EPA-600/1-76-025
July 1976 Environmental Health Effects Research Series
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environ-
mental Protection Agency, have beep grouped into five series. These five broad
categories were established to facilitate further development and application
of environmental technology. Elimination of traditional grouping was con-
sciously planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating to the
tolerances of man for unhealthful substances or conditions. This work is gener-
ally assessed from a medical viewpoint, including physiological or psycho-
logical studies. In addition to toxicology and other medical specialities, study
areas include biomedical instrumentation and health research techniques uti-
lizing animals—but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-76-025
July 1976
PESTICIDE INDUCED DELAYED NEUROTOXICITY
Proceedings of a Conference
February 19-20, 1976
Washington, D. C.
Sponsored by:
Environmental Protection Agency
and
National Institute of Environmental Health Sciences
Edited by:
Ronald L. Baron
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
U. S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N. C. 27711
LIBRARY
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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ACKNOWLEDGEMENTS
The objective of the Symposium was to bring to a specific open
forum a problem that has been recognized and investigated intensively
for almost half a century. It was hoped that the presentations and
discussions would stimulate new research to fully understand and control
the problem. This document is a compilation of the papers presented and
the discussion of the two day meeting.
There are many people to whom the EPA and NIEHS are indebted for
their work in putting this meeting together. Thanks are due to the
participants for attending and special acknowledgements are due Dr.
William Upholt and Dr. John Casida for taking the time to serve as
chairmen. In preparation for the meeting, the following were instrumental
in planning the agenda: Dr. William Durham, Dr. Orville Paynter, Mr.
August Curley, Dr. John Casida, and Dr. Florence Kinoshita. Additional
thanks are due to Mrs. Sandra Townsend of Kappa Systems, Inc. and Mr.
Frank Ayers of the Research Triangle Institute for their excellent
management of the planning sessions and the execution of the program.
I greatly acknowledge the help of all those responsible for reading
and putting together the final document. Special thanks are due to Mrs.
Donna Wicker for her assistance with the revisions and corrections that
were necessary. Without all of the above people, this meeting could not
have taken place.
Ronald L. Baron
m
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CONTENTS
Page
Introduction ............... . .................... Terri Damstra 1
Dedication .................................... Ronald L. Baron 2
Participants ................................................. 3
Discussants .................................................. 5
Recognition and Overview of the Organophosphorus Problem .....
................. John E, Casida and Ronald L. Baron 7
Reaction of Serine Esterases Relevant to the Delayed Neuro-
toxi city Problems .................... W. N. Aldridge 24
The Mechanism of Action of Neurotoxic Organophosphorus Esters
.................................. Martin K. Johnson 51
Discussion .............................................. 59
Structure/Activity Relationships Among Organophosphorus Esters
with Respect to Delayed Neuropathy ..................
................................... Martin K. Johnson 70
The Pathology of Delayed Neurotoxicity Due to Organo-Phosphates
...................................... W. A. Bradley 84
Persistent Effects of Organophosphate Exposure as Evidenced by
Electroencephalographic Measurements ...............
.Frank H. Duffy, James L. Burchfiel, and Van M. Sim 102
Discussion
Organophosphate Exposure from Industrial Usage, Electroneuro-
myography in Occupational Medical Supervision of Ex-
posed Workers ........................ Klaas W. Jager '52
Discussion ..............................................
Pesticide-Induced Delayed Neurotoxicity: Poison Control or
Medical Aspects .......................... John Doull 206
Discussion .............................................. 223
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CONTENTS
Page
Test Protocols and Limitations for Detection of Neurotoxicity
John P. Frawley 234
Di scussion 253
Organophosphate Exposure from Agricultural Usage...John Swift 264
Discussion 289
Pesticide Regulatory Responsibility Orville E. Paynter 291
Discussion 298
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INTRODUCTION
DR. TERRI DAMSTRA
Ladies and Gentlemen, welcome to the conference, Pesticide-Induced
Delayed Neurotoxicity, jointly sponsored by the U. S. Environmental
Protection Agency and the National Institute of Environmental Health
Sciences.
Recent investigations, have provided some exciting insights into
the possible mechanisms of action of organophosphorous esters induced
delayed neurotoxicity. We will hear about some of the primary bio-
chemical events, pathology, structure activity relationships, effects
on the EEG, and medical treatment. Hopefully, some of these beginning
insights into the mechanisms of action will also provide some very
practical benefits, such as the development of better test systems for
the accurate prediction of the potential of delayed neurotoxicity of
pesticides in both agricultural and industrial work places.
I'm sure that the presentations of the papers will stimulate a
great deal of discussion. We hope that the conference will at least
generate some new ideas and approaches that are necessary to continue
the advancement of both the understanding and elimination of the problem
of delayed neurotoxicity.
Once again, welcome, on behalf of the Environmental Protection
Agency, and the National Institute of Environmental Health Sciences.
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DEDICATION
The EPA consultant group that met in the summer of 1975 with
the responsibility of planning this Symposium was unanimous in pro-
posing that Dr. John M. Barnes of the Medical Research Council
Laboratories, Carshalton, England was the most appropriate scientist
to present the introduction on Recognition and Overview of the Organo-
phosphorus Delayed Neurotoxicity Problem. This was a very easy and
logical choice on considering the major contributions Dr. Barnes
made to this and many other areas of toxicology. With the death
of Dr. Barnes on September 24, 1975, this Symposium and the scientific
community as a whole suffered a severe loss. Dr. Barnes made a
tremendous number of significant contributions to science and was a
renowned leader in the field of toxicology. The neurotoxic syndrome
which is the subject of this Symposium was researched intensively
by Dr. Barnes. His contributions stimulated much of the work reported
to date. I suggest that this Symposium be dedicated to the memory of
Dr. John M. Barnes with the hope that the work he so ably began in
many areas will continue for the benefit of the peoples of all nations.
Dr. R. L. Baron
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PARTICIPANTS
W. N. Aldridge, Ph.D.
MRC Toxicology Unit
Medical Research Council Laboratories
Carshalton, Surrey
England
R. L. Baron, Ph.D.
Health Effects Research Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina 27711
W. A. Bradley, B.V.Sc.
Geigy Pharmaceuticals
Stanford Lodge, Altringham Road
Morley, Wi1mslow, Cheshire
England
0. L. Burchfiel, Ph.D.
Seizure Unit
Children's Hospital Medical Center
Boston, Massachusetts 02115
J. E. Casida, Ph.D., Chairman, Day 1
Department of Entomology
University of California
Berkeley, California 94720
T. Damstra, Ph.D.
Office of Programs
National Institute of Environmental Health Sciences
Research Triangle Park, North Carolina 27709
J. Doull, M.D., Ph.D.
Department of Pharmacology
University of Kansas Medical Center
Kansas City, Kansas 66103
F. H. Duffy, M.D.
Seizure Unit
Children's Hospital Medical Center
Boston, Massachusetts 02115
J. P. Frawley, Ph.D.
Hercules, Inc.
Wilmington, Delaware 19899
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K. W. Jager, M.D.
Shell Internationale Chemie, Mij BV.
The Hague
Netherlands
M. K. Johnson, Ph.D.
M.R.C. Toxicology Unit
Medical Research Council Laboratories
Carshalton, Surrey
England
0. E. Paynter, Ph.D.
Office of Pesticide Programs
Environmental Protection Agency
Washington, D. C. 20460
V. M. Sim, M.D.
Biomedical Laboratory
Edgewood Arsenal
Aberdeen Proving Ground, Maryland 21010
J. E. Swift, Ph.D.
Cooperative Extension
University of California
Berkeley, California 94720
W. M. Upholt, Ph.D., Chairman, Day 2
Office of Water and Hazardous Materials
Environmental Protection Agency
Washington, D. C. 20460
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DISCUSSANTS
M. D. Abou-Donia
Duke University
Durham, North Carolina
J. R. Albert
Shell Development Company
Modesto, California
V.K.H. Brown
Shell Research, Ltd.
Sittingbourne, Kent
England
R. Clyne
American Cyanamide Company
Princeton, New Jersey
A. M. Coehlo
Southwest Foundation for
Research and Education
San Antonio, Texas
F. Coulston
Albany Medical College
Albany, New York
T. Edwards
Environmental Protection Agency
Washington, D. C.
H. Feinman
Food and Drug Research Laboratories, Inc.
East Orange, New Jersey
S. H. Frazier
Food and Drug Administration
Washington, D. C.
J. Gehrich
National Institute for Occupational
Safety and Health
Salt Lake City, Utah
W. J. Hayes
Vanderbilt University School of Medicine
Nashville, Tennessee
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G. Kitimerle
Institut fur Toxikologie Bayer AG
Wuppertal
West Germany
D. Kuroda
Environmental Protection Agency
Washington, D. C.
C. C. Lee
Midwest Research Institute
Kansas City, Missouri
J. C. Olson
Food and Drug Administration
Washington, D. C.
E. Reiner
Institute for Medical Research and
Occupational Health
Zagreb
Yugoslavia
D. V. Roberts
University of Liverpool
Liverpool
England
J. R. Sanborn
Illinois Natural History Survey
Urbana, Illinois
G. Zweig
Environmental Protection Agency
Washington, D. C.
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RECOGNITION AND OVERVIEW OF THE
OR6ANOPHOSPHORUS INDUCED DELAYED NEUROTOXICITY PROBLEM
John E. Casida, Ph.D. and Ronald L. Baron, Ph.D.
Organophosphorus Biocides
Organophosphorus (OP) esters are now the major chemical weapons
used to combat pest insects and acarines of agricultural, veterinary
and medical importance. More than 100 OP biocides are currently in
use of which one or two dozen are major compounds. They collectively
account for an annual worldwide production exceeding 200 million
(2 x 108) pounds.
The insecticidal activity of OP compounds was discovered in 1937
by Gerhard Schrader who was instrumental in deriving a type formula
for the biocidal structures. They are acid anhydrides of the following
type:
alkyl
ami no
aryl
0-alkyl
S-alkyl
R
\
P
R
P=S
metaboli zed
to P=0
"acyl group or
acid residue
The P=S compounds are metabolically oxidized to the P=0 derivatives
before they are toxic. The first important OP insecticides and acaricides
were p_,0_-dimethyl or 0_,0-diethyl phosphates (P=0) or phosphorothionates
(P=S) optimized for high contact potency by varying the acyl group.
Subsequent developments led to the introduction of plant and animal
systemics, j_.e_. compounds absorbed and translocated in insecticidal
concentrations by crops and livestock.
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In the course of testing many tens of thousands of OP compounds
to optimize the insecticidal activity and reduce the hazard for
mammals (j_.ei. selective toxicity), other types of useful biocidal
activity were discovered, extensively studied, and often reduced to
practice. Thus, the OP compounds include interesting and/or important
nematocides, rodenticides, avicides, insect chemosterilants and
insecticide synergists, fungicides, herbicides and plant growth regulators.
These new compounds of widely divergent structures introduced new
problems. With the reduced mammalian toxicity, there was a greater
chance of demonstrable secondary effects at nonlethal dosages. With
compounds acting on plants and microorganisms, emphasis was placed on
the induction of biochemical lesions of different types than that
associated with the insecticidal and acaricidal activity. Thus, man
and his environment are now exposed to OP compounds that have the
potential for inducing a variety of biochemical lesions (Eto, 1974).
The toxicological tests that are made in evaluating the use safety
of OP biocides are generally the same as those with other types of
toxicants, especially those that are found to impact on man as inadvertant
food additives. In part, they include acute toxicity studies, life-time
feeding studies in at least 2 mammalian species, metabolic and bio-
chemical studies, and an evaluation of carcinogenic, mutagenic and
teratogenic potential.
Toxicology of Organophosphorus Compounds
The primary parasympathomimetic effects of the OP insecticides
and acaricides, in both the pest organisms and in mammals, are attributable
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in part or entirely to phosphorylation of a serine residue within
the active site of acetylcholinesterase (AChE), a critical enzyme in
the nervous system. The simplified reaction sequence is as follows:
A
R.,R2P(0)X + HO-serine-AChE -—* [El]
inhibitor (I) esterase (E)
CA + H20, -R1R2P(0)OH
B
> R1R2P(0)0-'serine-AChE
- HX
V
D + H20, -R^
HO(R2)P(0)0-serine-AChE
In the first step (A), the OP inhibitor (I) combines with the esterase
(E) to form an esterase-inhibitor complex (El). With a suitable fit
at the active site of AChE and sufficient reactivity of the OP compound,
a serine residue critical for the esteratic activity undergoes phos-
phorylation (B). The inactive phosphorylated enzyme which is formed
normally cleaves by dephosphorylation (C) to regenerate the active
esterase. However, a portion of the inhibited esterase may undergo
cleavage of the R,-P bond (D) in a process referred to as "ageing",
yielding an inactive enzyme with a very stable ionized acidic phosphorus
group on the esteratic site. The rate of each reaction is strongly
dependent on the configuration and reactivity of R-R and X on the
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nature and conformation of the active site of the esterase. Other
AChE inhibitors such as carbamates [R1R2NC(0)X] and sulfonates (RS02X)
react in steps A, B and C in an analogous manner. However, the
inhibited derivatives are more easily cleaved (step analogous to C)
and reaction D is not significant (Aldridge and Reiner, 1972).
Nerve AChE is only one of several esterases undergoing phosphory-
lation. Other forms of cholinesterase, in many tissues, are also
inhibited. Animals exposed to OP biocides are sometimes increased in
susceptibility to other toxicants because of inhibition of esterases
important in detoxifying the second compound. It appears likely that
a "teratogenic esterase" important in the synthesis of nicotinamide-
adenine dinucleotide in chicken embryos is inhibited by some teratogenic
OP and methylcarbamate biocides (Proctor and Casida, 1975). A "neurotoxic
esterase" is also phosphorylated by some OP biocides. This reaction is
central to the subject of delayed neurotoxicity and is considered in
detail in later contributions to this Symposium.
Most but not all OP biocides act as phosphorylating agents. Others
are mild to potent alkylating or acylating agents. The fungicidal
activity of some OP compounds appears to result from alkylation or
acylation. Many OP biocides react readily in mammals with glutathione,
catalyzed by glutathione S^transferase; the alkylation or arylation of
glutathione probably serves as a protective mechanism against more
deleterious lesions. The alkylating activity is also of interest in
relation to possible mutagenic and carcinogenic effects of OP compounds.
The OP insect chemosterilants, such as aziridinyl phosphorus compounds,
alter nucleic acid metabolism, but these chemosterilants are not used
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because of the hazards associated with such action. The alkylating
activity of OP insecticides is an area of definite interest and
concern, and one of a considerable volume of research literature
(Eto, 1974).
There are also other actions of OP biocides, two of which warrant
mention. Many phosphorothionates and 0_-(2-propynyl) phenylphosphonates
are potent but transient in y1yo_ inhibitors of hepatic microsomal mixed-
function oxidases important in zenobiotic metabolism (Casida, 1970;
De Matteis, 1974; Norman ejt al_., 1974). Some extremely toxic 4-alkyl-l-
phospha-2,6,7-trioxabicyclo[2.2.2]octane derivatives act as central
nervous system stimulants, possibly by blocking inhibitory neuronal
mechanisms (Casida, et^al_., 1976). These actions are not dependent on
the phosphorus substituent since non-phosphorus analogs are also active.
This background on the deversity of actions of OP biocides serves
to point out the complexity of problems in evaluating their toxicology.
It also serves to focus on the subject of greatest immediate interest
and concern, that of delayed neurotoxicity.
Delayed Neurotoxicity Induced by Chemical and Microbial Agents
Exposure to a wide variety of chemical and biological agents leads
to injury of the human nervous system involving axonal and/or myelin
disruption. Toxic neuropathies result from, among other causes, exposures
to the following agents: ino rga n ic chemica1s (especially salts of heavy
metals such as arsenic, thallium, mercury or lead); drugs [isoniazid,
£-bromophenylacetylurea, hydralazine, L-penicillamine, tetraethylthiuram-
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disulfide (antabuse), the nitrofuran group (m'trofurazone, nitrofurantoin,
furaltadone), clioquinol, plasmocid, distavol, thalidomide and several
vinca alkaloids]; fungicides (diethyldithiocarbamates); solvents (carbon
disulfide, methyl butylketone, trichloroethylene, hexane); polymer or
p1 asti c izer precursors (acrylamide, OP compounds); many biological agents
including viruses, toxins, etc. (Cavanagh, 1964, 1973). Other neuro-
pathological conditions resulting in human central or peripheral nervous
system disruption include such disorders as: amyotrophic lateral
sclerosis; Friedreichs ataxia; Werdnig-Hoffmann's disease; certain
vitamin deficiencies; and acute porphyria neuropathy.
Some OP biocides also have the potential to produce delayed neuro-
toxicity. This subject will be reviewed briefly here and in detail in
later Symposium papers.
Tri-o-Cresyl Phosphate (TOCP) and OP Delayed Neurotoxicity
A most unfortunate incident involving more than 20,000 human cases
of peripheral neuropathy occurred in the United States around 1930.
Researchers in the U. S. Public Health Service identified TOCP as the
etiological agent (Smith ejt al_., 1930), for the first time focusing
attention on OP delayed neurotoxicity. In this instance, the syndrome
was referred to initially as "ginger paralysis" or "ginger Jake" denoting
the source of beverage adulteration ("Jamaica Ginger") during prohibition
that led to the ataxia. The clinical signs were characterized as bilateral
and symmetrical flaccid paralysis of the distal muscles predominantly of
the lower extremities. Histological examination of central and peripheral
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nerve tissue preparations from early animal studies revealed extensive
disruption of the myelin sheath, suggesting a process of demyelination
as the principal mechanism leading to ataxia. The term "demyelination"
denotes the end result of a step-wise process involving sequential
block of an undefined biochemical reaction, axonal disruption presumably
starting at the distal portion, a dying-back process, and finally myelin
disruption. Clinical signs of this neurotoxic syndrome are produced
in man by a dose within the range of 2 to 40 mg TOCP/kg. The second
catastrophic incident of TOCP neurotoxicity occurred in Morocco in 1959
as the result of tricresyl phosphate from a lubricating oil normally
used in jet engines containing known amounts of TOCP which had been mixed
with olive oil for human consumption (Smith and Spalding, 1959). The
"ginger jake" and Morocco fiascoes account for most of the more than
40,000 known cases of OP delayed neurotoxicity in man. Incidents of
this type with TOCP, fortunately on a small scale, continue to appear
periodically.
TOCP requires bioactivation prior to exerting its neurotoxic
effect. The conversion of TOCP to a neurotoxic agent is due to a
two-step reaction, the first step involving mixed-function oxidase
hydroxylation of a methyl group to form a hydroxy intermediate and the
second a cyclization, catalyzed by albumin, liberating a cresyl group
and forming a saligenin cyclic phosphate.
0
CH,
0
H
\p-
.(..
CV
^
1) mixed function oxidase
2) albumin
3) -cresol
0
,o\j!/o
0
CH.
TOCP
saligenin cyclic phosphate
(the neurotoxic metabolite)
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TOCP continues to be used in laboratory experiments as the model compound
against which the effects of other materials are measured. The saligenin
cyclic phosphate metabolite and related compounds are used in studies of
the biochemical lesion.
Accidental poisoning with one candidate pesticide chemical (the
systemic insecticide Mipafox) resulted in three cases of human delayed
neurotoxicity (Bidstrup e_t a]_., 1953). This compound is closely related
to DFP, another chemical normally used in laboratory neurotoxicity studies.
(CH,),CHNH (CH,)9CHO
\//° \ //°
P P
/\F /\
(CH3)2CHNH h (CH3)2CHO h
Mipafox DFP
To date, no case of human neurotoxic involvement has been reported
as the result of recognized and authorized industrial and agricultural
use of OP compounds. However, accidental poisoning and gross misuse may
lead to severe consequences. It is clear that new OP compounds must be
evaluated for potency as delayed neurotoxins prior to their commercial
use as industrial or agricultural chemicals (Davies, 1963; Johnson,
1975a,b).
Tests on Laboratory Animals and Species Specificity for OP Delayed Neurotoxicity
The experimental production in animals of an OP-induced neuropathy,
resembling that in man, is complicated by marked species differences
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in susceptibility and clinical signs. The chicken appears to respond
most consistently in mimicing the dose- and time-dependent clinical signs
of human poisoning. Because of their availability, and ease of adapta-
tion to laboratory conditions, hens are routinely used as the test animals
with TOCP as the standard reference compound. The clinical signs after
acute TOCP administration to hens are such that the birds appear normal
for a latent period of 8 to 14 days; they then spend an increasing
proportion of time sitting in a characteristic position and, when
exercising, they display a clumsiness of gait accompanied by overt weakness;
by 15 to 20 days after treatment the birds are severely paralyzed in the
legs and lower extremities and the wings are detectably weakened. At
low neurotoxic doses, a weight loss observed over the 3-week period will
be reversed and the animal will maintain general good health although
not fully regain the use of its affected appendages. At doses producing
a severe clinical neuropathy, the animal loses its ability to eat and
subsequently dies following a severe weight loss.
There are fascinating and disturbing species differences in the
OP delayed neurotoxicity. In man and mature hens a single acute oral
administration is sufficient to initiate the irreversible clinical
response and these species are also susceptible to subacute poisoning.
There is no distinct response in young hens and variable responses in
non-human primates (squirrel and Rhesus monkey and baboon). In rats,
rabbits and other rodent species, the delayed signs of poisoning are
not readily induced. In this case TOCP administration yields cholinergic
signs of poisoning which are subsequently replaced within variable time
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intervals by fi:ccid paralysis of musculature followed by respiratory
paralysis and death. The guinea pig develops a delayed ataxic response
on extended subacute administration of DFP. In dogs, which are a
sensitive species, TOCP and DFP induce flaccid paralysis of the hind
quarters but only after an unusually extended delay period. Other
susceptible species include the cat, calf and cow, sheep and horse.
It is unfortunate that atypical responses are obtained with rats and
dogs, species used routinely in life-time feeding studies. In considering
the results of tests with laboratory animals it must be borne in mind
that man may be more sensitive than any species thus far examined, at
least to TOCP-induced delayed neurotoxicity (Davies, 1963; Johnson,
1975 a,b).
Structure-Activity in Relation to the Biochemical Lesion
It is extremely important to understand the relationship of
chemical structure and neurotoxicity, at both the organismal and
biochemical levels. It is clear that many of the delayed neurotoxins
require bioactivation (see above for TOCP) and that phosphorylation of
a critical nerve esterase is involved. The most extensive and successful
investigations on the biochemical lesion have been carried out by
Johnson, Aldridge and Barnes of the Medical Research Council Laboratories,
Carshalton, England (Johnson, 1975 a,b). Johnson has shown by an elegant
series of iji vivo and in vitro experiments with hens that inhibition
of a specific nerve esterase, the "neurotoxic esterase", is associated
with the delayed neurotoxicity. Further, he has proposed a simple in vitro
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enzyme assay to determine whether or not a new OP compound is likely
to induce delayed neurotoxicity. These findings are beginning to
clarify the seemingly confusing relationship of chemical structure
and delayed neurotoxicity. They may ultimately provide a basis for
understanding the species variations in response, which might result
from different sensitivities of the relevant enzymes or varying
significance of the inhibited pathways in maintaining normal nerve
function. Other workers have examined, with little or no success,
the possible involvement in the delayed neurotoxicity of various
vitamins (nicotinic acid, thiamine and tocopherol), steroids, cortisone,
lipids, metals (copper) and ceruloplasmin (Davies, 1963; Johnson,
1975a,b; Kimmerle and Loser, 1974).
The following pesticides (plus carbophenothion and trichlorfon)
that are currently in use or that have been subjected to widespread
trials are reported to have delayed neurotoxic properties on single or
multiple dose administration to hens:
CHJ)
C2H50^ S
« Cl
leptophos (Phosvel)
NO,
dichlorvos
C1CH2CH20-^ ^0
Haloxon
DMPA (Zytron)
DBF
[CH3(CH2)3S]3P
Merphos
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Many important OP biocides without delayed neurotoxic properties are
closely related to neurotoxic compounds (Eto, 1974; Johnson, 1975a,b).
A large number of very effective candidate pesticides have been dropped
by industry when their neurotoxic properties were discovered. Thus,
regulatory aspects are an important part of the consideration of OP
delayed neurotoxicity.
Regulatory Aspects of OP Biocides with Delayed Neurotoxic Activity
The year 1975 and the OP insecticide leptophos, which is registered
in several countries for use on cotton and food crops, have focused
attention on the regulatory aspects of OP delayed neurotoxicity. Two
actions must be considered, one by the United States EPA and the other
by a World Health Organization/Food and Agriculture Organization (WHO/FAO)
Commi ttee.
In mid-1975 the EPA issued a notice of proposed revocation of
tolerances previously established for leptophos (Ritch, 1975). Initial
neurotoxicity studies with leptophos were negative but further tests
showed the propensity for initiating the delayed neurotoxic response in
hens. In a move designed to insure the safety of man and his environment
and to assure that residues in food will not be hazardous, the EPA
proposed the revocation of tolerances for leptophos. The notice of
revocation stated:
"...a revaluation of the petition (for tolerances of 10 ppm
on lettuce and 2 ppm on tomatoes) and other data confirmed
that leptophos is an agent which produces delayed neuro-
toxicity in hens. Additional information on leptophos is
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necessary to evaluate the possible hazard to man and other
nontarget species from the potential effects of its use.
Based on the above information, there is a reasonable basis to
propose revocation of the established tolerances for residues
until adequate data are provided by the petitioner to show that
the use of leptophos on lettuce and tomatoes will not be
detrimental to the public health."
This appears to be the first time a pesticide tolerance established by a
U. S. regulatory authority has been recommended for revocation.
Also in 1975, the WHO Expert Committee on Pesticide Residues in
a Joint Meeting with the FAO Working Party of Experts on Pesticide Residues
considered the problem of delayed neurotoxicity, in conjunction with an
examination of the worldwide use of leptophos in agriculture and with
respect to its safety as a residue in food entering international commerce.
This consideration was in conjunction with the role of the Joint Meeting
in estimating an Acceptable Daily Intake (ADI) and recommending maximum
residue limits (tolerances) in food to the Codex Alimentarious Commission.
The Joint Meeting considered that:
"the potential hazards associated with delayed neurotoxicity
are two-fold: (1) exposure of occupationally or accidentally
exposed individuals who would be affected by high doses for
short periods; and (2) long term low level exposure and
possible build-up of the toxicant to threshold levels leading
to ataxia. Although the first principle does not fall into
the direct terms of reference of the Meeting, the toxicological
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hazard associated with such exposure must be considered. The
Meeting considered that delayed neurotoxicity is a parameter of
toxicology that appears to follow a dose-response relationship
and as such a no-effect level could be estimated following
acute or chronic exposure in a susceptible species. With an
adequate margin of safety, an ADI for man can be allocated as
far as pesticide residues in food are concerned, with a sufficient
degree of assurance."
The WHO/FAO Committee took a very significant stand on the problem
of delayed neurotoxicity. They concluded that OP-induced delayed neuro-
toxicity is a toxicological syndrome similar to other toxicological
parameters and subject to the same quantitative considerations given
other types of toxicants. The dosage-response relationship was considered
to be applicable to delayed neurotoxicity as it is to other modalities of
injury. The WHO/FAO Joint Meeting reported that sufficient toxicological
data had been presented for leptophos, including short and long-term
studies with rats and dogs, reproduction investigations, and carcino-
genesis studies. The only significant adverse toxicological parameters
reported were pup mortality at high levels in the reproduction study and
delayed neurotoxicity in hens. Based on an observed no-effect level in
rats and dogs and the no-effect level for delayed neurotoxicity in acute
studies with hens, a temporary ADI of 0 - 0.001 mg/kg body weight was
established. This group reflected its concern over the delayed neuro-
toxicity finding by applying a high safety factor to the experimental
no-effect levels in animals in arriving at the ADI for man. In conjunction
-20-
-------
with the establishment of an ADI, the WHO/FAO Joint Meeting recommended
the establishment of tolerances on 20 crops with values ranging from
0.05 to 2 mg/kg (ppm). These recommendations will now be considered
by the Codex Alimentarius Commission with respect to establishing
international tolerances for world trade.
While the two actions taken in 1975 (by the EPA and the WHO/FAO
Joint Meeting) may appear to be in conflict, they reflect the continuing
difficulties in evaluating the safety of toxicants and their direct and
indirect effect on man and his environment. The validity and implications
of these actions with respect to world trade will be discussed for many
years in select circles. This Symposium deals with both the basic
science and regulatory aspects of OP-induced delayed neurotoxicity.
They are critical and timely issues for the future of OP biocides and
the establishment of adequate safety standards.
-21-
-------
References Cited
Aldridge, W. N. and Reiner, E. Enzyme Inhibitors as Substrates. Inter-
actions of Esterases with Esters of Organophosphorus and Carbamic
Acids. New York: American Elsevier Pub!. Co., Inc., 1972.
Bidstrup, P. L., Bonnell, J. A. and Beckett, A. G. "Paralysis following
poisoning with a new organic phosphorus insecticide (Mipafox). Report
on two cases." Brit. Med. J. 1953 I: 1068-1072.
Casida, J. E. "Mixed-function oxidase involvement in the biochemistry
of insecticide synergists." J. Agr. Food Chem. 18 (1970): 753-772.
Casida, J. E., Eto, M., Moscioni, A. D., Engel, J. L., Milbrath, D. S.
and Verkade, J. G. "Structure-toxicity relationships of 2,6,7-trioxa-
bicyclo[2.2.2]octanes and related compounds." Toxicol. Appl. Pharmacol,
(1976): accepted.
Cavanagh, J. B. "The significance of the "dying-back" process in
experimental and human neurological disease." jrvt. Rev. Exp. Path.
3 (1964): 219-267.
Cavanagh, J. B. "Peripheral neuropathy caused by toxic agents." Crit.
Rev. Toxicol. 2 (1973): 365-417.
Davies, D. R. "Neurotoxicity of organophosphorus compounds." In
Cholinesterases and An t i c ho1i ne s te ras e Agents, Handbuch der
E xperimentenen Pharmakologie. Erganzungswerk, XV. (G. B. Koelle, Ed.).
Berlin: Springer-Verlag, 1963, Chap. 19, 860-882.
De Matteis, F. "Covalent binding of sulfur to microsomes and loss of
cytochrome P-450 during the oxidative desulfuration of several
chemicals." Mol. Pharmacol. 10 (1974): 849-854.
-22-
-------
Eto, M. Organophosphorus Pesticides: Organic and Biological Chemistry.
Cleveland, Ohio: CRC Press, 1974.
Johnson, M. K. "The delayed neuropathy caused by some Organophosphorus
esters: mechanism and challenge." Crit. Rev. Toxicol. 3 (1975a):
289-316.
Johnson, M. K. "Organophosphorus esters causing delayed neurotoxic effects.
Mechanism of action and structure/activity studies." Arch. Toxicol.
34_ (1975b): 259-288.
Kimmerle, G. and Loser, E. "Delayed neurotoxicity of Organophosphorus
compounds and copper concentration in the serum of blood." Environ.
Qual. Saf. 3 (1974):173-178.
Norman, B. J., Poore, R. E. and Neal, R. A. "Studies of the binding of
sulfur released in the mixed-function oxidase-catalyzed metabolism
of diethyl p_-nitrophenyl phosphorothionate (parathion) to diethyl
p_-nitrophenyl phosphate (paraoxon). Bipchem. Pharmac'"'. 23 (1974):
1733-1744.
Proctor, N. H. and Casida, J. E. "Organophosphorus and methyl carbamate
insecticide teratogenesis: diminished NAD in chicken embryos."
Science 190 (1975): 580-582.
Ritch, J. B., Jr. "Leptophos. Proposed revocation of tolerance."
Environmental Protection Agency. Fed. Reg. 40 (May 27, 1975): 22847.
Smith, H. V. and Spalding, N. M. K. "Outbreak of paralysis in Morocco
due to ortho-cresyl phosphate poisoning." Lancet 1959 jj'._: 1019-1021.
Smith, M. I., Elvove, E. and Frazier, W. H. "The pharmacological action
of certain phenol esters, with special reference to the etiology of
so-called ginger paralysis." U. S. Publ. Hlth. Rep. 45 (1930): 2509-2524.
-23-
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REACTION OF SERINE ESTERASES RELEVANT TO THE DELAYED NEUROTOXICITY
PROBLEMS
W. N. Aldridge, Ph.D.
This talk must be regarded as an introduction to Dr. Johnson's
lecture on the mechanism of delayed neurotoxicity. I shall discuss
very briefly those properties of the reaction of organophosphorus
compounds with esterases which have been used in the development of
his hypothesis. Some of this no doubt will be to many of you old
information but the emphasis will be directed towards the above purpose.
The first part concerns the reaction which seems to be an exact analogy
of the substrate - enzyme system. The aging reaction which has no such
analogy will be dealt with in more detail. (See Figure 1)
Evidence has been obtained for all the steps shown in the figure.
For most examples the rate of reaction of organophosphorus compounds
with esterase follows 1st order kinetics and the rate of the reaction
is directly proportional to the concentration of inhibitor. (See Figures
2 and 3)
In some instances there is indirect evidence for saturation
kinetics. Thus, the evidence for substrates of the existence of a
Michael is type complex is that a maximum rate of product formation is
reached - i.e. when the enzyme is all present as the reversible complex
with the substrate. In a similar way for some organophosphorus compounds
the first order constant for inhibition is not linearly related to the
concentration of inhibitor and the rate tends towards a maximum. This
-24-
-------
X
co
-f
LJJ
-t- <•
X
CO
OJ
-I-
CO
X
LJJ
/I
r—
+
V
CO
X
LJJ
O
-25-
-------
0 i .
CCH30)2PO-/ V
NO.
LOG°/oACT. pH=8*55
2-0
1-8
1-6
1-4
1-2
2>iM
= 0'4minr
1-0 2-0
TIME(min.)
INHIBITION OFChE
3-0
FIGURE 2
-26-
-------
0
N(CH3)
km= 21x1ff* M
r3
-2
-1
k, =
min"1
1.0 2.0 3.0
Time (min)
Q2 0.4 0.6 0.8 1.0
10'xCi(M)
Inhibition of ChE at pH 8.9
FIGURE 3
-27-
-------
is interpreted as evidence for the existence of a reversible Michaelis-
type complex between inhibitor and enzyme. (See Figure 4)
When this condition is found the bimolecular rate k constant
a
is not determined but K and k+2 may be separately measured. (See
Figure 5)
Numerous inhibitors have now been examined and constants
determined. A few examples are given from published work including that
of Dautermann & Main. (See Figures 6 and 7)
If the reaction is so similar to the reaction with substrates
then organophosphorus compounds which resemble substrates should be
better inhibitors. In general this is true and examples of inhibitors
and substrates of cholinesterase, chymotrypsin, and a brain esterase
are given. (See Figure 8)
In some instances the inhibited enzyme is unstable and is hydrolysed
at a measureable rate.
The reaction therefore is an exact analogy of the enzyme substrate
reaction, the major difference being the stability of the acylated
intermediate. For example, the half-life of various phosphorylated
acetylcholinesterases are 10 min or so, whereas the half-life of
acetylated cholinesterase, the intermediate in the hydrolysis of acetyl-
choline, is approximately 100 y sec. (See Figure 9)
The rate of reaction of organophosphorus compounds is influenced
by many factors, and these are outlined in the figure. (See Figure 10)
The fact, that the inhibited enzyme is normally stable and the
inhibition follows defined kinetics, has allowed different esterases
-28-
-------
co'
-f
LU
CO
CO
<
CO
X
DC
o
-29-
-------
FIGURE 5
Ku(M)
-4
Phosphostigmine(25°) 1.2x10 5.2
DFP (5°) 1.6x 10~3 U .9
Paruoxon (5°) 3.6xlU~ 42.7
Malaoxon(5°) 2.1xl(f3 67.0
Tetram(5°) 1.8 x 10"4 126
-30-
-------
CH.
NH R2
CH COO
N02
EtO
EtO
POO
NO.
5.7
EtO
POO
CH
N02 1.5 x 104
Hartley, Kilby (1950)
Becker et al. (1963)
FIGURE 6
-31-
-------
o
CH. P OCH CH N(CHJ,
a i — — o o
F
o x 10
8
EtO
EtO
PS CH9 CH? S (Et)2
9x 10
8x10
7
FIGURE 7
-32-
-------
o
o—P—o
CH,*
CH, O
(I)
(ID
FIGURE 8
-33-
-------
Stability of liiglkylphosphorylated true
cholinesterase
2 life (min)
Dimethyl Si
2-Chlorethyl 16
2 -Chloropropyl 32
3-Chloropropyl 11
2:3 - dichloropropyl 139
Aid ridge (1953)
Pickering, Malone (1966)
FIGURE 9
-34-
-------
Important Factors in Acylatinq
Inhibition of Esterases
Structure of inhibitor
Chemical reactivity of inhibitor
Type of esterase
Particular acyl group attached to esterase
Conditions of medium (pH, temp, ionic
strength and composition etc.)
FIGURE 10
-35-
-------
loo
ACT
I I i I I
I i i i
(> S
it- I
t-oc
|o
or £600
3oM*N. AT 3*7°
P «
V 3
FIGURE 11
-36-
-------
1-0
% fieri
(•4
I'X
CHICKEN
3 If.
OF &FP
3o HlN AT 3T*
FIGURE 12
-37-
-------
to be separated. Two examples are given - one a sensitive and insensi-
tive enzyme, (Figure 11), and the other of two esterases of differing
sensitivity (Figure 12).
