DOE
EPA
Department
of
Energy
Lawrence Livermore Laboratory
University of California
Livermore CA 94550
UCRL-81690, Rev. 2
United States
Environmental Protection
Agency
Office of Energy, Minerals, and
Industry
Washington DC 20460
EPA-600/7-79-172
August 1979
Research and Development
Estimating the
Potency of
Mutagens
Cytotoxicity as an
Obligatory
Consequence of
Mutagenicity
Interagency
Energy/Environment
R&D Program
Report
-------
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 INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from the
effort funded under the 17-agency Federal Energy/Environment Research and
Development Program. These studies relate to EPA's mission to protect the public
health and welfare from adverse effects of pollutants associated with energy sys-
tems. The goal of the Program is to assure the rapid development of domestic
energy supplies in an environmentally-compatible manner by providing the nec-
essary environmental data and control technology. Investigations include analy-
ses of the transport of energy-related pollutants and their health and ecological
effects; assessments of, and development of, control technologies for energy
systems; and integrated assessments of a wide range of energy-related environ-
mental issues.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/7-79-172
August, 1979
ESTIMATING THE POTENCY OF MUTAGENS
Cytotoxicity as an Obligatory Consequence of Mutagenicity
by
June H. Carver, Elbert W. Branscomb, and Frederick T. Hatch
Biomedical Sciences Division
Lawrence Livermore Laboratory
University of California
Livermore, California 94550
Department of Energy Contract No. W-7405-ENG-48
EPA-IAG-D5-E681-AN and AO
U.S. D.O.E. PROJECT DIRECTOR
U.S. E.P.A. PROJECT OFFICER
G. Stapleton
Office of Health Effects Research
Asst. Sec. for Environment
U.S. Dept. of Energy
Washington, D.C. . 20545
G. Rausa
Office of Energy, Minerals and
Industry
U.S. Env. Prot. Agency
Washington, D.C. 20460
-------
DISCLAIMER
This report has been reviewed by the Lawrence Livermore Laboratory, the
U.S. Department of Energy, and the 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.
ii
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FOREWORD
Short-term toxicologic testing, particularly methods for detecting
genetically active substances, has become a keystone of the Environmental
Protection Agency's efforts to detect and evaluate environmental hazards.
Among the most serious hazards to man's future are agents that damage the
genome, which is the information bank of cells and organisms, by mechanisms
that can increase the future population load of inherited structural defects
and genetic diseases or that can increase the incidence of cancer in the
current generation. Tests for environmental mutagens play a dual role by
virtue of their ability to detect mutagens and their presumptive ability to
detect carcinogens, owing to abundant laboratory evidence that most known
carcinogens respond positively to tests for mutagens.
The Biomedical Sciences Division of the Lawrence Livermore Laboratory
has for the past five years been heavily engaged in the development and
application of short-term tests for genetic toxicology. Specific examples
are as follows:
• basic studies of classes of mutations in mammalian cells in culture and
methods for selective recovery of mutant cells;
• development of suitable strains of cells for detection of forward
mutations at multiple loci, and of detailed experimental protocols for
mutagen detection;
• development and application of multiple modes of testing _iri vitro and ^ji
vivo for chromosomal damage and misrepair in the form of sister
chromatid exchange, together with simultaneous correlation with mutation
induction;
• development and application of highly sensitive tests for injury to
sperm cells in the male testis and to oocytes in the juvenile female
ovary;
• development of automated cytochemical methods for detection of rare
events of mutation and malignant transformation of cells in living
animals, including man;
• application of cytogenetic and automated cytopathologic methods to
workforce populations engaged with energy technology, and
lii
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• in collaboration with our Environmental Sciences Division, application
of a battery of the foregoing short-term tests to complex effluents from
coal gasification and oil shale retorting technologies.
