United States
Environmental Protection
Agency
Health Effect Research
Laboratory
Research Triangle Park NC 27711
X-/EPA
Research and Development
EPA-600/S1 -82-011 Sept. 1983
Project Summary
Chemistry and Mode of
Action of Insecticides:
Phase II
T. R. Fukuto, R. B. March, and T. A. Miller
Results gathered for the period
February 15, 1976-May 31, 1981 are
presented. Progress is reported in
areas dealing with the chemistry,
metabolism, and mode of action of
organophosphorus, carbamate, and
organochlorine insecticides. Among
the topics included are: insecticide
metabolism and mode of action; insec-
ticide selectivity; structure-activity
relationships; biological and environ-
mental alteration of insecticides;
effect of impurities on the potentiation
of organophosphorus insecticides;
delayed toxicity of trialkyl phosphoro-
thioates; delayed neurotoxicity of
organophosphorus esters; mode of
action of phosphoramidothioate esters;
gas chromatography of insecticides;
and neurophysiological studies on the
mode of action of insecticides.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park.
NC, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Insecticide Metabolism and
Mode of Action
Studies on the Ox/dative
Conversion of P=S to P=O
Esters and the Effect of
Chirality on Toxicological
Properties
The stereochemistry of P=S to P=O
conversion effected by chemical model
oxidation systems and mouse liver
mixed-function oxidases (MFO), using
the resolved isomers of fonofos (O-ethyl
S-phenyl ethylphosphonodithioate) was
examined. The stereochemical aspects
of the chemistry, metabolism, and mode
of action of the chiral isomers of fonofos
are presented.
The conversion of (5)-p- fonofos to (/?)p-
fonofos oxon by the action of m-
chJoroperoxybenzoic acid occurred
predominantly with retention of config-
uration of the phosphorus atom. Oxida-
tion to the rearrangement product, (/?)P-
phenyl ethyl(ethoxy)phosphinyl disulfide
took place with predominant inversion
of configuration. In the reaction leading
to the oxon, the stereochemicai course
was identical to that observed with
mouse liver MFO.
Toxicological data indicated a 2- to 4-
fold higher toxicity of (/?)p-fonofos to
mice and houseflies relative to (S)p-
fonofos. Compared to fonofos, the
difference in toxicity between the
fonofos oxon enantiomers was greater
and (S)p-oxon was 2.6- (i.p.) and 12-fold
more toxic to white mice and houseflies,
respectively, than the (/?)P-oxon.
The relative toxicities of (S)p- and
(/?)p-fonofos oxon to houseflies and
white mice were qualitatively in agree-
ment with their relative in vitro anti-
cholinesterase activities toward fly-
head, mouse-brain, and blood ChE.
Quantitatively the differences in anti-
cholinesterase activities between the
isomers were substantially greater (49-
to 60-fold) than the differences in
toxicities to houseflies and white mice.
At the same mtraperitoneal dosage of
oxon isomers (0.6 mg/kg), the level of
maximum inhibition of both brain and
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blood ChE obtained from mice treated
with (S)p-oxon was approximately 3-fold
greater than enzymes obtained from
mice treated with the (/?)p-enantiomer.
Maximum inhibition, however, oc-
curred at different time intervals after
treatment and the relationship was
further complicated by the differences
in recovery rates of the inhibited
enzymes. Nevertheless, agreement be-
tween in vivo inhibition of mouse ChE
and intraperitoneal toxicity was good.
The relationship between in vivo
inhibition of housefly-head acetylcho-
linesterase (HFAChE) and toxicity of
houseflies treated with the two isomers
was less satisfactory. Maximum reduc-
tion of HFAChE in flies treated with 5
fjg/g (/?)p-oxon was about 35% compared
to about 80% for flies treated with the
same amount of (S)p-oxon, a slightly
greater than 2-fold difference, a dif-
ference substantially smaller than the
12-fold difference observed for housefly
toxicity. Plots show that HFAChE inhi-
bited by (/?)p-oxon recovered to a signifi-
cantly greater extent (compare activities
at 12 h), and it is possible that ChE de-
pression over longer time periods
should be considered in relating in vivo
inhibition with toxicity.
Studies on the in vitro metabolism of
the two isomers in the presence of
mouse liver MFO showed that (R)p-
fonofos and (S)p-fonofos were convert-
ed to the respective (S)p-oxon and (R)p-
oxon with 70-80% stereospecificity.
Since (S)p-oxon is a stronger anti-
cholinesterase and is more toxic than
(/?)P-oxon, the order of toxicity of the
fonofos isomers is in the right direction.
Although metabolic conversion of
fonofos to the oxon occurred predom-
inantly with retention of configuration,
a significant amount of inversion appar-
ently also took place, leading to partial
racemization of the oxon. This result un-
doubtedly contributed to the smaller
toxicity ratios between the fonofos iso-
mers.
Studies on the relative rates of
metabolism revealed that while (R)p-
fonofos was metabolized at a rate
approximately 2-fold greater than (S)p-
fonofos, analysis of the products
showed that (/?)P-fonofos was converted
in greater proportion to the detoxication
product diphenyl disulfide (DPDS) than
its enantiomer. Thus, even though (/?)P-
fonofos was metabolized by mouse liver
MFO at a faster rate, the difference in
amounts of fonofos oxon formed from
the fonofos isomers was not large.
