*>EPA
Health  Effects
Research  Laboratory
Office of Health Research
Office of Research and Development

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              HEALTH EFFECTS RESEARCH LABORATORY


                            Office of Research and Development

                               Office of Health Research
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                                 Progress Report


                          October 1,1985 - September 30,1986
                                  . <
                          U.S. Environmental Protection Agency
                          tlbrnrv. Room 2404  PM-211-A
                          401 M Street, S.W.
                          WashingtOD, DC   20460

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Acknowledgements:

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
HERL Mission Statement	1
Organizational Chart	2
Divisional Functional Statements	3
Air and Radiation
  Research Objectives	9
  Accomplishments	17
  Publications	23
Water
  Research Objectives	83
  Accomplishments	89
  Publications	99
Pesticides and Toxics
  Research Objectives	183
  Accomplishments	191
  Publications	203
Hazardous Waste
  Research Objectives	279
  Accomplishments	283
  Publications	289
Future Perspectives  	.'	297
Index of Abstracts	303
Appendix A - List of Submitted Documents 	353
Appendix B - List of Presentation Abstracts	371

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            HEALTH EFFECTS RESEARCH LABORATORY

                           Dr. F. Gordon Hueter, Director
                        Dr. Judith A. Graham, Deputy Director
  HERLis 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. Smaller programs are being conducted concerning
hazardous wastes1 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 hazard 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
lexicological 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 approximately 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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                  HEALTH EFFECTS RESEARCH LABORATORY
                      DIVISION FUNCTIONAL STATEMENTS
                       INHALATION TOXICOLOGY DIVISION

                                Dr. Frederick Miller, Director
 The Inhalation Toxicology Division conducts research with humans and animals to determine the health
 effects of inhaled environmental pollutants. The research uses highly sophisticated human 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 lexicological
 methodologies are used, emphasis is also placed on the development of improved methods which enable
 significant 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 mechanisms against 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 hazardous 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 mechanism.^ 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
vitro 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, mobilization, biotransformation, and excretion. Shorter term, predictive toxicologic tests
are developed and compared Microbiologjc 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 analysts 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 statistical 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 programming 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 planning, 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 organization including functional and structural. In develop-
ing the necessary multidisciplinary approach to the study of neurotoxicity, the Division addresses both the
short-term needs (t.e., toxicity testing) and long-term goals (i.e., methods development and validation,
hazard 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 hazardous air pollutants are in progress);  (3) determinations of the sig-
nificance of neurotoxicological indicators for assessing risk in humans; (4) developmental neurotoxicol-
ogy (behavioral teratology) which evaluates the effects of perinatal toxicant exposure on the development
of the nervous system; and (5) mechanism of action studies.

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                                                   DIVISION FUNCTIONAL STATEMENTS
                       EXPERIMENTAL BIOLOGY DIVISION

                                Dr. Richard Phillips, Director
The Experimental Biology Division conducts and manages biological research to define and quantify the
effects of environmental pollutants, singly or in combination. Emphasis is given to the study of effects of
non-ionizing radiation and toxic substances. This involves laboratory studies of intact animals or cellular
and subceUular systems using controlled and simulated environments. Major research attention is
devoted to elucidation of mechanisms 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 immunologjcal status, growth, functional development of systems, pathological ef-
fects, biophysical and biochemical interactions.  In addition, this division plans and conducts special
studies in non-ionizing 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 pollutants. 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 unproved 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-
tially 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 making 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 (Oa), carbon monoxide (CO), participate matter (PM), sulfur dioxide
                (SO2), lead (Pb) and nitrogen dioxide (NOz).  For these pollutants, primary standards
                are set to protect human health. The CAA amendments also require EPA to review and
                regulate hazardous 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
                hazardous and require regulation.

 Oxidants - 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 Oj include respiratory tract problems such as difficult breathing and reduced lung
 function, reduced resistance to infection, and structural and biochemical changes of lung tissue. Health
 concerns related to NOj are similar; asthmatics are more responsive to NO2 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 Os, NOz, and
 PM is conducted. HERL scientists are conducting research in the following general areas in support of
 this effort.

 • Provide lexicological data on the health effects of acute and chronic exposure to Os and  NOj 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 damage 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 Planning 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.

 Approach: Human and animal 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 NOz 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 NO2 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 animal 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 NOa 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 humans 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 epldemlologfc 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, epidemic-logic 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 NCh. These questions include: 1) Does the presence of a gas
stove increase NOa exposure in the home? 2) If so, what are these exposure levels? 3) Does this level of
NCh 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 NO2 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 SO2 causes significant pulmonary responses in individuals with asthma. Fine par-
ticles, about the size of cigarette smoke particles, can cause temporary or permanent damage when they
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 AIR HEALTH
RESEARCH OBJECTIVES
 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 bettor models to extrapolate quantitatively from animal data to human pulmonary effects
    following exposure to sulfur dioxide (SOa) and particles as well as combinations of selected gases
    and particles.

 Rationale: Mathematical models predictive of respiratory tract dose patterns of SO2 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 SO2 alone and in
 combination with particles. Studies are being conducted to provide data on die relationship between par-
 ticle size and lung deposition in man.  Regional deposition data for animals are being developed and
 refined and lung casts are being used to examine 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 using 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 humans. 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 SO2 and participate 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 unmunological 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 unpaired 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 OBJECTIVES
AIR HEALTH
Approach: A four-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 examined 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
                                                              . t
• Work with Chinese scientists to determine the causes and contributing factors of lung cancer in
   Xuan Wei County, .Yunnan Province, Peoples Republic of China (PRC).

Rationale: Item DA, Annex I "U.S.-China Protocol for Scientific and Technical Cooperation..." calls for
a study of lung cancer in Xuan Wei 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 examined 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 paniculate 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 human 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-
vironmental 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 Emission Standards for Hazar-
dous Air Pollutants (NESHAPS) under the Clean Air Act for some hazardous 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 hazardous in support of appropriate
regulatory decisions.

 • Produce dose-response data on the toxic effects of HAFs.

Approach: The specific aim is to provide dose-response data on the lexicological effects of hazardous air
pollutants.  The lexicological 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 hazardous air pollutants, 2) to develop a comparative potency method for as-
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AIR HEALTH
RESEARCH OBJECTIVES
sessing cancer risk from source emission, and 3) to evaluate the contribution of source emissions to can-
cer risk.

•  Identify toxic components of air pollution.

Approach: Identify potentially carcinogenic, mutagenic, and teratogemc components b 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 (NOx), SOz, Os 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 emissions or from atmospheric transformation of precursor chemicals.

•  Evaluation of humans for genotoxlc effects following UV-B radiation.

Rationale: This research complements that mandated by the Clean 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 UV-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 Progams (ORP).

Background:    Executive Order 10831 of 1959 (24 Fed Reg. 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  Reorganization Plan also transferred certain
               regulatory and research responsibilities of the Bureau of Radiological Health as derived
               from the Public Health Service Act.

The radiofrequency 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 ORD 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 articles 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, hazardous 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 pulmonary function
 responses in some normal volunteers following acute inhalation 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 inflammation: Other studies with pulmonary cellular elements derived from the lungs of volunteers
 by bronchoalveolar lavage are underway to evaluate the association of local inflammation 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 inflamation 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 import ant 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 asthmatic
 subjects who were exposed to 0.3 ppm NOa with moderate exercise. Two other human studies were com-
 pleted in FY*86. One described the group concentration-response of mildly asthmatic subjects exposed
 to SOa concentrations between 0.25 and 1.0 ppm. Another showed that there was no interaction between
 SO2 and HaSO4 mist when mildly asthmatic subjects were studied.

 A chronic animal toxicology study of the effects of NOz and Oa 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 animals, thereby making 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 examined 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 examined. 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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Health  research in the  People's Republic  of China continued.  A report summarizing current
epidemiologtc, chemical, and toricologic evidence relating to Xuan Wei lung cancer etiology was
published k 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 U-S.-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 paniculate 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 - 47.4 ug/dl there was a significant negative
relationship between1 blood lead level and Stanford-Binet IQ, a measure of cognitive function. The IQ
decreased linearly as blood lead increased. An analysis of NHANES n 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 (0.5,1,2 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 Toxics Study (IATS) 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 FV86, p-xylene; toluene and phosgene exposures
were completed.   Neurotoxicity,  teratogenicity,  tmmunotoxicity, hepatotoxicity,  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 initiated during FV86. Pulmonary function and bronchial reactivity
to a methylcholine 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 hazardous air pollutants
(HAP) chemicals.
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 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. Neurotaxic 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 (1/2 LD50) 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 MNjQt, which is a combustion product of the fuel additive methylcyclopentadienvl manganese
 tricarbonyl (MMT), was conducted to evaluate the fate of inhaled particles translocated to the gut.
 Results indicate higher absorption and retention in young (preweaning) rats than in adults resulting in
greater exposure for younger animals and an increased possibility of toxicity.

Mutagenicity, 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 participate mutagenicity was from woodstoves
and 50% from automobiles.
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Issue:   Provide monitoring systems, control technologies, and data on the health effects and atmos-
        pheric processes of mobile source pollutants to support the mobile source regulatory program.

A study oa 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 FY1986. The
results indicate that, for this patient population hi general, a threshold for COHb apparently easts be-
tween these concentrations since the investigators were unable to detect significant physiologic or
symptomatic changes in these patients when the COHb was 4%; however, the symptoms reported were
significantly increased when the COHb was 6% as compared to the responses following exposure to clean
air. An additional study hi the Neurobehavioral Laboratory indicates that CO dimin«hfis hand-eye coor-
dination.
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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...

Programmatic Relevancy statements are italicized.

Other articles submitted for publication, but not published as yet, are listed in Appendix A.

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        DESIGN and MEASUREMENT CONSIDERATIONS for EXERCISE
       PROTOCOLS in HUMAN AIR POLLUTION INHALATION STUDIES

                                      S. M. Horvath

                           University of California at Santa Barbara

 Information 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 magnitude of the exercise load, the duration of the exercise period, and the timing 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, M J. Utell, J.D. Hackney, J. Van Ryrin 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. Bedi, Deborah M. Drechsler-Parks, and Steven M. Horvath

                            University of California-Santa Barbara

The results from this study provide the Office of Air Quality Planning and Standards (OAQPS) with infor-
mation pertinent to the determination of the National Air Qualify Standards (NAQS)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 (DAY1); 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 ven-
tilatory minute volume (Ve) of 27 L/min (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^, respiratory rate (fa), 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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Both ozone exposures induced significant (P<0.05) decrements in FVC; FEVi.o (forced expiratory
volume in 1 second); FEV3.o (forced expiratory volume in 3 seconds); FEF 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 DAY1, and averaged 7.2 percentage points greater
than those following the DAY1 exposure.

CITATION: Journal of American Industrial Hygiene Association 46(12): 731-734,December 1985.

A104 XX-84-022 Project Officer: Horstman
          RESPIRATORY RESPONSES of VIGOROUSLY EXERCISING
                   CHILDREN to 0.12 PPM OZONE EXPOSURE

                         William F. McDonnell,1 Robert S. Chapman,1
                   Margaret W. Leigh2, Gerald L. Strop?, and Albert M. Collier2

                        1HERL, USEPA, Research Triangle Park, NC
                        University of North Carolina School of Medicine

Epidemiologic studies had suggested that children may be more responsive to ozone exposure than com-
parably exposed adults. This study was performed to measure the respiratory responses of heavily exercising
children exposed to 0.12ppm 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 important to the Agency's mission of setting National Ambient Air Quality Standards that adequate-
ly protect the health of all population groups.

Changes in respiratory function have been suggested for children exposed to less than 0.12 ppm ozone
(Oa) 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 Oa in random order. Exposures were for 25 h and included 2 h of in-
termittent heavy exercise.  Measures of forced expiratory volume in one second (FEVi) and the symptom
cough were determined prior to and after each exposure. A significant decline in FEVi was found after
the Oa 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 Os 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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       OZONE RESPONSE RELATIONSHIPS in HEALTHY NONSMOKERS


              Thomas J. Kulle, Larry R. Sauder, J. Richard Hebet, and Marie D. Chatham

                        University of Maryland, Department of Medicine

 This study examined the respiratory effects in heavify exercising men of several levels of ozone above and
 below the present National Ambient Air Quality Standard.  This study is particularly important to the
 Agency's mission of setting a NAAQSfor ozone which will protect the public health. A previous study indi-
 cated that respiratory effects occurred following exposure to 0.12 ppm ozone. This present study found ef-
 fects at 0. IS ppm but not at 0.10ppm confirming 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 FVC, FEVi, FEF2S-75, SGaw, 1C, and TLC in 20
 healthy, nonsmoking volunteers exposed randomly to 0.00,0.10,0.15,0.20, and 0.25 ppm Oa. In addition,
 significant response changes for FVC, FEVi, and FEFas-ys were shown with time over the 2-h exposure.
 Intermittent, heavy exercise (VE, 68 I/mm) lasting 14 min was employed every 30 min 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 Oa. Six subjects experienced decreases >5% in FEVi or >15% in
 SGaw at 0.15 ppm. This concentration is only slightly higher than the 1-h Os 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-
 lishing a safe Oa exposure level.

 CITATION: American 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 Kehrl? and Milan Hazucha3

                        HlERL, USEPA, Research Triangle Park, NC
                          Environmental Monitoring Services, Inc.
                           ^University of North Carolina-Chapel Hill

The results pom this study provide the Office of Air Quality Planning and Standards (OAQPS) with infor-
mation pertinent to the determination of National Air Quality Standard for sulfur dioxide.

The purpose of this study was to describe for asthmatic subjects the distribution of individual bronchial
sensitivity to sulfur dioxide (SOz). 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 min exposures to 0.00,0.25, 0.50 and 1.00 ppm SO2 at
26°C, 70% relative humidity. During exposures, subjects breathed naturally and performed moderate ex-
ercise (VE, normalized for body surface area = 211/m2 x min). Before and 3 min after exposure, specific
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airway resistance (SRaw) was measured by body plethysmography. Those subjects whose SRaw was not
doubled by exposure to 1.00 ppm were also exposed to 2.00 ppm SOz. Dose response curves (relative
change in SRaw, corrected for change b clean air vs. SOa concentration) were constructed for each sub-
ject. Bronchial sensitivity to SO2[PC(SO2)], defined as the concentration of SCh which provoked an in-
crease in SRaw 100% greater than the response to clean air, was determined. Substantial variability in
sensitivity was observed: for 23 subjects, PC(SO2) ranged between 0.28 and 1.90 ppm, while for the
remaining 4 subjects, it was greater than 2.00 ppm SOi- The median PC(SO2) was 0.75 ppm SOz, and 6
subjects had a PC(SCh) of less than 050 ppm.  PC(SOz) was not related (r = 031) 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

                                       David A. Otto

                         HERL, USEPA, Research Triangle Park, NC

Children tire known to be more susceptible than adults to the neurotcodc effects of many chemicals such as
lead. This paper reviews electrophysiological test methods that are currently available for neurotaaddty test-
ing in children. Specific tests are described and the relevant neurotaadcology literature is renewed. This
methods renew should be helpful in the design of both intramural and extramural neurotosdcuy 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,
somatosensory) 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, Neurobehavioral Toxicology and Teratology, S. Schroeder, ed., 1986.

A101MS-86-036  Project Officer: Otto
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      THE RELATIONSHIP of EVENT-RELATED BRAIN POTENTIALS and
                                LEAD ADSORPTION:
                       A REVIEW OF CURRENT EVIDENCE


                                      David A. Otto

                         HERL, USEPA, Research Triangle Park, NC

 This renew 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 exposure.

 The results of human and animal studies of the effects of lead adsorption on sensory evoked and slow
 brain potentials 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 jig/dl were reported in two studies.
 Sensory evoked potentials 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 animals to clarify inconsistencies in the limited existing literature.

 CITATION: In: Lead Environmental Health: The Current Issues, 1986.

 A119 MS-S6-038 Project Officer: Otto
     5-YEAR FOLLOW-UP STUDY of CHILDREN with LOW-to-MODERATE
        LEAD ABSORPTION: ELECTROPHYSIOLOGICAL EVALUATION


                    D^L Otto,1  G. Robinson2, S. Baumanh2, S. Schroeder2,
                         P.Musha)?tD.Kleinbaum2,andL.Boone2

                        1HERL» USEPA, Research Triangle Park, NC
                      2University 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 is 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 exposure.
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Forty-nine children aged 6 to 12 years were evaluated for residual effects of lead exposure using
psychometric, electrophysiologjcal, and medical tests 5 years after initial assessment. The original range
of blood lead (PbB) levels was 6-59 (x = 28) jig/dl; the current range was 6-30 (x - 14) |ig/dl. A linear
relationship between PbB and slow brain wave voltage during sensory conditioning was observed at ini-
tial evaluation and at 2-year follow-up. No significant relationship between PbB 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 conditioning was suggested by exploratory
analyses. Another exploratory analysis revealed a significant linear relationship between the original PbB
levels and the latency of waves HI and V of the brainstem auditory evoked potential. The latency of both
waves increased as a function of original PbB. Increased latency of these waves is suggestive of subclini-
cal pathology of the auditory pathway rostral to the cochlear 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.

A119MS-85-086 Project Officer: Otto
        SEPARATING the EFFECTS of LEAD and SOCIAL FACTORS on IQ

                    .  Stephen & Schroeder1, Barbara Hawk1, David A. Otto2,
                              Paul Mushak1, and Robert E, Hicks1

                        University of North Carolina School of Medicine
                         2HERL, USEPA, Research Triangle Park, NC

This study supports the agency decision to phase down lead in gasoline, contributes to Lead Criteria Docu-
ment updating, and is 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 exposure.

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 jig/dl on IQ after con-
trolling for concomitant social factors, such as socioeconomic status, home environment, and maternal
IQ. The main concomitant variable was socioeconomic status, which was multicolinear with other social
factors. Five years later, when all blood lead levels were 30 |xg/dl or less, lead effects on IQ were no longer
significant. The correlation between maternal and child IQ, 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.

CTTA.TIO^: Environmental Research 38:144-154,1985.

A119 MS-85-156 Project Officer: Otto
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       TRIGGERING of a CYTOLYTIC FACTOR with TNF-LIKE ACTIVITY
                            FROM HUMAN MONOCYTES
                    HillelS. Koren,1 Karen P. McKnnon2 and Allen R. Chen2

                         1HERL,USEPA, Research Triangle Park, NC
                              2Duke University Medical Center

 The model system described in this paper will be used to assess the effect(s) of pollutants on human macro-
phages obtained by bronchoscopy and bronchoalveolar lavage.  Macrophages are important celts for im-
munity vs. microorganisms and tumors.

 Monocytes have a nonoridative mechanism of tumor cytolysis operative in CGD and in normal monocytes
 under anaerobic conditions. The absence of competitive inhibition provides negative evidence for a con-
 tact-independent mechanism 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 antigenically. Further work will be
 required to demonstrate its biochemical identity, its precise mechanism of toxicity, and the extent of its
 importance in vivo.

 CITATION: In: CytoUadc Lymphokines and Cancer Biological and Chemical Aspects', J.H. Ransom and
            J. Ortaldo, eds., Humana Press, Clifton, NJ, 1986.

A101MS-86-148 Project Officer: Koren
        OZONE INHIBITS PROSTACYCLIN SYNTHESIS in PULMONARY
                                   ENDOTHEUUM

                  Mitchell Friedman, Michael C. Madden, D. Stephen Sounders,
                    Kenneth Gammon, Gilbert C. White, II and Lester Kwock

                        University of North Carolina School of Medicine
           «.                             t  -:
This study provides some insight into alterations of prostacycline synthesis in the lung after exposure to
short bursts of ozone. Prostacycline, a metabolite ofarachidonic acid, is a precursor ofprostaglandin. This
class of compounds elicit potent actions on pulmonary cell permeability, blood pressure, and blood clot
formation. It is not presently known if continued alteration of these compounds, resulting from chronic
ozone exposure, would result in serious health problems. Research is 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 prostacycHn 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 examined 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, similar to the findings in dogs given in-
domethacin (15 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 inhalation affects pulmonary endothelial arachidonate metabolism in vivo as
well as in vitro.

CITATION: Prostoglandms 30(6): 1069-1083, December 1985.

A101XX-86-089 Project Officer: Goldstein
  EFFECTS of INHALATION of 0.12 and 0.25 PARTS PER MILLION OZONE on
      the PROXIMAL ALVEOLAR REGION of JUVENILE and ADULT RATS

                    BrendaE. Bony,1 Frederick J. Miller,2 and James D. Crapo1

                               1Duke University Medical Center
                         2HERL, USEPA, Research Triangle Park, NC

The degree of lung injury caused by prolonged inhalation of low levels of ozone (O$) is of interest since
urban environmental levels periodically reach 0.2 to 0.3 ppm.  Since the area of the junction of the conduc-
tive and respiratory regions of the lung has been reported as the major site of injury due to 03 inhalation,
techniques were devised to specifically study alveolar tissue from Ms region. One-day-old or six-week-old
male rats were exposed to either 0.12 or 0.25 ppm  0312 hours/day or to continuous room air for 6 weeks.
infrastructure morphometric analysis indicated significant changes  in the alveolar epithelium of the
proximal alveolar region of all exposed animals. The hyperplastic 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 suggest that low concentrations of 03 cause a chronic epithelial injury
in the proximal alveolar region consistent with  a pattern which could lead to chronic obstructive lung dis-
ease even at ambient urban 03 concentrations.  These findings bear upon the potential lack of a safety mar-
gin in the current ambient standard for Oa concentrations to which the public at large is exposed.

The degree of lung injury caused by prolonged inhalation of low levels of ozone (Oa) is of interest since
urban environmental levels periodically reach 0.2 to 03 ppm.  Since the area of die junction of the con-
ductive and respiratory regions of the lung has been reported as the major site of injury due to Os 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 Oa 12 hours/day or to continuous room air for 6
weeks. An additional group of 6-week-old rats were exposed to 0.12 ppm Oa 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 examination of the tissue revealed little observable damage to the proximal al-
veolar tissues. However, by infrastructure morphometric analysis, significant changes occurred in the al-
veolar epithelium of the proximal alveolar region of all exposed animals. In the animals exposed to 0.25
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 ppm Oa from 1 day of age (juvenile animals), the number of type 1 epithelial 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 Os 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 animals exposed to 0.12 ppm Oa 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 O3 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

              GaiyE. Hatch, Ralph Slade, MaiyJane K. Belgrade, and Andrew G. Stead

                         HERL, USEPA, Research Triangle Park, NC

A diet which is poor in vitamin Cmay increase sensitivity to nitrogen dioxide (NOi), a common indoor and
outdoor air pollutant. Guinea pigs fed a diet which is deficient in vitamin C for 7 days or longer showed in-
creased lung damage when exposed to 5 ppm N(h for 3 hr.  Vitamin E was found to be more easily
destroyed by N(h when vitamin C was lowered. Lungs which were damaged by JVOz synthesized another
protective agent, ghttathione. However the increased gtutathione 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 (NOa). The present study showed that exposure to NOa (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 sulfhydryls (NPSH) were increased to 114% of control.
Exposure of the guinea pigs to NO2 (45 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 NO2. 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 NO2 leads to oxidation of AT and pulmonary edema. Conditions in which NO2 produced edema were
accompanied by only a slight consumption of AT and no detectable oxidation of lung AA or NPSH.

CITATION: Toxicology and Applied Pharmacology 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

                     Ung-Yi Chang,1 Judith A. Graham,2 Frederick J. Miller,2
                            JeanJ. Ospital,3 and James D. Cnapo1

                              1Duke University Medical Center
                         2HERL, USEPA, Research Triangle Park, NC
                             Southern California Edison Company

As part of a larger investigation of the issue of age sensitivity to coddants, a study of nitrogen dioxide (NCty
was completed. Both neonatal (1-day-old) and young adult (6-wk-old) rats were exposed for 6 wk to an
urban pattern ofNOz (0.5 ppm, 23 hr/day, 7 days/wk on which were superimposed spikes of 1.5 ppm ATOa
for Ihrinthe morning and afternoon for 5 days/wk). Another group of adult rats were exposed to a higher
level ofNOi (a continuous baseline of 2 ppm with spikes to 6 ppm). The structure of the region of the lung
known to be most sensitive to NOi (the proximal alveolar region) was studied quantitatively using highly
advanced morphometric methods. Nitrogen dioxide caused morphometric changes; specifically epithelial
injury was observed, with die higher concentration ofNOz causing more effects. The adult rats were as sen-
sitive or more sensitive, depending on the specific endpoint, to NOz than were the rats exposed from 1 day
of age. This work, as well as associated studies, imply mat the developing king is not at increased risk to the
effects ofNCh,  Also this study is the first of its kind in which structural effects have been reported after ex-
posure to urban patterns ofNOi The research will be useful in reducing the uncertainties regarding the ef-
fects of low levels ofNOi on an important parameter and age sensitivity when EPA Devaluates the NO2
NAAQS.

Inhalation of nitrogen dioxide (NOa) 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 NOi One-day-old and six-week-
old rats were exposed to either room air or 0.5 ppm NO2 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 NOz for the same duration. Two daily how-
spikes to three times the background concentrations (0.5 to 1.5 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 (03 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 sectioning. Morphometric analysis revealed that epithelial injury occurred in all exposed animals.
The juvenile rats which had been exposed to 05 ppm NOz since 1 day of age exhibited changes in the
characteristics of type n epithelial cells. These cells spread to cover more alveolar surface and became
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 thinner. Adult animals exposed to 0.5 and 2.0 ppm NOa showed changes in alveolar macrophages and in
 the alveolar interstitium in addition to changes in the epithelium. Animals exposed to 0.5 ppm NO2
 showed spreading and hypertrophy of type n epithelial cells. Those animals exposed to the higher con-
 centrations of NOa had similar changes in type n 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 NO2 can cause injury to the alveolar epithelium indicated
 initially by spreading and hypertrophy of type n cells followed by differentiation into type I cells to com-
 pensate and repair the injury.  Adult rats were as sensitive or more sensitive to NO2 injury than were
 juvenile rats.

 CITATION: Toxicology and Applied Pharmacology 83(1): 46-61, March 1986.     '  'f

 A104 MS-85-2Q8 Project Officer: Graham                                                     :
           CONVERSION FROM RT-11 to MICRO-RSX for REAL-TIME
                        DATA COLLECTION and ANALYSIS

                                      Mitchell E.King

                                   Northrop Services, Inc.

This paper provides technical information relevant to the Pulmonary Functions Laboratory, computer sys-
tems operations within the Inhalation Toxicology Division of the Health Effects 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, multiterminal
support for concurrent data analysis and reduced costs per experimental setup. This paper presents
simple techniques for converting Fortran/Macro-11 single-user programs running under RT-11  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-11 routines from RT-11 with Micro-RSX. Tech-
niques for overlaying programs and setting up group global event flags for dual-task control will be il-
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.                                                         /  '       ;

A101XX-86-087 Project Officer: Costa
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        DOSIMETRY MODELING of INHALED TOXIC REACTIVE GASES


                               John Overton and Fred Miller                     t       '  .

                        HERL, USEPA, Research Triangle Park, NC

HERL is a co-sponsor of the Health Effects Institute (HEI). The chapter provides recommendations for
future HEI research and mil guide and influence HEI's decisions concerning dosimetry modeling by
promoting HERL and HEI program coordination and reducing program overlap.'

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 NOz and Oa, 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.

A101MS-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. Martonen,1 M. G. Menache^M. Lippmann,3
                             R.C. Graham? and D. M. Spektor*

                        *HERL, USEPA, Research Triangle Park, NC
                                ^Northrop Services, Inc.
                           ^ew York University Medical Center

Particulate size-selective sampling is an important consideration in determining ambient air quality stand-
ards. Hazard evaluations, as well as risk analyses, can benefit from an improved understanding of factors
affecting regional respiratory tract deposition of particles in man. Thoracic deposition and its component
parts were examined, as a Junction of paniculate size, for ventilation rates ranging from normal respiration
to heavy  exercise in individuals who are habitual mouth breathers and in those who normally employ
oronasal breathing when minute ventilations exceed approximately 35 L min'\ 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.

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 unproved.understanding of. factors affecting regional respiratory tract
 deposition of particles in man. Here, thoracic deposition and its component parts are examined, as a
 function of paniculate size, for ventilation rates ranging 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 + ea+PlogX]"1 to data from tests measuring extrathoracic(ET) and
 tracheobronchial (TB) deposition.  X was defined  as an impaction parameter (pd2Q) and as the
 aerodynamic diameter of the particle (Dae) 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 Particles Vf, Cambridge, England, September 1985.

 A101MS-86-082 Project Officer: F. Miller
        MODELLING of the UPPER and LOWER RESPIRATORY SYSTEMS

                                         A. L. Patra

                                    Northrop Services, Inc.

 The toxic expression of inhaled particles and vapors is highly dependent on the region of the respiratory tract
 on which the substance deposits or is absorbed. Silicone replica casts of the nose, larynx, trachea, and
 tracheobronchial airways were constructed and described for humans and standard laboratory animals to
 allow more accurate dosimetric extrapolation of pulmonary toxicity data. Differences in anatomy and de-
pendent airflow and particle deposition  across species were carefully quantitated with considerations for
sex, 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 human and for standard laboratory animals. Mor-
phometric measurements were made on nasopharyngeal and tracheobronchial 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 morphometric dimensions were shown to be highly sig-
nificant for use in extrapolating lexicological 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.

A101XX-86-122 Project Officer: F. Miller
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            AMBIENT SULFATE AEROSOL DEPOSITION in MAN:
              MODELLING the INFLUENCE of HYGROSCOPICITY

                 T. Blayne Martonen,1 Andrew E. Burnett,2 and Frederick J. Miller*

                        1HERL, USEPA, Research Triangle Park, NC
                                 Northrop Services, lac.

Some ambient contaminents of health effects concern are hygroscopic and mil absorb water vapor in the
humid environment of the human htng.  The uptake ofHyO will affect the deposition site and thus the toxic
potential of an inhaled aerosol. The mathematical model presented in this study will enable the Health Ef-
fects Research Laboratory (HERL) to more accurately predict regional aerosol deposition patterns within
the respiratory tract, and thereby to better assess the threat to human health following exposure to certain
hygroscopic, sulf ate pollutant aerosols.  •

Atmospheric sulfate aerosols [HaSQj, (NHOz SO4, and NH» HSO4] are of international concern because
of their global prevalence and potential irritant or tone effects on humans.  To assess hazards 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
         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 HjSO^ (NH4>2 SO4, and NH* 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

                             Vemon E. Steele and Julia T. Arnold

                                  Northrop Services, Inc.

A method was developed to isolate and maintain in long-term culture nasal epithelial cells from rats, rab-
bits and humans.  The method was developed as part of the extrapolation program of the Inhalation
Toxicology Division of HERL.  With this technique,  it is possible to make direct in vitro dose-response
comparisons of the effects of chemicals in man and animals. By linking in vitro and in vivo effects in
animals and cross-correlating to human in vitro effects, extrapolation becomes more possible, especially for
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 chemicals that cannot be tested in man for ethical reasons and chemicals that.deposit predominantly in the
 nasal region, e.g. coarse mode particles, formaldehyde.

 Nasal turbinate epithelial cells were isolated from rats, rabbits, and humans using either a surgical or an
 in situ enzyme incubation 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 20 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: Graham
            A NONPARAMETRIC VERSION of WILLIAMS'TEST for a
                           RANDOMIZED BLOCK DESIGN                      .

                                      Dennis E. House                             s

                         HERL.USEPA, Research Triangle Park, NC               '

The taxicity or other effects of various substances can be assessed by either in vitro or in vivo experi-
ments which have increasing doses of the  substance,  including a control or zero dose,  as the major factor
in the design. Often, the various doses  are given  to me same subject, animal or batch of cells'. One
method of statistical analysis of these experiments is the Williams' test which assumes a normal distribu-
tion of errors and equal variances in the various groups.  However, in  most HERL studies these assump-
tions are invalid which makes die Williams' test invalid.  In this paper, a new method of statistical analysis
is derived that is valid for the above described experiments when the normality and equal variance assump-
tions are not met.

A large-sample nonparametric 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,lE.Stallard,lM. Woodbury,1 J.P. Creason,2 andW. B. Riggm2

                                    *Duke University
                        2HERL, USEPA, Research Triangle Park, NC
                                                       t -
The contributing role of gases, paniculate, and other environmental exposures to disease and death is criti-
cally important.- Yet, the level of exposure changes over tone. Past models have not allowed for this change
during the period covered in the study.  This 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 mortality data. The results for the younger male cohort sug-
gests a leveling off and probable decline in male htng cancer mortality rate in the near future. The ability to
include changes over time in risk factors is essential in evaluating the effect of environmental pollutants on
morbidity or mortality. The inclusion of cohorts in the analysis may identify the direction of a change
before it is measurable in the total population. This is especially important in modeling the health effects
(cancer and other chronic diseases) from exposure over time to environmental pollutants including toxic
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
hazard 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 changing exposures. The model is fitted to U£. lung
cancer mortality data where for recent male cohorts there appears to be changing 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, February 1986.

A119 MS-85-204 Project Officer: Riggan
        LIMITED OCCUPATIONAL MORTALITY DATA and STATISTICS

                    Michael/. Symons, Donald L. Doerfler, and Yang C Yuan

                      University of North Carolina School of Pubic Health
                                 / .
The general problem of the appropriate statistical analysis model for interpretation of the mortality or mor-
bidity experience of a population cohort, with a common occupational or environmental characteristic is
considered.  Choice of an appropriate control group is discussed.  Advantages and disadvantages of the
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  A MULTTVARIATE ASSESSMENT of METEOROLOGICAL INFLUENCES on
                   INHALABLE PARTICLE SOURCE IMPACTS

                          George D. Thurston1 and John D, Spengfe?

                                  *New York University
                         2Harvard University School of Public Health

The article identifies the sources of fine and coarse inhalable particles at a site in Boston and investigates
their respective relationships with the weather, using both local surface observations and information
regarding the movement of large air masses. One of four proposed criteria is selected for use with these
specific types of pollutants and weather data. The results should allow EPA researchers to refine measure-
ment techniques and projections of short-term changes in inhalable particle 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 Scree test of eigenvalues is found to result in the most interpretable com-
ponents for the specific air pollution and meteorological data sets considered in this work.

CITATION: Journal of Climate and Applied Meteorology 24(\\\. 1246-1256, November 1985.

A119 XX-86-116 Project Officer: Hayes
           ESTIMATION of INDIVIDUAL OZONE EXPOSURES USING
                    MICROENVIRONMENT MEASUREMENTS

                  Charles F. Contant, Jr., Brenda M. Gehan, Thomas H. Stock,
                          Alfonso H. Holguin, and Patricia A. Buffler

                          University of Texas Health Science Center

The ability of a computer model to estimate personal exposure to ozone given information on ozone con-
centrations in three different environments and data on subjects' movements through these environments
was tested.  The model performed well though one-hour estimates were 7-12 ppb below the observed per-
sonal exposures. Sources of error 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 ranch 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
 mode! 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 Photochemical Oxidants Standards, H. England, 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. Penis, Jr., and John D. Spengler

                          Harvard University School of Public Health

 Problems in estimating human exposure to ambient air pollutants are discussed.   The infeasibility of
 measuring actual exposure levels is described in detail.  Specific problems include  time spent indoors,
 variety of particle sizes and chemical variation among particles. Conclusions point to the need to develop
 lightweight, accurate, and reliable personal monitors. Findings emphasis the need and difficulty of control-
 ling for indoor exposures when studying the health effects of criteria pollutants.

 Problems associated with estimation of human 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

                                      /. F. Goldstein

                         School of Public Health, Columbia University

Different 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 exposure and health respon-
ses.  These technologic advances together with rapid  developments in the field of biological markers
promise to close the gaps in our ability to draw firm 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: IK Proceedings of the 79th Annual Meeting Air Pollution Control Association, Minneapolis,
            MN, June 1986.

A101MS-86-107 Project Officer: Hayes
   EFFECTS of AMBIENT SULFUR OXIDES and SUSPENDED PARTICLES on
           RESPIRATORY HEALTH of PREADOLESCENT CHILDREN


                    /. H. Ware, B. G. Ferris, /r, D. W. Dockery, J. D. Spengler,
                               D.O. Strom, and F.E.Speizer

                          Harvard University School of Public Health

Across six cities, frequency of cough was significantly associated with levels of three different air pollutants.
However, within each city no exposure/disease relationships were seen. Results suggest for preadolescent
children no reduction in pulmonary health from moderately elevated levels of air pollutants under study.
Though the preliminary results reported here provide no evidence for revision of existing NAAQS ambient
standards for SOX or suspended particles, attention will be paid to future reports from the Six-Cities Study
when such revisions are 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 examination and a parent completed a standard questionnaire.  Of this cohort,

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 8^80 children were seen for a second examination one year later. An air pollution monitoring program
 was begun in each community at about the time of the first examination. For this report, measurements
 of Total Suspended Particulates (TSP), the sulfate fraction of TSP (TSO-O, and sulfur dioxide (SCfe) con-
 centrations at study-affiliated outdoor stations were combined with measurements at other public and
 private monitoring sites to create a record of TSP and SO2 concentrations b each of 9 air pollution
 regions during the one-year period preceding each examination and, for TSP, during each child's lifetime
 up to the time of testing. Follow-up of these children is continuing, and analyses of the longitudinal health
 data along with the more extensive aerometric data collected b later years will aid in the interpretation
 of these results.

 CITATION: American Review of Respiratory Disease 133(5): 834-842, May 1986.

 A101XX-86-123 Project Officer: Hayes
   PERSISTENCE of PEAK FLOW DECREMENT in CHILDREN FOLLOWING
           OZONE EXPOSURES EXCEEDING the NATIONAL AMBIENT
                              AIR QUALITY STANDARD

                          P.J.Lioy,T.A.Vollmuth,andM.Lippmann

                             New York University Medical Center

Studying children exposed to ozone (03) while resident at a summer camp near Mendham, New Jersey, re-
searchers found a general tendency for decreased lung function with increasing Oa exposure.  The present
reanafysis of the Mendham data suggest a persistent decrement in lung/unction lasting for as much as a
week after foe end of a four day smog period.  Observed effects were small but statistically significant
though biological implications are not known. Though it is impossible to determine whether observed ef-
fects were caused by 03 or acid aerosol, studies now underway may help resolve this question. However,
when changes in children's king/unction such as those reported here and elsewhere are reported at or below
levels of the current NAAQS, then the 03 standards may need to be lowered,

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 Oa 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 mainstream cigarette smoke or airborne chemicals from oc-
cupational or hobby activities. Such exposures could mask or confound the effects of the ambient air pol-
lutant exposures.

CITATION: Journal of the Air Pollution Control Association 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

                  Robert S. Chapman,1 Beatrice J. Setwyn,2 Thomas H. Stock,2
                            Robert/. Hardy,2 Frances A. Chan,2
                         Daniel E. Jenkins,  and Dennis 3. Kotchmar3

                        1HERL, USEPA, Research Triangle Park, NC
                         2University of Texas School of Public Health
                        ^CAO, USEPA, Research Triangle Park, NC

This study was designed to determine the degree of association between day-to-day changes in ambient
ozone exposure and corresponding changes in lungfitnction of adults after vigorous outdoor exercise. Thus,
this study provided a test in the ambient situation of research previously conducted mainly in the laboratory.
It therefore provided information relevant to the ozone standard-setting process.

The purpose of the analysis was to examine the association of changes in ambient ozone concentrations
with changes in the pulmonary function (PFT) 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

                              /. F. Goldstein and L. R. Andrews

                        School of Public Health, Columbia University

Findings on continuous exposure to NO2 by persons cooking a meal on a gas stove are presented. Also
reported are peak NO2 levels at different heights above the floor and at various distances from the stove
white range and oven are in use.  The possible use of continuous NOi monitoring in a study of health ef-
fects from short term exposure to high levels ofNOz among asthmatics and non-asthmatics is described.
Use of methods described here in future "natural experiments'" of NO2 exposure 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 exposure to criteria pollutants.
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 Findings on continuous exposure to nitrogen dioxide by persons cooking a meal on a gas stove are
 presented. In addition peak levels of NO2 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 ranging from 0.20 ppm
 to over 1.5 ppm on pulmonary function and respiratory symptoms in asthmatic and non-asthmatic 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 administered throughout the
 study period to assess both health effects, and changes in pulmonary function associated with the above
 exposures.

 CITATION: la: Proceedings of the 79th Annual Meeting Air Pollution Control Association, Minneapolis,
             MN, June 1986.

 A101MS-86-108 Project Officer: Hayes
   RESPIRATORY SYMPTOMS and PEAK FLOW ASSOCIATED with INDOOR
              and OUTDOOR AIR POLLUTANTS in the SOUTHWEST

                          Michael D. Lebowitz,1 Catharine J. Holberg,1
                               Batbara Bayer, * and Carl Hayes2

                          University of Arizona Health Sciences Center
                         2HERL, USEPA, Research Triangle Park, NC

Daily symptom diaries and peak flows of expelled 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 particulate levels; indoor CO was correlated with gas stove use. Indoor and out-
door factors affected asthmatic symptoms, after controlling for age, sex, smoking and other ambient en-
vironmental variables. Findings underscore the importance of limits on ambient 03 and NOz

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 bouses was performed for Oa ,TSP, RSP, CO, temperature (T), and relative humidity (RH).
Questionnaires determined type of stove and number of smokers in all households. Ambient pollutants
(Oa, TSP, CO, NOz), were monitored in or near the dusters, 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 (PEF) associated with outdoor 03 after
adjusting for other factors. Asthmatics' PEF was associated with smoking, gas stove use and outdoor
NO2, and with outdoor Os 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 D. Spender, Margaret P. Reed, Erik Lebret, Bei-Hung Chang
                   James H. Ware, Frank E. Speizer, and Benjamin G. Ferris, Jr.

                          Harvard University School of Public Health

Researchers describe subject sampling, pollution monitoring and health surveillance techniques employed
in a study of indoor air pollution.  Preliminary findings from the first city under investigation are reported.
Design modifications for subsequent study sites are discussed also.  The paper illustrates the need for and.
complexities of control for confounding the role of indoor exposures to cigarette smoke, gas stove use and
similar exposures in studies of ambient pollutants.

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/welhiess 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 NO2 and particle concentra-
tions within these strata the gas  stove and  smoker strata were oversampled. This paper reports the
preliminary findings 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 Meeting Air Pollution Control Association, Minneapolis,
            MN, June 22-27,1986.

A101XX-86-112 Project Officer: Hayes
           EFFECTS of PASSIVE SMOKING on HEALTH of CHILDREN

                  Benjamin G. Ferris, Jr.,1 James H. Ware,1 Catherine S. Berkey,2
                   Douglas W. Dockery,1 Avron Spin, ///,  and Frank E. Speizer1

                           Harvard University School of Medicine
                          2Harvard University School of Public Health

Data regarding the effect of passive smoking on preadolescent children demonstrate an exposure-response
relationship between the number of smokers in the household and reported rates of a variety of lung ill-
nesses and symptoms. Amount of air that children could expel in one second (FEV\) and rate of growth in
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 ¥EV\, were lower in children with smoking mothers, and these children were shorter (Han children of non-
 smoking 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. Similarly, 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. FEVi 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, remains 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 significance remains 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 GROWTH in
                           PREADOLESCENT CHILDREN

                     Catherine S. Berkey, James H. Ware, Douglas W. Dockery,
                          Benjamin G. Ferris, Jr., and Frank E. Speizer

                           Havard University School of Public Health

 Using 7,834 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 differences in growth were due to current.ex-
posures or to an effect of prenatal or early childhood exposure.  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 National Ambient Air Quality Standard (NAAQS) for NC>2 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 (p< 0.0001), and FEVi growth rates approximately 0.17% per year lower
(p=6.05). 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 ml/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 magnitude of the effect
on early childhood exposure on the course of development. The magnitude of the effect on FEV, 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 small 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.

A101XX-86-113 Project Officer: Hayes
     INDOOR AIR SAMPLING and MUTAGENICITY STUDIES RELATED to
               EMISSIONS from UNVENTED COAL COMBUSTION

                          /. L. Mumford,1 K Williams,1 D. B. Harris,2
                                J.CChuangfandM.Cooke3

                        ^ERL, USEPA, Research Triangle Park, NC
                        2AEERL, USEPA, Research Triangle Park, NC
                               %attelle-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 organic extrac-
table matter and mutagenicity of the filter samples collected by both med-vol and hi-vol were comparable.
For the med-vol sampling most mutagenicity and the biologically active compounds ofpofycyclic 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 onty in China Project but also used in the Integrated Air
Cancer Project and IndoorAir 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 /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 PMio size selective
inlet was used for comparison with the medium-volume sampler. The percent organic extractable and
polycyclic aromatic hydrocarbon (PAH) 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 smaller 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 PAHs, 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: la: Proceedings of the 79th Air Pollution Control Association, Minneapolis, MN, June 1986.

 A119 MS-86-190  Project Officer: Mumford
       SELECTED ORGANIC POLLUTANT EMISSIONS from UNVENTED
                                KEROSENE HEATERS

                          G. W. Traynor,lM. G.Apte,lH.A. Sokol,1
                              J. CChuang,2 and J.L. Mumford*

                              1Lawrence Berkley Laboratories
                               Battelle-Columbus Laboratories
                        'HERL, USEPA, Research Triangle Park, NC

Tliis study confirms 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; phtalates; alkyl benzenes; and pentachlorophenol.
Additional studies are needed to evaluate the health effects of the emissions 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-m3 chamber with a prescribed on/off
pattern. Organic compounds were collected on teflon-impregnated glass filters backed by XAD-2 resb
and analyzed by gas chromatography/mass spectrometry. Pollutant source strengths were calculated
using a mass-balance equation. The results show that kerosene heaters can emit polycyclic aromatic
hydrocarbons (PAHs), nitrated PAHs;  alkyl benzenes;  pentachlorophenol; phtalates; hydro naph-
thalenes; aliphatic hydrocarbons, alcohols, and ketones; and other organic compounds.

CITATION: In: Proceedings of the 79th Air Pollution Control Association, Minneapolis, MN, June 1986.

A101MS-86-191 Project Officer: Lewtas
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          THE EFFECTS of INHALATION of ORGANIC CHEMICAL AIR
                                   CONTAMINANTS
                        on MURINE LUNG HOST DEFENSES


          Catherine-Army,1 William J. O'Shea,1 Judith A. Graham,2 and Frederick/. Millet*

                                   hlT Research Institute
                         2HERL, USEPA, Research Triangle Park, NC

The potential health hazards of exposure to threshold limit value (TLV) concentrations of acetaldehyde,
acmlein, propylene oxide, chloroform, methyl chloroform, carbom tetrachloride, alfyl chloride, methylene
chloride,  ethylene trichloride, perchloroethylene,  benzene, phenol, monochlorobenzene, and benzyl
chloride, were investigated by single and multiple 3-hr inhalation exposures in mice to experimentally in-
duced  streptococcus  aerosol .infection and pulmonary  bactericidal activity  to inhaled  Klebsiella
pneumoniae.  Significant increases in susceptibility to respiratory streptococcus infection were observed
after a single 3-hr  exposure to methylene chloride,  perchloroethylene, and ethylene trichloride.  For
methylene chloride and perchloroethylene, these exposure conditions also resulted in significantly decreased
pulmonary bactericidal activity. These effects implicate several toxic air pollutants, as immunotoxic, 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  tetrachloride,  ally! chloride,
methylene chloride, ethylene trichloride, perchloroethylene, benzene, phenol, monochlorobenzene, and
benzyl chloride, compounds which may be present in the ambient or work room atmosphere were inves-
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  Klebsiella 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-hr exposure to TLV concentrations of methylene chloride, perchloroethylene, and
ethylene trichloride.: For methylene chloride and perchloroethylene, these exposure conditions also
resulted in significantly decreased pulmonary bactericidal activity.

CITATION: Fundamental and Applied Toxicology 6:713-720,1986.

A101MS-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. Stead, 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.PA.  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 hmg epithelium.  No simple relationship was observed between the body weights of the
 animals and the concentration-response curves 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 (COCb) in dif-
 ferent species of laboratory animals was made in order to better understand the influence of the choice
 of species in inhalation toricity studies. The effect of 4-h exposures to ozone at concentrations of 0.2,0.5,
 1.0, and 2.0 ppm, and to COCfe 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 Os- and COCb -induced pulmonary edema. All species had similar basal levels of LFP (250-
 350 iig/ml) 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 Oa and above, while rabbits responded
 only at 2.0 ppm Oa.  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 Oj but not COCh. Results of this study
 indicate that significant species differences are seen in the response to low levels of Oa and COCh. These
 differences do not appear to be related in a simple manner to body weight.

 CITATION: Journal of Toxicology and  Environmental Health 19:43-53,1986.

 A101MS-85-060 Project Officer: Hatch
   PULMONARY BIOCHEMICAL EFFECTS of INHALED PHOSGENE in RATS


                             Serafino Franch1 and Gary E. Hatch2

                              1North Carolina State University
                        2HERL, 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 (COCfe).  Rats were sacrificed (1)  immediately or
throughout a 38-d recovery period after inhalation of 1 ppm COCh for 4-h, (2) at intervals during a 7-h
exposure to 1 ppm phosgene, or (3) at several time points throughout a 17-d exposure to 0.125 and 0.25
ppm COCfe (4 h/d, 5 d/wk) and during a 21-d recovery period. Regimen 1 revealed significantly elevated
lung wet weight, lung nonprotein sulfhydryl (NPSH) content, and glucose-6-phosphate dehydrogenase
(G6PD) activity that stayed elevated for up to 14-d.  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 animals showed sustained elevations in lung wet weight, NPSH content,
and G6PD activity after 7-d of exposure. No significant changes in these endpoints were observed for the
0.12S ppm COCk group. No consistent elevation in hydroxyproline content was seen at either exposure
concentration.  Light microscopic examination of lung tissue exposed to 0.25 ppm COClj for  17-d
revealed moderate multifocal accumulation of mononuclear cells in the centriacinar region. In summary,
exposure to COCfc caused changes similar in most ways to those observed for other lower-respiratory-
tract irritants.

CITATION: Journal of Toxicology  and Environmental Health 19:413-423,1986.

