450R76101
THE REPORT OF THE
LEPTOPHOS ADVISORY COMMITTEE
TO
THE ADMINISTRATOR
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C
20460
OCTOBER 1976
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TABLE OF CONTENTS
Page
LEPTOPHOS ADVISORY COMMITTEE i
ACKNOWLEDGEMENTS ii
LIST OF TABLES iii
I INTRODUCTION 1
II CHEMISTRY 4
III METABOLISM 10
IV THE TOXIC EFFECTS OF OP INSECTICIDES 16
AND INHIBITION OF CHOLINESTERASES
V DELAYED NEUROTOXICITY 28
VI CARCINOGENESIS, MUTAGENESIS, AND TERATOGENESIS 53
(INCLUDING REPRODUCTION STUDIES)
VII POSITION STATEMENTS ON 'CHARGES 61
VIII CONCLUSIONS AND RECOMMENDATIONS ' 6?'
IX REFERENCES 69
X APPENDICES
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LEPTOPHOS ADVISORY COMMITTEE
Julius M. Coon,Ph.D., M.D., Chairman
Emeritus Professor of Pharmacology
Jefferson Medical College
Thomas Jefferson University
Philadelphia, Pennsylvania 19107
(Tel.215-829-7766)
Seymour L. Friess, PhD.
Chairman, Environmental Biosciences Department
Naval Medical Research Institute
Bethesda, Maryland 20014
(Tel. 301-295-1163)
Tetsuo R. ^ukuto, PhD.
Professor of Entomology, Chemistry,
and Insect Toxicology
Department of Entomology
University of California
Riverside, California 92502
(Tel. 714-787-5824)
Bernard P. McNamara, PhD.
Chief, Toxicology Division
Biomedicals Laboratory
U.S. Army Material Command
Edgewood Arsenal, Maryland 21010
(Tel. 301-671-3034)
Gerald M. Rosen, PhD.
Assistant Professor of Pharmacology
Department of Physiology and Pharmacology
_Duke University Medical Center
Durham, North Carolina 27710
(Tel. 919-684-6305)
I hereby certify that this report has been approved by
each of the members of the Leptophos Advisory Committee.
Chairman, Leptophos Advisory
Commit tee
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11
ACKNOWLEDGEMENTS
On behalf of the Leptophos Advisory Committee, the
chairman wishes to thank the many persons who have provided
e'xhibits, testimony, suggestions and other assistance which
has been most useful in the preparation of this report — those
named as well as others who gave their time.
Special credit goes to Mr. Jerry A. Moore of the Office
of Special Pesticide Reviews (OSPR), Office of Pesticide
Programs who took over as Secretariat of this Committee with
only 23 working days remaining to meet a regulatory deadline
of October 20, 1976. The former Secretariat, Mr. David
Bowen, is owed many thanks for his excellent work in organiz-
ing and starting the work of the Committee prior to his
leaving the Washington Office on a special assignment
for the Environmental Protection Agency.
Last, but not least, thanks go to Mr. Ronald E. Dreer,
/
Acting Director of the Office of Special Pesticide Reviews
who unselfishly provided the time of three persons from his
office who were responsible for getting the report together.
These are the Secretariat, Mr. Moore5 Ms. Jacqueline B.
Martin who is an accomplished typist, and Mr. Edward Thomas
who was responsible for the writer/editor task of this
report.
Julius M. Coon, Ph.D., M.D. Chairman, Leptophos
Advisory Committee
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Ill
LIST OF TABLES
Table I Percent of Samples with Detectable Residues 14
Table II Percent Distribution of Residue Components, 15
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I. INTRODUCTION
The Leptophos Advisory Committee was established under
the authority of Section 408 of the Federal Food, Drug and
Cosmetic Act to consider the proposal of the Environ-
mental Protection Agency (EPA) to revoke a regulation which
established tolerances for the insecticide leptophos on
lettuce and tomatoes. The committee was appointed by EPA's
Assistant Administrator for Water and Hazardous Materials
and is composed of candidates nominated by the National
Academy of Sciences - National Research Council. The
committee's charge as initially given was as follows:
The Advisory Committee is charged to con-
sider and evaluate all relevant scientific evi-
dence concerning the safety of leptophos, its
metabolites and degradation products (hereinafter,
leptophos). The Committee is to submit a report
and recommendation on the proposed revocation of
the tolerances on lettuce and tomatoes together
with all underlying data and a statement of the
reasons and basis for the recommendations.
Specifically the Committee should express its
opinion on the following:
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(1) Whether leptophos has a delayed neurotoxic
effect in hens, rabbits, sheep, water buffalo or any
other animal.
(2) Whether there are other toxic effects from
use of leptophos.
(3) Whether there is a potential for hazard to
man from ingestion of any amounts of leptophos.
(4) Whether there is a potential for hazard
to man from ingestion of leptophos residues on lettuce
of 10 ppm and on tomatoes of 2 ppm or from lesser resi-
dues .
(5) Whether leptophos bioaccumulates and is
persistent and therefore poses a hazard to man or the
environment.
(6) Whether exposure to leptophos causes adverse
effects which to the extent necessary to protect the
public health, make it unsafe to use.
Charges (5) and (6) of the foregoing were later changed
to read:
(5) Whether leptophos bioaccumulates in tissue
and/or is persistent and therefore poses a hazard to
man.
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-(6) Whether exposure to leptophos in food,
including raw agricultural commodities, causes adverse
effects to man which, to the extent necessary to
protect the public health, make it unsafe for use.
A tolerance for a pesticide must be established under
the Federal Food, Drug and Cosmetic Act before such pesticide
raay^ be lawfully marketed in commerce under the Federal
•
Insecticide, Fungicide, and Rodenticide Act for use where
there is any reasonable expectation that residues of such
pesticide may occur in or on raw agricultural commodities.
The committee was informed that leptophos had been used in
the United States in past years under the provision of a
oH j.xmZuCu cl C iT £ ci £ u U i_ *.iiat_ I c 1 1 a 3 HCt- u3£ii
registered for general use.
On May 31, 1974 the EPA issued a regulation published
in the Federal Register (39 FR 19208) establishing toler-
ances for leptophos and its metabolites on lettuce at 10 ppm
and tomatoes at 2 ppm. These tolerances were set on the basis of
information contained in the petition filed by the Velsicol
Chemical Corporation.
On May 27, 1975 the EPA announced in the Federal
Register (40 FR 22817) its proposal to revoke the aforemen-
tioned tolerances on the basis of a reevaluation of the
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petition and other information confirming that leptophos is
an agent which produces delayed neurotoxicity in hens. That
notice further stated:
"additional information on leptophos is
necessary to evaluate the possible hazard
to man and other non-target species from
the potential effects of its use."
Following that notice, the Velsicol Chemical Corpora-
tion requested that the matter be referred to an advisory
committee as was its right under the statute; however, it
sub sequerrt ly withdrew its request on August 18, 1976.
Nevertheless, the EPA informed the advisory committee on
August 27, 1976 of its desire that the committee furnish its
report and recommendations under the charge previously
given.
II. CHEMISTRY ,
A . C ompos i t ion.
The principal constituent of the organophosphorus
(OP) insecticide leptophos (trade name PHOSVEL, Velsicol
Chemical Corporation code number VCS-5Q6) is £-methyl
(4-bromo-2,5-dichloropheny1 phenyIphosphonothioate (struc-
tural formula below):
„__/"<,»
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The pure compound is' a white crystalline solid, m .p . 71.5-
o o
72.0 C, specific gravity (supercooled liquid) 1.53 at 25 C
(10) .
Technical leptophos is reported to have the following
typical composition (86):
Compound
leptophos
CH30
Cl
I
Cl
87.0%
0,0-dimethyl
phenylphosphonothioate
p(OCH3)2
3. 5%
0 ,0-bis- (4-bromo-
2, 5-dichlorophenyl)
phenylphosphonothioate
Cl
•(o
Cl
4 .0
Cl
4-brono-2,5-dichlorophenolBr
OH
Cl
Volatiles
0. 5%
Miscellaneous
Compo und s
2.5%
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In addition to the components listed above, desbrorao-
leptophos [ (0_-( 2 , 5-dich lor opheny 1) 0_-methyl phenyl phosphono-
thioate] and the S_-methyl isomeride [0_-( 2, 5-dichlor opheny 1)
»
S-methyl phenylphosphonothioate] also have been reported as
impurities in technical leptophos in minor amounts (65,86).
The presence of desbromo-leptophos is of particular interest
since this material is approximately 3-fold more effective
than leptophos as a neurotoxic agent (33).
B. Photoalteration.
Compared to most organophosphorus insecticides, lepto-
phos is relatively stable to photodegradation under atmos-
pheric conditions (86,65). Exposure of thin films of
leptophos on glass plates to sunlight outdoors for 77 days
resulted in significant degradation of leptophos to a
variety of products (65,86). The principal photodegra-
dation products detected by glc analysis were leptophos
oxon, desbromo-leptophos, 0-methyl phenylphosphonothioic
acid, 0-methyl phenyIphosphonic acid, 4-bromo-2,5-chloro-
phenol and 2,5-dichloropheno1. Evidence was also obtained
for the presence of minor amounts of the S-inethyl isomeride
of leptophos and other dehalogenated products of leptophos.
Pathways for the photodegrad at ion of leptophos are on the
next page :
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Cl
Cl
leptophos oxon (major)
0
CH3S
'/
Cl
Cl
^-methyl isomeride
(minor)
Br / \ ^fV"
CH30
Cl
leptophos
Cl
Cl
-fHO-^ VBr
Cl
(major)
CH30
minor amount of other
dehalogenated products
of leptophos
"desbromo"-leptophos
(major)
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8
Analogous studies with leptophos oxon standard showed that
it was slightly less stable to photolysis than leptophos, giving
comparable alteration products. One of the principal products of
leptophos oxon was desbrono-leptophos oxon.
Desbromo-leptophos is reported to be the major altera-
tion product observed after UV irradiation of a chloroform
solution containing leptophos (58). In acetone solution,
leptophos was converted to two major products, desbrono-
leptophos and another compound which is believed to be
^-methyl C^,P_-( 5-ch loro-2 , 2 ' -bipheny1ene)phosphonothioate
(structure below). (Velsicol Petition, Section D, Part
IV).
This substance may be formed from desbromo-leptophos by
a photo-indueed cyclization reaction in which HC1 is lost.
The toxicological properties of this compound are unknown but
it is structurally closely related to the salinigen phos-
phates which are potent neurotoxic agents. The cyclic
compound, however, has not been detected as a residue in any
crop treated with leptophos in the field.
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The photolytic transformation of leptophos to deobrocio-
leptophos may be of concern owing to tt.e increased neuro-
toxic activity of this material. Desbromo-leptophos also was
detected in the surface extract, although in minor amounts,
when leptophos applied to the surface of cotton leaves was
exposed to sunlight for 12 weeks (65).
C. Hydrolysis-.
Leptophos evidently is stable at ambient
temperature after prolonged exposure in acidic media but
hydrolyzes slowly under strongly alkaline conditions. The
effect of pH and temperature on the rate of hydrolysis of
leptophos has been studied (26). In acidic medium at 25°C
(pH range 1-7) leptophos was relatively stable, e.g. at
pH 1.0 only 0.2% of the initial concentration was detected
as hydrolytic products after 48 hours. Susceptibility to
hydrolysis increased with increasing pH and under neutral
conditions approximately 30% of the charged leptophos was
hydrolyzed after 4 days. Hydrolysis in the pH range 8-11
(buffer) was first order with respect to leptophos concentra-
tion.'-and from the 1st order rate cons t an t-r-the half-lives
I-*.-' '^is
(t 1/2) of leptophos (25°C) at pH 8.0, 9.0, 10.0 and 11.0
were calculated to be 160, 104, 84, and 28 hours, respec-
tively.
Decomposition products identified after acid hydrolysis
were desmethyl leptophos, phenylphosphonic acid, and 0_-methyl
pheny1phosphonothioic acid. Identified as alkaline hydroly-
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10
sis products were phenylphosphonic acid and £-methyl phenyl-
phosphonic acid. Surprizing ly, no 0-methyl phenylphos-
phonothioic acid was reported for alkaline hydrolysis. Since
32P-labeled leptophos was used in these studies, phenolic
products were not identified.