Rat and mouse serum were effective
in degrading fonofos oxon but not
fonofos. The in vitro degradation of
fonofos oxon by rat or mouse serum was
relatively slow compared to the oxidative
reactions, but results clearly showed
that (S)p-oxon was degraded at a faster
rate than the (/?)p-oxon (approximately
2-fold). The greater susceptibility of
(S)p-oxon to enzymatic degradation may
account for the smaller difference in
toxicities between the fonofos-oxon
enantiomers relative to their differences
in anticholinesterase activities (50- to
60-fold). Further, since (/?)p-fonofos is
preferentially metabolized to (S)p-oxon
and (S)p-fonofos to (/?)?- oxon, the faster
rate of degradation of (S)p-oxon may
contribute to the even smaller dif-
ferences in toxicities of the fonofos
enantiomers Thus, the smaller dif-
ference in toxicities between the
fonofos enantiomers compared to the
relative toxicities of the oxons and their
anticholinesterase activities may be ac-
counted for by the partial racemization
in the oxidative desulfuration reaction
and faster rate of degradation of (5)p-
oxon.
Studies on the Mode of
Action of Methamidophos
O,S-Dimethyl phosphoramidothioate
(methamidophos), although highly
effective with a bimolecular inhibition
constant of (k,) 9.2 * 102 M'1 min'1 for
the inhibition of fly-head AChE, is a
relatively weak anticholinesterase.
Studies were conducted on the toxico-
logical properties, in vitro and in vivo
anticholinesterase behavior, and me-
tabolism of methamidophos
Evaluation of the kinetics of housefly
ChE inhibition showed that its affinity
for the enzyme and its phosphorylation
and bimolecular inhibition rates are all
relatively low. In vivo metabolism
studies in houseflies provided evidence
that it is not activated to a more effective
ChE inhibitor and indirect evidence also
was obtained for its slow degradation In
vitro metabolism studies in housefly
and mouse tissues provided additional
evidence for its lack of activation and
slow metabolic degradation. Compared
to other effective organophosphorus
insecticides, methamidophos was slow
in producing acute symptoms of poison-
ing and ChE inhibition and required the
accumulation of comparatively high
internal levels for toxic effects. However,
in vivo ChE inhibition studies provided
evidence for the interrelationships of
ChE inhibition and toxic effects. Thus,
its relative stability and low in vivo |
degradation appeared to be of critical
importance in accumulating and main-
taining a sufficient internal concentra-
tion for a long enough period of time to
permit the development of its slowly
expressed toxicity.
Insecticide Selectivity
Selective Toxicity of N,N'-
Thiodicarbamates
A series of /V-(alkyl alkylcarbamoyl-
sulfenyl) derivatives of the methyl-
carbamate insecticides carbofuran, m-
isopropylphenyl methylcarbamate, pro-
poxur, aldicarb, methomyl, and oxamyl
were prepared and examined for toxicity
to houseflies, mosquito larvae, and
white mice. Compared to the parent
methylcarbamate, the derivatives were
generally of equal toxicity to the
housefly and substantially more toxic to
mosquito larvae With the exception of
the oxamyl derivatives, all other deriva-
tives were much less acutely toxic to the
white mouse The toxicities of the
derivatives to mosquito larvae and the
white mouse were correlated with their
octanol-water partition coefficients
Insecticidal Properties of the
N-Sulfonyl Derivatives of
Propoxur and Carbofuran
A series of substituted /V-benzene-
sulfonyl derivatives of carbofuran and
propoxur containing electron-with-
drawing substituents of the benzene
ring was synthesized and examined for
toxicity to houseflies and mosquito
larvae The derivatives were generally
nonmsecticidal when tested alone, but
some of the compounds were toxic to
houseflies when synergized with piper-
onyl butoxide. The poor insecticidal
activity and low mammalian toxicity of
these derivatives are attributable to
their high stability and their inability to
be converted into the parent methyl-
carbamate.
Selective Toxicity of N-
Arylsulfenyl Derivatives of
Methylcarbamate Insecticides
The lexicological properties of a
series of /V-sulfenylated derivatives of
commercial methylcarbamate insecti-
cides were studied with special focus on
the toxicological response observed in
honeybees. Work on the comparative
metabolism of one of these derivatives
was conducted in the honeybee and
housefly to determine the basis for its
selective toxicity.
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Of the 34 different sulfenylated de-
rivatives of propoxur, carbofuran, and
carbaryl evaluated, only three showed a
toxicity ratio greater than one although
many were safer to bees than the parent
methylcarbamate. The most notable
exception was the 2-methyl-4-f-butyl-
phenylsulfenyI derivative of propoxur (7)
which was more than 178-fold safer to
the honeybee than propoxur Although
the 4-f-butyl moiety in the ring appeared
to introduce the greatest amount of
honeybee safety in these derivatives, no
obvious relationship between the struc-
ture of the carbamate derivative and
honeybee toxicity was apparent
The pathways in the honeybee and
housefly, while qualitatively similar,
contain enough quantitative differences
to account for the difference in the
toxicity of (7) to the two insects.
Penetration of (7) and its subsequent
conversion into propoxur are apparently
the rate-limiting steps in the metabolism
of (7) in the honeybee Rapid conversion
of propoxur into secondary metabolites
results in its accumulation in a low,
steady-state level in the bee Conse-
quently, intoxication does not occur. In
houseflies, however, penetration and
conversion of (7) into propoxur was fast.
Intoxicating levels of propoxur accumu-
lated within a short period of time after
treatment.
Selective Toxicity of
Phosphoramidothioate Esters
A study of the comparative metabolism
of the hexanoyl derivatives and the
smaller chain acyl derivatives of meth-
amidophos in a mammal and insect was
undertaken to establish the basis for
their lexicological properties Metabolic
and lexicological data gathered for 0,5-
dimethyl propionyl- (2) and hexanoyl-
phosphoramidolhioaie (3)showlhat the
metabolism of the two compounds in
the while mouse and housefly is
qualilalively and quaniitalively different
The major difference in the metabolism
of (2) and (3) in the mouse appears lo be
in the larger amounts of methamidophos
formed from (3) compared to (2).
