United States
Environmental Protection
Agency
Environmental Monitoring
and Support Laboratory
PO Box 15027
Las Vegas NV 89114
EPA-600/9-79-004
January 1979
Research and Development
&EPA
Report of the
Workshop on Biological
Screening Tests
Las Vegas, Nevada
September 12-14, 1977
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EPA-600/9-79-004
January 1979
REPORT OF THE WORKSHOP ON
BIOLOGICAL SCREENING TESTS
Las Vegas, Nevada
September 12 - 14, 1977
by
Charles A. Bicking, Editor
Tracer Jitco, Inc.
Rockville, Maryland 20852
Contract No. CB-7-0913-B
Project Officer
Dr. John A. Santolucito
Monitoring Systems Research and Development Division
Environmental Monitoring and Support Laboratory
Las Vegas, Nevada 89114
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89114
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring and Support
Laboratory-Las Vegas, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or recom-
mendation for use.
ii
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FOREWORD
Protection of the environment requires effective regulatory actions
which are based on sound technical and scientific information. This infor-
mation must include the quantitative description and linking of pollutant
sources, transport mechanisms, interactions, and resulting effects on man
and his environment. Because of the complexities involved, assessment of
specific pollutants in the environment requires a total systems approach which
transcends the media of air, water, and land. The Environmental Monitoring
and Support Laboratory-Las Vegas contributes to the formation and enhancement
of a sound monitoring data base for exposure assessment through programs
designed to:
develop and optimize systems and strategies for
monitoring pollutants and their impact on the
environment
demonstrate new monitoring systems and tech-
nologies by applying them to fulfill special
monitoring needs of the Agency's operating
programs
This report contains recommendations for selecting substantially
predictive biological screening tests. The large number of chemicals
which can potentially impact human health and the environment precludes
the complete testing of each substance. In order to effect preliminary
chemical hazard ranking, initial tests must be standardized and validated
and the necessary quality control practices and techniques developed and
implemented.
This report contains recommendations for selecting substantially
predictive biological screening tests upon which the Agency's quality
assurance resources may be initially concentrated.
George/B./Moi
Jiredtor /
Environmental Monitoring and Support Laboratory
Las Vegas
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CONTENTS
Foreword lit
Abbreviations vi
Introduction 1
Conclusions and Recommendations 4
Report on Chemical/Physical Aspects of
Toxicity Testing 8
Report on Whole Animal Acute and Sub-
chronic Testing 15
Report on In-Vitro Testing 25
Report on Model Ecosystems 29
General Bibliography 36
List of Workshop Participants 42
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LIST OF ABBREVIATIONS
ABBREVIATIONS
AOAC — Association of Official Analytical Chemists
ASTM — American Society for Testing and Materials
BOD — biological oxygen demand
CFR — Code of Federal Regulations
CNS — central nervous system
COD — chemical oxygen demand
CPSC — Consumer Products Safety Commission
CSL — Chemical Systems Laboratory
DREW — Department of Health, Education, and Welfare
EMSL-LV — Environmental Monitoring and Support Laboratory-Las Vegas
EPA — U.S. Environmental Protection Agency
ERL — Environmental Research Laboratory
F2 — second generation effects
FDA — Federal Drug Administration
FR — Federal Register
NIH — National Institutes of Health
OMTS — Office of Monitoring and Support
ORNL — Oak Ridge National Laboratory
OSHA — Occupational Safety and Health Administration
OTS — Office of Toxic Substances
P — primary production
PR — primary production/respiration ratio
R — respiration
TOC — total oxygen concentration
TOSCA — Toxic Substances Control Act
w/w — ratio of gaseous to particulate
vi
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INTRODUCTION
The Workshop on Biological Screening Tests was cosponsored by the
Environmental Monitoring and Support Laboratory-Las Vegas (EMSL-LV) and the
Office of Toxic Substances, U.S. Environmental Protection Agency (EPA).
The Workshop objective was to identify and recommend screening tests
which are immediately applicable and substantially predictive of the impact
of chemical pollutants on biological systems.
Attendees included representatives of EPA laboratories involved in bio-
logical research, individuals from a number of other governmental agencies
engaged in similar work, academic experts in various fields of toxicity test-
ing, and individuals from Tracer Jitco, Inc., of Rockville, Maryland, who had
responsibility for convening the Workshop, assembling working group reports,
and preparing this report. A list of the participants is included at the
end of this document.
The Manufacturing Chemists Association was invited to send a represen-
tative to the Workshop and to give a presentation. Unable to do so, it
recommended a paper by Astill et al., as representing suitable industry input
on the subject matter of the Workshop: Astill, B. D., et al., 1977. A Tier
Testing Scheme. Presented at the Toxicological Forum, Institute of Pathology,
Aspen, Colorado, July 18-22, 1977.
As shown in the Workshop agenda on page 3, invited papers covering vari-
ous areas of toxicity testing of interest to the Workshop were presented on
the first day. The second and third days were devoted to separate meetings
of working groups on Chemical/Physical Testing, Whole Animal Acute and Sub-
chronic Testing, In Vitro Testing, and Model Ecosystems.
Although each of the working groups concluded that it was possible,
within the brief duration of the meeting, to identify applicable tests in
its area, the recommendation of a specific battery of tests was impractical
for a number of reasons. Among the reasons were: (1) the small amount of
information available that correlates short-term tests with long-term effects
of interest to the EPA; (2) the developmental stage in which a number of
promising tests remain; and (3) the desire of working group participants to
obtain a wider consensus within the scientific community on which of the
available tests should be selected.
The working groups felt that the range of substances of interest to the
EPA is so wide that no single set of tests could be prescribed which would
meet all conditions. They also felt there was a need to consider not only
toxicity to animals and humans (health effects) but also adverse effects in
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the whole environment (ecological effects).
Most of the Workshop participants felt that their view of the EPA's need
was too limited to warrant their recommending specific tests, and that the
EPA had the responsibility for selecting a battery or batteries of tests fron
the lists the Workshop participants supplied. This was not meant to preclude
further assistance from the scientific community in filling information gaps.
This report presents the conclusions and recommendations drawn from
individual reports of each working group which are also included.
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AGENDA FOR THE WORKSHOP ON BIOLOGICAL SCREENING TESTS
General Session
Monday. September 12, 1977
Welcome to Laboratory - George B. Morgan, Director, EMSL-LV
Opening Remarks - Albert C. Trakowski, Jr., OMTS, and William M.
Upholt, OTS
Workshop Charge - John A. Santolucito, EMSL-LV
Testing of Generic Groups of Chemicals - John M. Bryant, OSHA
Biological Monitoring of Available Toxic Materials in Soil - Robert D.
Rogers, EMSL-LV
Applicability of Animal-to-Human Correlation from Tissue Culture -
John F. Lontz, VA
Approaches to Screening Agents for Mutagenicity - Sidney Green,
Howard University
Whole Animal Toxicity Testing - Bernard P. McNamara, CSL
Model Ecosystems - Sidney Draggan, ORNL
Notices and Instructions for Working Groups - John Santolucito, EMSL-LV
Working Group Sessions
Tuesday, September 13, and Wednesday, September 14, 1977
I. Chemical/Physical Aspects of Toxicity Testing
II. Whole Animal Acute and Subchronic Testing
III. In Vitro Testing
IV. Model Ecosystems
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CONCLUSIONS AND RECOMMENDATIONS
CHEMICAL/PHYSICAL TESTING
The report of the Working Group on Chemical/Physical Aspects of Toxicity
Testing outlines the data that a manufacturer may be expected to submit in
complying with the Toxic Substances Control Act (TOSCA). These data will be
useful in evaluating the potential environmental impact of the chemical, in
establishing the priorities for biological testing of chemicals, and in selec-
ting appropriate biological test methods for each chemical.
Not every test listed in the report will be applicable to every product
examined as a result of a requirement of the TOSCA. The parameters measured
might vary greatly depending upon the nature of the substance and the prob-
lems of evaluating its ultimate environmental impact. Most of the data re-
quired should be available from standardized analytical tests and from good
manufacturing practices.
Structure/activity correlations have been attempted for many chemical
classes and, where data are available, they should be used judiciously and
to the extent possible in selecting biological tests to be performed.
In compliance with Sections 4, 5, and 8 of the TOSCA, representative
samples of proposed new products should be submitted to the EPA and it is
recommended that, should such sample collection be activated, the EPA estab-
lish a product repository for storing and distributing these reference samples.
There is a need for standardized tests for measuring biodegradability.
WHOLE ANIMAL TESTING
National laws and regulations indicate that it may be necessary to test
for any or all short- or long-term toxicological effects on body organs or
functions. Test areas and sources of test methods are tabulated in the report
of the Working Group on Whole Animal Acute and Subchronic Testing.
The practical situation of use may make the degree of exposure and risk
very different for different chemicals. Not all compounds need to be tested
for every effect and it is possible that some compounds may require no test-
ing.
