-EPA
Health Effects
Research  Laboratory
Office of Health Research
Office of Research and Development
PROGRESS
REPORT
October 1,1985  September 30,1986

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HEALTH EFFECTS RESEARCH LABORATORY

            Office of Research and Development
                Office of Health Research
                  Progress Report
           October 1,1985 - September 30,1986

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Acknowledge ments:
This report was prepared by the Information Management Staff of the Office of the Director, Health Ef-
fects Research Laboratory, Office of Health Research, Office of Research & Development, US Environ-
mental Protection Agency.
Many other individuals within the Health Effects Research Laboratory also contributed their time, ener-
gy and support to developing the report.
Systems Research and Development Corporation provided document preparation support.

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Table of Contents
Page Number
I—IE1 1..?vIissionStatenient . . . ..... . 1
OrganizationalChart .2
Divisional Functional Statements .3
Air and Radiation
ResearchObjectives 9
Accoiiiplisliiiients . . 17
Publications 23
Water
Research Objectives . . • . 83
Accoiiipiisiiiiients 89
l’ublicatioris 99
Pesticides and Toxics
Research Objectives 183
Accomplishments 191
I ubJications S. 203
Hazardous Waste
Research Objectives 279
Accornplisluiients 283
Publications 289
Future Perspectives 297
Iiidexofe .bstracts ...303
Appendix A - List of Submitted Documents 353
Appendix B - List of Presentation Abstracts 371

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HEALTH EFi kCFS RESEARCH LABORATORY
Dr. F. Gordon Hueter, Director
Dr. Judith A. Grnh ’im, Deputy Director
HERL is one of fourteen Office of Research and Development (ORD) laboratories
within the Environmental Protection Agency. HERL provides the health data base
which serves as the foundation for the health-related regulatory decisions of the Agen-
cy. In building this foundation, evidence is gathered, not only from internal research
studies, but also from cooperative agreements and contracts with universities and
private institutions, and through agreements with other governmental agencies.
Wide ranges of pollutants known or suspected to cause health problems are studied.
The research focuses on air pollutants, water pollutants (both chemical and microbial),
toxic substances, and pesticides. Sm l1er programs are being conducted concerning
hazardous wastes and mobile source pollutants. Non-ionizing radiation research is
also being conducted at a level consistent with legislative guidance.
The major mission of HERL is to provide h izard assessment-related research in
support of risk assessment. To achieve this in a fashion compatible with the four
program offices with which we work requires major research emphasis on hazard iden-
tification, dose-response studies, extrapolation, and dosimetry. In many instances,
being responsive to the Program Offices requires HERL to make significant advances
in the state-of-the-art, especially for model development and extrapolation. The
diversity of the needs of EPA has resulted in HERL’s developing a broad-based
program with multiple high priorities. This has led to HERL’s unique capability to
conduct research using oral, dermal, and inhalation routes of exposure; in vitro, animal
toxicology, human clinical, and epidemiological approaches; and a full range of
toxicological disciplines including neurotoxicology, reproductive toxicology, teratol-
ogy and perinatal toxicology, geriatric toxicology, pulmonary toxicology, im-
munotoxicology, cardiovascular toxicology, genotoxicology, hepatotoxicology, other
target organ toxicology, dosimetry, and microbiology.
While most of the facilities are located in the Research Triangle Park, North
Carolina, HERL has one division, the Toxicology and Microbiology Division, located
in Cincinnati, Ohio. Also, HERL has one of the nation’s few sophisticated human in-
halation exposure facilities which is located on the campus of the University of North
Carolina at Chapel Hill.
Organizationally, HERL consists of three staff offices and seven divisions with a
total of approximzitely 300 employees. In addition to the EPA staff, approximately 250
on-site contract and cooperative agreement employees provide an extension to
HERL’s in-house research program.
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INHALATION TOXICOLOGY
DIVISION
Dr Fredericic Miller
Clinical Research
Branch”
Dr John ONoft
Dosimetry and Cm’dlo.
pulmonary Section
Dr. DanIel Costa (Acting)
€OJ 1
Biochemical Toxicology
Section
Dr Gary Hatch
2651
Microbiology and immu-
notoxicology Section
Or. Mary Jane Sotgrade
Gamete Biology
Section
Or Saty Dazney
Dosimetry and
Metabolism Section
Dr Edward Massa,o
Molecular Biology
Section
Dr Clinton Kawanlehi
BIOMETRY
DIVISION
Dr. Robert Payne (ActIng)
5156
Blostatistlcs
Branch
Dr. John Creason
2598
Data Management
Branch
Dr Rcbert Payne
Data Systems
Support Section
Vacancy
TOXICOLOGY AND MICRO
BIOLOGY DIVISIONt
Dr. Elmer Aicki
741 ‘I
Chemical and Statistical
Support Services Branch
Dr Frededdi Kopiler
7451
EXPERIMENTAL
BIOLOGY DIVISION
Dr Richard Phil s
HEALTH EFFECTS RE
Program Planning
Technical Support Staff
Dr. Judith Graham ( Acting ) 2283
RCH LABORATORY
OFFICE OF THE DIRECTOR
Dr F. Gordon Huetor, Director
Dr Judith Graham
Deputy Director
2281
Administrative
Staff
Phyllis Wynne
2655
2283
Information
Management Staff
Ann Akland
NEUROTOXICOLOGY 1 DEVELOPMENTAL
DIVISION BIOLOGY DIVISION
Or Lawrence Rotter Dr. Richard Phtlpa ( Acting) - -
2602
2298
2883
GENETIC TOXICOLOGY
DIVISION
Dr Michael Waters
2537
Mutageneals and Cellular
Toxicology Branch
Dr Ma,lha Moore
3933
Carcinogonesls and
Metaboliam Branch
Dr. Stephen Nesnow
2647
Genetic Bioassay
Branch
Dr Joellon Lewtas
PhysIology
Section
Dr. Donald Hoqitnwn
__ 3806
2671
Behavioral Toxicology
Branch
Dr Robert MacPhail
2781
406 ’
Reproductive Toxicology
Branch
Dr. John Laskey
Cellular and Molecular
Biology Section
Dr Hillel S Karen
Neurophysiology
Branch
Dr Robert Dyer
I
276
7401
5127 3849
Developmental
Reproductive Biology
Section
Dr. Earl Gray
775
Aerobiology
Section
Dr. Tlmctiiy R Gerrity
MIcrobiology
Branch
Dr. Alfred Dufour
Systems EngineerIng
Branch
Dr Charles Woodley
2771
- 7871
Cellular Biophyalcs
Branch
Dr Joe Elder
2671
5156
Toxicology
Branch
Dr. DanIel Coats (Acting)
Target Organ
Toxicology Branch
Dr. Lyman Condle
3838
721
Bioassay
Branch
Dr. Bernard Daniel
Endocrinology and
Gerontology Section
Dr. Ralph Cooper
4084
Pednatal Toxicology
Branch
Dr. Robert Kavioclc
Molecular BIology
SectIon
Dr John Mis
2541
Immunoblology
Section
Dr Joe Elder (Acting)
Data Preparation
and Control Section
Vacancy
2771
2321
Tsratogsnesla Section
Dr. Robert Kavtock (Acting)
Biological Engineering
Branch
Or. Ronald Spiegel
.1
7542
232
EngineerIng
Section
Dr Joe All (Acting)
bDI
chronic Toxicity and
Exposure Section
Dr Elaine Groee
224C
317
2531
Physiology
Section
Dr EvaBwman
706t
2240
NOTE ThB number is the four digit telephone extension
Dai FTS (629) +4 dIgIts for North Carolina offIces
For Toxicology and MicrobIology Division and Branches,
dial FTS (684) +4 dIgits
t Located in Cinonnati, Ohio
Located In Chapel Hill. N.C
Immunology Section
Dr Ra h Smlalowlcz
5776

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HEALTh EFFECTS RESEARCH LABORATORY
DIVISION FUNCTIONAL STATEMENTS
INHALATION TOXICOLOGY DIVISION
Dr. Frederick Miller, Director
The Inhabition Toxicology Division conducts research with hflmans and s.nim i1c to determine the health
effects of bih ted environmental pollutants. The research uses highly sophisticated humpn and animal in-
halation exposure facilities and is designed to determine cause and effect relationships at pollutant con-
centrations which mimic those occurring in the environment. While conventional toxicological
methodologies are used, emphasis is also placed on the development of improved methods which enable
ignf1cant advances in the knowledge of the health effects of air pollutants. Intense investigations center
on the pulmonary and cardiovascular systems, the immune system, host defense 2g inct in-
fectious and neoplastic disease and other extrapulmonary systems which are also susceptible to inhaled
pollutants. Continual efforts are made to improve the correlation between animal and human studies and
extrapolation models are developed to enable better risk assessments to be made. The Division serves as
a primary technical resource within the Agency for activities requiring expertise in human and animal in-
halation studies and in the health effects of common air pollutants. Although the research of this Division
is focused on enhancing the health effects data base for pollutants regulated by National Ambient Air
Quality Standards, problems relevant to toxics, pesticides and ha ’ardous air and waste pollutants are also
addressed.
GENETIC TOXICOLOGY DIVISION
Dr. Michael Waters, Director
The research program of the Genetic Toxicology Division encompasses the fields of mutagenesis, car-
cinogenesis, and cellular toxicology. The program also includes and is centered around definitive studies
on the metabolic activation and detoxification of those agents which exert their effects via genetically-re-
lated mechanicmc. The Division possesses the capability of fully evaluating the mutagenic and oncogenic
potential of agents of environmental concern including pure chemicals and complex environmental mix-
tures. The process of evaluation of potential environmental genotoxicants is approached through the
step-wise application of bioassay methodologies involving short-term prescreening tests, confirmatory
short-term bioassays and, eventually, established whole animal mutagenesis and carcinogenesis bioassays.
The intramural research competence of the Division is directed principally toward the development,
validation, and application of short-term bioassays to meet the research and regulatory needs of the
Agency. Long-term mutagenesis and carcinogenesis bioassays are conducted through the use of ex-
tramural grants and contracts. The Division meets the research requirements of the Office of Research
and Development in responding to the needs of the EPA Program Offices.
TOXICOLOGY AND MICROBIOLOGY DIVISION
Dr. Elmer Akin, Director
The division performs studies to identify, characterize, and quantitate harmful effects that may result
from exposure to biological or chemical agents found in the environment. Appropriate animal model in
vitm test systems are utilized to identify overt or potential toxic effects of pollutants. Ingestion, dermal, in-
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DIVISION FUNCTIONAL STATEMENTS
halation, and multiple routes of exposure are used to determine toxic effects, and to quantify absorption,
distribution, mobili tio; biotransformation, and excretion. Shorter term, predictive toxicologic tests
are developed and compared. Microbiologic pathogens in the environment are identified and studied by
the appropriate disciplinary methodology, i.e., bacteriology, virology, parasitology, and/or immunology.
The division collaborates with the Centers for Disease Control (HHS) in the study of environmentally re-
lated disease outbreaks. An objective is to relate environmental quality indicators and pathogen trans-
mission to man.
BIOMETRY DIVISION
Dr. Robert Payne, Acting Director
The Biometry Division provides statistical, mathematical, and computer science competence to all com-
ponents of the Health Effects Research Laboratory. The division conducts research into new statistical
methodology required for the proper analysis of HERL projects. Such statistical research may be in-
tramural or extramural and involves such topics as time series methods, contingency table analysis, non-
parametric procedures, censored and missing data techniques, and general linear model development.
The division provides appropriate experimental design and st’athtical analysis of the epidemiological,
clinical, biological, toxicological, and other studies performed by HERL The division also designs, im-
plements, and documents data processing systems required for laboratory projects as well as provides
computer progrnmming and data processing capability. The division also advises HERL personnel on
statistical, computer science, and data processing topics and problems, performs and reports on in-
tramural research projects and directs extramural research on statistical and computer science topics
consistent with the objectives and needs of HERL assures that appropriate computer hardware and
software required by the laboratory are available, and maintains a computerized data bank of health ef-
fects information for completed studies.
NEUROTOXICOLOGY DIVISION
Dr. Lawrence Reiter, Director
The Neurotoxicology Division is the focal point for plnnning , conducting, coordinating, supporting and
evaluating a program aimed at studying the effects of physical and/or chemical agents on nervous system
function. In order to thoroughly examine toxicant-induced changes in nervous system function, scientific
investigation proceeds at all levels of neural orgnn tion including functional and structural. In develop-
ing the necessary multidisciplinaq approach to the study of neurotoxicity, the Division addresses both the
short-term needs (i.e., toxicity testing) and long-term goals (i.e., methods development and validation,
hs mTd assessment and establishment of a data base) of the Agency. The overall program strategy stres-
ses the development of cost-effective testing methods for evaluating neurotoxicity and for predicting risk
to humans. Within the framework of this strategy, five overall objectives have been identified. These are:
(1) methods development and validation, including evaluation of existing methods, design and evaluation
of new methods, and development of testing strategies; (2) toxicity evaluation (studies of the toxicity of
heavy metals, pesticides, and bs7Jlrdous air pollutants are in progress); (3) determinations of the sig-
nificance of neurotoxicological indicators for assessing risk in hnm inc ; (4) developmental neurotoxicol-
ogy (behavioral teratology) which evaluates the effects of perinatal toxicant exposure on the development
of the nervous system; and (5) mechnnicm of action studies.
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DIVISION FUNCFIONAL STATEMENTS
EXPERIMENTAL BIOLOGY DIVISION
Dr. Richard Phillips, Director
The Experimental Biology Division conducts and man ges biological research to define and quantify the
effects of environmental pollutnnts, singly or in combination. Emphasis is given to the study of effects of
non-ioni”ing radiation and toxic substances. This involves laboratory studies of intact ianimalc or cellular
and subcellular systems using controlled and simulated environments. Major research attention is
devoted to elucidation of mech nicmc of action of the toxicants under study, as well as engineering
developments and improvements in exposure systems and dosimetry. Biologic parameters studied in-
clude evaluations of immunological status, growth, functional development of systems, pathological ef-
fects, biophysical and biochemical interactions. In addition, this division plans and conducts special
studies in non-ioni ’ing radiation dosimetry and the radiation absorption characteristics of living tissue.
DEVELOPMENTAL BIOLOGY DIVISION
Dr. Richard Phillips, Acting Director
The Developmental Biology Division conducts research on the effects of environmental agents on age-re-
lated biological phenomena. The research identifies and utilizes appropriate laboratory animal species
as models to provide data for the assessment of potential hazards to humans resulting from exposure to
various environmental pollutant ’. The agents studied include pesticides, toxic substances, air pollutants,
and water contaminants. The biological parameters which are studied include teratogenesis, postnatal
alterations of organ function, reproductive physiology including sexually dimorphic characteristics, the
relationships of senescent changes to toxic insults, and the histopathological correlates of the above.
Major research emphasis is on developing new and improved methodologies for the assessment of perina-
tal dysfunction, reproductive impairment, or insults exacerbated by senescent changes. The research
generated contributes to the development of guidelines for the safe usage of pesticides and other poten-
tiafly toxic compounds as mandated by the Agency.
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Research Objectives
Air Health
Radiation

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AIR HEALTH RESEARCH PROGRAM
Objective: To improve the scientific basis for regulatory activities under the Clean Air Act.
Background: In 1963, Congress passed the Clean Air Act (CAA) which was strengthened considera-
bly when the Clean Air Act Amendments of 1970 were signed into law m dcing EPA the
focal point of the Federal effort. These amendments required EPA to set National Am-
bient Air Quality Standards for “criteria air pollutants.” EPA has identified six criteria
pollutants: ozone (03), carbon monoxide (CO), particulate matter (PM), sulfur dioxide
(S03), lead (Pb) and nitrogen dioxide (N02). For these pollutants, primary standards
are set to protect human health. The CAA amendments also require EPA to review and
regulate h rdous air pollutants. These pollutants are defined as those that can con-
tribute to an increase in mortality or serious illness but which are not already regulated
as criteria pollutants. Standards have been issued for several of these pollutants and
EPA is currently analyzing a number of other pollutants to determine whether they are
hii iardous and require regulation.
Oxldants - Ozone is the Nation’s number one air pollution problem. Many areas across the country con-
tinue to have levels of this pollutant which are higher than EPA’s ambient standard. Health concerns re-
lated to exposure to 03 include respiratory tract problems such as difficult breathng and reduced lung
function, reduced resistance to infection, and structural and biochemical changes of lung tissue. Health
concerns related to N02 are similar; asthmatics are more responsive to N02 than are healthy people. The
CAA requires that EPA periodically reevaluate the National Air Quality Standards. As part of this
process, new research on the health risk of exposure to the criteria pollutants, especially 03, N02, and
PM is conducted. HERL scientists are conducting research in the following general areas in support of
this effort.
• Provide toxicological data on the health effects of acute and chronic exposure to 03 and N02 ex•
posure using human and animal studies.
Rationale: Earlier studies have shown that exposure to oxidants, expecially in certain patterns, may cause
irreversible chronic lung tbivn ge and a series of acute effects. Also, certain subpopulations, such as
children and asthmatics, may be more sensitive to oxidant insult. The Office of Air Quality PLuming and
Standards (OAQPS) and the Office of Health and Environmental Assessment (OHEA) have requested
more research to reduce uncertainties in the risk assessment data base in order to make responsible
regulatory decisions.
Approachi Humiun and nimiut toxicology studies are being performed to describe changes in pulmonary
function, biochemistry, and immunology following acute and chronic exposure. There are ongoing
human studies to define the nature and significance of the acute response to ozone exposure. In addition,
the acute effects of NO2 are being evaluated in asthmatic subjects. In addition, a collaborative study is
underway with investigators at UCLA to evaluate the association between acute pulmonary responses to
ozone inhalation and the development of chronic pulmonary changes of individuals living in areas with
elevated ozone levels. An animal study is being performed to examine the lifetime effects of exposure to
patterns of N03 and 03 similar to those encountered in urban environments. Studies are also being done
to determine the effects of oxidant inhalation on epithelial cell barrier function.
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RESEARCH OBJECTIVES AIR HEALTH
• Provide models to quantitatively extrapolate animni data to humans.
Rationale: EPA must rely partly on animal test data in setting and revising standards for protecting
human health. Refined extrapolation models will assist OAQPS in predicting human health risk from
animal studies, thereby reducing the likelihood that EPA will set standards which are either too lax to
protect public health or too stringent to be enforced.
Approach: Theoretical models of respiratory tract deposition and uptake of N02 and 03 are being
developed. Species sensitivity to these pollutants is being compared by examining effects on pulmonary
host defense mechanisms at equivalent predicted concentrations. Experimental studies in human and
animals will be done to determine the amount of oxidant gas which is removed in the nasopharyngeal
region. Results of these studies will be used to refine the animal-to-human dosimetry model.
• DetermIne how and where epidemiologic research can contribute to an Improved scientific basis for
revising current air quality standards or developing new standards and to develop studies where
appropriate.
Rationale: Where feasible, epidemiologic studies can provide direct evidence of human health effects at
environmental exposure levels.
Approach: Studies are being done which attempt to answer questions raised in past epidemiological
studies, especially regarding the effects of N02. These questions include: 1) Does the presence of a gas
stove increase NOi exposure in the home? 2) If so, what are these exposure levels? 3) Does this level of
N02 increase susceptibility to respiratory infection? 4) Are some subpopulations, such as children and
asthmatics, at greater risk than the general population? 5) If health effects are seen, do they result strict-
ly from exposure to N02 or do other sources cause or contribute significantly to the effects?
Carbon Monoxide (CO) - CO is a colorless, odorless, and tasteless gas produced by the incomplete com-
bustion of fuel. When inhaled, it replaces oxygen in the bloodstream and can impair vision, alertness, and
other mental and physical capacities. It has particularly severe health effects for people with heart and
lung problems. EPA is required by the CAA to periodically reevaluate the carbon monoxide ambient air
quality standard. The objectives of HERL research in support of this effort are described below:
• Develop amd validate techniques to produce dose-response data and produce dose response data
on the toxic effects of CO.
Rationale: The Clean Air Act requires that the criteria and standard for CO be updated every five years.
This work will provide dose-response data as well as improved methods for obtaining dose-response data
for risk assessments needed by OAQPS.
Approach: Volunteers are being exposed to CO during rest and exercise to measure the appearance of
carboxyhemoglobin (COHb) in the blood and to validate equations which predict COHb formation. The
gamma camera is being used to measure left ventricular function in patients with coronary artery disease
and to quantitate changes produced by exposure to CO. The neurobehavioral effects of CO exposure are
also being evaluated.
Gases and Particles - Particles in air such as dust, smoke and aerosols may have both acute and long-term
health and environmental effects. These effects range from irritating the eyes and throat to causing mor-
tality. Inhalation of S02 causes significant pulmonary responses in individuals with asthma. Fme par-
ticles, about the size of cigarette smoke particles, can cause temporary or permanent damage when they
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AIR HEALTH RESEARCH OBJECI’IVES
are inhaled deeply and lodge in the lungs. Some particles are suspected of causing cancer. Since 1971,
EPA has had a National Ambient Air Quality Standard for Total Suspended Particulates. HERL re-
search is aimed toward providing health effects information for use in EPA’s periodic reevaluation of the
standard.
• Provide better models to extrapolate quantitatively from animni data to bum ? ” pulmonary effects
following exposure to sulfur dioxide (S02) and particles as well as combinations of selected gases
and particles.
Rationale: Mathematical models predictive of respiratory tract dose patterns of S02 and particles are es-
sential to extrapolate effective pollutant concentrations between animal and man. They are also useful
for predicting the regional pulmonary deposition of particles as a function of size in adults and children
Approach: Theoretical models are being developed of respiratory tract deposition of S02 alone and in
combination with particles. Studies are being conducted to provide data on the relationship between par-
ticle size and lung deposition in man. Regional deposition data for anmals are being developed and
refined and lung casts are being used to eramine deposition in children. Increased emphasis is being
placed on improving physiochemical data relevant to extrapolation models.
• Develop techniques and equipment to conduct research Into the physiologic response of humans to
air pollutants.
Rationale: Clinical studies ‘ icing accurate proven instrumentation and techniques are needed to provide
reliable data for assessing human health risk.
Approach: An aerosol physics laboratory is being designed and constructed to make possible the study
of respirable particle distribution and clearance in hunians . Instrumentation is being constructed and in-
stalled to 1) partition airways resistance into its proximal and distal portions using forced random noise
excitation of the airways, 2) generate and describe the size and mass of aerosols used to challenge airways,
3) measure lung water and diffusing capacity of the lungs, and 4) measure particle distribution, deposi-
tion, and clearance from the lungs.
• Provide health data on acute and chronic effects of exposure to gases and particles.
Rationale: Uncertainties in the health effects data base for gases and particles must be resolved prior to
future reviews of the S02 and particulate matter standards. There are almost no subchronic or chronic
health data on some common inhalable particles with toxic potential. Past studies indicate that particle
size affects deposition of particles in the lung and an organism’s ability to expel harmful compounds.
Approach: Data will be obtained on 1) the respiratory, biochemical and immunological effects of gases
and particles in normal and asthmatic subjects and patients with chronic obstructive lung disease, 2) the
effects of gases and particles (fine and coarse mode) on pulmonary function, morphology, biochemistry
and immunology of normal and impaired animals, and 3) neurobehavioral effects in lead-exposed
children and adults.
• Determine how and where epidemiologic research can contribute to an Improved scientific basis for
revising current air quality standards.
Rationale: Where feasible, epidemiologic studies can provide direct evidence of human health effects at
environmental exposure levels.
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RESEARCH OBJEC11VES AIR HEALTH
Approach: A lout-year prospective study is being conducted of 3,000 homes in Tuscon, Arizona, to
evaluate the correlation between exposure to air pollutants such as particles and gases and respiratory
responses. This study includes indoor and outdoor monitoring and some personal monitoring. Trends in
cardiovascular disease are being investigated. Acute effects of exposure to summertime haze episodes is
being elfimined in a population of young healthy persons, including joggers, in an urban area in the north-
east. These studies should provide data which can help illuminate the relationship between gases and
respirable particles and chronic cardiopulmonary effects.
China Research
• Work pith Chinese scientists to determine the causes and contributing factors of lung cancer In
Xuan Wel County, Ynnnnn Province, Peoples Republic of China (PRC).
Rationale: Item IIA, Annex I 1J.S.-China Protocol for Scientific and Technical Cooperation...” calls for
a study of lung cancer in Xuan Wel County. The work plan being undertaken has received approval by
both U.S. and Chinese officials in a meeting in Beijing. September 1982.
Approach: Epidemiologic, chemical and bioassay studies will be undertaken to assess the impact of
known and suspected risk factors, including domestic coal burning in Xuan Wei County. In addition,
non-malignant health effects will be ex imined epidemiologically.
Objectives include: 1) quantitation of the etiologic role of indoor coal smoke and of physical and chemi-
cal components thereof in producing lung cancer in Xuan Wei, 2) collection of particulate and gas phase
samples under simulated combustion conditions in the U.S., 3) development of an epidemiologic data col-
lection system to be used by the Chinese, 4) analysis of data gathered, 5) collection of air samples from
Xuan Wei, 6) bioassays of all samples to determine mutagenic potential, and 7) exchange of scientists.
Hazardous Air Pollutants (HAP) - Despite their low concentrations, toxic chemicals emitted into the air
by industrial and commercial processes and personal activities may have serious short- and long-term ef-
fects on hnman health and the environment. Most information on the direct human health effects of air-
borne toxicants comes from studies of industrial workers. Exposure to these substances in the work place
is generally much higher than in the ambient. We know relatively little about the specific health and en-
vironinental effects of most of these substances at the low levels at which they are found in ambient air or
at levels experienced during accidental releases. EPA has issued National Rmic .cion Standards for Hazar-
dous Air Pollutants (NESHAPS) under the Clean Air Act for some h rdous air pollutants and is as-
sessing risks and control options on others. HERL scientists are exploring the following areas of research
to provide health information on pollutants suspected of being h rdous in support of appropriate
regulatory decisions.
• Produce dose.response data on the toxic effects of HAP’s.
Approach: The specific aim is to provide dose-response data on the toxicological effects of 1 a ,’ nrdous air
pollutants. The toxicological effects include genotoxic effects (carcinogenesis and mutagenesis),
neurotoxic effects, physiological effects and pulmonary effects.
• Develop models which Improve our ability to use toxicological data in risk assessments.
Approach: 1) to develop and validate bioassays which are sufficiently sensitive and reliable to evaluate
the neurotoxic potential of h i72Tdous air pollutants, 2) to develop a comparative potency method for as-
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AIR HEALTH RESEARCH OBJECTIVES
sessing cancer risk from source emic .cion, and 3) to evaluate the contribution of source emic ions to can-
cer risk.
• Identify toxic components of air pollution.
Approack Identify potentially carcinogenic, mutagethc, and teratogenic components in ambient air and
source emissions and develop dose-response data from gene mutation and other genetic bioassays for use
in comparative risk evaluations. Studies will also be conducted to investigate the effects of nitrogen
oxides (NOxj, S02, 03 and other gases on the generation and decomposition of mutagens, carcinogens
and teratogens in air.
• Improve assessments of human cancer risk from HAP’s.
Approach: This program is the health component of a long-range research effort to address major scien-
tific uncertainties about the relationship between air pollution and human cancer. The program is
providing scientific methods and data to improve evaluations of human cancer risk from individual or
complex source emic sions or from atmospheric transformation of precursor chemicals.
• Evaluation of humans for genotoxic effects following 1W-B radiation.
Rationale: This research complements that mandated by the Qean Air Act owing to the inverse relation-
ship between stratospheric ozone depletion and atmospheric penetration of ultraviolet radiation at
wavelengths capable of biological damage . Data from this study can contribute information required in
order to allow sound regulatory decisions on the protection of stratospheric ozone.
Approach: Research is being conducted to determine the feasibility of testing humans, via the Lym-
phocyte Test Battery, to determine the significance of ill health effects after IJV-B exposure. This re-
search is to be conducted in the laboratory with the cooperation of the staff and patients of the Duke
University Dermatology Department’s Ultraviolet Therapy Section.
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RADIATION RESEARCH PROGRAM
Objective: To conduct a phase-out of the health effects research program that was established to
provide and to evaluate the scientific data on the health effects of radiofrequency radia-
tion in support of the guidance activities of the Office of Radiation Prog mc (ORP).
Background: Executive Order 10831 of 1959 (24 Fed. Re& 6669) established the Federal Radiation
Council (FRC). Statutory authority for the FRC is also contained in amendments to the
Atomic Energy Act of 1954 (42 U.S.C. Sec. 2021) (h). Reorganization Plan No.3 of
1970 transferred the FRC authorities to EPA. ORD’s radiation research program sup-
ports the Agency’s Federal Radiation Council authority. FRC authorities stipulate that
the Administrator is to advise the President with respect to radiation matters, directly
or indirectly affecting health, including guidance for all federal agencies in the formula-
tion of radiation standards and in the establishment and execution of programs of
cooperation with states. Further, the Reorg2ni7sltion Plan also transferred certain
regulatory and research responsibilities of the Bureau of Radiological Health as derived
from the Public Health Service Act.
The radiofrequcncy radiation health research program directly supports the activities of the Office of
Radiation Programs (ORP). ORP is developing environmental guidance for radiofrequency radiation
and has requested health effects data and evaluation. ORD scientists provided a background health ef-
fects review and assessment document, which serves as the primary health effects reference in develop-
ing the Federal Radiation Protection Guidance. The 01W report, Biological Effects of Radiofrequency
Radiation (EPA-600/8-83-026F), was published in 1984.
• A plan was developed to phase-out both the intramural and extramural research on the health ef-
fects of radiofrequency radiation. Intramural research in progress was allowed to continue until
publishable units of research were achieved; extramural projects are continuing as negotiated. Thus,
there will be some final contract reports and journal artides in FY87 and possibly in FY88. The staff
involved in the radiofrequency radiation program are being assigned work in other research areas
such as toxic substances, h rdous wastes and air toxics.
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Accomplishments
Air Health

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ACCOMPLISHMENTS AIR HEALTH
Issue Provide scientific assessments and data on the health and welfare effects of criteria air pol-
lutants to develop and review primary and secondary NAAQS
Investigators in the Clinical Research Branch have demonstrated significant adverse pulmonaiy function
responses in some normal volunteers following acute inhalRtion exposure to ozone. These acute respon-
ses are apparently not mediated by airway smooth muscle (broncho-constriction) but appears to be due
to a reflex inhibition which limits ones ability to inhale to the total lung capacity. This reflex is likely
mediated. In addition, the permeability of the respiratory epithelium is increased following ozone ex-
posure. This permeability increase appears to be greatest in those individuals having larger pulmonary
function responses. There is also evidence indicating that the epithelial changes may be associated with
local inThimm tion. Other studies with pulmonary cellular elements derived from the lungs of volunteers
by bronchoalveolar lavage are underway to evaluate the association of local infbinin’ntion with the acute
pulmonary responses to ozone. In addition, the association between pollutant exposure and resistance to
infection in the lungs is being evaluated for the function of specific cellular elements known to be impor-
tant in pulmonary host defenses. Studies employing nasal lavage techniques are being conducted to
evaluate the development of infl tmition following exposure to ozone in upper versus lower airway. This
technique has potential for application in field studies. Investigators at UCLA in collaboration with EPA
are studying the respiratory effects of long-term oxidant exposure and the nature of the association be-
tween the chronic effects and the magnitude of the acute effects of a single 03 exposure.
Studies have been completed which describe the amount of ozone removed in the nasal passages and in
the lungs of individuals while they breathe ozone. These studies provide information which is particular-
ly important for the determination of the dose of ozone reaching target tissues in the lungs and for risk as-
sessment analysis in man.
In addition, it was shown that significant airway responses were measured in a population of asthmstic
subjects who were exposed to 0.3 ppm N02 with moderate exercise. Two other hnnii n studies were com-
pleted in FY’86. One described the group concentration-response of mildly asthmiitic subjects exposed
to SO 2 concentrations between 0.25 and 1.0 ppm. Another showed that there was no interaction between
S02 and H2SO4 mist when mildly asthmatic subjects were studied.
A chronic animal toxicology study of the effects of N02 and 03 on a multitude of biological endpoints was
continued during FY86. The one week, 3 week and three month exposure durations were completed and
the 12 and 18 month exposure periods are in progress. The focus of these studies is the ability of oxidant
gases to cause chronic lung disease as reflected by biochemical, structural and functional changes in the
lung. Our understanding of immunological responses in the lung to oxidant exposure was greatly
facilitated by the development of natural killer cell and interferon studies. An animal viral infectivity
model was developed that is more analogous to what happens in man than conventional bacterial infec-
tivity models. The model was used to study the effects of 03 exposure on host defenses. The oxidant
dosimetry program continued to progress during the year with a major accomplishment being a refined
model for the uptake of ozone in the lungs of 2nmnk , thereby nis&ing available improved dosimetry com-
parisons for the ozone risk assessment to be conducted in FY87. Significant advances were made during
the year in the experimental portion of the dosimetry program, both using isotopic techniques and
physiologic mass balance studies. The basis for the Agency’s position on PMio, from a respiratory tract
deposition viewpoint, was significantly strengthened by simulations that e, ’n ’ined the influence of route
of breathing and of activity levels (from normal respiration to heavy exercise) on deposition in man. Also,
the deposition of hygroscopic particles as a function of age was ex2niined. One of the first studies specifi-
cally designed to meet the Agency’s need for quantitative extrapolation modeling was completed wherein
concentration response studies in multiple species were conducted using the same methodologies.
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AIR HEALTH ACCOMPLISHMENTS
Health research in the People’s Republic of China continuecL A report sununarizing current
epidemiologic, chemical, and toxicologic evidence reladng to Xuan Wei lung cancer etiolo ’ was
published in Science. The toxicologic and chemical evidence is consistent with the epidemiologic
hypothesis that indoor smoky coal burning is the prime determinant of Xuan Wei lung cancer, especially
in women. A joint US-China manuscript, documenting the successful field-testing of a modified
medium-volume indoor sampler in Xuan Wei, is nearing completion. Another joint manuscript present-
ing results of the 1985 personal monitoring pilot is in preparation; this documents the high particulate and
organic exposures that many Xuan Wei residents receive, and questions the utility in community studies
of some passive pollution collection devices developed for use in occupational studies. In other
epidemiological studies a series of papers have been published in several peer reviewed journals dealing
with the effects of ambient pollutants , indoor pollution and passive smoking on lung function growth and
respiratory symptoms.
A research project evaluating the neurotoxicity of lead in 75 three to seven year old children has been
completed. The results indicated that within the range of 63-4Th MJdl there was a idgnificant negative
relationship between blood lead level and Stanford-Binet 10, a measure of cognitive function. The 10
decreased linearly as blood lead increased. An analysis of NUANES II audiometric data was initiated to
investigate the relationship of blood lead levels and hearing thresholds. Results indicate that the prob-
ability of elevated hearing thresholds increase significantly with increasing blood lead levels at the fre-
quencies tested (03,1,2 and 4 KHz); this paper has been submitted for publication. A paper on the
neurophysiological effects of lead exposure in monkeys was also accepted for publication. This study,
which is part of a larger investigation of the effects of perinatal lead exposure, indicated prenatal or
postnatal exposure to lead resulted in abnormal neurophysiological processing of complex auditory
stimuli. Electrophysiolgical recordings from specific auditory areas of the brain showed that lead-exposed
monkeys discriminated between different sets of auditory stimuli in an abnormal and immature fashion.
Issue: Provide scientific assessments, monitorIng systems, control technologies, and data on the
health effects and atmospheric processes on non-criteria pollutants to support regulations for
hazardous air pollutants
An Interdivisional Air Tot Study (LATh) was initiated as a interdisciplinary project to study the health
effects of inhaled hazardous air pollutants. Compounds under study have been selected based on high
production and potential human exposure. During FY’84 p-xylene, toluene and phosgene exposures
were completed. Neurotoxicity, teratogenicity, immunotoxicity, hepatotozicity and biochemical
endpoints were investigated. Results indicated a concentration-related response of saccharine aversion
to p-xylene in rats. A teratogenic study on the effects of p-xylene exposure was completed. The effects
of subchronic exposure to toluene on bactericidal activity of alveolar macrophages and the resistance of
the host to bacterial infection were investigated.
Human exposures to toluene were itiltiated during FY’86. Pulmonary function and bronchial reactivity
to a methyicholine challenge were measured. Collaboration with the Environmental Monitoring Science
Laboratory is currently underway in development of an exposure system for nasopharyngeal uptake
studies of organic vapors in humans . These studies will provide significant data in determination of pul-
monary dose.
Neurotoxicological research has proceeded along two lines: (1) to develop and validate improved and
cost-effective laboratory methods; and (2) to evaluate the neurotoxic potential of hnardous air pollutants
(HAP) chemicals.
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ACCOMPLISHMENTS AIR HEALTH
1. Methods Development and Validation. Flash evoked potentials are commonly used to measure
neurotoxicity. Studies using discrete lesions in the visual cortex are beginning to elucidate the relation-
ships between neural structures in the visual system and individual components of the evoked poten-
tial. These data provide for a better understanding of the neurological basis of neurotoxicity produced
by HAP chemicals and have now been accepted for publication.
Studies to evaluate nervous system specific proteins (NSSP) as biochemical markers for neurotoxicity
continue to support its utility. A variety of NSSPs (synapsin I, GFAP, P-38, tubulin and N-200) have been
shown to respond to prototype neurotoxicants in a manner which is consistent with the accompanying
cytopathology thus indicating that NSSP can be used as biochemical indicators of neurotoxicity.
2. Neurotoxic Potential of HAP Chemicals. A report, “Thirty-day toluene exposure to rats: Experimental
evidence and exploratory studies,” was completed. This report describes a comprehensive evaluation
of rats exposed subchronically to either air or toluene (1000 ppm,6 hr/day, 5 days/week, for 30 days).
Results indicate that for this exposure regimen, toluene is not seriously debilitating to rats. Human
studies on neurobehavioral and pulmonary effects of toluene are continuing in the Chapel Hill facility.
A study to evaluate the effect of sulfolane (a high volume industrial solvent) on visual function was com-
pleted and submitted for publication. The results indicated that high dosages (112 LD5O) were required
to produce effects, thereby indicating that the visual system is not particularly sensitive to this compound.
These data are in contrast to previous work which has shown that neurotoxic effects such as increased sus-
ceptability to seizures are produced by lower dosages of sulfolane.
More cost effective and better predictive indicators of reproductive dysfunction are being developed to
evaluate potential HAPs. A series of papers were published in peer reviewed journals dealing with
reproductive effects of manganese in rats. The results indicated a delay in sexual development in males
with no apparent long term reproductive impact. An evaluation of age dependent gastrointestinal adsorp-
tion of MN304, which is a combustion product of the fuel additive methylcyclopentadienyl manganese
tricarbonyl (MMT), was conducted to evaluate the fate of inhaled particles translocated to the gut.
Results indicate highnr absorption and retention in young (preweaning) rats than in adults resulting in
greater exposure for younger animals and an increased possibility of toxicity.
Mutagethcity metabolism, bioavailability, dosimetry and DNA binding studies of several nitrated
polycyclic aromatic hydrocarbons, including 3-nitrofluoranthene and 1-nitropyrene, are being conducted
in whole animals and selected target cells and respiratory tissue. Lung intratracheal instillation studies of
the bioavailability of nitropyrene coated particles were completed. These studies show that nitropyrene
is rapidly released from the particles and appears in the blood within two hours after exposure. Compara-
tive mutagenicity and carcinogenicity studies on wood and coal combustion source emission samples are
in the final stages of completion. The two woodstove emission samples both showed a dose-related
tumorigenic response in the Sencar mouse skin tumor initiation-promotion assay. These two samples are
from an airtight woodstove burning hardwood (oak) or a soft-wood mixture.
Data analysis is in progress on bioassay data from the FY 85/86 sampling in Raleigh and Albuquerque as
part of the Integrated Air Cancer Project. Two manuscripts have been reviewed and submitted. Bioas-
say data has been used in the source receptor modeling analysis for the first time. Results from the Albu-
querque site show that on an average 50% of the ambient particulate mutagenicity was from woodstoves
and 50% from automobiles.
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AIR HEALTh ACCOMPUSHMENTS
Issue Provide monitoring systems, control technologies, and data on the health effects and atmos-
pheric proessses of mobile sourea pollutants to support the mobile sourea regulatol7 program.
A study on patients with ischemic heart disease who were exposed to carbon monoxide sufficient to raise
the blood carboxyhemoglobin (COHb) concentration to either 4 or 6% was completed in FY 1986. The
results indicate that, for this patient population in general, a threshold for COHb apparently exists be-
tween these concentrations since the investigators were unable to detect gnificant physiologic or
symptomatic duinges in these patients when the COHb was 4%; however, the symptoms reported were
significantly inereased when the COHb was 6% as compared to the responses following exposure to clean
air. An additional study in the Neurobehavioral Laboratory indicates that CO dimiithhes hand-eye cow-
dins tio n .
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Publications
Air Health
Radiation
This report includes abstracts of manuscripts for journal articles published and conference
proceedings papers presented between October 1, 1985 and September 30,1986...
Programm2tic Relevancy statements are italicized.
Other articles submitted for publication, but not published as yet, are listed in Appendix A.

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PUBLICATiONS AIR HEALTh
DESIGN and MEASUREMENT CONSIDERATIONS for EXERCISE
PROTOCOLS in HUMAN AIR POLLUTION INHAlATION STUDIES
S. M. Hoivath
University of California at Santa Barbara
Infonnation contained in this book chapter will assist other health scientists in the design of studies of the
health effects of exposure to air pollutants.
The impact on pulmonary functions of exercising at different intensities during pollutant exposures was
evaluated. It was apparent that there was considerable variation in exercise protocols. These variations
occurred in the miignitude of the exercise load, the duration of the exercise period, and the tiniing of the
exercise during exposure. It was also apparent that ventilation during rest and exercise was not always
determined; in many instances ventilation was estimated based on other criteria, such as heart rate. The
influence of ambient temperature conditions was frequently not considered. Determinations of pul-
monary functions were also made at various time intervals following exercise or subsequent rest periods.
All of these factors could result in some degree of misinterpretation of the consequences of pollutant ex-
posure.
CITATION: In: Inhalation Toxicology of Air Pollution. Clinical Research Considerations, R. Frank, JJ.
O’Neil, MJ. Utell, J.D. Hackney, J. Van Ryzin and P.E. Brubaker, eds., American Society
for Testing and Materials, 1985.
A104 XX-86-130 Project Officer: Horstman
DURATION of INCREASED PULMONARY FUNCTION SENSITIVITY
to an INITIAL OZONE EXPOSURE
John F. Bed4 Deborah M. Drechsler-Parks, and Steven M. Hoivath
University of California-Santa Barbara
The results from this study provide the Office of Air Quality Planning and Standards (OAQPS) with infor-
ination pertinent w the detemzination of the NationalAir Quality Standards (NA QS) for ozone.
The metabolic and pulmonary function effects were investigated in six non-smoking young adults who
were exposed for 2 hours (22°C WBGT) to: 1) filtered air (FA); 2)0.45 ppm ozone (DAYI); and 3) two
days later to a second exposure to 0.45 ppm ozone (DAY2). The subjects alternated 20-minute periods
of rest and 20-minute periods of bicycle ergometer exercise at a workload predetermined to elicit a yen-
tilatory minute volume (ye) of 27 Llmin (BTPS). Functional residual capacity (FRC) was determined
pre- and post-exposure. Forced vital capacity (FVC) was determined before and after exposure, as well
as 5 minutes after each exercise period. Heart rate was monitored throughout the exposure, and Ve,
oxygen uptake (Vo 2 ), respiratory rate (fR), and tidal volume (VT) were measured during the last 2
minutes of each exercise period. There were no changes in any variable consequent to FA exposure.
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AIR HEALTH PUBLICA11ONS
Both ozone exposures induced significant (PcO.05) decrements in FVC; FEV1.o (forced expiratory
volume in 1 second); FEV o (forced expiratory volume in 3 seconds); FE? 25-75% (average flow rate be-
tween 25% and 75% of FVC); and total lung capacity (TLC). The decrements following the DAY2 ozone
exposure were significantly greater than following DAY], and averaged 7.2 percentage points greater
than those following the DAY1 exposure.
CITATION: Journal of American Industrial Hj iene Association 46(12): 731-734,December 1985.
A104 XX-84-022 Project Officer: Horstmi’n
RESPIRATORY RESPONSES of VIGOROUSLY EXERCISING
CHILDREN to 0.12 PPM OZONE EXPOSURE
William F. McDonnel4 1 Robeit S. Chapman, 1
Margaret W. Leigh 2 , GeraldL Strope 2 , andAlbe,tM. Collier 2
‘HERL, USEPA, Research Triangle Park, NC
2 University of North Carolina School of Medicine
Epidemiologic studies had su ested that children may be more responsive to ozone exposure than corn-
parab y exposed adzdLc. This study was pe fornzed to measure the respiratory responses of heaviiy etercising
children exposed to 0.12 ppm ozone under the same conditions as adults exposed in a previous study and to
compare the results of the two groups. The finding that children did not appear to be more responsive than
adults is irnpo tant to theAgency’s musion of settingNazionalArnbientAir Quality S tandarde that adequate-
yprotec: the health of all population groups.
Changes in respiratory function have been suggested for children exposed to less than 0.12 ppm ozone
(03) while engaged in normal activities. Because the results of these studies have been confounded by
other variables, such as temperature or the presence of other pollutants or have been questioned as to the
adequacy of exposure measurements, we determined the acute response of children exposed to 0.12 ppm
03 in a controlled chamber environment. Twenty-three white males 8 to 11 yr of age were exposed once
to clean air and once to 0.12 ppm 03 in random order. Exposures were for 2.5 h and included 2 h of in-
termittent heavy exercise. Measures of forced expiratory volume in one second (FEy 1 ) and the symptom
cough were determined prior to and after each exposure. A significant decline in FEVi was found after
the 0.3 exposure compared to the air exposure, and it appeared to persist for 16 to 20 h. No significant
increase in cough was found due to 03 exposure. Forced vital capacity specific airways resistance,
respiratory frequency, tidal volume, and other symptoms were measured in a secondary exploratory
analysis of this study.
CITATION: American Review of Respiratory Disease 132(4): 875-879, October 1985.
A104 MS-85-067 Project Officer: McDonnell
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PUBLICATIONS AIR HEALTH
OZONE RESPONSE RELATIONSHIPS in HEALTHY NONSMOKERS
Thomas I. KuIIe Lany A Sauder, J. Richard Hebe4 and Mane D. Chathain
University of Maryland, Department of Medicine
This study examined the respiratory effects in heavi y exercising men of several levels of ozone above and
below the present National Ambient Air Quali y Standan’L This study is pwticularly impostant to the
Agency’s mission of setting a NAAQSJor ozone which will protect the public health. A previous study mdi-
cated that respiratory effects occwred following exposure to 0.12 ppm ozone. This present study found ef-
fects at 0.15 ppm but not at 0.10 ppm conflmzing that ozone does have some effect on the respiratory system
at or near the present NAAQS during heavy exercise.
Significant concentration responses were observed in PVC, FEV 1 , FEF 2 S.7 5 , SGaw, IC, and TLC in 20
healthy, nonsmoking volunteers exposed randomly to 0.00,0.10,0.15,0.20, and 0.25 ppm 03. In addition,
significant response changes for FVC, FEVi, and FEF25.75 were shown with time over the 2-h exposure.
Intermittent, heavy exercise (VE, 68 1/mm) lasting 14 niln was employed every 30 mm during exposure.
Inspection of the concentration and time response curves suggests that the threshold for the group
response is at or below 0.15 ppm 03. Six subjects experienced decreases >5% in FEVi or >1.5% in
SGaw at 0.15 ppm. This concentration is only slightly higher than the 1-h 03 National Ambient Air
Quality Standard. A dose-related response was also seen for cough, nose and throat irritation, and chest
discomfort. The work load, length of exposure, and individual sensitivity must be considered for estab-
liithing a safe 03 exposure level.
CITATION: Ame,ican Review of Respiratory Disease 132(1): 36-41, October 1985.
A104 XX-84-038 Project Officer: McDonnell
AIRWAY SENSITIVITY of ASTHMATICS to SULFUR DIOXIDE
Donald Horstman, 1 L. Jack Roger, 2 Howard Keh4 3 and Milan Haz cha 3
1 HERL , USEPA, Research Triangle Park, NC
2 Environmental Monitoring Services, Inc.
kiniversity of North Carolina-Chapel Hill
The results from this study provide the Office of Air Quality Planning and Standards (OAQPS) with infor-
mation peninent to the dezennination of NationalAir Quali y Standard for sulfur diaxide.
The purpose of this study was to describe for asthmatic subjects the distribution of individual bronchial
sensitivity to sulfur dioxide (S02). Subjects were nonsmoking male asthmatics (n = 27) who were sensi-
tive to inhaled methacholine. None of the subjects used corticosteroids or cromolyn sodium. Oral
medications were withheld for 48 hr, inhaled medications for 12 hr prior to all testing. Each subject par-
ticipated in four separate randomly ordered 10 mm exposures to 0.00, 0.25, 0.50 and 1.00 ppm S02 at
26°C, 70% relative humidity. During exposures, subjects breathed naturally and performed moderate ex-
ercise (Vs, normalized for body surface area = 21 1/rn 2 x miii). Before and 3 miii after exposure, specific
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AIR HEALTH PUBLICATIONS
airway resistance (SRaw) was measured by body pleth mograpby. Those subjects whose SRaw was not
doubled by exposure to 1.00 ppm were also exposed to 2.00 ppm S02. Dose response cutwes (relative
change in SRaw, corrected for dtange in clean air vs. S02 concentration) were constructed for each sub-
ject. Bronchial sensitivity to S02 [ PC(S02)], defined as the concentration of S02 which provoked an in-
crease in SRaw 100% greater than the response to clean air, was determined. Substantial variability in
sensitivity was obscrved for 23 subjects, PC(S02) ranged between 0.28 and 1.90 ppm, while for the
rem lining 4 subjects, it was greater than 2.00 ppm S02. The median PC(S02) was 0.75 ppm S02, and 6
subjects had a PC(S02) of less than 0.50 ppm. PC(S02) was not related (r = 0.31) to airway sensitivity
to methacholine.
CiTATION: Toxicology and Industrial Health 2(3): 1-25, September 1986.
A104 MS-84-065 Project Officer: Horstman
ELECTROPHYSIOLOGICAL ASSESSMENT of NEUROTOXICITY In
CHILDREN
DavidA. Otto
HERL, USEPA, Research Triangle Park, NC
Children are knoiwz to be more susceptible than adulLc to the neurotoxic effecLc of many chemicals such as
lewL This paper reviews elecfrophysiological test methods that ate cwrent y available for neuroloxicily test-
ing in children. Specific tests are described and the relevant neurotoxicolorj literature is revieweS This
methods review should be heipftsl in the de.cign of both intramural and rwnural neumta*ity studies in
children.
Electrophysiological measures appropriate for use in neurotoxicity testing in children are reviewed.
These methods include sensory evoked potentials (auditory brainstem, pattern-reversal visual,
somatoseusory) and event-related slow potentials (contingent negative variation and association/P300
wave). Selection criteria, strengths and weaknesses of individual tests are discussed. Slow potential and
auditory brainstem data from children exposed to lead are presented as examples.
CITATION: In: Mental Retardation, Neumbehavioral Toxicology and Teratology, S. Schroeder, ed., 1986.
AlOl MS-86-036 Project Officer Otto
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PUBLICATIONS AIR HEALTH
THE RELATIONSHIP of EVENT-RELATED BRAIN POTENTIALS and
LEAD ADSORPTION:
A REVIEW OF CURRENT EVIDENCE
DavidA. Otto
HERL, USEPA, Research Triangle Park, NC
This review supports the agency decision to phase down lead in gasoline contributes to Lead Criteria
Document updating and is directly relevant to ongoing cooperative agreements with the University of Cin-
cinnati (CR812875) and the University of Wisconsin (CR811664). These studies and the review seek to
clarify the neurobehavioral effect threshold level following neonatal or childhood lead esposure.
The results of hnm2n and glnim2l studies of the effects of lead adsorption on sensory evoked and slow
brain potenti i1s are reviewed. These studies of flash evoked potentials in rats produced conflicting
evidence of increased and decreased latencies, decreased amplitude, and increased or decreased corti-
cal excitability. Studies of slow wave voltage in children during sensory conditioning indicated a linear
relationship to blood lead level in two studies, an effect that could not be replicated in an independent
sample of children. Results of a fourth study indicated that slow voltage measures were more sensitive to
lead during active rather than passive conditioning. Conflicting evidence of lead effects on pattern-rever-
sal visual evoked potentials in children was found in three studies. Evidence of increased latencies of
brainstem auditory evoked potentials at blood lead levels above 25 1 igIdl were reported in two studies.
Sensory evoked potent Ic hold considerable promise as noninvasive, clinically valid, culture free
measures of the effects of lead exposure on sensory nerve conduction, but further study is needed in
humans and Rnims k to clarify inconsistencies in the limited existing literature.
CITATION: In: Lead Environmental Health. The Cu,rent Issues, 1986.
A119 MS-86-038 Project Officer Otto
5-YEAR FOLLOW-UP STUDY of CHILDREN with LOW-to-MODERATE
LEAD ABSORPTION: ELECTROPHYSIOLOGICAL EVALUATION
D.A. Otto, 1 G. Robinson 2 , S. Baumann 2 , S. Schmede, ,
P. Mushak 2 , D. Klein bawn 2 , andL Boone 2
1 HERL, USEPA, Research Triangle Park, NC
2 University of North Carolina School of Medicine
and School of Public Health
This study supports the agency decision to phase down lead in gasoline, contributes to Lead Criteria Docu-
ment updating and Lv directly relevant to ongoing cooperative agreements with the University of Cincinnati
(CR812875) and the University of Wisconsin (CR811664). These studies seek to clarify the
neurobehavioral effect threshold level following neonatal or childhood lead etposure.
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AIR HEALTH PUBLICATIONS
Forty-nine children aged 6 to 12 years were evaluated for residual effects of lead exposure using
psychometric, electrophysiotogical, and medical tests 5 years after initial assessment. The odgjnal range
of blood lead (PbB) levels was 6-59 (x = 28) pg dl the current range was 6-30 (x = 14) psJdl. A linear
relationship between PbB and slow brain wave voltage during sensory conditioning was observed at mi-
tial evaluation and at 2-year blow-np. No significant relationship between Ybli and slow wave voltage
during passive conditioning was found at the 5-year follow-up, although a linear increase in slow wave
negativity relative to the current PbB level during active condiiioning was suggested by exploratory
analyses. Another exploratory analysis revealed a igalficant linear relationship between the original PbB
levels and the latency of waves Ill and V of the brainstem auditory evoked potential The latency of both
waves increased as a function of original PhD. Increased latency of these waves is suggestive of subclini-
cal pathology of the auditory pathway rostral to the cocblear nucleus, although end-organ impairment
cannot be ruled out. No threshold for the effect of Pb on auditory function was apparent.
CITATION: Environmental Research, 38: 168-186,1985.
A119 MS-85-086 Project Officen Otto
SEPARATING the EFFECTS of LEAD and SOCIAL FACTORS on IQ
Stephen 11. Schroeder 1 Bathara Hawk 1 , DavidA . Ott ,),
Paul Mushak 1 , and RobertE. Hicks 1
1 University of North Carolina School of Medicine
2 HERL, USEPA, Research Triangle Park, NC
This study supportr the agency decision to phase down lead in garoline conbibutes to Lead Cñteria Docu-
ment updating and Lv tdy relevant to ongoing cooperative agreements with the Universi(y of Cincinnati
(CR812875) and the University of Wisconsin (CR811664). These studies seek to clafify the
neurobehaviora) effect threshold level following neonatal or childhood lead eiposuie.
Initial evaluations of 104 low-socioeconomic status black children screened by the local community health
departments in North Carolina showed significant effects of lead in the range 6-59 pzjdl on 10 after con-
trolling for concomitant social factors such as socioeconomic status, home environment, and maternal
10. The main concomitant variable was socioeconomic status, which was multicolinear with other social
factors. Five years later, when all blood lead levels were 30 gg/dl or less, lead effects on 10 were no longer
significant. The correlation between maternal and child 10, which had been suppressed initially in
children with higher lead levels, returned to expected levels when decreases in blood lead level occurred,
while concomitant variables remained stable over the 5-year period
CITATION: En vironmental Research 38: 144-154,1985.
AilS) MS-85-156 Project Officer: Otto
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PUBLICATIONS AiR HEALTH
TRIGGERING or a CYTOLYTIC FACTOR with TNF-LIKE ACTIVITY
FROM HUMAN MONOCYTES
Hillel S. Koren, 1 Karen P. McKbznon 2 andAllen
USEPA, Research Triangle Park NC
2 Duke University Medical Center
The model system described in this paper will be used to assess the effect(s) of pollutants on hwnan macro-
phages obtained by bronchoscopy and bronchoalveolar lavage. Macrophages are impolt ant cells for im-
munity c. microorganisms and tumors.
Monocytes have a nonoxidative met1i ni in of tumor cytolysis operative in COD and in normal monocytes
under anaerobic conditions. The absence of competitive inhibition provides negative evidence for a con-
tact-independent mech inism of cytolysis, confirmed by the demonstration of a soluble mediator secreted
independently of effector-target cell contact. This cytolytic monokine, a protein of apparent molecular
weight between 25,000 and 40,000, resembles TNF functionally and antigenicafly. Further work will be
required to demonstrate its biochemical identity, its precise mechanism of toxicity, and the extent of its
importance in vivo.
CITATION: 1n Cjeotoxic Lymphokines and Cancer Biological and ChemicalAspects, J.H. Ransom and
J. Ortaldo, eds., Hnm in Press, Clifton, NJ, 1986.
AlOl MS-86-148 Project Officer: Koren
OZONE INHIBITS PROSTACYCLIN SYNThESIS in PULMONARY
ENDOThELIUM
Mitchell Friedman, Michael C. Madden, D. Stephen Sawzder
Kenneth Gammon, Gilbert C White H and Lester Kwock
University of North Carolina School of Medicine
This study provules some insight into alterations of prostacycline synthesis in the lung after e.tposure to
short bursts of ozone. Frostacycline, a metabolite of arachidonic aci4 is a precursor ofprostaglandin. This
class of compounds elicit potent actions on pulmonaiy cell pemzeabili y , blood pressure, and blood clot
fomzation. It is not presendy known if continued alteration of these compounds, resulting from chronic
ozone exposure, would result in serious health problems. Research L v needed to investigate possible links
between acute effects as identified in this study and chronic illnesses.
The effects of ozone on lung arachidonate metabolism in vitro were studied in cultured bovine pulmonary
endothelial cells exposed for 2 hours to ozone in concentrations up to 1.0 ppm. A concentration-depend-
ent decrease in prostacyclin synthesis was found (90% decrease at the highest ozone level of 1.0 ppm).
The inhibition of prostacyclin synthesis was not due to a decreased release of arachidonic acid from
membrane lipids. We also crsiniined the hypoxic pulmonary vasoconstrictive response to 10% oxygen in-
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halation in anesthetized dogs in vivo after exposure to 1.0 ppm ozone for 1 hour. Pulmonary vascular
resistance was significantly increased after ozone exposure, siniibir to the fi di gs in dogs given in-
domethacin (IS mg/kg). The percentage change in the hypoxic pulmonary pressor response was similar
between the ozone exposure and indomethacin-treated groups, although due to the variance of the pul-
monary vascular resistance values during hypoxia the results did not reach statistical significance. These
results suggest that ozone inh th tion affects pulmonary endothelial arachidonate metabolism in vivo as
wellasin vitro.
CITATION: Plostaglandins 30(6): 1069-1083, December 1985.
AlOl XX-86-089 Project Officer: Goldstein
Ei ’IECTS of INHALATION of 0.12 and 0.25 PARTS PER MILLION OZONE on
the PROXIMAL ALVEOLAR REGION of JUVENILE and ADULT RATS
Brenda E. Bar r y, 1 Frede,ick I. Miller, 2 and James D. Crupo’
1 Duke University Medical Center
USEPA, Research Triangle Park, NC
The degpee of lunt injury caused by prolonged inhalation of low leveLs of ozone (03) is of interest since
wban en*onniental levels peiiodically reach 0.2 to a3ppm. Since the wra of the junction of the conduc-
tive and respfrutoiy regions of the lung has been repoited as the major site of injury due to 03 inhalation,
techniques were devised to speciflcal v study alveolar tissue from this region. One-day-old or fr-week-old
male TaLc were exposed to either 0.12 or 0.25 ppm 0312 hoursf day or to continuous room air for 6 weeks.
Ultrastiuciure moiphometi’ic analysis indicated significant changes in the alveolar epithelium of the
proximal alveolar region of all exposed animals. The hypeiplastic responses of the lungs of the juvenile abd
adult rats were similar except that the adults also showed mild interstitial inflammation which was con-
centration dependent. These results su est that low concentrations of 03 cause a chronic epithelial injury
Li the proximal alveolar region consistent ivith a pattern which could lead to chronic obstraclive lung dis-
ease even at ambient wban 03 concentrations. These findings bear upon the potential lack of a safety mar-
gin in the cun’ent ambient stan dard for 03 concentrations to which the public at laige is exposeS
The degree of lung injury caused by prolonged inh ib tion of low levels of ozone (03) is of interest since
urban environmental levels periodically reach 0.2 to 03 ppm. Since the area of the junction of the con-
ductive and respiratory regions of the lung has been reported as the major site of injury due to 03 inhala-
tion, techniques were devised to specifically study alveolar tissue from this region. One-day-old or 6-
week-old male rats were exposed to either 0.25 ppm 0312 hours/day or to continuous room air for 6
weeks. An additional group of 6-week-old rats were exposed to 0.12 ppm 03 for the same time period.
All lungs were fixed at the end of the exposure by intratracheal installation of buffered 2% glutaral-
dehyde. Cylinders of tissue containing a cross-section of a terminal bronchiole were punched out of lung
tissue slices using a sharpened cannula. These tissue cylinders were oriented, embedded in Epon, serial
sectioned until the first alveolar duct bifurcation was reached, and then thin sectioned for electron
microscopy. Qualitative ex uminiition of the tissue revealed little obseivable dam2gn to the proximal al-
veolar tissues. However, by ultrastructure morphometric analysis, significant changes occurred in the al-
veolar epitheium of the proximal alveolar region of all exposed aninl2I 5 . In the nhuuth exposed to 0.25
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ppm 03 from iday of age (juvenile pninials) , the number of type 1 epitheial cells doubled, their mean
surface area decreased 38%, and their mean thickness increased 24%. The number of alveolar type 2
epithelial cells increased, and the number of alveolar macrophages doubled. Adult animals exposed to
0.25 ppm 03 showed similar patterns of changes in the epithelium of the proximal alveolar region and in
addition had a doubling of interstitial macrophages, indicating a mild inflammatory stimulus in the inter-
stitium. Adult an ma1c exposed to 0.12 ppm 03 showed smaller, but statistically significant changes in the
alveolar type 1 epithelium, suggesting a relatively linear concentration-response relationship. The
change in number and size of type 1 cells in all exposed animals is consistent with an increased cell turn-
over rate due to prolonged 03 inhalation. These results suggest that low concentrations of 03 cause a
chronic epithelial injury in the proximal alveolar region and that the extent of these changes occurs in a
concentration-dependent manner, even at concentrations as low as 0.12 ppm. No statistically significant
age-dependent effects were found.
CITATION: Laboratory Investigation 53(6): 692, December 1985.
A104 MS-85-087 Project Officer: F. Miller
NITROGEN DIOXIDE EXPOSURE and LUNG ANTIOXIDANTS in
ASCORBIC ACID-DEFICIENT GUINEA PIGS
Gary E. Hatch, Ralph Slade Mary/one K Seigrade, and Andrew G. Stead
HERL, USEPA, Research Triangle Park, NC
A diet which is poor in vitamin C may increase sensitivity to nitrogen diau?de (N02), a common indoor and
outdoor air pollutant. Guinea pigs fed a diet which is deficient in vitamin Cfor 7 days or longer showed in-
creased lung damage when &posed to 5 ppm N02 for 3 hr. Vitamin E was found to be more easü)’
destroyed by N03 when vitamin C was lowereeL Lungs which were damaged by N02 synthesized another
protective agen4 glutathione. However the increased glutathione did not compensate for the lack of vitamin
C.
We have previously found that ascorbic acid (AA) deficiency in guinea pigs enhances the pulmonary
toxicity of nitrogen dioxide (N02). The present study showed that exposure to N02 (4.8 ppm,3 hr) sig-
nificantly increased lung lavage fluid protein (a sensitive indicator of pulmonary edema) only in guinea
pigs fed rabbit chow (a diet not supplemented with vitamin C) for at least 7 days, at which time lung AA
was about 50% of normal. The rabbit chow diet did not cause reduced body weight as did commercial
synthetic scorbutic diets, even when they were supplemented with AA. After 14 days of feeding rabbit
chow, lung AA was reduced to 15% of control At this time, a-tocopherol (AT) in the same lungs was
reduced to 85% of control, and lung nonprotein sulfliydryls (NPSH) were increased to 114% of control.
Exposure of the guinea pigs to N02 (4.5 ppm, 16 hr) increased wet lung weight and further altered the an-
tioxidants in deficient (but not normally fed) animals in the following manner: NPSH content was in-
creased to 130% of control, AT was decreased to 74% of control, and AA was increased from 15 to 50%
of control. These findings suggest that depletion of AA in guinea pigs removes an important defense
against N02. The lung appears to be able to partially compensate for the dietary lack of antioxidant by ac-
cumulating AA from other tissues and by increasing NPSH concentrations. However, sufficient exposure
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to N02 leads to oxidation of AT and pulmonary edema. Conditions in which N02 produced edema were
accompanied by only a slight consumption of AT and no detectable oxidation of lung AA or NPSH.
CITATION: Toncology and Applied Phwvnacology 82(2): 351-359, April 1986.
A104 MS-85-167 Project Officer: Hatch
EFFECTS OF SUBCHRONIC INHALATION of LOW CONCENTRATIONS of
NITROGEN DIOXIDE
I. THE PROXIMAL ALVEOLAR REGION of JUVENILE and ADULT RATS
Lln 7 Chang 1 ludithA. Graham, 2 Fredenckl. Miller, 2
Jean I. Ospi&4 3 and James D. Crapo 1
1 Duke University Medical Center
2 HERL, USEPA, Research Triangle Park, NC
3 Southern California Edison Company
As pa f of a la.vger investigation of the issue of age sensitivity to audants, a study of nitrogen diadde (NO,)
was completeS Both neonatal (1-day-old) and young adult (6-M*-old) luLs were exposed for 6 wk to an
urban pattern of N02 (O.Sppni, 23 hr/day, 7 days/wk on which were supeamposed spikes of 1.5 ppm N02
for 1 hr in the morning and afternoon for 5 dayslwk). Another group of adult rats were exposed to a higher
level of N02 (a continuous baseline of2 ppm with spikes to 6ppm). The structure of the region of the lung
known to be most sensitive to N02 (the prothnal alveolar region) was studied quantitatively using highly
advanced mo’phometric methods. Nitrogen dioxide caused morphometric changes; specifically epithelial
injwy was obse,ve4 with the higher concentration of NO 2 causing more effects. The adult rats were as sen-
sitive or more sensitive depending on the specific ei4 oint to N02 than were the rats exposed from I day
of age. This worlç as well as associated studies, imply that the developing lung is not at increased risk to the
effects of NO 2 . ALso this study is the first of its kind in which structural effects have been reported after ex-
posure to urban patterns of N02. The research will be useful hi reducing the uncertainties regarding the ef-
fects of low levels of N02 on an importa,u parameter and age sensitivity when EPA reevaluates the N02
NAAQS.
Inhalation of nitrogen dioxide (N02) produces injury to the epithelium of terminal airways and the alveoli
proximal to the airways. Techniques were devised to isolate alveolar tissue from this region for mor-
phometric studies to define the extent of alveolar septal injury caused by NO 2 . One-day-old and six-week-
old rats were exposed to either room air or 03 ppm N02 for 23 hr per day 7 days per wk for 6 weeks. An
additional group of 6-week-old rats were exposed to 2.0 ppm N02 for the same duration. Two daily hour
spikes to three times the background concentrations (03 to 13 ppm and 2.0 to 6.0 ppm) were applied
Monday through Friday. At the end of the exposure, rat lungs were fixed by intratracheally infusing buf-
fered 2% glutaraldehyde. Pieces of lung tissue were embedded in large plastic blocks which were sof-
tened with heat and thin (0.3 mm) sliced. Terminal bronchioles and their corresponding proximal al-
veolar regions were identified from the thin plastic slices, removed, and glued to cylindrical EM blocks
for thin sectionin& Morphometric analysis revealed that epithelial injury occurred in all exposed animals .
The juvenile rats which had been exposed to 03 ppm NO 2 since 1 day of age exhibited changes in the
characteristics of type II epithelial cells. These cells spread to cover more alveolar surface and became
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thinner. Adult animals exposed to 05 and 2.0 ppm N02 showed changes in alveolar macrophages and in
the alveolar interstitium in addition to changes in the epithelium. Animals exposed to 05 ppm N02
showed spreading and hypertrophy of type II epitheial cells. Those ‘nimiatc exposed to the higher con-
centrations of N02 had similar changes in type II epithelial cells and in addition showed an increase in
type I cell number. The type I epithelial cells were smaller and covered less alveolar surface area than
normal type I cells, suggesting a regenerating population of type I cells. These results suggest that
prolonged exposure to low concentrations of N02 can cause injury to the alveolar epitheliuni indicated
initially by spreading and hypertrophy of type II cells followed by differentiation into type I cells to com-
pensate and repair the injury. Adult rats were as sensitive or more sensitive to N02 injury than were
juvenile rats.
CITATION: Toxicology and Applied Phamiacology 83(1): 46-61, March 1986.
A104 MS-85-208 Project Officer: Gvlham
CONVERSION FROM RT-11 to MICRO-RSX for REAL-TIME
DATA COIL RCTION and ANALYSIS
MitcheilE. IGng
Northrop Services, Inc
This paper provides technical information relevant to the Pulmonary Functions Laboratory, computer ys-
ferns operations within the Inhalation Toxicology Division of the Health EffecLr Research Laboratory.
Many scientists with DEC microcomputers use the RT-11 operating system for the acquisition of real-
time data in the laboratory. For these researchers, the work required to learn a new operating system and
the time needed to reprogram software prevents them from upgrading their laboratory computer resour-
ces. However, there are several advantages in upgrading to Micro RSX, a multiuser, multitasking system.
Some advantages include: sharing of hardware resources by many experimental setups, multiterminfil
support for concurrent data analysis and reduced costs per experimental setup. This paper presents
simple techniques for converting Fortran/Macro-li single-user programs running under RT-i1 to run
concurrently under Micro-RSX. The concepts of using a shared common region that maps the I/O page
are introduced along with a simple method to use Macro-il routines from RT-1l with Micro-RSX. Tech-
niques for overlaying programs and setting up group global event flags for dual-task control will be II-
lustrated. Programs rewritten for Micro-RSX to acquire and analyze pulmonary physiology data will be
used as examples.
CITATION: In: Proceedings of the Digital Equipment Computer Users Society Symposium, April/May
1986.
AlOl XX-86-087 Project Officer: Costa
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DOSIMETRY MODEUNG of INHALED TOXIC REACTIVE GASES
John Oveilon and Fred Miller
HERL, USEPA, Research Triangle Park, NC
HERL Lc a co-sponsor of the Health Effects Institute (HEI). The chapter provides recommendations for
flaure HE! research and will guide and influence HErs decisions concerning dosimetry modeling by
promoting HERL and HFJ program coordination and reducing progrwn ovedap.
This chapter focuses on the physical, chemical, and biological processes and factors involved in the ab-
sorption of reactive gases. Emphasis is placed on the importance of these factors in developing dosimetry
models, special consideration being given to the role of lung fluids and tissues. Several dosimetry models
are discussed and illustrations of predicted results presented to demonstrate the application of the
models to the uptake of N02 and 03, and to demonstrate the use of models in determining the effects of
physical, chemical and biological parameters on dosimetry predictions. Gaps in our knowledge and un-
derstanding of the processes of dosimetry are pointed out and research recommendations made to in-
crease our understanding of the processes and to enhance the development of dosimetry models.
CITATION: Health Effects Institute, in press.
AlOl MS-86-197 Project Officer: F. Miller
INFLUENCE of BREATHING MODE and ACTIVITY LEVEL on the
REGIONAL DEPOSITION of INHALED PARTICLES and IMPLICATIONS
for REGULATORY STANDARDS
F. Miller, 1 T. B. Maitonen, 1 M. G. Menache 2 M. Lippmann, 3
A C. Grahani, 2 andD.M.Spektoi 3
1 HERL, USEPA, Research Triangle Park, NC
2 Northrop Services, Inc.
3 New York University Medical Center
Pwiiculate size-selective sampling is an impoitant consideration in determining ambient air quality stand-
aids. Hàzaid evaluations, as well as risk analyses, can benefit from an improved understanding of factors
affecting regional respiratoiy tract deposition of pailicles in man. Thoracic deposition and its component
pails were exwnine4 as a function ofpaniculase size, for ventilation iutes ranging from nonnal respiration
to hemy exercise in individuals who are habitual mouth breathers and in those who normally employ
oronasal breathing when minute ventilations exceed approthnatel v 35 L mind. Our ana ysas demonstrate
that the activity level of the exposed population should be taken into account to assess the potential health
consequences from ambient or workplace exposures.
Particulate size-selective sampling is an important consideration in determining ambient air quality
standards and threshold limit values for workplace exposures. Hazard evaluations, as well as risk
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analyses, can benefit from an improved understanding of factors affecting regional respiratory tract
deposition of particles in man. Here, thoracic deposition and its component parts are e nimined, as a
function of particulate size, for ventilation rates rangiiig from normal respiration to heavy exercise in in-
dividuals who are habitual mouth breathers and in those who normally employ oronasal breathing when
minute ventilations exceed approximately 35 Lmin 1 . Nonlinear regression techniques were used to fit
logistic models of the form Y = [ 1 + e a + Iog X 4 to data from tests measuring extrathoracic (ET) and
tracheobronchial (TB) deposition. X was defined as an impaction parameter (pd 2 Q) and as the
aerodynamic diameter of the particle (D ) for ET and TB deposition, respectively. The logistic models
yielded significantly improved fits of the experimental data compared to previously used linear regression
models. Our analyses demonstrate that the activity level of the exposed population should be taken into
account to assess the potential health consequences from ambient or workplace exposures.
CITATION: In: Proceedings of Inhaled Pc4icles VI, Cambridge, England, September 1985.
AlOl MS-86-082 Project Officer: F. Miller
MODELLING of the UPPER and LOWER RESPIRATORY SYSTEMS
A.LPatra
Northrop Services, Inc.
The toxic expression of inhaled particles and vapors u high y dependent on the region of the respiratory tract
on which the substance deposits or Lc absorbed. Silicone replica casts of the nose, laiynx trachea, and
tracheobronchial airways were constracted and described for humans and standrud laboratory animals to
allow more accurate dosimetric e.xirapolation of pulmonary toxici y data. Differences in anatomy and de-
pendent airflow and particle deposition across species were careful y quantitated with considerations for
s age and airway branching pattern. Studies such as this better refine the experimental data in animals
and their relationship to man for more accurate and defensible standard setting processes.
Casting of silicone rubber replica casts of upper respiratory airways including nose, larynx trachea,
and tracheobronchial airways is described for the hiim in and for standard laboratory nimilc . Mor-
phometric measurements were made on nasopharyngeal and tracheobroncb.ial airway casts. Cross-sec-
tional areas of nasopharyngeal airways were observed to be complex in nature; each species exhibited dif-
ferent branching patterns and numbers of lobes. Replica casts were used to understand anatomical dif-
ferences in nose and lung due to parameters such as age, sex airway branching patterns, and mor-
phometric dimensions. Effects of growth on niorphometric dimensions were shown to be highly sig-
nificant for use in extrapolating toxicological results from one age group to another. Species and strain
differences in morphometric dimensions were also observed. Use of replica casts for velocity distribution
pattern studies and for particle deposition efficiencies studies were investigated.
CITATION: In: Modeling of the Upper and Lower Respiratory Systems, Amit L. Patra, ed., Raven Press,
1986.
AlOl XX-86-122 Project Officer: F. Miller
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AMBIENT SULFATE AEROSOL DEPOSITION in MAN:
MODELUNG the INFLUENCE of IIYGROSCOPIC1TY
T. Wayne Manonen, 1 AndrewE. Barnet4 2 and Fredeñckf. Millers
1 HERL, USEPA, Research Triangle Park, NC
2 Northrop Services, Inc
Some ambient contwninents of health effects concern are hjgoscopic and will absorb water vapor hi the
humid environment of the human lung The uptake of Hi) will affect the deposition site and thus the toxic
potential of an inhaled aerosoL The mathematical model p.tsented hi th Is study will enable the Health Ef-
feas Research laboratory (HERL) to more accwuteiy predict regional aerosol deposition patterns within
the respinitory ba a, and thereby to better assess the threat to human health following eqoswe to certain
hjgoscopic, su ate pollutant aerosoLs.
Atmospheric sulfate aerosols [ IbSO4, (NH4)z 504, and NH4 HSO4 I are of international concern because
of their global prevalence and potential irritant or toxic effects on hnmanc . To assess hnsrds following
inhalation exposure, the total dose delivered to the human respiratory tract and its regional distribution
must be determined. The mass median aerodynamic diameter of the inhaled aerosol will influence the
sites of deposition in the respiratory tract. Atmospheric sulfate aerosols are hygroscopic and will have
changing particle sizes and densities as they absorb water vapor in the humid environment of the human
respiratory tract. Experimental and theoretical data that describe particle size as a function of tempera-
ture and relative humidity were used in computer subroutines of an aerosol deposition model in order to
calculate the dose dispersion of H2S04, (NH4)2 S04, and Nib HSO4 aerosols in man. Different
temperature and relative humidity environments that approximately correspond to nasal and oral breath-
ing were studied. The predicted deposition patterns are very different from those of nonhygroscopic
aerosols with identical inhaled mass median aerodynamic diameter values.
CITATION: Environmental Health Perspectives 63: 11-24, November 1985.
A119 MS-85-069 P.O. Martonen
ISOLATION and LONG-TERM CULTURE of RAT, RABBIT, and
HUMAN NASAL TURBINATE EPIThELIAL CELLS
Vernon E. Stee le and Julia T. Arnold
Northrop Services, Inc.
A method was developed to Lw/ate and maintain hi long-tenn culture nasal epithelial cells from rats, rab-
bits and hwnans. The method was developed as pan of the enrapolation program of the Inhalation
Toxicology Division of HERL With th Is technique, it Is possible to make direct in vitro dose-response
comparisons of the effects of chemicals hr man and animals. By llnldnghr vitro and hi vivo effects in
animals and cross-correlating to human in vitro effects, extrapolation becomes more possible, especi ally for
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chemicals that cannot be tested in man for ethical reasons and chemicals that deposit predomin antiy in the
nasal region, e.g. coarse mode paflicles, fonnaldehyde.
Nasal turbinate epithelial cells were isolated from rats, rabbits, and humans using either a surgical or an
in situ enzyme incul ’ation technique. The culture conditions that permit optimal cell attachment and
selective growth of the nasal epithelial cells were determined. These conditions will permit the long-term
culture of these cells where typically 2(1 to 30 population doublings were observed. Differences between
rat and human nasal epithelial cells were seen in substrate requirements, colony-forming efficiency, and
response to fetal bovine serum and bovine serum albumin. These methodology and results will permit
mechanistic studies of normal and abnormal cellular function and permit comparative response studies
between nasal epithelial cells from rats and humans .
CITATION: In Vitro Cellular and Developmental Biology 21(12): 681-687, December 1985.
A119 XX-85-054 Project Officer: Grpham
A NONPARAMETRIC VERSION of WILUAMS’ TEST for a
RANDOMIZED BLOCK DESIGN
Dennis E. House
HERL, IJSEPA, Research Triangle Park NC
The toxicity or other effects of various substances can be assessed by either in vitro or in vivo eJipen-
mails which have increasing doses of the substance, including a control orzei’o dose, as the major factor
in the design. Often, the various doses are given to the same subjec4 animal or batch of cells. One
method of statistical analysis of these e*perimenu is the Williams’ test which assumes a nonnal distribu-
tion of en’ors and equal variances in the vwious groups. However, in most HEPJ., studies these assump-
tions are invalid which makes the Williams’ test invalitt fn this papei . a new method of statistical analysis
r derived that is valid for the above described experiments when the noimality and equal vwiance assump-
tions are not met.
A large-sample nonparanietric version of Williams’ test for comparing increasing doses of a substance
with a zero-dose control response is given for observations from a randomized block experimental design.
The method is based on Friedman-type ranks. Williams’ test and the nonparametric equivalent for a one-
way design are briefly described. The validity of the new method is shown and a numerical example is
given.
CITATION: Biometrics 42(1): 187-190, March 1986.
A104 MS-85-158 Project Officer: House
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COMPARTMENT MODEL APPROACHES for ESTIMATING the
PARAMETERS of a CHRONIC DISEASE PROCESS UNDER CHANGING
RISK FACTOR EXPOSURE
K G. Manton, 1 E. Stallam 1 Al. Woodbuiy, 1 J. P. Creason, 2 and W. B. , 2
1 Duke University
2 HERL, USEPA, Research Trifingje Parlc NC
The contributing role of gases pai1iculate and other environmental cposw’es to disease and death Li cnti-
cal4 y impollanL Ye4 the level of ewasure changes over time. Part modeir have not allowed for this change
during the period covered in the stiuly. Thu model provides the mathematical and statistical procedure to
include changes in exposure to environmental pollutants over time and provides for cohort analysis. In this
report the model is fitted to U.S. lung cancer mo,wJity data The results for the younger male cohort sug-
gests a leveling off and probable decline in male lung cancer moJtali(y rate in the nearfuture. The ability to
include changes overtime in risk factors L i essential in evaluating the effect of en vironmental pollutants on
morbidity or mortality. The inclusion of cohorts in the analysis may identifr the direction of a change
before it Li measurable in the total population. This Li especially important in modeling the health effects
(cancer and other chronic diseases) from exposure over time to environmental pollutants including tosic
pollutants.
Compartment model approaches have been proposed for the analysis of the age incidence of specific
types of cancer. These models represented the age increase in incidence as the result of a compound
1i rd function where individual level risks were described by the Weibull hazard function and where the
population level hazard rate is a continuous mixture of the Weibull hazards. These formulations assumed
that the mixing function, which described differences in risk due to different exposure histories, was con-
stant after the age at which the model was first applied. In this paper we show how the mixing distribu-
tion can be allowed to change with time reflecting chiinging exposures. The model is fitted to U.S. lung
cancer mortality data where for recent male cohorts there appears to be di nging patterns of exposure
possibly related to recent declines in male smoking. The implications for future lung cancer mortality
trends in the U.S. are discussed.
CITATION: Computers and Biomedical Research 19: 151-169, Februaiy 1986.
A119 MS-85-204 Project Officer: Rigg n
LIMITED OCCUPATIONAL MORTALITY DATA and STATISTICS
Michaelj. Syinons, Donald L Doesfier, and Yang C Yuan
University of North Carolina School of Pubic Health
The general problem of the appropriate statistical analysis model for inteapretation of the mortality or mor-
bidity experience of a population cohort with a common occupational or environmental characteristic Li
considereS Choice of an appropriate control group L v discusseS Advantages and disadvantages of the
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standardized mostality ratio (SMR) and the propoitional and nonpropoiiional hazard models we
describeS The number and inspo,tance of potential applications for these results are increasing with the
growing interest in following studies of populations in prothni(y to environmental locations such as toxic
waste di sposal sites or nuclear power plants.
From principles of survival analysis statistical approaches are identified for a major problem with occupa-
tional mortality studies when few deaths, as with cause specific mortality, and no exposure data on the in-
dividuals are available. Specifically, this limitation is the general inadequacy of a geographic population
as a comparison group for employed populations. Qualitative aspects of the so-called healthy worker ef-
fect are briefly presented, while the essential features of statistical strategies for part of this issue are
sketched in detail. Although the occupation epidemiology literature focuses on selection bias as the
source of the problems with the standardized mortality ratio, the potential inadequacy of the proportion-
al h rda model for occupational mortality evaluations is emphasized here. A practical, nonproportion-
al h rds model, patterned after the slope-ratio design in bioassay, may provide a more sensitive ap-
proach.
CITATION: Irn Biostatistics: Statistics in Biorned ical , Public Health and Environmental Sciences, P.K.Sen,
ed., Elsevier Science Publishers, The Netherlands, pps. 195-218, 1985.
AilS XX-86-098 Project Officer: Creason
GEOGRAPHIC VARIATION in DECLINING ISCHEMIC HEART DISEASE
MORTALITY in the UNITED STATES, 1968-1978
1. RATES and CHANGE, WHITES AGED 35-74 YEARS
Wayne B. Dai& Ca. ’! G. Hayes, 2 Ma,i yn Knowles, 1 Wilson B. Ri an’ 2
John Van Bn en 2 andff.A. Tyroler 1
1 University of North Carolina School of Public Health
2 HERL, USEPA, Research Triangle Park, NC
Using maps of the continental United States, the percentage change in and the average rates of ischemic
hew! disease mortality for whites aged 35-74 are presented by county and state economic area for the penod
196 -7& These maps provide visual evidence of different average rates and different degrees of change
amoung these political and economic areas. Higher rates were most concentrated in the South and East.
Despite the well known geographic pattern for heart disease mortality, studies of the decline in ischemic
heart disease have not provided a comprehensive e, imin ’ tion of its spatial component. In this study, the
authors computed and mapped mean rates and per cent change in ischemic heart disease mortality for
whites aged 35-74 years, for the period of the Eighth Revision (1968-1978) of the International Classifica-
tion of Diseases. Visual evidence of clustering and markedly different spatial patterns were found for
mean rate and per cent change among the state economic areas of the United States.
CITATION: American Journal of Epidemiology 122(4): 657-672, October 1985.
A119 MS-85-O11 Project Officer: Hayes
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A MULTI VARIATE ASSESSMENT of METEOROLOGICAL INFLUENCES on
INHALABLE PARTICLE SOURCE IMPACTS
George D. Thwston 1 and John 1). SpengIei
‘New York University
2 Harvard University School of Public Health
The afli& identifies the sowres of fine and coarse inhalable particles at a site in Boston and investigates
their respective relationships with the weather, using both local swface observations and infonnadon
regarding the mov nent of large air masses. One of four proposed criteria is selected for use with these
specific t ,q,es of pollutants and weather data. The results should allow EPA researchers to refine measure-
ment techniques and projections of sho,t-teim changes in üth&ablepaflkle leveLs.
This paper identifies the sources of fine and coarse inhalable particles at a site in metropolitan Boston
and investigates their respective relationships to meteorological conditions. In this work, Principal Com-
ponent Analysis (PCA) is applied to: 1) particle mass elemental data; 2) coarse particle mass elemental
data, and 3) meteorological measurements (primarily collected at nearby Logan International Airport).
In addition to local surface observations, air mass trajectory information concerning each sampling day is
included in the meteoroLogical data set, allowing the consideration of air mass transport as one factor in
particle impacts. As part of these PCA analyses, four different objective component selection criteria are
examined and compared. The Sane test of elgenvalues is found to resuk in the most interpretable com-
ponents for the specific air pollution and meteorological data sets considered in this work.
CITATION: Journal of alinate and Applied Meteorology 24(11): 1246-1256, November 1985.
AU9 XX-86-116 Project Officer. Hayes
ESTIMATION of INDIVIDUAL OZONE EXPOSURES USING
MICROENVIRONMENT MEASUREMENTS
Qiades F. Contan4 Jr., Brenda M. Gehwi Thomas H. Stoclç
A fonso H. Holguin, and Pat,iciaA. Buffler
University of Texas Health Science Center
The ability of a computer model to estimate personal exposure to ozone given infonnation on ozone con-
centrations in three different environments and data on subjects’ movements through these environments
was testeS The rnodelpeifonned well mourn one-how estimates were 7-12 ppb below the observed per-
sonal erposwes. Sources of enor within the data were identified but deemed non-serious.
Estimates of an individual’s ozone exposure were generated for a panel of 51 asthmatic study subjects
using ambient ozone concentrations, the relationships observed between the ambient concentrations and
the concentrations found in each of two broad classes of environments and knowledge of the individual’s
movement through these locations. These estimates are compared to actual ozone concentrations ob-
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served during personal monitoring of 30 of the individuals. Earlier comparisons disclosed that the es-
timated hourly means were in much better agreement with the observed hourly means than were the am-
bient hourly values. The hourly maxima observed in the personal monitoring for each of three environ-
ments are compared to the exposure estimates. The ability of the exposure model to estimate average
hourly exposures for individu has been previously tested. In the present work, the ability of the exposure
model to estimate peak exposures within an hour is evaluated, using measurements of ozone concentra-
tion obtained at 5 minute intervals during the personal monitoring program as a standard. One-hour ex-
posure estimates based on fixed site measurements are evaluated as welL Sources of error within the data
set are identified but appear to not seriously affect the results. The exposure model produces one-hour
estimates which are 7-12 ppb below the observed personal exposures.
CITATION: In: Scientific Basis for Ozone and Other Photocheniical Oa dants Standards, H. Englund, ed.,
December 1985.
A104 XX-86-025 Project Officer: Hayes
PROBLEMS in the ESTIMATION of HUMAN EXPOSURE to COMPONENTS of
ACID PRECIPITATION PRECURSORS
Benjamin G. Fenis , Jr., and John D. Spengler
Harvard University School of Public Health
Problems in estimating human exposure to ambient air pollutants are discusseS The infeasibility of
measuring actual q,osure leveLs is described in detail. Specific problems include time spent indoors,
variety of particle sizes and chemical variation wnong particles. Conclusions point to the need to develop
lightweigh4 accurate, and reliable personal monitors. Findings emphasis the need and difficulty of control-
lingfor indoor exposures when studying the health effects of criteria pollutants.
Problems associated with estimation of hnm2n exposure to ambient air pollutants are discussed. Ideally,
we would prefer to have some indication of actual dose. For most pollutants this is not presently feasible.
Specific problems discussed are adequacy of outdoor monitors; the need to correct for exposures and
time spent indoors; the need to have particle size distributions described and the chemistry of the par-
ticles presented. These indicate the need to develop lightweight accurate and reliable personal monitors.
CITATION: Environmental Health Perspectives 63:5-9, November 1985.
A104 XX-85-067 Project Officer: Hayes
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DESIGN STRATEGIES for EPIDEMIOLOGIC STUDIES of ENVIRONMENTAL
IMPACTS on HEALTH
I. F. Goldstein
School of Public Health, Columbia University
D(fferent design strategies for epidemiologic studies of acute and chronic health effects are discussed and
examples used which illustrate the use of improved technologies to assess both osure and health respon-
ses. These technologic advances together with rapid developments in the field of biological markers
promise to close the gaps L ’ s our abiliiy to draw finn conclusions from environmental epidemiology studies
This paper describes epidemiologic designs and methods in studies of health effects of air pollution,
whose implications, however, can be extended to the detection of health effects of other environmental
exposures. Recent advances in measurement technology for the assessment of both exposure and health
responses and rapid developments in the field of biological markers of both exposure and health response
offer the opportunity to rectify some past weaknesses or gaps in our ability to draw firm conclusions from
environmental epidemiologic studies. Different design strategies for epidemiologic studies of acute and
chronic health effects are discussed with appropriate examples which incorporate the above mentioned
technological advances.
CITATION: In: Proceedings of the 79th Annual MeetingAir Pollution ContmlAssociation, Minneapolis,
MN, June 1986.
AlOl MS -86-107 Project Officer: Hayes
EFICECTS of AMBIENT SULFUR OXIDES and SUSPENDED PARTICLES
RESPIRATORY HEALTH of PREADOLESCENT CHILDREN
J.LW a r B. G. Fenis Jr WDockeiy,J.D. Spengler,
D. 0. Strom, and F. E. Speizer
Harvard University School of Public Health
Across sLy cities, fl quency of cough was significantly associated with levels of three different air pollutants
However, within each city no exposure/disease relationships were seen. Results sugest for preadolescent
children no reduction in puhnona y health from moderately elevated leveLs of air pollutants under study.
Though the preliminary results reponed here provide no evidence for revision of existing NAAQS ambient
standards for SO or suspended pw!icles, attention will be paid to future repoits from the Sir-Cities Study
when such revisions w made.
This paper reports results from an ongoing study of outdoor air pollution and respiratory health status of
children living in six cities in the eastern and midwestern United States. The study enrolled 10,106 white
preadolescent children between 1974 and 1977 in three successive annual visits to each city. Each child
received a spirometric cr mins ’tion and a parent completed a standard questionnaire. Of this cohort,
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8,380 children were seen for a second ex2mination one year later. An air pollution monitoring program
was begun in each community at about the time of the first e ramination. For this report, measurements
of Total Suspended Particulates (TSP), the sulfate fraction of TSP (TSO4), and sulfur dioxide (S02) con-
centrations at study- ffil ’ed outdoor stations were combined with measurements at other public and
private monitoring sites to create a record of TSP and S02 concentrations in each of 9 air pollution
regions during the one-year period preceding each e min tion and, for TSP, during each child’s lifetime
up to the time of testing Follow-up of these children is conthuing, and analyses of the longitudinal health
data along with the more extensive aerometric data collected in later years will aid in the interpretation
of these results.
CITATION: Amencan Review fRespi,utor Disease 133(5): 834-842, May 1986.
AlOl XX-86-123 Project Officer: Hayes
PERSISTENCE of PEAK FLOW DECREMENT in CHILDREN FOLLOWING
OZONE EXPOSURES EXCEEDING the NATIONAL AMBIENT
AIR QUALITY STANDARD
P. 1. Lioy, 7A. Vollnw* andM.Lippmaws
New York University Medical Center
Studying children exposed to ozone (03) while resident at a swwner camp near Mendham, New Jersey, re-
searchers found a general tendency for decreased lung function with increasing 03 exposure. The present
reanalysis of the Mendha,n data su est a persistent decrement in lung function lasting for as much as a
week after the end of a four day smog peiiod. Observed effects were small but statistically significant
though biological implications are not known. Though it is impossible to detemiine whether obse,ved ef-
fects were caused by 03 or acid aerosol; studies now underway may help resolve this question. However,
when changes in children’s lung function such as those reported here and elsewhere are repoiled at or below
levels of the cwrent NAAQS, then the 03 standards may need to be lowereS
Healthy, active young children in supervised summer recreational programs may be especially sensitive
to the effects of ambient ozone (03) for several reasons. First, they spend most of their time out of doors
during mid-day periods when the outdoor 03 concentrations are high and indoor concentrations are
lower. Second, they engage in vigorous physical activity, which increases their minute ventilation. The
activity increases the amount of 03 inhaled and its penetration into the distal airways where it has its
greatest effects on respiratory epithelia. Third, young children are much less likely than adolescents or
adults to have had significant exposures to m nctream cigarette smoke or airborne chemicals from oc-
cupational or hobby activities. Such exposures could ntsick or confound the effects of the ambient air pol-
lutant exposures.
CITATION: Journal of theAir Pollution ControlAssociation 35(10): 1068-1071, October 1985.
A104 XX-85-098 Project Officer: Hayes
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HEALTH EFFECTS of AMBIENT OZONE EXPOSURE in VIGOROUSLY
EXERCISING ADULTS
Robeit S. Chap man, 1 Beatrice!. Selwyn, 2 Thomas H. StocA 2
Robe,fJ. Hard , 2 FrwzcesA. chwt, 2
DanielE. Jenkins , and DennLsJ. Kotchma?
‘HERI.., USEPA, Research Triangle Park NC
2 University of Texas School of Public Health
3 ECAO, USEPA, Research Triangle Park NC
This study was designed to detemune the degree of association between day-to-day changes in ambient
ozone eqosure and con’esponding changes in lung function of adults after vigorous outdoor erercise. Thus,
this study provided a test in the ambient situation of research previous y conducted main y in the laboratomy.
It therefore provided infonnation relevant to the ozone standard-setting process.
The purpose of the analysis was to e mine the association of changes in ambient ozone concentrations
with changes in the pulmonary function (PFF) of healthy adults after exercising vigorously outdoors.
During May-October 1981,24 community residents (men and women) ran three miles twice a week some
time between 4:30 and 6:30 PM at a high school track near Houston, Texas. An air monitoring trailer
stood beside the track and monitored environmental variables continuously. The continuously
measured environmental variables in this analysis (ozone, temperature, and relative humidity) were
averaged over 15 minute intervals. Standard forced expiratory maneuvers were performed before and
after each run. Exercise exertion was standardized.
CITATION: In: Scientific Basis for Ozone and Other Photochemical Oxidants Standards, H. Englund, ed.,
Houston, Texas, November 1985.
A104 MS-86-020 Project Officer: Chapman
PEAK EXPOSURES to NITROGEN DIOXIDE and STUDY DESIGN to DETECT
THEIR ACUTE HEALTH EFFECTS
I. F. Goldstein and L R. Andrews
School of Public Health, Columbia University
Findings on continuous eq osure to N02 by peiwns cooking a meal on a gas stove are presenteL Also
reposied are peak N02 levels at different heights above the floor and at various distances from the stove
while range and oven are in use. The possible use of continuous N02 monitoming in a study of health ef-
fects from sho,t temi exposure to high levels of NO 2 among asthmatics and non-asthmatics Lv describeiL
Use of methods described here in future “natural eapenments” of N02 eaposure will allow the direct
measurement of acute health effects among humans and again emphasis the need to control for indoor ex-
posures when assessing the health consequences of e.*posure to criteria pollutants.
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Findingc on continuous exposure to nitrogen dioxide by persons cooking a meal on a gas stove are
presented. In addition peak levels of N02 at different heights above the floor and at various distances
from the stove while the range and oven are in operation are reported for 24 homes. A study design to
detect the health effects of short term exposures to high levels of nitrogen dioxide rnng ng from 0.20 ppm
to over 1.5 ppm on pulmonary function and respiratory symptoms in asthmatic and non-asthmntic sub-
jects is described. The continuous exposure to nitrogen dioxide of the study subjects before, during and
after cooking a dinner on a gas stove is determined using a continuous nitrogen dioxide monitoring instru-
ment. Lung function tests are performed and symptom questionnaires are ai lministered throughout the
study period to assess both health effects and changes in pulmonary function associated with the above
exposures.
CITATION: In: Proceedings of the 79th Annual Meeting Air Pollution Control Association , Minneapolis,
MN, June 1986.
AlOl MS-86-108 Project Officer: Hayes
RESPIRATORY SYMPTOMS and PEAK FLOW ASSOCIATED with INDOOR
and OUTDOOR AIR POLLUTANTS in the SOUTHWEST
MichaelD. Lebowit2 1 Cathannel. Holberg 1
Bwbara Boyer, 1 and Carl Hayd
1 University of Arizona Health Sciences Center
2 HE USEPA, Research Triangle Park NC
Dai y symptom diaries and pealc flows of erpelled air were obtained for 211 subjects over a two year period.
Data on outdoor and indoor pollutant levels, temperature and relative humidity were collected. Smoking
was correlated with indoor pafliculaze levels; indoor CO was correlated with gas stove use. Indoor and out-
door factors affected asthmatic symptoms, after controlling for age, se.i smoking and other ambient en-
vironmental va,iableL Findings underscore the importance of limits on ambient 03 and NO2 .
A symptom-stratified, geographic cluster sample of 117 middle class households was studied. Symptom
daily diaries and peak flows were obtained for 211 subjects over a two-year period. Indoor sampling in a
sample of houses was performed for 03 ,TSP, RSP, CO, temperature (T), and relative humidity (RH).
Questionnaires determined type of stove and number of smokers in all households. Ambient pollutants
(03, TSP, CO, NO 2 ), were monitored in or near the clusters, as were T and RH. Smoking in the household
was significantly correlated with TSP and RSP. Indoor CO was significantly correlated with gas stove
usage. Normal young adults under age 25 had daily peak flows (PEP) associated with outdoor 03 after
adjusting for other factors. Asthmatics’ PEF was associated with smoking, gas stove use and outdoor
N02, and with outdoor 03 and temperature, after controlling for other factors. Indoor and outdoor fac-
tors affected asthmatic symptoms, after controlling for age, sex, smoking and other ambient environmen-
tal variables.
CITATION: 12 Journal of the Air Pollution Control Association 35(11): 1154-1158, November 1985.
A119 MS-86-086 Project Officer: Hayes
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HARVARD’S INDOOR AIR POLLUTION/HEALTH STUDY
John I). Spengler, Margaret P. Ree4 Erik Lebre Bel-Hung Chang
James H. Ware, Frank E. Speizer, and Benjamin G. Ferns, Jr.
Harvard University School of Public Health
Researcheac describe subject sampling pollution monitoring and health swveillance techniques employed
in a study of indoor air pollution. mary fin dingr from the fl t city under investigation are reported
Design modifications for subsequent study sites are discussed alro. The paper illustrates the need for wad
complexities of control for confounding the role of indoor exposures to cigarette smoke, gas stove use and
similar e*posw s in studies ofwnbicntpollutants
An indoor air pollution/acute respiratory health study is being conducted by researchers at the Harvard
University School of Public Health. Upper and lower respiratory symptoms of 300 children living in
Watertown, Massachusetts, have been recorded on a daily diary by a parent. Every two weeks the parent
is called for the illness/wellness history. At the end of the month the calendar is returned by mail.
Families participated in this survey from the time of induction (after September 1, 1984) to August 31,
1985. Coincident with the symptom survey, indoor air pollution measurements are made in each home
and in the elementary schools of the participating student. Participants were selected by stratified ran-
dom sampling. The original pool of possible participants was all children in the 3rd and 4th grades from
Watertown who had been tested for lung function during the previous year. The questionnaire describ-
ing home characteristics provided information on type of cooking stove and number of smokers in the
home. Sampling was stratified by presence or absence of a gas stove and by presence or absence of a
smoker. Because previous work clearly shows unequal variance in indoor N02 and particle concentra-
tions within these strata the gas stove and smoker strata were oversampled. This paper reports the
preIiniin ry flndingc from the Watertown indoor survey. It describes the design modifications imple-
mented in the two cities currently being studied; Kingston, Tennessee and St. Louis, Missouri.
CITATION: In: Proceedings of the 79th Annual Meetbag Air Pollution Control Association, Minneapolis,
MN, June 22-27, 1986.
AlOl XX-86-112 Project Officer Hayes
EFFECTS of PASSIVE SMOKING on HEALTH of CHILDREN
Benjamin G. Fem Jr., 1 Jwne .y H. Ware Cathenne S. Berkey,
Douglas W. Docke,y, 1 Avron Spiro, 111 and FrankE. Speizer 1
1 Harvard University School of Medicine
2 Harvard University School of Public Health
Data regarding the effect of passive smo/drag on preadolescent children demonstrate an exposure-response
relationship between the number of smoke,v in the household and reported rates of a vari4y of lung ill.
nesses and .syinploms. Amount of air that children could expel in one second (FEV 1 ) and rate of growth in