A101MS-86-067 Project Officer: Hatch
    SUPERIOR COLLICULUS LESIONS and FLASH EVOKED POTENTIALS
                                 FROM RAT CORTEX

                             Martha I. Barnes and Robert 5. Dyer

                         HERL, USEPA, Research Triangle Park, NC

To  understand Julfy the  biological significance of changes in neurophysiological endpoints, the
Neurotoxicology Division of HERL must know the extent to which different brain regions contribute to
responses recorded from the scalp or cortex.  This paper indicates the effect of lesions in the superior col-
liculus,partofthe visual system on responses recorded from the visual cortex to a flash 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 by way 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).  FEP waveforms were obtained from all
animals prior to treatment, and 2 and 5 days 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 FEP 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.

 A101MS-86-010 Project Officer: Dyer
       TOXICITY of PARTICLES EMITTED from COMBUSTION of WASTE
                                   CRANKCASE OIL:
                           IN VITRO and IN VIVO STUDIES

                    /. L. Mumford,1 G. E. Hatch,1 R. E. Hall,2 M. A. Jackson,2
                               R. G. Merrill, Jr.,3 and J. Lewtas1

                         1HERL, USEPA, Research Triangle Park, NC
                       Environmental Health Research and Testing, Inc.
                        3AEERL, USEPA, Research Triangle Park, NC

 This paper reports that the emission particles from automotive waste crankcase oil combustion contained
 high levels of heavy metals, particularly Fb, and are highly toxic in in vitro and in vivo assays.  These emis-
 sion particles are comparable in overall potency to the most toxic 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 OSW and 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 hazards 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 atomizing 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 emission particles, VOB emission 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
 jig/mouse, whereas a similar dose of VOB ash produced significant elevations in mortality (61.1%).
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Emission 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: Fundamental and Applied Toxicology 7(1): 49-57, July 1986.

A101MS-86-Q26 Project Officer: Mumford
             S9-DEPENDENT ACTIVATION of 1-NITROPYRENE and
                             3-NITROFLUORANTHENE
                     in BACTERIAL MUTAGENICITY ASSAYS

                     L. M. Ball,11C Williams,2 M. /. Kohan,2 and/. Lewtas2

                         University of North Carolina at Chapel Hill
                        2HERL,USEPA, Research Triangle Park, NC

This paper provides relevant information on the mutagenicity and metabolism of two of the most impor-
tant NOtfAHs we have identified in ambient air and source emissions, 1-Nitropyrene and 3-Nitrofluoran-
thene.

Nitro-substituted polycyclic aromatic hydrocarbons (NOaPAH) 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 mammalian drug-metabolizing enzymes (S9 fraction) decreases
their activity in this system. In contrast, in an assay of forward mutation (to 8-azaguanine resistance) with
Salmonella typhimurium strain TM677, without S9 NFA has relatively low activity (10 to 20 mutants per
ICr survivors at 5 tig/ml) and NP only marginally doubles the background mutation rate. Addition of S9
protein enhances NP at SO tig/ml and 80 to 100 x 10"5 for NFA at 5 to 10 tig/ml. Therefore generation of
active genotoxic 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: Proceeding of the Third International Conference on Biological Reactive Intermediates,
            Pleason Press, New York, NY, 1985.

A101MS-86-035 Project Officer: Lewtas
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    METABOLISM of 1-NITROPYRENE by CULTURED RABBIT ALVEOLAR
              MACROPHAGES and RESPIRATORY TRACT TISSUES

                        Leon C. King,l Louise M. Ball,1 Marcus Jackson,2
                              Jeff P. fnmon,1 andJoellen Lewtas1

                         1HERL, USEPA, Research Triangle Park, NC
                       Environmental Health Research and Testing, Inc.

Pofycyclic organic matter (POM) associated with  combustion emissions and urban air particles is car-
cinogenic and mutagenic and is thought to account for a significant portion of the lung cancers attributable
to air pollutions.  The research reported in these manuscripts provided data critical to elucidating the
trachea as a target organ for  carcinogenesis by combustion particle emissions.The DNA binding data
provides important target cell dosimetry data for nitrated pofycyclic aromatic hydrocarbons, important con-
stituents of both products of incomplete combustion (PICs) and pofycyclic organic matter (POAfs) for
which die Office of Air Quality  Planning and Standards (OAQPS) and OMSAPG need assessment data.

The metabolism of l-nitro[MC]pyrene(14C-l-NP; 8.1 uM) was studied in cultured (20 hr) rabbit alveolar
macrophages, lung tissue, and trachea! tissue. Metabolites from the incubation medium and from the
macrophages and respiratory 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:  l-mtropyrene-4,5-dihydrodioI, N-acetyl-1-aminopyrene, 1-aminopyrene, and  10-
hydroxy-1-nitropyrene. Nitropyrene phenols (4-, 5-, 6-, 8- or 9-hydroxy-l-nitropyrene) and 3-hydroxy-l-
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 10-hydroxy-l-nitropyrene were detected in the macro-
phage incubation medium, and only minute quantities of lmitropyrene-4,5-dihydrodiol, 1-aminopyrene,
and 10-hydroxy-l-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  C, respective-
ly) than in the alveolar macrophages (6.3% of the recovered 14C). 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 Pharmacology 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. Mumford,1 Silvestre B. Tejada,2 Marcus Jackson? and Joellen Lewtas1

                        1HERL, USEPA, Research Triangle Park, NC
                        2ASRL, USEPA, Research Triangle Paark, NC
                       ^vironmental Health Research and Testing, Inc.

This paper is relevant to programmatic efforts (OHEA 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 l-nitropyrene vapor-coated fly ash as a model for such dosimetry and bioavailability studies.  In this
case, the mutagenic l-nitropyrene after being coated onto the particles was bioavailable in the culture
medium.                           -             .             ,

Alveolar macrophage cultures exposed to coal fly ash vapor-coated with l-nitropyrene were used as a
model system to study the bioavailability and the uptake of a nitroaromatic hydrocarbon from coal com-
bustion emissions.  Initially, 1-nitropyrene-coated fly ash and uncoated fly ash were examined for
cytotoxicity using rabbit alveolar macrophages and for mutagenicity in the Salmonella typhimurium plate
incorporation assay. .The results were compared to determine the effects of vapor deposition. The dis-
tribution and recovery of l-nitropyrene from macrophage cultures treated with coated fly ash were deter-
mined by using a reverse-phase high-performance liquid chromatography-fluorescence method. 1-
Nitropyrene  alone was not very toxic, nor did vapor deposition of l-nitropyrene onto coal fly ash sig-
nificantly affect the toritity of the fly ash. Most toxitity resulted from the original, 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 l-nitropyrene loss
was 2.7 ng/l(r macrophages for medium and 4.1 ng/106 macrophages for the whole culture.  The
mutagenic 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[14C]PYRENE TO DNA

                                 L. M. Ball1 and/. Lewtas2

                          ^University of North Carolina-Chapel Hill
                        2HERL, USEPA, Research Triangle Park, NC

This paper provides insight on the mechanism of metabolic activation of a nitrated pofycfycic aromatic
hydrocarbon  (NOz-PAH). In order to assess the potential icodcity and carcinogenkity of NOt-PAH's
found in ambient air, the Office of Health and Environmental Assessment (OHEA) and the Office of Air
and Radiation/Office of Air Quality Planning and Standards (OAR/OAQPS) need bask information on
the metabolism and dosimetry of these compounds in order to determine the DNA dosimetry resulting from
human exposure.

The recently characterized environmental mutagen and potential carcinogen l-nitropyrene(NP) is known
to bind DNAiaSalmonella typhimurium, 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 14C 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/ing 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 Na, 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-methylcholanthrene (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 N2 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 P-450 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,l L. C. King,2 L. M. Ball,3 S. Ghayourmanesh4
                                A. M. Jeffrey,4 and J.Lewtas2

                         1Environmental Health Research and Testing
                        2HERL, USEPA, Research Triangle Park, NC
                          ^University of North Carolina-Chapel Hill
                         4Columbia University Health Science Center

Pofycyclic organic matter (POM) associated with combustion emissions and urban air panicles is car-
cinogenic and mutagenic and is thought to account for a significant portion of the lung cancers attributable
to air pollutions. The research reported in these manuscripts provided data critical to elucidating the trachea
as a target organ for carcinogenesis by combustion particle emissions.  The DNA binding data provides im-
portant target cell dosimetry data for nitrated pofycyclic aromatic hydrocarbons, important constituents of
both products of incomplete combustion (PICs) and pofycyclic organic matter (POMs) for which the Office
of Air Quality Planning and Standards(OAQPS) and the Office of Mobile  Source Air Pollution Con-
trol(OMSAPC) need assessment data.

Binding of 1-nitro (14C)pyrene (NP) or its metabolites to cellular DNA and protein in cultures of rabbit
alveolar macrophages, lung tissue, and trachea! tissue was examined.  DNA binding in trachea! tissue (136
 ± 18.3 pmole NP/mg  DNA) was four to five times the levels measured in either lung tissue (38 ±9.4
pmole NP/mg DNA) or macrophages (26 ± 7.5 pmole NP/mg DNA). Adduct analysis of DNA isolated
from lung tissue incubated with l-nitrofH^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 tis-
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 14C-NP (380 ppm, 0.085 u,Ci/mg) 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 14C
bound to protein and no detectable levels of 14C 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
                       CIGARETTE SMOKE CONDENSATE


                    R. Williams,1 C. Sparacino,2B. Petersen,3J. Bumgamer*
                               R. H. lungers,4 and /. Lewtas 5

                      Environmental Health Research and Testing, Inc.
                               2Research Triangle Institute
                             •hiattelle Columbus Laboratories
                        4EMSL, USEPA, Research Triangle Park, NC
                        5HERL, USEPA, Research Triangle Park, NC

In order to develop strategies for regulating carcinogenic pofycyclic organic matter (POM) in the ambient
air, 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.  Capillary GC/MS 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 Environmental Analytical Chemistry 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 Mania G. Nishioka,2 and Bruce A. Petersen3

                       ^ERL, USEPA, Research Triangle Park, NC
                               Battelle Columbus Division
                                     3Sciex, 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, mammalian 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: Proceeding? of the Technology Transfer Conference No. 6, Toronto, Ontario, Canada,
            December 1985.

A101MS-86-042 Project Officer: Lewtas
 SURVIVAL CURVES and INCIDENCE of NEOPLASTIC and NON-NEOPLASTIC
                             DISEASE in SENCAR MICE

              CJ. Con^N. Clappt2AJ.P. Klein-Szanto,1 S. Nesnow,3 and T. j. Slaga1

                        lThe University of Texas System Cancer Center
                             2Oak Ridge Associated Universities
                        ^ERL, USEPA, Research Triangle Park, NC

 The SENCAR mouse has been used by HERL to develop an extensive data base on the tumorigenic
properties 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 byEPA's Office
of Air Quality Planning and Standards (OAQPS). This paper describes background studies on spon-
taneous tumor formation and survival of this sensitive strain of mice which will provide invaluable informa-
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 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: Carcinogenesis 6(11): 1649-1652, October 1985.

A101/L104 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
                                                                                   i
                                    Joellen Lewtas

                        HERL, USEPA, Research Triangle Park, NC

 This paper describes a new comparative approach to cancer risk assessment of complex mixtures which has
 been under development in the Office of Research and Development(ORD). The risk assessment process
 is critical to all of the EPA program offices. Since the risk assessment process and the current guidelines
 will be updated, it is important 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, animal tumorigenicdty 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: Toxicology and Industrial Health 1(4): 193-203, December 1985.

 A109MS-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 complex combustion
 emissions. It is highly relevant to the Office of Air and Radiation/Office of Air Quality 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 containing carcinogenic polycyclic organic compounds which are
 mutagenic in short-term genetic bioassays in microbial and mammalian 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 human cancer risk data for all the
various possible exposures to combustion emissions. Furthermore it is impossible to collect human can-
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car 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 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 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.

A109MS-85-249 Project Officer: Lewtas
  A STANDARD GUIDE for the SALMONELLA TYPHIMURIUM/MAMMALIAN
  MICROSOME TESTS for BACTERIAL MUTAGENICITY [ASTM STANDARD]

                    L. D. Oaxton,lK. E. Mortelmans,2 J.Allen? A. Auletta,4
                          E. Nestmann,5 E. Zeiger,6 andJ. McCann 7

                        aHERL, USEPA, Research Triangle Park, NC
                                    viRI International
                                 American Cyanamid Co.
                              'h'EB, USEPA, Washington, DC
                     Environmental Health Directorate, Ottawa, Canada
                     National Institute of Environmental Health Sciences
                              Lawrence Berkeley Laboratories

The Ames test is often used by and requested by most program offices. Its proper use and interpretation,
therefore, is of major importance.  This standard guide  provides a definitive guide for analyzers and
reviewers ofS, typhimurium mutagenicity data as well as for researchers who perform the test.

Since its development by Dr. Bruce Ames and his coworkers, the Salmonella typ/umurium/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 E47.09.01) and because of new developments
within the field of microbial mutagenesis tests, but it is not blended 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.

A101MS-86-220 Project Officer: Claxton
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      BIOASSAY DIRECTED CHEMICAL ANALYSIS in ENVIRONMENTAL
                                      RESEARCH


                            Dennis Schuetzle1 and JoellenLewtas2

                               ^ORD MOTOR COMPANY
                        2HERL, 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 Otis feature journal article describing the principles, anafytical 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 Chemistry 58:1060a, 1986.

 A101MS-86-199 Project Officer: Lewtas
             THE MUTAGENIC ACTIVITY of IRRADIATED C2H4/NO*
          MIXTURES in the PRESENCE of DIETHYLHYDROXYLAMINE

                T. E. F3eindienst\ E. O. Edney\ G. R. Namie1, and L. D. Claxton2

                                  Northrop Services, Inc.
                        2HERL, USEPA, Research Triangle Park, NC

In order to understand the source ofgenotojdcants within ambient air it is necessary to understand the role
of atmospheric transformation processes. By using the Salmonella mutagenicity bioassay it is possible to
monitor the creation and distntction of genotcadcants within irradiated ambient-like air.  This research will
aid decision makers such as EPA's Office of Air Quality Planning and Standards to understand the source
and relevance of hydrocarbon species within ambient air.
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Ethylene/oxides of nitrogen mixtures were  irradiated  in  the  absence and  presence of diethyl-
hydroxylamine. As previously reported, the presence of diethylhydroxylamine inhibited the photooxida-
tion 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 diethylhydroxylamine.  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
Salmonella typhimurium strain TA100. A significantly greater mutagenic activity was observed in the ir-
radiated ethylene/diethylhydroxylamine/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-
tion products.       .                  -                  '

CITATION: Atmospheric Environment 20(5): 971-978, May 1986.

A109 MS-85-192 Project Officer: Claxton
             SYNTHESIS and BIOLOGICAL ACTIVITY of a SERIES of
                NITROSUBSTITUTED CYCLOPENTA-FUSED PAH

                       /. M. Coloring, L. M. Ball, R. Sangaiah, and A. Gold

                      University of North Carolina School of Public Health

This paper reports the synthesis and mutagenicity of a series of novel nitratedpofycydie aromatic hydrocar-
bons, the cyclopenta-fused PAH nitrated on the eltheno bridge.  The nitroPAH in general are potent bac-
terial mutagens which may contribute substantially to the genotaxicity of ambient air paniculate matter.
The studies described here indicate that this particular group of nitroPAH are  relatively inactive as
mutagens in the Ames Salmonella 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
ultimately provide a rational basis for assessment of the genotaxicity of novel compounds.

PAH containing a peripherally fused cydopenta ring are genotosdcally active in Salmonella and mam-
malian cells and have been identified in combustion emissions. Since the cydopenta ring is predicted to
be susceptible to electrophilic attack, nitrosubstituted cydopenta-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 cydopenta (cd) pyrene and the cyclopenta-fused benzanthracene isomers were prepared by reac-
tion of the PAH 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 cydopenta ring in ac-
cord with expectation. Preliminary assay in 5. typhimurium indicates that 4-nitro-cycIopenta(cd)pyrene is
mutagenically active without exogenous metabolic activation in nitroreductase-competent strains. The
high resonance stability of the nitrenium ion formed from the putative hydroxylamine 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 nitroPAH.

 CITATION: In: Proceeding? of the Tenth International Symposium on Pofynuclear Aromatic Hydrocar-
            bons, W. M. Cooke & A. J. Dennis, eds., Battelle Press, Columbus, OH, October 1985.

 A101MS-86-211 Project Officer: Lewtas
     METABOLISM and ACTIVATION PATHWAYS of the ENVIRONMENTAL
                       MUTAGEN 3-NITROFLUORANTHENE

                       Louise M. Ball,1 MichaelJ. Rohan? Kay Williams?
                       M.G. ffishioka,3Avram Gold,l and Joellen Lewtas1

                           ^University of North Carolina-Chapel Hill
                         2HERL, USEPA, Research Triangle Park, NC
                               TSatteUe-CoIumbus Laboratories

 The results described in this symposium paper confirm that 3-nitrofluoranthene, 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 nitroreductase enzymes.  Preliminary identification of mammalian metabo-
 lites of 3-nitrofluoranthene is also reported. Such studies  will eventually allow characterization of the
 mutagenicity 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 polycyclic 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"1" 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-deficient strains of TA98 (TA98NR and TA98/1.8-DNP6), 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 proteio/hr). 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 Potynudear Aromatic Hydrocar-
            bons, edited by W.M. Cooke and A J. Dennis, Battelle Press, October 1985.

A101MS-86-127 Project Officer: Lewtas
    A RETROSPECTIVE REVIEW of the VALUE of SHORT-TERM GENETIC
     BIOASSAYS in PREDICTING the CHRONIC EFFECTS of DIESEL SOOT

                                  /. Lewtas and K, Williams

                         HERL, USEPA, Research Triangle Park, NC

This paper puts into perspective for all air program offices the utility of short-term bioassays.  This paper
brings into a much clearer focus that short-term mutagenicity bioassays are important in analysis proce-
dures because they are (1) capable of detecting and identifying carcinogenic material, (2) used in the iden-
tification of specific carcinogens within complex materials, (3) capable of demonstrating the bioavailabiluy
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) identifying NO2-PAHs 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 lexicological 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 "Taodcological Effects of Emissions from Diesel
            Engines", Tsukuba Science City, Japan, July 1986.

A101MS-86-217 Project Officer: Lewtas
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          EFFECTS of 60-HZ FIELDS on HUMAN HEALTH PARAMETERS

                       Mary R. Cook, Carl M. Month, and Harvey D. Cohen

                                  Midwest Research Institute

 The study reported here used quantitative exercise testing techniques to evaluate whether increases in meta-
 bolism, caused by moderate steady-state exercise prior to exposure to real or sham fields, would clarify
 potential field effects.: The findings indicated that the physical recovery 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 interbeat interval (IBI) (equivalent to a 3 beat/min decrease in heart
 rate) was found when subjects were exposed to the real field for 2 hr after sitting quietly prior to exposure.
 This replicates earlier research, in which IBI was increased following a longer period of field exposure. The
 results suggest 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 examined, only heart rate
 (cardiac interbeat interval) was altered by 2 hr of field exposure. A small, statistically significant decrease
 hi heart rate (3 beats/min) 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 human arousal and physiological activation.

 CITATION: U.S. Environmental Protection Agency, Final Report of Cooperative Agreement No. CR-
            812805, EPA-600/1-86-006, September 1986.

 F104 HERI^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. R. Rabinowitz, D. E. House,
                                      andW. T.Joines

                         HERL, USEPA, Research Triangle Park, NC

This manuscript represents a major breakthrough for clues to the mechanism for a non-thermal process in
radiofrequency-radiation biological-effects work. As such, it has garnered favorable publicity for EPA's
scientific programs as well as demonstrated to the EPA radiation program office the correctness of caveats
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in the bioeffects Review Document and in the Notice for Proposed Recommendations. Scientists in other
laboratories an now adjusting their working hypotheses to include the new results. Basically, the finding is
that to cause biological change, various frequencies ofradiofrequency radiation must be co-mixed with one
of a number of specific intensities of the earth's magnetic field.  This requirement appears to explain some
of the problems encountered in previous attempts to replicate reports of non-thermal effects, while also
providing the first substantial information 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
(LGF) at a density of 38 microTesla (jiT), 15- and 45-Hz electromagnetic signals (40 Vp-p/m in air) have
been shown to induce a change 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 signal can be rendered ineffective when the LGF
is reduced to 19 jiT with Helmholtz coils. In addition, the ineffective 30-Hz signal becomes effective
when the LGF is changed to ±25.3uT or to ± 76^T. These results demonstrate that the net intensity of
the LGF is an important variable. The results appear to describe a resonance-like relationship in which
the frequency of the electromagnetic field that can induce a change in efflux is proportional to a product
of LGF 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 signals.  In future investigations of this phenomenon, the
LGF vector should be explicitly described. If the. underlying mechanism involves a general property of
tissue, then research conducted in the ambient electromagnetic environment (50/60 Hz) may be subjected
to unnoticed and uncontrolled influences, depending on the density of the LGF.

CITATION: Bioelectromagnetics 6(4): 327-337, December 1985.

F104 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 renews a number of experiments conducted over several years indicating parameters of exposure
 to electromagnetic energy such as the air temperature, duration of exposure, animal exposed, and orienta-
 tion of the animal in the field may quantitatively otter the observed effect.  These results suggest ambient
 conditions must be considered in developing standards for allowable exposure to electromagnetic radiation.
 These results also illustrate a general principle of the need for caution in the extrapolation of taadcological
 data obtained under a limited set of environmental conditions.

 Alteration in animal 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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 don were kept constant.  Operant response rates were reduced and response durations were increased
 after overnight irradiations with 10 or 15 mW/cm 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 min. 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 magnetic field vectors during irradia-
 tion with 15 mW/cm2 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: In: Biological Effects of Electropollution, in press.

 F104 MS-86-125 Project Officer: Gage
   A THREE-DIMENSIONAL FINITE-DIFFERENCE THERMOREGULATORY
                          MODEL of a SQUIRREL MONKEY

                              R. I. Spiegel1 and M. B. E. Fatmi2

                         1HERL> USEPA, Research Triangle Park, NC
                                  2Northrop Services, Inc.

 To achieve experimental verification of a thermoregulatory model, and minimize risk of human experimen-
 tation under radiofrequency exposure conditions, an acceptable approach is to model an animal whose
 thermoregulatory system is similar to man's and then perform the necessary thermophysiologkal experi-
 ments on the animals. The verification of such an animal model based on these measurements would fur-
 nish substantial support for a similar human model. This 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 obtained.  The goal of Ms research is to provide scien-
 tifically credible data, methodologies, and assessments of non-ionizing radiation (NIR) for the Office of
 Radiation Programs (ORP) to use in regulatory decisions to modify and extend 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, skin, 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: Int. 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 (ARANEUSDIADEMATUS)
                                               V:

                          C G. Liddle, J. P. Putnam, O. L. Lewter,
                        J. Y. Lewis, B. Bell, M. W. West, and A. Stead

                        HERL, USEPA, Research Triangle Park, NC

Microwave at low and moderate levels had been reported to affect the central nervous system of laboratory
animals. The ability of the spider to spin an orb web has been used to screen drug? 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 exposed to
the microwave field. This information will be used to assist at establishing microwave exposure guidelines
for the general population.

Eight cross spiders (Araneus diadematus) 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/cm2 (estimated SARs 40.4,
and 0.4 mW/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, January 1986.

F104 MS-83-003 Project Officer: Liddle
      CIRCULATING ANTIBODY RESPONSE of MICE EXPOSED to 9-GHz
                        PULSED MICROWAVE RADIATION

                          C G. Liddle, J. P. Putman, O. H. Lewter,
                                 M. West, and G. Morrow

                        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 die antibody response of immunized mice.  This study involved the exposure of immunized
max to a lower level of microwaves. There was no increase in the antibody response in these animals. This
information 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 Streptococcus pneumoniae type III were exposed to
9-GHz pulsed microwaves (pulse repetition rate 970-1,000, pulse width 1.0 jis, peak power 1 W/cm ) at
an average incident power density of 1 mW/cm2 (calculated SAR as 0.47 W/kg) for 2 h per day for 5 days.
Circulating antibody tilers 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-GHz pulsed microwaves at 1 mW/cm do not alter the immune response of
mice immunized against $, pneumoniae.

CITATION: Bioelectramagrtetics 7(1): 91-94, January 1986.

F104 MS-83-244 Project Officer: Liddle
      BEHAVIORAL EFFECTS of MICROWAVE RADIATION ABSORPTION

                             . /. C. Monahan1 andJ. A. D'Andrea2

                               1Food and Drug Administration
                                    2University of Utah

This publication brings together in a singe source the major research finding? related to the behavioral con-
sequences of microwave exposure. In addition it attempts to provide a critical assessment of this informa-
tion and to provide a perspective upon which the reader can interpret 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 hi the Soviet Union and then proceeds to examine 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 humans, dose considerations, and problems inherent in
this type of research.

CITATION: Department of Health and Human Services Publication, FDA 85-8238,1985.

F104 HERL-0523 Project Officer: Elder
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              BRAIN TEMPERATURE MEASUREMENTS in RATS:
       A COMPARISON of MICROWAVE and AMBIENT TEMPERATURE
                                    EXPOSURES

                  Thomas R. Ward,1 David J. Svendsgaard,1 Ranald J. Spiegel,1
                     Earl T. Puckett,2 Meiritt D. Long,1 and James B. Khar

                        1HERL, USEPA, Research Triangle Park, NC
                                 2Northrop Services, Inc.

In research into the bioeffects of non-ionizing radiation, a major problem is defining the relationship be-
tween tissue heating induced by exposure  to OK radiation and tissue heating induced by conventional
methods.  This study addressed Otis problem. The work was done in support of the Office of Radiation
Programs' effort to develop a data base on the bioeffects of radiofreauency radiation. The Office of Radia-
tion Programs is actively involved in determining guidelines for public exposure to radiofreauency 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 substantial evidence for temperature differentials, or "hot spots," in the brain
of these animals. In the first study, after a 30-min 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 profiles were measured in
rats exposed to either 30 mW/cm2 or 36.2°C. In this study, the 30-min exposure period was divided into
seven intervals and the change in temperature during each period was analyzed. Only the cortex showed
significantly different heating rates between the air heating and microwave heating; however, this dif-
ference disappeared after the initial 5 min of exposure.

CITATION:  Bioelectromagnetics 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

                   Maria A. Stuchfy,1 Ronald J. Spiegel,2 Stanislaw S. Stuchfy,3
                                 andAndnej Kraszewsld3

                   Radiation Protection Bureau, Health and Welfare Canada
                       2HERL» USEPA, Research Triangle Park, NC
                           University of Ottawa, Ottawa, Canada

 The dose rate, or the rate at which radiofreauency (RF) energy is imparted in the body, defined as the
 specific absorption rate (SAR), is used in quantifying biological effects and formulating standards on ex-
 posure to RF fields.  It is also recognized that the spatial distribution of the SAR within the exposed body
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plays an essential role.  In this paper, the calculations and measurements of the spatial distribution and
average SARsfor various body parts for a full-scale model of man exposed to the 350 MHz radiation are
presented. The goal of this research is to provide scientifically credible data, methodologies, and assess-
ments of non-ionizing radiation (NIR) for the Office of Radiation Programs (ORP) to use in regulatory
decisions to modify and extend 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 MTT-34(i): 26-31, January 1986.

F104 MS-84-130 Project Officer: Spiegel
      TEMPORAL RESPONSE of NEURONS to AMBIENT HEATING in the
        PREOPTIC and SERIAL AREA of the UN ANESTHETIZED RABBIT


                         ' Christopher J. Gordon and Elizabeth C White

                         HERL, USEPA, Research Triangle Park, 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 thermoregulatory 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.,
  1-1/e) 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 < 70s.
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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.

F104 MS-82-087 Project Officer: Gordon
    THE MEASUREMENT of SMALL MECHANICAL VIBRATIONS of BRAIN
          TISSUE EXPOSED to LOW-FREQUENCY ELECTRIC FIELDS

                      R. J. Spiegel. J. S. AH, /. F. Peoples, and W. T, Joines

                         HERL, USEPA, Research Triangle Park, NC

Radiofrequency fields can interact with biological tissue both electrically and mechanically.  This study in-
vestigated the mechanical interaction between brain tissue and an extremely low frequency (ELF) field by
measuring the resultant vibrational amplitude.  It was found that the applied field caused the brain to
vibrate at very smalt amplitudes. This small vibration might have some influence on the association of cal-
cium ions near the brain's surface, and consequently could play a role in calcium-ion efflux from brain tis-
sue exposed to ELF fields.  The goal of this research is to provide scientifically credible data, method-
ologies, and assessments of non-ionizing radiation (NIR) for the Office of Radiation Programs (ORP) to
use in regulatory decisions to modify and extend radiofrequency 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 Hz; between
50 and 200 Hz resonant phenomena were observed; and above 200 Hz the amplitude fall-off is rapid.

CITATION: Bioelectromagnetics 7(3): 295-306, July 1986.

F104 MS-85-210 Project Officer: Spiegel
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               OFF-CENTER SPHERICAL MODEL for DOSIMETRY
                     CALCULATIONS in CHICK BRAIN TISSUE

                   Guillermo Gonzalez,1 lames C. Nearing,1 Ronald J, Spiegel,2
                                   and William T. Joined

                                    1 University of Miami
                         2HERL,USEPA, Research Triangle Park, NC
 •tn                                                          '
In order to understand the effect of amplitude modulated (AM)  radiofrequency radiation on calcium-ion
efflux from chick brain tissue, dosimetry information must be accurately determined. Current models do not
allow accurate determination 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 surface, with un-
equal amounts 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 Programs (ORP) 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-
tribution 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: Bioelectmmagnetics 7(2): 209-221, June 1986.

F104 MS-85-164 Project Officer: Spiegel                                                 '
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     A SYSTEM for MEASUREMENT of SMALL VIBRATIONS at MATERIAL
           INTERFACES INDUCED by ELECTROSTRICTTVE FORCES

                              Joseph S. All and William T. Joines

                         HERL, USEPA, Research Triangle Park, NC

Life scientists at EPA and elsewhere have observed the efflux of calcium tons from selected tissues includ-
ing brain tissue under various specific intensities and frequencies of amplitude-modulated radiofrequency
(RF) and extremely tow frequency (ELF) radiation exposure. The effect may have health implications be-
cause calcium ions play an important role in biological systems including the excitation and transmission
of nerve impulses.  Thus it is important to understand the underlying basis for this phenomenon. A
mechanism of interaction relying on the concept of nanometer range mechanical vibration influencing the
efflux of calcium  has been proposed  by others.  To investigate the validity of this model a system was
developed to. expose samples to ELF and amplitude-modulated RF fields while monitoring the induced
vibration by a noncontacting method.   '                                        . t

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 RF hearing and calcium ion efflux. 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: Ali
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     APPLICATION of a FINITE-DIFFERENCE TECHNIQUE to the HUMAN
                   RADIOFREQUENCY DOSIMETRY PROBLEM


                         R. J. Spiegel,1 M. B. E. Fatmi,2 andK. S. Kun?

                        1HERL, USEPA, Research Triangle Park, NC
                                  2Northrop Services, Inc.
    •          <       •   •     Lawrence Uvennore Laboratory

It is not well understood for humans where radiofrequency energy is deposited, but the deposition site has
important implications for the resultant biological effect.  Realistic models of the human body and
dosimetry studies are needed to enhance risk estimates.  This study described a finite-difference numerical
method to calculate specific absorption rates (SARs) in human models.  With this technique it is possible
to calculate SARs at higher frequencies and with greater spatial resolution than is possible with other
methods.  The goal of this research is to provide scientifically credible data, methodologies, and assess-
ments of non-ionizing radiation (NIR) for the Office of Radiation Programs (ORP) to use in regulatory
decisions to modify and extend radiofrequency radiation guidance.

A powerful finite-difference numerical  technique has been applied to the-human 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 human body is comprised of rectangular cells with
dimensions of 4x4x6 cm and dielectric properties that simulate average tissue (2/3 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 Ixlxl 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 Ixlxl cm cells. Computed results from the in-
homogeneous chest model are compared with the homogeneous model

CITATION: Journal of Microwave Power20(4): 241-254, December 1985.

F104 MS-85-168 Project Officer: Spiegel
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 RADIATION
PUBLICATIONS
        OVERVIEW of WHO ENVIRONMENTAL HEALTH CRITERIA 35 on
                 EXTREMELY- LOW-FREQUENCY (ELF) FIELDS

                                     Richard D. Phillips

                         HERL, USEPA, Research Triangle Park, NC

 This docoment summarizes World Health Criteria 35 as possible effects on humans of exposure to extreme-
 ly-low-frequency (ELF) electric and magnetic fields, the lowest frequency band in the nonioniang radiation
 spectrum. The author was a member of (he WHO Task Force who reviewed and revised a draft criteria
 document, made an evaluation of the health risks of exposure to ELF radiation, and considered rationales
, for the development of human exposure limits. The WHO Criteria Document is a valuable resource to the
 Office of Radiation Programs .by providing guidance on the assessment of risks from occupational and
 generalpopulaton exposure to nonumizatg 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 magnetic fields on human health and to give guidance on the
 assessment of risks from occupational and general population exposures. 'The Criteria Document in-
 cludes a review of biological effects on human beings and animals 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, mechanisms of.interaction, biological effects in animals
 and animal tissues, human 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 east in the literature and where further research is needed.  The goal was to provide useful infor-
 mation for the development of national protection measures against 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

                                       Joe A. Elder

                         HERL, USEPA, Research Triangle Park, NC

 The following topics are discussed in this report: 1) environmental exposure levels ofradiofrequency (RF)
 radiation; 2) Federal and other activities related to the control of exposure of the general public to RF radia-
 tion; 3) biological effects; 4) limitations in our knowledge on biological effects; and 5) research support,

 The question of human 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 W/kg, 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 University Symposium Proceedings, Washington, DC, September 9,1985.

 F104 MS-86-187 Project Officer: Elder
     IMPROVED TECHNIQUE for MONITORING ELECTROCARDIOGRAMS
            DURING EXPOSURE to RADIO-FREQUENCY RADIATION

                        William P. Watkinson and Christopher J. Gordon

                         HERL, USEPA, Research Triangle Park, NC

 The research described in this article  was conducted to support the Office of Radiation Programs'
 microwave radiation research. The primary documented effect of exposure to microwave radiation is an in-
 crease in body temperature.  The investigation of other potential related effects, such as cardiovascular ef-
fects, has been limited by the prohibition of metallic instrumentation within the microwave field. This study
 reports on newly-developed instrumentation and procedures which permit simultaneous monitoring ofther-
 moregulatory and cardiovascular parameters during microwave exposure.' The use of these procedures
 should open new areas of investigation and perhaps significantly 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 (Tco), 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 RF 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 Tro 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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 Objective:


 Background:
                      WATER HEALTH RESEARCH PROGRAM
To improve the scientific basis for regulatory activities under the Clean Water Act and
the Safe Drinking Water Act.

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 contamination 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 III of the CWA mandates that EPA control tone 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 OBJECTIVES
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 correlating 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 transmited 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 die 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  lexicological approaches  to fill
knowledge gaps in our understanding of health effects.  Bloassays 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, primariv the
   parasite ascaris 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 ascaris 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
                                             -86-

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 WATER
RESEARCH OBJECTIVES
 are pathogenic. Microbial contamination continues to be a concern because contaminated drinking
 water systems can rapidly spread diseases such as cholera, dysentery, and gjardiasis. In 1974 Congress
 passed the Safe Drinking Water Act because of concern about contaminants in drinking water and un-
 even State supervision of public drinking water supplies. Regulations under the law set maximum con-
 taminant 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 hazard of unregulated contaminants in drinking water.

 *  Provide the toxicologies! 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
    Ugh 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 lexicological, 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 disinfectants, 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 disinfectants, various treatments chemicals, and
 the construction of water supply sysems.

 Approach: The disinfectants themselves will be examined 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 examined lexicologically 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 contaminants.

 Approach: Data are being developed to elucidate chronic toxicological effects of drinking water con-
 taminants. Experiments are being conducted to determine if specific drinking water contaminants act as
 cancer initiators or as tumor promoters 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-
tion and for extrapolation from high to low doses are being developed for renal and hepatic toxicity. The
vast majority of drinking water supplies are contaminated by more than one chemical. A methodology
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RESEARCH OBJECTIVES
WATER
for predicting the hazards of exposure to complex mixtures can only be accomplished by examining the
impact of mixtures that act by the same or different mechanisms 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 identifying infectious disease agents from drink-
   ing water samples and human specimens for use in determining waterborne health hazards. These
   methods will be developed for bacteria, viruses, parasites and will address the problem of deter-
   mining infectivity and viability of these organisms 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 primary 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 human exposures.

Approach: The approach is primarily lab-based with some field work and focuses on known waterborne
pathogens, e.g., norwalk-like virus, giardia, legionella  and campylobacter 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 by ORD for EPA Regional
staff to respond to state and local health agency needs.

• Determine how and where epidemiologic research can contribute to an unproved scientific basis for
   revising current drinking water maximum contaminant 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 human 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
mirobiologjcal standards and disease outbreaks will be better defined. The contribution of indoor air ex-
posures from volatilization of organic contaminants 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 hi the lake was not due to human
 fecal contamination. Therefore, poor water quality was due solely to animal fecal contamination 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  02 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 staphyfococti 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 (LESS), 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 hi 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 serologjcal 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 refine 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 hazard 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-
tions 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 Salmonella/microsome 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 TA98 in a methylene chloride fraction was 120 net rever-
tants at a dose level of 10 mg of extract per plate. The imnrmmm 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 5 mg 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 organtcs was increased with time. This increased activity may be a result of biodegrada-
tion of inert organics resulting hi 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 identifying 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 Resources, 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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 ACCOMPLISHMENTS
WATER HEALTH
 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 infectivily 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 giardiasis 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-
 ministered 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 10"3 to 9 x Iff4 focus
 forming units (ffu) in buffered salt solution after consumption of SO ml of 4% NaHCO3. The dose of virus
 required to cause human infection or illness was between one and 10 ffu. Although pre-inoculation liters
 of serum neutralizing 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 classified 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 examined: 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
ACCOMPLISHMENTS
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 L. pneumophila 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 administration of cimetidine,  it was found that very low densities of L. pneumophila ad-
ministered per dose conferred a protective effect when these animals were challenged with 10 IDSO's by
the intraperitoneal route. These results show that ingestion of potable water might be a route of trans-
mission for Legionella, 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 against 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:  Examine utility  of an in  vitro model  for studying toxic interactions between 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 examine 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 of in vitro models at-
tractive.

Studies were conducted in an attempt to reproduce in cultured hepatocytes, the superadditive in vivo in-
teraction between chloroform (CHC13) 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 CC14 compared to minimally toxic responses observed with either CHC13 or CC14 ex-
posure alone. Toxicity was assessed with serum clinical chemistries and histopathology. These exposure
conditions i/i vivo cannot be duplicated exactlym vitro due to inherent limitations of cultured hepatocytes.
Cytochrome  P-450 enzymes, which are responsible for metabolizing CC14  and CHC13 to toxic inter-
mediate, decrease to less than 50% of control zero time levels in 24 hours. A 72 hour pretreatment with
CHC13 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 CHC13 and CC14 in vitro was to expose cul-
tured hepatocytes from untreated male rats to CHC13 and CC14 in combination and to compare the ef-
fects to cells treated with only CC14 or CHC13. Cells exposed to 0.5 -10 mM CC14 alone showed a dose-
related bcrease in toxicity after 24 hours.  Hepatocytes exposed to 0.5 -10 mM CHC13 for 24 hours
showed minimal toxicity.  When hepatocytes were exposed to combinations of CHC13 and CC14, toxicity
was not different from cells treated with CC14 alone. Thus, under these in vitro exposure conditions to
 CHC13 and CC14, 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 CHC13 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 05 - 5 mM CC14 from rats pretreated with CHC13 or corn oil revealed only a slightly en-
 hanced toxicity to CC14 in cells isolated from CHC13 pretreated rats.

 These studies underscore some of the considerations in attempting to reproduce, by in vitro methods, a
 chemical interaction observed in vivo. Initial attempts to validate an in vitro hepatocyte model for an in-
 teraction between CHC13 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 CHC13 and CC14 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 drinking 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 examining the effect of chlorinated drinking water in a validated, CHD-susceptible animal model
 was deemed necessary. Based on this need a subchronic study using the White Carneau Pigeon was in-
 itiated. .It was shown that chlorinated drinking water raises plasma total cholesterol levels and increases
 atherosclerotic plaque formation hi 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 using two species of non-human primates (Cercopithecus, Rhesus). In the first phase of the study
 the hyperiipemic response to 15% lard, 1% free cholesterol was established in 21 weeks. This was fol-
 lowed by changing 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 mg/L
NaOCl. 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/HDL 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 animals  (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 NaOCl did not immediately reverse the ef-
fect, rather indications of a very slow return to pre-chlorine baseline values were observed. At 5 ppm
NaOCl a detectable trend, however not statistically significant, occurred in 2 of  the 12 animals. (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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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 polyunsaturated:saturated (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.1

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 toxiclty 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 acids
were found in finished drinking water as by-products of chlorination.  Recent studies by the Toxicology
and Microbiology Division demonstrated the carcinogenic potential of dichloroacedc acid (DCA) and
trichloroacetic acid (TCA) in mice supplied drinking water containing these chlorinated acids. The acids
appear to be complete carcinogens since prior initiation did not increase tumor incidence.

Much of our work with by-products of drinking water disinfection 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 carcinogenicity of DCA and TCA was
demonstrated, no data were obtained to quantitate exposure to these chlorinated acids 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 acids.
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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 hi 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 drinking 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 NCI 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 hi 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 contaminants from agricultural runoff. An exposure history will be
 developed from the lifetime residential history and data on chlorine use and THMs levels.

 Because chloramination 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 disinfect.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 chloraminated 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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by age, gender, smoking, occupation and socio-economic status was controlled by multiple logistic regres-
sion.  Preliminary 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 chloraminated 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 italicized.

Other articles submitted for publication, but not published as yet, are listed in Appendix A.

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WATER HEALTH
           WATER DISINFECTION: MICROBES VERSUS MOLECULES
                            AN INTRODUCTION of ISSUES

                         James R, Fowle, III1 and Frederick C. Kopfler2

                              ^HR, USEPA, Washington, D.C
                               2HERL, USEPA, Cincinnati, OH

The disinfection of drinking water is one of the most widely practiced and most successful public health
programs in the US.  While it has dramatically reduced the incidence ofwaterbome illness, it has been
determined that the organic chemical contaminant occurring 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.

Because water is essential for life, humans 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 humans, 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 trade offs 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 thinking, 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 disinfectants
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 PATTERNS of
                         MORTALITY in MASSACHUSETTS

                        Sally Zierler, Robert A. Danley, and Lisa Feingold

                          Massachusetts Department of Public Health

The disinfection of drinking water with chlorine is widely practiced and has dramatically reduced the in-
cidence ofwaterbome illness in die 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.  Some water supplies disinfect with chlorine and am-
monia, producing chloramines which do not react to produce these organic by-products.  This paper com-
pares the mortality of. populations in Massachusetts consuming chlorinated and chloraminated drinking
water.

Patterns of mortality were examined for residents in Massachusetts who died from 1969-1983 and lived in
communities using drinking water that was disinfected 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  small 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
chloraminated 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 similar 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: In: Proceedings of the Second International Symposium on Health Effects of Drinking Water
            Disinfectants  nod Disinfection By-Products,  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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       EPIDEMIOLOGIC STUDIES of ORGANIC MICROPOLLUTANTS in
                                  DRINKING WATER

                                      GuntherF. Oaun

                               HERL, USEPA, Cincinnati, OH

The disinfection of drinking water with chlorine is widely practiced and has dramatically reduced the in-
cidence ofwaterbome 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 micropolhttants.

Epidemiologic studies have been conducted in order to make a quantitative statement about associations
between drinking water contaminants 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 assessing, 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
magnitude 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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     A SUMMARY of WATERBORNE ILLNESS TRANSMITTED THROUGH
                       CONTAMINATED GROUNDWATER

                                   GuntherF. Cmun  v

                             HERL, USEPA, Cincinnati, OH

Data are required to identify water supply deficiencies responsible for waterbome disease outbreaks to
prevent the recurrence of outbreaks.  The Office of Drinking Water (ODW) uses these data to develop
regulations for safe drinking water.

The use of contaminated, untreated or inadequately treated groundwater (a) was responsible for 51% of
all waterbome outbreaks and 40% of all waterborne illness reported in the United States during 1971-82.
Contaminated, untreated or inadequately disinfected 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 contamination, and surface runoff contamination. An increase in the number of
outbreaks resulting from the use of untreated, contaminated-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. Camann,1 P. J. Graham,2 M. N. GuentzeL3 H. I. Harding.1
                  1C T. KmbaU,1B. E. Moore.4 R. L. Northrop? N. L. Altman?
                R. B. Harrist,5A. H. Holguin,5 R. L. Mason,l C. Becker Popescu,2
                                  andC.A.Sorber4

                              Southwest Research Institute
                              University of Illinois-Chicago
                            •^University of Texas-San Antonio
                              University of Texas-Austin
                        5University of Texas School of Public Health

Evidence of seroconversion to enteric viruses was found in a population exposed to wastewater aerosols
through spray irrigation. Additional analyses of these data are being performed in a separate study. This
study adds to the data base onoccurrence and significance of microorganisms in wastewater aerosols. It
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should be of value to the Office of Water Program Operations in developing criteria and guidelines for tiie
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 epidemiologtcal 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 liters to 23 viruses, Legionelto audEntamoeba histofytica. Fecal specimens were collected to
isolate pathogens; electron microscopic examination was performed  to detect virus-like particles, and
tuberculin skin tests were administered.  Weekly illness information was provided by participants.
Microorganisms were measured in the wastewater, aerosols and drinking 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:  U.S. Environmental Protection Agency, cooperative agreement no. CR-807501,  EPA-
             600/S1-85-023, January 1986.

B101HERL-0526 Project Officer: Jakubowski
        MUNICIPAL SEWAGE SLUDGE APPLICATION on OHIO FARMS:
                                   HEALTH EFFECTS

                            C R. Dom, C. S. Reddy, D. N. Lamphere,
                 '•                J. V. Gaeuman, andR. Lanese

                                   The Ohio State University

This journal article 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
pathogens 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
(S3 families) participated by cooperating with monthly questionnaires concerning their health and their
animals' health, annual 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 animal 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.
                                                                  y .
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

                  BadriFattal, Yohanan Wax, Michael Danes, andHUlell. Shuval

                               Hebrew University of Jerusalem

This study suggested that sprinkler irrigation with poor microbial quality partially treated stabilization pond
effluent resulted in a higher enteric disease rate during irrigation periods m 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: Jakubowski
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WATER HEALTH
    COMPARISON of THREE STUDIES PERFORMED in ISRAEL on HEALTH
               RISK ASSOCIATED with WASTEWATER IRRIGATION

                 BadriFattal, Yochanan Wax, TatianaAgursky, andHittell. Shuval

                       The Hebrew University • Hadassah Medical School

 This prospective epidemiolo&cal 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. especially by spray
 application.

 Three epidemiologjcal studies were performed in Israel in order to determine the health risk associated
 with the utilization of wastewater effluent from stabilization ponds for agricultural use (Study I - Katzenel-
 son et al., 1976; Study II - Fattal et al., 1981 and Study m - Shuval et al., 1985). This paper compares the
 three studies regarding the incidence rates of shigellosis, 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 III 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 Ecosystem Stability, vol. m - A&B, Z. Dubinsky and Y. Stein-
            berger, eds., Bar-Dan 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


                                      JudyA.Stober

                              HERL, USEPA, Cincinnati, OH

The results of the research described in the attached workshop proceedings article further characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in lexicological 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.
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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 analysts 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 animal 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.

C104MS-86-113 Project Officer: Stober                                           -
        AN OVERVIEW of STATISTICS on ACUTE and CHRONIC WATER
                           CONTAMINATION PROBLEMS

         •                             GuntherF.Craun

                               HERL, USEPA, Cincinnati, OH

The disinfection of drinking water with chlorine is widely practiced and has dramatically reduced the in-
cidence ofwaterbome illness in the U.S. Under certain conditions, the reaction of chlorine with precursor
substances ifi water produces organic by-products which haw 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 micropollutants. The emphasis of this
paper is on the occurrence ofwaterborne-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.: 425,329 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 inves-
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 Quality Symposium, American Society of Agricultural Engineers
             and Water Quality Association, 1985.
                   i

 C104 MS-86-047 Project Officer: Craun
  SERUM BACTERICIDAL ACTIVITY AGAINST LEGJONELLA 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 pneumophila has also been shown to vary considerably in its virulence characteristics. Some
 strains are highly virulent and others are avirulent.  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 (UHl and RH1) were incubated in fresh human
 serum. The UHl strain was serum resistant, whereas the RH1 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-87-011 Project Officer: Dufour
       INTRATRACHEAL INFECTION withL. PNEUMOPHILA SUBTYPES

                      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 pneumophila has also been shown to vary considerably in its virulence characteristics. Some
strains are highly virulent and others are avirulent. This report covers areas related to Legionetta 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 tfiis organism in drinking water.

Two strains of Legionella pneumophila sero-group 1 (UHl and RH1) were injected intratracheally into
guinea pigs. There were no differences in infectivity or mortality, but there was evidence of disseminated
infections b more guinea pigs infected with the UHl 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 Laboratory Immunology 20(3): 119-120, March 1986.

