United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-81-045 July 1981
Project Summary
Potency Ranking of Chemicals
Based on Enhancement of
Viral Transformation
Bruce C. Casto
Treating primary hamster embryo
cells with various classes of chemical
carcinogens and mutagens leads to
enhancement of transformation by
simian adenovirus SA7. It appears
that carcinogenic chemicals render
the individual cells more sensitive to
viral transformation, thus increasing
the total number of cells integrating
SA7 DNA. Enhancement of viral
transformation appears to be a sensi-
tive indicator for chemical agents with
the potential to damage cell DNA by
either direct or indirect means and
thus may be useful as a screening tool
to detect these chemicals in the envi-
ronment.
The Project Report summarizes and
compares the results for 136 chemi-
cals (both carcinogenic and noncar-
cinogenic) assayed for enhancement
of SA7 transformation, chemical
transformation, and induction of DNA
strand breaks and DNA repair synthesis.
In addition, these chemicals are ranked
by lowest effective concentration in
the assay for enhancement of viraT
transformation.
This report was submitted in fulfill-
ment of Contract No 68-02-2566 by
Northrop Services, Inc.-Environ-
mental Sciences, under the sponsor-
ship of the U.S. Environmental Pro-
tection Agency. This report covers the
period from June 1980 to August
1980, and work was completed as of
August 1980.
This Project Summary was devel-
oped 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
information at back).
Introduction
The U.S. Environmental Protection
Agency (EPA) has been given the re-
sponsibility for regulating the release of
toxic chemicals into the environment
under the Toxic Substances Control Act,
Resource Conservation and Recovery
Act, and the Clean Air and Clean Water
Acts.
Assessment of the chronic effects of
chemical exposure is a complex task,
since the consequences of exposure
may appear long after the initial contact.
To determine these effects, lifetime
studies in animals must be conducted at
considerable expense using specialized
facilities and personnel. Single tests for
carcinogens may cost more than $300,000
and last for 2 to 3 years. Some 50,000
chemicals are commercially produced
in the United States and 700 to 1,000
new chemicals are introduced each
year, yet the present capacity to conduct
long-term animal studies is limited to
approximately 500 compounds per year.
Initial reliance on short-term tests for
the identification of toxic chemicals,
carcinogens, and mutagens is manda-
tory if the introduction of new chemicals
continues at its present rate and esti-
mates of 70-80% for environmentally-
induced cancer are valid.
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Several methods for in vitro testing
have been described and many new test
systems are being developed. It is
anticipated that a battery of tests will
eventually be available with a high
degree of reproducibility and correlation
with in vivo activity. These tests should:
generate no false-negatives and only a
low percentage of false-positives, be
responsive to the various classes of
chemical and physical agents, and lend
themselves to quantitation. All of these
attributes must be met if effective
judgments are to be made concerning
the relative risk of the test agent for
humans. In addition, other parameters
such as exposure, production volume,
lability of the chemical, dose-effect at
low concentrations, and the type of
population at risk must be considered.
Presently, the in vitro systems receiv-
ing the most attention for prediction of a
chemical's potential to cause chronic
effects are: mammalian cell cytotoxicity
and transformation; mutagenesis assays
in microbial and mammalian cells; and
analysis of DNA damage and repair. For
a number of chemicals of known activity,
the detection of carcinogens has been
stated to be as high as 90 percent with
systems such as microbial mutagenesis,
mammalian cell transformation, or
mammalian cell mutagenesis. However,
none of the above in vitro tests can be
used alone to predict the chronic toxicity
potential of suspect environmental
agents.
It has been proposed that testing of
environmental agents proceed through
a phased or tier approach. Such an
approach is presently being employed
by governmental and industrial labora-
tories, progressing from routine detec-
tion systems, involving microbial cells,
to more complex assays utilizing mam-
malian cell cultures, and subsequently
to whole-animal testing. Decisions
concerning the degree of testing to be
done are made at different levels of
testing (and cost) depending upon the
nature and use of the chemical being
evaluated. As a final assessment of risk
to man, the suspect agent is tested in
animals using genetic and pharmaco-
logical evaluations based on dose, route
of administration, and length of
exposure.
