Metabolism of the insecticide teflubenzuron in rats

Article abstract of DOI:10.1080/004982597240172

1. The metabolic fate of the insecticide teflubenzuron, orally dosed to the male Wistar rat, was investigated. Particular attention was paid to the metabolic fate of the benzoyl and aniline moiety after hydrolysis of the urea bridge. 2. The 0-48-h urinary and faecal metabolic patterns and recoveries showed that for a dose range of 4-53 μmol (1.5-20 mg) teflubenzuron, 90% of the dose was excreted in the faeces mainly in unmodified form, approximately 4.6% was absorbed from the lumen and excreted in the urine, and 5.4% was retained in the body. Metabolites excreted in the urine could be identified as benzoate and aniline derivatives originating from the two aromatic rings of teflubenzuron liberated from the parent molecule by hydrolysis of the urea bridge. 3. The amount of urinary benzoate-type metabolites was about eight times the amount of aniline-type metabolites, indicating significant differences in efficiency of urinary excretion of the benzoate moiety as compared with the aniline ring. 4. To investigate further the possible reason underlying this difference in urinary excretion efficiency between the two aromatic derivatives formed from teflubenzuron, dose-recovery studies of these aniline- and benzoate-type metabolites were performed. These studies confirmed the discrepancy observed between the urinary recovery of the benzoyl and the aniline moiety of teflubenzuron. 5. Additional results of the present study indicate that the above discrepancy can be explained by the fact that the benzoate derivative is excreted mainly in its unmetabolized form, whereas the aniline derivative needs additional phase I and II modifications before it can be excreted from the body, the former being a relatively slow reaction. Furthermore, conversion of the halogenated aniline derivative in phase I metabolism might result in a reactive benzoquinone-type or N-oxidized primary metabolite, which can be retained in the body due to reaction with cellular macromolecules.

Full text of DOI:10.1080/004982597240172

xenobiotica , 1997, vol. 27, no. 8, 801±817
M etabolism of the insecticide te¯ubenzuron in rats
J. KOERTS,* A. E. M. F. SOFFERS, J.-W. DE KRAKER, N. H. P.
CNUBBEN and I. M. C. M. RIETJENS
Department of Biochemistry, Agricultural University Wageningen, Dreijenlaan 3, 6703
HA Wageningen, The Netherlands
1. The metabolic fate of the insecticide te¯ubenzuron, orally dosed to the male Wistar
rat, was investigated. Particular attention was paid to the metabolic fate of the benzoyl and
aniline moiety after hydrolysis of the urea bridge.
2. The 0±48-h urinary and faecal metabolic patterns and recoveries showed that for a
±
dose range of 4±53 mol (1 5±20 mg) te¯ubenzuron, 90 % of the dose was excreted in the
faeces mainly in unmodi®ed form, approximately 4 6 % was absorbed from the lumen and
l
±
±
excreted in the urine, and 5 4 % was retained in the body. Metabolites excreted in the urine
could be identi®ed as benzoate and aniline derivatives originating from the two aromatic
rings of te¯ubenzuron liberated from the parent molecule by hydrolysis of the urea bridge.
3. The amount of urinary benzoate-type metabolites was about eight times the amount
of aniline-type metabolites, indicating signi®cant diåerences in eæciency of urinary
excretion of the benzoate moiety as compared with the aniline ring.
4. To investigate further the possible reason underlying this diåerence in urinary
excretion eæciency between the two aromatic derivatives formed from te¯ubenzuron,
dose±recovery studies of these aniline- and benzoate-type metabolites were performed.
These studies con®rmed the discrepancy observed between the urinary recovery of the
benzoyl and the aniline moiety of te¯ubenzuron.
5. Additional results of the present study indicate that the above discrepancy can be
explained by the fact that the benzoate derivative is excreted mainly in its unmetabolized
form, whereas the aniline derivative needs additional phase I and II modi®cations before
it can be excreted from the body, the former being a relatively slow reaction. Furthermore,
conversion of the halogenated aniline derivative in phase I metabolism might result in a
reactive benzoquinone-type or N-oxidized primary metabolite, which can be retained in
the body due to reaction with cellular macromolecules.
Introduction
Over the past 20 years the development of ¯uorinated agrochemicals has
increased considerably and an even further increase in ¯uorinated agrochemicals is
foreseen for the years ahead (Banks 1994). Replacement of an atom by a ¯uorine can
change the properties of a chemical and aåect its metabolism, chemical reactivity
and lipophilicity (Luteijn and Tipker 1986, Banks 1994, Park and Kitteringham
1994).
An example of such a series of ¯uorinated agrochemicals consists of the
benzoylphenyl urea insect growth regulants which act by inhibiting the enzyme
responsible for the chitin synthesis in larves (Verloop and Ferell 1977).
Di¯ubenzuron, which is the active component in the product named Dimilin (®gure
1), was introduced in 1975 and is used as a larvicide for fruit, cotton and soyabeans,
and it was the ®rst benzoylphenyl urea insect growth regulant commercialized.
Te¯ubenzuron, the active component in the insecticide commercialized as Nomolt
(®gure 1), was introduced in 1984, and is another benzoylphenyl urea larvicide used
for the protection of fruit and vegetables.
* Author for correspondence.
±
’
0049±8254}97 $12 00
1997 Taylor & Francis Ltd

802
J. Koerts et al.
Figure 1. Molecular structure of the insecticides di¯ubenzuron and te¯ubenzuron, and the proposed
scission products formed by hydrolytic cleavage of the phenylurea bridge of te¯ubenzuron,
pathway 1 being the major route (Verloop and Ferell 1977).
Upon development of an agrochemical it is important not only to investigate they
metabolic fate and disposition of the compound in target organisms (Verloop and
Ferell 1977, Van Laecke and Degheele 1991) but also in non-target organisms
(Metcalf et al. 1975, Verloop and Ferell 1977, Ivie 1978, Willems et al. 1980,
Opdycke et al. 1982a). Non-target organisms, including man, are not only exposed
to these chemicals upon the manufacture of these compounds but also when these
chemicals are released into the environment or are present as a residue in food
following crop treatment.
Previous studies on the bioavailability and metabolism of di¯ubenzuron in
various non-target species, i.e. rat, sheep, cattle, swine and chicken, showed that
"
upon a 4±10 mg kg (13±32 lmol kg) oral dose of di¯ubenzuron, 50 % of the dose
}
}
was excreted unchanged in the faeces (Ivie 1978, Willems et al. 1980, Opdycke et al.
1982a, b). The remainder of the dose was absorbed from the gastrointestinal tract.
Part of the di¯ubenzuron absorbed was metabolized to conjugated aromatic ring
hydroxylated di¯ubenzuron derivatives and excreted in the bile. The remainder of

