dioxide, total suspended particulates and suspended sulfates to observed
disease frequencies.
Chronic respiratory disease studies have indicated that the relative contri-
bution of air pollution alone ranged from one-third to one-seventh as strong as
that of cigarette smoking as a determinant of chronic bronchitis prevalence in
communities. The sum of the evidence suggests that, while personal cigarette
smoking is the largest determinant of bronchitis prevalence among parents of
school children, air pollution itself is a significant and consistent contributing
factor, leading to increased bronchitis rates in nonsmokers as well as smokers
from polluted communities.
A monograph "The Health Consequences of Sulfur Oxides: A Report from CHESS:
which summarizes the results of studies in New York City, Utah, and the Chicago
study areas was published in June 1974. ' Since then, a second series of indivi-
dual research reports summarizing result? from the eastern United States and an
additional year's effort in the New York metropolitan area have been assembled in
preliminary form and will be developed into the contents of a second monograph,
"The Health Consequences of Particulate Pollution". In general, these studies con-
firm the adverse effects of air pollution reported in the first monograph in that
the incidence or prevalence of diseases are higher in areas of highest population.
The data also indicate that lengths of residence from 3 to ii years apparently
increase the probability that adverse health effects will develop in a population
and that there are significant differences between the white and black races in
the manner in which they report adversities generally assumed to occur in conjunction
with environmental stresses.
The health parameters in the studies contained in the Particulate Monograph
are chronic respiratory disease, lower respiratory disease, pulmonary function,
acute respiratory disease, irritation of asthmatics, and irritation of symptoms
26
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reported by the general population during an acute air pollution episode. In each
instance increased rates of adverse health status were associated with increases
in the air pollution to which the population was exposed.
The preliminary data suggest that total suspended particulate levels of about
3 3
100 yg/m in the presence of sulfur dioxide at levels about 100 yg/m or less
were not associated with significant increases in respiratory morbidity in New
York City. Other data indicated that in the southeastern United States that total
3
suspended particulate at levels lower than 70 yg/m annual average in the presence
of low levels of sulfur dioxide also were not strongly associated with an elevated
incidence of chronic bronchitis. Studies of lower respiratory disease in New York
City show that higher incidences were associated with exposure of sulfur dioxide
concentrations of approximately 50 to 425 yg, total suspended particulate concen-
3
trations of 60 to 185 yg/m , and suspended sulfate concentrations ranging from 9
to 20 yg/m . In the southeastern United States excess acute lower respiratory
disease was associated with annual mean levels of suspended sulfates ranging from
3
10 to 13 yg/ , in the presence of sulfur dioxide concentrations of less than
3
25 yg/m . In the low pollution areas of the New York communities, respirable
particulate amounted to approximately 70% of the total suspended particulates
whereas in the Intermediate exposure areas the respirable particulate amounted to
between 54 and 70% of the total suspended particuiate matter. In the southeastern
part of the country in the low pollution area, respirable particulates amounted
to nearly 53% of the total suspended particulate and almost the same percentage
of the total suspended particulate consisted of fine particulate in the high
exposure area. Pulmonary function studies in the southeastern area indicated th-t
reduced function was associated with higher pollution. Inter-area differences in
mean FEVQ 7,- as follows: Differences were greater in females than in males, greater
in whites than in blacks, and greater in younger than in older children. In the
high pollution area, both older and younger children had received 50 to 70% higher
27
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lifetime doses of total suspended participate than had unildren in the low pollution
area. In the low pollution area, RSP levels fell about 20% from fall to winter
and rose about 10% in the spring. In the high pollution area, RSP levels fell
about 10% from fall to winter and rose about 25% in the spring. RSP levels in the
high pollution area for the entire school year averaged 35% higher than those in
the low pollution area.
A study of the incidence of acute respiratory disease in the southeastern
United States suggested that small but consistent increases in illness were
3
associated with annual geometric mean levels of TSP ranging from 81 to 93 yg/m ,
o
or annual geometric mean levels of RSP ranging from 34 to 45 yg/m .
Additional pulmonary data are available from studies on the short-term effects
due to air pollutants. From the New York metropolitan area, there was good evidence
that effects of air pollutants upon the health of elderly panelists appeared to have
the strongest effects upon the well elderly but similar health patterns were observed
for the subjects with lung illness, with heart illness, or with heart and lung ill-
3
ness. Best case threshold estimates developed from preliminary analyses were 40 yg/m
33 3 3
RSP, 75 yg/m TSP, 4 yg/m suspended nitrates, 8 yg/m suspended sulfates, 60 yg/m
o
S02, and 45 yg/m N02- In the New York metropolitan area, studies demonstrated
increases in irritation symptom reporting for days designated as higher pollution
exposure days when compared to a control period. However, no pattern of response
emerged in terms of relating specific irritation symptoms to increases in any
pollutant. The pollutants measured included total suspended particulates, respirable
particulates, the sulfate fraction of the particulates, the nitrate fraction of
the particulates, and sulfur dioxide. During the episode period, increases were
variable from community to community as was the pollutant exposure; but in most
areas total suspended particulate during the high pollution period was 80 to 120%
above that experienced during the control period. This was equally true for the
sulfate fraction of the particulate matter.
28
-------
While these data are preliminary and subject to revision, they do in the I"
present form suggest the general adequacy of the present primary air quality
3
standard for TSP (75 yg/m ) and indicate that a reasonable standard for RSP, if
3
it is determined that one is desirable, might be approximately 35 yg/m .
The Human Studies Laboratory has recently negotiated an option to the CHAMP
contract for the purpose of improving the monitoring of sulfate aerosols in all
CHESS areas by developing equipment and methods for measuring the sulfate fraction
of fine particulates. This same contractor under a separate agreement also is to
provide mobile field monitoring capability to permit better characterization of
actual human exposure to pollutants. Also, under contract the Human Studies Labora-
tory will be gathering information on the effects of catalytic converters on road-
side pollution levels and on the levels of materials deposited in human tissues.
Materials of greatest interest will include lead and noble metals. These studies
are just now getting under way.
Completion of a human exposure chamber is anticipated in the forseeable future.
This facility will provide the Clinical Studies Branch of the Human Studies Laboratory
with the capability of testing the effects of short-term exrc^re to pollutants,
singly or in combination, on both healthy and disease.i subjects. It is also anti-
cipated that mobile laboratory facilities will be -omp1 ted and delivered in the
next few months. These facilities will permit the ai>sossment of the effects of air
pollution in the general population using the soph 11 rated equipment and techniques
usually restricted to the Clinical Laboratory. Also, •/ pidemiologic studies will
be initiated. Some of these will be replications of (..,'ior studies; others will be
new studies, but all will' be designed to determine the relationship between expos He
to specific levels of air pollution and various acute or chronic health statuc indi-
cators. Indicators to be measured will include (a) prevalence of chronic respiratory
disease, (b) incidence of lower respiratory disease, (c) chromosomal abnormalities,
(d) aggravation of symptoms in asthmatics, and (e) pulmonary function.
29
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Other studies to be initiated in the current fiscal year include (a) validation
of the self-administered chronic respiratory disease questionnaire, (b) estimation of
populations at risk, and (c) biochemical or metabolic alterations associated with
exposure to NCL.
A final report has been received from Southwest Research Institute on the measure-
ment of body burdens of fuel additives in populations with high density of motor
vehicles. Analysis of heavy metals (Cd, Cu, Pb, MnT Zn) in human blood, urine, hair,
and feces were made on residents and persons in occupations that would reflect differen
levels of exposure. For example, policemen directing vehicular traffic, workers in
enclosed areas such as garages and tunnels participated in this study. This study
was also highly valuable in developing methodology for selection and participation of
human subjects and the conduct of pollutant burden studies on both autopsy tissues and
living populations. This study was undertaken to determine the relationship between
exposure levels and durations of exposure to automotive pollution under normal working
and living conditions to differentiate burden in human tissue specimens.
Following the decision on the part of automobile manufacturers tc use catalysts
to meet automobile exhaust emission standards imposed by Congress for 1975 year
models, Southwest Research Institute was requested to plan and implement a base-
line survey in persons occupationally exposed to these catalysts and living in areas
that exposure to catalytic exhaust emissions could be highest.
An initial three month planning and survey completed the following tasks:
• identification of prominent point sources of platinum and palladium;
• compilation of production and consumption data;
• estimation of amounts entering the environment under present
and projected future uses;
o documenting published human exposure cases of platinum and
palladium and their medical histories;
« summarizing toxic effects of noble metals in living organisms.
30
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This study provided a sound background to implement a major effort to obtain
effects of working in mining and processing plants for these catalysts and to
obtain base line data on populations that will be exposed to motor vehicles using
oxidation catalysts. This refers primarily to platinum and palladium.
In March 1974 Southwest Research Institute was contracted to initiate this
survey to obtain analysis of human hair, blood, urine and feces of residents living
in areas where there is an intense use of motor vehicles, and in workers mining
or processing these catalysts.
The project investigators are presently establishing volunteer participants
in the Los Angeles Basin. This area was selected for the initial survey for the
following reasons:
e plans for early introduction of catalysts in California;
« for the extensive data available in L.A. and currently being
generated by Federal, State and local agencies;
e heavy motor vehicles in use;
• adverse climatic conditions resulting in pollutant exposures
and photochemical reaction products.
It is planned to sample a total of 40 subjects from two refineries in New
Jersey and 40 subjects from the mining area. Adequate cooperation appears promising
from mining management. Some difficulties are being experienced in obtaining medical
data on platinum and palladium workers in processing plants.
Satisfactory arrangements are being worked out with residents in the L.A. Basin
area in completing guestionnaires pertaining to this survey and in providing hair,
urine, blood, and fecal samples for analysis.
Air, soil and water sampling activities are being coordinated with other
environmental sampling programs underway in the L.A. Basin by EPA, State, and
local agencies.
31
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Because of the proprietary nature of work areas in refineries, it is
probable that SwRI personnel will be prevented from placing high volume samplers
in work areas. Alternative procedures will be used whereby sampling devices and
instructions will be provided to the management for sample collection.
This catalyst exposure survey is exploratory in nature. Its purpose is:
e to identify which tissues are t!.c best indicators of
various facets of the body burden of platinum and palladium;
t to examine variables affecting tissue levels;
» to establish the relationship between air concentration;
• to analyze body tissues for these metals.
There is unavailable, at present, systematic data on the distribution of
platinum and palladium levels within an exposed population. Hence, it is pre-
mature to specify which statistical procedure will be used to draw inferences.
Actual statistical techniques to be used will be dictated, in part, by the nature
of data obtained.
Analysis of the refinery data will meet most of the current exposure study
objectives. This survey will attempt to identify those tissue specimens that are
the best indicators of chronic exposure, recent acute inhalation or ingestion of
these pollutants. For example, the best indicator of chronic inhalation will be
a tissue specimen meeting the dual criteria of a high mean concentration level
over all refinery workers and a minimal variance between two samples collected
from each worker. The optimal tissue selected for an indicator of body burden
level will be treated as the primary dependent variable in subsequent analyses.
Analysis of tissue specimens from volunteer participants will be identified with
air samplers in the most comparable situation. The relationship of air concen-
tration on body burden will be examined by histograms for the workers identified
with a particular air sampler. Other variables such as age of participant, smoking
habits, length of employment, sex, ethnic origin, will be assessed and factored
32
-------
into statistical analyses. Through this process major covariates will be
identified. If asthma, platinosis, and other allergic reactions that have been
linked to platinum exposures are evident or prevalent among refinery workers,
an effort will be made to correlate exposure levels to frequency and severity of
these diseases.
In addition, air, soil and water samples will be collected at various distances
from the refinery.
Analysis of mine data will be directed to the solubility question. Nearly
all airborne platinum and palladium produced by the refinery is expected to be in
the soluble form which can be absorbed by the human body via the lung, through the
skin or by oral ingestion. Much of the platinum and palladium in the mine is
expected to be in the insoluble metallic or oxide form. Thus, a comparison of
the body burden of workers exposed to the insoluble and soluble forms should be
possible. Histogram plots will also be used to exam the relationship of body
burden to exposure levels.
A literature survey was conducted in 1973 by Stewart Laboratories, Inc. to
ascertain whether a published analytical method existed for the determination of
platinum and palladium in biological tissues. The survey revealed that no such
method existed and that the anticipated levels of these metals in tissues would
likely be in the nanogram range. The objective, of course, was to be able to
ascertain current, pre-catalyst use levels of these metals in human autopsy tissue
samples and/or white blood thus providing a human burden base line. Subsequent
to this survey, a contract was let to Stewart Laboratories to:
« develop an analytical method for platinum in tissues which
would be 2 to 5 times more sensitive than the current "best"
method;
« conduct analysis of Pt, Pb, and Mn in animal tissues provided
by EPA.
33
-------
An analytical method for Pt in mammalian tissues has been developed which
represents an improvement in sensitivity of 5 times that of the current "best"
methods. The analysis of animal tissue burdens for Pt, Pb, and Mn were completed.
Stewart Laboratories also conducted a limited survey of human autopsy tissue
burdens of Pt. This is preliminary data and i<= summarized in the following table.
Medical files have not yet been received, so we cannot ascertain potential occupa-
tional or medical exposures to Pt relative to these autopsy tissues.
Table VI.
HUMAN AUTOPSY TISSUE BURDEN OF Pt
Micrograms Pt per gram
Sources Orqan of wet tissue
Birmingham, Ala. Liver 1.0
Kidney <0.015
Charlotte, N.C. Liver 1.5
Kidney .009
Liver 0.07
Kidney <0.015
Liver (6) <0.015
Kidney (6) <0.015
New York City Liver <0.015
Kidney <0.015
A more comprehensive analysis of human autopsy tissues for Pt will be made
in FY 75.
Hazleton Laboratories, Inc., Vienna, Va. has undertaken several studies using
experimental monkeys to determine lowest effective dose of sulfuric acid mists. '
In cynomolgus monkeys exposed for 78 weeks (22 hours per day) to a breathing
3
atmosphere of 0.99 mg/m of sulfuric acid mist of less than 1 micron mass median
q
diameter (MMD) combined with 0.53 mg/m fly ash of less than 5 micron MMD resulted
-------
in adverse microscopic changes in the lungs. These changes were present in the
bronchial and bronchiolar mucosa and in the walls of the bronchioles and were
characterized by focal areas of erosion and epithelial hypertrophy and hyperplasia.
In some instances the airways were ectatic. No adverse changes were observed in
3 3
monkeys exposed to 0.11 mg/m H?SCL and 0.53 mg/m fly ash. Fly ash was deposited
in both peribronchial lymph nodes and in the lungs from all exposed monkeys. No
adverse reaction was associated with this deposition of fly ash. In both sets of
experiments the size of the acid aerosols and fly ash particulates were equivalent.
to\
Later, published resultsv ' from this laboratory show that sulfuric acid aerosol
mist exposure in cynomolgus monkeys at levels of 2.43 and 4.79 mg/m with concentrations
3
of 2.43 to 4.79 mg/m and particle sizes of 3.60 micron and 0.73 micron respectively
produced definite deleterious effects on pulmonary structures and deterioration in
pulmonary function. At lower concentrations the effects were absent or less definite.
3
No adverse reaction was seen in the lungs from monkeys exposed to 0.11 mg/m
H2$0, with 0.53 mg/m fly ash. Fly ash was deposited in both peribronchial lymph
nodes and in the lungs from all of the exposed monkeys. No adverse reaction was
associated with this disposition of fly ash.
No microscopic alterations associated with exposure to sulfuric acid mist and
fly ash were seen in the trachea, .heart, liver or k (!;ieys.
Guinea pigs did not show an adverse effect at any of the above levels of
exposure to sulfuric acid mist and fly ash.
The National Acadmey of Sciences/NRC has two activities underway considering
the health aspects of introducing catalytic converters in motor vehicles.
A Panel on Noble Metals has been formed in the NAS Division of Biological
Sciences, Committee on Medical and Biological Effects of Environmental Pollutants.
This Panel, chaired by Dr. Hightower of Rice University, is considering the public
health aspects of platinum and palladium attrition products in exhaust emissions.
A report of this Panel will be issued in January 1975.
35
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A second group, also chaired by Dr. Hightower, is in the Division of Engi-
neering of NAS/NAE. A subpanel on catalytic converters under the Committee on
Mobile Vehicle has been formed to issue a report on the general health aspects of
catalysts to be used in motor vehicles. Special attention is being given to
V >4»
pollutants other than noble metal catalysts. For example, sulfuric acid mists
and oxidants of. hydrocarbons will be evaluated as to their possible impact on
public health and welfare.
36
-------
REFERENCES
1. Roberts, A. E. Platinosis, A Five-Year Study of the Effects of Soluble
Salts on Employees in an Platinum Laboratory and Refinery. Ind. Hyg.
and Occup. Med. 4_: pps. 549-559, 1951.
2. Toribara, T. The Ultrafilterable Calcium of Human Serum. J. Phar. Clin.
Invest. 36_: p. 738, 1957.
3. Friedman, M. E., et al. The Blocking of Tetrachloroplatinate II Inhibition
of Mai ate Dehydrogenase by Sulfur-Containing Ami no Acids. Biochemica et
Biophysica Acta. 341: pps. 277-283, 1974.
4. P. Melius, et al. Inhibition of Leucine Aminopeptidase and Malate Dehydro-
genase by Aquoplatinum (II) Complexes. Biochemica et Biophysica Acta.
268: pps. 194-198, 1972.
5. Oresteno, G. The Pharmacologic Actions of Palladium Chloride. Boll., Soc.
Ital. Biol. Sper. .8: pps. 1154-1156, 1933.
6. The Health Consequences of Sulfur Dioxides: A Report from CHESS, 1970-1971.
EPA-650/1-74-004, May 1974. Document available: U.S. Gov't. Printing Office,
Washington, D.C.
7. Chronic Exposure of Cynomolgus Monkeys to Sulfuric Acid Mist and Fly Ash
Mixtures. Project RP-74, Hazel ton Laboratories, Vienna, Va. Dec. 1972.
8. Alarie, Y., W. M. Busey, et al. Long-Term Continuous Exposure to Sulfuric
Acid Mist in Cynomolgus Monkeys and Guinea Pigs. Arch. Environ. Health.
27: pps. 16-24, July 1975.
37
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Appendix B4.2
SULFURIC ACID EFFECT ON DEPOSITION OF RADIOACTIVE AEROSOL IN
THE RESPIRATORY TRACT OF GUINEA PIGS
Glen A. Fairchild, S. Stults, ami D. L. Coffin
NATIONAL ENVIRONMENTAL RESEARCH CENTER
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
OCTOBER 10, 1974
38
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SUi-iMRY
The effect of inhalation of H?SCV aerosol on respiratory deposition of
radiolabelled streptococcus aerosol was investigated in guinea ?igs. A
60 minute exposure to 3^20 ugm/m^ H?SO^ (1.8 u Ci'iD) resulted in a 60% increase
in total respiratory deposition rate and a proximal shift in the regional
pattern of deposition to the nasopharynx. Dose-response experiments revealed
that a concentration of JO ugn/m-^ H_SO^ (o»25 u C/.D) also induced a proximal
shift in the regional pattern of particle deposition but in this instance the
shift was to the trachea. The interrelationship between mass concentration
of H SO. and its particle size to the interpretation of these results is
discussed. A hypothesis concerning the relationship of this data to previously
reported respiratory physiologic effects of HpSCV inhalation and to a patho-
physiologic mechanism which may underlie health effects attributed to sulfur
oxides by epiderciologic studies is described.
39
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Introduction
Recent epidemiologic investigations which have demonstrated positive
statistical associations between elevated levels of suspended sulphate and.
acute respiratory symptoms in man have stimulated re*:e-,»ed interest in the
1
health effects of sulfur oxides. The clinical syndromes which have been
observed most frequently during episodes of sulfur oxide type air pollution
have been increased incidence of asthma attacks and complaints which reflect
upper airway irritation. The pathophysiologic mechanisms which have been
associated with this type of air pollution appear to reflect predominantly
upper airway irritation.
One of the sulfur oxides which has been incriminated as a potent
respiratory irritant is sulfuric acid aerosol. The toxicologic effects
2
of sulfuric acid aerosol have been reviewed recently. A limited number
of experimental studies of human volunteers have demonstrated that H-SC,,
3-6
inhalation can be irritating to the upper airways. The most sensitive
indicator of response to H.SC/, inhalation is altered mechanics of respiration,
2 3,6,7
especially increased pulmonary airflow resistance. The guinea pig
7
responds to K_5C, inhalation in a similar manner as ...an.
2 •-*•
Altered mechnnics of respiration is a subtle, non-specific response
reflecting a type of respiratory irritation which is usually completely
reversible. It is questionable whether this physiolosic response per se
could explain the epidemioloric observations which have incriminated
sulfur oxides as respiratory irritants under ambient conditions. However
certain interrelationships exist between acute H2SC^ induced respiratory
function alterations, especially increased respiratory airflow resistance,
40
-------
observations of positive associations between sulfur oxides in air and
increased incidence of asthma and other symptoms of airway irritation.
An important physiologic correlary to air flow dynamics in the rest>-
8-12
iratory tract is its relationship to particle deposition. Alterations
in the normal pattern of regional deposition of particles in the respiratory
tract may have important pathophysiologic implications.
To the extent that H SCK inhalation may induce increased airflow
resistance in the respiratory system, the possibility of altered patterns of
regional deposition of particles within the system appears to be a likely
hypothesis. Indeed, a previous investigation has shown that cigarette
smoking is associated with increased airflow resistance and a proximal
12
shift in the deposition rate of particles in the respiratory tract of man.
In addition, increased respiratory particle deposition has been observed in
12
a limited number of individuals with bronchitis or asthma. This invest-
igation describes experimental conditions in which short term exposure to
HgSC^ aerosol induced increased total deposition of inhaled radioactive
streptococcus aerosol with a concomitant proximal shift in the pattern of
regional particle deposition in the respiratory tr.ict of guinea pigs. The
relationship of these observations to previously -<-• ported respiratory
physiologic effects of H SO,, inhalation and to their significance as a
2 /|
possible pathophysiolce:ical basis for health effects observed in recent
epidemiolofric studies of sulfur oxides are discussed.
-------
Methods
Aniir.als« Female Hartley strain guinea pigs of two different bodyweight
ranges(600-830 and 250-350 gms.) were used in these experiments. We observed
no differences in HLSO^ effect which could be attributed to the bodyweight
differences so no further allusion to bodyweight differences will be made.
In each experiment' the f^SC^ exposed guinea pig and its cohort control were
approxinately the sa.'e bodyweight. All animals were lightly sedated with
± 0.015 ng/gm bodyweight of sodium pentobarbital administered intraperitoneally
30 minutes before they were inserted into the body plethysmograph in order to
reduce excitement and struggling while in the apparatus.
Respiratory neasurements. The guinea pigs were housed irt a pressure
plethysmogpaph to measure respiratory ventilation (respiratory frequency,
tidal volume, and ir.ir.ute volume) throughout the experimental procedure (Figure l).
Thirty minutes after injection of-the sedative, two guinea pigs were inserted
into plethysmographs for apparatus acciimitation. The pressure changes inside
the plethysrr.ograph were sensed with a Stathair. Fi'il5 transducer and recorded
by a in>-v."ritir..T oncillio-raphic recorder. The tidal volume was determined
for each plethysr.Dcrraph from a calibration curvs determined by put.pins- knou'n
volur.es of air into the plethysmorraph (which contained a guinea pig phantom)
at several frequencies within the normal breathing frequency range. The
minute volume was calculated from the breathing frequency and tidal volume.
The ventilatory :ueasurements were obtained at 10 minute intervals
during the plethysmograph acciimitation and exposure to H^SCX aerosol and
continually throughout the ^-T labelled streptococcus aerosol exposure.
Sulfuric Acid A-rcs^l. An H^SC^ aerosol was generated by nebulizing a
10 X solution of H~2T., (diluted ir. dl3til]rj w^ier +o a predetermined concentration
v.hich was required t.: rro^cce T.hs cLo^irc^ ..a^.~ cr-r.ccr^r.'ition in ths L-uiiiea pig
42
-------
exposure zone. The nebulizer was operated at 14 psi and 5 LPM. The primary
aerosol was introduced into a secondary airstream of filtered room air up-
stream from the guinea pig exposure zone (Figure 2). Total exposure chamber
air flow was 20 LPK which was equivalent to approximately 0.7 air changes per
minute.
The ^SO^ aerosol size was measured with a right-angle light scattering
photometer. The photometer was calibrated periodically with 0.6 and 1.0
micron latex spheres. Air samples were collected by liquid impingement in
distilled water and the mass concentration of h^SO^ was determined by comparison
of the pH of an air sample of known volume with a calibration curve.
In each experiment the guinea pig was exposed to the H2SO/, aerosol for
60 minutes. Its respective cohart control was exposed to ambient air.
Radiolabelled Streptococcus Aerosol. The particle used for the respiratory
deposition studies was -^phosphorous labelled and killed Group C streptococci.
13
The radiolabelling procedure has been described previously. The labelled
'it
streptococci were washed in broth four times to remove unincorporated ^-'F.
After four washes only about 0.2^ of the radioactivity in a bacterial suspension
could be found in the super T.tent after centr-fusration. After the radiolatelled
streptococci were vrashec four tiir.es> they were resuopended in a 1% fetal
bovine serum-saline solution in preparation for aerosolization. This washed,
radiolabelled streptococcus suspension was used within 4 hours. Within this
period of tims leaching of the 33p radioactivity from the bacteria was minimal.
The size distribution of the stcreptococci aerosol was determined by
collecting the aerosol on gelatin overlaid af~ar plates in an eight-stage
1*
Andersen Viable Particle Sar.pler by a technique described previously. The
radioactivity of the streptococci collected on the ire la tin was determined by
-------
liquid scintillation counting. The particle size distribution of the aerosols
is shown in Figure 3.
Experimental Protocol* For each experiment two guinea pigs were placed
in body plethysmographs for JO minutes of apparatus acclimitation before the
exposure to an HpSO^ aerosol was begun. After this "acclimitation period,
one guinea pig was inserted into the exposure chamber and a 60 minute ^SO^
aerosol exposure was begun (Figure 2). Three air "samples were obtained for
HpSO. mass concentration and the aerosol size was monitored by a light
scattering photometer. After 60 minutes of ^SO^ exposure the generator was
stopped and the aerosol was exhausted. Two minutes later, the cohort control
(exposed to ambient air) was inserted into the exposure chamber adjacent to
the ^SO. exposed guinea pig.
Both guinea pigs were then exposed to a -^-T labelled streptococcal aerosol
for 4- minutes. A 1 UVi air sample was obtained on an 0.45 micron cellulose
acetate filter located between the heads of the two guinea pigs. During the
last 20 seconds of the bacterial exposure an air sar.ple was obtained for
particle size determination with an eight-stage Andersen Viable Particle
Sampler.
Immediately after the 33p labelled streptococci exposure the guinea pigs
were killed by carbon dioxide inhalation. The skin was disoctsd from the
larynx and the trachea and esophagus was clamped with a hemostat to prevent
mucociliary translocation of particles from the trachea to the pharynx
or gasterointestinal tract.
The skin was dissected from the head from the neck forward so as to
avoid contamination of the nasal specimens with radioactive particles which
might have been deposited on the fur of the face of the guinea pig. The
anterior and :.i:icile ; or1-ion of t/.o r.-ur.l cavity vcr3 rcrcvc'J ir~- -1:-." •c;-;1"
44
-------
with a bone saw and these tissues were crushed and digested with a tissue
solubilizer. The nasopharynx-pharynx-larynx (NF), trachea, lungs, esophagus,
and stoirach were obtained in that sequence; a procedure which took about
20 minutes. Each respective tissue was obtained alternately first from the
control guinea pig and then from the HoSC^ exposed animal.
. *
The ti5sues were crushed and/cr minced, and digested with a solublizer,
**
for 48 hours. A 2 ml aliquot of each tissue was then decolorized, a fluor
T-3 ***
was added and J P activity was determined by a liquid scintillation counter.
To minimize the influence of non-specific background effects in scintillation
counting one window of the counter was set at the peak of the -^F ionization
energy spectrur. and the statistical analysis of these data was based upon the
counts detected in the greater half of the energy spectrum. The counts detected
in the nose and lung specimens were generally at least J to 10 tines the normal
tissue background activity. The total amount of radioactivity in each specimen
was calculated after standardization for background activity in normal tissue,
specimen volume, and a quench correction factor(determined by the internal
standard method ] wh«n necessary.
Results
Aercsol cc'.icer.tration and size. The effect of ree different .".asc
concentrations of H?SC(j_ aerosol was investigated: °C2G ug.-./r.^ (high), J2.2
ugn/.i,^ (medium), and JO ugm/m^ (low) respectively. ".-••- zize cf these aerosols
also differed relative to their r.ass concentration. 7h_- size of the HoSd,
aerccols baser: ur.or ~r.eir count r.e';ir.r. cia.T.cttr deterr.ir.^d by lirht ccatterir.r
photometry was l.P, 0,7, ar.d C.25 micrcmeters resrectively^Figure j]. From th: ^e :at=
*Protonol/ ::?:•! -_r^~-.:v '."jclear Corp., Sector., .^,ss.
''."'•'- i r ."_": _-'•'_-' j ""•>_- _ -: .__'-',.. ." ^: .: ~ :.:,
-------
the particle size of each aerosol based upon weight was determined by the
formula utilized previously by Amdur and Corn:
where x = class i particle size by count
f = number of particles in class i
mean size by weight =
The particle size by weight of the high, medium, and low mass concentration
of HgSO, aerosol was 2.0, 0.9, and 0.38 micrometers respectively.
33
The P labelled streptococci aerosol to which all guinea pigs were exposed
was generated under the same experimental condition. The frequency distribution
of these aerosols based upon their aerodynamic diameter is also shown (Figure 3).
Ventilatory measurements. Keasurements of frequency of breathing, tidal
volune, and minute ventilation were made at 10 minute intervals during plethysmo-
graph acclimitation and the HpSC^ exposure, and throughout the b minute radiolabelled
streptococcal aerosol exposure. No significant alterations in any of these
ventilatory parameters was observed which could be attributed to the HpSC^ exposure.
These conclusions are exemplified in the ventilatory data obtained from the
guinea pigs which were exposed to the 3020 ugm/m^ (high) H?SC, aerosol (Figure **•).
The reduction in breathing frequency observed during the first few minutes
after insertion into the plethysmograph reflects acclimitation to the apparatus
whereas the slight increase in both the HpSC^ exposed and control guinea pigs
during the latter 30 minutes of the experiment reflects increased alertness
of the animals as they began to recover from the effects of the sodium pento-
barbltal induced sedation.
HpSC;. effects en respiratory particle deposition. The 3020 ugm/nr K?3C;,
aerosol (l.S u C/.D) induced both a si,=7: if leant ir.crea.~e in the1 total respiratory
deposition of radioactive streptococcus aerosol and a proximal shift in the
-------
respiratory deposition occurred in 9 of 10 guinea pigs. The mean increase in
particle deposition rate in the PUSO^ group was 60^ greater than their
respective control values. This difference was highly significant when
analyzed by the paired t test ( F=<10.01, t=4.12, 9 df, one tail).
The increase in the streptococcus aerosol deposition rate occurred as a
result of an increase in particle deposition in the nasopharyngeal and/or
laryngeal region. The increase in nasopharyngeal deposition occurred as a
result of both an increase in the absolute number of radioactive counts detected
in the nasopharynx and a proximal shift in the amount of radioactivity detected
in this region. As a correlary to this redistribution to the nasopharynx a
corresponding reduction in aerosol deposition occurred in the lung.
Dose-Response experiments of H?SO/, effects on particle deposition. A series
of three dose-response experiments were conducted to determine the effect of
different H?SC^ concentrations on respiratory particle deposition in guinea
pigs (Table 2). As the concentration of P^SO^ was reduced the aerosol size
diminished concomittently. Therefore, the effect of r^SC^ concentration,
based upon mass, cannot be ascertained independent of the aerosol size.
The analysis of the data in Table 2 confirmed the hi^h concentration
HgSC^ aerosol effect of increasing the total respiratory deposition of radio-
active aerosol and inducing a proximal shift in particle deposition to the
nasopharyngeal region. The differences between the high HpSO^ and control
values remained at the same level of statistical significance as in Table 1
when the data was compared to the mean values obtained from 39 guinea pigs who
provided the control data for all three experimental groups (Table 2).
No effect on total or regional dejcsition rates was observed at the 320
UOT./m (0.6 u Ci-'.D) H?SC,( concentration. However at 30 ugm/n^ (0.25 u CrD)
HpSC^ concentration i F-i^nirica.vL ir^r'."•.:••- in radioactivity '•—.-• . *"' ir, ^-.<-
47
-------
trachea. A concomitant decrease in radioactivity in the lung occurred. A
9% increase in the total respiratory deposition rate was also observed at
the low HgSQj, concentration but this difference was not different from control
values at the 5% level of statistical significance.
The 53% total respiratory deposition rate in control guinea pigs and the
48.6, 5.0, and 46.4^ regional deposition rate in'the head, trachea, and lung
respectively reflect normal values which could be anticipated from the exposure
to the 2.6 u Ci'D radiolabelled streptccoccal aerosol to which the guinea pigs
had been exposed . (Figure 3)«
DISCUSSICN
The increase in respiratory particle deposition induced by exposure to
sulfuric acid aerosol represents an important biologic response which a-ids
to the evidence of -the irritant effect of this chemical. In addition, the
proximal shift in the pattern of regional deposition of radiolabelled aerosol
provides indirect evidence of the site of action of the HoSC^, aerosols. While
the underlying; physiologic mechanism which caused this response has not "been
directly addressed in these experiments, this data, coupled with previous
investigations of the physiologic action of HpSC^, inhalation in man and cruinea
3,6~,?
pigs , provides .a reasonable basis for cautious speculation about the
explanation for the phenomena we have observed.