Melhamidophos is highly toxic to the
mouse (LDso 14 mg/kg), and is probably
the agent responsible for intoxication
when the mouse is trealed with either
(2) or (3). The small amounts of
melhamidophos formed from (2) readily
accounl for ils safety to mice. Similarly,
ihe relalively high toxicily of (3) to mice
may be atlribuled to the subslantial
quantily of melhamidophos formed in
Ihe mouse treated with (3)
Structure-Activity
Relationships
Hydrolytic and Toxicological
Properties of Ethyl a-
Cyanobenzaldoxime
Phosph oramidates
The desired order of selectivity
demonstraled by phoxim (0,0-diethyl
O-or-cyanobenzaldoxime phosphorothi-
oate) and the limited knowledge of the
relationship between chemical structure
and biological activily of phosphorami-
date esters led to sludies on phosphor-
amidates containing the a-cyanobenz-
aldoxime leaving group which is present
in phoxim.
A series of ethyl alkylphosphoramidale
eslers of a-cyanobenzaldoximes were,
Iherefore, synlhesized and examined
for alkaline hydrolysis, anlicholinester-
ase activity, and toxicity to houseflies
and mice The compounds showed
variable toxicily lo bolh houseflies and
mice, and were effective inhibitors of
acetylcholmesterase.
Satisfactory correlalion was obtained
between alkaline hydrolysis rale con-
stants and anticholmesterase activity
with Tafl's a* and Es values Toxicity to
houseflies was related to anticholine-
sterase activity and Hansch's n con-
stants Several of the compounds were
of low toxicity to Ihe while mouse but
were highly effective against the
housefly
Insecticidal Properties of
Phosphonamidothioate Esters
and Derivatives
A series of S-alkyl methyl- or ethyl-
phosphonamidothioate eslers analogous
lo melhamidophos (O,S-dimethyl phos-
phoramidothiale) and acephate (O,S-
dimelhyl /V-acetylphosphoramido-
thioate) was synthesized and evaluated
for lexicological properties. Although S-
methyl methyl- and ethylphosphonami-
dothioate were more effeclive against
the housefly than methamidophos, the
acylated denvalives showed variable
loxicily bui were all subslanlially less
effective than acephate. The acylated
derivatives, however, showed improved
mouse toxicity. S-methyl methyl- and
ethylphosphonamidodithioate were
less active against the housefly than
melhamidophos
Insecticidal Activity of DDT
Analogs
A series of DDT analogs whose
structures were based on a hypothetical
model for the DDT receptor site was
synthesized and examined for insecti-
cidal activity. Several of these com-
pounds showed activity; one of them,
2,2-bis(p-ethoxyphenyl)-1,1 -dichloropro-
pane showed activity equal to that of
DDT against houseflies and mosquito
larvae. This is the first case in which a
DDT analog with an a-methyl moiety in
place of hydrogen has been shown to
have insecticidal activity.
Effect of Ring Substituents
on the Inhibition of
Cholinesterase by Carbamate
Esters
A series of 27 substituted aryl N-
methoxy-/V-methyl carbamates was
synthesized. The ability of these com-
pounds to reversibly inhibit housefly-
head (HFAChE) and bovine erythrocyte
acetylcholmesterase (BAChE) and horse
serum cholinesterase (HSChE) was
determined. All were compelitive re-
versible inhibitors of BAChE, but some
showed mixed competitive inhibition
against HFAChE and HSChE. Dissocia-
tion constants (K,) as small as 9 9 * 1CT9
M and as large as 1.4 * 10~4 M were
observed. Satisfactory correlation be-
tween log K, for the inhibition of fly-head
acetylcholinesterase by the /V-methoxy-
/V-methylcarbamates and -log Uoforthe
inhibition of the same enzyme by ihe
corresponding methylcarbamates was
noted.
Biological and Environmental
Alteration of Insecticides
Characterization of the Bound
Phenthoate Residues in Citrus
The metabolism of phenthoate or
O,O-dimethylS-[a-(carboethoxy)benzyl]
phosphorodithioate (Cidial®) in Valencia
orange fruit was examined with empha-
sis on the characterization of bound
phenthoate residues in the fruit peel.
Data for the distribution of radioactivity
in the surface wash, peel, and pulp after
treatment with l4C-phenthoaie showed
a gradual decline in external fruit
surface wash with a gradual increase in
percentage of radioactivity recovered in
the internal acetone extract and peel
residue. Radioactive labeling was also
used to indicate the nature and distribu-
tion of metabolites from the different
fractions of orange fruit. The five
principal metabolites obtained were
phenthoate oxon, demethyl phenthoate,
phenthoate acid, ethyl mandelate, and
mandelic acid, along with unchanged
phenthoate.
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Metabolism of
Dibutylaminosulfenyl and
Morpholinosulfenyl
Carbofuran in Plants, Animals
and Soil
Metabolism in Corn and Cotton
Plants
The metabolism of 2,3-dihydro-2,2-
dimethyl-7-benzofuranyl (d\-n-buty\-
aminosulfenyl) (methyl)-carbamate
(DBSC or dibutylaminosulfenyl carbo-
furan) and 2,3-dihydro-2,2-dimethyl-7-
benzofurenyl(morpholinosulfenyl)(meth-
yl)-carbamate (MSC or morpholinosul-
fenyl carbofuran), two potentially useful
insecticides, in corn and cotton plants
was studied.
