PBS2-I32002
Possible Approaches to the Health Effects
Testing of Fuels and Fuel Additives
Southwest Foundation for Research and Education
San Antonio, TX
Prepared for
Health Effects Research Lab
Research Triangle Park, NC
Get 31
^
U.S. Department of Cm-amerce
National Technical lnformstiQ«_$?rvice
-------
-132CC?
EPA-600/2-81-235
POSSIBLE APPROACHES TO THE HEALTH EFFECTS TESTING
OF FUELS AND FUEL ADDITIVES
by
Emily M. Cause
Martin L. Heltz
Nathan D. Greene
Department of Behavioral and Environmental Sciences
Southwest Foundation for Research and Education
San Antonio, Texas 78284
Contract No: 68-02-2286
Modification No: 1
Project Officers
Joellen Lewtas
Donald Gardner
Health Effects Research Laboratory
and
Robert Jungers
Environmental Monitoring and Support Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
US ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NC 27711
-------
TECHNICAL REPORT DATA
' fijJ /mrr«r;:<.'N} tin In? /rrc/jr bflvrc Ct
\. REPORT NO.
-££^600/2^81-235.
13. RECimNVS A CC COS I ON NO.
_ORD_ Repo r t_
1. TITLE AND SUBTITLE
Possible Approaches to the Health Effects Testing of
Fuels and Fuel Additives
5 REPORT DATE
October 1981
7. AUTHOR(S)
Emily M. Gause, Martin L. Meltz, and Nathan D. Greene
0. PERFORMING ORGANISATION COOK
_3.28_Q52
8. PERFORMING ORGANISATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Behavioral and Environmental Sciences
Southwest Foundation for Research and Education
San Antonio, Texas 78284
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory and
Environmental Monitoring and Support Laboratory
U.S. Environmental Protection Agency
Research Triangle. Park, North Carolina 27711
10. PROGRAM ELEMENT NO.
C9GA1A
11. CONTRACT/GRANT NO.
Contract No: 68-02-2286
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-600/11
IS. SUPPLEMENTARY NOTES
EPA Project Officers: Joellen Lewtas Huisingh, Robert Jungers, and Donald Gardner
16. ABSTRACT
This document describes possible approaches to the testing of fuels and fuel
additives for potential health effects. Such health effects testing is required
of the manufacturer of a fuel or fuel additive. The health effects tests must
include but are not limited to carcinogenic, teratogenic, or mutagenic effects. In
order to determine the appropriate protocol for health effects testing, the following
areas arc discussed: (1) test materials, (2) relationship of physics! and chemical
properties of test materials to physiological distribution and biological activity,
(3) weighing factors involved in determining an approach to appropriate health
effects.testing, (4) route and mode of exposure.
The possible health effects tests described are organized into the following
areas: (1) toxicity (with an emphasis on pulmonary effects), (2) mutagenesis, (3)
carcinogenesis, (4) teratogenesis and reproductive performance.
The.final chapter describes two possible approaches to testing. This report is
a technical background document and is not intended to serve as a health effects
testing protocol for fuels and fuel additives.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
:. COSATl ! !rlii;Gr'oup
fuels, fuel additives, health effects,
bioassays, toxicology, mutagenesis,
carcinogenesis, testing approaches,
combustion products, emission products
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
SECURITY CLASS r/Vi,v
^UNCLASSIFIED.
20 SECURITY CLASS iTIiil
UNCLASSIFIED
21. NO. Of PAGES
216
EPA Form 2220-1 (Roy. 4-77} PREVIOUS EOITIOM is OOSOL.KTU
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publlcaticn. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products consti-
tute endorsement or recomaiendation for use.
il
-------
FOREWORD
The many benefits of our trodcrn, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relativ/e risk
of existing and new man-made environmental hazards is necessary for tha
establishment of sound regulatory policy. Ihese regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxi-
cology, epidemiology, and clinical studies using human volunteer subj-
ects. These stu'-ics address problems in air pollution, non-ionizing
radiation, environmental circinogenesis and the toxicology of pesticides
as well as other chemical pollutants. The Laboratory participates in
the development and revision of air quality criteria documents on pollu-
tants for which national ambient air quality standards exist or are
proposed, provides the data for registration of new pesticides or
proposed suspension of those already in use, conducts research on
hazardous and toxic materials, and is primarily responsible'for provi-
ding the health basis for non-ionizing radiation standards. Direct
support to the regulatory function of the Agency is provided in the form
of expert testimony and preparation of affidavits as well as expert
advice to the Administrator to assure the adequacy of health care and
surveillance of persons having suffered imminent and substantial endanger-
mcnt of their health.
This document addresses itself to the acquisition of data necessary
for determination by the Administrator as to whether or not an increased
health-risk would exist should the fuel or fuel additive in.question .
become a produce of commerce.
F. Gordon Hueter, Director .
Health Effects Research Laboratory
iii
-------
ABSTRACT
This document describes possible approaches co the testing of fuels
and fuel additives for potential health effects. Such health effects
testing is required of the manufacturer of a fuel additive. The health
effects tests must include but are not limited to carcinogenic, tera-
togenic, or mutagenic effects. In order to determine the appropriate
protocol for health effects testing, the following areas are discussed:
(1) test materials, (2) relationship of physical and chemical properties
of test materials to physiological distribution and biological activity,
(3) weighing factors involved in determining an approach to appropriate
health effects testing, and (4-) route and mode of exposure.
The possible health effects tests described are organized into the
following areas: (1) toxicity (with an emphasis on pulmonary effects),
(2) mutagenesis, (3) carcinogenesls, and (4) teratoger.esis and repro-
ductive performance.
The final chapter describes two possible approaches to testing.
This report is a technical background document and is not intended to
serve as a health effects testing protocol for fuel additives.
-------
TABin OF CONTENTS
Page
1\
>
Abstract iv
Background 1
Overview 3
Hazard Evaluation 10
I. Test Materials I.I
A. Uncombiisrc-d State 1.1
B. Combustion Products ' I. 2
1. Whole Exhaust 1. 3
2. Characterized- Emission Products 1.3
a. Physically Isolated Phases I. 4
b. Chemically Identifiable Fraction I. 5
3. Additive Pyrolysis Products 1.5
II. Relationship of Physical and Chemical Properties
of test Materials to Physiological Distribution
and Biological Activity II. 1
A. Physical Properties II. '.
B. Chemical Properties II. 2
C. Chemical Structure II. 2
III. Weighting Factors Involved in Determining an
Approach to Appropriate Health Effects Testing III. 1
A. Amount of Raw Material Produced III. 1
B. Amount of Potentially Toxic Material Emitted III. 1
C.' Persistence and Chemical Form : " '- " .- III. 1, < -
1. Persistence in the Environment and
Biological Organisms III. 2
2.- Bioaccuniulation III. 2
3. Biomagnification III. 2
4. Biotransformation to More Toxic Forms III. 2
D. Evaluation of Existing Health Data III. 2
E. Risk Assessment Analysis III. 3
IV. Route and Mode of Exposure IV. 1
A. Pulmonary IV. 1
1. Inhalation IV. 1
2. Intratracheal Instillation IV. 2
B. Oral IV. 3
C. Eye and Skin IV. 3
V. Health Related Tests V. 1
A. Toxicity V. 1 j
-------
1. General V. 1 '
a. Oral . V. 1
(1) LDSO Determination V. 1
(2) Blood/Urine Analysis V. 4
(5) Gross and Ilistopathology V. 6
b. Dermal V. 8
c. Lye V. 12
d. J_n \j_i££ To/.icity Tests V. 14 ]
(1) Tissue Slices V. 14 1
(2) Primary Cells . V. 15 ]
(3) Cell Lines V. 15
2. Pulmonary V. 19
a. LC$Q Determination V. 19
b. Host Defense Mechanisms V. 22
(1) Infcctivity Model System V. 22
(2) Mucociliary Function V. 23
Ciliary Activity V. 23
Mucociliary Transport V. 24
Mucus Production and Character-
istics V. 24
(3) Free Lung Cell Populations V. 24
(4) Aiveoinr Macrophage Function V. 25
JJ1 Vivo Exposure V. 25
i!i XAUP Exposure V. 26
(5) Immune Function V. 27
c( Pu Imo!"i3rv F\ir.c.t ion V. 40
d. Gross and Ilistopathology ' V. 43
e. Blood/Urine Analysis V. 43
3. Central Nervous System/Behavior V. 43
B. Mutagcncsis V. 46
1. Gene Mutations V. 46
a. Procaryotic Microorganisms (Bacteria) V. 47
(1; SaImonella typhiniurium V. 48
" ~ (2) JJ.schcr_i_ch_i_a col i 1VP2 '" "" ' V. 49
(3) 1-schcrichia coli K12 V. 49
b. Eukaryotic Microorganisms V. 56
(1) Nourospora crassa V. 56
(2) Map"loid Strains of Yeast V. 56
c. Plants V. 58
(1) Tradcscantia V. 58
d. Insect V. 60
(1) Drosophi la melanogg.stcr V. 60
e. Mammalian Cells V. 62
(1) Mouse Lymphoma, 1.5178Y V. 62
(2) Chinese llanster Lung, V79 V. 62
(3) Chinese Hamster Ovary, CI10 V. 62
f. Rodent. V. 65
(1) Mouse Specific Locus V. 65
(2) Mouse Isonyme Specific Locus V. 66
2. DNA Damage and Repair V. 68
a. Repair-defective Microorganisms V. 68
-------
(1) Reccmbinationless Mutants of
Bacteria V. 68
(2) D.NA Pol yinerasc-def icicnt Mutants
of Bacteria V. 69
(3) Normal versus Repair-deficient
Yeast V. 70
b. Mammalian Cells V. 72
(1) DNA Single-strand Breaks V. 72
(2) Unscheduled (Nonrcplicativc)
D.NA Synthesis V. 74
c. Rodent Germinal Cells V. 76
3. Chromosomal Effects V. 78 SJ
a. Yeast (Mitotic Recombination) V. 78 |
b. Insect (Drosophila) V. 79
(1) Dominant Lethality V. 79
(2) Chromosomal Rearrangements V. 80
c. Mammalian Cells V. 82
(]) Chromosomal Aberrations V. 82
(2) Sister Chromatid Exchange V. 84
d. Rodents . V. 86
(1) Cytogcnctic Analysis V. 86
Bone Marrow V. 86
Circulating Peripheral
Lymphocytes V. 86
Spermatocytcs V. 86
(2) Heritable Chromosomal Damage V. 87
Sex Chromosome Loss V. 87
Dominant Lethal Effects . V. 87
Heritable Translocation Test V. -88
4. Methodological Considerations V. 92
a. Metabolic Activation ' V. 92
(1) Iji vivo V. .92
(2) "hii vitro V. 93
'.- - b.- Treatment Conditions - *'*' " - ' V; 94 "
C. Carcinogcncsis V. 98
1. Selected Mutagcnesis Tests V. 98
2. Mammalian Cell .Ncoplastic (Oncogenic)
Transformation V. 98
a. Primary Cells V. 100
b. Continuous Lines . V. 102
3. Rodent Tests V. 104
a. Pulmonary Tissue Carcinogencsis V. 104
Aerosol inhalation V. 105
Vapor inhalation V. 105
Intrarrachcal instillation V. 105
b. Nori-pul,.ionary Target Tissue Carci.no-
gcnesis V. 106
Pulmonary exposure V. 106
Skin painting V. 106
Carcinogen liioassay - oral
administration V. 106
Vli
-------
4. Cocarcihogcncsis and Tumor Promotion V..110
a. Mouse Skin Papilloma V. 110
b. C3ll/10T!i Mammalian Cells in Promoter
Mode V. 110
. Tcratogencsis and Reproductive Performance V. 112
1. Reproductive Performance V. 112
2. Teratogenesis V. 112
Possible Testing Approaches VI. 1
A. Matrix Approach VI. 2
B. Heirarchical Approach . VI. 2
(
i
viii
-------
BACKGROUND
Section 211 of the Clean Air Act of 1970 which is amended to
Section 222 in 1977 by adding new subsections at the end thereof,
charges the Administrator of the Environmental Protection Agency
(EPA) with responsibility for the regulation of fuel and fuel
additives. Registration is required by the Agency of Fuels and
Fuel Additives prior to sale or introduction into commerce. The
administrator shall promulgate regulations not later than August 8,
1978, to implement the authority to require the manufacturer of
a fuel or fuel additive:
(A) to-conduct tests to determine potential public
health effects of .such fuel or additives (in-*(r
eluding, but not limited to, carcinogenic, tera-
togenic, or mutagenic effects); and
(B) to furnish the description of any analytical
techniques that can be used to detect and measure
any additive in such fuel, the recommended range
of concentration of such additive, and the recom-
mended purpose in use of such additives, and such
other information as is reasonable and necessary
to determine the emissions resulting from the use
of the fuel or additives contained in such fuel,
the effect of such fuel or additives on the
emission control performance of any vehicle or
vehicle engine, or the extent to which emissions
, affect the public health or welfare; with respect"
to each fuel or fuel additive which is registered
on the date of promulgation of such regulations
and with respect to each fuel or fuel additive fcr which
an application for registration is filed thereafter!.
In addition, all such manufacturers would be required to furnish
summaries of information in their possession about mechanisms of
action of additives, reactions between fuels and their additives,
effects of the additives on emissions, identification and measure-
ment of emission products, and effects of the emission products
on health, welfare, and emission control devices.
Additive manufacturers would be required to furnish additional
information about the chemical composition and structure of addi-
tives and methods of analysis, any impurities, and sales volumes.
Fuel manufacturers would be required to furnish additional infor-
mation on sucli properties as hydrocarbon composition, polynuclear
organic materials, sulfur, trace elements, vapor pressure,
-------
distillation temperatures, and cetane numbers, including methods
of analysis i'or each of these, information on variation by
geographic region, and production volume.
The registration of fuels and fuel additives is provided
for in 46 CFR Part 79, A Revision of 40 CFR Part 79, (Federal
Register, Volume 59, No. 46 (March 7, 1974). As revised, Part
79 designates certain fuels and additives as requiring registration:
namely, motor v hide gasolines, motor vehicle diesel fuel, and
additives for use in motor vehicle gasoline and motor vehicle
diesel faels, ar.d/or 'in motor vehicle crank case oil. These
substances are further identified and designated in paragraphs
79.31, 79.32, 79.33 of Part 79 (46 CFR 79).
-------
OVERVIEW
This document addresses itself to the acquisition of data
necessary for determination by the Administrator as to whether
or not an increased health risk would exist should the fuel or
fuel additive in question become a product of commerce.
Basic to our present day culture, and to that of all
economically developed areas of the world, is the generation of
energy from the combustion of fossil fuels. This base supports
both an advanced technology, and the mobility and livelihood of
an expanding population. Whether associated with the fixed
sources of industry and municipal utilities or the mobile sources
of personal and commercial transportation, all practicable com-
bustion processes result in incomplete combustion of the cai'bon-
containing fuel. Therefore, instead of the theoretical complete
combustion products of water and carbon dioxide, a complex
dynamically-variant mixture of many partial oxidation products
including those of other elements - such as nitrogen from the air
and sulfur and similar minor component elements -present in fu?l -
as well as those of carbon, hydrogen, and oxygen, is actually
produced. The exact composition of the combustion products varies
v;ith the character-.sties cf each individual fuel, combustion
conditions, environmental conditions, etc. Because the combur-tible
fuel substances are all natural products, it is not possible to
control their chemical composition or the attendant composition
of the combustion products.
Urban areas provide combinations of: (1) relatively high
atmospheric concentrations of emissions from multiple-source
combustion processes; (2) a man-made environment which restricts
the dissipation of these emissions; and (5) large numbers of people
who have to live and function in this environment. In this situa-
tion, motor vehicle emissions represent a ma.ior source of concern
with respect to the health and well being of human populations.
Consequently, a significant research effort has been directed
toward understanding health effects implications of emission
components and whole emissions. From the results of such studies,
it is possible to correlate certain specific biological effects
with the occurrence and concentration of specific chemical entities
in the emissions. Such knowledge is extremely valuable, but the
current data base falls short of enabling predictions of the health
effects of lifetime exposures to emissions for hetci'ogeneous human
populations.
However, it is now necessary to consider, (1) whether a given
fuel composition will have more or less, or better or worse, health
-------
effects than another fuel composition; and (2) whether the addition
of single or multiple optional ingredients, defined here as
"additives", could possibly alter the health effects associated
with the emissions in a manner which would result in increased
hazard to human populations. These optional additions to, or
alterations in, the basic hydrocarbon fuels consist mainly of
chemicals selected for the purpose of improving specific aspects
of engine performance, fuel stability, emissions characteristics,
etc. As such additives are synthetic chemicals with inherent
manufacturing costs, economic considerations dictate that they
will be used sparingly in high-volume bulk fuels. The very low
concentrations of these iptional additive substances along with
certain physical chemical properties pertinent to the choice of
chemical nature of such additives (e.g., low volatility) all
contribute to predictions of generally low levels of atmospheric
contamination by cither tiic combustion products of such additives
or the unbumed additive material itself in the exhaust gas stream.
In fact, at normal use levels for some of these additives, con-
ventional analytical methods are not able to detect any residual
unburncd additive compound in the emissions, while concentrations
of specific combustion products resulting from these same additives
may be extremely low.
However, conventional bulk analyses may not reflect biologically
effective concentrations as there has been found to be a surface
predominance of toxic trace elements in fine particles produced by
combustion processes, with an inverse- relationship between particle
size and trace element concentration. This inverse relationship
is thought to be due to the volatilization of trace elements or
their compounds followed by condensation onto particle surfaces as
the temperature drops (Linton e£ aj_., 1976). Polyalkyl gasoline
additives and reactions between ammonia, nitric oxide and soot have
both been linked to the occurrence of organic nitrogen-containing
bases on the surfaces of filter-collected particles. . . . -
The existence of such low-dose levels docs not necessarily
mean that human systems do not respond, but rather that resulting
effects are likely to be lost among the general variability of
measureablc responses. Low-dose response measurement and inter-
pretation is immensely complex and must be considereded within the
context of synergistic and potentiating effects, sub-toxic effects,
bioaccumulation, biomagnification, and many other factors related
to variability in health status of specific sub-groups within the
human population.
For motor vehicle emissions, the primary human target systems
are most likely to be the external organs of eye and skin and the
respiratory system. Exposure of eye and skin arc generally asso-
ciated with transient irritative effects, while insult to the
respiratory system lias much more serious implications over a much
wider temporal scale. For example, gaseous emission components
which cause irritation of eyes and skin will also cause inflammation
-------
of tissues in the respiratory tract, while the properties of the
gases (specifically, solubility and volatility) will determine
the depth of penetration into the respirator)' tract, and conse-
quently the nature of the symptoms produced.
In addition to possible overlapping effects of individual
gaseous components of emissions upon human respiratory systems,
the paniculate phase may also exert specific toxic effects -
the site of which will be largely determined by the physical
characteristics of the particulate. For example, size aid shape
will determine deposition site and clearance mechanism; while
the hygroscopicity, partition coefficient, and solubility in
lung fluids will determine particle growth and aggregation, lung
tissue uptake and binding, and retention time. Moreover, one
pollutant may influence the biological effect of another; for
example, normal respiratory clearance mechanisms for particulates
could be inhibited by gaseous components of emissions, thus
contributing to increased retention times for emission particulates
and increased retention of inhaled microorganisms along with
diminution of normal microbicidal capacity. These effects may
result in both exacerbation of particulate toxicity and increase
in susceptibility to infection.
In addition to rapid absorption of inhaled organic compounds
by respiratory tissues, there is a marked concentration of basic
lippphilic amines by the lung, even when the route of administration
is other than inhalation (Junod. 1974; Philnot e_r_ aiL, 1977).
This chemical class includes polyalkyl amine fuel additives and
potentially carcinogenic aromatic amine combustion products -
precisely those nitrogen-containing organic bases found to be
concentrated on the surfaces of respirable smog particles (N'ovakov
£t£l_., 1972).
Not only are certain hydrophobic substances absorbed or
.concentrated by lung tissue or localized in the respiratory tract
by deposition, but lung and respiratory tract tissues possess a
remarkable metabolic capacity as well; in fact, for some chemical
classes., lung is more active metabolically than liver. For example,
lung has almost 50 times the metabolic capacity for N-methyl
indoleamincs as liver (Brown, 1974); and although liver mixed
function oxidase activity is much higher than that of the lung, some
substrates such as benzene, dccane, benzphetamine, biphenyl, and
N-methyl-p-chloroaniline are metabolized by lung microsomes at rates
equal to or greater than those obtained with liver microsomes. In
fact, p-xylene is hydroxylated 4 times faster by rabbit lung micro-
somes than by rabbit liver microsomes on a weight of tissue protein
basis and 16-20 times faster on a mole of enzyme basis (Philpot et al.,
1977). Since metabolism of xenohiotic chemicals involves a cluster
of interrelated enzymes the number and specificity of which apparently
varies from crgan to organ, the overall pattern of metabolites pro-
duced by a given tissue iray well be the determining factor in target
organ carcinogenesis. Metabolites produced in the lung may be ei-
ther returned in the lung or distributed to other organs of the body
via the circulation, hence lung metabolic capacity assumes unique
importance.
-------
There are still other types of chemical structure for which
lung is the target organ for toxic effects regardless of route
of exposure; nitro-olefins are such a class, specifically, the
automotive emission constituent-olefins C2 to C7 which react
with atmospheric NOX to produce nitro-olefins (Deichmann et al.,
1958).
All of these factors are relevant to the biological effects
of inhaled motor vehicle emissions. For example, if the particulate
phase of emissions from a specific fuel, or fuel plus additive
combination, is of a roughly spherical shape and diameter less
than three microns, the particulates will be preferentially
deposited within the alveolar regions of the lung where clearance
processes can have half-lives of months to years for relatively
insoluble particles (Casarett, 1975: Kilburn, 1977). The numbers
of such small particles retained in the lung from a single exposure
may be quite large (Lippmann, 1977); therefore, if exposure condi-
tions are chronic, it is reasonable to expect that the mass of
such material will continue to accumulate. It is not unreasonable
to anticipate a situation in which a biologically-active trace co.n-
ponent of emissions could be absorbed upon the considerable surface
area of relatively insoluble particulates less than three microns
in diameter. Therefore, upon inhalation, these particles would be
deposited in the alveolar regions of the lungs subject to clearance
by alveolar macrophages and by dissolution in lung fluids, and other.
mechanisms. However, if the absorbed material is either cytotoxic
to rnacropadges, or insoluble in lung fluids, or botli, it would re-
sist clearance by either Phase III or IV-mechanisms and would con-
tinue to accumulate over long periods of time.* In this, or a
similar situation, even the most minute quantities of any given
chemical species take on a significance quite out of proportion to
its existence in barely-detectable concentrations in whole emissions;
particularly if the chemical species under consideration acts as
carcinogen, cocarcinogen, or tumor promoter, initiating irreversible
delayed toxic processes (cancer) with lead times of five to thirty
years.
*FOOTNOTE
"Phase I clearance rates, with half-times of minutes
to hours, reflect clearance of particulates from the
upper respiratory tract and the naso-pharyngeal regions.
Phases II and III both involve alveolar macrophages, .
but whereas Phase II phagocytic clearance rates have
half-times of the order of 2 to 6 weeks, Phase III
half-times are on the order of months and are consid-
ered to involve sequestration of materials. Phase
IV clearance when it occurs, is thought to depend en-
tirely on solubilization, and, depending upon the
chemical nature of the substance, may exhibit half-
times of months to years (Casarett, 1975)."
-------
7 .
In otlier words, no matter how low the concentration per unit
time reaching the target biological system is, if the normal turn-
over mechanisms of the body have not adapted or evolved to handle
this type of exogenous substance, it will accumulate. Since dose
is a product of concentration and time, the actual dose of trace
contaminant to which the target tissue is exposed may become quite
significant under conditions of chronic exposure. For this reason,
there is a real need for the development of screening tests
capable of predicting chronic health effects without requiring
chronic animal exposures.
There are other conditions under which a trace component,
or even an occasionally occurring trace component of emissions
could possess biological significance disproportionate to actual
target dose level. Such a chemical entity could act as inhibitor
or modulator of key enzymes, or interact with vital hormones 01
hormone receptors. The physiological level of these suns*ances
is generally in the nanomolar to picomolar (10"^ to 10"*- molar)
range or below, so low concentrations of a modulator substance
might be quite effective. Another possible role for a trace
toxicant could be as non-enzymatic catalyst of a nonphysiological
detrimental reaction; similarly, a trace pollutant could non-
enzymatically trigger a physiological cascade-type reaction
producing biologic amplification analogous to the complement
system of blood.
A concept of relevance to the present situation involves
consideration of human exposure to low levels of environmental
carcinogens in terms of life-shortening (Albert and Altshulcr,
1976). Epidemic!ogical evidence suggests that as much as 80%
of human cnacer is due to exposure to environmental carcinogens.
This incidence of cancer is considered to represent a background
or baseline of environmentally-caused cancer. The introduction
of a cocarcinogen, or an increase in the concentration of an
existing carcinogen may shortei the time-to-turaor appearance,
thereby increasing the incidence and shortening the lives of
affected individuals (WHO-IARC, 1976). The impact of the new
carcinogen can be evaluated from tumor incidence measurements
in animals, and related to the human condition.
Some elenent of public health risk associated with exposure
to combustion products is unavoidable because of the overriding
need for energy generated in the combustion process. In order
to determine whether a given fuel or fuel additive would present
an undue heaith risk, it will be necessary to make judgments based
upon: 1) whether its use would result in an identifiable incre-
mental increase in risk; 2) whether the increased risk is counter-
balanced by a worthwhile benefit.
The most logical way to approach an estimate of possible
increased public health risks appears to be by comparison of the
biological effects of the combustion products of a given fuel or
fuel additive with those effects resulting from the combustion
products of a standardized reference fuel and speci.fie'd engine
-------
system. In formulating these recommendations for drafting of
protocols for health effects evaluation, consideration has been
given to the following reports:
(]) PRINCIPLES FOR EVALUATING CHEMICALS IN
THi; ENVIRONMENT
National Academy of Sciences
Washington, D.C., 1975
(2) FUF.I.S A.NT) FUEL ADDITIVES FOR HIGHWAY
VEHICLES AND THE IK COMBUSTION PRODUCTS
National Academy of Sciences-National
Research Council, 1976
(3) Report - Working Conference on Health
Intelligence for Fuels and Fuel
Additives, 1973.
Convened by: Fuels and Fuel Additive
Registration Program, National Environ-
mental Health Research Center - Environ-
mental Protection Agency, Research Triangle
Park, North Carolina.
These scientific panels have unanimously stressed the
importance of performing health effects testing of fuels rind fuel
additives under conditions which are as realistic as possible.
These conditions would include: exposure route, biological
target systems, engine operation, realistic atmospheric concen-
trations of combustion products, and the full range of atmospheric
conditions such as irradiation with light of from 300 to 700
nanometers, the absence of such irradiation, varied humidity
conditions,-etc; - ' '.-..
Several of these advisory groups (NAS, 1975; NAS-NRC, 1976;
NEIIRC, 1973) have called attention to the necessity for priority
attention to be given to effects of subchronic and chronic exposures.
Exposures of this nature implicate such health effects as respiratory
infections, pulmonary diseases, and possible irreversible, delayed
effects such as carcinogcncsis, mutagencsis, or teratogcnesis.
It should perhaps be pointed out here that human population.1:
wi11 actually be exposed to the subject fuels and fuel additives
and their emissions products in the atmosphere, since the per- .
tinent legislation permits these substances to be registered,
marketed, and used during the period in which health effects
testing will be performed.
The scientific panels mentioned above recommend that health
effects testing must employ-in vitro and selected whole animal
-------
studies, as a preliminary screen of biological activity, in an
effort to limit the cost and time required per test material.
However, it is also emphasized that whole animal exposures which
are carefully designed and controlled should be used to confirm
screening bioassays.
-------
10
HAZARD EVALUATION
The projection and assessment of hazards to human health
which could result from the widespread use of a fuel or fuel
additive requires a great deal of specific data.
A. Preliminary estimate of exposure
In order to make a preliminary estimate of exposure of
human populations, it will be necessary to obtain data on:
(1) quantities of the substance to be released to
the environment a:' a result of production, use,
and disposal;
(2) physical and chemical properties of the sub-
stance;
(.3) the environmental transformation of the sub-
stance;
(4) likelihood of bioaccumulation.
B. Refinement of exposure estimate
In order to refin-e and improve the estimate of exposure,
it will be necessary to obtain a great deal of more specific
data for each fuel or fuel additive; specifically:
(1) physical and chemical properties, biological
activities of the products in all forms - i.e.,
active ingredients, commercial formulation,
' formulation in fuel;
(2) physical and chemical properties and biological
activities of products after combustion, both
in the presence and absence of irradiation by
light of wave lengths reaching the atmosphere;
(3) mechanisms and rate of appearance of original
or altered product in combustion products,
specifically: a) how much of substance comes
through engine unchanged (survival index),
b) mechanisms and rates of formation of combustion
products (plus or minus irradiation) - i.e.,
whether the rates of product appearance are
determined by chemical kinetics or by physical
conditions of the engine combustion process;
(4) quantitative estimation of concentrations likely
tc be delivered via all possible routes of human
exposure including: a) inhalation, the most
likely route of exposure for vapors of unburned
-------
11
material or material which goes through the
combustion process unchanged, or combustion
products; b) skin absorption and eye contact
due to consumer product use, accidental spillage,
or waste disposal; c) ingestion due to environ-
mental contamination. .
-------
I. 1
'
I. TEST MATERIALS
Since human exposure to fuels and fuel additives may occur
from both the initial chemical forms and the combustion products,
and since both states of matter may or may not be subjected to
irradiation in the environment, the potential health effects
of all forms of these substances must be considered for their
most likely routes of exposure. Therefore, at the discretion
of the Administrator, testing for specific health effects of any
or all of the forms of registered fuels, fuel "additives, or fuel/
fuel additive combinations described below may be required.
A. Uncombustcd State
1. Fuels
"Fuel" means any material which is capable of releasing
energy or power by combustion or other chemical or physical
reaction.
a. Motor Vehicle Gasoline
The types of gasoline being designated are: unleaded;
leaded, premium; and. leaded, nonpremium.
.b. Motor Vehicle Diesel Fuel
The designated grades of motor vehicle diesel fuels are:
grade 1-D and grade 2-D.
c. Motor Oil
Motor vehicle engine oils employed to lubricate cylinder
walls and intake and exhaust valves of both standard piston and
rotary engines are also defined as fuels in that a portion of
the oil is in fact combusted in the normal operation of the
engine.
2. Additives
"Additive" means any substance, other than one composed
solely of carbon and/or hydrogen, that is intentionally added to
a fuel named in the designation (including any added to a motor
vehicle's fuel system) and that is not removed prior to sale or
use.
\
\
-------
I. 2
a. Individual Ingredients
Individual ingredients are those separate chemical substances
incorporated into the additive product of commerce:
(1) purified - individual ingredients subjected
to chemical purification;
(2) technical grade - individual ingredient
chemical substance resulting from chemical
manufacturing processes without subsequent
purification.
b. Commercial Formulation
The additive product of commerce including all principal
and minor ingredients sucii as those listed in (1) and (2) above.
3. Combinations
a. F.Lv-1 Plus Single Addiuive (or Additive Package)
b. Single Fuel Plus Multiple-Additive Mixture
c. Multiple-Additive Mixture
B. Combustion Products
There appears to be-a consensus that the problems of fuel
;md fuel additive toxicology must include consideration of whole
exhaust effluent (p. 8, references (1) and (3)). This approach
would compare the toxicity of emissions from a reference fuel with
thoso from the same fuel plus additives (included at the concen-
tration prior to combustion specified by the manufacturer) or
with a test fuel. It is anticipated that the engine design, emis-
"sions control accessories,'Fuel composition range, engine operating
conditions, conditions of irradiation, and other pertinent var-
iables would be specified by the Administrator for each additive
in accord with current and future ambient Air Quality Standards.
The whole exhaust effluent, irradiated or non-irradiated,
represents the most relevant test material for study of effects of
alterations in fuel composition upon health effects associated with
motor vehicle emissions. However, for many of the accepted screen-
ing tests for biological activity, it will not be practical to
employ whole exhaust, but components, i.e., particulatcs and isolated
fractions can be tested. To some extent, the actual form of test
material employed may be dictated by requirements and limitations
of the test selected. .Therefore, to attempt to correlate biological
effect with additive presence, it will be necessary to determine
the fate of the additive upon combustion; specifically, with what
physical phase(s) or processed fraction(s) of the emissions is
-------
I. 3
either the unrcactcd additive, its combustion products or both
associated (Bradow and Sigsby, 1976)?
1. Whole Exhaust
The utilization of whole exhaust as a test material is
suitable for whole animal exposures if allowance is made for
cooling of the hot emissions and for diluting with air so that
non-toxic levels of carbon monoxide are obtained. Systems for
the generation and control of whole exhaust suitable for toxi-
cologic studies have been described.
Facilities for the generation of exhaust emissions repre-
sentative of that produced under actual motor vehicle traffic
conditions, and for the monitoring, irradiation, tempei ure
control, chemical and physical characterization, and delivery
of these emissions to biological test systems require a consider-
able investment of funds. Such facilities are of limited avail-
ability for health effects tes-ting.
The irradiation of whole exhaust significantly alters the
concentrations and character of the chemical species present
and causes a marked increase in irritant effects. For those
experiments involving irradiated exhaust, it is important that
the exhaust effluent be exposed to light containing wave lengths
from 280 nanometers to 700 nanometers, immediately upon exit -
i.e., simultaneously with or prior to dilution.
2. Characterized Emission Products
The individual components of exhaust emissions may be
described on the basis of cither their physical properties or
chemical properties. For those test situations in which whole,
freshly generated exhaust cannot be employed, it may be advisable
'to' tcst'all collectible or'isolatable fractions^ A given addi-'
tive could alter the emission from a specific fuel in a variety
of ways. For example, (1) additive combustion products may be
associated both-with particulate and/or vapor phase; (2) unburned
additives may be adsorbed on particulates; (3) the presence of
additives may alter the size cf particulates formed; and (4) the
presence of additives may alter the chemical nature of species in
cither, or both, vapor and particulate phase.
If the emissions generated from fuel containing a specific
additive are determined upon chemical analysis to be the same as
obtained from the same fuel in the absence of the additive (i.e.,
unchanged by the additive), no further testing is necessary. If
the additive or its combustion products are definitively determined
to exist solely in cither the vapor phase or the particulate phase,
that phase with which the additive is associated could be used as
the biological test material. It may be deemed appropriate by the
Administrator to screen for certain health effects on the basis
-------
I. 4
of either the physical properties - i.e., vapor phase versus
particulatc phase - or the chemical properties - for example,
oxygenated hydrocarbons, polycyclic hydrocarbons, acidic
exti-acts, etc.
a. Physically Isolated Phases
(1) Vapor Phase
Exhaust gas contains low molecular weight hydrocarbons,
some of which are present in the original fuel and others that :
are not. Specific hydrocarbons which are known to occur in emis-
sions include methane-, ethane, ethylene, acetylene, propylene, .
four-carbon (C^) .olefins, and sometimes propadiene and methyl
acetylene. These low molecular weight hydrocarbons may constitute
40-60% of the volume of the vapor phase. In addition, fuel com-
ponents include hydrocarbons heavier than butane and may number
over 100 different hydrocarbons. The hot exhaust also contains
vaporized aromatic hydrocarbons such as ethylbenzene, styrene,
benzene, benzaldehyde, and toluene. In addition, oxygenated
organic compounds such as aldehydes, ketones, alcohols, ethers,
esters, acids, and phenols are found in exhaust vapor. The total
oxygenate concentration is about one-tenth the total hydrocarbon
concentration. There are also epoxide and peroxide compounds,
which have apparently not been well characterized in exhaust, but
which might be particularly significant in terms of health effects.
In addition to the organic compounds, the vapor phase of emissions
may also contain oxides of carbon, sulfur, and nitrogen; oxhcr
sulfur compounds; other nitrogen compounds; and oxidant species.
Reference material dealing with the composition, analysis,
and sampling of motor vehicle vapor phase emissions are provided
in the references at the end of this section.
(2) Particulate Phase
Motor vehicle emissions contain particulates which-1r"esult
from both primary emissions and secondary reactions. In general
the primary particulates are below 0.08 microns, a size range
described as "Transient Nuclei Range" (or Aitken range). Secondary
particulates formed from the condensation of emission products in
the atmosphere range from 0.08 microns up to approximately'1 to 2
microns, a range known as the "Accumulation Range." The range
from approximately 1 to 2 microns, with a maximum of 6 microns, is
k/iown as the "Mechanical Aerosol Range" and includes particles
produced by mechanical processes. The polycyclic aromatic hydro-
carbons of emissions are primary particulates as are a variety of
metal particulates. A number of secondary particulates result
from gas-to-pavticle conversion processes occurring--*!! emissions-
containing atmospheres (Finlayson-Pitts and Pitts, 1976).
-------
I. 5
The biological effects 01 participates obtained .from both
irradiated and nonirradiatcd whole exhaust should be considered.
b. Chemically Identifiable Fraction
Por the purposes of these Guidelines, a chemically identi-
fiable fraction of whole test exhaust (irradiated and nonirradiated)
may be defined as:
(1) a single chemical compound established to be
a combustion product of experimental emissions
(reference fuel plus additive, reference fuel
plus test fuel, etc.) which is absent in
control emissions (reference fuel only);
(2) a physically or chemically derived fraction
or extract of both experimental emissions
and control emissions.
References describing the preparation, of such chemically
identifiable fractions are included in those listed at the end
of this section..
3. Additive Pyrolysis Products
In certain cases, additive pyrolysis products may be a
si'.itnblo preliminary test material. These should be generated
in an air atmosphere combustion chamber of a high-v-oumn system
with provision for immediate trapping - either cryogenic or
absorption (Axelrod and Lodge, 1976) - or filtration and trapping.
-------
- 1.6
I. B. References for Test Materials - Combustion Products
1. Whole Exhaust
Bradow, R. L. and Sigsby, J. E. , Jr. '(1976). Protocol to
assess the effect of fuel additives on control system performance.
ESRL, OALKU, ORD, EPA, RTP, N. C.
Hinners, R. (1962). Engineering the chronic exposure of animals
to laboratory produced automobile exhaust. J. Air.Pollut. Control
Assoc., 12: 527-530.
Burkhart, J. K., Hinners, R. G. and Malanchuk, M. (1973).
Catalytic converter exhaust emissions. Paper no. 74-129, 25 pp.
Air Pollution Control Assoc., Pittsburgh, Pennsylvania.
Rose, A. H., Jr., and Brandt, C. S. (1960). Environmental
irradiation test facility, J. Air Pollut. Control Assoc.,
10: 331-335.
Hurn, R., Allsup, J. and Cox, F. (1976). Effects of gasoline
additives on gaseous emissions (Part II). EPA-600/2-76-026,
65 pp. NTIS, Springfield, Virginia.
Hurn, R. W., Allsup, J. R., and Cox, F. (1974). Effect of
^3.SG.i. inG 3Ci>u i cj-VCS GTi (j£cl3CGu3 Cmio 3iCn^>. tlA ~ OjU/i. .'** UI'4»
NTIS, Springfield, Virginia.
Dietzmann, 11. (1975). Protocol to characterize gaseous emissions
as a function of fuel additive composition. EPA/600/2-75-048.
(PB-253 363/6GA). NTIS, Springfield, Virginia, 132 pp.
-------
I. 7
I. B. References for Test Materials - Combustion Products
(continued)
2. Characterized Emission Products
Bradow, R. L. and Sigsby, J. E., Jr (1976). Protocol to
assess the effect of fuel additives on control system performance.
ESRL, OALWU, ORD, EPA, RTP, N. C.
National Research Council. Committee on Biologic Effects of
Atmospheric Pollutants. (1972). Participate polycyclic
organic matter, Appendix B: Separation methods for polycyclic
organic matter; Appendix C: Detection, identification, and
quantitation, pp 261-276; 277-303. National Academy of Sciences,
Washington, D. C.
National Research Council. Panel on Vapor-Phase Organic
Pollutants. (1976). Vapor-phase organic pollutants: volatile
hydrocarbons and oxidation products. Appendix A: Collection
and sampling techniques for vapor-phase organic air pollutants;
Appendix B: Analytic techniques for vopor-phase organic air
pollutants, pp 287-304; 505-317. National Academy of Sciences,
Washington, D. C.
Axelrod, II. and Lodge, J., Jr., (1976). Sampling and calibration
of gaseous pollutants. In: Air pollution, vol. 3, 3d ed.,
A. C. Stern, ed., pp 156-15S. New York. Academic.
Guerin, M. R. (19J5J. Discussion: Identification of carcinogens,
tumor promoters, and cocarcinogens in tobacco smoke. (CONF 750633-2).
NTIS, Springfield, Virginia.
Guerin, M. R., Olerich,'G.'and Morton, A. D.' (1974). Routine
gas chromatographic component profiling of cigarette smoke for
the identification of biologically significant constituents.
J. Chromatogr. Sci., 12: 385-391.
Groh, K. (1965). Gas chromatography of cigarette smoke. Part III.
Separation of the overlap region of gas and particulate phase by
capillary columns. J. Gas Chroniatogr. 3: 52-56.
Conklc, J. , Register, J. and Worth, G. (1965). Multi-stage cryogenic
trapping system. Aerosp. Med., 36: 869-874.
Candeli, A., Morozzi, G., Paolacci, A., and Zoccolillo, L. (1975).
Analysis using th.in layer and gas-liquid chromatography of polycyclic
aromatic hydrocarbons in the cxhause products froraa European car
running on fuels containing a range of concentrations of these
hydrocarbons. Atmos. Environ., 9: 845-849.
-------
I. 8
I. B. References for Test Materials - Combustion Products
(continued)
2. Characterized Emission Products (continued)
Panalakis, T. (1976). Determination and identification of
polycyclic aromatic hydrocarbons in smoked and charcoal-broiled
food products by high pressure liquid chromatography and gas chrom-
atography. J. Environ. Sci. Health, Pt. B»ll, 4: 299-315.
Lee, R., Patterson, R., Crider, W., and Wagman, J. (1971).
Concentration and particle size distribution of particulate
emissions in automobile exhaust. Atmos. Environ., 5: 225-237.
Adams, D. F. (1976). Sulfur compounds. In: Air Pollution,
3d ed., vol. 3, A. C. Stern, ed., pp 213-258. New York. Academic.
Katz, M. (1976j. Nitrogen compounds and oxidants. In: Air
Pollution, 3rd ed., vol. 3, A. C. Stern, cd., pp 259-.306. New
York. Academic.
Gentel, J.. Manary, 0. and Valenta, J. (1974). Determination
of effect on particulate exhaust emissions of additives and
impurities in gasoline. EPA/650/2-74/061. (PB-253 910/4GA).
NTIS, Springfield, Virginia.
Giever, P. (1976). Particulate matter sampling and sizing.
In: Air Pollution. 3rd ed., vol. 3. A. C. Stern, ed., pp 43-50.