The factors controlling particle deposition in the respiratory tract
are related to both the chemical-physical properties of the aerosols (in our
experiments both the H^SC^ and radiolabelled streptococcus aerosols) and the
respiratory physiologic response of the animal to these aerosols. Among the
physiologic parameters affecting respiratory particle deposition both vantilatory
and respiratory mechanical factors nay be involved. However the predominant
;hy,:ljl: - L-, fic.cr ?.ffscr in--- ro.r:Jc]'3 c;.;,:.: ticn in th>3 urr?r airway:
5-12
of air flow.
48
-------
We believe that HpSO. alterations in air flow dynamics in the respiratory
tract of guinea pigs is the raost likely factor contributing to the increase
in particle deposition and proximal shift in the pattern of regional aerosol
deposition in our experiments. We measured the ventilatory parameters of
tidal volume, frequency of breathing, and minute ventilation in all of our
experiments. None of these parameters were altered by X^oC^ inhalation, even
at our highest r^SC^ concentration (3020 ugr/P - 1.8 u CrJ)). These
7
observations are consistent with previous results. We did not make
measurements of mechanics of respiration. However A.viur has observed that
a 60 minute exposure to 1.9 mg/irx H?SC^ of 0.8 u diameter by weight or
o
6 mg/ni H?EC, of 2.5 u diameter by weight induced a 50^ increase in
pulmonary airflow resistance in guinea pigs. It is estimated that in
normal guinea pigs V;^ of total pulmonary airflow resistance measured by
U
the intrapleural pressure method is contributed by nasal airflow resistance.
We therefore presume that increased airflow resistance was experienced by our
guinea rl^s who were extosed to the hirh r' SC. aerosol (3^20 ugr./m-'-l.S u C..L].
£ ~r
A substantial amount of aerosol of this size would be cereal .';i in the nose
and nasopharynx, even disregard ing the possibility : ~ : tr.is r.
aerosol r.i~ht increase in oize in *r.e mifh nur.i ; ?.- ic-r''. =r:
of the head ar.d thus ircrease the probability of -:.:.- . ..rl detcsiticr. in this
region. To the extent that this aerosol was irri- 'ttir.•- to the nasopharvr.ge'.l
epithelium, airway narrowing could also have occui .--'- • .' :h would further
increase the probability of particle impact ion in th° :.?.£cpr.?.r}T.x consequent
to locally increased airflow resist?.!.ce. ijxpccure of rulr.ea pirs to X^.IC
concentrations lirre enoueh to kill them (an ei~ht hour LIr~. = 18-50 m.r/m-'
j -1
depending upon co-dy>;ei=ht) did so as a result of asphexia which was thcaght to
-------
Experimental evidence has shown a good correlation between airflow
resistance, including nasal airflow resistance, and particle deposition in
8,18
that region. Cigarette smokers, bronchitics, and asthmatics experienced
a proximal shift in the pattern of regional aerosol deposition in the respiratory
12 - ,
tract. Cur observations suggest that a siini3?r phenomenon occurs in response
to HpSC^ inhalation.
The low H2SO^ exposure (30 ugia/m^ - 0.25 u Ci'J)) also induced a proximal shift
in the pattern of regional deposition but under these circumstances the increase
occurred in the trachea. Deposition in this lower region of the respiratory
tract is not surprising since this very small aerosol size would facilitate
deep lung deposition. However we believe that some of the radioactivity
detected in the trachea may have originated from particles deposited initially
in the primary and perhaps secondary bronchi which were rapidly translocated
by mucociliary clearance to the trachea before the specimens were obtained, ",'e
attempted to minimize this possibility by using a short (U minute) radioactive
aerosol exposure and by obtainir.fr the specimens as rapidly as possible after
exposure. However, about 15 minutes was nevertheless required to disect the
specimens from the ar.i: al and this may have been sufficient time to permit
some translocation of radioactive material to the trachea.
The fact that the intermediate H2SC^ concentration (302 ugm/m - 0.6 u Z;.~->]
did not induce any change in the particle deposition parameters is interpreted
as evidence of insufficient concentration to induce irritation to the upper
airways, especially in the head, but a particle size which was too large to
permit sufficient mass to penetrate to a depth in the lung where adequate
stimulus to induce airv?.y r.arrowir.- was available. Thus we infer from the
dose-response experiments that the lung is much more responsive to airway
50
-------
Cur inferences concerning the site of deposition of our H?30^ aerosols
must retrain speculative, since no direct data is available concerning the
site of deposition of sulfuric acid aerosol. The need for such information
has been made apparent by the results of our studies. It is clear that the
particle deposition data presently available concerning inert, non-hydroscopic
aerosols is not applicable to K SC, aerosols which can he irritating to the
respiratory epithelium and are hydroscopic; both are factors which r.ay
contribute to alterations in the ncrnal pattern of deposition cf particles
in the respiratory tract.
Cur observations on increased particle deposition in the respiratory
tract lead us to conclude that H?SC exposure can result in significant
pathophysiolosic effects of greater consequence than respiratory physiologic
alterations per se. To the extent that H?SC^ exposure increases respiratory
particle deposition of either viable or non-viable particles tne increased
deposition rate n.ay result in a more severe biologic response. However,
the shift of the pattern of regional deposition to sites hirher in the
respiratory tract complicates our atter.pt to assess the ul^'-ate bi loric
consequence cf this phenomena. For sor.e par:3 el-,-, "_rtic';lirly r.or.-
viable particles, w;:ose health effect ;viy be pr~'-,- y*"-~"-' cr. t: e deep,
alveolar rericn of ti.e lun~ or where the route ci" ry into the body occurs,
such a response ;i.ay tend to reduce the pathologic cor.GC7.uer.ee of inhalation
of such particles. Cn the other hand, other parti;_ '.specially certain
nicrcorrar.ior.s, r.ay induce a -ore severe response if ,r.r; particles are
deposited hipher in the respiratory tract, '..'hile cnly a lir.ited arour.t of
information is availa.ble or. this subject, studies of rhir.ovirus, coxsackie
]_C .
virus, and rasteurella pestis inf^c^ior. surest tr.at this r.a'y be true.
51
-------
that sulfur oxide type air pollution was positively associated with an
increased incidence of an illness characterized as the common cold infection
In residents of New York City. Cur data also indicate that lung deposition
is diminished by H SO • The proximal shift in regional deposition of
particles induced by H SCV inhalation raises the question of if the lung
may, in a sense, be "protected" from the irritant effect of this inhalant.
But the consequences of this response may only manifest itself in a different
and perhaps more indirect and subtle way. Thus the net biologic effect of
this shift in the pattern of regional deposition of inhaled particles has
yet to be determined.
A crucial question which must be applied to these experimental results
is whether this response in guinea pigs reflects a response which occurs in
man. While as yet no direct data exists to answer this question, several
important ancillary bits of information are available which justify
cautious speculation on this subject.
Spidemiologic studies of air pollution health effects in areas with
predomintantly sulfur oxide type pollutants have continually reported
that the principal, most subtle health effects are increases in the incidence
of asthma attacks, clinical sy~.pto.~s of upper airway irritation, and
exacerbation of pre-existing illness in individuals with chronic obstructive
lung disease, particularly chronic bronchitis. The primary anatomic site
involved in all of these health effects is the airways and adjacent tissues
in the respiratory tract. We believe that subtle physiologic responses
z.ay occur in so:.e individuals which lead to increased deposition of
particulate matter in the respiratory system and ultimately nay lead to/or
enhance the disease processes mentioned previously.
52
-------
than concentrations which viould be expected to develop in the atmosphere.
An H^SG, concentration of 240 ugm/m-' has been observed in the Los Angeles
17
basin. However, as yet HpSC^ is not co.Tsr.only monitored in ambient air
and epidemiologic data is lacking concerning the ambient concentrations
which r.ay be hazardous to human health. The results of our dose-respor.se
experiments suggest however, that relatively lo.v concentrations of H?SC-,
may indice alterations in host response which may be deleterous to health.
As a result of the demonstrated sensitivity of the guinea pig to
sulfuric acid aerosol and ether respiratory irritants, as measured by
respiratory mechanical alterations, Amdur has suggested that the guinea
pig nay be a particularly suitable lower animal analog to human population
22
groups which are particularly sensitive to air pollutants. Cur observation
of increased respiratory particle deposition from •%,£(! exposure is a
manifestation of an effect which is an important ccrrelary to respiratory
airflow rr.echanical alterations, and reflects an important pathophysiolcgic
7
sequel to the physiologic responses observed earlier by Amdur. These
observations have tread r-'ir.~ir.r implications for health re-crcrses tc ctrc-r
resriratcry irrit?,nts as '..'oil which should "^e £tu':.-,- b-tfcre the full
sigrific^r.ce of this resrcr.se c?.r.-be as
53
-------
REFERENCES
1. French, J. 0., G. Lowrir.ore, W. C. Nelson, J. F. Finklea, T. Ensrlish,
and n. Hertz, The effect of sulfur dioxide and suspended sulfates on
acute respiratory disease. Arch. Environ. Health, 1973, 27 s 129-133.
2. Lewis, T. R., iv. 0. Andur, A. D. Fritzhand, and X. I. Campbell, Toxicology
of atmospheric sulfur dioxide decay products, Publication ^AF-111,
Environrr.ental Protection Apency, Research Triangle Park, Korth Carolina,
July, 1972.
3. Air.dur, r.. C., -L. Silver.Vian, and P. Drinker, Inhalation of H?SO, rriist
by human subjects. A.n.A. Arch. Ind . HyFj. Cccup. r.ed., 195^» ^005-3^3'
k. Bushtueva, X. A., Determination of the liiT.it of allowable concentrations
of atmospheric pollutants, Bock 3* Pyazaruv, V. A. Levine, and B. S.
Levine, U. 5. L-ept. of Commerce, Office of Technical S&rvices, washinrton,
D. C., 1957, ?. 2C-36.
5. Sin., V. and R. Pattle, Effect of possible SILOS irritants on huirian
subjects, J. A. A. A., 1957, 165:19-3-1913.
6. Toyar.a, T., and K. Nakaruira, Syr.ereistic response of h^roren peroxide
aerosols and SCp on pulmonary airway resistance, Ind. Health, 19^4,
2:3^-45.
7. A.T.dur, i\. C., The respiratory response of guinea piss to sulfuric acid
rr.ist. A.n.A. Arch. Ind. Health, I-?5?-» lS:aC?-^l^.
8. Hounar., R. F., A. Black, and A. V.'alsh, Deposition of aerosol particles
in the nasoTharyn^eal region of the hur^an respiratory tract. Nature,
1Q69, 221:125^-1255.
Q. Li1:'".-.-.!-!'". , - ., I>.rc3iA 1 or. and cjenrarce of inhaled 'articles on the huran
ncse, '\rn. Ctcl. J-'nir. and L~ryr:~., lc,-7;"> TC:1-1C.
10. Olass, P. arc ~~ . F. Teller, Acsosir.^rt of dynamics cf :".soThar;/r.real
air flow. V.er. Rev. ?esp. Dis-*, 19:°, 99:^3?-^39«
11. Li?pro.nr,, .-.. and R. S. Albert, The effect of particle size on the
regional deposition of inhaled aerosols in the hur.an respiratory tract*
Amer. Ind. Hyp:. Assoc. J., 19^9, 30:257-275*
12. Li^rrp.nn, .•.., R. E« Albert, and H. T. Peterson, The regional deposition
of inhaled aerosols in man, In Inhaled rarticles ar.d vapors. III.
Unwin Brothers Limited, Surrey, Enrland , 1971, p. 105-120.
13, Fairchild, G. A., ?. Kane, £. Adams, and D. Coffin, Sulfuric acid aerosol
effects or. clearance of streptococci from the respiratory tract of mice.
!'3.i ior.al I- r.viro '. \cntal ?".::'_r?h C:r.-_ :r, ?er-:-arch Tri-r.rle Park, North
Carolina, ir. manuscript, Cctol-^r, I1?7-.
-------
15» Amdur, i-i. 0. and n. Corn, The irritant potency of zinc ammoniuru sulfate
of different particle sizes. Amer. Ind. Hyg. Assoc. J., 1963, 24:326-333.
l6« Andur, M. 0. and J. Mead, A riethod for studying the mechanical properties
of the lunjjs of unanesthetized aninals: Application to the study of respiratory
irritants. Proc. Third Nat. Air Pol. Synpos., Pasadena, Calif., Stanford
Research Institute, 1955, p. 150-15'J.
I?. Amdur, r,. 0., R. Z. Schulz, and F. Drinker, Toxicity of sulfuric acid
mist to guinea pigs. A.h.A. Arch. Ind. Hyg. Cccup. i-.ed., 1952, 5O18-329.
18. Kouna.Ti, R. F., A. Black, and v.. Walsh, The deposition of aerosol particles
in the nasopharyr.f/eal region of the huinan respiratory tract. In Inhaled
particles and vaoors. III. Unwin Brothers Limited, Surrey, England,
1971, P. 71-79.
19. Couch, R. 3., V. Knight, F. J. Gerone, T. R. Gate, R. G. Douglas, Factors
influencing response of volunteers to ir.oculaticr. with coxsackie virus A
type 21, Amer. Rev. Resp. Dis., 1969» 99:2^
20. Druett, H. A., J. >.. Robinson, D. W. Henderson, L. Fackinan, and S.
Feacock, Studies on respiratory infection. II. The influence of aerosol
particle size on infection of the guinea pig with Fasteurella pestis.
J. Hyg. (Cair.b.), 1956, 5^37
21. Thompson, D. J., A. Lebowitz, 2. J. Cassell, D. Wolter, J. I'.cCarroll,
Health and the urban environment. VIII. Air pollution, weather, and the
common cold. A.T.er. J. Pub. Health, 1970, 60:731-739«
22. The sr.og problem of Los Angeles County, Third Interim Report, Stanford
Research Institute, Stanford, California. Cited in Arr.dur, .-i. 0.,
Toxicity of sulfuric acid r.ist to guinea pigs, A.n.A. Arch. Ir.d. Hyg.
Occup. i-.ed., 1952, 5O18-329.
23. Amdur, .-.. C., The irpact of air pollutants on pr.ysioloric risponsas of
the respiratory tract. ?roc. Ar.er. Phil. 5oc.» 197^, ll~:3-3.
55
-------
Legend
Figure 1. Guinea pig plethysir.ograph.
Figure 2. Guinea pig exposure apparatus,
33 *' "
Figure 3« Sulfuric acid and JJf labelled streptococcus aerosol sizes.
Figure 4. Effect of sulfuric acid inhalation on respiratory ventilation
in guinea pigs.
56
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Appendix B4.3
Sulfuric Acid Aerosol Effects on Clearance of Streptococci
From the Respiratory Tract of Mice
Fairchild, G.A., P. Kane, B. Adams, and D. Coffin
From the
National Environmental Research Center
Research Triangle Park, North Carolina 27711
July 1, 1974
63
-------
A recent epidemic logic study reported positive correlations between
elevated levels of sulfur oxides, namely total suspended sulfate and sulfur
dioxide, and increased incidence of asthma and symptoms of upper airway
irritation, especially laryngotracheobronchitis. The health effects were
more highly correlated with suspended sulfate than sulfur dioxide. Sulfuric
acid aerosol (H^SO.) concentration was not measured in this study, but it is
likely that it co-existed with the other sulfur oxides during the study period.
While the exact health effects of ambient exposure to any specific sulfur oxide
has not yet been completely defined, it is commonly hypothesized that these
chemicals are direct respiratory irritants or manifest their effect indirectly
through exacerbation of preexisting respiratory disease. However, the assess-
ment of the biologic action of specific, single pollutants in a complex, physio-
chemical atmospheric environment has proven to be a difficult challenge for
epidemiologists.
Experimental studies have previously demonstrated that I-LSO, aerosol can
be a direct irritant to the respiratory tract and induce a greater effect than
2-5
sulfur dioxide (SO^) alone. Short term exposure to H?SO, aerosol was irritating
to the respiratory tract of man and guinea pigs, as reflected by alteration in
2-4
mechanics of respiration, especially increased air flow resistance. This
physiologic measurement is a sensitive indicator of acute response to sulfuric
acid aerosol; it is usually reversible and may be without pathologic sequella, if
the concentration is low.
Pathologic consequences of a respiratory irritant may be manifested through
altered clearance of particles from the respiratory tract which may render the
host more susceptible to respiratory infection and/or disease. SCL and cigarette
smoke are examples of respiratory irritants which diminish mucociliary clearance
of particles under certain conditions. ~ The normal bacteriocidal capacity of the
lung may be altered by cigarette smoke and gaseous irritants such as ozone and
614
-------
nitrogen dioxide, thus rendering laboratory animals more susceptible to bacterial
. 10-13
pneumonia.
This investigation was therefore conducted to determine if H-SCL aerosol
exposure alters the clearance rate of viable bacteria or the physical clearance
rate of killed, radiolabelled bacteria from the respiratory tract of mice. This
study may provide some insight into a mechanism of action of H^SO. aerosol on
the pathogenesis of respiratory disease.
Method s_
*
Mj_ce_. Female, specific pathogen-free mice originally derived from the
Swiss-Webster strain were used in all experiments. The mice were 7-9 weeks old
and their bodyweight ranged between 25-29 grams.
Streptococci cultivation and tissue assay. A Group C Streptococcus
originally isolated from a hamster and subsequented passaged in mice in whom it
exhibits only moderate virulence. A large seed stock of streptococci was
proprogotcd in Todd-h'ewitt broth (THB) culture and 1 ml aliquots of this stock
were lyopholized and used in these experiments to provide a greater degree of
uniformity in replication of experimental results.
_2
An aliquot of streptococci seed stock was diluted to 5 X 10 from the
a 3 "-I* volur.p
original concentrate and was mixed with an equal volume of killed, radiolabelled
streptococci for aerosolization, jhe preparation of mdiolatelled streptococci
is defcrir^G in the follcr-,-:,'r.- seetjon.
Mice were exposed for 30 minutes to an aerosol of streptococci generated by a
***
nebulizer. The nebulizer was operated at 12 psi filtered air pressure. The
<
nebulizer air flow was diluted by secondary filtered air to provide a total
unidirectional flow through the animal exposure chamber of 20 liters/minute.
The lungs and nose of mice were removed immediately after cessation of
aerosol exposure and at 2 and 4 hours post exposure, and the number of viable
streptococci were determined in each tissue by a colony count assay.
J - — - -„ _ _-
*CD-1 rAcr, Cn.'irlr? River ibuse farms, Wilmington, Mass.
a^ch ?nd I r..b Srcctro ?0, Rochester, New York
Vaponc:>iirin Cc D-iny, "> ev-' York, :',ew York.
65
-------
The nose was obtained by removing the skin from the head and cutting the nose
from the skull at the median canthus of the eye. The tissues were carefully
ground in TUB in Ten-Broeck grinders and 0.05 ml of supernatent was decanted
and cultured for streptococci on each of 3 b.lpod, agar petri plates per 10 fold
specimen dilution. The number of streptococcus colonies were enumerated after
24 hours incubation at 37°C. The total number of viable streptococci in each
tissue was calculated after standardization for specimen dilution, This
exposure method resulted in a dose of streptococci to the lung of control mice
cells.
at 0 time of 5 x 10 to 1 x 10 f\ Such a dose seldom- produces pulmonary disease
in normal mice of 7-9 weeks of age.
Radiolabell ing__a_n_d radioactive tissue assay methods. The physical
33
clearance rate of particles was determined through the use of killed, Phosphorous
labelled streptococci. Radiolabelled streptococci were prepared by adding
~* *3 *3 "3
250 uc of J P as NaH PO^ to a 10 ml culture of streptococci in the early log
phase of replication. After about 18 hours of incubation the heavy growth of
streptococci was concentrated further by centrifugation, resuspended to the
original volume in broth and killed by heating to 56°C for 30 minutes in a
33
water bath. The streptococci were freed of unincorporated P by washing the
cells 4 times with broth. Preliminary experiments indicated that after 4
cycles of washing and resuspension in saline, 99.5^ of the radioactivity in the
suspension was associated with the bacteria. About 5o of the radioactivity
«
leached from the bacteria over a 24 hour period.
The killed radiolabelled streptococci were mixed with the suspension of
viable streptococci immediately before the aerosol exposure was begun.
The radioactivity in the nose, trachea, lung, and stomach of the mice was
determined by liquid scintillation counting. The trachea and stomach were
66
-------
processed in their entirety and 1 ml aliquots of the lung and nose
tissue were processed and counted after they were tested for viable
*
streotococci. The specimens were digested with a solulibizer ,
decolorized with an optimal amount of benzoyl peroxide, a fluor was
added, and the specimens were counted by a liquid scintillation counter.
After decolorization tissue quenching was no longer a problem except in
a few stomach specimens. Since this tissue is not a principal concern
of these studies, a quench correction factor for the stomach specimens
has not been applied.
The total activity in each tissue was determined after standardization
for specimen dilution factors. The radioactivity in the lungs of mice
immediately post exposure was approximately 6-8 times background. All
clearance rates are expressed as percentage changes from 0 time values.
Sulfuric acid generation and assay method. Two types of sulfuric acid
aerosols were generated. A dilute acid aerosol was produced by
nebulizing a 3.0% solution of IN H2SO. in deionized water. The concen-
trated (IN H-SO.) aerosol was generated by nebulizing the concentrated
acid into a 10 liter mixing chamber anddirecting only small portion of
the aerosol through an inlet port to ch-^ annual exposure chamber;
the remaining acid aerosol was vented to exhaust air (Figure 1). The
aerosols were approximately the same concent-a'ion but differed in
particle size and number per volume of air. Thus, one aerosol
(concentrated) contained a relatively high concentration of acid in
each droplet and the second aerosol (dilute) contained a relatively small
amount of acid in each droplet.
* Protosol, New r.n;'.la;ul N'uclt-.ir Corn., lioston, Massachusetts.
** .Viuaso], ;,V'.; i:iif,l''"'l Nuclear Corp., Boston, Massachusetts.
67
-------
The aerosols were generated with a nebulizer* operated with
filtered compressed air (12 psi) at an air flow of 5 liters per minute.
This aerosol was mixed with secondary filtered air to make a total
chamber air flow rate of 20 liters per minute. The temperature and
relative humidity in the animal exposure zone was approximately 23°C
and 70% respectively.
The concentration of acid in the animal exposure zone was determined
by collecting air samples periodically during the exposure period.
Usually 3 samples were obtained during each 90 minute exposure. Air
samples were obtained by liquid impingement and the acid concentration
was determined by determining the pH of the air samples and comparing
these results with a calibration curve. The pH method was compared to
!
j the barium sulfate method in preliminary experiments and was found to
] !
| be within 10% agreement. A calculation of the volume of H?SO.
i generated during the exposure and the chamber air flow conditions was
also compared with the pH method and was found to be within 10% of the
calculated values.
The particle size of the sulfuric acid aerosol was determined
with a right angle light scattering photometer containing multiple
channels for particle size differentiation. The photometer was
calibrated periodically with polystyrene spheres of known size
(0.6 and 1.0 microns). The particle size distribution of the con-
centrated and dilute acid aerosols is shown in Figure 2.
Hi s t op a t ho1o gy. For histopathologic study animal heads and lungs were
fixed in formaldehyde, and the heads decalcified. Three coronal sections
through the nose were prepared; at the incisors, midway between the inci-
sors and medial canthi, and at the medial canthi. Tissue blocks were
embedded in paraffin, sectioned at 6;i and stained with hematoxylin and
eosin. Microscopic sections were examined without prior knowledge of
expos u_re_c;roup.
* \',-i])()jic]'lirin Co; ijxiiiy, Ncv: York, .','cw York.
68
-------
^Experimental protocol. In one protocol mice were exposed to either
dilute or concentrated H SO aerosol prior to bacterial aerosol exposure.
The exposure regimen was for 90 minutes once per day for 4 days. Immediately
following the 4th H So exposure, acid exposed mice and cohort controls
were exposed to the streptococcal aerosol. After a 30 minute bacterial
exposure, 3-5 mice from each treatment group were killed immediately
(0 time) and at the 2nd and 4th hour post streptococcus exposure. The
nose and lung were alternately obtained from mice in each treatment
group for quantitative assays for viable streptococci. In addition
to the nose and lung, the trachea and stomach (including the esophagus)
33
were obtained for determination of P activity by a liquid scintillation
technique described above.
A second exposure regime was a four hour exposure of mice to
concentrated H?SO. immediately following a streptococcus aerosol exposure.
Mice were killed immediately after bacterial exposure (0 time) and
after the 2nd and 4th hour of H SO. exposure. The assay for viable for
33
bacteria and P radioactivity was the same as that described previously.
Each experiment was repaated at least once. The results were
similar and the data was pooled and analyzed. Any differences in
treatment groups were considered different at P=<0.05.
RESULTS:
The amount of radioactivity detected in the tissues of the
respiratory tract and stomach of mice exposed to labelled streptococci
for 30 minutes is presented (Table 1). No statistical difference was
found in the amount of radioactivity in tissues of acid exposed mice
compared to control tissues. An estimate of the sensitivity of the
detection method was obtained by comparing the total amount of
69
-------
33
activity detected in the tissues to the P concentration in the
air and aerosol size. The mi nu te volume was assumed to be
1^
1.25 ml/gin bodyweight according to the observations of Guyton . The
aerosol concentration was calculated from the radioactivity found
on air filters operated at 1 LPM for 6 minutes. A calculated 81%
33
^recovery .rate of P in mice was observed. Since it can be
anticipated that not all of the inhaled aerosol (3.2 microns in size)
will be deposited, an 81% recovery rate was considered very satisfactory
for our purposes.
CJoararice of radiolabelled streptococci from the nose and lung. Exposure
to concentrated IUSCL (15 mg/m^ of 3.2 micron CMD) prior to streptococcus
33
exposure induced a reduction in the clearance rate of * P labelled
streptococcus Prom the nose of mice (Figure 3). The reduction in nasal
clearance rate was statistically significant at 2 hours post exposure
(P = <0.05). This difference represented a 50% increase above the
control values. A difference between the treatment and control groups
remained at 4 hours post streptococci but it was not significantly
different. A substantial interanimal group variation occurred through-
out the acid treated groups- suggesting that certain mice may have
been more severely affected by the acid aerosol. These differences
contributed to the relatively large variance estimates of the H?SO
exposed groups of mice. No effect of dilute H SO, aerosol was
observed on nasal clearance of radiolabelled streptococci.
A small but statistically insignificant reduction was observed
in the radiolabelled streptococcal clearance from the lungs of mice
70
-------
I
exposed to the concentrated and dilute acid aerosol (Figure 4).
3
When mice were exposed to H?SO. aerosol (15 mg/m — 3 micron size)
33r
• for 4 hours immediately after streptococcal exposure the JJP label was
s
'. cleared more slowly from both the nose and lung (Figure 5). As
1
| happened in experiments mentioned previously, a substantial interanimal
>
variation in clearance rate in acid exposed mice was observed. The
difference in intergroup variability in nasal radioactivity between
the 2 hour H?SO, group and the control group was significantly
different by the F test analysis (F= 3.20; 8,24 df , P=<0.05).
Two experiments were conducted in an effort to establish the minimal
H?SO, concentration which would alter the rate of clearance of viable and
radiolabelled streptococci from the respiratory tract of mice. While the
mass concentration was reduced the particle size also was reduced
concommitently. The interpretation of these data must therefore take into
account both the mass concentration and also the reduced aerosol size. No
effect on radiolabelled streptococci clearance from the lung or nose was
observed in mice exposed to small particle FLSCK aerosol
(1.5 mg/m --0.6 micron CMD) (Figure 6).
33
The rate kinetics for the early, rapic phase of clearance of P
labelled streptococci from the nose and lungs if mice can be estimated
from these studies. The 50". clearance time from the nose during this
stage was approximately 2 hours with a diminution in physical clearance
rate becoming apparent during the second to fourth hour (Figures 3,5, and 6)
71
-------
About 65-70% of the P radioactivity remained in the lungs.of
mice four hours after the bacteria] exposure (Figures 4,5, and 6).
It can be assumed that a portion of the 3.2 micron aerosol to which
t he mice were exposed would have been deposited on the ciliated
epithelium of the airways of the lung and would be cleared by
mucociliary .activity. The radioactivity which remained probably
represented in large part that fraction of the aerosol which was
deposited in the alveolar region of the lung.
In all of the experimental regimes cited previously studies on
the "clearance" of viable streptococci from the nose and lung were
conducted 0 time, 2 and 4 hours post streptococcus exposure. No
alteration in the clearance of viable streptococci from the nose or
lung was observed in any of the mice exposed to H2SO. concentrations
3
as high as 15 mg/m either before or after streptococcus exposure
(Figure 7-8).
The rate of clearance of viable streptococci from the nose and lung
of mice was rapid and essentially the same. An 85% reduction in viable
streptococci was observed in both the lung and nose 2 hours after
bacterial exposure.. In absolute terms this represents a reduction
from about 9 x 103 to 1.3 x 103 from the lung and .4.4 x 102 to 9.4 x 10
in the nose.
Not all of the reduction in viable streptococci from the lung and nose can
be attributed to bacteriocidal defense mechanisms since mucociliary clearance
would combine with bacteriocidal action to reduce the number of viable
bacterial during the period of observation. The physical removal of bacteria
at the 4th hour post bacterial exposure could account for about 35% of the ,•
removal from the lung and 65-7Q» removal from the nose (Figures 3-6).
72
-------
Discussion-
A short term, high concentration exposure to H~SO. aerosol
q
(15 nig/in - 3.2 u CMD) induced a reduced rate of clearance of nonviahle,
radiolabelled streptococci from the nose and lung of mice. However, this
exposure regime did not produce an effect on the clearance of viable
streptococci from either the lung or nose.
The lack of effect on the clearance of viable streptococci from thj
lung may be explained, in part, on the basis of the particle size of the
aerosol. Much of the aerosol mass would have been expected to have been
deposited on the ciliated portion of the respiratory tract, including the
upper airways and nose; thus the macrophages in the alveolar region of the
lung may not have received significant exposure, Indeed, this aerosol size
was generated with the principal intention of determining the effect, of
HpSO. on physical clearance of particles and thus determine if an impair-
ment in mucociliary activity would occur.
It was of interest to observe, however, that, a small particle aerosol
3
(0.6 u- 1.5 mg/m ) of H?SO, did not alter the physical or viable clearance
rate of streptococcus from either the lung or nose. Wnile the concentra-
tion was 10 fold lower than that the concentrated I-LSO. aerosol the small
size should facilitate deep lung penetrat'o.. II may be hazardous to
hypothesize about the expected site of deposition of H~SO, aerosol however,
since we have, as yet, no data demonstrating the site of deposition of this
3
hydroscopic aerosol. Amdur observed that at a c nrvntration 2 mg/m' guinea
pigs exposed to a 0.8 u MMD aerosol experienced a more rapid, and greater
increase in pulmonary air flow resistance than when they were exposed to u
4
2.5 u MMD aerosol. These observations provide indirect evidence for deeper
lung penetration of small particle H^SO, aerosol.
73
-------
We were not completely successful in our attempt to evaluate the
effect of an aerosol containing droplets of concentrated H?SCK
in contrast to an aerosol of the same mass concentration but composed
of droplets of dilute HpSO. in water. We attempted to create these
contrasting conditions in an effort to provide an inhaled dose of
H?SO. which would be deposited over a wider surface area of the
respiratory tract. While the total mass concentration was comparable,
the particle size of the dilute aerosol was smaller than the concentrated
H SO, aerosol (2.7 u compared to 3.2 u). The rate of regional
deposition of these two aerosols would therefore be different and the
resultant effects on particle clearance rate concommitently affected.
The size difference could have occurred because of the difference in
solute concentration of the H?SO. and consequent difference in the
rate of drying of the aerosols as they flowed through the apparatus.
Therefore while the concentrated acid aerosol had the greater effect
on diminution of particle clearance from the nose, this difference
may be attributed to a greater deposition rate in the nose because
of the larger particle size.
Few or no lesions were observed in the nasal or pulmonary
epithelium of mice exposed to the concentrated H?SO. aerosol. A
few areas of low grade epithelial inflammation were observed in the
nose of an occasional H SO. exposed mouse. A similar lesion was
occasionally observed in the nose of control mice which lends doubt
to the specificity of this lesion as an H?SO, effect. However this
low grade lesion which occurred sporadically may have contributed to
-------
33
the interanimal variability of P labelled particle clearance from
the nose. These results indicate that particle clearance is a more
sensitive indicator of H^SO. effect under the experimental conditions
used here, as effects on particle clearance were not accompanied
by morphologic evidence of tissue damage as determined by light
microscopy.
The alveolar macrophage has been shown to play a key bacteriocidal
role in protecting the alveolar region of the lung against infection.
Green and Kass have demonstrated this role of the alveolar macrophage
through studies of the rate kinetics of clearance of viable staphylo-
coccus aureus from the mouse lung in contrast to slow disappearance
of the radiolabelled bacteria from the lung reflective of mucociliary
15
clearance . Our data on the rate kinetics of clearance of viable
streptococci from the mouse lung is similar to that of Green and Kass.
33
However, the rate of removal of the "' phosporous label1 ed streptococci
33
from the lung was faster in our experiments. Since the rate of P
removal is an indicator of mucociliary activity, it can be inferred
that a higher-percentage of streptococci we' „ deposited in the airways
of the lung in contrast to the deposition rate in this site observed
in the experiments of Green and Kass. This hypothesis is consistent
with the observed differences in the bacterial aerosol size between
our experiments. Our streptococcal aerosol size was larger (3.2 u CMD)
whereas Green and Kass reported their aerosol size was 90% less than
15
3 u (no frequency distribution was reported).