One reaction which has no substrate - enzyme analogy is the aging
reaction. After the discovery of nucleophilic reactivators of phos-
phorylated cholinesterases it was found by Hobbiger that if a di-iso-
propyl phosphorylated cholinesterase was stored it became impossible
to reactivate. The diethylphosphoryl enzyme changes very slowly in
this way (Figure 13).
Work in Holland and here at Edgewood showed that the essential
feature is the loss of a group attached to the phosphorus atom after
carbonium ion formation (Figure 14); this was proved chemically for SOMAN
inhibited enzyme (Figure 15).
Many phosphorylated cholinesterases undergo this aging reaction
but at different rates (Figure 16); the reaction is acid catalysed and
considered to be a purely chemical transformation not involving the
participation of the active centre.
For some time the cholinesterases were the only enzymes which
were capable of undergoing this change, but it is now known to be a
more general property depending on the enzyme and the particular groups
attached to the phosphorus (Figure 17).
The general view that the sole mechanism involves carbonium ion
formation has now begun to look unlikely. For example, some straight
chain alkyl derivatives were shown to age and Lee & Turnbull in 1958
had shown that diphenylphosphoryl chymotrypsin released one mole of
phenol. The known difficulty of carbonium ion formation with phenyl
-38-
-------
10
20
30
40
50
60
70 .
Hobbiger (1956)
2 4 6 8 hr.
Enzyme reactivation after prolonged inhibition. Fresh human
red cells were first incubated with the inhibitor. Small samples
were taken after various periods of incubation and exposed for
1 hr. to 10~2M-PjAM. Enzyme reactivation was then determined
after a 10-fold dilution. Similar results were obtained when high
concentrations of AM were used for enzyme reactivation.
• •: enzyme inhibited by an organophosphate containing
a diethylphosphato group. O O: enzyme inhibited by an
organophosphate containing a difropropylphosphato group.
Abscissa: duration of inhibition before addition of reactivator.
Ordinate: % enzyme activity which cannot be restored by the
reactivator (logarithmic scale).
FIGURE 13
-39-
-------
CH CH
0
0
q
Wl '3 S~
CH3
V^l \\J
E
01 13
MZ
x* V^ 1
E
CH
ilife [pH7-4, 37°] 2-3 min
FIGURE 14
-40-
-------
Rate of ageing of methylisopropoxy
phosphorylated cholinesterase
Human erythrocyte, 37°
PH
6.0
7.1
7.2
8.0
8.5
10 x k(mi
10
4
3.8
3.0
1.2
Davies, Green (1956)
FIGURE 15
-41-
-------
Phosphorylated enzymes which age
Acetylcholinesterase
Cholinesterase
Chymotrypsin
Carboxylesterase (Sheep, chicken, pig, horse)
Esterase on CNS membrane
("Neurotoxic esterase"; phenyl n-valerate)
FIGURE 16
-42-
-------
pH 7.4, 37°
R!
(CHj)2CHO -
CH3
CH3
:H(cn CH .0-
R2
(CH3)2CHO-
(CH3)2 CH CH (CH3) 0-
(CH3)3 C CH (CH3) 0 -
CH. CHfCl) CH_ O-
\ life (i
2140
58
6
160
o
Cl CH2 CH2 CH20 - Cl CH2 CH? CH2 O -
COULT, MARSH, READ (1966)
PICKERING, MALONE (1966)
FIGURE 17
-43-
-------
groups and the fact that the best substrates for chymotrypsin contain
two aromatic rings suggests other possibilities.
Several examples are now known where aging and spontaneous
reactivation occur at comparable rates. (Figure 18)
The pH dependence of spontaneous reactivation is bell shaped and
for phenyl methylphosphoryl cholinesterases spontaneous reactivation
and aging both have bell shaped pH dependence. This contrasts with
the H+ catalysed reaction for branched chain alkyl derivatives. (Figures
19, 20 and 21)
I therefore propose that aging can take place by two different
mechanisms (Figure 22), one H+ catalysed through carbonium ion formation,
and the other through a part of the normal enzyme reaction hydrolysing
another ester bond. It will be clear from the structure-activity
relationships for neurotoxic agents and the current hypothesis for the
mechanism of delayed neurotoxicity necessitate this additional mechanism
of aging.
This thumb-nail sketch of the reactions of organophosphorus
compounds covers most of the background to the techniques and inter-
pretation of results which will be developed in the next lecture.
-44-
-------
Phosphorylated acetylcholinesterases
undergoing spontaneous reactivation and aging
Enzyme derivative
Methylphosphonyl -
Phenyl
4-methoxyphenyl
4-cyanophenyl
Diphenylphosphoryl-
Phenylchlorophosphoryl -
Dimethylphosphoryl -
Di 2-ch!oropropylphosphoryl
Di 3-chloropropy!phosphoryl
Hovanec & Lieske (1972)
Wins & Wilson (1974)
Skrinjaric-Spolar, Simeon
& Reiner (1973)
Pickering & Malone (1967)
FIGURE 18
-45-
-------
= CONHCH3
k3C°/o)
100
80
60
40
20
pK - 6-9
= PO(OC2H4Ct)
> x°
/ °x«
pK=9'85
9 10 11
RATE CONSTANTS OF REACTIVATION OF INHIBITED
ChE. THEORETICAL CURVE CALCULATED FROM
OBSERVED pK VALUES
FIGURE 19
-46-
-------
3.0n
2.5H
X 1.5r
ot
Jt
1.0-
.5-
i
7
pH
8
i
10
pH-rate profile for the spontaneous reactivation at 25°
of AChE inhibited with p-cyanophenyl methylphosphonochloridate
Hovanec and Lieske (1972)
FIGURE 20
-47-
-------
1.2.-,
1.0-
e
>c
.6-
.4-
.2-
56789
PH
10
pH-rate profile for the aging at 25° of AC HE inhibited by
p-cyanophenyl methylphosphonochloridate (IV).
Hovanec & Lieske (1972)
FIGURE 21
-48-
-------
Spontaneous
reactivation
(enzyme
catalysed)
Mechanism E aging
(enzyme catalysed)
Mechanism A aging
(acid catalysed)
Reactions of phosphorylated
cholinesterases
FIGURE 22
-49-
-------
References
A detailed discussion of the reaction of organophosphorus compounds
and esterases has been published.
Aldridge, W. N. & Reiner, E. 'Enzyme Inhibitors as Substrates'
interaction of esterases with esterase of organophosphorus
and carbamic acids. Amsterdam: North Holland Publishing
Co. (1975) p. 1 - 328.
A recent assessment of the aging phenomena was presented at an
International Symposium "Cholinesterase and Cholinoreceptors" at
Split, Yugoslavia.
Aldridge, W. N. Survey of Major Points of Interest about
Reaction of Cholinesterases, Croatica Chemica Acta, Zagreb,
(1975) p. 225-33.
-50-
-------
THE MECHANISM OF ACTION OF NEUROTOXIC ORGANOPHOSPHORUS ESTERS
M. K. Johnson, Ph.D.
Professor Casida, ladies and gentlemen. I think it right that
we acknowledge that the ability to get an understanding comes from
almight God. It's also very true that every scientific worker climbs
upon the shoulders of every other worker. In some cases other workers
grab him by the legs and try to pull him down, but in others they seize
him by the ankles and hoist him up! I'm fortunate in that I've been
in the situation of the latter at Carshalton. We have already heard
the tribute to the late Dr. John Barnes. Both he and Dr. Aldridge
have consistently and over many years hoisted me up in order to help
on the investigations which they set in motion.
I want to say three negative things to start with. Firstly, to
reemphasize the fact that we really ought not to talk or think in terms
of demyelination. It's true that if you wait long enough after a long
and severe assault, you will see myelin degeneration. But the primary
degeneration, as Professor Casida has already pointed out, is undoubtedly
degeneration of axons. And "demyelination" only serves to deflect both
the pathologist and the biochemist from looking for the right sort of
lesion.
Secondly, to remind us that not all anticholinesterases are inevitably
neurotoxic. And thirdly, to say that we're not speaking about the effect
which has been described as an immediate paralytic effect of one or two
pesticides, by Durham, Gaines, and Hayes (1956), and Witter and Gaines
p
(1963). I believe there is evidence in the original papers which can
-51-
-------
be interpreted to show that that is a prolonged, chronic cholinergic
effect. So we're talking about what I would like to call delayed
neuropathy.
Organophosphorus compounds which cause delayed neurotoxic effects
3
phosphorylate a characteristic nervous-tissue protein, and inhibit
its activity as an esterase. There is ample evidence that the phos-
phorylation measured radiochemically and the inhibition measured enzymatically
both represent the covalent reaction of the inhibitor at a single enzyme
active site. It is important to the understanding that we are not beguiled
into proposing a second site to explain the results with protective
agents (see below).
In vivo studies with hens using a variety of inhibitors of neurotoxic
esterase reveal that the toxic effect does not occur directly because
of the loss of esterase activity. The toxic response depends on the
c
chemistry of the inhibited enzyme. Two classes of inhibitors of neurotoxic
esterase have been defined (Figure 1). Class A compounds (phosphates,
phosphonates and phosphoramidates) are neurotoxic. Class B compounds
(phosphinates, sulphonyl fluorides and carbamates) are not only non-
neurotoxic but actually protect hens against subsequent challenge doses
567
of Class A compounds. ' It is probable that a process analogous to
the "aging" of inhibited cholinesterase occurs after neurotoxic esterase
has been inhibited by neurotoxic (Class A) agents. The charged acidic
group which then would be left attached to the membrane-bound neurotoxic
esterase could disrupt normal metabolism in the neurone. Speculations
-52-
-------
GROUP A
GROUP B
R-0 0
Ml
P-X
R'V
Phosphate
R-NH 0
II
/P-X
R'-NH
Phosphoramidate
R-0 0
\H
P-X
R'
Phosphonate
0
II
R-S-X
II
0
Sulphonate
R 0
\»
/P-X
R'
Phosphinate
R
0
\
R X
Carbamate
FIGURE 1. Compounds which belong in Group A and which are found to inhibit
neurotoxic esterase in vivo are neurotoxic. By contrast those of
Group B which will inhibit NTE in vivo are protective.
-53-
-------
q
have been put forward concerning how much disruption might occur.
The chemical processes involved in "aging" could not occur after
inhibition of neurotoxic esterase by protective (Class B) compounds.
We can now quantify the effect on the target protein of candidate
pesticides by a laboratory assay. This is an important advance from
the point of view of toxicological evaluation and in the fields of
therapy and of structure/activity relationships. Whether the discovery
of protective compounds can be applied to the cause of safety remains
to be seen.
A problem in the study of delayed neuropathy is that even when
the most favoured substrate is used the activity of neurotoxic esterase
accounts for only 6% of the total esterase activity of hen brain homogenate:
there are several other esterases which attack the substrate but are
distinct and separate from neurotoxic esterase. The physiological
significance of these other esterases is unknown. They are not sensitive
to the same spectrum of inhibitors as neurotoxic esterase and their
presence in a screening assay for neurotoxicity must therefore be excluded.
False conclusions will be drawn by monitoring the response of the whole
esteratic activity of hen brain to administration of test compounds.
A detailed protocol of the necessary differential assay of neurotoxic
esterase is laid down in Reference^. Phenyl valeate is the substrate
of choice for assays and mipafox is used as a selective inhibitor
to pick out neurotoxic esterase from irrelevant (paraoxon-sensitive)
esterases which also attack this substrate. Phenyl benzylcarbamate is
a safer but not completely selective alternative to mipafox. A better
-54-
-------
alternative is needed and I hope to submit a report soon to Toxicology
and Applied Pharmacology describing a safer and simpler assay which
will be suitable for screening of dosed hens: it is not suitable
however for checking active compounds in vitro.
The relationship of structure to activity in vivo has been explored
for a wide range of compounds. ' ' These are related both to potential
pesticides and also to the plasticiser-type tri-aryl phosphates.
It has been possible to indicate certain features of structure which
tend to be associated with low neurotoxic potential and some generalisations
are summarized in the next paper. The respone of single hens to test
doses of compounds can be assessed biochemically one day after dosing.
The effects of doses below those causing frank ataxia can be graded in
a fashion not possible in the all-or-none clinical test.
Investigations of the mechanism of action of neurotoxic organo-
phosphorus esters have been undertaken over a period of more than 40
years. A number of blind alleys have been explored, but it is certain
that the more recent successes would not have been achieved without the
earlier work. The way in which these fundamental studies would prove
relevant to toxicity testing and to safety could not be foretold. Enzyme
processes have been scrutinized in great detail, and studies have not
been restricted by the demands or criteria of short-term projects. It
is only recently that the practical benefits to toxicology have begun to
be appreciated. However the benefits are considerable - coherent
structure/activity studies have been performed with a suitable monitor;
prospective forecasts of activity are possible; chronic dosing can be
evaluated; preventive measures are possible; and therapy is being explored
-55-
-------
rationally; species variation and human sensitivity can be explored
more clearly. Many further practical investigations utilizing present
12
understanding have been proposed. This does not mean that mechanistic
studies should cease. Rather, efforts should be increased in the
confidence that further understanding of mechanism will be valuable,
and possibly in unforeseen ways. Further work is needed to
(a) confirm, if possible, the "aging" hypothesis;
(b) locate neurotoxic esterase intracellularly;
(c) purify and study the properties of this curious protein in
hens, man, and other species;
(d) discover its physiological function as distinct from its
apparently unnecessary esterase activity;
(e) elucidate the sequence of events between phosphorylation
of neurotoxic esterase and axonal degeneration;
(f) study the processes at work in maintaining connection of
nerve endings with their receptor organs;
(g) try to find links between neuron degeneration caused by
organophosphorus compounds and that caused by other chemicals
and disease agents. In spite of morphological similarities
in some cases, there is no evidence as yet that these neuro-
pathies share a common mechanism.
-56-
-------
References
1. Durham, W. F., Gaines, T. B. and Hayes, W. J., Jr. Paralytic
(1956) and Related Effects of Certain Organic Phosphorus Compounds.
Arch. Ind. Hlth. 1j;326-30.
2. Witter, R. F. and Gaines, T. B. Relationship Between Depression
(1963) of Brain or Plasma Cholinesterase and Paralysis in Chickens
Caused by Certain Organic Phosphorus Compounds. Biochem.
Pharmacol. V2:1377-86.
3. Johnson, M. K. A Phosphorylation Site in Brain and the Delayed
(1969) Neurotoxic Effect of Some Organophosphorus Compounds.
Biochem. J. Ill: 487-495.
4. Johnson, M. K. The Delayed Neurotoxic Effect of Some Organophosphorus
(1969) Compounds: Identification of the Phosphorylation Site as an
Esterase. Biochem. J. 114: 711-717.
5. Johnson, M. K. Organophosphorus and Other Inhibitors of "Neurotoxic
(1970) Esterase" and the Development of Delayed Neurotoxicity in Hens.
Biochem. J. 120:523-531.
6. Johnson, M. K. and Lauwerys, R. R. Protection by Some Carbamates
(1969) Against the Delayed Neurotoxic Effects of Di-isopropyl
Phosphorof1uoridate. Nature (London) 222:1066-1067.
7. Johnson, M. K. The Primary Biochemical Lesion Leading to the
(1974) Delayed Neurotoxic Effects of Some Organophosphorus Compounds.
J. Neurocnem. 23: 785-789.
8. Johnson, M. K. Mechanism of Protection Against the Delayed
(1976) Neurotoxic Effects of Organophosphorus Esters. Fed. Proc. 35:
73-74.
-57-
-------
9. Johnson, M. K. Structure/Activity Relationships for Substrates
(1975) and Inhibitors of Hen Brain Neurotoxic Esterase. Biochem.
Pharmacol. 24:797-805.
10. Johnson, M. K. Organophosphorus Esters Causing Delayed Neurotoxic
(1975) Effects: Mechanism of Action and Structure/Activity Studies.
Arch. Toxicol. 34:259-288. (Originally cited as Toe. Env. Chem.
Res.)
11. Johnson, M. K. The Initial Biochemical Events Leading to the
Delayed Neuropathy Caused by Some Organophosphorus Esters.
Proc. Eur. Soc. Toxicol. In the Press.
12. Johnson, M. K. The Delayed Neuropathy Caused by Some Organo-
(1975) phosphorus Esters: Mechanism and Challenge. Crit. Rev. Toxicol
3:289-316.
-58-
-------
Discussion
DR. J. ALBERT: We've talked for some time about species differences.
Have you looked at the rat and the chicken with respect to the enzyme
inhibitors?
DR. M. JOHNSON: Yes, I have looked. And quite clearly, the species
difference doesn't reside in this preliminary step. Rats are not sensitive
but have an inhibitable neurotoxic esterase as far as simple comparative
characterization goes. Baboons and sheep both have the enzyme.
Sheep are susceptible and the neurotoxic esterase is inhibited by the
appropriate neurotoxic dose of haloxon. In young baboons, the enzyme
is inhibited by the saligenin cyclic phosphates, but I've never had an
adult baboon for a clinical test: it was very young and so the testing
there wasn't really strictly relevant.
Young chickens, are also resistant apparently, but if you give
multiple doses, you can clearly get ataxic chickens. However, the first
dose was adequate for full inhibition. So it looked as though there
was some sort of an adaptive process. Perhaps in growing birds it's
easier to explain than actual differences between species. In the growing
bird, perhaps other axons take over and the bird adapts as it grows.
For the difference of species, guinea pigs are resistant to a single
dose as Professor Casida pointed out. However, there is evidence that
if several doses are given, you can get an ataxic guinea pig. It is
possible that the difference lies in differences in rate of ageing but
I wouldn't hang my hat on that. I think it's a difference in sheer
anatomy, physiology. Maybe size of axons. It seems to be the smaller
animals which are the least susceptible.
-59-
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For monkeys and some other primates, I don't think the testing
has been fair. Because it's really only been done with TOCP. My
conviction is that if you're really going to check something, you ought
to use a direct inhibitor rather than one which needs metabolic activation
and may also be poorly absorbed. You should use the cresyl or the
phenyl saligenin phosphate, if you really want to know about TOCP.
So the answer about species is we don't know and I'm making guesses.
DR. N ALDRIDGE: Could I just re-emphasize, without underestimating
the difficulty with the rat, that over the last five to ten years, I
think there has been a reorientation in our view. Ten years ago it was
thought that the chicken, cat and man were the only sensitive species.
There is now quite a catalogue of sensitive species, particularly,
as Dr. Johnson said, the larger species. Sheep, cows, horses, pigs,
and water buffalo, are all very sensitive. We are beginning to realize
that these compounds are attacking some fundamental property of the
central nervous system. We now should look to why the mouse and the
rat are exceptional.
DR. C. C. LEE: A comment on Professor Casida's remarks concerning
the rats and the dogs, and their being non-reactive to neurotoxic agents.
Three years ago we undertook a long-term evaluation of the di-methyl,
di-thio carbamates, sponsored by NIEHS.* After feeding rats high doses
of Ferbam or Thiram, commonly used fungicides in the United States
and overseas, for up to 18 months, a neurotoxicity developed, characterized
by ataxia, Parrota's a paralysis of the posterior part of the body,
degeneration of myelin sheath, and axon degeneration.
* National Institute of Environmental Health Sciences, Dept. HEW,
Research Triangle Park, North Carolina
-60-
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The animals, before developing the neurotoxic effect displayed,
a series of abnormal behavioral activity in tests, which might indicate
the subtle changes due to a neurotoxic effect.
DR. J. E. CASIDA: Could you restate the compound?
DR. C. C. LEE: A dimethyl-di-thiocarbamate. The compound is
ferbam, which is the ferric salt. This type of compound was developed
because interest in the rubber industry. It was subsequently discovered
that these molecules have medical uses and also as pesticides.
Antabuse, which is used in the treatment of alcoholism, belongs
to this group. Detailed information will be presented to a symposium
March 1st, 2nd, 3rd, 1976 at Research Triangle Park, N. C., sponsored
by WHO and NIEHS, concerning the chemicals of interest to the rubber
industry. The proceedings will be published in Environmental Health
Perspectives.
DR. N. ALDRIDGE: I think we must be absolutely clear that what is
being proposed by Dr. Johnson is the mechanism of the production of a
dying back lesion in the peripheral and central nervous system by
organophosphorus compounds. It is clear that other compounds will
produce dying back lesions in the peripheral nervous system by mechanisms
which are not the same. For example, acrylamide produces a beautiful
dying-back lesion in rats and other species which does not involve
neurotoxic esterase. As for the carbamates that have just been mentioned,
these compounds do not carbamylate, so they are compounds of a quite
different nature. There are probably many different mechanisms of
producing a dying back lesion in the central nervous system.
-61-
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DR. T. EDWARDS: I would like to ask about reversibility of
the harm from neurotoxic organophosphates. I was wondering if you've
done any work on the reversibility of this dying back effect, or whether
it's some other further complication that's caused persistence of the
lesion.
DR. M. JOHNSON: I think it's well known that in adults, both
men and hens, that once a severe ataxia comes on from organophosphorus
esters, then there appears to be really no improvement in clinical
state. This is different from the acrylamide neuropathy, for instance,
which Dr. Aldridge has just referred to.
In terms of the enzyme, we need to be clear that the initial
biochemical event, can be induced within one hour of dosing. The
earliest pathological, microscopic lesion is only seen after 6-8 days and
at that time resynthesis of enzyme has caused a significant return of
activity as you saw on one of the slides. I want to emphasize and perhaps
keep on emphasizing that it isn't the loss of enzyme activity that
matters. We have insulted the neuron in some way, as from one hour
after dosing, that it doesn't like. And at the moment there doesn't
seem to be any way of reversing that biochemical insult. And from then
on, everything progresses. It seems to me rational that, with the
understanding that we now have, it may be possible to construct reactivating
agents analogous to RAM, but appropriate to the active site of neuro-
toxic esterase. In fact I've listed some in that review in Critical
Reviews in Toxicology, which seemed to me appropriate. And we are
engaged actively in looking at some of these. So far, I would say, with
only moderate success whether, even if we did reverse the inhibition
-62-
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and pull that charged group off the enzyme, whether there would
be no onset of frank lesions remains to be seen.
DR. N. ALDRIDGE: I don't know whether there is a neuro-
pathologist who would comment on the well known fact that neuropathy
in the peripheral nerves recovers completely, whereas in the central
nervous system, if it once progresses to a disconnection, there appears
to be no recovery.
DR. T. EDWARDS: I don't have an explanation, but I have run across
some references where the difference in species would depend on the ratio
between sphingomyelin and lecithin in the nerve. And of course there
would be a difference between peripheral and brain as well as a difference
in peripheral nerves in various species.
DR. M. JOHNSON: I have discussed that idea of Rosenberg's in my
critical review: it is interesting but some data may not be too good.
DR. J. SANBORN: You commented on the fact that young chickens
aren't as susceptible to neurotoxic agents, except by multiple dosing.
Have you tried to characterize this enzyme in young chickens as opposed
to chickens that are older and does any of this have anything to do
with the chickens when they become old and are in full egg production?
Is there any kind of steroidal implication between the egg production
and the development of this esterase? The systems are, of course, very
different physiologically, but I'm curious about this.
DR. M. JOHNSON: Well, to answer the second part first, the same
age dependence is seen in man. Children in Morocco, for instance, were
far less severely affected, and they tended to improve in time. I think
in Canada there has been a publication in TAP awhile ago on the contrast
* Toxicology and Applied Pharmacology.
-63-
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between adult cats and young kittens with one of the saligenin phosphates.
And so I don't feel it's so much perhaps developmental in that sense,
but perhaps the nervous system is still developing and the axons are
certainly growing. In my simplistic view, I think that's more significant.
As regards the enzyme, the obvious characteristics in chicks and
adult hens appear to be identical. And indeed, I think it would be
hard to think of it otherwise because the chick goes through a gradation
where it begins to become somewhat sensitive and then fully sensitive.
And I find it hard to believe that the enzyme is changing in a graded
way. Also the half life of inhibition and return is just the same in
the young chick as the adult chick.
DR. J. DOULL: I'd agree with Dr. Johnson's comment on children.
There's another phenomenon there, and that's plasticity that occurs in
children, whereby an axon can switch from one cell body to another.
My question has to deal with therapy. I had hoped that you were going
to offer us some hope with the statements that you would make about the
sulphonates and the phosphinates and so on. But I thought I understood
you to say that when they were given after exposure, they were worthless.
Is that correct?
DR. M. JOHNSON: That is absolutely right. They are perfectly
effective as protective agents. But once the appropriate covalent
bond has been formed at the enzyme, there is nothing that another
acylating agent can do. You need a quite different class of compounds,
such as oximes tailored to this enzyme. Whether we shall ever find one,
remains to be seen. I live in some hopes.
-64-
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DR. J. DOULL: Let me rephrase my question. Do you have any
thoughts or recommendations that would be useful in the way of therapy
to modify that lesion before the axonal degeneration really gets set?
If we're saying, that there is a period there in which we could intervene
to prevent degeneration starting from the end of the axon, how would
we go about that?
DR. M. JOHNSON: I have absolutely no suggestion.
DR. N. ALDRIDGE: For reactivation of phosphorylated cholinesterase,
the oximes are tailored to utilize the active center, i.e., they have
positive charges. They are however totally ineffective when the enzyme
is aged. No method has been found to reactivate aged enzyme and it
would clearly require something quite different from an oxime.
DR. S. FRAZIER: What is the relationship with the changes in the
cholinesterase of the brain, serum and so forth, in determining neuro-
toxicity?
DR. M. JOHNSON: There is no correlation whatsoever. We are dealing
with two separate enzymes, each with its own structure/activity relation-
ships which can be characterized as you would characterize structure
activity for any other substrate or inhibitor or any other enzyme. There
is, however, of course, some overlap, so that some compounds which have
some anticholinesterase activity also have some anti-neurotoxic esterase
activity. But perhaps when we come to talk about structure activity a
little more, I can point out some of the chemical features which favor
either potent inhibition of neurotoxic esterase, or possibly discourage
it. You can run the whole gamut with OP compounds on the one hand you can
-65-
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inject a compound which will produce negligible cholinergic response
and yet cause delayed neuropathy. On the other hand there are many
compounds which only show the delayed neuropathy if you give really
quite heroic treatments to prevent them succumbing first to the acute
anticholinesterase effects.
DR. G. KIMMERLE: If you are dosing hens, orally or by other
routes of administration, and you give your animals different dosages,
a range from very low dosages to high dosages, and protect them with
oximes, do you see a dose relationships in your enzyme bio-assay?
In using compounds which are neurotoxic to chickens in doses from 1
to 1000 mg/kg, can you find a dose relationship in your bioassay?
DR. M. JOHNSON: Yes, I can find a dose relationship. You will
find some of the data in that review in Archives of Toxicology. In the
Tables there are a number of compounds which have been tested at various
dosages. And you will see a column which says, "Percentage Activity
Remaining of NTE". When you see 70% or 50% remaining, the compound is
not neurotoxic at that dose but when you find residual activity around
20% or less, then you'll always find a positive neurotoxic response
marked off in the next column. There is quite a clear dose-response
relationship.
DR. N. ALDRIDGE: I don't know whether the questioner was hinting at
another question which really has not been fully explored. As Dr. Johnson
says, his data indicates that at a certain level of inhibition you obtain
a clinical response in the hen. There is another question which can be
raised, which, perhaps later on in the meeting will be explored a bit more;
-66-
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whether the meaning of percentage inhibition is a statistical statement.
Does it mean if you have a 70% inhibition in one neuron that you have
100% in some and 30% in others. These are all sorts of quantitative
questions which maybe one can begin to answer. The ability to make
quantitative measurements on the receptor will greatly aid movement
forward in answering such questions.
DR. V. K. H. BROWN: I was very perturbed, sir, to see in your
early slide that you listed dichlorovos as being neurotoxic. I was
wondering whether any of the speakers could tell us the level of evidence
for this. As I understand it, from the work of Albert and the work
of Dr. Johnson, the long chain analogues, C5 longer chain analogues of
dichlorvos may be highly neurotoxic. But I understood that the dimethoxy
compound was not a cause of delayed peripheral neurotoxicity. Could I
have a comment on this, please?
DR. M. JOHNSON: I did react, perhaps in a slightly modified version
to yourself, seeing that on the slide. And in the sense that it is possible
under the most heroic conditions imaginable, making the hen house look
like a surgical operating theater and dosing with dichlorovos on two
successive days, at 100 milligrams per kilo on each occasion (each about
10 X unprotected LD5Q) with pre-dosing with eserine and atropine plus
oxime, you can just about get them through the acute effects and show
a paralytic effect. But this is very different from the other five
compounds which were listed on Professor Casida's slide: they all produce
a neurotoxic response with a far greater degree of ease in conditions where
acute cholinergic effects were much less severe. Now, I think we must
-67-
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allow the fact that, in a research laboratory, I have been trying to
squeeze the last bit of information out of structure/activity relation-
ships, and have therefore gone to quite unprecedented lengths, in order
to keep a bird alive. I found that, at a lower dose, dichlorvos did
produce some inhibition, and I wanted to know what would happen if
I could force the inhibition up. But I think you are fully justified
in pointing out that dichlorvos is really in a very different class
from EPN or leptophos, for instance.
DR. J. ALBERT: I would agree with Dr. Johnson that with heroic
efforts we can perhaps keep these birds going and obtain paralysis.
I would like to know if there was a distinct recovery phase on those
birds before they showed the paralysis and the degree of paralysis
that you did see after that period of time.
DR. M. JOHNSON: There was a distinct recovery phase. They had
recovered sufficiently within 24 hours after the first dose to be able to
survive a second identical dose on the second day and they were quite
o.k. two days later. May I shift my ground a little bit to another
dimethyl compound? With dimethylphosphorofluoridate, you can give a
dose such that they look almost dead within about three minutes of dosing.
Ten minutes later they're picking up their heads, and half an hour later
they're walking around the cage. With that compound also, it's necessary
to give two heroic doses. I think we could say that those two are the
only dimethyl compounds which are likely to have structures with much
fit for the enzyme. There is a clear recovery stage for both these compounds
and the hens walk around healthily for days before they become paralyzed.
-68-
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DR. 0. ALBERT: I have just one short technical question on
the phosphinates. Do you see inhibition of only the neurotoxic
esterase, or do you see also a paraoxon-like inhibition and an
additional inhibition beyond this.
DR. M. JOHNSON: Every one of the OP compounds I've tested
has got its own spectrum of specificity. A number of them are quite
good inhibitors of the paraoxon-sensitive enzymes, but I haven't always
checked those. I have gone straight to what I call the B minus C assay,
so I can tell you something about the effect on neurotoxic esterase
and on the residual seven percent. I don't know about phosphinates
on the paraoxon-sensitive enzymes.
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STRUCTURE/ACTIVITY RELATIONSHIPS AMONG ORGANOPHOSPHORUS ESTERS WITH
RESPECT TO DELAYED NEUROPATHY
M. K. Johnson, Ph.D.
In the Mechanism talk (see previously) I explained that the primary
target in organophosphate neuropathy is the esterase site on a nervous
system protein which we call neurotoxic esterase. It is possible to
correlate neurotoxic doses with high inhibition of neurotoxic esterase
assayed in homogenates of whole hen brain. It is clear that we are using
the brain (which is easily removable from a carcass) as a monitor for the
effect in spinal cord and peripheral nerve; lesions are most marked in
these latter tissues but the tissues are less convenient for routine
biochemical investigations. The validity of this practice of monitoring
brain is proven by the results. I have published recently an extensive
review of structure/activity relationships and my purpose here is to
state a few of the major generalisations which have emerged. For com-
pounds which are directly active against neurotoxic esterase in vitro
it has been possible to understand the relationships more easily since
we are not confused there by problems of distribution and differences in
2
activation and disposal mechanisms. I have reported on the relation-
ships in vitro for organophosphorus esters and also for carbamates and
other compounds which might be useful as protective agents or as analytical
tools.
On structure/activity, this meeting is, of course, committed to
interest in pesticides. But we've seen quite clearly that the mechanism
is common to both the pesticidal organophosphorus esters and to the tri-
-70-
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aryl phosphates which have been responsible for the majority of the
human cases of delayed neuropathy. And since there are some quite
pretty structure/activity relationships there, I thought it would be
instructive just to look at them briefly, before we go on to the
pesticidal compounds. The classical neurotoxic compound, tri-orthocresyl
phosphate, has been studied by many workers. But when we talk about
tri-orthocresyl phosphate, I think we ought to be perfectly clear that
probably 99.9% of all the work which has ever been done with tri-orthocresyl
phosphate has not, in fact, been done with pure compound. To get pure
orthocresol in former days from coal tar distillates was not a practical
proposition. One got preparations which were labelled "Orthocresol".
"Metacresol". "Paracresol", but they were all crude mixes, containing
varying percentages of the isomers plus some phenol or perhaps a sub-
stituted ethyl-phenol. Now, this is very important indeed, because there
are vast variations in activity of the triesters according to the variants
of the substituents on the three aryl groups. This has been thoroughly
documented by three different groups of people. First of all, Henschler
in Germany has done a colossal amount of work. And then also Bondy,
Field, Worden and Hughes, and also Dr. Barnes and Dr. Aldridge had a
look at substituents, in collaboration with Bondy of the Coalite Chemical
Company in Britain. It is now quite clear that if you compare the
symmetrical tri-esters having ortho substituents with esters which
have only one ortho-substituent, plus two, say, phenyl groups, or two
para-cresyl groups, you will get a compound which is much more neuro-
toxic. This means that if you start with a feedstuff which you thought
was pure orthocresol and make tri-ester with phosphorus oxychloride,
-71-
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you won't, in fact, get a hundred percent tri-orthpcresyl phosphate.
You'll get maybe 97 or 98% pure tri-ester and you'll also get varying
amounts of the dicresyl mono-phenyl ester, and diphenyl mono-orthocresyl
ester. So if you start with orthocresol contaminated with phenol,
you'll get a much more neurotoxic compound. And therefore when we talk
about comparing man and hen, as regards their sensitivity to triortho-
cresyl phosphate, we are on a very sticky wicket. It cannot really be
done. Nobody knows what was the actual chemical entity in ginger jake
or in the Moroccan cooking oil. Certainly tri-orthocresyl phosphate
was a substantial proportion of it. But it is also almost certain that
there were a number of other isomers of varying degrees of activity,
and in many cases much higher activity. I say that, by the by, because
obviously this business of species comparisons is important to us.
So, having said all that, let's go back to structure/activity.
The first obvious contrast which came along was that the ortho
compound was very neurotoxic, while the tri-paracresyl compound was not
neurotoxic, even on repeated doses. This is supported by the biochemical
test which shows high and negligible inhibition respectively one day after
dosing. However, when testers looked at the ethyl substituents, the
situation was reversed. The tri-orthoethyl compound was not neurotoxic
and the tri-paraethyl compound was neurotoxic! And there it was! There
was an impasse on the "score zero" versus "score one" test. The value
of the biochemical test in this situation is clearly seen in Table 1
which compares both clinical and biochemical test data for the isomeric
tri-ethylphenyl phosphates. Biochemical testing confirmed the neurotoxic
potency of the para isomer but it showed that the ortho isomer was by no
-72-
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means inactive (42% inhibition of neurotoxic esterase after a single
dose). Subsequent testing of 4 doses showed the parallel result of
high inhibition and clinical ataxia. In this case biochemical monitoring
revealed and predicted a hazard. In the case of the meta isomer the
test revealed negligible biochemical response after a single dose and
the Table shows that the consequent prediction that multiple doses of
this compound would also be ineffective has been confirmed. Thus the
brief biochemical test after a single dose has effectively distinguished
between two compounds as being hazardous and safe respectively, whereas
clinical testing alone did not reveal the neurotoxic potential of the
one compound and could never quantify the margin of safety for the other.
Now, it's true to say that tri-aryl phosphates are intended to be
inert chemicals. They are synthesized as plasticizers, hydraulic fluids
or flame retardants, and are not intended to have biological activity.
And therefore I don't think it's unreasonable to go looking for compounds
which give very low response in the biochemical test, even at repeated
doses. However, when we turn to pesticides, I'm not sure that the same
rigid rule can be applied. After all, to be a pesticide, an organo-
phosphorus ester has got, first of all, to have an ability to inhibit
esterases. It's tailored to inhibit acetylcholinesterase of the pest in
particular. It is always likely that it will have some activity against
other esterases. I think it is asking the impossible of the chemists and
of enzymology and of proteins, to imagine that you could ever get a
pesticide which would be highly potent against pest acetylcholinesterase
and inhibit neither mammalian acetylcholinesterase nor any other esterase
to any degree whatsoever. That's asking for the moon. What is done, is that.
-74-
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the compounds are tailored to have comparative toxic responses, high
anti-pest activity and lower mammalian toxicity. And we can do something,
in similar style, about segregating anti-cholinesterase activity from
anti-neurotoxic esterase activity.
Table 2 is a summary of tests I have done measuring both the clinical
and enzymic response in hens after administration of a single dose of
a variety of organophosphorus esters (usually in conjunction with prophylactic
and therapeutic treatment for anticholinesterase effects). Clinical
ataxis was never seen at 10-20 days after dosing without a concomitant
high inhibition of neurotoxic esterase at 1-2 days. The general impression
emerges that phosphonates (and their thio-precursors) have a higher
tendency to be neurotoxic than are phosphates. This is borne out by
observations of inhibitory power of analogous structures in vitro where
the phosphonates are more active against neurotoxic esterase by a factor
which considerably exceeds the factor of increased lability or general
reactivity: this trend is not true for anticholinesterase activity of
phosphates and phosphonates.
