SERA
United States
Environmental Protection
Agency
Great Lakes
National Program Office
77 West Jackson Boulevard
Chicago, Illinois 60604
EPA 905-R95-002
January 1995
Assessment and
Remediation of
Contaminated Sediments
(ARCS) Program
DETECTION OF GENOTOXINS IN
CONTAMINATED SEDIMENTS:
AN EVALUATION OF A
NEW TEST FOR COMPLEX
ENVIRONMENTAL MIXTURES
•) United States Areas of Concern
Ł ARCS Priority Areas of Concern
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DETECTION OF GENOTOXINS IN CONTAMINATED SEDIMENTS:
AN EVALUATION OF A NEW TEST FOR COMPLEX
ENVIRONMENTAL MIXTURES
Assessment Document
Great Lakes Program Office
U.S. Environmental Protection Agency
Project Officer: Rick Fox
1995
B. Thomas Johnson
National Biological Survey1
U.S. Department of the Interior
Columbia, MO
formerly the National Fisheries Contaminant Research Center, Fish &
Wildlife Service, U.S. Department of the Interior.
U.S. Environmental Protection Agency
Region 5, Library (PL-12J)
77 West Jackson Boulevard, 12th Floor
Chicago, IL 60604-3590
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Use of trade names does not constitute U.S. government endorsement of
products.
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DETECTION OF GENOTOXINS IN CONTAMINATED SEDIMENTS:
AN EVALUATION OF A NEW TEST FOR COMPLEX
ENVIRONMENTAL MIXTURES.
OVERVIEW
This document reviews a new approach to detect genotoxins in contaminated
freshwater sediments and summarizes the lessons learned from this investigation of the
Great Lakes Basin (Johnson, 1992a, 1992b, 1993a, 1993b). This study was conducted
as part of the U.S. Environmental Protection Agency, Great Lakes National Program
Office, Assessment and Remediation of Contaminated Sediment (ARCS) Program in
cooperation with the National Biological Survey Contaminant Research Center
(Johnson, 1993a). The objective was to detect genotoxic chemical contamination in
Great Lakes Basin sediments (Bro et al., 1987).
BACKGROUND
In the last few decades, genetic toxicology -- a new discipline -- has emerged
with the generally accepted view that some chemicals (genotoxins) can induce DNA
damage in cells that may result in lethality, mutagenesis, carcinogenesis, and potential
eco-genotoxicological expressions (Wurgler and Kramers, 1992). To ascertain
potential environmental hazards, numerous short-term qualitative tests (Epler, 1980;
Brockman and DeMarini, 1988; DeMarini et al., 1989) have been developed to detect
genotoxic agents. These bioassays, in most instances, were poorly suited for extensive
environmental surveys of complex mixtures in sediments because they were not well-
adapted to field applications, were costly, and required sophisticated technical
expertise. In addition, these bioassays encountered vexing cytotoxicity problems in
sample analysis that frequently negated their effective use in complex environmental
samples such as sediments. New tests were needed to evaluate environmental
mixtures.
One of NBS primary tasks was to develop and explore innovative techniques to
detect environmental genotoxins in complex mixtures associated with sediments. A
primary objective of this investigation was to search for a new genotoxicity bioassay
that was suited for field studies of large geographic areas, that was short-term and cost
effective, and that was simple to use. An assay developed by Microbics Corporation2
2Use of trade names does not constitute government endorsement of products.
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(Carlsbad, CA), under the trade name Mutatox™, that is currently undergoing trials in
laboratories throughout this country (Sun and Stahr, 1993; Ho et al., 1994) and Canada
(Khan et al. 1990; Legault and Blaise, 1994) satisfied these criteria. The
developmental goals in this project with the Mutatox™ assay were fourfold:
(1) to validate the relative sensitivity and selectivity of the assay using model
genotoxins and nongenotoxins in simple, binary and model complex mixtures
and to validate its use for biohazard assessment of complex pollutants in
freshwater sediments.
(2) to develop a protocol to detect and estimate chemical genotoxins in complex
environmental samples;
(3) to use this assay to determine the potential genotoxicity of contaminated
sediments (environmental samples) from selected areas of concern (AOC) sites
in the Great Lakes Basin; and
(4) to compare Mutatox™ performance for sensitivity, utility, and cost with the
well-validated Salmonella -- microsome mutagenicity test, frequently referred to
as the Ames test (Ames et al., 1973; Maron and Ames, 1983).