However, since the rate of clearance of viable streptococci
was the same in both investigations, despite our larger aerosol size,
75
-------
the possibility that bacteriocidal substances in the mucus lining
the airway epithelium plays a significant role in killing viable
streptococci is inferred. This is suggested by the observations
of the rate kinetics of viable and radiolabelled streptococcus
33
clearance from the nose. While the concentration of P labelled
streptococci was only 35% of 0 time control values at 4 hours post
streptococci exposure, only 10% of the viable streptococci remained
at 4 hours, a rate similar to that in the lung. Phagocytosis of
bacteria by neutrophils plays a role in clearance of viable bacteria
from the nose. Also, non-cellular substances in nasal secretions,
including lysozyme, may play a significant role in clearance of viable
streptococci from the nose in conjunction with mucociliary activity.
These data describe an experimental circumstance in which H?SO.
aerosol impaired the mucociliary defense mechanism of the respiratory
2
tract. The concentration which induced this impairment (15 mg/m )
is, however, much greater than concentrations which would be expected
to exist in ambient air, even during severe pollution episodes.
Whether this response can be associated with human responses to
H SO. remains debatable. It does, however, reflect an important
physiological response to a respiratory irritant that was not
accompanied by structural evidence of respiratory disease.
76
-------
References
1. French, J. G., G. Lcrinrore, W. C. Nelson, J. F. Finklea, T. English,
and M. Hertz. The effect of sulfur dioxide and suspended sulfat.es on
acute respiratory disease. Arch. Environ. Health 27_: 129-133, 1973.
2. Amdur, M. 0., L. Silverman, and P. Drinker. Inhalation of sulfuric
acid mist by human subjects. A MA Arch. Indust. Hyg. 6>:305~317, 1952.
3. Amdur, M. 0., R. Z. Schulz, and P. Drinker, Toxicity of sulfuric acid
mist-to guinea pigs. A MA Arch. Indust. Hyg. 5^:318-326, 1952.
4. Amdur, M. 0. The respiratory response of guinea pigs to sulfuric acid
mist. AMA Arch. Indust. Hyg. 18:407-414, 1958.
5. Amdur, M. 0., Toxicologic appraisal of particulate matter, oxides of
sulfur, and sulfuric acid, J. Air. Poll. Control Assoc., ]_6:638-644, 1969,
6. Andersen, I., G. R. Lundqvist, P. L. Jensen, and D. F. Proctor. Human
responses to controlled levels of sulfur dioxide. Arch. Environ.
Health 28:31-39? 1974.
7. Spiegelman, J. R., G. D. Hanson, A. Lazarus, R. J. Bennett, M. Lippmann,
and R. E. Albert. Effect of acute sulfur dioxide exposure on bronchial
clearance in the donkey. Arch. Environ. Health j_7_:321-326, 1968.
8. Albert, R. E., J. R. Spiegelman, O.Shatsky, and M, Lippmann, The effect
of acute exposure to cigarette smoke on bronchial clearance in the
miniature donkey. Arch. Environ. Health _1_8_:30-41, 1969.
9. Rylander, R. Lung clearance of particles and bacteria: Effects of
cigarette smoke exposure.A rch. Environ. Health 23_^321-326, 1971,
10. Green, G. M., and Carol in, D.: The depressant effect of cigarette
smoke on the jn_ vntro antibacterial activity of alveolar macrophages,
New Eng. J. Med." 276.:421-427, 1967.
11. Goldstein, £'. , W. S. Tyler, P. D. Hoeprich, and C. Eagle, Ozone and
the antibacterial defense mechanisms of the murine lung. Arch. Intern.
Med. J_27_: 1099-1102, 1971.
12. Coffin, D. L. and D. E. Gardner, Interaction of biological agents and
chemical air pollutants, Ann. Occup. Hyg., ]_5_:219-234, 1972.
13. Ehrlich, R., and M. C. Henry, Chronic toxicity of nitrogen dioxide, I.
Effect on resistance to bacterial pneumonia. Arch. Environ. Health,
17:860-865, 1968.
77
-------
14. Guyton, A. C. Measurement of the respiratory volumes of laboratory
animals. Amer. J. Physiol. 1^0,:70-77, 1947.
15. Green, G., and E. H. Kass. The role of the alveolar macrophage in the
clearance of bacteria from the lung. J. Exper, Med. 119:167-176, 1964,
16. Green, G. M. The response of the alveolar macrophage system to host
and"environmental changes. Arch. Environ. Health 18:548-550, 1969.
78
-------
LEGEND FOR FIGURES
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Animal exposure apparatus.
Aerosol size distribution.
Effect of H~SO. on clearance of radiolabelled streptococci
from the nose of mice: H?SO. before streptococci.
Effect of H-SO^ on clearance of radiolabelled streptococci
from the lungs of mice: H-SO. before streptococci.
Effect of HpSCL on clearance of radiolabelled streptococci
from the lung 2nd nose of mice: Concentrated H^SO, after
streptococci.
Effect of HpSCL on clearance of radiolabelled streptococci
from the lung and nose of mice: dilute H-SCL after streptococci.
Effect of HpSO. on clearance of viable streptococci from the
lung of mice: HpSO, before streptococci.
Effect of HpSO. on clearance of viable streptococci from the nose
of mice: H~SO, before streptococci.
79
-------
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Figure 1. Animal exposure apparatus.
1. COMPRESSED AIR
2. PRESSURE REGULATOR ;
3. FILTER |
4. VALVE !
5. NEBULIZER
6. MAGNEHELIC PRESSURE GAUGE
7. ORIFICE PLATE
8. ANIMAL CAGES
9. GAS WASHING BOTTLE
10. EXHAUST FAN
_j
: AEROSOL BYPASS —
HUMIDITY
TEMPERATURE
AEROSOL SIZING
81
-------
stribution
size
o
01
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CD
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Figure 3. Effect of H?S04 on clearance of radiolabolled streptococci
from the nose of mice: H-SO,, before streptococci.
c/o
O
UJ
EC
O
O
O
O
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a.
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a:
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o
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O CONTROL;_N__=JL4-l5:
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A DILUTED_H2S04; N =7
MEAN*SE
- \\ \
\\
30
20 _.
2 4
HOURS AFTER 33p STREPTOCOCCI EXPOSURE
83
-------
Figure 4. Effect of H0SO. on clearance of radio-labelled streptococci
from the luhgs of mice: HgSO^ before streptococci.
CD
z:
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o
o
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cc
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li.
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A DILUTE H2S04; N~=10~~
AMEAN±SE
2 4
HOURS' AFTER 33P STREPTOCOCCI EXPOSURE
-------
Figure 5. Effect of H?SO^ on clearance of radiolabelled streptococci
from the luilg and nose of mice: Concentrated M9SO. after
streptococci. *• 4
; SIGNIFICANT DIFFERENCE AT P = <0.05.
t 'FiTEST; SIGNIFICANT DIFFERENCE AT P = < 0,05.
; N=9
O NOSE-CONTROL; N =25
HOURS AFTER STREPTOCOCCI EXPOSURE
85
-------
Figure 6. Effect of H-SO. on clearance of radio!abelled streptococci
from the lung and nose of mice: dilute H?S(h after streptococci
100
O CONTROL,-LIK.'G; N =32
O HoSO* - LUNG; H =10
A'CONTROL-NOSE; N =32
N=10
WEAN ± SE;
: 1.5 mg/m3 H2S04
MICROMETER AEROSOL SIZE
4
HOURS AFTER STREPTOCOCCI EXPOSURE
86
-------
Figure 7.
Effect of H2>SO/
lung of mice:
clearance of viable streptococci from the
\ 1 r- . . .
on
HpSO. before streptococci.
-LI
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O CONTROL' N =14-15
; N_=8
DILUTE H2S4;N= 9-11
HOURS AFTER STRE-J'TOCQCC1 ! XF'OS'JRE
87
-------
figure 3. Effect of H^SO, on clearance of viable streptococci from the nose
of mice: HS0 before streptococci.
(H2S04 PRE STREP)
!DILUTE HSO/; N =7-9,
2 4
HOJfIS AFTER'STRL'PTOCOCC! EXPOSURE
88
-------
Appendix B4.4
AMMONIUM AND SULFATE ION RELEASE OF HISTAMINE
FROM LUNG FRAGMENTS
Jeffrey M. Charles, B.A., and
Daniel F. Menzel, Ph.D.
Departments of Physiology and Pharmacology and Medicine
Duke Medical Center
Durham, North Carolina 27710
89
-------
ABSTRACT
In vitro studies, presented here, with guinea pig lung fragments
"•""•--•• • "\
incubated with 10-20CmM concentrations of ammonium ion demonstrated the re-
lease of significant quantities of histamine. Of the cations tested with
ammonium ion, sulfate was the most potent, while nitrate and acetate ions
were of intermediary potency and chloride less potent. An osmotic effect is
unlikely since equal concentrations of sodium chloride failed to release
histamine. Isoproterenol, known to decrease anaphylactic histamine release,
and acetylcholine, known to increase histamine release, had no effect on the
ammonium sulfate mediated release of histamine. N ,2'-0-Dibutyryl adenosine
3',5' monophosphate (dibutyryl c-AMP) was also ineffective. These studies
suggest that the inhalation irritation associated with certain sulfate and
other salts, may be a function of their ability to release histamine in the
presence of ammonium ion.
90
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INTRODUCTION
Studies of atmosphere chemistry have shown that a portion of the sulfur
dioxide emitted into the atmosphere undergoes oxidation, leading to the for-
mation of sulfuric acid and sulfate particulates. Amdur et_ al_. (1), have
provided data demonstrating that some of these particulate sulfur oxidation
products have a greater irritant potential than sulfur dioxide itself. McJilton,
Frank and Charlson (2) showed that sulfur dioxide was more irritating with in-
creasing humidity suggesting that sulfuric acid or sulfate was responsible for
the irritation. Catalytic converters, to be installed on all new automobiles
in 1975, may become a new source of increased sulfates and sulfuric acid mist
in urban areas. The observations of Amdur and Corn (3) that ammonium sulfate
was highly irritating led us to examine the effects of ammonium sulfate and
other salts directly on the histamine release from fragments of guinea pig
lung in vitro.
METHODS AND MATERIALS
Male Camm-Hartley guinea pigs (Camm Laboratories, Wayne, N. J.), weighing
between 300-400 g, were anesthetized and the lungs excised. The lungs were
cut into fragments (75-150 mg), which were washed repeatedly with Tyrode's
solution until the fragments were free of blood. The Tyrode's solution con-
tained 0.9 g NaCl, 0.02 g KC1, 0.02 g CaCl2, 0.01 g MgClg, 0.1 g glucose, 0.1
g NaHC03, and 0.005 g NaH2P04 per 100 ml. One or two lung fragments were
employed in each test as a sample weighing 150-200 mg. The samples were then
suspended in 3.0 ml of Tyrode's solution (pH 7.4) containing varying concen-
trations (10-200mM) of the various salts under study. The fragments were
incubated in a shading v,'2tGr bath et 37°C for 30 minutes and ths supernatant
assayed for the histamine released. The salts studied in this manner were
91
-------
sodium chloride, sodium sulfate, ammonium chloride, ammonium sulfate, ammonium
nitrate and ammonium citrate. Isoproterenol and acetylcholine were tested by
preincubating the fragments for 5 minutes with 10" and 10~ M concentrations,
respectively, before the addition of the salts. Dibu^tyryl c-AMP was tested in
a similar manner at 10" M. Total histamine (pq/gm lung) was determined by boil-
ing fresh lung slices for 8 minutes and assaying the supernatant.
dj-losproterenol HC1 , acetylchol ine MCI, histamine diphosphate, dibutyryl
c-AMP and o-phthaldialdehyde v/ere obtained from Sigma Chemical Co. (St. Louis,
Mo.).
Histamine assay
Histamine was measured spectrophotofluorometrically (4,5). Two tenths
mi Hi liter of 70* perchloric acid was added to 2.0 ml of sample solution and
the mixture incubated for 30 minutes. One milliliter of the supernatant was
added to 0.75g Nad and then 1.5 ml n-butanol and 0.3 ml 5N NaOH were added
and mixed. The lower aqueous phase was removed by suction and 1.5 ml IN NaOH
saturated with NaCl was added to the remaining phase and mixed. One milliliter
of the upper butanol layer was transferred to 1.5 ml hexane and 1.25 ml 0.1N IIC1
and the mixture agitated. The upper organic phase was removed by suction and
0.45'ml of the acid phase was added to 0.1 NaOH and 0.05 ml of a 0.5% methanolic
solution of o-phthaldialdehyde. The reaction was stopped after 4 minutes by the
addition of 0.05 ml 3N HC1 . After the addition of 1.5 nil distilled water, the
fluorescence was measured at 450 nm by excitation at 350 nm.
RESULTS
Lung fracnents inci'bat?d v/i to ^rmem'in r.uH^tf, a ^ionium nitrpt^, c1 rioviiT'1
92
-------
in proportion to the concentration of the salts present (Fig. 1). Sodium sulfate
and sodium chloride, however', did hot release any detectable histamine. These
salts that did release histamine, had varying efficacies. The most efficacious,
ammonium sulfate, showed maximal histamine release at a concentration of lOOmM
after 30 minutes. The ammonium sulfate mediated release was equal to 97% of the
total histamine content of the guinea pig lung. The total histamine present in
the guinea pig lung was found to be 27.0 + 3.0 yg per gram of tissue. This
value for the total histamine content compares favorably with that reported by
Shore et_cil_. (4). The relative potencies of the various salts are listed in
Table 1, with ammonium sulfate arbitrarily assigned a value of 100%.
Guinea pig, monkey, and human lungs release histamine and slow reacting
substance of anaphylaxis (SRS-A) on stimulation by immunoglobulin E (IgE) medi-
ated antibody-antigen reaction (6,738). On the basis of studies with drugs known
to influence cellular levels of c-AMP, it appears that the c-AMP system may be
capable of modulating the imnunological release of histamine and SRS-A from human
and guinea pig lung fragments (7). The p-adrenergic agonists, isoproterenol and
epinephrine, which are known to increase cellular levels of c-AMP, inhibited the
antigen induced release of histamine and SRS-A (9,10,11). Predominantly a-adren-
ergic agonists such as phenylephrine and norepinephrine, probably decrease c-AMP
cellular levels and enhance the release of histamine and SRS-A (12). Acetylcholine,
working through the c-GMP system, has also been shown to increase the release of
these vasoactive substances (12).
In the studies presented here lung slices were preincubated in the presence
of 10" M isoproterenol for 5 minutes and the release of histamine determined up-
on the addition of ammonium r>ulfc;te, ^niionium chloride, or sodium sulfate, 30
minutes later. Table 2 shows that the presence of isoproterenol had no effect
on histamine release, as compared to incubations in its absence.
93
-------
Similarly, the presence of 10~'M acetylcholine was investigated and no
significant change in histamine release was noted (Table 3). Dibutyryl c-AMP
was preincubated with the lung fragments at a concentration of 10" M for 5
minutes before the addition of the salts and again no significant change in
total histamine release was seen (Table 4).
COMMENT 7
In order to estimate the hazard of inhalation of sulfur dioxide, sulfuric
acid and sulfate salts to man, the biological potency of these agents must be
known. Epidemiological studies suggest that the incidence of lower respiratory
diseases are more closely associated with suspended sulfates than sulfur dioxide
(13).
Measurements of ambient sulfate levels in the Hudson River Valley during
1970-1971 were conducted by cascade impacter collection. Sulfate residues were
found on plates 5 and 6 and the backup filter of an Anderson impacter. The mole
ratio of ammonia to sulfate was 1:1 indicating that ammonium sulfate is a prin-
cipal component of sulfate residues in the atmosphere (R. L. Bradow, Ph.D., per-
sonal communication). The present output of auto exhaust catalytic converters
is mainly as sulPuric acid. Ammonia present in the environment probably converts
this mist to a mixture of ammonium sulfate and sulfuric'acid. Present plans for
second generation auto exhaust devices include the addition of a reducing catalyst
to convert NO to nitrogen. Carbon monoxide serves as the source of electrons
A
and during the incomplete reduction of N0v ammonia is the favored product. Ammcnia
could also be supplied from the blood forcing ammonium sulfate.
/•;ndurs et al . (1) tnd /V.idur and Corn (3) studied the irricanL potency oT zinc
-------
was least potent, it was considerably more irritating than sulfur dioxide. The
.observations of McJilton, Frank and Charlson (2) support the concept that certain
sulfate salts are bronchoconstrictors. Nadel e_t al_. (14) have shown that a zinc
ammonium sulfate aerosol produces a physiological response similar to that pro-
duced by a histamine aerosol, but lesser in degree. The experiments presented
here are in agreement with these findings and indicate that the release of
histamine may be important in the mode of action of these salts.
Because sulfate residues exist mainly as particulates in the atmosphere,
the local alveolar concentration resulting from the dissolution of a respirable
parti cul ate could be high. It appears that ammonium sulfate, in the concentration
of lOOmM, releases approximately 100% of the stored histamine in the guinea pig
lung. An equal ammonium ion concentration (ZOOmM ammonium chloride) released
only half of the histamine stores. Since lung fragments in the presence of
200mM sodium chloride exhibited no detectable release of histamine, the histamine
released by ammonium chloride is ascribed to the ammonium ion. Similarly the
difference observed in histamine released between lOOmM ammonium sulfate and 200mM
ammonium chloride must be some function of the-sulfate anion.. The sulfate ion,
per se , had no inherent toxicity alone, since sodium sulfate at concentrations
*
ranging from 10 to 200mM caused no histamine release. The presence of ammonium
ion seemed to be a necessary factor for sulfate mediated histamine release.
Ammonium nitrate and acetate were of intermediary potency in histamine release.
These experiments also demonstrated that the histamine released by
ammonium and sulfate ions cannot be modulated by either c-AMP or c-GMP, since
isoproterenol , acetylcholine and dibutyryl c-AMP failed to influence histamine
release.
\. 1 j ; haf, c uoj-j';, L'_:d i.'u.'_ ino M»i.ii1
a variable number of 0-sulfate groups of the heparin of the mast cell granule
* * 95
-------
bind histamine. At high salt concentrations' the dissociation of the heparin-
histamine complex occurs. A similar release phenomenon may occur here also,.
Investigation in the cellular uptake of sulfate, in the presence and absence
of ammonium ion is now being undertaken to elucidate-the mechanism of the
observed histamine release.
96
-------
REFERENCES
\
1. Arndur MO: The imoact of air pollutants on physiologic responses of the
respiratory tract. Proc. Am. Phil . Soc. 14:3-8, Feb. 1970.
2. McJilton C, Frank R, Char!son R: Role of relative humidity in the
synergistic effect of a sulfur dioxide-aerosol mixture on the lung.
Science 182:503-4, 1973.
3. Amdur MO, Corn' M: The irritant potency of zinc ammonium sulfate of
different particle sizes. ATI. Ind. Hyg. Assoc. J. 24:326-333, July-
August 1963.
4. Shore PA, Burkhalter A, Cohn VH: A method for the fluorometric assay of
histamine in tissues. J. Pharrcacol. Exp. Ther. 127:182, 1959.
5. Tauber Al „ Kaliner M, Stechschulte DJ, et al : Imrnunologic release of
histar.iine and slow reacting substance of anaphylaxis from human lung;
V. Effects, of prostaglandins on release of histamine. J. of Inrnunol.
111:27, 1973.
6. Stechschulte DJ, Austen KF, Block KJ: Antibodies involved in antigen
induced release of slow reacting substance of anaphylaxis (SRS-A) in
in the guinea pig and rat. J. Exp. Mgd. 125:222-247, 1967.
7. Orange RP, Austen WG, Austen KF: Imnunological release of histamine and
SRS-A from human luna; I. Modulation by agents influencing cellular
levels of c-AMP. J/Exp. fled. 134:136S, 1971.
8. Ishizeka T, Ishizaka K, Orange RP, et al: The capacity of human IgE to
mediate the release of histamine and SRS-A from monkey lung. J. of
Irnmunol_._ 104:335, 1970.
9. Orange RP, Kaliner MA, Laraia PJ, et al: Iraiunological release of
histamine and slow reacting substance of anaphylaxis from human lung;
II. Influence of cellular levels of cyclic AMP. Fed. Proc. 30:1725-
29, 1970.
10. Lichenstein LM, M-:rgo~lis S: Mictamine release in vitro: Inhibition by
catecho!amines and methylxanthines. Science^ 161:902, 1968.
11. Schmutzlor W, Dei-wall R: Experiments en the role of c-AMP in auinea pig
anaphylaxis. Int. Arch. Allergy 45:120, 1973.
12. Kaliner- M, Orange RP, Auster KF: Immmologi cal release of histemins f.nd
slow rrrv.tinq subst; r.cc of rnr: ny hxis fV',, ivj.'ar; lu:,r:: IV. Fnhi.nc;;:':-:^.
by ci:ol ihcrgic arid a adrenergic stimulation. J. Exp. Med. ir.5:5J6, 1973.
97
-------
13. Finklea JF, Shy CM, Love GJ. et al: Health consequences of sulfur oxides:
Summary and conclusions based upon Chess studies of 1970-1971, in: Health.
Conse_quen_ces of Sul fur Oxides: A Report from Chess, 1970-71. U. S. %
ErwironrnGnTaT~Protection Agency, May 1974, p. 7-9.
14. Nadel JA: Location and mechanism of airway constriction after inhalation
of histanrine aerosol and inoroanic sulfate aerosol, in Davies CN (ed):
Proc Second Interna_tion_al__Sy:nposi urn on Inha_led Particles and Vapors .
Oxford~,~'Qrgamon Press, 1966, p."5o~ ~
15. Lagunoff D: Analysis of dye binding sites in mast'cell granules.
Biochemistry 13:3982-86, 1974.
98
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ACKNOWLEDGMENTS
We wish to thank Drs. David Coffin and Robert Frank for their encouragement
and support. We also wish to thank Dr. R. L. Bradow of the U.S.E.P.A. for shar-
ing with us his unpublished data on ammonia and sulfate residues in the environment
and for consulting with us on the chemistry of auto exhaust catalytic converters
which was invaluable. This work was supported by a contract from the U.S.E.P.A.
number 797576.
104
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APPENDIX B&. 5",
Toxiciry OF PALLADIUM, PLATINUM, AND THEIR CO.MPOUNDS
•
AN ANNOTATED BIBLIOGRAPHY
Kelga Gerstner
June 1973
TOXICOLOGY INFORMATION RESPONSE CENTER
Work supported by the Toxicology Information Program,
National Library of Medicine, National Institutes of Health,
• Department of Health, Education, and Welfare
under NLM Interagency Agreement "o. 40-274-71
(This report is reproduced on recycled paper.)
OAK RIDGE NATIONAL LABORATORY
Oak Ridge, Tennessee 37830
operated by
UNION CARBIDE CORPORATION
for the
U. S. ATOMIC ENERGY COMMISSION
105
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PREFACE
The Toxicology Information Response Center (TIRC) began
operation in 1971 to establish a national and international
center of toxicology information. Toxicology is defined in its
broadest sense as the science dealing with the adverse effects
of chemical and physical agents on living systems. TIRC gathers,
selects, and disseminates toxicology and peripherally oriented
information on drugs, food additives, pesticides, industrial
chemicals, and environmental pollutants..
Sponsored, by the National Library of Medicine's Toxicology
Information Program (NLM/TIP) and located at the Oak Ridge
National Laboratory (ORNL), TIRC is part of a larger scientific
and technical information division known as the Environmental
Information System Office (EISO). EISO coordinates the functions
and activities of environmentally oriented information units within
ORNL. This association affords a unique opportunity for extensive
scientific and technical interactions. It is in this atmosphere
that TIRC provides vast toxicology information.
Requests for information or literature searches should be
directed to:
Toxicology Information Response Center
Environmental Information System Office
Oak Ridge National Laboratory
Post Office Box Y, Building 9224
Oak Ridge, TN 37830
Telephone: Area Code 615 483-8611 Ext. 3-5296
FTS 615 -183-5296
106
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PART I
TOXICITY OF PALLADIUM AND PALLADIUM COMPOUNDS: AN ANNOTATED BIBLIOGRAPHY
TIP 73-182-1204
Compiled and Annotated by: Helga Gerstner
June 1973
Abstract
This annotated bibliography of 28 references was compiled by"the Toxicology
Information Response Center in answer to a specific request. Coverage of
the abstract literature spans the years 1937 to 1973 and yielded citations
from 1938 to 1972. The citations are divided into two sections, secondary'
references arranged by year and primary references arranged chronologically
by year and alphabetically by first author within each year.
Sources Searched:
Vol. 31: 1937 to Vol. 78(19):
Vol. 18: 1944 to Vol. 55(8):
MEDLINE Data Base
TOXLINE Data Base
Chemical Abstracts:
May 14, 1973
Biological Abstracts:
April 15, 1973
Cumulated Index Medicus: Vol. 1: 1960 to Vol. 12: '1971
and Vol. 23: 1938 to Vol. 48: 1950
Nuclear Science Abstract Journal: Vol. 24: 1970 to
Vol. 26: 1972
Toxicology Information Response Center's Library
Terms Searched: Palladium
Palladium Compounds
TIRC Keywords:
Toxicity
Metals
Exposures
NTIS Keywords:
Toxicity
Metals
Palladium
107
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Secondary References '
Anonymous
Encyclopaedia of Occupational Health and Safety, Vol. II,
p. 996
International Labour Office CH 1211, Printed in Tisk,
Czechoslovakia (1972)
Anonymous
Encyclopaedia of Occupational Health and Safety, Vol. I,
p. 270
International Labour Office CH 1211, Printed in Tisk,
Czechoslovakia (1972)
Christensen, H. E. (Ed.)
The Toxic Substances List, p. 387
U. S. Department of Health, Education, and Welfare; Health
Services and Mental Health Administration; National
Institute for Occupational Safety and Health;
Rockville, MD (1972)
LD of palladium chloride administered intravenously
to rabbits was 19 mg/kg.
Stecher, P. G. (Ed.) ' .
The Merck Index* 8th Ed., p. 778 •
Merck & Co., Inc., Rahway, NJ (1968)
LD of palladium chloride i.v. in rabbits is 18.6
mg/kg; does not cause the same skin sensitization
observed with platinum. Animal experiments indi-
cate low toxicity. _
Sax, N. I. (Ed.)
Dangerous Properties of Industrial Metals, 3rd Ed., pp. 990-1
Van Mostrand Reinhold Co., New York, NY (1968)
Palladium chloride, administered orally at "about 1
grain daily for treatment of tuberculosis f caused
no toxic effects; applied locally to skin,, palla-
dium chloride showed little or no irritation.
Intravenous injection to animals produced damage
to bone marrow, liver, and kidneys.
Patty, F. A. (Ed.)
Industrial Hygiene and Toxicology, 2nd Ed.
Vol. II, Toxicology, pp. 1125-31; Fassett, D. W. ; Irish, D. D. (Eds.)
Interscience Publishers, John Wiley & Sons, New York, NY (1967)
I As of 196J, no threshold limit had been set by an official
agency in the United States for any of the Pt group metals
including palladium or any of their compounds.
108
-------
, Primary References
' 1972
i
Christerisen, G. M.
Effects of metal cations and other chemicals upon the in vitro
activity of two enzymes in the blood plasma of the white sucker,
Catostornus coinrnersoni (Lacepede)
Chem-Biol. Interactions 4(5): 351-61 (1972)
Relative changes in the activity of glutamic oxalacetic
transaminase (GOT) and lactic dehydrogenase (LDH) in
blood plasma were determined after incubation with 49
compounds. GOT was most sensitive to Ag and Jig; LDH
to Pd and fjg.
Shishniashvili, D. M. ; Lystsov, V. M. ; Moshkovskii, Yu. Sh.
Spectrophotometric studies of the interaction of palladium
Soobshch. Akad. Wauk Gruz. SSR 66(1): 181-4 (1972) (Russ)
Spectral changes proved that the action of Pd was on
the electron system of purine and pyrimidine chains.
Shishniashvili, D. M.; Lystsov, V. N.; Moshkovskii, Yu. Sh.
Spectrophotometric studies of the interaction of palladium
ions with DNA and its components
Soobshch. Akad. Nauk Gruz. SSR 65(2): 457-60 (1972) (Russ)
Incubation of calf thymus DNA with Pd-2+ resulted in
degradation of the DNA molecule. With increasing pH,
the amount of degradation was reduced.
Ullmann, G. ; Hammerstein, J. . -
Inhibition of sperm motility in vitro by copper wire
Contraception 6(1): 71-6 (1972)
Effect of Cu, Pd, and stainless steel wires on
motility of human spermatozoa in cervical muscles .
was tested in vitro. Cu wire had an inhibitory
effect, Pd and stainless steel wires did not.'
0.971 .......
* " • • ...
Fujita, S. ••;• • " . - . -
Silver-palladium-gold alloys carcinogenicity and acid
mucopolysaccharides in the induced tumors
Shika Igaku 34(6): 918-32 (1971) • . - .
A 1 cm2 silver-palladium-gold plate for dental
use imbedded s.c. in rats caused tumors in 7 of
14 animals.
Schroeder, H. A.; Mitchener, M.
Scandium, chromium(VI), gallium, yttrium, rhodium, palladium,
indium in mice. Effects on growth and -life span
J. Nutr. 101(10): 1431-7 (1971) .
I Growth rate of mice given 5 ppm palladium in drinking
I water was suppressed. Malignant tumors were increased
* * in the Rh and Pd groups.
' 109
-------
lladium -
Shishniashvili, D. M. ; Lystsov, V. M.; Ulanov, B. P.;
Moshikovskii, Yu. S.
Study of the interaction of DMA v/ith palladium ions
Biofizika 16(6): 965-9 (1971) (Eng. sunm.)
The interaction of solutions of native calf DNA with
palladium chloride was titudied; kinetics of the decrease
of viscosity were determined and related to the mechanism
of interaction.
Shishniashvili, D. M.; Lystsov, V. N. ; tflahov, B. P.; '
Moshkovskii, Yu. Sh.
Reaction of DNA with palladium ions
Biofizika (6): 965-9 (1971) (Russ)
Treatment of calf thymus DNA with PdC12 led to a
marked decrease in viscosity, especially at low pH.
- - ' 1970 . . .
Dymond, A. M. ; Kaechele, L. F.; Jurist, J. M. ; Crandall, P. H.
Brain tissue reaction to some- chronically implanted metals
J. Neurosurg. 33: 574-80 (1970)
1969
Honda, T. ; Kawahata, K. ; Sakaki, T. ; Sugihara, R. ; Tsunami, N. ;
Tsutsumino, R. .
Experimental studies on carcinogenicity of physical stimuli:
I. Carcinogenicity of metal crowns set on the lower lateral
incisors in rats
J. Osaka Odontol. Soc. 32(2): 195-9 (1969)
No tumor developed grossly or microscopically on rat
oral tissue following 15 months placement of Ag-Pd-
Au alloy metal crowns on lower incisors.
Lewis, R. W.; Mack, R. W.; Dennis, M. J.; Woods, J. J.
Leukopenia-induction capacity of 6-MP palladium complex
in the chick
Proc. Soc. Exp. Biol. Med. 131(4): 1219-22 (1969)
In comparvson with 6-mercaptopurine (6-MP), the
6-MP-palladium complex was less toxic to 5-day
old White Leghorn chickens demonstrated by greater
weight gain and lower mortality. . - -
Munro-Ashman, D. ; Munro, D. D. ; Hughes, T. H. " ,
Contact dermatitis from palladium . x • '
Trans. St. John Hosp. Derm. Soc. 55: 196-7 (1969)
Parrot, J-L.; Herbert, R.; Saindelle, A.; Ruff, F.
Platinum and platinosis: Allergy and histamine release due to
some platinum salts . .
Arch. Environ. Health 19{5): 685-91 (1969)
I Platinosis is not caused by metallic ions but usually by
its complex salts, mainly chloroplatinates, an^ is mani-
. fested as cutaneous and^ respiratory allergies.
110 • ; - '
-------
r-j::--r. •v--'- j, H. ; Y; r.c •-";-», T,. ? T'o-'-c, J. I].; ."-.: .%••_-, V.
PiatiriUfa compounds: A new class of potent an ti tumor agents
Nature 222(5191): 385-6 (1969)
• lice were injected intraperitoneally with platinum
compounds; 5 to 10 r>j/kg cis-di-umrninedicnloroplatinum(II)
was a potent antilouksmic agent.
SpiKes, J. D. ; Hodgson, C. P.
j Enzyme inhibition by palladium chloride '
Biochen. Biophys. Res. Comiun. 35: 420-2 (1969)
19_66 ....
Kirschr.or. S. ; V.^i, Y-K. ; Francis, D; Be re/nan, J. G.
7mticar.c:er and potential rjitiviral aotivity of complex inorganic
compounds
J. Med. Chem. 9(3): 363-72 (1966)
Platinum(IV) and pallcidium(II) complexes of
G-Marcaotopurine *>;ere o.chiva actr-.ir.st adc-.ic-
carcinoma 755 and sarcoma 180,
1964
Matsushita, H. • •
Relation between physicocharnica! properties of niatal cations and
their acute L.D. to rabbit, rat, and mouse
Ind. Health 2(1): 1-10 (-1964) (Japan)
The log of the acute LD of inatal cations decreased
linearly with the increase of the log of the stability
constant o'f the netal-EDTA chelate and the standard
electrode potential.
1963
Bienvenu, P.; Norfe, C.; Cier, A.
Comparative general toxicity of metallic ions. A relation with
the periodic classification
Compt. Rend, 256: 1043-4 (1963)
!7*cute'toxicities, LD50/30, for aqueous solutions of 42
cations were determined by intraperitoneal-injection into
mice.
1951
VThite; J.; Munns, D. J.
Inhibitory effect of common elements towards yeast growth
J. Inst. Brewing 57: 175-9 (1951)
Of 50 elements tested for toxicity towards yeast, Cd,
Cu, Ag, Os, Hg, and Pd ranked as" very toxic in tha
order given.
? 111 . .-•-'.
-------
- u
Herrero, F. J.
/•-.ction of cations on the qrowth of bc'icteri^; a new method of study
Arch. Farm. Bio-juim. Tuonan 2: 239-318 (1945)
The inhibiting action of various metal chlorides was
tested on E. coli and Htaphylococcus aureus.