When we turn to consider the intrinsic activity in vitro of a
compound against neurotoxic esterase then factors such as metabolic dis-
posal and anticholinesterase activity do not interfere with measurements.
However one metabolic factor must be considered and this is activation.
Table 3 shows that the two thiophosphonates illustrated, although neuro-
toxic in the hen, are not active against the enzyme in vitro whereas the
oxon analogue is highly active both jn yitro and in vivo. It is well
known that anticholinesterase activity of thiophosphates is like !se
negligible until the molecule has been activated by conversion f* the
-75-
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Neurotoxic
Dose (mg/kg)
In Vitro MT.E.
l (pM)
400 - 500
N.S.
( > 100)
40 - 60
0.4
60 (max)
N.S.
Table 3 Comparison of the effectiveness of typical neuro-
toxic thiophosphonates and a related oxon in vivo
and in vitro. NTE - neurotoxic esterase;
NS - not significant at 100 ^M.
-77-
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oxon. Thus it is not easily possible to start assessing neurotoxic
potential of an organophosphorus ester by an exclusively in vitro
test unless the major probable antiesterase metabolites are available.
In most cases the identity of these is easily predicted, though DBF
(S,S,S-tributyl phosphorothiolate) is one example which comes to my
mind where I'm not sure how much is known of active metabolites. It is
possible that a combined liver microsomal metabolising system could be
included in an in vitro test system provided that interfering liver
esterases were effectively inhibited.
Let us now dissect the influence of the substituents in a typical
organophosphorus ester with the general structure
0
'(0)
R2'
If the detailed data in references (1) and (2) are analysed we can
make the further general observations:-
1) Phosphonates are more active than analogous phosphates while
phosphinates are yet more active but are, of course, pro-
tective rather than neurotoxic as pointed out in the Mechanism
talk.
2) Table 4 shows the marked increase in anti-neurotoxic esterase
activity in vitro and in vivo as the bulk and hydrophobic
1 2
character of R and R is increased up to the bis-n-pentyl homologue.
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2,2 ' -Dichlprovi nyl Phosphates
In Vitro sc Dose % NTE Activity
(mg/Kg) remaining
Me 70 30 64
Et 2 18* 25
aPr 0.05 2* 5
n.Pent 00003 2* 9
n.Hexyl 0.015
n.Octyl 0.07
a.Decyl 0.5-1
Neurotoxic dose
Table 4 The relationship of alkyl chain-length to
neurotoxic potential assessed in vitro and
in vivo. The I^s refers to neurotoxic
esterase not to acetylcholinesterase.
-79-
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The same trend is seen in Table 5. The non-neurotoxicity
of dimethyl phosphates is very marked. Joining the R and
2
R substituents into a cyclic structure appeared to reduce
neurotoxic potential in one compound I tested.
3) A comparison of Tables 4 and 5 shows the large effect of the
leaving group (X). Thus the activities in vitro of the
appropriate jvpropyl compounds differ by a factor of 2000
and this is reflected (with a smaller factor) in their
behaviour j_n vivo.
4) The most neurotoxic esters tend to have rather small and
flexible leaving groups (X) such as fluoride or dicholorovinyl.
Leaving groups which tend to lower neurotoxic potential
include 4-nitrophenyl, oxime groups, a variety of hetero-
cyclic groupings and groups with considerable steric hindrance
around the ester bond.
5) Linkage of the leaving group to phosphorus via a thioloester
bond rather than by oxygen drastically reduces the potency
of inhibitors in the substrate and carbamate series and seems
likely to do the same in phosphates.
6) Insertion of hydrophilic substituents into an aromatic ring
also reduces inhibitory power against neurotoxic esterase.
7) Use of R or X groups which are non-planar (cyclohexyl),
heterocyclic (pyridinyl) or very bulky (naphthyl, etc.)
tends to produce compounds which do not act as good substrates
or inhibitors of neurotoxic esterase.
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-81-
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Many more potential pesticides have been synthesized in commercial
research laboratories than ever reach the market. If these were re-evaluated
by in vitro testing against neurotoxic esterase much more detailed
structure/activity relationships could be worked out than those which
I have sketched. Comprehension of the target should enable a rapid
advance in synthesis of compounds concerning whose neurotoxic potential
we can have quantitative data and, hopefully, a firm assurance of
non-neurotoxicity. This is a great step beyond the assertion that
"it didn't paralyse the hens".
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References
1. Johnson, M. K. Organophosphorus Esters Causing Delayed Neurotoxic
(1975) Effects: Mechanism of Action and Structure/Activity Studies.
Arch. Toxicol. 34:259-288. (Originally cited as Tox. Env. Chem. Rev.),
2. Johnson, M. K. Structure/Activity Relationships for Substrates
(1975) and Inhibitors of Hen Brain Neurotoxic Esterase. Biochem.
Pharmacol. 24:797-805.
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THE PATHOLOGY OF DELAYED NEUROTOXICITY DUE TO ORGANO-PHOSPHATES
W. A. Bradley, B.V.Sc.
Delayed neurotoxicity caused by organo-phosphorus compounds has
been seen in many thousands of people. The most common group of compounds
producing this are the tri-cresyl group of phosphates containing ortho-
cresyl groups which have produced on occasions, epidemic-like outbreaks.
Much of the work identifying the various members of this group that are
neurotoxic was done by Bondy et al (1960), with the original work on
tri-orthocresyl phosphate (ToCP) done by Smith and his co-workers. Smith
and Elvove (1930) showed that Ginger Paralysis was caused by ToCP and
that similar symptoms, including histopathological lesions, could be
re-produced in the chicken (Smith, Engel and Stohlman 1932).
Other organo-phosphorus esters, particularly some of the fluorine-
containing esters, can also produce neurotoxicity. Many of these are
powerful cholinesterase inhibitors and as such, in some cases of poisoning,
the victims do not often survive the initial crisis. However, reports
have appeared showing that on occasions where the person has survived
the initial exposure, delayed neurotoxicity has been seen. In 1953
Bidstrup, Bonnell and Beckett reported on two cases caused by Mipafox
(bis-isopropyl aminofluorphosphine oxide) and Namba et al (1971) reported
on a case where the person involved had worked on the synthesis of
new organo-phosphorus compounds for 10 years. Generally, cases of
delayed neurotoxicity are rare after poisoning with commercial pesticides,
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although haloxon (di-2-chlorethyl-3-chloro-4-methylcoumarin-7-yl
phosphate) has produced neurotoxicity in the pig after an apparently
normal anthelmintic medication (Christian, 1975).
Ever since Smith, Engel and Stohlman (1932) reproduced neurotoxic
lesions in the hen which resembled those seen in the human with roughly
a similar dose range, the hen has been the species of choice. Other
advantages are that there appears to be no strain differences, the hen
is easily obtainable, easy to keep in fairly basic surrounds and responds
to a neurotoxic dose within a fairly short time (under 3 weeks usually).
While results generally are easily reproducible and predictable, we have
had two cases in our laboratories, using tri-aryl phosphates, where we
have had a more severe reaction than expected. One of these had multiple
fibrosarcoma nodules involving abdominal organs and tissues including
the liver and the other had multiple caseous nodules (cause not diagnosed)
throughout the liver. However, two cases are not sufficient to draw any
definite conclusions as it is possible to account for these as being at
the extreme of individual variation.
Apart from the hen, several other animal species have been shown to
be affected and these have included cattle, goats, pigs, cats, some
primates, pheasants and ducks. Other species such as the rat, rabbit,
partridge and quail, appear to be resistant (Johnson 1975a).
Barnes and Denz (1953) showed that there was an age susceptibility
in that young birds tended to be more resistant, although Johnson and
Barnes (1970) were able to overcome this resistance with repeated doses.
It is, therefore, advisable to use older birds. The United Kingdom
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Ministry of Agriculture, Fisheries and Food recommend hens older than
nine months for testing pesticides for neurotoxicity. However, when
looking for very low levels of neurotoxicity it may be advisable to
use birds in the two to three year-old age group. In fact I have been
able to show very low levels of toxicity using older birds instead of
younger ones and the results have correlated very well with those obtained
by Johnson (1975b).
Following poisoning in hens with a neurotoxic organo-phosphorus
compound there is initially, depression of cholinesterase and related
enzymes which will produce effects ranging from no apparent clinical
abnormality to sudden and rapid death. Assuming the hen survives the
initial crisis, signs of neurotoxicity are seen generally between 8-10
days (range 7-20 days) after the neurotoxic dose. There does appear to
be a critical dose level below which, signs do not appear but these lower
levels do seem to be cumulative if repeated, although below an even lower
level no toxicity can be produced (Smith, Engel and Stohlman 1932).
Clinically the neurotoxic symptoms in hens appear to be confined mainly
to the legs. Initially the birds appear to sit longer and when disturbed,
a very slight unsteadiness in their gait is occasionally seen. When a
dose above the minimal toxic dose is given the ataxia slowly develops.
The tail tends to become depressed slightly so as to maintain balance,
then walking becomes difficult and they stand with their hocks resting
on the ground. Eventually they are unable to use their legs for walking
though these are not completely paralysed and can still be vigorously
moved. This phase takes up to two weeks to develop after the initial
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symptoms have been seen. If left the hens may die, usually as the
result of starvation. Indeed, if body weight is monitored fairly
carefully, slight weight loss is noticed from about the tenth day,
particularly in birds that are kept in groups. There are two reasons
for this, one being the increasing difficulty in getting to food and
the other is the change in 'peck order1 because of their disability.
More often, providing they can obtain food and water, there will be a
variable period when there is no apparent recovery or further deterioration
before some recovery is seen. The less severe cases may recover completely,
the others will show varying degrees of spasticity and ataxia.
Basically the lesion is a 'dying back1 process or Wallerian
degeneration in the axons (Figure 1) although earlier work tended to
consider it a de-myelinating disease (Smith and Lillie 1931). Lesions
are not detected histologically until after the delay period which is
about the same time that clinical symptoms are noted. No changes are
found before this time even when using electron-microscopy (Bischoff
1970). These are seen in the long sensory and motor pathways of the
spinal cord and in the larger fibres of the peripheral nerves and restricted
to the distal end of the axons. In the peripheral nerves, degenerative
lesions are most often seen in the more distal muscles involving the
intramuscular nerve bundles and the sensory nerves from the muscle
spindle. In the hen lesions are very rarely seen much above the division
of the sciatic into the tibial and peroneal nerves. The pathology of
these lesions has been reviewed recently (Cavanagh 1973, Le Quesne 1975)
and the distribution of the lesions, particularly in the C.N.S. was covered
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FIGURE 1. Wallerian Degeneration in a Nerve Fibre in the Tibial Nerve.
L. S. . H. and E. Stain Magnification 1200
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earlier by Barnes and Denz (1952). Various methods for investigating
and identifying neurotoxic lesions have been described by Glees (1967).
Other degenerative lesions have been noted in other parts of the
peripheral nervous system (Vij and Kanagasuntheram 1972; Krishnamusti,
Kanagasumtheram and Vij 1972).
The neuroanatomy of the fowl has been described by Jungherr
(1969). The dorsal funiculus carries mainly ascending fibres, the
ventral funiculus, particularly near the ventral median fissure, has
both descending and ascending fibres and the lateral funiculi tend to
have ascending fibres in their lateral aspect and descending ones in
their medial aspect. Histologically neurotoxic lesions are seen most
easily in the long sensory and motor tracts at the distal end of the
axon. Hence lesions in the ascending tracts are seen in the cervical
region and in the descending tracts in the dorsal and lumbo-sacral region
of the spinal column as well as in the more distal portion of the peripheral
nerves (Figures 1, 2, and 3). Practically lesions have been seen at all
levels of the spinal cord but with a preponderance in the upper and
mid-cervical region. Lesions in the lumbo-sacral region tend to be fairly
sparse. Janyck and Glees (1966) found chromatolysis of the spinal neurone
in the lumbo-sacral region but generally this is not often seen.
Very rarely similar changes are seen in odd axons in normal hens.
These can be distinguished from the neurotoxic lesions in that they are
usually a single degenerating axon, often at a difference stage of
degeneration, whereas neurotoxic lesions are usually multiple, bilateral
in distribution and all of a similar 'age'. These odd single lesions are
more likely to be in older birds where the occasional neurone dies off.
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FIGURE 2. Degenerative Lesions in the Ascending Tracts of the Lateral
Funiculus at an Approximate Level of C6. T.S.. H. and E. Stain
Magnification 120
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FIGURE 3. Degenerative Lesions in the Descending Tracts of the Ventral
Funiculus at an Approximate Level of L. S. 1. T. S.. H. and E.
Stain Magnification 425
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While specialised histological techniques have been described for
examining nervous tissue, these are often time consuming and difficult.
Hence they are not really suitable for regular examination of tissues
from a number of birds, such as occurs in a routine toxicological study.
Fortunately haematoxylin and eosin is one of the better stains for
showing axon degeneration. The lesion seen in the spinal cord is
swelling of the axon which often breaks up to form globules as seen in
longitudinal sections (Figure 4). Those seen in transverse sections vary
in the size and the amount of degenerating axon that they contain, this
being dependent upon where the axon was sectioned. The lesions tend to
be solid eosinophilic masses, occasionally slightly basophilic and
sometimes foamy and faintly eosinophilic. In the peripheral nerve the
lesions are not so obvious, there is not so much swelling and where the
axon has degenerated it usually forms several small spherical eosinophilic
masses which do not differentiate well from the surrounding tissue
(Figure 1). When ataxia is present, histological lesions should always be
found at some level in the cord and possibly in the peripheral nerves,
unless some other cause for the ataxia is demonstrated. However, with
compounds that are of low neurotoxic potential and others dosed at minimal
effective doses, clinical symptoms are often not seen but, when examined
histologically, typical bilateral lesions are found in a few of the birds.
In our laboratories, all birds are killed twenty-one days after
the final dose as it is considered that any ataxia developing after this
time is not usually caused by the compound being tested (Johnson 1975a).
In looking for lesions it is important that good sampling from the spinal
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FIGURE 4. Degenerative Lesion in the Ascending Tracts of the Lateral
Funiciilus at an Approximate Level of C7-8. i .S , H and F.
Stain Maqnification 4?r;
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cord is carried out. To do this we anaesthetise the hen with pento-
barbitone sodium given intravenously, and kill by exsanguination after
cutting the posterior vena cava. We then perfuse with warm 10% formal
saline (approximately 40°C) until the musculature stiffens. The spinal
column is dissected out, with as much muscle as possible trimmed away,
and divided into five segments as follows: cervical region into three
equal parts, the dorsal region and the lumbo-sacral region (Table 1).
Small nicks are made into the vertebral canal through the inter-vertebral
spaces and the whole column is then immersed in 10% formal saline for at
least seven days. The distal end of the sciatic nerve, together with
the proximal part of the peroneal and tibial nerves are dissected from
each leg, laid on card and fixed in formalin. After fixing, the spinal
cord is carefully dissected out from the spinal column and each section,
apart from the lumbo-sacral region and peripheral nerve, is sampled as
follows: a transverse section is taken from close to each end of each
segment and a longitudinal section cut through the lateral spinal tracts
from the centre of each segment. The lumbo-sacral region has two transverse
sections taken, the most distal one being through the glycogen body.
The peripheral nerve is sectioned longitudinally as distal as possible
(Table 2). Three sections from each level are then cut by microtome at
10 ym with 15-20 ym between such each section. These sections are stained
with haematoxylin and eosin (Ziegler 1944).
The routine examination for delayed neurotoxicity produced by
organo-phosphorus esters in hens can easily be undertaken by using simple
clinical observations and fairly simple routine histological techniques.
By using these methods with tri-aryl compounds containing ortho-cresyl groups,
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Table 1. Vertebral Column of the Fowl
Region
Cervical
Dorsal (Thoracic)
Lumbo-sacral
Coccygeal
Number of Vertebrae
13 - 14
7 No. 2-5 anchylosed; 7 anchylosed to
1 limbo-sacral
14 All anchylosed
6-7 No. 1 anchylosed to lumbo-sacral
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Table 2. Sampling Levels for Section
Region
Transverse Sections
Cervical
Dorsal
Lumbo-sacral
Approximate Level Sections are Taken
1, 4, 6, 9, 10, 13.
2, 6
1, 5
Long i tudi na 1 Secti ons
Cervical
Dorsal
Peripheral Nerve
2-3, 7-8, 11-12.
3-4
Proximal end of the tibial and peroneal
nerves.
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we have been able to pick up esters showing the whole range of neurotoxicity
and where alternative biochemical methods have been used these have
confirmed our findings (Johnson 1975b). However, with many pesticides
organo-phosphates difficulties do arise in keeping the animal alive through
the initial crisis caused by the depression of cholinesterase. Oximes
and atropine are usually given to try to prevent death and the more
severe symptoms. Thus the neurotoxicity of some of these compounds with
very severe anticholinesterase activity may not be important unless it
can be shown that repeated exposure to a small non-fatal dose can produce
lesions of neurotoxicity.
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Summary
A brief description of the neuropathology of delayed neuro-
toxicity due to organo-phosphate esters in the hen is given together
with a method of sampling and examination that is suitable for routine
examinations involving several birds.
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References
Barnes, J. M. and Denz, F. A. "Demyelination with organo-phosphorus
(1953) compounds" J. Pathol. Bacteriol 65. 597.
Bidstrup, P. L., Bonnell, J. A. and Beckett, A. G. "Paralysis following
(1953) poisoning by a new organic phosphorus insecticide (Mipafox).
Report on two cases". Brit. Med. J. 1. 1068.
Bischoff, A. "Ultrastructure of tri-orthocresyl phosphate poisoning in
(1970) the chicken. II Studies on spinal cord alterations". Acta
Neuropathol. (Berl.) 15, 142.
Bondy, H. F., Field, E. J., Worden, A. N. and Hughes, J. P. W. "A study
(1960) on the acute toxicity of the tri-aryl phosphates used as plasticizers",
Br. J. Ind. Med. 17. 190.
Cavanagh, J. B. "Peripheral neuropathy caused by toxic agents". CRC
(1973) Grit. Rev. Toxicol. 2. 365.
Christian, M. "Paralysis in the sow" given at the Lancashire Veterinary
(1975) Association. A.G.M.
Glees, P. "A morphological and neurological analysis of neurotoxicity
(1967) illustrated by tri-cresyl phosphate intoxication in the chick".
Pro. Eur.-Soc. Study Drug Toxicity. Int. Cong. Series No. 118,
Neurotoxicity of Drugs, Excerpta Med. Found. Amsterdam 8. 136.
Namba, T., Nolta, C. G., Jackrel, J. and Grob, D. "Poisoning due to
(1971) organophosphorus insecticides. Acute and chronic manifestations."
Am. J. Med. 50. 475.
Smith, M. I. and Elvove, E. "Pharmacological and chemical studies of the
(1930) cause of so called Ginger Paralysis; Preliminary report."
Pub. Health Rep. 45. 1703.
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Smith, M. I. and Lillie, R. D. "The histopathology of tri-ortho-cresyl
(1931) phosphate poisoning." Arch. Neurol. Psychiatry 26, 976.
Smith, M. I., Engel, E. W. and Stohlman, E. F. "Further studies on the
(1932) pharmacology of certain phenol esters with special reference
to the relation of chemical constitution and physiologic action."
NIH Bull. 160, 111.
Vij, S. and Kanagasuntheram, R. "Effect of Tri-o-cresyl phosphate poisoning
(1972) on sensory nerve terminations of slow loris." Acta. neuropath.
(Berl.) 20, 150.
Ziegler, E. E. "Haematoxylin-Eosin tissue stain. An improved, rapid and
(1944) uniform technique." Arch. Path. 37, 68.
Janyck, H. H. and Glees, P. "Chromatolysing spinal neurones in the chick
(1966) following tri-cresyl phosphate poisoning". Acta Neuropathol.
(Berl.) 6, 303.
Johnson, M. K. "The delayed neuropathy caused by some organo-phosphorus
(1975a) esters; Mechanism and challenge." CRC Crit. Rev. Toxicol. 3,
282.
Johnson, M. K. Personal communication.
1975b
Johnson, M. K. and Barnes, J. M. "Age and sensitivity of chicks to the
(1970) delayed neurotoxic effects of some organo-phosphorus compounds."
Biochem. Pharmacol. 19, 3045.
Jungherr, E. L. "The neuroanatomy of the domestic fowl (Gallus domesticus)"
(1969) Avian Pis., Special issue April.
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Krishnamusti, A., Kanagasuntheram, R. and Vij, S. "The effect of ToCP
(1972) Poisoning on the pacinian corpuscles in the slow loris,"
Acta. neuropath. Berl.) 22, 345.
Le Quesne, P. M. "Toxic neuropathy" in Modern Trends in Neurology.
(1975) Vol. 6 Williams, D. Ed., Butterworth, London
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PERSISTENT EFFECTS OF ORGANOPHOSPHATE EXPOSURE AS EVIDENCED
BY ELECTROENCEPHALOGRAPHIC MEASUREMENTS
Frank H. Duffy, MD
James L. Burchfiel, PhD, and Van M. Sim, MD
Introduction
Organo-phosphate (OP) compounds are known to have potent effects
upon the nervous system of animal and man. They were first introduced
as chemical warfare agents during World War II (nerve gas) and are now
among the more common pest control agents (insecticides). Their extreme
toxicity appears to result from a persistent anticholinesterase action
at both the peripheral neuro-muscular junction and the central choli-
nergic synapse. OP compounds are still used clinically in the treatment
of glaucoma and myasthenia gravis where such a persistent action may be
advantageous.
Central nervous system (CNS) symptoms of acute OP exposure range
from irritability and difficulty concentrating at low doses to overt
convulsions at higher doses. Acute behavioral effects are usually
accompanied by marked changes in the electroencephalogram (EEG) consis-
ting of desynchronization and increased fast activity (beta) at lower
doses and spike-wave discharges at higher levels.
Low level sustained administration of OP compounds produces more
subtle but definite effects. There may be difficulty sustaining atten-
tion associated with slowing of intellectual and motor processes. Feel-
ings of being generally slowed down, confused, agitated, and tense are
reported. Grobe et al reported the additional symptoms of insomnia,
excessive dreaming, emotional lability, increased libido, paraesthesias,
visual hallucination, and tremor in subjects given daily injection of
o
diisopropyl fluorophosphate (DFP).
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Grishon and Shaw reported that of sixteen patients with prolonged low
level OP exposure, eight had memory difficulty, seven were severely
depressed, five demonstrated schizophrenic reactions, and four had
unstable emotional states. These lasted up to one year following cessa-
tion of the exposure. Similar, but less severe, long-termed sequelae
were reported by Tabershaw and Cooper. Rowntree et al suggested that
OP exposure may precipitate or worsen symptomatology in patients with
preexisting psychiatric problems.
Not as well studied are the long term effects of single or multi-
ple but spaced exposures to OP compounds. Rapid treatment of an acute
exposure with an anticholinergic compound (atropine) and a cholines-
terase activator (2-PAM CL) is usually effective in treating all but the
most severe exposures. It is generally believed that if the secondary
effects of hypoxia can be avoided that symptoms and signs do not persist
beyond the immediate exposure period. In most instances EEG abnormali-
ties have been reported to disappear within two weeks of acute exposure
267
or at termination of low level sustained exposure. ' ' However, other
studies indicate that the EEG effects of insecticide poisoning may
Q Q
persist for many weeks or months. Metcalf and Holmes suggested that
OP exposure may lead to chronic EEG changes. They examined EEGs from a
group of industrial workers with a past history of insecticide poisoning,
but with no recent exposures, and reported a high Incidence of abnormal
low-to-medium voltage slow-wave activity in the theta range (4-6 Hz).
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;'ne present study was undertaken lo search for and statistical iy
verify ionu tarm effe.Xs of OP exposure upon the primate brain as mea-
sured by dwnu.es in ELG activity. Tne OP, sarin, was chosen for study
as it was the Or agent with the highest Incidence of exposure *n the
-i .-,
Metes-' ai;a br.iie-:, . tud.v' ' (personal communica Jon). Sarin (GB; !so-
propyiine£h>;p-!Ci'iphonof]uor1dflLy;, is not used commercially. It "is ordi-
narily Uussi. ;-=u &=> (3 "nerve gas,,' however, it shares a similar Jiemi-
cal ;, tructure and pnai maco'logical action with more common OP insecti-
cides such as meiathion and parathion. Sarin differs from most commer-
cail OP compounds in two major aspects. First, it is fluorinateds
and, second- i*. is considerably more potent.
A two part study was uncV*taken, flrsjt, rhesus monkeys were
exposed tc sarin, EEGs fakfr before and ore year after exposure were
compared for f^rh a'-iiial, "he number of exposed animals showing signi--
fleant FCG changes a^ one year was compared to the number of unexposed
(control) monkeys showing such changes (if any). Second, the EEGs of
a peculation of industrial workers with histories of exposure to sarin
we>"e compared to a control poojlat^on, (report in preparation).
-------
Methods
A) Monkey Study
Twenty-two rhesus monkeys of both sexes, weighing 2.5-4.0 Kg
were used in this study. Dura! EEG electrodes were placed (under ge-
neral anaesthesia and asceptic conditions) over the frontal, central,
occipital and temporal cortices, bilaterally. Insulated leads from the
electrodes were soldered to two 9-pin Winchester-type miniature connec-
tors which were cemented to the skull with dental acrylic. Additional
acrylic was used to form a durable, protective cap over the skull cover-
ing all leads and electrodes.
Sarin for injection was prepared by dilution of an aliquot of
purified drug (stored at-55°F) with normal saline, Biological effective-
ness of this solution was determined routinely in rabbits and was never
used more than 2 hr after dilution. Chemical analysis of random samples
indicated that sarin was always 90-95°/0 pure.
Sarin was administered according to one of two schedules. In one,
animals received a single "large dose" of the drug: 5 ug/kg of sarin
i.v. In the other, animals received multiple "small doses": a series of
10 injections of 1 yg/kg of sarin, im, given at intervals 1 week apart.
Hereafter these will be referred to respectively as the "large-dose" and
"small-dose" schedules. These dose levels were arrived at through pilot
studies in which the attempt was made to find dose levels which would
approximate, respectively, (1) a serious but sublethal exposure showing
all the signs of poisoning and (2) a series of subclinical but near-
threshold exposures showing few, if any, signs of overt poisoning.
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At the large-dose level all animals had generalized seizures. To
prevent the possibility of secondary anoxic brain damage, animals were
paralyzed with gallamirie triethiodiHe, arid artificially respired. Sarin
was given by slow iv injection 0ve*» 3 min. Animals w*»e allowed to
recover from paralysis and breathe spontaneously when their EEG
revealed a cessation of seizure activity. The average total length
of paralysis for experimental animals was about 2.5 hr and never more
than 4 hr. A group of control animals was similarly paralyzed and
received an injection of an equal volume of drug diluent. The length of
paralysis for controls was matched as nearly as possible to that of the
experimentals. Paralysis was not necessary for the animals receiving
the multiple small doses.
Animals were randomly assigned to one of two drug-treatment groups:
(1) single, large-dose sarin; (2) multiple, small-dose sarin. There
were three animals in each treatment group. These groups were matched
with groups of control animals, Controls were subjected to the same
injection schedules,
Each animal in the study was subjected to a series of EEG recording
sessions. Prior to drug administration, three separate control sessions
were performed on different days. For the single, large-dose animals, a
recording was taken ?4 hr after administration. For the multiple,
small-dose animals, a recording was taken 24 hr after administration of
the last injection in the series. Subsequently, the EEGs of all animals
were periodically monitored to check the integrity of the recording
'106 -
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system and CNS activity. One year after exposure an additional three
recordings were performed for all animals on three separate days. During
the 1-yr interval the animals were maintained in separate cages in a
temperature-controlled environment; they received no other drugs.
During a recording session the animal sat in a primate-restraining
chair, with a mechanical head restraint, inside a closed, electrically
shielded chamber. EEGs were recorded on seven-channel FM magnetic tape
(AMPLEX FR-1300) via calibrated outputs of a Grass Model 78 polygraph
with a bandpass of 0.580 Hz. An individual recording session consisted
of the following runs: (1) Awake, alert in light, an epoch of at least
2-min duration in which the animal sat in a lighted chamber; alertness
was maintained by intermittent auditory distraction; (2) Awake, alert in
darkness, same as (1), but in darkness; (3) drowsy, an epoch of 15-30
min duration in which the animal sat in the darkened chamber and silence
was maintained.
For each run, recordings were obtained from the cortical leads in
the following bipolar pairings: frontal-central, central-occipital, and
occipital-temporal.
Spectral analysis of the EEG data was accomplished on a POP-12
computer (Digital Equipment Corp.) using the comprehensive SIGSYS-12
software system (Agrippa Data System-Boston). Signals were played back
from FM tape through a Khrone-Hite filter set to bandpass 0.5-50 Hz (24
db per octave) and analog-to-digltal converted by the computer at a rate
of 256 Hz. Fast-Fourier transforms (FFTs) were performed, without
smoothing, on 4-sec epochs of EEG (Computed on 1024 points over the
frequency range 0-128 Hz with a resolution of 1/4 Hz). After F'
-107-
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computation, the square root was taken of each frequency component to
yield a voltage spectrum. Epochs containing excessive eye blinks,
movement artifact, electrode artifact, or muscle activity were not
analyzed. Consecutive epochs were averaged to yield a spectrum repre-
senting 1 min of EEG; i.e., the spectra of 15 4-second epochs were
averaged. Two 1-min spectra were computed for each state in a recording
session (i.e., awake in light, awake in darkness, and drowsy). The 2-
min epoch of EEG most representative of drowsiness was selected by
visual inpection of the polygraph tracing obtained in parallel with the
analog tape recording. The selection of the drowsy epoch was made by an
experienced electroencephalographer (EEGer), based on the monkey EEG
12
criteria of Reite et al. The EEGer was unaware of a monkey's exposure
history at the time of selection.
For each spectrum, a calculation was made of the percentage of
total energy present in each of the classical EEG frequency bands de-
fined as follows: delta=0.5-3.75 Hz, theta=3.75-7.75 Hz, alpha=7.75-
13.0 Hz, beta 1=13-22 Hz, and beta 2=22-50 Hz. The band from 50 to 128
Hz was not analyzed.
For the final analysis, total spectra and frequency bands were
averaged respectively for the three pre-drug sessions, the 24-hr session,
and the three 1-yr sessions. This was done for each lead and each
state. Thus, a final datum consisted of an average spectrum and fre-
quency bands representing EEG activity from one electrode derivation,
-108-
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for one state, and for one set of sessions. Since two spectra were
derived for each state and lead in a recording session, the values of N
for the final averages were 6 for pre-drug, 2 for 24 hr, and 6 for 1 yr.
In computing the final averages, standard deviation was also calculated.
Statistical analysis was performed in a two-step procedure. First
a longitudinal comparison was made for each animal using a two-tailed
Student's t test. The average spectrum and frequency bands derived from
the control sessions (for a given lead and state) were compared first
with that derived from the session at 24 hr post-drug and next with that
from the sessions at 1 yr. For the average spectrum, the t test was
applied at each point. As might be expected, this produced a large
number of seemingly random "significant" differences. (Theoretically,
one would expect an average of 5% of the t tests to yield t values with
a probability <0.05 merely by chance.) Such differences at individual
points throughout the spectrum proved very difficult to interpret. We
felt that any criteria we might establish to determine when statistically
significant points constituted a physiologically significant difference
might suffer from subjective bias. For this reason we chose to base our
results upon analysis of integrated frequency bands. Therefore, at the
conclusion of the first step of statistical analysis, we had determined
for each animal which frequency ranges were different by t test at 24 hr
post-drug and at 1 yr post-drug as compared to the pre-drug control -
In the longitudinal comparison, each animal served as his own con-
-109-
-------
trol; data from animals within the same experimental group were not
pooled for comparison. The next question, therefore, was which dif-
ferences, if any, revealed by t test were consistently and exclusively
seen among the drug-treated animals as opposed to control animals. This
question was answered by the second step of the statistical analysis,
Fisher's test. For this analysis the question was asked, how many
drug-treated animals showed a significant change (by t test) in a parti-
cular frequency band and how many did not? The same question was asked
of the control animals. From these answers a 2 x 2 contingency table
was constructed and the exact probability of obtaining the resultant
distribution in the table, or one more extreme, by chance alone was
calculated. If this probability were <0.05, then it was concluded that
a significant change in frequency distribution of the EEG had occurred
in a drug-treated group as compared to the controls. Statistical analy-
sis among the various groups of control animals showed no consistent
differences; therefore, for Fisher's test all control animals were
considered as a single group.
B) Human Study
Three separate but related investigations were undertaken: (1)
spectral analysis of tape recorded EEG, (2) visual inspection of routine
clinical EEG, and (3) visual inspection of an all night sleep EEG.
-110-
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Seventy-seven industrial workers with histories of accidental
exposure to sarin were studied. Thirty-seven of these had 3 or more
exposures within the 6 years preceeding the study but no subject was
exposed during the year just preceeding the study. The exposure criteria
were defined as a history of exposure, with resultant clinical signs and
symptoms, and a demonstrable reduction in red blood cell cholinesterase
to a level at least 25% of the individual's pre-exposure baseline.
Thirty-eight workers from the same geographical area with no history
of OP exposure (chosen so that their age distribution, sex, race, and
socioeconomic background matched that of the exposed subjects)-were
used as controls. Until it became necessary to group the subjects for
statistical purposes, no one involved in data analysis knew the personal
or exposure history of the individual subjects.
Subjects were studied in a standard EEG laboratory. EEG activity
was recorded on an Ampex FR 1300 tapedeck via a Grass model 78
polygraph with a bandpass of 0.1-35 Hz. A simultaneous strip chart was
made to monitor for artifact and to assess level of consciousness.
Subjects were studied in the following states:
1. Eyes open - EO (2 minutes) - awake with eyes open in a lighted
environment and frequently alerted by an auditory stimulus.
2. Eyes closed - EC (2 minutes) - same as 1, but with instructions
to close eyes.
3. Drowsy - D (15 minutes) - drowsiness and sleep. Room lights
-111-
-------
were turned off and extraneous sounds were masked with white noise. One
minute of this 15-minute period was chosen to represent drowsiness on
the basis of the EEG strip chart. Drowsiness was defined as the state
beginning after the break-up of alpha activity and ending with the early
signs of vertex waves and spindling.
4, Hyperventilation - HV (5 minutes) - 5 minutes of deep breathing
with eyes open and room lights on. The third minute was chosen to
represent the HV state.
5. Recovery from hyperventilation - PHV (3 minutes) - the third
minute following cessation of HV was used to represent the PHV state
(eyes open and room lights on).
Off-line data analysis was performed on the EEG activity recorded
from the following bipolar linkages (10-20 international electrode
placement): 0 -C (occipital-central), F~-C~(frontal-central), and T.-T,-
(temporal). Signals were bandpassed between 0.5 and 32 Hz (24 db per
octave) at the time of computer analysis. Frequency spectra were formed
for each subject, state, and linkage. Spectral analysis was performed
in the same manner as for the monkey study with the exception that the
analysis was limited to the 0-30 Hz range. The band from 30-128 Hz was
not analyzed. For each spectrum the energy in ten 3-Hz segments was
calculated from 0.5 to 30 Hz. These segments may be related to the
standard EEG bands as follows:
Delta = 0.5-3 Hz, slow theta = 3-6 Hz, fast theta = 6-9 Hz, alpha =
-112-
-------
9-12 Hz, slow beta - 12-15 Hz, beta = 15-30 Hz.
To facilitate analysis the energy within each frequency band was
averaged across all subjects within a group for each state and linkage.
Variance and standard deviations were also calculated. Comparisons were
made between groups (for each state and linkage) on the basis of three
statistical tests: Student's t-test, Mann-Whitney U-test, and chi-
square analysis. For a test to be considered valid, it had to achieve
the 0.05 two-tailed level of significance. The two non-parametric tests
(U test and chi-square) were undertaken due to the relatively large
variance encountered in the various groups which we felt might interfere
with the parametric .t-test. The u-test is sensitive to the relative
rank of each member of a group but not its absolute value. Chi-square
analysis was performed as follows: the combined mean of the two groups
being compared was calculated; next, the number of members in each group
above and below the mean was placed in a 2 x 2 contigency table and chi
square was calculated. Due to the larger numbers in each cell (>5) we
did not have to use the Fisher test to analyze the 2 x 2 table.
Routine clinical EEGs were performed using a Grass Model 78 EEG
with a complete 24 electrode montage. Both monopolar and bipolar link-
ages were examined. In addition to the standard clinical "reading", the
EEGer designated as "normal" or "abnormal" 17 separate parameters (see
Table 6). The numbers of exposed workers or control workers within the
normal-abnormal category for each parameter were placed in a 2 x 2
-113-
-------
contengency table. Statistical comparison was made by Fisher's test as
individual entries were often below 5 in number. At the time of eval-
uating the EE6, the EEGer was unaware of the subject's exposure history.
Computer spectral analysis and visual readings were done at the Seizure
Unit, Childrens Hospital Medical Center at Harvard Medical School,
Boston.
All night sleep EEGs were performed during a subject's usual sleep
period. Three bipolar linkages of EEG (Oz-Cz, F3-C3, F4-C4), an electro-
myogram (EMG) of neck muscle tone, an electrooculogram (EOG) for eye
motion potentials, an electrocardiogram (EKG), and a bed movement trans-
ducer were recorded on the paper strip chart of a Grass Model 78 poly-
graph. These charts were then sent to the Sleep Laboratory of the Univ.
of Maryland (Dr. Althea Wagman) in Baltimore for scoring. Absolute and
percent times in rapid eye movement (REM), stage 1, stage 2, stage 3,
and stage 4 sleep were calculated for each subject. Comparisons between
groups were made by t-test and u test.
