Mutation Research 415 Ž1998. 59–67
In vitro and in vivo genotoxic activity of miral, an
organophosphorus insecticide used in Colombia
C.H. Sierra-Torres a , N. Cajas-Salazar a , L.S. Hoyos b, M. Zuleta c , E.B. Whorton a ,
W.W. Au a,)
a
Department of PreÕentiÕe Medicine and Community Health, The UniÕersity of Texas Medical Branch, GalÕeston, TX 77555-1110, USA
b
Department of Biology, UniÕersity of Cauca, Popayan,
´ Colombia
c
Department of Biology, UniÕersity of Antioquia, Medellın,
´ Colombia
Received 17 September 1997; revised 6 April 1998; accepted 9 April 1998
Abstract
Miral w 500 CS ŽCASa 42509-80-8., an organophosphorus insecticide, has been widely used in Colombia to fumigate
coffee plantations. Therefore, there is extensive human exposure to this pesticide. Miral’s mutagenic and genotoxic
activities, however, are not known. In this study, such activities of the pesticide were evaluated using the Salmonella
TA98rS9 test and the chromosome aberration assay in bone marrow cells of Swiss albino CD1 male mice. All doses tested
with Salmonella in the presence of S9 mix Ž3.2, 16, 80, 400 and 2000 m grplate. induced a mutagenic response that was
three times the spontaneous mutation frequency. The mutagenic response without S9 was twice the spontaneous frequency.
Based on a 4-day treatment Ži.p.. of mice with Miral, the median lethal dose ŽLD50. and the maximum tolerated dose
ŽMTD. were 912.5 mgrkg and 730 mgrkg, respectively. A significant dose-dependent cell cycle delay Ž r 2 s 0.85,
p - 0.01. was observed in bone marrow cells when mice were treated for 24 h with 73, 146, 219, 292, 365, 438, 511, 584,
657 and 730 mgrkg. Significant increases in mitotic indices Ž p - 0.02. and chromosome aberrations Ž p - 0.05. were
induced in bone marrow cells, when mice were treated for 18 h with the highest dose 511 mgrkg. Our results indicate that
Miral is a mutagenic compound in Salmonella and is capable of inducing chromosome aberrations at high doses in mice.
Additional genotoxicity studies in farmers exposed to Miral should be conducted to determine the potential human health
risk resulting from chronic low-dose exposures to this pesticide. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: Organophosphorus insecticide; Mutagenicity; Cytotoxicity; Genotoxicity; Salmonella typhimurium; Bone marrow cell
1. Introduction
Pesticides represent one of the greatest sources of
exposure to toxic chemicals for humans. Several
epidemiological studies have shown that farmers who
)
Corresponding author. Tel.: q1-409-772-1803; fax: q1-409772-9108; E-mail: william.au@utmb.edu
are exposed to a variety of pesticides have significantly elevated risks for developing cancers of the
blood and of the immune system w1,2x. In particular,
organophosphorus insecticide ŽOP. exposure is associated with risk of non-Hodgkin’s lymphoma w3x.
OPs, increasingly used by farmers, are responsible
for more poisonings than any other class of pesticides w4x, indicating the need for investigating their
potential hazard to human health.
1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 5 4 - 0
60
C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67
In Colombia, agriculture accounts for 21% of
gross national product and employs 40% of the labor
force w5,6x. According to the Colombian Agriculture
and Livestock Institute ŽICA., 40,000 tons of pesticides were used in 1990 w7x. It is estimated that up to
40% of the entire Colombian population is directly
exposed to pesticides w8x. Over the last few years, the
use of OP compared to organochlorates has increased in Colombia because organophosphates have
a shorter half life in the environment and do not
accumulate through the food chain. The OP, trade
formulation Miral w 500 CS ŽA.I. Isazophos, CAS
Reg. No. 42509-80-8. is widely used on coffee,
banana, and potato plantations in Colombia. As an
OP, it can inhibit the acetyl-cholinesterase enzyme
which is essential for normal neural transmission,
leading to respiratory arrest and muscular weakness
w4x. Although the general toxicity of this insecticide
has been described w9x, based on review of the
scientific literature and documents from EPA, and
inquiry to the manufacturer, Ciba-Geigy, in Colombia, the genotoxic potential of Miral has not been
investigated.
