United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
Research and Development
EPA/600/S1 -86/002 July 1986
4>EPA Project Summary
Final Report on the Evaluation of
Four Toxic Chemicals in an
In Vivo/In Vitro Toxicological
Screen: Acrylamide, Chlordecone,
Cyclophosphamide, and
Diethylstilbestrol
Joellen Lewtas
This document is an internal EPA
report of research conducted across
divisions within HERL and augmented
by on-site contract support personnel.
An in vivo/in vitro Toxicological Screen
(Tox Screen) has been developed for
screening large numbers of wastes for
biological activity. Emphasis is placed
on identifying a wide range of potential
toxic responses by employing diverse
test methods with toxic endpoints in
mutagenesis/carcinogenesis, general
toxicology, neurotoxicology, reproduc-
tive toxicology, teratology, and immu-
notoxicology. Oral administration of
waste material is given to rats for 10
consecutive days after which the whole
animal, body tissues and fluids are
evaluated for toxicity. The Tox Screen is
being validated to ensure that the pro-
tocol will be capable of detecting
biological activity and to identify those
assays which most readily detect toxic-
ity. The most accurate and sensitive
assays would be used as a Prescreen for
the entire protocol. Results of the
validation study with 4 toxic chemicals
are included. Acrylamide produced its
greatest effects in the neurotox assays
and in mutagenesis. The reproductive
assays were most sensitive in detecting
the toxicity of Chlordecone. The toxicity
of cyclophosphamide was most readily
identified by the immunological and
mutagenesis tests. Diethylstilbestrol
produced dose-response effects in all
disciplines. The Tox Screen is currently
being re-evaluated to answer questions
concerning the length of exposure and
the need to add and/or eliminate certain
tests to increase sensitivity and accu-
racy. In addition, several new areas of
research are being pursued in additivity/
synergism/antagonism, chronicity and
in vitro to in vivo extrapolation.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC. to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering in-
formation at back).
Technical Summary
An in vivo/in vitro Toxicological Screen
(Tox Screen) employs methods designed
to be rapid, inexpensive, capable of
screening large numbers of wastes for
biological activity, and identifying a wide
range of potential toxic responses asso-
ciated with each waste. The toxic end-
points to be identified include mutagen-
esis/carcinogenesis, general toxicology,
neurotoxicology, reproductive toxicology,
teratology, and immunotoxicology. The
protocol of Tox Screen involves oral
administration of waste material to rats
for 10 consecutive days. At the end of the
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10-day period, the whole animal and its
body tissues and fluids are evaluated for
toxicity. A major goal of Tox Screen is
maximization of the potential health
effects information obtained by exposing
metabolically competent intact animals
to complex waste mixtures.
Tox Screen is being validated through
use of a series of compounds of known
and defined toxicity. The purpose of the
validation study is to ensure that the
protocol will be capable of detecting
biological activity associated with each
compound and identifying those assays
which most readily detect the toxic poten-
tial of the compounds. A selected number
of the most accurate and sensitive assays
might constitute a prescreening test for
the entire protocol. The prescreening
could then be used (with an abbreviated
form of the in vivo exposure regime) as a
rapid means of prioritizing waste samples
to be evaluated in the full Tox Screen. It is
important to note that a large number of
compounds of known toxicity and no
toxicity must be evaluated in the Tox
Screen before bioassay tests for the
prescreening would be initiated. These
evaluation tests would constitute a vali-
dation study to establish the sensitivity,
specificity, and accuracy of Tox Screen.
Initial trials of the validation study with
acrylamide, chlordecone (kepone), cyclo-
phosphamide, and diethylstilbestrol (DES)
demonstrated the feasibility of the Tox
Screen for detecting biological activity of
chemicals.
Acrylamide, evaluated at five test con-
centrations ranging-from 3.75 to 60
mg/kg in rats, produced its greatest toxic
effects in motor activity, responses to
acoustic stimuli (neurotoxicology), and in
the induction of sister chromatid ex-
changes (mutagenesis). Most other tests
resulted in positive responses at the
highest concentration tested, a concen-
tration which produced 30% lethality.
The reproductive assays were most
sensitive in detecting the toxic potential
of chlordecone (evaluated at 0.625 to
10.0 mg/kg in rats). Testicular sperm was
markedly reduced, demonstrating that
chlordecone is a reproductive toxin. This
observation agrees with previously re-
ported studies of sterility in chemical
workers employed in chlordecone manu-
facturing in Hopewell, Virginia. Had this
sample been an unknown hazardous
waste mixture, the observed results would
suggest the presence of a potential
reproductive toxin and would indicate a
need for further research.