The following report represents an analysis of quantitative data on
cytotoxicity and mutation induction in mammalian cultured cell systems for
our laboratory and a review of the literature. The analysis revealed that
the cytotoxic potency of 22 chemical mutagens is linearly correlated to a
high degree with their mutagenic potency in five rodent and human cell
systems. This relationship indicates that the potential mutagenic potency
of chemicals can be reliably estimated from assays for their cytotoxicity.
Since the latter assays are rapid and relatively inexpensive, they are
suggested for wide application to screening of, for example, industrial
chemicals for potential genetic toxicity as a precursor to setting
priorities for further testing. Thus, a substance exhibiting high toxicity
(i.e., killing efficiency) for mammalian cells would be given priority for
mutagenesis and other genetic toxicology assays. A substance exhibiting low
cytotoxicity could be, at most, a weak mutagen and would have a lower
urgency for further testing.
Mortimer L. Mendelsohn, M.D., Ph.D.
Associate Director for Biomedical and
Environmental Research
Lawrence Livermore Laboratory
iv
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ABSTRACT
Rapid and reliable screening methods are required for identifying
environmental mutagens and estimating their mutagenic potency in preparation
for use of more elaborate tests to assess the genetic risk to man. In this
report, we show that the cytotoxic potency of 22 chemical mutagens is highly
correlated with their mutagenic potency as assayed in five rodent and human
in vitro cell systems. This relationship implies that the maximum potential
mutagenic potency of such compounds may be reliably estimated from rapid and
straight-forward measurements of their cytotoxic potency, the latter defined
as the failure of cultured cells to undergo continued cell division.
This report was submitted by the Lawrence Livermore Laboratory (Dept. of
Energy, Contract No. W-7405-ENG-48) in partial fulfillment of Interagency
Agreements IAG-D5-E681-AN and AO under the sponsorship of the U.S.
Environmental Protection Agency. This report covers a period from January,
1978 to January, 1979; work is still ongoing.
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CONTENTS
Foreword iii
Abstract v
Contents vi
List of Figures vii
List of Tables vii
1. Introduction 1
2. Discussion 2
References 9
vi
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FIGURES
Number
1 The relationship between induced mutation frequency per
viable cell per unit exposure dose and the reciprocal of
D37(M), i.e., the dose required to kill 63% of the
initial cell population
2a The induced mutation frequency per viable cell calculated
for the 0-37 exposure dose of 22 chemicals mutagens, plus
ultraviolet and ionizing radiation 7
TABLES
Number Page
1 Compounds evaluated for mutagenic potency 3
vii
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SECTION 1
INTRODUCTION
A variety of short-term mutagenesis assays employing both procaryotic
and eucaryotic systems are being used to identify potentially hazardous
agents in the environment. At present, the screening of a large number of
compounds is most effectively done with in vitro mutagenesis assays, of
which the main types use either bacterial or mammalian cells. Although
tests with mammalian cells are not as rapid and inexpensive as microbial
assays, they are needed to confirm and extend the bacterial results.
Indeed, because of known differences in genome organization and possible
differences in metabolic and repair processes, mutagenicity assays using
mammalian cells may provide a better assessment of potential risk to humans
than do microbial tests. It would be desirable to screen all potential
mutagens with mammalian cell mutagenicity assays, but this does not seem
feasible unless, as we propose, test compounds are first prescreened on the
basis of their cytotoxic potency.
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SECTION 2
DISCUSSION
On theoretical grounds, one expects a certain minimum cytotoxic potency
to correlate with mutagenic potency, particularly when the latter is
measured using forward mutations that result in inactive gene products. In
fact, since cells cannot divide unless a substantial fraction of their
genome is functionally intact, mutagenic agents should be obligatory
cytotoxic agents as well, with a given mutagenicity conferring a certain
irreducible cytotoxicity. Thus, assays of cytotoxic potency, which are
relatively easy to perform with mammalian cells, might provide a reliable
estimate of an agent's maximum potential mutagenic potency. The estimate is
necessarily a maximum one, since an agent may exert cytotoxic effects by
pathways independent of its mutagenic action.