New York. Academic. '
West, P. (1976). Analysis of inorganic particulates. In:
Air Pollution, 3rd ed., vol. 3, A. C. Stern, ed., pp 51-99.
New York. Academic.
McCrone, W. (1976). Microscopy and pollution analysis. In:
Air Pollution, 3rd. ed. ; vol:'3, A. C". Stern, ed. ,'pp 100-144.
New York. Academic.
Lintcn, R. W., Loh, A., Natusch, D. F. S., Evans, C. A., Jr.,
and Williams, P. (1975). Surface predominance of trace elements
in airborne particles. Science, 191: 852-854.
Novakov, T., Mueller, P. K., Alcocer, A. £., and Otvos, J. W.
(1972). Chemical composition of Pasadena aerosol by particle
size and time of day. III. Chemical states of nitrogen and sulfur
by photoelectron spectroscqpy. J. Colloid Interface Sci.,
39: 225-234.
-------
II. 1
II. RELATIONSHIP OF PHYSICAL AND CHEMICAL PROPERTIES OF
TEST MATERIALS TO PHYSIOLOGICAL DISTRIBUTION AND
BIOLOGICAL ACTIVITY
Exposure via the pulmonary route to a pollutant mixture such
as motor vehicle emissions is influenced markedly by a complex
interplay of variables relating to pollutant-component physical
and chemical properties, respiratory tract physiology, and host
defense mechanisms. Any alteration in properties of an emission
component(s) due to the inclusion of additive, or to the substi-
tution of a test fuel for the reference fuel, could result in
physiologically significant alterations in interactive mechanisms.
Some of the pollutant physical and chemical properties considered
most relevant to actual human inhalation exposures to fuel and
fuel additive test emissions are outlined here.
A. Physical Properties
Relevant physical properties of particulate pollutants are
size, shape, electrical charge, and a function known as aerodynamic
diameter (a parameter which incorporates both particle density and
drag, and can be described as the diameter of a unit density sphere
having the same terminal settling velocity as the particle in
question, whatever its size, shape, and density) (Lippmann, 1977).
Size of the particle serves as a general determinant of site of
deposition within the respiratory tract in that particles greater
than lOp are generally screened out in the nasal passages, while
only particles smaller than 3y reach the deep lung or alveolar
region. Shape of the particle is significant in that fibers of
narrow cross section but long length may reach much-deeper regions .
in the respiratory tract than would otherwise be expected. For
example, asbestos fibers 200u long have been observed in human lung
samples (Lippmann, 1977). Particles with electric charge can
exhibit enhanced respiratory tract deposition resulting from image
charges induced on the surface of air waves by the charged particles.
Aerodynamic diameter is the most appropriate parameter in terms of
particle deposition by impaction and sedimentation processes which
usually account for most of the deposition by mass in the head and
lungs (Lippmann, 1977). Aerodynamic size can be measured directly
for particles using an aerosol spectrometer.
Physical chemical properties affecting respiratory tract
deposition and retention are: hygroscopicity, -solubility in various
lung fluids, partition coefficient, and quantity and nature of
adsorptive surfaces of particulates. Hygroscopic particles may
aggregate or swell within the moist environment of the respiratory
tract producing a significant change in effective particle size.
-------
II. 2
B. Chemical Properties
Highly soluble gases and other materials affect primarily
the upper respiratory tract; examples are ammonia, alkaline dusts
and mists, hydrogen chloride, hydrogen fluoride, and sulfur
dioxide. Compounds of intermediate solubility affect both upper
respiratory tract and pulmonary tissue (such compounds include
ozone, oxygen, nitrogen dioxide, halogens, diethyl or dimethyl
sulfate, and phosphorus chlorides). However, certain .insoluble
materials (i.e., insoluble in aqueous media) such as ethyl ether
and many organic vapors, partition so readily into the lipophilic
tissues of the alveolar regions that they do not accumulate there,
but can accumulate in the upper respiratory tract and bronchi to
proc'.ace irritation (Casarett, 1975).
The solubility of so-called "insoluble" particles in lung
fluic:; is an extremely important factor, in that many organic
polycyclic aromatic hydrocarbons or metallo-organic compounds may
be quite insoluble in water, but be solubilized appreciably by
lipid surfactant materials, mucus secretions, or the serum fluids
of the alveolar regions of the lungs. For example, polycyclic
aromatic hydrocarbons are readily eluted from soot by serur proteins
(Falk ct_ £l_., 1958). It has been demonstrated that the clearance
half-times of many insoluble dusts in tlie lung are proportional
to their solubilities in physiological fluk's (Kilburn, 1977).
C. Chemical Structure
An extensive scientific literature relating variation in
ch°mical structure with variation in biological activity rxists
for the fields of medicinal chemistry, pharmacology, and physical
chemistry. A number of physicochcmical parameters such as partition
coefficient, the Hammett constant, a charge transfer constant, and
chromatographic hydrophobic parameters have,.been employed to compare ,
biological activity of structurally analogous or similar compounds.
In addition, such parameters can be employed to predict skin
permeability, cellular uptake, bioconccntration and similar effects
based on the partition coefficient (octanol-watcr system) or
specific lipid solubility. Many of the potential test substances
considered herein contain chemical groupings or structures amen-
able to some degree of predictive analysis by these methods with
respect to biological retention, distribution, and activity. For
those test substances for which chemical structure or chemical
class is known to the manufacturer, the scientific literature may
provide information on toxicity of the test substance or structurally
similar compounds.
The most important parameters related to chemical structure
and chemical properties for all pollutants deriving from fue.ls and
fuel additives are considered to be:
(1) partition coefficient in octanol-watcr (Ilansch and Elkins,
1971);
-------
II. 3
(2) aqueous solubility (Hague and Schmedding, 1975),
preferably at 37°, and pH of aqueous solution;
(3) chemical structure (if it is not possible to determine
chemical structure, then minimal chemical characteri-
zation should include as complete a description as
possible, e.g., aromatic or polycyclic hydrocarbon
compounds containing carbonyl oxygen, or cyclic nitro-
gen-containing ring structures); and
(4) lipid solubility (preferably at 37°).
The chemical parameters listed above apply to all physical
states of test materials - i.e., liquid, vapor, and solid. Addi-
tionally, if the test material is particulate in nature, it will
be most important also to characterize it as to certain physical
properties, specifically:
(S) size and shape;
(6) aerodynamic diameter (Lippmann, 1977); and
(7) hygroscopicity.
It would also be extremely valuable to determine the nature
of substances adsorbed on the surfaces of particulates as this is
a mechanism for concentrating and presenting to the respiratory
system potentially toxic chemical species formed or volatilized
during combustion (Linton et al_., 1976; Novakov et_ al_., 1972).
-------
III. 1
III. WEIGHTING FACTORS INVOLVF.D IN DETERMINING AN APPROACH
TO APPROPRIATE HEALTH EFFECTS TESTING
The dimensions and complexity of the task of evaluating the
impact of candidate fuels and fuel additives upon human health
dictate a need for flexibility of approach and a steady flow of
informed, knowledgeable decision making. The most important
decisions in the entire scheme of evaluation will be the choice
of which fuels and fuel additives to test. Guidance for such
decisions must come from as detailed an estimation of human
exposure conditions as possible. For a given fuel or fuel addi-
tive, the factors outlined here should be taken into consideration
in the selection of both test material and test approach. ' '
A. Amount of Raw Material Produced
The quantities of material involved in production, use, and
disposal constitute the limits of potential human exposure. These
data may be obtained from chemical industry bulk production volumes
and volume by use classifications.
B, Amount of Potentially TOY-JC Material Emitted
Data obtained according to the Emissions Characterization
Protocol (Bradow and Sigsby, 1976) and from tests performed by EPA
and other laboratories should be assessed in making estimates of
the amount of potentially toxic material which may be introduced
into the environment.
The amount of total toxic material "emitted is an"important - '
first consideration. However, it must be taken into account" that
if fuel additives do indeed survive the combustion process, they
could be volatilized and condense on the surfaces of particulates
as they form, resulting in essentially the total bulk volume of
additive being localized and concentrated on the surfaces of
respirable particles. In addition to the increase in effective
environmental concentration, the additives are in an optimal form
for presentation to human respiratory systems.
Taken together these data will indicate the potential for
biological activity and the likely deposition sites and retention
times for both human targets and the environment.
C. Persistence and Chemical Form
The degree of persistence and the chemical form of material
which persists become significant factors in considering human
-------
III. 2
exposure to fuel additives. An ideal approach to evaluating this
factor for a given additive would be to attempt a mass balance in
which a given charge of additive to an engine is followed through
the environment. However, for numerous reasons this is not a
practicable approach. Estimation of the basic processes involved
in environmental transport and assimilation, with subsequent
extrapolation, appears preferable. ..-.
- .
1. Persistence in the Environment and Biological Organisms j
Alterations within the environment may proceed through chemical,. j
photochemical, and microbial processes. j
1
2. Bioaccumulation ;
The potential of a given additive for bioaccumulation can be
estimated from its partition coefficient and its tendency to form
complexes with sulfhydryl and amino groups (NAS-NKC, 1975).
3. Biomagnification 5
i
The potential of a substance for biomagnification would !.
involve measurement of bioconcentration factors for food chain i
organisms. Aquatic organisms are able to concentrate (perhaps
1CP to 10° fold) some lipophilic organic compounds and heavy metals
into their tissues (DHEW-NIEH3, 1977).
j
, 4
4. Biotransformation to More Toxic Forms
11
Transformation of a substance by biological systems (i.e., ;
microorganisms) to a more toxic chemical form should be taken into
consideration. The biotransformation of mercury to the more toxic
methyl mercury in the environment is an example of the importance:
of this factor. - .-,*.-- .,--', *<- J
D. Evaluation of Existing Health Data
A search and evaluation of the scientific literature pertaining
to experimental and epidcmiological data relevant for the actual
exposure agent itself (if known) and for analogous chemical struc-
tures, emissions fractions, etc., should be performed. This search
and evaluation should include both the original test substance
itself as well as its combustion products, also the potential
biological activity of any chemical substituent groups, or structural
moieties. In addition, any information available on environmental
persistence or transformation, both chemical and biological, should
also be considered. This evaluation should include weighting of
the scientific evidence as to quality of data, adequacy of data, and
the kind of response induced.
-------
1
III. 3
E. Risk Assessment Analysis
The objective of the most- thorough and comprehensive of
testing schemes and collection of cpiclemiological information is
the assessment of risk to human populations. If at a ! 1 possible,
input to this assessment process should include both human data
and a range of experimental results. However, for the present
problem of risk assessment attendant to comparison of exposure to
exhaust emissions from a fuel with an additive versus those from
a fuel without a-rv additive, human data may be difficult to obtain. . ]
The potential for cumulative, synergistic, or potentiating
effects resulting from low-dose chronic exposures may be a com-
plicating factor in risk assessment analysis for fuel and fuel
additive health effects. Other complicating factors in this
particular risk assessment analysis involve the superimposition
of any test emissions effects upon: pulmonary disease states like
emphysema; metabolism of po-lycyclic aromatic hydrocarbon compounds
from tobacco smoke; effects of other inhaled pollutants; aging;
and genetic factors (e.g., alpha-1-antitrypsin deficiency, a familial
deficiency of a key protease inhibitor which predisposes affected
individuals to emphysema at an early age). Population groups into
which these factors segregate may be susceptible to effects of any
exhaust emissions, and it may not be possible to evaluate an increase
in susceptibility due to the inclusion of additives.
For the purposes of risk assessment, a toxic response observed
in animals (more than one species) should correlate with a corre-
sponding human health effect, otherwise extrapolation between
species is meaningless. A primary concern for human exposure to
fuel and fuel additive test emissions is induction or enhancement
of pulmonary carcinogenesis or cocarcinogenesis.
Once a background '"of acceptable quality animal response data ' r '"
for a given additive is obtained, extrapolation for purposes of
estimating low effect levels for humans and thereby defining expo-
sure limits can be undertaken. There are several mathematical
models employed for this type of extrapolation; these models are of
two basic types:
(1) dichotomous response models and
(2) "time-to-occurrence" as a function
of dose level models.
Dichotomous models include the "one-hit" model and the Mantel- ' / <
Bryan probit model; while examples of time-to-occurrence models are j
the lognormal and the Weibull (Peto et al. , 1972). Factors involved ]
in the applications of these various models for risk estimation |
have been considered in depth, and recommendations made as to interim , !
procedures for setting levels of qualitatively unavoidable chemicals-
in the environment by the Subcommittee on Estimation of Risks of
Irreversible, Delayed Toxicity to the DIll-W Committee to Coordinate
-------
III. 4
Toxicology and Related Programs (Iloel ^t al_., 1975). Additional
background discussion is provided by "Biological and Statistical
Considerations in the Assessment of Risk", Chapter V, in reference
NAS-NRC, 1975. A valuable discussion of problems attendant to
extrapolation of toxicity data from animals to man has also been
presented recently (Dixon, 1976).
i .
-------
""SI
I
IV. 1 i
IV. ROUTE AND MODE OF EXPOSURE
For now motor vehicle fuels and fuel additives, the major
route of human exposure will be by inhalation of air containing
emission products. These emission products may or may not have been
irradiated, depending upon time of day and weather conditions; they
will also be subject to a number of other environmental variables.
All of these factors will have a direct bearing on the nature and
range of health effects which could result from inhalation of
emission containing atmospheres, and from exposure of eye and skin.
There are other routes of human exposure which also should be
considered although they are anticipated t.o be infrequent and of
low magnitude compared to the inhalation of emissions. For example,
for unburned fuels, additive ingredients, additive formulations,
and fuels containing additives, human exposure may take place
through: (1) inhalation of vapors, (2) splashing onto skin and
into eyes, or (3) ingestion due to environmental contamination.
Photochemical reactions may or may not occur for all of these expo-
sure conditions. ' .
The considerations outlined in this section apply principally
to whole animal toxicity testing; however, in vitro systems most
relevant to each route of human exposure are indicated.
A. Pulmonary
1. Inhalation
For whole animal testing of fuel and fuel additive health
effects, the importance of exposure by the pulmonary route and
specifically, the inhalation mode, cannot be overemphasized.
Inhalation toxicology is considerably more complex than oral
toxicology, and it is essential that investigators demonstrate
knowledge of and capability in the application of inhalation toxi-
cology. More so than for any other route of exposure, the chemical
and physical properties of the specific individual test agent will
determine: the design of the system for generating and metering
the test atmosphere; design of the exposure apparatus; dose esti-
mation and calculation; exposure schedule; and the nature of the
biological effects produced. The interrelationships of the properties
of the toxicant with the nature of biological response are outlined
in Section V. The significance of the properties of the toxicant
with respect to exposure methodology and inhalation toxicologic
assessment is discussed in a number of references from the scientific
literature presented at the end of this section.
-------
1
IV. 2
Assessment of the health effects of fuel and fuel additives
emissions will necessitate inhalation exposure to atmospheres
containing participates. Considerations which are basic to the
performance of such tests include: behavior of aerosols; aerosol.
characterization; pulmonary deposition; particle clearance by the
lungs; and pulmonary responses. Aerosol behavior is determined
by factors such as particle size, particle size distribution,
sedimentation rates, coagulation., and diffusion. For aerosol
characterization, the most commonly measured parameters are particle
size and mass. As a general rule, the toxicity of inhaled materials
is most considerably related to the mass of material inhaled and
the number of particles inhaled, measured as the number of particles
per unit volume of air. Particle number is also occasionally
directly related to the effect observed, as is particle surface
area; however, the distribution of the mass of material is more
frequently relatable to toxic effect. In terms of methodology,
size or mass frequency distributions are tho most complete, repro-
ducible, and acceptable means 'of measurement and expression of
particle size. A knowledge of particle clearance pathways and
rates is necessary for selecting the proper exposure schedule and
duration.
Pulmonary deposition of particles, particle clearance, and
pulmonary responses are dealt with in Section II.
In addition to a wide variety of specific lung effects, t!ie
administration of a toxicant by inhalation could result in as many
systemic effects as toxicants given by the oral route. The systemic
effects may differ significantly between the two routes because of
different metabolites formed by the lung and by the liver, or the
magnitude of the effects may differ for a number ot reasons such
as different clearance mechanisms.
' "' Test'materials appropriate for administration to whole animals
via the inhalation mode include: whole exhaust (either fre^h or
aged, and with or without irradiation); volatile exhaust fractions;
or aerosolized fractions of whole exhaust or chemically-characterized
fractions.
2. Intratracheal Instillation
In some cases, intratracheal instillation may be the most .
logical mode of exposure for the pulmonary route. For example,
in cases where the amount of. available test material is very
limited, or is known to be very hazardous (such as contain a
known carcinogen), this would probably be the exposure mode of
choice. Other applications might include cocarcinogenesis testing
where a defined concentration of a known carcinogen is to be
applied along with emissions test material. Papers describing the
application of this technique are included in the references for
this section.
-------
IV. 3
B. Oral
Although the oral route is likely to be only a minor route
of human exposure, its possibility of occurrence dictates its
inclusion in these guidelines. This route has been employed for
the bulk of systemic toxicological studies for many years.
For administering test materials via the oral route two modes
may be employed: (1) incorporation into food or water; and (2)
gavage (stomach tube). Although (1) may be easier and simpler in
many cases, gavage offers closer control of dose of exposure agent,
less wastage of test materials, and may be the preferred mode of
oral exposure in many experimental situations.
C. Eye and Skin
For motor vehicle emissions, eye and skin contact are impor-
tant routes of human exposure. The health effects of exposure via
the eye and skin routes are generally considered to be primarily
transient irritation; however, the eye possesses a potentially
high sensitivity to toxic substances and should receive careful
attention as a potential target organ for fuels and fuel additives
both before and after combustion. For example, a splash of unbumed
test material or simply exposure to vapors of test material may
result in dulling of the cornea and loss of epithelial cells -
symptoms of exposure to organic solvents.
-------
IV. 4
IV. References for Route and Mode cf Exposure
Carpenter, C. P., Kinkead, E. R., Geary> D. L., Jr., Sullivan,
L. J., and King, J. M. (1975). Petroleum hydrocarbon toxicity
studies. I. Methodology. Toxicol. Appl. Pharraacol. 32: 246-262.
Pavia, D., Thomson, M. and Shannon, II. S. (1977). Aerosol
inhalation anc4 -iepth of deposition in the human lung. Arch.
Environ. Health, 32: 131-137.
Chamberlain, A. C., Clough, W. S., Heard, M. J., Newton, D.,
Stott, A. N. B. and Wells, A. C. (1975). Uptake of inhaled
lead from motor exhaust. Postgrad. Med. J., 51: 790-794.
Dennis, R (1976). Handbook on aerosols. (TID-26608). NTIS,
Springfield, Virginia. 148 pp. !.
i
Mercer, T. T. (1967). On the role of particle size in the j
dissolution of lung burdens. Health Phys. 13: 1211-1221. j
Mercer, T. T. (1973). Aerosol technology in hazard evaluation.
394 pp. New York. Academic.
Phalcn, R. F. (1976). Inhalation exposure of animals. Environ. !
Health Perspect., 16: 17-24.
Tillcry, M. I., Wood, G. 0. and Ettinger, H. J. (1976). Generation j
and characterization of aerosols and vapors for inhalation experiments. <
Environ. Health Perspect. 16: 25-40. i
Fraser, D. A. (1959). Exposure chambers for research in animal
inhalation: design, construction, operation and performance.
Public Health Monogr. no.- 57, U. S. Govt. Print. Off., Washington,
D. C. ' " - - '- - ' - - . ' -
Schcuplein, R. J. (1977). Permeability of the skin. In: Handbook
of physiology, sec. 9, Reactions to environ, agents, D. H. K. Lee,
cd., pp299-322. Bethcsda, Maryland. American Physiological Society.
-------
V. HEALTH RELATED TESTS
A. Toxicity
1. General
a. Oral
(1) LDjQ Determination
(2) Blood/Urine Analysis
(.3) Gross and Histopathology
b. Dermal
c. Eye
d. J_n Vitro Toxicity Tests
(1) Tissue Slices
(2) Primary Cells
(3) Cell Lines
2. Pulmonary
a. kCgQ Determination
b. Host Defense Mechanisms
(1) Infectivity Model System
(2) Mucociliary Function . ,
Ciliary Activity
Mucociliary Transport
Mucus Production and Characteristics
(3) Free Lung Cell Populations
(4) Alveolar Macrophage Function
jjQ. Vivo Exposure
J_n Vitro Exposure
(5) Immune Function
-------
c. Pulmonary Function
d. Cross and Hiscopathology
e. Blood/Urine Analysis
3. Central Nervous System/Behavior
-------
V. 1
V. HEALTH RELATED 'TESTS
A . Toxicity
1 . General
a. Oral
(1) LD$Q Determination
Acute toxicity tests are designed to determine the harmful
effects of an agent resulting from exposure to a single dose.
Selected effects (e.g., altered physiological, anatomical, or
behavioral parameters, or death) are monitor d for a specified
period of time. The most commonly used criterion for such a test
is the LL)50 (the dose which results in death of half of the exposed
animals) with its associated confidence limits. In order to most
efficiently determine this value, without the requirement to use
excessively large numbers of experimental animal groups, an initial
range-finding study is performed. Small groups of animals, usually
two per group, are administered the test agent, covering a wide
concentration range. It is generally recommended that the dosage
steps increase by a factor of 2 anJ uovt-r a sufficiently wide rtnige
so that a no-effect as well as a consistently lethal dosage can be
determined (Loomis, 1974).
For determination of acute toxicity by the oral ingestion
route or by skin penetration, standard procedures used for general
toxicological testing should be appropriate.
The Lpso test determines the dosage of test material which
results in death for 50 percent of the exposed animals in a specified
time period. Test animals, usually rats, are exposed to graded
dosages 'of the test material, the range of which has been previously
determined in a range-finding experiment. Dosage intervals are
generally chosen for mathematical or statistical convenience, i.e.,
increasing the dosage by a factor of 1.26 or the equivalent of 0.1
log interval, from which it is then simple to compute the LD^Q from
published formulas and tables.
The number of animals to be used at each dosage level should
be considered in terms of the type of information desired from the
test, i.e., statistical validity and slope and shape of the dose-
response line. Generally a minimum of 10 animals should be assigned
to each group, either of the same sex or an equal number of each
sex (EPA, Pesticides Guidelines, 1977).
-------
V. 2
In order to obtain the maximum amount of information from
the acute toxicity test which will be of value in designing
subsequent sub-acute testing, extensive evaluation of all harmful
effects should be performed. In addition to the obvious criterion
of lethality, monitoring of a variety of behavioral and physiological
symptoms such as described by Loomis (1974) would be appropriate.
The choice of animal species is generally based 0:1 previous
work in the area; thus, the most commonly used are rats and mice.
However, species variability in response should be considered
on the basis of known toxicity in various species of similar or
related compounds to the one being tested, and the choice of test
animal be made accordingly. Similarly, the length of the observation
period should be chosen based on best-available information on the
time response of similar or related materials. A 24-hour obseiva-
tion period is sometimes sufficient; .more commonly, 14 days is
employed. In any event, details of experimental materials and
methods should be clearly specified in the reporting of results.
-------
V. 3
V. A. 1. a. References for Oral
(1) ED Determination
*5 U
Committee on Toxicology, Arnold J. Lehman, Chairman. (1964)
Principals and procedures for evaluating the toxicity of
household substances. NAS-NRC Publ. No. 1158, Washington,
D. C.
EPA. (1977) Pesticides Guidelines Draft.
Litchfield, J. T., Jr., and Wilcoxon, F. (1949) A simplified
method of evaluating dose-effect experiments. J. Pharmacol.
Exp. Ther. 96:99-113.
Loomis, T. A. (1974) Essentials of toxicology, 2nd ed. Lea
and Febiger, Philadelphia, 223 pp.
Weil, C. S. (1952) Tables for convenient calculation of median-
effective dose (ED,., or ED_ .) and instructions in their use.
Biometrics 8:249-2&j.
Weil, C. S. (1972) Guidelines for experiments to predict the
degree of safety of a material for man. Toxicoi. Appl.
Pharmacol. 21:194-199.
Weil, C. S. and Wright,.G. J. (1976) Intra- and interlaboratory
comparative evaluation of a single oral test. Toxicoi. Appl.
Pharmacol. 11:378-588.
-------
V. 4
(2) Blood/Urine Analysis
For all exposure modes in which systemic toxic effects may
be expected, i.e., dermal, oral, and inhalation, alterations in
metabolism or physiology may occur. Such metabolic or physiological
changes, as well as direct cellular injury, may result in variations
from normal patterns of the blood and urine constituents. Con-
sequently, hematology, blood chemistry, and urinalysis are basic parts
of routine toxicological testing. Testing should include but not
be limited to the following:
Hematology - hematocrit, hemoglobin, erythrocyte count,
total and differential, leukocyte counts,
and reticulocyte count.
Blood Chemistry - calcium, sodium, potassium, chloride,
total serum protein, scrum protein electro-
phoresis, serum bilirubin, blood urea
nitrogen, fasting blood sugar, and serum
enzymes.
Urinalysis - pH, specific gravity, total protein, glucose,
ketones, bilirubin, and r.icroscopic exami-
nation of sediment.
-------
V. 5
V. A. 1. a. (cont.)
(2) Blood/Urine Analysis
Committee on Toxicology, Arnold J. Lehman, Chairman. (1964)
Principles and procedures for evaluating the toxicity of
household substances. NAS-NRC Publ. No. 1158, Washington,
"D. C.
EPA. (1977) Pesticides Guidelines Draft.
Loomis, T. A. (1974) Essentials of toxicology, 2nd ed. Lea
and Febiger, Philadelphia, 223 pp.
-------
V. 6
(3) Gross and Histopathology
Gross necropsy should bo performed on all animals which die
during the course of toxicology testing by all administration
routes;'and on all animals at the termination of testing. Gross
necropsy reveals evidence of overt injury or disease processes in
organ systems, and allows sampling of suspect tissues for subsequent
histopathologic examination. In all cases, a complete dissection
and necropsy should be performed, and should include examination
of the external body surface, orifices, and all thoracic and abdomi-
nal viscera.
Suspect tissues observed during the gross necropsy should be
prepared for histopathologic examination, following suitable
fixation and staining. Additionally, hlstopathologic examination \
should form a part of the overall toxicologic profile for all i
subacute and chronic testing routines, regardless of route of j
administration. Minimal examination should include: all gross
lesions, brain, spinal cord, eye, pituitary, salivary gland, heart,
thymus, thyroids, parathyroid, lungs, trachea, esophagus, stomach,
small and large intestine, adrenals, pancreas, liver, gall bladder,
kidneys, urinary bladder, aorta, testes, prostate, ovaries, corpus
and cervix uteri, spleen, at'least 2 lymph nodes, bone and marrow,
skeletal muscle, skin, sciatic nerve, and mammary gland. Additionally,
for inhalation route of exposure, the examination should include
particular attention to tissues of the respiratory tract, including
the trachea, bronchi, and paranasal sinuses.
Variations in requirements for histopathologic examination of
animals in different dosage groups are detailed elsewhere (Pesticides
Guidelines Draft, 1977) and appear to also be appropriate for
toxicity testing of fuels and additives. A valuable guide to the
methodology for pathological assessment of pulmonary toxicity is also
found in the 1976 review paper by Dungworth et al.
-------
V. 7
V. A. 1. a. (cont.)
(3) Gross and Histopathology
Uungworth, L). L., Schwartz, L. W., Tyler, W. S., and Phalen,
R. F. (1976) Morphological methods for evaluation of
pulmonary toxicity in animals. Annu. Rev. Pharmacol.
Toxicol. 16:381-589.
EPA.. (1977) Pesticides Guidelines Draft.
Loomis, T. A. (1974) Essentials of toxicology, 2nd ed. Lea
and Febiger, Philadelphia, 223 pp.
-------
1
V. 8
V. A. 1. b. Dermal
Assays for dermal toxicity must take into consideration
several aspects of possible tissue injury resulting from the
presence of the test material. These include corrosive action
or gross destruction of dermal tissue; primary irritation or a
local nonimmunological inflammatory response which is reversible
and does not lead to tissue destruction; and contact sensitization,
an inununologically mediated reaction resulting generally from
repeated exposure and manifested by inflammation, edema, and often
formation of blisters at the site of contact. Acute dermal toxicity
results from penetration of the skin by the test material, with
systemic spread of the material by the circulation resulting in
toxicity simi-lar to that caused by other routes of entry such as
oral or respiratory.
; Assays for primary irritation or corrosion generally use the
albino, rabbit as the test animal, although other animals, in par-
ticular the guinea pig, could be used. An albino animal is preferred
in order to aid visualization of responses. Comparison of the
rabbit and guinea piy (Roudabush et^ al_., 1965; Nixon et^ al_., 1975)
indicates that the latter can reliably serve as a substitute for
the rabbit, but there are indications that neither is a consistently
reliable indicator of human responses. This is particularly true
with respect to comparisons made using abraded skin and it has been
suggested (Nixon £t_ ajL_., 1975) that the abraded skin test be omitted,
since interpretation of results are made mach more difficult when
the test materials are applied to abraded :skin.
The use of a 4-hour test period rather than the 24-hour period
as originally described by Draize (1944) has been suggested as a
more pertinent indicator of actual human exposure to many poten-
tially hazardous materials. Critical evaluation of the 4-hour
test for skin penetration toxicity (Weil £t al^., 1971) indicated
that the shorter testing period correlated well with the longer
period, and that one can be reliably converted to the other using
mathematical computations.
Despite numerous examples in the literature, the use of animal
models to reliably predict human contact sensitivity to various
chemicals is still an inexact.science. This is the case largely
because of subtle differences in the immune response systems of
various animals, including man, reflected in different responses
of various species to a similar antigenic challenge. The usual
animal species used for determination of potential of a material
for contact sensitization is the albino guinea pig, since this
species develops excellent delayed type hypersensitivity responses
to many antigens. Confirmation of the skin sensitizing potential
of materials shown to be positive in the guinea pig is generally
made in. human volunteers. However, it should be appreciated that
sensitization of volunteers may have long-lasting effects, with the
result that subsequent contact with the sensitizer years later may
produce adverse symptoms.
-------
V. 9
Methods for the study of acute dermal toxicity, i.e.,
systemic toxicity arising from penetration through the skin by
materials applied topically, follow the general procedures for
acute toxicity testing by other routes. That is, initial range-
finding studies should be conducted in rats or mice, increasing
the dosage by cither geometric or logarithmic progression. Methods
for application of test material to the skin are described by
Draize et^ a_l_. (1944), and should be considered in the context of
the potential conditions of human exposure to the materials. That
is, duration of exposure to the material, potential for normal
evaporation from the skin, covering the test site with a semiper-
meable or impermeable dressing, etc., must be decided on the basis
of projected contact with the materials by consumers.
-------
V. 10
V. A. 1. b. References for Dermal
Brown, V. K. H. (1971) A comparison of predictive irritation
tests with surfactants on human and animal skin. J. Soc.
Cosmet. Chem. 22:411-4^0.
Committee on Toxicology, Arnold J. Lehman, Chairman. (1964)
Principles and procedures for evaluating the toxicity of
household substances. NAS-NRC Publ. No. 1138, Washington,
D. C.
Davies, R. F.., Harper, K. H., and Kynoch, S. R. (1972) Inter-
species variation in dermal reactivity. J. Soc. Cosmet.
Chem. 23:371-381.
Draize, J. H. (1965) Appraisal of the safety of chemicals in
foods, drugs, and cosmetics-dermal toxicity. Assoc. of
Food and Drug Officials of the U.S., Topeka, Kansas.
Draize, J. II., Woodard, G., and Calvery, H. 0. (1944) Methods
for the study of irritation and toxicity of substances
applied topically to the skin and mucous membranes. .J.
Pharmacol. Exp. Ther. 82:377-390.
Edwards, C. C. (1972) Hazardous substances. Proposed revision
of test for primary skin irritants. Federal Register 37:23,
635-23, 636.
Maibach, H. (1976) Cutaneous pharmacology and toxicology.
Rev. Pharmacol. Toxicol. 16:401-411.
Annu.
Nakaue, H. S. and Buhler, D. R. (1976) Percutaneous absorption
of hexachlorophene in the rat. Toxicol. Appl. Pharmacol.
, - -35:381-391. ..,--, r . -- . - , - <---
Nixon, G. A., Tyson, C. A., and Wertz, V.'. C. (1975) Interspecies
comparisons of skin irritancy. Toxicol. Appl. Pharmacol.
31:481-490.
Phillips, L., II, Steinberg, M., Maibach, H. I., and Akers, W. A.
(1972) A comparison of rabbit and human skin response to
certain irritants. Toxicol. Appl. Pharmacol. 21:369-382.
Roudabush, R. L. , Tcrhaar, C. J., Fassett, D. W., and Dziuba,
S. P. (1965) Comparative acute effects of some chemicals
on the skin of rabbits and guinea pigs. Toxicol. Appl.
Pharmacol. 7:559-565.
Weil, C. S., Condra, N. I., and Carpenter, C. P. (1971) Correla-
tion of four-hour versus twenty-four hour contact skin
penetration toxicity in the rat and rabbit, and use of the
-------
V. 11
V. A. 1. b. (cont.)
former for predictions of relative hazard of pesticide
formulations. Toxicol. Appl. Pharmacol. 18:734-742.
Ivester, R. C. and Maibach, H. I. (1975) Percutaneous absorption
in the rhesus monkey compared to man. Toxicol. Appl.
Pharmacol. 32:394-398.
-------
V. 12
V. A. 1. c. Eye
This is a measure of the potential of a chemical to induce
corrosive or irritative changes in a highly sensitive organ (eye)
which may also be an important target organ for human exposure.
The most commonly used procedure is adapted from the method of
Draize ct^ aj_. (1944) and involves the use of rabbits. A measured
quantity, usually 0.1 ml of liquids or 10.0 ing of powered insol-
uble chemicals, is placed in the conjunctival sac of rabbits. The
resulting ocular response is determined by visual observation at
24, 48, and 72 hours. Responses are graded according to a carefully
defined grading system based on damage to the various ocular
structures and duration of injury. A similar evaluation of eye
damage could also be performed following exposure of animals to
aerosolized test materials.
The use of rabbits as test animals lias been the subject of
some criticism, based generally on the premise that the eye of the
rabbit is not sufficiently similar to the human eye to permit
completely reliable extrapolation. A comparative study, using the
rhesus monkey and the rabbit and comparing the results to human
volunteers (Beckley ct_ a^. , 1969), confirmed the utility of the
monkey in predicting human response. However, cost, availability,
and other factors may preclude widespread use of nonhuman primates
for such testing. On the other hand, the use of smaller species
such as guinea pigs and rats may not be appropriate because of
constraints imposed by the small size of the eye. In the rabbit,
the relatively large size of the eye facilitates instillation of
test, materials and subsequent observation for tissue injury.
-------
V. 13
V. A. 1. c. References for Eye
Beckley, J. 11., Russell, T. J., and Rubin, L. F. (1969) Use
of the rhesus monkey for predicting human response to eye
irritants. Toxicol. Appl. Pharmacol. 15:1-9.
Browne, R. K., Anderson, A. N., II, Charvez, B. W., and Azzarello,
R. J. (1975) Ophthalmic response to chlorhexidine
digluconate in rabbics. Toxicol. Appl. Pharmacol. 52:621-627.
Committee on Toxicology, Arnold J. Lehman, Chairman. (1964)
Principles and procedures for evaluating the toxicity of
household substances. NAS-NRC Publ. No. 1138, Washington,
D. C.
i Draize, J. H., Woodard, G., :>".d Calvery, H. 0. (19-44) Methods
,'' ; for the study of irritation and toxicity of substances applied
'' ' topically to the skin and mucous membranes. J. Pharmacol.
Exp. Ther. 82:377-390.
/ Hazardous substances and articles; administration and enforcement
-v regulations. (1973) Test for eye irritants. Federal
; Register 38: p 27019.
Kikkawa, Y. (1972) Normal cornea! staining with fluorescein.
, '.'. Exp. Eye Res. 1.4:33-20,
«""" ** . '
' - McDonald, T. 0., Baldwin, H. A., and Beasley, C. H. (1973) Slit-
lamp examination of experimental animal eyes. I. Techniques
of illumination and the normal animal eye. J. Soc. Cosmet.
Chem. 24:163-180.
Weil, C. S. and Scala, R. A. (1971) Study of intra- and inter-
" " ' '' " " ' " laboratory variability in the "results of rabbit eye and skiri
irritation tests. Toxicol. Appl. Pharmacol. 19:276-360.
.
-------
V. 14
V. A. 1. d. In Vitro Toxicity Tests
In vitro toxicity tests can be performed with a large variety
of freshly isolated and/or continuously proliferating mammalian
cell systems. The types of toxicity tests can be divided into
four general categories, including a) cell growth inhibition tests,
b) cell survival studies, c) biochemical toxicity tests, and
d) functional toxicity tests. Individual assays from each of the
above categories can be applied to any given cell system; the
selection of cell system should be based on its relationship to
species (and metabolic capabilities of the cells of .that species),
tissue of origin (as close as possible to the expected i_n Vitro
route of exposure, e.g., lung cells), and desired endpoint.
Cell growth inhibition tests include effects of continuous
exposure of chemicals on the increase in concentration (cell/ml)
of cells in exponential growth after a defined period of time
(i.e., 48 or 72 hr), or on the accumulation of total cell protein
per container (e.g., flask, dish, or tube) of cells in exponential
growth.
Cell survival studies include measurement of the cloning
efficiency of cells which attach to solid substrates (e.g., glass
or plastic) or measurement of the ability of the cells to form
colonies in soft agar. The cells can be treated prior to the assay
for both of the above, or during the assay for surface attached
reli .lines. Dye exclusion procedures can be employed for nonpro-
liferating cell lines, although consideration must be given to
possible nonlethal membrane effects by the chemical'on'-the measure-
ment .
Biochemical toxicity tests include measurement of DNA precursor
incorporation (DNA synthesis) during a continuous treatment
interval or for a short period of time (rate study) immediately
"after a treatment period; UNA precursor incorporation (UNA synthesis)
for a short period of time (rate study) immediately after a treat-
ment period; or amino acid incorporation into protein for a short
period (rate study) immediately after a treatment period.
Functional toxicity tests are relevant to the specific cell
system selected, e.g., the alveolar macrophage, as described below.
(1) Tissue Slices
Some of the biochemical and functional toxicity tests mentioned
above could be performed on slices of appropriate tissues. An
example is the assay of effects of in vit-ro exposure upon ciliary
beating employing slices of tracheal tissue. (See Pulmonary
Toxicity, V. A. 2. b. (2)).
-------
V. .15
(2) Primary Ceils
Primary cells may be-utilized for in vitro determination of
functional and biochemical toxicity effects. An example is the
jji vitro alveolar mncrophage test system described under Pulmonary
Host Defense Mechanisms (See V. A. 2. b. (4)).
(3) Cell Lines
Continuous mammalian cell lines could be used for all four
categories of vn vitro toxicity tests described above. The cell
lines chosen should include a normal human diploid cell line for
reference purposes, but should also include a cell system appro-
priate to the cndpoint being measured or to previous standardization
experience. Consideration should be given to use of the cell.
lines employed by the in vitro screening program.of the National
Cancer Institute for cell growth inhibition studies and to the use
of the same mammalian cell lines recommended for iji vitro chemical
transformation and mutagenesis for the cell survival studies. These
same cell systems can of course be employed for biochemical toxicity
tests, provided they contain the appropriate enzymes necessary for
metabolic activation of potentially hazardous chemicals.
-------
V. 16
V. A. 1. d. References for In Vitro Toxicity Tests
(1) Tissue Slices
Barren, E. X., Miller, Z. B., and Bartlett, G. R. (1947) Studies
on biological oxidations. XXI. The metabolism of lung as
determined by a study of slices and ground tissue. J. Biol.
Chem. 171:791-800.
Levy, S. E. and Harvey, C. (1974) The effect of tissue slicing
on rat lung metabolism. J. Appl. Physiol. 37:239-240.
Lock, E. A., Smith, L. L. , and Rose, M. S. (1976) Inhibition of
paraquat accumulation in rat lung slices by a component of
rat plasma and a variety of drugs and endogenous ajnines.
Biochem. Pharmacol. 25:1769-1772.
O'Neill, J. J. and Tierney, D. F. (1974) Rat lung metabolism:
glucose utilization by isolated perfused lungs and tissue
slices. Am. J. Physiol. 226:867-873.
Rose, M. S., Smith, L. L., and Wyatt, I. (1976) The relevance of
pentose phosphate pathway stimulation in ra.t lung to the
mechanism of paraquat toxicity. Biochem. Pharmacol 25:1765-1767.
Sayeed, M. M., Senior, R. M., Chaudry, I. M., and Baue, A. E. (1975)
Characteristics of sodium and potassium transport in the lung.
Am. J. Physiol. 229:1073-1079.
Tierney, D. F. (1974) Intermediary metabolism of the lung. Fed.
Proc. 33:2232-2237.
Yeager, H., Jr., and Massaro, D. (1972) Glucose metabolism and
r glycoportein synthesis by lung slices. J. Appl. Physiol. 32:
477-482.
-------
V. 17
V. A. 1. d. (cont.)
(2) Primary cells
Aranyi, C. (1977) Cytotoxic effect of trace metals adsorbed onto
fly ash particles. Environmental Health Effects Research
' Scries, EPA-600/1-77-017, 21 pp.
Bowden, D. 11. (1975') The alveolar macrophage and its role in
toxicology. Grit. Rev. Toxicol. 2:95.
Elson, N. A. and Crystal, K. G. (1977) Cellular approaches to the
study of environmental pollutants. In: Biochemical effects
of environmental pollutants, S. D. Lee, Ed. Ann Arbor
Science, Ann Arbor, Michigan,' pp 7-25.
Cause, E. M., Greene, N. D., Meltz, M. L., and Rowlands, J. R.
(1977) ln_ vivo and j_n vitro effects of sulfur dioxide upon
biochemical and immunological pai-ameters. In: Biochemical
effects of environmental pollutants, S. D. Lee, Ed. Ann
Arbor Science, Ann Arbor, Michigan, pp 273-292.
Huisingh, J. L., Cambell, J. A., and Waters, M. D. (1976) Evalua-
tion of trace element interactions using cultured alveolar
macrophages. Hanford Biology Symp. 16:55.
Waters, M. D., Vaughan, T. 0., Abernethy, D. J., Garland, H. R.,
Cox, C. C., and Coffin, D. L. (1975) Toxicity of platinum
(IV) salts for cells of pulmonary origin. Environ. Health
Perspect. 12:45-56.
Waters, M. D., Gardner, D. E., Aranyi, C., and Coffin, D. L. (1975)
Metal toxicity for rabbit alveolar.macrophages in vitro.
Environ. Res. 9:32-47.
Witschi, H. (1975) Exploitable biochemical approaches for the
evaluation of toxic lung damage. In: Essays in toxicology,.
vol. 6, W. J. Hayes, Jr., Ed. Academic, New York, pp 125-191.