Metabolism of te¯ubenzuron
803
the dose absorbed was excreted in the urine mainly as derivatives from products
resulting from hydrolysis of the urea bridge, which were identi®ed especially as 2,6-
di¯uorobenzoic acid derived metabolites. However, there were relatively many
unidenti®ed metabolites in the urine and a lot of uncertainty remained about the
recovery and metabolic fate of the aniline moiety which must also have been formed
upon the hydrolytic cleavage of the parent compound. Metabolites of this second
scission product, 4-chlorophenylurea containing the aniline ring, could not be
detected in the urine or bile although unidenti®ed metabolites observed in the urine
were taken as being suggestive of further metabolism of the 4-chlorophenylurea
moiety (Willems et al. 1980).
Altogether the metabolic fate of benzoylphenylurea insecticides is not well
described and especially the fate of the aniline part of the insecticide upon hydrolysis
of the urea bridge has not been well investigated. Therefore, the aim of the present
study was to investigate the diåerences in the recovery and metabolic fate of the two
aromatic scission products of the benzoylphenyl urea insecticides formed upon
hydrolysis of the urea bridge. To achieve this, the benzoylphenyl urea larvicide
te¯ubenzuron was taken as the model compound not only because the presence of
¯uorine substituents in both the benzoate but also the aniline moiety (®gure 1)
provides a way actually to detect the metabolic fate of both parts of the molecule,
since the ¯uorine-containing metabolites can be identi®ed and quanti®ed by the use
"*
of F nmr (Malet-Martino and Martino 1992).
M aterials and m ethods
Chemicals
Te¯ubenzuron, the active compound in Nomolt (purchased from Shell BV, The Netherlands), was
obtained by extracting Nomolt with chloroform in a 1 :2 (v v) ratio. The chloroform phase was
evaporated and white powder obtained (7% purity) was dissolved in dimethyl sulphoxide. (3,5-
}
±
Dichloro-2,4-di¯uorophenyl)urea (99 %) and 3,5-dichloro-2,4-di¯uoroaniline (99 6%) were a generous
gift from Shell Forschung (Schwabenheim, Germany). 2,6-Di¯uorobenzamide (97 %) was purchased
"
from Aldrich Chemie (Steinheim, Germany). 2,6-di¯uorobenzamide ( 97 %) was obtained from
Fluorochem (UK). Synthesis of 2,6-di¯uorobenzoylglycine from 2,6-di¯uorobenzoic acid was performed
according to Vogel (1989). Synthesis of 2-amino-3,5-di¯uoro-4,6-dichlorophenylsulphate was carried
out according to a method for synthesis of O-aminophenyl sulphates described by Boyland et al. (1953).
In vivo metabolism
Male Wistar rats (350±400 g) were exposed to 1±53 mol (as indicated) te¯ubenzuron, 2,6-
l
di¯uorobenzoic acid, 2,6-di¯uorobenzamide, 3,5-dichloro-2,4-di¯uoroaniline or (3,5-dichloro-2,4-
di¯uorophenyl)urea, all administered in olive oil with 20 % dimethyl sulphoxide by oral gavage. After
dosing, 0±24, 24±48-h urine and 0±48-h faeces samples were collected.
±
For "* F nmr measurement, urine samples were either diluted once in 0 2
m
potassium phosphate
±
pH 7 6 or, when necessary, concentrated by freeze-drying and subsequent dissolving in 0 1 potassium
phosphate pH 7 6.
±
m
±
Faeces samples were homogenized by extensive (overnight) stirring in 25 ml ethyl acetate using a
magnetic stirrer. The homogenate obtained was ®ltered over glass mineral wool and the residue washed
three times with 25 ml ethyl acetate. The ethyl acetate ®ltrate was evaporated to dryness. The residue was
dissolved in 4 ml ethyl acetate and analysed by "* F nmr.
Acid and enzyme hydrolysis of urine samples
In order to identify phenolic metabolites excreted in the urine in their N-acetylated, glucuronidated
b
or sulphated form, urine samples were treated with acid, with -glucuronidase (from Escherichia coli K12
[Boehringer, Mannheim, Germany] [free of arylsulphatase]) or with arylsulphatase -glucuronidase
b
}
(from Helix pomatia [Boehringer, Mannheim, Germany]). Enzyme treatments were carried out
essentially as described before by Vervoort et al. (1990).
±
For acid hydrolysis of the urine samples 1 0 ml urine sample was incubated with 0 1 ml 37 % (12N)
HCl for 1 h in a boiling water bath. After acid hydrolysis the mixture was neutralized with 6 N NaOH
±
±
and the sample diluted once in 0 2 potassium phosphate pH 7 6 for analysis by F nmr.
±
m
"*

804
J. Koerts et al.
Microsomal preparations
Preparation of microsomes was carried out essentially as described previously (Vervoort et al. 1990).
Microsomes were obtained from perfused livers of male Wistar rats (300±400 g), which were exposed
to 150 mg kg body weight of the cytochrome P450 inducer isosafrole (Janssen Chimica, Beerse, Belgium)
}
by i.p. injection daily for 3 days using a stock solution of 100 mg ml in olive oil. The amount of
}
cytochrome P450 in the microsomes was measured according to the method described by Omura and
Sato (1964).
In vitro microsomal incubations
In vitro microsomal incubations contained (®nal concentrations): 0 1 potassium phosphate pH 7 6,
±
±
m
m
0±5 m 3,5-dichloro-2,4-di¯uoroaniline, added from a 100 times concentrated stock solution in dimethyl
sulphoxide and 2
lm
microsomal cytochrome P450. Microsomes from the isosafrole-pretreated rat were
used to allow comparison of the results to those of microsomal metabolism of aniline derivatives reported
in a previous study (Cnubben et al. 1994) which also demonstrated that these microsomes are most
eåective in metabolism of aniline derivatives (Cnubben et al. 1994, Koerts et al. 1996). The total sample
m
volume was 1 ml. The reaction was started by the addition of NADPH (1 m ®nal concentration) and the
°
mixture was incubated at 37 C for 10 min. The reaction was stopped by the addition of 0 3 ml 20 % (w v)
trichloroacetic acid after which the mixture was centrifuged for 3 min at 13 000 g.
±
}
Chemical assay for the detection of 4-hydroxy-3,5-dichloro-2-¯uoroaniline
4-Hydroxy-3,5-dichloro-2-¯uoroaniline present in the urine and microsomal incubations was
determined using the method of Brodie and Axelrod (1948) for chemical determination of 4-aminophenol
b
derivatives. Arylsulphatase -glucuronidase treated urine samples were extracted with ethyl acetate. To
}
l
40 l of this ethyl acetate fraction, 760 l demineralized water and 240 l trichloroacetic acid (20 % w v)
were added. To 800 l of this mixture 80 l phenol reagent (5 % w v phenol in 2 5 NaOH) and 160 l
l
l
}
±
l
l
m
l
}
±
2 5 Na CO were added. The absorbance at 630 nm was measured after 1 h at room temperature. Ethyl
acetate extract from non-enzyme treated urine was used as a blank.
m
#
$
l
l
For the in vitro microsomal incubations 100 l phenol reagent and 200 l Na CO were added to 1 ml
#
$
of the trichloroacetic acid mixture. A microsomal incubation with water instead of substrate was used as
blank.
e
of the 4-hydroxy-3,5-dichloro-2-¯uoroaniline derived indophenol is not known. Therefore,
’$!
based on a previous study on the cytochrome P450-catalysed 4-hydroxylation of halogenated aniline
e
derivatives in which the
(Cnubben et al. 1994), the
of various halogenated aminophenol-derived indophenols were determined
of the 4-hydroxy-3,5-dichloro-2-¯uoroaniline-derived indophenol was
’$!
e
’$!
estimated to be 20 m " cm ".
Õ
Õ
m
Data analysis
m
The apparent Michaelis±Menten constant K (m ) and the apparent maximum reaction rate V
max
(nmol 4-hydroxy-3,5-dichloro-2-¯uoroaniline min nmol cytochrome P450) for the in vitro microsomal
m
}
}
cytochrome P450-catalysed 4-hydroxylation of 3,5-dichloro-2,4-di¯uoroaniline were calculated by
m
®tting the obtained data of the in vitro incubations to the Michaelis±Menten equation
¯
V
[[S] (K -[S]). Fitting the experimental data to the Michaelis±Menten equation resulted in
}
m
max
"
±
0 97.
correlation coeæcients
"* F nmr measurements
"*
F nmr measurements were performed on a Bruker AMX 300 nmr spectrometer at 280 K. Proton-
decoupling was achieved with the Waltz-16 pulse sequence at ®20 dB from 50 W.
±
Samples contained 100 l H O to lock the magnetic ®eld and 10 l 8 4 m 4-¯uorobenzoic acid added
as an internal standard. The total sample volume was 1 71 ml. Enzyme and acid treated urine samples
l #
l
m
#
±
were made anaerobic by evacuating and ®lling with argon four times before analysis. This was done to
prevent autoxidation of the phenolic metabolites. Quanti®cation of the diåerent metabolites was done by
"*
comparison of the integral of the F nmr resonance of the added internal standard 4-¯uorobenzoic acid
to the integrals of the "* F nmr peaks of the metabolites. The ethyl acetate extract of the faeces was
#
analysed with a coaxial insert containing 4-¯uorobenzoic acid dissolved in H O. The total sample
#
±
volume was 1 6 ml. Chemical shifts are reported relative to CFCl .
$
Molecular orbital calculations
Semi-empirical molecular orbital calculations were performed on a Silicon Graphics Iris 4D 85
}
computer with the AM1 (Austin Method 1) Hamiltonian from the AMPAC program (Quantum
Chemistry Program Exchange, program no. 506, Indiana University, Bloomington, IN, USA).
Calculations were carried out with PRECISE criteria and self-consistent ®eld was achieved for all

