United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600/1-78-052
July 1978
Research and Development
v>EPA
Directory
of Short Term Tests
for Health
and Ecological
Effects
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/1-78-052
July 1978
DIRECTORY OF SHORT-TERM TESTS
FOR HEALTH AND ECOLOGICAL EFFECTS
Prepared for the
Office of Health and Ecological Effects
Office of Research and Development
U.S. Environmental Protection Agency
Washington, District of Columbia 21040
By the
Genetic Toxicology Program
Biochemistry Branch
Environmental Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
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 recommendation for use. Men-
tion of specific contractors does not necessarily indicate either exclusive
engagement or recommendation.
i i
-------
FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for which national
ambient air quality standards exist or are proposed, provides the data
for registration of new pesticides or proposed suspension of those already
in use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of
health care and surveillance of persons having suffered imminent and
substantial endangerment of their health.
Historically, the Health Effects Research Laboratory has made a strong
commitment to the development and implementation of short-term tests for
potential health effects of environmental agents.
This commitment is evidenced by the formation within this laboratory of
two new programs; the Genetic Toxicology Program and the Neurotoxicology
Program. These programs will require redirection and commitment of new
resources to develop techniques that rapidly evaluate pure chemicals and
complex environmental samples for possible genotoxic and neurotic hazard.
Research on short-term tests for health and ecological effects is ad-
vancing rapidly throughout the Office of Health and Ecological Effects. This
document should prove of significant value in maintaining coordination of the
research program during this growth period.
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory
I I I
-------
PREFACE
At the request of Dr. Delbert Barth, Deputy Assistant Administrator for
the Office of Health and Ecological Effects (OHEE), the first OHEE Workshop on
Short-Term Tests for Health and Ecological Effects was held January 18-20, 1978,
at the U.S. Environmental Protection Agency, Health Effects Research Laboratory,
Research Triangle Park (HERL-RTP), North Carolina.
The need for such a meeting, especially in the area of genetic toxicology,
was suggested by Dr. Alexander Malcolm of the U.S. Environmental Protection
Agency at Narrangansett, Rhode Island. The Workshop was planned and coordinated
by Dr. Frode Ulvedal and Dr. George Armstrong of OHEE with the assistance of
Dr. Malcolm and the staff of the Biochemistry Branch, HERL-RTP.
The following objectives of the Workshop were established and transmitted
with Dr. Earth's letter of invitation to the seven OHEE laboratories and to
the National Center for ToxicologicaI Research:
• Produce a directory listing OHEE's screening system efforts with the
address and telephone numbers of the key people associated with these
efforts;
• Initiate a continuing dialogue among the various investigators as well
as visitation among sister laboratories;
• Formulate an agreement for the coordination of the Agency's efforts in
health and ecological bioassays; and
• Identify OHEE's needs for future research in this area.
A major initiative of the Workshop was the preparation of a directory of
the short-term tests currently being performed throughout OHEE. This document,
the Directory of Short-Term Tests for Health and Ecological Effects, provides
I v
-------
PREFACE (continued)
information on the tests themselves, the laboratories where they are being per-
formed, and the key individuals involved. It should prove to be an important
Agency and interagency reference to this rapidly growing and challenging field
of scientific investigation. The Directory also seeks to further the intent
of the Workshop, namely to enhance communication, collaboration, understanding,
and appreciation of a major component of the U.S. Environmental Protection
Agency's research program.
Michael D. Waters, Ph.D.
Coordinator, Genetic Toxicology Program
Chief, Biochemistry Branch
Health Effects Research Laboratory
Research Triangle Park, North Carolina
-------
CONTENTS
Foreword ,
Preface
Abbreviations ix
Acknowledgment x
Note Regarding the Test Systems xi
1000 Short-Term Tests for Health Effects
MOO General Toxicity I
I I 10 In-Vitro Systems/Acute I
1120 In-Vivo Systems/Acute 21
1130 In-Vivo Systems/Subacute 25
1140 In-Vivo Systems/Inhalation Toxicology 35
1150 In-Vivo Systems/Neurobehavioral 47
1200 Genotoxicity 57
1210 Mutagenesis 57
1220 DNA Damage 73
1230 Carci nogenesi s 83
1240 Marine Applications 91
1300 Perinatal Toxicology 99
2000 Short-Term Tests for Ecological Effects 107
2100 Freshwater and Marine Algae 107
2200 Freshwater Animals and Plants
2210 Fish Toxicity
2220 Invertebrate Toxicity
2230 Plant Toxici ty/Resi due 123
2240 Ecosystem 125
(conti nued)
vi i
-------
CONTENTS (continued)
2300 Marine Animals 127
2310 Fish Toxicity 127
2320 Invertebrate Toxicity 131
2330 Ecosystem 137
2400 Terrestrial Animals and Plants 141
2410 Plants 141
2420 Ecosystem 145
2500 Physical-Chemical/Bio-Accumulation 149
Ind ices
X:
XI :
XI I :
XIII:
XIV:
XV:
Test Systems in Numerical Order 154
Test Systems in Alphabetical Order 160
Experimental Subject Listing 168
Contact Personnel in Alphabetical Order . 172
U.S. EPA Laboratories in Alphabetical Order 175
U.S. EPA Departmental Listing in Alphabetical Order 176
Grant/Contract Laboratories in Alphabetical Order 180
Status of Development of the Test Systems 184
Endpoints of the General and Perinatal Toxicity
Test Systems 185
Endpoint Distribution of the Genotoxicity
Test Systems 190
Endpoint Distribution of the Ecological Test Systems 191
Application Distribution Table 192
Sample Distribution of the General and Perinatal Toxicity
Test Systems 195
Sample Distribution of the Genotoxicity Test Systems 199
Sample Distribution of the Ecological Test Systems 203
VI I I
-------
ABBREVIATIONS
ERL-COR Environmental Research Laboratory-CorvaI Iis, Oregon
ERL-DUL Environmental Research Laboratory-DuIuth, Minnesota
ERL-GB Environmental Research Laboratory-Gulf Breeze, Florida
ERL-NAR Environmental Research Laboratory-Narrangansett, Rhode Island
HERL-CIN Health Effects Research Laboratory-Cincinnati, Ohio
HERL-RTP Health Effects Research Laboratory-Research Triangle Park, North
Caroli na
NCTR National Center for Toxicological Research, Jefferson, Arkansas
OAWM Office of Air and Waste Management
OEMI Office of Energy, Minerals, and Industry
OHEE Office of Health and Ecological Effects
OPP Office of Pesticide Programs
ORD Office of Research and Development
OTS Office of Toxic Substances
OWHM Office of Water and Hazardous Materials
U.S. EPA United States Environmental Protection Agency
ix
-------
ACKNOWLEDGMENT
The cooperation of each Workshop participant and each Directory contributor
are gratefully acknowledged.
Special thanks are extended to the three science editors, Dr. Shahbeg
Sandhu of HERL-RTP, Dr. Jeffery Charles of HERL-RTP, and Dr. James Mckim of
ERL-DUL, for their extensive efforts in screening and coordinating the contri-
butions to the subject areas of Health Effects-Genetic Toxicology, Health
Effects-General and Perinatal Toxicology, and Ecological Effects, respectively.
Our sincere appreciation is also extended to Northrop Services, Inc.,
for indexing, editing, and typing the Directory, and in particular to Olga
Wierbicki, who coordinated the effort.
-------
NOTE REGARDING THE TEST SYSTEMS
The test system information contained in this document was provided by
the workshop participants and their colleaques. The science editors, Dr.
Shahbeg Sandhu, Dr. Jeffrey Charles, and Dr. James Mckim, combined data where
possible, questioned obvious errors and missing information, and insured
uniformity to the extent possible in data sheets and indices. No effort was
made to restrict inclusion of any test system or related information. Selec-
tion and interpretation of terms related to status of development, applica-
tions, complexity (simplest to most complex on a scale of I to 4), program
office support, etc., were the responsibility of the submitters of the in-
formation.
xl
-------
CONTENTS: TEST SYSTEMS
1000 Short-Term Tests for Health Effects
I 100 General Toxicity
I I 10 In-Vitro Systems/Acute.
MM Integrated Systems: Lung Organ Culture System,
Tissue Homogenates, Purified Enzyme Systems . . 2
1112 Integrated System: Tissue Homogenates, Purified
Enzyme Systems 3
1113 Instrumental Methods of Detecting Functional and
Metabolic Damage to Target Tissues 4
1114 Trachea I Mucociliary Transport 6
1115 Platelet Secretion Measured by ATP Release. ... 8
IMS Mammalian Platelet and Fibroblast Impairment. . . 9
1117 Lymphocyte Cytotoxicity 10
1118 Impairment of Neutrophil Phagocytosis II
1119 Human Lung Fibroblasts (WI38) 12
II I 10 Chinese Hamster Ovary (CHO) Clonal Toxicity ... 14
Mill Rabbit Alveolar Macrophage (RAM) 16
I I I 12 Rat Hepatocyte (Liver Cell) 18
I I 113 Chinese Hamster Ovary (CHO) Cytoxicity and
Mutagenicity 19
120 In-Vivo Systems/Acute 21
-------
1111 INTEGRATED SYSTEM: LUNG ORGAN CULTURE SYSTEM, TISSUE HOMOGENATES,
PURIFIED ENZYME SYSTEMS
Biological Activity Detected: Toxicity.
Principle: Enzyme inhibition and/or induction.
Endpoints: Qua Ii tati ve: Alteration in enzyme activities ?,id/or
concentration of metabolites. Quantitative: Degree of alteration
in enzyme activities and/or concentration of metabolites.
Strengths: Excellent indicator for pulmonary fibrosis; Very sensitive
early indicators.
Weaknesses: Lacks in specificity in some cases; Terminal; Difficult
to extrapolate to human situation.
Status of Development: Being implemented.
Describe: Test systems have been fully developed. Data are being
coI Iected.
Applications: Multimedia.
Samples: Pure Chemicals: N02, S02, Hg, Cd, Mn, Zn, Cu. CompI ex
Mixtures: Transportation Related - diesel.
Duration: 5 years ending in 1979.
Cost/sample or chemical: $85.
Interpretation: This system is a very sensitive measure for the
degree of alteration relative to pulmonary fibrosis.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-C1N, Laboratory Studies Division,
Functional Pathology Branch, Biochemistry Section.
Persons to Contact: S.D. Lee, U.S. EPA, HERL-CIN, 26 W. St. Clair St.,
Cincinnati, OH 45268, (FTS 684-7442).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of California Medical Center, San Francisco, CA 94132,
R.S. Bhatnagar.
Program Office Support: OPP; OEM).
References: I) Bhatnagar, R.S. The Role of Superoxide in Oxidant-
Induced Pulmonary Fibrosis. In: Biochemical Effects of Environ-
mental Pollutants. S.D. Lee, ed., Ann Arbor Science Publishers,
Ann Arbor, Ml, 1977. 2) Hussain, M.Z., R.S. Bhatnagar, and S.D.
Lee. Biochemical Mechanisms of Interaction of Environmental
Metal Contaminants with Lung Connective Tissue. In: Biochemical
Effects of Environmental Pollutants. Ann Arbor Science Publishers,
Ann Arbor, Ml, 1977.
-------
1112 INTEGRATED SYSTEM: TISSUE HOMOGENATES, PURIFIED ENZYME SYSTEMS
Biological Activity Detected: Toxicity.
Principal: Enzyme inhibition and/or induction.
Endpoints: Qua Iitative: Alteration in enzyme activity and/or con-
centration of metabolites. Quantitative: Degree of alteration
in enzyme activity and/or concentration of metabolites.
Strengths: Early indicators; Probably more sensitive than any other
method.
Weaknesses: Lacks in specificity in many cases; Terminal; Difficult
to extrapolate to human situation.
Status of Development: Validated.
Describe: Data are being accumulated to validate toxic effects
of specific pollutants.
Applications: Multimedia.
Samples: Pure Chemica I s: 63, NC>2, Hg, Cd, SC>2. Complex Mixtures:
Ambient - 03 + S02.
Duration: 3 years ending June 1978.
Cost/sample or chemical: $75.
Interpretation: This system provides sensitive early indicators for
metabolic/cellular injury and recovery.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Biochemistry Section.
Persons to Contact: S.D. Lee. U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7442).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of California School of Medicine, Los Angeles, CA 90032,
M.G. Mustafa.
Program Office Support: OHEE; OPP.
References: I) Mustafa, M.G., and S.D. Lee. Pulmonary Biochemical
Alterations Resulting from Ozone Exposure. Ann. Occup. Hyg.,
19:17-26, 1976. 2) Mustafa, M.G., A.D. Hacker, J.J. Ospital,
N. Elsayed, and S.D. Lee. Prophylactic Effect of Dietary Vitamin
E on the Metabolic Response of Lung Tissue to Low-Level Ozone
Exposure. Amer. Rev. Resp. Dis., 113:98, 1976. 3) Hacker, A.D.,
N. Elsayed, M.G. Mustafa, J.J. Ospital, and S.D. Lee. Effects
of Short-Term Nitrogen Dioxide Exposure on Lung Collagen Syn-
thesis. Amer. Rev. Resp. Dis., 113:107, 1976. 4) Ospital, J.J.,
N. Elsayed, A.D. Hacker, M.G. Mustafa, and D.F. Tierney. Altered
Glucose Metabolism in Lungs of Rats Exposed to Nitrogen Dioxide.
Amer. Rev. Resp. Dis., 113:108, 1976. 5) Lee, S.D., and M.G.
Mustafa. Influence of Dietary Antioxidants in Low Level Oxidant
Exposure. Presented at 4th International Clean Air Congress,
Tokyo, Japan, May, 1977. 6) Mustafa, M.G., A.D. Hacker, J.J.
Ospital, M.Z. Hussain, and S.D. Lee. Biochemical Effects of
Environmental Oxidant Pollutants in Animal Lungs in Biochemical
Effects of Environmental Pollutants. S.D. Lee, ed., Ann Arbor
Science Publishers, Ann Arbor, Ml, 1977. 7) Mustafa, M.G., and
S.D. Lee. Biological Effects of Environmental Pollutants:
Methods for Assessing Biochemical Changes. In preparation.
-------
1113 INSTRUMENTAL METHODS OF DETECTING FUNCTIONAL AND METABOLIC DAMAGE TO
TARGET TISSUES
Biological Activity Detected: Toxicity.
Principle: Increased functional activity of a tissue requires energy.
Consequently, if a tissue's functional activity is stimulated,
ATP is hydrolyzed to ADP and P. which in turn stimulates oxida-
tion of substrate and resynthesis of ATP. These metabolic changes
may be observed as increases in oxygen consumption, substrate
utilization or as metabolic transients induced in the electron
carriers directly in tissues, in-vitro. The kinetics of these
metabolic responses to stimulation are sensitive to a wide variety
of chemical agents with varying mechanisms of action with both
in-vitro and in-vivo treatments.
Endpoints: To this point in time the test has only been developed
for brain tissue. Responses are measured in response to elec-
trical pulses or elevation in K concentrations. Qua Ii tati ve: N/A.
Quantitative: Transient redox changes in NAD(P)H, fp, cyt a, b,
c; Substrate utilization; Oxygen consumption; Lactic acid output;
Neurotransmitter release; Amino acid metabolism; Electrical
threshold; Frequency response.
Strengths: In-vitro results may be directly confirmed in-vivo with
same parameters; Applicable to a wide variety of mechanisms;
Applicable to all aerobic tissues; Involves measurement of the
kinetics of going from a resting to an excited state rather than
the steady state, thereby greatly increasing sensitivity; Ap-
plicable to very small tissue samples (2 to 3 mg).
Weaknesses: Does not lend itself to immediate identification of
mechanisms unless there is a direct effect on energy metabolism
proper.
Status of Development: Validated.
Describe: The test has been validated with a wide variety of
inhibitors of energy and membrane active compounds such as ouabain
and saxitoxin. In-vitro and in-vivo treatments with lead, methyl
mercury, and a Iky I tin compounds indicate equivalent or more
sensitive measures of effect than other parameters which have
been applied to these problems.
Applications: Multimedia.
Samples: Pure ChemicaIs: All classes. Complex Mixtures: Indus-
trial; Energy Related; Transportation Related.
Duration: 2 weeks/compound in-vitro.
Cost: Not precisely established, estimate $1,000 to $2,000/compound.
Interpretation: The test basically assesses potential for neuro-
toxicity. It indicates the effect of a chemical somewhere be-
tween functional activity and the metabolism induced by functional
activity. Further studies are required to determine if the effect
is on function or metabolism.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxicologicaI Assessment Branch.
Persons to Contact: R.J. BuI I, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
-------
1113 INSTRUMENTAL METHODS OF DETECTING FUNCTIONAL AND METABOLIC DAMAGE TO
TARGET TISSUES (continued)
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE.
References: I) Bull, R.J., and A.J. Trevor. J. Neurochem. 10:999-
1009, 1972. 2) Bull, R.J., and A.J. Trevor. J- Neurochem.,
19:1011-1022, 1972. 3) Cummins, J.T., and R.J. Bull. Biochem.
Biophys. Acta, 253:29-38, 1971. 4) Bull, R.J., and J.T. Cummins.
J. Neurochem., 21:923-937, 1973. 5) Bull, R.J., and S.D. Lutkenhoff,
Neuropharmacol., 14:351-359, 1975. 6) Bull, R.J. J. Neurochem.,
26:149-156, 1976. 7) Bull, R.J., P.M. Stanaszek, J.J. O'Neill,
and S.D. Lutkenhoff. Envir. Hlth. Perspect., 12:89-95, 1975.
-------
1114 TRACHEAL MUCOCILIARY TRANSPORT
Biological Activity Detected: Toxicity.
Principle: Particulates or gases which are deposited on the ciliated
epithelium of conducting airways could adversely affect the major
functions of this tissue — that of clearance of particulates and
effete cells from the lung.
Endpoints: Qua Iitative: Cytological and histological examination
of trachea exposed to air pollutants in-vivo or in-vitro.
Quantitative: Measurement of ciliary beating frequency after
in-vitro or in-vivo exposure.
Strengths: The in-vitro exposure model permits rapid dose-response
studies for ranking of toxicity which can be followed with rapid
dose-response studies after in-vivo exposure; The test is quite
sensitive; The in-vivo exposure testing dose-response effects
permits results to be used for standard setting and regulatory
purposes; The in-vitro exposure model requires small amounts of
polIutant sample.
Weaknesses: For the inhalation exposure, relatively large amounts
of pollutant sample are required.
Status of Development: Validated.
Describe: Both the in-vitro and in-vivo exposure model have been
successfully used for Ni, Cd, h^SO^, and carbon, and pollutant
mixtures. The model has not been used for screening purposes.
AppIications: Air.
Samples: fure ChemicaIs: Any chemical likely to be deposited
on conducting airways. Complex Mixtures: Ambient; Industrial;
Energy Related; Transportation Related; Other - any gas, partic-
uI ate or combination thereof.
Duration: In-vitro exposure: 2 weeks/dose-response of I chemical;
In-vivo exposure: 4 weeks/dose-response of I chemical.
Cost: Approx. $4,000/dose-response of I chemical in-vivo; $2,000/
dose-response of I chemical in-vitro.
Interpretation: Positive result predictive of damage to clearance
mechanisms of lung.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: J.A. Graham, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators:
I IT Research Institute, 10 West 35th Street, Chicago, IL 60616,
L. Schiff; Northrop Services, Inc., P. 0. Box 12313, Research
Triangle Park, NC 27709, B. Adkins; Ball State University,
Muncie, IN 47306, D. Adalis; U. of North Carolina, Chapel'mil.
NC 27514, A. Col I ier.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: DAdalis, D., D.E. Gardner, F.J. Miller, and D.L. Coffin.
Toxic Effects of Cadmium on Ciliary Activity Using a Trachea I
Ring Model System. Envir. Res., 13:111-120, 1977. 2) Collier,
A.M., and J.B. Baseman. Organ Culture Techniques with Mycoplasma.
Ann. N.Y. Acad. Sci., 225:277-289, 1973. 3) Donnelly, G.M., H.F.
-------
1114 TRACHEAL MUCOCILIARY TRANSPORT (continued)
McKean, C.S. Heird, and J. Green. Ciliostasis as a Bioassay
Arch. Envir. Hlth., 28:350-355, 1974.
-------
1115 PLATELET SECRETION MEASURED BY ATP RELEASE
Biological Activity Detected: Toxicity; Pharmacologic modulation.
Principle: Platelet function is important in thrombosis, shock, and
most inflammatory reactions. Platelet secretion accompanies the
more commonly measured aggregation response, and secretion is
more easi ly measured than aggregation.
Endpoints: Qua Iitative: N/A. Quantitative: ATP release
from platelet suspensions is measured by Lucifirin-Iuciferase
assay.
Strengths: Fast; Reproducible; Does not require expensive equipment;
Applicable to studies of human blood; Can employ both in-vivo and
in-vitro exposures.
Weaknesses: Have not yet been determined.
Status of Development: Developmental.
Describe: Procedures have not been fully developed.
Applications: Air; Water; Food.
Samples: Pure Chemicals: Most classes. CompI ex Mixtures:
Ambient; Industrial; Energy Related; Transportation Related.
Duration: I month to develop in-vitro technique; 3 months to
develop in-vivo technique; approx. I week to implement.
Cost: For development $15,000; to test chemical less than $5,0007
chemi caI.
Interpretation: A positive result is predictive of possible platelet
dysfunction.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: G. Hatch, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I) Charo, O.F., R.D. Feinman, and T.C. Detwiler. J.
Clin. Invest., 60:866-873, 1977.
-------
1116 MAMMALIAN PLATELET AND FIBROBLAST IMPAIRMENT
Biological Activity Detected: Toxicity.
Principle: Contaminant is added to platelets in-vitro. The relative
amounts of energy metabolism intermediates are measured. C14
adenine is used as a precursor.
Endpoints: Qua Iitative: Ratios of ATP, ADP, and AMP. Quantitative:
N/A.
Strengths: Quantitative; Rapid; Capable of direct interpretation.
Weaknesses: Sensitivity.
Status of Development: Developmental.
Describe: Testing with dilutions of pure compounds and extracts
of wastewater.
Applications: Water.
Samples: Pure ChemicaIs: Hydrocarbons. Complex Mixtures: Am-
bient - rivers; Other - wastewaters.
Duration: 3 years.
Cost/sample or chemical: $125.
Interpretation: Positive result suggests a possible interference
of platelet function in-vivo.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
logical Assessment Branch.
Persons to Contact: H. Pahren, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7217).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Colorado Medical Center, 4200 E. 9th Ave., Denver, CO
80262, C.C. Solomons.
Program Office Support: OHEE.
References: Not yet available.
-------
1117 LYMPHOCYTE CYTOFOXICITY
Biological Activity Detected: Toxicity.
Principle: Lymphocytes (T cells) have been shown to have cytotoxic
effector actions against neoplastic or other cells. The potential
exists that pollutants could adversely affect this function,
thereby increasing the risk of the host to the development of
neoplastic disease.
Endpoints: Qua Iitative: N/A. Quantitative: Measurements
of lymphocyte cytotoxic activity and lectin induced transforma-
tion will be made following in-vitro pollutant exposure.
Strengths: The in-vitro model would permit rapid screening for a
significant health parameter; Dose-response studies would permit
ranking of pollutant effects; Relatively small quantities of
pollutant would be required.
Weaknesses: The in-vitro model is not yet validated. Even after
validation, results would have to be confirmed with in-vivo
exposure studies, possibly of a chronic nature, before the data
could be useful for regulation.
Status of Development: Developmental.
Describe: Highly developmental. No pollutants have yet been
tested. Model to be completed March, 1979.
Applications: Multimedia.
Samples: Pure Chemicals: Potentially, any chemical. Complex
Mixtures: Ambient; Industrial; Energy Related; Transportation
Related; Other - any mixture that does not include gas.
Duration: Not yet determined.
Cost: $175,000/model development.
Interpretation: In-vitro model not validated at present.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: J.A. Graham, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators:
Rockefeller U., 1230 York Ave., New York, NY 10021, M. Bowers.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I) Kirchner, H., and R.M. Blaese. In Lymphocyte Recogni-
tion and Effector Mechanisms. Acad. Press, N.Y., 1974. pp.
357-361. 2) Lightbody, J., and J.C. Rosenberg. In Lymphocyte
Recognition and Effector Mechanisms. Acad. Press, N.Y., 1974.
pp. 363-367. 3) Perlman, P., and G. Holm. Adv. Immune I., II:
117, 1969.
10
-------
1118 IMPAIRMENT OF NEUTROPHIL PHAGOCYTOSIS
Biological Activity Detected: Toxicity.
Principle: Functional and metabolic disturbance of neutrophils
are measured after exposure to contaminant.
Endpoints: Qua Iitative: Percent phagocytosis and percent killing
power are measured. Quantitative: N/A.
Strengths: Rapid; Dose-response of procedure has been shown.
Weaknesses: Sensitivity may be a possible problem if low PPB
exposure is necessary.
Status of Development: Developmental.
Describe: Testing with dilutions of pure compounds and extracts
of wastewater.
Applications: Water.
Samples: Pure Chemicals: Hydrocarbons. Complex Mixtures: Am-
bient - rivers; Other - wastewaters.
Duration: 3 years.
Cost/sample or chemical: Not yet determined.
Interpretation: Positive result is predictive of possible damage
to neutrophils in-vivo.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
logical Assessment Branch.
Persons to Contact: H. Pahren. U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7217).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Colorado Medical Center, 4200 E. 9th Ave., Denver, CO
80262, W.L. Weston.
Program Office Support: OHEE.
References: I) Tan, J.S., et al. A Modified Assay of NeutrophiI
Function: Use of Lysostrophin to Differentiate Defective
Phagocytosis from Impaired Intracellular Killing. J. Lab. Clin.
Med., 78:316, 1971.
I I
-------
1119 HUMAN LUNG FIBROBLASTS (WI38)
Biological Activity Detected: Toxicity.
Principle: Toxicants alter biosynthetic processes leading to a
reduction in cell growth and division.
Endpoints: Qua Iitative: Morphology. Quantitative: Cell number
and viability; Total cell protein and DMA; Cell adenosine
triphosphate; Incorporation of radio-label led thymidine,
uridine, and leucine.
Strengths: Relatively inexpensive; Rapid; Fewer samples required
than for conventional whole animal bioassays; One of the best'
characterized diploid human cells available for toxicity bioassays.
Weaknesses: Not representative of intact animals, providing only
preliminary information about the potential health hazards of the
test chemicals; May be replaced by other cell strains as supplies
dwindle; The system currently can not be coupled with mutagenecity
testing unlike other mammalian cell systems.
Status of Development: Being implemented.
Describe: A number of pure compounds have been evaluated.
Applications: Air; Water.
Samples: Pure ChemicaIs: Inorganics, Organics, Heavy Metals.
Complex Mixtures: Industrial; Energy Related - fly ash;
Other - AWT effluent, metal-coated fly ash.
Duration: 20 hr.
Cost/sample or chemical: $500 to $1,000.
Interpretation: Alterations in the basic metabolic processes
and cellular structure indicate the potential toxicity of
the agent.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
logical Assessment Branch; HERL-RTP, Environmental Toxicology
Division, Biochemistry Branch.
Persons to Contact: N.E. KowaI, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7477); M.D. Waters, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
J.L. Huisingh, U.S. EPA, HERL-RTP, Research Triangle Park, NC 27711
(FTS 629-2537).
Grant/Contract Laboratory Involved and Principal Investigators:
Gulf South Research Institute, P. 0. Box 26518, New Orleans, LA
70186, N. Gruener; Northrop Services, Inc. P.O. Box 12313,
Research Triangle Park, NC 27709, N.E. Garrett.
Program Office Support: OHEE; OEM I.
References: I) Campbell, J.A., H.F. Stack, M.R. Williams, D. Tillery,
N. Custer, B.F. Russell, S.W. King, E.B. Siegel, and N.E. Garrett.
Cellular Toxicity of Four Liquid Effluent Samples from Textile
Mills: Studies on the Rabbit Alveolar Macrophage, WI38 Human
Fibroblast and Chinese Hamster Ovary Cell In-Vitro. Contract
Report ESG-TR-78-04 to the U.S. Environmental Protection Agency,
Northrop Services, Inc., Research Triangle Park, NC. February
1978. 2) Garrett, N.E., J.A. Campbell, J.L. Huisingh, and M.D.
Waters. The Use of Short-Term Bioassay Systems in the Evaluation
of Environmental Particulates. In: Proceedings of the Symposium
-------
1119 HUMAN LUNG FIBROBLASTS (W138) (continued)
on the Transfer and Utilization of Particulate Control Technology.
Denver, CO, July 24, 1978. In press. 3) Waters, M.D., T.O.
Vaughan, D.J. Abernathy, H.R. Garland, C.C. Cox, and D.L. Coffin.
Toxicity of Platinum (IV) Salts on Cells of Pulmonary Origin.
Envir. Hlth. Perspect., 12:45-56, 1975. 4) Waters, M.D., D.R.
Abernathy, H.R. Garland, and D.L. Coffin. Toxic Effects of Selected
Metallic Salts on Strain WI38 Human Lung Fibroblasts. In-Vitro,
10:342, 1974. 5) Waters, M.D., J.L. Huisingh, and N.E. Garrett. The
Cellular Toxicity of Complex Environmental Mixtures. In: Proceedings
of the Symposium on the Application of Short-Term Bioassays in the
Fractionation and Analysis of Complex Environmental Mixtures.
Wi I Iiamsburg, VA, 1978.
-------
11110 CHINESE HAMSTER OVARY (CHO) CLONAL TOXICITY
Biological Activity Detected: Toxicity.
Principle: The toxicity of the samples is evaluated from clonal
growth.
Endpoints: Qua Iitative: N/A. Quantitative: Colony formation.
Strengths: The CHO cell system is well characterized and able to
form discrete colonies from single cells; The cell is phagocyti-
cally active in culture and sensitive in toxic particulate materi-
als.
Weaknesses: The cell type may not be representative of metabolicaIly
active target cells which receive exposure to environmental
toxicants.
Status of Development: Being implemented.
Describe: Protocols are established and environmental samples
are being evaluated.
Applications: Air; Water.
Samples: Pure Chemicals: h^SiOs, Ni3S2- Complex Mixtures: Indus-
trial - textile mill effluents; Energy Related - fly ash; Other -
particulate materials: silica, fly ash, Dolomite.
Duration: 6 days.
Cost/sample or chemical: $500 to $1,000.
Interpretation: Biochemical changes in CHO cells exposed to the
particulates indicate toxicity.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch.
Persons to Contact: J.L. Huisingh, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2537).
Grant/Contract Laboratory Involved and Principal Investigators:
Northrop Services, Inc., P.O. Box 12313, Research Triangle Park,
NC 27709, N.E. Garrett.
Program Office Support: OEM I.
References: I) Hsie, A.W., et al. Quantitative Mammalian Cell Genetic
Toxicology: Study of the Cytotoxicity and Mutagenicity of Seventy
Individual Environmental Agents Related to Energy Technologies
and Three Subfractions of a Crude Synthetic OiTin the CHO/HGPRT
System. In: Proceedings of the Symposium on Short-Term Bioassays
in the Fractionation and Analysis of Complex Environmental Mix-
tures. Williamsburg, VA, 1978. 2) Campbell, J.A., H.F. Stack,
M.R. Williams, D. TiIlery, N. Custer, B.F. Russell, S.W. King,
E.B. Siegel, and N.E. Garrett. Cellular Toxicity of Four Liquid
Effluent Samples from Texti le Mi 1 Is: Studies on the Rabbit
Alveolar Macrophage, WI38 Human Fibroblast and Chinese Hamster
Ovary Cell In-Vitro. Contract Report ESG-TR-78-04 to the U.S.
Environmental Progection Agency, Northrop Services, Inc., Research
Triangle Park, NC. February, 1978. 3) Garrett, N.E., J.A. Campbel
J.L. Huisingh, and M.D. Waters. The Use of Short-Term Bioassay
Systems in the Evaluation of Environmental Particulates. In:
Proceedings of the Symposium on the Transfer and Utilization of
Particulate Control Technology. Denver, CO, July 24, 1978. In
press.
14
-------
11110 CHINESE HAMSTER OVARY (CHO) CLONAL TOXICITY (continued)
4) Waters, M.D., J.L. Huisingh, and N.E. Garrett. The Cellular
Toxicity of Complex Environmental Mixtures. In: Proceedings of
the Symposium on the Application of Short-Term Bioassays in the
Fractionation and Analysis of Complex Environmental Mixtures.
Wi I Iiamsburg, VA, 1978.
15
-------
11111 RABBIT ALVEOLAR MACROPHAGE (RAM)
Biological Activity Detected: Toxicity.
Principle: Toxic agents alter basic metabolic processes and cellular
structure of the macrophage.
Endpoints: Qua Iitative: Morphology. Quantitative: Cell number and
viability; Cell adenosine triphosphate; Phagocytic activity; Total
cell protein; Hydrolytic enzyme specific activities.
Strengths: The alveolar macrophage plays an important role in the
defense of the lung against inhaled particulate materials; This
cell type receives direct exposure to environmental toxicants.
Weaknesses: This in-vitro cell system approximates the response which
might be observed in the intact animal.
Status of Development: Being implemented.
Describe: The response of the RAM system to a variety of indus-
trial and energy-related particulates has been studied.
Applications: Air; Water.
Samples: Pure Chemicals: Metal chlorides and suI fates, Metallic
oxides. Complex Mixtures: Industrial - textile effluents,
aluminum refinery, copper smelter; Energy Related - coal gasifica-
tion and fluidized bed combustion; Other - metal-coated fly ash.
Duration: 20 hr.
Cost/sample or chemical: $500 to $900.
Interpretation: Changes in cellular adenosine triphosphate and
viability by dye exclusion indicate potential toxicity of tested
substance.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch.
Persons to Contact: M.D. Waters, HERL-RTP, Research Triangle Park,
NC 27711, (FTS 629-2693); J.L. Huisinqh, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2537).
Grant/Contract Laboratory Involved and Principal Investigators:
NT Research Institute, 10 West 35th Street, Chicago IL
60616, C. Aranyi: Northrop Services, Inc., P.O. Box 12313,
Research Triangle Park, NC 27709, N.E. Garrett.
Program Office Support: OHEE; OEM I; OAWM.
References: I) Huisingh, J.L., J.A. Campbell, and M.D. Waters.
Evaluation of Trace Element Interactions Using Cultured Alveolar
Macrophages. In: Pulmonary Macrophage and Epithelial Cells.
Conf-760972, Sanders, C.L., R.P. Schneider, G.E. Dagle, and H.A.
Hagan, eds. ERDA Symposium Series 43, Technical Information Center,
Energy Research and Development Administration, 1977. pp. 346-357.
2) Waters, M.D., J.L. Huisingh, and N.E. Garrett. The Cellular
Toxicity of Complex Environmental Mixtures. In: Proceedings of
the Symposium on the Application of Short-Term Bioassays in the
Fractionation and Analysis of Complex Environmental Mixtures.
Wi I Iiamsburg, VA, 1978. 3) Waters, M.D., D.E. Gardner, and D.L.
Coffin. Cytotoxic Effects of Vanadium on Rabbit Alveolar Macro-
phage In-Vitro. Toxicol. Appl. Pharmacol., 28:253-263, 1974.
4) Waters, M.D., T.O. Vaughan, J.A. Campbell, F.J. Miller, and
D.L. Coffin. Screening Studies on Metallic Salts Using the Rabbit
16
-------
mil RABBIT ALVEOLAR MACROPHAGE (RAM) (continued)
phage In-Vitro. Toxicol. Appl. Pharmacol., 28:253-263, 1974.
4) Waters, M.D., T.O. Vaughan, J.A. Campbell, F.J. Miller, and
D.L. Coffin. Screening Studies on Metallic Salts Using the Rabbit
Alveolar Macrophage. In-Vitro, 10:342-343, 1974. 5) Waters, M.D.,
D.E. Gardner, C. Arnyi, and D.L. Coffin. Metal Toxicity for
Rabbit Alveolar Macrophages In-Vitro. Envir. Res., 9:32-47, 1975.
6) Waters, M.D., T.O. Vaughan, D.J. Abernathy, H.R. Garland, C.C. Cox,
and D.L. Coffin. Toxicity of Platinum (IV) Salts for Cells of Pul-
monary Origin. Envir. Hlth. Perspect., 12:45-56, 1975.
17
-------
11112 RAT HEPATOCYTE (LIVER CELL)
Biological Activity Detected: Toxicity.
Principle: Toxic agents alter basic metabolic processes and cellular
structure and function of the hepatocyte.
Endpoints: Qua Iitative: Morphology. Quant!tati ve: Cellular via-
bility; Adenosine triphosphate content; Tyrosine aminotransferase
activity; Total cell protein.
Strengths: These primary liver parenchyonal cells resemble the adult
liver cell in-vivo morphologically and in many of the biochemical
parameters evaluated.
Weaknesses: These cells do not divide and must be isolated from a
rat prior to each assay. Since there is rat-to-rat variation,
cells from several rats should be used to evaluate each chemical.
Status of Development: Developmental; Being implemented.
Describe: New endpoints are being developed; however, the assay
is now being implemented with both inorganic and organic chemicals.