Previous studies have demonstrated that OPs have
mutagenic and clastogenic activities in several biological test systems w10–15x. These studies have
raised public concerns about the potential for adverse
genotoxic effects in humans. Therefore, additional
well-conducted in vitro and in vivo genotoxic studies
are necessary to assess possible health risks associated with the extensive use of OPs w16x. Short-term
genotoxic assays for the detection of potential human
carcinogens have been used by many investigators
and validated in international collaborative programs
w17x. The in vitro Salmonella typhimurium assay
with the rat-liver microsomal fraction S9 w18,19x is
one of the most frequently used tests for assessing
the mutagenic potential of both pure compounds w20x
and complex mixtures w21x. When the genotoxicity of
a chemical is unknown, as in the case of Miral, it is
advisable to conduct this in vitro mutation assay
using a wide range of doses w16x. In addition, the
U.S. Environmental Protection Agency recommends
that a positive in vitro response should be confirmed
by an in vivo assay, since some chemicals producing
positive responses in the Salmonella assay have
shown negative results after in vivo evaluations
w22,23x. Short-term test batteries, including combinations of the Salmonellarmicrosome assay together
with the chromosome aberration test in mammalian
cells, were found to improve significantly the sensitivity for detection of potential human carcinogens
w24x. The chromosome aberration assay is used extensively in population monitoring w25,26x since it is
recognized as a reliable biomarker for documentation
of genotoxic effects due to exposure w27–30x. Therefore, we have used both the Salmonella mutation
assay and the in vivo chromosome aberration assay
in this study to evaluate the genotoxic potential of
Miral.
Our testing shows that Miral induced a positive
mutagenic effect in Salmonella TA98 in the presence of metabolic activation ŽS9 mix.. In addition,
Miral was found to be cytotoxic and clastogenic in
the bone marrow cells of mice treated with this
pesticide.
2. Materials and methods
2.1. Chemicals
Miral w 500 CS wchloro-1-Ž1-methylethyl.-1 H1,2,4-triazol-3-yl.-0,0-diethyl phosphorothioatex ŽFig.
1., a yellow liquid, is formulated by Ciba-Geigy in
Colombia for use as an insecticide. 2-Aminofluorene
Ž2-AF. was obtained from Lancaster Synthesis. Glucose-6-phosphate, NADP, cyclophosphamide and
Fig. 1. Chemical structure and characteristics of Miral 500 CS.
C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67
vinblastine were from Sigma. All other chemicals
used were of the highest purity available commercially.
2.2. Mutagenicity testing in Salmonella
2.2.1. Bacterial strain
To evaluate the mutagenic activity of Miral, the
Ames S. typhimurium strain TA98 Ž hisD3052 . was
used. This tester strain Žgenerously provided by Dr.
Bruce Ames, Department of Biochemistry, University of California, USA. was maintained as frozen
stocks in liquid nitrogen and grown in media from
the master plate as described by Maron and Ames
w31x. The strain was periodically raised from a single
colony to check for the presence of the genetic
markers Ž uÕrB, rfa and pKM101.. S9 fraction was
prepared from the livers of Aroclor-1254 treated rats.
S9 mix was freshly prepared for each experiment
according to the method of Maron and Ames w31x.
2.2.2. Ames mutagenicity testing
Five doses of Miral Ž3.2, 16, 80, 400 and 2000
m grplate. were plated in duplicate with approximately 1 = 10 8 bacterial cells per plate. A total of
100 m lrplate of distilled water and 10 m grplate of
2-AF Ždissolved in DMSO 0.1 mgrml. were used as
negative and positive controls, respectively. The solutions were tested in the absence Ž0.5 mlrplate
phosphate buffer 1 M, pH s 7.4. and presence of S9
mix Ž0.5 mlrplate.. The mixture containing chemicals and bacteria with or without S9 was vortexed
and pre-incubated at 378C for 30 min as described by
Maron and Ames w31x. The mixture was then plated
with 2 ml of soft agar on glucose supplemented
minimal agar. After 48 h of incubation at 378C, the
plates were scored for revertant colonies Ž his q ..