This report relates a series of unique events -- protocol development, validation,
and field application -in the exploration of a genotoxicity assay for use in complex
mixtures. No effort was made to test any other short-term assays to detect genotoxins.
The Ames test served as a benchmark for comparisons. This report focused only on a
new bioluminescent procaryotic bioassay — Mutatox™ with its specific application to
detect genotoxins in complex environmental mixtures. These were the highlights of the
findings.
MUTATOX"1 ASSAY: THEORY
The Mutatox™ assay detects genotoxins with a dark mutant strain of the
luminescent bacterium Photobacterium phosphoreum. DMA-damaging substances are
recognized by measuring the ability of a test sample to restore the luminescent state in
the bacterial cells (Johnson, 1992a). Light produced by luminescent bacteria makes an
easy quantitative endpoint in the genotoxicity assay. The amount of light increase
indicates the genotoxicity of the sample. Various genotoxins, base-substitution or
frame-shift, DMA synthesis inhibitors, and DNA-intercalating agents, have been
detected with the Mutatox™ assay (Johnson, 1992a; A. A. Bulich, Microbics Corp.,
personal communication).
MUTATOX™ ASSAY: PROTOCOL
Mutatox™ protocol is very simple, requiring minimal expertise. The assay may
be initiated and completed in less than 24 hours. Prepackaged dehydrated media,
freeze-dried bacteria, and standard disposable reaction tubes require only a few
hydration, mixing, and dilution steps and eliminate the rigors, tedium and cost of sterile
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technique. Bacteria are nonpathogenic, clonal, and require no reisolation or
reculturing; in addition, they are completely disposable without causing any
environmental harm. Incubation time is short which reduces the possibility of cross-
contamination with bacterial contaminants. Quality assurance and quality control are
simply maintained because the protocol develops a clear paper trail and the archival
tester strains and test media are easily stored for inspection or audit. The volume of the
reactants is low; as a result, the quantity of toxic wastes and the cost of their disposal is
significantly reduced. The assay is conducted with exogenous metabolic activation,
traditionally a rat hepatic microsomal mixture. Results and confirmation of a suspected
genotoxic substance are obtained in <24 hours. These operational procedures require
minimal technical or microbiological training (Johnson, 1992a, 1993b). Most
importantly, Mutatox™ is unlike most genotoxicity assays because the assay is
available on demand and requires no preculture of test organisms.
Unlike traditional toxicity tests where lethal concentration or lethal dose is the
endpoint, metabolic activation systems are required in many genotoxicity tests. Most
environmental genotoxins are found in an inactive state (progenotoxin) and must be
metabolically activated to become DMA-damaging substances. The incorporation of a
mammalian (rodent) hepatic metabolic activation system - the postmitochondrial
supernatant fraction (commonly referred to as the S9 fraction) - into a genotoxicity test
(Ames et al. 1973) significantly improved the assay's sensitivity to a broader spectrum
of genotoxins. The use of fish S9 (Johnson, 1993c) has increased the ecological
relevancy of these tests in aquatic ecosystems. This addition of a hepatic metabolic
activation system has become an important milestone in the development of
environmental genotoxicity testing (Brusick, 1990).
MUTATOX™ ASSAY: VALIDATION
Validation experiments for Mutatox™ were performed with selected EPA priority
pollutants (Callahan et al. 1979; Richards, D.J. and W.K. Shieh. 1986.). The assay
detected the priority pollutants that are known to be found in organic sediment extracts
from complex environmental samples (Jacobs, 1993). For example, 2-
aminoanthracene (2-AA) and benzo(a)pyrene (BaP) were dose-responsive with a
maximum detected concentration (MDC) of 5 ^ig and 2.5 ng, respectively; a lowest
detected concentration (LDC) of 0.07[xg and 0.07|o,g respectively; and dose-response
numbers of 7 and 6 respectively (Figs.1 and 2 ). A chemical was identified as
genotoxic when there were three or more responses in each dilution series (dose-
response number ;>3). In general, the sensitivity of the Mutatox™ assay to these
priority pollutants was <:1 ng/cuvette (Table I). A partial list of chemicals evaluated
with Mutatox™ and Ames is compared in Table II.