Applications: Multimedia.
Samples: Pure ChemicaIs: Inorganic salts, Organic solvents, Organic
solids. Complex Mixtures: N/A.
Duration: 20 hr.
Cost/sample or chemical: $500 to $1,000.
Interpretation: Alterations in the basic metabolic processes and
cellular structure and function of the liver cells determine the
potential toxicity of the agent.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section.
Persons to Contact: J.L. Huisingh, HERL-RTP, Research Triangle Park,
NC 2771 I, (FTS 629-2537).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OTS.
References: I) Huisingh, J.L., J.P. Inmon, L.C. King, K. Williams,
and M.D. Waters. The Use of Rat Liver Parenchymal Cells in
Evaluating Cellular Response to Toxic Metals and Carcinogenic
Polycyclic Aromatic Hydrocarbons. In-Vitro, 13:182, 1977.
2) Waters, M.D., and J.L. Huisingh. In-Vitro Testing for Chemi-
cal Toxicity: Mammalian Target Cells. In-Vitro, 13:192, 1977.
-------
11113 CHINESE HAMSTER OVARY (CHO) CYTOTOXICITY AND MUTAGENICITY
Biological Activity Detected: Toxicity; Mutagenicity.
Principle: Toxicity is evaluated from effects on clonal growth.
Mutation at the hypoxanthine-guanine phosphor!bosyI transferase
(HGPRT) locus is determined from mutants isolated in 6-thioguanine'
containing media.
Endpoints: Qualitative: N/A. Quantitative: Colony formation
(cytotoxicity); Mutation frequency.
Strengths: Cytotoxicity and mutagenicity may be studied simultaneously.
Weaknesses: The cell type may not be representative of metabolicaIly
active cells which receive exposure to environmental toxicants.
Status of Development: Developmental.
Describe: This assay has been shown to be useful in studies of
70 individual environmental agents related to energy technologies
and 3 subfractions of a crude synthetic oil.
Applications: Air; Water.
Samples: Pure ChemicaIs: Polycyclic hydrocarbons, Metallic compounds,
Nitrosamines, Quincline compounds, Physical agents, A Iky I at ing
agents, Nitrogen mustards, and Aromatic amines. Complex Mixtures:
Energy Related - synthetic fuel.
Duration: 18 days.
Cost/sample or chemical: $500 to $1,000.
Interpretation: Decreasing clonal growth after exposure of CHO cells
indicates potential toxicity of test substance. Increasing numbers
of mutants with increasing concentration of the test substance in-
dicate the substance is a potential mutagen.
Level of Complexity: 2
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section.
Persons to Contact: J.L. Hu i s ingh, HERL-RTP, Research Triangle Park,
NC 2771 I, (FTS 629-2537).
Grant/Contract Laboratory Involved and Principal Investigators:
Northrop Services, Inc., P.O. Box 12313, Research Triangle Park,
NC 27709, N.E. Garrett.
Program Office Support: OHEE; OEM I.
References: I) Hsie, A.W., et al. Quantitative Mammalian Cell Genetic
Toxicology: Study of the Cytotoxicity and Mutagenicity of Seventy
Individual Environmental Agents Related to Energy Technologies
and Three Subfractions of a Crude Synthetic Oil in the CHO/HGPRT
System. In: Proceedings of the Symposium on Short-Term Bioassays
in the Fractionation and Analysis of Complex Environmental Mix-
tures. Wi I Iiamsburg, VA, 1978. 2) O'Neill, J.P., P.A. Brimer,
R. Machanoff, G.P. Hirsch, and A.W. Hsie. A Quantitative Assay of
Mutation Induction at the Hypoxanthine-Quanine Phosphor!bosyI
Transferase Locus in Chinese Hamster Ovary Cells: Development
and Definition of the System. Mutat. Res., 45:91-101, 1977.
3) O'Neill, J.P., D.B. Couch, R. Machanoff, J.R. San Sebastian,
P.A. Brimer, and A.W. Hsie. A Quantitative Assay of Mutation Induc-
tion at the Hypoxanthine-Guanine Phosphor!bosyI Transferase Locus
in Chinese Hamster Ovary Cells (CHO/HGPRT system): Utilization
with a Variety of Mutagenic Agents. Mutat. Res., 45:103-109, 1977.
-------
CONTENTS: TEST SYSTEMS
1120 In-Vivo Systems/Acute 21
I 121 LD50 22
1122 Whole Animal LD50 - Ora I , Dermal 23
1130 In-Vivo Systems/Subacute 25
21
-------
1121 LD50
Biological Activity Detected: Toxicity.
Principle: Based on preliminary range finding tests, 10 dose-levels
of the test compound are selected in the range from Q% to 100$
mortality. Each group would consist of 7 animals. The animals
receive one dose and then are observed for a period of 14 days.
After the 14-day period, a dose-response (cumulative mortality)
curve is plotted and the dose level producing 50% mortality is
i nterpolated.
Endpoints: Qua Iitative: Clinical signs of toxicity. Quantitative:
Cumulative mortality.
Strengths: Can determine a dose-response curve for nearly all test
compounds and from that establish a maximum tolerated dose level
to use in a multiple dose, longer-ranged study.
Weaknesses: Toxicity of vehicle; Solubility; Vehicle-compound
synergism and antagonism.
Status of Development: Validated.
Describe: The chemical is administered by route of interest,
condition of animals standardized, and observation period
specified. Decisions, however, must be made concerning the
type of solvent or vehicle that is most appropriate.
Applications: Multimedia.
Samples: Pure ChemicaIs: Most classes. Complex Mixtures: Drink-
ing water concentrates.
Duration: Variable.
Cost/sample or chemical: $500.
Interpretation: The establishment of a maximum tolerated dose.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxicologicaI Assessment Branch.
Persons to Contact: R.J. BuI I, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Loomis, T.A. Essentials of Toxicology, 2nd Ed. Lea
and Febiger, Philadelphia, PA, 1974. pp. 17-25.
22
-------
1122 WHOLE ANIMAL LD50 - ORAL, DERMAL
Biological Activity Detected: Toxicity.
Principle: Administration of test substance to a sufficient number
of rats, over a dosage range resulting in 0% to 100$ effects.
Endpoints: Qua Iitative: Clinical effects. Quantitative: Effective
dosage to produce effect, LD50 value.
Strengths: Estimates relative toxicity; Economical; Simple to
conduct.
Weaknesses: May not apply to all species; May not correspond with
data from other laboratories.
Status of Development: Being implemented.
Describe: Tests currently being conducted on limited basis on
relevant selected compounds.
Applications: Multimedia.
Samples: Pure Chemicals: AI I classes. Complex Mixtures: Other -
technical grade materials, formulated products.
Duration: 3 months.
Cost: $5,000/chemical for full battery of tests.
Interpretation: A rating of the toxicity of the various chemicals
is obtained.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology
Division.
Persons to Contact: R. Linden, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2701).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP.
References: I) Gainer, T.B. Acute Toxicity of Pesticides. Toxicol.
Appl. P'harmacol., I960.
-------
CONTENTS: TEST SYSTEMS
1130 In-Vivo Systems/Subacute 25
1131 Integrated System: General Clinical
Pathology 26
1132 Sleep-Time Study 27
1133 Metabolism of Chlorinated Hydrocarbons in
Subhuman Primates 28
1134 Metabolic Profiles 29
1135 Model Substrate Metabolism 30
I 136 Xenobiotic Mechanisms 32
1137 Oxidant Production by Leukocytes and Alveolar
Macrophages Measured by Chemi Iuminescence ... 33
1138 Cyclic Nucleotide Concentrations in Leukocytes
and Alveolar Macrophages 34
1140 In-Vivo Systems/Inhalation Toxicology 35
25
-------
1131 INTEGRATED SYSTEM: GENERAL CLINICAL PATHOLOGY
Biological Activity Detected: Systemic effects.
Principle: Measurement of serum constituents, proteins, enzyme
activities, hematologicaI parameters such as cell counts, cell
morphology in peripheral blood and In bone marrow, measurement
of special endocrinologic parameters such as thyroid and adrenal
functions, pituitary hormones, etc. Diagnosis of malignancies
by measurement of tumor marker proteins, serum isoenzyme patterns,
etc. Measurement of urinary constituents.
Endpoints: Qua Iitative: Technique dependent. Quantitative: Tech-
n ique dependent.
Strengths: High degree of quality control; Great amount of informa-
tion is available as to the diagnostic implications of abnormal
f indi ngs.
Weaknesses: Often not capable of signaling asymptomatic preclinical
toxic effects; Selection of tests must be done with care and
proper planning to ensure maximal effectivity.
Status of Development: Validated.
Describe: This entry encompasses approximately 800 different
varieties of tests that may be performed on animals in-vivo or
on biological specimens in-vitro. Usually, a battery of tests
will be performed examining various organ functions.
Applications: Multimedia.
Samples: Pure ChemicaIs: N/A. Complex Mixtures:
Other - biological specimens: blood, urine, bone marrow.
Duration: Continuous.
Cost/sample or chemical: Test dependent.
Interpretation: The totality of a battery is aimed at detecting
organ-specific toxic effects.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxicologicaI Assessment Branch, Systemic and Genetic Effects
Group; HERL-CIN, Field Studies Division, ToxicologicaI Assessment
Branch.
Persons to Contact: R.J. BuI I, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213); J.P. Bercz, U.S. EPA,
HERL-CIN, 26 W. St. Clair St., Cincinnati, OH 45268, (FTS 684-
7480).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: Theories and practical techniques as compiled in clinical
pathology textbooks: I) Henry and Davidson. Clinical Diagnosis
by Laboratory Methods. 2) Tietz. Clinical Chemistry. 3) Win-
trobe. Clinical Hematology.
26
-------
1132 SLEEP-TIME STUDY
Biological Activity Detected: Identifies the biological act-
ivity of a compound indicating a potential for interaction with
other compounds.
Principle: Chemicals that induce or inhibit MFO will alter the
pharmacological effects of drugs metabolized by MFO. Hexabarbital
and zoxogolamin are depressant drugs whose properties are well
known in this respect. Animals are given a single dose or multiple
doses of the test compound at a tolerated but effective level. Two
hours after the final dose they are challenged with an anesthetic
dose of hexobarbitaI. A control group receiving no test compound
also receives the hexobarbitaI. The time is measured from the
instant the animals lose their "righting reflex" (ability to right
themselves when laid flat on their back) to the time they regain
it.
Endpoints: Qua Iitative: Induction or inhibition of liver enzyme
activity. Quantitative: Measured sleep-time.
Strengths: Fast and presumptive assay to determine whether a compound
will induce or inhibit liver enzymes; Useful in planning more
extensive metabolism studies.
Weaknesses: Changes in rates of metabolism must be documented to
conclude the effect mediated via MFO.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: All classes. Complex Mixtures:
Industrial; Energy Related.
Duration: Variable depending on projected properties of test compounds.
Cost: Approx. $200/compound.
Interpretation: The results determine the ability of a compound to induce
or inhibit enzymatic systems.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Toxicologica1 Assessment Branch; HERL-RTP, Clinical Studies
Division, Biomedical Research Branch.
Persons to Contact: R.J. BuI I, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213); D.E. Gardner, U.S.
EPA, HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Conney, A.H., et al. Adaptive Increases in Drug
Metabolizing Enzymes Induced by Phenobarbital and Other Drugs.
J. Pharmacol. Exp. Ther., 130:1-8, I960. 2) LaDu, B.N., H.G.
Mandel, and E.L. Way. Fundamentals of Drug Metabolism and Drug
Disposition. The Williams and Wilkins Co., Baltimore, MD, 1971.
27
-------
1133 METABOLISM OF CHLORINATED HYDROCARBONS IN SUBHUMAN PRIMATES
Biological Activity Detected: Comparative metabolism.
Principle: In drug metabolism studies, the primate as a model more
often represents humans than any other animal model. It is
likely that this is the case for environmental contaminants
a I so.
Endpoints: Qua Iitative: Comparison of metabolites from various
animal species. Quantitative: Quantitative analysis of meta-
bolites from various animal species.
Strengths: The primate is most likely to be representative of man
in its metabolic activity toward environmental contaminants.
Weaknesses: Expense and difficulty of working with monkeys.
Status of Development: Validated.
Describe: After dosage, specific enzymes are tested for activity,
Excreta samples are chemically analyzed for metabolites.
Applications: Multimedia.
Samples: Pure ChemicaIs: Chlorinated aliphatic hydrocarbons,
Chlorinated aromatic hydrocarbons. Complex mixtures: N/A.
Duration: 3 months.
Cost: $15,000 to $18,000.
Interpretation: Comparison of metabolism from various animal systems.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Exposure Evaluation Branch,
Organics Metabolism Section.
Persons to Contact: R.D. Lingg, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7463).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Cincinnati, Cincinnati, OH 45221, C. Smith, (Commercial
513 872-5700).
Program Office Support: OHEE.
References: I) Smith, C., R.D. Lingg, and R.6. Tardiff. Comparative
Metabolism of Haloethers. Ann. N.Y. Aca. Sci., 298:111, 1977.
28
-------
1134 METABOLIC PROFILES
Biological Activity Detected: Toxicity; Biotransformation; Compara-
tive metabolism.
Principle: Identification and quantitative analysis of major meta-
bolites are obtained through the use of modern analytical tech-
niques. From these results, a metabolic pathway for a selected
compound can be proposed. The potential of a compound for inter-
action with macromolecules is better understood.
Endpoints: Qua Iitative: Identification of major metabolites.
Quantitative: Quantitative analysis of major metabolites.
Strengths: Leads to an understanding of the structural differences
in compounds which affect their metabolic disposition and
toxicity.
Weaknesses: Difficult to extrapolate to the human condition.
Status of Development: Being implemented.
Describe: Most separation and derivatization techniques have
been validated using g-Chloroethers and Trichlorobenzenes as
model compounds. Additional work needs to be done on processing
mass spectral data.
Applications: Multimedia.
Samples: Pure ChemicaIs: Halogenated aliphatic hydrocarbons,
Halogenated aromatic hydrocarbons. Complex Mixtures: N/A
Duration: 3 months.
Cost/sample or chemical: $10,000 to $15,000.
Interpretation: The determination of metabolic pathways leads to
an understanding of the differences in the toxication of a
selected chemical in various animal species.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Exposure Evaluation Branch,
Organics Metabolism Section; HERL-RTP, Environmental Toxicology
Division, Biochemistry Branch, Metabolic Effects Section.
Persons to Contact: R.D. Linqq, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7463); S. Nesnow, U.S. EPA,
HTRL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) R.D. Lingg, et al. Fate of Bis (2-ChloroethyI) Ether
in Rats after Acute Oral Administration. To be presented at the
Seventeenth Annual Meeting of the Society of Toxicology, Mar.
12-16, 1978, San Francisco, CA. 2) R.D. Lingg, W. Kaylor,
S.M. Pyle, and R.6. Tardiff. Thiodiglycolic Acid: A Major
Metabolite of Bis (2-ChloroethyI) Ether. Submitted to Toxicol.
and Appl. Pharmacol. Dec., 1977.
29
-------
1135 MODEL SUBSTRATE METABOLISM
Biological Activity Detected: Toxicity.
Principle: The model substrate assay attempts to determine the effect
of environmental chemicals on metabolic pathways in the living
animal. This test involves repeated exposure of the experimental
animals (6/treatment group) to the toxicants under investigation.
After this pretreatment period the animals receive a single oral
dose of the model substrate, llfC-l indane. A useful variation in
this assay involves the simultaneous administration of lindane
with the toxicants being studied.
Endpoints: Qua Iitative: Altered metabolite profiles serve as a
"fingerprint of toxicant exposure" since xeriobiotics have charac-
teristic effects on the metabolic pathways of the model substrate.
Quantitative: Induced or inhibited metabolism are measured by
GLC analysis and liquid scintillation counting of excreted pro-
ducts.
Strengths: Analysis of excreted metabolites provide direct measure
of overall metabolism in living animal; Changes in enzyme activity
due to disruption of phospholipid membranes, loss of permeability
barriers, and loss of local charge effect are avoided; Anomalous
enzyme alterations caused by accumulation of substrate and/or
metabolites are avoided since normal clearance mechanisms are
functional; Economical and practical since comparative activity
of multiple pathways are determined on individual animals; Daily
changes in the metabolic pathways of animals repeatedly exposed
to toxicants can be determined.
Weaknesses: Requires GLC standards of the model substrate metabolites
being determined.
Status of Development: Being implemented.
Describe: The model substrate assay has been successfully
employed to study interactions between a variety of environmental
chemicals, nutritional stress, and the enzyme systems that detoxify
or enhance the toxicity of xenobiotics. The correlation between
exposure to xenobiotics, whose metabolites produce the same
lesion, and their induction of a model substrate metabolite
profile with a common element is being investigated.
Applications: Multimedia.
Samples: Pure Chemicals: Ca, Organochlorine pesticides, Herbicides,
Insecticides, Fungosides, Drugs, Toxic substances.
Complex Mixtures: Not yet tested.
Duration: Test: 2-week exposure; Analysis: 3 weeks.
Cost: $2,700.
Interpretation: It is thought that the generation of unusual altera-
tions in the relative activity of various metabolic pathways of
a model substrate can signal toxic interactions.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology
Division, Biochemistry Branch.
30
-------
1135 MODEL SUBSTRATE METABOLISM (continued)
Persons to Contact: R.W. Chadwick, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2750); M.F. Copeland,
U.S. EPA, HERL-RTP, Research Triangle Park, NC 27711,
(FTS 629-2678).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OTS.
References: I) Chadwick, R.W., C.J. Chadwick, J.J. FreaI, and C.C.
Bryden. Comparative Enzyme Induction and Lindane Metabolism in
Rats Pre-treated with Various Organochlorine Pesticides. Xeno-
biotica, 7:235-246, 1977. 2) Chadwick, R.W., W.S. Simmons, C.C.
Bryden, L.T. Chuang, L.M. Key, and C.J. Chadwick. Effect of
Dietary Liquid and Dimethyl Sulfoxide on Lindane Metabolism.
Toxicol. and Appl. Pharmacol., 39:391-410, 1977. 3) Chadwick,
R.W., M.F. Copeland, and C.J. Chadwick. Enhanced Pesticide
Metabolism, a Previously Unreported Effect of Dietary Fiber in
Mammals. Food and Cosmetics Toxicol., 1978. In press.
-------
1136 XENOBIOTIC MECHANISMS
Biological Activity Detected: Capacity of chemical to act as a
synergist or antagonist.
Principle: Groups of 10 anima Is/sex/dose level are exposed to test
compounds for periods of 3 to 7 days, to test the potential of the
compound to induce or inhibit liver enzymes. Dose levels used
are the maximum tolerated dose (MTD), 1/2 MTD and 1/4 MTD. Fol-
lowing the dose regimen the animals are sacrificed and liver
samples are taken for the enzyme assays.
Endpoints: Qua Iitative: Hexabarital cytP-450 levels, cytPc reductase,
0-demethylase, and other microsomal enzyme activities. Quantitative:
Hexabarital cytP-450 levels, cytPc reductase, 0-demethylase, and
other microsomal enzyme activities.
Strengths: Quick assay to determine drugs' potential to cause liver
damage; Does not require large numbers of animals.
Weaknesses: No significant weaknesses.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: Xenobiotics. Complex Mixtures: N/A
Duration: Variable, I week to chronic, depending on whether testing
is acute or chronic.
Cost/sample or chemical: Variable, depending on test. I week at
$35,000/manyear equals approximately $700.
Interpretation: This test determines the potential for synergisms
and/or antagonisms mediated through altered xenobiotic metabolisms.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxicologicaI Assessment Branch; HERL-RTP, Environmental Toxi-
cology Division, Biochemistry Branch, Metabolic Effects Section.
Persons to Contact: R.J. Bu I I, U.S. EPA, HERL-CIN., 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213); S. Nesnow, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Hayakwa, T. A Simple Radioisotope Assay for Micro-
somal Aryl Hydroxylase. Anal. Biochem., 51:501-509, 1973. 2)
Dallner, G. Studies on the Structural and Enzymatic Organization
of Liver Microsomes. Acta Path. Scand., 166:7-41, 1963. 3) NeaI,
R.A. Studies on the Mechanisms of Detoxification of Cholinergic
Phosphorothioates. J. Pharmacol. Exp. Therap., 148:185-192, 1956.
4) Lucier, G.W. Microsomal Rat Liver UDP Glucuronyl Transferase:
Effects of Piperonyl Butoxide. Arch. Biochem. and Biophys., 145:
520-530, 1971. 5) Omura, T. The Carbon Monoxide Binding Pigment
of Liver Microsomes: I. Evidence for its Hemoprotein Nature.
J. Biol. Chem., 239;2370-2378, 1964.
32
-------
1137 OXIDANT PRODUCTION BY LEUKOCYTES AND ALVEOLAR MACROPHAGES MEASURED
BY CHEMILUMINESCENCE
Biological Activity Detected: Toxicity.
Principle's Oxidant production in alveolar macrophage microblcidal
activity is due to alveolar macrophage metabolic activation. Oxi-
dants are measured by measuring light produced by oxidation re-
actions.
Endpoints: Qualitative: N/A. Quantitative: Production
of light as measured in an ATP photometer. The amount of light
can be measured in response to purified macrophage stimulates
and particles.
Strengths: Very reproducible; Fast; Simple; Does not kill cells;
Requires very few cells; Applicable to human blood cells; Several
types of oxidant can be measured; In-vivo or in-vitro dose-response
tests can be made; In-vivo tests are suitable for standard-setting
and regulatory purposes.
Weaknesses: ChemiIuminescent reactions are susceptible to many
quenching effects and competing reactions; Adequate controls are
necessary; For in-vivo exposures, relatively large amounts of
pollutant sample are required.
Status of Development: Being implemented.
Describe: Effects of 63, NC>2, and various particles are being
determi ned.
Applications: Multimedia.
Samples: Pure ChemicaIs: All transparent chemicals which are
not readily oxidized. Complex Mixtures: All materials except
those which are very opaque.
Duration: In-vitro exposure: 2 weeks/dose-response of I chemical; in-
vivo exposure: 4 weeks/dose-response of I chemical.
Cost: In-vitro exposure: $5,000/chemicaI; In-vivo exposure: $IO,000/
chemicaI.
Interpretation: Defect in oxidant production predicts possible micro-
bicidal defect in the macrophages.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: G. Hatch, U.S. EPA, HERL-RTP, Biomedical
Research Branch, Research Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OEMI; OTS.
References: I) Hatch, G.E., D.E. Gardner, and D.B. Menzel. J. Exp.
Med., 147;183-195, 1978. 2) Rosen, H., and S.J. Klebanoff. J.
Clin! Invest., 58:50, 1976.
33
-------
1138 CYCLIC NUCLEOTIDE CONCENTRATIONS IN LEUKOCYTES AND ALVEOLAR MACROPHAGES
Biological Activity Detected: Toxicity; Presumptive oncogenicity;
Pharmacologic modulation of promotion.
Principle: Cyclic AMP and Cyclic GMP are intracelIular hormones
which modulate cellular functions, including cell proliferation,
secretion, and movement. The hormones are measured by radio-
immunoassay.
Endpoints: Qua Iitative: N/A. Quantitative: Concentrations
of cyclic nucleotide per cell correlate with cellular activity.
Strengths: Cyclic nucleotide concentrations are easily altered by
subtle means; Their concentrations are of central importance to
cellular activity; Applicable to human blood cells; In-vivo or
in-vitro dose-response studies can be done; In-vivo tests would
be suitable for standard setting and regulatory purposes.
Weaknesses: Time consuming; Tedious; Requires a lot of cells; Dif-
ficult to establish steady basal or control values; In-vivo
exposures will require relatively large amounts of pollutant
samples.
Status of Development: Being implemented.
Describe: Effects of N02, 03, and NO are being tested for effects
on Cyclic AMP and Cyclic GMP.
Applications: Air; Water; Food; Multimedia.
Samples: Pure ChemicaIs: Most chemicals. Complex Mixtures:
Ambient; Industrial; Energy Related; Transportation Related.
Duration: In-vitro exposure: 2 weeks/chemical for dose-response;
In-vivo exposure: 4 weeks/chemical for dose-response.
Cost: In-vitro exposure: $5,000/chemicaI; In-vivo exposure: $IO,000/
chemicaI.
Interpretation: Detection of alteration in cellular hormone metabolism
which is important to homeostasis.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: G. Hatch, U.S. EPA, HERL-RTP, Biomedical
Research Branch, Research Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I) Hatch, G.E., W.K. Nichols, and H.R. Hill. J. Immunol.,
I 19:450-456, 1977.
34
-------
CONTENTS: TEST SYSTEMS
1140 In-Vivo Systems/1nhaI at ion Toxicology 35
1141 Toxicity of Aerosolized Pollutants/Acute and
Subacute 36
1142 Toxicity of Gaseous and Vapor Phase/Acute and
Subacute 37
1143 Inhalation LC50 Tests 38
1144 Deposition and Clearance of Radioactive
Materials Following Inhalation Exposure .... 39
1145 Integrated System: Pulmonary Function and
Pulmonary Metabolism 40
1146 Pulmonary Function in Rats 41
1147 Pulmonary Function of Unanesthetized
Guinea Pigs 42
1148 Arterial Blood Gas Measurement in
Conscious Rats 43
1149 Infect!vity Model 44
11410 In-Vivo Alveolar Macrophage Cytotoxicity 45
1)50 In-Vivo Systems/NeurobehavioraI 47
35
-------
1141 TOXICITY OF AEROSOLIZED POLLUTANTS/ACUTE AND SUBACUTE
Biological Activity Detected: Toxic'ity.
Principle: Exposure begins with a relatively high concentration,
which results in 100$ mortality in I to 4 hours. The concentration
is decreased by factors of 10 until no deaths occur. Performed
in rats.
Endpoints: Qualitative: N/A. Quantitative: Deaths of
animals being exposed.
Strengths: Non-ambiguous positive or negative results.
Weaknesses: For moderately or low toxic substances the determination
of a LC50 may be impractical.
Status of Development: Being implemented.
Describe: The equipment required for aerosol generation in the
respirable range is being evaluated.
AppIications: Ai r.
Samples: Pure Chemicals: 'Pesticides. Complex Mixtures: N/A.
Duration: Variable, I week to 2 months.
Cost: $10,000 to $20,000/analysis.
Interpretation: A rating of the relative toxicity in mammalian species
is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Toxic Effects Branch, Inhalation Toxicology Group.
Persons to Contact: J. Charles, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2696).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP; OTS.
References: I) Hinners, R.G., J.K. Burkhart, and C.L. Punte. Arch.
Envir. Hlth., 16:194-206, 1968.
36
-------
1142 TOXICITY OF GASEOUS AND VAPOR PHASE/ACUTE AND SUBACUTE
Biological Activity Detected: Toxicity.
Principle: Exposure begins with a relatively high concentration
which results in 100$ mortality in I to 4 hours. The concentrations
are decreased by factors of 10 unti I no deaths occur. Performed
primarily in rats. Physiologic and biochemical parameters are
measured.
Endpoints: Qua IJtative: N/A. Quantitative: Death of
animals being exposed. Biochemical parameters, enzymes, and
substrate levels are assayed for in surviving animals.
Strengths: Non-ambiguous positive or negative results.
Weaknesses: For moderately or low toxic substances the determination
of an LC50 may be impractical.
Status of Development: Being implemented.
Describe: 5 chambers, 20 animals/chambers are in operation.
AppIications: Ai r.
Samples: Pure ChemicaIs: Gases, Vaporizable liquids, Pesticides,
Toxic substances in general. Complex Mixtures: N/A.
Duration: Variable, I week to 2 months.
Cost: $10,000 to $20,000/analysis.
Interpretation: A rating of the relative toxicity in mammalian
species is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Toxic Effects Branch, Inhalation Toxicology Group.
Persons to Contact: J. Charles, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2696).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP; OTS.
References: I) Drew, R.T., and S. Laskin. Methods of Animal Experi-
mentation, Vol. 4. N.Y. Academic Press, pp. 1-41. 2) Hinners,
R.G., J.K. Burkhart, and C.L. Punte. Arch. Envir. Hlth., 16:194-
206, 1968.
-------
1143 INHALATION LC50 TESTS
Biological Activity Detected: Toxicity.
Principle: Animals are exposed to different concentrations of the
agent, and lethality is determined over a preset period of time.
Endpoints: Qua Iitative: N/A. Quantitative: Concentration required
to cause death in 50% of the exposed animals.
Strengths: Provides initial data on toxicity.
Weaknesses: Cannot be used to determine subtle changes associated
with low level exposure.
Status of Development: Validated.
Describe: Most chemicals and complex mixtures can be examined;
extremely toxic or carcinogenic samples cannot be examined as
HERL-CIN does not have the necessary facilities.
Applications: Multimedia.
Samples: Pure ChemicaIs: Most chemicals. Complex Mixtures: Ambient,
Industrial; Energy Related; Transportation Related; Other.
Duration: 14 days/study.
Cost: $4,000 to $10,000, test dependent.
Interpretation: A rating of the relative toxicity in mammalian
species is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Functional Pathology Branch; HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: W. Moore, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7431); D.E. Gardner, U.S.
EPA, HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: ORD.
References: I) Moore, W., M. Malanchuk, W. Crocker, D. Hysell, A.
Cohen, and J.S. Stara. Whole Body Retention in Rats of Different
191Pt Compounds Following Inhalation Exposures. Envir. Hlth.
Perspec., 12:35, 1973. 2) Moore, W. , J.S. Stara, W. Crocker,
M. Malanchuk, and R. Mtis. Comparison of Cadmium Retention
in Rats Following Different Routes of Administration. Envir.
Res., 6:473, 1973.
38
-------
1144 DEPOSITION AND CLEARANCE OF RADIOACTIVE MATERIALS FOLLOWING INHALATION
EXPOSURE
Biological Activity Detected: Deposition; Trans location clearance.
Principle: Animals are exposed to radioactive aerosol for 15 to 30
minutes; then they are counted at various intervals of time.
Tissues are also taken for analysis.
Endpoints: Qua Iitative: N/A. Quantitative: Data on clearance,
trans location, distribution, and excretion as a factor of time.
Strengths: Provides metabolic parameters on the agent under study.
Weaknesses: Agents used are usually radioactive.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: Radioactive chemicals. Complex Mixtures:
N/A.
Duration: Variable, depending on the goals of the study.
Cost/sample: $3,000 to $5,000, depending on cost of compound.
Interpretation: A measure of the residence tissue in the lung and
body tissues for the compound is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Functional Pathology Branch; HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: W. Moore, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7431); D.E. Gardner, U.S.
EPA, HERL-RTP, Research Triangle Park, NC 27711 (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: ORD.
References: I) Moore, W., D. Hysell, W. Crocker, and J.S. Stara.
Biological Fate of 103Pd in Rats Following Different Routes of
Exposure. Envir. Res., 8:234, 1974.
39
-------
1145 INTEGRATED SYSTEM: PULMONARY FUNCTION AND PULMONARY METABOLISM
Biological Activity Detected: Toxicity.
Principle: Structural changes in lung airways and parenchyma can
be evaluated in-vivo by use of appropriate tests of pulmonary
function. Concommitant metabolic changes can be estimated in-
vitro.
Endpoints: Changes can be measured in intact animals. Qua Iitative
and Quantitative: Changes in pulmonary mechanics, spirometry, and
diffusion can be evaluated. Metabolic changes of lung tissue
can be estimated in-vitro to establish dose-response relationships.
Strengths: Measurements of pulmonary function accomplished by non-
invasive methods; In-vivo dose-response effects more applicable
for standard-setting and regulatory purposes.
Weaknesses: Expensive equipment; Time consuming; Requires several
pairs of hands; Some tests are still being validated; Parenchymal
changes (i.e., development of pulmonary fibrosis, pulmonary
emphysema or chronic bronchitis) are of great interest in terms
of health effects, but they are usually chronic diseases.
Status of Development: Developmental; Being implemented; Validated.
Describe: Different tests are in different stages of development.
We are presently measuring lung volumes and capacities (TLL, VC,
1C, FRC, RV, VT), breathing frequency, minute ventilation, dif-
fusing capacity for carbon monoxide, nitrogen washout, and quasi-
static pressure volume relationships of the lung and chest wall.
We are developing methods to measure dynamic compliance and re-
sistance, maximum flow volume relationships, compliance charac-
teristics of excised lungs, and the single breath oxygen test.
App I ications: Ai r.
Samples: Pure ChemicaIs: Oxides of N and S, Pulmonary toxicants
(e.g. Paraquat), Pesticides. Complex Mixtures: Ambient - N0£,
S02, 03; Energy Related - particulates and NO , SO , 03, organics,
others; Transportation Related - diesel; Other - Toxic substances,
Any compound which changes pulmonary physiology.
Duration: 3 month/dose-response of I chemical.
Cost: $30,000/dose-response of I chemical.
Interpretation: These tests are sensitive and can detect small
changes in lung physiology.
Level of Complexity: 3 to 4.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: J.J. O'Nei I, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2711/2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OEMI; OTS.
References: I) Koo, K.W., et a I. Respiratory Mechanics in Normal
Hamsters. J. Appl. Physiol., 40:936-942, 1976. 2) Diamond, L.,
and M. O'Donnell. Pulmonary Mechanics in Normal Rats. J. Appl.
Physiol., 43:942-948, 1977. 3) Takezawa, J., F. Miller, and
J.J. O'Nei I. Single Breath Diffusing Capacity and Lung Volumes
in Small Laboratory Animals. J. Appl. Physiol., 1979. In
preparation.
40
-------
1146 PULMONARY FUNCTION IN RATS
Biological Activity Detected: Toxicity.
Principle: Residual volume of the lung increases with many types of
obstructive and destructive lung damage. The slope of the static
compliance of the pressure-volume curve of the lungs increases
with fibres is and decreases with diseases such as emphysema
that destroy alveolar tissue.
Endpoints: Qua Iitative: N/A. Quantitative: Residual
volume (cc) and the slope of the static compliance curves.
Strengths: The measurements are sensitive indicators of lung volume;
They are relatively easy to perform.
Weaknesses: The measurements are conducted with rats; Pulmonary
anatomy and susceptibility of these animals may differ somewhat
from man.
Status of Development: Validated.
Describe: Rats have shown large changes in residual volume and
in the slope of the static compliance curve, after I to 14 days
of exposure to either 0.75 or 1.0 ppm ozone. A manuscript de-
scribing these results is being prepared.
Applications: Air; Water; Food.
Samples: Pure ChemicaIs: Sulfates, Oxidants, Heavy metals.
Complex Mixtures: Ambient - air; Industrial - coal dust; Energy
Related - stack gases; Transportation Related - diesel and gasoline
exhaust; Other - asbestos.
Duration: I day to 30 days.
Cost/sample or chemical: $1,000/animaI.
Interpretation: An increase in the slope of the static compliance
curve and/or an increase in residual volume is indicative of
either pulmonary edema or emphysema.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Functional Pathology Branch.
Persons to Contact: W.E. Pepelko, U.S. EPA, HERL-CIN, 26 W. St.
Clair St., Cincinnati, OH 45268, (FTS 684-7437).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) J. Appl. Physlol., 26:738-744, 1966. 2) Chest, 475-
481, 1967.
-------
1147 PULMONARY FUNCTION OF UNANESTHETIZED GUINEA PIGS
Biological Activity Detected: Toxlcity.
Principle: The method allows for measurement of respiratory and
cardiovascular response of an unanesthetized guinea pig to a
controlled test atmosphere.
Endpoints: Qua Iitative: ECG. Quantitative: Airway resistance,
lung compliance, tidal volume, minute volume, breathing rate,
heart rate.
Strengths: This system is most suitable for I to 3 hour exposures
to gases, aerosols, drug response, complex pollutants, etc.;
Each animal serves as its own control during testing.
Weaknesses: The system can be used for measuring response to a long
term exposure where chronic breathing damage is expected. How-
ever, there is wide variation between animals. So for reliable
results either a dramatic response is needed or a very large
number of animaIs.
Status of Development: Being implemented.
Describe: System has been used for catalyticaIly altered auto
exhaust study, sulfur dioxide exposure study, and diesel engine
auto exhaust study. Results are presently being evaluated.
Applications: Air; Multimedia.
Samples: Pure ChemicaIs: Sulfur dioxide. Complex Mixtures:
Transportation Related - catalyticaIly altered car exhaust,
diesel auto emissions.
Duration: 5 hours/animal.
Cost/sample or chemical: $500/sample. This refers only to the test
animals needed.
Interpretation: Reliable results require dramatic response differences.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN.
Persons to Contact: M.J. Wiester, U.S. EPA; HERL-CIN, 26 W. St.
Clair St., Cincinnati, OH 45268, (FTS 684-7424).
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE.
References: I) Amdur, M.O., and J. Mead. Mechanics of Respiration
in Unanesthetized Guinea Pigs. Am. J. Physiol., 192:364, 1958.
42
-------
1148 ARTERIAL BLOOD GAS MEASUREMENT IN CONSCIOUS RATS
Biological Activity Detected: Toxicity.
Principle: Pollutant inhalation resulting in lung damage can
decrease the ability of the animal to oxygenate the blood and
remove C02.