A correlation between cell survival and mutagenic response has been
noted for ionizing radiation mutagenesis^ and appears to be applicable to
ultraviolet radiation and certain chemicals as W6112-4. Roberts e± al.
have discussed the molecular aspects of cellular lethality and mutagenesis
in terms of damage to DNA5. To further test this relationship, we have
compiled data on the cytotoxicity and mutagenicity of the 24 chemical and
physical mutagens listed in Table 1. As the measure of cytotoxic potency,
we have chosen to use the 037 unit of survival, defined here as the dose
required to kill approximately 63% of the initial cell population. Unlike
DQ, estimation of 037 is usually possible from the limited survival data
that accompanies published mutation frequencies; 037 generally measures
survival within the linear portion of the dose response for mutation (lower
survivals are frequently out of the linear mutation range). Moreover, this
parameter reflects differences in repair capability or fidelity that may
influence both toxicity and mutagenicity.
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Table 1 Compounds evaluated for mutagenic potency
Compound na Ref.
Alkylating agents
Streptozotocin (SZ) 1 16
Dimethylnitrosamine (DMN) 2 22,30
Ethyl methanesulfonate (EMS) 15 6,10,11,12,14
15,25,27,28,34,41
Diethylsulfate (DES) 1 9
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 13 6,8,12,16,17,20
24,26,33,38,39
N-methyl-N-nitrosourea (MNU) 2 8,39
N-ethyl-N-nitrosourea (EMU) 1 8
2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) 2 26,35
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) 1 8
N-butyl-N-nitrosourea (BNU) 1 8
Methyl methanesulfonate (MMS) 2 9,29
Dimethylsulfate (DMS) 1 9
Polycyclic aromatic hydrocarbons (PAH)
Benzo(a)pyrene, diol epoxide (BPde) 1 23
3-methylcholanthrene (MC) 2 21
Benzo(a)pyrene (BP) 3 19,21
7,12-dimethylbenz(a)anthracene (DMBA) 2 21
7,bromomethylbenz(a)anthracene (MBA-7br) 1 18
7,12-dimethylbenz(a)anthracene, 5,6-oxide (DMBAox) 1 36
Dibenz(a,h)anthracene, 5,6-oxide (DBAox) 1 36
Benzo(a)pyrene, 4,5-oxide (BPox) 1 36
Frameshift mutagens
Heterocyclic nitrogen mustard compounds (iCR's) 5 13,39
Physical mutagens
X- and -irradiation 4 1,5,7,31,32
Ultraviolet irradiation (UV) 6 10,12,15,25,
37,40
Miscellaneous
4-nitroquinoline-l-oxide (NQO) 4 6
aNumber of determinations evaluated for each compound (some at multiple genetic
loci).
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In Fig. 1, we compare the frequency of forward mutations induced per
applied dose with the reciprocal of the molar dose required for 037
survival in three rodent and two human cell systems5~41> ^s plotted, the
data constitute a potency index, extending from weakly mutagenic and less
toxic compounds to highly mutagenic agents that are toxic at very low
concentrations. The relative increase in cytotoxicity is accompanied by a
proportional increase in mutagenicity, i.e., the slope of the regression
relationship is 1.03 j+ 0.03. The observed correlation between cytotoxicity
and mutagenicity suggests that a determination of the D37 dose of toxic
agents can be used to estimate the maximum potential induced mutation
frequency. In particular, these data permit the hypothesis that no agents
will be found whose location on this graph is significantly above the upper
dotted line. Whereas mutagenicity and cytotoxicity separately range over
six orders of magnitude, no agent can induce more than approximately one
forward mutation at a given locus per one hundred cells surviving at the
037 dose. If this hypothesis is valid, this is a biological limit to
mutagenic potency that is demonstrated by compounds whose cytotoxicity is
due solely to mutational events.