C104 XX-87-012 Project Officer: Dufour
               ORAL INGESTION t>t LEGIONELLA PNEUMOPHILA

               Joseph F. Plouffe, Michael F. Para, KathyA. Putter, and Gary E. Boffin

                         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 highfy virulent and others are avirulent.  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.
                                                                                  *
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. pneumophila seroconverted. Previously fed guinea pigs were then challenged
with a lethal intraperitoneal dose of L. pneumophila 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 Laboratory Immunology 20(3): 113-117, March 1986,

C104 XX-87-013 Project Officer: Dufour
  AEROSOLS CONTAINING LEGIONELLA PNEUMOPHILA GENERATED by
                  SHOWER HEADS and HOT-WATER FAUCETS

              GaryE. Bollin, Joseph F. Plouffe, Michael F. Para, and Barbara Hackman

                         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 highfy virulent and others are avirulent.  This report covers areas related to Legionella virulence
and the potential risk to individuals exposed to this organism. The information in Otis report wilt be useful
for making decisions releveant to maximum contaminant levels for this organism in drinking water.
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 Shower heads and hot-water faucets cotAaanojagLegionella pneumophila were evaluated for aerosoliztion
 of the organism 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
 running. Low numbers (3 to 5 CPU/IS ft3 [0.43 m3] 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. pneumophila recovered were trapped in aerosol particles be-
 tween 1 and 5 pjn in diameter. Air was collected 1 to 3 ft (30 to 91 cm) from 14 sinks while the hot water
 was running. Low numbers (1 tp 5 CFU/15 ft3 of air) were recovered from 6 to 19 air samples obtained.
 Approximately 50% (6 of 13 CPU) of the organisms recovered were trapped in aerosol particles between
 1 and 8 |jjn in diameter. Shower heads and hot-water taps containing L. pneumophila can aerosolize low
 numbers of the organisms 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 transmission of L,
 pneumophila from potable water to the patient.

 CITATION: Applied and Environmental Microbiology 50(5): 1128-1131, November 1985.

 C104 XX-87-010 Project Officer: Dufour
                     GJARDIA DETECTION in WATER SYSTEMS
                                       Judith F, Sauch

                                HERL, USEPA, Cincinnati, OH

 An immunofluorescence method to detect and identify Giardia cysts in water samples following sample
 concentration was developed. The immunofluorescence method selectively stains cysts and enchances con-
 trast between cysts and other sample constituents and, therefore, allows quick location of cysts by their green
fluorescence. This technique may be useful in confirming outbreaks ofgiardiasis as waterbome, providing
 guidance on treatment prosesses, 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 staining of cysts
 with aati-Giardia antiserum and a fiuorochrome 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 sue-
• cessfully tested during recent outbreaks in Pennsylvania and Massachusetts. However, when chlorinated
 water samples were tested, confirmation of presumptive Giardia cysts was difficult or impossible. Subse-
 quent laboratory exposure of Giardia 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: Sauch
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          WATERBORNE GIARDIASIS in the UNITED STATES 1965-1984

                                        G.F.Craun

                               HERL, USEPA, Cincinnati, OH

Giardia lamblia is the most frequently identified pathogen in waterbome outbreaks and data are required to
identify those water deficiencies which are responsible for these outbreaks. Most waterbome outbreaks of
giardiasis have occurred as a result of consuming contaminated surface water, and a regulation requiring
filtration of surface water supplies is 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 significant (15%) number of cases of giardiasis resulted from outbreaks caused by
contamination of water mains through cross-connections, damage of mains, and repair of mains.

CITATION: Lancet (8505): 513-514, 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 variety of animal species including
 man. In recent years it has increasingly become recognized as a significant cause of gastroenteritis.  It was
 first reported as the agent responsible for a U.S. waterbome outbreak in 1965. Since that date, 90 water-
 bome outbreaks with 23,776 cases of gastroenteritis have been attributed to this agent.  The Office of Drink-
 ing Water (ODW) is considering additional treatment regulations to control its transmission via drinking
 water.

 For about a century after Giardia was named by LambI in 1859, the occurrence of the organism in humans
 was not widely considered significant in explaining 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, drinking water became increasingly implicated as an important route of transmission. 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 giardiasis.  Sewage contamination of the well water supply was
 thought to be the source of the organism. Further, evidence for waterborne transmission of Giardia 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 gjardiasis were reported in the
 United States. This report discusses the etiologic agent and examines drinking water outbreak occur-
 rence and control.

 CITATION: In: Witter Science and Technology 18(10):219-226, August 1986.

 C104 MS-86-228 Project Officer: Akin
 USE of IMMUNOFLUORESCENCE and PHASE-CONTRAST MICROSCOPY for
                                     DETECTION
         and IDENTIFICATION of GIARDIA CYSTS in WATER SAMPLES

                                      Judith A. Sauch

                               HERL, USEPA, Cincinnati, OH

 Giardia lamblia is the most frequently identified pathogen in waterborne disease outbreaks in the U.S. The
 Safe Drinking Water Act requires EPA to establish standards for pathogens that present a public health
 hazard.  Thus, methods are needed to detect and identify Giardia cysts in water samples. Current methods
for detection of Giardia cysts involve tedious, time-consuming examination 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 bright green with Giardia antibody and a fluorescent tag.
 This procedure permits individual green cysts to be quickly located against a dark background even in water
 samples heavily contaminated with other microorganisms and debris Giardiai cyst confirmation 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 aatl-Giar-
 dia 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 microorganisms 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 outbreaks.

 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 manuscript mis presented as a lecture at the 1984 Annual Meeting of the American Water Works As-
sociation. It was intended to introduce the concept of selectively tagging Giardia cysts in 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 described at this manuscript
improve detection of organisms like Giardia that are present in small numbers in water sample sediments.
These methods make me of the unique and selective reaction between an antigen (ie., Giardia cyst), and
its antibody.. Because the antibody can be tagged with a fluorescent stain, cysts glow green among sample
sediments when illuminated by UV light. 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 transmitted via municipal water 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 microorganisms of similar size and shape
and against a background of debris:

CITATION: In: Proceedings of the 12th Annual American Water Works Association Water Quality Technol-
            ogy Conference, 79-86,1985.

C104 MS-85-049 Project Officer: Sauch
 COMPARISON of ANIMAL INFECTTVITY and EXCYSTATION AS MEASURES
            of GIARDIA MURIS CYST INACTTVATION by CHLORINE

                        /. C. Hoff,lK W. Rice,1 ahdF. W. Schaefer, uf-

                             aWERL, USEPA, Cincinnati, OH
                             2HERL, 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 inac-
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tivation studies indicate that in vitro excystation is an adequate indication of G. muris cyst infecdvity for
the host and can be used to determine the effects of disinfectants on cyst viability.

CITATION: Applied and Environmental Microbioloty 50(4): 1115-1117, October 1985.

C104 MS-85-101 Project Officer: Schaeffer
              GIARDIA MURIS: ULTRASTRUCTURAL ANALYSIS of
                              IN VITRO EXCYSTATION

                         James R. Cogpns1 and Frank W. Schaefer, II?

                                  University of Wisconsin
                              2HERL, USEPA, Cincinnati, OH

These procedures are to determine Giardia cyst nobility.  The Agency and the cooperative grantees have
used these procedures for disinfection studies in water treatment.

Giardia mum cysts were examined by TEM before treatment, post-induction, and at timed intervals
during incubation. Untreated G. muris cysts had a thick cyst wall 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, Golgj 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 closely adhering to the surface of the trophozoite.  These
vesicles appear to be of cvtoplasmic 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 endoplasmic reticulum. No more
than two nuclei were observed within each organism. The earliest excysted organisms 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: Experimental Parasitology 61:219-228, April 1986.

C104 MS-85-100 Project Officer: Schaeffer
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  BILIARY LIPIDS SUPPORT SERUM-FREE GROWTH otGIARDIA LAMBUA

                    Frances D. Gillin,1 MichaeM. Gault,1 Alan F. Hofinann,1
                           Devorah Gurantz,l and Judith F. Sauch

                      University of California-San Diego Medical Center
                              ^HERL, USEPA, Cincinnati, OH

The administration of toxic chemicals during earfy pregnancy in humans may cause undetected fetal loss.
In the present study control injections caused abortions during pregnancy in the rabbit by inhibiting ovarian
function.

Giardia latnblia has been grown in vitro only in media containing serum or serum fractions. How this
pathogen can grow in the human 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, phosphatidylcholine (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 l-palmitoyl-2-linoleoyi-PC or l-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. latnblia may help ex-
plain why this parasite colonizes the upper small intestine.

CITATION: Infection and Immunity 53(3): 641-645, September 1986.

B101MS-86-048 Project Officer: Sauch
         ANTIGEN DETECTION in the DIAGNOSIS of NORWALK VIRUS
                                 GASTROENTERITIS

                    /. E. Hermann,1 G. P. Kent,2 N. A. Nowak,lJ. Brondum,3
                                    andN.R.Blacklowl

                          University of Massachusetts Medical School
                                 Centers for Disease Control
                             3Rhode Island Department of Health

 The research described in the attached article details development of an antigen detection system for Nor-
 walk virus.  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 Nonvalk virus are
 being developed by one of the authors under that agreement.

 A study was undertaken to assess the suitability of monoclonal-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 1 day after specimen receipt, rather than the 4 to 6 weeks required
 for collecting convalescent sera.

 CITATION: The Journal of Infectious Diseases 154(3): 547-548, September 1986.

 C104 XX-86-107 Project Officer: Hurst
                            VIRUSES in DRINKING WATER

             Gabriel Bitton,1 Samuel R. Farrah,1 Clay L. Montague,1 and Elmer W. Akin2

                               Hjniversity of Florida-Gainesville
                               2HERL, USEPA, Cincinnati, OH  .

Human enteric viruses that may cause disease when ingested exist in waterways via fecal contamination.
Many factors contribute to their survival or inactivation in source water and treated drinking water. The ac-
tual extent of water contamination with viable viruses is unknown. This global survey for data on virus oc-
currence in drinking water is important to EPA in providing information on the potential hazard and need
for control technology for waterbome viruses.

Rising populations around the globe have resulted in an ever-increasing need for potable waters. Com-
munities obtain their potable water from surface or underground sources. The contaminations 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 against virus breakthrough? Are classical water quality parameters
adequate for guaranteeing safe drinking water from virological 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 finally
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 DETECTION of ROTAVIRUS in STOOL SPECIMENS

                                   John E. Hermann

                         University of Massachusetts Medical School

Rotaviruses are known to occur in water and to cause waterbome gastroenteritis outbreaks.  Yet the virus
has not yielded to routine detection procedures. The identification ofrotavirus as the etiological agent of
waterbome cases and outbreaks is hampered by the non-availability of a detection procedure. This project
has developed a detection procedure for rotavirus in stool specimens and will be used by the Agency in the
investigation of waterbome outbreaks.

Rotavirus infections are generally recognized as a major problem in young children; however, they have
also been associated with severe gastroenteritis in adults and neonates. Infections b neonates are usual-
ly asymptomatic, although the incidence of infection may be high. Adult infections also are often inap-
parent or mild, and thus, adults may be a reservoir of infection in pediatric wards. Diagnosis of infection
is now routinely performed by enzyme immunoassay (EIA) in preference to the more cumbersome
electron microscopy (EM). A commercial polyclonal EIA test, Rotazyme® (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 recommen-
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
            .   of MICROBIAL ANTIGENS and THEIR ANTIBODIES

                                    John E. Herrmann

                          University of Massachusetts Medical School

Many viral agents that are associated with waterbome enteric disease have been visualized by electron
microscopy but have not been detectable by routine cultivation techniques.  This study extended the
developments made on an alternative detection procedure, enzyme-linked immunoassays, that may be used
in the investigation of waterbome outbreaks in the absence of appropriate 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 radioisotopes 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 EIA to diagnosis of infec-
 tious diseases.  This includes tests for both antigen and antibody detection in viral, rickettsial, bacterial,
 and mycotic infections. EIA 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 (ROTAZYMEII) and a COMMERCIAL LATEX
                       AGGLUTINATION TEST (ROTALEX):
  COMPARISON with a MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

           Gary V. Doem, John E. Herrmann, Patricia Henderson, Darlene Stobbs-Walro,
                           Dorothy M. Perron, and Neil R. Blacklow

                         University of Massachusetts Medical Center

The research described in the attached article details development  of an antigen detection system for
rotavirus. This is a potential causative agent of waterbome gastroenteritis outbreaks.  The paper acknow-
ledges a cooperative agreement of this laboratory because test methods for identifying rotavirus virus are
being developed by the authors under that agreement.

A total of 176 human fecal specimens were examined for the presence of rotavirus by four different as-
says: a monoclonal antibody enzyme immunoassay, the original polydonal 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 examined for the
presence of  rotavinis by  electron microscopy,  immune  electron  microscopy,. and  RNA gel
electropboresis. 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, Rotazyme 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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PUBUCATIONS
               METHODS for RAPID IDENTIFICATION of VIRUSES

                                     John E. Herrmann

                          University of Massachusetts Medical School

Many viral agents that are associated with waterbome enteric 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 waterbome outbreaks in (he absence of appropriate virus 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 nucleic acid has recently sparked considerable interest. The current status
of these methods and potential means for improving thenfare presented in this report.

CITATION: In: Recovery of Viruses from the Environment, CRC Press, Boca Raton, FL, 1986.

C104XX-86-060 Project Officer: Akin     .
     VIRUS-LIKE PARTICLES WITH T=19 ICOSAHEDRAL SYMMETRY in
                       a HUMAN GASTROENTERITIS STOOL

                                     P.P. Williams, Jr.

                               HERL, USEPA, Cincinnati, OH

 Tliis article reports a new virus-tike particle observed in this laboratory.  The unexpected finding was a result
 of using negative-stain transmission electron microscopy for virus detection.  This methodology has been
 implemented in-house as part of a continuing project to develop methods for the identification of non-cul-
 tivable gastroenteritis viruses.

 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 EEM, although an antibody increase was not demonstrated.

 CITATION: Micron andAficroscopicaActa 16(3): 173-178,1985.
                                                                   r '
 C104 MS-85-102 Project Officer Williams
                     WATERBORNE VIRAL GASTROENTERITIS

                            Fred P. Williams, Jr. and Elmer W. Akin

                               HERL, USEPA, Cincinnati, OH

 The renew article, "Waterbome Viral Gastroenteritis," was written to provide responsible individuals within
 the water industry, through the Journal of the American Water Works Association, with state-of-the-art in-
 formation on Ms rapidly moving area of investigation relative to drinking 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 impli-
 cated in waterborae outbreaks of this illness. Another virus, the Snow Mountain agent, was first iden-
 tified from the investigation of one waterborae gastroenteritis outbreak.

 CITATION: Journal of the American Water Works Association 78(1): 34-39, January 1986.

 C104 MS-85-021 Project Officer: Williams
    EVALUATION of METHODS for CONCENTRATING HEPATITIS A VIRUS
                            ,   from DRINKING WATER

                 .   MarkD. Sobsey, Stephen E. Oglesbee, and Douglas A. 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 onfy recently been developed. This study used these HAV detection procedures to evaluate
the standardized virus concentration methodology developed for other enteric viruses in the recovery of
HAV. The availability of HAV concentration and detection procedures will provide the tools for the Agency
tojurther evaluate the role of water in the transmission of Ms major viral disease.

By using recently developed cultivation and assay systems, currently available methods for concentrating
enteric viruses from drinking 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 HAV (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 HAV was further concentrated efficiently by acid precipitation (organic flocculation) of
eluents containing beef extract made from powered, but not paste, sources. By using 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 contaminated drinking water with currently available methods should prove useful
in further studies to determine the role of drinking water in HAV transmission.

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. Gerba, Shri N. Singh,
                             Gary A. Toranzos, and Bruce Keswick1

                                    University of Arizona
                               University of Texas Science Center
                                                                                         t
Full conventional treatment, as used in this country to produce drinking waterfront polluted surface sour-
ces, is generally considered to be adequate for virus removal. Earlier non-EPA funded work has shown a
conventional plant in Mexico to allow virus penetration.  This EPA funded study was conducted to confirm
the earlier report and attempt to identify the factors that may have allowed virus penetration. This informa-
tion is important to  EPA in identifying conditions and needed controls concerning waterbome virus trans-
mission.

Reduction of enteroviruses and rotaviruses averaged 81 and 93%, respectively, at a full-scale 205-mgd
(776-ML/d) 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 prechlorination-clarification and final
chlorination.  Enteroviruses or rotaviruses occurred in 24% of the finished water  samples, which had
> 0.2 mg free chlorine/L and met coliform 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

                        /. E. Herrmann, N. A. Nowak, and N. R. Blacklaw

                          University of Massachusetts Medical School

 The research described in the attached article details development of an antigen detection system for Nor-
 walk mas.  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 Norwalk virus are
 being developed by the authors under that agreement.

 The development of a solid-phase microtiter enzyme immunoassay (EIA) 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 immunoas-
 says.  This indicates that antigen detection may be important for use in epidemiologjcal studies. Neither
 of the inununoassays gave positive reactions for. stools known to contain enteric adenovirus, rotavirus, or
 Hawaii virus, or in 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
                 GASTROENTERITIS: ROLE of NORWALK VIRUS

              Dale L. Morse,1 John J. Guzewich,2 John P. Hanrahan,1 Rachel Stricof,1
              MehdiShayegani,1 Rudolf Deibel,1 John C. Grabau,1 Nancy A. Nowak,1
                    John E. Herrmann,2 George Cukor,2 and Neil R. Blacklow2

                           *New York State Department of Health
                        2University of Massachusetts School of Medicine

The research described in the attached article details viral disease hazards associated with consumption of
improperly cooked shellfish, A series of outbreaks which occurred in 1982 were studied. The outbreaks in-
cluded 1017 cases of illness associated with eating clams and oysters.  Tins paper acknowledges a coopera-
tive agreement of this  laboratory because test methods for identifying Norwalk virus 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 December 31, there were
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103 well-documented outbreaks in which 1017 persons became ill: 813 cases were related to eating clams,
and 204 to eating oysters. The most common symptoms were diarrhea, nausea, abdominal cramps, and
vomiting. Incubation periods were generally 24 to 48 hours long, and the duration of illness was 24 to 48
hours, 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 IgM 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 dam 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 shellfish.

CITATION: New England Journal of Medicine 314(11): 678-681, March 1986.
               fr                                     -                                    .
C104 XX-86-070 Project Officer: Hurst
         INVESTIATION of PARASITES in SLUDGES and DISINFECTION
                                     TECHNIQUES

                  R. S. Reimers, M. D. Little, A. J, Englande, Jr., D. B. McDonelt,
                               D. D, Bowman, and/. M. Hughes

                 Tulane University School of Public Health and Tropical Medicine

This report provides the best available information on the concentration of parasites in municipal sludges
in the United States. It will be used by the Office of Water as an important source of data in the formula-
tion  of guidelines and regulations  governing the management of municipal sludge, particularly for the
development of technical regulations for the reuse and disposal of sewage sludge under Section 405(d) of
the Clean Water Act by the Office of Water Regulations and Standards.

The objectives of this research grant were to: 1) assess the presence and densities of resistant 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 examination of waste sludges. Sludge samples from aU
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: U.S. Environmental Protection Agency, cooperative agreement no. CR-806711, EPA-
            600/1-85-022, December 1986.

BID HERL-0522 Project Officer: Kowal
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       HEALTH EFFECTS of LAND APPLICATION of MUNICIPAL SLUDGE
                                                                                          I
                                                                                          i
                                     Norman E. Kowal

                               HERL, USEPA, Cincinnati, OH

 This report summarizes 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 Program Operations to '
 sewage treatment plant operators, and will provide a convenient resource for'the formulation of EPA \
 regutans, permits, and guidelines under the Clean Water Act and (he Resources Conservation and Recovery
 Act.                                                                                      i

 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 tone substances. The pathogens include bacteria, viruses,
 protozoa, and helminths; 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 animals,
 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. R. Dom, D. N. Lamphere, andJ. D. Powers

                                  The Ohio State University

This journal article 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
pathogens as a result of land application, and potential adverse health effects.

Transmission of infectious agents and translocation of Cd, Cu, Pb, and Zn from anaerobically digested
sludge to the tissues of farm animals grazing 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 Nematodirus spp., Strvngylus spp.,
Strongytoides spp., Trichuris spp.,Eimeria spp.,Ascaris spp., andAncylostomum spp. 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 animals. 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 grazing 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 grazing of cattle on pastures
receiving heavy sludge applications, especially with sludges containing 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

                              Christon J, Hurst and Tamara Goyke

                                HERL, USEPA, Cincinnati, OH

This paper describes in statistical terms the stability of indigenous enteric viruses during storage of
processed wastewater sludge samples. The research helps to fulfill HERL's mission to evaluate potential
health effects associated with disposed material from 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 extract elution-organic flocculation
concentration procedure, was studied at 2,23 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 logic 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 (HaO) and frozen on dry ice
(C02).

CITATION:  Canadian Journal of Microbiology 32:649-653, August 1986

B101MS-86-088 Project Officer: Hurst
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     DEVELOPMENT of METHODS to MEASURE VIRUS INACTTVATION in
                                   FRESH WATERS

                             Richard L. Ward and Pat E. Winston

                        James N. Gamble Institute of Medical Research

 The study described in this publication involved an identification of experimental design factors which
 could affect laboratory measurements of virus survival. This work is 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 wafers. It was found that viable microorganisms
 in an environmental water sample increased greatly after addition of small 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 radioactivety 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 contamination 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
                         WASTEWATER SLUDGE SAMPLES

                            Christon J. Hurst1 and Tamara Goyke2

                              *HEKL, USEPA, Cincinnati, OH
                              2EMSL, USEPA, Cincinnati, OH

Tliis paper describes in statistical terms the stability of indigenous viruses during 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 treatment 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 liter 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 £ 0.05) 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 liter 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 (HaO), and on dry ice
(C02).

CITATION:  Canadian Journal of Microbiology 32:645-648, August 1986.

B101MS-86-064 Project Officer: Hurst
    A COMPARISON of SEVEN METHODS for CONCENTRATING ORGANIC
            CHEMICALS from ENVIRONMENTAL WATER SAMPLES

                         F. C. Kopfler, H. P. Ringhand, andR. G. Miller
                                  ~*

                               HERL, USEPA, Cincinnati, OH

Assessment of the risk associated with consumption of drinking water containing organic chemicals win re-
quite that bioassays be performed on samples of the organic chemicals. This paper discusses the suitability
of various methods for preparing representative samples of these organics 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 eliminated 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 Society Advances in Chemistry Series No. 214, Advances in Sampling
            and Analysis of Organic Pollutants from Water, I.H. Suffet and M. Malaiyandi, eds., 1986.

C104 MS-86-097 Project Officer: Kopfler
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       COMPARISON of HIGH MOLECULAR ORGANICS ISOLATED from
                        DRINKING WATER in FIVE CITIES

                    £. 5. K. Chian,1 M. R Giabbai,2J. S. Km,1!. H. Ratter,3
                                    andF. C Kopfler4

                              1Georgia Institute of Technology
                          Environmental Science Laboratories, Inc.
                              School of Geophysical Sciences
                              4HERL, USEPA, Cincinnati, OH

 The findings indicate that the nonvolatile chemicals in drinking water which form when the humic acid in
 them is chlorinated are very similar. Thus results on toxicological testing of a limited number of samples
 may be safely extrapolated to the general case.  In 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 taxicological 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 (namely: New Orleans, Philadelphia, Miami, 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 Chemistry 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

                                R. J. Baker and 1. 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 reverse 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 b
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 similar to those of batch LLE.

CITATION: In: American Chemical Society Advances in Chemistry Series, (in press).

C104 XX-86-038 Project Officer: Ringhand
      PYROLYSIS-MASS SPECTROMETRY/PATTERN RECOGNITION on a
              WELL-CHARACTERIZED SUITE of HUMIC SAMPLES


          P. MacCarthy,1 S. /, Deluca,l K. J. Voorhees,l R. L. Malcolm,2 and E. M. Thtuman2

                                 1Colorado School of Mines
                                  2U.S. Geological Survey

It is now accepted that most of the organic material in chlorinated drinking water is composed of complex
products of reaction between  the chlorine and the humic and fulvic acids which occur naturally in the
source water.  The Safe Drinking Water Act, as amended in 1980, required the Administrator to conduct
studies an the health effects of these reaction products. The results described in  this paper will provide
some of the required background information on the similarity and differences of humic and fulvic acids
from different source waters 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 determined.

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): 2091-20%, 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, Jr.,1 Katherine Mazina,1 Daniel Sonenshine,1 and
                                    Frederick Kopfler1

                                  Old Dominion University
                              2HERL, 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 waterbome illness, it has been
 determined that the organic chemical contaminant occurring 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,

 Stomach fluid was recovered from Sprague-Dawley rats that had been fasted for 48 hours and ad-
 ministered 4 ml deibnized 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/1 as N. In separate ex-
 periments piperidine and gfycine were administered to Sprague-Dawley rats followed by a solution of
 aqueous hypochlorite. Stomach fluid was recovered from these animals and shown to contain the cor-
 responding chloramino products. Because stomach fluid already contains high concentrations of amino
 nitrogen, the percent'conversion of an exogenous amine to its chloramino compound on administration
 of hypochlorite is low. The actual yield of chloramine was determined by administration of tritium-labeled
 piperidine followed by aqueous hypochlorite. 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
 chloramine formed. For concentrations of hypochlorite ranging from 200 ppm to 1,000 ppm, yields of or-
 ganic chloramines 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 chloramine derivatives had formed. These derivatives were
 isolated by preparative liquid chromatography and their retention times correlated with known deriva-
 tives of chloramino acids by two chromatographic techniques. The identification of these compounds will
be discussed.

 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 69:259-265, 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 contaminants may be greater than exposure
through ingestion.  The Office of Drinking WaterfODW) should consider this route of exposure when
developing drinking water regulations.

Our field studies show that indoor air concentrations of volatilized trichloroethylene (TCE) can be sub-
stantial when TCE-contaminated 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 inhalation 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, Andelman

                                  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 exposure when
developing drinking 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 dhirnally 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 trichloroethylene (TCE) in showers using TCE contaminated 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 volatilization from a full range of typical water uses within the
home, calculations indicate that the expected air inhalation exposures can be substantially 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 inhalation from
 other much greater volume indoor uses of water should be considered as well.

 CITATION: Environmental Health Perspectives 62:313-318, October 1985.

 C104 XX-86-057 Project Officer: Craun
                EFFECT of CHLOROACETIC ACIDS on the KIDNEYS
                                                                                    :
                                        Mary 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 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 drinking water
result from it.   This symposium paper summarizes the results to date of the Agency's research into the •
health effects of these contaminants.

Previous studies on dichloroacetate have shown that its effects on intermediary metabolism have the
potential to interfere with the ability of renal mechanisms to maintain acid-base balance. Dichloroacetate
decreases gluconeogenesis from lactate, in part by activating pyruvate dehydrogenase, routing lactate to
acetoacctate 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 acid-base regulation by decreasing glutamate entry .into the tricarboxylic acid cycle, con-
commitantly decreasing 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 dichloroacetate and to determine if trichloroacetic acid has
similar effects. Exposure to lower concentrations of either dichloracetic or trichloroacetic acids did not
affect food consumption, growth, or urine output, whereas higher doses were associated with decreased
food consumption and either lowered body weight gam or weight loss. These effects were observed in
male rats with trichloroacetic acid given daily by gavage (300 mg/kg) or by exposure through water (3 g/1,
approximately 250 mg/kg/day).  Exposure to dichloroacetate in drinking water  impaired growth only at
the highest concentration (7.5 g/1) in males, however, female rats showed weight loss after exposure to
1.875 g/1. The higher exposures resulted in lower urine volumes and elevated urine osmolalities. Hip-
purate excretion was not increased in the groups exposed to dichloroacetic acid. This finding is sugges-
tive of altered acid-base regulation.

CITATION: In: Proceeding 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 EFFECTS in PEOPLE with a HIGH
                     INTAKE of OYSTERS in NEW ZEALAND
                           .i              • '                 **.
                         /. McKenrie,1 T. Kjellstrom,2 and R. Sharma1

                            University of Otago, New Zealand
                           2University of Auckland, New Zealand

This final report 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 cadmium, an important toxic 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 associated 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).
Questionnaires 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 pg cad-
mium/day at the end of the season; calculated intakes for Categories I, II and III were 34 ug/d, 75 ng/d
and 116 jxg/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,
ztnc 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 III and especially Category IV subjects was relatively higher than that for Categories I and n
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.

B113 HERL-0548 Project Officer: Kowal
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        HEALTH CONSIDERATIONS in APPLYING MINIMUM TREATED
                              WASTEWATER to LAND

                                    Norman E. Kowal

                              HERL, USEPA, Cincinnati, OH

 This is overview of the health effects resulting from the land application of municipal wastewater and
 sludge.  It is also a summary of two existing HERL publications:  "Health Effects of Land Treatment:
 Microbiological (EPA 600/1-82-007, May 1982) and "Health  Effects of Land Treatment:  Tcodcological
 (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 human exposure, i.e. aerosols, surface soil and plants, subsurface soil
 and groundwater, and animaK 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:  In: Proceedings of the Soil Science Society of America - Utilization, Treatment, 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 Stuart Simon,1 John L. Egte, Jr.,2 Robert W. Dougherty,1
                                 and Joseph F. Borzetleca2

                                   1Rhone-Powlenc Inc.
                            Virginia Commonwealth University

Chlordecone is a highly stable pesticide with the potential for causing prolonged environmental contamina-
tion. An estimate of its reproductive toxicity is, therefore, of principal importance to the health program.

Male rats received 3.6 or 11.4 mg/kg/day of chlordecone orally for 5 days. Some statistically significant
events were seen hi the reproductive data of females mated to males receiving chlordecone. However,
these events did not follow a consistent pattern and do not suggest the conclusion that chlordecone causes
dominant 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. Chlordecone was distributed throughout the reproductive tract. The des-
cending order of concentration was seminal vesicular fluid > prostate > vasdeferens > 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.

B101/C104/L104 XX-86-129 Project Officer: Smith
        DOMINANT LETHAL EFFECTS of SUBCHRONIC ACRYLAMIDE
              ADMINISTRATION in the MALE LONG-EVANS RAT

                     M.Kate Smith,1 Harold Zenick,2R. Julian Preston,3
                         Emma Lou George? and Richard E. Long*

           1HERL, USEPA, Cincinnati, OH, 2University of Cincinnati Medical College,
                3Oak Ridge National Laboratory, and 4Pathology Associates, Inc.

Acrylamide is widely used in its polymeric form in the treatment of waste and drinking waters. The tancity
profile of the residual monomer is, therefore, essential to the setting of health  advisories in the water
program.

Acrylamide, a widely used vinyl monomer, is well known as a neurotoan 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 neurotoricity. 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 173:273-277,1986.

C104 MS-85-229 Project Officer: Smith
 EVALUATION of CONVENTIONAL TREATMENT PROCESSES for REMOVAL
         of MUTAGENIC ACTIVITY from MUNICIPAL WASTEWATERS

                          John R. Meier1 and DolloffF. Bishop2

                      •   '    1HERL, USEPA, Cincinnati, OH
                            -*WERL, USEPA, Cincinnati, OH

The Office of Water Enforcement and Permits and the Office of Water Regulations and Standards are inter-
ested in the feasibility of using tcadcity bioassays in a monitoring program of practical utility to water pollu-
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 tion control agencies.. In the present study the Salmonella microsome mutagenicity assay was used to ex-
 amine the effectiveness of conventional treatment processes to remove mutagenic pollutants from two
 municipal wastewaters, one heavily industrialized and one primarily domestic.  The results indicated that
 the level of mutagenicity in the domestic effluent was substantially lower than in the industrial effluent.
 Furthermore, an appreciable portion of the responsible mutagens are relatively refractory to removal by con-
 ventional primary and activated sludge treatment.

 The present study was conducted to evaluate the mutagenicity of a domestic wastewater and a combined
 domestic/industrial 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. 5. typhimurium 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 S9 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 little in-
 fluence on the mutagenicity seen.

 CITATION: Journal of the Water Pollution Control Federation, 57(10): 999-1005, October 1985.
                         !
 B113 MS-85-095 Project Officer: Meier
        MUNICIPAL SEWAGE SLUDGE APPLICATION on OHIO FARMS:
                         ESTIMATION of CADMIUM INTAKE

                                  C S. Reddy and C R. Dom

                                  The Ohio State University

This journal article 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
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 gastrointestinal 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 jig/day for females and 8.87 to 18.52 p.g/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 jig/day higher than for non-smokers, the difference was not statistically significant.
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Cattle grazing on sludge-treated pastures consumed significantly 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 significantly contribute to the daily Cd intake in humans; cattle on such farms,
however, significantly increased their Cd consumption.

CITATION: Environmental Research 38(2): 377-388, 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. Zenick, E. Hope, J. M. Manson, E, L, George,1 andM. K. Smith1

                           University of Cincinnati Medical Center
                              ^ERL, USEPA, Cincinnati, OH

 Trichlorophenol is a contaminant detected widely in water supplies. Data concerning its reproductive ef-
fects are important in the setting of reference doses (RFDs) and health advisories.

The tenacity of chlorinated organic compounds which may be 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, 2,4,6-trichlorophenol (TCP), in male and female rats. Adult males were treated with either
0,100,500, or 1000 rag/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, die 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


                     M. Kate Smith,1 Harold Zenick,2 and Emma Lou George1

                              1WEKL, USEPA, Cincinnati, OH
                         University of Cincinnati College of Medicine

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 waterbome illness, it has been
determined (hat 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 Agency's research into the
health effect of these contaminants.

 The disinfection of surface water for potability results in reactions between the disinfectant and the or-
ganic compounds  and materials present at the source. The reproductive consequences of exposure to
several better-known reaction products have been examined.   Some  of the trihalomethanes  and
chlorophenols have been studied for teratogenic and reproductive effects,  and this information is
reviewed. We have examined other disinfection byproducts using an in vivo teratology screen in the
Long-Evans rat.  Pregnant 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 disinfection derivatives during gestation. We found that at doses
toxic to the dam (300,500 mg/kg), acetonitrile administered 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  utero toxicity compared among chloro-, dichloro-, trichloro-, dibromo-, and
bromochloroacetonitrile, 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 segment-JI study of .trichloroacetonitrile was performed.

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 69:177-182, November 1986.

C104 MS-86-178 Project Officer: Smith
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         REPRODUCTIVE EFFECTS of ALTERNATIVE DISINFECTANTS


                         Betsy D. Cartoon? Paul Bartlett,1 Ali Basaran,1
                         Kate Colling,1 Irene Osis,l and M. Kate Smith2

                               ^attelle-Columbus Laboratories
                               2HERL, 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 waterbome illness, it has been
determined 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 Agency's research 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 disinfectants 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 ex-
amined the reproductive effects of sodium chlorite, chloramine, and chlorine administered 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 vaginal patency and thyroid hormone
levels.  No differences were observed between control anifnals and rats exposed to chlorite, chlorine, or
chloramine when fertility, viability, litter size, day of vaginal patency, or sperm count were evaluated. A
significant (p< .01) dose dependent decrease k sperm direct progressive movement (urn/sec) was ob-
served among male rats exposed to 100 or 500 ppm chlorite. In addition, a significant (p < .001) increase
in abnormal sperm morphology was also observed in these animals. 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
chloramine-treated rats.

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 69:237-241, 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
                   t

                       J.R. Hayes,1 L.W. Condie, Jr.,2 andJ.F. Borzelleca1
                                                       •
                                Medical College of Virginia
                              2HERL, USEPA, Cincinnati, OH

 This paper describes a subchronic toxicology study in which tetrachloroethylene was administered to rats in
 drinking water.  These data win assist the Office of Drinking Water (ODW)  in setting standards for
 tetrachloroethylene,

 Tetrachloroethylene (perchloroethylene) has been identified in drinking water at a concentration of ap-
 proximately 1 rag/liter and in ambient air at levels of 0.18-4.5 ppb (0.0012-0.03 ng/m 3). Much of the avail-
 able lexicological data are from inhalation 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 1400 mg 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 hepatotoxicitv; 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 humans to reported
 levels of tetrachloroethylene in drinking water does not constitute a serious health hazard.

 CITATION: Fundamental and Applied Toxicology 7(1): 119-125, July 1986.

 C104 MS-85-153 Project Officer: Condie
 EPIDERMAL HYPERPLASIA in the MOUSE SKIN FOLLOWING TREATMENT
           .with ALTERNATIVE DRINKING WATER DISINFECTANTS

            Merrel Robinson,1 RichardL Bull,2 Marlene Schamer? and Richard E. Long3

                             ^ERL, USEPA, Cincinnati, OH
                                Washington State University
                                ^Pathology Associates, Inc.

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 waterbome illness, it has been
determined 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 Agency's research into the
health effect of these contaminants.
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 Female SENCAR mice were treated with aqueous solutions of HOCL, NaOCL, C102, and NH2C1 by
whole body exposure (except head) for a 10-minute period for 4 days in the first experiment and for 1 day
(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/1, for experiment #1 and 1,000 mg/1 for experiment #2. Thick-
ness of the interfollicular epidermis (IFE) for control animals was 15.4 ± LSujn.  After 4 days of treat-
ment at 1,000 mg/1, HOCL and ClQz increased thickness to 39 ± 7.0 and 402 ± 11.8, whereas NaOCl in-
creased thickness to 25.2 ± 6.1 pjn.  Only HOD and C102 were tested at 300 mg/1, and these yielded an
IFE increase of 30.0 ± 13.1 and 16.8 ±  0.8 jun, respectively. The response to HOC! was found to be
dose-related, with the  minimally effective dose being 100 mg/1. In earlier preliminary tests to determine
optimum treatment schedule, the response to HOC1 appeared to be mammal after 4 days of treatment
and tended to decrease with further treatment. The time course study following a single treatment of
1,000 mg/1 HOC!, however, showed a progression of IFE thickening of from 18.3 ± 1.4 at 1 day to 30.8 ±
8 days then decreasing to 19.1 ± 62 (un at 12 days. ClOa and NaOCl, when tested in this manner, did not
result in increased thickness of IFE with time, but rather gave a persistent level of increase that remained
for the 12 days. NHaCl reduced skin thickness to 13.6 ± 6.1 ^un.  Examining sections of skin treated with
HOC1 and C1Q2 indicated an increase in cell numbers.  HOCl and ClOa are therefore capable of induc-
ing hyperplastic responses in the mouse skin. The basis for the decrease in skin thickness resulting from
NHaCl treatment remains to be established.

CITATION: In: Proceeding? 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 69:293-300, November 1986.

C104 MS-86-177 Project Officer: Robinson
                    TOXICOLOGY OF HALOACETONITRILES

              „  Johnnie R. Hayes,1 Lyman W. Condie,2 and Joseph F. Borzelleca1

                                lMedical College of Virginia
                              2HERL, 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 ofwaterbome illness, it has been
determined 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 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. Dibromoacetonitrile (DBAN) was negative
in selected mutagenic assays; dichloroacetonitrile (DCAN) was mutagenic in 5. typhimurium, but not in
S. cerevisiae. Both DBAN and DCAN may be carcinogenic. There is a paucity of basic lexicological 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 LDso 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/kg/day 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/kg/day; 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 Over. 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 33 mg/kg/day.

 CITATION: In: Proceedings of the Second International Symposium on the Health Effects of Drinking
             Water Disinfectants and Disinfection By-Products, Cincinnati, 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

                      /. Peter Bercz, Lillian L. Jones, Robert M. Harrington,
                              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 ike US.  While it has dramatically reduced the incidence of waterbome illness, it has been
determined 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 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 covalentry 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 disinfectants may arise from disturbances of iodine metabolism and from the genera-
tion of wi situ toxic substances from otherwise nontoxtc nutrients and hormones. The extent and severity
of this phenomenon is probably proportional to the oxidizing power (redox potential) of the disinfectant
molecule.                                                   -

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 69:249-254, November 1986.

C104 MS-86-173 Project Officer: Bercz
           SHORT COMMUNICATION - TERATOGEN METABOLISM:
      THALIDOMIDE ACTIVATION is MEDIATED by CYTOCHROME P-450

                         A. G. Broun, F. A. Harding and S. L. Weinreb

                            Massachusetts Institute of Technology

The tumor cett attachment assay is undergoing validation as a teratogen screen. This paper describes its
response to the archetypal teratogen thalidomide.  Authentication of such a screen would greatfy reduce
need for costly teratology testing in all programmes involved in hazard evaluation.

A metabolite of thalidomide generated by hepadce microsomes inhibited the attachment of tumor cells
of concanavalin A-coated polyethylene.  Evidence that metabolite formation is mediated by microsomal
cytochrome P-450 is presented.  Microsomes incubated with thalidomide underwent a type I spectral
shift.  Metabolite formation was  reduced or eliminated by carbon monoxide, DKF-525A, meryrapone,
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 metabolite 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
b the drug's teratogenic activity remains to be shown.

CITATION: Toxicology and Applied Pharmacology 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

                                      Merrel Robinson
                                                          <*
                               HERL, USEPA, Cincinnati, OH

 The results of the research described in the attached workshop proceedings further characterizes the SEN-
 CAR mouse and its advantages and disadvantages as an animal testing model in taxicological and car-
 dnogenesis 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) histopaihology including treatment-induced and spontaneous lesions.

 Since the establishment 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 animal 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-including 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  aging
 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 proceedings.

 CITATION: Environmental Health Perspectives 68:1-250, September  1986.

 C104 HERL-0544 Project Officer: Robinson
              BARIUM in TEETH as INDICATOR of BODY BURDEN

                 Robert G. Miller,1 John D. B. Featherstone2Martin E. J. Canon2
                           TammyS. Mills,1 and.Carole P. Shields2

                              %ERL, USEPA, Cincinnati, OH
                                  Eastman Dental Center

The results of this research project concerning barium in drinking water was to provide support 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 ug Ba/g Ca in teeth of life-long residents drinking city water compared to teeth
levels of 7 fig Ba/g Ca in the children of the low level barium town which had a drinking water concentra-
tion of 0.2 mg/L barium.

CITATION: In: Advances in Modem Environmental Toxicology, Vol. 9: Inorganics in Drinking Water and
            Cardiovascular Disease (Chapter 19), EJ. Calabrese, 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

             Robert G. Miller, Daniel Greathouse, Richard J. Bull and James U. Doerger

                               HERL, USEPA, Cincinnati, OH

Tliis research project was to show the dietary effects of "hard water* minerals upon the absorption of lead,
thereby helping to establish chemical taxicities involving the cardiovascular system.  This ultimately would
provide support to regulatory development and revision ofMCL.

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 animal groups, while maintaining 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 animals 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 animals receiving 150 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: In: Advances in Modem Environmental Toxicology, Vol. 9: Inorganics in Drinking Water and
             Cardiovascular Disease (Chapter 25), EJ. Calabrese, R.W. Tuthill, and LAV. 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

                  KmberL. White, Jr.,1 Virginia M. Sanders,l Donald W. Barnes,2
                         < George M. Shopp, Jr.,1 and Albert E. Munson1

                                 Medical College of Virginia
                                  2East Carolina University'

 The adverse effects of chemicals on the fymphoreticular system have generated considerable lexicological
 interest. This paper describes the methods and general approach used in judging a chemical's potential risk
 to the immune system.  Risk'evaluation was approached utilizing acute, 14- and 90-day studies from ex-
posure to drinking water contaminants. The immune system was evaluated against a background of more
 standard toxicological parameters.  These techniques will assist researchers in evaluating the health 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 utilizing 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 included 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 determining the number of IgM 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 (PEC). 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: Drug and Chemical Toxicology 8(5): 299-331, October.1985. .

C104 XX-86-100 Project Officer: Condie
   A TEST BATTERY for SCREENING BEHAVIORAL TERATOGENS in MICE

                       May Jeanne Kallman1 and Lyman W. Condie, Jr?

                                 University of Mississippi
                              2HERL, USEPA, Cincinnati, OH
                                                                               t
Research was conducted to develop and validate a test battery for identifying tenttogens in mice.  The mice
were exposed to environmental contaminants by the oral route of exposure.  The techniques employed
should enable other drinking water contaminants to be evaluated for establishing their teratogens potential.

The assessment battery was developed for identifying teratogens in mice (CD-I, outbred strain) since
many non-behavioral lexicologists prefer working with the mouse and because the mouse is less costly to
house and feed. This approach emphasizes 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 includes the assessment of
multiple behavioral capabilities, including physical landmark^, reflex developments, motor capability and
passive avoidance learning. We have found the behavioral capabilities of the mouse to be similar to those
of the rat in variability and range of response. Our methodology has emphasized drinking water exposure
to chemicals since we were involved in the evaluation of drinking water contaminants, but the battery
should be amenable to any route of exposure.

CITATION: Neurobehavioral  Toxicology and Te/atofogy 7:727-731,1985.

C104 MS-86-089 Project Officer: Condie
           TOXICOLOGY of 1,1,2-TRICHLOROETHANE in the MOUSE

                 Kimber L. White, Jr.,1 Virginia M. Sanders,l Donald W. Barnes,2
                         George M. Shopp, Jr.,1 an d Albert E. Munson1

                                Medical College of Virginia
                                 2East Carolina University

This paper reports toxicity findings of a potential drinking water organic chemical contaminant.  1,1,2-
trichloroethane (TCE) was administered to male and female CD-I mice to evaluate its effect on standard
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 taxicologfcal parameters. Following determination of the acute lethal dose for 50% of animals and a 14-
 day range-finding study, a PtWay drinking water study was performed.  The liver was a target of TCE
 tenacity in both sexes as demonstrated by dose-dependent alterations in liver enzymes. This paper will assist
 the Office of Drinking Water (ODW) in setting Drinking Water Standards or Health Advisories for TCE.

 1,1,2-Trichloroethane (TEC) was administered to male and female CD-I mice to evaluate its effect on
 standard lexicological parameters. Following determination of the acute LD50 (378 mg/kg 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 significantly decreased hematocrit and hemoglobin levels.

 CITATION: Drug and Chemical Toxicology 8(5): 333-355, October 1985.

 C104 XX-86-101 Project Officer: Condie
    ACUTE TOXICITY of MONOCHLOROPHENOLS, DICHLOROPHENOLS
                   and PENTACHLOROPHENOL in the MOUSE

                  Joseph F.Borzetteca,1 JohnnieR. Hayes,1 LymanW. Condie,2
                                   andJohnL.Egfe.Jr.1

                                Medical College of Virginia
                              2HERL, USEPA, Cincinnati, OH

Research was conducted to determine the acute toaacity of monochlorophenols, dichlorophenols, andpen-
tachlorophenols.  It  was found that 2-chlorophenol and 3-chlorophenol  were more toxic  than  the
dichlorophenol series.  These studies may aid the Office of Drinking Water(ODW) in setting Health Ad-
visories.

Acute oral LD50 values were determined for 2-, 3-, and 4-chlorophenol; 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-
dichlorophenol and pentachlorophenol in male and female mice. LD50 values (mg/kg) ranged from 117
(females) and  177 (males) for pentachlorophenol to 2389 (females) and  2643 (males) for 3,5-
dichlorophenol. It was found that 2-chlorophenol and 3-chlorophenol were considerably more toxic than
the dichlorophenol series. Values for males and females were generally similar, the major differences
being with pentachlorophenol and 2,5-dichlorophenol where in both cases the female LD50 was lower.

CITATION: Toxicology Letters 29(1): 39-42, December 1985.

C104 MS-85-124 Project Officer: Condie
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           PRIMARY LIQUID INTAKE and URINARY STONE DISEASE

                   Jonathan Shuster, Birdwett Finlayson, Richard L. Scheaffer,
                      Robert Sierakowski, John Zoltek, and Sylvia Dzegede

                              University of Florida-Gainesville

This paper summarized the results of a study to determine if an association between water hardness and
urinary stone disease easts as previously reported,. If this can be shown to be a causal relationship, ODW
could propose regulations for the artificial softening of drinking water. Results of this study indicate there
are important associations between a person's primary liquid intake and urinary stone disease but no
causal relationship was implied.

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 Caucasian male patients from two
geographical regions, the Carolinas (both North and South) and the Rockies (including Colorado, Idaho,
Nevada, Montana, Utah and Wyoming) an important p <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

                       5. L. Herren-Freund,1 M. A. Pereira, 1R.E. Long.2
                                   and M. M. Khoury2

                             1HERL, USEPA, Cincinnati, OH
                                 Pathology Associates, Inc.
                                                                 i-V
The results demonstrate that juvenile rats (32 days of age) are more sensitive than adult rats (60 days of
age) to the induction of gamma-glutamyltranspeptidase (GGT)-foci, putative preneoplastic lesions.  This
type of study is important to optimize the conditions of the liver foci bioassayfor use in determining the car-
cinogenicity of environmental samples.

The induction of gamma-glutamyltranspeptidase (GGT)-foci by single and by split doses of dtethyl-
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/cm3 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 initiated 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 5
 daily doses of DENA starting on day 32 of age, the incidence of GGT-foci/cm3 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/cm3 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: Caninogenesis 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

                               HERL, USEPA, Cincinnati, OH

This review paper describes research conducted in the Toxicology and Microbiology Division of HERL and
sponsored by EPA regarding tcodcological effects of disinfectants and their by-products. This paper indi-
cates the current status of our research and should assist the Office of Drinking Water (ODW) in their
promulgation of drinking water standards for disinfectants.

The author reviews and summarizes the lexicological hazards associated with disinfecting drinking water
with either chlorine dioxide or chlorine. Adverse health effects seen in animal experiments as well as
human 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: Journal American 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. Revis,1 Paid McCauley,2 and George Holdsworth1

                               1Oak Ridge Research Institute
                             2HERL, USEPA, Cincinnati, OH

The disinfection of drinking miter is one of die most widely practiced and most successful public health
programs at the U.S. While it has dramatically reduced the incidence of waterbome illness, it has been
determined that there may be undesirable side effects of disinfection.  This symposium paper summarizes
one of these side effects.
             •                 "                 "                     '
The importance of dietary iodide on the reported hypothyroid effect of drinking water disinfectants 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 containing 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. Similar trends were observed in rabbits exposed to the same treatment; however, significant
hypothyroid effects were not observed in this animal 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: 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 69:243-248, November 1986.

C104MS-86-168 Project Officer: McCauley
    RESULTS of TOXICOLOGICAL TESTING of JEFFERSON PARISH PILOT
                                  PLANT SAMPLES

                    Robert G. Miller, Frederick C. Kopfler, Lyman W. Candle,
             Michael A. Pereira, John R. Meier, H. Paul Ringhand, and Bruce C. Casto1

                              HERL, USEPA, Cincinnati, OH
                       Environmental Health Research and Testing, Inc.