For all analyses, control subjects were compared to all the exposed
subjects (CxE analysis) and again to the SVB-group of subjects with
multiple (3 or more) exposures (CxM analysis). All data analysis was
performed at the Seizure Unit with a PDP-12 computer using the parametric
and non-parametric statistical packages of SIGSYS-12.
-114-
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RESULTS
A) Monkey Study
Long term (one year) alterations 1n EEG frequency spectra were
observed for both the single, large-dose and multiple, swall-dose
exposed monkeys.
Large dose. The single large dose of sarfn (5 yg/kg) produced a
persistent Increase 1n the relative amount of voltage 1n the beta fre-
quency bands (13-22 and/or 22-50 Hz) of the occipital-temporal (0-T) EEG
derivation. Figure 1 shows the data for a control monkey and Figure 2
for a large dose sarfn animal.
Figures 1 and 2 Illustrate some general findings which are appli-
cable to all the drug treatment groups. First of all, 1t can be seen in
comparing the control records from these animals that a considerable
degree of Individual variability exists in the EEG frequency distribu-
tions. However, for a given animal, the spectra tended to be reasonably
constant from day to day. The control animal shown in Fig. 1 is typical
of the data from the 10 untreated animals. There are no significant
differences among the spectra over the 1-yr period of the study. Be-
cause of the much greater degree of variability from animal to animal
than from day to day for the same animal we adopted the longitudinal
method of statistical analysis described in the methods section.
Similar degrees of variability were seen among the drug-treated animals.
However, the key factor among the large-dose sarln animals was that,
regardless of what other alternations in the spectra might occur, there
-115-
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CONTROL
BAND
D
T
A
Bl
B2
PERCENT
ENERGY S.E.
23.54
18.35
22.22
18.47
20.86
0.62
0.62
1.00
0.77
1.00
24 HOURS
1 YEAR
BAND
BAND
D
T
A
Bl
B2
PERCENT
ENERGY S.E.
22.74
17.02
22.26
20.68
20.86
1.87
0.42
1.56
0.53
0.42
PERCENT
ENERGY S.E.
23.46
18.00
21.95
20.14
19.94
1.57
0.75
0.95
0.97
0.72
t VALUE
vs CONTROL
0.56
1.16
0.02
1.55
0.01
t VALUE
vs CONTROL
0.05
0.36
0.20
1.34
0.75
FIGURE 1. Example of average voltage spectra and frequency bands for
one control animal. Occipital-temporal EEG derivation in the
state of awake in darkness. For the spectra, the ordinate
scale is in microvolts and the abscissa scale is in hertz.
Frequency bands give percentages of total voltage (0-50 Hz)
over the following ranges: D = delta (0.5-3.75 Hz); T = theta
(3.75-7.75 Hz); A = alpha (7.75-13.0 Hz); Bl = beta 1 (13-22 Hz);
B2 = beta 2 (22-50 Hz). The t value is the result of Student's
t test between the indicated band and the corresponding band of
the control spectrum., P £0.05*. P < 0.01**
-116-
-------
CONTROL
BAND
PERCENT
ENERGY S.E.
19.89
19.32
32.50
18.72
13.25
0.92
1.05
0.94
0.78
0.41
24 HOURS
BAND
D
T
A
Bl
B2
PERCENT
ENERGY S.E.
26.80 2.12
19.05 0.65
19.39 0.33
19.65 1.44
18.26 0.22
t VALUE
vs CONTROL
3.53**
0.14
7.64**
0.59
6.68**
1 YEAR
BAND
D
T
A
Bl
B2
PERCENT
ENERGY
21.05
17.16
30.36
18.64
16.29
S. E.
0.29
0.73
0.93
0.58
1.23
t VALUE
vs CONTROL
0.99
1.51
1.55
0.07
2.77*
FIGURE 2. An example of voltage spectra and frequency bands for a
large-dose sarin animal. See legend, Figure 1. Note
persistence of increased beta 2 at 1 year.
-117-
-------
was a consistent increase of relative voltage 1n the beta frequency
bands. For instance, in Fig. 2 at 24 hr post-drug there was a signi-
ficant increase in beta 2 which was still present 1 yr later. But, in
addition, there was a significant increase in Delta and a decrease in
Alpha at 24 hr. Neither of these changes persisted for one year,
however.
A summary of the results of t test analysis for increased relative
beta activity in the 0-T frequency spectrum is given in Table 1. Each
entry in the table is the proportion of animals (control of treated)
which showed a significant increase in beta activity by t test. For a
given post-drug session and recording state, the proportion of treated
animals showing an increase and the proportion of controls showing an
increase form a 2 x 2 contingency table. Beneath each pair of propor-
tions, in parentheses, is given the Fisher exact probability that such a
distribution could arise by chance alone (Where there is obviously no
difference, the probability is omitted). It can be seen that the in-
crease in relative beta voltage was most prominent in the state of awake
in darkness. At 24 hr after administration, all three drug-treated
animals showed a statistically significant increase in beta activity by
t test, whereas only two of ten controls showed such an increase. The
probability of this distribution arising by chance is 0.035. At 1 yr
after sarin exposure two of three treated monkeys still showed a rela-
tive increase in beta activity; none of the controls showed any increase.
This distribution has a chance probability of 0.0385.
-118-
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TABLE 1
SINGLE LARGE-DOSE ADMINISTRATION OF SARIN, OCCIPITAL-
TEMPORAL EEGa
Recording state
24 hr
Sarin
Control
1 yr
Sarin
Control
Awake in
light
0/3
0/10
2/3
2/10
Awake in
darkness
3/3
2/10 .
(p=0.035)D
2/3
0/10 .
(p=0.038)D
Drowsy
2/3
1/10 ,
(p=0. 105)D
3/3
2/10 ,
(p=0.035)D
Sarin 5 ug/kg, iv. Proportion of animals showing
a significant increase by t test in relative beta voltage.
A percentage of total voltage of spectrum in the frequency
bands 13-22 or 22-50 Hz of the EEG frequency spectrum at
24 hr and 1 yr post-drug.
Exact probability of distribution of drug-treated and
control animals occurring by chance (2x2 Fisher Test).
-119-
-------
For the drowsy state there was a significant increase in beta
activity for treated animals at 1 yr (all sarin animals showing an
increase as opposed to two of ten controls, p=0.035). However, at 24 hr
this change was not quite statistically valid (an increase in two of
three treated animals and in one of ten controls, p=0.105).
In the state of awake in light, there was no significant increase
of relative voltage in the beta bands. However, there would appear to
be a trend toward increased beta at 1 yr after exposure (two of three
treated animals showed a significant increase, while eight of ten con-
trols did not, p=0.188).
Small dose. The animals receiving the multiple small doses of
sarin (1 pg/kg once a week for 10 weeks) showed a persistent relative
increase in 0-T beta activity similar to that seen with the large-dose
animals. The results of t test analysis for increased beta activity in
all small-dose animals is given in Table 2. At 24 hr after the last
dose of sarin, all the treated animals showed a significant increase in
beta for both the states of awake in darkness and drowsy. These increases
were still present at 1 yr after exposure.
In addition to the alternation in 0-T EE6 spectra, the small-dose
animals also showed increases in relative beta activity in the frontal-
central (F-C) EEG (Table 2). Such F-C alterations were not seen with the
single large dose of sarin. The increase in F-C beta also differed from
the increase in 0-T beta in that it occurred only in the spectra at 1 yr
post-drug; at 24 hr there was no significant increase (Table 2).
-120-
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TABLE 2
MULTIPLE SMALL-DOSE ADMINISTRATION OF SARIN, OCCIPI-
TAL-TEMPORAL (0-T) AND FRONTAL-CENTRAL (F-C) EEGa.
Recording state
A.O-TEEG
24 hr
Sarin
Control
1 yr
Sarin
Control
B.F-CEEG
24 hr
Sarin
Control
1 yr
Sari n
Control
Awake in
light
0/3
0/10
2/3
2/10
0/3
1/8
3/3
1/8 ,
(p=0.024)D
Awake in
darkness
3/3
2/10 .
(p=0.035)D
2/3
0/10 h
(p=0.0385)D
0/3
2/8
3/3
1/8 ,
(p=0.024)D
drowsy
3/3
1/10 ,
(p=0.014)D
3/3
2/10 .
(p=0.035)D
1/3
1/8
3/3
1/8 ,
(p=0.024)D
Sarin, 10 injections of 1 ug/kg, im, given at 1-week
intervals. Also see footnote a, Table 1.
See Footnote b, Table 1.
-121-
-------
In contrast, the spectra at 24 hr after termination of the small-dose
schedule showed a consistent trend for all treated animals toward in-
creases in relative voltage in the delta and theta bands and decreases
in higher frequencies, particularly alpha. With time this frequency
distribution reversed such that beta was increased and delta and theta
decreased. The increase in relative beta at 1 yr was seen in all three
recording states (Table 2). In Table 3, all monkeys exposed to sarin
are grouped and compared to the control animals for the 0-T lead, beta
range, one year after exposure. Note the high significance level for
both states.
B) Human Study
(1) Spectral Analysis
Tables 4 and 5 show the results of the CxE and CxM analyses, res-
pectively. Note in Table 4 that that there were significant decreases
in delta in the Eyes Open (EO) state for the T3-T5 (temporal) and F3-C3
(frontal) linkages (P<.02). However, no other comparisons reached
significance. This raises the important question of how many individual
comparisons must be statistically significant to have physiological
significance. For example, the T3-T5 linkage in Table 4 has 50 compari-
sons - 5 states and 10 bands. At the .02 level, .02 x 50 or 1 compari-
son might have shown significance by chance. For the entire CxE
analysis of Table 4, .02 x 150 or 3 comparisons might show significance
by chance. As only one comparison in T3T5, one in F3C3, and two in the
entire CxE analysis were statistically significant we must conclude
-122-
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-124-
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Table 4
Results of the spectral analysis between the control subjects (c)
and all exposed subjects (E). The ten columns represent the 10
integrated frequency bands from 0.5 to 30 kg in 3 Hz steps. The
rows represent different recording locations and different clinical
states. E0=eyes open alert, EC=eyes closed alert, D=Drowsy, HV=
hyperventilation, pHV=after hyperventilation, T3T5=temperal, F3-C3=
frontal-central, 02-C2=occipital-central.
Each table entry represents the significance level of the CxE
comparison for that frequency band, lead, and state. The absence
of an entry indicates failure to reach the .05, 2 tailed, level of
significance. T=significant t-test, U=significant U-test, C=signifi-
cant chi-square test. The arrow indicates the direction of the effect
with reference to change from the C group mean.
In this table, eyes open, temporal, delta (0.5-3Hz) was signifi-
cantly decreased by t-test in T3-T5 (P <_ .02) and by t-test, U-test and
chi-square in F3-C3. No other comparisons were significant.
-125-
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-126-
-------
Table 5
Results of the spectral analysis between the control subjects
and the maximally exposed subjects (M). Same convention as
table 1. All significance levels are 0.05 2 tailed or better. Note
the clustering in the beta range (13-30Hz) of T3T5 and OZ-CZ and
the fact that all significant levels represent an increase of beta
activity in the maximum exposure group.
-127-
-------
that these may have arisen by chance alone and, therefore, have no
definite physiological significance.
On the other hand, the C x M analysis of Table 5 shows a different
picture. There are nine significant comparisons (P <_• 0.05} for T3T5,
three for F3C3 and twelve for OZCZ. For each linkage we might expect
2,5 comparisons significant by chance at the .05 level. For the entire
analysis we might expect 7.5 by chance. Clearly, we have exceeded the
chance level for all linkages (especially T3T5 and OZCZ) and the entire
C x M analysis (24>7.5). The physiological significance is increased by
the clear tendency for the results to cluster in the beta range and to
represent an i_n_cre_ase_ of beta in every case.
Thus, the results of the spectral analysis show (a) no differences
between the control and entire exposed populations (b) extremely signi-
ficant differences between the control and maximum exposure populations,
(c) a tendency of the differences to cluster in the beta range and
represent an increase in beta within the exposed group.
(2) Visual Inspection of Clinical EEG
Table 6 shows the results of the C x E and C x M analyses for the
parameters designated normal or abnormal by the EEGer. Note that there
is a significant decrease in background EEG voltage at the .015 level
and a decrease in alpha amplitude at the .04-.06 level for both analyses.
Thus the results of the visual suspection of EEG suggest a decrease
of background voltage and alpha.
-128
-------
TABLE 6
HUMAN EEG READINGS-EXACT PROBABILITY TEST
c
E
CxE
M
CxM
C
E
CxE
M
CxM
WAKING
20/13
39/54
.1331
13/14
.1306
PAROXYSMAL
ACTIVITY
24/9
45/28
.0971
17/10
.1591
DROWSY
18/15
37/36
.1556
14/13
.1987
FOCAL
ACTIVITY
32/1
70/3
.4132
24/3
.1979
SLEEP
23/10
38/26
.1093
14/9
.1781
ASYMMET
RIES
30/3
72/1
.0802
26/1
.3021
HYPER-
VENT1 N
22/11
43/30
.1299
15/12
.1439
DELTA
30/3
58/15
.1331
21/6
.1607
POST
HYPER-
VENTL'N
23/10
53/20
.1736
19/8
.2221
THETA
21/11
42/31
.1286
14/13
.1196
STROBE
30/3
61/12
.1547
22/5
.1722
ALPHA
27/6
48/25
.0458*
17/10
.0625
ENTIRE
RECORD
16/17
31/42
.1413
10/17
.1409
BETA
24/9
56/17
.1712
17/10
.1591
BKGND
VOLTAGE
32/1
51/14
.0110*"
17/6
. 01 44 **
DOCTORS
GUESS
19/14
36/37
.1228
13/14
.1584
BKGND
RHYTHMS
25/2
50/6
.2890
16/4
.1584
NORMAL/ABNORMAL
-129-
-------
Table 6
Results of the Fishers exact probability test on the Human
EEG data obtained by visual inspection of EE6. Numbers above repre-
sent the numbers of normally or abnormally classified subjects in
each group. C = control group, E = entire exposed group, M = maxi-
mally exposed group, CxE = comparison between control and exposure
group by Fisher's test C x M = same as C x E but for maximally ex-
posed group. Exact probability values are shown. Asterisks show
significant values. "Doctors Guess" refers for an attempt by the
EEGer to guess whether the record represented a control or exposed
subject. Significant probability levels are indicated by asterisks.
-130-
-------
(3) Visual Inspection of Sleep EEC
Table 7 shows the results of the C x E and C x M analyses of the
all night sleep EEG records. No significant differences are seen for
the four stages of slow wave sleep or total sleep time. On the other
hand, absolute time spent 1n REM 1s significantly Increased for the CxM
analysis (.02 level) and the % time 1n REM sleep Increased for the CxE
(P < .02) and C x M (P < .01) analysis.
The results of the all nlte sleep study show that the entire ex-
posed group showed an Increase in REM sleep and that this was of slight-
ly greater magnitude for the maximum exposure group.
Discussion
The results of the monkey study Indicate that a single symptomatic
exposure or a series of subcl1n1cal exposures to the OP, sarln, can
alter the frequency spectrum of the EEG for up to 1 year. The major
effect was a persistent increase 1n beta activity (13-50Hz) seen mainly
in the occlpltal-temperal (0-T) EEG during the states "awake in darkness"
and "drowsy". The small dose schedule also showed an increase in
the frontal-central (F-C) derivation.
The beta Increases in the large-dose animals were evident at 24 hr
post-drug and persisted. We feel that the large-dose beta increase is
the result of a persistence of acute actions of the drugs. Following
the slow iv injection of sarln at the large-dose level, the EEG decreased
in voltage and increased in frequency producing a record of almost
-131-
-------
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continuous low-voltage beta activity. This was followed by irregular
bursts of high-voltage sharp waves in the 6-10 Hz range intermixed with
the high-frequency background. Finally, there was almost continuous
seizure activity consisting of runs of spikes, polyspikes and spike-
waves separated by periods of relative electrical silence. This "burst-
suppression" pattern continued for about 1 hr in most animals and then
was replaced by a post-ictal record consisting of high-voltage slow
waves in the 1-3 Hz range. By 24 hr, the EEG was still dominated by
slow-wave activity, but the initial high frequency beta activity was
still present (see Fig 2). Our results would indicate, therefore, that
of the EEG alterations acutely produced by a single convulsive dose of
sarin, spike activity and slow waves are spontaneously reversible, but
the increased beta activity persists for up to a year,
A significant result of this study was the effect produced by mul-
tiple small-dose exposures. The dose levels used in these treatment
schedules did not produce the overt signs of poisoning seen with the
single, large doses. However, the multiple small-dose exposures pro-
duced the same type and degree of EEG alteration as the single large-
dose exposures. Indeed, for sarin, multiple small doses appeared to
produce more change than the large dose. This is surprising in view of
the fact that the multiple small-dose group showed no overt clinical
manifestations related to drug administration, whereas the sinqie largr-
dose group suffered a severe CNS insult (prolonged electrical convuls^ors'l.
It is to be noted that the total dose received by the multiple small-dose
-133-
-------
group was roughly twice that of the single-dose group. Thus long-term
EE6 changes may be more a function of cumulative drug dose than of
clinical manifestations at time of exposure. Note, also, that the small
dose schedule had a more widespread long term effect, as 1t involved
both 0-T and F-C derivations; the large dose schedule affected only the
0-T derivation.
The results of the human study demonstrate that workers with
histories of exposure to sarin have waking and sleep EEGs that differ
significantly from those of workers of no exposure history. These
differences were:
1. Spectral analysis - increased beta activity in the temporal and
occipital-central leads of the maximum exposure group (P <_ 0.05).
2. Visual reading of standard EEG - decreased background vol-
tage (P <_ 0.015), and decreased amounts of Alpha (P <_ 0.05).
3. Sleep EEG - increased amounts of REM sleep (P <. 0.01-0.02)
Spectral analysis failed to show statistically significant dif-
ferences between the control and exposure (CxE) groups, but did demon-
strate highly significant differences between the control and the maxi-
mum exposure (CxM) group. These differences tended to cluster in one
frequency range (beta, 12-30 Hz) and were found in both the temporal
(T3-T5) and occipital-central (OZ-CZ) electrode montages. This is the
very same frequency band and brain area where persistent EEG changes
were found for monkeys one year after sarin exposure.
-134-
-------
Statistical analyses of the visual EEG readings showed signifi-
cantly decreased background voltage and lower alpha amplitudes, without
a significant increase in background delta or theta slowing. This sug-
gested general background "desynchronlzatlon."
All night sleep EEGs demonstrated significant increases in the
percent of time in REM sleep for both experimental (P <_ 0.02) and maximum
(P <_ 0.01) groups. Absolute REM time was also Increased but just for
the maximum group. No other sleep stage, taken as percent or absolute,
showed significant differences. The fact that an increase 1n REM was
significant for the exposure group only when expressed as a percent
suggested that there must have been a slight decrease 1n slow wave sleep
for individuals in this group, not enough to be significant by itself,
but enough to increase the percent REM. Percent REM for all three of
our groups (Including the controls) fell below published normals for
14
middle aged men. This may be due to either or both of the following
reasons. First, REM 1s usually decreased during the first night of
sleep in a laboratory situation (first night effect). Second, most
REM tends to occur in the final third of a night's sleep. All our
subjects were awakened at 0530, approximately 30 to 45 minutes before
their usual waking time. Thus, most of this lost sleep may have been
REM. 0ur results confirm the finding of Metcalf and Holmes, who also
reported an increase of REM sleep in subjects with histories of OP
exposure.
-135-
-------
What Is the significance of these findings? De-synchronization in
general may be a sign of simple decrease in neuronal synchronization
(normal alert state), or it may signify functional neuronal depression
(hypothermia, drug effect), or neuronal depopulation (aging, dementia).
Following intravenous administration of a convulsive dose of Sar;n,
monkey EEGs show a brief period of desynchronization prior to the onset
of background slowing and seizure discharges. At subconvulsive dose
levels, desynchronization may persist for some time.
Increased beta activity may be due to any number of factors. Beta
is increased by drowsiness, sleep deprivation, and certain drugs. As
the
can be seen from Figure 2, a marked increase in/beta content of monkey
EEG follows intravenous sarin injection. Lower, subclinical doses of
sann produce longer periods of increased beta. This increase in beta
is quite consistent with the findings of desynchronization on visual
inspection. By definition, desynchronization usually includes an increase
in fust activity (beta) in addition to a decrease in background voltage
and a decrease in slow (delta, theta) activity. Fink has reviewed the
relationship between EEG and behavioral effects of drugs. Significantly,
many of the chronic symptoms reported for insecticide exposure are found
to L>O acutely associated with EEG patterns of increased beta activity
3:;d desynchronization. These include irritability, anxiety, memory
defects and thought disorder. Drugs producing acute patterns of increased
beta activity comparable to the chronic pattern induced by sarin include
••136-
-------
some of the most potent psychoactive agents such as amphetamine, be-
megride, dimethyltryptamine, LSD-25, mescaline and psilocybin.
The significance of increased amounts of REM sleep is unclear. Of
the 20 or more pathological states associated with clinical derangements
of sleep, as described by Williams, almost all involved no change or
decrease in REM. In a review of REM sleep pharmacology, Hartman
listed over 20 drugs causing a depression in REM (including barbiturates,
common tranquilizers, mood elevators, alcohol, and amphetamine), but
only four causing an increase in REM (Reserpine, L-tryptophane, 5-OH-
tryptophane, and LSD). Thus, it is unusual to find drugs that increase
REM. A significant exception to this are OP compounds. One patient
examined by the authors following accidental exposure to the OP, soman
(GD), demonstrated a strikingly desynchronized EEC and complaints of a
dream-like state of visual hallucinations associated with an inability
to fall deeper asleep (Unpublished Observations). Flurries of rapid eye
movements were noted during the 1st day after exposure period, although
no EEGs were obtained.
There have been several reports recently of a cholinergic link
18 21
involved in REM sleep mechanisms. Administration of cholinesterase
inhibitors elicited REM sleep in both intact animals and brain stem
20
preparations. It has been postulated that cholinergic mechanisms are
involved in REM initiation.18'19
What are the pharmacological mechanisms underlying the persistent
effect of OP exposure? Acute exposure to OP compounds produces pro-
-137-
-------
found CNS effects ranging from subjective changes 1n behavior at low
doses, to convulsions at higher doses. Furthermore, prolonged lower
level exposures may produce a psychiatric syndrome resembling schiz-
ophrenia. All these are believed to be secondary to reductions in
CNS cholinesterase levels. Accordingly, recovery is believed to par-
allel return of CNS cholinesterase to normal. On the other hand,
monkeys exposed to sarin showed increased temporal lobe beta one year
after sarin exposure. Studies of tissue cholinesterase in monkeys
usually demonstrate a return to normal within three months of exposure.22
Furthermore, the abnormalities demonstrated in the human study were
found in subjects with normal plasma and red blood cell cholinesterase
levels. Thus, evidence of CNS abnormality attributable to OP compounds
can be found in monkey and man at a time when tissue cholinesterase
levels are probably within normal limits.
One explanation may be that the low tissue cholinesterase levels at
the time of exposure induce long-term changes in synaptic morphology or
biochemical organization which do not completely reverse when tissue
cholinesterase levels return to normal. Evidence for or against this is
lacking.
Another explanation may be that sarin has actions beside that of a
potent anticholinesterase. Similar compounds, triorthocresylphosphate
(TOCP)'or difluorphosphate (DFP) are known to produce peripheral neuro-
pathies of delayed onset. Sarin can also produce such disturbances in
-138-
-------
23 24
peripheral nerves but only in the chicken, not in primates. ' Some
of the simpler OP compounds also produce central lesions of delayed
onset similar to the peripheral lesions. Sarin is not thought to do
this, but complete pathological studies are not available. The monkeys
which showed persistent EEC changes following sarin exposure were not
studied in this regard. Thus, one might speculate that the persistent
CNS effects in primates may not be related to anticholinesterase proper-
ties, but to some direct lesion of axons or myelin.
In either case, since the long-term findings (desynchronization,
increased beta, and increased REM) are also seen as part of the acute
exposure syndrome, the pathogenesis of the long-term effects probably
represents aji unexpected and possibly irreversible extension of the
primary drug effect.
Regardless of the pathogenic mechanisms, results of the current
study confirm the ability of OP compounds to induce persistent abnor-
malities in the electrical activity of the primate brain. Whether these
long-term neurophysiological changes can be directly related to reported
behavioral abnormalities is not known. On the other hand, the long-term
behavioral changes and long-term electrophysiological abnormalities
provide parallel evidence that OP exposure, even at subclinical levels,
can produce long term change in the brain function of monkey and man.
-139-
-------
References
1. Bowers, M.B., Goodman, E. and Sim, V.M. (1964). Some behavioral
changes in man following anticholinesterase administration. J.
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(1947). The administration of diisopropyl fluorophosphate (DFP)
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5. Rowntree, D.W., Nevin, S., and Wilson, A. (1950). The effects of
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7. Santolucito, J.A. and Morrison, G. (1971). EEG of rhesus monkeys
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8. Hoogendam, I., Versteeg, J.P.J. and De Vlieger, M. (1962).
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9. Dille, J.R. and Smith, P.M. (1964), Central nervous system effects
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neurological alterations In humans with organophosphate exposure.
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11. Burchflel, J.L., Duffy, F.H., and Sim, V.M. (1976). Persistent
effects of sarin and dleldrln upon the primate electroencephalogram.
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12. Reite, M.L., Rhodes, J.M., Kavan, E. and Adey, W.R. (1965). Normal
sleep patterns in macaque monkey. Arch. Neurol. 12:133-144
13. Fisher, R.A. (1934). Statistical Methods for Research Workers.
Oliver and Boyd, London.
14. Williams, R.L., Karacan, I.K. and Hursch, C.J. (1974). EEG
of human sleep. John Wiley and Sons, Inc., New York
15. Koo1, K.A. (1971). Fundamentals of electroencephalography.
Harper and Row, New York
16. Fink, M. (1969). EEG and human psychopharmacology. Annu. Rev.
Pharmacol. 9_: 241-258
17. Hartmann, E. (1968). On the pharmacology of dreaming sleep (the
D. state) J. Nerv. Ment. Dis. 146:165-173
18. Magherini, P.C., Pompelano, 0., and Thoden.U. (1972). Cholinergic
mechanisms related to REM sleep. I. Rhythmic activity of the
vestibulo-oculowater system induced by an anticholinesterase make
decerebrate cat. Arch. Ital. Biol. 110:234-259
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19. Thoden, U., Magherini, P.C., and Pompeiano, 0. (1972). Cholinergic
mechanisms related to REM sleep. II. Effects of an anticholinesterase
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20. Matsuzaki, M., Obada, Y., and Shuto, S. (1968). Cholinergic
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"neurotoxic esterase" and the development of delayed neurotoxicity
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-142-
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DR. F. COULSON: Dr. Bradley, I certainly appreciated very much
your presentation, and I think you've made several important points.
You've made it very clear, that there is a dose level, with all of
these compounds, where there is no effect on the central or peripheral
nervous system. Would you expect then that you could have a transitional
dose response until you reach an effect you can see as a pathologic
change.
DR. W. A. BRADLEY: Yes. I think there probably is a no-effect level.
But with a lot of these compounds, using repeated dosing, you get an
accumulative effect. This has particularly been seen with TOCP, as shown
by the early work done by Smith and his co-workers. They definitely
showed there was a no-effect level to a single dose, but when several
lower doses were given, delayed neurotoxicity could be produced. There
was an even lower dose, quite low down, where even with repeated dosing
they could get no effect. So the things to look for are: one, the effect
after a single dose, and two, the effect after repeated dosing. I think
with most of these you probably would find eventually that there is a
no-effect level, certainly as far as histological signs go.
DR. A. COELHO: This question is directed to Dr. Duffy. I have a
few very short questions dealing with your control group. First of all,
were your animals,your Macaca mulatta, feral or laboratory raised and how
large with respect to body weight were they?
DR. F. H. DUFFY: They were laboratory raised about three to four
kilograms and they received a dose of 5 milligrams per kilogram.
DR. COELHO: That's a fairly young juvenile. Were there both males
and females included:
DR. F. H. DUFFY: Yes.
-143-
-------
DR. A. COELHO: Were your controls real controls, or were they
basically reference groups in terms of EEG values? In other words,
did they receive sham injections?
DR. F. H. DUFFY: Yes, they were studied before and after sham
injections. They were also paralyzed and respired at the time of sham
drug administrations.
DR. N. ALDRIDGE: This certainly isn't my field and I'm not sure
whether I understood what you told us. What is the variation in the
controls of the monkey experiments over a very long period of time when
measurements are taken daily? And the second question, if you give monkeys
Sarin, where you get the changes in the REM sleep pattern? Now does this
follow with respect to time after dosage?
DR. F. H. DUFFY: Sarin usually produces an increase in REM sleep lasting
about 48 hours following a single dose, then the increase tails off.
There may be compensatory decrease in REM sleep for a few days thereafter.
Regarding the controls, we did not take just one measure. We studied them
for six separate recording sessions before the drug. The one year value
was based upon, six or eight separate recording sessions. If there had
been wide variation, this would have reflected itself as a large variance,
which would have rendered the T-test or any statistical comparison, non-valid.
The results were quite consistent, internally, among themselves.
Also, any change that came up in both the controls and the experimentals
would not have been significant.
DR. J. P. FRAWLEY: Under what set of circumstances were these 79
individuals exposed to Sarin? Was it laboratory exposure? Occupational
exposure? Human volunteer experiment?
-144-
-------
DR. F. H. DUFFY: These were accidental industrial exposures,
and their exposure was documented and treated in a clinical facility.
They were not volunteers. This is not a part of the Army's medical
volunteer program. These were industrial accidents.
DR. J. P. FRAWLEY: So in the year since Sarin exposure, because
of their occupation they might have been accidentally exposed to other
chemicals.
DR. F. H. DUFFY: They were very carefully screened, for exposure
to other toxic substances. We intentionally screened out any of those
who had histories of exposure to chlorinated hydrocarbon or exposures
to substances at the nearby Shell Chemical Company plant. No subjects
had documented exposures to any compound in the year prior to this study.
Our screening procedure left us with only a small number of subjects with
uncomplicated Sarin exposure. But of course, they could have had gardening
as their hobby, for all we know. This, of course, would apply to the
control group, too.
DR. J. P. FRAWLEY: But were they working in a chemical environment?
DR. F. H. DUFFY: Everyone was! They were all employed in the same
installation where Sarin was maintained. But the exposed group had
histories of exposure and of access. The controls were never in a position
where they would be exposed, barring a major catastrophe.
DR. J. GEHRICK: I'm interested in your experience with not being
able to classify these people as normal or abnormal. Yet, you found
with other computer processing of the data, there were some things that
were different. Would you care to reflect on what that means, in terms
of clinical abnormality or possible future clinical abnormalities?
-145-
-------
DR. F. H. DUFFY: Well, just to clarify again what we found on
the human study, a population of exposed individuals differed from a
population of control individuals. I'm sure if we had taken 79 people
off the street in Denver, and 79 people off the street, living at sea
level, we would have found differences. But we could not have attributed
them to any one factor. So the fact that we see differences in the
two groups is significant insofar only as we were carefully able to
control other important variables. We tried to think of every possible
item. We felt that the only difference between the control and exposure
groups was the history of exposure to Sarin. The results simply say the
populations are different. It is our presumption that this is due to
Sarin exposure.
I couldn't take a single individual and say anything by visual
inspection of his EEG recording or by looking at his spectrum. Now, it's
very hard to go from a population study to an individual basis. But
because we could do it with the monkeys and because we had the clue that
there was something there on the population level, we thought it would
then be interesting to then get much more involved in this question.
In other words, perhaps the only reason you can't take an individual
and say he belongs to the control or exposured group is due to the limitation
of your brain to make very subtle, multitudinous correlations. There
are very many factors to consider. Maybe the mind cannot appreciate
trends that are really there. That is where pattern recognition can be
helpful.
Here is how it is done. You develop 20 factors for each individual
of two populations. You feed data on all these individuals and their
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associated factors into a computer and you define which of the
people are exposed, and which are controls. The computer comes back and
says, for example, that only parameters one, five, nine and thirteen
are necessary to differentiate the two groups. So, then you measure
those factors for each individual, and you keep refining your parameters
until you discover, rather empirically, by interacting with the recognition
program, which parameters are of no use and which are good. It didn't
take very long before you were able, with only a ten percent error,
to take an individual and say from which group he came,
DR. J. GEHRICK: What do the things that you found, mainly increased
REM and increased fast activity in the exposed group, mean to you as
a clinician?
DR. F. H. DUFFY: That is a very hard question to answer. Low
background amplitude, increased fast activity and increased REM time
are the classic signs of a drug effect. I believe our study suggests
that individuals exposed to Sarinmay be under its pharmacological
influence for a much longer time than previously anticipated. However,
one cannot say that these individuals are brain damaged. Rather, our study
seems to provide support for material in the literature which suggests a
long term effect following organophosphate exposure. Maybe there is something
to the behavioral complaints that people come up with. Neither that nor
any other specific correlation was made in this study.
DR. W. N. ALDRIDGE: I'm very interested in pursuing this question
a bit more because of the implications of what you say. Do I understand
you that your technique involves taking these two populations of humans,
one of which has a history of exposure to Sarin, and the other has a history
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of working in the same factory with no exposure — that is the situation?
DR. F. H. DUFFY: That's correct.
DR. W. N. ALDRIDGE: You then have put into the computer your records,
and have been enabled by selection to get the situation that the groups
are always different.
DR. F. H. DUFFY: That's correct.
DR. W. N. ALDRIDGE: Now, what is the next stage? Do you then
have to go out and get another group of totally non-exposed people and
show that they always go into the control? You see, you're starting
with a kind of fixed population, two groups that you've selected by one
means, where you defined the parameters. But, they're restricted to
these groups. Do you now have to go out and take somebody who works
in a different occupational environment and see that they always
correspond to the controls?
DR. F. H. DUFFY: You see, we come to this question again. It isn't
enough to simply show a difference between two populations. This has
been a fault with many statistical studies. You have to be sure that
the only thing that these populations differ by is this measure and a
critical factor in their history. For example, the population that we're
concerned with is largely Mexican-American, in the age group between forty
and sixty, peaking at 45, living at 5,000 foot altitude. It would not
be correct to compare this population to a white middle class population
of both sexes living at sea level. Any statistically significant results
we might find could be attributed to a variable host of factors.
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DR. F. H. DUFFY: Just living an upper middle class life and not
being exposed to toxins of the slum may in fact be a more potent factor
in causing differences than exposure to Sarin. So, yes, if we had a
larger population unexposed to Sarin, but with everything else being the
same, that would make a much better control group but great care should
be taken to ensure a useful control population* We have saved another
group of exposed individuals, to retest this whole hypothesis. But we're
saving them for inclusion in the pattern recognition study.
DR. W. J. HAYES: I have two totally unrelated questions; Were
these exposures associated with illness? And, if so, how much or what
degree of illness? Has this exact kind of study you've made, ever been
done with people who might be expected to be concerned, for whatever
reason, about their situation? In other words, these people who had
been exposed the year before knew perfectly well they'd been exposed and
the controls knew perfectly well they were off in another part and were
no more exposed there, than you and I are here. So there had to be some
awareness on the parts of the individuals, even though they were removed
and exposed.
DR. F. H. DUFFY: I don't know how one could control that. This
approach has been used for other EEG based studies and other population
studies. Yes, there's no question that anxiety over the results of this
study might have affected the results. However, most of the individuals
did not seem to demonstrate anxiety, but were rather annoyed at having to
have this break in their routine. They were simply told that a new
regulation required EEG studies of all employees.
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To be entered in the study they had to have clinical signs of
exposure and more than a 25% reduction in their baseline cholinesterase
levels. This included everything from severe emergencies to treat-
and-go-home situations. We did not have a large enough population to
look at the changes versus the degree of exposure to quantify it and
look for trends.
DR. H. FEINMAN: In your animal studies, I believe there were some
statistically significant differences in the alpha, beta or delta or theta
wave patterns between treated and untreated animals that were still
demonstrable a year after exposure. Was any necropsy done at the time,
to determine whether these differences, on histopathological examination,
correlated with any demonstrated pathological lesions.
DR. F. DUFFY: There were no consistent changes in delta, alpha and
theta, only beta. There were just as many controls who would turn up
with increased or decreased alpha as experimentals that turned up with
decreases. In other words, there were random changes as well as the
specific ones. Post mortums were done on all animals but special stains
to look for demyelination were not used. There were no visible histological
lesions found with routine stains.
QUESTION: Did you imply that REM sleep is not a good thing?
DR. F. H. DUFFY: Not at all, it's necessary in the appropriate amount.
QUESTION: I would hope that anything that could prolong REM sleep
might be good for us. It's the best part of sleeping. It's when we make
all our nice dreams, have hallucinations about all sorts of things, and also
have persecutions. I didn't want the audience to get the impression that
we had interfered with this good part of sleeping.
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DR. F. H. DUFFY: I agree, REM sleep is the most enjoyable part.
It is to be recalled however that during REM sleep you have the highest
incidence of heart attacks and epileptic seizures, if these are the
underlying disease. REM sleep is necessary and desirable in appropriate
quantities. Too much or too little REM might be harmful and might also
signal underlying disease.