DBSC metabolites in cotton plants
were identified 1, 3, 6, and 10 days
following stem injection of ring-14C-
DBSC or carbonyl-14C-DBSC and con-
firmed by chromatography in several
solvent systems. Both sets of labeling
experiments showed similar results.
The principal speciesfound after 1 and 3
days was carbofuran, but 3-hydroxycar-
bofuran was equally important at 6 and
10 days.
The metabolism of MSC in cotton
plants was determined following stem
injection of carbonyl-14C-MSC. Overall,
results were similar to those found in
the metabolism of labeled DBSC.
DBSC metabolites in cotton plants
were also identified by treatment of the
foliage with carbonyl- and ring-labeled
DBSC. Absorption occurred steadily
over the 10-day test period; the same
metabolites as those identifiedfollowing
stem injection were observed. Overall,
the rate of metabolism after foliage
treatment appeared more rapid than
after stem injection
Stem injection of carbonyl-labeled
DBSC was used to identify the various
metabolites of DBSC in corn plants. The
same metabolites found in cotton were
found in corn, although DBSC was
converted to carbofuran more rapidly
than in cotton, and the carbofuran was
converted into subsequent metabolites.
The percentage of radioactivity remain-
ing in the residual straw was higher in
corn compared to cotton.
Metabolism of DBSC in the Rat
The metabolism of DBSC in the rat
was examined to determine the fate of
this compound in mammals and to
establish the basis for its lower mam-
malian toxicity relative to carbofuran.
Ring-labeled, carbonyl-labeled, or butyl-
labeled DBSC was administered in two
doses orally in male and female rats
The most important means of elimination
was via the urine for the ring-labeled
and dibutyl-labeled DBSC and via
expired carbon dioxide and urine for
carbonyl-labeled DBSC. The distribution
and identity of the metabolites was
determined by calculating percentages
of radioactivity recovered.
DBSC is initially metabolized by two
major pathways in the rat: via oxidation
of the sulfur to give sulfonyl DBSC, and
via N-S bond cleavage to give the actual
toxicant, carbofuran. Following the
initial metabolic degradation of DBSC to
carbofuran, carbofuran was detoxified
primarily via oxidation at the 3-carbon
position of the dihydrofuranyl ring. The
primary excreted metabolite was 3-
ketocarbofuran phenol, followed by 3-
hydroxycarbofuran.
While DBSC is converted to carbofuran
in the rat, it appears that the rate of
formation of carbofuran is slower than
expected from studies on its degradation
under different pH conditions. Based on
the high levels of DBSC present in the
blood 3 h after treatment, it appears that
carbofuran is released slowly and this
slow release, in part, accounts for the
lower mammalian toxicity of DBSC
Metabolism in Soil
The alteration of MSC and DBSC in
Cosad® sandy loam soil under aerobic
and anaerobic conditions was studied,
and the degradation of the major
primary metabolites, carbofuran and
dibutylamine, investigated.
Metabolism of ring-labeled carbonyl-
labeled DBSC occurred rapidly in both
types of soil conditions, a half-life of 2-3
days and a first-order thiolysis of DBSC
are reported. The major radioactive
component during the 30-day test
period remained carbofuran, which was
either oxidized at the 3-position of the
ring, or hydrolyzed at the carbamate
ester to form carbofuran phenol Bis-
carbofuran-disulfide, dibutylamine, and
at least 7 unidentified minor compounds
were also detected. Phenolic degradation
products appeared to be bound to the
soil humus by an oxygen-dependent
process. Ring cleavage was alsooxygen-
dependent.
Alteration of DBSC in a Water
Environment
A study was undertaken to determine
the reactions involved in the degradation
of DBSC and the resulting breakdown
products under different environmental
conditions. The breakdown of DBSC in
different buffer solutions (hydrolysis), in
buffer solutions containing sulfhydryl
reagents (thiolysis), and on the surface
of silica gel TLC plates was investigated.
In buffer solution and on thm-layer
plates, N-S bond cleavage readily oc-
curred to give carbofuran as a major
product, with minor amounts of bis-
carbofuran-/V,/V'-disulfide and -trisul-
fide. First-order kinetics was observed
for the hydrolysis reaction in buffer. An
unknown polar compound was also
obtained in these systems in significant
amounts. Thiolytic N-S bond cleavage
occurred in the presence of excess
cysteine and glutathione at pH 7.0 to
give carbofuran as the sole identifiable
product, and first-order kinetics was
observed.
Acid-Catalyzed Alteration of
DBSC
During the course of a study on the
behavior of DBSC under different
solvent conditions, DBSC was found
unstable in a dichloromethane-acetic
acid (9:1) mixture. In addition to
carbofuran, TLC analysis showed that
other reaction products were the
polysulfide derivatives of DBSC (CF-Sn-
NBu2 where n =2-4), biscarbofuran-/V,/V'-
disulfide (CF-S2-CF) and polysulfides
(CF-Sn-CF where n = 3-5), and dibutyl-
amine
NMRand massspectralanalysiswere
used to establish the overall structure of
each component in CF-Sn-NBu2and CF-
Sn-CF. It was not possible, however, to
determine the exact nature of the
polysulfide moiety. Poor insecticidal
activity was observed for CF-S2-CF, a
finding that was unexpected in light of
the good activity observed for the other
derivatives.
Analysis of carbonyl- and dibutylami-
no-14C-labeled DBSC degradation pro-
ducts showed that the amount of CF-Sn-
NBu2 gradually increased to a steady-
state level after 60-72 h, while amounts
of CF-S2-CF and CF-Sn-CF continued to
increase throughout the entire reaction
period of 120 h This finding suggests
that CF-Sn-NBu2 is an intermediate and
is slowly transformed into CF-Sa-CF and
CF-Sn-CF.