Because routine microbial assays
may be insensitive for detection of
certain toxic chemicals such as
hydrazine derivatives, inorganic metals,
steroid hormones, asbestos, and
chlorinated hydrocarbons that are
detected by selected mammalian cell
assays, some type of rapid mammalian
cell bioassay must be included in the
screening of suspect toxic environ-
mental chemicals.
A different approach to the in vitro
assay of carcinogens and mutagens has
been described in which the ability of
various chemicals to enhance adeno-
viral transformation is evaluated in
hamster embryo cells. Cells are either
pretreated for 2 or 18 hours with a
series of chemical dilutions prior to viral
inoculation or post-treated 5 hours after
viral infection. The cells a re transferred
for survival (cloning) and focus assays
(virus transformation) and maintained
for 8 days and 25-30 days, respectively.
To inhibit growth of normal cells and
promote growth of virus-transformed
cells, the cells for transformation assays
(employing adenovirus) are cultured in a
low calcium medium under 0.3% agar.
Foci do not develop in cells treated only
with chemicals since the conditions
favorable for virus transformation inhibit
the development of chemically-trans-
formed HEC.
The classes of chemicals assayed in
the enhancement assay include: alcohols
and phenols, aliphatic amines, alkyl
sulfates and sulfones, aromatic amines,
aryl halides, carbohydrates and deriva-
tives, hydrazines, hydroxylamines, metals
and derivatives, mycotoxins, and the
polycyclic hydrocarbons. The majority of
agents examined have been: (1) those
chemicals more commonly used in
other short-term in vitro assays, (2)
those used in various industrial applica-
tions, (3) chemicals annually produced
in large volumes and (4) inorganic metal
salts. Data from approximately, 105 test
performances in 29 different chemical
classes have been published from the
enhancement of viral transformation
assay. Unpublished or preliminary data
exist for approximately 100 other
chemicals.
The enhancement assay is a reflection
of the capacity of a chemical to damage
cell DNA by either direct or indirect
means. Mutation assays in micjobial
and mammalian cells or carcinogen
assays in mammalian cells with a
variety of short-term tests (inhibition of
DNA synthesis, induction of DNA repair,
breakage of cell DNA) also presume that
mutagenic/carcinogenic agents damage
or alter cell DNA.
The enhancement of viral transforma-
tion appears to be a sensitive indicator
for chemical agents with the potential
for da mag i ng cell DNA either by direct or
indirect methods and therefore may be
useful as a screening tool to detect
these chemicals in the environment.
Results and Discussion
Four assays for determination of
carcinogenic or mutagenic potential
have been conducted in Syrian hamster
embryo cells with a large number of
compounds. Chemicals were tested for
viral enhancement, induction of DNA
fragmentation or DNA repair, and mor-
phological transformation. With 136
negative or positive carcinogens tested,
94% agreed with their current classifi-
cation. Fifty chemicals were tested in all
four assays: stimulation of DNA repair
synthesis correctly classified 50%, DNA
fragmentation 72%, and chemical trans-
formation 92%.
Data for the 136 chemicals, carcino-
gens and noncarcinogens,obtained
from replicate experiments using the
enhancement of viral transformation
assay, have been ranked based upon the
least effective concentration (/jg/ml).
The upper limit for testing was usually 1
mg/ml unless solubility or toxicity
dictated using a lower dose.
For those compounds testing positive
the range of effective concentrations
varied by a factor of 2.5 x 105 with 7,12-
dimethylbenz(a)anthracene being the
most potent (0.004 ^ig/ml) and hydrazine
sulfate or nickel sulfide being the least
potent (1000/ug/ml).