Metabolism of te¯ubenzuron
805
Figure 2. For legend see page 807.

806
J. Koerts et al.
Figure 2. For legend see opposite.

Metabolism of te¯ubenzuron
807
calculations. Geometries were optimised for all bond lengths, bond angles and torsion angles using the
Fletcher±Powell criteria.
Results
Urine and faeces metabolite patterns of 2,6-di¯uorobenzoic acid, 2,6-
di¯uorobenzamide, 3,5-dichloro-2,4-di¯uoroaniline (3,5-dichloro-2,4-
di¯uorophenyl)urea and te¯ubenzuron
The in vivo metabolism of 2,6-di¯uorobenzoic acid, 2,6-di¯uorobenzamide, 3,5-
dichloro-2,4-di¯uoroaniline and (3,5-dichloro-2,4-di¯uorophenyl)urea was investi-
gated in order to identify the metabolites from te¯ubenzuron that result from
hydrolysis of the phenylurea bridge (®gure 1) and subsequent metabolism of the
scission products.
"*
2,6-Di¯uorobenzoic acid. Figure 2a shows the F nmr spectrum of the 0±24-h
urine of a rat exposed to 10 lmol 2,6-di¯uorobenzoic acid. Metabolites were
identi®ed on the basis of commercially available 2,6-di¯uorobenzoic acid and its
"*
synthesized glycine conjugate. The F nmr spectrum shows that urinary excretion
of 2,6-di¯uorobenzoic acid occurs mainly in the form of the unmodi®ed parent
compound 2,6-di¯uorobenzoic acid and to
a
minor extent as 2,6-
Õ
di¯uorobenzoylglycine (see table 1 for quantitative data). The amount of F
Õ
observed could be fully accounted for the by the F present in blank urine samples
from the unexposed rat (data not shown). The total urinary recovery was 86 %.
"*
The F nmr spectra of the ethyl acetate extracts of the 0±48-h faeces of the 2,6-
di¯uorobenzoic acid exposed rat show no resonance peaks (detection limit 1 lm for
"*
an o n run) ( F nmr spectrum not shown).
}
"*
2,6-Di¯uorobenzamide. Figure 2b shows the F nmr spectrum of the 0±24-h urine
of a rat exposed to 10 lmol 2,6-di¯uorobenzamide. Metabolites were identi®ed on
"*
the basis of commercially available or synthesized reference compounds. The
F
nmr spectrum shows that urinary excretion of 2,6-di¯uorobenzamide mainly occurs
in the form of 2,6-di¯uorobenzoic acid and a minor amount was excreted as 2,6-
di¯uorobenzoylglycine and unmodi®ed 2,6-di¯uorobenzamide (see table 1 for
Õ
qualitative data). The amount of F observed could be fully accounted for by the F
Õ
present in blank urine samples from unexposed rats (data not shown). The 24-h
urinary recovery was 100 %.
"*
The F nmr spectra of the ethyl acetate extracts of the 0±48-h faeces of the 2,6-
di¯uorobenzamide-exposed rat show no metabolite or substrate resonances (de-
"*
tection limit 1 lm for an overnight run) ( F nmr spectra not shown).
"*
3,5-Dichloro-2,4-di¯uoroaniline. Figure 2c shows the F nmr spectrum of the
"*
0±24-h urine of a rat exposed to 10 lmol 3,5-dichloro-2,4-di¯uoroaniline. F nmr
Figure 2. "* F nmr spectra of the 0±24-h urine samples of rats exposed to (a) 2,6-di¯uorobenzoic acid,
(b) 2,6-di¯uorobenzamide, (c) 3,5-dichloro-2,4-di¯uoroaniline, (d) (3,5-dichloro-2,4-di¯uoro-
phenyl)urea and (e) te¯ubenzuron. The resonance peak marked `a ’ in (e) belongs to a metabolite
derived from biotransformation of 3,5-dichloro-2,4-di¯uoroaniline, which is one of the scission
products of te¯ubenzuron after hydrolysis of the urea bridge. The ¯uorine resonances in the
spectra (a±d) are not similar in size due to the diåerent volumes of the 0±24-h urine samples
produced by the rats. The resonance peak `IS ’ is from the internal standard 4-¯uorobenzoic acid.
Chemical shifts are reported relative to CFCl .
$