Bodine, J. H. ; 7'ihr.usi.in, N.
The efj'ect of hoavy infttnl.^ on the activation and injury of the
on^yne tyrosinase • • - . •,-
Avch. 15 loch an. ?. : '103-11 (1943) : .
The ia?nornetric ncjthod was used to study the effect of
heavy netals on proLyrosina-'se (I) from the eggs of the
grasshopper, .'lalanoplus dif forentialis.
Ckanoto, K. , et al.
Biologic studies of netals (Cu, Ag, Au/ and Pb)
Tr. Soc. Path. Jap. 28: 577-80 (1938) (Ger)
112
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l\
PART II
TOXICITY OF PLATINUM AND PLATINUM COMPOUNDS: AN ANNOTATED BIBLIOGRAPHY
TIP 73-181-1203
Compiled and Annotated by: Helga Gerstner
June 1973
Abs_tract^
This annotated bibliography of 70 references was compiled by the Toxicology
Information Response Center in'answer to a specific request. Coverage of
the abstract literature spans the years 1937 to 1973 and yielded citations
from 1942 to 1973. The citations are divided into two sections, secondary
references arranged by year and primary references arranged chronologically
by year and alphabetically by first author within each year.
Sources Searched:
Vpl. 31: 1937 to Vol. 78(19):
Vol. 18: 1944 to Vol. 55(18):
MEDLINE Data Base
TOXLINE Data Base
Chemical Abstracts:
May 14, 1973
Biological Abstracts:
April 15, 1973
Cumulated Index Medicus: Vol. 1: 1960 to Vol 12: 1971
and Vol. 23: 1938 to Vol. 48: 1950
Nuclear Science Abstract Journal: Vol. 24: 1970 to
Vol. 26: 1972
Toxicology Information Response Center's Library
Terms Searched:
Platinum
Platinum Compounds
TIRC Keywords:
Toxicity
Metals
Exposures
NTIS Keywords:
Platinum
Toxicity
Metals
113
-------
Platinum i
i
i
Secondary References
! i
Anonymous ;
Encyclopaedia of Occupational Health and Safety, Vol. II
p. 1080 , j
International1 Labour Office CH 1211, Printed in Tisk,
Czechoslovakia (1972)
Anonymous
Documentation 'of the Threshold Limit Values for
Substances in Workroom Ai r, 3rd ed., pp. 213-14
Am. Conf. of Gov. Ind. Hyg., Cincinnati, Ohio (1971)
A TLV of 0.002 mg/m3 of platinum is recommended to
protect against respiratory diseases.
Dreisbach, R.H., (Ed.) . . . . , ..
Handbook of Poisoning, 7th ed. pp. 172-!*
Lange Medical Publication, Los Altos, Calif. (1971)
Platinum salts cause superficial des-truction of skin on
mucous membrane. .. ... .
ShubIk,.-P.; Hartwell, J.L,. . .
Platinum
Survey of.Compounds which have been tested for
Carcinogenici'ty Activity, Suppl . II, p. 58, GPO,
Washington (1969)
In implantation experiments wi til rats and mice,
during periods up to 29 months, sarcomas were
observed in 7 of 69 rats and G of 31 mice.
Patty, F.A., (Ed.) •
Industrial Hygiene and Toxicology, 2nd ed.
Volume II, Toxicology, pp. 1125-31 Fassett, D.W,.,
Irish, D.D., (Eds.) . . .
Interscience Publishers, John Wiley & Sons,
Mew York, N.Y. (1967)
As of 1967, no threshold limit had been set by
an official agency in the United States for any of the
Pt group metals or any of their compounds.
114
-------
PI atinum
Sax, N.I., (EH.)
Dangerous Properties of Industrial Metals, 3rd ed.,
pp. 1031-?.
Van Mos.tr.ind Reinliold Co., New York, N.Y. (1968)
Exposure to complex platinum salts causes asthmatic
symptoms; however, exposure to dust of pure metallic
platinum causes no intoxication.
Schwartz, L.; Tulipan, L.; Peck, S.M., (Eds.)
Occupational Diseases of the Skin, 2nd ed., p. 196
Lea & Febiger, Philadelphia, PA (19U7)
Exposure to dust or spray of p.latinum salts results
in asthmatic symptoms and dermatitis.
Stecher, P.G., (Ed.)
The Merck Index, 8th ed. (1968), p. 842
JV. Merck & Co., Inc. Rahway, N.J.. (1968)
Inhalation of dust of soluble platinum salts causes
irritation to the respiratory tract.
Thomson, A.J.; Wi.11 lams, R. J.P.; Reslova, S.
Chemistry of complexes related to cis-dichlorodiamine
platinum (II). Anti turner drug
Struct. Bonding (Berlin) 11: 1-US (1972)
Interaction of metal complexes with bioligical 1 igands
and nacromolecules, including amino acids, proteins,
, and nucleotides was reviewed; 90 references.
i
i . Primary References •
- . 1973
Cleare, M.J.; Hoeschele, J.D.
Antitumor platinum compounds. Relation between
structure and activity
Platinum Metals Res. 17(1): 2-13 (1973)
Antitumor activity of compounds containing alicyclic'
amines indicated that some Pt compounds are highly
selective.
115
-------
PI atlnum
Khan, A.; Hill/ J.M.
Suppression of lymphocyte b1 astogenesis in man
following ci s-pl ati nous di ainrni nodi ch 1 or i de
administration * .
Proc. Soc. Exp. Biol. Med. Ik2: 3214-6 (1973)
The lymphocyte blastogenic response to
phy tohemaggl ut i n urn was studied in patients
receiving ci s-pl at i nous di amini nodi ch 1 or i de;
the Pt compound produced significant inhibition of
blastogenesis.
Macquet, J.P.; Theophanides, T. • '
Complexes of hematoporphyrin IX with pi atinum-(I I).
Synthesis,interm^diates, spectra, and biological interest
Can. J. Chem. 51(2): 219-26 (1973) (Fr)
Biological application of HPH2PtCL2 for tumor therapy
was investigated.
1972 .
Howie, J.A.; Gale, G.R.; Smith, A.B.
A proposed mode of action of an ti turner platinum compounds
based upon studies with cis-dich1oro-((G-3H)dipyridine)
platinum (II)
Biochen._ Pharmacol. 21(10): lh6k-75 (1972)
Antitumor and antirnitotic activities of the title
compound were studied and related to its interaction
wi th DMA. . .
Khan, A.; Hill, J.M.
Suppression of g/afl-versus-host - react 1 on by cis~p1 atinum
(II) diami nodichloride
Transplantation 13(1): 55-7 (1972)
Relationship between the antitumor effect and
immunosuppression of the title compound was studied
i n nii ce. . .
116
-------
. l\ . .
Platinum
Levene, fi.M;
Platinum sensitivity
Br. J. Derrnatol. 85(6): 590-93 (1971)
BA 5«*: 52657
Exposure of workers to complex Pt salts over long periods
of time led to skin irritation and symptoms resembling
asthma and hayfever.
Pepys, J.; Pickering, C.A.; Hughes, E.G.
Asthma due to inhaled chemical agents. Complex salts of
pi ati num
Clin. Allergy 2U): 391-6 (1972) .
Inhalation tests for occupational exposure to dust from
mixtures of 40 mg of each Pt salt with 1 kg lactose
produced immediate asthmatic reaction in 8 of
11 persons.
Pickering, C.A.
Inhalat ion tests
of pi at i num
Proc, R. Soc. Med.
with chemical allergens:
65: 272-if (1972)
Complex salts
Rossof, A.M.; Slayton, R.E.; Perlia, C.P.
Preliminary clinical experience with cis-
diamminedichloroplatinum (II)
Cancer 30: 1U51-6 (1972)
Adverse effects of cis-diamminedichloroplatinum as an
antineoplastic agent were examined.
Shooter, K.V.; Howse, R.; Merrifield, R.K.; Robins, A.B.
Interaction of platinum (II) compounds with
bacteriophages T7 and R17
Chem.-Biol. Interactions 5(5): 289-307 (1972)
Inactivation of DNA-conta!ning bacteriophage T7
and RIJA-con tai ni ng bacteriophage R17 by three different
platinum (II) compounds was studied.
117
-------
PI atinum
Shooter, K.V.; Merrifield, R-.K.
Changes in the liydrodynami c properties of DMA
induced by interaction with platinum (II) compounds
Biochim. Biophys. Acta 287(1): 16-27 (1972)
Binding of the ethy 1enediamine platinum (11) to
native D!JA was studied and compared to the binding of
cis- and trans- dichlordiamine platinum (II).
Sodh i, A.
Regression of sarcoma-180 after ci.s-di ch 1 orod i amrni nepl at i num
(II). Fine-structural study
Proc. Electron Microsc. Soc. Amer. 30: 68-9 (1972)
DMA synthesis and mitotic acitivity stopped in sarcoma-180
in mice after a single i.p. injection of
cis-d?chlorodiammineplatinum (II).
Varkarakis, M.J.
Prostatic effects of cis-diamine-chloroplatinum in the dog
Res. Convnun. Chern. Pathol . Pharinacol . It(2): 1*33-8 (1972)
Dogs treated with the antitumor agent in toxic and
nontoxic doses showed no prostatic atrophy or other
histological changes.
Vonka, V.; Kutimova, L.; Drobnjk, J.; Brauerova, J.
Increase of Epstein-Barr-virus-positive cells in EB3
cultures after treatment with ci s-di chl orodi arrmi nepl at i nurn (II)
J. Nat. Cancer. Inst. 48(5): 1277-81 (1972)
Treatment of EB3 lyrnphoma cells caused a 2 to 3 fold increase
in the number of cells reactive to the indirect
i nimunof 1 uorescence test v/i th human ser.a possessing EB virus
antibody. . .
118
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Platinum
Zak, M.; Drobnlk, J.; Rezny, Z.
The effect of els-platinum (II) diamminodichloride on bone
ma r row
Cancer Res. 32(3): 595-9 (1972)
Changes in mouse bone marrow after i.p. administration of
8 mg/kg of the title compound corresponded to effects of
rather low doses of x-irradiat ion.
1971
Berenbaum, M.C.
Immunosuppression by platinum diamines
Brit. J. Cancer 25(1): 208-11 (1971)
Platinum diamine dichloride and tetrachloride inhibited
formation of antibody-forming cells in mouse spleen
after injection of sheep red cells.
Friedman, M.E.; Musgrove, B.; Lee, K.; Teggins, J.E.
Inhibition of malate dehydrogenase by platinum (II)
complexes
Biochim. Biophys. Acta 250(2): 286-96 (1971)
Enzyme inhibition by platinum (II) complexes was
studied through a pH range from 6.5 to 8.5.
Guthrie/ R.W.; Melius, P.; Teggins, J.E.
Inhibition of leucine aminopept5dase by halide complexes
of platinum
J. Med. Chem. li»: 75-G (1971)
Gale, G.R.; Howie, J.A.; Walker, E.M., Jr.
Anti tumor and antimi togeni c properties of
ci s-dichloro(di pyri di ne) platinum (II)
Cancer Res. 31: 950- 2 (1971)
Pharma'co logic activities of ci s-di chl oro( di py ri di ne)
platinum (II) were compared with those of Inorganic
platinum compounds; results showed the organic Pt
compound to be less potent.
Howie, J.A.; Thompson, H.S.; Stone, A.E.; Gale, G.R.
Cis-dichlorodi ammi nepl ati num (II): Inhibition of nucleic
acid synthesis in lymphocytes stimulated with
phytohemaggluti nln
Proc. Soc. Exp. Biol. Med 137(3): 820-25 (1971)
Using cultures of human lymphocytes,
cis-dichlorodiammineplat.!num (II) was a potent and
probably Irreversible inhibitor of DNA synthesis. > v
'•119
-------
PI at i nurn
Khan, A.; Hi)], J . M.
Inhibition of lymphocyte h 1 as togenes i s by platinum compounds
J. Surg. rjncol. 3: 565-7 (1971)'
i
Kociba, R.J.; Sleight, S.D.
Acute toxicologic and pathologic effects of
c i s~d i arnm i nod i ch 1 o ropl a t ? nurn in the male rat
Cancer Cheniother. Rep. Part I 55\1): 1-3 (1971)
The L050 of c i s-d i ai.vni nedi ch loropl at i num was 12.0 rng/kg
in rats; antitumor properties were attributed to its
cytoinhibitory effects.
Leonard, B. J. ; Eccleston, E.; Jones, D. ; Todd, P.; Wai pole, A.
Anti1eukemic and nephrotoxic properties of platinum compounds
Nature 23U5323): ^3~5 (1971)
The antileukemic and nephrotoxic action of Pt compounds
in rats inay be related to molecular configuration.
Levene, G.M.
Platinum sensitivity: treatment by specific hyposensitization
Clin. Allergy 1(1): 75-82 (1971)
Allergen was administered intraderna11y to a chemist with
respiratory tract and cutaneous allergies
to complex salts of Pt.
Melius, P.; Guthrie, R.W.; Teggins, J.u.
Inhibition of leucine aminopeptidase by halide complexes
of pi at i nun
J. hed. Chem., !<*(!): 75-G (1971)
Inhibition of swine leucine aninopeptidase by halide-
complexes of Pt increased with an increasing number
of halide ligands, which was closely related to their
anti tumor act i vi ty.
Popescu, M; Pascaru, A.; Nicolau, C.
Effect of c i s-d i ami nopl at i num chloride in viruses and '
virus-cell relations
Stud. Cercet. Inframicrobiol. 22(4): 383-9 (1971) (Rom)
Five rng/kg of c i s-di ami nopl a t i num given i.p. prolonged
the survival time of mice with Ehrlich ascites tumor;
it also inactivated the I and II type polio viruses
and the influenza virus AOPRS in vitro.
*" '
120 . vl
-------
PI nt in urn
Reslova, S.
Induction of lysogenic strains of Escherichia coli by
c i s-d i ch 1 oro-di ainini nepl at i num (II)
Chcm.-Biol. Interactions k(l) : 66-70 (1971)
A concentration of 50 uM of the title comoound
caused complete lysis of E. coli TAU-.
Rosenberg, B.
Biological effects of platinum compounds. New agents for the
control of tumors
Platinum Metals Rev. 15(2): U2-51 (1971)
Reviewed are antitumor activity, toxicological effects,
molecular biology of Pt complexes as well as the mode
of action of the antiturnor effects; 20 references.
Thompson, H.S.; Gale, G.R.
cis-Dich1orodiarmineplatinum (II): Hematopoietic effects in rats
Toxicol. Appl. Pharmacol. 19(4): 602-9 (1971)
Experimental studies revealed a decrease in reticulocytes
and 1 ymphocytes and changes in thyrnus and spleen following
injection of the Pt compound.
VJelsch, C.H.
Growth inhibition of rat mammary carcinoma induced by
cis-platinum diarnminodichloride-II
J. Hatl. Cancer Inst. 47: 1071-3 (1971)
Efficacy of cis-platinum d i amrni noch 1 or i de- 11 for treatment
of nai'itnary tumors in rats was discussed; weight loss
resulting from application of the Pt compound was analyzed.
1970
Brehm, G.
Platinum dermatitis of watchmaker , . "
Hautarzt 21: 388-9 (1970) (Ger)
Dymond, A.M.; Kaechele, L.E.; Jurist, J.M.; Crandall, P.H.
Brain tissue reaction to some chronically implanted metals
J. Neurosurg. 33: 57^-80 (1970)
121
-------
PI at i nurn
Harder, H.C.; Rosenberg, B.
Inhibitory effects of ant?-tumor platinum compounds on DNA,
RNA, and protein synthesis in marnrnal > nn cells in vitro
Ind. J. Cancer R: 207-16 (1970)
Several platinum compounds were tested on human arnn i on cells
i n vi t ro. ,»•
Howie, J.A.; Gale, G.R.
ci s--Di chlorodi ainrni nepl at inum (II): Cytological changed induced
i n Cscheri ch i a col i
J. Bact. 103: 258-9 (1970)
Changes in E. coli cells induced by
ci s-di chlorodi arnninepl at i num (II) were 'interpreted as a result
of selective inhibition of DNA synthesis.
Howie, J.A.; Gale, G.R.
cis-Dich1orodiamminep1 atinum synthesis in vivo
Siochem. Pharmacol. 19(10): 2757-R2 (1970)
The inhibitory action of c i s-d i ch 1 orodi ami nepl a t i num
on DMA was directly related to the chenotherapeutic efficacy;
therefore, a less frequent treatment regimen mav be as
effective as daily injections while evoking fewer
reactions.
Milne, J.E.
A case of platinosis
fled. J. Aust. 2: 119**-5 (1970)
Occupational exposure to platinum was
to respiratory and skin diseases.
analyzed in reference
Rosenberg, B.;
The successful
by platinum compounds
Cancer Res. 30(6): 1799-1302
Van Camp, L.
regression of
large solid sarcoma 180 tumors
(1970)
Cis-platinum (IV) diamminotetrachloride and cis-platinum (II)
di ammi nodi chlori de inhibited tumors in mice with no apparent
irreversible damage to the host.
Zakharenko, E.T.; Moshkooskii, lu. Sh.
St ructural
B i okli imi i a
changes i n
35: 917-21
DNA under the
(1970) (Russ)
influence of K2PtCl't
122
-------
P 1 at i num
1968
Freedman, S.O.; Krupey, J.
Respiratory allergy caused by platinum salts
J. Allerg. U2: 233-7 (1968)
Held, A.J.; Spi rgi, M.
Osteoperiostal response to various implants in
rabbi ts
Helv. Odont. Acta. 12: 1-1U (1963)
Platinum was one of the metals tested for adverse
effects on bones.
Lewis, R.W.; Rubin, B.A.; Davis, J'. ; VJoods, J.J.
Leukopenia induction capacity of G-mercaptopurine
platinum complex in the chick
Proc. Soc. Hxn. Biol. Med. 128(3): 857-62 (1968)
6-lisrcaptopuri ne and a platinum complex of this
compound were administered orally to white leghorn
chicks in equal doses; capacity to induce leukopenia
was the same; however, the platinum complex was less
tox i c.
1967
Beni ni, F.
The risk of carbon monoxide intoxication in platinum
catalyst workers
Lav. Umano 1-9(5): 195-9 (1967)
The mechanism of CO accumulation in the work environment
of platinum catalyst workers was analyzed.
Parrot, J.L.; Saindelle, A.; Ruff, F.
Platinum and platinosis. Histamine release by some
platinum salts and platinum allergy
Presse J-'.ed. 75(2817): 20-30 (1967)
Occupational diseases caused by platinum compounds
are discussed with respect to respiratory tract and
skin.
123
-------
PI .it i num
Renshaw, E.; Thomson, A.J.
Tracer studios to locate the site of platinum Ions
within filamentous inhibited cells of Escherichia coli
J. Bacteriol. 9'i(6): 1915-8 (19G7)
Cis-Pt(NH3)2CU pentrates the cell wal'l'of gram
positive B. cereus and S. aureus and subsequently
becoir.es bound by metabolic intermediates.
Rosenberg, B.; Renshaw, E.; Vancamp, I..; Hartwick, J.;
D ro b n i k, J .
PI atinum-induced filamentous growth in Escherichia coli
J. Bacteriol. 93(2): 716-21 (1967)
Certain metal compounds inhibited cell division in E.
coli; gram-negative bacilli were the nost sensitive;
gram-positive bacilli responded only at near-toxic
levels of the metal.
. 1966
Hebert, R.
Diseases caused by platinum compounds
Arch. Mai. Prof. 27: 877-35 (1956) (Fr)
Respiratory tract diseases were caused by occupational
exposure to platinum compounds.
Ki rschner, S.; V/e 1 , Y.K.; Francis, D.; Bergman, J.G.
Anticancer and potential antiviral activity of complex
inorganic compounds
J. Med. Chem. 9(3): 368-72 (19G6)
Platinum (IV) and palladium (II) complexes of
6-me reaptopurine were active against adenocarcinoma
755 and sarcoma 130.
i/oohsmann, U.;.Martrodt, U.
Histological and histochemical studies on the tissue
ability to tolerate depth electrodes and their insulation
in the central nervous system
Acta. Biol. ,'1ed. German 17: 207-1G (19CC) (Ger)
124
-------
PI at i num
- 1965
Mi 7. us hi ma, Y.; Okurnura, H.; Kasukawa, R.
Effects of gold and platinum on necrotizing factors, skin
sensitizing, antibody, and complement
Jap. J. Pharmacol. 15(2): 131-1* (1965)
Ionised platinum exhibited a weak inhibitory effect
on skin sensitising antibody; an t i compl ementa ry effect
of ionised gold and platinum was the strongest
among the metal ions.
Rosenberg, B.; Van Camp, L.; Thomas, K.
Inhibition of cell division in Escherichia col i by
electrolysis products from a platinum electrode
Nature 205(4972): 698-9 (1965)
Platinum and rhodium were equally effective in
cell inhibition studies; mechanism and
implications are discussed.
1963
Bienvenu, P.; Nofre, C.; Cier, A.
Comparative general toxicity of metallic ions. A
relation with the periodic classification
Compt. Rend. 256: 1043-U (1963)
Acute toxicities, LD50/30, for aqueous solutions
of 42 cations were determined by i n t raper i tonea 1
injection into mice.
Bi j 1 , von der, W. J.
Asthma as an occupational disease: Allergy to
platinum ammonium chloride
Allerg. Asthma (Leipzig) 9: 155-7 (1963) (Ger)
Parrot, J.L.; Saindelle, A.; Tazi, T.
Histamine release by sodium ch 1 oropl a t ima te
J. Physiol. (Paris) 55: 31U-5 (1963) (Fr)
Parrot/ J. L. ; Saindelle, A.; Tazi. T.
Release of histamine by action of platinum salts and
mechanism of their toxicity
Bull. Acad. Matl. fled (Paris) li»7( 22/2IO : 1*58-61 (1963)
The mechanism of poisoning by Pt salts was
investigated by i.v. injection into guinea pigs,
rats, and isolated organs; the toxic action
'was attributed to the release of toxic
mainly histamine.
125
-------
PI at i nuiii
1951
Robinson, F.R.; Johnson, M.T.
Histopathological studies of tissue reactions
various metals implanted in cat brains
USAF Aero Syst. Div. 61-397: 13 pp. (1961)
to
1952
f la t :ihal 1 , J .
As t lima
S
and
dermatitis caused by chloroplat
African fhd. Jour. 26(1): 8-9 (1952)
Sensitivity to ch loroplatinic acid with
respiratory and cutaneous manifestations
n i c acid
an d
sensitivity to platinum compounds are described
in a case report.
1950
Tietz, C.J.; Spiess, H.
Inhibition of growth of tubercle bacilli in vitro by
neans of peteosthor (pi at i num-thori urn preparation)
Klin. Wochenschri ft 28: U20-1 (1950).
1946
Singh, I.; Singh, I.S.
The effect of some metals, vitamins, anaesthetics,
and other substances on unstriated muscle
Proc. Indian Acad. Sci. Sect. B 23(6): 301-11 (1946)
Effects on excitability were investigated with
various metal compounds, including platinum
chloride.
Herrero, F.J.
Act ion of cat i ons
method of study
Arch. farm, bioquim.
19 4 5
on the growth of bacteria; a new
Tucuman 2: 239-318 (1945)
The inhibiting action of various metal chlorides
was tested on E. coli and staphylococcus aureus;
platinum had a rather high inhibitory effect.
126
-------
PI at i nuio
Hunter/ D. ; Hilton, R. ; Perry, K.M.A.
Asthma caused by the complex salts of platinum
Brit. J. Indust. Mod. 2: 92 (19U5)
Workers exposed to dust or spray of complex salts
of pi at 5num deve1 oped asthma syndrome; some also
suffered cutaneous lesions; methods for treatment
a re d i scus scd.
19't3
Bodine, J.H.; Tahmisian, T.fJ.
The effect of heavy metals on the activation and
injury of the enzyme tyrosinase
Arch. Biochem. 2: I>03-11 (19/»3)
The (nanometric method \vas used to study the effect
of heavy metals on protyrosinase (I) from the eggs
of the grasshopper. Me 1 anopl us d i f fe rent i al i s .
19 k 2
Braun-Stapoenbeck, M.
Suddeut. Apoth.-Zig. 82: 203-5 (19U2); Chcm. Zentr.
19U2 II, 1019
A brief summary in which the metals copper, iron,
chromium, molybdenum, cobalt, zinc, cadmi urn,
thallium, silver, gold, platinum, and lead are
cons i de red.
127
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Appendix B4.6
METHOD DEVELOPMENT AND
SUBSEQUENT SURVEY ANALYSIS
OF EXPERIMENTAL RAT TISSUES
FOR Ft, Mn, AND Pb CONTENT
CONTRACT NO. 68--02-0663
MODIFICATION NO. 3
PRESENTED
TO
ENVIRONMENTAL PROTECTION AGENCY
Research Triangle Park, NC 27711
EPA 03048
Submitted by
STEWART LABORATORIES, INC.
820 Tulip Avenue
Knoxville, Tennessee 37921
.A /
'v^
-------
BACKGROUND INFORMATION
On January 28, 1974, Stewart Laboratories, Inc. , submitted a pro-
posal to the Environmental Protection Agency for method development and
subsequent survey analysis of experimental rat tissues for platinum, man-
ganese, and lead content. The primary goal of the research was to develop
a method for Pt which would be two to five times more sensitive than the
standard Stewart Laboratories, Inc., emission spectrographic technique.
Al] analytical data for the study were to be delivered to EPA no later
than March 15, 1974.
TECHNICAL APPROACH
^ • JL^-ll? 1li.TIlojltILL_^T1 il^JL Ti ssues
Initial stages of the project were performed by The Department
of Biochemistry, School of Medicine, University of North Carolina,
under the direction of Dr. David J. Holbrook, Jr.
Experimental rats received controlled diets and measured dosages
of several solutions of platinum, manganese, or lead for varying
tirac1 periods. Preliminary data concerning this aspect of the study
are presented in this report with the respective analytical data
for each aninal group (pages 10-28).
Blood was drawn immediately after sacrifice, and the tissues of
interest were dissected and weighed. Each tissue specimen for
metal uptake or control level analysis was quick frozen in glass
129
-------
vials with aluminum insert plastic caps (screw top). All samples
remained frozen through receipt by Stewart Laboratories, Inc. (SLI)
B. Analytical Method Devejopinent
A custom emission spcctrographic technique was developed for the
analysis of Pt, Mn, and Pb in experimental rat tissues. The abso-
lute detection limits achieved using this custom method are 0.010
micrograms for Pt and 0.015 micrograms for Pb. The corresponding
analysis limits, based on a one-gram wet tissue sample size, are
0.015 micrograms for Pt and 0.020 micrograms for Pb. This repre-
sents an improvement in sensitivity for Pt of five times that of
SLl's existing standard emission spectrographic method and a 50
percent improvement for Pb.
The precision of the method, based on multiple analyst's of a syn-
thetic tin.sue san'ple, is - 10 peicent at the one-microgram level.
A statirftica] evaluation of the accuracy of the method, based on
Lin3 confj'rv in : .
-------
C. Sample Preparation and Analyses
^ __ - J
All specimens were coded and lyophilized upon receipt by SLI. In
those cases where sample size permitted, the lyophilized sample
was carefully mixed; and a portion of sample equivalent to 0.25
gram wet tissue was removed for confirming analyses by an inde-
pendent technique (atomic absorption). Sample splits used for the
confirming technique were burned by oxygen-flask combustion, and
the resulting solutions were analyzed by conventional atomic
absorption for manganese and platinum. In the case of lead, an
extraction procedure was used to concentrate the samples prior
to analysis.
Samples for emission analysis were ashed by a modified "wet"
ashing technique. The resulting ash was carefully mixed with
graphite; packed into a spectrographic electrode; and analyzed
by a total burn, d-c arc emission spectrochemical technique
utilizing a controlled atmosphere. The emission spectra were
recorded photographically and line intensities were determined
by standard densitometric procedures.
ANALYTICAL DATA
A • Em"*_§.?J.P.H_?'1OCJL1?^-Jl3 phic Analyses
All analytical data obtained by the emission spectrochemical method
are presented as direct computer output at the end of this report
(pages 10-28).
131
-------
B. C_p_nf_i_rriing Atomic Absorption Data
High levels of concentration of platinum and lead in some tissue
group) experiments permitted a confirmation analysis by atomic
absorption.
The residual concentration of manganese in biological tissues is of
sufficient magnitude to allow for ease of atomic absorption veri-
fication. Some representative atomic absorption data are shown
in Table 1.
DISCUSSION OF RESULTS
A. Abnormal Data
When initial sample size permits, it is customary to conduct a
ohc-ck analysis to confirm abnormal delta. Since this practice
couHl nor ho fnllc'ved in this study, a list of outliers due to
probable contamination is presented in Table II. A recommendation
ir. r -.do tb''. tho^e values be ta™od to avoid a bins of the data.
B. AnaJy_t_i_cnl. Prqb 1 ems VAIcountered
Analytical prohient> encountered resulted from (1) the inherently
low residual levels of the elements under study; (2) the limited
;:;-; u- ' ,^r ;„,'-;-,; .1,,, | ,-,-,,--,1^ tissues; < ;"! (3) the loss of certain
speci: ns due t>> a laboratory accident. The careful adherence
to trace analytical techniques was sufficient to overcome the
difficulties of (1) and (2).
132
-------
TABLE I. CONFIRMING ANALYSES BY ATOMIC ABSORPTION SPECTROSCOPY
Platinum Concentration, yg/gram Wet Tissue
SLI Code
FF 001
FF 002
FF Oil
FF 012
SLI Code
FF 073
FF 074
FF 075
FF 076
SLT Code
FF 071
FF 072
FF 073
FF 074
FF 075
FF 076
FF 125
FF 126
FF 127
FF J28
FF 173
FF 174
FF 175
FF 176
FF 227
Holbrook I. D.
Emission
Atomic Absorption
14-B-G-LV-b
14-B-P-LV-b
14-B-G-KL-b
14-B-P-KL-b
38.4
30.0
45.7
27.7
40.
32.
40.
<30.
Lead Concentration, yg/gram Wet Tissue
Holbrook I. D. Emission
16 G-KL-b
16- -P-KL-b
16- -R-KL-b
16- -W-KL-b
14.6
7.48
9.43
21.8
Manganese Concentration, yg/gram Wet
Holbrook T. D. Emission
16- -R-R-bl *0>97
16- -W-H-bJ
16 G-KL-b
16 P-KL-b
16 R-KL-b
16- -W-KL-b
12-3-C-LV-b
12-2-P-LV-b
12-1-R-LV-b
12-4-W-LV-b
17-2-G-LV-b
17-3-P-LV-b
17-1-R-LV-b
17-4-W-LV-b
9-1-R-BL-b
1.22
0.75
1.35
2.18
4.23
3.23
2.42
1.87
*3.75
*2.57
22.1
Atomic Absorption
16.5
9.00
10.4
24.4
Tissue
Atomic Absorption
0.98
0.72
1.54
0.87
•1.38
2.29
3.40
3.06
2.50
2.09
*3.23
*2.16
19.9
*Composite samples
133
-------
TABLE II. ABNORMAL VALUES PROBABLY DUE TO EXTRANEOUS CONTAMINATION
SLI Code
Ho]brook 1. D.
Element
FF
FF
FF
FF
FF
FF
131
131
J60
108
256
227
I
2-1-R-BL-b
12-]-R-BL-b
15- -W-TL-b
11-4-W-BL-b
11-1-R-BL-b
9-]-K-i;i,-i»
Mn
Pb
Pb
- Mn
Mn
Conccnrration, |ig/gm
7.02
2.00
23.3
17.0
22.1
~~ — Sniiipl r;.s used for composite analysis.
J
134
-------
However, the laboratory accident which occurred resulted in the
complete loss of the samples for 'two control studies—Group 18
and Group 24—and one platinum feeding study, Group 19, A block of
electrodes containing these samples was dropped as it was being
removed from a conditioning oven. Aliquots of some of the larger
specimens in Group 19 had previously been reserved for confirming
atomic absorption analyses. These samples were evaporated onto
graphite from an oxygen-flask solution, and an attempt was made
to analyze the Pt by the emission technique. A limited number of
analyses was possible from the solutions where sufficient sample
size and Pt concentration were concurrent (see Table III).
135
-------
TABLE III. PLATINUM DATA FOR SELECTED TISSUES—CROUP 19
SL1 Code
Holbrook I. D.
Pt, yg/grnm Wet Tissue
FF
FF
FF
FF
FF
FF
FF
FF
FF
FF
FF
TI-
FF
FF
FF
"TT^
283
284
285
286
287
288
289
290
299
300
30!
"io:
303
304
305
'•} />
19-
19-
19-
19-
19-
19-
19-
19-
]0-
19-
19-
]Q_
19-
19-
19-
19-
-C-LV-b
-P-LV-h
-R-LV-b
-W-LV-b
-G-BL-b
-P-BL-b
-R-BL-b
-W-BL-b
- 2.52
- 1.96
- 0.27
- 0.19
-G-KL-h 4.83
-P-KL-b 4.04
-U-KL-b 5.18
-V-K'L-b 4 . 98
-G-SP-b
-P-SP-b
-R-SP-b
-.' '. C |)_1,
r- 0.24
—= Samples used for composite analysis.
136
-------
CONCLUSIONS
A. The method, as developed, achieved the desired goal of improving
platinum sensitivity by a factor of five over the standard SLI
emission spectrographic technique.
B. A means was provided for evaluating initial exposure experiments
on test animals.
C. There is still a need for an analytical method which possesses
sufficient sensitivity to cope with and establish the statistical
variability inherent in dynamic biological systems at residual
platinum concentration levels.