DR. V. SIM: I might state that the animals also underwent extensive
psychological behavioral testing and were followed over a period of two
years with all the parameters that were available at that particular time.
There was nothing that we were able to pick up in the behavioral patterns
or the psychometric testing that gave us any lead as to what we saw,
as Dr. Duffy has discussed.
DR. F. H. DUFFY: These animals were tested at 24 hours after
exposure and they showed nothing on delayed performance test using
Wisconsin General Test Apparatus. We didn't think to test them a year
later because we didn't think we would be getting into the question of
delayed onset.
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ORGANOPHOSPHATE EXPOSURE FROM INDUSTRIAL USAGE, ELECTRONEUROMYOGRAPHY
IN OCCUPATIONAL MEDICAL SUPERVISION OF EXPOSED WORKERS
K. W. Jager, M.D.
In the first part of my paper I would like to deal specifically
with the problems of monitoring and controlling the exposure of industrial
workers to potentially neurotoxic compounds, which is a responsibility
of the industrial medical doctor. In the second part I propose to
review the literature concerned with neurotoxic effects, especially
in relation to organophosphate insecticides.
Electromyography (EMG) and electroneuromyography (ENMG) provide
sensitive and objective methods of detecting impairment of nerve and
muscle function. With these methods a.o. the conduction velocity of nerves
and the action-potential of muscles resulting from a controlled stimulus
on the nerve can be measured. Abnormal reaction patterns of the muscle
may be recognized and it is possible to measure reflex times.
In the past, EMG and ENMG were not suitable for health supervision
in occupationally exposed workers because needle electrodes had to be
introduced in the tissues near the nerve and in the muscle. More recently
newer methods have been developed in which surface electrodes can be used
and which give accurate and reproducible results. In general, a supramaximal
stimulus - this is a stimulus to the nerve which will evoke a maximum
reaction in all the muscle fibres - is given via electrodes placed on
the skin over the ulnar nerve, and the action potential and action pattern
of the adductor muscle of the thumb is recorded.
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This method was first used in the supervision of occupationally
exposed organophosphate workers, when the late Professor Andrew Wilson
of the University of Liverpool and Dr. D. V. Roberts of the same University
introduced it in the Industrial Medical Department of Shell's chemical
works in Rotterdam in The Netherlands. Groups of organochlorine and
organophosphate workers as well as a control group were then examined
and significant deviations from the norm were found in approximately 50%
2
of the workers exposed to organophosphate insecticides. This was the
more remarkable, because regular determination of the blood cholinesterase
activity in this group of workers, when compared with the results of
their individual pre-exposure value, only sporadically indicated possible
overexposure.
Since that time, a close scientific cooperation has been established
between Dr. Roberts of Liverpool and Dr. Ottevanger, who now supervises
the health of our pesticide workers, with the result that our organophosphate
insecticide workers have been checked regularly by means of ENMG in
addition to their normal medical examination and the regular determination
of blood cholinesterase activities.
On further periodical supervisory examinations we continued to find
in some of the exposed workers the same deviation from the norm, which
is qualitatively related to the intensity and the duration of the exposure,
neither of which can be measured directly. Initially, an effect of
base materials and intermediates could not be excluded, but later, similar,
though less pronounced changes were found in sprayers in the field, who
3 4
were exposed to the endproduct ' only.
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The changes found are the following:
1. A decrease in the voltage of the action potential of
the muscle. In non-exposed controls most values would
be between 11 and 12 mV, in the exposed workers the
range would be wider and values could occasionally be
as low as 4 mV.
2. A change in the pattern of the recorded electromyogram,
indicating that not all muscle fibers react at the same
time to the supramaximal stimulation.
3. A decrease in the conduction velocity of the nerve fibers.
In our organophosphate workers, all of whom have a full medical
examination at least once a year, we have never seen any signs and
symptoms or overt disease which could be related to the above findings.
One should, however, realize, that the complaints one might expect to
hear would be very unspecific, such as fatigue, weakness, etc.
Further results of the continued work of the same group have been,
or will shortly be published.206'207
The following actions are presently taken when workers show the
above-mentioned aberrations from the norm. The exposure situation is
closely scrutinized and where possible improved. Workers who show
progressively decreasing voltages on successive examinations would be
transferred to other work where they have no further contact with
organophosphate insecticides, until the reason for the effects has been
detected and corrected.
The workers in the plant are fully aware of the nature of this
monitoring procedure and welcome it. Experience over the last few years
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has shown, that the rapid feed-back of information derived from ENMG-studies
helps to maintain a high standard of industrial hygiene, and as a result
of this the incidence and severity of the abnormalities may be kept as
low as possible.
Transferred workers are followed up, and so far all abnormalities
have returned to normal. This generally takes a few weeks or months,
but sometimes up to a year.
This slow recovery and the changes found are in accordance with a
diagnosis of subclinical neuropathy. Neuropathy, no matter how slight,
must be regarded as a more direct parameter of human health than a quickly
reversible cholinesterase depression. The evaluation of the significance
of neuropathic changes is primarily a medical function.
Recently, very thorough reviews on peripheral neuropathy caused by
chemical agents '' have been published, as well as reviews of the
application of neurophysiological methods ' to occupational medicine.
It is well known, of course, that members of the group of organo-
phosphorus compounds can cause cholinesterase depression or may have
neurotoxic effects, and in some cases may do both. Currently one is
inclined to believe that members of this group of compounds in principle
have both capacities, but that they vary in the expression of these
effects.
In view of the well established neurotoxic effects of TOCP and some
other 0.P.-compounds, all organophosphate insecticides are tested for
possible neurotoxicity by a laboratory test on hens. However, we do not
really know how relevant the "hentest" is as regards the potential of
long term occupational exposure as a possible cause of neuropathy,
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From the medico-toxicological as well as from animal experimentation
literature complete studies covering all aspects of clinical signs and
symptoms, neurophysiology and pathology are scarce. They are, however,
an increasing number of papers relating to the subject which mention,
separately, clinical disease, neurophysiological abnormalities or
pathological changes, such as peripheral neuropathy, myopathy, visual
disturbances, cochlear and vestibular effects and behavioral changes in
relation to intentional or accidental, and sometimes occupational exposure
Q 7? 1 "\7 171
to a wide range of organophosphate insecticides and carbamates. ' '
pi C
Clear cases of peripheral neuropathy following intentional or accidental
overexposure have been published. Cases of clinical paralysis in man
have been described, even with "safe" products, such as malathion.
With a few compounds animal experiments have shown that both partial
demyelination and an effect on the axon cylinders occur. At times, these
changes have even progressed to necrosis in the muscle.
The neuropathy in experimental animals and man is by no means limited
to organophosphate insecticides. It can also be produced by a wide range
of chemicals, such as
T0cp 73-81, 172-174
- lead82'86' 175-177
- acrylamide87-88' 178-]79
carbondisulphide89' 18°-183
ethanol,
methyl butylketone and its metabolite
2,5-hexanedione90-92' 184
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•JQC
trichloroethylene,
hydrocarbon solvents such as hexane, heptane
93 11? 18fi
and low-boiling gasoline fractions '
triethyltin187'188
- methylmercury113-116' 189'193
194 195
arsenic compounds '
- hexachlorophene117' 118' 196'198
no i ?n
dithiocarbamatesuy'' u
- diphenyl121'199
- thalidomide122'200'201
but also by therapeutic agents, such as,
123
diazepam
chloroquine, and
dapsone
126 127 128
Diabetes, diphtheria, allergic reactions, as well as
129
compression and/or crushing of nerves and vibration by vibrating
130 202 203
tools ' ' may cause a similar neuropathy. Deficiency diseases,
208 209
certain virus diseases and idiopathic polyneuritis uo»tu:7 could be
added to this list.
There may be some qualitative differences in the effects caused
by these chemicals and other factors: some affect the sensory system
more than motor nerves, some have more central effects than peripheral
ones. The end-result, however, appears to be similar for all compounds
mentioned, although the quantitative aspects are of course quite different.
The list of substances and factors mentioned is by no means complete,
nor is the list of references at the end of this paper.
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In this group of chemicals lead is of special interest because
inthis case cause and effect are very well documented and quantified,
OO QC
bot from animal experiments and from human clinical experience.
It t known that in the past many cases of lead-paralysis have occurred.
Moretver, the ENMG changes found with lead closely resemble our data
with irganophosphate insecticide workers. From animal experiments we
know hat long-term oral exposure to lead may result in segmental
82
demyeTnation and axonal degeneration with typical ENMG-changes.
The sane ENMG-changes are found in workers with known heavy lead exposure
84
and a hstory of past lead poisoning. Similar ENMG-changes may be
present, albeit to a lesser extent in lead-workers who have never exceeded
acceptabe lead levels in blood and who have never had a lead-poisoning,
OC
nor any signs and symptoms.
With the organophosphate insecticides we appear to have a similar
situation; the parameter commonly used in the supervision of the workers,
the blood cholinesterase determination, is a measure for exposure. ENMG-
changes, hcwever, may occur unrelated to depressions in cholinesterase
activity and certainly there is a phase-difference between the two, for it
may take dajs or weeks for ENMG-changes to appear following over-exposure,
and it may take months to recover from it. As with lead, these changes may
develop in a situation which was believed to be "under control" using
parameters of exposure only. The WHO in a recent Technical Report on
"Early detection of health impairment in occupational exposure to health
hazards" also refers to this finding. The Pesticide Subcommittee of
the Permanent Commission on Occupational Health in its Workshop on
217
Cholinesterase in Cambridge in September 1975 came to a similar conclusion.
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The significance of these findings in relation to health is at
218
present still unknown. Our experience in man and other evidence
from the literature indicate, that there is probably a no-effect level.
Data obtained on control-groups demonstrate that the general population
is not affected.
Our data on organophosphate workers indicate that the changes are
reversible, but the literature shows that in more advanced stages which
are accompanied by clinical signs and symptoms this may not always be so.
Reversibility possibly depends upon both the magnitude and the duration
of exposure. Of course, with many organophosphate insecticides, exposure
is limited by the acute toxicity of the product, except in those cases
where tolerance has developed and the usual muscarinic warning signs and
symptoms may be diminished or absent.
We do not yet know at what point the individual or statistical trend
becomes of significance to health. We feel, however, that it is prudent
to assume, that if exposures, severe enough to cause such changes, are
allowed to continue unchecked, it might in the end lead to clinical
signs and symptoms.
Cholinesterase determinations and electroneuromyography both have
their own place in the medical supervision of organophosphate workers.
Which one gives the first indication of overexposure probably depends
on the timing of the test in relation to the exposure and the degree and
duration of exposure. ENMG-changes may be completely unrelated to
cholinesterase depression. Another esterase may be involved, or there
may be a completely different mechanism, as is possibly in the case of
other chemicals causin ENMG-changes.
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All this of course leads to the conclusion that further work is
required:
1. To elucidate the mechanism by which organophosphates cause these
ENMG-changes,
2. on the dose-response relation as related to the time factor,
3. on the reversibility of the effect.
4. No-effect-levels have to be determined in subchronic and long term
animal tests, using ENMG techniques, as well as light- and
electronmicroscopy of nerve tissues.
Although there is reason for caution and concern, a final decision
on the significance and acceptability of the ENMG-changes mentioned
will only be possible when some of these questions have been answered.
At the present time ENMG-monitoring has a practical value in that
it may be used in industrial hygiene supervision, as a check on the
efficiency of protective and safety measures, and as another source of
information on which the industrial medical director can base his clinical
judgement.
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DR. M. K. JOHNSON: I was impressed by your telling us all the
details. I think many people who, such as myself, don't know too much
about industrial exposure would be grateful if all companies were as
straightforward in declaring what they found and how they dealt with it.
I would like to ask whether if the route of exposure, were dermal
rather than systemic, might that mean the possible effects could arise
from a local inhibition of acetylcholinesterase without much effect being
seen on blood cholinesterase? Also, is it possible within the whole
group of organophosphoruous esters that your company produces, whether
you can, in any sense, correlate bigger or smaller effects with individual
compounds? In other words, if you can do sort of a mini-structure/activity
survey. And, if one were to think that perhaps one is looking for a
target, and you consider whether these are effects on acetylcholinesterase,
or on neurotoxic esterase, or on some other target altogether, one means
of coming to some decision might be to consider workers exposed to carbamates.
Because, as my hypothesis stands and as the observations go, it would seem
impossible to produce a typical organophosphate neuropathy with a carbamate.
But you could still get chronic anti-cholinesterase effects.
DR. K. JAGER: Thank you, Dr. Johnson. First, with respect to the
route of exposure, it is of course very difficult to tell what is the
major route of exposure. In a general way, we have the feeling that the
main route of exposure, at least in our organophosphate plant, is dermal.
Maybe with the exception of those few cases where we manufactured DDVP.
In those cases there may have been to a certain extent an inhalation exposure.
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With regard to the oral route, -- well, you cannot completely exclude
it, of course. But we have taken all sorts of measures to exclude that
route. In our opinion, it's mainly the dermal route of exposure.
As to the various compounds, I must say that we have seen this effect
with all the organophosphates which we manufacture. We do not manufacture
them all at the same time. Rather it's done blockwise. So we can really
look at it and say, well, now it's this substance and now it's that substance.
We have had an occasion where we had a developmental carbamate, which
was not manufactured on an industrial scale. But on the laboratory
scale, where, in fact, we also had these effects. And it had a very prolonged
effect.
With the carbamates, we thought that it would be impossible to
supervise employees in a proper way with the normal cholinesterase
determination method which we were using the Michel method, and that we
would have to use the Ph-stat method. Using electroneuromyography, we
found changes, which we checked regularly. It took months and months for
the recordings to become normal again. They became normal, but only after
a long time.
DR. R. CLYNE: I'm rather intrigued by your mention of the fact that
there were some people who allegedly developed paralysis as a result of
exposure to malathion. Could you elaborate on that and give us some
documentation? I think Dr. Coulston, many years ago in British Guinea,
had an opportunity to study people who in attempting suicide had horrendous
exposure to malathion. It was my understanding that there was no evidence
for paralysis in any of these people.
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DR. K. JA6ER: Well, I do not know how good this publication is.
It's a fairly old publication in the American Journal of Medicine in
1958 cited in my list of references. It was a massive overexposure!
A physician who used malathion in his garden thought that spraying with
25% concentrate every day would be better than doing it in a normal way.
So he went on and on and he got a paralysis and he died in the end.
DR. R. CLYNE: I can only say that with the experience we've had
with that compound over many years, close to 25 right now, including
people who had horrendous exposure in the holds of liberty ships in the
James River, where they had no protective clothing of any kind and where
they sprayed and were sprayed with, in many instances, the concentrate,
for up to four months, the people suffered no effects at all. There was
no depression of cholinesterase nor effects noted with any gross test. I
must confess we did not do ENMGs or EMGs on them. But, there were no
findings at all in the nervous system such as you allude to.
DR. K. OAGER: I would agree with you that this is a very exceptional
case. Reading through the case histories it's a very exceptional case.
I would not normally expect this to occur with malathion. On the other
hand, when you say that even in massive prolonged exposure we do not see
cholinesterase depression, this may be right. But it could be that if
you look at an ENMG, you would find changes. As I have mentioned, and
it has been mentioned earlier today, it could well be that very toxic, or
highly toxic organophosphates or carbamates, by the very effect of their
high toxicity are used more carefully, and you would see less ENMG changes
with those. Whereas, with the less toxic materials, you're less careful,
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you get a higher exposure, and you may see more ENMG effect. Working
on the assumption that it's an unrelated effect.
DR. J. SANBORN: This comment will be brief, since Dr. Johnson
has already reported on the neurotoxicity of desbromoleptophos, a
photodegredation product of leptophos. We have found the same results
at the same levels and Dr. Roy Fukuto has repeated and confirmed these
findings. With leptophos oxon, we found the same thing. In addition,
desmethyl leptophos, at 200 milligrams per kilogram, does not seem to
be neurotoxic. Dr. Fukuto has also tested it at 500 mg/kg, and it's not
neurotoxic.
One further note, not related to neurotoxicity; chickens given
25 milligrams desbromoeptophos in a single oral dose in gelatin capsules
are still excreting material in the eggs, if they still lay eggs, at
about half a part per million in the eggs. This indicates the stability
of this photo-degradation product, which is also a contaminant of technical
leptophos.
DR. J. E. CASIDA: What is the acute LDcn value for desbromo
bU
leptophos?
DR. J. SANBORN: I have no idea but it's not particularly insecticidal
as far as I know.
DR. J. E. CASIDA: In other words, is the acute toxicity:
delayed neurotoxicity ratio for desbromoleptophos similar to that for
leptophos.
DR. M. K. JOHNSON: No! It's much easier to produce
the neurotoxic syndrome with much less cholinergic response. I give
atropine, and I give P-2S, but it's very easy to show neurotoxicity with
desbromoleptophos and less easy with leptophos.
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DR. J. E. CASIDA: I would like to introduce Dr. Abou-Donia who
was the first to report in the literature the delayed neurotoxicity
of leptophos. I'm sure his comments will be of considerable interest.
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DR. M. ABOU-DONIA
I don't think that I can add any more to the information that
we just heard from the excellent speakers today. I have only a few
minutes to present two year's worth of work. However, you might
consider my presentation as an application or an example of the lessons
that were taught today on delayed neurotoxicity.
D
Chemically leptophos (Phosvel) is 0-(4-bromo-2,5-dichlorophenyl)
0-methyl phosphonothioate. My attention was first focused on leptophos
in 1971, when it was implicated in the poisoning of about 1,300 water
buffalos in the Nile Delta. The clinical condition was permanent ataxia
followed by paralysis and death. Two other pesticides were sprayed in
n
that area: Cyolane [2-(diethoxyphosphinylimino)-l,3 dithiolane and
p
Hinosan [0-ethyl SS-diphenyl phosphorodithioate]. The signs of delayed
neurotoxicity observed in the field were reproduced experimentally in
water buffalos fed forage sprayed only with leptophos. Subsequently,
we established that leptophos caused delayed neurotoxic effects in fowls
10-13 days after the oral administration, intubation, of a single dose
in corn oil (Abou-Donia, et al. 1974). A dose of 160 mg/kg, resulted
in "no effect" while higher doses (180-3,000 mg/kg) caused delayed neuro-
toxic effect in some cases. The most noticeable sign was a disturbance
in the control of movement of the legs with a change in the gait. In each
bird the earliest sign was a weaving of the gait, apparently secondary to
leg unsteadiness. This evolved into a gross ataxia. As time passed, the
signs progressed: legs sprawling out in front, inability to stretch the
legs, difficulties in moving the legs, inability to stand, and down on the
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hocks most of the time. An occasional dragging of the legs was observed.
The weakness of the legs was manifested by the unwillingness of the birds
to walk. When made to do so, the dragging of the legs was very prominent.
Once ataxia appeared, the bird's decline was rapid, and paralysis occurred
15-23 days after the administration of leptophos. A rapid irregular head
tremor was present in some birds and was especially pronounced in paralyzed
birds. After paralysis, all birds showed respiratory and swallowing
difficulties. Recovery was never observed in any bird having developed
ataxia. The severity of the neurotoxic signs and the number of birds
that developed the condition were dose-dependent; but no matter how great
the dose, the latent period before onset of neurotoxicity was never less
than 8 days.
Later, in collaboration with Dr. S. H. Preissig, we carried out an
investigation into the nature of the neurological lesions produced in hens
administered three neurotoxic doses of leptophos; 200, 400 and 800 mg/kg in
corn oil and gelatin capsules (Abou-Donia and Preissig, 1976).
Signs of neurological dysfunction were present 8-14 days after oral
administration of a single dose of leptophos. In general, the onset of
ataxia and paralysis was not markedly affected by the insecticide carrier.
All leptophos-treated birds lost weight. During the latent period,
between the administration of leptophos and the onset of ataxia, there
was virtually no change in body weight. Significant loss occurred in
the hens between the onset of ataxia and paralysis. The loss in weight
sharply increased during the time between paralysis and death or sacrifice.
At the beginning of the experiment, the hens acted normally and consumed
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amounts of water and feed comparable to control birds. Anorexia was
noted after the onset of ataxia, which increased as signs of neurotoxicity
progressed to paralysis. Paralyzed hens gradually stopped eating.
The number and weight of eggs was decreased in most of the treated hens.
Figure 1 shows hematoxylin and eosin (H&E) with Luxol fast blue
(LFB) stained longitudinal sections of sciatic nerve exhibiting numerous
foci of swollen myelin sheaths and fragments myelin (x650). These changes
were present in the majority of birds.
In Figure 2 the loss of myelin is seen in the anterior columns of
sections from the lumbar spinal cord.
Figure 3 is a diagram representing the pattern of degeneration of
myelin and axons (Black area) in the central nervous system of hens
administered leptophos. The lateral ascending tracts are involved in the
medulla. These lateral as well as the ascending posterior tracts are
damaged in the cervical cord. Only early degeneration in the anterior
descending tracts is seen in the thoracic cord. The lumbar cord shows
severe involvement of this anterior pathway.
H&E sections of muscles from the majority of hens were not significantly
different from controls. NADH diaphorase stain revealed clearing of the
central portion of a large proportion of muscle fibers; this represented
dissolution mitochondria, sarcoplasmic reticulum, and other subcellular
organelles. Wing muscle from chicken 4 showed several foci of small,
angulated atrophic fibers.
Initially leptophos appeared to inhibit red blood cell acetyl
cholinesterase significantly. In general, there was a dose dependent
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,.,.,».
•ngMB^fc^y*|g!;^,''-«t , *^jtf^yf'fft
''%»»»,.= . •• . ' ' ' ','^t, \..' W
^r-
: &$&•&! ^^^^^'j^^^^'^^^SW^'^^ *$FflSjiij$&!t.-<';-
.,.. . ,„, . ,,.« :-.---
FIGURE 1.
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FIGURE 2.
-198-
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MEDULLA
CERVICAL SPINAL
CORD
THORACIC SPINAL
CORD
LUMBAR SPINAL
CORD
FIGURE 3.
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inhibition of red blood cell acetylcholinesterase. The inhibition
was greatest 1 day after the administration of the compound. Spontaneous
recovery of the enzyme seemed to begin soon after maximal inhibition.
Plasma cholinesterase activity of birds at all treatment levels was
significantly reduced at the beginning of the experiment. Again, an
increase in dose produced an increase in enzymatic inhibition. There
was an initial depression of enzymatic activity produced by all doses
one day after administration of leptophos. There was a considerable
amount of spontaneous recovery of the enzyme. A secondary depression
of plasma cholinesterase followed by a second recovery of this enzyme
was noted in all treated hens. This recovery was followed by a third
depression of plasma cholinesterase beginning at about day 20-27.
Plasma cholinesterase activity values remained significantly lower than
those of the control, thereafter.
Our latest study has confirmed our original findings that the
delayed neurotoxic effect reported for leptophos resembles that of other
neurotoxic organophosphorus compounds.
The ability of long term low-dose administration of leptophos to
produce delayed neurotoxicity in hens was investigated. Table 1 shows
that 6 dose levels of leptophos were administered daily: 0.5, 1, 2.5,
5, 10 and 20 mg/kg in gelatin capsules. Three hens were given empty
gelatin capsules and served as controls. The dosing continued until
ataxia developed but no longer than 60 days.
Administration of small subneurotoxic doses of leptophos ranging
between 1 and 20 mg/kg could build up and cause neurotoxic effects. At
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Table 1
Sequence of intoxication, onset of clinical signs and histological
changes in tissues from hens fed a daily single dose of leptophos3
Days of Administration
nums*"! f\r\
Hen
A
B
C
D
E
F
G
H
I
J
K
L
M
N
0
P
Q
R
Dose
20.00
20.00
20.00
10.00
10.00
10.00
5.00
5.00
5.00
2.50
2.50
2.50
1.00
1.00
1.00
0.50
0.50
0.50
Ataxia
29
22
29
39
39
32
5S
58
59
25
65
65
68
62
62
—
--
— ••
Paralysis
Onset
39
26
35
58
56
61
__
-_
-_
-_
--
...
--
-_
68
__
—
•••
Recovery
*»•
_-
__
102
112
102
__
--
--
_—
_-
--
_-
__
73
__
«
~~
Sacrifice
85b
50
77b
108
216
216
216
59^
123b
103
103
103
160
160
160
78
78
78
L/VI 1 U W 1 X/l 1
of
Intoxication
56
28
48
69
177
184
157
1
64
78
38
38
92
98
98
__
—
•••
Histological Changes
Sciatic
Nerve
4-
+
NE
-
.
-
-
NE
-
+/-
-
NE
NE
NE
-
NE
NE
"•
Spinal
Cord
++
++
NE
+
+
+
+/-
NE
*/-
•»-/-
-
NE
NE
NE
-
NE
NE
**
The following abbreviations were used: NE, tissues not examined; -, changes absent;
•»•/-, changes equivocal; +, changes present; ++, severe degeneration.
Hen died shortly before dissection.
-201-
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these levels the dose was almost completely cumulative. The dose
of 1 mg/kg was a "threshold dose" below which no effects were produced.
A "no effect" daily oral dose of 0.5 mg/kg was found, which produced
no signs of ataxia, no histological changes and no marked variation in
the weight and number of eggs laid during 60 days of feeding. Duration
of administration of subneurotoxic doses of leptophos varied inversely
with the size of the daily dose. Also, the "total dose" that caused
ataxia was proportional to the size of the daily ingested dose. However,
this was not merely a cumulative effect, since at lower daily doses
there was a decrease in the total dose required to produce ataxia. This
finding may be attributed to a more efficient absorption and/or metabolism
of divided doses than of a single dose. This is certainly true with
larger doses, where one single dose of 160 mg/kg of leptophos was
ineffective, but 60 daily doses of 1 mg/kg were neurotoxic. Hens fed a
daily dose of 20 mg/kg of leptophos developed marked ataxia and paralysis
with no recovery observed. When smaller daily doses were fed, the condition
of the hens deteriorated during the first 2 weeks after onset of ataxia,
then remained stationary for a period of several weeks before gradually
beginning to improve. The pattern of myelin and axon degeneration in the
spinal cord and sciatic nerve in the long-term feeding of small doses
to hens was identical to that produced by a single dose of leptophos and
to that found in the classical picture of organophosphorus neurotoxicity.
A pharmacokinetic profile of [ C ] phenyl leptophos was determined
in laying hens following a single oral dose of 75 mg/kg (1.0 pCi).
Table 2 shows that most of the radioactivity was excreted into the urine
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Table 2
Cumulative Percentage Recovery of 14C Radioactivity
From Hens Given A Single 50mg/kg Oral Dose
(IpC-j) of ll|C-phenyl Leptophos
Days
h
2
4
8
20
Expired Air
0.05
0.16
0.40
1.03
1.25
Excreta
23.55
70.94
77.16
79.75
86.47
Egg
Al bumen
0.41
1.24
1.79
2.26
3.40
Egg
Yolk
0.24
0.78
1.34
2.05
2.54
Tissues
14.58
14.51
14.29
10.61
6.21
Contents
of G.I.
47.24
5.98
0.52
0.15
0.13
Total
91.06
93.61
95.51
96.05
100.00
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and feces. After 20 days, the total radioactivity recovered in the
combined urinary-fecal excretion was 86.47% of the administered dose
as compared to 2.54% in the egg yolk. Radioactivity was least excreted
in the expired C0?. The half-life for the disappearance of radioactivity
from the bird's body following the administration of [ C ] leptophos
was 11.55 days which is much longer than biologic half-life of most
organophosphorus pesticides.
Our studies have demonstrated that leptophos causes delayed neuro-
toxicity in chickens which resembles that of other neurotoxic organo-
phosphorus compounds. They have also shown that small subneurotoxic
doses of leptophos can build up to cause neurotoxic effects in hens.
Since it is assumed that a compound showing such activity might produce
the same effect in man, and in view of its long biologic half-life,
leptophos requires careful consideration before widespread use.
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References
1. Abou-Donia, M.B., Othman, M.A., Tantawy, G., Khalil, A.Z., Shawer, M.F.
(1974) Neurotoxic effect of leptophos, Experientia (Basel) 30:63-4
2. Abou-Donia, M.B. and Preissig, S. H. Delayed neurotoxicity of
(1976) leptophos; toxic effects on the nervous system of hens.
Toxicol. Appl. Pharmacol. 3^:269-282
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PESTICIDE-INDUCED DELAYED NEUROTOXICITY: POISON CONTROL OR MEDICAL ASPECTS
John Doull, M.D., Ph.D.
Introduction
Emergency room physicians and others concerned with the management
of acute pesticide poisoning were first alerted to the possibility that OP
insecticides might produce a "Ginger-Jake" like paralysis in 1952 by the
1 2
clinical note of Bidstrup and Hunter and their subsequent report on
three cases of Mipafox poisoning. Two of the three cases described by
2
Bidstrup, Bonnell and Beckett were severely poisoned and subsequently
developed paralysis whereas the third case exhibited less severe acute
symptoms and no delayed effects. In the first case, a 28 year old female
research chemist, the acute episode lasted for about four days and she was
then essentially symptom-free until leg weakness developed about ten days
later. During the next two weeks, her symptoms progressed and on readmission
she was found to have flaccid paralysis in both legs, loss of reflexes and
impairment of both the upper limb and trunk musculature. There did not
appear to be any CNS or sensory involvement although the patient later
exhibited some transient sensory changes. Motor function returned gradually
in this patient over the next six months with the legs being the last to
recover. She also exhibited severe muscle wasting in the hands and feet
and there was still slight evidence of this effect at eleven months after
exposure. The time course, signs and symptoms in the second patient, a
39 year old male chemical process worker, were similar to those of the
first patient although his acute poisoning episode did not appear to be
as severe as in the first patient. By ten months after the initial
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exposure, the second patient was able to return to work but he continued
to experience "cramp-like" pain in the right foot and calf muscles.
Although the case reports in this paper suggest that both of these patients
were overdosed with atopine during the acute episode, there is certainly
nothing in the way of symptoms, laboratory values or clinical course
which would distinguish the acute poisoning episode in these two cases
from that seen with other organic phosphate insecticides.
Since there were no other clinical reports of neuromuscular paralysis
following OP insecticide exposure with the possible exception of a
3
German greenhouse worker exposed to parathion , it seemed likely that
these two cases represented a specific effect of Mipafox rather than a
general effect of organic phosphate insecticide poisoning. This position
was supported by the results of chronic animal studies with OMPA and
E605 (parathion) which had been carried out in our laboratories in
Chicago ' and in the laboratories of Barnes and Denz at Carshalton .
However, in subsequent studies, which were stimulated by the Mipafox
paralysis report, Barnes and Denz were able to produce weakness, ataxia
and paralysis in chickens and weakness in rabbits with Mipafox. They also
demonstrated that similar effects could be produced with DFP which confirmed
8 9
and extended previous observations made by Model! et. <*]_• » Hunt and Riker
and by Koelle and Oilman ' . In evaluating their studies, Barnes and
Denz pointed out that although the effects and lesions produced in
chickens by exposure to these two fluorophosphates were similar to those
which they observed following tri-o-cresyl phosphate (TOCP) exposure, the
effects of Mipafox, DFP and TOCP could be distinguished from the effects
of other cholinesterase inhibitors by the marked species dependency, the
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relative insensitivity of young animals, the specificity of the neurologic
damage and the similarity of the lesions to those seen in cases of vitamin
B, deficiency. Thus, even at this early research stage in 1953, it was
evident that several significant advances had already occurred. First,
the potential hazard of delayed neuromuscular paralysis as a result of OP
insecticide exposure was recognized and there was concern as to whether
this represented a generalized clinical problem or was an isolated response
to fluorophosphates. Second, it was clear that although the response was
highly species-variable, it appeared that chickens would provide a satis-
factory model test system for detecting and investigating the response.
Third, it was evident that since cholinesterase inhibition could not be
directly implicated as the mechanism responsible for the neuromuscular
paralysis in either human or animal studies, it would be necessary to
look for other common or independent mechanisms to explain these effects.
During the next twenty years most of the research effort in this
area was focused on the same three problems: compound specificity or
structural-activity relationships, species variability and mechanism of
action. Numerous reviews of the progress which has been made in each of
1 ?-1R
these areas have appeared in recent years ~ and the current status of
each of these problems has been considered in the previous presentations
at this conference. The purpose of this presentation is to consider some
of the poison control or medical aspects of organic phosphate induced par-
alysis with particular emphasis on the three traditional aspects of poison
control: first, prevention; second, diagnosis or detection; and third,
treatment or management.
-208-
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Before doing this, however, I would like to comment briefly on some
of the research which was carried out at the University of Chicago Toxicity
Laboratory under the direction of the late Kenneth P. DuBois. Although we
were more concerned in these programs with the problem of characterizing
the structure-activity relationships responsible for the acute toxicity of
the organic phosphate insecticides, we did look for histologic evidence of
peripheral nerve damage in each of our chronic dog studies and, initially
at least, in some of the chronic rat studies. Included in these studies
were such compounds as Guthion (azinphos-methyl), Coral (coumaphos), Systox
(demeton), Meta-Systox-R (oxydementon-methyl), DiSyston (disulfoton),
Tiguvon (fenthion), Dasanit (fensulfonthion), Dylox (trichlorfon), Chlor-
thion and DEF (tributyl phosphorothioate). During this period we also
carried out chronic feeding studies with several carbamates such as Baygon
(propoxur) and Matacil (aminocarb) and with various Chemagro fungicides
such as Morestan (oxythioquinox), Eradex (thioquinox) and Dexon (fenamino-
sulf). We did not see delayed paralysis in any of the animals from these
studies and we were also unable to detect histologic evidence of damage in
the dog sciatic nerve sections. I should point out, however, that in the
majority of these studies we did not look at the distal end of the sciatic
nerve and that several of the compounds which we tested did turn out to be
neurotoxic when tested later on in chickens using the Barnes and Denz pro-
19
tocol . In addition to those agents which were subjected to chronic
toxicity studies, DuBois and his colleagues also investigated the toxicity
of many other phosphates and carbamates, and in many cases chickens were
included in the list of test species. Since these were regular oral LDr
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-------
tests with a two week observation period, one would not have expected
them to detect paralysis except perhaps with agents having low toxicity
and a very potent neurotoxic effect. During our studies on BFP or
dimefox, for example, we found it to be ten to fifteen times as toxic
20 21
as Mipafox to rats, mice, guinea pigs and dogs and DuBois suggested
on the basis of this toxicity and its weak cholinesterase activity that
22
it would not be neurotoxic. Subsequent studies by Davies e_t a]_. de-
monstrated neurotoxicity not only with dimefox but also with the ethyl
derivative which was even more toxic than dimefox. At this stage, the
"state of the art" in predicting neurotoxicity on the basis of structural
relationships was still pretty much at what Aldridge describes as the
23 24
"guess and test" level ' . It is really only in relatively recent years
that progress in understanding the biotransformation of these agents and
additional testing has enabled predictive toxicology based on structural-
25
activity relationships to become reality .
Prevention of Pesticide-Induced Delayed Neurotoxicity
As a first step in evaluating the poison control or medical aspects
of pesticide-induced delayed neurotoxicity, it is appropriate to consider
the real and potential magnitude of the problem. This subject has been
?fi ?7 ?R
reviewed by Hayes in 1969 and again by Namba in 1971 ' . If we restrict
our evaluation to the incidence of delayed neurotoxicity from organic phos-
phates and exclude the neurotoxic effects of other pesticides, such as the di-
thiocarbamates, and metal pesticides, such as arsenic, lead and mercury,
it is clear that delayed neurotoxicity is a rare and rather inconsistent
27
occurrence. In his review of the previous literature, Namba found only
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seven cases where he felt that neuropathy may have resulted from OP
insecticide exposure. In analyzing these and other possible cases,
28
Namba £t al_. point out that two of the three parathion cases exhibited
muscle weakness and decreased tendon reflexes for only two weeks , that both
29
of the cases described by Petty were exposed to mixtures of insecticides
(DDT in both and lead arsenate in one) and that in only one of the four cases
30 31 32 33
where malathion has been reported to cause neurotoxicity ' ' ' was
27
there a truly delayed effect. Namba also comments that he has not found
neuropathy among patients with acute poisoning by organophophorus insecti-
cides and cites the absence of neuropathy and myopathy in the five year
34
follow-up survey of 398 OP insecticide workers by Kovarik and Sercle and
in the survey by Davignon e£ a]_. of pesticide exposure in 441 apple
growers. There is an additional report of delayed neurotoxicity following
oc
parathion exposure but in this case the major sequalae (partial blindness
and focal epilepsy) were central rather than peripheral.
In an effort to check on the current poison control situation regarding
neurotoxic effects from pesticides, I went over the records of the 1300
poison control inquiries which we received last year. Twenty-seven of
these involved pesticides and all but two were subsequently seen either in
our emergency room or elsewhere. There were six cases of organic phosphate
poisoning which required hospitalization (3 malathion, 2 parathion and 1
DDVP), and although muscle weakness and fasciculation was present during
the acute phase in several of these patients, none of them exhibited any
sign of cranial nerve disfunction, peripheral neuropathy or persistant
muscle weakness when examined within a week or two of their exposure. I
also checked with Dr. Vernon Green, the head of the Toxicology Unit at the
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-------
Children's Mercy Hospital in Kansas City and with Dr. Barry Rumack,
the director of the Rocky Mountain Poison Center at Denver General
Hospital. Dr. Rumack estimates that they treat over 100 cases of
moderate to severe organic phosphate insecticide poisoning each year
but have seen no delayed neuromuscular paralysis in any of these
37
patients. With similar reports from poison control surveys in Nebraska ,
38 39
Arizona and Florida , it is tempting to simply agree with Namba's
28
evaluation that "Although studies have indicated neurotoxicity in some
organophosphate insecticides in certain species of experimental animals,
persistent neuropathy in man by poisoning due to commercially available
organophosphate insecticides seems exceptionally rare, considering the
large number of incidents of acute poisoning. Manifestations were variable
among reported patients and not comparable to the results in experimental
animals." Although I agree generally with this position, I do have two
problems. The first is that during the past few years there have appeared
a number of Russian and Rumanian papers reporting delayed paralysis or toxic
polyneuritis with trichlorofon primarily but also with other organic phos-
phate insecticides , and the second is that I think it is dangerous to
adopt generalizations about any class of pesticides which is as chemically
diverse as the organic phosphate insecticides.