2.3. Genotoxicity assays in mice
2.3.1. Animals and exposure route
Swiss albino CD1 mice, 10–12 weeks old Ž35 g,
approximately., were kindly provided to us free of
charge by Veterinaria de Colombia VECOL. Only
male mice were available for the study and were
acclimatized in our laboratory animal facility for 2
weeks before use. The mice were distributed in cages
61
under 12-h lightrdark photoperiod at constant conditions of temperature Ž22 " 38C. and with a diet
based on Soya chow and purified water. Animals
were injected intraperitoneally Ži.p.. with the test
solutions which were based on body weight Žb.w..
and never exceeded a volume of approx. 16.7 mlrkg
b.w. The choice of the doses was based on a 4-day
assay for the determination of the acute median
lethal dose ŽLD50. w32x and the maximum tolerated
dose ŽMTD. w33x. A value of 912.5 mgrkg b.w. was
determined as the LD50. The MTD of 730 mgrkg
b.w. was determined, based on 100% survival when
exposing the mice to 80% of the LD50.
2.3.2. Proliferation index
To study the effect of Miral on the bone marrow
cell proliferation kinetics, a total of 42 mice were
randomly divided into 10 groups ŽTable 2.. Each
group was exposed to either 73, 146, 219, 292, 365,
438, 511, 584, 657 or 730 mgrkg b.w. of Miral,
respectively. A negative control group Ž n s 6. was
injected with distilled water. A positive control group
Ž n s 6. was injected with 50 mgrkg b.w. of cyclophosphamide ŽCPA., diluted in 0.9% sodium chloride physiological solution 3.5 mgrml. A paraffincoated 5-bromo-2X-deoxyuridine tablet Ž50 mg
BrdUrtablet. was inserted into the abdominal region
of each animal 1 h prior to treatment for differential
staining of metaphase cells w34x. Vinblastine
Ž7 mgrkg b.w.. was injected into the animals 2 h
prior to harvesting as a cytokinesis blocker Ždiluted
in saline solution 1 mgrml.. Bone marrow preparations were made 24 h after treatment according to
the method of Hsu and Patton w35x. The slides were
stained following the FPG ŽFluorescence Plus
Giemsa. staining method as previously described
w36x. One hundred metaphase cells per animal were
analyzed to document the frequencies of these cells
in the first, second and third cycles. The data were
used to detect perturbation of cell proliferation by
changes in the proliferation index ŽPI..
2.3.3. Chromosome aberrations
From the PI study, three doses were chosen for
cytogenetic analysis: one dose with no significant
effect on PI Ž365 mgrkg b.w.., one with an intermediate effect Ž438 mgrkg b.w.., and one with a
significant effect Ž511 mgrkg b.w... Groups of five
62
C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67
mice each were exposed to either Miral, distilled
water, or CPA, and sacrificed after 18 h. Cytological
preparations were made from the femurs of each
mouse, and one hundred metaphase cells were scored
per animal. Numerical Žaneuploidy. and structural
Žchromatid and isochromatid. chromosome aberrations were scored. Complex aberrations Žtranslocations, dicentrics, etc.. and gaps Žchromatid and
isochromatid. were also scored. The mitotic index
ŽMI. was calculated by scoring 2000 cells per animal.
2.3.4. Statistical analysis
The computer software program ABSTAT Release 1.9 ŽAnderson-Bell. was used to analyze the
data. Evaluation of the difference in the means of the
PI among treated and control groups was conducted
using One-way analysis of variance ŽANOVA.. Multiple linear regression analysis was performed for the
PI. The frequency of chromosome aberrations in
treated animals was compared with the negative
control group using the Fisher’s exact test. MI means
were compared against the negative control by the
t-test. A probability Ž p . value less than 0.05 was
used as the criterion of significance.
3. Results
3.1. Mutagenicity testing in Salmonella
The observed mutagenic activity of Miral in S.
typhimurium is summarized in Table 1. In the pres-
Fig. 2. Dose–response effects of Miral 500 CS in S. typhimurium
TA98. Spontaneous revertants have been subtracted. Vertical bars
represent the mean"standard error among three independent experiments. Žl. qS9 mix; Ž`. -S9 mix.
ence of S9 mix, all tested doses of Miral demonstrated mutagenic activity. The number of his q
revertantsrplate Žranges 55.2 " 10.2 to 63.2 " 1.93.
was three times higher than the spontaneous frequency of 19 " 1.58. As illustrated in Fig. 2, an
increase in the revertant response over the spontaneous frequency was reached with the lowest dose
Ž3.2 m grplate., followed by a slight upward trend
towards a dose-dependent increase. In the assays
without S9, the bacteria exhibited a weak to moderate response Žranges 36.8 " 2.77 to 33.2 " 2.62., a
doubling over the spontaneous frequency Ž16 " 1.08..