Silica gel KCis reversed-phase TLC
proved to be a simple and convenient
method for separation of individual
components in the polysulfide mixtures
of DBSC and biscarbofuran disulfide
Separation of CF-Sn-NBu2 yielded at
least 8 components, and separation of
CF-Sn-CF, the biscarbofuran derivatives,
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7 components. A general correspon-
dence between R( values andthe number
of sulfurations was observed.
Effect of Impurities on the
Potentiation of
Organophosphorus
Insecticides
Identification and
Toxicological Properties of
Impurities
The major impurities commonly
present or those that may be developed
during storage in technical malathion
and acephate were studied. An evalua-
tion of the impurities' effect on the
mammalian toxicity of purified insecti-
cides was made
Impurities present in technical mala-
thion (—95% pure) and technical ace-
phate (—95% pure) were isolated by
column and thin-layer chromatography
Eleven structures in malathion and
seven in acephate were identified by
nuclear magnetic resonance, infrared
spectroscopy, and mass spectroscopy.
Eight of the compounds in technical
malathion were examined for their tox-
icologica I effects when added to purified
malathion. Data show that several of
the impurities potentiated rat toxicity.
The highest potentiation was observed
with 0,S,S-tnmethyl phosphorodithi-
oate and the S-methyl isomende of
malathion. Potentiating effects were
also decidedly greater in the rat than in
the mouse Of the impurities isolated
from acephate, O,O,S-trimethyl phos-
phorothioate showed slight potentiation
of mouse toxicity when added to purified
acephate but an antagonizing effect
was observed with 0,O-dimethyl-/V-
acetylphosphoramidothioate.
Storage of technical malathion for 3
to 6 months at 40°C resulted in
materials that were noticeably more
toxic to mice. A significant reduction in
mouse toxicity was observed after
storage of technical acephate under the
same conditions.
Mode of Potentiating Action
of Impurities Present in
Malathion
Results provided in vitro and in vivo
evidence that the impurities described
above dimmish the activities of serum
carboxylesterase and liver carboxylest-
erase (the enzymes involved in malathion
detoxication) and also diminish the
activity of serum cholinesterase The
compounds tested were, in order of
increasing potency, 0,0,5-tnmethyl
phosphorodithioate, O,0,S-tri methyl
phosphorothioate, O,S,S-tri methyl phos-
phorodithioate, and O,S-dimethyl S-
(1,2-dicarboethoxy)ethyl phosphorodi-
thioate. In vivo, the diminutions of
serum esterase activity caused by these
compounds were transient, with the
exception of those caused by 0,0,5-tri-
methyl phosphorothioate, which per-
sisted for at least 54 h.
The titer of malathion carboxylesterase
in liver and sera of rats, three strains of
mice, and five groups of pretreated mice
was correlated with the malathion LD5o
values measured in these groups. The
equation of a regression line relating
enzyme titer to toxicity was used to
predict the median lethal dosage of
purified malathion to humans. The
mean value, 3655 mg/kg, was compared
with dosages ingested in actual cases of
human malathion poisonings A dis-
crepancy was noted between LD5ovalue
predicted in this study and the life-
threatening doses of commercial mala-
thion reported in the clinical literature
A soluble, stable, and partially purified
preparation of human liver malathion
carboxylesterase was obtained by
detergent extraction and fractionation
of whole-liver homogenate. The activity
of this fraction was susceptible to
concentration-dependent mactivation
by isomalathion, the most potent of the
esterase inactivators, but was resistant
to three other impurities isolated from
technical malathion.
Properties of Rat Malathion
Carboxylesterase
Two distinct esterase fractions isolated
from rat liver microsomes were exam-
ined for enzymatic hydrolysis of [meth-
oxy-14C] malathion with special emphasis
on product analysis The effect of O,S,S-
tnmethyl phosphorodithioate and iso-
malathion as inhibitors of these ester-
ases was also investigated
Two malathion carboxylesterase
fractions, designated as esterase frac-
tions A and B, which hydrolyze malathion
were purified 13- and 18-fold, respec-
tively. Although the two enzymes could
not be distinguished from each other
kmetically, fraction A contained at least
one electrophoretic species not present
in fraction B. The molecular weight of
esterase fraction A was estimated at
50,000-60,000, but that of B was about
twice this value
Incubation of [methoxy-14C] malathion
with either fraction resulted ma mixture
of malathion or- and /3-monoacids, but
the composition of the mixture produced
by fraction A (a/ft ratio = 1.5) differed
from that produced by fraction B (a/ft
ratio - 0.2), indicating the presence of
multiple species of carboxylesterases in
mammalian liver microsomes.
Isomalathion was substantially more
potent as an inhibitor of both rat liver
and rat serum malathion carboxyl-
esterases than O,S,S-trimethyl phos-
phorodithioate. Isomalathion appeared
to be equipotent in inhibiting the rat
liver carboxylesterase-catalyzed reac-
tions leading to either a- or/3-monoacid,
while 0,S,S-trimethyl phosphorodithi-
oate preferentially diminished those
reactions. In contrast, the rat serum
carboxylesterase-catalyzed reactions
leading to either a- or /6-monoacid were
inhibited to approximately an equal de-
gree by either isomalathion or O,S,S-tri-
methyl phosphorodithioate
Delayed Toxicity of Trialkyl
Phosphorothioates
Delayed Toxicity of O,O,S-
Trimethyl Phosphorothioate
The delayed toxic effects observed in
rodents after treatment with simple
trialkyl phosphorothioates and phos-
phorodithioates were investigated
0,0,5-Tnmethyl phosphorothioate, an
impurity present in several technical
organophosphorus insecticides, admin-
istered orally to rats, caused delayed
mortality at single doses as low as 15
mg/kg, with death occurring 4-22 days
following treatment. Delayed toxic signs
were also observed in mice but in less
severity than in rats. 0,0,S-Tnethyl
phosphorothioate and O,S,S-tnmethyl
phosphorodithioate induced the same
signs of intoxication at slightly higher
doses.