In a very real sense, the emergency room physician is at the end
of the line in that he usually encounters these problems long after the
basic research and decision-making processes have taken place and often
the kind of practical information which he needs is not available. When
we see a report, for example, which indicates that leptophos causes neuro-
52
toxicity in chickens , it would be helpful to know whether leptophos
-212-
-------
is simply another OP insecticide which produces paralysis in hens but
does not cause similar effects in man or whether leptoohos is. in fact.
one that we reallv have to worrv about. Clinical toxicoloaists are also
committed to the idea that animal studies are predictive for man but all
of us would feel more secure about the OP delayed paralysis problem if the
reaistration requirements for these aaents included some tvoe of low dose
human studv couoled with a sensitive and sophisticated electromyoqraohic
search for neuromuscular effects. At the verv least, we need a mechanism
bv which the ER ohvsician can readilv obtain data on anv unusual health
problems observed in the workers enaaaed in the production, formulation
and use of such products. Optimally, this information should be a part
of the reqular poison control information systems such as the National
Clearinq House cards or the Poisindex microfiche.
One other suaaestion which has been made repeatedly in meetinqs of
the American Association of Poison Control Centers and the American Academy
of Clinical Toxicoloqy is that requlatorv aqencies should exert more pressure
on oesticide users to use the least toxic aqent which is capable of doinq
the .iob. This recommendation is based on the fact that most of our cases
of serious OP poisoninq are due to oarathion or methvl oarathion and that
other countries like Japan and Denmark have reduced the incidence of OP
poisonina bv leqislation and various tvoes of campaiqns to encouraqe users
to select the less toxic alternatives even thouqh they mav cost more monev.
Diagnosis and Treatment of Pesticide-Induced Delayed Paralysis
The symptoms and time course of the paralysis seen in the "Ginqer-
51 54
Jake" episode of 1931 were similar to those reported from Switzerland
in 194055. from Morocco in 195956 and from Fi.ii in 196957. In addition.
-213-
-------
most cases of Apiol poisoning (an extract of parsley dissolved in TOCP
and used as an abortifacient) also exhibited latency, flaccid symmetrical
paralysis of the limbs with minimal sensory and CNS effects. When these
effects are compared with the reports on the cases of delayed paralysis
following OP exposure, the only difference which seems significant, aside
from the immense difference in incidence, is that the recovery seems to
be more rapid and to be more likely to occur in the OP cases. Although
there have been attempts to classify TOCP as an upper motor neuron poison,
it is now clear that both TOCP and the paralysis producing organo-phosphate
insecticides affect initially and most severely the lower motor neurons
although spastic paralysis and other signs of upper motor neuron damage
may occur subsequently.
Cavanaugh has described the lesion as a "dying-back" process in
the peripheral nerves and suggests that this is metabolic in origin, and
12
Johnson has provided convincing biochemical evidence as to the nature
of the actual target for initiating the changes. The possibility that
many of the toxic agents which produce motor or sensory disfunction, and
perhaps even some of those that produce lesions higher in the nervous
system, may be involved in a common or at least related type of injury, is
exciting since it may result in new approaches to prevention and therapy
not only for the organic phosphates but for other toxic agents where the
problems are more immediate.
Finally, in regard to the therapy of phosphate paralysis, it is
evident that the only real hope for preventing or minimizing the delayed
sequela is early treatment. Most of the therapeutic measures previously
tried (atopine, oximes, vitamin B-,, cortisone, etc.) do not appear to be
-214-
-------
be particularly effective ' . However, Johnson ' has recently shown
that the prior injection of certain carbamyl, sulphonyl, and phosphinyl
esters can protect hens against the neurotoxic effects of DFP or TOCP and
these findings would appear to offer some therapeutic possibilities. It
would be interesting, for example, to see if these agents are as effective
against the neurotoxicity of agents producing neuropathy of rapid onset
(abate, azinophos, carbophenothion, coumaphos, crotoxyphos, crufomate,
dicapthon, dioxathion, disulfoton, EPN, ethion, fenthion, malathion, methyl
parathion, methyl carbophenothion, phorate, and ronnel) as they are against
the agents which produce delayed neuropathy like DFP, Mipafox, DEF, and
Dursban. From a poison control viewpoint, it would also be helpful to
know whether any of these agents are effective when given after the phosphate
exposure and, if so, the length of time during which they are effective.
In conclusion, I think it is fair to summarize the poison control
concerns about the problem of phosphate-induced delayed paralysis as more
of a sword of Damocles than as a serious current problem. This is not so
for other toxic agents or even for other pesticides and I think that the
most encouraging message that I will carry away from this conference is
that progress involving the problem of phosphate-paralysis may ultimately
improve our ability to prevent the neurotoxic effects of a whole variety
of toxic agents.
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1. Bidstrup, P. L. and Hunter, D., Toxic chemicals in agriculture,
(1952) Brit. Med. ,L , ]_(]_), 277.
2. Bidstrup, P. L., Bonnell, J. A., and Beckett, A. G., Paralysis
(1953) following poisoning by a new organic phosphorus insec-
ticide (Mipafox), Brit. Med. J_. , 1, 1068-72.
3. Petry, H., Polyneuritis durch E605, Zbl. Arbeitsmed.. 1, 86, Cited
(1951) in Arch. Ind. Hyg.. 6., 461.
4. DuBois, K. P., Doull, J., and Coon, J. M., Studies on the toxicity
(1950) and pharmacological action of octamethylphosphoramide (OMPA,
Pestox), J_. Pharm. Exper. Therap., 99^ 376-93.
5. DuBois, K. P., Doull, J., Salerno, P. R., and Coon, J. M., Studies
(1949) on the toxicity and mechanism of action of p-nitrophenyl
diethyl thionophosphate (parathion), J_. Pharm. Exp. Therap.,
95_, 79-91.
6. Barness J. M., and Denz, F. A., The chronic toxicity of p-nitrophenyl
(1951) diethyl thiophosphate (E605), J_. Hygiene, 49, 430-41.
7. Barnes, J. M., and Denz, F.A., Experimental demyelination with organo-
(1953) phosphorus compounds, J_. Path. Bact., 65, 597-605.
8. Modell, W., Knop, S., Hitchcock, P., and Riker, W. F., General
(1946) systemic actions of di-isopropylfluoride (DFP) in cats,
J_. Pharm. Exp. Therap., 8_7_, 400-412.
9. Hunt, C. C., and Riker, W. F., The effect of chronic poisoning with
(1947) di-isopropyl fluorophosphate on neuromuscular function in
the cat. J.. Pharm. Exp. Therap.. 91_, 298-305.
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10. Koelle, G. B., and Gilman, A., The chronic toxicity of di-isopropyl
(1946) fluorophosphate (DFP) in dogs, monkeys, and rats, J_.
Pharm. Exp. Therap., 87\ 435-48.
11. Koelle, G. B., and Gilman, A., Antiocholinesterase drugs, Pharm.
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12. Aldridge, A. N., and Johnson, M. K., Side effects of organophosphorus
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13. Aldridge, A. N., Barnes, J. M., and Johnson, M. K., Studies on
(1969) neurotoxicity produced by some organophosphorus compounds,
Ann. New York Acad. Sci., 160, 314-322.
14. Johnson, M. K., The delayed neuropathy caused by some organophosphorus
(1975) esters: mechanism and challenge, CRC Critical Reviews in
Toxicology. ^(3.), 289-316.
15. Johnson, M. K., Mechanism of protection against the delayed neuro-
(1976) toxic effects of organophosphorus esters, Fed. Proc_., 35, 73-4.
16. Johnson, M. K., Delayed neurotoxic action of some organophosphorus
(1969) compounds, Brit. M_ed. Bull., 25_, 231-35.
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19. Gaines, T. B., Acute toxicity of pesticides, Tox. Appl. Pharm.,
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20. Okinaka, A. J., Doull, J., Coon, J. M., and DuBois, K. P., Studies
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231-45.
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21. DuBois, K. P., Toxicological evaluation of the anticholinesterase
(1963) agents, Handbuch Exp. Pharmakol. Erg., Vol. XV_, Chap. 18,
833-59.
22. Davies, D. R., Holland, P., and Rumens, M. J., The delayed neuro-
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23. Hine, C. H., Dunlap, M. K., Rice, E. G., Coursey, M., Gross, R. M.,
(1956) and Anderson, H. H., The neurotoxicity and anticholinesterase
properties of some substituted phenyl phosphates, J_. Pharm.
Exp. Therap., 116, 227-36.
24. Davies, D. R., Holland, P., and Rumens, M J., The relationship between
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phosphorus compounds, Brit. J_. Pharmacol., 15, 271-78.
25. Johnson, M. K., Structure-activity relationships for substrates and
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Pharmacol. 24_, 797-805.
26. Hayes, W. J., Pesticides and human toxicity, Ann. New York Acad.
(1969) Sci. 160. 40-54.
27. Namba, T., Cholinesterase inhibition by organophosphorus compounds
(1971) and its clinical effects, Bull. WHO. 44_, 289-307.
28. Namba, T., Nolte, C. T., Jackrel, J. D., and Grob, D., Poisoning
(1971) due to crgancphosphate insecticides, Amer. 0_. Med., 50,
475-92.
29. Petty, C. S., Organic phosphate insecticide poisoning: residual
(1968) effects in two cases, Am. J_. Med., 2A_, 467-70.
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30. Goldman, H.5 and Teitel, M., Malathion poisoning in a 34-month old
(1958) child following accidental ingestion, J_. Red.. 52, 76-81.
31. Parker, G. F., and Chattin, W. B., A case of malathion intoxication in
(1955) a ten year old girl, J.. Indiana Med. Assoc.. 48_, 491-2.
32. Goldin, A. R., Rubenstein, A. H., Bradlow, B. A., and Elliot, G. A.,
(1964) Malathion poisoning with special reference to the effect of
cholinesterase inhibition on erythrocyte survival, New Eng.
J.. Med.. 271. 1289-93.
33. Healy, 0. K., Ascending paralysis following malathion intoxication: a
(1959) case report, Med. JL Australia. ]_, 765-7.
34. Kovarik, J., and Sercle, M., The influence of the organophosphate
(1966) insecticides on the nervous system of man, 15th International
Congress or^ Occupational Health, Abstracts of Papers, &(]), 209.
35. Davignon, L. F., St-Pierre, J., Charest, G., and Tourangeau, F. J.,
(1965) A study of the chronic effects of insecticides in man, Can.
Med. Assoc. J_. 92!, 597-602.
36. Urban, E., and Sando, M. J. W., Organic phosphate poisoning, Med.
(1965) 0. Australia, 52(2.), 313-16.
37. Mclntire, M. S., Angle, C. R., and Maragos, G., Insecticide poisoning
(1965) of childhood: follow-up evaluation, J_. Ped., 67., 647-8.
38. Harris, C. J., Willeford, E. A., Kemberling, S. R., and Morgan, D. P.,
(1969) Pesticide intoxication in Arizona, Ariz. Med., 26_, 872-6.
39. Davis, J. H., Davies, J. E., and Fisk. A. 0., Occurrence, diagnosis
(1969) and treatment of organophosphate pesticide poisoning in man,
Ann. New York Acad. Sci., 160, 383-92.
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40. Shutov, A. A., and Varankina, T. T., Neurological disorders in acute
(1969) chlorophos poisoning, Klinich. Med.. 47^9}, 140-2.
English abstract 70-0348 in Pesticides Abstracts, _3, 110, 1970.
41. Kosik, V. N., Chlorophos poisoning at home complicated by polyneuritis,
(1968) Vrachebnoe Delo., Bt 133-4. English abstract 71-0088 in
Pesticides Abstracts. 4L> 22, 1971.
42. Lobzin, V.S., and Tsinovoi, P. E., Neurological disorders in chlorophos
(1969) poisoning, Zh. Neuropatol. i Psihiatr. Neurochir.. 69_(5_), 679-83.
English abstract 71-0510 in Pesticide Abstracts, 4_, 140, 1971.
43. Vernik, A. Y., Polyneuritis from chlorophos, Sov., Med.. 9, 44-45.
(1971) English abstract 72-0543 in Pesticide Abstracts, 5_, 122, 1972.
44. Simkin, A. Z., and Mironov, Y. P., Acute poisoning with chlorophos,
(1971) Klin. Med. (Moscos). 49_, 133-4. English abstract 72-0547
in Pesticide Abstracts. 5., 123, 1972.
45. Voiculescu, V., Gheorghiu, M., Cioran, C., Dumitreascu, C., and
(1971) Plaiasu, D., Polyneuritis due to organophosphate insecticide
(parathion and dipterex) poisoning, Neurol. Psihiat.
Neurochir., 16J6J, 535-539. English abstract 73-0074 in
Pesticide Abstracts, 6_, 16, 1973.
46. Babchina, I. P., Nervous system damage in trichlorfon poisoning,
(1972) Vrach. Delo.. 2., 137-39. English abstract 73-2876 in
Pesticide Abstracts, 16, 650, 1973.
47. Semenov, I. A., Palamarchuk, Ye. S., Mudritskiy, V. D., and
(1972) Yaroshchuk, G. S., Emergency aid in acute poisoning with
organophosphorus compounds, Vrach. Delo., 10, 131-34.
English abstract 73-2877 in Pesticide Abstracts, 6^ 650, 1973.
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48. Kazakavich, R. L., Parkhomov, A. A., and Gorenshteyn, G. S., On
(1973) complications occurring following acute poisoning
with trichlorfon, Vrach. Delo., ]_0_, 134-36. English
abstract 73-2878 in Pesticide Abstracts, 6^ 650, 1973.
49. Petrova, N. I., Kogan, Ye. M., Botsyurko, V. I., and Krekhovetskiy,
(1972) Z. S., Acute Trichlorfon poisoning, complicated by poly-
neuritis, Vrach. Delo., 12, 114-15. English abstract
73-2879 in Pesticide Abstracts, 6., 651, 1973.
50. Vasilescu, C., Motor nerve conduction velocity and electromyogram in
(1972) in triorthocresyl-phosphate poisoning, Rev. Roum. Neurol.,
9.(|), 345-50. English abstract 74-0342 in Pesticide
Abstracts, 7., 77, 1974.
51. Pollinger, B., Cozma, V., and Oprisan, C., Clinical and electromyo-
(1973) graphic study of two cases of severe polyneuritis following
acute poisoning with Neguvon (organophosphoric pesticide),
Electroencephalogr. Clin. Neurophysiol.. 35_(4) , 433. English
abstract 75-0322 in Pesticide Abstracts, 8_, 78, 1975.
52. Abou-Donia, M. B., Othman, M. A. Tantawy, G., Khalil, A. Z., and
(1974) Shawer, M. F., Neurotoxic effect of leptophos, Experientia,
3p_d)» 63-4.
53. Smith, M. I., Elvove, E., Valaer, P. J., Frazier, W. H., and Mallory.,
(1930) G. E., Pharmacological and chemical studies of the cause
of so-called ginger paralysis, Public Health Reports,
45.(30), 1703-16.
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54. Smith, M. I., Elvove, E., and Frazier, W.H., The pharmacological
(1930) action of certain phenol esters with special reference
to the etiology of so-called ginger paralysis, Public
Health Reports, 45J42), 2509-24.
55. Jordi, A. W., Acute poisoning by tricresylphosphate, 0^ Aviation
(1952) Med., 23, 623-25.
56. Smith, H. V., and Spalding, J.M.K., Outbreak of paralysis in Morocco
(1959) due to ortho-cresyl phosphate poisoning, Lancet, 2^, 1019-21.
57. Sorokin, M., Orthocresyl phosphate neuropathy: report of an outbreak
(1969) in Fiji, Med. J.. Australia, Hi), 506-9.
58. Davies, D. R., and Holland, P., Effect of oximes and atropine upon the
(1972) development of delayed neurotoxic signs in chickens
following poisoning by DFP and sarin, Biochem. Pharmacol.,
21, 3145-51.
59. Johnson, M. K., The primary biochemical lesion leading to the delayed
(1974) neurotoxic effects of some organophosphorus esters, J_.
Neurochem., 23., 785-89.
60. Doull, 0. The treatment of insecticide poisoning, in INSECTICIDE
(In BIOCHEMISTRY AND PHYSIOLOGY, Ed. C. F. Wilkinson, Plenum
Press) Pub. Co., New York.
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DR. M. K. JOHNSON: Perhaps I could make a comment concerning the
possibility of contrast, between cresylphosphate and pesticide poisoning
in humans. The severe case, Mary Whittaker, who had the substantial
dosing with mipafox, has not recovered significantly. There is considerable
muscle wasting. She can travel about 100 yards, with effort and with
the aid of her crutches. So, I think it's rather unlikely that a
severe case of neuropathy from a pesticide would turn out to be much
different from cresyl phosphate neuropathy.
As regards human studies, we are setting up a very simple exercise
in our laboratory, to compare the general i_n_ vvtro behavior of human
neurotoxic esterase with hen neurotoxic esterase. This will tell us
nothing about the problems of different metabolic conversions, excretion,
or indeed the different sensitivity of the human nervous system to insult
at the enzyme active site. But it will at least tell us whether basically
the active site is similar in the two species. That I think is probably
about as far as we shall be able to go.
As regards the chlorophos cases, I did make the suggestion that the
Russian cases might be due to an impurity. Certainly the specimens of
chlorophos I've seen from various parts of Europe vary in their general
appearance and purity. However, in the laboratory, they do not appear
to differ in response when compared with pure trichlorophon. Now I
think I should say, a propos trichlorophon, the same as I said about
DDVP. It is only under the most heroic conditions that I have ever
been able to get an ataxic hen with trichlorophon treatment. This was
on double dosing with huge quantities, two or three days apart,
coupled with tremendous doses of atropine, and oximes, and very, very ill
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birds. The Russian sample that I managed to get, which is only one
out of many perhaps which are circulating, was very dirty. But in
biological activity, it seemed about the same. When I mentioned
impurity, what I had in mind was that if one happened to use an impure
preparation of methanol in the synthesis, one which contained ethanol
to some degree, that would increase the neurotoxic potential very
dramatically. One factory might do it occasionally and that was
a possibility.
Regarding antidotal treatment of the rapid onset paralysis, 1
haven't tried it. I have never produced that lesion in the laboratory.
I have never tried. And it was put to me quite forcefully last night
that I shouldn't make the assertion that it's different without showing
that it's different. However, let me state, the reasons why I think
it's different. First of all that with EPN oxon you can very easily
get the delayed neurotoxic effect without any rapid onset paralysis. I
find it hard to believe that the rapid onset paralysis is mediated by
anything other than a chronic release of the oxon. Secondly, as I read
the published papers, the brain cholinesterase was inhibited for a very
long time, more than two weeks after EPN and right down near the base
line. I therefore assumed that rapid onset paralysis is in fact chronic
cholinergic stimulation.
DR. J. DOULL: I appreciate that additional word on the one case,
Is that true also for the second case?
DR. M. K. JOHNSON: I'm honestly not sure! I've met Mary Whittaker.
I haven't met the other person. I've got the impression that with that
one, it wasn't a very severe case by comparison.
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DR. J. DOULL: Actually what that really does then for me is to
distinguish the mipafox cases even further from what has been reported
subsequently in the pesticide literature.
DR. F. H. DUFFY: As a clinician, I have to interject the speculation
that the neuropathic process may be more common that we think, because
in reality we rely upon overt clinical signs and symptoms to bring this
to our attention. Suppose someone comes with an acute cholinergic
crisis, a drop in cholinesterase, or a history of exposure and is at
risk. He's usually studied very intensely at this point and then sent
home. Now, if signs of a subtle disorder of peripheral nerve conduction
should develop, chances are very likely that this would not result in
symptoms of such severity for the patient to present himself to a physician
for reexamination -- I use as a model -- the spontaneously occurring
neuropathies, diabetic, alcoholic, tabetic, that we see very day in the
clinic. People will walk in with the complaint of a headache, or something
else, and you'll discover very pronounced neuropathic changes. Patients
may be totally unaware of this.
You can also look at people in their twenties who are at risk for
diabetes, for example, and discover lower nerve conduction velocities,
missing ankle jerks or signs of neuropathy of which they are totally
unaware. As a neurologist, I pick this up all the time.
So I suspect that if you did a study in time on someone exposed to
these compounds, you might see changes in the following 3-10 days especially
if an objective test of nerve conduction velocity were used. This kind
of approach to the problem is totally lacking, except in a few very well
equipped laboratories.
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What this means is you would probably need to do base line nerve
conduction studies on people at risk, maybe cortical conduction velocities
with evoked potential as well. Next one would study them immediately
after exposure and then serially for two weeks to see if there is any
change. And if there is a change, whether it comes back to normal.
Most of the studies have been "one shot". Is this person within
the range of normal? But you don't know whether he moved from high normal
to low normal. And whether or not subtle changes are seen across a
large group of exposures. So I suspect that there's a lot of epidemiological
work that needs to be done.
Point two is that the term neuropathy has been used consistently
here, but there's involvement in -- even in the chick -- in the spinal
cord. And the correct term for this is a radiculopathy or myelopathy.
So, if you do literature research on the subject, you should really look
at radiculopathy neuropathy, and/or myelopathy.
DR. J. DOULL: I did and I apologize for using the less precise
terminology.
I might say, in regard to the possibility that some of the people
we have treated in the emergency room and sent home have in fact developed
paralysis that we are not aware of. If they were seen in our hospital
and they return to any clinical service in our medical center, it is more
than likely that we would know of it through our consult mechanism.
Since this has not occurred, I think I can say reliably that none of our
cases of organic phosphate poisoning have developed delayed paralysis.
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I would agree with you about the need for more epidemiology
studies in this area. There is a fair amount of literature from California
and elsewhere concerning central nervous system changes after organic
phosphate exposure. Some of these studies reported effects such as
muscle weakness, fatigue, irritability, etc., which may involve peripheral
nerve disease, or may be due to central effects. I think we need to
look closer at both and I think we need to begin to pay more attention to
some of these low level chronic complaints that are mentioned in the
literature.
DR. W. N. ALDRIDGE: I'm interested in Dr. Doull's statement that
we have had no real problems with pesticides in this field, and I would
agree with him. But I think perhaps it would be useful to think if we
can understand why this is so. The fact is that most pesticides have
been designed, or in retrospect they appear to have been designed
primarily as inhibitors of cholinesterase. And it happens that the
structure/activity relationships, such as we can discern them, are the
inverse of one another for cholinesterase inhibition and the production
of this lesion, i.e., the production of this lesion depends on long, rather
large groups attached to the phosphorus. In contrast, for the cholin-
esterase, and therefore for the pesticides, by and large small groups are better,
Of course, there are some exceptions, e.g. EPN.
So I think this is perhaps largely the explanation for the difference
between the tri-ortho-cresyl phosphate (TOCP) series and the pesticide
field. In terms of clinical recovery as well, I think there are other
factors to consider. Dr. Doull mentioned, of course, the question of
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re-education. There are other features which may be important which
are compound determined. One is the particular relationship between
the peripheral and central damage. This appears to vary although
detailed information is sparse with the compound. It may vary with the
species and it may vary also with the dose, i.e. whether it's long term
or short term. And of course, if the damage is peripheral, then recovery
is likely to be almost complete.
The other thing which I would like to emphasize here, is what
Dr. Barnes kept hammering home. That we must pay attention if we get
worried about compounds to humans whom we know have had substantially
more exposure, than to the general population, i.e., occupationally
exposed people. We must pay very much attention to this and make as
many measurements as we can on these people.
DR. J. DOULL: I would like to support Dr. Aldridge's comment on
occupationally exposed workers. We need to have a mechanism whereby
industry could funnel information on all of the atypical reactions that
have occurred in their workers during the manufacture of each product.
Oftentimes that is exactly the kind of information that the emergency
room physician needs to manage difficult cases. I don't know what the
mechanism should be to accomplish this, but I've heard the same suggestion
from a lot of emergency room people.
DR. W. N. ALDRIDGE: I think it is quite clear that from the acute poisoning
point of view, organophosphorus pesticides really aren't much of a problem.
Poisoning is almost always due to inhibition of cholinesterase. There are
practically no examples, except for mipafox, which was not a commercial
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pesticide, of an acute episode of poisoning by pesticide leading to
delayed neurotoxicity. But nevertheless, the problem still stays in
our mind as to whether if there are repeated episodes of poisoning,
cumulative damage is produced. I hope that from this conference,
a variety of people will go away with the message that we now have a few
more parameters to measure. We can measure phosphorylation of a
particular protein, and in properly designed experimental situations,
in the hen, where this question can be properly explored.
DR. J. DOULL: I should have mentioned previously that I did talk
to a number of people from industries that are engaged in the manufacture
of phosphates, to ask them if they had had any neuromuscular-worker
health problems related to pesticide workers. I was unable to uncover any.
DR. F. COULSTON: I would like to point out that we must separate
the accidental poisoning episodes relating to the factory or occupationally
exposed individual from one of the real hazards, the presence of pesticide
residues in foods. I would like to comment on the annual joint meetings
of the FAO/WHO Working Party and Expert Committee on pesticide residues
in food. Annually this group reviews data on new and old OP compounds
that have been assessing the toxicological significance of residues in
and on food. It is the trend, year after year now, to study some of
these in man. And not only do they consider and evaluate animal data,
Lut invariably a 30-day test on man is considered. These are often administers
at dof.es based upon the residues or tolerances that might be recommende-l
to be in the food at the time of consumption,
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Now, what I'm driving towards is that heroic doses are all acceptable
for experimental purposes, to try and develop rationale for a mechanism
of action. I would even doubt this mechanism means much if you have to
use atropine and 2-PAM, and artificial ways to keep, in this case, the
chicken, alive. What is important is that studies be established, both
in animals and man, at reasonable doses or reasonable factors of the
intended use for that particular chemical.
Now, for example, it was at the beginning of the meeting that it
was pointed out that the recent 1975 FAO/WHO Joint Meeting on Pesticide
Residues did indeed recommend a temporary tolerance for leptophos. In
doing this, they took into account not only the positive chicken data,
but the fact that there was no effect in any other species, for example,
the dog, a susceptible species which was studied over a 2-year interval.
A comparative toxicology view was a necessity in this instance. Now,
giving leptophos a temporary tolerance and a temporary ADI entailed
allowing an enormous safety factor. Not as large as has been used in
some other compounds, but one 2,000 times the observed no effect level.
I would like to emphasize again, what is learned inside the factory and
by accidental means is important. But we should always keep in mind how
these compounds are to be used.
DR. J. DOULL: Thank you, Dr. Coulston. I think you and Dr. Duffy
and Dr. Aldridge and all of us are saying something important to the EPA.
I hope so.
DR. M. K. JOHNSON: Could I just say again I agree very much with
what Dr. Coulston has said concerning these ones which have been shown to be
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neurotoxic only under most heroic conditions. However, if 'I could just
raise a plea, I think the laboratory testing is tremendously important
to make us aware on a numerical sort of basis. If we can get a graded
response on sample biochemical testing, we are much more aware of that
with which we are dealing. And we must face the fact that although food
tolerances and residues may be important, they are very much a maybe.
We're not quite sure whether anybody has really suffered much from eating
food from plants treated with pesticides of any sort. But if there's an
accident, it would be a great pity to work like mad to get somebody through
the cholinergic symptoms and then to find that they're paralyzed for
the rest of their lives. We are dealing with a more or less irreversible
damage in really severe cases. And therefore ought to focus rather
carefully on accidental and severe exposures.
DR. G. ZWEIG: I just would like to ask Dr. Coulston a point of
information. What was that two thousand safety factor based upon?
On the no effect level of what symptom?
DR. F. COULSTON: The conclusions were based on the fact that in
the chicken a dose response curve could be established and a no-effect
level could be observed with leptophos. This is not realized by many
people, and I must say I wasn't quite sure of it. But that was one
of the cardinal principals involved. So, with a no-effect level in a
chicken, and with no-effect levels noted in many other species of animals
that were studied, it was possible to apply 2,000 times safety factor,
to the lowest no-effect level noted.
DR. G. ZWEIG: But was it cholinesterase or was it delayed neurotoxicity?
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DR. F. COULSTON: The FAO/WHO Joint Meeting suggested the rat
and the dog as sensitive species for evaluating cholinesterase in-
hibition. The actual no-effect level was based on plasma and red
blood cell cholinesterase depression values.
With the hens we based a neurotoxicity no-effect level or data
showing an acute dose of 50 mg/kg in hen. The doses for the rat and
dog were 30 and 60 ppm respectively. These doses are equivalent to
1.5 and 2.1 mg/kg body weight for the rat and dog respectively. From
this an ADI of 0.001 mg/kg was estimated which is a 2000-fold safety
factor.
The point to make is that, having seen the no-effect level based
on the most sensitive species with respect to cholinesterase depression
and applying a large safety factor there is a much greater margin of
safety than just basing an ADI on delayed neuro-toxicity dosage related
to the hen.
DR. R. J. RICHARDSON: Dr. M. K. Johnson's discovery of neurotoxic
esterase should have great practical benefit in neurotoxicity testing,
and I hope intelligent use of this assay will be made in assessing the
neurotoxic hazard of new compounds. However, I can see a potentially
greater benefit arising out of continued studies of this unusual neuronal
protein. When clinicians speak of 'neuropathy1 or indeed of a wide
variety of spontaneous neurological disorders, they are speaking of
diseases with unknown etiologies and unknown methods of cure. With
neurotoxic OP compounds, we have a chemical means to produce neurological
disease, and a defined molecular target, 'neurotoxic esterase', as
the triggering point of the neuropathological process. It would seem
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that a thorough study of neurotoxic esterase and its role in chemically-
induced neuropathy should prove extremely rewarding in the study of
neurological disease and in the understanding of long-term maintenance
processes in the normal nervous system. Our group at Michigan hopes
to continue along these lines, in collaboration with Dr. Johnson and
his group in Carshalton.
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TEST PROTOCOLS AND LIMITATIONS FOR DETECTION OF NEUROTOXICITY
John P. Frawley, Ph.D.
At this point in the Conference, it is unnecessary for me to provide
an introduction in the form of relevant research reported in the literature.
Other speakers have already thoroughly reviewed the most significant work
and have identified the area of concern which brings us together.
However, I do believe that you are entitled to an introduction of a
different type, because the data I will present are over twenty years old.
I believe these data are highly relevant to the selection of animal models
to evaluate the chronic neurotoxic effects from organic phosphate pesti-
cides and provided the bases for an FDA policy 20 years ago on evaluating
the safety of organic phosphate pesticide tolerances - a policy which I
suspect has long been forgotten.
By way of explanation, let me give you the ancient history of these
data. For a period of time in the late 40's and 50's, I worked for the
Division of Pharmacology of FDA, which many of us refer to as Dr. Arnold
Lehman's old Division. I was assistant to Dr. Garth Fitzhugh who was
Head of the Chronic Toxicity Branch. We became interested in the problem
of myelin degeneration as a result of the poisoning cases in Great Britain
by Mipafox reported by Lesley Bidstrup in 1953. Our interest was not
related to the potential health hazard to the manufacturing worker or the
agricultural applicator, which in those days were the responsibility of
the Public Health Service and Department of Agriculture. Our concern was
focused on the potential of the spray residue on food to produce myelin
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degeneration from long-term or chronic ingestion. To evaluate this potential,
we conducted a series of long-term chicken and pigeon feeding studies on a
series of organic phosphate pesticides. I gave a preliminary report on
these studies in the Spring of 1956 at the Society of Pharmacology meeting
and the abstract of that paper was published in the Federation Proceedings.
We terminated our studies later in 1956 and I left the Food and Drug in
December 1956. I cannot be certain why FDA or I did not publish the results
of the studies but I suspect it was the difficulty in those days to get
unsensational toxicological data published. In the meanwhile, everyone
who had participated in anyway in these studies or even had knowledge that
they had been conducted left FDA via retirement or for other reasons. To
make matters worse, the responsibility for regulation of pesticides was
transferred from FDA and apparently the policy on suitable protocols was
also lost in the shuffle.
When I was asked to present a paper at this symposium, I delayed
acceptance until I started digging to locate more of the data than that
contained in the brief 1956 abstract. Fortunately, I had taken copies of
much of the data, including pathology reports with me when I left FDA.
Presumably, I did this with the intent of someday writing a report. Dr.
Robert E. Zwickey, now with Merck Institute of Therapeutic Research, was
the pathologist who worked with me on this project and I wish to acknowledge
his contributions. Also collaborating with me on these studies were Henry
Fuyat, biologist and Dr. J. W. Lawson, toxicologist. Unfortunately, some
pieces of data are missing, most important of which are the cholinesterase
data on the chickens and pigeons. I'm afraid these are irretrievably lost
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in the files of FDA. With the above explanation in mind I will recon-
struct the experiments as best my memory and notes permit and present
the data obtained.
These studies were conducted in three phases:
(1) a range-finding and screening study with chickens,
(2) a long-term study with chickens, and
(3) a long-term study with pigeons.
I will describe them for you in that order.
Range-Finding and Screening Study
These are the studies which were reported at the 1956 Pharmacology
meeting and published only as an abstract in Federation Proceedings.
In all cases the compounds were dissolved in corn (maize) oil, mixed
into commercial laying mash, and made available to the birds ad libitum.
All chickens were laying hens approximately one year of age. My memory
tells me they were Rhode Island Red strain. Each insecticide was incor-
porated in the diet at a relatively low concentration and increased
after several weeks if clinical signs of neurotoxicity or severe toxicity
due to cholinesterase inhibition did not appear. In either case the
clinical signs were allowed to progress until the animals were sacrificed
in extermis. The compounds included in the screening study were tri-ortho-
cresyl phosphate (TOCP) as a positive control, EPN, parathion, malathion,
and demeton.
Table 1 gives an outline of the screening study showing the number
of chickens, highest dietary level achieved and the duration in weeks that
the chickens were fed the respective compounds. Because most of the chickens
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eventually became too weak to eat and became severely emaciated before
sacrifice, a separate group of control chickens were placed on a restricted
diet to duplicate any pathologic changes that might be due to malnutrition.
Table 2 shows the weight at sacrifice for the various groups in relation
to controls. It can easily be recognized that all of these levels are
high levels which would be expected to cause marked cholinesterase inhibi-
tion. My incomplete records show that the initial feeding levels of
parathion and demeton was 100 ppm and that the level was doubled every
2 or 3 weeks. Memory suggests that the initial level for EPN was 300 ppm,
750 ppm for malathion because of their lower acute toxicity. I believe
TOCP was started at the 2,500 ppm level.
It should be noted from Table 1 that the TOCP chickens reached an
extremis situation sooner than the others, and the EPN chickens at an
intermediate time to the others. It is also noteworthy that despite the
known lower toxicity of EPN to other species, chickens could not tolerate
as high a dietary level as the other pesticides.
The reason for this is shown in Table 3. The TOCP chickens developed
an inability to stand after 2 weeks and consequently were unable to reach
their feed and rapidly lost weight. The EPN chickens did not show the same
rapid onset of leg paralysis, but did develop clinical signs of muscle
weakness and inability to stand before other toxic symptoms predominated.
The malathion chickens gave a slight suggestion of specific leg weakness
after many weeks of feeding. Parathion and demeton chickens showed no
symptoms other than those expected from a cholinesterase inhibitor.
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At autopsy, the only gross pathologic change which was related
to treatement was emaciation which was graded from marked to extreme in
all birds except the TOCP and negative controls groups. Microscopic
changes which were related to treatment were observed only in voluntary
muscle, sciatic nerve and spinal cord. In addition to the usual formalin
fixative with hematoxylin-eosin stain for most organs including part of
the sciatic nerve and upper spinal cord, the rest of the sciatic nerve
and lower spinal cord was fixed with Orth solution and stained with osmic
acid. No treatment-related microscopic changes were detected except in the
TOCP and EPN groups. With both groups voluntary muscle tissue showed very
slight to slight shrinkage of muscle fibers and multiplication of nuclei.
The changes observed in nervous tissue are shown in Table 4. In the case of
TOCP, the sciatic nerve of all chickens showed fragmentation and lysis of
axons, and swelling of nerve fibers. The spinal cord of 2 chickens showed
similar changes with oxyphilic irregular masses, probably remnants of
degenerated axons. In the case of EPN, the sciatic nerves showed the
same changes as TOCP but moderate myelin degeneration was present in one
chicken. However, unlike TOCP the spinal cords of the EPN chickens were normal
At the completion of this study, we were convinced that we could
clinically diagnose leg weakness and paralysis caused by nerve damage
as distinguished from generalized weakness due to the pharmacological
action of anticholinesterase. Although we had fed excessively high levels,
we had demonstrated that neurotoxic effects could be produced by subacute
administration of TOCP and EPN. Some leg muscle weakness had been observed
with malathion, but no pathologic changes were found in the nervous tissues.