D. Thermal alteration.
Leptophos heated at 180°C for 5 hours resulted in
85% decomposition. At 208°C, decomposition was complete
after 2 hours. The major product was the S_-inethyl isocieride
of leptophos (86).
III. METABOLISM
A. Metabolism in mammals.
The metabolism of leptophos has been examined in
rats and mice (86,25)'. Leptophos administered orally to
rats and mice was rapidly absorbed and eliminated from the
body, mainly as water soluble urinary metabolites. Elimi-
nation of radioactivity from rats treated orally with 0.8
rog/kg phenyl ring-labeled (14C)-leptophos (phenylphosphono-
thioate ring) was virtually complete after 96 hours with
80-88% of the dose detected in urine and 11-12% in feces.
After 24 hours, recovery ranged from 98-103% with 75-81% of
the recovered radioactivity observed in the urine. Excre-
tion of radioactivity after oral administration with phenoxy
ring (l4C)-leptophos was highly erratic with 8-S5% of the
recovered radioactivity appearing in the urine and 8-84% in
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the feces 96 hours following treatment. These studies using
a total of 4 rats of each sex showed extremes in biological
variation with some animals excreting more in urine than in
feces and others reversing the trend. However, in all cases
elimination was rapid and a major portion of the applied
radioactivity was eliminated within 24 hours. Analysis of
skin and fat showed levels of radioactivity of about 1% of
the administered dose. In adipose tissue, the major residue
was leptophos along with traces of the phenol.
Analogous studies with mice (25) treated orally
at 25 mg/kg dosage with phenoxy (14C)- leptophos showed that
radioactivity was rapidly eliminated with virtually complete
elimination within 48 hours. The bulk of the recovered
radioactivity remained in the urine and about 4% was present
in feces. Compared to the phenoxy label, the rate of
elimination of pheny1-labeled radioactivity was notably
slower and rad io_ac t ivi ty was detected in the urine as long
as 6 days after treatment.
The products obtained from the metabolism of leptophos
in rats and mice were those normally expected of a phospho-
nothioate ester and the major portion of the metabolites
were identified as hydrolytic and oxidative products.
Although several different metabolites were detected in rat
excreta, their structures evidently were not confirmed.
Leptophos was identified in the feces but none was found in
urine.
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12
The major metabolites isolated from mouse urine after
oral administration of (14C )-leptophos were 0_-methyl phenyl-
phos phono thioic acid, £-tne thy 1- phenyl phos phonic- ac id ,
leptophos phenol and a conjugate of leptophos phenol. Small
amounts (1-2%) of leptophos and leptophos oxon were found in
feces. Evidence also was obtained for the presence of a
minor amount ( 1%) of 0-(4-bromo-2,5-dichloropheny1) phenyl-
phosphonic acid in urine. The pathways for metabolism of
leptophos in mice may be depicted as follows:
OH
Cl
(leptophos)
Cl
-t- HO -<' y- Br
kci
V
conjugate
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13
Thus, the metabolism of leptophos was straightforward and
unexpected metabolites were not observed.
B. Me t abo1i sm in plants.
Qualitatively, the degradation of leptophos in or
on cotton leaves o'f plants contained in a greenhouse was
similar to that occurring in the mouse (25). The major
component present in recoverable radioactivity one week
after application was unchanged leptophos (91-96%) and the
amount of this material gradually diminished with time to
about 29% after nine weeks. Most of the leptophos remained
on the leaf surface although significant but small amounts
were absorbed into the leaf. Only trace amounts of lepto-
phos oxon were observed during the 9-week sampling period.
Other metabolites observed were the phenol (in the form of
an unknown salt), 0_-methyl 'pheny 1 phosphonothioic acid,
0-methyl pheny1phosphonic acid and pheny1phosphonic acid. No
desbromo-leptophos was observed.
Determinations of residues of leptophos and metabolites
have been conducted on a wide variety of crops, including
corn, cotton, vegetable and agronomic crops. In most cases
quantitative and qualitative analyses of leptophos and
metabolites were achieved by gas chromatography. Leptophos,
leptophos oxon and the phenol have been detected by glc
analysis of corn treated with leptophos and milk from
cows fed treated corn (15,16). These materials also have
been detected in leptophos fieId-treated wheat (43),
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rapeseed grain (42), a wide variety of vegetables includ-
ing broccoli, celery, lettuce, carrots, onions and tomatoes
(40,18), grapes (18), and coastal Bermuda grass (12). In
some cases thin-layer chromatographic and cholinesterase
inhibition analyses were used to reaffirm the presence of
the oxon.
In addition to leptophos, leptophos oxon and the
phenol, the photoalteration product desbromo-leptophos also
was observed in a number of different crops (Velsicol Chea.
Corp.). This material undoubtedly is formed from leptophos
on the plant surface by the action of sunlight. A summary
of residue data, giving both pattern of detection and
residue composition, is presented in Table? T and TT. Rates
of application varied with the crops, ranging from 0.45 to
2.7 Ibs. actual material per acre.
Table I. Percent of Samples with Detectable Residues'"
Ty
pe of
S amp 1 e
Co
Co
Po
le crops
rn
Ke rnel
Ke rne 1 & cob
Cob & husk
Stalks & leaves
t atoes
Tomatoes
Le
Co
1 1 uce
ttonseed
Lepto
93.
18.
30.
100.
100.
30.
98.
100.
95.
oho s 0
8
1
2
0
0
8
0
0
0
3
2
6
7
2
xon
3.
9.
5.
6.
8.
3.
40.
6
9.
1.
3
0
6
7
8
1
2
0
5
Phe
59
0
2
66
100
84
70
73
1
nol
. 4
. 0
. 3
. 7
. 0
. 6
. 6
. 8
. 5
De sb
lept
3
8
9
4
7
r orao-
o pho s
8.
0.
2.
S.
5.
9.
4.
1.
2.
5
0
3
9
4
5
3
9
0
*Informat ion concerning the time at which analysis were
conducted was not available. (Velsicol Chemical Corp.
Petition, p. 1733-1748, Part IV).
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Table II. Percent of Distribution of Residue Components
Ty
pe
S amp
Co
Co
Po
To
Le
Co
le
rn
ta
ma
o
le
c
Ke
Ke
Co
St
to
f
.
rops
rnel
rnel & cob
b & husk
alks & leaves
es
toes
ttuc
tt
on
e
seed
De sb r omo-
Lep to
90.
100
100
95.
90.
12.
82.
94.
95.
pho s
A
4
7
4
8
5
0
Oxon
4.
0.
0.
1.
1.
13.
5.
2.
1.
8
0
0
6
4
0
5
1
5
Pheno 1 leptophos
2.
0.
0.
0.
7.
73.
8.
1.
1.
7
0
0
9
0
o -
7
6
5
2.
0.
0.
2.
0.
1.
3.
1.
2.
1
0
0
1
9
2
0
8
0
*Same as Table I.
The presence of desbroino-leptophos as residues in these
crops should be taken into account in the assessment of
hazards a I" 1° S i n cr from thp rnnc;umnt-i_rin r> f le^to^hos trSEted
crops.
C. Model E c o s y s t_gm Studies.
The fate of 0_-methyl ( 14C)-leptophos has been
investigated in a terres trial-aquatic model ecosystem (13).
Leptophos was exceptionally stable in this system and
persisted in different organisms, e.g. fish, snail, and
algae, over an experimental period of 49 days. Accumulation
in fish, G ambus i a a f f ini s, was about 2-to 200- fold greater
with leptophos compared to other typical organophosphorus
insecticides such as ch lorpyrifos, dyfonate, and parathion.
Ecological magnification, i.e. the ratio of the amount of
leptophos present in snails and in water, was approximately
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48,000. Th'is was 5-to 550- fold greater than the ecological
magnification observed with other organophosphorus insecti-
cides examined. Overall,'the results indicated that lepto-
phos is the most accumulative and persistent organophos-
phorus insecticide ever examined in the terrestrial-aquatic
model ecosystem. This is consistent with the long-term
persistence of leptophos reported in cotton leaves (9
weeks), tomatoes (7 weeks), and grapes (7 weeks) (25,18).
IV. TOXIC EFFECTS OF OP INSECTICIDES AND INHIBITION OF
CHOLINESTERASES
A. Signs and Syjnp_t_ocis of Acute Poisoning by OP
Insectic id e s
Signs and symptoms of acute systemic poisoning
by OP insecticides are predictable from their biochemical
mechanism of action. Thus • inhibition of acety1cho1ineste-
rase results in accumulation of endogenous acetyIcho1ine in
nerve tissue and effector organs with signs and symptoms
that mimic the muscarinic, nicotinic and central nervous
system actions of acetylcholine. The muscarinic receptors
are located at the effector organs of the parasympathetic
nervous system. The signs and symptoms of OP insecticides
poisoning that result from stimulation 'of these receptors
include: tightness of the chest and wheezing due to bron-
choconstriction and increased bronchial secretion, increased
salivation, increased sweating, increased gastrointestinal
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tone and peristalsis with consequent development of nausr.a,
vomiting, abdominal cramps, diarrhea bradycardia that
can progress to heart block, frequent and involuntary
urination, and raiosis.
Nicotinic signs and symptoms result from accumula-
tion of acetyIcholine at the endings of motor nerves and
autonomic ganglia. Muscular effects include easy fatigue
and mild weakness followed by involuntary twitching and
muscular weakness that affects the muscles of respiration
and contributes to dyspnea and cyanosis. Nicotinic actions
at autonomic ganglia may, in severe intoxication, mask some of
the muscarinic effects. Thus, tachycardia may result from
stimulctien cf sympathetic g?nglia to cvprrorip fhp usual
bradycardia due to muscarinic action on the heart. Other
effects like elevation of blood pressure and hyperglyceniia
are often reflected by nicotinic actions at sympathetic
ganglia.
Accumulation of acety1cho1ine in the central
nervous system is believed to be responsible for the tension,
anxiety, restlessness, insomnia, headache, emotional insta-
bility and neurosis, excessive dreaming and nightmares,
apathy and confusion in poisoning by OP insecticides.
The immediate cause of death in fatal OP poisoning is
asphyxia resulting from respiratory failure.
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18
^ • Local izeci^f f ec t s
Localized effects at the site of exposure may be
seen in the absence of obvious signs and symptoms of systemic
absorption as described earlier. Exposure to vapors can
exert local effects on smooth muscles of the eyes, resulting
in early miosis and blurred vision due to spasm of accomo-
dation.
Secretory glands of the respiratory tract may be
affected by minimal inhalation leading to watery nasal
discharge, nasal hyperemia, sensation of tightness in the
chest and prolonged wheezing respiration. Local effects of
dermal exposure include localized sweating and fasciculations
at the site of contact.
Gastrointestinal manifestations are usually the
first to appear after oral ingestion and are due to local
anticholinesterase action in the gastrointestinal tract.
C. Sys temic E f fec t s
Systemic effects are for the most part similar,
irrespective of the route of absorption, but the sequence may
differ. Respiratory and ocular symptoms would be expected
first after exposure to airborne OP insecticides. If oral
or dermal exposure is first, gastrointestinal symptoms and
•localized sweating would occur. The onset of symptoms
after exposure is usually rapid, within minutes to several
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19
hours. The duration of symptoms is generally from one
to five days. It should be recognized that, in addition to
the usual factors of route and exposure and concentrations of
active material, the quality of the signs and symptoms,
their rate of onset and their duration may differ markedly
depending on the OP insecticide, its metabolism and affini-
ties for acetylcholinesterase.
D * Metabolism-ToxicityRelationship
It became apparent when studying parathion and
its oxygen analog, paraoxon, that in addition to conferring
•greater stability against non-enzymatic hydrolysis, substitut-
ing P=S for P=0 altered the toxicity of the compound. It
was observed that parathion was less toxic to animals
than paraoxon even though both compounds inhibited acetyl-
cholinesterase and thus produced similar cholinergic signs
and symptoms of poisoning. However, in further studies,
highly purified parathion was found not to inhibit cholines-
terase activity, j.n vitro, and the inhibitory activity of
commercial parathion was attributed to contamination with
the S-ethyl and S-phenyl isoiners of parathior or with its
oxygen analog, paraoxon. Later studies indicated that
paraoxon is the active agent responsible for cho1inesterase
inhib it ion.