The Working Group did not attempt to assess the degree of risk; instead
it outlined, within current scientific knowledge, those tests which were
necessary and acceptable, leaving it to the EPA to specify the tests required
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for a chemical, based on use information supplied by manufacturers.
Problems associated with adherence to a strict experimental protocol for
each class of chemicals include:
o Pre- and post-natal exposure
o Selection of species and strain
o Sex, age, pre-existing or intercurrent disease
o Route of administration
o Frequency and level of dosing
o Dose-response relationships
o Interaction with other chemicals and environmental
factors which may produce synergistic, potentiating
or antagonistic effects.
Aquatic Environment Testing
An outline is presented showing, for various aquatic species, acute and
chronic tests with comments regarding their availability and use. The tests
are classified as readily available, or as new but promising. No specific
recommendations are made on tests to be run, but the information in the out-
line is adequate to enable the EPA to select a screening battery of available
tests which can be used to assess the toxicity of a chemical to a variety of
aquatic species.
Behavioral Toxicology
Relatively little data have accumulated on the neurophysiologic and
behavioral effects of environmental chemicals. However, standardized tests
are routinely used in screening drugs for central nervous system (CNS)
effects and could be similarly applied to environmental pollutants. A screen-
ing might include, therefore, activity changes, objective signs, reflex
changes, elicited responses, and body weight changes.
These screening tests should be followed by study of delayed effects
using longitudinal research design which is well standardized and reported.
Tests requiring minimal instrumentation and training time include motor
activity using a photocell cage or an activity wheel and Sidman avoidance
performance. Sensory function tests are also available.
Attention should be given to the feasibility of using behavioral tests
in a first-screening battery of tests for environmental toxicants. Rapidity
of response, relatively low cost, ease of administration, and repeatability,
all support this approach.
IN VITRO TESTING
The Working Group on In Vitro Testing focused its attention on endpoints
of mutagenicity, carcinogenicity and cytotoxicity. It was agreed that short-
term testing cannot be substituted for whole animal testing in any of these
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three areas at present.
In mutagenicity, full source documents exist evaluating the correlation
of short-term results with known in vivo results. In addition to published
articles, more recent drafts have summarized the state-of-the-art in muta-
genicity testing methodologies. No such full source documentation is avail-
able for carcinogenicity. Procedures available for consideration consist,
therefore, of short-term tests already in use in the mutagenicity area, plus
neoplastic cell transformation assays.
In cytotoxicity, evaluation of acute cellular toxicity in vitro provides
information regarding potential in vivo activity of chemicals, prosthetic
materials and devices, and biomedical implants. Although it would be very
difficult to replicate the entire range of cellular responses of the intact
animal by use of tissue culture methods, certain target cell types lend
themselves readily to maintenance in vitro. Exposure of these cell types to
chemical substances provides information on cellular toxicity and metabolism.
It was the consensus of the Working Group that in vitro assay or short-
term testing cannot be predictive for risk assessment of human populations.
In addition, this group could also agree with the following statements:
o The source documents and drafts on mutagenicity testing contain
the current state-of-the-art for a number of test systems.
This area is well explored in methodology and is documented.
o Toxicological decisions (e.g., carcinogenicity) are usually
on non-human studies; the same criteria should be applied to
decisions in the area of mutagenesis.
o The available procedures for detecting and characterizing the
effects of chemical mutagens are based upon our understanding
of mutational processes. Therefore, decisions in chemical
mutagenesis should now be considered as an integral area of toxi-
cology. Further, as in all other areas of toxicology, there is
a need for continual monitoring and upgrading of existing pro-
cedures. When interpreting the utility of short-term tests, we
must know the distribution of a chemical, specifically to the
gonads, in concert with applicable somatic effect, in vivo or
in vitro, including gene and chromosome effects; only then can
we rely on such testing as indicators of potential genetic
hazard.
In summary:
o Available short-term mutagenicity tests for use as an aid in
indicating potential carcinogenic hazard cannot at present
stand alone to identify a carcinogen—long-term animal assays
are still necessary.
o Neoplastic cell transformation assays, as yet not included in
mutagenicity studies, are a grouping of in vitro tests that
are still under evaluation.
o The whole area of cocarcinogenesis and promotion needs to be
recognized.
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o There is a possibility that different batteries of tests should
be set up for different classes of chemicals. For example,
the Office of Pesticide Programs (OPP) battery for pesticides is
considered to be very good.
The Working Group made the following recommendations: (1) convene a
group of experts to specifically consider the applicability of the Pesticide
Guidelines to the needs of the Office of Toxic Substances in using short-
term tests as indicators of mutagenic potential of chemicals, and (2) inter-
act with the other regulatory agencies [Federal Drug Administration (FDA),
Occupational Safety and Health Administration (OSHA), and Consumer Products
Safety Commission (CPSC)] in the development of a technology assessment docu-
ment [along the lines of the EPA Office of Pesticide Guidelines and the CPSC
and the Department of Health, Education, and Welfare (DREW) mutagenicity
documents] to evaluate the status and use of short-term tests as aids in
evaluating the carcinogenic potential of chemicals.
MODEL ECOSYSTEMS
According to the report of the Working Group on Model Ecosystems, screen-
ing tests in ecosystems were understood to be concerned with small replicas
(microcosms) of natural systems constructed either artificially or taken
intact from the field. These microcosms alone should not be expected to
provide assessments of chemical hazards to the environment.
Initial assessment might best be accomplished using chemical benchmark
information, simple short-term laboratory tests (e.g., EC5o,NLD5Q, or behav-
ioral test) and mathematical models of chemical behavior. Microcosm testing
provides information on chronic, long-term effects of hazardous chemicals on
fundamental natural processes such as energy flux, nutrient cycling and homeo-
static properties, and on species interactions.
The report includes tables giving information abqut microcosms that have
been constructed and how they have been used. Microcosms generally are
tailored to specific ecosystems, and questions about processes and observed
chemical behavior usually are not amenable to generalization to ecosystems.
Thus, there is no such thing as a standard microcosm.
A microcosm test should not be expected to have lower parameter vari-
ability than that of a natural environment. This variability is essential to
adequate simulation of the natural environment and may provide data within
the range of actual environmental occurrences. Standards for variability of
replication do not exist. It is not well known to what extent construction
of microcosms distorts field conditions, and methods for extrapolation from
the microcosm to the field are still being debated.
Microcosm tests should address specific questions and data needs.
Therefore, it is suggested that, based on the ecosystem type, tests of proven
success be selected to meet the need.
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REPORT ON
CHEMICAL/PHYSICAL ASPECTS OF TOXICITY TESTING
INTRODUCTION
Under Sections 5 and 8 of the Toxic Substances Control Act, a manufactur-
er may be required to submit chemical and physical data on the proposed new
product and/or process to assist the Administrator in evaluating the possible
risk to man and the environment associated with the manufacture, processing,
distribution, use, and disposal of the product. This Workshop has attempted
to catalog some of the important types of chemical and physical data that may
be needed for risk assessment.
It is expected that these data will be submitted by the manufacturer
with the initial notification. The data will be used in the evaluation of
the anticipated magnitude of human exposure to the product and the byproducts
of its manufacture, use, and dispersal into the environment and the potential
effects of the product and its byproducts on human health and the environment.
It is expected that some of these data may be of use in the selection of
relevant biological tests that should be performed on the product and/or its
byproducts. Structure/activity correlations have been attempted for many
chemical classes and should be used judiciously and to the extent possible in
the selection of biological tests that will be performed.
INFORMATION TO BE SUPPLIED BY THE MANUFACTURER
It is recognized that some of these data may not be pertinent or readily
accessible for some new products or processes. An effort has been made, how-
ever, to limit the data requested to those which should be available from
standardized quantitative tests and from good manufacturing practices. Thus,
it is anticipated that the Administrator will require these data, where
applicable, for a full evaluation of potential risk.
Much of this information would be required eventually, in any case,
under other laws and regulations administered by the EPA. The intent here is
to ensure that this information is gathered, reviewed, and evaluated to the
extent possible, before the production process begins, so that situations
presenting unacceptable risks can be prevented, rather than corrected after
damage has resulted.
It also is recognized that, in some cases, these data will be based on
extrapolation from pilot plant or even smaller scale operations, with consid-
erable attendant uncertainty. The Act recognizes this problem and provides
for follow-up after the process goes on line in the form of reports,
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inspections, and monitoring (under this law as well as other laws and regu-
lations).
It is anticipated, then, with the qualifications above, that the follow-
ing types of chemical and physical data should be supplied by the manufactur-
er:
I. Product
A. Chemical: The following data should be presented for
each chemical component accounting for 1 percent or
more of the total.