Chemicals have been placed into 3
groups depending upon the concentra-
tion necessary to produce a positive
enhancement response. Those showing
the highest enhancement activity (at 10
//g/ml or less) were classified as Group
I; Group II consisted of those compounds
active at 10-100 A/g/ml; Group III was
composed of those chemicals only
active at more than 100 yug/ml. Ap-
proximately 46 chemicals could be
classified as highly active (Group I), 22
as intermediate (Group II) and the
remaining as weak (Group III); the only
known false-positive (not carcinogenic
or mutagenic) was caffeine. Three
compounds were unclassified (±) due to
the failure to be consistently positive in
several experiments (e.g. zinc sulfate
was positive in 3/7 trials).
Known carcinogens testing negative
for enhancement (e.g. N-2-acetylamino-
fluorene, N-nitrosodimethylamine, N-
nitrosodiethylamine) were those appar-
ently not metabolized in vitro by hamster
fibroblasts since other tests (transforma-
tion, DNA breakage, and DNA repair)
were also consistently negative. Incor-
poration of an exogenous activating
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iystem (a liver S9 mix) or exposure to
ihemical in utero converts many of the
legative compounds to positive in en-
lancement assays or transformation
tssays, respectively. Other chemicals
legative for enhancement that are
tuspect carcinogens in humans or
mimals include: 1,2diethyl-2-thiourea;
richloroethylene; red dye #2; and 1,4
'ioxane.
Of 35 chemicals tested in both Sal-
nonella and the enhancement of viral
ransformation assays, there was agree-
nent between the two tests with 26 of
he compounds. Four carcinogens were
elected with the viral enhancement
issay, but not with Salmonella. The
emaining three required activation not
>rovided by hamster embryo cells and
vere positive in Salmonella in combina-
ion with a liver S9 mix.
Forty-two of the top 50 volume-
iroduced compounds have been tested.
Vith the exception of a few agents,
nost were tested at least twice in the
mhancement assay. Among these
iompounds, ethylene dichloride, pro-
lylene oxide and vinyl acetate have
:aused enhancement. Butadiene (1,3-)
he 27th ranked compound) has not
teen tested, but2-chloro-1,3-butadiene
mhances transformation. The temporal
leriod between viral and chemical
reatment with these compounds is
imilar to that found previously with
:affeine, MnCb, and Ara-C. In experi-
nents with ethylene dichloride, en-
lancement was only observed at the
lighest dose used (1 mg/ml). Routinely,
oncentrations higher than 1 mg/ml
re not tested, but with two other
hemicals, phthalazinone and ethylene
ibromide, enhancement continued to
ncrease when 2 mg/ml were used.
•thylene dichloride was also positive at
oses higher than 1 mg/ml. None of the
emaining chemicals from the top 50 list
ave shown any indication that they
lay cause enhancement when added
ither before or after virus.
The mutagenic and carcinogenic
ctivity of many of these chemicals has
een under investigation in recent
ears. Chlorobutadiene (CBD) was
eported to be negative in the Salmonella
ssay in some laboratories and positive
n others; additionally CBD has been
eported to cause chromosome aberra-
ions in lymphocytes of exposed workers
nd to be related to the appearance of
kin and lung tumors among rubber
'orkers.
Propylene oxide (PO) used in the
lanufacture of propylene glycols, poly-
glycols and propylene glycol esters, as a
fumigant herbicide, as a solvent for
cellulose nitrate or acetate and vinyl
chloride or acetate, and used on foods to
control spoilage, was produced (1974
data) at the rate of 1.78 billion Ibs/yr
and has been shown to be mutagenic in
Drosophilia and carcinogenic. In addition
to the enhancement of viral transforma-
tion, PO was shown to transform HEC in
the focus assay.
Studies in Salmonella by both have
been negative for vinyl acetate, and
have not found any carcinogenic activity
when VA was used in long-term bioassays
as a control for vinyl chloride. Neverthe-
less, VA-was positive in repeat experi-
ments for viral enhancement when
added to HEC after SA7 and was positive
for focus formation when tested alene.