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FEV 1 , were lower in children with smoking mothers, and these children were shorter than children of non-
smoldng women. The medical significance of the small differences reported remains to be defined.
Analysis of data on the effects of passive smoking obtained in preadolescent children from the Harvard
Six-Cities Study demonstrates an exposure-response relationship between the number of smokers in the
household and the reporting rates for doctor-diagnosed respiratory illness before age 2, history of
bronchitis, wheeze most days and nights apart from colds, and a composite of symptoms defined as the
lower respiratory index. Similiirly, when only the amount currently smoked by the mother was used, the
data indicated a relatively uniform increase in each of the reported diseases and symptoms. FEy 1 was
lower in children with smoking mothers compared to children of nonsmoking mothers. Rate of increases
in FEVi after adjusting for normal growth was significantly smaller in children of smoking mothers and
was related also to amount smoked. Notably the effect on level of FVC was not seen and this finding, con-
sistent in several studies, remI inc unexplained. Although children of smoking mothers were shorter on
the average than children of nonsmoking mothers, no on-going passive smoking effect on height growth
can be ascertained. All these differences are small and their medical sgnficance ren lins to be defined.
CITATION: Environmental Health Perspectives 62: 289-295, October 1985.
A119 XX-86-114 Project Officer: Hayes
INDOOR AIR POLLUTION and PULMONARY FUNCTION GROWFH in
PREADOLESCENT CHILDREN
Catherine S. Berkey, Iwnes H. Ware, Douglas W. Docker
Benjamin G. Ferns, Jr., and Frank E. Speizer
Havard University School of Public Health
Using Z834 children aged 6-10 years, the association of parental smoking and gas stoves with lung growth
and function was measured. Maternal smoking was associated with small but significant reduction in the
growth in lung volume and a slower increase in the amount of air that children could expel in one second.
From the available data researchers could not determine if thfferences in growth were due to current ex-
posures or to an effect of prenatal or early childhood etposure. The data did provide some evidence of an
association between gas stove exposure and lung function especially at younger ages. Possible effect of gas
stove exposure is relevant to the NationalAmbientAfr Quality Standard (NAAQS) for N02 though in itself
it does not provide strong evidence in support of the standard. The results emphasize the importance of
eliminating or controlling in the analysis for indoor exposures when studying the health effects of ambient
pollutants.
Results are reported from a study of the association between exposure to sidestream cigarette smoke or
gas stove emissions and pulmonary function level and growth rate of 7,834 children seen at 2-5 annual
visits between the ages of 6-10 years. Children whose mothers smoked one pack of cigarettes per day had
levels of forced expiratory volume in one second (FEVi) at age eight that were 0.81% lower than children
of nonsmoking mothers (pcO.0001), and FEVi growth rates approximately 0.17% per year lower
(p=O.OS). For a child of age eight with an FEVi of 1.62 liters, this corresponds to a deficit in rate of
change of FEVi of approximately 3 mI/annum and a deficit of 13 ml at age eight. Children whose mothers
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smoked one pack per day had levels of forced vital capacity (FVC) at age eight that were 033% higher
than children of nonsmokers (p = 0.12); however, their growth rates of FVC were 0.17% per year lower
(p = 0.04). Because few mothers changed their smoking habits during the course of the study, it was not
possible to determine whether the difference in rate of growth was due to current exposure or to an ef-
fect of prenatal and early childhood exposure on the course of development. The nuitg&tude of the effect
on early childhood exposure on the course of development. The magnitude of the effect on FEy, is con-
sistent with deficits in FEVi of up to 3% in early adult life due to childhood exposure to sidestream
cigarette smoke. The importance of this relatively nizall effect will be evaluated further through follow-
up of these children as they are exposed to other risk factors such as personal active smoking. The data
provide some evidence for an association between gas stove exposure and pulmonary function level, espe-
cially at younger ages, but no evidence for an effect of gas stove exposure on growth rate.
CITATION: American Journal of Epidemiology 123(2): 250-260, February 1986.
AlOl XX-86-113 Project Officer: Hayes
INDOOR AIR SAMPLING and MUTAGENICITY STUDIES RELATED to
EMISSIONS from UNVENTED COAL COMBUSTION
I. L Mumfor4 1 K H4lliams, 1 D. B. Ha r ri s, 2
.i. c cizuang 3 andM. Cooke 3
1 HERL, USEPA, Research Triangle Park, NC
2 AEERL, USEPA, Research Triangle Park, NC
3 Battdlle-Columbus Laboratory
The results demonstrate that the newly developed medium-volume sampler can be used for indoor air sam-
pling in the field to collect both particulate and semi-volatile organics. The percent of the o?ganic t1rac-
table matter and mutagenicity of the filter samples collected by both med-vol and hi-vol were comparable.
For the med-vol sampling most mutagenicily and the biologically active compounds of polycyclic aromatic
hydrocarbons were mainly detected in the filter (particulate) sample not in XAD (semi-volatiles) sample.
As result of this study, the med-vol was used not only in China Project but also used in the Integrated Air
Cancer Project and In doorAir Kerosene heater studies in the Agency. The performance of the sampler has
been very satisfactory in these studies.
The purpose of this study is to develop sampling strategies and bioassay methods for indoor air in homes.
The work reported here was conducted to prepare for a joint U.S.-China field study in Xuan Wei Coun-
ty, Yunnan Province, southern China, where the residents traditionally burn coal or wood for domestic
cooking and heating without flue ventilation. These residents are exposed daily to high levels of combus-
tion smoke and have unusually high lung cancer mortality rates, the women’s rate the highest in China,
and the men’s rate among the highest. This paper reports the chemical and biological characterization of
coal emissions from a simulated combustion laboratory. A medium-volume sampler (flow rate 0.11
m 3 /min) with a PMio size selective inlet, a filter, and an XAD-2 module was developed to collect the par-
ticulate matter and semivolatile organics. A high-volume particulate sampler with a PM 10 size selective
inlet was used for comparison with the medium-volume sampler. The percent organic extractable and
polycyclic aromatic hydrocarbon (PAN) content of the filter and XAD-2, as well as the distribution of the
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organic matter, where determined. The mutagenicity of the filter and XAD-2 samples was determined
by the Ames Salmonella mutagenesis assay. Most PAH compounds collected in the XAD-2 resin were
equal to or sm2Iler than 4-ring compounds and had much lower mutagenic activity than the filter sample.
The filter samples contained a wide range of compounds, including 3- to 7-ring PAils, and showed much
higher mutagenic activity than XAD-2 samples. The coal combustion emitted both direct and indirect
mutagens, most of which are frameshift mutagens.
CITATION: In: Proceedings of the 79th Air Pollution ControlAssocialion, Minneapolis, MN, June 1986.
A119 MS-86-190 Project Officer: Mumford
SELECTED ORGANIC POLLUTANT EMISSIONS from UNVENTED
KEROSENE HEATERS
G. W. Traynor,’ M. G.Apte, 1 H.A.Sokol 1
i. c. chuan 2 andJ. L. Mwnford 3
1 Lawrence Berkley Laboratories
2 Battelle-Columbus Laboratories
3 HERL, USEPA, Research Triangle Park, NC
This study conftnns the results of other studies, i.e., that the kerosene combustion process can emit PAHs
and nitrated-PAHs. In addition, kerosene heaters were found to emit many other organic compounds, in-
cluding aliphatic hydrocarbons, alcohols, and ketones; phtalazes; alkyl benzenes; and pentachiorophenol.
Additional stud ies are needed to evaluate the health effects of the em issions from the kerosene heater.
An exploratory study was performed to assess the semivolatile and nonvolatile organic-compound emis-
sions from unvented kerosene space heaters. A well-tuned radiant heater and a maltuned convective
heater were used for this study. Each heater was operated in a 27-rn 3 chamber with a prescribed on/off
pattern. Organic compounds were collected on teflon-impregnated glass filters backed by XAD-2 resin
and analyzed by gas chromatography/mass spectrometiy. Pollutant source strengths were calculated
using a mass-balance equation. The results show that kerosene heaters can emit polycydic aromatic
hydrocarbons (PAils), nitrated PARs; alkyl benzenes; pentachiorophenol; phtalates; hydro naph-
thalenes; aliphatic hydrocarbons, alcohols, and ketones; and other organic compounds.
CITATION: In: Proceedings of the 79th Air Pollution ControlAssociadon, Minneapolis, MN, June 1986.
AlOl MS-86-191 Project Officer: Lewtas
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THE EFFECTS of INHALATION of ORGANIC CHEMICAL AIR
CONTAMINANTS
on MURINE LUNG HOST DEFENSES
CathenneAranys,’ William I. O’Shet 1 Judith A. Graham, 2 and Frederick J. A(iller
1 IIT Research Institute
USEPA, Research Triangle Park, NC
The potential health hazards of exposure to threshold limit value (7LY) concentrations of acetaldehyde,
acrolein, propylene mide , chlorofonn, methyl chlorofonn, cwbom tetrachiodde, allyl chloride, methylene
chloride, ethylene bfchloride, perchloroethylene, benzene, pheno4 monochlorobenzene, and benzyl
chloride, vere investigated by single and multiple 3-hr inhalation e.*posures in mice to experimentally in-
duced streptococcus aerosol infection and puhnonaiy bactericidal activity to inhaled Kiebsiella
pneumoniae. Signiiftcant increases in susceptibility to respiratory streptococcus infection re obsewed
after a single 3-hr exposure to methylene chloride, perchloroethylene, and ethylene trichloride. For
methylene chloride and perchloroethylene, these exposure conditions also resulted in si ficantly decreased
pulmonary bactericidal activity. These effects implicate several toxic air pollutants, as immunotavie, at high
ambient or workplace concentrations.
The potential health hazards of exposure to threshold limit value (TLV) concentrations of acetaldehyde,
acrolein, propylene oxide, chloroform, methyl chloroform, carbon tetrachioride, allyl chloride,
methylene chloride, ethylene trichioride, perchioroethylene, benzene, phenol, monochlorobenzene, and
benzyl chloride, compounds which may be present in the ambient or work room atmosphere were hives-
tigated. The effects of single and multiple 3-hr inhalation exposures were evaluated in mice by monitor-
ing changes in their susceptibility to experimentally induced streptococcus aerosol infection and pul-
monary bactericidal activity to inhaled Kiebsiella pneumoniae. When significant changes in these
parameters were found, further exposures were performed at reduced vapor concentrations until the no-
measurable-effect level was reached. Multiple exposures on 5 consecutive days were then performed at
this concentration. Significant increases in susceptibility to respiratory streptococcus infection were ob-
served after single 3-br exposure to TLV concentrations of methylene chloride, perchioroethylene, and
ethylene trichioride. For methylene chloride and perchioroetbylene, these exposure conditions also
resulted in significantly decreased pulmonary bactericidal activity.
CITATION: Fundamental and Applied Toxicology 6: 713-720,1986.
AlOl MS-86-219 Project Officer: F. Miller
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SPECIES COMPARISON of ACUTE INHALATION TOXICITY of OZONE and
PHOSGENE
Gary E. Hatch, Ralph Slade Andrew G. Stea4 and Judith A. Graham
HERL USEPA, Research Triangle Park, NC
Improved extrapolation of animal toxicology data to humans is needed for risk assessment purposes of
E.P.A. One of the first questions to be asked in this area is “Can data be extrapolated between animal
species?” This paper attempted to answer this question by exposing rabbits, rats, guinea pigs, hamsters and
mice to the same concentrations of ozone and phosgene and comparing the effects of these gases on the
permeability of the lung epithelium. No simple relationship was observed between the body weights of the
animaLs and the concentration-response cwves obtained indicating that more advanced models are needed
which take into account nasal removal and differences in lung morphology.
A comparison of the concentration-response effects of inhaled ozone (03) and phosgene (COC12) in dif-
ferent species of laboratory animals was made in order to better understand the influence of the choice
of species in inhalation toxicity studies. The effect of 4-h exposures to ozone at concentrations of 0.2,0.5,
1.0, and 2.0 ppm, and to COd 2 and 0.1,0.2,0.5, and 1.0 ppm was determined in rabbits, guinea pigs, rats,
hamsters, and mice. Lavage fluid protein (LFP) accumulation 18-20 h after exposure was used as the in-
dicator of 03- and COCI2 -induced pulmonary edema. All species had similar basal levels of LFP (250-
350 paJml) when a volume of saline that approximated the total lung capacity was used to lavage the col-
lapsed lungs. Ozone effects were most marked in guinea pigs, which showed significant effects at 0.2 ppm
and above. Mice, hamsters, and rats showed effects at 1.0 ppm 03 and above, while rabbits responded
only at 2.0 ppm 03. Phosgene similarly affected mice, hamsters, and rats at 0.2 ppm and above, while
guinea pigs and rabbits were affected at 0.5 ppm and above. Percent recovery of lavage fluid varied sig-
nificantly between species, guinea pigs having lower recovery than other species with both gases. Lavage
fluid recovery was lower following exposure to higher levels of 03 but not COC12. Results of this study
indicate that significant species differences are seen in the response to low levels of 03 and COC12. These
differences do not appear to be related in a simple manner to body weight.
CITATION: Jounwj of Toxicology and Environmental Health 19:43-53, 1986.
AlOl MS-85-060 Project Officer: Hatch
PULMONARY BIOCHEMICAL EFFECTS of INHALED PHOSGENE in RATS
Serafino Frwich 1 and Gary E. Hatch 2
1 North Carolina State University
2 HERL USEPA, Research Triangle Park, NC
Phosgene is an important ambient air and industrial pollutant. This paper examined whether biochemical
changes in the lungs which have been observed previously following inhalation of ozone would also be ob-
served with phosgene inhalation. In most cases both gases produced similar biochemical effects, indicating
that these changes may not be specific to one type of gas but perhaps to tissue damage in generaL
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Three exposure regimens were used to study the time course of indicators of lung damage and recovery
to single or repeated exposures to phosgene (COC Ii). Rats were sacrificed (1) immediately or
throughout a 38-d recovery period after inhalation of 1 ppm COCk for 4-h, (2) at intervals during a 7-h
exposure to 1 ppm phosgene, or (3) at several time points throughout a 17-4 exposure to 0.125 and 025
ppm COCk (4 h/d, 5 d/wk) and during a 21-d recovery period. Regimen 1 revealed s ignificantly elevated
lung wet weight, lung nonprotein sulfliydryl (NPSH) content, and glucose-6-phosphate dehydrogenase
(G6PD) activity that stayed elevated for up to 14-4. A significant decrease in body weight and food in-
take was observed 1-d after exposure. Regimen 2 caused a slight depression in NPSH content but did not
affect G6PD activity. Regimen 3 nnnn1s showed sustained elevations in lung wet weight, NPSH content,
and (36PD activity after 7-d of exposure. No significant changes in these endpoints were observed for the
0.125 ppm COCk group. No consistent elevation in hydroxyproline content was seen at either exposure
concentration. Tight microscopic ennihiation of lung tissue exposed to 0.25 ppm COCk for 17-d
revealed moderate niultifocal accumulation of mononuclear cells in the centriacinar region. In summary,
exposure to COCk caused changes similar in most ways to those observed for other lower-respiratory-
tract irritants.
CITATION: Journal of To4cology and Environmental Health 19: 413-423,1986.
AlOl MS-86-067 Project Officer: Hatch
SUPERIOR COLLICULUS LESIONS and FLASH EVOKED POTENTIALS
FROM RAT CORTEX
Martha !. Barnes and Roben £ Dyer
HERL , USEPA, Research Triangle Park, NC
To understand ful’y the biological significance of changes in neurophysiological endpoinLs the
Neumtoxicolov Division of HERL must know the extent to which dIfferent brain regions contribute to
responses reconied from the sca4, or cona This paper indicates the effect of lesions in the superior col-
Ilculus, pan of the visual system on responses recorded from the visual cortex to aJlarh stimulus.
It is generally assumed that the primary response of the rat flash evoked potential (FEP) is activated by a
retino-geniculate path, and that the secondary response reflects input to the cortex byway of the superior
colliculus (SC) or other brainstem structures. In the present study, male Long-Evans rats were implanted
with monopolar screw electrodes placed over the left visual cortex and a pair of twisted monopolar depth
electrodes, which were used to produce electrolytic lesions, were placed in each SC. One half of the
animals did not receive the electrolytic treatment (controls). PEP waveforms were obtained from all
animals prior to treatment, and 2 and 5 clays after treatment. Histological analysis was performed to
verify electrode placement and determine lesion size. Electrolytic lesions resulting in massive destruction
of the SC produced no decrement in any portion of the rat PEP but did produce an increase in amplitude
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of the N2P3 component. The data show that the secondary response is not generated by Sc in rats, but
that SC may modulate amplitude of the response.
CITATION: Brain Research Bulletin 16: 225-230, 1986.
AlOl MS-86-010 Project Officer: Dyer
TOXICITY of PARTICLES EMITFED from COMBUSTION of WASTE
CRANKCASE OIL:
IN VITRO and IN VIVO STUDIES
J. L Mumfor4 1 G. E. Hatch, 1 R. E. Ha 11 2 M. A. Jackson, 2
R. G. Mernl4 Jr., 3 audi. Lewtas
1j j USEPA, Research Triangle Park, NC
2 Environmental Health Research and Testing. Inc.
3 AEERL, USEPA, Research Triangle Park, NC
This paper reports that the emission particles from automotive waste crankcase oil combustion contained
high leveLs of hea y metaLs, particularly Pb, and are highly toxic in in vitm and in vivo assays. These emis-
sion pwticles are comparable in overall pote&y to the most tozc combustion particles tested previously in
our laboratory. Several program offices, the Office of Air Quality Planning and Standards (OAQPS) and
the Office of Solid Waste (OSW) have been concerned that used automotive crankcase oil which may be
burned as a source of heat for garages, will be a serious source of toxic air pollutants. This report was the
result of research specifically requested by OSWand OAQPS of the Air and Energy Engineering Research
Laboratory (AEERL) and conducted in collaboration with the Health Effects Research Laboratory
(HERL).
The ever-rising cost of energy provides incentives for the utilization of waste crankcase oil (WCO) for
space heating. The potential health htl7llrds of emissions and waste products resulting from the combus-
tion of WCO are unknown. The toxicity of the emission particles and waste products from two different
types of burners, a Dravo atonth ing oil burner (AOB) and a Kroll vaporizing oil burner (VOB), are
evaluated using automotive WCO. Samples are characterized by performing elemental analysis and scan-
ning electron microscopy. Both burners emitted fine respirable particles. The AOB emission particles
contained high concentrations of toxic heavy metals, especially Pb. The VOB retained a significant
amount of heavy metals in the burner residue and emitted a much smaller quantity into the air. The
toxicity of AOB emis sion particles, VOB emiss ion particles, and VOB waste residue is evaluated in three
bioassay systems, i.e., a rabbit alveolar macrophage cytotoxicity in vitro assay, an intratracheal injection
infectivity assay, and a peritoneal irritancy test in mice. The emission particles from both burners and
leachate from VOB residue produce a dose-related reduction in viability and cellular adenosine triphos-
phate (ATP) in macrophages following 20-hr exposure. Mortality due to infection with aerosolized Strep-
tococcus sp. is not significantly elevated by intratracheal injection of AOB emission particles at 100
p .glmouse, whereas a similsir dose of VOB ash produced significant elevations in mortality (61.1%).
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Pmc ion particles from both burners are comparable in overall potency to the most toxic combustion
particles tested previously, with the exception of AOB particles in the infectivity assay.
CITATION: Fund WIIentOJ and Applied Toxicologj 7(1): 49-57, July 1986.
AlOl MS-86-026 Project Officer: Mumford
S9-DEPENDENT ACTIVATION of 1-NITROPYRENE and
3-NITROFLUORANTHENE
in BACTERIAL MUTAGENICITY ASSAYS
L M. Bal 1 K Williwns, 2 M. I . Kohan, 2 and I. Lewra?
1 University of North Carolina at Chapel Hill
USEPA, Research Triangle Park, NC
This paperprovides relevant infomwJion on the nwtagenici y and metabolism of two of the most impor-
tant NO PAHs ve have identified in ambient air and source emissions, 1-Nitropyrene and 3-NitroflUoiun -
thene.
Nitro-substituted polycycic aromatic hydrocarbons (NO2PAH) such as 1-nitropyrene (NP) and 3-
nitrofluoranthene (3-NFA), both widespread environmental mutagens, generally require activation by
bacterial nitroreductases for maximal expression of their mutagenicity in the Ames Salmonella histidine
reversion assay, addition of exogenous miimm 1iin drug-metabolbing enzymes (S9 fraction) decreases
their activity in this system. In contrast, in an assay of forward mutation (to 8-azaguanine resistance) with
Salmonella typhimudum strain TM677, without S9 NFA has relatively low activity (10 to 20 mutants per
i0 5 survivors at 5 p.g/ml) and NP only margin iHy doubles the background mutation rate. Addition of S9
protein enhances NP at 50 pg/ml and 80 to 100 x 10 for NFA at 5 to 10 /ml. Therefore generation of
active genotoidc intermediates in this forward mutation system may proceed through metabolic pathways
other than the previously-defined bacterial nitro-reduction. Previously identified oxidative metabolites of
NP are known to be mutagenic.
CITATION: In: Proceedings of the Third International Conference on Biological Reactive Intemtediates,
Pleason Press, New York, NY, 1985.
AlOl MS-86-035 Project Officcr Lewtas
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METABOLISM of 1-NITROPYRENE by CULTURED RABBIT ALVEOLAR
MACROPHAGES and RESPIRATORY TRACT TISSUES
Leon C. King 1 LoulseM. Ball Marcus Jackson, 2
Jeff P. Inmon and Joe/len Lewt&
1 HERL, USEPA, Research Triangle Park, NC
2 Environmental Health Research and Testin& Inc.
Polycyclic organic matter (POM) associated with combustion emLssions and urban air panic/es is car-
cinogenic and mutagenic and is thought to account for a significant ponion of the lung cancers attributable
to air poilulions. The research reponed in these manuscnpts provided data critical to elucidating the
trachea as a target organ for cwvinogenesis by combustion pa#icle emissions. The DNA binding data
provides unpoitant target cell doshneby data for nitrated po ycyclic aromatic hydrocarbons, impoMant con-
stituents of both products of incomplete combustion (P1Cc) and polycyclic organic matter (POMs) for
which the Office of Air Quality Planning and Standards (OAQPS) and OMSAPG need assessment data.
The metabolism of 1-nitro [ 14 qpyrene( 14 C-1-NP; 8.1 pM) was studied in cultured (20 hr) rabbit alveolar
macrophages, lung tissue, and tracheal tissue. Metabolites from the incubation medium and from the
macrophages and respiratoiy tract tissues were extracted and then analyzed and quantified by high-pres-
sure liquid chromatography. The following metabolites were detected in the lung and tracheal tissue in-
cubation medium: 1-nitropyrene-4,5-dihydrodiol, N-acetyl-1-aminopyrene, 1-aminopyrene, and 10-
hydroxy-1-nitropyrene. Nitropyrene phenols (4-, 5-, 6-, 8- or 9-hydroxy-1.nitropyrene) and 3-hydroxy-1-
nitropyrene were only detected in the lung and tracheal tissue and not in the incubation medium for these
tissues. Minor amounts of 1-aminopyrene and 1O-hydroxy-1-nitropyrene were detected in the macro-
phage incubation medium, and only minute quantities of 1-nitropyrene-4, 5-dihydrodiol, 1.aminopyrene,
and 10-hydroxy-1-nitropyrene were detected in macrophages. The total percentage of 1-NP metabolism
was significantly greater in the lung and tracheal tissue (28.0 and 23.0% of the recovered 14 C, respective-
ly) than in the alveolar macrophages (6.3% of the recovered 14 C). The tracheal tissue was found to have
the highest activity both in 1-NP metabolism and intracellular metabolite concentration. A major portion
of the 1-NP metabolites produced was released into the incubation medium. The majority of the meta-
bolites produced by tracheal and lung tissue, 70 and 84%, respectively, were ethyl acetate extractable.
The metabolites retained within the cells or tissues were also predominantly ethyl acetate extractable
rather than water soluble (83% for the macrophages and trachea, 95% for the lung tissue). The metabo-
lite profiles obtained demonstrate that metabolism by both nitro reduction and ring oxidation occurs in
respiratory tissue, and a degree of tissue specificity in the formation of metabolites exists. Ring oxidation
was demonstrated in the lung and tracheal tissue, but very little occurred in the macrophages.
CITATION: Toxicology and Applied Phannacology 82(2): 292-300, February 1986.
A109 MS-84-045 Project Officer: Lewtas
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BIOAVAILABILITY of 1. .NITROPYRENE from MODEL COAL FLY ASH and ITS
UPTAKE by ALVEOLAR MACROPHAGES
Judy L Mwnfor4 1 Silvestre B. Tejada 2 Marcus Jaclcron, 3 and Joellen Lewtas 1
1 }ffiRI, USEPA, Research Triangle Park NC
USEPA, Research TriangJe Paark NC
3 Environmental Health Research and Testing, Inc.
This paper is relevant toprogrwnmatic effoits (OHE.4 and OAR) to improve the assessment of human risk
from toxic air pollutants by improving the data input on dosimetry. This study showed that it is possible to
use 1-nitmp ne vapor.coated fly ash as a model for such dosimetry and bioavailabili(y studies. In this
case, the mutagenk 1-nitropyrene after being coated onto the pwticles was bioavailable in the culture
medium.
Alveolar macrophage cultures exposed to coal fly ash vapor-coated with 1-nitropyrene were used as a
model system to study the bioavailabillty and the uptake of a nitroaromatic hydrocarbon from coal com-
bustion emissions. Initially, 1-nitropyrene-coated fly ash and uncoated fly ash were e, imined for
cytotoxicity using rabbit alveolar macrophages and for mutagenicity in the Salmonella himunum plate
incorporation assay. The results were compared to determine the effects of vapor deposition. The dis-
tribution and recovery of 1-nitropyrene from macrophage cultures treated with coated fly ash were deter-
mined by acing a reverse-phase high-performance liquid chromatography-fluorescence method. 1-
Nitropyrene alone was not very toxic, nor did vapor deposition of 1-nitropyrene onto coal fly ash sig-
nificantly affect the toxicity of the fly ash. Most toxicity resulted from the orig n l, uncoated fly ash par-
ticles. 1-Nitropyrene after being coated onto the particles was bioavailable in agar and aqueous culture
medium. The coated fly ash showed mutagenic activity when the particles were tested directly, the un-
coated fly ash did not show mutagenic activity. 1-Nitropyrene recovery from alveolar macrophage cul-
tures exposed to the coated fly ash diminished as cell number increased. The rate of 1-nitropyrene loss
was 2.7 ngf 106 macrophages for medium and 4.1 ng/10 6 macrophages for the whole culture. The
mutagemc activity recovered from these macrophage cultures also decreased with increasing cell num-
ber.
CITATION: Environmental Research 40(2): 427-436, August 1986.
A109 MS-83-223 Project Officer: Mumford
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RAT LIVER SUBCELLULAR FRACTIONS CATALYZE AEROBIC BINDING
of 1-NITRO [ 14 C]PYRENE TO DNA
L. M. Ball 1 and!. Lewta?
1 University of North Carolina-Chapel Hill
2 HE USEPA, Research Triangle Park, NC
This paper provides insight on the mechanism of metabolic activation of a nitrated polyclycic aromatic
hydrocarbon (N02-PAH). In order to assess the potential toxicity and carcinogenici(y of N02-PAH’s
found in a,nbient air, the Office of Health and Environmental Assessment (OHE4) and the Office of Air
and Radiation/Office of Air Quality Planning and Standards (OAR/OAQFS) need basic information on
the metabolism and dosimet,y of these compounds in order to detennine the DNA dosimetiy resulting from
human &posure.
The recently characterized environmental mutagen and potential carcinogen 1-nitropyrene(NP) is known
to bind DNA inSaimonella typhimunu n , and also in anaerobic incubations catalyzed by purified xanthine
oxidase. In this study we show that rat liver S9 supernatant, microsomal and cytosolic subcellular frac-
tions are also able to catalyze the binding of 1-nitropyrene labeled with 14 C to calf thymus DNA in vitro.
In incubations conducted under air, S9 and microsomes from Charles River CD rats were the most active
fractions, and NADPH was required for maximum activity (25-100 pmole NP bound/mg DNA/mg protein
in 1 hr). S9 and microsomes had about one-fourth the activity under nitrogen, although less of this ac-
tivity was NADPH-dependent. Binding in cytosolic incubations was generally low (1 to 5 pmole NP/mg
DNA/mg protein in 1 hr), was somewhat enhanced under N and was more extensive in the absence of
NADPH. Treatment of rats (Harlan Sprague-Dawley) with the inducing agents phenobarbital (PB),
Aroclor 1254 (A), or 3-methyicholanthrene (3-MC) enhanced NADPH-dependent binding in aerobic S9
(2- to 5-fold) and microsomal (10- to 20-fold) incubations. The effects of induction regimen on binding
assays conducted under N were more equivocal: 3-MC produced a 2-fold increase in binding in both S9
and microsomes, while the other two agents decreased binding from 50 to 75%. These results indicate
that classic cytochrome P450 inducers were able to stimulate activation of NP, but that this activation is
not mediated solely by cytochrome P-450.
CITATION: Environmental Health Perspectives 62: 193-196, October 1985.
A109 MS-85-107 Project Officer: Lewtas
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NITROPYRENE: DNA BINDING and ADDUCT FORMATION in RESPIRATORY
TISSUES
M.A.Jackson, 1 L c g 2 L M. BaJ4 3 S. GhayounnanecIi 4
A. M. Jeffr ey, 4 and I. Lewtd
1 Environmental Health Research and Testing
2 j USEPA, Research Triangle Park, NC
3 lJniversity of North Carolina-Chapel Hill
4 Columbia University Health Science Center
Po ycyclic organic matter (POM) associated with combustion emtrsions and wban aL’ particles u car-
cinogenic and mutagenic and is thought to account for a significant portion of the lung cancers attributable
to airpollutions. The research reported in these man uscnptr provided data critical to elucidating the trachea
as a target organ for carcinogenesLs by combustion particle emissions. The DNA binding data provider un-
portent target cell dosimetry data for nitrated polycyclic aromatic hydrocarbons, important constituents of
both products of incomplete combustion (P1cc) and po ycyclic organic matter (POMs) for which the Office
of Air Qualizp Planning and Standardr(OAQPS) and the Office of Mobile Source Air Pollution Con-
trol(OMSAFC) need assessment data.
Binding of 1-nitro (‘ 4 C)pyrene (NP) or its nietabolites to cellular DNA and protein in cultures of rabbit
alveolar macrophages, lung tissue, and tracheal tissue was examined. DNA binding in tracheal tissue (136
± 18.3 pmole NP/mg DNA) was four to five times the levels measured in either lung tissue (38 ±9.4
pniole NP/mg DNA) or macrophages (26 ± 7.5 pmole NP/mg DNA). Adduct analysis of DNA isolated
from lung tissue incubated with 1.nitro [ H 3 ]pyrene in vitro resulted in the identification of 2 to 5% of the
NP adducts as C8-deoxyguanosine 1-aminopyrene. NP was also bound to cellular protein in tracheal th-
sue and lung tissue, and at a lower level in macrophages. Cocultivation of the macrophages with lung and
tracheal tissue decreased the DNA binding in tracheal tissue by 45%. Following intratracheal instillation
of diesel particles (5 mg) vapor-coated with 14 C-NP (380 ppm, 0.085 p.CWmg) particles into rats, 5-8% of
the radioactivity remained in the lungs after 20 hr. Most of the diesel particles were also deposited in the
lung. Examination of DNA and protein binding in this tissue showed 5 to 12% of the pulmonary 14 C
bound to protein and no detectable levels of 14 C bound to DNA.
CITATION: Environmental Health Perspectives 62:203-207, October 1985.
A109 MS-85-106 Project Officer: Lewtas

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COMPARATIVE CHARACTERIZATION of ORGANIC EMISSIONS from
DIESEL PARTICULATE, COKE OVEN MAINS, ROOFING TAR VAPORS, and
CIGARETI’E SMOKE CONDENSATE
A. Williams, 1( • Sparacino, 2 B. Petersen, 3 I. Bumgarner, 4
R. H. Jungers, 4 andJ. Lewtas 5
1 Environmental Health Research and Testing Inc.
2 Research Triangle Institute
3 Battelle Columbus Laboratories
4 EMSL, USEPA, Research Triangle Park, NC
5 HERL, USEPA, Research Triangle Park, NC
In order to develop strategies for requlating carcinogenic polycyclic organic matter (POM) in the ambient
au it is important to chemically characterize POM emissions and to identify the potential carcinogins in
these complex mixtures. The organic emissions characterized in this paper have also been extensively
evaluated for their mutagenic and carcinogenic activity in bioassay studies.
This paper reports the characterization of the extractable organics from diesel particle emissions com-
pared to other complex organics which have been reported to increase the risk of human lung cancer.
Class fractions of diesel, cigarette smoke condensate, roofing tar, and coke oven extracts were obtained
using liquid/liquid partitioning and silica gel chromatography. Capillaiy GCIMS was used to identify
compounds in each extract fraction. This manuscript reports the mass distribution after fractionation of
each extract, all identified fraction components and quantification of selected mutagenic and car-
cinogenic compounds.
CITATION: International Journal of EnvironmentalAnalytical Chemisby 26: 27-49, September 1986.
A109 MS-86-041 Project Officer: Lewtas
IDENTIFICATION and COMPARATIVE RISK ASSESSMENT of AIRBORNE
CARCINOGENS FROM COMBUSTION SOURCES
Joellen Lewtas, 1 Marcia G. Nishioka 2 and Bruce A. Petersen 3
1 HERL, USEPA, Research Triangle Park, NC
2 Battelle Columbus Division
3 Sciex, Inc.
This paper reviews research progress in identifying airborne carcinogens using short-term genetic bioassays.
The data presented and reviewed is important to the Office of Air and Radiation, Office of Air Quality
Planning and Standards in the assessment of air toxics.
The identification and assessment of airborne carcinogens has been significantly advanced by the use of
short-term genetic bioassays. Bioassay-directed fractionation coupled with new organic characterization
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methods has provided the tools needed to more efficiently identify potential carcinogens in complex com-
bustion emissions and urban air samples. These studies have shown that a significant portion of the
mutagenicity is found in fractions more polar than polynuclear aromatic hydrocarbons (PAHs). New
mass spectrometry techniques have been developed to identify and quantify the more polar mutagens in
these samples. The comparative mutagenicity and carcinogenicity of a series of combustion emissions has
been assessed using dose-response studies in bacteria, msinimiili in cells and rodents. This data base has
been used to develop a comparative potency risk assessment methodology for a series of combustion
emissions.
CITATION: In: Proceedings of the Technology Transfer Conference No. 6, Toronto, Ontario, Canada,
December 1985.
AlOl MS-86-042 Project Officer: Lcwtas
SURVIVAL CURVES and INCIDENCE of NEOPLASTIC and NON-NEOPLASTIC
DISEASE in SENCAR MICE
C i Cont4 N. aapp, 2 AJ.P. Xkin-Szant4 1 S. Nesnow, 3 and T. I. Slaga’
1 The University of Texas System Cancer Center
2 Oak Ridge Associated Universities
3 HERL USEPA, Research Triangle Park, NC
The SENCAR mouse has been used by HERL to develop an extensive data base on the tumorigenic
propeilies of environmental complex mixtures. This data base has been used to establish the “comparative
potency approach” to risk assessment with these mixtures which is being considered for use by EPA’S Office
of Air Quality Planning and Standards (OAQPS). This paper describes background studies on spon-
taneous tumor formation and sur&ival of this sensitive strain of mice which will provide invaluable infomsa-
tion for past and future studies with SENCAR mice.
The survival curves and the incidence of spontaneous diseases were studied in a population of SENCAR
mice, a stock derived by a selected breeding protocol for enhanced suseptibility to chemical car-
cinogenesis in the skin. SENCAR mice proved to be as long-lived as other mouse strains or stocks, in-
cluding one of their parental lines, Charles River CD-i. The most frequently occurring neoplasias in
SENCAR mice were lymphoma, myeloid leukemia and reticulum cell sarcoma. Other frequently occur-
ring neoplastic diseases included lung adenomas and carcinoma and mammary gland carcinoma.
However, the incidence of these tumors was not higher than the incidence in CD-i mice or other mouse
strains or stocks. A variety of non-neoplastic diseases, both infl mni toiy and degenerative, were also ob-
served in old mice. The most common were liver, spleen and kidney amyloidosis, pyelonephritis and
papillary necrosis. These data indicate that selective breeding for susceptibility to chemical car-
cinogenesis has not produced a concomitant increase in the incidence of spontaneous neoplastic and non-
neoplastic disease.
CITATION: CwvinogenesLr 6(11): 1649-1652, October 1985.
A1OIJL1O4 MS-86-052 Project Officer: Nesnow
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DEVELOPMENT of a COMPARATIVE POTENCY METHOD for CANCER
RISK ASSESSMENT of COMPLEX MIXTURES USING SHORT-TERM
IN VIVO and IN VITRO BIOASSAYS
Joellen Lewtas
HERL, USEPA, Research Triangle Park, NC
This paper describes a new comparative approach to cancer risk assessment of compl matures which has
been under development in the Office of Research and Development(ORD). The risk assessment process
L v crii’ical to all of the EPA program offices. Since the risk assessment process and the current guidelines
will be update4 it Lv imnpoMant for ORD to conduct and report on research to evaluate the assumptions
used in risk assessment.
A comparative potency method for cancer risk assessment has been developed based on a constant rela-
tive potency hypothesis. This method was developed and tested using data from a battery of short-term
mutagenesis bioassays, an maI tumorigenicity data and human lung cancer risk estimations. This data
base was developed for a series of complex mixtures including emissions from coke ovens, roofing tar
pots, cigarette smoke and automotive engines. The series of automobiles used in this study included both
diesel- and gasoline-powered vehicles. The assumptions inherent in this method are discussed, together
with the methods and data base used to test these assumptions.
CITATION: Toncologj and Industrial Health 1(4): 1 3-2O3, December 1985.
A109 MS-85-223 P.O.: Lewtas
COMPARATIVE POTENCY METHOD for CANCER RISK ASSESSMENT:
APPLICATION to the QUANTITATIVE ASSESSMENT of the CONTRIBUTION of
COMBUSTION EMISSIONS to LUNG CANCER RISK
Joellen Lewtas
HERL, USEPA, Research Triangle Park, NC
This paper reviews the comparative potency method for cancer risk assessment of complcc combustion
emissions. It Lv high y relevant to the Office of Air and Radiation/Office 0/Air Ouality Planning and Stan-
dards (OAR/OAQPS) to have the data presented in this paper which assesses the relative importance of
various combustion sources with respect to potential lung cancer risk
Combustion sources emit soot particles conta nng carcinogenic polycycic organic compounds which are
mutagenic in short-term genetic bioassays in microbial and m mmaIian cells and are tumorigenic in
animals. Although soot is considered to be a human carcinogen, soots from different combustion sour-
ces and fuels contain variable quantities of organic tars of somewhat different composition. This presents
a serious problem when assessing risk since it is not possible to collect hnm24n cancer risk data for all the
various possible exposures to combustion emissions. Furthermore it is impossible to collect human can-
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cer data for exposures to emissions from new technologies which have not previously been in the environ-
ment. The use of chronic animal testing for each new combustion technology developed would be
prohibitively expensive and time consuming. This problem has motivated us to develop and test a com-
parative potency risk assessment methodology which would utilize short-term mutagenesis and
tumorigenesis data. This method was developed and tested using data from a battery of short-term
mutagenesis bioassays, animal tuniorigenicity data and human lung cancer risk estimations. This data
base was developed for a series of complex mixtures including emktions from coke ovens, roofing tar
pots, cigarette smoke, and automotive engines. The assumptions inherent in this method are discussed
together with the methods and data base used to test these assumptions.
CITATION: Proceedings of International Environmental Mutagen Society Meeting, Stockholm, Sweden,
June 1985.
A109 MS-85-249 Project Officer: Lewtas
A STANDARD GUIDE for the SALMONELLA 7YPHIMURIUM/MAMMALIAN
MICROSOME TESTS for BACTERIAL MUTAGENICITY [ ASTM STANDARD]
L D. Clarton, 1 K E. Mosteim ans, 2 I. Allen, 3 A. Auletta, 4
E. Nest,nann, 5 E. Zeiger, 6 andl. McCann 7
1 }j USEPA, Research Triangie Park NC
2 SRJ International
3 Ainerican Cyananiid Co.
4 TEB, USEPA, Washington, DC
5 Environmental Health Directorate, Ottawa, Canada
6 National Institute of Environmental Health Sciences
7 Lawrence Berkeley Laboratories
The Ames test is often used by and requested by most program offices. Its proper use and intespretation,
therefore, is of major impoitance. This standard guide provides a definitive guide for analjaers and
reviewers of S. typhimwium mutagenicily data as well as for researchers who perfonn the test.
Since its development by Dr. Bruce Ames and his coworkers, the Salmonella typhimurium/mammalian
microsome mutagenicity assay has been used widely throughout the world. Many authors have suggested
various modifications and made recommendations in regards to this assay. Although the recommenda-
tions of a panel of experts was published 1979 by deSerres and Shelby, a committee of members of the En-
vironmental Mutagen Society (EMS) initiated this effort in response to the encouragement by the
American Society of Testing and Materials (Committee E47.09.O1) and because of new developments
within the field of microbial mutagenesis tests, but it is not intended for these recommendations to replace
or diminish the usefulness of presently available protocols and procedures.
CITATION: Proceedings of the American Society for Testing and Materials, 1986.
AlOl MS-86-220 Project Officer: Claxton
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BIOASSAY DIRECTED CHEMICAL ANALYSIS in ENVIRONMENTAL
RESEARCH
Dennis Schuetzle 1 and Joellen Lewtas 2
‘FORD MOTOR COMPANY
2 HE USEPA, Research Triangle Park, NC
Bioassay directed chemical analysis has become the leading methodology for identifying mutagens and car-
cinogens in complex mixtures. EPA scientists have been the leaders in developing this methodology as
reflected in this feature journal article describing the panciples, analytical logic and methodology for this
approach.
It became apparent in the late 1970’s that genetic bioassays could be used in combination with chemical
fractionation to greatly simplify the process of identifying significant mutagens in complex environmental
samples such as diesel particles, ambient air particles, complex fuels, woodsmoke, industrial effluents,
chemicals in drinking water, and commercial products. The use of short-term bioassay tests in conjunc-
tion with analytical measurements, constitute a powerful tool for identifying important environmental
contaminants. We have coined the terminology “bioassay directed chemical analysis” to best describe this
marriage of analytical chemistry and biology. The objective of this methodology is to identify key com-
pounds in various types of air pollutant samples. Once that task is completed, studies on metabolism,
sources, environmental exposure and atmospheric chemistry can be undertaken. The principles and
methodologies for “bioassay directed chemical analysis” are presented and illustrated in this paper. Most
of our work has been directed toward the characterization of ambient air and diesel particulates which
we will use as examples in this report to illustrate the analytical logic used for identifying the bio-active
components of complex mixtures.
CITATION: Analytical Chemist,y 58: 1060a, 1986.
AlOl MS-86-199 Project Officer: Lewtas
THE MUTAGENIC ACTiVITY of IRRADIATED C2H4JNO
MIXTURES in the PRESENCE of DIETHYLHYDROXYLAMINE
T. E. Kleindienst 1 , E. 0. Edney 1 , G. K Na,nie 1 , and L D. Cl&rton 2
1 Northrop Services, Inc.
2 HERL, USEPA, Research Triangle Park, NC
In order to understand the source of genotoxicants within ambient air it is necesswy to understand the role
of atmospheric transformation processes. By using the Salmonella mutagenicity bioassay it is possible to
monitor the creation and distmction of genotoxicants within irradiated ambient-like air. This research will
aid decision makers such as EPA’S Office of Air Quality Planning and Standardc to understand the source
and relevance of hydrocarbon species within ambient air.
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Ethylene/oxides of nitrogen nthctures were irradiated in the absence and presence of diethyl-
hydroxyismine. As previously reported, the presence of diethylhydroxybmbie inhibited the photooxida-
don of the hydrocarbon and nitric oxide and the onset of ozone formation. Once the diethyl-
hydroxylamine completely reacted, the ozone rose more rapidly, but to a lower level than in the absence
of diethyihydroxylamine. Peroxyacetyl nitrate was also produced with the addition of diethyl-
hydroxylamine. The reaction mixtures were tested for total mutagenic activity by gas-phase exposure to
Safrnonella typhimwium strain TAIOO. A sign ficantly greater mutagenic activity was observed in the ir-
radiated ethylene/diethythydroxylamine/oxides of nitrogen mixture relative to the irradiated ethylene/
oxides of nitrogen mixture. Only - 20% of the observed response could be accounted for by known reac-
don products.
CiTATION: Atmospheric Environment 20(5): 971-978, May 1986.
Al® MS-85-192 Project Officer: Claxton
SYNTHESIS and BIOLOGICAL ACTIVITY of a SERIES of
NITROSUBSTITUTED CYCLOPENTA-FUSED PAH
J. M. Goldzing L M. BaIj It Sangaiah andA. Gold
University of North Carolina School of Public Health
This paper reports the synthesis and mutagenic4y of a series of novel nitrated polycyclic aromatic hydrocar-
bona the cyclopenta-fused PAR nitrated on the eltheno bridge. The nitroPAR in general are potent bac-
terial mutagens which may contribute substantially to the genotoxici y of ambient air particulate matter.
The studies described hen indicate that this particular youp of nitroPAH are relatively inactive as
‘nutagens in the Ames Salm onella plate incorporation assay. These results contribute to the body of infor-
mation needed to establish valid relationships between structural features and mutagenic activity, which will
ultimate y provide a rational basis for assessment of the genotoxicity of novel compounds.
PAH containing a peripherally fused cyclopenta ring are genotoxically active in Salmonella and mam-
malian cells and have been identified in combustion emissions. Since the cyclopenta ring is Predicted to
be susceptible to electrophilic attack, nitrosubstituted cyclopenta-fused PAH may be formed in the
presence of NO’, particularly in diesel exhaust, and hence be present in the environment. Nitro deriva-
tives of cyclopenta (cd) pyrene and the cyclopenta-fused benzanthracene isomers were prepared by reac-
tion of the PA !! with dinitrogen tetroxide. The purified products were characterized by mass spectros-
copy, ‘H NMR, UV-vis and HPLC. In all cases, nitro substitution occurred on the cyclopenta ring in ac-
cord with expectation. Preliminary assay in £ typhimurium indicates that 4-nitro-cyclopenta(cd)pyrene is
inutagenically active without exogenous metabolic activation in nitroreductase-competent strains. The
high resonance stability of the nitrenium ion formed from the putative bydroxylamine intermediate may
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be correlated with the mutagenicity of these compounds. The results of this study have allowed us to test
this hypothesis and will aid in development of structure-activity relationships of nitroPAB.
CITATION: In: Froceedings of the Tenth International Symposium on Polynuclear Aromatic Hydrocar-
bons, W. M. Cooke & A. J. Dennis, eds., Battelle Press, Columbus, OH, October 1985.
AlOl MS-86-211 Project Officer: Lewtas
METABOLISM and ACTIVATION PATHWAYS of the ENVIRONMENTAL
MUTAGEN 3-NITROFLUORANTHENE
Louise Al. Ba 14 1 Michael I. Kohan, 2 Kay William?
MG. Nishioka, 3 Awarn Go 14 1 and Joellen Lewtas
1 University of North Carolina-Chapel Hill
2 HERL, USEPA, Research Triangle Park, NC
3 Battelle-Columbus Laboratories
The results described in this .syniposium paper conflnn that 3-nitrofluoronthene, an identified air pollutant,
is a potent bacterial mutagen. These results also indicate that its activation in bacterial assays is not
mediated solely by the bacterial nhtroreductase enzymes. Frelimina y identification of mainmaliwz metabo-
lites of 3-nitrofluoranthene is also repoMed. Such studies will eventually allow characterization of the
mutagenici(y of these metabolites, and of specific mammalian activation pathways for this compound.
This will aid in assessment of the potential genotoxic risk of this air pollutant to mammalian organisms,
and to human health.
Nitro-substituted polycydlic aromatic hydrocarbons are widespread environmental contaminants that are
mutagenic and potentially carcinogenic. 3-Nitrofluoranthene (3-NFA) has been found in diesel and am-
bient air particulate extracts; although less abundant than its isomer 1-nitropyrene (1-NP), its content in
these extracts correlates well with their direct-acting mutagenic potency in the Ames assay. 3-NFA is
more mutagenic towards Salmonella TA98 than 1-NP both without (2 500 His + rev/nmol vs. 350
rev/nmol) and with exogenous metabolic activation (250 vs. 60 rev/nmol). 3-Aminofluoranthene was less
active than 3-NFA both with and without S9 (100 and 40 rev/nmol respectively); 3-acetamidofluoranthene
was even less active with S9 (60 rev/nmol), and quite inactive without S9. In contrast, 1-acetamidopyrene
(400 rev/nmol +S9) was more active than the parent 1-NP. The activity of 3-NFA was substantially
reduced in the nitroreductase-deflcjent strains of TA98 (TA98NR and TA98/1,8-DNP 6 ), indicating that
the “classical” nitroreductase and the transacetylase enzymes are involved in activation of 3-NFA. The
presence of S9 reduced the activity of 3-NFA in all assays except the forward mutation assay in strain
TM677, where it was substantially enhanced. 3-NFA is readily metabolised by rodent hepatic subcellular
fractions (at rates of the order of 25 nmoles/mg protein/br). Hydroxylated NO2PAH in an ambient air
particulate extract have been tentatively identified as 3-NFA derivatives, and contribute significantly to
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the mutageni-city of this extract. Therefore as with 1-NP, oxidation may play a role in metabolic activa-
tion of 3-NFA.
CITATION: In: Proceedings of the Tenth International Symposium on Po ynuclear Ammatic Hydrocar-
bons, edited by W.M. Cooke and AJ. Dennis, Battelle Press, October 1985.
AlOl MS-86-127 Project Officer: Lewtas
A RETROSPECTIVE REVIEW of the VALUE of SHORT-TERM GENETIC
BIOASSAYS in PREDICTING the CHRONIC EFFECTS of DIESEL SOOT
J. Lewtas and K lliams
HERI., USEPA, Research Triangle Park NC
This paper puts into perspective for all air program offices the utili y of short-tenn bioassays. This paper
brings into a much clearer focus that shoit-teim mutagenici y bioassay: are impoitant in ana ysLs proce-
dures because they are (1) capable of detecting and identifying carcinogenic tnateria4 (2) used in the iden-
tification of specific carcinogens Within coinpia materials, (3) capable of demonstrating the bioavailability
of airborne carcinogens, and (4) can be used to compare technologies, pollution control devices, etc.
In retrospect, it is now safe to conclude that short-term mutagenicity assays were not only useful but in-
strumental in: (1) indicating that diesel soot was potentially carcinogenic and should be evaluated in
chronic animal cancer bioassays, (2) identif 4ng N02-PAH5 as potential carcinogens in this very complex
mixture, (3) providing initial evidence that the mutagens were bioavailable, and (4) estimating the relative
importance of various sources and fuels and other factors which can influence human exposure to car-
cinogens. This is not to say that short-term bioassays used alone can accomplish all of this. However,
used in combination with chemical/analytical methods and toxicological tools, short-term genetic bioas-
says have become a critical component of many environmental health studies. Although substantial ad-
vances in our knowledge of the toxicology of diesel emissions have been made since 1978 when the initial
observation that the organics extracted from diesel soot were mutagenic, a number of important questions
remain not only for diesel emissions but for other combustion sources as well. Are the chemicals which
induce positive results in the short-term bioassays the same agents which cause tumors in chronic animal
bioassays? Which phase of the diesel emissions (gaseous or particulate) is carcinogenic in the animal in-
halation studies? With advances in our understanding of the molecular mechanisms involved in produc-
ing chronic effects such as cancer, it is possible that new genetic tools and short-term bioassays will con-
tinue to contribute to our ability to answer these and other questions as they arise.
CITATION: In: Proceedings of International Symposium “Toxicological Effects of Emissions from Diesel
Engines”, Tsukuba Science City, Japan, July 1986.
AlOl MS-86-217 Project Officer: Lewtas
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EFFECTS of 60-HZ FIELDS on HUMAN HEALTh PARAMETERS
Marj R. Coolç Cwl M. Maresh, and Hwvey D. Cohen
Midwest Research Institute
The study repoifed here used quantitative exercise testing techniques to evaluate whether increases in meta-
bolisni caused by moderate steady-state exercise pnor to exposure to real or sham fields, would ciwify
potential field effects. The findings indicated that the physical recoverj process following moderate steady-
state exercise performed under no-exposure conditions, was the same in real and sham fields. Only statisti-
cally significant increase in cardiac inrerbeat inteival (IBI) (equivalent to a 3 beat/mm decrease in hewi
rate) was found when subjects were exposed to the real field for 2 hr after sitting quietly pnor to erposure.
This replicates earlier research, in which IBI was increased following a longer penod of field e*posure. The
results su est that future studies should examine a broader range of the continuum of human arousal and
physiological activation.
Research on the effects of exposure to 60-Hz electric and magnetic fields has provided contradictory
evidence for both increases and decreases in physiological and metabolic functioning, and specific results
have often been difficult to replicate. If biological responses to powerline fields occur, they are undoub-
tedly subtle, and research strategies must be specifically designed to enhance and clarify subtle effects.
The study reported here used quantitative exercise testing techniques to evaluate whether increases in
metabolism, caused by moderate steady-state exercise prior to exposure to real or sham fields, would
clarify potential field effects. This research showed that physical recovery processes following moderate
steady-state exercise were the same in real and sham fields. Of the variables e,ainined, only heart rate
(cardiac interbeat interval) was altered by 2 hr of field exposure. A small, statistically significant decrease
in heart rate (3 beats/mm) was found when subjects were exposed to the real field after sitting quietly
prior to exposure. This replicates our earlier research, in which heart rate showed a similar decrease after
a total of 6 hr of field exposure. The results suggest that future studies should examine a broader range
of the continuum of hiinian arousal and physiological activation.
CITATION: U.S. Environmental Protection Agency, Fmal Report of Cooperative Agreement No. CR-
812805, EPA-600/1-86-006, September 1986.
F104 HERL-0551 Project Officer: Blackman
A ROLE for the MAGNETIC FIELD in the RADIATION-INDUCED EFFLUX
of CALCIUM IONS from BRAIN TISSUE IN VITRO
C. F. Blackman, S. G. Benane, J. 11. Rabinowitzj D. E. House,
and W. T. Joines
HERI., USEPA, Research Triangle Park, NC
This manuscript represents a major breakthrough for clues to the mechanism for a non-thennal process in
radiofrequency-radiation biological-effects work As such, it has garnered favorable publici(y for EPA’S
scientific programs as well as demonstrated to the EPA radiation progrwn office the correctness of caveats
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hi the bioeffects Review Document and hi the Notice for Proposed Recommendations. ScientLits in other
laboratories are now adjusting their wodcing hypotheses to include the new results. Basically, the finding is
that to cause biological change, various frequencies of radiofrequency radiation must be co-mired with one
of a number of speciftc intensifies of the earth’s magnetic flelat This requirement appears to explain some
of the problems encountered hi previous attempts to replicate repo’tr of non-thermal effecz while also
providing thefl substantial infonnation upon which to devise and test modeLs for the mechanism of ac-
tion.
Two independent laboratories have demonstrated that electromagnetic radiation at specific frequencies
can cause a change in the efflux of calcium ions from brain tissue in vitro. In a local geomagnetic field
(LOP) at a density of 38 microTesla ( 1 ST), 15- and 45-Hz electromagnetic signaEc (40 Vp p/m in air) have
been shown to induce a chsinge in the efflux of calcium ions from the exposed tissues whereas 1- and 30-
Hz signals do not. We now show that the effective 15-Hz signs 1 can be rendered ineffective when the LOP
is reduced to 19 with Helmholtz coils. In addition, the ineffective 30-Hz signal becomes effective
when the LOP is changed to ± 253 T or to ± 76p.T. These results demonstrate that the net intensity of
the LOP is an important variable. The results appear to describe a resonance-like relationship in which
the frequency of the electrornsgnetic field that can induce a change in efflux is proportional to a product
of LOP density and an index; 2n +1, where n = 0,1. These phenomenological findings may provide a
basis for evaluating the apparent lack of reproducibility of biological effects caused by low-intensity ex-
tremely- low-frequency (ELF) electomagnetic signatc . In future investigations of this phenomenon, the
LOP vector should be explicitly described, If the underlying mechanism invokes a general property of
tissue, then research conducted in the ambient electromagnetic environment (50/60 Hz) maybe subjected
to unnoticed and uncontrolled influences depending on the density of the LOP.
CITATION: Bioelectronzagnetics 6(4): 327-337, December 1985.
P104 MS-85-070 Project Officer: Blackman
NON-ELECTROMAGNETIC FACTORS INFLUENCE BEHAVIORAL
EFFECTS of MICROWAVE EXPOSURE
Michael I. Gage
HERL, USEPA, Research Triangle Park, NC
The report reviews a num bet of experiments conducted over several years indicating parameters of exposure
to electromagnetic energy such as the air temperature, duration of exposure, animal eaposec4 and orienta-
tion of the animal in the field may quantitativeap alter the observed effect. There results su est ambient:
conditions must be considered hi developing stan daMs for allowable esposure to electromagnetic rad iation.
These results alto illustrate a general principle of the need for caution h i the esapolation of toxicological
data obtained under a limited set of environmental conditions.
Alteration in snimsi behavior both during and after microwave irradiation is well documented. This
report reviews a number of experiments in which specific behavioral effects in laboratory rodents were
quantitatively changed by aspects of the exposure such as ambient air temperature, duration of the ex-
posure, and orientation in the field while exposure parameters of 2450 MHz (CW) electromagnetic radia-
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tion were kept constant. Operant response rates were reduced and response durations were increased
after overnight irradiations with 10 or 15 mW/cm 2 when ambient air temperature during the exposure was
28°C but not when the temperature was 22°C. Operant behavior of rats which was reduced by 50% or
more after an overnight irradiation was not reduced after a similar exposure of only 55 mm. In another
experiment, response rates declined with time after exposures lasting 4 hrs., did not decline further after
exposures lasting from 4 to 8 hrs., but did decline further after exposures lasting longer than 8 hrs. Rats
did not change orientation or movement in relation to the electric or ni gnetic field vectors during irradia-
tion with 15 mW/cm 2 whereas mice showed increased movement but no preferred change in orientation
during exposures with a similar incident power level. During these exposures field orientation would not
alter the absorbed energy (specific absorption rate, SAR) in the rat but would in the mouse because of
the relationship of body size to wavelength of the radiation.
CITATION: 1n Biological Effects of Electropolludon, in press.
F104 MS-86-125 Project Officer: Gage
A THREE-DIMENSIONAL FINITE.DIFFERENCE THERM OREGULATORY
MODEL of a SQUIRREL MONKEY
R.J.Spiegel t andM.B.E.Falmi 2
‘HERI, USEPA, Research Triangle Park, NC
2 Northrop Services, Inc.
To achieve experimental verification of a themzoregulato y inode4 and minimize risk of hwnan expen’nen-
tation under radiofrequency exposure conditions, an acceptable approach Lc to model an animal whose
thennoregulatory system is similar to man’s and then peifonn the necesswy theimophysiological experi-
ments on the anjnwJc. The verification of such an animal model based on these measurements would fur-
ntsh substantial suppoir for a similar human modeL ThLc study chose a squirrel monkey as a surrogate to
study the accuracy of the thermal response model. It was found that reasonable agreement between calcu-
lated and available measured temperatures could be obtaineS The goal of this research iE to provide scien-
tiflcal y credible data, methodologies, and assessments of non-ionizing radiation (NIR) for the Office of
Radiation Flograms (ORP) to use in regulatory decLrions to modify and eiiend radiofrequency radiation
guidance.
A three-dimensional thermoregulatory model of a squirrel monkey, whose shape is approximated by 742
rectangular blocks of varying sizes, has been developed. The inhomogeneous model has four layers: a
core, a composite layer of muscle and fat, sicin, and fur. The model simulates the flow of heat into and out
of the body, including internal heat generation (metabolism) by the body, cooling and distribution of heat
by blood, thermal conduction throughout the body, evaporative heat loss from sweating, and radiation
and convection from the outer surface of the body. It also simulates dynamic thermoregulatory behavior
such as peripheral vasomotor responses (skin vasodilation and vasoconstriction) and variable sweating
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rates. Computed results are compared with available experimental data; the agreement is good, especial-
ly for ambient temperatures above 26°C.
CITATION: InL Journal of Radiation Oncology Biol. Phys. 12(6): 983-992, June 1986.
F104 MS-85-079 Project Officer: Spiegel
EFFECT of 9.6-GHZ PULSED MICROWAVES on the ORB WEB SPINNING
ABILITY of the CROSS SPIDER 4RANEUS DL4DEMA TUS)
C G. 1iddIe J.P.FUtuwn, aLLewter,
J.Y ewi , B. Beg MW Wes4 andA. Stead
HERL, USEPA, Research Triangle Park, NC
Microwave at low and moderate levels had been repoted to affect the central nervous system of laboratory
animals. The ability of the spider to spin an oth web has been used to screen drugs for central nervous sys-
tem effects. The present study showed that microwaves in the radar frequency range at low and moderate
leveLs did not affect the web spinning ability of spiders who spun their web while they were being e*posed to
the microwave flekL This information will be used to assist in establiøthig microwave eiposw guidelines
for the gen eral population.
Eight cross spiders (Araneus d iadematus) were exposed overnight (16 h) during web-building activity to
pulsed 9.6 GHz microwaves at average power densities of 10,1, and 0.1 mW/cm 2 (estimated SARs 40.4,
and 0.4 niW/g). Under these conditions, 9.6-GHz pulsed microwaves did not affect the web-spinning
ability of the cross spider.
CITATION: Bioelectromagnetics 7(1): 101-105, January1986.
F104 MS-83-003 Project Officer: Laddie
CIRCULATING ANTIBODY RESPONSE of MICE EXPOSED to 9-GHz
PULSED MICROWAVE RADIATION
C G. Liddle, J. P. FWman, a H. Lewte,
M. Wes4 and G. Mo,row
HERL USEPA, Research Triangle Park, NC
A previous study had shown that microwaves at moderate levels in the radar frequency range produced a
stimulation of the antibody response of immunized mice. This study involved the esposwe of immunized
mice to a lower level of microwaves. There was no increase in the antibody response in these animaLs. This
infonnation will be used to assist in establishing microwave exposure guidelines for the general population.
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Female CD-i mice immunized against the bacterium Strep tococcus pnewnoniae type I II were exposed to
9-0Hz pulsed microwaves (pulse repetition rate 9704,000, pulse width 1.0 jis, peak power 1 W/cm 2 ) at
an average incident power density of 1 mW/cm 2 (calculated SAR 0.47 W/kg) for 2 h per day for 5 days.
Circulating antibody titers for the microwave-exposed animals were not significantly different from those
of the sham-irradiated animals, and there were no differences in any of the hematological parameters
analyzed, indicating that 9-0Hz pulsed microwaves at 1 mW/cm 2 do not alter the immune response of
mice immunized against £ pneumoniae.
CITATION: Bicelectromagnetics 7(1 ): 91-94, Januaiy 1986.
P104 MS-83-244 Project Officer: lAddie
BEHAVIORAL EKEECTS of MICROWAVE RADIATION ABSORPTION
I. C. Monahan 1 and I ’. A. D’Andree?
1 Food and Drug Administration
2 University of Utah
ThLr publication brings together in a single source the major research findings related to the behavioral con-
sequences of microwave exposure. In addition it attempts to provide a critical assessment of this infonna-
don and to provide a perspective upon which the reader can intespret the findings.
The need for an understanding of the biological effects induced by exposure to microwave radiation has
increased in recent years because of increased usage and applications and also concerns about potential
adverse health effects. Although many research studies have been conducted to examine the question of
biological effects, the information is scattered in many diverse sources. This publication begins with a
review of behavioral-microwave research in the Soviet Union and then proceeds to exa mine the work of
researchers in the Western countries. Both learned and unlearned behaviors are examined in the context
of microwave induced effects. Other important areas which are covered include: selecting appropriate
animal models, extrapolation of animal data to hnmans , dose considerations, and problems inherent in
this type of research.
CITATION: Department of Health and Himian Services Publication, FDA 8S-8238 1985.
P104 HERL-0523 Project Officer: Elder
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BRAIN TEMPERATURE MEASUREMENTS in RATS:
A COMPARISON of MICROWAVE and AMBIENT TEMPERATURE
EXPOSURES
Thomas B. Wan 1 David!. Svendrgaar4 1 Ronaldf. Spie! ’
Earl T. FUcket4 2 Menilt D. Long’ and Jwnes B. Kinn
USEPA, Research Triangle Park, NC
2 Northrop Services, Inc
In research into the bioeffecls of non-ionizing radiation, a major problem is defining the relationship be-
tween tissue heating induced by exposure to the radiation and tissue heating induced by conventional
methods. This study addressed this problem. The work was done in suppom of the Office of Radiation
F ogranis’ effosi to develop a data base on the bloeffects of radiofrequency radiation. The Office of Radia-
tion F og,wns is activeJ v involved in nn ining guidelines for public e pasure to radiofrequency radiation.
In an effort to understand microwave heating better, regional brain and core temperatures of rats exposed
to microwave radiation (2450 MHz) or elevated air temperatures were measured in two studies. In
general, we have found no subst ntizi1 evidence for temperature differentials, or ‘hot spots,” in the brain
of these iinimalc . In the first study, after a 30-mm exposure, no temperature differences between brain
regions either after microwave or ambient air exposure were found. However, a highly significant cor-
relation between brain and core temperatures was found and this correlation was the same for both
microwave and ambient air heating. In the second study, time-temperature proffles were measured in
rats exposed to either 30 mW/cm 2 or 36.2°C. In this study, the 30-mm exposure period was divided into
seven intervals and the change in temperature during each period was analyted. Only the cortex showed
significantly different heating rates between the air heating and microwave heating; however, this dif-
ference disappeared after the inithl 5 mm of exposure.
CITATION: Bloelectromagnetics 7(3): 243-258, July 1986.
F104 MS-84-169 Project Officer: Ward
EXPOSURE of MAN in the NEAR-FIELD of a RESONANT DIPOLE:
COMPARISON BETWEEN THEORY and MEASUREMENTS
ManaA. Stuchly, Ronald I. Spieg4 2 Stanislaw S. Swch1y,
andAndrzej Cmurewski
1 Radiation Protection Bureau, Health and Welfare Canada
2 g USEPA, Research Triangle Park NC
3 University of Ottawa, Ottawa, Canada
The dose ute or the lute at which radiofrequency (RE) energy Ls impafled in the body, defined as the
specific absoiption rate (SAR), Ls used in quantifring biologwal effects and formulating standards on er-
posure to RE fields. II is also recognized that the spatial d&sti’ibution of the MR within the exposed body
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plays an essential role. In thLs paper, the calculations and measurements of the spatial distribution and
average SARs for various body pans for a full-scale model of man exposed to the 350 MHz radiation we
presentsS The goal of this research is to provide scientiflcal y credible data, methodologies, and assess-
ments of non-ioniring radiation (NIR) for the Office of Radiation Programs (OP.P) to use in regulatory
decisions to modify and e.dend radiofrequency radiation guidance.
The rate of the radio frequency energy deposition in a block model of the human body exposed in the
near-field of a resonant dipole at 350 MHz was calculated using the moment method. Detailed maps of
the electric field strength in a homogeneous model of a realistic shape under the same exposure condi-
tions were obtained using a computer-controlled scanning system and an implantable electric field probe.
A comparison of the measurement data with the calculations shows a relatively good agreement when
average values over relatively large volumes are concerned; however, the calculations do not show large
spatial gradients and tend to underestimate the magnitude of ‘hot spots” observed experimentally.
CITATION: IEEE Transactions on Microwave Theory and Techniques MTr-34(1): 26-31, January 1986.
F104 MS-84-130 Project Officer: Spiegel
TEMPORAL RESPONSE of NEURONS to AMBIENT HEATING in the
PREOPTIC and SEPTAL AREA of the UNANESTHETIZED RABBIT
Chi’istopherJ. Gordon and Elizabeth C. JThite
HERL, USEPA, Research Triangle Parlc NC
Understanding the neuronal control of body temperature is extremely useful in the interpretation of the
biological effects of pesticides and other toxic agents. By recording the response of single neurons in the
brainstem to peripheral heating we can better understand how thennoregulatory control may be influenced
by toxic agents.
• The firing rates of single neurons were recorded in the septal and preoptic areas of unanesthetized
rabbits during brief periods of ambient heating.
• The temporal response for neurons responsive to ambient temperature were calculated as the inter-
val of time between the onset of heating and the point at which the unit’s activity reached 63% (i.e.,
1-ife) of its total change in activity.
• Thirty-one neurons were isolated in 5 rabbits. Fourteen neurons were facilitated, 10 were inhibited
and 7 were unaffected by heating.
• Temporal responses ranged from <5 to 122s.
• We observed a bimodal relationship in the number of neuronal responses to ambient temperature vs
temporal response: a sharp 0- to <10-s peak comprising 22% of all responses and a relatively broad
peak with a mode of 60 to 
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• These data resemble the temporal response patterns of thermally excitable neurons throughout the
central nervous system.
CITATION: Comparative Biochemistry and Physiology 82A(4): 879-884, December 1985.
P104 MS-82-087 Project Officer: Gordon
THE MEASUREMENT of SMALL MECHANICAL VIBRATIONS of BRAIN
TISSUE EXPOSED to LOW-FREQUENCY ELECTRIC FIELDS
Al. Spiege4 I. S. A14 I. F. Peoples, and W. T. Joines
HERI USEPA, Research Triangle Park NC
RadiofrequencyfiekLs can interact with biological tissue both electdcally and mechanically. This study in-
vestigated the mechanical interaction between brain tissue and an e.Tdremely low frequency (ELF) field by
measuiing the resultant vibrational amplitude. It was found that the applied field caused the brain to
vibrate at very small amplihIdP This small vibration might have some influence on the association of cal-
cium ions near the brain’s surface and consequently could play a rok in calcium-ion efflux from brain tLc-
sue exposed to ELF flelds The goal of this research is to provide scientifically credible data, method-
ologies, and assessments 0/non-ionizing radiation (NIR) for the Office of Radiation hvgrams (ORP) to
use in regulatory decisions to modify and extend mdi ofrequ ency radiation guidance.
Electromagnetic fields can interact with biological tissue both electrically and mechanically. This study
investigated the mechanical interaction between brain tissue and an extremely low-frequency (ELF)
electric field by measuring the resultant vibrational amplitude. The exposure cell is a section of X-band
waveguide that was modified by the addition of a center conductor to form a small TEM cell within the
waveguide structure. The ELF signal is applied to the center conductor of the TEM cell. The applied
ELF electric field generates an electrostrictive force on the surface of the brain tissue. This force causes
the tissue to vibrate at a frequency equal to twice the frequency of the applied sinusoidal signal. An X-
band signal is fed through the waveguide, scattered by the vibrating sample, and detected by a phrase-sen-
sitive receiver. Using a time-averaging spectrum analyzer, a vibration sensitivity of approximately 0.2
nmpp can be achieved. The amplitude of the brain tissue vibrational frequencies below 50 H between
50 and 200 Hz resonant phenomena were observed; and above 200 Hz the amplitude fall-off is rapid.
CITATION: Bioclectromagnetics 7(3): 295-306, July1986.
P104 MS-85-210 Project Officer: Spiegel
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PUBLICATIONS RADIATION
OFF-CENTER SPHERICAL MODEL for DOSIMETRY
CALCULATIONS in CHICK BRAIN TISSUE
Guillermo Gonzale z 1 James C. Nearing 1 RonaldJ. Spiege4 2
and Williwn T. Joinel
‘University of Miami
USEPA, Research Triangle Park, NC
In order to understand the effect of amplitude modulated (AM) radiofrequency radiation on calcium-ion
effiur from chick brain tissue, dosimetry information must be accurately determined. Current models do not
allow accurate detennination of the surface electric field. This model more accurately represents the actual
exposure situation, since it allows the modeling of the chick brain as a continuous smooth suiface, with un-
equal wnowils of buffer solution on the top and bottom halves. The goal of this research is to provide
scientifically credible data, methodologies, and assessments of non-ionizing radiation (NIR) for the Office
of Radiation PØgrwns (OR?) to use in regulatory decisions to modify and extend radiofrequency radiation
guidance.
This paper presents calculations for the electric field and absorbed power density distribution in chick
brain tissue inside a test tube, using an off-center spherical model. It is shown that the off-center spheri-
cal model overcomes many of the limitations of the concentric spherical model, and permits a more realis-
tic modeling of the brain tissue as it sits in the bottom of the test tube surrounded by buffer solution. The
effect of the unequal amount of buffer solution above the upper and below the lower surfaces of the brain
is analyzed. The field distribution is obtained in terms of a rapidly converging series of zonal harmonics.
A method that permits the expansion of spherical harmonics about an off-center origin in terms of spheri-
cal harmonics at the origin is developed to calculate in closed form the electric field distribution. Numeri-
cal results are presented for the absorbed power density distribution at a carrier frequency of 147 MHz.
It is shown that the absorbed power density increases toward the bottom of the brain surface. Scaling rela-
tions are developed by keeping the electric field intensity in the brain tissue the same at two different fre-
quencies. Scaling relations inside, as well as outside, the brain surface are given. The scaling relation dis-
thbution is calculated as a function of position, and compared to the scaling relations obtained in the con-
centric spherical model It is shown that the off-center spherical model yields scaling ratios in the brain
tissue that lie between the extreme values predicted by the concentric and isolated spherical models.
CITATION: Bioelectromagnetics 7(2): 209-221, June 1986.
F104 MS-85- 164 Project Officer: Spiegel
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RADIATION PUBLICATIONS
A SYSTEM for MEASUREMENT of SMALL VIBRATIONS at MATERIAL
INTERFACES INDUCED by ELECTROSTRICTIVE FORCES
Joseph S. All and William T. Joiner
HERL, USEPA, Research Triangle Park NC
Life scientists at EPA and elsewhere have obse,ved the efflur of calcium ions from selected tissues includ-
ing brain tissue under various specific intensities and frequencies of amplitude-modulated radiofrequency
(RF) and eziremely low frequency (ELF) radiation e*posure. The effect may have health implications be-
cause calcium ions play an impoitant role in biological systems including the excitation and transmission
of ner ’e impuLses. Thus it is bnpo,tant to understand the underlying basis for thIs phenomenon. A
mechanism of interaction relying on the concept of nanometer iunge mechanical vibration influencing the
effiux of calcium has been proposed by others. To investigate the validity of this model a system was
developed to espose samples to ELF and amplitude-modulated RFfields while monitoring the induced
vibration by a noncontacting methoL
The mechanisms of interaction of ELF and ELF-modulated RF fields with biological systems is present-
ly an active area of research. Some models propose that field-induced forces may influence certain ob-
served biological effects such as EPhearing and calcium ion effiux. To investigate the validity of the field-
induced force model for the calcium-ion efflux effect, a system is needed which is capable of exposing
samples to ELF fields or to ELF-modulated RF fields. At the same time the induced vibration caused by
the forces of electrostriction must be monitored, preferably by a noncontacting method. A microwave
phase-sensitive receiver was designed to sense the small vibrations. Limitations on the receiver sensitivity
imposed by phase noise is discussed. Phase noise measurement systems were designed and used to
characterize the key receiver components. A limiting amplifier in the IF section of the receiver eliminates
the need for knowledge of the reflection coefficient of the object of interest for quantitative vibration
measurements. A special exposure cell is described which is a section of X-band waveguide which has
been modified by the addition of a center conductor to form a rectangular transmission line. The center
conductor with a sample placed under the waveguide broad wall is excited by a low-frequency or low-fre-
quency amplitude modulated signaL In addition an X-band signal is fed through the waveguide, scattered
by the sample and detected by the phase-sensitive receiver. The fields and the resulting forces on certain
standard shaped samples are derived. With the measurement of the Young’s modulus and Poisson’s ratio
of the samples, the resulting vibration amplitude of the field-induced vibration is estimated. Actual vibra-
tion measurements verified with a piezoelectric crystal indicate a vibration sensitivity of about 1
nanometer peak-to-peak. Vibrations in this range were predicted to occur in certain calcium-ion efflux
studies. This system therefore promises to help unravel some of the uncertainties surrounding the inter-
action of RF fields with biological systems.
CITATION: U.S. Environmental Protection Agency, EPA-600/1-85-021, November 1985. Available from
NTIS: PB86-116530/AS.
F104 HERL-0518 Project Officer: All
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PUBUCATIONS RADIATION
APPLICATION of a FINITE-D1FIERENCE TECHNIQUE to the HUMAN
RADIOFREQUENCY DOSIMETRY PROBLEM
R.J. Spieg4’ M. B. E. Fatms, 2 andK S. Kunz
1 }j j USEPA, Research Triangle Park, NC
2 Northrop Services, Inc.
3 Lawrence Livermore Laboratory
It is not well undeivtood for humans where radiojivquency eneigy Lv deposite4 but the deposition site has
unpo?lant implications for the resultant biological effect. Realistic models of the human body and
dosunetiy studies are needed to enhance nsk estimates. This study described a finite-difference numerical
method to calculate specific absorption inter (SAR) in human models. U th this technique it Lv possible
to calculate £4R at higher frequencies and with greater spatial resolution than Lv possible with other
methods. The goal of this research Lv to provide scientiflcal y credible data, methodologies, and assess-
ments of non-ionizing ,vAialion (NIR) for the Office of Radiation FTogiwns (ORP) to use in regulatorj
decisions to modify and estend radiofrequency radiation guidance.
A powerful finite-difference numerical technique has been applied to the huni in radiofrequency
dosimetry problem. The method possesses inherent advantages over the method-of-moments approach
in that its implementation requires much less computer memory. Consequently, it has the capability to
calculate specific absorption rates (SARs) at higher frequencies and provides greater spatial resolution.
The method is illustrated by the calculation of the time-domain and frequency-domain SAR responses at
selected locations in the chest. The model for the hwnan body is comprised of rectangular cells with
dimensions of 4x4x6 an and dielectric properties that simulate average tissue (213 muscle). Additionally,
the upper torso (chest) is configured by both homogeneous and inhomogeneous models in which this
region is subdivided into 20,736 cells with dimensions of ixIxi cm. The homogeneous model of the chest
consists of cells with average tissue properties, and the calculated results are compared with measure-
ments acquired from a homogeneous phantom model when the exposure frequency is 350 MHz. For the
inhomogeneous chest model the lungs and surrounding region (ribs, spine, sternum, fat, and muscle) are
modeled with as much spatial resolution as allowed by the Ixixi cm cells. Computed results from the in-
homogeneous chest model are compared with the homogeneous model
CITATION: Journal of Microwave Power 20(4): 241-254, December 1985.
F104 MS-85-168 Project Officer: Spiegel
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RADIATION PUBLICATIONS
OVERVIEW of WHO ENVIRONMENTAL HEALTH CRITERL& 35 on
EXTREMELY- LOW-FREQUENCY (ELF) FIELDS
Richard D. Phillips
HERL, USEPA, Research Thangle Park, NC
This docoment summw’izes World Health Cjitena 35 aspossthle effects on humans of esposwre to extreme-
ky-low -frequency (ELF) electdc and magnetic fields, the lowest frequency band in the nonionizing radIation
spectnlm. The author was a member of the WHO Task Force who reviewed and revised a draft c,ite,ia
docwnen4 made an evaluation of the health nskc of exposure to ELF radiation, and considered rationales
for the development of human exposure limits. The WHO Olterla Document is a valuable resowre to the
Office of Radiation F1ogn ms by providing guidance on the assessment of nsks from occupational and
genenilpopulaton exposure to nonionizing radiation.
This paper summarizes and reviews the document World Health Criteria 35. The purpose of the Criteria
35 is to provide information for health authorities and regulatory agencies on the possible effects of ex-
tremely-low-frequency (ELF) electric and m netic fields on humiin health and to give guidance on the
assessment of risks from occupational and general populatioq exposures. The Criteria Document in-
cludes a review of biological effects on human beings and inhiui1c exposed to ELF fields in the frequen-
cy range of 0-300 Hz with emphasis on power (50- and 60-Hz) electric fields. Subjects reviewed in the
document include the physical characteristics of ELF fields, measurement techniques and dosimetry,
levels of exposure from devices in common use, mech2ni m of interaction, biological effects in nim 1
and anims 1 tissues, hnm in studies, health risk evaluation, and guidance on the development of protective
measures such as regulations and safe-use guidelines. The document also identifies areas in which uncer-
tainties exist in the literature and where further research is nee4ed. The goal was to provide useful infor-
mation for the development of national protection measures sig imst ELF fields.
CITATION: In: Proceedings of the International Utility Symposium, September 16-19, 1986.
F104 MS-86-162 Project Officer: Phillips
RADIOFREQUENCY RADIATION: ACTiVITIES and ISSUES
JoeA. Elder
HERL, USEPA, Research Triangle Park, NC
The following topics are discussed in this repo 1: 1) environmenal exposure levels of radiofrequency (RF)
radiation; 2) Federal and other activities related to the control of exposure of the gen eral public to RF radia-
lion; 3) biological effects; 4) limitations in our knowledge on biological effects; and 5) research suppoit.
The question of hnmsin safety relative to exposure to RF radiation obviously predates the first ANSI
guideline established in 1966, but no enforceable Federal standards or guidlines exist for RF radiation ex-
posure; the ANSI guideline which was revised in 1982 is voluntary or advisory. EPA has been pursuing
the goal of promulgating guidance to control exposure of the public to RF radiation. In support of this
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PUBLICATIONS RADIATION
regulatory activity, a report entitled “Biological Effects of Radiofrequency Radiation” was published in
September 1984. The conclusion of the report was that biological effects occurred at a dose rate of about
1 WAg, and that these effects may be significant under certain environmental conditions. Examples of
adverse biological effects that occur in laboratory animaLs at dose rates of 2-6 W/kg are death and tem-
porary male sterility. These effects as well as the behavioral changes that are the basis for the ANSI
guideline can be attributed to heat stress in animals caused by absorption of RF energy. Some experimen-
tal results occur at very low exposure conditions that cause no significant thermal input; these responses
are called non-thermal effects. The mechanisms of interaction of non-thermal effects and their
physiological significance are a subject of scientific debate. RF radiation research budget reductions,
which reflect changes in funding priorities, will leave unresolved many of the questions concerning the
biological effects of RF radiation and their possible health implications.
CITATION: In: Howard Universi y Symposium Proceedings, Washington, DC, September 9, 1985.
F104 MS-86-187 Project Officer: Elder
IMPROVED TECHNIQUE for MONITORING LF CTROCARDIOGRAMS
DURING EXPOSURE to RADIO-FREQUENCY RADIATION
William P. Watldnson and ChristopherJ. Gordon
HERL, USEPA, Research Triangle Park , NC
The research described in this aiticle was conducted to suppoit the Office of Radiation Fmgrams’
microwave radiation research. The pdmaiy documented effect of exposure to microwave radiation is an in-
crease in body temperature. The investigation of other potential related effects, such as cardiovascular 4:
fects, has been limited by the prohibition of metallic instrumentation within the microwave fielS This study
repolu on new y -developed instrumentation and procedures which pennit simultaneous monitoring of ther-
‘noregulatoiy and cardiovascular parwne:ers during microwave e posure. The use of these procedures
should open new areas of investigation and perhaps sign iflcant y expand this area of research.
Studies were conducted using improved methodology that examined the effects of radio-frequency (RF)
radiation on heart rate (HR), deep body temperature (T ), and electrocardiographic (ECG) waveform
parameters in anesthetized rats. One group of animals was exposed to two power levels of continuous-
wave RF radiation averaging 1.0 and 7.4 W/kg at a frequency of 600 MHz. A second group of animals,
treated identically but not exposed to RF radiation, served as a controL The electrodes used for monitor-
ing the ECG during RF exposure were fabricated from carbon-loaded Teflon wire, a semiconductor
material that does not perturb the BY field. Analyses of the ECG were conducted by use of a recently
developed computer-assisted procedure that quantitates HR and waveform intervals over 25-40 in-
dividual ECG complexes. There were no artifacts or arrhythmias in the ECGs of the animals exposed to
RF radiation. There was a significant linear correlation between HR and T 0 in the RF-exposed group
that was not present in the control group.
CITATION: American Journal of Physiology 250(19): H320-H324, February 1986.
F104 MS-85-002 Project Officer: Watkinson
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Research Objectives
Water Health

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WATER HEALTh RESEARCH PROGRAM
Objective: To improve the scientific basis for regulatory activities under the Clean Water Act and
the Safe Drinking Water Act.
Background: EPA, in partnership with State and local governments, has responsibility for water
quality in three areas. The first is reducing pollution of surface water, i.e. rivers, lakes,
streams, coastal waters, oceans, and sensitive areas such as wetlands and estuaries. The
second is preventing cont min tion of ground waters, i.e., underground formations of
saturated rock and sand. The third is maintaining the purity of drinking water, i.e., the
surface and ground waters needed for human consumption. The principal law regulat-
ing toxic and conventional water pollution is the Clean Water Act (CWA). The Safe
Drinking Water Act (SDWA) protects underground sources of drinking water from
fluids injected into the ground. EPA’s approach to the problem of contaminated drink-
ing water focuses on providing safe water to users of public water systems. Under the
SDWA, EPA sets national standards for drinking water and requires routine monitor-
ing to ensure that drinking water is free of harmful levels of contaminants. Some of the
pollutants that affect water include organic waste, sediment, acid, bacteria and viruses,
protozoa, nutrients, oil and grease, heat, heavy metals such as mercury and lead, and or-
ganic chemicals, such as PCB’s, solvents, and pesticides. Some of these pollutants can
become serious threats to public health.
Water Quality
• Provide health effects testing to support the National Pollutant Discharge Elimination System
(NPDES).
Rationale: Title ifi of the CWA mandates that EPA control toxic pollutants, and under the CWA the
discharge of pollutants into the waters of the United States is prohibited unless a permit is issued by EPA
or a State under the NPDES. The Office of Water Regulations and Standards and the EPA regional of-
fies have identified 100 sites where high concentrations of water pollutants are of particular concern. In-
vestigators would like to use existing short-term health tests to determine whether or not a site receiving
a large number of chemical contaminants is a public health risk. Research is being conducted to evaluate
appropriate sampling, preservation, concentration, and bioassay techniques for estimating health hazards
from waste effluents. These data will be incorporated into a users manual. The manual will help inves-
tigators select and interpret the appropriate tests which directly support the agency’s change to a water
quality based approach to pollution control.
Approach: Health effect tests to determine the toxicity of chemically impacted effluents will be field
validated within a regulatory decision logic. Tier 1 screening carcinogenesis bioassays such as the Ames
assay and cytogenetic assay will be tested at numerous locations and with various types of effluents.
• Determine health effects of microbial water quality. Develop a health-effect related Indicator for
shellfish growing waters In cooperation with NOAA and FDA and extend studies on the enterococ.
cus Indicator system for recreational water.
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RESEARCH OBJECFIVES WATER
Rationale: A national goal of the Clean Water Act is water quality adequate for recreation and shellfish
production. State water quality standards based on established criteria are the designated means to
achieve this goaL Current shellfish growing water standards likely do not correlate with health effects
since the same standard did not correlate in recreational waters.
Approach: Methods are being evaluated to isolate and identify various microbiological indicators includ-
ing enterococcus in shellfish growing waters and shellfish meats. An epidemiological study is being con-
ducted to determine the occurrence of gastroenteritis in populations ingesting shellfish (oysters and
clams) harvested from approved shellfish harvesting areas of varying water quality and correlationg dis-
ease with water quality as determined by various indicator systems.
• Hazard evaluatIon of organic chemicals in municipal sludge. Provide data and appraisal docu-
ments on the health aspects of land application of municipal sludge. Emphasis will be on heavy
metals and organic chemicals that are transmlted from sludge and have the potential to cause ad-
verse health effects.
Rationale: This research is needed to assess the effects on human health resulting from the exposure to
heavy metals and organic chemicals contained in sludges applied to land. it is a major input to the for-
mulation of EPA regulations, permits and guidelines under the Clean Water Act.
Approach: State-of-the-art documents are being provided on the health effects of sludge disposal.
Research is being conducted using chemical, epidemiological and toxicological approaches to fill
knowledge gaps in our understanding of health effects. Bioassays will be used to assess complex chemi-
cal mixtures in sludges for mutagenicity, particularly for sludges applied to land and used in distribution
and marketing systems. More comprehensive appraisals will be made as the research data becomes avail-
able.
• Provide data on the occurrence, survival and transport of enteric pathogens in sludge, prlmariy the
parasite ascoAs and enteric viruses. Work has also begun on determining the microbiological
health hazard from distribution and marketing sludge products.
Rationale: This research is being done to improve the scientific and technical basis for regulatory and en-
forcement activities to assess the effects on human health resulting from the exposure to pathogens, con-
tained in sludges applied to land. it is a major input to the formulation of EPA regulations, permits and
guidelines under the Clean Water Act.
Approach: A series on the fate of sludge microorganisms applied to soil is being conducted to develop
the needed data to make the health effects appraisal. Research is being conducted using microbiological
and epidemiological approaches to fill knowledge gaps in health effects data. More comprehensive ap-
praisals will be made as the research data becomes available. The transport of viruses from sludge into
the soil matrix (and ultimately to groundwater) and the survival of ascarir eggs in mixtures sludge-
amended at field sites is being determined. An epidemiology feasibility study of the potential for disease-
causing organisms to occur in distribution and marketing products will be conducted if the microbiology
data indicate a hazard may exist.
Drinking Water Health Research Program - Along with controlling ground water contamination, the
most significant challenges facing EPA and the States to protect drinking water supplies are to (1)
eliminate disease-bearing organisms, primarily in small water supply systems, (2) develop standards for
toxic contaminants, and (3) control recontamination of distribution systems. All naturally occurring
water contains microbes, bacteria, viruses, and protozoa. While most such organisms are harmless, some
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WATER RESEARCH OBJECTIVES
are pathogenic. Microbial contamination continues to be a concern because cont2minqed drinking
water systems can rapidly spread diseases such as cholera, dysentery, and giardiasis. In 1974 Congress
passed the Safe Drhiking Water Act because of concern about cont lminnnts in drinking water and un-
even State supervision of public drinking water supplies. Regulations under the law set maximum con-
tanhinant levels (called MCL’s) for coliform bacteria, turbidity, and a number of organic, inorganic and
radioactive chemicals. EPA also publishes health advisories to help local and State officials determine
the health hz nTd of unregulated cont n1inzants in drinking water.
• Provide the toxicological information needed for the Office of Drinking Water (ODW) to develop
maximum contaminant level’s and health advisories for specific chemicals found in drinking water.
Rationale: Section 1412 of the Safe Drinking Water Act (SDWA) requires EPA to publish national
primary drinking water regulations for compounds that occur In sufficient locations and at levels
high enough to pose significant health effects. In addition, Section 1442 SDWA requires EPA to
provide guidance to the states on pollutants of regional interest or which are accidentally spilled
into the water supply. Approach: All health endpoints will be considered, but the most significant
endpoints for each pollutant will be selected for in-depth toxicological, dose-response characteriza-
tion in the appropriate in vivo or in vitro systems. The chemicals chosen and the priority given to
those selected will be determined by the drinking water research planning group. The dose
response data generated in the problem will be used in development of national primary drinking
regulations and health advisories.
• Develop the toxicological data needed by the Office of Drinking Water for evaluating the effects of
various dlsinfectants, other chemicals used in drinking water treatment and materials capable of
leaching from surfaces in contact with potable water.
Rationale: Section 1442 of SDWA requires EPA to conduct research to determine the health effects of
drinking water disinfectants and their by- products. Section 1412 of SDWA requires the agency to deter-
mine the impact to public health of drinking water in the treatment and distribution process. ODW will
use this information to develop guidance on the use of dit infectants, various treatments chemicals, and
the construction of water supply sysems.
Approach: The rlisinfectants themselves will be er imined to determine their potential carcinogenicity
and target organ effects. By-products formed by reaction of the disinfectants with background organic
material or in the body will be ex imined toxicologically to determine possible adverse health effects.
Treatment chemicals such as flocculant aids will be examined to develop dose-response data of the toxic
effects of these chemicals. Chemicals leaching from water contact surfaces such as lead and organotins
will be investigated for various target organ and carcinogenic endpoints.
• Develop methods for use in improving existing and proposed extrapolation models used to develop
standards for drinking water contaminants.
Rationale: Section 1442 of the SDWA provides for developing methods to identify and measure the
health effects of exposure to DW contanhinRnts.
Approach: Data are being developed to elucidate chronic toxicological effects of drinldng water con-
taminants. Experiments are being conducted to determine if specific drinking water contaminants act as
cancer initiators or as tumor promotors in order to indicate appropriate extrapolation model (linear vs.
non linear to use in risk assessments). Experimental work to improve models for cross-species extrapola-
don and for extrapolation from high to low doses are being developed for renal and hepatic toxicity. The
vast majority of drinking water supplies are cont minpted by more than one chemical. A methodology
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RESEARCH OBJECTIVES WATER
for predicting the h ’nrds of exposure to complex mixtures can only be accomplished by ez n’ining the
impact of mixtures that act by the same or different mech ini nic and using this information to predict the
degree of interaction likely based upon structure activity consideration. This research will provide better
methods and data to use in extrapolationg experimental data to conduct more accurate risk assessments.
• Develop methods for concentration, Isolating and ldentl1 4ng Infectious disease agents from drink-
ing water samples and hnm n specimens for use In determining waterborne health haiards. These
methods will be developed for bacterIa, viruses, parasites and will address the problem of deter-
mining Infectivity and viability of these orgnkms In the water environment. The occurrence of
acute waterborne disease outbreaks will be determined through surveillance and investigation.
Rationale: Section 1412 of the SDWA requires EPA to establish national primaiy drinking water stand-
ards to protect the public health. In order to review and revise the existing microbiological standard EPA
needs methods to measure the level of infectious agents in drinking water to determine hiim in exposures.
Approach: The approach is primarily lab-based with some field work and focuses on known waterborne
pathogens, e.g., norwalk-like virus, giasdia, legioneila and cwnpylobacter and opportunistic pathogens.
Outbreak surveillance and investigation will be conducted in cooperation with the Center for Disease
Control. Technical assistance capabilities will be developed and supported byORD for EPA Regional
staff to respond to state and local health agency needs.
• Determine how and where epidemiologic research can contribute to an Improved scientific basis for
revising current drinking water nIn Imum confmlnilnt levels (MCL) or developing new MCL’s and
conduct studies where appropriate.
Rationale: The Safe Drinking Water Act provides for research that will supply health effects data useful
for development of drinking water standards. Where feasible, epidemiologic studies can provide the
most direct evidence of humfin health effects at environmental exposure levels.
Approach: After evaluating current data, epidemiologic research will be conducted to resolve various
scientific issues associated with specific regulations or proposed regulation. Before initiation of research,
rigorous studies will be conducted to define these research problems and determine the feasibility of
using the epidemiologic approach. To the extent possible, research will be coordinated with the efforts
of other federal agencies. Effects of acute and chronic exposures will be considered. Where possible, re-
search will be coordinated with ongoing or proposed epidemiologic studies. The relationship between
mirobiological standards and disease outbreaks will be better defined. The contribution of indoor air ex-
posures from volatilization of organic contamin lnts and microbiological agents in drinking water will also
be assessed.
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Accomplishments
Water Health

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ACCOMPLISHMENTS WATER HEALTH
Issue: Provide research data on the microbiological health aspects of land application of municipal
sludge. Provide assessment of potential health effects associated with the discharge of sewage
effluents to surface waters.
The microbiological quality of surface waters is typically determined by indicator bacteria that could
originate from human (sewage) or nonhuman sources. In order to more accurately determine the health
hazard from exposure to contaminated surface waters, studies have been conducted relative to water
quality factors and infectious disease.
Investigations carried out by Yale University have shown that poor water quality resulting from storm
water runoff was not related to swimming-associated illness. Exposure of swimmers was studied at a
small man-made lake in central Connecticut fed by two small streams. A careful examination of the near-
by watershed indicated that any water quality degradation occurring in the lake was not due to human
fecal contamination. Therefore, poor water quality was due solely to animal fecal contsimination carried
to the lake with rain water runoff. The geometric mean fecal coliform density was 143 per 100 ml during
periods of rainfall greater than 0.2 inches, whereas under dry conditions, the geometric mean fecal
coliform density was 33 per 100 ml. Gastroenteritis observed in swimmers could not be associated with
heavy rainfall or high densities of bacterial indicators of fecal contamination that resulted from the rain-
fall. Illnesses were associated with high bather densities and high densities of staphylococci in the water,
indicating that illness may have been transmitted from bather to bather. These results indicate that non-
point sources of pollution may not pose the same risk to recreationists as do point sources of pollution.
The implication of these findings is that the source of pollution should be taken into account when recom-
mending limitations to water quality degradation.
A five-year study, known as the Lubbock Infection Surveillance Study (LISS), was completed in FY 86.
The project examined the possible adverse effects of pathogens in wastewater applied to land by spray ir-
rigation. The study incorporated various subjective and objective measures of disease and infection. Ex-
posure was characterized by measuring organisms in the wastewater and in the aerosols and an exposure
index was developed. No obvious connection between aerosol exposure and acute illness was noted but
the rate of viral infections, as determined by serological responses, was slightly higher among participants
who had a high degree of aerosol exposure. In addition, the association of viral infections with aerosol
exposure showed a dose effect. However, it could not be determined if aerosol exposure to wastewater or
possible alternative explanations were the actual risk factors in these infections. A study was begun in FY
86 to refme the statistical analysis of the data.
Work continued on a study of Ascaris ova survival in an aerobically digested sludge applied to agricul-
tural land in Texas, Louisiana and Ohio. Preliminary results suggest that ova die-off is greatest when the
sludge is applied to the surface of grass-covered plots as compared to tilling the sludge into the soil. Dif-
ferences in ova survival related to soil characteristics and to agricultural practices are also being noted.
Since the initial application of ova to grass plots in Texas took place in the wintertime when freeze-thaw
cycles occurred, ova were re-seeded on the grass plots in the summer of FY 86. A similar sharp drop in
ova survival was still noted, even under these conditions.
Collection of data progressed satisfactorily in a study of microorganisms in distributed and marketed
sludge products. Samples of these products produced at 26 different locations around the country are
being examined for indicator organisms and pathogens. Preliminary results show a high prevalence of
Salmonella in some products. In addition, high levels of Yersinia have been detected at some sites during
the winter months. Any potential microbial ha7ard would thus far appear to be limited to bacteria since
very few samples have been found positive for viruses or viable parasites.
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WATER HEALTH ACCOMPLISHMENTS
Technical assistance was provided to ECAO through the review of criteria documents on the land ap-
plication and ocean disposal of sewage sludge. Expertise was provided to OW by participation on the
Pathogen Equivalency Committee. The function of the committee is to consider and make recommenda-
(ions on applications for PSRP and PFRP equivalent processes.
Issue: Provide assessments of the mutagenic potential of municipal sludges and sludge amended soils
A research project has been completed evaluating the levels of mutagenic activity in municipal sludges
and the changes in this activity when sludges are applied to soil. Twelve municipal wastewater treatment
plant sludges were collected and extracted by sequential extractions with methylene chloride and
methanol on a Soxhlet apparatus. Each of three sludge fractions, i.e., methylene chloride, methanol, and
a combined fraction (36 fractions total) were tested in the Salznoneilafmicrosome assay. Only one of the
sludge fractions induced a positive response in the absence of metabolic activation, and twelve fractions
induced a doubling of revertant colonies at two or more consecutive dose levels with activation. The max-
imum mutagenic response observed with strain TA9S in a methylene chloride fraction was 120 net rever-
tants at a dose level of 10 mg of extract per plate. The maximum response induced by the methanol and
combined fractions was 89 net revertants, induced by a municipal sludge with 20% industrial contribu-
tion, and 61 net revertants, induced by a municipal sludge with 19% industrial contribution, respectively,
both tested with strain TA98 at a dose level of 5mg of extract per plate. These results indicate that there
will be substantial differences in the mutagenic potential of municipal sludges from a single source and
from different sources.
The analysis of sludge amended soil from a land application study indicated that the mutagenic potential
of the residual organics was increased with time. This increased activity may be a result of biodegrada-
tion of inert organics resulting in a greater expression of the residual mutagens, or some type of synergis-
tic interactions between the residual mutagens in the soil. The results of the present study indicated that
some sludges may persist in the soil for extended periods of time increasing the potential for leaching or
plant uptake of sludge-borne mutagens.
Issue: Provide methods for concentrating, Isolating and Ident i fying protozoa In drinking water.
Provide an assessment of potential exposures to protozoa, viruses and bacteria in raw source
and treated drinking waters.
A method for detecting Giardia cysts in water using fluorescence/phase microscopy was published in FY
86. The method simplifies the detection of cysfs against the background of debris normally found in fil-
ter concentrates of surface waters. Training in this methodology was given to representatives of the Pen-
nsylvania Department of Environmental Resource; the University of Pittsburgh and EPA Regions I and
x.
In a companion inhouse study, an antibody specific to Giardia cyst walls was developed in rabbits. Test-
ing of the antibody showed that it would react with cyst walls but not with either human or mouse
trophozoites. The antibody was subsequently used in cooperation with a University of California at San
Diego investigator in developing criteria for differentiating cysts from trophozoites. In this same study,
serum-free in vitro growth of Giardia trophozoites was obtained and in vitro encystation of Giardia was
achieved. Manuscripts on the serum-free growth and on the in vitro encystation were submitted for pub-
lication.
Cooperative agreements with the University of Minnesota and with Cleveland State University resulted
in two procedures that may have value in determining the viability of Giardia cysts. Both procedures use
fluorogenic dyes—in one case, fluoroboras and in the other, a combination of propidium iodide and
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fluoroscein diacetate. In addition, the University of Minnesota study also examined more than 1100
beaver and muskrat fecal specimens and demonstrated that more than 95% of muskrats are naturally in-
fected with Giardia. Furthermore, cross-species infectivity studies showed that both beaver and muskrat
could become infected with human-source Giardia.
HERL scientists assisted authorities in Pittsfield, MA, in the investigation of the largest waterborne out-
break of giardia sis reported to date. Water samples from three reservoirs serving the community were
shown to contain Giardia cysts. One reservoir was highly suspect as the cause of the outbreak and cysts
from this reservoir were demonstrated to be viable by inoculation into gerbils.
A clinical virus feeding study was conducted in order to assess the hazard of low level virus exposure as
may occur through environmental transmission of these infectious agents. Human rotavirus was ad-
ninktered orally to 62 adult volunteers in a study designed to determine the dose required to produce in-
fection with or without illness. Study participants ingested doses ranging from 9 x i0 to 9 x 10 focus
forming units (ifu) in buffered salt solution after consumption of 50 ml of 4% NaHCO3. The dose of virus
required to cause human infection or illness was between one and 10 ifu. Although pre-inoculation titers
of serum neutrali’ing antibody to challenge virus in the study subjects ranged from <1:2 to 1:1,600, the
concentration of serum antibody could not be correlated with protection from infection or illness in sub-
jects given an infectious dose of virus. The results of this study, which were published in the Journal of In-
fectious Diseases, defined two important findings, first, that the infective dose of rotavirus for humans is
very low and, second, there does not appear to be a protective immunity conferred on individuals by a
previous infection with this virus.
A study that examined the relationship between health effects and the use of point-of-use granular ac-
tivated carbon (GAC) filters has been completed. GAC filters are known to be colonized by
heterotrophic bacteria which may grow to very high densities and subsequently slough off into the filter
effluent. Many heterotrophs are dassified as opportunistic pathogens, organisms which cause disease in
compromised individuals such as those on immunosuppressive drugs and which may pose a risk to in-
dividuals exposed to high densities of these bacteria. Two study groups were followed for 18 months.
One group drank water filtered through GAC and the other unfiltered water. The effluent from the GAC
filters contained heterotrophic bacteria at a geometric mean density of 1000 to 2000 per ml. The
heterotrophic bacteria geometric mean density in unfiltered tap water was about 92 per mL Some of the
bacterial species isolated from GAC effluent samples were known opportunistic pathogens, such as
Acinetobacter, Flavobacterium and Pseudomonas species. Two types of filters were e niined: the faucet
type filter, which attached directly to the tap, and the bypass type filter, which tapped into the cold water
line and which delivered water through a third tap at the sink. The study group was comprised of about
one-half adults (>18 yrs old); 25%, children younger than six years old; 15%, 6-10 years old and 10%, 11
to 18 years old. About half were male and half were female. A number of participants exhibited
symptomatic illness such as vomiting, diarrhea, nausea or fever during the course of the study, but none
of the symptoms were significantly associated with GAC filter use. Similarly, none of the ill participants
contributed clinical specimens wherein bacterial isolates were obtained that could be linked to bacteria
growing in the GAC filter. The results of this study indicate that the use of point-of-use GAC filters and,
therefore, the increased exposure to heterotrophic bacteria does not pose an increased health risk to in-
dividuals using these devices.
Studies have been completed which examined the infectious dose, virulence characteristics and route of
transmission of Legionella pneumophila. The results of these studies have shown that some strains of
Legionella are less virulent than others and this is manifested by susceptibility to human serum, the
presence of plasmids and reactivity with monoclonal antibodies. The infectious dose of plasmidless
Legionella strains in guinea pigs by the intraperitoneal route was significantly lower than strains contain-
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WATER HEALTH ACCOMPLISHMENTh
ing 45 and 85 dalton plasmids by the intraperitoneal route. These differences in virulence could not be
shown when guinea pigs were challenged via the respiratory route. Ingestion challenge doses required in-
ordinately high densities of I .. pneumophlla before any effect was noted. The administration of
cimetidine, an antacid, prior to challenge lowered the dose required for an effect to be seen to levels
found in some potable water plumbing systems. In addition to the infective dose being lowered by con-
comitant admhustrafion of cimetidine, it was found that very low densities of L pneumophlla ad-
ministered per dose conferred a protective effect when these anhnatc were challenged with 1011)50’s by
the intraperitoneal route. These results show that ingestion of potable water might be a route of trans-
mission for Legione especially in cases where antacids are commonly used. They also show that if
small numbers of Legionella reach the small intestine, they can stimulate an immune response that con-
fers some protection again -ct subsequent challenge with high doses of this organism. Factors, such as
heterotrophic bacteria densities and types, and water temperatures were studied in five hospital com-
plexes. No correlation could be found with heterotroph types or densities. Water temperature was the
only physical factor showing a positive correlation to the presence of Legionella. A hospital using flash
water heating units and no water storage was free of Legionella, whereas hospitals using conventional
water heating units with storage tanks were more likely to be colonized by Legionella.
Issue: Pramlue utility of an in vito model for studyIng toxic interactions betwaen chlorinated
hydrocarbons
Liver is a common target organ for toxicity of chlorinated hydrocarbons which are found in drinking water
as chlorine disinfection by-products or industrial contaminants. Although regulation of chemicals in
drinking water is based upon data from single chemical exposure, it is possible that chemicals may inter-
act in producing toxicity under environmental exposure conditions. Studies are underway to e l s a mine the
potential use of isolated hepatocyte cultures in examining hepatotoxic interactions between binary com-
binations of chlorinated hydrocarbons. Of particular interest are those combinations of chemicals which
produce toxic interactions in a superadditive fashion. The relatively large expenditure of resources and
time in conducting in vivo chemical interaction studies make the possible utilization olin vito models at-
tractive.
Studies were conducted in an attempt to reproduce in cultured hepatocytes, the superadditive h i vito in-
teraction between chloroform (CHCI3) and carbon tetrachloride (CC14). When male rats are pretreated
with orally administered CHC13 for 72 hours, these animals show substantial hepatotoxicity after 24 hours
of treatment with CCI4 compared to minimally toxic responses observed with either CHCIS or CCI4 ex-
posure alone. Toxicity was assessed with serum clinical chemistries and histopathology. These exposure
conditions in vivo cannot be duplicated exactlyin vitro due to inherent limitations of cultured hepatocytes.
Cytochrome P450 enzymes, which are responsible for metabolizing CCI4 and CHCI3 to toxic inter-
mediate, decrease to less than 50% of control zero time levels in 24 hours. A 72 hour pretreatment with
CHCI3 in culture is not possible without adverse effects on the metabolic activation potential of cells.
Thus, it is important to expose hepatocytes to chemicals a few hours after isolation to ensure adequate
metabolic activation of chemicals to produce toxicity.
An initial approach to demonstrate an interaction between CHCI3 and CC14 in vitro was to expose cul-
tured hepatocytes from untreated male rats to CHCI3 and CCL4 in combination and to compare the ef-
fects to cells treated with only CC14 or CHCI3. Cells exposed to 03-10 mM CCI4 alone showed a dose-
related increase in toxicity after 24 hours. Hepatocytes exposed to 05-10 mM CHCI3 for 24 hours
showed minimal toxicity. When hepatocytes were exposed to combinations of CHCI3 and CC14, toxicity
was not different from cells treated with CCI4 alone. Thus, under these in vitro exposure conditions to
CHCI3 and CCI4, an interaction between these two chemicals could not be demonstrated.
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A second approach was to treat rats for 72 hours with CHCI3 or corn oil gavage vehicle alone, isolate
hepatocytes and expose cultured cells of either treatment to CC14 for 24 hours. A comparison of the in
vitro response to 0.5-5 mM CCI4 from rats pretreated with CHCI3 or corn oil revealed only a slightly en-
hanced toxicity to CCI4 in cells isolated from CHCI3 pretreated rats.
These studies underscore some of the considerations in attempting to reproduce, by in vitro methods, a
chemical interaction observed in vivo. Tniti d attempts to validate an in vitro hepatocyte model for an in-
teraction between CHCI3 and CC14 were not successful. Chemical exposure conditions will be modified
in an attempt to refine this in vitro system. It is possible that some important biological factors involved in
the superadditive interaction between CHCI3 and CCI4 observed in vivo are lost when liver tissue is dis-
associated into isolated cells. It is also possible that extrahepatic tissues may play a role in hepatic chemi-
cal interactions in vivo as well.
Issue Determination of the atherogenic potential of chlorinated water.
Suspicion has been raised in the past that chlorinated drinlcing water may be involved in the etiology of
atherosclerotic coronary heart disease (CHD). This hypothesis emerged from the claims of a military
physician, who noted unusual incidences of atherosclerotic plaques in young war casualties during the
Korean War. The commonality in all of these cases was the excessively chlorinated drinking water which
the soldiers drank under field conditions.
Thus ex imining the effect of chlorinated drinking water in a validated, CHD-susceptible animal model
was deemed necessary. Based on this need a subchronic study ‘ icing the White Carneau Pigeon was in-
itiated. It was shown that chlorinated drinking water raises pI&cm i total cholesterol levels and increases
atherosclerotic plaque formation in the birds when fed diets rich in cholesterol and saturated fatty acids.
In order to more closely approximate human health effects, we recently completed a 14 month chronic
study n ing two species of non-human primates (Cercopithecus, Rhesus). In the first phase of the study
the hyperlipemic response to 15% lard, 1% free cholesterol was established in 21 weeks. This was fol-
lowed by chpnging the drinking water of the monkeys to 30 mg/L free available chlorine as NaOCl for 11
weeks, followed by return to distilled water for 13 weeks. Finally for 12 weeks the animals received 5 mglL
NaOC1. The following lipid parameters were measured weekly in fasting serum: total cholesterol,
triglycerides, high density lipoprotein cholesterol by precipitation and lipoprotein electrophoresis
(agarose gel). Indices of atherosclerosis (as commonly applied for human risk) were computed, namely
cholesterol/H1)L ratio, LDL content, LDL/HDL ratio and sum of alpha bands/sum beta, pre-beta bands
ratio in electrophoresis pattern. For the purposes of statistical analysis each animal served as its own con-
trol.
Results of the study indicate that 30 ppm hypochlorite in the drinking water significantly elevated the
atherosclerotic indices of 12 out of 13 animsils (both species, two sexes.) In worst cases the
cholesterol/HDL ratio increased as much as 20 fold over the control values.
The data suggest that the increase in atherogenic indices was primarily due not to elevation in serum lipids
(e.g. cholesterol), but rather to decreased HDL (Alpha apolipoprotein equivalent) synthesis. This im-
plies that some enzymatic pathways, involved in HDL synthesis, e.g. the compaction of chylomicrons, may
be inhibited by chlorine ingestion. Cessation of exposure to NaOC1 did not immediately reverse the ef-
fect, rather indications of a very slow return to pre-chiorine baseline values were observed. At 5 ppm
NaOCl a detectable trend, however not statistically significant, occurred in 2 of the 12 sinim k . (Two
monkeys had to be sacrificed due to endometriosis, not related to treatment.) Concurrent with the animal
studies a clinical trial was recently conducted in human volunteers to determine whether chlorinated
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WATER HEALTH ACCOMPLISHMENTS
drinking water affects human lipid metabolism and, if it does, which lipoproteins and apolipoproteins are
so affected. The study consisted of a 15-week dose-response trial in healthy male volunteers on an in-
patient metabolic ward where all factors known to affect human lipid metabolism were controlled. Each
subject consumed 1.5 liters of study water daily and extra distilled water as needed. The doses of chlorine
were 0 ppm (3 weeks), 2 ppm (4 weeks), 5 ppm(4 weeks) and 10 ppm (4 weeks). The subjects consumed
a diet with a caloric distribution of 40% carbohydrate, 40% fat and 20% protein, with 600 mg of dietary
cholesterol! day, and with a polyunsaturatethaturated (P/S) fat ratio of 0.4, which is known to affect
human lipid metabolism. The diet is isocaloric, e.g. it is adjusted as necessary to maintain subjects at the
same weight. While this diet is relatively high in cholesterol and saturated fat, it is typical for many
Americans. Exercise and smoking were held constant, no alcohol intake was permitted. Serum
lipoprotein profiles consisting of plasma total cholesterol, triglycerides, high-density lipoprotein
cholesterol, calculated low-density lipoprotein cholesterol and apolipoproteins Al, A2 and B were deter-
mined on each subject daily for 4 days at the end of each dosage period. Thyroid function tests and a bat-
tery of safety tests were also performed periodically. Only minor effects on human lipid and thyroid me-
tabolism were seen during this first study. An additional study is currently being conducted with both
male and female volunteers at a higher chlorine concentration of 20 ppm.’
Presently it is concluded that while the etiology of atherosclerotic CHD cannot be connected with
chlorinated drinking water, further work needs to be done in elucidating the role of intra-alimentary
halogenation of nutrients with lipid and lipoprotein metabolism.
Issue: Determine mechanisms of carcinogenic toxicity of water disInfection by-products.
Interest in the carcinogenicity of chlorinated acetic acids arose because, as major metabolites of
chlorinated ethylenes, research suggested that their formation and accumulation in test species were the
basis for the carcinogenicity of these chemicals. Interest became concern when chlorinated acetic adds
were found in fin ished drinking water as by-products of chlorination. Recent studies by the Toxicology
and Microbiology Division demonstrated the carcinogenic potential of dichioroacetic add (DCA) and
trichloroacetic add (TCA) in mice supplied drinking water containing these chlorinated adds. The adds
appear to be complete carcinogens since prior initiation did not increase tumor incidence.
Much of our work with by-products of drinking water dishifection is concerned with developing a data
base on the mechanisms of action of chlorinated acids so that projections of the risk in human popula-
tions exposed to these chemicals can be estimated. While the carcinogenidty of DCA and TCA was
demonstrated, no data were obtained to quantitate exposure to these chlorinated adds or establish a
dose-response relationship. Experiments are underway to correct deficiencies in the earlier study using
the neonatal B6C3F1 mouse, an extremely sensitive test system.
Although the limited data did not allow a reliable estimate of the relationship between tumor burden
(number of carcinomas per liver) and the degree of peroxisome proliferation (palmitoyl CoA oxidase ac-
tivity) for both acids, the tumor burden per unit of peroxisome proliferation was much greater for DCA.
While our studies suggest a role for peroxisome proliferation in the carcinogenicity of chlorinated acids,
clearly other factors are also operating. We propose to examine some consequences of peroxisome
proliferation such as lipid peroxidation, generation of reactive oxygen species, and DNA damage to
evaluate the extent of the involvement of peroxisome proliferation in the carcinogenicity of the
chlorinated adds.
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Issue: Conduct epidemiologic studies of health effects associated with drinking water disinfection.
Since 1974, a number of statistical and epidemiologic studies have been conducted in the United States
to assess the relationship between cancer and drinking water quality. These studies differ markedly in
their design and in what they can reveal about this possible association. Because of the wide-spread ex-
posure to chlorination by-products, most of these studies have focused on the chlorination of drinking
water. Historical information on water sources and chlorination practices have primarily been used as
surrogate measures of exposure to THMs, but in some instances current levels of THMs have been used
in conjunction with various operational parameters to estimate historical exposure levels.
Associations between water chlorination and cancer of the bladder, stomach, large intestine and rectum
were suggested in a number of the early epidemiologic studies, which were of descriptive or ecologic
design. Since these studies did not provide information on diseases, exposures and potential confound-
ing characteristics for individual study participants, additional analytic epidemiologic studies were con-
ducted. A review of five case-comparison mortality studies completed through 1981 concluded that in-
formation provided by the studies strengthened the evidence for an association between rectal, colon and
bladder cancer and water chlorination but were not sufficient to establish a causal relationship between
chlorinated by-products in drinking water and cancer. Because of methodological limitations and the
small increased risks observed in these studies, it was not possible to separate possible associations from
potential confounding characteristics which could not be assessed or controlled.
Recently, results have been reported for a cohort study and several case-comparison studies. Although
incidence rates for cancer of the bladder among men were found to be nearly two-fold higher in a cohort
supplied with chlorinated surface water at home when compared with a cohort supplied with un-
chlorinated groundwater, the reported rates from the study were felt to be unstable and subject to ran-
dom variation due to the small numbers of individuals in each cohort. The two most recently reported
studies have employed traditional case-comparison study designs and have provided an indication of the
magnitude of the cancer risk associated with chlorinated drinldng water for colon and bladder cancer. A
weak to moderate association (RR = 1.4 to 3.4) between water chlorination and colon cancer was ob-
served in an elderly population, and the association was stronger among the elderly who had been ex-
posed to chlorinated water for more than IS years. A moderately strong association (RR = 2.3) between
chlorinated water and bladder cancer was observed in an otherwise low-risk population (nonsmokers)
who had received chlorinated water for 60 or more years.
To further study the observed associations between chlorinated water and both bladder and colon can-
cers, EPA initiated a collaborative case-comparison study with NC! and the University of Iowa in late
1985. Approximately 2500 incident cases of cancer of the colon, rectum, bladder, brain and pancreas
(liver and kidney, if feasible) and approximately 1500 population-based controls matched for race,
gender, age will be interviewed in Iowa for information on lifetime residential history, smoking, occupa-
tion, medical history, diet and socio-economic status to determine cancer risks associated with
chlorinated drinking water and water cont Inain lnts from agricultural runoff. An exposure history will be
developed from the lifetime residential history and data on chlorine use and THMs levels.
Because chlor iminsation has been shown not to produce THMs, epidemiologic studies have been con-
ducted in Massachusetts where chlorine and ammonia have been used since 1938 to dii infect surface
water provided to most communities in the Boston metropolitan area. A recently completed mortality
study showed a slight increase in bladder cancer in populations in Massachusetts receiving chlorinated
surface water compared with populations receiving chloraniinated surface water. To address problems in
interpretation of the results of this study, a case-comparison study of 614 individuals who had died of
primary bladder cancer and 1074 individuals who had died of other causes was conducted. Confounding
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WATER HEALTH ACCOMPLISHMENTS
by age, gender, smoking, occupation and socio-econornic status was controlled by multiple logistic regres-
sion. Pre1imin iry results confirm a high risk of bladder cancer mortality in populations receiving
chlorinated drinking water; the risk of bladder cancer mortality was found to be three times the risk of
those consuming chIoriniin ited drinking water.
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Publications
Water Health
This report includes abstracts of manuscripts for journal articles published and conference
proceedings papers presented between October 1, 1985 and September 30,1986...
Programmatic Relevancy statements are italicizeS
Other articles submitted for publication, but not published as yet, are listed in Appendix A.