1. Identity and molecular structure
2. Percent of each chemical compound in the product
and the anticipated range and variability for each
3. Analytical methods used in the identification of
each chemical compound in the product and in its
quantification
B. General characterization (e.g., physical state, color,
odor, crystalline form if applicable)
C. Reference sample: A representative sample of the product
should be submitted to the EPA. This sample could be used
by the EPA for cross-checking data submitted by the manufac-
turer, for further analytical and biological tests, and for
future reference (e.g., in evaluation of batch-to-batch
variation in toxic impurities). It is recommended that
the EPA establish a product repository for storing and
distributing these reference samples.
D. Physical properties
1. Melting point (if solid)
2. Boiling point (if liquid)
3. Vapor pressure
a. At ambient temperature and pressure
b. Under anticipated conditions of use
4. Flashpoint
5. Polarity
6. Solubility
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a. In water as a function of pH
b. In hexane
7. Octanol-water partition coefficient (polar/non-polar)
8. Conductivity of aqueous solutions
E. Chemical properties
1. Oxidation and reduction potentials (and product if
known)
2. Rate of hydrolysis (and products if known)
3. Photochemical reactions (oxidation, etc.)
4. Electrophilicity
5. Differential thermal analysis, with identification
of major off-gases
6. Chelating factor
7. Chemical oxygen demand (COD) (of aqueous solutions)
8. Chlorine demand (of aqueous solutions)
9. Corrosion
F. Biochemical properties
1. Biochemical tests: A suitable battery of in vitro bio-
chemical tests should be devised that would assist in
postulating the major metabolic processes and products
in animals and man.
2. Biodegradability: Information on biodegradability is
of obvious importance and should be provided whenever
possible. The Working Group noted the need for stan-
dardized tests for this parameter.
II. Process: A complete quantitative mass balance for the manu-
facturing process should be provided, including identification
and quantification of starting materials, products, byproducts,
and anticipated emissions and effluents. Process chemistry
should be outlined schematically, along with a careful descrip-
tion of pollution control technology and anticipated performance
standards. See Paragraph III for emission and effluent information
requirements.
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III. Emissions and Effluents
A. Emissions (air)
1. Total (gross) emission
a. Ratio of gaseous to particulate (w/w)
b. Particle-size distribution
c. Solubility in water
2. Individual components: The following data should be
presented for each chemical component in the air
emissions accounting for 1 percent or more of the
total (excluding air), by weight:
a. Chemical structure
b. Percent of total emission
c. Amount in pounds (kilograms) per day at capacity
d. Chemical properties
(1) Oxidation and reduction potentials (and
products if known)
(2) Photochemical reactions (oxidation, etc.)
(3) Electrophilicity
-e. Biochemical properties (see Paragraph I.F.I.)
B. Effluents (Water)
1. Total (gross) effluent
a. pH
b. Turbidity
c. Conductivity
d. Chemical oxygen demand (COD)
e. Biological oxygen demand (BOD)
f. Total oxygen concentration (TOG)
g. Chlorine demand
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2. Individual components: The following data should be
presented for each chemical component in the water
effluent accounting for 1 percent or more of the
total (excluding water), by weight:
a. Oxidation and reduction potentials (and products
if known)
b. Solubility
(1) In water as a function of pH
(2) In hexane
c. Partition coefficient (polar/non-polar)
d. Electrophilicity
e. Biodegradability
f. Biochemical properties (see Paragraph I.F.I.)
IV. Environmental Degradation Products: To the extent possible, the
following information should be submitted on all known and sus-
pected products of environmental degradation of the product
itself and important byproducts (see Paragraph III.A.2 and III.B.2)
A. Chemical structure
B. Source (degradation process) and where found (air, water,
soil; near manufacturing, formulating plants; in homes, etc.)
C. Anticipated quantities
D. Rates of appearance and of further degradation
E. Biodegradability
F. Leachability (from typical soils)
G. Solubility
1, In water as a function of pH
2. In hexane
H. Partition coefficient (polar/non-polar)
I. Photochemical reactions
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V. Waste Disposal: Recognizing the ultimate necessity of disposal
of most chemical products at some point in time and the attendant
potential risks to human health and the environment, the follow-
ing information on anticipated and recommended disposal practices
should be submitted, where applicable.
A. Solid waste
1. Anticipated nature and composition of solid wastes
2. Combustion
a. Conditions required for complete combustion
b. Products of complete and of incomplete combustion
3. Sanitary Landfill
a. Likely leachates; relative quantities
b. Likely volatiles
4. Recommended disposal procedures for solid wastes
B. Liquid waste
1. Anticipated nature and composition of liquid wastes
2. Combustion (see Paragraph A. 2 above)
3. Sanitary landfill (see Paragraph A. 3 above)
4. Discharge into sewage system
a. Toxicity/compatibility with sewage treatment systems;
at what levels?
b. See Paragraph III.B.
5. Discharge into receiving waters (navigable waters)
a. See recommendations of Aquatic Toxicology Testing Group.
6. Recommended disposal procedures for liquid wastes
EPILOG
This discussion of chemical and physical data which may be required for
risk assessment has focused on the product and the manufacturing process.
However, it is clear that the subsequent formulation, processing, distribu-
tion, and use of the product will often constitute the major portion of the
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risk to human health and the environment presented by a new product or pro-
cess. The Toxic Substances Control Act requires the manufacturer to provide
such information as is available on anticipated uses with the initial noti-
fication, but much of this will be conjecture until the product is actually
marketed.
Therefore, additional data will undoubtedly be required on formulation,
processing distribution, and uses after marketing. This might include a
periodic market profile and an evaluation of the potential for human expo-
sure from release of the chemical substance to the environment. Each major
use and each stage of the formulation and distribution process should be
evaluated.
It is anticipated that not every test would be applicable to every pro-
duct examined as a result of requirements of the Toxic Substances Control
Act. Indeed, the parameters might vary greatly, depending upon the nature
of the substance and the techniques available for evaluating its ultimate
environmental impact. Standard, or at least widely accepted, methods for
measuring many of the listed properties currently exist in the literature.
Standard reference sources such as the Association of Official Analytical
Chemists (AOAC) and the American Society for Testing and Materials (ASTM)
list recommended techniques for determinations such as conductivity, polar-
ity, BOD, COD, etc.
If standard or reference methods are not available, suitable techniques
may be found in the technical and experimental literature. In many cases,
new methods will have to be devised to make the required measurements.
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REPORT ON
WHOLE ANIMAL ACUTE AND SUBCHRONIC TESTING
INTRODUCTION
In order to protect the public against hazards resulting from exposure
to environmental chemicals, it is necessary to know the short- and long-term
(acute, subchronic, and chronic) toxicological effects of the chemical upon
all organs and functions of the exposed subjects. The practical situation
of use may make the degree of exposure and risk very different for different
chemicals. Not all compounds need to be tested for every effect and it is
possible that some compounds may require no testing.
The Toxic Substances Control Act provides for submission by the manufac-
turer to the EPA of information describing the merits and value of the chemi-
cal, the nature of usage, the amount to be produced, the size of the popula-
tion at risk, expected frequency and duration of exposure, any toxicity data,
predictions of hazard, and protocols.
On the basis of the information submitted, the EPA should specify the
tests required for a chemical within a given use or situation. The EPA can
specify the test methodology on the basis of the "state-of-the-art" and
established test methods. Within an established method, the EPA should
reserve the authority to specify the nature of the test sample, animal spe-
cies, routes of administration, dose levels, solvents, diluents, dilution
levels of test solutions, frequency and duration of dosing, and quality assur-
ance procedures. These modifications ensure that the methodology is appro-
priate to the operational situation.
The purpose of the Working Group is to outline, within current scientific
knowledge, those tests which are necessary and acceptable to demonstrate the
presence of toxic effects in body organs or functions. The adequacy of exist-
ing data should be determined by the EPA. The tests used to supply the need-
ed information should be selected from sources given below.
GUIDELINES FOR TOXICOLOGICAL TESTING
Requirements and guidelines for toxicological testing have been advanced
by regulatory agencies and expert groups having official or quasi-official
sponsorship or acceptance. They include the following:
1. Code of Federal Regulations (CFR) 46, Shipping. Jan. 1, 1972,
para. 146.25-10.
2. CFR 49, Transportation. Parts 100-199, Jan. 1972, para. 173.343.
15
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3. CFR 40, Protection of the Environment. Jan. 1, 1972, para. 162.8.
4. Federal Register (FR), Vol. 38, No. 187. Part II. Consumer Pro-
ducts Safety Commission, Federal Hazardous Substance Act Regulation,
Revision and Transfer. Sept. 27, 1973.
5. FR, Vol. 40, No. 129. Part II, para. 162.8. Environmental Protec-
tion Agency Pesticide Program. Registration, Reregistration and
Classification Procedures. July 3, 1975.
6. Appraisal of the Safety of Chemicals in Food, Drugs, and Cosmetics.
1959. Association of Food and Drug Officials of the U.S. Edito-
rial Office, 2411 N. Charles Street, Baltimore, MD.