-------
V. 18
V. A. 1. d. (cont.)
(3) Cell Lines
Blevins, R. D. and Dunn, W. C., Jr. (1975) Effects of carbaryl
and dieldrin on the growth, protein content, and phospholipid
content of HeLa cells. J. Agric. Food Chem. 23:377-382
tiagle, H. and Foley, G. E. (1958) Cytotoxicity in human cell
cultures as a primary screen for the detection of anti-tuinor
agents. Cancer Res. 18:1017-1025.
Geran, R. I., Greenberg, N. H., Macdonald, M. M. , Schumacher, A. M.,
and Abbott, B. J. (1972) Protocols for screening chemical
agents and natural products against animal tumors and other
biological systems .(3rd edition). Cancer Chemotherapy Reports,
Part 3,2:1-104.
Myhr, B. C. (1973) A screen for pesticide toxicity to protein and
RNA synthesis in HeLa cells. J. Agric. Food Chem. 21:362-367.
Pilotti, A., Ancker, K., Arrhenius, E., and F.nzell, C. (1975)
Effects of tobacco and tobacco smoke constituents on cell
multiplication in vitro. Toxicology 5:49-62.
Thayer, P. S., Himmclfarb, P., and Watts, G. L. (1971) Cytotoxicity
assays with L1210 cells 'in_ vitro: comparison with L1210 in_
vivo and KB cells in vitro. Cancer Chemotherapy Reports,
Part 2,2:1-25.
-------
V. 19
V. A. 2. Pulmonary
a. LCgQ Determination
Acute inhalation toxicity tests generally involve determina-
tion of the LC^Q and gross pathologic examination of animals
killed by exposure. Standard, routinely used test procedures should
suffice for obtaining this information for the uncombusted materials,
with particular attention being given to choice of solvents, solvent
controls, etc. In the case of exhaust products, difficulties may
be encountered in obtaining sufficiently high concentrations to
determine It-thai toxicity of specific exhaust components due to
the dilution necessary to reduce carbon monoxide- content to non-
lethal levels. The usual experimental animal for such studies is
the rat; exposure is usually for a minimum of 1 hour, followed by
daily observation for adverse symptoms and recording of mortality
for a subsequent 14-day period. Dead animals should be subjected
to complete gross pathologic examination, examining all appropriate
organ systems. At the end of the test period, all surviving test
animals are similarly subjected to complete gross pathologic exami-
nation. These tests provide information on the relative potential
of the test material to cause irreversible alterations in. various
organ systems. '"
-------
V. 20
A. 2. a. References for Pulmonary LC5Q Determinations
Binns, R. , Seven, J. L. , Wilton, L. V., and Lugton, W. G. D.
(1976) Inhalation toxicity studies on cigarette smoke.
II. Tobacco smoke inhalation dosimetry studies on small
laboratory animals. Toxicology 6:197-206.
Binns, R., Beven, J. L., Wilton, L. V., and Lugton, W. G; D.
(1976) Inhalation toxicity studies on cigarette smoke.
III. Tobacco smoke inhalation dosimetry study on rats.
Toxicology 6:207-217.
Binns, R. (1977) Inhalation toxicity studies on cigarette smoke.
IV. Expression oi" the dose of smoke particulate material
applied to the lungs of experimental animals. Toxicology
7:189-195.
Carpenter, C. P., Kinkead, B. R. , Geary, D. L., Jr., Sullivan,
L. J., and King, J. M. (1975) Petroleum hydrocarbon
toxicity studies. I. Methodology. Toxicol. Appl . Pharmacol.
32:246-262. .
Casarett, L. J. (1975) Toxicology of the respiratory system.
In : Toxicology, the basic science of poisons, L. J.
Casarett and J. Doull, Eds. Macmillan, New York, pp 201-224.
Cornish, 11. 11. (1975) Solvents and vapors. In : Toxicology,
the basic science of poisons, L. J. Casarett and J. Doull,
Eds. Macmillan, New York, pp 503-526.
Deichmann, W. B. , Keplinger, M. L., and Lanier, G. E. (1958)
Acute effects of nitro-olefins upon experimental animals.
A.M. A. Arch. Indust. Health 18:312:319. . ,-
Dungworth, D. L. , Schwartz, L. W. , Tyler, W. S. , and Phalen, R. F.
(1976) Morphological method? for evaluation of pulmonary
toxicity in animals. Annu. Rev. Pharmacol. Toxicol. 16:
381-399.
EPA.- (1977) Pesticides Guidelines Draft, Subpart F, Acute
Inhalation Toxicity, and references therein.
Kavet, R. I. and Brain, J. D. (1974) Reaction of lung to air
pollutant exposure. Life Sci. 15:849-861.
Kctkar, M. B. , Resnik, G. , and Mohr, V. (1977) Pathological
alterations in -Syrian golden hamster lungs after passive
exposure to cigarette smoke. Toxicology 7:265-273.
-------
v. v. :. .i.
-.i.".. i.i-int.-;. J. :-:..-:\. '..,i-.i . 'I-.-. Mn
.". f^ci-i'v . !;ir~/.e:.:i:.i. .-la-'1: .ip.ii , ~n
i ~-<:n-.im\ LI :. . ^V. : L^"" "nv ;:-. ;
L.Thala:i.:n .inii che :itj::rh of ojoi.s1~ i,:n in hunan 1'uin. .'r-:
hi. H. iL'J":;1 Pro! Lfir.ur. icn of Ty
.V rsvi-i'.v of j::inniGn r'jsri:nsijs in ;'.::<
iung daina.c-i Kroiiuced by ci.enJija.Ls. F'id. !Jr"<:. .:J : .i'J-^'i.
1
-------
V. A. 2. b. Host Defense Mechanisms
(1) Ir.fectivity Model System
Sterility of the lower airways is maintained through the
cooperative interactions of a complex system comprising both mechan-
ical and biological components. It has been shown that increased
susceptibility to respiratory infection results from inhalation of
pollutants capable of compromising the functional integrity of any
of the individual elements of the pulmonary defense system. The
increase in susceptibility may be effected by activation of latent
infections once surveillance and defense mechanisms are compromised,
or by the failure of host defense systems to respond adequately to
subsequent bacterial invasion. The consequences of the decrease
in resistance to respiratory infection can be life-threatening for
certain populations such as the elderly.
- Whether or not a specific pollutant atmosphere has a detri-
' mental effect upon the respiratory defense system can be determined
by small animal screening studies (Gardner ert a_l_., 1?77). For the
present situation of exposure of human populations to motor vehicle
emissions of varying composition, this test offers a variable means
of detecting and comparing direct effects of inhaled test materials.
Endpoints measured are usually alterations in mortality or survival
time of animals exposed to aerosols of viable bacteria.
If an alteration in resistance to infection is observed,
specific elements of the overall defense system can then be studied :
.to allow for identification of high-risk populations and possible .
protective or therapeutic measures. Separable elements of the
respiratory defense system which can be studied include: alveolar
macrophage function, mucociliary transport, ciliary beating, and
mucus physical properties. In performing these tests, exposure to
the bacteria may either precede or follow.exposure to the test ,. -. -
material, depending upon the nature of the experimental design.
Particular attention should be given to several details of the
,xposure protocol: selection of the infectious agent should be
made after consideration of consistency and reproducibility of
infective dose, lethality for the particular test animals being
used, logistics of handling and aerosolizing infective agents which
may be of human health significance, etc. The uniformity of bac-
terial exposures for all animals in a group should be confirmed by
determining bacterial loading of animals placed at different posi-
tions in the exposure chamber. Selection of the length of observa-
tion time should take into consideration the particular character-
istics of both the test animal and the infectious agent being used.
An observation time of 14 days is commonly used, during which time
the number of animals surviving at each day of observation is
determined and the mean survival time calculated by standard formulas.
Bacterial inactivation and killing, and rates of clearance, ».
can be ascertained by using radiolabeled bacteria. Following
-------
exr-osure or tne anir.a.s to tr.e la-eiec agents, totai :-acteriai
dose can be determined by ho-ogeni:ing the Lungs of exposed aninals,
determining the saount of radioactive label present, and extrapo-
lating the nurber of bacteria fr:s a --redetemir.ed standard,
relating acnount cf label vi-h nucncers of bacteria. At varicus
exposure to the labeled infectious agent, clearance cf bacteria
fro-n the lung can be similarly determined, as well as the effi-
ciency of killing of the bacteria by nacrcphages or other defense
nec'nanis.TiS. For determination of inactivation or killing efficiency,
Lungs are removed fron infected aninaii at varicus t i.~e intervals
after exposure, the amount of radioactivity (thus the total nuxter
of bacteria present] is detem.ir.ed, and the nuraber of viable .bacteria
determined by standard dilution and culture techniques ,Goldstein
et_ a I. , 1976).
(2) Mucociliary Function
S!uco
-------
V. 24
of measurement of ciliary rates is by means of high-speed photography,
or other objective methods using photoelectric or stroboscopic
techniques.
Mucociliary Transport
Mucociliary transport rates are determined by visually moni-
toring the rate of movement of particles placed within an excised
tracheobronchial system, or an incised and externalized JJH situ
system or by externally monitoring the movement of inhaled radio-
active particles in intact animals and humans. Visual methods
usually employ carbon particles, india ink, or graphite, the movement
of which is observed microscopically within the trachea. The human
subject or the experimental animal inhales radioactive particles
from an aerosol generator; sequential measurement of particle
locations are then made with a gamma-ray-detecting camera, such as
an Anger camera
Mucus Production and Characteristics
Rates of mucus production are measured by collecting secretions
from an anesthetized animal via an intratracheal or endotracheal
cannula, or by a method which involves isolation of a short segment
of cervical trachea of a dog and formation of the segment into a
subcutaneous pouch.
Increases in either viscosity or elasticity of mucus are
associated with reductions in mucociliary function, therefore mea-
sureme-t of visco-elastic properties of mucus as a function of
exposure to a given agent provide valuable information. The physi-
cochemical properties of mucus are due to large molecular weight
aggregates of several distinct glycoproteins which are in random
coil configuration in the aqueous solution. Irritant, water soluble
gases such as SC>2 have been,.shown to increase mucus viscosity (Litt
e_t_ aj_., 1976). An increase in mucus viscosity causes a slowing of
mucus flow resulting in abnormal retention and accumulation of
particulates.
(3) Free Lung Cell Populations
Following inhalation exposure, alterations in numbers and
types of free lung cells could provide information useful in pre-
dicting possible impairment in host defense against inhaled infectious
agents, or inflammatory or other tissue injury. Free cells generally
are comprised of predominantly alveolar macropiiages, with small
percentages of lymphocytes, polymorphonuclear leukocytes, and occa-
sionally other cell types. The numbers and distribution of these
cell types can be significantly altered following insults of various
kinds; induction of inflammatory changes may lend to marked increases
in polymorphonuclear leukocytes and macrophag3S, ant-igenic stimulation
may lead to increased lymphocyte numbers, participate stimulation
may lead to increased numbers of alveolar macrophages, etc. (Goldstein
et al., 1976).
-------
V. 25
For determination of numbers and distribution of lung cells,
procedures are available for routine lavage of the lungs of various
experimental animals. Particular attention should be given to the
composition of the lavage fluid; more complete recovery of free
cells is accomplished with the use of calcium and magnesium free
solution. Cells thus recovered can be washed, counted by standard
laboratory procedures (hemacytometer or particle counter), and
viability determined by ability to exclude vital dyes. Differerftial
determinations should be made following preparation of smears on
microscope slides and staining with standard hematological techniques
(Brain, 1970).
(4) Alveolar Macrophage Function
III Vivo Exposure
Alveolar macrophages function as primary effectors of host
defense against inhaled particulate matter such as bacteria, viruses,
and fungi, which are ingestod, killed, and degraded by the macro-
phages. Nonbiological particles such as dusts, soot, etc., are
also ingested and degraded to varying extents, unless their chemical
composition makes them cytotoxic to macrophage?, in which case the
particles are released from dead cells to be taken up. by other
macrophages and repeat the cycle. The separate stages of macrophage
function can be inhibited individually - i.e., metabolic inhibitors
could preferentially inhibit intracellular bacterial killing with-
out affecting phagocytosis, or could cause a delayed response such
as labilizution of lysosomal membranes leading to cell injury or
death without affecting phagocytosis rates or numbers of particles
taken up before sufficient quantities of hydrolytic enzymes had
leaked into the cytoplasm to injure the cell. However, phagocytosis
measurements are easily and readily performed, are useful for compar-
ative toxicity studies, and, if performed in conjunction with a
careful quantitative estimation of cell viability, will provide
an assessment of alterations in macrophage function.
Alterations in function of alveolar macrophagcs subsequent
to inhalation of test materials can be estimated on the basis of
ability of cells recovered from animals subsequent to exposure to
exclude dyes and capacity of the recovered cells for phagocytosis
of particles. Phagocytosis is frequently assayed on macrophages
which have been allowed to attach to a glass or plastic surface,
with the cells which did not attach within a given period of time
removed by decantation. The attachment process, however, may
represent a process of selection for metabolically activated cells
(i.e., those with a high energy charge) over those in a resting,
metabolically depleted, or immature stage, and since it is presently
not clear how this compares with the normal state of macrophages
within the lung, consideration should be given.to comparison of
phagocytic capabilities for both suspended and attached cells.
-------
V. 26
Phagocytosis can be expressed as numbers of particles invested
per cell and numbers of cells taking up particles, both of which
measurements have relevance to assessment of macrophage function.
Two methods of estimating impairment of endocytosis which may j
provide an index of cytotoxicity of particulate material or of !
other substances inhaled concurrently should be of particular value-
for fuels and fuel additives (Brain and Corkery, 1977; Graham £t
aK , 1975).
Another aspect of macrophage function which is likely to be
altered by inhalatiori, of test emissions is the activity of lysosomal
enzyme.-. The enzymes of the lysosomes represent the means by which
substances taken in by phagocytosis are degraded; or by-~which
physiological protein, lipid and carbohydrate molecules are broken
down. Extracellular release of lysosomal proteases as a consequence
of phagocytosis of particulates may produce emphysema-like lesions j
and may be the initiating factor in emphysema. Inhalation of test !
emissions could cause an increase in release of lysosomal enzymes |
either by cytotoxicity to macrophages resulting in cell lysis, or !
by the increased phagocytic load (Rogers ert al_., 1976; Janoff et !
aK, 1971; Traurig, 1976). |-
I_n_ Vitro Exposure
Alveolar macrophage function consequent to iji vivo exposure i
to pollutant atmospheres frequently-provides a sensitive index of ' !
pollutant effect. The capacity of a specific pollutant for affecting ,1
lung macrophage function can also be screened by exposing isolated ;j
macrophages in_ vitro. Specific parameters which are measured in the
macrophage test system of Waters el^ al_. (1975) include: phagocytosis, ''
viability, cell number. ATP concentration, depression of respiration, tj.
and lysosomal-enzyme activities. -.,.- - , -
(5) Immune Function ;
/
The possibility that exposure to various chemicals may lead ;
to compromised immunological capabilities is currently being recog-
nized as an important aspect of toxicol")gical assessment of hazard.
Of particular importance are toxic effects which may be manifested
as development of various degrees of immunosuppression, either by
virtue of interference with immunolo^ic recognition processes or
by indirect effects on the cell populations necessary for normal
immunolof;ical function. In order to adequately examine the effects
of such agents on immune systems, it is necessary to be able to
detect alterations in both 7ompartmerts of the immune response,
i.e., humoral and cell mediated. A variety of assay systems are
available for studying effects of perturbing agents on both of
these systems.
The usual procedure for assessing changes in humoral or antibody
mediated Lnmunity involves immunization of a suitable test animal,
-------
V. 27
frequently mice or rabbits, with heterologous erythrocytes or
another suitable antigen and subsequently measuring antibody titers,
or quantitating numbers of antibody producing spleen cells by a
plaque assay. The latter has the advantages of permitting deter- -
nination of both IgG and IgM producing cells, important parameters
which may be related to susceptibility to infection or development
of other disease processes; it also is generally done with mice,
thus minimizing the cost of the assay.
Evaluation of effects on the cellular immune system may take
many forms. These may include studies of hematological and histo-
pathological alterations in the processes responsible for maintenance
of effective cell populations in various organs .including blood and
lymph nodes; direct measurements of functional integrity of immuno-
logically relevant cells including lymphocytes and macrophages; or
endpoint measurements of response to immunization with antigens
known to evoke responses in the cell mediated compartment of the
immune system, e.g., skin sensitization to tuberculin. Under con-
ditions of suspected high risk of interference with the immane
system, it would be valuable to perform assessment of lymphocyte
and macrophage function, using such test procedures as: blastogenic
responsiveness of lymphocytes to both T and B cell mitogens and
defined antigens to which the animals have been sensitized; capa-
bility of lymphocytes to produce and release selected mediators of
cellular immunity such as macrophage migration inhibitory factor or
lymphotoxin; and assessment of the capability of macrophages from
test animals to function normally in processes related to immune
responsiveness, i.e., response to migration inhibitory factor,
phagocytic capability, and capability to co-operatively interact
with lymphocytes.
-------
', V. 28
V. A. 2. b. References for Host Defense Mechanisms
(1) Infectivity Model System
Casarett, L. J. (1975) Toxicology of the respiratory system.
In: Toxicology, the basic science of poisons, L. J. Casarett
and J. Qoull, Eds. Macmiilan, New York, pp 201-224.
Gardner, D. K. and Graham, J. A. (1977) Increased pulmonary
disease mediated through altered bacterial defenses. Proc.
16th Annual Hanford Biol. Symp., Richland, Washington.
Gardner. D. E., Miller, F. J., Illing, J. W., and Kirtz, J. M.
(1977) Alteration in bacterial defense mechanisms of the
lung induced by inhalation of cadmium. Bull. Eur. Physiol.
Pathol. Respir. 13:157-174.
Goldstein, E., Jordan, G. W., Mackenzie, M. R., and O.sebold, J. S.
(1976) Methods for evaluating tJie toxicologica] effects
of gaseous and particulate contaminants on pulmonary microbial
defense systems. Annu. Rev. Pharmacol. Toxicol. 16:447-465.
Green, G. M., Jakob , G. J., Low, R. B.,. and Davis, G. S. (1977)
Defense mechanisms of the respiratory membrane. Am. Rev.
Resp. Dis. 115:479-514.
Kim, M., Goldstein, E., Lewis, J. P., Lippert, W., and~Warshauer,
D. (1976) Murihe pulmonary alveolar macrophages: rates of
bacterial ingestion, inactivacion, and destruction. J.
Infect. L'is. 153:510-520.
*
-------
V. 29
V. A. 2. b. (cont.)
(2) Mucociliai-y Function
Adalis, D., Gardner, D. E., Miller, F. J., and Coffin, D. L.
(1977) Toxic effects of cadmium on ciliary activity using
a tracheal ring model system. Environ. Res. 13:111-120.
Bang, B. G and Bang, F. B. (1977) Nasal mucociliary systems.
In: Respiratory defense nechanisms, Part I., J. D. Brain,
D. F. Proctor, and L. M. Reid, Eds. (Lung biology in health
and disease, vol. 5, Part I). Marcel Dekker, Inc., New
York, pp 405-426.
Bang, F. B. and Bang, B. G. (1977) Mucous.membrane injury and
repair. In: Respiratory defense mechanisms, Part I., J. D.
Brain, D. F. Proctor, and L. M. Reid, Eds. (Lung biology in
health and disease", vol. 5, Part I). Marcel Dekker, Inc.,
New York, pp 453-488.
Goldstein, E., Jordan, G. W., MacKerzie, M. R., and Osebold, J. S.
(1976) Methods for evaluating xhe toxicological effects of
gaseous and partic'ilate contaminants on pulmonary microbial --
defense systems. Annu. Rev. Pharmacol. Toxicol. 16:447-463.
Kilbum, K. (1977) Clearance mechanisms ir. the respiratory tract.
In: Handbook of physiology, Sec. 9, Reactions to environmental
agents, D. H. K. Lee, Ed. American Physiological Society,
Bethesda, pp 243-262.
Lippmann, M., Albert, R. E., Yeates, D. B., Berger, J. M., Foster,
W. M., and Bohniug, D. E. (1977) Factors affecting tracheo-
jronchial mucociliary transport. In: Inhaled particles IV,
Part T, W. H. Walton, Ed. Pcrgamon, New York, pp 305-319.
Proctor, D. F., Andersen, I., and Lundqvist, G. (1977) Nasal
mucociliary functions in humans. In: Respiratory defense
mechanisms, Part I., J. D. Brain, D. F. Proctor, and L. M.
Reid, Eds. (Lung biology in health and disease, vol. 5,
Part I). Marcel Dekker, Inc., New York, pp 427-452.
Reid, L. (1570) Evaluation of model systems for study of airway
epithelium, cilia, and mucus. Arch. Intern. Med. 126:428-434.
.Sleigh, M. A. (1977) The nature and action of respiratory tract
cilia. In: Respiratory defense mechanisms, Part I., J. D.
Brain, D. F. Proctor, and L. M. Reid, Eds. (Lung biology in
health and disease, vol. 5, Part I). Marcel Dekker, Inc.,
N'ew York, pp 247-2SS.
Wolff, R. K., Dolovich, M., Obminski, G., and Newhouse, M. T.
(1977) Effect of sulphur dioxide on tracheobronchial
-------
V. 30
V. A. 2. b. (2) (cont.)
clearance at rest and during exercise. In: Inhaled
particles IV, Part 1, K. H. Walton, Ed. Pergamon, New York,
pp 321-332.
-------
V. 31
V. A. 2. b. (cont.)
(2) Mucociliary function
Ciliary Activity
III V'J-V° exposure
Adalis, D., Gardner, D. E., Miller, F. J., and Coffin, U. L.
(1977) Toxic effects of cadmium on ciliary activity using
a tracheal ring model system, linviron. Res. 15:111-120.
Dalhamm, T. (1956) Mucous flow and ciliary activity in the
trachea of healthy rats and rats exposed to respiratory
irritant gases (S0?, H,N, HCHO). Acta Physiol. Scand.
36:1 (Supplement 123). °
Ualhamm, T. and Rylander, R. (1964) Ciliastic action of smoke
from filter-tipped and non-tipped cigarettes, ii'ature 201:
401.
Kilburn, K. H. (1967) Cilia and mucus transport as determinants
of the response of lung to air pollutants. Arch. Environ.
Health 14:77-91.
Kilburn, K. 11. (1977) Clearance mechanisms in the respiratory
tract. In: Handbook of physiology, Sec. 9, Reactions to
environmental agents, D. H. K. Lee, Ed. American Physiological
Society, Bethesda, pp 245-252.
Sleigh, M. A. (1977) The nature and action of respiratory tract
cilia. In; Respiratory defense mechanisms, Part I., J. D.
Brain, U. F. Proctor, and L. M. Reid, Eds. (Lung biology in
. .health -and disease, vol. 5, Part I). Marcel Uekker, Inc.,
New York, pp 247-288.
I_n_ vitro exposure
Adalis, D., Gardner, D. E., Miller, F. J., and Coffin, D. L.
(1977) Toxic effects of cadmium on ciliary activity using
a tracheal ring model system. Environ. Res. 15:111-120.
Dalhamm, T. and SjOholm, J. (1965) Studies on S07, NO, and Ml :
effect on ciliary activity in rabbit trachea of single
^Ln_ vitro exposure and resorption in rabbit nasal cavity.
Acta Physiol. Scand. 58:287-291.
Kilburn, K.H. (1967) Cilia and mucus transport as determinants
of the response of lung to air pollutants. Arch. Environ.
Health 14:77-91.
-------
V. 32
V. A. 2. b. (2) (cont.)
Kilburn, K. H. (1977) Clearance mechanisms in the respiratory
tract. In: Handbook of physiology, Sec 9, Reactions to
environmental agents, D. li. 'K. Lee, lid. American Physiological
Society, Bethesda, pp 245-262.
Mucociliary Transport
Berr.field, P., Nixon, C. W., and Iloraburger, F. (1964) Studies
on the effect of irritant vapors on ciliary mucus transport.
Toxicol. Appl. Pharmacol. 6:105.
Goldhamer, R., Barnett, B., and Carson, S. (1964) A new technique
for the study of mucus flow in the intact animal. Fed. Proc.
25:406.
Goldstein, E., Jordan, G. W., Mackenzie, M. R., and Osebold, J. S.
(1976) Methods for evaluating the toxicological effects of
gaseous and particulate contaminants on pulmonary ndcrobial
defense systems. Annu. Rev. Pharmacol. Toxicol. 16:4
-------
V. 33
V. A. 2. b. (2) (cont.)
clearance at rest and during exercise. In: Inhaled particles
IV, Part 1, W. II. Walton, Ed. Pergamon, New York, pp 321-532.
Mucus Production and Characteristics
Andersen, I., Lundqvist, R., Jensen, P. L., and Proctor, D. F.
(1974) Human response to controlled levels of sulfur dioxide.
Arch. Environ, Health 28:31-39. .
Gallagher, J. T., Kent, P. W., Passatore, MY", Phipps, R. J., and
Richardson, P. S. (1975) The composition of tracheal mucus
! and the nervous control of its secretion in the cat. P'rpc.
' R. Soc. London. B. 192:49-76.
Keal, E. E. (1977) Physiological and pharmacological control of
airways secretions. In: Respiratory defense mechanisms,
Part I., J. D. Brain,T. F. Proctor, and L. M. Reid, Eds.
(Lung biology in health and disease, vol. 5, Par.t I). Marcel
Dekker, Inc., New York, pp 357-403.
Litt, M., Khan, M. A., Chakrin, L. W., Sosnowski, G., andWardell,
Jr., J. R. (1976) Effect of chronic sulfur dioxide inhala-
tion on Theological properties of tracheal mucus. Biorheology
13:107-114. '
Litt, M., Khan, M. A., and Wolf, D. P. (1976) Mucus rheology:
relation to structure and function. Biorheology 13:37-48.
Lopez-Vidriero, M. T., Das, I., and Reid, L. M. (1977) Airway
secretion: source, biochemical and rheological properties.
In: Respiratory defense mechanisms, Part I., J. D. Brain,
D. F. Proctor, and L. M. Reid, Eds. (Lung biology in health
and disease, vol. 5, Part I). Marcel Dekker, New York,
pp 289-356.
Meyer, F. A. (1976) Mucus structure: relation to biological
transport function. Biorheology 13:49-58.
Van As, A. and Webster, I. (1974) The ..'.orphology of mucus in
mammalian pulmonary airways. Environ. Res. 7:1-12.
Wolff, R. K., Dolovich, M., Eng, P., Rossman, C. M., and Jiewhouse,
M. T. (1975) Sulfur dioxide and tracheobronchial clearance
in man. Arch. Environ. Health 30:521-527.
-------
V. 34
V. A. 2. b. (cont.)
(3) Free Lung Cell Populations
Daniele, R. P., Altose, M. D., and Rowlands, D. T., Jr. (1975)
Imraunocompetent cells from the lower respiratory tract .of
normal human lungs. J. Clin. Invest. 56:986-995.
Elson, N. A. and Crystal, R. G. (1977) Cellular approaches to
the study of environmental pollutants. In: Biochemical
effects of environmental pollutants, S. D. Lee, Ed. Ann
Arbor Science, Ann Arbor, Michigan, pp 7-25.
Cause, E. M., Greene, N. D., Meltz, M. L., and Rowlands, J. R.
(1977) lr± vivo and i_n vitro effects of sulfur dioxide upon
biochemical and immunological parameters. In: Biochemical
effects of environmental pollutants, S. D. Lee, Ed. Ann
Arbor Science, Ann Arbor, Michigan, pp 273-292.
Gorenberg, D. J. and Daniele, R. P. (1976) Characterization of
immunocompetent cells recovered from the respiratory tract
and tracheobronchial lymph node of normal guinea pigs. Amer.
Rev. Resp. Dis. 114:1099-1105.
Kazmierowski, J. A., Fauci, A. S., and Reynolds, 11. Y. (1976)
Characterization of lymphocytes in bronchial lavage fluid
from monkeys. J. Immunol. 116:615-618.
Reynolds, H. Y., Fulmer, J. D., Kazmierowski, J. A., Roberts, W. C.,
Frank, M. M., and Crystal, R. G. (1977) Analysis of cellular
and protein content of bronchoalveolar lavage fluid' from
patients with idiopathic pulmonary fibrosis and chronic
hypersensitivity pneumonitis.. J..Clin. Invest. 59:165-175.
Reynolds, H. Y. and Newball, H. H. (1974) Analysis of proteins
and respiratory cells obtained from human lungs by bronchial
lavage. J. Lab. Clin. Med., 84:559-573.
Reynolds, H. Y. and Newball, H. H. (1976) Fluid and cellular
milieu of the human respiratory tract. In: Immunologic and
infectious reactions in the lung, C. H. Kirkpatrick and H. Y.
Reynolds, Eds. Marcel Dekker, New York, pp 3-27
-------
IS
f}.
:L~.:::,iin. ^. . .-.irk 1 ;< . V. r v,i_r:.-,- 7... :;-.,; Vi.= 5.il:. '..
j::.i.l .:u:;-. >: I-;:,i.lrc -:i:~.:: 1 ;.,. 1".', 'i.7~. .i.. V.
''i . . '."r^'rC'r. ''I.. C. . *-''lrl".I: '^. 1. . I."u < .T'v 1 i.".ll-: . ." . _
\~-.: :: ;n'/"i r'inincir; ^.i.l. TH 11 '."..IT. "..;. r. '. _.;.;_ I-.:. '.t;i
' :>: 1 rrc-r . 'm -JT7': r . ^^ ;."_.L l'i~ . ~r. '. ~':-.'.'.- '. ..'
.'"' i.iuniir:.~. ' ~~~. n.i;.7. I.~"'"i?-:r ..
-------
; \
.:i» . .1. .3. . inu
ing" :;n. ." . mil .'I.'. i.jc
r.: p;ir~". .: ^ JH . fi.)«r:;
in : "l^::cr.;::r;^ ::; 'i
:;:i;ne :n icu. .iydr-;
rLj;;vie inu ;:illu_j.r
of "he ^ul:nenary i J.-'. -».: L.i
'lui=taf:i, M. I., .iack^r. A.
.'.nn Xr'jor, Mi
V,'nbr:mdt, D. and
du::i-iu aid
-------
V. 37
V. A. 2. b. (cont.)
(4) Alveolar Macrophage Function
£n vitro exposure
Aranyi, C. (1977) Cytotoxic effect of trace metals adsorbed onto
fly ash particles, Environmental Health Effects Research
Scries, HERL, ORD, EPA, Research Triangle Park, N. C. 27711.
EPA-600/1-77-017 (March 1977), 31 pp, PB 267-078.
Elson, N. A. and Crystal, R. G. (1977) Cellular approaches to
the study of environmental pollutants. In: Biochemical
effects of environmental pollutants, S. D. Lee, Ed. Ann
Arbor Science, Ann Arbor, Michigan, pp 7-25.
Huisingh, J. L., Cambell, J. A., and Waters, M. D. (1976) Evaluation
of trace element interactions using cultured alveolar macrophages.
In: Han ford Biology Symposium 16:35.
Low, E. S., Low, R. B., and Green, G. M. (1977) Correlated effects
of cigarette smoke components on alveolar macrophage adenosine
triphosphatase activity and phagocytosis. Am. Rev. Rcsp. Dis.
115:963-970.
Waters, M. D., Vaughan, T. 0., Abernethy, P. J., Garland, H. R.,
Cox, C. C., and Coffin, D. L. (1975a) Toxicity of platinum
(IV) salts for cells of pulmonary origins. Environ. Health
Perspect. 12:45-56.
Waters, M. D., Gardner, D. E., Aranyi, C., and Coffin, D. L. (1975b)
Metal toxicity for rabbit alveolar macrophages in- -vitro. - -
Environ. Res. 9:32-47.
-------
V. 38
V. A. 2. b. References for Host Defense Mechanisms
(5) Immune Function
Graham, J. A., Miller, F. J., Daniels, M. J., Payne, E. A.,
and Gardner, D. E. (1977). Influence of Cd, Ni, and Cr
on primary immunity in mice. Environ. Res., in press.
Green, G. M., Jakab, G. J., Low, R. B., and Davis, G. S.
(1977). Defense mechanisms of the respiratory membrane.
Am. Rev. Resp. Dis., 115: 479-514.
Street, J. C. and Shrama, R. P. (1975). Alteration of induced
cellular and humeral immune responses by pesticides and chemical
of environmental concern: Quantitative studies of immunosuppression
by DDT, Aroclor 1245, Carbaryl, Carbofurnn, and Methylparathion.
Toxicol. Appl. Pharmacol., 32: 587-602.
Vos, J. G. and van Genderen, H. (1973). lexicological aspects
of immunosuppression. In: Pesticides and the environment,
W. D. Deichman, ed. New;York. Intercontinental Medical Book Corp.
Schneider, L. K. and Calkins, C. A. (1970). Sulfur dioxide-
induced lymphocyte defects in human peripheral blond, cultures.
Environ. Res., 3: 473.
Stupfel, M., Magnier, M., Romary, H., Iran, M. H., and Moutet,
J. P. (1973). Lifelong exposure of SPF rats to automotive exhaust
gas. Arch. Environ. Health, 26: 264-269.
Rylander, R. (1969). Alterations of lung defense mechanisms
against airborne bacteria. 'Arch. Environ. Health; 18': 551".
Rylander, R. (1970). Studies of lung defense to infections in
inhalation toxicology. Arch. Intern. Mod., .126: 496.
Valand, S. B., Acton, J. D., and Myrvik, Q. N. (1970). Nitrogen
dioxide inhibition of viral-induced resistance in alveolar
monocytes. Arch. Environ. Health, 20: 303.
Zarkower, A. (1972). Alterations in antibody response induced
by chronic inhalation of SO and carbon. Arch. Environ. Health,
25: 45.
1','arr, G. A. and Martin, R. R. (1974). Chemotactic responsivensss
of human alveolar macrophages. -Effects of cigarette smoking.
Infect. Immun., 9: 769.
-------
V. 39 j
' ' ' ' .';"!
V. A. 2. b. References for Host Defense Mechanisms j
(5) Immune Function (continued) '
j
Warr, G. A. and Martin, R. R. (1973). In vitro migration of !
human alveolar macrophages: Effects of cigarette smoking.
Infect. Immun., 8: 222. j
i
Martin, R. R. and Laughter, A. H. (1976). Pulmonary alveolar !
macrophages can mediate immune responses: Cigarette smoking >
impairs these functions. Fed. Proc., 55: 2811. j
Harrington, J. T., Jr. and Stasny, P. (1973). Macrophagc
migration from an agarose droplet: Development of a micromethod
for assay of delayed hypersensitivity. J. Immunol., 110: 752.
Nulsen, A., Holt, P. G., and Keast, D. (1974). Cigarette
smoking, air pollution, and immunity: A model system. Infect.
Immun., 10: 1226-1229.
Esber, H. J., Menninger, F. F., Jr., Bogden, A. E., and Mason,
M. M. (1973). Immunological deficiency associated with cigarette
smoke inhalation by mice. Arch. Environ. Health, 27: 99-104.
Green, G. M. and Carolin, D. (1967). The depressant-effect of
cigarette smoke on in vitro antibacterial activity of alveolar
macrophages. N. EngT J. Med., 276: 421-427.
Thomas, W. R., Hold, P. G., and Keast, D. (1973). Cellular
immunity in mice chronically exposed to fresh cigarette smoke.
Arch. Environ. Health, 27: 372-375.
Bloom, B. R. and David, J. R. (1976). In vitro methods in
cell-mediated and tumor immunity. New York. Academic Press.
Rose, N. R. and Friedman, H. (1976). Manual of clinical
immunology. Washington, D. C. Amer. Soc. for Microbiol.
-------
V. 40
V. A. 2. c. Pulmonary Function j
Assessment of pulmonary function following inhalation exposure
to fuels and/or fuil additives would provide information about
lung damage of a different nature than that derived from morpho-
logical studies alone. The complementary information tha:, could be
derived from a combination of morphology and lung function, in. those
experimental animal systems which lend themselves to those types of
studies, indicates that both types of studies should be -performed;
i.e., histopathological studies for overt tissue injury, and lung
function studies for detecting alterations which may provide evidence
of functional impairment. Measurement of changes in respiratory
rate alone is a simpler procedure for which several advantages can
be considered: the procedure is sufficiently simple to permit its
use in a screening program; the results are highly reproducible
and permit formulation of dose-response curves; and it is suffi-
ciently sensitive to permit- detection of irritating effects at very
low concentrations of test materials where pathological tissue
injury would not be expected to be seen.
Gaseous irritants such as sulfur dioxide, formaldehyde., acrolein,
formic acid, and sulfuric acid cause an increase in pulmonary flow
resistance, a slight decrease in compliance, and at higher concentra-
tions, a decrease in breathing frequency. Irritants produce inflam-
mation and inflammation of the upper respiratory tract leads to
rhinitis, pharyngitis, anil laryngitis; inflammaticr of the bronchi
results in bronchitis and bronchopneumonia; while inflammation of
the lungs leads to pulmonary edema and pneumonia.
A complete pulmonary function profile would include: volume
studies, including total lung capacity and its subdivisions, vital
capacity and functional losidual capacity - for detection of injury
compatible with emphysematous or fibrotic changes; pressure-volume
curve (cpmpliance) as a measure of elasticity; .airway flow char-
acteristics (conductance) to provide information about flow-volume
relationships; closing volume, which determines at what volu^a the
small airways close, thus detecting damage to smaller airways in
the basilar portion of the lung; and diffusing capacity which doter-
mines the characteristics of gas movement from the alveoli into the
blood and t'.ius provides a measure of damage to capillaries, the
alveolar units, or both which may result in impaired gas exchange.
Along with the pulmonary function studies, additional useful info"-
mation can be obtained by monitoring the levels of blood gases,
including 02, C02, f-'M the blood pH.
Determination of whether or not to require pulmonary function
studies would have to be made after consideration of such factors
as cost and applicability of the procedures for the particular
animal models selected. A complete wor!;up is difficult or impossible
to perform with small animals such as rats or hamsters; selected
types of information can be obtained, however. For larger animals,
such as dogs OT subhuman primates, complete pulmonary function
*
i
j
-------
V.
studies could be done, and could provide valuable information with
significant clinical relevance. -
i 'i
-------
V. 42
V. A. 2. c. References for Pulmonary Function
Amdur, M. 0., and Mead, J. (1958). Mechanics of respiration in
unanesthetized guinea pigs. Am. J. Physiol., 192: 564-368.
Koo, K. W., Leith, D. E., Sherter, C. B., and Snider, G. L.
(1976). Respiratory mechanics in normal hamsters. J. Appl.
Physiol. 40: 936-942.
Robinson, N. E., Gillespie, J. R., Berry, J. D., and Simpson, A.
(1972). Lung compliance, lung volume, and single-breath diffusing
capacity in dogs. J. Appl. Physiol., 33: 808-812.
-------
y. 43
V. A. 2. d. Gross and Histopathology
Refer to V. A. 1. a. (3).
V. A. 2. e. Blood/Urine Analysis
Refer to V. A. 1. a. (2).
V. A. 3. Central Nervous System/Behavior
Behavioral toxicology as a sensitive indicator of neurotoxicity
has developed rapidly within the past decade and is expected to
become an increasingly important arm of environmental toxicology
as its development continues. I- is recognized that neurotoxicants
introduced into the environment affect the central nervous system
(CNS) through a variety of mechanisms and that these CNS effects
aie manifested as modifications in behavior. The question now
arises as to what extent behavioral problems confronting society
may be due to actions of environmental contaminants. For example,
some childhood behavioral disorders (hyperkinesis, minimal brain
dysfunction, etc.) appear to be of epidemic character and could be
due to chemical factors in the environment; both lead and food .
additives have been proposed as causative factors. .Neurotoxicity
is recognized to be manifested as behavioral modification related
to severity of dose of exposure to heavy metals such as manganese,
lead, and mercury; organophosphate pesticides; organic solvents;
and carbon monoxide. At another level, more subtle psychological
affects appear to be related to environmental chemical exposure.
Occupational exposure to organophosphate pesticides has been linked
to higher anxiety levels (Levin £t aJL , 1975), and carbon disulfide
exposure, which produces a broad range; of behavioral and neurological
disorders, has been associated with increased risk of death- from -
coronary heart disease and suicide.
The susceptibility of the CNS to chemical toxicants is enhanced
in the immature and aged individual, and the CNS m^y be particularly
vulnerable in the fetal stage. In fact, behavioral teratology
may prove to be uniquely significant in the detection of: (1) re-
productive effects as « consequence of continuous, environmental
exposure of both parents and of the offspring pre- and postnatally;
and (2) mutagenic processes may first be detected as expressions
of behavior (DHEW-N1EIIS, 1977).
In considering the potential health erfects of fuel and fuel
additive exposures, it jhould be remembered that different neuro-
toxicanto whose behavioral expressions are mediated through a
common mechanism could result in additive effects which would hove
to be taken into account in the regulation of environmental levels.
-------
V. 44
The assessment of neurotoxicity of a given agent can be
approached in either of 2 ways: (1) through a sequential series
of tests of increasingly specific nature (Weiss crt aj_., 1975);
and (2) by a single sensitive, complex performance task. The
sequential approach is economically attractive for screening of
potentially neurotoxic agents; however, a compound with only
subtle effects would require a long series of t.csts with essen-
tially negative findings in this approach. On the other hand,
more complex behaviors and performances are more sensitive to
brain lesions and drugs than simpler behaviors and performances.
Therefore, if the most sensitive assay for detection of the lowest
level of exposure of environmental contaminant is sought, complex
performance measurement may be the most meaningful (DHEW-NIEHS,
1977). Disadvantages of the complex performance approach are the
inherent time and cost involved.
If a neurotoxicant interacts xvith the CNS strongly enough,
gross behavioral changes can be detected in small animalsi.as
convulsions, ataxia, and the loss of various reflexes. These
gross behavioral changes may be employed in acute, range-finding
experiments for at least some forms of fuel and fuel additive test
materials; additive ingredients, technical grade materials, and
use-formulations could be screened by such measurements. These
initial screening measurements include: (1) activity changes -
i.e., motor functions; (2) objective signs such'as tremors, ptosis,
salivation, lacrimation, defecation, convulsions, abnormal postural
changes, etc.; (3) reflex changes - hyper- or hyporeflexia;
(4) elicited responses - grasping, response to pinch, induced
convulsions, orientation, ef..; and (5) body weight changes,
immediate and b'rief weight loss and failure to gain normally.
These tests and other appropriate tests are used by the pharma-
ceutical industry as initial screens for CNS activity and could
be of significant predictive value for fuel and fuel additive
testing (NAS-NRC, 1975). If there initial screening tests show
CNS activity they should be follov:ed by more definitive behavioral
toxicology tests as described above.
-------
V. 45
V. A. 3. References for Central Nervous System/Behavior
Bokina, A. I., Ekslcr, N. D., Semenenko, A. D., and Merkuryeva,
R. V. (1976) Investigation of the mechanism of action of
atmospheric pollutants on the central nervous system and
comparative evaluation of methods of study. Environ. Health
Perspect. 13:37-42.