808
J. Koerts et al.
Table 1. 24-h Urinary metabolic pro®le of 2,6-di¯uorobenzoic acid, 2,6-di¯uorobenzamide, 3,5-
a
dichloro-2,4-di¯uoroaniline, (3,5-dichloro-2,4-di¯uorophenyl)urea and te¯ubenzuron.
Substrate
metabolite
% total metabolites
}
2,6-Di¯uorobenzoic acid
2,6-Di¯uorobenzoylglycine
Parent compound
±
±
5 4³2 5
±
±
94 6³2 5
2,6-Di¯uorobenzam ide
2,6-Di¯uorobenzoic acid
2,6-Di¯uorobenzoylglycine
Parent compound
±
±
97 1³2 4
±
±
±
2 3³1 9
±
0 6³1 0
3,5-Dichloro-2,4-di¯uoroaniline
± ±
0 4³0 8
41 3³5 7
2-Amino-3,5-di¯uoro-4,6-dichlorophenol
2-Amino-3,5-di¯uoro-4,6-dichlorophenylsulphate
2-Amino-3,5-di¯uoro-4,6-dichlorophenylglucuronide
4-Amino-2,6-dichloro-3-¯uorophenylsulphate
4-Acetamido-2,6-dichloro-3-¯uorophenol
±
±
±
±
±
±
16 7³5 1
15 2³2 1
±
±
0 2³0 4
± ±
16 0³2 1
4-Acetamido-2,6-dichloro-3-¯uorophenylsulphate glucuronide
Unidenti®ed
}
± ±
10 2³2 1
(3,5-Dichloro-2,4-di¯uorophenyl)urea
± ±
71 9³8 2
(3,5-Dichloro-2-¯uoro-4-phenylsulphatephenyl)urea
(3,5-Dichloro-2-¯uoro-4-phenylglucuronidephenyl)urea
±
±
28 1³8 2
Te¯ubenzuron
2,6-Di¯uorobenzoic acid
± ±
81 4³3 8
± ±
4 0³0 5
± ±
2 0³0 3
2,6-Di¯uorobenzoylglycine
2,6-Di¯uorobenzamide
± ±
2-Amino-3,5-di¯uoro-4,6-dichlorophenylsulphate
2-Amino-3,5-di¯uoro-4,6-dichlorophenylglucuronide
4-Amino-2,6-dichloro-3-¯uorophenylsulphate
4-Acetamido-2,6-dichloro-3-¯uorophenylsulphate
(3,5-Dichloro-2-¯uoro-4-phenylsulphatephenyl)urea
(3,5-Dichloro-2-¯uoro-4-phenylglucuronidephenyl)urea
2 8³0 5
±
±
1 2³0 3
±
±
±
±
1 2³0 4
0 6³0 5
±
±
±
±
0 3³0 6
5 2³2 4
b
± ±
1 3³0 7
Unidenti®ed
a
Results present the average metabolic pro®le of the indicated dose range as similar metabolic
pro®les were obtained for the diåerent tested doses.
b
Similar ppm as the unidenti®ed metabolite observed for 3,5-dichloro-2,4-di¯uoroaniline.
resonances of conjugated metabolites, i.e. sulphated, glucuronidated or acetylated
"*
products, were identi®ed on the basis of the shift of their F nmr resonance in the
spectrum upon arylsulphatase and or b-glucuronidase, or acid treatment of the
}
urine. The aromatic ring-hydroxylated product 2-amino-3,5-di¯uoro-4,6-
dichlorophenylsulphate was identi®ed on the basis of its synthesized reference
compound. Identi®cation of 4-amino-2,6-dichloro-3-¯uorophenol, formed from its
N-acetylated, sulphated and or glucuronidated conjugate upon acid, arylsulphatase
}
and or b-glucuronidase treatment of the urine, was achieved on the basis of the
}
chemical assay for detection of 4-aminophenol derivatives. The amount of 4-amino-
2,6-dichloro-3-¯uorophenol detected by this chemical assay correlated with the
"*
±
amount of the metabolite giving rise to the F nmr resonance at ®136 5 ppm, which
could thus be identi®ed. This latter result was supported by the following
observation. In a previous study it was demonstrated that the introduction of a
hydroxyl group meta with respect to a ¯uorine in ¯uorinated benzene derivatives
"*
±
±
results in a F nmr chemical shift of 1 8³1 2 ppm (Koerts et al. 1996). Based on this
"*
result, the F nmr chemical shift of 4-amino-2,6-dichloro-3-¯uorophenol was
±
±
"*
F
predicted to be ®136 3³1 2 ppm clearly supporting the identi®cation of the
±
nmr resonance at ®136 5 ppm as 4-hydroxy-3,5-dichloro-2-¯uoroaniline.

Metabolism of te¯ubenzuron
809
The results in Figure 2c show that the urinary excretion of 3,5-dichloro-2,4-
di¯uoroaniline is mainly in the form of the N-acetylated, sulphated and or
}
glucuronidated conjugates of its ortho and para-hydroxylated metabolites (see table
"*
1 for quantitative data). Only one of the metabolite peaks in the F nmr spectrum
could not be further identi®ed. However, its presence in the urine spectrum of the
3,5-dichloro-2,4-di¯uoroaniline-exposed rats clearly identi®es this metabolite as
derived from the aniline moiety of te¯ubenzuron. The total 24-h urinary recovery
±
was 59 5 % of the administered dose.
"*
The F nmr spectra of the ethyl acetate extracts of the 0±48-h collected faeces
reveal the presence of some parent aniline compound excreted in the faeces to about
"*
2 % of the dose administered ( F nmr spectrum not shown).
"*
(3,5-Dichloro-2,4-di¯uorophenyl)urea. Figure 2d shows the F nmr spectrum of
the 0±24-h urine of a rat exposed to 10 lmol (3,5-dichloro-2,4-di¯uorophenyl)urea.
Metabolites were identi®ed in a similar way as described for 3,5-dichloro-2,4-
di¯uoroaniline. The results in ®gure 2d demonstrate that (3,5-dichloro-2,4-
di¯uorophenyl)urea is excreted in the urine as the sulphated and glucuronidated
conjugates of (4-hydroxy-3,5-dichloro-2-¯uorophenyl)urea (see table 1 for quan-
±
titative data).The total 24-h urinary recovery was 54 5 % of the administered dose.
"*
The F nmr spectra of the ethyl acetate extracts of the 0±48-h faeces sample
reveal the presence of some parent compound excreted in the faeces to about 6 % of
"*
the dose administered ( F nmr spectrum not shown).
"*
Te¯ubenzuron. Figure 2e shows the F nmr spectrum of the 0±24-h urine of a rat
exposed to 53 lmol (20 mg) te¯ubenzuron. Based on the in vivo urinary metabolic
patterns of 2,6-di¯uorobenzoic acid, 2,6-di¯uorobenzamide, 3,5-dichloro-2,4-
di¯uoroaniline and (3,5-dichloro-2,4-di¯uorophenyl)urea (®gure 2a±d), metabolites
resulting from hydrolysis of the urea bridge of te¯ubenzuron and further metabolism
of the aromatic scission products could be identi®ed. The metabolic pro®le thus
identi®ed shows that the benzoate part of te¯ubenzuron is mainly excreted as 2,6-
di¯uorobenzoic acid, and, to a minor extent, as 2,6-di¯uorobenzoylglycine and 2,6-
di¯uorobenzamide. The aniline ring of te¯ubenzuron was excreted as the sulphated
and glucuronidated conjugates of (4-hydroxy-3,5-dichloro-2-¯uorophenyl)urea and
2-amino-3,5-di¯uoro-4,6-dichlorophenol, the sulphated conjugate of 4-amino-2,6-
dichloro-3-¯uorophenol and the glucuronidated conjugate of 4-acetamido-2,6-
dichloro-3-¯uorophenol (see table 1 for quantitative data). In addition to all these
"*
identi®ed metabolites, the F nmr spectrum of the urine of the te¯ubenzuron-
exposed rat shows several unidenti®ed resonance peaks, one of which has the same
chemical shift value as the unidenti®ed resonance peak observed in the urine of rats
exposed to 3,5-dichloro-2,4-di¯uoroaniline indicating that this metabolite can also
be ascribed to a metabolite derived from the aniline part of te¯ubenzuron. The
percentage of unidenti®ed peaks amounts to only 12 % of the total metabolic
pattern. These unidenti®ed metabolites might be ascribed to metabolites with the
te¯ubenzuron structure intact as these kind of metabolites were also observed in the
urine of di¯ubenzuron exposed rats, cattle and sheep (Willems et al. 1980, Ivie
1978). It is also possible that the unidenti®ed metabolites can be ascribed to
impurities and metabolites derived from these impurities present in the te¯u-
"*
benzuron dosed to the animals. The F nmr spectrum of the administered
"*
te¯ubenzuron dose indicates a purity of about 97 %. The F nmr resonances of