Endpoints: Qua I itatlve: N/A. Quantitative: Arterial blood Pn
pH, bicarbonate. U2
Strengths: A sensitive indicator of lung damage; An important
parameter as adequate PA levels are necessary to support life;
U2
A sample can be collected quickly and fairly easily.
Weaknesses: Rats are the only small laboratory animal to which this
method can be adapted; Extrapolation of results from rats to
humans may be subject to criticism; Each animal can be used
on ly once.
Status of Development: Validated.
Describe: Arterial blood gas measurements were carried out after
I, 3, 7, and 14 days exposure to either 0.75 or 1.0 ppm ozone.
Arterial blood P~ declined in proportion to lung damage. A
manuscript is in preparation.
Applications: Air; Water; Food.
Samples: Pure Chemica Is: Sulfates, Oxidants, Trace metals.
Comp lex Mixtures: Ambient - air; Industrial - coal and rock dust;
Energy Related - stack gas emission; Transportation Related -
auto and diesel emission; Other - food and water born pollutants
such as Paraquat.
Duration: 24 hours to 30 days.
Cost/sample or chemical: $200/animal.
Interpretation: A decrease in arterial P_ or an increase in
indicates lung damage. The type of damage is not specified
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Functional Pathology Branch.
Persons to Contact: W.E. Pepelko, U.S. EPA, HERL-CIN, 26 W. St.
Clair St., Cincinnati, OH 45268, (FTS 684-7437).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) J. Appl. Physiol., 38:581-587, 1975. 2) Ann. Rev.
Pharmacol. and Toxicol., 16:465-486, 1976. 3) Am. Rev. Resp.
Dis., 113:531-559, 1976.
43
-------
1149 INFECTIVITY MODEL
Biological Activity Detected: Toxicity.
Principle: Inhalation of a variety of gases and participates has
been shown to increase susceptibility to infectious pulmonary
disease. Evaluation of the infectivity model used for these
tests indicates that the model reflects the effects of a pol-
lutant on a number of host defense systems, thereby increasing
its sensitivity for detecting effects.
Endpoints: Qualitative: N/A. Quantitative: Mortality
from laboratory-induced bacterial pneumonia is measured.
Strengths: The model is established for inhalation toxicology; Prior
work has shown its exquisite sensitivity; The test is rapid; A
battery of related follow-up tests are available; Whole animal
inhalation exposures and dose-response studies are more directly
applicable to standard setting and regulatory action.
Weaknesses: Relatively large amounts of pollutant sample are re-
quired for inhalation studies.
Status of Development: Validated.
Describe: The model has been successfully used for 63, NC>2,
Cd, Ni, sulfates and pollutant combinations. Even though a
great potential exists it has not been used for screening purposes.
App I ications: Ai r.
Samples: Pure ChemicaIs: Any chemical likely to reach gaseous
exchange areas of the lung. Complex Mixtures: Ambient; Indus-
trial; Energy Related; Transportation Related; Other - any other
particulate, gas or combination of same.
Duration: 8 weeks/dose-response of I chemical.
Cost: $30,000 to $35,000/dose-response of I chemical.
Interpretation: Estimation of enhanced susceptibility to infectious
diseases due to pollutant.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: D.E. Gardner, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators:
I IT Research Institute, 10 West 35th St., Chicago, IL 60616,
R. Ehrlich; Northrop Services, Inc., P.O. Box 12313, Research
Triangle Park, NC 27709, B. Adkins.
Program Office Support: OHEE; OPP; OEMI; OTS.
References: I) Coffin, D.L., and D.E. Gardner. Ann. Occup. Hyg.,
15:219-234, 1972. 2) Coffin, D.L., et al. Envir. Hlth. Perspect.,
13:11-15, 1976. 3) Ehrlich, R. Bacteriol. Rev., 30:604-614, 1966.
4) Ehrlich, R., et al. Internat. Conf. Photo. Oxid. Pollut. and
Its Control, Proc. Vol I, EPA-600/3-77-OOIa, 1977- pp. 565-574.
44
-------
11410 IN-VIVO ALVEOLAR MACROPHAGE CYTOTOXICITY
Biological Activity Detected: Toxicity.
Principle: Cytotoxic effects of inhalation of environmental chemicals
will be measured using isolated alveolar macrophages. Any altera-
tion in these cells could increase the potential risk of respi-
ratory infections.
Endpoints: Qualitative: N/A. Quantitative: The following
measurements can be made: viability, phagocytic functioning,
bacteriocidal activity, enzymatic profile, morphology and other
biochemical parameters.
Strengths: A sensitive indicator of cytotoxicity using an in-vivo
model system; Data generated quickly, which can be used to vali-
date in-vitro cytotoxicity testing; Can serve as criteria for
standard setting and regulatory purposes.
Weaknesses: Requires a substantial quantity of the test chemicals
for generation of aerosols.
Status of Development: Validated.
Describe: The model has been successfully used for 03, NC>2, Cd,
Ni, Mn, cigarette smoke, and other metals.
AppIications: Ai r.
Samples: Pure ChemicaIs: Particulates, Gases, Mists, Any chemical
likely to reach gaseous exchange areas of the lung. CompI ex
Mixtures: Ambient; Industrial; Energy Related; Transportation
Re Iated.
Duration: 8 weeks/dose-response of I chemical.
Cost: $30,000/dose-response of I chemical.
Interpretation: Depending upon the particular endpoint, alteration
would indicate enhanced susceptibility to infectious disease
or potential for alteration of lung tissue.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: D.E. Gardner, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators:
I IT Research Institute, 10 West 35th St., Chicago, IL 60616,
C. Aranyi; U. of California, Davis, CA 95616, E. Goldstein;
Southwest Research Institute, San Antonio, Texas 78284, E.
Cause.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I) Gardner, D.E., et a I. J. Bacteriol., 98:1041-1043,
1969. 2) Hurst, D.J., et al. J. ReticuloendotheI. Soc., 8:288-
300, 1970. 3) Bingham, E. Arch. Envir. Hlth., 25:406-414, 1972.
4) Gardner, D.E. Thesis, U. of Cincinnati, 1971. 5) Warshauer,
D., et al. J. Lab. Clin. Med., 83:228-240, 1974. 6) Kass, E.H.,
et al. Bacteriol. Rev., 30:488-497, 1966. 7) Kim, M., et al.
J. Infect. Dis., 133:310-320, 1976.
45
-------
CONTENTS: TEST SYSTEMS
1150 In-Vivo Systems/Neurobehaviora I 47
1151 Integrated System: NeurobehavioraI
ToxicologicaI Assessment 48
1152 Integrated System: The Effects of Selected
Organic Contaminants in Drinking Water
on the Functions of the Reproductive,
Nervous, and Immune Systems 49
1153 Computer Automated Analysis of Patterned
Behavior in the Primate 51
1154 Computer Automated Analysis of Learning and
Memory in the Primate 52
1155 Integrated System: Behavioral Analysis of
Rats — Developmental, Locomotor, Explor-
atory, and Learned Behavior 53
1156 Integrated System: Instrumental Methods of
Detecting Functional and Metabolic Damage
to the Nervous System 54
1200 Genotoxicity 57
1210 Mutagenesis. 57
47
-------
1151 INTEGRATED SYSTEM: NEUROBEHAVIORAL TOXICOLOGICAL ASSESSMENT
Biological Activity Detected: Toxicity.
Principle: A battery of tests are being implemented which evaluate
various aspects of CNS functions.
Endpoint's: Qua I itati ve: Evaluate CNS Function. Qua I i tati \/e:
Thesa include: locomotor activity, neuromotor function, CNS
excitability, learning and memory.
Strengths: Provides rapid and sensitive broad spectrum evaluation
of CNS function.
Weaknesses: Requires skilled personnel; Real possibility of false
negatives.
Status of Development: Validated.
Describe: This battery has been implemented and is currently
being validated using known neurotoxins.
Applications: Multimedia.
Samples: Pure ChemicaIs: Heavy metal; Pesticides, Nonionizing
radifilm. Complex Mixtures: N/A.
Duration: Test: 2 weeks, assuming acute exposure; Analysis: 3 weeks.
Cost: $2,000/sample, assuming acute exposure.
Interpretation: The profile of change in the various functional
tests provide information on neurotoxicity of test compounds.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology Divi-
sion, Neurobiology Branch.
Persons to Contact: L. Reiter, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2671).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Reiter, L., et al. Residential Maze. Envir. Hlth.
Perspect., 12:119-123, 1975. 2) Archer, J. Anim. Behav., 21:205-
235, 1973. 3) Dunham, N.W., and T.S. Miya. Roto Rod. J. of
Amer. Pharmaceu. Asso., 46(3):208-209, 1957. 4) Gait, S., R.
Rushton, and H. Stellberg. Anim. Behav. and Drug Action, 207-
223, 1964. 5) Tremors, R.R., G.K. Chalmers, and W. Yim. Proc.
Soc. Exp. Biol. Med., 109:202-205, 1962. 6) Hornston, M. Startle
Reflex. Physio, and Rev., 3:839-844, 1968. 7) Reiter, L., et al.
Passive Avoidance Test. Toxlcol. and Appl. Pharm., 25:582-588,
1973. 8) Miczek, K., and H. Barr, II. Social Behavior. Behav.
PharmacoL, 176-257, 1976.
48
-------
1152 INTEGRATED SYSTEM: THE EFFECTS OF SELECTED ORGANIC CONTAMINANTS IN
DRINKING WATER ON THE FUNCTIONS OF THE REPRODUCTIVE, NERVOUS, AND IMMUNE
SYSTEMS
Biological Activity Detected: Reproductive, immune-, neuro-, and
behavioral toxicity; Mutagenicity.
Principle: Determine effects of experimental exposure to certain
selected organic contaminants on: I) Immune system and host re-
sistance capabilities; 2) NeurochemicaI processes and dynamics
in the brain; 3) Behavior; 4) Male reproductive function (includ-
ing dominant lethal mutagenicity assay); 5) General toxicity end-
points (lethality, body-organ weights, hematology, etc.).
Endpoints: Qua Ii tat i ve: N/A. Quantitative: I) Immune response and
host resistance: Humoral - serum antibody production to S. aureus;
Cell mediated - response to C. parvum; RES activity - global pha-
gocyt. index (vase, clearance) and tissue distribution of 14C S.
aureus; Susceptibility to pathogens (bact., virus, fungus) and
transplant tumor; 2) Brain neurochemistry and dynamics: In-vivo
and in-vitro (synapfosome) systems; Endogenous levels, uptake, re-
lease, metabolism...in Norepinephrine, dopamine, serotonin and
AcCh systems; 3) Behavioral toxicology: Operant behavior - sched-
uled and learned performance; Learning ability; Behavior develop-
ment; Other - elem. screen, spont. mot. activ., visual, swim,
maze, etc; 4) Reproductive performance and dom. lethal mutagen.
asso.: Antiferti Iity, reversibility, mutagenic potential, mode
of action, penetration of BT barrier (in-vivo); Penetration mech-
anism, spermatic nucleo/protein synth. spermatic uptake (in-vitro);
5) Preliminary and range finding shorter term toxicology: Leth-
ality, body/organ weights, hematology, etc.
Strengths: Provides much information on several toxicologic aspect areas in
coordinated manner; Readiness reduces time turnaround.
Weaknesses: Expensive.
Status of Development: Being implemented.
Describe: The tests are being implemented. Some are still
developmental, but most are validated. Current work applicable
to and aimed at drinking water contaminants, but techniques and
inferences applicable to other media.
Applications: Water.
Samples: Pure ChemicaIs: Trihalomethanes; Benzenes; Ethers;
Phenyls; Pesticides; PCB; Dioxin; 2, 4-DNT; (Many chemicals, but
not all are under test by a I I the test systems under present
project). Complex Mixtures: Not now under test but could be
implemented.
Duration: Existing grants, about 2 years, covering multiple chemical;
Individual tests Involve short (I to several days) to long-term
(3 months) exposures.
Cost/sample or chemical: Approx. $120,000 for the entire integrated
muItidiscipIinary workup involving multiple dose levels and ex-
posure periods lasting up to a year for completion of all phases.
However, application of selected portions, reduced exposures or
fewer dose levels, etc., would be less expensive.
-------
1152 INTEGRATED SYSTEM: THE EFFECTS OF SELECTED ORGANIC CONTAMINANTS IN
DRINKING WATER ON THE FUNCTIONS OF THE REPRODUCTIVE, NERVOUS, AND IMMUNE
SYSTEMS (continued)
Interpretation: Toxicity interpreted in terms of potential human
health hazards.
Level of Complexity: Complexity levels range from 3 to 5.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Toxicologic Assessment Branch.
Persons to Contact: K.I. CampbelI, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7481).
Grant/Contract Laboratory Involved and Principal Investigators:
Medical College of Virginia, Richmond, VA 23298: J.F. Borzelleca,
Project Manager (Coordinator) and Reproduction Studies; A.E.
Munson, Immune Systems, etc.; R.L. Balster, Behavioral Toxico-
logy; W.L. Dewey, Neurochemistry.
Program Office Support: ORD.
References: I) Grant document and report: R80470I. 2) Grant docu-
ment and report: R804290. 3) Deichmann, W., et a I. Toxicol. and
Appl. Pharmacol., 5:201, 1963. 4) Ball, H. J. Nat. Can. Inst.,
44:1070, 1966. 5) Szakol, L., and H. Hanna. Nat. Can. Inst.
Monographic., 35:173, 1972 6) For additional related references,
contact investigator.
50
-------
1153 COMPUTER AUTOMATED ANALYSIS OF PATTERNED BEHAVIOR IN THE PRIMATE
Biological Activity Detected: Toxicity.
Principle: Animal behavior is structured (patterned) and chemicals
which affect CMS function will disrupt this patterned behavior.
Endpoints: Using closed-circuit TV and on-line computing, various
components of primate behavior are defined and measured for
frequency, duration, and sequences. Qualitative: N/A.
Quantitative: Frequency of various motor items in experimental
period; Duration of various motor items per occurrence; Sequencing
of behavior.
Strengths: Should provide sensitive index of neurotoxicity which is
fully automated and applicable to a wide range of pollutant test-
ing; Also no pre-training is required.
Weaknesses: Expensive; Specialized equipment is required.
Status of Development: Developmental.
Describe: System has been developed and is being tested with
known psychoactive drugs.
Applications: Multimedia.
Samples: Pure ChemicaIs: Heavy metals. Complex Mixtures: N/A.
Duration: Test: I week, assuming acute exposure; Analysis: 2 weeks.
Cost: $200/subject.
Interpretation: Changes in frequency, duration, or patterning of
behavior are indicative of neurotoxicity.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology
Division, Neurobiology Branch.
Persons to Contact: L. Reiter, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2671).
Grant/Contract Laboratory Involved and Principal Investigators:
Iowa State U., Ames, IA 50010, W.E. Lloyde.
Program Office Support: OEM I.
References: I) Norton, S. Physiol. Behav., 11:181-186, 1973. 2)
Norton, S. J. Theoret. Biol., 21:229-243, 1962.
51
-------
1154 COMPUTER AUTOMATED ANALYSIS OF LEARNING AND MEMORY IN THE PRIMATE
Biological Activity Detected: Toxicity.
Principle: Chemicals affecting CNS function may interfere with the
learning process. The fact that primates rely heavily on the
visual sensory modality is utilized to determine pollutant
effects on visual discrimination learning.
Endpoints: Qua I 1tative: N/A. Quantitative: Two choice non-spatial
visual discrimination tasks examine animals' ability to distin-
guish between different visual patterns; Delayed response task:
evaluates memory coordination utilizing visual stimuli.
Strengths: Provides system index of chemical effects on learning
and memory; Provides information on behavioral effects in primates.
Weaknesses: Expensive; Requires skilled personnel to perform test;
Used only for toxicity testing of pure compounds when specific
information on primates is required.
Status of Development: Developmental.
Describe: System has been developed and is being tested with
psycho-active drugs.
Applications: Multimedia.
Samples: Pure ChemicaIs: Heavy metals. Complex Mixtures: N/A.
Duration: Test: 6 weeks, assuming acute exposure; Analysis: 2 weeks.
Cost: $200/subject.
Interpretation: Alterations in performace are indicative of neuro-
toxicity.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology Division,
Neurobiology Branch. '
Persons to Contact: L. Reiter, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2671).
Grant/Contract Laboratory Involved and Principal Investigators:
Iowa State U., Ames, IA 50010, W.E. Lloyde.
Program Office Support: OEMI.
References: I) Fletcher, H.J. The Delayed-Response Problem. In:
Behavior of Nonhuman Primates, A.M. Schrier, H.F. Harlow, and
F. Stollnitz, eds. Academic Press, New York, 1965. pp. 129-
165. 2) Miles, R.C. Discrimination-Learning Sets. In: Be-
havior of Nonhuman Primates, A.M. Schrier, H.F. Harlow, and
F. Stollnitz, eds. Academic Press, New York, 1965. pp. 53-54.
52
-------
1155 INTEGRATED SYSTEM: BEHAVIORAL ANALYSIS OF RATS -DEVELOPMENTAL,
LOCOMOTOR, EXPLORATORY, AND LEARNING BEHAVIOR
Biological Activity Detected: Toxicity; Behavioral.
Principle: Various levels of lead are known to affect the maturation
of energy metabolism in the cerebral cortex of rats. Studies
have shown that delays in the development of the nervous system
may affect behavioral responses observable in both the young and
adult animal. Specific affects need to be tested on the above
Ii sted i ndices.
Endpoints: Qua Iitative: Expression of learned behavior through use
of a water T-Maze - percentage of correct trials and amount of
time to reach criteria; Expression of locomotor activity and
exploratory behavior through the use of a Berylene Box - primarily
a measurement of frequency and duration spent on each parameter;
Expression of developmental behavior through locomotor activity
and use of ultrasonics - the measurement of general activity pat-
terns and monitoring of communication abilities dependent upon
development. Quantitative: Maze activity; Benylene box; General-
ized and specific locomotor activity; Frequency and duration of
ultrasonic vocalizations.
Strengths: Should provide sensitive behavioral tests which can then
be correlated with physiological data collected in previous
studies.
Weaknesses: Requires specialized instrumentation for testing; Time-
consuming to run the battery of tests for each designated dosage
I eve I.
Status of Development: Being implemented.
Describe: Most of the instrumentation has been constructed and
subjects are being treated with designated lead doses.
Applications: Water.
Samples: Pure ChemicaIs: Trace metals, Organic chemicals.
Complex Mixtures: Industrial; Energy Related.
Duration: I to 3 months.
Cost: Not yet established.
Interpretation: Altered physiological or neurological function as a
result of treatment chemical would indicate toxicity.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Toxicological Assessment Branch.
Persons to Contact: R.J. BuI I, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OWHM.
References: I) Relter, L.W., et a I. Envir. Hlth. Perspect., 12:119-
123, 1975. 2) Sobotka, T.J., et al. Toxicol., 5:175-191, 1975.
3) Brown, D.R. Toxicol. Appl. Pharmacol., 32:628-637, 1975. 4)
Padich, R., et al. Pharmacol. Biochem. Behav., 6:371-375, 1977.
5) Fox, D.A., etal. Toxicol. Appl. Pharmacol., 40:449-461, 1977.
6) Robblns, T., et al. Psychopharmacologia, 28:155-164, 1972.
7) Arose I, A., etal. Science, 197:786-788, 1977.
53
-------
1156 INTEGRATED SYSTEM: INSTRUMENTAL METHODS OF DETECTING FUNCTIONAL AND
METABOLIC DAMAGE TO THE NERVOUS SYSTEM
Biological Activity Detected: Toxicity.
Principle: Increased functional activity of a tissue requires energy.
Consequently, if a tissue's functional activity is stimulated,
ATP is hydrolyzed to ADP and P., which in turn stimulates oxida-
tion of substrate and resynthesis of ATP. These metabolic changes
may be observed as increases in oxygen consumption, substrate
utilization, or as metabolic transients induced in the electron
carriers directly in tissues, in-vitro. The kinetics of these
metabolic responses to stimulation have been shown sensitive to
a wide variety of chemical agents with varying mechanisms of
action with both in-vitro and in-vivo treatments.
Endpoints: To this point in time, an endpoint has only been developed
for brain tissue. Responses are measured in response to electrical
pulses (10 s) or elevation in K concentrations (3 to 30 mM).
Qua Iitative: N/A. Quantitative: Transient redox changes in
NAD(P)H, fp, cyt a, b, c. ; Substrate utilization; Oxygen con-
sumption; Lactic acid output; Neurotrarvsmitter release; Amino
acid metabolism.
Strengths: In-vitro results may be directly confirmed in-vivo with
the same parameters; Applicable to a wide variety of mechanisms;
Applicable to all aerobic tissues; Involves measurement of the
kinetics of going from a resting to an excited state rather than
the steady state greatly increasing sensitivity; Applicable to
very small tissue samplers (2 to 3 mg).
Weaknesses: Does not lend itself to immediate identification of
mechanisms unless there is a direct effect on energy metabolism
proper.
Status of Development: Validated.
Describe: The test system has been validated with a wide variety
of inhibitors of energy metabolism and membrane active compounds.
In-vitro and in-vivo treatments with lead, methyl mercury, and
a Iky I at ion compounds indicate equivalent or more sensitive measures
of effect than other parameters which have been applied to these
probI ems.
Applications: Multimedia.
Samples: Pure ChemicaIs: All classes. Complex Mixtures : Indus-
trial; Energy Related; Transportation Related.
Duration: 2 weeks to 3 months.
Cost: Not yet established.
Interpretation: This is a general system for determining neurotoxicity
It is capable of detecting non-specific damage to a variety of
systems (e.g., decreased membrane excitability, altered responses
to neurotransmitters, direct effects on energy metabolism). It
has also been used to detect delays in brain development, which
were subsequently confirmed by morphologic and behavioral methods.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxicologicaI Assessment Branch.
54
-------
1156 INTEGRATED SYSTEM: INSTRUMENTAL METHODS OF DETECTING FUNCTIONAL AND
METABOLIC DAMAGE TO THE NERVOUS SYSTEM (continued)
Persons to Contact: R.J. Bui I
St., Cincinnati,
OH 45268,
Grant/Contract Laboratory Involved
In-house.
Program Office Support: OHEE.
and
U.S. EPA, HERL-CIN, 26 W. St. Clair
(FTS 684-7213).
and Principal Investigators:
References: I) Bull, R.J., and A.J
1009, 1972. 2) Bui I, R.J., and
101 1-1022, 1972. 3) Cummins, J
, Trevor. J
A.J. Trevor. J,
,T., and R. BuI I
Neurochem., 19:999-
Neurochem., 19:
Biochem. Bio-
phys. Acta, 253:29-38, 1971. 4) Bull, R.J., and J.T. Cummins.
and S.D. Lutken^
I, R.J. J.
J. Neurochem., 21:923-937, 1973. 5) Bull
hoff. Neuropharmacol., 14:351-359, 1975.
Neurochem., 26:149-156,
J.J. O'NeiI I,
89-95, 1975.
R.J
6) Bui
1976. 7) Bull, R.J., P.M. Stanaszek,
and S.D. Lutkenhoff. Envir. Hlth. PerSpect., 12:
55
-------
CONTENTS: TEST SYSTEMS
1200 Genotoxicity 57
1210 Mutagenesis 57
1211 Salmonella typhimurium (Ames) 58
1212 Escherichia col i (WP2) 60
1213 Saccharomyces cerevi siae, Sch i zosaccharomyces
gombe, Forward and Reverse Mutation 61
1214 Body Fl u id Ana lysis 62
1215 Bacterial PI asm ids 63
1216 Mouse Lymphoma (L5I78Y) 64
1217 Chinese Hamster Ovary Cells (CHO) Drug
Resistance . 65
1218 Chinese Hamster Cells (CHO) Nutritional
Competency 66
1219 Chinese Hamster Lung Cells (V79) 67
121 10 Drosophila melanogaster, Sex Linked
Recessive Lethal 68
121 I I Tradescantia Stamen Hair 69
121 12 Maize Waxy Locus Assay 70
121 13 In-Vivo Cytogenetics in Mice 71
1220 DMA Damage 73
57
-------
1211 SALMONELLA TYPHIMURIUM (AMES)
Biological Activity Detected: Mutagenicity; Presumptive oncogenicity.
Principle: Histidine dependent strains of SaImoneI I a genet i caI Iy
engineered to increase their sensitivity and specificity, are
exposed to a test substance in the presence of mamma Iian meta-
bolic activating enzymes. The formation of bacterial colonies
in a histidine-free medium after treatment is considered a
criteria of the effectiveness of a test substance as a mutagen.
Endpoints: Qua Iitative: Spot test and plate incorporation test
are considered to be qualitative in nature. In spot tests no
attempt is made to count the number of colonies per plate. In
plate incorporation the number of colonies are counted but not
expressed as a fraction of survival. Quantitative: The induced
mutational frequencies may be expressed on the basis of units
of test material and survival in suspension.
Strengths: Genetically we I I-characterized system; Rapid; Inexpensive;
Well validated as a test for gene mutation; Works well with in-
vitro metabolizing microsome fractions; Can be used as indicator
organism in host-mediated assays.
Weaknesses: Reverse mutation assay requiring several strains to
permit detection of a broad spectrum of compounds; Requires
metabolic activation; Lacks pharmacological relevance; Prokaryotic
organization of genetic material.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure ChemicaIs: All major classes of chemicals except
metals and hormones. Con\p_ I ex Mi xt u re s : Ambient - air particu lates,
drinking water, and water concentrates; Industrial - effluents;
Energy Related - alternate effluents, shale; Transportation Re-
lated - auto/truck fuels; Other - human body fluids, extracts
from crops treated with sludge.
Duration: 3 weeks/study.
Cost: $300 to $650/compound for plate test; $1,000 to $ I,200/compound
for suspension test.
Interpretation: The growth of colonies in a hist idine-deficient
medium indicates genetic alteration.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section; HERL-CIN, Field
Studies Division, ToxicologicaI Assessment Branch; ERL-GB; National
Center for ToxicologicaI Research, Division of Mutagenesis,
Somatic Cell Section.
Persons to Contact: J_. L. Huisingh, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2948); L. Claxton, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2942);
M.D. Waters, U.S. EPA, HERL-RTP, Research Triangle Park, NC 27711,
(FTS 629-2693); J.P. Bercz, U.S. EPA, HERL-CIN, 26 W. St. Clair St.,
Cincinnati, OH 45268, (FTS 684-7432); H.R. Pahren, U.S. EPA,
HERL-CIN, 26 W. St. Clair St., Cincinnati, OH 45268, (FTS 684-
7217); L.J. McCabe, U.S. EPA, HERL-CIN, 26 W. St. Clair St.,
58
-------
1211 SALMONELLA TYPHIMURIUM (AMES) (continued)
Persons to Contact (continued): Cincinnati, OH 45268, (FTS 684-7211)
J.F. Stara, U.S. EPA, HERL-CIN, 26 W. St. Clair St., Cincinnati,
OH 45268, (FTS 684-7407); N. Richards, U.S. EPA, ERL-GB, Sablne
Island, Gulf Breeze, FL 32561, (FTS 686-9011); E. Lazear, NCTR,
Jefferson, AR 72079, (FTS 740-4573); D.A. Casciano, NCTR,
Jefferson, AR 72079, (FTS 740-4495).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA, V.F. Simmons; Litton
Biometics, Inc., Nicholson Lane, Kensington, MD, D.T. Brusick;
U. of Cincinnati Medical Center, J. Loper; Louisiana State U.
Medical School, W. Pelon; U. of Missouri, Columbia, MO, C.
Marianseld; U. of West Florida, J. Bazlls; U. of Texas, Medical
Branch, Galveston, TX 77550, M. Legator.
Program Office Support: OHEE; OPP; OEMI; OTS.
References: I) Ames, B.N., et al. Mutation Res., 31:347-364, 1975.
2) McCann, et a I. Proc. Natl. Acad. Sci., 70:782-786, 1975. 3)
McCann, and B.N. Ames. Proc. Natl. Acad. Sci., 73:950-954, 1976.
59
-------
1212 ESCHERICHIA COLI (WP2)
Biological Activity Detected: Mutagenicity.
Principle: Tryptophan dependent strains of Escherichia coli geneti-
cally engineered to increase their sensitivity and specificity,
are exposed to a test substance in the presence of mammalian
metabolic activating enzymes. The formation of bacterial colonies
in a tryptophan-free medium after treatment indicates the effec-
tiveness of a test substance as a mutagen.
Endpoints: Qua Iitative: Growth in a tryptophan-free medium.
Quantitative: N/A.
Strengths: Rapid; Inexpensive; Well validated test for gene mutation;
Works well with in-vitro metabolizing microsomal enzymes; Can be
used as indicator organism in host-mediated assays.
Weaknesses: Reverse mutation assay requiring several strains to
permit detection of a broad spectrum of compounds; Requires
metabolic activation; Lacks pharmacological relevance; Prokaryotic
organization of genetic material; Not as well characterized nor
as sensitive as SaImonella/microsome assay (1211). It detects
only base pair substitutions.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure ChemicaIs: All major classes of chemicals except
metals and hormones. Complex Mixtures: Ambient - air particulates,
drinking water, and water concentrates; Industrial - effluents;
Energy Related - alternate effluents, shale; Transportation Re-
lated - auto/truck fuels; Other - human body fluids, extracts
from crops treated with sludge.
Duration: 3 weeks.
Cost: $450/chemical.
Interpretation: The growth of tester strains in a tryptophan free
medium after treatment with a test substance indicates mutation.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch, Cellular Biology Section.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2693); L. Claxton, U.S. EPA, HERL-RTP,
Research Triangle Park, NC 27711, (FTS 629-2942); S.S. Sandhu,
U.S. EPA, HERL-RTP, Research Triangle Park, NC 27711, (FTS
629-2693); J.L. Huisingh, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2948).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, V.F. Simmons.
Program Office Support: OHEE; OPP.
References: I) Bridges, B.A. Lab Practice, 21:413-416, 1972.
2) Bridges, B.A., et a I. Mutation Res., 19:295-303, 1973.
3) Bridges, B.A., et aL Chem/Biol. Interactions, 5:77-84, 1972.
60
-------
1213 SACCHAROMYCES CEREVISIAE. SCHIZOSACCHAROMYCES POMBE, FORWARD AND REVERSE
MUTATION ~
Biological Activity Detected: Mutageniclty.
Principle: Gene mutation is detected by loss of function resulting
in nutritional requirements or resistance to toxic chemicals and
shift in color of cell colonies.
Endpoints: Qualitative: Growth in a selective medium; Change in
colony pigmentation. Quantitative: Mutation frequencies may be
adjusted for cytotoxic effects.
Strengths: Both forward and reverse mutation can be studied; Eukar-
yotic organization of genetic material; Fast; Relatively inexpen-
sive; Cells can be cultured as haploids; Fairly wide spectrum of
genetic events can be scored; Can be used as indicator organism
in host-mediated assays.
Weaknesses: Requires exogenous metabolic activation which has not
worked well with yeast systems; Lacks pharmacological relevance;
Chromosomes are too small to permit direct cytological observa-
tion; Relatively insensitive to some chemicals.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure ChemicaIs: Alkylating agents, Halogenated hydro-
carbons, Polycyclics, Carbamates. Complex Mixtures: Ambient -
water concentrated; Other - extracts from crops treated with
sludge, human body fluids.
Duration: 3 weeks.
Cost: $400 to $700/compound, depending on the test.
Interpretation: The appearance of pigmented colonies and growth in
selective mutation after treatment with test compound indicates
mutation.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch, HERL-CIN, Field Studies Division,
ToxicologicaI Assessment Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
J.P. Bercz, U.S. EPA, HERL-CIN, 26 W. St. Clair St., Cincinnati,
OH 45268, (FTS 684-7432); H.R. Pahren, U.S. EPA, HERL-CIN, 26 W.
St. Clair St., Cincinnati, OH 45268, (FTS 684-7217).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, V.F. Simmons.
Program Office Support: OHEE; OPP; OEMI.
References: I) Zimmermann, F.K. In: Chemical Mutagens: Principles
and Methods for Their Detection. Vol. 3. A. Hoilaender, ed.
Plenum Press., NY, 1973. pp. 209-239. 2) Parry, J.M. Muta-
tion Res., 46(3):165-176, 1977. 3) Brusick, D.J., and V.W.
Mayer. Envir. Hlth. Perspect., 6:83-96.
61
-------
1214 BODY FLUID ANALYSIS
Biological Activity Detected: Mutagenicity.
Principle: Promutagens which need mammalian metabolic activation
are biotransformed in the intact animal and are tested for muta-
genic activity in Salmonella, yeast, and Chinese hamster ovary
eel I test systems.
Endpoints: Qua Iitative: Appearance of revertant colonies in a
selective medium. Quantitative: The number of prototrophic
colonies in a histidine deficient medium, adjusted for cytotoxic
effects of the test chemical.
Strengths: Combines in-vivo metabolic activation with the in-vitro
microbial test system.
Weaknesses: Limited number of bacteria exposed; Recovery of bacteria
is problematic; Exposure time has not been standardized; Difficult
to quantitate the response.
Status of Development: Being implemented.
Describe: The basic experimental procedure for pure chemicals
has been established. But protocol for testing for mixtures has
not been developed. Furthermore, the test system needs valida-
tion by testing a wide variety of chemicals.
Applications: Multimedia.
Samples: Pure ChemicaIs: Mycotoxins, Nitrosamines, Aromatic
amines, Aromatic hydrocarbons. Complex Mixtures: Not yet tested.
Duration: 4 weeks.
Cost: $1,000 to $l,200/chemical.
Interpretation: The positive response indicates the ability of the
test chemical to be transformed into reactive electrophile by the
intact mammalian metabolic activation system.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch; HERL-CIN, Field Studies Division, Toxicolo-
gical Assessment Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
L. Claxton, U.S. EPA, HERL-RTP, Research Triangle Park, NC 27711,
(FTS 629-2942); J.F. Stara; U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7407).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Texas, Medical Branch, Galveston, TX 77550, M. Legator.
Stanford Research Institute, Menlo Park, CA 94205, V.F. Simmons.
Program Office Support: OHEE; OPP; OEMI.
References: I) Legator, M., et a I. Mutation Res. 26:456, 1974.
2) Legator, M., et al. In: Chemical Mutagens: Principles and
Methods for Their Detection. Vol. 4. A. Hollander, ed. Plenum
Press, NY, 1976. pp. 171-190.
62
-------
1215 BACTERIAL PLASMIDS
Biological Activity Detected: Mutagenicfty.
Principle: Purified pi asm id DMA is exposed to potential mutagen in
a cell-free system and is then analyzed for ability to infect a
host bacterium.
Endpoints: Qualitatlve: Ability of treated plasmid DMA to infect
bacterial cells. Quantitative: The number of colonies produced
per unit of test materials.
Strengths: May avoid problems of extreme cytotoxicity of many
chemicals since the exposure is in a cell-free system; Rapid;
EconomicaI.
Weaknesses: Problems with exposing purified DMA; Still early in
developmental stage; Requires exogenous metabolic activation
systems.
Status of Development: Developmental.
Describe: Initial pilot work with MNNG is encouraging.
Applications: Multimedia.
Samples: Pure ChemlcaIs: Organics. Complex Mixtures: Ambient;
Industrial; Energy Related; Transportation Related; Other.
Duration: I to 2 days.
Cost: Undetermined.
Interpretation: Loss of infectivity suggests damage to DMA.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxicologicaI Assessment Branch.
Persons to Contact: N. Clarke, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7411); B. Daniel, U.S.
EPA, HERL-CIN, 26 W. St. Clair St., Cincinnati, OH 45268, (FTS
684-7482).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: Not yet available.
63
-------
1216 MOUSE LYMPHOMA (L5178Y)
Biological Activity Detected: Mutagenicity. +,_
Principle: Forward mutation from thymidjne kinase competency (TK )
to thymidine kinase incompetency (TK ), resulting in resistance
to the base analogs BUdR or TFT.
Endpoints: Gene mutation. Qua Iitative: Formation of eel I colonies
in a selective medium. Quantitative: Induced mutation frequencies
are based on cell survival and cloning efficiency.
Strengths: Both forward and reverse mutation can be measured; Cell
will grow in suspension culture; Cells have short generation time;
Cells have stable, near-diploid chromosome number; High plating
efficiency; High recovery of mutant cells.
Weaknesses: Mutation measured at a single locus; Requires metabolic
activation; Requires additional validation; Lacks pharmacological
relevance; PPLO contamination is a serious problem and the cells
must be continuously monitored to ensure that they are PPLO-free.
Status of Development: Being implemented.
Describe: Problems of expression time and metabolic activation
have yet to be resolved. This bioassay has been contracted out
for validation by the National Cancer Institute.
Applications: Multimedia.
Samples: Pure ChemicaIs: Alkylating agents, Halogenated hydro-
carbons, Inorganic derivatives, N-Nitroso compounds, Metals,
Mycotoxins. Complex Mixtures: Ambient; Industrial.
Duration: 3 weeks.
Cost: $3,000/compound.
"Interpretation: Growth of heterozygous thymidine competent cells in
a medium containing TFT or BUdR suggests mutation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch, Cellular Biology Section.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2693); S.S. Sandhu, U.S.