Levin, H. S., Rodnitzkv, P.. L. , and Mick, D. L. (1975) Anxiety-
associated with exposure to organophosphate compounds. Arch.
Gen. Psychiat. 33:225-228.
Norton, S. (1975) Toxicology of the central nervous system. In:
Toxicology, the basic science of poisons, L. J. Casarett and
J. Doull, Eds. Macmillan, New York, pp 151-169.
Schuster, C.'R., Balster, R., Lipton, M., and Weiss, B. (1977)
Behavioral toxicology. In: Human health and the environment--
some research needs. Report of the Second Task Force for
Research Planning in Environmental Health Science. D11EW
Publication no. NIH 77'-1277.
Turner, R. A. (1965) Screening methods in pharmacology. Academic,
New York, 332 pp.
Weiss, B., Brozek, J., Hanson, 11., Leaf, R. C., Mello, M. R., and
Spyker, J. M.. (1975) Effects on behavior. In: Principles
for evaluating chemicals in the environment. N.A.S., Washington,
D. C., pp 198-216.
Weiss, B. and V. G. Laties, Eds. (1975) Behavioral toxicology.
Plenum, New.York, .445 pp.
Keis:s, B. and Lcvine, T. E. (1976) Studies in the behavioral
toxicology of environmental contaminants. Environ. Health
Perspect. 13:31-55.
-------
V. B. Mutagenesis
' 1. Gene Mutations '
a. Procaryotic Microorganisms (Bacteria)
(1) Salmonella typhimurium
(2) Escherichia coli WP2
(3) Escherichia coli K12
b. Eukaryotic Microorganisms
(.1) Neurospora crassa
(2) Haploid Strains of Yeast
c. Plants
(1) Tradescantia
d. Insect
(1) Drosophila melanogaster
e. Mammalian Cells
(1) Mouse Lymphoma, L5178Y
(2) Chinese Hamster Lung, V79
(3) Chinese Hamster Ovary, CilO
f. Rodent
(1) Mouse Specific Locus
(2) Mouse Isozyme Specific Locus
2. DNA Damage and Repair
a. Repair-defective Microorganisms
(1) Recombinationless Mutants of Bacteria
(2) DNA Poiymerase-deficient Mutants of Bacteria
(5) Normal versus Repair-deficient Yeast
b. Mammalian Cells
(1) DNA Single-strand Breaks
(2) Unscheduled (Nonreplicative) DNA Synthesis
-------
c. Rodent Germinal Cells
3. Chromosomal Effects
a. Yeast (Mitotic Recombination)
b. Insect (Drosuphila)
(1) Dominant Lethality
(2) Chromosomal Rearrangements
c. Mammalian Cells
(1) Chromosomal Aberrations
(2) Sifter Chromatid Exchange
' 'i
d. Rodents . |
: , j
. - i
(1) Cytogenetic Analysis . j
Bone Marrow . ;
Circulating Peripheral Lymphocytes
Spermatocytes ' i
(2) Heritable Chromosomal Damage i
i
Sex Chromosome Loss
Dominant Lethal Effects _ o j
i
Heritable Trarislocation Test j
i
4. Methodological Considerations
a. Metabolic Activation . i
i
(1) In vivo I
. : !
(2) In vitro j
b. Treatment Conditions !
-------
V. 46
V. B. Mutagcnesis
1. Gene Mutations
Induced gene mutations include dominant gene mutations and
recessive gene mutations. The molecular processes responsible for
either can be point mutations, including base-pair substitutions,
additions, or deletions, or small interstitial deletions, defined
as chromosomal deletions which are too small to be detected
microscopically, but which may involve several genes or part of
one gene. The latter is likely to result in limited expression,
even if recessive, while point mutations are rarely expressed
because most are recessive.
The number of gene:, (markers) in mammals currently available
for study are limited; the detection of either dominant or
recessive gene mutations in whole animals can require tens of
thousands of animals. This limits the use of whole ar.imals for
this type of mutagenicity testing, which can be performed with
lower organisms.
Induced gene mutations are detected as changes of specific
loci or as recessive lethals. Both tests can measure mutations
other than gene mutations. The former .is measured using forward
or reverse mutation, while the latter is usually detected as sex-
linked lethals.
In application of any in^ vir.ro mutagenesis system'," careful
attention should be given to the metabolic activation capability
of the system; and, if not inherently present in the specific
organism, an appropriate metabolic activation system must be
employed.
Reverse mutation assays can be performed with a number of
unicellular organisms. A major consideration in performing reverse
mutation assays is that a given chemical may cause mutation by
only one specific molecular mechanism, e.g., base-pair substitution
as compared to frameshift mutation, and if the indicator organisjn
only demonstrates reversion due to one of these mechanisms, poten-
tially mutagenic agents can go undetected. To eliminate this
difficulty, each system employed should allow for testing against
different strains of the same organism which respond to chemicals
acting by different mechanisms. In each case, these strains
chosen must be of documented genetic character, and a data base
must exist for its response to known chemicals acting by different
mechanisms.
Forward mutations can be detected in several unicellular
biological organisms; they offer the advantage, as compared to
reversion mutations, of being caused by several different mutagenic
mechanisms. This may be of advantage in determining relative
-------
V. 47
mutagenic potential of a series of chemicals, at least in that
organism; this relative ranking would be very difficult when a
series of different tester strains are employed for reversion mutation
assay. Bacterial systems for detection of forward mutation are
being developed.
a. Procaryotic Microorganisms-(Bacteria)
Reversion at different loci (e.g., histidine, tryptophan,
ar.d arginine loci) in different strains of Salmonella typhimurium,
Hscherichia coli, and Bacillus subtilis.
The currently available bacterial reverse mutation tests include .
the histidine reversion test in Salmonella typhimurium with associated
metabolic activation system (Ames et^ al_., 1973, 1975; McCann et_
al., 1975; McCann and Ames, 1976; Frantz and Mailing, 1975); reversion
tests in fcscherichia coli K-12, e.g., at the arginine locus (Mohn
et al., 1974); reversion in Escherichia coli WP2 at the tryptophan
locus (Bridges et_ al_., 1967; Bridges, 1972; Mitchell, 1974; Green
and Muriel, 1976). At the present time, the most extensive validation,
i.e., positive correlation with known carcinogens and .noncarcinogens,
has been performed with the S_. typhimurium histidine reversion assay
and reversion in Bacillus subtillis 168 I/V at leucine and valine
loci (Popper et^ al_., 1973). . .
The immediate advantage of the bacterial level reversion
assays ;is the low cost and rapidity of obtaining test results
which can indicate the potential of a chemical to cause mutations.
This is not, of course, to say tMat thfi chemical will be mutagenic
in man, but rather that it has the potential for being so. . Pre- |
suming appropriate selection of bacterial tester strain, and i£
vitro metabolic activation systems, consideration should also be
given to whether the"system can respond to insoluble components of
the test material (if a mixture), introduction, of turbidity into . ...
the test plates, and evaporation of volatile agents. The assay
will not indicate what effect different routes of entry have on :
in vivo mutagenesis.
In performing reversion assays the tester should use a
standardized and documented procedure, maintaining regular checks
of the genetic nature of the tester strains. Any modifications of
the procedure in order to take into account solubility or volatility
of the test agent should be specified, with listing of the criteria
for its selection; Lhis is particularly important in view of the .'-
possibility that directly or potentially biohazardous components
of complex mixtures may be present in small proportions.
Positive controls should be run with any metabolic activation
system, and, also, known chemicals (positive controls) must be run
in the presence of the test chemical (or mixture) to verify that
the latter is not inactivating the metabolic activation system.
-------
ir.e acvanra^e or reverie mutation is its convenience, i.e.,
Available bactrriai reverse nut a tier. tests include:
i,i) sal BO r.el I a typhimurium with Mammalian
MctabolL- Activati.-.z iysrem - Assay for
Kisricine Reversion
The plate incorporation form of -his assay esc loving the
improved tester strains is the method 'vhi:h hi< ::-ee^ emnicvic fjr
vaiidation. i.Xnes £££^-', i^"5j . Several Ji';di;i,:ition.s of thii
assay are particularly .relevant to the screemn.| of fuel and f-.i-.ai'
a-j-'viitive test ouitcrials:
Modification of .i_;s.iy for Volatile
; 1975; Xues et_ i_l_. , 1973}
' Modification of Assay for ^uantitaticn
, of Toxic ity and Mutagenicity by U-se of
Liquid Susper^i-:r.
. .Mailing, 1971; .ijie-5 et aj. . , 1975,
' Modification of Assay for Scrs-rriiiig of
, Maamaiian Urinary vierj.bolites
(Durston. and .-.Ties, 1974; CO'inp.on.er et ai.,
As pointed out elsewhere -in this document, by far the sest
extensively and rigorously validated of a11.in vitro Jiutajen-
detection methods is the- S.alaor.ella tyrhiniuri-jai st^indard plate"
assay of \-Tiei et ai. 1^1975, , *r.ic.~ in-o~orrites .na-nmaiian metabolic
activation". The basis fcr this validatizn 'is the s-reenin.j :f
some 500 caro-.nojenic i.I'NA- reactive; and ncnzarc-nCjeniC chemi:ai
compounds of various structural types, resulting in the detecti:n
of approximately 90;; of known caicinoiens i~ positive ^lutazens by
the assay' >!cCann et_ ai_. , 1975: N'cCann and .\jies, 197c). Further
information with respect tc vaiic'ation have also been published
The S_. t;'-phiJiuriuji assay is rapid, re-:u:rinz .1-5 days f ;r the
provides dose-response data :or T:Uta.jens -vLth a >ice rar,|e or
muta^-enic: potency i.Ajies , 19~o} . Of particular relevance to the..;
-------
V. 49
testing of the mutagenic capacity of fuel and fuel additive test
materials, this assay has been employed for mixtures (Kier et aj^,
1974; Ames, Kammen and Yamasaki, 1975), and for air-borne polycyclic
aromatic hydrocarbon particulatcs (Tokiwa _e_t a_l_., 1977).
The usual methodology of these assays, when a metabolic
activation system is employed, is to combine the latter in the
agar with the bacteria during the entire incubation period. As
indicated in several of the above references, and also by Bartsch
e£ aj_. (1976) and Herbold e_t a_l_. (1977), this "standard" plate
assay has many problems, and a suspension treatment prior to
incorporation into agar probably should be validated as the
"standard" assay (Bartsch...e_t a_l_., 1976; Sugimura et_ a_!L ,. 1976) ,
Other recent developments in bacterial mutageaesis iitc-lude application
of a fluctuation test (Greene £t^ al_., 1977) and use of concentration
gradient plates (Cline and McMahon, 1977).
(2) Cscherichia coli WP2 with Mammalian Metabolic
Activating Systems - Assay for Tryptophan
Reversion
(Bridges crt al_., 1967; Bridges, 1972;
Mitchell, 1974; Greene and Muriel, 1976)
WP2 is a tryptophan-requiring strain of £. coli which can be
caused to revert to tryptophan-independence by base substitution; ]
a number of derivatives of this strain are available which arc ; j
deficient in different"DNA repair mechanisms. These repair- ; .
deficient strains offer the advantages of: (1) detection of )j \
lower doses of mutagen for those strains in which the missing f .
repair process would normally remove the damaged section of DNA; - ]
and (2) hypersensitivity of agents damaging DNA for those strains ' '
for which the repair process is itself involved in the conversion
of DNA damage to the final mutated base sequence. An additional
advantage is that-it ts possible, to partly characterize the type , - .
of DNA damage produced;by a given agent from the pattern of response
of repair-deficient strains (Green and Muriel, 1976).
A disadvantage of the IYP2 system at present is the .lack of an
extensive data base required for validation; however, a data base
appears to be developing (Shirasu et^ al_., 1976; Brusick, 1977).
(3) Cschcrichia coli K12 with Mammalian Metabolic
Activating System - Assay for Reverse
Mutation at Arginine and Nicotinic Acid Loci
and Forward Mutation for Resistance Against
5-methyltryptophan and for Gal* Phenotypc
(Mohn ct^ a_K, 1974; Mohn and l;llenbergcr,
' 1977)
K12 (strain 343/113) is a multi-purpose strain of Ii. coli in ;
which both reversion and forward mutations, of several genes can
-------
V. 50
be detected. The assay ;can be performed in .spot tests, suspension
tests, with mammalian me.tabolic activation, and in mammalian host-
mediated tests (Moh'n e^ a^., 1974). In addition to allowing for
detection of forward mutation, an advantage of this assay of
particular pertinence to the testing of fuels and fuel additives
is that it also manifests greater sensitivity toward certain
carcinogens, such as nitroheterocylic compounds and dialkylnitros-
amiries, and it is also more suitable than the Salmonella system for
host-mediated assay (Sobels, 1977).
As to validation, the data base for this test appears to be
severely limited at this time (Brusick, 1977).
-------
V. 51
I
V. B. 1. a. References for Gene Mutations - Procaryotic j
Microorganisms (Bacteria) . . . {
«
I
Ames, B., McCann, J., and Yaroasaki, E. (1975) Methods for j
detecting carcinogens and mutagens with the Salmonella/mammalian- !
microsome mutagcnicity test. Mutat. Res. 31:347-364. 1
!
Ames, B., Lee, F., and Durston, W. (1973) An improved bacterial j
test system for the detection and classification of mutagens and ;
carcinogens. Proc. Natl. Acad. Sci. USA 70:782-786. i
McCann. J., Choi, E.~, Yamasaki, E. , and Ames, B. N. (1975) j
Detection of carcinogens as mutagens in the Salmonella/microsome ;
test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. USA 72: j
5135-5139. ' |
McCann, J. and Ames,'-B. N. (1976) Detection of carcinogens as >
mutagens in the Salnionella/microsome~test: Assay of 300 chemicals: :
Discussion. Proc. Natl. Acad. Sci. USA 73:950-954. V :
_ ' I:
Frantz, C. N. and Mailing H. V. (1975) The quantitative microsomal
mutagenesis assay method. Mutat. Res. 31:365-380.
Mohn, G., Ellenberger, J. and McGregor, D. (1974) Development of
mutagenicity 'cest.s using Eschericiiia coli K-12 as indicator organism.
Mui-at. Res. 25:187-196. : " : '' "-.
ri
Bridges, B. A., Dennis, R. E., Munson, R. J. (1967) Mutation in '
Escherichia coli B/r. WP2 try" by reversion or suppression of a cuain- : ;
terminating condon. Mutat. Res. 4:502-504.
.Bridges, B. A.,. (1972) S'.iple bacterial systems for detecting. ,- ,. .,,.-';
mutagenic agents. Lab. Fr~ ,tice 21:413-416.
Mitchell, I. de G. (1974) A comparison of the sensitivity and
specificity of microbial systems for assessing genetic damage.
Agents and Actions -t: 286-294.
Green, M. H. L. and Muriel, W. J. (1976) Mutagen testing using ;,
trp+ reversion in Escherichia coli. Mutat. Res. 38:3-32.
Popper, H., Czygan, P., Greim, H., ScKaffncr, F., and Garro, A. J.
(1973) Mutagenicity of primary and secondary carcinogens altered
by normal and induced hepatic microsomfis-. Proc. Soc. Exp. Biol.
-------
' j,
r
V. i-2 t
V. 8. I. a. (continued) :
:' '
(1) Salmonella typhiniurium Assay
Assay technique/validation:
3
Ames, B., McCann, J., and Yamasaki, E. (1975). Methods for
detecting carcinogens and mutagens with the Salmonel 1-i/mansna? ian-
microsjme '.nutagenicity test. Mut.at. Res., 31: 347-364.
McCann, J., Choi, E., Yamasaki, E., and Amos, B. N. (1975).
Detection of carcinogens as mutagens in the Salmonell.a/mcruscme
test: Assay of 300"chemicals. Proc. Natl. Acad- Sex., USA,
72: 5135-5139.
McCann, J. and Ames, B. N. (1976). Dececticn ot carcinogons as I
mutagens in the Salmonella/microsome test: Assav of 300 chenicals:
Discussion. Proc. Natl. Acad. Sci., USA, 73: 950-9S4'.
Frantz, C. N. and Mailing, H. V. (1975). The quantitative microoOtf.il
mutagenesis assay method. Mutat. Reb., 31: 365-380.
MaJaveille, C., Planche, G., and Bartsch, H. (1977). Factors for
efficiency of the Salmonella/microsome mutagenicity assay.
Chem.-Biol. Interactions, 17: 129-136. . ; ;
Environmental Protection Ager.cy.. (1977). Pesticides Guidelines,
draft revision. . ...
Pertinent experimental considerations/interpretation:
,Dur?ton, W: E. and Ames, B.- N. (1974). A simple method for the - - ' -
detection of mutagens in urine: Studies with the carcinogen
2-acetylaminofluorene. Proc. Natl. Acad. Sci., USA> 71: 73'/ 741.
Commoner, B., Vithayathil, A. J., ar.d ifenry, J. (1974). Detection ;
of metabolic .carcinogen intermediates i.r. urin.^ of Cuicinogen-feu :
rats by means of bacterial mutagenesis.. Nature. 249:^. C50-85?.
Bridges, B. A. (1976). Short term screening tests for -:arcinjgens. ';:
Nature, 261: 195-200.
Shirasu, Y., Moriya, M.. Kato, K... Furuhashi, A., and Kar»a, T. fl976). '-"
Mutagenicity screening of pesticides invthe microbia.i. system.
Mutat. Res., 40:. 19-30.
Rosei.kranz, H. S., Gutter, B., a!-.d Speck, W. T. (1976J. Mutagenicity \
and DNA-modifying activity: A comparison of two microbial assays.
Mutat." Res., 4.1 . 61-70. '' ;;
Drake, J. W., Aorahaniro.1, S., Crow, J. F., HoJlaender, A., Lederberg, S.,
Legato*!-, M. a., Neel, J. V., Shaw, M. W;, Button, H. E., von Borstel, R.,<
and i.iirn.-^ing, S. (.1^7^). Enviro..tnjntal mutagenic hazards. ^
Science, 187: 503-514. '..'-:. . ' '.f.
-------
V. 53
V. B. 1. a,, (continued)
/Vines, B. N. (1976). Carcinogenicity tests. Science, 191: 241-
245.
Kier, L. D. , Yanasaki, E., and Ames, B. N. (197-1). Detection
of nutagenic activity in cigarette sraoke condensates. Proc.
Nat. Acad. Sci.,USA, 71: 4159-4163.
« ^
Araes. B. N., Karaaien, II. 0., and Yamasaki, E. (1975). Hair dyes
are nutagenic: identification of a variety of mutagenic ingredients.
Proc. Kiir. Acad. Sci. USA, 72: 2423-2427.
Tokiwa, H., Morita, K., Takeyoshi, H., Takahashi, K., and Ohnishi, Y.
(1977). Detection of nutagenic activity iri paniculate air
pollutants. Mutat. Res., sS: 257-248.
Bartsch, H., Caaus, A., and Malaveille. C. (1976). Comparative
mutagenicity of N-nitrosaiaines in a semi-solid and in a liquid
incubation systea in the presence of rat or human tissue fractions.
Mutat. Res., 37: 149-162.
Herbold, B. A., Rohrbom, G., Buselnaier, W. (1977). Point-
mutation research: relevance for humans. J. Toxicol. Environ.
Health, 2: 1183-1191.
Sugimura, T., Yahagi, T., Nagao, M., Takeuchi, M., and Kuwachi, T.
(1976), Validity of tr.utagenicity tests using taicrobes as a rapid
screening method for environmental carcinogens. In: Screening
tests in cheraical carcinogenesis, R. Montcsano. H. Bartsch, and
L. Toaatis, eds., pp 81-101, IARC Scientific publications no. 12,
Lyoa, France. International Agency for Research on Cancer.
Green. M. H. L., Bridges, B. A., Rogers, A. M., Horspool, G.,
Muriel. W. J. t -Bridges-. J. W., and Fry, J.- R. ,(1977).- ffutagen ^
screening by a simplified bacterial fluctuation test: Use of
nicrosomal preparations and whole liver cells for metabolic
activation. Mutat. Res., 48: 287-294.
Clinc, .1. C. , and Mc.Mahon, R. P.. (1977). Detection of chemical
nsutagens: Use of concentration gradient plates in a high capacity
screen. Res. Comnum. Qicra. P.ithol. Phanaacol., 16: 523-533.
-------
V. 54
V. B. 1. a. (continued)
(2) . Eschcrichia coli W2/microsotae assay
Bridges. B. A., Dennis, R. I-., Munson, R. J. (1967). Mutation
in |--i.c.fo_e.rJLc.!lLa co1* B/r Wl>- tr>'~ b>' reversion or suppression of
a chain-terminating codon. Mutat. Res., 4: 502-504.
Bridges. B. A., (1972). Simple bacterial systems for detecting
mutagenic agents. Lab. Practice. 21: 413-416.
Mitchell, I. de G. (1974). A comparison of the sensitivity and
specificity of microbial systems for assessing genetic damage.
Agents and Actions, 4: 286-294.
Green. M. H. L. , and Muriel, W. J. , (1976). Mutagen testing
using trp* reversion in Escherichia coli . Mutat. Res., 3S:
3-32. . : :
Shirasu, Y. , Moriya, M. , Kato, K. , Furuhashi, A., and Kada.T.
(1976). f'fejtagenicity screening of pesticides in the microbial system.
. Res., 40: 19-.30.
Brusick, D. J. (1977). In vitro tautagencsis assays as predictors
of chemical carcinogencsis in mammals. In: Toxicology Annual, vol. 2,
C. L. Kinek and S. P. Shanor, eds. , pp 79-109. New York. Marcel
Dckker, Inc.
-------
v- 55
. - :-----:-f-:.-.'^-:^
V. B. 1. a. (continued) " : "!:£
(3) Eschcrichia coli K12/microsome assay " ""'"'
^ i
Mohn, G., Ellenberger, J., and McGregor, D. (1974). Development ;x
of mutagenicity tests using Escherichia coli K-12 as indicator /
organism. Mutat. Res., 25: 187-196.
Mohn, G. R. and Ellenberger, J. (1977). The use of Escherichia
coli K12/343/113 ( ) as a multipurpose indicator strain in
carious mutagenicity testing procedures. In: Handbook of
rautagenicity test procedures, B. J. Kilbey, M. Logator, W. Nichols
and C. RAmel, eds., Amsterdam. Elsevier/North Holland Biomedical
Press. 475pp.
Sobels, F. H. (1977). Some problems associated with the testing
for environmental mutagen3 and a perspective for studies in
"comparative mutagenesis". Mutat. Res., 46: 245-260.
Brusick, D. J. (1977). In. vitro mutagenesis assays as predictors
of chemical carcinogenesis in mammals. In: Toxicology annual, /
vol. 2, C. L. Kinek and S. P. Shanor, eds., pp 79-109. New York .//
Marcel Dekker, Inc.
-------
b. liukaryotic Mi-.roorganisnis
(1) Forced Hctcrokaryons of the Ascoraycetes
Ni'tirospora erassa
The induction oi: forward mutations in the ad-5 locus of a two-
compartment heterokaryon of Ncurospora erassa is considered to be
caused by a wide spectrum of genetic events. For the detection
of forward Mutation due to genetijc. alterations at the ad - 5 locus,
a wry simple procedure involving the appearance of purple colonies
is available (de Serres and Mailing, 1971). A published data
base is apparently limited.
Also employing the two-component heterokaryon of Ncurospora
erassa, it is possible to measure mutation induction resulting
from any kind of genetic damage that produces complete inactivation
over the entire genome. An estimate of the frequency of recessive
lethal damage over the entire genome can be obtained by determining
the percentage of heterokaryotic colonies with recessive lethal
damage in one of the two components. The recessive lethal mutations
in this system result from both point mutations and chromosome
deletions (de Serres and Mailing, 1971).
(2) Haploid Strains of Yeast
The forward mutation assay in haploid strains of yeast can
be performed by detecting loss of function., i.e., certain forward
mutations cause resistance to certain anti-metabolites which allow
for selective procedures. Alternatively, forward mutation at
the so-called red-adenine genes can be determined (Mortimer and
Manney, 1971; Brusick and Mayer, 1973; Zimmermann, 1973, 1975).
-------
V. 57
V. B. 1. b. References for Eukaryotic Microorganisms - -
i
(1) . Neurospora crassa
Ong, T-M., (1977). Use of the spot, plate and suspension
test systems for the detection of the rautagenicity of
environmental agents and chemical carcinogens in Nourospora crassa.
Ong, T-M., and de Serres, F. J. (1972). Mutagenicity of chemical
carcinogens in Neurospora crassa. Cancer Res., 32: 1890-1893.
de Serres, F. J. and Mailing, H. V. (1971). Measurement of
recessive lethal damage over the entire genome and at two
specific, loci in the ad-3 region of a two-component heterokaryon j
of Neurospora crassa. In: Chemical mutagens, Principles and j
methods for their detection, vol. 2, A. Hollaender, ed., pp 311-342. |
New York. Plenum. -' . f
»
J
(2) Haploid Strains of Yeast I
limmermann, F. K., (1975). Procedures used in the induction of
mitotic recombination and mutation in the yeast Saccharomyces
cerevisiae. Mutat. Res., 31: 71-86.
Zimmermann, F. K. (1973). Detection of genetically active
chemicals using various yeast systems. In: Chemical mutagens,
principles and methods for their detection, «ol. 3, A. Hollaender,
ed., pp 209-239. New York. Plenum.
Mortimer, R. K., and Manney, T.-R., (1971). Mutation induction"
in yeast. In: Chemical Mutagens, Principles and methods for
their detection, vol. 1, A. Hollaender, cd., pp 28:)-310. New
York. Plenum.
Brusick, D. J. and Mayer, V. W., (1973). New developments in
mutagcnicity screening techniques with yeast. Environ. Health
Perspect., 6: 83-9o.
-------
V. 5S
c. Plants
(1) Tradescantia - Pink Mutation of Stamen Hair
Cells
Tradcscantia clone 02 is a diploid plant (2n = 12), hcvcroz/gous
for flower color. A change from the normal blue color to pink
in cells of stan:c-n hair after chemical treatment is considered
a true mutational event because of this heterorygosity. Other
types of "aberration events" can also be scored. The exact mechanism
of the mutational event causing the change from blue to pink is
unknown; it nuy include deletion events.
The examination of Tradcscantia stamen hairs for "pink events"
as an indicator of mutation appears to be straight-forward, and
the system appears uniquely useful for gaseous exposures (Underbrink
et^ al_., 1973; Sparrow e£ al_., 1974; Nauaar. ct_ aj_., 1976; McNulty
e_t_ aj_., 1977). The available data base is limited; this system
is still under development.
-------
V. 59
V. B. 1. c. References for Plants
(I) Trndescuntia
Underbrink, A. G., Schairer, L. A., and Sparrow. A. H. (1973).
Trndcscantia stamen hairs: A radiobiological test system
applicable to chemical mutagencsis. In: Chemical mutagens,
Principles and methods for their detection, vol. 3, A. Hollaender,
ed., pp 171-207. New York. Plenum.
Sparrow, A. H., Schaircr, L. A., and Vi1lalobos-Pietrini, R.
(1974). Comparison of soraatic mutation rates induced in
Tradescantia by chemical and physical miitagens. Mutat. Res.,
26: 265-276.
Nauraan, C. II., Sparrow, A. rl. , and Schairer, L. A. (1976).-
Comparative effects of ionizing radiation and two gaseous
chemical mutagens on somatic nutation induction in one mutable
and two non-mutable clones of Tradescantia. Mutat. Res., 38: 53-70.
McNulty, P. J., Nauman, C. II., Sparrow, A. H., Schweramer, S. S.
and Schairer, L. A. (1977). Influence of x-ray-dose fractionation
on the frequency of somatic mutations induced in Tradcscantia
stamen hairs. Mutat. Res., 44: 235-246.
. v
-------
V. 60
d. Insect
(1) Drosophila mclanogastet
The sex-linked recessive lethal r.cst in Drosophila is considered
applicable to mutation screening since it can delect mutations
arising at many loci in the genome, and it can detect the more
common types of gene mutations.
In performing the test, genetically appropriate males arc
treated and mated with genetically appropriate females; daughter
females are then individually mated to several of their brothers.
The resulting generation is examined in the culture container
and the number of cultures which lack normal-eyed (non-Bar) males
are an estimate of the nuinter of treated X chromosomes which carry
one or more sex-linked recessive lethal mutants. This test system
can also be designed to study mutation in treated females (Abrahamson
and Lewis, 1971; Vogel and Leigh, 1975). Recently, attention has
been given to the capacity'of Drosophila to metabolically activate
pre-carcinogens (Sobels and Vogel, 1976; Baars ££ a_l_-, 1977).
Mutations in Drosophila,have been observed after gaseous exposures
(Garrett. and Fucrst, 1974); more attention should be given to
validation of this system.
-------
V. 61
V. B. 1. d. References for Gene Mutations - Insect
(1) Drosophila melanogaster
Vogel, E. (1976). The relation between rautational pattern
and concentration by chemical mutagens in Drosophila. In:
Screening tests in chemical carcinogenesis, R. Montesano,
H. Bartsch, and L. Tomatis, eds., Lyon, France. IARC
Scientific Publications no. 12. International Agency for
Research on Cancer.
Vogel, E. and Sobels, F. H. (1976). The function of
Drosophila in genetic toxicology testing. In: Chemical
mutagens, Principles and methods for their detection, vol. 4,
A. Hollaender, ed., New York. Plenum.
Abrahamson, S. and Lewis, E. B. (1971). The detection of
mutations in Drosophila melanogaster. In: Chemical mutagens,
Principles and methods for their detection, vol. 2, A. Hollaender,
ed., pp 461-487. New York. Plenum.
Vogel, E. and Leigh, B. (1975). Concentration-effect studies
with MMS, TEB, 2,4,6-TriCl-PDMT, and DEN on the induction of
dominant and recessive lethals, chromosome loss and translocations
in Drosophila sperm. Mutat. Res., 29: 383-396. .
Sobels, F. H.and Vogel, E. (1976). The capacity of Drosophila
for detecting relevant genetic damage. Mutat. Res., 41: 95-106.
Baars, A. J., Zijlstra, J. A., Vogel, E., and Breimer, D. D.
(1977). The Occurrence of cytochrome P-450 and aryl hydrocarbon
hydroxylase activity in Drosophila melanogaster microsomes,
.rand the. importance of-this metabolizing capacity for the screening '
of carcinogenic and mutagenic properties of foreign compounds.
Mutat. Res., 44: 257-268.
Garrett, S. and Fuerst, R. (1974). Sex-linked mutations in Drosophila
after exposure to various mixtures of gas atmospheres. Environ.
Res., 7: 286-293.
-------
V. 62
e. Mammalian Cells
Several icammalian cell culture systems are available for detecting
forward mutations. The use of mammalian cell systems is a ra^.dly
developing area, and it will be important to specify choice of
mutation system and criteria of selection. The nununalian cell
systems are either diploid or aneupl^id; they are more complex
biochemically and cietabolically than the bacterial level rautagenicity
systems, which are in several instances "sensitized" to chemical
penetration and damage response.
It will be necessary to determine which mammalian cell system
is most appropriate for a given test material, taking into consideration
the available data base for each system at the time of testing;
criteria for selection should be specified. Qther important considerations
are whether the system selected has the capability of metabolically
activating chemicals; and duration of treatment.times, since potentially
active chemicals may be present in small percentages in a mixture.
Currently available mammalian cell assay systems include:
(1) Mouse lymphoma, 1.517SY, TK +/- forward mutation at
the thymidine kinase locus - assay of Clive and Spector (iy75)
with and without metabolic activation.
(2) Chinese hamster lung, V79, HGP.rtT +/- forward mutation
at the hypoxanthine-guanine ohosphoribosyl-transferase locus
(Chi;, 1971; Newbold et^a.^.-, 1977; tPA Pesticide Guidelines, 1977).
(5) Chinese hamster ovary, CHO, HGPRT »/- forward mutation
at the hypoxanthine-guanine phosphoribosyi-transfcrase locus -
assay of llsie ej^ al. ^"^3) with and without metabolic
activation.
-------
V. 63
V. B. 1. e. References for Gene Mutations - Mammalian Cells
(1) 'Mouse lymphoma, L5178Y
Clive, D. Flarara, W. G. , and Patterson, J. B. (1973).
Specific locus rautational assay systems for mouse lymphoraa cells.
In: Chemical mutagcns, Principles and methods for their
detection, vol. 3, A. Itollacnder, ed., pp 79-103. New York.
Plenum.
Clive, D. (1977). A linear relationship between
tumorigenic potency i_n vivo and mutagenic potency at the hetero-
zygous thyraidine kinasc (TK +/-) locus of L5178Y mouse lymphoma
cells coupled with mammalian metabolism. Abstract no. Bb-3,
Presented at Environmental Mutagen Society Meeting, Feb. 1977,
Colorado Springs, Colorado.
Clive, D. and Spector, J. F. S. (1975). Laboratory procedure
for assessing specific locus mutations at the TK locus in
cultured L5178Y mouse lyraphoma cells. Mutat. Res. 31: 17-29.
(2) Chinese hamster lung, V79
Ihibcrman, E. and Sachs, L. (1976). Mutability of different genetic
loci in mammalian cells by inetabolically activated carcinogenic
polycyclic hydrocarbons. Proc. Natl. Acad. Sci.,USA, 73: 188-192.
Krahn, D. F. and Heidclburger, C. (1977). Liver horaoRenate-
mcdiatcd mutagenesis in Chinese hamster V79 cells by polycyclic
aromatic hydrocarbons and aflatoxins. Mutat. Res. 46: 27-44.
Chu, E. H. Y. , (1971). Induction and analysis of gene mutations
in raamni.ilian cells in culture. In: Chemical mutagens, Principles
and methods for their detection, vol. 2, A. Hollacnder, ed.,
pp 411-444. New York. Plenum.
Ncwbold, R. F., Wiglcy, C. B. Thompson, M. H., and Brookes, P.
(1977). Cell-mediated rmjtagcncsis in cultured Chinese hamster
cells by carcinogenic polycyclic hydrocarbons: Nature and
extent, of t'\c associated hydrocarbon-DNA reaction. Mutat. Res.,
43: 101-116.
Environmental Protection Agency, (1977). Pesticide guidelines,
draft revision.
-------
TJ'
V. 64
V. B. 1. c. (continued)
(5) Chinese Hamster Ovary, CHO
llsic, A. K. , Couch. D. B., Brimcr, P. A., O'Neill, J.P., .
and Machanoff, R. C1977). The utility of a quantitative
sensitive and versatile mutational assay at the hypoxanthine-
guanine phosphorlbosyl transferase locus of Chinese hamster
ovary cells (CHO/HGPRT assay) in determining fhe mutagenicity , j
of physical and chemical carcinogens. Abstract to be presented <
at the American Assoc. of Cancer Research Mtg. . j
O'Neill, J. P., Brimer, P. A., Machanoff, R., Ilirsch, G. P. ' \
and Hsic, K. W. (1977). A quantitative assay of mutation ; 1
induction at the hypoxanthine-guanine phosphoribosyl trans- |
fcrase locus in Chinese hamster ovary cells (CHO/HGPRT system): ;"
Development aiid definition of the system. Submitted ID Mutat. ; I
Res. - ; |
.1
O'Neill, J. P., Couch, D. B., Machanoff, R., San Sebastian, J. R., j
Brimer, P. A., and Hsie, A. W. (1977). A quantitative assay of ]
mutation induction at the hypoxanthine-guanine phosphoribosyl 1
transferase locus in Chinese hamster ovary cells (CHO/HGPRT ?
system): Utilization with a variety of mutagcnic agents. !
Submitted to Mutat. Res. i
Hsic, A. K., Li, A. P., and Machanoff, R. (1977). A fluence- j
response study of lethality and mutagenicity of white,;"black, J
and blue fluorescent light, sunlamp, and sunlight irradiation J
in Chinese hamster ovary cells. Mutat. Res., in press. *
O'Neill, J. P. and Hsic, A. W. (1977). Chemical mutagenesis { J
of-maronalian cells can be quantitated-. 'Nature; in press." " ' ' " j
; j
Hsic, A. W., Brimer, P. A., Mitchell, T. J., and Gosslce, D. G. . j
(1975). The dose-response relationship for ethyl mcthanesulfonate- j
induced mutations at the hypoxanthine-guanine phosphoribosyl }
transferase locus in Giinesc hamster ovary cells. Somatic j
Cell Gen., 1: 247-261. ]
Hsic. A. W., Brimcr, P. A., Mitchell, T. J.', and Gosslce, D. G. |
(1975). The dose-response relationship for ultraviolet-light- I
induced mutations at the hypoxanthine-guanine phosphoribosyl
transferase locus in Chinese hamster ovary cells. Somatic Cell
Gen., 1: 333-389.
i J
-------
V. 65
f. Rodent .
(1) Specific Locus Assay in Mice
This test determines point mutations in mammals, a class of
mutations of particular concern for human populations because the
genetic damage may be initially invisible and only show serious
consequences many generations later. Mouse coat color and morphologic
markers representing seven specific loci are observed to estimate
rates of forward mutation. After treatment wild-type males or
females are bred with non-treated mates homozygous for the seven
recessive genes; a viable mutation at any of the loci-can be detected
in the offspring allowing estimation of the rate of induction of
recessive traits. This is a valuable test sir.ce it provides a
direct measure of recessive mutations in mammals; it presently
is the basis for most of the estimates of mutagenic risk in humans
(Russell, 1951; Searle, 1975).
Advantages of this assay are: it is an intact ill vivo system
therefore different routes of exposure could be compared, and pharma-
cokinetic distribution mechanisms are operative; also most significantly,
effects upon germinal tissue are being tested, and mutant animals
can be visibly recognized at one week after birth. Disadvantages
are that 20,000 to 30,000 offspring must be scored of each dose
level of mutagen, making it a very expensive and tiir.c-ccnsunung
test; and that only a few loci are monitored and the nature of
genetic alteration cannot be identified in a majority of cases.
The test is considered marginally sensitive, and laboratories with
the necessary mouse colony facilities and scientific expertise
are limited.
-------
V. 66
(2) Mouse Isoiyrac Specific Locus Tesr.
/
This is another murine specific locus assay involving mutations
at ..aracteristic isozyrae loci - i.e., genes coding for variations
in protein structure of different forms of the sane cniyne. Specific
enzymes from different tissues of offspring of treated male and
female animals are compared for variation in electrophoretic mobility
profiles (isoen:yme variance) as compared to the iso;yae constitution
of the same enzymes of the untreated parents. This analysis allows
the detection of mutant phenotypes where the en:yae is not destroyed,
but rather has had its charge altered due to changes in amino acid
composition, or has undergone alteration in enzymatic activity. j
This assay requires selection of mouse strains with appropriate j
isoenzyrne differences (e.g., C57BL/6 and UBA/2 mice) ami is not j
yet as well developed as the preceding specific locus test (Valcovic j
and Mailing, 1973). j
-------
V. 67
V. B. 1. f, References for Gene Notations -.Rodent
(1) Specific Locus Assay/Mice
Russell, IV. L. (1951). X-ray-induced mutations in mice.
In: Genes and mutations, Cold Spring Harbor Symposia on
Quantitative Biology, vol. XVI, pp 327-336. Cold Spring
Harbor, L. I., New York. The Biological Laboratory.
Soarle, A. G. (1975). The specific locus test in the mouse.
Mutat. Res., 31: 277-290.
(2) Mouse Isozyme Specific Loous Assay
Valcovic, L. R. and Mailing, H. V. (1973). An approach to
measuring germinal mutations in the mouse. Environ. Health
Perspect., 6: 201-205.
-------
V. 68
2. UNA Damage and Repair
These assays do not measure mutagenic activity as such, but
indicate that a given chemical has entered the cell, and through
some indeterminate mechanism caused damage to the DNA. One type
of damage can be measured directly, i.e., by attempting to detect
DNA single strand breaks. Other types of damage can be detected
by testing for repair (by assay for nonreplicative synthesis) of
that damage. Finally, biological systems which require repair
of DNA for survival can be used to detect whether DNA damage has
been caused by the treatment.
a. Repair-defective Microorganisms
(1) Recombinationless Mutants of Bacteria
Bacterial strains can be generated which have impaired mechanisms
of DNA repair. The so-called "rec~ assay" involves screening chemical
agents for relative toxicity against cells lacking this recombination
capability (rec~) and cells with this capability (rec"1"). If the
agent is more toxic against the former than the latter, DNA reactivity
of the test chemical is indicated which is taken as indicative
of a potential for mutagenicity of the test agenf (Kada e_t aJL,
1972; Nagao and Sugimura, 1972, Ichinotsubo et_ ajk, 1977). These
systems are currently being extensively validated :'n Japan.
-------
V. 69
(2) DNA Polymerase-deficient Mutants
of Bacteria
Bacterial strains can be generated which are deficient in
UNA polymerase (pol A"), a DNA-synthesizing enzyme involved in
repair processes. Bacterial cells exposed to agents that react
with DNA can act to protect themselves by excising the altered
DNA sequence and resynthesizing the correct sequence. Those bacterial
strains which are polymerase deficient (pol A~) are more sensitive
to agents which modify DNA than those bacterial cells possessing
the DNA polymerase (pol A+). As in the case of the preceding assay,
"rec" assay, this assay can be indicative of the potential for ,
mutagenicity of a test agent, without employing a battery of tester [
strains or otherwise performing bacterial mutagenicity studies ;
(Slater e£ aj^., 1971; Rosenkranz et_ al_., 1976; Nagao and Sugimura, ;
,1972). |
-------
V. 70 j
(3) Normal versus Repair-deficient Yeast
Ultraviolet light-sensitive mutants of Saccharomyces cerevisiae j
can be generated and utilized as described above for sensitivity j
comparison with wild type cells as an indicator of the DNA reactivity
of chemical agents (Nagao and Sugimura, 1972).
-------
V. 71
V. B. 2. a. References for Repair Defective Microorganisms
(1) Recombinationless Mutants/Bacteria
Kada, T., Tutikawa, K., and Sadaie, Y. (1972). In vitro
and host-mediated "rec-assay" procedures for screening
chemical mutagens; and phloxine, a mutagenic red dye detected.
Mutat. Res., 16: 165-J74.
Nagao, M. and Sugimura, T. (1972). Sensitivity of repair-deficient
mutants to 4-nitroquinoline 1-oxide, 4-nitropyridine 1-oxide, and
their derivatives. Cancer Res., 32: 2369-2374.
Ichinotsubo, D., Mower, H. F., Setliff, .!., and Mandel, M.
(1977). The use of rec bacteria for testing of carcinogenic
substances. Mutat. Res., 46: 53-62.
Nishioka, H. (1975). Mutagenic activities of metal compounds in
bacteria. Mutat. Res., 31: 185-189.
(2) DNA Polymerase-deficient Mutants/Bacteria
Rosenkranz, H. S., Gutter, B., and Speck, W. T. (1976).
Mutagenicity and DNA-modifying activity: A comparison of
two microbial assays. Mutat. Res., 41: 61-70.
Slater, E. S., Anderson, M. D., and Rosenkranz, H. S. (1971).