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E . Results of C hoi ine s t e^ase S tud le_s
1. Studies with the Rabbit
a. Acute Toxicity - Definition: a single or
multiple exposure in which the observation period for
toxicity is less than one month.
Kamel et al. (23) have studied the anticho-
linesterase activity of wettable powder containing 50%
leptophos. The authors observed significant decreases
in serum cholinesterase activity 9-48 hours after a single
oral administration of this mixture at the LD-50 (124.2
mg/kg). Other cholinesterase activity also seemed to be
impaired since sever? cholinergic symptoms were apparent
(inus c ar inic 3 nicotinic and C.N.S.). The symptoms., increased
respiration rate, salivation, watery diarrhea, lack of
co-ordination and convulsions, started within three hours
post administration. Survivors of the LD-50 showed only
respiratory disturbances and sometimes wheezing.
Animals receiving 31 mg/kg showed no symptoms
of cho 1 ines ter ase inhibition (23). When the dose x^as
raised to 62.2 ing/kg and 93.1 mg/kg, the animals exhibited
symptoms of cho1inesterase inhibition, but to a lesser
degree than with the LD-50. The authors noted that all
animals given the LD-50 exhibited symptoms of cho1inesterase
inhibition even though serum cholinesterase was not inhi-
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bited significantly until 9 hours after administration. The
serum cholinesterase approached the normal level after 60
hours, the time when the death rate was quite low. After 72
hours, levels of pseudocholinesterase were at normal and no
more deaths were observed. The authors suggested that the
relationship of symptoms and serum enzyme activity may be
due to the selective nature of the compound towards differ-
ent cholinesterases.
2. Sub-acute Toxicity - Definition: a single or
multiple exposure in which the observation period for
toxicity is one to three months.
Johnson et al (16) studied the effects of
corn which had been sprayed before harvest with 0.56,
1.12 and 2.24 kg per hectare in 468 1 of water (residue of
leptophos was estimated to be 56-66% of amount applied).
After being ensiled for 62 days, the silages were fed to 16
Jersey cows for 56 days. Whole blood cholinesterase activity
was measured as a function of leptophos sprayed per hectare.
At the lowest level (0.56 kg/hectare) no apparent cholinester-
ase inhibition was observed and only 12% and 19% cholinester-
ase depressions were observed at the higher levels (1.12 and
2.24 kg/hectare). As in the case of the rabbit, depression
of blood cholinesterase was less than anticipated from the
symptoms observed.
-------
3. Studies with the Rat (86,65).
a.- Acute Studies
Groups of rats (both sexes) were fed
leptophos in the diet for 10 days at levels of 0, 3, 10,
a.
30, and 100 ppm. No depression of plasma chol ines ter'se was
observed. The red blood cell and brain cholinesterases were
depressed at the higher levels (30 ppm and above). When the
levels of leptophos were increased to 100, 250 and 500 ppn
for a 28 day regimen, a dose-dependent depression of cholines-
terase activity was observed.
In a second series of experiments, female
rats were fed levels of 0, 50 and 75 ppm for 14 days. Red
blood cell cholinesterase levels were decreased by 22% at 50
ppm and 32% at 75 ppm after 7 days, but inhibition of plasma '
cholinesterase was not apparent until the end of the second
week. This observation agrees well with other animal
studies.
b. Sub-acute Studies
Groups of rats (both sexes) were fed
leptophos in the diet at levels of 0, 1, 5, and 10 ppra for
90 days. Cholinesterase activity in red blood cells,
plasma, and the brain were examined after the 40th day and
90th day. In all cases, the cholinesterase activity levels
were not depressed.
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23
c. Chronic Toxicity - Definition: a single or
multiple exposure in which the observation period for
toxicity is greater than three months.
Groups of rats (both sexes) were fed
leptophos for two years at concentrations of 0, 10, 30, and
60 ppra. Cho 1 ines t er as e activity was measured in the brain
over the first 90 days and showed no reduction in activity
at any dose level. Erythrocyte cholinesterase was slightly
depressed at the lower levels of lepthphos tested but only
at 60 pptn was there a significant reduction (more than
25%).'
4. Studies with Steers (86,65).
a. Acute Studies
Groups of steers were fed leptophos at
levels of 0, 15, 45 and 150 ppm for 4 weeks. Daily observa-
tions showed no abnormal signs of poisoning. Depression of
whole blood and plasma cholinesterases was observed only at
the two higher dose levels. Within a week after cessation
of leptophos feeding the depressed levels returned to
normal .
5. Studies with Chickens
a. Acute Studies (36,65).
Groups of chickens were fed leptophos in
the diet at levels of 0, 0.03, 0.01, and 0.3 ppm for
4 'weeks. Eggs were collected and incubated during the
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24
latter 14 days of treatment. The chicks were observe'7 for
14 days post-hatching, during which they were fed leptophos
in the diet. No signs of cho1inesterase poisoning were
observed. No enzyme studies were cited.
b. Sub-acute Studies (74d).
Groups of chickens were fed one single oral
dose of leptophos (200, 400, and 800 mg/kg) and cholineste-
rase activity was determined daily for 42 days. A dose-depen-
dent inhibition (800 mg/kg produced 47% depression of red
blood cell cholinesterase) was obtained with the greatest
degree of inhibition observed within the first five days.
Plasma cho1inesterase activity was signifi-
cantly reduced at the beginning of the experiment (800 mg/kg
produced 49% depression of activity). As in the case of red
blood cell cholinesterase, recovery occurred during the
experiment. It was surprising to note that after the
plasma cho1inesterase activity recovered by day six, a
decrease in enzyme activity reoccurred and then continued
for the remainder of the experiment, with the lowest level
being reached at day 15.
Groups of chickens were fed a daily dose of
0.5, 1, 2.5, 5, 10 and 20 mg/kg until ataxia developed but
no longer than 60 days (35). Daily feeding of a single dose
of leptophos gave a dose-dependent inhibition of red blood
cell cholinesterase. After the feeding of leptophos was
-------
25
stopped, enzymatic activity remained low for about 10 days
and then rapidly recovered. As in the case of red blood
cell cholinesterase, the inhibition of plasma cholinesterase
was dose-dependent, but upon completion of the feeding
regimen, the enzymatic activity did not return to the
control level within 20 to 40 days, at which point the
observations were discontinued.
When the animals were exposed to 0.5 rag/kg
for 72 days, the plasma cholinesterase activity was decreased
by as much as 40% at the termination of the experiment (73).
6. Studies with Rats Given Leptophos Oxon
(86,65).
a. Acute Studies
Groups of rats (both sexes) were fed
leptophos oxon for 28 days. The concentration of leptophos
oxon in the diet was increased'at weekly intervals from 200
ppm to 300 ppm to 500 ppm and finally to 800 ppia. Cholines-
terase activities of the red blood cell and of the brain were
depressed throughout the test. Plasma cho1inesterase were
depressed in females throughout the test, while in males the
depression became evident only after 14 days.
Groups of female rats were fed lepto-
phos oxon for 4 weeks at 0, 1, 5, 10 and 20 ppm. Red blood
and plasma cholinesterase activity were measured at 14 and
28 days. Plasma cholinesterase activities was unaffected
while erythrocyte cholinesterase activity was depressed at
-------
26
28 days. Plasma cholinesterase activity was unaffected
while erythrocyte cholinesterase activity was depressed at
10 ppm and above at the 14th day. By day 28, erythrocyte
cholinesterase activity had practically recovered.
b. Sub-acute Studies
Groups of rats (both sexes) were fed
leptophos oxon for 90 days at levels of 0, 25, 50 and
500 ppm. Inhibition of plasma cholinesterase activity was
noted at all levels for females while males were
affected at only 500 ppm at the 84th day. Inhibition of
erythrocyte and brain cholinesterases was similar in both
sexes.
(83,83a,83b,83c,83d).
Thirty-four subjects were exposed to
leptophos under field conditions for four eight hour days.
Twenty three subjects showed significant erythrocyte cholin-
esterase inhibition (about 30%). Recovery of enzymatic
activity was generally slow. In fact, two weeks after the
last exposure, only four subjects had enzymatic activity
which approached their control levels.
In the case of plasma cholinesterase, only
50% of the subjects exhibited mild inhibition. Recovery was
rapid at the end of the experiment.
8. Studies on Humans Involved in the Manufactur-
ing, the Packaging and the Application of Leptophos (84).
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27
On February 12, 1976, Drs. Tenaca, Hatch
and Markel submitted their report on a preliminary survey of
the workers in Velsicol's Bayport plant located in Houston,
Texas. Twen.ty-six of the 30 employees were given physical
examinations with special emphasis on neurological signs and
symptoms. Two examinees presented some neurological signs
and symptoms. It was unknown, at that time, whether their
findings were associated with exposure to leptophos.
A more recent report, dated May 26, 1976,
was submitted by Dr. Tanaca. He noted that during the five
year period (August 1969 to August 1974) the monthly results
of cholinesterase activity were found to be either 75% or
100% of the normal level. There were a few occasions in
vhicb the cholinss'!?'r''<5 c *! i v •? t y dropped to 50^> but upon
rechecking the data, the value always returned to either 75%
or 10 0% of normal.
In September 1974, the company switched to
the Becton-Dickinson Unopette method for determining red
blood cell cho1inesterase activity. The normal range was
listed as 0.68 - 1.04 pH units/hr. This appears to be a
more reliable test than the one previously employed.
With this method, several of the employees
each month showed levels below the 0.68 level. It was
interesting to note that some employees who had an abnormal-
ly low level in one month did not return the next month. It
is not known whether they were fired or voluntarily resigned.
In January 1976, the laboratory expanded the normal
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28
range from.0.4 to 1.2 pH units/hr. No abnormal values
have been registered since that date.
F. Conclusions
Like other OP insecticides, exposures to
leptophos lead to inhibition of cho1inesterase activity.
The variations in degree of inhibition can be related to
species differences and the experimental design. It is
noteworthy that chickens exposed to as little as 0.5 mg/kg
daily for 72 days exhibited considerable (40%) inhibition
of plasma cholinesterase. Humans exposed to leptophos
exhibited typical signs and symptoms of OP poisoning. It
was surprising to note that inhibition of erythocyte cholines-
terase activity was considerable while plasma cholinesterase
activity was only mildly inhibited, if at all.
In both rats and man, leptophos oxon, unlike
paraoxon, initially inhibits red blood cell cholinesterase
and later plasma cholinesterase. Since leptophos is a poor
in vitro anticholinesterse agent, its oxon metabolite is
assumed to be directly responsible for the in vivo enzyme
inhibition of choliaesterase activity.
V. DELAYED NEUROTOXICITY
Within the past decade a considerable body of evidence
has been accumulating worldwide indicating that ingestion of
leptophos, alone or in the presence of certain of its
degradation products, by a broad range of animal species,
can lead to a progression of neurotoxic effects including
-------
29
sis, and ultimately death. The literature on neurotoxic
actions of leptophos has been reviewed thoroughly in a
recent World Health Organization (WHO) report (87),. so that
for present purposes it may be useful to limit further
discussion to the salient features of the intoxication
processes which are clearly dose related, and which stem
from chemical interactions with neural tissues leading to
either reversible or irreversible alteration of tissue
structure or function.
The analysis of leptophos toxicity in animal models is
complicated by the general observations that: (1) There is
a considerable variation in species sensitivity to chemical
iasulL uy this material, a b registered in the variety and
intensity of toxic responses; and (2) the onset and progres-
sion of neurotoxic signs.in susceptible species of animals
are quite sensitive to the ingestion exposure factors of
dose per unit weight of animal, and the division of dosages
over total times. Accordingly, it is convenient to treat
the time and dosage factors regulating leptophos responses
in animals by a division of the discussion into sections
dealing separately with acute, sub-acute and chronic neuro-
toxicity studies and their results.
In many reviews of the phenomenon known as delayed
neurotoxicity produced by certain OP compounds, with tri-cr-
cresyl phosphate (TOCP) taken as a representative agent of
this class, the point has been made (4,5,7) that the mature
-------
30
chicken (hen) and the cat are relatively sensitive to this
class of neurotoxic chemicals. Further, the neuropathy
observed in these sensitive species is remarkably similar to
that observed in man following accidental intoxications with
OP compounds such as TOCP (4,5,8), some of which have
occurred on a massive scale in the past forty years.
