Dialkylquinoneimine metabolites of chloroacetanilide herbicides induce sister chromatid exchanges in cultured human lymphocytes

Article abstract of DOI:10.1016/S1383-5718(97)00163-0

Some of the most widely-used herbicides are the chloroacetanilides exemplified by alachlor and butachlor (derived from 2,6-diethylaniline) and metolachlor and acetochlor (synthesized from 2-ethyl-6-methylaniline). This investigation tests the hypothesis that the previously-observed oncogenicity of these herbicides is due to genotoxic intermediates such as diethylbenzoquinoneimine, a purported alachlor metabolite. Syntheses are reported here for the corresponding 2,6-dialkylbenzoquinoneimines, selected chloroacetyldialkylbenzoquinoneimines and several other candidate or known metabolites. The possible mutagenicity of diethylbenzoquinoneimine was tested in Salmonella typhimurium strains TA98 and TA100 with a weakly-positive response in the TA100 strain indicating induction of base-pair substitution mutations. The frequency of sister chromatid exchange (SCE) in Chinese hamster ovary cells was increased by alachlor at 10 μM and diethylaniline but not ethylmethylaniline at 30 and 3 μM. Isolated and cultured peripheral lymphocytes (mostly T cells) were used from two human donors to study the effects of the chloroacetanilides and their metabolites on primary human cells. In tests at 10 μM, the SCE frequency was increased by alachlor and possibly acetochlor but not by butachlor, metolachlor, dimethachlor (a 2,6-dimethyl analog) and dimethenamid (an analog based on 2,4-dimethyl-3-thienylamine). At 0.3 μM in cultured human lymphocytes, alachlor, the corresponding chloroacetanilide (N-dealkyl-alachlor) and aniline metabolites (and their 4-hydroxy derivatives), and diethylbenzoquinone were inactive or active in only one of the two donors whereas at 0.1-0.3 μM the SCE ratio for treated cells divided by the controls was always higher for diethylbenzoquinoneimine than for ethylmethyl- and dimethylbenzoquinoneimines. All the tested compounds were toxic to lymphocytes, but the depression of the mitotic index and increased duration of the cell cycle were not directly linked with SCE induction. Previous investigations have suggested that chloroacetanilide herbicides such as alachlor derived from 2,6-dialkylanilines are metabolized to 2,6-dialkylbenzoquinoneimines and the present study provides the first direct evidence that these metabolites are genotoxic in human lymphocytes.

Full text of DOI:10.1016/S1383-5718(97)00163-0

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.
Mutation Research 395 1997 159–171
Dialkylquinoneimine metabolites of chloroacetanilide herbicides
induce sister chromatid exchanges in cultured human
lymphocytes
1
)
Anna B. Hill , Phillip R. Jefferies, Gary B. Quistad, John E. Casida
EnÕironmental Chemistry and Toxicology Laboratory, Department of EnÕironmental Science, Policy and Management, UniÕersity of
California, 114 Wellman Hall, Berkeley, CA 94720-3112, USA
Received 29 May 1997; revised 18 August 1997; accepted 18 August 1997
Abstract
Some of the most widely-used herbicides are the chloroacetanilides exemplified by alachlor and butachlor derived from
Ž
.
Ž
.
2,6-diethylaniline and metolachlor and acetochlor synthesized from 2-ethyl-6-methylaniline . This investigation tests the
hypothesis that the previously-observed oncogenicity of these herbicides is due to genotoxic intermediates such as
diethylbenzoquinoneimine, a purported alachlor metabolite. Syntheses are reported here for the corresponding 2,6-dial-
kylbenzoquinoneimines, selected chloroacetyldialkylbenzoquinoneimines and several other candidate or known metabolites.
The possible mutagenicity of diethylbenzoquinoneimine was tested in Salmonella typhimurium strains TA98 and TA100
with a weakly-positive response in the TA100 strain indicating induction of base-pair substitution mutations. The frequency
Ž
.
of sister chromatid exchange SCE in Chinese hamster ovary cells was increased by alachlor at 10 mM and diethylaniline
Ž
.
but not ethylmethylaniline at 30 and 3 mM. Isolated and cultured peripheral lymphocytes mostly T cells were used from
two human donors to study the effects of the chloroacetanilides and their metabolites on primary human cells. In tests at 10
mM, the SCE frequency was increased by alachlor and possibly acetochlor but not by butachlor, metolachlor, dimethachlor
Ž
.
Ž
.
a 2,6-dimethyl analog and dimethenamid an analog based on 2,4-dimethyl-3-thienylamine . At 0.3 mM in cultured human
Ž
.
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lymphocytes, alachlor, the corresponding chloroacetanilide N-dealkyl-alachlor and aniline metabolites and their 4-hydroxy
.
derivatives , and diethylbenzoquinone were inactive or active in only one of the two donors whereas at 0.1–0.3 mM the SCE
ratio for treated cells divided by the controls was always higher for diethylbenzoquinoneimine than for ethylmethyl- and
dimethylbenzoquinoneimines. All the tested compounds were toxic to lymphocytes, but the depression of the mitotic index
and increased duration of the cell cycle were not directly linked with SCE induction. Previous investigations have suggested
Abbreviations: BrdU, 5-bromo-2X-deoxyuridine; CHO, Chinese hamster ovary; CI, chemical ionization; DMSO, dimethyl sulfoxide; EI,
electron impact; GSH, glutathione; HBSS, Hank’s buffered saline solution; M1, M2 and M3, first-, second- and third-division metaphases;
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.
MI, mitotic index; MS, mass spectrometry; MTT, 3- 4,5-dimethylthiazol-2-yl -2,5-diphenyltetrazolium bromide; PRI, proliferation index;
SCE, sister chromatid exchange; SCE index, number of SCEs in treated cells % number of SCEs in control cells
)
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.
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.
Corresponding author. Tel.: q1 510 642-5424; Fax: q1 510 642-6497; E-mail: ectl@nature.berkeley.edu
1
Present address: Metabolex, Inc., 3876 Bay Center Place, Hayward, CA 94545, USA.
1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.
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.
PII S1383-5718 97 00163-0