 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 waterbome illness, it has been
determined that the organic chemical contaminants occurring at the highest concentration in drinking water
result pom 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, Louisiana. The facility design allows for collection of samples at several sites within five separate
 streams each treated with an alternative disinfectant (nondisinfection, ozone, chlorine, chlorine dioxide,
 chloramine) before treatment with granular activated carbon (GAC). The samples were collected and
 concentrated to either 400X by reverse osmosis or to l.OOOX by adsorption to and subsequent elution
 from macroreticular resins. Before initiation of lexicological tests, the resin extracts were further con-
 centrated to 4.000X. The lexicological 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//i Vivo Toxicity Test in Mice using 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 disinfectants chlorine, chlorine dioxide, and chloramine.
 However, after treatment with GAC in the chlorine stream, the samples were negative for mutagenic ac-
 tivity using the Ames Assay.  The remaining tests were conducted on samples (4,OOOX) collected by the
 resin process. The results of carcinogenicity tests were all negative.

 CITATION: In: Proceeding 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 69:129-139, November 1986.           :

 C104 MS-86-167 Project Officer: R. Miller
   .STUDIES of the TOXIC INTERACTIONS of DISINFECTION BY-PRODUCTS

            R- Dana Laurie, I. Peter Bens, Thomas K. Wessendarp, and Lyman W. Condie

                               HERL.USEPA, Cincinnati, OH

The disinfection of drinking water is  one of the most widefy 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  contaminants occurring 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.

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
dichloropropanones. Since the lexicological properties of this group have not been well characterized
and because  a number of investigators have shown that ketones potentiate  the  heptatoxicity of
haloalkanes, we investigated the toxicity of the dichloropropanones and their interaction with carbon
tetrachloride.  A variety of  experimental techniques  were used to  evaluate the toxicity of the
dichloropropanones 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 Drinking Water
            Disinfectants  and Disinfection  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 CYTOTOXTCTTY of
ACETAMINOPHEN in a PRIMARY CULTURE SYSTEM of RAT HEPATOCYTES

                   David B. Mitchell,1 Daniel Acosta,2 and James V. Bruckner3

                          1Miami Valley Labs, Proctor and Gamble
                                University of Texas-Austin
                               University of Georgia-Athens

This project was a part of an in vivo/in vitro comparison study of drinking water pollutants. This research
demonstrates the accuracy and feasibility of the in vitro testing method.

A primary culture system of postnatal  rat hepatocytes was utilized to  study the cytotoricity of
acetaminophen and the lexicological significance of glutathione (GSH) depletion.  The relative time of
onset and magnitude of GSH depletion, lipid peroxidation and cytotoxicity were contrasted in order to
gain insight into their interrelationships. Exposure of the hepatocytes to acetaminophen resulted in time-
and dose-dependent depletion of cellular GSH. The acetaminophen-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 GSH. 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 viability. Maximal LDH leakage in response to IAA and acetaminophen preceded
mammal malondialdehyde (MDA) formation, suggesting that lipid peroxidation may be a consequence
of cell damage as weU as GSH depletion. IAA 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 mechanisms of chemical toxicity.

CITATION: Toxicology 37:127-146, 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

                       /. V. Bruckner,1 W. F. MacKenae,2 S. MuraKdhara,3
                            R. Luthra,3 G, M. Kyle? andD. Acosta3

                                University of Georgia-Athens
                         2University of Texas Medical School-Houston
                                 University of Texas-Austin

Carbon tetrachloride is a common drinking water pollutant. • This study is in direct support of drinking
water regulations concerning safe carbon tetrachloride concentrations in potable water.

This investigation was conducted to characterize the acute, subacute, and subchronic toxic potency of in-
gested carbon tetrachloride (CCLt). 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 CCU/kg 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 CCU/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 significant increases in some serum enzyme levels.  In general, there was progressively
severe hepatic injury at each dosage level over the 11-day period. CCU 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 CCU/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 vacuolization; 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 CCU exposure. Microscopic examination of livers of the 33-
mg/kg rats revealed cirrhosis, characterized by bile duct proliferation, fibrosis, lobular distortion,
parenchyma! 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:
             DIFFERENTIATION from GRANULOCYTIC LEUKEMIA

                                  11                  2
                    Richard E. Long  Gary Knutsen, and Morel Robinson

                                ^Pathology Associates, Inc.
                             2HERL, USEPA, Cincinnati, OH

The results of the research desaibed in the attached workshop proceeding? article further characterizes the
SENCAR mouse and its advantages and disadvantages as an  animal testing model in lexicological and
carcinogenesis experimentation. The data deals with three categories 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) histopathology-incatding treatment-induced and spontaneous lesions.

The term myeloid hyperplasia 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 myeloid hy-
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 changes 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 73MAW-1^-DICHLOROETHYLENE

                  George M. Shopp, Jr., Virginia M. Sanders, KunberL. White, Jr.,
                                  and Albert E. Munson

                                Medical College of Virginia

This paper reports toxicity findings of a potential drinking water organic chemical contaminant. This study
assessed possible adverse immunological effects of trans-1,2-dichIoroethylene (DCE) on random-bred CD-
1 mice. A 14-day range-finding study was performed on male mice by gavage at doses 1/10 and 1/100 the
lethal dose for 50% of animals. No alterations in either humoral or cell- mediated immunity were observed
following this exposure. A 90-day study was conducted in which DCE was administered in drinking water
of male and female mice. No changes were observed in the cell-mediated immune status of either sex or in
(he humoral immune status of females. However, a marked suppression in humoral immune status was ob-
served 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. These data will assist the Office of Drinking Water (ODW) in setting Drinking Water
Standards and Heal A Advisories.

This study assessed possible adverse immunological effects of frons-l^-dichloroethylene (DCE) on ran-
dom-bred CD-I mice following 14 and 90 days of exposure. A 14-day range-finding study was performed
on male mice by gavage at doses 1/10 and 1/100 the LD50 (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 against sheep
erythrocytes (sRBC).. Macrophage function was depressed only in females, as indicated by the decreased
ability of thioglycollate-recniited peritoneal exudate cells (PEC) to phagocytize sRBC.

CITATION: Drug 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, KmberL. White, Jr.,
                           George M. Shopp, Jr., and Albert E. Munson

                                  1East Carolina University
                                 Medical College of Virginia

This paper reports toxicity findings of a potential drinking water organic chemical contaminant, Trans-1,2-
dichlotoethylene (DCE) was administered to male and female CD-I mice ui order to evaluate its effects on
standard toxicological parameters. Following an acute lethal dose for 50% of animals (LD50) determina-
tion 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 LD50.  The most noteworthy changes occurred
in the males exposed to the highest level of DCE, where there was a signficant decrease in glutathione levels,
and in the females exposed  to all three DCE levels, where there was a significant decrease in aniline
hydraxylase activity. These data will assist the Office of Drinking Water in setting Drinking Water Standards
and Health Advisories.

7>a/ts-l,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 LD50 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 LD50.
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 b 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 significant decrease in aniline hydroxjdase activity. These data served as
background for the immunotoricological evaluation presented in the following manuscript.

CITATION: Drug and Chemical Toxicology, 8(5): 373-392, October 1985.

C104XX-86-103 Project Officer: Condie
         HUMORAL and CELL-MEDIATED IMMUNE STATUS of MICE
                      EXPOSED to 1,1,2-TRICHLOROETHANE


                           Virginia M. Sanders, KmberL. White, Jr.,
                        •  GeorgeM. Shopp, Jr., and Albert E.  Munson

                                 Medical College of Virginia

This paper reports tenacity 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-trichloroethane 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 1/20 and 1/1000 the acute lethal dose for 50% of animals revealed no alterations in either
humoral or cell-mediated immune status. Following 90 days of exposure in the drinking water, a more
detailed series of immunological parameters was assessed. Cell-mediated immunity was unaltered in both
sexes, while humoral immune status was depressed in both sexes, particularly when determined by hemag-
glutination liters.  This paper wiU assist the Office of Drinking 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 (TCE) on ran-
dom-bred CD-I mice following 14 and 90 days of oral exposure. A lexicological 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-day immunological range-finding study in males exposed to doses 1/10 and 1/100 the
LD50 (38 and 3.8 rag/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
rug/kg for females), a more detailed series of immunological parameters was assessed. Cell-mediated im-
munity was unaltered in both sexes, while humoral  immune status was depressed in both sexes, particular-
ly when determined by hemaggludnation liters. Macrophage function was depressed only in the males as
indicated by the ability of thioglycolate-recruited  peritoneal exudate cells (PEC) to phagocytize sheep
erythrocytes (sRBC).

CITATION: Drug and Chemical Toxicology 8(5): 357-372, October 1985.

C104XX-S6-102 Project Officer: Condie
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    RESEARCH in PROGRESS: THE EFFECTS of CHLORINATED DRINKING
                     WATER on HUMAN LIPID METABOLISM


                              Robert Wanes and Charles Glueck

                          University of Cincinnati College of Medicine

The disinfection of drinking water is one of the most widely practiced and most successful public health
programs in the US. While it has dramatically reduced the incidence of waterbome illness, it has been
determined 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 Agency's research into the
health effect of these contaminants.

Studies in animals 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 human lip id, lipoprotein, and apolipoprotein effects of drinking water
chlorination. The initial research protocol will study 20 healthy men and women. On  admission to the
GCRC, each will have a complete history and physical examination, 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 600 mg of cholesterol daily with a polyunsaturated-
to-saturated-fat ratio of 0.4. The diets will also be mildly deficient b 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 2 ppm, 5 ppm, and 10
ppm chlorine, respectively.  These chlorine concentrations have shown biological effects in animals and
are not extraordinary for many treated drinking waters.

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 69:255-258, November 1986.

C104XX-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 this research has specifically focused on the task of determining the potential health hazards
of complex mixtures of organic compounds found in drinking and waste waters.  Samples were collected
from each stream that had been disinfected by use of either ozone, chlorine dioxide, mono-chloramine or
chlorine. These samples were subsequently examined in a battery of five short-term lexicological assays. In
addition to the EPA Compliance Policies to protect public health and the regulatory aspects of Otis re-
search, the toxicological testing results described in this report will provide EPA with information needed to
develop useful short-term in vivo and in vitro test systems that are capable of detecting adverse toxicological
effects caused by environmental pollutants.

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 human 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 hazards
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 typhimurium 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-
fillment 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 summary 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.

B101HERL-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
                             Mark A. Morse and Gary P. Carison

                                    Purdue University

The mouse skin bioassay system is being used extensively to assess the taxicity of chemicals isolated from
drinking water.  This test model is to be used in collecting data to establish allowable limits of con-
taminants in drinking -water for adherence to the mandate of the Safe Drinking Water Act calling for com-
prehensive study of public water supplies.
                                1  .           -        •    T
When benzo[a]pyrcne (BaP) is used as the initiator in initiation-promotion assays, the topical route of ad-
ministration 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 [^HJBaP was examined in the skin,
liver, lung, and stomach of SENCAR and BALB/c mice following topical or oral administration of BaP
at time periods ranging from 0.5 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-
ministration 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 epidermal 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 examined.
          : Journal of Toxicology and Environmental Health 16: 263-276, November 1985.

C104 XX-86-058 Project Officer: Robinson
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           NON-NEOPLASTIC LESIONS: USE of DATA FROM PRE- or
                           NON-NEOPLASTIC LESIONS
            THAT MAY INDICATE POTENTIAL for CARCINOGENESIS


                                      Judy A. Stober

                              HERL, USEPA, Cincinnati, OH

Development of statistical methods far carcinogenesis tiered testing screen will aid in the assessment of the
feasibility of combining dose response information and multiple in vivo short term assays to estimate rela-
tive carcinogenic potency.  These methods mil aid the Office of Drinking Water in evaluating the effects of
various disinfectants and single chemicals used for drinking water treatment, particularly for those cases
when a long term whole animal study is neither logfsticalfy possible 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 (CTM). 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 estimating 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 Power Research Institute Workshop on Investigation of New Ap-
            proaches to Use of Data in Cancer Risk Assessment, 1986.

C104 MS-86-145 Project Officer: Stober
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                           MOUSE LIVER TUMOR DATA:
                    ASSESSMENT of CARCINOGENIC ACTIVITY


                                     Michael A. Pereira

                               HERL, USEPA, Cincinnati, OH

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 carcinogenic ac-
tivity of a chemical in mouse liver can be used to predict carcinogenidty 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 carcinogenidty in humans. Many of
these mouse liver carcinogens lack genotoxic activity and, as such, have been proposed to be tumor
promoters. Two mechanisms 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
human 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 human health hazard, it is important to distinguish  between genotoxic and non-
genotoxic  mechanisms in. applying such  data.  Further study of  the  biochemical and molecular
mechanisms of chemical carcinogens is necessary to determine the relationship between their activity in
mouse liver and their activity in humans.

CITATION: Toxicology 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 B6C3F1 MICE by CORN OIL:
             IMPLICATIONS for CHLOROFORM CARCINOGENESIS


                     RichardJ. Bull,1 Janice E. Brown,2EarleA. Meierhenry,2
                    Ted A. Jorgenson,2 Merrel Robinson,3 and Judith A. Stober*

                                Washington State University
                                    ^RI International
                              3HERL, 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 waterbome illness, it has been
determined 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 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 at, 1985). The results obtained in the male, Osborne-Mendel rats were
comparable to those observed in an earlier study sponsored by the National Cancer Institute (NCI, 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 (NCI, 1976). The doses of chloroform used were
comparable to those which produced an 85 percent incidence in the NCI study. When administered in
corn oil, chloroform significantly decreased serum TG levels but did not effect this parameter when ad-
ministered 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 (5 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 animal$ administered corn oil without
chloroform nor in mice receiving chloroform in 2 percent  Emulphor. These data indicate  that ad-
ministration 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 (NCI, 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-Products,  Cincinnati,  Ohio, August  27-29, 1986.
            Published in: Environmental Health Perspectives 69:49-58, November 1986.

C104 MS-86-176 Project Officer: Robinson
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WATER HEALTH
     MUTAGENIC BY-PRODUCTS FROM CHLORINATION of HUMIC ACID

              John R. Meier, H. Paul Ringhand, W, Emile Coleman, Kathy M. Schenck,
                      Jean W. Munch, Robert P. Stretcher, William H. Kaytor
                                   and Frederick C. Kopfler

                               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 ofwaterbome illness, it has been
 determined 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 Agency's research into the
 health effect of these contaminants.

 Chlorination of humic and fulvic acid results in the formation of direct-acting mutagenicity, detectable b
 theSfl/mo/ie//o/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
 mutagenicity. Synergistic or antagonistic interactions among the identified components has been ruled
 out as a possible explanation 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 trichlorohydroxyfuranone isomers have been tentatively identified b 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 Symposium on the Health Effects of Drinking
            Water Disinfectants 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: Meier
  GROSS and MICROSCOPIC PATHOLOGY in the SENCAR MOUSE SKIN and
                  LUNG-INITIATION and PROMOTION STUDIES

                   Gary L. Knutsen,] Robert M. Kovatch, land Merrel Robinson2

                                 ^Pathology Associates, Inc.
                              2HERL, USEPA, Cincinnati, OH

The results of the research described in the attached workshop proceedings article further characterizes the
SENCAR mouse and its-advantages and disadvatages as an animal testing model in taadcological and car-
cinogenesis experimentation.  The data deals with three categories relevant to the response of the mouse in
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laboratory tests: (1) response to carcinogens, (2) alternate testing (other than skin tumor initiation/promo-
tion 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 Carrinogenesis Testing Matrix were examined. This included SENCAR
mice used in three different short-term bioassay protocols in which the Skin Papilloma Assay was used to
identify initiators, promoters 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 b both the skin and lung tissues. 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 neoplasms, 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 neoplasms 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 Adenoma 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 using 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.

C104MS-86-116 Project Officer: Robinson
        METABOLIC ACTIVATION of BENZO(a)PYRENE in SENCAR and
                   BALB/c MOUSE EMBRYO CELL CULTURES

                     William M. Baird, SaidM. Sebti, and Lori A. Reinsvotd

                            Purdue University School of Pharmacy

The results of the research described in the attached workshop proceeding? article further characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in taxicologfcal 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 BALB/c mice, a strain relatively resistant to two-stage tumorigenesis. Cultures from
both strains metabolized similar amounts of B(o)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-9,10-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(«)P-DNA ad-
 duct formed in B(a)P-treated cultures from both strains was the adduct formed by reactin of(+)anti-
 B(«)P-7,8-diol-9,10-epoxide (antf-B(a)PDE; the isomer with the epoxide and the benzylic hydroxyl on
 opposite faces of the molecule) with the exocydic 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

                   Robert M. Kovatch, * Gary L. Knutsen,1 and Morel Robinson*

                                Pathology Associates, Inc.
                              2HERL, USEPA, Cincinnati, OH

The results of the research described in the attached workshop proceedings article further characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in lexicological 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.

Lesions, that were considered naturally occurring, 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. Carlson, Anthony A. Fossa, Mark A. Morse, and
                                    Patrice M. Weaver

                           Purdue University School of Pharmacy

The results of the research described in the attached workshop proceedings article further characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in taxicologicai 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.

Previous investigators have determined that benzo(a)pyrene was much more effective in causing skin
papillomas 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 orally. In an attempt to understand the reason for this discrepancy in route
dependency, ii-benzo(a)pyrene,  C-urethane and  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 MYRISTATE ACETATE and CATECHOL as SKIN
                       COCARCINOGENS in SENCAR MICE

                 Benjamin L. Van Duuren, Susan Melchionne, andlrvin Seidman

                            New York University Medical Center

The results of the research described in the attached workshop proceedings article further characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in taxicologicai 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 tumor initia-
 tion/promotion assay), and (3) histopathology including treatment-induced and spontaneous lesions.

 The enhancement of the cartinogenicity of benzo(a)pyrene (BP) and of beta-propiolactone (BPL) by the
 mouse skin cocarcinogens phorbol myristate acetate (PMA) and catechol were examined 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 jig, respectively in all ex-
 periments. PMA was used at three doses, 25,1.0 and 0.5 jig, per application and catechol was used at
 one dose, 2 mg 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 carcinogens the most marked
 cocarcinogenic effects were observed at the lowest dose of PMA used, i.e. 0.5 jxg per application; this ap-
 plied for days to first tumor, animals 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 jtg per application 15 of 30 animals
 had 28 tumors, 5 of which were squamous carcinomas. In two-stage carcinogenesis experiments  with
 7,12-dimethyibenz(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.

 C104XX-86-085 Project Officer: Pereira                          ,                 .
                    SPONTANEOUS TUMORS in SENCAR MICE
                 Susan Melchionne, Irving Seidman, and Benjamin L. Van Duuren

                             New York University Medical Center
                                                          • ~*
The results of the research described in the attached workshop proceedings article further characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in lexicological and
carcinogenesis experimentation. The data deals with three categories relevant to die 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.

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, ie., 12 of 36 animals. Other frequently occurring tumors were leukemia and papillomas of the fores-
tomach.             -                     •                           i  '

CITATION: Environmental Health Perspectives 68:135-140, September 1986.

C104 XX-86-086 Project Officer: Pereira                                       ,       .
         HALOACETONITRILES: METABOLISM, GENOTOXICITY, and
                          TUMOR-INITIATING ACTIVITY
           *   *             »                                              *   p
                 Edith L. C. Lin, F. Bernard Daniel, SydnaL-Herren-Freund, and  -'
              :                       Michael A. Pereira

                              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 waterbome illness, it has been
determined 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 Agency»s research into the
health effect of these contaminants.

Haloacetonitriles (HAN) are drinking water contaminants produced during chlorine disinfection. We
evaluated the  metabolism, genotoxicity, 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 (DBAN)  > bromochloroacetonitrile (BCAN)  > chloroacetonitrile
(MCAN) > dichloroacetonitrile (DCAN) > trichloroacetonitrile (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 dimethylnitrosamine demethylase
(DMN-DM) activity. The most potent inhibitors were DCAN and BCAN, with Kj = 3 - 4 x HT5 M; the
next potent inhibitors were DCAN and TCAN, with Kj' = 2 x Iff4 M; and the least potent inhibitor was
MCAN, with Ki = 9 x 10'2 M. When administered orally, TCAN, but not DBAN, inhibited hepatic DMN-
DM activity. 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 >
DBAN > DCAN > MCAN, which was only marginally active.  DCAN reacted with polyadenylic acid
and DNA to form adducts in a cell-free system, however, the oral administration of DBAN 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 summary, 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: In: Proceedings of the Second International Symposium on Healtli Effects of Drinking Water
            Disinfectants and Disinfection By-Products,  Cincinnati,  Ohio, August  27-29,  1986.
            Published in: Environmental 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. Henen-Freund and Michael A. Pereira

                                HERL, USEPA, Cincinnati, OH

 The disinfection of drinking water is one of the most widely practiced and most successful public health
progams in the U.S. While it has dramatically reduced the incidence of waterbome illness, it has been deter-
mined 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 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 gamma-glutamyltrans-peptidase-positive foci (GGT-foci) as an indicator of car-
cinogenicity. The protocol for this assay consists of a two-thirds partial hepatect omy 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-acetylaminofluorene, aflatoxb BI, diethylnitrosamine, dimethyl-
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 Animals 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 concommUantly with week-
ly doses of DENA, it did enhance 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-tetrachloro-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 activity, although some byproducts appear to modulate
tumor promotion.

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 69:59-65, November 1986.

C104 MS-86-180 Project Officer: Herren-Freund
               EFFECTS OF CHRONIC TOPICAL APPLICATION of
        12-0-TETRADECANOYLPHORBOL-13-ACETATE on the SKIN and
                      INTERNAL ORGANS of SENCAR MICE

                Andres J. P. Klein-Szanto,l Claudia J. Conti,1 Claudio M. Aldaz,1
                     Neal Clapp,2 Stephen Nesnow,3 and Thomas J. Slaga1

            *            Hjniveristy of Texas System Cancer Center         v
                              2Oak Ridge National Laboratory
                        3HERL, USEPA, Research Triangle Park, NC

The results of the research described in the attached workshop proceedings article further characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in lexicological 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.

Repetitive topical applications of 2jig 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 interfoUicular epidermis.  The general
effects of the promoter or the animal^ probably through impairment of the immune system, decreased
markedly their longevity.

CITATION:  Environmental Health Perspectives 68:75-80, September 1986.

C104 MS-86-115 Project Officer: Nesnow
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       PERMEATION of WATER CONTAMINATIVE PHENOLS THROUGH
                               HAIRLESS MOUSE SKIN

                       A. S. Huq,l N. F. H. Ho,l N. Husari,1G. L. Ftynn,1
                              W. E. Jetzer,1 andL.W. Condie, Jr.2

                                  University of Michigan
                               2HERI^USEPA, Cincinnati, OH

 This paper mil aid in the assessment of exposure of water contaminants by the percutaneous route of ex-
 posure.

  As a means of determining the risk of absorption of water contaminative phenolic compounds through
 the skin, the permeabilities 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, Pvt, and the stratum corneum, Psc, estimated from the PC'S have been cor-
 related with their log Koctanol/mter partition coefficients. Pe and Psc values systematically increased with
 increasing phenol lipophilicity to limiting values of about 0.15 and 030 cm/h, respectively. The estimated
 value of Pvt is relatively constant at about 0.36 cm/h. Because of the inductive  effects of Cl and NOa 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, Pe, 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 Contamination and Toxicology 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/. Ruch, James E. fSaunig, Norman E. Schultz, Augusta B. Askari,
                    David A. Lacher, Michael A. Pereira,1 and Peter J. Goldblatt

                                  Medical College of Ohio
                              ^RL, USEPA, Cincinnati, OH

lite 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 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 symposium paper summarizes the results to date of the Agency's research into the
health effect of these contaminants.

Chloroform (CHCb) is formed as a byproduct of sewage and drinking water chlorination and has been
identified in public drinking water. CHCb is tone to mammalian 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 CHCb to produce trichloromethanol, which spontaneously
dehydrochlorinates to the toxic component, phosgene (CHOC1 2). It has been suggested that free-radi-
cal generation occurs during CHCb metabolism and may be important in CHCb toxicity. Therefore, we
examined the effects of the MFOS inhibitor, SKF-525A; the anti-oridants, x-tocopherolacetate (Vitamin
E) and NjN'-diphenyl-p-phenyldiamine (DPPD); and a depleter of cellular gluthathione (GSH); diethyl-
maleate (DEM), on CHCb toxicity to isolated male B6C3F1 mouse hepatocytes after 2,4, and 20 hours.
CHCb alone showed a dose-responsive toxicity at concentrations of 1 and 5 mM. SKF-525A (25 jjsl),
Vitamin E (100 jiM), DPFD (25 jtM), and DEM (0.75 jiM) were not toxic by themselves at any time
point. SKF-525A inhibited CHCb toxicity at 2,4, and 20 hours. Vitamin E and DPPD had no effect on
CHCb toxicity. DEM potentiated CHCb toxicity at all three time points.

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 69:301-305, November 1986.

C104 MS-86-172 Project Officer: Pereira
   EXCRETION and TISSUE DISPOSITION of DICHLOROACETONITMLE in
                                    RATS and MICE

               Mark R. Roby\ Sigrid Carle1, Michael A. Pereira2, and Dean E. Carter1

                                   University of Arizona
                              2HERL, 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 ofwaterbome illness, it has been
determined that the organic chemical contaminants occurring at the highest concentration in drinking water
result from it.  This symposium paper summarizes Oie results to date of the Agency's research into the
health effect of these contaminants.

Dichloroacetonitrile 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 dichloroacetonitrile radiolabeled on each of the carbon atoms to follow the
fate of the fragments of the molecule after metabolism. Male Fischer 344 rats (175-22Sg) were dosed
orally with aqueous solutions of dichloroacetonitrile at 0.1,0.01, and 0.001 LD50. Dosing solutions were
prepared by mixing (2114C) dichloroacetonitrile (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 03-0.4 ml. Animals
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 elimination 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 (1"14C) 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* C)
 dichloroacetonitrile, but the amount expired as COj (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: 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 69:215-220, November 1986.

 C104 MS-86-171 Project Officer: Pereira
   CHRONIC BIOASSAYS of CHLORINATED HUMIC SUBSTANCES in B6C3F1
                                          MICE
                                    -j

                Benjamin L. Van Duuren,1 Susan Melchionne,1 Irving Seidman,1 and '
                                     Michael A. Pereira2

                             *New York University Medical Center
                               2HERL, 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 waterbome illness, it has been
determined 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 Agency's research into the
health effect of these contaminants.

Humic acids (Fluka), chlorinated to C:C1 ratios of 1:1 and 1:03, 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/1. The mice
were 6 to 8 weeks old at the beginning of the bioassays. This dose was based on short-term (6 weeks)
evaluations for toxicity, palatability, and weight gain. The chronic bioassays included the following con-
trol groups:   nonchlorinated humic acids  (0.5 g/1), 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 humic 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 acids tested contained direct-acting alkylating agents, based on their reactivity
with p-nitro-benzylpyridine, and showed mutagenic activity in 5. typhimwium. However, they did not
produce a significant increase in tumor incidences compared to controls when administered to mice at
high doses.

CITATION: In: Proceeding 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 69:109rll7, 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 Michael A. Pereira2

                                 Medical College of Ohio
                              2HERI^USEPA, Cincinnati, OH

The disinfection of drinking water is one of the most widely practiced and most successful public health
programs in the KS. While it has dramatically reduced the incidence of waterbome illness, it has been
determined 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 Agency's research into the
health effect of these contaminants.                            '-

The liver-tumor-promoting activities of chloroform (CHCb), 1,1-dichloroethane (1,1-DCE), and 1,2-
dichloroethane (1,2,-DCE) were examined in male B6C3F1 mice. Four-week-old mice received 10 mg/kg
diethylnitrosamine (DENA) in their drinking water for 4 weeks followed by continuous exposure to either
CHCb, 1,1-DCE, or 1,2-DCE in the drinking water. In both protocols, the three chlorinated compounds
were examined at two dose levels (maximum tolerated dose MTD and 1/3 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 examined grossly and histologically for hepatic
tumors. Neither CHCb, 1,1-DCE, nor 1,2-DCE increased the incidence of liver tumors in mice either fol-
lowing DENA initiation 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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 phenobarbitat only. These results contradict those previously reported for CHCb, 1,1-DCE, and 1,2-
 DCE when administered in corn oil to B6C3F1 mice by gavage.

 CITATION: la: Proceeding? of the Second International Symposium on Health Effects of Drinking Water
            Disinfectants  and Disinfection By-Pmducts,  Cincinnati,  Ohio,  August 27-29, 1986.
            Published in: Environmental 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 aver contamination of our water supplies with toxic chemicals requires that research be
done to investigate the extent and magnitude of the problem.  The present study provides a focus on water
contamination bymutagenic chemicals from a variety of sources.  This research also provides a perspective
on the relative importance 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: In: Basic and Applied Mutagenesis, Amir Muhammend 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. /. Bull, J. R. Meier, M. Robinson, H. P. Ringhand,
                               R. D. Laurie, andJ. A* Stober

                             HERL, USEPA, Cincinnati, OH

Over the past decade, much of the emphasis on drinking water contamination by tone pollutants has
shifted towards the chemicals which are produced as by-products of chlorine disinfection.  Although
trihalomethanes are the most widely recognized disinfection by-products, haloacetonitriles have also been
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Identified in recent years as major chlorination by-products. This study examines the mutagenic and car-
cinogenic properties of several of the haloacetonitriles. The results indicate some members of this class of
compounds display activity as.genotaxic agents in vitro and/or as tumor initiators for mouse skin in vivo.
This data is relevant to the Office of Drinking Water (ODW) in providing additional information on poten-
tial hazards posed by chlorination of drinking 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), trichloroacetonitrile (TCAN), bromochloro-
acetonitrile (BCAN), and dibromoacetonitrile (DBAN) were tested for their ability (1) to produce point
mutations in the Sabnonellamicrosome assay, (2) to induce sister chromatid exchanges (SCE) in Chinese
hamster ovary (CHO)  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 CHO cells in 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 (p<0.02). These data indicate that the haloacetonitriles do display mutagenic 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/. Pamell, Loren D. Roller, Jerry H. Exon, andJeanene M. Amzen

                                     University of Idaho

 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 ofwaterbome illness, it has been
 determined 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 Agency's research into the
 health effect of these contaminants.

 Trichloroacetic (TCA) and dichloroacetic acid (DCA) are the major nonvolatile chlorinated products
 formed during chlorination of water containing natural humic 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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WATER HEALTH
 don of hepatocytcs. .The metabolic relationship of TCA with trichloroethylene (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 toricity, im-
 munotoxitity, and initiation/promotion activity of TCA will be presented and discussed.

 CITATION: In: Proceeding? 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 69:73-79, November 1986.

 C104 XX-86-118 Project Officer: Laurie
     A COMBINED CARCINOGEN BIOASSAY UTILIZING BOTH the LUNG
                 ADENOMA and SKIN PAPILLOMA PROTOCOLS

                      Merrel Robinson? Richard J. Bull,2 Gary L. Knutsen?
                             Robert P. Shields*andJudy Stober1

                 1HERL,USEPA, Cincinnati, OH, Washington State University,
                       ^Pathology Associates, Inc.,4University of Florida

The results of the research described in the attached workshop proceedings article further 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.

To test the feasibility of employing a combined lung adenoma/skin papiUoma assay for broader detection
of chemical carcinogenesis than realized with either bioassay done separately, four strains of mice; SEN-
CAR, BALB/c, A/J and ICR-Swiss, were administered carcinogens by either the oral or I.P. routes. The
carcinogens administered were ethyl carbamate (EC), benzo(a)pyrene (BP), N-[4-(5-nitro-2-furyl)-
thiazolyl]-formamide (FANFT), and acryUunide (ACR). Starting two weeks later, l-Sjtg (depending on
strain) of 12-0-tetradecanoyl-phorboH3-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 A/J 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, FANFT 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,
animals were treated with a single oral dose of diethylnitrosamine (DEN) followed by tri-weekly applica-
tion of 1.0 pg TPA. This treatment resulted hi 51/57 animals developing lung adenomas vs. 5/57 in the
control animals. 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 maybe broadened using,
a combined bioassay that examines both lung and skin responses.

CITATION: Environmental Health Perspectives 68:141-146, September 1986.

C104MS-86-119 Project Officer: Robinson
    CARCINOGENIC POTENTIAL of ARSENIC COMPOUNDS in DRINKING
                                        WATER

                    Donald Y. Shirachi, Shing-Hui Tit, and John P. McGowan

                                  University of the Pacific

Tltis is a report of a study to determine if arsenic compounds are carcinogenic in the rat 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 realistic estimates of the carcinogenic risk that is associated with the
various forms of arsenic found in drinking water, and therefore aid in the regulation of arsenic compounds
under the Safe Drinking Water Act.

The primary objective of this research project was to determine whether arsenite (AsHI), 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 ranging from 10ppm-2560ppm for 7 weeks, depending upon the arsenic studied.  The ap-
parent MTD determined were: MA, SOOppm; Asm, 160ppm; AsV, 160ppm; and DMA, SOppm. The cal-
culated LDso were DMA, 98J5ppm; Asm, 264.8ppm; AsV, 294.5ppm; and MA, 1160.1ppm. 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 (30mg/kg i.p.) and on day 7 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 (p < 0.05) in the number of
animals exhibiting tumors in the kidney in the  Asm promoted group, 7/10, as compared to the DENA
controls, 2/9 when treated for 25 weeks. In both protocols AsIH 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-
            600/1-86-003, May 1986.

B107 HERL-0543 Project Officer: Robinson
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WATER HEALTH
        ASSOCIATION of CARCINOMA YIELD with EARLY PAPILLOMA
                         DEVELOPMENT in SENCAR MICE

              Richard J, Butt1, Merrel Robinson2, R. Dana Laurie2, and Judith A. Stober2

                                Washington State University
                               2HERL, USEPA, Cincinnati, OH

The results of the research described in the attached workshop proceedings article further characterizes the
SENCAR mouse and its advantages and disadvantages as an animal testing model in toxicological and
cardnogenesis 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 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 malignant tumors. The chemicals were administered as initiators by four different routes; orally, in-
traperitoneally, subcutaneoush/ and topically. Following the initiating dose of carcinogen, the animals
were subjected to triweekly, topical applications of 1 jig 12-0-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-!
ministration. However, no route was clearly superior in that different chemicals gave greater responses
by different routes. Papilloma yield at 24 weeks following the start of the promotion schedule was clear-
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 papilloma
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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Research Objectives
                       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 Rodentitide 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 significant. 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 synergjstic 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 against 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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PESTICIDES/TOXICS
TSCA also assigns to EPA responsibility for assuring the safety for human health and the environment of
genetically-engineered organisms. This responsibility includes engineered organisms created for use as
pesticides or for other purposes and products of those organisms.
EPA has authority to establish the ma««m'" acceptable levels of pesticide residues in foods and animal
feed called "tolerance levels," that will protect human 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, animals, and eventually to humans. Finally, some pesticides exhibit evidence of causing 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,  mutagenesis/carcinogenesis
   (genotoxic effects), neurotoricology 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 utilization and sensitivity of the test methods.

Approach: This research will utilize both in vivo and in vitro tests with various animal species.  Tests will
be run using 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 animal 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 animals 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 administration 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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RESEARCH OBJECTIVES
   sible Utter effects are being developed for the quantification of chemical-induced risk to the
   developing conceptus and postnatal animal.  Multispecles, multidose teratology bioassays 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. OFF will use these models and management systems for evaluating
 compounds submitted for registration.                           ;

 Approach: Various animal species and in vitro systems will be utilized as candidate models to assess
 human 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 biotlnated 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 OFF 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 L5178YTK+/" mouse lymphoma assay to detect genetic damage, and develop methods
   to improve the sensitivity and broad spectrum response of bioassay systems toward the major clas-
   ses  of  carcinogens.  In vitro methods will also  be  developed to determine  biochemical and
   physiological effects, and bioassays 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-
   neurotoxiclty 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 animals 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.
                                            -187-

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 RESEARCH OBJECTIVES
PESTICIDES/TOXICS
 • Provide lexicological data on the adverse effects of inhaled asbestos and asbestos-like fibers
   (erionite, chryssotile, and croddolite) including the correlation or 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 toxiclty.

 Rationale:  OTS 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 put in whole animals 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 morphometric data on the pul-
   monary acinus region for use in mathematical dosimetry models and to determine the relationship
   of indicators of neurotoxiclty to disease states  and/or neurobehavioral dysfunction.

 Rationale: To make judgements about the degree of risk, it is necessary to extrapolate from animals 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 animal species and cell cultures
 as well as associated studies to determine pharmocokinetics and biotransformation. Areas of study will
 include mutagenetidty/cartinogenicity, immunotoxictty, reproduction/teratology and inhalation toxicol-
 ogy.

 » Carry out epidemiology studies on human population exposed to compounds of priority interest to
   OTS.  Emphasis will be given to the evaluation, in exposed human  population, of laboratory-
   developed biomonitoring methods in the areas of genetic toxicology, neurotoxicology, developmen-
   tal toxicology/teratology  and immunotoxicology.  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
 human populations.  Other epidemiologic studies will be made of people occupationally- or environmen-
 tally-exposed to compounds of priority interest to OTS.

 • Develop a method  to correlate chemical structure with toxicologic effect, including genetic, car-
   cinogenic, and other toxic activities using pattern recognition, statistical and thermodynamic 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-
                                             -188-

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RESEARCH OBJECTIVES
 tivity relationships can be developed for health, they will be most useful in TSCA, Section 5, PMN actions
 because of the lack of 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 utilising a variety of short-term tests for genotoric 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, generate 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 organisms 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 microorganisms. Health
 concerns are implicit when the inserted genes express products that have lexicological, 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 establishment of data base management system for assisting in the evaluation of potential
 hazards of genetically engineered material will be useful to the Agency in protecting human health and
 the environment.

 Approach: A compilation of known modified organisms 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 organisms and may enable the prediction of characteristics of newly created or-
ganisms.
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                      Pesticides
                   Toxic Substances

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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 organopbosphorus-induced
 delayed neuropathy (OPIDN) to replace the traditional chicken model. .Acceptance of the rat model
 would enable die 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 examining 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 neurotoxtc esterase (NTE), the putative
 target enzyme for OPIDN in the hen, is bdeed 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 OF 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 similar 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 23% 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 young/adult 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 corneum 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
                                            -193-

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ACCOMPLISHMENTS
the qualitative profiles 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 vitro and in vivo assays
employed. These 9 compounds include the structurally similar organophosphate insecticides, acephate,
demeton, monocrotophos and trichlorfon; the phthalimide fungicide analogues, captan and foipet; 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 2,4-D, 2,4-DB
and 2,4,5-T. The remaining part of Category 2 (14 compounds) consists of four  structurally similar or-
ganophosphate  insecticides;  'three similar 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.  LD50 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, chlordimeform, dichlofenthion, 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 LD50 value over a 1-year
period was examined for two typical insecticides. Six consecutive bimonthly oral LD50 determinations
for parathion and DDT in adults of both sexes indicated that the LD50 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 hi 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.
                                            -194-

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PESTICIDES
Issue;   Biotechnology: Mlcrobial and Biochemical Pest Control Agents

In continuing studies on the potential human health hazard 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 BTI.  BTI synthesizes a multicomponent, proteinaceous
inclusion body during sporulation. This protein is cytotoxic to most cells and exerts myotoxic and
neurotaric effects in both insects and  fnammaig  in our study, the 28 kDa peptide, which contains the
mammalian toxicity factor, was removed by affinity chromatography with a monoclonal antibody and the
effectiveness of the remaining protein on mosquitocidal activity was examined. Our results indicate that
the 28 kDa peptide is not the major mosquitocidal protein contained in the BTI inclusion 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 mosquitocidal 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-
munological probes.
                                           -195-

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TOXICS
 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 changing gut flora. To'determine the magnitude and extent of chemical-microbial
 interaction as well as the ontogeny of microfloral enzyme activity in the developing animal, freshly voided
 feces are incubated with various substrates. Metabolites of substrates •y-hexachlorocyclohexane, p,p'-
 DDT, p-nitrophenyl-p-D-glucuronide, p-hitrophenyl sulfate, and 3,4-dichloronitrobenzene are analyzed
 by EC gas chromatography and are used to assess dechlorinase, dehydrochlorinase, {J-glucuronidase, aryl
 sulf atase, and nitroreductase activity. The sensitivity of the analysis permits use of short incubation times,
 low substrate concentrations and small fecal samples.

 HERL 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 human
 exposure and demonstrated animal carcinogenicity. However, its genetic activity has been enigmatic: 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. Similar 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 damage. This work has direct
 application to OPP, OTS, OSW and Superfund.

 HERL developed a new in vivo mammalian germ cell bioassay for screening chemical genotoxic effects
 and for probing mechanisms of chromosomal alterations.  The project began in 1980 and is continuing.
 This methodology addresses many Agency needs expressed in die recently prepared EPA Mutagenesis
 Guidelines and provides an important tool for hazard 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 technical
 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 significantly 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 ^-^S^'^S'hexachlorobiphenyl (PCS) 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 1.3% 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 pharmacokinetic 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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ACCOMPLISHMENTS
HERL has developed methods for assessing cellular immune responses in the lung which are particular-'
ly useful for measuring the immunotoxic effects of inhaled compounds.  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 tone agents.

Issue:   Health: Markers, Doslmetry and Extrapolation

HERL studies on the tumorigenesis of mineral fibers have emphasized development of an in vitro 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 croctdolite, UICC
chrysotile to Chinese hamster V79 cells was compared. The results indicate that the minerals were tone
by showing more than 50% toxicity for at least one dose between 10 and 100 jig. 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 dear 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 10-300^ mol/mg (1.9-56.4 rag/kg) of EDB 21 and 4 hours before testing.  Liver
toxicity studies included tests for DMA damage (alkaline elution) and biochemical effect (glutathione,
cytochrome P-450 and ornithine decarboxylase, and serum alanine transaminase).  EDB doses of 10^
mol/kg (1.9 mg/kg) and above caused DNA damage as determined by the alkaline elution technique.
Only the highest dose of EDB (300 jjtmol/kg, 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 damage
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 neurotoricity 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-
ing of a workshop on computer-based testing protocols for humans. The test, now called the NES battery,
is currently being validated hi populations of painters, alcoholics, and others with suspected neurotoxicity.
                                            -198-

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TOXICS
 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 neurotoxtcants. Plans are underway to include sections of the battery in the upcoming
 NHANES-III 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 examination 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 animal 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 profiles 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 Epldemlologic 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.  Exppsure 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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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 human 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 b 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 genomic
   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 vivo
   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 being, 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 premamifacture 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 b
1985 and is presently undergoing modification and usage b collaboration with program offices and for
additional chemical classes and biological endpobts. Since the establishment of this system, SAR
methods associating the mutagenicity and structure of many classes of compounds can aid b the setting
of priorities for the identification and quantitation of these compounds b ambient Air, Hazardous Waste,
Superfund, and OTS. This is expected to have a major impact b these program offices.

Issue:   Biotechnology

To provide data on the potential human health risk of mutant and genetically engineered microorganisms
that are or will be released bto the environment, the microorganisms that degrade hazardous compounds
(e.g., pesticides, polychlorinated 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 recombbant microorganisms is bebg
developed. A manuscript is currently bebg prepared that describes the isolation and characterization of
the Pseudomonas spp. that metabolize  chlordecone. Polychlorinated biphenyl (PCB's) degrading
                                           -200-

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TOXICS
microorganisms have been isolated from a commercially available product designed for chemical waste
PCB degradation.  The following microorganisms have been isolated from the product and identified:
two Bacillus subtillis strains, an Enterobacter cloacae strain, three P. aerugtnosa, 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. coli strain. A variety of other microbial species are now being engineered for use
in the environment. The EPA needs established methods for evaluating these microorganisms 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 examined for the presence of the organism. At the highest dose, the organism was recoverable
24 hours after dosing. No dosed microorganisms were recoverable at the two lower doses. Preliminary
work is underway to enumerate microorganisms from the mouse intestines.  Models such as these will
allow a more proper assessment and regulation of engineering organisms.
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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 italicized.

Other articles submitted for publication, but not published as yet, are listed hi Appendix A.

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 PUBLICATIONS
                                             PESTICIDES
       ACUTE TOXICITY of PESTICIDES in ADULT and WEANLING RATS

                             Thomas B. Gaines and Ralph E. Under

                          HERL, USEPA, Research Triangle Park, NC

 This paper is the third and final summary ofLDSO studies of pesticides in the Sherman rat. Fifty-seven
 chemicals were tested by the oral route in both sexes of adults. Of these chemicals, 41 were also tested by
 the dermal route and 36 were tested by the oral route in weanlings. Under this program the acute toaddty of
 a total of 157pesticidal chemicals have been studied using the same methodology and the same strain of
 rat.  This has provided an extensive body of information on the relative toaddty of pesticides. The informa-
 tion obtained in this study may be useful in categorizing the risk from acute accidental exposures and in for-
 mulating regulations to ensure safe handling and use of pesticidal chemicals.

 LD50 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 weanl-
 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, chlordimefonn,
 dichlofenthion, leptophos) were more toxic to females than to males whereas famphur 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 LD50 value over a 1-year period was examined for
 two typical insecticides. Six consecutive bimonthly oral LD50 determinations for parathion and DDT in
 adults of both sexes indicated that the LD50 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: Linder
        EFFECTS on the FETUS of MATERNAL BENOMYL EXPOSURE in
                            the PROTEIN-DEPRIVED RAT

                Frances J. Zeman,1 Elizabeth R. Hoogenboom,1 Robert J. Kavlock,2
                              • i
   and Janet L.  Semple

University of California-Davis
                         'HERL, USEPA, Research Triangle Park, NC

Standard risk 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 rat on the teratogenicity of a common fungicide. The incidence of
benomyl-induced central nervous system malformations was reduced in the protein deprived group, suggest-
ing a protective effect of malnutrition. These results are being pursued to determine possible-mechanisms.
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The separate and combined effects of protein deprivation and benomyl [(methyl 1- butylcarbomoyl)-2-
benzimidazole carbamate] exposure were studied in the pregnant rat fed a diet containing 24% (control)
or 8% (deficient) casein throughout gestation. Within each diet group, subgroups were gavaged at 31.2
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-85-215 Project Officer: Kavlock
        EFFECTS on the FETUS of MATERNAL NITROFEN EXPOSURE in
                            the PROTEIN-DEPRIVED RAT


               Frances J.Zeman,1 Elizabeth R-Hoogenboom,1 Carol Chase-Deesing1
                            Robert J. Kavlock,2 and Janet L. Semple1

                                University of California-Davis                       .         ;
                         2HERL, USEPA, Research Triangle Park, NC

Standard risk assessment procedures generally utilize data generated from animal bioassays in which the ex-
posedpopulations receive optimum nutritional intakes. This paper presents a portion of the data derived from
a research project examining whether the teratogenicity of pesticides can be altered in populations receiving
less optimal dietary intakes. Utilizing the teratogenic herbicide nitrofen as a prototype compound and a low
protein diet, this study demonstrates a synergism of the two insults in terms of kidney, intestinal and pul-
monary development. However, these interactions occurred in only severely malnourished dams 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 125 (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,  intestine 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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 live mechanism negated by nitrofen exposure. The data suggest also that kidney, intestine and lung are
 particularly affected. Effects, however, occur primarily in young of severely malnourished dams receiv-
 ing a relatively high dose of nitrofen.

 CITATION: Tcgdcoto® 38(1): 55-68, January 1986.

 E104 MS-85-214 Project Officer: Kavlock
   EFFECTS on the FETAL RAT INTESTINE of MATERNAL MALNUTRITION
            and EXPOSURE to NITROFEN (2,4-DICHLOROPHENYL^-
                               NITROPHENYL ETHER)


                Soltanali Mahboob* Elizabeth & Hoogenboom,1 Robert J. Kavlock,2
                                   and Frances J. Zeman1

                                University of California-Davis
                         2HERL,USEPA, Research Triangle Park, NC

Standard risk assessment procedures generally utilize data derived from animal bioassays in which the ex-
perimental subjects receive optimal nutritional intakes. In order to evaluate the potential effects of dietary
insufficiencies on teratogenic effects, studies were desiffted to examine the effects of protein deprivation on
the developmental taxicity of the herbicide nitrofen. A previous study demonstrated an interaction between
these two treatments on postnatal viability of exposed litters.  This study, although demonstrating marked
effects of both treatments, indicates that the intestinal tract alterations are not involved with the increased
postnatal mortality.

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 containing 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 123 mg nitrofen in corn oil/kg/day 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 villi per length of intestine (VMM). Protein (horseradish peroxidase) and lipid ab-
sorption were studied histochemically. 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  (IW) and length (IL),  reduced IW/BW and tW/D
ratios, VH, and VMM. The VW 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, Carcinogenesis, andMutagenesis 6(6): 45-57, January 1986.

E104 MS-85-213 Project Officer: Kavlock
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 ISOLATION of a PRECURSOR and a NASCENT CHAIN FORM of GLUCOSES-
   PHOSPHATE DEHYDROGENASE from RAT UTERUS and REGULATION of
                   PRECURSOR PROCESSING by ESTRADIOL

                         Audrey M. Cummings and Kenneth L. Barker

                        Texas Tech University Health Sciences Center

The data described in this paper provide information about the mechanisms by which estrogen regulates the
synthesis of a specific protein, gtucose-6-phosphate dehydrogenase, in rat uterus.  This information is im-
portant as baseline data on hormone mechanisms in the female reproductive tract. In addition, the data
have specific relevance to current research on the toxic effects ofestrogenic pesticides, such as methoxychlor
and kepone, on reproductive function in female rats.

SDS-potyacrylamide gel electrophoresis of anti-giucose-6-phosphatc dehydrogenase immunoprecipi-
tates 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 predominant 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 [^SJmethionine,
labeled proteins A, B and C were isolated byimmunoprecipitation and electrophoresis. Each protein was
individually co-digested with authentic, pHjmethionine-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-terminal 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 containing  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 predominant form of the enzyme and enhances
the rate of translational conversion of protein C into higher molecular weight forms.

CITATION: Biochimica et Biophysics Acta 880(2/3): 226-241, February 1986.