Mutatox™ validation experiments delineated the assay's relative spectrum of
detectability, focused on pollutants of interest -- PAH types, and confirmed the ability of
Mutatox™ to detect expected genotoxins that could be encountered in contaminated
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sediments. Selection of specific models was based on sediment residue work of
Jacobs et al. 1993 and the EPA pollutant priority series (Callahan et al. 1979; Richards,
D.J. and W.K. Shieh. 1986). Limited experiments were performed to simulate the
interactions of complex mixtures and their potential influence on genotoxin detection.
The reader is cautioned that it is axiomatic in toxicology that there is always toxicity in
dosage. The term cytotoxicity is used in the traditional sense: lethality to the test cell.
Cytotoxicity is described here in relationship to sample doses and observed
genotoxicity responses. As expected, Photobacteria do show cytotoxicity to
environmental pollutants; however, they seem less effected than other bacterial tester
strains. It must be remembered that some environmental samples contain several
cytotoxic substances, which may or may not also be genotoxic, that may potentially
interfere with an assay's sensitivity.
MODEL COMPLEX MIXTURES
Genotoxins
Binary mixtures of four pollutants (2-AA + 2-aminofluorene (2-AF), 2-AA + BaP,
2-AA + pyrene (PY), 2-AF +BaP, 2-AF + PY, and BaP + PY) at concentrations of 10 to
0.6 |o,g/cuvette showed no evidence of inhibitory interactions (Table III) (Johnson,
1992b).
Non-genotoxins
The model complex mixture of carbofuran, di-2-ethylhexyl phthalate, malathion,
simazine, permethrin, and Aroclor 1254, representing six classes of potential aquatic
contaminants (both pesticides and industrial sources), showed no genotoxic response
or cytotoxicity at test doses of ^10 |u,g/cuvette, nor did the mixture interfere with the
genotoxic expression of known progenotoxins (Table III) (Johnson, 1992b).
ENVIRONMENTAL SAMPLES: ORGANIC SEDIMENT SAMPLES
Thirty-eight sediment samples were collected from 28 sites in three Great Lakes
priority AOCs (Grand Calumet River in Indiana, Buffalo River in New York, and
Saginaw River in Michigan), extracted with the solvent dichloromethane, and evaluated
for genotoxicity with the Mutatox™ assay (Johnson, 1992b). For example, 210
genotoxic measurements were made from seven sites along the Grand Calumet River.
All grab samples collected in August 1989 were genotoxic, with an average 5.5 (0.8)
dose-response number/site (Fig. 3). The MDC detected ranged from 50 to 12 mg eq.
sediment/mL and the LDC ranged from 0.7 to 0.09 mg eq. sediment/mL - a single data
set of site six in shown in Figure 4. Similar samplings were taken from Buffalo River in
New York and the Saginaw River in Michigan (Johnson, 1992b). The Saginaw extracts
(Fig. 5) demonstrated various genotoxic responses with two sites designated
"Negative," one "Suspect," and four "Genotoxic." The Mutatox™ assay clearly
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demonstrated the ability to detect environmental genotoxins in complex environmental
mixtures. Cytotoxicity was not observed in either spot-plate or tube turbidity tests;
positive controls (2-AA, 2-AF, BaP and PY) were within acceptable sensitivity limits. All
findings are reported as mean • standard deviations. These data show that large
geographic areas can be sampled for Mutatox™ determination of environmental
hazards.
QUALITATIVE TOXICITY TESTING
Mutatox™ is a qualitative toxicity test. The assay produces a yes-no answer to
the question: Is the sample genotoxic? Two arbitrary values are used to determine if a
test substance is genotoxic. First, a light emission value of or greater than 100
indicates that the single-dilution sample is genotoxic. Second, three or more dilution
series responses of or greater than 100 indicate that the sample is genotoxic. The
relative light responses are irrelevant because a simple yes or no designation -- a
qualitative designation (Figs. 1, 2, and 3) ~ is the assay's endpoint. The rationale for
qualitative toxicity evaluation is straightforward - there are no partially genotoxic
substances (although there frequently are suspects, samples that may require
additional testing.).
Other toxicity tests measure multiple organisms over time in a series of chemical
concentrations to determine the lethality of an aquatic community. The resulting dead
or immobile organisms are easily quantified, usually in the form of a 50% effect: e.g.,
the EC50 (Effective Concentration) or LC50 (Lethal Concentration). This number is
then used to estimate the acute toxicity of the test chemical. These assays provide a
quantitative answer.