EPA, HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-
2693); M.M. Brown, U.S. EPA, HERL-RTP, Research Triangle Park,
NC 2771 I, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, A. Mitchell.
Program Office Support: OHEE; OPP; OTS.
References: I) Clive, D., and J.F.S. Spector. Laboratory Procedure
for Assessing Specific Locus Mutations at the TK Locus in Cul-
tured L5I78Y Mouse Lymphoma Cells. Mutation Res., 31:17-29,
1975.
64
-------
1217 CHINESE HAMSTER OVARY CELLS (CHO) DRUG RESISTANCE
Biological Activity Detected: Mutagenicity; Presumptive oncogenicity.
Principle: Forward mutation assay measuring drug resistance at hypo-
xanthine-guanine-phosphoribolyltransferase (HGPRT) locus. The
HGPRT competent cells in presence of mammalian metabolic activa-
tion enzymes are exposed to a test substance. Induced frequency
of HGPRT deficiency is determined using a selective medium
containing base analogue 8-azaguanine (8 AZ) or 6-thioguanine
(6 TH).
Endpoints: Gene mutation. Qua Iitative: Formation of eel I colonies
in a selective medium. Quantitative: Induced mutation fre-
quencies are based on cell survival and cloning efficiency.
Strengths: Mammalian organization of genetic material; Forward muta-
tion assay; Fast generation time; Stable karyotype; Easy to cul-
ture; May be particularly well-suited to quantitation as HGPRT
locus mutants may not show replicative advantages/disadvantages
over wild-type cells under nonselective conditions.
Weaknesses: Mutation measured at a single locus; Requires metabolic
activation; Needs additional validation; Lacks pharmacological
relevance; Fairly high spontaneous mutation rate; Long optimal
expression period (7 days).
Status of Development: Being implemented.
Describe: Basic system with metabolic activation has been de-
scribed. Several compounds representing diverse classes of
chemicals have been tested. However, additional chemicals by at
least two laboratories have to be tested before it could be
considered as validated.
Applications: Multimedia.
Samples: Pure ChemicaIs: Alkylating agents, Nitrosamines, Organics,
PNA's, Metallic compounds. Complex Mixtures: Not yet determined.
Duration: 3 weeks/compound.
Cost: $2,000 to $3,000/compound, including dose-response.
Interpretation: Growth of HGPRT competent cells in a medium contain-
ing 8 AZ suggests mutation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch; National Center for ToxicologicaI Research,
Division of Mutagenesis, Somatic Cell Section.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2693); S.S. Sandhu, U.S. EPA, HERL-RTP,
Research Triangle Park, NC 27711, (FTS 629-2693); D.A. Casciano,
NCTR, Jefferson, AR 27079, (FTS 740-4495).
Grant/Contract Laboratory Involved and Principal Investigators:
Oak Ridge National Laboratory. Biology Division, P.O. Box Y,
Oak Ridge, TN 37830, A. Hsie.
Program Office Support: OHEE.
References: I) O'Neill, P.J., et al. Mutation Res., 45:91-101, 1977.
2) O'Neill, P.J., et al. Mutation Res., 45:103-109, 1977.
65
-------
1218 CHINESE HAMSTER CELLS (CHO) NUTRITIONAL COMPETENCY
Biological Activity Detected: Mutagenicity.
Principle: Isolation of auxotrophic mutants using 5-bromodeoxyuridine
(BUdR) and visible light as selective agents.
Endpoints: Gene and chromosomal mutation. Qua I Itative: Formation
of auxotrophic cell colonies. Quantitative: Can be used to
determine induced mutation frequencies based on cell survival
and cloning efficiency.
Strengths: System is clean (survivors are either auxotrophs or they
are not); Several loci are available for detection of genetic
changes; Forward mutation assay with very low spontaneous muta-
tion frequencies at available loci; Test populations can be
easily freed of spontaneous auxotrophs by growing cells three
days in minimal medium; Mammalian system in terms of organization
of genetic materials.
Weaknesses: Assay very insensitive (95% of auxotrophs lost during
selection to effects of starvation); Mutant identification is
tedious and not amenable to screening; Experiments require five
weeks to complete; Requires metabolic activation; Requires valida-
tion; Lacks pharmacological relevance; Nutritional mutants appear
to be at a replicative disadvantage in mixed populations under
nonselective conditions.
Status of Development: Developmental.
Describe: Improvement of technique is necessary for increased
sensitivity. Also necessary is the addition of an in-vitro meta-
bolic activation.
Applications: Multimedia.
Samples: Pure ChemicaIs: Alkylating esters, Base analogs, Nitro-
samines, Acridine, Mustards, Heavy metals, Hydroxylamine.
CompI ex Mixtures: Energy Related - petroleum oil extracts;
Transportation Related - jet fuel extracts; Other - ultraviolet
radiation, x-rays.
Duration: 5 weeks.
Cost: $2,000/assay.
Interpretation: Test agents inducing significant numbers of auxotrophs
may be regarded as potential mutagens/carcinogens for animals and
man.
Level of Complexity: 4 to 5.
OHEE Laboratory Involved: ERL-NAR, Toxicology Branch, Genetic Toxi-
cology Team.
Persons to Contact: A.R. Malcolm, U.S. EPA, ERL-NAR, South Ferry Rd.,
Narrangansett, Rl 02882, (FTS 838-4843 X247 or X238).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: 1) Kao, F.T., and T.T. Puck. Induction and Isolation
of Auxotrophic Mutants in Mammalian Cells. In: Methods in Cell
Biology. Vol. 3. D. Prescott, ed. Academic Press, NY, 1974.
pp. 23-39.
66
-------
1219 CHINESE HAMSTER LUNG CELLS (V79)
Biological Activity Detected: Mutagenicity.
Principle: Forward mutation assay measuring drug resistance at;hypo-
xanthine-guanine-phosphoribolyltransferase (HGPRT) locus. The
HGPRT competent cells in presence of mammalian metabolic activa-
tion enzymes are exposed to a test substance. Induced frequency
of HGPRT deficiency is determined by using a selective medium
containing base analogues 8-azaguanine (8 AZ).
Endpoints: Gene mutation. Qualitative; Formation of cell colonies
in a selective medium. Quantitative: Induced mutation fre-
quencies are based on cell survival and cloning efficiency.
Strengths: Mammalian organization of genetic material; Forward muta-
tion assay; Fast generation time; Stable karyotype; Easy to
culture; May be particularly well-suited to quantitation as
HGPRT locus mutants may not show replicative advantages/disad-
vantages over wild-type cells under nonselective conditions.
Weaknesses: Mutation measured at a single locus; Requires metabolic
activation; Requires additional validation; Lacks pharmacological
relevance; Fairly high spontaneous mutation rate (I 5 x 10 6);
Long optimal expression period (7 days).
Status of Development: Being implemented.
Describe: Needs additional validation.
Applications: Multimedia.
Samples: Pure Chemicals: N-Nitroso compounds, Alkylating agents,
Primarily organics mixtures. Complex Mixtures: Ambient; Indus-
trial; Energy Related.
Duration: 4 weeks.
Cost: $3,000.
Interpretation: Growth of the treated HGPRT competent cells in a
selective medium suggests genetic change.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemical Branch; HERL-CIN, Field Studies Division, Toxicologi-
caI Assessment Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
N.E. KowaI, U.S. EPA, HERL-CIN, 26 W. St. Clair St., Cincinnati,
OH 45268, (FTS 684-7477).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, A. Mitchell;
Gulf South Research Institute, P.O. Box 26518, New Orleans, LA
70186, N. Gruener.
Program Office Support: OHEE.
References: I) Krahn, D.F., and C. Heidelburger. Proc. Natl. Acad.
Sci., 73:188-192, 1977. 2) Artlett, et al. Mutation Res., 33:
261-278, 1975.
67
-------
12110 DROSOPHILA MELANOGASTER, SEX LINKED RECESSIVE LETHAL
Biological Activity Detected: Mutagenicity.
Principle: Wild type males are treated with the test chemical and
mated with untreated females with a marked chromosome. r-L females
are sib-mated and the progeny are scored for the presence of
x-I inked recessive lethals.
Endpoints: Qua Iitative: Change in sex ratios in F2 generation.
Quantitative: Point mutations and small delect ions may be scored
in germ eel Is.
Strengths: Higher organism, genetically well characterized; Multiple
loci available for detection of genetic alterations; Some metabolic
processes similar to that of mammals; Small number of large chromo-
somes; Broad spectrum of genetic events can be detected and
scored; Wealth of mutant strains makes possible detailed analysis
of induced genetic changes.
Weaknesses: Short life span makes organism unsuitable for chronic
exposure studies; Limited use for testing pesticides due to ex-
treme toxicity.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure ChemicaIs: Alkylating agents, Nitrosamines,
Halogenated ethers. Complex Mixtures: Protocols not yet devel-
oped.
•Duration: 4 to 6 weeks.
Cost: $4,000 to $6,500.
Interpretation: Change in the sex ratio in the F£ progeny of the
test population indicates mutation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2693); S.S. Sandhu; U.S. EPA, HERL-RTP,
Research Triangle Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, MenloPark, CA 94205, A. Mitchell.
Program Office Support: OHEE.
References: I) Vogel, E., and F.H. Sobels. The Function of Drosophila
in Genetic Toxicology Testing. In: Chemical Mutagens: Principles
and Methods for Their Detection. Vol. 4. A. Hollaender, ed.
Plenum Press, NY, 1976. pp. 93-132. 2) Sobels, F.H., and E.
Vogel. The Capacity of Drosophi la for Detecting Relevant Genetic
Damage. Mutation Res., 41:95-106, 1976. 3) Legator, M.S., and
S. Zimmering. Gen. Toxicol. Ann. Rev. Pharmacol., 387-408, 1975.
68
-------
12111 TRADESCANTIA STAMEN HAIR
Biological Activity Detected: Mutagenicity.
Principle: Mutation in petals and stamen hair in clones heterozygous
for flower color is detected as a change in pigmentation.
Endpoints: Qua Iitative: Change in stamen hair cells' color from
blue to pink. Quantitative: Mutational events per stamen hair
and dose-response relationship can be established.
Strengths: Can detect broad spectrum of genetic events; Can be used
to monitor in situ environment; Can detect mutagens in the gaseous
phase; Eukaryotic organization of genetic material; Many muta-
tions 1 events can be observed directly; System appears highly
sensitive to physical and chemical mutagens.
Weaknesses: Lacks pharmacological relevance; May not be suitable
for evaluating many compounds requiring mammalian metabolic
activation.
Status of Development: Developmental.
Describe: The Tradescantia system was initially developed (and
is well-suited) for study of radiation effects. The system is
applicable to at least some chemical mutagens and is currently
under development for that purpose.
Applications: Air; Water.
Samples: Pure Chemicals: Organics, Nitroso derivatives, Poly-
el ichydrocarbons, Nucleic acid bases, and analogs, Hydroxyl-
amines, Hydrasine. Complex Mixtures: Ambient - drinking water,
a i r; Industrie I - soi i.
Duration: 2 to 5 weeks.
Cost: $500 to $700, depending upon the test protocol.
Interpretation: The change in stamen hair cells' color from blue to
pink suggests mutagenicity.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section; HERL-CIN, Field
Studies Division, ToxicologicaI Assessment Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
L.J. McCabe, U.S. EPA, HERL-CIN, 26 W. St. Clair St., Cincinnati,
OH 45268, (FTS 684-721 I).
Grant/Contract Laboratory Involved and Principal Investigators:
Brookhaven National Laboratories, Long Island, New York, L. Shirer;
U. of Missouri, Columbia, MO 65201, J.T. O'Connor; Stanford
Research Institute, Menlo Park, CA 94205, G. Newell.
Program Office Support: OHEE; OPP.
References: I) Sparrow, A.H., et al. Mutation Res., 26:265-276,
1974. 2) Underbrink, A.G., et al. In: Chemical Mutagens:
Principles and Methods for Their Detection. Vol. 3. A. Hollaender,
ed. Plenum Press, NY, 1973. pp. 171-207. 3) McNulty, P.J.,
et al. Mutation Res., 44:235-246, 1977.
69
-------
12112 MAIZE WAXY LOCUS ASSAY
Biological Activity Detected: Mutagenicity.
Principle: The assay is based on the change from the ability of the
plants to synthesize amylose to the inability to synthesize this
compound. Pollen from the treated plants is stained with iodine.
Mutated pollen grains are stained purple.
Endpoints: Qua Iitative: Change in pollen grain color from yellow
to purple. Quantitative: Induced mutation frequency is expressed.
Strengths: Test is performed in-vivo representing relevant conditions
of exposure; Damage to germ cells is measured.
Weaknesses: Time consuming.
Status of Development: Developmental.
Describe: Few pure compounds or mixtures have been tested in
this system. This test system appears promising but needs
va I idation.
Applications: Air; Soil; Multimedia.
Samples: Pure Chemicals: Pesticides. Complex Mixtures: Industrial.
Duration: 8 weeks.
Cost: Unknown.
Interpretation: Change in pollen grain color after staining suggests
mutation.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Illinois, Urbana, IL 61801, M. Plewa.
Program Office Support: OPP.
References: I) Plewa, M., and J. Gentile. Maize Cooper. Newsletter,
50:44, 1976. 2) Plewa, M., and J. Gentile. Mutation Res.,
38:287-292, 1976.
70
-------
12113 IN-VIVO CYTOGENETICS IN MICE
Biological Activity Detected: Mutagenicity.
Principle: Chemicals are administered into mice through various
routes. After a specific period, treated animals are sacrificed.
Bone marrow and spermatogonial cells are analyzed for chromosomal
aberrations.
Endpoints: Qua Iitati ve : Observation of chromosomal and chromatid
breaks. Quantitative: Number of breaks/cell as compared to the
control, provides a quantitative assessment of treatment response.
Strengths: In-vivo bioassay provides the benefit of intact pharmo-
kinetics.
Weaknesses: Expensive; Requires a well trained personnel to perform
the test and interpret the data.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure ChemicaIs: A Iky I ating agents. Complex Mixtures:
Protocol not yet developed.
Duration: 3 weeks.
Cost: $3,000 to $6,500/chemicaI.
Interpretation: A significant increase in chromosomal aberrations
over the control suggests mutation.
Level of Complexity: 5.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section; HERL-CIN, Field
Studies Division, ToxicologicaI Assessment Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
L. Claxton, U.S. EPA, HERL-RTP, Research Triangle Park, NC
27711, (FTS 629-2942); J.F. Stara, U.S. EPA, HERL-CIN, 26 W. St.
Clair St., Cincinnati, OH 45268, (FTS 684-7407).
Grant/Contract Laboratory Involved and Principal investigators:
U. of Texas, Medical Branch, Galveston, TX 77550, M. Legator;
Stanford Research Institute, Menlo Park, CA 94205, G. Newell.
Program Office Support: OHEE, OPP.
References: I) Evans, H.J. In: Chemical Mutagens: Principles and
Methods for Their Detection. Vol. 4. A. Hollander, ed. Plenum
Press, NY, 1977. 2) Schmid, W. In: Chemical Mutagens:
Principles and Methods for Their Detection. Vol. 4. A. Hollander,
ed. Plenum Press, NY, 1977. pp. 31-52.
-------
CONTENTS: TEST SYSTEMS
1220 DNA Damage 73
1221 Baci I I us subti I Is rec~ 74
122! Escherichia col i, Pol A~ 75
1223 Mitotic Recombination and Gene Conversion
i n Saccaromyces cerevisiae 76
1224 Unscheduled DNA Synthesis (UDS) 77
1225 Sister-Chromatid Exchange Formation (SCE) .... 78
1226 In-Vivo Assessment of DNA Damage 79
1227 Intact Rodent Hepatocytes in Primary
Culture 80
1228 In-Vivo DNA Binding 81
1229 Chinese Hamster Cells (CHO) Unscheduled
DNA Synthesis (UDS) 82
1230 Carcinogenesi s 83
73
-------
1221 BACILLUS SUBTILIS REC"
Biological Activity Detected: Toxicity; Primary DNA damage.
Principle: The DNA recombinational repair deficient and proficient
strains are streaked out along the intersecting lines. The test
chemical is spotted at the intersection. The differential killing
between the repair deficient and proficient strains is used as a
criteria of DNA damage.
Endpoints: Qua Iitati ve: A comparison is made between the zone of
growth inhibition for the repair deficient and proficient strains.
Quantitative: N/A.
Strengths: Very rapid and versatile bioassay; Inexpensive.
Weaknesses: Requires fairly large amount of test substances for
testing; Not suitable for substances which do not diffuse readily
in agar.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: A Iky I at ing agents, Nitroso compounds,
Polynuclear aromatics, Nitroso derivatives; Pesticides. CompI ex
Mixtures: Ambient; Industrial; Energy Related; Transportation
Related; Other.
Duration: 2 to 3 weeks.
Cost: $200.
Interpretation: Relative sizes of killing zones in repair-proficient
and repair-deficient stains indicate primary damage to DNA.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); L. Claxton. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 277i I , (FTS 629-2942);
S.S. Sandhu, U.S. EPA, HERL-RTP, Research Triangle Park, NC
27711, (FTS 629-2693); J.L. Huisingh. U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2948).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, V.F. Simmons.
Program Office Support: OHEE; OPP.
References: I) Kada, T., K. Tutikawa, and Y. Sadaie. Mutation Res.,
16:165-174, 1972.
74
-------
1222 ESCHERICHIA COLI, Pol A".
Biological Activity Detected: Toxicity; Primary DMA damage.
Principle: The assay measures the di f ferentia I killing between DNA
repair proficient Escherichia coli strain_(W5l10, Pol A ) and
DNA repair deficient strain (P3478, Pol A ) as affected by environ-
mental toxicants.
Endpoints: Qua Iitative: Differential killing between DNA repair
proficient and deficient strain after treatment with a test-
substance. Quantitative: Liquid suspension test provides a,
quantitative measure of primary DNA damage.
Strengths: The assay is well suited for detecting chemicals causing
frame shift mutations; Adequate data base is present on this
bioassay; Genetically we 1 I-character!zed system; Rapid; Inexpensive;
Well validated as a test for gene mutation; Works well with in-
vitro metabolizing microsome fractions; Can be used as indicator
organism in host-mediated assays.
Weaknesses: Reverse mutation assay requiring several strains to
permit detection of a broad spectrum of compounds; Requires
metabolic activation; Lacks pharmacological relevance, Prokaryotic
organization of genetic material.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: A Iky I at ing agents, Nitroso compounds,
Polynuclear aromatics, Nitroso derivatives, pesticides. CompI ex
Mixtures: Ambient; Industrial; Energy Related; Transportation
Related; Other.
Duration: 2 to 4 weeks.
Cost: $500.
Interpretation: Relative sizes of killing zones in repair-proficient
and repair-deficient stains indicate primary damage to DNA.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); L. Claxton, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2942).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, V.F- Simmons.
Program Office Support: OHEE; OPP.
References: I) Rosenbranz, H.S., et al. Mutation Res., 41:61-70,
1976. 2) Rosenbranz, H.S. Ann. Res. Microbiol., 27:383-401,
1973. 3) Rosenbranz, H.S. Cancer Res., 33:458-459, 1973.
75
-------
1223 MITOTIC RECOMBINATION AND GENE CONVERSION IN SACCHAROMYCES CEREVISIAE
Biological Activity Detected: Primary DMA damage.
Principle: Recombination of reciprocal type, mitotic recombination,
and the non-reciprocal type mitotic gene conversion are used
for assessing the DNA damaging potential of environmental chem-
icals.
Endpoints: Qua Iitative: Appearance of twin spots and growth in
selective medium. Quantitative: The degree of mitotic crossing-
over is evaluated by the frequency of twin spot sectors and that
of mitotic gene conversion by the differential growth in a selec-
tive medmm..
Strengths: The diploid cells with eukaryotic chromosomal organization;
Rapid; Inexpensive.
Weaknesses: Less versatile due to problems associated with cell
wall permeability and with coupling of metabolic activation;
Inadequate data base showing the reliability of this assay system.
Status of Development: Being implemented.
Describe: This test system needs to be validated with a variety
of classes of compounds. Further work is needed in understanding
the mechanism and significance of mitotic recombination and
cross i ng-over.
Applications: Multimedia.
Samples: Pure ChemicaIs: Alkylating agents, Nitrosamines, Carbamates,
Nucleic acid bases and analogs, Aromatic amines. Complex Mixtures:
Protocol not yet developed.
Duration: 2 to 3 weeks.
Cost: $200 to $500.
Interpretation: The appearance of twin spots and growth in a selective
medium suggests mutation.
Level of Complexity: 1.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, V.F. Simmons.
Program Office Support: OHEE; OPP.
References: I) Zimmermann, F.K. Mutation Res., 31:71-86, 1975.
2) Brusick, D.J., and V.W. Mayer. Envir. Hlth. Prospect., 6:83,
1973.
76
-------
1224 UNSCHEDULED DNA SYNTHESIS (UDS)
Biological Activity Detected: Primary DNA.
Principle: This assay evaluates the test compounds for their ability
to induce unscheduled DNA synthesis (UDS) in human diploid WI38
fibroblasts blocked in the GI phase.
Endpoints: Qua Iitative: Incorporation of 3H thymidine. Quantitative:
dpm 3H thymidine per yg or ymole of DNA.
Strengths: DNA repair can be measured in human cells in culture;
Similar studies can be performed in animals using peripheral
leucocytes; The latter permit comparison between in-vitro and in-vivo
exposures to carcinogens or mutagens.
Weaknesses: The precise type of molecular binding between carcinogens
and DNA which triggers excision repair is unknown; DNA repair
synthesis does not measure residual damage to DNA.
Status of Development: Validated.
Describe: The mechanism of UDS is still not completely understood.
Applications: Multimedia.
Samples: Pure Chemicals: Alkylalois; A Iky I at ing agents, Nitroso
compounds, Polynuclear aromatics. Complex Mixtures: Ambient;
Industrial; Energy Related; Transportation Related; Other.
Duration: 4 to 6 weeks.
Cost: $350 to $2,000.
Interpretation: The incorporation of labeled nucleotide precursors
into the cells arrested in the GI phase after treatment is used
as a criteria of the ability of the test material to cause pri-
mary DNA damage.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, A. Mitchell.
Program Office Support: OHEE; OPP.
References: I) Stich, H., and S. Laighes. DNA Repair and Chemical
Carcinogenesis. Pathobiol. Ann., 3:342-376, 1973. 2) San, R.H.,
and H.F. Stich. DNA Repair Synthesis of Cultured Human Cells as
a Rapid Bioassay for Chemical Carcinogens. Int. J. Cancer, 16:
284-291, 1975. 3) Williams, G.M. Detection of Chemical Carcino-
gens by Unscheduled DNA Synthesis in Rat Liver Primary Cell
Cultures. Cancer Res., 37:1845-1851, 1977. 4) Simmon, V.F.,
A.D. Mitchell, and T.A. Jorgenson. Evaluation of Selected
Pesticides as Chemical Mutagens: In-vitro and In-vivo Studies.
Ann. Rep., Envir. Hlth. Effects Series, EPA-600/1-77-028, 1977.
77
-------
1225 SISTER-CHROMATID EXCHANGE FORMATION (SCE)
Biological Activity Detected: Primary DMA damage.
Principle: SCE involves a reciprocal exchange between sister-chro-
matids which does not result in a change in the overall chromo-
sonal morphology. SCE may be observed as darkly staining and
lightly staining chromatids after growth in BUdR for two succes-
sive cell generations and subsequent-staining with fluorochrome
dyes.
Endpoints: Qua Iitati ve: Observation of sister-chromatid exchanges
of metaphase. Quantitative: SCE/cell are expressed.
Strengths: Rapid; Relatively economical; Very sensitive; Can be
tested in-vivo or in-vitro.
Weaknesses: Mechanism and significance of SCE is not understood;
No clear relationship between SCE and chromosomal breaks has
been estabIi shed.
Status of Development: Being implemented.
Describe: The validation of this system is near completion in
several laboratories.
Applications: Multimedia.
Samples: Pure Chemicals: A Iky I at ing agents, Mycotoxins, Halo-
genated hydrocarbons, Ureas and Thioureas, Nitro derivatives.
Complex Mi xtures: Protocol not yet developed.
Duration: 2 to 4 weeks.
Cost: $1,000 to $1,200.
Interpretation: Test agents inducing signigicant numbers of sister-
chromatid exchanges may be regarded as potential DNA-damaging
agents for animals and man.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch; ERL-NAR, Toxicology Branch, Genetic Toxicology
Team.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu, U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
A.R. Malcolm, U.S. EPA, ERL-NAR, South Ferry Rd., Narragansett, Rl
02882, (FTS 838-4843); G.G. Pesch, U.S. EPA, ERL-NAR, South
Ferry Rd., Narragansett, Rl 02882, (FTS 838-4843).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, V.F. Simmons.
Program Office Support: OHEE; OPP.
References: I) Perry, P., and H.J. Evans. Nature, 258;121-125,
1975. 2) Latt, S.A. Proc. Natl. Acad. Sci., 70:3395-3399, 1973.
3) Popescur, N.C., et al. F. Natl. Cancer Inst., 59:289-293,
1977.
78
-------
1226 IN-VIVO ASSESSMENT OF DNA DAMAGE
Biological Activity Detected: Primary DNA damage.
Principle: Detection of in-vivo DNA repair activity that is stimulated
by chemical carcinogens.
Endpoints: Qua Iitative: Initial endpoint-measurement of DNA molecular
weight change. Quantitative: Molecular weight distribution of
cleaved DNA strands.
Strengths: Detects DNA damage in-vivo; Assay is done on biopsy
material; Non-destructive, animal sampled can later be scored
for tumors to validate assay.
Weaknesses: Not yet apparent; Mainly technical.
Status of Development: Developmental.
Describe: Still in early stages of development.
Applications: Multimedia.
Samples: Pure Chemicals: Organic compounds, Inorganics, Heavy
metals. Complex Mixtures: Ambient - water; Industrial; Energy
Related; Transportation Related; Other.
Duration: Variable, but not to exceed 3 months once validated.
Cost: Not yet determined.
Interpretation: Mutagenic and carcinogenic agents act through damage
of DNA. This method will quantitate DNA damage.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxicologicaI Assessment Branch.
Persons to Contact: R.J. Bui I, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators:
Ohio State University, Chemical Biomedical Environmental Research
Group, Columbus, OH 43210, R.W. Hart, (FTS 940-9375).
Program Office Support: OHEE; OWHM.
References: I) Brash, et al. N.Y. Acad. J.C., 1977. 2) Brash and
Hart, R.W. Envir. Hlth. Perspect., 1978. In press.
79
-------
1227 INTACT RODENT HEPATOCYTES IN PRIMARY CULTURE
Biological Activity Detected: Toxicity; Presumptive mutagenicity;
Oncogenicity; DMA damage.
Principle: Detects interaction of chemical agents which result in
DNA damage. This interaction is detected as unscheduled DMA
synthesis using radiotracer, centrifugal and autoradiographic
techniques.
Endpoints: Qua Iitative: A positive response suggests potential to
act as a mutagen or carcinogen. Quantitative: Can quantify the
number of grains/nucleus which increases with increased dose.
Strengths: Rapid; Economical; The entire genome is the target;
Maintains several functions of tissue of origin, thereby activat-
ing many different chemicals; Not limited to direct acting com-
pounds.
Weaknesses: Represents only a single target organ; System lacks
validation; May not detect promoters or co-carcinogens.
Status of Development: Developmental.
Describe: This system is now in the process of validation in
several laboratories. It has not yet been applied to unknown
compounds.
Applications: Multimedia.
Samples: Pure Chemicals: Aromatic amines, Polycyclics, Alky-
lators, Hormones. Complex Mixtures: Not yet determined.
Duration: 2 days to 2 weeks, depending on technique.
Cost: $500 to $2,000.
Interpretation: This test is considered a reliable screen for
prioritization of chemical testing in more complex in-vivo
systems. It suggests that the chemical reaches the genome and
requires further analysis.
Level of Complexity: 3 to 4.
OHEE Laboratory Involved: National Center for ToxicologicaI Research,
Division of Mutagenesis Research, Somatic Cell Section; HERL-RTP,
Environmental Toxicology Division, Biochemistry Branch, Cellular
Biology Section.
Persons to Contact: D.A. Casciano, National Center for ToxicologicaI
Research, Jefferson, AR 72079, (FTS 740-4573); J.L. Huisinqh,
U.S. EPA, HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-
2948).
Grant/Contract Laboratory Involved and Principal Investigators:
American Health Foundation, Naylor Dana Institute for Disease
Prevention, Hammond House Road, Valhalla, NY 10595, G.M. William.
Program Office Support: OHEE.
References: 1) Kitagawa, T., et al. Cancer Res., 35:3682-3692,
1975. 2) Michalopoulos, G., et al. Life Sciences, 18:1139-1144,
1976. 3) Williams, G.M. Cancer Letters, 1:231-236, 1976. 4)
San, R.H.C., and H.F. Stich. Intl. J. Cancer, 7:65-74, 1971.
-------
1228 IN-VIVO DNA BINDING
Biological Activity Detected: Binding of chemical to DNA.
Principle: This test system attempts to correlate binding with DNA
repair and tumorigenicity. Various rodent stains will be treated
with the chemical carcinogen DNA from presumptive target tissue
analyzed for DNA-carcinogen adducts by radiometric and/or fluoro-
metric techniques, measuring the rate of excision of the bound
chemi caI.
Endpoints: Qua Iitative: Adducts will be determined by chromatographic
procedures. Quantitative: Can quantify amount of carcinogen
bound/unit DNA (y mole carcinogen/mole DNA-phosphate).
Strengths: A direct measurement of the extent of carcinogen inter-
action with DNA; Allows measurement of total DNA damage.
Weaknesses: A considerable portion of the DNA-carcinogen adducts may
have little or no biological relevance.
Status of Development: Developmental.
Describe: Initial experiments are being carried out to provide
background for development of in-vitro DNA repair assay.
Applications: Multimedia.
Samples: Pure Chemi caIs: Organic-metallic compounds, Organics.
Complex Mixtures: N/A.
Duration: 2 weeks.
Cost: Not yet determined.
Interpretation: This test measures the direct interaction of the
test compound with genetic material as an indication of carcino-
genic and mutagenic activity.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxicologicaI Assessment Branch.
Persons to Contact: B. Daniel, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7482).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OWHM.
References: Not yet available.
-------
1229 CHINESE HAMSTER CELLS (CHO) UNSCHEDULED DNA SYNTHESIS (UDS)
Biological Activity Detected: DNA Repair.
Principle: Repair of induced damage to DNA is detected as unscheduled
DNA synthesis via incorporation of 3H thymidine.
Endpoints: Qua I 1tative: Unscheduled DNA synthesis is measured and
compared to controls. Quantitati ve: Amount of unscheduled DNA
synthesis per cell may be determined.
Strengths: Rapid; Relatively low cost; DNA repair is probably a
more sensitive detector of DNA damage than are chromosomal
aberrations.
Weaknesses: The in-vitro cell assay lacks pharmacological relevance;
Requires metabolic activation.
Status of Development: Being implemented.
Describe: The test is presently being applied to standard com-
pounds and some selected unknowns. It is still being refined
and modified.
Applications: Water; Multimedia.
Samples: Pure Chemicals: Organics (EMS), Heavy metals, UV radia-
tion. Complex Mixtures: Energy Related - JP-5 jet fuel.
Duration: 2 to 3 weeks.
Cost: $1,000 to $1,200/compound.
Interpretation: Agents inducing significant unscheduled DNA synthesis
represent potential carcinogens/mutagens for animals and man.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-NAR, Toxicology Branch, Genetic
Toxicology Team.
Persons to Contact: E. Jackim. U.S. EPA, ERL-NAR, South Ferry Rd.,
Narragansett, Rl 02882, (FTS 838-4843 X229, X3IO); A.R. Malcolm,
U.S. EPA, ERL-NAR, South Ferry Rd., Narragansett, Rl 02882,
(FTS 838-4843 X238, X247).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: San, R.H.C., and H.F. Stitch. Int. J. Cancer, 16:284-
291, 1975. 2) Martin, C.N., et al. Cancer Letters, 2:355-360,
1977. 3) Trosko, J.E., and J.D. Yager. Exp. Cell Res., 88:47-
55, 1974.
82
-------
CONTENTS: TEST SYSTEMS
1230 Carcinogenesi s 83
1231 C3HIOTI/2CL8 Mouse Embryo Fibroblast
Oncogenic Transformation with Exogenous
Metabol ic Activation 84
1232 C3HIOTI/2CL8 Mouse Embryo Fibroblast
Oncogen ic Transformation 85
1233 BHK-21 Mammalian Cell Oncogenic
Transformation 86
1234 Syrian Hamster Embryo Oncogenic
Transformation (Focus Assay) 87
1235 BALB 3T3 Oncogenic Transformation and
Mutagenesis with Exogenous Metabolic
Activation 88
1236 BALB 3T3 Oncogen ic Transformation 89
1237 Pulmonary Adenoma Bioassay in Mice 90
1240 Marine Applications 91
83
-------
1231 C3H10T1/2CL8 MOUSE EMBRYO FIBROBLAST ONCOGENIC TRANSFORMATION WITH
EXOGENOUS METABOLIC ACTIVATION
Biological Activity Detected: Presumptive oncogenicity.
Principle: Normal cells in log phase are treated with the test
agent. Four weeks after the cells have attained confluence they
are scored for morphologically transformed foci (clones of cells).
These transformed cells will give rise to tumors when injected
into immunosupressed syngeneic animals.
Endpoints: Qua Iitative: Appearance of morphologically transformed
foci. Quantitative: Simultaneous cytotoxicity experiments are
performed to obtain the lethal toxicity of the agent and trans-
formation is then adjusted for that toxicity.
Strengths: This system is easy to score; Has an extremely low back-
ground of spontaneous transformation; Is particularly sensitive to
PAH and their derivatives; Has metabolic activation capability;
Can be used to detect tumor promoters and initiators.
Weaknesses: These mouse embryo cells are aneuploid; Requires 6 weeks
to complete the experiment; Seems to be somewhat refractory to
the carcinogenic effects of alkylating agents, aflatoxin BI and
some aromatic amines.
Status of Development: Developmental.
Describe: The C3HIOTI/2 system is being modified by the addition
of exogenous metabolic activation capability, giving, therefore,
increased sensitivity towards a broad range of chemical carcinogens.
AppIications: Ai r.
Samples: Pure ChemicaIs: Polycyclic aromatic hydrocarbons.
Complex Mixtures: N/A.
Duration: 5 to 6 weeks.
Cost/sample or chemical: $5,000 to $7,000.
Interpretation: The appearance of morphologically altered clones
of cells indicates oncogenic transformation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Metabolic Effects Section.
Persons to Contact: S. Nesnow, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
Microbiological Associates, Bethesda, MD 20014, R. Kouri,
L. Schectman.
Program Office Support: OEMI.
References: I) Reznikoff, et al. Cancer Res., 33:3231-3249, 1973.
2) Nesnow, et al. Cancer Res., 36:1801-1808, 1976. 3) Mondal,
et al. Cancer Res., 36:2254-2260, 1976. 4) Bertram. Cancer Res.,
37:514-523, 1977. 5) Benedict, et a I. Cancer Res., 37:2202-
2208, 1977.
84
-------
1232 C3H10T1/2CL8 MOUSE EMBRYO FIBROBLAST ONCOGENIC TRANSFORMATION
Biological Activity Detected: Presumptive oncogen! city.
Principle: Normal cells in log phase are treated with the test agent.
Four weeks after the cells have attained confluence they are
scored for morphologically transformed foci (clones of cells).
These transformed cells will give rise to tumors when injected
into immunosupressed syngeneic animals.
Endpoints: Qua 1 itati ve: Appearance of morphologically transformed
foci. Quantitative : Simultaneous cytotoxicity experiments are
performed to obtain the lethal toxicity of the agent and the
transformation is then adjusted for that toxicity.
Strengths: This system is easy to score; Has an extremely low back-
ground of spontaneous transformation; Is particularly sensitive
to PAH and their derivatives; Has metabolic activation capability;
Can be used to detect tumor promoters and initiators.
Weaknesses: These mouse embryo cells are aneuploid; Requires 6 weeks
to complete the experiment; Seems to be somewhat refractory to the
carcinogenic effects of alkylating agents, aflatoxin BI , and some
aromatic amines.
Status of Development: Being implemented.
Describe: The C3HIOTI/2 system is being modified so that its
metabolic activation capability is increased, giving, therefore,
increased sensitivity towards a broad range of chemical car-
cinogens.
Applications: Multimedia.
Samples: Pure Chemical s
Polycyclic aromatic hydrocarbons,
aromatic amines, pesticides. Comp lex Mixtures
Aromatic azo dyes,
Model parti cu I ates.
Duration: 5 to 6 weeks.
Cost/sample: $5,700 to $7,000.
Interpretation: The appearance of morphologically altered clones
indicates oncogen ic transformation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental
Biochemistry Branch, Metabolic Effects Section
Persons to Contact: S. Nesnow, U.S. EPA, HERL-RTP
Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
I n-house.
Program Office Support: OHEE; OPP.
References: I) Reznikoff, et al. Cancer
Toxicology Division,
Research Triangle
Res., 33:3231-3249, 1973.