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PUBLICATIONS WATER HEALTH
WATER DISINFECTION: MICROBES VERSUS MOLECULES
AN INTRODUCTION of ISSUES
James R. Fowle, HI 1 and Fredenck C. Kopfler 2
1 OHR, USEPA, Washington, D.C.
2 HE USEPA, Cincinnati, OH
The disinfrcdon of d inIdng waler is one of the most wide y practiced and most successful public health
programs in the U.S. While it has drwnatically reduced the incidence of waterbome illness, it has been
detennined that the organic chemical contaminant occurring at the highest concentration in drinking water
result from it. This symposium paper summwizes the results to date of the Agency’s research into the
health effect of these contaminants.
Because water is essential for life, hIlm2nc have taken measures to insure its quality for thousands of years.
It is known that human and other activities result in the introduction of a multitude of microbes into our
drinking water. A number of these are pathogenic to hiimi ns , causing a variety of effects ranging from
mild gastrointestinal distress to systemic disease and sometimes death. We are capable of providing
finished water to the customer that is essentially pathogen free by treatment with various chemical sub-
stances. However, many of these substances are capable of causing adverse biological effects or they
react with other substances present in the water to form by-products capable of causing adverse biologi-
cal effects. This introduces a tension and a paradox. If the chemicals used to rid drinking water of dis-
ease-causing microbes are themselves potentially harmful, is drinking water safe? What tradeoffs are ac-
ceptable with respect to water quality, microbial vs. chemical? This conference deals with current think-
ing about these topics and as much as the conference will contribute to the future, the subjects discussed
reflect the evolution of tlinking, both scientifically and socially, about how to best supply the public with
safe, pure potable water. The goal of this chapter is to introduce the issues associated with dkinfectants
and disinfectant byproducts in water. This will be done by presenting an historical overview of the use of
chemical disinfectants to purify drinking water and the subsequent awareness of potential health con-
cerns.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Drinking Water
Disinfectants and Disinfection By-Products, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69:3-6, November 1986.
C104 MS-86- 164 Project Officer: Kopfler
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TYPE of DISINFECTANT in DRINKING WATER and PATIERNS of
MORTALITY in MASSACHUSETTS
Sally Zierler, RobeitA. Dan fry, and Lisa Feingold
Massachusetts Department of Public Health
The disinfection of drinking water with chlorine is widely practiced and has dnunatically reduced the in-
cidence of waterbome illness in the U.S. Under ceitain conditions, the reaction of chionne with precursor
substances in water produ ce.r organic by-products which have been found to be caicmogenic at high doses
in animal studies and mutagenic in bacterial tests. Some water supplies disinfect with chlorine and am-
monia, producing chioramines which do not react to produce these organic by-products. This paper com-
pares the monali y of populations in Massachusetts consuming chlorinated and chioraminated drinking
water.
Patterns of mortality were e mniined for residents in Massachusetts who died from 1969-1983 and lived in
communities Ilcing drinking water that was dkinfected either by chlorine or a combination of chlorine and
ammonia. Comparison of type of disinfectant among 51,645 cases of deaths due to selected cancer sites
and 214,988 controls who died from cardiovascular, cerebrovascular, or pulmonary disease, or from lym-
phatic cancer showed smnll variation in the patterns of mortality. Bladder cancer was found to be
moderately associated with residence at death in a chlorinated community (mortality odds ratio = 1.7)
from a logistic regression analysis using controls who died from lymphatic cancer. A slight excess of
deaths from pneumonia and influenza was observed in communities whose residents drank
chlornminnted water compared to residents from chlorinated communities, as well as to all Mas-
sachusetts residents. These results are intended to be preliminary and crude descriptions of the relation-
ship under study. The serious potential for misclassification of exposure status and errors in death cer-
tificate classification of cause of death affect the interpretability of the overall evidence that patterns of
mortality are imi1nr according to disinfectant in drinking water. These data, however, provide justifica-
tion to conduct a bladder cancer case-control study to determine if bladder cancer cases are more likely
to have resided in communities using chlorine as a disinfectant compared to chlorine and ammonia. This
study is currently being planned.
CITATION: 1n Proceedings of the Second International Symposium on Health Effects of Drinking Water
Disinfectants nad Disinfection By-hoducts, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69: 275-279, November 1986.
C104 XX-86-117 Project Officer: Craun
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PUBLICATIONS WATER HEALTh
EPIDEMIOLOGIC STUDIES of ORGANIC MICROPOLLUTANTS in
DRINKING WATER
GuntiserF. Craun
HERL, USEPA, Cincinnati, OH
The disinfection of drinking water with chlonne is wide y practiced and has drainaticaUy reduced the in-
cidence of wate*orne illness in the US. Under ceitain conditions, the reaction of chloiine with precursor
substances in water produces organic by-products which have been found to be carcinogenic at high doses
in animal studies and mutagenic in bacterial tests. This paper summanses the results to date of the
Agency’s epidemiologic studies of the health effects of these organic micropollutants.
Epidemiologic studies have been conducted in order to make a quantitative statement about associations
between drinking water cont nhini nts and disease. The basic measures of the association are a rate ratio
or relative risk and rate difference or attributable risk. The appropriateness of this measure is depend-
ent on components of study design, data collection, and the analysis of epidemiologic data, and these must
be evaluated for each study to determine precision (lack of random error) and validity (lack of systematic
error). Internal validity includes considerations for preventing selection bias, minimizing observation
bias, and asse.c.cing, preventing and controlling confounding bias within a particular study. No single
epidemiologic study is likely to provide a definitive answer, and the results of epidemiologic studies must
be interpreted in the context of other scientific information. Epidemiologic studies of orgainc micropol-
lutants in drinking water have been reviewed and are summarized based on these considerations. The
two most recently reported case-comparison epidemiologic studies have provided an indication of the
m gniIude of the cancer risk associated with chlorinated water for colon and bladder cancer. A weak to
moderate association between water chlorination and colon cancer was observed in an elderly popula-
tion, and this association was found to be stronger among the elderly who had been exposed to
chlorinated water for more than 15 years. A moderately strong association between chlorinated water
and bladder cancer was observed in an otherwise low-risk population of nonsmokers, who had received
chlorinated surface water for 60 or more years. Additional case-comparison or cohort studies are re-
quired to confirm these findings and are currently being planned. Any interpretation as to the casual as-
sociation suggested by these studies should await the completion of these planned studies and other
relevant research in this area.
CITATION: The Science of the Total Environment 47:461-472, November 1985.
C104 MS-86-074 Project Officer: Craun
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WATER HEALTH PUBLICATIONS
A SUMMARY of WATERBORNE ILLNESS TRANSMITI’ED ThROUGH
CONTAMINATED GROUNDWATER
Gunther F. Craun
HERL, USEPA, Cincinnati, OH
Data are required to identify water supply deficiencies responsible for waterbome disease outbreaks to
prevent the recunrnce of outbreaks. The Office of Drinldng Water (ODW) uses these data to develop
regulations for safe diinldng water.
The use of cont imin ted, untreated or inadequately treated groundwater (a) was responsible for 51% of
all waterborne outbreaks and 40% of all waterborne illness reported in the United States during 1971-82.
Contfimin2ted, untreated or inadequately di nfected groundwater caused 65% of the waterborne out-
breaks and 66% of the waterborne illness which occurred in noncommunity and individual water systems
but only 32% of the outbreaks and 31% of illness in community water systems. Illnesses most frequently
transmitted through groundwater included acute gastroenteritis of undetermined etiology, chemical
poisonings, hepatitis A, shigellosis, and viral gastroenteritis. Waterborne outbreaks in water systems
using untreated well water were caused primarily by the overflow or seepage of sewage from septic tanks
or cesspools, chemical cont min ’tion, and surface runoff cont mrnition. An increase in the number of
outbreaks resulting from the use of untreated, contiin’inated well water was noted during the summer
months.
CITATION: Journal of Environmental Health 48(3): 122-127, Nov/Dec 1985.
C104 MS-85-078 Project Officer: Craun
THE LUBBOCK LAND TREATMENT SYSTEM RESEARCH and
DEVELOPMENT PROJECT VOLUME IV LUBBOCK INFECTION
SURVEILLANCE STUDY(LISS)
D. E. Q n, 1 P.1. Grahwn, 2 M. N. Guentze4 3 H. I. Hardin& 1
K T. Kl,nbaI4 1 B. E. Moore 4 R. L Noithrop, N. L Albnan,
R. B. Hazr&s4 5 A. H. Holguin,’ R. L Mason, 1 C. Becker Popescu, 2
and C. A. Sojber 4
1 Southwest Research Institute
2 University of Illinois-Chicago
kJniversity of Texas-San Antonio
4 University of Texas-Austin
kiniversity of Texas School of Public Health
Evidence of seroconversion to enteric viruses was found in a population exposed to wastewater aerosols
through spray imgation. Additional analyses of these data are being perfonned in a separate study. This
study adds to the data base onoccun nce and significance of microorganisms in wastewater aerosols. It
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PUBLICATIONS WATER HEALTH
should be of value to the Office of Water Operations in developing cnteria and guidelines for the
safe treatment and disposal of domestic waste.
This study was conducted to identify possible adverse effects on human health from slow-rate (sprinkler)
land application of wastewater which contained pathogens. An epidemiological study of 478 area resi-
dents and farm workers was maintained during the first 20 months of sprinkler operation and during the
20-month period immediately preceding initiation of wastewater irrigation. Blood samples were analyzed
for antibody titers to 23 viruses, Legionella and Entamoeba histolytica. Fecal specimens were collected to
isolate pathogens; electron microscopic e,mmination was performed to detect virus-like particles, and
tuberculin skin tests were administered. Weekly illness information was provided by participants.
Microorg2nisms were measured in the wastewater, aerosols and drinicing water. An index of relative
cumulative exposure of each participant to the wastewater aerosol was calculated. High levels of bacteria
and enteric viruses were present in the wastewater obtained via pipeline from the treatment plant.
Enteroviruses were found in the wastewater aerosols. Disease surveillance did not disclose any obvious
connection between acute illness and aerosol exposure but three different statistical approaches indi-
cated that the rate of viral infections was slightly higher among participants who had a high degree of
aerosol exposure. However, it could not be determined whether aerosol exposure or alternative explana-
tions were the actual risk factor(s) in these infections. The association of viral infections with aerosol ex-
posure showed a dose effect.
CITATION: US. Environmental Protection Agency, cooperative agreement no. CR-807501, EPA-
600/S1-85-023, January 1986.
BlOl HERL-0526 Project Officer: Jakubowski
MUNICIPAL SEWAGE SLUDGE APPLICATION on OHIO FARMS:
HEALTH EFFECTS
C. R. Dom, C. S. Reddy, D. N. Lainphere,
J. V. Gaeuman, and R. Lanese
The Ohio State University
This journal an icle contains results from HERL’s research program on the health aspects of the land ap-
plication of municipal sludge. It deals with the human exposure to heavy metals, organic chemicals, and
paihogens as a result of land application, and potential adverse health effects.
A 3-year prospective epidemiologic study was conducted on 47 farms receiving annual applications of
treated sludge (average of 2-10 dry metric tons/ha/year) and 46 control farms in three geographic areas
of Ohio. On the sludge-receiving farms 164 persons (78 families) and on the control farms 130 persons
(53 families) participated by cooperating with monthly questionnaires concerning their health and their
animals’ health, annuaJ tuberculin testing, and quarterly blood sampling for serological testing. The es-
timated risks of respiratory illness, digestive illness, or general symptoms were not significantly different
between sludge farm and control farm residents. Similarly, there were no observed differences between
disease occurrence in domestic animals on sludge and on control farms. No conversions from negative to
positive tine test results occurred after sludge had been applied to the farms. The frequency of serologi-
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cal conversions (fourfold or greater rise in antibody) to a series of 23 test viruses and the frequency of as-
sociated illnesses were similar among persons on sludge and control farms. The absence of observed
human or anim2l health effects resulting from sludge application in this study of Ohio farms was as-
sociated with low sludge application rates which were in accordance with Ohio and U. S. Environmental
Protection Agency guidelines. Caution should be exercised in using these data to predict health risks as-
sociated with sludges containing higher levels of disease agents and with higher sludge application rates
and larger acreages treated per farm than used in this study.
CITATION: Environmental Research 38(2): 332-359, December 1985.
B113 XX-86- 127 Project Officer: Kowal
HEALTH RISKS ASSOCIATED with WASTEWATER IRRIGATION:
AN EPIDEMIOLOGICAL STUDY
Radii Fatta4 Yohanan Ww Michael Davies , and Hillel L Shuval
Hebrew University of Jerusalem
This study su ested that sprinkler inigation with poor microbial quality paflial y treated stabilization pond
effluent resulted in a higher enteaic disease rate diving irrigation periods hi the 0 to 4 year-old age group.
The significance of these findings with respect to non-kibbutz communities is not clear.
An analysis of morbidity was made in 11 kibbutzim (cooperative agricultural settlements), with a total
population of 3,040, that had switched from nonwastewater to wastewater sprinkler irrigation or vice
versa. Generally, partially treated stabilization pond effluent of poor microbial quality was used for ir-
rigation. Vegetables or salad crops were not irrigated with effluent. The results showed that a seasonal,
twofold, excess risk of “enteric disease was found in the 0 to 4 year-old age group during the summer ir-
rigation months in those years in which wastewater was used for irrigation, compared with the parallel
summer months of nonwastewater irrigation years in the same kibbutz. On the year-round rates basis, lit-
tle or no excess enteric disease was found in wastewater irrigating communities.
CITATION: American Journal of Public Health 76(8): 977-979, August 1986.
B113 XX-86- 140 Project Officer: Jalcubowski
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COMPARISON of THREE STUDIES PERFORMED in ISRAEL on HEALTh
RISK ASSOCIATED with WASTEWATER IRRIGATION
Bad f Fatta4 Yochwzan Ww Ta naAgu,cky, and Hillel L Shuval
The Hebrew University - Hadassah Medical School
ThLc prospective epidemiologicoJ study of residents of 30 KIbbutzim in Israel was conducted to obtain data
on the infectious desease hazards associated with the land application of wastewater. especial4y by spray
application.
Three epidemiological studies were performed in Israel in order to determine the health risk associated
with the u ill tion of wastewater effluent from stabilization ponds for agricultural use (Study I - Katzenel-
son Ct a!., 1976; Study II- Fattal et aL, 1981 and Study ifi - Shuval et aL, 1985). This paper compares the
three studies regarding the incidence rates of shigeliosis, salmonellosis and viral hepatitis in populations
of agricultural communities using wastewater effluent for irrigation with those not using wastewater ef-
fluent. In no case was effluent used for irrigation of edible crops, and it is assumed that the potential
routes of exposure were aerosolized wastewater or direct contact with wastewater or with wastewater ex-
posed workers. Only in Study I was a statistically significant excess of shigellosis, salmonellosis and viral
hepatitis found in the wastewater-irrigating kibbutzim. However, Study I had serious methodological
problems and does not, in our opinion, permit drawing firm conclusions. No significant excess of the
three diseases were found for wastewater irrigating kibbutzim in Studies II and ifi for any age-group,
neither for the irrigating period nor for the entire year. We consider the “switch” study (part of Study II),
based on a natural “before and after” experiment of switching irrigation from wastewater to nonwas-
tewater and vice versa, and Study III, a prospective study with strict quality assurance, to be the more reli-
able. Based on these two studies, we conclude that there is little or no health risk associated with was-
tewater utilization for the general population exposed to wastewater irrigation.
CITATION: In: Environmental Quality and Eco ystenz Stability, vol. ifi - A&B, Z. Dubinsky andY. Stein-
berger, eds., Bar-ilan University Press, Ramat-Gan, Israel, 783-793,1986.
B113 XX-87-032 Project Officer: Akin
METHODOLOGY for STATISTICAL ANALYSIS of
SENCAR MOUSE SKIN ASSAY DATA
/udyA. Stober
HERL, USEPA, Cincinnati, OH
The results of the research described in the attached workshop proceedings article fluiher characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in toxicological and
carcinogenesis erperimentalion. The data deals with three categories relevant to the response of the mouse
in Iaborato y tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initia-
tion/promotion assay), and (3) histopathology including treat,nent-induced and spontaneous lesions.
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Various response measures and statistical methods appropriate for the analysis of data collected in the
sencar mouse skin assay are examined. The characteristics of the tumor response data do not readily lend
themselves to the classical methods for hypothesis testing. The advantages and limitations of convention-
al methods of analysis and methods recommended in the literature are discussed. Several alternative
response measures are described which were developed specifically to answer the problems inherent in
the data collected in the sencar bioassay system. These measures take into account nnimsil survival, tumor
multiplicity and tumor regression. Statistical methods for the analysis of these measures testing for a
positive dose response and a dose response relationship, are discussed. Sample data from representative
initiation/promotion studies are used to compare the response measures and methods of analysis.
CITATION: Environmental Health Perspectives 68: 5-10, September 1986.
C104 MS-86-11.3 Project Officer: Stober
AN OVERVIEW of STATISTICS on ACUTE and CHRONIC WATER
CONTAMINATION PROBLEMS
Gunther F. Craun
HERL, USEPA, Cincinnati, OH
The disinfection of drinking water with chlorine Lv widety practiced and has dramatical y reduced the in-
cidence of wuierbome illness in the U.S. Under certain conditions, the reaction of chlorine with precursor
substances in water produces organic by-products which have been found to be carcinogenic at high doses
in animal studies and mutagenic in bacterial tests. This paper summarizes the results to date of the
Agency’s epidemiologic studies of the health effects of these organic micropollutanLv. The emphasis of this
paper Lv on the occunence of wateibome-disease outbreaks and epidemiologic studies of the association of
drinking water contaminants with cardiovascular disease and cancer. During 1920 through 1983, 1531
waterbome outbreaks were reported in the U.S.: 413 329 cases of illness and 1083 deaths were associated
with these outbreaks.
Two case-comparison epidemiologic studies have provided an indication of the magnitude of the cancer
risk associated with chlorinated water for colon and bladder cancer. Additional case-comparison or
cohort studies are required to confirm these findings and are currently being planned. Any interpreta-
tion as to the causal association suggested by these studies should await the completion of these planned
studies and other relevant research in this area. The major cause of outbreaks was the use of con-
taminated, untreated groundwater, but the greatest number of cases of waterborne disease resulted from
outbreaks caused by deficiencies in the treatment of surface water sources. Since 1957 numerous hives-
tigators have reported an association between drinking water quality and cardiovascular disease. In
general, epidemiologic studies have found lower cardiovascular disease mortality in areas where the hard-
ness of drinking water is high. Additional evidence to support this association has been obtained from
recent epidemiologic studies in the United States and the United Kingdom, but additional research is re-
quired before specific regulations can be developed on the advisability of not removing calcium or mag-
nesium by softening drinking water supplies which are hard. Recently, results have been reported for
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several analytic, epidemiologic studies of the relationship between water chlorination and cancer in the
United States.
CITATION: In: Fourth Domestic Water Quali y Symposium, American Society of Agricultural Engineers
and Water Quality Association, 1985.
C104 MS-86-047 Project Officer: Craun
SERUM BACTERICIDAL ACTIVITY AGAINST LEGIONELILA PNEUMOPHILA
Joseph F. Plouffe, Michael F. Para and KathyA. Fuller
The Ohio State University School of Medicine
Disease outbreaks caused by Legionella pneumophila are frequently linked to potable water supplies.
Legionella pnewnophila has also been shown to vary considerably in its virulence characteristics. Some
strains are highly virulent and others are avindent. This report covers areas related to Legionella virulence
and the potential risk to individuaLs exposed to this organism. The information in this report will be useful
for making decisions relevant to maximum contaminant levels for this organism in drinking water.
Two strains of Legionella pneumophila serogroup 1 (UH1 and RH1) were incubated in fresh human
serum. The UH1 strain was serum resistant, whereas the RHI strain was serum susceptible. The bac-
tericidal activity of fresh serum was abrogated by heating. Serum resistance of L. pneumophila strains
may correlate with increased virulence.
CITATION: Journal of Clinical Microbiology 22(5): 863-864, November 1985.
C104 XX-87-O11 Project Officer: Dufour
INTRATRACHEAL INFECTION with L PNEUMOPHILA SUBTYPES
Joseph F. Plouffe, Michael F. Pare, and Kathy A. Fuller
The Ohio State University School of Medicine
Disease outbreaks caused by Legionella pneumophila are frequently linked to potable water supplies.
Legionella pneumophila has also been shown to vary considerably in its virulence characteristics. Some
strains are highly virulent and others are avindent. This report covers areas related to Legionella virulence
and the potential risk to individuals exposed to this organism. The information in this report will be useful
for making decisions releveant to maximum contaminant levels for this organism in drinking water.
Two strains of Legionellapneumophila sero-group 1 (UH1 and RH1) were injected intratracheally into
guinea pigs. There were no differences in infectivity or mortality, but there was evidence of disseminated
infections in more guinea pigs infected with the UH1 strain. Guinea pigs that received sublethal inocula
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were then rechallenged intratracheally and had a lower mortality rate than did previously uninfected con-
trols.
CITATION: Journal of Clinical Laboratorj Immunology 20(3): 119-120, March 1986.
C104 XX-87-012 Project Officer: Dufour
ORAL INGESTION of LEGIONELLA PNEUMOPHILA
Joseph F. Plouffe Michael F. Para Kathy A. Fuller, and Garj E. Bollin
The Ohio State University School of Medicine
Disease outbreaks caused by Legionella pnewnophia we frequently linked to potable waler supplies.
Legionella pneumophila has also been shown to vwy considerably in its vi,ulence characteristics. Some
strains we highly vindent and others are aviiulent. This report covers areas related to L4onella vindence
and the potential risk to individuals exposed to this organism. The infomiation fr i this report will be useful
for making decisions releveant to maximum contaminant levels for this organism in drinldng water.
Guinea pigs were fed L. pneumophila through an orogastric tube. Gastric acid was rapidly cidal to the
organisms. Serial necropsies demonstrated that organisms in the colon and blood stream at 1 hr. Guinea
pigs fed large doses of L pnewnophila seroconverted. Previously fed guinea pigs were then challenged
with a lethal intraperitoneal dose of L. pnewnophila and were protected in a dose-dependent fashion.
Pretreatment of the guinea pigs with cimetidine lowered the dose of oral L. pneumophila required for
protection. This model may be useful in studying the immune response after oral ingestion of L
pneumophila.
CITATION: Journal of Clinical Laboratoiy Immunology 20(3): 113-117, March 1986.
C104 XX-87-013 Project Officer: Dufour
AEROSOLS CONTAINING LEGIONELL4 PNEUMOPHILA GENERATED by
SHOWER HEADS and HOT-WATER FAUCETS
Gaiy E. Bollin, Joseph F. Plouffe Michael F. Para and Bwbara Hackinan
The Ohio State University School of Medicine
Disease outbreaks caused by Legionella pneumophila are frequently linked to potable water supplies.
Legionella pnewnophila has also been shown to vaay considerably in its vindence characteristics. Some
strains we highly vindent and others we avinilent. This report covers areas related to Legionella vindence
and the potential jisk to individuals exposed to this organism. The information in this report will be useful
for making decisions releveant to maximum contaminant levels for this organism in ddnking water.
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Shower heads and hot-water faucets contz iningLegionellapneumophila were evaluated for aerosoliztion
of the org lnism with a multistage cascade impaction air sampler. Air was collected above two shower
doors and from the same rooms approximately 3 ft (91 cm) from the shower doors while the hot water was
rilnning Low numbers (3 to 5 CPU/iS ft 3 [ 0.43 m 3 J of air) of L pneumophila were recovered above both
shower doors, but none was recovered form the air in either room outside the shower door. Ap-
proximately 90% of (7 of 8 CPU) of the L pnewnophila recovered were trapped in aerosol particles be-
tweenland5M.mindiameter. Airwascollectedlto3ft(3Oto9lcm)froml4sinkswhilethehotwater
was running . Low numbers (1 tp 5 CFU/15 ft 3 of air) were recovered from 6 to 19 air samples obtained.
Approximately 50% (6 of 13 CPU) of the organcms recovered were trapped in aerosol particles between
1 and 8 p.m in diameter. Shower heads and hot-water taps conuuining L pneumophila can aerosolize low
numbers of the orgiintcms during routine use. The aerosol particle size is small enough to penetrate to
the lower human respiratory system. Thus, these sites may be implicated as a means of tr2ncmcsion of L.
pnewnophila from potable water to the patient.
CITATION: Applied and Environmental Microbiology 50(5): 1128-1131, November 1985.
C104 XX-87.010 Project Officer: Dufour
GJARDL4 DETECTION in WATER SYSTEMS
Judith F. Sauch
HERL., USEPA, Cincinnati, OH
An iinmunofluorescence method to detect and identify Giardia cysts in water samples following sample
concentration was developed. The ininiunofluorescence method selectively stains cysts and enchances con-
trast between cysts and other sample constituents an4 then fore, allows quick location of cysts by their green
fluorescence. This technique may be useful fr i confinning outbreakr of gia,d iasis as wate,bome, providing
guidance on treabnentprosesses, and establishing baseline data on the prevalence of Giardia cysts in water
supplies.
A method has been developed combining immunofluorescence and phase-contrast to microscopically lo-
cate and identify Giardia cysts among particulates filtered from water samples. Selective st uining of cysts
with anti-Giardia antiserum and a fluorochrome enhances the contrast between cysts and other sample
constituents and allows quick location of cysts by their green fluorescence. Their identity can then be con-
firmed with phase-contrast observation of size, shape, and internal morphology. This method was suc-
cessfully tested during recent outbreaks in Pennsylvania and Massachusetts. However, when chlorinated
water samples were tested, confirmation of presumptive Glare/ia cysts was difficult or impossible. Subse-
quent laboratory exposure of Glare/ia cysts to various chlorine levels showed that although these cysts
could still be detected by flurorescence, their internal morphology had been destroyed. These methods
for detecting and identifying Giardia cysts may be useful in confirming outbreaks, providing guidance on
treatment processes, and establishing baseline data on the prevalence of Giardia cysts in water supplies.
CITATION: In: Proceedings of the 3rd Conference on Progress in Chemical Disinfection , Binghamton, NY,
April 3-6, 1986.
C104 MS-86-252 Project Officer: Saudi
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WATERBORNE GIARDIASIS in the UNITED STATES 1965-1984
G. F. Crawi
HERI., USEPA, Cincinnati, OH
Giardia lamblia Lv the most frequ ent4 v idenufied pathogen in wateibosne outbreaks and data we required to
identify those water deficiencies which are responsible for these outbreaks. Most waterbome outbreaks of
giardiasLc have occwred as a result of consuming contaminated surface water, and a regulation requinng
filtration of swf ace water supplies Lv under consideration by the Office of Drinking Water (ODW).
Ninety waterborne outbreaks and 23,776 cases of giardiasis have been reported in the United States
through 1984. Approximately 69% of the outbreaks and 87% of the cases occurred in community water
systems. Most outbreaks (57%) and cases (52%) resulted from the use of contaminated surface water
which had not been treated or had been treated only by simple chlorination. Fifteen outbreaks (17%) and
7,440 cases (31%) were associated with operational problems on inadequate design of water filtration
plants. A small but signficant (15%) number of cases of giarLs ’cis resulted from outbreaks caused by
contamination of water msinc through cross-connections, d2niagc of mains, and repair of miiins .
CITATION: Lancet (8505): 51.3-514, August 1986.
C104 MS-86-231 Project Officer: Craun
DRINKING WATER TRANSMISSION of GIARDIASIS in the UNITED STATES
E. W. Akin, and W. Jakubowski
HERL, USEPA, Cincinnati, OH
Giardia is a microorganism (protozoan) that has a life cycle in a wide vwie y of animal species including
man. In recent years ft has increasingiy become recognized as a significant cause of gastroenteii& It was
first reported as the agent responsible for a U.S. waterbome outbreak in 1965. Since that date, 90 water-
borne outbreaks with 23 776 cases of gastroente itis have been attributed to this agent. The Office of Drink-
ing Water (ODW) Lv considering additional treatment regulations to control its transmission via drinking
water.
For about a century after Giardia was named by Lamb l in 1859, the occurrence of the organism in hnnl2ns
was not widely considered significant in expkin ng the etiology of disease. This organism is now recog-
nized as a cause of disease that ranges from mild to severe and debilitating gastroenteritis. Beginning in
1965, drhiking water became increasingly implicated as an important route of transinis.cion. An outbreak
occurred in a Colorado ski resort in that year. A survey of visitors to the resort revealed that 123 persons
experienced acute enteritis, possibly giardinsis Sewage contamination of the well water supply was
thought to be the source of the organicm Further evidence for waterborue trancmkcion of Giwdia came
in 1975 with the first isolation of the organism from a water supply implicated in an outbreak A cyst was
isolated from the water supply during an extended outbreak that resulted in 359 confirmed cases over a
7-month period. Subsequent outbreaks have clearly established the role of water in Giardia transmission.
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From 1965 through 1984,90 waterborne outbreaks with 23,776 cases of giardiasis were reported in the
United States. This report discusses the etiologic agent and e mhies drinking water outbreak occur-
rence and control.
CITATION: In: Water Science and Technology 18(1O):219-226, August 1986.
C104 MS-86-228 Project Officer: Akin
USE of IMMUNOFLUORESCENCE and PHASE-CONTRAST MICROSCOPY for
DETECTION
and IDENTIFICATION of GIARDL4 CYSTS in WATER SAMPLES
Judith A. Sauch
HERL, USEPA, Cincinnati, OH
Giardia lwnblia is the most frequently identified pathogen in waterborne disease outbreaks in the U.S. The
Safe Thinking Water Act requires EPA to establish standards for pathogens that present a public health
hazard. Thus, methods are needed to detect and ident ify Giardia cysts in water samples. Current methods
for detection of Gia,dia cysts involve tedious, time-consuming esaminalion of water sample sediment by
microscope. This study has decreased the time and improved the ease with which Giardia cysts can be
detected because the cysts are selectively stained bug/it green with Giardia antibody and a fluorescent tag.
This procedure pennits individual green cysts to be quickly located against a daric background even in water
samples heavily contaminated with other microorganisms and debris Giardiai cyst conflnnation by
visualization of this unique internal morphology is made possible by viewing detected cysts with phase-con-
trast microscopy.
A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used
for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giar-
die cyst antiserum and fluorescein conjugate were applied to known Giardia cysts on membrane filters,
the cysts fluoresced bright green when they were illuminated by UV light. This procedure permitted in-
dividual cysts to be quickly located even in samples heavily contaminated with other microorgzanicms and
debris. The identity of presumptive Giardia cysts located in this way could then be confirmed by observ-
ing characteristic internal morphological features with phase-contrast microscopy. With this method,
Giardia cysts were detected and their identities were confirmed in samples taken from raw and finished
surface water supplies during several recent outbreak
CITATION: Applied and Environmental Microbiology 50(6): 1434-1438, December 1985.
C104 MS-85-138 Project Officer: Sauch
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DETECTION and IDENTIFICATION of GIARDIA CYSTS USING
IMMUNOFLUORESCENCE and PHASE CONTRAST MICROSCOPY
Judith A. Sauch
HERL, USEPA, Cincinnati, OH
This manuscnpt was presented as a lecture at the 1984 Annual Meeting of the American Water Works As-
sodation. It was intended to introduce the concept of selective’y tagging Giardia cysts h i water samples
with Giardia antibody and a fluorescent dye. Giardia cysts in drinking water pose a public health hazard
and therefore must be regulated by the EPA. Fluorescent antibody methods as desaibed in this manuscript
improve detection of organisms like Giardia that are present in small numbers in water sample sediments.
These methods make use of the unique and selective reaction between an antigen (i.e., Giardia cyst), and
its antibody. Because the antibody can be taxed with a fluorescent stain, cysts glow green among sample
sediments when illwninated by UV llght Identification of detected presumptive Giardia cysts by fluores-
cent antibody methods is then possible by phase-contrast microscopy which allows visualization of the in-
ternal morphological characteristics unique to Giardia cysts.
The flagellated protozoan, Giardia, recognized now as the most common intestinal parasite in the U.S.,
can be trsnsmitted via municipal waler supplies and mountain streams. Coliform counts are unreliable
as indicators for the presence of Giardia cysts in water supplies because the cysts are much more resis-
tant to disinfection. In the absence of a cultural method, detection and identification of these protozoans
has been accomplished by direct microscopic observation of sample concentrates. In this method, the ob-
server must locate and identify Giardia cysts among numerous microorgsnicms of cimilsir size and shape
and against a background of debris.
CITATION: In: Proceedings of the l2thAnnualAmencan Water WorlcsAssociation Water Quality Technol-
og, Conference, 79-86,1985.
C104 MS-85-049 Project Officer: Saudi
COMPARISON of ANIMAL INFECTIVITY and EXCYSTATION AS MEASURES
of GL4RDL4 MUPJS CYST INACTIVATION by CHLORINE
J. C. Hoff, 1 E. WRice, 1 andF. WSchaefer, III
l j 1 L, USEPA, Cincinnati, OH
2 HE USEPA, Cincinnati, OH
These procedures are to determine Giardia cyst viability. The Agency and the cooperative grantees have
used these procedures for disinfection studies in water treatment.
In this study, in vitro excystation and mouse infectivity were compared as methods for quantitatively deter-
mining the viability of Giardia muris cysts before and after exposure to free residual chlorine. The mouse
infectivity results show that very few cysts (1 to 15) constitute an infectious dose. The results of the mac-
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tivation studies indicate that in vitro excystation is an adequate indication of G. mwis cyst infectivity for
the host and can be used to determine the effects of disinfectants on cyst viability.
CITATION: Applied and Environmenlal Microbiology 50(4): 1115-1117, Octobcr 1985.
C104 MS-85- 101 Project Officer: Scbaeffer
GIARDL4 MURtS: ULTRASTRUCTURAL ANALYSIS of
IN VITRO EXCYSTATION
James R. Co ins 1 and Frank W. Schaefer, jj2
1 University of Wisconsin
2 HE USEPA, Cincinnati, OH
These procedures are to detennine Giardia cyst viability. The Agency and the cooperative grantees have
used these procedures for dLcinfection studies in water treatment.
Giardic muris cysts were exi mined by TEM before treatment post-induction, and at timed intervals
during incubation. Untreated G. niwis cysts had a thick cyst wali composed of a fibrous outer wall and a
thin, electron dense inner membrane which extended from the trophozoite plasma membrane. The
cytoplasm was devoid of endoplasmic reticulum, Golgi bodies, and mitochondria. Numerous large
vacuoles were present within the ectoplasm just beneath the plasma membrane in untreated cyst. Follow-
ing induction these cysts lacked ectoplasmic vacuoles. Concurrently, numerous membrane-bound
vesicles were seen in the peritrophic space dosely adhering to the surface of the trophozoite. These
vesicles appear to be of cytoplasmic origin. The cytoplasm of fully excysted trophozoites lacked ectoplas-
mic vacuoles but displayed well developed ribbons of microtubules, probably precursors of the ventral
disk, lateral flange, and median bodies and also contained extensive endoplasniic reticulum. No more
than two nuclei were observed within each organism. The earliest excysted organism c were observed 0-5
minutes after incubation had begun, and most organisms had excysted within 10 minutes. Cytokinesis oc-
curred only after excystation was complete.
CITATION: Erpen nental Pai’asitology 61: 219-228, April 1986.
C104 MS-85-100 Project Officer: Schaeffer
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BILIARY LIPIDS SUPPORT SERUM-FREE GROWHI of GL4RDIA LAMBLL4
Frances D. Gillin, Michael I. Gaul4 1 Alan F. H nann, 1
Devorah Gurangz; 1 and Judith F. Sauch
‘Universip of California-San Diego Medical Center
HERI., USEPA, Cincinnati, OH
The administration of toxic chemicals dwing ea4y pregnancy in humans may cause undetected fetal loss.
In the present study control injections caused aboitions during pregnancy in the rabbit by inhibiting ovarian
function.
Giardia lamblia has been grown in vitro only in media containing serum or serum fractions. How this
pathogen can grow in the hIlm2n small intestinal lumen without serum is not known. We found that
samples of human hepatic or gall bladder bile maintained G. lambliai survival for 24 to 48 h in medium
without serum but did not support growth. By contrast, an artificial biliary lipid dispersion containing six
bile salts, phosphatidylchollne (PC), and cholesterol, in the ratios characteristic of human bile, supported
parasite growth in medium without serum or serum fractions. To define the requirements, we showed
that 1-palmitoyl-2-linoleoyl-PC or 1-palmitoyl-2-oleoyl-PC (which predominate in human bile) satisfied
the requirement for PC. Moreover, either glycocholate or glycodeoxycholate could be substituted for the
bile salt mixture. The finding that biliary lipids can support serum-free growth of G. lam blia may help ex-
plain why this parasite colonizes the upper small intestine.
CITATION: Infection and Immunity 53(3): 641-645, September1986.
BlOl MS-86-048 Project Officer: Sauch
ANTIGEN DETECTION in the DIAGNOSIS of NORWALK VIRUS
GASTROENTERITIS
I.E. Heirmann, 1 G. P. Ken4 2 N.A.. Nowalç 1 J. Brondum, 3
and N. K Blacklow t
t University of Massachusetts Medical School
2 Centers for Disease Control
3 Rhode Island Department of Health
The research described in the attached article details development clan antigen detection system for Nor-
wallc vinis. This is a known causative agent of wate,bome gastroententis outbreaks. The paper acknow-
ledges a cooperative agreement of this laborato,y because test methods for identifying Norwalk viius are
being developed by one of the authors under that agreement.
A study was undertaken to assess the suitability of inonoclonal-based enzyme immunoassays for diagnos-
ing Norwalk virus associated gastroenteritis outbreaks. The assay system was found sufficiently sensitive
to use alone for outbreak diagnosis without aid of radio-immunoassays. In addition the enzyme im-
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munoassay test permits diagnosis witin iday after specimen receipt, rather than the 4 to 6 weeks required
for collecting convalescent sera.
CITATION: The Journal of Infectious Diseases 154(3): 547-54 September 19&i
C104 XX-86-107 Project Officer: Hurst
VIRUSES in DRINKING WATER
Gabriel Bitton, 1 SamuelR. Farrah, 1 Clay L. Montague 1 and Elmer W Akin 2
‘University of Florida-Gainesville
2 HE USEPA, Cincinnati, OH
Human entenc viruses that may cause disease when ingested e.ust in waterways via fecal contamination.
Many factors contribute to their susvival or inactivation in source water and treated drinldng water. The ac-
tual ent of water contwnination with viable vi,uses is unknown. This global sur ’ey for data on vinis oc-
cu,rence in drinking water is impoilant to EPA in providing information on the potential hazard and need
for control technology for watethome viruses.
Rising populations around the globe have resulted in an ever-increasing need for potable waters. Corn-
munities obtain their potable water from surface or underground sources. The contan,inations of these
source waters with viruses and other pathogens and parasites has been well documented. The source
waters, particularly groundwater, are sometimes consumed without any treatment or after being only dis-
infected. They are also subjected to the full treatment provided by conventional water treatment plants.
Do these plants provide a safe barrier ag inct virus breakthrough? Are classical water quality parameters
adequate for guaranteeing safe drinking water from virologica] standpoint? What are the priorities with
respect to research on viruses in drinking water? To attempt to answer some of these questions, we will
review the available literature dealing with virus monitoring of drinking water and then make comments
on detection methods, indicators, effect of water treatment processes on virus removal and fmally
epidemiological health significance of viruses in drinking water.
CITATION: Environmental Science and Technology 20(3): 216-222, March 1986.
C104 MS-86-046 Project Officer: Akin
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ENZYME IMMUNOASSAY WITH MONOCLONAL ANTIBODiES
for the DETECFION of ROTAVIRUS in STOOL SPECIMENS
John E Hemnann
University of Massachusetts Medical School
Rotavintse.c are known to occur in water and to cause waterbome gastroenteiitis outbreaici. Yet the vines
has not yielded to routine detection procedures. The identification of rotavints as the etiological agent of
waterbome cases and outbreaks is hwnpered by the non-availability of a detection procedure. This project
has developed a detection procedure for rotavbus in stool spechnens and will be used by the Agency in the
investigation of wate,bome outbreaks.
Rotavirus infections are generally recognized as a major problem in young children; however, they have
also been associated with severe gastroenteritis in aduks and neonates. Infections in neonates are usual-
ly asymptomatic, although the incidence of infection may be high. Adult infections also are often map-
parent or mild, and thus, adults maybe a reservoir of infection in pediatric wards. Diagnosis of infection
is now routinely performed by enzyme iznmunoassay (EIA) in preference to the more cumbersome
electron microscopy (EM). A commercial polyclonal EtA test, Rotazyine® (Abbott Laboratories, North
Chicago, IL), has been widely used for this purpose. The efficacy of this assay in adults has not been
described, and reports of false-positive reactions in samples from neonates have resulted in recomnien-
dations that Rotazyme not be used with neonatal stool specimens. False-positive results in neonates have
also been reported for other rotaviral EIA tests based on polyclonal sera.
CITATION: Journal of Infectious Diseases 152(4): 830-832, October 1985.
C104 XX-86-043 Project Officer: Akin
ENZYME.LINKED IMMUNOASSAYS for the DETECTION
otMICROBIALANTIGENS and THEIR ANTIBODIES
John E. Hemnanu
University of Massachusetts Medical School
Many vimi agents that are associated with waterbome ente,ic disease have been visualized by electron
microscopy but have not been detectable by routine cultivation techniques. This study ertended the
developments made on an alternative detection procedure enzyme-linked imnlunoassays, that may be used
in the investigation of waterbome outbreaks in the absence of appropnate virus cultivation procedures.
The advantages of enzyme-immunoassay (EAI) over radioactive assay techniques are mainly convenience
in use, in that the labelled immunoreagents are stable for long periods, and the precautions and disposal
procedures required for radloisotopes are unnecessary. In addition, the use of chromogenic substrates
for the enzyme labels permits visual interpretation of test results in some cases. The only real disad-
vantages of EIA tests are the loss of antibody reactivity that may result from conjugation to enzymes, and
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the limits of substrate detection. For example, use of enzymes that have molecular weights higher than
that of IgG molecules such as B-D-galactosidase (mw 540,000 daltons) can cause steric hindrance of an-
tibody activity. With regard to limits of substrate detection, improvement of enzyme detection by use of
fluorogenic, luminescent, or radioactive substrates has been proposed. The major emphasis of this report
will be on current developments in EIA methodology and the application of ETA to diiignosis of infec-
tious diseases. This indudes tests for both antigen and antibody detection in viral, rickettsial, bacterial,
and mycotic infections. ETA tests for diagnosis of parasitic agents, hormones, and other antigens have
been described but are not discussed here.
CITATION: In: Advances in Applied Microbiology, Academic Press, New York, NY, 1986.
C104 XX-86-061 Project Officer: Akin
DETECTION of ROTAVIRUS with a NEW POLYCLONAL ANTIBODY
ENZYME IMMUNOASSAY (ROTAZYME II) and a COMMERCIAL LATEX
AGGLUTINATION TEST (ROTALEX):
COMPARISON with a MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY
Gary V. Doern, John E. Hermzann, Patricia Henderson, Darlene Stobbs-Walro,
Dorothy M. Perron, and Neil A. Blacklow
University of Massachusetts Medical Center
The researeh described in the attached wticle details development of an antigen detection system for
rotavi ‘us. Thls is a potential causative agent of waterborne gastroenreritis outbreaks. The paper acknow-
ledges a cooperative agree?nenr of this laboratory because test methods for identifying rotavuus viras are
being developed by the authors under that agreement.
A total of 176 human fecal specimens were ex2mined for the presence of rotavirus by four different as-
says: a monoclonal antibody enzyme immunoassay, the original polyclonal antibody enzyme immunoas-
say marketed by Abbott Laboratories, North Chicago, IL (Rotazyme I); a modification of this assay which
is now commercially available (Rotazyme II); and a latex agglutination test (Rotalex) recently introduced
by Medical Technology Corp., Somerset, NJ. In addition, selected specimens were ex imined for the
presence of rotavirus by electron microscopy, immune electron microscopy, and RNA gel
electrophoresis. A total of 40 specimens were positive in the monoclonal antibody enzyme immunoassay,
and 136 were negative. Using the results obtained with this procedure as the reference standard, we
found the sensitivities of the Rotazyme I, Rotazyine II, and Rotalex tests to be 97.4, 100, and 81.6%,
respectively. The specificities of these three procedures were 88.8,83.9, and 100%, respectively.
CITATION: Journal of Clinical Microbiology 23(2): 226-229, February 1986.
C104 XX-86-041 Project Officer: Hurst
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METHODS for RAPID IDENTIFICATION of VIRUSES
John E. Hemnann
University of Massachusetts Medical School
Many viral agents that are associated with wateibome enteiic disease have been visualized by electron
microscopy but have not been detectable by routine cultivation techniques. This study extended the
developments made on alternative detection procedures such as immunoassays and genetic probes that
may be used in the investigation of waterbonze outbreaks in the absence of appropriate vints cultivation pro-
cedures.
The progress made in the last few years in methods for direct detection of viruses in clinical samples,
without the need for in vitro culture, suggests that they will be applicable to at least some types of environ-
mental samples. The methods for detecting viral antigen include: radioimmunoassay, enzyme immunoas-
say, agglutination tests, fluorescent antibody techniques and electron microscopic procedures. The use
of probes for detecting viral nudeic acid has recently sparked considerable interest. The current status
of these methods and potential means for improving them are presented in this report.
CITATION: In: Recoveiy of Vuusesftvm the Environment, CRC Press, Boca Raton, FL, 1986.
C104 XX-86-060 Project Officer: Akin
VIRUS-LIKE PARTICLES WITH T=19 ICOSAHEDRAL SYMMETRY in
a HUMAN GASTROENTERITIS STOOL
F. P. Williams, Jr.
HER!.., USEPA, Cincinnati, OH
This aaticle reports a new vinis-likepaiiicle obser?ed in this laboratory. The unexpected finding was a result
of using negative-stain transmission electron microscopy for viius detection. This methodology has been
implemented in-house as part of a continuing project to develop methods for the identification of non-cu!-
tivable gastroenteritis vinises.
Virus-like particles not previously described were observed in a human gastroenteritis stool using nega-
tive-stain TEM. The stool was among a number of acute-phase illness stools which had been collected in
Egypt during 1980. The particles measured 65-70 nm in diameter, and it was possible to detect individual
capsomeres on many of these particles. The capsomeric pattern identified on the particles corresponded
to an icosahedrally symmetric T= 19 capsid. Distinctive five-fold vertices, usually appearing as darker
spots on the capsid, were an additional feature of these particles. The capsid structure, which is skew,
could readily be distinguished from the T= 25 capsid of adenovirus and the holey capsids of rotavirus and
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reovirus. Antibody to the particles was detected in both the shedding individual’s acute- and convales-
cent-phase serum specimens using IEM, although an antibody increase was not demonstrated.
CITATION: Micron and Microscopica Ada 16(3): 173-178,1985.
C104 MS-85-102 Project Officer: Williams
WATERBORNE VIRAL GASTROENTERITIS
Fred P. JWliamv, Jr. and Elmer W. Akin
HERI., USEPA, Cincinnati, OH
The review article, “Waterborne Viral Gastroenteritis,” was written to provide responsibk individuaLr within
the water industiy through the Journal of the American Water Workc Association, with state-of-the-art in-
fonnalion on this rapidly moving area of investigation relative to diinldng water health.
In studying the causes of human gastroenteritis, electron microscopy and related techniques have led to
the identification of new viral agents that had previously escaped detection by routine cell culture proce-
dures. Efforts to characterize and study these agents further are currently being made by researchers in
many areas of the world. Two of the best-known agents, rotavirus and Norwalk virus, have been inipli-
cated in waterborne outbreaks of this illness. Another virus, the Snow Mountain agent, was first iden-
tilled from the investigation of one waterborne gastroenteritis outbreak.
CITATION: Journal of the Ameifcan Water Works Association 78(1): 34-39, January 1986.
C104 MS-85-021 Project Officer: WiUiams
EVALUATION of METHODS for CONCENTRATING HEPATITIS A VIRUS
from DRINKING WATER
MarkD. Sobsey, Stephen E. Oglesbee, and DouglasA. Wait
University of North Carolina School of Public Health
Hepatitis A virus (HAV) is the classic waterbome viral agent. However, in vitro detection and cultivation
procedures have only recently been develope L This study used these HAVdetection procedures to evaluate
the standardized virus concentration methodology developed for other enteric viruses in the recovery of
HAy. The availability of FL4Vconcentration and detection procedures will provide the tools for the Agency
to further evaluate the role of water in the tra,umLrsion of this major viral disease.
By nsng recently developed cultivation and assay systems, currently available methods for concentrating
enteric viruses from drinldng water by adsorption to and subsequent elution from microporous filters fol-
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lowed by organic flocculation were evaluated for their ability to recover hepatitis A virus (HAV). Cell
culture-adapted HAy (strain HM-175) in seeded tapwater was efficiently adsorbed by both electronega-
tive (filterite) and electropositive (Virosorb IMDS) filters at pH and ionic conditions previously used for
other enteric viruses. Adsorbed HAV was efficiently eluted from these filters by beef extract eluents at
pH 9.5. Eluted HAY was further concentrated efficiently by acid precipitation (organic flocculation) of
eluents cont dning beef extract made from powered, but not paste, sources. By ining optimum adsorp-
tion conditions for each type of filter, HAV was concentrated > 100-fold from samples of seeded tap-
water, with about 50% recovery of the initial infectious virus added to the samples. The ability to recover
and quantify HAV in cont niinated drinking water with currently available methods should prove useful
in further studies to determine the role of drinking water in HAV tr nsmicsion.
CITATION: Applied and Environmental Microbiology 50(6): 1457-1463, December 1985.
C104 XX-86-039 Project Officer: Akin
ISOLATING VIRUSES FROM FINISHED WATER
Joan B. Rose Charles P. Ge,ba, SM N. Sing i,
GwyA. Toranzos, and B,uce j( .j 1
University of Arizona
t University of Texas Science Center
Full conventional treatmen4 as used in this country to produce d inking water from polluted surface soui-
ces, is generally considered to be adequate for vuus removal. Earlier non-EPA funded woi* has shown a
conventional plant in M&ico to allow vhus penetration. This EPA funded study was conducted to confinn
the earlier repoit and attempt to identify the factors that may have allowed vints penetration. This infomia-
tion is inzpoi’twit to EPA in identifying conditions and needed controls concerning waterborne vuus buns-
mission.
Reduction of enteroviruses and rotaviruses averaged 81 and 93%, respectively, at a full-scale 205-mgd
(776-MUd) plant whose treatment train includes chemical flocculation, sand filtration, and chlorination.
The highest reduction of enteroviruses occurred during prechlorination-flocculation and filtration,
whereas the highest reduction of rotaviruses occurred during prechiorination-clarification and final
chlorination. Enteroviruses or rotaviruses occurred in 24% of the finished water samples, which had
>0.2mg free chlorine/L and met coliforin bacteria (1/100 mL) and turbidity (1 ntu) standards. Although
major plant deficiencies may have been responsible for the occurrence of viruses in the finished water,
the results of this study indicate that finished water, with measurable levels of free residual chlorine and
meeting standards for coliform bacteria and turbidity cannot be assumed to be virus free.
CITATION: Journal of the American Water Works Association 78(1): 56-61, January 1986.
C104 XX-86-036 Project Officer: Akin
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DETECTION of NORWALK VIRUS in STOOLS by ENZYME IMMUNOASSAY
I. E. Hemnann, N. A. Nowalç and N. A Blacklow
University of Massachusetts Medical School
The research described in the attached wiicle details development of an antigen detection system for Nor-
walk vints. This is a known causative agent of waterbome gastroenteritis outbreaks. The paper acknow-
ledges a cooperative agreement of this laboratory because test methods for identifying Noiwalk viius are
being developed by the authors under that agreemenL
The development of a solid-phase microtiter enzyme immunoassay (EJA) for detection of Norwalk virus
antigen in stool samples is described. The EIA was compared with a previously developed radioim-
munoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 volunteers who
received Norwalk virus. The EIA detected viral antigen in 15. Seroconversion was a more sensitive in-
dicator of infection in some patients. However, two samples from volunteers who were clinically ill but
did not show seroconversion to Norwalk virus were positive for Norwalk virus antigen by both iinmunoas-
says. This indicates that antigen detection may be important for use in epidemiological studies. Neither
of the immunoassays gave positive reactions for stools known to contain enteric adenovirus, rotavirus, or
Hawaii virus, orin stools from patients with acute diarrhea of unknown cause. The stability of the EIA
reagents and ease of use should provide a means for more extensive testing of Norwalk virus in outbreaks
of gastroenteritis.
CITATION: Journal of Medical Virology, 17: 127-133, October 1985.
C104 XX-86-042 Project Officer: Hurst
WIDESPREAD OUTBREAKS of CLAM- AND OYSTER- ASSOCIATED
GASTROENTERI’HS: ROLE of NORWALK VIRUS
DaleL Morse 1 JohnJ. GUzeWi& John?. Hanrahan, 1 Rachel Stricof, 1
Mehdi Shayeganz, 1 Rudolf De1b4 1 John C. Grabau 1 Nancy A. Nowalc 1
John E. Hemnann, 2 George Qikor, 2 and Neil R. Blacklov?
1 New York State Department of Health
2 University of Massachusetts School of Medicine
The research described in the attached article details viral disease hazards associated with consumption of
improperly cooked shel ftsh. A series of outbreaks which occurred in 1982 were studieS The outbreaks in-
cluded 1017 cases of illness associated with eating clams and oysters. This paper acknowledges a coopera-
tive agreement of this laboratory because test methods for identifying Norwalk vi,us in environmental
samples and clinical specimens were developed by one of the authors under that agreement.
Consumption of raw shellfish has long been known to be associated with individual cases and sporadic
outbreaks of enteric illness. However, during 1982, outbreaks of gastroenteritis associated with eating raw
shellfish reached epidemic proportions in New York State. Between May 1 and December31, there were
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103 well-documented outbreaks in which 1017 persons became 111:813 cases were related to eating riams ,
and 204 to eating oystert The most common symptoms were diarrhea, nausea, abdominal eramps, and
vomitin& Incubation periods were generally 24 to 48 hours long, and the duration of illness was 24 to 48
hon Bacteriologic analyses of stool and shellfish specimens did not reveal a causative agent. Norwalk
virus was implicated as the predominant etiologic agent by clinical features of the illness and by serocon-
version and the formation of 1gM antibody to Norwalk virus in paired serum samples from persons in five
(71%) of seven outbreaks in which testing was done. In addition, Norwalk virus was identified by
radioimmunoassay in clam and oyster specimens from two of the outbreaks. Determining the source of
the shellfish was not always possible, but northeastern coastal waters were implicated. The magnitude,
persistence, and widespread nature of these outbreaks raise further questions about the safety of consum-
ing raw shel1flcIi
CITATION: New England .Iownoi of Medicine 314(11): 678-681, March 1986.
C104 XX-86-070 Project Officer: Hurst
INVESTIATION of PARASITES in SLUDGES and DISINFECTION
TECHNIQUES
R. £ Reimers M. D. Little A. J. England.e, Jr., D. B. McDonell
D. D. Bowman, and I. M. Hughes
Tulane University School of Public Health and Tropical Medicine
This report provides the best available infonnation on the concentration of parasites in inwnic4pal sludges
in the United States. It will be used by the Office of Water as an iinpoitant source of data in the fonnula-
Lion of guidelines and regulations goveming the management of municipal sludgc particularly for the
development of technical regulations for the reuse and disposal of sewage sludge under Section 405(d) of
the Clean WaterAct by the Office of WaterRegulasions and Standards.
The objectives of this research grant were to: 1) assess the presence and densities of re ci ctant stages of
parasites in municipal wastewater sludges (sewage) in northern United States; 2) compare the results of
this study with the results of our previous study of sludges in southern United States; 3) to evaluate several
decontamination techniques for their effectiveness in inactivating parasites in waste sludges; and 4)
develop a standard method for the parasitologic eramination of waste sludges. Sludge samples from all
phases of treatment (i.e., primary, etc.) were collected during the fall, winter and summer from 48
municipal wastewater treatment plants located in New York (13 plants), Ohio (12 plants), Minnesota (11
plants), and Washington (12 plants).
CITATION: US. Environmental Protection Agency cooperative agreement no. CR-806711, EPA-
600/1-85-022, December 1986.
B113 HERL-0522 Project Officer Kowal
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HEALTH EFFECTS of LAND APPLICATION of MUNICIPAL SLUDGE
Nonnan E. Kowal
HERL, USEPA, Cincinnati, OH
This repon’ suminwizes the literature, and provides a broad overview of the potential health effects of the
land application of municipal sludge. It will be distributed by the Office of Water Pwgrani Operations to
sewage treatment plant operators, and will provide a convenient resource for the fonnulation of EPA
regulans, permits, and guidelines under the aean WaterAct and the Resowves Conseri’ation and Recovery
Act.
The potential health effects arising from the land application of municipal sludge are examined, and an
appraisal of these effects made. The agents, or pollutants, of concern from a health effects viewpoint are
divided into the categories of pathogens and toxic substances. The pathogens include bacteria, viruses,
protozoa, and helmiuths; the toxic substances include organics, trace elements, and nitrates. For each
agent of concern the types and levels commonly found in municipal wastewater and sludge are briefly
reviewed. A discussion of the levels, behavior, and survival of the agent in the medium or route of poten-
tial human exposure, i.e., aerosols, surface soil and plants, subsurface soil and groundwater, and inimi Lc ,
follows as appropriate. Infective dose, risk of infection, and epidemiology are then briefly reviewed.
Finally, some general conclusions are presented.
CITATION: U.S. Environmental Protection Agency, EPA-600/1-85-015, November 1985.
B113 HERL-0499 Project Officer: Kowal
MUNICIPAL SEWAGE SLUDGE APPLICATION on OHIO FARMS:
TISSUE METAL RESIDUES and INFECTIONS
C. S. Reddy, C. K Dom, D. N. Lamphere, and I. D. Powers
The Ohio State University
This journal an’icle contains results from HERL’s research program on the health aspects of the land ap-
plication of municipal sludge. It deals with the human exposure to heaiy metals, organic chemicals, and
pathogens as a result of land application, and potential adverse health effects.
Transmkcion of infectious agents and translocation of Cd, Cu, Pb, and Zn from anaerobically digested
sludge to the tissues of farm animals grw’ing on pastures to which sludge was applied (2-10 metric tons
per hectare) were studied on eight farms. No significant health risk associated with the possible presence
in sludge of Salmonella spp., or of common animal parasites including Nernatodinis spp., Strongylus spp.,
Strongyloides spp., Trichuris spp., Eimeria spp.,Ascwis app., and Ancylostomurn app. was noted. Caudal
fold as well as cervical tuberculin testing indicated no conversions from negative to positive following ex-
posure of cattle to sludge. Significantly higher fecal Cd concentrations were detected in samples col-
lected from cattle soon after being placed on sludge-treated pastures compared to preexposure values in
the same inimsak . Significant Cd and Pb accumulations were found in the kidneys of calves grazing
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sludge-treated pastures compared to control calves. Although older cows graiing sludge-treated pas-
tures had significantly higher blood Pb levels, no metal accumulation was observed in other tissues. Statis-
tically significant accumulations of Cd and Pb in the kidney of calves grazing these pastures for a relative-
ly short period suggest that caution should be. exercised to avoid prolonged gr ing of cattle on pastures
receiving heavy sludge applications, especially with sludges cont inbig high concentrations of heavy me-
tals.
CITATION: Environmental Research 38(2): 360-376, December 1985.
B113 XX-86-126 Project Officer Kowal
STABILITY of VIRUSES In WASTEWATER SLUDGE ELUATES
Chiiston 1. Hurst and Twnara Goyke
HERI USEPA, Cincinnati, OH
This paper descñbes in statistical tenns the stabili(y of indigenous enteric vuuses hiiing storage of
processed wastewater slu4çe samples The research helps tofu fili HERL’s mission to evaluate potential
health effectc associated with disposed inaterialfivm wastewater treatment processes.
The survival of indigenous enteric viruses in samples of unconcentrated and concentrated wastewater
sludge eluates, which had been prepared using a combination beef e ract elution-organic flocculation
concentration procedur; was studied at Z 23 and -70°C. Changes of virus titer occurring in the samples
were followed during an 84-day observation period, with rates of change then calculated by least-squares
regression. Virus survival in both types of eluates was statistically dependent (p <0.05) upon storage
temperature. Based upon the observed rates of inactivation the average times which would be required
for a 90 percent decrease (one logio unit) in virus titer for unconcentrated eluates are 27 days at 23°C, 198
days at 2°C, and 375 days at -70°C. The calculated average times required for a 90 percent decrease in
virus titer for concentrated eluates are 22 days at 23°C, 132 days at 2°C, and 246 days at -70°C. In both
types of eluates the rates of virus inactivation at 2°C were statistically different from those observed at
23°C, but not different from those observed at -70°C. The three study temperatures were selected to ap-
proximate holding of samples in an air conditioned room, fluid on wet ice (1120) and frozen on dry ice
(C02).
CITATION: Canadian Journal of Microbiology 32:649-653, August 1986
BlOl MS-86-088 Project Officer: Hurst
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DEVELOPMENT of METHODS to MEASURE VIRUS INACTIVATION in
FRESH WATERS
Jlichwd L Ward and Pat E. Winston
James N. Gamble Institute of Medical Research
The study described in thLc publication involved an identification of erpeiimental design factors which
could affect laboratory measurements of vinis survival. This work Lv important to that part of our research
programs which addresses persistence of contaminating viruses in environmental waters. The findings
presented in this publication have already been applied in viral survival studies being conducted by our
group on an in-house basis.
This study concerns the identification and correction of deficiencies in methods used to measure inactiva-
tion rates of enteric viruses seeded into environmental w*rs. It was found that viable microorg inkms
in an environmental water sample increased greatly after addition of sm dI amounts of nutrients normal-
ly present in the unpurified seed virus preparation. This burst of microbial growth was not obsevered
after seeding the water with purified virus. The use of radioactively labeled poliovirus revealed that high
percentages of virus particles, sometimes 99%, were lost through adherence to containers, especially in
less turbid waters. This effect was partially overcome by the use of polypropylene containers and by the
absence of movement during incubation. Adherence to containers clearly demonstrated the need for
labeled viruses to monitor losses in this type of study. Loss of viral infectivity in samples found to occur
during freezing was avoided by addition of broth. Finally, microbial contfimin tion of cell cultures during
infectivity assays was overcome by the use of gentamicin and increased concentrations of penicillin, strep-
tomycin, and amphotericin B.
CITATION: Applied and Environmental Microbiology 50(5): 1144-1148, November 1985.
B113 XX-85-114 Project Officer: Brashear
SURVIVAL of INDIGENOUS ENTERIC VIRUSES DURING STORAGE of
WASTE WATER SLUDGE SAMPLES
Christon J. Hw st and Tamara Goyke 2
1 HE USEPA, Cincinnati, OH
2 EMSL USEPA, Cincinnati, OH
This paper descnbes in statistical terms the stabili(y of indigenous viruses diving storage of processed was-
tewater sludge samples. The research helps to fulfill HERL’s mission to evaluate potential health effects
associated with disposed material from wastewater lreatm ent processes.
The stability of indigenous enteric viruses in samples of settled primary and mixed-liquor activated
sludges was studied at 2,23 and -70°C. Changes of virus titer which occurred in these samples were fol-
lowed during an 84-day observation period, with rates of change then calculated by least-squares regres-
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sion. Virus survival was found to be statistically dependent (p sO.05) upon storage temperature but not
sludge solids content. Based upon the observed rates of inactivation, the average times which would be
required for a 90 percent decrease in virus titer are 26 days at 23°C, 180 days at 2°C, and 163 days at -70°C.
As a group, the rates of virus inactivation observed at 2°C were statistically different (p s 0.05) from those
observed at 23°C, but not different from those observed at -70°C. The three study temperatures were
selected to approximate holding of samples in an air conditioned room, on wet ice (1120), and on dry ice
(C02).
CITATION: Canadian Journal of Microbiology 32 645 648, August 1986.
BlOl MS-86-064 Project Officec Hurst
A COMPARISON of SEVEN METHODS for CONCENTRATING ORGANIC
CHEMICALS from ENVIRONMENTAL WATER SAMPLES
F. CKopfler,HP.Ringhand andR.G.Miler
HERI ., USEPA, Cincinnati, OH
Assessment of the risk associated with consumption of drinldng water containing organic chemicals will re-
quire that bioassay: be performed on samples of the organic chemicaLr. This paper discusses the suitability
of various methods for prepanng representative samples of these o ganics of sufficient size for biological
testing.
Because there are no quantitative analytical techniques for the complex organic matter that occurs in
chlorinated water, it is not possible to directly determine the efficiency of techniques for isolating them.
Seven methods capable of isolating gram quantities of organic matter from water samples were evaluated
by determining the ability of each to recover a set of model compounds possessing a wide variation in
polarity, functional groups, water solubility and molecular weight. No single method appeared to be su-
perior overall, based on the recovery of the model solutes, but some could be elimin2ted from field ap-
plication for the present time since the adsorbents required were not commercially available, field ap-
plication of two methods were undertaken and the samples collected are being tested in several bioassays.
CITATION: In: American Chemical Socie(yAdvances hi ChemLctiy Series No. 214 Advances in Sampling
and Ana ysis of Organic Pollutants from Water, I.H. Suffet and M. Malalyandi, eds., 1986.
C104 MS-86-097 Project Officer: Kopfier
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COMPARISON of HIGH MOLECULAR ORGANICS ISOLATED from
DRINKING WATER in FIVE CITIES
E. S. K chian,’ M. F. Giabba4 2 I. S. KIm, 1 1. H. Reuter, 3
andF. C.Kopfler 4
1 Georgia Institute of Technology
2 Environmental Science Laboratories, Inc.
3 School of Geophysical Sciences
4 HERL, USEPA, Cincinnati, OH
The findings indicate that the nonvolatile chemicals in drinking water which form when the humic acid in
them Ls chlorinated we veiy similar. Thus results on toxicological testing of a limited number of samples
may be safely extrapolated to the general case. fn contrast, the commercially obtained humic acid yield a
totally different substance upon chlorination; then some caution should be taken in attempting to extrapo-
late from results of toxicological testing of this material.
Since the previously unidentifiable non-volatile fraction of the chlorinated organics in drinking water is of
significant health concern, the physical and chemical characteristics of the high molecular weight organics
isolated from drinking water in five cities (nanieIy New Orleans, Philadelphia, Mipmi , Seattle, and Ot-
tumwa) were compared. From the results of this study, they may be considered as part of a single group
of natural organic compounds. Although the respective molecular structures of this group of compounds
are still unknown, they showed, however, great similarities in terms of molecular, solubility and function-
al group among the samples collected from the five cities. This indicates chlorinated organics from a
limited number of drinking water sources.
CITATION: In: Advances in che nLc1iy Series, No. 214, American Chemical Society , 1986.
C104 MS-86-243 Project Officer: Kopfler
EVALUATION of a CONTINUOUS LIQUID-LIQUID EXTRACTOR for
ISOLATION and CONCENTRATION of NONPOLAR ORGANICS for
BIOLOGICAL TESTING in the PRESENCE of HUMIC MATERIALS
A J. Baker and I. H. Suffet
Drexel University
Methods for preparing representative concentrates of the organic matter in aqueous samples are needed for
contaminant identification and for biological testing. The continuous liquid-liquid extraction system
evaluated was judged to be sufficiently reliable for long-term composite sampling. Coupled with ieve’ce os-
mosis the system can produce in the field a volume of concentrate adequate for animal feeding studies.
A continuous liquid-liquid extraction system (CCLE) for concentrating trace organics from water into
methylene chloride for analysis was designed, built and evaluated. The CCLE uses Teflon coils for phase
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contact and gravity phase separation. The system includes a self-contained excess solvent distillation
chamber, so the methylene chloride is recovered and recycled. Fourteen organic model compounds in
the presence of humic material were used as test probes to evaluate the CLLE. Recovery values were
determined for these compounds using CLLE and batch LLE (separatory funnel liquid-liquid extrac-
tion), and CLLE recoveries were found to be imibir to those of batch LLE.
CITATION: In: American Chemical Socie yAdvance$ in Chemistsy Senes, (in press).
C104 XX-86-038 Project Officer: Ringb’ iid
PYROLYSIS-MASS SPECTROMETRY/PATFERN RECOGNITION on a
WELL-CHARACTERIZED SUITE of HUMIC SAMPLES
P. MacCarthy, 1 S. !. Deluca 1 IC !. Voorhees, 1 R. L Malcohn 2 andE. M. Thunnan 2
1 Colorado School of Mines
kJ.S. Geological Survey
It Lv now accepted that most of the organic material in chlorinated drinking water Lv composed of complex
products of reaction between the chlothze and the humic and fuMc acids which occur natzuul y in the
source water. The Safe Drinking Water Act; as amended in 1980, required the Administrator to conduct
studies on the health effects of these reaction products. The results described in this paper will provide
some of the required backgmwid infomzalion on the similan y and differences of humic and fulvic acids
fivm different source wate so the number of different sources required to provide adequate information so
that general conclusions about the health effects of the reaction products can be deterininesL
A suite of well-characterized humic and fulvic acids of freshwater, soil and plant origin was subjected to
pyrolysis-mass spectrometry and the resulting data were analyzed by pattern recognition and factor
analysis. A factor analysis plot of the data shows that the humic acids and fulvic acids can be segregated
into two distinct classes. Carbohydrate and phenolic components are more pronounced in the pyrolysis
products of the fulvic acids, and saturated and unsaturated hydrocarbons contribute more to the humic
acid pyrolysis products. A second factor analysis plot shows a separation which appears to be based
primarily on whether the samples are of aquatic or soil origin.
CITATION: Geochimica et CosmochimiaActa 49(10): 2091-2096, October 1985.
C104 XX-86-040 Project Officer: Kopfler