Rats treated with O,O,S-tnmethyl
phosphorothioate refused food and
water within 24 h after treatment until
time of death. Neither injection of nu-
trient solution nor atropine reduced or
blocked intoxication The isomeric
0,0,0-trimethyl phosphorothioate was,
however, a potent antagonist of the
toxicity of O, O,S-tnmethyl phosphoro-
thioate As little as 1% of the 0,0,0-tn-
methyl isomer protected rats from the
intoxicating effects of the O,0,S-tri-
methyl isomer at doses as high as 200
mg/kg. Rat serum carboxylesterase and
ChE were inhibited for prolonged per-
iods following a single oral dose of
0,0,S-tnmethyl phosphorothioate but
the duration of inhibition was signifi-
cantly less when the toxicant contained
1% of the 0,0,0-trimethyl isomer
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Structure-Delayed Toxicity
Relationships
Analogs of 0,O,S-tnmethyl phos-
phorothioate were examined for acute
and delayed toxicity to rats as well as for
anticholinesterase activity. The O,0-
dimethyl S-alkyl phosphorothioate
esters containing small S-alkyl moieties
(methyl, ethyl, ^-propyl) were highly
toxic to the rat. An increase in the S-
alkyl chain length beyond propyl,
however, caused a precipitous drop in
rat toxicity Of the smaller S-alkyl 6,0-
dimethyl esters, the S-methyl and S-
ethyl analogs clearly caused delayed
toxic effects with death occurring at
long time intervals after treatment Rats
treated with the S-/7-propyl analog, the
most toxic ester, died relatively quickly
and it was difficult to differentiate acute
from delayed effects.
Anticholinesterase measurements
show that the dimethyl esters are
inactive as cholmesterase inhibitors,
being 105-to 106-fold less effective than
such strong inhibitors as paraoxon and
tetraethyl pyrophosphate Bimolecular
rate constants (k,) for the inhibition of
either bovine erythrocyte acetylcholme-
sterase (BAChE) or rat serum cholme-
sterase (RSChE) were essentially the
same for all the straight-chain 0,0-
dimethyl S-alkyl esters (CH3 to /?-C6Hi3),
and the range in rat toxicity for these
compounds was 12 to > 750 mg/kg.
The striking difference between rat
toxicity and anticholinesterase activity
suggests a noncholmergic mechanism
of intoxication.
Delayed Toxicity and Delayed
Neurotoxicity of Phosphoro-
and Phosphonothioate Esters
Simple dialkyl phenylphosphonothi-
oates such as O,0-diethyl phenylphos-
phonothioate, its corresponding oxon,
and 0,O-diethyl 4-chlorophenylphos-
phonothioate, impurities present in
technical-grade ethyl leptophos [O-(4-
bromo-2,5-dichlorophenyl) O-ethyl
phenylphosphonothioate], caused de-
layed neurotoxicity in hens The mode of
action of delayed neurotoxic com-
pounds is still uncertain but clinical
symptoms associated with delayed
neurotoxicity are not seen until 8 to 14
days after exposure to the toxic agent
The delayed toxic response described in
rats treated with the simple trialkyl
phosphorothioates suggested the pos-
sibility that some of those compounds
may cause delayed neurotoxicity in
hens. It was also possible that dialkyl
phenylphosphonates may cause de-
layed toxicity in rats. Therefore, tri-
methyl phosphate, trimethyl phos-
phorothioates, and a series of methyl
and ethyl esters of methyl-, ethyl-, and
phenylphosphonates and phosphono-
thioates were examined for delayed
neurotoxicity to hens and delayed toxic-
ity to rats.
Comparison of the toxic properties of
those compounds that are delayed-toxic
to rats with those that are delayed-
neurotoxic to hens suggests that the
two types of toxicity are different. At
least compounds that cause delayed
toxicity in rats are not delayed-neurotoxic
to hens and vice versa Of the 15
compounds examined in the present
study, only 0,0-diethyl phenylphos-
phonothioate and its oxon, 0,0-diethyl
phenylphosphonate, were delayed-
neurotoxic
0,S-Diethyl ethylphosphonothioate
produced cholmergic symptoms in rats
and most of the rats lost weight for the
first two orthree days. Asthecholinergic
symptoms subsided, rats that lost less
than 25% of their initial body weight
completely recovered while those that
lost more than 25% of their initial
weight died Death occurred up to 6
days after treatment when the weight
loss was 34 to 37% The symptomology
of poisoning appears to indicate a
possible similarity between the effects
caused by the two types of compounds
Mode of Action of Delayed-
Toxic Action of O,O,S-
Trimethyl Phosphorothioate
Efforts to determine the mode(s) of
action of O,O,S-trimethyl phosphoro-
thioate have focused on the following
areas the effects of this compound on
serum electrolyte; serum enzyme and
urinary electrolyte levels, determinations
of elimination, respiration, and tissue
distribution of radiochemical label in
animals treated with 14C-O, O,S-tn-
methyl phosphorothioate (labeled in the
OCH3 moiety), pathological studies of
tissue damage following treatment with
the compound; and the definition of
pharmacokinetic parameters through
the use of cannulae implanted in the
exterior jugular vein of test animals.