7. National Academy of Sciences-National Research Council. 1964.
Principles and Procedures for Evaluating the Toxicity of Household
Substances. Publication 1138.
8. National Academy of Sciences-National Research Council. Revised,
1977. Principles and Procedures for Evaluating the Toxicity of
Household Substances. Publication 1138.
9. Food and Drug Administration Advisory Committee on Protocols for
Safety Evaluation: Panel on Reproduction Studies in the Safety
Evaluation of Food Additives and Pesticide Residues. Toxicol. ami
Appl. Pharmacol. 16: 264-296. 1970 .
10. National Academy of Sciences-National Research Council. 1975.
Principles for Evaluating Chemicals in the Environment.
11. Department of Health, Education and Welfare Committee to Coordinate
Toxicology and Related Programs. April, 1977. Approaches in
Determining the Mutagenic Properties of Chemicals: Risks to Future
Generations.
12. Department of Health, Education and Welfare. Guidelines for Car-
cinogen Bioassay in Small Rodents. (DHEW) Publication No. (NIH)
76-801.
13. R. M. Graziano's Tariff No. 25. Hazardous Materials Regulations of
the Department of Transportation including specifications for Ship-
ping Containers, para. 173.343, 173-240. July 28, 1972.
14. CFR 21. Food and Drugs. Parts 147 to 199, para. 191.1. Jan. 1,
1972.
15. Health, Education, and Welfare - Canada. 1975. The Testing of
Chemicals for Carcinogenicity, Mutagenicity, and Teratogenicity.
p. 28.
16. FR Vol. 40, No. 123. Environmental Protection Agency Pesticide
Program, Guidelines for Registering Pesticides in the U.S. Part
II, para. 162.81. Proposed Rules. 1975.
17. National Institutes of Health, Third Printing, 1977. Guide for
the Care and Use of Laboratory Animals. DHEW NIH 74-23.
TOXICOLOGICAL TEST AREAS
Established laws (National Environmental Policy Act of 1969, Clean Air
Act Amendments of 1977, Toxic Substances Control Act 1976) and the guidelines
for toxicological testing which have been established or proposed by the Food
and Drug Administration, Department of Transportation, Environmental Protec-
tion Agency, Consumer Products Safety Commission, National Cancer Institute
and the National Academy of Sciences-National Research Council indicate that
it may be necessary to test for any or all short- or long-term toxicological
16
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effects on body organs for functions. Test areas appropriate to the guide-
lines are given in Table 1.
The table also cites the references, on pages 15 and 16, to acceptable
methodologies which may be used to evaluate toxicological effects on the
designated organ, organ system, or physiological function.
TABLE 1. TOXICOLOGICAL TEST AREAS
Toxicological Area
Reference
I. Acute Toxicity (single dosing)
A. General Toxicology
1. LDso (by appropriate routes)
II.
III
LC50
2. Clinical signs
3. Autopsy findings (gross)
B. Local Tissue Effect
1. Skin and eye irritation
2. Skin sensitization test
Subchronic and Chronic Toxicity Tests
A. Metabolism
B. Pharmacokinetics
Hematology
Clinical Chemistry Tests
Pathology
Pharmacodynamics
Behavior
Neurological Effects
Endocrinology
Reproduction
Teratogenesis
Mutagenesis
Dominant lethal
Cytogenetic
Carcinogenicity
1. Short-term
2. Long-term
Care and Use of Laboratory Animals
C.
D.
E.
F.
G.
H.
I.
J.
K.
L.
M.
1.
2.
1,2,3,4,5,6,7,8,10,14,15
1,2,3,4,5,6,7,8,10,14,16
7,8,16
5,6,8,10,16
5,6,8,10,16
1,2,3,4,5,6,7,8,10,12,14,16
4,5,6,8,10,14,16
6,8,10,16
6,10
5,6,8,10
5,6,8,10
5,6,8,10,16
6
6,8,10,16
6,10,16
6
5,6,8,9,10,16
5,6,8,10,15,16
6,8,10,11,16
6,8,10,11,16
8,11,16
5,8,10,12,15,16
8,10,15
6,10,12,15,16
17
17
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GENERAL PROBLEMS IN FOLLOWING THE TESTING PROCEDURES OUTLINE
The Working Group recognizes that certain problems would emerge as a
result of strict experimental protocols for every class of chemicals that may
warrant testing. Examples of these include: selection of species and strain
of test organism, other variables such as sex, age, pre-existing or inter-
current disease, route of administration, frequency and level of dosing, and
dose response relationships.
These and related problems are compounded in carcinogenicity testing
because of the long latent period for development of cancer. They include
pre- and post-natal exposure, differences in biochemical and biological com-
petence between strains, sexes and species, interaction with other chemicals,
and environmental factors which may produce synergistic, potentiating or
antagonistic effects.
While some credence can be given to suspecting toxicity, it should not
be assumed that qualitative and quantitative prediction of toxicity can be
made on the basis of structural similarity to another compound; note for
example, the difference in toxicity between kepone and mirex, isomers of
benzene hexachloride, neurotoxic effects of organophosphates, and pharmaco-
kinetic behavior of different halogenated compounds.
Finally, the Working Group recognizes the need for the inclusion of
behavioral studies to reveal subtle dysfunctional effects on the central
nervous system and the development of better animal models for extrapolation
to humans.
MARINE ENVIRONMENT TESTING
I. Phytoplankton Bioassays
A. Laboratory Assessment: Short-term, many species. Growth
Rate, Carbon-14 uptake, and Chlorophyll _a are parameters.
Methods already being routinely used for screening by
EPA contractors.
B. Field Assessment: In situ Carbon-14, Adenine triphosphate,
and Chlorophyll £ measures. Standard oceanographic tech-
niques. Routinely used in assessment of damage from powerplants.
These methods are applicable to acute hazard detection but offer, as
presently applied, little information on bioconcentration and long-term
hazard.
References'. 1. Marine Bioassay Workshop Proceedings. 1974.
API, EPA, MTS: Geraldine Cox, ed.; Marine
Technology Society
2. Bioassay Procedures for the Ocean Disposal
Permit Program. EPA-600/9-76-010; May 1976.
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II. Zooplankton Bioassays
Laboratory Assessment: Acartia tonsa/Acartia clausi, sensitive
indigenous marine species, presently used in Ocean Disposal and
Dredge Spoil Programs.
Short-term static test is routinely run. Parameter is survival.
Method amenable to other zooplankton (colcenoids).
Flow-through short-term assay method available but not tested
outside the EPA's Environmental Research Laboratory-Narragansett
(ERL-Narragansett). Potentially offers broad applicability and
sensitivity.
III. Crustacean Larval Bioassay
Generally using larvae of macrocrustaceans. There are no
routinely standardized methods here to point to. However,
both static and flow-through exposure systems and protocols
for Lobster larvae that are amenable to other decapod larvae
are available.
IV. Static and Flow-through Bioassay Procedures for Grass Shrimp
and Larvae (Paleomonetes)
Both procedures have been used effectively by in-house research
laboratories but not extensively by contracting laboratories.
Until further testing, they are not recommended as routine tests,
V. Static and Flow-through Bioassay on Mysids
This method, while recent in development, is being rapidly
adopted for routine screening and also for whole life cycle
chronic testing. It is currently being contracted and the
EPA has interlab calibration in progress. Offers the oppor-
tunity to compare acute with "safe" levels of pollutant with
short-life cycle species (18-30 days). Detailed methods are
currently being prepared for publication by the EPA/Gulf-
Breeze. Multiple parameter and bioaccumulation in the second
generation effects (F2).
VI. Macroalgal Bioassays
New technique developed and applied at the ERL-Narragansett.
Includes fertilization and growth. Developmental abnormalities
are sensitive measures of acute hazard, while growth rate
changes reflect chronic long-term exposure. Method also has
field assessment potential. Method available from the ERL-
Narragansett. Not currently contractable.
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VII. Chronic Bioassay-Cyprinondon variegatus (Sheepshead Minnow)
Whole life cycle bioassay—widely used within the EPA and by
contractors. Currently the only chronic exposure test system
available using marine fish. Excellent for estimating a variety
of parameters including bioconcentration and F2 effects.
VIII. Embryo-Fry Bioassay
Available for many species of fish, particularly suitable for
indigenous species. Not widely used yet.
A. Cyprinodon variegatus (Sheepshead Minnow): Available from
Gulf-Breeze, Florida, and contractable.
B. Flounder Species: Available from the ERL-Narragansett in
winter and summer. Require special holding and spawning
conditions. Therefore, not used by contractors.
C. Miscellaneous Species: Menidia menidia (Atlantic Silver-
side), Fundulus heteroclitus (Rollifish). Species can be
studied but not routinely at this point. Could use more
development. Also requires spawning and holding condi-
tions.
IX. Laboratory Bioaccumulation Studies on Molluscs
These methods are not generally being used by contractors.