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QUANTITATION and IDENTIFICATION of ORGANIC N-CHLORAMINES
FORMED In STOMACH FLUID on INGESTION of AQUEOUS HYPOCHLORITE
Frank E. Scully, J,. 1 J( ,,the,ine Mazin4 1 Daniel Sonenshin4 1 and
Frederick Kopfler
1 01d Dominion University
USEPA, Cincinnati, OH
The disinfection of diinldng water is one of the most widely practiced and most successful public health
programs in the U.S. While it has dramatically reduced the incidence of waterbome illness, it has been
determined that the organic chemical contaminant occurring at the highest concentration in drin!dng water
result from it. This symposium paper summarizes the results to date of the Agency’s research into the
health effect of these contaminants.
Stomach fluid was recovered from Sprague-Dawley rats that had been fasted for 48 hours and ad-
ministered 4 ml deionized water. It contained high concentrations of total Kjeldahl nitrogen. The
chlorine demand of the stomach fluid was found to range between 40 and 80 mg/I as N. In separate ex-
periments piperidine and glycine were administered to Sprague-Dawley rats followed by a solution of
aqueous hypochiorite. Stomach fluid was recovered from these animsils and shown to contain the cor-
responding chioramino products. Because stomach fluid already contains high concentrations of amino
nitrogen, the percent conversion of an exogenous amine to its chloramino compound on adniinistration
of hypochiorite is low. The actual yield of chioramine was determined by administration of tritium-labeled
piperidine followed by aqueous hypochiorite. The quantity of N-chloropiperidine formed was deter-
mined by direct liquid chromatography of the compound. This quantity combined with the average
“chlorine demand” determined for stomach fluid as described above was used to calculate the yield of
chioramine formed. For concentrations of hypochiorite ranging from 200 ppm to 1,000 ppm, yields of or-
ganic chioramines ranged from 50 percent to 75 percent of the theoretical amount expected for these
compounds. Details of this determination will be presented. Stomach fluid was recovered from
laboratory rats, chlorinated, and derivatized with dansyl sulfinic acid. Chromatography of the derivatized
stomach fluid revealed that many apparent chiorainine derivatives had formed. These derivatives were
isolated by preparative liquid chromatography and their retention times correlated with known deriva-
tives of chloraniino acids by two chromatographic techniques. The identification of these compounds will
be discussecL
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Drinking Water
Disinfecta,us and Disinfection By-Products, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69:259-265, October 1986.
C104 MS-86- 165 Project Officer: Kopfler
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INHALATION EXPOSURE in the HOME to VOLATILE ORGANIC
CONTAMINANTS of DRINKING WATER
Julian B. Andelman
University of Pittsburgh, Graduate School of Public Health
The inhalation route of exposure for volatile organic water contwninants may be greater than exposure
through ingestion. The Office of Drinldng Water(ODW) should consider this route of ctposure when
developing drinldng water regulations.
Our field studies show that indoor air concentrations of volatilized trichioroethylene (TCE) can be sub-
stantial when TCE-cont min ted water is used domestically. Using a model shower, increases in TCE
water concentrations, water temperature and drop path (time) increased the steady-state air TCE con-
centrations. Volatilization was incomplete and the rates were comparable to predicted ones. Indoor air
models show that the inh ition route of exposure for such chemicals has the potential for being much
greater than by direct ingestion. This should be considered in developing regulations to limit adverse
health impacts from contaminants of potable water.
CITATION: The Science of the Total Environment 47:443-460, November 1985.
C104 XX-86-059 Project Officer: Craun
HUMAN EXPOSURES to VOLATILE HALOGENATED ORGANIC
CHEMICALS in INDOOR and OUTDOOR AIR
Julian B. Andebnan
University of Pittsburgh
The inhalation route of exposure for volatile organic water contaminants may be greater than exposure
through ingestion. The Office of Drinking Water (ODW) should consider this route of posure when
developing drinldng water regulations.
Volatile halogenated organic chemicals are found in indoor and outdoor air, often at concentrations sub-
stantially above those in remote, unpopulated areas. The outdoor ambient concentrations vary con-
siderably among sampling stations throughout the United States, as well as diurnally and daily. The vapor
pressures and air-water equilibrium (Henry’s Law) constants of these chemicals influence considerably
the likely relative human exposures for the air and water routes. Volatilization of chemicals from indoor
uses of water can be a substantial source of exposure, as shown for radon-222. Measurements of air con-
centrations of trichioroethylene (TCE) in showers using TCE contnminated groundwater show increases
with time to as high as one-third of occupational threshold limit values. Using a scaled down experimen-
tal shower, such volatilization and subsequent decay in air was also demonstrated. Using a simplified in-
door air model and assuming complete vol fili”ation from a full range of typical water uses within the
home, calculations indicate that the expected air inhsilstion exposures can be substantinfly higher than
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those from ingestion of these chemicals in drinking water. Although the regulation of toxic chemicals in
potable water supplies has focused traditionally on direct ingestion, the volatilization and inh2I tion from
other much greater volume indoor uses of water should be considered as well.
CITATION: Envbonmental Health Perspectives 62: 313-318, October 1985.
C104 XX-86-057 Project Officer: Craun
EFFECT of CHLOROACETIC ACIDS on the KIDNEYS
Maiy E. Davis
West Virginia University Medical Center
The disinfection of drinking water is one of the most widely practiced and most successful public health
programs in the (1.5. While it has dramatically reduced the incidence of wateibome illness, it has been
determined that the organic chemical contaminant occurring at the highest concentration in drinking water
result from it. This symposium paper summanzes the results to date of the Agency’s research into the
health effects of these contaminants.
Previous studies on dichioroacetate have shown that its effects on intermediary metabolism have the
potential to interfere with the ability of renal mechs ncni to maintain acid-base balance. Dichloroacetate
decreases gluconeogenesis from lactate, in part by activating pyruvate dehydrogenase, routing lactate to
acetoacetate rather than through the tricarboxylic acid cycle. Dichloroacetate also decreases uptake of
lactate by kidneys, augmenting the reduction of the tricarboxylic acid cycle metabolism. This process can
interfere with add-base regulation by decreasing glutamate entry into the tricarboxylic acid cycle, con-
commitantly decre cing ammonia formation from glutamate. The accumulated glutamate is an inhibitor
of phosphate-dependent glutaminase, which is activated during acidosis to increase ammonia synthesis
from glutamine. During acidosis, y-glutamyl transpeptidase activity shifts to that of phosphate-inde-
pendent glutaminase; this change in activity is mediated by the binding of hippurate to the transpeptidase.
The present studies were designed to determine the extent and dose-response relationships of these me-
tabolic derangements during exposure to dichioroacetate and to determine if trichioroacetic acid has
similar effects. Exposure to lower concentrations of either dichioracetic or trichioroacetic acids did not
affect food consumption, growth, or urine output, whereas higher doses were associated with decreased
food consumption and either lowered body weight gain or weight loss. These effects were observed in
male rats with trichloroacetic acid given daily by gavage (300 mg I kg) or by exposure through water (3 g/l,
approximately 250 mg/kg/day). Exposure to dichioroacetate in drinking water impaired growth only at
the highest concentration (75 gfl) in males, however, female rats showed weight loss after exposure to
1.875 gIl. The higher exposures resulted in lower urine volumes and elevated urine osmolalities. Hip-
purate excretion was not increased in the groups exposed to dichioroacetic acid. This finding is sugges-
tive of altered add-base regulation.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Drinking Water
Disinfectants and Disinfection By-Products, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69:209-214, November 1986.
C104 XX-86-119 Project Officer: Laurie
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CADMIUM INTAKE VIA OYSTERS and HEALTH EFFECTS in NEW ZEALAND
CADMIUM INTAKE, METABOLISM and EI F ECTS in PEOPLE with a HIGH
INTAKE of OYSTERS in NEW ZEALAND
L McKenzQ T. ICjelLctroni, 2 andR. Shamia’
‘University of Otago, New Zealand
2 University of Auckb.nd, New Zealand
This final repon’ contains results from HERL’s research program on the health aspects of the land applica-
tion of municipal sludge. It deals with the potential adverse health effects of the high rate of consumption
of cadmiuni an iinpon’ant tazic contaminant of sludge.
The aim of this study was to confirm the high dietary intakes of cadmium and other trace elements from
oysters in a population assoaated with the oystering industry, and to determine 1) the impact of those high
intakes on cadmium concentrations in accessible tissues of the study subjects and 2) the occurrence of
health effects in the population resulting from their dietary exposure to cadmium. Methods for the
analysis of cadmium in whole blood, urine and hair by flameless atomic absorption spectrophotometry
were established. Seventy-eight subjects participated; from forty-eight of them samples of faeces (3 day),
blood, urine (overnight) and hair were obtained both pre-season and end-season (i.e. 6 months later).
Questionns ires on oyster intake were administered; dietary and medical questionnaires were ad-
ministered at the end of the season when height, weight and blood pressure were measured. The faecal
output of cadmium confirmed the high intakes; e.g. Category IV subjects were ingesting about 250 i.g cad-
mium/day at the end of the season; calculated intakes for Categories 1,11 and ifi were 34 p..g/d, 75 g/d
and 116 ig/d, respectively, at the end of the season. The concentration of cadmium in whole blood was
higher in the smokers than in the non-smokers. In the non-smokers the increase in whole blood cadmium
due to oyster consumption was only 1.2 ng/ml for Category IV. The concentrations of cadmium, zinc and
B2-microglobulin in the urine were unaffected by oyster consumption; there was no indication of
glycosuria or proteinuria that could have been attributed to a high intake of cadmium. Hair cadmium,
zinc and copper also appeared to be unaffected by oyster consumption. There were no indications of any
medical problems that could have been attributed to a high cadmium intake; although the blood pressure
of Category Ill and especially Category IV subjects was relatively higher than that for Categories I and II
and they also had a greater body size.
CITATION: U.S. Environmental Protection Agency, grant no. R-807058, EPA-600/1-86-004, July 1986.
B1L3 HERL-0548 Project Officer: Kowal
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HEALTH CONSIDERATIONS in APPLYING MINIMUM TREATED
WASTEWATER to LAND
Nonnan E. Kowal
HERL USEPA, Cincinnati, OH
This is overview of the health effects resulting jlvm the land application of municipal wartewater and
sludge. it is also a summaly of two existing HERL publications: “Health Effects of Land Treatment
Microbiological (EPA 60011-82-007, May 1982) and “Health Effects of Land Treatment Toxicological
(EPA 600/1-84-303 Jan 1985).
This paper is primarily a summary of two previously published detailed reports on the health effects of
land treatment (Kowal 1982,1985), and the reader is encouraged to seek there the supporting data and
arguments for the generalizations made here. In those reports the types and levels commonly found in
municipal wastewater and the efficiency of preapplication treatment (usually stabilization pond) are
briefly reviewed for each agent of concern. A discussion of the levels, behavior, and survival of the agent
in the medium or route of potential hiini n exposure, i.e. aerosols, surface soil and plants, subsurface soil
and groundwater, and s nin1 Ic , follows as appropriate. For the pathogens, infective dose, risk of infec-
tion, and epidemiology are then briefly reviewed. Finally, conclusions and research needs are presented.
CITATION: 1n Proceedings of the Soil Science Society of America - Utilization, Treatmen4 and Disposal
of Waste on Land, Chicago, IL, December 6-7, 1985.
B113 MS-87-031 Project Officer: Kowal
DOMINANT LETHAL ASSAY of CHLORDECONE and ITS DISTRIBUTION
in the MALE REPRODUCTIVE TISSUES of the RAT
Glenn Stua t Simo , 1 John L Egle, Jr., 2 RobeM W. Doughefly, 1
and Joseph F. Borzelleca 2
1 Rhone-Poulenc Inc.
2 Virginia Commonwealth University
Chiordecone is a highly stable pesticide with the potential for causing prolonged environmental contamina-
tion. An estimate of its reproductive toxicity Lr, therefore ofpnncipal impotance to the health program.
Male rats received 3.6 or 11.4 mg/kg/day of chiordecone orally for 5 days. Some statistically ignificant
events were seen in the reproductive data of females mated to males receiving chiordecone. However,
these events did not follow a consistent pattern and do not suggest the condusion that chiordecone causes
dominjrnt lethal effects. Male rats received a single oral dose (40 mg/kg) of chlordecone and were killed
at 1,2, 3,5, 7, 14 or 21 days. Chiordecone was distributed throughout the reproductive tract. The des-
cending order of concentration was semin I vesicular fluid > prostate > vas deferens > seminal vesicle
> unwashed sperm > washed sperm. it is concluded that chlordecone is well distributed throughout the
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reproductive tract of the male rat, appears in the ejaculate, and does not appear to produce dominant
lethal effects.
CITATION: TOxicOlOgy Letters 30:237-245, March 1986.
B1OJJCIO4IL1O4 XX-86-129 Project Officer: Smith
DOMINANT LETHAL EFFE S of SUBCHItONIC ACRYLAMIDE
ADMINISTRATION in the MALE LONG-EVANS RAT
A L Kate Smith, 1 Ha,old Zenicl4 2 It Julian Fleston, 3
E mma Lou Geo ’ge ’ Wed Richard E. Long4
1 HERL, USEPA, Cincinnati . OH, 2 University of Cincinnati Medical College,
3 Oak Ridge National Laboratory, and 4Pathology Associates, Inc.
Aciylwnide is wide(y used in its poipnedcfonn in the treatment of waste and drinking waters. The tau’city
pmfile of the resithial monomer is, therefore, essential to the setting of health advisories h i the water
program.
Acnflamide, a widely used vinyl monomer, is well known as a neurotoxin but inactive as a mutagen in bac-
terial test systems. The experiments reported demonstrate that after subchronic oral dosing in the male
rat, acrylamide induced significant elevations in both pre- and post-implantation loss following dominant
lethal testing. These effects were seen at doses which failed to produce clinical or pathological evidence
of neurotoxicity. In an accompanying cytogenetic study, no increase in chromosome aberrations was ob-
served in spermatogonia or spermatocytes of treated animals . When spermatocytes from treated sper-
matogonial stem cells were analyzed, reciprocal translocations (4) were observed in the treated animals
and not in the control, but the significance of this result still needs to be established.
CITATION: Mutation Research 173: 2’73-277, 1986.
C104 MS-85-229 Project Officer: Smith
EVALUATION of CONVENTIONAL TREATMENT PROCESSES for REMOVAL
of MUTACENIC ACT WITY from MUNICIPAL WASTEWATERS
/dm 11. Meier 1 and Dolloff F. Bishop 2
1 }{flfl , USEPA, Cincinnati, OH
2 WERL., USEPA, Cincinnati, OH
The Office of Water Enforcement and Pent its and the Office of Water Regulations and Standards are inter-
ested in the feas ibility of using toxicity bioassays in a monitoring program of practical utility to water pollu-
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lion control agencies. In the present study the Salmonella microsome mutagenicigi assay was used to a-
amine the effectiveness of conventional treatment processes to remove inutagenic pollutants from twv
municipal wastewaters, one heavi y industrialized and one pdmad y domestic. The results indicated that
the level of mutagenici(y in the domestic effluent was substantialty lower than in the industrial effluent.
Fwthennorç an appreciable portion of the responsible mutagens are relative y refractory to removal by con-
ventionalprbnary and activated sludge treatment
The present study was conducted to evaluate the mutagenicity of a domestic wastewater and a combined
domesticllndustrial wastewater at various stages of conventional treatment Influent (raw wastewater)
and effluent samples were subjected to sequential acid/base liquid-liquid extractions with methylene
chloride and the extracts were assayed for mutagemic activity in the Ames test. £ typhimudum strain
TA98 gave the highest response of the tester strains to mutagens present (up to approximately 20,000
revertants/liter), but activity was also detectable with strains TA1537 and TA100. The majority of the ac-
tivity was direct-acting but in some cases significant enhancement of the activity was seen after activation
with a rat liver 59 fraction. Mutagen levels based on TA98 activity in the industrial/domestic effluents
were 7 to 45-fold higher than in the domestic effluent. Despite the consistent efficient removal ( > 90%)
of extractable organics by activated sludge treatment, the removal of compounds with mutagenic activity
was relatively poor and highly variable as evidenced by increases of 4 to 20-fold in the specific
mutagenicities of the extracts on a per mg basis. Chlorination at the secondary effluent stage had litt le in-
fluence on the mutagenicity seen.
CITATION: Joumal of the Water Pollution Control Federation, 57(10): 999-1005, October 1985.
BUS MS-85-095 Project Officer: Meier
MUNICIPAL SEWAGE SLUDGE APPLICATION on OHIO FARMS:
ESTIMATION of CADMIUM INTAKE
C. S. Reddy andCR. born
The Ohio State University
This journal article contains results jivm HERL’s research program on the health aspects of the land ap-
plication of municipal sludge. It deaLt with the human exposure to heay metals, o’ganic chemicals, and
pathogens as a result of land application, and potential adverse health effects.
This study was designed to estimate the contribution of municipal sewage sludge exposure and smoking
to the daily Cd intake in Ohio farm residents, based on the Cd concentrations in 24-hr fecal samples. The
fecal samples were analyzed for Cd and the daily Cd intakes were calculated by correcting for absorption
(4.6%) in the gastrointestina] tract. Fecal samples from cattle grazing on sludge-treated pastures were
also similarly treated except that published 24-hr fecal weights and a 2% absorption correction were used.
Fecal weights and the daily Cd intakes, in humans, calculated from these data were significantly lower in
females than in males with a female/male ratio of 0.77/1. Daily Cd intakes calculated from these data
ranged from 537 to 13.31 pg/day for females and 8.87 to 18.52 pg/day for males. No significant increase
in daily Cd intake resulted from exposure of humans to sludge on the farmlands. Although daily Cd in-
take for smokers was 1 pg/day higher than for non-smokers, the difference was not statistically significant.
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Cattle grii ing on sludge-treated pastures consumed cignificantly more (up to 3 times) Cd than cattle on
control pastures. It was concluded that application of sewage sludge on farmlands at rates of 2-10 dry
metric tons/ha did not cignilicantly contribute to the daily Cd intake in hflmnTLc ; cattle on such farms,
however, sgnil candy increased their Cd consumption.
CITATION: Environmental Research 38(2): 371-388, December 1985.
B113 XX-86-125 Project Officer: Kowal
EVALUATION of the REPRODUCTIVE TOXICOLOGY of
2,4,6-TRICHLOROPHENOL in MALE and FEMALE RATS
K Blackburn, H. Zeniclç E. Hope I. M. Maitson, E. L George 1 and M. K Smith 1
University of Cindnnnti Medical Center
‘HERL., USEPA, Cincinn iti, OH
Tnchlorophenol Lc a contaminant detected ii4de y in water supplies. Data concerning its reproductive ef-
fectr are impomtwzt in the setting of reference doses (RFDs) and health advisoiies.
The toxicity of chlorinated organic compounds which maybe generated as a by-product of drinking water
chlorination has been an issue of increasing concern. Relatively few data are available concerning their
reproductive toxicity. The present study was designed to evaluate the reproductive effects of one of these
compounds, 24,6-trichlorophenol (TCP), in male and female rats. Adult males were treated with either
0,100,500, or 1000 mg/kg of TCP (po) for 10 weeks, at which time semen evaluations were conducted on
ejaculates recovered from the genital tract of receptive females. Fertility was assessed in the 0- and 1000-
mg/kg groups. Females were treated with identical doses for 2 weeks prior to pregnancy then throughout
gestation. Dams were allowed to litter and pup development was monitored until Day 42 postpartum.
TCP had no effect on any sperm parameter or male fertility. Treatment of females with 1000 mg/kg of
TCP produced gross maternal toxicity as reflected in increased lethality and decreased weight gains in the
dams. However, no treatment-related differences were seen in litter sizes or pup survival. Male and
female birth weights were significantly depressed in the 500- and 1000-mg/kg groups; these differences
disappeared by Day 4 postpartum, suggesting that they were a reflection of maternal toxicity. To this ex-
tent, the reproductive processes of male and female rats do not appear to be a primary target for the ef-
fects of TCP.
CITATION: Fundamental and Applied Toxicology 6(2): 233-239, February 1986.
C104 MS-85-129 Project Officer: Smith
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REPRODUCTIVE TOXICOLOGY of DISINFECTION BY-PRODUCTS
Al. Kale Smith, 1 Harold Zeniclç 2 and Emma Lou George 1
‘HERL, USEPA, Cincinnsati, OH
2 University of Cincinnati College of Medicine
The disinfection of drinldng water is one of the most widely practiced and most successfid public health
programs in the U.S. While it has dramatically reduced the incidence of watethome illness, it has been
detennined that the organic chemical contaminants occwring at the highest concentration in dAnldng water
result from it This symposium paper summanzes the results to date of the Agency’s researeh into the
health effect of these contaminants.
The dicinfection of surface water for potability results in reactions between the dWnfectant and the or-
ganic compounds and materials present at the source. The reproductive consequences of exposure to
several better-known reaction products have been ernmined. Some of the trilialomethanes and
chiorophenols have been studied for teratogenic and reproductive effects, and this information is
reviewed. We have e nmined other dicinfection byproducts n- ing an in vivo teratology screen in the
Long-Evans rat. Pregnzint females were exposed during gestation to a single high dose (maximum
tolerated dose MTD) of a substance and allowed to litter. The pups were then evaluated for weight and
number on post partum days 1 and 4. Selected pups were then retained and in some cases exposed to the
compound of interest until puberty. We have also investigated the developmental consequences of in-
gesting acetonitrile and its halogenated dtcnfection derivatives during gestation. We found that at doses
toxic to the dam (300,500 mg/kg), acetonitrile adminictered prenatally showed no effect on parameters
measured for pup neonatal growth and survival. Haloacetonitriles, however, at maternally toxic doses (55
mg/kg) were fetotoxic, with gestational exposure resulting in reduction of both pup birth weight and
postnatal growth. The in utem toxicity compared among chioro-, dichioro-, trichloro- , dibromo-, and
bromochioroacetonitrile, was enhanced with increasing substitution at the a carbon. The in utero toxicity
of acetonitrile is considered to be largely due to cyanide release. In assessing our comparative data,
cyanide is not likely to be the fetotoxic agent for the haloacetonitriles. In view of the results of this screen,
a seg ment-I l study of trichioroacetonitrile was performed.
CITATION: In: Proceedings of the Second Inteinational Symposium on Health Effects of Drinking Water
Disinfectants andDisinfection By-Products, Cincinnati, Ohio, August 27-29, 1986. Published
in: Environmental Health Perspectives 69: 177-182, November 1986.
C104 MS-86-178 Project Officer: Smith
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REPRODUCTIVE EFFECTS of ALTERNATIVE DISINFECTANTS
Betsy D. Carlto j 1 PaulBwtlett, 1 Ail Baranm, 1
Kate Collin& Irene Osis, t and M. Kate Smith 2
1 Battelle-Columbus Laboratories
2 HERL, USEPA, Cincinnati, OH
The disinfection of ddnidng water Lr one of the most widely practiced and most successful public health
programs in the U.S. W7sile it has dramatically reduced the incidence of wateibome illness it has been
determined that the oiganic chemical contaminants occun’ing at the highest concentration in ddnklng water
result — it. This symposium paper summasizes the results to date of the Agency’s reseasch into the
health effect of these contaminants.
Potential health hazards are posed by organohalides formed through the reaction of chlorine and organic
compounds in natural and waste waters. For this reason, alternative water &tinfectants that do not form
organohalides are being investigated with great interest. However, data on the health effects of these
compounds, in particular, their reproductive toxicity effects, are limited. In our laboratory, we have a-
amined the reproductive effects of sodium chlorite, cbloramine, and chlorine adnih ctered in the drink-
ing water or directly by gavage in Long-Evans rats. Animals were treated for a total of 66 to 76 days.
Males were treated for 56 days and females for 14 days before breeding and throughout the 10-day breed-
ing period. Following breeding, the males were necropsied and evaluated for sperm parameters and
reproductive tract histopathology. Females were treated throughout gestation and lactation. Adult
females and some pups were necropsied at weaning on postnatal day 21. Other pups were maintained
and treated post-weaning, These pups were evaluated for the day of ngina ? patency and thyroid hormone
levels. No differences were observed between control aniniak and rats exposed to chlorite, chlorine, or
chloramine when fertility, viability, litter size, day of vaginal patency, or sperm count were evaluated. A
s ignificant (pc .01) dose dependent decrease in sperm direct progressive movement (um/sec) was ob-
served among male rats exposed to 100 or 500 ppm chlorite. In addition, a significant (p cOOl) increase
in abnormal sperm morphology was also observed in these animaIs No significant differences in any male
reproductive parameter were observed for rats exposed to chlorine. A change in thyroid hormone levels
was observed for rats exposed to chlorite. Hormone levels are currently being examined for chlorine- and
ch loramine-treated rats.
CITATION: In: Proceedings of the Second Intemational Symposiwn on Health Effects of Ddnldng Water
Disi4ectantr and Disinfrction By-Products, Cincinnati, Ohio, August 27-29, 1986.
Published it Environmental Health Perspectives 69: 237-241, November 1986.
C104 MS-86- 179 Project Officer. Smith
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THE SUBCHRONIC TOXICITY in RATS of
TETRACHLOROETHYLENE(PERCHLOROETHYLENE)
ADMINISTERED In the DRINKING WATER of RATS
IS. Hayes,’ LW. Condie, Jr ,, 2 and iF. Borzelleca 1
1 Medical College of Virginia
2 HERL, USEPA, Cincinnati, OH
Thi, paper describes a subchronic toxicology study in which tetrachloroethylene was administered to rats in
drinking wa1es These data will assist the Office of Drinldng Water (ODW) in setting standards for
tet rach loro ethylene.
Tetrachioroethylene (perchloroethylene) has been identified in drinking water at a concentration of ap-
proximately 1 mg/liter and in ambient air at levels of 0.18-4.5 ppb (0.0012-0.03 &g/m 5. Much of the avail-
able toxicological data are from inhsiktion of gavage studies. This study provides data on the effects of
tetrachloroethylene in drinking solutions. The acute oral LD .so was determined in male and female Char-
les River rats and found to be 3835 mg/kg for males and 3005 mg/kg for females. Male and female rats
received theoretical daily doses of 14,400 and 1400mg tetrachloroethylene/kg bw/day for 90 consecutive
days. There were no compound related deaths. Body weights were significantly lower in male and female
rats at the higher doses. There were no consistent dose related effects on any of the hematological or
urinalysis parameters. 5’Nucleotidase activity was increased in a dose dependent manner, suggesting
possible bepatotoxieity other blood chemistry parametere were unaffected by the treatment. There were
no gross pathological effects observed. Liver and kidney ratios were elevated at the higher doses. There
was no apparent evidence of accumulation. These data suggest that exposure of hnnizinc to reported
levels of tetrachloroethylene in drinking water does not constitute a serious health hazard.
CITATION: Fundamental andApplied Toxicology 7(1): 119-125, July 1986.
C104 MS-85-L53 Project Officer: Condie
EPIDERMAL HYPERPLASIA in the MOUSE SKIN FOLLOWING TREATMENT
with ALTERNATIVE DRINKING WATER DISINFECTANTS
Merrel Robinson, 1 Richard I. Bul 2 Marlene Schasner, 3 and Richard E. Long 3
1 HERL, USEPA, Cincinnat i , OH
2 Wnshhigton State University
3 Pathology Associates, Inc
The disinfection of drinking water is one of the most wid4i practiced and most successful pub lk health
programs in the U.S. MUle it has dramaticaI y reduced the incidence of wate,tome illness, it has been
detennined that the organic chemical contaminants occuning at the highest concentration in drinking water
result /k im it. This symposium paper swnmarizes the results to date of the Agency’s research into the
health effect of these contaminants.
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Female SENCAR mice were treated with aqueous solutions of HOCI.., NaOCL C102, and NH2C1 by
whole body exposure (except head) for a 10-minute period for 4 days in the first experiment and for iday
(except NH2C1) in the second experiment. Animals were sacrificed the day following the last treatment
(experiment #1) or on day 1,2,3,4,5,8,10, and 12 following treatment (experiment #2) and skin thick-
ness was measured by light microscope at x400 using an eyepiece micrometer. Concentrations of disin-
fectants were 1,10,100,300, and 1,000 mg/I, for experiment #1 and 1,000 mg/I for experiment #2. Thick-
ness of the interfollicular epidennis (IFE) for control animals was 15.4 ± lSp.in. After 4 days of treat-
ment at 1,000 mg/I, HOCL and ClOi increased thickness to 39±7.0 and 402±11.8, whereas NaOC1 in-
creased thickness to 25.2 ± 6.1 pm. Only HOCI and C102 were tested at 300 mg/I, and these yielded an
IFE increase of 30.0 ± 13.1 and 16.8 ± 0.8 pm, respectively. The response to HOCI was found to be
dose-related, with the minimally effective dose being 100 mg/i. In earlier preliminaTy tests to determine
optimum treatment schedule, the response to HOC1 appeared to be maximal after 4 days of treatment
and tended to decrease with further treatment. The time course study following a single treatment of
1,000 mg/I HOC I, however, showed a progression of WE thickening of from 18.3 ± 1.4 at 1 day to 30.8±
8 days then decreasing to 19.1 ± 6.2 m at 12 days. C102 and NaOC I, when tested in this manner, did not
result in increased thickness of WE with time, but rather gave a persistent level of increase that remained
for the 12 days. NH2C1 reduced skin thickness to 13.6 ± 6.1 p.m. Examining sections of skin treated with
HOCI and C102 indicated an increase in cell numbers. HOC1 and C102 are therefore capable of induc-
ing hyperplastic responses in the mouse skin. The basis for the decrease in skin thickness resulting from
NH2CI treatment remains to be established.
CITATION: In: Proceedings of the Second International Symposiwn on Health Effects of Drinking Water
DLcinfectants and Disinfection By-Pioducis, Cincinnati, Ohio, August 27-29, 1986.
Published in: En vironmental Health Perspectives 69: 293-300, November 1986.
C104 MS-86-177 Project Officer: Robinson
TOXICOLOGY OF HALOACETONITRILES
Johnnie R. Hayes, 1 Lyman W. condie , 2 and Joseph F. Borzelleca 1
1 Medical College of Virginia
USEPA, Cincinnati, OH
The disinfection of drinking water is one of the most widely practiced and most successful public health
programs in the U.S. While it has dramatically reduced the incidence of watethorne illness, it has been
detemuned that the organic chemical contaminants occurnng at the highest concentration in d*iking water
result from it. This symposium paper summanzes the results to diite of the Agency’s research into the
health effect of these contaminants.
Haloacetonitriles are by products of water chlorination and may form in vivo from the reaction of residual
chlorine with endogenous compounds such as amino acids. Dibronioacetonitrile (DBAN) was negative
in selected mutagenic assays; dichioroacetonitrile (DCAN) was mutagenic inS. yphirnurium, but not in
S. cerevisiae. Both DBAN and DCAN may be carcinogenic. There is a paucity of basic toxicological data
for these compounds. The studies described were conducted to determine the acute, subacute, and sub-
chronic toxicity of DBAN and DCAN. The acute oral LD 0 values (mg/kg) in mice and rats are: DBAN
- mice: 289 (M), 303 (F); rats: 245 (M), 361 (F); DCAN - mice: 270(M), 279 (F); rats: 339 (M), 330 (F).
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Death was preceded by slowed respiration, depressed activity, prostration, and coma. There were no ap-
parent compound-related gross pathological effects. DBAN (in corn oil) was administered by gavage to
male and female CD rats for 14 or 90 days at levels of 23,45, 90, and 180 mg/kglday or 6,23, and 45
mg/kg/day, respectively. Mortality was 100 percent at 180 mg/kg and 40 percent (M) and 20 percent (F)
at 90 mg/kglday, there were no deaths during the 90-day study. No consistent, significant, adverse com-
pound-related effects on any of the parameters evaluated were evident. Possible target organs might be
spleen, thymus, and liver. The no-observed-adverse-effect level (NOAEL) for 14 days was 45 mg/kg/day
and for 90 days, was 23 mg/kg/day. DCAN (in corn oil) was administered by gavage to male and female
CD rats for 14 or 90 days at levels of 12,23,45, and 90 mg/kg/day or 8,33, and 65 mg/kg/day, respective-
ly. There were no deaths during either study. Body weights were significantly lower at 90 and 65
mg/kg/day; weight and ratios of spleen and gonads and cholesterol levels were significantly lower at 90
mg/kg/day. No consistent, significant adverse compound-related effects on any of the parameters
evaluated were evident. The NOAEL for 14 days was 45 mg/kg/day and for 90 days, was 33mg/kg/day.
CITATION: 1n Proceedings of the Second International Symposium on the Health Effects of Drinldng
Water Disinfectants and Disinfection By-Products, Cindnrnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69:183-202, November 1986.
C104 MS-86-175 Project Officer: Condie
MECHANISTIC ASPECTS of INGESTED CHLORINE DIOXIDE on THYROID
FUNCTION: IMPACT of OXIDANTS on IODINE METABOLISM
I. Peter Berc Lillian L. Jones, Robe, M. Harnngton,
Rohit Bawa, and Lyman W. Condie
HERL, USEPA, Cincinnati, OH
The disinfection of drinking water is one of the most widely practiced and most successful public health
programs in the U.S. While it has dramatically reduced the incidence of waterborne illness, it has been
detennined that the organic chemical contaminants occurring at the highest concentration in drinldng water
result from it. This symposium paper summarizes the results to date of the Agency’s research into the
health effect of these contaminants.
The etiology of thyroid inhibitory effects of ingested chlorine dioxide in multiple experimental animal
models is apparently connected to disturbances of iodine metabolism induced by the oxidant chemical.
in vivo as well as in vitro studies with radio tracer iodide provide evidence that ingestion of disinfectants
(both C102 and OC1) induce excessive and abnormal organification of dietary iodine in the alimentary
tract. Both pulsed and steady-state administration of radioiodide in feed and concurrent administration
of the disinfectants via drinking water resulted in the immobilization of the isotope on the oral and gastric
mucosal surfaces as well as in accelerated fecal loss of iodide. A major part of the fecal isotope content
was covalently bound to nondigestable particles. Thus it appears that ingestion of disinfectants induces
a dose-and-time-dependent iodine deficiency in the organism , albeit probably insufficient in magnitude
to explain the decrease in thyroid hormone synthesis. More importantly, there is evidence that the intra-
alimentary organification of iodide involves some nutrient or hormone molecules, some of which may pos-
sess extensive metabolic inhibitory properties after covalent iodination. Furthermore, based on the
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polarizability of the carbon-iodine covalent bond, it is postulated that some of the products of the or-
ganification process are genotoxic and may be carcinogenic. From our experiments it appears that the
principal effects of dicinfectants may arise from disturbances of iodine metabolism and from the genera-
don of in situ toxic substances from otherwise nontoxic nutrients and hormones. The extent and severity
of this phenomenon is probably proportional to the oxidi”ing power (redox potential) of the disinfectant
molecule.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Drinldng Water
Disinfectants and Disinfr clion By-Products, Cmdnn2ti, Ohio, August 77-29, 1986. Published
in: Environmental Health Perspectives 69:249-254, November 1986.
C104 MS-86-173 Project Officer: Bercz
SHORT COMMUNICATION - TERATOGEN METABOLISM:
THALIDOMIDE ACTIVATION is MEDIATED by CY OCHROME P-450
A. G. Brawi F. A. Harding and S. L Weinreb
Massachusetts Institute of Technology
The tumor cell attachment assay is undergoing validation as a teratogen screen. ThLs paper describes its
response to the w-chelypal teratogen thalidomide. Authentication of such a screen would greatly reduce
need for costly teratology testing in all programmes involved in hazard evaluation.
A metabolite of thalidomide generated by hepatice microsomes inhibited the attachment of tumor cells
of concanavalin A-coated polyethylene. Evidence that metabolite formation is mediated by niicrosomal
cytochrome P-450 is presented. Microsomes incubated with thalidomide underwent a type I spectral
shift. Metabolite formation was reduced or elhninnted by carhon monoxide, DKF-525A, metyrapone,
and N-octylamine. Superoxide dismutase treatment had no effect. Metabolite formation inquired
microsomes and NADPH and was dependent on the length of 37°C incubation. The metaboite could be
isolated by successive hexane and chloroform extractions. It is likely the inhibitory thalidomide metabo-
lite was generated by a minor cytochrome P-450 species. Whether this thalidomide metabolite is involved
in the drug’s teratogenic activity remains to be shown.
CITATION: Toxicology andApplied Phannacology 82(1): 175-179, January 1986.
C104 XX-86-088 Project Officer: Smith
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PROCEEDINGS of the WORKSHOP on the SENCAR MOUSE in
TOXICOLOGICAL TESTING
Meirel Robinson
HERL, USEPA, Cincinnati, OH
The results of the research descnbed in the attached workshop proceedings fiuther charactenzes the SEN-
CAR mouse and its adwantages and disadvantages as an animal testing model in toxicological and car-
cinogenesis experimentation. The data deals with three categories relevant to the response of the mouse in
laboratory tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initiation/promo-
tion assay), and (3) histopatholorj including treabnent-induced and spontaneous lesions.
Since the estabiWimeut of the more tumor susceptible SENCAR stock of mice, it has been used exten-
sively in carcinogenesis assays by government agencies,-academia, and commercial laboratories. The
model has facilitated substantial cancer research involving mechanistic studies, as well as testing of
various classes of chemicals, and complex mixtures such as organic extracts of drinking water, automobile
exhaust, and consumer products, all of which are of particular concern to the regulatory agencies. The
purpose of this EPA Workshop on the SENCAR Mouse in Toxicological Testing is to provide a forum
for the discussion of research data related to the use of this i nim d model. The workshop critically ad-
dresses the performance of the SENCAR mouse as a carcinogenesis assay model. Presentations of data
by noted researchers deal with three categories relevant to the response of this mouse in laboratory tests:
(1) Response to carcinogens-including strain and stock comparison and methodology for statistical
analysis of mouse skin assays; (2) Alternate testing in the SENCAR--induding in vitro comparison of
SENCAR and BALB/c primary epidermal cells, metabolic activity in embryo cells, distribution studies,
and modified initiation/promotion studies; and (3) Histopathology of the SENCAR mouse--including
treatment-induced and spontaneous lesions, chronic effect of promoters, and pathology of g ng
SENCARS. The workshop concluded with an afternoon devoted to a round table discussion involving all
presenters as well as members from the audience. A summary of this discussion follows the individual
papers in these proceedingc.
CITATION: Environmental Health Perspectives 68:1-250, September 1986.
C104 HERL-()544 Project Officer: Robinson
BARIUM in TEETH as INDICATOR of BODY BURDEN
Robe,t G. Miller, 1 John D. B. Featherstone, 2 Mwf in E. I.
Tammy S. Mills, 1 and Carole P. Shields
1 IffiRL USEPA, Cincinnati, OH
2 Eastman Dental Center
The results of this research project concerning banum in drinking water was to provide suppon to regulatory
development and revision of MCL Data indicates that barium from drinking water is absorbed in
deciduous teeth and is an indicator of body burden.
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A study was conducted to determine the biological availability of naturally occurring barium in a
municipal drinking water by the analysis of barium in deciduous teeth of children. The grade school
children of two Illinois towns were chosen for this study. The towns were chosen based upon the barium
content of their drinking water supply, geographic proximity, population, ethnic composition, and
socioeconomic status of its residents. The high barium town had an average drinking water level of 10
mg/L and a mean of 36 p.g Ba/g Ca in teeth of life-long residents drinking city water compared to teeth
levels of 7 g Ba/g Ca in the children of the low level barium town which had a drinking water concentra-
tion of 0.2 mgIL barium.
CITATION: In: Advances in Modem Environmental Taucology, VoL 9: Inorganks in D*zldng Water and
Cardiovascular Disease (Chapter 19), EJ. Calabresn, R.W. Tuthill, and L.W. Condie, eds.,
Princeton Scientific Publishing Co., 211-219, 1986.
C104 MS-84-090 Project Officer: R. Miller
ABSORPTION of LEAD FROM DRINKING WATER with VARYING
MINERAL CONTENT
Robeit G. Miller, Daniel Greathouse Richwrlj. Bull and Iwnes U. Doerger
HERI USEPA, Cincinnati, OH
This research project was to show the dietaiy effects of “hard water” mineraLs upon the absorption of lea4
thereby helping to establish chemical toxicities involving the cardiovascular system. This ultfrnate y would
provide suppo 1 to regulatory development and revision of MCL
Lead (Pb) (200 ppm) was administered via drinking water to rats for nine weeks. In addition, the rats were
grouped so that they received 75,100,150 and 250% of the minimum daily requirements (MDR) of cal-
cium (Ca), iron (Fe), and magnesium (Mg) as required for normal growth. The exposures were arranged
so that no more than one element was varied within the same nnim ii groups, while maintpining 100% of
the MDR of all other elements and nutrients. Blood lead analyses were performed at 0,1,2,3,7,8 and 9
weeks after exposure. Food and water consumptions and body weights were measured each week (0-9).
Some gross differences were seen both within and among the groups with respect to milliliters water con-
sumed vs. body weight and grams of food consumed vs. body weight. During the first few weeks of ex-
posure, the mean blood lead in nnizniils fed 75% MDR of Ca, Fe, and Mg appeared less than those receiv-
ing 100% MDR of the corresponding elements. There was a decrease in mean blood lead concentration
in nnim als receiving ISO and 250% of the MDR of Fe at the 7-9 week time period; however, this was
probably due to a slight decrease in water consumption during this time period. There seemed to be no
appreciable differences in mean blood lead concentrations of groups exposed to high concentrations of
Ca and Mg. However, analysis of the data after correcting for varying Pb dose points to the conclusion
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that dietary magnesium has no effect on uptake of lead whereas dietary iron decreases blood lead con-
centration and calcium only increases blood lead concentration after extended exposure (7-9 weeks).
CITATION: 1n Advances in Modem Envimtunental Toxicology, VoL 9: Inorganics m Drinldng Water and
CanliovascularDLcease (Chapter 25), EJ. Calabrese, R.W. Tuthill , and L.W. Condie, eds.,
Princeton Scientific Publishing Co., 289-297, 1986.
C104 MS-84-099 Project Officer: R. Miller
IMMUNOTOXICOLOGICAL INVESTIGATIONS in the MOUSE:
GENERAL APPROACH and METhODS
KimberL Thite, Jr., 1 Vuginia M. Sanders, 1 Donald W. Barnes, 2
George M. Shopp, Jr., 1 andAlbe ,! E. Munson 1
1 Medical College of Virginia
2 Carolina University
The adverse effects of chemicals on the ymphoreticular system have generated considerable toxicological
interesL This paper descnbes the methods and general approach used in judgjng a chemical’s potential nsk
to the immune system. Risk evaluation was approached utilizing acute, 14- and 90-day studies porn &-
pasure to thinking water contaminants. The immune system was evaluated against a backgpound of more
standard toxicological pararn eters. These techniques will assist researchers in evaluating the heafth impact
of drinking water contaminants.
The adverse effects of chemicals on the lymphoreticular system have generated considerable toxicologi-
cal interest. In this series of papers, the effects of selected environmentally relevant compounds are
reported. This first paper describes the methods and general approach used in judging a chemical’s
potential risk to the immune system. Risk evaluation was approached utilinng acute, 14.- and 90-day
studies. Both sexes of the CD-i random-bred mouse were employed. The immune system was evaluated
against a background of more standard toxicological parameters, which induded fluid consumption, body
and organ weights, hematology, serum and liver chemistries, hepatic microsomal enzyme activities and
blood coagulation. Bone marrow status was evaluated by assessing DNA synthesis. Humoral immunity
was evaluated by determnng the number of 1gM spleen antibody-forming cells (AFC) to sheep
erythrocytes (sRBC), the serum antibody level to sRBC, and spleen lymphocyte response to the B cell
mitogen, lipopolysaccharide (LPS). The status of cell-mediated immunity was assessed by quantitating
the delayed type hypersensitivity (UTH) response to sRBC, proliferation of the popliteal lymph node,
and the spleen cell response to the T lymphocyte mitogen, Concanavalin A (Con A). Macrophage func-
tion was evaluated by measurement of the vascular clearance rate and distribution of radiolabeled sRBC
in the liver, spleen, lungs, and thymus, and recruitability, adherence, chemotaxis, and phagocytic activity
of peritoneal exudate cells (FEC). Historical control data from six 14- and 90-day studies conducted over
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a one-year period are given. The data resulting from these types of studies can provide a basis for the in-
itial evaluation of a chemical’s adverse effect on the immune system.
CITATION: Drag and Chemical Toxicology 8(5): 299-331, October 1985.
C104 XX-86-100 Project Officer: Condie
A TEST BAT lERY for SCREENING BEHAVIORAL TERATOGENS in MICE
Mary Jeanne Kalbnan 1 and Lyman W. Condie, Jr. 2
1 University of Micsic cippi
2 HERL, USEPA, Cincinn2ti, OH
Research was conducted to develop and validate a test battery for identifying teni togens in mice. The mice
were exposed to envuvnmental contaminants by the oral route of exposure. The techniques employed
should enable other d,inldng water contaminants to be evaluated for establishing their teratogens potentiaL
The assessment batteiy was developed for identifying teratogens in mice (CD-i, outbred strain) since
many non-behavioral toxicologists prefer working with the mouse and because the mouse is less costly to
house and feed. This approach emphsacwes the importance of adult toxicity data in the selection of ex-
posure levels for teratologic studies and for interpreting behavioral teratogenic effects. Potential
teratogen exposure is both prenatal and postnatal with the goal of identifying the need for more costly and
time consuming, cross-fostering and critical period designs. The test battery indudes the assessment of
multiple behavioral capabilities, including physical landmarks, reflex developments, motor capability and
passive avoidance learning. We have found the behavioral capabilities of the mouse to be siniil r to those
of the rat in variability and range of response. Our methodology has emphpsized drinking water exposure
to chemicals since we were involved in the evaluation of drinking water contanhinnnts, but the battery
should be amenable to any route of exposure.
CITATION: Neurobehavioral Toxicology and Teratology 7:727-731,1985.
C104 MS-86-089 Project Officer: Condie
TOXICOLOGY of 1,1,2-TRICHLOROETHANE in the MOUSE
ICImberL W 1 zite, Jr., 1 Virg inia M. Sanders, 1 Donald W. Ban ies, 2
GeorgeM. Shopp, Jr., 1 andAlbei’tE. MwLson 1
1 Medical College of Virginia
2 Carolina University
This paper repo tc toxicity findings of a potential di’inldng water organic chemical contaminant. 1,1,2-
t,ichloroethane (TCE) was administered to male anti ftmale CD-i mice to evaluate its effect on standard
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toxicological parameters. Following detennination of the acute lethal dose for 50% of animaLs and a 14-
day range-finding study, a 90-day dsinkhig waler study was pe fonneS The liver was a talget of TCE
toxicity in both seses as demonstrated by dose-dependent alterations in liver enzymes. ThLc paper will assLct
the Office of Thinking Waler (ODW) in setting Drinking Water Standards or Health AdvLsodes for TCE.
1,1,2-Tricliloroethane (TEC) was admini ctered to male and female CD-i mice to evaluate its effect on
standard toxicological parameters. Following determination of the acute LD5O (378 mgflcg in males and
491 mg/kg in females), and a 14-day range-finding study, a 90-day drinking water study was performed in
which the doses consumed were 4.4,46, and 305 mg/kg for males and 3.9,44, and 384 mg/kg for females.
The liver was a target of TCE toxicity in both sexes as demonstrated by dose-dependent alterations in
hepatic microsomal enzyme activities and serum enzyme levels. The erythroid element of the female mice
was also affected, as indicated by ignificantly decreased hematocrit and hemoglobin levels.
CiTATION: Drug and Chemical Toxicology 8(5): 333-355, October 1985.
C104 XX-86-1O1 Project Officer: Condie
ACUTE TOXICITY of MONOCHLOROPHENOLS, DICHLOROPHENOLS
and PENTACHLOROPHENOL in the MOUSE
Joseph F. Borzelleca, 1 Johnnie A Hayes, 1 Lyman W. Condie 2
and John L. Egle, Jr. 1
1 Medical College of Virginia
2 HE USEPA, Cincinnati, OH
Research was conducted to detennine the acute toxicity of monochlorophenols, dichiorophenols, and pen-
tachiorophenols. It was found that 2-chiorophenol and 3-chiorophenol were more toxic than the
dichiorophenol series. These studies may aid the Office of Drinking Water(ODW) in setting Health Ad-
v&sojes.
Acute oral LD5O values were determined for 2-, 3-, and 4-chiorophenol; 23-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-
dichiorophenol and pentachiorophenol in male and female mice. LD5O values (mg/kg) ranged from 117
(females) and ill (males) for pentachiorophenol to 2389 (females) and 2643 (males) for 3,5-
dichiorophenol. It was found that 2-chiorophenol and 3-chiorophenol were considerably more toxic than
the dichiorophenol series. Values for males and females were generally similar, the major differences
being with pentachiorophenol and 2,5-dichlorophenol where in both cases the female LD5O was lower.
CITATION: Toxicology Letters 29(1): 39-42 , December 1985.
Ci04 MS-85-124 Project Officer: Condie
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PRIMARY LIQUID INTAKE and URINARY STONE DISEASE
Jonathan Shuster, Birdwell Finlayson, Richard L. Scheaffer,
Robeit Sierakowski , John Zolte!c and Sylvia Dzegede
University of Florida-Gainesville
This paper summarized the results of a study to detemiine if an association between water hardness and
unna.’y stone disease erLvts asprevious y repoiteS. If this can be shown to be a causal relationship, ODW
could propose regulations for the wtificial softening of drinking water. Results of this study indicate there
are impoitant associations between a person’s primary liquid intake and unnaly stone disease but no
causal relationship was implieS
This investigation indicates that there are important associations between urinary stone disease and a
person’s primary liquid intake. Based on data collected from 2295 cauc c’ n male patients from two
geographical regions, the Carolinas (both North and South) and the Rockies (including Colorado, Idaho,
Nevada, Montana, Utah and Wyoming) an importantp <0.01) positive association was found between
urinary stone disease and soda (carbonated beverage) consumption within both geographical regions. It
was also found that negative associations exist between urinary stone disease and both beer consumption
and coffee consumption in the Rockies and that no important associations exist between urinary stone
disease and any of milk, water, or tea, when these beverages represent a person’s primary liquid intake.
Moreover, soda can be viewed almost synonymously as sugared cola, since few subjects had diet sodas or
sugared non-cola soda as primary fluid. No cause/effect relationships are implied in this paper.
CITATION: Journal of Chronic Diseases 38(11): 907-914, November 1985.
C104 XX-83-087 Project Officer: Greathouse
THE EFFECT of SINGLE VERSUS SPLIT DOSES of DIETHYLNITROSAMINE
on the INDUCTION of GAMMA-GLUTAMYLTRANSPEPTIDASE..FOCI
in the LIVERS of ADULT and JUVENILE RATS
S. L Heiren-Freun4 1 MA. Pereira 1 it E. Long 2
andM.M. g, , 2
1} J , USEPA, Cincinnati, OH
2 Pathology Associates, Inc.
The results demonstrate that juvenile ‘aIr (32 days of age) are more sensitive than adult rats (60 days of
age) to the induction of gamma-glutwnyWanspepildase (GGT)-fo4 putative preneoplastic lesions. This
type of study is important to optimize the conditions of the liver foci bioassay for use in delemiining the car-
cinogen icily of environmental samples.
The induction of g linimi -glutamyltranspeptidase (GGT)-foci by single and by split doses of diethyl-
nitrosamine (DENA) was evaluated in the livers of juvenile and young adult male, Sprague-Dawley rats.
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A single dose of DENA was administered at either 32,41 or 52 days of age and followed by a promoting
regimen of sodium phenobarbital in the drinking water. In 32 day old rats, DENA resulted in a higher
incidence of GGT-foci/cin 3 than were observed when it was administered to 41 or 46 day old rats. The
same dose of DENA was inactive in 52 or 60 day old rats. In rats initipted with DENA on day 32 of age,
starting the phenobarbital promotion at 41 days of age resulted in a higher incidence of GGT-foci than
when the phenobarbital treatment was begun at 60 days of age. When the dose of DENA was split into S
daily doses of DENA starting on day 32 of age, the incidence of GGT-foci/cin 3 was equivalent to the single
dose given on day 32 of age. However, when the five daily doses were started on 48 days of age, the in-
cidence of GGT-foci/cni 3 was less than the five doses of DENA started on day 32 of age. Hence, juvenile
rats are more sensitive to both single and split doses of DENA than adult rats. The dose-response
relationship was also determined in juvenile rats for a single and for up to 64 daily doses of 3 mg/kg DENA
started on day 32 of age. The dose-response relationship for a single dose of 3 to 96 mg/kg DENA was
equivalent to the relationship of one to 32 daily 3 mg/kg doses. Thus, low daily doses of DENA were ad-
ditive when started in juvenile rats. Neither a threshold nor a plateau was obtained for the dose-response
relationship of DENA initiation of GGT-foci.
CITATION: Carcinogenesic 7(7): 1107-1110, July 1986.
C104 MS-86-129 Project Officer: Herren-Freund
TOXICOLOGICAL PROBLEMS ASSOCIATED with CHLORINE DIOXIDE
and CHLORINE
Lyman W. Condie
HERI., USEPA, Cincinnati, OH
This re view paper descnbes research conducted in the Toxicology and Microbiology Divirion of HERL and
sponsored by EPA regarding toxicological effects of disinfectantc and their by-products. This paper indi-
cates the cwvent status of our research and should assist the Office of Drinking Water (OD 9 in their
promulgation of drinking water standards for disinfectwzts.
The author reviews and summarizes the toxicological hazards associated with disinfecting drinking water
with either chlorine dioxide or chlorine. Adverse health effects seen in inm i1 experiments as well as
humiin poisoning incidents are reported. Toxicological effects, which are associated with the disinfec-
tants themselves as well as with the products formed when disinfectants react with organic material
present in water, are considered in this paper.
CITATION: JournalAmerican Water Works Association 78: 73-78, June 1986.
C104 MS-86-105 Project Officer: Condie
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RELATIONSHIP of DIETARY IODIDE and DRINKING WATER
DISINFECTANTS to THYROID FUNCTION in EXPERIMENTAL ANIMALS
Nathaniel W. RevLs Paid McCauley, 2 and George Holds wor&
‘Oak Ridge Research Institute
2 HE USEPA, Cincinnati, OH
The dLcinfeciion of drinking water £c one of the most Mdely practiced and most succesful public health
programs in the U.S. J Ile it has dranwtically reduced the incidence of waterbome ilbiess it has been
detejmined that there may be undesinzble side effects of disinfection. This symposium paper .swnmwizes
one of these side effec&c
The importance of dietary iodide on the reported hypothyroid effect of drinking water dicinfectants on
thyroid function was investigated. Previous studies have also showed differences in the relative sensitivity
of pigeons and rabbits to chlorinated water. Pigeons and rabbits were exposed for 3(300 ppb) levels of
iodide and to drinking water cont nh ’g two levels of chlorine. Results showed that the high iodide diet
prevented the hypothyroid effect observed in pigeons given the low-iodide diet and chlorinated drinking
water. Simibir trends were observed in rabbits exposed to the same treatment; however, significant
hypothyroid effects were not observed in this nim i1 model. The factor associated with the observed ef-
fect of dietary iodide on the chlorine-induced change in thyroid function is unknown, as is the relative sen-
sitivity of rabbits and pigeons to the effect of chlorine. Several factors may explain the importance of
dietary iodide.and the relative sensitivity of these species.
CITATION: 1n Proceedings of the Second International Symposium on Health Effects of Drinking Water
Disinfectants and Dicinftciion By-Products, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69: 243-248, November 1986.
C104 MS-86-168 Project Officer: McCauley
RESULTS of TOXICOLOGICAL TESTING of JEFFERSON PARISH PILOT
PLANT SAMPLES
Robe,t G. Miller, Frederick C Kop/ler, Lyman W. Condie,
MichaelA. Pereira John 11. Meler, H. Paul Pjnghan4 and Bnsce C Casio 1
HERJ.., USEPA, Cincinnati, OH
t Environmental Health Research and Testing Inc.
The dirinftction of drinking water is one of the most widely practiced and most successful public health
programs in the U.S. Wlzik it has dramatically reduced the incidence of wateabome illness, it has been
detennined that the organic chemical contaminants occurnag at the highest concentration in drinking water
result from it. This symposium paper summarizes the results to date of the Agency’s research into the
health effect of these contaminants.
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Drinking water samples were collected from the alternative disinfection pilot plant facility at Jefferson
Parish, Loiikis iui The facility design allows for collection of samples at several sites within five separate
streams each treated with an alternative dicinfectant (nondisinfection, ozone, chlorine, chlorine dioxide,
chloraniine) before treatment with granular activated carbon (GAC). The samples were collected and
concentrated to either 400X by reverse osmosis or to 1,000X by adsorption to and subsequent elution
from macroreticular resins. Before initiation of toxicological tests, the resin extracts were further con-
centrated to 4,000X. The toxicological assays conducted were Ames Assay, In Vivo Toxicity Test in Mice,
SENCAR Mouse Skin Bioassay, Rat Liver Foci Assay, and Lung Adenoma Assay in Strain “A” Mice. The
results of the In I4vo Toxicity Test in Mice ncng the 400X samples derived by reverse osmosis process in-
dicates significant differences occurred in the growth rate pattern among some of the exposure groups.
The results of the Ames Assay on the samples concentrated by reverse osmosis were negative for
mutagenic activity. Those samples that were deemed positive in the Ames Assay were collected by XAD
resins from the streams treated with the tlicinfectants chlorine, chlorine dioxide, and chioramine.
However, after treatment with GAC in the chlorine stream, the samples were negative for mutagenic ac-
tivity nsing the Ames Assay. The remnining tests were conducted on samples (4,000X) collected by the
resin process. The results of carcinogenicity tests were all negative.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Drinking Water
DLrinfectants and Dtsinfrction By-Fmducts, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69:129-139, November 1986.
C104 MS-86-167 Project Officer: R. Miller
STUDIES or the TOXIC INTERACTIONS of DISINFECTION BY-PRODUCTS
A Dana Laurie, J. Peter Bercz , Thomas K Wessendaip, and Lyman W. Condie
HERL, USEPA, Cincinnati, OH
The disinfection of diinldng water is one of the most widely practiced and most successful public health
programs in the (iS. Julie it has drwnasically reduced the incidence of waterbome illness, it has been
detennined that the organic chemical contaminants occwring at the highest concentration in drinking water
result from it. This rymposium paper summarizes the results to date of the Agency’s research into the
health effect of these contaminants.
A large number and variety of compounds are formed in the process of chlorinating drinking water. The
classes of compounds formed include trihalomethanes, haloacetic acids, haloacetonitriles, halophenols,
and haloacetones. Many of the compounds have been shown to be toxic and are currently being further
evaluated by the EPA. One group of haloacetones found in chlorinated drinking water is the
dichioropropanones. Since the toxicological properties of this group have not been well characterized
and because a number of investigators have shown that ketones potentiate the heptatoxicity of
halonlknnes, we investigated the toxicity of the dichioropropanones and their interaction with carbon
tetrachioride. A variety of experimental techniques were used to evaluate the toxicity of the
dichioropropanones and their interaction with carbon tetrachloride-cytochrome P450, reduced
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glutathione levels, thiobarbituric acid reactive materials, pentane generation, clinical chemistries, calcium
levels, and histopathology. The findings of these studies will be presented in detail at this meeting.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Dr inking Water
Dicinfectants and Dtsinfection By-Products, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69:203-207, November 1986.
C104 MS-86-166 Project Officer Laurie
ROLE of GLUTATHIONE DEPLETION in the CUOTOXICITY of
ACETAMINOPHEN in a PRIMARY CULTURE SYSTEM of RAT HEPATOCYTES
David B. Mitchel4 t DanielAcosQ and James V. Bnsckne?
1 Miami Valley Labs, Proctor and Gamble
2 University of Texas-Austin
kJniversity of Georgia-Athens
This project was a putt of an in vivolin vitro comparison study of drinking water pollutants. This research
demonsnrues the accuracy and feasibility of the in vitro tasting method
A primary culture system of postnatal rat hepatocytes was utilized to study the cytotoxicity of
acetaminophen and the toxicological significance of glutathione (GSH) depletion. The relative time of
onset and magnitude of GSH depletion, lipid peroxidation and cytotoxicity were contrasted in order to
— insight into their interrelationships. Exposure of the hepatocytes to acetaminophen resulted in time-
and dose-dependent depletion of cellular GSH. The acetaniinophen-induced GSH depletion and ensu-
ing lactate dehydrogenase (LDH) leakage were quite modest and delayed in onset; in contrast to that
caused by iodoacetamide (IAA) and by diethylmaleate (DEM), 2 well-known depletors of OSH. There
was comparable LDH leakage irrespective of drug treatment; when GSH levels decreased to about 20%
of normal. Reduction of GSH levels below the 20% threshold by IAA treatment resulted in marked LDH
leakage and loss of viabilit Maximal LDH leakage in response to IAA and acetaminophen preceded
maximal malondialdehyde (MDA) formation, suggesting that lipid peroxidation may be a consequence
of cell damage as well as GSH depletion. LA S and DEM produced a comparable, modest accumulation
of MDA, yet IAA was much more cytotoxic. These findings indicate that lipid peroxidation does not play
a central role in hepatocellular injury by compounds which deplete GSH, although it may contribute to
degeneration of the cell. As events in the cultured postnatal hepatocytes paralleled those reported in
vivo, the system can be a useful and valid model with which to study mechsnkms of chemical toxicity.
CITATION: Toxicology 37: m-146 October 1985.
C104 XX-86-056 Project Officer: McCauley
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ORAL TOXICITY of CARBON TETRACHLORIDE:
ACUTE, SUBACUTE, and SUBCHRONIC STUDIES in RATS
I. V Biuckner, 1 W. F. MacKenzie, 2 S. Muraiidhare, 3
R. LutFuri, G. M. Kyle, 3 and D. Acosta 3
t University of Georgia-Athens
k niversity of Texas Medical School-Houston
3 University of Texas-Austin
Carbon tetnichionde Lr a common drinldng water pollutant. ThLr study is in direct suppoit of drinking
water regulations concerning safe cwbon tetrachlo,ide concentrations in potable water.
This investigation was conducted to characterize the acute, subacute, and subchronic toxic potency of in-
gested carbon tetrachloride (CCI4). In the first acute and subacute toxicity study, male Sprague-Dawley
rats of 300-350 g were gavaged with 0,20,40, or 80 mg CCI4Ikg once daily for 5 consecutive days, rested
for 2 days, and dosed once daily for 4 additional days. Rats of 200-250 g were gavaged with 0,20,80 or
160 mg CCLi/kg according to the same dosage regimen in the second acute and subacute study. In the
first and second studies one group of rats at each dosage level was sacrificed for clinical chemistry and
histopathological evaluation at 24 hr, 4 days, and 11 days after initiation of dosing. Single 20- and 40-
mg/kg doses had no apparent toxic effect at 24 hr, although 80 mg/kg caused mild hepatic centrilobular
vacuolization and ignificant increases in some serum enzyme levels. In general, there was progressively
severe hepatic injury at each dosage level over the 11-day period. CCLI was more hepatotoxic to the 200-
250-g rats than to the 300-350-g rats. In the subchronic study, rats initially 200-250 g were gavaged 5 times
weekly for 12 weeks with 0, 1, 10, or 33 mg CCI4/kg. Body weight and clinical chemistry indices were
monitored during the 12 weeks of dosing and 2 weeks after cessation of dosing. A dose of 1 mg/kg had
no apparent adverse effect; 10 mg/kg produced slight, but statistically significant increases in sorbitol
dehydrogenase activity and mild hepatic centrilobular vacuolizadon 33 mg/kg caused marked
hepatotoxicity. Serum enzyme levels remained elevated during the 12-week dosing period, but returned
toward normal within 13 days of cessation of Ccl i exposure. Microscopic examination of livers of the 33-
mg/kg rats revealed cirrhosis, characterized by bile duct proliferation, fibrosis, lobular distortion,
parenchymal regeneration, hyperplastic nodules, and single-cell necrosis. The fibrosis was not reversed
within the 13-day recovery period.
CITATION: Fundamental and Applied Toxicology 6:16-34,1986.
C104 XX-85-006 Project Officer: McCauley
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MYELOID HYPERPLASIA in the SENCAR MOUSE:
DIY1 ’ERENTL&TION from GRANULOCYFIC LEUKEMIA
Richard E. Long’ Gary KnuLcen 1 and Merrel Robinson 2
1 Pathology Associates, Inc.
2 HERL, USEPA, Cincinnati, OH
The results of the research described in the attached workshop proceedings afliclefiuiher characterizes the
SENCAR mouse and iLs advantages and disadvantages as an animal testing model in taricological and
carcinogen esis expenmentation The data deals with three categones relevant to the response of the mouse
laboratory tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initiation/promo-
tion assay), and (3) hLstopathology-including treatment-induced and spontaneous lesions.
The term myeloid h erplasia has been used interchangeably with many other terms to describe an in-
creased production of granulocytes in the spleen and other organs in the mouse. This process is oc-
casionally misdiagnosed as granulocytic leukemia. This paper reviews some of the terms used interchan-
geably with myeloid hyperplasia and describes criteria which can be used in differentiating mye loid by-
perplasia from granulocytic leukemia. Additionally, the results of a study in which myeloid hyperplasia
was induced following the formation of skin tumors is discussed. In this study, positive correlations were
found between skin lesions, the spleen weight and histologic appearance of the spleen. The liver rarely
showed microscopic dianges of myeloid hyperplasia unless the spleen weighed at least 1.0 percent of the
body weight.
CITATION: Environmental Health Perspectives 68: 117-123, September 1986.
C104 MS-86-118 Project Officer: Robinson
HUMORAL and CELL-MEDIATED iMMUNE STATUS of MICE
EXPOSED to TRANS-1,2-DICHLOROETHYLENE
George M. Shopp, Jr., Virginia Al. Sanders, Kim ber L 1 ite, Jr.,
andAlbeitE. Munson
Medical College of Virginia
ThLc paper repo u to4cisy findings of a potential drinking water organic chemical contaminanL This study
assessed possible adverse immunological effects of tmns-1,2-dichlomethylene (DCE) on random-bred CD-
1 mice. A 14-day range-finding study was pe’fomied on male mice by gavage at doses 1110 and 1/100 the
lethal dose for 50% of animals. No alterations in either humoral or cell- mediated immunity were obs&ved
following thIs e.iposure. A 90-day study was conducted in which DCE was administered in drinking water
of male and ftmale mice. No changes ware obse ved in the cell-mediated immune status of either sex or in
the humoral immune status of fern ales. However, a marked suppression in humoial immune status was ob-
seri’ed in male mice exposed to all three levels of DCE, as indicated by a decreased ability of spleen cells to
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produce antibody. There data will assist the Office of Drinking Water (ODW) in setting Drinldng Water
Standards and Health Advisories.
This study assessed possible adverse immunological effects of trans-1,2-dichloroethylene (DCE) on ran-
dom-bred CD-i mice following 14 and 90 days of exposure. A 14-thy range-finding study was performed
on male mice by gavage at doses 1/10 and 1/100 the LD5O (210 and 21 mg/kg). No alterations in either
humoral or cell-mediated immunity were observed following this exposure. A 90-day study was con-
ducted in which DCE was administered in the drinking water of male and female mice. The levels of DCE
in the drinking water were calculated to deliver levels equivalent to, and higher than, those delivered for
14 days (17, 175, and 387 mg/kg for males and 23,224, and 452 mg/kg for females). No changes were ob-
served in the cell-mediated immune status of either sex or in the humoral immune status of females.
However, a marked suppression in humoral immune status was observed in male mice exposed to all three
levels of DCE, as indicated by a decreased ability of spleen cells to produce antibody agninst sheep
erythrocytes (sRBC). Macrophage function was depressed only in females, as indicated by the decreased
ability of thioglycollate-recruited peritoneal exudate cells (PEC) to phagocytize sRBC.
CITATION: Drag and Chemical Toxicology 8(5): 393.407, October 1985.
C104 XX-86-104 Project Officer: Condie
TOXICOLOGY of TRANS -1,2-DICHLOROETHYLENE in the MOUSE
Donald W. Barnes, 1 Virginia M. Sanders, KimberL White, Jr.,
George M. Shopp, Jr., andAThe E. Munson
‘East Carolina University
Medical College of Virginia
This paper reports toxicity findings of a potential thinking water organic chemical contaminant. Tmns-1,2-
dichioroethylene (DCE) war administered to male and female CD-i mice in order to evaluate its effects on
standard toxicological parameters. Following an acute lethal dose for 50% of animals (LDSO) detennina-
tion arid a 14-day range-finding study, a 90-day drinking water study was performed using leveLs of DCE
calculated to deliver approximately 1/104 1/10, and 1/5 the LD5O. The most noteworthy changes occwred
in the males exposed to the highest level of DCE, where there was a signflcant decrease in glutathione levels,
and in the frmales exposed to all three DCE levels, where there was a significant decrease in aniline
hydnrtylase activity. These data will assist the Office of Drinking Water in setting Drinking Water Standards
and Health Advisories.
Trans-1,2-dichloroethyelene (DCE) was administered to male and female CD-i mice in order to evaluate
its effects on standard toxicological parameters. Following an acute LD5O determination (2122 mg/kg in
males and 2391 mg/kg in females) and a 14-day range-finding study, a 90-day drinking water study was
performed using levels of DCE calculated to deliver approximately 1/100, 1/10, and 1/5 the LD5O.
Various toxicological assessments were made, including body and organ weights, hematology, serum
chemistries, and hepatic microsomal activities. Few alterations were observed in either sex following 90
days of exposure. The most noteworthy changes occurred in the males exposed to the highest level of
DCE, where there was a significant decrease in glutathione levels, and in the females exposed to all three
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DCE levels, where there was a aignificant decrease in aniline hydroxylase activity. These data served as
background for the immunotoxicological evaluation presented in the following manuscript.
CITATION: Diug and Chemical Taucology, 8(5): 373-392, October 1985.
C104 XX-86-103 Project Officer: Condie
HUMORAL and CEWMEDIATED IMMUNE STATUS of MICE
EXPOSED to 1,1,2-TRICHLOROETIIANE
Virginia M. Sanders, KknberL. Thite, Jr.,
George it Shopp Jr., andAlbeit E Munson
Medical College of Virginia
This paper reports to tici(y findings of a potential drinking water organic chemical contaminant The pur-
pose of this study was to assess the immunological effects of 1,1,2-tiichloroethane on random-bred CD-I
mice following 14 and 90 days of oral exposure. The 14-day immunological range-finding study in males
exposed to doses 1/10 and 1/1000 the acute lethal dose for 50% of animaLs revealed no alterations in either
humoral or cell-mediated immune status. Following 90 daw of ctposure in the drinking water a more
detailed series of immunological parameters was assesseS Cell-mediated immunity was unaltered in both
sexes, while hwnoral immune status was depressed in both sexes, particularly when dete,vnined by henzag-
glutinalion titers. This paper will assist the Office of Drinldng Water (ODW) in setting Drinking Water
Standards and Health Advisories.
The purpose of this study was to assess the immunological effects of 1,1, 2-trichloroethane (‘FCE) on ran-
dom-bred CD-i mice following 14 and 90 days of oral exposure. A toxicological evaluation conducted at
the same time revealed the target organs to be the liver of both sexes and the erythroid elements of the
females. The 14-thy immunological range-finding study in males exposed to doses 1110 and 1/100 the
LD5O (38 and 3.8 mg/kg) revealed no alterations in either humoral or cell-mediated immune status. Fol-
lowing 90 days of exposure in the drinking water (4.4,46, and 305 mg/kg for males and 3.9 44, and 384
mg/kg for females), a more detailed series of immunological parameters was asseswL Cell-mediated im-
munity was unaltered in both sexes, while humoral immune status was depressed in both sexes, particular-
ly when determined by hemi gglutination titers. Macrophage function was depressed only in the males as
indicated by the ability of thioglycolate-recruited peritoneal exudate cells (PEC) to phagocytize sheep
erythroc tes (sRBC).
CITATION: Drug and Chemical Tancology 8(5): 357-372, October 1985.
C104 XX-86-102 Project Officer: Condie
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RESEARCH in PROGRESS: THE EFFECTS of CHLORINATED DRINKING
WATER on HUMAN UPID METABOLISM
Robert Wones and Charles Glueck
University of Cincinnati College of Medicine
The disinfection of drinldng water is one of the most widely practiced and most successful public health
programs in the U.S. While it has dramatically reduced the incidence of waterbome illness, it has been
determined that the organic chemical contwninants occumng at the highest concentration in drinking water
remit from it. This symposium paper summarizes the results to date of the Agency’s research into the
health effect of these contaminants.
Studies in nm i1c suggest that chlorination of drinking water may increase total serum cholesterol.
Human data on this question are limited, particularly for the lipoproteins and apolipoproteins, and are
insufficient to formulate guidelines or regulations. Therefore, the U.S. Environmental Protection Agen-
cy and the General Clinical Research Center (GCRC) of the University of Cincinnati have initiated a
cooperative agreement to study the hnm in lipid, lipoprotein, and apolipoprotein effects of drinking water
chlorination. The int 1 research protocol will study 20 healthy men and women. On admission to the
GCRC, each will have a complete history and physical e,amination, chest x-ray, EKG, determination of
their serum lipids, lipoproteins, and apolipoproteins, and a battery of other common metabolic tests in-
cluding thyroid function tests. For the duration of the study, the subjects will eat all their meals in the
GCRC. Their diet will be isocaloric with 20%, 40%, and 40% of the calories coming from protein, fat ,
and carbohydrate, respectively. The diet will contain 600mg of cholesterol daily with a polyunsaturated-
to-saturated-fat ratio of 0.4. The diets will also be mildly deficient in calcium. The subjects will receive
all their drinking water and other liquids from the GCRC. For the first 3 weeks of the study, each person
will drink only nonchlorinated water and other nonchlorinated liquids. Thereafter, there will be 3 con-
secutive 4-week periods during which the subjects will receive drinking water with 2ppm,5 ppm, and 10
ppm chlorine, respectively. These chlorine concentrations have shown biological effects in anim lc and
are not extraordinary for many treated drinking waters.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Dnnlcing Wales
DLrinfectwzts and Disinfection By-F vducts, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Pe cpectives 69: 255-258, November 1986.
C104 XX-86- 120 Project Officer: Condie
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TOXICOLOGICAL TESTING of ORGANIC SUBSTANCES from
CONCENTRATED DRINKING AND WASTE WATERS
Linda K Snow and Bruce C Casto
Environmental Health Research and Testing Inc
The results of thLc research has specificali v focused on the task of detennining the potential health hazards
of complex mixiwes of organic compounds found in drinking and waste waters. Samples were collected
fivm each stream that had been disinfected by use of either ozone chlorine diaude mono-chloramine or
chlo,ine. These swnples were subsequent’y examined in a balterg of five short-tenn toxicological assays. In
addition to the EPA Compliance Policies to protect public health and the regulatory aspects of this re-
search, the toxicological testing results described in this report will provide EPA with infonnation needed to
develop useful short-term in viwi and in vitro test systems that are capable of detecting adverse toxicological
effects caused by environmental pollut ants.
The U.S. Environmental Protection Agency has been given the responsibility for regulating the release of
toxic chemicals into the environment. Accordingly, the EPA presently conducts an extensive and com-
prehensive research program to determine the adverse effects of environmental factors on hnn’jin health.
A considerable amount of this research activity is directed toward toxicological testing and test develop-
ment. The Health Effects Research Laboratory of the EPA, in Cincinnati, Ohio, has specifically focused
on the task of determining through the use of appropriate short-term assays, the potential health hawds
of the complex mixtures of organic compounds found in drinking and waste waters. A battery testing ap-
proach is being employed by the EPA at Cincinnati for the toxicological testing of these waters, progress-
ing from routine detection systems in microbial cells to more complex systems in mammalian cells and
subsequently to short-term animal testing. In support of the EPA’s inhouse research and testing a study
was conducted employing a battery of five short-term assays for the toxicological testing of coded con-
centrated drinking and waste water samples. The five assays performed were: (1) the in vivo toxicity test,
(2) Salmonella typhimunum mutagenesis assay, (3) Sencar mouse skin bioassay, (4) rat liver focus assay,
and (5) lung adenoma assay. The Final Report for this project gives the results of the testing of a total of
40 coded water samples in one or more of the above assays. This project summary was submitted in ful-
flilnient of Contract No. 68-03-1840 by Environmental Health Research and Testing Inc. under the spon-
sorship of the U.S. Environmental Protection Agency. This snnlnlrny covers a period from February 1984
to September 30, 1985, and work was completed as of September 30, 1985.
CITATION: U.S. Environmental Protection Agency, contract no. 68-03-1840, EPA-600/1-86-005, July
1986.
BlOl HERL-0549 Project Officer: R. Miller
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DISTRIBUTION and MACROMOLECULAR BINDING of BENZO [ a] PYRENE
in SENCAR and BALB/c MICE FOLLOWING TOPICAL and ORAL
ADMINISTRATION
MaricA. Morse and Gwy P. Cadson
Purdue University
The mouse sldn bioassay system Ls being used extensively to assess the toxicity of chemicals Lsolated from
drinldng water. This test model is to be used in collecting data to establish allowable limits of con-
taminants in drinking water/or adherence to the mandate of the Safe Drinking WaterAct calling for com-
prehensive study ofpublic water supplies.
When benzo [ a]pyrene (BaP) is used as the initiator in initiation-promotion assays, the topical route of ad-
minictration has been shown to produce a greater epidermal tumor incidence than do other routes of ad-
ministration, particularly the oral route. In addition, different strains of mice exhibit varying degrees of
susceptibility to two-stage epidermal tumorigenesis using BaP. The SENCAR strain is known to be far
more sensitive to epidermal tumor formation following BaP initiation than are other strains, such as the
BALB/c strain. To investigate the possible contribution of distribution and binding to DNA in such route
and strain differences, the distribution and macromolecular binding of ( 3 H]BaP was e mined in the skin,
liver, lung, and stomach of SENCAR and BALBIc mice following topical or oral administration of BaP
at time periods r1m ng from 03 to 48 h. Levels of labeled material in skin were higher, and the binding
of BaP to epidermal DNA was greater following topical administration than following oral administration
for mice of both strains. The much greater binding of BaP to epidermal DNA following topical ad-
minii tration may account for the much greater epidermal tumor incidence in mice following topical ad-
ministration of BaP. Following topical administration, BALB/c mice had generally higher levels of
labeled material in whole skin than did SENCAR mice, and the binding of BaP to epidernial DNA at 48
h was greater in BALB/c mice than in SENCAR mice following either route of administration. Thus, the
known differences between these two strains in susceptibility to epidermal tumor formation when BaP is
used as an initiator cannot be explained on the basis of differences in tissue distribution or the amount of
binding to epidermal DNA at the time periods ei zimined.
CITATION: Journal of Toxicology and Environmental Health 16:263-276, November 1985.
C104 XX-86-058 Project Officer: Robinson
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NON-NEOPL4STIC LESIONS: USE of DATA FROM PRE. or
NON-NEOPLASTIC LESIONS
THAT MAY INDICATE POTENTIAL for CARCINOGENESIS
JudyA. Stober
HERL, USEPA, Cincinnati, OH
Development of statistical method, for carcinogenesic tiered testing screen will aid in the assessment of the
feasibili(y of combining dose response infonnalion and multiple in vivo shost teim assays to estimate rela-
tive carcinogenic potency. These methods will aid the Office of Drinking Water in evahwting the effects of
vanous disinfectants and single chemicals used for drinking water treatsnen4 paflicularly for those cases
where a long tenn whole animal swdy is neither logistically possthle nor economically feasible. The test
matrix methods also will aid in the evaluation of complex mixtures where chemical species are unknown.
The Toxicology and Microbiology Division of the Health Effects Research Laboratory has initiated a re-
search program to develop a matrix of short-term tests to distinguish carcinogens from non-carcinogens
among genotoxic substances and to develop methods for predicting relative carcinogenic potency based
on the results of short-term in vivo bioassays. This paper includes a basic description of the project and
outlines the proposed statistical methods for evaluating the Carcinogenesis Testing Matrix (CrM). Suc-
cessful development, testing and implementation of the test matrix system is very dependent on statistical
design, analysis and prediction procedures. Since this is an extension of the usual application of a single
screen to predict a qualitative effect, considerable statistical development and testing is needed. Three
short-term in vivo bioassays have been proposed for the CTM, the sencar mouse skin bioassay, the mouse
lung adenoma bioassay and the liver foci bioassay. Appropriate statistical methods in terms of statistical
power and robustness for analyzing the data from each assay are being developed and evaluated. Methods
for estimi ting and comparing potencies from the long-term studies and short-term test systems are being
developed and evaluated. The proposed methods include procedures for comparing potencies, estima-
tion of confidence limits and assessment of statistical significance. The methods will be evaluated in terms
of statistical considerations, adaptability to laboratory test situations, ease of application, efficiency and
predictive power.
CITATION: In: Proceeding, of the Electric PowerResearch Institute Workshop on Investigation of NewAp-
proaches to Use of Data in Cancer PjskAssessmeszt, 1986.
C104 MS-86-145 Project Officer: Stober
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MOUSE LIVER TUMOR DATA:
ASSESSMENT of CARCINOGENIC ACTIVITY
MichaelA. Pereira
HERL USEPA, cincinnati, OH
Significant nwnbei s of chemicals have been shown to be carcinogenic in mouse liver althoum they do not
exhibit carcinogenic activity in other organs or tissues of mice or rats. ThLs review focuses on the reasons for
the unique susceptibility of the mouse liverto these carcinogens and the dent to which the carcinogenic ac-
tivhy of a chemical in mouse liver can be used to predict carcinogenicity in humans.
Significant numbers of chemicals have been shown to be carcinogenic in mouse liver although they do not
exhibit carcinogenic activity in other organs or tissues of mice or rats. This review focuses on the reasons
for the unique susceptibility of the mouse liver to these carcinogens and the extent to which the car-
cinogenic activity of a chemical in mouse liver can be used to predict carcinogenicity in hum nc . Many of
these mouse liver carcinogens lack genotoxic activity and, as such, have been proposed to be tumor
promoters. Two mecluinicmc that may explain the action of nongenotoxic carcinogens in mouse liver are
reviewed. These are: (1) direct action on precursor cancer cells, either to accelerate their growth or to
prevent their death and (2) the selective growth advantage, resulting from regenerative hyperplasia of
precursor cancer cells in response to the necrosis of normal cells produced by hepatotoxins. Estimating
hiiins n health risks on the basis of mouse liver tumor data is believed to differ for nongenotoxic and
genotoxic carcinogens in two fundamental ways. The first involves intraspecies extrapolation and the
second involves low-dose extrapolation. In conclusion, although mouse liver tumor data are seen to be of
value in estimating hunu n health hazard, it is important to distinguish between genotoxic and non-
genotoxic mech nicm in applying such data. Further study of the biochemical and molecular
meduiniunc of chemical carcinogens is necessary to determine the relationship between their activity in
mouse liver and their activity in bnm ins .
CITATION: Taucolorj and Industrial Health 1(4): 311-333, 1985.
C104 MS-85-040 Project Officer: Pereira
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ENHANCEMENT of the HEPATOTOXICITY of CHLOROFORM in
FEMALE B6C3FI MICE by CORN 01L
IMPLICATIONS for CHLOROFORM CARCINOCENESIS
RichwdL Bul4 1 lanice E. Brown, 2 EarieA. Meietheni y, 2
TedA. Jorgenson, 2 Metre! Robinson, 3 and Judith A. Stobe?
1 Washington State University
2 SRI International
3 HERL, USEPA, Cincinnati, OH
The disinfection of thinking water is one of the most vAd4v practiced and most successfid public health
programs in the U.S. JThile it has dramatica1 p reduced thE incidence of waterbome illness, it has been
detennined that the organic chemical contaminants occuning at the highest concentration in drinking water
result from it. This symposium paper swnmwires the results to date of the Agency’s research into the
health effect of these contaminants.
A recent study of the ability of chloroform in drinking water to produce cancer reported that male Os-
borne-Mendel rats developed renal tumors, but that female B6C3F1 mice failed to develop hepatocel-
lular carcinomas (Jorgenson et 41985). The results obtained in the male, Osborne-Mendel rats were
comparable to those observed in an earlier study sponsored by the National Cancer Institute (Na, 1976).
On the other hand, the lack of an increased incidence of hepatocellular carcinomas in the female B6C3F1
mice was in sharp contrast to previously reported results (NC!, 1976). The doses of chloroform used were
comparable to those which produced an 85 percent incidence in the Na study. When administered in
corn oil, chloroform significantly decreased serum TO levels but did not effect this parameter when ad-
minktcred in 2 percent Emulphor. Chloroform decreased body weight and increased liver weight when
administered with either vehicles, but the effects were significantly greater when it was administered in
corn oil. Mice administered chloroform in corn oil displayed a significant degree of diffuse parenchymal
degeneration (S of 10 males and 1 of 10 females) and mild to moderate cirrhosis (5 of 10 males and 9 of
10 females); pathological lesions were not observed in the ainnls administered corn oil without
chloroform nor in mice receiving chloroform in 2 percent Emuiphor. These data indicate that ad-
min i stration of chloroform by corn oil gavage results in more marked hepatoxic effects than those ob-
served when it is provided in an aqueous suspension. A major difference between the two recent car-
cinogenesis bioassays of chloroform in this same mouse strain was in the vehicles used: corn oil, in the
study that yielded an increased incidence of hepatocellular carcinoma (NC!, 1976), and drinking water,
in the negative bioassay reported by Jorgenson et al. (1985).
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Drinking Water
Disinfectants and Disinfection By-Froths cts , Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 6% 49-58 November 1986.
C104 MS-86-176 Project Officer: Robinson
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MUTAGENIC BY-PRODUCrS FROM CHLORINATION of HUMIC ACID
John A Meier, H. Paul Ringhan4 W. Eniile Coleman, Kathy M. Schenclç
Jewi W Munch, Robe,rP. Streicher, Miliam H. Kaylor
and Fredenck C. Kopfler
HERL, USEPA, Cincinnati, OH
The disinfection of thinking water is one of the most widely practiced and most successful public health
programs in the U.S. Thile it has dramatically reduced the incidence of waterbome illness, it has been
determined that the organic chemical contaminants occurnng at the highest concentration in drinking water
remit from it. This s)mposiwn paper summarizes the results to date of the Agency’s research into the
health effect of these contwnjnangs.
Chlorination of humic and fulvic acid results in the formation of direct-acting mutagenicity, detectable in
the Salmonella/microsome assay (Ames test). This mutagenicity is being characterized as part of an over-
all effort aimed at evaluating potential health risks associated with the presence of mutagenic chemicals
in drinking water. A number of chlorinated organic compounds have been identified and quantified in
diethyl ether extracts of the chlorinated humic acid solutions, including several known mutagens.
However, the total mutagenicity of these compounds accounts for only about 7 percent of the original
mutagenidty. Synergistic or antagonistic interactions among the identified components has been ruled
out as a possible expbin ition for the failure to account for a higher percentage of the activity. Recent
progress has been made to separate the activity into neutral and strong acid fractions. Further isolation
of the strong acids by high pressure liquid chromatography (HPLC) has resulted in the purification of the
mutagenicity into a major peak of activity with a specific mutagenicity of about 20,000 TA100 revertants
per mg. Several trichiorobydroxyfuranone isomers have been tentatively identified in this fraction. The
contribution of these types of compounds to the mutagenicity of chlorinated humic acid is under inves-
tigation.
CITATION In: Proceedings of the Second International Symposiwn on the Health Effects of Drinking
Water Disinfectanas and Disinfection By-Products, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69 101-107, November 1986.
C104 MS-86-174 Project Officer Meler
GROSS and MICROSCOPIC PATHOLOGY in the SENCAR MOUSE SKIN and
LUNG-INITIATION and PROMOTION STUDIES
Gasy L. Kiwtsen, 1 Robert M Kovatch, 1 and Merrel Robinson 2
1 Pathology Associates, Inc.
2 HE USEPA, Cincinnati, OH
The insults of the research described fri the attached workshop proceedings aiticle fiuiher characterizes the
SENCAR mouse and its advantages and dicadvasages as an animal testing model in toxicological and car-
cinogenesis experimentation. The data deals with three categories relevant to the response of the mouse in
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laborato’y tea: (1) sesponse to carcinogens, (2) altern ate testing (other than skin tumor initiation , promo-
don assay), and (3) histopathology including treatment-induced and spontaneous lesions.
During the past three years, the skin and lung tissues from SENCAR mice utilized as part of the Environ-
mental Protection Agency’s Carcinogenesis Testing Matrix were examined. This included SENCAR
mice used in three different short-tenn bioassay protocols in which the Skin Papilloma Assay was used to
identify initiators, promotors and complete carcinogens. Also included were the pathology findings from
SENCAR mice used in the combined bioassay in which both the skin assay and the Lung Adenoma Assay
were conducted simultaneously. The gross and microscopic features of treatment-associated and spon-
taneous lesions of the skin and lung of the SENCAR mouse used in these studies are defined and the
lesions most commonly observed are described. There was generally a poor correlation between gross ob-
servations and microscopic findings in both the skin and lung tissue& Although there are several definite
criteria on which gross interpretations of the various skin and lung lesions can be made, it was found that
with the exception of pedunculated squamous cell papillomas and the classic squamous cell carcinomas,
the various lesion types had a wide variety of clinical presentations that severely compromised the ac-
curacy of gross diagnosis. Further, in the case of benign skin neoplactn c, it was found that malignant trans-
formation of these tumors most often occurred at the base of the lesion and was initially hidden from gross
observation. As a result, approximately 50% of the neoplanns interpreted clinically as benign tumors
(papillomas and keratoacanthomas) were actually malignant neoplasms. Moreover, many lesions deter-
mined to be non-tumorous grossly were in fact neoplastic microscopically. The SENCAR mouse was
found to be more responsive in the Lung Adenonia Assay than other strains examined with exception of
the Strain A. Although accurate interpretation of the lung lesions in the SENCAR was compromised by
non-neoplastic treatment-associated and/or spontaneous lesions, the feasibility of acing the SENCAR
skin and lung as target tissues in two stage combined carcinogenesis studies merits further consideration.
CITATION: Environmental Health Perspectives 68: 91-104, September 1986.
C104 MS-86-116 Project Officer: Robinson
METABOLIC ACTIVATION of BEN ZO(a)PYRENE in SENCAR and
BALB/c MOUSE EMBRYO CELL CULTURES
Williwn M. Baird, Said Al. Sebt4 and IAMA. Reinsvold
Purdue University School of Pharmacy
The results of the research described in the attached workshop proceedings ankle jluther characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in toxicological and
carcinogenesis experimentation. The data deaLs with three categories relevant to the response of the mouse
in laboratory tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initia-
tion (promotion assay), and (3) histopathology including treatment-induced and spontaneous lesions.
The metabolism and DNA-binding of benzo(a)pyrene (B(a)P) were compared in early passage mouse
embryo cell cultures prepared from SENCAR mice, a strain especially susceptible to two-stage
tumorigenesis, and BALBIc mice, a strain relatively resistant to two-stage tumorigenesis. Cultures from
both strains metabolized similar amounts of B(a)P, however the proportion of water-soluble metabolites
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formed was higher in the BALD/c cultures than in the SENCAR cultures. The major metabolites formed
in cultures from both strains were B(a)P-9,1O-diol, B(a)P-7,8-diol and the glucuronic acid conjugate of 3-
hydroxy-B(a)P. The level of binding of B(a)P to DNA was greater in the SENCAR mouse embryo cell
cultures than in the BALB/c cultures after 5,24 and 48 hours of exposure. The major B(a)P-DNA ad-
duct formed in B(a)P-treated cultures from both strains was the adduct formed by reactin of (+ )anti-
B(a)P-7,8-diol-9,1O-epoxide (anti-B(a)PDE; the isomer with the epoxide and the benzylic hydroxyl on
opposite faces of the molecule) with the exocydlic amino group of deoxyguanosine. Immobilized
boronate chromatography followed by high-performance liquid chromatography demonstrated the
presence of small amounts of syn-B(a)PDE (the isomer with the epoxide and benzylic hydroxyl on the
same face of the molecule)-DNA adducts: the proportions of these were similar in cultures from both
strains. The results suggest that SENCAR mouse embryo cell cultures may convert less B(a)P to water-
soluble metabolites and more to DNA-binding metabolites than BALB/c mouse embryo cells. If similar
differences exist in B(a)P metabolism in the skin of these strains of mice, it could help to explain the rela-
tively high sensitivity of the SENCAR mice to tumor induction by the two-stage protocol.
CITATION: Enviromental Health Perspectives 68:45-52, September 1986.
C104 XX-86-083 Project Officer: Robinson
NATURALLY OCCURRING NON-NEOPLASTIC PAThOLOGY in the
FEMALE SENCAR MOUSE
Robes M. Kovatck 1 Ga yL Knutsen, 1 and Me,re l Robinson 2
1 Pathology Associates, Inc.
USEPA, Cincinnati, OH
The results of the research described in the attached workshop proceedings a ,!icle fwther characteiizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in toxicological and
carcinogen esis experimelualiolL The data deaLs with three categones relevant to the response of the mouse
in laboratory tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initia-
tion/promotion assay), and (3) histopathology including treatment-indaced and spontaneous lesions.
Lesions, that were considered naturally occurrin& were surveyed in female SENCAR mice utilized in
short term bioassays. These lesions were encountered in protocol designated target organs, and organs
and tissues with gross lesions. The genitourinary system was the most frequent site for lesions where cys-
tic ovaries, cystic endometrial hyperplasia and glomerulonephritis were commonly encountered.
CITATION: Environmental Health Perspectives 68:105-116, September 1986.
C104 MS-86-117 Project Officer: Robinson
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BINDING and DISTRIBUTION STUDIES in the SENCAR MOUSE of
COMPOUNDS DEMONSTRATING a ROUTE DEPENDENT EFFECT
Gary P. Can!son, AnthonyA Fossa, Mai*A. Morsç, and
Pat,ice Al. Weaver
Purdue University School of Pharmacy
The results of the research descsibed in the attached workshop proceedings article fiuther characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model vi toxicological and
carcinogen esis experimentation. The data deals with three categories relevant to the response of the mouse
in laboratory tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initia-
tion/promotion assay), and (3) hLctopathology including treatment-induced and spontaneous lesions.
Previous investigators have determined that benzo(a)pyrene was much more effective in canithig Skin
papillonias if applied topically than when administered orally in the initiation-promotion assay in Sencar
mouse whereas urethane and acrylamide caused a higher percentage of mice with papillomas and more
tumors per mouse when given orall ,. In an attempt to understand the reason for this discrepancy in route
dependency, 3 H-benzo(a)pyrene, 4 C-urethane and 14 C-acrylamide were administered as single doses
orally or topically to male SENCAR mice. Distribution in skin, stomach, liver and lung was determined
for time periods up to 48 hours. The binding of these compounds to DNA, RNA and protein in these tis-
sues was determined 6 and 48 hours after administration. For all three compounds, high concentrations
were found in the skin following topical application, but very little material reached this target organ fol-
lowing oral administration. In contrast, the internal organs generally contained more material after oral
administration. The binding of labelled compound to DNA, RNA and protein generally reflected the dis-
tribution data with more compound being bound in the stomach, liver and lung after oral administration
compared to topical application and the opposite being true for the skin. This was particularly evident for
the benzo(a)pyrene. The results suggest that differences in distribution to the skin and binding to mac-
romolecules following oral or topical administration cannot explain the greater tumorigenicity of
urethane and acrylamide after oral administration in the Sencar mouse.
CITATION: Environmental Health Perspectives 68: 53-60, September 1986.
C104 XX-86-084 Project Officer: Robinson
PHORBOL MYRI STATE ACETATE and CATECHOL as SKIN
COCARCINOGENS in SENCAR MICE
Benjamin L Van Duuren, Susan Melchionne and I,vin Seidman
New York University Medical Center
The results of the research described hi the attached workshop proceedings aiticle further characterizes the
SENCAR mouse and its advantages and disadvantages as an anunal testing model in toxicological and
carcinogenesis experimentation. The data deaLs with three categories relevant to the response of the mouse
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in laboratory tests: (1) response to carcinogens, (2) alternate testing (other than skin twnor initia-
don/promotion assay), and (3) histopathology including treaiment-inchtced and spontaneous lesions.
The enhs n cement of the carcinogenicity of benzo(a)pyrene (BP) and of beta-propiolactone (BPL) by the
mouse skin cocarcinogens phorbol myristate acetate (PMA) and catechol were e mined in female SEN-
CAR mice, thirty per group. The carcinogen and cocarcinogen were applied simultaneously, three times
weekly for 490-560 days. BP and BPL were used at constant doses of 5 and 50 g, respectively in ali ex-
periments. PMA was used at three doses, 2.5, 1.0 and 0.5 p.g, per application and catechol was used at
one dose, 2mg per application. Control groups included animals which received carcinogen only, cocar-
cinogen only, acetone only and no treatment groups. The carcinogenicity of BP and BPL were enhanced
by the cocarcinogens, particularly in terms of tumor multiplicity. For both cardnogens the most marked
cocarcinogenic effects were observed at the lowest dose of PMA used, i.e. 0.5 .g per application; this ap-
plied for days to first tumor, nhnidc with tumors, tumor multiplicity and incidence of malignant skin
tumors. Catechol applied alone did not induce any tumors; with PMA alone there were significant in-
cidences of benign and malignant tumors, e.g., at a dose of only 0.5 p.g per application 15 of 30 animals
had 28 tumors, 5 of which were squamous carcinomas. In two-stage carcinogenesis experiments with
7,12-dimethylbenz(a)anthracene as initiator and PMA as promoter SENCAR mice showed a greater sus-
ceptibility to tumor induction when compared to ICR/Ha mice used in earlier work. This was most
notable for rate of tumor appearance and tumor multiplicity.
CITATION: Environmental Health Perspectives 68:33-38, September 1986.
C104 XX-86-085 Project Officer: Pereira
SPONTANEOUS TUMORS in SENCAR MICE
Susan Melchionne living Seidman and Benjamin L. Van Duu,en
New York University Medical Center
The results of the research described in the attached workshop proceedings aMicle further charactenzes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in toxicological and
carcinogenesis experimentation. The data deals with three categories relevant to the response of the mouse
in Iaborwoiy tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initia-
tion/promotion assay), and (3) histopathology including treatment-induced and spontaneous lesions.
A total of 305 female and 36 male SENCAR mice in six different groups were observed for 540 to 875 days
for tumors as well as non-neoplastic lesions. Three of these groups were observed for their lifespans.
One hundred and twenty three females and 36 males, observed for their lifespans, 800 to 875 days, showed
median survival times ranging from 730 to 745 days. The average peak body weights were 42.4 grams for
females and 50.0 grams for males. In the animals observed for their lifespans, the total tumor incidence
was 65.6% for females and 69.4% for males. Both sexes showed a high incidence of papillary tumors of
the lung, 16-39%, which was independent of aging Females showed a 5.1 to 21.0% incidence of mam-
mary tumors which increased with aging. Males showed a high incidence of hyperplastic nodules of the
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liver, it., 12 of 36 animals• Other frequently occurring tumors were leukemia and papillomas of the fores-
tomach.
CITATION: Environmentoi Health Perspectives 68: 135-140, September 1986.
C104 XX-86-086 Project Officer: Pereira
HALOACETONITRILES: METABOLISM, GENOTOXICITY, and
TUMOR-INITIATING ACTIVITY
Edith LX. Lki, F. Bernard Danie4 Sydna L &nvn-Freun4 and
MichaelA. Pereira
HEW.., USEPA, Cincinnati, OH
The disinfection of drinking water it one of the most wideay practiced and most successfisl public health
programs in the U.S. W hile it has dramatical(y reduced the incidence of waterbome ilne&ç it has been
detennined that the oganic them ical contwninanLc occuning at the hiveu concentration in dzinldng water
result from it. This symposium paper summazires the results to date of the Agencyn research into the
health effect of these contaminants.
Haloacetonitriles (HAN) are drinking water contnn 4 nnnts produced during chlorine disinfection. We
evaluated the metabolism, genotmicity, and tumor-initiating activities of these chemicals. The HAN,
when reacted with the electrophilic trapping agents 4-(p-nitrobenzyl)pyridine, exhibited the alkylating
potential of dibromoacetonitrile (DEAN) > broniochioroacetonitrile (ECAN) > chloroacetonitrile
(MCAN) > dich loroacetonitrile (DCAN) > trich loroacetonitrile (TCAN). When administered orally to
rats, the HAN were metabolized to cyanide and exreted in the urine as thiocyanate. The extent of
thiocyanate excretion was 14.2% of the dose for MCAN, 7.7% to 12.8% for the three dihaloacetonitriles
and 2.25% for TCAN. Haloacetonitriles inhibited in vitro microsomal dimethylnitrosarnine demethylase
(DMN-DM) activ4 The most potent inhibitors were DCAN and ECAN, with IC =3-4 x 1O M; the
next potent inhibitors were DCAN and TCAN, with Kj = 2 x io 4 M; and the least potent inhibitor was
MCAN, with K = 9 x 1& M. When administered orally, TCAN, but not DEAN, inhibited hepatic DMN-
DM actMty The haloacetonitriles produced DNA strand breaks in cultured human lymphoblastic
(CCRF- CEM) cells, an in vitro assay for genotoxicity. TCAN was the most potent DNA strand breaker,
producing twice as many breaks as the genotoxic methylating agents methylmethanesulfonate and methyl-
nitrosourea. The other HAN were much less potent strand breakers, and their activity was BCAN>
DEAN> DCAN > MCAN, which was only margjnally active. DCAN reacted with polyadenylic acid
and DNA to form adducts in a cell-free system, however, the oral administration of DEAN or DCAN to
rats did not result in detectable adduct formation in the liver DNA. None of the HAN initiated gamma-
glutamyl-transpeptidase positive foci in the rat liver foci bioassay. In s mmny the HAN were
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demonstrated to be genotoxic in vitro, but appeared after oral administration to lack genotoxic and tumor-
initiating activity in rat liver.
CITATION: 1n Proceedings of the Second International Symposium on Health Effects of Drinldng Water
Disinfectants and DLrinfection By-F oducLc, Cincinnati, Ohio, August 27-29, 1986.
Published in En vtronmental Health Perspectives 69:67-71, November 1986.
C104 MS-86-181 Project Officer: Lin
CARCINOGENICITY of BY-PRODUCTS of DISINFECTION in
MOUSE and RAT LiVER
Sydna L Heiren-Freund and Michael A. Pereira
HERL, USEPA, Cincinnati, OH
The disinfection of diinldng water is one of the most widely practiced and most successful public health
progwns hi the U.S. While it has dramatically reduced the incidence of waterbome illness, it has been deter-
mined that the organic chemical contaminants occurnng at the highest concentration in drinking water
result from iL This symposium paper summwizes the results to date of the Agency’s research into the
health effect of these contaminants.
Byproducts of disinfection were tested in a rat liver initiation promotion bioassay that employs an in-
creased incidence of g imm i -ghdamy1trans -peptidase -posidve foci (GGT-foci) as an indicator of car-
cinogenicity. The protocol for this assay consists of a two-thirds partial hepatectomy followed by 18 to 24
hours later by the administration of the initiator. Seven days after initiation, the rats start to receive the
promoter in their drinking water. The promoter treatment is continued for at least 10 weeks. The rat
liver foci bioassay is being validated for inclusion in a battery of tests that would be used to demonstrate
the carcinogenic activity of environmental samples of complex mixtures such as drinking water. During
this validation it has been determined that the assay can detect the initiating activity of both hepatic and
nonhepatic carcinogens such as 2-acetylziminofluorene, aflatoxin Bi, diethylnitrosamine, dimetliyl-
hydrazine, and urethane. Therefore, we used this assay to determine the tumor initiating activity of
byproducts of disinfection including chloramine, halogenated humic acids, halogenated ethanes,
halogenated acetonitriles, halogenated methanes, halogenated ethylenes, and N-Cl-piperidine. These
compounds did not include GGT-foci, which would indicate that they do not possess significant ability to
initiate carcinogenesis. Chemicals that have been tested in this assay for their ability to promote the oc-
currence of GGT-foci and tumors initiated by diethylnitrosamine (DENA) include halogenated benzenes
and chloroform. Chloroform (1800 ppm in the drinking water) administered to nimi 1s previously in-
itiated with DENA 18 hours after a partial hepatectomy either did not promote or inhibit the occurrence
of GGT-foci. However, when the chloroform was given in the drinking water concommitantly with week-
ly doses of DENA, it did enhmce the formation of liver tumors. Of 20 halogenated benzenes tested, only
1,2,4,5-tetrachlorobenzene and hexachlorobenzene promoted DENA-initiated GGT-foci.
Hexachlorobenzene promoted GGT-foci in both male and female rats, whereas 1,2,4,5-tetrachioro-ben-
zene only promoted GGT-foci in male rats. Thus in rat liver, the tested byproducts of drinking water dis-
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infection did not demonstrate tumor-initiating activit although some byproducts appear to modulate
tumor promotion.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of D*ildng Water
Disinfectants and Disinfection By-Floductc, Cincinnati, Ohio, August 21-29, 1986. Published
in Environmental Health Perspectives 69: 59-65, November 1986.
C104 MS-86-180 Project Officer: Herren-Freund
ElF ECTS OF CHRONIC TOPICAL APPLICATION of
12-O-TETRADECANOYLPHORBOL-13-ACETATEon the SKIN and
INTERNAL ORGANS of SENCAR MICE
Andres 1. P. Klein-Szwzt4 1 Claudio J. Conti Cka4dio M. Akl&
Neal Clapp, 2 Stephen Nesnow, 3 and Thomas J. Slaga 1
1 Univeristy of Texas System Cancer Center
2 Oak Ridge National Laboratory
3 HERL, USEPA, Research Triangle Park, NC
The results of the research desaibed in the attached workshop proceedings wticle fiather charactenzes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in to4cological and
carcinogenesis experimentation. The data deaLs with three categories relevant to the response of the mouse
in laboratory tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initia-
tion/promotion assay), and (3) histopathology including treaUnent-indaced and spontaneous lesions.
Repetitive topical applications of 2 .g TPA twice weekly for 37 to 52 weeks induced a sustained epider-
mal hyperplasia, hyperplasia of hair follicles and increased dermal cellularity in SENCAR mice. In addi-
tion, after 52 weeks of protracted promoter treatment most animals developed generalized amyloidosis
involving liver and spleen, as well as, interstitial nephritis. Severe pyelonephritis and papillary necrosis
were also frequently seen. Reactive lymphoid hyperplasia was also a frequent finding Chronic ad-
ministration of TPA is not an inocuous treatment affecting only the interfollicular epidermis. The general
effects of the promoter or the nim k , probably through impairment of the immune system, decreased
markedly their longevity.
CITATION: EnvironmentalHealth Perspectives 68:75-80, September 1986.
C104 MS-86-1L5 Project Officer: Nesnow
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PERMEATION of WATER CONTAMINATIVE PHENOLS THROUGH
HAIRLESS MOUSE SKIN
A.S. Huq 1 N. F. H. Ho, 1 N. Hiasw G.LF ynn, 1
W.E.Jeizer, 1 andL W. Condie, Jr. 2
1 University of Michigan
2 HEpj ,, USEPA, Cincinnati, OH
This paper will aid in the assessment of e.*posure of water containinanLs by the percutaneous route of a-
posure.
As a means of determining the risk of absorption of water cont niinative phenolic compounds through
the skin, the pernieabilities of phenol and certain of its analogs, all on the EPA’s list of priority pollutants,
through hairless mouse skin have been studied using in vitro diffusion cell methods. Experimentally
determined effective permeability coefficients for whole and stripped skin, Pe’s, and permeability coeffi-
cients for the viable tissue layer, Pvc, and the stratum corneuni, Psc, estimated from the P 5 ’s have been cor-
related with their log Koct o j , ter partition coefficients. P and values systematically increased with
incre1a ing phenol lipophilicity to limiting values of about 0.15 and 0.30 cm/h, respectively. The estimated
value of P ,t is relatively constant at about 0.36 cm/h. Because of the inductive effects of Cl and N02 sub-
stituents on the aromatic ring, phenolic analogs containing these functions are quite acidic and, conse-
quently, their overall skin permeabilities tend to be highly pH dependent in the range of pH values seen
for surface waters. The free acid permeability coefficient, P , of 4-nitro phenol was seen to be exception-
ally low. It appears from the data pattern that it might intermolecularly associate. The fluxes of free
(unionized) species existing at low pH were, as expected, greater than fluxes of ionized forms no matter
the phenol. Nevertheless, anionic phenolic species have measurable fluxes across intact skin. With the
exceptions of relatively polar phenol and the mono-nitro phenols, the free acid forms of all the phenols
studied permeated skin with ease and at rates approaching those of stratum corneum free skin.
CITATION: Archives of Environmental Conta,nination and Toxicolo 15:557-566, August 1986.
C104 MS-86-133 Project Officer: Condie
MECHANISMS of CHLOROFORM and CARBON TETRACHLORIDE
TOXICITY in PRIMARY CULTURED MOUSE HEPATOCYTES
Randall I. Ruch, James E. Klaunig Nomian E. Schultr Augusta B. As/carl,
DavidA. Lacher, MichaelA. Pereira 1 and Peter I. Goldblatt
Medical College of Ohio
1 HERL, USEPA, Cincinnati, OH
The disinfection of drinking water is one of the most wide y practiced and most successful public health
programs in the U.S. While it has drain atically reduced the incidence of waterbome illness, it has been
determined that the organic chemical contaminants occurring at the highest concentration in drinking water
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result from it. This syniposiwn paper summaji.zes the results to date of the Agency’s research into the
health effect of these contwninanL
Chloroform (CHCL3) is formed as a byproduct of sewage and drinking water chlorination and has been
identified in public drinking water. CHC13 is toxic to mpmmalian liver in vivo and to isolated hepatocytes
and is metabolized in the liver by the mixed-function oxygenase system (MFOS). A cytochrome P-450
monooxygenase oxidizes the C-H bond of CHCI3 to produce trichioromethanol, which spontaneously
dehydrochiorinates to the toxic component, phosgene (CHOC1 2). It has been suggested that free-radi-
cal generation occurs during CHC1S metabolism and may be important in CHCI3 toxicity. Therefore, we
exz mined the effects of the MFOS inhibitor, SKF-525A; the anti-oxidants x-tocopherolacetate (Vitamin
E) and N,N’ -diphenyl -p-phenykli imine (DPPD); and a depleter of cellular gluthathione (GSH), diethyl-
maleate (DEM), on CHC13 toxicity to isolated male B6C3F1 mouse hepatocytes after 2,4, and 20 hours.
CHCI3 alone showed a dose-responsive toxicity at concentrations of 1 and 5 mM. SKF-525A (25 g.M),
Vitamin E (100 pM), DPPD (25 pM), and DEM (0.75 pM) were not toxic by themselves at any time
point. SKF-525A inhibited CHCI3 toxicity at 2,4, and 20 hours. Vitamin E and DPPD had no effect on
CHC13 toxicity. DEM potentiated CHC1S toxicity at all three time points.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Drinldng Water
Disinfectants and Disinfection By-Prodacts, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69:301-305, November 1986.
C104 MS-86-172 Project Officer: Pereira
EXCRETION and TISSUE DISPOSITION of DICHLOROACETONITRILE in
RATS and MICE
Mark R. Roby 1 , Sigrid Cane 1 , MichaelA. Pereira 2 , and Dean E. Ca er 1
1 University of Arizona
2 HERL , USEPA, Cincinnati, OH
The disinfection of drinking water is one of the most widely practiced and most successful public health
programs in the U.S. While it has dramatically rethiced the incidence of watethorne illness, it has been
detemiined that the organic chemical contaminants occurnng at the highest concentration in drinldng water
result from it. This symposium paper summarizes the results to date of the Agency’s research into the
health effect of these contaminants.
Dichioroacetonitrile is found in drinking water as a byproduct of chlorination. The toxicokinetics of this
compound are under study in rats and mice. During metabolism, the molecule may split into one-carbon
fragments. Our studies use dichioroacetonitrile radiolabeled on each of the carbon atoms to follow the
fate of the fragments of the molecule after metabolism. Male Fischer 344 rats (l75-225g) were dosed
orally with aqueous solutions of dichioroacetonitrile at 0.1,0.01, and 0.001 LD5O. Dosing solutions were
prepared by mixing (21 14 C) dichioroacetonitrile (specific activity, 4.04 mCi/mm) with unlabeled com-
pound in water to make a 15, 1.5, and 0.15 mg/kg body weight dose in a volume of 0.3-0.4 ml. Anini a1s
eliminated 84 percent of the dose in 48 hours, and total recoveries in tissue and excretia were greater than
95 percent. The major routes of elimirnition were by expired CO2 and urine in approximately equal
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amounts (33 percent). Tissues contained about 15 percent of the dose after 48 hours, with liver (5 per-
cent), muscle (4 percent), and blood (3 percent) containing the majority of the retained dose. Animals
dosed orally with (r’ 4 C) dichloroacetonitrile (5.0 mCi/mm) at 0.1 LD50 were dosed as described pre..
viously. The rats required 6 days to eliminate 70 percent of the dose. Total recoveries in tissues and ex-
cretia were greater than 89 percent. Urinary excretion (32 percent) was similar to that found for (2’ 4 C)
dichioroacetonitrile, but the amount expired as C02 (9 percent) was substantially lower. Tissues con-
tained about 19 percent of the dose after 6 days, with blood (7 percent), muscle (4 percent), skin (3 per-
cent), and liver (2 percent) containing the majority of the retained dose. The data suggest different me-
tabolic fates for each of the carbon atoms since both the rate of excretion and the route of excretion are
different for these two labeled compounds. These results will be compared with data from male B6C3F1
mice.
CITATION: In: Proceedingc of the Second International Symposium on Health Effects of Drinking Water
Disinfectants and Dicinfection By-FlvducLc, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Peivpectiver 69:215-220, November 1986.
C104 MS-86-171 Project Officer: Pereira
CHRONIC BIOASSAYS of CHLORINATED HUMIC SUBSTANCES in B6C3F1
MICE
Benjamin L. Van Duuren, 1 Susan Melchionne 1 living Seidinan, 1 and
MichaelA. Pereira 2
1 New York University Medical Center
2 HE , USEPA, Cincinnati, OH
The disinfection of ddnldng water is one of the most widely practiced and most successful public health
prog ’ains in the U.S. While it has dramatically reduced the incidence of waterborne illness, it has been
detennined that the organic chemical contaminants occuning at the him est concentration in ddnldng water
result from it. This symposium paper summaiizes the results to date of the Agency’s reseaivh into the
health effect of these contaminants.
Humic acids (Fluka), chlorinated to C:Cl ratios of 1:1 and 1:0.3, were administered to B6C3F1 mice, 50
males and 50 females per group, in the drinking water at a total organic carbon level of 0.5 g/l. The mice
were 6 to 8 weeks old at the beginnhig of the bioassays. This dose was based on short-term (6 weeks)
evaluations for toxicity, palatabilit and weight gain. The chronic bioassays included the following con-
trol groups: nonchiorinated huniic acids (05 gfl), no-treatment (100 males and 100 females),
dibromoethane (DBE, 2mM in drinking water; positive control) and 0.4 percent sodium chloride in
drinking water, i.e., at the same concentration as that of the drinking water for mice receiving chlorinated
humic acids. The chlorinated humic acids were prepared freshly and chemically assayed once per week.
All chemicals were, with the exception of DBE, administered for 24 months; DBE was administered for
18 months. The volumes of solutions consumed were measured once per week. All experimental groups
showed normal weight gain except the DBE group. At the completion of exposure, the animals were
sacrificed and necropsied, and tissue sections were taken for histopathology. No markedly significant in-
crease in the incidences of tumors was observed in any of the organs and tissues examined in the
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chlorinated humic acid groups compared to nonchlorinated huniic acids and the no-treatment control
groups. DBE caused the expected high incidence of squamous carcinomas of the forestomach. The frac-
tions of chlorinated humic adds tested contained direct-acting alkylating agents based on their reactivity
with p-nitro-benzylpyridine and showed mutagenic activity in S. typhfrnwium. However, they did not
produce a significant increase in tumor incidences compared to controls when administered to mice at
high doses.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Drinking Water
Disinfectantr and DLsinfection By-Products, Cincinmiti, Ohio, August 27-29, 1986.
Published irn Environmental Health Perspectives 69: 109-117, November 1986.
C104 MS-86-170 Project Officer: Pereira
CARCINOGENICITY of CHLORINATED METHANE and ETHANE
COMPOUNDS ADMINISTERED in DRINKING WATER to MICE
James E. Klaunig 1 Randall J. Ruch, 1 and MichaelA. Pereira 2
1 Medical College of Ohio
2 HE USEPA, Cincinnati, OH
The disinfection of drinking water is one of the most widely practiced and most successful public health
programs in the U.S. While it has dramatically reduced the incidence of wateibome illness, it has been
detennined that the organic chemical contaminants occurring at the highest concentration in drinking water
result from it. This symposium paper summarizes the results to date of the 4gency’s research into the
health effect of these contaminants.
The liver-tumor-promoting activities of chloroform (CHC13), 1,1-dichioroethane (1,1-DCE), and 1,2-
dichloroethane (1,2,-DCE) were e imined in male B6C3F1 mice. Four-week-old mice received 10 mg/kg
diethylnitrosamine (DENA) in their drinldng water for4 weeks followed by continuous exposure to either
CHCI3, 1,1-DCE, or 1,2.DCE in the drinking water. In both protocols, the three chlorinated compounds
were e,nmined at two dose levels (maximum tolerated dose MTD and 113 MTD). Control mice received
DENA followed by untreated drinking water or no treatment. A positive promoter experimental group
consisted of phenobarbital (500 ppm) in the drinking water. Mice were sacrificed after 6 months (10 mice
per group) and 12 months (25 mice per group) and exsimined grossly and histologically for hepatic
tumors. Neither CHC13, 1,1-DCE, nor 1,2-DCE increased the incidence of liver tumors in mice either fol-
lowing DENA initintipn or when administered without DENA initiation. DENA/phenobarbital-treated
mice displayed an increase in the number of hepatic tumors over animals treated with DENA only and
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phenobarbital only. These results contradict those previously reported for CHCIS, 1,1-DCE, and 1,2-
DCE when adminictered in corn oil to B6C3F1 mice by gavage.
CITATION: In: Proceedings of the Second Intenwiional Symposium on Health Effects of Drinking Water
Disinfectants and Duuzfection By-Products, Cincinnati, Ohio, August 27-29, 1986.
Published in: En vironmental Health Perspectives 69:89-95, November 1986.
C104 MS-86-169 Project Officer: Pereira
WATER CONTAMINATION and ENVIRONMENTAL MUTAGENS
John C Loper
University of Cincinnati College of Medicine
Public concern over contamination of our water supplier with toxic chemicaLs requires that research be
done to investigate the edent and magnitude of the problem. The present study provides a focus on water
contamination by mutagenic chemicals from a vane y of sources. This research also provides a perspective
on the relative i npoitance of the problem in both industrialized and developing countries.
Citizens of industrialized and developing nations share a common concern for safe water sources, but
each group must contend with different priorities and problems. Examples of pollution involving surface,
ground and irrigation water are presented. The Salmonella mutagenesis assay has proven to be a valu-
able bioassay for detection and isolation of unknown waterborne mutagens, and is useful in monitoring
the levels of mutagenic pesticides.
CITATION: 1n Basic and Applied Mutagenesis, Amir Mnliammend and R.C. von Borstel, eds., Plenum
Press, New York, 43-61, 1985.
C104 XX-86-029 Project Officer: Meier
EVALUATION of MUTAGENIC and CARCINOGENIC PROPERTIES of
BROMINATED and CHLORINATED ACETRONITRILES:
BY-PRODUCTS of CHLORINATION
R. I. Bu14 I. K Meier, M. Robinson, H. P. Ringhan4
RD. Lawie andJ.A. Stober
HERL USEPA, Cincinnati, OH
Over the past decade, much of the emphasis on drinking water contamination by toxic pollutants has
shifted towards the chemicals which are produced as by-products of chlorine disinfection. Although
tiihalonzethanes w the most wid4 ’ recognized disinfection by-products, haloacetoniojies have also been
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identified in recent yean as major chlorination by-products. Th Is study exam fr ies the mutagenic and car
cinogenlcpmpe’ties of several of the haloacetonitriles. The results indicate some members of this class of
compounds display activity as genotoaic agents hi vitro and/or as tumor initiators for mouse skin in viva
This data Is relevant to the Office of Drinking Water (ODW) in providing additional information onpoten-
tie! hazards posed by chlorination of thinking water.
The present study was undertaken to determine if chlorinated and brominated acetonitriles formed
during the chlorination of drinking water possess mutagenic and/or carcinogenic properties.
Chloroacetonitrile (CAN), dichloroacetonitrile (DCAN), trichioroacetonitrile (TCAN), bromochioro-
acetonitrile (BCAN), and dlbronxoacetonitrile (DBAN) were tested for their ability (1) to Foduce point
mutations in the Salmonellamicrosome assay, (2) to induce sister chromatid exchanges (SCE) in Chinese
hamster ovary (CR0) cells in vitro, (3) to produce micronuclei in polychromatic erythrocytes in CD-i
mice, and (4) to act as tumor initiators in the skin of Sencar mice. DCAN and BCAN were found to be
direct-acting mutagens in Salmonella. All five haloacetonitriles induced SCE in CR0 cells h i vitro. This
activity paralleled the extent of chlorine substitution and was further enhanced in the dihaloacetonitrile
series when bromine was substituted for chlorine. None of the haloacetonitriles showed evidence of ac-
tivity in the mouse micronucleus assay. DBAN, BCAN, and CAN initiated tumors in the mouse skin with
topical applications followed by a 20-week promotion schedule of 12-0 tetradecanoylphorbol-13-acetate
applications (pcO.02). These data indicate that the haloacetonitriles do display mutagenie and car-
cinogenic properties in some test systems and the hazard associated with their occurrence in drinking
water and production within the gastrointestinal tract require further evaluation.
CITATION: Fundamental and Applied Toxicology 5:1065-1074,1985.
C104 MS-85-131 Project Officer: Robinson
TRICHLOROACETIC ACID EFFECTS on RAT LIVER PEROXISOMES and
ENZYME-ALTERED FOCI
Michael I. Famel4 Loren D. Koller, Jerry H. Exon, and Jean ene At Anizen
University of Idaho
The disinfection of drinldng water is one of the most widely practiced and most successful public health
programs hi the US. JThile it has dramatically reduced the incidence of waterborne illness, it has been
detennined that the organic chemical contaminants occwring at the hishest concentration in drinking water
result from it. This symposium paper summarizes the results to date of the Agency’s research into the
health effect of these contaminants.
Trichioroacetic (TCA) and dichloroacetic acid (DCA) are the major nonvolatile chlorinated products
formed during chlorination of water containing natural huniic material. Although TCA and DCA are
structurally similar , chlorination studies of fulvic and humic acids indicate that TCA formation does not
proceed through a DCA intermediate, but that both compounds form independently. The acute toxicity
of TCA and DCA is rather high, although long- term, chronic toxicity has not been established. Neither
TCA nor DCA exhibit mutagenicity in the Ames assay. The numerous hepatic effects of TCA and DCA
suggest the liver as a possible target organ. Both compounds are thought to induce peroxisomal prolifera-
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tion of hepatocytes. The metabolic relationship of TCA with trichioroethylene (TCE), a hepatic car-
cinogen, and DCA to other chlorinated organic hepatic toxicants and carcinogens suggests these two
agents may be either potential initiators or promoters of hepatic carcinogenesis. The toxicity , im-
munotoxicity and initiation/promotion activity of TCA will be presented and discussed.
CITATION: In: Proceedings of the Second International Symposium on Health Effects of Drinking Water
DirinfectanLc and Dirinfection By-Products, Cincinnati, Ohio, August 27-29, 1986.
Published in: Environmental Health Perspectives 69: 73-79, November 1986.
C104 XX-86-1IS Project Officer: Laurie
A COMBINED CARCINOGEN BIOASSAY UTILIZING BOTH the LUNG
ADENOMA and SKIN PAPILLOMA PROTOCOLS
Meirel Robinso n , 1 RichardJ. Bull, 2 Gary L Knutsen, 3
Robert P. Shields 4 and Judy Stober 1
1 HERL,USEPA, Cindnnati, OH, 2 Ws shington State University
3 Pathology Associates, inc.,kjniversity of Florida
The results of the research described in the attached workshop proceedings article further characterizes the
SENCAR mouse and its advantages and dfradvantages as an animal testing model in toxicological and
carcinogenesis experimentation. The data deals with three categories relevant to the response of the mouse
in laboratory tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initia-
tion/promotion assay), and (3) hlstopathology including treatment-induced and spontaneous lesions.
To test the feasibility of employing a combined lung adenoma/skin papilloma assay for broader detection
of chemical carcinogenesis than realized with either bioassay done separately, four strains of mice; SEN-
CAR, BALB/c, NJ and ICR-Swiss, were administered carcinogens by either the oral or I.P. routes. The
carcinogens administered were ethyl carbamate (BC), benzo(a)pyrene (BP), N- [ 4-(5-nitro-2-furyl)-
thiazolyl]-formnmde (PANFr), and acrylamide (ACR). Starting two weeks later, 1-5p.g (depending on
strain) of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in 0.2 ml acetone/mouse was applied 3 times
weekly to the shaved back for 20 weeks. All strains displayed increases in the yield of lung adenomas in
response to EC at 32 weeks. BP increased lung adenomas in only the SENCAR and NJ strain. Only the
SENCAR and ICR-Swiss mice gave positive responses in the skin. In the SENCAR this was seen with all
of these 4 chemicals, however, FANF gave an inconsistent response. The ICR-Swiss responded with an
increased skin papilloma yield only to EC. In a separate experiment involving only SENCAR mice,
sninuitc were treated with a single oral dose of diethylnitrosamine (DEN) followed by tn-weekly applica-
tion of 1.0 jig TPA. This treatment resulted in 51/57 animals developing lung adenomas vs. i i in the
control nimsis No treatment related skin tumors resulted with DEN. Histopathologically confirmed
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lesions indicate that the spectrum of chemicals detected in the SENCAR mouse may be broadened using
a combined bioassay that er mines both lung and skin responses.
CITATION: Environmental Health Perspectives 68:141-146, September 1986.
C104 MS-86-119 Project Officer: Robinson
CARCINOGENIC POTENTIAL of ARSENIC COMPOUNDS in DRINKING
WATER
Donald Y. Shirachi, Shing-Hui Tu, and John P. McGowan
University of the Pacific
This is a report of a study to determine if arsenic compounds are carcinogenic in the rut liver. Work was
done to gain data to clarify some of the apparent contradiction between human and animal data and
provide the basis for arriving at more realLctic estimates of the carcinogenic risk that Lv associated with the
various forms of arsenic found in dii nldng water, and therefore aid in the regulation of arsenic compounds
under the Safe Drinking WaterAcL
The primary objective of this research project was to determine whether arsenite (Asifi), arsenate (AsV),
dimethylarsinic (DMA) and monomethylarsonic acid (MA) were initiator carcinogens and/or promoters
of DENA-initiated tumors in the rat liver. The maximum tolerated doses (MTD) of the arsenics to be
used in this study were determined by treating male Wistar rats in the drinking water increasing con-
centrations rRnging from lOppm-2560ppm for 7 weeks, depending upon the arsenic studied. The ap-
parent MTD determined were: MA, 800ppm; Ash, l6Oppm; AsV, l6Oppm; and DMA, Soppm. The cal-
culated LD were DMA, 98.Sppm; Ash, 264.8ppm; MV, 294.Sppm; and MA, 1160.lppm. To deter-
mine whether the arsenic compounds had an initiator or promoter activity, male Wistar rats were partial-
ly hepatectomized, treated with a single dose of diethylnitrosamine (3omglkg i.p.) and on day7 started
treatment with the MTD of each arsenic for 7,25 and 43 weeks in the drinking water. This was the
promoter protocol. The initiator protocol did not include the diethylnitrosamine treatment. There were
no significant numbers of animals with tumors in the liver in any of the arsenic treated animals as com-
pared to their controls at the three time periods of treatment with either the initiation or promotion
protocols. However, in the promotion protocol there was significant increase (J) <0.05) in the number of
animals exhibiting tumors in the kidney in the Ash promoted group, 7/10, as compared to the DENA
controls, 2/9 when treated for 25 weeks. In both protocols MI I I treated animals showed a significant
decrease in thymus gland weights. This was suggested to be a contributory factor in the renal
tumorigenesis observed in this study.
CITATION: U.S. Environmental Protection Agency, cooperative agreement no. CR-807235, EPA-
60011-86-003, May 1986.
B107 HERL-0543 Project Officer: Robinson
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ASSOCIATION of CARCINOMA YIELD with EARLY PAPILLOMA
DEVELOPMENT in SENCAR MICE
Rkhwdf. Bull 1 , Meirel Robinson 2 , A Dana Lawie , and Judith A. Stobe?
1 Ws chington State University
2 HE USEPA, Cincinnati, OH
The results of the research described in the attached workchop proceedingr asticle further charactenzes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in todcological and
carcinogenesis espenmentation. The data deals with three categories relevant to the response of the mouse
in laboratoiy tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initia-
tion/promotion assay), and (3) histopathology including treatment-induced and spontaneous lesions.
The responsiveness of the SENCAR mouse skin to 20 different chemicals with known carcinogenic
properties was assessed in initiation/promotion experiments. The purpose of these experiments was to 1)
evaluate the extent of false negative responses in mouse skin initiation/promotion protocols and 2) deter-
mine the extent to which early papilloma development can be used to predict the eventual development
of ni iIigrv nt tumors. The chemicals were administered as initiators by four different routes; orally, in-
traperitoneally, subcutaneously and topically. Following the initiating dose of carcinogen, the inim a1s
were subjected to triweekly, topical applications of 1 j.i.g 12-O-tetradecanoyl-phorbol-13-acetate (TPA)
for a period of twenty weeks. The yield of papillomas at 24 weeks was selected as a potential predictor of
carcinoma yields at 52 weeks following the start of the promotion schedule. Positive responses were ob-
served with only eight of the compounds tested. Where positive results were observed there was some
evidence that the response could depend both qualitatively and quantitatively upon the route of ad-
m n tration. However, no route was clearly superior in that different chemicals gave greater responses
by different routes. Papilioma yield at 24 weeks following the start of the promotion schedule was dear-
ly related to the development of carcinomas at 52 weeks. There was not a simple linear relationship be-
tween papilloma yield and carcinoma development in that the number of malignant tumors per papillonia
decreased with increasing papilloma yields. The relationship between papilloma and carcinoma yields
appeared to be independent of the carcinogen employed. These data indicate that there are some limita-
tions in the use of mouse skin initiation/promotion experiments as the sole basis of identifying substances
with carcinogenic activity. However, the test does perform well within certain classes of compounds.
Within the limits of these chemical classes, the use of papilloma yields to predict carcinoma yield appears
to be justified.
CITATION: Environmental Health Perspectives 68:11-18, September 1986.
C104 MS-86-114 Project Officer: Robinson
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Pesticides
Toxic Substances