Table 1
Mutagenicity of Miral 500 CS in S. typhimurium TA98
Treatment
DW Ž m l.
Miral 500 CS
2-AF
a
Concentration per plate Ž m g.
100
3.2
16
80
400
2000
10
Mean his q revertantsrplate Ž"S.E.. a
q S9 mix
yS9 mix
19.0 " 1.58
55.2 " 10.2
55.3 " 3.70
57.2 " 3.63
61.3 " 7.72
63.2 " 1.93
395.5 " 77.78
16.0 " 1.08
36.8 " 2.77
41.6 " 5.41
40.8 " 5.58
37.8 " 4.08
33.2 " 2.62
38.8 " 4.50
The values are means" standard error of three separate experiments, each one in duplicate.
DW: distilled water.
C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67
63
However, the number of revertantsrplate showed an
initial increase followed by a slight decrease at higher
doses ŽFig. 2.. For comparison, the response with
2-AF Ž10 m grplate., the positive control, was 395.5
" 77.78 with S9 and 38.8 " 4.50 without S9 ŽTable
1..
Miral ŽTable 2.. However, this problem did not
occur in the genotoxicity study, where all the animals survived the selected doses Ž365 to 511 mgrkg
b.w.. ŽTable 3..
3.2. Cytotoxicity assay in mice
The frequency of chromosome aberrations and the
percentage of aberrant cells per treatment are summarized in Table 3. The observed aberrations were
chromatid breaks and chromatid gaps. The tested
doses Ž365, 438 and 511 mgrkg. induced a 3-, 4and 7-fold increase in the number of gaps, respectively, compared to the negative control. Based on
the observation of chromatid and isochromatid breaks
Žexcluding gaps., only the highest dose Ž511 mgrkg
b.w.. induced a significant number of chromosome
aberrations Ž p - 0.05.. In addition, a few polyploid
metaphase cells Ž1%. were observed at this concentration ŽTable 3.. No significant differences in mitotic indexes were found between the 365 or the 438
mgrkg b.w. doses of Miral and the negative control.
The dose 511 mgrkg b.w., however, produced a
significant increase in the MI Ž p - 0.02. compared
to the negative control ŽTable 3..
3.3. Genotoxicity assays in mice
The cytotoxic effect of Miral in bone marrow
cells of treated animals is summarized in Table 2.
There were not significant differences in PI from
mice treated with 73 through 365 mgrkg b.w. Miral
Žranges 1.82 " 0.04 to 1.98 " 0.17. when compared
to the negative control Ž1.88 " 0.12, p ) 0.05.. Increasing the dose to 438 mgrkg b.w. induced a
significant reduction in PI Ž1.48 " 0.02, p - 0.05..
At the four highest doses Ž511, 584, 657 or 730
mgrkg b.w.., Miral caused a significant delay in cell
proliferation Ž p - 0.01., stopping the cells in the
first cell cycle. Multiple linear regression analysis of
the PI data indicates that the effect of Miral in
reducing the proliferation of bone marrow cells was
dose-dependent Ž r 2 s 0.85, p s 0.02.. Some of the
mice died in the groups treated with high doses of
Table 2
Effect of miral on bone marrow proliferation index of mice ŽPI.
Miral 500 CS Žmgrkg.
DW Ž16.7 mlrkg.
73
146
219
292
365
438
511
584
657
730
CPA Ž50 mgrkg.
a
Treated
Dead
MI
M II
M III
6
3
3
3
3
5
5
4
5
5
6
6
0
0
0
0
0
2
2
1
2
2
4
0
16
15
15
17
21
14
50
100
100
100
100
56
80
72
76
79
76
8
48
0
0
0
0
43
4
13
9
4
3
6
2
0
0
0
0
1
The values are means" standard error between animals.
p - 0.05 ŽANOVA..
c
p - 0.01 ŽANOVA..
DW: distilled water; CPA: cyclophosphamide
PI: proliferation index s ŽMI = 1 q MII = 2 q MIII = 3.rtotal metaphases.
b
PI " S.E.a
Cells on replication cycle Ž%.