The methods are well developed for a wide variety of species
(i.e., oysters, scallops, mussel, quahog, etc.). Methods
are currently being prepared for Ocean Disposal Manual to be
published in January 1978. Methods apply to a wide variety
of toxicants both in the presence and absence of substratum.
X. Benthic Bioassays using Polychaetes
In house flow-through methods for Neanthes and Arenicola are
available but not currently being used outside research
facility. Methods will become available in January 1978
in Ocean Disposal Manual. Chronic and acute measures in
sediment system also useful in dredge spoils.
XI. Field Assessment Techniques using Mytilus edulis
Mussel Watch Program of ERL-Narragansett using biological
measure of pollutant accumulation from water. Method details
currently being prepared for publication. Has major use as
monitoring tool in hazard assessment.
XII. Benthic Bioassay for Sediment
New technique with details in Dredge Soil Manual.
20
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The General Methods described above highlight principal assessment tools
available. There are potentially many variations within any category.
Sources of method descriptions are:
1. Environmental Protection Agency-660/3-75-009. Methods for Acute
Toxicity Tests with Fish, Macroinvertebrates, and Amphibians.
2. Environmental Protection Agency-660/9-76-010. Bioassay Procedures
for the Ocean Disposal Permit Program.
3. Marine Technology Society. 1974. Marine Bioassay. Workshop Pro-
ceedings.
4. EPA/Army Corps of Engineers. 1977. Ecological Evaluation of
Proposed Discharge of Dredged Material into Ocean Waters.
FRESHWATER ENVIRONMENT TESTING
I. Acute Toxicity Tests (static and flow-through)
Routinely used by many laboratories with many species of fish,
macroinvertebrates and amphibians. (Committee on Methods for
Toxicity Tests with Aquatic Organisms, 1975.)
II. Life Cycle Toxicity Tests (survival, growth, and reproduction)
Routinely used by many laboratories with Daphnia roagna, fathead
minnow, and brook trout. Has been successfully conducted by
some laboratories with bluntnose minnow, bluegill sunfish, green
sunfish, Daphnia pulex, rainbow trout, midge, mayfly, flagfish,
and catfish.
III. Embryo-Larval Test (fish)
Has been routinely used with fathead minnows and brook trout.
Can be used with almost any species of fish for which eggs are
available. Results are good indicators of results of life
cycle tests.
IV. Bioaccumulation
Has been routinely used with fathead minnows, brook trout,
and bluegill sunfish. Can be used with almost any species
of fish and some invertebrates.
V. Flavor Impairment (fish)
Has been routinely used with brook trout, rainbow trout, and
catfish. Can be used with almost any species of fish over
about 20 grams.
21
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VI. Toxicity Tests with Algae
Has been routinely used with some species of algae by some labora-
tories.
BEHAVIORAL TOXICOLOGY
In the study of environmental toxins, there must be a concern with tran-
sient or permanent functional effects in addition to the documentation of
manifest disease or morbidity. Functional deficits, more often than not,
involve the central nervous system which plays a major integrating role in
the physiology and function of an organism. Because of this integrative role,
a toxic effect in many of the organ systems is also reflected in a change in
the functioning of the nervous system. This functional capacity, however,
cannot be evaluated by pathological, biochemical, or even physiological stud-
ies independent of a behavioral analysis.
The techniques with which to study the influences of chemicals on behav-
ior are available largely from psychopharmacology. The somewhat standard-
ized approach to central nervous system (CNS) drug screening in the pharma-
ceutical industry may not transfer directly or totally to the study of envi-
ronmental agents. Weighing risks against therapeutic benefits requires
different decisions for an agent intended for use under limited conditions
for a relatively prescribed time, than for a chemical that might be dispersed
widely, linger for many years, and expose large segments of the population.
A decision tree can be constructed to provide some guidelines for a
sequential testing protocol for behavioral effects. The first step in this
sequence should be an elementary screening program to determine approximate
dose response functions for readily observable CNS effects. This screen can
be adopted for both acute and subchronic studies. The elements in the screen
must be determined by the class of compounds to be studied but might include:
(a) Activity changes: locomotion of a standard distance; spontaneous
or exploratory behavior, abnormal gait.
(b) Objective signs: tremors, ptosis, salivation, convulsions,
defecation, etc.
(c) Reflex changes: hyper- or hyporeflexia of cornea and pinna
righting.
(d) Elicited responses: gasping, hanging, nose or tail pinch,
orientation to external stimulus.
(e) Body weight changes: immediate and brief weight losses;
failure to grow in relation to species and colony norms.
Instead of stopping with these observations, however, the evaluation of
an environmental agent should continue for possible delayed effects. Many
agents (for example, the heavy metals) do not reveal their damage immediately
after exposure but only following a prolonged latent period. Any agent that
22
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manifests delayed or residual toxic effects must be considered for rejection
because of the possibility of exposing a large segment of the population.
Individuals with dormant damage may be less capable of withstanding further
CNS deterioration as might result from normal phenomena such as aging. Many
elements in the preliminary screen can be used repeatedly to look for delayed
or persistent effects.
Prenatal exposure is another potential danger with environmental agents.
The combination of an immature, developing nervous system with a lack of ade-
quate detoxification mechanisms makes the fetus and neonate especially sus-
ceptible. Sometimes the consequences of such exposure can remain dormant
and not manifest themselves until later life as a behavioral disorder, mental
deficiency, or overt functional impairment. Determination of long-term or
delayed effects of a particular developmental influence on biological or be-
havioral functions requires the use of a longitudinal research design. This
protocol follows specific individuals from birth through maturity. A series
of standardized tests appropriate for such longitudinal studies has been pub-
lished by J. Werboff in Principles of Psychopharmacology, edited by W. G.
Clark and J. Del Guidice (1970, New York, Academic Press). It is strongly
recommended that even chemicals that show no obvious CNS effects on the pre-
liminary screen be studied for delayed and, especially, prenatal effects.
Particularly if the effects are delayed—so a long latent period supervenes
between exposure and effect—the chemical must be presumed to offer hidden
dangers that, except in rare instances, will not be worth contending with,
despite many other possible benefits.
If CNS effects are revealed in the preliminary screen, they should be
characterized more precisely and more quantitatively, yet with minimal instru-
mentation and training time. This behavioral assay might include: (a) quan-
titative measures of spontaneous motor activity using photocell cage or an
activity wheel; and (b) stable Sidman avoidance performance. The latter
procedure is especially useful since the performance of the untreated animal
does remain stable and reproducible over many months of testing. An avoid-
ance procedure that uses a warning stimulus can be used as a first approxi-
mation to the study of sensory function.
An analysis of the nature of specific behavioral deficits permits some
guess at the site or mechanisms of toxic action and may provide valuable sug-
gestions as to which behavioral parameters should be monitored in exposed
human populations. An investigation in depth, however, requires a substan-
tial investment in time, talent, and money. Sometimes, of course, such an
assessment is inescapable, as in the case of chemicals that get into the
food chain.
Sensory function
Analysis of sensory function cannot be done independently of behavioral
tests. All facets of visual functioning, viz, pattern and brightness dis-
crimination and hue, acuity, scotopic and photopic curves, can and have been
studied in a wide variety of animal species. Similar tests are available
for auditory and tactile discrimination. The techniques have been important
tools for studying various drugs (kanamycin and neomycin for their ototoxic
23
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effects; pheniprazine for producing red-green color-blindness; and methyl
mercury for its constriction of visual fields). Many of these techniques
have been described in Animal Psychophysics, edited by W. Stebbins (1970,
New York, Appleton, Century and Crofts).
Motor control
Behavioral methods for the detection of gross changes in motor control
and coordination are well documented in the pharmacological literature.
Two examples of methods that can be used to assess environmental toxicants
are rotorod (length of time an animal can remain on a rotating rod) and time
an animal can remain suspended from an inverted screen.
A more sensitive system for assessing fine motor control in animals has
been developed by John Falk and his associates (Physiology and Behavior,
1969, V4, 421-427). A strain gauge is used to measure force exerted on a
lever and the force output must remain within a prescribed bandwidth. Con-
current electromyograms (EMG) can also be measured.
Learning and memory
Learning and memory are critically important functions in the discharge
of human affairs. Any significant disruption of these functions can have
serious consequences on the quality of human life.
Maze learning has been used widely to study the effects of many varia-
bles such as cretinism, heavy metals, cerebral lesions, pesticides, anesthetic
gases.
The delayed match-to-sample task (D'Amoto, The Psychology of Learning
and Memory, edited by G. Bower) has been used extensively to study memory
in both animals and humans, especially children. It has been used to eval-
uate marijuana constituents, nicotine, heavy metals, and extra low frequency
magnetic fields.
Affective behaviors
Many early effects of environmental poisoning have been reported as
vague, non-specific subjective complaints reflecting emotional lability. In
psychopharmacology, three basic procedures have been used to assess the
effects of anxiety in experimental animals: (1) Sidman avoidance condition-
ing; (2) the Estes-Skinner conditioned anxiety procedure; and (3) the
Geller-Seifter conflict procedure. Whether these techniques are applicable
to the study of environmental toxins remains to be determined.