Animals intoxicated with 0,O,S-
trimethyl phosphorothioate showed
several physiological and pathological
changes, the most important of which
are lowered serum K+ levels, severe
hemato-concentration resulting from
diarrhea and excessive urination, and
damage to the liver and kidneys at
moderate-to-high doses of the material
Initial 14C-labeling studies showed I
rapid uptake and stabilization in the
body with slow decline of total radioac-
tivity up to and including 3 h following
treatment. Relative levels were much
higher following i.p. and i.v. treatments
when compared to oral treatment. At 24
h, however, a higher percentage of the
initial maximum 14C-level in the blood
remained following oral treatment
when compared to levels from i.p
treatment
Metabolism leads primarily to excre-
tion in the urine with a half-lifeof 20-24
h. Of the 63%of total applied radioactivity
excreted in the urine, only 1.6% was
isolated as unchanged material Little of
the 14C-label was excreted as C02 or in
fecal matter
Delayed Neurotoxicity of
Organophosphorus Esters
Effect of Structure on the
Delayed Neurotoxicity of O-
AlkylO-Aryl
Phenylphosphonothioate
Analogs Related to Leptophos
A systematic study of pesticidal
activity versus delayed-neurotoxic
potential of structural analogs of
leptophos was conducted. Analogs of ,
leptophos [O-(4-bromo-2,5-dichloro-
phenyl) 0-methyl phenylphosphono-
thioate] were examined for acute
toxicity to mice and houseflies, and for
delayed-neurotoxic activity in adult
hens following administration of a
single oral dose Development of ataxia
after recovery from acute poisoning was
the criterion for delayed-neurotoxic
activity. All mono- and dichlorophenyl
analogs were delayed-neurotoxic, the
2,5-dichlorophenyl analog being the
most potent delayed-neurotoxic com-
pound tested. Substitution for the
methyl by an ethyl group abolished
delayed-neurotoxic activity in both
leptophos and the 2,5-dichlorophenyl
analog at doses of 1000 mg/kg. The/?-
propyl and r?-butyl analogs of the latter
were also nondelayed-neurotoxic at
500 mg/kg and 333 mg/kg, respec-
tively. Substitution of methyl for ethyl in
EPN [0-p-mtrophenyl O-ethyl phenyl-
phosphonothioate] did not alter its de-
layed neurotoxicity.
Effect of Chirality on the
Toxicological Properties of
Leptophos and
Desbromoleptophos
The resolution and determination of
the absolute configuration of the chiral
-------
isomers of leptophos and desbromo-
leptophos and the lexicological prop-
erties of these compounds were inves-
tigated
The chiral isomers of 0-methyl
phenylphosphonothioic acid, 0-(4-
bromo-2,5-dichlorophenyl) 0-methyl
phenylphosphonothioate (leptophos),
and 0-(2,5-dichlorophenyl) 0-methyl
phenylphosphonothioate (desbromo-
leptophos) were prepared and their
absolute configurations established by
x-ray diffraction analysis of the (-)-a-
methylbenzylammonium salt. The abso-
lute configurations of the enantiomers
of leptophos and desbromoleptophos
were assigned by relating them to the
configurations of the corresponding O-
methyl phenylphosphonothioic acids.
The (R)p(+) isomers were more acutely
toxic to the housefly and white mouse,
while the (S)p(-) isomers were more
delayed-neurotoxic to the hen
Effect of Impurities on the
Delayed Neurotoxicity of
Ethyl L ep toph os
The delayed neurotoxicity of ethyl
leptophos, the effect of organophos-
phorus (OP) impurities in the technical-
grade material on delayed neurotoxicity,
the relationship of dose and time on
delayed neurotoxicity of technical
versus purified material with multiple
doses, and the inherent delayed neuro-
toxicity of significant OP impurities
were investigated.
The delayed neurotoxicity of technical
and purified 0-(4-bromo-2,5-dichloro-
phenyl) 0-ethyl phenylphosphonothioate
administered orally to hens was com-
pared under different conditions. The
technical material was neurotoxic at
750 mg/kg while the purified compound
was neurotoxic at 1000-1250 mg/kg.
The'chemical composition of the tech-
nical material was analyzed for organo-
phosphate contaminants. Impurities
present as more than 0.1% were
examined for delayed-neurotoxic activity.
O,O-Diethyl phenylphosphonothioate,
its oxon analog, and O,O-diethyl 4-
chlorophenylphosphonothioate were 5-
10 times more potent as delayed
neurotoxins than the parent compound.
These impurities evidently potentiate
the delayed neurotoxicity of ethyl
leptophos and may potentiate the
delayed neurotoxicity of other O-ethyl
phenylphosphonothioate pesticides as
well.
Mode of Action of
Phosphoramidothioate Esters
The mode of action of methamidophos
(O,S-dimethyl phosphoroamidothioate)
was studied A poor anticholmesterase
agent, it is yet highly toxic to animals,
which die of typical cholmergic symp-
toms of poisoning. The study was
designed to determine the nature of the
phosphorylatmg moiety when electric
eel acetylcholinesterase (EEAChE) is
inhibited by methamidophos
Separation of EEAChE from metham-
idophos was first established using a
Sephadex G-25 column. Enzyme inhi-
bition studies were conducted using
both [0-methyl 14C]- or [S-methyl 14C]-
methamidophos. Results provide strong
evidence for P-S bond cleavage in the
reaction leading to the inhibition of
acetylcholinesterase by methamidophos
Gas Chromatography of
Insecticides
The development of a novel gas
chromatographic column (surface-mod-
ified support), obtained by high-temper-
ature conditioning and subsequent
exhaustive methanol extraction of 6%
Carbowax 20M on HCI-extracted Chro-
mosorb W, was applied to insecticides
and extended to other pesticides. An
evaluation of other support phases for
the gas Chromatography of pesticides
was also made.