Accordingly, the evidence for delayed neurotoxicity in
animal models as induced by ingestion of leptophos will be
examined starting with the most sensitive species tested, to
be followed by results obtained from studies with other
laboratory model animals and with farm animals potentially
subject to exposure to insecticide residues.
A. S tudies with the Chicken
Since early studies of the delayed neurotoxicity
syndrome induced by OP compounds in this species had shown
that the .mature hen (aged about 18 months) is the most
sensitive target of these chemicals, the bulk of published
studies with leptophos acting on the chicken employed the
hen. However, it should be noted that the male is also
susceptible, but to a lower degree, and has been used in
several leptophos studies.
1. Acute Studies
The literature contains a series of studies in which
chickens have received one or more doses of leptophos by
oral intubation or gelatin capsule, and then, been observed
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31
for onset of the classical delayed neurotoxicity syndrome.
The syndrome is characterized by a lag tine of some 10-18
days, followed by development of peripheral weakness,
ataxia, weight loss, paralysis and death. Most histological
studies have confirmed that the syndrome involves swollen
and degenerating inyelin sheaths and axons in the spinal
cord, and in peripheral nerves such as the sciatic.
However, interpretations have varied with regard to the
intensity of the effects observed.
Franklin (67) has reviewed the toxicological
evidence recently, beginning with the earliest acute studies.
In a feeding study using White Leghorn chickens and 0.30 ppm
of ieptophos in the food tor 28 days, Veisicoi Chemical
Corp. (10) found no abnormal behavior patterns or toxic
signs in the treatment groups. Using single doses of 200,
400 and 800 rag/kg of insecticide administered by gelatin
capsule to mature laying hens, Abou-Donia and Preissig
(14,29) observed the development of all signs of the delayed
neurotoxicity syndrome, beginning 8-14 days after dosing.
Signs developed at all doses, along with fragmentation of
myelin sheaths and degeneration of axons in cord tissues and
the sciatic nerve. This work extends a previous study by
Abou-Donia et al. (28) in male chickens showing that
single, high doses of Ieptophos (180-3000 rag/kg) produce the
delayed neurotoxic effect, with the incidence of signs
incresing at the higher dose levels. A collaborative
study (29,51,62,63,66,68,69,70,71) involving officials of
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32
the Environmental Protection Agency (Dr. Richardson),
Industrial Bio-Test Corp. (Dr. Gordon) and Velsicol Chemi-
cal Corp. (Mr. Calo) also involved a single dosage leptophos
design in the mature hen, with intubation at levels of 200,
100 and 75 mg/kg. All exposed groups developed the delayed
neurotoxic response, with accompanying evidence of degene-
rative lesions in myelinated nerve fibers. Additionally,
Kimmerle (22) has reported on acute studies in White
Leghorn hens (aged 15-18 months) at single dose levels in
the range 50-5000 mg/kg, with administration via either the
oral intubation route or by injection into the abdominal
cavity. The development of neurotoxic signs (ataxia,
paralysis) occurred after 8-12 days at all dose levels >^
100 mg/kg for orally dosed birds, and within 7-10 days for
intraperitoneally dosed birds. Barnes (36) has also con-
firmed that leptophos administered to hens produces the
classical delayed neurotoxic effect.
It is also of interest that major impurities and photo-
decomposition products of leptophos found in technical
preparations and in crop residues are capable of initiating
the delayed neurotoxicity syndrome in the chicken.
Using White Leghorn pullets and single oral doses via
gelatin capsules, Sanborn et al. (33) have determined that
desbromo leptophos and leptophos oxon are approximately
»
three and two times, respectively, more potent neurotoxic
agents than the parent compounds. Note should also be taken
-------
33
of the results of a recent neur'o toxic ity study with lt:ptophos
in chickens by Calandra et al. (60), which is especially
extensive. Mature birds were orally dosed at levels of
5, 10, 15, 30, 50, 75, 100 and 200 rng/kg of technical
leptophos, and observed for development of the neurotoxicity
syndrome over a period of 18 days. The majority of test
group birds exhibited generalized weakness, anorexia and
slight to severe ataxia within a few hours after dosing,
and birds at the three highest dosing levels displayed
varying degrees of neurotoxic signs post-dosing. High
dosage birds were killed on test day 18 and cord, brain and
sciatic nerve tisslies taken for study by light and electron
microscopy. The remaining birds were redosed, and observed
for a further 21-day period. In general, high-dosage birds
exhibited weight loss or depressed weight gain, and the
development of neurotoxic signs in dose-related intensity.
Retrograde degeneration of axons and degeneration of myelin
/
sheaths in the sciatic nerve were noted in about 50% of birds
dosed at the 200 mg/kg level. However, some criticism has
been leveled (49) at the technical quality of the histo-
pathology specimens prepared and examined in the 1975
Industrial Bio-Test Laboratory study (60).
2. Sub-Acute Studies.
Previous mention has been made of a 1976 study by
Abou-Donia and Preissig (14) in which mature hens were
orally dosed with technical grade leptophos at 200, 400 and
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34
800 mg/kg, using oral intubation or gelatin capsules, *nd
then observed for periods up to 30 days in. most instances,
and up to 77 days for a single 400 mg/kg animal monitored
for improvement from paralysis. Leptophos produced the full
delayed neurotoxic syndrome, with onset of signs 8-14 days
after dosing and development of paralysis within 30 days.
The one hen followed for 77 days showed partial recovery
from paralysis. Histological examination of tissues from
paralyzed birds revealed marked axon and myelin degeneration
of the sciatic nerve and spinal cord. Tais work has now
been extended by these investigators (31,35) to sub-acute
experiments in which low levels of leptophos were adminis-
tered to mature hens, orally in gelatin capsules, for 60
days or until ataxia' developed. Doses were 1, 2.5, 5, 10
and 20 mg/kg of technical grade leptophos. It was found
that the development of the delayed neurotoxicity syndrome
was dependent on the size of the daily dose, with temporary
paralysis resulting from the smaller doses and permanent
paralysis from the larger. The duration of administration
before onset of ataxia and the total dose causing ataxia are
also dependent on the size of the daily dose; in general,
the total dose required to produce ataxia decreased as the
magnitude of the daily dose was decreased. The neurotoxic
effects of repetitive dosing were cumulative. From the
histology of spinal cord and sciatic nerve tissues from
intoxicated birds, it appears that fully paralyzed birds
display very marked changes (degeneration of myelin and
axons) while ataxic birds exhibit only mild changes.
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35
A portion of the 1975 study by Calandra et al . (60)
using mature hens and oral intubation of leptophos also
falls into the sub-acute category, since the single doses
were repeated on day 21 for all animals not killed on day
18, and observations were continued for a further 21 days.
Despite criticisms leveled at this study (49,61), it is
clear that the delayed neurotoxicity syndrome does develop
in birds redosed at the 75 and 100 mg/kg level, and followed
for the full 42 day duration of the study. The adequacy of
the histopathologic procedures employed in the study of
tissue sections may be open to some question.
3. Chronic Studies.
None have appeared.
4. Comments.
From the bulk of literature evidence registered in
acute and sub-acute studies with leptophos in the chicken,
it is justifiable to conclude that this ester and certain of
its degradation products (desbromo derivative, oxon) can
elicit the delayed neurotoxicity syndrome in both male and
female animals. The syndrome is of the classical "delayed
neurotoxicity" type involving an induction interval and the
"dying back" process characteristic of the action of TOCP
and other OP compounds (4,9).
-------
"" Studies in the C_at
Despite"1iterature observations (4,8) that the cat
is similar to the chicken and man in its sensitivity to the
delayed neurotoxicity syndrome evoked by administration, of
certain organophosphorus compounds, it is unfortunate that
no published data are available for the specific effects of
leptophos in this species. However, very recently a report
of a preliminary study (89d) on acute effects of Leptophos
in several test species including the cat has become avail-
able.
1. Acute Studies
CuulsLuu e L al. (39uy iidVe reporLtiu oa some
preliminary studies in which female cats were subjected to
intraperitoneal injection of leptophos solutions. Animals
that received doses greater than 50 mg/kg died within 2 to 4
days despite administration of repeated doses of atropine.
These animals suffered from immediate onset of cholinergic
distress.
Of two animals given 50 mg/kg, one died on the
second day after the dose, but the second recovered slowly
from the toxic effects of the compound, including ataxia.
However, from day 13 to day 25 after dosing, this animal
t
exhibited a tendency toward extension of the hind limbs
which persisted until the animal was killed on day 25.
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37
One animal given 45 nig/kg was ataxic for about
10 days, but recovered thereafter.
• «•
2. Sub-Acute Studies.
None have been recorded.
3. Chronic Studies.
None have been recorded.
4. Comments.
The data on acute effects of leptophos in the
cat are quite preliminary and scanty. Yet, present indica-
tions are that single intraperitoneal doses near 50 nig/kg
are able to initiate toxic interactions that culminate in
development of peripheral neuropathy.
C. Studies in the Dog
1. Acute Studies.
None have been recorded.
2. Sub-Acute Studies.
Calandra et al. (10) have reported on a 90-day
feeding study with beagle dogs (both sexes) maintained on a
diet containing 0, 10 or 30 ppm of leptophos. Behavior and
growth were not affected over this interval, and gross and
microscopic observation of tissues at the conclusion of the
study revealed no abnormalities.
-------
3. Chronic Studie-s. •
Calandra et al. (10) have also conducted a
two-year feeding study with leptophos in the beagle at
levels of 0, 10, 20, 30 and 60 ppm in the diet. The group
fed 60 ppm had first been on diet containing 5 ppm for 180
days, and was then shifted to 60 ppm for the remainder
of the study. During the entire two years, no mortality
occurred, and growth and behavior were normal. No clinical
signs of toxicity were observed, and no abnormalities were
found in neural tissues (optic and sciatic nerves) at the
conclusion of the study.
4. Comments .
Although the dosage range in this pair of
feeding studies was rather limited, it seems quite clear
that the dog is resistant to any neurotoxic effects of
leptophos at levels up to 60 ppm in the diet.
D * S tudies in the Rat
1. Acute Studies.
This species is known to be resistant to the
neurotoxic effects of organophosphorus esters. Therefore, no
acute studies have been recorded for leptophos in the rat in
which the experimental design was directed at observations
of the delayed neurotoxicity syndrome.
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39
2. Sub-Acute Studies.
Groups of male and female rats were fed lepto-
phos in the diet at levels of 0, 1, 5 and 10 ppn for 90
»
days. It was reported (10) that growth and behavior were
not affected by leptophos, and that gross and microscopic
examination of tissues showed no leptophos-re lat ed lesions.
3. Chronic Studies.
Groups of albino rats (male and female) were
fed leptophos in the diet for two years at concentrations of
0, 10, 20, 30 and 60 ppm (10). The animals on the 60 ppn
diet were initially fed a 5 ppm diet for the first seven
.
Cl .1. A. 11 , W 1
neurotoxic signs, it was found that growth was unaffected
and that gross and microscopic examination of tissues
failed to indicate any lesions attributable to leptophos.
4. Comment s .
As expected for this resistant species, the
feeding experiments cited produced no evidence for neurotoxi-
city elicited by long-term ingestion of low levels of lepto-
phos in the diet.
t
E. S tudies in the House
1. Acute Studies.
None
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40
2. Sub-Acute Studies
None
3. Chronic Studies.
In a study designed to assess the carcinogenic
potential of leptophos in mice (10), male and female Swiss
white mice were fed leptophos in the diet for 18 months at
dose levels of 0, 50, and 100 ppm. At six months and at 18
months animals were killed and tissues, including the sciatic
nerve examined for lesions and tumors. No leptophos-
related lesions were found in any tissues of test animals.
4. Comments .
As expected in the rodent, resistance to
neurotoxicity was observed in the mouse in the single
chronic feeding study available.
F. Studies in the Rabbit
A single acute study with leptophos in the rabbit
has been recorded in which toxic effects leading to death
are described (23).
1. Acute Studies.
Karael et a 1. (23) have described an acute
intoxication study in which Baladi rabbits (both sexes, 0.8
- 1.0 kg, 2-3 months of age) were given graded oral doses
(31-178 mg/kg) of leptophos wettable powder dissolved in
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41
distilled water, and then observed for development of toxic
signs for 72 hours.. Animals began exhibiting toxic signs
within three hoars after administration of the compound,
starting with manifestations of acute cholinergic distress.