Rapid detection of mutagens and carcinogens. Cancer Res., 31:
970-973. ':'.'< -,..'--:.- -, - - - , . * - -
Nagao, M. and Sugimura, T. (1972). Sensitivity of repair-deficient
mutants and similar mutants to 4-nitroquinoline 1-oxide, 4-nitropyridine
1-oxide, and their derivatives. Cancer Res., 32: 2369-2574.
(3) Normal versus Repair-deficient Yeast
Nagao, M. and Sugimura, T. (1972). Sensitivity of repair-defi-
cient mutants and similar mutants to 4-nitroquinoline 1-oxide,
4-nitropyridine 1-oxide, and their derivatives. Cancer Res.,
32: 2369-2374.
-------
V. 72
b. Mammalian Cells
(1) DNA Single-strand breaks
Several techniques are available for detecting single strand
breaks in the DNA of treated cells; these include alkaline sucrose
gradient centrifugation (Laishes and Stj-ch,. .1973; Stich ct a 1 . ,
1975) and the alkaline elution technique (Fornace ct_ a_K , 1976;
Kohn et_ al_. , 1976; Swenberg et_ al_. , 1976; Ewfg and Kohn, 1977).
The latter technique offers the additional advantage of detecting
DNA-protein cross-links (Fornace and Kohn, 1976) , It is important
to determine the sensitivity of a given technique for the detection
of single strand breaks. Attention should also be given, as for
other mammalian cell assays, to the possible requirement for a
metabolic activation system. Criteria for selection of a particular
mammalian cell system for a given test material should be justified.
-------
V. 73
V. B. 2. b. References for DNA Damage/Repair - Mammalian Cells
CD DNA Single Strand Breaks
Laishes, B. and Stich, H. (1973). Repair synthesis and
sedimentation analysis of DNA of human cells exposed to
diraethylnitrosamine and activated dimethylnitrosanine.
Biochem. Biophys. Res. Comraun., 52: 827-833.
Stich, H., Kieser, B., Lnishes, R. and Warren, P. (1975).
DNA repair of human cells as a relevant, rapid, and economic
assay for environmental carcinogens. GANN Monogr. Cancer Res.,
17: 3-15.
Pomace, A. J., Jr., and Kohn, K. W. (1976). DNA-protein
cross-linking by ultraviolet radiation in normal human and
xerodenna pignentosura fibroblasts. Biochim, Biophys. Acta,
435: 95-103.
Fomacc, A. J. , Jr., Kohn, K. W. , and Kann, H. E., Jr. (1976).
PNA single-strand breaks during repair of UV damage in
human fibroblasts and abnormalities of repair in xerodenaa
pigcientosuia. Proc. Nat. Acad. Sci.,USA, 73: 39-43.
Kohn, K. K., Erickson, L. C., t'wig, R. A. G., and Friedman,
C. A. (1976). Fractionation of DNA from mammalian cells by
alkaline elution. Biochem. 15: 4629-4637.
Swenberg, J. A., Petiold, G. L., and Harbach, P. R. (1976).
In vitro DNA damage/alkaline elution assay for predicting
carcinogenic potential. Biochem. Biophys. Res. Conmun.,-
72: 732-738.' "
Stich, H. F., Lam, P., Lo, L. W., Koropatnick, D. J. and
San, R. H. C. (1975). The search for relevant short terra bioassays
for chemical carcinogens: The tribulation of a modern Sisyphus.
Can. J. Genet. Cytol., 17: 471-492.
l>ig, R. A. G., and k'ohn, K. W. (1977). DNA damage and
repair in mouse leukemia L1210 cells treated with nitrogen
mustard, 1,3-bis(2-chloroethyl)-I-nitrosourca, and other
nitrosourcas. Cancer Kes., 37: 2114-2122.
-------
V. 74
(2) Unscheduled (N'careplicativc) DNA
Synthesis
Several techniques are available for detecting the occurrence
of DNA repair, defined as unscheduled or nonreplicative,DNA synthesis.
These include: (a) incorporation of the DNA precursor °H-thymidine !
into the acid-precipitable DNA of the cells which are in GQ stage '.;
(nonraitotic; nonproliferatirig, e.g., peripheral lymphocytes); (b) ?
unscheduled DNA synthesis, wherein autoradiographic techniques
are employed to detect cells which incorporate ^H-thymidine into .
their DNA at a lower level during the labeling period than occurs
in cells replicating their DNA. This technique detects repair |
synthesis in non-S phase cells; (c) repair replication; the technique j
involves alkaline density gradient certrifugation procedures to
separate normal density repair-replicated DN'A (labeled with 3||_ ' ;..
BrUdR or ^H-thymidine plus nonradioactive BrUdR) from serai-conservatively i
synthesized BrUdR density-shifted DNA; and (d) photolysis of DNA
incorporating S-bromo-deoxyuridine into repair-replicated regions ;
of the DMA. j
The choice of cell system employed should be justified. The ?
cell should either have its own metabolic capability for chemical * {
activation, or one must be provided (e.g., normal human diploid
cells with aa u± vitro metabolic activation system for unscheduled
synthesis determination). At this time, the most extensive validation
of any test exists for WI-38 system with metabolic activation
(Sinunon et^ al., 1977).
I i
-------
V. 75
V. B. 2. b. References for DNA Damage and Repair - Mammalian
Cells
(2) Unscheduled DNA Synthesis
San, R. H. C., and Stich, H. F. (1975). DNA repair synthesis
of cultured human cells as a rapid bioassay for chemical
carcinogens. Int. J. Cancer, 16: 284-291. '.
Williams, G. M. (1977). Detection of chemical carcinogens by
unscheduled DNA synthesis in rat liver primary cell cultures. '-
Cancer Res., 37: 1845-1851.
Simmon, V. F., Mitchell, A. D., and Jorgenson, T. A. (1977). ;
Evaluation of selected pesticides as chemical mutagens: In
vitro and in vivo studies. Annu. rep., Environ. Health Effects
Series, EPAT600/1-77-028, May, 1977. 1
i
-------
V. 76
c. Rodent Germinal Cells (Mouse Spermatocytes)
Particularly relevant for test materials for which DNA repair
is detected is the determination of. whether or not the agent actually
readies germ cells and causes damage to' the DiNA of these cells.
This can be accomplished by using nonreplicative DNA synthesis
techniques, such as are described in the references given. Since
one of the initial steps is the isolation and purification of sperm,
a number of the above described DNA repair procedures might, also
be applicable. Whichever system is employed will require an appropriate
data base, with adequate positive controls and all other ^n vivo
criteria suitably specified (Sega ct al., 1976) .
-------
V. 77
V. B. 2. c. References for DNA-Damage and Repair - Rodent
Germinal Cells
Sega, G., Owens, J. and Gumming, R. (1976). Studies on
DN'A Repair in early spermatid sta^r* of male mice after
in vivo^ treatment with methyl-ethyl, propyl, and isopropyl
methancsulfonate. !>titat. Res., 36: 193-212.
-------
3. Chromosomal Effects
V. 78 ', . .
; i
Chromosomal effects are defined as breaks within chromosomal
structures (chromosomes or chrumatids) with or without rejoining,
or changes in chromosome number or any part of total morphology.
It is important to differentiate between breaks and gaps, which
may be unstable and heritable only to a slight degree; and chromosomal
translocations and inversions (as well as other stable rearrangements),
which are more likely to be heritable. Such induced chromosomal
effects can be measured in a variety of test systems, ranging from
microorganisms to the tissues of animals and man.
a. Yeast (Mitotic Recombination)
Mitotic recombination is defined as either reciprocal crossing-
over or nonreciprocal gene conversion. The possible result of
mitotic recombination is the generation of a new genotype by exchange
of genetic material between homologous chromosomes followed by
appropriate segregation of the products of mitosis. The result
of such an event in a cell is the expression of a recessive phenotype
in the homozygous condition which would otherwise go undetected
in the heterozygous state (Brusick and Mayer, 1973).
Saccharomyccs ccrevisiac - Metabolic activation is
required; test results may be dependent upon both
time and pli.
-------
V. 79
b. Insect (Drosophila)
(1) Dominant Lethality
The measure of dominant lethal induction in Drosophila involves
comparison of the percentage of eggs hatching in a control versus
a treated series of pair raatings. The failure of the zygote to
develop beyond the embryonic stage is associated with the loss
or gain of one of the major autosomes, or of large portions of
such chromosomes or of the X chromosome. Since the presence of
unhatched eggs can be due to factors other" than chromosome breakage
or chromosome loss (e.g., to direct effects on the gametes, or to
interference with the matir.g performance of the flics) , the occurrence
of a positive test result in the very simple Drosophila dominant
lethal test should be followed by other, available genetic tests
in Drosophila.
-------
V. 80
(2) Chromosomal Rearrangements
Tests for chromosomal rearrangement would not i'.ecessarily
be performed as an initial test, but rather as a confirmatory test
for dominant or recessive lethal positives in Drosophila. This
would allow a determination as to whether the autagen is acting
by point mutations or by breaking chromosomes as well.
Available tests include genetic tests for reciprocal translocations,
position-effect tests for chromosomal rearrangement, and cytological
tests for chromosomal rearrangement.
-------
V. 81
V. B. 3. References for Chromosomal Effects
a. Yeast
Brusick, D. and Mayer, V. (1973). New developments in mutagenicity
screening techniques with yeast. Environ. Health Per-spect.,
6: 83-96.
Zimmerman, F. (1975). Procedures used in the induction ot mitotic
recombination and mutation in the yeast Saccharomyces cerevisiae.
Mutat. Res., 31: 71-86. ~
Zimmerman, F. (1973). Detection of genetically active chemicals
using various yeast systems. In: Chemical mutagens, Principles
and methods for their detection, vol. 3, A. Hollaender, ed.,
pp 209-239. New York. Plenum.
Mortimer, R. and Manney, T. (1971). Mutation induction Ln
yeast. In: Chemical mutagens, Principles and methods i'.r their
detection, vol. 1, A. Hollaender, ed, pp 289-310. New York.
Plenum.
b. Insect
(1) Dominant Lethality
Abrahamson, J. and Lewis, E. (1971). The detection of mutations
in Drosophila melanogaster. Inr Chemical mutagens", Principles
and methods for their detection, vol. 2, A. Hollaender, ed.,
pp 461-487. New York. Plenum.
Vogel, E. and Leigh, b. (1975). Concentration-effect studies
with MMS, TEB, 2,4,6-triCl-PDMT, and DEN on the induction of
dominant and recessive lethals, chromosome loss and translocation
in Drosophila sperm. Mutat. Res., 29: 383-396.
(2) Curomosomal Rearrangements
See references for (1) Dominant Lethality above.
-------
V. 82
c. Mammalian Cells
(1) Chromosomal Aberrations
Mammalian cells in culture provide an _in_ vitro model system,
for assessing chromosomal damage, which may~also be indicative
of the potential of the agent for inducing gene mutations. The
lesion can be classified as a chromatid lesion involving only one
of the two chromatids of the chromosome or a chromosomal lesion
in which both chromatids are involved. A chromatid lesion is likely
to occur if damage occurs after the DNA of that chromosome has
been replicated, while a chromosomal lesion results when damage
occurs before the chromosome had replicated its D.\A,. Abnormalities
seen include breaks, gaps, and rearrangements, with the latter
requiring continual cellular metabolic function to occur. Stable
effects such as deletions and translocations are considered most
significant.
In vitro cytogenetic studies of mammalian cells can employ
either continuously proliferating cell lines or freshly isolated
peripheral leukoyctes from a variety of species. If continuous
cell lines are employed, the cytogenetic character of the cell
line should be well documented, and previous experience of effects
of other agents, including x-irradiation, should be available.
The ploidy must be constant.
These cell lines can be exposed either as an asynchronous,
rapidly proliferating population, where collection and cytogenetic
analysis of the cells at the first mitosis after treatment results
in indication of chromosome damage induced at all stages of the
cell cycle; or they,can be exposed as a synchronised population
of cells, where the tester must indicate why he has so chosen specific-
stages (e.g., increased sensitivity) for testing.
When leukocytes are treated prior to raitogen stimulation,
the cytogenetic analysis of the cells, if performed at the first
mitosis after treatment, will be indicative of chromosome damage
induced pre-DNA synthetic (S) period. The tester can alternatively
choose to treat with test agent during the period of mitogen
stimulation.
Criteria for selection of cell line (and species), protocol
for treatment, protocol for collection of mitotic figures, and
protocol for analysis and scoring and for statistical evaluation
should be specified. He should state how his controls were handled,
and how the experiments were repeated. It is also important to
employ "double-blind" studies to avoid observer bias, and to describe
test controls and replications.
-------
V. 83
V. B. 3. References for Chromosomal Effects (continued)
c. Mammalian Cells
(1) Chromosomal Abberations
Cohen, M. and Hirschhorn, K. (1971). Cytogenetic studies
in animals. In: Chemical mutagens, Principles and methods
for their detection, vol. 2., A. Hollaender, ed. New York.
Plenum.
Brewer, J. and Preston, R. (1974). Cytogenetic effects of
environmental mutagens in mammalian cells and the extrapolation
to man. Mutat. Res., 26: 297-505.
Nichols, W. (1973). Cytogenetic techniques in mutagenicity
testing. Agents Action, 3:' 86-92.
Ishidate, M., Jr. and Odashima, S. (1977). Chromosome
tests with 154 compounds on Chinese hamster cells in_ vitro -
a screening for chemical carcinogens. Mutat. Res., 48: 337-354.
-------
V. 84
(2) Sister Chromatid Exchange
Sister chromatid exchange (SCE) involves a symmetrical exchange
at one locus between sister chromatids, which does not result in
an alteration of overall chromosome morphology.
The measurement of sister chromatid exchanges can be performed
with proliferating mammalian cells in culture; the availability
of fluorescent techniques eliminates thw need for radioactivity.
The procedure requires less time and skill than analysis and scoring
of chromosome aberrations, but it is important to remember that
this test involves cells in the mitosis of the second cell cycle
after initiation of treatment and therefore selects for only those
treated cells which can pass through the first mitosis and.all
of the second cell cycle. Chromosome alterations, in contrast,
can be investigated at the first and/or second mitosis. The occurrence
of SCE seems to be related more closely, when different chemical
mutagen effects are compared, to chromosomal abnormalities in eukaryotes
than to induction of point mutations in bacteria. The sensitivity
of SCE appears to be greater for some chemicals than morphological
chromosome damage detection, but SCE may not be as responsive as
the latter to other chemicals.
SCE can be detected using either continuous cell lines or
freshly-isolated peripheral lymphocytes, which are cultured .in
vitro and mitogen stimulated. It is important to specify: criteria
for selection of cell system (and/or species); protocol for treatment;
and protocol for analysis and scoring. Analysis should be performed
using "double-blind" procedures.
-------
V. 85
V. B. 3. References for Chromosomal Effects (continued)
c. Mammalian Cells
(2) Sister Chromatic! Exchange
Perry, P. and Evans, H. J. (1975). Cytological detection of
mutagen-carcinogcn exposure by sister chromatid exchange.
Nature, 258: 121-125.
Stetka, D. G. and Wolff, S. (1976). Sister chromatid exchange
as an assay for genetic damage induced by mutagen-carcinogens.
II. In vitro test for compounds requiring metabolic activation.
Mutat. Res., 41: 343-350.'
Stetka, D. G. and Wolff, S. (1976). Sister chromatid exchange
as an assay for genetic damage induced by mutagen-carcinogens.
I. In vivo test for compounds requiring metabolic activation.
Mutat. Res., 41: 333-342.
Taylor, J. (1958). Sister chromatid exchanges in tritium-labeled
chromosomes. Genet., 43: 515-529.
Perry, P. and Wolff, J. (1974). New giemsa method for the
differential staining of sister chromatiJs. Nature, 251: 156-158.
Latt, M. (1976). Analysis of human chromosome structure, replication
and repair using Brd V-33258 Hoechst techniques. J. Reprod. Med.,
17: 41-52. , , . p. . _ r, _ . . ., . ..
Solomon, E. and Borrow, M. (1975). Sister chromatic exchanges -
a sensitive assay of agents damaging human chromosomes. Mutat.
Res., 30: 273-278.
-------
V. 86
d. Rodents
(1) Cytogenetic Analysis
In performing cytogenetic analysis upon in_ vivo exposure,
three systems are of primary interest. These are the cells of
the bone marrow, circulating peripheral lymphocytes, and spermatocytes
(after exposure as spermatogonia). In performing ijT. vivo cytogenetic
tests, attention should be given to selection of species (sensitivity,
number and morphology of chromosomes, pharmacology), spontaneous
incidence of chromosome aberrations, effects of age, effects of
other environmental factors, optimum time of examinations, and
duration of treatment. In performing these tests consideration
should be given to application of SCE studies and the micronucleus
test.
Bone Marrow
While proposed herein for studres of induction of chromosome
and chromatid aberrations, the cytogenetic analysis of bone ^narrow
is the best source of information dealing with direct effects on
mitosis (Mitotic index); no in vitro culture is involved. Such
studies can be performed after gaseous exposures.
. Circulating Peripheral Lymphocytes
Circulating peripheral lymphocytes provide the best system
for showing effects of in vivo exposure on cells in G., since most
of these cells are in the pre-DNA synthetic phase while in circulation.
The study of aberration induction in these cells offers great potential
.for use during chronic exposure since~the exposed animals need '- '
not necessarily be terminated to perform this analysis. Tests
can also be performed after (or during) gaseous exposure.
Special attention should be given to the time of analysis
after mitogen stimulation, i.e. 48 vs. 72 hours, so as to distinguish
cells in first mitosis from those in second.
Spermatocytes
The study of gamctocytes can be of considerable importance,
since it is the only means to detect potential genetic damage without
actually studying the offspring.
In performing these tests, it may be necessary to investigate
a complete cycle of spermatogenesis so as to include effect differences
or. specific stages.
-------
V. 87
(2) Heritable Chromosomal Damage
Sex Chromosome Loss
This test can be performed in Drosophila, with treatment of
tho male parent being performed. The test available should be
performed with caution, however, since the spontaneous frequency
of loss of a given chromosome is much more subject to external
modification than is the spontaneous frequency of mutation of a
given gene. In performing this study, F. progeny of treated males
are scored for entire sex-chromosome loss and for partial loss
of the Y chromosome.
Dominant Lethal Effects
Dominant lethal mutations can be used as indicators of major
genetic damage in mammals. The genetic basis for dominant lethality
is mainly the induction of structural and numerical chromosomal
anomalies, such as translocation and aneuploidies. These in turn
may induce a) preimplantation losses of nonviable zygotes; b) early
fetal deaths; and c) sterility in F. progeny.
The advantages of this procedure are that a) the test organism
is a laaaaial and therefore has some biochemical arid metabolic resemblances
to man; b) the test cell is a germ cell; and c) the results are
obtained before the first generation is born. Disadvantages are:
a) there is no definitive proof that a given lethal embryo is killed
by genetic damage; b) the type of damage is chromosomal, which
may not be related to gene mutation; c) the damage in evidence
will most likely only be related to post-meiotic stages of spermatogencsis,
even if earlier stages are damaged;-and-d) the limitations of the - - -
biochemical and pharmacological relationship of the mouse to man.
It is important to specify exposure protocol and state criteria
for the selection of mode of treatment. The schedule of raatings
will have to be specified, as will the criteria for choosing a
given mating schedule. The data recorded can include a) the number
of pregnant fenmlcs per number of mated males; b) the total number
of implantations; c) the total number of corpora letea (preimplantation
losses); d) dead implantations; e) proportion of females with either
one or more, or two or more, dead implantations; and f) dead implantations
per total implantations.
Criteria for the data recorded and presented should be indicated.
-------
V. 88
Heritable Translocation Test
The heritable translocation test can be differentiated from
'.he spermatocyte test on treated males in that the assay is performed
by testing for sterility and semi-sterility in FI male progeny
of the treated male mice. After confirmation by a second mating
of the semi-sterility or sterility of the FJ male progeny, the
primary speriaatccytes of the latter are analyzed cytogenetically
to confirm the presence of a heritable chromosome translocation.
The evidence currently available indicates that many translocations
occurring prior to ineiosis may be eliminated by this process, which
makes the FI translocation assay valuable primarily for measuring
effects induced in postmeiotic stages of sperma.togenesis.
The major advantage of the heritable translocation assay is
that it measures an explicit genetic event, with possible limitation
to assessment of postmeiotic chromosomal damage. Live progeny
are observed, another advantage, but a large number of animals
are required and a six-month period is necessary to complete the
assay. The cost per assay is also very high, while response to
known mutagens may be low.
The mouse appears to be the species of choice. In general,
translocation heterozyoot.es are detected by testing for sterility
and semi-sterility in Fj male progeny of treated male mice. Generally,
two or three females are mated with each Fj male and necropsied
at mid-terra to determine the number of viable embryos. Those males
that produce a significant reduction in the number of livii^ embryos
arc then remated to another series of females for additional evidence
of semi-sterility. Finally, semi-sterile and sterile males are
examined cytogeneticaliy by analyzing primary spermatocytes to
"confirm"the presence of a heritable chromosome" translocation.
-------
V. 89
V. B. 3. References for Chromosomal Effects (continued)
d. Rodents
(1) Cytogenetic Analysis
Leonard, A. (1976). Heritable chromosome aberrations in
mammals after exposure to chemicals. Rad. Environ. Biophys.,
13: 1-8. .
Legator, M. S., Palmer, K. A., and Adlsr, I. (1973). A collaborative
study of in vivo cytogenetic analysis. I. Interpretations of
slide preparations. Toxicol. Appl. Pharmacol., 24: 337-350.
Frohberg, H. (1973). Critique of in vivo cytogenetic test
systems. Agents Actions, 3: 119-123.
Schmid, W. (1975). The micronucleus test. Mutat. Res., 31: 9-15.
Vogel, W. and Bauknecht, T. (1976). Differential chromatid
staining by i£ vivo treatment as a mutagenicity tes't system.
Nature, 260: 448-449.
i
Stetka, D. G. and Wolff, S. (1S7G). Sis.-^r chromatid exchange as an !
assay for genetic damage induced by mutagen-carcinogens. I. IP
vivo test for compounds requiring metabolic activation. Mutat.
Res., 41: 333-342.
Allen, J. and Latt, S. (1976). Analysis of sister chromatid
exchange formation in_ vivo in mouse spermatagonia as a new
-test system for environmental, mutagens. Nature, 260:^449-451.
Cohen, M. and Hirschhorn, K. (1971). Cytogenetic studies in animals.
In: Chemical mutagens, Principles and methods for their detection,
vol. 2, A. Hollaender, ed., pp 515-534. New York. Plenum.
Osterberg, R. E., Murphy, J., Bierbower, G., and Moreland, S.
(1975). An evaluation of the mutagenic potential of an aerosol
spray adhesive in the rat. Mutat. Res., 31: 169-173.
Gooch, P., Luippold, H., Creasia, D., and Brewen, J. (1977).
Observation on mouse chromosomes following nitrogen dioxide
inhalation. Mutat. Res., 48: 117-120.
Gooch, P., Creasia, D., and Brewen, J, (1976). The cytogenetic
effects of ozone: Inhalation and in vitro exposures. Environ.
Res., 12: 188-195.
Friedman, M. A. and Staub, J. (1977). Induction of micronuclei
in mouse and hamster bone-marrow by chemical carcinogens. Mutat.
Res., 43: 255-262.
-------
V. 90
V. B. 3. d. (continued)
(1) Cytogenetic Analysis (continued)
Leonard, A. (1973). Observations on meiotic chromosomes of the
male mouse as a test of the potential mutagenicity of chemicals
in mammals. In: Chemical mutagens, Principles and method;, for
their detection, vol. 3, A. Hollaender, ed., pp 21-56. New York.
Plenum.
(2) Heritable Chromosomal Damage
Generoso, W. M., Cain, K. T., Huff, S. W., and Gosslee, D. G.
(1977). Heritable translocation test in mice. In: Chemical
mutagens, Principles and methods for their detection, vol. 5,
A. Hollaender, ed. New York. Plenum.
Leonard, A. (1975). Tests for heritable translocations in
male mammals. Mutat. Res., 31: 291-298.
Leonard, A. (1976). Heritable chromosome aberrations in mammals
after exposure to chemicals. Rad. Environ. Biophys., 13: 1-8.
Epstein, S., Arnold, E., Andrea, J., Bass, W., and Bishop, Y.
(1972). Detection of chemical mutagens by the dominant lethal
assay in the mouse. Toxicol. Appl. Pharmacol., 23: 288-325.
Bateman, A. (1973). The dominant lethal assay in the mouse.
Agents Actions, 3:73-76.
"Ehling", U.' '(1976). 'Mutagenicity testing-and'risk estimation
with mammals. Mutat. Res. 41: 113-122.
Green, S., Zeiger, E., Palmer, K., Springer, J., and Legi "»rt M.
(1976). Protocols for the dominant lethal test, host-media ed assay,
and ir± vivo cytogenetic test used in the Food and Drug
Administration's review of substances in the GRAS (Generally
Recognized as Safe) list. J. Toxicol. Environ. Health, 1: 921-928.
Bateman, A. and Epstein, S. (1971). Dominant lethal
mutations in mammals. In: Chemical mutagens, Principles
and methods for their detection, vol. 2, A. Hollaender, ed.,
pp 541-568. New York. Plenum.
Green. S., Moreland, F., and Flamm, W. (1977). A new approach
to dominant lethal testing. Toxicol. Appl. Pharmacol., 39:
549-552.
-------
V. <)
V. B. 3. d. (continued)
(2) Heritable chromosomal damage (continued)
Anderson, D., McGregor, D. B., Purchase, I. F. H., Hodge, M.C.E.,
and Cuthbert, J. A. (1977). Dominant-lethal test results with known
mutagens in two laboratories. Mutat. Res., 43: 231-246.
-------
V. 92
4. Methodological Considerations '
*!
a. Metabolic Activation j.i
i|
The need for metabolic activation of pre -carcinogens to an \>
ultimate carcinogenic form is well documented. In attempting to j
perform any type of bioassay, one must first determine if the system *
being employed as the indicator has its own inherent capability (j
to perform this metabolic activation, and secondly, if it can achieve jjj
a sufficient amount of activation duiing the period of the treatment jj
with the test agent. Related to the latter is the question of j!
whether or not the necessary activation system is pre-inducible
in the test system to obtain higher levels of activity.
If the system does not possess its own metabolic activation ji
system, then an appropriate mechanism to allow for the necessary i]
activation must be included in the assay. Met.ibolic activation ij
for i£ vitro assay systems will generally require the use of human jj
or animal tissue. Exceptions may b;; Drosophila and plant (Tradescantia) |!
systems. Attention should also be given to the factors of species, j]
strains, age, sex, and tissue of origin of metabolicaliy capable H
preparations. Jj
(1) I_n v i vo |
Considering the necessity for metabolic activation/ deact.ivation j
of test materials, one approach is to employ the whole animal as
the metabolic factory'. The route of administration can be varied,
and the cheiucals v.-jll !>e metabolicaliy activated (or detoxified)
in thor.c tissues in which they localize and which have the enzymatic
capability.
. .Two avenues for testing are then available. .-The first is. , -. ,
to collect urine from treated animals, and after appropriate treatment
of the urine to dcconjugatc potentially active metabolites, to
then treat the test organisms with the urine. The second approach, |j
known as "host-mediated ass-iy", involves locating the test organism '|
or cell in particular tissues or organs of an animal, treating ',
the whole animal with the test agent (using various routes of administration) , :. i
and recovering and subsequently assaying in vitro for a defined ',;
endpoint effect. This procedure lias been employed with bacterial,
yeast, and mammalian cell tests. ',
It will be necessary to consider the best currently available '
information, and to specify criteria for selection of species,
strain(s), age(s), sc.x(es), and tissue of the animal employed. !
Attention must be given to the availability of a data base for
the selected system, and to insure that the activation system is '
pre-induced to a high metabolic capability. j
-------
V. 93
(2) jji vitro
In addition to all of the considerations described above, . J
the tester can select from a variety of in vitro metabolic activation
systems that are potentially useful, including: 1) the S-9 fraction;
2) isolated microsoraes; 5) postmitochondrial supernatant; 4) tissue
homogenate; or 5) a mammalian cell feeder layer, tach of these
systems has advantages and disadvantages; which should be considered
in relation to the endpoint indicator organism being employed.
It is important to include positive known controls in all tests
in order to ensure that the test material iias not inactivated the
metabolic acti1 ation system.
-------
V. 94
b. Treatment Conditions
It is realized that the test materials pertinent to fuels
and fuel additives will generally not be soluble in aqueous solutions
and may be complex mixtures of variable and indetei'minant chemical
composition. It will therefore be essential that every effort
is made to ensure that all components of a given test material
actually do contact the cells of the test organism and are taken
up_ by the cells.
These considerations are particularly important for _in vitro
tests with unicellular organisms. For bacterial systems,""spot
tests or plate tests can be performed, but neither will be satisfactory
if the chemical is insoluble in aqueous medium, and cannot diffuse
through semisolid agar during the continuous exposure period. Spot
tests for mutagenicity are simplest, but would only be recommended
for strong or volatile mutagens, since only a minority of cells
on the plate would be exposed .to an effective level of mutagen.
Suspension treatmment of bacteria otherwise appear to be required
(with and without metabolic activation), but consideration should
be given to the relative shortness of exposure time in suspension
treatments as compared to the plate methods; i.e., can enough chemical
be metabolized to an active form during a short exposure period
to provide a high enough concentration oi activated ingredient
to produce a significant effect. Volatile components or gaseous
exposures require additional considerations, and methodological
variations of the best available design should be employed.
In general, for any procedure, a major consideration is the
effective level of the mutagen to be employed. If the concentration
of a mutagen is too,low, too few mutants will be induced-to be-
detected. If the concentration is too high, too many mutants will
be killed for any to be detected. The test concentration should
not exceed 90% killing, and should in general range from 0-^0%.
Further discussion of the various techniques for bacteria can be
found in the references listed.
The use of spot test procedures or treatment in suspension
are also employed for studies with Saccharomyces cerevisiac, with
the above considerations being relevant; forced heterokaryons of
Neurospora crassa are usually treated in suspension. For mammalian
cells, the treatment protocol employed may have to be varied from
that recommended in the references provided in the appendix; for
example, chemical transformation experiments generally allow for
a continuous 72.hours of treatment; for mixtures, this might iiave
to be prolonged. The standard protocol for at least one mammalian
cell mutagenesis assay is defined as two hours; this also may have
to be prolonged. Appropriate judgments will have to be made as
to length of exposure versus linearity of microsomal activation
in_ vitro when the latter is required in conjunction with mammalian
cell assay system. Modifications for gaseous exposure will have
to be developed.
-------
V. 95
Requirements for utilization of specific bacterial test systems
for detection of mutagenic activity are not detailed in these draft
protocols; and indeed, the variety and usefulness of these systems
is continually expanding, so that specific selection guidelines
will require informed judgmental decisions. However, attention
is directed to one bacterial system which might have unique application
for fuels and fuel additives. This is the Escherichia coli K12
system developed by Mohn et_ al. (1974, 1975), which is unique in
that it manifests greater sensitivity towards certain carcinogens,
such as nitroheterocyclic compounds and dialkylnitrosamines than
do the Salmonella strains. In addition, this E_. coli strain allows
for the forward detection of several genes (whereas Salmonella
systems detect only reverse mutations') and is more suitable for
the intra-sanguineous host-jaediated assay (Sobels, 1977).
For L'rosophila, which is an iji vivo system, considerations
must be given to the possibility that the flies can voluntarily
close their spiracles, end therefore prevent entry of a chemical
in aerosol form.
-------
V. 96
V. B. 4. References for Methodological Considerations
(a) Metabolic Activation
Mohn, G., Ellenberger, J., McGregor, D. and Merker, H. (197S).
Mutagenicity studies in microorganisms ^n_ vi.tro, with extracts
of mammalian organs, and with the host-mediated assay. Ntitat.
Res., 29: 221-233.
Brusick, D. (1977). In vitro mutagenesis assays as predictors
of chemical carcinogenesis in mammals. Toxicol. Annu., 2: 79-109.
Frant-, C. and Mailing, H. (1976). Bromodeoxyuridine resistance
induced in mouse lymphoma cells by microsomal activation of
dimethylnitrosaraine. J. Toxicol. Environ. Health, 2: 179-187.
Frantz, C. and Mailing, H. (1975). The quantitative microsomal
mutagenesis assay method. Mutat. Res., 31: 365-380.
Malaveillc, C., Planche, G. and Bartsch, H. (1977). Factors
for. efficiency of the Salmonolla/microsome mutagenicity assay..
Chcm.-Biol. Interactions, 17': 129-136.
Brusick, D., Jagannath, D. anu hVekes, U. (iy/'o). .The utilization
of in vitro mutagenesis techniques to explain strain, age and
sex related" differences in dimethylnitrosamine tumor susceptibil-
ities in mice. Mutat. Res., 41: 51-60.
Jensen, E. M. , I.aPolla, R. J., Kirby, P. E. , and Haworth, S. R.
(1977). In vitro studies of chemical mutagens and carcinogens.
I. Stability studies in cell culture medium. J. Natl. Cancer Inst.
59: 941-944.
Newbold, R. F., Wigley, C. B., Thompson, M. II., and Brookes, P.
(1977).' Cell-mediated mutagenesis in cultured Chinese hamster
cells by carcinogenic polycyclic hydrocarbons: Nature and extent
of the associated hydrocarbon-DNA reaction. Mutat. Res., 45:
101-116.
(b) Treatment Conditions
Malaveille, C.,Planche, G. and Bartsch, H. (1977). Factors
for efficiency of the Salmonclla/microsome mutagenicity assay.
Chcm.-Biol. Interactions, 17: 129-156.
Purchase, I.F.H., Ix>ngstaff, E. , Ashby, .)., Styles, J. A.,
Anderson, D., Lefevre, P. A. and Kestwood, F. R. (1976).
Evaluation os six short term tests for detecting organic chemical
carcinogens and recommendations for their use. Nature,. 264: 624-627.
-------
V. 97
V. B. 4. References for Methodological Considerations
(b) Treatment Conditions (continued)
Cline, J. C. and McMahon, R. E. (1977). Detection of
chemical mutagens, Use of concentration gradient plates
in a high capacity screen. Res. Commun. Chem. Pathol. Pharmacol.
16: 523-533.
Ames, B. N., McCann, J. and Yamasaki, E. (1975). Methods
for detecting carcinogens and mutagens with the Salmonella/ir.ic-
rosome tnutagenicity test. Mutat. Res., 31: 3-17-364.
Tokiwa, H., Morita, K., Takeyoshi, H., Takahashi, K., and
Ohnishi, Y. (1977). Detection of mutagenic activity in particulate
air pollutants. Mutat. Res., 48: 237-248.
Kier, L. D., Yamasaki, E., and Ames, B. N. (1974). Detection
of mutagenic activity in cigarette smoke condensates. Proc.
Nat. Acad. Sci., USA, 71: 4159-4163.
Easier, A., llerbold. B., Peter, S. and Rohrborn, G. (1977).
Mutagenicity of poiycyclic hydie.carbcr.s. II. Monitoring
genetical hazards of chrysene in vitro and vivo. Mutat. Res.,
48: 249-254.
environmental Protection Agency. (1977). Pesticides guidelines,
draft revision and references therein.
-------
I/, 9 7
V. C Carcinogenesis
1. Selected Mutagenesis Tests
2. Mammalian Cell Neoplastic (Oncogenic) Transformation
a. Primary Cells
b. Continuous Lines
3. Rodent Tests
a. Pulmonary Tissue Carcinogenesis
Aerosol inhalation
Vapor inhalation
Intratracheal instillation
b. Non-pulmonary Target Tissue Carcinogenesis
Pulmonary exposure
Skin painting
Carcinogen bioassay - oral administration
4. Cocarcinogenesis and Tumor Promotion
a. Mouse Skin Papilloma
-' " b. C3H/10T^ Mammalian Cells in Promoter Mode
-------
V. 98
V. C. Carcinogenesis-
1. Selected Mutagenesis Tests
Chemicals which are either carcinogenic or mutagenic are
capable of entering cells and reacting or interacting with DNA.
If the cell within which the chemical "hits" the DNA is a germ
cell, mutagenesis may be induced, while if the involved cell is a
somatic cell, carcinogenesis may result. The potential for damage
to DNA which characterizes both mutagens and carcinogens can be
employed to advantage for rapid and inexpensive iri vitro screening
of environmental chemicals. The quickest and least expensive of
these in_ vitro screening tools are the bacterial mutation detection
assays, which have an impressive record for predicting carcinogenic
potential when coupled with mammalian metabolic activation systems.
For evaluation of fuel and fuel additive test materials for which
the primary route of exposure is inhalation and pulmonary carcinogenesis
is a priority concern, it may be advisable to consider utilization
of both mammalian lung and liver activation systems in order to
account .for any significant differences in metabolic toxification
or detoxification capacity between the two organs.
The current status of the utilization of bacterial .mutagenesis
assays for detection of carcinogenic potential has recently been
reviewed (Bridges, 1976).
2. Mammalian Cell Neoplastic (Oncogenic) Transformation
For purposes of short-term, iri vitro predictive screening for
carcinogenic potential in a mammalian system, mammalian cells in
culture have been used. The endpoint sought is transformation to
a malignant state by treatment with a test material. -The only
"absolute indicator for this endpoint is the ability of cells to
produce a tumor when inoculated into an appropriate host animal;
however, there are various secondary growth characteristics which
correlate with malignancy, and can be visually monitored in culture.
Transformed cells may have a number of distinguishing characteristics,
including: 1) morphological alteration; 2) ability to form colonies
in a semi-solid medium without anchorage; 3) ability to form multi- >.
layered clones under conditions where normal cells remain as a
monolayer; 4) ability to form clones on a monolayer of normal cells;
5) ability to grow in low concentrations of serum; 6) ability to ^
grow more rapidly than non-transformed cells, particularly in low
concentrations of serum; 7) ability to survive under conditions
where normal cells tend to senesce and die ("immortalization");
8) presence of altered membrane properties, including lectin binding .
and lectin-induced agglutination, etc. (Martin and Anderson, 1976;
Steuer et al_., 1977). This list is not inclusive; biochemical ;
indicators such as the ability to secrete a proteinase involved in :
fibrinolysis and the release of plasminogen activators can also
be distinguishing markers, and other markers are continuously
-------
V. 99
being developed. Host important, however, is the demonstration
and documentation, for whatever endpoint of transformation is
utilized, that, the selected endpoint correlates with the generation
of tumors in animal hosts upon injection of transformed cells.
-------
V. TOO
V. C. 2. a. Primary Cells
Primary or secondary cultures of cells from Syrian hamster
embryos can be transformed quantitatively by exposure to carcinogens
in culture. Transformation in culture as judged by morphological
parameters correlates with the production of sarcomas when the
cells are inoculated into hamsters (DiPaolo ct_ al_., 1971, 1972).
Good correlations have been obtained between carcinogenic
activities of various compounds and their ability to transform
these cells in culture (Meidelbcrger, 1975; and references therein).
A recently published study describes a 90.8% positive correlation
for 82 chemicals known to be either carciiu^ens or non-carcinogens
(Pienta et aj_., 1977). These cells have th; capacity to metabolically
activate some classes of carcinogens such as polycylic hydrocarbons
and alkylating agents, although other carcinogen classes arc not
activated by early passage cultures - i.e., primary or secondary
cultures (Pienta et^ ca, 1977; DiPaolo c_t aj_., 1972). For screening
of fuel and fuel additive test materials, it would therefore be
advisable to screen test materials both with and without mammalian
metabolic activating systems. This assay discriminates between
structurally related carcinogens and noncarcinogcns; no false
positives were obtained when noncarcinogcns were assayed; and the
system is sensitive to metal carcinogens (Pienta e_£ al., 1977).
Methods for optimising and standardizing this assay for ijn_ vitro
carcinogenesis screening are outlined by Pienta ejt al;. (1977) and
Casto ct al. (1977).
-------
V. 101
V. C. 2. a. References for Primary Cells
Casto, B. C., Janosko, N., and DiPaolo, J. A. (1977) Development
of a focus assay model for transformation of hamster cells in_
vitro by chemical carcinogens. Cancer Res. 37:3508-3515.
DiPaolo, J. A., Donovan, P. J., and Nelson, R. L. (1971) ]in
vitro transformation of hajnster cells by polycylic hydrocarbons:
factors influencing the number of cells transformed. Nature
(New Biol.) 250:240-242.
DiPaolo, J. A., Nelson, R. L., and Donovan, P. J. (1972) ln_
vitro transformation of Syrian hams.ter embryo cells by diverse
chemical carcinogens. Nature 235:278-280.
DiPaolo, J., Nelson, R., Donovan, P., and Evans, C. (1973) Host-
mediated i£ vivo - in. vitro assay for chemical carcinogenesis.
Arch. Pathol. 95:380-385. ''
Pienta, R. J., Poiley, J. A., and Lebherz, W. 3., Ill (1977)
Morphological transformation of early passage golden Syrian
hamster embryo cells derived from cryopreserved primary
cultures as a reliable iri vitro bioassay for identifying
diverse carcinogens. Int. J. Cancer 19:642-655.
-------
V. 102
V. C. 2. b. Continuous Lines
Continuous cell.lines have been derived from rodent embryo
cells, offering the advantage of working with a homogeneous cloned
cell population rather than the heterogenous population of primary
cell cultures. Again, good correlations have been observed
between carcinogenic activity and the extent of transformation
produced in cultured lines. The lines specified here have been
shown to exhibit good stability in culture and .a high degree of
sensitivity to post-confluence inhibition of division (Reznikoff
et al., 1973a, 1975b). The capacity for metabolic activation of
carcinogens of various chemical classes varies from one clone to
another for a given cell line and probably as a function of length
of passage in culture also. It would therefore be wise to screen
fuel and fuel additive test materials both in the presence and
the absence of supplementary mammalian metabolic activating systems.
Other rodent cell lines have been employed for detection of
oncogenicity in culture, specifically rat embryo cell lines (Sekely <
et^ aJL, 1973; Rhim and Huebner, 1973) and a baby hamster kidney
line, BHK21 (Di Mayorca e£ al., 1973).
Mouse embryo fibroblast line, C3H/10TVCL8 j
This system has been characterized (Reznikoff et_ al., . 1
1973 a, 1973b) and modified for two stage oncogenesis |S
(Mondal et al.. 1976). \
: ('
Mouse embryo fibroblast line, BALB/3T3, clone A31-1 j
Application of this system to the assay of carcinogenic I
activity has been described by Kakunaga (1973, 1974) and j
UiPaolo et al. (1972). ' j
-------
V. 103
V. C. 2. b. References for Continuous Lines
Benedict, W. F., Banerjee, A., Gardner, A., and Jones, P. A.
(1977) Induction of morphological transformation in mouse
C3H/10T%/CLS cells and chromosomal damage in hamster
A(Tj)Cl-3 cells by cancer chemotherapeutic agents. Cancer
Res. in press.
DiPaoio, J. A., Takano, K., and Popescu, N. C. (1972) Quantitation
of chemically induced neoplastic transformation of BALB/3T3
cloned cell lines. Cancer Res. 32:2686-2695.