810
J. Koerts et al.
these impurities were not observed in the faeces and urine of the te¯ubenzuron
exposed rats, indicating that these unknown compounds might be absorbed from the
lumen, metabolized in the liver and excreted in the urine and or via the bile in the
}
faeces. Since the actual structure and nature of these impurities are unknown,
suggestions for the nature of their possible urine metabolites can not be given.
Although a similar amount of benzoate and aniline metabolites should originate
from the hydrolytic cleavage of te¯ubenzuron, the amount of benzoate derived
metabolites identi®ed in the urine was 87 % of the total identi®ed metabolic pattern
whereas the amount of aniline derivatives was 13 %, indicating that the amount of
benzoate-derived metabolites was about seven times the amount of aniline derived
metabolites. Thus, the 12 % of unidenti®ed minor metabolites does not alter the
conclusion that there is a substantial diåerence in urinary recovery of the two
aromatic moieties derived from te¯ubenzuron. The 24±48-h urine sample of the rat
exposed to 53 lmol te¯ubenzuron was also analysed in order to investigate whether
due to retarded excretion the remainder of the aniline moiety was excreted in the
24±48-h urine. However, the amount of metabolites in this 24±48-h urine was only
±
1 5 % of the amount excreted in the 0±24-h urine. Altogether, these data show that
part of the aniline moiety is retained in the body. Since total recovery in 0±24-h
±
urine, 24±48-h urine and 0±48-h faeces amounted to 4 5, 0 1 and 90 % respectively,
±
±
approximately 5 4 % of the dose of te¯ubenzuron could not be accounted for.
Taking into account that recovery of the dose of te¯ubenzuron administered as
±
benzoate and aniline-derived urinary metabolites amounts to approximately 7 5, and
±
0 9 % respectively, this indicates that a signi®cant amount of the dose retained in the
body in the ®rst 48 h may indeed be ascribed to aniline moiety containing
metabolites.
"*
The F nmr spectra of the ethyl acetate extracts of the 0±48-h faeces sample of
the te¯ubenzuron-exposed rat showed mainly (91 %) unchanged te¯ubenzuron.
Scission products of te¯ubenzuron or metabolites derived from them were not
observed. The recovery of te¯ubenzuron in the faeces was about 90 % of the dose.
Dose±recovery studies of te¯ubenzuron, 2,6-di¯uorobenzoic acid, 2,6-
di¯uorobenzamide, 3,5-dichloro-2,4-di¯uoroaniline and (3,5-dichloro-2,4-
di¯uorophenyl )urea
To investigate whether the amount of te¯ubenzuron administered and of the
scission products derived from it would aåect the extent of absorption and excretion
via urinary and faecal excretion routes, dose±recovery studies of te¯ubenzuron and
its possible scission products were performed. Analysis of the 0±48-h urinary
samples showed that over the whole dose-range tested, i.e. 4±53 lmol for
te¯ubenzuron (n ¯ 1±2) and 1±10 lmol for the scission products (n ¯ 1), their
urinary recoveries did not vary signi®cantly. For the scission products this dose
range of 1±10 lmol was chosen because the results obtained from the dose±recovery
±
±
study of te¯ubenzuron showed that about 0 5±4 0 lmol te¯ubenzuron was absorbed
from the gastrointestinal tract and excreted in the 0±24-h urine indicating that the
range of 1±10 lmol of aromatic scission products would be in the range expected to
be formed in the rats exposed to 4±53 lmol te¯ubenzuron.
±
At all dose-levels of te¯ubenzuron, about 4 5 % of the dose is excreted in the
0±24-h urine mainly as metabolites derived from the scission products resulting
from hydrolysis of the urea bridge of te¯ubenzuron, whereas about 90 % of the dose

Metabolism of te¯ubenzuron
811
Figure 3. Dose±recovery curves of (_ ) 2,6-di¯uorobenzoic acid, (¬) 2,6-di¯uorobenzamide, (E ) 3,5-
dichloro-2,4-di¯uoroaniline and ) (3,5-dichloro-2,4-di¯uorophenyl)urea. The recovery is
presented as the percentage of the dose excreted in the urine 24 h after an oral dose of the chemical
(
U
(n ¯ 1).
is excreted in the faeces mainly as unmodi®ed te¯ubenzuron. Furthermore, the
urinary recovery of the aniline moiety was only about 12 % of the excreted benzoate
moiety over the entire 4±53 lmol dose range.
The dose±recovery studies of the benzoate and aniline scission products of
te¯ubenzuron (®gure 3) show that, for a dose range of 1±10 lmol, the 0±24-h urinary
recovery of 2,6-di¯uorobenzoic acid and of 2,6-di¯uorobenzamide was always
!
±
0 001) higher and about twice the recovery observed for similar
signi®cantly (p
doses of 3,5-dichloro-2,4-di¯uoroaniline or (3,5-dichloro-2,4-di¯uorophenyl)urea.
This two-fold diåerence thus explains only part of the 8-fold diåerence in recovery
of the benzoate and aniline moiety in case of their formation from te¯ubenzuron.
This discrepancy might be due to the fact that at the doses tested the rate-limiting
phase I metabolism of the aniline-type scission products is still operating at almost
saturating aniline derivative concentrations, whereas upon their formation in situ by
hydrolysis of te¯ubenzuron their steady-state concentration for phase I metabolism
is below V
conditions. Because dose±recovery studies at lower doses of the aniline
max
"*
derivatives were not feasible due to the detection limit of the F nmr urine analysis,
further experiments were performed to determine the kinetic values for this phase I
aromatic hydroxylation of the aniline derivative.
In vitro microsomal incubations
Figure 4 presents the Michaelis±Menten plot for the microsomal cytochrome
P450-catalysed 4-hydroxylation of 3,5-dichloro-2,4-di¯uoroaniline. The K and
m
± ± ± ±
derived from these data were 0 58³0 06 mm and 0 24³0 01 nmol min nmol
V
}
}
max
cytochrome P450 respectively.
Molecular orbital computer calculations
Since the microsomal conversion of (3,5-dichloro-2,4-di¯uorophenyl)urea could
not be followed by the sensitive chemical assay available for the aniline derivatives,
support for its equally low V
was derived from theoretical studies. Molecular
max
orbital computer calculations on 3,5-dichloro-2,4-di¯uoroaniline and (3,5-dichloro-
2,4-di¯uorophenyl)urea were performed to ®nd theoretical support for the low rate
of phase I aromatic hydroxylation of these te¯ubenzuron-derived splitting products.