Respiratory distress, profuse salivation, diarrhea and
urination were followed by development of unsteadiness, lack
of coordination, scattered muscular twitches, convulsions,
motor paralysis, loss of strength and failure to stand.
Hitopathology after'72 hours of exposure to leptophos in
surviving animals revealed that the cerebellum had been
affected, with paralysis attributable to derayelination in
the white matter of this tissue of animals given 124 rag/kg.
The ataxia produced during the appearance of toxic signs was
attributed to degenerative changes in the Purkinje ceils in
thecerebellum.
2. Sub-acute Studies.
None .
3. Chronic Studies.
None .
4. Comment s.
It is clear that the rabbit is sensitive to
neurotoxic actions of leptophos on an acute basis.
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42
G. Studies in Cattle
1. Acute Studies.
"In a short-term feeding study, groups of steers
were fed leptophos in the diet at levels of 0, 15, 45 and
150 ppm for 4 weeks (10,87). At this point, sone of the
animals were sacrificed and tissues subjected to analysis,
while other animals were shifted to normal, untreated food
for a further 14 days, and then sacrificed. Throughout tie-
study, daily observations showed no abnormal behavior or
toxic signs of poisoning. No gross or microscopic lesions
were observed in tissues at the conclusion of the study.
Tfi an^hVin-y o^nfo ct"ii^v (10 $ 7 ' cr T r» M n c r» f 1 a r* t" ;3 f" —"
• « *~~~ •"*•"*"•* — — — — — ~ _ .j ^ _ _ , _ « , , o _ _ „ c » _ ~ _ _ _
ing dairy cows were fed leptophos in the diet at a.nalyzed
levels of 0, 3.2, 10.0 and 37.4 ppm for 28 days, and main-
tained for a further 14 days on normal control diet.
Appearance, behavior and general condition of the animals
were normal over the entire period. Body weights of several
animals treated with leptophos were reduced over the
study period, but there were no remarkable tissue anomalies
observed at necropsy.
2. Sub-acute Studies.
None .
3. Chronic Studies.
None .
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43
4. Comment s.
From the results of the two brief feeding
studies in cattle, it can only be concluded that oral intake
of leptophos in this animal at levels of 150 ppm or lower for
a month does not lead to a cumulative dosage for onset
of neurotoxic signs.
H. Studies in Water Buffaloes
Stemming from an apparent epidemic of paralysis in
water buffaloes in Egypt during 1971 (19) in the course of
which some 1200-1300 animals died, epidemiologic investiga-
tion led to the suspicion that the agricultural use of
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*.—£...»£-.. „ ~ — .J. - ... .. » .. „ _ . « . *, — Q ,- .* . . -._,.,.. ^, w £,.. I. » ^ VM.~V*.
of the epidemic was not firmly established, the observation
that the disease was characterized in the initial stages by
a loss of coordination in the hindquarters, followed by the
development of paralysis and ultimately death, pointed
strongly to the possibility of a chemically induced, delayed
neuropathy akin to that produced by TOCP in a variety of
animal species.
Accordingly, in 1971-1972 (29,59,87) and again in
1974 (48,87) toxico logic a 1 experiments were undertaken in
Egypt under the auspices of the Egyptian Ministry of Agricul-
ture in which corn plants sprayed with technical leptophos
were fed to test animals. The water buffaloes were then
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44
monitored for the development of the delayed neurotoxicity
syndrome. Each experiment had the character of a sub-acute
trial, as described below:
1. Acute Studies.
None.
2. Sub-acute Studies.
In the 1971-1972 experiment, two full grown
(400 kg) animals were used for the leptophos feeding test
Corn plants were sprayed with leptophos at a rate of
675 g. per acre, and then collected for feeding to test
animals. Each animal was fed twice a day, receiving 5 kg of
treated plants each time. After 46 days of this regimen,
each daily diet was supplemented with 25 kg of clover and
cotton seed cake for the next 5 days, and then supplemented
daily with 50 kg of clover only for the final 9 days of the
total 60-day experiment. The initial deposit of leptophos
on the corn plants was calculated at 15 ppra.
Both animals developed severe neurotoxic signs.
One developed paralysis of the hindquarters by the 48th day,
with loss of all sensation. During the period 51-60 days
this animal developed difficulty in breathing, trembling in
the forelimbs, loss of ability to urinate or defecate, and
severe irritation of the eyelids. By the final day, the
animal had no control on urination. The second animal
began its neurotoxicity syndrome with some lack of balance
-------
45
in the hindquarters, collapse of the hind legs, problems
with urination and defecation, loss of sensation in the
abdominal area, and inability to stand by the 54th day. The
first animal was sacrificed in extremis on the 65th day, and
the second died on the 70th day.
In the 1974 experiment, a highly complex feeding
regimen was used over a total time period of 60 days,
starting with the first day of feeding leptophos-sprayed
corn. The corn was sprayed at a rate of 2.25 1. of leptophos
2
30% concentrate per 4200 m . Various batches of corn
ranged in content of leptophos from 6-40 ppm. Animals (two
per group) were fed 10 kg of corn per day, divided into two
equal portions, plus daily supplements of 3 kg of dry fodder
plus 8 kg of dry straw. The feeding protocol was: (a) Group
one animals were fed for 45 days with corn sprayed only
once; (b) Group two animals were fed daily for 15 days with
corn sprayed only once, and then for another 30 days on corn
sprayed twice; (c) Group three animals were exposed to
direct spraying drift and then fed for 45 days on corn
sprayed only once; (d) Group four animals were exposed to
direct spraying drift and then fed only untreated corn;
and (e) Group five animals (controls, 3 in number) were fed
only untreated corn. From 45 days to 60 days the animals
were under close observation, and then examined carefully on
the 60th day. Surprisingly, in view of of the results of
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46
the 1971-1972 experiment, the following conclusions were
reached by the Committee directing the 1974 experiment: (a)
no paralysis of the hind quarters occurred in any animal;
(b) animals fed on corn sprayed only once showed no notice-
able toxic signs; (c) one animal fed on corn sprayed twice
showed "insignificant" lessening in sensation and "insignifi-
cant" stiffness in a hind leg; (d) the animals exposed to
drift spray and fed on corn sprayed once showed "insignifi-
cant" signs; and (e) the animals exposed to drift spray
only showed no noticeable signs at 60 days.
3. Chronic Studies.
None.
D. Comments
It is difficult to reconcile the disparate results
of the two sub-acute feeding studies with leptophos-sprayed
corn and the water buffalo. Undoubtedly, the lack of
precise knowledge of ingested amounts contributes to this
disparity, but it is doubtful that the ingestion dosages
could have varied so widely as to produce a very powerful
delayed neurotoxicity syndrome in the 1971-1972 experiment
and only a hint of the beginning of such a syndrome in the
1974 test. Nevertheless, the evidence must be taken on
balance in assessing whether leptophos can induce the
delayed neuropathy syndrome in this species following
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47
oral ingestion, given that the dosages are sufficient to
initiate the toxicological response. In view of the epide-
miological evidence, the very positive results of the
1971-1972 experiment, and the negative or marginal evidence
from the 1974 trail, it must be taken as probable that the
insecticide formulation can indeed initiate the delayed
neurotoxicity pattern of responses in the water buffalo on
prolonged 'feeding of corn plants sprayed with a sufficient
quantity of leptophos.
I. S tudies in the Sheep
Stemming from experience with leptophos and the water
buffalo, Egyptian investigators have begun experiments with
>
other farm animals aimed at development of a more convenient
and suitable livestock model for study of the effects of
ingestion of insecticide-sprayed silage. Some recent
experiments (52,56) have employed the Egyptian sheep and
leptophos intoxication, as described below.
1. Acute Studies
In an acute study with one 25 kg ram, 5 ml of 30%
leptophos concentrate was administered orally without any
carrier diluent. The animal (crossbred, Rahinany X Marino)
receiving this dose, estimated at 60 mg/kg for the leptophos
ingredient, died 20 hours later. No observations were
recorded with respect to possible toxic signs leading to
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48
death. El-Sebae et al.(56) seated that the sheep must be
considered more sensitive to the toxic actions of leptophos
than the rat.
2. Sub-acute Studies.
Again using one ram as subject (crossbred, 30 kg,
aged one year), the exp-er iment al design (56) involved
feeding a ration of sprayed, freshly cut clover at the rate
of 5 kg per day for 40 days. The growing clover was sprayed
every other day with 30% leptophos concentrate, resulting
in an estimated ingestion dosage of 90 mg leptophos per day.
The animal developed the toxic signs of the delayed neuro-
pathy syndrome starting at 59 days after the s.tart of
feeding, beginning with ataxia in the hind limbs and followed
by failure of the ability to stand on the rear legs. No
further pathophysiological signs were recorded in this
report.
3. Chronic Studies.
None
4. Comments.
It is clear that the Egyptian crossbred sheep as a
species, subjected to ingestion of leptophos in the diet, is
sensitive to the delayed neurotoxic ity syndrome evoked by
this organophosphorus compound in other mammalian and avian
species of animals.
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49
J. S t u d i e s i n t h e Rhesus Monkey
A preliminary study (89d) includes some initial
observations -on effects following intraperitoneal injection
of leptophos solutions into male Rhesus monkeys. One animal
received 40 mg/kg in one dose, and a second received the
first of such doses on day 1 and the second on day 21 of the
experiment. Despite initial signs of cholinergic distress
in the form of miosis followed by mydriasis, neither animal
developed any signs of peripheral neuropathy at the end of
32-43 days.
K. S ummary of Del ayed Neurotoxicity Results
From extensive experimental evidence, it is clear
that the chicken (especially the mature hen) is a very
susceptible species in regard to delayed neurotoxicity
induced by ingestion of leptophos, on an acute or sub-acute
basis. The rabbit is also quite susceptible to development
of the full spectrum of neurotoxic signs following oral
ingestion of leptophos acute experiments. Preliminary
evidence on effects of leptophos in the sheep, by oral
intubation or by ingestion of clover sprayed with the
compound, indicates that this animal species does develop
delayed neurotoxicity in response to the agent. The evi-
dence for development of the delayed neurotoxicity syndrome
in the water buffalo is somewhat equivocal, but a balanced
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50
consideration of the epidemiological results' and laboratory
based experiments leads to a tentative affirmative assign-
ment of susceptibility in this species when exposed to
leptophos via residues in its feed. In cattle, oral intake
of leptophos in the feed at 150 ppm or lower for a month
does not lead to neurotoxic signs. Rodents such as the rat
and mouse appear to be resistant to the neurotoxic effects
of leptophos, as is the dog when fed the material at levels
up to 60 ppm in the diet.
It is presently unclear whether the delayed neurotoxi-
city syndrome induced in sensitive species by leptophos is
the result of a general mechanism (6) elaborated for the
common action of neurotoxic organophosphorus esters.
However, the striking similarity of toxic signs across a
broad spectrum of such esters makes it likely that at least
one common mechanism applies, with the bulk of present
evidence pointing to primary inhibition by phosphory1 ation
of a neurotoxic esterase at loci in the brain and spinal
cord, followed after a lag time by the systematic "dying-back1
of peripheral motor nerves in retrograde fashion starting at
the myoneural junctional areas on motor muscles.
L. E pidemiolog ical Evidence for Neurotoxicity in the
Human
Epideraiological evidence pointing to the senstivity
of man to the delayed neurotoxic actions of such OP com-
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51
pounds as tri-o-cresy1 phosphate is widespread (4,5,6,7,8).
Yet, specific epidemiological information on sequelae of
exposures of humans to leptophos is quite scanty, despite
the fact that this insecticide has been employed agricultur-
ally on a worldwide basis. One such piece of information
has been reported (19) from Egypt after the crop growing
season of 1972 in which leptophos was used as an insecticidal
spray. Long after the spraying season was over, six people
were discovered to have symptoms of neurotoxicity; traces of
leptophos were found in their tissues. No information
exists as to how these people contacted the chemical.
More recent clinical reports ( 83, 83a , 83b ,.83c , 83d) on a
series of 65 Egyptian patients exposed during 1975 to
varying levels of leptophos include one fatality following
acute intoxication. The toxic symptoms were those of acute
cholinergic distress typical of organophosphorus ester
poisoning, accompanied in certain cases by reversible (inild)
hepatocellular injury and temporary interference with
glomerular filtration. No symptoms of neurological dysfunc-
tion were recorded in these reports.