RECOMMENDATIONS
A. From an analytical standpoint, the use of larger test animals in
most desirable. Increased sample sizes would result in a number
of itpprovfwnts in the precision and accuracy of the analytical
tint a. .1 ii ,'sd-J i t i; ,•, ;:11 .-vlyticv;! operations would be s ir';-'1 i f i i-d .
B. J.I would be uuijiiuble Lo analyze the proposed feeds and test
solutions prior to the start of future animal feeding experiments.
137
-------
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Proposed Final Report
Appendix B4.7
EPA Contract No. 68-02-1205
Title: Assessment of Fuel Additives Emission Toxicity via Selected
Assays of Nucleic Acid and Protein Synthesis
University of North Carolina
Office of Research Administration
South Building
Chapel Hill, N.C. 27514
Author: David J. Holbrook, Jr., Ph.D.
(919) 933-7862
Department of Biochemistry
School of Medicine
University of North Carolina
Chapel Hill, N.C. 27514
Date of Preparation: August 23, 1974.
Prepared for the Environmental Protection Agency, Research Triangle
Park, N.C. 27711
157 '
-------
Abstract.
Various parameters of toxic ity have been studied for salts of manga-
nese, lead, palladium and platinum. Following intraperitoneal injection,
the acute toxicities (LD-50 doses, 14-day observation period, doses ex-
pressed in molar quantities) in decreasing order, were: PtCl,> Pt (SO. )„ '
^ M* PdCl^A^O >Pt (S04>2 'Al^O (from K and K Lab-
oratories), MnCl2'4H20>PdS04> PtCl2> PbCl2. Following oral administration,
the acute toxicities (LD-50 doses), in decreasing order, were: PtCl, >
, PbO,
PdO,
Following dietary (via drinking fluid) admini ration of oluble salts
2+ 4+
of Pb (PbCl2) or Pt (PtCl^ or Pt (SO, )'4H20) , the highest concentra-
tions of metallic cations occurred in the kidney, intermediate levels in
the liver, and generally lower levels in the spleen, heart, testes, brain
and blood. In rats which survived for 14-days following the administration
of approximately the oral LD-50 and intraperitoneal LD-50, the kidneys con-
tained approximately 16 and 37 ugPt/g wet tissue, respectively, and the
livers contained approximately 2 and 34 ug Pt/g wet tissue, respectively.
Weights of five organs (liver, kidney, spleen, heart, testes) were
measured in rats which had been treated with various metallic salts in the
diet (either drinking fluid or solid feed). The organ weights were ex-
MnCl2'4H20 at a level of
pressed as a percentage of body weight. Rats which received.1.6 or 3.7
g/liter (8.3 or 18.6 mmoles/liter) for 90 days did not show statistically
significant changes in the weights of any organs. PbCl2, at a level of
1.0 g/liter (3,7 mmole/liter) for 30 or 90 days, consistently increased the
kidney weights of treated rats (17% and 23% above control in the 90-day
experiments). The use of saturated solutions of PdCl^t^O (8 days) or of
PdSO, (8 or 30 days) as the drinking fluid did not cause a consistent
158
-------
change in any of the organ weights of the treated rats.
If PtCl, was added to the drinking fluid at 183 mg/liter (0.5 mmoles/
liter) for 30 or 90 days, or at 550 mg/liter (1.6 mmoles/liter) for 8 days,
no consistent changes were observed in the organ weights. If the concen-
tration-duration of PtCl, was increased to 550 mg/liter (1.6 mmoles/liter)
for 30 days or 825 mg/liter (2.4 mmoles/liter) for 9 days, the kidneys were
increased in weight by approximately 6% in each of four experiments and the
testes were increased by approximately 11% in each of four experiments; how-
ever, in each tissue in each individual experiments, the differences between
control and metal-treated animals showed statistically significant differences
(p<0.05) or trends (p<0.10) in only about one-half of the experiments.
After experimental rats were maintained on metal-containing diets for
approximately 8, 30 or 90 days, hepatic microsomes were isolated and the fol-
lowing parameters related to in vitro drug metabolism were measured: yield
of microsomal protein/g liver; in vitro activities of aniline hydroxylase
and aminopyrine demethylase; content of cytochromes P-450 and b5/mg micro-
somal protein. Treatment with MnCl^AH^O, 1.6 g/liter (8.3 mmoles/liter)
or 3.7 g/liter (18.6 mmoles/liter) for 90 days did not alter any of the
studied parameters of drug metabolism. The administeration of low levels
2+
of Pd (saturated solution of PdCl2'2H20 for 8 days or saturated solution
of PdSO, for 8 or 30 days) in the drinking fluid resulted in somewhat de-
creased activities of aniline hydroxylase and aminopyrine demethylase.
PtCl2 (saturated solution as drinking water) did not produce consistently
statistically different levels of aniline hydroxylase or aminopyrine
demethylase.
4+
A wide range of dose levels-duration of soluble salts of Pt did not
cause consistent changes in the levels of aniline hydroxylase or aminopyrine
159
-------
demethylase in liver tissue; dosages and durations used included 0.5 mmoles/
liter (183 mg PtCl,/liter) for 30 and 90 days; 1.6 nunoles (550 mg PtCl,/
liter or 750 mg Pt(SO, )2''4H20/liter for 8 days or for 30 days (PtCl, only).
Work has been completed on the development of. a rapid and convient
method for the analysis of ribosomal RNA in studies of RNA synthesis.
160
-------
Introduction.
A. Contract. These studies were conducted pursuant to contract num-
ber 68-02-1205 and project number DU-73-B439 with the Enviornmental Pro-
tection Agency.
B. Importance of compounds. Lead salts are an emission product from
mobile (or automotive) emission sourses due to the addition of tetraethyl
lead to gasoline. Because of known toxic properties of lead salts, it has
been proposed that alkyl manganese compounds be substituted as a fuel addi-
tive for tetraethyl lead. With the introduction of platinum and palladium
in the catalytic converters of 1975-model year vehicles, it is of concern
to determine the quantities of platinum and palladium metal and salts which
will be in emission products and the biological effects of these compounds
on mammalian tissues.
c- Studies undertaken. Consequently, experiments were undertaken
in this laboratory to study the effects of various metal salts on the fol-
lowing: acute lethal dose following oral and intraperitoneal administra-
tion, growth of animals receiving the salts in feed or drinking fluid, the
tissue concentration of some of the metals in various organs, the size of
selected organs, the in vitro activity of two representative microsomal
drug-metabolizing enzymes and the cytochrome P-450 and b5 concentrations
in liver, the activity in vitro of eucaryotic DNA polymerases, and RNA syn-
thesis in vivo.
161
-------
Materials and Methods
All experimental studies were conducted with male Sprague-Dawley
rats. The animals were received at 3-3.5 weeks of age and were main-
tained for 1-1.5 weeks before use. The mean body weights were usually
100-110 g when the rats were used for the lethal-dose experiments or
started on the diets.
In the lethal dose experiments, the salts were administered orally
(via stomach tube) or intraperitoneally. The rats were observed through
a 14-day observation period. In the completed experiments, the LD-50
values were calculated by the method of Litchfield rnd Wilcuxon (1).
In the diet experiments, four rats were maintained per cage.
The metallic salt under study was dissolved in the drinking fluid.
Animals consumed feed and drinking fluid ad libitum. Analyses for
metals were performed on samples from three lots of feed (Purina
Laboratory Chow). The feed contained (mean + std. dev.): 56 + 5 rag
Mn/kg feed and 0.99 + 0.07 rag Pb/kg feed; the analyses of the three
lots for platinum were 0.09, < 0.02, and ^0.02 mg Pt/kg, feed. Measure-
ments were made of the body weights of individual rats and feed and fluid
consumption per cage of four rats at 7-day intervals during the course of
each diet experiment.
At the termination of the dietary experiments, samples of liver
were used for the isolation of microsomes. Aniline hydroxylase was
measured by the method of Imai et al. (2), modified by the addition of
HgCl2 (3). Aminopyrine demethylase was measured by the formation of
formaldehyde (Nash reaction) (4).
162
-------
The analyses of the rat tissues for platinum, lead and manganese
were carried out by Yoakura, Stewart and Sterrett (5) of Stewart
Laboratories, Inc. by an emission spectrochemical method.
Lethal dose studies.
The rats used in these studies usually had a mean body weight of 100-
110 grams. The LD-50 values and the 95% confidence limits were determined
by the method of Litchfield and Wilcoxin (1).
163
-------
A- Oral adminis tration . The summary of the lethel dose studies fol-
lowing oral administration are given in Table 1.
In terms of the LD-50 (dose lethal to 50% of the animals within the
14-day observation period), the acute toxicities (.expressed in molar quan-
tities), in decreasing order, were: PtCl^ !• Pt(S04)2'4H20£
RuCl3> MnCl2J4H20>PdS04, PtCl2>Pt02, PbO, PbCl2>Mn02, PdO.
4+
Thus, the two soluble Pt salts were found to be the most toxic salts
following oral administration. As anticipated, the poorly absorbed, insol-
uble salts, namely PbCl9, PtCl7, PbO, Pt09 , PdO and Mn09, were the least
the latter * t * /
toxic. In named cases, doses could not be increased sufficiently to
attain 50% lethality in the experimental rats and still maintain the volume
administered to 2% or less of the body weight.
In each of these cases, the LD-50 dose is greater than the 5,000 mg/kg
body weight which the National Institute for Occupational Safety and Health
uses as a criterion for inclusion as a toxic substance in the Toxic Substances
List. 1972 edition; thus, in terms of the acute LD-50, the salts PbCl0,
^
PbO, PdO, Pt02, and Mn02 would be considered "non-toxic" by this standard.
Also included in Table 1 are the LD-10 and LD-90, i.e., the doses
which cause the death of 10% and 90%, respectively, of the rats in the 14-
days after oral administration. In those cases where data are available,
the order of toxicities for the oral LD-10, "the minimal lethal dose", is
the same as that given above for the order of the LD-50 values.
The slopes of the toxicity curves can be compared by the ratio of the
LD-90 to LD-10 (Table 1). For example, MnCl2'4H20 is relative non- toxic
and the oral LD-10 dose is 6.3 mmoles/kg body weight. However, if the dose
was increased 1.4-fold to 9.0 mmoles/kg, 90% of the treated rats died. In
contrast, PtCl, was orally the most toxic of the salts tested; the ratio
of the LD-90 to LD-10 was approximately 5.0. The other soluble or partially-
164
-------
Table 1.
Lethal Doses Following Oral Administration
Unit
Compound
Ptci4
Pt(S04)2'4H20
PdCl2-2H20
RuCl3
MnCV4H20
PdS04
(per kg body
weight)
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
LD-50
(957. confidence limits)
0.70 (0.51-0.96)
240 (171-320)
136 (99-188)
2.2 (1.57-3.1)
1010 (720-1400)
430 (310-600)
2.7 (2.2-3.4)
590 (470-730)
290 (240-360)
3.2 (2.4-4.0)
650 (500-830)
310 (240-400)
7.5 (7.0-8.1)
1490 (1380-1610)
410 (380-450)
>7.5
>1500
>790
LD-10
0.31
104
60
1.37
630
270
1.56
330
166
1.78
370
180
6.3
1260
350
--
--
--
LD-90
1.57
530
310
3.5
1620
690
4.8
1030
520
5.4
1130
550
9.0
1780
490
--
--
__
LD-90
LD-10
5.1
2.6
3.1
3.0
1.4
--
165
-------
Table 1 (continued)
Unit
(per kg body
LD-50
Compound weight)
PtCl2 mmoles
mg salt
mg cation
PtO~ mmoles
mg salt
mg cation
PbO mmoles
mg salt
mg cation
PbCl9 mmoles
mg salt
mg cation
PdO mmoles
mg salt
mg cation
MnO« mmoles
mg salt
mg cation
(95% confidence limits)
>8
>2,000
>1,400
>35
>8,000
>6,900
>45
>10,000
>9,300
»35
»9,600
»7,200
» 82
» 10,000
>> 8,700
(est.) 135
(est.) 12,000
(est.) 7,400
LD-10
--
-:
--
--
--
--
«.„
<6,700
<6,200
21-35
5,800-9,600
4,300-7,200
--
--
--
(est.) 60
(est.) 5,200
(est.) 3,300
LD-90
LD-90
LD-10
166
-------
soluble salts tested had intermediate LD-90 to LD-10 ratios of 2.5-3.1.
acute
If the^toxicities following oral administration are expressed as mg
of cation/kg of body weight, the LD-50 values were in the following de-
creasing order: PtCl4> PdCl2'2H20, RuCl3> MhCl^l^O, Pt(S04)2'4H20 >
PdS04>PtCl2> Mn02, Pt02, PbCl2> PbO, PdO.
Likewise, if the LD-10 values are expressed in terms of mg of cation/
kg, the acute toxicities are in the following decreasing order:
PdCl2'2H20, RuCl3>Pt(S04)2'4H20>MnCl2'4H20»Mn02, PbCl2, PbO.
167
-------
B. Intraperitoneal injection. The summary of the lethal dose studies
following intraperitoneal injection are given in Table 2. Values are given
each
for the LD-50, LD-10 and LD-90 and parameter is expressed in terms of mmoles/
kg body weight, mg salt/kg body weight, and mg cation/kg of body weight.
In terms of the LD-50, the acute toxicities (expressed in molar quantities)
in decreasing order, were: PtCl,> Pt(SO, )„'4H90 (Goldsmith)> PdCl7 -2H70 £.
CiSb
Pt(S04)2-4H20 (K andR), MnCl2'4H20> PdS(>4> PtCl2 > PbCl2-
On a molar basis, the LD-10 are in the following decreasing order:
£—Pt(S04)2'4H20 (Goldsmith) (K and K)>
PtCl4>'VPdCl2-2H20>MnCl2-4H20, Pt(S04)2'4H20A PdS04 > PbCl2 . This was exactly
the same order as the molar LD-50 values.
The limiting dosages differentiating toxic and nontoxic substances used
for inclusion of a substance in the Toxic Substances List is 2,000 mg/kg
following an intraperitoneal injection in rats. According to this standard,
all of the tested compounds are "toxic" after intraperitoneal injection.
If the LD-50 following intraperitoneal injection is expressed in terms
of mg cation/kg body weight, the acute toxicities of the compounds decrease
in the following order: PtCl4> MnCl2-4H20 >PdCl2-2H20, Pt(S04)2'4H20 (Gold-
smith) > Pt(S04)2-4H20 (K and K Lab), PdS04> PtCl2 > PbCl2.
The intraperitoneal LD-10 values, expressed in mg cation/kg body weight,
are in essentially the same sequence as the eight most toxic compounds
listed for the intraperitoneal LD-50 values.
C. Duration of survival.
The rapidity of death in non-surviving rats following oral administration
varied widely. For example, rats receiving approximately the oral LD-50
survived for *1.0 day, PtCl4> Pt(SO^^l^O; 1-2.5 days, RuCl3; 3.5-4 days,
MnCl2'4H20; or 5 days, PdCl2'2H20. For rats which received approximately
the intraperitoneal LD-50, the non-surviving rats lived for<1.0 day,
MnCl2-4H20; 3.0-3.5 days, Pt(S04)2'4H20, PdCl2'2H20; and 4.0-4.5 days, PdS04.
168
-------
Table 2.
Lethal Doses Following Intraperitoneal Injection
Unit
(per kg body
• • . v
LD-50
Compound
ptci4
Pt(SO,)2-
4H20
(K and K Lab)
MnCl2-4H20
Pt(S04)'4H20
(Goldsmith)
PdCl2'2H20
PdS04
PbCl2
ptci2
weight)
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
mmoles
mg salt
mg cation
(957. confidence limits)
0.11' (0.09-0. 15)
38 (29-50)
22 (17-29)
0.68 (0.60-0.76)
310 (280-350)
132 (117-149)
0.70 (0.61-0.80)
138 (120-159)
38 (33-44)
0.3-0.4
138-184
59-78
0.57 (0.45-0.72)
121 (95-154)
60 (48-77)
1.42 (1.11-1.81)
290 (220-370)
151 (118-193)
8.5 (5.0-14.4)
240 (1400-4000)
1760 (1050-3000)
2.5 (1.58-4.0)
670 (420-1060)
490 (310-770)
LD-10
0.56
260
110
0.56
111
31
0.2-0.3
92-138
39-59
0.39
84
42
(est.)
(est.)
(est.)
1.6
440
330
LD-90
0.82
380
160
0.87
172
48
0.4-0.6
184-280
78-117
0.82
175
87
0.77 1.8
156 370
82 195
16.8
4700
3500
LD-90
LD-10
1.5
1.6
2.1
2.4
10
169
-------
In rats which survived for 7 to 14 days after administration of doses which
were two-thirds or less of the oral or intraperitone'al LD-50, significant lack
of weight gain was noted during days 0-7 in rats receiving most of the compounds.
However, weight gain, (expressed in grams) T*as approximately the same in the
the
treated and control rats during days 7-14 of the observation period.
Two samples of Pt(SO, )2'4H20 were tested in the intraperitoneal lethal
dose studies; one was purchased from ICN-K and K Laboratories and a second
sample from D. F. Goldsmith Chemical and Metal Corp. The two samples differed
in their acute toxicities by approximately 2-fold. It has not besn possible
to identify the cause of the differences.
Detailed data on the acute toxicities are given in the attached Appendix.
The data include the duration of survival by all animals and non-surviving
animals and the weight gain during the two weeks following metal administration
to the surviving animals. Also included are the plots of the probits versus
log dose which were used in the method of Litchfield and Wilcoxon.(l) to
evaluate the LD-10 and LD-90 values.
170
-------
Metal content in various tissues.
Analyses for lead, manganese and platinum were conducted by
Yoakum, Stewart and Sterrett (5). In a series of rats treated for
90-91 days, the control rats ingested approximately 0.15 g of
manganese (from the solid feed). The tissue concentration of Mn
was 1.4 and 1.0 pg Mn/g wet tissue in the liver and kidney, respect-
ively. In Mn-treated rats, which received 8.3 mM MnCl .4H 0 as the
drinking fluid and ingested approximately 2.3 g of Mn per rat
during the 90-91 day interval, the concentration of Mn was somewhat
increased, namely 2.8 and 1.6 ;ig Mn/g of wet tissue in the liver and
kidney, respectively. The Mn concentration in spleen, heart, testes
and blood was not increased in the tissues of Mn-treated rats.
A second group of rats received 3.6 mM PbCl in the drinking
water for 90-91 days and ingested approximately 3 g of lead per rat
during the interval; control rats ingested ^ 0.01 g of Pb in the
solid feed during the same interval. Kidney showed a marked accumula-
tion of Pb (to 11.1 jig Pb/g of wet tissue) in the lead-treated rats;
in the same rats the concentration in liver was 1.2 jig Pb/g of wet
tissue. The corresponding levels in the control rats were approximately
171
-------
0.3 pg Pb/g of wet tissue in both kidney and liver. The other
tissues -- spleen, heart, testes and blood -- did not exhibit
appreciably higher levels of Pb in the Pb-treated rats.
Soluble Pt salts were included in the drinking fluid of rats
for 8-9 day intervals. The approximate total Pt intake (mg Pt per
rat) and data on the tissue concentration of Pt in various tissues
are presented in Table 3. Although the Pt concentrations in tissues
of untreated control rats often attain level? measurable by the
technique used by Stewart Laboratories, Inc., the levels are low
and are generally less than 0.1 p.g Pt/g of wet tissue. For the higher
4+
levels of Pt intake in the Pt-treated rats, the highest tissue
concentrations of Pt occurred in the kidney and ranged from 4.5-5
/ag Pt/g of wet tissue. High levels, ranging from 0.7-2.5 ;ug Pt/g,
also occurred in the liver. In contrast, brain showed only a very
low level of Pt which may reflect a contribution from the blood.
Separate experiments were conducted on the .tissue concentrations of
Pt in rats which received a saturated solution of PtCl as the
2
drinking fluid for 30-31 days. In the PtCl -treated rats, the mean
Pt concentration for liver, kidney and spleen were < 0.08 jug Pt/g
of wet tissue.
In Table 4 are presented the Pt concentration of tissues
removed from rats which had survived for the 14-day observation
period in lethal-dose experiments. The doses of Pt(SO ) .4H 0
42 2
administered by both the oral and intraperitoneal routes were
approximately 90% of the LD-50 values by the respective routes.
172
-------
Table 3. Pt-content of tissues of rats maintained on drinking
fluid containing Pt salts.
Diet (drinking Control Pt(SO^)2.AH20
PtCl,
riuia;
Pt salt concn.
(mg Pt/lit)
Duration of diet
(days)
Total Pt intake ^0.01
106
8
26
319
9
80
319
8
60
Tissue Tissue concentration of Pt
(ug Pt/g wet tissue)
Liver 4. 0.02
+ 0.02
Kidney < 0.23
± 0.45
Spleen ^ 0.08
+ 0.08
Heart < 0.02
+ 0.01
Testes < 0.014
± 0.010
Brain
Blood 0.10
+ 0.13
0.07
(0.04-
0.09)
0.26
+ 0.05
0.02
(0.01-
0.01)
0.02
0.04
± 0.05
-
0.05
0.85
(0.73-
0.97)
4.6
(4.5-
4.7)
0.13
0.25
-
0.015
+ 0.002
0.22
(0.09-
0.36)
2.2
(2.0-
2.5)
4.8
+ 0.5
0.24
-
-
-
0.23
(0.19-
0.27)
Control rats are those from diet experiments after approximately
8 or 30 days; 5-7 values for blood, spleen and heart, 13-16 values for
liver, kidney and testes. +, standard deviation is given for means
with 4 values; ranges are indicated in parentheses for means of 2 values,
173
-------
Table 4. Pt concentration in rat tissues following the
administration of single high doses of Pt(SO,)2.4H20
Treatment
Route
Dose of Pt
(mg Pt/kg)
Tissue
Liver
Kidney
Spleen
Controls
Oral
-
Pt(S04)2.4H20
Oral I. p.
382 113
Tissue Concentration of Pt
(ug Pt/g wet weight of tissue)
< 0.01
(0.004-0.006)
< 0.008
(0.004-0.004)
^ 0.013
(0.007-0.011)
2.3 34
(1.2-3.5) (30-38)
16 37
(13-19) (28-46)
3.3 16
(2.3-4.2) (12-20)
Heart
Testes
Brain
Blood
0.02
0.011
(0.009-0.013)
0.01
0.008
0.8
0.5
3.0
1.2
(0.4-0.6) (0.9-1.5)
0.10 0.6
(0.07-0.14)(0.07-1.1)
3.3
1.0
Range of 4 values for control liver, kidney and spleen,
and range of 2-3 values of all other tissues are given
in parentheses. Control values are the mean values of
Pt concentration in 2 rats which received orally NaCl.
174
-------
During the two-week observation period, the rats gained weight at a
rate from one-third to three-fourths the rate of the control rats.
In the orally treated rats, the highest concentration of Pt occurred
in the kidney (approximately 16 ug Pt/g) and appreciable levels of
. Pt also occurred in liver and spleen (range, 1-4 jig Pt/g of wet
tissue). In the intraperitoneably treated rats, the kidney, liver
and spleen showed very high levels of Pt in the range of 10-40 ug
of Pt/g of wet tissue.
In a comparable lethal dose experiment, rats were treated
orally with a dose of MnCl .4H 0 equivalent to 100% of the oral
LD-50 value and the tissues were analyzed in surviving rats at the
end of the 14-day observation period. In contrast to the finding
with the Pt salt, the oral administration of a single, large but
nonlethal dose of MnCl .4H 0 to rats did not result in the retention
2 2
after 14 days of excess concentrations of Mn in any of the tissues
analyzed (Table 5). Due to low levels of absorption and/or a
high capacity for excretion of the Mn, the tissue Mn levels of the
experimental rats were approximately equal to the levels found in
control rats.
These studies show that in rats treated with soluble Pt
salts, appreciable levels of the metal can be found in the kidney,
liver and spleen. Further studies will be necessary to determine the
effects of the Pt and other metals on various biochemical reactions.
175
-------
Table 5. Mn c.oncent rat ions in rat tissues following
the oral administration of a single large dose of
Treatment Controls3 MnCl2t4H20
Dose of Mn — 416
(mg Mn/kg)
Tissue Tissue Concentration of Mn
(pg Mn/g wet weight of tissue)
Liver
Kidney
Spleen
Heart
Testes
1.60
+ 0.87
0.75
± 0.50
1.46
+ 1.99
0.55
+ 0.35
0.44
+ 0.35
1.9
(1.3-2.
1.3
(1.0-1.
1.3
(1.1-1.
0.7
0.5
(0.4-0.
5)
5)
5)
5)
Brain 0.3 0.03
Blood 0.86 0.4
+ 0.44 (0.2-0.6)
Control values are from rats treated orally.with NaCl, and rats on
diet experiments for approximately 8 or 30 days. Means + standard
deviations are given for 6-7 samples of spleen, heart and blood and for
13-18 samples of liver, kidney and testes from control rats; ranges are
given in parentheses where two values are available from Mn-treated rats,
176
-------
Organ weights
Weights of five organs (liver, kidney, spleen, heart and testes) were
measured in rats which had been treated with various metallic salts in the
The organ weights were
diet (either drinking fluid or solid feed), ^ expressed as the percentage
of the body weight. In the discussions which follow, no consistent changes
following
in organ weights occurred unless specifically noted. In general, treatment
with most metallic salts (at the doses used) weight changes were not observed
in liver, spleen and heart; several metals changed the kidney weight and a
few salts changed the testes weight.
MnCl2'4H20. MnCl2'4H20, in the drinking fluid at 8.3 or 18.6 mmoles/
liter (1640 or 3690 mg/liter) for 90 days did not bring about major changes
in organ weights, although there was some enlargement (12, 13, and 87o above
control; not statistically significant) in the spleen of all three experi-
mental groups of animals. None of the other organs of rats receiving
MnCl7'4H90 in the drinking fluid showed consistent changes.
/ In rats
PbCU. A which received 3.7 mmoles/liter (1022 mg/liter) for 30
days, or for 90 days> the size of the liver was increased (6-12%
above control; only one statistically significant) in three of the four
experiments. In contrast, the kidney size was increased in both the 30-day
dietary experiments (77» and 67> above control; not statistically significant)
and the 90-day experiments (1770 and 237o above control; each statistically significant.)
Consistent changes in organ weights were not noted in the cases of spleen,
heart and testes although spleen showed increased size in two of four experi-
ments. In a single experiment, PbCl? (8.3 mmoles/liter or 2300 mg/liter)
for 30-days caused 187o and 257<, (neither statistically significant) increases
in kidney and spleen size, respectively. The increase in kidney size due to
2+
treatment with Pb is consistent with the data by Hirsch (6) and by others.
PdCl2 (anhydrous) and PdCl '2H20. Although the addition of PdCl2 (an-
hydrous) to the feed (13.2 mmoles/kg or 2345 mg/kg ; 30-days) caused
177
-------
changes in several organ weights, the pattern was not consistent to that
found in a second experiment. The use of a saturated solution of PdCl9'
2H20 as the drinking fluid (8-days) also did not cause a consistent change
in any of the organ weights.
PdSO,. The use of a saturated solution of PdSO, as the drinking fluid
for 8 or 30 days did not cause a consistent pattern of changes in the organ
weights of the experimental rats. In a single experiment in which solid
PdSO, was added for 30-days to the feed at a level of 5.9 mmoles/kg feed
(1.19 g salt/kg feed), no changes were observed in organ weights; each rat
received a mean of 3.8 mmoles of Pd salt (0.40 g cr Pd% duri ^ the total
diet period.
PtCl, . When this salt was added to the drinking fluid at 0.5 mmoles/
liter (183 mg salt/liter) for 30 days or 90 days, or at 1.6 mmoles/liter
(550 mg salt/liter) for 8 days, no consistent changes were detected in the
weights of the five organs. If the concentration was increased to 1.6 mmoles/
liter (550 mg salt/liter) for 30 days or to 2.4 mmoles/liter (825 mg salt/
liter) for 9 days, the weight of the kidneys were increased by approximately
6% in each of 4 experiments but only two experiments gave p<0.1.
In addition, in rats treated at the higher levels (1.6 mmoles/liter
for 30 days or 2.4 mmoles/liter for 9 days), the testes were increased in
weight by approximately 11% in each of the 4 experiments but the difference
was statistically significant (p
-------
Drug metabolism in vitro.
After experimental rats were maintained on metal-containing diets
for approximately 8, 30 or 90 days, hepatic microsomes were isolated
and parameters related to drug metabolism were measured. Data on pre-
sented in Table 6. Microsomal protein was expressed as mg microsomal
protein/g liver; aniline hydroxylase activity, nmoles p-aminophenol pro-
duced/mg microsomal protein/20 min; aminopyrine demethylase, nmoles form-
aldehyde produced/mg microsomal protein/10 min; cytochrome P-450 and
cytochrome b5, nmoles/mg microsomal protein.
Treatment with MnCl?'4H_0 for approximately 90 days at either 8.3
mmoles (1.6 g salt)/liter or 18.6 mmoles (3.7 g salt)/liter did not ap-
(Table 6A),
pear to consistently change any of the parameters^ Treatment with PbCl_
at 3.7 mmoles (1.0 g salt)/liter for 90 days or 8.3 mmoles (2.3 g salt)/
liter for approximately 30 days did not affect recovery of microsomal
protein or the activity of aniline hydroxylase; for the other three para-
meters, it was not possible to detect a trend since data were obtained for
only one experiment each.
PdCl '2H_0, a slightly soluble salt, was administered to rats as a
saturated solution as the drinking fluid for 8 days and a marked decrease
was noted in the activities of aniline hydroxylase and aminopyrine demethy-
(Table 6B),
lase^ However, caution must be exercised in the interpretation of this
trend. PdSO, , which is also a slightly soluble salt, was administered
as a saturated solution as the drinking fluid for 8 days or for approxi-
mately 30 days. In each of these experiments no trend of changes was ob-
(Table 6C).
served in the activities of the two enzymes^ At this time, it is not pos-
sible to explain the apparent differences as a result of the administration
2+
the two slightly soluble salts of Pd
179
-------
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2+
PtCl9, an "insoluble" salt of Pt , was administered to rats as a
saturated solution as the drinking fluid for approximately 30 days. No
consistent changes were observed in the activities of aniline hydroxy-
lase and aminopyrine demethylase although the former enzyme showed de-
creased activity in two of the three experiments (Table 6D).
4+
The Pt salts, PtCl, and Pt(SO,)2'4H?0, are very soluble and were
included in the drinking water. A wide range of dosages and durations
were used in the dietary experiments: 0.54 mmoles (183 mg PtCl,)/liter
for 30 and 90 days, and 1.6 mmoles (550 mg PtCl, or 750 mg Pt(SO, )2'4H2 0'liter)
for 8 days and for 30 days (PtCl, only). Under none of the conditions
were consistent changes observed in the level of the drug metabolizing
enzymes (aniline hydroxylase or aminopyrine demethylase), or in the re-
covery of microsomal protein in the liver (Tables 6E, 6F).
It is apparent that the activities of the two representative drug
metabolizing enzymes and amounts of cytochromes P-450 and b5 are not
4+ 2+
extremely sensitive to lower levels of dietary Pt and Pd . In order
4+ 2+
to administer in the diet sufficient Pt and Pd to affect these para-
meters of drug metabolism, it will be essential or preferable to administer
the metallic salts in the solid feed rather than in the drinking fluid.
Obviously, the use of higher doses of the insoluble or slightly soluble
salts will require administration in the solid feed. Such studies are
currently in progress.
183
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Studies on RNA synthesis in vivo.
Two major rRNA-containing peaks resulted from centrifugation of
resuspended hepatic polysomes on a sucrose gradient containing 0.1 M
NaCl and 0.001 M EDTA. The peaks had sedimentation coefficients of
approximately 27S and 43S. A0,A/A00,. ratios of the isolated peaks in-
/bu /ou
dicated a greater content of protein than in corresponding peaks pre-
pared from phenol-SDS or SDS-extracted rRNA. When compared with profiles
of phenol-SDS or SDS-extracted rRNA, material treated with only EDTA
and NaCl exhibited greater homogeniety and/or tighter conformation.
Increases in gradient NaCl concentration (0.25 M or 0.5 M) resulted in
additional peaks, all having S values greater than 27S. As indicated
by labeling studies for 1 h and 24 h, the EDTA-NaCl procedure was com-
parable to the SDS extraction procedure for observation of rRNA. Ribo-
somal RNA from RNase-treated polysomes (5 mg or 11 mg RNA/0.1 ug RNase)
showed greater structural integrity following EDTA-NaCl treatment than
following SDS extraction. When compared with diethylpyrocarbonate as
a means of improving resolution of rRNA following RNase degradation of
polysomes, the EDTA-NaCl procedure gave equally satisfactory results
with significantly greater convenience. A manuscript describing this
study has been prepared.
187
-------
References
1. Litchfield, J. T. , and Wilcoxon, F. A simplified method of evaluating
dose-effect experiments. J. Pharmacol. Exp. Ther. 96: 99 (1949).
2. Imai, Y., Ito, A., and Sato, R. Evidence for biochemically different
types of vesicles in the hepatic microsomal fraction. J. Biochem.
(Tokyo) 60: 417 (1966).
3. Chhabra, R. S., Gram, T. E., and Fouts, J. R. A comparative study of
two procedures used in the determination of hepatic microsomal aniline
hydroxylation. Toxicol. Appl. Pharmacol. 22: 50 (1972).
4. Schenkman, J. B., Remmer, H., and Estabrook, R. W. Spectral studies
of drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol.
J3: 113 (1967).
5. Yoakum, A. M., Stewart, P. L., and Sterrett, J. E. Method development
and subsequent survey analysis of biological tis--- les for platinum, lead
and manganese content. Environ. Health Perspect. (preceding
paper) (1974).
6. Hirsch, G. H. Effect of chronic lead treatment on renal function.
Toxicol. Appl. Pharmacol. 25: 84 (1973).
7. Christensen, H. E. (ed.), National Institute for Occupational Safety and
Health Toxic Substances List, 1972 Edition (1972).