812
J. Koerts et al.
Figure 4. Michaelis±Menten plot for the microsomal cytochrome P450-catalysed 4-hydroxylation of
3,5-dichloro-2,4-di¯uoroaniline (n ¯ 2). From these data the K and V were derived.
m
max
The energy of the HOMO (highest occupied molecular orbital) of 3,5-dichloro-
±
±
2,4-di¯uoroaniline and (3,5-dichloro-2,4-di¯uorophenyl)urea is ®9 0 and ®9 3 eV
respectively, indicative of a V for (3,5-dichloro-2,4-di¯uorophenyl)urea that can
max
not be higher than that obtained for the aniline derivative (Cnubben et al. 1994)
since the lower E(HOMO) indicates an even lower reactivity of this compound for
+
an electrophilic attack by the activated (FeO) species of cytochrome P450.
$
Discussion
The aim of the present study was to investigate the biotransformation of the
insecticide te¯ubenzuron in rat. Particular attention was focused on the metabolic
fate of the aniline part of te¯ubenzuron because in previous studies on the
biotransformation of di¯ubenzuron (an analogue of te¯ubenzuron) a lot of
uncertainty remained about the recovery and metabolic fate of the aniline derivative
formed after hydrolysis of the urea bridge (Verloop and Ferell 1977, Ivie 1978,
Willems et al. 1980, Opdycke et al. 1982a, b).
Male Wistar rats were orally exposed to diåerent dose levels of te¯ubenzuron
"*
"*
and urine samples as well as faecal extracts were analysed by F nmr. The F nmr
spectra of urine samples of rat orally exposed to te¯ubenzuron (®gure 2e) mainly
showed metabolites derived from products resulting from hydrolysis of the urea
bridge. Analysis of faecal extracts of the te¯ubenzuron-exposed rat revealed the
presence of mainly unmodi®ed te¯ubenzuron. Within 48 h almost the total dose of
±
te¯ubenzuron was recovered, partly as metabolites in the urine (4 6 % of the dose
administered) and mainly in unmodi®ed form in the faeces extract (90 % of the dose
administered). Independent of the dose of te¯ubenzuron, the amount of urinary
aniline derived metabolites observed amounted to only 12 % of the amount of
benzoate-derived metabolites, in spite of the fact that hydrolytic cleavage of the
urea bridge in te¯ubenzuron will generate equal amounts of benzoate and aniline
derivatives. This observation of ineæcient excretion of the aniline part of
te¯ubenzuron, formed upon hydrolytic cleavage of the urea bridge in the
benzoylphenyl urea agrochemical, is in agreement with the qualitative results
described previously for di¯ubenzuron (Verloop and Ferell 1977, Ivie 1978, Willems
et al. 1980, Opdycke et al. 1982a, b).

Metabolism of te¯ubenzuron
813
Further experiments described in the present study were performed to ®nd a
rationale for this signi®cant diåerence between the urinary recovery of the benzoate-
and aniline-type scission products.
First, it was investigated to what extent the observed discrepancy might be
ascribed to a diåerence in excretion eæciency of the benzoate and aniline derivatives
themselves. Dose±recovery studies were performed for the four possible scission
products, i.e. 2,6-di¯uorobenzoic acid, 2,6-di¯uorobenzamide, 3,5-dichloro-2,4-
di¯uoroaniline and (3,5-dichloro-2,4-di¯uorophenyl)urea. The results obtained
showed a diåerence in the excretion eæciency between the benzoate-type derivatives
on the one hand, which were, at all dose levels investigated, almost fully excreted
within the ®rst 24 h following administration, and the aniline-type derivatives on the
other hand, which were excreted, also independent of the dose, to about 50 % of the
dose administered. Based on these data, excretion eæciencies of the aniline-type
compounds are demonstrated to be at least two-fold lower than that of the benzoates.
However, the diåerence observed in the excretion eæciency of the benzoate- and
aniline-type compounds when compounds are formed in situ from te¯ubenzuron is
8-fold. This discrepancy between the excretion eæciency of the benzoate- and
aniline-type compounds of te¯ubenzuron compared with the experiments when the
compounds were dosed as such might be explained on the basis of the following
additional observations of the present study.
First, is of importance to note that the benzoate moiety is almost exclusively
excreted in its unmodi®ed form and to a minor extent as its glycine conjugate,
suggesting that after hydrolysis no further metabolism is required to obtain eæcient
excretion of the 2,6-di¯uorobenzoate or the 2,6-di¯uorobenzamide as was shown
before for the metabolism of di¯ubenzuron (Willems et al. 1980) and a series of
¯uorinated benzoic acids (Ghauri et al. 1992). In contrast, the aniline part of
te¯ubenzuron,
i.e.
3,5-dichloro-2,4-di¯uoroaniline
or
(3,5-dichloro-2,4-
di¯uorophenyl)urea require further metabolism in both phase I and II metabolism
before they can be excreted from the body. The aromatic hydroxylation of such
aniline derivatives catalysed by cytochromes P450 to give the aminophenol
metabolites suitable for phase II conjugation and formation of the products that can
be eæciently excreted, could be a relatively slow reaction. It is likely that the rate of
this cytochrome P450-catalysed aromatic ring hydroxylation is relatively low
because cytochrome P450-catalysed aromatic hydroxylation is often the rate-
limiting step in biotransformation and generally much slower than Phase II
conjugation reactions (Cnubben et al. 1994, Soåers et al. 1994).
If the rate of cytochrome P450 conversion of the aniline derivative would indeed
be the rate-limiting factor for its excretion, the actual concentrations of the aniline
derivative in relation to the K for the reaction might be a crucial factor in¯uencing
m
the actual eæciency of the conversion to a large extent. Especially when the
concentration is around or below the K , variation in the actual concentration of the
m
compound will aåect the eæciency of its conversion and excretion. This may then
be the explanation for the fact that upon direct administration of 1±10 lmol of the
aniline derivatives their excretion is more eæcient, then when a similar dose is
formed over a long time interval upon the hydrolytic cleavage of te¯ubenzuron. The
latter might result in a lower time averaged concentration of the compound.
Comparison of the actual K and V
determined for the cytochrome P450-
m
max
catalysed C4-hydroxylation of 3,5-dichloro-2,4-di¯uoroaniline in the present study,
to an estimate of the concentrations expected in the liver, supports this latter