On the other hand, we believe the absence of points below the lower
dotted line to be artifactual, representing the bias of reported mutagenesis
studies for compounds whose ratio of mutagenicity to cytotoxicity is
relatively high. Compounds are known that would graph somewhere in this
lower range, e.g., a few derivatives of ICR frameshift mutagensl^ a
metabolite of a polycyclic hydrocarbon^, and an inhibitor of DNA
synthesis^ are highly toxic, but apparently are nonmutagenic in mammalian
j.n vitro test systems. Also, certain chemicals yield multiphasic cell
survival curves that cast doubt on the 037 concept for these mutagens.
The shaded area in Fig. 1 reflects values below the detection limit for the
systems studied. The cytotoxicity data for non-mutagens would also fall
within this range. Thus, the dynamic range for forward mutations in
mammalian ir\ vitro systems is approximately four orders of magnitude (from
the lowest detectable level to the highest possible mutagenic potency at the
037 level of cytotoxicity). Compounds near the bottom of this range will
presumably be of greater concern as cytotoxins than as mutagens.
The relative mutagenicity of different compounds at a given cytotoxicity
(Fig. 1) is not significantly related to the class of mutagen or the forward
mutation marker scored. This is shown in Fig. 2, which, displays the
observed values of the mutation frequency per locus at the 037 dose for
different classes of mutagens at different genetic loci. This variation is
also not due to systematic differences among cell systems (data not shown).
\
Cytotoxicity, as defined (Fig. 1), may result from genetic (i.e.,
DNA-related) and nongenetic mechanisms of injury. Agents that induce the
highest mutation frequencies at the D37 dose may have the tightest
coupling between genetic injury and lethality, i.e., the cytotoxicity
related to DNA damage predominates over other forms of general toxicity.
Agents inducing lower mutation frequencies at the 037 dose may produce a
larger proportion of non-genetic injury.
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104
103
0)
I 1°1
3
I 10"
3
TJ
10-1
~i—rrn—i—rrn—i—rrn—i—rrn—r
i ' iii — nr
DMBA-
./MOOT BPox'
nMOA ' E
X DMBAox«»
DBAOX
102 103 104 105 106 107 108
1
D3?(M)
Fig. 1. The relationship between induced mutation frequency per viable cell
(M.F). per unit exposure dose (M, mole liter~l) and the
reciprocal of D37 (M), i.e., the dose required to kill 63% of the
initial cell population. Killing is defined as the inability of a
cell to undergo continued cell division in vitro resulting in a
viable cell colony. The D37 values were estimated from (1)
authors' precise survival responses; (2) computer fits in ref. 14;
(3) published tabular data (occasionally over limited dose
ranges). In some cases, the values are approximated but individual
estimates generally vary no more than +_ 20% and never more than ^
50% (in two cases). Mutation frequency data usually included at
least two dose points in the most linear range/ except for a few
reports having only limited data where a single dose and zero dose,
i.e., spontaneous frequency, were used. Data included in this plot
are those for mutations at the hgprt locus (hypoxanthine-guanine
phosphoribosyltransferase, EC 2.4.2.7), aprt locus (adenine
phosphoribosyltransferase, EC 2.4.2.8), and the ^jc locus (thymidine
kinase, EC 2.7.1.75). Results in Chinese hamster ovary cells6-13
(n = 25), V79 hamster cells!4-26 (n _ 18)f L5178Y mouse lymphoma
cells27"30 (n = 4), normal human diploid fibroblasts31"37 (n =
9), and human lymphoblasts38-39 (n - 4) were combined and
expressed as the induced frequency of mutants resistant to
azaguanine, AGr (n = 27); thioguanine, TGr (n = 27);
azaadenine, AAr (n = 4); and bromodeoxyuridine, BUdR^ (n = 2).