The analysis of behavior is still an infant science and most of its
procedures have not been standardized with respect to specific application.
By and large, the procedures have been sufficiently delineated to be used by
various laboratories with consistent, reproducible results. Mention of
specific procedures should not be taken as an endorsement but rather as one
portal of entry into a particular problem area.
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REPORT ON
IN VITRO TESTING
Since the membership of this Working Group was limited, and the amount
of time available was also limited, it was felt that the most profitable
utilization of time would be to document the current references with regard
to short-term assays for mutagenicity and oncogenicity and to make recom-
mendations to the Office of Toxic Substances regarding the course of action
that will enable them to meet their goals in these areas. Short-term tests
for cytotoxicity were also briefly discussed.
USE OF SHORT-TERM TESTS FOR EVALUATION OF MUTAGENIC POTENTIAL
It was the general consensus of the committee that there are several
adequate references available documenting the current status of the use of
short-term tests to assess potential mutagenic hazards of chemicals (1-7).
In particular, four documents (4-7), either completed or nearing completion,
represent the efforts of several committees, and it was generally felt that
this area has been reasonably explored and appropriately documented.
The available procedures for detecting and characterizing chemical
mutagens are probably as good if not better than other toxicological end-
points in that not only can a mutational process be directly detected,
but frequently an understanding regarding the mechanism(s) of action of the
mutagen can be determined on a molecular level. It was noted that since
toxicological decisions in general are usually based upon non-human studies,
it is reasonable to apply the same criteria to decisions in the area of
mutagenicity. It is thus recommended that chemical mutagenesis testing
should be an integral part of any toxicological evaluation of chemicals and
should form a portion of the data upon which regulatory decisions be made.
USE OF SHORT-TERM TESTS FOR EVALUATION OF CARCINOGENIC POTENTIAL
Studies utilizing short-term assays for oncogenicity have been exten-
sive, and in general a good correlation between positive results in various
systems to known carcinogenicity in vivo has been found. However, an in-
depth evaluation of the various methodologies has not yet been undertaken
by an expert committee. Several expert committees have assessed the use-
fulness of short-term tests in evaluating the potential mutagenicity of
chemicals (as discussed above). Although there is considerable overlap
between evaluating the status of short-term tests for predicting muta-
genicity and oncogenicity, several areas involved in assessing the value of
short-term tests for predicting carcinogenicity of chemicals have not yet
been approached by any expert committee: (a) It can be determined if a
25
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chemical is really a carcinogen by testing it in animals (there are also data
in humans for about 20 carcinogens). Thus the ability of the various short-
term tests to detect carcinogens should be checked. (b) There are several
types of short-term tests for carcinogenicity, notably in vitro transforma-
ion tests, which have not been evaluated by the expert committees on muta-
genesis since their endpoints are not mutations. (c) The problem of how
short-term tests for carcinogenicity should fit into the overall scheme of
the toxicological evaluation of regulated substances is again a problem that
has not been considered. Since animal carcinogenicity tests remain the best
indicators of the potential human carcinogenicity of chemicals, it is essen-
tial to determine how short-term test results influence whether or not an
animal cancer test is done, and how short-term test results are weighed
against animal cancer test data in the overall evaluation of the potential
human hazard of a chemical.
These are decisions which should ultimately be made at policy-making
levels within the Agency. However, an analysis of the scientific background
relevant to these decisions is crucial to assist the policy makers in coming
to reasonable and scientifically credible decisions. It was strongly urged
that such an in-depth study be undertaken. It was also noted that there have
been recent reviews on in vitro studies for oncogenesis which may be particu-
larly pertinent (8-9) and that certain organizations such as the Food and
Drug Administration and the Clearing House on Carcinogens at the National
Cancer Institute are planning to undertake evaluation of the current status o
the methodologies and results in these areas. However, until such an overall
evaluation is completed by these or similar organizations, the relative
importance of various assays for predicting carcinogenicity in vivo, and
particularly in man will remain undetermined. It was emphasized, neverthe-
less, that there are sufficient data to make such an evaluation at the presen
time.
It was the consensus of the group that although a number of tests are
available as an aid in indicating potential carcinogenic hazards, such tests
cannot at present stand alone as identifying a substance as a carcinogen,
i.e., long-term animal carcinogenicity studies are still necessary. It was
further recognized that areas of cocarcinogenesis and promotion of onco-
genesis need further research. There is a very small data base in these
areas.
CYTOTOXICITY
Although cytotoxicity studies were not explored at length it was noted
that systems for evaluation of acute cellular toxicity in cell culture have
provided useful information regarding the potential in vivo activity of
certain classes of agents such as antitumor drugs, prosthetic materials and
devices, biomedical implants, and antibiotics. Such tests consider transient
(reversible) morphological and biochemical alterations as well as irreversib]
effects leading ultimately to cellular destruction. These changes can be
quantitated and hence permit reasonable estimates of cytotoxic potency.
Although it is obviously impossible to replicate the entire range of cellulai
responses of the intact animal by the use of tissue culture methods, certain
target cell types utilizing activation systems can be maintained in vitro
26
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and provide useful information regarding cytotoxicity and metabolism.
RECOMMENDATIONS OF THE WORKING GROUP TO THE OFFICE OF TOXIC SUBSTANCES
1. Convene a group of experts to consider specifically the applica-
bility of the Pesticide Guidelines to the need of the Office of Toxic Sub-
stances in using short-term tests as indicators of mutagenic potential of
chemicals.
2. Interact with the other regulatory agencies (FDA, OSHA, CPSC) in the
development of a technology assessment document (along the lines of the EPA
Office of Pesticide Guidelines, and the CPSC and DREW mutagenicity documents),
to evaluate the status and use of short-term tests as aids in evaluating the
carcinogenic potential of chemicals.
It should be emphasized that this committee felt strongly that its only
functions could be those of reviewing and acknowledging those documents which
should provide significant source material for the EPA and others, as well as
making certain suggestions regarding further needs, such as the formation of
more complete documents on short-term oncogenicity studies.
REFERENCES
1. World Health Organization. 1974. Assessment of the Carcinogenicity and
Mutagenicity of Chemicals. Technical Report Series No. 546.
2. Health and Welfare Ministry Canada. 1975. The Testing of Chemicals for
Carcinogenicity, Mutagenicity and Teratogenicity.
3. J. W. Drake, Chairman, Prepared by Committee 17 of the Environmental
Mutagen Society. 1975. Environmental Mutagenic Hazards. Science,
187:503-514.
4. Working Group of the Sub-Committee on Environmental Mutagenesis. 1977.
Approaches to Determining the Mutagenic Properties of Chemicals: Risk
to Future Generations. Prepared for the DREW Committee to Coordinate
lexicological and Related Programs.
5. Office of Pesticides Program, Environmental Protection Agency, Draft,
July 12, 1977. Mutagenicity. Testing Requirements Section of the FIFRA.
Registration Guidelines for Hazard Evaluation of Humans and Domestic
Animals.
6. Office of Pesticides Program, Environmental Protection Agency, Draft,
August 31, 1977. Criteria for Evaluating the Mutagenicity of Chemicals.
7. National Academy Sciences. 1977. Principles and Procedures for Eval-
uating the Toxicity of Household Substances. Chapter VJ, Mutagenicity
Tests.
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8. Montesano, R., H. Bartsch, and L. Tomatis, eds. 1976. Screening Tests
in Chemical Carcinogenesis. International Agency for Research on Cancer,
Lyon, IARC Scientific Publ. No. 12, 1976.
9. U. Saffiotti and H. Autrup, eds. 1977. In Vitro Carcinogenesis, Guide
to the Literature, Recent Advances and Laboratory Procedures. National
Cancer Institute Carcinogenesis Report Series, DREW Publication No. (NIH)
78-844.
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REPORT ON
MODEL ECOSYSTEMS
MICROCOSMS AS SCREENING TOOLS FOR TOXIC CHEMICALS
DEFINITION
Microcosms are relatively small experimental units that contain the
major components and exhibit the major processes of natural ecosystems.
Conceptually, microcosms are functionally similar to the natural ecosystems
they represent; however, the two may differ in origin and structure. Micro-
cosms may be constructed artifically from stock components or, intact, co-
adapted communities and their abiotic substrates may be excised from a
natural system (1). From the outset, it should be realized that microcosms
should not be expected to provide initial assessments of chemical hazards in
the environment. Initial assessments and hazard rankings of chemicals might
best be accomplished using available chemical benchmark information, simple
short-term laboratory tests (e.g., lethality, ECso, LD50), and predictive
mathematical models of chemical behavior. Information gathered from these
initial assessments of potential chemical behavior in the environment can aid
in the design of subsequent microcosm studies, and in the interpretation of
microcosm-derived data. It is not expected that microcosm studies will be
used to provide information on the toxic effects of all chemicals. However,
they may provide information on the chronic, long-term effects of chemicals
on fundamental ecosystem processes (e.g., energy flux, nutrient cycling,
homeostatic properties, species interactions).