MUTATOX™ AND AMES TEST COMPARISON
The Mutatox™ assay compared favorably with the Ames Salmonella
Mutagenicity Test (Tables II and IV)(Johnson, 1992a). Parallel Mutatox™ and Ames
bioassays with EPA priority pollutants and other model genotoxins showed comparable
spectra of sensitivity (Table II). In complex environmental mixtures, Mutatox™ assays
and Ames tests compared favorably with 96% (27/28) site agreement in detecting
evidence of genotoxic substances in all three priority areas: Grand Calumet River,
Buffalo River, and Saginaw River (Johnson, 1992b).
Importantly, the Mutatox™ system showed low cytotoxicity in testing complex
environmental mixtures (Johnson, 1992b). Toxic chemicals did not induce bacterial
cytotoxicity in the Mutatox™ assay at the test dosage. The Mutatox™ tester strain
Photobacterium is not a nutritional auxotroph; therefore, it does not require a tedious
and time-consuming confirmation test. In contrast the Ames test needs to demonstrate
the reversion from auxotroph to prototroph of the histidine-deficient tester strain
Salmonella.
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Unlike the Ames test, which requires a 12 to 18-hours preincubation period of
the tester strain, Mutatox™ is available on demand. The use of prepackaged test
materials makes conducting Mutatox™ simpler. Mutatox™ results were obtained in <24
hours whereas the Ames test required >68 hours. The liquid wastes from the Mutatox™
assay were only 2% of the volume from the Ames test. The Mutatox™, however, was
easier and more rapid to perform with environmental samples and, as a result, more
affordable than the Ames test. The three most common indexes of performance --
sensitivity, selectivity, and predictability (Purchase 1982) - demonstrated that the
Mutatox™ assay is a valuable monitoring tool for detection of complex environmental
genotoxins, and that it should be considered for routine assessment of contaminant
toxicity.
HAZARD EVALUATION: POTENTIAL VERSUS BIOAVAILABILITY OF
ENVIRONMENTAL GENOTOXINS
Most toxicological bioassays, procaryotic or eucaryotic, single cell or
multicellular, lack an important ingredient: the element of in situ exposure to the real
world. The best test can only simulate. Most genotoxins recovered from chemically
contaminated sediments are mobilized with strong organic solvents, concentrated from
large soil samples, and dissolved in assay-compatible solvents. Therefore,
genotoxicity findings must be prefaced with the word potential, i.e., existing in
possibility, not in actuality.
The bioavailability of genotoxins in freshwater sediments -- how they move in
pore water, how they sorb onto sediment components, and how they move through the
food-chain -- is still poorly understood and worthy of further investigation. The
widespread occurrence of anthropogenic polyaromatic hydrocarbons (PAHs) in the
environment and the high sensitivity of Mutatox™ to detect these substances
undoubtedly will create difficulties both in terms of scientific and political conclusions
for governmental regulators and resource managers in their efforts to eliminate
environmental hazards from chemical contaminants. The quantitative and qualitative
toxicity testing of organic extracts from contaminated sediment offers only estimates of
the true environmental hazards influencing the freshwater ecosystem.
CONCLUSIONS AND RECOMMENDATIONS
Validation experiments showed that Mutatox™ is a useful genotoxicity test with
important field applications.
• It is sensitive: environmental genotoxins are detected in the low ^g (<10 \i
g/cuvette) range.
• It is selective: non-genotoxins are differentiated; no false positives were
observed with models in simple, complex or environmental mixtures.
• It is predictable: relative sensitivity and selectivity over time remained a
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laboratory constant.
• It is rapid: the assay is available on demand; the incubation time is short;
results are obtained in <24 hours.
• It is simple; minimal technical or microbiological training is required.
• It is economical; cost of large environmental samplings is minimal.
• It is cost effective; the volume of the reagents is low, reducing the quantity of
toxic wastes and the cost of their disposal.
• It is quality assurance and quality control friendly; archival tester strains and
test media are easily stored for inspection or audit.
These results suggest that Mutatox™, used with traditional organic extraction
methods, shows potential as a tier I genotoxicity screening tool for hazard assessment
of large freshwater bodies. The Mutatox™ Test System needs additional field sampling
and laboratory testing to confirm its value as an effective and practical screening tool to
detect environmental genotoxins.