Forty-four stationary phases were
prepared as surface-modified supports
and evaluated Several of these supports
exhibit excellent characteristics for the
direct gas Chromatography of organo-
phosphorus and carbamate insecticides'
low bleed, very good peak symmetry and
resolution, short retention times at
relatively low temperatures, and high
surface mactivation with little decom-
position of labile compound.
The most effective supports for both
phosphorothionates and phosphates
were prepared with AN 600, Reoplex
400, SILAR-9CP, NPGS, EGSP-Z, and
BDS These columns are effective in
resolving fenthion, fenoxon, and their
sulfoxides and sulfones at a single
isothermal temperature. The SILAR-
9CP column is interesting because it
shows the greatest retention differences
between phosphorothionates and their
oxygen analogs The NPGS surface-
modified support is the only support
examined so far that successfully
chromatographs azinphosmethyl oxon,
a compound which pyrolyzes on-
column above 205°C but requires an
oven temperature of at least 185°C for
volatilization.
Surface-modified supports were
evaluated for the direct gas Chromatog-
raphy of 12 carbamate insecticides of
diverse chemical structures and physical
properties, and for the temperature-
programmed separation of impurities in
their technical products. Promising
results were achieved with a 2-ft x 2-
mm Nickel 200 column containing a
surface-modified Carbowax 1540 sup-
port Effective isothermal separation of
Isolan, aldicarb, 11203, MIPC, and
propoxur with sharp symmetrical peaks
was obtained at column temperatures
of 100-130°Cand an injection tempera-
ture of 110°C. Less volatile carbamates
carbofuran, mexacarbate, 9699, Pyrolan,
and carbaryl produced broader, less
symmetrical peaks at these tempera-
tures but, except for carbaryl, which
showed increased pyrolysis, higher
column temperatures appear promising
for improving peak shape and symmetry.
Temperature-programmed Chromatog-
raphy of the technical insecticides
from 50 to 130°C at 10°C/mm appears
promising for the separation of impuri-
ties for further identification studies
Triton X-305 and EGSP-Z also show
promise for the direct Chromatography
of a number of carbamate insecticides
Neurophysiological Studies
on the Mode of Action of
Insecticides
A series of neurophysiological studies
on the mode of action of insecticides
was conducted. Pyrethroid insecticides
were found to poison the nervous sys-
tem by acting at specific sites on neu-
rons. On motor nerves, all poisoning
was confined to presynaptic nerve term-
mals, in sensory nerves the sensory
endings were poisoned.
Picrotoxinin, a natural plant convul-
sant, was toxic to the desheathed
housefly central nervous system (CNS),
and was much less toxic to the intact
CNS
A series of substituted /V,/V'-thiodi-
carbamates was found to poison intact
houseflies with the same latency from
application to disruption of flight motor
neuron coordination Evidence sug-
gested that the sulfenylated carbamate
analogs were converted very rapidly at
the cuticle to the parent carbamate and
that this conversion was not rate-
limiting.
A housefly strain was obtained
containing the kdr factor, which rendered
the strain resistant to pyrethroid and
-------
DDT insecticides. The entire nervous
system of the kdr housefly was resistant
to direct perfusion of pyrethroids and
DDT. Both the central and peripheral
nervous systems were refractory to
compounds. In addition, the perineunal
sheath on the CNS made little difference
to bioassay responses whether intact or
removed, and, therefore, played no role
in the resistance. An experimental
procedure was outlined which measures
quantitatively the amount of kdr present
in mosquitoes and houseflies.
"Slow" and "fast" motor units of
Dipteran maggots are mediated by
different neurotransmitters, thataspartic
acid mimics the "slow" neurotrans-
mitter, and glutamic acid the "fast"
neurotransmitter However, a major
problem was encountered with accepting
any ammo acid as a neurotransmitter
when it was shown that normal,
neurally-evoked synaptic transmissions
occurred in the presence of slightly
elevated calcium concentrations, which
also rendered the preparations com-
pletely insensitive to perfused or
iontophoretically-applied asparate or
glutamate under the same conditions.
Attempts were made to distinguish
between DDT and pyrethroid actions on
the insect nervous system A list of
criteria was reported which allowed
analogs to be categorized as DDT-like or
pyrethroid-like.
The consequences of insecticide
poisoning were investigated by compar-
ing the histological appearance of the
thoracic ganglion of houseflies which
had been treated with doses causing
mortality in 50% of a population
Lindane treatment was found to cause
"vacuoles" in the thoracic ganglion that
were correlated in number with the
amount of disability measured behav-
iorally. The disability type was correlated
with the vacuolization site.
Examination of CNS activity in the
housefly was accomplished by recording
the comparing motor unit activity
before, during, and after poisoning.
Measurements were made without
dissection and during performance of
uninhibited movement, the only restric-
tions being a tether and wires inserted
through the cuticle in a few sites on the
thorax. This approach allowed the first
unambiguous description of overt poi-
soning symptoms based on nervous
activity.
T. R. Fukuto, R. B. March, and T. A. Miller are with University of California,
Riverside. CA 92521
William F. Durham is the EPA Project Officer (see below).
The complete report, entitled "Chemistry and Mode of Action of Insecticides:
Phase II," (Order No. PB 83-247 213; Cost: $20.50, subject to change) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
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