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Abbreviations for the mutagens are given in Table 1. The linear
regression line is given by the equation (log M.F./M) = 1.03 (- log
D37) - 3.49, with a correlation coefficient of 0.93. The dotted
lines represent boundaries of the 95% confidence band for Y data
points estimated from X. The physical mutagens were entered on the
figure after calculating their potencies relative to EMS, based on
the ratios of the number of lesions induced in the DNA of a cell at
the 037 doses (UV-induced dinners40 and total ionization
lesions5) to the number of alkylations41 induced by EMS at its
037. The "molar equivalent 037" values were obtained by
multiplying the D37 for EMS (6 x 10~3 M) by the potency ratios
for UV (0.18) and ionizing radiation (1.4 x 10~3) . The "molar
equivalent mutation frequencies" were obtained by multiplying the
geometric means of the observed mutation frequencies per 037 for
UV and ionizing radiation by the respective "molar equivalent
037" values. The calculated parameters were added to the graph,
but were not included in the regression calculation. The lower
detection limit of the assays (stippled area) was estimated,
assuming that (a) at frequencies below 10~5 induced mutations per
viable cell, statistically-significant determinations of M.F. are
generally not practical, and (b) the maximum feasible exposure dose
is usually 10 fold higher than the 037 concentration.
The remarkable similarity among mutation frequencies at equitoxic
concentrations has been noted by others for several mutagens5,16,36. The
correlation between cytotoxicity and mutagenicity need not be due solely to
the same type of cellular damage causing both lethal and mutational
events4; factors that influence the effective exposure of the cells to
chemical mutagens (toxification, detoxification), may affect both end points
proper tionally.
Additional data are needed to establish that partitioning of genetic and
nongenetic toxicity induced by a mutagenic agent can be estimated from the
magnitude of mutation induced at the 037 dose. However, the data in Fig.
1 indicate that cytotoxicity as measured by the 037 dosage provides a
quantitative estimate of potential mutagenicity. Where appropriate, more
specific bioassays can then be applied to determine precisely the in vitro
mutagenic potency.
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Alkylating
agents
PAH
NQO, UV
X/y
Alkylating
agents
Frameshift
NQO, UV
X,7
Alkylating
agents
PAH
NQO, UV
M 'M
1
M
^^^
1 1 1
i L i 1
1 1
§8£$i-:'':-:-:"x-: ";::-:-:-:-:-:-
1 1 1
l( |l 1
1 1 1
^^^f^^^
1 1 1
AGr,
AAr
TGr,
BUdRr
OUAR
10
6
Mutation frequency per locus at the D37 dose
Fig. 2. The induced mutation frequency per viable cell (M.F.) calculated
for the D37 exposure dose of the physical and chemical mutagens
-------
listed in Table 1. The loci include hgprt (AGr>TGr),aprt,
(AAr ), and _tk (BUdRr), as well as resistance to ouabain
(OUAR)involving presumed lesions in the Na+-K+-ATPase enzyme (EC
3.6.1.3). The inner mark within bars represents the geometric mean
of data for all systems, with the range of reported values
indicated by the ends of each bar. The arrows refer to the
authors' data for the Chinese hamster ovary multiple marker
system^. According to the assumptions listed in Fig. 1, the
lower limit for determining the M.F. at the D^-j would be 10"^.
-------
REFERENCES
1. Thacker, J., Stretch, A. & Stephens, M.A. Mutat. Res. 42, 313-326
(1977).
2. Chadwick, K.H., Leenhouts, H.P., Szumiel, I. & Nias, A.H.W. Int. J.
Radiat. Biol. 30, 511-524 (1976).
3. Parodi, S. & Brambilla, G. Mutat. Res. 47, 53-74 (1977).
4. Munson, R.J. & Goodhead, D.T. Mutat. Res. 42, 145-160 (1977).
5. Roberts, J.J. in Advances in Radiation Biology (eds Lett, J.T. & Adler,
H.) 212-436 (Academic Press, New York, 1978); Roberts, J.J., Sturrock,
J.E. & Ward, K.N., in Chemical Carcinogenesis, Part A (eds Ts'o, P.O. &
Dipaolo, J.A.) 401-425 (Marcel Dekker, Inc., New York, 1974).