2)
et
Nesnow, et al. Cancer Res., 36:1801-1808, 1976. 3) Mondal
al
Cancer Res., 36:2254-2260, 1976. 4) Bertram. Cancer Res.
37:514-523,
2208, 1977.
1977. 5) Benedict, et al. Cancer Res., 37:2202-
85
-------
1233 BHK-21 MAMMALIAN CELL ONCOGENIC TRANSFORMATION
Biological Activity Detected: Presumptive oncogenicity.
Principle: Normal baby hamster kidney fibroblasts do not grow in
soft agar. After treatment with carcinogens these cells do grow
in soft agar and will give tumors when injected into syngeneic
animaIs.
Endpoints: Qua Iitative: Cells which grow in soft agar are considered
transformed. Quantitati ve: Colony formation is scored.
Strengths: Not yet known.
Weaknesses: Not yet known.
Status of Development: Developmental.
Describe: Testing of unconcentrated and concentrated wastewater
samples.
Applications: Water.
Samples: Pure Chemi caIs: PAH. Complex Mixtures: Wastewaters.
Duration: 3 weeks. '
Cost/sample or chemical: Not yet known.
Interpretation: A positive result suggests a possible carcinogenic
materi a I.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
logical Assessment Branch.
Persons to Contact: H. Pahren, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7217).
Grant/Contract Laboratory Involved and Principal Investigators:
Syracuse Research Corporation, MerriI I Lane, Syracuse, NY
13210, J. Saxena.
Program Office Support: OHEE.
References: I) Bouck, N., and G. diMayorca. Nature, 264:722-727
1976.
86
-------
1234 SYRIAN HAMSTER EMBRYO ONCOGENIC TRANSFORMATION (FOCUS ASSAY)
Biological Activity Detected: Presumptive oncogen!city.
Principle: Freshly isolated cells from hamster fetuses are seeded
into dishes, passaged twice, and then treated for two consecutive
three drug treatments with the test agents. Toxicity is scored
I to 2 days after the experiment is begun and the transformation
(appearance of morphologically transformed foci) is scored 10 days
after toxicity.
Endpoints: Qua Iitati ve: Appearance of morphologically transformed
foci. Quantitat 1ve: Simultaneous cytotoxicity experiments are
performed to obtain the lethal toxicity of the agent and the trans-
formation is then adjusted for that toxicity.
Strengths: These cells have high metabolic activation capability;
Are diploid; Respond to a wide variety of different chemical
agents; Few false positives are known.
Weaknesses: Variability within the assay due to variations in
obtaining and preparing viable primary cell cultures; Observable
spontaneous transformation background; Difficulty in scoring.
Status of Development: Being implemented.
Describe: The Syrian hamster embryo biassay is being evaluated
for use in the evaluation of particulate samples.
App1ications: Ai r.
Samples: Pure Chemicals: Polycyclic aromatic hydrocarbons.
Complex Mixtures: Model particulates.
Duration: I month.
Cost/assay: $4,000 to $5,000.
Interpretation: The appearance of morphologically altered clones
of cells indicates oncogenic transformation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Metabolic Effects Section.
Persons to Contact: S. Nesnow, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2693); M.D. Waters. U.S. EPA, HERL-RTP,
Research Triangle Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE.
References: I) Casto, B.C., N. Janosko, and J.A. DiPaolo. Cancer Res.,
37:3508-3515, 1977.
87
-------
1235 BALB 3T3 ONCOGENIC TRANSFORMATION AND MUTAGENESIS WITH EXOGENOUS
METABOLIC ACTIVATION
Biological Activity Detected: Mutagenicity; Presumptive oncogenicity.
Principle: Mammalian cell clones of BALB 3T3 clone AI3 undergo
malignant transformation upon treatment with known carcinogens.
Extent of transformation is expressed in focus formation and al-
tered morphology, increased saturation density and enhanced
plating efficiency in soft agar. Simultaneously these cells also
undergo a permanent genetic change at the ouabain locus. Tumor
induction is observed in recipient animals by reinjecting trans-
formed ceI Is.
Endpoints: Qua Iitative: Appearance of morphological transformed
foci for transformation and clones of cells which grow in ouabain
supplanted media. Quantitati ve: Number of observed foci adjusted
for the cytotoxic effect of the agent.
Strengths: BALB 3T3 cells are mouse embryo fibroblasts which have
the capability to activate 3-methyIcholanthrene to metabolites
which transform these cells; Direct acting alkylating agents such
as MNN6 are also effective transforming agents.
Weaknesses: These cells are not transformed by the carcinogens
benzolalpyrene or 6-aminochrysene and possibly other potent car-
cinogens unless exogeneous metabolic activation is provided.
Status of Development: Developmental.
Describe: The addition of rat liver preprarations to activate
carcinogens and mutagens to make this assay system more sensi-
tive is currently underway.,
AppIications: Ai r.
Samples: Pure ChemicaIs: PAH, Aromatic amines. Complex Mixtures:
Transportation Related.
Duration: 5 to 6 weeks.
Cost/sample or chemical: $5,000 to $7,000
Interpretation: Morphologically altered clones of cells indicate
oncogenic transformation. Cells growing in the presence of
ouabain are indicative of a mutagenic change.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Metabolic Effects Section.
Persons to Contact: S. Nesnow, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 277II, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
Microbiological Associates, Bethesda, MD 200I4, L. Schectman,
R. Kouri.
Program Office Support: OEMI.
References: I) Kakunaga, T. A Quantitative Assay for Malignant
Transformation by Chemical Carcinogens Using Clone from BALB
3T3. Int. J. Cancer, 12:463, 1973.
-------
1236 BALE 3T3 ONCOGEN1C TRANSFORMATION
Biological Activity Detected: Presumptive oncogenicity.
Principle: Mammalian cell clones of BALE 3T3 clone AI3 undergo
malignant transformation with known carcinogens. Extent of trans-
formation is expressed in focus formation and altered morphology,-
increased saturation density, and enhanced plating efficiency in
soft agar. Tumor induction is observed in recipient animals by
reinjecting transformed cells.
Endpoints: Qua Iitati ve: Appearance of morphological transformed foci.
Quantitative: Number of observed foci adjusted for the cytotoxic
effect of the agent.
Strengths: BALB 3T3 cells are mouse embryo fibroblasts which have
the capability to activate 3-methyIcholanthrene to metabolites
which transform these cells; Direct acting alkylating agents such
as MNNG are also effective transforming agents.
Weaknesses: These cells are not transformed by the carcinogens benzo-
(a)pyrene or 6-aminochrysene and possibly other potent carcinogens.
Status of Development: Being implemented.
Describe: Currently BALB/T3 cell culture is used for routine
testing, but other cell lines (e.g. epithelial) are being in-
vestigated in an effort to increase sensitivity.
Applications: Water.
Samples: Pure Chemicals: Organics. Complex Mixtures: Ambient -
drinking water; Other - advanced waste treatment, concentrate
eff1uent.
Duration: 5 to 6 weeks.
Cost/sample or chemical: $5,000 to $7,000.
Interpretation: A positive result indicates possible carcinogenesis.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
logical Assessment Branch; HERL-CIN, Laboratory Studies Division,
Toxicological Assessment Branch.
Persons to Contact: J.P. Bercz, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7432); N.E. KowaI, U.S. EPA,
HERL-CIN, 26 W. St. Clair St., Cincinnati, OH 45268, (FTS 684-
7477); R.J. Bui I, U.S. EPA, HERL-CIN, 26 W. St. Clair St.,
Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Cincinnati, Medical Center, Cincinnati, OH 45221,
J.C. Loper and D. Lang; Gulf South Research Institute, P.O. Box
26518, New Orleans, LA 70186, N. Gruener.
Program Office Support: OHEE.
References: I) Kakunaga, T. A Quantitative Assay for Malignant
Transformation by Chemical Carcinogens Using Clone From BALB
3T3. Int. J. Cancer, 12:463, 1973.
89
-------
1237 PULMONARY ADENOMA BIOASSAY IN MICE
Biological Activity Detected: Presumptive oncogenicity.
Principle: After 13 weeks no untreated strain A mice develop lung
tumors; however, after I year 100$ develop lung tumors. When
treated with a carcinogen, lung tumors start developing within
13 weeks.
Endpoints: Qua Iitative: Tumor formation. Quantitative: Average
number of tumors/mouse lung in the treated animals as compared
to the positive controls (urethane) and untreated controls.
Strengths: Relatively rapid in-vivo carcinogenesis bioassay.
Weaknesses: The adenoma (pulmonary) has no counterpart in human
neoplastic pathology.
Status of Development: Being implemented.
Describe: The system has been developed and used in other
laboratories, see references.
Applications: Multimedia.
Samples: Pure ChemicaIs: Metalic compounds, Food additives,
chemotherapeutic agents. Complex Mixtures: Industrial; Trans-
portation Related - diesel particulate.
Duration: 13 to 30 weeks.
Cost/sample or chemical: Not yet known.
Interpretation: The formation of visually observable lung nodules
indicates oncogenicity of the test substance.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Functional Pathology Branch.
Persons to Contact: J. Orthoefer, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7434).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Shimkin and Stoner. Lung Tumors in Mice: Application
to Carcinogenesis Bioassay. Adv. in Cancer Res., 21:1-58, 1975.
2) Stoner, et a I. Test for Careinogenicity of Metallic Compounds
by the Pulmonary Tumor Response in Strain A Mice. Cancer Res.,
36:1744-1747, May 1976.
90
-------
CONTENTS: TEST SYSTEMS
1240 Marine Applications 91
1241 Tumor Induction in Massive Crustaceans,
Molluscs, and Tel lost Fish 92
1242 MFC Induction as an Indicator of Toxicity
Exposure 93
1243 Limb Regeneration System 94
1244 Isogenic Fish 95
1245 Integrated System: Development of Mutagen/
Carcinogen Activation, Concentration,
Separation, and Weathering Systems 96
1246 Biphenyl Hydroxylase 97
1300 Perinatal Toxicology 99
91
-------
1241 TUMOR INDUCTION IN MASSIVE CRUSTACEANS, MOLLUSCS, AND TELIOST FISH
Biological Activity Detected: Presumptive oncogenicity.
Principle: Animals are exposed to known carcinogens under laboratory
conditions and histopathology is performed. Also, feral animals
are surveyed for histopathologicaI abnormalities and correlations
are established with tissue residues and water concentrations.
Endpoints: Qua Iitative: Not supplied. Quantitative: Correlation
of exposure/response.
Strengths: May have value as sentinel system for water quality and
as a model system.
Weaknesses: Field correlation requires laboratory validation.
Status of Development: Being implemented.
Describe: Not supplied.
Applications: Water.
Samples: Pure ChemicaIs: PNA's. Complex Mixtures : Ambient -
water; Industrial - water.
Duration: Not supplied.
Cost: Not suppIied.
Interpretation: Not supplied.
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: J. Couch, U.S. EPA, ERL-GB, Sabine Island, Gulf
Breeze, FL 32561, (FTS 686-90 I I).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Oregon, Eugene, OR 97403, M. Mix; U. of Southern Mississippi
Hattiesburg, Ml 39401, B.J. Martin.
Program Office Support: OEMI; OHEE.
References: Not supplied.
92
-------
1242 MFO INDUCTION AS AN INDICATOR OF TOXICITY EXPOSURE
Biological Activity Detected: Toxicity; Mutagenicity; Presumptive
oncogenicity.
Principle: Fetal and laboratory reared animals are exposed to ambient
environmental conditions as sentinal organisms.
Endpoints: Qua Iitative: Not supplied. Quantitative: Degree of
induction of MFO.
Strengths: Pre-pre screen method for water quality; Provides guidance
for chemical analysis programs.
Weaknesses: Nonspecific.
Status of Development: Developmental.
Describe: Not supplied.
Applications: Water.
Samples: Pure ChemicaIs: PNA's; Complex Mixtures: Ambient -
estuarine/marine.
Duration: Not supplied.
Cost: Not supp I ied.
Interpretation: Positive test indicates that the test animal has
recently been exposed to inducer(s) of MFO systems.
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: N. Richards, U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011); P. Schoor, U.S. EPA, ERL-
GB, Sabine Island, Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of West Florida, Pensacola, FL 32504, R. Rao.
Program Office Support: OHEE.
References: Not supplied.
93
-------
1243 LIMB REGENERATION SYSTEM
Biological Activity Detected: Differentiation/teratology.
Principle: Crustacean limbs are removed at predetermined breakpoints
and regenerated by a precise sequence of biochemical events.
Endpoints: Qua Iitative: Interference with limb regeneration -
determined by gross morphology and histopathology. Quantitative:
Not suppIied.
Strengths: Appropriate for marine samples.
Weaknesses: Difficult to extrapolate to humans.
Status of Development: Being implemented.
Describe: Not supplied.
Applications: Water.
Samples: Pure Chemicals: PCP, Colchicine, PNA's. Complex
Mixtures: Ambient - estuarine/marine; Energy Related - drilling
fluids.
Duration: Not supplied.
Cost: Not supplied.
Interpretation: Pre-screen for teratogens.
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: N. Richards, U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of West Florida, Pensacola, FL 32504, R. Rao.
Program Office Support: OEMI.
References: Not supplied.
94
-------
1244 ISOGENIC FISH
Biological Activity Detected: Presumptive oncogen!city; Teratology.
Principle: Isogenic fish provide uniform progeny with predictable
life stages, and an opportunity to genetically engineer genotypes
for susceptabi Iity to carcinogens.
Endpoints: Qua Iitative: Not supplied. Quantitative: Interference
with development; Melanoma/melanin proliferation.
Strengths: In-vivo; Rapid.
Weaknesses: Toxic substances may kill test animal before oncogenic/
carcinogenic response is elicited; Substances may not be permeable
to eggs.
Status of Development: Developmental.
Describe: Not supplied.
Applications: Water.
Samples: Pure ChemicaIs: PNA's, Nitrosamines, Aflotoxin, etc.
Complex Mixtures: Not supplied.
Duration: I month.
Cost: Not yet determined.
Interpretation: Test results indicate presumptive teratogen, presump-
tive carcinogen, or toxic substance.
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: N. Richards, U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of North Carolina, Chapel Hill, NC 27514, D. Humm.
Program Office Support: OEM I.
References: Not supplied.
95
-------
1245 INTEGRATED SYSTEM: DEVELOPMENT OF MUTAGEN/CARCINOGEN ACTIVATION,
CONCENTRATION, SEPARATION, AND WEATHERING SYSTEMS
Biological Activity Detected: Toxicity; Mutagenicity.
Prinicple: The above procedures are being developed for use with
quick screen tests. Biological, physical, and chemical methods
are being used to concentrate, separate, and activate compounds
which interfere with testing.
Endpoints: Qua Iitative: Sample concentrated, freed of interfering
substances, activated, prepared for testing. Quantitative: Not
suppI led.
Strengths: Separation of toxic components from complex mixtures
will allow detection; Concentration of dilute genotoxics will
allow their detection; Weathering may assist in prediction of the
environmental fate; Marine activation systems aid in predicting
biological fate and food web relationships.
Weaknesses: Extensive exploratory research is required to validate
the methods.
Status of Development: Developmental.
Describe: Not supplied.
Applications: Water; Food.
Samples: Pure ChemicaIs: PNA's; Complex Mixtures: Ambient - •
estuarine/marine h^O; Energy Related - shale; Other - tissue
resi dues.
Duration: Not supplied.
Cost: Not supplied.
Interpretation: These methods may be useful for all in-vitro and
in-vivo methods to expand their application to complex samples.
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: N. Richards, U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Gulf South Research Institute, E. Kline; U. of West Florida,
Pensacola, FL 32504, R. Rao.
Program Office Support: OEM I; OHEE.
References: Not supplied.
96
-------
1246 BIPHENYL HYDROXYLASE
Biological Activity Detected: Presumptive oncogen!city.
Principle: Pre-pre screen for carcogenicity depends on correlation
of interference with hydroxyI at ion.
Endpoints: Qua Iitative: Not supplied. Quantitative: Interference
with enzyme reaction.
Strengths: Extremely rapid; Inexpensive.
Weaknesses: Requires extensive development, modification and valida-
tion; Current data based on correlation only; Mechanism unknown.
Status of Development: Developmental.
Describe: Requires extensive development, modification, and
vaIidation.
Applications: Water; Multimedia.
Samples: Pure ChemicaIs: Multiple classes being screened.
CompI ex M i xtu res: Not supplied.
Duration: Not supplied.
Cost: Not supplied.
Interpretation: After validation and development, the test may be
useful as a rapid, inexpensive pre-pre screen.
Level of Complexity: 0.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: N. Richards, U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Denver Research Institute, J. Schmidt-Coderis.
Program Office Support: OEMI.
References: Not supplied.
97
-------
CONTENTS: TEST SYSTEMS
1300 Perinatal Toxicology 99
1301 Mammalian Teratology 100
1302 Perinatal Toxicology 101
1303 Fetal Toxicity in Rats, Mice, Guinea Pigs/
Hamsters 102
1304 Developmental Toxicity in Neonatal Rats 103
1305 Teratology In-Vivo/In-Vitro 104
1306 Direct Spectral Measurement of the
Biochemical Development of the
Nervous System 105
2000 Short-Term Tests for Ecological Effects 107
2100 Freshwater and Marine Algae 107
99
-------
1301 MAMMALIAN TERATOLOGY
Biological Activity Detected: Teratology.
Principle: Determine the potential of the compound to produce
anatomical malformations.
Endpoints: Qua Iitative: N/A. Quantitative: Number and type of
ma I formations.
Strengths: Biologically active compounds are readily detected;
Same techniques used for most species.
Weaknesses: Only gross anatomical malformations are noted.
Status of Development: Validated.
Describe: Protocols and methods well established. Compounds
are being tested on a routine basis.
Applications: Multimedia.
Samples: Pure Chemicals: All classes. Complex Mixtures: Ambient;
Industrial, Energy Related; Transportation Related; Other.
Duration: 6 weeks for small rodents, start to report. Additional
gestational time needed for larger animals.
Cost: $6,000/specie/compound.
Interpretation: The teratogenic potential of compounds can be
estabIished.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Toxic Effects Branch, Perinatal Toxicology Section.
Persons to Contact: K.D. Courtney, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2370).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP.
References: I) OPP Guidelines for Teratology.
100
-------
1302 PERINATAL TOXICOLOGY
Biological Activity Detected: Postnatal development.
Principle: Establish biochemical markers to determine normalacy of
postnatal development. Isozyme profiles of lactic dehydrogenase
(LDH) and creative phosphokinase (CPK) show definite develop-
mental patterns from day 7 to 14 in the postnatal mouse.
Endpoints: Qua Iitative: N/A. Quantitative: LDH and CPK total
activities and isozyme profiles are determined in postnatal mice
after prenatal and/or actational exposure. Results are entered
into computer by key punch cards and analyzed by established pro-
gram.
Strengths: Establish a measurable parameter of postnatal development
that can be quantified; Determining the cardiac isozymes permits
correlation with clinical human data; Comparative species studies
use same techniques and interpretations.
Weaknesses: Must wait for animals to be born.
Status of Development: Validated.
Describe: The postnatal isozyme profiles of LDH and CPK have
been established and treated animals are being evaluated.
Applications: Multimedia.
Samples: Pure ChemicaIs: AM classes. Complex Mixtures: Ambient;
Industrial; Energy Related; Transportation Related; Other.
Duration: Gestation pI us 4 weeks, start to report.
Cost: $3,000/compound.
Interpretation: Postnatal development is evaluated with the same
techniques that are used in human medicine so that results are
directly comparable.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Toxic Effects Branch, Perinatal Toxicology Section.
Persons to Contact: K.D. Courtney, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2370).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP-
References: Not yet available.
101
-------
1303 FETAL TOXICITY IN RATS, MICE, GUINEA PIGS/HAMSTERS
Biological Activity Detected: Fetal toxicity including teratogenicity.
Principle: Pregnant females are treated with chemical (or other
agent) during the period of major organogenesis of their litters.
The animals are sacrificed before term and the fetuses subsequently
examined for signs of toxicity including visual and skeletal de-
fects.
Endpoints: Qualitative: Descriptions of defects and anomalies
encountered. Quantitative: Degree of fetal toxicity, and
incidence of defects encountered.
Strengths: A fairly rapid test, 30 to 45 days to final report; Much
historical background.
Weaknesses: Difficulty in assessing form and degree of fetal toxi-
city; Difficulty in extrapolation of data to human species.
Status of Development: Validated.
Describe: Standardized protocols have been available for over
10 years.
Applications: Multimedia.
Samples: Pure Chemicals: AM classes. Complex Mixtures: Drinking
water contaminants.
Duration: 45 days from initiation of treatment to completion of
analysi s.
Cost: $ 0,000/species.
Interpretation: A positive response in a species that has some
metabolic similarities to man would suggest that the compound
or mixture in question has the potential to induce abnormal
development in humans.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology Division,
Neurobiology Branch.
Persons to Contact: N. Chernoff, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2326); R. Kavlock. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2326).
Grant/Contract Laboratory Involved and Principal Investigators:
Contract currently under negotiation for the testing of 5
pesticides/year by established protocols.
Program Office Support: OPP; ORD.
References: I) Pesticide Registration Guidelines. In preparation.
2) Testing of Chemicals for Carcinogenicity, Mutagenicity, and
Teratogenicity. Published by Minister of Health and Welfare,
Canada, 1973.
102
-------
1304 DEVELOPMENTAL TOXICITY IN NEONATAL RATS
I
Biological Activity Detected: Developmental toxicity.
Principle: Pregnant rats are treated with compounds from the time
of implantation continuing through lactation. The neonatal rats
are then examined for a number of developmental milestones.
This design allows for the continuous exposure of a mammalian
organism through its most sensitive periods of development.
Endpoints: Qua 1itati ve: Growth and viability of neonatal rats.
Quantitative: Measurement of developmental milestones in early
postnatal life including reflex and morphological development;
Also measurement of open field behavior in young adults.
Strengths: Exposure to organisms during the perinatal period
maximizes the possibility of producing alterations in the
morphological and behavioral aspects of the exposed animal.
Weaknesses: Testing is a labor-intensive operation; Lack of
standardized procedures by various investigations; Difficult to
extrapolate to humans.
Status of Development: Developmental.
Describe: Investigators in this f?eld are evaluating the
reliability and sensitivity of various test procedures.
Applications: Air; Water; Food.
Samples: Pure Chemi caIs: AM classes. Complex Mixtures: N/A.
Duration: 3 to 4 months, start to report.
Cost: $IO,000/species.
Interpretation: The production of growth disturbances/behavioral
anomalies in the young postnatal animal is one of the more
sensitive indicators of developmental toxicity. The implication
of this test is that positive results may point to the induction
of behavioral disturbances in humans subsequent to perinatal
exposure. This link-up to human effects, however, has yet to
be empirically demonstrated.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology Division,
Neurobiology Branch.
Persons to Contact: R. Kavlock, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2326); N. Chernoff, U.S. EPA, HERL-RTP,
Research Triangle Park, NC 27711, (FTS 629-2326).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP; ORD.
References: I) Wiess, B., and J. Spyker. Behavioral Implications
of Prenatal and Early Postnatal Exposure to Chemical Pollutants.
Pediatrics, 53(5):Part III, May 1974. 2) Final Report of the
Committee on Postnatal Evaluation of Animals Subjected to Insult
During Development. C. Kimmel, Chairperson. Funded by the
Nat. Inst. of Envir. Hlth. Sci., Mar. 1977.
103
-------
1305 TERATOLOGY IN-VIVO/IN-VITRO
Biological Activity Detected: Teratology; Toxicology.
Principle: Treat pregnant rats from day 6 to 9 of gestation, then
remove embryos and grow them in tissue culture for 24 to 48 hrs.
During the culture interval, determinations of anatomical and
biochemical growth are made.
Endpoints: Qua Iitative: Determine degree and normalacy of neurala-
tion and somite development; Quantitative: Measure growth in-
dicators, DNA, and protein.
Strengths: The maternal animal is treated; The embryo is accessible
for a long enough period to make some measurements; Zero time
measurements can use embryos from the same litter; Delays in
growth during this gestational stage could result in malformations
or abnormal development such as extra ribs; Embryos from the
same litter are available for residue determination.
Weaknesses: The culture of the embryos is limited to 48 hours.
Status of Development: Being implemented.
Describe: Embryos from treated animals are being grown in culture.
Methods for growing control embryos have been established.
Applications: Multimedia.
Samples: Pure ChemicaIs: All classes. Complex Mixtures: Ambient;
Industrial; Energy Related; Transportation Related; Other.
Duration: 4 weeks, start to report.
Cost: $2,000/compound.
Interpretation: Toxic as well as teratogenic evaluators are deter-
mined for the embryo.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Toxic Effects Branch, Perinatal Toxicology Section.
Persons to Contact: K.D. Courtney, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2370).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Michigan, School of Medicine, Department of Anatomy:
Ann Arbor, Ml 48104, A. Beaudoin.
Program Office Support: OTS.
References: Not yet available.
104
-------
1306 DIRECT SPECTRAL MEASUREMENT OF THE BIOCHEMICAL DEVELOPMENT OF THE
NERVOUS SYSTEM
Biological Activity Detected: Delayed and arrested development.
Principle: A large change in the capacity of the nervous system for
oxidative metabolism occurs with development. This increase may
be followed directly in slices of tissue by taking advantage of
the known spectral properties of the cytochromes using dual-wave-
length spectroscopy. During brain development there are quantita-
tive and qualitative differences in the way the tissue will re-
spond to stimulation (e.g., electrical, elevated K ) metabolicaIly
that may be observed using polarographic, spectral, and enzymatic
ana lyses.
Endpoints: Qua Iitati ve: Developmental changes in: Cytochrome levels;
Oxygen uptake in response to stimulation; Lactic acid output;
Uptake and release of neurotransmitters; Amino acid concentrations;
Redox changes produced in tissue pyridine nucleotides, flavopro-
teins, and cytochromes in response to standard stimulants.
Quantitative: Measurements of: Cytochrome levels; Oxygen uptake
in response to stimulation; Lactic acid output; Uptake and re-
lease of neurotransmitters; Amino acid concentrations; Redox
changes produced in tissue pyridine nucleotides, fIavoproteins,
and cytochromes in response to standard stimulants.
Strengths: Spectral measurements can be applied to as little as 3 mg
of tissue; Responses of tissues dependent upon the integrity of
a wide variety f systems within the tissue (e.g., cell excit-
ability, Na , K -ATPase, neurotransmittance, uptake, release and
intrinsic activity, glycolytic and TCA cycle enzymes, etc.). The
responses to K are multiphasic, one phase probably being appli-
cable to neuronal responses, the other to glial responses. Con-
sequently, the system is unique in that it will detect a wide
variety of types of damage.
Weaknesses: Principle weakness is that it does not lend itself to
immediate identification of mechanism unless there is a direct
effect on energy metabolism proper so that crossovers can be
identified. However, this disadvantage is overcome by the fact
that delays in development resulting from early exposure to Pb
can be almost perfectly correlated with delays in morphological
development (e.g., synaptogenesis).
Status of Development: Being implemented.
Describe: The basic developmental patterns for the cerebral
cortex have been established for the spectral measurements of
cytochrome concentrations and form of the metabolic responses.
Spectral changes induced by K have been correlated with respira-
tory changes, lactic acid output, and changes in tissue adenine
nucleontides. Delays in development have been demonstrated in
these parameters with Pb and correlated with delayed synaptogenesis
in the rat.
Applications: Multimedia.
Samples: Pure Chemicals: Organics; Inorganics. Complex Mixtures:
Industrial; Energy Related; Transportation Related; Other.
105
-------
1306 DIRECT SPECTRAL MEASUREMENT OF THE BIOCHEMICAL DEVELOPMENT OF THE
NERVOUS SYSTEM (continued)
Duration: As short as 3 weeks to as long as 3 months if animals are
to be taken to sexual maturity.
Cost: $2,500 to $25,000, depending upon duration and number of
rep Iicat ions.
Interpretation: Delays in biochemical development of the brain have
been well correlated with delays in morphological development
(synaptogenesis) and behavioral development (activity away from
mother during lactation) in lead-treated animals. With these data
as support, delayed biochemical development may be interpreted as
presumptive evidence of retarded mental development.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Toxicologica1 Assessment Branch.
Persons to Contact: R.J. Bui I, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE.
References: I) Himwich, W.A. Developmental Neurobiology. C.C.
Thomas, Springfield, IL, 1970. pp. 22-46, 311-330, 331-369,
370-392. 2) Bull, R.J., and S.D. Lutkenhoff. J. Neurochem. ,
21:913-922, 1973. 3) Bull, R.J., and J.T. Cummins. J. Neuro-
chem., 21:923-937, 1973. 4) Bull, R.J., and J.J. O'Neill.
Psychopharmacol. Commun., 1:109-115, 1975. 5) Bull, R.J., P.M.
Stanaszek, and S.D. Lutkenhoff. Envir. Hlth. Perspect., 12:
89-95, 1975. 6) Bull, R.J. J. Neurochem., 26:149-156, 1976.
106
-------
CONTENTS: TEST SYSTEMS
2000 Short-Term Tests for Ecological Effects 107
2100 Freshwater and Marine Algae 107
2101 Freshwater Algal Assay Bottle Test 108
2102 Marine Algal Assay Bottle Test 109
2200 Freshwater Animals and Plants Ill
2210 Fish Toxicity Ill
107
-------
2101 FRESHWATER ALGAL ASSAY BOTTLE TEST
Biological Activity Detected: Toxicity; Stimulation.
Principle: Standard test alga (Selenastrum capricornutum) is added to
a test water, cultured under standard light, temperature, and gas
exchange conditions, and evaluated for inhibitory (toxic) or
stimulatory response to pollutant stress.
Endpoints: Qua Iitative: Can be used to screen stimulatory or
inhibitory properties of point and non-point source pollutants.
Quantitative: Response in mg dry wt I -1 of test alga either
stimulatory (% S14) or inhibited (% 114) at day 14. Results are
expressed as % stimulation or % inhibition as compared to control
vs waste concentration.
Strengths: Detection of bioreactive components in a test water,
or of waste discharge; Identification of toxic or stimulatory
components.
Weaknesses: Insufficient application of test to relate to potential
health effects of a pollutant.
Status of Development: Validated.
Describe: N/A.
Applications: Water.
Samples: Pure Chemicals: Nutrients, N03, N02, NH3, Ortho-P,
Tot-P, Heavy metals, Pesticides, Herbicides, Insecticides.
Complex Mixtures: Ambient - receiving water; Industrial - waste
discharges; Energy Related - coal storage, leachates; Other - new
product formulations, i.e., detergents.
Duration: Test: 14 days; Analysis: 5 days; Total: 21 days maximum
in most cases.
Cost: $400 to evaluate a compound or complex waste.
Interpretation: Test data can be used to define nutrient limitation,
heavy metal toxicity, and inhibitory or stimulatory properties of
complex wastes, as they effect ecology of aquatic systems.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-COR, Assessment Criteria Development
Division, Special Studies Branch; ERL-DUL, Newtown Fish Toxi-
cology Station.
Persons to Contact: W.E. Mi Iler. U.S. EPA, ERL-COR, 200 SW 35th St.,
Corvallis, OR 97330, (FTS 420-4775, Commercial 503 757-4775);
T. Shiroyama, U.S. EPA, ERL-COR, 200 SW 35th St., Corvallis, OR
97330, (FTS 420-4776, Commercial 503 757-4776); J.C. Greene, U.S.
EPA, ERL-COR, 200 SW 35th St., Corvallis, OR 97330, (FTS 420-4764,
Commercial 503 757-4764); E. Robinson. U.S. EPA, ERL-DUL, New-
town Fish Toxicology Station, 341 i Church St., Cincinnati, OH
45244, (FTS 684-8601).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OTS.
References: I) Algal Assay Procedure Bottle Test. U.S. EPA, Aug.
1971. 2) Standard Methods for the Examination of Water and
Wastewater. 14th edition, 1975. To be published by ASTM.
108
-------
2102 MARINE ALGAL ASSAY BOTTLE TEST
Biological Activity Detected: Toxicity; Stimulation.
Principle: Standard test algae are added to a test water, cultured
under standard light, temperature, and gas exchange conditions
and evaluated for inhibitory (toxic) or stimulatory response to
polIutant stress.
Endpoints: Qua Iitative: Can be used to screen potential stimulatory
or toxic_properties of pollutants. Quantitative: Response in mg
dry wt I * of the stimulatory (% Sm> or inhibitory (% l^)
response as compared to control vs control to which a waste
concentration has been added.
Strengths: Detection of stimulatory and/or inhibitory bioreactive
components in a test water, and of waste discharge.
Weaknesses: Insufficient application of test (round Robin); Inade-
quate to predict possible health effects of specific pollutants.
Status of Development: Validated.
Describe: Interlaboratory calibration is necessary, as are broad
application studies, i.e., complex wastes, organic compounds,
etc.
Applications: Water.
Samples: Pure Chemicals: Nutrients, i.e., Nitrogen and Phosporous,
Heavy metals, PCB's, Chloramines, Free chlorine. Complex Mixtures:
Ambient - receiving waters; Industrial - waste discharges, dredge
spoil, monochlorinated organics detergent builders.
Duration: Test: 14 days; Analysis: 5 days; Total: 21 days.
Cost: $400 to evaluate a compound of complex waste.
Interpretation: Test data can be used to define nutrient limitation,
heavy metal toxicity, and inhibitory or stimulatory properties of
complex wastes, as they effect ecology of marine ecosystems.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-NAR, Toxicology Branch, Marine Toxi-
cology Team; ERL-COR, Marine and Freshwater Branch; ERL-GB,
Experimental Environments Branch.
Persons to Contact: J. Gentile, U.S. EPA, ERL-NAR, South Ferry Rd.,
Narragansett, Rl 02882, (FTS 838-4843 X244); D. Specht, ERL-
COR, 200 SW 35th St., Corvallis, OR 97330, (FTS 420-4675);
G. Walsh, U.S. EPA, ERL-GB, Sabine Island, Gulf Breeze, FL 32561
(FTS 686-901 I).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OTS.
References: I) Marine Algal Assay Procedure Bottle Test. U.S. EPA,
Dec. 1974. Bioassay Procedures for the Ocean Disposal Permit
Program. EPA-600/9-78-010, U.S. EPA, 1978.
109
-------
CONTENTS: TEST SYSTEMS
2200 Freshwater Animals and Plants
2210 Fish Toxicity Ill
2211 Acute Fish Toxicity Test, Static and
Flow-Through, All Freshwater Species 112
2212 Subchronic Embryo-Larva I, Early Juvenile
Fish Toxicity Test 113
2213 Chronic Fish Toxicity Test, American
Flagfish (Jordanella floridae) 114
2214 Fish Respiratory Activity Toxicity Test,
Electrode Chamber Method 115
2215 Fish Avoidance Test, Gradient Tanks 116
2220 Invertebrate Toxicity 117
I I
-------
2211 ACUTE FISH TOXICITY TEST, STATIC AND FLOW-THROUGH, ALL FRESHWATER SPECIES*
Biological Activity Detected: Toxicity.
Principle: Determine the 96-hr LC50 of various pure compounds and
complex mixtures on freshwater organisms.
Endpoints: Qua Iitative: Behavior. Quantitative: Mortality (LC50).
Strengths: A "hard" number on lethality.
Weaknesses: The LC50 is not protective of aquatic species. There-
fore, one must estimate or test for lower, no-effect concentra-
tions.
Status of Development: Validated.
Describe: The 96-hr flow-through and static aquatic toxicity
tests have been conducted for many years with some cross valida-
tion between laboratories; Some methods were written by Standard
Methods and ASTM.
Applications: Water.
Samples: Pure Chemica1s: Insecticides, Herbicides, Heavy Metals,
PCB's. Complex Mixtures: Industrial - effluents; Energy Related -
dri 1 Ii ng muds, oils.
Duration: Test: 96 hours; Analysis: 96 hours.
Cost: Static test: $300; Flow-through test: $650.
Interpretation: From the tests the lethal effects of a toxicant
along with a statistically valid 95% confidence interval can be
determi ned.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch.
Persons to Contact: C. Stephan, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804 (FTS 783-9510, Commercial 218 727-6692
X570).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) Methods for Acute Toxicity Tests with Fish, Macro-
invertebrates and Amphibians. EPA-600/3-75-009, U.S. EPA, 1975.
*This test is also applied to marine fish. See 2311
I 12
-------
2212 SUBCHRONIC EMBRYO-LARVAL, EARLY JUVENILE FISH TOXICITY TEST
Biological Activity Detected: Toxicity.
Principle: This test involves a 30-day exposure of the embryo-larval,
early juvenile stages of development.
Endpoints: Qua Iitative: Behavior. Quantitative: Survival; Growth;
Deformities; Determination of an estimated maximum acceptable
toxicant concentration (MATC).
Strengths: Direct measure of an MATC; Gives an excellent estimate
of chronic toxicity in I month. Chronic tests themselves would
require 12 months to complete.
Weaknesses: Requires 30 days to complete test; Requires a supply of
fish embryos.
Status of Development: Validated.
Describe: These tests have been run successfully by several
contract laboratories as well as routinely by several EPA
laboratories.
Applications: Water.