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160
A.B. Hill et al.rMutation Research 395 1997 159–171
that chloroacetanilide herbicides such as alachlor derived from 2,6-dialkylanilines are metabolized to 2,6-dial-
kylbenzoquinoneimines and the present study provides the first direct evidence that these metabolites are genotoxic in
human lymphocytes. q 1997 Elsevier Science B.V.
Keywords: Acetochlor; Alachlor; Butachlor; Herbicide; Metolachlor; Sister chromatid exchange
1. Introduction
Three chloroacetanilide herbicides alachlor, ace-
The four oncogenic chloroacetanilide herbicides
Ž
are derived from 2,6-diethylaniline for alachlor and
Ž
.
Ž
butachlor or 2-ethyl-6-methylaniline for acetochlor
.
and metolachlor and vary in the N-alkoxyalkyl sub-
stituents Fig. 1 . A related herbicide dimethachlor
is derived from 2,6-dimethylaniline Fig. 1 and is
used for weed control in oilseed rape 6 . Two
analogs important as herbicides with limited evi-
.
tochlor and metolachlor are used in large amounts
Ž
.
Ž
.
Ž
Ž
.
)100 000 000 lbsryr for economical weed control
in corn, soybean, sorghum and other major crops and
.
w x
Ž
.
a fourth chloroacetanilide butachlor is an important
Ž
w
.
rice herbicide Fig. 1 . These compounds are onco-
genic in rats 1–6 and alachlor is classified as a
probable human carcinogen 1–3 . Alachlor manu-
facturing workers had possible elevated rates of col-
x
w x
dence of carcinogenicity are dimethenamid 11 and
w
x
w x Ž
.
propachlor 6 Fig. 1 . Although these relationships
focus attention on the 2,6-dialkylaniline moiety, there
is a notable lack of structure-activity data on the
genotoxicity of these herbicides.
w
x
orectal cancer 7,8 . Residues of the chloroac-
etanilide herbicides are present in groundwater, in-
cluding that used in some cases for human consump-
Metabolic activation may be important in the
genotoxicity of alachlor and possibly of some of its
analogs. Although several mechanisms have been
w
x
tion 1,9,10 . It is, therefore, important to consider
possible mechanisms for the genotoxicity of these
four chloroacetanilide herbicides based on metabolic
considerations and short-term assays.
Ž
.
considered for alachlor see Section 4 , the favored
w x
proposal 3 is that the activity in inducing nasal
turbinate tumors in rats is due to metabolic activation
to the electrophilic 3,5-diethyl-p-benzoquinone-4-
Ž
.
imine diethylquinoneimine which readily binds to
w
x
cellular proteins 12–14 . More generally, it appears
possible that the oncogenic activity of the 2,6-dial-
kylaniline-derived herbicides may be due to di-
alkylquinoneimine metabolites. This hypothesis can
be examined directly by comparing the genotoxicity
of the relevant herbicides and their major metabo-
lites.
Selecting a genotoxicity assay for alachlor and its
analogs and metabolites is facilitated by extensive
w
x
studies on alachlor 1,2 which have used bacteria
w
x
w
x
15 , mouse bone marrow 16,17 , Chinese hamster
Ž
.
w x
ovary CHO cells 18 , and human lymphocytes
w
x
16,19–22 as experimental models. The latter stud-
w
ies show that alachlor induces genotoxic effects sis-
ter chromatid exchanges SCEs , chromosomal aber-
Ž
.
x
rations and micronuclei formation in human lym-
w x
phocytes at 4–138 mM. Acetochlor 4 , butachlor
w
x
w x
23 and metolachlor 24 also induce chromosomal
aberrations in human lymphocytes. Studies on SCE
induction are particularly convenient for potential
structure-activity investigations on analogs and
Ž
Fig. 1. Structures and names for five 2-chloro-N- 2,6-dial-
.
Ž
.
kylphenyl -N- alkoxyalkyl acetamide and two other chloroac-
etamide herbicides studied.

(
)
A.B. Hill et al.rMutation Research 395 1997 159–171
161
Fig. 2. Metabolic activation of alachlor to diethylquinoneimine showing possible intermediates from oxidative and hydrolytic pathways. For
Ž
brevity the compounds are designated by chemical type deleting the diethyl portion. Additional metabolic reactions are also involved see
.
Ž .
Section 4 . Potencies for induction of sister chromatid exchanges in cultured human lymphocytes are rated from negative y to distinctly
positive qqq at 0.3 mM. A compound rated negative y at 0.3 mM may be positive at a higher concentration, e.g. alachlor is rated as
Ž
.
Ž .
Ž
.
qqq at 10 mM.
metabolites because of high sensitivity and ease of
alachlor as an example, and the names used are
shown in Fig. 2.
w
x
scoring 25 . In addition, peripheral lymphocytes cir-
culate for long periods and are a useful indicator of
cytogenetic effects of occupational exposure to pesti-
2. Materials and methods
w
x
cides 26,27 .
The studies reported here have examined whether
diethylquinoneimine is mutagenic in bacteria and
whether the oncogenic chloroacetanilide herbicides
and their analogs and metabolites induce SCEs in
CHO cells and human lymphocytes. The relation-
ships between the candidate metabolites, using
2.1. Chemicals
Ž
Sources for the chemicals and their CAS num-
.
Ž
.
Ž
bers were: alachlor 15972-60-8 , butachlor 23184-
.
Ž
.
66-9 , metolachlor 51218-45-2 and propachlor
Ž
.
Ž
.
1918-16-7 from Chem Service West Chester, PA ;
Fig. 3. General synthesis scheme for selected metabolites of alachlor and its analogs.