E104 MS-85-227 Project Officer Cummings
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 MEASUREMENT of EPIDIDYMAL SPERM MOTILITY as a TEST VARIABLE in
                                         theRAT

                    Ralph E, Under,1 Lillian F Strader,2 and W. Keith McElroy1

                         1HERL, USEPA, Research Triangle Park, NC
                                   2Northrop Services, Inc.

Effects an sperm motility in commonly used test species such as the rat are useful in evaluating effects on
the male reproductive system. This study examines variables that impact on the motility assay in dose-
response studies of chemical tenacity.  Standardization of sample collection and handling is emphasized as
a means of improving precision. Dose-related effects on sperm motility in rats treated with dinoseb (a pes-
ticide) and a-chlorohydrin are presented. The technique developed in this research will improve the quan-
titative assessment of sperm motility in the rat and contribute to improved methodology in the detection of
chemicals which cause reproductive changes in the male.

Several environmental contaminants, notably dibromochloropropane (Whorton et 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 emphasize the need for adequate testing of chemicals for effects on
the male reproductive system. Although important in clinical 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
       p 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 tracking of multiple motile spermatozoa is circumvented by counting only nonmotile cells. An
index of motility is calculated from the difference in the nonmotile 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-dinkrophenol), a testicular toxicant in the rat (Under et al., 1982) and a-
chlorohydrin (3-chloro-l,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: Under
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 NEW STANDARD GUIDE for PERFORMING the MOUSE LYMPHOMA ASSAY
                     for MAMMALIAN CELL MUTAGENICITY

                    D. dive,1 W. Caspary,2P.E.Kirby,3R.Krehl*M.Moans*
                                J. Novak,5 and T. J. Oberty,6

     Burroughs Wellcome Company, 2NIEHS, ^SITEK Research Laboratories, 4HERL, USEPA,
            Research Triangle Park, NC, ^The Upjohn Company, %li Lilly & Company

This paper is the result of an ASTM committee consensus on guidelines for performing the mouse fym-
phoma assay.  This assay is one of the most widely used for in vitro mammalian mutagenesis. The Office
of Toxic Substances (OTS) receives test data in the Premanufacturing Notice (PMN) process. These
guidelines were developed by representatives of six research laboratories 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 LS178Y/TK+/' 3.7.2C mouse Jymphoma
mutageniclty 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
mammaBan 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-
   malian cell mutagenicity test.

*  Provide generalized criteria by which researchers can evaluate if they are property performing, utiliz-
   ing, and interpreting this assay.

*  Provide criteria by which individuals responsible for evaluating mouse rymphoma 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 mammalian cell
   mutagenicity test.

CITATION: In: Proceedings of the American Society for Testing and Materials, 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, October 1985.

 The role of nutrition in the etiology of human 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 die 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 Conference on Antimutagenesis and Anticarcinogenesis, D. Shankel, P.
            Hartman, and A. Hollaender, eds., Plenum Press, New York, NY, 1986.

 E104MS-86-128 Project Officer: Sandhu
 NEUROTOXICOLOGY in the FETUS and CHILD - CONFERENCE SUMMARY


                                    Lawrence W. Reiter

                         HERL, USEPA, Research Triangle Park, NC

The conference "Neurotaxicology of the Fetus and Child" addressed issues of 1) normal development of the
nervous system; 2) methods for evaluating chemically-induced damage to the developing nervous system;
and 3) the effects of chemical exposures on the developing nervous system. Dr. Reiter was invited to sum-
marize the conference and this paper contains that summary.

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 organism.  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: Neurotoodcology 7(2): 665-670, May 1986.

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

Considerable evidence suggests that some substances display age-related neurotoxicity. The very young are
often most sensitive to neurotaodcants.   To enable EPA to predict and understand age-dependent
neurotoxicity, it is important to understand the consequences of disrupting major events in the development
of the nervous system. This article revwws recent findings on the development of the somatosensory system
in the rat and the implications of these findings for studies of developmental neurotoxicity.

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: Neumtoxkotogy 7(2): 169-182, May 1986.

E104MS-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

                          David J.Svendsgaard1 and Kevin M.Crofton2

                         1HERL, USEPA, Research Triangle Park, NC
                           2University of North Carolina-Chapel Hill

 Traditionally, it has been thought thatpyrethroidpesticides that cause convulsions accomplish this through
 action on the sodium channel m the brain.  Recently, it has been noted that certain pyrethroid pesticides
 bind strongty at a site m the brain that acts on the chloride channel  The paper discusses some mathemati-
 cal models and statistical methods to aid in determining whether the sodium channel action or the chloride
 channel action  is correct. The models are fit to neurobehavioral activity data from rats exposed to mixtures
 of both types of pyrethroids and drugs with known mechanism of action.  Such information is relevant to
 the Office Pesticide Programs (OPP).  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 changes are
 not involved in the production of clinical symptoms of interest that these toxins cause. By learning the
 mechanism 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 classified as being either Type I or Type H based upon
 several factors: (1) in vivo toxic  signs in rats (Verschoyle and Aldridge, 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 Narahashi, 1983); and (3) interactions at the •y-aminobutyric acid
 (GABA) receptor complex (Gammon et al., 1982; Lawrence and Casida, 1983). Structurally, the Type II
 pyrethroids usually contain an a-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 the American Statistical Association, October 1985.

 E104 MS-86-001 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 taxkity 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 (Le., neuroblastomas, gliomas, and pheochromocytomas) to organotypic explants
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provide rapid, inexpensive models for either screening putative neurotoodcants or addressing their
mechanism(s) of action. A chemical's toxic potential can best be addressed using a tier approach in which
neurotcodc assessment progresses from the simple, inexpensive and less specific assay (e.g., cell lines) to the
more complex, technically 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 tcodcity
studies, can strengthen our assessment of a chemical's neumtadc hazard.

Many lexicologists in academic, industrial and governmental sectors consider in vitro (i.e., cell and tissue
culture) alternatives to animal toxitity testing as inevitable given the sheer volume of new chemicals and
the time and cost confines of conventional methodologies (Roofe, 1971; Nardone, 1977; Tardiff, 1978;
Stammatti et al., 1981; Ekwall, 1983; Goldberg, 1984; Grisham and Smith, 1984). For neurotoricologists,
in vitro systems ranging from tumor cell lines (i.e., neuroblastomas, gUomas, and pheochromocytomas) to
organotypic explants provide rapid, inexpensive models for either screening putative neurotoricants 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 neurotoric end-points. Assuming this
accessibility, a chemical's toxic potential can best be addressed using a tier approach in which neurotoric
assessment progresses from the simple, inexpensive and less specific assay (e.g., cell lines) to the more
complex, technically 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 toricity, usually in
terms of cell viability. The next level of inquiry addresses the pathogenesis or mechanisms of injury and
is designed to describe neurotoxicity in morphological, biochemical, and in some cases, electrophysiologi-
cal terms.                                        •

CITATION: In: A Tex&ook of Neurotaxtcotogy, 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

                             Rosanne Novak1 and Stephanie Padilla2

                                   Northrop Services, Inc.
                          2HERL> USEPA, Research Triangle Park, NC

Organophosphate-induced delayed neuropathy (OPIDN), a distal axonopathy of the larger-diameter and
longer fibers of the central and peripheral nervous systems, is known to develop after acute or chronic ex-
posures to some organophosphorus (OP) esters. Studies of OPIDN in the chicken have led to the iden-
tification of a putative biochemical  lesion:  the inhibition and "aging' of a  carboxylesterase, termed
neurotoxic esterase or neuropathy target enzyme (NTE). The chicken is traditionally used as a model for
OPIDN.  Recent studies, however, have demonstrated that the rat does develop a neuropathy that is
topographically and qualitatively similar to the chicken model of OPIDN in the absence ofataxia. In order
to tighten the linkage between the research and regulatory sectors of the Agency by promoting a mammalian
species, rather Aon an avion species for toxtcity testing ofOPs, a validation of the rodent model of OPIDN
was begun. Previous work by research scientists at the Neurotoxkology Division had shown that, like the
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hen, the brain and spinal cord inhibition ofNTE correlates with the incidence of neuropathic response in
OP treated rats. The study described in this journal article was undertaken to investigate the similarities be-
tween rat and chicken NTE with respect to amount, inhibitor sensitivities and molecular wei&ti.  It was
found that the NTE from both species was very similar with only a few quantitative differences-a further
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)-hydrolyang carboxyiesterases showed that rat esterases were more sensitive than
chicken to paraoxon inhibition at concentrations <, 1 jiM and superimposable with chicken esterases at
concentrations of ZS-1000 jiM. Mipafox titration of the paraoxon-resistant esterases at a fixed paraoxon
concentration of 100 jiM (mipafox concentration: 0-1000 jiM) resulted in a mipafox ISO of 73 piM for
chicken brain NTE and 11.6 nM 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 similar showing broad,  slightly alkaline  optima from  pH 7.9 to 8.6
[HiJDiisopropyl phosphorofraoridate (DFP)-labeled NTE from the brains of both species bad an ap-
parent mol wt of 160,000 measured by sodium dodecyl sulfate pofyacrylamide gel electrophoresis. In con-
clusion, NTE from both species was very similar, with the mipafox ISO for rat NTE within the range of
reported values for chicken and human NTE, and the inhibitor parameters of the chicken NTE assay were
applicable for the rat NTE assay.

CITATION: Fundamental and Applied Toxicolo& 6(3): 464-471, April 1986.

E104 MS-85-201 Project Officer: PadUla
  THE CORRELATION BETWEEN NEUROTOXIC ESTERASE INHIBITION and
             MIPAFOX-INDUCED NEUROPATHIC DAMAGE in RATS


                   Bellina Veronesi,1 Stephanie S, Padilla,1 and Donald Lyerfy 2

                         1HERL, USEPA, Research Triangle Park, NC
                                  Northrop Services, Inc.

The ability to link a critical percent inhibition of NTE and neuropathology in the rat has been demonstrated
with another model organophosphate compound, Mipafox.  This demonstration has strengthened the
validity of the rodent model of OPIDN for the testing of putative neurotcodc compounds.

The correlation between neuropathic damage and inhibition of neurotoxic esterase or neuropathy target
enzyme  (NTE)  was  examined  in  rats acutely exposed to  Mipafox  (N^T-diisopropvlphos-
phorodiamidofluoridate), a neurotoxic organophospate,  Brain and spinal cord NTE activities were
measured in Long-Evans male rate 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 a 67% of control values produced severe ( £ 3) cervical cord
pathology in 85% of the rats. In contract, dosages of Mipafox ( s 5 mg/kg) which inhibited mean NTE
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activity in spinal cord £ 61% and brain £,60% produced this degree of cord damage in only 9% of the
animals.  These data indicate that a critical percentage of NTE inhibition in brain and spinal cord
sampled shortly after Mipafox exposure can predict neuropathic damage in rats several weeks later.

CITATION: Neurotcodcotogy 7(1): 207-216, April 1986.

E104MS-85-170 Project Officer: Veronesi               •   •         t
       PHENYLMETHYLSULFONYL FLUORIDE PROTECTS RATS from
                 MIPAFOX-INDUCED DELAYED NEUROPATHY •

                            Bellow Veronesi and Stephanie Padilla

                        HERL, USEPA, Research Triangle Park, NC

These experiements demonstrate that in the rat, as in more conventional animal models of organophos-
phorus tccdcity, the initiation of the delayed neuropathy OPIDN is a multistaged event involving inhibition
and aging of the target enzyme. Because of (his, animals can be protected from the neuropathy bypretreat-
ment with non-agreeable organophosphorus compound. These data strengthen the use of rats over chick-
ens as an alternative experimental 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 "aging" 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 against 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 (15 mg/kg, 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 ± 23%. 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 to Mipafox
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 a 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 damage 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 aging, and that these stages are experimentally separable.

CITATION: Toxicology and Applied Pharmacology 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


                     Bettina Veronesi,1 Stephanie Padilla,1 and Dale Newland 2

                         1HERL, USEPA, Research Triangle Park, NC
                                  Northrop Services, Inc.

 Triphenylphosphite is an organophosphorus compound chemically related to the model OPIDNproducing
 chemical, TOCP.  When injected into chickens, TPP produces symptoms commonly associated with
 OPIDN (e.g. ataxia, 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 parts, NTE is not inhibited and the pathology is clearly different from that seen
 when the animals are injected with TOCP.  These data indicate that TPP neuropathy is masked m the
 chicken when fust the conventional end-points of OPIDN are employed but clearly differentiated in the rat.

 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 ml/kg (1184 rag/kg) injections (sc) of
 TPP spaced 1 week apart and sampled for biochemical and neuropathological examination. At the time
 of sampling, rats displayed dysfunctional changes including tail rigidity, circling, and hindlimb paralysis.
 Neuropathic damage was confined to the lateral  and ventral columns of all spinal levels and consisted of
 myelin ellipsoids and giant axonal swelling filled with smooth endoplastic reticulum. Wallerian-like
 degeneration was observed in the spinal roots, the sciatic nerve, and tibial branches. Biochemical assess-
 ment of brain acetylcholinesterase (AChE) and neurotoxic esterase (NTE) activity was determined 1,4,
 24, and 72 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 Pharmacology 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 Robert C. MacPhail

                         HERL, USEPA, Research Triangle Park, NC

Formamidines represent an agriculturally and economically important class of pesticide of which chlor-
dimeform is widely considered the prototype agent. Using schedule-controlled performance as a measure of
cognitive function, this experiment showed that there are important differences in the behavioral effects of
fomamidines.  These results suggest there  are differences in the neural mechanism(s) by which for-
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mamidines produce behavioral effects. As a consequence, caution should be exercised in labeling effects as
being formamidine-like1" or chlordimeform-Ske\

Chlordimefonn (CDM), amitraz (AMZ), and formetanate (FMT) are members of the formamidine dass
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 daring 1-hr sessions to lever-press under a multiple fixed-interval (FI)-l-
min FI-5-min schedule of milk reinforcement.  Dose-effect determinations for each compound  ad-
ministered ip, 20 min presession, were carried out in each subject.  The dose ranges were: CDM HC1,
0.3-20 mg/kg; FMT HO, 0.03-0.75 mg/kg; and AMZ, 5-75 mg/kg. Under baseline conditions response
rates were higher under the FI-1-min than under FI-5-min, and index of curvature (IOC) values (a
measure of within-interval response patterning)'were generally higher under FI-5-min. All compounds
produced dose-dependent decreases in response  rate.  CDM significantly decreased only FI-1-min
response rates; a similar 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 both components, with IOC decreased more under FI-5-min than under FI-1- min. AMZ
produced significant decreases in IOC only under FI-5-min. 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. Formamidine pesticides produce differential effects of FI schedule-con-
trolled behavior that are in turn modulated by the parameter value of the FI schedule.

CITATION: Toxicology and Applied Pharmacology 84:315-324, October 1986.

E104 MS-85-112 Project Officer: MacPhail
  MICROSPORIDIAN TAXONOMY: APPLICATION OF ELECTROPHORETIC
                       and IMMUNOLOGICAL TECHNIQUES

                                      CY.Kawanishi

                         HERL, USEPA, Research Triangle Park, NC

The review summarizes the utility of electrophoretic and immunological methods for identifying and class-
ifying Microsporidia.  This is important for regulating microbial pesticides containing protozoans under
FIFRA for the following reasons: (1) The true taxonomic relationship of pesticide agents to similar mam-
malian pathogens is unclear due to the lack of definitive criteria for classification, (2) There is a need for
methods for detecting and identifying protozoan agents in test animals and environmental samples for
preregistration testing.  This review shows that electrophoretic and immunological parameters can be util-
ized for registration data interpretation.

A review of investigations utilizing 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 classify strains and closely related species. Certain
immunological 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: In: Proceedings of the 4th International Colloquium on Invertebrate Pathology, The Nether-
            lands, 1986.

 E104 MS-86-109 Project Officer: Kawanishi
       DEMONSTRATION of MULTIPLE ANTIGENIC DETERMINANTS on
           MYCOPLASMA PNEUMONIAS ATTACHMENT PROTEIN by
                           MONOCLONAL ANTIBODIES

                    P.Hu,lC.Huang,1 Y. Huang,2A. Collier,1 and W. Cfydel

                       University of North Carolina School of Medicine
                        2HERL, USEPA, Research Triangle Park, NC

 This study contributed to the basic knowledge of monclonal antibodies and their antigenic determinant
recognition. The development of mouse clonal antibodies is a major part of our biorational pesticides
program.  It provides a more specific and quick assay to determined the microbial pesticides. In addition,
Mycoplasma pneumoniae which causes respiratory infection,  has been used as infectivity model, to study
the effect of air pollutant on diseased animals.                                         \

Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been
identified by limited proteolytic cleavage using specific monoclonal antibodies. Western blots prepared
from the gek containing the cleaved fragments were probed with antiserum against M. pneumoniae or
monclonal antibodies. Five distinct bands with intact antigenic determinants were detected by the an-
tiserum of which two band were each readable 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.

E104MS-85-169 Project Officer: Huang
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    IMMUNOBLOT ANALYSIS of EXOSPORE POLYPEPTIDES from SOME
                        ENTOMOPHILICMICROSPORIDIA
                      4                      •.                          .<             *
                  W. S. Irby,l Y. 5. Huang,2 C. Y. Kawanishi2 andW.M. Brooks *

                              ^orth Carolina State University
                        2HERL, USEPA, Research Triangle Park, NC

A basic problem with many dosses of microbialpesticidal agents is the lack of reliable indentification and
quantification techniques that are usable in preregistration hazard evaluation tests. The Microsporidia, a
protozoan group to which a registered pestiddal agent belong, is one such group.  In this feasibility study,
microsporidians were tested to determine' whether enough antigenic differences occurred 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 by immunoblot
analysis.  Exospore polypeptides from 2 Nosema spp., three Vairimorpha spp., and 2 undescribed
Vammojpfio-like spp. were analyzed. Gel electrophoresis and immunoblot analysis revealed that a
variety of polypeptides, most between 15,000 and 90,000 molecular weight, were present on th exospore.
The three Vairimorpha spp. were highly related scribed spp. The two Nosema spp. immunologically dis-
tant from each other and from the Vairimorpha spp. Indirect evidence, however, indicated that may in-
ternal spore polypeptides present in both genera are similar. Cross reactivity between exospore polypep-
tides from entomophilic microsporidia and antisera to a mammalian microsporidhim, 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 microsporidia are sufficiently diverse to be of immunodiagnostic value.

CITATION:  Journal of Protozoology 33(1): 14-20, January 1986.

E104MS-85-110 Project Officer: Kawanishi                         -    '          '
PESTICIDE POISONINGS REPORTED by FLORIDA CITRUS FIELDWORKERS

                       Jack Griffith,1 Robert Duncan,2 and Janet Konefal2

                        1HERL, USEPA, Research Triangle Park, NC
                         2University of Miami(FL) School of Medicine

The Office of Pesticide Programs requires scientific data to support or reject the registration of pesticide
products, or the suspension or cancellation of existing products,

In a 1981 survey of 1811 Florida citrus fieldworkers, 25 pesticide related poisoning incidents involving 29
fieldworkers were reported. Suspected poisonings were categorized into possible and confirmed poison-
ings, and from these reports it was possible to project an estimated 438 possible poisonings, and 73 con-
firmed poisonings among all citrus fieldworkers. Confirmed pesticide poisonings were developed into an
incidence rate of 34 poisonings per 10,000 permanent and semi-permanent fieldworkers. The number of
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 possible and confirmed poisonings, for all fieldworkers, was then developed into an incidence rate of 160
 poisonings 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 CHLOROBENZILATE RESIDUES in CITRUS FIELDWORKERS

                             Jack Griffith1 and Robert C Duncan2

                        1HERL> USEPA, Research Triangle Park, NC
                            University of Miami School of Medicine

 The Office of Pesticide Programs (OPP) requires scientific data to support or reject the registration of pes-
 ticide products, or the suspension or cancellation of existing products.

 In May 1976 the U.S. Environmental Protection Agency (EPA) issued a notice of rebuttable presumption
 against 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 chlorobenzilate, 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
 et 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'-dichlorobenzophenone (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

                          Robert C Duncanl and Jack Griffith 2

                         University of Miami School of Medicine
                       2HERL, USEPA, Research Triangle Park, NC

The Office of Pesticide Programs (OPP) requires scientific data to support or reject the registration of pes-
ticide products, or the suspension or cancellation of easting products.

A cross-sectional monitoring study was made of citrus fieldworkers employed during an entire citrus
growing season in Florida. A survey questionnaire was administered to 1,811 fieldworkers 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 fieldworkers 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 DETP 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 fieldworkers, 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 Toxicology and Environmental Health 16(3&4): 509-521, November 1985.

E104 MS-86-184 Project Officer: Griffith
    COMPARISON of PLASMA CHOLINESTERASE DEPRESSION AMONG
    WORKERS OCCUPATIONALLY EXPOSED to ORGANOPHOSPHORUS
               PESTICIDES as REPORTED by VARIOUS STUDIES

                    Robert C Duncan,1 Jack Griffith,2 and Janet Konefal *

                         1University of Miami School of Medicine
                       2HERL, USEPA, Research Triangle Park, NC

The Office of Pesticide Programs (OPP) requires scientific data to support or reject the registration of pes-
ticide products, or the suspension or cancellation of existing 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 insight
into the use history—c.g., mixing, loading, application, and harvesting—that might lead to a hazardous
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 (Le., 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 fltilfojng 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 Toodcologf and Environmental Health 18(1): 1-11, May 1986.

£104 MS-86-183 Project Officer: Griffith
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                MEIOTIC ORIGIN of ANEUPLOIDY: AN OVERVIEW

                             Lorraine C. Backer and James W. Allen

                          HERL, USEPA, Research Triangle Park, NC

 Aneuploidy arising at meiosis is associated with spontaneous abortions and various disabling syndromes of
 livebom.  The clinical significance of this cytogenetic defect has become increasingly recognized, as
 evidenced by the Agency's Mutagenesis Guidelines and the 1985 Symposium on Aneuploidy. Yet, very little
 is understood about toe mechanistic aspects of aneuploidy generation in germ cells.  This chapter sum-
 marizes some of toe 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 mechanism.  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 similar 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 sumr"a"7e 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).

 L104MS-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. Carrano,3 and R. Julian Preston 4

                        1HERL, USEPA, Research Triangle Park, NC
                                2University of Texas-Houston
                          ^Lawrence Livermore National Laboratory
                              4Oak Ridge National Laboratory

Germ 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
reports concerning chemical-induced aneuploidy in mammalian male germ cells and recommendations for
further work. It is a major review coordinated with, or responsive to, broad Agency efforts (aneuploidy sym-
posium, 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 mammalian 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 embryonic 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 chiasma frequencies, univalent frequencies, and spermatid/sperm sex
chromosome body counts.

CITATION: Mutation Research 167(1&2): 123-137, January/March 1986.

L104MS-S5-239 Project Officer: Allen
             DETECTION of CHEMICALLY INDUCED ANEUPLOIDY
                             with PLANT TEST SYSTEMS

                  ShahbegS. Sandhu,1 Baldev K. Vig,2 and Milton J. Constantin 3

                         1HERL, USEPA, Research Triangle Park, NC
                                2University of Nevada-Reno
                                  'Phyton Technologies, Inc.

The utility of plant test systems far detecting chemically-induced aneuploidy was evaluated by using papers
published in peer-review journals. A total of 147 papers was provided to the group by the Environmental
Mutagen Information Center. Based on the criteria established by the Gene-Tax Committee (Waters and
Auletta, 1981), 22 papers were selected for in-depth review. EPA has an active program to develop and
apply short-term bioassays to detect and characterize chemically-induced aneuploidy.  This review will
provide scientific information 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
cytogenedc analysis, only a few  have been employed for this purpose.  In this review, Allium cepa was
found to be the most commonly used test system. Other species used less frequently were Viciafabe, Hor-
deum vulqare, Sorgham vulgare, and Pennisetum americanum. 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 for detecting chromosomal malsegregation in meiotic or mitotic cells
is presented.

CITATION: Mutation Research 167(1&2): 61-69, January/March 1986.

L104 MS-8S-146 Project Officer: Sandhu
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        INHIBITION of AROMATASE CYTOCHROME P-450 (ESTROGEN
          SYNTHETHASE)  by DERIVATIVES of a-NAPHTHOFLAVONE

                  James T. KelKs, Jr.,1 Stephen Nesnow,2 and Larry E. Vickeryl

                               University of California-Irvine
                        2HERL, USEPA, Research Triangle Park, NC

One of the most important enzyme systems in the mammal is the cytochrome P-450 complex.  This system
usually junctions as a detoxification system removing harmful foreign chemicals and drugs 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 cytochrome P-450 enzyme Junctions: the conversion ofandrogens to estrogens. 7,8-
Benzoflavone, a material similar to dietary flavones found in citrus fruits, was used to study this important
enzyme complex.

a-Naphthoflavone (ANF; 7,8-benzoflavone) is a potent competitive inhibitor of human aromatase
cytochrome P-450 [J. T. Kellis, Jr. and L. E. Vickery, Science 225:1032,1984].  We have further inves-
tigated inhibition of aromatase by several derivatives of ANF. Using human placenta! microsomes and
40nM androstenedione as substrate, the compounds tested and their 150 values were: ANF, 0.07(iM; 2-
(2-naphthyl)-4//-naphtho[l12-b]pyran-4-one, l.OpJVl; 7,8-benzoisoflavone, «=100jxM; and 2-phenyl-4/f-
naphtho[l,2b]furan, > lOOjiM. 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 exocyclic 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 (Iso = 20 nM).
Inhibition by 9-hydroxy-ANF was competitive, and its K{ value of 5 nM indicates a higher affinity for the
enzyme than the natural steroid subtrates- theXm values for androstenedione and testosterone under
these conditions are 10 and 80 nM respectively. 9-Hydroxy-ANF also induced a change in the absorption
spectrum of the aromatase cytochrome P-450 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 1986. -

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

                 George G. Hatch,1 Patricia M. Conklin,1 Carlo C Christansen,1
                 Tara M.Anderson,1 Robert Langenbach,2 and Stephen Nesnow2

                                Northrop Services, Inc.
                       2HERL, USEPA, Research Triangle Park, NC

Ethylene oxide is a high volume production organic intermediate which is used, in addition as feedstock
item, as a sterilization agent for Pharmaceuticals and related materials. Hospital and industrial workers
can be exposed to high levels of this material.  This study determined the mutagenic and oncogmic poten-
tial of ethylene oxide using mammalian cells as target ecus and exposing these cells to the gaseous form of
ethylene oxide. These results of this study strongfy suggest that ethylene oxide is mutagenic and potentially
carcinogenic to mammalian cells.

Ethylene oxide is a classical mutagen and a carcinogen based on evidence from studies in experimental
animals. It is widely distributed in industrial, research, hospital, and food environments. In an effort to
explore the use  of newly developed methods for exposing mammalian 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. Similarly, primary Syrian hamster 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 nontoric concentrations. After 2-hr ethylene oxide treatment at 625-2,500 ppm a significant en-
hancement of virus transformation was observed. At 20 hr after treatment no enhancement was observed.
Treatment of hamster cells with ethylene oxide hi both bioassay systems yielded concentration-related,
quantitative results.

CITATION: Environmental Mutagenesis 8:67-76, January 1986.

L104 MS-83-101 Project Officer: Nesnow
   EVALUATION of the EFFECT of AGAR on the RESULTS OBTAINED in the
                       L5178Y MOUSE LYMPHOMA ASSAY

                 Mary Meyer,1 Karen Brock,l Kay Lawrence,1 Bruce Casto, 1and
                                   Martha M. Moore2

                      Environmental Health Research and Testing, Inc.
                        2HERL, USEPA, Research Triangle Park, NC

The.LSlTSYmouse tymphoma assay is one of the most widely used in vitro mammalian mutagenesis as-
says.  This assay is particularly useful 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 •
 cells — and resulted in a significant technical improvement in the assay.
• the agar used for cloning
 The L5178Y TK+/" moose lymphoma assay is widely used in mutagenicity testing, Trifluorothymidine-
 resistant (TFT1) mutants are quantitated following growth in agar-supplemented cloning 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 cloned 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 S3 small colonies) and 69 mutants from
 untreated cultures (24 large colonies and 45 small colonies) and tested them for TFT 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.

 L104MS-86-006 Project Officer: Moore
            GENOTOXICITY STUDIES of BENZ(L)ACEANTHRYLENE

                   A.D. KKgerman,lM.M. Moore,2 G.L. Erexson,1 K.H. Brock,1
                            CLDoerr,1 J.W.Allen* and S.Nesnon?    '

                       Environmental Health Research and Testing, Inc.
                         2HERL, USEPA, Research Triangle Park, NC

Benz(L)aceanlhrylene is a member of a unique class ofpofycyclic aromatic hydrocarbons, the cyclopenta-
fused PAHs. Various cyclopenta-fused PAHs have been identified as air pollutants andB(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 information on the pofycyclic aromatic hydrocarbons.

The  genotoxicity of the cyclopenta-fused polycyclic  aromatic hydrocarbon, benz(L)aceanthrylene
[B(L)A], was evaluated in vitro using the L5178Y/TK+/" mouse lymphoma assay and in vivo using the
mouse peripheral blood lymphocyte (PBL) culture system. The mutagenicity and sister chromatid ex-
change (SCE) inducing potential of B(L)A was then compared to that of benzo(a)pyrene [B(a)P]. 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 dastogenic as well as mutagenic.  Gross
chromosome aberration analysis of treated L5178Y/TK+/" mouse lymphoma cells confirmed the clas-
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togenicity of B(L)A in vitro. In the mouse PBL system, after administration by gavage, B(L)A was more
cytoloxic and produced a sharper elevation b SCE frequency than B(a)P.

CITATION: Cancer Letters 31(2): 123-131, May 1986.

L104 MS-86-076 Project Officer: Moore
                   GENOTOXICITY of GAMMA IRRADIATION in
                        L5178Y MOUSE LYMPHOMA CELLS

                  Martha M. Moore,1 Amanda Amtower,2 GaryH.S. Strauss,1 and
                                      Carolyn Doerr2

                         1HERL, USEPA, Research Triangle Park, NC
                       Environmental Health Research and Testing, Inc.

 We have been investigating the ability of the L5178Y mouse fymphoma assay to detect and differentiate
 compounds which induce single gene mutations and chromosomal mutations. Gamma irradiation is con-
 sidered (he "classic" agent which induces chromosome mutations.  These studies confirm our hypothesis
 that the assay can detect both types of damage and thus is important to the Office of Toxic Substances
 (OTS) at evaluating test data from this assay.

 The L5178Y mouse tymphoma assay has been widely used in short-term mutagenicity testing. Research
 into the types of genetic damage detected at the thymidine kinase 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 damage is based upon the observation that
 the mutants can be separated by colony size, using an automatic colony counter, into a, small colonies and
 X, large colonies.   In addition a mutants  appear to represent chromosomal alterations to that
 chromosome 11 carrying the TK locus, while X mutants may represent smaller-scale, single-gene muta-
 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 X mutant frequencies. Several compounds known
 to be clastogens have already been shown to induce a significant proportion of a mutants.  These include
 methyl   methanesulfonate,     benzo(a)pyrene,   2-acetylaminofluorene,   N-methyl-N'-nitro-N-
 nitrosoguanidine, and dimethylnitrosamine. A study of classic clastogenic agents would be incomplete
 without evaluating either X-ray or gamma irradiation. We report here the results of experiments analyz-
 ing gross aberrations, sister chromatid exchange, and thymidine kinase gene mutation (including a and X
 mutant frequency) following exposure of TK   3.7.2C mouse lymphoma cells to gamma irradiation.

 CITATION: Mutation Research 174(2): 149-154, June 1986.

 L104MS-86-004 Project Officer: Moore
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 SURVIVAL CURVES and INCIDENCE of NEOPLASTIC and NON-NEOPLASTIC
                            DISEASE in SENCAR MICE


              CJ. Conti,l N. Oapp,2AJJt. Klein-Szanto,l S. Nesnow,3 and T, J. Slaga1

                       *The University of Texas System Cancer Center       *•
                           .2Oak Ridge Associated Universities
                       3HERL, USEPA, Research Triangle Park, NC

 The SENCAR mouse has been used by HERL  to develop an extensive data base on the tumorigmic
properties 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 Ah- Quality Planning and Standards  (OAQPS).  This paper describes background studies on spon-
 taneous tumor formation and survival of this sensitive strain of mice which will provide invaluable informa-
 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 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-
 rinogenesis has not produced a concomitant increase in the incidence of spontaneous neoplastic and non-
neoplastic disease.

CITATION: Cardnogenesis 6(11): 1649-1652, October 1985.

A101/L104 MS-86-052 Project Officer: Nesnow
   COMPARISON of the TUMORIGENIC RESPONSE of SENCAR and C57BL/6
          MICE to BENZO(a)PYRENE and the INTEREXPERIMENTAL
                  VARIABILITY OVER a THREE-YEAR PERIOD
                                       »

                   Stephen Nesnow,1 Hinda Bergman,1 and Thomas f. Slaga2

                       ^HERL, USEPA, Research Triangle Park, NC
                       2The University of Texas System Cancer Center

This paper describes an extensive data base on the historical tumor incidence and histopathologtc charac-
terization of tumors found in the carcinogen sensitive SENCAR mouse. The SENCAR mouse has been used
extensively to characterize the tumorigenic properties of complex mixtures and pure chemicals particularly
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those associated with automotive exhaust The knowledge of both the range of expected tumor incidence for
standard reference carcinogens and the range of spontaneous tumor incidence data is crucial to interpreting
test data pom this mouse.  This work wins supported in part by the Office of Air Quality Planning and Stan-
dards (OAQPS) and the Office of Toxic Substances (OTS) and is used in the lexicological evaluation of en-
vironmental chemicals.     .                   .                ...

SENCAR and C57BL/6 mice were compared for their ability to produce tumors after benzo(a)pyrcne
[B(a)P] initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion.  SENCAR mice in-
itiated with 101 jig/mouse B(a)P and promoted with TPA (2 (jig/mouse, twice weekly) produced large
numbers of papillomas, whereas CS7BL/6 mice produced none after 26 weeks of promotion. Continued
treatment of the B(a)P-initiated C57BL/6 mice with TPA up to 52 weeks did not induce any papillomas
nor did higher doses of B(a)P. Application of increased doses of TPA (10 jig/mouse, twice weekly) to
B(a)P-initiated C57BL/6 mice (404 u,g/mouse) for 50 weeks produced few papillomas. Substantial papil-
loma formation in C57BL/6 mice was observed after weekly treatment with B(a)P (101 fig/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 mammal papilloma formation occurring
12 to 13 weeks earlier. Therefore, C57BL/6 mice exposed to B(a)P are capable of producing papillomas
under certain  experimental conditions.   The  inter-experimental  variability of B(a)P-induced (50.5
jig/mouse) papilloma formation after 30 weeks of TPA promotion (2 tig/mouse, twice weekly) was ex-
amined in SENCAR mice over a 37-month period. Low statistical variation was observed in papilloma
multiplicity, papilloma incidence, or papilloma latency. Male and female SENCAR mice produced equal
values in the three parameters: 4.4 ± 1.6 papillomas/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 com-
parisons of mouse skin tumor initiation results between SENCAR and other strains or stocks of mice and
with other chemicals.

CITATION: Environmental Health Perspectives 68:19-25, September 1986.

L104 MS-85-172 Project Officer: Nesnow
                   CYTOGENETIC CHARACTERIZATION of the
              L5178Y TK+/' 3.7 JC MOUSE LYMPHOMA CELL LINE

                Jeffrey Sawyer, * Martha M. Moore? Donald Clive? and John Hozier1
                                      Institute of Technology
                        2HERL, USEPA, Research Triangle Park, NC
                              Burroughs Wellcome Company

The mouse fymphoma assay is one of the most widely used in vitro mammalian mutagenesis assays.  In
order to interpret test data from this assay (which is often submitted to the Office of Toxic Substances for
Premanufacturing Notices (PMNs)), it is necessary to understand the genetic basis for the assay. These two
papers provide information which characterize 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,
 utilizing 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 staining 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 (TK) locus in this cell line. Chromosome analysis of the TK+/" 3.7.2C cell
 line and derived TK"'" mutants has become important in demonstrating that the TK+/TK"/* assay may
 detect and Hi$ri«gtiigli 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 L5178YTKV'
                 MUTANTS EARLY in THEIR CLONAL HISTORY

                         John Horier,1 Jeffrey Sawyer,1 Donald Clive,2
                                  and Martha M. Moore3
                                      Institute of Technology
                               Burroughs Wellcome Company
                         3HERL, USEPA, Research Triangle Park, NC

 The mouse lymphoma 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 Toxic 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 mutants induced in the assay.

 We have developed a cytogenetic technique that allows observation of chromosome rearrangements as-
 sociated with TiT' mutagenesis of the L5178Y/TK"1"'* 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 (a) mutants
 studied have chromosome 11 rearrangements (the chromosome containing the thymidine kinase gene)
 while large-colony (X) mutants do not have detectable chromosome rearrangements. In addition, we find
 among the chromosome abnormalities in                 •

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


           /. M. Siegfried? K. Rudo,1 B. J. Bryant? S. Ellis,1 M. J. Mass,zandS. Nesnow2

                      Environmental Health Research and Testing, Inc.
          '              2HERL,USEPA, Research Triangle Park, NC

 This manuscript describes the metabolism of the environmental carcinogen benzo(a)pyrene (BP) in cell
 cultures of bronchial epithelium from humans. Large interindividual variations in metabolic profiles and
 metabolic rates were found which did not correlate with the cytotcodcity of BP to the cultured cells. The
 paper is relevant to Program Office needs on metabolism of carcinogenic xenobiotics.

 Benzo(a)pyrene [B(a)P] metabolism was measured in monolayer cultures of human bronchial epithelial
 cells derived from 18 specimens of explanted tissue. Bronchial epithelial cells converted B(a)P to
 dihydrodiols, phenols, quinone derivatives, and polyhydrpxylated 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
 epithelial 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: CancerResearch 46:4368-4371, September 1986.

 L104 MS-86^081 Project Officer:  Mass
    PRENEOPLASTIC TRANSFORMATION of RESPIRATORY EPITHELIAL
        CELLS by COMPLEX ORGANIC MIXTURES in a CLONAL ASSAY

                       M. J. Mass,1C M. Marr, HI? andJ. L. Mumford1

                        ^RL, USEPA, Research Triangle Park, NC
                      Environmental Health Research and Testing,* Inc.

The paper describes the use of an in vitro rat tracheal cell transformation assay to assess carcinogenidty of
complex mixtures from heating fuel emissions. The assay was able to detect carcinogenic activity in three
fuel exhaust extracts but the relative potencies of the extracts could not be determined because the complex
mixtures were not completely soluble. The assay is one tool that might be used in assessing health risks of
combustion products.
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 In this study complex organic mixtures that were extracts of paniculate emissions from 3 heating sources
 were tested for the presence of agents that induce preneoplastic transformation of rat trachea! epithelial
 (RTE) cells in an in vitro 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 "smokey" coal in an
 open indoor pit as the sole source of heat and cooking fuel under unvented conditions. Dichloromethane
 extracts of particulate  samples (<10 um in size) of "smokey" 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 "smokeless" coal, and wood as fuels.
 These samples were also analyzed by gas chromatography/mass spectrometry for polynudear aromatic
 hydrocarbon (PAH):content. The organic extracts of smokey coal emissions produced significant in-
 creases in the number of transformed RTE cells exposed in vitro, in a reproducible manner. 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 similar, 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: In: Health and Environmental Research on Complex Organic Mixtures, in press.

 L104 MS-86-011 Project Officer: Mass
   QUANTITATIVE ANALYSIS of the METABOLISM of BENZO(a)PYRENE by
      TRANSFORMABLE C3H10T1/2CL8 MOUSE EMBRYO FIBROBLASTS

                     Kenneth Kudo,1 Scott Ellis,1 BJ. Bryant,1 Kay Lawrence,2
                     Gaynetle Curtis,1 Helen Garland,2 and Stephen Nesnow2,

                       Environmental Health Research and Testing, Inc. '
                         2HERL, USEPA, Research Triangle Park, NC

One of the major short-term bioassays which predict animal carcinogenicity is the C3H10T1/2CL8 mouse
embryo transformation bioassay.  This assay is used by the Office of Pesticides and Toxic Substances
(OPTS) to explore the genotaac potentials of chemicals.  This study fully characterizes the metabolic
capacity of these cells for future use of this bioassay system.

The metabolism of benzo(a)pyrene [B(a)P] to organic soluble and water soluble metabolites by transfor-
mable C3H10T1/2CL8 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 iiM. As cell density increased,
the metabolic rate decreased for organic-soluble and water-soluble products between 6,300 and 54,000
cells/cm2 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 p-glucuronidase and arjd sulfatase to identify specific conjugated products. The sulfate
conjugated metabolites identified were B(a)P-7,8-diot, B(a)P-pre-9,10-diols, B(a)P-9,10-diol, and B(a)P-
3,6-quinone. The f)-glucuronic acid metabolites identified were B(a)P-pre-9,10-diok, 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 C3H10T1/2 cells with respect to incubation time and cell density.

CITATION: Teratogenesis, Carcinogenesis, and Mutagenesis 6(4): 307-319, August 1986.
                                       •»
L104 MS-85-178 Project Officer: Nesnow
 INVESTIGATION of POSSIBLE AGE EFFECTS on MEIOTIC CHROMOSOMAL
        RECOMBINATION and SEGREGATION in ARMENIAN HAMSTER
                                  SPERMATOCYTES

                           James W. Allen and Carolyn W. Gwaltney

                         HERL, USEPA, Research Triangle Park, NC

Aneuploidy in gem cells is of particular concern for its heritable clinical consequences.  Age effects upon
meiotic  chromosomal recombination  and segregation have been shown to be important in female
aneuploidy and are suspect in male aneuploidy. The present study has attempted to define such age effects
in male Armenian hamsters.  These animals are being used for various mechanistic studies concerning age
and mutagen influences to result in aneuploidy induction. This research area is relevant to program offices
concerned with risk assessment, in particular, toxic chemical effects and heritable mutagenesis.

Male Armenian hamsters were evaluated for age effects upon meiotic recombination and aneuploidy in-
cidence. Primary spermatocytes from young and old animals revealed similar chiasma frequencies.  The
incidence of terminal-type chiasmata in sex bivalents was also measured and found to be unaffected by
age. Percentages of univalents and of hyperploid metaphase n cells were evaluated by chromosome
groups based upon individual chromosome morphologies.  Regardless of age, the larger dichiasmate
chromosomes were seldom  observed as univalents, or as extra chromosomes in hyperploid cells.  The
monochiasmate small autosomes and sex chromosomes revealed univalent frequencies  approximating to
5.5-63% 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 animals (0.9% incidence in young and 2.4% in old hamsters). The events
leading to higher aneuploidy levels hi older male Armenian hamsters are not dear. 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 mechanisms of age effects to increase
aneuploidy levels is indicated.

CITATION:  Cytobios 43(172): 225-232, December 1985.

L104MS-85-238 Project Officer: Allen
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  CONSERVED CHROMOSOMAL POSITIONS of DUAL DOMAINS of the £75
                PROTOONCOGENE in CATS, MICE and HUMANS

          Dennis K. Watson,1 MaryJ. McWUliams-Smith,1 Christine Kozak,2 Roger Reeves,3
              John Geamart? Michael F. Nunn,4 William Nash,1 John R. Fowle, III,5
                    Peter Duesberg4 Tolas S. Papas,1 and Stephen /. O'Brien1

                                National Cancer Institute
                     2National Institute of Allergy and Infectious Diseases
                          3The Johns Hopkins University Hospital
                             ''University of California-Berkeley
                             5OHR, USEPA, Washington, DC

Transforming genes, or protooncogenes, are cellular cancer genes that may play an important role in
tumorigenesis. There are a limited number of them described to date, and each exists in a number of af-
ferent species, including humans. This paper contributes to the knowledge of the chromosomal location of
the ets cancer gene. Knowledge of the chromosomal location of cancer genes in conjunction with informa-
tion  about metabolic function and possible role  in carcinogenesis is essential for understanding
mechanisms of chemically induced carcinogenesis and for assessing risk.  Risk assessments  based on
molecular understanding of carcinogenesis will enable better regulatory decision-making under TSCA (e.g.,
Section 6) and FIFRA(e#, Section 3).

The mammalian protooncogene homologue of the avian v-est sequence from the E26 retrovirus consists
of two sequentially distinct domains located on different chromosomes. Using somatic cell hybrid panels,
we have mapped the mammaliam homologue of the 5* v-etr-domain to chromosome 11 (J5T51) in man, to
chromosome 9 (ETS-1) in mouse, and to chromosome Dl (ETS1) in the domestic cat. The mammalian
homologue of the 3" v-ets domain was similary mapped to human chromosome 21 (£732),  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 mammalian or-
ders indicate that these two genes have been separate since before the evolutionary divergence of mam-
mals.

CITATION: Proceedings of Ote National Academy of Science 83:1792-1796, March 1986.

L104 MS-86-218 Project Officer: Fowle
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  MORPHOLOGICAL TRANSFORMATION and CHROMOSOME DAMAGE by
                  AMSACRINE in C3H/10T1/2 CLONE 8 CELLS

                 Lynnette R. Ferguson,l Pierre van Zijl,1 and Stephen Nesnow 2

                  1University of Auckland School of Medicine, New Zealand
                       2HERL, USEPA, Research Triangle Park, NC

Morphological transformation of mammalian cells is a process which is closefy allied to the tumorigenic
process in mammals. The study of the effects of chemicals in morphological tranformation bioassays can
give insight into the processes by which the chemical induces tumors in animals. This study examines the
effects of amsacrine, a unique antitumor agent which has structural similarities to some industrial organics,
in a mouse cell morphological transformation system. This study finds that amsacrine may be a potential
carcinogen and may be deleterious to mammalian cells.

Morphological transformation, cell survival, chromosomal aberrations and micronuclei were measured
in C3H/101/2CL8 cells after 24 h exposure to amsacrine. A weak but dose-related increase in the percent-
age of dishes containing transformed foci occurred. As previously reported for alleviating agents, this ef-
fect was increased by treating 5 days instead of 1 day after plating. There was no evidence for gene muta-
tion at the Na/K ATPase locus, although amsacrine induced micronuclei in a large percentage of cells and
chromosomal aberrations, including interchange events and double minute chromosomes, in dividing
cells. It would appear that transformation and chromosomal events may be related in amsacrine-trcated
C3H/101/2CL8 cells. The results strongly suggest that amsacrine has carcinogenic potential, possibly re-
lated to its chromosome-breaking 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 HALOGENATED HYDROCARBONS FOUND in the
                                  GREAT LAKES
                                              i
                            Kirk T. Ktchin 1 and Sam Kacew 2

                       1HERL, USEPA, Research Triangle Park, NC
                              2University of Ottawa, Canada

The Great Lakes ecosystem is contaminated with many lipid soluble compounds. The neonatal toxicology
of ten of these compounds, eight chlorinated benzenes, hexachlorobutadiene, and octachlarostyrene, are
presented in this book chapter.  Emphasis is placed on pharmacokmetic and metabolic factors which in-
fluence their biological effects.  Where data permit, human poisoning episodes, environmental levels and
human body burdens are discussed.
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 Industrialization has resulted in the emission of a wide variety of organic chemicals into the biosphere.
 Despite the original source of the organic contaminant, these chemicals have managed to enter drinking
 water supplies and bioacculute in food chains. Of particular interest are the organic contaminants 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 bioaccumuladon of these chemicals, this chapter will focus on just ten chemicals, principally
 chlorinated benzenes, octachlorostyrene and hexachlorobutadiene.   The adult, fetal and neonatal
 toxicities of these compounds are discussed with emphasis on metabolic and pharmacokinetic factors
 which influence them.

 CITATION:  In: Toadcologtc and Phamacologic Principles in Pediatrics, Sam Kacew and Simon Lock,
             eds.,, Hemisphere Publishing 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 study, spematogenic  cells from the  mouse  testis were stained  with  various DNA specific
ftuorochromes.  The cells were then analysed by flow cytometry for differences in staining characteristics.
This method gives a profile of the relative number of cells of each spermatogenic cell type and has been
used successfully in quantifying and characterizing toxicant-induced lesions in the testis. The present study
demonstrates the relative advantages and disadvantages of each of die dyes tested. 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 orange, propidium
iodide,  ellipticine,  Hoechst 33342, mithramycin, chromomycin  As,  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 HCI.   For histone-containing
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, ellip-
ticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6-
to 4.0-fold after HCI treatment. In sharp contrast, HCI treatment of vas sperm did not increase the stain-
ing level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other
intercalating dyes (13- to 15-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 staining 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 context of chromatin alterations related to the unique events of
meiosis and protein displacement and replacement during sperm differentiation.

CITATION: Cytometry 7:45-53, January 1986.

L104 XX-85-085 Project Officer: Darney
 MOUSE TESTTCULAR and SPERM CELL DEVELOPMENT CHARACTERIZED
   from BIRTH to ADULTHOOD by DUAL PARAMETER FLOW CVTOMETRY

                      Frank C. Janca, Loma K. Jost, and Donald P. Evenson

                               South Dakota State University

In this study, cellular changes which occur during the development of the mouse testis were quantified using
flow cytometry. The results are important to the program offices in that they establish baseline data for fu-
ture testing of toxicant-induced changes in  or arrest of testicular development.  In addition,  this study
demonstrates that the first sperm populations to arise in the testis are somewhat abnormal when compared
to those in adults.  This finding may be important in risk assessment since it suggests that young animals
(humans?) may differ from 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. Animals 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 acid 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 acid denaturation of DNA in situ. Sperm
from cauda epididymis and vas deferens were examined 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 HEPATOCVTES: IN VITRO
                         DETECTION of a PROTERATOGEN

                   .  Linda A. Oglesby,1 Marian T, Ebrort,2 Patricia E. Beyer,1
                            Brenda D. Carver? and Robert J. Kavlocl?                 .