A very recent report (84,85) from the U.S. Public Health
Service summarizing the results of a preliminary health
survey of the Velsicol Chemical Corp. plant in Bayport,
Texas, casts some interesting light on the occupational
safety and health status of workers employed in the manufuac-
ture of leptophos. This plant was engaged in the manufac-
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52
ture and packaging of the insecticide during the interval
•
1971-1976, but medical information on the neurological
status of its workers is documented only from the survey
results in early 1976. Of 28 current workers in 1976 whose
medical records and physical examinations were reviewed, 26
were neurologically normal and two displayed some neurologi-
cal symptoms. One of these affected workers was classified
as presenting symptoms of toxic myelopathy; the second
showed signs classified in the domain of borderline abnorma-
lity. Additionally, incomplete evidence (84) points to the
possibility that people previously employed at the plant
have also noted signs of neurotoxicity associated with their
work.
Amplification of this PHS health survey is contained in
a Velsicol Chemical Corporation report (dated August 12,
1976) which summarizes the medical findings on a total of 42
Bayport employees evaluated for neurological function at
the Texas School of Medicine at Houston. Of these employees,
40 were characterized by neurological findings that were
either completely normal or only insignificantly deviant
from normal. However, two employees who are presumably
identical with the two mentioned in the Public Health
Service survey (84) displayed symptoms of peripheral
neuropathy. In one worker, the involvement has resulted in
permanent and total paralysis in the lower extremities. In
the second, the toxic myelopathy is reflected in partial
paralysis of the lower extremities.
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53
In addition to these 42 workers for whom neurological
examination results are at hand, Velsicol Chemical Corpora-
tion has also reported (August 12, 1976) that their files
contain informtaion on 12 other individuals who were no^t
employees of the Company at the time neurological examina-
tions were given, but had previously been involved in
leptophos manufacturing or formulation. These individuals
have complained of or demonstrated symptoms of neurological
dysfunction.
The epidemiological evidence cited above is indeed
fragmentary and brief. Yet, from the positive indications
given, it would be prudent to take future precautions based
on the probability that sufficiently high doses of leptophos
in man can induce the syndrome of delayed neurotoxicity.
VI. CARCINOGENESIS, MUTAGENESIS, AND TERATOGENESIS (INCLUD-
ING REPRODUCTION STUDIES)
A. Reproduction Studies in the Rat (87)
In two studies, 8 male and 16 female rats per
group, were fed leptophos in the diet at levels of 0, 10,
30, 40 and 60 ppm. There were 2 control groups of 8 males
and 16 females each. These animals were subjected to a
standard three generation, 2 litter per generation reproduc-
tion study. The 60 ppm level was discontinued because of
pup mortality after two litters of the first generation
were born. A second study was initiated at 0 and 5 ppm.
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54
After one generation the 5 ppn level was changed to the 40
ppm level (noted above) and maintained at this level for 2
further generations.
Survival data on pups, including viability and
lactation indices, were significantly decreased at 60 ppm
but were similar to control values at 40, 30, and 10 ppm.
Mean body weight data for pups at weaning at all dose levels
(including 60 ppm) were similar. Parental body weight and
reproductive performance were similar in the 60 ppm group
to the control group. Reproductive indices, including
mating, pregnancy, fertility and parturition showed no
differences between the control and all treated rats. A
no-effect level for rat reproduction was stated to be 30
ppm.
B * Effects in Lactat ing Cows (87)
Groups of lactating dairy cows (3 cows/group fed
leptophos and 1 cow/control group) were fed leptophos in the
diet. Concentrations were calculated to be 0, 5, 15 and 50
ppm. The actual concentrations fed as assessed by cheraical
analysis were 0, 3.2, 10.0 and 37.4 ppm. The animals were
dosed for 28 days and maintained for a further 14-days on
control diets to measure changes in leptophos levels in
tissues. Appearances, behavior and general condition were
normal over the study. Body weights of several animals
tested with leptophos were reduced during the study. Milk
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55
production was reduced in the low and intermediate group but
not at the high-level of leptophos. Traces of leptophos
were noted in fat at the conclusion of the 28 day feeding
interval at levels ranging from 0.2 to 0.5 ppm at the high
(37.4 ppm) dose level. After two weeks, these levels were
reduced to 0.2 ppm. (79,80,81)
C . Teratogenici ty in Rabbits (87 )
Groups of 10-13 pregnant New Zealand rabbits were
given 0, 1, and 3 mg/kg leptophos orally from day 6 to day
18 of gestation. Another group of rabbits received 37.5
mg/kg of thalidomide over this same period as a positive
rabbits. Leptophos, at a dose of 3 mg/kg administered
orally to rabbits during organogenes i s elicited no teratologi-
cal response. Skeletal or somatic abnormalities were noted
with thalidomide.
D. C a r c i n o g e n i c i t y i n H i c e (87)
Groups of 65 male and 65 female Swiss white mice
were fed leptophos in the diet for 18 months at dose levels
of 0, 50, and 100 ppm to test for carcinogenic potential.
Two positive control groups were fed N-ni trosod ie thy 1 amine
at levels of 10 pptn (50 males and 50 females) and 40 ppm (15
males and 15 females). After 6 months, the mice of the high
positive control group and 15 of each sex in the leptophos
group were sacrificed and subjected to gross and microscopic
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56
examination for tumor formation. At 18 months, all remain-
ing animals were sacrificed and 10 animals of each sex
(or group) were examined.
The positive controls sacrificed at 6 months (40
ppm) showed definite evidence of lung adenoma or carcinoma.
At 18 months, the positive controls (10 ppm) again showed a
positive response to the carcinogen. No evidence of lung
lesions were noted at 100 ppm leptophos in the diet. There
were no leptophos- re lated lesions or tumors in any of the
tissues and organs examined in this study.
E . Carcinogenicity in Rats (87)
p v, o — i *> »
50 femal es /group ) were fed leptophos in the diet. for two
years at concentrations of 0, 10, 20, and 30 ppm. Another
group was initially fed 5 ppm for the first seven months
and 60 ppm for an additional 17 months. Mortality was
unaffected by leptophos in the diet, although at the conclu-
sion of the study very few animals in all groups were alive.
Food consumption and growth were not affected. The experimen-
tal and control values were similar in the hematology blood
chemistry or urine parameters examined. Gross and micro-
scopic examination of tissues and organs failed to indicate
any pathologic disorders attributable to leptophos.
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57
F' Mutagenicity S tudies (87)
1. Dominant Lethal Study
A dominant lethal study was conducted in which
male mice (8 mice/group) were given a single oral or
intraperitoneal dose of 0, 15, or 30 mg leptophos/kg. Each
male was mated with 3 females per week for 6 weeks during
the period of spermatogenesis. Positive control studies
were performed with methyl me thanesulfonate. Leptophos had
no effect on mating or reproduction including preimplantation
loss, early resorption or embryo viability. In this test,
leptophos did not induce mutagenic changes in male germinal
2. Recombination Assay (79)
Chemicals may alter DNA without producing
rautagenic effects on cells. Recombination assays measure
/
the effect of chemicals on DNA. Leptophos in concentrations
of 10, 50, 100, 500, or 1000 ug per 10 ul of solvent,
produced no inhibition on Rec + or Rec - genotypes of 15
subt i1i s. The solvents used did not inhibit either genotype.
Known positive mutagens (hydroxylamine, ethy me thane-sulfo-
nate, N-methy1-N-nitro-N-nitrosoguanidine, and 4-nitroquino-
line oxide did inhibit one or both genotypes.
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58
3. Host-Mediated Assay (80)
Host-mediated assays for mutagenicity were
conducted in both albino rats and albino mice. The rats
were treated by gavage for 5 consecutive days with either 3
or 10 mg/kg of leptophos; albino mice were treated in a
similar manner with 10 or 30 mg/kg of body weight.
The animals were then injected intraperi-
toneally with a histidine-dependent strain of _S_. typh imurium
(strain G 46). After a 3 hour exposure, the bacteria were
recovered and the number of bacteria no longer dependent
on an external source of histidine was determined. Positive
contro-1 rats received a single intramuscular injection of
100 mg/kg of dime thy Ini trosaiaine and the positive control
mice received a single intramuscular injection of 30 mg/kg
of N-methyl-N-nitro-N-nitroso guanidine prior to intraperi-
toneal injection with bacteria. One rat from the 10 ng/kg
treatment group died. No other deaths occurred. Rats
exposed to 10 mg/kg leptophos exhibited severe tremors and
significant weight losses after 1 dose. Slight hyperactivity
occurred among rats given 3 mg/kg and among mice exposed to
30 rig/kg. No other untoward behavioral reactions were
observed.
The number of revertants (mutants) obtained
from either rats or mice treated with leptophos revealed no
consistent differences from the number of revertants in the
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59
control animals. Known mutageno produced increased mutation
rates in both r ?. t s and mice.
It was concluded that leptophos does not
produce a mutagenic response in S_. typhimurium (strain G 46)
in a host-mediated assay using rats and mice.
4. Reverse Mutation Studies (81)
Leptophos was examined for mutagenic activity
in a series of in vitro microbial assays using Salmonella
and Sacchar oiny ces indicator organisms. The test material
was tested directly and in the presence of liver microsomal
enzyme preparations from Aroclor-induced rats. The dose
lonrolc \J a V e* 0 ^ ^ 0 10 0 an/1 SO 0 it £ no-r-
. v . w ^ V • M _ v , -f J — 4 - f _ - . — ^ v>**-~- — — » *. ~~O "" "™ ^«-^^^^J*.«^.— -J-^-fc
plate in the activation test. The highest dose levels
produced some physiological effect. Dose levels of 250 and
500 ug/plate produced complete toxicity in the nonactivation
assays. The 2 lower dose levels in both tests were below
the level of significant toxicity.
The results obtained with the positive control
materials demonstrate that the test system", are functional
with known mutagens. None of the indicator strains exhibited
a response to leptophos in the nonactivation test (81).
A slight increase in the mutant count was noted
at 250 ug/plate with TA-135 in the activation test. This
increase was less than f°.u_£_-££.ld g.rea_t_££ than the control
-------
count and there was no increase at 500 ug/plate. The
results of the activation test were considered to be nega-
t ive .
It was concluded that leptophos did not exhibit
genetic activity in any of the in vitro assays employed in
this investigation.
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61
VII. POSITION STATEMENTS ON CHARGE
Charge #1: ^Wh ether leptophoshasadelayed neuro toxic
effect in hens , r abb i ts , _ sh_eep ,___ wa t er bu f f alo or any other
s ' "
From extensive experimental evidence, it is clear
that the chicken (especially the mature hen) is a very
susceptible species in regard to delayed neuro toxic i ty
induced by ingestion of leptophos, on an acute or sub-acute
basis* The rabbit is also quite susceptible to development
of the full spectrum of neurotoxic signs following oral
ingestion of leptophos in acute experimentS4 Preliminary
evidence on effects of leptophos in the sheep, by oral
incubation or by ingestion of clover sprayed with the
compound, indicates that this animal species also develops
delayed neurotoxic effects. The evidence on development of
the delayed neuro toxic ity syndrome in the water buffalo is
somewhat equivocal, but a balanced consideration of the
epidemiolog ical results and laboratory based experiments
leads to a tentative affirmative assignment of susceptibi-
lity in this species when exposed to leptophos via residues
in its feed. In cattle, an oral intake of leptophos in the
feed at 150 ppm or lower for a month does not lead to
neurotoxic signs. Rodents such as the rat and mouse appear
to be resistant to the neurotoxic effects of leptophos, as
is the dog when fed the material at levels up to 60 ppm in
the diet,
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62
Charge #2: Whether there are other toxic,^e f feet s f roii use
of leptophos *
The answer to Charge #2 is "yes*"
The two primary to'xic effects are:
1. Depression of blood and brain cho1inesterases;
2. Acute cholinergic distress syndrome.
Leptophos was exceptionally stable in this system and
persisted in different organisms, e.g. fish, snail, and
algae, over an experimental period of 49 days. Accumulation
in fish, G ambus ia a ffinis, was about 2-200-fold greater with
leptophos compared to other typical organophosphorus insecti-
cides such as chlorpyrifos, dyfonate, and parathion. Ecologi-
cal magnification, i.e. the ratio of the amount of leptophos
present in snails and in water, was approximately 48,000.