188
-------
Data on Lethal Dose Levels
Values are the means (or weighted means) ± (upper) standard devia-
tions ± (lower) standard errors; the number of values is given in paren-
theses. NA, not applicable or not applied. In the column of "percentage
survival", the underlined values were used for the determination of the
LD-50, LD-10 and LD-90 doses by the method of Litchfield and Wilcoxon.
The standard deviations and standard error in the percentage survival
column are calculated from the products of the percentage survival and
the number of rats in each experiment in which that dose was tested.
Statistical analyses (student's t-test) were applied only to weekly weight
gains: ***, p<0.001; **, p^O.Ol; *, p<0.05; m, 0.05* p< 0.10; no mark-
ing used were p>0.10. Percentage changes of the weight gains are indi-
cated only where p 0.10 or where the percentage was less than 90% or
more than 110% of the control values.
o
"Corrected value from Table 1 of Litchfield and Wilconon for meas-
ured 0% or measured 100% survival.
"Expected value" was <0.01% or >99.99% by method of Litchfield and
Wilconon and not used in determination of lethal doses.
£
Value not used in determination of lethal doses.
Value approximated by assumption of 0.25 survivors/number tested
where 070 survival was measured, and 0.25 non-survivors/number tested
where 1007. survival was measured.
189
-------
MnCl?'4H20 / Intraperitoneal injection
Dose
(mg salt
/kg
body
weight) Expt.
Controls 21,25,
32,34
400 2,21,
25
200 2,21,
25
141.4 32,34
100 2,21,
25
50 21,25
Number
survivors
/number Percentage
tested survival
10/10 NA
100.0
±0.0
±0.0
0/11 NAb
0.0
±0.0
±0.0
0/10 2.5d
0.0
±0.0
±0.0
4/12 33.3
33.3
±0.0
±0.0
11/11 97. 7d
100.0
±0.0
±0.0
11/11 NAb
100.0
±0.0
+0.0
Duration of
survival (hr)
all
animals
336
±0
±0
(10)
2
±2
±1
(11)
8
±4
±1
(10)
120
±159
±46
(12)
336
±0
±0
(11)
336
±0
±0
(ID
non-
survivors
NA
2
±2
±1
(11)
8
±4
±1
(10)
12
±3
±1
(8)
NA
NA
Body
weight
(g) on
day 0
117
±8
±3
(10)
112
±12
±4
(11)
112
±9
±3
(10)
114
±13
±4
(12)
112
±8
±2
(11)
112
±8
±2
(ID
Weight gain (g)
days
0-7
63
±11
±3
(10)
NA
NA
18
±34
±17
(4)
*
297.
33
±19
±6
(9)
***
527o
45
±11
±3
(11)
**
71%
days
0-14
110
±13
±4
(10)
NA
NA
56
±57
±28
(4)
m
517o
81
±21
±7
(9)
**
747o
96
±15
±4
(11)
*
877»
day
7-1
48
±9
±3
(10
NA
NA
38
±25
±12
(4
ns
79
48
±12
±4
(9
ns
51
±12
±4
(11
ns
190
-------
MnCl_.4H20 / Intraperltoneal injection
e
-------
MnCl?'4H20 / Oral administration
Dose
(mg salt
/kg
body
weight) Expt.
Controls 5,8,
14,23
2500 1,5,
14,23
2000 5,8,
14,23
1750 8,14,
23
1500 5,8,
14,23
1250 1,8,
14,23
1000 5,8,
14,23
Number
survivors
/number Percentage
tested survival
14/14 NA
1/12 NA°
8.3
±15.1
±4.3
0/12 1.0a
0.0
±0.0
±0.0
2/12 16.7
16.7
±14.7
±4.2
6/12 50.0
50.0
±28.9
±8.3
10/11 90.9
90.9
+12.6
+3.8
12/12 99. 9a
100.0
±0.0
±0.0
Duration of
survival (hr)
all non-
animals survivors
336
±0
±0
(14)
40
±94
±27
(12)
22
±23
±7
(12)
106
±114
±33
(12)
213
±133
±38
(12)
307
+97
+29
(11)
336
±0
±0
(12)
NA
13
±11
±3
(1J-)
22
±23
±7
(12)
60
±43
±14
(10)
90
±48
±19
(6)
NA
NA
Body
weight
(g) on
day 0
107
±10
±3
(14)
109
±9
±2
(12)
103
±7
±2
(12)
109
±8
±2
(12)
103
±14
±4
(12)
111
+10
+3
(11)
105
±7
±2
(12)
Weight gain
days days
0-7 0-14
51
±10
±3
(14)
NA
NA
NA
22
±22
±9
(6)
**
43%
39
+25
+9
(7)
ns
76%
40
±19
±5
(12)
m
78%
100
±31
±8
(14)
NA
NA
NA
84
±26
±10
(6)
ns
84%
103
+26
+10
(7)
ns
99
±20
±6
(12)
ns
(g
da
7-
4
±2
±
(1
N
N
N,
6:
±'
±
(<
I
12
6<
+1(
+<
C
r
13
5!
±li
&
(i:
n:
12(
192
-------
MnCl-.AH-O / Oral administration
> «•>
3
CO
-------
MnCL / Oral administration
Dose
(mg salt
/kg
body
weight) Expt.
Controls 33,38
10,000 33,38
6,666 38
Number
survivors
/number Percentage
tested survival
6/6 NA
100.0
±0.0
±0.0
6/10 60.0
60.0
±0.0
±0.0
9/11 81.8
81.8
Duration of ,
survival (hr) we°g£t
all
a n ima 1 s
336
±0
±0
206
±168
±53
(10)
285
±113
±34
(11)
non- (g) on
survivors day 0
NA 99
±9
±4
(6)
11 103
±8 ±10
±4 ±3
(4) (10)
NA 99
±10
±3
(11)
Weight gain (g
days
0-7
68
±11
±4
(6)
58
±15
±6
(6)
ns
85%
51
±18
±6
(9)
*
75%
days
0-14
128
±12
±5
(6)
120
±16
±6
(6)
ns
102
±44
±15
(9)
ns
80%
da
7-
6
±
±
(
6
±
±
(
n
5
±2
±
(
n
8
194
-------
MnO_ / oral aamiruatration
« s
-------
PbCl9 / Intraperitoneal injection
Dose
(mg salt
/kg
body
weight)
Controls
2083
1250
750
450
270
Expt.
12,21,
28,41,
48
28,41,
48
21,28,
41,48
12,21,
28,41,
48
12,21,
28,41
21,28
Number
survivors
/number Percentage
tested survival
13/13 NA
100.0
±0.0
±0.0
9/16 56.2
56.2
±14.3
±3.6
11/16 68.8
68.8
±27.1
±6.8
12/16 75.0
75.0
±29.8
±7.5
11/12 91.7
91.7
±12.3
±3.6
7/7 NA
100.0
±0.0
±0.0
Duration of
survival (hr)
all
animals
336
±0
±0
(13)
227
±130
±33
(16)
257
±126
±31
(16)
304
±74
±18
(16)
325
±39
±11
(12)
336
±0
±0
(7)
non-
survivors
NA
87
±39
±15
(7)
82
±63
±28
(5)
206
±101
±50
(4)
NA
NA
Body
weight
(g) on
day 0
111
±9
±3
(13)
101
±6
±2
(16)
110
±9
±2
(16)
106
±9
±2
(16)
111
±6
±2
(12)
116
±4
±2
(7)
Weight gain (g)
days
0-7
55
±19
±5
(13)
30
±24
±8
(10)
*
55%
31
±18
±5
(12)
#*
56%
27
±18
±5
(15)
***
49%
29
±25
±7
(12)
**
53%
29
±16
±6
(7)
**
53%
days
0-14
125
±17
±5
(10)
76
±36
±12
(9)
**
61%
63
±37
±11
(11)
•kit-it
50%
57
±37
±11
(11)
***
46%
54
±44
±16
(8)
***
43%
74
±30
±11
(7)
**
59%
day
7-1
62
±11
±4
(10
41
±21
±7
(9!
*
665
31
±30
±9
(ID
**
50%
32
±16
±5
(11)
*•**
52%
28
±24
±9
(8)
•ft*
45%
44
±16
±6
(7)
*
71%
196
-------
PbCl-, / Intraperitoneal injection
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Dose (log)(mg salt/kg body weight)
1Q7
-------
PbCl2 / Oral administration
Dose
(mg salt
/kg
body
weight) Expt.
Controls 51
9645 51
5787 51
Number
survivors
/number Percentage
tested survival
2/2 NA
100.0
9/10 90.0
90.0
9/10 90.0
90.0
Duration of ,
survival (hr) . ?;..
weight
all
animals
NA
305
±97
±31
(10)
325
±35
±11
(10)
non- (g) on
survivors day 0
NA NA
NA 118
±12
±4
(10)
NA 116
±10
±3
(10)
Weight gain (g
days
0-7
NA
44
±13
±4
(9)
54
±27
±9
(10)
days
0-14
NA
107
±12
±4
(9)
115
±31
±10
(9)
da;
-i _
N>
6.'
±f
±J
(<
5f
±U
±i
V
198
-------
PbO / Oral administration
Dose
mg salt
/kg
body
2ight) Expt.
jntrols 33,39
.0,000 33,39
6,666 39
Number
survivors
/number Percentage
tested survival
6/6 NA
100.0
±0.0
±0.0
7/11 63.6
63.6
±15.7
±4.7
6/9 66.7
66.7
Duration of
survival (hr)
all
animals
336
±0
±0
(6)
217
±165
±50
(ID
228
±161
±54
(9)
non-
survivors
NA
9
±4
±2
(4)
13
±4
±2
(3)
Body
weight
(g) on
day 0
105
±6
±2
(6)
103
±10
±3
(11)
-97
±4
±1
(9)
Weight gain (g)
days
0-7
64
±15
±6
(6)
32
±19
±7
(7)
**
507=
44
±12
±5
(6)
i(
697.
days
0-14
106
±33
±13
(6)
96
±15
±6
(7)
ns
105
±14
±6
(6)
ns
days
7-14
43
±18
±7
(6)
64
±8
±3
(7)
*
149%
61
±12
±5
(6)
m
142%
199
-------
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CHAMPION LINE NO. 674 - PROBABILITY X SO
Dose (log)(mg salt/kg body weight)
200
-------
PdCl2'2H20 / Intraperitoneal injection
)ose
; salt
'kg
)ody
Lght) Expt.
itrols 17,24,
37
79 17,24
86 17,24,
37
24 17,24,
37
82.7 24,37
55.1 24,37
Number
survivors
/number Percentage
tested survival
8/8 NA
100.0
±0.0
±0.0
0/5 NA
0.0
±0.0
±0.0
1/11 9.1
9.1
±8.7
±2.6
4/11 36.4
36.4
±19.5
±5.9
11/11 92. 6a
100.0
±0.0
±0.0
11/11 NA
100.0
±0.0
±0.0
Duration of
survival (hr)
all
animals
336
±0
±0
(8)
17
±6
±3
(5)
59
±94
±28
(ID
172
±138
±42
(11)
336
dbO
±0
(11)
336
±0
±0
(11)
non-
survivors
NA
17
±6
±3
(5)
32
±22
±7
(10)
78
±58
±22
(7)
336
±0
±0
(11)
336
±0
±0
(11)
Body
weight
(g) on
day 0
97
±7
±2
(8)
95
±6
±2
(5)
96
±5
±2
(ID
101
±4
±1
(11)
98
±8
±2
(11)
97
±5
±2
(11)
Weight gain (g)
days
0-7
60
±8
±3
(8)
NA
NA
33
±19
±9
(5)
*
55%
59
±8
±2
(ID
ns
57
±11
±3
(ID
ns
days
0-14
118
±15
±5
(8)
NA
NA
86
±8
±4
(4)
***
73%
119
±11
±3
(11)
ns
116
±21
±6
(11)
ns
days
7-14
57
±9
±3
(8)
NA
NA
46
±6
±3
(4)
*
81%
60
±8
±2
(11)
ns
59
±13
i4
(11)
ns
201
-------
PdCl2.2H20 / Intraperitoneal injection
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S 4- — ri-:---4----±
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to
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J = = = = = = = = = = :I = == = = = == = = = = | = = =^ = = == = = = r 2 =
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..._..,., . .. - .LI - - L
si I L .JIM II ILI
s 1.9 2.0 2.1
*
"•• -"T S
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00
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^Tr
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±:±-|:|::: :-::::::::r::::::::::::::::::
-,«-«_ -i- ™-»__^«^«™. ,„__«._ j_ « wv^™ „_».„«_ ...__...,._ o
j|||j||^
i \ !
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NTlUflr In IT' 1 TiTms
ittr ' "t
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^:~":: _ :n "::~±" _ " "4^4 -"" " "
M
1 ^
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--X± ILL ""«*
:J:£:p_:;_;::±i:d :_:::;;::::::;::_::::;;:: 8
4_ -4_ 1^-, | o
XL 1 :
-L ^j_; J» JJ. -Lj j J» -L i- J. S
2.2 ? . 1 ? . 4 rf
CHAMPION LINE NO. 67A - PROBABILITY X BO
Dose (log) (nig salt/kg body weight)
-------
PdCl2-2H20 / Oral administration
Dose
(mg salt
/kg
body
weight) Expt.
Controls 15,16,
18,25
1410 16,18,
25
940 15,16,
18,25
627 15,16,
18,25
418 15,16,
18,25
279 15,16
18,25
186 15
'
Number
survivors
/number Percentage
tested survival
9/9 NA
. 100.0
±0.0
±0.0
0/11 2.2a
0.0
±0.0
±0.0
2/11 18.2
18.2
±31.2
±9.4
5/11 45.5
45.5
±32.6
±9.8
7/10 70.0
70.0
±29.2
±9.2
11/11 96. 2a
100.0
±0.0
±0.0
3/3 NA
100.0
Duration of
survival (hr)
all
animals
336
±0
±0
(9)
58
±57
±17
(ID
125
±108
±32
(11)
213
±130
±39
(11)
276
±100
±32
(10)
336
±0
±0
(11)
336
±0
±0
(3)
non-
survivors
NA
58
±57
±17
(ID
79
±31
±10
(9)
110
±76
±31
(6)
136
±52
±30
(3)
NA
NA
Body
weight
(g) on
day 0
108
±13
±4
(9)
109
±11
±3
(11)
102
±11
±3
(11)
106
±8
±3
(11)
103
±8
±3
(10)
107
±11
±3
(11)
98
±5
±3
(3)
Weight gain (g)
days
0-7
57
±8
±3
(9)
NA
NA
14
±23
±9
(6)
***
25%
11
±20
±7
(8)
***
35%
37
±28
±8
(11)
*
65%
49
±10
±6
(3)
ns
86%
days
0-14
115
±13
±4
(9)
NA
NA
69
±31
±14
(5)
**
60%
57
±40
±15
(7)
**
50%
89
±30
±9
(11)
*
77%
106
±14
±8
(3)
ns
days
7-14
59
±9
±3
(9)
NA
NA
51
±13
±6
(5)
ns
86%
42
±23
±9
(7)
m
71%
52
±9
±3
(11)
ns
88%
57
±4
±3
(3)
ns
203
-------
PdCl2.2H20 / Oral administration
o
s mil nil. mi. -ii _i iiiimii: "~_-iii-_"ii
o — _. — _ _
o
CM
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tft
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« .________., _ _ III ,_. „ ___..-..---., _ - _ . . _ _ _ _ ...___
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•rl _ - - - „_-
r :::::": _ : : :: : : :: :
g s -
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s ^"EiiziEE i""-"--" :E":i -""-•- :""-:- ;-:--
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8 2.4 2.5 2.6 2.7
8
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— • s
-f_ .... ___--»m
Tj- *
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/
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„. __- .. - _^ . _-
: i: - - - ± Jr. j ±.±_.:::;: o
" " ji"-^ IT " — ". r _ iXt ; :±:: •>
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E = EE = EEE=E = |EEEEE^^{EEEpEE = = EEEEEEEE = = EEe
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rf
a 2 _ _ _, _ _.„ _„ _- . i
L^.X ,2_. o
~f
-- r- ^-- S
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,
----- ---------- .-_-_---_- --*_-.---_. .„_„-_- _- O
^ — : -;^:::--- ::-----:- ±: ----- ±---:-:--
t. _ . --- -.- -.--.- .. _ - j. _ -_-+-- - - -
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± li±t T .-
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2.8 2.9 3.0 3.1
CHAMPION LINE NO. 674 - PROBABILITY X 9O
Dose (log)(mg salt/kg body weight)
204
-------
PdO / Oral administration
Dose
(mg salt
/kg
body
weight) Expt.
Number
survivors
/number
tested
Duration of „ .
survival (hr) . •;.
v weight
Percentage
survival
all
animals
non- (g) on
survivors day 0
Weight gain (g)
days
0-7
days
0-14
days
7-14
Controls 33,54
10,000 33,54
5/5
6/6
NA
1.00.0
±0.0
±0.0
336
±0
±0
(5)
336
±0
±0
(5)
NA
108
±7
±3
(5)
69
±12
±5
(5)
130
±10
±4
(5)
62
±7
±3
(5)
NA
103
±13
±5
(6)
ns
50
±18
±7
(6)
m
72%
98 48
±35 ±17
±14 ±7
(6) (6)
m ns
75% 77%
205
-------
PdSO, / Intraperitoneal injection
Dose
(mg salt
/kg
body
weight) Expt.
Controls 22,40,
47
450 40
300 40,47
245 47
200 22,40,
47
133.3 22,40
Number
survivors
/number Percentage
tested survival
9/9 NA
100.0
±0.0
±0.0
0/4 NA
0.0
5/10 50.0
50.0
±21.5
±6.8
5/9 55.6
55.6
8/10 80.0
80.0
±17.2
±5.4
4/4 NA
100.0
±0.0
±0.0
Duration of
survival (hr)
all
an -(.-.a Is
336
±0
±0
(9)
52
±52
±26
(4)
219
±127
±40
(10)
245
±114
±38
(9)
294
±88
±28
(10)
336
±0
±0
(4)
non-
survivors
NA
52
±52
±26
(4)
101
±40
±18
(5)
132
±60
±30
(4)
NA
NA
Body
weight
(g) on
day 0
107
±4
±2
(9)
101
±6
±3
(4)
i03
±7
±2
(10)
110
±10
±3
(9)
105
±6
±2
(10)
104
±13
±6
(4)
Weight gain (g)
days
0-7
62
±10
±3
(9)
NA
44
±10
±4
(5)
**
717,
38
±16
±7
(6)
**
61%
31
±16
±6
(8)
***
50%
34
±21
±10
(4)
*
55%
days
0-14
126
±19
±6
(9)
NA
89
±39
±17
(5)
m
71%
86
±33
±15
(5)
*
68%
75
±34
±12
(8)
**
60%
90
±33
±17
(4)
m
71%
days
7-14
64
±12
±4
(9)
NA
45
±30
±13
(5)
ns
70%
46
±18
±8
(5)
m
72%
44
±25
±9
(8)
m
69%
56
±14
±7
(4)
ns
88%
206
-------
PdSO, / Intraperitoneal injection
V*
O
s :::::::::::::::::::: : ::::::::.::::..: :.;
o
e*
o '
Ml
e ~
- .:::...: - - H
r-4 ._ _
eri o ...._..__. ...... .„.».-. ..___.
S *> I ! I ! !_"!!!!"!!"___" _ . """_ ~
< ::::__:..:: - _ _- _
& $ . 4J4UUIIIW tttm
3 *
ra
g
a) HI ILL ill
. Q ;;;;; — ; : : ; : i ; : :_ . ::..... .: :
4J X .......... -..-.----- ......_._. ._._..
d . "i i .."".." '-..' . -. _ . ; ~ _. :
y o ;::;:::::: :*:; ;: _. ;;;_.«'._;
u "~ "~ • ""«'* ""
PM • :"ii: :::::."::: .":.. :~ :~ .: ,*•." " " "
* ^
S::::::, ::::::,:: = ::, ::::;: = = = ( fcr:= : = ::: :::::
1 Li rl H MM M"T 1
g t
S. . . . . ..---_ . .-_-- .
S :
8 ::::::::::::::::::::::::::::::::—:
•o
S - -- -
0>
8.
8 2.2 2.3
s
a
s
«i
" "" S
::;::;;:;;;:::;:=:::;:::::;::::::;::::::::::: g;
- ^ - -. - - - - - - - ~ - ~ - • go
-.-. _.- --. __.__..«.--.»«
~ ~ " ~ " ~ " -~ ~~ Z m--~ ^ m mZ '. O
t *
r •- g
j. -,j ;. o
-li& 3''
-J \f _,,?
:--::: : S
- - -- -
. . ._. «
o
. . . . «•{
o
jillllill^^ 1 °
^4
2.4 2.5 rf
CHAMPION LINE NO. 674 - PROBABILITY X SO
Dose (log)(mg salt/kg body weight)
207
-------
PtCl2 / Intraperitoneal injection
Dose
(mg salt
/kg
body
weight) Expt.
Controls 18,52
53
1111 52,53
741 52,53
578 52,53
450 18,53
250 18,53
Number
survivors
/number Percentage
tested survival
6/6 NA
100.0
±0.0
±0.0
1/4 NA
25.0
±16.6
±8.3
3/9 33.3
33.3
±18.9
±6.3
3/6 50.0
50.0
±38.7
±15.8
9/10 90.0
90.0
±5.3
±1.7
8/10 80.0
80.0
±10.5
±3.3
Duration of
survival (hr)
all
annals
336
±0
±0
(6)
89
±165
±82
(4)
151
±167
±56
(9)
175
±177
±72
(6)
307
±91
±29
(10)
329
±18
±6
(10)
non-
survivors
NA
7
±6
±3
(3)
5b
±118
±48
(6)
13
±21
±12
(3)
NA
NA
Body
weight
(g) on
day 0
104
±24
±10
(6)
108
±21
±11
(4)
112
±12
±4
(9)
107
±8
±3
(6)
117
±12
±4
(10)
117
±16
±5
(10)
Weight gain (g)
days
0-7
66
±18
±7
(6)
NA
6
±23
±12
(4)
**
9%
16
±12
±7
(3)
**
24%
39
±16
±5
(9)
**
597»
53
±11
±3
(10)
ns
80%
days
0-14
124
±17
±7
(6)
NA
53
±52
±30
(3)
m
43%
58
±27
±15
(3)
**
47%
100
±25
±8
(9)
*
81%
107
±21
±7
(8)
ns
86%
days
7-14
57
±4
±2
(6)
NA
46
±24
±14
(3)
ns
817o
43
±15
±9
(3)
ns
75%
61
±12
±4
(9)
ns
56
±12
±4
(8)
ns
208
-------
PtCl, / Intraperitoneal injection
Dose
(mg salt
/kg
body
weight) Expt.
Controls 42,45,
50
108 42,45
64.8 42,45,
50
38.9 45,50
23.3 45,50
14.0 45
Number
survivors
/number Percentage
tested survival
6/6 NA
100.0
±0.0
±0.0
0/6 NA
0.0
±0.0
±0.0
1/10 10.00
10.0
±12.2
±4.1
2/10 20.00
20.0
±0.0
±0.0
10/10 97. 5d
100.0
±0.0
±0.0
5/5 NA
100.0
±0.0
±0.0
Duration of
survival (hr)
all non-
animals survivors
336
±0
±0
(6)
0
±0
±0
(6)
34
±106
±34
(10)
107
±123
±39
(10)
336
±0
±0
(10)
336
.±0
±0
(10)
NA
0
±0
±0
(6)
1
±0
±0
(9)
50
±29
±10
(8)
NA
NA
Body
weight
(g) on
day 0
95
±12
±5
(6)
105
±7
±3
(6)
101
±9
±3
(10)
100
±6
±2
(10)
100
±9
±3
(10)
102
±8
±3
(5)
Weight gain
days
0-7
61
±6
±3
(6)
NA
NA
NA
22
±22
±7
(10)
***
57
±11
±5
(5)
ns
days
0-14
120
±11
±5
(6)
NA
NA
NA
87
±26
±8
(10)
**
73%
96
±6
±3
(5)
**
80%
(8)
days
7-14
59
±7
±3
(6)
NA
NA
NA
62
±14
±4
(10)
ns
38
±11
±5
(5)
**
64%
209
-------
PtCl, / Oral administration
Dose
(mg salt
/kg
body
weight) Expt.
Controls 31,43,
46
660 29
440 29,46
293.3 31,43,
46
195.6 31,43,
130.4 31,43,
46
86.9 31,43
Number
survivors
/number Percentage
tested survival
7/8 NA
87.5
±23.1
±8.2
0/6 NA
0.0
1/9 11.11
11.1
±10.5
±3.5
4/10 40.0
40.0
±22.0
±6.9
8/10 80.0
80.0
±18.3
±5.8
7/10 70.0
70.0
±31.6
±10.0
2/3 NA
66.7
Duration of
survival (hr)
all
ani-.als
310
±72
±26
(8)
0
±0
±0
(6)
38
±112
±37
(9)
140
±170
±53
(10)
270
±138
±44
(10)
236
±162
±51
(10)
240
±166
±96
(3)
non-
survivors
NA
0
±0
±0
(6)
1
±3
±1
(8)
9
±10
±4
(6)
NA
1
±0
±0
(3)
NA
Body
weight
(g) on
day 0
100
±15
±5
(8)
98
±9
±4
(6)
100
±6
±2
(9)
109
±10
±3
(10)
103
±12
±4
(10)
102
±9
±3
(10)
106
±15
±9
(3)
Weight gain (g)
days
0-7
65
±18
±7
(7)
NA
NA
16
±38
±19
(4)
*
25%
NA
57
±13
±5
(7)
NA
days
0-14
108
±35
±13
(7)
NA
NA
81
±34
±17
(4)
ns
75%
NA
115
±22
±8
(7)
NA
days
7-14
44
±29
±11
(7)
NA
NA
65
±14
±7
(A)
ns
148%
NA
58
±22
±8
(7).
NA
210
-------
PtCl, / Oral administration
5R
8!
S
m
3
CO
*J
o
2.0 2.1 2.2
CHAMPION LINE NO. 674 - PROBABILITY X SO
2.3
2.4
2.5
2.6
2.7
Dose (log)(rag salt/kg body weight)
211
-------
Pt02 / Oral administration
Do QP
JL/wOC
(mg salt
/kg
body
weight) Expt.
Controls 9,20,
33,51
8000 9,33,
51,52
4444 20,52
Number
survivors
/number Percentage
tested survival
9/9 NA
100.0
±0.0
±0.0
5/7 71.4
71.4
±48.8
±18.4
5/6 83.3
83.3
±12.9
±5.3
Duration of _ ,
survival (hr) *°dy
weight
all
animals
336
±0
±0
(9)
253
±143
±54
(7)
326
±25
±10
(6)
non- (g) on
survivors day 0
NA 106
±16
±5
(9)
NA 118
±11
±4
O
NA 115
±21
±9
Weight gain (g)
days
0-7
66
±20
±7
(9)
68
±15
±7
(5)
ns
62
±18
±7
(6)
ns
days
0-14
117
±35
±12
(9)
114
±17
±8
(5)
ns
106
±36
±16
(5)
ns
90%
days
7-14
51
±18
±6
(9)
47
±10
±4
(5)
ns
37
±28
±13
(5)
ns
73%
212
-------
Pt(SO,)2'4H20 / Intraperitoneal injection
- Dose
(mg salt
/kg
body
weight) Expt.
Controls 14,22,
24,32,
600 14,22,
24
400 14,22,
24
326.6 32,34
267 14,22,
24
178 14,22,
24
Number
survivors
/number Percentage
tested survival
16/16 NA
100.0
±0.0
±0.0
0/10 NAb
0.0
±0.0
±0.0
1/11 9.1
9.1
±20.2
±6.1
2/12 16.7
16.7
±0.0
±0.0
10/11 90.9
90.9
±8.7
±2.6
11/11 NAb
100.0
±0.0
±0.0
Duration of
survival (hr)
all
animals
336
±0
±0
(16)
28
±16
±5
(10)
67
±92
±28
(11)
117
±106
±31
(12)
308
±93
±28
(11)
336
±0
±0
(11)
non-
survivors
NA
28
±16
±5
(10)
41
±23
±7
(10)
73
±31
±10
(10)
NA
NA
Body
weight
(g) on
day 0
106
±10
±3
(16)
100
±4
±1
(10)
102
±11
±3
(ID
108
±11
±3
(12)
101
±8
±2
(ID
104
±11
±3
(11)
Weight gain (g)
days
0-7
59
±7
±2
(16)
NA
NA
NA
39
±17
±5
(10)
**
66%
36
±14
±4
(11)
***
61%
days
0-14
114
±14
±4
(16)
NA
NA
NA
96
±26
±8
(10)
m
84%
94
±32
±10
(ID
m
82%
days
7-14
55
±13
±3
(16)
NA
NA
NA
56
±11
±3
(10)
ns
58
±21
±6
(11)
ns
213
-------
Pt(S04)2.4H20 / Intraperitoneal injection
b
ss
o . . _ ..
fM
b
in
b
^ ~
o „____,...___. ._._...__, . _,_.._._, _, .-__-.
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Dose (log)(mg salt/kg body weight)
21 '4
-------
Pt(SO,)2'4H20 (Goldsmith) / Intraperitoneal injection
Dose
(mg salt
/kg
body
weight) Expt.
Controls 56,57
400 55
267 55,56
57
119 56,57
52.7 56
Number
survivors
/number Percentage
tested survival
4/4 NA
100.0
±0.0
±0.0
1/3 NA
33.3
0/7 3.6d
0.0
±0.0
±0.0
6/7 85.7
85.7
±17.8
±6.7
4/4 NA
100.0
Duration of ,
survival (hr) ? ^
weight
all
animals
336
±0
±0
114
±167
±96
(3)
76
±13
±5
(7)
307
±78
±29
(7)
336
±0
±0
(4)
non- (g) on
survivors day 0
NA 105
±18
±9
(4)
NA 121
±16
±9
(3)
76 112
±13 ±13
±5 ±5
(7) (7)
NA 114
±7
±3
(6)
NA 120
±18
±9
(4)
Weight gain (g)
days
0-7
52
±12
±6
(4)
NA
NA
8
±31
±13
(6)
*
16%
56
±16
±8
(4)
ns
days
0-14
106
±15
±8
(4)
NA
NA
56
±34
±14
(6)
*
52%
117
±24
±12
(4)
ns
days
7-14
54
±5
±2
(4)
NA
NA
47
±19
±8
(6)
ns
88%
61
±9
±4
(4)
ns
113%
215
-------
Pt(S04)2'4H20 / Oral administration
Dose
(mg salt Number
/kg survivors
body /number Percentage
weight) Expt. tested survival
Controls 14,16, 11/11 NA
23,25 100.0
±0.0
±0.0
2025 14,16, 1/10 10.0
23,25 10.0
±31.6
±10.0
1350 14,16, 1/10 10.0
23,25 10.0
±31.6
±10.0
900 14,16, 5/10 50.0
23,25 50.0
±45.1
±14.3
600 14,16, 9/10 90.0
23,25 90.0
±16.1
±5.1
400 14,16, 7/7 99. 8a
23 100.0
±0.0
±0.0
Duration of
survival (hr)
all
api:.\ctls
336
±0
±0
(11)
46
±104
±33
(10)
43
±103
±33
(10)
172
±173
±55
(10)
307
±92
±29
(10)
336
±0
±0
(7)
non-
survivors
NA
13
±24
±8
(9)
11
±10
±3
(9)
8
±4
±2
(5)
NA
NA
Body
weight
(g) on
day 0
106
±12
±3
(11)
109
±13
±4
(10)
109
±11
±3
(10)
109
±10
±3
(10)
108
±11
±3
(10)
109
±7
±3
(7)
Weight gain (g)
days
0-7
50
±8
±2
(11)
NA
NA
27
±33
±15
(5)
ns
54%
37
±25
±8
(9)
ns
74%
52
±15
±6
(7)
ns
days
0-14
108
±16
±5
(11)
NA
NA
78
±48
±21
(5)
ns
72 %
106
±17
±6
(9)
ns
116
±20
±8
(7)
ns
days
7-14
58
±11
±3
(11)
NA
NA
51
±19
±8
(5)
ns
88%
70
±29
±10
(9)
ns
64
±6
±2
(7)
ns
110%
216
-------
Pt(SOy)0.4H00 / Oral administration
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Dose
(mg salt
/kg
body
weight) Expt.
Controls 4,7,
35,36
1350 35,36
900 35,36
600 7,35,
36
400 4,7,
35,36
200 4
Number
survivors
/number Percentage
tested survival
8/9 NA
88.9
±22.0
±7.3
0/11 4.8a
0.0
±0.0
±0.0
3/11 27.3
27.3
±12.2
±3.7
5/12 41.7
41.7
±28.9
±8.3
12/12 90. la
100.0
±0.0
±0.0
2/2 NA
100.0
Duration of
survival (hr)
all
an imp Is
335
±4
±1
(9)
36
±16
±5
(11)
139
±132
±40
(ID
150
±164
±47
(12)
336
±0
±0
(12)
336
non-
survivors
NA
36
±16
±5
(11)
65
±45
±16
(8)
18
±17
±7
(7)
NA
NA
Body
weight
(g) on
day 0
114
±16
±5
(9)
110
±11
±3
(11)
109
±7
±2
(11)
102
±10
±3
(12)
114
±13
±4
(12)
132
Weight gain (g)
days
0-7
57
±11
±4
(8)
NA
NA
44
±7
±4
(3)
*
777=
45
±12
±5
(6)
m
797=
NA
days
0-14
113
±16
±6
(8)
NA
10
±31
±18
(3)
***
102
±19
±8
(5)
ns
907=
101
±26
±7
(12)
ns
897o
NA
days
7-14
52
±17
±6
(7)
NA
NA
71
±4
±2
(3)
*
1377o
56
±11
±5
(6)
ns
NA
218
-------
RuCl_ / Oral administration
a
a
CO 8
-------
Appendix B4.8
DETERMINATION OF NO-EFFECT LEVELS OF PT-GROUP AND
BASE METAL COMPOUNDS USING MOUSE INFECTIVITY MODEL
by
Life Sciences Research Division
IIT Research Institute
10 West 35th Street
Chicago, Illinois 60616
Prepared for
Environmental Protection Agency
Research Triangle Park, North Carolina 27711
November 1974
220
-------
DETERMINATION OF NO-EFFECT LEVELS OF PT-GROUP AND
BASE METAL COMPOUNDS USING MOUSE INFECTIVITY MODEL
!_. INTRODUCTION
The objective of this phase of the program is to determine
the levels of platinum and palladium compounds that alter the
susceptibility to respiratory infection of experimental animals.