ACKNOWLEDGMENT
This research was supported in part by the Environmental Protection Agency,
Great Lakes National Program Office, Chicago, IL I thank A. A. Bulich, Microbics
Corp., Carlsbad, CA, for the tester strain and supplies for the Mutatox™ assay.
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.Ames, B.N., W.E. Durston, E. Yamasaki, and F.D. Lee. 1973. Carcinogens are
mutagens: a single test system combining liver homogenates for activation and
bacteria for detection. Proc. Natl. Acad. Sci. (USA) 70:2281-2285.
Bro, K.M., W. C. Sonzogni, and M. E. Hanson. 1987. Relative cancer risks of chemical
contaminants in the Great Lakes. Environ. Manage. 11:495-505.
Brockman, H.E. and D.M. DeMarini. 1988. Utility of short-term tests of genetic toxicity
in the aftermath of the NTP'S analysis of 73 chemicals. Environ. Mol. Mutagen.
11:421-435.
Brusick, D.J., 1990. Environmental mutagenesis: an assessment of the past twenty
years. Mutation and the environment, Part A, Wiley-Liss, Inc., New York, NY, pp.
1-16.
Callahan, M.A., M. Slimak, N. Gbel, I. May, C. Flower, R. Freed, P. Jennings, R.
DuPree, F. Whitmore, B. Maestri, B. Holt, and C. Gould. 1979. Water-related
environmental fate of 129 priority polutants. EPA-44014-79-029a,b, NTIS.
DeMarini, D.M., J. Lewtas, and H.E. Brockman. I989. Utility of short-term tests for
genetic toxicity. Cell Biol. Toxicol. 5:189-200.
Epler, J.L I980. The use of short-term tests in the isolation and identification of
chemical mutagens in complex mixtures. In F.J. deSerres and A. Hollaender,
eds., Chemical Mutagens, Principles and Methods for Their Detection, Vol. 6.
Plenum Press, New York, NY, pp. 239-270.
Ho, K.T., LJ. Mills, C. Mueller, and S.C. Anderson. 1994. The influence of sediment
extract fractionation methods on bioassay results. Environ. Toxicol. Water Qual.
9:1-10.
Jacobs, M.W.. J.A. Coates, J.J. Delfino, G. Bitton, W.M. Davis, and K.L Garcia. 1993.
Comparison of sediment extract Microtox toxicity with semi-volatile organic
priority pollutant concentrations. Arch. Environ. Contam. Toxicol. 24:461-468.
Johnson, B.T. 1992a. An evaluation of a genotoxicity assay with liver S9 for activation
and luminescent bacteria for detection. Environ. Toxicol. Chem. 11:473-480.
Johnson, B.T. 1992b. Potential genotoxicity of sediments from the Great Lakes.
Environ. Toxicol. Water Qual. 7:373-390.
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Johnson, B. T. 1993a. "Potential genotoxicity of sediments from the Great Lakes." In
Chapter VII. Genotoxicity; US Fish and Wildlife Service and Battelle Final Report
for the USEPA GLNPO Assessment and Remediation of Contaminated
Sediments, ARCS Project: Biological Assessment of Contaminated Great Lakes
Sediment, C.G. Ingersoll, D.R. Buckler, E.G. Crecelius, and T. W. LaPoint, eds.
Johnson, B.T. 1993b. Activated Mutatox assay for detection of genotoxic substances.
Environ. Toxicol. Water Qual. 8:103-113.
Johnson, B. T. 1993c. Genotoxicity testing with fish hepatic S9 for evaluation of
complex mixtures in the aquatic environment: the use of channel catfish as a
model. Aquat. Toxicol. 27:293-314.
Kwan, K.K., B.J. Dutka, S.S. Rap and D. Lui. 1990. Mutatox Test: A new test for
monitoring environmental genotoxic agents. Environ. Pollut. 65:323-332.
Legault, R., C. Blaise, D. Rokosh, and R. Chong-Kit. 1994. Comparative assessment of
the SOS Chromotest Kit and the Mutatox test with the Salmonella plate
incorporation (Ames test) and fluctuation tests for screening genotoxic agents.
Environ. Toxicol. Water Qual. 9:45-57.
Maron, D.M., and B.N. Ames. 1983. Revised methods for the Salmonella mutagenicity
test. Mutat. Res. 113:173-215.