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)
162
A.B. Hill et al.rMutation Research 395 1997 159–171
Ž
.
Ž
Ž
.
Ž
.
acetochlor 34256-82-1 and dimethachlor 50563-
etate 140 mg in ethyl acetate 5 ml at 0–58C.
.
Ž
.
Ž
.
Ž
36-5 from Zeneca Agrochemicals Richmond, CA ;
dimethenamid 87674-68-8 from Sandoz Agro, Inc.
Palo Alto, CA ; 2,6-diethylaniline 579-66-8 , 3,5-
dimethylphenol 108-68-9 and 3-ethylphenol 620-
After completion 10 min 8% NaHCO3 solution 2
ml was added. The organic phase gave dimeth-
ylquinoneimine, mp 84–858C cream needles from
Ž
.
Ž
.
.
Ž
Ž
.
Ž
1
.
Ž
. Ž
w
x
.
ether literature 33 mp 81–838C . H-NMR d 6.33
s, H-2 and -6 , 2.15 s, Me . 13C-NMR d 187.2
.
Ž
.
Ž
.
Ž
.
17-7 from Aldrich Chemical Co. Milwaukee, WI .
Ž
.
Ž
Ž
.
Ž
.
Ž
.
Ž
2-Chloro-N- 2,6-diethylphenyl acetamide 6967-29-
. Ž
C-1 , 167.6 C-4 , 145.8 br C-3 and -5 , 130.8 C-2
and -6 , 17.5 Me . MS CI , mrz 136 Mq1
purity 99% .
.
referred to as the chloroacetanilide metabolite
.
Ž
.
Ž
.
Ž
.
9
w
x Ž
.
Ž
.
was prepared as described 28 purity 99% . NMR
spectra were measured with a Bruker AM300 spec-
Ž
.
Ž .
trometer CDCl3 . Chemical ionization CI and
2.2.5. 3-Ethyl-5-methyl-p-benzoquinone-4-imine
3-Ethyl-5-methyl-4-aminophenol was oxidized
with lead tetraacetate as described above to give
Ž
.
Ž
.
electron impact EI mass spectra MS were ob-
tained in the Department of Chemistry University of
California at Berkeley . Purities of the synthetic
compounds were determined by TLC and H-NMR
Ž
.
Ž
ethylmethylquinoneimine, mp 63–648C needles
1
1
.
Ž
from hexane . H-NMR d 6.35 and 6.32 ss, H-2
.
Ž
.
Ž
.
Ž
and they were stored anhydrous at y208C.
and -6 , 2.17 s, Me , 2.57 q, Js7 Hz and 1.19 t,
Js7 Hz Et . 13C-NMR d 187.4 C-1 , 166.9
. Ž
.
Ž
.
(
)
Ž
.
Ž
.
Ž
.
2.2. Syntheses Fig. 3
C-4 , 150.8 br C-3 , 146.2 br C-5 , 130.6 and
Ž
.
Ž
.
Ž
.
128.7 C-2 and -6 . MS CI , mrz 150 Mq1
(
)
Ž
.
2.2.1. 3,5-Dimethyl-4-aminophenol 3096-70-61
purity 99% .
w
x
Following the general method 29 , 3,5-dimethyl-
phenol was treated with diazotized sulphanilic acid
and the azo dye reduced with fresh sodium hydrosul-
fite at 658C. The product was purified on silica with
(
2.2.6. 3,5-Diethyl-p-benzoquinone-4-imine 108451-
) (
)
26-9 quinoneimine
Oxidation of 3,5-diethyl-4-aminophenol as above
Ž
Ž
CHCl3-methanol mixtures, mp 181–1838C plates
gave diethylquinoneimine, mp 35–368C prisms from
1
. Ž
w
x
. Ž
.
Ž
.
.
from CHCl3 literature 29 : 183–1848C purity
hexane at y208C . H-NMR d 6.33 s, H-2 and -6 ,
.
Ž
.
Ž
Ž
99% .
2.56 q, Js7.2 Hz and 1.18 t, Js7.2 Hz, Et .
13
.
Ž
.
Ž
C-NMR d 187.4 C-1 , 166.2 C-4 , 150 br C-3
(
.
Ž
.
Ž .
2.2.2. 3-Ethyl-5-methyl-4-aminophenol 112730-64-
and -5 , 128.4 C-2 and -6 , 23.2 and 12.2 Et . MS
)
8
Ž
.
Ž
. Ž
.
CI , mrz 164 M q 1 purity 99% . Dieth-
ylquinoneimine in pH 7.4 100 mM phosphate at
258C has a half-life of about 2 h but reacts com-
pletely within a few minutes on addition of a slight
w
x
3-Ethyl-5-methylphenol 30 was treated as above
to give 3-ethyl-5-methyl-4-aminophenol, mp 167–
1708C plates from ethyl acetate as reported 31
Ž
.
w x
Ž
.
Ž
.
purity 99% .
molar excess of glutathione GSH .
(
) (
(
)
2.2.3. 3,5-Diethyl-4-aminophenol 108451-25-8 hy-
2.2.7. 2,6-Diethyl-p-benzoquinone 50348-20-4
)
(
)
droxyaniline
quinone
Chromatography of diethylquinoneimine on silica
3,5-Diethylphenol was obtained from 3-ethylphe-
w
x
Ž
.
nol using a published method 32 modified by in-
creasing the aluminum chloride by 30%. The dieth-
ylphenol was treated as above to give 3,5-diethyl-4-
with ethyl acetate–hexane 20:1 gave diethylbenzo-
Ž
.
quinone, mp 36–378C yellow needles from pentane
. Ž
Ž
w
x
.
literature 34 : mp 358C purity 99% .
Ž
aminophenol, mp 105–1078C needles from
.
w
x Ž
.
toluene–hexane as reported 12 purity 99% .
2.2.8. N-Chloroacetyl-3,5-dimethyl-4-aminophenol
3,5-Dimethyl-4-aminophenol 100 mg in toluene
3 ml , tetrahydrofuran 0.3 ml , and triethylamine
0.75 ml were cooled to 08C and chloroacetyl chlo-
ride 0.35 g in toluene 0.7 ml was added dropwise
with stirring. After 10 min, the solution was washed
Ž
.
.
Ž
Ž
.
Ž
2.2.4. 3,5-Dimethyl-p-benzoquinone-4-imine
132298-15-8
3,5-Dimethyl-4-aminophenol 30 mg in ethyl ac-
(
)
.
Ž
.
Ž
.
Ž
.
Ž
.
etate 3 ml was treated dropwise with lead tetraac-