                                   Northrop Services, Inc.
                         2HERL, USEPA, Research Triangle Park, NC

 The study of chemical teratogenicity and subsequent interspecies hazard extrapolation is, 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 exposed. This paper reports
 on the development of an in vitro system which utilizes the whole embryo culture 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 possible to determine
 whether a unique, bioactive metabolite of thalidomide is produced by the human which affects develop-
 ment regardless of the species of the embryo, or whether the  rodent embryo does not possess the potentially
 harmful'thalidomide receptor.*

 The technique of whole embryo culture developed by New [Environ Health Perspect. 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 in vitro 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 similar numbers of hepatocytes were used for metabolic activation of
CP the induced embryotoxicity was greater in the presence of rabbit and hamster hepatocytes than with
rat hepatocytes.  Development of procedures for the culture of rat embryos with hepatocytes from other
species.

CITATION: Teratogenesis, Carcinogenesis, andMutagenesis 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 John M. Rogers? Robert J. Kavlock,l
                    Joseph S. Ostby,JanetM. FemttfandKatrinaL. Graf

                       1HERL,USEPA, Research Triangle Park, NC
                                 2Northrop Services Inc.

This study on dinocap was conducted at the request of the program office in support of their evaluation of
the teratogenicity of this compound.  This study was the first in the literature to demonstrate that dinocap
induced malformations in mice. The neurological and behavioral abnormalities produced by dinocap have
never been reported before as a consequence of a prenatal toxic insult.

The present study is an evaluation of the developmental toxicity of dinocap in three rodent species using
an in vivo teratology screen.  Our protocol uses postnatal viability, weight gain, and morphological and
behavioral development through weaning to assess the developmental toxicity of compounds.  Dinocap
administered orally on days 7 to 16 of gestation to the CD-I mouse resulted in increased postnatal mor-
tality at 25 mg/kg/d (80% in block 1 and 40% in block 2). Many of the treated pups that died during the
neonatal period were "ballooned" and had cleft palates. Although there was no treatment related mor-
tality in the 12 mg/kg/d dosage group, 6% (14/226) 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 walking. 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-flips, 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 (SO 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-85-1% Project Officer: Gray
       POSTNATAL ALTERATIONS in DEVELOPMENT RESULTING from
                     PRENATAL EXPOSURE to PESTICIDES

                     R. /. Kavlock, J. M. Rogers, N. Chemoff, andL. E. Gray

                        HERL, USEPA, Research Triangle Park, NC

This article reviews research done in the Developmental Biology Division relating to postnatal manifesta-
tions ofprenatalfy induced teratogenic  outcomes, especially as they relate to the effects of pesticides on
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 developing organisms. It presents an argument for the eventual inclusion of a postnatal component to a
 standard teratology bioassay and given support to the Agency's guidelines for developmental taxicity.

 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 examinations 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 Chemistry, 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 CHEMICAL&EFFECTS of CYTOSINE ARABINOSIDE, DINOCAP,
                             NITROFEN, and VITAMIN A
                      L. E. Gray, Jr., & J. Kavlock, L S. Ostby, /. Af. Ferrell,
                                  J.M. Rogers, andK. Gray

                         HERL, USEPA, Research Triangle Park, NC

Prenatal exposure to drugs and xenobiotics (methylmeraay) can result in abnormal human behavioral
development. However, test protocols to screen chemicals for such toxicity have not been developed and,
for this reason, a postnatal behavioral assessment  of developmental toxicity is rarefy conducted.  The
present study is an effort 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*5.
After this incident protocols were  developed to examine the potential of a chemical to cause abnor-
malities in experimental animals (Workd Health Organization 1967). In these studies, pregnant
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 DECARBOXYLASE in
           BRAIN and OTHER TISSUES of FETAL and NEONATAL RAT

          Theodore A, SloOdn,1 Steven Packman,1 Robert J. Kavlock? and Jorge Bartotome1

                             1Duke University Medical Center
                        2HERL, USEPA, Research Triangle Park, NC

Omithine decarbaxylase (ODC) is an enzymatic regulator of potyamines.  Pofyamine biosynthesis has been
closely associated with the ability to synthesize macromolecules (e.g.t DNA, RNA and protein).  To ex-
amine whether this enzyme could be used as a marker for chemically induced alterations in development,
rats were exposed prenataUy to the prototype teratogen methylmerctay. Analysis of multiple tissues at
various time points of development pointed to a specific effect of this chemical on the development of the
cerebellum.  The methylmercury-mduced alterations in ODC activity were found at  earlier times and at
lower doses than previously reported. The results indicate that this enzyme may be a simple, yet highly sen-
sitive indicator of organ-specific developmental tcodcity, 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 administration of methylmercury (0.5 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 methylmercury by 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 methylmer-
cury.

CITATION: Teratology 32(2): 195-202, October 1985.

L104MS-85-063 Project Officer: Kaviock
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 CONGENITAL RENAL HYPOPLASIA: EFFECTS on BASAL RENAL FUNCTION
                               in the DEVELOPING RAT

                         & J. Kavlock, B. F. Rehnberg, andE. H. Rogers

                         HERL, USEPA, Research Triangle Park, NC

Manifestations of developmental taxicity include death, altered growth, malformations, and functional
deficits. Validated methodologies to assess the last manifestation are lacking a major function of our re-
search program is to address these deficiencies. In Otis paper, we demonstrate that alterations in renal
growth, induced by toe prototype teratogen chloramsucU, 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 is suspicion of renal morphological alterations in a developmental tenacity test.

Administration of chlorambucil to pregnant rats on day 11 of gestation induced dose-related alterations
in renal growth and function in the postnatal offspring. These effects occurred above and beyond the
reductions in body growth and were evident in animals 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 increasing 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 chlorambucil
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: Kavlock
     AMPHOTERICIN B- and FOLIC ACIDJNDUCED NEPHROPATHIES in
                                 DEVELOPING RATS

                    Robert J. Kavlock, Blair F. Rehnberg, and Ellen H. Rogers

                         HERL, USEPA, Research Triangle Park, NC

Techniques have been developed by (his laboratory to evaluate renal function in neonatal animals follow-
ing prenatal exposure to xenobiotic agents. In this report, 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 amphotercin B, where not
only was function altered in the absence of histological damage, but also a new mechanism of renal taxicity
was hypothesized on the basis 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 program office to request specific, validated tests of
organ functional competence will allow greater characterization of biological effects and hence improve the
ability to assess risk.

The kidneys of newborn rats, which are both morphologically and physiologically immature, have been
shown to be relatively insensitive  to the nephrotoxic effects of several chemicals.  To examine the
specificity of these age-related differences, pups received sc injections of either 20 rag/kg of amphotericin
B or 250 mg/kg folk acid, two known nephrotoxins in adult animals, on postnatal Day 1,8, or 15. Renal
function was examined by a basal clearance test and a hydropenia challenge at 1,2, or 5 (6 in the case of
amphotericin B) days after treatment. We observed no difference in degree of renal toxicity with age, but
repair of renal damage 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 change 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 folk 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 insensittvity 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: Toxicology and Applied Pharmacology 81(3): 407-415, December 1985.

L104 MS-85-085 Project Officer: Kavlock
     DESCRIPTION and IMPLICATIONS for ANALYSIS of BRAIN GROWTH
                                  in SUCKLING MICE

                       Dennis House, Ezra Herman, and Hershell B. Carter

                          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 curve 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 report 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 earfy 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 mam 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 49:426-438, December 1985.
                                                               *• »
 L104 MS-85-116 Project Officer: Herman
         KIDNEY MORPHOLOGY and FUNCTION in the YOUNG of RATS
     MALNOURISHED and EXPOSED to NITROGEN DURING PREGNANCY

                  Carol Chase-Deesing, * Robert J. Kavlock,2 and Frances J, Zeman1

                                University of California-Davis
                         2HERL, USEPA, Research Triangle Park, NC

In the report, the potential interaction between prenatal protein malnutrition and exposure to a teratogenic
herbicide was. evaluated.  As migrant farm workers often receive nutritionally inadequate diets and also
receive relatively high level exposure to pesticides, the importance of potential synergism between these two
insults is obvious.  This study demonstrated a strong interaction between the two treatments in terms of off-
spring viability. 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-
ficiently marked to be the primary cause of death. * Overall, the study indicated that malnourished popula-
tions may be at greater risk to exposure to pesticides.

The separate and combined effects of prenatal protein deficiency (6% casein) and prenatal nitrofen (2,4-
dichlorophenyl-/>-nitropheny! ether) exposure (12.5 mg/kg on gestational d 7-21) on renal morphology in
the 21-d fetal and postnatal rat were examined. 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
         RETENTIONof PARTICULATE MANGANESE OXIDE in the RAT

                      Georgia L. Rehnberg, Jay F, Hem, Susan D. Carter, and
                                      JohnW.Laskey

                         HERL, USEPA, Research Triangle Park, NC

It is well known that the developing organism is uniquely sensitive to the toxic effects of heavy metals such
as lead and manganese. One of the malar routes of exposure to heavy metals is through inhalation, with
subsequent translocation of inhaled panicles of greater than 1 micron from the upper respiratory tract.  In
previous studies, with paniculate MnjQ*, we found that a portion of this age susceptibility was due to in-
creased absorption of manganese by the young animal. We suspected that one of the factors responsible
was the immaturity 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 ofparticulate material intheGI
tract ofthepre- 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 rat Addi-
tionally, the overall retention time of the paticulate manganese  oxide was considerably longer in the pre-
weanling animal.  The overall half-time ofparticulate manganese oxide in the pre-weanling rat was ap-
proximately 27hours as compared to 2.7hours in the 24 day old rat. This study demonstrates that the over-
all maturity of the GI tract is a significant factor in the assessment of the enhanced sensitivity of the young
animal to many potential toxicants.

Translocation of inhaled particulates from the nasopharynx and upper tracheobroncbial area to the
gastrointestinal tract is a major route of exposure for particles with a mass median diameter of greater
than 1 |un. Previous studies in this laboratory with particulate MnsO4 have shown that preweanling rats
have substantially higher tissue Mn concentrations than similarly treated adults, indicating possible dif-
ferences in uptake or elimination or both. This study was conducted to evaluate changes in gastrointes-
tinal movement and retention of particulate Mn3O4 in the preweanling and weaned rat. SSr-labeled
microspheres were used to evaluate gastrointestinal transit rate (TR), while particulate MnsO4 was used
to evaluate particulate retention at selected ages. The results show that stomach retention time in the
preweanling is at least twice that of the postweanling (90 nun versus 42 min). 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 preweanling 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 com-
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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 Toxicology 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. Copeland, R.W. Chadwick, N. Cooke,
                                    Whitehouse, and D.M. Hill
                         HERL, USEPA, Research Triangle Park, NC

 The metabolism of foreign compounds at the developing animal are not well known.  This study was con-
 ducted to investigate the distribution, metabolism, and excretion of lindane in the developing rat. Rat pups
 aged 2, 9, 16, and 23 days were dosed with lindane and the distribution, metabolism,  and excretion of Un-
 done was determined. Results from this study indicate that (1) the metabolism of Undone 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) oxidative reactions increase significantly with age, while reductive dechlorination decreased
 significantly with age; (4) conjugation reactions are age dependent, with sulfate and ghitathione conjugation
 decreasing and gatcuronide conjugation increasing with age and (S)different isazymes may be involved in
gatcuronidation of we alcohol and chlorophenol metabolites of lindane.  Studies of this nature may help to
 evaluate hepatic function in the developing animal.

 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 mg/kg containing 0.5 jtCi [U~
  CJlindane 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 I reactions increased significantly, while anerobic reductive
 dechlorination of lindane to 4-chlorophenylmercapturic acid  decreased significantly with age. Phase II
sulfate and glutatbione 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 I 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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      EFFECTS of AGE and OBESITY on the METABOLISM of LINDANE by
                BLACK a/a, YELLOW A ^/a, and PSEUDOAGOUTI
                A ^/a PHENOTYPES of (YS x VY)Fi HYBRID MICE

                  R.W. Chadwick,1 M.F. Copetond,1 G. L. Wolff? N. Cooke,1  .
                             D. A. Whitehouse,1 and M. L. Mole1

                        1HERL, USEPA, Research Triangle Park, NC
                     2National Center for Toxicologjcal Research, DHHS

Some strains of truce are susceptible to liver tumors after chronic exposure to the organochlorine insecticide
Undone while other strains are not. This work was part of a collaborative study with NCTR to investigate
the effects of age, genetic influence and altered metabolism on susceptibility to Undone treatment  It was
found that age and obesity had significant effects on the tissue storage and biotransformation of Undone.

Lindane (•y-hexachlorocydohexane) has been shown to produce hepatomas 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^/a and black a/a phenotypes of
(YSxVY)Fi hybrid female mice at 8,17,30,56, and 86 wk of age. At 24 h prior to sacrifice the mice were
dosed p.o. with 18 mg lindane (containing 55 jJ.Ci[U ~14C]Iindane/kg). Aging altered the biotransforma-
tion of lindane such that while the excretion of lindane 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 dechlorinase activity compared to that of their pseudoagouti A^7a and black a/a si-
blings.

CITATION: Journal of Toxicology and Environmental Health 16(6): 771-7%, December 1985.

L104 MS-85-029 Project Officer: Chadwick
  INVESTIGATION of HCB as a METABOLITE from FEMALE RATS TREATED
                        DAILY for SIX DAYS with LINDANE


                            R. W. Chadwick andM. F. Copeland

                        HERL, USEPA, Research Triangle Park, NC

Widespread occurrence of the fungicide hexachlorobenzene (HCB) in human milk samples,  the
transplacental transfer of HCB to the fetus in several species and the teratogenicity of this pesticide in rats
and mice constituted a potential hazard to mothers and their developing offspring in 1984 when it was
reported that lindane, an organochlorine insecticide was metabolized by rats to hexachlorobenzene. Con-
cern over another source of HCB exposure led to the study of the metabolism of Undone to HCB in female
rats. Results from this study indicated that there was no significant biotransformation of lindane to HCB in
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 rats.  This work enabled the agency to meet its deadlines on the exposure assessment of HCB without
 changing the resources required.

 The biotransformation of lindane to hexachlorobenzene (HCB) by male rats was recently reported. Since
 HCB has been widely detected in human milk samples, and since the transplacental 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-hr
 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-METABOLIZING
                                  ENZYME SYSTEM


                   Robert W. Chadwick,l M. Frank Copeland,1 Gary P. Carlson,2
                            Bruce A. Treto,2 and Bernard M.Mos?
                                                                                    t
                        1HERL, USEPA, Research Triangle Park, NC
                                    2Purdue University
                                  Northrop Services, Inc.

The effect of exposure to environmental chemicals on the integrity of the hepatic drug-metabolizing enzyme
system has generally been evaluated in vitro using isolated liver preparations.  Uncertainty over how well
changes observed in vitro reflect those which occur in the live animal has resulted in the testing of a nonin-
vasive technique for the determination 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 vivo index of
xenobiotic-induced alterations in the hepatic drug-metabolizing enzymes.  The model substrate assay
described in this paper will permit the study of unanticipated, persistent, and latent side effects from pre- 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-metabolizing 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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                                                                       PUBLICATIONS
 COMPARISON of IN VIVO and IN VITRO METHODS for ASSESSING EFFECTS
                         of ALLYL ALCOHOL on the LIVER

                    Bruce A. Trela,1 Gary P. Carlson,1 Robert W. Chadwick,2
                      ,           and M. Frank Copeland2

                                   1Purdue University
                        2HERL, USEPA, Research Triangle Park, NC

 The effect of exposure to environmental chemicals on the Integrity of the hepatic drug-metabolizing enzyme
system has generally been evaluated in vitro using isolated liver preparations.  Uncertainty over how well
changes observed in vitro reflect those which occur in the live animal has resulted in the testing of a nonin-
 vasive technique for the determination 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 drug-metabolizing enzymes.  The model substrate assay
 described in this paper will permit the study of unanticipated, persistent, and latent side effects from pre- or
postnatal exposure to environmental chemicals.

 Allyl alcohol was administered 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 minimally elevated. No dose-related chan-
 ges were observed in hexobarbital oxidation, aniline hydroxylation, or ethylmorphine  demethylation.
 Aldrin epoxidation was slightly elevated. Naphthol glucuronidation and glutathione-5-transferase ac-
 tivity with l,2-dichloro-4-nitrobenzene were increased. Results from in vivo studies on the metabolism of
 lindane were in dose agreement with the in vitro measurements suggesting that daily treatment for one
 week with ally! alcohol at doses of 3,10 and 30 mg/kg has no significant effect on phase I pathways, has a
 selective effect on  phase n pathways  and, under the conditions of this experiment, has minimal
 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 M77?O METHODS and the IN VIVO METABOLISM
 of LINDANE for ASSESSING the EFFECTS of REPEATED ADMINISTRATION
                 of ETHANOL on HEPATIC DRUG METABOLISM

                    Bruce A. Trela,l Gary P. Carlson,[ Robert W. Chadwick,2
                                 andM. Frank Copeland2

                                    Purdue University
                        2HERL» USEPA, Research Triangle Park, NC

 Tlie effect of exposure to environmental chemicals on the integrity of the hepatic drug-metabolizing enzyme
 system has generally been evaluated in vitro using isolated liver preparations. Uncertainty over how well
 changes observed 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 determination 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 drug-metabolizing enzymes.  The .model substrate assay
 described in this paper will permit the study of unanticipated, persistent, 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 ml/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 minimal effect of
 ethanol on drug metabolism at low levels of administration.

 CITATION:  Toxicology Letters 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

          Bruce A. Trela,1 Gary P. Carlson,1 Robert W. Chadwick,2 andM. Frank Copeland 2

                             Purdue University School of Pharmacy .
                         2HERL, USEPA, Research Triangle Park, NC

 The effect of exposure to environmental chemicals on the integrity of the hepatic drug-metabolizing enzyme
system has generally been evaluated in vitro using isolated liver preparations. Uncertainty over how well
changes observed in vitro reflect those which occur in the live animal has resulted in the testing of a nonin-
vasive technique for the determination 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 vivo index of
xenobiotic-induced alterations  in the hepatic drug-metabolizing enzymes.   The model substrate assay
described in this paper will permit the study of unanticipated, persistent, and latent side effects from pre-or
postnatal exposure 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) alkaline phosphatase, glutamic-pyruvic transaminase (GPT) and
glutamic-oxalacetic transaminase (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 aldrin epoxidase determined in vitro were not inhibited by mercuric chloride al-
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though aniline hydroxylase activity was decreased.  Of the four phase-II reactions measured, only the
glucuronidation of chloramphenicol 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 dose 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. Watldnson,1 M. Ann Brice,1 and Kathy S. Robinson 2

                         ^RL, USEPA, Research Triangle Park, NC
                                   Northrop Services, Inc.

The research described in this article was conducted to support the programs of the Office of Toxic Sub-
stances (OTS).  There was a need to develop methodology to enable research lexicologists to efficiently
identify potentially toxic agents.  In addition, there was a need to establish a program within the EPA with
the capability to evaluate the cardiovascular effects induced by exposure to such toxic agents. The develop-
ment of the computer-assisted electrocardiographic analysis system described in this article provided a solu-
tion to both problems. Thus, monitoring the effects of substances on heart rate and electrocardiograms of
animal models servces 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 permit more in-depth
study 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. ECG signals 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 signal playback rate and permits operator review of the  slowed signal 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 complex. 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 animals; (2) enhanced sensitivity-the use of functional 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 animals, 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: Journal of Toxicology and Environmental Health 15:713-727, November 1985.

 L104 MS-83-042 Project Officer: Watkinson
                        EFFECTS of CHLORDIMEFORM on
                CARDIOVASCULAR FUNCTIONAL PARAMETERS:
   PART 1. LETHALITY and ARRHYTHMOGENICITY in the GERIATRIC RAT

                                    William P. Watkinson

                         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 formamidine pesticide chlordimeform (CDM) is currently being studied by several
 laboratories within HERL, with primary emphasis on its neurotoxicological and thermoregulatory effects.
 Relatively little is known of its effects on other organ systems.  The data presented in this particular study
 describing the effects of CDM on cardiovascular functional parameters serves a dual purpose. First, these
 results provide a significant quantity 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 observed effects. Second, these data serve to validate previously'developed methodol-
 ogy based upon computer-assisted electrocardiographic analytical procedures.

 Chlordimeform  (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
 60 mg/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 animals.  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 13-DIPHENYLGUANIDINE in
                                         RATS

                    P. V. Shah,lM. R. Stonier,1 Y. M. loannou,2H. L. Fisher,3
                                      and L.L. Half

                                  Northrop Services, Inc.
                                        2NffiHS
                        ^ERL, USEPA, Research Triangle Park, NC

1,3-diphenylguanidine (DPG) is a chemical widely used in the processing of rubber and in food packaging.
Studies were undertaken to assess the potential of DPG to be absorbed through the skin as this route is of
major concern on exposure to many occupational and agricultural chemicals. Even though DPG shows
slow dermal 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 packaging, was studied in adult female Sprague-
Dawley rats. DPG shows 10% penetration through dipped 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(Ti/2 = 33.6
d). Approximately 13% of the absorbed dose remained in the body in 5 d. Retention in skin, muscle, liver,
intestine and fat contributed most to the body burden of DPG-derived radioactivity in Sd. 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 15(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 is part of a workshop proceedings in which some of the perinatal toxicology work performed in
 HERL was summarized. 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 function have been
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 observed, these alterations an reversible with normal levels of immune function achieved after a period of
 time following termination of exposure. These results are useful to the Agency in that they provide a data
 base for the immunotasdc potential of certain chemicals on a select population, i.e., the very young.

 The most profound toxic effects of many physical and chemical agents are observed when exposure oc-
 curs during mammalian development. The development of immunocompetence in mammals 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 utero and/or during early postnatal life to the following agents:
 cycloposphamide, nonionizing radiation, cyclospprin A, urethan, di-n-octyltin dichloride or NiCfe. At
 immunological maturity (i.e., 6-8 weeks of age) animals 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 activity, primary antibody responses to sheep erythrocytes, delayed hypersensitive responses,
 and resistance to tumor development. The results of these studies are summarized.

 CITATION: In: Proceeding of the Indo-US.  Workshop on the Role of Predisposing Conditions of Health,
            Nutrition, and Environment on Safety of Drugs and Chemicals, Lucknow, India, February 25-
            28,1986.

 L104 MS-86-251 Project Officer: Smialowicz
         IMMUNOLOGICAL STUDIES in MICE FOLLOWING IN UTERO
                                  EXPOSURE to NiCl2

                     Ralph J, Smialowicz, Ronald R. Rogers, Marie M. Riddle,
                            Denise G. Rowe, and Robert W. Luebke

                         HERL, USEPA, Research Triangle Park, NC

Nickel has been shown to inhibit certain immune functions in adult mice. Since it has been reported that
the most profound effects of chemicals that modulate the immune system of animals occur when dosing of
the compound begins during the development of the fymphoid system, mice were exposed to nickel during
gestation. Results of mis work, however, indicate that in utero exposure to nickel does not adversely affect
the immune junction 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 toxicuyforuse by the Agency in making risk assessments.

The effect that NiCb has on the development of immune function in mice was examined 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 p.g/g NiCfc. The pumps delivered NiCh to the dams through day 19 of gestation. At 8-10 weeks of
age the offspring of NiCb-dosed dams were evaluated for immune function. No consistent significant al-
terations were observed between control and treated offspring for the following: lymphoid organ or body
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weights; the lymphoprolifcrativc  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 to sheep red blood cells. Natural killer (NK) cell activity was
reduced in offspring exposed to NiCfe in utero; however, the biological relevance of these reductions is
questionable because of the failure to demonstrate an increased susceptibility to the B16-F10 syngeneic
tumor. The results indicate that under the conditions and doses employed it appears that NiCb 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: Smialowicz
   IMMUNE FUNCTION of YOUNG ADULT MICE FOLLOWING IN UTERO
                       EXPOSURE to CYCLOPHOSPHAMIDE

                   '  R. W. Luebke, M, M. Riddle, R. R. Rogers, R. 7. Gamer,
                              D. G. Rowe, and R. /. Smialowicz

                         HERL, USEPA, Research Triangle Park, NC

The sensitivity of the developing immune system to perturbations by xenobiotic agents is as yet not well
characterized. Alteration of the normal developmental sequence has the potential to be expressed postna-
talfy as significant immune deficiencies postnatalfy.  The work described in this paper describes studies
designed to investigate the effects of in utero exposure to the well known adult animal anmunotaacant
cyclophosphamide.  The information from this work contributes to the data base upon which the Agency
can determine risk assessment as it relates to perinatal 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 jig/g ' d intravenously on d 9-12 or 14-17 or gestation; (2) 5 n,g/g intravenously on 12 of
gestation; (3) 1,23 or 5 ug/g ' d intraperitoneally on d 12 of gestation; or (4) 5; 10, or 20 jtg/g in-
traperitoneally on d 17 of gestation. There were no surviving pups born to mothers administered 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 dams.
Reduced body weight in 5- and 8-wk-old progeny was noted after exposure to 20 jtg/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
toCYat20 (jig/gond 17 of gestation. The T-lymphocyte parameters were relatively unaffected by in 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 C57BL/6J MICE FOLLOWING EXPOSURE to
                         URETHAN PRE- or POSTNATALLY


                    Robert W. Luebke,1 Marie M. Riddle,1 Ronald R. Rogers,1
                   Denise G. Rowe,2R.John Gamer,1 and Ralph J. Smialowicz1

                     .   1HERL, USEPA, Research Triangle Park, NC
                                  2Northrop Services, Inc.

 The effects ofxenobiotics on the developing immune system are not well known.  This study was conducted
 as part of our model development/validation effort in developmental immunotoxicology. Mice were ex-
posed to the weak carcinogen urethan either pre- or postnataUy and evaluated immunologicalfy at an age
 when full immunologic competence should have been attained. In this study, differences in adult levels of
 immune Junction were observed when chemical exposure occurred during different periods of immune 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 2.0 mg URE/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 leukocyte counts and a trend toward increased
spleen and thymus size in offspring of treated mothers.  Humoral immune function, as measured by the
IgM response to sheep erythrocytes, was suppressed in pups from dams injected with a total of 1.0 mg/g
URE. These results indicate that marked differences in immunopharmacologic effects maybe observed
if chemical exposure occurs at different times during the ontogeny of the immume system.

CITATION: Journal of Immunopharmacology 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 /. Smiaiowicz, Robert W. Luebke, Ron R. Rogers,
                            Marie M. Riddle, and Denise G. Rowe

                         HERL, USEPA, Research Triangle Park, NC

Ordram®, a thiocarbamate herbicide, has been reported to affect the immune system of experimental
animals. Following a request from the Office of Pesticide Programs (OPP), the work reported in Oils paper
was performed. The results of this work indicate that this herbicide does not adversely affect the immune
system of mice under the exposure conditions described. The information from this work contributes to the
database upon which the Agency can determine 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-
mune parameters measured were the following: body and lymphoid organ weights; splenic natural killer
(NK) cell activity; lymphoproliferative responses to B and T lymphocyte mitogens and allogeneic 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 examined. 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-85-157 Project Officer: Smiaiowicz
    STRATEGIES for the SELECTION of TEST METHODS for SCREENING in
                            BEHAVIORAL TERATOLOGY

                            Mark A. Geyer and Lawrence W. Reiter

                         HERL, USEPA, Research Triangle Park, NC

Tiiis publication recommends that several factors and criteria be considered when designing a test battery
for assessing developmental neurotoxicity.  The major recommendation is that testing procedures be objec-
tive and simple, to insure reliability of results. Other recommendations 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 neurotoxicity in the Neurotoodcology Division of HERL and are consistent with the current
Office of Toxic Substances (OTS) guideline for neurotoxicity 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

                    /.Adams,1/. Buelke-Sam,1 C.A. Kimmel,1 C.J. Nelson,1
                   L. W. Reiter? T. Sobotka,1 H.A. Tilson,1 andB. K. Nelson1

                            Children's Hospital Research Center
                        2HERL, USEPA, Research Triangle Park, NC

 This publication describes the design of testing procedures used in the NCTR Collaborative Behavioral
 Teratology Study. In this study, intra- and inter-laboratory reliability of numerous test methods were ex-
 amined and the importance 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
 neurotoxicity 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 Kimmel
 and Buelke-Sam (1985) and the preliminary work which  resulted in the design of the CBTS has been
 presented (Adams, Buelke-Sam, Kimmel, 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, Ruppert, Karen F. Dean, and Lawrence W. Reiter

                         HERL, USEPA, Research Triangle Park, NC

The results cf studies reported in this publication indicate that there are social and experiential factors
which determine the profile of the development of motor activity in figure-8-mazes. These studies highlight
important design considerations for test methods utilized in the Neurotaxicology 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 examined.  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 min,
30 min, or 1 hr/day, and (4) pups tested daily for 30 min/day either singly in a maze, 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, minimal 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. Veronesi1 andS. Bondy2

                         ^ERL.USEPA, Research Triangle Park, NC
                                         2NIEHS

 Triethyltin (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 entorhinal region. Damage to this population
 of celts results in associated changes in the hippocampal region, which is involved in memory and learning.

 Neuropathological and biochemical effects of neonatal exposure to the alkyl metal triethyltin were ex-
 amined in Long Evans juvenile male rats. Rats were injected intraperitoneally on post-natal day 5 with 6
 ink/kg of triethyltin bromide and sampled on day 20. The brains of tin-treated animals 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 immunocytochemical staining of astrocytes. Hippocampal involvement was further evidenced
 by a protrusion of the molecular layer of the dentate gyms, and an abnormal staining pattern of acetyl-
 cholinesterase in this layer. Sections stained by the Timm's method for the deposition of heavy metals
 showed a marked reduction in the staining of the hippocampal CA4-2 sectors and an absence of stained
 laminae 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 triethyitin produces severe
 neuronal damage in the posterior cortex and a derangement of hippocampal afferent circuitry.

 CITATION: Neumtaodcology 7(1): 69-80, April 1986.

 L104 MS-85-179 Project Officer: Veronesi
          MACROPHYSIOLOGICAL ASSESSMENT of ORGANOMETAL
                                   NEUROTOXICITY

                                       Roberts, Dyer

                         HERL, USEPA, Research Triangle Park, NC

Part of the Neurotoxicology Division's mission is evaluation of methods for detection and characterization
of neurotoxicity. The various program offices, particularly the Office of Toxic Substances, need validated
test methods to discharge their responsibility for requiring testing and test data*  This chapter provides a
critique of 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 analysts for microphysiological methods is cellular or subcellular (e.g. 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: Neurotancants 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
                                 ofNEUROTOXICITY

                                   James P. O'Callaghan

                         HERL.USEPA, Research Triangle Park, NC

Reviews progress of a research approach designed to provide the Agency with a simple, cost-effective testing
methodology, that can be used to evaluate the potential neurotaxic 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 neurotoxicity.

CITATION: In: Neurotcodcology, 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, Ivor Walaas, Angus C. Nairn, 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 efforts to develop and validate nervous-system-specific
proteins as biochemical indicators of neurotoxicity.

 The present study reports the existence of Purkinje cell-specific phosphoprotein, Mr 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
 caldum/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 Neurosdence 6(4): 954-961, April 1986.

 L104 XX-86-138 Project Officer: O'Caflaghan
  THERMOREGULAT1ON in MICE FOLLOWING ACUTE CHLORDIMEFORM
                                 ADMINISTRATION

                   Christopher J. Gordon, Metritt D. Long and Andrew G. Stead

                        . HERL, USEPA, Research Triangle Park, NC

 Chlordimeform has been shown to exert a variety of toxic effects including visual dysfunction and bradycar-
 dia.  Chlordimeform also promotes a reduction in body temperature which may indirectly be responsible for
 the toxic effects.  Hence, it is important to understand the mechanisms by which Chlordimeform affects
 body temperature. This study demonstrated that Chlordimeform affects both the autonomk and behavioral
 control of body temperature.  The study also found that the thermoregulatory response to intoxication by
 Chlordimeform may be crucial to survival.

 CBA/J mice were injected intraperitoneally (i.p.) with the formamidine insecticide Chlordimeform
 (COM) 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
 min after administering CDM doses of 0,15,30,60, and 75 nag/kg. The hypothermia 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

                    fSmi S. Strek, Merritt D. Long, and Christopher J. Gordon

                        HERL, USEPA, Research Triangle Park, NC

Many pesticides and other toxic chemicals affect the control of body temperature in rodents. It is possible
that the action of these compounds on the thermoregulatory centers of the central nervous system could be
anesthetic-tike in nature. Hence, this study 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 thermoregulation 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 rag/kg and male CBA/J mice were given
doses of 0, S, 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 IS mg/kg and then placed into a temperature-controlled environmental chamber set at 3p°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 wanner Ta.

CITATION:  Pharmacology Biochemistry and Behavior 24(4): 1147-1150, April 1986.

L104 MS-85-193 Project Officer: Gordon
        ACUTE BEHAVIORAL TOXICITY of SULFOLANE: INFLUENCE of
                                   HYPOTHERMIA

                            Patricia H. Ruppert and Robert S. Dyer

                         HERL, USEPA, Research Triangle Park, NC

Sulfolane is a high volume solvent of interest to the Office of Toxic Substances (OTS).  Previous work
showed behavioral and neurophysiological consequences of acute exposure to this compound. This paper
reports that concurrent hypothermia may account for the severity of some of the behavioral 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
 rag/kg sulfolane. Motor activity in figure-of-eight mazes was assessed 1 h after dosing in testing rooms
 maintained at either 20.8°C or at 323°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 HYPOTHERMIA on the IN VIVO MEASUREMENT of RAPID
             AXONAL TRANSPORT in the RAT: A CAUTIONARY NOTE

                            Stephanie Padilla1 and Donald Lyerfy 2

                         1HERL, USEPA, Research Triangle Park, NC
                                    Northrop Services, Inc.

 Changes in axonal transport parameters are often used to assess the functional integrity of both central and
peripheral nervous system neurons. This paper is basically a quality control paper. To date, several studies
 have had conflicting results when measuring axonal transport profiles in drug- or toxicant-treated animals.
 The findings presented in this manuscript, i.e., that body temperature had a significant influence on the
 measurement of axonal transport in mammals, should be taken as a cautionary note for those investigators
 exploring perturbations of axonal transport in toxicant-treated animals. The experimenter must be very care-
ful not to confuse changes.in axonal transport due to body temperature with neurotoxic changes due to the
 toxicant exposure.

 Rapid axonal transport of glycoproteins was examined in the retinofugal projections of hypothermic and
 normothermic adult male Long-Evans hooded  rats previously  receiving intraocular injections  of
 [3H]fucose. The amount of retinal fucosylation appeared normal in the hypothermic animals 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 Neurochemistry 46(4): 1227-1230, April 1986.

L104 MS-85-205 Project Officer: Padilla
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     YOHIMBINE ATTENUATES the DELAYED LETHALITY INDUCED in
                MICE by AMITRAZ, A FORMAMIDINE PESTICIDE

                           Virginia C Moser and Robert C MacPhail

                         HERL, USEPA, Research Triangle Park, NC

Amitraz, a commercially used formamidine pesticide, produced a delayed lethality in mice that differs
greatfyfrom the time course action of the prototype formamidine chlordimeform. The finding that yohim-
bine significantly blocked lethality suggest that amitraz exerts this action via stimulation ofatpha-adrenergic
receptors.  This finding should help OPP in regulating amitraz 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 examine this phenomenon, adult albino mice of both sexes were treated with
either yohimbine (10 mg/kg, i.p.) or deionized water (6 ml/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 o2-adrenergic
mechanism is involved in the delayed lethality produced by amitraz.

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

            RaefynJanssen,1 VemonA. Benignus,1 Laura M. Grimes,2 and Robert S. Dyer1

                        1HERL, USEPA, Research Triangle Park, NC
                                  2Northrop Services, Inc.

The selection of filter 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.  While filter distortion is a known phenomenon, this paper reports for the first time that substantial
distortion can occur even when the selected bandpass is outside the spectrum of the waveform. This distor-
tion can result in the temporal reversal of slow and fast peaks in the waveform. Phase shifts resulting from
the use of a typical analogue filter set are quantified, and the spectrum of the brainstem auditory evoked
response of the rat is presented.  The paper recommends filtering as little as possible in order to preserve in-
terpretability of physiological responses.

Analog filtering of the brain-stem auditory evoked response (BAER) and synthetic wave forms using
steeply sloped filters are shown to produce significant 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, Robert C. MacPhail and Jackie 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 experiment showed that the neurotoxicant thallium sulfate in-
duced robust conditioning flavor aversions when given orally but not when given intraperitonealfy. These
results highlight the importance of a methodological detail (route 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 toxicuy 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 route 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-m access to a 0.1% saccharin solution followed 20-m 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.-administered 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 administered.

.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 NEUROTOXICITY:
                  SENSITIVITY to NITROUS OXIDE EXPOSURE

                         B. Greenberg, P. Moore, R. Letz, and E. Baker

                         Harvard University School of Public Health

Results of Otis study are relevant to project objectives and demonstrate the sensitivity of the computerized
test battery to a potentially neurotonic gas, nitrous oxide.

We recently developed a flexible, portable, computer-based neurobehavioral evaluation system (NES) to
standardize data collection: in epidemiologjc  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 drug 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 phannacologjc agents.

CITATION: Clinical Pharmacology and Therapeutics 38(6): 656-660, December  1985.

L104 XX-86-072 Project Officer: Otto
     NEUROPHYSIOLOGICAL CONSEQUENCES of ACUTE EXPOSURE to
                                METHYLPYRIDINES

            Robert S. Dyer,1 Linda /. Burdette,2 Raefyn Janssen,l and William K. Boyes1

                        ^ERL, USEPA, Research Triangle Park, NC .
                                  Northrop Services, Inc.

At the request of the Office of Toxic Substances (OTS), the Neurotoxicology Division evaluated the effects
of acute or 30 day exposure to substituted methylpryridines. This paper reports the effects of acute exposure
to these compounds. All compounds were significantly neuroactive at 1/2 the i.p. LD50, 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. LDSO.  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 similar to those seen
following administration 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 Toxicology 5(5): 920-932, October 1985.

L104 MS-84-127 Project Officer: Dyer
  DIETHYLDITHIOCARBAMATE INCREASES DISTRIBUTION of CADMIUM to
        BRAIN BUT PREVENTS CADMIUM-INDUCED NEUROTOXICITY

                          James P. O'Callaghan1 and Diane B. Miller*

                         1HERL, USEPA, Research Triangle Park, NC
                                  2Northrop Services, Inc.

The results of this study show that a known neurotoxicant, cadmium, affects known nervous-system-specific
proteins'in the predicted manner. By validating the use of nervous-system-specific proteins in this manner,
the Agency mil be provided with a simple, specific and cost-effective means for detecting and characterizing
neurotoxicity.

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 examine 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'Callaghan
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 PHARMACOLOGIC and IMMUNOLOGIC APPROACHES to the PROBLEMS of
   POST/TRAUMATIC GLIAL PROLIFERATION FOLLOWING CNS DAMAGE

                    M. L. Billingsley,1 C D. Bataban,1 andJ. P. O'Callaghan2

                            1The Pennsylvania State University
                        2HERL» USEPA, Research Triangle Park, NC

 this paper reviews recent progress in validating the use of nervous-system-specific proteins as biochemical
 indicators of neurotoxicity. Validation of this neurotoxicity testing scheme will provide the Agency with a
simple, specific and cost-effective means for the detection and characterization of neurotcadcity.

 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 ^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 damage and subsequent gliosis. Preliminary results suggest that these toxicants reduce the im-
 munocylochemical 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 anitimitotic agents.

 CITATION: In: NATO Advanced Research Workshop: Glial-Neuronal Communication in Development
            and Regeneration, 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 GaryM. Brittenham1 and Victor Hasselblad2

                             xCase Western Reserve University
                        2HERL> 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. Somepes-
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ticide residue values are recorded as in intervals of values or as being below the minimum detectable level
of measurement,. In these recurrences, 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 easts. This statistical methods 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 samples ob-
tained from SO 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 missing information technique.  Results of the one-sample chi-*
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 expectation-maximization 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. KJopman, and H. S. Rosenkranz

                               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 refy 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 utilizing 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 misclassifications 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 extrathennodynamic variables are especially 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:  Environmental Mutagenesis 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,lR. Contreras,1 H. S. Rosenkranz,1 andM. D. Water?

                              k^ase Western Reserve University
                         2HERL» 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 short-term genetic bioassays applied to
pesticide chemicals. The study successfully delineated two chemical fragments, a methoxyphosphinyl and a
chlorovinyl group, as the common structural subunits responsible for the biological activities detected in the
battery of tests employed.  This methodology should, therefore, be very valuable to the Office of Pesticide
Programs in determining which test systems to  apply or to require in the evaluation ofgenotoxic 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
genotoxlc 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 chlorovinyl group appear as the com-
mon structural subunits responsible for the activities detected in the battery composed of the Salmonella
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 typhimurium histidine reversion assay, the mouse lymphoma gene mutation assay and recombination in
 die yeast Saccharomyces cerevisiae.

 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

                       J.R. Rabinowitz,1 K. Namboodri,2 and H. Weinstein2
                         'HERLjUSEPA, Research Triangle Park, NC
                                2Qty University of New York

 The molecular electrostatic potential (MEP) is a measure of the capacity of a molecule to interact with its
 surroundings and has been found to provide useful insight into the differential biological activity within
families of chemicals. It could provide an important parameter for the assessment of potential chemical
 tenacity from chemical structure.  The difficulty in using the MEP in a structure activity study for tcodcity is
 the difficulty in its computation and characterization. This paper derives and tests a rapid method for com-
putation of the MEP and provides methods for its characterization.  These advances will facilitate its use in
structure activity (SAR) 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
1.5 A from any atom in the molecule. In contrast, rearrangement of the terms  into an'expansion cor-
responding only to Mulliken 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. Slouch and P. 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 be performed when all the relevant bioassay
information is not available. In these cases, quantitative structure activity relationships that use pattern
recognition techniques like ADAPT can provide valuable assistance. An important characteristic used in
these methods and in other relevant methods is the volume and shape of chemicals. This paper presents a
method for quantifying and using these characteristics in structure activity studies.

A conceptually and computationally simple method for the definition, display, quantification, and com-
parison of the shapes of three-dimensional mathematical molecular models is presented. Molecular or
solvent-accessible volume and surface area can also be calculated. Algorithms, programming considera-
tions, accuracy, and time and storage requirements are discussed.  The method requires no extensive
programming 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. Jaeger and P. C. JUTS

                              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 be performed when all the relevant bioassay
information is not available. In these cases, quantitative structure activity relationships that use pattern
recognition techniques like ADAPT can provide valuable assistance. This paper develops a new method to
compare the structural similarity of chemicals by using the relative frequency of all unique atom pairs in
each chemical being compared.

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 similar 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 training 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:  In: Proceeding; of the 28th .ORNL-DOE Conference on Analytical Chemistry in Energy and
             Technology, Knoxville, TN, October 1985.

 L104 XX-86-081 Project Officer: Rabinowitz
    PATTERN RECOGNITION STUDIES of COMPLEX CHROMATOGRAPHIC
                                        DATA SETS

                             P. C. Jurs, B. K. Lavine, and T, R. Slouch

                               The Pennsylvania State University

In the premanufacture notification program of the Office of Toxic Substances (OTS) and in other cir-
cumstance, the assessment of potential chemical tenacity must be performed when all the relevant bioassay
information is not available.  In these cases, quantitative structure 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 bio-effects data.

Chromatographic fingerprinting of complex biological samples is an active research area with a large and
growing literature. Multivariate 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, discriminants 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 using 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 examined. 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 Bureau of Standards 90(6): 543-549, November/Decem-
            ber 1985.

L104XX-86-076Project Officer. Rabinowitz                                 
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TOXICS
 THE EFFECTS of MATERNAL MURINE CYTOMEGALOVIRUS INFECTION on
        the MOUSE CONCEPTUS at DIFFERENT GESTATIONAL STAGES

                    Yuan-Shen Huang Neil Chemoff, Robert J. Kavlock, 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 microbial pesticidal agents which include viruses.

 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 animals 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 Mgnifinynt increase b 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 13 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 animals 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:  Teratogenesis, 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, andL.D. Claxton

                        HERL, USEPA, Research Triangle Park, NC

In order to develop health effects models for genetically engineered microorganisms (GEMS) that decade
hazardous substances, it is sometimes necessary to develop the chemical methods to monitor for the
microbialdegredation products.

High performance liquid chromatography (HPLC) was used to separate chlordecone, Kepone, and two
of its metabolites, hydrochlordecone and dihydrochlordecone. Elution of the three peaks occurred after
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the solvent concentration reached 100% and was maintained at 100% methanol for approximately five
minutes. The method was linear for chlordecone in the concentration range of 10 to 100 jig.

CITATION: Chromatopaphia 22(1-6): 165-168, June 1986.

U04MS-86-092 Project Officer: Claxton
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Research Objectives
                   Hazardous Waste

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                   HAZARDOUS WASTE RESEARCH PROGRAM
To improve the basis for regulatory activities under the Resource Conservation and
Recovery Act (RCRA).
Objective:
Background:    The RCRA was created in 1976 to mlnlmne. risks from hazardous 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 bioassays for screening wastes for designation
   as hazardous.  Bioassay screens are being evaluated for the  following health effects.  General
   tcnddty, cardnogenldty, neurotoxidty, immunotoxidry, teratogenicity and reproductive effects.

Rationale: Section 3001 of the RCRA requires EPA to promulgate criteria for identifying the charac-
teristics of hazardous wastes and the listing and delisting of hazardous 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
prioritizing which wastes are most important for lexicological 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 animal exposure protocol followed by a closely integrated matrix of short-term lexicological
tests. This test battery will be  evaluated with dose-response validation studies using well-characterized
chemicals previously tested in animal studies.

• Provide improved methodologies for the assessment and prediction of toxicity of the halogenated
   dioxtas and related compounds.

Rationale: The poryhalogenated 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 making health hazard evaluations at waste sites where dioxin-like materials
are present.  .

Approach: Methodologies to be studied will include both in vitro and in vivo 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, PCDPs, and mixtures to in-
duce AHH/EROD enzymes, to  bind specific receptors, and to cause XB cell keratinization will be
studied.  The molecular electrostatic potential and other key features of recognization processes of
selected dioxin-like chemicals will be computed and analyzed.
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                  Hazardous Waste

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HAZARDOUS WASTE
 Issue:  Waste Characterization of the Bioassay Screen.

 An in vivo/in vitro Toxicological Screen (Tox 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-
 munotoxtcology. The protocol, called the Toricological Screen, involves the oral administration of waste
 material to rate for 10 consecutive days.  At the end of the 10-day period the whole animal, body tissues
 and fluids are evaluated for toxirity. 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 animals to complex waste mixtures.

 The Toxicological Screen is being validated by using 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 Prescrecn could then be used (with an abbreviated form of the in
 vivo 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, chlordecone (kepone), cydophoshamide, and
 dtethylstilbestrol (DES) demonstrate the feasibility of the Toxicological Screen for detecting biological
 activity of chemicals.

 Acrylamide, evaluated at five test concentrations ranging 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 chlordecone is
 a reproductive toxin.  This observation agrees with  previously reported studies of sterility in chemical
 workers employed in chlordecone manufacturing in HopeweU, Virginia.  Had this sample been an un-
 known hazardous 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 cydophosphamide (15 to 24 mg/kg in rats) was most readily identified by the immunologi-
 cal and mutagenesis tests. Most other tests responded at the highest concentration tested, which resulted
 in 70% lethalities.  Cydophosphamide produced mutagenic metabolites  in the urine of treated rats.
 These metabolites were detectable using the Salmonella histidine reversion assay. Increases in sister
 chromatid exchanges 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
 cydophosphamide. 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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The exposure of animals 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 mg/kg. 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 establishing 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 biologically 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 (assuming 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 vitro 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 vitro 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 vivo/in vitro screening methods in the areas of liver toxicol-
   ogy, neurotoxicology, immunotoxtcology, and developmental biology for addition to the integrated Tox
   Screen.

3. The investigation and resolution of some of the basic lexicological problems related to complex mix-
   tures and the screening assay (i.e., acute vs. chronic effects and comparison of in vivo and in vitro
   methodologies).

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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HAZARDOUS WASTE
Issue:  Dioxia

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 using prominent biomarkers as
endpoints. The receptor binding, the induction of AHH and EROD, both in vivo and in vitro were deter-
mined which led to the following:  1) there is a definite structure-activity-relationship relative to the
toxitity 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 vitro and in vivo assays. Additionally, it was determined
that the in vitro 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-
tiated or were additive.
                                          -287-

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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 VIVO/IN VITRO
          TOXICOLOGICAL SCREEN: ACRYLAMIDE, CHLORDECONE,
              CYCLOPHOSPHAMIDE, and DIETHYLSTILBESTROL


                     A Andon,1 T. K. Rao,1 B. Most,2!. AUen.3E. Sermon,3
                       N. Oiemoff? K. Dean? D. DeMarini,3 J. Laskey?
                         R. MacPhail?L. Reiter,3 and R. Smialowicz3

                       Environmental Health Research and Testing, Inc.
  1 •                    .           Northrop Services, Inc.
                        3HERL, USEPA, Research Triangle Park, NC

This document describes a method to identify biologically active hazardous waste samples and process
residuals. The method is capable of screening large numbers 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 toxicity, reproductive toxicity, teratology, neurotoxicity and immunotoxscity.  Such a bioassay screen-
ing procedure for identifying biologically active wastes provides an important tool to EPA for setting stand-.
ards in the management of hazardous wastes and process-residuals.

An in vivo/in vitro 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 hi mutagenesis/carcinogenesis, general toxicol-
ogy, neurotoxicology, reproductive toxicology, teratology, and immunotoxicology. Oral administration of
waste material is given to rats for 10 consecutive days after which the whole animal, 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 chlor-
decone.  The toxicity of cyclophosphamide was most readily identified by the immunological and
mutagenesis tests. Diethylstilbestrol 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 eliminate certain tests to increase sensitivity and accuracy. In addition, several new areas of re-
search are being pursued in additivity/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
                                         -291-

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HAZARDOUS WASTE
PUBLICATIONS
  SCREENING COMPLEX HAZARDOUS WASTES for MUTAGENIC ACTIVITY
        USING a MODIFIED VERSION of the TLC/SALMONELLA ASSAY

                        Virginia Stewart Houk,1<2 and Larry D. Qaxton1

                        ^ERL, USEPA, Research Triangle Park, NC
                          2University of North Carolina-Chapel Hill

This research demonstrates the potential usefulness of an inexpensive and simple fractionation/bioassay
scheme that couples thin layer chromatography with the Salmonella tnutagenesis assay.  The study showed
that crude hazardous wastes can be successfully bioassayedformutagenic components in this manner; 7 of
10 hazardous wastes demonstrated mutagenic activity when tested by this method. The assay can be applied
to any number of complex mixtures, and should help program offices to quickly and inexpensively assay en-
vironmental samples for mutagenic constituents.