Purchase, I.F.H. 1982. An appraisal of predictive tests for carcinogenicity. Mutat. Res.
99:53-71.
Richards, D.J. and W.K. Shieh. 1986. Biological fate of organic priority pollutants in the
aquatic environment. Wat. Res. 20:1077-1090.
Sun, T.S.C. and H. M. Stahr. 1993. Evaluation and application of a bioluminescent
bacterial genotoxicity test. JOAC International 76: 893-898.
Waters, M.D., H.F. Stack, A.L Brady, P.H.M. Lohman, L. Haroun and H. Vaino. I988.
Use of computerized data listings and activity profiles of genetic and related
effects in the review of 195 compounds. Mutat. Res. 205:295-312.
Wurgler, F.E., and P.G.N. Kramers. 1992. Environmental effects of genotoxins (eco-
genotoxicology). Mutagenesis 7:321-327.
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List of tables and figures.
Table I. List of selected EPA priority pollutants detected with Mutatox"
(Photobacterium-rat hepatic S9).
Table II Partial list of chemicals evaluated with Mutatox and Ames Test for
genotoxicity
Table III Assay sensitivity and selectivity: Genotoxicity of progenotoxic chemicals
in model complex mixtures determined with and without activation in
Mutatox™ (Photobacterium-rat hepatic S9).
Table IV Comparison: Mutatox™ assay and Ames testa.
Fig. 1. Genotoxicity of 2-aminoanthracene determined with Mutatox.
Fig. 2. Genotoxicity of benzo(a)pyrene determined with Mutatox.
Fig. 3. Genotoxicity of sediment extracts from Grand Calumet River in Indiana
determined with Mutatox (sensitivity < 1 /vg/tube.). Dose-response number
= the mean (dark bar) of three replicates of a ten-tube dilution series with
standard deviation (white bar).
Fig. 4. Single genotoxicity data set of sediment extracts from site 6 of Grand
Calumet River in Indiana determined with Mutatox.
Fig. 5. Genotoxicity of sediment extracts from Saginaw River in Michigan
determined with Mutatox (sensitivity < 1 /yg/tube.). Dose-response number
= the mean (dark bar) of three replicates of a ten-tube dilution series with
standard deviation (white bar).
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Table I
List of selected EPA priority pollutants detected with Mutatox"
(Photobacterium-ra\. hepatic S9).
acenaphthene
acenaphthylene
2-aminoanthracene
2-aminofluorene
anthracene
benz(a)anthracene
benzo(a)pyrene
chrysene
fluoranthrene
fluorene
naphthalene
phenanthrene
pyrene
a. Sensitivity <, 1 pig/cuvette
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Table II. Partial list of chemicals evaluated with
Mutatox and Ames Test for genotoxicity.
Compound Mutatox Ames
Aflatoxin B1
2-Aminoanthracene
2-Aminoflurorene
9-Aminoacridine
Benzene*
Benzidine
Benzoin*
Benzo(a)pyrene
Captan
2-Chloroethanol*
Cyclophosphamide
1,2-Dichloropropane
1 ,3-Dichloropropene
Dioxane
Ethylene glycol
8-Hydroxyquinoline*
Lindane
Monuron*
3-methylcholanthrene
Nalidixic acid
Pyrene
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Positive
Negative
Postive
Postive
Positive
Postive
Positive
Positive
Positive
Positive
Negative
Positive
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Positive
Negative
Negative
Positive
Negative
Negative
Designated National Toxicology Program Chemical
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Table III
Assay sensitivity and selectivity: Genotoxicity of progenotoxic chemicals3 in model
complex mixtures determined with and without activation in Mutatox™
(Photobacterium-rat hepatic S9).
CHEMICAL
HEATED"
S9
TREATMENT
WITHOUT
S9
WITH
S9
PROGENOTOXINS NDC
NON-GENOTOXINSd ND
PROGENOTOXINS +
NON-GENOTOXINS ND
CONTROL"
CONTROL*
ND
ND
ND
ND
ND
ND
ND
GENOTOXIC
ND
GENOTOXIC
ND
v
ND
aProgenotoxins: As single and binary mixtures: ( 2-aminoanthracene (2-AA) + 2-
aminofluorene (2-AF), 2-AA + benzo(a)pyrene (BaP), 2-AA + pyrene (PY), 2-AF
+ BaP, 2-AF + PY, and BaP + PY).
bBoiling water for 15 seconds.