(
)
A.B. Hill et al.rMutation Research 395 1997 159–171
163
Ž .
cology Laboratory Menlo Park, CA with two
with water, dried and evaporated. TLC of the prod-
Ž
.
Ž
Salmonella typhimurium tester strains TA98 and
uct on silica in CHCl3-methanol 50:1 gave the
. w
x
corresponding hydroxychloroacetanilide, mp 168–
TA100 35 for diethylquinoneimine at 0, 1, 3, 10,
30 and 100 mgrplate in the absence of metabolic
activation using three plates per dose. TA100 reveals
base-pair substitution mutations and TA98 detects
both frameshift and base-pair mutations. The sample
1
Ž
.
Ž
1708C prisms from CHCl3 . H-NMR d 6.37 s,
H-2 and -6 , 4.07 s, COCH2 , 2.02 Me . 13C-NMR
CDCl3rCD3OD d 166.7 C5O , 156.4 C-1 ,
137.1 C-3 and -5 , 115.2 C-2 and -6 , 42.0 C-2 ,
.
Ž
.
Ž
.
.
Ž
.
Ž
Ž
.
.
X
Ž
.
Ž
.
Ž
Ž
Ž
.
Ž
.
q. Ž
Ž
.
17.3 Me . MS EI , mrz 213, 215 M purity
stored at y208C was dissolved and diluted in
.
Ž
.
99% .
dimethyl sulfoxide DMSO and immediately added
to the test system. Solvent and positive controls were
within acceptable limits and sterility controls showed
no microbial contamination.
2.2.9. N-Chloroacetyl-3,5-diethyl-4-aminophenol
(
)
hydroxychloroacetanilide
This compound was prepared as for the dimethyl
Ž
analog, mp 146–1488C plates from ethyl acetate–
2.4. Cells assays
1
.
Ž
.
Ž
hexane . H-NMR d 6.42 s, H-2 and -6 , 4.29 s,
.
.
Ž
.
Ž
COCH2 , 2.53 q, Js7.5 Hz and 1.19 t, Js7.5
. Ž
167.3
2.4.1. CHO cells and cultures
All tissue culture supplies and reagents were from
Grand Island Biological Co. Grand Island, NY .
Hz Et . 13C-NMR CDCl3rCD3OD
d
.
Ž
Ž
.
.
Ž
.
Ž
.
Ž
C5O , 156.4 C-1 , 137.1 C-3 and -5 , 115.2 C-2
Ž
.
Ž
.
Ž
.
Ž .
and -6 , 42.7 COCH2 , 25.0 and 17.6 Et . MS EI ,
mrz 241, 243 M purity 99% .
CHO cells from American Tissue Type Culture Col-
Ž
q. Ž
.
Ž
.
lection Rockville, MD were cultured in Hamm’s
F12 medium and 10% fetal bovine serum. Alachlor
cytotoxicity was measured using a colony formation
2.2.10. N-Chloroacetyl-3,5-dimethyl-p-benzo-
quinone-4-imine
N-Chloroacetyl-3,5-dimethyl-4-aminophenol was
treated with lead tetraacetate as above. The noncrys-
talline product hydrolyzed to the quinone on at-
Ž
.
assay and a 3- 4,5-dimethylthiazol-2-yl -2,5-diphen-
. Ž
Ž
yltetrazolium bromide MTT Sigma Chemical Co.,
.
w x
St. Louis, MO dye reduction procedure 36 for
mitochondrial activity in viable cells. For colony
formation studies 100 cells were plated in 100 mm
dishes and the next day the medium was removed
and fresh medium added with varying concentrations
Ž
.
tempted chromatography silica, florisil but was pu-
rified by precipitation of impurities from ether with
hexane and sublimation. H-NMR d 6.46 s, H-2
and -6 , 4.32 s, COCH2 , 2.15 s, Me . MS EI ,
1
Ž
.
Ž
.
Ž
.
Ž .
Ž
of alachlor dissolved in DMSO up to 1% final
q
Ž
M
. Ž .
mrz 211, 213
204 M–Cl . Minor peaks
.
concentration . After 6 days incubation the colonies
1
Ž
Ž
.
10% were observed in the H-NMR at 6.6 and 4.2
were fixed by 3:1 methanol: acetic acid and stained
.
purity 90% .
Ž
.
with 1% Giemsa pH 7.4 ; visible colonies were
scored and an LD50 was determined. For MTT stud-
ies, 2500 cells per well were seeded in 96-well
plates. The medium was removed after 24 h and
replaced with medium containing varying concentra-
tions of alachlor. After incubation for 72 h viable
cells were assayed with MTT. For cytogenetic stud-
ies, 5=105 cells were plated in T-25 flasks. After
2.2.11. N-Chloroacetyl-3,5-diethyl-p-benzoquinone-
4-imine chloroacetylquinoneimine
Oxidation of N-chloroacetyl-3,5-diethyl-4-
aminophenol gave chloroacetyldiethylquinoneimine
which was purified by sublimation. 1H-NMR d 6.43
(
)
Ž
.
Ž
.
Ž
s, H-2 and -6 , 4.32 s, COCH2 , 2.41 q, Js7.3
.
Ž
.
Ž .
Hz and 1.15 q, Js7.3 Hz, Et . MS EI , mrz
24 h the cultures were treated with 10 mM 5-bromo-
q
Ž
.
Ž
.
Ž
.
239, 241 M , 204 M–Cl . Minor peaks 10%
X
Ž
. Ž
1
2 -deoxyuridine BrdU Aldrich Chemical Co., Mil-
Ž
were observed in the H-NMR at 6.6 and 4.2 purity
.
Ž
waukee, WI and with test compounds added in
.
90% .
.
DMSO; 1% final concentration then incubated for
48 h at 378C with 5% CO2. The 48-h assay time was
chosen to obtain a high percentage of second-divi-
2.3. Bacterial assays
Ž
.
The preincubation modification of the standard
Ames test was performed at SRI International Toxi-
sion metaphases M2 in both the control and treated
cultures, i.e. 64–66% and 72–92%, respectively.