Samples: Pure Chemicals: Inorganics, Organics. Complex Mixtures:
Industrial; Energy Related.
Duration: Test: 30 days; Analysis: Included in the 30 days.
Cost: $6,000/test.
Interpretation: This test gives an excellent estimate of the chronic
(life-cycle) toxicity of individual toxicants or complex mixtures
to fish.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physiological
Effects of Toxicants Section.
Persons to Contact: J.M. McKim, U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9567, Commercial 218 727-6692 X567);
W.A. Brungs, U.S. EPA, ERL-DUL, 6201 Congdon Blvd., Duluth, MN
55804, (FTS 783-9546, Commercial 218 727-6692 X546).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) McKim, J.M. Evaluation of Tests with the Early Life-
Stage of Fish for Predicting Long-Term Toxicity. J. Fish Res.
Bd. Can., 34(8):I 148-1154, 1977. 2) U.S. EPA. Proposed Recom-
mended Bioassay Procedure for Egg and Fry Stages of Freshwater
Fish (manuscript). U.S. EPA, Duluth, MN. 3) Sauter, S., K.S.
Buxton, K.J. Macek, and S.F- Petrocelli. Effects of Continuous
Exposure to Lead, Chromium, Copper, and Cadmium on Eggs and Fry
of Selected Freshwater Fish. Ecol. Res. Series, U.S. EPA, Duluth,
MN, 1976.
-------
2213 CHRONIC FISH TOXICITY TEST, AMERICAN FLAGFISH (JORDANELLA FLORIDAE)
Biological Activity Detected: Toxicity.
Principle: Determine the impact of toxicants on survival, growth,
and reproduction of a freshwater fish with a rapid life cycle.
Endpoints: Qua Iitative: Behavior. Quantitative: Growth; Survival;
Reproduction success (fecundity, hatchabi Iity); Determination of
a maximum acceptable toxicant concentration (MATC).
Strengths: Similar in sensitivity to commonly tested freshwater fish,
i.e., brook trout and fathead minnow; Short life cycle compared
to most fish; Data generated on all stages of life cycle including
those stages considered to be most sensitive.
Weaknesses: Fish is semi tropical and may not have direct application
to most U.S. waters.
Status of Development: Validated.
Describe: Many people at ERL-DUL have run this test successfully.
Applications: Water.
Samples: Pure Chemicals: Pesticides, Metals. Complex Mixtures:
Waste oil.
Duration: Test: 6 months; Analysis: Included in 6 months.
Cost: $16,000.
Interpretation: This test can be used as an indicator of potential
chronic fish effects.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physiological
Effects of Toxicants Section.
Persons to Contact: W.A. Brunqs. U.S. EPA, ERL-DUL, 6201 Congdon Blvd.
Duluth, MN 55804, (FTS 783-9546, Commercial 218 727-6692 X546);
R. Spehar. U.S. EPA, ERL-DUL, 6201 Congdon Blvd., Duluth, MN 55804
(FTS 783-9521, Commercial 218 727-6692 X52I).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: I) Smith, W.E. A Cyprinodont Fish, Jordanella floridae,
as a Reference Animal for Rapid Chronic Bioassays. J. Fish.
Res. Bd. Can., 39:329-330, 1973. 2) Spehar, R.L. Cadmium and
Zinc Toxicity to Jordanella floridae. J. Fish. Res. Bd. Can.,
33:1939-1945, 1976. 3) U.S. EPA Committee on Aquatic Bioassays.
Recommended Bioassay Procedures for Brook Trout, Bluegill, Fat-
head Minnow, and Flagfish Chronic Tests. U.S. EPA, Duluth,
MN, 1972.
tV 14
-------
2214 FISH RESPIRATORY ACTIVITY TOXICITY TEST, ELECTRODE CHAMBER METHOD
Biological Activity Detected: Toxicity.
Principle: Bioelectric signals generated by respiratory activities
are electronically amplified and recorded on stripchart records.
Endpoints: Gill purge (cough) and ventilation rates. Qua Iitative:
Behavior. Quantitative: Rate of increase/unit of time.
Strengths: Rapid; Predictive.
Weaknesses: Stripchart records must be interpreted.
Status of Development: Being implemented.
Describe: The test results have been published (see references)
and other researchers are starting to use this method.
Applications: Water.
Samples: Pure Chemicals: Multi. Complex Mixtures: Industrial.
Duration: Test: 4 days; Analysis: I day.
Cost: $750/chemical.
Interpretation: Concentrations which do not cause a statistically
significant increase in gill purge rates are not likely to cause
long-term adverse effects.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physiological
Effects of Pollutants Section.
Persons to Contact: R. Drummond, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804, (FTS 783-9511, Commercial 218 727-6692
X5II; R. Carlson, U.S. EPA, ERL-DUL, 6201 Congdon Blvd., Duluth,
MN 55804, (FTS 783-9591, Commercial 218 727-6692 X59I).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Spoor, W.A., et al. Trans. Am. Fish. Soc., 1971. 2)
Drummond, R., et al. J. Fish. Res. Bd. Can., 1973. 3) Drummond,
R., et al. Trans. Am. Fish. Soc., 1974. 4) Carlson, R., and
R. Drummond. Water Res., 1978. 5) Drummond, R., and R. Carlson.
Ecol. Rep. Series, U.S. EPA, 1978.
15
-------
2215 FISH AVOIDANCE TEST, GRADIENT TANKS
Biological Activity Detected: Toxicity; Physical environment.
Principle: Levels of environmental variables and toxicants avoided
by fish are determined.
Endpoints: Qua Iitative: Avoidance behavior. Quantitative. Loco-
motor activity/unit of time.
Strengths: Short-term tests; Endpoints easy to determine; Collection
of data can be automated.
Weaknesses: Needs validation.
Status of Development: Developmental.
Describe: Laboratory investigation underway.
Applications: Water.
Samples: Pure Chemicals: Dissolved gases, Oxygen, Metals, Organics.
Complex Mixtures: Industrial; Energy Related.
Duration: Test: 4 hours; Analysis: I hour.
Cost: $150.
Interpretation: Avoidance has ecological significance.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Office of the Director.
Persons to Contact: W.A. Spoor, U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9506, Commercial 218 727-6692 X506).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: ORD.
References: I) Spoor, W.A., and R. Drummond. Trans. Am. Fish. Soc.,
101:714-715, 1972.
I 16
-------
CONTENTS: TEST SYSTEMS
2220 Invertebrate Toxicity M7
2221 Acute Invertebrate Toxicity Test, Static
and Flow-Through, All Freshwater Species ... I 18
2222 Subchronic Invertebrate Toxicity Test,
Stream Insects 119
2223 Subchronic Invertebrate Toxicity Tests,
Chironomid (Tanytarsus dissimilis) 120
2224 Chronic Invertebrate Toxicity Test,
Water Flea (Daphnia maqna) 121
2230 Plant Toxicity/Residue 123
I 17
-------
2221 ACUTE INVERTEBRATE TOXICITY TEST, STATIC AND FLOW-THROUGH, ALL
FRESHWATER SPECIES*
Biological Activity Detected: Toxicity.
Principle: Determine the 48-hr LC50 or EC50 of various pure com-
pounds and complex mixtures on freshwater organisms.
Endpoints: Qua Iitative: Behavior. Quantitative: Mortality (LC50)
or Immobilization EC50.
Strengths: A "hard" number on lethality, or measurable effect is
determi ned.
Weaknesses: The LC50 or EC50 is not protective of aquatic species.
Therefore, one must estimate or test for lower, no-effect con-
centrations.
Status of Development: Validated.
Describe: The 48-hr and 96-hr flow-through and static aquatic
toxicity tests have been conducted for many years. Some methods
were written by Standard Methods and ASTM.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides, Heavy Metals,
PCB's. Complex Mixtures: Industrial - effluents; Energy Related
dri I Ii ng muds, oils.
Duration: Test: 48 or 96 hours; Analysis: 48 hours.
Cost: Static test: $300; Flow-through test: $650.
Interpretation: From the tests, the lethal effects of a toxicant
along with a statistically valid 95% confidence interval can be
determi ned.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch;
ERL-DUL, Extramural Program Branch.
Persons to Contact: C. Stephan, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804 (FTS 783-9510, Commercial 218 727-6692
X570); K.E. Biesinqer, U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9524, Commercial 218 727-6692 X524).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) Biesinger, K.E., and G. Christensen. Effects of
Various Metals on Survival, Growth, Reproduction, and Metabolism
of Daphnia maqna. J. Fish Res. Bd. Can., 29(2):1691-1700, 1972.
2) Methods for Acute Toxicity Tests with Fish, Macro!nvertebrates
and Amphibians. EPA-600/3-75-009, U.S. EPA, 1975.
*This test is also applied to marine invertebrates. See 2321
I I;
-------
2222 SUBCHRONIC INVERTEBRATE TOXICITY TEST, STREAM INSECTS
Biological Activity Detected: Toxicity.
Principle: The insects are exposed for 4 weeks. The time length
allows for a good estimate of toxicity.
Endpoints: Qua Iitative: Behavior. Quantitative: Survival LC50;
B i oaccumuI at i on.
Strengths: Allows testing of stream invertebrates.
Weaknesses: Requires a "clean" collecting site near test facility;
Requires good water source since it is a flow-through system.
Status of Development: Developmental.
Describe: System is developed and can be used if the need was
established (i.e., could be implemented but isn't).
Applications: Water.
Samples: Pure Chemicals: Heavy metals, Pesticides. CompI ex
Mixtures: Not tested.
Duration: Test: 4 week exposures; Analysis: Could be included in
test time.
Cost: $2,800 at 4 weeks at $35,000/manyear.
Interpretation: It is a procedure which allows exposure for a time
longer than acute (4-day) tests. This allows a better prediction
of toxic effects.
Level of Complexity: I to 2.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physical Pol-
Iutant Section.
Persons to Contact: R.L. Anderson, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804, (FTS 783-9565, Commercial 218 727-6692
X565).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: I) Spehar, R.L., R.L. Anderson, and J.T. Fiandt. Toxi-
city and Bioaccumulation of Cadmium and Lead in Aquatic Inverte-
brates. Envir. Pollut., 15:195, 1978. 2) Anderson, R.L., and
D. DeFoe. Toxicity and Bioaccumulation of Endrin and Methoxychlor
by Aquatic Invertebrates. 1979. In press.
119
-------
2223 SUBCHRONIC INVERTEBRATE TOXICITY TEST, CHIRONOMID (TANYTARSUS DISSIMILIS)
Biological Activity Detected: Toxicity.
Principle: The animals are exposed from egg to 2nd or 3rd instar
for 10 to 12 days of total exposure.
Endpoints: Qualitative: N/A. Quantitative: Survival; Growth;
LC50 and EC values.
Strengths: Exposure through molting; Can also measure growth effects.
Weaknesses: Static, has only been tested with metals.
Status of Development: Developmental.
Describe: A report is being prepared regarding exposure to cadmium,
lead, copper, and zinc.
Applications: Water.
Samples: Pure Chemicals: Heavy metals. Complex Mixtures: N/A.
Duration: Test: 10 to 12 days exposure; Analysis: Up to 2 weeks
including preparation and clean-up.
Cost: 2 weeks at $35,000/manyear equals approximately $1,400; Chemical
analysis might be additional if complex compounds were used.
Interpretation: The system exposes an insect during embryogenesis,
hatching, growth, and molting. These 4 events are critical to
the survival of the animal.
Level of Complexity: 2 to 3
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physical Pol-
Iutant Section.
Persons to Contact: R.L. Anderson, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804, (FTS 783-9565, Commercial 218 727-6692
X565).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: None, in-house development.
120
-------
2224 CHRONIC INVERTEBRATE TOXICITY TEST, WATER FLEA (DAPHNIA MAGNA)
Biological Activity Detected: Toxicity; Reproduction.
Principle: Animals are exposed for 3 to 4 weeks. The exposure period
includes molting and reproduction.
Endpoints: Qua Iitative: N/A. Quantitative: Survival (LC50);
Reproduction.
Strengths: Low equipment cost; Manpower requirements.
Weaknesses: Renewal system.
Status of Development: Being implemented.
Describe: Procedure has been published and is now being used
with variation in many places.
Applications: Water.
Samples: Pure Chemicals: Metals, Organics. Complex Mixtures:
Industrial; Energy Related.
Duration: Test: 3 to 4 weeks may be a good estimate. This would
include data analysis and perhaps chemical analysis if single
or low number mixtures are used.
Cost: 3 weeks at $35,000/manyear equals approximately $2,100.
Interpretation: Test allows fairly rapid screening of pollutants
with a zooplankton representative.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch.
Persons to Contact: C. Stephan, U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9510, Commercial 218 727-6692 X5IO).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: I) Biesinger, K., and G. Christensen. Effects of Various
Metals on Survival, Growth, Reproduction and Metabolism of Daphnia
magna. J. Fish. Res. Bd. Can., 29(12):1691-1700, 1972. 2) ASTM
DRAFT. Proposed Standard Practice for Conducting Life-Cycle
Toxicity Tests with the Daphnid, Daphnia maqna.
121
-------
CONTENTS: TEST SYSTEMS
2230 Plant Toxicity/Residue 123
2231 Acute Plant Toxicity Test,
Duckweed (Lemma minor) . , , 124
2240 Ecosystem 125
123
-------
2231 ACUTE PLANT TOXICITY TEST, DUCKWEED (LEMNA MINOR)
Biological Activity Detected: Toxicity; Residue.
Principle: Exposure of growing plants for I week.
Endpoints: Qua Iitative: N/A. Quantitative : Growth as reflected in
frond count; EC values.
Strengths: Only flow-through system available for aquatic plants;
Fast.
Weaknesses: Not developed to a point where weakness can be adequately
descri bed.
Status of Development: Developmental.
Describe: Exposure to copper has been completed. Exposure with
other compounds is projected. Completion of procedure is pro-
jected for 1979.
Applications: Water.
Samples: Pure Chemicals: Metals, Organics. Complex Mixtures:
Industrial; Energy Related.
Duration: Test: I week exposure, perhaps I to 2 weeks for prepara-
tion and clean-up.
Cost: 2 weeks at $35,000/manyear equals approximately $1,400; Analysis
cost may be included if simple compounds are used.
Interpretation: This is the only system which allows exposure of an
aquatic plant. Toxicity and bioaccumulation data should be
obtainable from the procedure.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physical Pol-
lutant Section.
Persons to Contact: R.L. Anderson, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804, (FTS 783-9565, Commercial 218 727-6692
X565).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: I) Walbridge, C.T. A Flow-Through Testing Procedure with
Duckweed (Lemna minor). Ecol. Res. Series, EPA 600/3-77-108,
U.S. EPA, 1977.
124
-------
CONTENTS: TEST SYSTEMS
2240 Ecosystem 125
2241 Laboratory Ecosystem 126
2300 Marine Animals 127
2310 Fish Toxicity 127
125
-------
2241 LABORATORY ECOSYSTEM TEST
Biological Activity Detected: Toxicity.
Principle: To determine the effects of toxicants on ecosystem
processes.
Endpoints: Qua Iitative: N/A. Quantitative: Modifications in rates
and components of organic carbon budget includes water inflow
and outflow; System photosynthesis and respiration; Periphyton
growth; Benthic macro!nvertebrates; Sediment accumulation and
degradation; Macrophyte decomposition; MATC, based on change in
the ecosystem processes.
Strengths: More realistic than single species testing; Ihcludes
species and environment interaction; Includes impact of environ-
ment on toxicant; Can follow accumulation of toxicants in food
web; Includes some organisms not traditionally included in fresh-
water toxicology.
Weaknesses: Labor intensive; Difficult to obtain degree of replica-
tion necessary for statistical treatment of data; Complex inter-
actions make data evaluation and interpretation difficult; Must
extrapolate to natural situations.
Status of Development: Developmental.
Describe: Development of preliminary non-toxicant methods has
just been initiated.
Applications: Water.
Samples: Pure Chemicals: Inorganics, Organics. Complex Mixtures:
N/A.
Duration: Test: 3 to 5 months; Analysis: An additional 2 to 3 months.
Cost: $35,000.
Interpretation: Data can be used to identify sensitive ecosystem
processes as well as to determine potential assimilation capacity.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-DUL, Newtown Fish Toxicology Station.
Persons to Contact: S.F. Hedtke, U.S. EPA, ERL-DUL, Newtown Fish
Toxicology Station, 3411 Church Street, Cincinnati, OH 45244,
(FTS 684-8601).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: ORD.
References: Work is presently in developmental stages. Information
on test system has not been published. See Persons to Contact.
126"
-------
CONTENTS: TEST SYSTEMS
2300 Marine Animals 127
2310 Fish Toxicity 127
2311 Acute Fish Toxicity Test, Static and
Flow-Through, All Marine Species 128
2312 Subchronic Embryo-Larval Fish Toxicity Test,
Sheepshead Minnow (Cyprinodon varieqatus). ... 129
2313 Chronic Fish Toxicity Test, Sheepshead
minnow (Cyprinodon varieqatus) 130
2320 Invertebrate Toxicity . 131
127
-------
2311 ACUTE FISH TOXICITY TEST, STATIC AND FLOW-THROUGH, ALL MARINE SPECIES*
Biological Activity Detected: Toxicity.
Principle: Determine the 96-hr LC50 of various pure compounds and
complex mixtures on marine organisms.
Endpoints: Qua Iitative: Behavior. Quantitative: Mortality (LC50).
Strengths: A "hard" number on lethality.
Weaknesses: The LC50 is not protective of aquatic species. There-
fore, one must estimate or test for lower, no-effect concentra-
tions.
Status of Development: Validated.
Describe: The 96-hr flow-through and static aquatic toxicity
tests have been conducted for many years with cross validation
by many laboratories; Some methods were written by Standard
Methods and ASTM.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides, Heavy Metals,
RGB's. Complex Mixtures: Industrial - effluents; Energy Related
dri I Ii ng muds, oiIs.
Duration: Test: 96 hours; Analysis: 96 hours.
Cost: Static test: $300; Flow-through test: $650.
Interpretation: From the tests the lethal effects of a toxicant
along with a statistically valid 95% confidence interval can be
determi ned.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch.
Persons to Contact: C. Stephan, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804 (FTS 783-9510, Commercial 218 727-6692
X570).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) Methods for Acute Toxicity Tests with Fish, Macro-
invertebrates and Amphibians. EPA-600/3-75-009, U.S. EPA, 1975.
'This test is also applied to freshwater fish. See 2211
128
-------
2312 SUBCHRONIC EMBRYO-LARVAL FISH TOXICITY TEST, SHEEPSHEAD MINNOW
(CYPRINODON VARIEGATUS)
Biological Activity Detected: Toxicity; Growth; Pathologic effects.
Principle: To determine the effects of a toxicant on the early life
stages of the sheepshead minnow.
Endpoints: Determine concentrations of a toxicant which affect
survival, growth, behavior, and pathologic effects. Qua Iitative:
Behavior. Quantitative: LC50 values; Significant differences can
be established from experimental and control survival, and growth;
Determination of an estimated maximum acceptable toxicant con-
centration (MATC).
Strengths: A good estimate of toxicity,- particularly chronic toxicity,
can be made in many instances.
Weaknesses: Duration is generally 28 days, or more.
Status of Development: Being implemented.
Describe: Embryo/fry studies have been conducted by ERL-6B and
a private laboratory.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides, PCB's,
Pentachlorophenol. Complex Mixtures: N/A.
Duration: Test: 28 days; Analysis: 28 to 40 days if a chemical analysis
is required for bioconcentration.
Cost: $6,000 to $7,000, depending upon whether or not chemical analyses
are required.
Interpretation: From these tests the concentrations of a pollutant
that affects survival, growth, etc. of a sensitive life stage of
an estuarine fish can be determined.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: D.J. Hansen, U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, EG & G, Rt. 6, Box 1002, Pensacola, FL 32507,
P.R. Parrish.
Program Office Support: OHEE; OPP; OWHM.
References: I) Schimmel, S.C., P.R. Parrish, D.J. Hansen, J.M.
Patrick, Jr., and J. Forester. Endrin; Effects on Several
Estuarine Organisms. Proc. 28th Annu. Conf. Southeast. Asso.
Game Fish Comm., 1974.
129
-------
2313 CHRONIC FISH TOXICITY TEST, SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)
Biological Activity Detected: Toxicity; Growth; Pathologic effects;
Fecund ity.
Principle: to determine the chronic effects of a pollutant on an
estuarine fish.
Endpoints: Qua Iitative: Behavior. Quantitative: Growth; Survival;
Fecundity; Pathologic effects.
Strengths: A good estimate of toxic affects.
Weaknesses: Cost; Time.
Status of Development: Being implemented.
Describe: Both ERL-GB and Bionomics, Pensacola, a private labora-
tory, have conducted the tests, although both laboratories have
not completed full chronic tests on the same chemical for com-
parison.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides. CompI ex
Mixtures: N/A.
Duration: Test: 4 to 5 months; Analysis: 6 to 7 months.
Cost: $35,000 with analytical back-up.
Interpretation: One of the best available estimates of the effects
of a pollutant on an estuarine fish is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: D.J. Hansen, U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, EG & G, Rt. 6, Box 1002, Pensacola, FL 32507.
P.R. Parrish.
Program Office Support: OHEE; OPP; OWHM.
References: I) Hansen, D.J., S.C. Schimmel, and J. Forester. Endrin:
Effects on the Entire Life Cycle of a Salt Water Fish. J.
Toxicol. Envir. Hlth., 3:721-733, 1977.
130
-------
CONTENTS: TEST SYSTEMS
2320 Invertebrate Toxicity 131
2321 Acute Invertebrate Toxicity Test, Static
and Flow-Through, All Marine Species 132
2322 Acute Toxicity Test, Benthic Assemblages 133
2323 Chronic Invertebrate Toxicity Test,
Estuarine Shrimp (Pa laemonetes pugio) 134
2324 Chronic Invertebrate Toxicity Test,
Estuarine mysid (Mysidopsis bahia) 135
2330 Ecosystem 137
-131
-------
2321 ACUTE INVERTEBRATE TOXICITY TEST, STATIC AND FLOW-THROUGH, ALL
MARINE SPECIES*
Biological Activity Detected: Toxicity.
Principle: Determine the 48-hr LC50 or EC50' of various pure com-
pounds and complex mixtures on marine organisms.
Endpoints: Qua Iitative: Behavior. Quantitative: Mortality (LC50)
or EC50.
Strengths: A "hard" number on lethality, or measurable effect, is
determined.
Weaknesses: The LC50 or EC50 is not protective of aquatic species.
Therefore, one must estimate or test for lower, no-effect con-
centrations.
Status of Development: Validated.
Describe: The 48-hr flow-through and static aquatic toxicity
tests have been conducted for many years with cross validation
by many laboratories. Some methods were written by Standard
Methods and ASTM.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides, Heavy Metals,
PCB's. Complex Mixtures: Industrial - effluents; Energy Related
driI I ing muds, oils.
Duration: Test: 48 hours; Analysis: 48 hours.
Cost: Static test: $300; Flow-through test: $650.
Interpretation: From the tests the lethal effects of a toxicant
along with a statistically valid 95% confidence interval can be
determi ned.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch.
Persons to Contact: C. Stephan, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804 (FTS 783-9510, Commercial 218 727-6692
X510).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) Methods for Acute Toxicity Tests with Fish, Macro-
invertebrates and Amphibians. EPA-600/3-75-009, U.S. EPA, 1975.
*This test is also applied to freshwater invertebrates. See 2221
132
-------
2322 ACUTE TOXICITY TEST, BENTHIC ASSEMBLAGES
Biological Activity Detected: Toxicity.
Principle: The acute effects of toxic substances on a macrofunal
genthic microcosm representative of natural benthic assemblages
in the Pacific Northwest will be examined.
Endpoints: Qua Iitative: Behavior, observe animals able to bury in
dredge sediment. Quantitative: Count of survivors and count
of animals able to bury in substrate in relation to water con-
centration provides EC values.
Strengths: Test organism is a sensitive amphipod species, Paraphoxus
epi stomus.
Weaknesses: New species not frequently used in aquat.ic testing.
Status of Development: Developmental.
Describe: This test procedure has only been conducted at the
Newport, Oregon, facility.
Applications: Water; Soil.
Samples: Pure Chemicals: Heavy metals, i.e., Ca, Zn, Cr.
Complex Mixtures: Dredge sediment; Complex wastes.
Duration: Test: 96 hours.
Cost: Not yet determined.
Interpretaton: From this test one can determine if a dredge soil
is acutely toxic to estuarine benthic animals.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Marine and Freshwater Branch, Newport Field Station.
Persons to Contact: R. Swartz, U.S. EPA, ERL-COR, Newport Field
Station, Marine Science Center, Newport, OR 97365, (FTS 423-4111,
Commercial 503 867-4041).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OTS.
References: I) U.S. EPA, Corps of Engineers. Appendix F. Guidance for
Performing Solid State Bioassays. In: Report on Ecological
Evaluation of Proposed Discharge of Dredged Material on Ocean
Waters. Tech. Committees on Criteria for Dredge and Fill
Material. U.S. Army Waterways Station, Vicksburg, MS, July 1977.
133
-------
2323 CHRONIC INVERTEBRATE TOXICITY TEST, ESTUARINE SHRIMP (PALAEMONETES PUGIO)
Biological Activity Detected: Toxicity; Growth; Reproduction.
Principle: Determine chronic effects of a pollutant on the entire
life cycle of Grass Shrimp, Palaemonetes pugio.
Endpoints: Qualitative: N/A. Quantitative: LC50 values;
Significant differences in survival, growth, and fecundity in
experimental animals compared to controls.
Strengths: LC50 values; Significant differences in survival; Deter-
mination of growth and reproduction. Data give good indication
of the effects of a toxicant over the animal's entire life cycle.
Weaknesses: Duration is long, about 5 months for chronic test; 2 to
3 months for partial-chronic.
Status of Development: Developmental.
Describe: Chronic tests have only been conducted at ERL-GB.
Applications: Water.
Samples: Pure Chemi caIs: Pesticides. Complex Mixtures: N/A.
Duration: Test: Approximately 5 months.
Cost: $25,000, including analytical back-up.
Interpretation: A maximum acceptable toxicant concentration can be
established for this estuarine invertebrate.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: D.B. Tyler-Schroeder, U.S. EPA, ERL-GB, Sabine
Island, Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Tyler-Schroeder, D.B. Use of the Grass Shrimp,
Palaemonetes pugio, in a Life Cycle Toxicity Test. Symposium
on Aquatic Toxicology and Hazard Evaluation. ASTM, 1978. In
press. 2) Bioassay Procedures for the Ocean Disposal Permit
Program. EPA-600/9-78-010, U.S. EPA, 1978.
134
-------
2324 CHRONIC INVERTEBRATE TOXICITY TEST, ESTUARINE MYSID (MYSIDOPSIS BAHIA)
Biological Activity Detected: Toxicity; Fecundity; Growth.
Principal: Determine the chronic effects of toxicants on the entire
life cycle of the crustacean species.
Endpoints: Qua Iitative: N/A. Quantitative: LC50 values;
Significant differences in growth, reproduction, and survival.
Strengths: An estimate of chronic toxicity can be determined.
Weaknesses: Relatively high cost and duration of test; Animals are
not always available throughout the year.
Status of Development: Being implemented.
Describe: ERL-GB and Bionomics EG & G, a private laboratory,
have conducted these tests.
Applications: Water.
Samples: Pure Chemicals: Pesticides; Metals. Complex Mixtures:
N/A.
Duration: Test: Approximately 28 days.
Cost: $7,000, including analytical back-up on pure chemicals.
Interpretation: A maximum acceptable toxicant concentration (MATC)
can be established for a marine/estnarine invertebrate in this
test.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: D.W. Nimmo, U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics EG & G, Rt. 6, Box 1002, Pensacola, FL 32507, P.R.
Parrish.
Program Office Support: OHEE; OWP,
References: I) Nimmo,-'D.W., L.H. Bhaner, R.A. Rigby, J.M. Sheppard,
and A.J. Wilson. Mysidopsis bahia: An Estuarine Species Suitable
for Life-Cycle Toxicity Tests to Determine the Effects of a
Pollutant. In: Aquatic Toxicology and Hazard Evaluation,
Mayer, Hamelink, eds. ASTM, STP 634:109-1 16, 1977.
135
-------
CONTENTS: TEST SYSTEMS
2330 Ecosystem 137
2331 Estuarine Microcosym I 138
2332 Estuarine Microcosmy II 139
2333 Estuarine Communities 140
2400 Terrestrial Animals and Plants 141
2410 Plants 141
137
-------
2331 ESTUARINE MICROCOSMY I
Biological Activity Detected: Fate.
Principle: This test assesses transport and degradation potential
of aquatic pollutants.
Endpoints: Qua Iitative: Types of transport; Localization; Mechanisms
of degradation and character of degradation products. Quantitative:
Rates of above processes; Effect of environmental parameters
on rates; EC vaIues.
Strengths: Use of environmental substrates; Mass balance analysis
with radiolabeled pollutants; Versatility; Short turn-around
time.
Weaknesses: Sealing factors from laboratory systems to the environ-
ment.
Status of Development: Developmental.
Describe: Systems have been designed and are in operation. Fate
of pesticides has been tested. Optimization of systems are now in
progress. Field validation is being initiated.
Applications: Water.
Samples: Pure Chemicals: Pesticides, Toxic Organics. CompI ex
Mixtures: Industrial - effluents; Energy Related -oil.
Duration: Test: 4 to 8 weeks/chemical pollutant.
Cost: $2,000/month, not including senior investigator time.
Interpretation: This test supplies data on transport and degrada-
tion in natural system.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-GB, Processes and Effect Branch.
Persons to Contact: A.W. Bourquin, ERL-GB, Sabine Island, Gulf
Breeze, FL 32561, (FTS 686-9011); R.L. Garnas, ERL-GB, Sabine
Island, Gulf Breeze, FL 32561, (FTS 686-9011); P.M. Pritchard,
ERL-GB, Sabine Island, Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OTS; OEMI.
References: I) Bourquin, A.W., R.L. Garnas, P.M. Pritchard, F.G.
Wilkes, C.R. Gripe, and N.I. Rubinstein. Interdependent
Microcosms for the Assessment of Pollutants in the Marine
Environment. Internat. J. of Envir. Studies., 1978. In press.
138
-------
2332 ESTUARINE MICROCOSMY II
Biological Activity Detected: Toxiclty.
Principle: This test assesses the toxicity of pollutants to microbial
growth and microbial degradation processes.
Endpoints: Qua Iitative; Types of microorganisms affected; Total
biomass reduction; Selection of species; Physiological indies
affected; Mechanisms of toxicity. Quantitative; EC values.
Strengths: Quick screen which uses natural assemblages of micro-
organisms.
Weaknesses: Extrapolation from laboratory systems to the environ-
ment; Requires analytical supports.
Status of Development: Being implemented.
Describe: Pesticides and toxic organics have been tested.
Applications: Water.
Samples: Pure Chemicals: Pesticides, Toxic organics, Heavy
metals. Complex Mixtures: Industrial - effluents; Energy Re-
lated -oil.
Duration: Test: 4 to 8 weeks/chemical pollutant.
Cost: $1,500/month, not including senior investigator time.
Interpretation: This test determines toxicant effects on microbial
assemblages.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-GB, Processes and Effect Branch.
Persons to Contact: A.W. Bourquin, ERL-GB, Sabine Island, Gulf
Breeze, FL 32561, (FTS 686-9011); R.L. Garnas, ERL-GB, Sabine
Island, Gulf Breeze, FL 32561, (FTS 686-9011); P.M. Pritchard,
ERL-GB, Sabine Island, Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Georgia State U., Atlanta, GA 30303, D.G. Ahearn; Gulf Coast
Research Laboratory, P.O. Box 26518, New Orleans, LA, W. W. Walker.
Program Office Support: OEM I.
References: I) Bourquin, A.W., P.H. Pritchard, and W.R. Mahaffey.
Effects of Kepone on Estuarine Microorganisms. Developments in
Industrial Microbiology. 1978. In press. 2) Bourquin, A.W.
Effects of Malathion on Microorganisms of an Artificial Salt-Marsh
Environment. J. Envir. Quality, 6:383-378, 1977.
/139
-------
2333 ESTUARINE COMMUNITIES
Biological Activity Detected: Toxicity.
Principle: This test determines concentrations of a toxicant which
offset the settling and development of benthic estuarine com-
munities. These-communities develop in sand substrate from
larvae in unfiltered seawater.
Endpoints: Qua Iitative: N/A. Quantitative: Measure of species
diversity; Number of sensitive phyla, biomass, growth, and total
number of species; EC values.
Strengths: From this test we can see how a toxicant changes the
community makeup by limiting sensitive groups and promoting
growth of others.-
Weaknesses: Duration is long (2 to 4 months); The investigator
must have a good taxonomic background.
Status of Development: Developmental.
Describe: Only ERL-GB and one contractor have completed this
type of community study.
Applications: Water.
Samples: Pure Chemica ks: Pesticides. Complex Mixtures: Energy
Related - drilling muds.
Duration: Test: 2 to 4 months to conduct test; Analysis: another
2 to 4 months to identify the animals.
Cost: $6,000.
Interpretation: From these studies we are able to predict the con-
centration of a toxicant that wilt adversely affect the recruit-
ment and development of estuarine benthic communities.
Level of Complexity: 3 to 4.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: M. Tagatz, ERL-GB, Sabine Island, Gulf Breeze,
FL 32561, (FTS 686-901 I).
Grant/Contract Laboratory Invo-lved and Principal Investigators:
Florida State U., Tallahassee, FL 32306, B. Glasson.
Program Office Support: OHEE; ORD.
References: I) Hansen, D.J. Aroclor 1254: Effect on Composition
of Developing Estuarine Animal Communities in the Laboratory.
Marine Sci., 18:19-33, 1974.
140
-------
CONTENTS: TEST SYSTEMS
2400 Terrestrial Animals and Plants.
2410 Plants 141
2411 Stress Ethylene Bioassay in Plants 142
2412 Measurement of Nitrogenase Activity by
Acetylene Reduction in Nodulated Plants .... 143
2420 Ecosystem 145
141
-------
2411 STRESS ETHYLENE BIOASSAY IN PLANTS
Biological Activity Detected: Alteration of physiological process.
Principle: Environmental stresses cause plants to produce large
amounts of ethylene. The elevated ethylene occurs before and
often in the absence of visual injury.
Endpoints: Qua I itative: Observation of increase of ethylene produc-
tion; In some cases modification of plant growth form.
Quantitative: Extent of increase for comparison with reference
plants and chemicals.
Strengths: More sensitive than visual assessment; Less subject
to variabi Iity.
Weaknesses: Short duration of the phenomena (approx. 48 hours);
The test is designed as a flow-through test and not as a static
system.
Status of Development: Being implemented.
Describe: The test has been used to examine effects of ozone and
chlorine on a variety of plants ranging from pine trees to
potatoes.
AppIications: Ai r.
Samples: Pure ChemicaIs: Gaseous pollutants: Ozone, S02, NO ,
CO. Complex Mixtures: Ambient - air; Industrial - air pollutants;
Energy Related - air pollutants; Transportation Related - air
pollutants; Other - acid rain.
Duration: Test: 3 to 5 days exclusive of plant rearing; Analysis:
Simultaneous with test.
Cost: $2,500.
Interpretation: This test provides evidence of tissue injury. Excess
ethylene production may result in defoliation.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Terrestrial Ecology Branch.
Persons to Contact: D.T. Tinqey, ERL-COR, 200 SW 35th St., Corvallis,
OR 97330, (FTS 420-4621).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Tingey: D.T., C. Standley, and R.W. Field. Stress
Ethylene Evolution, a Measure of Ozone Effects on Plants.
Atmos. Envir., 10:969-974, 1976.
142
-------
2412 MEASUREMENT OF NITROGENASE ACTIVITY BY ACETYLENE REDUCTION IN
NODULATED PLANTS
Biological Activity Detected: Alteration of physiological process.
Principle: Measure reduction of acetylene to ethylene.
Endpoints: Qua Iitative: N/A. Quantitative: Amount of ethylene
produced in relation to pollutant concentration.
Strengths: Requires minimal facilities or equipment; Rapid analysis.
Weaknesses: Needs further evaluation.
Status of Development: Being implemented.
Describe: This test is used to assess the impact of heavy metals
(cadmium) on soybean, alder, and alfalfa nitrogen fixation systems,
AppIicat ions: Soi I.
Samples: Pure ChemicaIs: Heavy metals. Complex Mixtures:
Industrial - sludge.
Duration: Test: 10 to 15 days exclusive of time to grow plants;
Analysis; Immediately following test.
Cost: $5,000/chemical.
Interpretation: This test provides information on the ability of
nodulated plants to fix nitrogen in the presence of stress.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, terrestrial Ecology Branch.
Persons to Contact: C. Wickliff, U.S. EPA, ERL-COR, 200 SW 35th St.,
Corvallis, OR 97330, (FTS 420-4622).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Wickliff, C. PhD Thesis, Oregon State U. 1977.
2) Fishbeck, K., H.J. Evans, and L.L. Boersma. Agronomy J.,
65:429-433, 1973. 3) Huang, Chi-Ying, F.A. Bazzaz, and L.N.