ADVANTAGES OF MICROCOSMS
Microcosm experimental units afford the researcher control of ecosystem
complexity relating to both biotic and abiotic ecosystem components. They
are low in cost and easily replicable from a given ecosystem, and allow con-
trol of selected environmental conditions, such as temperature, humidity or
light. Microcosms are attractive since they obviate the need for contami-
nation of natural environments (2). Microcosms fall into two general cate-
gories: (1) Process-oriented (simple) microcosms, and (2) Integrative
(complex) microcosms. Simple microcosms allow the measurement of chemical
transfer rates and coefficients over short experimental time periods. How-
ever, they may provide only order of magnitude estimates of more complex
systems. Complex microcosms, containing more of the functional components of
natural ecosystems, include more of the complexity and events that may be
caused by biotic-biotic and biotic-abiotic interactive processes.
29
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DISADVANTAGES OF MICROCOSMS
There is a real difficulty in extrapolating results from processes
measured in isolation to actual environmental behavior of chemicals. It is
also difficult to evaluate the comparability of similar, but not-identical
microcosm systems (need for cross comparison). Microcosms are generally
tailored to specific ecosystems and specific questions about ecosystem pro-
cesses and chemical behavior. Therefore, results obtained from them are
usually not amenable to generalization of other ecosystems. It is expected
that microcosms will best be used to evaluate specific situations rather than
as a general screening tool.
Environmentally, realistic microcosms should be expected to exhibit
parameter variability the same as that exhibited by the natural environment.
Currently, it is unknown to what degree ecosystem function is distorted by
fabrication or field excision of microcosms, and accepted methods for extra-
polation to field situations do not exist.
PROCESSES AMENABLE TO STUDY BY MICROCOSM INVESTIGATION (3)
1. Mobility
a. Physical transport pathways
b. Biological uptake, accumulation, food chain biomagnification
2. Partitioning Among Microcosm Components
3. Degradation to Simple Substances
a. Abiotic and physicochemical
b. Biotic and metabolic
4. Transformation
a. Physicochemical (photolysis, redux reactions)
b. Biogenic Transformation
5. Ecosystem Effects
a. Behavior of organisms (populations)
b. System metabolism (energy, primary production (P), respiration
(R), P/R ratio, carbon dioxide evolved oxygen utilized)
c. Nutrient cycling
d. Community dynamics (multi-species interactions)
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UTILITY OF MICROCOSMS IN TOXIC COMPOUND SCREENING APPROACH
It is strongly emphasized that the lack of any "standard microcosm"
underscores the requirement that the microcosm itself address specific
questions and data needs.
Microcosm experiments are intended to bring out results to be expected
only when separate components are combined into a system. Lowered magnitudes
of responses may result from the buffering effect of the prtesence of many
components, or increased responses may result from multiple pathways or
cumulative effects. Were it not for the possibility of unforeseeable re-
sults, there would be little reason for testing toxic chemicals in micro-
cosms.
Interpretation of results from microcosms involves at least changes in
scale. Components of microcosms are present because they are considered to
be important components in real world systems. Yet it is virtually impos-
sible to include these components in the same proportion to each other as
expected in any real world system. Therefore data on behavior of chemicals
and responses of organisms must be differentially scaled when the results
are applied to the real world. We term this scale of results "projection."
Although we emphasize that no "standard" is now accepted or proposed,
there are traditional studies which parallel the microcosm concept. For
example, pond studies of productivity, weed control or, in some cases, chemi-
cal compound effects have been conducted for decades and give some background
for methodology and statistical analysis.
An appropriate microcosm experimental approach should then be designed,
phased, and sampled according to previously identified extrapolation mecha-
nisms. Microcosm testing or screening lends itself to certain kinds of
questions of effects or fate of specific compounds. It is important to
design the microcosm test in terms of real world environmental types, so
that results of this operation bracket real world questions in a relevant
or relatable way. These questions of relevance guide in the kind or dosing
of the microcosm test applied.
Examples of the kinds of questions to be considered include:
1. Characteristics of toxic substances to be tested.
2. Processes to be considered in the test (e.g., transformation,
transport, uptake or partitioning, population impacts, etc.).
3. The ecosystem(s) of concern which microcosm(s) test(s) should
address; in other words, the breadth of environmental concern
involved.
4. What "scale" of microcosm test is most appropriate?
5. What duration(s) should test be run?
The next question should be considered before executing the test!
6. What interpretation mechanisms should be used to extrapolate
results from test to prediction or estimation of real world
environments ?
31
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Projection of microcosm outputs may be accomplished in at least two ways:
Subjective projection: interpretation of microcosm outputs in a direct
manner assuming that the microcosm used is an accurate representation or
simulation of its real world counterpart. This requires careful calibration
of microcosm parameters to environmental counterparts. These data still
become weighed, prioritized and integrated into the decision matrix of the
regulatory user, and are therefore subjectively judged with toxicological,
socioeconomic or other factors in producing the decision or recommendation.
Use of models in place of a subjective interpretation mechanism does not
fundamentally change the role of the regulator. Information enters the
decision matrix also to be evaluated along with toxicity, socioeconomic and
other factors. Model-derived information is merely more specific and some-
what more quantitative; therefore, it can be weighed more objectively.
Objective projection; an alternative technique, objective projection,
is to use mathematical models representing the fate of the material in the
microcosm as a tool for projection. Such a model would be based upon physi-
cal, chemical, and biological characteristics of the chemical, together with
description of the microcosm. Models of this sort can reasonably well pre-
dict the fate of the chemical in the microcosm. Change of scale of the
environmental factors to describe an environment of interest will then yield
a projection of the microcosm results to that environment. In this manner
results of microcosm studies can be much more widely applied.
It should be emphasized that while prediction of fate by models based
upon physical chemical principles is feasible, there are few principles that
can be employed to predict effects. To the extent that dose-response rela-
tionships exist, however, expected effects can be computed based upon results
predicted by models.
Chemical fate simulation should focus on conceptualization of the
systems being studied and on the development of a set of equations for the
description and prediction of major chemical transport pathways among eco-
system and microcosm components. A preliminary model for chemical transport
in a terrestrial ecosystem is shown on the following page.
This model is, of course, subject to revision as experimental results
become available to validate the essentiality of each component. It is
expected that not only will the experimental results influence the conceptual
model but the set of equations depicting transport and transformation will
influence experimental design.
The sensitivity of model parameters and hypothesized chemical transport
between system components may be evaluated with critical pathway analyses.
In addition, sets of equations describing transport may be solved in order
to compare predicted compartmental values with time-series measurements of
microcosm parameters. With replication of microcosms, the chemical trans-
port models developed may be validated independently of the data used to
estimate the model parameters. Transport models may also be used in com-
parative-predictive evaluation of large-scale natural ecosystem models similar
32
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to those developed under other programs. (2,4,5)
PRIMARY
PROCEDURES
ATMOSPHERE
DETRITUS
SOIL
_ MICROBES
INVERTEBRATE
CONSUMERS
SOIL MOISTURE
AND LEACHATE
(Modified from Draggan 1976) (2)
Many types of microcosms and methods of study have been developed for
assessing chemical behavior in various ecosystems. Tables 1 and 2 give
examples of applications. (3)
TABLE 1. TYPES OF MICROCOSMS
Terrestrial
Terrestrial - Aquatic
River
Aquatic - Batch
Aquatic - Continuous Culture
Chemostate and Turbiodostat
Special Microcosms
Species Defined (Gnotobiotic)
In situ Bioassay
Closed (Biorengenerative Life Support)
Naturally Occurring Microcosms
TABLE 2. METHODS COMMONLY USED IN MICROCOSM STUDIES
I. Types of Inputs
A. Solution
1. Single entry, point source
2. Single entry, mixed into system
(Continued)
33
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TABLE 2. METHODS COMMONLY USED IN MICROCOSM STUDIES (Continued)
3. Multiple entry, point or mixed
A. Continuous entry
B. Biological entity
1. Live, e.g., a labeled prey
2. Dead, e.g., tagged leaf litter
C. Nonbiological entity
1. Solid, e.g., labeled fly ash
2. Liquid, e.g., labeled rain
3. Gas, e.g., nitrogen-15
II. Timing of Measurements
A. Initial sampling (validation of initial input)
B. Periodic sampling, using subsaraples of replicate(s)
C. Terminal sampling on portion of the replicates (e.g.,
destructive sampling of 1/3 of the replicates at
3 different durations)
D. Terminal sampling of all replicates simultaneously
III. Compartments Measured
A. One [i.e., species of interest (fish) or system
output (leachate)]
B. Special
C. All compartments except container surfaces and
atmospheric gases
D. All compartments including container surfaces
and atmospheric gases
IV. Entity Measured per Compartment
A. Chemical of interest (e.g., radionuclide or DDT)
B. Carrier (or stable isotope) mass
C. Degradation products (e.g., DDD, DDE)
D. Compartment mass (e.g., dry weight)
V. Calculated Values
A. Concentration of entity in a compartment
B. Transfer rates between compartments (first order,
(Continued)
34
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TABLE 2. METHODS COMMONLY USED IN MICROCOSM STUDIES (Continued)
function of mass in donor compartment; second order,
function of mass in donor and recipient compartments)
requiring sequence of measurements and model of inter-
actions
C. Specific activity
D. Concentration factor, also called biological or
ecological magnification, bioaccumulation factor
or index (ratio of concentration in recipient
compartment/donor compartment—donor may be
water or food)
E. Biodegradability index (ratio of concentration of
breakdown productions/parent compounds, e.g., polar/
nonpolar labeled compounds, Metcalf et al., 1971b)
F. Distribution among major compartments, e.g., % in
soil, water, algae, snails
G. Total budget studies
VI. Special Types
A. Species defined (gnotobiotic)
B. In situ bioassay
C. Naturally occurring microcosms
D. Closed (bioregenerative life support)
REFERENCES
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protection of the environment. Sci. Total Environ., 9:63-74.