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A.B. Hill et al.rMutation Research 395 1997 159–171
Ž
2.4.2. Human lymphocytes and cultures
croplate Reader Molecular Devices, Menlo Park,
.
For human lymphocyte studies, white blood cells
CA .
were isolated from two different non-smoking males
Ž
.
30–40 years old , neither of whom was taking
2.4.3. Chromosome and cell cycle analyses
Ž
.
medication. The blood 20 ml from each was drawn
into heparinized tubes and diluted 1:1 with Ca2q
and Mg2q-free Hank’s buffered saline solution
In both CHO and lymphocyte experiments, col-
Ž
.
-
cemid 0.1 mgrml was added during the last 2 h of
incubation to arrest mitotic division for metaphase
chromosome examination. Chromosome preparations
Ž
.
HBSS . Isolated lymphocytes were used rather than
Ž
whole blood since the two donors differed signifi-
cantly in white blood cell numbers and the same
number of cells could then be innoculated for each
culture. Diluted blood was centrifuged in
Lymphoprepw tubes and the white blood cells in the
upper phases were then washed with HBSS, and the
cell number determined by staining with Trypan
Blue, followed by hemocytometer counting. RPMI
made by standard procedures hypotonic treatment,
.
followed by methanol: acetic acid fixation were
stained using the fluorescence plus Giemsa technique
38 . Twenty-five M2 cells were used for each treat-
ment to score SCEs unless noted otherwise. Each
w
x
lymphocyte experiment was designated by a letter
Ž
.
Ž
.
A–H and donor number 1–2 .
A two-tailed Student’s t-test determined whether
w
1640 medium supplemented with 15% fetal bovine
the SCE frequency of treated cultures differed from
that in the corresponding controls. SCE induction in
lymphocytes was quantitated by calculating an SCE
Ž
serum, penicillin-streptomycin 100 unitsrml and
.
100 mgrml, respectively , 2 mM glutamine, and 1%
phytohemagglutinin M was used for lymphocyte
cultures. Although both B and T cells were present
in the isolated lymphocytes, phytohemagglutinin M
x
Ž
index number of SCEs in treated cells % number of
.
Ž
. Ž
SCEs in control cells . The mitotic index MI per-
cent of mitotic cells was determined for human
.
Ž
.
is primarily a T-cell mitogen 25 resulting in a
lymphocytes using 2 000 cells per culture. Fifty
dividing cell population highly enriched in T-
metaphases per treatment were used to determine the
6
Ž
.
Ž
.
lymphoblasts. Isolated lymphocytes 1.0–1.5=10
percent value of cells in the first division M1 ,
Ž
.
Ž
.
were seeded in flasks, 25 mM BrdU was added, and
cells were incubated in 5 ml of medium for 24 h at
378C with 5% CO2. Then the test herbicides and
metabolites were added as fresh solutions in DMSO.
second division M2 and third division M3 in both
CHO and lymphocyte cultures. The proliferation in-
Ž
.
dex PRI was calculated using the following for-
w Ž .x
.
Ž
.
Ž
mula: PRI s 1 %M1 q 2 %M2 q 3 %M3 r100
26 . PRI values can range from 1.00 all metaphases
Ž
.
w
x
Ž
Mitomycin C Sigma freshly dissolved in water was
used as a positive control for SCE induction. Lym-
phocyte cultures were incubated for 72 h after addi-
Table 1
Ž
tion of test compounds or carrier solvent alone con-
Diethylquinoneimine as a mutagen in Salmonella typhimurium
strains TA98 and TA100
.
trol . This duration allowed comparison of M2
Ž
metaphases in controls and all treated cultures dis-
Compound
Dose, mgrplate Revertantsrplate,
mean"SE
.
cussed later .
A lymphocyte-based MTT assay of cytotoxicity
was developed from the protocol of Schiller et al.
TA98
29"3
22"2
28"2
28"3
TA100
Diethylquinoneiminea
2-Nitrofluorene
0
159"12
170"4
335"12
w
x
37 . Briefly, lymphocytes were incubated with the
test compounds for 72 h as above, then cells from an
10
30
0
Ž
.
aliquot 1 ml of each culture were collected by
Ž
.
centrifugation at 1000=g. Fresh medium 200 ml
Ž
.
positive control
Ž
.
containing MTT 0.1 mgrml was added to the cells
in each tube followed by incubation for 2 h at 378C
in 5% CO2. The cells were recovered by centrifuga-
tion and 200 ml DMSO added to dissolve the purple
5
0
1884"11
Sodium azide
167"6
Ž
.
positive control
5
1152"43
a Diethylquinoneimine was not significantly different from the
Ž
.
formazan product from reduction of MTT for mea-
surement at 570 nm using a UVmax Kinetic Mi-
Ž
.
control 0 mg when tested at 1 and 3 mg and toxic at 100 mg.

(
)
A.B. Hill et al.rMutation Research 395 1997 159–171
165
Table 2
Alachlor and two dialkylanilines as inducers of sister chromatid exchanges and effects on mitotic index and cell cycle progression in
Chinese hamster ovary cells
Treatment
mM
SCErchromosome"SEa
Cell cycleb
Ž
.
Ž
.
Ž
.
M1 %
M2 %
M3 %
PRI
Control
Alachlor
Diethylaniline
–
0.31"0.01
0.40"0.01) )
0.40"0.02)
0.37"0.02)
0.35"0.01
0.36"0.02
2
4
2
0
2
2
64
66
72
78
92
60
34
30
26
22
6
2.32
2.26
2.24
2.22
2.04
2.36
10c
30
3
Ethylmethylaniline
30
3
38
a Based on 25 second-division metaphases. Exposure for 48 h.
b Based on 50 metaphases.
c No metaphases at 30 mM.
) pF0.05; ) ) pF0.01.
Table 3
Alachlor and analogs as inducers of sister chromatid exchanges in cultured human lymphocytes; structures are given in Fig. 1
Compound
Controls
mM
Expt. donor
SCErcell"SEa
SCE indexb
0
0
0
0
0.01
0.01
0.01
F1
4.36"0.30
5.56"0.36
6.00"0.35
G1
G2
H1
F1
G1
G2
4.44"0.32
8.00"0.44) ) )
17.9"1.3) ) )
20.5"1.3) ) )
1.83
3.22
3.42
Ž
.
Mitomycin C positive control
Ž
.
Derivatives of 2,6-dialkylanilines and alkyl substituents
Alachlor Et,Et
10
10
10
3
F1
G2
H1
F1
G2
F1
G2
F1
G2
F1
G2
F1
G2
F1
G2
6.16"0.36) ) )
7.68"0.48) )
6.96"0.46) ) )
5.20"0.33
6.72"0.51
4.00"0.33
5.89"0.44
4.56"0.36
6.00"0.35
6.40"0.57) )
6.68"0.50
4.84"0.28
5.96"0.37
4.72"0.39
5.60"0.31
1.41
1.28
1.57
1.19
1.12
0.92
0.98
1.05
1.00
1.47
1.11
1.11
0.99
1.08
0.93
Ž
.
3
1
1
Ž
.
Butachlor Et,Et
10
10
10
10
10
10
10
10
Ž
.
Acetochlor Me,Et
Ž
.
Metolachlor Me,Et
Ž
.
Dimethachlor Me,Me
Derivatives of other arylamines
Dimethenamid
10
10
1
F1
G2
G1
3.88"0.27
5.56"0.29
7.48"0.47) )
0.89
0.93
1.35
Propachlorc
a ns25 except G2 for alachlor at 3 mM ns19. Values by experiment and donor as mean"SE.
b Mean for treatment % mean for corresponding control.
c
Ž
.
Ž
.
No metaphases at 10 mM F1 and G1 and 1 mM G2 .
) ) pF0.01; ) ) ) pF0.001