Styrene was negative in two experi-
ments (-18 hr and + 5 hr treatment peri-
ods). Produced in the US at the rate of 6
billion Ibs/yr and used in the production
of plastics or resins and in styrene-
butadiene rubber, styrene may be me-
tabolically converted to the mutagenic
form styrene oxide. In a series of exper-
iments with styrene and styrene oxide,
using the yeasts S. cerevisiae or S.
pombe and Chinese hamster cells,
styrene oxide was uniformly mutagenic
whereas styrene was negative even in
the presence of a liver microsome
activating system. In a host-mediated
assay, styrene was weakly mutagenic
for S. pombe when Swiss mice were
treated with 1 gm/Kg. Styrene and
styrene oxide have been tested in 5
strains of Salmonella (TA98, TA100,
TA1535, TA1537, TA1538) and again
styrene was negative; styrene oxide
induced mutations in TA100 (Milvy and
Garro, 1976).
The findings of certain chemicals
such as vinyl acetate, positive in the en-
hancement assay but negative in the
Salmonella assay is not uncommon. For
example, several recognized mutagens
or carcinogens have not been mutagenic
for Salmonella, even when incubated
with a S-9 activating system, but were
positive for viral enhancement including:
thioacetamide, lUdR, 1,2-dimethylhy-
drazine, hydroxylamine phthalazinone,
and the metal carcinogens or mutagens.
A total of 46 metal salts have been
tested in the viral enhancement assay.
Positive enhancement was found with
salts of antimony, arsenic, beryllium,
cadmium, cobalt, copper, iron, lead,
manganese, molybdenum, nickel, sele-
nium, platinum, thallium, vanadium and
zinc. Negative metals include the acetate,
chloride or sulfate salts of aluminum,
barium, calcium, lithium, magnesium,
potassium, stronthium, titanium and
zirconium. Both the positives and nega-
tives from above are in excellent agree-
ment with the in vitro infidelity of DNA
synthesis assay; the only exception
being FeCI2 that was positive in the viral
enhancement assay and negative for
introducing copy error in the infidelity of
DNA synthesis assay. Although metallic
iron has not been shown to be car-
cinogenic iron dextran will induce
tumors in rats, mice, and hamsters. In
contrast to the above, ferrous chloride
or ferric chloride and sulfate were
earlier found to induce point mutation in
E. co//.
The enhancement data with metals
also agree with data obtained using rec-
assays with B. subtilis. In these studies
of 56 metal salts, arsenic, cadmium,
chromium, mercury, manganese and
molybdenum were considered positive.
Three of the strongly positive metals,
arsenic, chromium and molybdenum,
were also mutagenic in E. coli. The rec-
assays, however, failed to detect beryl-
lium, copper, iron, lead, nickel, antimony
or zinc. There are several other reports
on the carcinogenic and mutagenic ac-
tivity of the metals shown to be positive
in the viral enhancement assay.
Because of the use of metals in
various industrial applications and the
increasing evidence that many are
involved in human carcinogenesis,
sensitive and reliable assays for poten-
tial mutagenic or carcinogenic activity
are necessary. The good agreement
between the viral enhancement assay
in HEC and the mutagenic or carcino-
genic activity of the metals in other
systems justifies the use of the SA7
transformation assay as one of the tests
to be included in assays for potential
environmental mutagenic or oncogenic
metal complexes.
(, US. GOVERNMENT PRINTING OFFICE 1M1 .757-012/7212
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Bruce C. Casto is with Northrop Services, Inc.—Environmental Sciences,
Research Triangle Part, NC 27709.
Stephen Nesnow and Michael D. Waters are the EPA Project Officers (see
below).
The complete report, entitled "Potency Ranking of Chemicals Based on En-
hancement of Viral Transformation,"(OrderNo. PBS 1-210 080; Cost: $6.50,
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 Officers can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
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