This was 5 to 500-fold greater than the ecological magnifica-
/•
tion observed with other organophosphorus insecticides
examined. Overall, the results indicated that leptophos is
the most accumulative and persistent organophosphorus
insecticide ever examined in the terrestrial-aquatic model
ecosystem. This is consistent with the long-term persistence
of leptophos reported in cotton leaves (9 weeks), tomatoes
(7 weeks),-and grapes (7 weeks) (25,18).
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63
Charge No 3. Whether there is a potential for hazard to man
from ingestion of any amount s of leptophos.
»
The answer to this charge is unequivocally y^js.
However, a yes response would apply to virtually any insecti-
cide owing to the words "any amounts" in the charge. From
epidemio1ogical information presently available on the effect of
leptophos on man, evidence can be cited pointing to the
induction of the acute cholinergic distress syndrome and the
onset of processes leading to delayed neurotoxicity.
Charge No. 4. Whether there is a potential for hazard to
man from the injestion of leptophos residu e s on lettuc e o f
10 ppm and on tomatoes of 2 ppm or from lesser residues.
For the purposes of this charge, we define the term
potential to be the probability that irreversible damage to
cell structure and/or function will occur.
In order to assess the potential harm for man, we
assume that the toxicological effects of leptophos on the
most senstive species, the chicken, will parallel those that
would be observed in man. Because leptophos causes
irreversible delayed neurotoxicity, we have chosen a safety
«
factor of 1000 between the no effect level exhibited
in a chronic test on the most sensitive animal species and
the estimated daily intake by the human fron residues
on lettuce and tomatoes.
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64
At present, the most reliable data suggest that the no-
effect level in_the chicken for cholinesterase inhibition
is less than 0.5 mg/kg per day after a 72-day feeding
regimen, and for delayed neurotoxicity the level is less
than 0.5 mg/kg per day after a 60-day feeding exposure.
From these no-effect levels and the given minimum
safety factor of 1,000 we conclude that the residue levels
cited could produce an intake by the human at least 10 times
greater than the acceptable daily intake.
We conclude that the potential for hazard to man from
the ingestion of leptophos residues on lettuce and tomatoes
is • rpa 1 .
Charge No. 5. Whether leptophos bioaccumulates' in tissue'
and/or is persistent and there fore poses a hazard to man.
As indicated in Section III, compared to other OP
insecticides leptophos was found to be highly persistent
in the aquatic food chain in studies conducted in a
terrestrial-aquatic model ecosystem. Briefly reiterated,
the biological magnification of leptophos (amount in organism
relative to that present in water) was 1,443 in mosquito
fish (G arabu.s ia af ini s) and > 48,000 in water snails (P hy s a) .
Another study with bluegills has shown that, on continous
exposure 'to water containing leptophos, residues of lepto-
phos and its metabolites build up in fish (Velsicol Petition,
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65
Section D, Part. Ill, p. 1548). Based on these studies, it.
is apparent that leptophos bioaccumulates in biological
tissue and therefore poses a hazard to man via his food
chain.
Low levels of leptophos residues have been found in
the fat, tissues, and organs of dairy and beef cattle
treated orally with leptophos (Note: see Section D, Part
II, pp. 2070-2079, Velsicol petition).
Response to Charge No. 6. "Whether exposure to leptophos in
food , including raw agricultural commodities causes adverse
effects in man wh ich, to the extent necessary to protect the
pub lie health, make it unsafe for use."
This question is interpreted to bear on the question- of
the safety of the consunpt ion of food or raw agricultural
commodities containing leptophos, and not on the safety of
the handling of such materials.
There have been no reports of injury to man caused by
the consumption of food or raw agricultural commodities
containing leptophos. Therefore, it cannot be categorically
stated that leptophos in these materials causes "adverse
effects to man" which make it unsafe for use.
However, since it must be assumed, until it is shown
otherwise, that man is as sensitive as the chicken to the
adverse effects of the chronic consumption of leptophos, and
since definitive chronic toxicity studies in the chicken
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66
. have not yet been reported, there is no basis for 'cone lud i.ng
*
that leptophos in food or raw agricultural commodities
does not cause "adverse effects to nan" and is safe for
use .
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67
VIII. CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS:
1. From existing evidence, exposure to leptophos in feed
has caused the death of water buffalo and other animal
species.
2. There are indications that neurolo'gical effects have occurred
in workers involved in the manufacturing, packaging and
application of leptophos.
3. It is concluded that no scientifically supportable "no
effect" dose or tolerance limits can be established at
the present time because of insufficient data on chronic
toxicity in asensitive species, i.e., the chicken.
RECOMMENDATIONS:
1. The existence of tolerances implies that the specified
limits are safe. This cannot be proven at the present
time. Consequently, the currently existing tolerances
should be revoked.
2. Additional toxicity data should be obtained before
new tolerances are established. New experiments to
establish a "no effect" dose should be performed in
chickens, and in a proven sensitive mammalian species
during a major portion of its life span.
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68
3. We suggest that commercial leptophos be sprayed on
plants under normal field conditions for at least one
full growing season, and then the residues on the plants
and in the soil be analyzed for composition. This
mixture with this residue composition should then be
used in a chronic feeding study in the chicken and
a sensitive mammalian species, for example, the cat.
The positive control experiment sho.uld be with commer-
cially pure leptophos. Feeding should be for a lifetime,
beginning at hatch or birth and continuing to maturity.
Because there are possibilities of an induced resistance
in young animals, some studies should begin at maturity
and continue for the life span of the species.
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69
REFERENCES
1. Proposed Toxicological Guidelines
2. Issuance of Tolerance on Lettuce and Tomatoes
3. Proposed Revocation of Tolerance
4. Davis, D.R. "Neurotoxicity of Organophosphorous
Compounds," H andbuch der Experiments lien
Pharmako1ogie. Berlin :Springer-Verlag,
I9T37
5. Cavanagh, John B. "Peripheral Neurophathy Caused
by Chemical Agents," C R C Critical Rev Jews in
Toxicology, June, 1973>~p~p~!365-417~.
6. Johnson, M.K. "The Delayed Neuropathy Caused by
Some Organophosphorus Esters: Mechanisms
and Challenge," CRC Critical^ Rey i ew s in Tox i-
cology, June, 1975, pp. 289-316.
7. Aldridge, W.N. et al. "Studies on Delayed Neurotox-
icity Produced by Some Organophosphorous Com-
pounds ," J__. of the New York Academy of Sciences ,
June, 1969, pp. 314-322.
8.- Cavanagh, J.B. "The Significance of the 'Dying Back1
Process in Experimental and Human Neurological
Diseases," IttLeriiaL^uaal Review of Exp. Pac'n^,
No. 3, 19647 pp. 219-T6I" '
9. Cavanagh, J.B. "The Toxic Effects of Tri-Ortho-
Cresyl Phosphate on the Nervous System: An
Experimental Study on Hens, " J. of Neurol.
N eurosurg . P_ s y c h i a t. , vol. 17, 1954, pp.
163-172."
10. Velsicol Chemical Corporation, Chicago, 111., Pho s-
ve 1 Insecticide General Bulletin, no. 09-070-
601 A, FetTi 19TI7
11. Leuck, D.B. et al. "Residues of Velsicol VCS-506:
Their Persistence and Degradation in Forage
Corn," J. of Economic Entomology, vol. 62, no.
6, Dec ember 19~6~9^ ppT~l4 58-14 6 1 .
12 Leuck, D.C. et al . "Persistence of Velsicol VCS-
506 (0-(4-bromo-2, 5-dich1 oropheny1 0-methyl
phenylphosphonothioate), Its Oxygen Analogue
and its Phenol in Coastal Bermuda Grass Pasture,"
Scientific Notes , August 1970, pp. 1346-1347.
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layed Neurotoxicity of Leptophos: Toxic Effects
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-------
70
15. Bowman, Malcolm C. and Bero'za, Morton. "Determination
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506), Its Oxygen Analog and Its Phenolic Hy-
drolysis Product in Corn and Milk by Gas Chroma-
tography," J.ofAgr. Food C hem. , 1969, pp.
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17. "Tomato Growers Spray Less with New Insecticide^"
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71
29. Materials and Viewpoints of EOF, Velsicol request for
Advisory Committee and others.
30. Suggested Protocols for Assessment of the Possible
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Se cond St udy .
31. Abou-Donia, M.B. and Preissig, S.H. "Neurotoxicity
Produced by Long-Term and Low-Level Feeding of
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gist , vol. 17, no. 2, Fall 1975.
32. Metcalf, R.L. and Sanborn, J.R. "Pesticides and En-
vironmental Quality in Illinois," N at. Hist. S urvey
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33. Sanborn, James R. "The Neurotoxicity of 0-(2,5-
Dichlorophenyl) Q-Methyl Phenylphosphonothi-
oate, an Impurity and Photoproduct of Lepto-
phos (PhosvelR) Insecticides." Submitted to
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34. Hassan, Alad in et al . "Chemistry and Toxicology of
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in the Rat." Lectures at IUPAC Third Inter-
national Congress of Pesicide Chemistry, July
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35. Abou-Donia, M.B. and Preissig, S.H. "Neurotoxicity of
Long-Term Low-Dose Feeding of Leptophos to Hens."
Prepublication , Duke University Medical Center, 1976,
36. Barnes, J.M. (Medical Research Council, Carshalton,
Surrey, England). Communication to Dr. Richard-
son (EPA) dated July 11, 1975.
37. Pesticide Safety Precautions Scheme Agreed Between
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minster House, London, April 1967.
38. Certification of Usefulness Pesticide Petition 2F
1228 (per communication from P.C. Williams,
EPA, to Drew M. Baker,EPA, dated August 23,
1972) .
39. Braun, H.E. "Gas-Liquid Chroraotographic Determination
of Residues of Chlorpyriphos and Leptophos, Includ-
ing their Major Metabolites, in Vegetable Tissue,"
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7:Abst.#74-0990, 1974) .
40. Braun, H.E. et al . "Residues of Leptophos and its Me-
tabolites Following Application to Various Plants,"
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90-95. (2 refernces) (ABST:HAPAB 8:Abst.#75-
0782, 1975) .
41. Braun, H.E. et al . "Residues of Chlorpyriphos and
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Abst . # 75-1040, 1975) .
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42. Currie, R.A. "Determination of Leptophos, Lepto-
phos Oxon and a Possible Phenolic Photoconver-
sion Metabolite in Rapeseed Grain, " J_. of the
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Abst . # 75-0227, 1974) .
43. Struble, D.L. and McDonald, S. "Residue Analysis of
Leptophos, its 'Oxygen Analogue, and its Phenol
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mol . , vol. 66, no. 6, 1973, pp. 1321-1325.(13
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44. Townsen, L.R. and Specht , H.B. "Organophosphorous
and Organochlorine Pesticide Residues in Soil
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(ABSTrHAPAB :Abst . // 75-2332, 1975).
45. Certain Knowledgeable Individuals on Leptophos.
46. Johnson, M.K. "Structure-Activity Relationships
for Substrates and Inhibitors of Hen Brain Neuro-
toxic Esterase," Biochemical Pharmacology, vol.
24, 1974, pp. 797-805.
47. Sanborn, James R. (Illinois Natural History Survey).
Communication to Edwin Johnson (EPA) dated Feb-
ruary 16, 1976.
48. El-Sayed, Mohammed. Report on Bagour Experiment to
De Le i'ni iu e Effects of Phosvcl Use on. Gs.tuGU3 (Water
Buffalos). Dokki,Cairo, Egypt: Ministry of Agri-
culture, Central Pesticide Library.
49. Richardson, Howard (EPA). Phosvel-Demyelination
Studies, memo dated August 27, 1974.
50. Shafik, T.M. et al. Trip Report (Egypt) SFCP # 3-
545-1, 3-545-2, 3-545-3, memo to Dr. Donald Oak-
ley, dated May 21, 1975.
51. Ho tell ing, Virginia (American Association of Path-
thologists) . Communication to Dr. Gunter Zweig
(EPA) dated December 18, 1975 and including one
ab strac t .
51a. Richardson, M.E. et al. Morphologic Lesions in the
Sciatic Nerve of Chickens. Abstract, Washington,
D.C.: U.S. Environmental Protection Agency and
Armed Forces Institute of Pathology, 1975.