(
)
166
A.B. Hill et al.rMutation Research 395 1997 159–171
.
Ž
undergoing first division to 3.00 all metaphases
ylquinoneimine is weakly mutagenic in the TA100
but not the TA98 strain.
.
undergoing third division .
3.2. Cytotoxicity and genotoxicity of alachlor and
metabolites in CHO cells
3. Results
3.1. Bacterial mutagenicity studies
The LD50 of alachlor was )30 mM in both the
MTT and colony formation assays. To test for possi-
ble genotoxicity, alachlor and two dialkylanilines
were incubated for 48 h in exponentially-growing
Two Salmonella tester strains were used to deter-
mine whether diethylquinoneimine reverts hisy mu-
tations to hisq Table 1 . In both, 100 mgrplate was
toxic. In TA98, diethylquinoneimine at 10 and 30
mgrplate was not significantly different than the
CHO cultures with 10 mM BrdU Table 2 . Mitotic
index depression was a more sensitive measure of
cytotoxicity than the MTT assay since no mitotic
figures were observed with alachlor at 30 mM; how-
ever, they were present with alachlor at 10 mM and
diethylaniline and ethylmethylaniline at 30 and 3
Ž
.
Ž
.
negative control whereas
5
m g of 2-
Ž
.
nitrofluorenerplate positive control resulted in a
mutation frequency that was 65-fold greater than the
negative control. In contrast, TA100 showed a two-
fold increase in mutation frequency with dieth-
ylquinoneimine at 30 mgrplate; because this dose
induced some toxicity, representative colonies were
grown on hisy plates, confirming the heritability of
Ž
.
mM. Diethylaniline 30 and 3 mM and ethylmeth-
Ž
.
ylaniline 30 mM increased the percent of M2 cells,
with a corresponding decrease of M3 cells, and
decreased the PRI values which indicate that these
treatments extend the cell cycles. The SCE rate for
Ž
.
Ž
the mutations. Sodium azide 5 mgrplate as a posi-
tive control for TA100 gave a 7-fold increase in
hisq revertants. It is therefore concluded that dieth-
M2 cells was increased by alachlor at 10 mM p-
.
Ž
.
0.01 and by diethylaniline p-0.05 but not ethyl-
methylaniline at 30 and 3 mM.
Table 4
Selected alachlor metabolites as inducers of sister chromatid exchanges in cultured human lymphocytes; structures are given in Fig. 2
Compound
Controls
mM
Expt. donor
SCErcell"SEa
SCE indexb
0
0
0
A1
B1
E2
E2
A1
E2
A1
E2
A1
E2
A1
A1
E2
A1
E2
A1
B1
E2
B1
4.80"0.40
4.04"0.30
5.84"0.37
13.2"0.8) ) )
4.88"0.35
5.20"0.30
6.12"0.47)
toxic
5.88"0.42
5.76"0.21
5.92"0.37)
6.08"0.37)
5.08"0.37
5.32"0.36
5.92"0.42
5.28"0.42
4.48"0.25
5.88"0.48
3.56"0.21
Ž
.
Mitomycin C positive control
Alachlor
0.01
0.3
0.3
0.3
0.3
0.3
0.3
0.1
0.3
0.3
0.3
0.3
0.1
0.3
0.3
0.03
2.26
1.02
0.89
1.28
Chloroacetanilide
Hydroxychloroacetanilide
1.23
0.99
1.23
1.27
0.87
1.11
1.01
1.10
1.11
1.01
0.88
Aniline
Hydroxyaniline
Quinone
a ns25. Values by experiment and donor as mean"SE.
b Mean for treatment % mean for corresponding control.
) pF0.05; ) ) pF0.001.

(
)
A.B. Hill et al.rMutation Research 395 1997 159–171
167
Table 5
3.3. Genotoxicity of alachlor and analogs in human
lymphocytes
Dialkylquinoneimines and chloroacetyldialkylquinoneimines as in-
ducers of sister chromatid exchanges in cultured human lympho-
cytes. Structural types are given in Fig. 2
Compound mM Expt. donor SCErcell"SEa SCE indexb
Ž
The first experiments each with two different
.
blood donors determined whether alachlor and six
analogs butachlor, acetochlor, metolachlor,
dimethachlor, dimethenamid and propachlor in-
duced SCEs using 10 mM as the test dose Table 3 .
Metaphase chromosomes were observed in all treat-
ments except for cultures incubated with propachlor
at 10 mM for both donors and 1 mM for donor 2.
M2 cells were used to quantitate SCEs. Only alachlor
at 10 mM consistently and significantly increased the
SCE rate while acetochlor at 10 mM and propachlor
Controls
0
0
0
0
0
0
A1
B1
C2
D1
D2
E2
4.80"0.40
4.04"0.30
4.36"0.34
5.68"0.37
4.20"0.20
5.84"0.37
Ž
.
Ž
.
Ž
.
Dialkylquinoneimines 2,6-Substituents
Et, Et
0.3 A1
B1
7.80"0.70) ) ) 1.63
6.00"0.31) ) ) 1.49
6.56"0.43) ) ) 1.50
C2
D1
D2
7.45"0.96) )
1.31
6.32"0.27) ) ) 1.50
Ž
.
at 1 mM only donor 1 were less active. Butachlor,
metolachlor, dimethachlor and dimethenamid did not
induce SCEs at 10 mM. Positive controls treated
with 0.01 mM mitomycin C showed a 2.8-fold in-
crease in SCEs.
0.1 A1
D1
6.88"0.63) )
1.43
8.48"0.53) ) ) 1.49
6.56"0.26) ) ) 1.56
D2
0.03 B1
E2
0.3 C2
D1
4.24"0.23
6.08"0.47
5.60"0.37)
6.48"0.37)
1.05
1.04
1.28
1.14
Et, Me
3.4. Cytotoxicity and genotoxicity of alachlor and
selected metabolites in human lymphocytes
D2
6.28"0.34) ) ) 1.50
0.1 C2
D1
D2
0.3 C2
D1
D2
0.1 C2
D1
5.32"0.38
6.52"0.61
1.22
1.15
Two alachlor metabolites, the chloroacetanilide
and the aniline, were highly toxic to primary lym-
phocytes with LD50 s of approximately 1 mM based
on the MTT assay. To directly compare the genotox-
icity of alachlor with selected metabolites, the test
concentrations used were therefore 0.3, 0.1 and 0.03
5.48"0.31) ) ) 1.30
6.00"0.33) ) ) 1.38
Me, Me
6.96"0.44)
5.64"0.39) )
5.92"0.38) )
7.52"0.54) )
4.92"0.45
1.23
1.34
1.36
1.32
1.17
D2
Ž
.
mM Table 4 . Alachlor was not genotoxic at 0.3
Ž
mM whereas three metabolites chloroacetanilide and
Ž
.
Chloroacetyldialkylquinoneimines 2,6-Substituents
aniline at 0.3 mM and hydroxychloroacetanilide at
Et, Et
0.3 B1
C2
6.16"0.41) ) ) 1.52
5.28"0.44
6.12"0.24
4.72"0.25
3.84"0.21
5.52"0.27)
5.96"0.46
4.40"0.18
1.21
1.08
1.12
0.95
1.27
1.05
1.05
.
0.1 but not at 0.3 mM induced a small but signifi-
D1
D2
0.03 B1
0.3 C2
D1
Ž
cant increase in SCEs in donor 1 cells experiment A
only . The chloroacetanilide was toxic to donor 2’s
.
lymphocytes and the hydroxyaniline and quinone did
not increase SCEs with either donor. These results
indicated weak genotoxicity for three of the alachlor
metabolites.
Diethylquinoneimine is proposed to be a metabo-
lite of alachlor and possibly therefore of butachlor.
By analogy, ethylmethylquinoneimine is a candidate
metabolite of acetochlor and metolachlor and
dimethylquinoneimine of dimethachlor. Their geno-
toxicity and that of the corresponding
chloroacetylquinoneimines is therefore of special in-
Me, Me
D2
a ns25 except C2 for the first compound ns18 and D1 for the
third compound ns20. Values by experiment and donor as
mean"SE.
b Mean for treatment % mean for corresponding control.
) p-0.05; ) ) pF0.01; ) ) ) pF0.001.
Ž
.
diethyl and dimethyl were compared at 0.03–0.3
mM for activity in induction of SCEs Table 5 . The
SCE index was always higher for diethylquinonei-
Ž
Ž
.
terest. Dialkylquinoneimines diethyl, ethylmethyl
.
and dimethyl and chloroacetyldialkylquinoneimines