6. Carver, J.H., Wandres, D.L., Adair, G.M. & Branscomb, E.W. Mutat. Res.
j>3, 96-97 (1978) .
7. Carver, J.H., Dewey, W.C. & Hopwood, L.E. Mutat. Res. 34, 465-480
(1976).
8. Couch, D.B. & Hsie, A.W. Mutat. Res. 57, 209-216 (1978).
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10. Hsie, A.W., Brimer, P.A., Mitchell, T.J. & Gosslee, D.S. Somat. Cell
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11. Jones, G.E. & Sargent, P.A. Cell 2, 43-54 (1974).
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C.M. Mutat. Res. 3J3, 261-278 (1975) .
-------
16. Bhuyan, B.K., Peterson, A.R. & Heidelberger, C. Chem.-Biol.
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17. Davies, P.J. & Parry, J. Genet. Res. 24, 311-314 (1974).
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10
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NOTICE
This report was prepared as an account of work sponsored by the United
States Government. Neither the United States nor the United States
Department of Energy, nor any of their employees, nor any of their
contractors, subcontractors, or their employees, makes any warranty,
express or implied, or assumes any legal liability or responsibility for the
accuracy, completeness or usefulness of any information, apparatus,
product or process disclosed, or represents that its use would not infringe
privately-owned rights.
Reference to a company or product name does not imply approval or
recommendation of the product by the University of California or the U.S.
Department of Energy to the exclusion of others that may be suitable.
11
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing]
1. REPORT NO.
EPA-600/7-79-172
4. TITLE AND SUBTITLE
Estimating the Potency of Mutagens: Cytotoxicity as an
Obligatory Consequency of Mutagenicity
6. PERFORMING ORGANIZATION CODE
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
August, 1979
7. AUTHOR(S)
June II. Carver, Elbert W. Branscomb, Frederick T. Match
8. PERFORMING ORGANIZATION REPORT NO
Dept. of Energy
UCRL-81690, Rev. 2
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Lawrence Livermore Laboratory
Biomedical Sciences Division
University of California
Livermore. CA 94550
10. PROGRAM ELEMENT NO.
1NE 625F
11. CONTRACT/GRANT NO.
EPA-IAG-D5-E681-AN and AO
12. SPONSORING AGENCY NAME AND ADDRESS
U. S. Environmental Protection Agency
Washington, CS 20460
13. TYPE OF REPORT AND PERIOD COVERED
Interim 1/78 thru 1/79
14. SPONSORING AGENCY CODE
EPA/600/17
15. SUPPLEMENTARY NOTES
This project is part of the EPA-planned and coordinated Federal Interagency
Energy/Environment R & D Program.
16. ABSTRACT ~~
Rapid and reliable screening methods are required for identifying
environmental mntap.ons anrl estimating tlieir mutagenic potency in preparation for use
of more elaborate tests to assess the genetic risk to man. On theoretical grounds,
one expects a certain minimum cytotoxic potency to correlate with mutagenic potency,
particularly when the latter is measured using forward mutations that result in
inactive gene products. Furthermore, as cells cannot divide unless a substantial
fraction of their genome is functionally intact, mutagenic agents should also be
obligatory cytotoxic agents, with a given mutagenicity conferring a certain
irreducible cytotoxicity. We show here that the cytotoxic potency of 22 chemical
niutagens is highly correlated with their mutagenic potency as assayed in five rodent
and human in vitro cell systems. This relationship implies that the maximum potential
mutagenic potency of such compounds may be reliably estimated from rapid and
straightforward measurements of their cytotoxic potency, the latter defined as the
failure of cultured cells to undergo continued cell division. The estimate is
necessarily a maximum one, as an agent may exon cytotoxic effects by pathways
indpendt;nt of its mutagenic action.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
environmental tnutagens
cytotoxic agents
cytotoxic potency
mutagenic potency
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
11
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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