°ND = not detected (genotoxic)
dNon-genotoxins: complex mixture of carbofuran (carbamate insecticide), di-2-
ethylhexyl phthalate (plasticizer), malathion (organophosphate insecticide), sim-
azine (triazine herbicide), permethrin (synthetic pyrethroid insecticide) and
Aroclor 1254 (PCB product).
eControl sediment = methylene chloride sediment extract (Florissant, MO)
'Control solvent = dimethylsulfoxide
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Table IV
Comparison: Mutatox™ assay and Ames test3.
MUTATOX™
AMES
TEST ORGANISM
BACTERIAL
REQUIREMENT
ENDPOINT
EXOGENOUS
ACTIVATION
TEST DURATION
TEST TEMPERATURE
RELATIVE
SENSITIVITY6
STERILITY
PROCEDURE
INSTRUMENTATION
COST
Labor
Materials
Disposal
SCIENTIFIC
DEVELOPMENT
Photobacterium
One isolate
Light emission
Optional
16-24 h
23 ± 2°C
<1.0/vg/tube
Optional
Simple
Luminometer
Low
Validation
Salmonella
Usually one to
four isolates
Colony formation
Optional
48-72 h
37°C
<1.0 /yg/plate
Essential
Complex
Particle counter0
High
In common use
a. Photobacterium-act\vat\or\ and Sa/mone//a-activation genotoxicity assays.
b. Rat S9 activation with 2-acetamidofluorene, aflatoxin B1 , 2-amino-
anthracene, 2-aminofluorene, 2-aminonapthalene, benzo(a)pyrene,
3-methylcholanthrene, and pyrene.
c. Particle counter is essential for enumeration of large samples.
d. Extensive literature and validation.
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Fig. 1. Genotoxicity of 2-aminoanthracene (2-AA) determined with
Mutatox.
[Raw Data Set: Light Emission Values Recorded]
2-Aminoanthracene
Ten-tube dilution series (microgram per cuvette)
Concentration
2-AA
Control
10 5 2.5 1.2 0.6 0.3 0.15 0.07 0.03
0 1060 930 410 380 280 170 140 80
0 2233356 6
Dose-Response
Cone (ug/t)
Summary:
Maximum detected concentration = 5 micrograms per cuvette
Lowest detected concentration = 0.07 microgram per cuvette
Dose-response number (DRN) = 7
Conclusion: 2-Aminoanthracene is genotoxic.
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Fig. 2. Genotoxicity of benzo(a)pyrene (B(a)P) determined with
Mutatox.
[Raw Data Set: Light Emission Values Recorded]
Benzo(a)pyrene
Ten-tube dilution series (microgram per cuvette)
Concentration
B(a)P
Control
10 5 2.5 1.2 0.6 0.3 0.15 0.07 0.03
0 80 950 920 190 280 300 106 56
0 2233356 6
Dose-Response
.c
o>
Cone (ug/t)
Summary:
Maximum detected concentration =2.5 micrograms per cuvette
Lowest detected concentration = 0.07 microgram per cuvette
Dose-response number (DRN) = 6
Conclusion: Benzo(a)pyrene is genotoxic.
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0)
CO
c
o
Q.1-
(0 0>
0) A
tr E
o
Q
Genotoxic
2-
4567
Site Number
10
Fig. 3. Genotoxicity of sediment extracts from Grand Calumet River in Indiana
determined with Mutatox (sensitivity < 1 ^g/tube.). Dose-response number
= the mean (dark bar) of three replicates of a ten-tube dilution series with
standard deviation (white bar).
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e
^o
'5
J=
J
Genotoxic
100 50 25 12 6 3 1.5 0.7 0.3 0.1
Sediment Extract [mg eq/cuvette]
Fig. 4. Single genotoxicity data set of sediment extracts from site 6 of Grand
Calumet River in Indiana determined with Mutatox.
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8 -i
6-
0)
c
o
0.1-
0)
0> A
DC E
o
Q
4 •
2-
Genotoxic
5 6
Site Number
16
24
Fig. 5. Genotoxicity of sediment extracts from Saginaw River in Michigan
determined with Mutatox (sensitivity < 1 //g/tube.). Dose-response number
= the mean (dark bar) of three replicates of a ten-tube dilution series with
standard deviation (white bar).
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