Di Mayorca, G., Greenblatt, M., Trauthen, T., Soller, A., and
Giordano, R. (1973) Malignant transformation of BlI^i clone
13 cells in_ i'itro by nitrosarcines--a conditional state. Proc.
Natl. AcadT Sci. USA 70:46-49.
Heidelberger, C. (1975) Chemical carcinogcncsis. Annu. Rev.
Biochem. 44:79-121.
Kakunaga, T. (1973) A quantitative system for assay of malignant
transformation by chemical carcinogens using a clone derived
from BALB/5T3. Int. J. Cancer 12:463-473.
Kakunaga, T. (1974) Requirement for cell replication i" the
fixation and expression of the transformed state in mouse
cells treated with 4-nitroquinoline-l-oxide. Int. J. Cancer
14:736-742.
Mondal, S., Brankow, D. W., and Heidelberger, C. (1976) 'Two-stage
chemical oncogenesis in cultures of CSH/lOT's cells. Cancer
Res. 36:2254-2260.
Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. (1973a)
Establishment and characterization of a cloned line of C3H
mouse embryo cells sensitive to postconfluence inhibition of
division. Cancer Res. 33:3231-3238.
Rcznikoff, C. A., Bertram, J. S., Brankow, G. W., and Heidelberger,
C. (1975b) Quantitative and qualitative studies of chemical
transformation of cloned C5H mouse embryo cells sensitive to
postconfluence inhibition of cell division. Cancer Res. 33:
3259-3249.
Rhim, J. and lluebncr, R. (1973) Transformation of rat embryo
cells in vitro by chemical carcinogens. Cancer Res. 33:695-700.
Sekely, L. I., Malejka-Giganti, D., Gutmann, H. R., and Rydell,
R. L. (1973) Malignant transformation of rat embryo fibro-
blasts by carcinogen.ic fluorenylliydroxamic acids in vitro.
J. Natl. Cancer Inst. 50:1337-1345.
-------
V. 104
V. C. 3. Rodent
a. Pulmonary Tissue Carcinogenesis
Motor vehicle emissions are known to contain a number of
compounds which act as chemical carcinogens when tested in animals
These emission components include all three types of chemical
carcinogens:
(1) direct-acting or primary carcinogens, such as
aryl epoxides, alkylene epoxides and lactones and
sulfate esters;
(2) secondary or procarcinogens, such as benz(a)
anthracene and benz(a)pyrcne; and
(3) co-carcinogens or promoters, such as phenols and
glycols.
Primary carcinogens frequently act to produce tumors at the
points of application since they do not require metabolic activation,
although they are subject to detoxification and excretion; however,
they may also act in remote tissues. Secondary carcinogens
usually do not act at the point of application, but often involve
specific target tissues; they require metabolic activation, but
also are detoxified. Cocarcinogens arc not carcinogenic alone,
but can markedly potentiate carcinogen action. They do not
necessarily have to be administered simultaneous]y with, or ^ven
by the same route as the carcinogen. A comparatively larger
amount of cocarcinogen can promote, tumors from an extremely small
amount of carcinogen. This promoter effect may be very important
for human exposure to motor vehicle emissions as the carcinogenicity
of exhaust condensate has been found to be greater than can be
accounted for on the basis of known carcinogen concentration (Va.n
Duuren, 1976; Hoffman and Wynder, 1976). '.
Inhalation exposure is characterized by: (1) large intake
volumes; (2) repetitive intake; (5) large tissue surface area in
rapid equilibrium with the entire blood circulation; (4) ability
of respiratory tract tissues to metabolically activate secondary
carcinogens; and (5) specific binding and retention by lung tissue
of certain chemical classes of potential carcinogens such as
hydrophobic amines (Philpo.t ct^ aJL, 1977). N'ot only are hydrophobic
amines concentrated in lung, regardless of the route of administration,
but lung tissue has a capacity for N-o:cidation of such amines that
is equal to that of liver, .in spite of a lower concentration of
P_45Qthan the liver (Brown, 1974). However, N-hydroxylation is
thought to be the major pathway by which aromatic amines and their
nitro derivatives are converted to the ultimate carcinogen form
and the occurrence or concentration of this enzyme activity in
various organs may explain why aromatic amines are predominantly
bladder carcinogens. The metabolism of aromatic amines, and their
subsequent carcinogenicity are affected by hormonal changes also
(Clayson and Garner, 1976).
-------
V. 105
Specific lung carcinogens include bis (chloromethyl) ether
(BCMC). BCME, a vapor thought to be forced from the reaction of
formaldehyde and hydrochloric acid, produced a high incidence of
lung cancer in rats exposed one time to 0.1 ppm BCME. The powerful
lung-specific carci.'iogenicity for humans of BCME has been confirmed
by epidemiological evidence from chemical industry workers exposed
to BCME and commercial chloromethyl ether which contains 1-7%
BCME (Farkes, 1976).
In considering the capacity of test emissions for contributing
to the incidence of cancers of the respiratory tract in human
populations, it is important to evaluate:
primary carcinogenicity;
secondary carcinogenicity;
' (after netabclism)
; and cocarcinogcn.Lcity;
(direct and two-stage).
The effects of sex, hormonal status, diet, age, and organ-specific
metabolic activation/deactivation should also be considered.
Aerosol inhalation
Tiis is the preferred method of exposure because it . ,
parallels the human exposure situation, and is therefore ( j
essentially free of experimentally-introduced artefacts \ I
when whole exhaust is the test material. Solid fractions I i
of exhaust may also be aerosolized with due experimental j j
caution. Complete pathological study of the entire f ]
respiratory tract should be performed. ! j
Vapo r i nha1 a t i on i j
If whole exhaust is not employed as the material for < -f. f
aerosol inhalation, then the carcinogenicity and ; |
eocarcinogenicity of the vapor phase should be evaluated. : )
1 !
Intratracheal instillation , j
Intratrachcal instillation is a useful method of
exposure for those cases whore: the amount of test
ma^^rial (liquid or solid) is limited; or the test
material is known or suspected to be toxic, flammable, ;
explosive, or otherwise hazardous.
-------
V. 106 |
V. C. 3. b. Non-pulmonary Target Tissue Carcinogenesis i
Pulmonary exposure |
The incidence of tiunors at sites other than in the |
respiratory tract as a result of pulmonary exposure
should be observed. This would logically be combined
with the evaluation of respiratory tract carcinogenesis.
Skin painting
The potential of a given test material for carcinogenicity
may be evaluated by the application of solutions of
the test material in an appropriate nontoxic solvent
to mouse skin. This assay should be coupled with one
which screens the carcinogenic potential of the vapor-
phase of emissions.
Carcinogen bioassay - oral administration I
The test material may be administered by gavage (stomach -i
tube), preferably; or mixed into food and the amount ,1
consumed monitored. 'I
*
All significant tissues, including those of the respiratory
tract, should be examined pathologically as detailed in the NCI
Guidelines for Carcinogen Bioassay in Small Rodents (Sontag, Page, |
and Saffiotti, 1976), along with other parameters of chronic ;|
toxicity. If any fuel or fuel additive test materials are .;
evaluated for chronic toxicity by any route of administration, f|;
this carcinogenesis bioassay should also be included. It would «$!
also be advisable to consider observation of all animal test groups 4
for purposes of determining differences in time-to- tumor development .j
as well as differences in tumor incidence (Hoel et al., 1975). »
-------
V. 107
V. C. 3. References for Carcinogenesis - Rodent
a. Pulmonary Tissue Carcinogenesis
Philpot, R. M., Anderson, M. W., and Eling, T. E. (1977).
Uptake, accumulation, and metabolism of chemicals by the
lung. In: Metabolic functions of the lung, Y. S. Bakhle
and J. R. Vane, eds., pp 123-171. (Lung biology in health
and disease, 4: 123-171.) New York. Dekker.
Brown, E. (1974). The localization, metabolism, and effects of
drugs and toxicants in the lung. Drug Metab. Rev., 3: 35-87.
Hoffmann, D. and Wynder, E. L. (1968). Chemical analysis and
carcinogenic bioassays of organic particulate pollutants. '
In: Air Pollution, 2nd ed., vol. II, A. C. Stern, ed., |
pp 187-247. New York. Academic. !
Van Duuren, B. L. (1976). Tumor-promoting and co-carcinogenic j
agents in chemical Carcinogenesis. In: Chemical carcinogens, |
C. E. Searle, ed., pp 24-51. Washington, D. C. American \
Chemical Society. . '.
i
Ciayson, D. B. and Garner, R. C. (19/6). .Carcinogenic .aromatic !.
amines and related compounds. In: Chemical carcinogens, C. E. !
Searle, ed., pp 366-461. Washington, D. C. American Chemical j
Society. " >
Parkes, H. (1976). The epidemiology of the aromatic amine '
cancers. In: Chemical carcinogens, C. E. Searle, ed., pp 462-
480. Washington, D. C. , American Chemical Society. ' - - c
Saffiotti, U., Cefis, F., and Kolb, L. H. . (1968). A method ;
for the experimental induction of bronchogenic carcinoma. Cancer '
Res., 28: 104-124. ].
f
Reid, W. D., Ilett, K. F., Click, J. M., and Krishna, G. (1973). I
Metabolism and binding or aromatic hydrocarbons in the lung. \
Am. Rev. Respir. Dis., 107: 539-551. \.
Feron, V. J. (1972). Respiratory tract tumors in hamsters after
intratracheal instillations of benzo(a)pyrene alone and with
furfural. Cancer Res., 32: 28-36.
Feron, V. J., de Jong, D., and Rijk, M. A. H. (1976). Clearance
of benzo(a)pyrene from the respiratory tract of hamsters following
its intratrrcheal instillation with or without ferric oxide.
Zentralbl. oakteriol. Parasitenkd. Infektionskr. Hyg. Abt. Orig.
Reihe. B 163: 441-447.
-------
V. 108
V. C. 3. References for Carcinogenesis - Rodent (continued)
a. Pulmonary Tissue Carcinogenesis (continued)
Schntahl, D.and Schmidt, K. G. (1974). Conception and methods
of experimental studies in Germany to estimate the carcinogenic
gurden by air pollution in man. In: Experimental lung cancer,
E. Karbe and J. I. Park, eds., pp 139-145. New York.
Springer-Ver lag .
Farrell, R. L. and Davis, G. W. (1974). Effect of particulate
benzo(a)pyrene carrier on Carcinogenesis in the respiratory
tract of hamsters. In: Experimental lung cancer, E. Karbe
and J. F. Park, eds. pp 186-198. New York. Springer-Verlag.
Farrell, R. L. and Davis, G. W. (1974). The effects of
particulates on respiratory Carcinogenesis by diethylnitrosamine.
In: Experimental lung cancer, E. Karbe and J. F. Park, eds.,
pp 219-233. New York. Springer- Verl.ag.
Montesano, R. , Saffiotf, U., Ferrero, A. and Kaufman, D.
(1974). Brief Communication: Synergistic effects of benzo(a)-
pyrene and diethylnitrosamine of respiratory Carcinogenesis
in hamsters. J. Natl. Cancer last., 53: 1395-1397.
Sellakumar, A. R. , Montesano, R. , Saffiotti, U. , and Kaufman, D. G.
(1973). Hamster respiratory Carcinogenesis induced by benzo(a)-
pyrene and different dose levels of ferric oxide. J. Natl.
Cancer Inst., 50: 507-510.
Saffiotti, U., Montesane, R., Sellakumar, A. R. , Cefis, F., and
Kaufman, D. G. (1972). Respiratory tract Carcinogenesis in ham-
sters induced by different numbers of administrations of benzo-
(a)pyrene and ferric oxide. Cancer Res., 32: 1073-1081.
Saffiotti, U. , Montesano, R. , and Sellakumar, A. R. and Kaufman,
D. G. (1972). Respiratory tract Carcinogenesis induced in
hamsters by different dose levels of benzo(a)pyrene and ferric
oxide. J. Natl. Cancer Inst., 49: 1199-1204.
Cohen, G. M. and Moore, B. P. ( 1975). Metabolii.m of
(a)pyrene by different portions of the respiratory tract.
Pharmacol., 25 :. 1623-1629.
Crersia, D. A., Poggenburg, J. K. , Jr. and Nettesheim, P. (1976).
Elut.ion of benzo(a)pyrene from carbon particles in the respiratory
tract of mice. J. Toxicol. Environ. Health, 1: 967-975.
Biochem.
-------
V. 109
V. C. 3. References for Carcinogenesis - Rodent (continued)
a. Pulmonary Tissue Carcinogenesis (continued)
Davis, B R., Whitehead, J. K., Gill, M. E., Lee, P. N.,
Butterworth, A. D., and Roe, F. J. (1975). Response of
rat lung to inhaled vapour phase constituents (VP) of tobacco
smoke alone or in conjunction with smoke condensate or fractions
of smoke condensate given by intratracheal instillation.
Br. J. Cancer, 31: 462-468.
Rappaport, S. M., Morales, R., Weeks, R. W., Jr., Campbell, E. K.,
and Ettinger, H. J. (1975). Development of sampling and analytical
methods for carcinogens. Los Alamos Scientific Laboratory of the
University of California. Los Alamos, New Mexico. Progress
Report LA-6387-PR. 19 pp.
Shabad, L. M., Pylev, L. N., and Kolesnichenko, T. S. (1963).
Importance of the deposition of carcinogens for cancer induction
in lung tissue. J. Natl. Cancer Inst. 33: 135-141.
b. Non-pulmonary Target Tissue Carcinogenesis
Lee, P. N. and O'Neill, J. A. (1971). The effect both of time
and dose applied on tumour incidence rate in benzopyrene skin
painting experiments. Br. J. Cancer, 25: 759-770.
Berenblum, I. and Shubik, P. (1949). The persistence of latent -,'.,
tumor cells induced in the mouse's skin by single application
of 9,10-dimethyl-l,2-benzathracene. Br. J. Cancer 3: 3S4-3S6.
Sontag, J. M., Page, N. P., and Saffiotti, U. (1976). Guidelines i
for carcinogen bioassay in small rodents. N'ati.onal Cancer Institute '
Carcinogenesis Technical Report. Series no. 1, 65 pp. (DllEW !
pub. no. (NIH) 76-801.) U. S. Govt. Print. Off., Washington, D. C. !.
Hoel, D. G., Gaylor, D. W., Kirschstein, R. L., Saffiotti, U., '
and Schneiderman, M. A. (1975). Estimation of risks of irrever-
sible delayed toxicit.y. J. Toxicol. Environ. Health, 1: 153-151.
-------
V. 110
V. C. 4. Cocarcinogencsis and Tumor Promotion
Cocarcinogenesis is the augmentation of tumor production over
that produced by the carcinogen alone. If the test substance is
applied at the same time as the carcinogen and enhancement of
tumor production is observed, the substance is considered a
cocarcinogen. If the test substance produces an increase in ;
tumorigenesis when administered subsequently to the carcinogen,
this is termed promotion, or two-stage carcinogencsis. Promotion
may occur if the promoter is administered through a different route
than the carcinogen. For screening of fuels and fuel additives .
health effects, cocarcinogencsis occurring in the respiratory j
tract is of greatest interest, and some studies have dealt with :
this (Mohr et_ al_., 1976); however, the most widely employed and j
best characterized system for studying cocarcinogenesis is that I
employing mouse skin (Berenblum and Shubik, 1949; Van Duuren, 1967). !
An i.n_ vitro assay for promoter activity has recently been described |
(Mondai et_ al_., 1976).
Appropriate forms of test material should be screened for
cocarcinogenic and tumor promotion activities with one or more of
the polyaromatic hydrocarbon carcinogens known to o'ccur in motor |
vehicle emissions. If only one carcinogen is employed, it should . 5
be that occurring in highest concentration in emissions. j
a. Mouse Skin Papilloma (Two-stag'e Oncogenesis)
An assay for tumor promotion activity, which is defined as !
enhancement of tumor formation by low doses of a carcinogen due to j
subsequent action of the promoter, has been described by Berenblum
and Shubik (1949). jj
In addition to tumor promoters, a number of chemicals such as i
various aliphatic hydrocarbons have been shown to be cofactors for »
carcinogcnesis, markedly enhancing carcinogenicity when applied to 'J
mouse skin simultaneously with u carcinogen (Van Duuren, 1976; ]
Wcinstein and Troll, 1977). - '
Fuel and fuel additive test materials should be evaluated as j
both promoters and cofactors for carcinogcnesis.
b. C3H/10TJs Mammalian Cells in Promoter Mode
It has recently been demonstrated that substances which act
as tumor promoters i_n_ vivo also display promoter activity rn
yitro by enhancing transformation of C51I/10T-4, a mouse embryo cell
line, when administered 4 days after a non-transforming dose of
carcinogen (Mondai et_ a_^., 1976).
-------
V. Ill
V. C;- 4." References for Cocarcinogenesis and Tumor Promotion
Berenblum, I. (1969) A re-evaluation of the concept of cocarcinogenesis.
Prog. llxp. Tumor Res. 11:21-30.
Bertnblum, I. and Shubik, P. (1949) The persistence of latent tumor
cells induced in the mouse's skin by single application of
9,10-diniethyl-l,2-benzanthracene. Br. J. Cancer 3:384-386.
Mondal, S., Brankow, D. W., and Heidelberger, C. (1976) Two-stage
chemical oncogenesis in cultures of CSH/lOTij cells. Cancer
Res. 36:2254-2260.
Van Duuren, B. (1967) Tumor-promoting agents in two-stage carcino-
genesis. Prog. Exp. Tumor Res. 11:31-68.
Van Duuren, B. (1976) Tumor-promoting and cocarcinogenic agents
in chemical carcinogenesis. In: Chemical carcinogens, C. E.
Searle, Ed. Am. Chem. Soc., Washington, D. C., pp 24-51.
Van Duuren, B. and Sivak, A. (1968) Tumor-promoting agents from
Croton tiglium L. and their mode of action. Cancer Res. 28:
2349-2356.
Weinstein, I. B. and Troll, W. (1977) National Cancer Institute
Workshop on tumor promotion -..id cofactors in carcinogenesis.
Cancer Res. 37:3461-3463.
-------
V. D. Teratogenesis and Reproductive Performance
1. Reproductive Performance
2. Teratogenesis
-------
V. 112
V. D. Tcratogenesis and Reproductive Performance
1. Reproductive Performance
Reproductive performance is concerned with any action of an
environmental agent at any time relative to the conception process
which can affect the ability of parents to successfully produce
offspring, and would include effects occurring during the reproductive
cycle(s).
In performing studies to evaluate the effects of long-term,
continuous, low-dosage exposures on overall reproductive integrity,
the so-called "three generation test" recommended by the Food and
Drug Administration appears appropriate. These tests provide an
opportunity for accumulation of substances or insults to the point
where deleterious effects may be observed. As described by the
Food and Drug Administration Advisory Committee or. Protocols for
Safety Evaluations (1970), the study can give information on
fertility and pregnancy in the parent F(0) generation. "Observations
of the F(l) generation would determine the effects of a substance
upon the uterine environment and upon lactation as well as on
postweanijig growth and development. The F(l) animals which have
been exposed continuously to this substance from the time of
conception would reveal changes in reproduction characteristics
acquired during the periods of embryogenesis, infancy, puberty,
and reproductive maturity. The accumulation of a potentially toxic
substance would be determined by the reproductive performance of
the F(l) generation and observation of the growth and development
of the F(2) generation." Apparently, some question exists as to
the information to be gained on studying an additional F(3)
generation.
Descriptions of current-methodology and critiques of these c
procedures can be found in the references. Attention should be
given to criteria for selection of species, number of species,
strain, exposure, and especially to selection of endpoints for
evaluation of effect and statistical analysis of the effect. The
numbers of animals must be selected with attention to the latter.
2. Teratogenesis
Teratogenesis is concerned with the occurrence of developmental
errors, both structural and functional, which are initiated at
any time between fertilization and postnatal maturation. Because
teratogenic effects can be masked in a variety of ways during a
continuous exposure (e.g., by chemical alteration in the parent
of metabolism of the treatment chemical), establishment of teratogenic
risk should be investigated by short-term expo.sux'" during a period
of high susceptibility. The embryonic period when early organ
formation is in progress appears to be the time when the developing
organism is most sensitive to adverse influences; it is recommended,
-------
V. 113
however, that attention also be given to exposure during late
pregnancy.
Descriptions of current methodology and critiques of these
procedures can be found in the references listed. Attention should
especially be given to specification of criteria for the selection
of species and number of species, as well as exposure levels,
route(s) of exposure, times and intervals of exposure, and for
endpoints for evaluation of effect. The numbers of animals studied
and procedure for statistical analysis should be justified.
-------
V. 114
V. D. References for Teratogenesis and Reproductive Performance
1. Reproductive Performance
Food and Drug Administration Advisory Committee on Protocols
for Safety Evaluations: Panel on Reproduction report on
reproduction studies in safety evaluation of food additives
and pesticide residues. (1970). Toxicol. Appl. Pharmacol.
16: 264-296.
Wilson, J. (1975). Reproduction and teratogenesis: Current
methods and suggested improvements. J. Assoc. Off..Anal. Chem.,
58: 657-667.
Wilson, J. G. (1975). Environment and birth defects. New York.
Academic. 305 pp.
2. Teratogenesis
Food and Drug Administration Advisory Committee on Protocols
for Safety Evaluations: Panel on Reproduction report on
reproduction studies in safety evaluation of food additives
and pesticide residues. (1970). Toxicol. Appl. Pharmacol.,
16: 264-296.
Becker, B. (1975): Teratogens. " In: Toxicology,"The basic
science of poisons, L. J. Casarett and J. Doull, eds., pp 313-
332. New York. Macmillan.
Clcgg, D. (1971). Teratology. Annu. Rev. Pharmacol., 2: 409-
424.
Grabowski, C. T. (1977). Atmospheric gases: Variations in
concentration and some common pollutants. In: Handbook of
Teratology, vol. 1, General principles and etiology, J. G. Wilson
and F. C. Fraser, eds., pp 405-420. New York. Plenum.
Report of a WHO Scientific Group. (1967). Principles for the
testing of drugs for teratogenicity. WHO Tech. Rep. Ser., 364:
18PP.
Robens, J. (1974). Teratogenic techniques and agents and
mutagenicity. In: Handbook of laboratory animal science,
vol. 1, E. C. Mclby, Jr. and N. T. Altman, eds., pp 177-190.
Cleveland, Ohio. CRC.
-------
. \. 115 ..,
V. D. References for Teratogenesis and Reproductive Performance
(continued)
2. Teratogenesis (continued)
/
Wilson, J. G. (1977). Environmental chemicals. In: Handbook
of teratology, vol. 1, General principles and etiology, J. G. Wilson ;
and F. C. Fraser, eds., pp 357-385. New York. Plenum. /'
Wilson, J. G. (1975). Reproduction and teratogenesis: Current /
methods and suggested improvements. J. Assoc. Off. Anal. Chen., j
58: 657-667. . J
<
r
-------
Vi. POSSIBLE TESTING APPROACHES
A. Matrix^ Approach
B. Heirarchical Approach
-------
VI. 1
VI. POSSIBLE TESTING APPROACHES
Kith the increasing realization that human exposure to chemicals
may contribute to a significant health risk, federal agencies and
other groups have formulated recommendations and guidelines regarding
evaluation of these chemicals and the health risk they may present.
Guidelines have been published for the testing of pesticides, food
additives, and household substances. Recent concern regarding the
increased incidence of cancer and potential genetic.hr.23rd to future
generations due to mutagenic a.nd carcinogenic chemicals in the
environment has led to recommendations for mutagenicity-carcinogenicity
screening of chemicals. These guidelines and recommendations for
health effects testing of chemicals can be classified as involving
either a matrix (battery) approach or a hierarchical (tier) approach.
Either of these approaches could be implemented with a fixed set
of tests or could employ a flexible set of tests which would be
selected based on the chemical structure of the test material, pre-
existing data base end other weighting factors discussed in Chapter
III.
Examples of these two approaches are shown in Tables 1 and 2.
The particular tests listed in each table are shown only as examples.
The final determination of which specific tests and approach to
testing will be implemented would take into account many of the
following factors:
1. The purpose of such tests (e.g., to detect potential
biological activity versus making regulatory decisions). - '-''
2. The weighting factors discussed in Chapter III.
5. Consultation by scientific experts as to which tests are
most appropriate.
4. Desirability for uniform health effects testing requirements
within EPA and across severa- regulatory agencies.
5. The number of fuels and fuel additives or test materials
derived from them that would require tnstini?.
6. The cost/benefit considerations for including specific
tests.
-------
VI. 2
A. Matrix Approach
The matrix approach to health effects testing which, for
example, is utilized in the registration of pesticides, involves
the employment of a prescribed battery of tests which may be
sufficient for human risk assessment. Under these guidelines all
tests'are completed prior to decision making. Generally acute
tests are utilized in this approach to determine exposure conditions
for the chronic studies. The matrix of tests employed can be |
flexible as shown in the example (Figure 1, mutagenicity column)
where 8 tests must be completed from three different areas, but
there is some choice in the specific test employed. Alternatively,
the entire matrix could be fixed such that a specified number of
tests would be required for registration of the substance.
; The matrix approach to health effects testing has been widely
used and accepted as an approach which permits safety evaluation.
This approach is most applicable to the evaluation of relatively
small number:; of chemicals which are expected to present a signi-
ficant human exposure, as in the case of drugs, food additives and «
pesticides. This approach has not been used with combustion products ]
nor have the matrices previously employed emphasized inhalation j
exposures which would be moit useful in a health risk assessment 3
of fuels and fuel additives. The matrix approach would be the most
definitive as well as the most expensive approach to testing '.aels
and fuel additives.
B. Heirarchical Approach
The heirarchical approach to health effects testing has been
recommended as an approach to testing large numbers of compounds.
A tiered battery of tests is employed-in a step-wise fashion with ,
results from each of the first tier of screening tests determining
whether further tests should be used. In this approach, after
prioritizing chemicals based on chemical structure (Tier 0), a
large number of chemicals would be screened in rapid, sensitive
jjn vitro or acute i_n_ vivo tests. The first tier or level of tests
detects biological activity in the areas of toxicity, mutagenicity,
carcinogenicity, and teratogenicity. The second tier of testing
employs confirmatory or subchronic tests while the rhird tier of
tests involve chronic whole animal exposures which should allow
risk assessment. .
It is important that the tests in the first tier detect as
few false negatives as possible since confirmation (Tier 2) tests
will generally be employed only on those test materials detected
as potentially hazardous. This approach therefore tests larger
numbers of compounds in the earliest tiers with less expensive and
less time consuming tests. Only a small percentage of the compounds
in Tier 1 would be expected to show sufficient biological activity
-------
VI. 3
to warrant Tier 2 or 3 testing. The later (higher) levels of
tests are more time consuming and expensive. The entire tier would
need to be completed prior to a human risk assessment.
-------
VI. References for Possible Testing Approaches
Environmental Protection Agency Pesticide Program. (1975).
Guidelines for registering pesticides in the United States.
Fed. Reg., 40: 1, 2, 3, June 25.
Food and Drug Administration Advisory Committee on Protocols
for Safety Evaluation. (1971). Panel on Carcinogenesis report on
cancer testing in the safety evaluation of food additives and pest-
icides. Toxicol. Appl. Pharmacol., 20: 419-438.
National Academy of Sciences - National Research Council. (1964).
Committee on Toxicology. Principles and procedures for
evaluating the toxicity of household substances. NAS Publ.
no. 1138. .Washington, D. C.
DHEW. (1977). Approaches to determining the mutagenic properties
of chemicals: Risk to future generations. Prepared for the
DHEW Committee to Coordinate Toxicology and Related Programs by
the working group of the Subcommittee on Environmental Mutagenesis.
In preparation, 1977.
Food and Drug Administration. (1976). Criteria for evaluation of
the hfcalLii aspects uf using flavoring substances as food ingredients.
Prepared for Bureau of Foods, FDA. Life Sciences Research Office,
Federation of American Societies for Experimental Biology,
Bethesda, Md.
Bridges, B. A. (1973). Some general principles of mutagenicity
screening and a possible framework for testing procedures.
Environ. Health Perspect., 221-227. , - , , ., , f,
Bridges, 13. A. (1974). The three-tier approach to mutagenicity
screening and the concept of radiation-equivalent dose. Mutat.
Res., 26: 335-340.
Flanun, W. G. (1974). A tier system approach to mutagen testing.
Mutat. Res., 26: 329-333.
Bridges, B. A. (1976). Use of a three-tier protocol for evaluation
of long-term toxic hazards particularly mutagenicity and
careinogcnicity. In: Screening tests in chemical Carcinogenesis,
R. Montcsano, H. Bartsch, and L. Tomatis, eds., pp 549- 559.
(IARC public, no. 12, Lyon, France).
Sobcls, F. H. (1977). Some problems associated with the testing
for environmental mutagens and a perspective for studies in "Com-
parative Mutagenesis". Mutat. Res., 46: 245-260.
-------
TOXIC ITT
t"ioner:il
A'.:ute i>i uivri
/V/jm,-7/./m/r1
Chronii in vivo
ink-.lation
Pulmonary
Inhalation in vivo
a<:nte
uub-vhi'on'iv
Central Nervous System
MUTAGENICITY
Gene Mutation
3 of these 5
lirmtet'ia
F.ukariiatc
Inanet
MrvTalian cells
Hodent (specific locus)
Chromosonal Effects
3 of these 4
Inane-1
Rodent cijtorjenetic test
Rodent dominant ,lethzl
Rodent heritable trcmslocation
DNA Damage and Repair
2 of these 4
Bantcria
Yeaat tnitotic recombination
and nune conversion
Miitmalian cell DNA repair
Kamalian cell eiater-chrornitid
exvfange
CARCINOCENICITY
Chronic? in vivo
inhalation
TERATOGKNICITY
Tcratogenesis
test
HHALTH RISK ASSESSMENT
1 .
AH KXAMPLE OF A HATKJX APPROACH' TO TESTING
-------
TlERl
(Level 1)
TOXICITY
r.encrnl
l^iilnonary
fic-.ttn i'l'iiiila i i on
Toxicity Toxic:
TIER 2
(Level 21
TIER 3
(Level })
HUTACI-SICITY
Com: Mutation
PNA Repair
Clirociosomal Hffects
y^cj/i t
Neg.-itlvc Posi
,-u
l:i
laon.'iry
,,:,
ntral Nervous
Syslcn
C!h runic
In uC'J
' '""'
Ci-nc Mutation
H-jmnlian calls
UNA Repair
MTxmlian sells
Chromosomal liffects
(icne Mutation
Rodcnta ,
Chronosoiaal Effects
1HEALTH RISK ASSESS tNT
CARCINOGI-NICITY
Mut.igenes is
Tier 1 teats
Noga t i vc
Posi
ivc
Ncopl ast ic
Trunitforoation
in
vitro
J
Ch ron i c
:"
fffffin
>i :w
TliRATOCnMCITY
Teraf ogcncsis
in
JIVO
BIO'tJQSlCAl,
ACTivrrr !.\L-U
TESTS (Cub-
chronic)
RISK
,'/;/!.'' :.. /iff f'XAM.'K i'f AX uiKRih."ita;:;. AIriioAi'i! TO rt::;ri:i
-------
BIBLIOGRAPHY
-------
BIBLIOGRAPHY
Abrahamson, S., and Lewis, E. B. (1971) The detection of mutations
in Drosophila melanogaster. Chera. Mutagens 2:461-487.
Adalis, D., Gardner, D. E., Miller, F. J., and Coffin, D. L.
(1977) Toxic effects of cadmium on ciliary activity using
a tracheal ring model system. Environ. Res. 13:111-120.
Adams, D. F. (1976) Sulfur compounds. I_n_: Air pollution, 3rd
ed., vol. 3, A. C. Stern, Ed. Academic, New York, pp 213-258.
Alarie, Y. (1966) Irritating properties of airborne materials
to the upper respiratory tract. Arch. Environ. Health
13:433-449. -
Alarie, Y. (1973) Sensory irritation of the upper airways by
airborn chemicals. Toxicol. Appl. Pharmacol. 24:279-297.
Alarie, Y. (1973) Sensory irritation by airborne chemicals.
CRC Grit. Rev. Toxicol. 2:299-363.
Albert, R. E. and Altshuler, B.. (1976) Assessment of environmental
carcinogen risks in terms of life shortening. Environ.
Health Perspect. 13:91-94.
Albert, R. E., Train, R. E., and Anderson, E. (1977) Rationale
developed by the environmental protection agency for the
assessment of carcinogenic risks. J. Natl. Cancer Inst.
58:1537-1541.
Allen: J. W. and Latt, S. A. (1976) Analysis of sister chromatid
exchange formation in vivo in mouse spermatogonia as a new
test system for environmental mutageris. Nature (London)
260:449-451.
Altschuler, B., Yarmus, L., Palmes, E. U., and Nelson, N. (1957)
Aerosol deposition in the human respiratory tract. I. Experi-
mental procedures and total deposition. Arch. Ind. Health
15:293-303.
Altshuller, A. P. (1968) Analysis or organic gaseous pollutants.
In: Air Pollution, vol. 2, A. C. Stern, Ed. Academic, New
York, pp 115-145.
Altshuller, A. P. (1976) Hydrocarbons and carbon oxides. In:
Air pollution, 3rd ed., vol. 3, A. C. Stern, Ed. Academic,
New York, pp 183-211.
Amdur, M. 0. (1975) Air Pollutants. In: Toxicology, the basic
science of poisons, L. J. Casarett and J. Doull, Eds.
MacMillan, New York, pp 527-554.
-------
Amdur, M. 0. and Mead, J. (1958) Mechanics of respiration in
unanesthetized guirea pigs. Am. J. Physiol. 192:564-368.
Ames, B. N. (1976) Carcinogenicity tests. Science 191:241-245.
Ames, B. N., Kaminon, H. 0., and Yamasaki, E. (1975) Hair dyes
are mutagenic: identification of a variety of mutagenic
ingredients. Proc. Natl. Acad. Sci. USA 72:2423-2427.
Ames, B. N., Lee, F. D., and Durston, W. E. (1973) An improved
bacterial test system for the detection and classification
of mutagens and carcinogens. Proc. Natl. Acad. Sci. USA
70:782-786.
Ames, B. N., McCann, J., and Yamasaki, E. (1975) Methods for
detecting carcinogens and mutagens with the Salmonella/
mammalian-microsome mutagenicity test. Mutat. Res. 31:
347-363.
Andersen, I., Lundqvist, G. R., Jensen, P. L., and Proctor, D. F.
(1974) Human response to controlled levels of sulfur dioxide.
Arch. Environ. Health 28:31-39.
Anderson, M. W. and Eling, T. E. (1976) Studies on the uptake,
metabolism, and release of endogenous and Rxogenws che.micals
by use of the isolated perfused lung.- Environ. Health
Perspect. 16:77-81.
Andreyeshchcva, N. G. (1976) Predicting biological effect as a
function of the chemical structure and the primary physical
and chemical properties of organic compounds. Environ.
Health Perspect. 13:27-30.
Aranyi, C. (1977) Cytotoxic effect of trace metals adsorbed onto
fly ash particles. Environmental Health Effects Research
Series, HERL, OKI), EPA, Research Triangle Park, N. C. 27711.
EPA-600/1-77-017 (March 1977), 31 pp, PB 267-078.
Axelrod, II. D. and Lodge, J. P., Jr. (1976) Sampling and cali-
bration of gaseous pollutants. In: Air pollution, 3rd ed.,
vol. 3, A. C. Stern, Ed. Academic, New York, pp 145-182.
Axelrod, 11. D. and Lodge, J. P., Jr. (1976) Sampling and cali-
bration of gaseous pollutants. In: Air pollution, 3rd ed.,
vol. 5, A. C. Stern, Ed. Academic, New York, pp 156-158.
-------
Baars, A. J., Zijlstra, J. A., Vogel, E., and Breimer, D. U.
(1977) The occurrence of cytochrome P-450 and aryl hydrocarbon
hydroxylase activity in Drosophila melanogaster microsoraes,
and the importance of this metabolizing capacity for the
screening of carcinogenic and mutagenic properties of foreign
compounds. Mutat. .Res. 44:257-268.
Bang, B. G. and Bang, F. B. (1977) Nasal mucociliary systems.
In: Respiratory defense mechanisms, Part I., J. D. Brain,
D. F. Proctor, and L. M. Reid, Eds. (Lung biology in health
and disease, vol. 5, Part I). Marcel Dekker, Inc., New
York, pp 405-426.
Bang, F. B. and Bang, B. G. (1977) Mucous membrane injury and
repair. In: Respiratory defense mechanisms, Part I., J. U.
Brain, D. F. Proctor, and L. M. Reid, Eds. (Lung biology in
health and disease, vol. 5, Part I). Marcel Uekker, Inc.,
New York, pp 455-488.
Barron, E. S., Miller, Z. B., a/id Bartlett, G. R. (1947) Studies
on biological oxidations. XXI. The metabolism of lung as
determined by a study of slices and ground tissue. J. Biol.
Chem. 171:791-800.
Bartsch, H., Camus, A., and Malaveille, C. .(1976) Comparative
mutagenicity of N-nitrosamincs in a semi-solid and in a liquid
incubation system in the presence of rat or human tissue
fractions. Mutat. Res. 37:149-162.
Bartsch, H., Malaveille, C., and Montesano, R. (1975) Human,
rat and mouse liver-mediated mutagcnicity of vinyl chloride
in S_. Typhimurium strains. Int. J. Cancer 15:429-437.
Bateman, A. J. (1973) The dominant lethal assay in the mouse.
Agents Actions 5:73-76.
oateman, A. J. and Epstein, S. S. (1971) Dominant lethal mutations
in mammals. Chem. Mutagens 2:541-568.
Becker, B. (1975) Teratogens. In: Toxicology, the basic science
of poisons, L. J. Casarett and J. Doull, Eds. Macmillan,
New York, pp 313-332.
Beckley, J. H., Russell, T. J., and Rubin, L. F. (1969) Use of
the rhesus monkey for predicting human response to eye irri-
tants. Toxicol. Appl. Pharmacol. 15:1-9.
Bend, J. R. and Hook, G. E. R. (1977) Hepatic and extrahepatic
mixed-function oxidases. In: Handbook of physiology, Sec.
9, Reactions to environmental agents, D. H. K. Lee, Ed.
American Physiological Society, Bethesda, Maryland, pp 419-440.
Berenblum, I. (1969) A re-evaluation of the concept of cocar-
cinogenesis. Prog. Exp. Tumor Res. 11:21-30.
-------
Berenblum, 1. and Shubik, P. (1949) The persistence of latent
tiuiior cells induced in the mouse's skin by single application
of 9,10-diraethyl-l,2-benzanthracene. 13r. J. Cancer 3:384-386.
Bernfield, P., Nixon, C. W., and Homburger, F. (1964) Studies
on the effect of irritant vapors on ciliary mucus transport.
Toxicol. Appl. Pharmacol. 6:103.
Biller, W. 1:. and llarkins, J. (1976) Measurement, testing and
surveillance of air pollution from mobile sources. IJK
Air pollution, 3rd ed., vol. 3, A. C. Stern, Lid. Academic,
New York, pp 647-716.
Bingham, E., Barkley, W., Murthy, R., and Vassalo, C. (1977)
Investigation of alveolar macrophages from rats exposed to
coal dust. In: Inhaled particles, IV, part 2, W. H. Watton,
Ed. Pergamon, New York, pp 543-550. \
Binghara, E., Niemeier, R., and Dalbey, W. (1976) Metabolism of
environmental pollutants by the isolated perfused lung. Fed.
Proc. 35:81-84.
Bingham, 13., Pfitzer, E. A., Barkiey, W., and Radford, E. P.
(1968) Alveolar macrophages: reduced number in rats after
prolonged inhalation of lead sesquioxide. Science 162:
1297-1299.
Binns, R. (1977) Inhalation toxicity studies on cigarette smoke.
IV. Expression of .the dose of smoke ^articulate material
applied to the lungs of experimental animals. Toxicology
7:189-195.
Binns," R.; Beven, J. L., Wilton/L. Vr, and Lugton, W. G. D.-
(1976) Inhalation toxicity studies on cigarette smoke.
II. Tobacco smoke inhalation dosimetry studies on small
laboratory animals. Toxicology 6:197-206.
Binns, R., Beven,.J. L., Wilton, L. V., and Lugton, W. G. D.
(1976) Inhalation toxicity studies on cigarette smoke.
III. Tobacco smoke inhalation dosimetry study on rats.
Toxicology 6:207-217.
Blevins, R. D. and Dunn, W. C., Jr. (1975) Effects of carbaryl
and dicldrin on the growth, protein content, and phospholipid
content of HcLa e.ells. J. Agric. Food Chem. 23:377-582.
Bloom, B. R. and J. R. David, Eds. (1976) In vitro methods in
cell-mediated and tumor immunity. Academic, New York, 748 pp.
Bocek, K. (1976) Relationships among activity coefficients,
partition, coefficients and solubilities. Experientia Suppl.
23:231-240.
-------
Bokina, A. I., Eksler, N. D., Semenenko, A. D., and Merkuryeva,
R. V. (1976) Investigation of the mechanism of action of
atmospheric pollutants on the central nervous system and
comparative evaluation of methods of study. Environ. Health
Perspect. 13:37-42.
Bowden, D. 11. (1976) The pulmonary macrophage. Environ. Health
Perspect. 16:55-60.
Bradow, R. L. and.Sigsby, J. E., Jr. (1976) Protocol to assess
the effect of fuel additives on control system performance.
ESRL, EPA, Research Triangle Park, N. C.
Brain, J. D. (1970) Free cells in the lungs, some aspects of
their role, quantitation and regulation. Arch. Int. Med.
126:477-487.
Brain, J. D. and Corkery, G". C. (1975) The effect of increased
particles on the endocytosis of radiocolloids by pulmonary
macrophages i£ vivo: competitive and toxic effects. Inhaled
Particles 4 (2):551-564.
Brewen, J. G. and Preston, R. J. (1974) Cytogenetic effects of
environmental mutagens in mammalian cells and the extrapola-
tion to man. Mutat. Res. 2^:297-305.
Bridges, B. A. (1972) Simple bacterial systems for detecting
mutagenic agents. Lab. Praet. 21:413-416.
Bridges, B. A. (1976) Short terra screening tests for carcinogens.
Nature 261:195-200.
Bridges, B. A., Dennis, R. ,E., and Munson, R. J. (1967) Muta.- .-
tion in Escherichia coli B/r WP2 try" by reversion or
suppression of a chain-terminating condon. Mutat. -Ites. 4:
502-504.
Brown, E. A. B. (1974) The localization, metabolism, and effects
of drugs and toxicants in the lung. Drug. Mctab. Rev. 3:
33-87.
Brown, V. K. H. (.1971) A comparison of predictive irritation
tests with surfactants on human and animal skin. J. Soc.
Cosmet. Chcjn. 22:411-420.
Browne, R. K., Anderson, A. N., II, Charvez, B. W., and Azzarello,
R. J. (1975) Ophthalmic response to chlorhexidine diglu-
conate in rabbits. Toxicol. Appl. Pharmacol. 52:621-627.