(
)
168
A.B. Hill et al.rMutation Research 395 1997 159–171
Table 6
Diethylquinoneimine and chloroacetyldiethylquinoneimine effects on mitotic index and cell cycle progression in cultured human lympho-
cytes
Treatment
Expt. donor
mM
Cell cyclea
MIb
%M1
%M2
%M3
PRI
Controls
D2
E2
D2
E2
E2
D2
D2
E2
D2
–
–
1.35
1.25
0.20
2.55
2.60
0.65
1.20
1.20
0.80
12
20
N.D.c
12
6
34
34
10
26
24
12
64
68
2.52
2.48
N.D.c
2.54
2.78
2.08
2.08
2.60
2.16
N.D.c
22
N.D.c
66
Ž
.
Mitomycin C positive control
0.2
0.1
0.01
0.3
0.1
0.03
0.3
10
24
24
20
84
42
42
70
Diethylquinoneimine
Chloroacetyldiethylquinoneimine
32
42
a Based on 50 metaphases.
b Mitotic cellsr100; based on 2000 cells.
c Too few metaphases to analyze.
mine than for ethylmethyl- and dimethylquinonei-
mines at 0.3 and 0.1 mM, while diethylquinoneimine
was not genotoxic at 0.03 mM. Chloroacetyldiethyl-
and dimethylquinoneimines were not consistently
genotoxic at 0.3 mM, indicating that the chloroacetyl
group lowers the genotoxicity of the di-
alkylquinoneimines.
from those of cells treated with 0.03 mM dieth-
ylquinoneimine and 0.1 mM mitomycin C, which at
0.01 mM gave a substantially higher PRI value than
Ž
.
control cells Table 6 . Significant cell cycle delay
Ž
.
PRI values of 2.08–2.16 was observed in cultures
treated with 0.1 and 0.3 mM diethylquinoneimine
and 0.3 mM chloroacetyldiethylquinoneimine. Thus,
Ž
.
a long incubation time with test compound 72 h
was necessary to have an adequate number of M2
3.5. Effects of diethylquinoneimine and
chloroacetyldiethylquinoneimine on mitotic index and
cell cycle progression in human lymphocytes
Ž
.
cells PRI was reduced for careful analysis of SCEs.
With mitomycin C a significant number of SCEs can
be induced without cell cycle delays. We conclude
that diethylquinoneimine and chloroacetyldiethyl-
quinoneimine induce cell cycle delays to the same
extent, even though the genotoxicity is much higher
for diethylquinoneimine. Thus, depression of the MI
and increased duration of the cell cycle by dieth-
ylquinoneimine are not directly linked with SCE
induction.
Metaphases from two experiments were used
to determine MI values and percentages of M1, M2
and M3 cells for cultures treated with dieth-
ylquinoneimine, chloroacetyldiethylquinoneimine
Ž
. Ž
.
and mitomycin C positive control Table 6 . Dieth-
ylquinoneimine and chloroacetyldiethylquinoneimine
at 0.3 mM, but not the former compound at 0.1 and
0.03 mM, inhibited mitosis by 40–50%. In contrast,
mitomycin C increased the MI by two-fold at 0.01
and 0.1 mM but depressed it by 85% at 0.2 mM.
Diethylquinoneimine at 0.1 and 0.3 mM and
chloroacetylquinoneimine at 0.3 mM reduced the M3
cells to 42% from 64–68% in the controls. Mito-
mycin C, a potent inducer of SCEs, did not delay
cell division at 0.1 mM or lower.
4. Discussion
Alachlor is genotoxic in CHO cells and human
lymphocytes based on our studies determining SCE
frequency and other investigations showing in-
creased SCEs 20,22 , chromosomal aberrations
16,18,21 and micronuclei formation 19 with
w
x
Ž
.
w
x
w x
The proliferation index PRI is another measure
of cell cycle delay. Control values were not different
Ž
.
alachlor at a high level ca. 4–40 mM . Single