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PESTICIDES AND TOXIC SUBSTANCES RESEARCH PROGRAM
Objective: To improve the scientific basis for regulatory activities under the Federal Insecticide,
Fungicide and Rodenticide Act (FIFRA), Toxic Substance Control Act (TSCA).
Background: More than 60,000 chemical substances are manufactured in the United States for use in
an almost unlimited number of products. The benefits derived from such extensive use
of chemicals are cignificant. However, we now know that a number of chemicals may
cause cancer, birth defects, reproductive failures, and other irreversible effects. Often
chronic health effects can result only after long-term exposure to toxic chemicals. In
other cases, such effects may develop many years after a single exposure. Furthermore,
these toxic substances can work together or in combination with other substances such
as those in cigarette smoke, to greatly increase the likelihood of chronic health effects.
We understand little about the synergistic effects of these substances.
Since its inception, EPA has had regulatory responsibility for pesticides under the 1947
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). This law has been
amended to encompass all pesticides used in the United States and requires a balanc-
ing of a pesticide’s benefits agninct its health and environmental risks. In 1976 Congress
passed the Toxic Substances Control Act (TSCA). This legislation has given EPA the
tools to identify and control unreasonable risks. Under regulatory authority from
TSCA, EPA requires a manufacturer to submit a ‘premanufacture notice’ that contains
relevant information about environmental and health effects of chemicals. EPA may
prohibit or limit the production and use of a new chemical if it presents an unreasonable
risk to health or the environment. EPA has similar authority to review and control sig-
nificant new uses of existing chemicals or chemicals newly imported into the United
States. For existing commercial chemicals, TSCA gives EPA the authority to regulate
the manufacture, processing, distribution, use and disposal if there are unreasonable
public health or environmental risks.
Regulatory tools range from labelling and use restrictions to outright bans on their
manufacture. TSCA also authorizes EPA to require that industry test a chemical when
there is insufficient data to assess the risks and there is likely to be substantial exposure.
Under FIFRA, EPA has authority to control the risks of pesticides primarily through a
registration process. All proposed uses of a pesticide must first be approved by EPA.
This process involves a comprehensive review of potential health and environmental
risks. The approved uses of a pesticide must be clearly indicated on the product’s label.
EPA may restrict or cancel some uses of an existing registered pesticide or completely
remove the product from the market if it is found to pose unreasonable risks.
Obviously, in these programs involving toxicology test procedures to assure compound
safety, great importance attaches to methodology. HERL research emphasizes toxicol-
ogy test methods development with the objective of providing more sensitive, more
rapid, and cheaper methods.
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TSCA also iccign to EPA responsibility for assuring the safety for hIIm2n health and the environment of
genetically-engineered orgi nicms. This responsibility includes engineered organisms created for use as
pesticides or for other purposes and products of those org nkms.
EPA has authority to establish the maximum acceptable levels of pesticide residues in foods and nim i1
feed called “tolerance levels,” that will protect hrnniin health while allowing for production of an “ade-
quate” wholesome and economic food supply.
Pesticides - If not handled properly, some pesticides can cause serious health problems, even death if
spilled on the skin, inhaled or otherwise misused. Some of the earlier pesticides also persist in the en-
vironment over long periods of time, moving up through the food chain--from plankton or insects to birds,
fish, srnimalc , and eventually to humans. Finally, some pesticides exhibit evidence of cansing chronic
health effects such as cancer or birth defects. HERL scientists are conducting research in the following
areas in support of this effort:
• Develop test methods In the areas of reproduction/teratology, mutagenesls/cardnogenesls
(genotoxic effects), neurotoxicology and Immunotoxicology. This research Is designed to provide
test methodology for inclusion in test guidelines for use by registrants and for use in Section 4,
TSCA testing requirement.
Rationale: This research is being done to improve the scientific and technical aspects of the tests EPA
requires industry to use in submitting data. Also, to reduce the number of animals required per test,
decrease the time and cost of the test, and to increase the util tion and sensitivity of the test methods.
Approach: This research will utilize both in vwo and hi vitro tests with various animal species. Tests will
be run sing a series of known toxic agents and dose levels. The results obtained will be used to compare
the various endpoints for their predictability and to develop quantitive dose-response relationships
among various test systems. Estimates of exposure and dosimetric studies of DNA adduct formation will
help in providing the quantitive basis for the extrapolation of data from nimsi1 to humans.
• Provide endpoint specific dose response data on pesticides of interest to the Office of Pesticide
Programs (OPP). Such data will Include information about carcinogenic, reproductive, teratologi-
cal and neurotoxicological effects.
Rationale: OPP needs independent verification of toxicological effects produced by pesticides within the
registration/tolerance-setting process. Also these data will provide improved scientific basis for ex-
trapolation from high to low dose, from nim k to man, and therefore improve the reliability of the risk
assessment process.
Approach: Standard research protocols and methodologies are utilized. Laboratory species are exposed
to test compounds by appropriate routes of admini- tration and the response of test populations is
monitored. Three or more dose levels are studied ranging from maternal toxic to “apparent no effect”
levels. Research to characterize the neurotoxic properties of various pesticides involves studies to deter-
mine dose and time effect relationships, subchronic effects and age-dependent effects. The multidiscipli-
nary approach includes assessment of behavior, including motor, cognitive and sensory function.
• Provide models for evaluating health risks of pesticides on the basis of laboratory animal studies.
This research is on reproductive and teratologic effects mechanisms involved in the expression of
agent-induced toxic responses (teratologic and reproductive primarily). These effects are being
studied by a variety of biochemical mechanisms. Also, mathematical models which factor in pos-
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sible litter effects are being developed for the quantification of chemical-induced risk to the
developing conceptus and postnatal animal. Multispecies, multidose teratology bloassays will be
performed In order to provide the necessary data base for evaluation of mathematical models. A
computerized data management system which stores genetics Information is under development.
Rationale: This research is being done to improve our ability to assess risk from pesticides by taking ad-
vantage of the state-of-the-art in biological testing and computer generated models based on the
biochemical and biomedical data. OPP will use these models and n1 n gement systems for evaluating
compounds submitted for registration.
Approach: Various nimia1 species and in vitro systems will be utilized as candidate models to assess
linnign risk from pesticide teratologic and reproductive effects.
• Provide data on the Immunological effects of biological pesticide agents on mammalian cells.
Specific goals are: (1) the determination of the abIlity of biological agents to replicate, and (2) to
provoke Immune responses In non-target (mammAlian) hosts. Also, methods will be developed
using monoclonal antibodies and blotinated DNA probes to enable the Identification of genetic
material from biological pesticides In nontarget sItes such as mammalian cells both in vitro and in
vivo. These methods are needed as a prelude to the validation of critical sections of the Subpart M
guIdelines for testing microbial pesticides.
Rationale: This research is being done to provide OPP with the state of the art scientific and technical
information on new generation of biological pesticides and, thus, improve OPP’s basis for making
regulatory/standard decisions.
Approach Evidence for replication, multiplication or toxicity will be determined by a variety of techni-
ques including blot immunoassays, DNA scission techniques and DNA molecular hybridization, quan-
titative culturing techniques or by cytolytic assays.
Toxic Substances — Agency need is for a program to identify, evaluate, and control the human health
risks from currently used toxic chemicals and those compounds proposed for manufacture. To this end,
HERL studies include the following projects:
• Develop and validate methods to detect pulmonary carcinogens and promoters, characterize the
ability of the L5178YrK mouse lymphoma assay to detect genetic damage, and develop methods
to Improve the sensitivity and broad spectrum response of bioassay systems to rd the major clas-
ses of carcinogens. In vitro methods will also be developed to determine biochemIcal and
physiological effects, and bloassays will be developed for detection of adverse alterations In the
developmental and reproductive processes of animals. Bioassays to screen and evaluate chemicals
for Immunotoxic effects as well as the selection of methods which best detect and characterize
neurotoxicity will be studied and evaluated.
Rationale: This research is being done to provide more sensitive toxicology test methods for use in Sec-
tion 4 and Section 5 test requirements under TSCA.
Approach: Both laboratory ini,rnik and cell and organ culture methodologies will be utilized to develop
more sensitive, reliable and efficient procedures for evaluating adverse health effects of toxic chemicals.
These studies include methods to improve the sensitivity of mouse cells toward carcinogens, the develop-
ment of adequate bioassays which test for reproductive and/or developmental effects, and the study of
cell mediated and humoral immune system parameters.
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RESEARCH OBJECI’IVES PESTICIDES/TOXICS
• Provide toxicological data on the adverse effects of Inhaled asbestos and asbestos-like fibers
(erlonlte, chryssotile, and crocidollte) Induding the correlation of short-term tests with in vivo car-
cinogenesis studies and testing of fibers of priority Interest to the Office of Toxic Substances.
Research will also Include studies In the Influence of fiber geometry on toxlcIt
Rationale: 0Th is required to revise the control strategies for asbestos. This research supports the
revisions of control strategies for asbestos which is presently regulated under TSCA.
Approach: Studies will be carried out in whole sinininlc and through the use of tissue cultures. Addition-
ally, newly-developed procedures to separate fibers of different lengths will be utilized. Intratracheal in-
stillation in rats using samples of sized fiber lengths will be carried out.
• Develop and validate models on new approaches to perform toxicology risk assessments. Test sys-
tems aimed at assessing chromosomal effects In germ cells after exposure to mutagens/carcinogens
will be studied. Additionally, tests will be performed to assess the effects of hepatic, cardiovascular,
and hormonal alterations on general health status, to obtain better morphometrlc data on the pal-
monaly adnus region for use In mathematical dosimetry models and to determine the relationship
of Indicators of neurotoxldty to disease states and/or neurobehavloral dysfunction.
Rationale: To make judgements about the degree of risk, it is necessary to extrapolate from nn m lc to
man and from high dose to low dose. Improved methods for such extrapolations will reduce some of the
uncertainties associated with present methodology.
Approach: Both in vivo and in vitro tests will be conducted using various nnimal spades and cell cultures
as well as associated studies to determine pharmocokinedcs and biotransformation. Areas of study will
include mut enetidy/carcinogenicity immunotoxicity, reproduction/teratology and inhalation toxicol-
ogy.
• Carry out epidemiology studies on human population exposed to compounds of priority Interest to
0Th. Emphasis will be given to the evaluation, In exposed human population, of laboratory-
developed blomonitoring methods In the areas of genetic toxicology, neurotoxicology, developmen-
tal toxicology/teratology and Immunotoxlcology. The objective is to develop biochemical proce-
dures to serve both as measures of human exposure and as early Indicators of disease processes.
Current emphasis focuses on genetic toxicology.
Rationale: The Toxic Substances Control Act calls for research that will supply health effects data use-
ful for development of regulations controlling toxic substances. Where feasible, epidemiologic studies
can provide the most direct evidence of human effects at environmental exposure levels.
Approach: Laboratory-developed methods for determining genetic toxicity will be evaluated in exposed
humsn populations. Other epidemiologic studies will be made of people occupationally- or environmen-
tally-exposed to compounds of priority interest to 0Th.
• Develop a method to correlate chemical structure with toxicologic effect, Including genetic, car-
dnogenlc, and other toxic activities using pattern recognition, statistical and therinodysamic tech-
niques. Construct chemical data bases In several areas of toxicological response and separately re-
late them to genetic and carcInogenic activitIes.
Rationale: The Office of Toxic Substances is required to estimate health effects of new chemicals as part
of the requirement of TSCA, Section 5, the premanufacturing notification action (PMN). If structure ac-
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tivity relationships can be developed for health, they will be most useful in TSCA, Section 5, PMN actions
bec e of the lackof actual test data on these chemicals to use in evaluation.
Approach: The research will involve study of structure/activity relationships for various active and inac-
tive compounds utli” ng a variety of short-term tests for genotoxic effects. Computer searches of the
gene-tox data base will be carried out on structural analogues in an attempt to define SAR relationships
for additional compounds.
• Biotechnology health test methods - Study of the potential dispersal capability of genetically en-
gineered genes between bacterial extrachromosomal genetic elements, genomic DNA strains, sub-
strains and related species. From this research suitable models to assess these factors will be
developed using well characterized species.
Rationale: New health concerns arise when genetically engineered organisnis are created for broadcast-
ing into the environment as agents. The health and ecological consequences are difficult to assess because
little or no data are available on the fate of new genetic combinations that are created. These new genetic
combinations are potentially free to interact with a variety of diverse nontarget microorganicmc. Health
concerns are implicit when the inserted genes express products that have toxicological, pharmacological
or developmental effects.
Approach: Vertebrate control cells will be compared to a recombinant baculovirus containing a marker
gene. Evidence for replication, gene expression and host-virus DNA exchange will be studied using a
variety of techniques including DNA probes, immunoassays, and quantitative assays.
• Biotechnology health risk methods - develop a computer-oriented database which compiles the
known characteristics of genetically engineered biological material.
Rationale: The establLclniient of data base mnnagement system for assisting in the evaluation of potential
h27Jards of genetically engineered material will be useful to the Agency in protecting huninn health and
the environment.
Approach: A compilation of known modified org inisnic based upon proper microbiological nomencla-
ture, species specificity, molecular characteristics, biological properties, and nucleotide sequence will be
created. From this it may be possible to correlate known biological properties with the possible
pathobiologic nature of orgnnismc and may enable the prediction of characteristics of newly created or-
ganisms.
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ACCOMPLISHMENTS PESTICIDES
Issue: Develop and Validate Test Methods for FIFRA Registration and Enforcement Program
An effort was begun three years ago to promote the rat as a test-species for organophosphorus-induced
delayed neuropathy (OPIDN) to replace the traditional chicken model. Acceptance of the rat model
would enable the researcher to use a mammalian species, phylogenically closer to humans , rather than an
avian species for screening of potentially neuropathic organophosphorus compounds--providing a more
solid foundation on which to base dose-effect extrapolation to human exposure conditions. A series of
studies, each eiamining a different aspect of OPIDN in the rat, was carried out to validate the rodent
model of OPIDN from a biochemical aspect. It was found that neurotoxic esterase (NTE), the putative
target enzyme for OPIDN in the hen, is indeed present in the rat and that the degree of inhibition of NTE
within 48 hours of organophosphate (OP) dosing predicts the degree of neuropathic damage in the spinal
cord of the animals 2 weeks later. This is true whether the OP compound does or does not require hepatic
activation for its neuropathic potency. With these studies, similar to previous studies using the hen as a
test species, NTE inhibition was established as a potential biochemical marker for the development of
OPIDN in the rat. In an entirely in vitro study comparing rat with hen NTE, it was established that the
two enzymes were very SimilaT with respect to inhibitor sensitivities and molecular weight. Hens dosed
with neuropathic levels of an OP compound do not develop OPIDN if they are dosed previously with the
serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), which is thought to block the active site
of NTE. Again , comparable results were found when rats were used rather than hens as the experimen-
tal animals . As a negative validation of the relationship between NTE inhibition and the subsequent
development of OPIDN, it was shown that in rats dosed with triphenyl phosphite, an OP compound used
mainly as a plasticizer, there was very little NTE inhibition in the brain. There was, however, neuropathic
damage distributed throughout the nervous system in a pattern not reported in animaLs with OPIDN. This
neuropathy, therefore, cannot be classified as an OPIDN; a conclusion predicted by the lack of early NTE
inhibition. Thus, it appears that rat brain NTE inhibition measured within 48 hours of dosing can be used
not only to detect but to predict and characterize OPIDN neurotoxicity.
Skin penetration of fourteen pesticides representing eight groups with widely differing physical and
chemical properties was studied in young and adult rats. Young (33-day old) and adult (82-day old)
female Fischer 344 rats, with previously clipped mid-dorsal skin, were treated with three dosages of
labeled pesticides in acetone. The treated area was 2.3% of the body surface area and was protected by
a plastic blister. Skin penetration was determined by measuring the radioactivity remaining at the ap-
plication site. Skin penetration of the pesticides in young animals ranged from 0.9 to 90% and in the adult,
it ranged from 1.0 to 93%. Chlorpyrifos, dinoseb, mono- and disodium methanearsonate did not show a
significant effect of dosage on dermal penetration. Dinoseb, mono- and disodium methanearsonate dis-
played constant fractional penetration in both young and adult animals over the dosage range examined.
The other compounds showed dose-dependent dermal absorption although the total amount absorbed
usually increased with dose. Dose response curves for young and adult animals were not parallel in eight
of the fourteen pesticides studied. Eleven of the fourteen pesticides showed significant age-dependent
differences in skin penetration. Four of these eleven compounds had greater absorption in the young
than adult at some dose, the highest and lowest significant youngladult penetration ratios were 1.53 and
0.19, respectively. With this heterogeneous group of chemicals, the correlation between skin penetration
and octanol/water partition coefficients was marginal. Stratum corneuin thickness did not appear to be
a factor in the penetration differences between young and adult rats.
Issue: Develop Models to Assess Risks from Pesticides
The Genetic Toxicology Division has previously reported the qualitative results of a major study on 65
pesticides. Dose information from this investigation (either lowest effective or highest ineffective dose
tested) has now been incorporated into a computerized data management system. This report focuses on
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the qualitative proffles of genetic activity produced by these pesticides and our efforts to classify them ac-
cording to their genotoxic effects and chemical structures. Three main categories may be distinguished
based on the qualitative results: Category 1 pesticides were active in most of the in vitm and in vivo assays
employed. These 9 compounds include the structurally similar organophosphate insecticides, acephate,
demeton, monocrotophos and trichlorfon; the phthallmide fungicide analogues, captan and folpet; and
the thiocarbamate herbicide analogues, diallate, sulfallate and triallate. The 26 Category 2 compounds
demonstrated fewer positive results and may be subdivided into two parts, one of which contains 12
halogenated aromatic or heterocyclic ring compounds, including the phenoxy herbicides 24-D, 2 ,4-DB
and 2,4,5-T. The remaining part of Category 2(14 compounds) consists of four structurally smilar or-
ganophosphate insecticides; three siniHar ethylenebisdithiocarbamate fungicides; three similar
pyrethroid insecticides, and four structurally diverse compounds. The third category of 30 pesticides
gave negative results in all tests and represents structurally diverse compounds.
A study of the acute toxicity of pesticides which has extended over several years was completed and
published. LD5O values were determined for 57 pesticides administered by the oral or dermal route to
adult male and female Sherman rats. Thirty-six of the chemicals were also tested by the oral route in one
sex of weanlings. Nine pesticides tested by the oral route (bufencarb, cacodylic acid, dialifor, del-
tamethrin, dicamba, diquat, quintozene, phoxim, pyrazon) and four tested by the dermal route (bufen-
carb, chiordimeform, dichiofenthion, leptophos) were more toxic to females than to males whereas fam-
phur and 2,4,5-T (oral route) were less toxic to females. Eighteen of the test chemicals were more toxic
to the adult than to the weanling and four compounds (leptophos, methidathion, pyrazon, and sulfoxide)
were more toxic to the weanling. In additional studies, the variability of the LD5O value over a 1-year
period was examined for two typical insecticides. Six consecutive bimonthly oral LD5O determinations
for parathion and DDT in adults of both sexes indicated that the LD5O values were little affected by the
time of year that the tests were done.
Workers in a pesticide formulation plant in Egypt were exposed to Leptophos as recently as 1975 and to
EPN more recently. In 1985, production workers from this plant and two reference plants were screened
for OPIDN and cholingeric effects. Biochemical assays included serum cholinesterase and lymphocyte
neurotoxic esterase (LNTE). Neurobehavioral effects were determined by interview, examination and
the Santa Ana Dexerity Test, the Block Design Test and the Optacon threshold voltage (optacon), which
measures tactile sensitivity. The optacon correlated well with the total of both interview and exam find-
ings relating to OPIDN. The logs of the optacon and the LNTE means were significantly higher among
the pesticide workers than among same-city reference plant workers, but were not significantly correlated
with each other. Twenty-five (11%) of the pesticide workers’ LNTE was inhibited more than 50% rela-
tive to the mean of the reference plant workers. One year follow-up data is now being collected.
Gestational exposure to the fungicide dinocap produced behavioral abnormalities in the house mouse
that were not apparent at birth but became obvious at weaning. Torticollis (head-tilting) appeared in the
treated offspring at three weeks of age and, during a test of swimming ability many of the mice sank below
the surface or were unstable and swam on their side in the water. These behavioral abnormalities are the
result of agenesis of the otoliths in the inner ears. These behavioral and neurological alterations were the
only developmental defects noted in the 12 mg/kg/d dosage group. In this group 4.4% of the mice dis-
played torticollis, 9.2% did not swim normally, 19% were missing one or more whole otoliths and partial
agenesis of the crystalline materials was seen in an additional 11.6% of the mice. The frequency of be-
havioral and inner ear defects increased in the higher (25 mg/kg/d) dosage group, but the order of sen-
sitivity of the effects did not change. These results indicate that this compound causes behavioral and
morphological malformations in the offspring of dams exposed during pregnany. This data has been used
by the Agency in negotiation with the manufacturer.
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lssue Blotechnology Microbial and Biochemical Pest Control Agents
In continuing studies on the potential human health 1 rd of microbial and viral pesticides, HERL
scientists have studied the effect of removal of the cytolytic factor of Bacillus thuringiensis subsp. is-
raelensis (BTI) on the mosquitocidal activity of BTL BTI synthesizes a multiconiponent, proteinaceous
inclusion body during sporulation. This protein is cytotoxic to most cells and exerts myotoxic and
neurotoxic effects in both insects and nunnm k . In our study, the 28 Wa peptide, which contains the
nisimm i1iiin toxicity factor, was removed by pffinity chromatography with a monoclonal antibody and the
effectiveness of the remi hihig protein on mosquitocidal activity was eviimned. Our results indicate that
the 28 Wa peptide is not the major mosquitocidal protein contained in the BTI indusion and its absence
did not significantly influence the level of mosquito toxicity of this toxin. Thus, these results suggest that
it may be possible to obtain a preparation which would retain the inosquitocidal activity but not be toxic
to mammals.
Research is in progress to assist the Office of Pesticide Programs in development and standardization of
protocols to test for the potential health effects of candidate microbial pesticides. These protocols will
be included in Subdivision M of the pesticide assessment guidelines. This work includes development,
standardization and validation of an intranasal/intratracheal application protocol to assess the effects of
inhalation exposure, refinement of an acute oral test protocol, and provision of a mammalian tissue cul-
ture test protocol for viral pesticides, including the development of assays utilizing nucleic acid and im-
inunological probes.
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Issue: Develop and Validate Test Methods to Identify Hazards Under TSCA
A sensitive, non-invasive assay to determine altered xenobiotic metabolism has been developed at HERL.
This work is important since chronic ingestion of environmental chemicals can lead to a selectively altered
population of GI microflora. Chemical-microbial interactions are particularly important in young
animals due to rapidly chnnging gut flora. To determine the magnitude and extent of chemical-microbial
interaction as well as the ontogeny of microflorai enzyme activity in the developing sinimn1 freshly voided
feces axe incubated with various substrates. Metabolites of substrates y-hexachlorocyclohexane, p,p’-
DDT, p-nitrophenyl- -D-glucuronide, p-nitrophenyl sulfate, and 3,4-dichloronitrobenzene are analyzed
by EC gas chromatography and are used to assess dechiorinase, dehydrochiorinase, frglucuronidase, aryl
sulfatase, and nitroreductase activity. The sensitivity of the analysis permits use of short incubation fimes
low substrate concentrations and small fecal samples.
HER.L has developed in vivo cytogenetic model systems for evaluating “problem” environmental chemi-
cal carcinogens. Urethane (ethyl carbamate) has been a public health concern because of its wide huni n
exposure and demonstrated zininial carcinogenicity. However, its genetic activity has been enignl2tic: as-
says of genotoxicity have given conflicting results. Methodological improvements to assess new tissues at
sites of direct exposure or pathology, and advantageous combinations of assays have allowed for im-
proved sensitivity to detect and interpret the significance of genetic effects. The application of these
methods have implicated vinyl carbamate in ethyl carbamate genotoxicity and carcinogenicity and have
helped to clarify the relationships between these two events. Simil2r studies to characterize the effects of
other human exposure chemicals important to the Agency, e.g., methyl isocyanate, butadiene, styrene,
etc., have also been performed. The methods developed and applied in this research project affords the
continuing capability to screen chemicals for the induction of cytogenetic diun ige. This work has direct
application to OFF, OTS, OSW and Superfund.
HERL developed a new in vivo mammalian germ cell bioassay for screening chemical genotoxic effects
and for probing mechani.cmc of chromosomal alterations. The project began in 1980 and is continuing.
This methodology addresses many Agency needs expressed in the recently prepared EPA Mutagenesis
Guidelines and provides an important tool for h nrd evaluation in risk assessment. Despite the critical
public health concerns associated with chemical-induced damage to germ cells and possible heritable
consequences analytical methods have suffered from limitations in sensitivity and also from tecimical
constraints. As a result of this research project, many such problems have been overcome, and the
Agency’s capability to screen chemicals and evaluate how they might cause potentially heritable
chromosome damage should be sign Wicantly improved. Validation trials and various applications of the
new methodology are presently being performed. This work has direct application to OPP, OTS, OSW
and Superfund.
Penetration of 14 C-2,4,5,2’,4’,S’hexachlorobiphenyl (PCB) through skin of young (33 days) and adult (82
days) female Fischer 344 rats was determined in vivo and by two in vitro methods. In vivo dermal penetra-
tion at 120 hours was 45% in young and 43% in adults. At 72 hours, in vivo dermal penetration was 35%
in young and 26% in adults compared to 13% for young and 1.4% for adult as measured with a con-
tinuous flow in vitro system and 2.5% for young and 1.2% for adults as measured with a static in vitro sys-
tem. Most of the absorbed PCB remained in the body as only 4.9% and 2.6% of that absorbed was ex-
creted by young and adult rats respectively by 120 hours. The excretion of PCB into feces was ap-
proximately six times higher than into urine. A physiological phannacokinetic model was fit to the organ
and tissue distribution data. Parameters in the model determined from dermal dosing of female Fischer-
344 rats were in reasonable agreement with those obtained from i.v. dosing of adult male Sprague Dawley
rats. Differences in the model parameters between young and adult rats were found.
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HERL has developed methods for assessing cellular immune responses in the lung which are particular-
ly useful for measuring the immunotoxic effects of inhaled compound& Application of these methods
have shown that phosgene depresses immune defenses in the lung which would not have been predicted
by conventional immunotoxicity tests. Also, murine cytomegalovirus and influenza infection in mice have
been developed as host resistance models to add relevance to immunotoxicity testing schemes. These
models are currently being validated with such compounds as phosgene, xylene, and tributyltin oxide.
These test methods are important to the Agency because public awareness that the integrity of the im-
mune system is critical in maintaining health has been heightened considerably by the AIDS epidemic,
and complaints of increased susceptibility to infectious disease (real or imagined) are common in popula-
tions exposed to toxic agents.
Issue: Health: Markers, Doshnetry and Extrapolation
HERL studies on the tumorigenesis of mineral fibers have emphasized development of an in viro method
which could provide results comparable with the more costly and time consuming animal bioassay proce-
dures. The cytotoxicity of four tumorigenic minerals, erionite (w), erionite(c), UICC crocidolite, UICC
chrysotile to Chinese hamster V79 cells was compared. The results indicate that the minerals were toxic
by showing more than 50% toxicity for at least one dose between 10 and 100 p.g. This confirms our pre-
vious findings that V79 cytotoxicity corresponds to the in vivo tumorigenic potentials of the mineral fibers.
Higher potency of erionite, however, was not evident in this system when the dose considered was ex-
pressed in mass units. On the other hand, when the degree of cytotoxicity was considered per number of
mineral fibers, it was clear that fewer erionite fibers of all three dimensions than those of UICC
crocidolite and UICC chrysotile were needed to produce similar toxicity. This work suggests that the
dose in number of fibers may be a better parameter than the total mass dose as a correlate of tumorigenic
potential.
In order to determine the lowest dose of EDB that produced a toxic effect on the liver of the rat in vivo,
rats were dosed with lO- 300 p. mol/mg (1.9-56.4 mg/kg) of EDB 21 and 4 hours before testing. Liver
toxicity studies included tests for DNA damage ( alkaline elution) and biochemical effect (glutathione,
cytochrome P-450 and ornithine decarboxylase, and serum abinine transaminase). EDB doses of 10
mol/kg (1.9 mg/kg) and above caused DNA tlarn ge as determined by the alkaline elation technique.
Only the highest dose of EDB (300 p moVkg 56.4 mg/kg) caused other biochemical effects. This mini-
mum toxic effect for EDB is 40-fold lower than that which has been demonstrated to cause DNA damsige
in rats in previous studies and 20-fold lower than that known to cause cancer in rats.
HERL developed evoked potential methods for evaluating the visual system of rats and humans exposed
to toxic substances. The project began in 1978. Completion of the rat model was marked by publication
of the paper: Dyer, R.S. The Use of Sensory Evoked Potentials in Toxicology. Fund. AppL Toxicol.,
1985, 5:24-40. The human model is nearly complete, as reflected by presentation of results in abstract
form (Hudnell and Boyes, Neurosci. Abs., 1986,12). The rat model provides a rapid and sensitive method
for evaluating effects of exposure to neurotoxic agents on visual function. Owing to this long-term
methods research, visual evoked potentials have already been used to evaluate the neurotoxicity of sol-
vents (toluene, sulfolane), organotins, and, at the request of OTS, methyl pyridines. Because these similar
models are now available for evaluating rat and human visual function, it will be possible to extrapolate
directly from one test to the other.
HERL has developed a battery of tests for evaluating cognitive function in humans with known or
suspected exposure to toxicants. The project began in 1981, and was completed in 1986 with the conven-
big of a workshop on computer-based testing protocols for humans. The test, now called the NES battery,
is currently being validated in populations of painters, alcoholics, and others with suspected neurotoxicity.
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It is also being used in-house as part of controlled laboratory studies of carbon monoxide toxicity. Be-
cause this battery was developed, it will be possible to evaluate on-site the outcome of accidental exposure
to suspected neurotoxicants. Plans are underway to include sections of the battery in the upcoming
NHANES-Ifl protocol, in order to establish an extensive database.
HERL staff helped develop the OTS testing guideline for the functional observational battery (FOB),
which is a neurological e amination for rats. In late 1985, HERL implemented a much broader FOB and
undertook a series of validation experiments using eight chemicals that were selected to produce different
states of intoxication. The validation scheme and data analysis will be completed in February 1987. On
the basis of preliminary evidence, the FOB appears to be a stable, sensitive and efficient test for rapidly
identifying the neurotoxic potential of compounds. As a consequence, HERL has strongly recommended
use of the FOB to evaluate several compounds of current concern to OTS. In addition, on the basis of
this work, HERL can now respond rapidly to requests from OTS and other program offices in assessing
the neurotoxic potential of priority chemicals.
HERL has underway a major research program to improve significantly the basis for quantitatively ex-
trapolating between animals and man, concentrations of selected chemicals that cause pulmonary effects.
Achieving this goal is necessary if apimal data are to be used more directly in risk assessments of toxic
chemicals where exposure is by the respiratory route. Development of anatomically correct physical
models of the lung airways and of the gas exchange region of the lung is an essential first component of
extrapolation modeling and is the focus of this project. In addition, microtoxicological data are needed
to focus evaluation of injury to specific sites in the respiratory tract. Efforts are underway to create path-
length proffles for the mouse acinus similar to those that were obtained for rat lung reconstructions. Fur-
ther work on the partitioning of gas flow in the rat lung has provided new insights on relationships be-
tween structure and function that will be important factors to consider in evaluating toxicity data. The use
of position emission topography is being examined for validation of mathematical dosimetry models since
an overlay between delivered dose and lung injury can be obtained in conjunction with three-dimensional
reconstruction techniques.
Issue: Develop, Validate and Conduct Epidemiologic Studies Among Populations Exposed to Toxic
Chemicals
A cooperative project is currently underway at Mt. Sinai School of Medicine to investigate asbestos re-
lated abnormalities in a group of 1200 female school teachers who were employed in schools with asbes-
tos surfacing material at least 20 years ago. Prevalences of X-ray, pulmonary function and other manifes-
tation of asbestos exposure will be compared with a control “unexposed” group of 600 teachers with other-
wise similar work histories. Comparisons will also be made with other groups of women exposed to as-
bestos non-occupationally and for whom prevalence of asbestos related X-ray abnormalities are avail-
able. Exposure estimates are being derived from teachers’ description of the usage and condition of as-
bestos in the schools(s) where they have been employed. These descriptions will be augmented by
photographs of representative conditions which are available from previous work by the principal inves-
tigator. Numerical ratings of potential exposure will be generated through evaluating school asbestos
conditions. In some schools where quantitative measurements of asbestos concentrations are available,
these data will be used to supplement teachers’ assessments. Also, in those schools where asbestos
materials have been assessed previously or are still in place, independent assessments will be used to
validate teachers estimates.
The research program in epidemiology is relatively new in comparison with our other health research ac-
tivities. For this reason, most of the epidemiology projects have not yet matured into the data producing
stage but a number of interesting and valuable studies are in progress:
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TOXICS ACCOMPLISHMENTS
Development of Methodology for the Use of Biochemical Markers in Epidemiologic Studies.
Through this cooperative agreement with the University of North Carolina, we will produce a
monograph that represents a detailed overview of biochemical markers as they relate to use in
epidemiologic studies. The monograph should enable epidemiologists to identify markers that are
viable tools for epidemiologic studies, and present suggested methodologic design considerations for
their use. Additional work will be undertaken in a field situation to test the feasibility of selected
markers in a hnm,in population.
• Research Strategy for an Integrated Study of Biomarker Formation and Mutagenic Profile in Animal
and Human Cells. In this cooperative agreement, we intend to work with the Oak Ridge National
Laboratory to identify populations exposed to a selected group of mutagenic chemicals, and to con-
duct in vitro and in vivo studies of a selected number of these chemicals in animal and human
populations exposed to these mutagens. In addition, the work will ultimately develop estimates of
the degree of correspondence between bioindicator measurements and measurements of genoniic
damage.
• Fumigants and Grain Workers: An Evaluation of Genotoxicity. Through this cooperative agree-
ment with the University of Minnesota, we intend to evaluate the effects of occupational exposure to
phosphine, malathion, and carbon tetrachloride on the sister chromatid-exchange rates, and on
chromosomal aberrations. Parallel studies of human lymphocytes exposed to the toxicants in viw
will be compared with in vitro generated genetic responses to exposure.
• Through an Interagency Agreement with NIOSH, we intend to evaluate how well hemoglobin alkyla-
tion and the frequency of chromosomal micronuclei correlate with occupational exposure to
ethylene oxide. The premise bein& that if the correlation between exposure and effect is high, these
genetic factors may provide sensitive dosimeters for measuring exposure.
Issue: Develop Predictive Techniques In Support of PMN Review
Development and Evaluation of the SAR System, ADAPT: HERL participated in the development and
evaluation of the correlative structure activity method, ADAPT, to aid identification of potential car-
cinogens and mutagens associated with premanufacture review of toxic chemicals. This computational
technique evaluates the potential for biological activity of a chemical from its structure and physical
properties is useful to the Program Offices as a prescreen or to assess the potential hazards of chemicals
that are not available for bioassay. The development of the present system began in 1981 and ended in
1985 and is presently undergoing modification and usage in collaboration with program offices and for
additional chemical classes and biological endpoints. Since the establishment of this system, SAR
methods associating the mutagenicity and structure of many classes of compounds can aid in the setting
of priorities for the identification and quantitation of these compounds in ambient Air, Hazardous Waste,
Superfund, and OTS. This is expected to have a major impact in these program offices.
Issue Biotechnoloirv
To provide data on the potential human health risk of mutant and genetically engineered microorganisms
that are or will be released into the environment, the microorganisms that degrade hazardous compounds
(e.g., pesticides, polychiorinated biphenyls (PCB’s), etc.) are being examined. Microbes that degrade
hazardous compounds are being isolated for this study so that a wide array of compound utilizers can be
examined. An assay to evaluate the health effects of mutant and recombinant microorganisms is being
developed. A manuscript is currently being prepared that describes the isolation and characterization of
the Pseudomonas spp. that metabolize chlordecone. Polychiorinated biphenyl (PCB’s) degrading
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ACCOMPLISHMENTS TOXICS
microorgiuthms have been isolated from a commercially available product designed for chemical waste
PCB degradation. The following microorg nicm c have been Isolated from the product and identified:
two Bacillus subtillis strains, an Enterobacter cloacae strain, three P. aeruginosa, and one P. maltophilia
strain. Of particular Interest are the Pseudomonads since they are the mutant species that degrade
PCB’s. Because earlier recombinant DNA studies, gut colonization studies, etc. have been done with a
well-characterized E. coil strain. A variety of other microbial species are now being engineered for use
in the environment. The EPA needs established methods for evaluating these microorganicni s for any
potential health effects whether the effects be direct or indirect. The approach being developed attempts
to establish screening methods for looking for both acute toxicity and Indirect health effects. As an ex-
ample, a mouse model system is being developed to study the colonization and competition potential of
the PCB degraders in the mouse. Mice were dosed by gavage with one of the P. aeruginosa strains and
their feces e, mned for the presence of the orgiinLcni. At the highest dose, the organism was recoverable
24 hours after dosing. No dosed microorg nicms were recoverable at the two lower doses. Prelimin2ry
work is underway to enumerate microorganisms from the mouse Intestines. Models such as these will
allow a more proper assessment and regulation of engineering organic” c .
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Publications
Pesticides
Toxic Substances
This report includes abstracts of manuscripts for journal articles published and conference
proceedings papers presented between October 1, 1985 and September 30,1986...
Programmatic Relevancy statements are italicizeS
Other articles submitted for publication, but not published as yet, are listed in Appendix A.

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PUBLICATIONS
ACUTE TOXICITY of PESTICIDES in ADULT and WEANLING RATS
Thomas B. Gaines and Ralph E. Linder
HERL, USEPA, Research Triangle Park, NC
This paper is the third and final summwy of LD5O studies of pesticides in the Shennan rat. F(fly-seven
chemicals were tested by the oral route in both seses of adults. Of these chemicals, 41 were also tested by
the dennal mute and 36 were tested by the oral mute in weanlings. Under this program the acute toxicity of
a total of 157 pesticidoi chemicals have been studied using the same methodology and the same strain of
rat. This has provided an extensive body of infomiation on the relative toxici(y of pesticides. The infomia-
lion obtained in this study may be useful in categorizing the nskfivm acute accidental exposures and in for-
mulating regulations to ensure safe handling and use of pesticidal chemicals.
LD5O values were determined for 57 pesticides administered by the oral or dermal route to adult male
and female Sherman rats. Thirty-six of the chemicals were also tested by the oral route in one sex of weani-
ings. Nine pesticides tested by the oral route (bufencarb, cacodylic acid, dialifor, deltamethrin, dicamba,
diquat, quintozene, phoxim, pyrazon) and four tested by the dermal route (bufencarb, chiordimeform,
dichiofenthion, leptophos) were more toxic to females than to males whereas famphur and 2,45-T (oral
route) were less toxic to females. Eighteen of the test chemicals were more toxic to the adult than to the
wezinhing and four compounds (leptophos, methidathion, pyrazon, and sulfoxide) were more toxic to the
wesrnling. In additional studies the variability of the LD5O value over a 1-year period was e mined for
two t ical insecticides. Six consecutive bimonthly oral LD5O determin tions for parathion and DDT in
adults of both sexes indicated that the LD5O values were little affected by the time of year that the tests
were done.
CITATION: Fundamental and Applied Toxicology 7(2): 299-308, August 1986.
E104 MS-86-018 Project Officer: Under
EIIECTS on the FETUS of MATERNAL BENOMYL EXPOSURE in
the PROTEIN-DEPRIVED RAT
Francesj. Zeman, 1 Elizabeth R Hoogenbooni, 1 RobrnJ. Kavlock 2
and JanetL Semple 1
1 University of California-Davis
2 HERL, USEPA, Research Triangle Park, NC
Standard ,isk assessment procedures generally utilize data generated from animal bioassays in which the
exposed populations are receiving optimal nutritional intakes. Situations exist in the human population,
however, in which nutritional intakes are inadequate yet pesticidal exposures high. This study examined the
effect of protein deprivation in the nit on the terafogenicity of a common fungicide. The incidence of
benomyl-inthiced central nervous system malfomiations was reduced in the protein deprived group, suWst-
ing aprotective effect of malnutrition. These results are being pursued to detennine possible mechanisms.
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The separate and combined effects of protein deprivation and benomyl [ (methyl 1- butylcarbomoyl)-2-
ben.imidazole carbamate] exposure were studied in the pregnant rat fed a diet containing 2.4% (control)
or 8% (deficient) casein throughout gestation. Within each diet group, subgroups were gavaged at 312
mg/kg body weight with benomyl or corn-oil carrier only on d 7-16 or 7-21 of gestation. No effects on the
skeleton were seen. Benomyl exposure in the last 2 wk in dams fed the 24% casein diet resulted in a high
incidence of fetal brain anomalies. This effect did not occur in those with benomyl exposure during the
period of organogenesis only and was reduced in groups fed the protein-deficient diet. Exposure to
benomyl in the last 2 wk in the protein-deprived rat resulted in a decrease in the weight of the fetal heart
in excess of that attributable to diet alone. Lungs were a smaller portion of body weight in fetuses of
benomyl-treated dams in both diet groups. The teratogenic effect on the brain in animals exposed to
benomyl in wk 2 and 3 of gestation suggests that screening for teratogenic effects during organogenesis
only may be insufficient.
CITATION: Journal of Toxicology and Environmental Health 17(4): 405-417, April 1986.
E104 MS-85-213 Project Officer: Kaviock
EFFECTS on the FETUS of MATERNAL NITROFEN EXPOSURE in
the PROTEIN-DEPRIVED RAT
Frances I. Zeman, 1 Elizabeth R. Hoogenboom, 1 Carol Chase-Deesing 1
Robert I. and Janet L Semple 1
1 University of California-Davis
USEPA, Research Triangle Park, NC
Standard nsk assessm ent procedures generally utilize data generated from animal bioassays in which the ex-
posed populations receive optimum nubitionalintaker. This paperpresents aportion of the data deiived from
a research project examining whether the teratogenici y of pesticides can be altered in populations receiving
less optimal dietary intakar. Utilivng the teratogenic hethicide nitrofen a aprotoI q,e compound and a low
protein die4 this study demonstrates a syneigism of the two insults in ternis of kidney, intestinal and pul-
monary developmenL However, these interactions occuned in only severely malnourished darns receiving a
relatively high dose of nitrofen. Further studies will examine the nature of the interaction in greater detaiL
The separate and combined effects of protein deprivation and nitrofen exposure were studied in the preg-
nant rat. Animals were fed diets containing 24,8,6 or 4% casein throughout gestation. Within each diet
group, subgroups were gavage-fed with 12.5 (lower dose) and 25 (higher dose) mg nitrofen/kg body
weight or with oil carrier only on days 7-21 of gestation. Dams were weighed and food intake was
measured daily. On day 21 of gestation, cesarean-derived pups were examined for congenital anomalies
and dissected for determination of organ weights. Skeletons were alizarin-stained and examined for
skeletal anomalies and developmental stage. No effects on the skeleton or gross congenital anomalies
were seen. Fetal size and weights of liver, kidney, intestine, heart, lung and brain were reduced with
decreasing casein content of the diet and as a result of the higher dose of nitrofen. An effect of interac-
tion between diet and nitrofen exposure was shown in the kidney, intesthe and lung weights. Specific
toxicity affecting organ size was shown to occur in the intestine and lung An interaction between diet and
nitrofen specifically affected kidney and intestine. Brain size tended to be preserved, a possible protec-
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the mechanism negated by nitrofen exposure. The data suggest also that kidney, intcslne and lung arc
particularly affected. Effects however, occur primarily in young of severely mialnourished dams receiv-
ing a relatively high dose of mtrofen.
CITATION: To.ucologj 38(1): 55-68, January1986.
E104 MS-85-214 Project Officer: Kaviock
EFFECTS on the FETAL RAT INTESTINE of MATERNAL MALNUTRITION
and EXPOSURE to NITROFEN (2,4.DICHLOROPHENYL-p-
NITROPHENYL EThER)
Soltanali Mahboob, 1 Elizabeth A Hoogenbooni 1 Roben’ 1. Kavloclç 2
and Frances I. Zeman 1
1 University of California-Davis
2I .1., USEPA, Research TriangJ Park, NC
Standard ,isk sment procedures genenzli y utilize data deiivedfroin animal bioassays in which the es-
pedmental subjects receive optimal nutritional intakes. In order to evah ate the potential effects of dietwy
insufficiencies on teratogenic effects, studies were designed to examine the effects of protein deprivation on
the developmental toxicity of the heibicide nitrofen. A previous study demonstrated an interaction between
these two treatments on postnatal viability of exposed iltiess. This study, although demonstrating mw*ed
effects of both treaIments indicates that the intestinal tract alterations are not involved with the increased
postnatal mo,tali(y.
The effects of maternal protein-energy malnutrition and exposure to nitrofen on selected aspects of in-
testinal morphology and function were studied in the fetal rat. Pregnant rats were fed, throughout gesta-
tion, diets cont ainbig 24% or 6% casein as the sole source of protein. Reduced total food intake
produced protein-energy malnutrition (PEM). Each diet group was divided in half and gavaged with
either 12.5 mg nitrofen in corn oil/kglday or corn oil carrier only from days 7 to 21 of gestation. Body
weight intestinal weight, length, and diameter were measured as were villus length (VL), villus width
(VW), and number of viii per length of intestine (VMM). Protein (horseradish peroxidase) and lipid ab-
sorption were studied histochemicaily. Lactase and dipeptidase activities were determined in proximal,
medial, and distal thirds of the intestine. Results showed that the restricted maternal diet resulted in
reduced fetal body weight (BW), intestinal weight (1W) and length (IL), reduced IW/BW and 1W/il
ratios, VH, and VMM. The W/ was reduced only in the distal third. Protein and lipid absorption were
unaffected. Lactase and dipeptidase activities were reduced. Maternal nitrofen exposure resulted in
reduced body weight, intestinal size, and lipid absorption, with some evidence of interaction with the diet
effects on enzyme activities. It is concluded that effects of maternal malnutrition were extensive, but that
nitrofen exposure, at this dosage level, is not likely to contribute to the postnatal fetal mortality rate in
either adequately nourished or malnourished rats.
CITATION: Teratogenesis, CarcinogenesLs and Mutagen ens 6(6): 45-57, January1986.
E104 MS-85-213 Project Officer: Kaviock
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ISOLATION of a PRECURSOR and a NASCENT CHAIN FORM of GLUCOSE .6-
PHOSPHATE DEHYDROGENASE from RAT UTERUS and REGUlATION of
PRECURSOR PROCESSING by ESTRADIOL
Audrey M. Qunmings and Kenneth L. Badcer
Texas Tech University Health Sciences Center
The data desciibedin thLr paper provide infomiation about the mechanLcms by which estrogen regulates the
s w*thesLc of a specific protein, glucose-6-phosphaze dehydmgenase, in ,at utems. Thu infonnation u im-
portant as baseline data on hoimone mechanisms in the female reproductive tract. In addition, the data
have specific relevance to cuirent research on the tauc effectc of e.strogenic pesticides, such as methatychlor
and kepone, on reproductive function in female iuLc.
SDS-polyacrylamide gel electrophoresis of anti-glucose-6-phosphate dehydrogenase immunoprecipi-
tales from radiolabeled uterine tissue extracts previously revealed three proteins: A, B and C, which were
tentatively identified as a 60-64 kDa precursor form, a 57 kDa predomirnint form, and a 40-42 kDa nas-
cent peptide form of the enzyme, respectively. A peptide-mapping technique was used to examine struc-
tural homologies among A, B and C. Following the labeling of uterine proteins with ( S]methonine,
labeled proteins A, B and C were isolated bX immunoprecipitation and electrophoresis. Each protein was
individually co-digested with authenti; [ ‘H1methionine-labeled glucose-6-phosphate dehydrogenase
using papain, the resulting peptides were resolved by isoelectric focusing and the peptides from the two
sources on each gel were compared using double-label counting methods. Proteins A, B and C had at
least eight peptides in common, both proteins A and C had two additional peptides in common that were
not present in protein B, and B protein had two peptides that were either absent or present in reduced
amounts in digests of proteins A and C. The extensive structural homology and immunoreactivity of these
proteins indicated that proteins A, B and C were all related to glucose-6-phosphate dehydrogenase. The
presence of two extra peptides in proteins A and C suggested that these peptides may be derived from a
common NH2-termin5ll leader sequence which was present in both the precursor and nascent peptide
chains. The presence of two peptides that were present in protein B and absent from proteins A and C
is easiest to explain if they are derived from the two ends of the molecule, with the corresponding pep-
tides in proteins A and C conts ining additional peptide sequences that are “normally removed by en-
dogenous proteolytic processing enzymes. Based on the relative time-course of synthesis of the three
glucose-6-phosphate dehydrogenase-related proteins in control and estrogen-treated uteri, it appears
that estradiol promotes an increase in the relative rate of transfer of label from protein A into B by
stimulating the rate of processing of the precursor to the predominsnt form of the enzyme and enhances
the rate of translational conversion of protein C into higher molecular weight forms.
CITATION: Biochimica et BiophysicaAcsa 880(2/3): 226-241, February 1986.
E104 MS-85-227 Project Officec Cnmmings
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MEASUREMENT of EPIDIDYMAL SPERM MOTILITY as a TEST VARIABLE in
the RAT
Ralph E. Linde ,, 1 Lillian FSbader, 2 and W. Keith McEfroy 1
1 IiEPI., USEPA, Research Triangle Park NC
2 Northrop Services, Inc.
Effects on spemi motility in common y used test species such as the rat are useful in evaluating effects on
the male reproductive s ctenL This study exwnines variables that impact on the motility assay in dose-
response studies of chemicoi toxicity. Standardization of sample collection and handling is emphasized as
a means of improving precision. Dose-related effects on sperm motility vi rats treated with din oseb (apes-
ticide) and a-chlorohyddn are presentet The technique developed in this research will improve the quan-
titative assessment of spenn motility in the rat and contribute to improved methodology in the detection of
chemicals which cause reproductive changes in the male.
Several environmental contfinlirnints, notably dibromochioropropane (Whorton Ct al., 1977) and kepone
(Taylor et aL, 1978; Cannon et al., 1978) have been implicated in sperm deficiencies among occupation-
ally exposed males. These incidents emph lci7e the need for adequate testing of chemicals for effects on
the male reproductive s tem. Although important in dinical diagnosis, the evaluation of sperm motility
has not been used extensively as a tool in chemical toxicology, particularly in commonly used small
laboratory species. Measurements of sperm motility parameters are complicated by several variables in-
cluding sample manipulation, temperature, cell concentration, time factors and the method of quantita-
tion. In the present study, a simple objective procedure to estimate the proportion of motile epididymal
spermatozoa in the rat has been developed. Emphasis is given to details of handling the sperm sample to
minimi intersample variation due to external factors. Quantitation of progressively motile spermatozoa
is achieved simply by standard manual and electronic blood cell counting techniques. The difficulty in
visual trarldng of multiple motile spermatozoa is circumvented by counting only nonmotile cells. An
index of motility is calculated from the difference in the nonniotile and total counts, each made on
separate aliquots of a sperm suspension. The procedure described is an elaboration of a technique used
by Mason and Thompson, 1977. The modified technique is demonstrated in rats given selected doses of
dinoseb (2-sec-butyl 4,6-dinitrophenol), a testicular toxicant in the rat (Under et aL, 1982) and a-
chiorohydrin (3-chloro-1,2-propanediol) whose antimotility effects have been documented in several
species (Jones, 1983).
CITATION: Bulletin of Environmental Contamination and Toxicology 36(3): 317-324, March 1986.
E104 MS-85-166 Project Officer: Linder
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NEW STANDARD GUIDE for PERFORMING the MOUSE LYMPHOMA ASSAY
for MAMMALIAN CELL MUTAGENICITY
D. Cilvç 1 W. Caspwy, 2 P.E.Iby, 3 AKrehi 1 M.Moore 4
LNovalç 5 andr.J. ObeNy, 6
1 Burroughs Welcome Company, 2 NIEHS, 3 SITEK Research Laboratories, 4 HERL, USEPA,
Research Thangle Park NC, 5 The Upjohn Company, 6 Eli Lilly & Company
This paper Li the result of an ASTM committee consensus on guidelines for perfomung the mouse
phoma assay. This assay Li one of the most wide’y used for in vitro mammalian mutagenests. The Office
of To4c Substances (OTS) receives test data in the Premanufacw4ng Notice (PMN) process. These
guidelines were developed by representatives of SLt research laboratoiies using the assay.
The purpose and scope of this standard guide is to present background material and to establish criteria
by which protocols and procedures for conducting the L5178Y!FK 3.7.2C mouse lymphoma
mutagenicity assay (MLA) can be properly understood and evaluated. These guidelines are also in-
tended to aid researchers and others to gain a better understanding of the critical elements involved with
m ammri1is n cell mutagenicity testing. More specifically, this document is intended to provide a guide to
researchers that will accomplish the following goals:
• Provide an understanding of the critical procedures (steps) involved in the performance of this mam-
nuili an cell mutagenicity test.
• Provide generalized criteria by which researchers can evaluate if they are properly performin utiliz-
ing, and interpreting this assay.
• Provide criteria by which individuals responsible for evaluating mouse lymphoma mutagenicity data
can determine if the experiments have been properly performed and interpreted.
• Provide a basis from which new procedures and developments in testing procedures can be
evaluated.
• Provide an understanding of the genetic principles involved in the dual mutational endpoint [ i.e.,
gene and chromosomal mutations affecting the thymidine kinase (TK) gene] of this m mm2lian cell
mutagenicity test.
CITATION: In: Proceedings of theAmeri can Socie(y for Testing and Mateiials, 1986.
E104 MS-86-012 Project Officer: Moore
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MECHANISMS of CARCINOGENICITY and ANTICARCINOGENICITY:
ROLE OF DIETARY COMPONENTS
ShahbegS. Sandhu and Michael D. Waters
HERL, USEPA, Research Triangle Park, NC
This is an introduction to a session of a conference on Antimutagenesis and Anticarcinogenesis held at
Kansas State University, Lawrence Kansas, October1985.
The role of nutrition in the etiology of hiim in disease, including cancer, has been recognized for a very
long time. This knowledge and, perhaps, personal preference are reflected by the choice and balance of
edibles included in the daily diet. Considerable progress has been made in identifying the cellular targets
involved in interaction between cells and initiating carcinogens and promoters. It has been evident since
the work of Miller and Miller (1969) that modification of cellular DNA (leading to mutagenesis) is an im-
portant event in initiating carcinogenesis. There are, of course, exceptions to the correlation between
mutagenicity and carcinogenicity. Some chemicals show strong mutagenicity when tested in in vitro test
systems, but they do not induce cancer in animals. Several chapters of this volume are devoted to the un-
derstanding of the mechanisms of carcinogenicity and anticarcinogenicity, as well as the role of diet or
hormones in the induction or inhibition of carcinogenesis.
CITATION: In: Proceedings of the Confrrence on Antimutagenesis andAnhicarcinogenesis, D. Shankel, P.
Hartman, and A. Hollaender, eds., Plenum Press, New York, NY, 1986.
E104 MS-86-128 Project Officer: Sandhu
NEUROTOXICOLOGY in the FETUS and CHILD -- CONFERENCE SUMMARY
Lawrence W. Reiter
HERL, USEPA, Research Triangle Park, NC
The conference Weurotoxicology of the Fetus and Child” addressed issues of 1) nonnal development of the
neri’ous system; 2) methods for evaluating chemically-induced damage to the developing nervous system;
and 3) the effects of chemical ccposures on the developing nervous system. Dr. Reiter was invited to sum-
manze the conference and this paper contains that summaly.
The stated objectives of this conference, “Neurotoxicology of the Fetus and Child”, was to present papers
and focus discussions on both clinical and experimental issues related to the effects of environmental in-
fluences on the structure and function of the developing org2nism. I will focus my summarizing remarks
on three general issues which were repeatedly emphasized throughout the conference: 1) the need for re-
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search in this area; 2) the types of research which are currently being conducted; and 3) future research
needs.
CITATION: NeUFOtOnCOIOgY 7(2): 665-670, May1986.
E104 MS-86-054 Project Officer: Reiter
ALTERED THALAMOCORTICAL AXON MORPHOLOGY FOLLOWING
NEONATAL PERIPHERAL NERVE DAMAGE:
IMPLICATIONS for the STUDY of DEVELOPMENTAL NEUROTOXICITY
Karl Jensen
HERL, USEPA, Research Triangle Park NC
Considenible evidence su ests that some substances display age-related neumto ciIy. The very young are
often most sensitive to neuroto4cants To enable EPA to predict and undentand age-dependent
neuroto4ci y, it Lc unpoltant to undeistand the consequences of disnepling major evenLc in the development
of the nervous system. This aitick reviews recent findings on the development of the somatosenso,y system
in the ut and the implications of these fin ding, for studies of developmental neumtoxici y.
Toxicant effects on central connectivity have not been extensively characterized. Studies of developmen-
tal neurotoxicity have typically described malformations arising from damage to neuronal precursors.
Toxicants may also affect later stages of neural development. In particular, they may disrupt the postna-
tal development of sensory systems. The purpose of this paper is to describe the effects of postnatal
damage to peripheral sensory nerves on the trigeminal pathway of the rodent somatosensory system.
These studies, which have demonstrated that neonatal peripheral nerve damage produces persistent al-
terations in central connections, have implications for the study of developmental neurotoxicology.
CITATION: Neurotoxicology 7(2): 169-182, May1986.
E104 MS-86-059 Project Officer: Jensen
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USING ASHFORD’S GENERAL MODEL to AID in the UNDERSTANDING of a
PESTICIDE’S NEUROTOXIC EFFECT ThROUGH PESTICIDE-DRUG
MIXTURE EXPERIMENTS
Davidi. Svendsgawd 1 and Kevin M. Crofton 2
1 }j J USEPA, Research Triangle Park NC
2 University of North Carolina-Chapel Hill
Traditionally, it has been thought that pyrethroid pesticides that cause convulsions accomplish this through
action on the sodium channel in the brain. Recently, it has been noted that certain pyrethroid pesticides
bind strongly at a site in the brain that acts on the chloride channel. The paper discusses some mathemati-
cal models and statistical methods to aid in detennining whether the sodium channel action or the chloride
channel action is correct. The models are fit to neurobehavioral activity data from rats exposed to mülures
of both types of pyrethroidA and drugs with known mechanism of action. Such information is relevant to
the Office Pesticide Fmgrwns (OFF). For example, knowledge of the correct mechanism is important
when considering which in vitro method to use for screening pyrethroid pesticides for convulsant effects.
Many changes can be measured in the brain after exposure to a neurotoxin. Some of these rJ inges are
not involved in the production of clinical symptoms of interest that these toxins cause. By learning the
mechsrnicm of action of a class of pesticides we are equipped with the necessary knowledge for determin-
ing the relevant in vitro tests for the assessment of risk to a particular disease state of interest. Pyrethroid
insecticides are potent neurotoxicants. They are dassified as being either Type I or Type II based upon
several factors: (1) in vivo toxic signs in rats (Verschoyle and Aidridge, 1980), mice (Lawrence and
Casida, 1982), and insects (Gammon et al., 1981); (2) duration of prolongation of sodium current in nerve
axons (Narahashi, 1962; Lund and Nar ih chi , 1983); and (3) interactions at the y-aminobutyric acid
(GABA) receptor complex (Gammon Ct aL, 1982; Lawrence and Casida, 1983). Structurally, the Type II
pyrethroids usually contain an u-cyanophenoxybenzyl moiety, whereas the Type I pyrethroids lack this
component. Currently, a major question is whether the observed in vivo effects of pyrethroids involve ac-
tion primarily at the axonal sodium channel or the GABA receptor complex.
CITATION: In: Proceedings of theAmeiican StatisticalAssociation, October 1985.
E104 MS-86-O01 Project Officer: Svendsgaard
IN ViTRO MODELING in NEUROTOXICOLOGY
Bellina Veronesi
HERL, USEPA, Research Triangle Park, NC
Many toxicologists in academic, industrial and governmental sectors consider in vitro (i.e., cell and tissue
culture) alternatives to animal toxicity testing as inevitable given the sheer volume of new chemicals and the
time and cost confines of conventional methodologies. For neurotoxicologists, in vitro systems ranging
from tumor cell lines (i.e., neumblastomas, gliomas, and pheochmmocytomas) to organotypic explants
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provide nzpi4 inexpensive modeLs for either screening putative neurotoxicanLv or addressing their
mechanism(s) of action. A chemical’s toxic potenli a! can best be addressed using a tier approach in which
neurotoxic asseisment proRresses from the simplej inexpensive and len specific assay (e.g., cell lines) to the
more coinple.i technicalô’ demanding and definitive assay (e.g., tissue explant). Understanding how and
when these techniques should be used either as a supplement or alternative system to intact animal toxicity
studies , can strengthen our assessment of a chemical’s newotadc hazaid.
Many toxicologists in academit; industrial and governmental sectors consider in vitro (i.e., cell and tissue
culture) alternatives to nnmiil toxicity testing as inevitable given the sheer volume of new chemicals and
the time and cost confines of conventional methodologies (Roof; 1971; Nardone, 1977; Tardiff, 1978;
Stammzitti Ct aL, 1981; Ekwall, 1983; Goldberg, 1984; Gridisam and Smith, 1984). For neurotoxicologists,
in vitro systems r’ ng ng from tumor cell lines (i.e., neuroblastomas, gliomas, and pheochromocytomas) to
organotypic explants provide rapid, inexpensive models for either screening putative neurotoxicants or
addressing their mechanism(s) of action. Ideally, a variety of methodologies are available to the inves-
tigator, since no single technique will be able to detect all possible neurotoxic end-points. Assuming this
accessibility, a chemical’s toxic potential can best be addressed using a tier approach in which neurotoxic
assessment progresses from the simple, inexpensive and less specific assay (e.g., cell lines) to the more
complex, te hnically demanding an definitive assay (e.g., tissue explant). The first tier acts as the prelimi-
nary screen for putative neurotoxicants and comments on a chemical’s non-specific toxicity, usually in
terms of cell viability. The next level of inquiry addresses the pathogenesis or mechsuiicms of injury and
is designed to describe neurotoxidtyin morphological, biochemical, and in some cases, electrophysiologi-
cal terms.
CITATION: In: A Textbook of Neurotoxicology, M.B. Abou-donia, ed, Elsevier Science Publishers, The
Netherlands, 1986.
E104 MS-86-030 Project Officer: Veronesi
AN IN VITRO COMPARISON of RAT and CHICKEN BRAIN NEUROTOXIC
ESTERASE
Roswme Novak 1 and Stephanie Fadilla 2
1 Northrop Services, Inc.
USEPA, Research Triangle Park NC
Organophosphate-induced delayed neuropathy (OPIDN), a distal axonopathy of the larger-diameter and
longer fibers of the central and penpheiul nervous systems, is known to develop after acute or chronic ex-
poswes to some oiganophosphoius (OF) esters. Studies of OPIDN in the chicken have led to the iden-
tification of a putative biochemical lesion: the inhibition and ‘aging’ of a caiboxylesterase, teimed
neurotoxic esterase or neuropathy target enzyme (NTE). The chicken is traditionally used as a model for
OPIDN. Recent studies , howeve, have demonstrated that the rat does develop a neuropathy that is
topographically and qualitatively similar to the chicken model of OPIDN in the absence of atwie. In order
to tighten the linkage between the research and regulatorj sectors of the Agency by promoting a mammalian
species, rather than an avian species for toxicity testing of OPs, a validation of the rodent model of OPIDN
was begun. F evious work by research scientists at the Neumtoxicology Division had shown that, like the
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hen, the brain and spinal coid inhibition of NTE coirelates with the incidence of neuropathic response in
OP treated TaLc. The study daccsibed in this jownal article was undertaken to investigate the sinzila,iIies be-
tween rat and chicken NTE with respect to ainoun4 inhibitor sensitivities and molecular weigliL It was
found that the NTEfioni both species was very similar with onty a few quantitative differences-a fiuther
validation of the rodent model of OPIDN.
A systematic comparison was undertaken to characterize neurotoxic esterase (NTE) from rat and chick-
en brain in terms of inhibitor sensitivities, pH optima, and molecular weights. Paraoxon titration of
phenyl valerate (PV)-hydrolyzing carboxylesterases showed that rat esterases were more sensitive than
chicken to paraoxon inhibition at concentrations  1 jr.M and superimposable with chicken esterases at
concentrations of 2.5-1000 p.M. Mipafox titration of the paraoxon-resistant esterases at a fixed paraoxon
concentration of 100 p.M (niipafox concentration: 0-1000 pM) resulted in a mipafox 150 of 7.3 pM for
chicken brain NTE and 11.6 p.M for rat brain NTE. NTE (i.e., paraoxon-resistant, mipafox-sensitive
esterase activity) comprised 80% of chicken and 60% of rat brain paraoxon-resistant activity with the
specific activity of chicken brain NTE approximately twice that of rat brain NTE. The pH maxima for
NTE from both species was mibir showing broad, slightly iIk 1ine optima from pH 7.9 to 8.6
( 3 H]Diisopropyl phosphorofluoridate (DFP)-labeled NTE from the brains of both species had an ap-
parent mol wt of 160,000 measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In con-
clusion, NTE from both species was very simibir , with the mipafox ISO for rat NTE within the range of
reported values for chicken and hiim in NTE, and the inhibitor parameters of the chicken NTE assay were
applicable for the rat NTE assay.
CITATION: Fundamental and Applied Toxicology 6(3): 464-471, April 1986.
E104 MS-85-201 Project Officer Padilla
THE CORRELATION BETWEEN NEUROTOXIC ESTERASE INHIBITION and
MIPAFOX-INDUCED NEUROPATHIC DAMAGE in RATS
Beilina Veronesi, 1 Stephanie S. Padiia, 1 and Donald Lyerly 2
1 H USEPA, Research Triangle Park NC
2 Northrop Services, Inc.
The abili(y to link a cnticalpeirent inhibition of NTE and neuropathology in the rat has been demonstrated
with another model organophosphate compoun4 Mipafat This demonstration has strengthened the
validity of the rodent model of OFIDN for the testing of putative neurotoxic compound.c.
The correlation between neuropathic damage and inhibition of neurotoxic esterase or neuropathy target
enzyme (NTE) was examined in rats acutely exposed to Mipafox (N,N’-diisopropylphos-
phorodianiidofluoridate), a neurotoxic organophospate, Brain and spinal cord NTE activities were
measured in Long-Evans male rats 1 hr post-exposure to various dosages of Mipafox (ip, 1-15 mg/kg).
These data were correlated with histologically scored cervical cord damage in a separate group of similar-
ly dosed rats sampled 14-21 days post-exposure. Those dosages ( 10 mg/kg) that inhibited mean NTE
activity in the spinal cord  73% and brain  67% of control values produced severe (  3) cervical cord
pathology in 85% of the rats. In contract, dosages of Mipafox ( 5 mg/kg) which inhibited mean NTE
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activity in spinal cord  61% and brain 60% produced this degree of cord dnm gn in only 9% of the
nim i1 . These data indicate that a critical percentage of NTE inhibition in brain and spinal cord
sampled shortly after Mipafox exposure can predict neuropathic d nii ge in rats several weeks later.
CITATION: Neurotadcology 7(1): 207-216, April 1986.
E104 MS-85-170 Project Officer: Veronesi
PHENYLMETHYLSULFONYL FLUORIDE PROTECTS RATS from
MIPAFOX-INDUCED DELAYED NEUROPATHY
Bellina Veronesi and Stephanie Padilla
HERL, USEPA, Research Triangle Park, NC
These ezpeñemenLr demonstrate that in the rat, as in more conventional animal models of organophos-
phonis tad city, the initiation of the delayed neuvopathy OPIDN Lc a multLctaged event invoMng inhibition
and aging of the target enzyme. Because of this, animaLc can be protected from the neuropathy by pretreat-
ment with non-agreeable oiganophosphonis conipounS These data strengthen the use of rats over chick-
ens as an alternative expeiimental model of OPIDN.
Initiation of organophosphorus-induced delayed neuropathy (OPIDN) is thought to consist of two
molecular events involving the phosphorylation of the target enzyme, neurotoxic esterase, or neuropathy
target enzyme (NTE), and a subsequent “ ‘g ng ’ reaction which transforms the inhibited NTE into a
charged moiety critical to the neuropathic process. Compounds that inhibit NTE but cannot age because
of their chemical structure abort this two-stage initiation process, and when administered before a
neurotoxic organophosphorus compound (OP), protect againct the neuropathy by blocking NTE’s active
site (Johnson, 1970). In support of this, we report that prior exposure to a nonaging NTE inhibitor,
phenylmethylsulfonyl fluoride (PMSF), protects rats from neurological damage after subsequent ex-
posure to a neurotoxic OP, Mipafox. Adult, male, Long-Evans rats were exposed to either PMSF (250
mg kg , sc) or to Mipafox (IS mgfkg, ip) and a time course of brain NTE inhibition and recovery was
defined. A separate group of PMSF-treated rats was exposed to Mipafox when brain NTE inhibition was
87.7 ± 2.3%. Conversely, another group of rats, pretreated with Mipafox was dosed with PMSF when
NTE inhibition was 90.2 ± 0.8%. A third group of animals , treated with PMSF, was exposed toMipafox
14 days later, when NTE activity had recovered to within 10 ± 4.2% of control amounts. Histopathologi-
cal survey (14 to 21 days post-exposure) indicated severe cervical cord damage (damage score  3) in the
following frequencies: PMSF, 0%; Mipafox, 85%; PMSF-4 hr-Mipafox, 0%; Mipafox-4 hr-PMSF, 100%;
PMSF-14 days-Mipafox 75%; controls, 0%. These data indicate that PMSF pretreatment protects rats
against Mipafox-induced neurological danvigc and that the timing of administration and order of presen-
tation are critical to this protection. These results support the hypothesis that the initiation of OPIDN is
a multistage event involving inhibition and agjng and that these stages are experimentally separable.
CITATION: Todcology andApplied Phannacologj 81(2): 258-264, November 1985.
E104 MS-85-108 Project Officer: Veronesi
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BIOCHEMICAL and NEUROPATHOLOGICAL ASSESSMENT of TRIPHENYL
PHOSPHITE in RATS
Beiina Veronesi, 1 Stephanie Padille, 1 and Dale Newland 2
USEPA, Research Triangle Park, NC
2 Northrop Services, Inc.
Tnphenylphosphite Lv an organophosphonis compound chemically related to the model OPlDNprothscing
chemica4 TOCP. When injected into chickens, TPP produces symptoms commonly associated with
OPIDN (e.g. at &da, NTE inhibition). And yet on close inspection of the neuropathological damage, TPP
produces a topography of neuropathy distinct from OPIDN in the chicken. This study demonstrates that
when TPP is injected into pwts, NTE is not inhibited and the pathology Lv clearly different from that seen
when the animaLv are injected with TOCP. These data indicate that TPF neuropathy Lv masked in the
chicken when just the conventional end-points of OPIDN are employed but clearly differentiated in the tat.
The putative neurotoxicity of the organophosphorus compound triphenyl phosphite (TPP) was examined
in Long Evans, adult male rats. Animals were exposed to two 1.0 mI/kg (1184 mg/kg) injections (sc) of
TPP spaced 1 week apart and sampled for biochemical and neuropathological eys.mination. At the time
of sampling, rats displayed dysfunctional changes induding tail rigidity, circling, and hindlimb paralysis.
Neuropathic daninge was confined to the lateral and ventral columns of all spinal levels and consisted of
myelin ellipsoids and giant axonal swelling tilled with smooth endoplastic reticulum. Walierian-like
degeneration was observed in the spinal roots, the sciatic nerve, and tibial branches. Biochemical assess-
ment of brain acetyicholinesterase (AChE) and neurotoxic esterase (NTE) activity was determined 1,4,
24, and fl hr after the second TPP treatment. Both enzyme activity concentrations were depressed max-
imally at 48 hr postexposure by 30 and 39%, respectively. Serum cholinesterase, sampled 48 hr after the
second TPP exposure was depressed by 33%. Data from this study indicated that subchronic exposure
to the organophosphite TPP results in severe neurotoxic consequences which differ from those previously
described in rats with organophosphorus-induced delayed neuropathy.
CITATION: Toxicology and Applied Phamsacology 83(2): 203-210, April 1986.
E104 MS-85-202 Project Officer: Veronesi
DIFFERENTIAL EFFECTS of FORMAMIDINE PESTICIDES on
FIXED-INTERVAL BEHAVIOR in RATS
Virginia C. Moser and Robeit C. MacPhail
HERL, USEPA, Research Triangle Park, NC
Fonnamidines represent an agriculturally and economically impoitant class of pesticide of which chior-
dimefoms Lv Widely considered the prototype agent. Using schedule-controlled perfonnance as a measureof
cogn itive function, this experiment showed that there are unpottant differences in the behavioral effects of
fonnamidines. These results su est there are differences in the neural mechanism(s) by which for-
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mwnidine.r produce behavioral effectc. As a consequence caution should be exercLced L’z labeling effectc ar
being 7onnamidine-lLke orchlordimefonn4ike”.
Chiordimeform (CDM), amitraz (AMZ), and formetanate (FMT) are members of the formamidine class
of pesticides. To date, effects on operant behavior have been determined only for CDM. This experi-
ment compared the effects of CDM, AMZ, and FMT on schedule-controlled responding. Nine male
Long-Evans rats were trained during 1-hr sessions to lever-press under a multiple fixed-interval (Fl)- !-
mm FI-5-min schedule of milk reinforcement. Dose-effect determinations for each compound ad-
minictered ip, 20 mm presession, were carried out in each subject. The dose ranges were: CDM HCI,
0.3-20 mg/kg. FMT HCI, 0.03-0.75 mg/kg. and AMZ, 5-75 mg/kg. Under baseline conditions response
rates were higher under the Fl-i-mm than under FI-5-min, and index of curvature (IOC) values (a
measure of within-interval response patterning) were generally higher under P1-5-mm. All compounds
produced dose-dependent decreases in response rate. CDM ignificantly decreased only Fl-i-mm
response rates; a simibw effect of AMZ was seen only at an intermediate dose. FMT decreased respond-
ing to the same extent in both components. CDM produced pronounced changes in the pattern of
responding in bcth components, with IOC decreased more under P1-5-mm than under FT-i- mm. AMZ
produced significant decreases in bC only under P1-5-nun. FMT did not appreciably decrease IOC in
either component. High doses of AMZ produced general signs of poor health that persisted for several
days. In addition, a greater effect on response rates and IOC in both components was obtained when
AMZ (75 mg/kg) was given more than 10 days following another dose compared to when it was given 7
days or less after another dose. Form midine pesticides produce differential effects of F! schedule-con-
trolled behavior that are in turn modulated by the parameter value of the Pb schedule.
CITATION: Toxicology and Applied Phannacology 84:315-324, October 1986.
E104 MS-85-112 Project Officer: MacPhail
MICROSPORIDIAN TAXONOMY: APPLICATION OF ELECTROPHORETIC
and IMMUNOLOGICAL TECHNIQUES
C Y. Kawanishi
HERL, USEPA, Research Triangle Park, NC
The review swnmwizes the utility of electrophoretic and invnwzological methods for identifying and class-
(fying Microsporidia. This is imponant for regulating microbial pesticides containing protozoans under
FIFPA for the following reasons: (1) The tiue tasonomic relationship of pesticide agents to similar main-
malian pathogens as unclear due to the lack of definitive criteria for classification, (2) There L v a need for
methods for detecting and identifying protozoan agents in test animatc and environmental samples for
preregistration testing. This review shows that electrophoretic and immunological parameters can be sail-
ized for registration data inteipretation.
A review of investigations utili?ing electrophoretic and immunological methods for identification and
classification of microsporidians, the group to which the first protozoan microbial pesticide belongs, in-
dicate that these methods can be successfully used to eJ ssify strains and dosely related species. Certain
immunologica] methods are also useful for genera and families. The limitations and phylogenetic range
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of usefulness of other methods are discussed and suggestions made about future studies with
Microsporida.
CITATION: 1n Proceedings of the 4th International Colloquiwn on Inveitebroie Pathology, The Nether-
lands, 1986.
E104 MS-86-109 Project Officer: Kaw inkhi
DEMONSTRATION of MULTIPLE ANTIGENIC DETERMINANTS on
MYCOPLASMA PNEUMONIAE AT ACIIMENT PROTEIN by
MONOCLONAL ANTIBODIES
P. H ’CHuang 1 Y.Huang 2 A. Collier, 1 and W. ( 1
1 University of North Carolina School of Medicine
USEPA, Research Triangle Park NC
This study cont,Ibuted to the basic knowledge of monclonal antibodies and their antigenic determinant
recognition. The development of mouse clonal antibodies Lv a major pwt of our biorational pesticides
program. It provides a more specific and quick assay to determined the microbial pesticides. In addition,
Mycoplasma pneumoniae which causes respiratoiy infection, has been used as infectivity model to study
the effect of air pollutant on diseased animals.
Distinct multiple antigenic deterniirnints of the attachment protein of Mycoplasmapnewnoniae have been
identified by limited proteolytic cleavage ncing specific monoclonal antibodies. Western blots prepared
from the gels oontslinng the cleaved fragments were probed with antiserum ag unct M. pnewnoniae or
monclonal antibodies. Five distinct bands with intact antigenic determirnrnts were detected by the an-
tiserum of which two band were each reactable with two monoclonal antibodies. A sequential binding
assay suggested that these monoclonal antibodies recognized different antigenic sites of each band.
These results demonstrate the existence of multiple antigenic sites on attachment protein and the
described procedures should prove useful for identifying those antigenic sites critical to the specific at-
tachment of M. pneumoniae.
CITATION: Journal of Infection and Immunity 50(1): 292-296, October 1985.
E104 MS-85-169 Project Officer: Huang
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IMMUNOBLOT ANALYSIS of EXOSPORE POLYPEPTIDES from SOME
ENTOMOPHIUC MICROSPORIDIA
WS.Iib 1 YS.Huag 2 CY.KawanLth i, 2 wzdWM.Broob 1
t North Carolina State University
USEPA, Research Triangle Park, NC
A basic problem with many classes of microbial pesticidal agents L v the lack of reliable indentification and
quantification techniques that are usable hi preregutration hazani evaluation testy. The Microsposidia a
protozoan group to which a registered pesticidoj agent belongs, is one such group. In thLsfeasibili(y study,
microsporidians re tested to detennine whether enough antigenic differences occwred in the surface
proteins of spores to serve as a potential basis for immunological differentiation of species.
Immunological relationships between genera and species of microsporidia were examined byimmunoblot
analysis. Exospore polypeptides from 2 Nosema spp., three Vammoipha spp., and 2 undescribed
Vaiiimnoipha-like spp. were analyzed. Gel electrophoresis and immunoblot analysis revealed that a
variety of polypcptides, most between 15,000 and 90,000 molecular weight, were present on th exospore.
The three Vainmoipha spp. were highly related scribed spp. The two Nosema spp. immunologicaliy dis-
tant from each other and from the Vainmoipha spp. Indirect evidence, however, indicated that may in-
ternal spore polypeptides present in both genera are c1miI r . Cross reactivity between exospore polypep-
tides from entomophilic microsporidia and antisera to a m mmali n microsporidium, Encephalitozoon
cuniculi was very limited. These results indicate that immunoblot analysis exospore polypeptides may be
employed to investigate the interrelatedness of microsporidian species, and that exospore polypeptides
of some rnicrosporidia are sufficiently diverse to be of immunodiagnostic value.
CITATION: Journal of Protozoolog 33(1): 14-20, January 1986.
E104 MS-85-110 Project Officer: Kawanishi
PESTICIDE POISONINGS REPORTED by FLORIDA CITRUS FIELDWORKERS
Jack Griffith, 1 Robert Duncan , 2 and Janet Konefal 2
1 lffiRl, USEPA, Research Triangle Park, NC
2 University of Mi iini(FL) School of Medicine
The Office of Pesticide Progiwns requires scientific data to support or reject the registration of pesticide
products, or the suspension or cancellation of isiing products.
In a 1981 survey of 1811 Florida citrus fleidworkers, 25 pesticide related poisoning incidents involving 29
fleidworkers were reported. Suspected poisoningc were categorized into possible and confirmed poison-
ings, and from these reports it was possible top an estimated 438 possible poisoningc and 73 con-
firmed poisonings among all citrus fleidworkers. Confirmed pesticide poisonings were developed into an
incidence rate of 34 poisonings per 10,000 permanent and semi-permanent fleidworkers. The number of
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possible and confirmed poisoningc, for all fleldworkers, was then developed into an incidence rate of 160
poisoning,c per 10,000 fieldworkers.
CITATION: Journal of Environmental Science and Health B20(6): 701-727, December 1985.
E104 MS-87-090 Project Officer: Griffith
URINARY CHLOROBENZLLATE RESIDUES in CITRUS FIELD WORKERS
Jack G,ifflth 1 and Robed C. Duncan 2
1 }ffi USEPA, Research Triangle Park, NC
2 University of M’ ’mi School of Medicine
The Office of Pesticide Programs (OFF) requires scientific data to suppoll or reject the registration of pes-
ticide products, or the suspension or cancellation of existing products.
In May1976 the U.S. Environmental Protection Agency (EPA) issued a notice of rebuttable presumption
gain t registration (RPAR) of pesticide products containing chlorobenzilate, based on a presumptive
cancer risk, in the Federal Register (1976). On June 30, 1978, the EPA published a notice of determina-
tion to conclude the RPAR process in the Federal Register (1978) and in 1979 moved to cancel all non-
citrus uses of chlorobenzilat; limiting use to citrus crops in California, Texas and Florida. At the time of
the EPA decision several methods were available for monitoring CB residues on crops and soil (George
Ct aL, 1961; Blinn and Gunther, 1963; Gordon et aL, 1963). However, methods for human exposure as-
sessment were relatively undeveloped. Subsequent to the EPA decision Brady et al. (1980) published a
method whereby urinary levels of CB analyzed as p, p’-dichlorobenzophcnone (DBP) could be deter-
mined. A study, on a small number of workers, by Levy et al. (1981) suggested that the method was
suitable for monitoring citrus field workers for CB exposure. The objective of the current study was to
apply the method developed by Brady et aL (1980) to the monitoring of a large number of CB exposed
citrus fieldworkers employed in Florida and Texas, in an effort to assess exposure levels experienced
during actual work activities.
CITATION: Bulletin of Environmental Contamination and Toxicology 35(4): 496-499, October 1985.
E104 MS-85-117 Project Officer: Griffith
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MONITORING STUDY of URINARY METABOLITES and SELECtED
SYMPTOMATOLOGY AMONG FLORIDA CITRUS WORKERS
Robeft C. Duncan t andlackGnffith 2
‘University of Mm mi School of Medicine
2 u, USEPA, Research Triangle Park, NC
The Office of Pesticide PWgmins (OPP) requires scientific data to suppon’ or reject the regisnution of pes-
ticide products, or the suspension or cancellation of tAsting products.
A cross-sectional monitoring study was made of citrus fleidworkers employed during an entire citrus
growing season in florida. A survey questionnaire was admini flered to 1,811 fle ldworkers employed as
applicators, mixer, loaders, tractor drivers, general combination workers, and pickers on 436 citrus groves
throughout Florida. The study was designed to evaluate exposure to organophosphorus pesticides by
analyzing the urine of citrus fleldworkers for dimethyl phosphate (DMP), dimethyl thiophosphate
(DMTP), dimethyl dithiophosphate (DMDTP), diethyl phosphate (DEP), diethyl thiophosphate
(DEFT), and diethyl dithiophosphate (DEDTP), and to relate the findings to the reported occurrence of
selected health symptoms associated with pesticide intoxication. Urine samples were collected for
analysis from 332 spray-season workers and 265 harvest-season workers. Measureable DEP residue
values were found in 68% of the spray season workers and 43% of the harvesters. However, with the ex-
ception of DEP and DEW values in the urine of spray-season workers, metabolite residues were low,
often at, or just above, detection limits (which were usually 0.02 ppm but occasionally rose higher, with a
few at 0.03 ppm and a very few at 0.04 ppm). Although there was clearly exposure to organophosphates
among the citrus fleidworkers, there was no apparent association between the reported health symptoms
and the relatively low levels of organophosphate metabolities found in the urine of the workers.
CITATION: Journal of To.ricolov and Environmental Health 16(3&4): 509-521, November 1985.
E104 MS-86-124 Project Officer: Griffith
COMPARISON of PLASMA CHOUNESTERASE DEPRESSION AMONG
WORKERS OCCUPATIONALLY EXPOSED to ORGANOPHOSPHORUS
PESTICIDES as REPORTED by VARIOUS STUDIES
Robot C Duncan, 1 Jack Gnffith, 2 and Janet Konefal’
‘University of Miam i School of Medicine
USEPA, Research Triangle Park, NC
The Office of Pesticide F)’ogmms (OPP) requires scient ific data to n ippo n or reject the registration of pes-
ticide products, or the suspension or cancellation of eâsting products.
A number of studies have reported on the inhibitory effects of organophosphorus pesticides (OPs) on the
enzyme cholinesterase (ChE) among agricultural workers. With the increasing use of OPs, surveys of
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blood ChE activity on exposed workers may help to identify workers at greatest risk and to provide in igI t
into the use history--e.g., mixing, loading, application, and harvesting--that might lead to a 1uwJ Tdous
situation. Although it does appear that measurements of ChE activity are valuable in worker surveillance
programs, it is difficult to interpret findings from various studies since they are dependent on the method
of assay and the emphasis is usually placed on statistical tests (i.e., p values) that depend on the number
of subjects studied. In the present paper a method is presented to compare ChE values reported in
several studies utilbing various methods and units of measurement, and to assess the impact of OP ex-
posure as a percentage of subjects with ChE values depressed below the normal limits.
CITATION: Journal of Toxicology and Environmental Health 18(1): 1-11, May 1986.
E104 MS-86-183 Project Officer. Griffith
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MEIOTIC ORIGIN of ANEUPLOIDY: AN OVERVIEW
Lorraine C. Backer and Jwnes W. Allen
HERL, USEPA, Research Triangle Park, NC
Aneuploidy arL ring at meiosis is associated with spontaneous aboiiions and various disabling syndromes of
livebom The dinical significance of thLc cytogenetic defect has become increasing1y recognize4 as
evidenced by the Agency’s Mutagenesir Guidelines and the 1985 Symposium on Aneuploidy. Ye4 veiy little
is understood about the mechanistic aspects of aneuploidy generation in ge,m cells. This chapter sum-
marizes some of the current major concepts of meiotic aneuploidy development.
Aside from its adaptive potential, germ cell aneuploidy is usually considered a “mistake’ with clinical con-
sequences. The prevalence of aneuploidy in spontaneous abortions and various disabling syndromes of
liveborn testifies to the importance of abnormal chromosome numbers arising at meiosis. While some
fraction of these conditions may be attributable to embryonic chromosomal errors, it is believed that
meiotic nondisjunction represents the primary mechani, m Apparently, meiotic trisomies arise from
nondisjuncition at metaphase I more often than at metaphase II. This is not surprising in view of the uni-
que chromosomal activities occurring in the primary meiocytes which may be vulnerable to alterations
predisposing these cells to aneuploidy. At this stage, errors in bivalent formation, crossing over, or
segregation may lead to aneuploidy. Secondary meiocyte division is imibir to mitosis and presumably is
susceptible to the same types of anomalous segregation that can lead to aneuploidy in somatic cells. The
intent of this review is to summarize and integrate some of the current major concepts of meiotic
aneuploidy development.
CITATION: In: Progress in Topics in Cytogenetic Aneuploidy Incidences and Etiology, Allen R. Liss
Publishers, New York, NY (in press).
L104 MS-86-206 Project Officer: Allen
REVIEW of LITERATURE on CHEMICAL-INDUCED ANEUPLOIDY in
MAMMALIAN MALE GERM CELLS
James W. Allen, 1 Jan C. Liang 2 Anthony V. Ca,rano, 3 and it Julian Preston 4
‘HERI., USEPA, Research Triangle Park, NC
2 University of Texas-Houston
3 Lawrence Livermore National Laboratory
4 Oak Ridge National Laboratory
Genn cell aneuploidy is a major concern in risk assessment. The Agency sponsored an International Sym-
posium in 1985 on Aneuploidy, Mechanisms, and Etiology. This review represents a survey of literature
repoltc concerning chemical-induced aneuploidy in mammalian male genn cells and recommen dations for
ftuther work It Lv a major review coordinated with, or responsive to, broadAgency effo,ts (aneuploidy sym-
pasium aneuploidy review committees, Mutagenesis Guidelines) to assess the problem of aneuploidy.
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Eighty papers published between 1970 and 1984 were evaluated for results pertaining to chemical-in-
duced aneuploidy in m mmatb n male germ cells. Diverse assays and endpoints were represented. The
assays considered to involve direct measures of aneuploidy were based upon chromosome counts in
premeiotic, meiotic, and embiyonic cells, and the male pronucleus, or upon phenotypic expression of X-
linked genetic markers. Assays in which indirect measures were interpreted as evidence for aneuploidy
included those primarily assessing chi cm frequencies, univalent frequencies, and spermatid/sperm sex
chromosome body counts.
CITATION: Mutation Research 167(1&2): IZ-137, Januaiy/March 1986.
L104 MS-85-239 Project Officer: Allen
DETECTION of CHEMICALLY INDUCED ANEUPLOIDY
with PLANT TEST SYSTEMS
Shahbeg S. Swulhu, 1 BoJdev K 7 2 and Milton I. Constwffin 3
‘HERL, USEPA, Research Tri nwJe Park NC
2 University of Nevada-P .eno
3 Phyton Technologies, Inc.
The utility of plant test systems for detecting cheinicalty-induced aneuploidy was evaluated by using papers
published in peer-review jouma&. A total of 147 papers was provided to the group by the Environmental
Mutagen Infonnation Center. Based on the ciitena established by the Gene-Tox Committee (Waters and
Auletta 1981), 22 papers were selected for in-depth review. EPA has an active program to develop and
app y sho,1-tenn bioassay: to detect and characterize chemically-induced aneuploidy. This review will
provide scienftfic infonnation in developing this program.
The utility of plant test systems for detecting chemically-induced aneuploidy was evaluated by using
papers published in peer reviewed journals. A total of 147 papers was provided to the group by the En-
vironmental Mutagen Information Center. Based on the criteria established by the Gene-Tox Commit-
tee (Waters and Auletta, 1981), 22 papers were selected for in-depth review. Only those papers listing
addition, deletion, or a lagging chromosome(s) in the meiotic or mitotic cells were included in this review.
Although most plant test systems may be developed to utilize either mitotic or meiotic cells for
cytogenetic analysis, only a few have been employed for this purpose. In this review, Alliwn cepa was
found to be the most commonly used test system. Other species used less frequently were Vicia fabe Hor-
dewn vuiqare, Sorghwn vulgare, and PennLsetum amencanum. None of the plant test systems have been
sufficiently utilized to warrant judgment for its sensitivity and specificity for detecting induced
aneuploidy A suggested protocol (or detecting chromosomal malsegregation in melotic or mitotic cells
is presented.
CITATION: Mutation Research 167(1&2): 61-69, JanuarylMarch 1986.
L104 MS-85-146 Project Officer: Sandhu
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INHIBITION of AROMATASE CYTOCHROME P.450 (ESTROGEN
SYNTHETHASE) by DERiVATIVES of a-NAPHTHOFLAVONE
James T. KelILc, Jr., 1 Stephen Nesnow, 2 and Lany E. Vickeiy 1
‘University of California-Irvine
USEPA, Research Triangle Park NC
One of the most iinpo tant enzyme systems in the mammal is the cytochrome P450 compkt This system
usually functions as a deta iflcadon system removing harmful foreign chemicals and diugc from the body.
In some instances this enzyme complex can activate chemicals to highly reactive mutegenic and car-
cinogenic forms which may cause irreversible damage. This study reports on investigation designed to
characterize one of the cytochrorne P-450 enzyme functions: the conversion of androgens to estrogens. Z8-
Benzoflavone, a mateAal similar to dietar, flavones found in citnisfiidtr, was used to study this important
enzyme complet
a-Naphthoflavone (ANF; 7,8-benzoflavone) is a potent competitive inhibitor of huninn aromatase
cytochrome P .450 [ J. T. Kellis, Jr. and L. E. Vickery, Science 225: 1032,1984]. We have further hives-
tigated inhibition of aromatase by several derivatives of ANF. Using human placental microsomes and
4OnM androstenedione as substrate, the compounds tested and their I s o values were: ANF, 0.07p.M; 2-
(2-naphthyl).41f-naphtho 1,2-b]pyran-4-one, 1.Op.M; 7,8-benzoisoflavone, 100&i.M; and 2-phenyl-4F1-
naphtho [ 1,2bjfuran, > 100p.M. These findings show the necessity of the keto group of ANF in its bind-
ing to the enzyme and the importance of size and position of substitution of the exocydlic phenyl ring.
Derivatives of ANF with hydroxyl substitution at positions 5,6,7,8,9, and 10 were also screened. 9-
Hydroxy-ANF, a known metabolite of ANF in liver microsomes, was the most effective (I s o = 20 nM).
Inhibition by 9-hydroxy-ANF was competitive, and its 14 value of 5 nM indicates a higher affinity for the
enzyme than the natural steroid subtrates-- the Km values for androstenedione and testosterone under
these conditions are 10 and 80 nM respectively. 9-I-Iydroxy-ANF also induced a change in the absorption
spectrum of the aromatase cytochrome P450 indicative of substrate displacement. Based on these data
we propose a model for the binding of 9-hydroxy-ANF in which the 7,8-benzochromone ring system of
the ANF derivatives occupies the steroid ring binding site of the enzyme.
CITATION: Biochemical Pharmacology 35(17): 2887-2891, April 198t1
L104 MS-87-092 Project Officer: Nesnow
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MUTATION and ENHANCED VIRUS TRANSFORMATION of CULTURED
HAMSTER CELLS by EXPOSURE to GASEOUS ETHYLENE OXIDE
Gecige a Hatck’ Patricia M. Conklin, Coda C Chdstansen, 1
Tarn At. Anderson, 1 Robe,? Langenbach, 2 and Stephen Nesnow 2
1 Northrop Services, Inc.
2 HER USEPA, Research Triangle Park, NC
Ethylene oxide Lv a high volume production organic intemzedkte whkh Lv use4 h i addition as feedstock
item, as a sterilization agent for phamsaceuticais and related materials. Hospital and industrial woi*ers
can be exposed to high levels of this materiaL This study determined the mutagenic and oncogenicpoten-
tial of ethylene adde using mwnnsalian cells as target cells and eqroshvg these celLs to the gaseous fonn of
ethylene ovide . These results of this study strvng{y sugest that ethylene adds Lv mutagenic and potentially
carcinogenic to mammalian cells.
Ethylene oxide is a classcal mutagen and a carcinogen based on evidence from studies in experimental
snima1c It is widely distributed in industrial, research, hospital, and food environments. In an effort to
explore the use of newly developed methods for exposing msmnn lbtn cells to gaseous or volatile
mutagens/carcinogens, Chinese hamster V79 cells were treated for 2 hr with gaseous ethylene oxide, in
sealed treatment chambers, and assayed for survival and mutagenic response by analysis of induced resis-
tance to 6-thioguanine or ouabain. Significant numbers of mutants were produced at both genetic
markers by 1 250-7,500 ppm ethylene oxide. Simi1arl primary Syrian llamcter embryo cells were treated
for 2 or 20 hr with gaseous ethylene oxide in sealed treatment chambers and subsequently assayed for sur-
vival and increased sensitivity to SA7 virus transformation. Treatment concentrations extended from toxic
to several nontoxic concentrations. After 2-hr ethylene oxide treatment at 625-2,500 ppm a cignificant en-
hancement of virus transformation was observed. At 20 hr after treatment no enhancement was observed.
Treatment of hancter cells with ethylene oxide in both bioassay systems yielded concentration-related,
quantitative results.
CITATION: Environmental MutagenesLv 8:67-76, January 1986.
L104 MS-83-101 Project Officer: Nesnow
EVALUATION of the EFFEa of AGAR on the RESULTS OBTAINED in the
L5178Y MOUSE LYMPHOMA ASSAY
Masy Meyei’ Karen Brvclç t Kay Lawrence 1 Bncce Cast4 1 and
Martha At Moore?
1 Environmental Health Research and Testin& Inc.
2 HERL, USEPA, Research Triangle Park, NC
The L5178Y mouse lymphoma assay is one of the most widely used in vitro mammalian mutagenesLv as-
says. This assay Lv particularly usefid since it can detect compounds that induce both single gene and
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chromosomal damage. This study investigated a technical aspect of the assay — the agar used for cloning
celLs - and resulted in a sigaificant technical improvement in the assay.
The L5178Y TK 4 ’ mouse lymphoma assay is widely used in mutagenicity testing Trifluorothymidine-
resistant (rFT ) mutants are quantitated following growth in agar-supplemented doning medium. In an
attempt to evaluate the effect of agar on plating efficiency, we have tested several lots of Difco Noble agar
(cat. No. 0142-01-8; normally used in this assay) and compared it with Baltimore Biological Laboratory
(BBL) agar (cat No.11849). We find that BBL agar gives a higher and less variable plating efficiency than
any of the Noble lots tested. Colonies plated in BBL agar tend to appear significantly earlier on the plates
than those doned in Noble agar. The absolute mutant number and the induced mutant frequency quan-
titated from a treated culture is generally higher in BBL compared to Noble agar. To determine if this
higher frequency is due to increased mutant recovery rather than “sneak through” of nonmutant cells, we
isolated 97 mutants from treated cultures (44 large colonies and 53 small colonies) and 69 mutants from
untreated cultures (24 large colonies and 45 small colonies) and tested them for TFF resistance. All but
one (a large colony from an untreated culture) were found to be TFTr, indicating that the mutant frequen-
cy is due to an increased mutant recovery. The spontaneous mutant frequency was quantitated for 122
untreated cultures. Showing little variation within and between experiments, the spontaneous mutant fre-
quency yielded a mean of 57.7, with a standard deviation of 14.4. Under our laboratory conditions, BBL
agar gave reliable results, and we prefer it for use in cloning L5178Y mouse lymphoma cells.
CITATION: Environmental Mutagenesis 8:727-740,1986.
L104 MS-86-006 Project Officer Moore
GENOTOXICITY STUDIES of BENZ(L)ACEANTHRYLENE
A.D. Kligennan, 1 M.M. Moore 2 G.L Ere con, 1 KH. Broclç 1
CL Doe,r, 1 J.WAllen, 2 andS. Nesnor?
1 Environmental Health Research and Testing, Inc.
2 HERL, USEPA, Research Triangle Park, NC
Benz (L)aceanthiylene is a member of a unique class of polycyclic aromatic hydrocarbons, the cyclopenta-
fused PAils. Various cyclopenta-fused PAHS have been identified as air pollutants and B(L)A has recent-
ly been found to be a component of wood smoke. In these studies B(L)A was found to be mutagenic, clas-
togenic (able to break chromosomes), and also to induce sister chromatid exchanges. These studies add to
our infomiation on the polycyclic aromatic hydrocarbons.
The genotoxicity of the cydopenta-fused polycyclic aromatic hydrocarbon, benz(L)aceanthrylene
[ B(L)A], was evaluated in vitro using the L5178Y!rK mouse lymphoma assay and in vM using the
mouse peripheral blood lymphocyte (PBL) culture system. The mutagenicity and sister chroinatid ex-
change (SCE) inducing potential of B(L)A was then compared to that of benzo(a)pyrene [ B(a)PJ. B(L)A
appeared to be slightly less mutagenic than B(a)P at the TK locus, and each compound produced both
small - and large-colony mutants indicating that they are clastogenic as well as mutagenic. Gross
chromosome aberration analysis of treated L5178Y!rK mouse lymphoma cells confirmed the clas-
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togenicity of B(L)A in vitro. In the mouse PBL system, after adminictration by gavage, B(L)A was more
cytotoxic and produced a sharper elevation in SCE frequency than B(a)P.
CITATION: Cancer Letters 31(2): 1Z3-131, May 1986.
L104 MS-86-076 Project Officer: Moore
GENOTOXICITY of GAMMA IRRADIATION in
L5178Y MOUSE LYMPHOMA CELLS
Mwiha M. Moore, 1 41 da Amtower, 2 Gwy H.S. 1 and
2
1 }f J , USEPA, Research Triangle Park NC
2 Environmental Health Research and Testing, Inc
We have been investigating the abilily of the L5178Y mouse ymphoma assay to detect and differentiate
compounds which induce single gene mutations and chromosomal mutations. Gamma imidiation Lc con-
sidered the ‘classic” agent which induces chromosome mutations. These studies conflnn onr hj uotherLc
that the assay can detect both types of damage and thus Lr impon’ant to the Office of Tauc Substances
(OTS) in evalUating test data from thu assay.
The L5178Y mouse lymphoma assay has been widely used in short-term mutagenicity testing. Research
into the types of genetic d im ge detected at the thyinidine kmnase locus indicates that the assay may be
capable of evaluating not only the potential gene mutagenicity but also the clastogenicity of a test chemi-
cal. The ability to distinguish between the two types of genetic d msaige is based upon the observation that
the mutants can be separated by colony size, using an automatic colony counter, into a’, small colonies and
A, large colonies. In addition a’ mutants appear to represent chromosomal alterations to that
chromosome 11 carrying the TK locus, while A mutants may represent snisaller-scale, single-gene inuta-
tions. The process of evaluating this hypothesis includes the testing of agents of known clastogenicity and
an evaluation of the relative proportions of the a and A mutant frequencies. Several compounds known
to be clastogens have already been shown to induce a significant proportion of o’ mutants. These include
methyl methanesulfonate, benzo(a)pyrene, 2-acetyfaminofluorene , N-methyl-N’-nitro-N-
nitrosoguanidine, and dimethylnitrosamine. A study of classic clastogenic agents would be incomplete
without evaluating either X-ray or gcmma irradiation. We report here the results of experiments analyz-
ing gross aberrations sister chromatid exch n e, and thymidine kin c gene mutation (including a and A
mutant frequency) following exposure of TK 3.7.2C mouse lymphoma cells to gammp irradiation.
CITATION: Mutation Reseanh 174(2): 149-154, June 1986.
L104 MS-86-004 Project Officer: Moore
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SURVIVAL CURVES and INCIDENCE of NEOPLASTIC and NON-NEOPL4STIC
DISEASE in SENCAR MICE
C.!. Conti N. aapp, 2 AJ.P. Klein-Szanto, 1 S. Nesnow, 3 and T. I. Slaga’
1 The University of Texas System Cancer Center
2O Ridge Associated Universities
3 HERL, USEPA, Research Triangle Park, NC
The SENCAR mouse has been used by HERL to p an extensive data base on the lumoñgenic
propmies of environmental complex mixtw s. This data base has been used to establish the “comparative
potency approach” to risk assessment with these midures which is being considered for use by EPA’s Office
of Air Quality Planning and Standards (OAQPS). This paper descnl,es background studies on spon-
taneous tumor fonnadon and swvival of this sensitive strain of mice which will provide invaluable infonna-
don for past and future studies with SENCAR mice.
The survival curves and the incidence of spontaneous diseases were studied in a population of SENCAR
mice, a stock derived by a selected breeding protocol for enhanced suseptibility to chemical car-
cinogenesis in the skin. SENCAR mice proved to be as long-lived as other mouse strains or stocks, in-
cluding one of their parental lines, Charles River CD-i. The most frequently occurring neoplasias in
SENCAR mice were lymphoma, myeloid leukemia and reticulum cell sarcoma. Other frequently occur-
ring neoplastic diseases included lung adenomas and carcinoma and InnmmaTy gland carcinoma.
However, the incidence of these tumors was not higher than the incidence in CD-i mice or other mouse
strains or stocks. A variety of non-neoplastic diseases, both inflammatory and degenerative, were also ob-
served in old mice. The most common were liver, spleen and kidney amyloidosis, pyelonephritis and
papillary necrosis. These data indicate that selective breeding for susceptibility to chemical car-
cinogenesis has not produced a concomitant increase in the incidence of spontaneous neoplastic and non-
neoplastic disease.
CITATION: Ca.’cinogenesss 6(11): 1649-1652, October 1985.
A1Ol/L104 MS-86-052 Project Officer: Nesnow
COMPARISON of the TUMORIGENIC RESPONSE of SENCAR and C57B116
MICE to BENZO(a)PYRENE and the INTEREXPERIMENTAL
VARIABILITY OVER a THREE-YEAR PERIOD
Stephen Nesnow, 1 Hinda Bergman, 1 and Thomas!. Slaga 2
1 HERL , USEPA, Research Triangle Park, NC
2 The University of Texas System Cancer Center
This paper descnbes an extensive data base on the histoñcal tumor incidence and histopathologic charac-
le,izagion of tumors found in the carcinogen sensitive SENCAR mouse. The SENCAR mouse has been used
extensively to chanictesize the tumwigenk propeities of complex mirtures and pure chemicals pailicularly
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those associ wed with automotive exhaust. The knowledge of both the range of expected tumor incidence for
standard reference cwvinogens and the range of spontaneous tumor incidence data is aucial to interpreting
test data from th&v mouse. This woi* was supported ii part by the Office ofAir Quality Planning and Stan-
danLr (OAQPS) and the Office of Toxic Substances (OTS) and Lv used in the toxicological ewthwtion of en-
vironmental chemicals.
SENCAR and C57B1.16 mice were compared for their ability to produce tumors after benzo(a)pyrene
[ B(a)P] initiation and 12-O4etradecanoylphorbol-13-acetate (TPA) promotion. SENCAR mice in-
itiated with 101 pg/mouse B(a)P and promoted with TPA (2 pglmouse, twice weekly) produced large
numbers of papillomas, whereas C57B116 mice produced none after 26 weeks of promotion. Continued
treatment of the B(a)P-initiated C57B116 mice with ‘ IPA up to 52 weeks did not induce any papillomas
nor did higher doses of B(a)P. Application of increased doses of TPA (10 pg/mouse, twice weekly) to
B(a)P-initiated C57BL/6 mice (404 pg/mouse) for 50 weeks produced few papillomas. Subst nthaI papil-
loma formation in C57B116 mice was observed after weekly treatment with B(a)P (101 pg/mouse), with
maximal production occurring at weeks 39 to 41 of treatment. In contrast, SENCAR mice treated accord-
ing to the same protocol produced an equivalent response with maximal papilloma formation occurring
12 to 1.3 weeks earlier. Therefore, C57BL16 mice exposed to B(a)P are capable of producing papillonias
under certain experimental conditions. The inter-experimental variability of B(a)P-induced (50$
pg/mouse) papilloina formation after 30 weeks of TPA promotion (2 pg/mouse, twice weekly) was ex-
amined in SENCAR mice over a 37-month period. Low statistical variation was observed in papilloma
multiplicity, papilioma incidence, or papilloma latency Male and female SENCAR mice produced equal
values in the three parameters: 4A ± 1.6 papiliomas/mouse, 87% ± 10% of the mice bearing papillomas,
and 9.6±13 weeks (time at which 10% of the mice bore papillomas). The numbers of papillomas per
mouse did not follow a Poisson distribution. These data can serve as a reference source for future coni-
parisons of mouse skin tumor initiation results between SENCAR and other strains or stocks of mice and
with other chemicals.
CITATION: Emvonrnental Health Perspectives 68: 19-25, September 1986
L104 MS-85-172 Project Officer: Nesnow
CYTOGENETIC CHARACTERIZATION of the
LS178Y TK 3.7.2C MOUSE LYMPHOMA CELL LINE
Jeffi y Sa e, Martha M. Moore , 2 Donald dive 3 and John Hozie?
t Florida Institute of Technology
USEPA, Research Triangle Pak NC
3 Burroughs Welcome Company
The mouse tymphoma assay is one of the most wide y used in viüo mammalian mutagenexis assays. In
order to interpret test data from this assay (which is often submitted to the Office of Toxic Substances for
Premanufaauiing Notices (PMNs)), it Lv necessary to understand the genetic basis for the assay These two
papers provide information which charactense the cell line and mutants induced in the assay
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The cytogenetic characterization of the L5178Y TK 3.7.2C mouse lymphoma cell line was carried out,
utilbing G-banded metaphase chromosomes, to provide a karyotypic basis for the precise delineation of
induced rearrangements in TK mutants. Band-pattern measurements were used to construct
ideograms which represent the position, number, size and sVaining intensity of the chromosome bands.
The TK 3.7.2C cell line has been shown to provide quantitation of forward mutations induced at the
autosomal thymidine kinase K) locus in this cell line. Chromosome analysis of the TK +1- 3.7.2C cell
line and derived TK mutants has become important in demonstrating that the TK FK assay may
detect and distinguish between chromosomal events and smaller, perhaps point-mutation, events in
mutant colonies.
CITATION: Mutation Research 147(5): 243-253, October 1985.
L104 MS-84-114 Project Officer: Moore
CHROMOSOME 11 ABERRATIONS in SMALL COLONY L5178Y TK
MUTANTS EARLY in THEIR CLONAL HISTORY
John Ho7Jer, 1 Jeffrey Saiiyer,’ Donald aive , 2
and Ma,Yha M. Moore 3
1 Florida Institute of Technology
2 Burroughs Welcome Company
3 HERL, USEPA, Research Triangle Park, NC
The mouse lyrnphoma assay is one of the most widely utilized in vitro mammalian mutagenesis assays. In
order to interpret test data from this assay (which is often submitted to the Office of Tozc Substances
(OTS) for Premanufacturing Notices (PMNs)), it is necessary to understand the genetic basis for the assay.
This paper provides information which characterizes the cell line and mutantc induced in the assay.
We have developed a cytogenetic technique that allows observation of chromosome rearrangements as-
sociated with TK mutagenesis of the L5178Y!FK 3.7.2C cell line early in mutant clonal history. For
a series of mutagenic treatments we show that the major proportion (93%) of small-colony (or) mutants
studied have chromosome 11 rearrangements (the chromosome conUlining the thymidine kinace gene)
while large-colony (k) mutants do not have detectable chromosome rearrangements. In addition, we find
among the chromosome abnornui!ities in a mutants a significant proportion (34%) with dicentric
chromosomes involving chromosome 11. These potentially unstable chromosome rearrangements may
help to explain the karyotypic instability and heterogeneity among chromosome 11 aberrations previously
noted in a mutants when they are analyzed later in their clonal history
CITATION: Mutation Research 147(5): 237-242, October 1985.
L104 MS-84-115 Project Officer: Moore
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METABOLISM of BENZO(a)PYRENE in MONOLAYER CULTURES of
HUMAN BRONCHIAL EPITHELIAL CELLS from a SERIES of DONORS
I. M. Siegfrie4 1 K Rudo,’ B. 1. Bryan4 1 S. Ellis, 1 M. I. Mass, 2 andS. Nesnow 2
1 Environmental Health Research and Testing, Inc.
USEPA, Research Triangle Parlç NC
This lnanuscnpt describes the metabolism of the environmental carcinogen benzo(a)pyrene (BP) in cell
cultures of bronchial epithelium from humans. Lwge inte,individual vanations in metabolic profiles and
metabolic razes were found which did not co,relate with the cytoto ci y of BP to the cultured cells. The
paper is relevant to Fmgnun Office needs on metabolism of carcinogenic xenobiotics.
Benzo(a)pyrene [ B(a)P] metabolism was measured in monolayer cultures of hlInl2n bronchial epithelial
cells derived from 18 specimens of explanted tissue. Bronchial epithelial cells converted B(a)P to
dihydrodiols, phenols, quinone derivatives, and polyhydroxylated forms. Sulfate and glucuronide con-
jugates of B(a)P metabolites were also detected. Both total metabolism and distribution of metabolites
showed a 10-fold or greater variation in cultures from different specimens. When the data were divided
according to smoking status, however, no differences in total metabolism, extent of conjugation, or dis-
tribution of metabolites could be demonstrated between the two groups. Wide variation (over 1000-fold)
in the cytotoxicity of B(a)P towards cells derived from different specimens was demonstrated but could
not be directly correlated to the extent of metabolic activation. The results suggest that human bronchial
epitheial cells which are newly grown from explanted tissue of smokers in culture do not demonstrate en-
zymatic induction. Variation among individuals observed in these studies probably represents basal dif-
ferences in metabolic capability.
CITATION: Can cer Research 46: 4368-4371, September 1986.
L104 MS-86-081 Project Officer: Mass
PRENEOPLASTIC TRANSFORMATION of RESPIRATORY EPITIIELIAL
CELLS by COMPLEX ORGANIC MIXTURES in a CLONAL ASSAY
M.L Mass, 1 C.M.Marr, jjj,2 and J.LMumfod
USEPA, Research Triangle Park NC
2 Environmcntal Health Research and Testing, Inc.
The paper describes the use of an in vitro rat tracheal cell transfomwiion assay to assess carcinogenici(y of
complex mirtures from heating fuel emissions. The assay was able to detect carcinogenic activi(y in three
fuel exhaust e .xtiucts but the relative potencies of the racts could not be detennined because the complex
mixtures were not complete y soluble. The assay is one tool that might be used in assessing health nsks of
combustion products.
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In this study complex organic mixtures that were extracts of particulate emissions from 3 heating sources
were tested for the presence of agents that induce preneoplastic transformation of rat tracheal epkhelial
(RTE) cells in an in vi t o clonal assay. The samples were derived from homes in Xuan Wei County, China
where extremely high incidences of lung cancer are found 4-fold in excess of the national average. In
some communes, the cancer mortality rates are as high as 19 to 30 times the national average rate. This
is postulated to result from inhalation exposures produced by the routine burning of a coal in an
open indoor pit as the sole source of heat and cooking fuel under unvented conditions. Dichloromethane
extracts of particulate samples (<10 ta in size) of “smokef coal obtained from the respirable air in
homes burning this fuel were tested in the RTE cell transformation assay. In addition, the response was
compared to that obtained from particulate emissions of 2 other fuel types used in communes in China
that have low associated lung cancer incidences communes that use ½mokeless’ coal, and wood as fuels.
These samples were also analyzed by gas chromatography/mass spectrometry for polynuclear aromatic
hydrocarbon (PAIl) content. The organic extracts of smokey coal emissions produced significant in-
creases in the number of transformed R I ’S cells exposed in vito, in a reproducible Tn anner. Extracts of
smokeless coal exhaust and woodsmoke exhaust produced variable levels of transformation but were
definitely positive in this assay. The difficulty in reproducibility is assumed to result from the insoluble
nature of these complex organic mixtures in the culture medium. The chemical analyses of selected PAH
in the 2 types of coal were quite cintilar , but very different from woodsmoke exhaust extract. The results
are discussed in relation to the level of particulate emissions, the profile of selected PAH and the cancer
incidences in the respective homes burning these fuels.
CITATION: 1n Health and Environmental Research on Complex Ozganic Mitures, in press
L104 MS-86-011 Project Officer: Mass
QUANTITATIVE ANALYSIS of the METABOUSM of BENZO(a)PYRENE by
TRANSFORMABLE C3H1OT1/2CL8 MOUSE EMBRYO FIBROBLASTS
Kenneth Rudo, 1 Scott E R i c 1 BJ. 3 41 ICayLa-eence 2
Gayneile Cbnfr, Helen Gadan4 2 and Stephen Nesnow 2
t Environmental Health Research and Testing, Inc.
2 HERL., USEPA, Research Triangle Park, NC
One of the ma/or short-term bioassays which predict animal carcinogenicfty is the C3H1OT1F2CL8 mouse
embiyo transfonnation bioassay. This assay Li used by the Office of Pesticides and Toxic Substances
(OPTS) to explore the genotoxic potentials of chemicals. This study frl4v characterizes the metabolic
capacig of these cells for future use of this bioassay system.
The metabolism of benzo(a)pyrene [ B(a)PI to organic soluble and water soluble metaboiltes by transfor-
mable C3HLOTII2CL8 mouse embryo fibroblasts was studied as a function of time, B(a)P concentration,
and cell density. The total formation of organic-soluble and water-soluble metabolites increased with in-
cubation time from 4 to 48 h and with B(a)P concentration from 4 to 40 j iM. As cell density increased,
the metabolic rate decreased for organic-soluble and water-soluble products between 6,300 and 54,000
cells/cm 2 probably due to decreases in B(a)P concentrations to values below saturation. Specific organic-
soluble metabolites identified were B(a)P-pre-9,10-diols, B(a)P-9,10-diol, B(a)P-7,8-diol, B(a)P-3,6-
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quinone, B(a)P-3-phenol, and B(a)P-9-phenol. Water-soluble metabolites were subjected to enzymatic
hydrolysis with frglucuronidase and aryL sulfatase to identify specific conjugated products. The sulfate
conjugated metabolites identified were B(a)P-7,8-diol, B(a)P-pre-9,1O-diols, B(a)P-9,10-diol, and B(a)P-
3,6-quinone. The p-glucuronic acid metabolites identified were B(a)P-pre-9,1O-diols, B(a)P-3,6-quinone,
and B(a)P-3-phenol. Patterns of metabolite formation rates are discussed as to their possible effect on
morphological transformation rates in C3H1(ff1t2 cells with respect to incubation time and cell density.
CITATION: TeratogenesLv Cwruiogenesis, and Mutagenesis 6(4): 307-319, August 1986.
L104 MS-85-178 Project Officer: Nesnow
INVESTIGATJON of POSSIBLE AGE EFFECTS on MEIOTIC CHROMOSOMAL
RECOMBINATION and SEGREGATION in ARMENIAN HAMSTER
SPERMATOCYTES
lamer W. Allen and Carolyn W. Gwallney
HERI, USEPA, Research Triangle Park, NC
Aneuploidy us germ celLs is of particular concern for its heritable clinical consequences Age effects upon
meiotic chrornosomal recoinbination and segregation have been shown to be important in female
aneuploidy arid are suspect in male aneuploidy The present study has attempted to define such age effects
in male Annenian hamsters. These animals are being used for various mechanistic studies concerning age
and nwtagen influences to result in aneuploidy induction. This research area irrelevant to program offices
concerned with risk assessmen4 in particular, tatsc chemical effects and heritable mutagenesis.
Male Armenian hamsters were evaluated for age effects upon meiotic reconibination and aneuploidy in-
cidence. Primary spermatocytes from young and old animals revealed shnili r chiasma frequencies. The
incidence of termirnal-type chia cm2ta in sex bivalents was also measured and found to be unaffected by
age. Percentages of urnvalents and of hyperploid metaphase II cells were evaluated by chromosome
groups based upon individual chromosome morphologies. Regardless of age, the larger dichiasm te
chromosomes were seldom observed as univalents, or as extra chromosomes in hyperploid cells. The
monochiasmate ni Il autosomes and sex chromosomes revealed univalent frequencies approximating to
55-63% in animals from both age groups. However, only the small autosomes appeared to be sig-
nificantly implicated in hyperploidy. Although still a rare event, metaphase II hyperploidy was sig-
nificantly increased in older inim L (0.9% incidence in young and 2.4% in old h ini ters). The events
leading to higher aneuploidy levels in older male Armenian hamsters are not clear. A role for reduced
meiotic recombination as a function of age cannot be ruled out in the present study. However, there was
no support for his hypothesis, and consideration of other possible mechsini mc of age effects to increase
aneuploidy levels is indicated.
CiTATION: Cytobios 43(lfl): 225.23Z, December 1985.
L104 MS-85-238 Project Officer: Allen
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CONSERVED CHROMOSOMAL POSITIONS of DUAL DOMAINS of the ETS
PROTOONCOGENE in CATS, MICE and HUMANS
Dennis K Watson,’ MwyJ. McWilliams-Smi& Roger Reeves, 3
John Geadw,t 3 Michael F. Nunn, 4 Williwn Nash, 1 John R. Fowk jjj,5
Peter Duecbeig 4 Takis £ Papas,’ and Stephen J. O’Bnen 1
1 National Cancer Institute
2 National Institute of Allergy and Infectious Diseases
3 The Johns Hopkins University Hospital
4 University of California-Berkeley
5 OHR, USEPA, Washington, DC
Transforming genes, or protooncogenes, are cellular cancer genes that may play an important role in
Iwnorigenesis. There we a limited number of them described to date, and each exists in a number of th7 .
ferent species including humans. This paper contributes to the knowledge of the chromosomal location of
the eta cancer gene. Knowledge of the chromosomal location of cancer genes in conjunction with infonna-
tion about metabolic function and possible role in carcinogen esis L essential for understanding
mechanisms of cheniical y induced carcinogenesis and for assessing risk. Risk assessments based on
molecular understanding of carcinogenesis will enable better regulatory decision-making under 7 Ci4 (e.g.,
Section 6) and FIFRA (e.g., Section 3).
The nA1nn l1hln protooncogene homologue of the avian v-est sequence from the E26 retrovirus consists
of two sequentially distinct dom iins located on different chromosomes. Using somatic cell hybrid panels,
we have mapped the niammaliam homologue of the 5’ v-etc-domain to chromosome 11 (ETS1) in man, to
chromosome 9 (ETS-1) in mouse, and to chromosome Dl (ETS1) in the domestic cat. The m imm ilian
homologue of the 3’ v-etc domain was similsiiy mapped to human chromosome 21 (ETS2), to mouse
chromosome 16 (ETS -2), and to feline chromosome C2 (ETS2). Both protooncogenes fell in syntenic
groups of homologous linked loci that were conserved among the three species. The occurrence of two
distinct functional protooncogenes and their conservation of linkage positions in the three muimm i1iiin or-
ders indicate that these two genes have been separate since before the evolutionary divergence of main-
mais.
CITATION: Proceedings of the NadonalAcademy of Science 83: 1792-1794 March 1986.
L104 MS-86-218 Project Officer: Fowle
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MORPHOLOGICAL TRANSFORMATION and CHROMOSOME DAMAGE by
AMSACRINE in C3H/1OT1/2 CLONE 8 CELLS
Lynnette R. Ferguson, 1 Pienv van Ziji, 1 and Stephen Nesnow 2
1 University of Aucirband School of Medicine New Zealand
USEPA, Research Thangle Park, NC
Mo phological t,wLcfonnation of mwnmalian cells Lv a process which Lv close allied to the tumosigenic
process in mammals. The study of the effects of chemicals in moiphological tranfonnation bioassays can
give insight into the processes by which the chemical induces tumo’ in animals. This study exwnines the
effects of amsacrine a unique anti turn or agent which has stnictural similanties to some industñal organics,
in a mouse cell moiphological transfonnasion system. This study finds that amsac,ine may be a potential
carcinogen and may be deleterious to mammalian cells.
Morphological transformation, cell survival, chromosoinal aberrations and micronuclci were measured
in C3H/1O1aCLS cells after 24 h exposure to s’n c amne. A weak but dose-related increase in the percent-
age of dishes contkining transformed foci occurred. As previously reported for alkylating agents, this ef-
fect was increased by treating 5 days instead of lday after plating There was no evidence for gene muta-
tion at the Na/K ATPase locus, although amsacrine induced inicronuclei in a large percentage of cells and
chromosomal aberrations, including interclu nge events and double minute chromosomes, in dividing
cells. It would appear that transformation and chromosomal events may be related in mncacrine-treated
C3H/1OIaCLS cells. The results strongly suggest that amsacrine has carcinogenic potential, possibly re-
lated to its chromosome -bre iking properties.
CITATION: Mutation Research 170:133-143, April 1986.
L104 MS-87-091 Project Officer: Nesnow
SOME PHARMACOKINETIC and METABOLIC FACTORS AFFECTING the
NEONATAL TOXICITY of HALOGENATEI) HYDROCARBONS FOUND in the
GREAT LAKES
ICh* T. Kltchin 1 and 2
‘HERL, USEPA, Research Triangle Park, NC
2 University of Ottawa, Canada
The Great Lakes ecosystem is contaminated with many lipid soluble compounds. The neonatal to. colorj
of ten of these compounds, eight chlorinated benzenes, hexachlorobutadiene and octachloros1yrene are
presented in this book chapter. Emphasis Lv placed on phannacokinetic and metabolic factors which in-
fluence thefr biological effects. Where data penni4 human poisoning episodes, environmental levels and
human body burdens are dLccussetL
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ndustri iIii tion has resulted in the emission of a wide variety of organic chemicals into the biosphere.
Despite the orig nfiI source of the organic contaminant, these chemicals have managed to enter drinking
water supplies and bioacculute in food ch aini . Of particular interest are the organic cont minsants which
have been identified in fish and wildlife in the Great Lakes Basin. As this waterway system functions as
an important source of drinking water for North Americans, concern has been raised with respect to
toxicity and bioaccumulation of these chemicals, this chapter will focus on just ten chemicals, principally
chlorinated benzenes, octachiorostyrene and hexachiorobutadiene. The adult, fetal and neonatal
toxicities of these compounds are discussed with emphasis on metabolic and pharmacokinetic factors
which influence them.
CITATION: 1n To4cologic and Phwmacologic Principles in Pediatrics, Sam Kacew and Simon Lock
eds., Hemisphere Publiching Co., (in press).
L104 MS-86-240 Project Officer: Kitchin
CHANGES in ACCESSIBILITY of DNA to VARIOUS FLUOROCHROMES
DURING SPERMATOGENESIS
Donald P. Evenson
South Dakota State University
In this sWd spemzatogenic cells from the mouse testis were stained with various DNA specific
fluorochromes. The cells vere then analysed by flow cytomeby for differences in staining charactenstics.
This method gives a profile of the relative number of cells of each spennatogenic cell type and has been
used successfully fr i quantifying and characterizing tadcant-induced lesions in the testis. The present study
demonstrates the relative advantages and disadvantages of each of the dyes testeS These results are impor-
tant to the program offices in the area of methods applications in reproductive toxicology.
Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orang; propidium
iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4’6-diamidino-2-phenylindole
(DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permealized
cells were either stained directly or after pretreatment with 0.06 N HC1. For histone-cont ining
tetraploid, diploid, and round spermatid cells, HCI extraction of nuclear proteins caused an approximate-
ly sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For
these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, cHip-
ticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6-
to 4.0-fold after HC1 treatment. In harp contrast, HCI treatment of vas sperm did not increase the stain-
ing level of 7-ainino-AMD, DAPI, or propidium iodide but did increase the staining level for the other
intercalating dyes (1.3- to 13-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain
a mixture of protein types including histones, transition proteins, and protamines demonstrated the
greatest variability of stnining with respect to type of stain and effect of acid extraction of proteins. In
general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a
decreased level of stainability relative to the same unit DNA content of diploid cells. The observed dif-
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ferential staining is discussed in the conte of chrovnatin alterations related to the unique events of
meiosis and protein displacement and replacement during sperm differentiation.
CITATION: Cytometsy 7:45-53, January 1986.
L104 XX-85-085 Project Officer: Darney
MOUSE TESTICULAR and SPERM CELL DEVELOPMENT CHARACTERIZED
from BIRTH to ADULTHOOD by DUAL PARAMETER FLOW CYFOMETRY
Frank C Janca Loma K .Tos4 and Donald F. Evenson
South Dakota State University
In this study, cellular changes which occurthuing the development of the mouse testis were quantified using
flow cyto?netiy. The results are important to the program offices in that they establish baseline data forfu-
ture testing of to4cant-induced changes in or an’est of testicular development. In addition, this study
demonstrates that the first speim populations to wise in the testis are somewhat abnormal when compared
to those in adults. This finding may be important in risk assessment since it su ests that young wthnatc
(humans?) may differ fivm adults in their response to reproductive insult.
Dual parameter flow cytometry was used to investigate cellular changes in male germinal tissue during
normal postpartum maturation in B6C3Fi/J mice. Anininic were killed at 2-day intervals from 2 to 42 days
postpartum and at 48,64,72,93 and 100 days postpartum. Testicular, cauda epididymis and vas deferens
cell suspensions were stained with the metachromatic fluorochrome acridine orange and measured by
flow cytometry for red and green fluorescence levels after excitation by blue laser light. Intensities of red
and green fluorescence reflect amounts of single- and double-strand nucleic add sites available for
acridine orange staining, respectively, and were used to classify cells on the basis of ploidy level, RNA
content, and chromatin structure, as defined by susceptibility to add denaturation of DNA in situ. Sperm
from cauda epididymis and vas deferens were crammed by light microscopy to determine frequency of
abnormal sperm head morphology, fluorescence data derived from acridine orange-stained testicular
cells quantified the sequential changes in 1) proportions of haploid, diploid and tetraploid cell types
during the first round of spermatogenesis, and 2) proportions of round, elongating, and elongated sper-
matids during the first round of spermiogenesis. Ratios of the three major testicular populations
(haploid, diploid, and tetraploid) reached adult levels by 48 days postpartum. Sperm cells were first
detected in the cauda epididymis and vas deferens on 30 and 36 days postpartum, respectively. Early
sperm populations, compared to adult sperm, exhibited up to 89% abnormalities in sperm head morphol-
ogy that correlated with significant levels of abnormal chromatin structure. Percentage of sperm head ab-
normalities and chromatin structure in the cauda epididymis and vas deferens approached normal adult
levels by 42 and 48 days postpartum, respectively.
CITATION: Biology of Reproduction 34:613-623, May 1986.
L104 XX-85-118 Project Officer: Darney
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CO-CULTURE of RAT EMBRYOS and HEPATOCYI’ES: IN VITRO
DETECTION of a PROTERATOGEN
Linda A. Oglesby, 1 Mwian T. Ebron, 2 Patricia E. Beyer, 1
Brenda D. Carver 2 and Robe tJ. Kavlock 2
1 Northrop Services, Inc.
USEPA, Research Triangle Park NC
The study of chemical teratogenicily and subsequent interspecies hazard extrapolation Lc, at times, troubled
by the wide variations in sensitivity to teratogenicity that one sees across species (e.g., thalidomide). These
species differences must be due either to inherent differences in the response of the various embryos to
chemicals or to differences in the types of metabolites to which the embryos are exposeS This paper reports
on the development of an in vitro system which utilizes the whole embryo cu tm lure technique in combination
with a metabolic activating system (primary hepatocytes) from a heterologous species in an effort to deter’
mine the contribution of the latter possibility to interspecies variations in response to teratogens. By com-
bining for example, human hepatocytes in culture with rat embryos, it may be possibk to determine
whether a unique, bioactive metabolite of thalidomide Lv produced by the human which affects develop-
ment regardless of the species of the emb ry4 or whether the rodent embryo does not possess the potentially
harmful “thalidomide receptor.”
The technique of whole embryo culture developed by New [ Environ Health Per’spect. 18:105-110, 1976]
provides a sensitive assay to evaluate the effects of a test chemical on embryo development independent
of maternal influences. To detect proteratogens, this assay must be coupled with an exogenous metabo-
lic activation system. We have developed methods for the co-cultivation of rat embryos with primary
hepatocytes, which offers several advantages over subcellular fractions when providing metabolic activa-
tion for fr i vit,o assays. In the present study, rat embryos removed from the dam on day 10 of pregnancy
were co-cultivated in vitro with primary cultures of rat, rabbit, or hamster hepatocytes. Embryos co-cul-
tivated with hepatocytes developed normally, as did embryos exposed to a test chemical, cyclophos-
phamide (CP) in the absence of hepatocytes. When embryos were co-cultivated with hepatocytes and ex-
posed to CP, a dose-related embryotoxicity was observed, indicating metabolic activation of the
proteratogen. Using hepatocytes isolated from rats pretreated in vivo with phenobarbital, we observed an
increase in CP-induced malformations and embryotoxicity compared to those of embryos exposed to CP
in the presence of uninduced hepatocytes. The teratogenic bioactivation of CP was inhibited in vitro by
the addition of metyrapone. When siniib r numbers of hepatocytes were used for metabolic activation of
CP the induced embryotoxicity was greater in the presence of rabbit and h nister hepatocytes than with
rat hepatocytes. Development of procedures for the culture of rat embryos with hepatocytes from other
species.
CITATION: Teratogenesis, Cwvinogenesi and Mutagenesis 6(2): 129-138, March 1986.
L104 MS-85-080 Project Officer: Kavlock
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PRENATAL EXPOSURE to the FUNGICIDE DINOCAP CAUSES BEHAVIORAL
TORTICOLLIS, BALLOONING and CLEFT PALATE in MICE, but NOT
RATS or HAMSTERS
L Earl Gray, Jr 1 1 John M. Roge 1 Robert I. Kavloclç 1
Joseph S. Ostby, Janet M. Ferrel4 and Kairina L Gray
1 HERI, USEPA, Research Triangle Park, NC
2 Northrop Services Inc.
This study on dinocap was conducted at the request of the program office in support of their evaluation of
the teratogenici y of this compowuL This study s the fli rt in the literature to demonstrate that dinocap
induced malformations in mice. The neurological and behavioral abnonnalities produced by dinocap have
never been reported before as a consequence of a prenatal tauc insult.
The present study is an evaluation of the developmental toxicity of dinocap in three rodent species ‘ icing
an in W v teratology screen. Our protocol uses postnatal viability, weight gain, and morphological and
behavioral development through wennng to assess the developmental toxicity of compounds. Dinocap
adminktered orally on days 7 to 16 of gestation to the mouse resulted in increased postnatal mor-
talityat25mgfkgld(80%inblockl and40%inblock2). Manyofthetreatedpupsthatdiedduringthc
neonatal period were wbalioonedu and had cleft palates. Although there was no treatment related mor-
tality in the 12 mg/kg/d dosage group, 6% (14t226) of these mice and 24% (23/96) of the survivors from
the 25 mg/kg/d dosage group displayed torticollis (a twisting of the neck resulting in an abnormal tilting
of the head). These tilted-head mice held the head and forepart of the body tilted constantly to one side,
both when resting and wnlking The tilt was in either direction but was always constant for a given animal;
in different mice, the angle varied considerably from almost 0 to 30°. Some mice circled repeatedly in one
direction in the home cage, others bobbed their heads and did back-ifips, while others rolled over, always
rolling in the same direction. In the hamster, developmental toxicity was seen at (100 and 200 mg/kg/d)
or near (50 mg/kg/d) maternally toxic doses but no behavioral alterations were noted and none of the pups
were ballooned.
CITATION: Teratogenesis, Carcinogenesis and Mutagenesis 6(1): 33-43, January 1986.
L104 MS-85- 196 Project Officer: Gray
POSTNATAL ALTERATIONS in DEVELOPMENT RESULTING from
PRENATAL EXPOSURE to PESTICIDES
R.J.Kavlocl J.M.Rogers, N. Chemoff, and L. E. Gray
HERL., USEPA, Research Triangle Park, NC
This article reviews research done hi the Developmental Biology Division relating to postnatal manifesta-
tions of prenatally induced teratogenic outcomes, especially as they relate to the effects of pesticides on
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developing oiganisms. It presents an ai ument for the eventual inclusion of a postnatal component to a
standa,d teratology bioassay and given suppoitto theAgency’s guidelines for developmental tadcity.
Alterations in the developmental processes of embryos resulting from exposure to chemicals are not
limited to morphological abnormalities that can be observed in the near term fetus. In our research on
the developmental toxicology of pesticides we have noted morphological defects (cataracts, ablation of
Harderian gland development, malformation of the otoliths) in postnatal offspring exposed prenatally
that could not be detected in fetal e aminstions because of the immature nature of these structures at that
stage of development. In addition, other effects on physiological functions have been detected
(locomotor hyperactivity, and decreased renal concentrating ability) that have no obvious morphological
basis. Thus, a postnatal component of a standard teratology bioassay can yield critical new information
in the overall assessment of developmental toxicity.
CITATION: In: Proceedings of the Congress of Pesticide Chemistiy, Ottawa, Canada, August 10-15, 1986.
L104 MS-86-208 Project Officer: Kavlock
AN EVALUATION of FIGURE-EIGHT MAZE ACTIVITY and GENERAL
BEHAVIORAL DEVELOPMENT FOLLOWING PRENATAL EXPOSURE to
FORTY CHEMICALS:E1cFECTS of CYTOSINE ARABINOSIDE, DINOCAP,
NITROFEN, and VITAMIN A
L E. Gray, Jr., R.J. Kavloclc I. S. Ostby, J. M. Ferrel4
/. M. Rogers, and K Gray
HERL, USEPA, Research Triangle Park, NC
Prenatal exposure to thugs and xenobiotics (methylmercury) can result in abnormal human behavioral
development. However, test protocols to screen chemicals for such toxicity have not been developed an4
for this reason, a postnatal behavioral assessment of developmental toxicity is mre y conducteL The
present study is an effoif to develop such a protocol and reports results on over 40 treatments.
Although it has been widely recognized that the developing organism is uniquely susceptible to some
chemicals and diseases, the science of developmental toxicity is in its infancy. Presently, it is recognized
that chemicals should be tested for their ability to produce defects during the prenatal period. This
awareness arose as a consequence of the thalidomide-induced birth defects in children in the 1950’s.
After this incident protocols were developed to examine the potential of a chemical to cause abnor-
malities in experimental inimak (Workd Health Organi7ation 1967). In these studies, pregnant animalc ,
usually rodents, are exposed to the chemical, and their fetuses are examined near term for skeletal and
soft-tissue defects. Limiting the examination to a gross morphological evaluation of the fetus is of con-
cern, because it is becoming increasingly apparent that prenatal exposure to chemicals may occasionally
cause functional deficits without producing malformations in the offspring. Such effects have been ob-
served in studies using rodents which have evaluated postnatal behavioral (Vorhees et al., 1979; Olson et
al., 1980a,b; Osborne et aL, 1980; Nelson et aL, 1980, 1981; Abel, 1980; Schalock et al., 1981; Gray et al.,
1981; Rodler, 1976; Zagon et aL, 1979), and reproductive development (Walker, 1981; McLachlan et al,
1985). In addition, the morphological developmental landmarks such as eye opening (Gray et al., 1982,
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1983), incisor eruption, and vaginal opening, which are regulated by diverse physiological processess, can
also be altered by prenatal exposure to a chemical (Gray and Kavlock, 1984).
CITATION: Neurotoxicology 7(2): 449-462, May 1986.
L104 MS-86-050 Project Officer: Gray
EARLY BIOCHEMICAL DETECTION of ADVERSE EFFECTS of a
NEUROBEHAVIORAL TERATOGEN: INFLUENCE of PRENATAL
METHYLMERCURY EXPOSURE on ORNITHINE DECARBOXYLkSE in
BRAIN and OThER TISSUES of FETAL and NEONATAL RAT
Theodore A. Slotldn, 1 Steven Pach,nan, 1 Robe,tJ. and Jorge Bwtolome 1
1 Duke University Medical Center
USEPA, Research Trianaje Park, NC
Omithine decaiborjlase (ODC) is an enzymatic regulator of polywnines. Polywnine biosynthesis has been
closely associated with the abili(y to synthesize macromolecules (e.g., DNA, RNA and protein). To er-
amine whether this enzyme could be used as a marker for chemically induced alterations in developmen4
rats were &posed prenatally to the pmtot e teratogen methybnercwy. Analysis of multiple tissues at
vwious time points of development pointed to a specific effect of this chemical on the development of the
cerebellum. The methylmercuiy-induced alterations Li ODC activhy were found at earlier times and at
lower doses than previously repo?feS The results indicate that this enzyme may be a wnple yet highly sen-
sitive indicator of orgwz-spec ftc developmental toxicity, thus perhaps allowing it to be utilized as a
biomarker of altered organ development.
Ornithine decarboxylase (ODC), an enzymatic regulator of macromolecule synthesis, has proven useful
as a biochemical marker for teratologic events. Daily adminktration of methylmercury (03 or 1 mg/kg
s.c.) to pregnant rats during the second and third trimesters had a profound effect on ODC in whole fetus
that was detectable as early as 13 days of gestation. Levels of enzyme activity in fetal brain also showed a
marked increase centered about gestational day 17 as well as a significant elevation during early postna-
tal life; in the latter case, the cerebellum appeared to be a major target for methylmercury-induced aber-
rations. These effects were accompanied by little or no alteration in general growth rate, brain weights,
or weights of other tissues (liver, heart, lung). Furthermore, no other tissue displayed such dramatic ef-
fects on ODC activity. Lowering the dose of methylmercuryby an order of magnitude (0.05 to 0.1 mg/kg),
levels which are associated with almost purely neurobehavioral effects of the teratogen, still resulted in
clear-cut elevations of both whole fetus ODC and fetal and neonatal brain ODC. These results indicate
that a sensitive biochemical detection procedure used in the fetus/neonate can successfully predict the
subsequent tissue-specific damage to neurotransmitter systems and behavior resulting from methyhner-
cury.
CITATION: Teratology 32(2): 195-202, October 1985.
L104 MS-85-063 Project Officer: Kaviock
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CONGENITAL RENAL HYPOPLASIM EFFECTS on BASAL RENAL FUNCTION
in the DEVELOPING RAT
It J. Kavloclc B. F. Rehnbe#g. and E. H. Rogers
HERI, USEPA, Research Triangle Park, NC
Manifestations of developmental toxicity include death, altered vwth, malfonnations, and functional
deficits. Vallidated methodologies to assess the last man (festation are lacking a major fimction of our re-
search program is to address these deflciencies In this paper, we demonstrate that alterations in renal
growth, indaced by the prototype teratogen chlorwnsucil, affected specific elements of renal function in the
offspring. Thus, the tests that were developed to assess renal function in the neonatal animals are sensitive
enough to detect functional alterations. Methodologies such as these could be applied to specific chemicals
when there Lc suspicion of renal mosphological alterations in a developmental toxicity test
Adminictration of chiorambucil to pregnant rats on day 11 of gestation induced dose-related alterations
in renal growth and function in the postn tRl offspring. These effects occurred above and beyond the
reductions in body growth and were evident in nini k that displayed no overt malfunction of the urogeni-
tal tract. Reductions in the overall growth amounted to 0,6 and 15% in the 3,4.5 and 6 mg/kg groups
respectively, while kidney weights were reduced by 7,15 and 23%. The weights of the kidneys relative to
the body were reduced 5,9 and 10% with incre ing dose. Although basal renal function was not affected
by the degree of hypoplasia seen in the low dose group, reduced glomerular and tubular function were
evident following a basal clearance test in the 2 highest dose groups. The data indicate that chiorambucil
induced renal hypoplasia results in reductions in renal function that persist for at least the first 3 weeks
after birth in the rat and that physiological assessment of development toxty can provide an extremely use-
ful addendum to the more classical morphological criteria.
CITATION: Toxicology 40(3): 247-258, September 1986.
L104 MS-85-212 Project Officer: Kaviock
AMPHOTERICIN B- and FOLIC ACID-INDUCED NEPHROPATHIES in
DEVELOPING RATS
Robe J. Kavloclç Blair F. Rehnberg and Ellen H. Rogers
HERL, USEPA, Research Triangle Park, NC
Techniques have been developed by thLc laboratoiy to evaluate renal function in neonatal animals follow-
ing prenatal exposure to xenobiotic agents. In this lepon, two of the techniques were evaluated for their
ability to detect the functional effects of two prototype nephrotoxicants on the developing rat pup. The data
clearly demonstrated the utility of the tests in detecting dysfunction, especially for ainphotercin B, where not
only was function altered in the absence of histological damage, but also a new mechanism of renal toxicity
was hypothesized on the bath of the observed alterations in physiology. These techniques are now ready for
routine application in situations where alterations in renal development are suspected following prenatal or
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early postnatal chemical exposure. The ability of a prog ain office to request specific, validated tests of
organ functional competence will allow greater characterization of biological effects and hence improve the
ability to assess nsk
The kidneys of newborn rats, which are both morphologically and physiologically imniature, have been
shown to be relatively insensitive to the nephrotoxic effects of several chemicals. To eramine the
specificity of these age-related differences, pups received sc injections of either 20 mg/kg of amphotericin
B or 250 mg/kg folic acid, two known nephrotoxins in adult animaLS on postnatal Day 1,8, or 15. Renal
f uncdonwas e r aininedbyabasalclearancetestandahydropeniachallengeatl,2 ,or5(6inthecaseof
amphotericin B) days after treatment. We observed no difference in degree of renal toxicity with age, but
repair of renal dam ge tended to proceed slower at the youngest age. Amphotericin treatment produced
uremia, increased fractional excretion of water and sodium, a decreased fractional excretion of urea, and
a diminished hydropenia response but no ehange in creatinine clearance and no renal pathology. The ob-
served pattern of renal toxicity may be attributed to an inability to maintain urea gradients in the distal
segment of the nephron. Folic acid treatment resulted in greatly increased kidney weights with marked
pathology, uremia with decreased creatinine clearance, increased fractional excretion of water, and a
decreased hydropenia response. Unlike the renal toxicity observed following amphotericin treatment,
renal toxicity from folic acid appears to be a nonspecific response to cell injury within the renal tubules.
The data indicate that, in general, neonates do not possess a relative insensitivity to nephrotoxins and that
renal physiological measurements which can be performed in neonatal rats are useful in evaluating and
interpreting alterations in renal function.
CITATION: To4cology and Applied Pharmacology 81(3): 407-415, December 1985.
L104 MS-85-085 Project Officer: Kaviock
DESCRIPTION and IMPLICATIONS for ANALYSIS of BRAIN GROWTH
in SUCKUNG MICE
Dennis House, Evu Bennan, and Hersheil B. Caner
HERL, USEPA, Research Triangle Park NC
The growth curve is a common parameter for evaluating the developmental and maturational effects of en-
vironmental agents. Growth curves are usually considered to be nonlinear. However, this study shows that
the growth curie resulting from measurements of mass (in this case, brains of mice 1-21 days of age) over
time can often be described by two straight lines. The repo?t describes the two straight lines for brain mass
in nursing mice and estimates the point where the two lines meet. This method of analysis should make a
significant contribution to understanding early brain growth and allow more confident evaluation of agents
that alter the rate of brain growth.
The growth pattern of the brains of normal CD-i mice was studies from day 1 through day 21 after birth.
The main purpose of the study was to decide on suitable methods of statistical analysis of these kinds of
data including whether to analyze brain weight or the brain-to-body weight ratio. A secondary purpose,
which aided the main purpose, was to find functions that describe the relationships between growth
parameters. Linear-linear segmented polynomials were found to describe well the relationships between
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(a) brain weight and body weight, (b) brain weight and age, (c) brain-to-body weight ratio and body
weight and (d) brain-to-body weight ratio and age. The analyses indicated that brain weight alone is the
preferred variable to analyze. Regression analysis of brain weight with sex and up to the first three powers
of body weight and age as independent variables would be a suitable method of analysis. Utter size per
se had no effect on brain weight.
CITATION: Growth 49:426-4* December1985.
L104 MS-85-116 Project Officer: Berman
KIDNEY MORPHOLOGY and FUNCTION in the YOUNG of RATS
MALNOURISHED and EXPOSED to NITROGEN DURING PREGNANCY
Carol Chase-Deesing 1 RobeitJ. Kavloclç 2 and Frances J. Zeman 1
1 University of California-Davis
USEPA, Research Triangle Park, NC
In the report, the potential interaction between prenatal protein malnutrition and posure to a teratogenic
he’bicide was evaluateiL As migrant farm workers often receive nutiitionalày inadequate diets and aLso
receive relative y high level atposwe to pesticides, the importance of potential synergism between these two
insults is obvious. This study demonstrated a strong interaction between the two treatments in teims of off-
spring viabili(y. Postnatal survival was reduced to 22% in the combination treatment versus 80-90% with
each alone. Renal dysfunction may have contributed to the increased mortality, but the effects were not suf-
ftcient y marked to be the primary cause of death. Oven,l4 the study indicated that malnowishedpopula-
tions may be at greater risk to cq,asure to pesticides.
The separate and combined effects of prenatal protein deficiency (6% casein) and prenatal nitrofen (2,4-
dichlorophenyl-p-nitrophenyl ether) exposure (12.5 mg/kg on gestational d 7-21) on renal morphology in
the 21-d fetal and postnatal rat were e mined. Body weights and kidney weights were reduced in prena-
tally protein-deprived (PPD) pups at birth and on postnatal day (PND) 10. Numbers of mature
glomeruli, creatinine clearance, water diuresis, and response of antidiuretic hormone (ADH), but not the
concentrating ability, were lower in the PPD neonates. These changes suggest that prenatal protein
deficiency delays renal development and possibly results in a decrease in glomerular clearance and in
tubular response to a water load and to antidiuretic hormone. Prenatal nitrofen exposure reduced body
weight and kidney size on PND 0 and 10. An increased incidence of hydronephrosis was indicated in the
nitrofen-exposed fetus. Prenatal nitrofen exposure depressed the ability to excrete excess water, the
response to ADH, and urine-concentrating ability. The functional deficits indicate tubular dysfunction,
but little or no effect on glomerular function, as indicated by the absence of an effect on creatinine
clearance. Postnatal survival was reduced to 22% by PND in the PPD plus nitrofen pups. Also, prenatal
nitrofen exposure increased the susceptibility of the glomeruli in the gestational day (GD) 21 PPD fetus
to the adverse effects of prenatal protein deficiency. By PND 10 the toxic effects were of the same order.
Renal dysfunction may contribute to the increased mortality in PPD plus nitrofen pups by reducing the
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ability to respond to stress, but the effects are not sufficiently marked to be considered the primary cause
of death.
CITATION: Journal of Toxicology and Environmental Health 19(1): 1-21, 1986.
L104 MS-86-039 Project Officer: Kaviock
AGE-DEPENDENT CHANGES in GASTROINTESTINAL TRANSPORT and
RETENTIONoI PARTICULATE MANGANESE OXIDE in the RAT
Georgia L Rehnbeig Joy F. Hem, Susan D. Carter, and
John W. Laskey
HERL, USEPA, Research Triangle Park, NC
It Lc well known that the developing organism is unique’y sensitive to the toxic effects of heavy metals such
as lead and manganese. One of the major routes of posure to heavy metals Li through inhalation, with
subsequent translocation of inhaled panicles of greater than 1 micron from the upper respiratory tracL In
previous studies, with particulate Mn3O , ve found that a portion of this age susceptthili y was due to in-
creased absorption of manganese by the young animaL We suspected that one of the factors responsible
was the immatwity of the GI tract of the pre-weanling animaL This study was designed to investigate the
rate of movement of material through the GI tract and the retention time of particulate material in the GI
tract of thepre- and post-weanling rat. We found that half-time of stomach emptying and transit rate in the
distal segment of the small intestine was longer in the pre-weanling rat than in the post-weanling rut. Addi-
tional y, the overall retention time of the paticulate manganese oxide was considerab y longer in the pre-
weanling animal. The overall half-time of particulate manganese oxide in the pre-weanling rat was ap-
proximate y 27 hours as compared to 2.7 hours in the 24 day old rat. This study demonstrates that the over
all maturity of the GI tract Li a sign ificant factor in the assessment of the enhanced sensitivi(y of the young
animal to many potential toxicants.
Translocation of inhaled particulates from the nasophaqnx and upper tracheobronchial area to the
gastrointestinal tract is a major route of exposure for particles with a mass median diameter of greater
than 1 p.m. Previous studies in this laboratory with particulate Mn304 have shown that preweanling rats
have subsv ntb 11y higher tissue Mn concentrations than siniib r1y treated adults, indicating possible dif-
ferences in uptake or elimination or both. This study was conducted to evaluate changes inrtrointes
final movement and retention of particulate Mn304 in the preweanling and weaned rat. Sr-labeled
microspheres were used to evaluate gastrointestinal transit rate (TR), while particulate Mn304 was used
to evaluate particulate retention at selected ages. The results show that stomach retention time in the
prewennling is at least twice that of the postweanllng (90 mm versus 42 mm). In general, intestinal TR
was not different in any of the ages evaluated, while transit time increased as intestinal length increased.
Analysis of the Mn data demonstrated that the prewezinhing rat had a two-component retention curve with
half-times of between 2 and 6 h for the short component and of between 24 and 26 h for the long corn-
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ponent. In the postweanling rat, only one component was identified, with a half-time of between 2 and 5
h.
CiTATION: Journal of Tozcolog,i and Environmental Health 16(6): 887-899, December 1985.
L104 MS-82-116 Project Officer: G. Rehnberg
THE USE of a MODEL SUBSTRATE (LINDANE) to DETERMINE the
ONTOGENY of METABOLISM in the DEVELOPING RAT
M.F. Copelan4 R.W. Chadwiclç N. Cooke
DA. Whitehouse, and D.M. Hill
HERI USEPA, Research Triangle Park, NC
The metabolism of foreign compounds in the developing animal are not well known. This study was con-
ducted to investigate the distiibudon, metabolism, and excretion of lindane in the developing rut. Rat pups
aged Z 9,14 and 23 days were dosed with lindane and the distribution, metabolism, and e.xcretion of lin-
dane was determineS Results .fivm this study indicate that (1) the metabolism of lindane is low at birth
and increases significantly with age; (2) the lung may have a greater role in the metabolism of lindane in
young rats; (3) audasive reactions increase significantly with age while reductive dechlorination decreased
significantly with age; (4) conjugation reactions are age dependen4 with su fate and glutathione conjugation
decreasing and glucuronide conjugation increasing with age and (5)different isozymes may be involved in
glucuronidation of the alcohol and chiorophenol metabolites of lindane. Studies of this nature may help to
evaluate hepatic function in the developing an irnaL
The compound lindane y-hexachlorocyclohexane) has been used to study the ontogeny of metabolism in
the developing Fischer 344 rat. The distribution and metabolic fate of lindane at 2,9,16, and 23 d of age
was investigated following subcutaneous administration of lindane at 20 mgfkg contsiining 0.5 p.Ci [ IY
14 C]linciane in peanut oil. Groups of 10 pups (5 male and 5 female) were sacrificed at 4-h intervals during
the 24-h period following dosing. Adrenals, blood, brain, heart, lung, liver, and kidneys were analyzed for
radioactivity. Urine samples were analyzed for radioactivity and metabolites of lindane. There was a sig-
nificant age-dependent increase in the metabolism of lindane in the rat. High levels of radioactivity in the
lung and increased reductive dechlorination suggest that the lung may play a greater role in metabolism
of lindane by young rats. Oxidative phase! reactions increased significantly, while anerobic reductive
dechlorination of lindane to 4-chiorophenylmercapturic acid decreased significantly with age. Phase I I
sulfate and glutathione conjugations decreased significantly and glucuronide conjugation increased sig-
nificantly with age. Metabolism and excretion of lindane appear to parallel development of the hepatic
enzymes involved in phase! and phase II reactions.
CITATION: Journal of Toxicology and Environmental Health 18(4): 527-542, August 1986.
L104 MS-85-245 Project Officer: Copeland
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EF1 CTS of AGE and OBESITY on the METABOUSM of LINDANE by
BLACKa/a,YELLOWA ’/a, and PSEUDOAGOUTI
A /a PHENOTYPES of (YS x VY)Fi HYBRID MICE
K W. Chadwiclç 1 M.F.Copelan4’ G.L.Wolff, 2 N. Cooke, 1
D.A. Whitehouse, 1 andM. L Mole 1
‘HERL, USEPA, Research Triangle Park, NC
2 National Center for Toxicological Research, DHHS
Some stirsins of mice are susceptible to liver tumors after chronic etposure to the oiganochlonne insecticide
hndwie while other strains are not. This woik was pan of a collaborative study with NC R to investigate
the effects of age, genetic influence and altered metabolism on susceptibility to lindane freaDnent It was
found that age and obesity had significant effects on the tissue storage and biotransfornw4on of lindane.
!Jndane (y-hexachlorocyclohexane) has been shown to produce hepatonias in some strains of mice but
not in others. Genetic factors and/or altered metabolism may play a role in the susceptibility to lindane-
induced hepatomas. This study reports the effect of age and obesity on the comparative metabolism and
disposition of lindane in obese yellow A’ ’/a and in lean pseudoagouti A” 7 /a and black a/a phenotypes of
(YSxVY)Fi hybridfcmaleniice at8, 17,30,56, and86wk of age. At 24hprior to sacrificethemicewere
dosed p.o. with 18 mg lindanc (Conh ining 55 Ci [ U 4 C]lindane/kg). Aging altered the biotransforma-
tion of lindane such that while the excretion of linLlane and its metabolites declined, the proportion of
conjugated and polar metabolites increased. Tissue storage was elevated in older animals. In the yellow
A /a mice, which are known to have a predisposition to the formation of hepatomas, there was ac-
celerated and prolonged growth, reduced metabolite excretion, a greater proportion of conjugated me-
tabolites, and higher dechiorinase activit , compared to that of their pseudoagouti A ’/a and black a/a si-
blings.
CITATION: Journal of Toxicology and Environmental Health 16(6): 771-796, December 1985.
L104 MS-85-029 Project Officer: Chadwick
INVESTIGATION of HCB as a METABOLITE from FEMALE RATS TREATED
DAILY for SIX DAYS with UNDANE
R. W. Chadwick and M. F. Copeland
HERL, USEPA, Research Triangle Park, NC
Widespread occuirence of the fungicide hexachiorobensene (HCB) in human milk samples, the
transplacental transfer of HCB to the fetus in several species and the teralogenicity of this pesticide in rats
and mice constituted a potential hazard to mothers and their developing offspnng in 1984 when it was
reponed that lindane, an organochiorine insecticide was metabolized by rats to hexachlorobenzene. Con-
cern over another source of HCB e.*posure led to the study of the metabolism of lindane to HCB in female
rats. Results from this study indicated that there was no s g,uftcant biotransfonnation of lhidane to NCR in