Brusick, D. J. (1977) In vitro mutagenesis assays as predictors
of chemical carcinogencsis in mammals. Clin. Toxicol. 10:
79-109.
-------
Brusick, D. J. and Mayer, V. W. (1973) New developments in
mutagenicity screening techniques with yeast. Knviron.
Health Perspect. 6:83-96.
Burkhart, J. K., Hinners, R. G., and Malanchuk, M. (1973) Cata-
lytic converter exhaust emissions, Paper no. 74-129. Air
Pollution Control Assoc., Pittsburgh, Pennsylvania, 25 pp.
-------
Candeli, A., Morozzi, G., Paolacci, A., and Zoccolillo. (1975)
Analysis using thin layer and gas-liquid chromatography of
polycyclic aromatic hydrocarbons in the exhaust products
from a European car running on fuels containing a range of
concentrations of these hydrocarbons. Atmos. Environ. 9:
843-849.
Capizzi, R. L., Papirmeister, B., Mullins, J. M., and Cheng, E.
(1974) The detection of chemical mutagens using the L5178Y/
Asn-murine leukemia in vitjro_ and in a host-mediated assay.
Cancer Res. 34:3073-3082.
Carpenter, C. P., Kinkead, E. R., Geary, U. L., Jr., Sullivan,
L. J., and King, J. M. (1975) Petroleum hydrocarbon
toxicity studies. I. Methodology. Toxicol. Appl. Pharmacol.
32:246-262.
Casarett, L. J. (1971) Toxicology:The respiratory tract. Annu.
Rev. Pharmacol. 11:425-446.
Casarett, L. J. (1975) Toxicology of the respiratory tract. In:
Toxicology, the basic science of poisons, L. J. Casarett and
J. Doull, Eds. Macmillan, New York, pp 201-224.
Casarett, L. J. and J. Doull, Eds. (1975) Toxicology, the basic
science of poisons. Macmillan, New York, 768 pp.
Casto, B. C., Janosko, N., and DiPaolo, J. A. (1977) Development
of a focus assay model for transformation of hamster cells
in vitro by chemical carcinogens. Cancer Res. 57:3508-3515.
Chamberlain, A. C., Clough, W. S., Heard, M. J., Newton, D.,
- Stott, A. N. B., and Wells, A.-C. (1975) 'Uptake of inhaled
.lead from r.'.otor exhaust. Postgrad. Mod. J. 51:790-794.
Chandra, S. V. and Shukla, G. S. (1976) Role of iron deficiency
in inducing susceptibility to manganese toxicity. Arch.
Toxicol. 55:319-323.
Chu, E. H. Y. (1971) Induction and analysis of gene mutations
in mammalian cells in culture. Chem. Mutagens 2:411-444.
Clayson, D. B., and Garner, R. C. (1976) Carcinogenic aromatic
amines and related compounds. In: Chemical carcinogens,
C. E. Searle, Ed. American Chemical Society, Washington,
D. C., pp 366-461.
Cleaver, J. E. (1975) Methods for studying repair of UNA damaged
by physical and chemical carcinogens. Methods Cancer Res.
11:123-165.
Clegg, D. J. (1971) Teratology. Annu. Rev. Pharmacol. 11:
409-424.
-------
Cline, J. C. and McMahon, R. E. (1977) Detection of chemical
mutagens: Use of .concentration gradient plates in a high
capacity screen. Res. Commun. Chem. Pathol. Pharmacol. 16:
523-533.
Clive, D. (1977) A linear relationship between turaorigenic
potency in vivo and mutagenic potency at the heterozygous
thyir.idine kinase (TK +/-) locus of L5178Y mouse lymphoma
cells coupled with mammalian metabolism. Abstract no. Bb-3,
presented at Environmental Mutagen Society Meeting, Feb. 1977,
Colorado Springs, Colorado.
Clive, D., Flamm, W. G., and Patterson, J. B. (1973) Specific-
locus mutational assay systems for mouse lymphoma cells.
Chem. Mutagens 3:79-103.
Clive, D. and Spector, J. F. S. (1975) Laboratory procedure for
assessing specific locus mutations at the TK locus in
cultured L5178Y mouse lymphoma cells. Mutat. Res. 31:17-29.
Coffin,.D. L. and Blommer, E. J. (1967) Acute toxicity of
irradiated auto exhaust. Its indication by enhancement of
mortality from streptococcal pneumonia. Arch. Environ.
Health 15:56-38.
Coffin, D. L., Gardner, D. E., HoJzraan, R. S,, and Wolock, F. J.
(1968) Influence of ozone on pulmonary cells. Arch.
Enviorn. Health 16:633-636.
Cohen, M. M. and Hirschhorn, K. (1971) Cytogenetic studies in
animals. Chem. Mutagens 2:515-534.
Cohen, G. M. and Moore, B. P. (1975) Metabolism of (3H)benzo(a)-
pyrene by different portions of the respiratory tract.
Biochem. Pharmacol. 25:1623-1629.
Committee for the Working Conference on Principles of Protocols
for Evaluating Chemicals in the Environment. (1975)
Principles for evajuatiftg -ehemicals in che environment,
Part Three: Huwan health effects, Chapter XI, Effects of
behavior, N. Nelson, Chairman. National Academy of Sciences,
Washington, D. C., pp 198-216.
Commoner, U., Vithayathil, A. J., and Henry, J. I. (1974) Detec-
tion of metabolic carcinogen intermediates in urine or
carcinogen-fed rats by means of bacterial mutagenesis.
Nature (London) 249:850-852.
Conkle, v. P., Register, J. W., and Worth, G. L. (1965) Multi-
sta/e cryogenic trapping system. Aeorsp. Med. 36:369-874.
-------
Cornish, H. H. (1975) Solvents and vapors. In: Toxicology,
the basic science of poisons, L. J. Casarett and J. Doull,
Eds. Macmillan, New York, pp 503-526.
Coulston, F. and Wills, J. H. (1975) Mutagenic, carcinogenic,
and teratogenic effects of pollutants in respect to man.
Pure Appl. Clie,u. 42:209-222.
Creasia, D. A., Poggenburg, J. K., Jr., and Nettesheira, P. (1976)
Elution of benzo(a)pyrene from carbon particles in the
respiratory tract of mice. J. Tuxicol. Environ. Health
1:967-975.
-------
Dalhanvm, T. (1956) Mucous flow and ciliary activity in the
trachea of healthy rats and rats exposed to respiratory
irritant gases (S02, lisN, HCHO). Acta Physiol. Scand.36:l
(Supplement 125).
Dalharaia, T. and Rylander, R. (1965) Ciliastic action of smoke
from filter-tipped and non-tipped cigarettes. Nature 201:401.
Davies, R. E., Harper, K. H. and Kynoch, S. R. (1972) Inter-
species variation in dermal reactivity. J. Soc. Cosmet.
Chem. 23:371-581
Davis, B. R., Khitehead, J. K., Gill, M. E., Lee, P. N., Butterworth,
A. D., and Roe, F. J. R. (1975) Response, of Tat lung to
3,4-benzpyrene administered by intratracheal instillation
in infusine with or. without carbon black. Br. J. Cancer
51:443-452. ' ;
Davis, B. R., Khitehead, J. K., Gill, M. E., Lee, P. N. Butterworth,
A. D., and Roe, F. J. R. (1975) Response of rat lung to
inhaled vapour phase constituents (VP) of tobacco smoke
alone or in conjunction with smoke condensate or fractions
of smoke condensate given by intratracheai instillation.
Br. J. Cancer 31:462-468.
Deichmann, W. B., Keplinger, M. L., and Lanier, G. E. (1958)
Acute effects of nitro-olefins upon experimental animals.
AMA Arch. Ind. Health 18:312-319.
Dennis, R. (1976) Handbook on aerosols (TID-2660-S). Nl'IS,
Springfield, Virginia, 148 pp.
de Serres, F. J.°"and Mailing, H. V." " (1971) Measurement'of
recessive letha.1 d^jnage over the entire -riome and at two
specific loci in the AD-3 region of a twr-component hetero-
karyon of N'curospora crassa. Chem. Muta -ens 2:511-3-52.
de Serres, F. J. (1976) Mutagenicity of cu;;:::.ercially available
hair dyes. J. Toxicol. Environ. Health 1:5.59-541.
Dietzmunn, H. E. (1975) Protocol to characterize gaseous emis-
sions as a function of fuel and additive composition. EPA/
600/2-75/048 (PB-255365/6GA). NTIS, Springfield, Virginia,
132 pp.
DIPaolo, J. A., Nelson, R. L., Donovan, P. J., and Evans, C. H.
(1!?75) Host-mediated in_ vivo-ir vitro assay for chemical
carcinogenesis. Arch. Pathol. 95:580-385.
DiPaolo, J. A., Takano, K., and Popescu, N. C. (1972) Quantita-
tion of chemically induced neoplastic transformation of
BALB/3T5 cloned cell lines. Cancer Res. 32:2686-2695.
-------
Dixon, R. L. (1976) Problems in extrapolating toxicity data for
laboratory animals to man. Environ. Health Perspect. 13:
43-50.
Dixon, R. L. (1972) Toxicology of environmental agents: A blend
of applied and basic research. Environ. Health Perspect. 2:
103-116.
Doull, J. (1975) Factors influencing toxicology. In: Toxicology,
the basic science of poisons, L. J. Casarett and J. Doull,
Eds. MacMillan, New York, pp 133-147.
Draize, J. H. (1965) Appraisal of the safety of chemicals in
foods, drugs, and cosmetics--dermal toxicity. Assoc. of
Food and Drug Officials of the U.S., Topeka, Kansas.
Draize, .J . H.', IVoodard, G., and Calvcry, H. 0. (1944) Methods
for the study of irritation and toxicity of substances
applied topically to the, skin and -aucous membranes. J.
Pharmacol. Exp. Ther. s:': 577-390.
Drake, J. W., Abraharason, S., Crow, J. F., Hollaender, A., Lederberg,
S., Legator, M. S., Neel, /. V.. Shaw, M. W. , Sutton, H. E.,
von Borstel, R., and Zinuncri i£ , S. (1975) Environmental - '-.
aoentc hazards. Science 187:503-514.
Dungworth, D. L, Schwartz, L. W., Tyler, W. S., and Phalen, R. F.
(1976) Morphological methods for evaluation of pulmonary
toxicity in animals. Annu. Rev. Pharmacol. Toxicol. 16:
381-399.
Durston, W. E. and Ames, B. N. (1974) A simple metho.j for .the
detection of mutagens in urine: Studies with the c.ircinogen
2-acetylaminofiuorone. Proc. Natl. Acad. Sci. USA 71:
737-741.
-------
Eagle, H. and Foley, G. E. (1958) Cytotoxicity in human cell
cultures as a primary screen for the detection of anti-tumor
agents. Cancer Res. 13:1017-1025.
Edwards, C. C. (1972) Hazardous substances. Proposed revision
of test for primary skin irritants. Fed. Regist. 3?:
27635-27636.
Edwards, H. W., Rosenvold, R. J., and Wheat, H. G. (1975)
Sorption of organic lead vapor on atmospheric dust particles.
In: University of Missouri's annual conference on Trace
Substances in Environmental Health, 9th, Columbia, Missouri,
June 10-12, 1975, D. D. Hemphill, Ed., pp 197-205.
Elson, N. A. and Crystal, R. G. (1977) Cellular approaches to
the study of environmental pollutants. In: Biochemical
effects of environmental pollutants, S. D. Lee, Ed. Ann
Arbor Science, Ann Arbor, Michigan, pp 7-25.
Ehling, U. H. (1976) Mutagenicity testing and risk estimation
with mammals. Mutat. Res. 41:113-122.
EPA (1977) Pesticides Guidelines Draft.
Epstein, S. S., Arnold, C., Andrea, J., Bass, W., and Bishop, Y.
(1972) Detection of chemical mutagens by the dominant lethal
assay in the mouse. Toxicol. Appl. Pharmacol. 23:288-325.
Esber, H. J., Menninger, F. F'.'j Jr., Bogden, A. E., and Mason,
M. M. (1973) Inimunological deficiency associated with
smoke inhalation by mice. Primary and secondary hemagglutinin
response. Arch. Environ. Health,27:99-104.
Ewig, R. A. G. and Kohn, K. W. ;1977) DNA damage and repair in
mouse leukemia L1210 cells treated with nitrogen mustard,
1,3-bis (2-chl-roethyl)-1-nitrosourca, and other nitrosourcas'.
Cancer Res. 37:2114-2122.
-------
Fahrig, R. (1975) Development of host-mediated mutagenicity
tests--ycast systems. II. Recovery of yeast cells out of
testes, liver, lung, and peritoneum of rats. Mutat. Res.
31:381-393.
Falk, H. L., Miller, A., and Kotin, P. (1958) Elution of 3,4-benz-
pyrene and related hydrocarbons from soots by plasma proteins.
Science 127:474-475.
Farrell, R. L. and Davis, G. W. (1974) Effect of particulate
benzo(a)pyrene carrier on carcinogenesis in the respiratory
tract of hamsters. In: Experimental lung cancer, E. Karbe
and J. F. Park, Eds. Springer-Verlag, New York, pp 186-198.
Farrell, R. L. and Davis, G. W. (1974) The effects of particulates
on respiratory carcinogenesis by diethylnitrosamine. In:
Experimental lung cancer, E. Karbe and J. F. Park, Eds.
Springer-Verlag, New York, pp 219-235.
Feron,.V. J. (1972) Respiratory tract tumors in hamsters after
intratracheal instillations of benzo(a)pyrene alone and
with furfural. Cancel Res. 32:28-36.
Feron, V. J., de Jong, D., and Rijk, M. A. H. (1976) Clearance
of benzo(a)pyrene from the respiratory tract of hainsters
following its intratracheal instillation with or without
ferric oxide. Zentralbl.- Bakteriol. Parasitenkd. Infektionskr.
Hyg. Abt. Grig. Reihe. B 163:441-447.
Food and Drug Administration Advisory Committee on Protocols for
Safety Evaluations. (1971) Panel on Carcinogenesis report
on cancer testing in the safe'y evaluation of food additives.
"and pesticides. Toxicol. Appl. Pharmacol. 20:419-438.
Food and Drug Administration Advisory Committee on Protocols for
Safety Evaluations. (1970) Panel on Reproduction report on
reproduction studies in safety evaluation 01 food additives
and pesticide residues. Toxicol. Appl. Pharmacol. 16:264-296.
Fornace, A. J., Jr. and Kohn, K. W. (1976) DNA-protein cross-
linking by ultraviolet radiation in normal human and xeroderma
pigmentosuin fibroblasts. Biochim. Biophys. Acta 455:95-105.
Forance, A. J., J,r., Kohn, K. K., and Kann, H. E., Jr. (1976)
DNA single-strand breaks during repair of UV damage in human
fibroblasts and abnormalities of repair in xeriv-erma pigmen-
tosu-n. Proc. N'atl. Acad. Sci. USA 75:39--!-.
i-'rant3, C. N. and Mailing, H. V. (1976) Brer ..voxyuriJinc
resistance induced in mouse lymphoma cei-.-. !>y ir.iciv' ;onial
activation of di.nerhylnitrosaminc. J. ;. xicoi. !.m : ron.
iiealth 2.: 179- 187.
-------
Frantz, C. N. and Mailing, H. V. (1975) The quantitative micro-
somal mutagenesis assay method. Mutat. Res. 31:365-379.
Fraser, D. A. et_ al. (1959) Exposure chambers for research in
inhalation: design, construction, operation, and performance.
(Public Health Monogr.) U. S. Govt. Print. Off., Washington,
D. C.
Freeman, A. E., Igel, 11. J., and Price, P. J. (1975) Carcino-
genesis in_ vitro. 1. In vitro transformation of rat embryo
cells: Correlations with the known tumorigenic activities
of chemicals in rodents. In Vitro 11:107-116.
Freireich, E. J., Gehan, E. A., Rail, D. P., Schmidt, L. H., and
Skipper, H. E. (1966) Quantitative comparison of toxicity
cf anticancer agents in mouse, rat, hamster, dog, monkey,
and man. Cancer Chemother. P.ep. 50:219-244.
Frohberg, H. (1973) Critique of in vivo cytogenetic test systems.
Agents Actions 3:119-123.
B.H
-------
Gallagher, J. T,, Kent, P. W., Passatore, M., Phipps, R. J., and
Richardson, P. S. (1975) The composition of tracheal mucus
and the nervous control of its secretion in the cat. Proc.
R. Soc. London (Ser. B) 192:49-76.
Gardner, D. E. and Graham, J. A. (1977) Increased pulmona-ry
disease mediated through altered bacterial defenses. Proc.
16th Annual Hanford Biol. Symp., Richland, Washington.
Gardner, D. E., Holzman, R. S., and Coffin, D. L. (1969) Effects
of nitrogen dioxide on pulmonary cell population. J.
Bacteriol. 98:1041-1043.
Gardner, D. E., Miller, F. J., Illing, J. W. Kirtz, J. M. (1977)
Alteration in bacterial defense mechanisms of the lung induced
by inhalation of cadmium. Bull. Eur. Physiol. Pathol.
Respir. 13:157-174. .. : .
Garrett, S. and Fuerst, R. (1974) Sex-linked mutations in
Drosophila after exposure to various mixtures of gas atmospheres.
Environ. Res. 7:286-293.
Cause, E. M., Greene, N. D., Meltz, M. L., and Rowlands, J. R.
(1977) In^ vivo and ir± vitro effects of sulfur dioxide upon
, biochemical and immunological parameters. In: Biochemical
effects of environmental pollutants, S. D. Lee, Ed. Ann
i Arbor Science, Ann Arbor, Michigan, pp 273-292.
| - Gentel, J. E., Manary, 0. J., and Valenta, J. C. (1974) Deter-
f mination of effect of particulate exhaust emissions of
additives and impurities in gasoline. EPA/650/2-74/061
I (PB-255910/4GA). NT1S, Springfield, Virginia, 228 pp.
f - . , . c . - . - . " r ,,..<- ,
Geran, R. I., Greenberg, N. H., Macdonald, M. M., Schumacher, A. M.,
and Abbott, B. J. (1972) Protocols for screening chemical
agents and natural products against animal tumors and other
biological systems, 3rd ed. Cancer Chemotherapy Reports,
part 3, 3:1-104.
Giever, D. M. (1976) Particulate matter sampling and sizing.
In: Air pollution, 3rd cd., vol. 3, A. C. Stern, Ed. Academic,
New York, pp 43-50.
Goldstein, E., Jordan, G. W., MacKenzie, M. R., and Osebold, J. W.
(1976) Methods for evaluating the toxicological effects of
gaseous and .particulate contaminants on pulmonary microbial
defense systems. Annu. Rev. Pharmacol. Toxicol 16:447-463.
Gooch, P. C., Creasia, D. A., and Brewer., J. C. (1976) The
cytogenetic effects cf ozone: Inhalation and in vitro
exposures. Environ. Res. 12:188-195.
-------
Gooch, P. C., Luippold, H. E., Creasia, D. A., and Brewen, J. G.
(1977) Observation on mouse chromosomes following nitrogen
dioxide inhalation. Mutat. Res. 48:117-119.
Graham, J. A., Gardner, D. E., Waters, M. D., and Coffin, D. L. \
(1975) Effect of trace metals on phagocytosis by alveolar 'j
macrophagcs. Infect. Immun. 11:1278-1283. i
Graham, J. A., Miller, F. J., Daniels, M. J., Payne, E. A., and j
Gardner, D. E. (1977) Influence of Cd, Ni, and Cr on <
primary immunity in mice. Environ. Res., in press. .1
Green, G. M. and Carolin, D. (1967) The depressant effect of j
cigarette smoke on the iji vitro antibacterial activity of \
of alveolar macrophages. N. Engl. J. Med'. 276:421-427. j
S
Green, G. M., Jakob, G. J., Low, R. B., and Davis, G. S. (1977) i
Defense mechanisms of the respiratory membrane. Am. Rev. 1
Respir. Dis. 115:479-514. . f
it
Green, G. M. and Kass, E. H. (1964) Factors influencing the *j
clearance of bacteria by the lung. J. CJin. Invest; 43: |
769-776. . I
Green, M. H. L., Bridges, B. A., Rogers, A. M., Horspool, G., |
Muriel, W. J., Bridges, J. W., and Fry, J. R. (1977) .3
Mutagen screening by a simplified bacterial fluctuation test: ]?
Use of microsomal preparations and whole liver cells for s
metabolic activation. Mutat. Res. 48:287-294. f
"»
%
Green, M. H. L. and Muriel, W. J. (1976) Mutagen testing using \
TRP"1' reversion in Escherichia coli. Mutat. Res. 38:3-32. |.
' .- . .- , ...... ,-'-,. 0 , r-V. , - -. --.- "3 \\
Green, S., Moreland, F. M., and Flamm, W. G. (1977) A new ;
approach to dominant lethal testing. Toxicol. Appl. . :
Pharmacol. 39:549-552.
Green, S., Zieger, E., Palmer, K. A., Springer, J. A., and Legator,
M. S. (1976) Protocols for the dominant lethal test, host-
mediated assay, and .in vivo cytogenetic test used in the
Food and Drug Administration's review of substances in.the
GRAS (generally recognized as safe) list. J. Toxicol. Environ.
Health 1:921-928. .
Grabowski, C. T. (1977) Atmospheric gases. Variations in con-
centration and some common pollutants. In: Handbook of
teratology, vol. 1, General principles and etiology, J. G.
Wilson and F. C. Fraser, Eds. Plenum, New York, pp 405-420.
Grob, K. (1965) Gas chromatography of cigarette smoke. Part
III. Separation of the overlap region of gas and particulate
phase by capillary columns. J. Gas Chromatogr. 5:52-56.
-------
Cuerin, M. R. (1975) Discussion: Identification of carcinogens,
tumor promoters, and cocarcinogens in tobacco smoke. (Conf.
750633-2). NTIS, Springfield, Virginia, 13 pp.
Guerin, M. R., Olerich, G., and Morton, A. D. (1974) Routine
gas chromatographic component profiling of cigarette smoke
for the identification of biologically significant constituents.
J. Chromatogr. Sci. 12:585-591.
Guess, H., Crump, K., and Peto, R. (1977) Uncertainty .estimates
for low-dose-rate extrapolations of animal carcinogenicity
data. Cancer Res. 37:3475-3483.
-------
Hansch, C. (1969) A quantitative approach to biochemical
structure-activity relationships. Ace. Chem. Res. 2:232-239.
Hansch, C. (1976) On the structure of medicinal chemistry. J.
Med. Chem. 19:1-6.
Hansch, C. and Fujita, T. (1964) p-o-ir Analysis. A method for
the correlation of biological activity and chemical structure.
J. Am. Chem. Soc. 86:1616-1626. .
Haque, R. and Schmedding, D. (1975) A method of measuring the
water solubility of hydrophobic chemicals: Solubility of
five polychlorinated biphenyls. Bull. Environ. Contam.
Toxicol. 14:13-18.
Harington, J. S. and Allison, A. C. (1977) Tissue and cellular
reactions to particles, fibers, and aerosols retained after
inhalation. In: .Handbook of physiology, Sec. 9, D. H. K.
Lee, Ed. . American Physiological Society, Bethesda, Maryland,
pp 263-283.
Harper, C., James, M. 0., Devereux, T. R., Patel, J. M., Bend,
J. R., and Fouts, J. R. (1976) Characteristics and development
of drug metabolism by pulmonary microsomes. Agents Actions
6:527-530.
Harrington, J. T., Jr. and Stastny,.P. (1973) Macrophage migra-
tion from an agarose droplet: Development of a micromethod
for assay of delayed hypersensitivity. J. Imraunol. 110:
752-759. ; >
Hazardous substances and articles; administration and enforcement
regulations. Test for eye-irritants." (1975) Fed. Regist.
38:27019.
Herbold, B. A., I'ohrborn, G., and Buselrnaier, W. (1977) Point-
mutation ressarch: Relevance for humans. J. Toxicol.
Environ. Health 2:1183-1191.
Herrold, K. M. and Dunham, L. J. (1961) Induction of carcinoma
and papilloma of the tracheobronchial mucosa of the Syrian
hamster by intratracheal instillation of benzo(a)pyr'ine.
J. Natl. Cancer Inst. 28:467-491.
Hetnarski, B. and O'Brein, R. D. (1975) The charge-transfer
constant. A new substituent constant, for structure-activity
relationships. J. Med. Chem. 18:29-33. __.,..
llilding, A. C. (1956) On cigarette smoking, bronchial carcinoma
and ciliary action. IV. Ciliary streaming through Inrynx
and distribution of laryngeal epithelium. Ann. Otol.
Laryngol.^65:736-746.
ii. '\ '
-------
Hilcling, A. C. (1957) Ciliary streaming in the bronchial tree
arid the time element carcinogenesis. N. Engl. J. Med. 256:
634-640.
Hinners, R. G. (1962) Engineering the chronic exposure of animals
to laboratory produced automobile exhaust. Air Pollut. Control
Assoc. 12:527-530.
Hirsch, J. A., Swenson, E. W., and Wanner, A. (1975) Tracheal
mucous transport in beagles after long-term exposure to 1 ppm
sulfur dioxide. Arch. Environ. Health 30:249-253.
Hoel, D. G., Gaylor, D. W., Kirschstein, R. L., Saffiotti, U., and
Schneiderman, M. A. (1975) Estimation of risks of
irreversible delayed toxicity. J. Toxicbl. Environ. Health
1:153-151.
Hoel, D. G. and Waiburg, H. E., Jr. (1972) Statistical analysis
of survival experiments. J. Natl. Cancer Inst. 49:361-372.
Hoffmann, D. and Wynder, E. L. (1968) Chemical analysis and
carcinogenic bioassays of organic particulate pollutants.
In: Air pollution, 2nd ed., vol. 2, A. C. Stern, Ed.
Academic, New York, pp 187-247.
Hoffmann, D. and Wynder, E. L. (1976) Environmental respiratory
carcinogenesis. In: Chemical carcinogens, C. E. Searle, Ed.
American Chemical Society, Washington, D. C., pp 324-365.
Hopfinger, A. J. and Battershell, R. D. (1976) Application of
SCAP to drug design. 1. Prediction of octanol-water
partition coefficients using solvent-dependent conformational
analyses.. -J. Med. Chem. 19:569-573. - - ,. . r
Hsie, A. W., Brimer, P. A., Mitchell, T. J., and Gosslee, D. G.
(1975). The dose-response relationship for ethyl methanesul-
fonate-induccd mutations at the hypoxanthine-guanine phos-
phoribosyl transferuse locus in Chinese hamster ovary cells.
Somatic Cell Genetics. 1:247-261.
Hsie, A. IV., Brimer, P. A., Mitchell, T. J., and Gosslee, D. G.
(1975) The dose-response relationship for ultraviolet-
light-induced mutations at the hypoxanthine-guanine phos-
phoribosyltransferase locus in Chinese hamster ovary cells.
Somatic Cell Genetics 1:383-389.
Hsie, A. W., Couch, D. B., Brimer, P. A., O'Neill, J. P., and
Machanoff, R. (1977) The utility of a quantitative sensitive
and versatile mutational assay at the hypoxanthine-guanine
phosphoribosyl transferase locus of Chinese hamster ovary
cells (CHO/HGPRT assay) in determining the mutagenicity of
physical and chemical carcinogens. Abstract to be presented
at the Am. Assoc. Cancer Res. Mtg.
-------
llsie, A. N., Li, A. P., and Machanoff, R. (1977) A fluence-
response study of lethality and mutagenicity of white, black,
and blue fluorescent light, sunlainp, and sunlight irradiation
in Chinese hamster ovary cells. Mutat. Res., in press.
i
Huberman, E. and Sachs, L. (1976) Mutability of different genetic |
loci in mammalian cells by metabolically activated carcinogenic ;
polycyclic hydrocarbons. Proc. Natl. Acad. Sci. USA 73: '
188-192. '
Huisingh, J. L., Canibell, J. A., and Waters, M. D'. .(1976) Evaluation j
of trace element interactions using cultured alveolar macrophages. {
llanford Biology Symposium 16:35. . !
Hum, R. W., Allsup, J. R., and Cox, F. (1974) Effect of j
gasoline additives on gaseous emissions. EPA/650/2-75-014. \
NTIS, Springfield, Virginia, 64 pp. ;
Hum, R. W., Cox, F. W., and Allsup, J. R. (1976) Effect of :
gasoline additives on gaseous emissions (Part II). EPA/ |
600/2-76-026. NTIS, Springfield, Virginia, 65 pp. j
Hurst, D. J., Gardner, D, F.., and Coffin, D. L. (1970) Effect ;
of ozone on acid hydrolases of the pulmonary alveolar
macrophage. J. Reticuloesidoiiiei. Soc. 8:288-300. ' !
Hyde, R. M. (1975) Relationship between the biological and ,
physicochemical properties of series of compounds. J.-Med.
Chem. 18:231-233. .
ft
-------
Ichinotsubo, D., Mower, H. F., Setliff, J., and Mandel, M.
(1977) The use of rec~ bacteria for testing of carcinogenic
substances. Mutat. Re0,. 46:53-62.
Ivanovic, V., Geacintov, N. E., and Weinstein, I. B. (1976}
Cellular binding of benzo(a)pyrene to DNA characterized by
low temperature fluorescence. Biochem. Biophys. Res.- Conunun.
70:1172-1179.
-------
Janoff, A., Rosenberg, R., and Galdston, M. (1971) Elastase-
like, estcroprotease activity in hmnan and rabbit alveolar
macrochage granules. Proc. Soc. Exp. Biol. Med. 136:1054-1058.
Junod, A. F. and R. de Mailer, Eds. (1975) Davos Symposium, 5th,
1974, Proceedings, Lung metabolism: proteolysis and anti-
proteolysis biochemical pharmacology, handling of bioactive
substances. Academic, New York, pp 219-230.
-------
Kada, T., Tutikawa, K., and Sadaie, Y. (1972) In vitro and' host- ]
mediated "rec-assay" procedures for screening chemical mutagens; |
and'phljxine, a mutr.genic red dye detected. Mutat. Res. 16: i
165-174. ]
Kakur.nga, T. (1973) A quantitative system for- assay pf. malignant ]
transformation by chemical carcinogens using a clone derived . i
from BALB/3T3. Int:. J. Cancer 12:463-473. j
Katz, M. (1968) Analysis or inorganic gaseous pollutants. In: j
Air pollution, vol II, chpt. 17, A. C. Stern,'Ed. Academic, j
New York, pp 53-114: . . 1
i
Katz, M. (1976) Nitrogen compounds and oxidant". In: Air " '
pollution, 3rd ed., vol. 3, A. C. Stern, Ed. Academic, New i
York, pp 259-306. f >
Kaufman, D. G. and Madison, R. M. (1974) Synergistic effects of . - !
benzo(a)pyrene and n-methyl-n-nitrosourea on respiratory ' i
carcinogenesis in Syrian golden hamsters. In: Experimental ' j
lung cancer, E. Karbe an i J: F. Parks, Eds, Springer-Verlag, ' i
New York, pp 207-218. ;' ^ j
Kavet, R. I. and Brain, J. D. (1974) Reaction of the lir to !
air pollutant exposure. Life Sci. 15:849-861. ' ;
:; <. .H
Keal, E. IE..- (ly?7) Physiological and pharmacological control ^ j
of airways secretions. In: Respiratory defense mechanisms, ;' !
Part I, J. D. Brain, D/ F. Proctor, and L. M. Reid, F.ds. S 1
flung biology in health and disease, vol. 5, Part I). , Marcel ^. ]
Oekker, Inc., New York, pp 357-404. . ~ j
Ketkar, M. B., Reznik, G., and Mohr, U. (i977) Pathological | j
alterations in-Syrian golden hamster lungs after passive' " " '''?.-_!
exposure to cigarette smoke. Toxicology 7:265-273. : j
Ki-jr, L. B., Murray, IV. J., and Hall, L. H. (1975) Molecular ;
connectivity. 4. Relationships to biological activities.
J. Med. Che:n. 18:1272-1274. ;
Kier, L. D., Yamasaki, E., and AJT.^S, B. N. (1974) Detection of \' ;
mutagenic activity in cigaret!e smoke condensates. Proc.
Natl. Acad. Sci. USA 71:4159-4163. . ':
Kikkawa, Y. (1972) Normal cornea.], staining with f luorescein. :'-
Exp. Eye Res. 14:13-20. .
Kilburn, K. H. (1967) Cilia and mucus transport as determinants
of the response of lung to air pollutants. Arch. Environ.
Health 14:77-91. . '. .'"'
-------
Kilburn, K. H. (1977) Clearance mechanisms an the respiratory
tract. In: Handbook of physiology. Sec. 9, Reactions to
environmental agents, D. II. K. Lee, lid. American Physiological
Society, Bethcsda, Maryland, pp 245-262.
Kilburn, K. H. (1977) The nature and action of respiratory tract
cilia. In; Respiratory defense mechanisms, Part 1., J. D.
Brain, D. F. Proctor, and L. M. Reid, lids. (Lung biology in
health and disease, vol. 5, Part I). Marcel Dekkcr,-Inc.,
New York, pp 247-288.
Kim, M., Goldstein, E., Lewis, J. i'., Lippert, W., and Warshauer,
D. (1976) Marine pulmonary alveolar macrophages: Rates
of bacterial ingestion, inactivation, and destruction. J.
Infect. Dis. 153:310-520.
Klaassen, C. D. (1975) Absoi-ption, distribution, and excretion
of toxicants, ^n: Toxicology, the basic science of poisons,
L. J. Casarett and J. Doull, Eds. Maoaillan, New York, pp 26-44.
Klimisch, H.-J. and Dontenwill, W. (1977) Investigations of
cigarette smoke dosages in inhalation experiments with Syrian
hamsters. I. Concentration cigarette smoke in the inhalation
chamber and of carbon monoxide in the blood. J. Natl. Cancer
Inst. 58:931-933.
Klimisch, H.-J. and Dontenwill, W. (1977) Investigations of
cigarette smoke dosages in inhalation experiments with Syrian
hamsters. II. Uptake of cigarette smoke by the animals;
comparisons of the influence of the gas phase and the whole
smoke of different cigarettes. J. Natl. Cancer Inst. 58:
935-939.
Kohn, K. W., Erickson, L. C., twig, R. A. G., and Friedman, C. A.
(1976) Fracttonation of D.NA from mammalian cells by alkaline
.clution. Biochemistry 15:4629-4637.
Koo, K. K., Lcith, D. E., Shertcr, C. B., and Snider, G. L. (1976)
Respiratory mechanics in normal hamsters. J. Appl. Physio!.
40:956-942.
Krahn, D. F. and lleidelburger, C. (1977) Liver homogcnate-
mcdiatcd mutagcncsis in Chinese hamster V79 cells by polycyclic
aromatic hydrocarbons and aflatoxins. Mutat. Res. 46:27-44.
Krstulovic, A. M., Rosie, D. M., and Brown, P. R. (1977) Distri-
bution of some' atmospheric polynuclear aromatic hydrocarbons.
. Am.'Lab. 9:11-12, .14, 16-18.
Kruyssc, A. and Fcron, V. J. (1976) Repeated exposure to cyclo-
pcntcnonc vapour: Long-term study in Syrian golden hamsters.
-------
Zcntralbl. Bakteriol. 1'urasitenkd. Infcktionskr. Hyg. Abat.
Orig. Reihe. B 163:448-457.
Kubinyi, 11. and Kehrhahn, 0. H. (1976) Quantitative structure-
activity relationships. 1. The modified Free-Wilson
approach. J. Med. Cheia. 19:578-586.
Kubinyi, H. (1976) Quantitative structure-activity relationships.
2. A mixed approach, based on Hansch and Free-Wilson
analysis. J. Med. Chem. 19:587-600.
Kuroki, T., Huberraan, E., Marquardt, H., Selkirk, J. K., Heidelberger,
C., Grover, P. L., and Sims, P. (1971/72) Binding of K-region
epoxidcs and other derivatives of benz(a)anthracene and
dibenz(a,h)anthracene to UNA, RNA, and proteins of transformable
cells. Chcm.-Biol. Interactions 4:389-397.
-------
Laishes, B. A. and Stich, H. F. (1973) Repair synthesis and
sedimentation analysis of DNA of human cells exposed to
dimethylnitrosaraine and activated diraethylnitrosamine.
Biochem. Biophys. Res. Commun. 52:827-833.
Latt, S. A. [1976) Analyses of human chromosome structure,
replication and repair using Brd U-33258 Hoechst techniques.
J. Reprod. Med. 17:41-52.
Lauwerys, R. (1975) Biological criteria for selected industrial
toxic chemicals: A review. Scand. J. Work. Environ. Health
1:139-172.
Lee, P. N. and O'Neill, J. A. (1971) The effect both of time and
dose applied on tumor incidence rate in benzopyrene skin
painting experiments. Br. J. Cancer 25:759-770.
Lee, R. E., Jr., Patterson, R. K., Crider, W. L., and Wagman, J.
(1971) Concentration and particle size distribution of
particulate emissions in .automobile exhaust. Atmcs. Environ.
5:225-237.
Lcgator, M. S. and Mailing, H. V. (1971) The host-mediated assay,
a practical procedure for evaluating potential mutagenic
agents in mammals. Chem. Mutagens 2:569-589.
Leo, A., Hansch, C., and Elkins, D. (1971) Partition .coefficients
and their uses. Chem. Rev. 71:525-616.
Leonard, A. (1976) Heritable chromosome aberration in mammals
after exposure to chemicals. Radiat. Environ. Biophys. 13:
1-8.
Leonard, A. (1973) Observations on meiotic chromosomes of the
male mouse as a test of the potential mutagenicity of chemicals
in mammals. Chem. Mutagens 3:21-56.
Leonard, A. (1975) Tests for heritable translocations in male
mammals. Mutat. Res. 31:291-297.
Levin, H. S., Rodnitzky, R. L., and Mick, D. L. (1976) Anxiety
associated with exposure to organophosphate compounds. Arch.
Gen. Psychiatry 33:225-228.
Levy, S. E. and Harvey, E. (1974) The effect of tissue slicing
on rat lung metabolism. J. Appl. Physiol. 37:239-240.
Lieberman, M. W. (1975) Discussion paper: Quantitative aspects
of using DNA repair to detect mutagens and carcinogens. Ann.
N. Y. Acad. Sci. 269:37-42.
-------
1
i
i
Linton, R. W., Loh, A., Natusch, D. F. S., Evans, C. A., Jr., and '
Williams, P. [1975) Surface predominance of trace elements j
in airborne particles. Science 191:852-854. |
i
Lippmann, M. (1977) Regional deposition of particles in the human !
respiratory tract. In: Handbook of physiology, Sec. 9, D. H. J
K. Lee, Ed. American Physiological Society, Bethcsda,
Maryland, pp 213-232.
Lippmann, M. (1970) "Respirable" dust sampling. Am. Ind. Hyg.
Assoc. J. 31:138-149.
Lippmann, M., Albert, R. E., Yeates, D. B., Berger, J. M., Foster,
W. M., and Bohning, D. E. (1977) Factors affecting tracheo-
bronchial mucociliary transport. Ij^: Inhaled particles IV,
Part I, V,'. II. Walton, Ed. Pergamon, New York, pp 305-319.
Litchfield, J. T., Jr. and Wilcoxon, F. (.1949) A simplified
method of evaluating dose-effect experiments. J. Pharmacol.
Exp. Ther. 96:99-113.
Litt, M., Khan, M. A., Chakrin, L. W., Sosnewski, G., and Wardell,
J. R., Jr. (1976) Effect of chronic sulfur dioxide inhal-
ation on Theological properties of tracheal mucus. Biorhe-
ology 13:107-114. .
Litt, M., Khan, M. A., and Wolf, D. P. (1976) Mucus rheology:
Relation to structure and function. Biorheology 13:37-48.
Lock, E. A., Smith, L. L., and Rose, M. S. (1976) Inhibition of
paraquat accumulation in rat lung slices by a component of
rat plasma and a variety of drugs and endogenous amines.
Biochem. Pharmacol. .25:1769-1772.
Loomir,, T. A. (1974) Essentials of toxicology, 2nd ed. Lea (,
Febiger, Philadelphia, 223 pp.
Lopez-Vidriero, M. T., Das, I., and Reid, L. M. (1977) Airway
secretion: source, biochemical and rheological properties.
In: Respiratory defense mechanisms, Part I, J. D. !5rain,
D. F. Proctor, and L. M. Reid, Eds. (Lung biology in health
and disease, vol. 5, Part I). Marcel Dekker, Inc., New York,
pp 289-356.
-------
MacEwen, J. D. and Vernot, E. H. (1974) Toxic Hazards Research
Unit, Annual Technical Report: 1974 (AD-A011 559). Nl'IS,
Springfield, Virginia, 188 pp.
MacFarland, H. (1975) Inhalation toxicology. J. Assoc. Off.
Agric. Chem. 58:689-693.
Maibach, H. (1976) Cutaneous pharmacology and toxicology. Annu.
Rev. Pharmacol. Toxicol. 16:401-411.
Malaveille, C., Planche, G., and Bartsch, II. (1977) Factors for -.
efficiency of the Salmonella/microsome mutage.iicity assay.
Chem.-Biol. Interact. 17:129-156.
Marshall, R. (1976) Pharmacological and pahtological responses
of the human pulmonary circulation to drugs'. Pharmacol.
Ther. B. 2:289-312.
Martin, R. G. and Anderson, J. L. (1976) Death and transformation.
In: Biology of radiation carcinogenesis, J. M. Yahas, R. K.
Tennant, and J. D. Regan, Eds. Raven Press, New York,
pp 289-300.
Martin, R. R. and Laughter, A. H. (1976) Pulmonary alveolar
macrophages can mediate immune responses: Cigarette smoking
impairs these function. Fed. Proc. 35:716 (2811).
Matula, R. A. (1974) Consultant report on emissions and fuel-
economy test methods and procedures (PB 242 093).
. Springfield, Virginia, 163 pp.
McCann, J. and Ames, B. N. (1976) Detection of carcinogens as
mutagens in the Salmonella/microsome test: Assay of 300
chemicals: Discussion. Proc. Natl. Acad. Sci. USA 73:950-954;
McCann, J., Choi, K., Yamasaki, E., and Ames, B. N. (1975)
Detection of carcinogens as mutagens in the Salmonella/microsome
test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. USA
72:5135-5159.
McCrone, W. C. (1976) Microscopy and pollution analysis. In:
Air pollution, 3rd ed., vol. 3., A. C. Stern, Ed. Academic,
New York, pp 99-144.
McDonald, T. 0., Baldwin, H. A., and Beasley, C. H. (1973) Slit-
lamp examination of experimental animal eyes. I. Techniques
of illumination and the normal animal eye. J. Soc. Cosmet.
Chem. 24:163-180.
McKenzie, W. H., Knelson, J. H., Rummo, N. J., and House, D. E.
(1977) Cytogenetic effects of inhaled ozone in man. Mutat.
Res. 48:95-102.