814
J. Koerts et al.
Figure 5. Proposed reaction pathway for the cytochrome P450-catalysed dehalogenation of 4-
halogenated anilines (Cnubben et al. 1995, Riefjens and Vervoort 1991).
hypothesis. Assuming a liver volume of 5±10 ml, and full absorption and transport
of a 1±10 lmol aniline dose from the gastrointestinal tract to the liver, a maximum
±
concentration of 3,5-dichloro-2,4-di¯uoroaniline in the liver of about 0 1±2 0 mm
±
can be foreseen. This is around the K for the cytochrome P450-catalysed oxidative
m
±
±
C4-dehalogenation determined to be 0 58³0 06 mm. In the te¯ubenzuron-exposed
animals, the same amount of aniline is liberated from the parent compound over a
time interval that is, for example, four times longer than the time interval required
for the eæcient uptake of the single dose of the aniline, and therefore will result in
lower liver concentrations that will be below the K resulting in lower rates of
m
conversion. Such an eåect could explain that in the te¯ubenzuron-exposed rat the
eæciency for excretion of the aniline-type metabolites is 8-fold lower than for the
benzoate-type of metabolites whereas in the direct exposure experiments this
diåerence is only 2-fold.
Although this diåerence in excretion eæciency of the benzoate and aniline-type
compounds can explain the observed 8-fold diåerence in their urinary recovery
when formed from te¯ubenzuron, one can not neglect the fact that even upon a
single dose of the aniline derivative about 50 % of the dose can not be recovered in
the 0±48-h urine or faeces. This means that part of the aniline derivative remains in
the body. An explanation for this observation might be related to the mechanism by
which the cytochrome P450-catalysed oxidative dehalogenation of these halogenated
anilines to 4-aminophenol derivatives occurs. This oxidative dehalogenation
represents the ®rst step required for formation of the 4-hydroxylated urinary
metabolites observed. Figure 5 presents the reaction pathway suggested for this
reaction. The primary product formed in such a cytochrome P450-catalysed
oxidative dehalogenation of a C4-halogenated aniline derivative is the reactive
benzoquinoneimine and not its two-electron reduced aminophenol form (Cnubben
et al. 1995, Rietjens and Vervoort 1991). Rapid binding of this reactive
benzoquinoneimine to tissue macromolecules instead of its chemical and or
}
enzymatic reduction to the non-toxic aminophenol, might cause the compound to be
retained in the body. In addition, the cytochrome P450-catalysed N-oxidation of the
aniline derivative might be another explanation for the observed low urinary
recovery of this compound. Results of previous studies (Kiese 1963, Eadsforth et al.
1984, Cnubben et al. 1996) showed that methaemoglobinaemia is induced in rats
which were orally exposed to C4-substituted aniline derivatives. In one of these
studies (Cnubben et al. 1996) the extent of methaemoglobinaemia was shown to
correlate with the rate of N-oxidation of the aniline derivatives in vitro. In vivo these
N-oxidized metabolites were not observed probably due to their high reactivity and
abundant possibilities to react with components in the body. The extent of the

Metabolism of te¯ubenzuron
815
cytochrome P450-catalysed N-oxidation of the 3,5-dichloro-2,4-di¯uoroaniline
derivative of importance in the present study can be estimated based on previous
results for the in vitro cytochrome P450-catalysed conversion of C4-halogenated
anilines (Cnubben et al. 1995). The metabolic data on the total in vitro cytochrome
P450-catalysed conversion of aniline, 4F-, 4Cl-, 4Br- and 4l-aniline show that the
sum of aromatic ring and N-hydroxylation of the tested halogenated anilines was
similar but that the ratio between N- and para or ortho-hydroxylation varied with
the substituent patterns. When a halogen substituent hampers mono-oxygenation at
C4, as can also be expected for the 3,5-dichloro-2,4-di¯uoroaniline derived from
te¯ubenzuron, the extent of N-hydroxylation equals 1±11 times the extent of C4-
oxidative dehalogenation. This means that lower possibilities for aromatic ring
hydroxylation result in a relatively higher extent of N-oxidized products. This
means that the extent of cytochrome P450-catalysed N-oxidation of the aniline
cleavage product of te¯ubenzuron, 3,5-dichloro-2,4-di¯uoroaniline, can be
expected to result in a signi®cant extent of N-oxidized products that at least equals
the amount of aromatic C2 and or C4 hydroxylated metabolites observed in urine.
}
Since binding of the reactive N-oxidized metabolites to tissue molecules hampers
their excretion, the extent of N-oxidation of 3,5-dichloro-2,4-di¯uoroaniline can be
an additional factor in causing the lower urinary recoveries. However, whether the
formation of these N-oxidized metabolites will result in measurable levels of
methaemoglobinaemia is uncertain as the amount of the absorbed te¯ubenzuron
from the lumen is only 10 % of the dose given and likely to result in concentrations
of N-oxidized aniline metabolites that are below the doses required for induction of
methaemoglobinaemia (Cnubben et al. 1996).
The possibility that reaction pathways leading to reactive benzoquinoneimine
and N-oxidized metabolites cause covalent binding of the aniline part of
te¯ubenzuron to tissue macromolecules seems to be supported by several other
±
observations. First, about 5 4 % of the dose of te¯ubenzuron could not be recovered
±
in urine or faeces. At least part of this 5 4 % was expected to be excreted in the form
of urinary aniline-type of metabolites since the urinary recovery in the form of
±
±
benzoate-type metabolites was 3 9 % compared with only 0 7 % in the form of
aniline-type of metabolites. Furthermore, the amount of the ¯uorine in the 24-h
urine of (3,5-dichloro-2,4-di¯uorophenyl)urea-exposed rat was above the amount
that could be accounted for by the ¯uorine originating from food and or drinking
}
water and from the amount of dehalogenated products observed. This observation
suggests that part of the aniline moiety is dehalogenated but not excreted from the
body. Finally, the absence of full excretion of the urea agrochemical from the body
would be in line with the results obtained in a study with rats orally exposed to
5 mg kg di¯ubenzuron for which 4 % of the dose was eventually recovered in the
}
carcass (Willems et al. 1980). Finally, the observation of the present study that the
24±48-h urine recovery of te¯ubenzuron was signi®cantly below the 0±24-h recovery
to some extent argues against the slow cytochrome P450-catalysed conversion of the
aniline moiety as the main reason for its relatively low recovery. Slow metabolism of
a circulating amount of 3,5-dichloro-2,4-di¯uoroaniline would imply that even after
24 h, metabolism and excretion would continue at a level comparable with that
observed in 0±24-h sample. Since this is not what is observed, the explanation for
low aniline recovery due to covalent interaction of a reactive metabolite derived from
it with tissue macromolecules provides the best explanation for this phenomenon.
Altogether, the mechanism depicted in ®gure 5 and or N-oxidation might provide
}