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rats. ThLc , v,* enabled the agency to meet its deadlines on the posure assessment of HCB without
changing the resowvxs required.
The biotransformation of lindane to hexachlorobenzene (HCB) by male rats was recently reported. Since
HCB has been widely detected in hi,nisin milk samples, and since the transpiacental transfer of HCB to
the fetus has been demonstrated in several species, the metabolism of lindane to HCB in female rats was
investigated. Young adult female Fischer 344 rats were dosed p.o. with either 20 mg lindane/kg/day or an
equivalent volume of the peanut oil vehicle. Feces samples were collected daily for two consecutive 4-br
intervals and a 16-hr interval. Twenty-four hours after the final treatment, all rats were sacrificed and
adipose tissue samples were excised at necropsy. Extracts of fat and feces samples were analyzed by gas-
liquid chromatography (GLC) on column packings of different polarity. Results of this study indicated
that no significant biotransformation of lindane to HCB occurred in the female Fischer 344 rat.
CITATION: Journal of Analytical Toxicology 9(6): 262-266, November/December 1985.
L104 MS-85-175 Project Officer: Chadwick
COMPARISON of IN VIVO and IN ViTRO METHODS for ASSESSING
the EFFECTS of PHENOBARBITAL on the HEPATIC DRUG-METABOUZING
ENZYME SYSTEM
Roben W. CltadwicA 1 M. Frank Copelan4 1 Gary P. Carlson, 2
BngceA. Trela, 2 and Bernard M. Mos?
1 HERL, USEPA, Research Triangle Park NC
2 Purdue University
3 Northrop Services, Inc.
The effect of eiposure to environmental cheinkals on the integñty of the hepatic drag-metabolizing enzyme
system has generally been evaluated in vitro using isolated liver preparations. Unceitainty over how well
changes obseived in vitro reflect those which occur in the live animal has resulted in the testing of a nonin-
vasive technique for the detenninadon of hepatic enzyme activity in living animaLs. This paper investigates
the applicability of using the metabolite profile from the model substrate lindane as an in vim index of
xenobiotic-inthiced alterations in the hepatic thug-metabolizing enzymes. The model substrate assay
descnbed in this paper will pennit the study of unanticipate4 persisten4 and latent side effects from pie- or
postnatal exposure to environmental chemicaLs.
The effect of daily i.p. injections of 0, 1,10 and 80 mg/kg phenobarbital for 1 week on the activity of the
hepatic drug-metaboliI’ing enzyme system was measured in the rat by a model substrate assay employing
lindane (y-HCH) and by a battery of in vitro enzyme assays. Comparison of the dose-response curves of
the in vivo and in vitro assays indicated that urinary metabolites of lindane provided a good index of
phenobarbital-induced change in both phase I and phase II reactions.
CITATION: Toxicology Letters 29(2&3): 95-105, December 1985.
L104 MS-85-217 Project Officer: Chadwick
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COMPARISON ofIN VIVO and IN VITRO METHODS for ASSESSING EFF ECTS
of ALLYL ALCOHOL on the LIVER
BnsceA . Trela, 1 Gwy P. Cwfton, 1 Robert W. O adwick 2
andM. Frank Cope! wd 2
1 Purdue University
USEPA, Research Triangle Park, NC
The effect of exposure to environmental chemicaLs on the integrity of the hepalic hu metabolithzg enzyme
system has general y been evaluated in vitro using tsolated liver preparalion& Uncertainty over how well
changes observed in vitro reflect those which occur in the live animal has resulted in the testing of a nonm-
vasive technique for the detennination of hepatic enzyme activity in living animals. This paper investigates
the applicability of using the metabolite profile from the model substrate lindane as an L’z viva index of
xenobiotic-induced alterations in the hepatic drag-metabolizing enzymes. The model substrate assay
described in this paper will pesmit the study of unanticipate4 persisten4 and latent side eff ects from pre- or
postnatal posure to environmental chemicaLs.
Allyl alcohol was adniinstered intraperitoneally (i.p.) to female Fischer 344 rats at doses of 0,3, 10 and
30 mg/kg daily for 7 days. Plasma sorbitol dehydrogenase was minim lIy elevated. No dose-related chan-
ges were observed in hexobarbital aiddation, nilinie hydroxylation, or ethylmorphine demethylation.
Aidrin epoxidation was slightly elevated. Naphtho l glucuronidation and glutathionc-S-transferase ac-
tivity with 1,2-dichloro-4-nitrobenzene were increased. Results from in viva studies on the metabolism of
lindane were in close agreement with the in vitro measurements suggesting that daily treatment for one
weekwithallyIalcoholatdosesof3,10and30maJkgbasnosignff9ca n teffectonphaseIpathwaysj a
selective effect on phase LI pathways and, under the conditions of this experiment, has minim 1
hepatotoxic effects in these rats.
CITATION: Toxicology Letters 29(2&3): 77-84, December 1985.
L104 MS-85-195 Project Officer: Chadwick
COMPARISON of IN VITRO METHODS and the IN VIVO METABOLISM
of LINDANE for ASSESSING the EFFLCTS of REPEATED ADMINISTRATION
of ETHANOL on HEPATIC DRUG METABOLISM
BniceA. Trela 1 Gasy P. Carlson, 1 Robert W Chadwick; 2
and M. Frank Copeland 2
1 Purdue University
2 HERL, USEPA, Research Triangle Park, NC
The effect of exposure to envirownental chemicals on the integrity of the hepatic dnig-metabolwng enzj ne
system has generaiI y been evaluated in vitro using isolated üver preparations. Uncertainty over how well
changes obse d in vitro reflect those which occur in the live animal has resulted in the testing of a nonin-
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vasive technique for the detennination of hepatic enzyme activity in living animaLc. This paper investigates
the applicability of using the metaboilte profile from the model substrate lindane as an in vivo index of
xenobiotic-induced alterations in the hepalic thug-metabolizing enzymes. The model substrate assay
descnbed in thLc paper will pennit the study of unanticipate4 pe?Nisten4 and latent side effects from pre- or
postnatal exposure to environmental chemicals.
In vitro methods of assessing alterations in drug metabolism and the measurement of lindane metabolites
in urine were compared for their ability to determine the influence of ethanol on drug metabolism.
Ethanol was administered intraperitoneally (i.p.) to young adult female rats daily for 7 days at doses of
0.12 0.60 and 3.0 mI/kg. No alterations were observed in ethylmorphine demethylation, hexobarbital
oxidation or glucuronyltransferase. Aniline hydroxylation was decreased at the high dose level and aldrin
epoxidation was increased at the intermediate dose. In vivo only the high dose of ethanol produced sig-
nificant changes with significant increases observed for the oxidation of lindane to alcohol metabolites,
the glucuronidation of the alcohol but not the chlorophenol metabolites, and glutathione conjugation.
The latter increase was also observed in vitro. The in vivo and in vitro data suggest a minimd effect of
ethanol on drug metabolism at low levels of administration.
CITATION: To colog Lette, c 29(2&3): 85-93, December 1985.
L104 MS-85-228 Project Officer: Chadwick
A COMPARISON of IN VITRO and IN VIVO METHODS for EVALUATING
ALTERATIONS in HEPATIC DRUG METABOLISM FOLLOWING MERCURIC
CHLORIDE ADMINISTRATION
BnsceA. Trel€ Gary P. Carson, 1 Robeii W. ChadwicA 2 and M. Frank Copeland 2
1 Purdue University School of Pharmacy
2 HERL, USEPA, Research Triangle Parlç NC
The effect of exposure to environ,nental chemicals on the integrity of the hepatic diug-metabolizing enzyme
system has generally been evaluated in vitro using isolated liver preparations. Unceitainty over how well
changes observed in vitro reflect those which occur in the live animal has resulted in the testing of a nonin-
vasive techn ique for the detennination of hepatic enzyme activity in living animals. This paper investigates
the applicability of using the metabolite profile from the model substrate undone as an in vivo index of
xenobiotic-induced alterations in the hepatic diug-metabolizing enzymes. The model substrate assay
described in this paper will perm it the study of unanticipate4 persisten4 and latent side effects from pre- or
postnatal ciposure to environmental chemicals.
Mercuric chloride was administered once i.p. to female Fischer-344 rats at doses of 0, 0.2, 0.6 and 1.8
mg/kg. Although there were no alterations in the urinary excretion of lactate dehydrogenase, significant
elevations in the activities of urinary (U) lilk2line phosphatase, glutamic-pyruvic transnniinnse (OPT) and
glutaniic-oxalacetic trausaminase (GOT) indicated that mercuric chloride was nephrotoxic. There was
no evidence of hepatotoxicity as hepatic glucose-6-phosphatase and serum sorbitol dehydrogenase were
essentially unaffected by mercuric chloride administration. The activities of ethylmorphine demethylase,
hexobarbital oxidase and aidrin epoxidase determined in vitro were not inhibited by mercuric chloride al-
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though lniline hydroxylase activity was decreased. Of the four phase-fl reactions measured, only the
glucuronidation of chioramphenicol was diminished by treatment with mercuric chloride. Results from
the in vivo studies on the metabolism of lindane, which indicated no change in the excretion of free of con-
jugated metabolites, were in close agreement with the in vitro data suggesting that the nephrotoxic effects
of mercuric chloride do not alter the urinary excretion of the model substrate lindane.
CITATION: Toxicology Letters 32(1&2): 133-140, July/August 1986.
L104 MS-86-077 Project Officer Chadwick
A COMPUTER-ASSISTED ELECTROCARDIOGRAPHIC ANALYSIS SYSTEM:
METHODOLOGY and POTENTIAL APPLICATION to CARDIOVASCULAR
TOXICOLOGY
William P. Watkinson, 1 M. Ann BAce, 1 and Kathy S. Robinson 2
USEPA, Research Triangie Park, NC
2 Northrop Services, Inc.
The research descnbed in thLc wiicle was conducted to suppoit the programs of the Office of Toxic Sub-
stances (OTS). There was a need to develop methodology to enable research toxicologists to efficiently
ident ify potentially toxic agent.c In addition, there was a need to establish a program within the EPA with
the capability to evaluate the cardiovascular effects induced by e. posure to such toxic agents. The develop-
ment of the computer-assisted electrocardiographic analysis system described in this wiicle provided a solu-
tion to both problems. Thus, monitoring the effects of substances on hewt rate and electrocardiograms of
animal modeLs se,vces as an effective screening procedure and permits investigators to differentiate between
substances which pose a significant systemic toxic threat and those which do not. In addition, the extended
capabilities compatible with standard cardiovascular methodologies and thereby pennit more in-depth
swdy of specific cardiovascular effects of identified toxic agents. The development and general availability
of the methodology described in this article should significantly expand the area of cardiovascular
toxicological research.
An automated analysis of electrocardiographic (ECG) waveforms, based on a precise one-dimensional
analysis of features within a generalized computer-enhanced ECG waveform, has been developed in our
laboratory. ECU sign Lc are monitored, amplified, and recorded using standard techniques. The re-
corder output signal is distributed to a microcomputer system. Software developed for the microcom-
puter slows the sgn ll playback rate and permits operator review of the slowed signsd for arrhythmia
analysis. The analysis program identifies and superimposes 10-40 individual ECG complexes, depending
on the heart rate, and generates an “ensembled” waveform. Operator interaction permits delineation of
specific points on the displayed waveform and calculation of heart rate and duration of components
within the ECG compleL The primary advantages of this system include (1) extensive automation-com-
puter support decreases analytical time, increases precision, and permits rapid screening of large num-
bers of nima1s ; (2) enhanced sensitivity-the use of functionnJ parameters should provide a more sensitive
index of toxicity than morphological parameters; (3) broad utility-this system provides the capability to
utilize a variety of nim i1c , both anesthetized and unanesthetized, ranging in age from fetuses to
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geriatrics, and permits studies of block as well as longitudinal design; and (4) ease of replication-stand-
ardization of equipment and techniques facilitates replication by other Laboratories.
CITATION: Jow wJ of Toxwology and Environmental Health 15:713-727, November 1985.
L104 MS-83-042 Project Officer: Watldnson
EFFECTS of CHLORDIMEFORM on
CARDIOVASCULAR FUNCTIONAL PARAMETERS:
PART 1. LETHALITY and ARRIIYI’HMOGENICITY in the GERIATRIC RAT
William F. Watldnson
HERL, USEPA, Research Triangle Park NC
The research described in this article was conducted to support the programs of the Office of Toxic Sub.
stances (OTS). The fonnamidine pesticide chlordimefonn (CDM) is currently being studied by several
laboratones within HERL, with prvna?y emphasis on its neurotoxicological and thennoregulatory effects.
Relatively little is known of its effects on other organ systems. The data presented in this particular study
descnbing the effects of CDM on cardiovascular functional parameters serves a dual purpose. Firstj these
results provide a significant quanthy of new data describing both the general systemic and specific car-
diovascular toxic effects of CDM, with additional insight provided regarding the mechanisms of actions
responsible for these obser. ed effects. Second, these data serve to validate previously developed inethodol-
o based upon computer-assisted electrocardiographic analytical procedures.
Chiordinieform (CDM), a formamidine pesticide, had a profound effect on the cardiovascular function
of geriatric rats. Two-year-old pentobarbital-anesthetized Sprague-Dawley rats (n = 8) received sequen-
tial intravenous CDM injections of 5,10,30, and 60 mg/kg. A control group of rats (n = 8) received mul-
tiple injections of normal saline vehicle over a similar time period, followed by a single CDM injection of
60mg/kg. Heart rate (HR), arterial blood pressure (BP), and electrocardiogram (ECG) were monitored
for all animals. CDM produced abrupt decreases in HR and BP at all doses. In addition, striking chan-
ges in ECG waveforms and intervals and various conduction-related arrhythmias were observed. These
acute effects triggered a reflex-mediated overshoot in HR and BP above preinjection levels, followed by
a delayed, persistent depression of these parameters. Three animals from the CDM-treated group died
after injection of the 30-mg/kg dose, while 60 mg/kg was lethal in the remaining s nini ik In the majority
of cases, death appeared to be due to cardiac arrest.
CITATION: Journal of Toxicology of Environmental Health 15:729-744, November 1985.
L104 MS-87-072 Project Officer: Watkinson
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DERMAL ABSORPTION and DISPOSITION of 1,3-DIPHENYLGUANIDINE in
RATS
P. V. Shah, M R . Swnler, 1 Y. M. Ioannou, 2 H. L Fisher, 3
andLL Hall 3
1 Northrop Services, Inc.
2 NIEHS
3 HERL, USEPA, Research Triangle Park, NC
1,3-diphenylguwzidine (DPG) Lv a chemical widely used in the processing of nibber and in food packaging.
Studies were undertaken to assess the potential of DPG to be absorbed through the skin as this route Lv of
major concern on exposure to many occupational and agricultural chemicaLs. Even though DPG shows
slow dennal penetration, this route of exposure needs to be considered in the risk assessments by the Office
of Pesticides and Toxic Substances (OPTS) because of the suspected long-term toxicity of DPG.
Dermal absorption, distribution, and metabolism of 1,3-diphenylguanidine (CAS 102-06-7) (DPG), wide-
ly used as an accelerator in processing rubber and in food pack iging, was studied in adult female Sprague-
Dawley rats. DPG shows 10% penetration through clipped back skin of the rats in 5 d. The first-order
dermal absorption rate constant as determined by least square method was 0.021 ± 0.002 d 1 (T1/2 = 33.6
d). Approximately 13% of the absorbed dose remained in the body in 5 ci. Retention in skin, muscle, liver,
intestine and fat contributed most to the body burden of DPG-derived radioactivity in 5d. All tissues
showed tissue to blood ratios greater than 1, with liver and intestine ratios of 26 at 5 d. Approximately
61% of the absorbed dose was eliminated into urine and 27% into feces in 5 d showing rapid clearance of
absorbed DPG from the body. High-pressure liquid chromatography (HPLC) analysis of urine revealed
two major peaks [ parent compound and metabolite(s)]. Within 72 h, approximately 50% of the DPG-
derived radioactivity excreted in the urine was parent compound. After 72 h, the DPG-derived radioac-
tivity in the urine was present in the form of a single metabolite, and no parent compound was detected.
No parent compound was detected in feces. Two metabolites, neither of which occurred in urine, were
detected in feces. The HPLC analysis of the radioactivity at the application site showed only parent com-
pound. Even though DPG shows slow dermal penetration, this route of exposure needs to be considered
in the risk assessments because of the suspected chronic toxicity of DPG.
CITATION: Journal of Toxicology and Environmental Health 1.5(5): 623-633, October 1985.
L104 MS-84-170 Project Officer: Hall
IMMUNE STATUS in NEWBORNS and THEIR VULNERABILITY
Ralph Smialowicz
HERL, USEPA, Research Triangle Park, NC
This paper Lv part of a workshop proceedings in which some of the pennatal toxicology work performed in
HERL was sunvnwizei Basically, we have found that alterations in immune function of rodents exposed
to a variety of agents are difficult to induce. In those cases where alterations in immune fwzction have been
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obsen’e4 these alterations are reversible wuh normal levels of immune function achieved after a penod of
time following temiination of e osure. These results are useful to the Agency in that they provide a data
base forthe hnmunotoxic potential of ce,tain chemicals on a select population, i.e., the veJy young.
The most profound toxic effects of many physical and chemical agents are observed when exposure oc-
curs during m nimalian development. The development of immunocompetence in m mm ih involves a
precise sequence of steps which begins during gestation and is completed during postnatal life. Pertur-
bation or abrogation of this developmental sequence of events may lead to life threatening dysfunctions
as exemplified by certain heritable defects in immune system development. In light of this we have ex-
amined the effects that prenatal and/or early postnatal exposure on immune function in young adult ro-
dents. Mice and/or rats were exposed in utem and/or during early postnatal life to the following agents:
cycloposphamide, nonion ng radiation, cyclosporin A, urethan, di-n-octyltin dichioride orNiCl2. At
immunological maturity (i.e., 6-8 weeks of age) 2nimldc exposed to these agents were assessed for immune
system functional integrity. Immune function tests included the following: lymohoid organ and body
weights, peripheral blood counts, lymphoproliferative responses to mitogens and allogeneic cells, natural
killer cell acfivity primary antibody responses to sheep erythrocytes, delayed hypersensitive responses,
and resistance to tumor development. The results of these studies are summarized.
CITATION: In: Proceedings of the Indo-U.S. Workshop on the Role of Fredisposing Conditions of Health,
Nutrition, and Environment on Safety of Drugs and Chemicals, Lucknow, India, February 25-
28,1986.
L104 MS-86-251 Project Officer: Smialowica
IMMUNOLOGICAL STUDIES in MICE FOLLOWING IN UTERO
EXPOSURE to NiC12
Ralph I. Smialowic Ronald P. Rogers, Mane M. Riddle,
Denise G. Rowe, and Robed W. Luebke
HERL, USEPA, Research Triangle Park, NC
Nickel has been shown to inhibit ce,iain immune functions in adult mice. Since it has been repofled that
the most profound effects of chemicals that modulate the immune system of animals occur when dosing of
the compound begins diving the development of the lymphoid system, mice were exposed to nickel dwing
gestation. Results of this worlç however, indicate that in utero ciposure to nickel does not adversely affect
the immune fun clion of young adult mice. Although these results were negative they have provided informa-
tion and experience in developmental immunotoxicology. This work also has added to the database on
nickel toxicity/or use by the4gency in making risk assessments.
The effect that NiCI2 has on the development of immune function in mice was ex2mined in the offspring
of dams implanted with mini-osmotic pumps during pregnancy. Time bred C57BL/6J mice were im-
planted subcutaneously on day 5 of gestation with mini pumps which delivered a total dose of from 9.1 to
73.2 paJg N1CI2. The pumps delivered NiC12 to the dams through day 19 of gestation. At 8-10 weeks of
age the offspring of NiCl2-dosed damc were evaluated for immune function. No consistent &gnH cant al-
terations were observed between control and treated offspring for the following: lymphoid organ or body
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weights; the lymphoprohicrative response to B or T lymphocyte mitogens; the lymphoproliferative
response to allogeneic spleen cells in the mixed lymphocyte reaction; the development of syngeneic
tumors; or the primary antibody response tosheep red blood cells. Natural killer (NK) cell activity was
reduced in offspring exposed to N1CI2 in utero; however, the biological relevance of these reductions is
questionable because of the failure to demonstrate an increased susceptibility to the B16-F1O syngeneic
tumor. The results indicate that under the conditions and doses employed it appears that NiCI 2 does not
adversely affect the developing immune system of the mouse.
CITATION: Toxicology 38(3): 293-303, March 1986.
L104 MS-85-190 Project Officer: Smiaiowicz
IMMUNE FUNCTION of YOUNG ADULT MICE FOLLOWING IN UTERO
EXPOSURE to CYCLOPHOSPHAMIDE
t W. Luebke M. M. Riddle It it Rogers, R. J. Gamer,
D. G.Rowe andR.LSmialowicz
HERL, USEPA, Research Triangle Park, NC
The sensitivity of the developing immune system to peflutharions by xenobiotic agenLs Lv as yet not well
charactenze€L Alteration of the nonnal developmental sequence has the potential to be expressed postna-
tally as significant immune deficiencies postnatally. The work described in this paper describes studies
designed to investigate the effects of in utero exposure to the well known adult animal uninunotoxicant
cyclophospharnide. The information from this work contributes to the data base upon which the Agency
can determine risk assessment as it relates topennatal exposure to toxic chemicals.
Long-lasting organic damage has been reported following in utero exposure to certain environmental or
therapeutic agents. The sensitivity of the developing immune system to chemical insult during or-
ganogenesis or histogenesis was evaluated in mice employing the known immunosuppressive agent
cyclophosphamide (CY). Experiments were conducted employing one of the following treatment
regimens: (1) 1 p Jg d intravenously on d 9-12 or 14-17 or gestation; (2) 5 p g/g intravenously on 12 of
gestation; (3) 1,2.5 or 5 p .g/g d intraperitoneally on d 12 of gestation; or (4) 5, 10, or 20 p.g/g in-
traperitoneally on d 17 of gestation. There were no surviving pups born to mothers alministered CY by
schedule 2; otherwise, numbers of surviving offspring were not affected by drug treatment, and no gross
terata were observed. Employing this variety of exposure protocols, consistent enhancement or suppres-
sion of cell-mediated or humoral immune function was not observed in offspring of treated danis.
Reduced body weight in 5-and 8-wk-old progeny was noted after exposure to 20 ).Lg/g on gestational d 17.
Increased in vitro B-lymphocyte blastogenic response to lipopolysaccharide occurred in 5-wk-old
animals, and production of antibody to sheep erythrocytes was increased in 8-wk-old offspring exposed
to CY at 20 p gIg on d 17 of gestation. The T-lymphocyte parameters were relatively unaffected byin utero
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exposure to CY, suggesting either that cell-mediated immune function was not affected by treatment or
that homeostasis was restored prior to immunologic evaluation of offspring.
CITATION: Journal of Toxicology and Environmental Health 18(1): 25-39, July 1986.
L104 MS-85-182 Project Officer: Smialowicz
IMMUNE FUNCTION in ADULT C57B116J MICE FOLLOWING EXPOSURE to
URETHAN PRE- or POSTNATALLY
Robert W. Luebke 1 Mane M. Rid€lle, 1 Ronald R. Rogers, 1
Denise G. Rowe 2 11. John Gamer, 1 and Ralph I. Smialowicz 1
USEPA, Research Triangle Parlç NC
2 Northrop Services, Inc.
The effects ofxenobioilcs on the developing immune system are not well known. This study was conducted
as pair of our model development/validation effort in developmental uninunotoxicology. Mice were &-
posed to the weak caivinogen w!than either pre- orpostnatally and evaluated iinmunological y at an age
when full immunologic competence should have been attaineL In this study, differences in adult levels of
immune fwzcdon were observed when chemical erposure occurred diuing different penods of immwze sys-
tem ontogeny.
Administration of urethan (URE or ethyl carbamate) to mice results in the development of a variety of
tumors, and, in certain strains of mice, marked suppression of the immune response. Perinatal exposure
of mice to URE has been found to result in increased tumor induction compared to exposure of adult
animals. In the present study, the effects of perinatal exposure to URE on the development of im-
munocompetence was investigated. Pregnant mice were injected with total doses of either 0.5 or 1.0 mg
URE/g of body weight over days 7-16 of gestation or pups of nontreated dams were administered a total
dose of 2.0mg URF/g of body weight over postpartum days 5-14. Postnatal exposure to URE suppressed
NK (natural killer) cell activity but left intact other measured parameters of the host defense system.
Prenatal exposure, on the other hand, resulted in elevated leulcocyte counts and a trend toward increased
spleen and thymus size in offspring of treated mothers. Humoral immune function, as measured by the
1gM response to sheep erythrocytes, was suppressed in pups from dams injected with a total of 1.0 mgfg
URE. These results indicate that marked differences in immunopharmacologic effects may be observed
if chemical exposure occurs at different times during the ontogeny of the immume system.
CITATION: Journal of Immunophannacology 8(2): 243-257,1986.
L104 MS-85-233 Project Officer: Smialowicz
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EVALUATION of IMMUNE FUNCTION in MICE EXPOSED to ORDRAM®
Ralph I. Smialowicz Robeit W. Luebke Ron R. Rogers,
Mane M. Riddle, and Denise G. Rowe
HERL, USEPA, Research Triangle Park, NC
Ordram®, a thiocarbanwie herbicide, has been reponed to affect the immune system of e.*perimental
animals. Following a request from the Office of Pesticide F og,wns (OFF), the wo,k repoifed in this paper
was performeS The results of this work indicate that this herbicide does not adversely affect the immune
system of mice under the esposure conditions described. The infonnation from this work contributes to the
database upon which the Agency can detennine risk assessment.
The potential effects that the thiocarbamate herbicide Ordram® has on the immune system of mice was
evaluated following 12 days of acute dosing by oral gavage. Dosages of Ordram® ranging from 20 to 320
mg/kg/day had no consistent significant effects on a variety of immune parameters investigated. The im-
mime parameters measured were the following: body and lymphoid organ weights; splenic natural killer
(NK) cell activity; lymphoproilferative responses to B and T lymphocyte mitogens and allogencic spleen
cells in a one-way mixed lymphocyte reaction; and delayed-type hypersensitivity and antibody responses
to sheep red blood cells (SRBC). The effects that the immunosuppressant cyclophosphamide has on
these immune parameters was also ex imh’ed. The results indicate that Ordram® does not appear to af-
fect key parameters of the immune system of mice under the conditions of exposure employed.
CITATION: Toxicology 37(3&4): 307-314, December 1985.
L104 MS-85-L57 Project Officer: Smialowicz
STRATEGIES for the SELECTION of TEST METHODS for SCREENING in
BEHAVIORAL TERATOLOGY
MaricA. Ge randLawrence W. Reiter
HERL, USEPA, Research Triangle Park, NC
This publication recommends that several factors and criteria be considered wizen designing a test balle,y
for assessing developmental neurotoxicity. The major recommendation is that testing procedures be objec-
tive and simple, to insure reliability of results. Other recom,nendations are that tests be non-invasive, allow
longitudinal testing of the same animals, and lend themselves to statistical analysis. Automated-measures
are recommended because they meet the above criteria and are also cost-effective. These recommendations
are relevant to the standardization of behavioral research such as testing procedures currently utilized in the
evaluation of neurotoxicizy in the Neurotoxicology Division of HERL and are consistent with the current
Office of Toxic Substances (OTS) guideline for neurotoxicily testing.
The purpose of this Workshop was to discuss factors and criteria relevant to the selection of behavioral
test methods in the context of the screening of compounds for the purposes of safety evaluation. Rather
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than attempt to achieve a consensus on a recommended set of behavioral tests, the group focused on the
strategies most appropriate for the selection of such tests.
CITATION: In: Neurobehavioral Toxicology and Teratology, in press.
L104 MS-86-008 Project Officer: Reiter
COLLABORATIVE BEHAVIORAL TERATOLOGY STUDY:
PROTOCOL DESIGN and TESTING PROCEDURES
J.Ada,ns, 1 J. Buelke-Sa,n 1 C.A. Kmm4 1 C. J. Nelson 1
L. W. Reiter, 2 T. Sobotka H. A. Tilson, 1 andB. K Nelson 1
1 Children’s Hospital Research Center
USEPA, Research Triangle Park, NC
This publication describes the desigs of testing procedures used in the NCTR Collaborative Behavioral
Teratology Study. In this study, intra- and interlaborazory reliability of nwnemus test methods were a-
amined and the impo tance of major factors such as experience, gender and litter were examined. This
publication has agency relevance because several test methods which were utilized in this study are currently
being proposed for inclusion in the Office of Toxic Substances (OTS) guideline for developmental
neurofoxicity testing.
The Collaborative Behavioral Teratology Study (CBTS) was conceived as a direct means of evaluating the
intra- and interlaboratory reliability and sensitivity of several behavioral test methods applied under
standardized conditions. Background information relevant to this study has been published by ICimniel
and Buelke-Sam (1985) and the preliminary work which resulted in the design of the CBTS has been
presented (Adams, Buelke-Sam, ICimmel, Nelson, and Miller, 1985). The purpose of this paper is to
present information on the test agents and the behavioral methods used in the CBTS, as well as the ex-
perimental design and procedures used in its conduct.
CITATION: In: Neurobehavioral Toxicology and Teratology, in press.
L104 MS-86-007 Project Officer: Reiter
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DEVELOPMENT of LOCOMOTOR ACTIVITY of RAT PUPS in
FIGURE.EIGHT MAZES
Patricia H. Ruppen Karen F. Dean, and Lawrence W. Reiter
HERL, USEPA, Research Triangle Park NC
The results of studIes reported In this publication indicate that there are social and expeiiential factors
which detennine the proftk of the development of motor activity infigure-8-mazes. These studies highlight
important design considerations for test methods utilized in the Neuroto4cology Division of HERL and
recommended for use in the Office of Toxic Substances test guidelines for motor activity measurements.
In a series of four experiments, social and experiential factors that influence the development of motor
activity in rat pups were eyamined. Motor activity was monitored from postnatal Days 13 to 21 as
photocell interruptions in figure-eight mazes and comparisons were made between (1) pups maintained
in a nest box containing a dam and siblings and allowed access to the maze for 23 hr/day, (2) pups tested
daily for 1 hr/day vs pups tested only on postnatal Days 15,18, or 21, (3) pups tested daily for either 5 mm,
30 mm, or 1 hr/day, and (4) pups tested daily for 30 mm/day either singly in a ni ,e , paired with a litter-
mate, or paired with an anesthetized pup of the same age. A monotonic increase in activity was seen for
nest-box testing, minim il developmental change was seen for pups tested on only a single day or for pups
tested with an anesthetized pup, whereas all other groups showed an inverted U-shaped profile of activity
which was influenced by the duration of testing and/or the presence of a littermate. These data emphasize
the relevance of environmental factors as determinants of preweaning behavior.
CITATION: Developmental Psychobiology 18(3): 247-260, May 1985.
L104 MS-85-017 Project Officer: Reiter
TRIEThYLTIN-INDUCED NEURONAL DAMAGE in NEONATALLY
EXPOSED RATS
B. Veronesi 1 and S. Bond?
1 HERL,USEPA, Research Triangle Park NC
2 ?JffiHS
Tdethyltin (TET) has been used as a model neurotoxic compound to study myelin edema in the adult
brain. The importance of this study rests in the demonstration that when TET is exposed to the developing
brain, it produces selective damage to cortical neurons in the ento,hinal region. Damage to this population
of cells results in associated changes in the hippocwnpal region, which Li involved in memory and learning.
Neuropathological and biochemical effects of neonatal exposure to the ailcyl metal triethyltin were ex-
amined in Long Evans juvenile male rats. Rats were injected intraperitoneally on post-natal dayS with 6
ink/kg of triethyltin bromide and sampled on day 20. The brains of tin-treated aniniak weighed sig-
nificantly less than either saline or starved controls and exhibited a marked anterior-posterior atrophy.
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Histologically, neuronal necrosis was noted in the enthorhinal and transitional cortex an observation con-
firmed by imniunocytochemical st2ining of astrocytes. Hippocampal involvement was further evidenced
by a protrusion of the molecular layer of the dentate gyrus, and an abnormal shlining pattern of acetyl-
choilnesterase in this layer. Sections stained by the Tinun’s method for the deposition of heavy metals
showed a marked reduction in the st2ining of the bippocampal CA4-2 sectors and an absence of stained
L minae in the outer molecular layer of the dentate gyrus. Receptor binding assays indicated a selective
depression of the benzodiazepine receptor in the hippocampus of tin-treated pups compared to starved
controls. Taken in concert, these data indicate that neonatal exposure to triethyltin produces severe
neuronal diunage in the posterior cortex and a derangement of hippocampal afferent circuitry.
CITATION: Neurotoxicolorj 7(1): 69-80, April 1986.
L104 MS-85-179 Project Officer: Veronesi
MACROPHYSIOLOGICAL ASSESSMENT of ORGANOMETAL
NEUROTOXICITY
Robert S. Dyer
HERL, USEPA, Research Triangle Park, NC
Part of the Neurotoxicology Division’s mission is evaluation of methods for detection and characterization
of neurotoxicily. The vanous program offices, pafliculady the Office of Toxic Substances, need validated
test methods to discharge their responsibility for requiring testing and test data. This chapter provides a
critique 0/certain neurophysiological methods, as they have been used to study organometallic compounds.
Neurophysiological methods fall into two broad categories, macrophysiological and microphysiological.
The unit of analysis for microphysiological methods is cellular or subcellular (e.& membrane or channel)
whereas the unit of analysis for macrophysiological methods is a population of neurons. Each set of
methods may be used to great advantage or disadvantage, depending upon the experimental question at
hand. The purpose of this chapter is to focus on studies which have used macrophysiological methods to
investigate the neurotoxicity produced by organometals. For each study to be discussed, the issues under
consideration will be (1) appropriateness of the method to answer the experimental question; (2) ade-
quacy of experimental design in uSing the method; (3) importance of the findings; and (4) identification
of unresolved issues. Only a few organometallic compounds have been investigated using macro-
physiological techniques, and it will become evident that these investigations have been incomplete. A
future goal of this chapter is to stimulate interest in pursuing these studies, so that we might achieve a
more complete understanding of the compounds and the methods we use to study them.
CITATION: In: Neumtoxicanty and Neurobiological Function, Tilson and Sparber, eds., in press.
L104 MS-86-009 Project Officer: Dyer
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NEUROTYPIC and GLIOTYPIC PROTEINS as BIOCHEMICAL INDICATORS
of NEUROTOXICITY
James F. O’Callaghan
HERL, USEPA, Research Triangle Parlç NC
Reviews progress of a research approach designed to provide the 4gency with a simple; cost-effective testing
methodology, that can be used to evaluate the potential neurotoxic effects of broad classes of environmental
pollutants.
Exposure of the developing or mature nervous system to neurotoxic xenobiotics results in complex be-
havioral, physiological and morphological alterations. With few exceptions, a biochemical basis for these
toxicant-induced changes has yet to be described (Damstra and Bondy, 1982). In this chapter I will brief-
ly address the issues to be considered when developing a biochemical approach to neurotoxicity assess-
ment and I will review the progress of work concerning one strategy: the use of neurotypic and gliotypic
proteins as biochemical indicators of neuroto dcity.
CITATION: In: Neurotoxicology, M.B. Abou-donia, ed., Oxford University Press (in press).
L104 MS-86-209 Project Officer: O’Callaghan
PCPP-260, A PURKINJE CELL-SPECIFIC CYCLIC AMP-REGULATED
MEMBRANE PHOSPHOPROTEIN of Mr 260,000
S. Ivar Walaas, Angus C Naim, and Paul Greengard
The Rockefeller University
This paper reviews recent progress in identifying and characterizing the protein composition of the nervous
system, and as such it complements ongoing effoirs to develop and validate nervous-system-specific
proteins as biochemical indicators of neurotoxicity.
The present study reports the existence of Purkinje cell-specific phosphoprotein, M 260,000 (PCPP-
260), a neuronal membrane phosphorprotein, in cerebellar Purkinje cells. PCPP-260, which on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis has an apparent molecular mass of 260,000 Da, has
been found to be phosphorylated in particulate preparations by endogenous or added exogenous cyclic
AMP-dependent protein kinase, but not by cyclic GMP-dependent, calcium/calmodulin-dependent or
calcium/phospholipid-dependent protein kinases. The protein has been found in high concentrations in
all mammalian cerebella so far analyzed, including human cerebellum. One- and two-dimensional
electrophoretic and peptide mapping analyses of proteins in other brain regions show that a closely re-
lated 265,000 Da phosphoprotein also exists, albeit in low concentrations, outside, the cerebellum.
Analysis of cerebella from mutant mice, deficient in either Purkinje cells or in granule cells, indicates that
PCPP-260 within the cerebellum is restricted to Purkinje cells. Furthermore, subcellular fractionation of
rat cerebella indicates that the protein is an integral menbrane protein. The cAMP-regulated
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phosophorylation of PCPP-260 is presumably involved in membrane functions important to Purkinje
cells.
CITATION: Journal of Neuroscience 6(4): 954-961, April 1986.
L104 XX-86-138 Project Officer: O’Cslbaghan
THERMOREGULATION in MICE FOLLOWING ACUTE CHLORDIMEFORM
ADMINISTRATION
Christopher 1. Gordon, Memtt D. Long andAndrew G. Stead
HERL, USEPA, Research Triangle Park, NC
Chlordimefonn has been shown to &ert a vaiie y of toxic effects including visual dysfunction and bradycar-
dia. Chlordimefonn also promotes a reduction in body temperature which may indirect’y be responsible for
the toxic effects. Hence it is impoitant to understand the mechanisms by which chlordimefonn affects
body temperature. This study monstiuted that chiordimefoim affects both the w4onomic and behavioral
control of body temperaIw . The study also found that the thennoregulator, response to intoxication by
chlordhneform may be cntcial to survivaL
CBA/J mice were injected intraperitoneally (i.p.) with the form imidine insecticide chiordimeform
(CDM) while colonic temperature, preferred ambient temperature (Ta), and lethality were monitored.
In the first experiment there was a dose-dependent decrease in colonic temperature when measured 60
mm after administering CDM doses of 0,15,30,60, and 75 mg/kg. The hypothermic effect of CDM was
more pronounced at a Ta of 20°C than at 30°C. In the second experiment, CDM at doses 30 mg/kg caused
a dose-dependent reduction in preferred Ta from the normal value of approx. 30°C to approx. 22°C. Thus,
the CDM-treated mouse lowered body temperature by selecting a cool Ta which accelerated the
hypothermic effect. In the final experiment, a 90 mg/kg i.p. injection of CDM (the approximate LDso
dose) caused 10% and 0% mortality at a Ta of 20 and 30°C, respectively, and 80% mortality at a Ta of
35°C. It is concluded that the physiological and behavioral response to CDM administration, i.e., select-
ing a cool Ta and lowering body temperature, may be beneficial to survival.
CITATION: Toxicology Letters 28(1): 9-15, October 1985.
L104 MS-85-093 Project Officer: Gordon
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EFFECT of SODIUM PENTOBARBITAL on BEHAVIORAL
THERMOREGULATION in RATS and MICE
Kim! S. Strelç Menitt D. Long and Christopher I. Gordon
HERL, USEPA, Research Triangle Park, NC
Many pesticides and other toxic chemicals affect the control of body temperature in rodentc. It is possible
that the action of these compounds on the thennoregzdato,y centers of the central ner.’ous system could be
anesthetic-like in natw . Hence this swdy was designed to assess the effect of an anesthetic (sodium pen-
tobarbital) on the control of body temperature and to compare these effects to that of toxic chemicals. It
appears that the action of toxic chemicals on thennoregulation is quite unique compared to the action of
anesthetics.
In this study on behavioral thermoregulation, male Sprague-Dawley rats were given intraperitoneal (IF)
injections of sodium pentobarbital in doses of 0,1,5,10 or 15 mg/kg and male CBA/J mice were given
doses of 0,5,10,15 or 30 mg/kg. The animals were immediately placed in a temperature gradient which
allowed them to select their preferred ambient temperature (Ta). The preferred Ta of rats increased fol-
lowing an injection of 10 mg/kg sodium pentobarbital, whereas, the barbiturate had no effect on the
preferred Ta of mice. In another study, male rats and mice were given sodium pentobarbital in doses of
0,5,10 and 15 mg/kg and then placed into a temperature-controlled environmental chamber set at 30°C
for mice and 25°C for rats (i.e., their approximate preferred Ta when dosed with sodium pentobarbital).
Colonic temperatures were taken one hour after injection. Sodium pentobarbital induced dose-depend-
ent hypothermia in rats at 25°C and hyperthermia in mice at 30°C. These data suggest a direct or indirect
block of heat gain/conserving effectors in rats treated with sodium pentobarbital which results in
hypothermia and an appropriate compensatory selection of a warmer Ta.
CITATION: Phannacology Biochemistry and Behavior 24(4): 1147-1150, April 1986.
L104 MS-85-193 Project Officer: Gordon
ACUTE BEHAVIORAL TOXICITY of SULFO LANE: INFLUENCE of
HYPOTHERMIA
Patricia H. Ruppeii and Robeat S. Dyer
HERL, USEPA, Research Triangle Park, NC
Sulfolane is a high volume solvent of interest to the Office of Toxic Substances (OTS). Flevious work
showed behavioral and neumphysiological consequences of acute aposure to this compound. This paper
repoats that concwrent hypothermia may account for the severi(y of some of the behaviorai findings.
Sulfolane is a solvent which produces hypothermia and decreased oxygen consumption following acute
exposure. In the present experiment, we investigated effects of sulfolane on a behavioral measure of
toxicity at ambient temperatures which would either prevent or facilitate the development of hypother-
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mia. Adult male Long-Evans rats (N = 10/dose) received a single i.p. injection of saline, 200,400 or 800
mg/kg sulfolane. Motor activity in figure-of-eight m s was assessed 1 h after dosing in testing rooms
maintained at either 20.8°C or at 32.3°C. At the warm ambient temperature, sulfolane produced hypoac-
tivity but not hypothermia. At the cooler temperature, sulfolane-induced hypoactivity was more
pronounced, and rats were hypothermic. Therefore, a behavioral change could be detected at sublethal
dosages of sulfolane in the absence of hypothermia.
CITATION: Toxicology Letters 28(2&3): 111-116, November 1985. -
L104 MS-85-099 Project Officer: Dyer
EFFECTS of HYPOTHERMLAon the IN VIVO MEASUREMENT of RAPID
AXONAL TRANSPORT in the RAT: A CAUTIONARY NOTE
Stephanie PadilIa 1 and Donald Lyerly 2
‘HER! ., USEPA, Research Triangle Park NC
2 Northrop Services, Inc.
C7uznges in aronal Iranspo?t parameters are often used to assess the functional integ,i y of both central and
peripheral nen’ous system newons. This paper Lv basical y a quality control paper. To date, several studies
have had conflicting results when measuring &mnal tianspob’t profiles in dnig- or toxicant-treated animals.
The findings presented in thLc manuscifp4 i.e., that body temperature had a significant influence on the
measurement of axonal transpolt in mammals, should be taken as a cautionwy note for those investigators
exploring peiturbations of &wnal bunspolt in toxicant-treated animals. The experimenter must be very care-
ful not to confuse changes in axonal transpolt due to body temperature with neurotoxic changes due to the
toxicant ctposure.
Rapid axonal transport of glycoproteins was examined in the rethofugal projections of hypothermic and
normothermic adult male Long-Evans hooded rats previously receiving intraocular injections of
[ 3 H]fucose. The amount of retinal fucosylation appeared normal in the hypothermic anim iLc 3.5 h after
isotope injection, but glycoprotein transport was reduced relative to normothermic controls. This reduc-
tion was especially pronounced in the most distal structure of the retinofugal tract (superior colliculus).
We conclude that rapid axonal transport decreases with reductions in mammalian body temperature.
This finding emphasizes the importance of controlling body temperature in in vivo studies of mammalian
axonal transport.
CITATION: Journal of Neurochemisüy 46(4): 1227-1Z30, April 1986.
L104 MS-85-205 Project Officer: Padilla
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YOHIMBINE Al IENUATES the DELAYED LETHALITY INDUCED in
MICE by AMITRAZ, A FORMAMIDINE PESTICIDE
Virginia C. Moser and Robert C. MacPhail
HERI,, USEPA, Research Triangle Park, NC
Amitra a commercially used forrnanzidine pesticide, produced a delayed lethali y in mice that differs
gj’eatly from the time course action of the prototype fonnwnidine chlordimefonn. The finding that him-
bine significantly blocked kthality su est that amitraz exerts this action via stimulation of alpha-adrenergic
receptors. This finding should help OFF hi reg dadng aniitraz by providing a better understanding of the
basis of its toxicity.
We have found that a single dose of amitraz, a formamidine pesticide, produces death in mice 2-5 days
after dosing, To further e nrnine this phenomenon, adult albino mice of both sexes were treated with
either yohimbine (10 mg/kg, i.p.) or deionized water (6 mi/kg, i.p.), immediately before an injection of
amitraz (600 mg/kg, i.p.) and twice daily thereafter for 8 days. Male mice treated with water were more
susceptible than water-treated females to the lethal effects of amitraz. In addition, yohimbine treatment
significantly decreased the number of deaths in both sexes. These data suggest that an a2-adrenergic
mech nisin is involved in the delayed lethality produced by amitraL
CITATION: Toxicology Letters 28: 99-104, November 1985.
L104 MS-85-118 Project Officer: MacPhail
ANALOG FILTERING and INTERPRETING the BRAINSTEM AUDITORY
EVOKED RESPONSE in LABORATORY RATS
Raelyn Janssen, 1 Vemon A. Benignus, 1 Lawu At Games, 2 and Robert S. Dyer’
‘HERL, USEPA, Research Triangle Park, NC
2 Northrop Services, Inc.
The selection offliter bandpass in physiological recording is critical to the resulting waveform and its inter-
pretation. In the development of methods in toxicology, choosing an appropriate bandpass is particularly
important because the distortion due to filtering can mask or decrease the method’s sensitivity to toxic ef.
fects. I37 ile filter distortion is a known phenomenon, this paper repoitc for the first time that substantial
distortion can occur even when the selected bandpass is outside the spectiwn of the waveform. This distor-
tion can result in the temporal reversal of slow and fast peaks in the waveform. Phase shifts reiulting from
the use of a typical analogue filter set are quantifie4 and the specrnun of the brainstem auditoty evoked
response of the rat ispresenteS The paper recommends filtering as little as possible in order to preserve in-
terpretability ofphysiological responses.
Analog filtering of the brain-stem auditory evoked response (BAER) and synthetic wave forms using
steeply sloped filters are shown to produce sign ificant distortion even when filter cut-off frequencies are
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well removed from the wave form spectrum. The degree of distortion is such that it may result in er-
roneous identification of peaks in the BAER. Reversal of the order of peaks may occur with high pass
settings at only 1/4 the lowest constituent frequency. Filter effects were identified as a major source of
cross-laboratory differences in BAERs recorded from laboratory rats. Filter transfer functions of a typi-
cal analog filter set were derived for both gain and phase as a function of frequency. Filtering of synthetic
wave forms was used to elucidate and highlight distortion effects. A typical Long-Evans rat BAER wave
form was spectrum analyzed and conclusions were drawn with respect to appropriate bandpass frequen-
cies.
CITATION: Electroencephalography and Clinical Neurophysiology 65(3): 203-211, May 1986.
L104 MS-85-127 Project Officer: Janssen
FLAVOR AVERSIONS INDUCED by THALLIUM SULFATE:
IMPORTANCE of ROUTE of ADMINISTRATION
David B. Peele, Robeit C. MacPhail and Jacide D. Fanner
HERL, USEPA, Research Triangle Park, NC
Flavor-aversion conditioning has been widely used as an efficient, reliable and sensitive method for deter-
mining the noxious effects of chemicals. This expenment showed that the neurotoxicwzl thallium sulfate in-
duced robust conditioning flavor aversions wizen given orally but not when given intrapentoneally. These
results highlig zt the importance of a methodological detail (mute of exposure) in assessing toxicants with
flavor-aversion conditioning techniques. These findings are relevant to the Office of Toxic Substances
(OTS) and the Office of Pesticide Programs (OPP) in their efforts to evaluate the toxicity of chemicals
based on behavioral assay data. Our results indicate that a failure to identify noxious properties of a
chemical using this type of aversion-learning technique may be due to the mute of exposure rather than to
the intrinsic safety of the chemicaL
Flavor aversions induced by intraperitoneal (i.p.) and oral (p.o.) administration of thallium sulfate were
compared in a repeated trial, two-bottle preference test. Male Long-Evans rats (N = 6/group) were given
30-rn access to a 0.1% saccharin solution followed 20-rn later by either i.p. or p.o. thallium sulfate (2.5,5,
10 or 20 mg kg), vehicle or nothing. Non-treated and vehicle-treated rats consistently preferred the sac-
charin solution, with relative saccharin intakes ranging from 0.65 to 0.85 over the three choice trials. On
the first choice trial, flavor aversions produced by i.p. -administered thallium sulfate were marginal and
occurred only at the highest dosage. In contrast, on the first choice trial, p.o.-adniinistered thallium sul-
fate led to pronounced aversions at all but the lowest doasge. Saccharin preferences on the second and
third choice trials resembled those obtained on the first choice trial. These results suggest that failure to
obtain toxicant-induced flavor aversions may be due in part to the particular route by which the toxicant
is admnistered.
CITATION: Neurobehavioral Toxicology and Teratology 8(3): 273-277, May/June 1986.
L104 MS-85-184 Project Officer: MacPhail
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COMPUTERIZED ASSESSMENT of HUMAN NEUROTOXICJTY:
SENSITIVITY to NITROUS OXIDE EXPOSURE
B. Greenbe g P. Moore, B. Letz, andE. Baker
Harvard University School of Public Health
Results of this study are relevant to project objectives and demonstrate the sensitivity of the computerized
test baste,y to a potentially neuroto.ric gas, nitrous wide.
We recently developed a flexible, portable, computer-based neurobehavioral evaluation system (NES) to
standardize data collection in epid miologic field studies of individuals at risk for neurobehavioral
toxicity. The current study was performed to examine the system’s sensitivity to subtle neurobehavioral
impairment induced pharmacologically in normal subjects. Twelve men 18 to 36 years old were tested
with the NES three times, in an initial training session followed by separate chug and control sessions in
randomized order. During drug sessions subjects received a combination of 20% nitrous oxide and 80%
oxygen through a nasal mask. In control sessions subjects received 100% oxygen. Nine NES tests evaluat-
ing psychomotor performance, visuospatial ability, memory, and mood were administered in ap-
proximately 40 minutes. Nitrous oxide exposure impaired performance on three tests: continuous perfor-
mance, symbol-digit substitution, and finger tapping. Another test, pattern memory, showed a trend
toward impairment, while the remainder were not affected by the drug. These data are consistent with
previous reports that the threshold effect of nitrous oxide is a decrement in psychomotor speed. The find-
ings indicate that our NES is sensitive to short-term impairment in central nervous system function of the
type induced by a number of neuroactive agents. In view of its efficiency in data acquisition and analysis,
computerized neurobehavioral testing appears to be a very promising method for evaluation of effects of
a wide range of pharmacologic agents.
CITATION: Clinical Phannacolorj and Therapeutics 38(6): 656-660, December 1985.
L104 XX-86-072 Project Officer: Otto
NEUROPHYSIOLOGICAL CONSEQUENCES of ACUTE EXPOSURE to
METHYLPYRIDINES
Robeit S. Dyei Linda .1. Bunlette, 2 Raelyn Janssen, 1 and William K Boyes 1
1 HERL, USEPA, Research Triangle Park NC
2 Northrop Services, Inc.
At the request of the Office of Toxic Substances (OTS), the Neurotoxicolog Division evaluated the effects
of acute or 30 day exposure to substituted methylp,yridines. This paper reports the effects of acute exposure
to these compounds. All compounds were sign ificant y neuroactive at 1/2 the i.p. LDSO, although the mag-
nitude of effect at these dosages was low.
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A series of neurophysiological tests was performed on Long-Evans hooded rats treated with either 2-,3-
or 4-methylpyridine at dosages of lOOmg(kg, approximately 1/2 the i.p. LD5O. The tests contained
measures of sensory function (paired pluse flash evoked potentials, pattern reversal evoked potentials,
brainstem auditory evoked responses) and cerebral excitability (pentylenetetrazol (PTZ) seizures and
hippocampal afterdischarges). In general, rats treated with 2- and 3-methylpyridine were more affected
than those treated with 4-methylpyridine. The changes observed were in many ways simlar to those seen
following adninktration of depressant compounds: increased latency of evoked potentials and increased
latency to PTZ seizures. Not all findings, however, were consistent with previously observed patterns of
CNS depression.
CITATION: Fundamental and Applied To4cology 5(5): 920-932, October 1985.
L104 MS-84-127 Project Officer: Dyer
DIETHYLDIThIOCARBAMATE INCREASES DISTRIBUTIONof CADMIUM to
BRAIN BUT PREVENTS CADMIUM-INDUCED NEUROTOXICITY
lamer P. O’Callaghan 1 and Diane B. MUle?
‘HERL, USEPA, Research Triangle Park, NC
2 Northrop Services, Inc.
The results of this study show that a known neurotoxican4 cadmium, affects known nervous-system -specific
proteins in the predicted manner. By validating the use of neivous- ystem-speaj1c proteins in this manner,
the Agency will be provided with a simple, specific and cost-effective means for detecting and charactelizing
neurotoxicily.
Dithiocarbamates exhibit metal-binding properties which have been exploited in a variety of applications,
one of which is chelation therapy for heavy metal toxicity. Such therapy, however, promotes the ac-
cumulation of metals in the brain, a side effect which may result in neurotoxicity. To e lmine this pos-
sibility we used morphological and biochemical indices to assess the effects of diethyldithiocarbamate
(DDC) on cadmium-induced neurotoxicity in the newborn rat. Co-administration of DDC prevented the
neurotoxic effects of cadmium while causing a persistent increase in the distribution of cadmium to brain.
CITATION: Brain Research 370(1): 354-358, April 1986.
L104 MS-85-199 Project Officer O’Ca1Lighi n
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PHARMACOLOGIC and IMMUNOLOGIC APPROACHES to the PROBLEMS of
POSTFRAUMATIC GLIAL PROLIFERATION FOLLOWING CNS DAMAGE
M. L Billingsley, 1 C. D. Balaban, 1 and I. P. O’Cailaghan 2
1 The Pennsylvania State University
USEPA, Research Triangle Park, NC
This paper reviews recent progress in validating the use of ne vous-systein-speciflc proteins as biochemical
indicators of neurotoxzci y. Validation of this neurotoxici y testing scheme will provide the Agency with a
simple, specific and cost-effective means for the detection and characterization of neuroto.ucity.
We have devised a pharmacologic approach to block the proliferation of glial cells (gliosis) which follows
various forms of trauma to nervous tissue. A method was devised using the incorporation of 3 H-
thymidine incorporation into DNA of glial cells as a proliferative index following mechanical trauma.
Both biochemical and histologic analysis revealed that treatment of lesioned rats with cell-cycle specific
antimitotic agents such as cytosine arabinoside reduced glial proliferation as measured by both DNA syn-
thesis and morphologic quantitation of cell numbers around the lesion. Treatment with cytosine
arabinoside resulted in the appearance of numerous unusual cells near the lesion, with indications of
aborted mitotic spindles in many of these cells. Other anitimitotic drugs such as cyclophosphamide and
vincristine were also able to inhibit glial proliferation. Additional experiments suggested that im-
munosuppression prior to lesioning greatly reduced the proliferative response of glial cells. We are also
using treatments with neurotoxicants such as kainic acid and trimethyl tin to produce non-mechanical
neuronal d imige and subsequent gliosis. Preliminary results suggest that these toxicants reduce the un-
munocytochemical appearance of neuronotypic proteins, and that glial fibrillary acidic protein is marked-
ly elevated in tissues which lose neurons. The relative merits of both mechanical and chemical models for
inducing gliosis will be discussed, as will the therapeutic possibilities of selected anitiinitotic agents.
CITATION: In: NATO Advanced Research Workihop: Glial-Neuronal Communication in Development
and Regen eration, West Germany, 1985.
L104 MS-86-043 Project Officer: O’Callaghan
ANALYSIS of the VOLUME of RED BLOOD CELLS: APPLICATION of the
EXPECTATION-MAXiMIZATION ALGORIThM to GROUPED DATA FROM the
DOUBLY-TRUNCATED LOGNORMAL DISTRIBUTION
Christine E. McLaren, 1 Ga,y M. Brittenham 1 and Victor Hasselblad 2
1 Case Western Reserve University
2 HERL, USEPA, Research Triangle Park, NC
The statistical estimation techniques developed and applied to this red blood cell volume data are equally
effective in dealing with any set of grouped doubly-truncated data. For example some studies have
recorded blood lead values as being inside a range of values rather than giving an exact number. Some pes-
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licide resithie values are recorded as in inteivals of values or as being below the minimum detectable level
of measure nent. In these recuirences, this mathematical model and parameter estimation technique will
provide a methodology for a complete and effective analysis of such data sets where no other reliable tech-
nique eüsU. This statistical method s development should therefore help program offices to effectively use
chemical burden data that was previously of quite limited value because no such estimation procedure ex-
isted for the problem prior to this approach.
In accordance with general principles recommended by the International Committee for Standardization
in Haematology (1982, Journal of Clinical Pathology 35: 1320-1322), we have developed statistical
methods for the analysis of red cell volume distributions. To select an appropriate reference distribution
for goodness-of-fit testing, we derived a mathematical model of erythropoiesis that predicted a lognormal
form for the distribution of erythrocyte volumes. Model predictions were then tested using samplesob-
tamed from 50 healthy individuals. Each grouped red cell volume distribution was doubly-truncated to
eliminate artifactual frequency counts. Distribution parameter estimates were computed using the ex-
pectation-maximization algorithm, a mkcng information technique. Results of the one-sample cl ii-
square goodness-of-fit test showed a fairly even distribution of P-values over the interval [ 0,1]. Examples
of the application of these statistical procedures to distributions from patients with anemia are given. Our
results suggest that, for the analysis of red blood cell volumes, (i) parameter estimation should be made
with the ex cttion-m imi7Jation method, and (ii) the truncated lognormal distribution should be used
as a reference distribution for goodness-of-fit testing. This method could be applied to any set of grouped
doubly-truncated data which, after transformation, follows the normal model.
CITATION: Biometrics 42:143-158, March 1986.
L104 MS-84-150 Project Officer: Creason
STRUCTURE ACTIVITY RELATIONSHIPS (SARs) AMONG MUTAGENS
and CARCINOGENS: A REVIEW
M. R. Frierson, G. Klopman, and H. S. Rosenloanz
Case Western Reserve University
This paper reviews methods for obtaining structure-activity relationships, particularly those that have been
applied to mutagenicity and carcinogenicity. It discusses methods that rely on both physical properties and
molecular connectivity. It explores their potential usefulness.
This review is an introduction to methods for evaluating structure-activity relationships (SARs), and, in
particular, to those methods that have been applied to study mutagenicity and carcinogenicity. A brief
history and some background material on the earliest attempts to correlate molecular structure and
biological activity are included. Most of the discussion focuses on modern methods utilinng extrather-
modynamic and physical property variables such as the Hansch method and SIMCA, and approaches
based on molecular connectivity such as the ADAPT, CASE, and Enslein methods. In general, the latter
class is potentially the most useful in the study of the large and structurally diverse databases so often en-
countered in the study of mutagenicity and carcinogenicity. They also are not very sensitive to lab-to-lab
variances in reported activities and outright misciassifications in activities of some compounds. This is
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chiefly because the statistical treatments used in these methods tend to dilute the importance of outliers.
The methods using physicochemical and extrathermodynaniic variables are especiafly important in rela-
tively small, congeneric databases and can help fine-tune the role of physicochemical properties in
mechanistic hypotheses. All of the above methods have been used to look at mutagenicity and car-
cinogenicity and some of the results reported in the literature are reviewed here. As far as specific
methods go, ADAPT, CASE, SIMCA and the Enslein approach all seem to have similar classification
powers (in the range of 75-95%), depending very much on the database studied. The emphasis in this
review is on showing that the use of these computer-aided storage, retrieval and analysis techniques is a
timely approach to predicting and even understanding the toxicity of environmental substances.
However, each of the methods discussed is still under development, and their potential usefulness for
predictive purposes is still being explored.
CITATION: EnvironmentalMutagenesis 8(2): 283-327, March 1986.
L104 XX-86-106 Project Officer: Rabinowitz
STRUCTURE-GENOTOXIC ACTiVITY RELATIONSHIPS of PESTICIDES:
COMPARISON of the RESULTS FROM SEVERAL SHORT-TERM ASSAYS
G. Klopman, 1 R. Contreras, 1 H. S. Rosenk,unr, andM. D. Waters 2
1 Case Western Reserve University
2 HERL, USEPA, Research Triangle Park, NC
This is the first example of the application of the CASE Computer-Automated Structure Evaluation
program to examine the chemical structural basis for the response of shoit-tenn genetic bioassays applied to
pesticide chemicals. The study successfully delineated two chemical fragments, a methoxyphosphinyl and a
chiorovinyl group, as the common structural subunits responsible for the biological activities detected in the
battery of tests employed. This methodology shoul4 therefore, be very valuable to the Office of Pesticide
R’ogmms in detennining which test systems to apply or to require in the evaluation of genotoxic hazards of
pesticide chemicals. By analogy, the same methodology should be of value to other program offices in
evaluating chemicals in their sphere of regulatory concern.
The Computer-Automated Structure Evaluation (CASE) program has been applied to the analysis of the
genotoxic activity of 54 pesticides (31 insecticides, 15 herbicides and 8 fungicides) in 5 different short-
term test systems measuring gene mutation and DNA damage. The database contains compounds
presenting diverse structures including carbamates, thiocarbamates, organophosphates, halo-aromatics
and other functionalities. Some significant relationships between common structural features and the
genotoxic activity displayed by these chemicals have been found. Among the most relevant fragments,
automatically selected by the program, a methoxyphosphinyl and a chiorovinyl group appear as the com-
mon structural subunits responsible for the activities detected in the battery composed of the Salmonella
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lyphiinw’ium histidine reversion assay, the mouse lymphoma gene mutation assay and recombination in
the yeast Saccharomyces ceievisiae.
CITATION: Mutation Research 147:343-356, December 1985.
L104 MS-86-045 Project Officer: Waters
A FINITE EXPANSION METHOD for the CALCULATION and
INTERPRETATION of MOLECULAR ELECTROSTATIC POTENTIALS
JR. Rabinowitz; 1 K Nambood4 2 and H. Weinstein 2
1 HERL, USEPA, Research Triangle Park, NC
2 City University of New York
The molecular electrostatic potential (MEP) is a measure of the capacity of a molecule to interact with its
sunuundmgs and has been found to provide useful insight into the differential biological activity within
families of chemicals. It could provide an important parwneter for the assessment of potential chemical
toxicity from chemical struciwe. The d iculty in using the MEP in a structure activity study for toxicity Ls
the dIfficulty in its computation and characterization. This paper derives and tests a rapid method for com-
putation of the MEP and provides metho& for its characterization. These advances will facilitate its use in
structure activity (&4R) studies.
Because it is useful to have the molecular electrostatic potential as an element in a complex scheme to as-
sess the toxicity of large molecules, efficient and reliable methods are needed for the calculation and
characterization of these potentials. A multicenter multipole expansion of the molecular electron charge
density calculated with a limited Gaussian basis set is shown here to have only a finite number of nonzero
terms from which the molecular electrostatic potential can be calculated. The discrete contributions to
the electrostatic potentials from the terms of this expansion provide a physically meaningful decomposi-
tion of the potential and a means for its characterization. With pyrrole as an example, the electrostatic
potential calculated from this finite expansion of the electron density is compared to that obtained from
exact calculations from the same wave function. Good agreement is obtained at distances greater than
13 A from any atom in the molecule. In contrast, rearrangement of the terms into an expansion cor-
responding only to Mullilcen atomic charges and dipoles yields a decomposition that produces electros-
tatic potentials which agree less well with the exact potential. This discrepancy is attributable to the
neglect of terms due to higher moments.
CITATION: International Journal of Quantum Chemistry 29(6): 1697-1704, June 1986.
L104 MS-85-150 Project Officer: Rabinowitz
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A SIMPLE METHOD for the REPRESENTATION, QUANTIFICATION and
COMPARISON of the VOLUMES and SHAPES of CHEMICALS
T.R.StouchandP. C Jurs
The Pennsylvania State University
In the premanufacture notification program of the Office of Toxic Substances (OTS) and in other cir-
cumstances, the assessment of potential chemical toxicity must bepe fonned when all the relevant bioassay
infonnalion Lv not available. In these cases, quantitative stnscture activity relationships that use pattern
recognition techniques like ADAPT can provide valuable assistance. An important characteristic used in
these methods and in other relevant ,nethoiLs is the volume and shape of chemicals. This paper presents a
method for quantifying and using these characteristics in s1mctw activity studies.
A conceptually and computationally simple method for the definition, display, quantification, and corn
parison of the shapes of three-dimensional mathematical molecular models is presented. Molecular or
solvent-accessible volume and surface area can also be calculated. Algorithmc, progrslmming considera-
tions, accuracy, and time and storage requirements are discussed. The method requires no extensive
progr mmhig skills and could be implemented on a desk-top computer.
CITATION: Journal of Chemical Information and Computer Sciences 26(1): 4-12, February 1986.
L104 XX-86-077 Project Officer: Rabinowitz
USE of a GRAPH THEORETIC SIMILARITY INDEX in PREDICTION
STUDIES of LINEAR DISCRIMINANTS and MODELS
E.P.JaegerandP. C.Jurs
The Pennsylvania State University
In the premanufaciure notification program of the Office of Toxic Substances (OTS) and in other cir-
cumstances, the assessment of potential chemical toxicity must beperfoimed when all the relevant bioassay
infonnazion Lv not available. In these cases, quantitative st?ucture activity relationships that use pattern
recognition techniques like ADAFTcan provide valuable assistance. This paper develops a new method to
compare the sbuctural similarity of chemicals by using the relative frequency of all unique atom pairs in
each chemical being compareS
A goal of many structure-activity and structure-property studies is to develop the capability to predict the
activity or property of interest for previously untested compounds. A quantitative model or a dis-
criminant is developed from a training set of molecules with known activities or properties. The best test
of the predictive ability of a model or a discriminant is to predict the activities or properties of a set of un-
known compounds, so the agreement between predicted and observed values can be judged. Unknown
compounds which are structurally simibir to those of the training set stand the best chance of being
predicted accurately. Structural similarity can be assessed with atom pairs. The metric is based on the
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relative frequencies of all unique atom pairs in each compound being compared. The method can be used
to compare two molecules, or it can be used to characterize a set of compounds, or it can be used to com-
pare a few compounds (a prediction set) to a large collection of compounds (a trnining set). The charac-
teristics of the atom pair similarity measure are discussed and examples of its use in the selection of
prediction set members in several structure-activity and structure-property studies are shown.
CITATION: 1n Proceedings of the 2&h ORNL-DOE Conference on Ana ytical Chemirby in Energy and
Technology, Knoxville, TN, October 1985.
L104 XX-86-081 Project Officer: Rabinowitz
PArr1 RN RECOGNITION STUDIES of COMPLEX CHROMATOGRAPHIC
DATA SETS
P. C. Jurs, B.KLavine and T. R. Stouch
The Pennsylvania State University
In the premanufacture noftfication pmgmnz of the Office of Toxic Substances (OTS) and in other cir-
cwnstance, the assessment of potential chemical toxicity must be performed when all the relevant bioassay
information is not available. In these cases, quantitative stnscture activity relationships that use pattern
recognition techniques like ADAPT can provide valuable assistance. These pattern recognition techniques
may also be used to identify chemicals in complex chromatographic data sets. It discusses Monte Carlo
simulations to assess the probability of chance classification of data which is also important for pattern
recognition studies of bic-effects data.
Chromatographic fingerprinting of complex biological samples is an active research area with a large and
growing literature. Mukivariate statistical and pattern recognition techniques can be effective methods
for the analysis of such complex data. However, the classification of complex samples on the basis of their
chromatographic profiles is complicated by two factors: 1) confounding of the desired group information
by experimental variables or other systematic variations, and 2) random or chance classification effects
with linear discriminants. We will treat several current projects involving these effects and methods for
dealing with the effects. Complex chromatographic data sets often contain information dependent on ex-
perimental variables as well as information which differentiates between classes. The existence of these
types of complicating relationships is an innate part of fingerprint-type data. ADAPT, an interactive
computer software system, has the clustering, mapping, and statistical tools necessary to identify and
study these effects in realistically large data sets. In one study, pattern recognition analysis of 144
pyrochromatograms (PyGCs) from cultured skin fibroblasts was used to differentiate cystic fibrosis car-
riers from presumed normal donors. Several experimental variables (donor gender, chromatographic
column number, etc.) were involved in relationships that had to be separated from the sought relation-
ships. Notwithstanding these effects, discriniins nts were developed from the chromatographic peaks that
assigned a given PyGC to its respective class (CF carrier vs normal) largely on the basis of the desired
pathological difference. In another study, gas chromatographic profiles of cuticular hydrocarbon extracts
obtained from 179 fire ants were analyzed acing pattern recognition methods to seek relations with social
caste and colony. Confounding relationships were studied by logistic regression. The data analysis tech-
niques used in these two example studies will be presented. Previously, Monte Carlo simulation studies
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were carried out to assess the probability of chance classification for nonparametric and parametric linear
discriminants. The level of expected chance classification as a function of the number of observations, the
dimensionality, and the class membership distributions were ex’ niined. These simulation studies estab-
lished limits on the approaches that can be taken with real data sets so that chance classifications are im-
probable.
CITATION: Journal of Research of the National Bwvau of Standards 90(6): 543-549, November/Decem-
ber 1985.
L104 XX-86-076 Project Officer: Rabinowitz
A NUMERICAL INDEX for CHARACTERIZING DATA SET SEPARATION
D. H. Lafeinina andP. C. lw’s
The Pennsylvania State University
In the premanufacture notification program of the Office of Tozc Substances (OTS) and in other cir-
cwnstances, the assessment of potential chemical toxicity must beperfonned when all the relevant bioassay
info,, nation is not available. In these cases, quantitative sliucture activity relationships that use pattern
recognition techniques like ADAPT can provide valuable assistance. In performing stracture activity
studies with ADAPT, both positive and negative data are needed. This paper descnbes a method/or detect-
ing the relative degree of separation between positive and negative chemicals in the space of the descriptors
used.
A method is reported for assessing the degree of separation between two dusters of points representing
chemical data. The method, based on trend vectors, has been tested on both randomly generated and ac-
tual data sets. The index is intended to be a quick method for detecting the relative degree of separation
between data sets (i.e., suitability of descriptors being used) in structure-activity studies or other pattern-
recognition studies.
CITATION: Journal of Chemical Information and Computer Sciences 25(4): 386, November 1985.
L104 XX-86-075 Project Officer: Rabinowitz
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THE EFI i CTS of MATERNAL MURINE CYTOMEGALOVIRUS INFECTION on
the MOUSE CONCEPTUS at DIL(IERENT GESTATIONAL STAGES
Yuan-Shen Huang Neil Chenzoff, Robe,! I. Kaviock, and
Clinton Y. Kawanishi
HERL, USEPA, Research Triangle Park NC
This research demonstrates that the teratological screening protocol developed in this laboratory for testing
chemical toxicants can be used for assessing the prenatal effects of viral infections. The study is relevant to
the development and standardization of tests for microbloipesticidal agents which include viiuses.
The effects of murine cytomegalovirus (MCMV) on the prenatal development of the CD-i mouse were
investigated. Two sets of experiments were performed. In the first, mice were inoculated with different
doses of MCMV on gestational day 7, and in the second, pregnant inimi 1s were inoculated with a sub-
acute injection dose at different gestational stages. The effects of maternal infection on pregnancy in
terms of maternal sickness, embryo lethality, date of parturition, litter size, postnatal death, and pups’
body weight on d 1 and d 3 postpartum were investigated. High-dose MCMV infection on d 7 of preg-
nancy resulted in a significant increase in early embryo resorption and also in a reduction of the neonatal
body weight of surviving pups. Two gestational stages were identified as being especially susceptible to
MCMV infection. Most embryonic death as indicated by resorption rates was found after treatment on
d 9, whereas perinatal death was most frequent when treatment was done on d 1.3 of gestation. Still births
and neonatal death within 24 h of birth were found commonly in this latter group, which also showed the
most pronounced growth retardation. The phenomenon of delay in time of parturition was noted and
found to be most significant in groups of inimzi1s that were inoculated on d 3 or d 13. This investigation
suggests that the effects of MCMV on the CD-i mouse vary greatly with the age of the embryo and the
course of the infection.
CITATION: Teratogenesu, Carcinogenesis, and Mutagenesis 6(4): 331-338 August 1986.
L104 MS-86-002 Project Officer: Huang
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SEPARATION of
CHLORDECONE and ITS METABOLITES
S.E. George, L.C. KIng and LD. Cloxton
HERL, USEPA, Research Triangle Park, NC
In order to develop health effects modeLc for genetically engineered microorganisms (GEMS) that degrade
hazardous substances, it Lv sometimes necesswy to develop the chemical methods to monitor for the
microbial degredation proda cis.
High performance liquid chromatography (HPLC) was used to separate chiordecone, Kepone, and two
of its metabolites, hydrochlordecone and dihydrochlordecone. Elation of the three peaks occurred after
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the solvent concentration reached 100% and was maintained at 100% methanol for approiimately five
minutes. The method was linear for chiordecone in the concentration range of 10 to 100 gig.
CITATION: Owomato uphia 22(1-6): 165-168, June 1986.
L104 MS-86-092 Project Officer Claxton
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Research Objectives
Hazardous Waste