-------
McNulty, P. J., Nauman, C. H., Sparrow, A. H., Schwemraer, S. S.,
and Schairer, L. A. (1977) Influence of x-ray dose
fractionation on the frequency of somatic mutations induced
in Tradesc.-mtia stamen hairs. Mutat. Res. 44:235--?46.
Mena, I. (1974) The role of manganese in human disease. Ann.
Clin. Lab. Sci. 4:487-491.
Menon, G. K. and Cammarata, A. (1977) Pattern recognition.
II: Investigation of structure-activity relationships. J.
Pharm. Sci. 66:304-314. .
Mercer, T. T. (1967) On the role of particle size in the dissolu-
tion of lung burdens. Health Phys. 13:1211-1221.
Mercer, T. T. (1973) Aerosol technology in hazard evaluation.
Academic, New York, 394 pp.
Merz, T., Bender, M. A., Kerr, H. D., and Kulle, T. J. (1975)
Observations of aberrations in chromosomes of lymphpcytes
from human subjects exposed to ozone at a concentration of
0.5 ppm for 6 and 10 hours. Mutat. Res. 31:299-302.
Meyer, F. A. (1976) Mucus structure: Relation to biological
transport function. Biorheology 13:49-58.
Miller, E. C. and Miller, J. A. (1976) The metabolism of chemical
carcinogens .to reactive electrophiles and their possible
mechanisms of action in carcinogencsis. In: Chemical
carcinogens, C. E. Searle, Ed. American Chemical Society,
Washington, D. C., pp 737-762.
Mitchell, I. de G. (1974) A comparison of the sensitivity and
specificity of microbial systems for assessing genetic damage.
Agents Actions 41:286-294.
Mohn, G. R. and Ellenberger, J. (1977) The use of Escherichia
coli K12/343/113 (A).as a multipurpose indicator strain in
various mutagenicity testing procedures. In: Handbook of
inutagenicity test procedures, B. J. Kilbey, M. Legator, W.
Nichols, and C. Ramel, Eds. Elsevier/North Holland Biomedical
Press, Amsterdam, 475 pp.
Mohn, G., Ellenberger, J.. and McGregor, D. (1974) Development
of mutagenicity tests using Escherichia coli K-12 as indicator
organism. Mutat. Res. 25:187-196.
Mohn, G., Ellenberger, J., McGregor, D., and Merkcr, H.-J. (1975)
Mutagenicity studies in microorgansims in vitro, with extracts
of mammalian organs, and with the host-mediated-assay. Mutat.
Res. 29:221-233.
-------
f*~ >£=*f -w«yKjftfi-y*;
Mohr, U., Reznik-Schuller, H., Reznik, G., Grimmer, G., and Misfeld,
J. (1976) Investigations,on the carcinogenic burden by
air pollution in man. XIV. Effects c, : automobile exhaust
condensate on the Syrian golden hamster lung. Zentrabl.
Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. Orig. Keihc. B
163:425-432.
Mondal, S., Brankow, D. K., and Heidelberger, C. (1976) Two-
stage chemical oncogenesis in cultures of C3H/10T1/2 cells.
Cancer Res. 56:2254-2260.
Montcsano, R., H. Bartsch, and L. Tomatis, lids. (1976) Screening
tests in chemical carcinogcnesis. International Agency for
Research on Cancer, Lyon, France, 661 pp.
Montesano, R., Saffiotti, U., ''errero, A., and Kaufman, D. G.
(1D74) Synergistic effects of benzo(a)pyrene and diethyl-
nitrosaminc on respiratory carc'nogenesis in hamsters. J.
Natl. Cancer Inst. 55:1395-1397.
Morrow, P. E., Mehrhof, E., Casarett, L. J., and Morken, D. A.
(1958) An experimental study of aerosol deposition in human
subjects. AMA Arch. Ind. Health 18:292-298.
Mortimer, R. K. and Manhey, T. R. (1971) Mutation induction in
yeast. Chem. Mutugens 1:289-310.
Murray, K. J., Kier,. L. B., and Hall, L. H. (1376) Molecular
connectivity. 6. Examination of the parabolic relationship
between molecular connectivity and biological activity. J.
Med. Chem. 19:573-578.
Mustafa, M. G., Hacker, A. D., Ospital, J. J., Hussai.n, M. 2., and
Lee, S. D. (1977) Biochemical effects' of environmental
oxidant pollutants in animal lungs. In; Biochemical effects
or environmental pollutants, S. D. Lee, Ed. Ann Arbor Science,
Ann Arbor, Michigan, pp 59-96.
Myhr, B. C. (1973) A screen for pesticide toxicity to protein
and RNA synthesis in HeLa cells. J. Agr. Food Chem. 21:
362-367.
-------
Nagao, M. and Sugiraura, T. (1972) Sensitivity of repair-deficient
mutants and similar mutants to 4-nitroquinoline 1-oxide,
4-nitropyriiiine 1-oxide, and their derivatives. Cancer Res.
52:2369-2574.
Nakaue, II. S. and Buhler, D. R. (1976) Percutaneous absorption
of hexachlorophene in the rat. Toxicol. Appl. Pharmacol
55:581-591.
National Cancer Advisory Board Subcommittee on Environmental
Carcinogenesis (1977) General criteria for assessing the
evidence for carcinogenicity of chemical substances: report
of the subcommittee on environmental carcinogencsis, National
Cancer Advisory Board. J. Natl. Cancer Inst. 58:461-465. :
National Environmental Research Center, Cincinnati, 'Ohio, 1972,
Annual Report. (1975) (:;PA-670/9-75-OS5). NT1S, Springfield, ':
Virginia, 94 pp.
National Research Council. Committee on Biologic Effects of -f
Atmospheric Pollutants. (1975) Manganese, Appendix: i
Environmental sampling and anlaysis of manganese. National i
Academy of Sciences, Washington, L). C., pp 141-148. ;
National Research Council. Committee on Biologic Effects of
Atmospheric Pollutants (1972) Particulate polycyclic
organic matter, Appendix B: Separation metiiOuS for poly- j
cyclic organic matter; Appendix C: Detection, identification, <
and quantitation. National Academy of Sciences, Washington, |
D. C., pp 261-276; 277-305. ]
National Research Council. Committee on Toxicology (1964) J
Principles and procedures for evaluating the toxicity of |
household substances, Arnold J. Lehman, Chairman, et al. \
(National Research Council Publ. no. 1138). National Academy
of Sciences-National Research Council, Washington, U. C., 29 pp.
National Research Council. Panel on Vapor-Phase Organic Pollutants. '
(1976) Vapor-phase organic pollutants: volatile hydrocarbons :;
and oxidation products. Chapter 6: Biologic effects of ;
vapor-phase organic pollutants in man and other mammals. ]
Naticnal ;\cademy of Sciences, Washington, U. C., pp 181-211.
National Research Council. Panel on Vapor-Phase Organic Pollutants.
(1976) Vapor-phase organic pollutants: volatile hydrocarbons
and oxidation products. Appendix A: Collection and sampling
techniques for vapor-phase organic air pollutants; Appendix
B: Analytic techniques for vapor-phase organic air pollutants.
National Academy of Sciences, Washington, D. C., pp 287-504;
505-517.
-------
Nauman, C. H., Sparrow, A. H., and Schairer, L. A. (1976) Com-
parative effects of ionizing radiation and two gaseous
chemical mutagens on somatic mutation induction in one
mutable and two non-mutable clones of Tradescantia. Mutat.
Res. 38:53-70.
N'eely, W. B., Branson, D. R., and Blau, G. E. (1974) Partition
coefficient to measure bioconcentration potential of organic
chemicals in fish. Enviorn. Sci. Technol. 8:1113-1115.
Nettesheim, P. (1974) Review and introductory remarks: Multi-
factorial respiratory carcinogenesis. In: Experimental lung
cancer, E. Karbe and J. F. Park, Eds. Springer-Verlag, New
York, pp 157-160.
Nettesheim, P., Creasia, D. A., and Mitchell, T. J. (1975) Car-
cinogenic and cocarcinogenic effects of inhaled synthetic
smog and ferric oxide particles. J. Natl. Cancer Inst. 55:
159-169.
Newbold, R. F., Wigley, C. B., Thompson, M. H., and Brookes, P.
(1977) Cell-mediated mutagenesis in cultured Chinese hamster
cells by carcinogenic hydrocarbons: Nature and extent of the
associated-DNA reaction. Mutat. Res. 43:101-116.
Nichols, W. W. (1973) Cytogenetic techniques in mutagenicity
testing. Agents Action 3:86-92. .
Niemeier, R. W. (1976) Isolated perfused rabbit lung: A critical
appraisal. Environ. Health Perspect. 16:67-71.
Nishioka, H. (1975) Mutagenic activities of metal compounds in
bacteria. Mutat. Res. 31:185-189.
Nixon, G. A., Tyson, C. A., and Wertz, W. C. (1975) Interspecies
comparisons of skin irritancy. Toxicol. Appl. Pharmacol.
31:481-490.
Norton, S. (1975) Toxicology of the central nervous system.
In: Toxicology, the basic science of poisons, L. J. Casarett
aiTd J. Doull, Eds. MacMillan, New York, pp 151-169.
Nulsen, A., Holt, P. G., and Keast, D. (1974) Cigarette smoking,
air pollution, arid immunity: A model system. Infect. Imhiun.
10:1226-1229.
-------
_ ~a
O'Neill, J. J. and Tierney, D. F. (]974) Rat lung metabolism:
glucose utilization by isolated perfused lungs and tissue
slices. Am. J. Physiol. 26:867-873.
O'Neill, J. P., Brimmer; P. A., Machanoff, R., Hirsch, G. P., and
Hsie, A. W. (1977) A quantitative assay of mutation induction
at the hypoxanthine-guanine phosphoribosyl transfcrase locus
in Chinese hamster ovary cells (CHO/11GPRT system): Development
and definition of the system. Submitted to Mutat. Res.
O'Neill, J. P., Couch, D. B., Machanoff, R., San Sebastian, J. R.,
Brimer, P. A., and Hsie, A. W. (1977) A quantitative assay
of mutation induction at the hypcxanthine-guanine phosphoribosyl
transferase locus in Chinese hamster ovary cells (CHO/HGPRT
system): Utilization with a variety of mutagenic agents.
Submitted to Mutat. Res.
O'Neill, J. P. and Hsie, A. W. (1977) Chemical mutagenesis of
mammalian cells can be quantitated. Nature, in press.
Ong, T.-M. (1977) Use of the spot, plate and suspension test
systems for the detection of the mutagenicity of environmental
agents and chemical carcinogens in Neurospora crassa. To
be published.
Ong, T.-M. and de Serres, F. J. (1972) Mutagenicity of chemical
carcinogens in Ncurospora crassa. Cancer Res. 32:1890-1893.
Ong, T.-M. and Mailing, H. V. (1975). Microsomal activation of
dimethylnitrosamine to metabolites mutagenic in Neurospora
crassa. Mutat. Res. 31:195-196.
Osterberg, R. E., Murphy, J. C., Bierbower, G. W., and Sauro, F. M.
(1975) An evaluation of the mutagenic potential of an aerosol
spray adhesive in the rat. Mutat."Res. 31:169-173." -r
A
-------
Panalaks, T. (1976) Determination and identification of poly-
cyclic aromatic hydrocarbons in smoked and charcoal-broiled
food products by high pressure liquid chromatography and gas
chroraatography. J. Environ. Sci. Health 11:299-315.
Parkes, II. (1976) The epidemiology of the aromatic amine cancers.
In: Chemical carcinogens, C. E. Searle, Ed. American Chemical
Society, Washington, D. C., pp 462-480.
Pavia, D., Thomson, M. , and Shannon, H. S. (1977) Aerosol inhala-
tion and depth of deposition in the human lung. Arch.
Environ. Health 32:131-137.
Perry, P. and Evans, 11. J. (1975) Cytological detection of
mutagen-carcinogen exposure by sister chromatid exchange.
Nature (London) 258:121-125. .
Perry, P. and Wolff, S. (1974) New Giemsa method for the differ-
ential staining of sis-ter chromatids. Nature (London) 251:
156-158.
Peto, R. and Lee, P. (1973) Weibull distributions for continuous-
carcinogenesis axperiments. Biometrics 29:457-470.
Peto, R., Lee, P. N., and Paige, W. S. (1972) Statistical analysis
of the bioassay of continuous carcinogens. Br. J. Cancer. 26:
258-261. .
Phalen, R. F. (1976) Inhalation exposure of animals. Environ.
Health Perspect. 16:17-24. .
Phillips, L., 11, Steinberg, M., Maibach, H. I., and Akers, W. A.
(1972) A comparison of rabbit and human skin response to
certain irritants. Toxicol. Appl. Pharmacol. 21:369-382. .
Philpot, R. M., Anderson, M. W. , and Eling, T. E. (1977) Uptake,
accumulation, and metabolism of chemicals by the lung. In:
Metabolic functions of the lung, Y. S. Bakhle and J. R. Vane,
Eds. (Lung biology in health and disease, 4.) Marcel Dckkcr,
Inc., New York, pp 123-171.
Picnta, R. J., Poilcy, J. A., and Lebherz, IV. B., III. (1977)
Morphological transformation of early passage golden Syrian
hamster embryo cells derived from cryopreservcd prin.ary
cultures as a reliable in_ vitro bioassay for identifying diverse
carcinogens. Int. J. Cancer 19:642-655.
Pilotti, A., Ancker, K., Arrhcnius, E., and Enzell, C. (1975)
Effects of tobacco and tobacco smoke constituents on cell
multiplication in vitro. Toxicology 5:49-62.
-------
Popper, H., Czygan, P., Greim, 11., Schaffner, F., and Garro, A. J.
(1973) Mutagenici'ty of primary and secondary carcinogens
altered by normal and induced hepatic microsomes. Proc. Soc.
Exp. Biol. Med. 142:727-729.
Potts, A. M. and Gonasun, L. M. (1975) Toxicology of the eye.
In: Toxicology, the basic science of poisons, L. J. Casarett
and J. Doull, Eds. Macraillan, New York, pp 275-509.
Proctor, D. F., Andersen, I., and Lundqvi.st, G. (1977) Kasal
mucociliary function in humans. In: Respiratory defense
mechanisms, Part I, J. D. Brain, I). F. Proctor, and L. M.
Reid, Eds. (I.ung biology in health and disease, vol. 5,
Part I.) Marcel Dekker, Inc., New York, pp 427-452.
h
-------
^ «T*aC^*SS3'S?!r\WV"i?*^3V^^
Quinn, F. R., Driscoll, J. S., and Hansch, C. (1975) Structure-
activity correlations among rifamycin B amides and hydrazides.
J. Med. Chem. 18:332-359.
s 1
ij
-------
Rappaport, S. M., Morales, R., Weeks, R. N., Jr., Campbell, E. E.,
and Ettinger, H. J. (1975) Development of sampling and
analytical methods for carcinogens. Los Alamos Scientific
Laboratory of the University of California, Los Alanos, New
Mex-co. Progress Report LA-63S7-PR. MIS, Springfield,
Virginia, 19 pp.
Regan, J. D. arid Setlow, R. B. (1973) Repair of chemical damage
to human DNA. Chem. Mutagens 3:151-170.
Reid, L. (1970) Evaluation of model systems for study of airway
epithelium, cilia, and mucus. Arch. Intern. MeU. 126:428-434.
Reid, W. D., Ilett, K. F., Click, J. M., and Krishna, G. (1973)
Metabolism and binding of aromatic hydrocarbons in the lung.
Relationship to experimental bronchiolar necrosis. Am. Rev.
Respir. Dis. 107:559-551
Reznikoff. C. A., *Brankow, D. W., and Heidelberger, C. (1973)
Establishment and characteriiation of a cloned line of C3H
mouse embryo cel.ls sensitive to postconfluence inhibition of
division. Cancer Res. 33:3231-3238.
Rhim, J. S. and Huebner, R. J. (1973) Transformation of rat
embryo cells iji vitro by chemical carcinogens. Cancer Res.
33:(>9S-700.
Rhoadcs, R. A. (1976) Perfused lung preparation for studying
altered gaseous environments. Environ. Health Perspect. 16:
73-75.
Rivera, J. A. (1962) Cilia, ciliated epithelium and ciliary
activity. Pergainon, Oxford, 167 pp.' '
Robens, J. Tcratogenic techniques and agents and mutagcnicity.
In; handbook of laboratory animal science, E. C. Melby, Jr.
and N. T. Altman, Eds. CRC, Cleveland, Ohio, pp 177-190.
Robinson, N. E., Gillespie, J. R., Berry, J. D., andiSimpson, A.
(1972) Lung compliance, lung volume, and single-breath
diffusing capacity in dogs. J. Appl. Physiol. 35:808-812.
Rogers, A., Mcllors, A., and Safe, S. (1976) Lysosoinal membrane
labili:ation by DDT, DDE and polychlorinatcd biphcnyls (PCB).
Res. Cdmmun. Chera. Pathol. Pharmacol. 13:541-344.
Rose, A. H., Jr. and Brandt, C. S. (1960) Environmental irradia-
tion test facility. J. Air Pollut. Control Assoc. 10:
551-335.
Rose, M. S., Smith, L. L., and Kyatt, I. (1976) The relevance of
pcntosc phosphate pathway stimulation in rat lung to the
mechanism of paraquat toxicity. Biochcm. Pharmacol. 25:
1763-1767. .
-------
Rose, N. R. and H. Friedman, Eds. (1976) Manual of clinical
immunology. /Vnerican Society for Microbiology, Washington,
D. C., 93 2 pp.
Rosenkranz, 11. S., Gutter, B., and Speck, IV. T. (1976) Mutagen-
icity and DNA-modifying activity: A comparison of two
microbial assays. Mutat. Res. 41:61-70.
Roudabush, R. L., Terharr, C. J., Fassett, D. W., and Dziuba, S. P.
(1965) Comparative acute effects of some chemicals on the :
skin of rabbits and guinea pigs. Toxicol. Appl. Pharmacol.
7:559-565.
Rubin, H. and Ames, B. N. (1976) Carcinogenicity tests. Science
191:241-245. ::
;'
Rushton, P. S. (1969) The effects of 5-fluorodeoxyuridine on .
radiation-induced chromatid aberrations in Tradescantia
micropores. Radiat. Res. 38:404-413. j
Russell, W. L. (1951) X-ray induced mutations in mice. Cold *
Spring Harbor Symp. Quant. Biol. 16:327-336. (
Rylander, R. (1S69) Alterations of lung defense mechanisms
against airborne bacteria. Arch. Environ. Health .18:551-555.
,
Rylander, R. (1968) Pulmonary defence mechanisms to airborne 3
bacteria. Acta Physiol. Scand. Suppl. 306:1-89. . f
Rylander, R. (1970) Studies of lung defense to infections in \
inhalation toxicology. Arch. Intern. Med. 126:496-499. ' I-
Rynbrandt, D^'and Kleinerman, J. (1977) Nitrogen dioxide and " ?
pulmonary proteolytic enzymes. Arch. Environ. Health 32:
165-172.
Ryttman, H. and Zetterberg, G. (1976) Induction of mitotic
recombination with N-methyl-N'-nitro-N-nitrosoguanidine \
(MNNG) in Saccharomyces cerevisiae. A comparison betwe-en
treatment rn vitro and in the host-mediated assay. Mutat.
Res. 34:2017215.
-------
Saffiotti, U. (1967) Experimental respiratory tract carcinogenesis. =
Prog. Exp. Tumor Re's. 11:302-333.
Saffiotti, U., Cefis, F., and Kolb, L. H. (1968) A method for ;
the experimental induction of bronchogenic carcinoma. Cancer '
Res. 28:104-124. 3
Saffiotti, U., Montesano, R., Sellakumar, A. R., Cefis, F., and i
Kaufman, D. G. (1972) Respiratory tract carcinogenesis in :
hamsters induced by different numbers of administrations of ":
benzo(a)pyrene and ferric oxide. Cancer Res. 32:1073-1081. "'-
i
Saffiotti, U., Montesano, R., Sellakumar, A. R., and Kaufman, D. G. *
(1972) Respiratroy tract carcinogenesis induced in hamsters
by different dose levels of benzo(a)pyrene and ferric oxide. '1
J. Natl. Cancer Inst. 49:1199-1204. :!
San, R. H. C. and Stich, H. F. (1975) DNA repair synthesis of I
cultured human cells as a rapid bioassay for chemical f
carcinogens. Int. J. Cancer 16:284-291. <|
. 1
Santora, N. J. and Anyang, K. (1975) Non-computer approach to .1
structure-activity study. An expanded fibonacci search applied ]
to structurally diverse types of compounds. J. Med. Chem. \
18:959-963. ; J
$
Sayeed, M. M., Senior, R. M., Chaudry, I. H., and Baue, A. E. H
(1975) Characteristics of sodium and potassium transport in g
the lung. Am. J. Physiol. 229:1073-1079. ^
Scheuplein, R. J. (1977) Permeability of the skin. In: Handbook '\
of physiology, Sec. 9, Reactions to environmental agents, ^
"D. H. K. Lee, Ed. American Physiological Society, Bethesda, " 'I
Maryland, pp 299-322. . .'
Schmahl, D..and Schmidt, K. G. (1974) Conception and methods of .
experimental studies in Germany to estimate the carcinogenic
burden by air pollution in man. In;. Experimental lung cancer,
E. Karbe and J. F. Park, Eds. Sp-ringer-Verlag, New York,
pp 139-145.
Schmid, W. (1975) The micronucleus test. Mutat. Res. 31:9-15.
Schneider, L. K., and Calkins, C. A. (1970) Sulfur dioxide-induced
lymphocyte defects in human peripheral blood cultures.
Environ. Res. 3:473-483.
Schoor, W. P. (1973) In vivo binding of p,p'-DDE to human serum
proteins. Bull. Environ. Contain. Toxicol. 9:70-74.
Schuster, C. R., Balster, R., Lipton, M., and Weiss, B. (1977)
Behavioral toxicology. In: Human health and the environment--
-------
some research needs. Report of the Second Task Force for
Research Planning in Environmental Health Science. DHEW
Publication no. NIH 77-1277.
Searle, A. G. (1975) The specific locus test in the mouse.
Mutat. Res. 31:277-290.
Searle, C. E. (1976) Chemical carcinogens. (ACS Monograph 173).
American Chemical Society, Washington, D. C., 788 pp.
Sega, G. A., Owens, J. G., and Gumming, R. B. (1976) Studies on
DNA repair in early spermatid stages of male mice after i.n_
vivo treatment with methyl-, ethyl-, propyl-, and isopropyl
methanesulfonate, Mutat. Res. 36:193-212.
Sellakumar, A. R., Montesar.o, R., Saffiotti, U., and Kaufman, D. G.
(1973) Hamster respiratory carcinogenesis induced by
benzo(a)pyrene and different dose levels of ferric oxide. J.
Natl. Cancer Inst. 50:507-510.
Sellakumar, A., Stenback, F., and Rowland, J. (1975) Effects of
different dusts on respiratory carcinogenesis in hamsters
induced by benzo(a)pyrene and diethylnitrosamine. Eur. J.
Cancer 12:312-319.
Shabad, L. M., Pylev, L. N., and Kolesnichenko, T. S. -(1963)
Importance of -the deposition of carcinogens for cancer indue-
tion in lung tissue. J. Natl. Cancer Inst. 33:135-141.
Shabad, L. M. (1975) On the so-called MAC (maximal allowable
concentrations) for carcinogenic hydrocarbons. Neoplasma
22:459-468.
* .. - , - . - . .
Shih, C. K., Litt, M., Khan, M. A., and Wolf, D. P. (1977) E'ffect
of nondialyzable solids concentration and viscoelasticity
on ciliary transport of tracheal mucus. Am. Rev. Respir.
Dis. 115:989-995.
Shirasu, Y., Moriya, M., Kato, K., Furuhashi, A., and Kada, T.
(1976) Mutagenicity screening of pesticides in the microbial
system. Mutat. Res. 40:19-30.
Siebert, D. (1973) A new method for testing genetically active
metabolites. Urinary assay with cyclophosphamide (Endoxan,
Cytoxan) and Saccharomyces cerevisiae. Mutat. Res. 17:307-314.
Simmon, V. F., Mitchell, A. D., and Jorgenson, T. A. (1977)
Evaluation of selected pesticides as chemical mutagens: in
vitro and rn vivo studies. Annu. Report in Environmental
Health Effects Research Series. EPA-600/1-77-028.
-------
Slater, E. E., Anderson, M. D., and Rosenkranz, H. S. (1971)
Rapid detection of mutagens and carcinogens. Cancer Res.
31:970-973.
Sleigh, M. A. (1977) The nature and action of respiratory tract
cilia. In: Respiratory defense mechanisms, Part I, J. D.
Brain, D. F. Proctor, and L. M. Reid, Eds. (Lung biology in
health and disease, vol. 5, Part I.) Marcel Dekker, Inc.,
New York, pp 247-288.
Smith, H. H. and Lotfy, T. A. (1954) Comparative effects of
certain chemicals on Tradescantia chromosomes as observed at
pollen tube mitosis. Am. J. Botany 41:589-593.
Sobels, F. H. (1977) Somu problems associated with the testing
for environmental mutagens and a perspective for studies in
"comparative mutagenesis". Mutat. Res. 46:245-260.
Sobels, F. H. and Vogel, E. (1976) The capacity of Dr.osophila
for detecting relevant genetic damage. Mutat. Res. 41:
95-106.
Solomon, E. and Bobrow, M. (1975) Sister chromatid exchanges - A
sensitive assay of agents damaging human chromosomes.' Mutat.
Res. 30:273-277.
Sontag, J. M., Page, N. P., and Saffiotti, U. (1976) Guidelines
for carcinogen bioassay in small rodents. National Cancer "' <
Institute Carcinogenesis Technical Report Series No. 1 (DHEW j
Pub. No. (NIH) 76-801). U. S. Govt. Print. Off., Washington, |
D. C. 65 pp. . j
i
Sorokin, S. P. and Brain, J.'D. '(1975) Pathways of'clearance in ' |
mouse lungs exposed to iron oxide aerosols. Anat. Rec. 181: |
581-625. ]
\
Sparrow, A. H., Schairer, L. A., and Villalobos-Pietrini, R. I
(1974) Comparison of somatic mutation rates induced in i
Tradescantia by chemical and physical mutagens. Mutat. Res. !
26:265-276.
Stenback, F., Sellakumar, A., and Shubik, P. (1975) Magnesium
oxide as a carrier dust in benzo(a)pyrene-induced lung
carcinogenesis in Syrian hamsters. J. Natl. Cancer Inst.
54:861-865.
Stetka, D. G. and Wolff, S. (1976) Sister chromatid exchange as
an assay for genetic damage induced by mutagen-carcinogens.
I. In vivo test for compounds requiring metabolic activation.
Mutat. Res. 41:333-342.
-------
Steuer, A. F., Rhim, J. S., Hentosh, P. M., and Ting, R. C. (1977)
Survival of human cells in the aggregate form: Potential
index of in vitro cell transformation. J. Natl. Cancer Inst.
58:917-921.
Stevens, M. T. (1977) An laternative method for the evaluation
of organ weight experiments. Toxicology 7:275-281.
Stich, H. F., Hammerberg, 0., and Casto, B. (1972) The combined
effect of chemical mutagen and virus on DNA repair, chromosome
aberrations and r.eoplastic transformation. Can. J. Genet.
Cytol. 14:911-917.
Stich, H. F., Kieser, D., Laishes, B. A., San, R. H. C., and
Warren, P. (1975) DNA repair of human cells aj a relevant,
rapid, economic assay for environmental carcinogens. Gann
Monogr. Cancer Res. 17:3-15.
Stich, H. F., Lam, P., Lo, L. W., Koropatnick, D. J., and San,
R. H. C. (1975) The search for relevant short term bioassays
for chemical carcinogens: The tribulation of a modern Sisyphus.
Can. J. Genet. Cytol. 17:471-492.
Stich, H. F., San, R. H. C., and Kawazoe, Y. (1971) DNA repair
synthesis in mammalian cells exposed to a series of oncogenic
and non-oncogenic derivatives of 4-nitroquinoiiue 1-uxide.
Nature 229:416-419.
Street, J. C. and Sharma, R. P. (1975) Alteration of induced
cellular and humoral immune responses by pesticides and
chemicals of environmental concern: Quantitative studies of
immunosuppression by DDT, Aroclor 1254, Carbaryl, Carbofuran,
and Methylparathion, Toxicol. Appl. Pharmacol 32:587-602.
Strother, A., Soong, S. L., Dev, V., and Sadri, M. (1977)
Structure, activity relationship of lidocaine type local
anesthetics. Life Sci. 21:71-81.
Stuart, B. 0. (1976) Deposition and clearance of inhaled particles.
Environ. Health Perspect. 16:41-53.
Stupfel, M., Mangier, M., Romary, F., Tran, M.-H., and Moutet,
J.-P. (1975) Lifelong exposure of SPF rats to automotive
exhaust gas. Arch. Environ. Health 26:264-269.
Sugimura, T., Yr.hagi, T., Nagao, M., Takeuchi, M., and Kawachi, T.
(1976) Validity of mutagenicity tests using microbes as a
rapid screening method for environmental carcinogens. In:
Screening tests in chemical carcinogenesis, R. Montesano,
H. Bartsch, and L. Tomatis, Eds. IARC Scientific publications
no. 12, Lyon, International Agency for Research on Cancer,
pp 81-101.
-------
Swenberg, J. A., Petzold, G. L., and Harbach, P. R. (1976) In
vitro DNA damage/alkaline elution assay for predicting
carcinogenic potential. Biochem. Biophys. Res. Conunun. 72:
732-738.
-------
Task Force for Research Planning in Environmental Health Science,
Second. (1977) Human health and the environmentSome
research needs. Chapter 13: Behavioral toxicology. (DREW
Publ. No. (NIH) 77-1277). U. S. Govt. Print. Cff., Washington,
D. C., pp 329-350.
Taylor, J. H., (1958) Sister chromatid exchanges in tritium-
labeled chromosomes. Genetics 43:515-529.
Thayer, P. S., "Himmelfarb, P., and Watts, G. L. (1971) Cytotoxicity
assays with L1210 cells iii vitro: comparison with L1210 i_n_
vivo and KB cells iji vitro. Cancer Chemotherapy Reports
(Part 2) 2:1-25.
Thomas, W. R., Holt, P. G., and Keast, D. (1973) Cellular immunity
in mice chronically exposed to fresh cigarette smoke. Arch.
Environ. Health 27:372-375.
Thompson, W. R. (1947) Use of moving averages and interpolation
to estimate median-effective dose. I. Fundamental formulas,
estimation of error, and relation to other methods. Bacteriol.
Rev. 11:115-145.
Tierney, D. F. (1974) Intermediary metabolism of the lung. Fed.
Proc. 33:2232-2237.
Tillery, M. I., Wood, G. 0., and Ettinger, H. J. (1976)
Generation and characterization of aerosols and vapors for
inhalation experiments. Environ. .Health Perspect. 16:
25-40.
Tokiwa, H., Morita, K., Takeyoshi, H., Takahashi, K., and Ohnishi,
Y. ' (1977) Detection of mutagenic activity in particulate
air pollutants. Mutat. Res. 48:237-248.
Tomlinson, E. (1975) Chromatographic hydrophobic parameters in
correlation analysis of structure-activity relationships.
J. Chromatogr. 113:1-45.
Traurig, H. H. (1976) Lysosomal acid hydrolase activities in
the lungs of fetal, neonatal, adult, and senile mice.
Gerontology 22:419-427.
Tuchmann-Duplessis, H. (1972) Teratogenic drug screening.
Present procedures and requirements. Teratology 5:271-285.
Turner, R. A. (1965) Screening methods in pharmacology, vol. 1.
Academic, New York, 332 pp.
Tute, M. (1971) Principles and practice of Hansch analysis: A
guide to structure activity correlation for the medicinal
chemist. Adv. Drug Res. 6:1-77.
!? ^
-------
Underbrink, A. G., Schaircr, I.. II., and Sparrow, A. II. (1973)
Tradcscnntia stamen hairs: A radiobiological test system
applicable to chemical mutagenesis. Chem. Mutagens 3:171-207.
-------
Valand, S. B., Acton, J..D., and Myrvik, Q. N. (1970) Nitrogen «''
dioxide inhibition of viral-induced resistance in alveolar
monocytes. Arch. Environ. Health 20:303-309.
Valcovic, L. R. and Mailing, H. V. (1973) An approach to
measuring germinal nutations in the mouse. Environ. Health '
Perspect. 6:201-205. }
Van As, A. and Webster, I. (1974) The morphology of mucus in
mammalian pulmonary airways. Environ. Res. 7:1-12. i
Van Duuren, B. L. (1967) Tumor-promoting agents in two-stage I
carcinogenesis. Prog. Exp. Tumor Res. 11:31-68. ;
j
Van Duuren, B. L. (1976) Tumor-promoting and co-carcinogenic 2
agents in chemical carcinogenesis. In: Chemical carcinogens, i
C. E. Searle, Ed. American Chemical Society, Washington, I
D. C., pp 24-51. I
I
Van Duuren,- B. L., Katz,-C., Shimkin, M. B., Swern, D., and Wieder, |
R. (1972) Replication of low-level carcinogenic activity "|
bioassays. Cancer Res'. 32:880-881. |
- t>
Van Duuren, B. L. and Sivak, A. (1968) Tumor-promoting agents *
from Crcton tiglium L. and their mode of action. Cancer Res. '\
28:2.349-2356- . ,|
'": I
Vogel, E. (1976) The relation between mutational pattern $
and concentration by chemical mutagens in Drosophila. In: |:
Screening tests in chemical carcinogenesis, R. Montesanp, H. |
Bartsch, and L. Tomatis, Eds. IARC Scientific Publications ?.
no. 12, International Agency for Research on Cancer, Lyon, J
France. - I'
Vogel, E. und Leigh, B. (1975) Concentration-effect studies with
MMS, TEB, 2,4,6-TriCl-PDMT, and DEN on the induction of
dominant and recessive lethals, chromosome loss and trans-
location in Drosophila sperm. Mutat. Res. 29:383-395. j.
Vogel, E. and Sobels, F. H. (1976) The function of Drosophila
; in genetic toxicology testing. In: Chemical mutagens, ;
principles and methods for their detection, vol. 4, A.
Hollaender, Ed. Plenum, New York.
Vogel, W., and Bauknecht, T. (1976) Differential chromatid '
staining by rn vivo treatment as a inutagenicity test system. "-..
Nature (London) 260:448-449.
Vos, J. G. and van Genderen, H. (1973) Toxicological aspects of :
immunosuppression. In: Pesticides ami- the environment, W. B.
Deichmann, Ed. Intercontinental Medical Book Corp., New
York, 'pp 527-545. -i
-------
" *--. -. f f
Wang, C. Y. and Lee, L. H. (1976) Mutagenic activity of carcino-
genic and noncarcinogenic nitrofurans and of urine of rats
fed these compounds. Chem. Biol. Interact. 15:69-75.
Warr, G. A. and Martin, R. R. (1974) Chemotactic responsiveness
of human alveolar raacrophages. Effects of cigarette smoking.
Infect. Inunun. 9:769-771.
Warr, G. A. and Martin, R. R. (1973) In vitro migration of human
alveolar raacrophages. Effects of cigarette smoking. Infect.
Immun. 8:222-227.
Watson, G. S. (1977) Age incidence curves for cance- Proc.
Natl. Acad. Sci. USA 74:1341-1342.
Waters, M. D., Gardner, D. E., Aranyi, C., and Coffin, D. L.
(1975) Metal toxicity for rabbit alveolar macrophages in_
vitro. Environ. Res. 9:32-47.
Waters, M. D., Gardner, D. E., and Coffin, D. L. (1974) Cytotoxic
effects of vanadium on rabbit alveolar macrophages in vitro.
Toxicol. Appl. Pharmacoi. 28:253-263.
Waters, M. D., Vaughan; T. 0., Abernethy, p. J., Garland, H. R., . .
Cox, Ct. C., and Coffin, D. L. (1975) Toxicity of platinum >
(IV) salts for cells of pulmonary origin. Environ. Health I
Perspect. 12:45-56. ' . I
Weil, C. S. (1972) Guidelines for experiments to predict the ?
degree of safety of-a material for man. Toxicol. Appl. |
Pharmacoi. 21:194-199. . \
Weil, C. S'.- '(1952) Tables for convenient calculation "of median- " - r- t'
effective dose (LDso or EDso) and instructions in their use. ;'
Biometrics 8:249-263. .
Weil, C. S., Condra, N. I., and Carpenter, C. P. (1971)
Correlation of 4-hour vs 24-hour contact skin penetration
toxicity in the rat. and rabbit and use of the former for
predictions of relative hazard of pesticide formulations.
Toxicol. Appl. Pharmacoi. 18:734-742. . :
Weil, C. S. and Scala, R. A. (1971) Study of intra- and inter-
laboratory variability in the results of rabbit eye and skin
irritation tests. Toxicol. Appl. Pharmacoi. 19:276-360.
Weil, C. S. and Wright, G. J. (1976) Intra- and interlaboratory
comparative evaluation of a single oral test. Toxicol. Appl.
Pharmacoi. 11:378-388.
;;
Weinstein, I. B. and Troll, W. (1977) National Cancer Institute
Workshop on tumor promotion and cofactors in carcinogenesis.
Cancer Res. 37:3461-3463. .
-------
Weiss, B., Brozek, J., Hanson, H., Leaf, R. C., Hello, M. R., and
Spyker, J. M. (1975) Effects on behavior. In: Principles
for evaluating chemicals in the environment. N.A.S., Washington,
D. C., pp 198-21b.
Weiss, B. and V. G. Laties, Eds. (1975) Behavioral toxicology
(.Environ. Sci. Pes.,, vol. 5). Plenum, New York, 969 pp.
Weiss, B. and Levin, T. E. (1976) Studies in the behavioral
toxico.logy of environmental contaminants. Environ. Health
Perspect. 13:31-35..
West, P. W. (1976) Analysis of inorganic particulates. In:
Air pollution, 3rd ed., vol. 3, A. C. Stern, Ed. Academic, J
New York, pp 51-97. . '
Wester, R. C. and Maibach, H. I. (1975) Percutaneous absorption
in the rhesus monkey compared to ;nan. Toxicol. Appl.:.
Pharmacol. 32:594-398 :- \
Whitmire,.C. E., Demoise, C. F., and Kouri, R. E. (1974) -The j
role of the host in the development of iji vivo models1 for \
carcinogenesis studies. In: Experimental lung cancer, E. *-
Karbe and J. F. Park, Eds. Springer-Verlag, New York, pp 20-47.
WHO Scientific Group Report. (1967) Principles for the testing .A
of drugs for teratogenicity. WHO Tech.-Rep. Ser. 364:18 pp. ;|
* 3
"*?
WHO-IARC. (1976) Screening tests in chemical carcinogenesis, R. $
Montesano, H. Bartsch, and L. Tomatis, Eds. IARC Scientific ;:|
Publication, no. 12, 661 pp. 'J
; - 3.
Williams, G. M. (1977) .Detection of chemical..carcinogens by ;«f
unscheduled DNA synthesis in rat liver primary cell cultures.
Cancer Res. 37:1845-1851. ; >
Wilson, J. G. (1973) Environment and birth defects. Academic,
New York, 305 pp.
Wilson, J. G. (1977) Environmental chemicals. In: Handbook of
teratology, vol. I, .General principles and etiology, chapter 9,
Environmental chemicals, J. G. Wilson and F. C. Fraser, Eds.
Plenum, New York, pp 357-385.
Wilson,'J. G. (1975) Reproduction and teratogenesis: Current
methods and suggested improvements. J.'Assoc. Off. Anal.
Chem. 58:657-667.
Witschi, H." (1975) Exploitable biochemical ..approaches for the
evaluation of toxic lung damage. Essays Toxicol. 6:125-191. :;
Witschi, H. (1976) Proliferation of Type II alveolar cells: A .
review of common responses in toxic lung injury. Toxicology
5:267-277. (
-------
Witschi, 11. and Cote, M. G. (1976) Biochemical pathology of
lung damage produced by chemicals. Fed. Proc. 35:89-94.
Kold, S. (1976) Some comments on possible types of analogy models
in the study of relations between chemical structure and
biological activity. F.xperientia Suppl. 23:87-96.
Wolff, R. K., Dolovich, M., Eng, P., Rossman, C. M., and Newhousc,
M. T. (1975) Sulfur dioxide and tracheobronchial clearance
in man. Arch. Environ. Health 30:521-527.
Wolff, R. K., Dolovich, M., Obminski, G., and Newhouse, M. T.
(1977) Effect of sulphur dioxide on tracheobronchial clearance
at rest and during exercise. In: Inhaled pavticles IV,
Part I, W. H. Walton, Ed. Pergamon, New York.. pp321-332.
Kolkowski, Z. W., Moccatti, D., Heymans, F., and Godfroid, J.-J.,
Jr. (1977) ' A quantitative structure-activity approach to
chemoreception: Importance of lipophilic properties. J.
Theor. Biol. 66:181--193.
Working Group of the Subcommittee on Environmental Mutagenesis,
April, 1977. (Prepared for the UHEW Committee to Coordinate
Toxicology and Related Programs.) Approaches to determining
the mutagenic properties of chemicals: Risk to future . ..
generations. '
Wynder, E. L. and Gori, G. B. (1977) Contribution of the'environ-
ment to cancer incidence: An epidemiolc?ic exercise.- J.
Natl. Cancer Inst. 58:825-832.
-------
Yanysheva, N. Ya. and Antomonov, .Yu. G. (1976) Predicting the
risk of tumor occurrence under the effect of small doses of
carcinogens. Environ. Health Perspcct. 13:95-99.
Yeager, H., Jr. and Massare, D. (1972) .Glucose metabolism.and
glycoprotein synthesis by lung slices. J. App.l. Physiol.
32:477-482.
Yeh, H. C., Phalen, R. F., and Raabe, 0. G. (1976) Factors
influencing the deposition of inhaled particles. . Environ.
Health Perspect. 15:147-156.
Young, S. L. (1976) An isolated perfused rat lung preparation.
Environ. Health Perspect. 16:61-66.
l.i
-------
Zarkower, A. (1972) Alterations in antibody response induced by \
chronic inhalation of SO-> and carbon. .Arch, linyiron. Health
25:45-50.
Zimmenaann, F. K. (1973) Detection of genetically active chemicals
using various yeast systems. Chcm. Mutagens 3:209-239.
Ziramermann, F. K. (1975) Procedures used in the induction of
mitotic recombination and mutation in the yeast Saccharomyces
cerevisiae. Mutat. Res.-31:71-86.
-------
- «S
<** "?v
,.*** >-/v*»-
' t ", u '"j
- ,', ;.,.«.«»* in
1"^.
r u *!v . '
f ;t?'%1
. Yv^j^5.
..", :^%
, ^*r*48?fcf
- ,v',?:-*-.'
. ^
, « *,'/"» -*
'y tv <; , 1$
^ k> " - ^
,,. ._>- ?' .gsr-^f^i.,- ^M^^«t*JT;';?^/;- - &*
- ' A3L^iH**^'^t' 'v^^^^V-'ft^1'^?1?*
.,Jl^J^^.^:^Ai^lA^
------- |