816
J. Koerts et al.
the best explanations for the low recovery of the C4-halogenated aniline part of the
molecule, both in case of te¯ubenzuron, but also in case of di¯ubenzuron, where the
C4 position in the aniline moiety is chlorinated. Clearly such a process resulting in
formation of a reactive benzoquinoneimine or N-oxidized metabolite may also be of
importance from a toxicological point of view, especially when taking into account
that di¯ubenzuron was reported to produce hepatocellular changes in mouse
(Young et al. 1986).
Acknow ledgem ents
We gratefully acknowledge Mr G. van Tintelen for assistance with animal
handling. Support from the TMR large scale NMR facility grant no. ERB-CHGE-
CT94-0061 is also acknowledged.
References
anks
B
B
B
, R. E. (ed.), 1994, Fluorine in Agriculture 9±11 January 1995. In Conference Papers University
of Manchester 1995 (Fluorochem).
oyland
anson
ims
, P., 1953, The preparation of O-aminophenyl sulphates. Journal of
, E., M
the Chemical Society, 3623±3628.
xelrod
, D. and S
rodie
, B. B. and A
N-acetyl p-aminophenol and p-aminophenol (free and conjugated) in biological ¯uids and tissues.
94
, J., 1948, The estimation of acetanilide and its metabolic products, aniline,
Journal of Pharmacology and Experimental Therapeutics, , 22±28.
nubben eelen orst ervoort eeger
1994, Molecular orbital-based quantitative structure-activity relationship for the cytochrome
iefjens
, I. M. C. M.,
C
, N. H. P., P
, S., B
, J.-W., V
, J., V
, C. and R
7
P450-catalyzed 4-hydroxylation of halogenated anilines. Chemical Research in Toxicology,
590±598.
,
nubben
ervoort
oersma
iefjens
, I. M. C. M., 1995, The eåect of
C
C
E
, N. H. P., V
varying halogen substituents on the cytochrome P450-catalyzed dehalogenation of 4-halogenated
49
, J., B
, M. G. and R
anilines to aminophenol metabolites. Biochemical Pharmacology, , 1235±1248.
nubben an den erg iefjens
, I. M. C. M., 1996, Relationships between the
, N. H. P., V
regioselectivity of the hydroxylation of C4-substituted 2-¯uoroaniline derivatives and their toxic
141
B
, C. L. and R
endpoints. Toxicology and Applied Pharmacology,
, 403±415.
, P. A., 1984, 2,4-Di¯uoroaniline and
4-¯uoroaniline exposure : monitoring by methaemoglobin and urine analysis. International
54
adsforth ogan orrison arburton
, C. V., L
, C. J., M
, B. J. and W
Archives of Occupational Environmental Health, , 223±232.
hauri
lackledge
len
weatman
indon
eddell
, C. R.,
, J. K., 1992, Quantitative structure-metabolism relationships for
G
, F. Y., B
ilson
, C. A., G
, R. C., S
, B. C., L
, J. C., B
icholson
W
, I. D. and N
44
substituted benzoic acids in the rat. Biochemical Pharmacology, , 1935±1946.
, G. W., 1978, Fate of di¯ubenzuron in cattle and sheep. Journal of Agriculture and Food Chemistry,
vie
I
26
, 81±89.
, M., 1963, The eåect of certain substituents upon the N-oxidation of aniline in vivo. Naunyn-
244
iese
K
K
Schmiedeberg’s Arch. exp. Pathologie und Pharmakologie,
oerts oeren ervoort eiss eeger
, 387±404.
iefjens
, I. M. C. M., 1996,
, J., B
, S., V
, J., W
, R., V
, C. and
R
Interaction between the substrate and the high-valent iron-oxo porphyrin cofactor as a possible
factor in¯uencing the regioselectivity of cytochrome P450-catalysed aromatic ring hydroxylation
99
of 3-¯uoro(methyl)anilines. Chemico-Biological Interactions, , 129±146.
uteijn ipker
L
, J. M. and T
activity and environmental chemistry of benzoylphenylureas. Pesticide Sciences, , 456±458.
artino artino
, J., 1986, A note on the eåects of ¯uorine substituents on the biological
17
, R., 1992, Magnetic resonance spectroscopy : a powerful tool for
74
alet
M
M
-M
, M. C. and M
drug metabolism studies. Biochimie, , 785±800.
etcalf owlus
u
,
R. L.,
di¯uorobenzyl)-3-(4-chlorophenyl)urea. Journal of Agriculture and Food Chemistry, , 359±364.
mura ato
L
,
P. and
B
,
S., 1975, Degradation and environmental fate of (2,6-
23
, R., 1964, The carbon monoxide pigment of liver microsomes, I. Evidence for its
239
O
O
O
, T. and S
haemprotein nature. Journal of Biological Chemistry,
pdycke iller enzer
, 2370±2378.
, R. E., 1982a, Metabolism and fate of di¯ubenzuron in
30
, J. C., M
, R. W. and M
swine. Journal of Agriculture and Food Chemistry, , 1223±1227.
pdycke iller enzer
, R. E., 1982b, In vivo and liver microsomal metabolism of
, J. C., M
, R. W. and M
30
di¯ubenzuron by two breeds of chickens. Journal of Agriculture and Food Chemistry,
1227±1233.
,
ark
, B. K. and K
itteringham
P
, N. R., 1994, Eåects of ¯uorine substitution on drug metabolism :
26
pharmacological and toxicological implications. Drug Metabolism Reviews, , 605±643.

Metabolism of te¯ubenzuron
817
iefjens
ervoort
R
, I, M. C. M. and
quinoneimines as primary reaction products. Chemico-Biological Interactions, , 263±281.
offers eeger iefjens
V
, J., 1991, Bioactivation of 4-¯uorinated anilines to benzo-
22
, I. M. C. M., 1994, In¯uence of the type of halogen
substituent on in vivo and in vitro phase II metabolism of 2-¯uoro-4-halophenol metabolites
24
S
, A. E. M. F., V
, C. and R
formed from 3-halo-¯uorobenzenes. Xenobiotica , , 759±774.
an aecke egheele
, D., 1991, Detoxi®cation of di¯ubenzuron and te¯ubenzuron in the
V
V
V
V
W
L
, K. and D
larvae of beet armyworm (Spodoptera exigua) (Lepidoptera : Noctuidae). Pesticide and Biochemical
40
Physiology, , 181±190.
erloop erell
, A. and F
chitin deposition. In Pesticide Chemistry in the 20th Century, vol. 37, edited by J. R. Plimmer, pp.
, C. D., 1977, Benzoylphenyl ureasÐa new group of larvicides interfering with
237±270.
ervoort e
nmr method for studies on the in vivo and in vitro metabolism of 2-¯uoroaniline. Xenobiotica ,
ager
teenbergen iefjens
, J. and R
"*
, I. M. C. M., 1990, Development of a F-
, J., D
J
, P. A., S
20
,
657±670.
ogel
, A. J., 1989, In Vogel’s Textbook of Practical Organic Chemistry, edited by B. S. Furniss, A. J.
Hannaford, P. W. G. Smith and A. R. Tatchell (London : Longman Scienti®c and Technical),
pp. 1074, 1274.
illems
vermars
cherpenisse
e
ange
ost
,
,
A. G. M.,
Di¯ubenzuron: intestinal absorption and metabolism in the rat. Xenobiotica , , 103±112.
rombetta arson
O
,
H.,
S
,
P.,
D
L
,
N. and
P
L. C., 1980,
10
, S., 1986, Eåects of di¯ubenzuron on the mouse liver.
Journal of Applied Toxicology, , 343±348.
oung
Y
, M. F., T
, L. D. and C
6