The toxicity of cyclophosphamide (1.5
to 24 mg/kg in rats) was most readily
identified by the immunological and mu-
tag'enesis tests. Most other tests respond-
ed at the highest concentration tested,
resulting in 70% lethalities. Cyclophos-
phamide produced mutagenic metabolites
in the urine of treated rats. These metabo-
lites were detectable using the Salmonella
histidine reversion assay. Increases in
sister chromatid exchanges were identi-
fied in the bone marrow at each exposure
concentration (1.5 to 24.0 mg/kg in rats).
Dose-response relationships were estab-
lished for each of the immunotoxicity
tests with cyclophosphamide. Also, there
were significant reductions in thymus
and spleen weights at all concentrations
tested. Body weight reductions were
observed only at the two highest doses.
These results indicate that generalized
body weight loss is not predictive of
potential immunotoxicity.
The exposure of animals to DES also
resulted in the generation of a number of
dose-response relationships in several
areas. Almost all parameters evaluated in
general toxicology, neurotoxicology, im-
munotoxicology, reproductive toxicology,
and teratology were affected at the lowest
concentration tested, 31.25 mg/kg. The
only test category which did not detect
the toxicity of DES was mutagenesis,
which is not surprising since this toxin is
not a bacterial mutagen nor does it induce
SCEs.
The results of the validation study to
date suggest that Tox Screen is capable of
detecting a range of biological activity of
the pure compounds as well as the type of
activity associated with the pure com-
pounds. Four compounds, however, are
insufficient for establishing the required
sensitivity (0.9), specificity (0.75), and
accuracy specified by OSW or to select
the best tests for the abbreviated pre-
screen. The number of compounds re-
quired to identify prescreen assays and to
establish the accuracy of Tox Screen
(with a 90% sensitivity) requires a mini-
mum of 15 to 20 biologically active
compounds for each of the six disciplines
of Tox Screen (assuming no overlap in
toxicity) and 15 to 20 control compounds
without biological activity (assuming
100% overlap in toxicity across end-
points). This results in a total of 105 to
140 compounds. The evaluation of this
many compounds may be a prohibitive
task because of both time and cost
limitations.
As a result, Tox Screen is being re-
evaluated with respect to the treatment
protocol and the toxicological assays
which it employs. Several questions must
be answered as part of this re-evaluation
process and before validation of Tox
Screen can be designed.
1. Is a 10-day exposure of a waste
material adequate for predicting
chronic effects? Should a 30- or 90-
day exposure regime be employed?
2. Are there toxicity tests which could
be added or deleted to improve the
overall performance and sensitivity
of the Tox Screen?
3. Can certain in vivo tests be replaced
with in vitro tests to reduce cost
without reducing sensitivity or accu-
racy?
4. Is 90% sensitivity and 75% accuracy
actually required?
In addition to addressing the above
questions, several new areas of research
are being pursued as part of an ongoing
developmental program in the evaluation
of complex mixtures.
1. The development of short term in
vitro genetic mutation and liver toxic-
ity bioassays for the identification of
hazardous waste samples possessing
acute and chronic biological effects.
2. The development of new and im-
proved in vivo/in vitro screening
methods in the areas of liver toxi-
cology, neurotoxicology, immunotox-
icology, and developmental biology
for addition to the integrated Tox
Screen.
3. The investigation and resolution of
some of the basic toxicological prob-
lems related to complex mixtures and
the screening assay (i.e., acute vs.
chronic effects and comparison of in
vivo and in vitro methodologies).
This report covers a period from April 8,
1983 to June 22, 1985 and work was
completed as of April 21,1986.
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The EPA author Joellen Lewtas is with the Health Effects Research Laboratory,
Research Triangle Park, NC 27711.
The complete report, entitled "Final Report on the Evaluation of Four Toxic
Chemicals in an In Vivo/In Vitro Toxicological Screen: Acrylamide, Chlordecone,
Cyclophosphamide. and Diethylstilbestrol," (Order No. PB 86-195 260/AS;
Cost: $9.95, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
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U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Agency
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Information
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Official Business
Penalty for Private Use $300
EPA/600/S1-86/002
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