Vanderhoff. Plant Physiology, 54:122-124, 1974.
143
-------
CONTENTS: TEST SYSTEMS
2420 Ecosystem 145
2421 Terrestrial Microcosm Chamber 146
2422 Soil Core Microcosm 147
2423 Soil/Litter Microcosm 148
2500 Physical-Chemical/Bio-accumulation 149
145.
-------
2421 TERRESTRIAL MICROCOSM CHAMBER
Biological Activity Detected: Toxicity; BioaccumuI at ion; Biomagnifi-
cation; Community processes.
Principle: Radiolabeled pesticides at or below accepted field ap-
plication rates are applied to assess fate, species and population
effects.
Endpoints: Qua Iitative: Microcosms should be viewed as a tool
which at present provide only trends in fate or effects.
Quantitative: N/A.
Strengths: Higher link between bench and field; Lower cost than
field studies; Provides indices of distribution.
Weaknesses: Cost/unit; Ambiguity of results; Not validated.
Status of Development: Developmental.
Describe: At present ERL-COR is developing a testing protocol
consisting of not one system, but a methodology that utilizes
"benchmark" data as well as various microcosms depending on the
information required.
Applications: Air; Water; Soil.
Samples: Pure Chemicals: Heavy metals, Pesticides, Gaseous
pollutants. Complex Mixtures: Industrial - effluents; Energy
Related - air pollutants; Transportation Related - air pollutants.
Duration: Test: 2 months; Analysis: 3 months.
Cost: $50 to $100,000, depending on the compound.
Interpretation: Fate results utilized ecological mag: bioaccumuI at ion,
biodegradabi Iity and degradation. Effects' results are sti I I open
to discussion other than acute toxicity.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Terrestrial Ecology Branch.
Persons to Contact: J.D. Gi |e, U.S. EPA, ERL-COR, 200 SW 35th St.,
Corvallis, OR 97330, (FTS 420-4649); J.W. Gi Ilett, U.S. EPA,
ERL-COR, 200 SW 35th St., Corvallis, OR 97330, (FTS 420-4622).
Grant/Contract Laboratory Involved and Principal Investigators:
U: of Michigan, Ann Arbor, Ml 48104, E. Goodman; U. of Wisconsin,
Madison, Wisconsin 53706, P. Lichtenstein.
Program Office Support: OHEE; OPP.
References: I) Gi Ilett, J.W., and J.D. Gi le. Pesticide Fate in
Terrestrial Laboratory Ecosystems. Intern. J. Envir. Studies,
10:15-22, 1976.
146
-------
2422 SOIL CORE MICROCOSM
Biological Activity Detected: Monitor community processes.
Principle: The use of an intact system provides a more realistic
representation of a natural system. The test relies on the
production of CC>2 and nutrient loss as indicators of community
f itness.
Endpoints: Qua Iitative : N/A. Quantitative: Rate of C02 production;
Loss of nutrients through leachate.
Strengths: Small size; Low cost/unit; Simple design.
Weaknesses: High degree of variability between units, due to natural
variabi Iity.
Status of Development: Developmental.
Describe: To date, only inorganics have been examined in the
soil core. ERL-COR is currently evaluating the system for use
with a broad spectrum of toxics.
AppI ications: Soi I.
Samples: Pure ChemicaIs: Heavy metals, Pesticides. CompI ex
Mixtures: Industrial - waste effluents.
Duration: Test: 3 months; Analysis: I to 2 months.
Cost: $2,000 to $3,000.
Interpretation: This test reflects the ability of a community to
decompose material. It also reflects the impact of a chemical
on nutrient cycling.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Terrestrial Ecology Branch.
Persons to Contact: J. Gi le, U.S. EPA, ERL-COR, 200 SW 35th St.,
Corvallis, OR 97330, (FTS 420-4649).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OTS.
References: I) Draggon, S. Soil Core Microcosm. Proceedings of
EPA Workshop on Terrestrial Microcosms. J.W. Gi Ilett, ed.
Corvallis, OR, 1977. In press.
147
-------
2423 SOIL/LITTER MICROCOSM
Biological Activity Detected: Monitor decomposition process.
Principle: Carbon dioxide production and 02 consumption are monitored as
indicators of microbial respiration.
Endpoints: Qua!itative: 02; CC>2; Microbiota; Microarthropods; Nema-
todes. Quantitative: Change in respiratory rate with changing
chemical concentration.
Strengths: Low cost/unit; Simple analytical technique and equipment;
Overall simplicity of system.
Weaknesses: Limited type of data generated.
Status of Development: Developmental.
Describe: Soil is tested in I qt. mason jars with respiration
C02 and 02 measured by simple titrametric or gasometric techniques.
AppIications: Soi I.
Samples: Pure ChemicaIs: Heavy metals, Pesticides. CompI ex
Mixtures: Ambient; Industrial; Energy Related.
Duration: Test: I to 4 weeks; Analysis: Simultaneous with test.
Cost: $500/chemical.
Interpretation: Changes in respiration may reflect the ability of a
community to decompose organic matter.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Terrestrial Ecology Branch.
Persons to Contact: B. Lighthart, U.S. EPA, ERL-COR, 200 SW 35th St.,
Corvallis, OR 97330, (FTS 420-4832).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Soil/Litter Microcosm. In: Proceedings of EPA
Workshop on Terrestrial Microcosms. J.W. GiIlett, ed. U.S. EPA,
Corvallis, OR, 1977. In press.
148
-------
CONTENTS: TEST SYSTEMS
2500 Physical-Chemical/Bio-accumulation 149
2501 Bioconcentration Studies, Marine and
Freshwater Animals 150
2502 Reverse-Phase High Pressure Liquid
Chromatography (HPLC) 151
Indices.
153
149
-------
2501 BIOCONCENTRATION STUDIES, MARINE AND FRESHWATER ANIMALS
Biological Activity Detected: Bioconcentration.
Principle: To determine the uptake and depuration rates of pure
chemicals in tissues.
Endpoints: Qua Iitative: Flesh tainting. Quantitative: Uptake
rates; Depuration rates; Bioconcentration factors (uptake of
a pure chemical in tissues divided by the concentration of the
chemical in the exposure water).
Strengths: A good estimate of the uptake, depuration rates of a
pure chemical, plateau level, and bioconcentration factors.
Weaknesses: Duration at least 1 month; Requires complicated chemical
analyses; Does not always reflect a true picture of bioconcentration
in the field because of uptake from other sources (of food and
sediment).
Status of Development: Being implemented.
Describe: Various laboratories within EPA and private industry
are now using this method. The procedure has not yet been
accepted by ASTM as it is still being revised.
Applications: Water.
Samples: Pure ChemicaIs: Insecticides, Herbicides, PCB's,
Chlorinated hydrocarbons, Radiolabeled material. Complex Mixtures:
N/A.
Duration: Test: 30 to 60 days; Analysis: 30 to 60 days.
Cost: $4,000.
Interpretation: From the above tests the potential of contaminants
reaching man's seafoods can be determined.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Chemical Pollutant
Section; ERL-GB, Experimental Environments Branch.
Persons to Contact: G.D. Veith, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9534); S.C. Schimmel, ERL-GB, Sabine
Island, Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, EG & G, Rt. 6, Box 1002, Pensacola, FL 32507,
P.R. Parrish.
Program Office Support: OHEE; OPP.
References: I) ASTM Committee on Bioconcentration of Test Materials
in Fishes and Oysters. (E-35 Committee on Pesticides). In prepara-
tion.
150
-------
2502 REVERSE-PHASE HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)
Biological Activity Detected: Bi©concentration.
Principle: HPLC retention time correlates with the logarithm of the
partition coefficient which correlates with the bioconcentration
factor of organic chemicals in fish tissue.
Endpoints: Qua Iitative: N/A. Quantitative; Provides bioconcentra-
tion potential of organic chemicals in animal tissue.
Strengths: Rapid; Inexpensive.
Weaknesses: Requires a HPLC.
Status of Development: Being implemented.
Describe: Chemical analysis is utilizing HPLC to obtain Log P
values, which can be correlated with water solubility and bio-
concentration factors for organic chemicals.
Applications: Water.
Samples: Pure Chemicals: Organic; Inorganic. Complex Mixtures:
Industrie I.
Duration: Test: 10 to 20 min; Analysis: 10 to 20 min.
Cost: $100.
Interpretation: This work sets forth a rapid, inexpensive method for
screening chemicals for their bioconcentration potential in the
envi ronment.
Level of Complexity: 0.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physiological
Effects of Pollutants Section.
Persons to Contact: G.D. Veith, U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9534, Commercial 218 727-6692 X534).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: I) Veith, G.D., and Morris. Ecol. Rept. Series, U.S. EPA,
1978. In press. 2) Lee, Huges, and G.D. Veith. Water, Air, and
Soil Pollution, 8:749-484, 1977. 3) Veith, G.D., and N.M.
Austin. Detection and Isolation of Bioaccumulable Chemicals
in Complex Effluents. In: Identification and Analysis of
Organic Pollutants in Water. L.A. Keith, ed. Ann Arbor
Science Publishers, Ann Arbor, Ml, 1976. pp. 297-304.
151
-------
INDICES
153
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER
NUMBER NAME OF TEST SYSTEM PAGE
MM Integrated System: Lung Organ Culture System,
Tissue Homogenates, Purified Enzyme Systems 2
1112 Integrated System: Tissue Homogenates, Purified
Enzyme Systems ... 3
1113 Instrumental Methods of Detecting Functional and
Metabolic Damage to Target Tissues 4
1114 Trachea I Mucoci Iiary Transport 6
IM5 Platelet Secretion Measured by ATP Release 8
1116 Mammalian Platelet and Fibroblast Impairment 9
IM7 Lymphocyte Cytotoxicity 10
1118 Impairment of Neutrophil Phagocytosis I I
MI9 Human Lung Fibroblasts (WI38) 12
I I MO Chinese Hamster Ovary (CHO) Clonal Toxicity. "|4
Mill Rabbit Alveolar Macrophage (RAM) 16
II I 12 Rat Hepatocyte (Liver Cell) 18
I II 13 Chinese Hamster Ovary (CHO) Cytotoxicity and
Mutagenicity 19
1121 LD50 22
1122 Whole Animal LD50 - Ora I , Dermal 23
1131 Integrated System: General Clinical 26
1132 Sleep-Time Study 27
1133 Metabolism of Chlorinated Hydrocarbons in
Subhuman Primates 28
(continued)
154
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
NAME OF TEST SYSTEM PAGE
1134 Metabolic Profiles 29
1135 Model Substrate Metabolism 30
1136 Xenobiotic Mechanisms 32
1137 Oxidant Production by Leukocytes and Alveolar
Macrophages Measured by ChemiIuminescence 33
1138 Cyclic Nucleotide Concentrations in Leukocytes
and Alveolar Macrophages 34
1141 Toxicity of Aerosolized Pollutants/Acute and
Subacute 36
1142 Toxicity of Gaseous and Vapor Phase/Acute and Subacute. . 37
1143 Inhalation LC50 Tests 38
1144 Deposition and Clearance of Radioactive Materials
Following Inhalation Exposure 39
1145 Integrated System: Pulmonary Function and Pulmonary
Metabolism 40
I 146 Pulmonary Function in Rats 41
1147 Pulmonary Function of Unanesthetized Guinea Pigs 42
M48 Arterial Blood Gas Measurement in Conscious Rats 43
1149 Infectivity Model 44
I 1410 In-Vivo Alveolar Macrophage Cytotoxicity. . 45
||5I Integrated System: NeurobehavioraI ToxicologicaI
Assessment 48
152 Integrated System: The Effects of Selected Organic
Contaminants in Drinking Water on the Functions
of the Reproductive, Nervous, and Immune Systems. ... 49
153 Computer Automated Analysis of Patterned Behavior
in the Primate 51
(conti nued)
55
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
NAME OF TEST SYSTEM PAGE~
1154 Computer Automated Analysis of Learning and Memory
in the Primate 52
1155 Integrated System: Behavioral Analysis of Rats —
Developmental, Locomotor, Exploratory, and Learned
Behavior 53
1156 Integrated System: Instrumental Methods of Detecting
Functional and Metabolic Damage to the Nervous
System 54
1211 Sa Imone I I a typhimuri urn (Ames) 58
1212 Escherichia col i (WP2) 60
1213 Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Forward and Reverse Mutation 61
1214 Body Fluid Analysis 62
1215 Bacterial Plasmids 63
1216 Mouse Lymphoma (L5I78Y). 64
1217 Chinese Hamster Ovary Cells (CHO)
Drug Resistance 65
1218 Chinese Hamster CelIs (CHO)
Nutritional Competency 66
1219 Chinese Hamster Lung Cells (V79) 67
12110 Drosophila melanogaster, Sex Linked Recessive Lethal ... 68
121 I I Tradescant i a Stamen Hair 69
121 12 Maize Waxy Locus Assay 70
121 13 In-Vivo Cytogenetics in Mice 7|
1221 Baci I lus subti I is rec~ 74
1222 Escherichia col i, PoI A~ 75
1223 Mitotic Recombination and Gene Conversion in
Saccaromyces cerevi siae 76
(conti nued)
156
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
E$NUMBEREMNAME OF TEST SYSTEMPAG?
1224 Unscheduled DNA Synthesis (UDS) 77
1225 Sister-Chromatid Exchange Formation (SCE) 78
1226 In-Vivo Assessment of DNA Damage 79
1227 Intact Rodent Hepatocytes in Primary Culture 80
1228 In-Vivo DNA Binding 81
1229 Chinese Hamster Cells (CHO)
Unscheduled DNA Synthesis (UDS) 82
1231 C3HIOTI/2CL8 Mouse Embryo Fibroblast Oncogenic Trans-
formation with Exogenous Metabolic Activation 84
1232 C3HIOTI/2CL8 Mouse Embryo Fibroblast Oncogenic Trans-
formation 85
1233 BHK-21 Mammalian Cell Oncogenic Transformation 86
1234 Syrian Hamster Embryo Oncogenic Transformation
(Focus Assay) 87
1235 BALB 3T3 Oncogenic Transformation and Mutagenesis
with Exogenous Metabolic Activation 88
1236 BALB 3T3 Oncogenic Transformation 89
1237 Pulmonary Adenoma Bioassay in Mice 90
1241 Tumor Induction in Massive Crustaceans, Molluscs,
and Teliost Fish 92
1242 MFO Induction as an Indicator of Toxicity Exposure. ... 93
1243 Limb Regeneration System 94
1244 Isogenic Fish 95
1245 Integrated System: Development of Mutagen/Carcinogen
Activation, Concentration, Separation, and
Weathering Systems 96
1246 Biphenyl Hydroxylase 97
(conti nued)
157
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
NAME °F TEST SYSTEM PAGE
1301 Mammalian Teratology 100
1302 Perinatal Toxicology 101
1303 Fetal Toxicity in Rats, Mice, Guinea Pigs/
Hamsters 102
1304 Developmental Toxicity in Neonatal Rats 103
1305 Teratology In-Vivo/In-Vitro 104
1306 Direct Spectral Measurement of the Biochemical Develop-
ment of the Nervous System 105
2101 Freshwater Algal Assay Bottle Test 108
2102 Marine Algal Assay Bottle Test 109
2211 Acute Fish Toxicity Test, Static and Flow-Through,
Al I Freshwater Species : I 12
2212 Subchronic Embryo-Larval, Early Juvenile Fish Toxicity
Test 113
2213 Chronic Fish Toxicity Test, American Fiagfish
(JordaneI I a flori dae) 114
2214 Fish Respiratory Activity Toxicity Test, Electrode
Chamber Method 115
2215 Fish Avoidance Test, Gradient Tanks 116
2221 Acute Invertebrate Toxicity Test, Static and Flow-
Through, AlI Freshwater Species 118
2222 Subchronic Invertebrate Toxicity Test, Stream Insects . . 119
2223 Subchronic Invertebrate Toxicity Test, Chironomid
(Tanytarsus d iss imi I is) 120
2224 Chronic Invertebrate Toxicity Test, Water Flea
(Daphnia magna) 121
2231 Acute Plant Toxicity Test, Duckweed (Lemma minor) .... 124
2241 Laboratory Ecosystem. 126
(conti nued)
158
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
E NUMBE™ NAME OF TEST SYSTEM PAGE
2311 Acute Fish Toxicity Test, Static and Flow-Through,
All Marine Species 128
2312 Subchronic Embryo-Larva I Fish Toxicity Test, Sheepshead
Minnow (Cyprinodon variegatus) 129
2313 Chronic Fish Toxicity Test, Sheepshead Minnow
(Cypri nodon variegatus) 130
2321 Acute Invertebrate Toxicity Test, Static and Flow-
Through, Al I Marine Species 132
2322 Acute Toxicity Test, Benthic Assemblages 133
2323 Chronic Invertebrate Toxicity Test, Estuarine Shrimp
(Pa I aemonetes pugio) 134
2324 Chronic Invertebrate Toxicity Test, Estuarine Mysid
(Mysidopsi s bah ia) 135
2331 Estuarine Microcosmy I. ...... 138
2332 Estuarine Microcosmy I I 139
2333 Estuarine Communities 140
2411 Stress Ethylene Bioassay in Plants 142
2412 Measurement of Nitrogenase Activity by Acetylene
Reduction in Nodulated Plants 143
2421 Terrestrial Microcosm Chamber 146
2422 Soil Core Microcosm 147
2423 Soil/Litter Microcosm 148
2501 Bioconcentration Studies, Marine and Freshwater Animals . 150
2502 Reverse-Phase High Pressure Liquid Chromatography
(HPLC) 151
159
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER*
NAME OF TEST SYSTEM ^NUMBER™
Acute Fish Toxicity Test, Static and Flow-Through,
Al 1 Freshwater Species . .
Acute Fish Toxicity Test, Static and Flow-Through,
Al 1 Marine Species
Acute Invertebrate Toxicity Test, Static and Flow-
Through, Al 1 Freshwater Species
Acute Invertebrate Toxicity Test, Static and Flow-
Through, Al 1 Marine Species
Acute Plant Toxicity Test, Duckweed (Lemma minor) . . .
Acute Toxicity Test, Benthic Assemblages
Algal Assay Bottle Test, Freshwatert
Algal Assay Bottle Test, Mar i net .
Alveolar Macrophage Cy totox i c i ty , In-Vivot
(Ames) or Salmonella typhimuriumt
Arterial Blood Gas Measurement in Conscious Rats. . . .
Bacillus subtil is rec~
Bacterial Plasmids. ....
BALB 3T3 Oncogen ic Transformation
BALB 3T3 Oncogen ic Transformation and Mutagenesis
with Exogenous Metabolic Activation
(continued)
22 1 1 ....
2311 ....
2221 ....
2321 ....
2231 ....
2322 ....
2101 ....
2102 ....
1 1410 ....
1211 ....
1148 . . . .
1221 ....
1215 ....
1236 ....
1235 ....
PAGE
1 12
128
1 18
132
124
133
108
109
45
58
43
74
63
89
88
* Integrated Systems are listed twice: once under "Integrated Systems" and once
under the title proper.
tThis test is listed under two titles.
160
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™ PAGE
Behavioral Analysis of Rats —Developmental, Loco-
motor, Exploratory, and Learned Behavior:
Integrated System I 155 .... 53
BHK-21 Mammalian Cell Oncogen ic Transformation 1233 .... 86
Bioconcentration Studies, Marine and Freshwater
Animals 2501 .... 150
Biphenyl Hydroxylase 1246 .... 97
Body Fluid Analysis 1214 .... 62
C3HIOTI/2CL8 Mouse Embryo Fibroblast Oncogenic
Transformation. 1232 .... 85
C3HIOT1/2CL8 Mouse Embryo Fibroblast Oncogenic
Transformation with Exogenous Metabolic
Activation 1231 .... 84
Chinese Hamster Cells (CHO)
Nutritional Competency 1218 .... 66
Chinese Hamster Cells (CHO)
Unscheduled DNA Synthesis (UDS) 1229 .... 82
Chinese Hamster Lung Cells (VF9) 1219 .... 67
Chinese Hamster Ovary Cells (CHO)
Clonal Toxicity Assay I M 10 .... 14
Chinese Hamster Ovary Cells (CHO)
Cytotoxicity and Mutagenicity Assay I I I 13 .... 19
Chinese Hamster Ovary Cells (CHO)
Drug Resistance 1217 .... 65
Chronic Fish Toxicity Test, American Flagfish
(Jordanel la f loridae) 2213 .... 114
Chronic Fish Toxicity Test, Sheepshead Minnow
(Cyprinodon variegatus) 2313 .... 130
Chronic Invertebrate Toxicity Test, Estuarine Mysid
(Mysidopsis bajij_aj. 2324 .... 135
(conti nued)
161
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™ PAGE
Chronic Invertebrate Toxicity Test, Estuarine Shrimp
(Pa I aemonetes pugio) 2323 .... 134
Chronic Invertebrate Toxicity Test, Water Flea
(Daphnia magna) 2224 .... 121
Computer Automated Analysis of Learning and Memory
in the Primate 1154 .... 52
Computer Automated Analysis of Patterned Behavior
in the Primate 1153 .... 51
Cylic Nucleotide Concentrations in Leukocytes
and Alveolar Macrophages I 138 .... 34
Cytogenetics in Mice, In-Vivot 121 13 .... 71
Deposition and Clearance of Radioactive Materials
Following Inhalation Exposure 1144 .... 39
Development of Mutagen/Carcinogen Activation,
Concentration, Separation, and Weathering
Systems: Integrated System 1245 .... 96
Developmental Toxicity in Neonatal Rats 1304 .... 103
Direct Spectral Measurement of the Biochemical
Development of the Nervous System 1306 .... 105
DNA Binding, In-Vivot 1228 .... 81
DNA Damage, In-Vivo Assessmentt 1226 .... 79
Drosoph ij_a me lanogaster, Sex Linked Recessive Lethal. . 121 10 .... 68
Effects of Selected Organic Contaminants in Drinking
Water on the Functions of the Reproductive, Ner-
vous, and Immune Systems: Integrated System 1152 .... 49
Escherichia coj_i_ (WP2) 1212 .... 60
Escher ich ia col i, Po I A 1222 .... 75
Estuarine Communities 2333 .... 140
Estuarine Microcosmy I 2331 .... 138
(conti nued)
162
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™ PAGE
Estuarine Microcosmy II 2332 .... 139
Fetal Toxicity in Rats, Mice, Guinea Pigs/Hamsters. . . 1303 .... 102
Fish Avoidance Test, Gradient Tanks 2215 .... 116
Fish Respiratory Activity Toxicity Test, Electrode
Chamber Method 2214 .... 115
Freshwater Algal Assay Bottle Testt 2101 .... 108
General Clinical Pathology: Integrated System 1131 .... 26
Human Lung Fibroblasts (WI38) 1119 .... 12
Impairment of Neutrophi I Phagocytosis . 1118 .... II
Infectivity Model 1149 .... 44
Inhalation LC50 Tests 1143 .... 38
Instrumental Methods of Detecting Functional and
Metabolic Damage to the Nervous System:
Integrated System I 156 ....
Instrumental Methods of Detecting Functional and
Metabolic Damage to Target Tissues 1113 .... 4
Intact Rodent Hepatocytes on Primary Culture 1227 .... 80
Integrated System: Behavioral Analysis of Rats —
Developmental, Locomotor, Exploratory, and
Learned Behavior I 155 .... 53
Integrated System: Development of Mutagen/Carcinogen
Activation, Concentration, Separation and Weathering
Systems ........ 1245 .... 96
Integrated System: The Effects of Selected Organic
Contaminants in Drinking Water on the Functions of the
Reproductive, Nervous, and Immune Systems ...... 1152 .... 49
Integrated System: General Clinical Pathology .... 1131 .... 26
(conti nued)
163
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™ PAGE
Integrated System: Instrumental Methods of Detecting
Functional and Metabolic Damage to the Nervous
System I 156 .... 54
Integrated System: Lung Organ Culture System, Tissue
Homogenates, Purified Enzyme Systems MM .... 2
Integrated System: NeurobehavioraI Toxicologica1
Assessment 1151 .... 48
Integrated System: Pulmonary Function and Pulmonary
Metabolism 1145 .... 40
Integrated System: Tissue Homogenates, Purified
Enzyme Systems 1112 .... 3
Isogenic Fish 1244 .... 95
In-Vivo Alveolar Macrophage Cytotoxicityt I 1410 .... 45
In-Vivo Assessment of DNA Damaget 1226 .... 79
In-Vivo Cytogenetics in Micet 12113 .... 71
In-Vivo DNA Bindingt 1228 .... 81
Laboratory Ecosystem „ . 2241 .... 126
LD50 M2I .... 22
Limb Regeneration System 1243 .... 94
Lung Organ Culture System, Tissue Homogenates,
Purified Enzyme Systems: Integrated System MM .... 2
Lymphocyte Cytotoxicity 1117 .... 10
Maize Waxy Locus Assay 121 12 .... 70
Mammalian Platelet and Fibroblast Impairment 1116 .... 9
Mammalian Teratology 1301 .... 100
Marine Algal Assay Bottle Testt 2102 .... 109
(cont i nued)
164
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™ PAGE
Measurement of Nitrogenase Activity by Acetylene
Reduction in Nodulated Plants 2412 .... 143
Metabolic Profiles 1134 .... 29
Metabolism of Chlorinated Hydrocarbons in Subhuman
Primates 1133 .... 28
MFO Induction as an Indicator of Toxicity Exposure. . . 1242 .... 93
Mitotic Recombination and Gene Conversion in
Saccaromyces cerevi si ae 1223 .... 76
Model Substrate Metabolism 1135 .... 30
Mouse Lymphoma (L5I78Y) 1216 .... 64
NeurobehavioraI ToxicologicaI Assessment:
Integrated System 1151 .... 48
Oxidant Production by Leukocytes and Alveolar Macro-
phages Measured by Chemi I uminescence 1137 .... 33
Perinatal Toxicology 1302 .... 101
Platelet Secretion Measured by ATP Release 1115 .... 8
Pulmonary Adenoma Bioassay in Mice 1237 .... 90
Pulmonary Function and Pulmonary Metabolism:
Integrated System 1145 .... 40
Pulmonary Function in Rats 1146 .... 41
Rabbit Alveolar Macrophage (RAM) Mill .... 16
Rat Hepatocyte (Liver cell) I I I 12 .... 18
Pulmonary Function of Unanesthetized Guinea Pigs. ... 1147 .... 42
Reverse-Phase High Pressure Liquid Chromatography
(HPLC) 2502 .... 151
Saccharomyces_ cerevisiae, Sch i zosaccharomyces pombe,
Forward and Reverse Mutation 1213 .... 61
(conti nued)
165
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™PAGE
Salmonella typh imuri urn (Ames)t 1211 .... 58
Sister-Chromatid Exchange Formation (SCE) 1225 .... 78
Sleep-Time Study 1132 .... 27
Soil Core Microcosm 2422 .... 147
Soil/Litter Microcosm 2423 .... 148
Stress Ethylene Bioassay in Plants 2411 .... 142
Subchronic Embryo-Larval, Early Juvenile Fish
Toxicity Test 2212 .... 113
Subchronic Embryo-Larval Fish Toxicity Test,
Sheepshead Minnow (Cyprinodon varieqatus) 2312 <. . . . I291
Subchronic Invertebrate Toxicity Test, Chironomid
(Tanytarsus dissimi I I is) 2223 .... 120
Subchronic Invertebrate Toxicity Test, Stream Insects . 2222 .... I 19
Syrian Hamster Embryo Oncogenic Transformation
(Focus Assay) 1234 .... 87
Teratology In-Vivo/ln-Vitro 1305 .... |Q4
Terrestrial Microcosm Chamber 2421 .... 146
Tissue Homogenates, Purified Enzyme Systems:
Integrated System 1112 .... 3
Toxicity of Aerosolized Pollutants/Acute and Subacute . 1141 .... 36
Toxicity of Gaseous and Vapor Phase/Acute and Subacute. 1142 .... 37
Trachea I Mucociliary Transport 1114 ....
6
Tradescantia Stamen Hair 121 I I .... 69
Tumor Induction in Massive Crustaceans, Molluscs, and
Tel iost Fish 1241 .... 92
Unscheduled DNA Synthesis (UDS) 1224 .... 77
(conti nued)
166
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™ PAGE
Whole Animal LD50 -Oral and Dermal 1122 .... 23
Xenobiotic Mechanisms 1136 .... 32
167
-------
INDEX III: EXPERIMENTAL SUBJECT LISTING
ORGANISM - SPECIES
TEST TYPE
IN-VITRO
IN-VIVO
OTHER
BACTERIA
Bad I I us subti I is
Escherichia coli
SaImoneI I a typh imurium
PLANTS
Algae
Duckweed
Mai ze
Plants - genera I
Tradescantia
Yeast
INSECTS
Fru it Fl ies
Stream Insects
FISH
American Flagfish
Freshwater - general
1221
1212
1222
121 I
1214
1215
2101
2102
1215
1214
1223
2502
TEST SYSTEM NUMBER*
2231
121 12
241 I
2412
121 I I
1213
J2I4
1223
121 10
2222
2213
221 I
2212
2214
2215
2501
(conti nued)
*An underlined number indicates that the test system is being applied to more
than one organism/species/test type.
168
-------
INDEX III: EXPERIMENTAL SUBJECT LISTING (continued)
ORGANISM - SPECIES TEST TYPE
IN-VITRO IN^VIVO OTHER
TEST SYSTEM NUMBER
FISH (continued)
Marine - general 2502 1242
231 I
2501
Sheepshead Minnow 2312
2313
Teliost 1241
(conti nued)
169
INVERTEBRATES
Ch i ronomi d 2223
Crustaceans 1241
Estuarine Mysid 2324
Estuarine Shrimp 2323
Freshwater - general 2221
2501
Marine - general 2321
2501
Mollusc 1241
Water Flea 2224
MAMMALS
Chinese Hamster I I I 10
I I I 13
1217
1218
1219
1225
1229
Guinea Pig I'45
1147
1303
Hamster 1233 1303
Man IM9
1224
Mouse 1216 121 I 13
1224 1226
1225 1237
1227 1301
-------
INDEX III: EXPERIMENTAL SUBJECT LISTING (continued)
ORGANISM - SPECIES
MAMMALS (continued)
Mouse
Rabbit
Rat
Subhuman Primates
Syrian Hamster
TEST TYPE
IN-VITRO IN-VIVO OTHER
TEST SYSTEM NUMBER
1228 1302
1231 1303
1232 1305
1235
1236
1 1 1 1
1 1 12
1 1 13
1 1 14
1 1 15
1 1 16
1 1 17
1 1 18
II 1 12
1 1 1 1
137
138
1410
121
122
131
132
134
135
136
141
142
143
144
145
146
148
149
151
152
153
155
156
303
304
306
133
153
154
1225
1234
OTHER
Benth ic AssembI age
Estuarine Community
(conti nued)
170
2322
2333
-------
INDEX III: EXPERIMENTAL SUBJECT LISTING (continued)
ORGANISM - SPECIES
IN.mkQ
QJHER
TEST SYSTEM NUMBER
OTHER (continued)
Estuarine Microcosmy
Laboratory Ecosystem
Soil Core Microcosm
Soil/Litter Microcosm
Terrestrial Microcosm Chamber
2331
2333
2241
2422
2423
2421
\i\,
-------
INDEX IV: CONTACT PERSONNEL IN ALPHABETICAL ORDER
CONTACT PERSONNEL
Anderson, R.L.
Bercz, J.P.
Biesinger, K.E.
Bourquin, A.W.
Brown, M.M.
Brungs, W.A.
Bull , R.J.
Campbel 1 , K. 1 .
Carlson, R.
Casciano, D.
Chadwick, R.W.
Char 1 es, J .
Chernoff, N.
Clarke, N.
Claxton, L.
Copeland, M.F.
Couch , J .
Courtney, K.D.
Daniel , B.
Drummond, R.
Gardner, D.E.
U.S. EPA LABORATORY
ERL-DUL
HERL-CIN
ERL-DUL
ERL-GB
HERL-RTP
ERL-DUL
HERL-CIN
HERL-CIN
ERL-DUL
NCTR
HERL-RTP
HERL-RTP
HERL-RTP
HERL-CIN
HERL-RTP
HERL-RTP
ERL-GB
HERL-RTP
HERL-CIN
ERL-DUL
HERL-RTP
(conti nued )
TEST SYSTEM NUMBER*
2222,
1 131 ,
1236.
2221 .
2331 ,
1216.
2212,
1 1 13,
1 132,
1 156,
1306.
1 152.
2214.
121 1,
1 135.
1 141,
1303,
1215.
121 1,
121 13,
1 135.
1241.
1301,
1215,
2214.
1132
1 144,
2223.
1211, 1213,
2332.
2213.
1121, 1131,
1136, 1155,
1226, 1236,
1217, 1227.
1 142.
1304.
1212, 1214,
1221, 1222.
1302, 1305.
1228.
1136 1143,
1 149, 1 1410.
*An underlined number indicates that the test system is used by more than one
of the above indicators.
172
-------
INDEX IV: CONTACT PERSONNEL IN ALPHABETICAL ORDER (continued)
CONTACT PERSONNEL
Garnas, R.L.
Genti le, J.
Gi le, J.D.
Gi 1 left, J.W.
Graham, J .A.
Green, J.C.
Hansen, D.J.
Hatch, G.
Hedtke, S.F.
Huisingh, J . L.
Jackim, E.
Kavlock, R.
Kowa 1 , N . E .
Lazear, E.
Lee, S.D.
Lighthart, B.
Linder, R.
Li ngg , R.D.
Malcolm, A.R.
McCabe, L.J.
McKim, J.M.
Mi 1 ler, W.E.
Moore, W.
Nesnow, S.
N i mmo , D . W .
O'Nei 1 , J .J.
Orthoefer, J.
Pahren, H.
U.S. EPA LABORATORY
ERL-GB
ERL-NAR
ERL-COR
ERL-COR
HERL-RTP
ERL-COR
ERL-GB
HERL-RTP
ERL-DUL, NFTS
HERL-RTP
ERL-NAR
HERL-RTP
HERL-CIN
NCTR
HERL-CIN
ERL-COR
HERL-RTP
HERL-CIN
ERL-NAR
HERL-CIN
ERL-DUL
ERL-COR
HERL-CIN
HERL-RTP
ERL-GB
HERL-RTP
HERL-CIN
HERL-CIN
TEST SYSTEM NUMBER*
2331, 2332.
2102.
2421, 2422.
2421.
1114, II 17.
2101.
2312, 2313.
1115, 1137, 1138.
2241 .
1119, 1 1 MO, 1 1 1 1 1 ,
1 1 1 12, 1 1 1 13, 121 1,
1212, 1221.
1229.
1303, 1304.
1119, 1219, 1236.
121 1.
MM, 1112.
2423.
1 122.
1133. 1134.
1218, 1225, 1229.
121 1 , 1211 1.
2212.
2101.
1143, 1144.
1134, 1231, 1232,
1234, 1235.
2324.
1 145.
1237.
1116, II 18, 121 1 ,
1213, 1233.
(conti nued)
173
-------
INDEX IV: CONTACT PERSONNEL IN ALPHABETICAL ORDER (continued)
CONTACT PERSONNEL U.
Pepelko, W.E.
Pesch, G.G.
Pritchard, P.H.
Reiter, L.
Richards, N.
Robinson, E.
Sandhu, S.S.
Sch imme 1 , S .C.
Schoor, P.
Sh i royama, T.
Specht, D.
Spehar, R.
Spoor, W.A.
Stara, J.F.
Stephan, C.
Swartz, R.
Tagatz, M.
Tingey, D.T.
Ty ler-Schroeder , D.B.
Veith, G.D.
Walsch, G.
Waters, M.D.
Wickl iff, C.
Wi ester, M.J .
.S. EPA LABORATORY
HERL-CIN
ERL-NAR
ERL-GB
HERL-RTP
ERL-GB
ERL-DUL, NFTS
HERL-RTP
ERL-GB
ERL-GB
ERL-COR
ERL-COR
ERL-DUL
ERL-GB
HERL-CIN
ERL-DUL
ERL-COR
ERL-GB
ERL-COR
ERL-GB
ERL-DUL
ERL-GB
HERL-RTP
ERL-COR
HERL-CIN
TEST SYSTEM NUMBER*
1146, 1148.
1225.
2331, 2332.
1151, 1153, 1154.
1211, 1242, 1243,
1244, 1245, 1246.
2101 .
1212, 1213, 1214,
1216, 1217, 1219,
121 10, 121 1 | , 121 12,
12113, 1221, 1223,
1225.
2501.
1242.
2101.
2102.
2213.
2215.
1211, 1214, 12113.
2211, 2221, 2224,
2311, 2321.
2322.
2333.
241 1.
2323.
2501, 2502.
2102.
1119, 11111,1211,
1212, 1213, 1214,
1216, 1217, 1219,
121 10, 1211 1 , 121 12,
12113, 1221, 1222,
1223, 1224, 1225,
1234.
2412.
1 147.