Platinum dioxide (Pt02) and palladium oxide (PdO) were investi-
gated at various concentrations in single and multiple doses using
intratracheal and intragastric instillation. In addition, mice
were exposed to PtO£ aerosols.
II. MATERIAL AND METHODS
Experimental Animals. Male Cr: RGH (SYR) Syrian golden
hamsters, 6 weeks old, were used for the intratracheal studies.
Male ARS-2 (CF1) mice, 5-6 weeks old, were used in the intra-
gastric and aerosol studies. Both species of animals were procured
from ARS/Sprague Dawley. Animals were held in quarantine for
at least 1 week before use in the experiments. Throughout the
experiments food and water were provided ad libitum.
Infectious Agents. Infectious challenge in the res-
piratory studies was with Klebsiella pneumoniae Type 1 (A).
Salmonella enteritidis ser. infantis was used in the intragastric
study. The identity of the microorganisms was verified in our
laboratories.
Preparation and Administration of Compounds. The
compounds, obtained from Platinum Chemicals, Ashbury Park,
New Jersey, were prepared as 1 to 10 y particles by personnel of
IITRI Chemistry Research Division. The compounds were suspended
in sterile phosphate-buffered saline (PBS) containing 0.57» gelatin
(PBSG). The suspensions were treated with an ultrasonic probe
221
-------
to assure the disruption of any aggregates that formed during
preparation. The sterility of the suspensions was checked prior
to use. For the intratracheal and intranasal instillations the
animals were anesthetized with carbon dioxide.
Aerosol Exposures. The chamber used for exposure to
the metallic aerosols is a plastic container 85 liters in volume
(30.5 x 30.5 x 91.4 cm) with aluminum end plates. Two blowers
are located in the chamber, one at the top side of the chamber
where the particulates are introduced and the second at the
opposite end on the bottom side. To assure proper distribution
of the particulates in the chamber the fan speed is regulated
by a variable transformer.
For exposure, the animals are positioned in the center of
the chamber on a platform to assure proper air circulation around
the exposure cage. The cage is constructed of extruded aluminum,
with dividers within the cage to provide individua.1 holding of
the animals during the exposure. The compounds, disseminated in a dry
form by compressed air with an IITRI-designed disseminator, are assayed
utilizing a membrane filter system and gravimetric analysis.
Two 47-mm diameter, 0.45 y membrane filters that have been pre-
weighed are placed in a filter holder back to back. A sample
of the chamber air is taken at a constant flow rate during the
entire animal exposure period. At the end of the sampling
period the flow rate is checked with a wet test meter to assertain
that the flow rate remained constant. The filters are removed and
weighed and the amount of aerosolized material per liter of
chamber air is estimated. If any change in tare weight of the
back-up filter is noted, the weight is applied as a correction
factor in determining the concentration of the compound.
222
-------
To monitor the particle size of the aerosol, 5- to 10-
second air samples are taken at various time intervals with a 25 mm
diameter membrane filter (0.45y) operated at a low flow rate. The
filters are examined microscopically using a calibrated measuring
eyepiece to determine the mean particle size. The filters are
stored, in the event that further analysis of particle size
distribution is necessary.
Infectious Aerosol Challenge. Infectious challenge was
conducted in a 350 liter plastic aerosol chamber (60 x 60 x 95 cm)
installed within a microbiological safety cabinet. A DeVilbiss
atomizer was used to disseminate the infectious agent by means of
filtered compressed air. For the challenge, mice were placed in
the aerosol chamber and exposed for 5 to 10 min to the airborne
infectious agent. After challenge the animals were air washed
for 10 min, removed from the chamber and held for 14 days in an
isolated animal room.
Relative Mean Survival Rate (RMSR). The RMSR was cal-
culated according to the equation
RMSR = S(AB) + (dL)
n
where A is the last day in which any individual animal is alive;
B is the number of animals surviving A days; d is the last day of
observation; L is the number of animals alive on day d; and n is
the original number of animals in the experimental group.
Lung Edema. Lung edema was expressed as the ratio of
weight of lungs immediately upon removal from the animals and after
desiccation to constant weight. The lungs were held in a vacuum
desiccator and reweighed at 24-hr intervals until no additional
weight loss was apparent.
223
-------
III. RESULTS
A. Multiple Dose Study
In these studies, the animals were given six doses of
Pt02 over a 2-week period. At 1, 24, 48, 96, and 192 hr after the
final dose of compound was administered, the animals were challenged
with the infectious agent.
In the intragastric studies the total quantities administered
were 6.0 and 60.0 mg of Pt02- Dose levels were 1.0 or 10.0 mg per
0.5 ml of PBSG per mouse. The infectious challenge was with
Salmonella enteritidis was by the intraperitoneal route. In general,
mice given a total dose of 6 or 60 mg of PtC>2 in PPSG and challenged
with Salmonella showed somewhat lower mortality rates and increased
survival times when compared to mice infected with Salmonella and
given PBSG (Table 1) .
A somewhat different pattern was apparent when infected mice
given Pt02 in PBSG or PBSG alone were compared to those challenged
with Salmonella only. When given 6 mg of PtO£ or six intragastric
intubations of PBSG, no specific trend of mortality rates was
noted. For example, mice infected 1 hr after the last PtC>2 or
PBSG instillation showed lower mortality rates than those given
Salmonella only, at the 24-hr interval, mortalities were slightly
increased, and at the 48-hr interval mortality rates were the
same in mice given PtC^ or Salmonella but increased in those
given PBSG.
When compared to Salmonella controls, increased mortalities
were seen in mice given either 60 mg of PtC>2 or six intragastric
intubations of PBSG and challenged with Salmonella 1, 96, and 192
hr after the last treatment. When challenged with Salmonella at
24 and 48 hr, mice given PBSG showed increased mortalities but
224
-------
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No mice died as a result of intragastric instillation of PBSG
or PtC>2 only.
In intratracheal studies the total amounts of Pt02
administered to hamsters were 0.6 and 6.0 mg. The dose level
of each intratracheal instillation was 0.1 or 1.0 mg per 0.5 ml
of PBSG per hamster. The infectious challenge with K. pneumoniae
was by intranasal instillation. In general, hamsters given
either 0,6 or 6.0 mg of PtC>2 and challenged with K. pneumoniae
had mortality rates similar to those given PBSG and K. pneumoniae
(Table 2). No significant differences were found in mortality
rates, mean survival time, and lung edema throughcat the study.
However, hamsters given Pt02 or PBSG and challenged at the
various time intervals with K. pneumoniae showed lower mortality
rates, longer mean survival times, and a lower lung edema than
those challenged with the infectious agent only. No hamsters
died as a result of intratracheal instillations of PBSG or Pt02
only. A possible explanation for these decreased mortalities
is that the multiple instillations of PtC^ and PBSG stimulates
the defense mechanism, thus making the hamster more resistant
to K. pneumoniae infection.
B. Retention Study
This study was conducted in an attempt to determine the
amount of compound retained in the lungs of experimental animals.
A single dose of 0.1 mg or 1.0 mg PtC>2 was introduced by intra-
tracheal instillation into each of 20 hamsters. Two hamsters
were sacrificed at 1, 6, 24, 48, and 72 hr and the lungs removed
and partially prepared for atomic absorption analysis. The
ashed samples were submitted to Dr. D. Gardner, EPA, for analysis.
226
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In addition, the above scheme was followed in preparing
specimens for histopathological examination. Controls for this
group included two normal hamsters and two hamsters from each
time period given PBSG only. At the various time periods the
hamsters were anesthetized witli carbon dioxide, the lungs
removed, fixed in a 10% phosphate buffered solution, sectioned,
and stained. The microscopic examination of the slides has not
been completed.
C. Aerosol Studies
Two experiments have been initiated in which mice were
exposed for 1 or 3 hr to Pt02 aerosol followed 1 and 24 hr later
by an aerosol challenge with K. pneumon-ae. The oncentrations
of Pt02 aerosol were 31 mg and 23 mg/m^ for the 1 and 3 hr
exposure. These concentrations are equivalent to 37.2 yg for
the 1 hr and 82.8 yg for the 3 hr exposures. The mortality
rates, mean survival rates, and lung edema measurements are
being made.
IV. FUTURE STUDIES
Studies will continue in which mice will be exposed to
aerosols of both platinum and palladium. The exposures will be
multiple as well as single.
228
-------
Appendix B4.8
DETERMINATION OF NO-EFFECT LEVELS OF PT-GROUP
AND BASE METAL COMPOUNDS USING MOUSE INFECTIVITY MODEL
by
Life Sciences Research Division
IIT Research Institute
10 Nest 35th Street
Chicago, Illinois 60616
Prepared for
Environmental Protection Agency
Research Triangle Park, North Carolina 27711
August 1974
229
-------
DETERMINATION OF NO-EFFECT LEVELS OF PT-GROUP AND
BASE METAL COMPOUNDS USING MOUSE INFECTIVITY MODEL
INTRODUCTION
The objective of this pnase of the program is to determine
the levels of platinum and palladium compounds that will alter sus-
ceptibility to respiratory infection in mice. Platinum dioxide
(Pt02) and palladium oxide (PdO) were investigated at various
concentrations using intratracheal and intragastric instillation.
Platinum metal has been obtained and will be included in the
future studies.
II. MATERIALS AND METHODS
Experimental Animals
Male Cr:RGH(SYR) Syrian golden hamsters, 6 weeks old,
were used in the intratracheal studies. Male ARS-2(CFI) mice,
5-6 weeks old, were used in the intragastric studies. Both
species were supplied by ARS/Sprague Dawley. Throughout the
experiments food and water were provided ad libitum.
Infectious Agents
Influenza virus A/PR/8 strain was prepared by rapid
passage through hamsters. Within 24 hr after intranasal
inoculation, lungs were removed aseptically and a 20% lung sus-
pension prepared in phosphate-buffered saline. After three
rapid passages, the influenza virus was inoculated into a
sufficiently large number of hamsters to provide a stock virus
adequate to complete this phase of the program. The influenza
virus, was identified by the use of reference reagent antiserum
obtained from NIAID, Bethesda, Maryland.
230
-------
Bacterial challenge in the intragastric studies was with
Salmonella enteritidis ser. infantis 6, 7:r-l,5, somatic group
C, IITRI No. 120.
Preparation and Administration of Compounds
The compounds Pt02 and PdO were obtained from Platinum
Chemicals, Ashbury Park, New Jersey and prepared to a 1- to 10-
micron size range by the IITRI Chemistry Division. The compounds
were suspended in 0.5 ml sterile phosphate-buffered saline (PBS)
containing 0.5% gelatin (PBSG) resulting in dose levels containing
10, 20, 40, 50, or 100 mg. The suspensions were checked for
sterility prior to use. The animals were anesthetized with carbon
dioxide and the particulate suspensions introduced by intra-
tracheal or intragastric instillation.
Pulmonary Consolidation. Lung consolidation of all
sacrificed animals were determined and recorded using the
following scale:
0 = no consolidation
1 = 1-25% consolidation
2 = 26-50%
3 = 51-75%
4 = 76%-100%
A value of 5 was assigned to mice dying during the experiment.
Relative Mean Survival Rate (RMSR). The RMSR was cal-
culated according to the equation
RMSR= S(AB)+(dL)
n
where A is the last day on which any individual animal is alive;
B is the number of animals surviving A days; d is the last day of
observation; L is the number of animals alive on day d; and n is
the original number of animals in the experimental group.
231
-------
III. RESULTS
At 1, 24, and 48 hr after intranasal viral challenge,
hamsters were given the various concentrations of test compounds
intratracheally. All dose levels were not tested at all time
periods because of a shortage of material. Platinum dioxide,
tested 1 and 24 hr after virus challenge, did not influence the
mortality rates, lung lesion consolidation or the relative mean
survival rate (Table 1). It is interesting to note in the 24-hr
interval study that PBS or PBSG given individually did not cause
mortalities, whereas a combined insult with the two (PBS-Pt02 in
PBSG, 0 mg) killed 22% of the hamsters. When PBSG was given 24
hr after virus challenge, 90% of the hamsters died, a number
similar to that observed in hamsters given PtC>2 '-4 hr after
virus challenge. This increased mortality due to PBSG given
intratracheally 24 hr after virus challenge has been noted pre-
viously in studies with nickel oxide (IITRI Report L6061-8,
October 1972).
When the interval between virus challenge and Pt(>2
instillation was extended to 48 hr, there appeared to be a
general increase in mortality (79% to 100%) and lung consolidation
(4.21 to 5.00) when compared with those hamsters given virus and
PBSG (78% and 3.70). In addition, the relative mean survival
rate was somewhat reduced for the experimental hamsters.
Histopathological examination of lungs of hamsters
exposed to 10 and 40 mg Pt02 was performed at 14 days post-
exposure to Pt02. In general, the PtC>2 was distributed in the
immediate vicinity of the bronchioles. The particles were
phagocytized by macrophages present in respiratory bronchioles
and alveoli. In some lobes, the PtC>2 particles were not
232
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phagocytized and appeared as large aggregations occluding
bronchioles. The number of particles present: in the lungs was
related to the initial dose.
In those animals receiving 40 mg of the metal particulate,
a moderate amount of bronchoalveolar proliferation was present.
*
This change was found only in association with retained particles,
and was not present in those animals receiving 10 mg Pt02.
In studies with PdO, results ^similar to those obtained
with PtC>2 were obtained (Table 2). When the interval between
virus challenge and PdO instillation was 24 hr, mortality ranged
from 89 to 100%. The mortality in hamsters challenged with the
virus and PBSG was 90%, however. When the interval was extended
to 48 hr, the mortality in the experimental hamsters ranged from
73 to 100%, whereas the mortality rate in control hamsters given
virus and PBSG was 60%. Lung consolidation was also greater in
the experimental hamsters and the survival time was markedly
shorter than in the controls. When the data were analyzed by
the Student t-test, only those hamsters given 10 mg of PdO had
significantly greater mortality and lung consolidation, with
significantly shorter survival time. In all groups but one
(100 mg), the relative mean survival rate was significantly
shorter than in hamsters given virus and PBSG.
The intragastric studies were conducted in a manner
similar to the intratracheal study. The same experimental plan
was used with the exception that the particulate suspensions were
introduced by intragastric instillation into mice which were then
challenged intraperitoneally with Salmonella. Body temperatures
were taken with a Tele-Thermometer (Yellow Springs Instrument Co.)
2 to 3 hr after the infectious challenge. The data, summarized in
Tables 3 and 4, indicate that Pt02 and PdO did not increase sus-
ceptibility to Salmonella infection as measured by mortality,
mean survival times, and body temperature.
234
-------
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IV. FUTURE STUDIES
Studies will be initiated using aerosol exposure of
animals to the particulates. In addition, a study is in progress
using multiple intratracheal instillations of the compounds
at lower concentrations.
238
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Appendix B4.9
Status Report
ROAP 26AAE, Task 007
Description of Progress under Contract 68-02-1204,
"Exposure of Tissue Culture Systems to Air Pollutants
under Conditions Simulating Physiologic Status of
Lung and Conjunctiva."
The objective of this contract is the development of a system for
exposure of living cells and tissues to airborne environmental pollu-
tants under conditions similar to those encountered in the intact animal.
The tissues being considered include the epithelium, or interior sur-
face layer, of the respiratory passages and lung, and of the eye.
The first year of trials in developing this system has led to the
selection of a stationary cell culture growing in a filter surface
with the cell culture being nourished from below the filter for expo-
sure to atmospheric pollutants from above. The cell culture is contained
in an exposure chamber within an incubator. By means of a pump, it is
possible to simulate the continual renewal of the overlying fluid that
normally "protects" tissue in the intact animal representing tears,
in the case of the eye, or mucous, in the case of the respiratory
passages. The exposure system is now ready to be tested by use of
oxidant gases, Oo and N0~, and subsequently will be modified for use
with suspended particulates. Biological reactions will be measured
in such a way as to identify immediate and delayed toxicity, precancerous
and cytogenetic changes.
The critical value of this system is that it facilitates more direct
extrapolation of data from purely in vitro studies to the intact animal
and man. An important complexity has been added to the conventional
239
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tissue culture, that of the "protective" nutrient and/or washing fluids
that normally overlay the surface cells of the respiratory passages,
lung, and tissue surrounding the eye. It is undoubtable that these
fluid layers are very important in modifying the response of cells and
tissues exposed to environmental pollutants. Hence, the test system
developed is expected to be of significant value in understanding the
chemical and biological reactions that occur at the tissue surface.
240
-------
Appendix B4.9
Environmental Protection Agency
Contract Number 68-02-1204
"Exposure of Tissue Culture Systems to Air Pollutants under
Conditions Simulating Physiologic States of Lung $ Conjunctiva"
Quarterly Progress Report
Fifth Quarter
Contractor:
Investigators:
Regents of the University of California
University of California, Irvine
T.| Timothy Crocker, M.D.
G. Scott Samuelsen, Ph.D.
••..September 30, 1974
Prepared for the Envirowicnta] Protection Agency,
Research Triangle Park, North Carolina 277]1
241
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II. PROGRESS OF WORK
A. Cell Culture System . . I
During the past three months, efforts have been directed towards
solving the problems that were listed in the fourth quarterly report.
*
These problems are:
(1) Keeping the millipore filters flat without sagging. Sagging
caused unequal distribution of medium and irregularity in the growth
of cells. . 'I
(2) Removal of air bubbles trapped under the filter. These air
bubbles prevented the diffusion of culture medium through the filter
to nourish the cells growing on the filter.
(3) Production of clones. Cloning efficiency has been very poor
on millipore filters. For this reason filter cultures were limited
to experiments on monolayers.
V- .
1. Solution of Problems 1 and 2
These problems were typical of work on membranes. Their solution
was probably valuable as part of learning how to deal with membrane
cultures.
a. Cross shaped filter support
In the millipore filter culture unit described earlier (see
fourth quarterly report) the filter rested on a flat porous disc.
Small air bubbles were trapped in the pores as well as in between
the filter and the disc. Mien the circular disc was replaced with
•
a cross-shaped support (made of autoclavable lexan) small air bubbles
did not form beneath the filter; however, the sagging was not satis-
factorily corrected. This observation made it necessary to use a
242
-------
circular porous disc and to explore new method^ to remove air
i
i
bubbles. . .
b. Effects of autoclaving . . . 1
.1
The filter was mounted in the filter holder and autoclaved.
This avoided sterilizing the filter holder and the filter separate-
ly before assembling. However, for unknown reasons (possibly currents
of steam under high pressure) the filter surface became uneven after
sterilization in the autoclave.
/
c. Phase II of Mounted Filter Culture Unit
Because of the adverse effects produced during the autoclaving
of the completely assembled culture unit, the filter and the filter
holder were treated separately. The filter holder (described in
detail in the fourth quarterly report) was wrapped in aluminum foil
and autoclaved separately. Filters were boiled in Hank's Balanced
Salt Solution for ten minutes to remove detergents impregnated in
filters, washed well in two changes of sterile distilled water and
mounted in the autoclaved filter holders aseptically. To insure
maximum sterility the assembled culture unit was exposed to steriliz-
&
ing UV for twenty minutes on each side. This was done in sterile
glass culture dishes that were specially made from crystallizing
dishes to contain the culture unit (the standard plastic or glass
petri culture dishes are not large enough for this culture .unit ).
•
After sterilization, the dish was covered with a lid made of sterile
aluminum foil because of the non-availability of suitable lids to
'cover the dishes.
243
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d. Plating cells and removal of air bubbles in vacuo
Sufficient culture medium was delivered in the culture dish
i
' so that it wet the underside of the filter and diffused through
the filter from below. An inoculum of 2 ml containing a known
number of cells was delivered onto the upper surface of the filter
making use of the well formed by the filter and the upper element of
the filter holder, (Fig. 1). The filter remained flat because of the
• supporting disc, but air bubbles appeared inside the pores of the
disc and between the underside of the filter and the disc. These
air bubbles interfered with nutrient access to the filter and the
cells on the upper surface of the filter. Bubbles were removed in
a vacuum dessicator.
The culture dish containing the nutrient medium together with
the mounted filter culture unit was placed in a vacuum dessicator
which had been washed well and swabbed with 70% ethanol. The vacuum
outlet of the dessicator was connected by tygon tubing to a vacuum
aspirator which sucked the air bubbles up through the filter and
laterally into the medium in the culture dish around the edges of the
filter holder. A safety valve was introduced in the vacuum line for
the careful return'of air to normal atmospheric pressure without
disturbing the mounted 'filter culture unit, and to prevent the influx
of water droplets. By this method the air bubbles were removed while
the filter remained flat.
Cells (10*/2 ml) were planted on the filter by adding 2 ml of
cell suspension. Cultures were then transferred to the incubator for
growth preparatory to future reduction in overlying medium during
244
-------
HOLE. A
o
-in-
OLLTEP, BODY
• VAj &toT B
A
PARTS /);B,*C ARE
ASSEMBLED/ "WITH
'. A t,B MUD i urn WiLi_f
,H GL/^TS MARI'
.;-
HOLES
2/J5
-------
exposure to gases.
e. Growth of cells on milliporc filters
r*
Culture units were prepared as described above and 10 cells
were plated in standard culture medium (Dulbecco's modified Eagle
medium + 101 fetal calf serum.") Those cultures are identified as"
"mounted filters",-Table 1. Controls consisted of cultures on
millipore filters that were tacked to the botto'n of standard
plastic tissue culture dishes. These cultures are identified as
"immersed filters", Table 1. Growth was measured by fixing, staining
and counting of cells after different tir.~ intervals (Table 1). The
procedures for all these processes were described earlier.
Type of Culture
- mounted filters
immersed filters
dish control
*
mean of six counts
TABLE 1
v number of
cells plated
105
.- io5
105
of sample grid areas
,,. •
in situ cell counts
on the fifth day
*
. 283
523
>700
Results . .
Millipore filter cultures are less cellular than cultures on plastic.
Growth on the mounted filters is less than on filters immersed "in the
medium.
246
-------
Variation was noticed within an experiment and among different ex-
,perimcnts. One apparent reason for this variation is the variations
in the volume of medium overlying the cells on the mounted filters.
The mounted filter cultures had been incubated with the same volume
(2 ml) used in planting, cells. Reduction in this volume with time
indicated that there was evaporation from the cell side of the filter
without fluid replacement from below. On the assumption that better
humidification would produce improvement, cultures were placed inside an
air-tight dessicator containing sterile distilled water in the bottom
compartment which made the inside of the dessicator saturated with
humidity. The interior of the dessicator was also saturated with 5%
CO- and 95% air. Although this, did show slight improvement, cells did
not grow at a rate comparable to those in immersed cultures. It became
obvious that diffusion of nutrient across the millipore filter was not
sufficient to maintain maximum cell growth either by replacing nutrient
in the thin layer over cells or by downward diffusion of metabolic
products from the (cell side to the nutrient side of the filter.
2. Phase III. Membrane Culture system
i
a. Positive pressure;
Perfusion cultures are used in several laboratories to improve
I
cell growth and cell yield. In this system, fresh nutrient is con-
i
tinuously made available to the cells and this renewal might also
eliminate growth inhibiting metabolites. These systems are not open
i
to the atmosphere and could not be used unmodified. Millipore filter
247
-------
assemblies such as the Aseptic Sterifilter are operated using a.
vacuum for the purpose of filtration. We therefore examined the
effect of forcing fluids across the filter by means of positive
pressure on the nutrient side of the filter. By adjusting the height
of a column of fluid sufficient pressure can be maintained to produce
'weeping1 of the nutrient medium across the filter.
The commercial Aseptic Sterifilter has been used for preliminary
trials. But this- has some disadvantages for routine use on a large
scale: (a), large volume (250 ml) of medium is necessary to support
a 47mm culture substratum, (b) the seal at the junction between the
filter and the filter holder is not leak-proof and therefore the
medium wells up along the periphery; (c) the medium cannot be drained
after it has crossed the filter; (d) because of its large size, only
one dish equivalent can be used inside a Rochester chamber. For
these reasons other alternatives were sought.
b. Modified Rose Chamber
Rose chambers (Fig. 2) are used for enclosed cell cultures and
can have periodic medium changes. In a standard Rose chamber the
ti
medium is contained in a chamber bounded by 2 large coverslips on
v
either side of a space in a thick rubber gasket. The cells grow on
one glass coverslip. To complete the chamber assembly, metal plates
outside both coverslips are connected by screws that compress the
coverslips against the gasket. Four units of a pressure chamber
have been made and tested as a modification of the Rose chamber
(Figs 3,4). In both cases the culture medium is contained by a space
in a silicone rubber gasket and is driven by hydrostatic pressure from
248
-------
73 mm
75mm
. 1. Drawing of a Rose inuHipuvposD cliambcr with its component parts.
:. i
-.1
c <
V)^''
-------
2.2.
• O
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• a "fluid reservoir. Medium pusses by forced diffusion through
the milliporc filter into the well formed by the filter and top ' •
piece. The latter well serves as a planting dishi for starting
i
cultures on the filter in a horizontal position. ',
After cell growth has been started, the pressure chambers will
be placed vertically on a support frame. Medium will flow slowly
through the filter at a rate set by the hydrostatic pressure.
Excess medium will flow into a container common for a group of Rose
chambers. At least five Rose chambers can be placed on each of two
shelves in a Rochester chamber to provide 10 culture samples for
exposure to test gases. This unit is being i,e_>ted f^r support of
cell growth. The basic concept is promising and modifications are
possible. If the vertical position of the filter is not satisfactory,
for example, the filter could be held horizontal while the nutrient
pressure is raised, then lowered, to flush the cell surface gently.
3. Cloning
It was noticed earlier that cloning efficiency was poor on millipore
filters. This has been attributed to the special surface properties of
the filter. However, further studies have yielded promising results.
Since a feeder layer always enhanced the cloning efficiency, a feeder
was pi-epared by plating irradiated cells on millipore filters fixed to
the bottom of plastic culture dishes. The procedure for preparing irra-
diated feeder cells .was described in detail earlier. Fifty thousand
irradiated hamster embryo cells were used as feeder on a 47mm filter.
Twenty-four hours after plating the feeder cells, cells were plated in
different numbers for producing clones. The culture medium consisted of
252
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2 parts of standard medium and 1 part of conditioned medium. After
10 days the cultures were fixed, stained and examined. Results
(Table 2) showed that plating efficiency for clones can be increased
to workable levels on a millipore filter bearing a feeder layer.
TABLE 2
Type of Substratum
Millipore filter
it ii
i. i.
Tissue culture dish
ii ii it
• «i -ii ii
Number cells plated
200
s 500
2000
200
500
2000
. Number clones
3.3
11.3
32.6
24.6
46.3
.Overlapping clones,
unable to count accurately
It has also been found that soaking the millipore filter in standard
culture medium, in" lieu of using a feeder layer, slightly improves
'cloning efficiency. Addition methods will be used to provide good
clonal growth without feeder cells.
Summary
The objective of providing a cell sheet that can be exposed to
airborne components through a minimal layer of fluid is slowly being
I
reached. Each new trial.seems potentially promising and the present
I
development appears especially likely to meet most criteria that, were
i
set. One always wishes {he most recent approach had been made long ago.
253 •
-------
Each stage in the past 6 months has produced information needed for
work with cell growth on a filter membrane, hence progress lias been
appropriate. . .
B. Exposure System • '
1. Description of individual tasks
a. installed three more Blue M incubators. Fabricated necessary
tubing to connect incubators to primary and secondary flow control boards
b. Installed, checked out, and certified a laminar flow hood.
c. Set up a laminar flow incubator. Fabricated necessary tubing
. to provide filtered air, water and CCL to incubator.
d. Assembled and checked out a Brady Array humidity sensing system.
e. Set up a thermal converter in the vent line from 'the exposure
chambers for the purpose of converting NO to NO prior to water extraction.
Li
2. Testing : .
'The following tests have been completed:
a. Humidity check on the water-saturated air delivery system. By
»
using the Brady humidity sensor, it has been determined that relative
humidity inside the Rochester environmental chamber can be maintained in
the range of 92-951.
b. Sterility tests
1) The humidification system and Rochester chamber with all
associated tubing were sterilized and assembled inside an incubator.
Plates of bacterial culture medium were exposed for 96 hours in the
environmental chamber. No indication of airborne mold or bacterial
• 254
-------
contamination in the test area was present at the conclusion of the
. test period.
2) After obtaining the necessary certification on the laminar
. flow hood, two 24-hour sterility checks revealed no: contamination
due to airborne mold or bacteria.
c. Gas mixing test
Preliminary testing of the use of flow restrictors to control the
concentration of NCL has been completed. " - •
3.' Contemplated work •
The following tasks will be started and/or completed during the next
reporting period.
a. Data acquisition - multipoint recorder on order. All read-outs
will be connected and checked out.
b. NO- delivery system - upon receipt of heated vent hose, tests using
*j ,
both dry and humidified air-NO_ can be run to determine loss and uptake
in the system.
• • y
c. The Bendix NO, N0», NO analyzer on loan from the Environmental
it X
Protection Agency will be checked out and put en stream.
d. Total system check-out - when above items are successfully completed,
a total'check-out of the gas and humidity delivery system, the exposure
system and the data'acquisition system will be carried out. •• -
255
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Appendix B4.10
A Comparative Study of the Effect of Inhalation of Platinum, Lead and Other
Base Metal Compounds Utilizing the Pulmonary Macrophage as an Indicator of Toxicity
Principal Investigator: E. Bingham Mattheis, Ph.D.
Progress Report
The purpose of this study is to evaluate the cellular response of
the lung, especially the pulmonary alveolar macrophages, in animals subjected
to the inhalation of various platinum and other base metal compounds.
Aerosols of a platinum chloride solution were produced by using a
Vaponefrin nebulizer. The fumes were passed through a tube ft'" aace, heated
to 800°C., where decomposition took place. The resulting particles were iden-
tified by x-ray diffractometry as platinum metal (platinum black), with a mass
median diameter of 0.04 urn (See Particulate Figure).
Rats were exposed to the inhalation of platinum fumes for six hours
3
per day at an average concentration of 150 ug Pt/m . The chamber was monitored
daily by collecting the sample on a Millipore filter and measured by atomic
absorption spectrometry. Animals were removed from the chamber and the total
number of alveolar macrophages determined. The following table illustrates
the number of macrophages, the percentage of cells found viable and the metal
found in the lungs.
Total cells Metal found in
Exposure Concentration g.lung x 10 7, Viable lung ug/g
Control Rats (8) —- — 2.91 97.0 <1.6
3
Platinum Rats (6) 16-24 days 150ug Pt/m 3.82 95.8 3.6
256
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Appendix B4.ll
A. CONTRACT NO. 68-02-1299
TITLE: Compare Pulmonary Carcinogenesis of Platinum Group
Metal Compounds and Lead Compounds in Association
with Polynuclear Aromatics Using in vivo Hamster
System.
Quarterly Technical Progress Narrative No. 1
Period Covered; June 14, 1974 to Sept. 30, 1974
Prepared by; Marvin Kuschner, M.D.
Department of Pathology
State University of New York
at Stony Brook
Stony Brook, N.Y. 11790
Date of Publication; September 30, 1974
Prepared for; Environmental Protection Agency
Research Triangle Park, N.C. 27711
B. Submitted to;
Dr. David L. Coffin Vincent E. Mason
Project Officer Contracting Officer
Chief, Pathobiology Research Branch
Experimental Biology Laboratory
258
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Section 1
Introduction; The purpose of this contract is to evaluate the potential role
of platinum and lead compounds as cofactors in respiratory carcinogenesis. The
data derived from this investigation will be useful to EPA in evaluating the
human health hazard associated with platinum compounds emitted into the environ-
ment in automotive exhaust. Such emissions have been suggested to be a po-
tential consequence of catalyst attrition in automobiles equipped with platinum
based catalytic exhaust converters. Results will aid in assessing the poten-
tial hazards of airborne particulate lead derived from combustion of gasoline
containing lead compounds as anti-knock additives. These metals are to be
evaluated for their potential cofactor role in the induction of neoplasia, par-
ticularly of the respiratory tract, in conjunction with the ubiquitous environ-
mental contaminant, and known carcinogen, benzo (a) pyrene. The contract ef-
fort is designed to permit an assessment of the relative hazard of platinum
compounds in comparison to lead compounds as environmental contaminants de-
rived from automotive exhaust.
The scope of work for this contract effort entails repetitive administration
by intratracheal instillation of the metal compounds under study to Syrian
Golden Hamsters. Each metal compound is to be administered alone and in com-
bination with benzo (a) pyrene as a saline suspension. Instillations of benzo (a)
pyrene alone, and of the combination of benzo (a) pyrene with iron oxide (Fe O )
*• O
are to be administered. The latter combination has been extensively characterized
in the past, arid is expected to be a potent inducer of pulmonary neoplasia when
administered via the endotracheal route. Test compounds are to be administered
to animals once weekly for at least 16 weeks, after which animals will be main-
tained for their natural lifetime. Gross and histopathologic evaluation of lesions
produced will provide the endpoint of the study.
Short term studies will evaluate the suitability of particle preparations for
intratracheal instillation, and establish the animals' ability to tolerate the
selected dose levels during long term administrations.