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HAZARDOUS WASTE RESEARCH PROGRAM
Objective: To improve the basis for regulatory activities under the Resource Conservation and
Recovery Act (RCRA).
Background: The RCRA was created in 1976 to minimi7. risks from h rdous wastes from genera-
tion to disposal.
Hazardous Wastes - HERL scientists are conducting the following research in support of this legislation.
• Develop and evaluate short-term in vivo and in vitro bloassays for screening wastes for designation
as h rdous. Bioassay screens are being evaluated for the following health effects. General
toxicity, carclnogenlclty, neurotox1cIty immunotoxicity, terategeniclty and reproductive effects.
Rationale: Section 3001 of the RCRA requires EPA to promulgate criteria for identifying the charac-
teristics of hnim dous wastes and the listing and delisting of h rdous wastes. Because of the large num-
ber of wastes to be screened, the Office of Solid Waste needs a rapid, inexpensive bioassay to use in
prioriti ing which wastes are most important for toxicological characterization and where they should in-
vest limited resources.
Approach: Developed bioassays will be assembled into a screening protocol and a prescreen protocol
using a brief uiini l exposure protocol followed by a closely integrated matrix of short-term toxicological
tests. This test battery will be evaluated with dose-response validation studies using well-characterized
chemicals previously tested in .nim aI studies.
• Provide improved methodologies for the assessment and prediction of toxicity of the balogenated
dioxins and related compounds.
Rationale: The polyhalogenated dioxins and their congeners rank at or near the top among the most haz-
ardous materials encountered at waste sites. Improved toxicity assessment and prediction methodology
will be useful to the Agency in m iklng health hn rd evaluations at waste sites where dioxin-like materials
are present.
Approach: Methodologies to be studied will include both in vitro and in viw procedures along with struc-
ture activity research. Female marmosets will be exposed to TCDD throughout gestation and fetuses ex-
amined by standard tertologic procedures. The capacity of various PCDD’s, PCDFs, and mixtures to in-
duce AHH/EROD enzymes, to bind specific receptors, and to cause XB cell ker’tini’ntion will be
studied. The molecular electrostatic potential and other key features of recogni’ ’tion processes of
selected dioxin-like chemicals will be computed and analyzed.
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Accomplishments
Hazardous Waste

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ACCOMPLISHMENTS HAZARDOUS WASTE
Issue Waste Characterization of the Bioassay Screen.
An in vim/in viDo Toxicological Screen ox Screen) has been developed to use methods that are
designed to be rapid, inexpensive and capable of screening large numbers of wastes for biological activity.
Emphasis is placed on identifying a wide range of potential toxic responses associated with each waste by
employing diverse test methods. The toxic endpoints identified by the screen include mutagenesis/car-
cinogenesis, general toxicology, neurotoxicology, reproductive toxicology, teratology, and im-
munotoxicology. The protocol, called the Toxicological Screen, involves the oral adnuinictration of waste
material to rats for 10 consecutive days. At the end of the 10-day period the whole inimziI , body tissues
and fluids are evaluated for toxicity. A major goal of the Toxicological Screen is to maximize the amount
and type of potential health effects information that can be obtained by exposing metabolically competent
intact to complex waste mixtures.
The Toxicological Screen is being validated by ‘ icing a series of compounds of known and defined toxicity.
The validation study ensures that the protocol will be capable of detecting biological activity associated
with each compound and identifying those assays which most readily detect the toxic potential of the com-
pounds. A selected number of the most accurate and sensitive assays could be used to constitute a
Prescreen for the entire protocol. The Prescreen could then be used (with an abbreviated form of the in
vim exposure regime) as a rapid means of prioritizing waste samples to be evaluated in the full Tox
Screen. it is important to note here that a large number of compounds of known toxicity and no toxicity
must be evaluated in the Tox Screen before bioassay tests for the Prescreen would be initiated on wastes.
These tests would constitute a validation study to establish the sensitivity, specificity and accuracy of the
Tox Screen.
Initial trials of the validation study with acrylamide, chiordecone (kepone), cyclophoshamide, and
diethylsti lbcstrol (DES) demonstrate the feasibility of the Toxicological Screen for detecting biological
activity of chemicals.
Acrylamide, evaluated at five test concentrations renging from 3.75 to 60 mg/kg in rats, produced its
greatest toxic effects in motor activity, responses to acoustic stimuli (neurotoxicology), and in the induc-
tion of sister chromatid exchanges (mutagenesis). Most other tests resulted in positive responses at the
highest concentration tested, a concentration which produced 30% lethality.
The reproductive assays were most sensitive in detecting the toxic potential of chlordecone (evaluated at
0.625 to 10.0 mg/kg in rats). Testicular sperm was markedly reduced, demonstrating that chiordecone is
a reproductive toxin. This observation agrees with previously reported studies of sterility in chemical
workers employed in chlordecone manufacturing in Hopewell, Virginia. Had this sample been an un-
known h rdous waste mixture the observed results would suggest the presence of a potential reproduc-
tive toxin and would indicate a need for further research.
The toxicity of cyclophosphamide (1.5 to 24mg/kg in rats) was most readily identified by the inimunologi-
cal and mutagenesis tests. Most other tests responded at the highest concentration tested, which resulted
in 70% leth i1ities . Cyclophosphamide produced mutagenic metabolites in the urine of treated rats.
These metabolites were detectable using the Salmonella histidine reversion assay. Increases in sister
chromatid eichknges were identified in the bone marrow at each exposure concentration (1.5 to 24.0
mg/kg rat). Dose-response relationships were established for each of the immunotoxicity tests with
cyclophosphamide. Also, there were significant reductions in thymus and spleen weights at all concentra-
tions tested. Body weight reductions were observed only at the two highest doses. These results indicate
that generalized body weight loss is not predictive of potential immunotoxicity.
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HAZARDOUS WASTE ACCOMPLISHMENTS
The exposure of lnim2Ic to DES also resulted in the generation of a number of dose-response relation-
ships in several areas. Almost all parameters evaluated in general toxicology, neurotoxicology, im-
munotoxicology, reproductive toxicology, and teratology were affected at the lowest concentration tested,
31.25 mg4 The only test category which did not detect the toxicity of DES was mutagenesis which is
not surprising since this toxin is not a bacterial mutagen nor does it induce SCEs.
The results of the validation study to date suggest that the Tox Screen is capable of detecting a range of
biological activity of the pure compounds as well as the type of activity associated with the pure com-
pounds. Four compounds, however, are insufficient for establidiing the required sensitivity (0.9),
specificity (0.75), and accuracy specified by OSW or to select the best tests for the abbreviated prescreen.
The number of compounds required to identify prescreen assays and to establish the accuracy of the Tox
Screen (with a 90% sensitivity) requires a minimum of 15 to 20 biologicafly active compounds for each of
the six disciplines of the Tox Screen (assuming no overlap in toxicity) and 15 to 20 control compounds
without biological activity (assnniing 100% overlap in toxicity across endpoints). This results in a total of
105 to 140 compounds. The evaluation of this many compounds may be a prohibitive task because of both
time and cost limitations.
As a result the Tox Screen is being re-evaluated with respect to the treatment protocol and the toxicologi-
cal assays which it employs. Several questions must be answered as part of this re-evaluation process and
before validation of the Tox Screen can be designed.
1. Is a 10 day exposure of a waste material adequate for predicting chronic effects? Should a 30 or 90 day
exposure regime be employed?
2. Are there toxicity tests which could be added or deleted to improve the overall performance and sen-
sitivity of the Tox Screen?
3. Can certain in vivo tests be replaced with in vitio tests to reduce cost without reducing sensitivity or ac-
curacy?
4. Is 90% sensitivity and 75% accuracy actually required?
In addition to addressing the above questions, several new areas of research are being pursued as part of
an ongoing developmental program in the evaluation of complex mixtures.
1. The development of short term in vitio genetic mutation and liver toxicity bioassays for the identifica-
tion of hazardous waste samples possessing acute and chronic biological effects.
2. The development of new and improved in vivolin vitm screening methods in the areas of liver toxicol-
ogy, neurotoxicology, immunotoxicology, and developmental biology for addition to the integrated Tox
Screen.
3. The investigation and resolution of some of the basic toxicological problems related to complex mix-
tures and the screening assay (i.e., acute vs. chronic effects and comparison of in vivo and in vitm
methodo1onies .
This report covers a period from April 8, 1983 to June 22, 1985 and work was completed as of April 21,
1986.
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ACCOMPLISHMENTS HAZARDOUS WASTE
lssue Dioxin
Many isomers and chemicals related to 2,3,7, 8-TCDD and 2,3,7, 8-TCDF were synthesized to deter-
mine the structure activity relationship of the numerous chemicals itcing prominent biomarkers as
endpoints. The receptor binding, the induction of AHH and EROD, both in vivo and in vibo were deter-
mined which led to the following: 1) there is a definite structure-activity-relationship relative to the
toxicity of the various dioxins and furans with the most toxic being the 2,3,7,8- configuration of the tetra-
penta-, and hexa- TCDDs and TCDFs in both in vWo and in vivo assays. Additionally, it was determined
that the in vi o assays could be used to determine the toxicity of compounds and mixtures in terms of
dioxin equivalents. Combinations of the TCDd and TCDf with related chemicals tested in the in vivo and
in vitro systems determined that some combinations inhibited the effect while other combinations poten-
tinted or were additive.
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Publications
Hazardous Waste
This report includes abstracts of manuscripts for journal articles published and conference
proceedings papers presented between October 1, 1985 and September 30,1986...
Programmatic Relevancy statements are italicized.
Other articles submitted for publication, but not published as yet, are listed in Appendix A.

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PUBLICATIONS HAZARDOUS WASTE
THE EVALUATION of FOUR TOXIC CHEMICALS in an IN VWO/IN VITRO
TOXICOLOGICAL SCREEN: ACRYLAMIDE, CHLORDECONE,
CYCLOPHOSPHAMIDE, and DIETHYLSTILBESTROL
B. 7 K B. Mos4 2 1. Ailei 3 E. Berman, 3
N. chernoff, 3 K Dean, 3 D. DeI%lañnz, 3 J. Lasice,y ,
R. MacPho4 3 L. Reite, 3 and R. Smialowicf
1 Environmental Health Research and Testing Inc.
2 Northrop Services, Inc.
3 HERL, USEPA, Research Triangle Park, NC
This docwnent descnbes a method to identify biologicali y active hazardous waste samples and process
residuals. The method Lv capable of screening laige nwnbers of wastes in a cost- and time-effective manner
by using a diverse but integrated battery of in vivo/in vitro tests to assay for mutagenesis/carcinogenesis,
general toxIci4 reproductive toxicity, teratology neurotoxicity and immunotoxicity. Such a bioassay screen-
ing procedure for identifying biological y active wastes provides an important tool to EPA for setting stand-
anis in the management of hatardous wastes and process residuals.
An in vivo/in vWo Toxicological Screen (Tox Screen) has been developed for screening large numbers of
wastes for biological activity. Emphasis is placed on identifying a wide range of potential toxic responses
by employing diverse test methods with toxic endpoints in mutagenesis/carcinogenesis, general toxicol-
ogy, neurotoxicology, reproductive toxicology, teratology, and immunotoxicology. Oral adrnnktration of
waste material is given to rats for 10 consecutive days after which the whole nim i1 , body tissues and fluids
are evaluated for toxicity. The Tox Screen is being validated to ensure that the protocol will be capable
of detecting biological activity and to identify those assays which most readily detect toxicity. The most
accurate and sensitive assays would be used as a Prescreen for the entire protocol. Results of the valida-
tion study with 4 toxic chemicals are included. Acrylamide produced its greatest effects in the neurotox
assays and in mutagenesis. The reproductive assays were most sensitive in detecting the toxicity of chior-
decone. The toxicity of cyclophosphamide was most readily identified by the immunological and
mutagenesis tests. Diethyistilbestrol produced dose-response effects in all disciplines. The Tox Screen
is currently being re-evaluated to answer questions concerning the length of exposure and the need to add
and/or elimin itc certain tests to increase sensitivity and accuracy. In addition, several new areas of re-
search are being pursued in add tivity/synergism/antagonism, chronicity and in vitro to in vivo extrapola-
tion.
CITATION: U.S. Environmental Protection Agency, EPA-600/1-86-002, May 1986.
D109 HERL-0542 Project Officer: Lewtas
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HAZARDOUS WASTE PUBLICATIONS
SCREENING COMPLEX HAZARDOUS WASTES for MUTAGENIC ACTIVITY
USING a MODIFIED VERSION of the TLCISALMONELLA ASSAY
Virginia Ste wait Houlç 1 ’ 2 and Lw y D. Claxton 1
USEPA, Research TriangJe Park, NC
2 University of North Carolina-Chapel Hill
This research demonstrates the potential usefulness of an in ipensive and simple fractionation/bioassay
scheme that couples thin layer chromatography with the Salmonella mutagenesis assay. The study showed
that cnide hazardous wastei can be successfully bioasstryedformutagenic components in this manner; 7 of
10 hazardous wastes demonstrated mutagenic activi(y when tested by this metho4 The assay can be applied
to any number of complex mLt res, and should help progrwn offices to quickly and ine pensively assay en-
vuvnmental samples for mutagenic constituents.
Ten complex hazardous wastes were tested for mutagenic activity using a modified version of the
TL( f Salmonella assay developed by Bjørseth et aL (1982). This fractionation/bioassay scheme couples
thin-layer chromatography (TLC) with the Salmonella/miamm iIi n microsome (Ames) assay for the
detection of mutagenic constituents in complex mixtures. Crude (unadulterated) h rdons wastes and
selected hi 7i Tdous waste extracts were fractionated on commercially available cellulose TLC plates.
Mutagenicity testing was performed in situ by applying a single overlay of minim al growth agar, tester
stain TA98 or TA100, and the optional metabolic activation system directly onto the developed
chromatogram. A mutagenic effect was indicated either by the appearance of localized clusters of rever-
tant colonies or by an increase in total revertant growth vis-a-vis control plates. Seven of 10 hJl7ilrdous
wastes (including tars, emulsions, sludges, and spent acids and caustics) demonstrated mutagenic activity
when tested by this method. To assess the sensitivity of the modified TLC!Salmonella assay, 14 Salinonel-
Ia mutagens from a wide range of chemical classes and polarities were tested. Selected compounds in-
cluded heterocydlics, aromatic amines, alkylating agents, antitumor agents, a nitrosamine and a
nitroaromatic. Eleven of the 14 mutagens were positive in this test system. The three compounds refrac-
tory to analysis included a polycyclic aromatic hydrocarbon and two volatiles.
CITATION: Mutation Research 169(3): 81-92, March 1986.
D109 MS-85-L55 Project Officer: aazton
OBSERVATIONAL BATFERIES and MOTOR ACTIVITY
Robest C MacPhail
HERL, USEPA, Research Triangle Park, NC
This paper describes the needs for and approaches to screening chemicaLs for neurotauci(y, and should be
of benefit to the Fmgram Offices as a background document
The U.S. EPA estimates that there currently are as many as 60,000 chemicals used in this country and that
as many as 1000-2000 new ones are introduced into commerce each year. Unfortunately, the ability to as-
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PUBUCATIONS HAZARDOUS WASTE
sess and predict the possible health hazards associated with exposures has not kept pace with the produc-
don of new compounds. As a consequence, there is growing concern that many chemicals introduced
originally to improve our lives may in fact be producing a range of organ-related toxicities and disease
states. A recent publication of the National Research Council (1984), for example, estimated that for the
most chemicals sufficient toxicity information was not available to conduct an adequate health-hazard as-
sessment. As a result the toxic potential of many chemical is often not appreciated until large numbers of
the population have been exposed to them. Under these conditions incidents such as the Ginger Jack
poisoning episode in the United States, and the methylmercury poisoning episodes in Japan and Iraq are
poignant reminders of the extent to which the nervous system may be at risk for exposure to environmen-
tal chemicals. Clearly, then, there is a pressing need for methods suitable for rapidly evaluating the
neurotoxic potential of a broad array of chemicals.
CITATION: In: Symposium on Neurobehavioral Method.s in SafetyAssessment of Chemicals and Thugs,
Dusseldorf, Germany, December 16-19, 1985.
D109 MS-86-093 Project Officer: MacPhail
A COMPARISON of the TOXICITY of ACRYLAMIDE, CYCLOPHOSPHAMIDE,
CHLORDECONE, and DIETHYLSTILBESTROL in CHINESE HAMSTER
OVARY (CHO) CELLS with THEIR TOXICITY IN VIVO
Jane Ellen Simmons, 1 Marcus Jackson, 2 Joellen Lewuzs, 3 and Ezra Bennan 3
1 HERL, USEPA, NRC Associate
2 Environmental Health Research & Testing, Inc.
3 HERL , USEPA, Research Triangle Park NC
This paper descabes and compares the toxicity of four chemicals (acayiwnide, chlordecone, cyclophos-
phamide, and diethyistiThestrol) in vivo, after 10 days of repeated dosin& with their toxicity in vitro, in a
Chinese hamster ovasy cytotoxicity assay. This study provided data to interpret and understand the results
of this in vim toxicity assay as they could apply to the in vivo situation. This woik also points out the need
to devise and utilize in vitro toxicity assays with metabolic capabilities that closely approximate the metabo-
lic capability of the intact animaL
In order to compare in vitro toxicity with in vim toxicity, four chemicals that have been tested in the in
vivo/in vitro toxicological screen proposed by the Health Effects Research Laboratory, EPA were tested
in a Chinese Hamster Ovary (CHO) cytotoxicky assay. Viability index, cellular ATP concentration and
rate of protein synthesis were decreased by exposure to acrylamide (AC), chlordecone (CH), diethyistil-
bestrol (DES), and cyclophosphamide (CY) (P < 0.00001). All of these chemicals were active in the
CHO cytotoxicity assay. The in vitro toxicity rankings (from most to least toxic) based either on cellular
ATP concentration or rate of protein synthesis were identical: DES CH < AC CY. The toxicity
ranking based on viability index was: DES CH < AC < CY. All of these chemicals were toxic in vivo
following 10 days of repeated dosing. The in vim toxicity rankings (from most to least toxic) based either
on mortality or on liver weight were identical: CY < AC < DES (unable to rank CH since highest con-
centration tested produced no change from control). The toxicity ranking for body weight at sacrifice was:
CH CY < AC (unable to rank DES since lowest concentration tested reduced body weight). The in
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HAZARDOUS WASTE PUBLICATIONS
vitro toxicity vinkingc did not correlate with in viva toxicity rankings based on death, body weight, or liver
weight. These results demonstrate the potential usefulness of the CHO cell assay to detect biological ac.
tivity but raise a question concerning the usefulness of this assay to predict in viva toxicity rankings. The
limited number of chemicals that we tested does not allow us to answer this question.
CITATION: In.AkemativeMetho&in Toxicol ogv Series, vol. 5:In Iitro Toxicology-Approaches to Valida-
tion, 1986.
D109 MS-86-163 Project Officer: Berman
INFLUENCE of CORTISOL on PROSTAGLANDIN SYNTHESIS by FETAL
MEMBRANES, PLACENTA and UTERUS of PREGNANT RABBITS
Randall P. Reynofrlr, and Patricia F. Noden
North Carolina State University
The adminLctnztion of toxic chemicals during early pregnancy in humans may cause undetected fetal loss.
This study shows that corlisol injections during pregnancy caused abon ions in the rabbit by inhibiting
ovarian function. Exposure of hwnan females to harardous chemicaLs could similarly alter ovarian fisnc-
tion and cause very early pregn ancy loss and aboilions
Two experiments were designed to assess the effects of cortison on prostaglandin-forming
cyclooxygenase in 4 gestational tissues of rabbits. Cortisol treatment (12 mg/kg body wt/h) was initiated
on day 21 of pregnancy and continued for a 24-h period. Each experiment included 5 treated and 5
vehicle-injected controls, sacrificed at 48 (Experiment 1) or 62 h (Experiment 2) after the initial injection.
At sacrifice, microsomes were prepared from amnion, yolksac splanchuopleure, uterus, and placenta,
and the prostaglandin-forming cyclooxygenase activity determined. In Experiment 2, pls cmsI drawn at
12-h intervals was qu2ntifled for prost gbrndin F (PGF), and progesterone. Cortisol, PGF, and PGE 2
were qunntified in imniotic fluid. In cortisol-treated rabbits, plasma progesterone decreased (P <.01)
from 72 ± 0.8 ng/ml on day 2 (pretreatment) to 1.6 ± 0.2 ng/ml on day 23,48 hrs after the initiation of
cortisol treatment. By 62 h, PGF, PGE2, and cortisol concentrations were all significantly higher (P <.05)
in the amniotic fluid of treated Rnbi1 1c• However, cortisol treatment did not increase prostaglandin-form-
ing cyclooxygenase activity in fetal or maternal tissues at either 48 or 62 h. Therefore, even though in-
creased prost g1andin production may be responsible for the cortisolinduced abortion, increased
cyclooxygenase activity in the gestational compartment was not detected.
CITATION: Biology of Reproduction
D109 XX-85-112 Project Officer: Gray
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PUBLICATIONS HAZARDOUS WASTE
EVALUATION of the IMMUNOTOXIC POTENTIAL of CHLORDECONE
with COMPARISON to CYCLOPHOSPHAMIDE
J. Smialowicz , R.WLuebke M. M. Riddle, R. R. Rogers, and D. G. Rowe
HERL, USEPA, Research Triangle Park, NC
This study was condo cted as past of an effoit by HERL to develop a panel of tests to assess the potential
health effects of complex hazardous waste mistures present in the environmenL Several biological systems,
including the immune system, have been examined as potential endpoints in this screen. The results
presented in this paper represent the initial steps using a se,ies of pure compounds (e.&, cyclophosphaniide)
with known biological effects and compounds of environmental concern (e.g., chiordecone) toward the
design validation and selection of a protocol that can be applied to assess complex waste mL*iures which
are found in the envimwnent This woik was perfomied at the request c/the Solid Waste Program.
The immunotoxic potential of chlordecone was evaluated in male Fischer 344 rats following 10 days of
dosing by oral gavage. These results were compared with a comparable dosing regimen with the known
immunosuppressive drug cyclophosphainide. Significant changes in the immune parameters crnniined,
which included spleen and thymus to body weight ratios, mitogen responsiveness to the lymphocyte
mitogen conconavalin A and natural killer (NK) cell activity gainst allogeneic WIFu-Gl rat lymphoma
target cells and xenogeneicYAC-1 mouse lymphoma target cells were observed only at the highest dosage
(10 mgJkgl day). A significant decrement in body weight also occurred at this dosage which suggests that
the observed changes in the immune parameters measured were most likely due to the overt toxicity of
chiordecone at this dosage. Rats dosed over 10 days by oral gavage with cyclophosphamide showed sig-
nificant decreases in spleen and thymus to body weight ratios at a dosage as low as 15 mglkgJday. Body
weight decrements were observed at dosages of 12 mg/kg/day or greater. At this same dosage of
cyclophosphamide significant decreases were observed for the following: 1) total leukocyte and absolute
lymphocyte counts; and 2) lymphoproliferative responses to both T- and B-lymphocyte mitogens. Sig-
nificant suppression of NK cell activity was observed at dosages of 6 mg/kg/day or greater. It appears that
T and B lymphocytes are more sensitive than are NK lymphocytes to the immunosuppressive effects of
cyclophosphamide. These results indicate that chiordecone did not affect the immune parameters ex-
ainined in the absence of overt toxicity.
CITATION: Journal of Toxicology and Envfronmental Health 15(5): 561-574, October 1985.
D109 MS-84-128 Project Officer: Smialowicz
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Future
Perspectives

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FUTURE PERSPECTIVES
Emerging environmental issues have increased complexity that can only be successfully achieved by inter-
disciplinary and multi-media approaches, both intra- and inter-laboratory. These issues indude indoor
air, hazardous waste, superfund, municipal waste incinerators, and biomarkers. Simultaneously, HERL
must continue to address other, equally important congressional mandates under the Clean Air Act,
TSCA, and FIFRA and the Water Acts. Both efforts require the development and application of ad-
vanced toxicological technology by HERL. The following summarizes these new directions.
Future research of the Toxicology and Microbiology Division (TMD) will emphasize the further iden-
tification of toxic products formed during drinking water treatment and determination of their health
hazard. This research program will include epidemiological, clinical and laboratory studies directed to
the development and evaluation of short term bloassays and markers that predict toxicological and car-
cinogenic responses in man from specific chemicals as well as mixtures present in drinking water. As this
work further clarifies the health luward from water didnfectants and their by-products, a companion
program to elucidate the potential ha rd from waterborne microorganism under reduced water disin-
fection conditions will be conducted. Animal models and in vitro virulence tests will be used to evaluate
the health h27ard of microorganisms transmitted under various water treatment criteria. Infectious
agents associated with waterborne outbreaks will be indentifled and studied in order to develop control
strategies.
The Neurotoxicology Division will continue to focus on an integrated research program to: (1) develop
methods for evaluating the neurotoxic potential of environmental chemicals (Hazard Identification); (2)
determine the relationships between toxicant-induced cellular/molecular changes and the resulting
physiological/behavioral alterations that are essential for establishing risk assessment methodologies; and
(3) apply these methodologies to programmatically relevant chemicals (e.g., pesticides, air toxics). Major
emphasis will be placed on the following categories of research: (1) biochemical markers for neurotoxicity
applying monoclonal antibody techniques to nervous system specific proteins; (2) quantitative mor-
phometric techniques for evaluating neuropathology in the adult and developing nervous system; (3) in
vitro methods for assessing neurotoxic potential; (4) electrophysiological approaches which are ap-
plicable to evaluating sensory dysfunctions in laboratory animals and man; and (5) methods for evaluat-
ing complex behaviors (Le.,learning/sensory/performance) in both adult and developing animal 5 . In-
herent in this research program will be the establishment of both qualitative and quantitative extrapola-
tion methods including endpoint extrapolation and dose extrapolation.
A recognized leader in the field of epidemiology is presently being recruited to man e the Biometry
Division. Plans are underway to reorgani7e and place additional emphasis on the epidemiology program.
Capabilities for adequate exposure assessment in conjunction with epidemiology studies will be
strengthened by developing and using new techniques available for biological exposure monitoring and
iochemkal markers of exposure. A new program in biochemical epidemiology is being expanded. It’s
goal is to develop and evaluate sensitive markers of both carcinogenic and noncarcinogenic disease in
human populations. The program is strongly linked to bench laboratory efforts where such techniques
are under initial development. Early detection of effects of pollutants at environmental levels of exposure
should prove useful. On-going relationships with such groups as the University of Pittsburgh Center for
Environmental Epidemiology and the University of North Carolina Epidemiology Program will be
strengthened. Research needs will be identified from efforts such as the National Academy of Sciences
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FUTURE PERSPECTIVES
Report on Epidemiology and Air Pollution, the Clean Air Scientific Advisory Committee, and research
phinning workshops.
Responding to the programmatic needs of the Agency, both current and future, the research program of
the Inhalation Toxicology Division (ITD) will continue to advance. This will involve a major emphasis on
extrapolation modeling to allow a more direct use of animal toxicological data in the standard setting
procesa, especially for NAAQS pollutants. Species sensitivity issues will receive increased emphasis since
such information, together with dosimetry data, forms the basis for quantitative extrapolation of
endpoints for which health data are being obtained. Currently ongoing chronic animal toxicological
studies will continue and will form the cornerstone of much of the Agency’s future health information on
chronic lung disease. Recently initiated hunia research programs in the areas of aerobiology and
molecular and cellular biology will be accelerated to complement existing capabilities in lung physiology
this expanded human research program will be applied to issues of interest to air toxics and will also focus
on building the bridges which improve our understanding of acute changes in animals and man.
One of the major challenges that will face EPA over the next 5-10 years will be the identification and con-
trol of hazardous waste. it is only with the identification of hazardous wastes that proper management
procedures can be utilized to ensure public health and the safety and integrity of the environment. Cur-
rent methods for identifying and classifying hazardous wastes are expensive and time synergistic or an-
tagonistic effects of a pollutant in the intact animal . An in vivo orin vivo/in vitro Toxicological Screen may
provide the best first approximation of the toxicity of a complex mixture. Research is in progress and will
continue over the next 2-3 years to broaden the scope of the toxicological screen developed at HERL and
test chemicals of known toxicity to validate the sensitivity and accuracy of the test protocoL The goal is
to provide OSW with a fully tested and validated protocol for assessing the toxic characteristics of was-
tes.
Research directions in the field of perinatal toxicology within the Developmental Biology Division (DBD)
are becoming more molecularly oriented and research projects are increasing!y targeted at exploring the
action of teratogens at the genomic level. Given the major research findings of the recent times in gene
regulation, including the discovery of homeoboxes in mammalian embryos, this promises to be a very ex-
citing area as we enter the 1990’s. Research efforts are also being expanded in the areas of risk extrapola-
tion for teratogenesis and in structure-activity relationships for chemically induced developmental
toxicity. The functional teratology group will be initiating experiments exploring trophic influences of the
sympathetic nervous system on the development of peripheral organ systems and how these influences
may be modulated by teratogenic exposure.
The long term goals in the reproductive toxicology program within the DBD include the development of
cost-effective and sensitive reproductive assessment procedures to assist the Program Offices in identify-
ing compounds with the potential to adversely effect reproductive integrity. The approach consists of
evaluating assessment procedures for the hypothalamic-pituitary-gonadal axis (from the simple observa-
tional to the complex state-of-the-art), and identifying those which are predictive of reproductive damage.
These procedures would then be integrated into an approach designed to first identify potential
reproductive hazards from those with no apparent reproductive impact. Following identification,
prioritization procedures will be identified to give the dose response, chronicity recovery and human im-
pact characteristics. These testing schemes will then be integrated and validated by commercial
laboratories with known positive and negative reproductive toxins.
Future Genetic Toxicology Division research will emphasize the development of methodology for ex-
trapolation, exposure evaluation (biomarkers), dosimetry, and complex mixture research. The Division
research will emphasize the evaluation of techniques to extrapolate effects in rodents to humans , includ-
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FUTURE PERSPECTIVES
ing tissue and route specificity. In addition, the relevance of genotoxic endpoints to human health will be
explored and defined. The major endpoints for extrapolation will indude both somatic and germ cell ef-
fects. Computational methods to predict toxicity of chemicals based on chemical structure will be a
prominent part of future Division research. In the past, the Genetic Toxicology Division has been recog-
nized in the Agency for its innovative complex mixture research program. Efforts will continue in this
area with specific regard to methods for chemical identification, multimedia/ multisourcetmtegrated as-
sessment, dose receptors, source apportionment, and comparative potency. Emphasis will also be placed
on the analysis of indoor air and lIa7ardous waste complex mixtures.
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Index of Abstracts
of Published Documents

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Index
Air Health
Page No .
Aranyi, Catherine, Wi11i im J. O’Shea, Judith A. Graham, and Frederick J. Miller. THE
EFFECTS of INHALATION of ORGANIC CHEMICAL AIR CONTAMINANTS
on MURINE LUNG HOST DEFENSES. Fundamental and Applied Toxicology 6:
713-720, 1986.
MS-86-219 52
Ball, L M.and 3. Lewias. RAT LIVER SUBCELLULAR FRACTIONS CATALYZE
AEROBIC BINDING of 1-NITRO [ 14CJPYRENE TO DNA. Environmental Health
Perspectives 62:193-196, October 1985.
MS-85-107 59
Ball, Louise M., Michael J. Kohan, Kay Williams, M.G. Nishioka, Awam Gold, and Joellen
Lewtas. METABOLISM and ACTIVATION PATHWAYS of the
ENVIRONMENTAL MUTAGEN 3-NITROFLIJORANTHENE. 1n Proceedings
of the Tenth International Symposium on Polynuclear Aromatic Hydrocarbons,
edited by W.M. Cooke and AJ. Dennis, Battelle Press, October 1985.
MS-86-127 67
Ball, L. M., K. Willisims, M. J. Kohan, and J. Lewtas. S9-DEPENDENT ACTIVATION of
1-NITROPYRENE and 3-NITROFLUORANTHENE in BACTERIAL
MUTAGENICITY ASSAYS. 1n Proceedings of the Third International Conference
on Biological Reactive Intermediates, Pleason Press, New York NY, 1985.
MS-86-035 56
Barnes, Martha I. and Robert S. Dyer, SUPERIOR COLLICULUS LESIONS and FLASH
EVOKED POTENTIALS FROM RAT CORTEX. Brain Research Bulletin 16:
225-230, 1986.
MS-86-O10 54
Barry, Brenda E., Frederick J. Miller, and James D. Crapo. EFFECTS of INHALATION of
0.12 and 0.25 PARTS PER MILLION OZONE on the PROXIMAL ALVEOLAR
REGION of JUVENILE and ADULT RATS. Laboratory Investigation 53(6): 692,
December 1985.
MS-85-087 32
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Be4 John E, Deborah M. Drechsler-Parks, and Steven M. Horvath. DURATION of
INCREASED PULMONARY FUNCTION SENSITIVITY to an INITIAL OZONE
EXPOSURE. Journal of American Industrial Hygiene Association 46(12):
731-734,December 1985.
XX-84-022
Berkey, Catherine S., James IL Ware, Douglas W. Dockery, Benjamin G. Ferris, Jr., and
Frank E. Speizer. INDOOR AIR POLLUTION and PULMONARY FIJNCFION
GROWTh! in PREADOLESCENT CHILDREN. American Journal of
Epidemiology 123(2): 250-260, February 1986.
XX-86-113 49
Chang, Ling-Yi, Judith A. Grahiun , Frederick J. Miller, Jean J. Ospital, and James D. Crapo.
EFFECTS OF SUBCHRONIC INHALATION of LOW CONCENTRATIONS of
NITROGEN DIOXIDE I. THE PROX1MAL ALVEOLAR REGION of
JUVENILE and ADULT RATS. Toxicology and Applied Pharmacology 83(1):
46-61, March 1986.
MS-85-208 34
Chapman, Robert S., Beatrice J. Selwyn, Thomas H. Stock, Robert J. Hardy, Frances A. Chan,
Daniel E. Jenkins, and Dennis J. Kotchmar. HEALTH EFFECTS of AMBIENT
OZONE EXPOSURE in VIGOROUSLY EXERCISING ADULTS. It Scientific
Basis for Ozone and Other Photochemical Oxidants Standards, H. Englund, ed.,
Houston, Texas, November 1985.
MS-86-020 46
Claxton, L D., K. E. Mortehnans, J. Allen, A. Auletta, K Nestmann. E. Zeiger, and J.
McCann. A STANDARD GUIDE for the SALMONELLA
TYPHIMURIUMJMAJ,fM jLoj4 MICROSOME TESTS for BACTERIAL
MUTAGENICITY IASTMSTANDARDJ. Proceedings of the American Society for
Testing and Materials, 1986.
MS-86-220 64
Contant, Charles F. Jr., Brenda M. Gehan, Thomas H. Stock, Alfonso H. Holguin, and
Patricia A. Buffler. ESTIMATION of INDIVIDUAL OZONE EXPOSURES
USING MICROENVIRONMENT MEASUREMENTS. In: Scientific Basis for
Ozone and Other Photochemical Oddants Standards, H. Englund, ed., December
1985.
XX-86-025 42
Conti, C. J., N. Clapp, A. J. P. IUein-Szanto, S. Nesnow, and T. J. Slaga. SURVIVAL
CURVES and INCIDENCE of NEOPLASTIC and NON-NEOPLASTIC DISEASE
in SENCAR MICK Carcinogenesis 6(11): 1649-1652, October 1985.
A1O1/L104 MS-86-052 62
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Davis, Wayne B., Carl G. Hayes, Marilyn Knowles, Wilson BJ igg in 1 John Van Bruggen, and
H. A. Tyroler. GEOGRAPHIC VARIATION in DECLINING ISCHEMIC
HEART DISEASE MORTALITY in the UNiTED STATES, 1968-1978. American
Journal of Epidemiology 122(4): 657-672, October 1985.
MS-85-O11 41
Ferris, Benj amin G., Jr., and John D. Spengler. PROBLEMS in the ESTIMATION of
HUMAN EXPOSURE to COMPONENTS of ACID PRECIPiTATION
PRECURSORS. Environmental Health Perspectives 63:5-9, November 1985.
XX 85-067 43
Ferris, Benjamin G., Jr., James H. Ware, Catherine S. Berkey, Douglas W. Dockery, Avron
Spiro, m, and Frank E. Speizer. EFFECTS of PASSIVE SMOKING on HEALTH
of CHILDREN. Environmental Health Perspectives 62 289-295, October 1985.
XX-86-114
Franch, Serafino and Gary E. Hatch. PULMONARY BIOCHE?bfICAL EFFECTS of
INHALED PHOSGENE in RATS. Journal of Toxicology and Environmental
Health 19:413-423,1986.
MS-86-067 53
Friedman, Mitchell, Michael C. Madden, D. Stephen Saunders, Kenneth Gammon, Gilbert C.
White, II and Lester Kwock. OZONE INHIBITS PROSTACYCLIN SYNTHESIS
in PULMONARY ENDOTHELIUM. Prost g!andins 30(6): 1069-1083, December
1985.
31
Goidring, J. M., L M. Ball, R. Sn’ig ah, and A. Gold. SYNTHESIS and BIOLOGICAL
ACTIVITY of a SERIES of NITROSUBSTITUTED CYCLOPENTA-FUSED
PAH. In: Proceedings of the Tenth International Symposium on Polynuclear
Aromatic Hydrocarbons, W.M. Cooke & AJ. Dennis, eds., Battelle Press, Columbus,
OH, October 1985.
MS -86 -211 66
Goldstein, I. F., DESIGN STRATEGIES for EPIDEMIOLOGIC STUDIES of
ENVIRONMENTAL IMPACTS on HEALTH. 1n Proceedings of the Air Pollution
Control Association, Minneapolis, MN, June 1986.
MS-86-107 44
Goldstein, I. F. and L. R. Andrews. PEAK EXPOSURES to NITROGEN DIOXIDE and
STUDY DESIGN to DETECT THEIR ACUTE HEALTH EFFECTS. In:
Proceedings of the Air Pollution Control Association, June 1986.
MS -86-108 46
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Hatch, Gary E., Ralph Slade, MaryJane K. Seigrade, and Andrew G. Stead. NiTROGEN
DIOXIDE EXPOSURE and LUNG ANTIOXIDANTS in ASCORBIC
ACID-DEFICIENT GUINEA PIGS. Toxicology and Applied Pharmacology 82(2):
351-359, April 1986.
MS-85-167 33
Hatch, Gary E., Ralph Slade, Andrew 0. Stead, and Judith A. Gr h im. SPECIES
COMPARISON of ACUTE INHALATION TOXICITY of OZONE and
PHOSGENE. Journal of Toxicology and Environmental Health 19:43-53,1986.
MS-85-060 53
Horstnuin, Donald, L Jack Roger, Howard Kebri, and Milan Hazucha AIRWAY
SENSITIVITY of ASTHMATICS to SULFUR DIOXIDE. Toxicology and
Industrial Health 2(3): 1-25, September 1986.
MS-84-065
Horvath, S. M. DESIGN and MEASUREMENT CONSIDERATIONS for EXERCISE
PROTOCOLS in HUMAN AIR POLLUTION INHALATION STUDIES. In:
Inhalation Toxicology of Air Pollution: Clinical Research Considerations, R. Frank,
JJ. O’Neil, Mi. Utell, J.D. Hackney, J. Van Ryun and P.E. Brubaker, eds., American
Society for Testing and Materials, 1985.
XX-86-130 25
House, Dennis E. A NONPARAMETRIC VERSION of WILLIAMS’ TEST for a
RANDOMIZFI) BLOCK DESIGN. Biometrics 42(1): 187-190, March 1986.
MS-85-158 39
Jackson, M. A., L. C. King, L. M. Ball, S. Ghayourmanesh, A. M. Jeffrey, and J. Lewtas.
NITROPYRENE: DNA BINDING and ADDUCE FORMATION in
RESPIRATORY TISSUES. Environmental Health Perspectives 62: 203-207,
October 1985.
MS-85-106
King, Leon C., Louise M. Ball, Marcus Jackson, Jeff P. Inmon, and Joellen Lewtas.
METABOLISM of 1-NITROPYRENE by CULTURED RABBIT ALVEOLAR
MACROPHAGES and RESPIRATORY TRACT TISSUES. Toxicology and
Applied Pharmacology 82(2): 292-300, February 1986.
MS-84.045
King, Mitchell E. CONVERSION FROM RT-11 to MICRO-RSX for REAL-TIME DATA
COLLECTION and ANALYSIS. In: Proceedings of the Digital Equipment
Computer Users Society Symposium, April/May 1986.
XX-86-087 35
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K1eindienst T. E., E. 0. Edney, 0. R. Namie, and L D. Claxton THE MUTAGENIC
ACTIVITY of IRRADIATED C2H4/NOx MIXTURES in the PRESENCE of
DIETHYLHYDROXYLAMINE. Atmospheric Environment 20(5): 971-978, May
1986.
MS-85- 192 65
Koren, Hillel S., Karen P. McKlnnon and Allen R. Chen. TRIGGERING of a CYTOLYTIC
FACTOR with TNF-LIKE ACTIVITY FROM HUMAN MONOCYTES. In:
Cytotoxic Lymphokines and Cancer: Biological and Chemical Aspects, J.H. Ransom
and J. Ortaldo, eds., Humana Press, Clifton, NJ, 1986.
MS-86-148 31
Kulle, Thomas J., Larry R. Sauder, J. Richard Hebel, and Marie D. Chathiim OZONE
RESPONSE RELATIONSHIPS in HEALTHY NONSMOKERS. American
Review of Respiratory Disease 132:36-41, October 1985.
XX-84-038 27
Lebowitz, Michael D., Catharine J. Holberg, Barbara Boyer and Carl Hayes.
RESPIRATORY SYMPTOMS and PEAK FLOW ASSOCIATED with INDOOR
and OUTDOOR AIR POLLUTANTS in the SOUTHWEST. Journal of the Air
Pollution Control Association 35(11): 1154-1158, November 1985.
MS-86-086 47
Lewtas, Joellen. COMPARATIVE POTENCY METHOD for CANCER RISK
ASSESSMENT: APPUCATION to the QUANTITATIVE ASSESSMENT of the
CONTRIBUTION of COMBUSTION EMISSIONS to LUNG CANCER RISK
Proceedings of IEMS Meeting, Stockholm, Sweden, June 1985.
MS-85-249 63
Lcwtas, Joellen. DEVELOPMENT of a COMPARATIVE POTENCY METHOD for
CANCER RISK ASSESSMENT of COMPLEX MIXTURES USING
SHORT-TERM IN VIVO and IN VITRO BIOASSAYS. Toxicology and Industrial
Health 1(4): 193-203, December 1985.
MS-85-223 63
Lewtas, Joellen, Marcia G. Nishioka, and Bruce A. Petersen. IDENTIFICATION and
COMPARATIVE RISK ASSESSMENT of AIRBORNE CARCINOGENS FROM
COMBUSTION SOURCES. In: Proceedings of the Technology Transfer
Conference No.6, Toronto, Ontario, Canada, December 1985.
MS-86-042 61
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Lewtas, J. and K. Wilhi ms. A RETROSPECTIVE REVIEW of the VALUE of
SHORT-TERM GENETIC BIOASSAYS in PREDICTING the CHRONIC
EFFECTS of DIESEL SOOT. In: Proceedings of International Symposium
rI oxicologicaI Effects of Emissions from Diesel Engines”, Tsukuba Science City,
Japan, July 1986.
MS-86-217 68
Lioy, P. J., T. A. Voilmuth, and M. Lippni inn PERSISTENCE of PEAK FLOW
DECREMENT in CHILDREN FOLLOWING OZONE EXPOSURES
EXCEEDING the NATIONAL AMBIENT AIR QUALITY STANDARD. Journal
of the Air Pollution Control Association 35(10): 1068-1071, October 1985.
XX-85-098 45
Manton, K. G., E. Stallard, M. Woodbury, J. P. Creason, and W. B. Riggan.
COMPARTMENT MODEL APPROACHES for ESTIMATING the
PARAMETERS of a CHRONIC DISEASE PROCESS UNDER CHANGING
RISK FACTOR EXPOSURE. Computers and Biomedical Research 19: 151-169,
February 1986.
MS-85-204 40
Martonen, T. Blayne, Andrew E. Barnett, and Frederick I. Miller. AMBIENT SULFATE
AEROSOL DEPOSITION in MAN: MODPIIJNG the INFLUENCE of
HYGROSCOPIC1TY. Environmental Health Perspectives 63: 11-24, November
1985.
MS-85-069 38
McDonnell, WiIIi2m F., Robert S. Chapman, Margaret W. Leigh, Gerald L. Strope, and
Albert M. Collier. RESPIRATORY RESPONSES of VIGOROUSLY
EXERCISING CHILDREN to 0.12 PPM OZONE EXPOSURE. American Review
of Respiratory Disease 1.32(4): 875-879, October 1985.
MS-85-067
Miller, F., T. B. Martonen, M. G. Menache, M. Lippmann, R. C. Grah n and D. M. Spektor.
INFLUENCE of BREATHING MODE and ACTIVITY LEVEL on the
REGIONAL DEPOSITION of INHALED PARTICLES and IMPLICATIONS for
REGULATORY STANDARDS. In: Proceedings of Inhaled Particles VI,
Cambridge, EngI nd, September 1985.
MS-86-082 36
Mumford, J. L., G. E. Hatch, R. E. Hall, M. A. Jackson, R. G. Merrill, Jr. and J. Lewtas.
TOXICITY of PARTICLES EM1TFED from COMBUSTION of WASTE
CRANKCASE OIL: IN VITRO and IN VIVO STUDIES. Fundamental and
Applied Toxicology 7(1): 49-57, July 1986.
MS-86-026 ss
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Mumford, Judy L., Silvestre B. Tejada, Marcus Jackson, and Joellen Lewtas.
BIOAVAILABILITY of 1-NITROPYRENE from MODEL COAL FLY ASH and
rI ’S UPTAKE by ALVEOLAR MACROPHAGES. Environmental Research 40(2):
427-436, August 1986.
MS-83-223 58
Mumford, J. L., K. WilIbin ’c D.B. Harris, J. C. Chuang and M. Cooke. INDOOR AIR
SAMPLING and MUTAGENIC1TY STUDIES RELATED to EMISSIONS from
UNVENTEI) COAL COMBUSTION. 1n Proceedings of the 79th Air Pollution
Control Association, Minneapolis, MN, June 1986.
MS-86-190 50
Otto, David A. ELECFROPHYSIOLOGICAL ASSESSMENT of NEUROTOXICITY in
CHILDREN. In Mental Retardation, Neurobehavioral Toxicology and Teratology,
S. Schroeder, ed, 1986.
I’ ’fS—86—036 28
Otto, David A., THE RELATIONSHIP of EVENT-RELATED BRAIN POTENTIALS
and LEAD ADSORPTION: A REVIEW of CURRENT EVIDENCE. In: Lead
Environmental Health The Current Issues, 1986.
MS-86-038 29
Otto, D.A., G. Robinson, S. Baumjann, S. Schroeder, P. MushaJc, D. Kleinbaum, and L Boone.
5-YEAR FOLLOW-UP STUDY of CHILDREN with LOW-to-MODERATE
LEAD ABSORPTION: ELECFROPHYSIOLOGICAL EVALUATION.
Environmental Research, 38:168-186,1985.
MS-85-086 29
Overton, John and Fred Miller. DOSIMETRY MODELING of INHALED TOXIC
REACI’IVE GASES. Health Effects Institute, (in press).
MS-86-197 36
Patra, A. L. MOD LTJNG of the UPPER and LOWER RESPIRATORY SYSTEMS. In:
Modeling of the Upper and Lower Respiratory Systems, Amit L. Patra, ed., Raven
Press, 1986.
XX-86-122 37
Schroeder, Stephen R., Barbara Hawk, David A. Otto, Paul Mushak, and Robert E. Hicks.
SEPARATING the EFFECTS of LEAD and SOCIAL FACTORS on 10.
Environmental Research 38:144-154,1985.
MS-85- 156
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Schuet7le, Dennis and Joellen Lewtas. BIOASSAY DIRECTED CHEMICAL ANALYSIS
in ENVIRONMENTAL RESEARCH Analytical Chemistry 58: 1060a, 1986.
MS-86-199
Spengler, John D., Margaret P. Reed, Erik Lebret, Bei-Hung Chaiig James H. Ware, Frank
E. Speizer, and Benjamin G. Ferris, Jr. HARVARD’S INDOOR AIR
POLLUTION/HEALTH STUDY. 1n Proceedings of the Air Pollution Control
Association, Minneapolis, MN, June 22-27, 1986.
XX-86-112 48
Steele, Vernon E. and Julia T. Arnold. ISOLATION and LONG-TERM CULTURE of
RAT, RABBIT, and HUMAN NASAL TURBINATE EPITHELIAL CFT 1.5• In
Vitro Cellular and Developmental Biology 21(12): 681-687, December 1985.
XX-85-054 38
Symons, Michael J., Donald L Doerfier, and Yang C. Yuan. LIMITED OCCUPATIONAL
MORTALITY DATA and STATISTICS. 1n Biostatistics Statistics in Biomedical,
Public Health and Environmental Sciences, PL Sen, ed., Elsevier Science Publishers,
The Netherlands, pps. 195-218, 1985.
XX-86-098 40
Thurston, George D. and John D. Spongier. A MULTWARIATE ASSESSMENT of
METEOROLOGICAL INFLUENCES on INHALABLE PARTICLE SOURCE
IMPACTS. Journal of Climate and Applied Meteorology 24(11): 1246-1256,
November 1985.
XX-86-116 42
Traynor, G. W., M. G. Apte, H. A. Soko, J. C. Chuan& and 3. L. Mumford. SELECTED
ORGANIC POLLUTANT EMISSIONS from UN VENTED KEROSENE
HEATERS. 1n Proceedings of the 79th Air Pollution Control Association,
Minneapolis, MN, June 1986.
MS-86-191 51
Ware, 3. H., B. G. Ferris, Jr., D. W. Docker J. D. Spengler, D. 0. Strain, and F. E. Speizer.
EFFECTS of AMBIENT SULFUR OXIDES and SUSPENDED PARTICI FS on
RESPIRATORY HEALTH of PREADOLESCENT CHILDREN. American
Review of Respiratory Disease 133(5): 834-842, May 1986.
XX-86-123 44
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Williams, R., C. Sparacino, B. Petersen, J. Binng rner, R. H. Jungers, and J. Lewtas.
COMPARATIVE CHARACFERJZATION of ORGANIC EMISSIONS from
DIESEL PARTICULATE, COKE OVEN MAINS, ROOFING TAR VAPORS, and
CIGARETTE SMOKE CONDENSATE. International Journal of Environmental
Analytical Chemistry 26: 27-49, September 1986.
M s-86 -041 61
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Index
Non-Ionizing Radiation
Page No .
All, Joseph S. and William T. Jot A SYSTEM for MEASUREMENT of SMALL
VIBRATIONS at MATERIAL INTERFACES INDUCED by
ELECTROSTRICI’IVE FORCES. U.S. Environmental Protection Agency,
EPA-600/1-85-021, November 1985. Available from NTIS: PB86-116530/AS.
}{ERL4J5 18 78
Blackman, C. F., S. G. Benane, I. R. Rabinowit D. E. House, and W. T. Joints. A ROLE for
the MAGNETIC FIELD in the RADIATION-INDUCED EFFLUX of CALCIUM
IONS from BRAIN TISSUE IN VITRO. Bioelectromagnetics 6(4): 327-337,
December 1985.
MS-85-070 69
Cook Mary R., Carl M. Maresh, and Harvey D. Cohen. EFFECTS of 60-HZ FIELDS on
HUMAN HEALTH PARAMETERS. U.S. Environmental Protection Agency,
cooperative agreement no. CR412805, EPA-600/1-86-006, September 1986.
HERL-0551 69
Elder, Joe A. RADIOFREQUENCY RADIATION: ACTIVITIES and ISSUES. In:
Howard University Symposium Proceedings, Washington, DC, September 9, 1985.
MS-86-187 80
Gag; Michael I. NON-ELECTROMAGNETIC FACTORS INFLUENCE BEHAVIORAL
EFFECTS of MICROWAVE EXPOSURE. In: BiOlOgiCal Effects of
Electropollution, (in press).
MS-86-125 70
Gonzalez , Guillermo, James C. Nearing, Ronald J. Spiegel, and William T. Joines.
OFF-CENTER SPHERICAL MODEL for DOSIMETRY CALCULATIONS in
CHICK BRAIN TISSUE Bioelectromagnetics 7(2): 209-221, June 1986.
MS-85-164 77
Gordon, Christopher J. and PIi pbeth C. White. TEMPORAL RESPONSE of NEURONS to
AMBIENT HEATING in the PREOPTIC and SEFTAL AREA of the
UNANESTHETIZED RABBIT. Comparative Biochemistry and Physiology 82A(4):
879-884, December 1985.
MS-82.087 75
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Page No .
Liddle, C. G., J. P. Putnam, 0. L Lewter, J. Y. Lewis, B. Bell, M. W. West, and A. Stead.
EFFECT of 9.6-GHZ PULSED MICROWAVES on the ORB WEB SPINNING
ABILITY of the CROSS SPIDER (ARANEUS DIADEMATUS).
Bioclectronifignetics 7(1): 101-105, January 1986.
MS-83-003 72
Liddle, C. G., J. P. Putmsni, 0. H. Lewter, M. West, and G. Morrow. CIRCULATING
ANTIBODY RESPONSE of MICE EXPOSED to 9-GHz PULSED MICROWAVE
RADIATION. Bloelectromagnetics 7(1): 91-94, January 1986.
MS-83-244 72
Mon h n, J. C. and J. A. D’Andrea. BEHAVIORAL EFFECFS of MICROWAVE
RADIATION ABSORPTION. DHHS Publication, FDA 85-8238 1985.
HERL-0523 73
Phillips, Richard D. OVERVIEW of WHO ENVIRONMENTAL HEALTH CRiTERIA 35
on EXTREMELY-LOW-FREQUENCY (ELF) FIELDS. In: Proceedings of the
International Utility Symposium, September 16-19, 1986.
MS-86-162 80
Spiegel, R. J., J. S. All, J. F. Peoples, and W. T. Joines. THE MEASUREMENT of SMALL
MECHANICAL VIBRATIONS of BRAIN TISSUE EXPOSED to
LOW-FREQUENCY ELECTRIC FIELDS. Bioelectromagnetics 7(3): 295-306, July
1986.
MS-85-210 76
Spiegel R. J. and M. B. E. Fatmi. A THREE-DIMENSIONAL FINITE-DIFFERENCE
THERMOREGULATORY MODEL of a SQUIRREL MONKEY. International
Journal of Radiation Oncology Biol. Phys. 12(6): 983-992, June 1986.
MS-85-079 71
Spiegel, R. J., M. B. E. Fatmi, and K. S. Kunz. APPLICATION of a FIN1TE-Dwi’i RENCE
TECHNIQUE to the HUMAN RADIOFREQUENCY DOSIMETRY PROBLEM.
Journal of Microwave Power 20(4): 241-254, December 1985.
MS-85-168 79
Stuchly, Maria A., Ronald J. Spiegel, St nicbiw S. Stuchly, and Andrzej Kraszewski.
EXPOSURE of MAN in the NEAR-FIELD of a RESONANT DIPOLE:
COMPARISON BEFWEEN THEORY and MEASUREMENTS. IEEE
Transactions on Microwave Theory and Techniques MTF-34(1): 26-31, January 1986.
I tS—84—13O 74
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Ward, Thomas R., David J. Svendsgaard, Ronald J. Spiegel, Earl T. Puckett, Merritt D. Long
and James B. Kinn. BRAIN TEMPERATURE MEASUREMENTS in RATS: A
COMPARISON of MICROWAVE and AMBIENT TEMPERATURE
EXPOSURES. Bioelectroinrignetics 7(3): 243-258, July 1986.
MS-84-169 74
Watkinson, Wi1Ii iin P. and Christopher J. Gordon. IMPROVED TECHNIQUE for
MONiTORING ELECFROCARDIOGRAMS DURING EXPOSURE to
RADIO-FREQUENCY RADIATION. American Journal of Physiology 250(19):
H320-H324, February 1986.
MS-85-002 81
-317-

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Index
Water Health
Page No .
Akin, E. W. and W. Jakubowski. DRINKING WATER TRANSMISSION of GIARDIASIS
in the UNiTED STATES. In: Water Science and Technology, August 1986.
MS-86-228 112
AndelmAn , Julian B. HUMAN EXPOSURES to VOLATILE HALOGENATED
ORGANIC CHEMICALS in INDOOR and OUTDOOR AIR. Environmental
Health Perspectives 62 313-318 October 1985.
XX-86-057 132
Ande1m n , Julian B. INHALATION EXPOSURE in the HOME to VOLATILE ORGANIC
CONTAMINANTS of DRINKING WATER. The Science of the Total Environment
47:4-43-460, November 1985.
XX-86-059 132
Baird, Wi11is rn M., Said M. Sebti, and Lori A. Reinsvold. METABOLIC ACI1VATION of
BENZO(a)PYRENE in SENCAR and BALB/c MOUSE EMBRYO CELL
CULTURES. Enviromental Health Perspectives 68:45-52, September 1986.
XX-86-083 166
Baker, R. J. and I. H. Suffet. EVALUATION of a CONTINUOUS LIQUID-LIQUID
EXTRACTOR for ISOLATION and CONCENTRATION of NONPOLAR
ORGANICS for BIOLOGICAL TESTING in the PRESENCE of HUMIC
MATERIALS. In: American Chemical Society Advances in Chemistry Series (in
press).
X-86-038 129
Barnes, Donald W., Virginia M. Sanders, Kimber L White, Jr., George M. Shopp, Jr., and
Albert E. Munson. TOXICOLOGY of TRANS-i, -DICHLOROETHYLENE in the
MOUSE. Drug and Chemical Toxicology, 8(5): 373-392, October 1985.
XX-86-103 157
Berc J. Peter, Iiflusin L. Jones, Robert M. Harrington, Rohit Bawa, and Lyman W. Condie.
MECHANISTIC ASPECTS of INGESTED CHLORINE DIOXIDE on ThYROID
FUNCTION: IMPACT of OXIDANTS on IODINE METABOLISM. In:
Proceedings of the Second International Symposium on Health Effects of Drinking
Water Disinfectants and Disinfection By-Products, Cincinnati, Ohio, August 27-29,
1986. Published in: Environmental Health Perspectives 69: 249-254, November 1986.
MS-86-173 143
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Page No .
Bitton, Gabriel, Samuel R. Farrah, Clay L Montague, and Elmer W. Akin. VIRUSES in
DRINKING WATER. Environmental Science and Technology 20(3): 216-222,
March 1986.
MS-86-046 117
Blackburn, K., H. Zenick, B. Hope, J. M. Manson, B. L George, and M. K. Smith.
EVALUATION of the REPRODUCTIVE TOXICOLOGY of
2,6-TRlCHI OROPHENOL in MALE and FEMALE RAfl Fundamental and
Applied Toxicology 6(2): 233-239, February 1986.
MS-85-129 138
Bollin, Gary B., Joseph F. Plouffe, Michael F. Pan, and Barbara Haclrmnn AEROSOLS
CONTAINING LEGIONELLA PNEUMOPHILA GENERATED by SHOWER
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Hurst, Christon 3. and Tamara Goyke. STABILITY of VIRUSES in WASTE WATER
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Morse, Mark A. and Gary P. Carlson. DISTRIBUTION and MACROMOLECULAR
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Pereira, Michael A. MOUSE LIVER TUMOR DATA: ASSESSMENT of
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Plouffe, Joseph F., Michael F. Para, and Kathy A. Fuller. INTRATRACHEAL INFECTION
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Plouffe, Joseph F., Michael F. Para, and Kathy A. Fuller. SERUM BACTERICIDAL
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Plouffe, Joseph F., Michael F. Para, Kathy A. Fuller, and Gary E. Bollin. ORAL
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FORMED in STOMACH FLUID on INGESTION of AQUEOUS
HYPOCHLORITE. In: Proceedings of the Second International Symposium on
Health Effects of Drinking Water Disinfectants and Disinfection By-Products,
Cin inrniti, Ohio, August 27-29, 1986. Published in: Environmental Health
Perspectives 69: 259-265, October 1986.
MS-86-165 131
Shirachi, Donald Y., Shing-Hui Tu, and John P. McGowan. CARCINOGENIC POTENTIAL
of ARSENIC COMPOUNDS in DRINKING WATER. U.S. Environmental
Protection Agency, cooperative agreement no. CR-807235, EPA-600/1-86-003, May
1986.
HERL-0543 180
Shopp, George M., Jr., Virginia M. Sanders, Kimber L. White, Jr., and Albert E. Munson.
HUMORAL and CELL-MEDIATED IMMUNE STATUS of MICE EXPOSED to
TRANS-1,2 ,-DICHLOROETHYLENE. Drug and Chemical Todcology 8(5):
393-407, October 1985.
XX-86-104 156
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Page No .
Shuster, Jonathan, Birdwell Finlayson, Richard L Scheaffer, Robert Sierakowski, John
Zokek and Sylvia Dzegede. PRIMARY LIQUID INTAKE and URINARY
STONE DISEASE. Journal of Chronic Diseases 38(11): 907-914, November 1985.
XX-83-087 150
Simon, Glenn Stuart, John L. Egle, Jr., Robert W. Dougherty.and Joseph F. Borzelleca.
DOMINANT LETHAL ASSAY of CHLORDECONE and ITS DISTRIBUTION.
in the MALE REPRODUCTIVE TISSUES of the RAT. Toxicology Letters 30:
237-245, March 1986.
B10I/C104/L104 XX-86-129 135
Smith, M. Kate, Harold Zenick, and Fmm a Lou George. REPRODUCTIVE
TOXICOLOGY of DISINFECTION BY-PRODUCTS. In: Proceedings of the
Second International Symposium on Health Effects of Drinking Water Disinfectants
and Disinfection By-Products Cincinnati, Ohio, August 27-29, 1986. Published in
Environmental Health Perspectives 69: 177-182, November 1986.
MS-86-178 139
Smith, M. Kate, Harold Zenick, R. Julian Preston, Emma Lou George, and Richard E. Long
DOMINANT LETHAL EFFECTS of SUBCHRONIC ACRYLAMIDE
ADMINISTRATION in the MALE LONG-EVANS RAT. Mutation Research 173:
273-277, 1986.
MS-85-229 ho
Snow, Linda K. and Bruce C. Casto, TOXICOLOGICAL TESTING of ORGANIC
SUBSTANCES from CONCENTRATED DRINKING and WASTE WATERS.
U.S. Environmental Protection Agency, contract no. 68-03-1840, EPA-60011-86-005,
July 1986.
HERL-0549 i o
Sobsey, Mark D., Stephen E. Ogiesbee, and Douglas A. Wait. EVALUATION of METHODS
for CONCENTRATING HEPATITIS A VIRUS from DRINKING WATER.
Applied and Environmental Microbiology 50(6): 1457-1463, December 1985.
121
Stober, Judy A. METHODOLOGY for STATISTICAL ANALYSIS of SENCAR MOUSE
SKIN ASSAY DATA. Environmental Health Perspectives 68:5-10, September 1986.
MS-86-113 107
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Stobcr, Judy A. NON-NEOPLASTIC LESIONS: USE of DATA FROM PRE- or
NON-NEOPLASTIC LESIONS THAT MAY INDICATE POTENTIAL for
CARCINOGENESIS. In: Proceedings of the EPRI Workshop on Investigation of
New Approaches to Use of Data in Cancer Risk Assessment, 1986.
MS-86-145 162
Van Duuren, Benjamin L, Susan Meichionne, and Irvin Seidman. PHORBOL MYRISTATE
ACETATE and CATECHOL as SKIN COCARCINOGENS in SENCAR MICE.
Environmental Health Perspectives 68: 33-38, September 1986.
XX-86-085 168
Van Duuren, Benjamin L., Susan Meichionne, Irving Seidm n, and Michael A. Pereira.
CHRONIC BIOASSAYS of CHLORINATED HUMIC SUBSTANCES in B6C3F1
MICE. In: Proceedings of the Second International Symposium on Health Effects of
Drinfring Water Disinfectants and Disinfection By-Products, Cincinnati, Ohio,
August 27-29, 1986. Published in Environmental Health Perspectives 69:109-117,
November 1986.
MS-86-170 175
Ward, Richard L and Pat E. Winston. DEVELOPMENT of METHODS to MEASURE
VIRUS INACI’IVATION in FRESH WATERS. Applied and Environmental
Microbiology 50(5): 1144-1148, November 1985.
XX-85-114
White, Kimber L, Jr., Virginia M. Sanders, Donald W. Barnes, George M. Shopp, Jr., and
Albert E. Munson. IMMUNOTOXICOLOGICAL INVESTIGATIONS in the
MOUSE: GENERAL APPROACH and METHODS. Drug and Chemical
Toxicology 8(5): 299-331, October 1985.
XX-86-100 147
White, Kimber L., Jr., Virginia M. Sanders, Donald W. Barnes, George M. Shopp, Jr., and
Albert E. Munson. TOXICOLOGY of 1,2,-TRICHLOROETHANE in the
MOUSE. Drug and Chemical Toxicology 8(5): 333-355, October 1985.
XX-86-101 148
Wi11 ni F. P.,Jr. VIRUS-LIKE PARTICLES with T = 19 ICOSAHEDRAL SYMMETRY
in a HUMAN GASTROENTERITIS STOOL. Micron and Microscopica Acta
16(3): 173-178, 1985.
MS-85-102 120
Williams, Fred P., Jr. and Elmer W. Akin. WATERBORNE VIRAL GASTROENTERITIS.
Journal of the American Water Works Association 78(1): 34-39, January 1986.
MS-85-021 121
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Wones, Robert and Charles Glueck. RESEARCH in PROGRESS: THE EFFEC’I’S of
CHLORINATED DRINKING WATER on HUMAN LIPID METABOLISM. In:
Proceedings of the Second International Symposium on Health Effects of Drinking
Water Disinfectants and Disinfection By-Products, Cindnn iti, Ohio, August 27-29,
1986. Published in: Environmental Health Perspectives 69:255-258, November 1986.
XX-86-120 J59
Zierler, Sally, Robert A. Danley, and Lisa Feingold. TYPE of DISINFECTANT in
DRINKING WATER and PArrERNS of MORTALITY in MASSACHUSETfS.
In: Proceedings of the Second International Symposium on Health Effects of
Drinking Water Disinfectants nad Disinfection By-Products Cincinnati, Ohio,
August 27-29, 1986. Published in Environmental Health Perspectives 69:275-279,
November 1986.
XX86117 io
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Index
Pesticides
Page No .
Clive, D., W. Caspary, P. E. Kirby, R. Krehl, M. Moore, J. Novalç and T. J. Oberly. NEW
STANDARD GUIDE for PERFORMING the MOUSE LYMPHOMA ASSAY for
MAMMALIAN CELL MUTAGENICITY. In: Proceedings of the American Society
for Testing and Materials, 1986.
MS-86-012 208
Cummings, Audrey M. and Kenneth L. Barker. ISOLATION of a PRECURSOR and a
NASCENT CHAIN FORM of GLUCOSE-6- PHOSPHATE DEHYDROGENASE
from RAT UTERUS and REGULATION of PRECURSOR PROCESSING by
ESTRADIOL. Biochimica et Biophysica Acta 880(2/3): 226-241, February 1986.
MS—85—227 206
Duncan Robert C. and Jack Griffith. MONITORING STUDY of URINARY
METABOLITES and SELECTED SYMPTOMATOLOGY AMONG FLORIDA
CITRUS WORKERS. Journal of Toxicology and Environmental Health 16(3&4):
509-521, November 1985.
MS-86-184 220
Duncan, Robert C., Jack Griffith, and Janet KonefaL COMPARISON of PLASMA
CHOLINESTERASE DEPRESSION AMONG WORKERS OCCUPATIONALLY
EXPOSED to ORGANOPHOSPHORUS PESTICIDES as REPORTED by
VARIOUS STUDIES. Journal of Toxicology and Environmental Health 18(1): 1-11,
May 1986.
MS -86 - 183
Gaines, Thomas B. and Ralph E. Under. ACUTE TOXICITY of PESTICIDES in ADULT
and WEANLING RATS. Fundamental and Applied Toxicology 7(2): 299-308,
August 1986.
MS-86-018 203
Griffith, Jack and Robert C. Duncan. URINARY CHLOROBENZILATE RESIDUES in
CITRUS FH T DWORKERS. Bulletin of Environmental Contamination and
Toxicology 35(4): 496-499, October 1985.
MS-85-117 219
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Griffith, Jack, Robert Duncan, and Janet Konefal. PESTICIDE POISONINGS REPORTED
by FLORIDA CITRUS FTRLDWORKERS. Journal of Environmental Science and
Health B20(6): 701-727, December 1985.
MS-87-090 218
Hu, P., C. Hn n& Y. Ha ing, A. Collier, and W. Clyde DEMONSTRATION of MULTIPLE
ANTIGENIC DETERMINANTS on MYCOPLASMA PNEUMONIAE
ATTACHMENT PROTEIN by MONOCLONAL ANTIBODIES. Journal of
Infection and Immunity 50(1): 292-296, October 1985.
MS-85-169 217
Irby, W. S., Y. S. Huan& C. Y. Kaw ini hi. and W. M. Brooks. IMMUNOBLOT ANALYSIS
of EXOSPORE POLYPEPTIDES from SOME ENTOMOPHILIC
MICROSPORIDIA. Journal of Protozoology 33(1): 14-20, January 1986.
MS-85-110 218
Jensen, KarL ALTERED THALAMOCORTICAL AXON MORPHOLOGY
FOLLOWING NEONATAL PERIPHERAL NERVE DAMAGE:
IMPLICATIONS for the STUDY of DEVELOPMENTAL NEUROTOXICITY.
Neurotoxicology 7(2): 169-182, May 1986.
MS-86-059 210
Kawzinishi, C. Y. MICROSPORIDIAN TAXONOMY: APPLICATION OF
ELEC ROPHORETIC and IMMUNOLOGICAL TECHNIQUES. In:
Proceedings of the 4th International Colloquium on Invertebrate Pathology, The
Netherlands, 1986.
MS-86-109 216
Under, Ralph E., 1iI1i in F Strader, and W. Keith McElroy. MEASUREMENT of
EPIDIDYMAL SPERM MOTILITY as a TEST VARIABLE in the RAT. Bulletin
of Environmental Cont imin iHon and Toxicology 36(3): 317-324, March 1986.
MS-85-166 207
Mahboob, Solt n, li, P1t ibeth R. Hoogenboom, Robert J. Kaviock, and Frances J. Zeman.
EFFECI’S on the FETAL RAT INTESTINE of MATERNAL MALNUTRITION
and EXPOSURE to NITROFEN (2,4-DICHLOROPHENYL-p- NITROPHENYL
ETHER). Teratogenesis, Carcinogenesis, and Mutagenesis 6(6): 45-57, January1986.
MS-85-213 205
Moser, Virginia C. and Robert C. MacPhail. DIFFERENTIAL EFFECI’S of
FORMAMIDINE PESTICIDES on FIXED-INTERVAL BEHAVIOR in RATS.
Toxicology and Applied Pharmacology 84:315-324, October 1986.
MS-85-1 12 215
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Page No .
Novak, Rosanne and Stephanie Padilla. AN IN VITRO COMPARISON of RAT and
CHICKEN BRAIN NEUROTOXIC ESTERASE. Fundamental and Applied
Toxicology 6(3): 464-471, April 1986.
MS-85-201 212
Reiter, Lawrence W. NEUROTOXICOLOGY in the FETUS and CHILD --
CONFERENCE SUMMARY. Neurotoxicology 7(2): 665-670, May 1986.
MS-86-054 209
Sandhu, Shabbeg S. and Michael D. Waters. MECHANISMS of CARCINOGENICITY and
ANTICARCINOGENICITY: ROLE OF DIETARY COMPONENTS. In:
Proceedings of the Conference on Antimutagenesis and Anticarcinogenesis, D.
Shankel, P. Hartman, and A. Hollaender, eds., Plenum Press, New York, NY, 1986.
MS-86-128 209
Svendsgaard, David J. and Kevin M. Crofton. USING ASHFORD’S GENERAL MODEL to
AID in the UNDERSTANDING of a PESTICIDE’S NEUROTOXIC EFFECT
THROUGH PESTICIDE-DRUG MIXTURE EXPERIMENTS. In: Proceedings of
the American Statistical Association, October 1985.
MS-86-O01 211
Veronesi, Bellina. IN ViTRO MODELING in NEUROTOXICOLOGY. In: A Textbook of
Neurotoxicology, M.B. Abou-donia, ed., Elsevier Science Publishers, The
Netherlands, 1986.
MS-86-030 211
Veronesi, Beilina and Stephanie Padilla. PHENYLMETHYLSULFONYL FLUORIDE
PROTECTS RATS from MIPAFOX-1NDUCED DELAYED NEUROPATHY.
Toxicology and Applied Pharmacology 81(2): 258-264, November 1985.
MS-85-108 214
Veronesi, Bellina, Stephanie S. Padilla, and Donald Lyerly. THE CORRELATION
BETWEEN NEUROTOXIC ESTERASE INHIBITION and
MIPAFOX-INDUCED NEUROPATHIC DAMAGE in RATS. Neurotoxicology
7(1): 207-216, April 1986.
MS-85-170 213
Veronesi, Belilna, Stephanie Padilla, and Dale Newland BIOCHEMICAL and
NEUROPATHOLOGICAL ASSESSMENT of TRIPHENYL PHOSPHITE in
RATS. Toxicology and Applied Pharmacology 83(2): 203-210, April 1986.
MS-85-202 215
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Zeman, Frances I ., Pifrsbetli R. Hoogenboom, Carol Chase-Deesing. Robert 3. Kavlock, and
Janet L Semple. EFFECTS on the FETUS of MATERNAL N1TROFEN
EXPOSURE in the PROTEIN-DEPRIVED RAT. Tozicolo 38(1): 55-68, January
MS-85 -214 204
Zeman, Frances J., Elizabeth R. Hoogenboom, Robert J. Kaviock, and Janet L Semple.
EFPECTS on the FETUS of MATERNAL BENOMYL EXPOSURE in the
PROTEIN-DEPRiVED RAT. Journal of Toxicology and Environmental Health
17(4): 405-417, April 1986.
IvLS—85—215 203
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Index
Toxics
Page No .
Adams, J., J. Buelke-Sam, C. A. ICinimel, C. J. Nelson, L. W. Reiter, T. Sobotka, H. A. Tilson,
and B. K Nelson. COLLABORATWE BEHAVIORAL TERATOLOGY STUDY:
PROTOCOL DESIGN and TESTING PROCEDURES. In: Neurobehavioral
Toxicology and Teratology, (in press).
MS-86-007 259
Allen James W. and Carolyn W. Gwaltney. INVESTIGATION of POSSIBLE AGE
EFFECTS on MEIOTIC CHROMOSOMAL RECOMBINATION and
SEGREGATION in ARMENIAN HAMSTER SPERMATOCYTES. Cytobios
43(lfl): fl5-23Z December 1985.
MS -85-238
Allen, James W., Jan C. Iii n& Anthony V. Carrano, and R. Julian Preston. REVIEW of
LITERATURE on CHEMICAL-INDUCED ANEUPLOIDY in MAMMALIAN
MALE GERM CPIIR. Mutation Research 167(1&2): 123-137, Januaiy/March
1986.
MS -85-239 223
Backer, Lorraine C. and James W. Allen. MEIOTIC ORIGIN of ANEUPLOIDY: AN
OVERVIEW. In: Progress in Topics in Cytogenetic Aneuploidy Incidences and
Etiology, Allen R. Liss Publishers, New York, NY (in press).
MS-86-206 2z3
BilHngsley, M. L, C. D. Balaban, and J. P. O’Callaghan. PHARMACOLOGIC and
IMMUNOLOGIC APPROACHES to the PROBLEMS of POSTFRAUMATIC
GLIAL PROLIFERATION FOLLOWING CNS DAMAGE. In: NATO Advanced
Research Workshop: Glial-Neuronal Communication in Development and
Regeneration, Germany, 1985.
MS-86-043 270
Chadwick R. W. and M. F. Copeland. INVESTIGATION of HCB as a METABOLITE from
FEMALE RATS TREATED DAILY for SIX DAYS with LINDANE. Journal of
Analytical Toxicology 9(6): 262-266, November/December 1985.
MS-85-175 248
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Chadwick, Robert W., M. Frank Copeland, Gary P. Carlson, Bruce A. Trela, and Bernard M.
Most. COMPARISON of IN V1VO and IN VITRO METHODS for ASSESSING
the EFFEC1’S of PHENOBARBITAL on the HEPATIC
DRUG -METABOLIZING ENZYME SYSTEM. Toxicology Letters 29(2&3):
95-105, December 1985.
MS.85-217 249
Chadwick, R. W., M. F. Copeland, U. L Wolff, N. Cook; D. A. Whitehouse, and M. L. Mole.
EFFECFS of AGE and OBESITY on the METABOLISM of LINDANE by BLACK
aia, YELLOW A vy/a, and PSEUDOAGOUTI A vy/a PHENOTYPES of (YS x
VY)fl HYBRID MICE. Journal of Toxicology and Environmental Health 16(6):
771-796, December 1985.
MS-85-029 248
Chase-Deesing, Carol, Robert I. Kaviock, and Frances J. Zeman. KIDNEY MORPHOLOGY
and FLJNCflON in the YOUNG of RATS MALNOURISHED and EXPOSED to
NITROGEN DURING PREGNANCY. Journal of Toxicology and Environmental
Health 19(1): 1-21, 1986.
MS-86.039 245
Conti, C. J., N. Clapp, A. J. P. Klein-Szanto, S. Nesnow, and T. I. Slap. SURVIVAL
CURVES and INCIDENCE of NEOPLASTIC and NON-NEOPLASTIC DISEASE
in SENCAR MICE. Carcinogenesis 6(11): 1649-1652, October 1985.
A1O1/L104 MS-86-052 229
Copeland, M. K, R. W. Chadwick, N. Cook; D. A. Whitehouse, and D. M. Hill. THE USE of
a MODEL SUBSTRATE (LINDANE) to DETERMINE the ONTOGENY of
METABOLISM in the DEVELOPING RAT. Journal of Toxicology and
Environmental Health 18(4): 527-542, August 1986.
MS-85-245 247
Dyer, Robert S. MACROPHYSIOLOGICAL ASSESSMENT of ORGANOMETAL
NEUROTOXICITY. IL Neuroto dcants and Neurobiological Function, Tilson and
Sparber, eds., (in press).
MS-86-009 261
Dyer, RobertS, Linda J. Burdetle, Raelya Janssen, and William K. Boyes.
NEIJROPHYSIOLOGICAL CONSEQUENCES of ACUTE EXPOSURE to
METHYLPYRIDINES. Fundamental and Applied Toxicology 5(5): 920-932,
October 1985.
MS-84-127 268
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Evenson, Donald P. CHANGES in ACCESSIBILITY of DNA to VARIOUS
FLUOROCHROMES DURING SPERMATOGENESIS. Cytometry 7:45-53,
January 1986.
XX-85-085 237
Ferguson, Lynnette R., Pierre van Ziji, and Stephen Nesnow. MORPHOLOGICAL
TRANSFORMATION and CHROMOSOME DAMAGE by AMSACRINE in
C3H/10T112 CLONE 8 CELLS . Mutation Research 170: 133-143, April 1986.
MS-87-091 236
Frierson, M. R., G. Klopman, and H. S. Rosenkranz. STRUCTURE-ACTIVITY
RELATIONSHIPS (SARs) AMONG MUTAGENS and CARCINOGENS: A
REVIEW. Environmental Mutagenesis 8(2): 283-327, March 1986.
XX-86-106 271
George, S. E., LC. King, and LD. Claxton. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY SEPARATION of CHLORDECONE and ITS
METABOLITES. Chromatographia 22(1-6): 165-168, June 1986.
MS-86-092 277
Geyer, Mark A. and Lawrence W. Reiter. STRATEGIES for the SELECTION of TEST
METHODS for SCREENING in BEHAVIORAL TERATOLOGY. In:
Neurobchavioral Toxicology and Teratology, (in press).
MS-86-008 258
Gordon, Christopher J., Merritt D. Long and Andrew G. Stead. THERMOREGULATION
in MICE FOLLOWING ACUTE CHLORDIMEFORM ADMINISTRATION.
Toxicology Letters 28(1): 9-15, October 1985.
MS-85-093 263
Gray, L. Earl, Jr., John M. Rogers, Robert J. Kaviocic, Joseph S. Ostby, Janet M. Ferrell, and
Katrina L. Gray. PRENATAL EXPOSURE to the FUNGICIDE DINOCAP
CAUSES BEHAVIORAL TORTICOWS, BALLOONING and CLEFT PALATE
in MICE, but not RATS or HAMSTERS. Teratogenesis, Carcinogenesis, and
Mutagenesis 6(1): 33-43, January 1986.
MS-85-196 240
Gray, L. E., Jr., R. J. Kaviock, J. S. Ostby, J. M. Ferrell,J. M. Rogers, and K. Gray. AN
EVALUATION of FIGURE-EIGHT MAZE ACTIVITY and GENERAL
BEHAVIORAL DEVELOPMENT FOLLOWING PRENATAL EXPOSURE to
FORTY CHEMICALS: EFFECTS of CYFOSINE ARABINOSIDE, DINOCAP,
N1TROFEN, and VITAMIN A. Neurotoxicology 7(2): 449-462, May 1986.
MS-86 -050 241
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Page No .
Greenberg B., P. Moore, R. Lctz, and E. Baker. COMPUTERIZED ASSESSMENT of
HUMAN NEUROTOXIC1TY: SENSITIVITY to NiTROUS OXIDE EXPOSURE.
Clinical Pharmacology and Therapeutics 38(6): 656-660, December 1985.
XX-86 -072 268
Hatch, George G., Patricia M. Conklin, Carla C. Christansen, Tara M. Anderson, Robert
Lanrnbach, and Stephen Nesnow. MUTATION and ENHANCED VIRUS
TRANSFORMATION of CULTURED HAMSTER CELLS by EXPOSURE to
GASEOUS ETHYLENE OXIDE. Environmental Mutagenesis 8:67-76, January
1986.
MS-83 . .101 226
House, Dennis, Ezra Berman, and Hershel B. Carter. DESCRIPTION and
IMPUCATIONS for ANALYSIS of BRAIN GROWFH in SUCKUNG MICE.
Growth 49:426-438, December 1985.
MS-85-116
Hozier, John, Jeffrey Sawyer, Donald Clive, and Martha M. Moore. CHROMOSOME 11
ABERRATIONS in SMALL COLONY L5178Y TK-/- MUTANTS EARLY in
THEIR CLONAL HISTORY. Mutation Research 147(5): 237-242, October 1985.
MS-84-115 231
HuJ n& Yuan-Shen, Neil Chernoff Robert J. Kaviock, and Clinton Y. Kawanishi. THE
EFFECTS of MATERNAL MURINE CYTOMEGAL() VIRUS INFECTION on
the MOUSE CONCEPTUS at DIFFERENT GESTATIONAL STAGES.
Teratogenesis, Carcinogenesis, and Mutagenesis 6(4): 331-338, August 1986.
MS-86-002
Jaeger E. P. and P. C. Jurs. USE of a GRAPH THEORETIC SIMILARITY INDEX in
PREDICTION STUDIES of LINEAR DISCRIMINANTS and MODELS. In:
Proceedings of the 28th ORNL-DOE Conference on Analytical Chemistry in Energy
and Technology, Knoxville, TN, October 1985.
XX .86 -081 274
Janca, Frank C., Lorna K. Jost and Donald P. Evenson. MOUSE TESTICULAR and
SPERM CELL DEVELOPMENT CHARACTERIZED from BIRTH to
ADULTHOOD by DUAL PARAMETER FLOW CYTOMETRY. Biology of
Reproduction 34:613-623, May 1986.
XX-85-118
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Page No .
Janssen, Raelyn, Vernon A. Benignus, Laura M. Grimes, and Robert S. Dyer. ANALOG
FILTERING and INTERPRETING the BRAINSTEM AUDITORY EVOKED
RESPONSE in LABORATORY RATS. Electroencephalography and Clinical
Neurophysiology65(3): 203-211, May1986.
MS-85-127 266
Jurs, P. C., B. K. Lavine, and T. R. Stouch. PAIThRN RECOGNITION STUDIES of
COMPLEX CHROMATOGRAPHIC DATA SETS. Journal of Research of the
National Bureau of Standards 90(6): 543-549, November/December 1985.
XX-86-076 275
Kaviock, Robert 3., Blair F. Rehnberg, and Ellen H. Rogers. AMPHOTERICIN B- and
FOLIC ACID-INDUCED NEPHROPATHIES in DEVELOPING RATS.
Toxicology and Applied Pharmacology 81(3): 407-413, December 1985.
MS-85-085 243
Kaviock, R. 3., B. F. Rehnberg. and E. H. Rogers. CONGENITAL RENAL HYPOPLASIA:
EFFECTS on BASAL RENAL FUNCTION in the DEVELOPING RAT.
Toxicology 40(3): 247-258, September 1986.
MS-85-212 243
Kaviock, R. J., J. M. Rogers, N. Chernoff and L. E. Gray. POSTNATAL ALTERATIONS in
DEVELOPMENT RESULTING from PRENATAL EXPOSURE to PESTICIDES.
In: Proceedings of the Congress of Pesticide Chemistry, Ottawa, Canada, August
10-15,1986.
MS-86-208 240
Kellis, James T. Jr., Stephen Nesnow, and Larry E. Vickery. INHIBITION of AROMATASE
CYTOCHROME P450 (ESTROGEN SYNTHETHASE) by DERIVATIVES of
a-NAPHTHOFLAVONE. Biochemical Pharmacology 35(17): 2887-2891, April 1986.
L104 MS-87-092 225
Kitchin, Kirk T. and Sam Kacew. SOME PHARMACOKINETIC and METABOLIC
FACTORS AFFECTING the NEONATAL TOXICITY of HALOGENATED
HYDROCARBONS FOUND in the GREAT LAKES. In: Toxicologic and
Pharmacologic Principles in Pediatrics, Sam Kacew and Simon Lock, eds.,
Hemisphere Publishing Co., (in press).
MS-86-240 236
Kligerman, A. D., M.M. Moore, G.L. Erexson, K.H. Broclç C.L. Doerr, J.W. Allen, and S.
Nesnow. GENOTOXICITY STUDIES of BENZ(1)ACEANTHRYLENE. Cancer
Letters 31(2): 123-131, May 1986.
MS-86-076 227
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Klopman, G., R. Contreras, H. S. Rosenkranz , and M. D. Waters.
STRUCFURE-GENOTOXIC ACTIVITY RELATIONSHIPS of PESTICIDES:
COMPARISON of the RESULTS from SEVERAL SHORT-TERM ASSAYS.
Mutation Research 147: 343-356, December 1985.
MS-86-045 272
Lafeniin D. H. and P. C. Jurs. A NUMERICAL INDEX for CHARACFERIZING DATA
SET SEPARATION. Journal of Chemical Information and Computer Sciences
25(4): 386, November 1985.
XX-86-075 276
Luebke, R. W., M. M. Riddle, R. R. Rogers, R. J. Garner, D. G. Rowe, and R. J. SmialowicL
IMMUNE FUNCTION of YOUNG ADULT MICE FOLLOWING IN UTERO
EXPOSURE to CYCLOPHOSPHAMIDE. Journal of Toxicology and
Environmental Health 18(1): 25-39, July 1986.
MS-85-182 256
Luebke, Robert W., Marie M. Riddle, Ronald R. Rogers, Denise G. Rowe, R. John Garner,
and Ralph I. Smialowicz. IMMUNE FUNCTION in ADULT C57BLI6J MICE
FOLLOWING EXPOSURE to URETHAN PRE- or POSTNATALLY. Journal of
Immunopharmacology 8(2): 243-257,1986.
MS-85-233 257
Mass M. J., C. M. Man, ifi, and J. L Mumford. PRENEOPLASTIC TRANSFORMATION
of RESPIRATORY EPITHELIAL CELLS by COMPLEX ORGANIC
MIXTURES in a CLONAL ASSAY. In: Health and Environmental Research on
Complex Organic Mixtures, (in press).
MS-86-011 232
McLaren, Christine E., Gary M. Brittenhs m1 and Victor Hasselbiad. ANALYSIS of the
VOLUME of RED BLOOD CELLS: APPLICATION of the
EXPECTATION-MAXIMIZATION ALGORIThM to GROUPED DATA FROM
the DOUBLY-TRUNCATED LOGNORMAL DISTRIBUTION. Biometrics 42:
143-158, March 1986.
MS-84-150 270
Meyer, Mary., Karen Brock, Kay Lawrence, Bruce Casto, and Martha M. Moore.
EVALUATION of the EFFECT of AGAR on the RESULTS OBTAINED in the
L5178Y MOUSE LYMPHOMA ASSAY. Environmental Mutagenesis 8: 727-740,
1986.
MS-86-006 226
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Page No .
Moore, Martha M., Am mda Amtower, Gary H. S. Strauss, and Carolyn Doerr.
GENOTOXICITY of GAMMA IRRADIATION in L5178Y MOUSE
LYMPHOMA CRLLS. Mutation Research 174(2): 149-154, June 1986.
MS-86-004
Moser, Virginia C. and Robert C. MacPhaiL YOHIMBINE ATIENUATES the DELAYED
LETHALITY INDUCED in MICE by AMITRAZ, a FORMAMIDINE
PESTICIDE Toxicology Letters 28: 99-104, November 1985.
MS-85-118 266
Nesnow, Stephen, Hmda Berginm, and Thomas J. Slaga. COMPARISON of the
TUMORIGENIC RESPONSE of SENCAR and C57BL/6 MICE to
BENZO(a)PYRENE and the INTEREXPERIMENTAL VARIABILITY OVER a
THREE-YEAR PERIOD. Environmental Health Perspectives 68(1): 19-25,
September 1986.
MS-85- lfl 229
O’Csi1bighs n, James P. NEUROTYPIC and GLIOTYPIC PROTEINS as BIOCHEMICAL
INDICATORS of NEUROTOXIC1TY. In: Neurotoxicology, M.B. Abou-donia, ed.,
Oxford University Press (in press).
MS-86-209 262
O’C iHaghan, James P. and Diane B. Miller. DIETHYLDITHIOCARBAMATE
INCREASES DISTRIBUTIONof CADMIUM to BRAIN BUT PREVENTS
CADMIUM-INDUCED NEUROTOXICITY. Brain Research 370(1): 354-358,
April 1986.
MS-85-199 269
Oglesby, Linda A., Marian T. Ebron, Patricia E. Beyer, Brenda D. Carver, and Robert J.
Kaviock. CO-CULTURE of RAT EMBRYOS and HEPATOCYTES: IN VITRO
DETECTION of a PROTERATOGEN. Teratogenesis, Carcinogenesis, and
Mutagenesis 6(2): 129-138, March 1986.
MS-85-080 239
Padifia, Stephanie and Donald Lyerly. EFFECFS of HYPOTHERMIA on the IN VIVO
MEASUREMENT of RAPID AXONAL TRANSPORT in the RAT: A
CAUTIONARY NOTE. Journal of Neurochemistry 46(4): 1227-1230, April 1986.
MS-85-205 265
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Page No .
Peele, David B., Robert C. MacPhail and Jackie D. Farmer. FLAVOR AVERSIONS
INDUCED by THALLIUM SULFATE: IMPORTANCE of ROUTE of
ADMINISTRATION. Neurobehavioral Toxicology and Teratology 8(3): 273-277,
MaylJune 1986.
MS-85-184 267
Rabinowitz, J. R., K. Namboodri, and H. Weinstein. A FINITE EXPANSION METHOD for
the CALCULATION and INTERPRETATION of MOLECULAR
ELECTROSTATIC POTENTIALS. International Journal of Quantum Chemistry
29(6): 1697-1704, June 1986.
MS-85-150 273
Rehnber& Georgia L., Joy F. Hem, Susan D. Carter, and John W. Laskey.
AGE-DEPENDENT CHANGES in GASTROINTESTINAL TRANSPORT and
RETENTION of PARTICULATE MANGANESE OXIDE in the RAT. Journal of
Toxicology and Environmental Health 16(6): 887-899, December 1985.
MS-82-116 246
Rudo, Kenneth, Scott Ellis, BJ. Bryant, Kay Lawrence, Gaynelle Curtis, Helen Garland, and
Stephen Nesnow. QUANTFFATIVE ANALYSIS of the METABOLISM of
BENZO(a)PYRENE by TRANSFORMABLE C3H1OTI/2CLS MOUSE EMBRYO
FIBROBLASTS. Teratogenesis, Carcinogenesis, and Mutagenesis 6(4): 307-319,
August 1986.
MS-85-178 233
Ruppert, Patricia H., Karen F. Dean, and Lawrence W. Reiter. DEVELOPMENT of
LOCOMOTOR ACTIVITY of RAT PUPS in FIGURE-EIGHT MAZES.
Developmental Psychobiology 18(3): 247-260, May 1985.
MS-85-017 260
Ruppert, Patricia H. and Robert S. Dyer. ACUTE BEHAVIORAL TOXICITY of
SULFOLANE: INFLUENCE of HYPOTHERMIA. Toxicology Letters 28(2&3):
111-116, November 1985.
MS-85-099 264
Sandhu, Shabbeg S., Baldev K. Vig, and Milton J. Constanth. DEFECFION of
CHEMICALLY INDUCED ANEUPLOIDY with PLANT TEST SYSTEMS.
Mutation Research 167(1&2): 61-69, January/March 1986.
MS-85- 146
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Page No .
Sawyer, Jeffrey, Martha M. Moore, Donald Clive, and John Hoñer. CYTOGENETIC
CHARACTERIZATION of the L5178Y TK + I- 3.7.2C MOUSE LYMPHOMA
CELL LINE Mutation Research 147(5): 243-253, October 1985.
MS-84-114 230
Shah, P. V., M. R. Sumler, Y. M. Ioannou, H. L Fisher, and L. L. Hall. DERMAL
ABSORPTION and DISPOSITION of 1,3-DIPHENYLGUANIDINE in RATS.
Journal of Toxicology and Environmental Health 15(5): 623-633, October 1985.
MS-84-170 254
Siegfried, J. M., K. Rudo, B. J. Bryant, S. Ellis, M. J. Mass, and S. Nesnow. METABOLISM
of BENZO(a)PYRENE in MONOLAYER CULTURES of HUMAN
BRONCHIAL EP1THELIAL CELLS from a SERIES of DONORS. Cancer
Research 46:4368-4371, September 1986.
MS-86-081 232
Slotkin, Theodore A., Steven Pachm in. Robert J. Kaviock, and Jorge Bartolome. EARLY
BIOCHEMICAL DETECTION of ADVERSE EFFECTS of a
NEUROBEHAVIORAL TERATOGEN: INFLUENCE of PRENATAL
METHYLMERCURY EXPOSURE on ORNITHINE DECARBOXYLASE in
BRAIN and OTHER TISSUES of FETAL and NEONATAL RAT. Teratology
32(2): 195-202, October 1985.
MS-85-063 242
Smialowicz, Ralph. IMMUNE STATUS in NEWBORNS and THEIR VULNERABILITY.
In: Proceedings of the Indo-U.S. Workshop on the Role of Predisposing Conditions
of Health, Nutrition, and Environment on Safety of Drugs and Chemicals, Lucknow,
India, February 25-28, 1986.
MS-86-25 1 254
Smialowicz, Ralph J., Robert W. Luebke, Ron R. Rogers, Marie M. Riddle, and Denise G.
Rowe. EVALUATION of IMMUNE FUNCTION in MICE EXPOSED to
ORDRAM®. Toxicology 37(3&4): 307-314, December 1985.
MS-85-1.57 258
Smialowic Ralph J., Ronald R. Rogers, Marie M. Riddle, Denise G. Rowe, and Robert W.
Luebke. IMMUNOLOGICAL STUDIES in MICE FOLLOWING IN UTERO
EXPOSURE to N1CI2. Toxicology 38(3): 293-303, March 1986.
MS-85-190 255
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Page No .
Stouch T. R. and P. C. Jurs. A SIMPLE METHOD for the REPRESENTATION,
QUANTIFICATION and COMPARISON of the VOLUMES and SHAPES of
CHEMICALS. Journal of Chemical Information and Computer Sciences 26(1): 4-12,
February 1986.
XX-86-077 274
Strek, Kimi S., Merritt D. Long, and Christopher J. Gordon. EFFECT of SODIUM
PENTOBARBITAL on BEHAVIORAL THERMOREGULATION in RATS and
MICE. Pharmacology Biochemistry and Behavior 24(4): 1147-1150, April 1986.
MS-85-193 264
Trela, Bruce A., Gary P. Carlson, Robert W. Chadwick, and M. Frank Copeland. A
COMPARISON of IN VITRO and IN VIVO METHODS for EVALUATING
ALTERATIONS in HEPATIC DRUG METABOLISM FOLLOWING
MERCURIC CHLORIDE ADMINISTRATION. Toxicology Letters 32(1&2):
133-140, July/August 1986.
MS-86-077 251
Trela, Bruce A., Gary P. Carlson, Robert W. Chadwick, and M. Frank Copeland.
COMPARISON of IN VITRO METHODS and the IN VIVO METABOLISM of
LINDANE for ASSESSING the EFFECTS of REPEATED ADMINISTRATION of
ETHANOL on HEPATIC DRUG METABOLISM. Toxicology Letters 29(2&3):
85-93, December 1985.
MS-85-228 250
Trela, Bruce A., Gary P. Carlson, Robert W. Chadwick, and M. Frank Copeland.
COMPARISON of IN V1VO and IN VITRO METHODS for ASSESSING
EFFECTS of ALLYL ALCOHOL on the LIVER. Toxicology Letters 29(2&3):
77-84, December 1985.
MS-85-195 250
Veronesi, B. and S. Bondy. TRIETHYLTIN-INDUCED NEURONAL DAMAGE in
NEONATALLY EXPOSED RATS. Neurotoxicology 7(1): 69-80, April 1986.
MS-85-179 260
Walaas, S. Ivar, Angus C. Nairn, and Paul Greengard. PCPP-260, A PURKINJE
CELL-SPECIFIC CYCLIC AMP-REGULATED MEMBRANE
PHOSPHOPROTEIN of Mr 260,000. Journal of Neuroscience 6(4): 954-961, April
1986.
XX -86-138 262
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Page No .
Watkinson, William P. EFFECFS of CHLORDIMEFORM on CARDIOVASCULAR
FUNCTIONAL PARAMETERS: PART 1. LETHALITY and
ARRHYTHMOGENICITY in the GERIATRIC RAT. Journal of Toxicology of
Environmental Health 15:729-744, November 1985.
MS-87-072 253
Watldnson, William P., M. Ann Brice, and Kathy S. Robinson. A COMPUTER-ASSISTED
ELECTROCARDIOGRAPHIC ANALYSIS SYSTEM: METHODOLOGY and
POTENTIAL APPLICATION to CARDIOVASCULAR TOXICOLOGY. Journal
of Toxicology and Environmental Health 15: 713-727, November 1985.
MS-83-042 252
Watson, Dennis K., Mary J. McWilliRm -Smith, Christine Kozak, Roger Reeves, John
Gearhart, Michael F. Nunn, Wi1li m Nash, John R. Fowle, Ill, Peter Duesber& Takis
S. Papas, and Stephen 3. O’Brien. CONSERVED CHROMOSOMAL POSITIONS
of DUAL DOMAINS of the ETS PROTOONCOGENE in CATS, MICE and
HUMANS. Proceedings of the National Academy of Science 83: 1792-1796, March
1986.
MS-86-218 235
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Index
Hazardous Waste
Page No .
Andon, B., T. K. Rao, B. Most, J. Alien, E. Berman, N. Chernoff, K. Dean, D. DeMarini, J.
Laskey, R. MacPhail, L. Reiter, and R. Smialowicz. THE EVALUATION of FOUR
TOXIC CHEMICALS in an IN VIVO/IN VITRO TOXICOLOGICAL SCREEN:
ACRYLAMIDE, CHLORDECONE, CYCLOPHOSPHAMIDE, and
DIETHYLSTILBESTROL. U.S. Environmental Protection Agency,
EPA-600/1-86-002 , May 1986.
HERL-0542 291
Houk Virginia Stewart and Larry D. Claxton. SCREENING COMPLEX HAZARDOUS
WASTES for MUTAGENIC ACTIVITY USING a MODIFIED VERSION of the
TLC/SALMONELLA ASSAY. Mutation Research 169(3): 81-92, March 1986.
MS-85-155 292
MacPhail, Robert C. OBSERVATIONAL BATTERIES and MOTOR ACTIViTY. In:
Symposium on Neurobehavioral Methods in Safety Assessment of Chemicals and
Drugs, Dusseldorf, Germany, December 16-19, 1985.
MS-86-093 292
Reynolds, Randall P. and Patricia F. Noden. INFLUENCE of CORTISOL on
PROSTAGLANDIN SYNTHESIS by FETAL MEMBRANES, PLACENTA and
UTERUS of PREGNANT RABBITS. Biology of Reproduction (in press).
XX-85-112 294
Simmons, Jane Ellen, Marcus Jackson, Joellen Lewtas, and Ezra Berman. A COMPARISON
of the TOXICITY of ACRYLAMIDE, CYCLOPHOSPHAMIDE
CHLORDECONE, and DIETHYLSTILBESTROL in CHINESE HAMSTER
OVARY (CHO) CELLS with THEIR TOXICITY IN VIVO. In: Alternative
Methods in Toxicology Series, vol.5: In Vitro Toxicology-Approaches to Validation,
1986.
MS-86-163 293
Smialowicz, Ralph J., Robert W. Luebke, Ron R. Rogers, Marie M. Riddle, and Denise G.
Rowe. EVALUATION of IMMUNE FUNCTION in MICE EXPOSED to
ORDRAM®. Toxicology 37(3&4): 307-314, December 1985.
MS-85-157 295
-351-

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Appendix A
Submitted
Documents
Submitted for publication between October 1, 1985 and September 30,1986 and awaiting publication.
Abstracts for these documents are not included in this report.