Ten complex hazardous wastes were tested for mutagenic activity using a modified version of the
TLC/Salmonella assay developed by Bjgrseth et al. (1982). This fractionation/bioassay scheme couples
thin-layer chromatography (TLC)  with the  SalmoneUa/mammalian-microsome (Ames) assay for the
detection of mutagenic constituents in complex mixtures. Crude (unadulterated) hazardous wastes and
selected hazardous waste extracts .were fractionated on commercially available cellulose TLC plates.
Mutagenicity testing was performed in situ by applying a single overlay of minimal 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-4-vis control plates.  Seven of 10 hazardous
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/SalmoneUa assay, 14 Salmonel-
la mutagens from a wide range of chemical classes and polarities were tested. Selected compounds  in-
cluded heterocyclics, aromatic  amines, alkylating agents,  antitumor agents,  a nhrosamme 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-85-155 Project Officer: Claxton
              OBSERVATIONAL BATTERIES and MOTOR ACTIVITY

                                    Robert C MacPhait

                        HERL, USEPA, Research Triangle Park, NC

This paper describes the needs for and approaches to screening chemicals for neurotoxicity, and should be
of benefit to the Program 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-
                                          -292-

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 PUBUCATIONS
HAZARDOUS WASTE
 sess and predict the possible health hazards associated with exposures has not kept pace with the produc-
 tion 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 an Neurobehavioral Methods in Safety Assessment of Chemicals and Drugs,
             Dusseldorf, Germany, December 16-19,1985.

 D109MS-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 Iff VIVO

             Jane Ellen Simmons, Marcus Jackson? Joetten Lewtas,  and Ezra Herman

                              ^ERL, USEPA, NRC Associate
                       Environmental Health Research & Testing, Inc.
                         3HERL, USEPA, Research Triangle Park, NC

This paper describes and compares the toxicity of four chemicals (acrylamide, chlordecone, cyclophos-
phamide, and diethylstilbestrol) in vivo, after JO days of repeated dosing with their toxicity in vitro, in a
Chinese hamster ovary cytotoxicity assay. This study provided data to interpret and understand the results
of this in vivo toxicity assay as they could apply to the in vivo situation.  This work 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 vivo toxicity, four chemicals that have been tested in the in
vivo/in vitro lexicological screen proposed by the Health Effects Research Laboratory, EPA were tested
in a Chinese Hamster Ovary (CHO) cytotoxicity assay.  Viability index, cellular ATP concentration and
rate of protein synthesis were decreased by exposure to acrylamide (AC), chlordecone (CH), diethylstil-
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 s CY. The toxicity
ranking based on viability index was: DES s CH < AC < CY. All of these chemicals were toxic in vivo
following 10 days of repeated dosing. The in vivo 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 as CY < AC (unable to rank DES since lowest concentration tested reduced body weight). The in
                                          -293-

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HAZARDOUS WASTE
PUBLICATIONS
vitro tenacity rankings did not correlate with in vivo 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 vivo toxicity rankings. The
limited number of chemicals that we tested does not allow us to answer this question.
        *                   *                                                              i
CITATION: In: Alternative Methods in Toxicology Series, vol. 5: In Vitro Toxicology-Approaches to Valida-
            tion, 1986.

D109 MS-86-163 Project Officer: Herman                                           .
    INFLUENCE of CORTISOL on PROSTAGLANDIN SYNTHESIS by FETAL
        MEMBRANES, PLACENTA and UTERUS of PREGNANT RABBITS

                          Randall P. Reynolds, and Patricia F. Noden

                               North Carolina State University

The administration of toxic chemicals during earfy pregnancy in humans may cause undetected fetal loss.
This study shows that cortisol injections during pregnancy caused abortions in the rabbit by inhibiting
ovarian function.  Exposure of human females to hazardous chemicals could similarly alter ovarian func-
tion and cause very earfy pregnancy loss and abortions

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 splanchnopleure, uterus, and placenta,
and the prostaglandin-forming cyclooxygenase activity determined. In Experiment 2, plasma drawn at
12-h intervals was quantified for prostaglandin F (PGF), and progesterone. Cortisol, PGF, and PGEa
were quantified in amniotic fluid. In cortisol-treated rabbits, plasma progesterone decreased (P< .01)
from 7.2 ±  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 animals 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  prostaglandin production may be responsible for the cortisol-induced abortion, increased
cyclooxygenase activity in the gestational compartment was not detected.

CITATION: Biology of Reproduction

D109 XX-85-112 Project Officer: Gray
                                          -294-

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PUBLICATIONS
HAZARDOUS WASTE
     EVALUATION of the IMMUNOTOXIC POTENTIAL of CHLORDECONE
                  with COMPARISON to CYCLOPHOSPHAMIDE

            R. J. Smialowicz, R. W. Luebke, M. M. Riddle, R. R. Rogers, and D. G. Rowe

                         HERL, USEPA, Research Triangle Park, NC

This study was conducted as part of an effort by HERL to develop a panel of tests to assess the potential
health effects of complex hazardous waste mixtures present in the environment. 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 series of pure compounds (e.g., cyclophosphamide)
with known biological effects and compounds of environmental concern (e.g.,  chlordecone) toward the
design, validation and selection of a protocol that can be applied to assess complex waste mixtures which
are found in the environment.  This work was performed at the request of 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 cyclophosphamide. Significant changes in the immune parameters examined,
which included spleen and thymus to body weight ratios, mitogen responsiveness to the lymphocyte
mitogen conconavalin A and natural killer (NK) cell activity against allogeneic-W/Fu-Gl rat lymphoma
target cells and xenogeneic YAC-1 mouse lymphoma target cells were observed only at the highest dosage
(10 mg/kg/day). A signifip^nr 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
chlordecone 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 mg/kg/day. 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-Iymphocyte 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 chlordecone did not affect the immune parameters ex-
amined in the absence of overt toxicity.

CITATION: Journal of Toxicology and Environmental Health 15(5): 561-574, October 1985.

D109 MS-84-128 Project Officer: Smialowicz
                                          -295-

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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 include 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 F1FRA and the Water Acts. Both efforts require the development and application of ad-
vanced toHCological 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 bioassays and markers that predict lexicological and car-
cinogenic responses in man from specific chemicals as well as mixtures present in drinking water. As this
work further clarifies the health hazard from water disinfectants and their by-products, a  companion
program to elucidate'the potential hazard from waterborae microorganism under reduced water disin-
fection conditions will be conducted. Animal models and in  vitro virulence tests will be used to evaluate
the health hazard of microorganisms transmitted under various water treatment criteria.  Infectious
agents associated with waterborne outbreaks will be indentified and studied in order to develop control
strategies.

The Neurotoricology 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.4earning/sensory/performance) in both adult and developing animals. 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 manage the Biometry
Division. Plans are underway to reorganize 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
biochemical 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
                                            -299-

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FUTURE PERSPECTIVES
Report on Epidemiology and Air Pollution, the Clean Air Scientific Advisory Committee, and research
planning 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 lexicological data in the standard setting
process, especially for NAAQS pollutants. Species sensitivity issues will receive increased emphasis since
such information, together with dosimetry  data, forms die 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 human 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 or in 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 k the field of perinatal toxicology within the Developmental Biology Division (DBD)
are becoming more molecularly oriented and research projects are increasingly 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-
                                            -300-

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                                                                  FUTURE PERSPECTIVES
ing tissue and route specificity. In addition, the relevance of genotoric endpoints to human health will be
explored and defined. The major endpoints for extrapolation will include both somatic and germ ceil 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/ multisource/integrated as-
sessment, dose receptors, source apportionment, and comparative potency. Emphasis will also be placed
on the analysis of indoor air and hazardous waste complex mixtures.
                                           -301-

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   Index of Abstracts




of Published Documents

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                                    Index
                                 Air Health
                                                                       Page No.

Aranyi, Catherine, William 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 J. Lewtas. RAT LIVER SUBCELLULAR FRACTIONS CATALYZE
       AEROBIC BINDING of 1-NTTRO[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, Avram Gold, and Joellen
       Lewtas. METABOLISM and ACTIVATION PATHWAYS of the
       ENVIRONMENTAL MUTAGEN 3-MTROFLUORANTHENE. In: 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. Williams, M. J. Kohan, and J. Lewtas. S9-DEPENDENT ACTIVATION of
       l-NITROPYRENE and 3-NTmOFLUORANTHENE in BACTERIAL
       MUTAGENICITY ASSAYS. In: Proceedings of the Third International Conference
       on Biological Reactive Intermediates, Pleason Press, New York, NY, 1985.
       MS-86-035	..-	i	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-86-010	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	1	32
                                     -305-

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                                                                        Page No.

Bedi, John F., 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	25


Berkey, Catherine S.; James H. Ware, Douglas W. Dockery, Benjamin G. Ferris, Jr., and
       Frank E. Speizer. INDOOR AIR POLLUTION and PULMONARY FUNCTION
       GROWTH in PREADOLESCENT CHILDREN. American Journal of
       Epidemiology 123(2): 250-260, February 1986.
       XX-86-113	49

Chang, Ling-Yi, Judith A. Graham, Frederick J. Miller, Jean J. Ospital, and James D. Crapo.
       EFFECTS OF SUBCHRONIC INHALATION of LOW CONCENTRATIONS of
       NITROGEN DIOXIDE I. THE PROXIMAL 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. In: 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. Mortelmans, J. Allen, A. Auletta, E. Nestmann, E. Zeiger, and J.
       McCann. A STANDARD GUIDE for the SALMONELLA
       TYPfflMimiUM/MAMMAUAN MICROSOME TESTS for BACTERIAL
       MUTAGENICITY [ASTM STANDARD]. 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 Oxidants Standards, H. Englund, ed., December
       1985.
       XX-86-025	42

Conti, C. J., N. Clapp, A. J. P. Hein-Szanto, S. Nesnow, and T. J. Slaga. SURVIVAL
       CURVES and INCIDENCE of NEOPLASTIC and NON-NEOPLASTIC DISEASE
       in SENCAR MICE. CarcinogenesLs 6(11): 1649-1652, October 1985.
       A10VL104MS-86-052	62
                                      -306-

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                                                                        Page No.

Davis, Wayne B., Carl G. Hayes, Marilyn Knowles, Wilson BJtiggan, 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-85-011	41

Ferris, Benjamin 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	48


Franch, Serafino and Gary E. Hatch. PULMONARY BIOCHEMICAL 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, H and Lester Kwock. OZONE INHIBITS PROSTACYCUN SYNTHESIS
       in PULMONARY ENDOTHELIUM. Prostaglandins 30(6): 1069-1083, December
       1985.
       XX-86-089	'.	.31


Goldring, J. M., L. M. Ball, R. Sangaiah, and A. Gold. SYNTHESIS and BIOLOGICAL
       ACnVTTY of a SERIES of NITROSUBSTTTUTED CYCLOPENTA-FUSED
       PAH. In: Proceedings of the Tenth International Symposium on Polynuclear
       Aromatic Hydrocarbons, W.M. Cooke & A J. Dennis, eds., Battelle Press, Columbus,
       OH, October 1985.
       MS-86-211	66


Goldstein, LF., DESIGN STRATEGIES for EPIDEMIOLOGIC STUDIES of
       ENVIRONMENTAL IMPACTS on HEALTH. In: 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
                                     -307-

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                                                                        Page No.

Hatch, Gary E., Ralph Slade, MaryJane K. Selgrade, 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 G. Stead, and Judith A. Graham. SPECIES
       COMPARISON of ACUTE INHALATION TOXICITY of OZONE and
       PHOSGENE. Journal of Toxicology and Environmental Health 19:43-53,1986.
       MS-85-060	,	53
Horstman, Donald, L. Jack Roger, Howard Kehrl, and Milan Hazucha AIRWAY
       SENSinVITY of ASTHMATICS to SULFUR DIOXIDE. Toxicology and
       Industrial Health 2(3): 1-25, September 1986.
       MS-84-065	
.27
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, M J. Utell, J.D. Hackney, J. Van Ryzin 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
       RANDOMIZED 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 ADDUCT FORMATION in
       RESPIRATORY TISSUES. Environmental Health Perspectives 62:203-207,
       October 1985.
       MS-85-106	60


King, Leon C, Louise M. Ball, Marcus Jackson, Jeff P. Inmon, and Joellen Lewtas.
       METABOLISM of 1-NrrROPYRENE by CULTURED RABBIT ALVEOLAR
       MACROPHAGES and RESPIRATORY TRACT TISSUES. Toxicology and
       Applied Pharmacology 82(2): 292-300, February 1986.
       MS-84-045	57


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
                                      -308-

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                                                                       Page No.

Kleindienst, T. E., E. O. Edney, G. 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. McKinnon 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. Chatham. OZONE
       RESPONSE RELATIONSHIPS in HEALTHY NONSMOKERS. American
       Review of Respiratory Disease 132:36-41, October 1985.
       XX-84-038	;....	27
              A
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: APPLICATION 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


Lewtas, Joeflen. 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 .'
                                    -309-

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                                                                       Page No.

Lcwtas, J. and K. Williams. A RETROSPECTIVE REVIEW of the VALUE of     .     .   . -
      SHORT-TERM GENETIC BIOASSAYS in PREDICTING the CHRONIC
      EFFECTS of DIESEL SOOT. In: Proceedings of International Symposium
      "Toricological Effects of Emissions from Diesel Engines", Tsukuba Science City,
      Japan, July 1986.
      MS-86-217	68


Lioy, P. J., T. A. Vollmuth, and M. Lippmann. 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 J. Miller. AMBIENT SULFATE
      AEROSOL DEPOSITION in MAN: MODELLING the INFLUENCE of
      HYGROSCOPICITY. Environmental Health Perspectives 63:11-24, November
      1985.
      MS-85-069	38

McDonnell, William 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 132(4): 875-879, October 1985.
      MS-85-067	'....:	26


Miller, F., T. B. Martonen, M. G. Menache, M. Lippmann, R. C. Graham, and D. M. Spektor.
      . INFLUENCE of BREATHING MODE and ACTIVTTY LEVEL on the
      REGIONAL DEPOSITION of INHALED PARTICLES and IMPLICATIONS for
      REGULATORY STANDARDS. In: Proceedings of Inhaled Particles VI,
      Cambridge, England, 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.
      TOXICTTY of PARTICLES EMITTED 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	55
                                     -310-

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                                                                       Page No.

 Mumford, Judy L., Silvestre B. Tejada, Marcus Jackson, and Joellen Lewtas.
       BIOAVAILABILITY of 1-NTTROPYRENE from MODEL COAL FLY ASH and
       ITS UPTAKE by ALVEOLAR MACROPHAGES. Environmental Research 40(2):
       427-436, August 1986.
       MS-83-223; .......................................... ........ ................. 58

 Mumford, J. L., K. Williams, D. B. Harris, J. C. Chuang and M. Cooke. INDOOR AIR
       SAMPLING and MUTAGENICITY STUDIES RELATED to EMISSIONS from
       UNVENTED COAL COMBUSTION. In: Proceedings of the 79th Air Pollution
       Control Association, Minneapolis, MN, June 1986.
       MS-86-190 ...... ................ . ............................................. 50

 Otto, David A. ELECTROPHYSIOLOGICAL ASSESSMENT of NEUROTOXICITY in
       CHILDREN. In: Mental Retardation, Neurobehavioral Toxicology and Teratology,
       S. Schroeder, ed., 1986.
       MS-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, DA., G. Robinson, S. Baumann, S. Schroeder, P. Mushak, D. Kletnbaum, and L. Boone.
       5-YEAR FOLLOW-UP STUDY of CHILDREN with LOW-to-MODERATE
       LEAD ABSORPTION: ELECTROPHYSIOLOGICAL EVALUATION.
       Environmental Research, 38: 168-186, 1985.
       MS-85-086 [[[ . ........... 29
Overton, John and Fred Miller. DOSIMETRY MODELING of INHALED TOXIC
       REACTIVE GASES. Health Effects Institute, (in press).
       MS-86-197	'I	
.36
Patra, A. L. MODELLING 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.

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                                                                        PageNo.

Schuetzle, Dennis and Joellen Lewtas. BIOASSAY DIRECTED CHEMICAL ANALYSIS
       in ENVIRONMENTAL RESEARCH Analytical Chemistry 58:1060a, 1986.
       MS-86-199	..	".	65


Spengler, John D., Margaret P. Reed, Erik Lebret, Bei-Hung Chang, James H. Ware, Frank
       E. Speizer, and Benjamin G. Ferris, Jr. HARVARD'S INDOOR AIR                  '
       POLLUTION/HEALTH STUDY. In: Proceedings of the Air Pollution Control
       Association; Minneapolis, MN, June 22-27,1986.             •                      :
       XX-86-112	 v	48


Steele, Vernon E. and Julia T. Arnold. ISOLATION and LONG-TERM CULTURE of
       RAT, RABBIT, and HUMAN NASAL TURBINATE EPITHELIAL CELLS. In         :
       Vitro Cellular and Developmental Biology 21(12): 681-687, December 1985.
       XX-85-054	38


Symons, Michael J., Donald L. Doerfler, and Yang C. Yuan. LIMITED OCCUPATIONAL
       MORTALITY DATA and STATISTICS. In: Biostatistics: Statistics in Biomedical,
       Public Health and Environmental Sciences, PJC Sen, ed., Elsevier Science Publishers,       ',
       The Netherlands, pps. 195-218,1985.                                             :
       XX-86-098	...	40  ,

Thurston, George D. and John D. Spengler. A MULTIVARIATE 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. Chuang, and J. L. Mumford. SELECTED
       ORGANIC POLLUTANT EMISSIONS from UNVENTED KEROSENE
       HEATERS. In: Proceedings of the 79th Air Pollution Control Association,
       Minneapolis, MN, June 1986.
       MS-86-191	51


Ware, J. H., B. G. Ferris, Jr., D. W. Dockery, J. D. Spengler, D. O. Stram, and F. E. Speizer.
       EFFECTS of AMBIENT SULFUR OXIDES and SUSPENDED PARTICLES on
       RESPIRATORY HEALTH of PREADOLESCENT CHILDREN. American
       Review of Respiratory Disease 133(5): 834-842, May 1986.
       XX-86-123	44
                                      -312-

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                                                                    Page No.

Williams, R., C. Sparacino, B. Petersen, J. Bumgarner, R. H. Jungers, and J. Lewtas.
      COMPARATIVE CHARACTERIZATION of ORGANIC EMISSIONS from
      DIESEL PARTICULATE, COKE OVEN MAINS, ROOFING TAR VAPORS, and
      CIGARETTE SMOKE COMPENSATE. International Journal of Environmental
      Analytical Chemistry 26:27-49, September 1986.
      MS-86-041	61
                                   -313-

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                                    Index
                          Non-Ionizing Radiation
                                                                      Page No.
Ali, Joseph S. and William T. Joines. A SYSTEM for MEASUREMENT of SMALL
       VIBRATIONS at MATERIAL INTERFACES INDUCED by
       ELECTROSTRICnVE FORCES. U.S. Environmental Protection Agency,
       EPA-600/1-85-02L November 1985. Available from NTE: PB86-116530/AS.
       HERL-0518	
.78
Blackman, C. F., S. G. Benane, J. R. Rabfaowitz, D. E. House, and W. T. Joines. 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. CR-812805, 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


Gage, 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 Elizabeth C. White. TEMPORAL RESPONSE of NEURONS to
       AMBIENT HEATING in the PREOPTIC and SEPTAL AREA of the
       UNANESTHETTZED RABBIT. Comparative Biochemistry and Physiology 82A(4):
       879-884, December 1985.
       MS-82-087	75
                                     -315-

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                                                                       Page No.

Uddle, C. G., J. P. Putnam, O. 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).
      Bioelectromagnetics 7(1): 101-105, January 1986.
      MS-83-003.	72

                   *             j.     -  »
Uddle, C. G., J. P. Putman, O. H. Lewter, M. West, and G. Morrow. CIRCULATING
      ANTIBODY RESPONSE of MICE EXPOSED to 9-GHz PULSED MICROWAVE
      RADIATION. Bioelectromagnetics 7(1): 91-94, January 1986.
      MS^83-244	:	72
Monahan, J. C. and J. A. D'Andrea. BEHAVIORAL EFFECTS 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. Ali, 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, Jury
      1986
      MS-85-210	...}	76
                                     i

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 FINITE-DIFFERENCE
      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, Stanislaw S. Stuchfy, and Andrzej Kraszewski.
      EXPOSURE of MAN in the NEAR-FIELD of a RESONANT DIPOLE:
      COMPARISON BETWEEN THEORY and MEASUREMENTS. IEEE
      Transactions on Microwave Theory and Techniques MTT-34(1): 26-31, January 1986.
      MS-84-130	.....74
                                     -316-

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                                                                     Page No.

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. Bioelectromagnedcs 7(3): 243-258, July 1986.
      MS-84-169	74


Watkinson, William P. and Christopher J. Gordon. IMPROVED TECHNIQUE for
      MONITORING ELECTROCARDIOGRAMS 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


Andelman, Julian B.  INHALATION EXPOSURE in the HOME to VOLATILE ORGANIC
       CONTAMINANTS of DRINKING WATER. The Science of the Total Environment
       47:443-460, November 1985.
       XX-86-059	 .132

Baird, William M, Said M. Sebti, and Lori A. Reinsvold. METABOLIC ACTIVATION 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).
       XX-86-038	129

Barnes, Donald W., Virginia M. Sanders, Kimber L. White, Jr., George M. Shopp, Jr., and
       Albert E. Munson. TOXICOLOGY of TRANS-1, -DICHLOROETHYLENE in the
       MOUSE. Drug and Chemical Toxicology, 8(5): 373-392, October 1985.
       XX-86-1Q3	157


Bercz, J. Peter, Lillian 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
                                     -319-

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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, E. Hope, J. M. Manson, E. L. George, and M. K. Smith.
      EVALUATION of the REPRODUCTIVE TOXICOLOGY of
      2,6-TRICHLOROPHENOL in MALE and FEMALE RATS.  Fundamental and
      Applied Toxicology 6(2): 233-239, February 1986.
      MS-85-129	138


Bollin, Gary E., Joseph F. Plouflfe, Michael F. Para, and Barbara Hackman. AEROSOLS
      CONTAINING LEGIONELLA PNEUMOPHILA GENERATED by SHOWER
      HEADS and HOT-WATER FAUCETS. Applied and Environmental Microbiology
      50(5): 1128-1131, November 1985;
      XX-S7-010	110


Borzelleca, Joseph F., Johnnie R. Hayes, Lyman W. Condie, and John L. Egle, Jr. ACUTE
      TOXIOTY of MONOCHLOROPHENOLS, DICHLOROPHENOLS and
      PENTACHLOROPHENOL in the MOUSE. Toxicology Letters 29(1): 39-42,
      December 1985.                         ,-
      MS-85-124	.-	149
Braun, A. G., F. A. Harding, and S. L. Weinreb. SHORT COMMUNICATION -
       TERATOGEN METABOLISM: THAUDOMIDE ACTIVATION is MEDIATED
       by CYTOCHROME P-450. Toxicology and Applied Pharmacology 82(1): 175-179,
       January 1986.               .       •
       XX-86-088.
.144
Bruckner, J. V., W. F. MacKenzie, S. Muralidhara, R. Luthra, G. M. Kyle, and D. Acosta.
       ORAL TOXICITY of CARBON TETRACHLORIDE: ACUTE, SUBACUTE, and
       SUBCHRONIC STUDIES in RATS. Fundamental and Applied Toxicology 6:16-34,
       1986.
       XX-85-006	155


Bull, Richard J., Janice E. Brown, Earle A. Meierhenry, Ted A. Jorgenson, Merrel Robinson,
       and Judith A. Stober. ENHANCEMENT of the HEPATOTOXICITY of
       CHLOROFORM in FEMALE B6C3F1 MICE by CORN OIL: IMPLICATIONS for
       CHLOROFORM CARCINOGENESIS. 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 69:49-58, November 1986.
       MS-86-176	164
                                      -320-

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                                                                         Page No.
 Bull, R. J., J. R. Meter, M. Robinson, H. P. Ringhand, R. D. Laurie, and J. A. Stober.
       EVALUATION of MUTAGENIC and CARCINOGENIC PROPERTIES of
       BROMINATED and CHLORINATED ACETRONTTRILES: BY-PRODUCTS of
       CHLORINATION. Fundamental and Applied Toxicology 5:1065-1074,1985.
       MS-85-131	177

 Bull, Richard J., Merrel Robinson, R. Dana Laurie, and Judith A. Stober. ASSOCIATION of
       CARCINOMA YIELD with EARLY PAPILLOMA DEVELOPMENT in
       SENCAR MICE. Environmental Health Perspectives 68:11-18, September 1986.
       MS-86-114	181
 Camann, D. E., P. J. Graham, M. N. Guentzel, H. J. Harding, K. T. Kimball, B. E. Moore, R.
       L. Northrop, N. L. Altaian, R. B. Harrist, A. H. Holguin, R. L. Mason, C. Becker
       Popescu and C. A. Sorber. THE LUBBOCK LAND TREATMENT SYSTEM
       RESEARCH and DEVELOPMENT PROJECT VOLUME IV - LUBBOCK
       INFECTION SURVEILLANCE STUDY(LISS). U.S. Environmental Protection
       Agency, cooperative agreement no. CR-807501, EPA-600/S1-85-023, January 1986.
       HERL-0526..*.	
.104-
Carlson, Gary P., Anthony A. Fossa, Mark A. Morse, and Patrice M. Weaver. BINDING and
       DISTRIBUTION STUDIES in the SENCAR MOUSE of COMPOUNDS
       DEMONSTRATING a ROUTE DEPENDENT EFFECT, Environmental Health
       Perspectives 68:53-60, September 1986.
       XX-86-084	168


Carlton, Betsy D., Paul Bartlett, Ali Basaran, Kate Colling, Irene Osis, and M. Kate Smith.
       REPRODUCTIVE EFFECTS of ALTERNATIVE DISINFECTANTS. 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 69:237-241, November 1986.
       MS-86-179	140


Chian, E. S. K., M. F. Giabbai, J. S, Kim, J. H. Reuter, and F. C. Kopfler. COMPARISON of
       HIGH MOLECULAR ORGANICS ISOLATED from DRINKING WATER in
       FIVE CITIES. In: American Chemical Society Advances in Chemistry Series, No.
       214,1986.
       MS-86-243	;i29


Coggins James R. and Frank W. Schaefer, ID. GIARDIA MURIS: ULTRASTRUCTURAL
       ANALYSIS of IN VITRO EXCYSTATION. Experimental Parasitology 61:219-228, '*
       April 1986.                          -
       MS-85-100	115
                                     -321-

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                                                                        Page No.
Condie, Lyman W. TOXICOLOGICAL PROBLEMS ASSOCIATED with CHLORINE
      DIOXIDE and CHLORINE. Journal American Water Works Association 78:73-78,
      June 1986.
      MS-86-105	.....151


Craun, Gunther F. A SUMMARY of WATERBORNE ILLNESS TRANSMITTED
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Craun, Gunther F. AN OVERVIEW of STATISTICS on ACUTE and CHRONIC WATER
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Craun, Gunther F. EPIDEMIOLOGIC STUDIES of ORGANIC MICROPOLLUTANTS in
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Gillin, Frances D., Michael J. Gault, Alan F. Hofmann, Devorah Gurantz and Judith F. Sauch.
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Herren-Freund, Sydna L. and Michael A. Pereira. CARCINOGENICITY of
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Herren-Freund, S. L., M. A. Pereira, R. E. Long, and M. M. Khoury. THE EFFECT of
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Herrmann, John E. ENZYME IMMUNOASSAV WITH MONOCLONAL ANTIBODIES
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Herrmann, John E. ENZYME-LINKED IMMUNOASSAYS for the DETECTION of
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Herrmann, J. E., G. P. Kent, N. A. Nowak, J. Brondum, and N. R. Blacklow. ANTIGEN
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Hurst, Christon J. and Tamara Goyke. SURVIVAL of INDIGENOUS ENTERIC VIRUSES
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Kallman, Mary Jeanne and Lyman W. Condie, Jr. A TEST BATTERY for SCREENING
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Knutsen, Gary L., Robert M. Kovatch, and Merrel Robinson. GROSS and MICROSCOPIC
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       November 1986.
       MS-86-170	175


Ward, Richard L. and Pat E. Winston. DEVELOPMENT of METHODS to MEASURE
       VIRUS INACTTVATION in FRESH WATERS. Applied and Environmental
       Microbiology 50(5): 1144*1148, November 1985.
       XX-85-114	127


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 b the
       MOUSE. Drug and Chemical Toxicology 8(5): 333-355, October 1985.
       XX-86-101	148


Williams, F. P.,Jr. VIRUS-LIKE PARTICLES with T = 19ICOSAHEDRAL SYMMETRY
       b a HUMAN GASTROENTERITIS STOOL. Micron and Microscopica Acta
       16(3): 173-178,1985.
       MS-85-102	120


Williams, Fred P., Jr. and Elmer W. Akb. WATERBORNE VIRAL GASTROENTERITIS.
      Journal of the American Water Works Association 78(1): 34-39, January 1986.
      MS-85-021	121
                                      -333-

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                                                                            Page No.
Wones, Robert and Charles Glueck. RESEARCH in PROGRESS: THE EFFECTS of
       CHLORINATED DRINKING WATER on HUMAN UPID 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 69:255-258, November 1986.
       XX-86-120	159
                                              t
Zierler, Sally, Robert A. Danley, and Lisa Feingold.  TYPE of DISINFECTANT in
       DRINKING WATER and PATTERNS of MORTALITY in MASSACHUSETTS.
       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.
      'XX-86-117	       . 102
                                       -334-

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                                   Index
                                 Pesticides
                                                                     Page No.

Clive, D., W. Caspary, P. E. Kirby, R. Krehl, M. Moore, J. Novak, 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	1	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	220

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 FIELDWORKERS. Bulletin of Environmental Contamination and
      Toxicology 35(4): 496-499, October 1985.
      MS-85-117	219
                                    -335-

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                                                                      Page No.
Griffith, Jade, Robert Duncan, and Janet Konefal. PESTICIDE POISONINGS REPORTED
      by FLORIDA CITRUS FffiLDWORKERS. Journal of Environmental Science and
      Health B20(6): 701-727, December 1985.
      MS-87-090	 .218


Hu, P., C. Huang, Y. Huang, 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. Huang, C. Y. Kawanishi, 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 NEUROTOXICrrY.
      Neurotoxicology 7(2): 169-182, May 1986.
   -   MS-86-059	210


Kawanishi, C. Y. MICROSPORIDIAN TAXONOMY: APPLICATION OF
      ELECTROPHORETIC and IMMUNOLOGICAL TECHNIQUES. In:
      Proceedings of the 4th International Colloquium on Invertebrate Pathology, The
      Netherlands, 1986.
      MS-86-109	216


Under, Ralph E., Lillian F Strader, and W. Keith McElroy. MEASUREMENT of
      EPIDIDYMAL SPERM MOT1LITY as a TEST VARIABLE in the RAT. Bulletin
      of Environmental Contamination and Toxicology 36(3): 317-324, March 1986.
      MS-85-166	207

Mahboob, Sottanali, Elizabeth R. Hoogenboom, Robert J. Kavlock, and Frances J. Zeman.
      EFFECTS on the FETAL RAT INTESTINE of MATERNAL MALNUTRITION
      and EXPOSURE to NTTROFEN (2,4-DICHLOROPHENYL-p- NITROPHENYL
      ETHER). Teratogenesis, Carcinogenesis, and Mutagenesis 6(6): 45-57, January 1986.
      MS-85-213	205

Moser, Virginia C. and Robert C. MacPhail. DIFFERENTIAL EFFECTS of
      FORMAMIDINE PESTICIDES on FIXED-INTERVAL BEHAVIOR in RATS.
      Toxicology and Applied Pharmacology 84:315-324, October 1986.
      MS-85-112	215
                                     -336-

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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, Shahbeg S. and Michael D. Waters. MECHANISMS of CARCINOGENICITY and
       ANTICARCINOGENICrrY: 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 MDCTURE EXPERIMENTS. In: Proceedings of
       the American Statistical Association, October 1985.
       MS-86-001	211

Veronesi, Bellina.  IN VITRO MODELING in NEUROTOXICOLOGY. In: A Textbook of
       Neurotoxicology, M.B. Abou-donia, ed., Ekcvier Science Publishers, The
       Netherlands, 1986.
       MS-86-030	211


Veronesi, Bellina and Stephanie Padilla. PHENYLMETHYLSULFONYL FLUORIDE
       PROTECTS RATS from MIPAFOX-INDUCED 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, Bellina, 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
                                     -337-

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                                                                        Page No.
Zeman, Frances J., Elizabeth R. Hoogenboom, Carol Cbase-Deesing, Robert J. Kavlock, and
      Janet L. Semple. EFFECTS on the FETUS of MATERNAL NITROFEN
      EXPOSURE in the PROTEIN-DEPRIVED RAT. Toxicology 38(1): 55-68, January
      1986.
      MS-85-214	204


Zeman, Frances J., Elizabeth R. Hoogenboom, Robert J. Kavlock, and Janet L. Semple.
      EFFECTS on the FETUS of MATERNAL BENOMYL EXPOSURE in the
      PROTEIN-DEPRIVED RAT. Journal of Toxicology and Environmental Health
      17(4): 405-417, April 1986.
      MS-85-215	i	203
                                      -338-

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                                    Index
                                    Toxics
                                                                       PageNo.

Adams, J., J. Buelke-Sam, C. A. Kimmel, C. J. Nelson, L. W. Reiter, T. Sobotka, H. A. Tilson,
       and B.K. Nelson. COLLABORATIVE 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(172): 225-232, December 1985.
       MS-85-238	234


Allen, James W., Jan C. Liang, Anthony V. Carrano, and R. Julian Preston. REVIEW of
       LITERATURE on CHEMICAL-INDUCED ANEUPLOIDY in MAMMALIAN
      MALE GERM CELLS. Mutation Research 167(1&2): 123-137, January/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 ... /.	223

Billingsley, M. L., C. D. Balaban, and J. P. O'Callaghan. PHARMACOLOGIC and
      IMMUNOLOGIC APPROACHES to the PROBLEMS of POSTTRAUMATIC
      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
                                     -339-

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                                                                      Page No.
Chadwick, Robert W., M. Frank Copeland, Gary P. Carlson, Bruce A. Trela, and Bernard M.
      Most. COMPARISON of IN VIVO and IN VITRO METHODS for ASSESSING
      the EFFECTS 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, G. L. Wolff, N. Cooke, D. A. Whitehouse, and M. L. Mole.
      EFFECTS of AGE and OBESITY on the METABOLISM of UNDANE by BLACK
      a/a, YELLOW A vy/a, and PSEUDOAGOUTIA vy/a PHENOTYPES of (YS x
      VY)F1 HYBRID MICE. Journal of Toxicology and Environmental Health 16(6):
      771-796, December 1985.                              '
      MS-85-029	..	,	248


Chase-Deesing, Carol, Robert J. Kavlock, and Frances J- Zeman. KIDNEY MORPHOLOGY
      and FUNCTION 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. J. Slaga. SURVIVAL
      CURVES and INCIDENCE of NEOPLASTIC and NON-NEOPLASTIC DISEASE
      in SENCAR MICE. Carcinogenesis 6(11): 1649-1652, October 1985.
      A101/L104 MS-86-052	229


Copeland, M. F., R. W. Chadwick, N. Cooke, 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
      NEUROTOXICTTY. In: Neurotoxicants and Neurobiological Function, Tilson and
      Sparber, eds., (in press).
      MS-86-009	."	261

Dyer, Robert S., Linda J. Burdette, Raelyn Janssen, and William K. Boyes.
      NEUROPHYSIOLOGICAL CONSEQUENCES of ACUTE EXPOSURE to
      METHYLPYRIDINES.  Fundamental and Applied Toxicology 5(5): 920-932,
      October 1985.
      MS-84-127	268
                                     -340-

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                                                                      Page No.
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 Zijl, and Stephen Nesnow. MORPHOLOGICAL
       TRANSFORMATION and CHROMOSOME DAMAGE by AMSACRINE in
       C3H/10T1/2 CLONE 8 CELLS. Mutation Research 170:133-143, April 1986.
       MS-87-091..!	.".	236

Frierson, M. R., G. Ktopman, and H. S. Rosenkranz. STRUCTURE-ACnVITY
       RELATIONSHIPS (SARs) AMONG MUTAGENS and CARCINOGENS: A
       REVIEW. Environmental Mutagenesis 8(2): 283-327, March 1986.
       XX-86-106	'.	271
George, S. E., L.C. King, and L.D. Clarion. 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:
       Neurobehavioral 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. Kavlock, Joseph S. Ostby, Janet M. Ferrell, and
       Katrina L. Gray. PRENATAL EXPOSURE to the FUNGICIDE DINOCAP
       CAUSES BEHAVIORAL TORTICOLLIS, 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. Kavlock, J. S. Ostby, J. M. FerrelLJ. M. Rogers, and K. Gray. AN
       EVALUATION of FIGURE-EIGHT MAZE ACTIVrrY and GENERAL
       BEHAVIORAL DEVELOPMENT FOLLOWING PRENATAL EXPOSURE to
       FORTY CHEMICALS: EFFECTS of CYTOSINE ARABINOSIDE, DINOCAP,
      NTTROFEN, and VITAMIN A. Neurotoricology 7(2): 449-462, May 1986.
      MS-86-050	241
                                    -341-

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                                                                      Page No.
Greenberg, B., P. Moore, R. Letz, and E. Baker. COMPUTERIZED ASSESSMENT of
      HUMAN NEUROTOXICITY: 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
      Langenbach, 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 Herman, and Hershell B. Carter. DESCRIPTION and
      IMPLICATIONS for ANALYSIS of BRAIN GROWTH in SUCKLING MICE.
      Growth 49:426-438, December 1985.
      MS-85-116	244


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


Huang, Yuan-Sheii, Neil Chernoff, Robert J. Kavlock, and Clinton Y. Kawanishi. THE
      EFFECTS of MATERNAL MURBNE CYTOMEGALOVIRUS INFECTION on
      the MOUSE CONCEPTUS at DIFFERENT GESTATIONAL STAGES.
      Teratogenesis, Cardnogenesis, and Mutagenesis 6(4): 331-338, August 1986.
      MS-86-002	277


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	238
                                     -342-

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                                                                       Page No.
Janssen, Raefyn, 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
       Neurophysiology 65(3): 203-211, May 1986.
       MS-85-127	266


JUTS, P. C, B. K. Lavine, and T. R. Slouch. PATTERN 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


Kavlock, Robert J., Blair F. Rehnberg, and Ellen H. Rogers. AMPHOTERICIN B- and
       FOUC ACID-INDUCED NEPHROPATHIES in DEVELOPING RATS.
       Toxicology and Applied Pharmacology 81(3): 407-415, December 1985.
       MS-85-085	243

Kavlock, R. J., 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


Kavlock, 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 P-450 (ESTROGEN SYNTHETHASE) by DERIVATIVES of
       a-NAPHTHOFLAVONE. Biochemical Pharmacology 35(17): 2887-2891, April 1986.
       L104MS-87-092	225


Kitchin, Kirk T. and Sam Kacew. SOME PHARMACOKINETIC and METABOLIC
       FACTORS AFFECTING the NEONATAL TOXKTTY 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

Kligennan, A. D., M.M. Moore, G.L. Erexson, K.H. Brock, C.L. Doerr, J.W. Allen, and S.
       Nesnow. GENOTOXICTTY STUDIES of BENZ(1)ACEANTHRYLENE.  Cancer
       Letters 31(2): 123-131, May 1986.
       MS-86-076	227
                                     -343-

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                                                                      Page No.
Klopman, G., R. Contreras, H. S. Rosenkranz, and M. D. Waters.
      STRUCTURE-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


Lafemina D. H. and P. C. JUTS. A NUMERICAL INDEX for CHARACTERIZING 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. Smialowicz.
      IMMUNE FUNCTION of YOUNG ADULT MICE FOLLOWING IN UTERO
      EXPOSURE to CYCLOPHOSPHAM1DE. 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 J. Smialowicz. IMMUNE FUNCTION in ADULT C57BL/6J 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. Marr, HI, 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. Brktenhaml and Victor Hasselblad. 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
                                                        »•
                                .      .i*      • ^   . .
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
                                     -344-

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                                                                      Page No.
Moore, Martha M., Amamda Amtower, Gary H. S. Strauss, and Carolyn Doerr.
       GENOTOXIOTY of GAMMA IRRADIATION in L5178Y MOUSE
       LYMPHOMA CELLS. Mutation Research 174(2): 149-154, June 1986.
       MS-86-004...	228


Moser, Virginia C. and Robert C. MacPhail. YOHIMBINE ATTENUATES the DELAYED
       LETHALITY INDUCED in MICE by AMTTRAZ, a FORMAMIDINE
       PESTICIDE. Toxicology Letters 28:99-104, November 1985.
       MS-85-118	266


Nesnow, Stephen, Hinda Bergman, 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-85-172	229


O'Callaghan, James P. NEUROTYPIC and GLIOTYPIC PROTEINS as BIOCHEMICAL
       INDICATORS of NEUROTOXICITY. In: Neurotoxicology, M.B. Abou-donia, ed.,     ;
       Oxford University Press (in press).
       MS-86-209	262


O'Callaghan, James P. and Diane B. Miller. DIETHYLDITHIOCARBAMATE
       INCREASES DISTRIBUTION©* CADMIUM to BRAIN BUT PREVENTS
       CADMIUM-INDUCED NEUROTOXICITY. Brain Research 370(1): 354-358,
       April 1986.
       MS-85-199	f	269


Oglesby, Linda A., Marian T. Ebron, Patricia E. Beyer, Brenda D. Carver, and Robert J.
       Kavlock. 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


Padilla, Stephanie and Donald Lyerly. EFFECTS 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
                                    -345-

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                                                                      Page No.
Peek, 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,
      May/June 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

Rehnberg, Georgia L., Joy F. Hein, 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, B J. Bryant, Kay Lawrence, Gaynelle Curtis, Helen Garland, and
      Stephen Nesnow. QUANTITATrVE ANALYSIS of the METABOLISM of
      BENZO(a)PYRENE by TRANSFORMABLE C3H10T1/2CL8 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, Shahbeg S., Baldev K. Vig, and Milton J. Constantin. DETECTION of
       CHEMICALLY INDUCED ANEUPLOIDY with PLANT TEST SYSTEMS.
       Mutation Research 167(1&2): 61-69, January/March 1986.
       MS-85-146	224
                                     -346-

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                                                                       Page No.
Sawyer, Jeffrey, Martha M. Moore, Donald Clive, and John Hozier. CYTOGENETIC
       CHARACTERIZATION of the L5178Y TK+/- 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. loannou, H. L. Fisher, and L. L. Hall. DERMAL
       ABSORPTION and DISPOSITION of 1,3-DIPHENYLGUANIDINE b 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 EPITHELIAL CELLS from a SERIES of DONORS. Cancer
       Research 46:4368-4371, September 1986.
       MS-86-081	232


Slotkin, Theodore A., Steven Pachman, Robert J. Kavlock, and Jorge Bartolome. EARLY
       BIOCHEMICAL DETECTION of ADVERSE EFFECTS of a
       NEUROBEHAVIORALTERATOGEN: INFLUENCE of PRENATAL
       METHYLMERCURY EXPOSURE on ORNTTHINE 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-86-251	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-85-157	258


Smialowicz, Ralph J., Ronald R. Rogers, Marie M. Riddle, Denise G. Rowe, and Robert W.
      Luebke.  IMMUNOLOGICAL STUDIES in MICE FOLLOWING IN UTERO
      EXPOSURE to NiCl2.  Toxicology 38(3): 293-303, March 1986.
      MS^85-190	255
                                     -347-

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                                                                      Page No,
Stouch T. R. and P. C Jure. 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, Kind 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
                     .     ' v
Trela, Bruce A., Gary P. Carlson, Robert W. Chadwick, and M. Frank Copeland.
      COMPARISON of IN VIVO 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
                                     -348-

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                                                                      Page No.
Watkinson, William P. EFFECTS 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


Watkinson, 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
                                                                          i

Watson, Dennis K., Mary J. McWilliams-Smith, Christine Kozak, Roger Reeves, John
      Gearhart, Michael F. Nunn, William Nash, John R. Fowle, III, Peter Duesberg, Takis
      S. Papas, and Stephen J. O'Brien. CONSERVED CHROMOSOMAL POSITIONS
      of DUAL DOMAINS of the ETS PROTOONCOGENE in CATS, MICE and
      HUMANS. Proceedings of the National Academy of Science 83:1792-17%, March
      1986.
      MS-86-218	235
                                    -349-

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                                    Index
                             Hazardous Waste
                                                                      Page No.

Andon, B., T. K. Rao, B. Most, J. Allen, E. Herman, N. Cbernoff, 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 ACTIVrrY 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.

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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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A101-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, JD; Miller, FJ. Effects of Inhalation of 0.25 ppm Ozone on the
    Terminal Bronchioles of Juvenile and Adult Rats. Submitted: American Journal of Pathology.
    MS-86-185

Benignus, VA; Midler, 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

Claxton, LD. A Guide for the Salmonella rjpAimurium/Mammalian Microsome Tests for Bacterial
    Mutageniciry. Submitted: Mutation Research.
    MS-86-203

Cooper, RL; 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

Goldring,JM; BalI,LM; Sangaiah,R;  Gold, A. Mutagenic Activity of Nitre-Substituted
    Cyclopenta-Fused Polycyclic Aromatic Hydrocarbons Towards Salmonella typhimurium.
    Submitted: Mutation Research.
    XX-86-143

Goldstein, GM; Raggio, L; House, DE. Factors Influencing Carboxyhemoglobin Stability.
    HERL-0532       *   .