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The only compounds which demonstrated any tendency to neurotoxicity
subacutely were those compounds which had been demonstrated previously
to respond to an acute dosage.
Long-Term Study with Chickens
Based on the clinical and histopathological observations of the
range-finding study, it was decided to conduct a long-term feeding study
with chickens at a series of lower feeding levels with TOCP and EPN.
These experiments were initiated in March and April 1956 and were termi-
nated in November and December after 7 months. Because we had recently
discovered the phenomenon of potentiation of the cholinergic properties
between EPN and malathion, we wondered whether or not potentiation might
extend to neurotoxicity. Therefore, later in August 1956, we added groups
of chickens on dietary combinations of EPN/TOCP and EPN/malathion. These
were also terminated in November and December after approximately 4 months.
In this experiment, female chickens approximately 2 years of age
were used. They weighed an average of 2.7 kg and experimental groups
consisted of 4 birds each. The experimental compounds were incorporated
into the diet in corn oil solutions and fresh diets were made available
three times a week ad libitum. All chickens were autopsied but unfortunately
no microscopic pathology reports can be located. I do not consider this a
serious loss because in the range-finding study, we had been able to
correlate clinical symptoms with histologic changes in nervous tissue.
In fact, histologic changes were not detected in any animal which had not
exhibited clinical signs of neurotoxicity.
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Table 5 lists the feeding levels administered to the chicken for
TOCP, EPN and the combinations, show the mortality observed with each
group and the time of death. The highest level of TOCP, 800 ppm, produced
rapid onset of neurotoxicity and death in 4 to 7 weeks. One of the
chickens on the highest level of EPN, 400 ppm, and one on the combination
of 50 ppm of EPN and 50 ppm of TOCP also developed early symptoms and
died in 4 to 6 weeks. One 400 ppm EPN chicken also died early due to
an accident. All of these birds lost weight rapidly and died in a
severely emaciated condition. All other chickens which developed symptoms
and died, exhibited a more delayed pattern, with death occurring
generally after 3 to 6 months on diet. In some of these later deaths,
the first symptoms of neurotoxicity did not appear until 5 months of
feeding, both with TOCP and EPN. No chickens developed initial symptoms
later than 5 months.
Table 6 lists the progression of symptoms as they developed from
"slight" to "moderate" and to "severe". This is the basis for reporting
the severity of symptoms that are reported in Table 8. The most
characteristic early symptom we observed was curling back of the toes.
This would interfere with the chicken's ability to stand erect and to
walk normally. Soon after this the chicken would start to rest on its
tarsus or hocks, and food intake would decrease presumably because
walking was difficult. Frequently, diarrhea would be present at this
moderate stage. As the symptoms progressed, the chicken would no longer
be able to maintain this upright position on its hocks and would roll over
on its side. Even with help it would be unable to stand and food
intake would be reduced to almost zero. The bird would develop severe
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emaciation and death was probably due to starvation.
Table 7 provides a tabulation of the average weight gain or loss
for each group and the approximate food intake. These are terminal
weights and reflect the severe emaciation which developed in the two
highest groups of TOCP and EPN. In these cases body weight loss was
between 30 and 54 percent. Food intake was appreciably low throughout
the experiment for these groups.
Table 8 shows the severity of symptoms which developed for each
bird in each group. Obviously, the response is dose related and at the
lowest levels for TOCP and EPN, the symptoms are slight. With one
exception, only occasional slight symptoms were noted with all of the
combination diets. Unfortunately, the combination diets were administered
for only 4 months, and delayed symptoms were observed as late as 5 months
with the individual compounds.
On the basis of 7 months feeding to chickens, these experiments
confirm a minimum neurotoxic effect at dietary levels of 100 ppm TOCP or
50 ppm EPN. On the basis of 4 months feeding, a combination diet of
25 ppm of TOCP plus 25 ppm EPN as well as 20 ppm EPN plus 500 ppm malathion
was with no neurotoxic effect. No evidence of potentiation of neurotoxic
effects was uncovered.
Pigeon Feeding Study
The purpose of this study was to develop sufficient information to
determine whether or not pigeons could be substituted for chickens as a
suitable animal model for neurotoxicity. The potential advantage from
their smaller size would be the opportunity to use large number of animals.
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Accordingly, three groups of ten birds each were fed diets
containing TOCP at 750, 225 and 75 ppm. Five groups of ten birds each
were fed EPN at dietary levels of 200, 100, 30, 10 and 3 ppm in laying
mash. The pigeons were provided the diets for 5 months unless they
died or were sacrificed sooner. Microscopic pathology was performed
on representative pigeons exhibiting leg paralysis. Table 9 summarizes
the results by presenting the incidence of symptoms and time of onset.
Most of the pigeons fed 750 ppm TOCP showed no symptoms until about six
weeks when leg paralysis developed. The other feeding levels produced
no symptoms in the 5 months of feeding.
The pigeons fed 200 ppm EPN developed leg paralysis more rapidly
and all died or were sacrificed within 4 weeks. An average of 20% loss
in body weight was seen in those birds which did not die promptly. At
100 ppm two pigeons were apparently paralyzed in 10 and 14 days, but
the remainder showed no symptoms throughout the study.
Microscopic examination failed to confirm any significant changes
in sciatic nerve or spinal cord either with hematoxylin/eosin or osmic
acid stained sections.
In general, the symptoms observed with pigeons did not appear to
progress through stages as with the chickens. The pigeons were either
lying down or not, apparently with leg paralysis. In view of the absence
of confirmatory lesion by microscopic pathology, the symptoms observed
may have been due to cholinergic properties, rather than specific
neurotoxicity. We concluded that the pigeon was less sensitive than
the chicken to neurotoxic effects of organic phosphates and consequently
a less suitable species than the chicken.
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Conclusions
It was concluded that:
1. Acute studies using near lethal doses was an adequate
qualitative screen for identifying organic phosphates capable of
producing neurotoxicity.
2. Neurotoxicity was a dose related toxic effect which was
cumulative in chickens up to 5 months.
3. The minimum neurotoxic dose of EPN was higher than the
minimum dose producing cholinesterase inhibition. Tolerances for EPN
residues on food had been established on the basis of cholinesterase
inhibition and thus had been based on the most sensitive index of
toxicity.
4. There was no evidence of potentiation of neurotoxic effects
from simultaneous exposure to combinations of organic phosphates.
5. Pigeons were less sensitive than chickens to the neurotoxic
action of organic phosphates and a less suitable animal model for man.
On the basis of these studies, FDA adopted a policy that any
organic phosphate which produced delayed neurotoxic effect in the acute
screen should be studied in a long-term (6 month) chicken study to define
the no toxic effect level. Such studies would not be required on those
which failed to produce neurotoxic effects in the acute screen.
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Table 1. Outline of Screening Study
Compound
Control
Control R.D.*
TOCP
EPN
Parathion
Malathion
Derneton
No. of
Chickens
9
3
2
4
4
4
4
Highest
Level -ppm
0
0
2,500
600
1,600
10,000
1,600
Duration
Weeks~
7-40
7-10
3-4
5-14
13-17
15-17
14-20
*Restricted Diet
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Table 2. Sacrifice Weight of Chickens
Control
Control R.D.
TOCP
EPN
2.7 kg
1.1 kg
1.9 kg
1.0 kg
Parathion
Malathion
Demeton
1.1 kg
1.2 kg
1.1 kg
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Table 5. Clinical Effects Noted
Control; no clinical abnormality
Control R.D.; progressive loss in weight, generalized muscle weakness,
emaciation, sacrificed in extremis
TOCP: inability to stand after 14-15 days, although able to move legs;
muscle response to stimuli decreased thereafter, prostrate with
slow, gasping respiration at sacrifice
EPN: progressive muscular weakness and ataxia, some tremors and inability
to stand; emaciation and little muscular response to stimuli at
sacrifice
Parathion: progressive muscular weakness," loss of balance, emaciation,
sacrificed in extremis
Malathion: progressive muscular weakness, sudden inability to stand,
emaciation, sacrificed in extremis
Demeton: delayed muscular weakness, emaciation, sacrificed in extremis
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Table 4. Pathologic Observations - Sciatic Nerve and Spinal Cord
Control: normal
Control R.D.: normal
TOCP: sciatic nerve of all showed fragmentation and lysis of axons,
swelling of nerve fibers (trace to marked)
spinal cord of 2 chickens showed swelling, fragmentation of
axons, oxyphilic, hyaline-like spheres and irregularily shaped
masses principally near anterior median fissure.
EPN: sciatic nerve of all showed fragmentation and lysis of axons,
swelling of nerve fibers (trace to marked), myelin degeneration
in one chicken
spinal cord normal
Parathion: normal
Malathion: normal
Demeton: normal
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Table 5. Mortality - Long-Term Chicken Study
Compound
TOCP
EPN
TOCP/EPN
EPN/MAL
CONTROL
Feeding
Level
(ppm)
800
400
200
100
400
200
100
50
50/100
50/50
25/25
10/10
40/1000
20/500
-
Mortality
Ratio
4/4
3/5
0/4
0/4
4/4
3/4
2/4
1/4
0/4
1/4
0/4
0/3
0/4
0/4
1/9
Time of
Death
(weeks)
4,4,5,7
15,16,19
-
-
4,5,13,14
9,12,17
20,20
24
6
-
-
;
8
-248-
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Table 6. Description of Progressing Symptoms of Delayed Neurotoxicity
Slight
Moderate
Severe
Uncoordinated gait
Failure to stand on toes
Toes curled
Rests on hocks (tarsus)
Diarrhea
Reduced food consumption
Rests on side
Uncoordinated movements
Emaciation
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Table 7. Long-Term Chicken Study - Body Weight and Food Intake
>mpound
TOCP
EPN
TOCP/EPN
EPN/MAL
CONTROL
Feeding Level
(ppm)
800
400
200
100
400
200
100
50
50/100
50/50
25/25
10/10
40/1000
20/500
Weight
Gain/ Loss
(%)
-35
-30
-17
+ 7
-54
-42
-11
- 4
- 8
-10
+ 3
+ 18
-19
0
0
+ 11
Approximate
Food Intake
(g/day)
60
90
160
170
60
90
130
140
-
-
-
-
160
150
-250-
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Table 8. Clinical Symptoms Observed in Long-Term Chicken Study
Compound
TOCP
EPN
TOCP/EPN
EPN/MAL
Feeding Level
(ppm)
800
400
200
100
400
200
100
50
100/50
50/50
25/25
10/10
40/1000
Symptoms
severe
severe
severe
moderate
slight
none
severe
accidental
death
severe
slight but
severe emac.
severe
moderate
slight
none
moderate
slight
none
slight
none
severe
slight
none
none
none
slight
none
4/4
5/5
2/4
2/4
1/4
3/4
3/4
1/4
3/4
1/4
1/4
1/4
1/4
1/4
1/4
2/4
1/4
3/4
1/4
1/4
1/4
1/4
0/4
0/3
1/4
3/4
20/500
none
0/4
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Table 9. Pigeon Feeding Study - Five Months Mortality and Symptoms
Compound
TOCP
EPN
Control
Feeding
Level (ppm)
750
225
75
200
100
30
10
3
_
Time of
Symptoms Symptoms (weeks)
10/10 6
0/10
0/10
10/10 2-4
2/10 2
0/10
0/10
0/10
0/16
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DR. J. SANBORN: Could not some of the weight loss that you observe(
have been due to the pal ability of the food? I venture to say that with
a V/o incorporation of some of these things in the diet, I don't think tco
many of us would find that very palatable.
DR. J. P. FRAWLEY: These levels were not levels that had not beer
accepted by other laboratory species. In all cases -- rats and dogs,
at least, in our own laboratory, had ingested levels approaching those.
I can't deny that it's possible that some of the emaciation might have
been due to food refusal. But the chickens, in nearly all cases where
the greatest emaciation occurred, were laying around not showing much
interest in doing anything else. I suspect they were just sick.
DR. F. H. DUFFY: Has anyone tried electrophysiological studies on
the chickens? It seems to me that if they can be used, they might save
you a lot of time and trouble in becoming chicken clinicians. It
doesn't cost very much to get a device that will shock a chicken's
foot and measure resultant nerve potentials from the neck. One could
determine a combined nerve and spinal cord conduction time. This
correlates, usually, very well with the neuropathology, and would seem
to be more sensitive than watching a chicken stagger. You could probably
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pick up conduction velocity abnormalities prior to the onset of any
clinically evident material. It's not a very expensive or difficult
thing to do. I just wondered if anyone has tried it in the chicken
and correlated it with the clinical results?
DR. J. P. FRAWLEY: Not to my knowledge. But I think it's a fine
suggestion.
DR. S. H. FRAZIER: You said that the dose for neurotoxicity
is cumulative. Are you saying that you can give a certain level and
on continuing to give that level the material would accumulate in terms
}f not metabolizing out and you would get neurotoxicity? Or are you
using the term cumulative in another context?
DR. J. P. FRAWLEY: I'm not talking about biochemical accumulation
0- of a material constantly building up in the body with absolutely
nc elimination. Cumulative toxicity doesn't necessarily mean one plus
one plus one equals three. You get some increased pharmacological or
toxicological response from subsequent doses. But the cumulation levels
off, as we've seen in these animals, and it does so in most cases of
cumulative toxicity. You reach some equilibrium point where metabolism
is compensating for the intake, and you have no further, either biochemical
accumulation or lesion taking place in the body.
DR. S. H. FRAZIER: In consideration of the safety of residues
in foods, from your results is the neurotoxicity parameter one of a
more primary concern than cholinesterase inhibition, as far as ADI is
concerned?
DR. J. P. FRAWLEY: I don't know of any case where neurotoxicity
has occurred at a lower level than causes that causing cholinesterase
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inhibition. I was maintaining that the ADI's that the WHO has set,
the tolerances that FDA and EPA has set, have usually been based on
the most sensitive index of toxicity, which is cholinesterase inhibition,
not neurotoxicity. So that if you're protecting against cholinesterase
effect, it seems to me that you're not allowing any neurotoxicity to
take place.
DR. OLSON: Many of the symptoms that you described in your birds
strongly resemble a great many of the symptoms of nutritional deficiency
seen in poultry in the early nutrition research days from the late '30's.
I think a lot of your emaciation would induce this, and perhaps to such
an extent that your pathology and other observations might have been
complicated by nutritional deficiencies. This might not be seen so much
with a modern day type of diet. I'd appreciate your comments on that.
DR. J. P. FRAWLEY: Well, certainly at the high levels these animals
did become emaciated. At the intermediate levels they were eating well.
I don't think that they were suffering from severe nutritional deficiencies,
Certainly I agree with you that at high levels those birds were down. And
perhaps if someone wanted to repeat this kind of a study today, it might
be better to intubate the animal to administer the material.
DR. M. K. JOHNSON: I was encouraged to find you bringing this
information out. It agrees extremely well, I think, with that which
Professor Cavanagh has quoted as unpublished work, which I have then
pirated and quoted in the review in Critical Reviews in Toxicology.
With 800 parts per million they got gross ataxia in 16 days. And the
birds had then only had about 0.45 mg/kg, which means something about
0.6 gm/kg. So there was very great cumulative effect. That's not
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much more than the single acute dose.
DR. J. P. FRAWLEY: This is TOCP?
DR. M. K. JOHNSON: Yes! And then, when they got down to 100 ppm,
in agreement with you, they couldn't find an effect in 150 days. I
calculated the dose as about 0.7 ml/kg (equal to 0.84 mg/kg) at that
stage, for which they showed no effect. I think it's interesting,
as you've pointed out that there is a quite rapid cutoff as one goes
down, to a point where there is no effect.
Now, to make two points about that. First, that if you spread
the doses out instead of giving daily doses then you get to the stage
where there appears to be no cumulative effect at all of substantial
doses which are more than three weeks apart. Dr. Barnes found this
because in routine testing he usually waited three weeks to see if
anything would happen, and then gave another dose. Whenever doses were
more than four weeks apart we never saw anything happen in response to
the second dose which hadn't happened on the first dose.
Secondly, it seems to me it would be nice, as you pointed out,
to know what is happening on the enzymic level in these chronic feeding
cases. You could put the question in this form. Does chronic feeding,
say, of 200 ppm or something like that level, keep the enzyme at 50%
inhibited for a long period, or does it manage to work its way up,
slowly increasing inhibition until you get up to 80 to 90 percent
inhibition? And I think it would be nice if anyone were willing to
repeat such a chronic study, if they could at intervals monitor the
enzyme levels.
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I think perhaps on a thing like that it might be necessary to
go directly and monitor, say, the spinal cord rather than the brain,
just in case there were a small variation in the actual percentage
inhibition. And I have one question to raise. Your data and Dr. Barnes's
data agree very well. Dr. Kimmerle has recently noted histological
damage obtained on chronic feeding at only 10 ppm. It does bring to
my mind this question about what exactly do we mean when we say TOCP.
I guess neither you nor Dr. Barnes would be able to say precisely what
was the composition. I wonder if the material which Dr. Kimmerle has
used has been chemically defined very precisely. And whether he could
tell us if those effects -- or how they related to the single dose activity.
In other words, was it in fact an unusually active preparation in both
respects.
DR. 0. P. FRAWLEY: I certainly don't have any idea on the purity
of the TOCP we used in those days, and I doubt if the sample could ever
be retrieved. But I would like to reflect for a minute on one of the
things that John Casida brought up earlier. That these compounds inhibit
a whole series of esterases. I think to properly investigate the
toxicology of these compounds, you should be looking at the effect on
the whole series of esterases that we can identify. That should include
not only the pseudo and true cholinesterases, but the aliphatic esterases,
to see whether or not there's a propensity for interfering with metabolism
with some other compounds as well as the neurotoxic esterase system.
I think it's a whole series of esterases. When we know how these
compounds affect them, we should look at them, before deciding on a safe level
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Does Dr. Kimmerle want to comment on the TOCP purity situation
in his studies?
DR. 6. KIMMERLE: I don't know the actual purity. It was a
technical mixture.
DR. M. D. ABOU-DONIA: I would like to comment on the effect of
leptophos, since EPN is very similar to leptophos in structure, both
being phenyl phosphonothioate compounds. From our experiments with
hens, we found that in both single dose administration and in long-term
chronic oral administration of leptophos, we obtained a dose response
relationship. However, there were distinct differences in both
treatments. In the case of a single oral dose, the no-effect level was
140-160 mg/kg body weight. An acute oral dose of 180 mg/kg resulted in
neurotoxicity. Those chickens that developed ataxia, developed a paralysis
and ultimately died. Clinical neurological signs of poisoning coincided
with positive histological findings on examination of nerves.
The basic difference between the single dose and the long-term
chronic administration is that in the case of a single dose that there
was always an all or none effect. Either there is no observable effect
or if there is an effect it will always proceed along the line of ataxia,
paralysis and death. In all cases, the clinical conditions correlated
with histopathological changes. In the case of the chronic feeding, there
were in some instances at the lower level, some recovery from paralysis.
In the case of a single dose, ataxia always led to paralysis and to death.
In those levels of chronic dosing where there was a recovery from paralysis
following ataxia, no histological changes were evident. The effect could
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have been solely at the molecular or biochemical level which had not
progressed to be observed on histopathology.
We did examine the effect on the enzymes and found that on the
pseudo cholinesterase enzyme was definitely inhibited. Even though
after we stopped the dosing of the chickens, the inhibition still continued.
DR. J. P. FRAWLEY: Well, I don't see that anything you've described
is particularly unusual in toxicology. I mean, in an acute dose you're
overwhelming the system. When you overwhelm a system it's hard to pick
up individual variations in animals. In your sub-acute study you're
titrating from the bottom and you're going to find the effects of
individual members of a strain or a species. So, you'll have more
variation in a sub-acute feeding of that nature than when you just
overload the whole system as with 180 milligrams per kilogram.
DR. M. B. ABOU-DONIA: What was interesting was the fact that the
effect was not just cumulative, but more than cumulative, much more of
an effect.
DR. J. P. FRAWLEY: Well, again, that's not unusual to see in
sub-acute and chronic studies.
DR. A. W. BRADLEY: I notice you found de-myelination in one of
your birds. I would suggest that this was probably a false positive,
actually. When you look for demyelination lesions, fixation is important.
I purposely didn't mention this in my discussion previously because
these are specialized techniques that are not really suitable for routine
examination. You need proper perfusion, and this means putting between
six to either litres of perfusion fluid through the bird, which takes from
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two to three hours to do. Hence, to look for demyelination in a
preparation that's not been properly fixed, will often lead to false
positives.
The common staining technique that has been used is the Marchi
method. When using a technique like the Marchi method, if you use
paraffin sections you will not show demyelination in carefully prepared
control sections.
Another comment on demyelination, some people are still referring
to delayed neurotoxicity as a demyelinating disease. It isn't! I think
it's twenty years since Cavanaugh showed it was an axonal degeneration,
and yet, people are still looking for demyelination which I don't think
is really applicable. It's far better to look for axonal degeneration
which is easiest to spot with some fairly simple techniques.
DR. D. V. ROBERTS: Could I just take advantage of your open
invitation for comments to say that what we are doing with the ENMG
is basically, to provide another bit of information concerning a
physiological parameter of nerve and muscle function. We believe the
real value of the ENMG at the moment is a practical one, in the sense
that we can measure this parameter and then assess whether or not changes
have occurred. Then we can go quickly back to the plant and say to the
people working there, "We believe that your exposure is more than it
should be. Would you like to check the working conditions?" We find
that when we do this, time and time again, they are able to come up with
an explanation of the overexposure and to correct it. Then if we examine
the workers over the next few weeks we find that they come back into the
normal range. And of course, there is no hazard to health in all this.
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It is right at the subclinical level. From our experience we feel
we are catching the effects long before they ever get to the state
of irreversible changes, and to us, this is the main point of the
exercise. The other thing that I would like to say is that the effects
we see are not specific for cholinergic action. Nor are they specific
for delayed neurotoxic effects. They reflect functional changes which
may be produced by many compounds. The method has a wide application
and should not be seen to relate more to delayed neurotoxic effects
than to other factors influencing nerve and muscle function.
DR. E. REINER: I am myself not involved in the EMG studies on
workers exposed to pesticides. But, as this was one of the topics
discussed here, I may perhaps say a few words about Dr. Anica Jusic's
work. She is a colleague in our institute in Zagreb, who is trying
to establish if there are some alterations in the EMGs of workers exposed
to pesticides. What she has done is to use needle electrode techniques
to measure the EMG. As far as I know, she has found no alterations in
the workers who were exposed for a long time to pesticides, particularly
OP compounds and carbamates, when she compared the EMGs of those exposed
people with the control group.
At the same time, she did her examinations, the cholinesterase
levels of these people were measured. These levels were decreased only
by about ten or fifteen percent of their own controlled pre-exposure
values. So one can argue, have these people absorbed any compound to
any appreciable extent? A decrease of something like 10 or 15 percent
can also occur normally in people. It doesn't necessarily mean an
inhibition due to absorption of an anti-cholinesterase compound.
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But, if one does assume that they have absorbed the compound,
then Dr. Jusic's conclusion is that one cannot find EMG changes in
those people who worked for a considerable time with anti-cholinesterase
pesticides.
DR. D. V. ROBERTS: This is an interesting difference between two
techniques, one using surface electrodes, in which we are essentially
looking at a population of nerve and muscle fibres, and the intramuscular
technique used by Dr. Jusic in which observation is restricted to a
very small number of muscle fibres. And it may well be that, in fact,
the ENMG changes that we see, which are perhaps due to changes in nerve
function, would not be reflected in the parameters of EMG by the con-
ventional needle technique. There is one additional point and that
is that Dr. Erik Stolberg of Uppsala has a nice technique which is a
very sensitive one for detecting abnormalities in function, particularly
of the nerve terminals. He has found evidence in a small number of
workers who have been engaged in spraying a variety of compounds of the
organophosphorus type, of a denervation type of lesion. I mention this
because we need to bring all the information together. If we use
different techniques we may, in fact, get different bits of information
but these will, in the end, make up a complete picture. I think that
Dr. Jusic's work is useful but it has to be taken in the context of
intramuscular recording, which of course means that needles have to be
put into muscles and the chances of doing a sequence of recordings over
many weeks is not quite as good as it is with the surface recording
which we use.
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DR. E. REINER: It is difficult for me to discuss Dr. Jusic's
results, but if I recall correctly, Dr. Jusic claims that she can't
get very reproducible results if she uses surface electrodes. If she
examines and re-examines a given person several times, it is a different
group of fibers in a different position which she does record. And she
claims that even in a control group where there's no record of any
exposure to pesticides, there's a tremendous variability in the EMGs
which she does record when using surface electrodes. Therefore, the
variability is such that in the exposed group, any alterations will
somehow disappear due to the alterations of the method. This is the
reason why she used needle electrodes, where she says she can get much
more reproducible results and be certain that it is just the potential
of one particular fiber which she can identify by putting her needle into
it.
That's her interpretation and I can't discuss whether it's correct
or not.
DR. D. V. ROBERTS: Yes. Reproducibility is important. I happen
to know that Dr. Jusic does not, in fact, use the same muscle that we
do, which we chose because with it, we can get more reproducible results.
In fact, if you take a number of non-exposed individuals and do their
EMG's once a day for three weeks, the average coefficient of variation
of values you obtain is seven percent, which is not too bad for a
biological function.
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ORGANOPHOSPHATE EXPOSURE FROM AGRICULTURAL USAGE
John E. Swift, Ph.D.
This discussion is supposed to be concerned only with the exposure
of humans to organophosphate compounds and the results of these
exposures. However, it is not possible for me to confine this subject
to just organophosphorous compounds and cover the problem of pesticide
exposure and related illnesses. The type and number of injuries is
most often determined by the type of work a person is doing at the
time of exposure.
These remarks will be confined to the situation in California as
that is where these statistics are from and also I read in a recent
EPA summary that Region IX has reported the greatest number of pesticide
related illnesses. The reasons for this are hard to define; is it
because we are more careless than others, use more pesticides, do a
better job of reporting illnesses or do we have special environmental
or climatic conditions that influence some episodes of pesticide poisoning?
I am inclined to believe that all of these factors may contribute to
this problem.
The reporting system as directed by the California Workman's Com-
pensation Law states that each physician who attends an employee who is
ill or injured as a result of a work practice must file a report on that
injury, this is with the California Department of Industrial Relations,
this is called the "Doctor's First Report of Work Injury". This agency
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then routes these reports to the California Department of Health who
compile these statistics, assign classifications of work injury with
the Department of Food and Agriculture investigate as many cases as
possible they then publish an annual report. Under this system about
85 percent of the injuries in the work force are covered, but it does
not include the farmer, unpaid family labor, self-employed pest control
operators, those not under Workman's Compensation which may exclude
some of the non-U.S. citizen field workers.
Some definitions are in order to clarify some of the points that
will be covered during this discussion: for this reporting a systemic
illness refers to a generalized illness that involves more tissues
than eyes or skin and usually two or more body systems; however, this
classification can involve only the respiratory system or a generalized
allergic reaction. Skin condition or dermatitis refers to a reaction
of the skin, excluding abrasions and thermal burns. Eye condition
refers to any condition of the eye caused by a chemical substance.
Eye and skin condition refers to cases that have both of these.
The occupation or job requirements appear to be almost as critical
in injury cases as the chemicals involved. The classification of
agricultural workers in regards to their potential exposure and injury
is as follows: ground applicator; mixers and/or loaders; gardeners;
field worker exposed to pesticide residues; nursery or greenhouse workers;
soil fumigators in agriculture; tractor drivers or irrigators; cleaners
and/or mechanics of application equipment; worker exposed to drift from
the application site; aerial applicators (pilots), flaggers for aerial
application and others.
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The workers most frequently reported as being ill from pesticide
poisoning are the ground applicators and the mixers and loaders. In
some cases one person could be doing all of these jobs; however, for
reporting purposes such injuries are divided into these two job
specialties.
In order to bring out the type and incidence of poisonings as they
relate to job specialties and chemicals involved, I will briefly go
through some of the accidents in some of these categories in 1974.
There were a total of 1,157 reported cases to the Department of Health
in 1974 which was a reduction of 117 cases from 1973; however, I hardly
believe this is significant. These incidences are not investigated
in detail except for those workers exposed to pesticide residues at
harvest or some other job involving intimate contact with the treated
foliage.
Ground applicators have had more illnesses than any other agricultural
occupation. The following will show some of these occurrences.
(1) GROUND APPLICATORS - 229 CASES - 1974
SYSTEMIC CASES - 92
Organophosphates 55 Roundup 1 Dowpon 1
(Parathion or
Phosdrin-37) Ansar 1 2,4-D 1
Carbamates 8 Kelthane 1 Ro-neet 1
Dinitro 3 Princep + Simazine 1 Mixtures 5
Paraquat 2 Sulfur 1 Unknown 11
Organophosphates most commonly involved in more serious systemic illnesses.
Spilling concentrate on skin while mixing is a major problem.
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Other factors:
1. Drift onto operator while spraying
2. Lack of safety equipment
3. Excessive heat which causes the worker not to use the
safety equipment
4. Poor training
5. Long working hours
6. Intimate contact with the spray materials and concentrates.
(2)
Weed Oil
Om i te
Comi te
Treflan
Dinitro
Ansar
Sulfur
Trysben
GROUND APPLICATOR - 1974
DERMATITIS - 66 CASES
13
4
3
1
1
1
3
1
Paraquat
Eptam
Alfa-tox
DD
Zinc
Lime
Dibrom + Omite
Omite + Sulfur
3 Omite + Guthion 1
1 Maiathi on+Acaraben 1
1 Dormant Oil +~~ 1
1 Bluestone
1 Paraquat + Keramax 1
1 Omite + ZNP 1
1 Mixture 3
1 Unknown 20
Omite and Comite involved in a large percentage of cases in 1974 and
even more in 1975.
Drift of spray on applicator caused 10 cases.
Lack of safety equipment caused 7 cases.
Material splashed on applicators.
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(3)
GROUND APPLICATORS - 1974
EYE INJURIES - 67 CASES
Sulfur
Weed Oil
Paraquat
Contact
Dinitro
Ansar
Bravo
2,4-D
10
8
2
2
3
1
2
1
Dow General
Weed Killer
Till am
Balan
Omite
Toxaphene
Eptam
Plictran
Drift in eyes of applicator a major problem - refused goggles,
Repairing hose and adjustments while equipment in operation.
Operator rubbing eyes without washing first.
Splashing material into eyes.
1 Difolatan
Pa rath ion
1 Diazinon
1 Omite + Benlate
1 Omite + Dibrom
1 Methyl bromide
1 Knox-out
1 Mixture
Unknown
1
2
1
1
1
1
1
3
20
(4)
GROUND APPLICATORS - 1974
EYE AND SKIN INJURY - 13 CASES
Om i te
Comi te
Weed Oil
3
1
1
Enide 1
Omite + Sulfur 1
Unknown 6
Common cause of injury drift of pesticide from point of application back
to and on applicator.
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It is obvious that the most serious illnesses are caused by the
organophosphorous compounds, but carelessness with other materials
causes some illnesses. It should be noted that parathion and phosdrin
are the worst offenders of the organophosphorous materials. Lack of
protective equipment and clothing is the basis for a number of injuries
and the excessive heat certainly contributes to this. The other types
of illnesses seem to be caused by almost anything that is used, and
the refusal to wear long-sleeved shirts, goggles and a hat apparently
is a contributing factor.
The mixer/loader group has the next highest number of illness and
apparently for the same reasons as the applicator except since they do
not do actual spraying they are not bothered by the spray drifting
on them. Their intimate contact with the concentrates is a major factor
in these illnesses.
(5) MIXER AND/OR LOADER - 142 CASES - 1974
SYSTEMIC ILLNESSES - 75 CASES
Organophosphates 46 Lead Arsenate 1 Sulfur 1
(Parathion &
Phosdrin-39) Dinitro 1 Herbicide 1
Carbamates 8 DD 1 Mixture 3
Broadside 1 Paraquat 1 Unknown 11
Organophosphates most frequently implicated in severe illnesses.
Most illnesses caused by direct contact with the concentrate.
Lack of use of safety equipment - most refuse goggles and gloves.
Inhaling dust from W. P. concentrates.
Splashing or spilling the concentrate on themselves during the mixing
or loading process.
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(6)
MIXER LOADER- 1974
DERMATITIS - 20 CASES
Tel one
Om i te
Dibrom
Sulfur
2
2
1
4
Ordram
Weed Oil
Nemagon
Chlordane
Dyrene
Carbamate
Paraquat
Unknown
Most injuries are a result of spillage on the skin.
1
1
1
4
(7)
MIXER LOADER - 1974
EYE INJURY - 40 CASES
Sulfur
Lannate
Om i te
Comite
Weed Oil
Ansar
Paraquat
Roundup
Dinitro
2
1
3
1
3
1
2
1
3
Eptam
Til lam
Thimet
Copper mono-
hydrate
Urea
Ethyl
Di bromide
Sodium
Arsenate
Phosdrin
Parathion
4
1
1
1
1
1
1
1
1
Tel one 1
Hydrated Lime 1
ToxapheneR 1
Difolatan + 1
Sevin
Eptam + Tref- 1
Ian
Derinol + 1
Planivin
Organophos- 1
phate
Mixture 1
Unknown 3
Herbicides caused a number of problems.
Fourteen injuries caused by splashing concentrate or dilution onto
worker - refusal to wear goggles.
Inhalation W.P. concentrate - Hose malfunction and carelessness.
(8)
Weed Oil
Dinitro
Thiodan
LOADER/MIXER - 1974
EYE AND SKIN INJURIES - 7 CASES
Sulfur
Mixture
Unknown
1
1
2
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Field workers exposed to pesticide residues may be the most
difficult situation to rectify. These people are those who thin,
prune and harvest the crops and have substantial contact with treated
foliage. They enter a field to do a specific job and frequently they
do not know that the plants have been treated with a pesticide. Even
if they did, there is not too much they can do to protect themselves
except to wear protective clothing, long sleeved shirts, hats and gloves.
Occasionally there are a large number of people made ill at one time and
we don't have an answer yet except in all recorded cases organophos-
phorous compounds are involved and usually ethyl parathion is the
chemical usually implicated. I will discuss this later.
Excluding systemic injury as a result of organophosphorous compounds,
both eye and skin injury frequently involved sulfur.
(9)
FIELD WORKERS EXPOSED TO PESTICIDES RESIDUES - 117 CASES - 1974
SYSTEMIC ILLNESSES - 11 CASES
Guthion + Zolone 2 Parathion + Malathion 2
Sevin 1 Unknown 3
Sulfur 3
Four cases occurred during the picking of grapes.
Two cases followed a pruning operation of grapes.
Fortunately we had no serious cases of systemic poisonings during 1974.
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(10) FIELD WORKERS EXPOSED TO PESTICIDE RESIDUES - 1974
DERMATITIS - 77 CASES
Sulfur 18 Ben!ate + Dithane 1 Cryolite 1
Ben! ate 1 Acaraben + Um'cide 1 Dyrene 1
Benlate + Cygon 1 Plictran + Imidan 1 Seven + Sulfur 1
Benlate + Omite 1 Difolatan 1 Mixture 3
Benlate + Plictran 1 Dalpon 1 Unknown 44
Pruning and tying grape vines accounted for a number of illnesses.
Several cases of injury occurred in the harvesting of crops: 5 in vine-
yards; 3 strawberries; 2 lemons, 2 celery; 1 squash; 1 pear, 1 cauli-
flower.
(11) FIELD WORKERS EXPOSED TO PESTICIDE RESIDUES - 1974
EYE INJURY - 18 CASES
Sulfur 11 Sulfur + Captan 1
Omite + Diazinon 1 Unknown 3
Sulfur + Sevin
Six injuries occurred during pruning and harvesting grapes and five
during celery harvesting.
As noted, sulfur is involved in almost every case.
(12) FIELD WORKERS EXPOSED TO PESTICIDE RESIDUES - 1974
EYE & SKIN INJURY - 11 CASES
Dyrene 4 Sulfur + Captan 1
Sulfur 2 Unknown 4
Most occurred during harvest of fruit crops.
In unknown injuries sulfur could still be the main problem.
Gardeners are members of another occupation with a high incidence
of pesticide caused illnesses. These are professional gardeners, not
home gardeners. Some professional gardeners in California are quite
careless or uninformed; most of them use hand operated equipment for
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spraying and they usually are short on protective equipment.
Fortunately, however, serious poisonings within this group do not
frequently occur because they usually do not have access to the
highly toxic materials such as parathion, phosdrin, other organophos-
phates and some carbamates. In California these chemicals are
restricted-use chemicals and require a permit for purchase, possession
and use (Table I). In 1974, there was no case of systemic poisoning
involving organophosphate compounds; however, there was one reported
case of dermatitis involving parathion (which is questionable). There
were five other cases of dermatitis or eye injury involving other
organophosphate compounds which are not on California's restricted-use
list -- specifically diazinon, malathion and metasystox. Most of the
materials available to professional gardeners are in the moderate to
slightly toxic categories. However, it seems obvious that if there
is a way to inflict self-injury by using a chemical, man will find it.
(13) GARDENERS - 101 CASES - 1974
SYSTEMIC ILLNESSES - 5 CASES
2,4-D 2 Weed Oil 1
Vapam 1 Mixture 1
Highly toxic organophosphorus compounds are not allowable in most work
of this type.
One injury was from a spill, a second from drift of his own spray and
the third from not using any safety equipment.