7-4
-------
INDEX V: U.S. EPA LABORATORIES IN ALPHABETICAL ORDER
U.S. EPA LABORATORY
TEST SYSTEM NUMBER1*
Environmental Research Laboratory-
Corva Mis, Oregon
Environmental Research Laboratory-
Duluth, Minnesota
Environmental Research Laboratory-
Gulf Breeze, Florida
Environmental Research Laboratory-
Narrangansett, Rhode Island
Health Effects Research Laboratory-
Ci nci nnati, Oh io
Health Effects Research Laboratory-
Research Triangle Park,
North Caroli na
2101, 2102, 2322, 2411, 2412,
2213, 2214,
2223, 2224,
232 I, 2501,
1243, 1244,
2312, 2313,
2332, 2333,
1218, 1225, 1229, 2102.
2421,
2101,
2215,
2231,
2502.
121 1,
1245,
2323,
2501 .
2422,
22 II,
2221,
2241,
1241,
1246,
2324,
2423.
2212,
2222,
2311,
1242,
2102,
2331,
IN,
119,
134,
147,
211,
2111,
236,
114,
1 1 1 1,
134,
141,
149,
211,
217,
21 13,
225
235,
304.
112,
121,
136,
148,
213,
21 13,
238,
M5,
1 1 12,
135,
142,
1410,
212,
219,
221,
227,
301,
113,
131,
143,
152,
214,
226,
306.
117,
1 1 13,
136
143,
151,
213,
21 10,
222,
231,
302,
1 1 16,
1 132,
1 144,
1 155,
1215,
1228,
1 1 19,
1 122,
1 137,
I 144,
1 153,
1214,
1211 1,
1223,
1232,
1303,
1 1 18,
1 133,
1 146,
1 156,
1219,
1233,
1 1 1 10,
1 132,
1 138,
1 145,
1 154,
1216,
121 12,
1224,
1234,
1304,
National Center for ToxicologicaI
Research-Jefferson, Arkansas
1211, 1217, 1227.
*An underlined number indicates that the test system is used by more than one
laboratory.
175
-------
INDEX VI: U.S. EPA DEPARTMENTAL LISTING IN ALPHABETICAL ORDER
DEPARTMENT U.S. EPA LABORATORY CONTACT PERSONNEL
ENVIRONMENTAL RESEARCH LABORATORY-CORVALLIS
200 SW 35th Street
Corvallis, Oregon 97330
Assessment Criteria Division
Special Studies Branch J.C. Green
W.E. Mi 1ler
T. Sh i royama
Ecological Effects Research Division
Marine and Freshwater Branch D. Specht
Newport Field Station . . . R. Swartz
Terrestrial Ecology Branch S. Draggon
J.D. Gi le
J.W. Gi Ilett
B. Lighthart
D.T. Tingey
C. Wickliff
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
6201 Congdon Boulevard
Duluth, Minnesota 55803
Extramural Program Branch K.E. Biesinger
Newtown Fish Toxicology Station S.F. Hedtke
E. Robinson
Research Branch
Chemical Pollutant Section G.D. Veith
Physical Pollutant Section R.L. Anderson
Physical Effects of Toxicants Section W.A. Brungs
R. Carlson
R. Drummond
J.M. McKim
R. Spehar
(conti nued)
176
-------
INDEX VI: U.S. EPA DEPARTMENTAL LISTING IN ALPHABETICAL ORDER (continued)
DEPARTMENT U.S. EPA LABORATORY CONTACT PERSONNEL
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
(conti nued)
Technical Assistance Branch C. Stephan
ENVIRONMENTAL RESEARCH LABORATORY-GULF BREEZE
Sabine Island
Gulf Breeze, Florida 32561
Experimental Environments Branch D.J. Hansen
D.W. N i mmo
S.C. Schimmel
M. Tagatz
D.B. Tyler-Schroeder
C. Walsch
Office of the Director J. Couch
(Carcinogenic Research Team) N. Richards
W.A. Spoor
Processes and Effects Branch A.W. Bourquin
R.L. Garnas
P.H. Pritchard
ENVIRONMENTAL RESEARCH LABORATORY-NARRANGANSETT
South Ferry Road
Narrangansett, Rhode Island 02882
Toxicology Branch
Genetic Toxicology Team E. Jackim
A.R. Malcolm
G.G. Pesch
Marine Toxicology Team J. Genti le
HEALTH EFFECTS RESEARCH LABORATORY-CINCINNATI
26 West St. Clair Street
Cincinnati, Ohio 45268
Field Studies Division
ToxicologicaI Assessment Branch .... J.P- Bercz
N.E. Kowal
L.J. McCabe
H. Pahren
J.F. Stara
(conti nued)
177
-------
INDEX VI: U.S. EPA DEPARTMENTAL LISTING IN ALPHABETICAL ORDER (continued)
DEPARTMENT U.S. EPA LABORATORY CONTACT PERSONNEL
HEALTH EFFECTS RESEARCH LABORATORY-CINCINNATI
(continued)
Laboratory Studies Division
Exposure Evaluation Branch R.D. Lingg
Functional Pathology Branch S.D. Lee
J. Orthoefer
W. Moore
W.E. Pepelko
M.J. Wiester
ToxicologicaI Assessment Branch R.J. Bull
K.I. Campbel I
N. Clarke
B. Daniel
HEALTH EFFECTS RESEARCH LABORATORY-RESEARCH TRIANGLE PARK
Research Triangle Park
North Carolina 2771 I
Clinical Studies Division
Biomedical Research Branch D.E. Gardner
J.A. Graham
G. Hatch
J.J. O'NeiI
Experimental Biology Division
Developmental Biology Branch N. Chernoff
R. Kavlock
Neurobiology Branch L. Reiter
Environmental Toxicology Division
Biochemistry Branch M.M. Brown
R.W. Chadwick
L. Claxton
M.F. Copeland
J.L. Huisingh
S. Nesnow
S.S. Sandhu
M.D. Waters
(conti nued)
178
-------
INDEX VI: U.S. EPA DEPARTMENTAL LISTING IN ALPHABETICAL ORDER (continued)
DEPARTMENT U.S. EPA LABORATORY CONTACT PERSONNEL
HEALTH EFFECTS RESEARCH LABORATORY-RESEARCH TRIANGLE PARK
(conti nued)
Toxic Effects Branch J. Charles
K.D. Courtney
R. Linder
NATIONAL CENTER FOR TOXICOLOGICAL RESEARCH
Jefferson, Arkansas 72079
Division of Mutagenesis Research
Somatic Ce I I Section D.A. Casciano
E. Lazear
79
-------
INDEX VII: GRANT/CONTRACT LABORATORIES IN ALPHABETICAL ORDER
GRANT/CONTRACT LABORATORY
PRINCIPAL INVESTIGATOR
TEST SYSTEM NUMBER
American Health Foundation
Naylor Dana Institute for
Disease Prevention
Hammond House Road
Va I ha I la, New York 10595
Ball State University
Muncie, Indiana 47306
Bionomics EG & G
Route 6, Box 1002
Pensacola, Florida 32507
Bionomics, Inc.
Wareham, Massachusetts
Brookhaven National Labo-
ratories
Long Island, New York
California, University of
Davis, California 95616
California, University of
School of Medicine
Los Angeles, California 90032
California, University of
Medical Center
San Francisco, California 94132
Cincinnati, University of
Cincinnati, Ohio 45221
Cincinnati University of
Medical Center
Cincinnati . Ohio 43221
G.M. Wi1 Iiams
D. Ada I is
P.R. Parrish
K.S. Macek
S. Sauter
L. Shirer
E. Goldstein
M.G. Mustafa
R.S. Bhatnagar
C. Smith
J.C. Loper
D. Lang
(conti nued)
1227.
I I 14.
2501, 2312, 2313,
2324.
2211, 2212, 2221,
2311, 2321.
221
121 I,
1236.
2212, 2221
2311, 2321.
121 I I.
I 1410.
I I 12.
I I I I.
I 133.
1236.
180
-------
INDEX VII: GRANT/CONTRACT LABORATORIES IN ALPHABETICAL ORDER (continued)
GRANT/CONTRACT LABORATORY
PRINCIPAL INVESTIGATOR
TEST SYSTEM NUMBER
Colorado, University of
Medical Center
4200 East 9th Avenue,
Denver Colorado 80262
Denver Research Institute
Denver, Colorado 80210
Florida State University
Tallahassee, Florida 32306
Georgia State University
Atlanta, Georgia 30303
Gulf Coast Research Laboratory
P.O. Box Drawer AC
Ocean Springs, Mississippi 39564
Gulf South Research Institute
P.O. Box 26518
New Orleans, Louisiana
NT Research Institute
10 West 35th Street
Chicago, Illinois 60616
I II i noi s, Un iversity of
Urbana, I I Iinois 61801
Iowa State University
Ames, Iowa 50010
Litton Biometics, Inc.
Nicholson Lane
Kensington, Maryland
Louisiana State University
Medical School
New Orleans, Louisiana 70112
Medical College of Virginia
Richmond, Virginia 23298
C.C. Solomons
W.L. Weston
J. Schmidt-Coderis
B. Glasson
D.G. Ahearn
W.W. Walker
N. Gruener
E. Kl ine
C. Aranyi
R. Ehrlich
L. Schiff
M. Plewa
W.E. Lloyde
D.T. Brusick
W. Pel on
R.L. Balster
J.F. BorzeIleca
W.L. Dewey
A.E. Munson
I I 16.
I I 18.
1246.
2333.
2332.
2332.
1119, 1219, 1236.
1245.
II I I 1, I 1410.
I 149.
I I 14.
12112.
1153, 1154.
121 I.
121 I.
I 152.
I 152.
I 152.
I 152.
(conti nued)
-------
INDEX VII: GRANT/CONTRACT LABORATORIES IN ALPHABETICAL ORDER (continued)
GRANT/CONTRACT LABORATORY
PRINCIPAL INVESTIGATOR
TEST SYSTEM NUMBER
Michigan, University of
Ann Anbor, Michigan 48104
Microbiological Associates
Bethesda, Maryland 20014
Missouri, University of
Columbia, Missouri 65201
North Carolina, University of
A. Beaudoin
E. Goodman
R. Kouri
L. Schectman
C. Marianseld
J.T. O'Conner
A. Col Iier
Chapel Hill, North Carolina 27514 D. Humm
Northrop Services, Inc.
P.O. 'Box 12313
Research Triangle Park,
North Carolina 27709
B. Adkins
N.E. Garrett
1305.
2421.
1231, 1235, 1237.
1231, 1235, 1237.
121 I.
121 I I.
I I 14.
1244.
1114, 1149.
1119, I I I 10, I I I I I,
I I I 13.
Oak Ridge National Laboratory
P.O. Box Y
Oak Ridge, Tennessee 37830
Ohio State University
Columbus, Ohio 43210
Oregon, University of
Eugene, Oregon 97403
Rockefeller University
1230 York Avenue
New York, New York 10021
Southern Mississippi,
University of
Hattiesburg, Mississippi 39401
Southwest Research Institute
San Antonio, Texas 78284
Stanford Research Institute
Menlo Park, California 94025
A. Hsie
1217.
R.W. Hart
M. Mix
M. Bowers
1226.
1241.
1 1 17.
B.J. Martin
E. Cause
A. Mitchel I
G. Newel I
V.F. Simmons
(conti nued)
1241.
I 1410.
1216, 1219, 12110,
1224.
121 I I, 121 13.
1211, 1212, 1213,
1214, 1221, 1222,
1223, 1225.
182
-------
INDEX VII: GRANT/CONTRACT LABORATORIES IN ALPHABETICAL ORDER (continued)
GRANT/CONTRACT LABORATORY
PRINCIPAL INVESTIGATOR
TEST SYSTEM NUMBER
Syracuse Research Corporation
Merri I I Lane
Syracuse, New York 13210
University of Texas
Medical Branch
Galveston, Texas 77550
University of West Florida
Pensacola, Florida 325Q4
Wisconsin, University of
Madison, Wisconsin 53706
J. Saxena
M. Legator
J. Bazlis
R. Rao
P, Lichtenstein
1233.
1211, 1214, 12113.
121 I.
1242, 1243, 1245.
2,421.
183
-------
INDEX VIII: STATUS OF DEVELOPMENT DISTRIBUTION OF THE TEST SYSTEMS
STATUS OF DEVELOPMENT
Developmenta 1 :
Being implemented:
Val i dated:
TEST SYSTEM
115, II 16, 1117, II 18,
215, 1218, I2III, 12112,
233, 1234, 1242, 1244,
1222, 2223, 2231, 2241,
M2I, 2322, 2423.
Ill, 1119, 1 II 10, 1 1 1 1 1,
137, 1138, 1141, 1142,
152, 1155, 1214, 1216,
229, 1232, 1234, 1236,
307, 2214, 2224, 2312,
2412, 2501, 2502.
112, II 13, 1114, 1 121,
143, 1144, 1146, 1148,
212, 1213, 12110, 12113,
302, 1303, 2101, 2102,
2311, 2321.
NUMBER
1 1 1 12,
1226,
1245,
2322,
1 1 1 12,
1 145,
1217,
1237,
2313,
1 131,
1 149,
1221,
221 1,
1 1 1 13,
1227,
1246,
2323,
1 122,
1 147,
1219,
1241 ,
2324,
1 132,
141 10,
1222,
2212,
1 153,
1228,
1304,
2331,
1 134,
1 1410,
1223,
1243,
2332,
1 133,
1 156,
1224,
2213,
1 154,
1231,
2215,
2333,
1 135,
1 151,
1225,
1305,
241 1 ,
1 136,
121 1,
1301,
2221 ,
184
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TOXICITY TEST SYSTEMS
ENDPOINTS TEST SYSTEM NUMBER
Adrenal function 1131.
Airway resistance 1145, 1147.
Alveolar macrophage - bacterial activity 1137.
Alveolar macrophage - cytotoxicity 11410.
Alveolar macrophage - enzymatic profile I 1410.
Alveolar macrophage - morphology I 1410.
Arterial blood PCO I 148.
Arterial blood P I 148.
U2
ATP, ADP, AMP tissue levels 1113, 1116, Mill, I I I 12,
1156, 1306.
ATP release from platelets I I 15.
Bicarbonate concentration in the blood 1148.
Biochemical development of the nervous 1156, 1306.
system
Blood pH I 148.
Brain neurochemistry 1152, 1156.
Breathing frequency 1145, 1147.
cAMP N38.
Cardio-vascular response 1147.
Cel I number 1119, HIM.
cGMP N38.
(continued)
185
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TOXICITY TEST SYSTEMS
(continued)'
ENDPOINTS
TEST SYSTEM NUMBER
Ci I iary beating
Cl in icaI symptoms
Colony formation
Comparative metabolism
Creative phosphokinase
CytologicaI
Cytochrome P-450
CNS Function
Deposition
Dose-response curve
Electrocardiograms (EC6)
Enzyme activity
FetaI anomaIies
Fetal malformation
Fetal toxicity
Growth of neonataI rats
Heart-rate
Hematology
Hi stoIogy
Hydrolytic enzyme activity
Immune response and host resistance
(conti nued)
186
I I 14.
1121, 1122.
I I I 10, I I I 13.
1133, 1134, 1135.
1302.
1114, 11410.
1136, 1156, 1306.
1151, 1152, 1156.
1114, 1144.
1122, 1141, 1142, 1143,
I 145.
I 147.
MM, MI2, 1132, 1133,
1142, 1145, I 1410.
1301, 1303, 1304, 1305,
1306.
1301, 1303.
1301, 1302, 1303, 1304,
1305, 1306.
1304.
I 147.
I 152.
IM4, 1141, 1142.
I II10.
I 152.
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TOX,ICITY TEST SYSTEMS
(continued)
ENDPOINTS
Infectivity model
1 sozyme prof i les
LC50
LD50
Learning behavior in rats
Learning in primates
Lethal ity
Liver enzymes
Locomotor activity
Lung clearance
Lung comp 1 iance
Lymphocyte cytotoxicity
Mammalian teratology
Maximum tolerated dose (MTD)
1/2 MTD
1 /4MTD
Metabol i tes
Memory in primates
Minute volumes
Morphology
Motor and sensory activity
TEST
1 149.
1 131,
1141,
1 121,
1 151,
1 151,
1 141,
1 152,
1305,
1136.
1 151,
1144.
1 145,
1 1 17.
1301.
1 136.
1 136.
1 136.
1 1 1 1,
1 134,
1 151,
1 145,
1 1 19,
1 151,
SYSTEM NUMBER
1302.
1142, 1143.
1 122.
1 152.
1153, 1154.
1142, 1143, 1149,
1301, 1303, 1304,
1306.
1152, 1153, 1304.
1147.
1112, 1113, 1133,
1135, 1136, 1142.
1153, 1154.
I 147.
1 1 1 1 1, 1 1 1 12.
1152, 1153, 1154.
(conti nued)
187
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TOXICITY TEST SYSTEMS
(continued)
ENDPOINTS
Mutation frequency
NADH
NADPH
Neutrophil phagocytosis
Oxidatant production in alveolar macrophages
Phagocytic index
Platelet function
Post natal development
Protein determinations
Pulmonary mechanics
Residual lung volumes
Righting reflex
Sensory moda 1 ity
Sequencing of behavior
Serum constituents
Serum isoenzyme patterns
S 1 eep-t ime
Somite development
Static compliance curves
Task discrimination
Teratogen ic i ty
Thyroid function
TEST
1 1 1 13.
1 1 13,
1 1 13,
1 1 18.
1 137.
1 1 18.
1 1 15,
1302,
1 1 19,
1 145,
1 145,
1 132.
1 151 ,
1151,
1 131.
1 131,
1 132.
1305.
1 145,
1 151,
1301,
1305,
1 131.
SYSTEM NUMBER
1 156.
1 156.
INN.
1 1 16.
1304, 1305, 1306.
1305.
1146, 1147.
1146, 1147-
1152, 1153.
1152, 1153.
1302.
1146, 1147.
1 154.
1302, 1303, 1304,
1306.
(conti nued)
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TQXICITY TEST SYSTEMS
(continued)
• . .-I- -I I- ..I., ,. I. - ,,!,..• . .^— I.) •! .. . - - ..I. ....
—— ''"''- ... • •• • . I f . f—. ..^.•1.. . •• —
ENDPOINTS TEST SYSTEM NUMBER
Tidal volume I 145, I 147.
Total eel I protein I I 19, I I I I I, 11112.
Urinary constituents 1131.
Viability of neonatal rats 1304, 1306.
189
-------
INDEX X: ENDPOINT DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS
ENDPOINTS
IN-VITRO
TEST TYPE"
IN-VIVO
OTHER
Care i nogenes i s
Mutagenes i s
Chromosomal aberrations
1231
1232
1233
1234
1235
1236
1225
TEST SYSTEM NUMBER*
1237
1241
1242
1246
121 13
1225
1218
1219
121 10
Point mutation 1211
1212
1214
1216
1217
1218
1219
Primary DMA Damage 1215
1218
1219
1221
1222
1224
1227
1229
1213
1214
121 10
121 1 1
121 12
1242
1245
1223 1245
1226
1228
1242
endpoint.
190
-------
INDEX XI: ENDPOINT DISTRIBUTION OF THE ECOLOGICAL TEST SYSTEMS
ENDPOINT
TEST SYSTEM NUMBER*
Bioconcentration Factors (BCF)
Effect Concentration (EC)
50% Effect Concentration (EC50)
50% Lethal Concentration (LC50)
Maximum Acceptable Toxicant
Concentration (MATC)
2501, 2502.
2101, 2102, 2212, 2214,
2215, 2223, 2224, 2231,
2312, 2313, 2322, 2331,
2332, 2333, 2411, 2412,
2421, 2422, 2423.
2221, 2222, 2321.
221 I, 2221, 2222, 2223,
2224, 231 1, 2312, 2321,
2323, 2324.
2212, 2213, 224lt, 2312.
*An underlined number indicates that the test system has more than one
endpoi nt.
tThis endpoint is based on change within the ecosystem processes,
-------
INDEX XII: APPLICATION DISTRIBUTION TABLE
APPLICATION:
TEST SYSTEM
NUMBER:
AIR
1 1 14
1 1 15
i 1 19
II MO
MM!
WATER SOIL
1 1 15
1 1 16
1 1 18
1 1 19
II MO
1 1 II 1
FOOD MULTIMEDIA
MM
1 1 12
1 1 13
1 1 15
1 1 17
3
I 138
I 141
I 142
I 145
I 146
I 147
I 148
I 149
I 1410
II M3
I 138
138
I 146
I 148
I 152
I 155
(continued)
i 146
I 148
II M2
I 121
I 122
I 131
I 132
I 133
I 134
I 135
I 136
I 137
I 138
143
144
147
I 151
I 153
I 154
192
-------
INDEX XII: APPLICATION DISTRIBUTION TABLE (continued)
APPLICATION: AIR WATER SOIL FOOD MULTIMEDIA
TEST SYSTEM
NUMBER: 1156
121 I
1212
1213
1214
1215
1216
1217
1218
1219
121 10
121II 121 I 1
12112 12112 12112
121 13
1221
1222
1223
1224
1225
1226
1227
1228
1229 1229
1231
1232
1233
1234
1235
1236
1237
1241
1242
1243
1244
1245 1245
1246 1246
1301
1302
1303
1304 1304 1304
1305
1306
2101
2102
221 I
(conti nued)
193
-------
INDEX XII:-•• APPLICATION DISTRIBUTION TABLE (continued)
APPLICATION: AIR WATER SOIL FOOD MULTIMEDIA
TEST SYSTEM
NUMBER: 2212
2213
2214
2215
2221
2222
2223
2224
223!
2241
231 I
2312
2313
2321
2322 2322
2323
2324
2331
2332
2333
241 I
2412
2421 2421 2421
2422
2423
2501
2502
194
-------
INDEX XIII: SAMPLE DISTRIBUTION OF THE GENERAL AND PERINATAL
TEST SYSTEMS
SAMPLES TEST SYSTEM NUMBER
Aerosols 1141.
Air I I 13, I I 14, I I 15, I 141,
1142, 1145, 1146, 1147,
1148, 1149, 11410.
Alkylating agents IIII3.
Aromati c ami nes I I i I 3.
Asbestos 1146.
AWT effluent I I 19.
Benzene I I 52.
Cadmium MM, M 12, 1114, Mill,
1149, II4IOT
Chlorinated aliphatic hydrocarbons 1133, 1134.
Chlorinated aromatic hydrocarbons 1133, 1134.
Coal dust H46, 1148.
Copper \\\\ .
Diesel fuel 'Ml, "13, 1145, 1146,
1147, 1148.
Dioxin "52, 1301.
Dolomite I I MO.
Drugs M35-
(conti nued)
195
-------
INDEX XIII: SAMPLE DISTRIBUTION OF THE GENERAL AND PERINATAL TOXICITY
TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
Energy related pollutants
Ethers
Fly ash
Gases
Gasoline exhaust
Heavy metals
Hydrocarbons
Industrie I pol1utants
I norgan i cs
Manganese
Mercury
Nickel
Nitrogen oxides
Nitroso amines
NO
Non-ionizing radiation
1113, 1114, II15, I I 17,
I I I 10, I I I I I, I 132, I 138,
1145, 1146, 1149, I 1410,
1 155,
1 152.
1 1 19,
MM,
1 1 13,
II II,
1 151,
1 1 16,
1 1 13,
1 1 19,
1 138,
1 148,
1 156,
1 1 19,
1303,
MM,
II 1 1,
1 1 14,
MM,
1 156,
1 1 1 10,
1 145,
1 1 14,
1 1 19,
1 154,
1 1 18.
1 1 14,
II 110,
1 141 ,
1 149,
1306.
II 1 12,
1304,
II II 1,
1 1 12.
Mill,
1 138,
1306.
1 II 1 1.
1149, 11410.
1115, M46.
II 113, 1146,
1303, 1306.
1 II 13.
1115, II 17,
Mill, M 32,
1142, 1146,
11410, 1155,
1301, 1302,
1306.
1 1410.
1149, 11410.
1 145.
11113.
I 138.
II I I,
1 145,
1112, 1137, 1138,
1149, 11410.
151.
(conti nued)
196
-------
INDEX XIII: SAMPLE DISTRIBUTION OF THE GENERAL AND PERINATAL TOXICITY
TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
Organ ics
Oxidants
Ozone
Paraquat
Parti cu 1 ates
Pest i ci des
Pheny Is
Platium
Radioactive aerosols
Rock dust
Silicic aci d
so2
Stack gases
Su 1 fates
Su 1 f uri c aci d
Su 1 fur oxi des
Technical grade materials
1 1 16,
1 122,
1301,
1305,
1 1 12,
1 1 12,
1 148,
1 145,
1 1 1 10,
1 149,
1 122,
1 151,
1303,
1 152.
1 1 1 1 1.
1 144.
1 148.
1 11 10.
II 1 1,
1 148.
1 146,
1 145,
1 1 14,
MM,
1 122,
1119, 1 II 12, 1 I2|,
1131, 1145, 1155,
1302, 1303, 1304,
1306.
1146, 1148.
1137, 1138, 1145,
1149, 11410.
1 148.
1137, 1141, 1145,
1 1410.
1135, 1141, 1142
1152, 1301, 1302,
1304, 1305.
1112, 1145, 1147,
1 148.
1146, 1148, 1149.
1 145.
1 145.
1141, 1142, 1143.
(conti nued)
197
-------
INDEX XIII: SAMPLE DISTRIBUTION OF THE GENERAL AND PERINATAL TOXICITY
TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
Toxic substances
Trace metals
Transportation related pollutants
Tri halomethanes
Vaporizable liquids
Water
Xenob iotics
Zi nc
I II 10, I 121, I 122, I 131.
1132, 1135, 1141, 1142,
1143, 1145, 1151, 1152,
1301, 1302, 1303, 1304,
1305.
IIII, 1119, 11111,1148,
11410, 1151, 1155, 1303,
1306.
1113, 1114, II 15, I I 17,
1138, 1145, 1146, 1147,
I 148, I 149, I 1410, I 155,
1156, 1306.
I 152.
I 142.
1115, II16.
1135, 1136.
MM.
198
-------
INDEX XIV: SAMPLE DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS*t
SAMPLES
- - 1 • '-11 • r~ ' ' • i
ALKYLATING AGENTS
Al dehydes
Alkane sulfonic esters
Alkyl and a 1 kane halides
Alkylsulfates
Ary Id ia 1 ky 1 triazenes
Az i rid i nes
Azoxy and hydrazo alkanes
Di azoa 1 kanes
Epoxides
Lactones
Nitrogen, sulfur,
and oxide mustards
Phosphoric acid esters
TEST SYSTEM NUMBER
12
12
II,
19,
121 1 ,
1219,
1223,
12
12
12
12
12
M,
19,
II,
19,
12,
121 1,
1219,
1223.
12
12
12
M,
II,
19,
121 i,
1219,
|223.
12
12
II,
19,
121 1 ,
1219.
1224.
12
12
M,
1 10,
12
12
12,
1 10.
1212,
121 10,
1224.
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12,
110,
12,
NO,
1 10,
12,
i 10,
1 10,
12,
1 10.
12,
110,
12,
110,
12,
NO,
12,
Ml,
12
12
12
12
12
13,
13,
HI,
13,
111,
1213,
1221,
12
12
12
III.
13,
III,
12
12
12
16,
16,
113,
1216,
1221 ,
1216,
1222,
12
12
16,
113,
1217,
1217,
1221,
1217,
1222,
1217,
1223.
1217,
1221,
1218
1218
1222
1218
1223
1218
1218
1222
9
9
9
9
t
9
9
1224.
12
12
12
13,
13,
HI,
1213,
1221,
12
12
13,
113,
1216,
1221 ,
12
12
12
16,
16,
113,
1216,
1222,
1216,
1221,
1217,
1222,
1217,
1217,
1221 ,
1217,
1223,
1217,
1222,
1218,
1223.
1218
1218
1222
1218
1224
1218
1223
1219
9
9
9
9
9
9
9
9
SuI tones
1211, 1212, 1213,
1224.
1221, 1222, 1223.
(cont inued)
*The data base "on test systems 1214, 1215, and 121 12 is not yet available.
tThe scope of this index extends beyond the test system texts.
199
-------
INDEX XIV: SAMPLE DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
ALKYLATING AGENTS (continued]
Triazines
AROMATIC AMIDES
AZO DYES
HALOGENATED ETHERS AND
HALOHYDRINS
HALOGENATED HYDROCARBONS
AND RELATED DERIVATIVES
FIuorocarbons
Halogenated aromatics
Vinyl and v inyI id ine
deri vati ves
HETEROCYCLICS
Acridines and quinicrines
Benz imidazoles
Cycled ienes
Di benzo-p-d ioxins
Di carboximi des
FIuorenones
Fuorocoumari ns
Phenoth iazines
Th ioxanth ines
Other
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, 121 I I, 12113, 1224.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, I2III, 12113, 1224.
1211, 1212, 1216, 1217, 1218, 1219,
121 10, 1224.
1211, 1212, 1216, 1217, 1218, 1219,
121 10, 121 I I.
1211, 1212, 1213, 12110.
121 I, 1212, 12110, 121 I I.
1211, 1212, 1216, 1218, 1219, 12110.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, 1221, 1222, 1223, 1224.
1211, 1212, 12110.
1211, 1212.
1211, 1212.
1211, 1212.
1211, 1212, 1216, 1217, 1218, 1219,
12 NO, 1221, 1222, 1223, 1224.
1211, 1212.
1211, 1212.
1211, 1212, 1213, 1216, 1217, 1218,
1219.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 121 10, 121 I I.
(conti nued)
200
-------
INDEX XIV: SAMPLE DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS (continued).
SAMPLES
TEST SYSTEM NUMBER
HYDRAZINES, HYDROXYLAMINES
CARBAMATES, HYDRAZIDES, AND UREAS
Carbamates
Hydrazides
Hydrazi nes
HydroxyI amines
Ureas and thioureas
INORGANIC DERIVATIVES
Halogens and derivatives
Meta I and meta Moid
deri vati ves
Ozone
Sulfur and nitrogen oxides
NATURAL PRODUCTS
Antibotics
Mycotoxi ns
Aflatoxin
Other
PyrroI izidine alkaloids
Steroi ds
Xanthines
NITRO DERIVATIVES
121 I,
1219,
121 I,
121 I ,
121 I ,
1219,
121 I,
1219,
121 I,
121 10.
121 I,
1219,
1212,
121 I ,
1222,
121 I,
1219,
121 I,
12110,
121 I ,
1224.
12110,
121 I,
1219,
121 I,
1219,
1224.
121 1,
1219,
1212, 1213, 1216, 1217, 1218,
12110, I2III, 1221, 1222, 1223.
1212, I2III.
1212, 12110.
1212, 1213, 1216, 1217, 1218,
12110, I2III, 1221, 1222, 1223.
1212, 1213, 1216, 1217, 1218,
12110, 121 I I.
1212, 1216, 1217, 1218, I2I9:
1212, 1213, 1216, 1217, 1218,
121 10, 121 I I, 1221, 1222, 1223.
12110, 121 I I.
1212, 1213, 121 10, 121 II, 1221,
1223.
1212, 1213, 1216, 1217, 1218,
121 10, 1221, 1222, 1223, 1224.
1212, 1216, 1217, 1218, 1219,
1221, 1222, 1223, 1224.
1212, 12110, 1221, 1222, 1223,
1221, 1222, 1223.
1212, 1213, 1216, 1217, 1218,
12110.
1212, 1213, 1216, 1217, 1218,
12110, 121 I I, 1221, 1222, 1223.
1212, 1213, 1216, 1217, 1218,
12110, 121 I I.
(continued)
201
-------
INDEX XIV: SAMPLE DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
NITRO DERIVATIVES (continued)
Nitrofurans
N itroimidazoIes
Nitroquino Iines and
compounds
N-NITROSO COMPOUNDS
N itrosamides
N itrosamines
N itrosoureas
NUCLEIC ACID BASES AND ANALOGS
ORGANIC PEROXIDES
ORGANO-METALLICS
Organo lead derivatives
Organo mercury derivatives
Other
POLYNUCLEAR AROMATICS
OTHER
Esters and anhydrides
N-ox i des
Quaternary ammonium
compounds
Ou i nones
Su I f ites
1211, 1212, 1216, 1217, 1218, 1219,
121 10, 1221, 1222, 1223.
1212, 12110.
1211, 1212, 1213, 12110, 1221, 1222,
1223, 1224.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 121 10, 121 I I, 1221, 1222, 1223,
1224.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12 MO, 121 I I, 1221 , 1222, 1223,
1224.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110.
1211, 1212, 1213, I2I6-, 1217, 1218,
1219, 12110, I2III, 12113.
1211, 1212, 12110.
1221, 1222, 1223.
1211, 1212, I2III, 1221, 1222, 1223.
1211, 1212, 12110, 1221, 1222, 1223.
1211, 1212, 1216, 1217, 1218, 1219,
12110, 1221, 1222, 1223, 1224.
121 I, 1212, 1213, 121 10, 121 I I.
1211, 1212, 1213, 12110.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110.
1211, 1212, 1216, 1217, 1218, 1219,
121 10, 121 I I.
1212, 1213, 12110, I2III.
202
-------
INDEX XV: SAMPLE DISTRIBUTION OF THE ECOLOGICAL TEST SYSTEMS
SAMPLES
TEST SYSTEM NUMBER
Ac i d ra i n
Mr pol I utants
Amblent
Ambient a i r
Chlorami nes
Chlorinated hydrocarbons
Chlorine
CO
CoaI storage
Complex wastes
Detergents
Di ssolved gases
Dredge sediment
Drilling muds
Energy related
Gaseous pollutants
Heavy metals
241 I.
•24II ,
2101,
241 I .
2102.
2501.
2102.
241 I.
2101.
2322.
2101 .
2215.
2102,
221 I ,
2333.
2101,
2221,
2321,
241 I ,
2421.
2102, 2411, 2423,
2322.
2221, 2311, 2321,
2211, 2212, 2215,
2224, 2231, 2311,
2331, 2332, 2333,
2423.
2411, 2421.
2101, 2102, 2211, 2221,
2222, 2223, 2311, 232],
2322, 2332, 2412, 2421,
2422, 2423, 2501.
(continued)
203
-------
INDEX XV: SAMPLE DISTRIBUTION OF THE ECOLOGICAL TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
Herbicides
Industrie I
Industrial air pollutants
Industrial effluents
Industrial sludge
Industrial waste discharges
Industrial waste effluents
Inorgan ics
Insectici des
Leachates
MetaIs
MuIti chemicaIs
Monochlorinated organics
NH-
3
NO.
2
NO,
NO
x
Nutrients
2101, 2211, 2221, 23"I,
2312, 2313. 2321.
2101, 2102, 2211, 2212,
2214, 2215, 2221, 2224,
2231, 2311, 2321, 2331,
2332, 2411, 2412, 2421,
2422, 2423, 2502.
241 I.
2211, 2221, 2311, 2321,
2331, 2332, 2421.
2412.
210!.
2422.
2212, 2241, 2502.
2101, 2211, 2221, 2311,
2312, 2313. 2321, 2501.
2101.
2213, 2215, 2224, 2231,
2324.
2214.
2102.
2101.
2101.
2101.
241 I.
2101, 2102.
(continued!
204
-------
INDEX XV: SAMPLE DISTRIBUTION OF THE ECOLOGICAL TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
Oi Is
Organ ics
Ortho-P
Other
Oxygen
Ozone
RGB's
PentachlorophenoI
Pesticides
Radiolabeled material
Receiving waters
so2
Tox-P
Toxic organics
Transportation related
Waste oi I
2211, 2221, 2311, 2321,
2331, 2332.
2212, 2215, 2224, 2231,
2241, 2331, 2332, 2502.
2101.
2101.
2215.
241 I.
2102, 2211, 2221, 2311,
2312, 2321, 2501.
2312.
2101, 2213, 2222, 2323,
2324, 2331, 2332, 2333,
2421, 2422, 2423.
2501.
2101,
241 I.
2101 .
2331 ,
241 I ,
2213,
2102.
2332.
2421 .
205
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. 2
EPA-600/1-78-052
4. TITLE AND SUBTITLE
DIRECTORY OF SHORT TERM TESTS FOR HEALTH AND
ECOLOGICAL EFFECTS
7. AUTHOR(S)
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Biochemistry Branch
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park,NC 27711
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory RTP,NC
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
15. SUPPLEMENTARY NOTES
3. RECIPIENT'S ACCESSI ON- NO.
5. REPORT DATE
July 1978
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1LA629, EHE625, 1AA601
11. CONTRACT/GRANT NO.
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/11
16. ABSTRACT
This directory provides basic information on the short term tests for
health and ecological effects being performed by various U.S. EPA Laboratories
through the Office of Health and Ecological Effects. The test systems are
cross-indexed.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS b.lDENTIFI
environmental tests
, , . short t
laboratories
biological laboratories
directories
indexes (documentation)
13. DIS I RIB UT ION STATEMENT 19. SECURI
"RFTEASE TO PTIBT If UNCLAS
iVE.L.JLri^JL 1U rUI^-L^ 20. SECURI
UNCLAS
ERS/OPEN ENDED TERMS C. COS ATI Field/Group
erm tests 06 F, T
TY CLASS (This Report) 21. NO. OF PAGES
SIFTED 218
TY CLASS (Thispage) 22. PRICE
SIFIED
EPA Form 2220-1 (9-73)
206
------- |