259
-------
Section II
Progress made during the first quarter of this newly initiated contract effort
centered around procurement of necessary test chemicals and materials, securing
experienced and competent technical assistance to participate in the investi-
gation outlined, and initiation of preliminary range finding and tolerance
studies involving lead. Written communication of June 25, 1974, from Dr. David
Coffin, Project Officer, specified the specific lead and platinum compounds
to be employed in this contract. These had not been previously defined by EPA.
.These are platinum dioxide , (PtO ) and the monoxide of lead (PbO). On the basis
of this we have now obtained specially prepared, finely particulate platinum
oxide from Platinum Chemicals, Inc., Asbury Park, N.J. Other necessary com-
pounds, except for benzo (a) pyrene (see below) and equipment necessary to conduct
the outlined research are available to us.
The communication of June 25, from Dr. Coffin also outlined the provisions ini-
tiated by EPA contract for preparation of defined particulate materials, in-
cluding polynuclear organic carcinogens, to be employed by in-house EPA investi-
gators as well as by various contract investigators in order to promote com-
parability of results. On the basis of this,' we have supplied to the Fine
Particles Division of IIT Research Institute in Chicago the necessary metal com-
pounds for use in our project. We understand that the IIT laboratory is as of
this date conducting the necessary analysis and procedures for size separating
the metal compounds for use in our animal test system. We anticipate receipt
of sized preparations of the lead and platinum within the next two weeks. The
IIT laboratory has already furnished us with sized, finely particulate iron
oxide.
Preliminary studies have been initiated and are now underway to evaluate animal
tolerance of two dose levels of particulate lead oxide (PbO) administered intra-
tracheally. The preparations for instillation of lead (PbO - Fisher Scientific
99.7% purity) have been made by hand grinding lead in a mortar with a pestle
for 30 minutes. Transmission electron microscopic examination of particle
preparations before and after grinding indicates size reduction in our ground
preparations. However, heterogeneity of particle size and retention of numerous
larger particles render these preparations less -stable in suspension than would
be anticipated with the preparations to be supplied by IIT Research Institute,
260
-------
in which actual size separation will have been carried out. The lead is sus-
pended in sterile, normal saline to which 0.5% gelatin is added. Male Syrian
Golden Hamsters (75-90 grams), obtained from Lakeview Hamster Colony, are
presently being exposed to lead oxide at levels of 5mg per intratracheal dose.
The suspension contains 25mg lead oxide per l.Occ., and the volume administered
is 0.2cc. One group of 20 hamsters is receiving this dose once per week, a
second group of 20 is receiving the 5mg dose twice per week. A control group
of 10 animals receives the gelatin-saline vehicle alone twice per week. The
intratracheal administrations are carried out in animals anesthetized with an
intraperitoneal injection of approximately 6mg per 100 grams body weight of So-
dium Methohexital (Brevane-Elanco Products Div., Eli Lilly). Animals are being
weighed and observed for evidence of adverse effects. Lead alone is being sub-
ject to initial range finding study as it is' anticipated that this would be
the compound most likely to present a problem of toxicity with impaired animal
tolerance during long term repeated administrations.
Section III
Current problems center around availability of test compounds. Aldrich Chemical
Company has not yet been able to supply the required single large batch of
Benzo (a) pyrene ordered in July. 'However, recent communication with that vendor
indicates that we should anticipate delivery of our order soon. We are cognizant
of the fact that other laboratories have also experienced unusual delays in ob-
taining this chemical from Aldrich in the past several months. Apparently Al-
drich is in the'process of replenishing their exhausted supplies. We have not
considered alternative arrangements at this time because of our understanding
that this obstacle will be overcome in the very near future. Importantly, we
seek to secure all of the Benzo (a) pyrene to be used in the entire contracted
project in one single batch from the same lot of chemical in order to obviate
any loss of comparability of results due to changes of properties of the supplied
organic carcinogen. In addition, Aldrich has been the chief supplier of B.P.
for related research and we seek to maintain uniformity of source. Although the
IIT laboratory is currently preparing test materials for our use, these will
be employed in short term, range finding and acute toxicity studies only. We
will await the availability of carcinogen and metal-carcinogen preparations em-
ploying the specified single batch of B.P. before initiating long term, large
scale protocols.
261
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Section IV
The work to be performed during the next reporting period includes the con-
tinuation of the ongoing intratracheal administration of lead to evaluate
animal tolerance of repeated exposure by this route to the selected dose
levels. The anticipated forthcoming availability of the definitive, refined
particulate metal and metal - Benzo (a) pvrene preparations will permit
initiation of the full exposure protocol, employing eight separate test groups
which will be:
1. B.P. alone
2. B.P. + Platinum Oxide
3. B.P. + Lead Oxide
4. B.P. + Iron Oxide
5. Platinum Oxide alone
6. Lead Oxide alone
7. Iron Oxide alone
8. Gelatin-Saline control
It is planned that the first group subject to long term exposure to each of these
eight compounds or combinations will contain 60 animals. Thus, by the completion
of the next reporting period it is expected that long-term, repetitive instilla-
tions, with appropriate controls will have been initiated on 480 animals.
262
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Appendix B4.12
STATUS OF INTERAGENCY AGREEMENT WITH LAWRENCE LIVERMORE LABORATORY/ATOMIC
ENERGY COMMISSION, LAWRENCE, CALIFORNIA TO CONDUCT STUDIES ON THE METHYLATION
CHEMISTRY OF NOBLE METALS. TASK 22, 21BCE.
Background: There is little or no information available regarding
the toxicology of the noble metals. However, the etiology, epidemiology,
and pathology of environmental diseases associated with trace metals
notes the significance of methylation as an important factor associated
with both direct and indirect exposure routes. Methylation can occur
under mild reaction conditions (pH, temperature) and is common to microbial
organisms, plants and animals. Health hazards arise from both free
metals that are methylated in vivo and metals that are methylated in the
environment (in vitro). The physiological stability of the methyl metal
bond can determine the nature and severity of the pathological response,
e.g. methylrnercury is essentially stable in vivo and is considered
primarily a "neurotoxin" while inorganic and aromatic (phenyl mercurials)
are largely renal poisons. In addition to mercury, arsenic, selenium
and tellurium, recent evidence indicates that thallium, platinum and
gold can be methylated by the common methyl-cobalamin reaction. Interference
in Vitamin B^ (methyl-cobalamin) metabolism imparts disturbances in the
oxidation of odd-carbon fatty acids, the biosynthesis of essential amino
acids and is associated with clinical pernicious anemia. Platinum-group
metals have also been reported to be mildly carcinogenic (leukemia,
lymphoma) in laboratory animals under chronic /subchronic exposure
conditions. These considerations impart a necessity to determine and
compare the capacity of noble metals to be methylated. Evaluation of
stable methylated products of metallic automotive emissions for their
relative toxicity is of primary importance to environmental health
considerations.
Purpose: The purpose of this agreement is to investigate and
compare the methylation chemistry of metals associated with emissions
arising from the use of automotive fuels, fuel additives and emission
control devices.
The primary objective of this project is to determine and compare
the methylation chemistry of platinum, palladium, manganese and lead
using methyl-cobalamin (Me-Vit. B,2)- Specific materials of interest
and relevant to the Fuel and Fuel additive Program are the oxides of
platinum, palladium, manganese and lead chlcrobromi-ie (PbCl, Br)2. The
secondary objective is to determine the reaction of methylated metal
products with important precursors to nucleic acid synthesis (nucleosides)
with subsequent/concomitant comparative analysis of the toxic effects of
these compounds.
263
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Significance to 21BCE; Elimination of known hazardous agents from
exhausts must be evaluated relative to potentially new health adversities
that may arise from exposure to modified and/or new emission products
associated with the use of such control devices. Therefore, it is prudent
and imperative to evaluate the environmental impact and potential toxicology
that may arise from exposure to noble metals prior to the widespread use
and distribution of automotive catalytic converters. Methylated metallic
compounds may pose a threat to the immediate and subsequent populations
by direct (pulmonary skin) and indirect exposure (food chain continuation).
Reports: Quarterly: Within fifteen (15) calendar days after the
end of each three (3) months of work under the agreement, the Lawrence
Livermore Laboratory shall provide the EPA project officer fifteen (15)
copies of a technical progress narrative report.
Status: Agreement approved by EPA and the Lawrence Livermore
Laboratory/Atomic Energy Commission on February 6, 1974 and work began
1n-April 1974. There have been two progress reports submitted to date,
a third is due in January 1974. A site visit to the La".re;ice Livermore
facilities was made by the Program Analyst of the FAA program in October,
1974.
264
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BACKGROUND CHEMISTRY
The methylation (alkylation) of various metals from a theoretical
chemistry point of view is as follows:
CH3+ (Methyl cation) + M° —^ CH3M+
CH3* (Methyl radical) + M+1 =» CHgM*
CH3:" (Methyl carbanion) + M+2 =» CH3M+ (RM+)
CH3: (Methyl carbanion) + RM+ => CH3MR
M = Metal; R = alkyl group
Among these reactions, methylation of a metal (M) and a monomethylated
organometallic ion (RM ) involving the methyl-carbanion donor (CH-:~)
are favored, i.e. a Grignard-like reaction. Methyl-cobalamin (Methyl
Vitamin B,2) is the only Grignard reagent found in nature capable of
methylating various metals. It has been shown to be involved in the
methylation of mercury. Therefore, this task has focused upon the
methylation chemistry of noble metals of the existing and new metallic
constituents of mobile source emissions, namely, platinum and palladium,
manganese and lead.
'Factors Known Jjo_ Influence Methylation
In addition to the oxidation state of metallic reactants and reactant
concentrations, there are several other factors that influence the
methylation reaction. The majority of the comments that follow refer to
mercury. It remains to be determined whether these conditions influence
the methylation of the metals of concern.
265
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Acidity: Labpratory and field studies have shown that the
loner the pH (higher acidity) the greater amount of methyl
mercury accumulates in aquatic organisms (fish). This observation
suggested that the methylation rate of mercury was pH-dependent.
In addition, the presence of specific methylated products also
appear to be pH-dependent. Dimethyl mercury (CH-HgCH.,) formation
is favored at higher pH levels (alkaline conditions) while
monomethylmercury formation predominates at lower pH values
(acidic conditions) in aqueous media. Dimethyl mercury is a
compound that readily volatizes from the water into the
atawsphere since it has a low boiling point (94°C).
Natural ambient water conditions tend to favor the production
of monomethylmercury by methanogen bacteria rather than dimethyl-
mercury. Therefore, the presence of methanogen bacteria,
proper pH and differences in physical-chemical properties of
the methylated products can serve as determinants regarding
their presence in a preferred environmental media (air, water,
soil). It is of equal importance in this task to assess the
i ,i
stability of methylated products, the pH-dependence of reaction
kinetics and to evaluate by virtue of analysis of reaction
conditions, reaction stoichiometry, and physical-chemical
properties of reaction products the anticipated contamination
of particular environmental media.
266
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'Anaerobic and Aerobic Conditions
In addition to acidity, the presence (aerobic) or absence (anaerobic)
of oxygen is an additional factor that tends to Influence the methylation
reaction. Mercury methylation that involves methyl donation from methyl -
cobalamln (Methyl Vit. B,2*» Grignard reagent) apparently occurs only
under strict anaerobic conditions in the absence of oxygen. However,
this does not exclude the formation of methylated metallic compounds in
natural waters. Methylation can also occur by other reactions under
aerobic conditions. Reduction of oxygen in many lakes and streams
promotes the formation of hydrogen sulfide, and in the case of mercury,
subsequently yields mercuric sulfide. Mercuric sulfide can be methylated
under aerobic conditions. Therefore, in fresh waters, high rates of
methylation may occur under aerobic conditions by biological (methanogen
bacteria) and non-biological (purely chemical) reactions as a consequence
of variation in oxygen content.
It is equally important to note that within biological systems (a
cell, organ) a complex can form between a metal in various oxidation
states and such endogenous biological molecules as homocysteine (amino
acid; protein constituent). In this complex, the metallic ion may be
methylated in preference to homocysteine in the ligand. This reaction
yields methylmercury homocysteine in the case of mercury ions. This
process is also not restricted to anaerobic conditions. Therefore,
while methyl-cobalamin is prevalent in nature and may be very reactive
under anaerobic conditions, methylation can occur by other reactions
in biological and non-biological systems.
267
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'Enzymatic and Non-Enzymatic Methylation
Initial analysis of mercury methylation within the cell or a whole
organism suggested the reaction was catalyzed enzytnatically. It has now
been shown to occur non-enzymatically. Methyl-cobalamin relinquishes
its methyl group in biological systems to divalent mercury ion quantitatively
under both aerobic and anaerobic conditions.
It is noteworthy that investigations concerned with the etiology of
the Minamata Bay disaster, involved, among other sources, the possible
contribution of an acetaldehyde factory. These investigations revealed
that methylmercury could be formed through a reaction between metallic
mercury as well as mercuric acetate and acet Idehyde. e;etaldehyde
serves as a methyl donor. Therefore, methylation of various metals may
occur in the absence of an enzyme (protein catalysis) and, in the
absence of methyl-cpbalamin as previously mentioned. While methyl-
cobalamin is sensitive to light that may minimize methylation under
exogenous environmental circumstances (outside the body), it is not
relevant to endogenous in vivo conditions (within the body or organisms).
It is also important to emphasize that other methylation reactions can
occur in the absence of methyl-cobalamin. Furthermore, the metals of
interest and aldehydes are present in mobile source emissions.
Agnes e^ i]_. published a paper in 1971 entitled "Methylation by
Methyl Vitamin B,^" (Chemical Communications, p. 850). In this report
methyl-cobalamin (methyl vitamin B,2) was shown to be capable of transferring
methyl groups to mercury, thallium, platinum, and gold. Palladium,
silver, cadmium and lead were also investigated. The reactions were
268
-------
studied spectrophotometrically by adding controlled amounts of metal
Ions to solution of methyl-cobalamin in the presence of various anions
between pH 0 and 5. The fate of the methyl group was determined by
gas-liquid chromatography. Two very different mechanisms were found by
which methyl-cobalamin can be demethylated and the methyl group transferred.
The first reaction by which methyl groups can be released from methyl-
cobalamin is an acid attack (electron donor) by divalent mercury or
trivalent thallium. Divalent cadmium and lead were not effective in
demethylating methyl-cobalamin. The second reaction involves a two-
electron redox switch resulting in demethylation and methyl group transfer.
Platinum and gold are characterized the second reaction. In this type
of reaction, both stable valence states of each metal are required.
The first step of the reaction results in the production of the methylated
metal while the second step results in transfer of the methyl group to
an acceptable nucleophilic agent such as a halide (chloride). The
overall reaction involving platinum is as follows:
1. MeB12 + Pt+4 —*> Aqueous B]2 + MePt*4
2. MePt+4 C1 —> Pt+2 + MeCl
+2
Methyl group transfer requires divalent platinum (Pt ). This transfer
probably occurs through the methyl-cobalamin divalent platinum complex
as follows:
3. Pt+48 + Pt+2MeB12 Pt+2J + Pt^Me + Aqueous B]2
The reaction rate of the overall reaction is expressed as follows:
Reaction rate = K [MeB]2] [Pt+2] [Pt*4]
The reaction rate is determined spectrophotoirietn'rally by following
the change in absorbance of wavelengths specific far each reactant
269
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as the reaction proceeds.
Reactant Wavelength (nm)
Aqueous B,^ 350
K2Pt C16; Pt+4 262
MeB]2 325
Photolysis
Methyl-cobalamin is sensitive to light and undergoes photolysis
resulting in disruption of the cobalt-methyl group bond in the corrin
ring system (Figure 1). Photolysis produces a methyl radical that in
turn could transfer to metallic ions present in its environment. Photolysis
is of concern in the ambient environment but ha^ little or no relevance
with regard to physiological reactions.
270
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(III II
(HI M
NI1.COCIU
CH
Th« rorrin
rin« system
V.vl sump
CM.CII.CONII,
S.D-DiiiirllivllxMv/iniiilar.ole
ribniiuc.loolidc
(.onin
ring system
VilfiMiiii II,, 1ri>linf(iinin| niul its
(Irrn'nlivi'S In c:y;inn< ii-
rlilnrm-.oli.il.imin K ^ t hlnnilr
Cnl»il(iiiiin /onus similarly niiincd
coinjilrxps tvilli su/fulc, livilmxyl. find
nilrili; ions. In HIP corn/yiiin K 15 the
5'-(lcoxyfi(/(;nosv/ group (in color
allow J. Tlic nontp n/ (lie coi;nzynie is o/(nn
ol)lirpviul<;d ns IMciiWoiiun.
Figure 1
271
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Progress Reports: First Quarter: April through June 1974
Platinum
1. The work of Agnes et al_ (Chem. Corrni., p. 850, 1971) briefly
described above with respect to the reaction of platinum with methyl-
cobalamin has been confirmed. The mechanism and products of reaction
reported by Agnes and described below have yet to be confirmed. Agnes
et, al_. described a two-step oxidation reduction reaction in which
platinum is methylated and the methyl group subsequently transferred to
a nucleophile receptor, thus:
1. MeB]2 + Pt+4 - - -^ Aqueous B,2 + MePt"1"4
2. MePt"1"4 + Cl" - => Pt+2 + MeCl
2. The reaction is favored by the presence of catalytic amounts of
divalent platinum. The specific reaction used in this task:
K2PtCl4 (Pt+2)
MeB12 + K2PtC1g - => Aqueous B]2 + MePt"1"4
40 yM (Pt+4) pH 2.0 optimum, dark
100 pM
In the presence of divalent platinum, the rate appearance of aqueous
B10, measured spectrophotometrically by the increase of absorbance at
I c. •
350 nM indicated that the reaction between- methyl-cobalamin and quadravalent
platinum does not obey second order reaction rate kinetics (the initial
concentration of both reactants are changing with time and the reaction
rate is expressed as the reciprocal of the product concentration with
time, e.g. liters per mole per minute (M" min * ). The amount of
divalent platinum (K,,PtCl4) used in catalytic amounts in this experiment,
272
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one fliicromole (pM), stimulates a 30 to 40 fold increase in reaction
rate. Irrespective of the kinetic mechanism, methyl-cobalamin and quadravalent
platinum reaction obeys a 1:1 stoichiometry. In the presence of excess
methyl-cobalamin and divalent platinum, the amount of aqueous B,2 that
ultimately forms corresponds exactly to the amount of quadravalent
platinum in (K^PtClg) that was added. Quadravalent platinum is
irreversibly and quantitatively consumed in the demethylation of
methyl -cobal ami _n_._
3. This reaction is favored under acidic conditions with an
optimum observed at pH 2.0 and is independent of whether the reaction
is carried out in hydrochloric acid (HC1), perchloric acid (HC10.),
sulfuric acid (HpSO.) or potassium phosphate buffer.
4. The reaction either does not occur or is so slow that it is
Insignificant above pH 5.5 following two hours observation at 22°C.
5. Divalent platinum does not demethylate methyl-cobalamin. No
significant demethylation was detected at any pH level using 100 micromoles
of KgPtCl^ therefore, the presence of divalent platinum is apparently
required only to assist in forming a methyl-cobalamin
complex that facilitates methyl group transfer to quadravalent platinum
ion as described by Agnes et al.
6. There are a number of other alkylated forms of cobalamin (B12)
that may serve as alkyl group donors to metallic ions, e.g. ethyl-
cobalamin (Et-B^). propyl-cobal ami n (pR-B,^)- The reaction of divalent
mercury with these various alkyl-corrinoids (alkyl cobalamin compounds)
has shown a high specificity for methyl-cobalamin, the natural alkyl-
273
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corrinoid found in the environment. The reaction of TOO pM of K2PtClg
(Pt+ ' with various alkyl-corHnoids was Investigated. The results of
reaction performed in the dark are as follows:
K2PtCl Alkyl-corrinoid Relative reaction rates
at pH 2.0, 22°C
100 pM 40 pM x 104 pM/sec
Methyl-B12 1.0
Me - cobinamide - B1? Q.02
Ethyl - B]2 0.012
Pr - cobinamide °'01
Propyl - B12 0.006
Therefore, the divalent platinum-promoted reaction of quadrivalent
platinum shows the same specificity for methyl-cobalamin as divalent
mercury that yield toxic methyl mercury.
7. Methyl-cobalamin is sensitive to light. It is well known that
the methyl-cobalt bond in methyl-cobalamin is readily photolyzed by
light at many wavelengths. Methyl radicals and B,0 radicals (cobalamin)
are generated. The structure and some properties of Vitamin B,2 are
presented to aid in understanding photolysis and methylation of metallic
ions (Figures 1 and 2).
Methyl-cobalamin is the Vitamin B-tp structure with the carbon atom
of the methyl group associated with cobalt (Figure 1). It is this bond
that is cleaved during photolysis and demethylation. Apart from the
'+2
methyl radical, a B,2 (Co ) ion is formed that subsequently yields
aqueous B,2 ( i.e. water) is now attached to cobalt where the carbon
atom of the methyl group used to be. The overall photolysis reaction
274
-------
(III II
,
l/ll II '.'
.V-di'oxv.ulrimsyl
«:,,
CH,CH,CONH,
The corn n
"»R system
5.6-Dimrlhvlbi*n/iiiii(lazale
ribonucloolide
The corrin
ring system
Ql, CH,CH,CONH,
, Vitumin II,, |c»lKi/(irnin) anil its
(/iTivfilin-s. lit ryiinrn:n-
b.ilnmin K^cyiini
-------
1s as follows:
+3
RCH2: Co(B12) fl>
-------
8. Questions remain regarding confirmation of a methylated platinum
compound. Preliminary experiments have been carried out with carbon-
14 labeled methyl-cobalamin synthesized from cyanocobalamin (CNB^) and
methylated C methyl iodide. A radioactive carbon atom in the methyl
group permits tracking of methyl vgroup transfer during the methylation
of platinum.
There appears to be a difference between the methylation reaction
of platinum and methyl-cobalamin carried out in the dark and the
photolysis-mediated reactfon with respect to the platinum-methyl B12
reaction intermediate. When 40 yM of Carbon-14 labeled methyl -
cobalamin was allowed to react to completion (total depletion of methyl-
cobalamin) in the dark with 100 yM of quadravalent platinum (KpPtClg)
at pH 2.0, the carbon-14 (methyl group) removed from cobalamin could not
be removed by lydphilization. This indicated the methyl group was not
1n a free or soluble state and suggested that it is complexed or reacted
with platinum i.e. methylated platinum. Further evidence in support of
this tentative conclusion is obtained from chromatographic analysis of
the lyophilizatTon residue of the reaction mixture. Paper chromatographic
analysis using an organic solvent-water mixture revealed that virtually
all of the carbon-14 (methyl group) migrated ahead of the aqueous B-.^
and carrier methyl-cobalamin in a distinct sharply defined zone indicating
the presence of a distinctly different chemical compound different from
the reactants (methyl-cobalamin and Pt ) and one of the products;
aqueous B^- Therefore, by the process of elimination, this radioactive
labeled band is probably methyl-platinum; the expected product of the
reaction.
277
V
-------
*
There was no distinct reaction product observed when the reaction
was carried out of pH 7.0 in the presence of light. When carbon-14
labeled methyl-cobalamin underwent photolysis under neutral conditions,
approximately 40% of the methyl group (carbon-14) was lost upon removal
of water (lyophilization). The reason for this difference in two reactions
has not been investigated.
9. Synthesis of methylated platinum for toxicological studies.
Preliminary experiments have been conducted to synthesize methylated
platintfin (MePt ) from methyl magnesium chloride and both divalent and
quadravalent compounds (KgPtCl^ and K2PtClg). Small amounts of black
material thought to be reduced metallic platinum have been observed
where divalent platinum (ICPtCl.) was added to the two reaction mixtures.
Reaction mixtures containing quadravalent platinum (K?PtCl,-) result in
the production of small amounts of an impure platinum product that has
yet to be identified. Identification depends upon preparation, separation
and identification of known methyl platinum chloride compounds to be
used as reference standards. Determination of their stability and
skeletal properties is essential to establishing the nature of any
methylated platinum which may arise from the demethylation of methyl -
cabal ami n.
Palladium _
1. Initial studies indicate that palladium forms a spectrally
distinct complex with methyl cobalamin at pH 2.0 (0.01 MHC1; K^PdClg,
quadravalent palladium). It is not known at this time whether this
complex breaks dawn to yield aqueous B,2 and methylated palladium.
Corp] ex formation was not observed at pH 7.0 in 0.1 M potassium phosphate
buffer.
278
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2. It is clear even after 16 hours, the complex formed between
methyl-cobalamin and quadravalent palladium still has an intact methyl-
cobalt. Therefore the methyl group does not appear to have been transferred
or methyl-cobalamin demethylated.
3. The reaction of divalent palladium with 40 yM levels of methyl -
cobalamin did not yield new distinctive spectral characteristics that
would be indicative of complex formation.
279
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Progress Report: Second Quarter July-September
Platinum:
1. First order rate constants for each reactant in the methyl-
cobalamin platinum reactions. The appearance of reaction products or
the disappearance of industrial reaetants is monitored by determining
changes in absorbance of wavelengths specific to each reactant or product.
Reaction Rate * K [MeB12] [Pt+4] [Pt+2J
2. The values obtained for reaction rates determined at 22°C with
50 yM of methyl-cobalamin and quadravalent platinum is as follows:
pH 1.0 1.8 - 2.2 + Pt*2 (30 pM)
pH 3.3 2.2 - 2.3 + Pt+2 (30 yM)
pH 3.3 0.2 - Pt+2
3. All rates were linear or first order with respect to both the
appearance of aqueous B,~ and the disappearance of quadravalent platinum.
4, The rate of the methyl-cobalamin-quadravalent platinum reaction
decreased with increasing pH (acidity) in the absence of divalent platinum.
5. Attempts to isolate and characterize any methylated platinum-
chloride compounds have not as yet been successful. The reaction employed
1n the attempt to prepare reference methylated platinum compounds involves
reactions with methyl magnesium chloride and not methyl-cobalamin. The
reference material would be used to identify and confirm methylated
platinum compounds in the methyl-cobalamin-platinum reaction.
The reactions investigated thus far are as follows:
280
-------
+4
Pt compounds
" MeMgCI +
MeMgCI +
MeMgCI + Na2PtClg + NaPtClg
MeMgCI + H2PtClg
Organic extracts (benzene, chloroform, carbon tetrachlorlde) of the
reaction products all contained platinum. Water soluble fractions are
also being analyzed.
6. Work will continue on identification of the reaction products
on hand as well as large scale reactions between methyl -cobal ami n and
quadra valent platinum will be conducted in order to isolate and identify
any stable methyl platinum chloride compounds.
7. It has been shown that divalent platinum in catalytic quantities
stimulates a ten-fold increase in the demethylation of methyl -cobal ami n
in the dark. Questions arise regarding the specificity of this effort
and if there is an influence of the halogen (chloride) atoms in the
reaction mixture. The following compounds were compared at pH 2.0 in
the presence and absence of divalent platinum in the demethylation .
reactions.
Relative methyiation
reaction rates
-Pt~
0.1
0.1
0.09
0.07
Compound
HC104
K^tClg
K2PtBr6
Platinic Acid
Amount
TOO yM
100 pM
100 yM
100 yM
±Pt2
~ 1.0
1.0
0.85
0.65
281
-------
Therefore, the reaction in each case was 10-fold slower in the absence
of divalent platinum. The reaction between Methyl-cobalamin and quadravalent
platinum does not require that the platinum compound be associated with
a halogen compound or a halogen platinate form.
8. Identification of methyl platinate compound from the reaction
of quadravalent platinum and methyl-cobalamin. Tentative evidence indicates
that the methyl group of methyl-cobalamin is indeed transformed to an
intermediate complex material, methylated platim'c reaction products, or
both.
Two reactions were performed. A control reaction containing radioactive
carbon-14 methyl-cobalamin (40 pM) hydrochloric acid (0.01 M) and an
experimental reaction in which the labeled methyl-cobalamin is reacted
with 40 yM KpPtClg (quadravalent platinum) and 0.01 M H.C1 in the dark.
The mixtures were lyophilized to dryness and then reconstituted in 0.1
ml of water. Approximately 65-85% of the carbon-14 (methyl) group has
been recovered following lyophilization and was not lost on drying with '
regard to the experimental reaction with quadravalent platinum. ,.--"" r
Nearly all (95-,100%) of the carbon-14 (methyl-cobalamin) was recovered
after lyophilization (drying) from the control reaction. Aliquots (20 j
yl) from both mixtures was analyzed by ascending column chromatography }<
I
in the dark (solvent phase: H^O: n-butyl alcohol: iPrOH and acetic !.
acid; 100: 100: 70: 1; respectively). When the chromatogram was J
dried and cut into 1 centimeter strips, the migration was as follows: f {
% 14C % Pt Migration j
Reaction mixture migrating migrating distance in cm j
-Pt control mixture 100 0 6-8 (carrier MeB^ zone) j
[Me"14C] MeB]2 + PtIV j
reaction mixtures 4 0 6-8 (carrier MeB^zone) I •
Reaction Mixtures 69 100 11-12 f
Reaction Mixtures 77 0 14-15 >
Reaction Mixtures 10 0 20-21 \
282 j
t
1 , j
-------
In order to confirm that that the association between the carbon-
labeled methyl group migration and the associated migration of platinum
is not coincidental, a second separation using strip electrophoresis
MS performed (acetic acid, pH 2.5). About 93% of the carbon-14
labelled methyl group obtained from the methyl-cobalamin quadravalent
platinum reaction remained at the origin and did not migrate in an
electric field. Unlabeled and unreacted methyl-cobalamin migrates about
5.0 cm from the origin toward the cathode pole. Furthermore, a second
ascending chromatographic analysis was performed that further revealed
distinct separation of the unreacted methyl-cobalamin and the product of
the methyl-cofaalamin quadravalent platinum reaction.
Palladium:
1. Additional spectral studies of the interaction of methyl -
cobalami n and quardravalent palladium reveals that a stable complex is
forned In which the methyl-cobalt bond remains intact.
2. Due to the stability of the complex, studies were made to
titrate its formation as a function of the molar ratios (relative concentration)
of the reactants. Monitoring absorbance changes, the experiments show
quite clearly that complex formation is maximal when the concentration
of palladium is twice that of methyl-cobalamin.
3. The complex was formed at pH 2.0 when the dimethylbenzimidazolyl
base is separated from the corrin ring system (Figure 2.). The molar
ratio of 2:1 K-PdClg to methyl-cobalamin at pH 2.0 in the base-off
state suggests that the complex is formed directly without an 1:1 intermediate
conplex expected to result with equal amounts of reactants present.
283
-------
' 4. Divalent palladium has not been observed in complex formation
with methyl-cobalamin.
5. Spectral analysis of the reaction involving methyl-cobalamin
and quadravalent palladium is different since the complex formed has a
spectral maxima identical to that of aqueous B,2 (350 pM). Therefore,
complex formation interferes with monitoring formation of a reaction
product. It is difficult to determine spectrophotometrically the breakdown
of the complex to aqueous B^ or alternatively, a slow transmethylation
between platinum and methyl-cobalamin that may occur independent of
complex formation. Therefore, photolysis studies using radioactive
carbon-14 labelled methyl-cobalamin was used. The carbon-14 labeled
methyl group released from the methyl-cobalamin cobalt atom would yield
C-14 HpO (labeled formaldehyde) upon photolysis. In this way, demethylation
rate of methyl-cobalamin caused by the palladium ion could be determined.
The results of these experiments indicate that approximately 25% of
the methyl-cobalamin (40 yM) demethylated with 60 minutes at 22°C in the
presence of quadravalent palladium (100 pM). Divalent palladium (100
pM) did not demethyTate methyl-cobalamin within a 60 minute period.
After 22 hours in the dark, approximately 55% of the methyl-cobalamin
iwas demethylated in the presence of quadravalent palladium compared to
o«Ty 23% for divalent palladium.
284
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9. PERFORMING ORGANIZATION NAME AND AOORESS
Health Effects Research Laboratory
Office of Research & Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
EPA-600/3-75-010 q
I. TITIE AND SUBTITLE
ANNUAL CATALYST RESEARCH PROGRAM REPORT
Appendices, Volume VI
5. REPORT DATE
September 1975
G. PERFORMING ORGANIZATION CODE
7. AUTHORliT"
0. PE/WORMING ORGANIZATION REPORT NO.
Criteria and Special Studies Office
10. PIU'GMAM ELEMENT NO.
1AA002 _
i». CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Same as above
13. TYPE OF RE PORT ANOPtniOO COVERED
Annual Program Status 1/74-9/7
14. SPONSORING AGENCY CODE
EPA-ORD
tb. SUPPLEMENTARY NOTES
This is the Summary Report of a set (9 volumes plus Summary).
See EPA-600/3-75gQ10 a thru OIQf & OlOh thru OlOj. Report to Congress.
• T n A f T ~ -fc-*"~ ' -^—^^—^^— _ —«^^—^^^—»™^—^^—™. _ --—.._
16. ABSTRACT
This report constitutes the first Annual Report of the ORU Catalyst Research
Program required by the Administrator as noted in his testimony before the
Senate Public Works Committee on November 6, 1973. It includes all research
aspects of this broad multi-disciplinary program including: emissions charac-
terization, measurement method development, monitoring, fuels analysis,
toxicology, biology, epidemiology, human studies, and unregulated emissions
control options. Principal focus is upon catalyst-generated sulfuric acid
and noble metal particulate emissions.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Catalytic converters
Sulfuric'acid
Uesulfurization
Catalysts
Sulfates
Sulfur
Health
l).IDENTIFIEHS/OrEN ENDED TERMS
Automotive emissions
Unregulated automotive
emissions
Health effects (public)
U. COSATI I lilil/dllillp
i-i. uisrwounoN STATLMCNT
Available to public
19. SECURITY CLASS f I Iti.i
21. NO. Of PAGES
290
Unclassified
22. PRICE
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