Goto.S; Williams, K; Lewtas, J. A Micro-Forward Mutation Assay in Salmonella typhimurium,
    Modifications and Application to Complex Environmental Emissions. Submitted: Mutation
    Research.
    MS-86-075
                                         -355-

-------
Grose, EC; Graham, JA; Jaskot,RH; Richards, JH; Menache,MA; Dauterman, WC. A
    Comparative Study of the Effects of Inhaled Cadmium Chloride and Cadmium Oxide, Pulmonary
    Response. Submitted: Toxicology and Applied Pharmacology.
    MS-86-061

Grose, EC; Graham, JA; Richards, JH; Jaskot,RH; Menache,MG; Dauterman, WC. Glutathione
    (GSH) Peroxidase and GSH Transferase Activity in Rat Lung and Liver Following Cadmium
    Inhalation. 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 Inhaled Cadmium Chloride and Cadmium Oxide, Hepatic
    Response. Submitted: Toxicology and Applied Pharmacology.
    MS-86-0%

Horstman, DH;  Carver, BD; McDonnell, WF; Kehrl,HR; Seal, EG; Chapman, RS; Massaro, EJ.
    Pulmonary Effects in Asthmatics Exposed to 030 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;  Graham, DG; Henderson, FW. Ozone Effects on
    Alpha-1-Proteinase Inhibitor In Vivo - Blood Plasma Inhibitory Activity is Unchanged. Submitted:
    Experimental Lung Research.
    MS-86-212

Kehrl, HR; Horstman, DH; Vincent, L;  Kowalsky, R;  McCartney, W; Bromberg, PA. Ozone
    Exposure Increases Respiratory Epithelial Permeability in Man. Submitted: Science.
    MS-86-037

King,LC; Jackson, M; Ball,LM;  Lewtas, J. Metabolism and DNA Binding of l-Nitro[14C]pyrene by
    Isolated Rabbit Tracheal Epithelial Cells. Submitted: Carcinogenesis.
    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 PAHs 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-

-------
 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,JL; He,X; Chapman,RS;  Cao,S; Harris,DB; Li,*; Xian, Y; Jiang, W; Xu,C; Chuang,
    JC Lung Cancer and Indoor Air Pollution in Xuan Wei, China, D. Indoor Air Characterization.
    Submitted: Science.
    MS-86-051                                           '

 Mumford,JL; Williams, 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; Graham, RC. A Model of the Regional Uptake of Gaseous Pollutants in the
    Lung. n. The Sensitivity of Ozone Uptake in Laboratory Animals to Anatomical and Ventilatory
    Parameters. Submitted: Toxicology and Applied Pharmacology.
    MS-86-106

 Fatra, 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; Shaka,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

 Selgrade, MK; Hatch, GE;  Grose, EC; Hling, JW; Stead, AG; Miller, FJ; Graham, 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, JD; 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, FT; Ratajczak, HV; Aranyi, C;  Graham, JA. Effects of
    Inhalation of Ethylene Bichloride on Pulmonary Defenses of Mice and Rats. Submitted:
    Toxicology and Applied Physiology.
    MS-86-102

 Shoaf, CR;  Wolpert, RL; Menzel, DL. Factors Controlling Nitrosamine 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-

-------
Stevens, MA; Fitzgerald, S; Menache, MG; Costa, 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; Graham, JA. Pulmonary Function in Juvenile
    and Young Adult Rats Exposed to Low Level NOz with Diurnal Spikes. Submitted: Journal of
    Toxicology and Environment Health.
    MS-86-142

Strong, AA; Lundgren, DS; Hazucha,MJ; 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.
    MS-86-090

Tepper, JS; Weber, MF; Wiester, MJ; Costa, DL; Watkinson, WP. Cardiopulmonary Effects in
    Awake Rats Four and Six Months After Exposure to Methyl Isocyanate. Submitted:
    Environmental Health Perspectives.
    MS-86-182

Wiester, MJ. Report on the Effects of Inhalation of Low Concentrations of Ozone (Oa) and Nitrogen
    Dioxide (NQz) 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
B101 - WASTEWATER
Hurst, CJ; 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, MK. Evaluation of the Teratogenic Effects of
    Tri-Ortho-Cresyl Phosphate in the Long-Evans Hooded Rat. Submitted: Fundamental and
    Applied Toxicology.
    MS-86-215
C104 - DRINKING WATER
Baker, RJ; Gibs,J; Weng,AK; Suffet, IH. 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-

-------
 Bercz,JP; Melnikoff, MS; Morgan,!; Gabriel, KL. Effect of Hexametaphosphate in Drinking Water
    on the Levels of Manganese, Chromium, Copper & Zinc in the Bones of Rats. Submitted:
    Fundamental and Applied Toxicology.
    MS-86-253

 Bernstein, DI; Ziegler.J; Ward,R;  Gamble, JN. Rotavirus Fecal IGA Antibody Response in Adults
    Challenged with Human 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

 Carlton,BD; Basaran,AH; Mezza,LE; Smith, MK. Examination of the Reproductive Effects of
    Tricresyl Phosphate to Long-Evans Rats. Submitted: Toxicology.
    MS-86-232

 Carter, AM;  Pacha, RE; Clark, GW; Williams, EA. Seasonal Occurrence of Campylobacter spp. in
    Surface Waters and Then* Correlation with Standard Indicator Bacteria. Submitted: Applied and
    Environmental Microbiology.   .
    XX-86-109

 Chriswell, 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, PJ; Millette, JR; Allenspach, AL; McCauley,PT; Washington, 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; Bemrick, WJ. Giardia Speciation, Some Evidence for a New Species Giardia Psittaci.
    Submitted: Journal of Parasitology.
    XX-86-141

 Gillin,FD; Reiner, DS; Gault,MJ; Douglas, H; Wunderlich, A; Sauch, JF. Encystation and
    Expression of Cyst Antigens by Giardia Lambia In Vitro. Submitted: Nature.
    MS-86-151                                                  r

 Gault, MI; Gillin, FD;  Zenian, AJ. Giardia Lamblia • Human Intestinal Mucus and Epithelial Cells
    Stimulate Growth in Serum-Free Medium. Submitted: Experimental Parasitology.
    XX-86-142

Harrington, RM;  Shertzer, HG; Bercz, JP. The Effects of CIO2 on Thyroid Function in the African
    Green Monkey and the Rat. Submitted: Journal of Toxicology and Environmental Health.
    MS-86-023
                                          -359-

-------
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. Submitted: Journal of Medical Primatology.
    MS-86-024

Hayes, JR; Condie, LW; Borzelleca,JF. The Acute and Subchronic Toxicity in Rate 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; Huq, AS; Norman, F; Ho, H; Flynn, GL; Duraiswamy, N; Condie, LW. Partitioning
    Dependencies for the Permeation of Skin and Silicone Rubber Membranes by Phenols. Submitted:
    Journal of Pharmaceutal Sciences.
    MS-86-241

Kallman, MJ; Condie, LW; Hayes, JR; Borzelleca, JF. 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

Kopfler, FC; Craun, GF. Environmental Epidemiology: The Importance of Exposure Assessment.
    Submitted: 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 Lamblia.
    HERL-0550

Lukasewycz, MT; Bieringer, CM; Liukkonen, RJ; Fitzsimmons, ME; Corcoran, HF; Lin, S; Carlson,
    RM. The Analysis of Inorganic and Organic Chloramines Derivatization with
    2-Mercaptobenzothiazole. Submitted: Environmental Science and Technology.
    XX-86-051

Millette,JR; Stober,JA; Weiler,C; Black, D; Mills, T. Documentation of EPA Version of Simplex
    Program Package for Determining Peak-to-Continuum Ratios from Energy Dispersive X-ray
    Spectra from Biological Tissue.
    HERL-0531

Pacha, RE; Clark, GW; Williams, 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; Scheffcimaier, JJ; Debassachene, P. Small
    Rodents and Other Mammals Associated with Mountain Meadows as Reservoirs of Giardia spp.
    and Campylobacter spp. Submitted: Applied and Environmental Microbiology.
    XX-86-110

 Ringhand, HP; Meier, JR; Kopfler, FC; Schenck, KM; Kaylor, WH; 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 Diacetate/Propidium Iodide Staining and Animal Infectivity. Submitted:
    Applied and Environmental Microbiology.
    XX-86-048

 Witherell, LE; Duncan, RW; Stone, KM; Stratton, LJ; Orciari, L; Kappel, S; Jillson, DA.
    Investigation ofLegLonellapneumophila 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, J; Brusick, D J. 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 Tetrachloride Detected as a Mutagen in
    Salmonella Strain TA102. Submitted: Mutation Research.
    XX-86-001
E1Q4- PESTICIDES
Beyer, PE; Cheraoff, N. The Induction of Supernumerary Ribs in Rodents, Role of Maternal Stress.
    Submitted: Teratogenesis, Carcinogenesis, and Mutagenesis.
    MS-86-139

Carter, SD; Hess, RA; Laskey, JW. The Fungicide Methyl 2-Benzimidazole Carbamate Causes
    Infertility in Male Sprague-Dawley Rats. Submitted: Biology of Reproduction.
    MS-86-062
                                         -361-

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Cheraoff, N; Kavlock,RJ; Beyer, P; Miller, DB. The Potential Relationship of Maternal Toricity,
    General Stress, and Fetal Outcome. Submitted: Fundamental and Applied Toxicology.
    MS-S6-201

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. Pyrethroid 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. Methoxychlor Affects the Decidual Cell Responses of the Uterus but Not
    Other Progestational Parameters in Female Rats. Submitted: Toxicology and Applied
    Pharmacology.
    MS-86-259

Garrett, NE; Stack, HF; Waters, MD. Evaluation of the Genetic Activity Profiles for Sixty-Five
    Pesticides. Submitted: Mutation Research,
    MS-86-101

Jensen, KF; Killackey, HP. Terminal Arbors of Axons Projecting to the Somatosensory Cortex of the
    Rat. n. The Altered Morphology of Specific Thalamocortical Afferents Following Neonatal
    Infraorbital Nerve Cut. Submitted: Journal for Neuroscience.
    MS-86-195

Jensen, KF; Killackey, HP. Terminal Arbors of Axons Projecting to the Somatosensory Cortex of the
    Rat. I. The Normal Morphology of Specific Thalamocortical Afferents. Submitted: Journal for
    Neuroscience.
    MS-86-198

Libbus,BL; Perreault, SD; Johnson, LA; Pinkel,D. Incidence of Chromosome Aberrations in
    Mammalian Sperm Stained with Hoechst 33342 and UV-Laser-Irradiated During Flow Sorting.
    Submitted: Mutation Research.
    MS-86-239

Moser, VC; MacPhail, RC. Cholinergjc 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 Fertilizing 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 Vitro Interactions with Rat Brain
    Neurotoxic Esterase. Submitted: Toxicology and Applied Pharmacology.
    MS-86-060
                                          -362-

-------
 Perreault, SD; Naish,SJ; Zirkin,BR. The Timing of Hamster Sperm NudearDecondensation and
    Male Pronudeus Formation is Related to Sperm Nuclear Disulfide Bond Content. Submitted:
    Biology of Reproduction.
    MS-86-083

 Phelps, PV; Laskey, JW. Comparison of Age Related Changes in In Vivo and In Vitro 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 m the
    Rat. Submitted: Toxicology Letters.
    MS-86-229

 Semple,JL; Ellis, WG;  Hoogenboom, ER; KavIock,RJ; 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 Damage.
    Submitted: Journal of Neuropathology.
    MS-86-134         *       -           '

 Zeman, FJ; Heng,H; Hoogenboom, ER; Kavlock,RJ; Mahboob, S. Cell Number and Size in
    Selected Organs of Fetuses of Rats Malnourished and Exposed to Nitrofen. Submitted:
    Teratogenesis, Cartinogenesis, and Mutagenesis.
    MS-86-027
F104 - RADIATION
Ali, JS; Elliott, DJ. Control of Energy Absorption Rate in Transmission Line RF Exposure Systems.
    Submitted: Bioelectromagnetics.
    MS-86-161

Blackman, CF; Benene,SG; Wood,AR; House, DE; Elliot, DJ. Influence of Electromagnetic Fields
    on the Efflux of Calcium Ions From Brain Tissue In Vitro, Frequency Response Up to 510 Hz.
    Submitted: Bioelectromagnetics.
    MS-86-103
.1        -              -              •               ^               .

Blackmail, CF; House, DE; Benane.SG; Joines,WT; Spiegel, RJ. Effects of Ambient Levels of
    Power-Line-Frequency Electric Fields on a Developing Vertebrate. Submitted:
    Bioelectromagnetics.
    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, CJ. 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 Mechanism 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 pneumoniae. Submitted: Bioelectromagnetics.
    MS-86-034

Spiegel, RJ; Joines, WT. A Quasi-Static Method for Calculating Electric and Magnetic Fields in TEM
    Cells. Submitted: IEEE Trans, on Electromagnetic Compatibility.
    MS-86-094

Wood, AW; Joines, WT; Blackman, CF. The Characteristics of Transverse Electric and Magnetic
    Field (TEM) Cells at Extremely Low Frequencies. Submitted: Bioelectromagnetics.
    MS-86-158
L104-TOXICS
Allen, JW. 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 Carbamate 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'Callaghan, 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-

-------
 Browning, MD; Huang, C; Greengard, P. Similarities Between Protein fflA and Protein HEB, Two   .
    Prominent Synaptic Vesicle-Associate Phosphoproteins. Submitted: Journal of Neuroscience.
    XX-86-139                                                                    .     \

 Burdette, LJ; Dyer, RS. Differential Effects of Caffeine, Picrotonn, and Pentylenetetrazol on
    Hippocampal Afterdischarge Activity and Wet Dog Shakes. Submitted: Experimental Neurology.
    MS-86-234

 Burleson,GR; Fuller, LB; Menache, M; Graham, JA. Poly(I), Poly(C)-Enchanced Alveolar and
    Petitioned Macrophage Phagocytosis, Quantification by a New Method Utilizing Fluorescent
    Beads. Submitted: Proceedings of the Society of Experimental Biology and Medicine.
    MS-86-120

 Campbell, JA; Eppersimons, CF; Kligennan, 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, GR;  Graham, JA; Selgrade, MK. Effects of NiCb and CdCb
    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; Kavlock, RJ. Toxicity of Mercuric Chloride to the Developing
    Rat Kidney. HI. Distribution and Elimination of Mercury During Postnatal Maturation. Submitted:
    Toxicology and Applied Pharmacology.
    MS-86-067

 DeMarini,DM; Brock, KH; Doerr, CL; Moore, MM. MutagenicityandClastogenicityofTeniposide
    (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 O-amsa in Mammalian 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-Benzomidazol-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/n Vitro Dermal Penetration and
    Pharmacokinetics of 2,4,5,2',4'^'-Hcxachlorobipnenj4 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 hi 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, CJ. Relationship Between Behavioral and Autonomic Thermoregulation in the Guinea Pig.
    Submitted: Physiology and Behavior.
    MS-86-213

Gray, JA; Kavlock, RJ. Pharmacologic Probing of Mercuric Chloride Induced Renal Dysfunction in
    the Neonatal Rat. Submitted: Journal of Pharmacology and Experimental Therapeutics.
    MS-86-226

Gray, LE. 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.JS; Kavlock, RJ;  Ferrell,JM;  Gray, KL. Prenatal Dinocap Exposure
    Alters the Swimming 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 Proceedings, Cincinnati,
    OH, July 10-13,1984. Submitted: Journal of Occupational Medicine.
    HERL-0557

Henry, DR; Lavine, BK;  JUTS, PC. Electronic Factors and Acridine Frameshift Mutagenicity a Pattern
    Recognition Study. Submitted: Mutation Research.
    XX-86-079

Kavlock, RJ; Chernoff, N; Short, RD. Further Evaluation of an In Vivo Teratology Screen. Submitted:
    Teratogenesis, Cartinogenesis, and Mutagenesis.
    MS-86-202
                                           -366-

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Kavlock, RJ; Rehnberg, BF; Rogers, EH. Consequences to the Kidneys of Fetal, Neonatal, and
    Weanling 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.
    Submitted: Toxicology and Applied Pharmacology.
    MS-86-230

Kligennan, AD; Campbell, JA; Erexson,GL; Allen, JW; Shelby, MD. Sister Chromatid Exchange
    Analyses in Lung and Peripheral Blood Lymphocytes of Mice Exposed to Methyl Isocyanate by
    Inhalation. Submitted: Environmental Mutagenesis.
    MS-86-130

Lau,C; Cameron, A; Irsula,O; 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;  Smialowicz, RJ. Selective Immunotoxic
    Effects in Mice Treaded with the Adenosine Deaminase Inhibitor 2'-Deoxycofonnycin. Submitted:
    Immunopharmacology.
    MS-86-121

McGinty, JF; Kanamatsu, T; Obie, J; Mitchell, C; Hong, JS; Dyer, RS. Amygdaloid Kindling
    Increases Enkephalin-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;  Sangaiah, R; Gold, A; Nesnow, S.
    Morphological Transforming Activity and Metabolism of Cyclopenta-Fused Isomers of
    Benz(a)anthracene in Mammalian Cells. Submitted: Cancer Research.
    MS-86-065

Moore, MM; Brock, KH; Doerr, CL;  DeMarini, DM. Mutagenesis of L5178Y/TK"I"/"3.7.2C Mouse
    Lymphoma Cells,by die Clastogen Ellipticine. Submitted: Environmental Mutagenesis.
    MS-86-149

Moore, MM; Amtower, A; Doerr, CL; Brock, KH; Dearfield, KL. Genotoxicity of Acrylamidein
    L5178Y Mouse Lymphoma Cells. Submitted: Environmental Mutagenesis.
    MS-86-150

Nesnow, S; Bergman, HB; Bergman, H; Chu, K; Frith, C; Helmes, T; McGaughy, R; Ray, V; Slaga,
    TJ; Tennant, 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
                                          -367-

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Nesnow, S; Garland, H; Curtis, G. Inhibition or Enhancement of Morphological Transformation of
    CSHlOrVlCW Mouse Embryo Cells by Mutiple Carcinogen Treatment. Submitted: Cancer
    Letters.
    MS-86-254

Osman,R; Namboodiri, K; Webstein, 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, JF; Most, BM; Coffin, DL. Metaphase and Anaphase Analysis of V79 Cells
    Exposed to Erionite, UICC Chrysotile, and UICC Crocidolite. Submitted: Carcbogenesis.
    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, JD; MacPhail, RC. Conditioned Flavor Aversions, Applications in Assessing the
    Efficacy of Chelators 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, JD. Neurotoxicologic and Behavioral Effect of Aluminum and Aluminum Compounds in Man
    and Animals. Submitted: Neurobehavioral Toxicology and Teratology.
    MS-86-066

Reiter, LW. Neurotoxicology in Regulation and Risk Assessment. Submitted: Pediatric Pharmacology.
    MS-86-246

Richard, AM; Rabinowitz, JR. Modified Molecular Charge Similarity Indices for Choosing Molecular
    Analogues. Submitted: International Journal of Quantum Chemistry.
    MS-86-154

Rogers, JM; 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
                                          -368-

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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-Coffins, 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,RJ; Cowdery.T; Orband,L; Bartolome,M; Gray, JA; Rehnberg, BF;
    Bartolome, J. Functional Consequences of Prenatal Methylmercury Exposure, Effects on Renal
    and Hepatic Responses to Trophic Stimuli and on Renal Excretory Mechanisms. Submitted:
    Toxicology.
    MS-86-079

 Slotkin,TA; KavIock,RJ;  Cowdery.T; Orband,L; Whitmore,W; Bartolome,!. Effects of Neonatal
    Methylmercury Exposure on Adrenergic Receptor Binding Sites in Peripheral Tissues of the
    Developing Rat. Submitted: Toxicology.
    MS-86-058

 Slotkin,TA;  Orband,L; Cowdery.T;  Kavlock,RJ; Bartolome, J. Prenatal Exposure to
    Methymercury Alters Development of Adrenergic Receptor Bonding Sites in Peripheral
    Sympathetic Target Tissues. Submitted: Toxicology.
    MS-86-153

 Smialowicz, RJ. Immunologic Effects of Nonionizing Electromagnetic Radiation. Submitted: IEEE
    Engineering in Medicine and Biology.
    MS-86-003

 Smialowicz, RJ;  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; Superczynski, 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 Nonparametric Linear Discriminant Studies. Submitted:
    Quantitative Structure Activity Relationships.
    XX-86-Q82

TiIson,HA; Rogers, B; Grimes, LM; Harry, GJ; Peterson, NJ; Hong, JS; Dyer, RS.
    Time-Dependent Neurobiological Effects of Colchicine Administered Directly Into the
    Hippocampus of Rats. Submitted: Brain Research.
    MS-86-233
                                         -369-

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Valtortoa,F; Schiebler.W; Greengard, P; Jahn,R; Gxxarelli, B. A Solid-Phase Assay for the
    Phosphorylation of Proteins Blotted on Nitrocellulose Membrane Filters. Submitted: Analytical
    Biochemistry.
    XX-86-132

Watkinson, WP; Gordon, CJ. Effects of Chlordimeform on Heart Rate and Body Temperature of
    Unanesthetized Unrestrained Rats. Submitted: Toxicology Letters.
    MS-86X)17
                                           -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-86-011

 Andrews, JE; Courtney, KD; Donaldson, WE. Impairment of Calcium Homeostasis 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, J. Hepatic Metabolism of the Environmental Mutagen
    2-Nitrofluoranthene. Proceedings of Fall Meeting of American Society for Pharmacology and
    Experimental Therapeutics, August 19-221986.
    AB-86-237

 Cooper, RL; Goldman, JM; Rehnberg, GL; Hein, JF;  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; Lehmann, JR; Stevens, MA; Fitzgerald, S. A Rat Model of Mild COPD for Use in
    Toxicologic Studies. Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-042

 Davies, DW; Andrews, JM; Milliard, HG. A Coarse Mode Aerosol Generator for Rodent Inhalation
    Exposures. AAAR Meeting, Albuquerque, New Mexico, November 18-22,1985
    AB-85-147

Fitzgerald, S; Lehmann, 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, TE; 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 Olfactory and Parietal
    Areas of Rat Brain. Society for Neuroscience Meeting, Dallas, Texas, October 20-24,1985
    AB-85-137
                                         -373-

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Grose, EC; Graham, JA; Richards, JH; Jaskot, RH; Dauterman, WC. The Relationship Between
    Pulmonary Glutathione Peroxidase and Glutathione Transferase Following Inhalation of Cadmium
    Chloride. ISSX Meeting, Key Biscayne, Florida, November 17-22,1985
    AB-85-151

Grose, EC; flling, JW; Stead, AG; Stevens, MA; Jaskot, RH; Hardisty, J. Pulmonary and Hepatic
    Health Effects of-Inhalation of Petroleum Smoke. Society of Toxicology Meeting, New Orleans,
    LA, March 3-7,1986.
    AB-86-026

Hatch, GE; Slade, R. Effects of Buthionine Sulfoximine (BSO) Treatment or of Ascorbic Acid (AA)
    Deficiency on the Edemagenic Effect of Inhaled Oa, NO2, and COCfe. Society of Toxicology
    Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-019

Hatch, GE; Slade, R; Koren, HS. 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. Mechanism of Action of Ozone on the Human Lung.
    American Thoracic Society, Kansas City, MO, May 11-14,1986.
    AB-86-128

Horstman, DH; Folinsbee, LJ; Vorona, RD; 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

Horstman, DH; Folinsbee, LJ. Airway Reactivity and Obstruction, Relationship to Pollutant Induced
    Broachoconstriction in Asthmatics. FASEB Meeting, St. Louis, MO, April 13-18,1986.
    AB-86-127

Jaskot, RH; Most,B; Menache,MG; Williams, T; Grose, EC; Roycroft,JH. The Uptake of Methyl
    Bromide in the Rat Following Inhalation Exposure. Society of Toxicology, New Orleans, LA,
    March 3-7,1986.
    AB-86-028

Kehri, 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 Society, Kansas City, MO, May 11-14,1986.
    AB-86-129

King, LC; Jackson, M; Ball, LM;  Lewtas, J. Metabolism of l-Nitro(14C)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 l-Nitro(14C)pyrene by Primary Tracheal
    Epithelial Cells. GEMS Meeting, Durham, NC, November 7,1985.
    AB-86-005
                                          -374-

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King, LC; Jackson, M; Ball, LM; Lewtas, J. Metabolism of l-Nitro[14CJpyrene by Primaiy Tracheal
    Epithelial Cells. Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-085

Korea, 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. VIICT 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; Graham, 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-Salaam, S;  Howe,DE; Hazucha, MJ. Airway Reactivity Assessment by
    Single Dose and Dose-Response Challenge to Methacholine. American Thoracic Society, Kansas
    City, MO, May 11-14,1986.
    AB-86-123

Milholland, VS; Horstman, DH; Koren, HS. Changes in Mononuclear Cell Populations in Exercised
    and Ozone Exposed Human Subjects. Clinical Application of Cytometry Annual Meeting,
    Charleston, SC, September 30-Qctober 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

Mumford, JL; Harris, BD; Williams, K; Chuang, JCC; Cooke, M. Indoor Air Pollution Related to
    Emission from Unvented Coal Combustion. 79th Annual Air Pollution Control Association
    Meeting, Minneapolis, MN, June 22-27,1986.
    AB-86-001

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
                                          -375-

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Otto, DA; Robinson, G; Baumann, SB. Electrophysiological Assessment of Sensory & Cognitive
    Function in Children with Undue Lead Absorption. International Workshop on the Effects of
    Lead Exposure on Neurobehavioral Development, Scotland, September 1986.
    AB-86-217

Overton, JH. Biological Disposition of Airborne Vehicle Emissions, III. Reactive Gases. Workshop on
    Health Effects Research and Motor Vehicles, Dearborn, MI, October 31 - November 1,1985.
    AB-86-070

Overton, JH; Graham, 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 (SO2) on Asthmatic Subjects. California Air Resources
    Board Meeting, March 18-20,1986.
    AB-86-180

Padilla,SS; Boyes, WK; Lyerly, D. Tunicamycin Blockage of Axonal Transport in the Rat Optic
    System Proceeds Graded Electrophysiological Changes. Conference on Axonal Transport,
    University of Calgary, Calgary, Alberta, Canada, July 13-19,1986.
    AB-86-183

Patra, AL; Shaka, N. Particle Deposition in Casts of Human 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;  MenzeLDB. 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

Riggan, WB. Mortality Variation and Trend by County, Race and Sex 2nd Conference of EPA
    Statisticians, Williamsburg, VA, November 5-8,1985.
    AB-86-003
                                          -376-

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 Riggan, 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, LJ; Horstman, DH; KehrI,HR. Absence of Change in Sensitivity to Inhaled Methachoiine in
    Asthmatics Exposed to NO2. 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, Washington, DC, March 1986.
    AB-86-230

 Schreiber, RT; McDonnell, WF; Horstman, DH; Ives,P; Abdul-Salaam, S; Bromberg, PA. The
    Relationship of Inhaled Citric Acid Sensitivity and Responsiveness to Ozone. American Thoracic
    Society, Kansas City, MO, May 11-14,1986.
    AB-86-144

 Schwartz,!; 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 Optimal Battery of Genetic Bioassays to
    Screen for Carcinogenicity. 2nd Conference of EPA Statisticians, Williamsburg, VA, November
    5-8,1985.
    AB-86-010

 Steele, VE;  Morgan, DL; Nixon, JC; Humphreys, JE;  Hatch, GE. An Animal 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, S; 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,MJ; Costa, DL; Weber, MF; King, ME. Adaptation to Ozone in F-344 Rats.
    Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-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; Hein,JF; McElroy,WK; Goldman, 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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WiesteiyMJ; Miller, FJ; Williams, TB; King ME; Menache, MG. Respiratory Dosimetry of Ozone
    (Oa) 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; Tyroler, HA; Hayes, CG; Riggan, WB. Geographic
    Variation in the Onset of the Decline of IHD Mortality in the United States. American Heart
    Association Council of Epidemiology, San Francisco, CA, March 3-5,1986.
    AB-86-169
B101-WASTEWATER
Camann, DE. Association of Viral Infections with Spray Irrigation of Municipal Wastewater. APHA
    Annual Meeting,!Washington, DC, November 17-21,1985
    XA-85-012

Donnelly, KC; 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-86-011

Dufbur, 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, CJ; Benton, WH. 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; Blazak,WF; Riccio.ES; Rushbrook, CJ; Stewart, BE; Condie, LW. Geaotoxicity and
    Subchronic Toncity Studies of Municipal Wastewater Effluents. Environmental Mutagen Society
    Meeting, Baltimore, MD, APRIL 9-13,1986.
    AB-86-142
C104 - DRINKING WATER
Bercz,JP; Condie, LW; Harrington, RM; Zimmer, H.Toricity of lodinated 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,RJ; 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
                                          -378-

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 Carlton, BO; Barlett, PB; Smith, MK, Reproductive Effects of Chloramine Administered by Gavage
    to Long-Evans Rats. Abstracts of the 25th Anniversary Meeting 6(1): 293, March 1986.
    AB-86-079

 Carlton, BD; Barlett, PB; Smith, MK. Reproductive Toricity 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-Dibromoethane 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 Vitro. ASPET-SOT Meeting, Baltimore, MD, August 1986.
    XA-86-016                                                      '      .        •

 Coleman, WE; Munch, JW; Streicher, RP; Hodakievic, 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; Bercz, JP. Effect of Corn Oil Vehicle on Subchronic
    Hepatotoadtity of Carbon Tetrachloride (CT) in CD-I Mice. Abstracts of the 25th Anniversary
    Meeting 6(1): 183, March 1986.
    AB-86-066

 Condie, LW; Berzc, JP. Health Effects of Drinking Water Disinfectants and Disinfection By-Products.
    Conference on Trace Substances in Environmental Health, Columbia, MO, June 2-6,1986.
    AB-86-168

 Conolfy, RB; Cramer, J; Andersen, ME. A Physiologically-Based Model for Rat Hepatic Glutathione
    (GSH) - Its Orcadian Oscillation and Interaction with Halogenated Hydrocarbons. ASPET-SOT
    Annual Meeting, Baltimore, MD, August 18-22,1986.
    XA-86-025

Davis, ME. Comparison of Subacute Tenacities of Dichloroacetate and Trichloroacetate in Female
    Rats. Abstracts of the 25th Anniversary Meeting 6(1): 161, March 1986.
    XA-86-012

DeAngelo, AB; Herren-Freund, SL; Pereira, MA; SchuIts,NE; Haunig, JE. Species Sensitivity to
    the Induction of Peroxisome Proliferation by Trichloroethylene and Its Metabolites. Abstracts of
    the 25th Anniversary Meeting 6(1): 113, March 1986.
    AB-86-086

DuMouiin,GC; Coleman, EC; Tsang,AY;  Stottmeier, KD. Contamination of Drinking Water
    Supplies with Mycobacterium Avium Complex (MAC). Interscience Conf. on Antimicrobial
    Agents and Chemotherapy, New Orleans, Lousiana, September 28- October 1,1986
    XA-87-007
                                          -379-

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George, EL; Smith, MK; Zenick, H; Hastings, L. Developmental Toxicity Associated with
    Acrjiamide (ACR) Exposure in Long-Evans Rate. Abstracts of the 25th Anniversary Meeting
    6(1): 90, March 1986.
    AB-86-087

Gillin, FD; Gault,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.KE; PfohLRJ; Laurie, RD. Exposure to Trichloroethvlene 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 Vivo 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 Dichloroacetic
    Acid (DCA), in Mouse Liver. American Association for Cancer Research, Los Angeles, CA, May
    7-10,1986.
    AB-86-191

Jakubowski, 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; Borzelleca, 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, RJ."Effects of Paniculate of Coal Tar Paint in A/J Mice. Abstracts of
    the 25th Anniversary Meeting 6(1): 233, March 1986.
    AB-86-107             ' •                  '

Laurie, RD; Robinson, M; Bercz, JP;  Mobley, SA; Long, RE; Condie, LW. Toxicity of
    1,2,3-Trichloropropane. ASPET-SOT 1986 Annual Meeting, Baltimore, MD, August 18-22,1986.
    AB-86-238
                                          -380-

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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 Drinking Water. Environmental Mutagen Society Meeting,
    Baltimore, MD, April 9-13,1986.
    AB-86-141

 Lin, 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

 Lykins, BW; Koffskey, W; Miller, RG. Pilot Scale Investigations at Jefferson Parish, Louisiana.
    Second National Conference on Drinking Water (Treatment for Organic Contaminants) --
    Edmonton, Alberta, Canada, April 7-8,1986.
    AB-86-205
                                           t
 Mather, G; ParneO,MJ; Exon,JH; Roller, LD, Promotion of Carcinogenesis by Trichloroacetic Acid
    (TCA). Abstracts of the 25th Anniversary Meeting 6(1): 231, March 1986.
    XA-86-002

 Meier, JR;  Rudd, CJ; Blazak, WF; Riccio, ES; Miller, RG. Comparison of the Mutagenic Activities
    of Water Samples Disinfected with Ozone, Chlorine Dioxide, Monochloramine, 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 Subchronic Oral
    Toxicity Studies of 1,1-DichIoroethane (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-Dichloropropane (DCP), Acute, Subacute and Subchronic Oral Exposures in Rats.
    Abstracts of the 25th Anniversary Meeting 6(1): 183, March 1986.
    XA-86-003
                                          -381-

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Nagy, B; Bercz, JP. Screening Tests in Tenacity or Drug Effect Studies with Use of Centrifichem
    General-Purpose Spectrophotometric Analyzer. Joint Meeting of the American Society of
    Biochemicals and the American Chemical Society, Washington, DC, June 7-14,1986.
    AB-86-166

Noland-Gerbec, EA; Pfohl,RJ; Taylor, DH; Bull, RJ. Glucose Uptake in the Developing Rat Brain
    upon Pre- and Post-Natal Exposure to Trichloroethyiene. Society of Neuroscience, Dallas, Texas,
    October 20-24,1985
    AB-85-165
                                     •j
Nowak,NA; Cubitt,WD; Herrmann, JE; Blacklow,NR.ImmunologicalRelatednessofNorwalk
    Virus and Human Calitivirus. American Society for Microbiology, Washington, DC, March 23-28,
    1986.
    XA-8WM7
                                             t
Fereira,MA;  Herren-Freund, SL; Olson, G; 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; Ruch, RJ. 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-86-006

Richmond, RE; Pereira, MA. Immunohistbchemical Detection of FES, 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

Ringhand, HP; Meter, JR; Kopfler.FC; Schenck,KM; Kaylor.WH; Mitchell, DE. Influence of pH
    And Chlorine Residue on die Recovery of Mutagenicity from Drinking Water by XAD Resins.
    Environmental Mutagen Society Meeting, Baltimore, MD, April 9-13,1986.
    AB-86-124

Ringhand, HP; Meier, JR; Kopfler,FC; Schenck,KM; 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, RJ. Carcinogenic Activity Associated with Chlorinated Acetones and
    Acroleins  hi the Mouse Skin Assay. Abstracts of the 25th Anniversary Meeting 6(1): 234, March
    1986.
    AB-86-067
                                         -382-

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 Sauch, JF; Monnile, MR. Bouyant Density Profiles of Giardla Lamblia Cysts from Mongolian Gerbils,
    Comparison to Human Giardia And G. Muris Cysts. American Society for Microbiology,
    Washington, DC, March 23-28,1986.
    AB-86-068

 Sauch, JF. Recovery and Detection of Giardia Cysts from Drinking Water Samples. Greater Cincinnati
    Parasitology & Topical Medicine Society, December 5,1985.
    AB-86-125

 Sauch, JF. 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; Guion,C; DeAngelo, AB; Chang, L;  Danner, R. Further Studies on the Mechanism of
    Chloroform Stimulation of Rat Hepatic Ornithine 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, JK Chloroform Induction of
    Ornithine Decarboxylase-Antizyme (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; Preston, RJ; George, EL; Long, RE. Dominant Lethal Effects of Subchronic
    Acrylamide Administration 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
    Trichloroacetonitrile 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 Trichloroethylene 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, July 27 - 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 Trichloroethylene
    on Hippocampal Development in Rat Pups. Abstracts of the 25th Anniversary Meeting 6(1): 122,
    March 1986.
    AB-86-075
                                          -383-

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Sublet, VH; Smith, MK; Randall, J; Zenick, H. Spermatogenic Stages Associated with Acrylamide
    (ACR) Induced Dominant Lethality. Abstracts of the 25th Anniversary Meeting 6(1): 292, March
    1986.
    AB-86-078

Toth, GF; Smith, MK. Effect of CIO2 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 Human Lipid
    Metabolism. American Chemical Society Meeting, New York, NY, April 1986.
    XA-86-017

Wones,RG; Mieczkowski, L; Glueck, CJ. Effect of Chlorinated Drinking Water on Human Lipid
    Metabolism. American Chemical Society Meeting, Anaheim, CA, September 7-12,1986.
    XA-86-018
D109-SOLID WASTE
Acedo, GN; Sandhu, SS. Detection of Chemically-Induced Aneuploidy in Vicia Faba Roots.
    Environmental Mutagen Society Meeting, Baltimore, MD, April 9-13,1986.
    AB-86-113

DeMarinijDM; Doerr, CL; Meyer, MK; Moore, MM. Mutagenitityof M-amsaandO-amsain
    Mammalian Cells is Due to the Clastogenic Effects of the Compounds. Environmental Mutagen
    Society Meeting, Baltimore, MD, April 9-13,1986.
    AB-86-110

Ebron, MT; Beyer, PE; Oglesby, LA. In Vitro Culture of Post-Implantation Hamster Embryos - A
    Second Approach. Society of Toxicology 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, GG; Edens,F; Rodgers,L; Most,B; Berman, E. Embryotoxic and Potential Tumorigenic
    Effects in Japanese Quail Inoculated In Ovo by Chemical Carcinogens and Noncarcinogens.
    Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-041

Houk, VS; DeMarint, DM. Use of the Microscreen Phage-Induction Assay to Assess the Genotoxicity
    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
                                         -384-

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Rodgers,L; Hatch, GG; Edens,F; Most,B; Berman, E. Embryotoric 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 9-13,1986.
    AB-86-112

Simmons, JE; Lewtas, J. Detecting Toxicity 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 Colliculus of
    Adult Long-Evans Rats Following Monocular Enucleation. Society for Neuroscience Meeting,
    Dallas, Texas, October 20-24,1985
    AB-85-140

Chernoff, N. Importance of Assessment of the Aging Animal in Toxicology Studies. 38th Annual
    Meeting Gerontological Society of America, New Orleans, Louisiana, 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; Mailman, 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.
    AB-86-060
                                          -385-

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Crofton, KM; Peek, 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 Detidual Cell Response of the Female Rat by Methoxychlor. 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 15-171986.
    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 Potentials
  ,  (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; Sunder, MR; Hall, LL. Chlordecone 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

Goldman, JM; Cooper, RL; Rehnberg, GL; Hein,JF; McElroy.WK; Gray, LE. The Influence of
    Methoxychlor (MXC) Upon the Hypothalamic-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-Dihydroxytryptamine. 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

Kawanishi, CY. Use of Monoclonal Antibodies for Classification and Identification of Insect
    Pathogens. IVth Interaation Colloquium on Invertebrate Pathology, Veldhoven, 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
                                           -386-

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 Moser, VC; MacPhail, RC. Cholinergjc Involvment in the Behavioral Actions of Formetanate, A
    Formamidine 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; Zirkii^BR. Ability of the Fertalizing Hamster Sperm Nucleus
    to Serve as Template for DNA Synthesis. American Society for Cell Biology Annual Meetbg,
    Atlanta, Georgia, November 1985
    AB-85-183

 Padilla, SS; Grizzle, T; Lyerly, D. A Comparison of In Vitro aadln 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 Decqndensation 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, HP; Brady, AL; Waters, MD. Genetic Activity Profile Methodology. Environmental Mutagen
    Society Meeting, Baltimore, 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-Induced 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
                                          -387-

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F104 -RADIATION
Allis,JW; Sinha-Robinson, RL; Stead, AG. Analysis of the Microwave Radiation Induced Inhibition
    of Na+/K+ATPase by Response Surface Methodology. Bioelectromagnetics Society, Madison,
    WI, June 1-5,1986.
    AB-86-135

Baumann, SB; Joines,WT; Herman, E; All, JS. Development of a Passive Transponder to Monitor
    Temperature within the Uterus of Freeh) Roaming Mice in Radiofrequency Reids.
    Bioelectromagnetics Society, Madison, WI, JUNE 1-5,1986.
    AB-86-130

Blackmail, CF; Benane,SG; House, DE;  Joines,WT; Spiegel, RJ. Effect of Power-Line-Frequency
    Electric Fields on a Developing Organism Bioelectromagnetics Society, Madison, WI, June 1-5,
    1986.
    AB-86-136

Blackmail, CF; Benane, SG; House, DE. Frequency-Dependent Responses in Brain Tissue to ELF
    Electromagnetic Fields and the Influence of the Local Geomagnetic 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, January 21,1986.
    AB-86-139

Elder, JA. Radiofrequency Radiation - Activities and Issues. Health Physics Society Annual Meeting,
    Pittsburgh, PA, June29-July 3,1986.
    AB-86-219

Fatmi, MBE;  Spiegel, RJ. A High Resolution Finite-Difference Model of A Squirrel Monkey to
    Predict Thermo-Physiologic Response Due to RF Radiation. Bioelectromagnetics Society,
    Madison, WI, June 1-5,1986.
    AB-86-131

Liddle, CG; Putnam, JP; Lewter, OH. Survival of Mice Exposed Chronically 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.
    Bioelectromagnetics Society, Madison, WI, June 1-5,1986.
    AB-86-134

O'Connor, ME; Tremble, D; Peterson, T; KeowSeng,N; Cooper, B. Thermal Dose-Response
    Measures in Rats  and Mice At Specific Absorption Rates Between 2 and 8 w/kg.
    Bioelectromagnetics Society, Madison, WI, June 1-5,1986.
    XA-86-008                                                           !
\
                                          -388-

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Wood, AW; Blackmail, CF; Joines, WT. The Use of a Transverse Electric and Magnetic Held (TEM)
    Ceil 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
Allen, JW; Stoner,G; Pereira,MA; Hatch, GG; Nesnow,S; Campbell, J; Sharief, Y. CeD
    Transformation, Tumorigenesis, and SCE Induction by Ethyl Carbamate and Vinyl Carbamate.
    Environmental Mutagen Society, Las Vegas, Nevada, February 1986
    AB-85-184

Amtower, AL; Brock, KH;  Doerr, 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, MJ. Effects of the Promoters TPA and Derivatives On Colony
    Formation in Culture of Human, Hamster, and Rat Respiratory Epithelial Cells. Gems Meeting,
    Durham, NC, November 7,1985.
    AB-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, LJ; 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, GR; Fuller, LB; Graham, JA. Poly (I), Poly (C)-Mediated Abrogation of Inhaled
    Ozone-Impaired Phagocytosis. Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-048

Catigani, JC; Burleson, GR; Selgrade, MK. Relationship Between Depressed Cytochrome P-4SO .
    Levels Observed During Murine Cytomegalovirus Infection and Virus Induced Interferpn
    Response. Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-045

Catignani, JC; Burleson, GA; Selgrade, MK. Interferon Response to Murine Cytomegalovirus
    Infection, Effect On Xenobiotic Metabolism. American Society for Microbiology Meeting,
    Washington, DC, March 23-28,1986.
    AB-86-012
                                         -389-

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Claxton, LD; Walsh, D; Stead, AG. Analysis, Including Correlation with Cartinogenicity, of the
   Salmonella tyhimurium Bioassay Data Bases by Chemical Class. Environmental Mutagen Society
   Meeting, Baltimore, MD, April 9-13,1986.
   AB-86-120

Clegg, MS; Rogers, JM; 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; Sandhu, SS. Development and Validation of Human Cell Culture
   Assay for the Detection of Chemically-Induced Aneuploidy. Environmental Mutagen Society
   Meeting, Baltimore, MD, April 9-13,1986.
   AB-86-118

Cooper, RL; Goldman, JM;  Rehnberg, GL; Booth, KC; McElroy, WK; Hein, J. Effect of
   Reproductive Toxins on Brain-Pkuitary-Gonadalans. EPA/Army Workshop, Charleston, SC,
   November 13-15,1985.
   AB-86-092

Copeland,MF; 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

Ciimmings, AM. The Decidual Cell Response and Maternal Toxicity. EPA/Army Workshop,
   Charleston, SC, November 13-15,1985.
   AB-86-098

Doerr, CL; Brock, KH; Demarini, DM; Moore, MM; Analysis of Gross Aberrations in Mouse '
   Lymphoma Cells Exposed to Various Genotoric Agents. Environmental Mutagen Society
   Meeting, Baltimore, MD, April 9-13,1986.
   AB-86-115

Erexson, GL; Kligennan, AD; Allen, JW. Development of a Mouse Peripheral Blood Lymphocyte
   (PBL) Micronudeus Test Using Cytochalasin B. Environmental Mutagen Society Meeting,
   Baltimore, MD, April 9-13,1986.
   AB-86-1I?

Fuller, LB; Burleson, GR; Graham, JA. Poly (I), Poly (C) Enhanced Phagocytosis of Rat Alveolar
   and Peritoneal Macrophages as a Model for Local Pulmonary and Systemic Immunotoxicology.
   Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
   AB-86-049

Goldman, JM; Cooper, RL;  McElroy, WK; Hein, J; Rehnberg, GL. The Effect of Methoxychlor
   Upon the Concentration and Release of Gonadotropin- Releasing Hormone from Mediobasal
   Hypothalamus. EPA/Army Workshop, Charleston, SC, November 13-15,1985.
   AB-86-093

Gray.JA; Rehnberg, BF; Slotkin,T; Kavlock,RJ. Effects of Exposure to A-Difluromethylornithine
   (DFMO) During Discrete Prenatal Periods On Postnatal Growth and Renal Function in the Rat.
   Teratology Society Meeting, Boston, MA, July 1986.
   AB-86-155
                                         -390-

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Gray, LE; Gray, K; Ferrell, JM; Ostby, JS. Methoxychlor-Induced (M) Alterations of Reproductive
    Development in Rats. Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-030

Gray, LE; Ferrell, JM; Gray.K; Ostby, JS. Alterations in Reproductive Development in Hamsters
    Induced by Methoxychlor. Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-031

Gray.LE; Rogers, JM; Ostby, JS; Gray,KL; Ferrell, JM. Torticollis and Swimming Behavior
    Abnormalities Develop in Mice After Prenatal Dinocap Administration 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, JS; Gray, K; Ferrell, JM. 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

HaU,LL; Fisher, HL; Sumler.MR; Shah, Pv. Age Related Percutaneous Penetration of Carbaryl,
    Determined by In Vivo and In Vitro Methods. Society of Toxicology Meeting, New Orleans, LA,
    March 3-7,1986.
    AB-86-038

Hein, J; Cooper, RL; Rehnberg, GL; Goldman, JM; McElroy, WK. Effects of Methoxychlor On
    Pituitary Prolactin. EPA/Army Workshop, Charleston, SC, November 13-15,1985.
    AB-86-101

Hess, RA. Histological Methods for Evaluating 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.JW. Fertility and Histopathology in Male
    Rats Following Carbendazim Exposure. EPA/Army Workshop, Charleston, SC, November 13-15,
    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, June 30 - July 2,1986.
    AB-86-220

Kavlock, RJ. Functional Aspects of Developmental Toxicky of Pesticides. International Conference on
    Pesticides, Lucknow, India, October 19885
    AB-85-187

Kavlock, RJ. Postnatal 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
                                          -391-

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Kavlock, RJ; Rehnberg, BF. Consequences to the Kidneys of Fetal, Neonatal and Weanling Rats of
    Prenatal Exposure to Adriamycin. Teratology Society Meeting, Boston, MA, July 7-11,1986.
    AB-86-157

Kligerman, AD; Campbell, JA; Erexson,GL; Shelby, MD;  Allen, JW. Cytogenetic Analysis of
    Peripheral Blood Lymphocytes (PELS) and Lung Cells of Mice Exposed to Methyl Isocyanate
    (MIC) by Inhalation. Environmental Mutagen Society, Baltimore, MD, April 9-13,1986.
    AB-86-143

Kohan, MJ; King, LC; Claxton, UD. The Influence of Extraction and Concentration Techniques on
    the Isolation of 1-Nitropyrene Bacterial Metabolites. American Society for Microbiology Meeting,
    Washington, DC, March 23-28,1986.
    AB-86-119

Koren, HS; Harris, DT; Gianriolo, GJ; Snyderman, R. Suppression of Natural Killer (NK) Cells by a
    Synthetic Peptide Homologous to Retroviral P15E. Conference On Growth Factors, Tumor
    Promoters, ect., Steamboat Springs, CO, April 1986.
    AB-86-089

Laskey, JW; Laws, SD. In Vitro Assessment of Leydig Cell Function FoUowing Metal Cation
    Treatment EPA/Army Workshop, Charleston, SS, November 13-15,1985.
    AB-86-100

Lau, C; Station, TA. Control of AdrenomeduUary Development by Central Biogenic Amine Systems.
    American Society for Neurochemistry, Montreal, Canada, March 16-21,1986.
    XA-86-004

Lau,C; Cameron, A; Irsula,O; Robinson, KS. Some Aspects of the Teratogenic Effects of Nitrofen
    Exposure in the Rat. Teratology Society Meeting, Boston, MA, July 7-11,1986.
    XA-86-010

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; Smialowtcz, RJ; Rowe, DG. Effects of
    5-Chloro- and 5-Fluorouracil on Immune Function. Society of Toxicology Meeting, New Orleans,
    LA, March 3-7,1986.
    AB-86-025
       l
Macphail, RC;  Cook, LL; Walker, QD. Acute Behavioral Toricity of Cyclotrimethylenetrinitramine
    (RDX). Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-061

Massaro, EJ. Tune-Dependent Tissue Distribution of Methyhnercury in the BALB/C Mouse and Its
    Hairless Homolog, the HRS/j Mouse. Society of Toxicology, New Orleans, LA, March 3-7,1986.
    AB-86-027

Massaro, EJ; 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
                                          -392-

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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, Richland, Washington, October 21-24,1985
    AB-85-134

 Mass, MJ; Siegfried, JM; Hozier, JC. Genes for Tumor Markers are Clustered Near Cellular
    Oncogenes on the Human Chromosome Map. American Association for Cancer Research, Los
    Angeles, CA, May 7-10,1986.
    AB-86-137

 Mass, MJ. Genes for Tumor Markers Are Clustered Near Cellular Oncogenes. UCLA Molecular
    Biology Symposium, Steamboat Springs, CO, January 20-22,1986.
    AB-86-138

 Maxwell, HD; FerreO,JM; 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.
    AB-86-097

 McElroy, WK; Cooper, RL; Rehnberg, GL; Goldman, JM; :Hein, J. Effects of Estrogenic
    Compounds On Food Intake and Body Weight. EPA/Army Workshop, Charleston, SC, November
    13-15,1985.
    AB-86-102

Milo, GE; Kurian,P; Nesnow, S. Characterization and Validation of Carcinogen-Induced
    Transformation of Human Cells In Vitro. 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; Sangaiah, R; Gold, A.
    Morphological Transformation of Mammalian Cells by Cyclopenta-Fused Isomers of
    Benz(a)anthracene 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.
    AB-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, LE. 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
                                         -393-

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Otto, DA. Development of Computerized Behavioral and Electrophysiological Batteries for Human
    Neurotoricity Testing. U.S.-Sweden Toxicology Workshop, NffiHS/RTP, April 14-16,1986.
    AB-86-179

Palekar, LD; Coffin, DL. Significance of Mineral Fibers in Mesothelioma 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; Under, 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 Altered Reproductive Function. EPA/Army
    Workshop, Charleston, SC, November 13-15,1985.
    AB-86-094          •    .       .    .     .

Reinhard, JF; O'Callaghan, JP. MPTP Decreased Dopamine Levies and Increases the Levels of Glial
    Fibrillary Acidic Protein in Mouse Striatum - Evidence for Glial Reaction to Injury. American
    Society for Pharmacology & Experimental Therapuetics, Baltimore, MD, August 1986.
    AB-86-212

Richard, AM; Rabinowitz, JR. Modified Molecular Charge Similarity Indices for Choosing Chemical
    Analogues. International Symposium on Quantum Biology and Quantum Pharmacology, St.
    Augustine, FL, March 6-8,1986.
    AB-86-149        .              .             ;

Rogers, JM; Carver, BD; Gray.LE;  Gray.JA; Kavlock, RJ. Teratogenic Effect of the Fungicide
    Dinocap in the Mouse. Society of Toxicology Meeting, New Orleans, LA, March 3-7,1986.
    AB-86-023

Rogers, JM; Gray, LE; Carver, BD;  Kavlock, RJ. 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, RS. Application and Limitations of a Mammalian Cell Culture Test System for
    Detecting Chemically-Induced Aneuploidy. International Symposium on Short-Tenn Tests for
    Genotoxicity, Pisa, Italy, April 5-8,1986.
    AB-86-172
                                          -394-

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 Selgrade, MK. Use of Susceptibility to Murine Cytomegalovirus as an Indicator of Immunotoxicity.
    Association of Official Analytical Chemists Meeting, Washington, DC, October 28,1985
    AB-85-155

 Selgrade, MK. Susceptibility to Murine Cytomegalovirus (MCMV) - A Host Resistance Model for
    Immunotoxicity Testing. Immunotoxicology Discussion Group Meeting, Chicago, Illinois, October
    9-10,1985
    AB-85-172

 Selgrade, MK; Catignani, 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, MK. Disease Susceptibility Models Under Investigation at EPA. Eastern Regional
    Symposium on Mechanisms of Immunotoxicity, Williamsburg, VA, September 4-5,1986.
    AB-86-241

 Shah,PV; Fisher, HL; Sumler, MR; Hall, 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-85-153

 Smialowicz, RJ; 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

 Smialowicz, RJ. 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, HAJ; 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, Rockville, MD, April 22-23,1986.
    AB-86-177

Viana, MA; Brock, TO; O'Callaghan, JP. 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
                                          -395-

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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

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

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                                          -396-

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