Mice
1.88 " 0.12
1.98 " 0.17
1.94 " 0.02
1.88 " 0.04
1.82 " 0.04
1.92 " 0.01
1.48 " 0.02 b
1.00 " 0.00 c
1.00 " 0.00 c
1.00 " 0.00 c
1.00 " 0.00
1.45 " 0.04
C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67
64
0
0.2
0.2
1
1.2
97
95.8
98.6
94.4
92.8
3
3.4
0.6
3.2
5.4
0
0.6
0.6
1.4
0.6
a
b
0.4"0.40
0.8"0.37
0
2.6"0.75d
22.8"3.56 d
0.4
0.8
0
2.6 d
34.6 d
0
0
0
0
1
0.4
0.8
0
2.6
33.6
0
0
0
0
0
5
5
5
5
5
DW
365
438
511
CPA
Total
Dead
Isochromatid
Chromatid
Treated
Results of five mice treated for each concentration Ž100 cellsranimal.;
Total values of register in each concentration Ž500 cellsrdose.;
c
2000 cells per animal scored;
d
p- 0.05 ŽFisher’s exact test.; e p- 0.02 Ž t-test.;
DW: distilled water Ž16.7 mlrkg.; CPA: cyclophosphamide Ž50 mgrkg.; MI: mitotic index Ž% metaphase cells..
0
0
0
0
3
0
0
0
2
3
1
3
4
7
31
Polyploid
40
39
38
Chromatid
Aberrant cells
Ž%"S.E..
Chromosome aberrations
per 100 cells a
Mice
Miral 500
CS Žmgrkg.
Table 3
Clastogenic and cytotoxic effects of Miral on mouse bone marrow cells
Complex
aberrations
Gapsb
Isochromatid
Cells with indicated
chromosome number Ž%.
MI"S.E.c
Ž%.
2.27"0.18
2.32"0.59
2.96"0.60
3.82"0.44 e
1.87"0.18
4. Discussion
In the present study, we show that Miral 500 CS
was able to induce G–C base pair mutations w31x
causing a frameshift reversion of the histidine dependent tester strain ŽTA98. to the wild type Ž his q ..
The moderate number of revertantsrplate induced by
Miral without S9 activation is considered to be a
mutagenic response, since this effect was reproducible according to the criteria employed by Ames
et al. w19x. This response also indicates that Miral by
itself is a direct-acting mutagen. The mutagenic activity was enhanced in the presence of metabolic
activation with S9, suggesting that Miral may also
contain indirect-acting mutagenic metabolites. In both
conditions Žwith and without S9., the dose–response
curves were non-linear ŽFig. 2.. One explanation for
this response is that Miral may be cytotoxic at high
doses. In general, mutagens at high concentrations
can be toxic to bacteria, thus, decreasing the number
of revertants on the plate w31x. Another explanation
may be due to hydrolysis of Miral Žwater solubility
69 ppm. w37x in the aqueous media. Hydrolytic reactions of organophosphorus compounds eventually
lead to inorganic phosphate which is non-mutagenic
w38x. Consequently, Miral may not have been sufficiently persistent in the plate to increase the number
of revertantsrplate, a phenomenon which was also
reported by Ivanovic et al. using other organophosphorus compound w39x. Our observed mutagenic effect was also accompanied by wider standard errors
for some doses, which may have been due to the
accumulation of Miral in some regions of the plate
causing cell death. This effect has been observed in
other studies using the Ames test w40x.
The mutagenic activity of Miral may be due to the
existence of electrophilic sites in the Miral molecule
or its intermediates, which are capable of binding to
nucleophilic sites in the bacterial DNA. This is
consistent with previous reports showing the ability
of some OPs to bind DNA w37,38x and cause mutation w41–44x. For example, chloracetophone induced
base pair substitution mutations in TA100 both with
and without metabolic activation w41x. According to
RTECS w42x, Carbofuran was reported to be mutagenic in Salmonella and in other organisms. Vlckova
et al. w43x detected direct mutagenicity of Phosmet
Žthe active ingredient of Decemtione EK20., which
C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67
induced frameshift mutations in TA97 and base pair
substitutions in TA100. Water samples contaminated
with the organophosphates dimethoate and methyl
parathion exhibited a significant degree of mutagenicity with TA102, TA100 and TA98 strains w44x.
It is well established that mutation plays an important role in the initiation phase of the multi-stage
carcinogenic process. There is increasing awareness
that not only DNA alterations, but also cytotoxicity
may also play an important role in carcinogenesis
w45–48x. The results from our study indicate that
Miral can be considered a cytotoxic agent since it
induced dose-dependent cell proliferation delay ŽTable 2. and mitotic arrest ŽTable 3. in mouse bone
marrow cells. The latter observation indicates blockage of cells in mitosis, therefore, Miral may act as a
mitotic poison Že.g., as do colcemid and vinblastine..