52. El-Sebae, A.H. Effects of Insecticides on Animals
and Plants . Technical Report, EPA PR 3-545-
1 (Summary of Activities), 1975.
53. En an, O.H. et al. Interaction Between Insecticides
in their Toxicity to White Mice, a report. Alex-
andria, Egypt: University of Alexandria, 1975.
54. Abou Akkada, A.R. et al. Effect of Phosvel on the
Metabalism, Performance and General Conditions
of Sheep, a report. Al exand r ia , Egyp t : Un iv .
of Alexandria, 1975.
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55. Abou Akkada, A.R. et al. Toxic Effects of Insecti-
cides on Farm Animals-E£fect of Some Insecticides *
on Artif-iclal Rumen M 1 cro- o r g an isms of Sheep , {
a report. Alexandria, Egypt: Univ. of Alex-
andria , 1975.
56. El-Sebae, A.H. et al. Acute and Delayed Neuro-
toxic ity , a report. Alexandria, Egypt: Univ. of « \
Alexandria, 1975.
57. El-Sayed, M.M. Levels of Toxicants in the Egyptiau
Environment . ^Technical Report (Foreign Research i
Agreement PR 3-545-3). Dokki, Cairo, Egypt: Min- !
istry of Agriculture, Central Agricultural Pesti-
cides Toxicological Laboratory, 1975.
58. Hassan, Alad in. A Study of Health Hazards of Pesti-
cides in Egypt. Report(EPA PR If 3-545-2). Dokki,
Cairo, Egypt: Pesticide Tox icolog ical Lab., 1975.
5 9. Report on Water Buffaloes Ex perinent in Egypt .
60. Industrial Bio-Test Laboratories, Inc.,, Northbrook,
II1 . Neurotoxicity Study with Technical Lepto~
phos in Chickens, Report to Velsicol Cheni . Corp.
IBT. No. 651-05789, 1975. ;
61. Scotti, Thomas M. (EPA). Communication to Gunter j
Zweig (EPA) , comments concerning Pathologic :
Findings in Leptophos Neurotoxicity Study, ;
dat^d December. 10. 1975. '
62. Scotti, Thomas M. (EPA). Communication to August
Curley (EPA), Review of Histologic Sections of
Sciatic Nerves of Chickens, received from AFPI,
EPA, dated April 26, 1976.
63. Kimbrough, Renate. Evaluation of LBT No. 651-
05789, Neurotoxicity Study of Technical Lepto-
phos in Chickens. P.O. No. C 64812, April 1976.
64. U.S. Environmental Protection Agency, Technical and
Literature Research Section. Pesticides Biblio-
graphies- Leptophos, February, 1976.
65. March, R.B. and Fukuto , T.R. R.eport to VelsicolCorp.
on Studies on the Photolysis and Metabolism of
P hosvel . Unpublished, 1975.
66. Scotti, Thomas M. (EPA). Communication to Dr. Gun-
ter Zweig (EPA) dated March 24, 1976.
67. Franklin, C.A. Report on the Neurotoxicity of Lepto~
£jh_p_s . Ot t awa : En v irontnen tal Health Directorate.
Presented at an Informal Tripartite (US, UK,
Canada) Meeting on pesticides, Washington, D.C.,
November 12-12, 1976.
68. Richardson, Howard L. (EPA). Communication to Donald
H. Jenkins (Wedge's Creek Research Farm, Neills-
ville, Wise.) dated July 29, 1975.
69. Jenkins, Donald H.(Wedge's Creek Research Farm, Neills-
ville, Wise .) . Communication to Dr . Mary E.
Richardson (EPA) dated August 18, 1975.
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74
70. Richardson, Howard L. (EPA). Communication to Hen-
ry Korp (EPA) dated November 15, 1974.
71. Photocopies of Photomicrography of tissues of birds
treated with TOCP and Leptophos.
72. Anderson, Ralph R. (Velsicol Chem. Corp.). Communi-
cation to David Bowen (EPA) dated June 23, 1974
and including two reports.
72a. Whitacre, D.M. et al. Metabolism of 14C-4-Brorao-
2 , 5-dichloropheno1 in Rats; A Multiple Dosing
Study. Chicago: Velsicol Chetn. Corp. Research
Dept. , March 1975.
72b. E.G. and G. Bionomics Toxicology Laboratory, Ware-
ham, Mass. Kinetics of 14C-PhosvelR in a Model
A c[u atic Ecosyst em , Nov ember 1975.
73. Abou-Donia, M.B. (Duke Univ. Med. Center). Communi-
cation to David Bowen (EPA) dated July 8, 1976
and including three abstracts and two published
reports.
73a. Abou-Donia, M.B. "Pharmacokinetics of a Sub Neurotoxic
Dose of Leptophos". Abstract, reprinted from
Fed e r at ion Proceedings, vol. 35, no. 3, March
1976.
73b. Abou-Donia, M.B. and Preissig, S.H. "Neurotoxicity Pro-
duced by Long-Term Low-Level Feeding of Leptophos."
Abstract, reprinted from Pharnacologist , vol. 17,
no. 2, Fall 1975.
73c. Abou-Donia, M.B. and Preissig, S.H. "Studies on the
Delayed Neurotoxicity Produced by Leptophos."
Abstract, reprinted from Federation Proceedings.,
vol. 34, no. 3, March 1, 1975.
74d, Abou-Donia, M.B. and Preissig, S.H. "Delayed Neurotox-
icity of Leptophos: Toxic Effect on the Nervous Sys-
tem of Hens," Applied Pharmacology, vol. 35, 1976, pp.
269-282.
74e. Abou-Donia, M.B. et al. "Neurotoxic Effects of Lepto-
phos," Separatum Experientia, vol. 30, 1974,
pp. 63-64.
75. missing
76. Hamilton, Peter B. (Williams, Connolly, and Califano).
Communication to Andrew W. Breidenbach (EPA) dated
July 15, 1976.
77. Breidenbach, Andrew W. (EPA). Communication to Peter
B. Hamilton (Williams, Connolly, and Califano) dated
July 19, 1976.
78.missing
79. Industrial Bio-Tests Laboratories, Inc., Northbrook, 111.
Recombination Assay of Phosvel Using Two Genotypes
of Bacillus Subtilis, Marburg Strain. IBT No. 633-
07858-B, February 2, 1976.
80. Industrial Bio-Tests Laboratories, Inc., Northbrook, 111.
Host-Mediated Assay for Detection of Mutation In-
duced by Phosv el _ Tec hn ical. IBT _lc. 623-07846,
February 27, 1976.
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75
81. Industrial Bio-Tests Laboratories, Inc., North-
brook, 111. Reverse Mutation Studies with
Phosvel- in Five Salmonella Strains and One
Sacc h a r omyces Strain. 1ST. No. 633-07848,
February 23, 1976.
82. Industrial Bio-Tests Laboratories, Inc., North-
brook, 111. N euro to_x ic i ty Study with Tech-
nical Leptophos in Chickens. I3T. No . 651-
05789, May 6, 1975.
83. Curley, August (EPA, Research Triangle Park, N.C.).
Communication to David I. Brandwein (EPA) dated
August 3, 1976 and including four unpublished
reports .
83a . Hassan, Aladin, PhD. Chemistry and Toxicology of
Pesticide Chemicals, VIII. Control data
necessary for evaluation of human exposure to
organophosphate chemicals. Mimeographed.
Cairo, Egypt: M.E. Regional Radioisotope Cen-
ter and Atomic Energy Authority in conjunction
with SFCP, EPA, Washington, B.C.
83b . Hassan, Aladin, PhD. et al . Chemistry and Toxicology
of Pesticide Chemicals, IX. Clinical observations
and biochemical studies on humans ^exposed to
Phosvel. Mimeograped. Cairo, Egypt: M.E.
Regional Radioisotope Center, Atomic Energy
Authority, and Al-Azhan Univ. in conjunction
with SFCP, EPA, Washington, D.C.
83c. Hassan, Aladin, PhD. et al. Chemistry and Toxicology
of Pesticide Chemicals, X. Hepatic, Renal, and
Neurohormonal function in humans exposed to Phos-
vel . Mimeographed. Cairo, Egypt: M.E. Regional
Radioisotope Center in conjunction with SFCP, EPA,
Washington, D.C.
83d. Hassan, Aladin,-PhD. et al. Chemistry and Toxicology
_of_Pesticide Chemicals , XI . Human intoxication
by P hosvel . Cairo, Egypt: M.E. Regional Radio-
isotope Center and Al-Azhar Univ. in conjunction
with SFCP, EPA, Washington, D.C.
84. Markel, Harold L. (DHEW, Region VI). Communication
to William A. Felsing, Jr. (DHEW) dated April
2, 1976 and including a preliminary survey re-
port concerning health conditions at the Bayport
PI ant , Texas .
85. Tanaka, Shiro, M.D. Communication to James A. Hacker
(Velsicol Chemical Corp.) dated August 5, 1976.
86. World Health Organization. Pesticide Residues in
Food , Report of the 1975 Joint Meeting o£ the
FAQ Working Party of the Experts on Pesticide
Residues and the WHO Expert Committee on Pesti-
c i d_e Residues. Geneva: World Health Organiza-
tion, Technical Rept. Series No. 592, 1976.
87. Leptophus, chapter from above (86).
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76
88. Baron, Ronald L., ed. Pest icide Delayed M eurotox ic i ty,
Proceedings of a Conference, Feb. 19-20, 1976.
Washington, D.C.: U.S. Environmental Protection
Agency, pub. no. EPA-600-1-76-025, July 1976.
89.' Anderson, Ralph F. (Velsicol Chemical Corp.) Com-
munication to David Bowen (EPA) dated August
18, 1976 and including various materials prev-
iously requested by the Leptophos Advisory Com-
mittee.
89a. Velsicol Chemical Corp. Technical Leptophos
Manufacturing-Bayport, Tex as:Report to the
U .S . _Environmental Protection Agency. Au-
gust 12, 1976.
89b . Levitan, Stephen R. Industrial Hygiene Survey:
Exposure to Phosvel. Painesville (Ohio):
Diamond Shamrock Corp., Environmental Labs,
1976.
89c. Medical Screening Clinics, Inc. Pasadena, Texas.
Results of Routine Cholinesterase Depression
Tests Using Phosvel at a U.S. Manufacturing
Facility. May 25, 1976.
89d. Coulston, Frederick, M.D. et al. Preliminary Study
of the Effects of Leptophos on White Leghorn
Chickens, Cats, and Rhesus Monkeys. Albany:
Al ben" Medical Co 1 1 c~ c T~3tit"*"c o S Coni~ciru~
tive and Human Toxicology.
89e. Wills, J.H., PhD. Communication to Dr. David
Whitacre (Velsicol Chemical Corp.) concerning
aspects of the study cited in 89d.
89f. Velsicol Chemical Corp. Protocol for One-Year
Chronic Neurotoxicity and Reproduction Study
with Leptophos in Adult Hens .
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APPENDICES
Appendix A
LEPTOPHOS ADVISORY COMMITTEE*
MEMBERS PRESENT:
JULIUS COON, CHAIRMAN
SEYMOUR FRIESS
TETSUO FUKUTO
BERNARD MC NAMARA
GERALD ROSEN
OTHERS PRESENT:
DAVID JBOWEri
JOHN L Y 0 M
FRANKLIN GEE
WILLIAM UPHOLT
ORVILLE PAYNTER
GEORGE WHITMORE
PETER HAMILTON
CHARLES CALA
DAVID WHITACRE
NEIL MITCHELL
CLAIRE FRANKLIN
DR. ZWEIG
* Persons present at the Leptophos Advisory Committee
Meeting (Public Session), July 20, 1976, Washington,
D.C.
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APPENDIX B *
EXHIBIT
REFERENCE NUMBER
L#2
L#5
L#6
L#7
L#8
L#9
L#10
L#ll
L#12
L#13
L#14
L#15
L#16
L#17
L#19
L#20
L#21
L#23
L#24
L#25
L#27
L#28
L#29
L#30
L#33
Lf 34
Lf>35
L#36
Lfrl37
L#38
L£39
L#40
L#41
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
*Each exhibit listing to the left has a corresponding reference
number to its right. Reference numbers are used in the report
instead of exhibit nunbers for citational purposes.
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