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3. Witherspoon, J. P., E. A. Bondietti, S. Draggan, F. B. Taub, N. Pearson,
and J. R. Trabalka. 1976. j>tate-of-the-Art and Proposed Testing for
Environmental Transport of Toxic Substances, Report No. EPA-560/5-76-001.
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35
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Mutation Res., 31:291-298.
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40
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Tracer Jitco, Inc. 1977a. Sample Biological Assay Protocols
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o Acute Inhalation Study in Rats
o Subchronic Inhalation Study in Rats
o Chronic Inhalation Study in Rats ( In Utero Exposure)
o 24-Month Inhalation Study in Mice
o 14-Day Multiple Dose Inhalation Study in Rats
o 1/10 Lifetime Inhalation Study in Rats
o Mammalian Reward and Motivation Study
o Teratogenicity in Rats
o Teratogenicity in Rabbits
o Three-generation Reproduction Study in Rats
o Microbial Assay (Ames Test)
o In-vitro Transformation of BALB/3T3 Cells
o Mouse Lymphoma Forward Mutation Assay
o Unscheduled DNA Synthesis (UDS) in Human WI-38 Cells
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41
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LIST OF WORKSHOP PARTICIPANTS
Name
Delbert S. Earth
Werner F. Beckert
Joseph V. Behar
William F. Benedict
Charles A. Bicking
Erich W. Bretthauer
John M. Bryant
Lawrence A. Burns
Lome A. Campbell
William P. Davis
Sidney Draggan
Dom V. Finocchio
Donald E. Gardner
Organization and Location
Office of Health and Ecological Effects, U. S.
Environmental Protection Agency, Washington, D. C.
Environmental Monitoring and Support Laboratory,
U. S. Environmental Protection Agency, Las Vegas,
Nevada
Environmental Monitoring and Support Laboratory,
U. S. Environmental Protection Agency, Las Vegas,
Nevada
Children's Hospital of Los Angeles, Los Angeles,
California
Tracer Jitco, Inc., Rockville, Maryland
Environmental Monitoring and Support Laboratory,
U. S. Environmental Protection Agency, Las Vegas,
Nevada
Occupational Safety and Health Administration, U. S.
Department of Labor, Austin, Texas
Environmental Research Laboratory, U. S. Environmental
Protection Agency, Athens, Georgia
Tracer Jitco, Inc., Rockville, Maryland
Environmental Research Laboratory, U. S. Environmental
Protection Agency, Bears Bluff, South Carolina
Environmental Sciences Division, Oak Ridge National
Laboratory, Oak Ridge, Tennessee; now National
Science Foundation, Washington, D. C.
Department of Pathology, University of Washington
School of Medicine, Seattle, Washington
Health Effects Research Laboratory, U. S. Environmental
Protection Agency, Research Triangle Park, North
Carolina
(continued)
42
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Name
Organization and Location
John Gentile
Sidney Green
Harry W. Hays
Robert M. Hehir
Renate Kimbrough
Robert R. Kinnison
John H. Kreisher
Roy R. Lassiter
Marvin S. Legator
John F. Lontz
Russel Malcolm
Irving Mauer
Joyce McCann
Craig McFarlane
Bernard P. McNamara
George B. Morgan
Stephen S. Olin
Bioassay Systems Laboratory, U. S. Environmental
Protection Agency, Narragansett, Rhode Island
Department of Pharmacology, Howard University
College of Medicine, Washington, D. C.
Agricultural Research Service, U. S. Department of
Agriculture, Washington, D. C.
Consumer Product Safety Commission, Bethesda,
Maryland
Center for Disease Control, Department of Health,
Education, and Welfare, Atlanta, Georgia
Environmental Monitoring and Support Laboratory,
U. S. Environmental Protection Agency, Las Vegas,
Nevada
Council of Tobacco Research, New York, New York
Environmental Research Laboratory, U. S. Environmental
Protection Agency, Athens, Georgia
University of Texas Medical Branch, Galveston, Texas
Veterans Administration Center, Wilmington, Delaware
Environmental Research Laboratory, U. S. Environmental
Protection Agency, Narragansett, Rhode Island
Tracor Jitco, Inc., Rockville, Maryland
University of California, Berkeley, California
Environmental Monitoring and Support Laboratory,
U. S. Environmental Protection Agency, Las Vegas,
Nevada
Chemical Systems Laboratory, Edgewood, Maryland
Environmental Monitoring and Support Laboratory,
U. S. Environmental Protection Agency, Las Vegas,
Nevada
Tracor Jitco, Inc., Rockville, Maryland
(continued)
43
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Name
Ruth Pertel
Robert D. Rogers
Organization and Location
Office of Pesticides Programs, U. S. Environmental
Protection Agency, Washington, D. C.
Environmental Monitoring and Support Laboratory,
U. S. Environmental Protection Agency, Las Vegas,
Nevada
John A. Santolucito
Richard E. Stanley
Thomas W. Stanley
Charles S. Stephan
Robert G. Tardiff
Richard L. Titus
A. C. Trakowski
William M. Upholt
Environmental Monitoring and Support Laboratory,
U. S. Environmental Protection Agency, Las Vegas,
Nevada
Environmental Monitoring and Support Laboratory,
U. S. Environmental Protection Agency, Las Vegas,
Nevada
Office of Monitoring and Technical Support, U. S.
Environmental Protection Agency, Washington, D. C.
Environmental Research Laboratory, U. S. Environ-
mental Protection Agency, Duluth, Minnesota
Health Effects Research Laboratory, U. S. Environ-
mental Protection Agency, Cincinnati, Ohio
Chemistry Department, University of Nevada, Las
Vegas, Nevada
Office of Monitoring and Technical Support, U. S.
Environmental Protection Agency, Washington, D. C.
Office of Assistant Administrator, Toxic Substances,
U. S. Environmental Protection Agency, Washington,
D. C.
Lawrence R. Valcovic
Michael Waters
National Institute of Environmental Health Services,
Research Triangle Park, North Carolina
Health Effects Research Laboratory, U. S. Environ-
mental Protection Agency, Research Triangle Park,
North Carolina
Jack A. Winstead
National Academy of Science, Washington, D. C.
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/9-79-004
RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
REPORT OF THE WORKSHOP ON BIOLOGICAL SCREENING TESTS
REPORT DATE
January 1979
PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Charles A. Bicking
3. PERFORMING ORGANIZATION REPORT NO.
3. PERFORMING ORGANIZATION NAME AND ADDRESS
Tracor Jitco, Inc.
Rockville, Maryland 20852
0. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
CB-7-0913-B
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency-Las Vegas, NV
Office of Research and Development
Environmental Monitoring and Support Laboratory
Las Vegas, NV 89114
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/07
15. SUPPLEMENTARY NOTES
EMSL-LV Project Officer for this report is John A. Santolucito.
Commercial Telephone (702) 736-2969, x 276 or FTS 595-2969, x 276
16. ABSTRACT ~~ ~~" '
This report contains recommendations for selecting substantially predictive
biological screening tests. The large number of chemicals which can potentially
impact human health and the environment precludes the complete testing of each
substance. In order to effect preliminary chemical hazard ranking, initial tests
must be standardized and validated and the necessary quality control practices
and techniques developed and implemented.
This report contains recommendations for selecting substantially predictive
biological screening tests upon which the Agency's quality assurance resources may
be initially concentrated.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Toxicology
Hazardous materials
Physiological effects
Ecological effects
Environmental biology
Biological screening
tests
06T
06F
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
52
!O. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
A04
EPA Form 2220-1 (9-73)
U.S. GOVERNMENT PRINTING OFFICE: 1979-749-903/12
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