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(14)
Weed Oil
Roundup
Dibrom
Dalapon
Chlordane
Dinitro
Caseron
GARDENERS - 1974
DERMATITIS - 40 CASES
5
1
1
2
1
1
1
Phytar
Parathion
Diquat
Paraquat
Amizol
Isotox
Difolatan
1
1
1
2
1
1
1
Diazinon + Princep 1
Dizinon + 1
Mercuric chloride
Malathion + Kelthane 1
Fungicide 1
Mixture 4
Unknown 12
Four illnesses from equipment malfunction. Four from spills of concentrate.
Three due to lack of safety equipment. Some from drift of application.
One-from contact of previously sprayed plants.
(15)
Weed Oil
Paraquat
Dowpon
Diquat
2,4-D
Princep
Metasystox
Insect repellent
Chlorothalonil
(Bravo)
GARDENERS - 1974
EYE INJURIES - 53 CASES
15 Copper sulfate
4 Sulfur
1 Simazine
1 Amizol
1 Aquathol
1 Krovar
1 Ben!ate
1 Rololind
1 Vapam
2 Amitrole 1
1 Diazinon 2
1 Carbaryl + chlordane 1
1 Dalapon + Weed Oil 1
1 Roundup + Pramitol 1
1 Sodium chlorate + 1
1 Metaborate
1 Pramitol 1
1 Mixture 1
Unknown 8
Equipment malfunction caused a number of injuries (over 15). Drift of
spray back into the eyes of the applicator accounted for several cases.
Goggles were not used in most cases. Splashes or spills on the operator
also caused a number of injuries.
(16)
GARDENER
EYE & SKIN ILLNESSES - 3 CASES
Daconil
Sulfur
Unknown
The last category I want to discuss is the nursery and greenhouse
workers. This group has a relatively high incidence of pesticide induced
illnesses.
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(17) NURSERY & GREENHOUSE WORKERS - 75 CASES - 1974
SYSTEMIC ILLNESSES - 11 CASES
Temik
Dinitro
Lannate
Metasystox
Paraquat
3
1
1
1
Dexon
Malathion
Amitrol + Lannate
Unknown
1
1
1
1
Three of these cases can be attributed to lack of any protective equipment.
The others are partly attributed to the mixing process and to spills.
(18)
Weed Oil
Benlate
Zectran
Diazinon
Paraquat
2,4-D
Kelthane
NURSERY AND GREENHOUSE WORKERS
DERMATITIS - 47 CASES
3 Copper sulfate
3 Chlordane
1 Actidione
2 Dithane
2 Regulain
1 Cygon
1 Dexon + Benlate
Temik, Captan +
Benlate
Captan, Metasystox
+ Dithane
Lannate
Benlate
Mixture
Unknown
Benlate
Dithane
1
1
1
1
22
Twelve employees became ill after handling treated plants. Several ill-
nesses occurred after the person had been spraying the material. Benlate
was frequently implicated.
(19)
Lime
Rololind
Metasystox
Dexon
NURSERY OR GREENHOUSE WORKERS
EYE INJURIES - 16 CASES
Dibrom
Azodrin
Weed-all
Banrot
Cygon
Temi k
Benlate + Dazatrol
Benlate + Omite
Unknown
1
1
1
1
4
Four injuries by contacting treated plants. Four injuries associated with
accidental spills.
One eye and skin injury cause not known.
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Considering the conditions of work and the types of materials frequently
used this group has a relatively good record and they also use restricted
materials. Supervision in nurseries and greenhouses is usually better
than among other areas and this may account for the relatively good record.
A number of other occupationally exposed workers are listed according
to their work speciality and these are:
OTHER PESTICIDE ILLNESSES ACCORDING TO OCCUPATION - 1974
1. Soil Fumigators in Agriculture 29
2. Equipment Cleaners and Mechanics 28
3. Tractor Drivers and Irrigators 23
4. Workers Exposed to Drift 22
5. Pilots 17
6. Flaggers 6
Those doing soil fumigation most frequently report illness from
methyl bromide and chloropicrin or other fumigants, no organophosphates
were involved.
Tractor drivers and irrigators reported 22 cases of injury and
5 incidences of systemic poisoning. Four out of these 5 cases involved
parathion and 1 thimet. There were no other injuries involving OP
compounds. The same was true for those who clean and repair equipment.
For those who are injured while cleaning equipment, for the most part
it is the result of carelessness or the owner or supervisor did not
advise them of the hazard involved in cleaning out the residue associated
with these chemicals. If the owner of the equipment properly advised
them as to the material in the equipment, there should be few accidents.
In the case of the mechanic repairing a piece of equipment the
illness is the result of the applicator or equipment owner or not notifying
the mechanic of the type of material that was last used in the machine.
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In both cleaning and repairing equipment, 9 of the 24 cases were
systemic poisonings and all involved organophosphorous compounds with
parathion or phosdrin causing all but one incident. In the repair
of equipment most cases involved a welding torch or some other heat
source which vaporized the material and resulted in a pesticide illness
almost immediately. This can be stopped if a little more respect is
afforded these chemicals.
Workers exposed to drift are those who are in the area but not
doing one of the jobs previously mentioned. In 1974, eight cases were
as a result of aerial application and 12 cases were from ground application.
Of the 10 systemic illnesses 9 were caused by organophosphorous compounds
and one by a carbamate. For other types of illnesses the offending
chemicals were the same as previously reported.
Amongst pilots, 13 of the 17 reported illnesses were systemic
poisonings and all but 3 of these were caused by organophosphorous
compounds involving parathion, phosdrin, systox, phosvel and guthion;
two of the other three were carbamates; the third unknown. Most injuries
were the result of equipment malfunctions in the airplane or crashes.
One would think that flaggers would be subject to many exposures
and illnesses caused by pesticides, but this is not the case. There
were only 6 cases of poisoning in 1974 and 3 of these were systemic
caused by parathion or a combination of parathion plus something else.
Organophosphorous compounds were not involved in the other three cases.
Flaggers move so that drift is always away from them, they move out
of the path of the plane as soon as the pilot gets on course and as a
result is one of the safest jobs in agriculture where chemicals are involved.
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The problem associated with most of the pesticide induced illnesses
can be avoided. Of course it is recognized certain types of accident
such as an airplane or truck crashes, explosion or fires cannot be
anticipated but many others can and proper measures taken to reduce
such illnesses. However, the problem associated with worker illnesses
due to exposure to pesticide residues on various crops is one that will
require considerably more research. Such cases have been recorded in
California since 1949 and while re-entry intervals imposed in 1971
(Table II) have reduced the number of such poisonings. They still are
occurring only. Only now are the mechanisms by which such poisonings
occur, the environmental factors involved and the extent of the problem
are being thoroughly studied.
Of the cases recorded since 1949 there have been a total of 26
incidences occurring in 13 different years (Table III). That is to say
some years there were no cases and others 3, 4, or 5 incidences per year.
The crops involved have been pears, citrus, grapes, peaches, olive,
prune and lettuce. Of significance in this report is that out of
26 incidences, 17 involved parathion, 4 parathion plus some other
organophosphorous compound, 4 other organophosphorous compounds and
1 unknown. To further complicate the picture, 18 of the incidences
involved citrus and 8 the rest of the crops mentioned. A clue to this
situation might be the distribution of these incidences in California.
The first occurrence was in the Sacramento Valley, then two cases in
the Riverside area and all the rest in the San Joaquin Valley.
The one factor in common here is the weather or climatic condition
except for an occasional situation, occur during extremely dry conditions
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from June to September, the humidity is low and usually a large
build up of dust on the foliage of the crop. Dr. Spear and others
have found that the weathered residue of parathion decay into
paraoxon and is associated with the dust on the leaves which termed
a dislodgable residues, and this gets onto the upper body of the
worker as he harvests the fruit, prunes or some similar job. Paraoxon
is also formed in the soil and it appears that a possible contamination
of the whole body may occur. It has been determined that poisoning
is a result of paraoxon being absorbed through the skin in sufficient
quantitites to cause intoxication. Spear et al., so far have concluded
that paraoxon is the principal toxic constituent of the weathered
residue. The absorbed dose is almost entirely dermal. In citrus
crops the fallout of foliage residues is probably of more importance
than direct foliar contact.
As a result of studies like this by Dr. Spear and others we may
eventually get to the cause and solution of this problem.
As a further study regarding exposure of field workers to organo-
phosphorous residues -- Peoples, Knaak & Maddy studied in 1975 -- collected
blood samples of 1,166 persons during the growing season of specific
crops in the San Joaquin Valley. These included male and female field
workers and non-field workers, but all from the same farming community.
There were 416 male and females who acted as controls. This monitoring
was done at the height of the harvest season for lettuce, grapes,
peaches, and citrus. The farm workers were selected by physicians
at Union farm worker clinics. Or those who volunteered to a sign
posted in the clinic printed in both English and Spanish saying "Obtain
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a free test today for pesticide exposure". This means of selecting
subjects should insure a relatively representative sample of the farm
worker population. Analysis of these results are still under way but
so far there is no difference in cholinesterase value between the
field workers and the controls. The largest number of persons involved
occurred in September at the end of the grape harvest and these did not
show any difference from the controls.
Many things are going on in California and the United States in
general to reduce the problem of worker injury to pesticide exposure.
Some of the significant things are the research by people like
Spear, Maddy and Kahn. Injury and accident investigation, re-entry
intervals that are imposed after the use of certain chemicals on certain
crops, employers' responsibility for training his employees, emergency
medical care and medical supervision for those working with certain
organophosphorous or carbamate compounds, it is now illegal for a
pilot to assist in loading operations, the future requirement of a
closed mixing system, shielding of flexible hoses if they go through
any vehicle used for ground or aerial application, and monitoring the
farm worker population, etc.
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ACKNOWLEDGMENT
I would like to acknowledge the help I received from the California
Department of Health, the California Department of Food and Agriculture
and the University of California Department of Environment Health Sciences,
My special thanks goes to Dr. Ephraim Kahn, M.D., of the California
Department of Health, K. T. Maddy, D.V.M., of the California Department
of Food & Agriculture and R. C. Spear, Ph.D., of University of California
Environmental Health Sciences.
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STATE OF CALIFORNIA
DEPARTMENT OF FOOD AND AGRICULTURE
TABLE I
RESTRICTED MATERIALS
Permit required for possession and use
(a) Certain pesticides containing
arsenic
IT) S"odium arsenite**
(2) Other pesticides containing
inorganic arsenic*
(b) Pesticides containing cadmium*
(c) Pesticides containing mercury
(d) Carbamates
(1) Aldicarb (Temik)
(2) Carbaryl (Sevin)*
(3) Carbofuran (Furadan)****
(4) Methomyl (Lannate)
(e) Fumigants
(1) Chloropicrin*
(2) Methyl Bromide*
(3) Aluminum phosphide
(4) Carbon bisulfide
(f) Mercury treated seeds
(g) Endrin treated conifer seeds
(h) Avjc'i'deV
(1) Avitrol
(2) Starlicide
(3) Strychnine
(i) Rodenticides
IT]Sodium fluoracetate
(Compound 1080)
(2) Strychnine*
(3) Zinc phosphide*
(j) Organic Phosphorus Compounds
TT)Azinphosmethyl (Guthion)
(2) Carbophenothion (Trithion)
(3) Bidrin
(4) Azodrin
(5) Monitor
(6) Supracide
(7) Demeton (Systox)
(8) Disulfoton (Di-Syston)*
(9) EPN
(10) Ethion
(11) Methyl Parathion
(12) Mevinphos (Phosdrin)
(13) Parathion
(14) Phorate (Thimet)
(15) Phosphamidon
(16) Schrardan (OMPA)
(17) Sulfotepp
(18) TEPP
(19) Dialifor (Torak)
(k) C hi on' na ted Hydroca rbons
(1) Aldrin*
(2) Benzene hexachloride (BHC)*
(3) Chlordane*
(4) ODD (TDE)
(5) DDT
(6) Dieldrin*
(7) Endosulfan (Thiodan)
(8) Endrin
(9) Heptachlor*
(10) Lindane*
(11) Toxaphene*
(1) All other pesticides registered for
use in the form of a dust, except
those products containing only
exempt materials.***
(m) Other Pesticides
TH Paraquat
See next page for explanation of asterisks (*).
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TABLE I
RESTRICTED HERBICIDES
Permit required for possession and use except
as provided for in Section 2451 (Admin. Code)
(a)
(b)
(c)
(d)
(e)
2,4-D
2,4,5-T
MCPA
2,4-DP
Si 1 vex
(f) 2,4-DB
(g) Plcloram
(h) Propanil
(i)Dicamba (Banvel)
PERMIT REQUIREMENTS
Restricted Materials
No permit required for home use, structural pest control,
industrial and institutional uses, and uses by certain
public agencies.
(A) Pesticides containing
arsenic other than
sodium arsenite as
specified in Section
2460 (a) (1)
(B) Pesticides containing
cadmium
(C) Pesticides containing
mercury
(D) Carbaryl (Sevin)
(E) Chloropicrin
(F) Methyl bromide
(G) Disulfoton (Di-Syston)
(H) Aldrin
(I) Benzene hexachloride (BHC)
(J) Chlordane
(K) Dieldrin
(L) Endosulfan (Thiodan)
(M) Heptachlor
(N) Lindane
(0) Strychnine (Rodenticide
uses only)
(P) Toxaphene
(Q) Zinc Phosphide
(R) Pesticides registered
for use in the form of
a dust included in (1)
on reverse side.
**
***
****
*****
No permit required for ready-to-use syrups or dry baits.
No permit required when packaged in containers holding 25 pounds
or less or for use in enclosed areas such as greenhouses.
No permit required for granular formulations containing not
more than 5% carbofuran.
No permit required for paraquat for home use only when possessed
and used in accordance with registered labeling.
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TABLE I
General
(1) Permits to possess restricted materials/herbicides shall
not be required of economic poison registrants or pesticide
dealers when operating under their licenses, or by govern-
mental agencies or by commercial carriers to transport
such materials.
(2) The person in charge of the property to be treated or the
pest control operator or both may apply for a permit, but
no permit shall be valid for possession or use by any
operator or person not named in the permit.
(3) A permit to use restricted materials/herbicides shall have
an expiration date no later than the calendar year for which
issued and shall be valid for the period specified unless
sooner revoked or suspended. A copy of each permit shall
be retained by the issuing officer.
(4) The person named in a restricted materials permit is
authorized to possess materials for which the permit was
valid after such permit expires, provided it is stored
in accordance with Section 3136.
REFER TO REGULATIONS FOR SPECIFIC PERMIT REQUIREMENTS.
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CALIFORNIA DEPARTMENT OF FOOD AND AGRICULTURE
TABLE II
Field Re-entry Safety Intervals
Safety intervals have been established by regulation between the
time certain pesticides are applied to citrus, grapes, peaches, nectarines,
and apples, and the time workers may be allowed to enter treated areas
to engage in an activity requiring substantial body contact with treated
foliage. In addition to the intervals specified by regulation, numerous
other safety intervals are included in pesticide labeling and must be
complied with. In cases where the safety interval specified in the
pesticide labeling differs from the safety interval specified in the
regulations, the longer interval must be followed.
Safety Intervals in Days for Citrus, Peaches & Nectarines, Grapes, Apples
Citrus
Peaches &
Nectarines
Grapes Apples
Azinphosmethyl (Guthion) 30
Carbophenothion (Trithion) 14
Demeton (Systox) 5
Diazinon 5
Dimecron (Phosphamidon) 14
Dimethoate (Cygon) 4
Dioxathion (Delnav) 30
EPN 14
Ethion 30
Malathion 1
Mevinphos (Phosdrin) 4
Naled (Dibrom) 1
Parathion-ethyl 21
30
45
Parathion-methyl
Phosalone (Zolone)
Imidan
Sulphur 1
TEPP 4
(a)
(b)
(c)
14
14
7
5
30
14
14
1
4
1
21
21
21
5
1
4
21
14
7
5
4
30
14
14
1
4
1
21
21
21
5
1
14
14
14
14
Footnotes: (Note these must not be confused with preharvest intervals)
No more than 4 pounds of actual parathion per acre in a single
(a)
(b)
(c)
application.
More than 4 pounds of actual parathion per acre, but no more than
10 pounds per acre, in the pat 12 months.
More than 8 pounds of actual parathion per acre per application or
more than 10 pounds per acre in the past 12 months.
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TABLE III
THE
PROBLEM
Since the introduction of the organophosphate (OP) pesticides
into California agriculture in the late Forties, it has become increasingly
clear that it is possible to become poisoned from sufficient exposure
to residues of these pesticides on foliage and in soils. The population
occupationally exposed to such residues is comprised of agricultural
fieldworkers engaged in thinning, pruning or harvesting operations. OP-
treated citrus crops appear to present a particular hazard but multiple
poisonings have also been reported in grapes, peaches, pears, olives
and lettuce crops. These incidents led to the imposition of regulations
aimed at limiting the exposure of fieldworkers which came into force
in the State in 1971 (California Administrative Code, Title 3, Article
2475). However, the mechanisms by which such poisonings occur, the
environmental factors involved and the extent of the problem are now
being studied.
Incidence of multipje systematic illness from exposure to OP-pesticides
for agricultural workers in California, 1949-1974
Date
7/8/49
6/27/51
8/27/52
7/6/53
11 /53
H /53
/ /59
10/5/61
8/9/63
6/29/66
7/8/66
7/21/66
8/2/66
8/11/66
9/2-23/67
9/14-16/67
5/5/70
5/25/70
5/27-28/70
9/14-17/70
10/1/70
8/16-24/71
5/6/72
9/15/72
9/9/72
8/30/73
Location
Marysvitle
Delano
Riverside
Riverside'
Riverside
Bryn Mawr
Whole State
Terra Bella
Hughson
Terra Bella
Porterville-
Lindsay
Navelencia
Terra Bella
Hughson
Ballico
Porterville
Lindsay
Terra Bella
McFarland
Orosi
Orange Cove
Lindcove
Exeter
Huron
fowler
No.
ill
20-25
16
11
7
_.
—
275
10
94
9
6
3
11
9
24
3
3
2
8-11
35
11
8
3
9
4
27
Probable
no.
exposed
56
24
30
—
—
—
—
—
—
15
11
30
22
28
—
—
30
22
—
35
55
9
—
22
31
32
Previous aoolication*
Crop and
activity
Pears
Grapes
Oranges
Oranges
Citrus
Citrus
Citrus
Lemons
Peaches
Oranges
Oranges
Oranges
Oranges
Oranges
Peaches
Peaches
Lemons
(prune)
Oranges
Oranges
Oranges
Oranges
Olives
(prune)
Oranges
Oranges
Lettuce
(weed)
Grapes
Pesticide
implicated
Parathion
Parathion
Parathion
Parathion
Parathion
Parathion
Parathion
Parathion
Parathion
Parathion
Parathion
Parathion
Parathion-
malathion
Parathion-
ethion
Azinphosmethyl
ethion
Azinphosmethyl
Dioxathion
naled
Parathion-
ethion
Azinphosmethyl
ethion
Parathion
Parathion
malathion
Parathion
Parathion
Parathion
Parathion
' 1
AIAk
2.50
1.87
2.00
—
—
—
3.00
2.00
1.87
1.33
2.00
13. 5p
3.75p
1.5a
2.0e
1.5
6d
In
7.5p
6.75e
12a
4e
9.00
3p
6.00
2.5
5.00
2.50
—
Entry
time1
12
33
16
17
34
33
—
17
14-38
15
32
13
28
46
38-47
66
1
14
8
11
34-37
31
31
21
12
1
41
Spray used
—
—
—
Parathion
Parathion
—
—
*
—
Dicofbl
TEPP
Dicofol
—
Parathion
Azinphosmethyl
Dioxathion
Azinphosmethyl
—
—
—
Parathion
—
Interval1*
— —
19
__
„
—_
—
97
36-110
—
— •
38-47
15-30
38
17
10-12
120
180
—
—
—
4-25
—
•Unless otherwise indicated in "Crop" column, workers are engaged in picking operation. 'Active ingredient per acre expressed! in Ib.
Days postapplication. d Days prior to most recent application.
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References
Maddy, K. T. "Worker Re-entry Safety. IV. The Position of the
(1975) California Department of Food and Agriculture on Pesticide
Re-entry Safety Intervals". Residue Reviews, Vol. 62.
Maddy, K. T. "Field Worker, Pesticide Safety Activities - Planned and
(1975) Underway in the Southern San Joaquin Valley - July-October, 1975".
California Department of Food and Agriculture. Mimeo.
Maddy, K. T. "Current Considerations on the Relative Importance of
(1976) Conducting Additional Studies on Hazards of Field Worker
Exposure to Pesticide Residues as Compared to Studying Other
Occupational Safety Hazards on the Farm". Mimeo.
Maddy, K. T. and Betts, W. "Pesticide Safety Problems in California".
(1976) Mimeo.
Popendorf, W. J. and Spear, R. C. "Incidence of Multiple Systematic
(1974) Illness from Exposure to OP-Pesticides for Agricultural
Workers in California, 1949-1974". Environmental Science and
Technology, Vol. 9.
Popendorf, W. 0. and Spear, R. C. "Preliminary Survey of Factors Affecting
(1974) the Exposure of Harvesters to Pesticide Residues". American
Industrial Hygiene Association Journal, 374-380.
Popendorf, W. J., Spear, R. C. and Selvin, S. "Collecting Folia Pesticide
(1975) Residues Related to Potential Airborne Exposure of Workers".
Environmental Science and Technology, Vol. 9 583-585.
Spear, R. C., Jenkins, D. L., and Milby, T. H. "Pesticide Residues and
(1974) Field Workers". Environmental Science and Technology, Vol. 9.
Spear, R. C., Popendorf, W. J., Leffingwell, J. R. and Jenkins, D.
(1975) "Parathion Residues on Citrus Foliage. Decay and Composition
-287-
-------
as Related to Worker Hazard". Journal of Agriculture and
Food Chemistry, Vol. 23.
Spear, R. C., Popendorf, W. J., Leffingwell, J. T., Milby, T. H.,
Davies, J. E., and Spencer, W. F. "Exposure and Response
of Fieldworkers to Weathered Residues of Parathion".
Archives of E ny 1ronmenta1 Health (In Press).
Swift, J. E. California Worker Safety Training Courses. University
(1974) of California, Berkeley.
California Department of Food and Agriculture. "Agricultural Pest
(1974) Control Operations and Other Interested Persons". Mimeo.
California Department of Food and Agriculture. "Restricted Materials
(1975) List". Mimeo.
California Department of Food and Agriculture. "Pesticide Worker Safety
(1974) Regulations". Mimeo.
California Department of Food and Agriculture. "Survey of San Joaquin
(1974) Valley During the Summer of 1974 for Worker Safety Hazards from
Pesticide Residues". Mimeo.
California Department of Health and California Department of Food and
(1975) Agriculture. "Occupational Illnesses Due to Exposure of Persons
to Pesticides or Their Residues in California in 1974". Mimeo.
California, Department of Public Health, Bureau of Occupational Health and
(1970) Environmental Epidemiology. "Occupational Disease in California
Attributed to Pesticides and Other Agricultural Chemicals".
Annual Report.
National Center for Health Statistics. Mortality Statistics - Special
Reports, Accident Fatalities, Division of Vital Statistics.
"Total Deaths from Accidental Poisonings - 1968-1973".
-288-
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DR. D. KURODA: In California, you have many lettuce fields.
Suppose, for example, a field is sprayed with a delayed neurotoxin.
How is a migrant worker going to know that his onset of symptoms are
due to an exposure two weeks ago? Has California had any experience
with gathering accident data on delayed neurotoxic pesticides?
DR. J. E. SWIFT: With delayed neurotoxic pesticides, as far as
I've been able to find out, and I asked persons who should know about
it, -- there are no known cases having occurred in California.
If you're asking about leptophos, yes it was used, but its usage
has been stopped for the present time. EPN is a material that is no
longer used. You can hardly even buy it in the state anymore. It
did not prove to be effective as a pest control agent. With most of
these pesticides I'm talking about -- at least the organophosphates
and the carbamates, if they're used on a field, the field has to be
posted in both English and Spanish in letters that can be read at least
25 yards away. It's posted on all four sides of the field and the entrance
place. Until the re-entry period that has been settled upon has passed.
After that they have to be taken down. This applies to any sort of a
crop, if certain compounds are used. EPN was put on this category where
it had to be used in posted fields. This was done as long ago as the
early 1950's. EPN is just not used anymore.
DR. W. J. HAYES: I think this has been a very valuable contribution
to hear about these occupationally connected cases from California.
These statistics have been available for many years. There's nothing
quite comparable to them in any of the other states. So, it's very
valuable to hear these reports of, in most instances, very mild effects,
with reporting stimulated by a compensation system.
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I thought it might be of interest to you, since I've had to
look into it, to get a little perspective on how this compares with
the other parts of the country and the world, and even with California
itself. In other statistics issued by the state, but published
separately for the last several years, to give you some idea of the
kind of thing that they will record, the number of cases of poisoning
by poison ivy is considerably in excess of those by pesticides.
Now, in other states and countries where they count only those
accidents that are of a more serious nature, and, at least in the
state of New York, are compensable, what one finds is that the number
of agriculturally related injuries is very much higher for equipment,
and even hand tools, than it is for pesticides. The number of pesticides
used are really quite small. If you take that kind of a base, then
you get essentially identical statistics from places as far removed
as New York State, the United Kingdom and Hungary. I have been searching
for some years to get comparable figures from California, but without
success.
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PESTICIDE REGULATORY RESPONSIBILITY
Orville E. Paynter, Ph.D.
Perhaps the only contribution a paper on pesticide regulatory
responsibilities might make to these proceedings is to give some
insight into the growing requirements of regulatory agencies for
scientific input and indicate how this has come about. The extent of
the authority exercised by such agencies is governed by the way elected
officials interpret society's needs and take action through enactment
of appropriate laws. In the past pesticide regulation had proceeded
at a leisurely pace, laws were not very complex and the scientific
input required to make them effective was minimal. Today this is no
longer true.
Federal Regulation of pesticides in the U. S. began with the
Insecticide Act of 1910 which prohibited the Interstate sale of any
insecticide or fungicide which was adulterated or misbranded. This Act
was concerned with the effectiveness of products and deceptive labeling
and did not require registration of pesticides. It can be seen that the
concepts of pesticide regulation were not very complex and the scientific
input, if any, was confined to rather simple chemical, entomological
and perhaps microbiological considerations.
In 1947, 37 years later, The Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA) established registration requirements for all
pesticides entering interstate trade, but authority to deny registration
applications was not provided until 1964. As with the 1910 Act, FIFRA's
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primary thrust was the protection of consumers from ineffective products
and deceptive labeling. However, FIFRA did require that pesticide labels
contain precautionary warnings and statements which would protect the
consumer from certain adverse effects. For the most part, these warnings
and statements were concerned with the acute toxic effects to humans and
domestic animals. The law had become more complex as society's percep-
tion of the problems had developed and changed. In addition to the
scientific input of previously mentioned deciplines, the inputs from
pharmacology, toxicology and various health sciences were now required
to make the law effective.
In 1972, FIFRA was drastically amended by the Federal Environmental
Pesticide Control Act (FEPCA). Additional amendments also occurred late
in 1975. Congress found the greatest need for the 1972 revision of FIFRA
to be in the areas of strengthening regulatory controls on the uses and
users of pesticides, speeding up procedures for barring pesticides found
to be undesirable, stremlining procedures for making valueable new measures,
procedures, and materials broadly available; strengthening enforcement
procedures to protect against misuse of pesticides; and creating an
administrative and legal framework under which continued research could
provide more knowledge about better ways to use existing pesticides as
well as developing alternative materials and methods of pest control.
These amendments, 65 years after the first law to control pesticides,
produced a complete metamorphasis of the Federal pesticide regulatory
scheme and completely redefined its thrust. FIFRA was changed from a
labeling law into a comprehensive regulatory statute controlling the
manufacture, distribution and use of pesticides. While FIFRA as
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amended is still concerned with the older concepts of 1910 and 1947,
that is the effectiveness of pesticides, deceptive labeling and
registration of pesticides; its thrust is primarily oriented toward
the protection of man and the environment from unreasonable adverse
effects caused by pesticide use. This orientation produced a
reordering of priorities in the sense that human health effects and
environmental effects are of primary concern in pesticide registration
and the regulatory actions taken by EPA.
Two other laws also provide safeguards for the protection of
man from potential long term or chronic adverse effects of pesticides
and add their authority to controlling misuse by requiring the establish-
ment of pesticide tolerances and providing enforcement procedures.
Federal regulatory control of pesticide residues in food and animal
feeds is exercised through the Federal Food, Drug and Cosmetic Act
(1938) primarily administered by the Food and Drug Administration (FDA).
EPA has the responsibility for establishing the pesticide tolerances and
FDA's responsibility is that of enforcement. Like FIFRA, this Act has
undergone a series of amendments over many years and has progressed from
concerns with adulteration and misbranding to regulation and control
of useful chemicals in the food supply. The Pesticide Amendment of
1954, which inserted Sec. 408 into the Act, provided a simplified
mechanism for the establishment of tolerances for pesticide residues
in or on raw agricultural commodities. In an important departure from
previous food legislation, it provided only that the additive pesticide
be certified as useful (not required) in production of the food. A
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Federal Register notice of March 11, 1955, contained a large number
of tolerances established under authority of Sec. 408 arising from the
findings of fact from a 1950 Spray Residue Hearings. All subsequent
tolerances were pursued through individual pesticide petitions as
prescribed in the Procedural Regulations for Sec. 408. The 1958
Food Additives Amendment inserted Sec. 409 into the FFDC Act. This
Amendment provides extremely broad regulatory coverage to additives
in foods but its important impact on pesticides is that it provided
the mechanism to regulate pesticide residues in processed foods to
complement the authority under Sec. 408 on raw agricultural commodities.
The Meat and Poultry Inspection Act is administered by the U. S.
Department of Agriculture. The act does not itself provide for the
establishment of pesticide residue tolerances in meat or poultry.
It does, however, provide enforcement procedures for compliance with
tolerances for pesticides in meat and meat products established by
EPA under the applicable provisions of the FFDC Act.
This very brief history of pesticide regulatory authority should
provide some insight into the various kinds of regulatory responsibilities
shared by many individuals within EPA and other agencies interested
in pesticide problems. The scientific inputs required to effectively
administer the amended FIFRA and establish tolerances are enormous and
are seriously straining the ability of the Registration Division's
technical resources to keep pace with developments impacting mandated
responsibilities.
Perhaps the individuals most affected by the reorientation of
priorities toward human and environmental effects, are those individuals
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we might call regulatory lexicologists and environmental hazard
evaluators. These designators should be interpreted in the broadest
sense, since many diverse scientific disciplines are involved.
These individuals bear the responsibility for examining and evaluating
pesticide data submitted to them from various sources; rendering an
opinion as to the adequacy and scientific integrity of the submitted
data; making a judgment as to the relevance of the data to the
perceived risks to human health and the environment; and ultimately
recommending some type of regulatory action. It is obvious that
the quality of the judgments made and the quality of the regulatory
actions recommended are dependent upon the adequacy of the submitted
data, the adequacy of the available data base to which the submitted
data are compared, and the training, competence, and experience of
the individuals involved. Therefore, the continued development of
adequate data bases by basic research in various health and environ-
mental disciplines, the sharing of opinions as the present state of
the art (as we have been doing at this conference) and the strength-
ening of benefit/risk analysis are essential if society is to benefit,
in the long run, from pesticide regulatory decisions.
A second important insight which the brief history of regulatory
authority should produce, is that while the regulatory scientist
endeavors to render judgments and opinions consistent with his
training, experience and the available data base, he is not entirely
free of the legal influences which are an essential part of a regulatory
agency's mandate and environment. For this reason, he is frequently
involved in actions of regulatory importance even though the basic
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sciences have not yet provided a firm base for sound regulatory
policy or action. This is especially true in the broad and complex
areas of environmental effects, carcinogenicity, and mutagenicity.
These examples are by no means exhaustive, since the subject of this
conference falls into the same category and more examples could be
put forward. When society demand regulatory action under these
circumstances, the vacuum is filled somehow, the results are usually
controversial to say the least, and the action taken might be inadequate
in the long run.
This conference was planned nearly a year ago to prevent, if
possible, this type of situation from occurring in the area of delayed
neurotoxicity. It is of course too early to judge the results in this
respect, but I believe that it has been successful in several ways.
While gross observations for neurotoxicity are an integral part of
almost every toxicological study, this conference reemphasized the fact
that the various species generally used in these tests are not especially
sensitive indicators of delayed neurotoxicity. It has indicated that
while knowledge of the possible mechanisms of action for some organo-
phosphates has increased, the techniques, procedures and test systems
relied upon by regulatory agencies in many respects have remained static.
More important than these benefits, this conference has gone a long way
in satisfying the regulatory needs for procedures which can measure
dose effect relationships. Still to be addressed is the questions of
potential cumulative effects of long term, low level exposure such as
might be received through the food supply or from long term occupational
exposure. It is possible, by application of the results of this
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conference, that regulatory scientists can arrive at an intelligent
solution of this problem. As many of you know, the EPA is currently
revising the Guidelines for Registering Pesticides in the U. S. The
results of this conference could improve the Methods for Study of
Neurotoxicity section in the Appendix of the Guidelines in a signifi-
cant way. We in the sponsoring organizations thank each of you for
these contributions.
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DR. M. 1C JOHNSON: I have been questioned quite a bit about what
one does about translating from the fundamental investigations to the
regulatory aspects. I have one suggestion which I think might be
worthy of being discussed, a propos of guidelines which were discussed.
I think it requires that we accept three presuppositions. First,
we must accept that the acute effects of organophosphate insecticides
are basically a reflection of their anti-cholinesterase activity, and
also that the delayed neurotoxic effects, as and when they appear, are
a function of that anti-neurotoxic esterase activity. Second, we must
accept that some sort of test on hens is going to be worthwhile, and
last, we accept that for most OP pesticides, the active antiesterase
molecule is in fact the oxon. I realize this, perhaps for some
materials, may be a problem, but for a majority I think we would accept
that the active anti-esterase molecule is the oxon. Could I suggest
then that, as a minimum, it would be worthwhile baseline information
to measure, in vitro, the activity of the appropriate oxon against acetyl
cholinesterase and neurotoxic esterase, presumably in hen brain or some
appropriate tissue just to have those numbers in front of one when
thinking about neurotoxic potential.
I make no suggestion as to whether one number should be larger
than the other or what one does about the fact that from cholinesterase
poisoning there is a chance of recovery and treatment with atropine and
RAM, and with neurotoxicity there doesn't seem to be much chance. But
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it seems to me to have those numbers available for intelligent
thinking might well be useful.
DR. 0. E. PAYNTER: I think that what you have just said, is
a significant contribution to this meeting. I think that's the
way that regulatory agencies would want to go although I would have
one caution. Numbers, in some places, become quite holy and sacred
and set in concrete. I noted yesterday, you made a plea that hopefully,
some regulatory agency wouldn't set some level and say, anything above
this is banned, nasty, or something else. Hopefully that won't happen.
But, I suggest that it may. I would not discourage anyone from using
these two systems, the hen test and the particular test system that you
are recommending. I think that's the way to go.
DR. J. E. CASIDA: I think the suggestion is quite a useful one,
but the number of compounds that would have to be tested will be quite
large when you consider, in some of the commercial compounds, all
of the metabolites that contain anti-esterase activity and all of the
photo products that do the same. So, that it might not be quite as
simple, as they discovered in mutagenesis tests, where they had to make
ramifications to accomodate such features.
DR. W. UPHOLT: Ladies and Gentlemen, thank you for your comments
and discussion. My thanks also to the speakers for their excellent
presentations. There are a number of conclusions that can be drawn
from these two days. But what I prefer to think is that in order to
avoid serious pitfalls, we must be constantly alert to the hazards, and
also must have a better understanding of cause and effect. You have
helped us by your participation in this conference, over these categories,
and we do appreciate it.
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I want to especially thank those distinguished scientists who
have come from abroad. They have added immeasurably to this
conference.
If there are no more comments, we stand adjourned, and thank
you very much for participating.
*U.S. GOVERNMENT PRINTING OFFICE: 1976-641-317/5517 Region No. 4
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-76-025
3. RECIPIF.NTT- ACCESSION-NO.
4. TITLE ANDSUBTITLE
Pesticide Induced Delayed Neurotoxicity
Proceedings of a Conference
5. REPORT DATE
July 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Ronald L. Baron (Editor)
8. PERFORMING ORGANIZATION REPORT NO,
. PERFORMING ORGANIZATION NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
Conference held February 19-20, 1976, Washington, D.C.
National Institute of Environmental Health Sciences.
Co-sponsored by the
16. ABSTRACT
A delayed neurotoxicity syndrome, shown to be induced by certain organo-
phosphorus esters, was first reported almost 50 years ago. The 1975 Guidelines
for Registering Pesticides in the United States reflect a continuing concern
for this problem in the recommendations made for evaluating the delayed neurotoxicit
hazard associated with organophosphate pesticide use.
This conference was designed to bring together those individuals interested
in the organophosphorus-ester-induced neurotoxicity syndrome to stimulate dis-
cussions and to evaluate the latest scientific results. With the aid of formal
and informal presentations, new pathways for investigation are becoming apparent
which may lead to the understanding and elimination of this problem.
KEY WORDS AND DOCUME-NT ANALYSIS
DESCRIPTORS
Toxic Diseases
Nervous System Disorders
Pesticides
Proceedings
h.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI l'iuld/(imup
06 T
. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
306
20. SECURITY CLASS (Thispage!
UNCLASSIFIED
22 PRICE
EPA Form 2220-1 (9-73)
301
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