Such cellular toxicity may induce heteroploid cells,
resulting from karyotypic instability, that can later on
progress to cancer cells w49x.
The observed lethality in the cytotoxicity assay
was not expected ŽTable 2.. However, this could be
attributed to a synergistic toxic effect from the multiple chemical exposure Žether, BrdU implantation and
vinblastine., the prolonged exposure to high doses of
Miral Ž24 h., or simply dosing accidents Žrupture of
liver, spleen or digestive tract by i.p. injection..
Lethality was not observed in the chromosome aberration studies ŽTable 3., where mice were exposed to
Miral for a shorter period of time Ž18 h.. In the bone
marrow assay, Miral induced a significant clastogenic effect only at the highest dose Ž511 mgrkg
b.w... Among mice treated with this dose, 1% of the
metaphase cells Ž n s 5. were polyploid ŽTable 3..
This observation is associated with a significantly
high mitotic index ŽTable 3, p - 0.02. due to cell
accumulation in the first cell division cycle ŽTable
2.. It is apparent that the registering of polyploid
cells was caused by an overlapping of metaphase
cells which were blocked in mitosis. Lower concentrations of Miral did not induce a sufficient number
of chromosome aberrations to show detectable clastogenicity. Clastogenic effects induced by OPs have
been documented by other investigators. For instance, the available evidence indicates that technical
grade malathion has the potential to produce genotoxic effects in several mammalian systems, including humans w50x. The total percentage of cells with
65
structural aberrations in curacron-treated mice increased significantly over controls w51x. Significantly
increased chromosomal aberrations, micronuclei and
sperm abnormalities were observed for Hinosan w15x.
Studies conducted in lymphocytes of OP-exposed
farm workers have shown an increase in chromosome aberrations compared to control subjects w52–
55x.
Kaufmann w56x proposed that gaps are the consequence of the production of lesions in the DNA
template, which inhibit DNA polymerase during
DNA replication. Formation of chromatid-type aberrations has been suggested to be a consequence of
failure of post-replication repair pathways to eliminate the gaps w56x. Although in our study gaps were
not considered in the statistical analysis as chromosome aberrations, we detected a substantial dose-dependent increase in gap frequency when mice were
exposed to Miral ŽTable 3.. Previous cytogenetic
studies have revealed a significant increase in chromatid breaks and gaps in blood lymphocytes among
workers exposed to pesticides w53,57x. The close
similarity of gaps and breaks, despite the difference
in their frequency, is interesting, and might indicate
a closer relationship between these two end-points
than we have, perhaps, allowed w58x.
The Ames test was performed in S. typhimurium
TA98 as this was the only bacterial strain available
to us in Colombia. Based on the results of this study,
the formulation Miral 500 CS can be characterized
as a compound having both direct and indirect in
vitro mutagenic activities. However, testing with
other Salmonella strains may provide additional information about the mechanism of mutagenesis of
this organophosphorus insecticide. In vivo, Miral
was clastogenic at the highest dose tested Ž511
mgrkg b.w... At this concentration, Miral also caused
a delay in cell proliferation and an increase in mitotic arrest. From our review of the literature and
U.S. EPA documents, this compound has not yet
been tested in a standard cancer bioassay. However,
our data suggests that this compound may be potentially carcinogenic after exposure to relatively high
doses. Therefore, it is advisable for farmers to follow
the Product Label instructions and use excellent
safety measures when applying Miral and to reduce
environmental contamination as much as possible. In
addition, the potential genotoxic hazard in humans
66
C.H. Sierra-Torres et al.r Mutation Research 415 (1998) 59–67
after exposure to low concentrations of Miral for a
long period of time, or exposure in combination with
other pesticides, should be investigated.
w12x
w13x
Acknowledgements
w14x
The authors wish to thank Dr. Sherif Abdel-Rahman for his helpful review of the manuscript and
critical comments. We also wish to thank professor
Jaime Calle and the staff at the Laboratory of Mutagenesis and Carcinogenesis at the University of Antioquia, and professor Silvio Carvajal and the staff in
the Genetic Toxicology and Cytogenetics Unit at the
University of Cauca for their invaluable assistance in
this study.
w17x
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