(
)
A.B. Hill et al.rMutation Research 395 1997 159–171
169
stranded DNA breaks are also evident in primary rat
an activation factor of about 100-fold, i.e. there is a
similar SCE index of 1.4–1.5 at 10 versus 0.1 mM
and inactive levels of 3 versus 0.03 mM, respec-
tively. The potencies of other alachlor metabolites
relative to the starting material and dieth-
ylquinoneimine do not clearly define an activation
sequence. The observed potency for dieth-
ylquinoneimine is a balance of activation and detoxi-
fication pathways. It has been observed as a metabo-
lite of diethylaniline by rat liver microsomal en-
w
x
w x
hepatocytes 39 and Chinese hamster V79 cells 20
exposed to G100 mM alachlor.
The metabolic activation of alachlor to dieth-
ylquinoneimine is illustrated in Fig. 2 based on the
candidate metabolites tested in this research for in-
duction of SCEs in cultured human lymphocytes.
Ž
Alachlor is active at high concentration 10 but not 3
.
mM possibly due to metabolic activation, which is
known to occur with cyclophosphamide and
w x
w
x
benzo a pyrene in purified human lymphocyte cul-
zymes 12 but not of alachlor in isolated rat hepato-
w
x
w
x
tures 40 . The findings support the proposal that
diethylquinoneimine is the reactive intermediate not
only for derivatizing GSH and nasal turbinate pro-
cytes 39 . A portion of the diethylquinoneimine
Ž
undoubtedly undergoes chemical degradation see
Section 2.2 , reacts in the medium e.g. with fetal
bovine serum or undergoes conjugation with GSH
in the cytosol. Lymphocytes are very low in GSH
.
Ž
w
x
.
teins 12–14 but also for induction of SCEs. Addi-
tional pathways possibly relevant to genotoxicity
from alachlor involve GSH conjugation to detoxify
w x
content 4 which otherwise would detoxify dieth-
w
x
w
x
diethylquinoneimine 12–14 , oxidation of dieth-
ylaniline to diethylnitrosobenzene 41 , and fragmen-
tation of the methoxymethyl moiety 42 . Although
the overall metabolic pathway is very complex 43 ,
that portion leading to diethylquinoneimine has spe-
cial toxicological relevance.
ylquinoneimine 12 . The potency of the
quinoneimine for inducing SCEs would presumably
be lower in systems with more GSH or other detoxi-
fication factors; on an analogous basis, acetochlor
induces a greater number of chromosomal aberra-
tions in isolated T-cells compared to T-cells cultured
w
x
w
x
w
x
w x
The oxidative bioactivation of alachlor and sev-
eral of its analogs and metabolites has some similari-
ties to that of the analgesics 4X-hydroxyacetanilide
in the presence of whole blood 4 .
Alachlor is more active than its chloroacetanilide
metabolite and herbicidal chloroacetamide analogs in
inducing SCEs in human lymphocytes. This may
result from serving as a better metabolic precursor
for dialkylquinoneimine formation. The higher activ-
ity of alachlor than butachlor, and of acetochlor than
metoachlor and dimethachlor, may be related to the
ease of metabolically removing the alkoxyalkyl moi-
Ž
.
w
x
acetaminophen and phenacetin 44,45 , the latter
w
x
causing nasal carcinoma in rats 46 . 2,6-Dial-
kylquinoneimines are the purported ultimate geno-
toxicants in the herbicide series and N-
acetylquinoneimine is the proposed hepato- and
nephrotoxicant from the analgesics and in each case
direct reaction of the quinoneimine or
acetylquinoneimine with GSH is a major detoxifica-
tion mechanism.
w
x
ety 42 . The greater potency of diethyl- than ethyl-
methyl- or dimethylquinoneimine may also be a
factor. Although acetochlor, butachlor and meto-
lachlor are less active than alachlor in inducing
SCEs, they are genotoxic with the more sensitive
Alachlor induces nasal turbinate tumors in rats but
possibly not man based on comparative metabolism
w
x
w
x
studies with rats and monkeys 3,14 . This proposal
can now be tested more directly comparing nasal
endpoint of chromosomal aberrations 4,23,24,48 .
The low activity or inactivity of dimethenamid is not
surprising since it could not generate a metabolite
directly analogous to the dialkylquinoneimines. The
observed cytotoxicity and genotoxicity of propachlor
may result from an unrelated mode of action.
w
x
mucosa cells of rats and humans 47 for genotoxic
effects of alachlor and its analogs and metabolites.
The structure-activity relationships observed for
inducing SCEs in human lymphocytes are inter-
pretable in part in relation to activation and detoxifi-
cation of the herbicides and their metabolites within
the cells. Relative potencies are consistent with
metabolic formation of diethylquinoneimine as the
reactive metabolite of alachlor since this constitutes
The mechanism by which SCEs are induced by
alachlor and diethylquinoneimine and some of their
analogs this study involves DNA damage and re-
pair, possibly leading to long-lived genetic change
and mutation 48 . The DNA damage may be associ-
Ž
.
w
x

(
)
170
A.B. Hill et al.rMutation Research 395 1997 159–171
Ž
.
alachlor well water survey, Environ. Sci. Technol. 26 1992
935–943.
w x
ated with protein derivatization 4 . The weak muta-
Ž
genicity of diethylquinoneimine and of diethylani-
w
w
w
x
10 U. Natarajan, R. Rajagopol, Surveying the situation, Environ.
w
x.
line with activation 41,49 in S. typhimurium strain
TA100 indicates induction of base-pair substitution
mutations. In summary, these studies show that when
cultured human lymphocytes are exposed to alachlor
and some of its analogs and metabolites, the result-
ing DNA damage and repair induce an increased
frequency of SCE.
Ž
.
Test. Anal. 2 1993 40–50.
x
11 Environmental Protection Agency, Pesticide tolerances for
Ž
.
dimethenamid, Fed. Reg. 61 1996 10681–10684.
x
12 P.C.C. Feng, S.J. Wratten, In vitro oxidation of 2,6-diethyl-
aniline by rat liver microsomal enzymes, J. Agric. Food.
Ž
.
Chem. 35 1987 491–496.
w
x
13 P.C.C. Feng, A.G.E. Wilson, R.H. McClanahan, J.E.
Patanella, S.J. Wratten, Metabolism of alachlor by rat and
mouse liver and nasal turbinate tissues, Drug Metab. Dispos.
Ž
.
18 1990 373–377.
w
x
14 A.A. Li, K.J. Asbury, W.E. Hopkins, P.C.C. Feng, A.G.E.
Acknowledgements
Wilson, Metabolism of alachlor by rat and monkey liver and
Ž
.
nasal turbinate tissue, Drug. Metab. Dispos. 20 1992 616–
618.
The study described was supported by Grant P01
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