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MO! - AIR HEALTH
Andrews, JE; Courtney, KD; Donaldson, WE. Impairment of Calcium Homeostasis by
Hexachlorobenzene Exposure in Fischer 344 Rats. Submitted: Toxicology and Applied
Pharmacology.
MS-86-256
Barry, BE; Mercer, RR; Crapo, 3D; Miller, FJ. Effects of Inhalation of 0.25 ppm Ozone on the
Terminiil Bronchioles of Juvenile and Adult Rats. Submitted: American Journal of Pathology.
MS -86-185
Benignus, VA; Muller, KE; Barton, CN; Prah, JD. Effect of Low Level Carbon Monoxide on
Tracking and Monitoring. Submitted: Neurobehavioral Toxicology and Teratology.
MS-86-245
Buffler, PA. Health Effects of Air Pollution in Texas Gulf Coast Area. US Environmental Protection
Agency report.
HERL -0521
aaxton, U). A Guide for the Salmonella ,himurium/Mammalian Microsome Tests for Bacterial
Mutagenicity. Submitted: Mutation Research.
MS-86-203
Cooper, R I; Goldman, JM; Rehnberg, GL. Pituitary Function Following Treatment with
Reproductive Toxins. Submitted: Environmental Health Sciences.
MS-86-146
Currie, WD; Hatch, GE; Frosolono, MF. Pulmonary Alterations in Rats Due to Acute Phosgene
Inhalation. Submitted: Fundamental and Applied Toxicology.
MS -86-136
Davies, DW; Walsh, L; Hiteshew, ME; Menache, MA. An Automated Chronic Gaseous Animal
Inhalation Exposure Facility. Submitted: American Industrial Hygiene Association Journal.
MS-86-055
Goidring. JM; Bali, LM; Sangaiah , R; Gold, A. Mutagenic Activity of Nitro-Substituted
Cydopenta-Fused Polycydic Aromatic Hydrocarbons Towards Salmonella lyphimunum.
Submitted: Mutation Research.
XX-86-143
Goldstein, GM; Raggio, L; House, DE. Factors Influencing CarboxyhemogJobin Stability.
HERL-0532
Goto, S; Williams, K; Lewtas, J. A Micro-Forward Mutation Assay in Salmonella lyphimurium,
Modifications and Application to Complex Environmental Emissions. Submitted: Mutation
Research.
MS-86-075
.355..

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Grose, EC; Gr h m, JA; Jaskot, RH; Richards, JH; Menache, MA; Dauterman, WC. A
Comparative Study of the Effects of lnhiiled Cadmium Chloride and Cadmium Oxide, Pulmonary
Response. Submitted: Toxicology and Applied Pharmacology.
MS-86-061
Grose, EC; GrallMm, JA; Richards, JH; Jaskot, RH; Menache, MG; Dauterman, WC. Glutathione
(GSH) Peroxidase and GSH Transferase Activity in Rat Lung and Liver Following Cadmium
Inh its.tion. Submitted: Toxicology Letters.
MS-86-095
Grose, EC; Graham, JA; Richards, JH; Jaskot, RH; Menache, MG; Dauterman, WC. A
Comparative Study of the Effects of lnluile4 Cadmium Chloride and Cadmium Oxide, Hepatic
Response. Submitted: Toxicology and Applied Pharmacology.
MS-86-096
Horstmzrn, DH; Carver, BD; McDonnell, WF; Kehrl, HR; Seal, EG; Chapman, RS; Massaro, EL
Pulmonary Effects in Asthmatics Exposed to 0.30 ppm Nitrogen Dioxide During Repeated
Exercise. Submitted: American Review of Respiratory Disease.
MS-86-140
Houk VS; Claxton, LD; Zweidinger, RB. The Mutagenicity of Teflon-Coated Glass Fiber Filters, a
Potential Problem and Solutions. Submitted: Environmental Science and Technology.
MS-86-111
Johnson, DA; Winters, RS; Woolley, T; Grah, m, DG; Henderson, FW. Ozone Effects on
Alpha-l-Proteinase Inhibitor In J’ivo - Blood Plasma Inhibitory Activity is Unchanged. Submitted:
Experimental Lung Research.
MS-86-212
Kehrl, HR; Horstm n, DH; Vincent, L; Kowaisky, R; McCartney, W; Bromberg, PA. Ozone
Exposure Increases Respiratory Epithdllal Permeability in Man. Submitted: Science.
MS-86 .037
King, LC; Jackson, M; Ball, LM; Lewtas, J. Metabolism and DNA Binding of 1-Nitro [ 14 C]pyrene by
Isolated Rabbit Tracheal Epithelial Cells. Submitted: Carcinogenesi&
MS-86-257
Lewtas, J; King, LC. Mutagenicity, Metabolism and DNA Dosimetry of Nitrated Polycyclic Aromatic
Hydrocarbons and Other POMS in Complex Mixtures Metabolism, Mutagenicity and Dosimetry
of Selected PAils and Nitro-PAHs Found in Air. US Environmental Protection Agency in-house
report, 1986.
HERL-0559
MacPhail, RC; Peele, DB. Behavioral Effects of Sulfolane - Motor Activity and flavor-Aversion
Conditioning. Submitted: Neurobehavioral Toxicology and Teratology.
MS-86-188
MacPhail, RC. Effects of Acute and Short-Term Repeated Administration of Sulfolane on the Motor
Activity of Mice. Submitted: Neurobehavioral Toxicology and Teratology.
MS-86-189
-356-

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Morse, PA; Molfese DL Laughin. NK Categorical Perception for Voicing Contrasts in Normal and
Lead-Treated Rhesus Monkeys Electrophysiological Indices. Submitted: Brain and Language.
XX-86-105
Mumford, 3L He, X; Chapman, RS; Cao, S; Harris DB; Li, X Xian, Y; hang, W; Xu, C; Chu2ng,
JC. Lung Cancer and Indoor Air Pollution in Xuan Wel, China, U. Indoor Air Characterization.
Submitted: Science.
MS-86-051
Mumford, JL; Wilibims, K Chuang, JC; Cooke, M. Development of a Medium-Volume Sampler for
Indoor Air Sampling and Mutagenicity Studies. Submitted: Environmental Science and
Technology.
MS-86-192
Overton, JH; Miller, FJ; Gr 1hfim 1 RC. A Model of the Regional Uptake of Gaseous Pollutants in the
Lung. IL The Sensitivity of Ozone Uptake in Laboratory Animals to Anatomical and Ventilatory
Parameters. Submitted: Toxicology and Applied Pharmacology.
MS-86-106
Patra, AL; Gooya, A; Morgan, KT. Airflow Characteristics in a Baboon Nasal Passage Cast.
Submitted: Journal of Applied Physiology.
XX-86-049
Patra, AL; Menache M; Shslrn , NB; Gooya, A. A Morphometric Study of Nasal-Pharyngeal Growth
for Particle Deposition in Rat. Submitted: Journal of American Industrial Hygiene.
XX-86-121
Schwartz, J; Otto, DA. Blood Lead Levels, Hearing Thresholds and Neurobehavioral Development in
Children and Youth. Submitted: Archives of Environmental Health.
MS-86-223
Seigrade, MK; Hatch, GE; Grose, EC; HUng . 3W; Stead, AG; Miller, PJ; Gr Ii im, JA; Stevens, MA;
Hardisty, JA. Pulmonary Effects Due to Short Term Exposure to Oil Fog. Submitted: Journal of
Toxicology and Environmental Health.
MS-86-227
Shelburne, 3D; Ingram, P; Linton, RW. Ultrastructure, X-ray and Ion Microanalysis of Macrophages
Exposed to Non-Criteria Pollutants. US Environmental Protection Agency report.
HERL-0519
Sherwood, RL; O’Shea, W; Thomas, PT; Ratajczak HV; Aranyi, C; Grsil m, JA. Effects of
Tnh dg tion of Ethylene Dichioride on Pulmonary Defenses of Mice and Rats. Submitted:
Toxicology and Applied Physiology.
MS-86-102
Shoaf, CR; Wolpert, RL; Menzel, DL. Factors Controlling Nitros imine Formation in the Lung. a
Unique Uptake System. Submitted: Lung.
XX-86-071
Slade, R; Hatch, GE. Role of Lung Nonprotein Sulfhydryls and Ascorbic Acid in Ozone, Nitrogen
Dioxide and Phosgene Toxicity. Submitted: Toxicology.
MS-86-053
-357-

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Stevens, MA; Fitzgerald, 5; Menache, MG; Cost; DL; Bucher, JR. Functional Evidence of
Persistent Airway Obstruction in Rats Following a Two-Hour Inhalation Exposure to Methyl
Isocyanate (MIC). Submitted: Environmental Health Perspectives.
MS-86-141
Stevens, MA; Menache, MG; Crapo, JD; Miller, FJ; Grahant, JA. Pulmonary Function in Juvenile
and Young Muk Rats Exposed to Low Level 1402 with Diurnal Spikes. Submitted: Journal of
Toxicology and Environment Health.
MS-86-142
Strong, AA; Lundgren, DS; Hazucha, MI; Cerini, ER. An Aerosol Generator System for Inhalation
Delivery of Pharmacological Agents. Submitted: Journal of Medical Instrumentation.
MS-86-157
Tepper, JS; King, ME; Wiester, Mi; Costa, DL Comparison of Cardiopulmonary Function in Awake
Fischer-344 and Sprague-Dawley Rats Exposed to Carbon Dioxide, a Computerized Technique.
Submitted: Fundamental and Applied Toxicology.
5-86-090
Tepper, JS; Weber, MF; Wiester, Mi; Costa, DL; Watkinson, VIP. Cardiopulmonary Effects in
Awake Rats Four and Six Months After Exposure to Methyl Isocyanate. Submitted
Environmental Health Perspectives.
MS-86-182
Wiester, MI. Report on the Effects of Inhalation of Low Concentrations of Ozone (03) and Nitrogen
Dioxide (1402) on the Proximal Alveolar Region of Juvenile and Adult Rats. US Environmental
Protection Agency, cooperative agreement no. CR807255, EPA-600/X-86-254, 1986.
HERL-0560
BlOl - WAS ’I IEWATER
Hurst CI; Brashear, DA. Laboratory Study on Leaching of Indigenous Viruses from Wastewater
Sludge. Submitted: Applied and Environmental Microbiology.
MS-86-049
Tocco DR; Randall, JL; York, RG; Smith, MIC. Evaluation of the Teratogenic Effects of
Tri-Ortho-Cresyl Phosphate in the Long-Evans Hooded Rat. Submitted: Fundamental and
Applied Toxicology.
MS-86-21.5
C104 - DRINKING WATER
Baker, LU; Gibs, I; Weng, AK; Suffet, III. Evaluation of a Teflon Helix Liquid-Liquid Extractor for
Concentration of Trace Organics from Water Into Methylene Chloride. Submitted: Journal of
Water Research.
XX-86-032
-358-

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Bercz, JP; Melnikoff, MS; Morgan, 3; Gabriel, XL. Effect of Hexametaphosphate in Drinking Water
on the Levels of Mkriglnese , Chromium, Copper & Zinc in the Bones of Rats. Submitted:
Fundamental and Applied Toxicology.
MS-86-253
Bernstein, DI; Ziegler, 3; Ward, R; Gamble, JN. Rotavirus Fecal IGA Antibody Response in Adults
Challenged with Hninsin Rotavirus. Submitted: Journal of Medical Virology.
XX-86-047
Black, DA Stober, JA; Mills, T; Millette, JR. An Application of the Sequential Simplex Procedure.
Submitted: The American Statistician.
MS-86-068
Canton BD; Basaran, AH; Meria, LE; Smith, MK. P min ition of the Reproductive Effects of
Tricresyl Phosphate to Long-Evans Rats. Submitted: Toxicology.
MS -86 -232
Carter, AM; Pacha, RE; Clark, GW; Wilhi2mc, EA. Seasonal Occurrence of Cwnpylobacterspp. in
Surface Waters and Their Correlation with Standard Indicator Bacteria. Submitted: Applied and
Environmental Microbiology.
XX-86-109
ChrisweH, CD; Coleman, WE. Accumulation of Organic Compounds from Chlorinated Humic Acid
Solutions by Absorption on Silicalite Molecular Sieve. Submitted: Environmental Science and
Technology.
MS-86-071
Clark, P3; Millette, JR; Allenspach, AL; McCauley, PT; Ws shington, IS. Liver Tissue Preparation
Using a Modified Cryoultramicrotomy Kit. Submitted: Electron Microscopy in Forensics
Occupational and Environmental Health Sciences.
MS-86-122
Condie, LW. Percutaneous Absorption of Chemical Contmainants. US Environmental Protection
Agency report.
HERL-0555
Erlandsen, SL; Bemnick, WJ. Giardia Speciation, Some Evidence for a New Species Giardia Psittaci.
Submitted: Journal of Parasitology.
XX-86 -141
Gillin, FD; Reiner, DS; Gault, MI; Douglas, H; Wunderlich, A; Sauch, iF. Encystation and
Expression of Cyst Antigens by Giardia Lambia In Pitro. Submitted: Nature.
MS-86-151
Gault, Ml; Gum, FD; Zenian, Al. Giardia Lamblia - Human Intestinal Mucus and Epithelial Cells
Stimulate Growth in Serum-Free Medium. Submitted: Experimental Parasitology.
XX-86-142
Harrington, RM; Shertzer, HG; Berc JP. The Effects of C10 2 on Thyroid Function in the African
Green Monkey and the Rat. Submitted: Journal of Toxicology and Environmental Health.
MS-86-023
-359-

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Harrington, RM; Shertzer, HG; Bercz, JP. Basal Thyroid Parameters in the African Green Monkey
(c. aethiops). a New Method for Determination of Radioactive Iodide Uptake in Nonhuman
Primates. Submittet Journal of Medical Primatology.
MS-86-024
Hayes, JR; Condie LW; Borzelleca, IF. The Acute and Subcbronic Toxicity in Rats of Trans-1,2
Dichloroethylene in Drinking Water. Submitted: Fundamental and Applied Toxicology.
MS-86-132
Herren-Freund, SL Pereira, MA; Olsen, G. The Carcinogenicity of Trichloroethylene and its
Metabolites, Tricholoroacetic Acid and Dichloroacetic Acid, in Mouse Liver. Submitted:
Toxicology and Applied Pharmacology.
MS-86-205
Jetzer, WE; Hu% AS; Nonnan, F; Ho, H; Flynn, GL Duraiswainy, N; Condi; LW. Partitioning
Dependencies for the Permeation of Skin and Silicone Rubber Membranes by Phenols. Submitted:
Journal of Pharmaceutal Sciences.
MS-86-2.41
Kaliman, Mi; Condie LW; Hayes, IR; Borzelleca, IF. Subchronic Oral Toxicity of N-Hexane and
Methyl Ethyl Ketone in CD-i Mice. Submitted: Toxicology Letters.
MS-86-025
Kopfler, FC. Second International Symposium on Health Effects of Drinking Water Disinfectants and
Disinfection By-Products.
HERL-0546
Kopifer, FC; Cram, GE Environmental Epidemiology. The Importance of Exposure Assessment.
Submitte& Proceedings of the 190th National Meeting American Chemical Society, Chicago, IL,
September 8-13, 1985.
HERL-0558
Lindquist, HD. Attachment and Replication of Viruses in Giardia Lwnblia.
HERL-0550
Lukasewycz, MT; Bieringer, CM; Liulckonen, U; Fitisimnions, ME; Corcoran, HF; Liii, S; Carlson,
RM. The Analysis of Inorganic and Organic Chloramines Derivatization with
2-Mercaptobenzothiazole. Submitte& Environmental Science and Technology.
XX-86-051
Millette, JR; Stober, JA; Weller, C; Black, D; Mills, T. Documentation of EPA Version of Simplex
Program Package for Determining Peal-to-Continuum Ratios from Energy Dispersive X-ray
Spectra from Biological Tissue.
HERL-0531
Pacha, RE; Clark, GW; Wil liams, EA; Carter, AM. Migratory Birds of Central Washington as
Reservoirs of Campylobacter Jejuni. Submitted: Applied and Environmental Microbiology.
XX-86-108
-360-

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Pacha, RE; Clark, GW; Williams, EA; Carter, AM; Scheffclmaier, ii; Debassachene, P. Small
Rodents and Other MammaI Associated with Mountain Meadows as Reservoirs of Giaidia spp.
and Cantpylobacter spp. Submitted: Applied and Environmental Microbiology.
XX-86-11O
lThighi nd, HP; Meier, JR; Kopfler, FC; Schenck, KM; Kaylor, WI !; Mitchell, DE. Importance of
Sample pH on the Recovery of Mutagenicity from Drinking Water by XAD Resins. Submitted:
Journal of Environmental Science and Technology.
MS-86-224
Schupp, DG; Erlandsen, SL A New Method to Determine Giardia Cyst Viability Correlation
Between Fluorescein DiacetatelPropidium Iodide St ning and Animal Infectivity. Submitted:
Applied and Environmental Microbiology.
XX 8
Witherell, LE; Duncan, RW; Stone, KM; Stratton, LI; Orciari, L; Kappel, S; Jilison, DA.
Investigation of Legionellapneumophila in Drinking Water. Submitted: Journal of American
Water Works Association.
XX-86-037
D109 - HAZARDOUS WASTE
DeMarini, DM. Topoisomerase-Mediated Chemical Clastogenesis and Mutagenesis. EMS Newsletter.
AB-86-216
DeMarini, DM. Risky Business. Submitted: Environmental Mutagenesis.
MS-86-087
DeMarini, DM; Lewtas, I; Brusick, DJ. Use of Limited Protocols to Evaluate the Genotoxicity of
Hazardous Wastes in Mammalian Cell Assays. Submitted: Journal of Toxicology and
Environmental Health.
MS-86-214
Stewart, KR; Mortelmans, KE; Dellarco, VL. Carbon Tetrachioride Detected as a Mutagen in
Salmonella Strain TA1O2. Submitted: Mutation Research.
XX-86-OO1
E104 - PESTICIDES
Beyer, PE; Chernoff, N. The Induction of Supernumerary Ribs in Rodents, Role of Maternal Stress.
Submitted: Teratogenesis, Carcinogenesis, and Mutagenesis.
MS-86-139
Carter, SD; Hess, PA; Laskey, 1W. The Fungicide Methyl 2-Ben7imidazole Carbaniate Causes
Infertility in Male Sprague-Dawley Rats. Submitted: Biology of Reproduction.
MS-86-062
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Chernoff, N; Kaviock, RJ; Beyer, P; Miller, DB. The Potential Relationship of Maternal Toxicity,
General Stress, and Fetal Outcome. Submitted: Fundamental and Applied Toxicology.
MS-86-201
Crofton, KM; Reiter, LW; Mailman, RB. Pyrethroid Insecticides and Radioligand Displacement from
the GABA Receptor-Chloride lonophore Complex. Submitted: Toxicology Letters.
MS-86-072
Crofton, KM; Reiter, LW. Pyretbroid Insecticides and the GABA Receptor Complex Locomotor
Activity and the Acoustic Startle Response. Submitted: Journal of Pharmacology and
Experimental Theraputics.
MS-86-073
Cummings AM; Gray, LE. Methoxychior Affects the Decidual Cell Responses of the Uterus but Not
Other Progestational Parameters in Female Rats. Submitted: Toxicology and Applied
Pharmacolnav.
Garrett, NE; Stack, HF; Waters, MD. Evaluation of the Genetic Activity Profiles for Sixty-Five
Pesticides. Submitted: Mutation Research.
MS-86-1O1
Jensen, KF; Killackey, HP. Termin ,il Arbors of Axons Projecting to the Somatosensory Cortex of the
Rat. II. The Altered Morphology of Specific Thalamocortical Afferents Following Neonatal
Infraorbital Nerve Cut. Submitted: Journal for Neuroscience.
MS-86-195
Jensen, KF; Killackey, HP. Termin,il Arbors of Axons Projecting to the Somatosensory Cortex of the
Rat. I. The Normal Morphology of Specific Th mocortical Afferents. Submitted: Journal for
Neuroscience.
MS-86-198
Libbus, BL; Perreault, SD; Johnson, LA; Pinkel, D. Incidence of Chromosome Aberrations in
M imni i1i,,n Sperm Stained with Hoechst 33342 and UV-Laser-Irradiated During Flow Sorting.
Submitted: Mutation Research.
MS-86-239
Moser, VC; MacPhail, RC. Cholinergic Involvement in the Actions of Formetanate on Operant
Behavior in Rats. Submitted: Neurobehavioral Toxicology and Teratology.
MS-86-112
Naish, SJ; Perreault, SD; Foehner, AL; Zirkin, BR. DNA Synthesis in the Fertili7ing Hamster Sperm
Nucleus, Sperm Template Availability and Egg Cytoplasmic Control. Submitted: Biology of
Reproduction.
MS-86-123
Padilla, SS; Grizzle, TB. Triphenyl Phosphite, In Vivo and In PTtm Interactions with Rat Brain
Neurotoxic Esterase. Submitted: Toxicology and Applied Pharmacology.
MS-86-060
-362-

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Perreault, SD; Naish, SJ; Zirkin, BR. The Timing of Hamster Sperm Nuclear Decondensation and
Male Pronudeus Formation is Related to Sperm Nuclear Disulfide Bond Content. Submitted:
Biology of Reproduction.
MS-86-083
Phelps, PV; Laskey, 3W. Comparison of Age Related Changes inin Vivo and In Vibo Measures of
Testicular Steroidogenesis After Acute Cadmium Exposure. Submitted: Journal of Toxicology.
MS-86-244
Rosen, M; Crofton, K; Chernoff, N. Postnatal Evaluation of Prenatal Exposure to P-Xylene in the
Rat. Submitted: Toxicology Letters.
MS-86-229
Semple, JL; Ellis , WG; Hoogenboom, ER; Kavlock RI; Zeman, FJ. Central Nervous System
Anomalies in Fetuses of Benomyl-Exposed Adequately Nourished or Protein-Deprived Rats.
Submitted: Toxicology.
MS-86-040
Veronesi, B; Dvergsten, C. Triphenyl Phosphite Neuropathy, Distribution of Spinal Cord D2m ge.
Submitted: Journal of Neuropathology.
MS-86-134
Zeman, FJ; Hen& H; Hoogenboom, ER; Kav1ock RJ; Mahboob, S. Cell Number and Size in
Selected Organs of Fetuses of Rats Malnourished and Exposed to Nitrofen. Submitted:
Teratogenesis, Carcinogenesis, and Mutagenesis.
MS-86-027
F104 - RADIATION
All, JS; Elliott, DJ. Control of Energy Absorption Rate in Transmission Line RF Exposure Systems.
Submitted: Bioelectromagnetics.
MS-86-161
Blackimin, CF; Benene, SG; Wood, AR; House, DE; Elliot, DJ. Influence of Electromagnetic Fields
on the Efflux of Calcium Ions From Brain Tissue In I’7bv, Frequency Response Up to 510 Hz.
Submitted: Bioelectroniagnetics.
MS-86-103
Blackimin, CF; House, DE; Benane, SG; Joines, WT; Spiegel, LI. Effects of Ambient Levels of
Power-Line-Frequency Electric Fields on a Developing Vertebrate. Submitted:
Bioelectrom gnetics.
MS-86-255
Elder, JA. Radiofrequency Radiation Activities and Issues, a 1986 Perspective. Submitted: Health
Physics Journal.
MS-86-236
-363-

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Gordon, Ci. Thermal Effects of Radiofrequency Radiation, Consequences of Total Body Surface
Area. Submitted: Bioelectromagnetics.
MS-86-057
Gordon, CJ. Substitution of Metabolic Heat for Energy Absorbed from Exposure to Radiofrequency
Radiation in the Rat. Submitted: Journal of Applied Physiology.
MS-86-104
Lewen, G; Lindsay, SM; Tao, NJ; Weidlick, T; Graham, RJ. A Mecluinism for the Large Anisotropic
Swelling of DNA Films. Submitted: Biopolymers.
XX-86-030
Liddle, CG; Putnam, JP; Lewter, OH. Effects of Microwave-Exposure and Temperature on Survival
of Mice Infected with Streptococcus pneumon iae. Submitted: Bioelectromagnetics.
MS-86-034
Spiegel, RI; Joines, WT. A Quasi-Static Method for Calculating Electric and Magnetic Fields in TEM
Cells. Submitted: Ii I t Trans. on Electromagnetic Compatibility.
MS-86-094
Wood, AW; Joines, WT; Blacfrmzin, CF. The Characteristics of Transverse Electric and Magnetic
Field (TEM) Cells at Extremely Low Frequencies. Submitted: Bioelectromagnetics.
MS-86-L58
L104 - TOXICS
Allen, 1W. Report on Needs and New Methods Concerning Assessment of Environmental Chemical
Effects to Induce Potentially Heritable Genetic Damage in Mammalian Germ Cells. US
Environmental Protection Agency, in-house report, EPA-600/X-86-236.
HERL-0554
Allen, JW; Stoner, GD; Pereira, MA; Backer, LC; Sharief, Y; Hatch, GG; Campbell, JA; Stead,
AG; Nesnow, S. Tumorigenesis and Genotoxicity of Ethyl Carbaniate and Vinyl Carbamate.
Submitted: Cancer Research.
MS-86-138
Bergman, HB; Nesnow, S. Report on Azoreductase Studies II. US Environmental Protection Agency
report.
HERL-0556
Brock, KH; Moore, MM; Oglesby, LA. Development of an Intact Hepatocyte Activation System for
Routine Use with the Mouse Lymphoma Assay. Submitted: Environmental Mutagenesis.
MS-86-100
Brock, TO; O’Ca1b ghm , JP. Quantitative Changes in the Synaptic Vesicle Proteins, Synapsin I and
P38, and the Astrocyte Specific Protein, GFAP, are Associated with Chemical-Induced Injury to
the Rat CNS. Submitted: Journal of Neuroscience.
MS-86-070
-364-

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Browning. MD; Hnirng C; Greengard, P. Similarities Between Protein mA and Protein 11th, Two
Prominent Synaptic Vesicle-Associate Phosphoproteins. Submitte& Journal of Neuroscience.
XX-86- 139
Burdette, U; Dyer, RS. Differential Effects of Caffeine, Picrotoxin, and Pentylenetetrazol on
Hippocampal Afterdischarge Activity and Wet Dog Shakes. Submitteck Experimental Neurology.
MS-86-234
Burleson, OR; Fuller, LB; Menache, M; JA. Poly(l), PoIy(C)-Enchanced Alveolar and
Peritioneal Macrophage Phagocytosis, Quantification by a New Method Utili7ing Fluorescent
Beads. Submitted: Proceedings of the Society of Experimental Biology and Medicine.
MS-86-120
Campbell, JA; Eppersimons, CF; Kligerman, AD; Petro, AB; Sharief, Y; Allen, JW. Sister
Chromatid Exchange Analysis in Cultured Primary Lung, Liver, and Kidney Cells of Mice
Following In Vivo Exposure to Vinyl Carbamate. Submitted: In Vitro.
MS-86-013
Chadwick, RW; Copeland, MF; Wolff, GL; Stead, AG; Mole, ML; Whitehouse, DA. Saturation of
Lindane Metabolism in Chronically Treaded (Ys x Vy) Fl Hybrid Mice. Submitted: Journal of
Toxicology and Environmental Health.
MS-86-196
Daniels, MJ; Menache, MG; Burleson, OR; Graham, JA; Seigrade, MK. Effects of NiCI2 and CdC1 2
on Susceptibility to Murine Cytomegalovirus and Virus-Augmented Natural Killer Cell and
Interferon Responses. Submitted: Fundamental and Applied Toxicology.
MS-86-152
Daston, GP; Rehnberg, BF; Hall, LL; Kaviock, RJ. Toxicity of Mercuric Chloride to the Developing
Rat Kidney. ifi. Distribution and Rumination of Mercury During Postnatal Maturation. Submitted:
Toxicology and Applied Pharmacology.
MS-86-067
DeMarini, DM; Brocic, KH; Doerr, CL; Moore, MM. Mutagenicity and Clastogenicity of Teniposide
(Vm-26) in L5178Y -3.7.2c Mouse Lymphoma Cells. Submitted: Mutation Research.
MS-86-159
DeMarini, DM; Doerr, CL; Meyer, MK; Brock, KH; Hozier, JC; Moore, MM. Mutagenicity of
M-amsa and 0-ainsa in M ’mm ilb n Cells Due to Clastogenic Effects. Submitted: Cancer
Research.
MS-86- 186
Dyer, RS; Boyes, WK Clark, CC. Surface Distribution of Flash-Evoked and Pattern Reversal-Evoked
Potentials in Hooded Rats. Submitted: Experimental Brain Research.
MS-86-091
Evenson, DP; Janca, FC; Jost, LK. Effects of the Fungicide Methyl-Benzoinidazol-2-yl Carbamate
(MBC) on Mouse Germ Cells as Determined by Flow Cytometry. Submitted: Journal of
Toxicology and Environmental Health.
XX-86-069
-365-

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Fisher, HL Hall, LL Shah, PV; Sumler, ML In Vivo and In Vitro Dermal Penetration and
Pharmacokinetics of 2,4,5, 2’,4’,5’-Hexachlorobiphen)4 in Young and Adult Rats. Submitted:
Journal of Applied Pharmacology.
MS-86-248
George, SE; Claxton, LD. Degradation of Chlordecone by Pseudomonas Species. Submitted: Applied
and Environmental Microbiology.
MS-86-194
Gordon, CJ; Long, MD; Fehlner, KS. Relationship Between Autonomic and Behavioral
Thermoregulation in the Golden Hamster (Mesocricitus Auratus). Submitted: Physiology and
Behavior.
MS-86-014
Gordon, CJ; Stead, AG. Effect of Nickel and Cadmium Chloride on Autonomic and Behavioral
Thermoregulation in Mice. Submitted: Neurotoxicology.
MS-86-019
Gordon, CJ; Stead, AG. Effect of Alcohol on Behavioral and Autonomic Thermoregulation in Mice.
Submitted: American Journal of Physiology.
MS-86-078
Gordon, CL Relationship Between Behavioral and Autonomic Thermoregulation in the Guinea Pig.
Submitted: Physiology and Behavior.
MS-86-213
Gray, JA; Kaviock, RJ. Pharmacologic Probing of Mercuric Chloride Induced Renal Dysfunction in
the Neonatal Rat. Submitted: Journal of Pharmacology and Experimental Therapeutics.
MS-86-226
Gray, LB. Compound-Induced Developmental Reproductive Abnormalities in Man and Rodents, a
Review of Effects in Males. Submitted: Journal of Toxicology and Environmental Health.
MS-86-200
Gray, LE; Rogers, JM; Ostby, iS; Kaviock, R i; Ferrell, JM; Gray, KL Prenatal Dinocap Exposure
Alters the Swmming Behavior of Mice Due to Agenesis of Otholiths in the Inner Ear. Submitted:
Science.
MS-86-225
Halperin, WE; Schulte, PA; Greathouse, DG. Conference on Medical Screening and Biological
Monitoring for the Effects of Exposure in the Workplace. Conference Proce ding Cincinnpti,
OH, July 10-13, 1984. Submitted: Journal of Occupational Medicine.
HERL-0557
Henry, DR; Lavine, BK; Jurs, PC. Electronic Factors and Acridine Frameshift Mutagenicity a Pattern
Recognition Study. Submitted: Mutation Research.
XX -86-079
Kaviock, Ri; Chernoff, N; Short, RD. Further Evaluation of an In I’Tvo Teratology Screen. Submitted:
Teratogenesis, Carcinogenesis, and Mutagenesis.
MS-86-202
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Kaviocic, RI; Rehnberg, BF; Rogers, EH. Consequences to the Kidneys of Fetal, Neonatal, and
We inIing Rats of Prenatal Exposure to Adriamycin. Submitted: Teratology.
MS-86-247
Kitchin, KT; Sanyal, MK; Schmid, BP. Rodent Whole-Embryo Culture as a Teratogen Screening
Method. Submitted: Methods and Findings in Experimental and Clinical Pharmacology.
MS-86-022
Kitchin, KT; Brown, JL. Biochemical Effects of Two Promoters of Hepatocarcinogenesis in Rats.
Submitteth Toxicology and Applied Pharmacology.
MS-86-230
Kligerman, AD; Campbell, JA; Erexson, GL; Allen, 3W; Shelby, MD. Sister Chromatid Exchange
Analyses in Lung and Peripheral Blood Lymphocytes of Mice Exposed to Methyl Isocyanate by
1nh2b tion. Submitted: Environmental Mutagenesis.
MS-86-130
Lau, C; Cameron, A; Irsula, 0; Robinson, KS. Effects of Prenatal Nitrofen Exposure on Cardiac
Structure and Function in the Rat. Submitted: Toxicology and Applied Pharmacology.
XX-86-055
Luebke, RW; Lawson, LD; Rogers RR; Riddle, MM; Smialowkz, RI Selective Immunotoxic
Effects in Mice Treaded with the Adenosine Deaminase Inhibitor 2’-Deoxycoformycin. Submitted:
immunopharmacology.
MS-86-121
McGinty, JF; IC in matsu, T; Obie, I; Mitchell, C; Hong, JS; Dyer, RS. Amygdaloid Kindling
Increases Enkeph ilin-Like Immunoreactivity but Decreases Dynorphin .A-Like Immunoreactivity
in Rat Hippocampus’l. Submitted: Neuroscience Letters.
MS-86-044
Mohapatra, N; MacNair, P; Bryant, BJ; Ellis, S; Rudo, K; Siing ’b’h , R; Gold, A; Nesnow, S.
Morphological Transforming Activity and Metabolism of Cyclopenta-Fused Isomers of
Benz(a)anthracene in M2mnvilian Cells. Submitted: Cancer Research.
MS-86-065
Moore, MM; Broclc, KH; Doerr, CL; DeMarini, DM. Mutagenesis of L5178Y/TK 3.7.2C Mouse
Lymphoma Cells by the Clastogen Ellipticine. Submitted: Environmental Mutagenesis.
MS-86-149
Moore, MM; Amtower, A; Dow, CL; Brock, KH; Dearfield, KL. Genotoxicity of Acrylamide in
L5178Y Mouse Lymphoma Cells. Submitted: Environmental Mutagenesis.
MS-86-150
Nesnow, S; Bergmpn, HB; Bergman, H; Chu, K; Frith, C; Helmes, T; McGaughy, R; Ray, V; Slaga,
TJ; Tenni nt, R. Chemical Carcinogens - A Review and Analysis of the Literature of Selected
Chemicals and the Establishment of the Gene-Tox Carcinogen Data Base. Submitted: Mutation
Research.
MS-86-193
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Nesnow, S; Garland, H; Curtis, G. Inhihition or Enhancement of Morphological Transformation of
C3H1OT 1 /2C18 Mouse Embryo Cells by Mutiple Carcinogen Treatment. Submitted: Cancer
Letters.
MS-86-254
Osman, R; Namboodiri, K; Weinstein, H; Rabinowitz, JR. Reactivities of Acrylic and Methacrylic
Acids in a Nucleophilic Addition Model of Their Biological Activity. Submitted: Journal of
American Chemical Society.
MS-86-258
Palekar, L; Eyre, iF; Most, BM; Coffin, DL Metaphase and Anaphase Analysis of V79 Cells
Exposed to Erionite, UICC Chrysotile, and UICC Crocidolite. Submitted: Carcinogenesis.
MS-86-249
Palekar, LD; Most, BM; Coffin, DL Significance of Mineral Fibers in the Correlation of V79
Cytotoxicity with Tumorigenic Potential of Mineral Fibers. Submitted: Environmental Research.
MS-86-242
Peele, DB; Farmer, 3D; MacPhail, RC. Conditioned Flavor Aversions, Applications in Assessing the
Efficacy of C nlators in the Treatment of Heavy Metal Intoxication. Submitted: Toxicology and
Applied Pharmacology.
MS-86-084
Peele, DB; Baron, SP. Effects of Selection Delays on Radial Maze Performance, Acquisition and
Effects of Scopolamine. Submitted: Pharmacology, Biochemistry and Behavior.
MS-86-204
Prah, 3D. Neurotoxicologic and Behavioral Effect of Aluminum and Aluminum Compounds in Man
and Anim il . Submitted: Neurobehavioral Toxicology and Teratology.
MS-86-066
Reiter, LW. Neurotoxicology in Regulation and Risk Assessment. Submitted: Pediatric Pharmacology.
MS-86-246
Richard, AM; Rabinowitr, JR. Modified Molecular Charge Similarity Indices for Choosing Molecular
Analogues. Submitted: International Journal of Quantum Chemistry.
MS-86-154
Rogers, 3M; Hurley, LS. Effect of Zinc Deficiency on Morphogenesis of the Fetal Rat Eye. Submitted:
Journal of Embryology and Experimental Morphology.
MS-86-160
Rogers, JM. Comparison of Maternal and Fetal Toxic Dose Responses in Mammals. Submitted:
Fundamental and Applied Toxicology.
MS-86-207
Rogers, JM; Gray, LE; Carver, BD; Kavlock, RJ. The Developmental Toxicity of Dinocap in the
Mouse is Not Due to Two Isomers in the Major Active Ingredients. Submitted: Teratogenesis,
Carcinogenesis and Mutagenesis.
MS-86-250
-36

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Sawyer, JR; Hozier, JC. High Resolution of Mouse Chromosomes - Banding Conservation Between
Man and Mouse. Submitted: Science.
XX-86-111
Shah, PV; Sumler, MR; Month, NJ; Fisher, HL; Hall, LL Dermal Penetration of Carbofuran in
Young and Adult Fischer 344 Rats. Submitted: Journal of Toxicology and Environmental Health.
MS-86-235
Sharief, Y; Brown, AM; Backer, LC; Campbell, JA; Westbrook-Collins, B; Stead, AG; Allen, JW.
SCE and Chromosome Aberration Analyses in Mice After In Vivo Exposure to Acrylonitrile,
Styrene, or Butadiene Monoxide. Submitted: Environmental Mutagenesis.
MS-86-016
Slotkin, T; Kavlock, RI; Cowdery, T; Orband, L; Bartolome, M; Gray, JA; Rehnberg BF;
Bartolome, I. Functional Consequences of Prenatal Metliyhnercury Exposure, Effects on Renal
and Hepatic Responses to Trophic Stimuli and on Renal Excretory Mech2nisms. Submitted:
Toxicology.
MS-86-079
Slotkin, TA; Kaviocic, RI; Cowdery, T; Orband, L; Whitmore, W; Bartolome, J. Effects of Neonatal
Methylmercury Exposure on Adrenergic Receptor Binding Sites in Peripheral Tissues of the
Developing Rat. Submitted: Toxicology.
MS-86-058
Slotkin, TA; Orba.nd, L; Cowdery, T; Kaviock, RI; Bartolome, J. Prenatal Exposure to
Methymercury Alters Development of Adrenergic Receptor Bonding Sites in Peripheral
Sympathetic Target Tissues. Submitted: Toxicology.
MS-86-153
Sinialowicz, R I. Immunologic Effects of Nonionb ng Electromagnetic Radiation. Submitted: IEEE
Engineering in Medicine and Biology.
MS-86-003
Smialowicz, RI; Rogers, RR; Rowe, DG; Riddle, MM; Luebke, RW. The Effects of Nickel on
Immune Function in the Rat. Submitted: Toxicology.
MS-86-237
Stoner, GD; You, M; Morgan, MA; Superczynslci, MJ. Lung Tumor Induction in Strain A Mice with
Benzotrichloride. Submitted: Toxicology and Applied Pharmacology.
XX-86-124
Stouch, TR; Jurs, PC. Chance Factors in Nonparanietric Linear Discriminant Studies. Submitted:
Quantitative Structure Activity Relationships.
XX -86-082
Tilson, HA; Rogers, B; Grimes, LM; Harry, GJ; Peterson, NJ; Hong, JS; Dyer, RS.
Time-Dependent Neurobiological Effects of Coichicine Administered Directly Into the
Hippocampus of Rats. Submitted: Brain Research.
MS-86-233
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Valtortoa, F; Schiebler, W; Greengard, F; Jahn, R; Ceccardlli B. A Solid-Phase Assay for the
Phosphorylation of Proteins Blotted on Nitrocdilulose Membrane Filters. Submitted Analytical
Biochemistry.
XX-86-132
Watkinson, WP; Gordon, CL Effects of Chiordimeform on Heart Rate and Body Temperature of
Unanesthetized Unrestrained Rats. Submitted: Toxicology Letters.
MS-86-017
-370-

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Appendix B
Presentation
Abstracts
Abstracts for presentations made between October 1, 1985 and September 30,1986.
These abstracts are not included in this report.

-------
AIR HEALTh
Andrews, JE; Courtney, KD; Donaldson, WE. The Effects of HCB on Calcium Metabolism and the
Vitamin D Endocrin System. Burroughs Wellcome/NCSU Symposium, Research Triangle Park
NC October 15, 1985.
AB-86-O11
Andrews, JE; Courtney, l CD; Donaldson, WE. Impairment of Calcium Hoineostasis by
Hexachlorobenzene Exposure in Fischer 344 Rats. Society of Toxicology Meeting, New Orleans,
LA, March 3-7, 1986.
AB-86-059
Ball, LM; Gold, A, Nishioka, MG; Kohan, MJ; Williams K; Lewtas, J. Metabolism and Activation
Pathways of the Environmental Mutagen 3-Nitrofluoranthene. Tenth International Symposium on
Polynuclear Aromatic Hydrocarbons, Columbus, Ohio, October 27-30, 1985.
AB-85-149
Ball, LM; Nishioka, MG; Lewtas, 3. Hepatic Metabolism of the Environmental Mutagen
2-Nitrofluoranthene 1 Proceedings of Fall Meeting of American Society for Pharmacology and
Experimental Therapeutics, August 19-22 1986.
AB-86-237
Cooper, RL; Goldman, JM; Rehnberg, GL Hem, IF; McElroy, WK. Altered Response to a
Reproductive Toxin in the Aging Male Rat. Society of Toxicology Meeting, New Orleans, LA,
March 3-7, 1986.
AB-86-033
Costa, DL; Lehniann, JR; Stevens, MA; Fitzgerald, S. A Rat Model of Mild COPD for Use in
Toxicologic Studiet Society of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-042
Davies, DW; Andrews, 3M; Hilliard, HG. A Coarse Mode Aerosol Generator for Rodent Tnhalation
Exposures. AAAR Meeting, Albuquerque, New Mexico, November 18-fl 1985
AB-85-147
Fitzgerald, S; Lchmann, JR; Stevens, MA, Costa, DL Alpha-1-Antiproteinase (2-1-pi) Deficiency in
Rats Treated with 2,1-Ethionine. Society of Toxicology Meeting, New Orleans, LA, March 3-7,
1986.
AB-86-043
Graedel, It; Hawkins, DT; Hill, DM; Claxton, LD. Atmospheric Chemical Compounds; Sources,
Occurrence, and Bioassay. American Geophysical Union, San Francisco, CA, December 9, 1985.
AB-86-006
Grimes, LM; Dyer, RS. Conditioning-Induced Waveforms Recorded over Olfactoiy and Parietal
Areas of Rat Brain. Society for Ncuroscience Meeting, Dallas, Texas, October 20-24, 1985
AB-85-137
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Grosej EC; Grshsim. JA; Richards, JU; Jaskot, RH; Dauterman, WC. The Relationship Between
Pulmonary Glutathione Peroiddase and Glutathione Transferase FOllOwing Inhalation of Cadiniunt
Chloride. ISSX Meeting, Key Biscayne, florida, November 17-fl, 1985
AB-85-151
Grose BC; Th ing , JW; Stead, AG; Stevens, MA; Jaskot, RH; Hardisty, J. Pulmonary and Hepatic
Health Effects of Tnhsthtion of Petroleum Smoke. Society of Toxicology Meeting, New Orleans,
LA, March 3-7, 1986.
AB4 Th
Hatch, GE ; Slack, R. Effects of Buthionine Sulfoximine (BSO) Treatment or of Ascorbic Acid (AA)
Deficiency on the Edemagenic Effect of Inhaled O , NO , and COC12. Society of Toxicology
Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-019
Hatch, GE; Slade, R; Koren, Nt Electrochemically Active Substances in Lavage Fluid and Cells of
Rats and Humans. Society of Toxicology Meeting, New Orleans, LA, March 3-71986.
AB-86-021
Hazucha, MJ; Bates, DV; Bromberg, PA. Meclmncm of Action of Ozone on the Human Lung.
American Thoracic Society, Ilancas City MO, May 11-14, 1986.
AB-86-128
Horstinan. DII; Folinsbee, LI; Vorona, PD; Prince, JM. Ozone Induced Lung Function Changes Are
Not Related to Decreased Maximum Respiratory Effort. Congress of International Union of
Physiological Sciences, Vancouver, Canada, July 13-19, 1986.
AB-86-122
Horsunan, DII; Folinsbee, LI. Airway Reactivity and Obstruction, Relationship to Pollutant Induced
Bronchoconstriction in Asthmatics. FASEB Meeting, St. Louis, MO, April 13-18,1986.
AB-86-127
Jaskot, RH; Most, B; Menache, MG; Wi llia m s, T; Grose, EC; Roycroft, il-I. The Uptake of Methyl
Bromide in the Rat Following Inhalation Exposure. Society of Toxicology, New Orleans, LA,
March 3-7, 1986.
AB-86-028
Kehrl, HR; Vincent, L; Kowalsky, R; Horstman, DH; McCartney, W; O’Neil, JJ; Bromberg, PA.
Ozone-Induced Increased Respiratory Epithelial Permeability (REP) Correlates with FEVi
Decrements. American Thoracic Sodety Kansas City, MO, May 11-14, 1986.
AB-86-129
King, LC; Jackson, M; Ball, LM; Lewtas, J. Metabolism of 1-Nkro( 14 C)pyrene Cultures of
Respiratory Tract Tissues and Isolated Tracheal Epithelial Cells. 10th International Symposium on
Polynuclear Aromatic Hydrocarbons, Columbus, OH October 1, 1985.
AB-86-004
King, LC Jackson, M; Ball, LM; Lewtas, J. Metabolism of 1-Nitro(’ 4 C)pyrene by Primary Tracheal
Epithelial Cells. GEMS Meeting, Durham, NC, November 7, 1985.
AB-86-005
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King LC; Jackson, M; Ball, LM; Lewtas, J. Metabolism of 1-NitroI 14 C]pyrene by Primary Tracheal
Epithelial Cells. Society of Toxicology Meeting New Orleans, LA, Mardi 3-7, 1986.
AB-86-08S
Koren, HS. The Effects of Ozone on Functional and Biochemical Characteristics on Pulmonary
Macrophages. Immunotoxicology Discussion Group, Bethesda, MD, June 23-24, 1986.
AB-86-192
Lewtas, J. A Retrospective View of the Value of Short-Term Genetic Bioassays in Predicting the
Chronic Effects of Diesel Soot. VI icr Satellite Meeting ‘Toxed”, Tsukuba Science City, Japan,
July 26-28, 1986.
AB-86-193
Lewtas, J; King, LC; Jackson, M; Ball, LM. Bioavailability of Nitropyrene from Diesel Soot. IV
International Congress of Toxicology Meeting Tokyo, Japan, July 15-21, 1986.
AB-86-194
Martonen, TB. Hygroscopicity - Its Effect on Inhaled Aerosols. GHEF & AAAR Joint Conference,
Berlin, Germany, September 22-26, 1986.
AB-86-073
Martonen, TB; Grahani 1 RC; Patra, AL. The Effect of Age on Regional Aerosol Deposition in Man.
American Association of Aerosol Research, Albuquerque, New Mexico, November 18-22, 1985
McDonnell, WF; Abdul-Salaani, S; Howe, DE; Hazucha, MI. Airway Reactivity Assessment by
Single Dose and Dose-Response Challenge to Methachoilne. American Thoracic Society, Kansas
City, MO, May 11-14, 1986.
AB-86-123
Milholland, VS; Horshnan, DH; Koren, HS. Changes in Mononuclear Cell Populations in Exercised
and Ozone Exposed Humfin Subjects. Clinical Application of Cytometry Annual Meeting
Charleston, SC, September 30-October 3,1986.
AB-86-225
Morgan, D; Steele, V; Nixon, J; Humphries, J; Hatch, GE. Extrapolation of Inhaled Particle Toxicity
Data from Experimental Animals to Humans. Society of Toxicology Meeting, New Orleans, LA,
March 3-7, 1986.
AB-86-020
Muinford, JL; Harris, BD; Williams, K; Chnang JCC; Cooke, M. Indoor Air Pollution Related to
Emis.cion from Unvented Coal Combustion. 79th Annual Air Pollution Control Association
Meeting Minneapolis, MN, June 22-27, 1986.
AB-86-O01
Otto, DA. The Relationship of Late Positive ERP’s, Age and Intelligence in Socioeconomically
Disadvantaged Children. 8th Conference on Event Related Potentials of the Brain, Stanford, CA,
June 22-28, 1986.
AB-86-206
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Otto, DA; Robinson, G; Baun’ nn, SB. Electrophysiological Assessment of Sensory & Cognitive
Function in Children with Undue Lead Absorption. International Workshop on the Effects of
Lead Exposure on Neurobebavioral Development, Scotland, September 1986.
AB-86-217
Overton, JH. Biological Disposition of Airborne Vehicle Emissions, ifi. Reactive Gases. Workshop on
Health Effects Research and Motor Vehicles, Dearborn, M I, October 31- November 1, 1985.
B -86 -070
Overton, JH; Grs h im, RC. The Influence of Upper Respiratory Tract Models on Simulated Lower
Respiratory Tract Uptake of 03. American Association for Aerosol Research, Albuquerque, New
Mexico, November 18-22, 1985
AB-85-150
O’Callaghan, JP; Miller, DB. Diethyldithiocarbamic Acid Facilities Entry of Cadmium into Brain but
Prevents Cadmium-Induced Neurotoxicity. Society for Neurotoxicity Meeting Dallas, Texas,
October 20-24, 1985
AB-85-133
O’Neil, JJ. Lung Research Activities at the Environmental Protection Agency Interagency Technical
Committee (IATC) Working Group on Lung Diseases.
AB-86-159
O’Neil, JJ. Health Effects Of Sulfur Dioxide (S02) on Asthmatic Subjects. California Air Resources
Board Meeting March 12-20,1986.
AB-86 -180
Padilia, SS; Boyes, WK; Lyerly, D. Tunicamycin Blockage of Axonal Transport in the Rat Optic
System Preceeds Graded Electrophysiological Changes. Conference on Axonal Transport,
University of Calgary, Calgary, Alberta, Canada, July 1.3-19, 1986.
AB-86-183
Patra, AL; Shaka, N. Partide Deposition in Casts of Hiimiin Tracheobronchial Tree. ACEMB
Conference, Baltimore, MD, September 13-16, 1986.
XA-86-013
Patra, AL; Gooya, A. Deposition of Particles in a Baboon Nose Cast. ACEMB Conference, Baltimore,
MD, September 13-16, 1986.
XA-86-014
Post, GB; Keller, DA; Menzel, DB. Variations in Glutathione Content of Human Lung Cells. Third
International Congress on Toxicology, San Diego, CA, August 1986.
XA-86-021
Post, GB; Keller, DA; Menzel, DB. Effects of Growth Time and Serum Concentration on Glutathion
Content in A549 Cells. FASEB Annual Meeting St. Louis, MO, April 13-16, 1986.
XA -86-022
Riggiin WB. Mortality Variation and Trend by County Race and Sex. 2nd Conference of EPA
Statisticians, WiII intcburg VA, November 5-8, 1985.
AB-86-003
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Riggan 3 WB. Mortality Variation and Trend by County Race and Sex. Workshop for Use of Empirical
Bayesian Procedure for Generating Stable Rates, Duke University, Durham, NC, March 27-28,
1986.
AB-86 -207
Roger, U; Horshlian, DH; Kehrl, HR. Absence of Change in Sensitivity to Tnbi 1ed Methacholine in
Asthnisitics Exposed to NO . Society of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-015
Ross, PD; Gardner, DE; Menzel, DB. Removal of Nitrate Ion from the Airways of Rat Lung. Society
of Toxicology, Wachington, DC, March 1986.
AB-86 -230
Schreiber, RT; McDonnell, WF; Horstm in DH; Ives, P; Abdul -Siihrnni, S; Bromberg, PA. The
Relationship of Tnh i1ed Citric Acid Sensitivity and Responsiveness to Ozone. American Thoracic
Society, Kansas City, MO, May 11-14, 1986.
AB-86-144
Schwartz, J; Otto, DA. Blood Lead, Hearing Thresholds and Neurobehavioral Development in
Children and Youths. ASPET/SOT Meeting, Baltimore, MD, August 20, 1986.
AB-86-243
Stead, AG. Using Stepwise Discriminant Analysis to Find an Optimpl Battery of Genetic Bioassays to
Screen for Carcinogenicity. 2nd Conference of EPA Statisticians, Williamsburg, VA, November
5-8, 1985.
AB-86-O10
Steele, yE; Morgan, DL; Nixon, JC; Humphreys, JE; Hatch, GE. An Animil to Human
Extrapolation Model for Inhalation Toxicology. IV International Congress Toxicology Meeting
Tokyo, Japan, July 21-25, 1986.
AB-86-182
Stevens, MA; Costa, DL; Fitzgerald, 5; Bucher, JR; Graham, JA; Menache, MG. Functional Airway
Obstruction in Male F-344 Rats Exposed to Methyl Isocyanate (MIC). Society of Toxicology
Meeting New Orleans, LA, March 3-7,1986.
AB-86-016
Tepper, JS; Wiester, Mi; Costa, DL; Weber, MF; King, ME. Adaptation to Ozone in F-344 Rats.
Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
B -86-022
Valentini, JE; Deal, DL; Menzel, DB. Ozone Increases Lung Permeability to Nitrate Ion. FASEB
Annual Meeting St. Louis, MO, April 13-16, 1986.
XA-86-023 Rehnberg. GL; Hem, JF; McElroy, WK; Golelman, JM; Cooper, RL. Endocrine
Events Associated with Altered Reproductive Function. Society of Toxicology Meeting, New
Orleans, LA, March 3-7, 1986.
AB-86-036
Watts, RR; Cupitt, LT. Sample Accountability Quality Assurance for the EPA “Integrated Air Cancer
Project” Research Program. Annual Meeting of the Association of Official Analytical Chemists,
Scottsdale, AZ, September 15-18, 1986.
AB-86-231
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Wiester, MJ; Miller, FJ; Williams, TB; King, ME; Menache, MG. Respiratory Doshnehy of Ozone
(03) in Awake Sprague Dawley Rats. Society of Toxicology Meeting, New Orleans , LA, March
3-7, 1986.
AB-86-029
Wing, S; Heiss , G; John, E; Knowles, M; Tholec HA; Hayes, CU; Rigg in 1 WB. Geographic
Variation in the Onset of the Decline of 11W Mortality in the United States. American Heart
Association Council of Epidemiology, San Francisco CA, March 3-5, 1986.
AB-86-169
BlOl - WASTEWATER
Camann , DE. Association of Viral Infections with Spray Irrigation of Municipal Wastewater. APHA
Annual Meeting, WaitMngton, DC, November 17-21,1985
XA-85-012
Donnefly, ICC; Brown, KW. Variability and Mutagenicity of Municipal Sludges. Symposium on the
Chemical & Biological Characterization of Municipal Sludge Sediments, Dredge Spoils & Drilling
Muds, Netherland Plaza, Cincinnati, OH, May 20-22, 1986.
XA-86-O11
Dufour, AP; HopkinS, L .A Membrane Filter Procedure for Enumerating Bacteria in Oysters.
American Society for Microbiology Annual Meeting, Washington, DC, March 23-28, 1986.
AB-86-069
Hurst, C. !; Benton, W I !. Effects of 5-Iododeoxyuridine Treatment and Mixed Cell Monolayers Upon
Enteric Virus Plaque Assay Titers. American Society for Microbiology Annual Meeting.
Washington, DC, March 23-28, 1986.
AB-86-063
Meier, JR; Bla’sk, WE; Riccio, ES; Rushbrook, a; Stewart, BE; Condie, LW. Genotoxicity and
Subtonic Toxicity Studies of Municipal Wastewater Effluents. Environmental Mutagen Society
Meeting, Baltimore, MD, APRIL 9-13, 1986.
AB-86-142
C104- DRINKING WATER
Bercz, iF; Condie, LW; Harrington, RM; 7 immer, H. Toxicity of Iodinated Nutrients Formed in the
Alimentary Tract Following Ingestion of Chlorine-Based Disinfectants Via Drinking Water.
Abstracts of the 25th Anniversary Meeting 6(1): 88, March 1986.
AB-86-065
Bull, RI; Robinson, M; Laurie, RD. Tumor Initiating Activity of Structural Analogs of Acrylamide.
Abstracts of the 25th Anniversary Meeting 6(1): 231, March 1986.
AB-86-109
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Canton, BD; Barlett, PB; Smith, ML Reproductive Effects of Chioramine Administered by Gavage
to Long-Evans Rats. Abstracts of the 25th Anniversary Meeting 6(1): 293, March 1986.
B -86 -079
Canton, BD; Banlett, PB; Smith, MK. Reproductive Toxicity of Tricresyl Phosphate in Long-Evans
Rats. Abstracts of the 25th Anniversary Meeting 6(1): 292, March 1986.
AB -86-080
Chang, LW; DeAngelo, AB; Pereira, MA. Binding of 1,2-Dibromoetbane to Plasma Membrane
Lipids of Rat Livers. Abstracts of the 25th Anniversary Meeting 6(1): 272, March 1986.
AB-86 -083
Coleman, JB; Lamb, RG. Role of CC14 Metabolism in the Activation of Hepatocellular
Phospholiphase C In VWo. ASPET-SOT Meeting, Baltimore, MD, August 1986.
XA-86-016
Coleman, WE; Munch, 3W; Streiclier, RP; Hodaldevic, PA. Artifacts Resulting from Extraction and
GC/MS Analysis of Aqueous Chlorinated Humic Acid Solutions. 192nd National American
Chemical Society Meeting, Anaheim, CA, September 7-12, 1986.
AB-86-171
Condie, LW; Laurie RD; Robinson, M; Berc JP. Effect of Corn Oil Vehicle on Subchronic
Hepatotnii 4 Iy of Carbon Tetrachioride (Cl ’) in CD-i Mice. Abstracts of the 25th Anniversary
Meeting 6(1): 183, March 1986.
AB46
Condie, LW; Berzc, JP. Health Effects of Drinking Water Disinfectants and Disinfection ByProducts.
Conference on Trace Substances in Environmental Health, Columbia, MO, June 2-6, 1986.
AB-86-168
Conolly, RB; Cramer, 3; Andersen, ME. A Physiologically-Based Model for Rat Hepatic Glut ithione
(GSH) - Its Circadian Oscillation and Interaction with Halogenated Hydrocarbons. ASPET-SOT
Annual Meeting, Baltimore, MD, August 18-22, 1986.
XA -86 25
Davis, ME. Comparison of Subacute Toxicities of Dichioroacetate and Trichioroacetate in Female
Rats. Abstracts of the 25th Anniversary Meeting 6(1): 161, March 1986.
XA -86-012
DeAngelo, AB; Herren-Freund, SL Pereira, MA; Schults, NE; Klaunig. JE. Species Sensitivity to
the Induction of Peroxisome Proliferation by Trichioroethylene and Its Metabolites. Abstracts of
the 25th Anniversary Meeting 6(1): 113, March 1986.
B-86 -086
DuMoulin, GC; Coleman, EC; Tsang, AY; Stottmeier, KD. Cont2nlinntion of Drinking Water
Supplies with Mycobacterium Avium Complex (MAC). Interscience Conf. on Antimicrobial
Agents and Chemotherapy, New Orleans, Loiicinn , September 28- October 1, 1986
A-87 -007
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George, EL; Smith, MX; Zenick, H; Hastings, L. Developmental Twdcity Associated with
Acrylamide (ACR) Exposure in Long-Evans Rats. Abstracts of the 25th Anniversary Meeting
6(1): 90, March 1986.
B-86-087
Gillin, PD; GauIt MJ; Zenian, A; Reiner, DS; Sauch, JF. Binary Lipids and Mucus Permit Growth
of Giardia Lanblia in Serum-Free Medium. American Society of Tropical Medicine and
Hygiene/Society of Tropical Veterinary Medicine-Joint Meeting Miami, Florida, November 3-7,
1985
AB-85- 160
Goldey, E; Taylor, DH; Lagory, KB; Pfohl, Ri; Laurie, RD. Exposure to Trichioroethylene During
Early Development Induces Learning Anomalies in Mature Rats. Abstracts of the 25th
Anniversary Meeting 6(1): 221, March 1986.
AB-86-074
Harrington, R; Shertzer, HG; Bercz, JP. An in I’7vo Method for Determining Inhibition of Iodide (I-)
Organification in the Rat Thyroid Gland. Abstracts of the 25th Anniversary Meeting 6(1): 88
March 1986.
AB-86-064
Herren-Freund, SL; Pereira, MA; Olsen, G; DeAngelo, AB. The Carcinogenicity of
Trichloroethylene (TCE) and Its Metabolites, Trichloroacetic Acid (TCA) and Dichioroacetic
Acid (DCA), in Mouse Liver. American Association for Cancer Research, Los Angeles, CA, May
7-10, 1986.
AB-86-191
Jakubowsici, W. Current Microbiological Concerns in Drinking Water and Evolving Concepts in
Regulation. 114th Annual APHA Meeting, Las Vegas, NV, September 28-October 1,1986.
AB-86- 175
Kopfler, FC; Coleman, WE; Melton, RG. Occurrence of Organic Substances in Drinking Water from
Eight Locations. American Chemical Society National Meeting Anaheim, CA, September 7-12,
1986.
AB-86-187
Lamb, RG; Coleman, JB; Condie, LW; Borzeileca, JF. Influence of Chlorinated Hydrocarbons on
Cultured Hepatocyte Function. Abstracts of the 25th Anniversary Meeting 6(1): 116, March 1986.
AB-86-108
Lamb, RG; Bush, SR; Condie, LW; Borzelleca, JF. Influence of Chlorinated Hydrocarbon Mixtures
on Cultured Hepatocyte Function. ASPET-SOT Meeting Baltimore, MD, August 1986.
AB-86-208
Laurie, RD; Robinson, M; Bull, Ri. Effects of Particulate of Coal Tar Paint in NJ Mice. Abstracts of
the 25th Anniversary Meeting 6(1): 233, March 1986.
AB-86-107
Laurie, 1W; Robinson, M; Bercz JP; Mobley, SA; Long, RE; Condie, LW. Toxicity of
1,2,3-Trichioropropane. ASPET-SOT 1986 Annual Meeting, Baltimore, MD, August 18-22, 1986.
AB-86-238
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Lee, PS; Rudd, CJ; Meier, JR. Use of the Mouse Lymphoma Mutagenesis Assay to Compare the
Activities of Concentrates of Treated Drinlcng Water. Environmental Mutagen Society Meeting,
Baltimore, MD, April 9-13, 1986.
AB-86-141
Liii, LC; Mattox, J; Danner, RM; Pereira, MA. Comparison of the Uptake, Distribution and
Macromolecular Binding in Mice of 1,2 -Dibromoethane and Chloroform Administered in Corn
Oil or Water. Abstracts of the 25th Anniversary Meeting 6(1): 261, March 1986.
AB-86-081
Lykuns, BW; Koffskey, W; Miller, RG. Pilot Scale Investigations at Jefferson Parish, Louisiana.
Second National Conference on Drinfring Water (Treatment for Organic Cont lmin2nts) —
Edmonton, Alberta, Canada, April 7-8, 1986.
AB-86-205
Mather, G; Parnell, MJ; Exon, iii; Koller, LD. Promotion of Carcunogenesis by Trichioroacetic Acid
(TCA). Abstracts of the 25th Anniversary Meeting 6(1): 231, March 1986.
XA-86-002
Meier, iR; Rudd, CJ; Bbl7 k WF; Riccio, ES; Miller, RG. Comparison of the Mutagenic Activities
of Water Samples Disinfected with Ozone, Chlorine Dioxide, Monochloranuine, or Chlorine.
Environmental Mutagen Society Meeting, Baltimore, MD, April 9-13, 1986.
AB-86-126
Miller, RG; Doerger, JU; Kopfler, FC; Read, E. Long-Term Stability of Drinking Water Samples for
Trace Metal (PB) Analysis. American Chemical Society National Meeting, Anaheim, CA,
September 7-12, 1986.
AB -86-186
Mobley, SA; Taylor, DH; Laurie, RD. Delays in Behavioral Development of Rat Pups Following
Indirect Exposure to Chlorine Dioxide and Chlorite. Midwest Regional Animal Behavioral
Meeting, Mt. Pleasant, MI, April 18-21, 1986.
AB-86- 181
Mobley, SA; Taylor, DH; Laurie, RD; Lagory, KE. The Effects of Chlorine Dioxide and Chlorite on
the Development of Locomotor Activity of Rat Pups. Society of Environmental Toxicology and
Chemistry, Saint Louis, Missouri, November 10-13, 1985
AB-85-186
Muralidhara, S; Ramanathan, R; Dallas, CE; Bruckner, JV. Acute, Subacute and Subchromc Oral
Toxicity Studies of 1,1-Dichloroethane (DCE) in Rats. Abstracts of the 25th Anniversary Meeting
6(1): 313, March 1986.
XA-86-001
Muralidhara, S; Dallas, CE; Kim, HJ; Bruckner, JV. Evaluation of the Hepatorenal Toxic Potential
Of 1,2-Dichioropropane (DCP), Acute, Subacute and Subchronic Oral Exposures in Rats.
Abstracts of the 25th Anniversary Meeting 6(1): 183, March 1986.
XA-86-003
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Nagy, B; Berc JR Screening Tests in Toxicity or Drug Effect Studies with Use of Centrifichem
General-Purpose Spectrophotometric Analyzer. Joint Meeting of the American Society of
Biochemicals and the America Chemical Society Washington, DC, June 7-14, 1986.
4.2-86-166
Noland-Gerbec EA Pfohl, LI; Taylor, DH; Bull, RI. Glucose Uptake in the Developing Rat Brain
upon Pre- and Post-Natal Exposure to Trichioroethylene. Society of Neurosdence Dallas, Texas,
October 20-24, 1985
AB-85-165
Nowak NA; Cubitt, WD; Herrmann, ia Blacklow, NR. Immunological Relatedness of Norwalk
Virus and Hnnan Calicivirus. American Society for Microbiology, Washington, DC ; March 23-28,
1986.
XA46M07
Pereira, MA; Herren-Freund SL4 Olson, U; Long, RE. induction by Halogenated Acetonitriles
(HA) And Ethanes (HE) of Nuclear Anomalies (NA) in the Gastrointestinal Tract of Rats and
Mice. Abstracts of the 25th Anniversary Meeting 6(1): 234, March 1986.
AB-86-084
Pereira, MA; Klaunig, JE; Herren-Freund, SL; Ruth, R I. The Effect of Phenobarbital (PB) on the
Development of Liver Tumors in Mice of Different Strains American Association for Cancer
Research, Los Angeles, CA., May 7-10, 1986.
AB-86-146
Perron, DM; Stobbs-Walro, D; Herrmann, JE; Blacklow, NR. Monoclonal Antibodies to Enteric
Adenoviruses, Characterization and Use in Immunoassays. American Society for Microbiology,
Washington, DC, March 23-28, 1986.
XA-8&006
Richmond, RE; Pereira, MA. Imniunohistochemical Detection of PBS, SRC, and MYC Oncogene
Proteins (OP) in Normal and Neoplastic Rat Liver. American Association for Cancer Research,
Los Angeles, CA, May 7-10, 1986.
AB-86-145
Ringband, HP; Meier, JR; Kopfler, PC; Schenck, 1CM; Kaylor, WH; Mitchell, DE Influence of pH
And Chlorine Residue on the Recovery of Mutagenicity from Drinking Water by XAD Resins.
Environmental Mutagen Society Meeting, Baltimore, MD, April 9-fl 1986.
AB-86-124
Ringhand, HP; Meier, JR; Kopfler, PC; Schenclç 1CM; Kaylor, WH; Mitchell, DE. Importance of
Sample pH on the Recovery of Mutagenicity from Drinking Water By XAD Resins. American
Chemical Society National Meeting, Anaheim, CA, September 7-12, 1986.
AB-86-185
Robinson, M; Laurie, RD; Bull, RI. Carcinogenic Activity Associated with Chlorinated Acetones and
Acroleins in the Mouse Skin Assay. Abstracts of the 25th Anniversary Meeting 6(1): 234, March
1986.
AB-86-067
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Sauch, JF; Mormile, MR. Bouyant Density Profiles of Giardia Lwnblia Cysts from Mongolian Gerbils,
Comparison to Hiini n Giardia And G. Muns Cysts. American Society for Microbiolo ,,
Washington, DC, March 23-28, 1986.
AB-86-068
Sauch, iF. Recovery and Detection of Giardia Cysts from Drinking Water Samples. Greater Cincinnati
Parasitology & Topical Medicine Society, December 5, 1985.
AB-86-125
Sauch, iF. Giardia Detection in Water Systems. Proceedings of the 3rd Conference on Progress in
Chemical Disinfection, State University of New York, Binghamton, NY, April 3-5, 1986.
AB-86-165
Savage, RE; Onion, C; DeAngelo, AB; Ch ng , L; Danner, R. Further Studies on the Mecharnsm of
Chloroform Stimulation of Rat Hepatic Ornitliine Decarboxylase (ODC). Abstracts of the 25th
Anniversary Meeting 6(1): 273, March 1986.
AB -86-088
Savage, RE; Pereira, MA; DeAngelo, AB; Danner, RM; Mattox, 3K. Chloroform Induction of
Ornithine Decarboxylase-Antizynie (ODC-AZ) in Rat Liver. American Association for Cancer
Research Annual Meeting Los Angeles, CA, May 7-10, 1986.
AB-86-147
Smith, MK Zenick, H; Presto; RJ; George, EL; Long, RE. Domin int Lethal Effects of Subchronic
Acrylamide Adminictration in the Male Long-Evans Rat. Abstracts of the 25th Anniversary
Meeting 6(1): 296, March 1986.
AB-86-077
Smith, MK; Randall, JL; ford, LD; tocco, DR; York, RG. Teratogenic Effects of
Trichioroacetonitrile in the Long-Evans Rat. Teratology Society Meeting Boston, MA, July 6-10,
1986.
AB-86-164
Steel, VL; Taylor, DH; Laurie, RD. Effects of Maternal Exposure to Trichioroethylene on Locomotor
Activity and Circulating Corticosterone Levels in Rat Pups. Abstracts of the 25th Anniversary
Meeting 6(1): 221, March 1986.
AB-86-076
Stober, JA; Crump, KS. Statistical Methods for Analyzing Mouse Lung Adenoma Bioassay Data.
International Biometric Conference, Seattle, WA, July27 - August 1, 1986.
AB-86-170
Stober, JA. Non-Neoplastic Lesions, Use of Data from Pre- or Non-Neoplastic Lesions that May
Indicate Potential for Carcinogenesis. EPRI Workshop on “Investigation of New Approcahes to
Use of Data in Cancer Risk Assessment”, Denver, CO, March 24-25, 1986.
AB-86-184
Strain, ME; Pfohl, RJ; Taylor, DH; Laurie, RD. Effects of Maternal Exposure to Trichioroethylene
on Hippocampal Development in Rat Pups. Abstracts of the 25th Anniversary Meeting 6(1): 122,
March 1986.
AB-86-075
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Sublet, VH; Smith 1 MK Randall, 3; Zenick, H. Spermatogenic Stages Associated with Acrylainide
(ACR) Induced Dominfint Letluility. Abstracts of the 25th Anniversary Meeting 6 (1): 292, March
1986.
AB-86-078
Toth, GP; Smith , MK. Effect of C10 2 on Cell Proliferation and Protein Synthesis in Developing Rat
Brain. Abstracts of the 25th Anniversary Meeting 6(1): 193, March 1986.
AB -86-082
Wones, RG; Mieczkowski, L; Glueck, CJ. Effect of Chlorinated Drinking Water on Hwn n Lipid
Metabolism. American Chemical Society Meeting New York, NY, April 1986.
XA-86-017
Wones, RG; Mieczkowski, L; Glueck, Cl. Effect of Chlorinated Drinking Water on Hmnnn Lipid
Metabolism. American Chemical Society Meeting Anaheim, CA, September 7-12, 1986.
XA-86-018
D109 - SOLID WASTE
Acedo ON; Sandhu, SS. Detection of Chemically-Induced Aneuploidy in Vicia Faba Roots.
Environmental Mutagen Society Meeting Baltimore, MD, April 9-13, 1986.
AB-86-113
DeMarini, DM; Doerr, CL Meyer, MK; Moore, MM. Mutagenicity of M-amsa and O -2m in
Mammalian Cells is Due to the Clastogenic Effects of the Compounds. Environmental Mutagen
Society Meeting Baltimore, MD, April 9-13, 1986.
AB-86-11O
Ebron, MT; Beyer, PE; Oglesby, LA. In Vitro Culture of Post-Implantation Hamster Embryos - A
Second Approach. Society of To dcology Meeting New Orleans, LA, March 3-7,1986.
AB-86-024
George, SE; King LC; Claxton, LD; Lewtas, J. Degradation of Kepone by Pseudomonas Species.
American Society for Microbiology Annual Meeting, Washington, DC, March 23-28, 1986.
AB-86 -072
Hatch, GO; Edens, F; Rodgers, L; Most, B; Berman, E. Embryotoxic and Potential Tumorigenic
Effects in Japanese Quail Inoculated In Owi by Chemical Carcinogens and Noncarcinogens.
Society of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-041
Houk, VS; DeMarim, DM. Use of the Microscreen Phage-Induction Assay to Assess the Genotoxiaty
of Selected Pesticides and Hazardous Wastes. Environmental Mutagen Society Meeting
Baltimore, MD, April 9-13, 1986.
AB-86-121
Inmon, JP; DeMarini, DM. Towards An Understanding of the Salmonella Microsuspension Assay,
How and Why It Works. GEMS Meeting Durham, NC, November 7, 1985.
AB-86-071
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Rodgers, L; Hatch, GG; Edens, F Most, B; Berman, E. Embryotoxic and Post Hatch Effects
Produced in Chickens by In Ovo Inoculation with Diverse Chemicals. Society of Toxicology
Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-046
Sandhu, SS; Acedo, GN. Mutagenicity Testing of Complex Mixtures in Arabidopsis. Environmental
Mutagen Society Meeting Baltimore, MD, April 943, 1986.
AB-86-112
Simmons, JE; Lewtas, J. Detecting To dcity Using Primary Hepatocyte Cultures. Society of Toxicity
Meeting New Orleans, LA, March 3-7, 1986.
AB-86-091
E104 - PESTICIDES
Boyes , WK; Moser, VC. Time Course of Visual Evoked Potential Effects of Acute Amitraz Exposure
in Rats. Society of Toxicology Meeting New Orleans, LA, March 3-7, 1986.
AB-86-052
Boyes , WK; Padilla, SS. Alterations in Electrophysiology and Fast Axonal Transport Induced by
Intraocular Injections of Tunicamycin. Society of Toxicology Meeting, New Orleans, LA, March
3-7, 1986.
AB-86-054
Boyes , WK; Veronesi, B. Time Course of Electrophysiological Changes in the Superior Colilculus of
Adult Long-Evans Rats Following Monocular Enucleation. Society for Neuroscience Meeting
Dallas, Texas, October 20-24, 1985
AB-85-140
Chemoff N. Importance of Assessment of the Aging Animal in Toxicology Studies. 38th Annual
Meeting Gerontological Society of America, New Orleans, Lorn ’ ina, November 22-26, 1985
AB-85-135
Chernoff, N. The Standard Teratology Protocol; Problems Associated with the Interpretation of Some
Types of Fetal Anomalies. International Conference On Pesticides, Toxics, Safety & Risk
Assessment, Lucknow, India, October 28, 1985.
AB-86-002
Crofton, KM; Reiter, LW; Mz iImz n, RB. Pyrethroid Insecticides and Radioligand Displacement from
the GABA Receptor Chloride lonophore Complex. Society of Toxicology Meeting, New Orleans,
LA, March 3-7, 1986.
AB-86-058
Crofton, KM; Reiter, LW. Differential Effects of Type I and II Pyrethroids On Motor Activity and the
Acoustic Startle Response. Society of Toxicology Meeting New Orleans, LA, March 3-7,1986.
B-86-060
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Crofton, KM; Peele , DB. Pyrethroid Effects On Schedule-Controlled Behavior, time and Dosage
Relationships. Society of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-062
Cummings , AM. Inhibition of the Decidual Cell Response of the Female Rat by Methoxychior. Society
of Toxicology Meeting New Orleans, LA, March 3-7, 1986.
AB-86-032
Cummings , AM; Gray, LE. Maternal Toxicity of Dibutyl Phthalate in Rats. Society for Study of
Reproduction Meeting, Cornell University, Ithaca, NY, July 15-17 1986.
AB-86-161
Dyer, RS; Jensen, KF; Boyes, WK. Focal Lesions of Visual Cortex - Effects on Flash Evoked
Potentials (FEPS), Paired FEP Recovery Functions, and Pattern Reversal Evoked Potentiils
(PREPS) in Rats. Society for Neuroscience Meeting, Dallas, Texas, Octoger 20-24,1985
AB-85-138
Dyer, RS; Rigdon, GC. Acute Neurotoxicity of Triadimefon. Society of Toxicology Meeting, New
Orleans, LA, March 3-7, 1986.
AB-86-050
Fisher, HL; Shah, PV; Sumler, MR; Hall, I L Chiordecone Kinetics After Dermal Exposure. Society
of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-040
Garrett, NE; Stack, HF; Waters, MD. An Evaluation of the Genetic Activity Profiles of 65 Pesticides.
Environmental Mutagen Society Meeting, Baltimore, MD, April 9-13, 1986.
AB-86-106
Goldm2n, JM; Cooper, RL Rehnberg, GL Hem, JF; McElroy, WK; Gray, LE. The Influence of
Methoxychior (MXC) Upon the Hypoth mic-Pituitary Reproductive Axis. Society of Toxicology
Meeting New Orleans, LA, March 3-7, 1986.
AB-86-035
Jensen, KF. Alterations in the Pattern of Serotonin Projections Following Neonatal Exposure to
5,7-Dihydroxytrypt2mine. Society of Toxicology Meeting New Orleans, LA, March 3-7, 1986.
AB-86-056
Jensen, KF; Killackey, HP. The Effect of Neonatal Infraorbital Nerve Section on the Pattern of
Thalamocortical Projections to the Somatosensory Cortex of the Adult Rat. Society for
Neuroscience Meeting, Dallas, Texas, October 20-25, 1985
AB-85-145
Kaw nWii , CY. Use of Monoclonal Antibodies for Classification and Identification of Insect
Pathogens. IVth Internation Colloquium on Invertebrate Pathology, Veidhoven, Netherlands,
August 18-22, 1986.
AB-86-232
Keefer, CF; Brackett, BG; Perreault, SD. Behavior of Bull Sperm Nuclei Following Microinjection
into Hamster Oocytes. Society for Study of Reproduction Meeting, Cornell University, Ithaca, NY,
July 15-17, 1986.
AB-86-160
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Moser, VC; MacPhail, RC. Cholinergic Involvment in the Behavioral Actions of Formetanate, A
Forn1Rm dine Pesticide. Society for Neuroscience Meeting Dallas, Texas, October 20-24, 1985
AB-85-141
Moser, VC; MacPhail, RC. Prolonged Behavioral and Neurochemical Effects of Acute Amitraz
Exposures in Rats. Society of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-053
Naish, SJ; Foehner, A; Perreault, SD; Zirlcin, BR. Ability of the Ferhi1i ng Hamster Sperm Nucleus
to Serve as Template for DNA Synthesis. American Society for Cell Biology Annual Meeting
Atlanta, Georgia, November 1985
AB-85-183
Padilla, SS; Grirale, T; Lyerly, D. A Comparison of In J’Tfro and In Vivo Neurotoxic Esterase
Inhibition by Triphenyl Phosphite (TPP). Society of Toxicology Meeting New Orleans, LA, March
3-7,1986.
AB-86-051
Perreault, SD; Under, RE; Hess, RA; Strader, LF. Infertility and Partial Recovery After A Single
Exposure to 1,3-Dinitrobenzene (DNB) in the Male Rat. Society of Toxicology Meeting New
Orleans, LA, March 3-7, 1986.
AB-86-034
Perreault, SD; Barbee, RR. Sperm Nuclear Decondensation Depends on Gluthathione Synthesis in
Maturing Hamster Oocytes. Society for Study of Reproduction Meeting, Cornell University,
Ithaca, NY, July 15-17, 1986.
AB-86-162
Stack, HF; Brady, AL; Waters, MD. Genetic Activity Profile Methodology. Environmental Mutagen
Society Meeting, B iltimore, MD, April 9-13, 1986.
AB-86-111
Strauss, GHS. Methods Development - A Test Battery Approach to the Measurement of Toxic,
Immunotoxic, and Genotoxic Effects in Lymphocytes Exposed to Mutagens. Environmental
Mutagen Society Meeting Baltimore, MD, April 9-13,1986.
AB-86-151
Strauss, GHS; Stanford, WL; Berkowitz, SJ. The Development of An In-Laboratory Microcomputer
System for Genetic Toxicology Experimentation and Testing. Environmental Mutagen Society
Meeting Baltimore, MD, April 9-13, 1986.
AB-86-152
Veronesi, B; Padilla, SS; Dvergsten, CL. Triphenyl Phosphite (TPP) Does Not Produce
Organophosphorus-Induceij Delayed Neurophathy (OPIDN) in Rats. Society of Toxicology
Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-055
Waters, MD; Sandhu, SS. Genetic Activity of Selected Pesticides in Short-Term Bioassays.
International Symposium on Short-Term Tests for Genotoxicity, Pisa, Italy, April 5-8,1986.
AB-86-173
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F104 - RADIATION
AlIis JW; Sinha-Robinson, RL Stead, AG. Analysis of the Microwave Radiation Induced Inhibition
of Na /K ATPase by Response Surface Methodology. Bioelectrom gnetics Society Madison,
WI, June 1-5, 1986.
AB-86-L35
Baiim ’nn, SB; Joines, WT; Berman, E All, JS. Development of a Passive Transponder to Monitor
Temperature within the Uterus of Freely Ro2’ning Mice in Radiofrequency FieId&
Bioelectromagnetics Society, Madison, WI, JUNE 1-5, 1986.
AB-86-130
Blac1rm n, CF; Benane, SG; House, DE; Joines, WT; Spiegel, RJ. Effect of Power-line-Frequency
Electric Fields on a Developing Org nicm Bioelectronii gnetics Society, Madison, WI, June 1-5,
1986.
AB-86-136
Blackman, CF; Benane, SG; House, DE. Frequency-Dependent Responses in Brain Tissue to ELF
Electromagnetic Fields and the Influence of the Local Geoms gnetic Field. Electrochemical
Society Annual Meeting, Boston, MA, May 4-9, 1986.
AB-86-140
Elder, JA. Radiofrequency Radiation Activities and Issues. N.C. Chapter of the Physics Society,
Chapel Hill, NC, January21, 1986.
AB-86-1.39
Elder, JA. Radiofrequency Radiation - Activities and Issues. Health Physics Society Annual Meeting
Pittsburgh, PA, June 29-July 3,1986.
AB-86-219
Fatmi, MBE; Spiegel, Ri. A High Resolution Finite-Difference Model of A Squirrel Monkey to
Predict Thermo-Physiologic Response Due to RF Radiation. Bioelectroms gnetics Society
Madison, WI, June 1-5, 1986.
AB-86-131
Liddle, CG; Putn im , JP; Lewter, OH. Survival of Mice Exposed Chronicaily to 2.45 GHZ CW
Microwaves. Bioelectromagnetics Society, Madison, WI, June 1-5, 1986.
AB-86-133
Liddle, CG; Putnam, JP; Lewter, OH. Effects of Microwaves on Rat Brain Histopathology.
Bioelectrom gnetics Societ Madison, WI, June 1-5, 1986.
AB-86-134
O’Connor, ME; Tremble, D; Peterson, T; Keow Seng, N; Cooper, B. Thermal Dose-Response
Measures in Rats and Mice At Specific Absorption Rates Between 2 and 8w/kg
Bioelectrom gnetics Society, Madison, WI, June 1-5, 1986.
XA-8 OO8
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Wood, AW; Blacbnnn. CF; Joines, WT. The Use of a Transverse Electric and Magnetic Field (TEM)
Cell for the Study of Electromagnetic Fields On Cellular Systems At Extrememly Low Frequencies
(ELFS). 7th National Congress of the Australian Institute Physics, Adelaide, Australia, August
25-29, 1986.
AB-86-222
L104 - TOXICS
Alien, JW; Stoner, G; Pereira, MA; Hatch, GG; Nesnow, S; Campbell, 3; Sharief, Y. Cell
Transformation, Tumorigenesis, and SCE Induction by Ethyl Carbamate and Vinyl Carbamate.
Environmental Mutagen Society Las Vegas, Nevada, February 1986
AB-85-184
Amtower, AL; Brocic, KH; Dow, CL; Dearfield, KL; Moore, MM. Genotoxicity of the Three
Acrylate Compounds in L5178Y Mouse Lymphoma Cells. Environmental Mutagen Society
Meeting Baltimore, MD, April 9-13, 1986.
AB-86-114
Beeman, DK; Siegfried, JM; Mass, Mi. Effects of the Promoters TPA and Derivatives On Colony
Formation in Culture of HnnI2n, Hamster, and Rat Respiratory Epithelial Cells. Gems Meeting,
Durham, NC, November 7, 1985.
B -86-008
Brock, KH; Oglesby, LA; Moore, MM. Further Characterization of an Intact Hepatocyte Activation
System for Use with the Mouse Lymphoma Assay. Environmental Mutagen Society Meeting,
Baltimore, MD, April 9-13, 1986.
AB-86-116
Burdette, U; Dyer, RS. Alterations in Hippocampal Afterdischarge Activity Following
Administration of Picrotoxin, Pentylenetetrazol or Caffeine. Society for Neuroscience, Dallas,
Texas, October 20-24, 1985
AB-85-136
Burleson, OR; Fuller, LB; Gr lh2m, JA. Poly (I), Poly (C)-Mediated Abrogation of Inhaled
Ozone-Impaired Phagocytosis. Society of Toxicology Meeting New Orleans, LA, March 3-7, 1986.
B-86 -048
Catigani, JC; Burleson, GR; Selgrade, MK. Relationship Between Depressed Cytochrome P450
Levels Observed During Murine Cytomegalovirus Infection and Virus Induced Interferon
Response. Society of Toxicology Meeting New Orleans, LA, March 3-7, 1986.
AB-86-045
Catignani, JC; Burleson, GA; Seigrade, ML Interferon Response to Murine Cytomegalovirus
Infection, Effect On Xenobiotic Metabolism. American Society for Microbiology Meeting
Waslungton, DC March 23-28, 1986.
AB-86-012
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Claxton, LD; Walsh, D; Stead, AG. Analysis, Including Correlation with Carcinogenicity, of the
Salmonella tyhimwiUm Bioassay Data Bases by Chemical Clas& Environmental Mutagen Society
Meeting, Baltimore, MD, April 9-13, 1986.
AB-86- 120
Clegg, MS; Rogers, 3M; Zucker, RM; Hurley, LS; Keen, CL Flow Cytometry Analysis of Cycle
Stages in Zinc Deficient Fetal Rat Brain. FASEB, St. Louis, MO, April 13-18,1986.
AB-86-148
Cole, RS; Kulak, K Athwal, RS; Sandia; 55. Development and Validation of Hnmsn Cell Culture
Assay for the Detection of Chemically-Induced Aneuploidy. Environmental Mntr Society
Meeting, Baltimore MD, April 9-LI, 1986.
AB-86-118
Cooper, RL Goldman, 3M; Rehnberg, GL Booth, KC McElroy, WK lieS, I. Effect of
Reproductive Toxins on Brain-Pituitaiy-Gonadalaxi& EPA/Army Workshop, Charleston, SC,
November 13-15, 1985.
AB4
Copeland, NIP; Chadwick, RW; Cooke, N; Whitehouse, DA; Hill, DM. The Use of a Model
Substrate (Lindane) to Determine the Ontogeny of Metabolism in the Developing Rat. Society of
Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-014
Cl lmmin&c , AM. The Decidual Cell Response and Maternal Tmicity. EPA/Army Workshop,
Charleston, SC, November 13-15,1985.
AB-86-098
Doerr, CL; Brock, KIT; Deniarini, DM; Moore, MM; Analysis of Gross Aberrations in Mouse
Lymphoma Cells Exposed to Various Genotoxic Agents. Environmental Mutagen Society
Meeting, Baltimore, MD, April 9-13, 1986.
AB-86-11S
Erexson, GL; Kligerman, AD; Allen, JW. Development of a Mouse Peripheral Blood Lymphocyte
(PBL) Micronucleus Test Using Cytodintacin B. Environmental Mutagen Society Meeting,
Baltimore, MD, April 9-13,1986.
AB-86-111
Fuller, LB; Burleson, GR; Grahnn, IA. Poly (I), Poly (C) Enhanced Phagocytosis of Rat Alveolar
and Peritoneal Macrophages as a Model for Local Pulmonary and Systemic Immunotoxicokw.
Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
AB-86-049
Goldman, 3M; Cooper, RL; McElroy, WK Hem, 3; Rehnberg, GL The Effect of Methoxychlor
Upon the Concentration and Release of Gonadotropin- Releasing Hormone from Medithasal
Hypothabnius. EPA/Army Workshop, Charleston, SC, November 13-15, 1985.
Gray, IA; Rehnberg, BF Slotkin, T; Kavlock RJ. Effects of Exposure to A-Difiuromethylornithine
(DFMO) During Discrete Prenatal Periods On Postnatal Growth and Renal Function in the Rat.
Teratology Society Meeting, Boston, MA, July 1986.
AB-86-155
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Gray, LE; Gray, K; Fend, 3M; Ostby, JS. Methoxychior-Induced (M) Alterations of Reproductive
Development in Rats. Society of Toxicology Meeting New Orleans, LA, March 3-7, 1986.
AB-8 6 -030
Gray, LE; Ferrell, JM; Gray, K; Ostby, IS. Alterations in Reproductive Development in Hamsters
Induced by Methoxychior. Society of Toxicology Meeting New Orleans, LA, March 3-7, 1986.
AB-86-031
Gray, LE; Rogers, 3M; Ostby, IS; Gray, KL Ferrell, 3M. Torticollis and Swimming Behavior
Abnorni 1ities Develop in Mice After Prenatal Dinocap Mmintstration Due to Complete
Agenesis of the Otoliths in the Inner Ear. Teratology Society Annual Meeting, Boston, MA, July
6-10, 1986.
AB-86-153
Gray, LE; Ostby, IS; Gray, K; Ferrell, 3M. An Assessment of Methoxychlor-Induced Alterations of
Estrous Cycle Dependent Running Wheel Activity in the Female Rat. Society for Study of
Reproduction Meeting Cornell University, Ithaca, NY, July 15-17, 1986.
AB-86-163
Hall, LL; Fisher, HL Sunder, MR; Shah, Pv. Age Related Percutaneous Penetration of Carbaryl,
Determined byln Vivo and In Vitro Methods. Society of Toxicology Meeting New Orleans, LA,
March 3-7,1986.
B-86-038
Hem, 3; Cooper, RL; Rehnberg, GL; Goldmnn JM; McElroy, WK. Effects of Methoxychior On
Pituitary Prolactin. EPA/Army Workshop, Charleston, SC, November 13-15, 1985.
AB-86-1O1
Hess, RA. Histological Methods for EvaluMing Testicular Pathology in a Toxicology Screen.
EPA/Army Workshop, Charleston, SC, November 13-15, 1985.
AB-86-096
Hess, RA; Carter-Laws, SD; Under, R; Strader, L Laskey, 3W. Fertility and Histopathology in Male
Rats Following Carbend i74m Exposure. EPA/Army Workshop, Charleston, SC, November 13-13,
1985.
AB-86-105
Huang, YS. Expression Vector with Promoter of Polyhedrin Gene From Insect Baculovirus First
SCBA International Symposium and Workshop, San Francisco, CA, June30 - July 2, 1986.
AB-86-flO
Kavlock, LU. Functional Aspects of Developmental Toxicity of Pesticides. International Conference on
Pesticides, Lucknow, India, October 19885
AB-85-187
Kavlock, RI. Pastiuitnl Alterations in Development Resulting from Prenatal Exposure to Pesticides.
Sixth International Conference On Pesticide Chemistry, Ottawa, Ontario, Canada, August 10-15,
1986.
AB-86-156
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Kaviock, Li; Rehnberg. BF. Consequences to the Kidneys of Fetal, Neonatal and We n1htg Rats of
Prenatal Exposure to Adriamycin. Teratology Society Meeting Boston, MA, July 7-11, 1986.
AB-86-157
Kilgerman, AD; Campbell, JA Erexson, GL Shelby, MD; Allen, JW. Cytogenetic Analysis of
Peripheral Blood Lymphocytes (PBLS) and Lung Cells of Mice Exposed to Methyl Isocyanate
(MIC) by Tn1 1jation. Environmental Mutagen Society, Baltimore, MD, April 9- 1986.
AB-86-143
Kohan, MJ; King, LC; Claxton, LD. The Influence of Extraction and Concentration Techniques on
the Isolation of 1-Nitropyrene Bacterial Metabolites. American Society for Microbiology Meeting,
W i hington, DC, March 23-28, 1986.
AB-86-119
Koren, HS; Harris, DT; Gianxiolo, GJ; Snyderman, R. Suppression of Natural Killer (NK) Cells by a
Synthetic Peptide Homologous to Retroviral PI5E. Conference On Growth Factors, Tumor
Promoters, ect., Steamboat Springs, CO. April 1986.
AB-86-089
Laskey, JW; Laws, SD. In J’Ttm Assessment of Leydig Cell Function Following Metal Cation
Treatment EPA/Army Workshop, Charleston, SS, November 13-15, 1985.
AB-86-1(X)
Lau, C; Slotkin, TA. Control of Adrenomedullary Development by Central Biogenic Am n Systems.
American Society for Neurochemistry, Montreal, Canada, March 16-21, 1986.
XA-86-004
Lau, C; Cameron, A; Irsula, 0; Robinson, KS. Some Aspects of the Teratogemc Effects of Nitrofen
Exposure in the Rat. Teratology Society Meeting, Boston, MA, July 7-11, 1986.
XA-86-O1O
Under, R; Strader, L Measurement of Sperm Quality and Quantity as a Level 1 Test in the Rat and
Hamster. EPA/Army Workshop, Charleston, SC, November 13-15, 1985.
AB-86-103
Luebke, RW; Lawson, LD; Rogers, RR; Riddle, MM; Smialowic7, Ri; Rowe, DO. Effects of
5-Chloro- and 5-Fluorouracil on Immune Function. Society of Toxicology Meeting, New Orleans,
LA, March 3-7, 1986.
B-86-025
Macphail, RC; Cook, LL Walker, QD. Acute Behavioral Toxicity of Cydotrimethylenemnuramine
(RDX). Society of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-061
Massaro, EJ. Time-Dependent Tissue Distribution of Metliylmercuty in the BALB/C Mouse and Its
Hairless Homolog, the HRSIj Mouse. Society of Toxicology, New Orleans, LA, March 3-7, 1986.
AB-86-027
Massaro, El; Massaro, T; Miller, G. Low-Level Affects Lead Exposure During Latent Learning in the
Rat Neonatal Development. IV International Congress of Toxicology, Tokyo, Japan, July 21-25,
1986.
AB-86-167
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Mass, MJ; Mumford, JL Marr, CM; Shouren, C. Proposed Method for Detection of Carcinogens in
Complex Environmental Mixtures by a Respiratory Epithelial Cell Culture Transformation Stytem.
Complex Mixtures Meeting. Ric1 L nd, W hngton, October 21-24, 1985
AB-85-134
Mass, MJ; Siegfried, 3M; Hozier, JC. Genes for Tumor Markers are Clustered Near CeUular
Oncogenes on the Hiini in Chromosome Map. American Association for Cancer Research, Los
Angeles, CA, May 7-10, 1986.
AB-86-137
Mass, MJ. Genes for Tumor Markers Arc Clustered Near Cellular Oncogenes. UCLA Molecular
Biology Symposium, Steamboat Springs, CO, January 20-22, 1986.
AB-86-138
Maxwell, ND; Ferrell, 3M; Gray, LE. Estrogenic Stimulation by Kepone of Ornithine Decarboxylase
and Glucose Metabolism in the Immature Rat Uterus. EPA/Army Workshop, Charleston, SC,
November 13-15, 1985.
B-86-097
McElroy, WK; Cooper, RL Rehnberg, GL Goldman, 3M; Hehi, J. Effects of Estrogenic
Compounds On Food 1ntnk and Body Weight. EPA/Army Workshop, Charleston, SC, November
13-15,1985.
AB-86-102
Mio, GE; Kurian, P; Nesnow, S. Characterization and Validation of Carcinogen-Induced
Transformation of Hnniiin Cells In Pi(m. NCI/NIOSH/EPA Workshop, Rockville, MD, April
22-23, 1986.
AB-86-176
Mohapatra, N; Nesnow, S; Macnair, P; Bryant, BJ; Ellis, S; Rudo, K; Sangai h, R; Gold, A.
Morphological Transformation of Mamm i1i in Cells by Cyclopenta-Fused Isomers of
Benz(a)nnthfacene and Their Metabolism. GEMS Meeting. Durham, NC, November 7, 1985.
AB-86-009
Nesnow, S; Mohapatra, N; Ross, J; Gold, A; Bryant, BJ; Rudo, K; MacNair, P; Ellis, S; Gupta, R.
Genotoxic Activities of Aceanthrylene and Acephenanthrylene. GEMS Meeting. Durham, NC,
November 7, 1985.
B-86 -007
Nesnow, S; Kakunaga, T; Yamasaki, H. International Workshop On “the Use of In Vitro
Transformation of Established Cell lines for the Prediction of Carcinogenic Chemicals”.
NCI/NIOSH/EPA Workshop, Rockville, MD, April 22-23, 1986.
AB-86-174
Ostby, J; Gray, LB. An Assessment of Methoxychlor-Induced Alterations of Estrous Cycle Dependent
Running Wheel Activity in the Female Rat. EPA/Army Workshop, Charleston, SC, November
13-15,1985.
AB-86-099
Ostby, JS; Gray, LE. The Teratogenic Effects of Two Benzidine-Based Dyes on the Testes of the
Mouse. Society of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-039
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Otto, DA. Development of Computerized Behavioral and Electrophysiological Batteries for Hirnuin
Neuroto,dcity Testing U.S.-Sweden Toxicology Workshop, NIEHS/RTP, April 14-16 1986.
AB-86-179
Palekar, LD; Coffin, DL. Significance of Mineral Fibers in Mesotheioma Induction and V79
Cytotoxicity. Society of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB-86-047
Perreault, SD. Additional Measures of Sperm Production and Function. EPA/Army Workshop,
Charleston, SC, November 13-15, 1985.
AB-86-095
Perreault, SD; Strader, L; Barbee, R; Linder, R. Fertilizing Ability of Spermatozoa from
Sprague-Dawley Rats After Acute Exposure to a Testicular Toxicant, 1,3-Dinitrobenzene (DNB).
EPA/Army Workshop, Charleston, SC, November 13-15, 1985.
AB-86-104
Rabinowitz, JR; Little, SB. Multipole Expansion Techniques for the Calculation and Characterization
of Molecular Electrostatic Potentials. International Symposium on Quantum Biology and
Quantum Pharmacology, St. Augustine, FL, March 6-8, 1986.
AB-86-150
Rehnberg, GL Endocrine Events Associated with Akered Reproductive Function. EPA/Army
Workshop, Charleston, SC, November 13-15, 1985.
AB-86-094
Reinhar4, iF; O’Callagh2n, JP. MF P Decreased Dopamine Levies and Increases the Levels of Gilal
Fibrillary Acidic Protein in Mouse Striatum - Evidence for Glial Reaction to Injury. American
Society for Pharmacology & Experimental Therapuctics, Baltimore, MD, August 1986.
AB-86-212
Richard, AM; Rabinowitz, JR. Modified Molecular Charge SimibiTity Indices for Choosing Chemical
Analogues. International Symposium on Quantum Biology and Quantum Pharmacology, St.
Augustine, FL, March 6-8, 1986.
AB-86-149
Rogers, 3M; Carver, BD; Gray, LE; Gray, JA; Kavlock, Ri. Teratogenic Effect of the Fungicide
Dinocap in the Mouse. Society of Toxicology Meeting, New Orleans, LA, March 3-7, 1986.
AB -86-023
Rogers, 3M; Gray, LE; Carver, BD; Kavioc1c Ri. Comparison of Material and Fetal in Mice, Rats
and Hamsters Treated with the Fungicide Dinocap. Teratology Society Meeting, Boston, MA, July
7-11, 1986.
AB-86-158
Sandhu, SS; Athwal, PS. Application and Limitations of a M inim ’Han Cell Culture Test System for
Detecting Chemicafly-Induced Aneuploidy. International Symposium on Short-Term Tests for
Genotoxicity, Pisa, Italy, April 5-8,1986.
AB-86- lfl
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Seigrade, MX. Use of Susceptibility to Murme Cytomegalovirus as an Indicator of Immunoto dcity.
Association of Official Analytical Chemists Meeting, Washington, DC, October 28, 1985
AB-85-155
Seigrade, MK Susceptibility to Murine Cytomegalovirus (MCMV) - A Host Resistance Model for
Immunotoxicity Testing, Immunotoxicology Discussion Group Meeting Chicago, Illinois, October
9-10, 1985
AB-85-lfl
Seigrade, MK Catign ni, JC; Burleson, GR. Effects of Murine Cytomegalovirus (MCMV) and Poly
I,C on Sensitivity to Parathion Poisoning. Society of Toxicology Meeting New Orleans, LA, March
3-7, 1986.
AB-86-044
Selgrade, MX. Disease Susceptibility Models Under Investigation at EPA. Eastern Regional
Symposium on Mechanisms of Immunotoxicity, Wlllbimsburg, VA, September 4-5, 1986.
AB-86-241
Shah, PV; Fisher, HL; Sumler, MR; Hail, LL. Dermal Penetration of Carbofuran in Young and Adult
Fischer-344 Rats. Society of Toxicology Meeting New Orleans, LA, March 3-7, 1986.
AB-86-037
Sheets, L; Reiter, LW. Ontogeny of the Rat’s Auditory Startle Response with Evidence of
Serotonergic in Involvement. Society of Toxicology Meeting New Orleans, LA, March 3-7, 1986.
AB-86-057
Smialowicz, RJ; Leubke, RW. Evaluation of Immune Function in Young Adult Rodents Exposed to
Known or Suspected Immunomodulators During Gestational and/or Early Postnatal Life.
Association of Official Analytical Chemists Meeting, Washington, DC, October 28-29, 1985
AB-85-L53
Smialowicz, RI; Rogers, RR; Riddle, MM; Luebke, RW; Fogelson, LD; Rowe, DG. Immunologic
Effects of Perinatal Exposure to Dioctyltin Bichloride. Society of Toxicology Meeting, New
Orleans, LA, March 3-7, 1986.
AB-86-013
Smialowic RI. Immune Status in Newborns and Their Vulnerability. Workshop on the Role of
Predisposing Conditions of Health, Nutrition and Environment On Safety of Drugs and Chemicals,
Lucknow, India, February 25-28,1986.
AB-86- 132
Stoner, GD; Shivapurkar, N; Schut, HAY; Klaunig JE; Mass, MJ. Metabolism of
4,4’-Methylene -Bis(2-Chloroaniline) in Explant Cultures of Human and Dog Bladder and Dog
Liver Cell Cultures. NCI/NIOSH/EPA Workshop, Rockviile, MD, April 22-23, 1986.
AB-86-177
Viana, MA; Brock, TO; O’Callaghan, ii’. Quantification of the Synaptic Vesicle Proteins, Synapsin I
and P38, and the Astrocyte Specific Protein, Glial Fibrillary Acidic Protein, Following
Trimethyltin-Induced Injury to the Rat Central Nervous System. Society for Neuroscience
Meeting Dallas, Texas, October 20-24, 1985
AB-85-139
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Waters, MD. Advances in the Use of Short-Term Tests for Prediction of Mutagenic and Carcinogenic
    Potential. Science Writer's Colloquium, Bologna, Italy, October 6-10,1985
    AB-85-185

Waters, MD. An Investigation of the Parallelogram Concept in Genetic Monitoring and Risk
    Estimation. U.S.-Sweden Workshop on Toxicology, NIEHS/RTP NC, April 14-16,1986.
    AB-86-178

Watitinson, WP. Effects of Chlordimeform on Heart Rate and Body Temperature of Unanesthetized,
    Unrestrained Rats. FASEB, St. Louis, MO, April 13-18,1986.
    AB-86-090
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